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https://openalex.org/W2334493322	https://amt.copernicus.org/articles/8/579/2015/amt-8-579-2015.pdf	English	null	First measurements of continuous δ&lt;sup&gt;18&lt;/sup&gt;O-CO&lt;sub&gt;2&lt;/sub&gt; with a Fourier Transform InfraRed spectrometer in Heidelberg, Germany	null	2,014	cc-by	13,095	First continuous measurements of δ18O-CO2 in air with a Fourier transform infrared spectrometer S. N. Vardag1, S. Hammer1, M. Sabasch1, D. W. T. Grifﬁth2, and I. Levin1 1Institut für Umweltphysik, Heidelberg University, Heidelberg, Germany 2Department of Chemistry, University of Wollongong, Wollongong, Australia Correspondence to: S. N. Vardag (svardag@iup.uni-heidelberg.de) Received: 15 May 2014 – Published in Atmos. Meas. Tech. Discuss.: 3 July 2014 Revised: 27 October 2014 – Accepted: 7 January 2015 – Published: 4 February 2015 Received: 15 May 2014 – Published in Atmos. Meas. Tech. Discuss.: 3 July 2014 Revised: 27 October 2014 – Accepted: 7 January 2015 – Published: 4 February 2015 highly resolved CO2, δ13C-CO2 and δ18O-CO2 records may provide in terms of better understanding regional scale con- tinental carbon exchange processes. Abstract. The continuous in situ measurement of δ18O in atmospheric CO2 opens a new door to differentiating be- tween CO2 source and sink components with high tempo- ral resolution. Continuous 13C–CO2 measurement systems have already been commercially available for some time, but until now, only few instruments have been able to pro- vide a continuous measurement of the oxygen isotope ratio in CO2. Besides precise 13C/12C observations, the Fourier transform infrared (FTIR) spectrometer is also able to mea- sure the 18O / 16O ratio in CO2, but the precision and ac- curacy of the measurements have not yet been evaluated. Here we present a ﬁrst analysis of δ18O-CO2 (and δ13C-CO2) measurements with the FTIR analyser in Heidelberg. We used Allan deviation to determine the repeatability of δ18O- CO2 measurements and found that it decreases from 0.25 ‰ for 10 min averages to about 0.1 ‰ after 2 h and remains at that value up to 24 h. We evaluated the measurement preci- sion over a 10-month period (intermediate measurement pre- cision) using daily working gas measurements and found that our spectrometer measured δ18O-CO2 to better than 0.3 ‰ at a temporal resolution of less than 10 min. The compati- bility of our FTIR-spectrometric measurements to isotope- ratio mass-spectrometric (IRMS) measurements was deter- mined by comparing FTIR measurements of cylinder gases and ambient air with IRMS measurements of ﬂask samples, ﬁlled with gases of the same cylinders or collected from the same ambient air intake. Two-sample t tests revealed that, at the 0.01 signiﬁcance level, the FTIR and the IRMS mea- surements do not differ signiﬁcantly from each other and are thus compatible. We describe two weekly episodes of ambi- ent air measurements, one in winter and one in summer, and discuss what potential insights and new challenges combined Atmos. Meas. Tech., 8, 579–592, 2015 www.atmos-meas-tech.net/8/579/2015/ doi:10.5194/amt-8-579-2015 © Author(s) 2015. CC Attribution 3.0 License. 1 Introduction Quantitative understanding of the processes governing the carbon cycle is vital in order to assess the impact and fate of increasing anthropogenic CO2 emissions to the atmosphere. The stable isotopes in CO2 can provide information about the ﬂuxes between the different carbon reservoirs, such as the atmosphere, the biosphere and the oceans. 13CO2 mea- surements can be used to distinguish between terrestrial bio- sphere and marine ﬂuxes (Keeling et al., 1989; Ciais et al., 1995), and are also used as a tracer for anthropogenic emis- sions, as most fossil fuel CO2 emissions are depleted in 13C relative to those of the biosphere (Tans, 1981). The interpre- tation of atmospheric δ18O-CO2 is more complex, since 18O in CO2 is strongly coupled to the water cycle (e.g. Francey and Tans, 1987; Farquhar et al., 1993; Cuntz et al., 2003a; 2003b, Buenning et al., 2014). During CO2 exchange with soil and leaves, the 18O isotopes of CO2 are exchanged with those of H2O (Hesterberg and Siegenthaler, 1991). Carbonic anhydrase facilitates the equilibration with leaf water (Gillon and Yakir, 2001; Farquhar et al., 1993). The isotopic com- position of soil water is determined by the isotopic compo- sition of precipitation, which itself has strong spatial varia- tions (IAEA/WMO GNIP database available at http://isohis. iaea.org). Since precipitation at higher latitudes is depleted in 18O (Dansgaard, 1964), the soil water and consequently 2 FTIR measurement principle and calibration procedure The in situ FTIR analyser used in Heidelberg was devel- oped and built at the University in Wollongong, Australia and is described in detail by Grifﬁth et al. (2012) and Ham- mer et al. (2013a). It was used during two travelling instru- ment campaigns by Hammer et al. (2013b) and Vardag et al. (2014) for CO2, CH4 and N2O measurements. Brieﬂy, the FTIR spectrometer obtains a broadband transmittance spec- trum of the sample air as the ratio of the infrared spectra measured with and without a sample in the optical cell. The measured transmittance spectrum is ﬁtted by non-linear least squares using the program MALT (Multi-Layer Absorption Transmittance) to model the spectrum (Grifﬁth, 1996; Grif- ﬁth et al., 2012). The model adjusts sample composition and instrument parameters to obtain the best ﬁt to the measured spectrum, and the best-ﬁt sample concentrations are taken as the retrieved values. The analyser is a prototype of the now commercially avail- able Spectronus FTIR trace gas analyser (Ecotech, Knox- ﬁeld, Australia). While functionally equivalent, there are some component differences. The FTIR spectrometer is an IRcube (Bruker Optics, Ettlingen, Germany) coupled to a glass multipass White cell (model 24 PA, IRanalysis Inc., Anaheim, CA) with 3.5 L volume and 24 m optical path. Spectra were recorded at 1 cm−1 resolution and typically co- added to 3 min averages (approximately one cell exchange time at the typical ﬂow rate). The sample handling system comprises four selectable inlets, an optional dryer (Naﬁon, Permapure PD-100T-24SS), followed by a granulated mag- nesium perchlorate trap), two mass ﬂow controllers (model D-5111, Bronkhorst, Germany) and a four-head diaphragm vacuum pump (model MV2, Vacuubrand, Germany). One mass ﬂow controller upstream of the cell controls sample air- The ﬁrst step to understanding the 18O-CO2 ﬂuxes to and from the terrestrial biosphere is to make reliable and com- parable measurements at high temporal resolution. However, measurements via isotope-ratio mass-spectrometry (IRMS) are elaborate and time-consuming, limiting the number of continuous records of 18O in CO2 that exist to date (Flana- gan et al., 1997; Langendörfer et al., 2002; Bowling et al., 2003; Pataki et al., 2003). A quantum cascade laser-based absorption spectrometer measuring 12C16O16O,13C16O16O and 12C16O18O with a high temporal resolution provided ﬁrst continuous records (Tuzson et al., 2011; Sturm et al., 2013). 12C16O16O and 13C16O16O have also been deter- mined by Fourier transform infrared (FTIR) spectroscopy in several previous studies (e.g. Published by Copernicus Publications on behalf of the European Geosciences Union. (2014) have studied the susceptibility of atmospheric δ18O-CO2 to environmental parameters, such as precipita- tion, relative humidity, temperature, solar radiation and cloud cover, and estimated the inﬂuences of these parameters on the atmospheric δ18O-CO2 using regional and global scale mod- els. They also assessed the effect of the isotopic composi- tion of precipitation and water vapour. They found that many of these parameters should not be neglected when quantify- ing biospheric gross 18O-CO2 ﬂuxes. They also highlight the complexity and the large uncertainties of the processes and sensitivities inﬂuencing atmospheric δ18O-CO2. Thus, in or- der to understand atmospheric δ18O-CO2 measurements in all their complexity, information about the regional isotopic composition of precipitation, environmental parameters such as temperature and water vapour deﬁcit and a comprehensive land-surface model are necessary (Yakir and Wang, 1996; Ciais et al., 1997; Langendörfer et al., 2002; Cuntz et al., 2003a; Buenning et al., 2014). The scope of this manuscript is to answer two important questions: ﬁrst, is it possible to measure δ18O-CO2 using FTIR spectroscopy, and if yes, how well can we measure it in terms of precision, accuracy and compatibility to con- ventional IRMS observations? Second, what insight into re- gional scale carbon exchange processes can one gain from a highly resolved δ18O-CO2 record (along with the continuous CO2, CO and δ13C-CO2 records) in the catchment area of our measurement site? Published by Copernicus Publications on behalf of the European Geosciences Union. Thus, in or- der to understand atmospheric δ18O-CO2 measurements in all their complexity, information about the regional isotopic composition of precipitation, environmental parameters such as temperature and water vapour deﬁcit and a comprehensive land-surface model are necessary (Yakir and Wang, 1996; Ciais et al., 1997; Langendörfer et al., 2002; Cuntz et al., 2003a; Buenning et al., 2014). The ﬁrst step to understanding the 18O-CO2 ﬂuxes to and al., 2008; Cambaliza, 2010; Grifﬁth et al., 2012). In prin- ciple, FTIR spectroscopy can also provide continuous mea- surements of 12C16O18O. However, in their original study, Esler et al. (2000) remarked that the degree of precision is too poor for a useful determination in natural abundances us- ing a 1 cm−1 resolution spectrometer. Given improvements in the instrumentation and spectral analysis methods since that time, we have revisited the practicality of continuous measurements of δ18O in CO2 using FTIR spectroscopy. the CO2 from root respiration and heterotrophic respiration is also depleted in 18O at higher latitudes (Farquhar et al., 1993). The soil invasion ﬂux will further inﬂuence the ap- parent soil respiration signature as the CO2 diffuses into the soil, partially equilibrates with soil water and retro-diffuses out of the soil with a new isotopic composition (Tans, 1998; Miller et al., 1999). Isotopic exchange during soil invasion might even be enhanced due to carbonic anhydrase in soils (Wingate et al., 2009). Miller et al. (1999) reported that in most settings and especially in dry ground and for short res- idence times of air close to the soil surface (corresponding to high boundary layer mixing heights), the effect will be smaller than 5 ‰. Due to 18O enrichment during evapotran- spiration, the plant leaf water is enriched in 18O relative to the soil water (Farquhar et al., 1993). During photosynthesis, CO2 equilibrates with leaf water and about two-thirds of the CO2 retro-diffuses into the atmosphere without being assimi- lated (Tans, 1998). The retro-diffused CO2 changes the atmo- spheric δ18O-CO2 value, depending on the isotopic signature of the leaf water. In central Europe, we expect the discrimi- nation against 18O during net CO2 assimilation to be positive (Farquhar et al., 1993; Cuntz et al. 2003b; Wingate et al., 2009). Still et al. (2009), Welp et al. (2011) and Buenning et al. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 580 the CO2 from root respiration and heterotrophic respiration is also depleted in 18O at higher latitudes (Farquhar et al., 1993). The soil invasion ﬂux will further inﬂuence the ap- parent soil respiration signature as the CO2 diffuses into the soil, partially equilibrates with soil water and retro-diffuses out of the soil with a new isotopic composition (Tans, 1998; Miller et al., 1999). Isotopic exchange during soil invasion might even be enhanced due to carbonic anhydrase in soils (Wingate et al., 2009). Miller et al. (1999) reported that in most settings and especially in dry ground and for short res- idence times of air close to the soil surface (corresponding to high boundary layer mixing heights), the effect will be smaller than 5 ‰. Due to 18O enrichment during evapotran- spiration, the plant leaf water is enriched in 18O relative to the soil water (Farquhar et al., 1993). During photosynthesis, CO2 equilibrates with leaf water and about two-thirds of the CO2 retro-diffuses into the atmosphere without being assimi- lated (Tans, 1998). The retro-diffused CO2 changes the atmo- spheric δ18O-CO2 value, depending on the isotopic signature of the leaf water. In central Europe, we expect the discrimi- nation against 18O during net CO2 assimilation to be positive (Farquhar et al., 1993; Cuntz et al. 2003b; Wingate et al., 2009). Still et al. (2009), Welp et al. (2011) and Buenning et al. (2014) have studied the susceptibility of atmospheric δ18O-CO2 to environmental parameters, such as precipita- tion, relative humidity, temperature, solar radiation and cloud cover, and estimated the inﬂuences of these parameters on the atmospheric δ18O-CO2 using regional and global scale mod- els. They also assessed the effect of the isotopic composi- tion of precipitation and water vapour. They found that many of these parameters should not be neglected when quantify- ing biospheric gross 18O-CO2 ﬂuxes. They also highlight the complexity and the large uncertainties of the processes and sensitivities inﬂuencing atmospheric δ18O-CO2. 2 FTIR measurement principle and calibration procedure Esler et al., 2000; Mohn et Atmos. Meas. Tech., 8, 579–592, 2015 www.atmos-meas-tech.net/8/579/2015/ S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 581 Wavenumber [cm -1] Wavenumber [cm -1] Figure 1. (a) Spectra of CO2 isotopologues and N2O in the 2150–2320 cm−1 region. The coloured traces show the individual isotopologues, the black spectra are a measured air spectrum (black line), calculated best-ﬁt spectrum (+ symbols) and the ﬁtting residual (black, upper panel). (b) Spectrum including CO2 and H2O near 3600 cm−1. The individual trace gas and isotopologue spectra are shifted by 0.2 upwards for clarity. Wavenumber [cm -1] Wavenumber [cm -1] Wavenumber [cm -1] Figure 1. (a) Spectra of CO2 isotopologues and N2O in the 2150–2320 cm−1 region. The coloured traces show the individual isotopologues, the black spectra are a measured air spectrum (black line), calculated best-ﬁt spectrum (+ symbols) and the ﬁtting residual (black, upper panel). (b) Spectrum including CO2 and H2O near 3600 cm−1. The individual trace gas and isotopologue spectra are shifted by 0.2 upwards for clarity. aligned condition as part of the Total Carbon Column Ob- serving Network (TCCON, Wunch et al., 2011) and its ILS is well characterised through high resolution test cell mea- surements (Hase et al., 2013) to be very close to the theoret- ically ideal shape calculated in the MALT model (modula- tion efﬁciency > 0.99, phase error < 0.5◦). Fitting these IFS 125/HR spectra resulted in residuals very similar in shape and magnitude to that in Fig. 1a (upper panel), which indi- cates that imperfect ILS is not the primary cause of the lack of ﬁt. To investigate the possible effects of non-Voigt molec- ular line shapes, typical FTIR analyser spectra were ﬁtted with two independent spectrum ﬁtting models, GFIT (Ge- off Toon, Jet Propulsion Laboratory) and PROFITT (Frank Hase, Karlsruhe Institute of Technology). These spectrum models optionally extend to non-Voigt line shapes including effects of speed dependent cross sections, line narrowing and line mixing (e.g. Ngo et al., 2013). Using several different line shape models did not remove the spectral residuals – in some cases they were slightly reduced or of different shape, but total residuals were reduced by at most 25 %. ﬂow, while the other downstream of the cell actively controls pressure via a proportional–integral software control loop to better than ±0.1 hPa. 2 FTIR measurement principle and calibration procedure The FTIR housing and the cell are both thermostated and stable within 0.01 ◦C (1σ). A Windows PC controls sample ﬂow, spectrum collection and online analy- sis. Sample air is delivered to the analyser at 1500–1800 hPa pressure through a clean diaphragm pump (model N86K.18, KNF Neuberger, Freiburg, Germany). In this work all mea- surements of both air samples and tank gases were dried (< 10 µmol mol−1 water vapour) and made at 1100 hPa pres- sure, 30 ◦C and a ﬂow of 1 SLPM (standard litre per minute). The measurements were performed in the laboratory under stable temperature conditions (±1 ◦C). Figure 1a shows the CO2 isotopologue components (coloured traces) of the infrared absorption spectrum of air in the 2150–2320 cm−1 spectral region routinely used for CO2 FTIR analysis by the analyser (Grifﬁth et al., 2012). The black traces show a measured spectrum and typical ﬁt to the composite air spectrum including 12C16O2, 13C16O2 and 12C18O16O. Although the 12C18O16O isotopologue is heavily overlapped by the parent and 13C16O2 isotopologues, its contribution to the total absorption is signiﬁcant and re- peatable and provides the basis for quantiﬁcation of this iso- topologue. The upper panel of Fig. 1a shows a typical spec- tral residual which is well above the detector noise level above 2240 cm−1. This residual is systematic and constant in shape from spectral ﬁt to ﬁt. The MALT spectrum calcula- tion model is not able to improve this ﬁt, which may be due to either (or both) an imperfect instrument line shape (ILS) or actual line shapes, which are not Voigt shaped as assumed in the model. To investigate the ILS contribution further, we have recorded spectra of air under the same conditions (tem- perature, pressure, resolution, cell path length) in a Bruker IFS 125/HR spectrometer at the University of Wollongong. This high resolution spectrometer is maintained in a well- Thus, from these two tests we conclude that the residuals are not primarily due to an imperfect instrument line shape, but rather due to the inadequacy of currently available line shape models for the calculation. The imperfect ﬁt is exacer- bated by the fact that the residuals are dominated by absorp- tion in the line wings of strongly absorbed lines, which are the least accurately modelled. We must therefore accept the imperfect ﬁts as unavoidable until further advances in line shape models become available. www.atmos-meas-tech.net/8/579/2015/ Atmos. Meas. Tech., 8, 579–592, 2015 2 FTIR measurement principle and calibration procedure If 12C18O16O is removed from the ﬁt, the residuals are two to three times larger; in this case, the least squares ﬁt routine adjusts the amounts of 12C16O2 and 13C16O2 in the ﬁt by unrealistic amounts to attempt to minimise the residual. The results in this paper show that the 12C18O16O amount retrieved from ﬁtting these www.atmos-meas-tech.net/8/579/2015/ Atmos. Meas. Tech., 8, 579–592, 2015 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 582 340 360 380 400 420 440 460 480 15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8 17.0 320 340 360 380 400 420 440 460 480 500 -0.10 -0.05 0.00 0.05 0.10 CO2 [µmol mol -1] δ 13C-CO2,raw CO2 [µmol mol -1] (a) (b) residuum δ 13C-CO2 340 360 380 400 420 440 460 480 15.4 15.6 15.8 16.0 16.2 16.4 16.6 16.8 17.0 340 360 380 400 420 440 460 480 55 60 65 70 75 80 85 90 95 320 340 360 380 400 420 440 460 480 500 -0.10 -0.05 0.00 0.05 0.10 320 340 360 380 400 420 440 460 480 500 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 CO2 [µmol mol -1] CO2 [µmol mol -1] CO2 [µmol mol -1] δ 13C-CO2,raw CO2 [µmol mol -1] (a) (c) δ 18O-CO2,raw (b) residuum δ 18O-CO2 residuum δ 13C-CO2 (d) Figure 2. CO2 dependence of raw δ13C-CO2 and δ18O-CO2 (a and b) and their residuals from the cubic ﬁt (b and d). The experimental results shown here were obtained in August 2012; the same experiment was repeated in March 2014 and showed no signiﬁcant difference to the earlier measurements. 340 360 380 400 420 440 460 480 55 60 65 70 75 80 85 90 95 320 340 360 380 400 420 440 460 480 500 -0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 CO2 [µmol mol -1] CO2 [µmol mol -1] (c) δ 18O-CO2,raw residuum δ 18O-CO2 (d) residuum δ 18O-CO2 CO2 [µmol mol -1] Figure 2. CO2 dependence of raw δ13C-CO2 and δ18O-CO2 (a and b) and their residuals from the cubic ﬁt (b and d). The experimental results shown here were obtained in August 2012; the same experiment was repeated in March 2014 and showed no signiﬁcant difference to the earlier measurements. 2.1 Data evaluation and calibration In the following, we describe the data evaluation and calibra- tion procedure for the isotopologue ratio δ18O-CO2, but the procedure is analogous for δ13C-CO2. 2 FTIR measurement principle and calibration procedure of the common isotopologue: of the common isotopologue: of the common isotopologue: spectra, despite the residuals, provides a consistent basis for quantiﬁcation of this isotopologue. δ18O-CO2HITRAN =   12C18O16Oraw 12C16O16Oraw sample RHITRAN −1  · 1000 ‰ (1) Total CO2 can also be retrieved from the region around 3600 cm−1 without isotopic discrimination. The ﬁt to this re- gion is shown in Fig. 1b, from Grifﬁth et al. (2012). Retrieval of CO2 from this region is more precise (i.e. lower noise, better repeatability) than that of 12C16O2 near 2300 cm−1, ﬁrstly because the bands are not saturated and are of near op- timum absorption (50 %), and secondly, because the whole bands have lower temperature sensitivity due to the inclusion of both high and low-J lines with both positive and negative temperature sensitivity. As detailed below, the total CO2 re- trieval, scaled if required, can be used as a proxy for 12C16O2 in isotopic calculations with acceptable accuracy. (1) with RHITRAN = 0.0040104 (Rothman et al., 2005). For 13C, the equivalent value of RHITRAN is 0.0112372. Following Coplen (2011) and common usage, we use the terminology δ18O-CO2, even though the δ-notation is originally deﬁned with the isotope ratio (in contrast to isotopologue ratio). The FTIR analysis implicitly uses the HITRAN scale (Rothman et al., 2005), which is re- ferred to Vienna Pee Dee Belemnite (VPDB) for δ13C and to Vienna Standard Mean Ocean Water (VSMOW) for δ18O; during the calibration (step 3) the ﬁnal reference scale of the calibrated data can be changed to any other scale. We chose the VPDB scale for δ13C-CO2 ((13C16O2 /12C16O2)VPDB = 0.0112372) and VPDB-CO2 scale for δ18O-CO2 ((12C18O16O/12C16O2)VPDB−CO2 = 0.0041767) following Allison et al. (1995). We abbreviate δ13C-CO2 and δ18O-CO2 on the VPDB-CO2 scale with δ13C-CO2,VPDB and δ18O-CO2,VPDB respectively. S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR Table 1. Interspecies interference and cross-sensitivity correction factors for δ13C-CO2 and δ18O-CO2 used in Eqs. (2) and (3). Refer- ence values were Tref = 31.8 ◦C, Fref = 1.0 SLPM, Pref = 1100 hPa and H2Oref = 0 µmole mole−1, for temperature, ﬂow, pressure and water vapour content, respectively. Table 1. Interspecies interference and cross-sensitivity correction factors for δ13C-CO2 and δ18O-CO2 used in Eqs. (2) and (3). Refer- ence values were Tref = 31.8 ◦C, Fref = 1.0 SLPM, Pref = 1100 hPa and H2Oref = 0 µmole mole−1, for temperature, ﬂow, pressure and water vapour content, respectively. δ13C-CO2 δ18O-CO2 dCraw/dT [‰ ◦C−1] 0.127 4.256 dCraw/dF [‰ SLPM−1] −0.91424 −2.92166 dCraw/dP [‰ hPa−1] 0.00249 −0.18694 dCraw/dH2O [‰ (µmole mole−1)−1] 0 0 a [‰] −10.344 −252.786 b [‰ (µmole mole−1)−1] 0.0461902 1.162269 c [‰ (µmole mole−1)−2] −0.0000658108 −0.00179787 d [‰ (µmole mole−1)−3] 0.000000034299 0.000001093919 δ13C-CO2 δ18O-CO2 dCraw/dT [‰ ◦C−1] 0.127 4.256 dCraw/dF [‰ SLPM−1] −0.91424 −2.92166 dCraw/dP [‰ hPa−1] 0.00249 −0.18694 dCraw/dH2O [‰ (µmole mole−1)−1] 0 0 a [‰] −10.344 −252.786 b [‰ (µmole mole−1)−1] 0.0461902 1.162269 c [‰ (µmole mole−1)−2] −0.0000658108 −0.00179787 d [‰ (µmole mole−1)−3] 0.000000034299 0.000001093919 to 470 µmol mol−1 for CO2 mole fraction, a δ13C-CO2,VPDB range from −8.7 to −12.8 ‰ and a δ18O-CO2,VPDB range from −1.9 to −5.0 ‰ as determined by the Heidelberg IRMS (Neubert, 1998). tion bands in the ﬁt. However, small second order effects re- main due to real imperfections in temperature and pressure measurements, spectrometer instrumental line shape and the assumption of the MALT models (such as Voigt line shapes, see above), necessitating small empirical corrections to the raw measured mole fraction (Craw) (Grifﬁth et al., 2012; Hammer et al., 2013a). A cross-sensitivity correction for sample temperature (T ) and pressure (P ), H2O amount and ﬂow rate (F), as well as an interspecies-sensitivity correc- tion for CO2 mole fraction (corr(CO2)) is applied for every measurement following Eq. (2): Step 4: Smoothed working standard correction We have found that regular measurements of different cylin- der gases on the FTIR analyser show small but correlated sub-weekly variations of δ18O-CO2. One can thus use a smoothed working standard correction in order to account for these small instrumental variations on a sub-weekly time scale. For this purpose, we smooth daily working gas mea- surements using a 10-point moving average and interpo- late the residual variation to the date of sample measure- ment using a cubic spline interpolation. We then subtract the smoothed residual variations from the long-term mean value of this gas vs. the reference standards from all sample mea- surements. By performing this correction, typically less than 0.2 ‰, the standard deviation of a weekly measured target or surveillance gas reaches about 0.2 ‰ for δ18O-CO2. Step 4 is not obligatory, but further increases the precision of the mea- surement. In the data presented in Sect. 4, we have applied this smoothed working standard correction. Ccorr = dCraw dT · (T −Tref) −dCraw dF · (F −Fref) −dCraw dP · (P −Pref) −dCraw dH2O · (H2O −H2Oref) 2 −corr(CO2) (2) (2) Where Pref, Tref, etc. are the reference values to which pres- sure, temperature etc. are corrected, and the CO2 correction follows: corr(CO2) = a + b · Craw + c · Craw2 + d · Craw3 (3) (3) Table 1 lists all cross-sensitivity parameters and CO2- interspecies interference corrections. g Figure 3 illustrates the application for the entire calibration procedure. For δ 13C-CO2, Fig. 3 shows raw (a), corrected (b) and calibrated (c) FTIR measurements against IRMS ref- erence values of δ13C-CO2, and Fig. 3d shows the difference between calibrated FTIR measurements and IRMS values (FTIR-IRMS) against CO2 mole fractions. Figure 3e–h show corresponding data for δ18O-CO2. The cross-sensitivity cor- rection forces δ13C-CO2 and δ18O-CO2 onto a linear regres- sion line (Fig. 3b and f), so that we can then apply a linear calibration. The large correction for δ18O-CO2 is most likely related to the systematic residual in the ﬁtting of the spectra (Fig. 1a). Hammer et al. (2013a) describe in detail the set-up of the experiment to determine the CO2 sensitivity. We use a cubic ﬁt to describe the CO2 interspecies correction (Fig. 2a and b; coefﬁcient of determination R2 = 0.99 for δ13C-CO2 and for δ18O-CO2), with residuals showing no further concentration dependence (Fig. 2b and d). Step 3: Calibration The cross-sensitivity corrected data are calibrated on the VPDB gas scale using a linear instrument response function (typically linear to the degree of R2 = 0.9998). We derive the calibration response function weekly from three refer- ence tanks with known values for CO2, δ13C-CO2 and δ18O- CO2. Our reference standards span ranges from about 370 Atmos. Meas. Tech., 8, 579–592, 2015 Step 2: Cross-sensitivity and interspecies interference corrections The FTIR computes the raw δ18O-CO2 value using the ratio of the raw value of the rare isotopologue and the raw value To ﬁrst order, the ﬁtting software MALT takes into account pressure, temperature and interspecies overlapping absorp- Atmos. Meas. Tech., 8, 579–592, 2015 www.atmos-meas-tech.net/8/579/2015/ S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 583 Atmos. Meas. Tech., 8, 579–592, 2015 2.2 Remarks on the calibration procedure al. (2012): 12C16O2 = CO2 X (4) (4) 2.2.1 Using total CO2 instead of 12C16O2 to calculate δ13C-CO2 and δ18O-CO2 where X is an isotopic partition sum with a value very close to unity. S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 584 -13 -12 -11 -10 -9 -8 16 18 20 22 -13 -12 -11 -10 -9 -8 16 18 20 22 -13 -12 -11 -10 -9 -8 -13 -12 -11 -10 -9 -8 350 400 450 500 -0.2 -0.1 0.0 0.1 0.2 -5 -4 -3 -2 -1 65 70 75 80 85 90 -5 -4 -3 -2 -1 75 76 77 78 79 80 81 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 350 400 450 500 -0.4 -0.2 0.0 0.2 0.4 δ 18O-CO2 (h) (c) (f) (e) (d) (g) (b) (a) FTIR - IRMS δ13C-CO2 [ ] Calibrated Uncalibrated Raw FTIR δ13C-CO2 [ ] IRMS δ13C-CO2 [ ] FTIR - IRMS δ18O-CO2 [ ] Calibrated Uncalibrated Raw FTIR δ18O-CO2 [ ] δ 13C-CO2 CO2 [µmol mol-1] IRMS δ18O-CO2 [ ] CO2 [µmol mol-1] Figure 3. (a) Raw, (b) cross- and interspecies corrected (but still un-calibrated) and (c) calibrated δ13C-CO2 measurements and (e) raw, (f) cross- and interspecies corrected (but still un-calibrated); (g) calibrated δ18O-CO2 measurements of different target cylinders against the IRMS measurement of the same cylinders. Lowest panels: (d) calibrated FTIR δ13C-CO2 value minus reference value measured by the Heidelberg IRMS, (h) same as (d) for δ18O-CO2, both plotted versus the CO2 mole fraction of the samples. The red lines in the lowest panels give the mean difference between the FTIR and the IRMS measurements. Grey areas illustrate the standard deviation of the differences. -13 -12 -11 -10 -9 -8 16 18 20 22 -13 -12 -11 -10 -9 -8 16 18 20 22 -13 -12 -11 -10 -9 -8 -13 -12 -11 -10 -9 -8 350 400 450 500 -0.2 -0.1 0.0 0.1 0.2 (c) (d) (b) (a) FTIR - IRMS δ13C-CO2 [ ] Calibrated Uncalibrated Raw FTIR δ13C-CO2 [ ] IRMS δ13C-CO2 [ ] δ 13C-CO2 CO2 [µmol mol-1] -5 -4 -3 -2 -1 65 70 75 80 85 90 -5 -4 -3 -2 -1 75 76 77 78 79 80 81 -5 -4 -3 -2 -1 -5 -4 -3 -2 -1 350 400 450 500 -0.4 -0.2 0.0 0.2 0.4 δ 18O-CO2 (h) (f) (e) (g) FTIR - IRMS δ18O-CO2 [ ] Calibrated Uncalibrated Raw FTIR δ18O-CO2 [ ] IRMS δ18O-CO2 [ ] CO2 [µmol mol-1] (but still un-calibrated) and (c) calibrated δ13C-CO2 measurements and (e) raw, 18 Figure 3. www.atmos-meas-tech.net/8/579/2015/ S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTI S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR (a) Raw, (b) cross- and interspecies corrected (but still un-calibrated) and (c) calibrated δ13C-CO2 measurements and (e) raw, (f) cross- and interspecies corrected (but still un-calibrated); (g) calibrated δ18O-CO2 measurements of different target cylinders against the IRMS measurement of the same cylinders. Lowest panels: (d) calibrated FTIR δ13C-CO2 value minus reference value measured by the Heidelberg IRMS, (h) same as (d) for δ18O-CO2, both plotted versus the CO2 mole fraction of the samples. The red lines in the lowest panels give the mean difference between the FTIR and the IRMS measurements. Grey areas illustrate the standard deviation of the differences. Raw, (b) cross- and interspecies corrected (but still un-calibrated) and (c) calibrated δ13C-CO2 measureme 3.1 Allan deviation with (12C18O16O/12C16O2)VPDB−CO2 = 0.0041767 (Allison et al., 1995), which takes into account that CO2 contains two oxygen atoms. We performed an Allan deviation repeatability test (Werle et al., 1993; Werle et al., 2011) on the FTIR system over 6 days from 17 September 2011 to 23 September 2011, with ﬂowing sample supplied from a reference gas cylinder with a δ13C-CO2 value of about −10.1 ‰ and a δ18O-CO2 value of about −3.7 ‰. We used the Allan deviation as a measure for the repeatability (following JCGM, 2008) as shown in Fig. 4. Allan deviation is the standard deviation of the pairwise dif- ferences between adjacent measurements averaged over dif- ferent averaging periods. In the absence of drift and with only white (random) noise, the Allan deviation will decrease with the square root of the averaging time. We found that the Al- lan deviations after 10 min were δ13C-CO2 = ± 0.03 ‰ and δ18O-CO2 = ± 0.25 ‰ (Fig. 4a and b). After 30 min, the Al- lan deviations decrease to δ13C-CO2 = ± 0.02 ‰ and δ18O- CO2 = ± 0.15 ‰. From 2 hours to up to 1 day, the Allan deviations stayed below about δ13C-CO2 = ± 0.02 ‰ and δ18O-CO2 = ± 0.10 ‰. No signiﬁcant increase in Allan de- viation could be observed within 1 day, since drifts on this time scale are small compared to the noise. Further, we can conﬁrm that the frequency of smoothed working standard correction is adequate, since between daily working standard gas measurements the system remains stable within 0.02 ‰ for δ13C-CO2 and 0.10 ‰ for δ18O-CO2. In Heidelberg, a typical diurnal variation of δ18O-CO2 is of the order of 1 ‰ (see Sect. 4). Thus, the system is stable enough to resolve diurnal ambient δ18O-CO2 variations (see Sect. 4). In principle, both methods should lead to the same re- sults, but they are sensitive to errors in different ways (Grif- ﬁth et al., 2012). In practice, we ﬁnd they differ by about 0.11 ± 0.03 ‰ for δ13C-CO2 and by 0.08 ± 0.15 ‰ for δ18O- CO2 (mean ± standard deviation for a 2-month period in 2014). The discrepancy between both calibration methods is most likely due to small inaccuracies in interspecies interfer- ence corrections. The ratio calibration requires a large CO2- interspecies interference correction over a large CO2 range (see Fig. 2c). 3.1 Allan deviation Only if the CO2 interspecies interference cor- rection is well determined can we obtain a reliable δ18O- CO2 value from the ratio method. For the independent iso- topologue calibration, no explicit interspecies CO2 correc- tion is required, but a very accurate determination of all CO2 isotopologue calibration equations is vital. The decision on which method to use should thus be based on which correc- tion can be performed with higher accuracy. In this work, we have found for the Heidelberg spectrometer that the empirical calibration method better ﬁts the Heidelberg IRMS values. S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR correct the isotopologue amounts 16O12C16O, 16O13C16O and 16O12C18O (step 2), calibrate them individually (step 3) and ﬁnally compute δ18O-CO2 and δ13C-CO2 from the cali- brated amounts, i.e. correct the isotopologue amounts 16O12C16O, 16O13C16O and 16O12C18O (step 2), calibrate them individually (step 3) and ﬁnally compute δ18O-CO2 and δ13C-CO2 from the cali- brated amounts, i.e. more details on the IRMS, see Neubert (1998). A two-sample t test reveals that, at the 0.01 signiﬁcance level, the means of the FTIR and the IRMS measurements (Fig. 3d and h) for δ13C-CO2 and for δ18O-CO2 do not differ signiﬁcantly and thus, are compatible. δ18O −CO2,VPDB =  12C18O16O 12C16O16O sample 12C18O16O 12C16O16O VPDB−CO2 −1 · 1000 ‰ (5) 2.3 Direct cylinder comparison to mass spectrometric values In order to check the FTIR calibration as well as the compat- ibility of the FTIR and the Heidelberg IRMS Finnigan MAT 252, we analysed measurements of different test cylinders in March and April 2014 on both instruments. The IRMS val- ues are linked to the VPDB scale via three pure CO2 ref- erence gases (RM8562, RM8563 and RM8564). The FTIR reference cylinders were calibrated by the IRMS and thus the FTIR and the IRMS are on the same scale. For all cylin- der measurements with the IRMS, we ﬁlled cylinder air into evacuated ﬂasks from an intermediate transfer volume; we then analysed these ﬂasks by both techniques like regular ﬂask samples, since pressure regulator effects have often dis- turbed the IRMS analyses. The precision of the IRMS is about 0.02–0.03 ‰ for δ13C-CO2 and 0.05–0.1 ‰ for δ18O- CO2 (standard deviation of repeated ﬂask measurements). Further, Wendeberg et al. (2013) have shown that the Hei- delberg IRMS scale does not exhibit any signiﬁcant scale contraction errors or errors through cross contamination be- tween sample and standard measurements in the IRMS. For 3 Characterisation of δ18O-CO2 and δ13C-CO2 measurements with the Heidelberg FTIR (5) 2.2.2 Direct isotopologue calibration As pointed out above, the precision of total CO2 measure- ment in the 3600 cm−1 range is signiﬁcantly higher (∼50 %) than that of 12C16O2 in the region of 2300 cm−1, due to an optimum absorption strength and a lower temperature sen- sitivity. 12C16O2, 13C16O2 as well as 12C16O18O absorb in this region, but the minor isotopologue absorptions are weak and are barely distinguishable. Thus, we calculate the raw δ13C-CO2 and δ18O-CO2 values using total CO2 from the 3600 cm−1 region instead of 12C16O2. There is a small bias between measurements of CO2 and12C16O2, but as long as the isotopic composition of the sample is close to the isotopic composition of the reference standards, the bias in δ13C- CO2 and δ18O-CO2 is negligible (< 0.03 ‰ for δ13C-CO2 and < 0.05 ‰ for δ18O-CO2) after calibration (step 3). How- ever, for strongly depleted cylinder gases, as may be the case for synthetic gas mixtures, the biases may become as large as 0.2 ‰. If necessary, the bias introduced by total CO2 can be corrected iteratively using Eqs. (8) and (9) of Grifﬁth et Grifﬁth et al. (2012) described two methods for calibration of isotopic fractionations, either Grifﬁth et al. (2012) described two methods for calibration of isotopic fractionations, either a. the isotopologue amounts are calibrated independently and the isotopologue δ values calculated directly from the calibrated isotopologue amounts, or b. the isotopologue δ values are calculated from raw mea- surements of the isotopologues and the calibration is carried through on the δ-values. These methods were referred to as “absolute” and “empiri- cal” calibration respectively by Grifﬁth et al. (2012), but to avoid ambiguity we will refer to them here as (a) isotopo- logue calibration and (b) ratio or δ-calibration. The correc- tion and calibration method described above and used in this work is the ratio calibration, (b). In principle, it is equally valid to use (direct) isotopologue calibration. In this case, we Atmos. Meas. Tech., 8, 579–592, 2015 www.atmos-meas-tech.net/8/579/2015/ 585 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR www.atmos-meas-tech.net/8/579/2015/ 3.3 Compatibility of ambient air measurements -10.7 -10.6 -10.5 -10.4 Dez Jan Feb Mrz Apr Mai Jun Jul Aug Sep Okt -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 δ 13C-CO2 (a) δ 18O-CO2 Year 2012/2013 (b) In the previous sections, we have evaluated the repeatabil- ity, as well as the intermediate measurement precision of the FTIR measurements. The results make us conﬁdent that the FTIR spectrometer is of sufﬁcient precision and stabil- ity to resolve atmospheric signals, such as the diurnal varia- tion of δ13C-CO2 and δ18O-CO2. Further, we have shown in Sect. 2.3 that the FTIR cylinder gas measurements are com- patible to those of the Heidelberg IRMS. In order to show that not only the direct cylinder measurements, but also the am- bient air measurements are compatible with the IRMS anal- yses, we compared in situ ambient air samples, which we measured with both instruments. Year 2012/2013 For this purpose, an automated ﬂask sampler (Neubert et al., 2004) collected dried (dew point −40 ◦C) ambient air from the same intake line as the FTIR into 2.5 litre glass ﬂasks. Every ﬂask was ﬂushed with a ﬂow rate of about 1.1 SLPM for 2 h and then pressurised to 2000 hPa absolute pres- sure and closed. Then the automated ﬂask sampler opened, ﬂushed and ﬁlled the next ﬂask to 2000 hPa. Pressurising the ﬂasks took about 5 min. With this procedure, we were able to capture a diurnal isotopic proﬁle with a 2-hourly resolution in the ﬂasks, which could be analysed by mass spectrome- try. We then compared these values to the continuous values measured by the FTIR spectrometer; the results are shown in Fig. 6. We used 9 min averaged values from the FTIR spec- trometer to compare them to the ﬂask results to account for atmospheric variability and to minimise differences due to lack of temporal synchronisation between the event sampler and the FTIR, and to reduce the noise on the FTIR measure- ment. We found that the mean residual and standard error is 0.01 ± 0.02 ‰ for δ13C-CO2 and 0.08 ± 0.14 ‰ for δ18O- Figure 5. Repeated daily working gas measurements (9 min aver- ages) depict an intermediate measurement precision of (a) ±0.04 ‰ for δ13C-CO2 and of (b) ±0.27 ‰ for δ18O-CO2 (b) for the period from December 2012 to October 2013. Red lines: mean values, grey areas: standard deviation. 3.2 Intermediate measurement precision We monitored the intermediate measurement precision (fol- lowing JCGM, 2008) by measuring standard gases every day or week under reproducible conditions. The averaging time for each cylinder measurement was 9 min. We used the stan- dard deviation of the 9 min cylinder gas averages to estimate the intermediate measurement precision of our instrumental set-up. For δ13C-CO2 and δ18O-CO2, we found that the in- termediate measurement precision was 0.04 ‰ and 0.27 ‰, respectively for the period from December 2012 to Octo- ber 2013 (see Fig. 5a and b). The Allan deviation at 9 min is very close to the standard deviation of the daily working cylinder measurements, which shows that for our system and laboratory conditions the repeatability dominates the inter- mediate measurement precision. www.atmos-meas-tech.net/8/579/2015/ Atmos. Meas. Tech., 8, 579–592, 2015 Atmos. Meas. Tech., 8, 579–592, 2015 586 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR Elapsed time [min] Elapsed time [min] Figure 4. Allan deviation of δ13C-CO2 (left) and δ18O-CO2 (right) measured over the course of 6 days in September 2011 with the FTIR. Figure 4. Allan deviation of δ13C-CO2 (left) and δ18O-CO2 (right) measured over the course of 6 days in Se f δ13C-CO2 (left) and δ18O-CO2 (right) measured over the course of 6 days in September 2011 with the FTIR Figure 4. Allan deviation of δ13C-CO2 (left) and δ18O-CO2 (right) measured over the course of 6 days in September 2011 with the FTIR. -10.7 -10.6 -10.5 -10.4 Dez Jan Feb Mrz Apr Mai Jun Jul Aug Sep Okt -4.5 -4.0 -3.5 -3.0 -2.5 -2.0 δ 13C-CO2 (a) δ 18O-CO2 Year 2012/2013 (b) Figure 5. Repeated daily working gas measurements (9 min aver- ages) depict an intermediate measurement precision of (a) ±0.04 ‰ for δ13C-CO2 and of (b) ±0.27 ‰ for δ18O-CO2 (b) for the period from December 2012 to October 2013. Red lines: mean values, grey areas: standard deviation. 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg In this section, we illustrate how we might potentially use a highly resolved δ18O-CO2 record at a typical European mon- itoring station, such as Heidelberg, in order to disentangle re- gional scale carbon exchange processes. Note, however, that for a quantitative evaluation, we would require explicit infor- mation on local CO2 source signatures and on the exchang- ing water reservoirs. We look here at two very different peri- ods in which the FTIR measured δ18O-CO2 along with δ13C- CO2, total CO2 and CO in Heidelberg (see Fig. 7). g g In order to interpret the atmospheric δ18O-CO2 variation, we must estimate the isotopic signature or discrimination of the processes inﬂuencing the isotopic content. The Hei- delberg catchment area is typical of many European urban areas with the most important CO2 ﬂuxes associated with plant photosynthesis, leaf and soil respiration, as well as fos- sil fuel burning. In the greater catchment area, discrimina- tion during photosynthesis tends to enrich atmospheric CO2 with respect to 13C and 18O (Cuntz et al., 2003b). Typical mean δ13C fractionation relative to the atmosphere during photosynthesis is about – (2–8) ‰ for C4 plants and about – (12–20) ‰ for C3 plants (Mook, 1994). As a ﬁrst ap- proximation, the 13CO2 / 12CO2 ratio captured during pho- tosynthesis is released during respiration, which leads to an overall depletion of the atmospheric 13CO2 / 12CO2 ratio. In addition, 18O discrimination during respiration tends to de- plete the atmosphere in its δ18O-CO2 value. Neubert (1998) measured the isotopic composition of soil-respired CO2 in the surroundings of Heidelberg and found values of δ18O- CO2,VPDB ≈−10 ‰ with a tendency of slightly more de- pleted values in winter (−15 ‰) than in summer (−5 ‰) and δ13C-CO2,VPDB ≈−25 ‰. For the discrimination dur- ing photosynthesis, typical mean values for the central Euro- pean continent are between 0 and +20 ‰ for 18O (Farquhar et al., 1993; Cuntz et al., 2003b). Further, the invasion ﬂux will inﬂuence the apparent soil respiration signature (Tans, 1998; Miller et al., 1999), but we cannot quantify the mag- nitude of this effect for our catchment area without inten- sive sampling and isotopic soil ﬂux modelling. Therefore, we only consider the invasion ﬂux in a sense that a larger range must be attributed to the signature of the apparent soil res- piration ﬂux when qualitatively discussing our atmospheric δ18O-CO2 records here. Figure 6. S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR There are the storage effect of the ﬂasks themselves, which could be slightly wet and thus alter the δ18O value of the CO2 in the ﬂask, or some other possible interference of the auto- mated ﬂask sampler (i.e. varying integration time due to ﬂow and pressure variations). CO2 [µmol mol -1] 3.3 Compatibility of ambient air measurements Note that in our calibration procedure we now use the daily measured cylinder (working standard gas) in a ﬁnal correc- tion step (step 4) to account for sub-weekly variations of the instrument response. Since we only recognised the need to correct for this variability well after commencement of the measurements, we do not yet have a long-term record for a real surveillance cylinder. Therefore, Fig. 5 displays the working standard measurements without any sub-weekly smoothing applied, and thus gives an upper estimate of the intermediate measurement precision of real measurements where we apply step 4 of our calibration procedure in ad- dition. www.atmos-meas-tech.net/8/579/2015/ Atmos. Meas. Tech., 8, 579–592, 2015 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 587 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR 400 425 450 475 -12 -10 -12 -10 -12 -10 -0.2 -0.1 0.0 0.1 0.2 -4 -3 -2 -1 0 -4 -3 -2 -1 0 -4 -3 -2 -1 0 15:00 21:00 03:00 09:00 -1.0 -0.5 0.0 0.5 1.0 FTIR averages Flask samples measured at the GC or IRMS (a) Continuous FTIR data CO2 [µmol mol -1] δ 13C-CO2 (b) ∆δ 13C-CO2 (c) (d) δ 18O-CO2 ∆δ 18O-CO2 3 rd/4 th March 2014 (e) Figure 6. Diurnal cycle event sampled on the 3–4 March 2014 at the Institut für Umweltphysik in Heidelberg. Red: GC concentration (in case of CO2) or IRMS isotopologue value (in case of isotopologues) of ﬂasks samples; blue: 9 min averaged values from FTIR; black: continuous 3 min values from the FTIR. (a) CO2 mole fraction; (b) δ13C-CO2 value; (c) residual of 9 min average δ13C-CO2 FTIR and IRMS measurement (FTIR - IRMS); (d) δ18O-CO2 value; (e) residual of 9 min averaged δ18O-CO2 FTIR and IRMS measure- ment (FTIR – IRMS). All error bars on the (blue) averaged FTIR data are the standard deviation during the 9 min of averaging time. The error bars on the (red) IRMS values show the typical inter- mediate measurement precision of our IRMS measurements. The residual (FTIR-IRMS) has an error bar, which combines the IRMS uncertainty and the FTIR uncertainty and the variability of atmo- spheric signal during the ﬂask ﬁlling time. 400 425 450 475 -12 -10 -12 -10 -12 -10 -0.2 -0.1 0.0 0.1 0.2 -4 -3 -2 -1 0 -4 -3 -2 -1 0 -4 -3 -2 -1 0 15:00 21:00 03:00 09:00 -1.0 -0.5 0.0 0.5 1.0 FTIR averages Flask samples measured at the GC or IRMS (a) Continuous FTIR data CO2 [µmol mol -1] δ 13C-CO2 (b) ∆δ 13C-CO2 (c) (d) δ 18O-CO2 ∆δ 18O-CO2 3 rd/4 th March 2014 (e) There are the storage effect of the ﬂasks themselves, which could be slightly wet and thus alter the δ18O value of the CO2 in the ﬂask, or some other possible interference of the auto- mated ﬂask sampler (i.e. varying integration time due to ﬂow and pressure variations). www.atmos-meas-tech.net/8/579/2015/ 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg Diurnal cycle event sampled on the 3–4 March 2014 at the Institut für Umweltphysik in Heidelberg. Red: GC concentration (in case of CO2) or IRMS isotopologue value (in case of isotopologues) of ﬂasks samples; blue: 9 min averaged values from FTIR; black: continuous 3 min values from the FTIR. (a) CO2 mole fraction; (b) δ13C-CO2 value; (c) residual of 9 min average δ13C-CO2 FTIR and IRMS measurement (FTIR - IRMS); (d) δ18O-CO2 value; (e) residual of 9 min averaged δ18O-CO2 FTIR and IRMS measure- ment (FTIR – IRMS). All error bars on the (blue) averaged FTIR data are the standard deviation during the 9 min of averaging time. The error bars on the (red) IRMS values show the typical inter- mediate measurement precision of our IRMS measurements. The residual (FTIR-IRMS) has an error bar, which combines the IRMS uncertainty and the FTIR uncertainty and the variability of atmo- spheric signal during the ﬂask ﬁlling time. CO2 (FTIR – IRMS). We tested the compatibility between the FTIR and the IRMS ambient air measurements with a two-sample t test and found that at the 0.01 signiﬁcance level, the means of the FTIR and the IRMS measurements in ambient air do not differ from each other for δ13C-CO2 or for δ18O-CO2. Note, that the standard deviation of the differences between the FTIR and the IRMS is 0.05 ‰ for δ13C-CO2 and 0.42 ‰ for δ18O-CO2 and with that the stan- dard deviation for δ18O-CO2 differences is higher than ex- pected from the combined Allan deviation (0.25 ‰ for 9 min averages) and the uncertainty of the IRMS measurement (ca. 0.05–0.1 ‰). The slightly larger variability in δ18O-CO2 ambient air comparison than in cylinder gas comparisons (Sect. 2.3) re- ﬂects the fact that there are more contributions to the differ- ence between the FTIR and the IRMS ﬂask measurement. For the isotopic signature of fossil fuels, most stud- ies assume a common δ18O fossil fuel signature of δ18O- CO2,VPDB ≈−17 ‰, corresponding to the ambient oxy- www.atmos-meas-tech.net/8/579/2015/ Atmos. Meas. Tech., 8, 579–592, 2015 588 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR -12 -10 -8 -12 -10 -8 -4 -2 -4 -2 200 400 600 350 400 450 500 -0.8 -0.4 0.0 19 20 21 22 23 -2 -1 0 1 2 ∆δ 18 δ 18O-CO2 δ 13C-CO2 CO [nmol mol -1] CO2 [µmol mol -1] meas.-constr. 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg ∆δ 13 December 2012 (a) (b) (c) (d) (e) (f) -12 -10 -8 -12 -10 -8 -2 0 2 -2 0 2 200 400 600 350 400 450 500 -0.4 0.0 0.4 1 2 3 4 5 6 -3 -2 -1 0 1 δ 13C-CO2,measured δ 13C-CO2,constructed δ 18O-CO2,measured δ 18O-CO2,constructed July 2013 (g) (h) (i) (j) (k) (l) Figure 7. Trace gas records in winter (left panel) and summer (right panel) in Heidelberg. (a) and (g) show the measured (dark blue) and artiﬁcially constructed (light blue) δ13C-CO2 value, (b) and (h) the measured (red) and artiﬁcially constructed (burgundy) δ18O-CO2 value, (c) and (i) the measured CO value, (d) and (j) the measured CO2 value. Panels (e) and (k) give the difference between the mea- sured and constructed δ13C-CO2 value with a mean isotopic source signature of δ13C-CO2,VPDB ≈−25 ‰ in the wintertime and δ13C- CO2,VPDB ≈−27 ‰ in the summertime. Panels (f) and (l) give the difference between the measured and constructed δ18O-CO2 value with a mean isotopic signature of δ18O-CO2,VPDB ≈−28 ‰ in the wintertime and δ18O-CO2,VPDB ≈−12 ‰ in the summertime. Grey vertical bars indicate the “reference periods”, in which the isotopic source signature for artiﬁcially constructed δ13C-CO2 and δ18O-CO2 was deter- mined from Keeling plots of about 20 individual atmospheric 3 min average measurements. The dashed vertical bar in the right panel shows a period of high precipitation. Grey horizontal bars in (f) and (l) mark the 1σ-uncertainty of the isotope measurements. -12 -10 -8 -12 -10 -8 -2 0 2 -2 0 2 200 400 600 350 400 450 500 -0.4 0.0 0.4 1 2 3 4 5 6 -3 -2 -1 0 1 δ 13C-CO2,measured δ 13C-CO2,constructed δ 18O-CO2,measured δ 18O-CO2,constructed July 2013 (g) (h) (i) (j) (k) (l) Figure 7. Trace gas records in winter (left panel) and summer (right panel) in Heidelberg. (a) and (g) show the measured (dark blue) and artiﬁcially constructed (light blue) δ13C-CO2 value, (b) and (h) the measured (red) and artiﬁcially constructed (burgundy) δ18O-CO2 value, (c) and (i) the measured CO value, (d) and (j) the measured CO2 value. Panels (e) and (k) give the difference between the mea- sured and constructed δ13C-CO2 value with a mean isotopic source signature of δ13C-CO2,VPDB ≈−25 ‰ in the wintertime and δ13C- CO2,VPDB ≈−27 ‰ in the summertime. 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg Panels (f) and (l) give the difference between the measured and constructed δ18O-CO2 value with a mean isotopic signature of δ18O-CO2,VPDB ≈−28 ‰ in the wintertime and δ18O-CO2,VPDB ≈−12 ‰ in the summertime. Grey vertical bars indicate the “reference periods”, in which the isotopic source signature for artiﬁcially constructed δ13C-CO2 and δ18O-CO2 was deter- mined from Keeling plots of about 20 individual atmospheric 3 min average measurements. The dashed vertical bar in the right panel shows a period of high precipitation. Grey horizontal bars in (f) and (l) mark the 1σ-uncertainty of the isotope measurements. ing sources is difﬁcult. Therefore we used the following approach: We ﬁrst constructed an artiﬁcial δ13C-CO2 and δ18O-CO2 record using the slope (aref) and offset (bref) of so-called “Keeling plots” (Keeling, 1958), determined from measured atmospheric δ- and CO2 concentration values in an exemplary and short nighttime reference period (grey bars in Fig. 7) according to: gen isotopic signature, but incomplete combustion can lead to a range of different isotopic signatures. The 18O sig- nature of fossil fuel emissions varies from about δ18O- CO2,VPDB ≈−11 to −40 ‰ (Schumacher et al., 2011). Traf- ﬁc exhausts tend to be less depleted in 18O relative to other fossil fuel CO2 emissions (δ18O-CO2,VPDB ≈−15 ‰), fol- lowed by natural gas burning (δ18O-CO2,VPDB ≈−28 ‰). Combustion of coal, on the other hand, leads to a δ18O- CO2 value of about −38 ‰ (Schumacher et al., 2011). To our knowledge, the potential range of these values is not well known. For δ13C, typical signatures are δ13C- CO2,VPDB ≈−29 ‰ for trafﬁc exhausts, −25 ‰ for coal combustion and −39 ‰ for natural gas emissions (Widory and Javoy, 2003; Kaul, 2007). With these examples of isotopic signatures, we can now look at our atmospheric CO2 records that show values of δ13C-CO2 between δ13C- CO2,VPDB ≈−8 and −12 ‰, while δ18O-CO2 varies be- tween δ18O-CO2,VPDB ≈−2 to −4 ‰ in winter and 0 to −2 ‰ in summer (Fig. 7). δmeas = aref · 1 CO2 + bref (6) (6) Note that in the nighttime reference periods, for which the reference slope and offsets were calculated, we can neglect photosynthetic sinks. Therefore, we can interpret the δ13C source signature of the reference period as the ﬂux-weighted average of all sources (Miller and Tans, 2003). www.atmos-meas-tech.net/8/579/2015/ 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg It is thus crucial to study also the hydrological conditions, such as precipitation and its isotopic signature, in order to quantitatively use the δ18O-CO2 records for carbon cycle research. erence period in which the Keeling plot slopes and offsets were derived, the Keeling plot had a high correlation coefﬁ- cient (r2 > 0.85) and showed an isotopic 13C and 18O source signature that was typical for the respective season (source δ13C-CO2 ≈−25 ‰ in the winter and −27 ‰ in the summer period, δ18O-CO2 ≈−28 ‰ in the winter and −12 ‰ in the summer period). To identify inﬂuences from enriching or de- pleting sources and sinks relative to those in the reference period, we then calculated the difference between the mea- sured and the artiﬁcially constructed (Eq. 6) δ13C-CO2 and δ18O-CO2 record (Fig. 7e, k, f and l): (8) 1δ(t) = δmeas(t) −δconstr(t). (8) Negative 1δ values occur in periods when the apparent sources are more depleted than in the reference period and positive values occur when apparent sources are more en- riched than in the reference period. During photosynthetic CO2 uptake, the equilibration of back-diffusing CO2 with enriched leaf water leads to an enrichment of atmospheric δ18O-CO2 and thus to positive 1δ18O values. We now have a tool that allows differentiation between more and less de- pleted ﬂuxes relative to the reference period. In the wintertime, relative fossil fuel contributions in the Heidelberg catchment area are higher than in the summer- time (Levin et al., 2003). Fossil fuel CO2 emissions lead to high concentration of CO2 (Fig. 7d) and deplete atmo- spheric CO2 in its heavy isotopes 13C and 18O (original mea- surements: dark blue and red in Fig. 7a, b). During incom- plete combustion of fossil fuels, CO (Fig. 7c) is often emit- ted as well. A typical example of a pollution event is shown in Fig. 7 (left panel) on 21 December 2012. The difference between the measured and artiﬁcially constructed δ13C-CO2 (Fig. 7e) decreases rapidly on 21 December. Environmen- tal parameters such as relative humidity, global radiation and temperature (not shown here) remain constant during the event, but low wind speed leads to an atmospheric inversion and, accompanied with a slight change of wind direction, to a more local source (mix), which is more depleted in 13C than during the reference period (δ13Cref = −25 ‰). 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg We then ap- plied the parameters (aref and bref) from the reference period to the entire CO2 record to calculate artiﬁcially constructed δ13Cconstr and δ18Oconstr values: Note that in the nighttime reference periods, for which the reference slope and offsets were calculated, we can neglect photosynthetic sinks. Therefore, we can interpret the δ13C source signature of the reference period as the ﬂux-weighted average of all sources (Miller and Tans, 2003). We then ap- plied the parameters (aref and bref) from the reference period to the entire CO2 record to calculate artiﬁcially constructed δ13Cconstr and δ18Oconstr values: δconstr(t) = aref · 1 CO2(t) + bref, (7) (7) Since all CO2 sources with a negative isotopic signature relative to atmospheric CO2 lead to δ13C-CO2 or δ18O- CO2 depletion, a differentiation between different deplet- Fig. 7a, b, g and h show the constructed δ13C-CO2 and δ18O- CO2 records in burgundy and light blue. During the ref- Atmos. Meas. Tech., 8, 579–592, 2015 www.atmos-meas-tech.net/8/579/2015/ 589 S. N. Vardag et al.: Continuous δ18O-CO2 measurements in air using FTIR In the summertime, we expect biosphere ﬂuxes to be much larger than during winter and at the same time fossil fuel (es- pecially residential heating) emissions to be smaller than in winter. In fact, we do not ﬁnd large deviations in δ13CO2 from those determined in the reference period (−27 ‰), pointing towards a relatively constant mixture of biogenic and fossil fuel emissions. On the other hand, the measured δ18O-CO2 decreased rapidly on 3 July, compared to the ref- erence period with a source isotopic signature of ≈−12 ‰. This decrease is not accompanied by changes of any other tracer, such as CO, δ13CO2 or CO2, and also not by drastic changes of environmental parameters such as relative humid- ity, temperature or wind speed (not shown here). A possible explanation for the decrease is a change in the hydrologi- cal conditions. After 4 dry days, a sudden heavy rain oc- curred in Heidelberg on 3 July (see dashed bar in Fig. 7, right panel). The rainfall replenished the water reservoirs with an 18O-depleted signature (Daansgard et al., 1964) and equili- bration between the soil and leaf water reservoirs and CO2 most probably caused the atmospheric δ18O-CO2 to become depleted relative to the reference period. This example illus- trates the close coupling between δ18O in the water and car- bon cycle. 5.1 Instrumental performance The main scope of this work was to ascertain whether the FTIR analyser is capable of measuring δ18O-CO2 in the at- mosphere and if so, to assess how well it performs. We have seen that the FTIR succeeds in measuring atmospheric δ18O- CO2 with a high repeatability (Allan deviation after 1 day: 0.1 ‰) and good intermediate measurement precision (δ18O- CO2 = 0.27 ‰ for daily repeated working standard gas mea- surements on 9 min averages over 10 months). We were also able to conﬁrm a good compatibility to the IRMS. Some opti- misation, concerning the calibration, the ﬁtted spectral win- dows and the theoretical spectrum modelling could lead to improved results. However, the current performance of the spectrometer sufﬁces to quantify typical diurnal and synoptic variations at an urban site, which is an important step towards quantiﬁcation of gross biospheric ﬂuxes using FTIR-based δ18O-CO2 measurements. 4 Example period of continuous trace gas and stable isotopologue measurements in Heidelberg The strong inﬂuence of a more 13C depleted source mix points towards a high contribution from fossil fuel sources, including do- mestic heating (natural gas). At the same time, the isotopic signature of δ18O-CO2 is very close to the isotopic signature during the reference period (−28 ‰) and increases during the pollution event. The different behaviour of δ13C and δ18O in CO2 points towards a larger inﬂuence from trafﬁc or natural gas combustion, as both sources are slightly more enriched in 18O, but less enriched in 13C with respect to coal-ﬁred com- bustion (Schumacher et al., 2011). One can see that the fact that different fossil fuel types inﬂuence both stable isotopes 13C and 18O in CO2 in a different way can potentially be used to differentiate between different emission groups in situa- tions when biogenic ﬂuxes are low (i.e. in winter). However, for a quantitative analysis we must know the exact isotopic signatures of all ﬂuxes in the area of inﬂuences. www.atmos-meas-tech.net/8/579/2015/ 6 Summary and conclusion The analysis of δ18O in CO2 using FTIR spectroscopy is novel. We evaluated the measurements of 18O in CO2 us- ing the FTIR with respect to repeatability, intermediate mea- surement precision and compatibility. The Allan deviation test showed that the instrument measures δ18O-CO2 with good stability over the course of a day (the frequency of the working standard measurement) to within 0.1 ‰. Averages of 9 min show a standard deviation of about 0.25 ‰, which is in agreement with the intermediate measurement precision based on daily working standard gas measurements. Ciais, P., Tans, P. P., White, J. W. C., Trolier, M., Francey, R. J., Berry, J. A., Randall, D. R., Sellers, P. J., Collatz, J. G., and Schimel, D. S.: Partitioning of ocean and land up- take of CO2 as inferred by δ13C measurements from the NOAA Climate Monitoring and Diagnostics Laboratory Global Air Sampling Network, J. Geophys. Res., 100, 5051–5070, doi:10.1029/94JD02847, 1995. Ciais, P., Denning, A. S., Tans, P. P., and Berry, J. A.: A three-dimensional synthesis study of δ18O in atmospheric CO2 1.surface ﬂuxes, J. Geophys. Res., 102, 5857–5872, doi:10.1029/96JD02360, 1997. Evaluation of diurnal ambient air variations is therefore possible using, for example, 30 min averages. The high tem- poral resolution of the FTIR measurement is a major advan- tage over the IRMS analyses. Even though the FTIR preci- sion does not reach the WMO inter-laboratory compatibility targets (WMO, 2012), a number of interesting scientiﬁc ap- plications seem possible using FTIR spectroscopy. In partic- ular, investigation of the processes that govern the δ18O-CO2 variability of atmospheric CO2 on the regional scale seem very promising if comprehensive knowledge on the isotopic signature of different CO2 sources and sinks, as well as of the inﬂuencing water reservoirs, is available. Coplen, T. B.: Guidelines and recommended terms for ex- pression of stable-isotope-ratio and gas-ratio measurement results. Rapid Commun. Mass Spectrom., 25, 2538–2560, doi:10.1002/rcm.5129, 2011. 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https://openalex.org/W3143154601	https://aprp.msal.ru/jour/article/download/2714/1642	Russian	null	Measures to Improve Financing of “Megascience” Class Projects	Aktualʹnye problemy rossijskogo prava	2,021	cc-by	4,598	* Арзуманова Лана Львовна, доктор юридических наук, доцент, профессор кафедры финансового права Московского государственного юридического университета имени О.Е. Кутафина (МГЮА) Садовая-Кудринская ул., д. 9, г. Москва, Россия, 125993 llarzumanova@msal.ru ФИНАНСОВОЕ ПРАВО DOI: 10.17803/1994-1471.2021.124.3.028-035 Л. Л. Арзуманова, А. А. Ситник* © Арзуманова Л. Л., Ситник А. А., 2021 Ситник Александр Александрович, кандидат юридических наук, доцент, доцент кафедры финансового права Московского государственного юридического университета имени О.Е. Кутафина (МГЮА) Садовая-Кудринская ул., д. 9, г. Москва, Россия, 125993 aasitnik@msal.ru Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Aleksandr A. Sitnik, Cand. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 aasitnik@msal.ru Aleksandr A. Sitnik, Cand. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 aasitnik@msal.ru Abstract. The paper is devoted to researching the mechanism of financing “megasience” class projects, as well as the development of measures to improve financing of projects under consideration. It is determined that at present the main source of financing for the “megasience” class installations is the money allocated from the budgets of the budget system of the Russian Federation, primarily from the federal budget. At the same time, extrabudgetary funds are also envisaged at the normative level for the implementation of major scientific projects under consideration. However, the authors highlight that the mechanism for attracting extrabudgetary money to finance the scientific field has not been developed at present. On the contrary, the attractiveness of private capital involvement in financing scientific research is, indeed, not obvious. In this regard, the authors make proposals to provide preferential taxation to business entities involved in co-financing of the projects. In the authors’ view, this will have a positive impact on improving financing in general. Keywords: megascience; science; innovative science; national projects; budgets; budget system; taxes; benefits; extrabudgetary sources; financing. Cite as: Arzumanova LL, Sitnik AA. Mery po sovershenstvovaniyu poryadka finansirovaniya nauchnykh proektov klassa «megasayens» [Measures to Improve Financing of “Megascience” Class Projects]. Aktualnye problemy rossiyskogo prava. 2021;16(3):28-35. DOI: 10.17803/1994-1471.2021.124.3.028-035 (In Russ., abstract in Eng.). П также рост вклада науки и техники в улучше- ние государственных экономических показа- телей3. П олноценное существование экономики и общества любого государства возможно лишь с развитием теоретических и прак- тико-ориентированных исследований в различ- ных направлениях науки, влияние на которые непосредственным образом оказывается со стороны государства, в том числе посредством реализации политики в области научно-техни- ческого развития как части социально-эконо- мической политики Российской Федерации. 3 Федеральный закон от 23.08.1996 № 127-ФЗ «О науке и государственной научно-технической политике» // СЗ РФ. 1996. № 35. Ст. 4137. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс»1 Аннотация. Статья направлена на исследование механизма финансирования крупных научных установок класса «мегасайенс», а также на выработку мер по совершенствованию порядка финансирования указан- ных проектов. Определено, что в настоящее время основным источником фиксирования научных устано- вок класса «мегасайенс» являются денежные средства, выделяемые из бюджетов бюджетной системы Российской Федерации, в первую очередь из федерального бюджета. Между тем на нормативном уровне предусмотрено также привлечение внебюджетных средств в целях реализации рассматриваемых крупных научных проектов. Однако следует констатировать, что механизм привлечения внебюджетных денег на финансирование научной сферы в настоящее время не разработан, а привлекательность участия част- ного капитала в финансировании научных исследований крайне неочевидна. В этой связи высказываются предложения по предоставлению льготного режима налогообложения субъектам бизнес-сообщества, участвующим в софинансировании проектов такого уровня, что, по мнению авторов, окажет позитивное воздействие на совершенствование порядка финансирования в целом. Ключевые слова: мегасайенс; наука; инновационная наука; национальные проекты; бюджет; бюджетная система; налоги; льготы; внебюджетные источники; финансирование. Для цитирования: Арзуманова Л. Л., Ситник А. А. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» // Актуальные проблемы российского права. — 2021. — Т. 16. — № 3. — С. 28–35. — DOI: 10.17803/1994-1471.2021.124.3.028-035. 1 Исследование выполнено при финансовой поддержке РФФИ в рамках научного проекта № 18-29-15036. 2 The reported study was funded by RFBR according to the research project № 18-29-15036. 1 Исследование выполнено при финансовой поддержке РФФИ в рамках научного проекта № 18-29-15036. 2 Th t d t d f d d b RFBR di t th h j t №18 29 15036 Ситник Александр Александрович, кандидат юридических наук, доцент, доцент кафедры финансового права Московского государственного юридического университета имени О.Е. Кутафина (МГЮА) Садовая-Кудринская ул., д. 9, г. Москва, Россия, 125993 aasitnik@msal.ru Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 28 Арзуманова Л. Л., Ситник А. А. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» Measures to Improve Financing of “Megascience” Class Projects2 Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru — независимость научно-технического по- тенциала, которая позволяет участникам доктринальных исследований определять формы и методы разрешения поставленных задач и тем самым усиливает ответственный подход к полученным результатам деятель- ности; — системность, способствующая выработке качественно инновационных технологий и образованию новых продуктов и услуг; — концентрация ресурсов в целях содействия развития научным исследованиям и раз- работкам; В свою очередь, перед Правительством РФ в рамках реализации национального проекта в области развития науки поставлены цели, кото- рых следует достигнуть к 2024 г., в частности: В свою очередь, перед Правительством РФ в рамках реализации национального проекта в области развития науки поставлены цели, кото- рых следует достигнуть к 2024 г., в частности: — организовать российское участие среди пяти стран-лидеров, осуществляющих научные исследования и разработки в тех сферах дея- тельности, которые признаны приоритетны- ми с точки зрения научно-технологического прогресса; — рациональный баланс, включающий под- держку со стороны государства для выпол- нения основных задач научно-технологиче- ского развития; — организовать российское участие среди пяти стран-лидеров, осуществляющих научные исследования и разработки в тех сферах дея- тельности, которые признаны приоритетны- ми с точки зрения научно-технологического прогресса; — открытость, которая сводится к взаимодей- ствию между обществом и государством при условии приоритета национальных интере- сов; — создать благоприятные условия в целях при- влечения российских и зарубежных молодых ученых-исследователей и их последующего профессионального роста; — адресная поддержка и конкурентно-свобод- ная среда, направленная на обеспечение доступа к государственным ресурсам субъ- ектов научно-технической и инновационной деятельности вне зависимости от формы их собственности и организационно-правовой формы. — заложить необходимый уровень финансиро- вания на научные разработки посредством мобилизации различных источников покры- тия финансовых затрат (п. 10). Таким образом, в современных реалиях государство поставило перед наукой абсо- лютно новые задачи — от их решения зави- сит построение прогрессивной инфраструк- туры в науке, включая образование сети установок уникального характера класса «мегасайенс». Предполагается, что именно полученные результаты такого рода иссле- дований могут способствовать достижению Провозглашенные цели исполнительная власть предполагает достичь благодаря реше- нию следующих задач: — сформировать прогрессивную базу для про- ведения разработок, включая научные уста- новки мегасайенс-класса; — модернизировать наполовину инфраструк- туру флагманских организаций, выполняющих 4 Указ Президента РФ от 01.12.2016 № 642 «О Стратегии научно-технологического развития Российской Федерации» // СЗ РФ. 2016. № 49. Ст. 6887. 5 Указ Президента РФ от 07.07.2011 № 899 «Об утверждении приоритетных направлений развития науки, технологий и техники в Российской Федерации и перечня критических технологий Российской Федера- ции» // СЗ РФ. 2011. № 28. Ст. 4168. Актуальные проблемы российского права. 2021. Т. 16. 4 Указ Президента РФ от 01.12.2016 № 642 «О Стратегии научно-технологического развития Российской Федерации» // СЗ РФ. 2016. № 49. Ст. 6887. 5 Указ Президента РФ от 07.07.2011 № 899 «Об утверждении приоритетных направлений развития науки, технологий и техники в Российской Федерации и перечня критических технологий Российской Федера- ции» // СЗ РФ. 2011. № 28. Ст. 4168. 4 Указ Президента РФ от 01.12.2016 № 642 «О Стратегии научно-технологического развития Российской Федерации» // СЗ РФ. 2016. № 49. Ст. 6887. Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Следует отметить, что в настоящее время на официальном уровне выделяется два основных исторических этапа государственной научно- технической политики: — первый (1991–2001 гг.) ознаменован пере- ходом к рыночной экономике, направленным на дальнейшее развитие научно-техническо- го потенциала общества, и воспроизводством на этой основе инновационных методов под- держания научно-технической мощи, а также точечным финансированием организаций и учреждений в сфере науки; С учетом обозначенных приоритетов можно выявить некоторые цели, которые поставле- ны во главе проводимой органами публичной власти научно-технической политики. К ним, в частности, относятся создание, разумное размещение и результативное применение потенциала научно-технического характера, а второй (2001 г. — н. в.) связан с переориен- тацией на экономику инновационного типа, 3 Федеральный закон от 23.08.1996 № 127-ФЗ «О науке и государственной научно-технической политике» // СЗ РФ. 1996. № 35. Ст. 4137. Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 29 ФИНАНСОВОЕ ПРАВО сопровождаемым ростом объемов финанси- рования национальной науки4. сопровождаемым ростом объемов финанси- рования национальной науки4. достойных результатов для России как в фун- даментальных, так и в прикладных областях знаний. При этом можно выделить несколько базо- вых направлений, на которых основана поли- тика научно-технологического прогресса: Президентом РФ были определены прио- ритетные направления развития технологий и техники на современном историческом этапе5. К ним, в частности, были отнесены: безопас- ность и противодействие терроризму; индустрия наносистем; информационно-телекоммуника- ционные системы; науки о жизни; перспектив- ные виды вооружения, военной и специальной техники; рациональное природопользование; комплексы (системы) военного, специального и двойного назначения; транспортные и косми- ческие системы; энергоэффективность, энерго- сбережение, ядерная энергетика. 4 Указ Президента РФ от 01.12.2016 № 642 «О Стратегии научно-технологического развития Российской Федерации» // СЗ РФ. 2016. № 49. Ст. 6887. 5 Указ Президента РФ от 07.07.2011 № 899 «Об утверждении приоритетных направлений развития науки, р 5 Указ Президента РФ от 07.07.2011 № 899 «Об утверждении приоритетных направлений развития науки, технологий и техники в Российской Федерации и перечня критических технологий Российской Федера- ции» // СЗ РФ. 2011. № 28. Ст. 4168. Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru № 3 (124) март 30 Арзуманова Л. Л., Ситник А. А. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» Арзуманова Л. Л., Ситник А. А. Арзуманова Л. Л., Ситник А. А. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» р у , Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» исследования в этих направлениях научной области; к управлению публичными деньгами, отмечая взаимосвязь финансирования из соответству- ющего бюджета с достижением положитель- ного результата7. С учетом изложенного следует обратить внимание на систему бюджетирова- ния, ориентированного на результат, о которой стали активно писать начиная еще с середины 2000-х гг. исследования в этих направлениях научной области; исследования в этих направлениях научной области; — организовать работу исследовательских цен- тров мирового масштаба, включая между- народные исследовательские центры; — учредить 15 центров научно-образователь- ного толка, соответствующих мировым трен- дам, путем координации действий универ- ситетов, научных учреждений и организаций реального сектора экономики, что соответ- ствует трендам современности, когда раз- работки ведутся не только ради науки, но и отталкиваются от интересов и запросов, поставленных обществом и конкретными компаниями, заинтересованными в тех или иных результатах проводимых иссле- дований; — учредить 15 центров научно-образователь- ного толка, соответствующих мировым трен- дам, путем координации действий универ- ситетов, научных учреждений и организаций реального сектора экономики, что соответ- ствует трендам современности, когда раз- работки ведутся не только ради науки, но и отталкиваются от интересов и запросов, поставленных обществом и конкретными компаниями, заинтересованными в тех или иных результатах проводимых иссле- дований; Бюджетирование, ориентированное на результат (БОР), представляет собой такую организацию бюджетного процесса и государ- ственного (муниципального) управления, при которой расходование бюджетных ассигнова- ний проводится с учетом взаимосвязи с полу- чаемыми результатами. Важным направлени- ем реализации БОР стала переориентация на программную форму формирования бюджета, предполагающая, что бо́льшая часть расходов бюджетов бюджетной системы Российской Федерации (около 75–90 %) осуществляется в рамках государственных программ8. — благодаря комплексному подбору задач про- возглашается еще одна магистральная пози- ция, ориентированная на развитие системно- го обучения педагогических кадров, которые должны принять участие на начальном этапе всей цепочки и обеспечить достаточными и необходимыми навыками молодых ученых для проведения научных исследований и разработок, включая возможность их участия в создаваемых с этой целью научных лабо- раториях и участие в конкурентоспособных коллективах. Президента РФ от 07.05.2018 № 204 «О национальных целях и стратегических задачах развития йской Федерации на период до 2024 года» // СЗ РФ. 2018. № 20. Ст. 2817. Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru — благодаря комплексному подбору задач про- возглашается еще одна магистральная пози- ция, ориентированная на развитие системно- го обучения педагогических кадров, которые должны принять участие на начальном этапе всей цепочки и обеспечить достаточными и необходимыми навыками молодых ученых для проведения научных исследований и разработок, включая возможность их участия в создаваемых с этой целью научных лабо- раториях и участие в конкурентоспособных коллективах. Благодаря реализации государственных про- грамм выделяется финансирование и на науку в Российской Федерации. Таким образом, про- граммный метод направлен на решение постав- ленных перед государством глобальных задач: ленных перед государством глобальных задач: — сосредоточение денежных средств на базо- вых направлениях научных исследований; — стабилизация субсидирования определен- ных научных наработок; В силу Указа Президента РФ от 07.05.2018 № 2046 воспроизводство и последующая реа- лизация мегасайенс-проектов входит в состав научного национального проекта. — организация проведения научно-исследо- вательских работ в обозначенные сроки и обеспечение результативного контроля за целевым применением ассигнований из бюджета федерального значения. Таким образом, бюджетирование мегаса- йенс-установок проводится на базе государ- ственных проектов и благодаря принимаемым на федеральном уровне целевым программам. Например, А. С. Матненко пишет о националь- ных проектах как о совершенно другом подходе Примером реализации такого метода пере- распределения денежных средств стал нацио- нальный проект «Наука»9. К числу основопо- лагающих целей этого проекта относятся: ненко А. С. Приоритетные национальные проекты и бюджетная деятельность государства. Омск : 2007. С. 20. rzumanova L. L. National experience in funding scientific megascience projects: legal modeling // 2020 s.: Conf. Ser. 1685 012008. 9 Паспорт национального проекта «Наука» утвержден президиумом Совета при Президенте РФ по стра- тегическому развитию и национальным проектам, протокол от 24.12.2018 № 16. Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 31 ФИНАНСОВОЕ ПРАВО — присутствие нашей страны в числе пяти передо- вых в рассматриваемом направлении государств; ческой инфраструктуры для проведения науч- ных исследований на территории Российской Федерации. — организация условий для привлечения моло- дых ученых как внутри страны, так и извне; При этом предполагается, что финансирова- ние нацпроекта будет осуществляться из различ- ных источников: — подъем затрат на проведение научных раз- работок посредством использования денеж- ных фондов разного характера. — 404,8 млрд руб. — деньги федерального фонда; Сейчас в фонде нацпроекта заложено 636,0 млрд руб., из которых 350,0 млрд руб. предлагается потратить на создание технологи- — 231,2 млрд руб. — иные источники финанси- рования (см. рисунок). 10 URL: https://futurerussia.gov.ru/nauka (дата обращения: 25.11.2020). 11 Обеспечение реализации национальных проектов на региональном уровне (материалы семинара- совещания руководителей аналитических служб аппаратов законодательных (представительных) и исполнительных органов государственной власти субъектов Российской Федерации) // Аналитический вестник. 2019. № 14. Ч. 1. 10 URL: https://futurerussia.gov.ru/nauka (дата обращения: 25.11.2020). Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Объем финансового обеспечения по годам реализации (млрд руб.)10 Объем финансового обеспечения по годам реализации (млрд руб.)10 Таким образом, заявленные суммы и пред- ставленный график финансирования демон- стрируют, что реализация исследований в обла- сти науки, а тем более возможность разработки проектов класса «мегасайенс», возможна лишь при соответствующем уровне финансовой под- держки, в первую очередь со стороны государ- ства, но и с привлечением финансов из иных источников. Аналогичной позиции придерживается и Счетная палата России, которая заявила, что получение надлежащих итогов в рамках проекта «Наука» находится в зависимости от привлече- ния средств внебюджетных фондов, которые на сегодняшний день четко не проработаны и носят хаотичный порядок поступления11. Следует отметить, что в рамках рассматри- ваемого национального проекта предусматри- 10 URL: https://futurerussia.gov.ru/nauka (дата обращения: 25.11.2020). 11 Обеспечение реализации национальных проектов на региональном уровне (материалы семинара- совещания руководителей аналитических служб аппаратов законодательных (представительных) и исполнительных органов государственной власти субъектов Российской Федерации) // Аналитический вестник. 2019. № 14. Ч. 1. Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 32 32 Арзуманова Л. Л., Ситник А. А. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» Арзуманова Л. Л., Ситник А. А. осуществлением на ее основе высокотехнологи- ческой деятельности ее участниками. вается постепенное повышение доли финанси- рования из внебюджетных источников: если на 2019 г. общий объем таких денежных средств должен был составить 36 % от запланирован- ного общего объема финансового обеспечения, то к 2024 г. предполагается, что он выйдет за пределы 50 %. При этом очевидно, что для при- влечения внебюджетных средств необходимы серьезные меры поддержки инвесторов. Между тем следует констатировать, что механизм при- влечения внебюджетных денег на финансиро- вание научной сферы всё еще не разработан, а привлекательность участия частного капитала в финансировании научных исследований крайне неочевидна. Позже, 25 июля 2019 г., был издан Указ Президента РФ № 35613, обязавший Прави- тельство РФ разработать Федеральную науч- но-техническую программу развития синхро- тронных и нейтронных исследований и иссле- довательской инфраструктуры на 2019–2027 гг., предусматривающую создание нескольких установок мегасайенс14. Названная программа финансируется за счет денежных средств бюд- жетов федерального и регионального уровня и средств внебюджетных фондов. Следует отметить, что, по данным РАН, объем затрат, связанных с финансированием науки, на современном этапе развития нацио- нальной экономики составляет около 86 % от соответствующего объема денежных средств, который выделялся в РСФСР в 1991 г. (в сопоста- вимых ценах). При этом внутренние затраты на исследования и разработки в России в расчете на одного исследователя составляют 102,9 тыс. долл. США в год. 12 СЗ РФ. 2019. № 151 (ч. III). Ст. 1750. 13 Указ Президента РФ от 25.07.2019 № 356 «О мерах по развитию синхронных и нейтронных исследований и исследовательской инфраструктуры в Российской Федерации» // СЗ РФ. 2019. № 30. Ст. 4923. 14 Полный перечень приведен в документе. 15 Доклад о реализации государственной научно-технической политики Российской Федерации в 2018 г. М. : Российская академия наук, 2019. 16 См.: Болтинова О. В. Расходы федерального бюджета на создание установок класса «мегасайенс» в национальных проектах Российской Федерации // Актуальные проблемы российского права. 2020. № 5. Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru В США, например, такие затра- ты составляют 359,9 тыс. долл., в Швейцарии — 406,7 тыс. долл., в Южной Корее — 219,6 тыс. долл., в Японии — 253,4 тыс. долл. в год15. Заметим, что проекты класса «мегасайенс» реализовываются в первую очередь в интере- сах неопределенного круга лиц, т.е. в публич- ном интересе. Обуславливается это тем, что магистральной целью создания такого рода проектов является получение новых знаний и инновационных технологий для их использо- вания в общественных интересах. Достижение поставленной цели, однако, не является при- влекательным для частного сектора, ввиду того что в ее рамках решаются глобальные задачи, которые не всегда «заточены» под получение прибыли в короткие сроки. Таким образом, показатели действующих про- грамм и подпрограмм свидетельствуют о недо- статочном финансировании научных исследова- ний мегасайенс-проектов, которые нуждаются во внебюджетных источниках. В этой связи можно поддержать идею создания в составе расходной части целевого бюджетного фонда, средства ко- торого рассчитаны на эффективное финансиро- вание проектов уникальных научных установок16. В рамках проводимого исследования следует упомянуть еще один документ — постановле- ние Правительства РФ от 29.03.2019 № 377, на основании которого утверждена программа «Научно-технологическое развитие Российской Федерации»12, частью которой является под- программа «Инфраструктура научной, научно- технической инновационной деятельности». На первоначальном этапе предполагалось, что про- грамма будет использоваться для обеспечения необходимой инфраструктуры с последующим Более того, в целях привлечения частных инвестиций можно предложить перераспре- деление бюджета на науку в пользу иннова- ционных компаний, а также предоставление Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 33 ФИНАНСОВОЕ ПРАВО льготного режима налогообложения научным организациям и организациям, не являющимся научными, но осуществляющим инвестиции в соответствующую деятельность. В состав расходов, связанных с производ- ством и реализацией (уменьшающих налого- облагаемую базу), отнесены расходы на науч- ные исследования и опытно-конструкторские разработки (пп. 4 п. 1 ст. 253 НК РФ). Говоря о налоговых льготах для научных ор- ганизаций, следует отметить следующее. В ходе осуществления научно-технической деятель- ности организации пользуются налоговыми льготами, установленными отдельными видами налогов. Однако не всеми налогами, платель- щиками которых являются данные организации, установлены льготы в отношении той деятель- ности, которую последние осуществляют. Тем не менее рассмотрим некоторые виды нало- гов, в отношении которых применим льготный режим их уплаты научными организациями, в том числе осуществляющими деятельность в области мегасайенс-проектов. В пункте 1 ст. 17 СЗ РФ. 2017. № 31 (ч. I). Ст. 4765. Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март БИБЛИОГРАФИЯ 1. Болтинова О. В. Расходы федерального бюджета на создание установок класса «мегасайенс» в нацио- нальных проектах Российской Федерации // Актуальные проблемы российского права. — 2020. — № 5. — С. 42–47. 2. Доклад о реализации государственной научно-технической политики Российской Федерации в 2018 г. — М. : Российская академия наук, 2019. — 620 с. 2. Доклад о реализации государственной научно-технической политики Российской Федерации в 2018 г. — М. : Российская академия наук, 2019. — 620 с. 3. Матненко А. С. Приоритетные национальные проекты и бюджетная деятельность государства. — Омск : Русь, 2007. — 144 с. 4. Обеспечение реализации национальных проектов на региональном уровне (материалы семинара- совещания руководителей аналитических служб аппаратов законодательных (представительных) и исполнительных органов государственной власти субъектов Российской Федерации) // Аналитический вестник. — 2019. — № 14. — Ч. 1. 4. Обеспечение реализации национальных проектов на региональном уровне (материалы семинара- совещания руководителей аналитических служб аппаратов законодательных (представительных) и исполнительных органов государственной власти субъектов Российской Федерации) // Аналитический вестник. — 2019. — № 14. — Ч. 1. 5. Arzumanova L. L. National experience in funding scientific megascience projects: legal modeling // 2020 J. Phys.: Conf. Ser. 1685 012008. Материал поступил в редакцию 10 сентября 2020 г. Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru Л., Ситник А. А. получить соответствующую оценку и должна быть серьезно рассмотрена высшими должност- ными лицами государства. В первую очередь подобные льготы должны быть предусмотрены в отношении субъектов, непосредственно при- нимающих участие в финансировании мегаса- йенс-проектов. Подобный шаг, по нашему мне- нию, значительно повысит для частных инвесто- ров привлекательность участия в реализации проектов класса «мегасайенс». получить соответствующую оценку и должна быть серьезно рассмотрена высшими должност- ными лицами государства. В первую очередь подобные льготы должны быть предусмотрены в отношении субъектов, непосредственно при- нимающих участие в финансировании мегаса- йенс-проектов. Подобный шаг, по нашему мне- нию, значительно повысит для частных инвесто- ров привлекательность участия в реализации проектов класса «мегасайенс». задач больше денежных средств. Кроме того, налоговые льготы имеют и важное психологиче- ское значение — вводя их, государство признает социальную значимость научной деятельности и важность профессии ученого для общества. В то же время в налоговом законодательстве Российской Федерации отсутствуют льготы, которые непосредственным образом были бы связаны с реализацией мегасайенс-проектов. задач больше денежных средств. Кроме того, налоговые льготы имеют и важное психологиче- ское значение — вводя их, государство признает социальную значимость научной деятельности и важность профессии ученого для общества. В то же время в налоговом законодательстве Российской Федерации отсутствуют льготы, которые непосредственным образом были бы связаны с реализацией мегасайенс-проектов. Между тем возможность их введения должна Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март Lana L. Arzumanova, Dr. Sci. (Law), Associate Professor, Full Professor, Department of Financial Law, Kutafin Moscow State Law University (MSAL) ul. Sadovaya-Kudrinskaya, d. 9, Moscow, Russia, 125993 llarzumanova@msal.ru 262 НК РФ определены рас- ходы на научные исследования и (или) опыт- но-конструкторские разработки, под которыми признаются расходы, относящиеся к созданию новой или усовершенствованию производимой продукции (товаров, работ, услуг), к созданию новых или усовершенствованию применяемых технологий, методов организации производства и управления. Льготное налогообложение в виде освобо- ждения от уплаты налога на имущество орга- низаций закреплено также в отношении иму- щества организаций, которым присвоен статус государственных научных центров; для органи- заций, получивших статус участника проекта в соответствии с Федеральным законом «Об ин- новационных научно-технологических центрах и о внесении изменений в отдельные законо- дательные акты Российской Федерации», — в отношении имущества, учитываемого на их балансе и расположенного на территории инно- вационного научно-технологического центра, в течение 10 лет начиная с месяца, следующего за месяцем постановки на учет указанного иму- щества (п. 15 и 28 ст. 381 НК РФ). Так, не входит в зону обложения НДС выпол- нение ряда работ, в частности: научно-иссле- довательских / опытно-конструкторских работ; научно-исследовательских / опытно-конструк- торских работ организаций в образовательной сфере деятельности; выполнение научно-иссле- довательских / опытно-конструкторских / техно- логических работ, которые отнесены к воспро- изводству инновационных продуктов; воспро- изводство новых технологий; создание опытных образцов машин, оборудования, материалов, обладающих принципиальными особенностями (пп. 16 и 16.1 п. 3 ст. 149 НК РФ). Аналогичное освобождение от уплаты зе- мельного налога предусмотрено для органи- заций, признаваемых фондами в соответствии с Федеральным законом «Об инновационных научно-технологических центрах и о внесе- нии изменений в отдельные законодательные акты Российской Федерации», — в отношении земельных участков, входящих в состав террито- рии инновационного научно-технологического центра (п. 13 ст. 395 НК РФ). В соответствии с п. 1 ст. 246.1 НК РФ органи- зации, получившие статус участников проекта по осуществлению исследований, разработок и коммерциализации их результатов в соответ- ствии с Федеральным законом от 28.09.2010 № 244-ФЗ «Об инновационном центре “Сколково”» либо участников проекта в соот- ветствии с Федеральным законом от 29.07.2017 № 216-ФЗ «Об инновационных научно-техно- логических центрах и о внесении изменений в отдельные законодательные акты Российской Федерации»17, в течение 10 лет со дня получе- ния ими указанного статуса приобретают право на освобождение от исполнения обязанностей налогоплательщиков. Подводя итог сказанному, отметим, что на сегодняшний момент налоговые льготы явля- ются важным инструментом стимулирования научной деятельности — они позволяют снизить фискальную нагрузку научных организаций и ученых, позволяя тратить на решение научных Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 34 Арзуманова Л. Л., Ситник А. А. Меры по совершенствованию порядка финансирования научных проектов класса «мегасайенс» Арзуманова Л. REFERENCES (TRANSLITERATION) 1. Boltinova O. V. Raskhody federal’nogo byudzheta na sozdanie ustanovok klassa «megasajens» v nacional’nyh proektah Rossijskoj Federacii // Aktual’nye problemy rossijskogo prava. — 2020. — № 5. — S. 42–47. 1. Boltinova O. V. Raskhody federal’nogo byudzheta na sozdanie ustanovok klassa «megasajens» v nacional’nyh proektah Rossijskoj Federacii // Aktual’nye problemy rossijskogo prava. — 2020. — № 5. — S. 42–47. 2. Doklad o realizacii gosudarstvennoj nauchno-tekhnicheskoj politiki Rossijskoj Federacii v 2018 g. — M. : Rossijskaya akademiya nauk, 2019. — 620 s. 3. Matnenko A. S. Prioritetnye nacional’nye proekty i byudzhetnaya deyatel’nost’ gosudarstva. — Omsk : Rus’, 2007. — 144 s. 4. Obespechenie realizacii nacional’nyh proektov na regional’nom urovne (materialy seminara-soveshchaniya rukovoditelej analiticheskih sluzhb apparatov zakonodatel’nyh (predstavitel’nyh) i ispolnitel’nyh organov gosudarstvennoj vlasti sub"ektov Rossijskoj Federacii) // Analiticheskij vestnik. — 2019. — № 14. — Ch. 1. 5 A L L N ti l i i f di i tifi i j t l l d li // 2020 J 4. Obespechenie realizacii nacional’nyh proektov na regional’nom urovne (materialy seminara-soveshchaniya rukovoditelej analiticheskih sluzhb apparatov zakonodatel’nyh (predstavitel’nyh) i ispolnitel’nyh organov gosudarstvennoj vlasti sub"ektov Rossijskoj Federacii) // Analiticheskij vestnik. — 2019. — № 14. — Ch. 1. 5. Arzumanova L. L. National experience in funding scientific megascience projects: legal modeling // 2020 J. Phys.: Conf. Ser. 1685 012008. Актуальные проблемы российского права. 2021. Т. 16. № 3 (124) март 35
https://openalex.org/W1846825472	https://ojrd.biomedcentral.com/counter/pdf/10.1186/s13023-015-0295-9	English	null	Chronic myelomonocytic leukemia as a cause of fatal uncontrolled inflammation in familial Mediterranean fever	Orphanet journal of rare diseases	2,015	cc-by	3,722	© 2015 Awad et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Awad et al. Orphanet Journal of Rare Diseases (2015) 10:76 DOI 10.1186/s13023-015-0295-9 Awad et al. Orphanet Journal of Rare Diseases (2015) 10:76 DOI 10.1186/s13023-015-0295-9 Open Access * Correspondence: serge.amselem@inserm.fr; ohermine@gmail.com; sonia. karabina@trs.aphp.fr †Equal contributors 1Sorbonne Universités, UPMC University Paris 06, INSERM UMR_S933, Hôpital Armand-Trousseau, Paris F-75012, France 3Service d’Hématologie clinique, AP-HP, Hôpital Necker, Université Paris Descartes – Sorbonne Paris Cité, Imagine Institute, INSERM UMR 1163 et CNRS ERL 8254, Paris, France Full list of author information is available at the end of the article Chronic myelomonocytic leukemia as a cause of fatal uncontrolled inflammation in familial Mediterranean fever Fawaz Awad1†, Sophie Georgin-Lavialle1,2†, Anne Brignier3, Coralie Derrieux4, Achille Aouba3, Katia Stankovic-Stojanovic2, Gilles Grateau1,2, Serge Amselem1,5, Olivier Hermine3 and Sonia-Athina Karabina1* Abstract We report on a familial Mediterranean fever (FMF) patient homozygous for p.M694V in the MEFV gene who developed chronic myelomonocytic leukemia (CMML) leading to an uncontrolled and fatal inflammatory syndrome. Plasma levels of IL-6 and IL-18 were found to be very high, as compared to healthy controls and CMML-free FMF patients. Our study unveils the interplay between two different disorders involving the same target cells, suggesting that in myelodysplasia with inflammatory manifestations, mutations in genes causing autoinflammatory syndromes, like MEFV, can be present and thus could be sought. Early chemotherapy with interleukin inhibitors could be proposed in such unusual situations. Keywords: FMF, MEFV, Inflammation, CMML, Interleukin inhibitors, Monocytes, Interleukin 18 Correspondence colchicine-resistant patients or in those presenting ad- verse events [4]. In addition, blocking the IL-1 pathway is safer, in terms of infectious risk, in comparison with other biological treatments [5, 6]. Letters to the Editor: Cytokines implicated in the pathophysi- ology of FMF such as IL-1β, IL-18 and IL-6 are import- ant for the regulation of immune and inflammatory responses. These cytokines are also involved in the pathophysiology of inflammatory anemia due to erythro- poiesis blockade [11, 12]. It is therefore tempting to speculate that the transformation of refractory anemia into CMML, which resulted in an increased number of circulating monocytes in our FMF patient, could be responsible for the persistent inflammatory syndrome. In keeping with this hypothesis, the progressive monocyto- sis was accompanied by a progressive elevation of CRP levels (Fig. 1b) and persistence of refractory anemia. Six months later (t = 6), he presented with asthenia, fever and biological inflammatory syndrome although he took the same dose of colchicine. Serum amyloid A (SAA) as well as C-reactive protein (CRP) levels were elevated (226 and 20 mg/L respectively), and Hb was low (6.9 g/dL). Neither infection nor profound neoplasia was detected. He had no proteinuria, and salivary gland biopsy revealed no amyloidosis. Colchicine was increased to 1.5 mg per day, but because of occurrence of diarrhea –a well-known side effect of colchicine therapy–, the dose was finally maintained 1mg/day. During the following 3 months, his general status got worse: he lost 10 kg, fever and in- flammatory syndrome persisted and required weekly red blood cell transfusions because of severe anemia (Hb = 5.5 g/dL). His condition suddenly deteriorated, with elevated fever and fatigue. Further investigations showed a progressive increase in monocyte count above 1 × 109/L with persistent anemia (Hb = 6.8 g/dL) and severe inflammatory syndrome (SAA = 327 mg/L; CRP 80 mg/L; Fig. 1b). A new bone marrow smear confirmed CMML (Fig. 1a, III&IV). He died 6 months later from We measured the plasma levels of IL-1β, IL-18 and IL-6 in the patient [see Patients and Methods in Additional file 1], 10 months (t = 10) after t = 0, and compared them with cytokine levels from FMF patients carrying unambiguous MEFV mutations (n = 8) [see Table S1 for genotype, inflammatory and clinical status of the FMF b c d a I II III IV Fig. 1 Morphological features of CMML in patient #9 and cytokine profiles in patients and controls. Letters to the Editor: Familial Mediterranean fever (FMF) is an autosomal recessive autoinflammatory disorder caused by muta- tions in the MEFV gene, mainly p.M694V in exon 10 [1, 2]. It is the commonest hereditary fever syndrome with recurrent episodes of fever accompanied by abdom- inal, chest and joint pain. MEFV encodes pyrin, a pro- tein expressed in neutrophils and monocytes [3] and is involved in the regulation of inflammation. Daily and life-long colchicine administration can prevent both attacks and occurrence of inflammatory amyloidosis. Ex-vivo studies performed with monocytes from FMF patients have demonstrated the importance of increased secretion of the potent pyrogenic cytokine interleukin (IL)-1β. Subsequently, IL-1 inhibitors have been pro- posed as alternative or supplementary treatment in Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic stem cell disorder classified as a mye- lodysplastic/myeloproliferative neoplasm [7, 8]. CMML is characterized by absolute monocytosis (>1 × 109/L) in per- ipheral blood persisting for at least 3 months [8]. The me- dian age of CMML diagnosis is 70 years [7] and current treatment includes hydroxyurea and/or 5-azacitidine [8]. We report a case of an 84-year-old man who had typ- ical FMF since his childhood. The diagnosis of FMF was confirmed by identification in the MEFV gene of the M694V mutation in the homozygous state. Lifelong col- chicine therapy (1 mg/day) for 40 years abolished febrile crises. No other medical problem was reported. At the age of 83 (t = 0), he started complaining of general weak- ness and a blood test showed profound macrocytic anemia, with no other cytopenia (Hb = 7.2 g/dL; MCV = 104 fL; leukocytes 7.6 × 109/L; platelets 228 × 109/L). Additional la- boratory tests showed no signs of hemolysis, inflammatory disease, hypothyroidism or deficiency in vitamin B12 or B9. Thus, a bone marrow smear was performed and Awad et al. Orphanet Journal of Rare Diseases (2015) 10:76 Page 2 of 4 pneumonia in the context of persistent profound anemia (Hb = 6.5 g/dL) and inflammation. revealed refractory anemia without blast excess (Fig. 1a, I&II) and with a normal karyotype. He received itera- tive red blood cell transfusions and vitamin D. FMF patients have been shown to display high plasma levels of proinflammatory cytokines [9]. Their mono- cytes are spontaneously activated and secrete high levels of IL-1β [10]. Letters to the Editor: a Bone marrow smears from patient #9 with CMML and FMF (see Additional file 1: Table S1) (I) showing dysgranulopoiesis (hypogranular cytoplasm and Döhle bodies indicated with the arrow) at t = 0; (II) dysplastic binucleate erythroblasts at the stage of myelodysplastic syndrome (t = 0); (III) persistence of myeloid dysplasia (indicated with an arrow) at t = 6; (IV) appearance of a monocytosis (solid arrow) with excess of blasts (dashed arrow) when progression to CMML at t = 6. b Blood monocyte counts and serum CRP levels at the indicated time points in patient #9. t = 0 is first time the patient complained about symptoms worsening. The solid black arrow indicates the time of CMML diagnosis and the dashed arrow indicates when plasma was drawn for cytokine measurements. c IL-6 levels in plasma samples from patient #9, FMF patients and apparently healthy controls, as quantified by ELISA. d IL-18 levels in plasma samples from patient #9, FMF patients and apparently healthy controls, as quantified by ELISA b b a I II III IV a d c d Fig. 1 Morphological features of CMML in patient #9 and cytokine profiles in patients and controls. a Bone marrow smears from patient #9 with CMML and FMF (see Additional file 1: Table S1) (I) showing dysgranulopoiesis (hypogranular cytoplasm and Döhle bodies indicated with the arrow) at t = 0; (II) dysplastic binucleate erythroblasts at the stage of myelodysplastic syndrome (t = 0); (III) persistence of myeloid dysplasia (indicated with an arrow) at t = 6; (IV) appearance of a monocytosis (solid arrow) with excess of blasts (dashed arrow) when progression to CMML at t = 6. b Blood monocyte counts and serum CRP levels at the indicated time points in patient #9. t = 0 is first time the patient complained about symptoms worsening. The solid black arrow indicates the time of CMML diagnosis and the dashed arrow indicates when plasma was drawn for cytokine measurements. c IL-6 levels in plasma samples from patient #9, FMF patients and apparently healthy controls, as quantified by ELISA. d IL-18 levels in plasma samples from patient #9, FMF patients and apparently healthy controls, as quantified by ELISA Fig. 1 Morphological features of CMML in patient #9 and cytokine profiles in patients and controls. Letters to the Editor: a Bone marrow smears from patient #9 with CMML and FMF (see Additional file 1: Table S1) (I) showing dysgranulopoiesis (hypogranular cytoplasm and Döhle bodies indicated with the arrow) at t = 0; (II) dysplastic binucleate erythroblasts at the stage of myelodysplastic syndrome (t = 0); (III) persistence of myeloid dysplasia (indicated with an arrow) at t = 6; (IV) appearance of a monocytosis (solid arrow) with excess of blasts (dashed arrow) when progression to CMML at t = 6. b Blood monocyte counts and serum CRP levels at the indicated time points in patient #9. t = 0 is first time the patient complained about symptoms worsening. The solid black arrow indicates the time of CMML diagnosis and the dashed arrow indicates when plasma was drawn for cytokine measurements. c IL-6 levels in plasma samples from patient #9, FMF patients and apparently healthy controls, as quantified by ELISA. d IL-18 levels in plasma samples from patient #9, FMF patients and apparently healthy controls, as quantified by ELISA Awad et al. Orphanet Journal of Rare Diseases (2015) 10:76 Page 3 of 4 Page 3 of 4 patients, in Additional file 1] and healthy controls. IL-6, a cytokine known to induce acute phase proteins, was not detected in the controls’ plasma but was present at a con- centration of 64 pg/mL in the CMML patient and also easily detectable in 4 other FMF patients (3–177 pg/mL), of whom 3 were under colchicine therapy (Fig. 1c). IL-1β is produced by monocytes and tissue macrophages as a pre- cursor, which is then processed to its active form by the inflammasome, an intracellular multiprotein complex [13]. IL-1β is mainly detected in supernatants of monocyte cul- ture after stimulation with Toll-Like receptor agonists like lipopolysaccharide [10]. Although ex-vivo studies demon- strated a major role of IL-1β in the pathogenesis of FMF, serum levels have been reported normal or even decreased in FMF patients [14]. Accordingly, IL-1β was not detected in the patient’s plasma, in controls, and in 6 out of the 8 FMF patients (data not shown). IL-18, which is also secreted by monocytes and regulated by inflammasome, was found at much higher levels in the patient’s plasma (7647 pg/mL) than in other patients (187–4527 pg/mL) or controls (172–246 pg/mL) (Fig. 1d). References Ibrahim J-N, Jounblat R, Delwail A, Abou-Ghoch J, Salem N, Chouery E, et al. Ex vivo PBMC cytokine profile in familial Mediterranean fever patients: Involvement of IL-1β, IL-1α and Th17-associated cytokines and decrease of Th1 and Th2 cytokines. Cytokine. 2014;69:248–54. 9. Ibrahim J-N, Jounblat R, Delwail A, Abou-Ghoch J, Salem N, Chouery E, et al. Ex vivo PBMC cytokine profile in familial Mediterranean fever patients: Involvement of IL-1β, IL-1α and Th17-associated cytokines and decrease of Th1 and Th2 cytokines. Cytokine. 2014;69:248–54. 10. Omenetti A, Carta S, Delfino L, Martini A, Gattorno M, Rubartelli A. Increased NLRP3-dependent interleukin 1β secretion in patients with familial Mediterranean fever: correlation with MEFV genotype. Ann Rheum Dis. 2014;73:462–9. Acknowledgements Fawaz Awad (F.A) was supported from a grant from the French government and Alquds University, Palestine and from the “Fondation pour la Recherche Médicale” (FDT20130928419). We thank the Department Hospitalo-Universitaire - Inflammation Immunopathology - Biotherapy (DHU I2B) for supporting our work. Authors’ contributions Authors contributions Conception and design: FA, SGL, S-AK, OH, SA. Cytokine measurements: FA, S-AK. Provision of study materials: OH, SGL, GG, AB, KSS, AA, CD. Data collection and analysis: SGL, OH, FA, S-AK, SA. Manuscript writing: All authors read and approved the final manuscript. Letters to the Editor: As these cytokines are known to be involved in chronic inflammation in FMF [9, 10], their presence could readily explain the un- controlled inflammation seen in the patient. Author details 1 1Sorbonne Universités, UPMC University Paris 06, INSERM UMR_S933, Hôpital Armand-Trousseau, Paris F-75012, France. 2Centre de référence de la fièvre méditerranéenne familiale, DHU I2B, Service de médecine interne, Hôpital Tenon, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris, France. 3Service d’Hématologie clinique, AP-HP, Hôpital Necker, Université Paris Descartes – Sorbonne Paris Cité, Imagine Institute, INSERM UMR 1163 et CNRS ERL 8254, Paris, France. 4Laboratoire d’hématologie biologique, Hôpital Necker, 149 rue de Sèvres, 75015 Paris, France. 5Service de Génétique, Assistance q Publique-Hôpitaux de Paris (AP-HP), Hôpital Trousseau, F-75012 Paris, France. Publique-Hôpitaux de Paris (AP-HP), Hôpital Trousseau, F-75012 Paris, France. Received: 25 March 2015 Accepted: 9 June 2015 Received: 25 March 2015 Accepted: 9 June 2015 From a more general viewpoint, our study unveils the interplay between two different disorders involving the same target cells. More specifically, it suggests that in myelodyspla- sia with inflammatory manifestations [15, 16], mutations in genes causing autoinflammatory syndromes, such as those found in MEFV, can be present and thus could be sought [17]. In this context it is interesting to note that an allogenic bone marrow transplantation on a young patient who had congenital dyserythropoietic anemia and FMF was once re- ported to significantly improve/treat the FMF symptoms [18] due to the hematopoietic involvement of the two disorders. Additional file 1: Patients and Methods. Table S1. Genotype, inflammatory and clinical status of the FMF patients. Competing interests The authors declare that they have no competing interests. Competing interests The authors declare that they have no competing interests. References 1. French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet. 1997;17:25–31. 1. French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet. 1997;17:25–31. 1. French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet. 1997;17:25–31. 2. Consortium TIF. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. The International FMF Consortium. Cell. 1997;90:797–807. 3. Centola M, Wood G, Frucht DM, Galon J, Aringer M, Farrell C, et al. The gene for familial Mediterranean fever, MEFV, is expressed in early leukocyte development and is regulated in response to inflammatory mediators. Blood. 2000;95:3223–31. 4. Cantarini L, Volpi N, Galeazzi M, Giani T, Fanti F, Lucherini OM, et al. Colchicine myopathy and neuromyopathy: two cases with different characteristics. J Clin Rheumatol Pract Rep Rheum Musculoskelet Dis. 2010;16:229–32. Our data suggest that among elderly FMF patients, with CMML, a severe inflammatory syndrome may appear and has to be treated. CMML diagnosis should be confirmed by bone marrow aspiration; and cytotoxic chemotherapy and/ or a demethylating agent should be considered in order to reduce monocytosis. This is even more important, as monocytosis is a key factor in the pathogenesis of FMF and the subsequent production of proinflammatory cytokines. In these rare cases of FMF or other autoinflammatory dis- eases combined with CMML, interleukin inhibitors (against IL-1, IL-6 or IL-18) alone or associated with a demethylat- ing agent could represent a valuable therapeutic strategy to decrease a potentially fatal inflammatory syndrome. 5. Selmi C, Ceribelli A, Naguwa SM, Cantarini L, Shoenfeld Y. Safety issues and concerns of new immunomodulators in rheumatology. Expert Opin Drug Saf. 2015;14:389–99. 6. Cantarini L, Lopalco G, Caso F, Costa L, Iannone F, Lapadula G, et al. Effectiveness and tuberculosis-related safety profile of interleukin-1 blocking agents in the management of Behçet’s disease. Autoimmun Rev. 2015;14:1–9. 7. Itzkson R, Fenaux P, Solary E. Chronic myelomonocytic leukemia: myelodysplastic or myeloproliferative? Best Pract Res Clin Haematol. 2013;26:387–400. 7. Itzkson R, Fenaux P, Solary E. Chronic myelomonocytic leukemia: myelodysplastic or myeloproliferative? Best Pract Res Clin Haematol. 2013;26:387–400. 8. Patnaik MM, Parikh SA, Hanson CA, Tefferi A. Chronic myelomonocytic leukaemia: a concise clinical and pathophysiological review. Br J Haematol. 2014;165:273–86. 8. Patnaik MM, Parikh SA, Hanson CA, Tefferi A. Chronic myelomonocytic leukaemia: a concise clinical and pathophysiological review. Br J Haematol. 2014;165:273–86. 9. Abbreviations FMF F ili l M FMF: Familial Mediterranean fever; CMML: Chronic myelomonocytic leukemia; CRP: C reactive protein; IL: Interleukin; SAA: Serum amyloid A. FMF: Familial Mediterranean fever; CMML: Chronic myelomonocytic leukemia; CRP: C reactive protein; IL: Interleukin; SAA: Serum amyloid A. 11. Macdougall IC, Cooper AC. Erythropoietin resistance: the role of inflammation and pro-inflammatory cytokines. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc. 2002;17 Suppl 11:39–43. Awad et al. Orphanet Journal of Rare Diseases (2015) 10:76 Additional file 11. Macdougall IC, Cooper AC. Erythropoietin resistance: the role of inflammation and pro-inflammatory cytokines. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc. 2002;17 Suppl 11:39–43. 11. Macdougall IC, Cooper AC. Erythropoietin resistance: the role of inflammation and pro-inflammatory cytokines. Nephrol Dial Transplant Off Publ Eur Dial Transpl Assoc - Eur Ren Assoc. 2002;17 Suppl 11:39–43. Page 4 of 4 Page 4 of 4 12. Maury CPJ, Liljeström M, Laiho K, Tiitinen S, Kaarela K, Hurme M. Anaemia of chronic disease in AA amyloidosis is associated with allele 2 of the interleukin-1beta-511 promoter gene and raised levels of interleukin-1beta and interleukin-18. J Intern Med. 2004;256:145–52. 13. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10:417–26. 14. Gang N, Drenth JP, Langevitz P, Zemer D, Brezniak N, Pras M, et al. Activation of the cytokine network in familial Mediterranean fever. J Rheumatol. 1999;26:890–7. 15. Braun T, Fenaux P. Myelodysplastic Syndromes (MDS) and autoimmune disorders (AD): cause or consequence? Best Pract Res Clin Haematol. 2013;26:327–36. 16. Mekinian A, Braun T, Decaux O, Falgarone G, Toussirot E, Raffray L, et al. Inflammatory arthritis in patients with myelodysplastic syndromes: a multicenter retrospective study and literature review of 68 cases. Medicine (Baltimore). 2014;93:1–10. 17. Jo T, Horio K, Migita K. Sweet’s syndrome in patients with MDS and MEFV mutations. N Engl J Med. 2015;372:686–8. 18. Milledge J, Shaw PJ, Mansour A, Williamson S, Bennetts B, Roscioli T, et al. Allogeneic bone marrow transplantation: cure for familial Mediterranean fever. Blood. 2002;100:774–7. 12. Maury CPJ, Liljeström M, Laiho K, Tiitinen S, Kaarela K, Hurme M. Anaemia of chronic disease in AA amyloidosis is associated with allele 2 of the interleukin-1beta-511 promoter gene and raised levels of interleukin-1beta and interleukin-18. J Intern Med. 2004;256:145–52. 13. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10:417–26. 13. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10:417–26. 14. Gang N, Drenth JP, Langevitz P, Zemer D, Brezniak N, Pras M, et al. Activation of the cytokine network in familial Mediterranean fever. J Rheumatol. 1999;26:890–7. 14. Gang N, Drenth JP, Langevitz P, Zemer D, Brezniak N, Pras M, et al. 12. Maury CPJ, Liljeström M, Laiho K, Tiitinen S, Kaarela K, Hurme M. Anaemia of chronic disease in AA amyloidosis is associated with allele 2 of the interleukin-1beta-511 promoter gene and raised levels of interleukin-1beta and interleukin-18. J Intern Med. 2004;256:145–52. 13. Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol Cell. 2002;10:417–26. 14. Gang N, Drenth JP, Langevitz P, Zemer D, Brezniak N, Pras M, et al. Activation of the cytokine network in familial Mediterranean fever. J Rheumatol. 1999;26:890–7. 15. Braun T, Fenaux P. Myelodysplastic Syndromes (MDS) and autoimmune disorders (AD): cause or consequence? Best Pract Res Clin Haematol. 2013;26:327–36. 16. Mekinian A, Braun T, Decaux O, Falgarone G, Toussirot E, Raffray L, et al. Inflammatory arthritis in patients with myelodysplastic syndromes: a multicenter retrospective study and literature review of 68 cases. Medicine (Baltimore). 2014;93:1–10. 17. Jo T, Horio K, Migita K. Sweet’s syndrome in patients with MDS and MEFV mutations. N Engl J Med. 2015;372:686–8. 18. Milledge J, Shaw PJ, Mansour A, Williamson S, Bennetts B, Roscioli T, et al. Allogeneic bone marrow transplantation: cure for familial Mediterranean fever. Blood. 2002;100:774–7. Additional file Activation of the cytokine network in familial Mediterranean fever. J Rheumatol. 1999;26:890–7. 15. Braun T, Fenaux P. Myelodysplastic Syndromes (MDS) and autoimmune disorders (AD): cause or consequence? Best Pract Res Clin Haematol. 2013;26:327–36. 15. Braun T, Fenaux P. Myelodysplastic Syndromes (MDS) and autoimmune disorders (AD): cause or consequence? Best Pract Res Clin Haematol. 2013;26:327–36. 16. Mekinian A, Braun T, Decaux O, Falgarone G, Toussirot E, Raffray L, et al. Inflammatory arthritis in patients with myelodysplastic syndromes: a multicenter retrospective study and literature review of 68 cases. Medicine (Baltimore). 2014;93:1–10. 16. Mekinian A, Braun T, Decaux O, Falgarone G, Toussirot E, Raffray L, et al. Inflammatory arthritis in patients with myelodysplastic syndromes: a multicenter retrospective study and literature review of 68 cases. Medicine (Baltimore). 2014;93:1–10. 17. Jo T, Horio K, Migita K. Sweet’s syndrome in patients with MDS and MEFV mutations. N Engl J Med. 2015;372:686–8. 17. Jo T, Horio K, Migita K. Sweet’s syndrome in patients with MDS and MEFV mutations. N Engl J Med. 2015;372:686–8. 18. Milledge J, Shaw PJ, Mansour A, Williamson S, Bennetts B, Roscioli T, et al. Allogeneic bone marrow transplantation: cure for familial Mediterranean fever. Blood. 2002;100:774–7. 18. Milledge J, Shaw PJ, Mansour A, Williamson S, Bennetts B, Roscioli T, et al. Allogeneic bone marrow transplantation: cure for familial Mediterranean fever. Blood. 2002;100:774–7. 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https://openalex.org/W4361265583	https://figshare.com/articles/journal_contribution/Supplementary_Figure_1_from_Patterns_of_Known_and_Novel_Small_RNAs_in_Human_Cervical_Cancer/22366475/1/files/39811517.pdf	English	null	Supplementary Figure 2 from Patterns of Known and Novel Small RNAs in Human Cervical Cancer	null	2,023	cc-by	244	0% 20% 40% 60% 80% 100% A 100% 80% 60% 40% 20% 0% SW756 C4I C33A CaSki SiHa ME-180 (n=1125) (n=1099) (n=1110) (n=749) (n=358) (n=660) 0% 20% 40% 60% 80% 100% A 100% 80% 60% 40% 20% 0% SW756 C4I C33A CaSki SiHa ME-180 (n=1125) (n=1099) (n=1110) (n=749) (n=358) (n=660) 0% 20% 40% 60% 80% 100% A 100% 80% 60% 40% 20% 0% SW756 C4I C33A CaSki SiHa ME-180 (n=1125) (n=1099) (n=1110) (n=749) (n=358) (n=660) B NC1 NC2 NC3 NC4 NC5 (n=421) (n=556) (n=269) (n=696) (n=261) 100% 80% 60% 40% 20% 0% Class III (candidate small RNA without significant hairpin ) Class II (candidate small RNA with non-canonical hairpin) Class I (candidate miRNA) previously identified miRNAs * mRNA repeat mitochondrial sn/sno/misc-RNA A 0% 20% 20% 0% SW756 C4I C33A CaSki SiHa ME-180 (n=1125) (n=1099) (n=1110) (n=749) (n=358) (n=660) B NC1 NC2 NC3 NC4 NC5 (n=421) (n=556) (n=269) (n=696) (n=261) 100% 80% 60% 40% 20% 0% tRNA rRNA Class III (candidate small RNA without significant hairpin ) Class II (candidate small RNA with non-canonical hairpin) Class I (candidate miRNA) previously identified miRNAs * not mapped/ unknown mRNA repeat mitochondrial sn/sno/misc-RNA B NC1 NC2 NC3 NC4 NC5 (n=421) (n=556) (n=269) (n=696) (n=261) 100% 80% 60% 40% 20% 0% B tRNA rRNA Class III (candidate small RNA without significant hairpin ) Class II (candidate small RNA with non-canonical hairpin) Class I (candidate miRNA) previously identified miRNAs * not mapped/ unknown mRNA repeat mitochondrial sn/sno/misc-RNA previously identified miRNAs
https://openalex.org/W3191238440	https://opus.lib.uts.edu.au/bitstream/10453/150713/2/A_Compact_Dual-Band_and_Dual-Polarized_Millimeter-Wave_Beam_Scanning_Antenna_Array_for_5G_Mobile_Terminals.pdf	English	null	A Compact Dual-Band and Dual-Polarized Millimeter-Wave Beam Scanning Antenna Array for 5G Mobile Terminals	IEEE access	2,021	cc-by	8,202	Received July 10, 2021, accepted July 25, 2021, date of publication July 28, 2021, date of current version August 10, 2021. Received July 10, 2021, accepted July 25, 2021, date of publication July 28, 2021, date of current version August 10, 2021. Received July 10, 2021, accepted July 25, 2021, date of publication July 28, 2021, date of current version August 10, 2021. Digital Object Identifier 10.1109/ACCESS.2021.3100933 YUQI HE 1, (Student Member, IEEE), SIHAN LV 1, LUYU ZHAO 1, (Senior Member, IEEE), GUAN-LONG HUANG2, (Senior Member, IEEE), XIAOMING CHEN 3, (Senior Member, IEEE), AND WEI LIN 4, (Senior Member, IEEE) INDEX TERMS 5G, dual-band, dual-polarization, mm-Wave, mobile phone antennas, microstrip antenna, phased array INDEX TERMS 5G, dual-band, dual-polarization, mm-Wave, mobile phone antennas, microstrip antenna, phased array. YUQI HE 1, (Student Member, IEEE), SIHAN LV 1, LUYU ZHAO 1, (Senior Member, IEEE), GUAN-LONG HUANG2, (Senior Member, IEEE), XIAOMING CHEN 3, (Senior Member, IEEE), AND WEI LIN 4, (Senior Member, IEEE) YUQI HE 1, (Student Member, IEEE), SIHAN LV 1, LUYU ZHAO 1, (Senior Member, IEEE), GUAN-LONG HUANG2, (Senior Member, IEEE), XIAOMING CHEN 3, (Senior Member, IEEE), AND WEI LIN 4, (Senior Member, IEEE) 1Key Laboratory of Antennas and Microwave Technologies, Xidian University, Xi’an 710071, China ( ) 1Key Laboratory of Antennas and Microwave Technologies, Xidian University, Xi’an 710071, China 2School of AI-Guangdong & Taiwan, Foshan University, Foshan, Guangdong 528225, China 3School of Information and Communications Engineering, Xi’an Jiaotong University, Xi’an 710049, China 4Global Big Data Technologies Centre, The University of Technology Sydney, Ultimo, NSW 2007, Australia Corresponding author: Luyu Zhao (lyzhao@xidian.edu.cn) This work was supported in part by the National Key Research and Development Program of China under Grant 2019YFF0216603, in part by the Key Research and Development Program of Shaanxi under Grant 2020ZDLGY15-03, in part by ZTE Corporation under Grant HC-CN-20191227012, in part by the Natural Science Foundation of China under Grant 61801300, and in part by the Fok Ying-Tong Education Foundation, China, under Grant 171056. ABSTRACT This paper presents a compact dual-band and dual-polarized millimeter-wave patch antenna array with satisfactory performance on element mutual coupling and beam scanning capabilities. Using capacitive feed technique and stacked conﬁguration with extra parasitic strips, the proposed antenna array is able to achieve a wide operating bandwidth in both the low- and high-bands. In order to reduce the array’s footprint, and to enhance the beam scanning performance in both bands, the element spacing is shrunk to less than 0.36 wavelength at 26 GHz. To improve the isolation between array elements due to their small spacings, two effective decoupling approaches are adopted, which result in a 6-dB isolation enhancement. The overall size of the proposed antenna array is only 18.2 mm × 4.1 mm × 1.07 mm, which is smaller than some industrial mm-Wave antenna modules released recently. Our simulation shows that the antenna array can fully cover the 5G NR bands of n258∼n261 simultaneously. The four-element array provides ±60◦ and ±45◦beam scanning performance in the low- and high-bands, respectively. The experimental data of reﬂection coefﬁcient, mutual coupling, and radiation patterns conﬁrm with the simulated results, rendering the proposed array to be a good candidate for 5G mm-Wave communications. INDEX TERMS 5G, dual-band, dual-polarization, mm-Wave, mobile phone antennas, microstrip antenna, phased array. under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VOLUME INDEX TERMS 5G, dual-band, dual-polarization, mm-Wave, mobile phone antennas, microstrip antenna, phased array. I. INTRODUCTION From another aspect, the spacing between the neigh- boring antenna elements in the array must be less than 0.5 λ so as to avoid grating lobes at large scanning angles. However, too close spacing will inevitably lead to strong coupling between antenna elements. Though many decou- pling methods have been reported in recent years [23]–[25], their application scenarios in mm-Wave terminals are limited. FIGURE 1. IMT mm-Wave bands allocated by WRC-19. TABLE 1. Specifications of 5G mm-Wave Bands. TABLE 1. Specifications of 5G mm-Wave Bands. TABLE 1. Specifications of 5G mm-Wave Bands. TABLE 1. Specifications of 5G mm-Wave Bands. TABLE 1. Specifications of 5G mm-Wave Bands. array is also demanded. Consequently, the mm-Wave anten- nas in mobile phone are expected to offer multi-band cov- erage with dual-polarized function, meanwhile they must be constructed in a limited size to form a compact antenna module. Such design speciﬁcations are very challenging for not only the academia but also the industry. Apart from the academic research, industries also provide some competitive antenna solutions. The latest mm-Wave module QTM052 from Qualcomm R⃝covers a bandwidth of 800 MHz including Bands n258, n260 and n261 [26] with a size of 19.03 mm × 4.81 × 1.7 mm3. It is reported that the mm-Wave module of Apple’s newly released iPhone 12 can cover Bands n260 and n261 with dual-polarization [27]. According to the public information, the module adopts the design strategy of separating low- and high-band antennas, which results in a relatively large module size. y y At present, there are already many innovative antenna designs proposed for mm-Wave mobile termial applications. In [7], a broadband coverage of 27-29 GHz is achieved by three well designed slot arrays. In [8], the proposed beam controllable collinear dipole array can operate from 22 GHz to 34 GHz. In [9], a mm-Wave magneto- electric monopole antenna with vertically polarized end ﬁre radiation is presented, which achieves both low proﬁle and broadband coverage of 23.5-44 GHz characteristics. In [10], a multi-beam antenna array with differential feed is proposed with an operating bandwidth of 26.8-29.2 GHz. In [11], hybrid mode technique is used to improve the bandwidth of the cavity backed mm-Wave slot antenna. In [12], a 5G mm-Wave phased array antenna conﬁgured with novel end- ﬁre planar folded slot antennas (PFSA) is proposed to operate at 37-39 GHz. The above-mentioned designs all demonstrate effecvtive array solutions in mm-Wave band. I. INTRODUCTION frequency bands bring additional design speciﬁcations and challenges for mm-Wave antennas compared with conven- tional Sub-6 GHz antennas, especially in the mobile terminal scenario [2]. To overcome the relatively high path-loss in the mm-Wave band and to increase the beam coverage capability, phased arrays with wide scanning angles are a preferred solu- tion in mm-Wave antenna designs for mobile phone applica- tions [3]–[6]. Meanwhile, the mainstream antenna arrays tend to cover as much 5G NR (New Radio) bands (cf. Table 1) as possible. Hence, dual-band/multi-band designs of antenna array become a necessity. On the other hand, to enable multiple-input multiple-output (MIMO) operations and alle- viate the multipath fading effect, a dual-polarized antenna Millimeter-wave (mm-wave) technologies for 5G applications attract more and more attentions after the 2019 World Radiocommunication Conference (WRC-19), where a series of globally uniﬁed resolutions on mm-Wave frequency bands for International Mobile Telecommunica- tions (IMT) are accomplished. As shown in Fig. 1, currently, the major 5G mm-Wave bands licensed worldwide include 24.25-27.5 GHz, 37-43.5 GHz, 45.5-47 GHz, 47.2-48.2 GHz, and 66-71 GHz [1]. Therefore, these newly-released The associate editor coordinating the review of this manuscript and approving it for publication was Wei Feng . VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array FIGURE 1. IMT mm-Wave bands allocated by WRC-19. TABLE 1. Specifications of 5G mm-Wave Bands. FIGURE 1. IMT mm-Wave bands allocated by WRC-19. polarizations [15]–[18]. Furthermore, for a phased array integrated in a limited volume of a mobile terminal, it is still necessary to reduce the communcation blind zone and achieve wide-angle beam steering [19]. As presented in [20], a compact patch antenna array operating in the band of 26-31.4 GHz is able to provide ±42◦beam coverage via beam switching. A dual-polarized mm-Wave antenna array operating in the band of 24.4-29.5 GHz with a scanning angle from −34◦to 33◦was proposed in [21]. Though these designs achieve certain promising performance, the scanning angle still needs to be extended in practical applications. The antennas proposed in [13] and [22] perform good perfor- mance with more than ±60◦beam scanning angle, yet they all occupy a large volume and are infeasiable for mobile phones as the mainstream mm-Wave antenna modules are installed in the side frames which are only several centimeters wide. I. INTRODUCTION However, they only operate in single band, which cannot comply with the IMT regulations that at least two bands have to be covered. In [13], a design scheme of mm-Wave broadband coverage using capacitance-feed and simple structure is proposed, which covers a broadband of 24-28 GHz in the form of single polarization. In [14], a dual-band and single-polarized antenna array operating at 28/38 GHz bands is obtained by utilizing novel photonics-based reconﬁgurable strategy. Nevertheless, to mitigate the multipath fading and to enable the MIMO operation for higher data rates, the antenna array in a mobile terminal is preferable to generate two orthogonal This paper mainly discusses an effective solution to meet the above-mentioned requirements and proposes a com- pact dual-band and dual-polarized patch antenna array with decoupling structures, which operates in the frequency bands of 26 GHz and 38 GHz. A 4-element antenna array based on this solution is able to provide ±60◦and ±45◦beam scanning capability in the two frequency bands, respectively. Compared to the existing mm-Wave antenna array designs, the design presented in this paper has the follow unique features: 1) Dual-Polarized characteristic with multiband coverage is enabled by advanced feeding design and elaborate parasitic structures. 2) The array is very compact in size due to the innovative decoupling approach utilized in the low-band. 3) A better beam scanning capability is achieved and experimentally veriﬁed for practical application, especially for the low-band. The remaining part of this paper is organized as follows: Section II introduces the design of the dual-band antenna element; Section III illustrates the antenna array design with a focus on the decoupling technique; Section IV presents 109043 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array TABLE 2. Detailed Dimensions of the Proposed Antenna (unit: mm). TABLE 2. Detailed Dimensions of the Proposed Antenna (unit: mm). the fabrication details of the antenna prototype as well as its performance evaluation; Conclusion will be drawn in Section V. II. DESIGN OF ANTENNA ELEMENT In this section, a dual-band and dual-polarized antenna element is designed in the 5G NR bands. The low-band (LB) and high-band (HB) cover 24.25-28.35 GHz and 37-43.5 GHz, respectively. The structural conﬁguration of the proposed antenna element is shown in Fig. 2. It is a stacked patch structure, where the bottom large patch (Fig. I. INTRODUCTION 2(b)) is used to generate a lower resonant frequency while the smaller patch on the top (Fig. 2(c)) is for higher resonant frequency. Dual-probe feeding method is utilized to achieve ±45◦linear polarizations. As is well-known, the probe-feed method would introduce additional inductive effect to the antenna [28]. Therefore, to increase the operating frequency band, power is coupled from the two probes to the bottom patch through two circular plates, which are realized by cutting two ring slots on the bottom patch. Compared with directly connecting to the patch, such indirect feeding brings additional capacitance to counteract the inductiveness from the probe-feed. Moreover, four corners of the bottom square patch are cut-off to further ﬁne tune its resonant frequency and reduce the footprint of the patch. The upper patch works at 39 GHz band. Better impedance matching and higher gain are achieved by opening a square aperture in the middle of the patch. Additionally, four bent parasitic strips are intro- duced around the upper patch to generate an extra resonant mode near the 41 GHz band so as to broaden the operating bandwidth in the HB. The antenna element is implemented by using the printed circuit board (PCB) technology. Rogers RO4350B (εr= 3.52) is used for the double-layer dielectric substrate, and Rogers RO4450F with a thickness of 4 mils is functioned as a prepreg (pp) layer which is utilized to bond different layers together. Detailed dimensions of the antenna element are list in Table 2. The antenna element is modeled by Ansys HFSS R⃝, and its scattering parameters (S-parameters) in both LB and HB are superposed in Fig. 3. The results indi- cate a better than −10 dB reﬂection coefﬁcient (represented by \|S11\|) over the frequency bands of 24.2-27.7 GHz and 36.2-43.8 GHz is achieved, which can fully cover the required 5G NR mm-Wave frequency bands. In addition, Fig. 3 shows the reﬂection coefﬁcient performance of the element without capacitance-feeding structure, from the comparison of which it can be seen that the operating bandwidth of the antenna is greatly increased from 1.6 GHz to 3.5 GHz in the LB. Also, the S-parameters of the HB element with and without the parasitic structures and the opening square aperture are shown in Fig. 3, from which one can observe that these structures greatly improve the impedance matching and port isolation in the HB. Fig. 4 and Fig. I. INTRODUCTION 5 shows the current distributions of different structures of the antenna element at different operating frequencies when the port 1 and 2 is excited. Fig. 4 (a) and Fig. 5 (a) are the current distribution of the LB antenna at 26 GHz, Fig. 4 (b) and Fig. 5 (b) show the current distribution of the HB antenna at 38 GHz, while Fig. 4 (c) and Fig. 5 (c) display the current distribution of the parasitic strips at 41 GHz. FIGURE 2. Geometrical configuration of the proposed mm-Wave antenna element. (a) 3D view. (b) Sketch of the HB patch. (c) Sketch of the LB patch. 109044 FIGURE 2. Geometrical configuration of the proposed mm-Wave antenna element. (a) 3D view. (b) Sketch of the HB patch. (c) Sketch of the LB patch. excited. Fig. 4 (a) and Fig. 5 (a) are the current distribution of the LB antenna at 26 GHz, Fig. 4 (b) and Fig. 5 (b) show the current distribution of the HB antenna at 38 GHz, while Fig. 4 (c) and Fig. 5 (c) display the current distribution of the parasitic strips at 41 GHz. FIGURE 3. Simulated S-parameters of the antenna element at two operating bands. 109044 VOLUME 9, 2021 FIGURE 3. Simulated S-parameters of the antenna element at two operating bands. VOLUME 9 2021 FIGURE 2. Geometrical configuration of the proposed mm-Wave antenna element. (a) 3D view. (b) Sketch of the HB patch. (c) Sketch of the LB patch. FIGURE 3. Simulated S-parameters of the antenna element at two operating bands. VOLUME 9, 2021 VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array FIGURE 4. Current distribution of different structure of antenna element when the port 1 is excited. (a) Low band antenna at 26 GHz. (b) High band antenna at 38 GHz. (c) Parasitic strips at 41 GHz. FIGURE 6. Simulated radiation patterns of the two orthogonal polarizations (pol.) of the antenna elements at LB and HB. (a) +45◦pol. at 26 GHz. (b) −45◦pol. at 26 GHz. (c) +45◦pol. at 39 GHz. (d) −45◦pol. at 39 GHz. FIGURE 4. Current distribution of different structure of antenna element when the port 1 is excited. (a) Low band antenna at 26 GHz. (b) High band antenna at 38 GHz. (c) Parasitic strips at 41 GHz. FIGURE 6. I. INTRODUCTION Simulated radiation patterns of the two orthogonal polarizations (pol.) of the antenna elements at LB and HB. (a) +45◦pol. at 26 GHz. (b) −45◦pol. at 26 GHz. (c) +45◦pol. at 39 GHz. (d) −45◦pol. at 39 GHz. FIGURE 6. Simulated radiation patterns of the two orthogonal polarizations (pol.) of the antenna elements at LB and HB. (a) +45◦pol. at 26 GHz. (b) −45◦pol. at 26 GHz. (c) +45◦pol. at 39 GHz. (d) −45◦pol. at 39 GHz. FIGURE 4. Current distribution of different structure of antenna element when the port 1 is excited. (a) Low band antenna at 26 GHz. (b) High band antenna at 38 GHz. (c) Parasitic strips at 41 GHz. FIGURE 5. Current distribution of different structure of antenna element when the port 2 is excited. (a) Low band antenna at 26 GHz. (b) High band antenna at 38GHz. (c) Parasitic strips at 41 GHz. III. DESIGN OF THE ANTENNA ARRAY A. CONSIDERATION OF MODERATE AND COMPACT ELEMENT ARRANGEMENTS In 5G mobile terminals, very limited space is reserved for mm-Wave phased arrays because of the existence of the Sub-6 GHz antennas, as well as the metal frames and full display screen. Both the miniaturized design of the antenna unit as discussed in the previous section and the reduced spacing of antenna elements will effectively contribute to a size reduction of the mm-Wave antenna array. However, for a dual-band antenna array, there is always a dilemma between the element spacing and array performance. For instance, once the physical distance between the array elements is around λ/2 at 26 GHz in the LB, the size of the array will be relatively large in the HB, and grating lobes will be generated during beam scanning since the element spacing in the HB is now greater than 0.75λ at 39 GHz. Such situation is called moderate arrangement in this work. In contrast, if the element spacing is chosen to be about λ/2 at 39 GHz, the mutual coupling in the LB will signiﬁcantly increase as the corre- sponding spacing in the LB is only 0.36λ at 26 GHz. This case is called compact arrangement. Therefore, according to the above analysis, the moderate arrangement of array elements will lead to large grating lobes in the HB while the compact arrangement will result in strong mutual coupling in the LB, which inevitably affects the antenna radiation efﬁciency and causes scanning blindness. In order to balance the array size and performance, the compact arrangement is chosen while effective decoupling approaches are taken in this work to deal with the resulting mutual coupling in the LB. FIGURE 5. Current distribution of different structure of antenna element when the port 2 is excited. (a) Low band antenna at 26 GHz. (b) High band antenna at 38GHz. (c) Parasitic strips at 41 GHz. Fig. 6 shows the simulated far-ﬁeld radiation patterns of the antenna element. The maximum gain value is found to be 5.8 dBi at 28 GHz and 6.2 dBi at 39 GHz, with at least 20 dB XPD (cross-polarization discrimination) at 26 GHz and 15 dB XPD at 39 GHz. The half-power beamwidth (HPBW) of the antenna element are 112◦at 26 GHz and 98◦at 39 GHz. The radiation pattern of the back lobe is larger at HB, which is caused by the larger size of the high-frequency parasitic strips structure relative to the ground plane. B. DECOUPLING STRUCTURE DESIGN In order to clearly demonstrate the effectiveness of the two proposed structures on mutual coupling suppression between the antenna elements, the current distributions of the two ele- ments in the proposed antenna array with respect to different conﬁgurations are given in Fig. 8, respectively. As shown in Fig. 8 (a), when Port 2 is excited, it is observed that a direct and strong coupling (S1) appears between Element A and Element B. To start with, the coupling between the two antenna elements can be effectively reduced by etching the C-shaped split ring slot etched on the ground plane, as shown in Fig. 8 (b), at this time the coupling between antenna Elements A and B is reduced to S2. Besides the C-shaped SRSs, I-shaped parasitic resonators are also introduced between the LB antenna elements to fur- ther improve their isolations, as shown in Fig. 8 (c), the typ- ical electrical length of the parasitic I-shaped resonators is around half wavelengths at the center frequency of the LB. In addition, both ends of the I-shaped parasitic resonator are bent to minimize its electrical length, providing a capaci- tive load to control the magnitude of coupling between the adjacent antenna elements. While in Fig. 8 (c), a new cou- pling route (S3) can be artiﬁcially generated through the I-shaped parasitic resonator. By changing the width (W2) and lengths (L6 & L7) of the I-shaped parasitic resonator, the amplitude and phase of the coupling introduced by the I-resonator can be adjusted properly so as to cancel out the existing coupling S1, the remaining coupling is marked as S3 in Fig. 8 (c). FIGURE 8. Vector current distribution of the two elements in the proposed antenna array. (a) Without the I-shaped resonator and the C-shaped SRSs. (b) With the C-shaped SRSs only. (c) With the I-shaped resonator only. (d) With the I-shaped resonator and the C-shaped SRSs. are added simultaneously. Eventually, the proposed antenna array with two decoupling structures could further reduce the coupling to a much lower level as shown in Fig.8 (d). As can be seen from the EM simulation results shown in Fig. 9, after introducing the I-shaped resonator between the antenna elements and the C-shaped SRS etched on the ground plane, the cross-polarization (x-pol.) isolation levels can be improved by around 6 dB. Fig. 10 shows the co-polarization (co-pol.) isolation in the array. III. DESIGN OF THE ANTENNA ARRAY A. CONSIDERATION OF MODERATE AND COMPACT ELEMENT ARRANGEMENTS As shown in Fig. 7, the array is formed by four dual- band antenna elements with an element spacing of 4.2 mm 109045 109045 109045 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array FIGURE 8. Vector current distribution of the two elements in the proposed antenna array. (a) Without the I-shaped resonator and the C-shaped SRSs. (b) With the C-shaped SRSs only. (c) With the I-shaped resonator only. (d) With the I-shaped resonator and the C-shaped SRSs. FIGURE 7. 4-element patch antenna array with decoupling structures. (a) I-shaped resonator. (b) C-shaped split ring slot. FIGURE 7. 4-element patch antenna array with decoupling structures. (a) I-shaped resonator. (b) C-shaped split ring slot. (equivalent to 0.36 λ at 26 GHz and 0.5 λ at 39 GHz). The overall dimension of the antenna array is 18.2 mm × 4.1 mm × 1.07 mm. Two sets of decoupling structures are adopted, i.e., the modiﬁed I-shaped parasitic resonators between the elements (see Fig. 7(a)) and the C-shaped split ring slot (SRS) etched on the ground plane (see Fig. 7(b)). B. DECOUPLING STRUCTURE DESIGN In addition, the co-polarization isolation between antenna elements is increased by 11 dB at most. It is worth mentioning that the two decoupling methods have no impact on antenna matching performance, and it will be shown in the next session that isolations among all elements in the array are better than 15 dB, demonstrating that In addition, when two different decoupling structures are added separately, the induced currents in antenna Element B are in opposite directions (S2 and S3), which allows the currents to cancel each other when two decoupling structures 109046 VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array FIGURE 9. Impact of the decoupling approaches on the cross-polarization isolation improvement between the antenna elements. (a) Isolation between Port 2 and Port 3. (b) Isolation between Port 1 and Port 4. FIGURE 10. Impact of the decoupling approaches on the co-polarization isolation improvement between the antenna elements. (a) Isolation between Port 1 and Port 3. (b) Isolation between Port 2 and Port 4. FIGURE 10. Impact of the decoupling approaches on the co-polarization isolation improvement between the antenna elements. (a) Isolation between Port 1 and Port 3. (b) Isolation between Port 2 and Port 4. FIGURE 9. Impact of the decoupling approaches on the cross-polarization isolation improvement between the antenna elements. (a) Isolation between Port 2 and Port 3. (b) Isolation between Port 1 and Port 4. FIGURE 11. Structure and layer stack of the proposed 4-element antenna array. they are effective decoupling techniques for the mm-Wave application. IV. PERFORMANCE EVALUATION AND DISCUSSION In order to validate the simulated results, a prototype of the proposed array antenna is fabricated and measured. It should be noted that the size of the mini-SMP (SMPM) connectors used in the mm-Wave measurement is compa- rable to the antenna itself. Therefore, for successful install- ment of the connectors, the ground plane is expanded to be 30 mm × 185 mm. An explosive-view and the layout of the bottom layer of the proposed antenna array are demon- strated in Fig. 11 and Fig. 12, respectively. As can be seen from Fig. 12, the footprints of the SMPM connectors will affect the integrity of the ground plane 2. Therefore, to ensure that the antenna array has a complete reﬂection ground plane, a two-layer grounding structure is constructed, which are connected by metallized vias as shown in Fig. 11. In these layers, the ground plane 1 is used to prevent the FIGURE 11. Structure and layer stack of the proposed 4-element antenna array. joint package affecting the antenna performance. In ground plane 1, eight metallized vias are constructed for signal transmitting from the SMPM connectors to the LB antenna elements. In addition, 301 metallized shorting vias are drilled between grounding layers 1 and 2 to maintain excellent elec- trical contact. All the dielectric substrates 1, 2 and 3 are Rogers 4350B. Detailed dimensions of the antenna array are list in Table 3. VOLUME 9, 2021 109047 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array ompact Dual Band and Dual Polarized mm Wave Beam Scanning Antenna Array FIGURE 14. Measured S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. FIGURE 15. Simulated S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. Keysight N5225A network analyzer. During the measure- ment, when the two orthogonal ports of one antenna element are under test, the other six ports are terminated with 50 VOLUME 9, 2021 p g y FIGURE 12. Layout of the bottom layer of the array prototype. TABLE 3. Detailed Dimensions of the Antenna Array (unit: mm). FIGURE 13. Prototype and measurement setup. (a) Prototype of the proposed 4-element antenna array. (b) Measurement setup of S-parameters. (c) Array under test with a phase modulator (BBox). (d) Measurement setup of radiation patterns. According to the array stacked ﬁgure shown in Fig. IV. PERFORMANCE EVALUATION AND DISCUSSION 11, an array prototype is fabricated and measured, as shown in Fig 13 The antenna array is measured by a two-port FIGURE 14. Measured S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. FIGURE 15. Simulated S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. Keysight N5225A network analyzer. During the measure- ment, when the two orthogonal ports of one antenna element are under test the other six ports are terminated with 50 FIGURE 14. Measured S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. FIGURE 12. Layout of the bottom layer of the array prototype. URE 12. Layout of the bottom layer of the array prototype. TABLE 3. Detailed Dimensions of the Antenna Array (unit: mm). FIGURE 13. Prototype and measurement setup. (a) Prototype of the proposed 4-element antenna array. (b) Measurement setup of S-parameters. (c) Array under test with a phase modulator (BBox). (d) Measurement setup of radiation patterns. FIGURE 14. Measured S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. FIGURE 14. Measured S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. FIGURE 13. Prototype and measurement setup. (a) Prototype of the proposed 4-element antenna array. (b) Measurement setup of S-parameters. (c) Array under test with a phase modulator (BBox). (d) Measurement setup of radiation patterns FIGURE 13. Prototype and measurement setup. (a) Prototype of the proposed 4-element antenna array. (b) Measurement setup of S-parameters. (c) Array under test with a phase modulator (BBox). (d) Measurement setup of radiation patterns. FIGURE 15. Simulated S-parameters of the proposed antenna. (a) Reflection coefficient. (b) Isolation. Keysight N5225A network analyzer. During the measure- ment, when the two orthogonal ports of one antenna element are under test, the other six ports are terminated with 50 According to the array stacked ﬁgure shown in Fig. 11, an array prototype is fabricated and measured, as shown in Fig. 13. The antenna array is measured by a two-port According to the array stacked ﬁgure shown in Fig. 11, an array prototype is fabricated and measured, as shown in Fig. 13. The antenna array is measured by a two-port 109048 109048 VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array FIGURE 16. 2D beam scanning patterns of the antenna array in the plane of theta = 90◦. IV. PERFORMANCE EVALUATION AND DISCUSSION (a) Simulated patterns of +45◦polarization at 26.6 GHz for proposed antenna array. (b) Simulated patterns of −45◦polarization at 26.6 GHz proposed antenna array. (c) Simulated patterns of +45◦ polarization at 38 GHz proposed antenna array. (d) Simulated patterns of −45◦polarization at 38 GHz proposed antenna array. (e) Simulated patterns of +45◦polarization at 26.6 GHz for antenna array without decoupling structure. (f) Simulated patterns of −45◦polarization at 26.6 GHz for antenna array without decoupling structure. (g) Measured patterns of +45◦polarization at 26.6 GHz. (h) Measured patterns of −45◦ polarization at 26.6 GHz. FIGURE 17. Simulated realized gain with respect to the +45◦polarization and −45◦polarization of the antenna array. FIGURE 17. Simulated realized gain with respect to the +45◦polarization and −45◦polarization of the antenna array. proposed array is evaluated by both simulation and measure- ment. As shown in Figs. 16(a) and (b), the EM simulation shows that the scanning angle of the proposed antenna array can reach ±63◦at 26.6 GHz, and the scanning angle is about ±45◦at 38 GHz as plotted in Figs. 16(c) and (d). In addition, the simulation patterns of the antenna array with- out decoupling structures are given in Figs. 16(e) and (f). It can be seen that both the scanning angle and the gain of the proposed antenna array are improved, and the sidelobe levels of the radiation patterns are also reduced. The beam scanning ability of the antenna array is also measured with the help of TMYTEK R⃝phase modulator, BBOX One [29], at 26 GHz to verify the consistency between the simulation and actual measurement. The antenna pattern measurement environment is already shown in Fig. 13(c). Figs. 16(g) and (h) display the measured beam scanning diagrams of the antenna array at 26.6 GHz, which shows less than 0.2 dB gain discrepancy compared to the simulated ones. It should be noted that due to the symmetrical arrangement of the antenna array, the beam scan- ning performances of the two polarizations are basically the same. Also, the beam steering characteristics of the array for plus/minus (+/−) scanning angles are basically symmetric. Therefore, for simpliﬁcation, only the beam scanning results of one direction are given in Fig. 16. Simulated realized gains shown in Fig. 17 are found to be around 10 dBi over the two band. FIGURE 16. 2D beam scanning patterns of the antenna array in the plane of theta = 90◦. [2] W. Hong, K.-H. Baek, and S. Ko, ‘‘Millimeter-wave 5G antennas for smartphones: Overview and experimental demonstration,’’ IEEE Trans. Antennas Propag., vol. 65, no. 12, pp. 6250–6261, Dec. 2017. [1] World Radiocommunication Conference 2019 (WRC-19), Final Acts. Accessed: Nov. 2019. [Online]. Available: https://www.itu.int/pub/R-ACT- WRC.14-2019 pp [3] B. Xu, K. Zhao, Z. Ying, D. Sjöberg, W. He, and S. He, ‘‘Analysis of impacts of expected RF EMF exposure restrictions on peak EIRP of 5G user equipment at 28 GHz and 39 GHz bands,’’ IEEE Access, vol. 7, pp. 20996–21005, 2019. IV. PERFORMANCE EVALUATION AND DISCUSSION (a) Simulated patterns of +45◦polarization at 26.6 GHz for proposed antenna array. (b) Simulated patterns of −45◦polarization at 26.6 GHz proposed antenna array. (c) Simulated patterns of +45◦ polarization at 38 GHz proposed antenna array. (d) Simulated patterns of −45◦polarization at 38 GHz proposed antenna array. (e) Simulated patterns of +45◦polarization at 26.6 GHz for antenna array without decoupling structure. (f) Simulated patterns of −45◦polarization at 26.6 GHz for antenna array without decoupling structure. (g) Measured patterns of +45◦polarization at 26.6 GHz. (h) Measured patterns of −45◦ polarization at 26.6 GHz. For a more visualized perception of the beam scanning capability, the proposed antenna array is placed on the short edge of a rectangular metallic board whose size mimics the actual size of a 5G ﬂagship mobile phone. Fig. 18 and Fig. 19 show the three-dimensional beam scanning patterns of the 4-element antenna array in free space operating in different bands. SMPM loads, as shown in Fig. 12(b). The measured and simulated S-parameters are shown in Fig. 14 and Fig. 15, respectively, which are in good agreement with each other. The passive experimental results validate that the antenna array can well cover the bands of 24-28 GHz and 36-42 GHz simultaneously with all port isolations better than 15 dB. Furthermore, to emphasize the strength of the proposed mm-Wave antenna array, a comparison between the proposed design and several mm-Wave arrays reported recently is shown in Table 4. Some key antenna characteristics includ- ing operating band, antenna size, polarization diversity and beam scanning range are highlighted. After the compari- son, it is clear that the proposed design not only achieves a As mentioned previously, beam scanning capability is a key ﬁgure of merit for mm-Wave antenna arrays in a 5G mobile terminal. Therefore, the beam scanning angle of the 109049 VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array FIGURE 18. 3D beam scanning patterns of +45◦polarization at 26 GHz with different phi angles in the plane of theta = 90◦. (a) 0◦. (b) 15◦. (c) 30◦. (d) 45◦. (e) 60◦. TABLE 4. Comparison between the Proposed Design and Other Reported Works. TABLE 4. Comparison between the Proposed Design and Other Reported Works. V. CONCLUSION In this paper, a dual-band and dual-polarized mm-Wave antenna array with decoupling structures has been proposed for 5G mobile terminal applications. The proposed design starts with the investigation of the antenna element, followed by two decoupling approaches for mutual coupling reduc- tion in the low-band, the operating principle of which has also been analyzed in detail. Subsequently, the antenna array performances have been conﬁrmed with the experimental results, which show that the isolation levels among all the eight ports are more than 15 dB in both the 24-28 GHz and 36-42 GHz bands, with reﬂection coefﬁcients less than −10 dB. The beam scanning angles of the antenna array are about ±60◦and ±45◦in the LB and the HB, respectively. The satisfactory performance reveals the proposed antenna array can be a promising candidate for 5G mm-Wave mobile terminals. FIGURE 18. 3D beam scanning patterns of +45◦polarization at 26 GHz with different phi angles in the plane of theta = 90◦. (a) 0◦. (b) 15◦. (c) 30◦. (d) 45◦. (e) 60◦. FIGURE 19. 3D beam scanning patterns of +45◦polarization at 38 GHz with different phi angles in the plane of theta = 90◦. (a) 0◦. (b) 15◦. (c) 30◦. (d) 45◦. ACKNOWLEDGMENT The authors would like to express our gratitude to Xi’an Lambda Communication Technology Company Ltd., for providing the mm-Wave single probe near/far ﬁeld mobile antenna measurement system, and TMY Technology Inc., for the help on the phase modulator BBOX. They would also like to thank Prof. Long Zhang and Prof. Sai-Wai Wong for preparation of the samples and helpful discussions. [1] World Radiocommunication Conference 2019 (WRC-19), Final Acts. Accessed: Nov. 2019. [Online]. Available: https://www.itu.int/pub/R-ACT- WRC.14-2019 [2] W. Hong, K.-H. Baek, and S. Ko, ‘‘Millimeter-wave 5G antennas for smartphones: Overview and experimental demonstration,’’ IEEE Trans. Antennas Propag., vol. 65, no. 12, pp. 6250–6261, Dec. 2017. [3] B. Xu, K. Zhao, Z. Ying, D. Sjöberg, W. He, and S. He, ‘‘Analysis of impacts of expected RF EMF exposure restrictions on peak EIRP of 5G user equipment at 28 GHz and 39 GHz bands,’’ IEEE Access, vol. 7, pp. 20996–21005, 2019. REFERENCES FIGURE 19. 3D beam scanning patterns of +45◦polarization at 38 GHz with different phi angles in the plane of theta = 90◦. (a) 0◦. (b) 15◦. (c) 30◦. (d) 45◦. larger bandwidth in dual-band operation, but also provides a larger scanning angle in the two orthogonal polarizations, which can be a promising candidate for 5G mm-Wave mobile terminals. 109050 109050 VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array [4] W. Hong, K.-H. Baek, Y. Lee, Y. Kim, and S.-T. Ko, ‘‘Study and proto- typing of practically large-scale mmWave antenna systems for 5G cellular devices,’’ IEEE Commun. Mag., vol. 52, no. 9, pp. 63–69, Sep. 2014. [24] X. Shen, Y. Liu, L. Zhao, G.-L. Huang, X. Shi, and Q. Huang, ‘‘A miniaturized microstrip antenna array at 5G millimeter-wave band,’’ IEEE Antennas Wireless Propag. Lett., vol. 18, no. 8, pp. 1671–1675, Aug. 2019. [5] J. Helander, K. Zhao, Z. Ying, and D. Sjöberg, ‘‘Performance analysis of millimeter-wave phased array antennas in cellular handsets,’’ IEEE Antennas Wireless Propag. Lett., vol. 15, pp. 504–507, Mar. 2016. g [25] J.-N. Hwang and S.-J. Chung, ‘‘Isolation enhancement between two packed antennas with coupling element,’’ IEEE Antennas Wireless Propag. Lett., vol. 10, pp. 1263–1266, 2011. [6] J. Helander, D. Sjöberg, M. Gustafsson, K. Zhao, and Z. Ying, ‘‘Character- ization of millimeter wave phased array antennas in mobile terminal for 5G mobile system,’’ in Proc. IEEE Int. Symp. Antennas Propag. USNC/URSI Nat. Radio Sci. Meeting, Vancouver, BC, Canada, Jul. 2015, pp. 7–8. [26] Qualcomm QTM052 mm-Wave Antenna Modules. Accessed: Jul. 2018. [Online]. Available: http://www.qualcomm.com/products/qtm052- mmwave-antenna-modules [27] Apple’s Mobile Phone-iPhone 12. Accessed: Oct. 2020. [Online]. Avail- able: http://www.apple.com/iphone-12/specs/ [7] S. Zhang, X. Chen, I. Syrytsin, and G. F. Pedersen, ‘‘A planar switchable 3-D-coverage phased array antenna and its user effects for 28-GHz mobile terminal applications,’’ IEEE Trans. Antennas Propag., vol. 65, no. 12, pp. 6413–6421, Dec. 2017. [28] H. Wong, K.-L. Lau, and K.-M. Luk, ‘‘Design of dual-polarized L-probe patch antenna arrays with high isolation,’’ IEEE Trans. Antennas Propag., vol. 52, no. 1, pp. 45–52, Jan. 2004. [8] D. Psychoudakis, Z. Wang, and F. Aryanfar, ‘‘Dipole array for mm-wave mobile applications,’’ in Proc. IEEE Antennas Propag. Soc. Int. Symp. (APSURSI), Orlando, FL, USA, Jul. 2013, pp. 660–661. [29] TMYTEK Phase Modulator-BBOX One. Accessed: Mar. 2020. [Online]. Available: http://www.tmytek.com/bbox [9] J. Wang, Y. Li, J. Wang, L. Ge, M. REFERENCES Chen, Z. Zhang, and Z. Li, ‘‘A low- proﬁle vertically polarized magneto-electric monopole antenna with a 60% bandwidth for millimeter-wave applications,’’ IEEE Trans. Antennas Propag., vol. 69, no. 1, pp. 3–13, Jan. 2021. YUQI HE (Student Member, IEEE) received the B.S. degree from Xidian University, Xi’an, China, in 2019, where he is currently pursu- ing the M.D. degree in electromagnetic wave and microwave technology. His research interests include millimeter wave array, phased antenna array, and metamaterial-based antenna arrays. [10] Q. Yang, S. Gao, Q. Luo, L. Wen, Y.-L. Ban, X. Ren, J. Wu, X. Yang, and Y. Liu, ‘‘Millimeter-wave dual-polarized differentially fed 2-D multibeam patch antenna array,’’ IEEE Trans. Antennas Propag., vol. 68, no. 10, pp. 7007–7016, Oct. 2020. [11] H. Li, Y. Cheng, L. Mei, and F. Wu, ‘‘Dual-polarized frame-integrated slot arrays for 5G mobile handsets,’’ IEEE Antennas Wireless Propag. Lett., vol. 19, no. 11, pp. 1953–1957, Nov. 2020. [12] J. Park, H. Seong, Y. N. Whang, and W. Hong, ‘‘Energy-efﬁcient 5G phased arrays incorporating vertically polarized endﬁre planar folded slot antenna for mmWave mobile terminals,’’ IEEE Trans. Antennas Propag., vol. 68, no. 1, pp. 230–241, Jan. 2020. [13] M. Stanley, Y. Huang, H. Wang, H. Zhou, A. Alieldin, and S. Joseph, ‘‘A capacitive coupled patch antenna array with high gain and wide coverage for 5G smartphone applications,’’ IEEE Access, vol. 6, pp. 41942–41954, 2018. SIHAN LV is currently pursuing the master’s degree with the National Key Laboratory of Science and Technology on Antennas and Microwaves, Xidian University. He has co- invented three Chinese patents. His main research interests include 5G millimeter wave terminal antennas and millimeter wave MIMO antenna technology. [14] I. F. da Costa, S. A. Cerqueira, D. H. Spadoti, L. G. da Silva, J. A. J. Ribeiro, and S. E. Barbin, ‘‘Optically controlled reconﬁgurable antenna array for mm-wave applications,’’ IEEE Antennas Wireless Propag. Lett., vol. 16, pp. 2142–2145, 2017. [15] Y. He, M. Rao, Y. Liu, G. Jing, M. Xi, and L. Zhao, ‘‘28/39-GHz dual- band dual-polarized millimeter wave stacked patch antenna array for 5G applications,’’ in Proc. Int. Workshop Antenna Technol. (iWAT), Bucharest, Romania, Feb. 2020, pp. 1–4. [16] R. M. Moreno, J. Ala-Laurinaho, A. Khripkov, J. Ilvonen, and V. Viikari, ‘‘Dual-polarized mm-wave endﬁre antenna for mobile devices,’’ IEEE Trans. Antennas Propag., vol. 68, no. 8, pp. 5924–5934, Aug. 2020. [17] W. Hong, S.-T. Ko, Y. Lee, and K.-H. REFERENCES Baek, ‘‘Multi-polarized antenna array conﬁguration for mmWave 5G mobile terminals,’’ in Proc. Int. Workshop Antenna Technol. (iWAT), Seoul, South Korea, Mar. 2015, pp. 60–61. LUYU ZHAO (Senior Member, IEEE) was born in Xi’an, China, in 1984. He received the B.Eng. degree from Xidian University, Xi’an, in 2007, and the Ph.D. degree from The Chinese University of Hong Kong, Sha Tin, Hong Kong, in 2014. in Xi an, China, in 1984. He received the B.Eng. degree from Xidian University, Xi’an, in 2007, and the Ph.D. degree from The Chinese University of Hong Kong, Sha Tin, Hong Kong, in 2014. From 2007 to 2009, he was with the Key Lab- oratory of Antennas and Microwave Technology, Xidian University, as a Research Assistant, where he was involved in software and hardware imple- mentation of RF identiﬁcation (RFID) technolo- gies. From 2014 to 2015, he was a Postdoctoral Fellow at The Chinese University of Hong Kong. From October 2015 to October 2016, he was with Wyzdom Wireless Company Ltd., where he was a Co-Founder and the CTO. He has been an Associate Professor with the National Key Laboratory of Antennas and Microwave Technology, Xidian University, since 2016. He has also been with Lambda Communication Company Ltd., since 2019. His current research interests include design and application of multiple antenna systems for next generation mobile communication systems, innovative passive RF and microwave components and systems, millimeter wave and terahertz antenna array, and meta-material-based or inspired antenna arrays. Dr. Zhao was a recipient of the Best Student Paper Award of 2013 IEEE 14th HK AP/MTT Postgraduate Conference, the Honorable Mention Award of 2017 Asia-Paciﬁc Conference on Antenna and Propagation, and the Best Paper Award of IEEE ICEICT 2019. [18] R. M. Moreno, J. Kurvinen, J. Ala-Laurinaho, A. Khripkov, J. Ilvonen, J. van Wonterghem, and V. Viikari, ‘‘Dual-polarized mm-wave endﬁre chain-slot antenna for mobile devices,’’ IEEE Trans. Antennas Propag., vol. 69, no. 1, pp. 25–34, Jan. 2021. From 2007 to 2009, he was with the Key Lab- oratory of Antennas and Microwave Technology, Xidian University, as a Research Assistant, where he was involved in software and hardware imple- mentation of RF identiﬁcation (RFID) technolo- gies. From 2014 to 2015, he was a Postdoctoral Fellow at The Chinese University of Hong Kong. From October 2015 to October 2016, he was with Wyzdom Wireless Company Ltd., where he was a Co-Founder and the CTO. REFERENCES He has been an Associate Professor with the National Key Laboratory of Antennas and Microwave Technology, Xidian University, since 2016. He has also been with Lambda Communication Company Ltd., since 2019. His current research interests include design and application of multiple antenna systems for next generation mobile communication systems, innovative passive RF and microwave components and systems, millimeter wave and terahertz antenna array, and meta-material-based or inspired antenna arrays. Dr Zhao was a recipient of the Best Student Paper Award of 2013 IEEE [19] W. Hong, Z. H. Jiang, C. Yu, J. Zhou, P. Chen, Z. Yu, H. Zhang, B. Yang, X. Pang, M. Jiang, and Y. Cheng, ‘‘Multibeam antenna technologies for 5G wireless communications,’’ IEEE Trans. Antennas Propag., vol. 65, no. 12, pp. 6231–6249, Dec. 2017. [20] K. Klionovski, M. S. Sharawi, and A. Shamim, ‘‘A dual-polarization- switched beam patch antenna array for millimeter-wave applica- tions,’’ IEEE Trans. Antennas Propag., vol. 67, no. 5, pp. 3510–3515, May 2019. [21] H. Li, Y. Li, L. Chang, W. Sun, X. Qin, and H. Wang, ‘‘A wideband dual-polarized endﬁre antenna array with overlapped apertures and small clearance for 5G millimeter-wave applications,’’ IEEE Trans. Antennas Propag., vol. 69, no. 2, pp. 815–824, Feb. 2021. [22] N. Ojaroudiparchin, M. Shen, S. Zhang, and G. F. Pedersen, ‘‘A switchable 3-D-coverage-phased array antenna package for 5G mobile terminals,’’ IEEE Antennas Wireless Propag. Lett., vol. 15, pp. 1747–1750, 2016. [23] F. Liu, J. Guo, L. Zhao, G.-L. Huang, Y. Li, and Y. Yin, ‘‘Dual-band metasurface-based decoupling method for two closely packed dual-band antennas,’’ IEEE Trans. Antennas Propag., vol. 68, no. 1, pp. 552–557, Jan. 2020. 109051 VOLUME 9, 2021 VOLUME 9, 2021 Y. He et al.: Compact Dual-Band and Dual-Polarized mm-Wave Beam Scanning Antenna Array Xi’an Jiaotong University, Xi’an. His research interests include MIMO antennas, over-the-air testing, reverberation chambers, and hardware impairments and mitigation. He received the IEEE Outstanding AE Awards, in 2018, 2019, and 2020, and the International Union of Radio Science (URSI) Young Scientist Award, in 2017. He serves as an Associate Editor for IEEE ANTENNAS AND WIRELESS PROPAGATION LETTERS. GUAN-LONG HUANG (Senior Member, IEEE) received the B.E. degree in electronic information engineering from Harbin Institute of Technology, Harbin, China, and the Ph.D. degree in electri- cal and computer engineering from the National University of Singapore, Singapore. GUAN-LONG HUANG (Senior Member, IEEE) received the B.E. REFERENCES degree in electronic information engineering from Harbin Institute of Technology, Harbin, China, and the Ph.D. degree in electri- cal and computer engineering from the National University of Singapore, Singapore. y g p g p He is currently a Full Professor with Foshan University, Foshan, Guangdong, China. He is also a Joint-Researcher with Peng Cheng Laboratory, Shenzhen, Guangdong, China. Prior to join the current university, he has been with Shenzhen University as an Associate Professor, Nokia Solutions and Networks System Technology as a Senior Antenna Specialist, and the Temasek Laboratories, National University of Singapore, as a Research Scientist, from 2011 to 2020. He has authored or coauthored more than 100 papers in journals and conferences. His research interests include design and implementation of high-performance antenna arrays, 5G base-station and mobile RF front-end devices/antennas, lens antenna, phased antenna arrays, liquid metal antenna, and 3-D printing technology in microwave applications. He was a TPC member and special session organizer of several international conferences. He was a recipient of the Fok Ying-Tong Education Foundation Award from the Ministry of Education of the People’s Republic of China, in 2020, the Best Reviewer Award of IEEE ANTENNAS AND WIRELESS PROPAGATION LETTERS and IEEE TRANSACTIONS ON ANTENNAS AND PROPAGATION from the IEEE Antenna and Propagation Society, in 2019 and 2020, respectively, and the Foundation for Distinguished Young Talents in Higher Education of Guangdong Province, China, in 2017. WEI LIN (Senior Member, IEEE) received the bachelor’s and master’s degrees in electronic engineering from South China University of Technology, Guangzhou, China, in 2009 and 2012, respectively, and the Ph.D. degree in elec- tronic engineering from the City University of Hong Kong, Hong Kong, in 2016. He worked as a Research Associate with Nanyang Technological University, Singapore, from August 2012 to August 2013, and a Postdoc- toral Research Associate with The University of Technology Sydney, Ultimo, NSW, Australia, from October 2016 to September 2018, where he is currently a Chancellor’s Postdoctoral Research Fellow with the Global Big Data Technologies Centre, Faculty of Engineering and IT, School of Electrical and Data Engineering. His research interests include the designs of circularly polarized antennas, electrically small antennas, reconﬁgurable antennas, HF antennas, satellite antennas, millimeter-wave antennas, wireless power transfer, terahertz devices, and their applications. REFERENCES He worked as a Research Associate with Nanyang Technological University, Singapore, from August 2012 to August 2013, and a Postdoc- toral Research Associate with The University of Technology Sydney, Ultimo, NSW, Australia, from October 2016 to September 2018, where he is currently a Chancellor’s Postdoctoral Research Fellow with the Global Big Data Technologies Centre, Faculty of Engineering and IT, School of Electrical and Data Engineering. His research interests include the designs of circularly polarized antennas, electrically small antennas, reconﬁgurable antennas, HF antennas, satellite antennas, millimeter-wave antennas, wireless power transfer, terahertz devices, and their applications. Dr. Lin has received many academic awards, which mainly include the Australia Research Council (ARC) Discovery Early Career Researcher Award (DECRA2021), the 2019 Raj Mittra Travel Grant (RMTG) from the IEEE AP-Society, the Best Paper Award (First Prize) at the International Symposium on Antennas and Propagation (ISAP 2018), the Best Young Professional Paper Award (First prize) at the 3rd Australian Microwave Symposium (AMS2018), the Best Poster Paper Award at the 2nd Inter- national Conference on Electromagnetic Materials and Technologies for the Future (EM-MTF2017), the Talent Development Scholarship from Hong Kong Government, and the Young Scientist Award at the IEEE Region 10 Conference (TENCON2015). He was a recipient of an Outstanding Reviewer Award from the IEEE ANTENNAS AND WIRELESS PROPAGATION LETTERS, in 2018, and the IEEE TRANSACTIONS ON ANTENNAS AND PROPAGATION, in 2020. XIAOMING CHEN (Senior Member, IEEE) received the B.Sc. degree in electrical engineering from Northwestern Polytechnical University, Xi’an, China, in 2006, and the M.Sc. and Ph.D. degrees in electrical engineering from Chalmers University of Technology, Göthenburg, Sweden, in 2007 and 2012, respectively. From 2013 to 2014, he was a Postdoctoral Researcher at Chalmers University of Technology. From 2014 to 2017, he was with Qamcom Research and Technology AB, Göthenburg. Since 2017, he has been a Professor with 109052 VOLUME 9, 2021 VOLUME 9, 2021
https://openalex.org/W1591466803	https://www.biodiversitylibrary.org/itempdf/120352	English	null	A catalog of cultivated woody plants of the southeastern United States /	null	1,994	public-domain	138,850	~ Agriculture * Agricultural United States Department of Research Service United States National Arboretum Contribution. Number 7 ~ Agriculture * Agricultural United States Department of Research Service United States National Arboretum Contribution. Number 7 — \ or PRUNUS mume Sieb. & Zucc. ‘Peggy Clarke’ [illustrator Lillian Nicholson Meyer] PRUNUS mume Sieb. & Zucc. ‘Peggy Clarke’ [illustrator Lillian Nicholson Meyer] 4355) SAM el) tal A Catalog of Cultivated Woody Plants of the a Southeastern United States 4355) SAM el) tal By Frederick G. Meyer, Peter M. Mazzeo, and Donald H. Voss By Frederick G. Meyer, Peter M. Mazzeo, and Donald H. Voss By Frederick G. Meyer, Peter M. Mazzeo, and Donald H. Voss To file a complaint, write the Secretary of Agricul- ture, U.S. Department of Agriculture, Washing- ton, DC, 20250, or call (202) 720-7327 (voice) or (202) 720-1127 (TDD). USDA is an equal employ- ment opportunity employer. Cover: IDESIA polycarpa Maxim. [illustrator Susan M. Johnston] Abstract The United States Department of Agriculture (USDA) prohibits discrimination in its programs on the basis of race, color, national or igin, sex, religion, age, disability, political beliefs, and marital or fa- milial status. (Not all prohibited bases apply to all prog rams.) Persons with disabilities who require alternative means for communication of program information (Braille, large print, audio tape, etc.) should contact the USDA Office of Communica- tions at (202) 720-5881 (voice) or (202) 720-7808 (TDD). Meyer, Frederick G., Peter M. Mazzeo, and Donald H. Voss. 1993. A Catalog of Cultivated Woody Plants of the Southeastern United States. U.S. Department of Agriculture, Agricultural Research Service, U.S. National Arboretum Contribution No. 7, 338 pp. Meyer, Frederick G., Peter M. Mazzeo, and Donald H. Voss. 1993. A Catalog of Cultivated Woody Plants of the Southeastern United States. U.S. Department of Agriculture, Agricultural Research Service, U.S. National Arboretum Contribution No. 7, 338 pp. This work catalogs cultivated landscape trees, shrubs, and woody climbers in a 13-state area of the southeastern United States, together with the District of Columbia. Included are approximately 5,000 entries—covering species, subspecies, varieties, forms, and cultivars—based on more than 14,000 documented voucher specimens. The burgeoning nursery business, along with the continued introduction of new cultivated plants, has added to the need for accurate application of botanical nomenclature. Many plants are misidentified in the nursery trade and among researchers, students, and home gardeners. The catalog serves as a reference guide for these groups, as well as for horticulturists, botanists, extension agents, educators, germplasm conser- vators, and those who work in botanic gardens and arboreta. Keywords: climbing plants, cultivars, gardens, horticulture, landscaping, names (botanical), nurseries, plant geography, plant names, plant nomenclature, plant taxonomy, shrubs, trees, woody vines. While supplies last, single copies of this publica- tion may be obtained at no cost from the Curator of the Herbarium, U.S. National Arboretum, 3501 New York Avenue, N.E., Washington, DC 20002- 1958. While supplies last, single copies of this publica- tion may be obtained at no cost from the Curator of the Herbarium, U.S. National Arboretum, 3501 New York Avenue, N.E., Washington, DC 20002- 1958. Copies of the publication may be purchased from the National Technical Information Service, 5285 Port Royal Road, Springfield, VA 22161. February 1994 iv iv Contents HIORE WONG wena rsere ese ais siac ohne seiien sae aceasta eet cose tisca css vii FRCKMOWIEASTIMCTUS -..eecccens oc odenceoesesebescseccesteeee vii PAHO GUC OMG asses scecenerine ec ecsuareseisiecsessscresesesonsee 1 Symbols;and abbreviations.............0....+.ss--00-2s0 IL Catalog of plants and their collection sites ...... 12 Ap PeGixeAy AUtMOLS CltEG fie scsseesn-css5 sce eecans: 218 Appendix B: Plant collection sites, cited by ACEOMYTIMG Paracecr ssaenasceetaes cases 237 Appendix C: Glenn Dale azalea cultivars documented in the U.S. National Ne KORA HEIN IS eoiahbboey ceocosoddasooe 248 Appendix D: Plants listed by botanical names andicultivarmames) \ass-ss-.-ce5-55-- 249 Appendix HE: Vernacular names ................-.- 313 GEMER AL 7 essacsias sotesstoncseseataes 313 Species and cultivars ......... 314 Seleciedsoibliognaply eensastescsccenscoseseseseeetaceos 326 Illustrations IDE STAG DOW CATPG) veccs-2.0- scecs--ecsacsssoeerreseo>s cover PRUNUS mume ‘Peggy Clarke’ ........ frontispiece VAN GAIN UID VAN GG UL GU ecxcct cos scaceueton=eeecueestecetes eee 22 PAISEDES VAC GU ULONre coececccaseceecemensreccerenecececoeeceee 24 BIGNONIA capreolata ...........::2cccseeeeeseeeeeeeeees 31 BRUNFELSIA Qustrdlis .........:.:1cccceeeceeseeeeeeeee 33 CALYCANTHUS floniGus <..2....0..2022..-00-022--020° 38 CHIMONANTHUS prae€cox .........cseeceeeeeneeeneees 50 DAVIDIA involucrata var. vilmoriniana ......... 63 DISANTHUS cercidifolius ............:cccseeeeeeeeeeeee 66 JROMMIGIOIRGHUL/AN TMOHOlP cecccencccconsceouescseaIDeGeIS3Ce 74 EVAILESSTAGCGTOUNG aecesescereceteeieeereccssectenseesees 77 FVAIVIA\MISTET Sim Olli) ete eece-ossecceesecceeessoeeeeceee ee 79 TEKOWADINI UA GRITCHS), “easoceecosccaocasqosoconsaaoaebacdeeecaeseC 88 HYDRANGEA anomala ssp. petiolaris ......... 89 TEI CIO Maflonicl aiuuinnre-cscsc-tacee-oeeees-oeeee ee aace 110 TERICIOM parviflonuim e...22s.2-..c2-22206+00222-ee02 111 JASMINUM nuiflorum .......cceeceseceeeeceneeeneees 113 JUWGEANSTCGIG ies secccesccosscecesceceesseseeseesoees: 114 KOELREUTERIA bipinnat ........:..cseceeeeeeeeee 120 HIORE WONG wena rsere ese ais siac ohne seiien sae aceasta eet cose tisca css vii FRCKMOWIEASTIMCTUS -..eecccens oc odenceoesesebescseccesteeee vii PAHO GUC OMG asses scecenerine ec ecsuareseisiecsessscresesesonsee 1 Symbols;and abbreviations.............0....+.ss--00-2s0 IL Catalog of plants and their collection sites ...... 12 Ap PeGixeAy AUtMOLS CltEG fie scsseesn-css5 sce eecans: 218 Appendix B: Plant collection sites, cited by ACEOMYTIMG Paracecr ssaenasceetaes cases 237 Appendix C: Glenn Dale azalea cultivars documented in the U.S. National Ne KORA HEIN IS eoiahbboey ceocosoddasooe 248 Appendix D: Plants listed by botanical names andicultivarmames) \ass-ss-.-ce5-55-- 249 Appendix HE: Vernacular names ................-.- 313 GEMER AL 7 essacsias sotesstoncseseataes 313 Species and cultivars ......... 314 Seleciedsoibliognaply eensastescsccenscoseseseseeetaceos 326 Illustrations IDE STAG DOW CATPG) veccs-2.0- scecs--ecsacsssoeerreseo>s cover PRUNUS mume ‘Peggy Clarke’ ........ frontispiece VAN GAIN UID VAN GG UL GU ecxcct cos scaceueton=eeecueestecetes eee 22 PAISEDES VAC GU ULONre coececccaseceecemensreccerenecececoeeceee 24 BIGNONIA capreolata ...........::2cccseeeeeseeeeeeeeees 31 BRUNFELSIA Qustrdlis .........:.:1cccceeeceeseeeeeeeee 33 CALYCANTHUS floniGus <..2....0..2022..-00-022--020° 38 CHIMONANTHUS prae€cox .........cseeceeeeeneeeneees 50 DAVIDIA involucrata var. vilmoriniana ......... Contents 63 DISANTHUS cercidifolius ............:cccseeeeeeeeeeeee 66 JROMMIGIOIRGHUL/AN TMOHOlP cecccencccconsceouescseaIDeGeIS3Ce 74 EVAILESSTAGCGTOUNG aecesescereceteeieeereccssectenseesees 77 FVAIVIA\MISTET Sim Olli) ete eece-ossecceesecceeessoeeeeceee ee 79 TEKOWADINI UA GRITCHS), “easoceecosccaocasqosoconsaaoaebacdeeecaeseC 88 HYDRANGEA anomala ssp. petiolaris ......... 89 TEI CIO Maflonicl aiuuinnre-cscsc-tacee-oeeees-oeeee ee aace 110 TERICIOM parviflonuim e...22s.2-..c2-22206+00222-ee02 111 JASMINUM nuiflorum .......cceeceseceeeeceneeeneees 113 IANO RUS OLDIES a.4060650000000000020000 33000 C RECO DIKE 123 MAGNOLIACaASIRCs (fimutit) ereesseses-eseeeacconencee se: 129 MAGNOLIA macrophylla (fruit) ...........2.....+ 129 MAGNO LIAGt IPCI Gmirenesrecsttestterssneet costae 133 IMDKCISI DUAN, F{10[0). <ardcséecineqanba0des20pn0eCCH AOD ICCRROHOTEC 138 PARROTIOPSIS jacquemontiana ............00- 143 PSEUDOLARIX GM@DilisS ..........c.-..000eeee00-000" 161 SCHIZOPHRAGMA hydrangeoides ............. 191 SINAC [RTO UES. sconanancousacaaseccodeacdneca9e00000 197 D> ACLOVAS IS) SUMEMSUS) cepacdcedooaneacadssanoneseeancen6ce 198 IRON SUES coooodcoscccbonboncqcxooncoccocsonTnCeCGe 206 WEMUSipanvifoliay ecc-nsrestecccesee eee ete see 209 IANO RUS OLDIES a.4060650000000000020000 33000 C RECO DIKE 123 MAGNOLIACaASIRCs (fimutit) ereesseses-eseeeacconencee se: 129 MAGNOLIA macrophylla (fruit) ...........2.....+ 129 MAGNO LIAGt IPCI Gmirenesrecsttestterssneet costae 133 IMDKCISI DUAN, F{10[0). <ardcséecineqanba0des20pn0eCCH AOD ICCRROHOTEC 138 PARROTIOPSIS jacquemontiana ............00- 143 PSEUDOLARIX GM@DilisS ..........c.-..000eeee00-000" 161 SCHIZOPHRAGMA hydrangeoides ............. 191 SINAC [RTO UES. sconanancousacaaseccodeacdneca9e00000 197 D> ACLOVAS IS) SUMEMSUS) cepacdcedooaneacadssanoneseeancen6ce 198 IRON SUES coooodcoscccbonboncqcxooncoccocsonTnCeCGe 206 WEMUSipanvifoliay ecc-nsrestecccesee eee ete see 209 Errata for A Catalog of Cultivated Woody Plants of the Southeastern United States Page 180, in Rosa: pone Ait. should read banksiae Ait-f. Page 188, under Salix xlaestadiana Hartm.: S. cineria should read S. cinerea. Page 188, under Salix x pontederana Willd.: S. cineria should read S. cinerea. Page 27, in Arecastrum: = Syagrus romanzoffianum should read = Syagrus romanzoffiana. Page 196, in Syagrus: romanzoffianum should read romanzoffiana. Page 32, in Buddleja: farreri Balf.f. & W.W.Sm. should read farreri Balf.f. & W.W.Sm. = crispa Benth. var. farreri (Balf.f. & W.W.Sm.) Hand.-Mazz. Page 199, in Syringa: ‘Charles Giant’ should read ‘Clarke’s Giant’. Page 32, in Buddleja: farreri Balf.f. & W.W.Sm. should read farreri Balf.f. & W.W.Sm. = crispa Benth. var. farreri (Balf.f. & W.W.Sm.) Hand.-Mazz. Page 199, under Syringa Xchinensis Willd.: [S. xX persica X S. vulgaris] should read [S. persica X S. vulgaris]. Page 54, in Comus: ‘Gold Star’ [C. kousa Hance] should read ‘Gold Star’ (Buerger ex Miq.) Hance. Page 203, in Thuja: ‘Cuprea’ [T. plicata D.Don] should read ‘Cuprea’ [T. plicata J.Donn ex D.Don]. Page 69, under Euodia daniellii (Benn.) Hemsl.: include the note E. daniellii still appears widely in horticultural works, but T.G. Hartley’s revision of the genus Tetradium moves it to Tetradium daniellii (Benn.) Hartley. Page 203, in Thuja: plicata J.Donn ex G.Don should read plicata J.Donn ex D.Don. Page 273, under Hibiscus: syriacus ‘Tamausagi’ should read syriacus ‘Tama usagi’. Page 275, under Ilex: aquifolium ‘Scotia’ should read aquifolium ‘Scotica’. Foreword Many individuals, too numerous to mention, generously facilitated our collection efforts at botanical gardens and arboreta, experiment stations, college campuses, nurseries, and private gardens. The herbarium of the U. S. National Arboretum is one of the few major herbaria to emphasize cultivated plants together with their wild progeni- tors. Its specimens support scientific research on these plants not only within the Department of Agriculture but also worldwide through a loan program. The ongoing work of the herbarium staff in addressing problems in plant taxonomy and nomenclature assists researchers, germplasm conservators, and others in plant identification and contributes to the efforts of nurseries to identify correctly the plant material they propagate and sell. We specifically acknowledge the invaluable assistance of the following specialists for their help in identifying plant specimens: W.A. Ander- son (Malpighiaceae), George Argus (Salix), Helen L. Blake (old roses), Barbara Briggs (Callistemon), C.E. Codd (Carissa macrocarpa), David W. Hall (miscellaneous Florida plants), James W. Hardin, Jr. (Aesculus), Elizabeth McClintock (Hydrangea and Myrtaceae), A. Edward Murray, Jr. (Acer), Eliane Norman (Buddleja), J.B. Phipps (Crataegus), the late Sigmund Solymosy (miscel- laneous Louisiana plants), and Charles A. Walker (old roses). T. R. Dudley contributed many voucher specimens cited in the catalog and compiled the lists of Ilex and Caprifoliaceae. A Catalog of Cultivated Woody Plants of the Southeastern United States represents the culmi- nation of more than 20 years of field work begun in 1967 that covers 13 southeastern states and the District of Columbia. The catalog has been prepared as a reference guide for nurseries, landscape architects, horticulturists, botanical and horticultural libraries, botanists, extension agents, educators, germplasm conservators, botanical gardens and arboreta, propagators, students, various units of the U.S. Department of Agriculture, plant societies, and the gardening public. It will be useful not only in the south- eastern states, but also in other parts of the United States where many of the plants listed can be found in cultivation. We express appreciation to the Friends of the National Arboretum (FONA) and to the Southern Nurserymen’s Association for financial support that greatly enhanced our ability to collect speci- mens in the southeastern United States. We also thank Carol A. Rahbar and Franziska Walczak as volunteer collaborators for their substantive assistance in coordinating data related to the computer data base, the list of site acronyms, and attending to many other details that contributed enormously to the accuracy of the catalog. Contents Page 203, in Thuja: ‘Rogersii’ [T. plicata D.Don] should read ‘Rogersii’ [T. plicata J.Donn ex D.Don]. Page 128, in Magnolia: include the entry foetida Sarg. = M. grandiflora (L.) after ‘Elizabeth’ [M. acuminata <x M. denudata]. Page 203, in Thuja: ‘Zebrina’ [T. plicata D.Don] should read ‘Zebrina’ [T. plicata J.Donn ex D.Don]. Page 134, in Malus ‘Aldenhamensis’: ‘Niedzwetzkeyana’ should read ‘Niedzwetzkyana’. Page 212, in Viburnum Mt. Fuji’: [V. plicatum Fort. f. tomentosum (Thunb. ex J.A.Murrt.) Rehd.] should read [V. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.]. Page 135, in Malus: include the entry niedzwetzkyana Dieck = M. domestica ‘Niedzwetzkyana’ after ‘Naragansett’ [M. / hybrid #28/ X M. ‘Wintergold’]. Page 213, in Viburnum ‘Shoshoni’: [V. plicatum Fort. f. tomentosum (Thunb. ex J.A.Murt.) Rehd.] should read [V. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.]. Page 255, under Buddleja: farreri Balf.f. & W.W.Sm. should read farreri Balf.f. & W.W.Sm. = crispa Benth. var. farreri (Balf.f. & W.W. Sm.) Hand.-Mazz. Page 156, in Prunus: ‘Albo-plena’ [P. glandulosa Thunb.] should read ‘Albo-plena’ [P. glandulosa Thunb. ex J.A. Murr.]. Page 160, in Prunus ‘Yae murasaki zakura’: (Yato zakura group) should read (Sato zakura group). Page 273, under Hibiscus: syriacus ‘Tamausagi’ should read syriacus ‘Tama usagi’. Page 275, under Ilex: aquifolium ‘Scotia’ should read aquifolium ‘Scotica’. Page 160, in Pseudotsuga: include the entry taxifolia (Lamb.) Britton = P. menziesii (Mirb.} Franco after ‘Oudemansii’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco]. Page 301, in Rosa: include the entry Xodorata (Andr.) Sweet ‘Old Blush’ after odorata (Andre.) Sweet ‘Fun Jwan Lo’. Page 301, in Rosa: include a X before the three odorata entries. Page 302, in Rosa: ir- lude the entry ‘The Fairy’ after ‘Sombreuil’. Page 305, in x Sorbopyrus: ‘Bulbiformis’ should be indented under auricularis. Page 306, under Syagrus: romanzoffianum should read romanzoffiana. Page 307, under Syringa: ‘Charles Giant’ should read ‘Clarke’s Giant’. Page 321, under Palm, Queen: Syargus romanzoffianum should read Syargus romanzoffiana. Page 264, under Cotoneaster racemiflorus, soongoricus should read songoricus. Page 321, under Palm, Queen: Syargus romanzoffianum should read Syargus romanzoffiana. Page 264, under Cotoneaster racemiflorus, soongoricus should read songoricus. Scope a large number of plants not previously listed or reported as cultivated in the United States are included as a result of recent introductions. This catalog presents a comprehensive reference list, based on documented material, of the trees, shrubs, suffruticose plants, and woody climbers cultivated in the southeastern United States. A list of acronyms identifies the collection sites for each entry. While the southeastern United States is well served by botanical manuals and other floristic works that identify native plants, such works on the cultivated woody plants (indigenous and introduced) of the southeastern states have been much neglected. The catalog includes many unusual or rare plants from the living collections of the U. S. National Arboretum not well represented else- where in this country. Between 1961 and 1990, 125 cultivars in 18 genera were released by the National Arboretum to the horticultural indus- try—cultivars of Buxus, Camellia, Clematis, x Cupressocyparis, Eurya, Hibiscus, Ilex, Kalmia, Lagerstroemia, Magnolia, Malus, Metasequoia, Platanus, Pyracantha, Pyrus, Rhododendron, Ulmus, and Viburnum. The catalog also lists for the first time most of the dwarf and slow-growing conifers in the Gotelli Collection at the U.S. National Arboretum. The list of Ilex, the largest of any genus in the catalog, is based primarily on the extensive collections of living plants and voucher herbarium specimens at the U.S. Na- tional Arboretum. The catalog contains about 5,000 entries— species, subspecies, varieties, forms, and culti- vars—in 112 plant families and 493 genera. These are based on more than 14,000 voucher herbarium specimens, most collected specifically for this project. The primary focus of the project has been a critical review for correct identifica- tion and nomenclature of material in the list. This material, now permanently deposited in the herbarium of the U. S. National Arboretum, was collected at nearly 700 sites in Alabama, Arkan- sas, Delaware, the District of Columbia, northern Florida, Georgia, Louisiana, Maryland, Missis- sippi, North Carolina, South Carolina, Tennes- see, southeastern Texas, and Virginia. This area covers about one-sixth of the continental United States and spans Plant Hardiness Zones 6b through 9a (U.S. Department of Agriculture 1990). The collection sites include botanical gardens and arboreta, cemeteries, college cam- puses, experiment stations, nurseries, private gardens, and roadway plantings. Meyer is a supervisory botanist (retired) and Mazzeo is a botanist, U.S. National Arboretum, Agricultural Research Service, U.S. Department of Agriculture, Washington, DC 20002; Voss, of Vienna, Virginia, prepared the computer data base and programming for the catalog and assisted in the checking of nomenclature. Foreword The catalog is based on more than 14,000 docu- mented voucher specimens critically reviewed for correctness of identification and nomenclature. With about 5,000 entries, both botanical names and cultivar names, the catalog is a comprehen- sive enumeration of woody plants cultivated in the southeastern United States. The authors are grateful for the privilege of using artwork by the late Lillian Nicholson Meyer, Susan M. Johnston, and Peggy K. Duke. The catalog is unique, being based wholly on documented material deposited in the U. S. National Arboretum herbarium and including a guide to sites where collections were made of plants growing in cultivation. It is hoped that the catalog will, by example, encourage similar documentation of the woody landscape plants in other areas of the country and the deposit in appropriate herbaria of voucher specimens collected for permanent reference. Allan K. Stoner Acting Director, U. S. National Arboretum A Catalog of Cultivated Woody Plants of the Southeastern United States by Frederick G. Meyer, Peter M. Mazzeo, and Donald H. Voss by Frederick G. Meyer, Peter M. Mazzeo, and Donald H. Voss Need for the Catalog In recent years, the nursery industry has become increasingly active in offering new plant introduc- tions, as a result of renewed interest in plant exploration and germplasm diversity. Each year introductions appear in ever-increasing numbers from all parts of the world. People are now planting a greater diversity of woody landscape plants than ever before in U.S. history. The U.S. National Arboretum has contributed significantly to this flow of new introductions from its plant breeding program and from expeditions to Japan, the People’s Republic of China, and the Republic of Korea. Some woody plants totally unknown in cultivation only a few years ago are now best- sellers in garden centers; these include Pyracan- tha ‘Mohave’ and Hibiscus syriacus ‘Diana’, both of which resulted from the U.S. National Arbore- tum plant breeding program. Scope Although we have not indicated the geographic origins of the plants, the cultivated woody flora of the southeastern states represents a wide diver- sity of germplasm from many countries, in particular the southeastern United States, Europe, and eastern Asia. In lesser numbers, the catalog lists plants from temperate areas of South America (southern Brazil, Uruguay, Para- guay, northern Argentina, and northern Chile), including Araucaria araucana, Brunfelsia austra- lis, Butia capitata, Calliandra tweediei, Erythrina crista-galli, Sesbania punicea, Tipuana tipu, and others. The diversity of woody landscape plants now in cultivation from South America and Central America suggests that renewed germplasm exploration in these areas would no doubt turn up other plants worth introducing for landscape use in the southeastern United States. Other areas represented in the catalog include Mexico, with Antigonon leptopus, Dioon edule, Parkinsonia aculeata, and some others; South Africa, with Carissa macrocarpa, Ochna atropurpurea, Phygelius capensis, and Tecomaria capensis; and Australia, with Casuarina cunninghamiana, Callistemon (six species), Callitris columellaris, Eucalyptus (six species), and Westringia rosmariniformis. We have observed, for example, that dwarf and slow-growing conifers are frequently misnamed in the nursery trade and by the specialists who grow them. Because of this confusion, the need is pressing to investigate the taxonomy and nomenclature not only of landscape plants but also of food, medicinal, and agronomic plants as a much-neglected aspect of horticultural botany. Access to voucher herbarium specimens makes identification of these plants much easier and far more accurate. Unfortunately, too few technically qualified botanists or horticulturists are available and willing to tackle the identification and nomencla- ture of landscape plants, leaving many plant groups in a state of perpetual taxonomic and nomenclatural confusion. Although botanical gardens and arboreta often maintain small herbaria to document the plants in their collec- tions, very few of the world's major herbaria are willing to incorporate specimens of cultivated plants for scientific study. The value of voucher herbarium specimens has not been sufficiently appreciated by horticulturists. Many problems related to the taxonomy and nomenclature of the plants in this catalog simply could not have been effectively investigated and resolved without the benefit of herbarium specimens. Scope The authors are solely responsible for the names and identifications of the plants listed in the catalog, but in difficult genera such as Acer, Aesculus, Callistemon, Crataegus, Hydrangea, Ilex, Salix, and Viburnum, we had the expertise of specialists who generously provided names and verified identifications. In this introduction, most of the botanical names are cited without authorities, since these are properly listed with the names in the catalog; but sometimes authori- ties are cited to substantiate a point. The catalog is arranged alphabetically by the internationally accepted botanical and cultivar names, including some synonyms. Vernacular, or common, names are also included for many entries. When a species is subdivided, the epithet for the infraspecific taxon that includes the type of the species name is an exact repeti- tion of the specific epithet (an autonym) and is cited without the author's name. The list of collection-site acronyms in Appendix B identifies the sources of the documented voucher speci- mens, thereby providing information on the abundance, distribution, and hardiness range of the plants listed. While comprehensiveness was an objective, the catalog makes no claim to completeness, since new plants are constantly being introduced to horticulture; and from a practical standpoint we were unable to visit every possible site. In genera such as Camellia, Rosa, Rhododendron, Hedera, and some others with a vast number of cultivars, completeness was simply not possible. However, Although we have not indicated the geographic origins of the plants, the cultivated woody flora of the southeastern states represents a wide diver- sity of germplasm from many countries, in particular the southeastern United States, Europe, and eastern Asia. In lesser numbers, the catalog lists plants from temperate areas of South America (southern Brazil, Uruguay, Para- guay, northern Argentina, and northern Chile), including Araucaria araucana, Brunfelsia austra- lis, Butia capitata, Calliandra tweediei, Erythrina crista-galli, Sesbania punicea, Tipuana tipu, and others. The diversity of woody landscape plants now in cultivation from South America and Central America suggests that renewed germplasm exploration in these areas would no doubt turn up other plants worth introducing for landscape use in the southeastern United States. Other areas represented in the catalog include Mexico, with Antigonon leptopus, Dioon edule, Parkinsonia aculeata, and some others; South Africa, with Carissa macrocarpa, Ochna atropurpurea, Phygelius capensis, and Tecomaria capensis; and Australia, with Casuarina cunninghamiana, Callistemon (six species), Callitris columellaris, Eucalyptus (six species), and Westringia rosmariniformis. Plant Origins The area covered by the catalog encompasses much the same range as J.K. Small’s Manual of the Southeastern Flora (1933). This vast region supports the richest ligneous flora in temperate North America and may be compared only with the highly diverse woody flora of the People’s Republic of China. More than 250 species of indigenous trees occur in the southeastern United States, many of them grown as landscape plants. One of the best known and admired is Magnolia grandiflora, which ranks among the most valuable indigenous landscape trees of the United States. Now widely cultivated abroad, this magnificent flowering tree is one of America’s great gifts to the gardens of the world. Because of climatic similarities between eastern Asia and the eastern United States, countries such as Japan, Korea, and the People’s Republic of China, with Taiwan, are heavy contributors of woody landscape plants now widely cultivated in the southeastern United States. Some well- known plants of east Asian origin include The influx of new plants has compounded the problems of plant identification and nomencla- ture in the nursery trade, as well as among researchers, students, and home gardeners. Many plants found in the nursery trade are misidentified. The same plant may appear under various names in different nursery catalogs, a problem that leads to nomenclatural confusion. Camellia japonica, Cryptomeria japonica, Ilex crenata, Juniperus chinensis, Lagerstroemia indica, Ligustrum japonicum, L. lucidum, Magnolia denudata, Rhododendron (many species), Ternstroemia gymnanthera, Wisteria floribunda, and W. sinensis. Camellia japonica, Cryptomeria japonica, Ilex crenata, Juniperus chinensis, Lagerstroemia indica, Ligustrum japonicum, L. lucidum, Magnolia denudata, Rhododendron (many species), Ternstroemia gymnanthera, Wisteria floribunda, and W. sinensis. probably did not introduce the camellia to Middleton Place in 1785 as long assumed. We have identified one of the four original plants attributed to him on this site as C. japonica ‘Anemoniflora’, a cultivar that only reached England in the first decade of the 19th century and was illustrated in Curtis’s Botanical Maga- zine, t. 1654, 1814. It is more likely that the camellias at Middleton Place reached America from Great Britain after 1814 and not earlier. From a horticultural perspective, the southeast- ern United States is one of the most interesting areas in the country. The oldest landscaped gardens in the United States, dating from the late 17th and early 18th centuries, are found in Virginia, the Carolinas, and Georgia. Trees The distinction between trees and shrubs is often controversial from various points of view. No two definitions totally agree. We know that some shrubs become treelike under certain growing conditions. A tree, by standard dictionary defini- tion, is a perennial woody plant usually having a single self-supporting trunk and ranging in height from 10 to more than 300 feet. Many trees fit this definition, but others do not. Indig- enous species such as Cornus florida and Vibur- num prunifolium rarely attain the stature of a tree in their native woodland habitats, but in an open sunny location these same species often become treelike. In another example, the red buckeye (Aesculus pavia) may grow to 30 feet as a single- trunked tree in the open but not more than a shrub 5 to 6 feet high in a woodland setting. It is recorded that about 20 to 25 exotic trees and shrubs were already in cultivation in gardens at Williamsburg, VA, by the middle of the 18th century, including cornelian cherry (Cornus mas), fig (Ficus carica), littleleaf linden (Tilia cordata), weeping willow (Salix babylonica), and the com- mon lilac (Syringa vulgaris). At the same time, about 80 species of native American trees and shrubs were cultivated at Williamsburg, includ- ing flowering dogwood (Cornus florida), American beech (Fagus grandifolia), common catalpa (Catalpa bignonioides), red buckeye (Aesculus pavia), Carolina cherry laurel (Prunus caroliniana), southern live oak (Quercus virginiana), and American holly (Ilex opaca). Examples of indigenous trees of the southeastern United States, with their maximum height, include: Acer saccharinum, silver maple, 70 ft Aesculus flava, yellow buckeye, 70 ft Fagus grandifolia, American beech, 70 ft Fraxinus americana, American ash, 80 ft Ilex opaca, American holly, 100 ft Liriodendron tulipifera, yellow poplar, tulip tree, 100 ft Magnolia grandiflora, southern magnolia, 90 ft Magnolia macrophylla, bigleaf magnolia, 50 ft Platanus occidentalis, American sycamore, 80 ft Quercus alba, white oak, 70 ft Quercus phellos, willow oak, 70 ft Quercus velutina, black oak, 70 ft Quercus virginiana, southern live oak, 70 ft. The introduction of the parasol tree (Firmiana simplex) about 1780 at Charleston, SC, is attrib- uted to André Michaux, a French plant explorer who lived near Charleston. American ships returning from China often landed at Charleston, and Michaux may have obtained seeds of the parasol tree from one of these ships. From the evidence at hand, it is doubtful that the common camellia (C. Plant Origins Since plants follow people, we know that many of the earlier introductions were brought by European immigrants to their new homes in America. Early plantation life in the southern colonies promoted an interest in the cash crops of to- bacco, rice, and indigo. By the end of the 17th century, ornamental plants such as boxwood (Buxus sempervirens) and garden flowers intro- duced from Europe were cultivated. The gardens at Middleton Place near Charleston, SC, date from about 1730 as one of the oldest landscaped gardens in America. About the same period, the earliest nurseries were established in Charleston. The first plant-introduction garden in America, called the “Founder’s Garden,” was established at Savannah soon after the founding of the Georgia colony in 1733 by James Oglethorpe. Examples of trees introduced into the region, with their maximum height, include: the spring, the previous year’s leaves are lost, and the tree becomes essentially leafless for a short period until the new leaves have fully expanded. But some oaks are, indeed, perma- nently evergreen, such as Quercus myrsinifolia, Q. glauca, and Q. acuta—all from Japan. An- other well-known tree, Magnolia grandiflora, remains fully evergreen throughout the year. In this species, leaves normally remain on the tree for 2 years before falling in spring of the second year, but the tree is never leafless. Ailanthus altissima, tree-of-heaven, 70 ft, People’s Republic of China Ailanthus altissima, tree-of-heaven, 70 ft, Ailanthus altissima, tree-of-heaven, 70 ft, People’s Republic of China Broussonetia papyrifera, paper mulberry, 40 ft, eastern Asia to Polynesia Cedrus deodara, deodar cedar, 100 ft, Nepal and adjacent People’s Republic of China Cunninghamia lanceolata, China fir, 70 ft, People’s Republic of China, with Taiwan Firmiana simplex, parasol tree, 60 ft, People’s Republic of China Morus alba, white mulberry, 40 ft, People’s Republic of China Paulownia tomentosa, princess tree, 50 ft, People’s Republic of China Platanus <acerifolia, London plane, 60 ft, garden origin Salix babylonica, weeping willow, 60 ft, People’s Republic of China(?) Sapium sebiferum, Chinese tallow tree, 80 ft, People’s Republic of China Sequoia sempervirens, coast redwood, 100 ft, California. Ailanthus altissima, tree-of-heaven, 70 ft, People’s Republic of China People’s Republic of China People’s Republic of China Broussonetia papyrifera, paper mulberry, 40 ft, eastern Asia to Polynesia Broussonetia papyrifera, paper mulberry, 40 Broussonetia papyrifera, paper mulberry, 4 ft, eastern Asia to Polynesia Broussonetia papyrifera, paper mulberry, 40 ft, eastern Asia to Polynesia ft, eastern Asia to Polynesia Cedrus deodara, deodar cedar, 100 ft, Nepal and adjacent People’s Republic of China Cunninghamia lanceolata, China fir, 70 ft, People’s Republic of China, with Taiwan Firmiana simplex, parasol tree, 60 ft, People’s Firmiana simplex, parasol tree, 60 ft, People’s Republic of China Trees japonica) came directly to America on board a ship returning from the Orient. Michaux Examples of trees introduced into the region, with their maximum height, include: Examples of trees introduced into the region, with their maximum height, include: Shrubs Republic of China Morus alba, white mulberry, 40 ft, People’s Republic of China Morus alba, white mulberry, 40 ft, People’s Republic of China Typically, shrubs differ from trees primarily in being multitrunked from the base and shorter than trees. Shrubs are seldom more than 20 feet in height; some of the largest may be single- trunked and treelike. Stature alone is not an absolute measure of shrub classification. A good example is illustrated by Magnolia virginiana. The northern aspect of this species, distributed from Massachusetts to North Carolina, is typi- cally deciduous, multitrunked, and shrubby, with a spreading habit that fits the stature of a large shrub. In cultivation and in the wild, it sometimes attains a height of 30 feet but seldom more. The southern aspect, often referred to as var. australis, occurs from North Carolina to southern Florida and west to Tennessee, Arkan- sas, and southeastern Texas, where it becomes a lofty evergreen tree with one or two trunks and sometimes reaches 100 feet in height. In cultiva- tion, the southern plant consistently remains evergreen and treelike at least as far north as Washington, DC, even in the coldest winters. Paulownia tomentosa, princess tree, 50 People’s Republic of China People’s Republic of China(?) Sapium sebiferum, Chinese tallow Woody climbers, or lianes Celastrus orbiculatus, Oriental bittersweet, Japan, Korea, and People’s Republic of China Gelsemium sempervirens, Carolina jessamine, The term scandent, from the Latin scandens, refers to woody and herbaceous plants that climb by various means. The term liana, or liane, refers to a woody climbing plant, especially in the tropics, but lianes occur in all climates except polar areas. Woody climbers include plants in five categories according to their mode of climb- ing, as follows: Gelsemium sempervirens, Carolina jessamine, southeastern United States Kadsura japonica, kadsura vine, Japan, Kadsura japonica, kadsura vine, Japan, People’s Republic of China, with Taiwan Lonicera sempervirens, trumpet honeysuckl Lonicera sempervirens, trumpet honeysuckle, eastern United States Trachylospermum jasminoides, Confederate Lonicera sempervirens, trumpet honeysuckle, eastern United States Trachylospermum jasminoides, Confederate Trachylospermum jasminoides, Confederate jasmine, Japan, Korea, and People’s Republic of China Wisteria spp. (species twine either to the right Tendril-climbers with auxiliary filiform leaf- less and usually branched organs that attach to another body and curl around it for support Camellia japonica, common camellia, 20 ft, Japan Chionanthus virginicus, old-man's beard, 12 ft, Camellia japonica, common camellia, 20 ft, Japan Chionanthus virginicus, old-man's beard, 12 f Chionanthus virginicus, old-man's beard, 12 ft, eastern United States Morphologically, a tendril may be a modified stem, a modified branch, a petiole, a stipule, ora leaflet of a compound leaf. Occasionally, as in Parthenocissus tricuspidata, the filiform branch tips are provided with terminal cups called holdfasts. Examples include: Kalmia latifolia, mountain laurel, 20 ft, eastern United States. Kalmia latifolia, mountain laurel, 20 ft, eastern United States. United States. Twiners that climb spirally for support Actinidia arguta, tara vine, Japan, Korea, and northeastern Asia Actinidia deliciosa var. deliciosa, Chinese goose- berry or kiwi fruit, People’s Republic of China Akebia quinata, chocolate vine, Japan, People’s Akebia quinata, chocolate vine, Japan, People’s Republic of China, and Korea. Berchemia scandens, Alabama supplejack, Vinca major, greater periwinkle, Europe Vinca minor, common periwinkle, Europe. Berchemia scandens, Alabama supplejack, eastern United States Celastrus orbiculatus, Oriental bittersweet, Ampelopsis arborea, peppervine, eastern United States Chrysanthemum nipponicum, Nippon daisy, Japan Indigofera spp., indigo (listed species) Lespedeza spp., bush clover (listed species) Nierembergia scoparia, tall cup-flower, Uruguay and Argentina Pachysandra procumbens, Allegheny spurge, southeastern United States Pachysandra terminalis, Japanese spurge, Japan, People's Republic of China, and Korea Teucrium chamaedrys, European germander, Europe Vinca major, greater periwinkle, Europe Vinca minor, common periwinkle, Europe. Chrysanthemum nipponicum, Nippon daisy, Japan Indigofera spp., indigo (listed species) Lespedeza spp., bush clover (listed species) Nierembergia scoparia, tall cup-flower, Uruguay and Argentina Pachysandra procumbens, Allegheny spurge, southeastern United States Pachysandra terminalis, Japanese spurge, Japan, People's Republic of China, and Korea Teucrium chamaedrys, European germander, Europe Vinca major, greater periwinkle, Europe Vinca minor, common periwinkle, Europe. Examples of smaller trees, both native and introduced, include: Examples of smaller trees, both native and introduced, include: Albizia julibrissin, mimosa tree, 30 ft, Iran to People's Republic of China, Japan, and Korea Albizia julibrissin, mimosa tree, 30 ft, Iran to People's Republic of China, Japan, and Korea Amelanchier arborea, serviceberry, 35 ft, eastern United States Amelanchier arborea, serviceberry, 35 ft, eastern United States Amelanchier arborea, serviceberry, 3 Asimina triloba, pawpaw, 35 ft, eastern United States Buxus sempervirens, common boxwood, 25 ft, Europe Although the distinction of deciduous or ever- green habit and the differences in stature can be used to separate the northern and southern aspects in the living state, no discernable taxo- nomic characters can be found to consistently distinguish these variants morphologically. Moreover, the two variants of M. virginiana are often intermediate as to evergreenness and stature, factors that further complicate their identification. For these reasons, var. australis is not recognized in this catalog as a distinct entity. Cercis canadensis, North American redbud, 25 ft, eastern United States eastern United States Chionanthus retusus var. serrulatus, 25 ft, People's Republic of China, with Taiwan Cornus florida, flowering dogwood, 30 ft, Cornus florida, flowering dogwood, 30 ft, eastern United States eastern United States Cotinus obovatus, American smoke tree, 40 ft, eastern United States Cyrilla racemiflora, titi, 25 ft, southeastern United States Magnolia ashei, Florida bigleaf magnolia, 25 ft, northern Florida. Examples of common native and introduced shrubs cultivated in the southeastern United States include: Trees are deciduous, evergreen, or sometimes semi-evergreen, depending upon climate and species. A well-known tree indigenous to the southeastern United States is the southern live oak (Quercus virginiana), usually regarded as evergreen. In fact, this oak is not truly evergreen in the strict sense. As the new leaves emerge in Abelia x grandiflora, glossy abelia, 6-8 ft, a hybrid of Chinese parentage Agarista populifolia, pipe plant, 12-15 ft, southeastern United States Tendril-climbers with auxiliary filiform leaf- less and usually branched organs that attach to another body and curl around it for support Morphologically, a tendril may be a modified stem, a modified branch, a petiole, a stipule, ora leaflet of a compound leaf. Occasionally, as in Parthenocissus tricuspidata, the filiform branch tips are provided with terminal cups called holdfasts. Examples include: Tendril-climbers with auxiliary filiform leaf- less and usually branched organs that attach to another body and curl around it for support Subshrubs A subshrub, or undershrub, is an intermediate category of a half-woody, or suffruticose, plant. The lower part of the stem, from which the new growth emerges each year, is woody and persis- tent. These are not simply half-hardy plants but represent a stage between herbaceous and the truly woody condition. Examples of subshrubs in the catalog include: Ampelopsis arborea, peppervine, eastern United States Ampelopsis glandulosa var. brevipedunculata, porcelain-berry, Japan and People’s Republic of China Bignonia capreolata, cross vine, southeastern United States Parthenocissus quinquefolia, Virginia creeper, eastern United States Parthenocissus tricuspidata, Boston ivy, Japan and People’s Republic of China Vitis spp., grape vine. Ampelopsis arborea, peppervine, eastern United States Ampelopsis glandulosa var. brevipedunculata, porcelain-berry, Japan and People’s Republic of China Bignonia capreolata, cross vine, southeastern United States Parthenocissus quinquefolia, Virginia creeper, eastern United States Parthenocissus tricuspidata, Boston ivy, Japan and People’s Republic of China Vitis spp., grape vine. Ampelopsis arborea, peppervine, eastern United States Ampelopsis glandulosa var. brevipedunculata, porcelain-berry, Japan and People’s Republic of China Bignonia capreolata, cross vine, southeastern United States Parthenocissus quinquefolia, Virginia creeper, eastern United States Parthenocissus tricuspidata, Boston ivy, Japan and People’s Republic of China Vitis spp., grape vine. Root-climbers with holdfast roots and disc- like attachments Wisteria spp. (species twine either to the right or left). Campsis radicans, trumpet vine, eastern United States Decumaria barbara, wood-vamp, southeastern United States Euonymus fortunei, wintercreeper spindle- bush, Japan, People’s Republic of China, and Korea Hedera helix, English ivy, Europe Hydrangea anomala ssp. petiolaris, climbing hydrangea, Japan. Campsis radicans, trumpet vine, eastern United States Scramblers with long straggling branches that support themselves on other branches with- out fastening in any active manner; prickles or thorns may assist climbing, as in roses and brambles Scramblers with long straggling branches that support themselves on other branches with- out fastening in any active manner; prickles or thorns may assist climbing, as in roses and brambles Decumaria barbara, wood-vamp, southeastern United States Decumaria barbara, wood-vamp, southeastern United States Decumaria barbara, wood-vamp, southeastern United States Euonymus fortunei, wintercreeper spindle- Euonymus fortunei, wintercreeper spindle- bush, Japan, People’s Republic of China, and Korea Hedera helix, English ivy, Europe Rosa laevigata, Cherokee rose, People’s Republic of China Rubus calycinoides, Taiwan Rubus cockburnianus, People’s Republic of China. Rosa laevigata, Cherokee rose, People’s Republic of China Rubus calycinoides, Taiwan Hydrangea anomala ssp. petiolaris, climbing hydrangea, Japan. Hydrangea anomala ssp. petiolaris, climbing hydrangea, Japan. Genus Clematis spp. and hybrids. Clematis spp. and hybrids. Clematis spp. and hybrids. Genus (plural, genera) is the first major taxo- nomic category under the family. The catalog is arranged alphabetically by genus, beginning with Abelia R.Br., assigned to the family Genus (plural, genera) is the first major taxo- nomic category under the family. The catalog is arranged alphabetically by genus, beginning with Abelia R.Br., assigned to the family Caprifoliaceae and its common name, honey- suckle family. Genera of Caprifoliaceae in the catalog include: Abelia, Diervilla, Dipelta, Heptacodium, Kolkwitzia, Lonicera, Sambucus, Symphoricarpos, Viburnum, and Weigela. Some families are monotypic, comprising a single genus, such as Ginkgo in the Ginkgoaceae and Cercidiphyllum in the Cercidiphyllaceae. For the convenience of the user, the species, subspecies, varietas, forma, and cultivar names are listed alphabetically under each genus without regard to botanical rank. A supplementary list (Appen- dix D) groups the names according to botanical rank. Plant Nomenclature The guiding principles for the regulation of plant nomenclature are embodied in two contemporary documents: (1) the International Code of Botani- cal Nomenclature, 1988 edition (called the Botani- cal Code) and (2) the International Code of No- menclature for Cultivated Plants, 1980 edition (called the Cultivated Code). The Botanical Code “governs the use of botanical names in Latin form for both cultivated and wild plants, except for graft chimaeras” (Brickell et al. 1980: 11). The Cultivated Code regulates nomenclatural matters related exclusively to agricultural, horti- cultural, and silvicultural cultivars. Reasons for changing plant names are rooted in the rule of priority stated in Article 11 of the Botanical Code. For a genus, the correct epithet is the earliest legitimate one at the same rank, except for a conserved name (nomen conservan- dum); for example, Pseudolarix Gordon (1858) not Chrysolarix H.E. Moore (1965). In categories below the rank of genus, the correct name is the earliest legitimate name at the same rank— species, subspecies, varietas, or _forma—to which the plant is assigned. A plant has only one correct name, generally a binary name or bino- mial, that consists of a genus name (for example, Magnolia) followed by a specific epithet (for example, grandiflora). To this binary name is appended the name of the authority(ies) who published the name. The correct citation of the southern magnolia is Magnolia grandiflora L. (“L.” stands for Carl Linnaeus, who first described this species in 1759). Other names for this plant are treated as synonyms (Magnolia foetida Sarg., for example) or homonyms but are illegitimate for purposes of scientific nomenclature. The binary name must be in Latin form regardless of its derivation. Plant nomenclature contains many names adapted from other languages, especially from Greek. Hierarchy of Rank Categories Leaf-petiole climbers that hold the stem in place by twining around an adjacent branch Subspecies Before the term cultivar was adopted by the Cultivated Code in 1953, the category forma had long been in use for many garden plants and also for some wild plants. Examples include Sophora japonica L. f. pendula (Sweet) Zabel and Pseudotsuga menziesii (Mirb.) Franco var. glauca (Beissn.) Franco f. glauca pendula Rueppel that were automatically given cultivar status after 1953. These plants are now properly cited as Sophora japonica L. ‘Pendula’ and Pseudotsuga menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Glauca Pendula’. The subspecies, abbreviated ssp. or subsp., follows the rank of species and is often used to categorize geographic or ecological variants of a polymorphic species into generally disjunct morphological units. These units may be iso- lated from each other by latitude, longitude, altitude, and by ecological or geographical fac- tors, as well as by morphological characters. Botanists still disagree on the application of the subspecies category, especially when the distinc- tions among variants are ill defined. Examples of subspecies in the catalog include Acer negundo L. ssp. mexicanum (DC.) Wesmael, Hydrangea anomala D. Don ssp. petiolaris (Sieb. & Zucc.) McClint., and Koelreuteria elegans (Seemann) A.C. Sm. ssp. formosana (Hayata) F.G. Meyer. Species In nursery lists and in many publi- cations on landscape plants, authorities for botanical names are usually omitted, but this is a mistake and leads to confusion. The citation of authorities clearly identifies the botanical name with its correct author(s), for example, Aesculus flava Solander, not of Aiton. In another example, the name Acer parviflorum was applied by Ehrhart in 1789 to A. spicatum Lamarck (1786) and later by Franchet & Savatier in 1879 to the plant now known as A. nipponicum Hara. Be- cause of Ehrhart’s earlier use of the specific epithet (though illegitimate because of Lamarck’s priority), the Franchet & Savatier name was a “later homonym” and therefore illegitimate. Without an author citation, a reference to the name A. parviflorum would not inform the reader whether A. spicatum or A. nipponicum was meant. In horticultural works, authorities for cultivar names are not required according to the Culti- vated Code. In nursery lists and in many publi- cations on landscape plants, authorities for botanical names are usually omitted, but this is a mistake and leads to confusion. The citation of authorities clearly identifies the botanical name with its correct author(s), for example, Aesculus flava Solander, not of Aiton. In another example, the name Acer parviflorum was applied by Ehrhart in 1789 to A. spicatum Lamarck (1786) and later by Franchet & Savatier in 1879 to the plant now known as A. nipponicum Hara. Be- cause of Ehrhart’s earlier use of the specific epithet (though illegitimate because of Lamarck’s priority), the Franchet & Savatier name was a “later homonym” and therefore illegitimate. Without an author citation, a reference to the name A. parviflorum would not inform the reader whether A. spicatum or A. nipponicum was meant. Forma The category forma (abbreviated f.) has long been applied to distinguish transitory variation with- out persistent biological significance that is found randomly within populations of wild plants. Such variation includes flower color, leaf size, leaf shape, plant habit, vesture (hairiness, for example), and other characters. The following examples illustrate the use of forma in three well- known native woody plants cultivated in the southeastern United States: pink flowering dogwood (Cornus florida L. f. rubra (Weston) Schelle), yellow-fruited flowering dogwood (Cornus florida L. f. xanthocarpa Rehd.), and the yellow-fruited American holly (Ilex opaca Ait. f. xanthocarpa Rehd.). Cultivar The 1980 edition of the Cultivated Code provides guidelines “to promote uniformity, accuracy, and fixity in the naming of agricultural, horticultural, and silvicultural cultivars (varieties).” The term cultivar (derived from cultivated variety) was included when the Cultivated Code was drawn up in 1953; it denotes “an assemblage of culti- vated plants which is clearly distinguished by any characters (morphological, physiological, cytological, chemical, or others) and which, when reproduced (sexually or asexually), retains its distinguishing characters.” Species Biologically, the species is the basic taxonomic unit employed in the recognition and classifica- tion of plants. For purposes of this catalog, Cronquist’s general definition of species is useful: “Species are the smallest groups that are consis- tently and persistently distinct, and distinguish- able by ordinary means” (1988, p.71). Criteria for the recognition of species may include factors of reproductive isolation, chromosome number, phenetic or morphological discontinuity, geo- graphic isolation, and ecogeographic coherence. As the basic unit of biology, the species is subor- dinate to the genus. As the next higher major category, the genus consists of one or more species. The number of species varies from genus to genus. The monotypic genus Ginkgo, for example, consists of a single species, biloba, but the genus Quercus consists of many species, perhaps 450 worldwide, and some genera are even larger. While botanists argue the relative importance of species criteria, the viability of the species as a fundamental biological unit is attested by the presence of over 1,000 Linnean names in the catalog more than 200 years after the death of Linnaeus. Names of plants are based on nomenclatural types, usually type specimens, which are docu- mented, dried, and pressed specimens deposited in an herbarium for permanent reference. In the absence of a specimen, an accurate illustration may be substituted for purposes of typification. Botanical names published since 1935 must be accompanied by a Latin diagnosis to be botani- cally legitimate. The word species is either singular or plural (specie refers to coinage). It is abbreviated sp. when singular and spp. when plural. In this catalog, species and cultivar names are cited in strict alphabetical order under the name of the 6 categories. As indicated by Cronquist, “Nowa- days one taxonomist’s subspecies is likely to be another's variety” (1988, p. 99). Varieties are recognized in many genera in this catalog. In the genus Acer, for example, we recognize Acer rubrum L. var. trilobum T. & G. ex K. Koch, A. rubrum L. var. drummondii (Nutt.) Sarg., A. palmatum Thunb. ex J.A. Murr. var. amoenum (Carr.) Ohwi, and A. saccharum Marsh. var. rugelii (Pax) Rehd. genus. The entries under the genus Abelia, for example, include the species chinensis, followed by the cultivar ‘Edward Goucher’, the hybrid x grandiflora, and the cultivars ‘Prostrata’ and ‘Sherwoodii’. In horticultural works, authorities for cultivar names are not required according to the Culti- vated Code. Sexual hybrids Cultivars are without rank in the hierarchy of botanical nomenclature. In woody plants, culti- vars are usually perpetuated as clones, that is as a genetically uniform assemblage of plants derived originally from a single individual and reproduced asexually as cuttings, divisions, or grafts, or by obligate apomixis. Seed-reproduced cultivars occur infrequently among woody plants (for example, in Acer and Betula). They are, however, well known in herbaceous genera, for example, Impatiens, Phlox drummondii ‘Sternenzauber’, and Zea maus. In interspecific hybrids, a multiplication sign is placed before the hybrid epithet as in Abelia x grandiflora, a hybrid of A. chinensis and A. uniflora. In intergeneric hybrids, a multiplication sign is placed in front of the hybrid genus name as in XCupressocyparis leylandii, a hybrid of Chamaecyparis nootkatensis and Cupressus macrocarpa. Varietas The category varietas, or botanical variety (abbre- viated var.) was long used as the only infraspe- cific category generally recognized by botanists. Because of difficulties in evaluating morphologi- cal characters in plants, the categories subspe- cies and varietas have been used interchangeably by different workers. There is no common agree- ment among botanists on the application of these Graft chimeras As defined in the Cultivated Code, “Graft-chimae- ras are composed of tissues in intimate associa- tion from two different plants. They originate by grafting and are not sexual hybrids.” There is no combining of cell contents or nuclei as in the case of hybrids; thus the word hybrid is inappro- priate for these plants. In a graft chimera, the tissue of a shoot originating at the callus of the graft union contains at least one cell from the scion and one from the root stock. Periclinal chimeras, in which cells from the scion and cells from the stock are in different layers, are persis- tent when propagated. Chimeras may exhibit characters typical of the scion or the stock, ora range of intermediate forms. Cultivar names, to be legitimate, must be regis- tered through the appropriate international registration authority or published with an adequate description in a recognized publication, such as a dated nursery catalog or journal. Cultivar names simply listed in a nursery catalog or other publication are without valid status. These practices are important for ensuring the validity and stability of cultivar names. Cultivar names published on or after January 1, 1959, may no longer be in Latin form but must be fancy names in the vernacular, that is, in English or another modern language. Cultivar names may be designated by the abbreviation cv. pre- ceding the name, or, as in this catalog, by single quotation marks as in the following examples: Abelia ‘Edward Goucher’, Hibiscus syriacus ‘Diana’, and Magnolia ‘Galaxy’. However, cultivar names in Latin form published before January 1, 1959, such as Acer palmatum ‘Atropurpureum’ and Ilex aquifolium ‘Argentea Marginata’, must be retained, according to the Cultivated Code. The graft chimeras listed in the catalog are of the periclinal type. Graft chimeras are most unusual in the simultaneous production of flowers from the scion and from the stock. They are known worldwide by only a very few well- documented examples; two are listed in the catalog, designated with a plus (+) sign before the name, as follows: Our method of listing cultivar names alphabeti- cally facilitates easy identification of the binary name (Latin scientific name) associated with a cultivar name, especially in those genera for which the user of the catalog does not know the species of a particular cultivar. saccharum Marsh.]. In some cases, the cultivar is identified only as to genus. How Plant Names Change The names of plants are not fixed in time but are subject to change for various reasons. It is not difficult to find examples of plants long known under established names that are incorrect according to the Botanical Code. Name changes for some well-known woody landscape plants listed in the catalog are discussed below. The name Plumbago auriculata Lam. is the correct binary name for a now widely cultivated South African species. Published by Lamarck in 1786, this name has an 8-year priority over the name Plumbago capensis Thunb. that was long used for this plant. In another example, the name Cladrastis lutea (Michx.f.) K. Koch dates from 1813 and is well known; but in 1811 Dumont de Courset had published the specific epithet kentukea for the same plant. According to the rule of priority, the name Cladrastis kentukea (Dum.-Cours.) Rudd has date priority as the correct name for yellow-wood. From time to time, various specific epithets have been applied to the Douglas fir—including Pseudotsuga douglasii Carr. (1867), P. lindleyana Carr. (1868), P. taxifolia (Lamb.) Britton (1889), and P. mucronata (Raf.) Sudworth (1895). But all of these must give way to the name Pseudotsuga menziesii (Mirb.) Franco (1950), based on Pinus menziesii Mirb. (1825), as the valid name for this well-known tree of the western United States. A well-known tree indigenous to the southeast- ern United States, the yellow buckeye, should be cited as Aesculus flava Sol. (1784), not A. flava Ait. (1789). This may appear as a small detail, but it illustrates the application of the rule of priority that exists to promote stability and accuracy in plant nomenclature. The name A. octandra Marsh. (1785), published later and long used for this tree, is a synonym without taxo- nomic validity of A. flava Sol. Changes in plant names sometimes may not conform with the facts, causing more confusion than existed before the change was made. Two examples illustrate name changes too hastily made. Under Magnolia in Hortus Third (Bailey 1976), the entries M. heptapeta (Buc’hoz) Dandy and M. quinquepeta (Buc’hoz) Dandy are listed for two well-known magnolias, M. denudata and M. liliiflora, respectively. In 1779, Buc'hoz pub- lished Lassonia heptapeta and L. quinquepeta for these taxa. In 1934, Dandy transferred the Buc'hoz epithets to the genus Magnolia. Ina paper by F. G. Meyer and E. Graft chimeras The following examples from the genus Acer illustrate the usual method of citing cultivars, first listing the cultivar name and, in square brackets, identify- ing the species: ‘Akaji Nishiki’ [A. palmatum Thunb. ex J.A. Murr.]; ‘“Schwedleri’ [A. platanoides L.]; and “‘Temple’s Upright’ [A. saccharum Marsh.]. In some cases, the cultivar is identified only as to genus. e Camellia +‘Daisy Eagleson’ = C. sasanqua ‘Maiden’s Blush’ + C. japonica. Camellia sasanqua ‘Maiden’s Blush’ is the rootstock and C. japonica, the scion. At flowering time, this evergreen shrub displays simultaneously on different branches the single flowers of C. sasanqua and the semi-double flowers of C. Japonica. e +Laburnocytisus adamii = Cytisus purpureus + Laburnum anagyroides. This small deciduous tree combines species of two genera of the bean family (Fabaceae). Branches of the yellow flowers of L. anagyroides and the dull purple flowers of C. purpureus bloom simultaneously on the same plant. Until relatively recently, two species of Sequoia were recognized. A detailed study by J.T. Buchholz showed that only the coast redwood (Sequoia sempervirens (D. Don) Endl.) should be retained in the genus, while the giant sequoia of the high Sierras of California (formerly known as Sequoia gigantea Lindl.) belongs to the closely related genus Sequoiadendron with the binary name Sequoiadendron giganteum (Lindl.) J. Buchholz. Until relatively recently, two species of Sequoia were recognized. A detailed study by J.T. Buchholz showed that only the coast redwood (Sequoia sempervirens (D. Don) Endl.) should be retained in the genus, while the giant sequoia of the high Sierras of California (formerly known as Sequoia gigantea Lindl.) belongs to the closely related genus Sequoiadendron with the binary name Sequoiadendron giganteum (Lindl.) J. Buchholz. How Plant Names Change McClintock pub- lished in 1987, the evidence shows that the combinations of Dandy were totally misapplied and without botanical validity. The original Chinese illustrations on which these names were based are artist’s renditions, probably of Magno- lia, but the details are totally incorrect and the drawings may not be properly assigned to the genus Magnolia or, indeed, to any known living plant. Meyer and McClintock concluded that the earliest valid names for these magnolias, M. denudata and M. liliiflora, were correctly pub- lished by Desrousseaux in 1791 and stand as the correct names for these plants. Confusion reigns in the names applied to two evergreen shrubs planted in the southeastern states, one of them commonly grown and the other one rare. The well-known Florida native Illictum parviflorum Michx. ex Vent. is often misidentified in nurseries as I. anisatum L., a Japanese plant. Although the Japanese species is barely known in most areas, its name has long been associated with the wrong plant. Another common shrub in nurseries, and one widely cultivated in the southeastern United States, is Ternstroemia gymnanthera (Wight & Arn.) Sprague of the tea family (Theaceae). It has long been sold in nurseries under the name Cleyera japonica Thunb., a different though closely related plant. Many nurseries are now aware of the correct identification of these plants and are labeling them accordingly. The true Cleyera japonica is an attractive, small evergreen tree with fragrant white flowers and black fruit, but it is rarely cultivated. The name Toona sinensis (Endl.) M.J. Roem. applies to a tree native to eastern Asia, replacing the name Cedrela sinensis Juss. This follows an earlier taxonomic decision that the name Toona should apply to the Asiatic species, while the New World species (although closely related) are correctly placed in the genus Cedrela. The name Anisostichus capreolatus (L.) Bur. (1884) can no longer be used for the cross vine, because the earlier name Bignonia capreolata L. w) (1753) is a nomen conservandum, or conserved name, for this woody climber of the southeastern United States. name Hymenanthes japonica Blume to Rhodo- dendron japonicum (Blume) Schneider. This was, in fact, the correct name until very recently the Committee for Spermatophyta of the Interna- tional Botanical Congress determined that the specific epithet japonicum correctly applies to the deciduous azalea. The name R. degronianum ssp. Homonyms According to the Botanical Code, “A name, unless conserved (Art. 14) or sanctioned under Art. According to the Botanical Code, “A name, unless conserved (Art. 14) or sanctioned under Art. 13.1(d), is illegitimate if it is a later homonym, that is, if it is spelled exactly like a name based on a different type that was previously and validly published for a taxon of the same rank.” Many homonyms exist among plant names found in the literature. For example, Linnaeus in 1753 pub- lished Ilex aquifolium as the name for English holly (in the Aquifoliaceae, or holly family). In 1784, Thunberg used Ilex aquifolium for a Japa- nese plant subsequently recognized as being in the Oleaceae, or olive family. Placing the Japa- nese plant in the genus Osmanthus of the Oleaceae, Siebold in 1846 adopted the specific epithet from Thunberg's name and published Osmanthus aquifolium. But this name is illegiti- mate, because Thunberg's name is a later hom- onym, and the nomenclatural type for Ilex aquifolium is the English holly named by Linnaeus. Because the earliest available specific epithet for the Japanese plant is that from G. Don's Ilex heterophylla (1832), the legitimate name for the familiar holly olive is Osmanthus heterophyllus (G.Don) P.S.Green var. heterophyllus. As noted, a later homonym may be conserved by formal action under the Botanical Code. For example, the genus name, Torreya Arn. (1838) of the yew family (Taxaceae) is conserved, and the earlier homonym Torreya of Rafinesque (1818) applied to a plant in the mint family (Lamiaceae) is rejected. How Plant Names Change heptamerum (Maxim.) Hara is now the correct name for the elepidote rhododendron long known as R. metternichii. The illegitimate name R. metternichii Sieb. & Zucc., however, continues to be used widely in horticultural circles. For more than a century, nurseries confused two commonly planted species in the genus Ligustrum—L. japonicum Thunb., Japanese privet; and L. lucidum Ait.f., wax-leaf privet. The name L. lucidum was misapplied to the Japanese privet, while the name L. japonicum was mistakenly applied to the wax-leaf privet. These errors were repeated year after year in nursery catalogs until Roland Jefferson (1976) corrected them. The problem was a simple case of applying the right name to the right plant. The deciduous azalea involved in this confusion of names was described by Asa Gray as Azalea Japonica, in the Luteum subseries from Japan. In 1908, Suringar transferred the specific epithet to Rhododendron as R. japonicum (A.Gr.) Suring. Suringar’s publication of this combination was, however, interpreted by the author of a revision of the genus as provisional, making the name illegitimate as applied to the azalea and requiring its use for the elepidote rhododendron. Because the resulting name changes would have undesir- able consequences, a formal proposal was made to reject R. japonicum (Blume) Schneider. After detailed re-examination of Suringar’s work, the Committee for Spermatophyta concluded that the Suringar combination (using japonicum for the azalea) was indeed validly published. The name R. japonicum (Blume) Schneider for the elepidote rhododendron is therefore illegitimate as a later homonym and the proposal to reject became moot. Symbols and Abbreviations R. prinophyllum (Small) Millais. Acronyms are cited alphabetically under each plant entry to identify the collection sites, for example, DCNA = District of Columbia/National Arboretum; FLUF = Florida/University of Florida, Gainesville; and GAUG = Georgia/University of Georgia. Synonyms periclymenoides (Michx.) Shinners R. periclymenoides (Michx.) Shinners Rhododendron metternichii Sieb. & Zucc. = R.degronianum Carr. ssp. heptamerum R.degronianum Carr. ssp. heptamerum (Maxim.) Hara Rhododendron roseum (Loisel.) Rehd. = R. prinophyllum (Small) Millais. Vernacular (Common) Names The botanical (Latin-form) names and registered cultivar names of plants have universal applica- tion in the world scientific community. They are, moreover, instructive in many ways. By con- trast, vernacular names are simply names of convenience for local application. Over a wide area some plants may have several common names, all of equal value, as in laurel magnolia, bay, sweet bay, swamp bay, beaver tree, and laurel for Magnolia virginiana. Some plants, especially the exotics, are often without a com- mon name until one is invented. In this catalog we include one or two common names in general use for many of the listed plants. These names are summarized in Appendix E. Cultivar names are spelled with initial capital letters in roman type and are enclosed with single quotation marks: Abelia ‘Edward Goucher’, Magnolia ‘Galaxy’. Synonyms Many plants have been repeatedly redescribed by different botanists, thus creating a long, compli- cated synonymy that is often difficult to recon- cile. The synonyms of Magnolia denudata Desr. (1791), for example, include M. conspicua Salisb. (1806), M. precia Correa ex Vent. (1803), and M. yulan Desf. (1809). The name Negundo Jfraxinifolium de Vos (1887) is a synonym and invalid, because its nomenclatural type is the same as that of Acer negundo L. (1753), boxelder, a common North American tree that spans the continent east to west and is cultivated world- wide. Another significant confusion occurred in the names of two well-known species in the genus Rhododendron, one an elepidote rhododendron and the other a deciduous azalea. In 1826, Blume applied the name Hymenanthes japonica to the elepidote rhododendron. In 1835, Siebold and Zuccarini transferred the name Hymenanthes to Rhododendron as a generic synonym and named the species in question Rhododendron metternichii Sieb. & Zucc.; however, by citing the earlier name H. japonica Blume as a synonym they rendered R. metternichii illegitimate. In 1909, Camillo Schneider transferred the earlier 10 Common taxonomic synonyms are listed in the catalog to identify invalid names found in nurs- ery catalogs and in the horticultural and botani- cal literature, such as the following: suckle refers only to species of Lonicera of the honeysuckle family (Caprifoliaceae). The com- mon name for Rhododendron periclymenoides is pinxterbloom in Maryland, purple honeysuckle in the Carolinas, and election pink in some other areas of the southeast. In the United States, the indigenous species of Tilia are called basswood or linden, while in the United Kingdom members of this genus are called lime trees. The use of common names should be discouraged because of the endless possibilities for misidentification and confusion. Common taxonomic synonyms are listed in the catalog to identify invalid names found in nurs- ery catalogs and in the horticultural and botani- cal literature, such as the following: Common taxonomic synonyms are listed in the catalog to identify invalid names found in nurs- ery catalogs and in the horticultural and botani- cal literature, such as the following: Cladrastis lutea (Michx.f.) K. Koch = C. kentukea (Dum.-Cours.) Rudd Magnolia foetida (L.) Sarg. = M. grandiflora L. Rhododendron nudiflorum (L.) Torr. = R. periclymenoides (Michx.) Shinners Rhododendron metternichii Sieb. & Zucc. = C. kentukea (Dum.-Cours.) Rudd Magnolia foetida (L.) Sarg. = M. grandiflora L. Rhododendron nudiflorum (L.) Torr. = R. Other symbols and abbreviations follow. umbellata (Mayr) Wils. = A. xumbellata koreana Wils. Korean F. DCNA nobilis (Dougl.) Lind]. = A. procera nordmanniana (Steven) Spach Nordmann F. DCAE MDLT NCBE VABF NCKK numidica de Lannoy ex Carr. Algerian F. DCNA pectinata DC. =A. alba pinsapo Boiss. Spanish F. ABELIA ABELIA R.Br. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) chinensis R.Br. Chinese A. DCNA MDFM SCWI ‘Edward Goucher’ [A. < grandiflora < A. schumannii] ALBH GAUG SCCU TXRS GAJS MDGC TNEN x grandiflora (Andre) Rehd. Glossy A. [A. chinensis < A. uniflora] DCCG MDJH MSEP NCSM FLMG MDKN NCBE SCCU LASL MDLT NCDU TNBM MDDF MDNA NCGP ‘Prostrata’ [A. < grandiflora (Andre) Rehd.] [A. chinensis < A. uniflora] DCNA ‘Sherwoodii’ [A. < grandiflora (Andre) Rehd.] [A. chinensis < A. uniflora] GAUG SCBR ABELIOPHYLLUM Nakai OLEACEAE Olive Family distichum Nakai White Forsythia DCNA MDLT ABIES Mill. Fir PINACEAE Pine Family SS 2 SS Ee RE SS ee alba Mill. Silver F. GABS NCTE VAWP balsamea (L.) Mill. Balsam F. DCNA brachyphylla Maxim. = A. homolepis cephalonica Loud. Greek F. DCNA MDLT ABELIA R.Br. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) chinensis R.Br. Chinese A. DCNA MDFM SCWI ‘Edward Goucher’ [A. < grandiflora < A. schumannii] ALBH GAUG SCCU TXRS GAJS MDGC TNEN x grandiflora (Andre) Rehd. Glossy A. [A. chinensis < A. uniflora] DCCG MDJH MSEP NCSM FLMG MDKN NCBE SCCU LASL MDLT NCDU TNBM MDDF MDNA NCGP ‘Prostrata’ [A. < grandiflora (Andre) Rehd.] [A. chinensis < A. uniflora] DCNA ‘Sherwoodii’ [A. < grandiflora (Andre) Rehd.] [A. chinensis < A. uniflora] GAUG SCBR ABELIOPHYLLUM Nakai OLEACEAE Olive Family distichum Nakai White Forsythia DCNA MDLT ABIES Mill. Fir PINACEAE Pine Family SS 2 SS Ee RE SS ee alba Mill. Silver F. GABS NCTE VAWP balsamea (L.) Mill. Balsam F. DCNA brachyphylla Maxim. = A. homolepis ABELIA R.Br. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) chinensis R.Br. Chinese A. DCNA MDFM SCWI ‘Edward Goucher’ [A. < grandiflora < A. schumannii] ALBH GAUG SCCU TXRS GAJS MDGC TNEN x grandiflora (Andre) Rehd. Glossy A. [A. chinensis < A. uniflora] DCCG MDJH MSEP NCSM FLMG MDKN NCBE SCCU LASL MDLT NCDU TNBM MDDF MDNA NCGP ‘Prostrata’ [A. < grandiflora (Andre) Rehd.] [A. chinensis < A. uniflora] DCNA ‘Sherwoodii’ [A. < grandiflora (Andre) Rehd.] [A. chinensis < A. uniflora] GAUG SCBR ABELIOPHYLLUM Nakai OLEACEAE Olive Family distichum Nakai White Forsythia DCNA MDLT ABIES Mill. Other symbols and abbreviations follow. ssp. = subspecies: Hydrangea anomala D. Don ssp. petiolaris (Sieb. & Zucc.) McClint. ssp. = subspecies: Hydrangea anomala D. Don ssp. petiolaris (Sieb. & Zucc.) McClint. var. = varietas (botanical variety): Acer palmatum Thunb. ex J.A. Murr. var. amoenum (Carr.) Hara While there are problems associated with com- mon names, many people prefer the vernacular. Some complain about Latin names of plants as too awkward and difficult to pronounce. Ap- proached with an open mind, scientific names are not difficult. Familiar names used in the vernacular, such as anemone, catalpa, chrysan- themum, gladiolus, iris, magnolia, petunia, phlox, rhododendron, sassafras, wisteria, yucca, and zinnia are, in fact, Latin genus names that have entered vernacular usage. x = multiplication sign used for hybrids: interspecific hybrids are identified with the multiplication sign placed in front of the hybrid epithet, as Abelia x grandiflora (Andre) Rehd.; in bigeneric hybrids the multiplication sign is placed in front of the hybrid genus name, as * Cupressocyparis leylandii (Dallim. & Jacks.) Dallim. + = symbol for graft chimera, as Camellia +'Daisy Eagleson’ + = symbol for graft chimera, as Camellia +'Daisy Eagleson’ [] enclose botanical names of cultivars and names of parents of hybrids and cultivars * designates plant introductions from the U. S. National Arboretum breeding program ? indicates uncertainty as to parentage. + = symbol for graft chimera, as Camellia +'Daisy Eagleson’ Once established, common names are often useful and convenient for trees such as Douglas fir, deodar cedar, white oak, and American elm. However, unlike botanical names, common names are sometimes misleading and confusing and may impede clear communication and understanding, since nothing about these names relates to name stability or to classification. In the southeastern United States, for example, the native deciduous azaleas are commonly known as “honeysuckle,” but to many people honey- [] enclose botanical names of cultivars and names of parents of hybrids and cultivars ll ll ABIES ABIES chensiensis Tieghem ssp. salouenensis (Bord.-Rey. & Gaussen) Rushforth Shensi F. DCNA cilicica (Ant. & Kotschy) Carr. Cilician F. VAMP concolor (Gord. & Glend.) Lindl. ex Hildebr. White F. MDLT NCBE VAMP ernestii Rehd. = A. chensiensis ssp. salouenensis firma Sieb. & Zucc. Momi F. ALOS MDLT TNWF DCNA NCBE fraseri (Pursh) Poir. Fraser F. NCAS TNBV VAFR VASG ‘Glauca’ [A. pinsapo Boiss.] DCNA holophylla Maxim. Manchurian F. DCNA homolepis Sieb. & Zucc. Nikko F. DCNA homolepis Sieb. & Zucc. var. Other symbols and abbreviations follow. Fir PINACEAE Pine Family SS 2 SS Ee RE SS ee alba Mill. Silver F. GABS NCTE VAWP balsamea (L.) Mill. Balsam F. DCNA brachyphylla Maxim. = A. homolepis cephalonica Loud. Greek F. DCNA MDLT ABIES procera Rehd. Noble F. DCNA x umbellata (Mayr) Liu [A. firma X< A. homolepis] DCNA x vilmorinii Mast. [A. cephalonica < A. pinsapo] DCNA ABUTILON Mill. Flowering Maple MALVACEAE Mallow Family > = RE ee ee SS ae SS) pictum (Gillies ex Hook. & Arn.) Walp. LAMP striatum Dicks. ex Lindl. = A. pictum ‘Thompsonii’ [A. pictum (Gillies ex Hook. & Arn.) Walp.] GACG ACACIA Mill. Wattle, Mimosa FABACEAE (Mimosoideae) Bean Family Fae EE TS TOS Oe ee ae ae pe ee Se) berlandieri Benth. TXSE farnesiana (L.) Willd. Sweet A. FLUF LAMP TXJS LAHG SCKS TXSE rigidula Benth. LASL roemeriana Scheele TXSE wrightii Benth. TXPS ACALYPHA lL. EUPHORBIACEAE Spurge Family SS Se EE DO EE ee ee ee ee hispida Burm.f. Chenille Plant ALBG sanderi N.E.Br. = A. hispida ACER ABIES procera Rehd. Noble F. DCNA x umbellata (Mayr) Liu [A. firma X< A. homolepis] DCNA x vilmorinii Mast. [A. cephalonica < A. pinsapo] DCNA ABUTILON Mill. Flowering Maple MALVACEAE Mallow Family > = RE ee ee SS ae SS) pictum (Gillies ex Hook. & Arn.) Walp. LAMP striatum Dicks. ex Lindl. = A. pictum ‘Thompsonii’ [A. pictum (Gillies ex Hook. & Arn.) Walp.] GACG ACACIA Mill. Wattle, Mimosa FABACEAE (Mimosoideae) Bean Family Fae EE TS TOS Oe ee ae ae pe ee Se) berlandieri Benth. TXSE farnesiana (L.) Willd. Sweet A. FLUF LAMP TXJS LAHG SCKS TXSE rigidula Benth. LASL roemeriana Scheele TXSE wrightii Benth. TXPS ACALYPHA lL. EUPHORBIACEAE Spurge Family SS Se EE DO EE ee ee ee ee hispida Burm.f. Chenille Plant ALBG sanderi N.E.Br. = A. hispida ABIES ABIES ABIES ACANTHOPANAX (Becne. & Planch.) Mig. = ELEUTHEROCOCCUS ACANTHOPANAX (Becne. & Planch.) Mig. = ELEUTHEROCOCCUS ACANTHOPANAX (Becne. & Planch.) Mig. = ELEUTHEROCOCCUS ACCA O.Berg MYRTACEAE Myrtle Family sellowiana (O.Berg) Burret Pineapple Guava FLMG LAHG NCGP SCBY FLRH MSEP NCOP SCLO FLUF MSRN NCWM VANB sellowiana (O.Berg) Burret (Leaves variegated) DCCG GAJS MSEN VAWP DCNA MDNA VACW FLUF MDRP VASC ACER L. Maple ACERACEAE Maple Family ‘Aconitifolium’ [A. japonicum Thunb. ex J.A.Murr.] DCNA GACG MDJS MDPJ ‘Akaji nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Aka washi no o’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Ornatum’ ‘Akikaze nishiki’ [A. truncatum Bunge] DCNA ‘Albo-limbatum’ [A. rufinerve Sieb. & Zucc.] = ‘Hatsuyuki’ ‘Albo-variegatum’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Versicolor’ ‘Almira’ [A. platanoides L.] DCNA amplum Rehd. = A. longipes ssp. amplum ‘Aokii’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Versicolor’ ‘Aoyagi’ [A. palmatum Thunb. ex J.A.Murr.] Mig. = ELEUTHEROCOCCUS sellowiana (O.Berg) Burret Pineapple Guava 13 ACER ‘Asahi zuru’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Atropurpureum’ [A. palmatum Thunb. ex J.A.Murr.] Bloodleaf M. (Atropurpureum group) ALEH GACD MDJH NCOP ANAND) GACG MDJS NCPL ARSN GATS MDLA TNMB DCNA GAUG MDMM TNWF DEWG MDDF MDNA VAAC FLMG MDGG £NCBE VACW GAAB MDHN #£NCDU VAGW ‘Atropurpureum’ [ A. pseudoplatanus L.] Purpleleaf Sycamore M. DCNA ‘Atropurpureum Superbum’ [A. palmatum Thunb. ex J.A.Murr.] (Atropurpureum group) DCNA ‘Aureo-variegatum’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Aureum’ [A. cappadocicum Gleditsch] Golden Colosseum M. TNTV ‘Aureum’ [A. japonicum Thunb. ex J.A.Murr.] Golden Full-moon M. MDKN SCCC ‘Aureum’ [A. palmatum Thunb. ex J.A.Murr.] MDDF ‘Autumn Glory’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Bloodgood’ [A. palmatum Thunb. ex J.A.Murr.] DCNA GAEC GAFN VATA ‘Brilliantissimum’ [A. pseudoplatanus L.] DCNA buergerianum Miq. Trident M. DCWR GAUG LXCE VATA ACER ACER ACER ‘Asahi zuru’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Atropurpureum’ [A. palmatum Thunb. ex J.A.Murr.] Bloodleaf M. (Atropurpureum group) ALEH GACD MDJH NCOP ANAND) GACG MDJS NCPL ARSN GATS MDLA TNMB DCNA GAUG MDMM TNWF DEWG MDDF MDNA VAAC FLMG MDGG £NCBE VACW GAAB MDHN #£NCDU VAGW ‘Atropurpureum’ [ A. pseudoplatanus L.] Purpleleaf Sycamore M. DCNA ‘Atropurpureum Superbum’ [A. palmatum Thunb. ex J.A.Murr.] (Atropurpureum group) DCNA ‘Aureo-variegatum’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Aureum’ [A. cappadocicum Gleditsch] Golden Colosseum M. TNTV ‘Aureum’ [A. japonicum Thunb. ex J.A.Murr.] Golden Full-moon M. MDKN SCCC ‘Aureum’ [A. palmatum Thunb. ex J.A.Murr.] MDDF ‘Autumn Glory’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Bloodgood’ [A. palmatum Thunb. ABIES ex J.A.Murr.] DCNA GAEC GAFN VATA ‘Brilliantissimum’ [A. pseudoplatanus L.] DCNA buergerianum Miq. Trident M. DCWR GAUG LXCE VATA GACG TNFE TXMS GAFN TNPW VAAC GAME TNA VABF ACER ‘Burgundy Lace’ [A. palmatum Thunb. ex J.A.Murr.] DCNA GACG ‘Butterfly’ [A. palmatum Thunb. ex J.A.Murr.] MDBG MDJS MDKN TXHL campbellii Hiern ssp. flabellatum (Rehd.) E.Murr. DCNA campbellii Hiern ssp. oliverianum (Pax) E.Murr. = A. oliverianum ssp. oliverianum campestre L. Hedge M. DCCG DCWH TNDR VASC DCLC GAUG VAAC DCNA NCBE VACW campestre L. var. leiocarpum (Opiz) Wallroth Hedge M. MDCP NCCA VABF NCBE NCDU capillipes Maxim. DCNA TNPW cappadocicum Gleditsch Colosseum M. DCCG DCGP DCNA cappadocicum Gleditsch ssp. truncatum (Bunge) E.Murr. = A. truncatum carpinifolium Sieb. & Zucc. Hornbeam M. TNWF ‘Chas. F. Irish’ [A. platanoides L.] DCNA ‘Chirimen nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Chitoseyama’ [A. palmatum Thunb. ex J.A.Murr.] DCNA cissifolium (Sieb. & Zucc.) K.Koch VABF ‘Compactum’ [A. campestre L.] ‘Atropurpureum’ [A. palmatum Thunb. ex J.A.Murr.] Bloodleaf M. (Atropurpureum group) ‘Aureum’ [A. japonicum Thunb. ex J.A.Murr.] Golden Full-moon M. MDKN SCCC 14 ACER ‘Filicifolium’ [A. japonicum Thunb. ex J.A.Murr.] = ‘Aconitifolium’ ‘Filigree’ [A. palmatum Thunb. ex J.A.Murr.] SCCC flabellatum Rehd. = A. campbellii ssp. flabellatum ‘Flavescens’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Dissectum Flavescens’ xfreemanii E.Murr. [A. rubrum < A. saccharinum] DCNA ‘Garnet’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) GAEC ‘Gerling’ [A. rubrum L.] DCNA ginnala Maxim. Amur M. DCNA SCCU TNSC GAUG TNCT VACW MDJH TNDR VATA ‘Globosum’ [A. campestre L.] = ‘Nanum’ griseum (Franch.) Pax Paperbark M. DCNA GAWH #£=MODLT VAGS DEWG MDBG TNHT VATA GALA MDGD TNSN ‘Hagaromo’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDKN MDMG SCEE ‘Hatsuyuki’ [A. rufinerve Sieb. & Zucc.] DCNA MDBG ‘Heptalobum’ [A. palmatum Thunb. ex J.A.Murr.] = A. palmatum var. amoenum ‘Hessei’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDJS MDKN ‘Higasayama’ [A. palmatum Thunb. ex J.A.Murr.] DCNA GACG MDJS MDKN ACER ACER ACER ‘Corallinum’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Crimson King’ [A. platanoides L.] DCNA MDJH TNSC VATA ‘Crimson Queen’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) DCNA davidii Franch. David's M. DCNA MDBG TNRT GATS TNFE diabolicum Bl. ex K.Koch Devil M. DCNA VABF ‘Discolor Versicolor [A. palmatum Thunb. ex J.A.Murr.] = ‘Versicolor’ dissectum Thunb. ex J.A.Murr. = A. palmatum ‘Dissectum’ ‘Dissectum’ [A. palmatum Thunb. ex J.A.Murr.] Laceleaf M. (Dissectum group; green-leaved) DCCG GACG TNDR DCNA GAEC ‘Dissectum Flavescens’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Dissectum Palmatifidum’ [A. palmatum Thunb. ex J.A.Murr.] DCNA SCCC ‘Dissectum Paucum’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Dissectum Palmatifidum’ ‘Dissectum Rubrifolium’ [A. palmatum Thunb. ex J.A.Murr.] DCNA DCWH MDJS NCBE ‘Dissectum Variegatum’ [A. palmatum Thunb. ex J.A.Murr.] MDKN ‘Drummondii’ [A. platanoides L.] DCNA ‘Faassen’s Black’ [A. platanoides L.] ‘Filicifolium’ [A. japonicum Thunb. ex J.A.Murr.] = ‘Aconitifolium’ ‘Filigree’ [A. palmatum Thunb. ex J.A.Murr.] SCCC flabellatum Rehd. = A. campbellii ssp. flabellatum ‘Flavescens’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Dissectum Flavescens’ xfreemanii E.Murr. [A. rubrum < A. saccharinum] DCNA ‘Garnet’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) GAEC ‘Gerling’ [A. rubrum L.] DCNA ginnala Maxim. Amur M. DCNA SCCU TNSC GAUG TNCT VACW MDJH TNDR VATA ‘Globosum’ [A. campestre L.] = ‘Nanum’ griseum (Franch.) Pax Paperbark M. DCNA GAWH #£=MODLT VAGS DEWG MDBG TNHT VATA GALA MDGD TNSN ‘Hagaromo’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDKN MDMG SCEE ‘Hatsuyuki’ [A. rufinerve Sieb. & Zucc.] DCNA MDBG ‘Heptalobum’ [A. palmatum Thunb. ex J.A.Murr.] = A. palmatum var. amoenum ‘Hessei’ [A. palmatum Thunb. ex IL: ACER ‘Hogyokw’ [A. palmatum Thunb. ex J.A.Murr.] GACG ‘Ichigyo ji’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘lijima sunago’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Inaba shidare’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) DCNA ‘Itaya’ [A. japonicum Thunb. ex J.A.Murr.] DCNA MDKN ‘Itaya meigetsu’ [A. japonicum Thunb. ex J.A.Murr.] = A. japonicum japonicum Thunb. ex J.A.Murr. Japanese M., Full-moon M. DCNA MDJS ‘Jiro shidare’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Kagiri nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Killarney’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Kingsville Red’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Kinugasayama’ [A. japonicum Thunb. ex J.A.Murr.] (Sometimes placed in A. sieboldianum Miq.) DCNA ‘Kocho nishiki’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Butterfly’ ‘Kohauchina kaido’ [A. japonicum Miq.] DCNA ACER ACER ‘Laceleaf [A. palmatum Thunb. ex J.A.Murr.] = ‘Filigree’ ‘Linearilobum’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDBG longipes Franch. ssp. ACER amplum (Rehd.) Jong DCNA ‘Lutescens’ [A. palmatum Thunb. ex J.A.Murr.] MDGD MDJS ‘Maiko’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Mai kujakw’ [A. japonicum Thunb. ex J.A.Murr.] = ‘Aconitifolium’ mandshuricum Maxim. Manchurian M. MDKN ‘Masukagami’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Matsukaze’ [A. palmatum Thunb. ex J.A.Murr.] DCNA maximowiczianum Miq. Nikko M. DCNA MDDF VAHM ‘Mikasayama’ [A. sieboldianum Miq.] DCNA ‘Mino yatsufusa’ [A. buergerianum Miq.] TNSN ‘Mioun’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) DCNA miyabei Maxim. Miyabe M. MDGD ‘Miyasama’ [A. buergerianum Miq. ssp. formosanum (Hayata) E.Murr. & Lauener] DCNA MDKN TNSN ‘Mizu kuguri’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Jiro shidare’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Kagiri nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Killarney’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Kingsville Red’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Kinugasayama’ [A. japonicum Thunb. ex J.A.Murr.] (Sometimes placed in A. sieboldianum Miq.) DCNA ‘Kocho nishiki’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Butterfly’ ‘Kohauchina kaido’ [A. japonicum Miq.] DCNA ‘Kohauchina kaido’ [A. japonicum Miq.] DCNA ‘Koshimino’ [A. palmatum Thunb. ex J.A.Murr.] MDMG ‘Kurabeyama’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Koshimino’ [A. palmatum Thunb. ex J.A.Murr.] MDMG 16 ACER ACER mono Maxim. = A. truncatum monspessulanum L. Montpellier M. VABF ‘Monumentale’ [A. saccharum Marsh.] = ‘Temple’s Upright’ ‘Monzukushi’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Moonfire’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Mure hibari’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Musashino’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Nomura’ ‘Nanum’ [A. campestre L.] Dwarf Hedge M. DCNA TNDR TNTV GACG TNSN VAGS ‘Naruo nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Nauto kaede’ [A. buergerianum Miq.] TNSN negundo L. Boxelder ALAU DCNA DEEM negundo L. ssp. latifolium (Pax) Schwerin = A. negundo ssp. negundo var. texanum negundo L. ssp. mexicanum (DC.) Wesmael MDGD negundo L. ssp. negundo var. texanum Pax FLUF MSEN TXHA MDSJ NCCA ‘Newton Sentry’ [A. saccharum Marsh.] MDJS nigrum Michx. = A. saccharum ssp. nigrum ACER ‘Nomura’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Nomura nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA oblongum Wall. ex DC. Evergreen M. FLUF ‘October Glory’ [A. rubrum L.] DCNA ‘Oekonomierat Stoll’ [A. platanoides L.] DCNA ‘Ogino nagare’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Ogon sarasa’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘O isami’ [A. japonicum Thunb. ex J.A.Murr.] DCNA ‘O kagami’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Okushimo’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDBG MDKN GACG MDJS oliverianum Pax DCNA FLUF GAIS ‘Omato’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Oo shi rini’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Oregon Sunset’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Orido nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Ornatum’ [A. palmatum Thunb. ex J.A.Murr.] mono Maxim. = A. truncatum monspessulanum L. Montpellier M. VABF 7 ACER ‘Osakazuki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDKN ‘Oshio beni’ [A. palmatum Thunb. ex J.A.Murr.] DCNA GACG ‘Oshu shidare’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘O take’ [A. japonicum Thunb. ex J.A.Murr.] DCNA ‘Palmatum’ [A. saccharinum L.] FLUF palmatum Thunb. ex J.A.Murr. var. amoenum (Carr.) Ohwi ALBH GAUG SCCEe VACW DCNA LAHG SCHS GATS MDJS TNDR palmatum Thunb. ex J.A.Murr. var. heptalobum Rehd. = A. palmatum var. amoenum palmatum Thunb. ex J.A.Murr. var. matsumurae (Koidz.) Makino DCNA palmatum Thunb. ex J.A.Murr. var. palmatum Japanese M. ARWS GAEC MDCP SECE DECECG GAOH MDHN SCNR DCNA GAUG MDJS VACW FLMG GAWP MSEP VATA GACG MDBG NCBE pensylvanicum L. Striped Maple VAGW pictum Thunb. ex J.A.Murr. = Kalopanax septemlobus ‘Pixie’ [A. palmatum Thunb. ex J.A.Murr.] (Name of doubtful status) DCNA platanoides L. Norway Maple ALAU DCWR MDNA VACW DCCG DEMC MDPJ VATA DCNA MDHN MDSJ VAWR DCWH MDJS SCKG ACER ACER ACER pseudoplatanus L. Sycamore Maple DCCG DESR TNFE VARO DCNA MDLA VACS pseudosieboldianum (Pax) Komar. Purplebloom Maple DCNA MDJS ‘Pyramidale’ [A. rubrum L.] DCSE ‘Reticulatum’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Shigitatsu sawa’ ‘Ribbon Leaf’ [A. palmatum Thunb. ex J.A.Murr.] (Atropurpureum group) DCNA ‘Roseo-maculatum’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Versicolor’ ‘Roseo-marginatum’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Kagiri nishiki’ ‘Rubrifolium’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Dissectum Rubrifolium’ rubrum L. Red Maple DCCG DCWH MDLA VAGG DCNA DEWG VACW rubrum L. var. drummondii (Nutt.) Sarg. Drummond Maple FLUF SCMP TXCT rubrum L. var. tridens A.Wood = A. rubrum var. trilobum rubrum L. var. trilobum T. & G. ex K.Koch ALAU MDHN VACW ALOS SCUC VAGW FLMG VAAC VAWR rufinerve Sieb. & Zucc. Redvein Maple DCNA GAEC saccharinum L. Silver Maple ALAU DCNA GAFN TNWF ARRB DEMC GAVI VACM DCCG FLUF SCEM VACW 18 ACER ACER ‘Seiryu’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) ay DCNA ‘Sessilifolium’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Hagoromo’ ‘Sherwood Flame’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Shigitatsu sawa’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDJS MDKN ‘Shigurezome’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Shino buga oka’ [A. palmatum Thunb. ex J.A.Murr.] MDJS shirasawanum Koidz. MDJS TNSN ‘Shirigosan’ [A. palmatum Thunb. ex J.A.Murr.] (Linearilobum group) DCNA ‘Shishigashira’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDJS MDKN SCCC ‘Shojo’ [A. palmatum Thunb. ex J.A.Murr.] DCNA sieboldianum Miq. Siebold Maple DCNA DEWG spicatum Lam. Mountain Maple GAEC ‘Stollii’ [A. platanoides L.] = ‘Oekonomierat Stoll’ ‘Sweet Shadow Cut-Leaf’ [A. saccharum Marsh.] DCCG ‘Tamukeyama’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) DCNA saccharum Marsh. ssp. floridanum (Chapm.) Desm. Florida Maple ALEH GAAB TXLL FLUF NCSM TXMS saccharum Marsh. ssp. leucoderme (Small) Desm. Chalk Maple ALEH GAEC GAWD saccharum var. monumentale (Temple) Rehd. = ‘Temple’s Upright’ saccharum Marsh. ssp. nigrum (Michx.f.) Desm. Black Maple DCNA TNWF VAMP saccharum Marsh. var. rugelii (Pax) Rehd. DCCG TNSC VAWD TNHG VACW VAWR saccharum Marsh. ssp. saccharum Sugar Maple ARRB MDHN TNWF VAHC DCCG MDMM VABF VAWR DCNA MDNA VAGG ‘Sagara nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Sango kaku’ [A. palmatum Thunb. ex J.A.Murr.] DCEL FLMG MDKN DCNA MDBG SCCC ‘Sanguineum’ [A. palmatum Thunb. ex J.A.Murr.] = A. palmatum var. palmatum ‘Sazanami’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Schlesingeri’ [A. rubrum L.] DCNA ‘Schwedleri’ [A. platanoides L.] Schwedler M. DCEL GAOH MDNA VACW GALA MDCP TNFE VAWR ‘Scolopendrifolium’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Linearilobum’ rg ACER ACER ACER ‘Tatsuta’ [A. palmatum Thunb. ex J.A.Murr.] DCNA tegmentosum Maxim. Manchurian Striped M. DCNA DCPP MDLT TNTV ‘Temple’s Upright’ [A. saccharum Marsh.] DCNA DEWG MDJS MDPJ ‘The Bishop’ [A. palmatum Thunb. ex J.A.Murr.] DCNA MDKN ‘Tilford’ [A. rubrum L.] DCNA ‘Tokiwa nishiki’ [A. truncatum Bunge] DCNA ‘Toyama nishiki’ [A. palmatum Thunb. ex J.A.Murr.] (Dissectum group) DCNA trifidum Hook. & Arn. = A. buergerianum ‘Trompenburg’ [A. palmatum Thunb. ex J.A.Murr.] DCNA truncatum Bunge Shantung Maple DCNA MDKN TNUT VABP GAUG TNDR VABF truncatum Bunge f. dissectum Wesmael DCNA ‘Tsuchi uno’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Tsuku bane’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Tsukushi gata’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Tsuri nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Utsu semi’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Variegatum’ [A. negundo L.] ALTD LAGN MDKN VANP ‘Variegatum’ [A. palmatum Thunb. ACER DCCG MDHN NCTE VAKH DCNA MDJH TNFE VAMP DEEM MDNA TNSC VAWP MDBP NCBE VACP MDCP NCKH VACW flava Ait. = A. flava Sol. glabra Willd. Ohio B. ARHS DCWR MDJH TNFE DCNA DEWG MDLS VAMP DCWH MDHN SCWI x glaucescens Sarg. [A. flava x A. sylvatica] DEWG hippocastanum L. H. DECG DEWG MDMM VAPS DCNA MDBG VACW DCSE MDCP VAGW DCWH MDLA VAMP ACOELORRHAPHE H.Wendl. ARECACEAE Palm Family = Tee ar Se ae er an eT) wrightii (Griseb. & H.Wendl.) H.Wendl. ex Becc. FLUF ACOELORRHAPHE H.Wendl. ARECACEAE Palm Family = Tee ar Se ae er an eT) wrightii (Griseb. & H.Wendl.) H.Wendl. ex Becc. FLUF ACOELORRHAPHE H.Wendl. ARECACEAE Palm Family = Tee ar Se ae er an eT) wrightii (Griseb. & H.Wendl.) H.Wendl. ex Becc. FLUF ACTINIDIA Lindl. ACTINIDIACEAE Actinidia Family a Oe ee SE OT oe TC SES Vn Se SS SET] arguta (Sieb. & Zucc.) Planch. ex Miq. Tara Vine DCDO DCNA MDGD VATA ‘Chico’ [A. deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa] VATA chinensis Planch. = A. deliciosa var. deliciosa chinensis Planch. var. deliciosa (A.Chev.) A.Chev. = A. deliciosa var. deliciosa deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa Kiwi Fruit, Chinese Gooseberry GAAB NCDU x fairchildii Rehd. [A. arguta < A. chinensis] MDGD VATA kolomikta (Maxim. & Rupr.) Maxim. VAGS polygama (Sieb. & Zucc.) Maxim. Silver Vine DCNA purpurea Rehd. VATA ADINA Salisb. RUBIACEAE Madder Family Ce ee EE ae EET ee a See SS Se] rubella Hance Chinese Buttonbush ALAU SCCU LASL VACW VAHM SSG FETT Pe he eae Sh eed ‘Baumanii’ [A. hippocastanum L.] Double-flowered H. DCNA MDBG DCSE MDCP NCBE NCDU ‘Briotii’ [A. x carnea Hayne] [A. hippocastanum x A. pavia] DCNA DEWG MDPJ DCWR MDKN VACW < bushii Schneid. [A. glabra x A. pavia] MDBP californica (Spach) Nutt. California B. DCNA SCWI x carnea Hayne Red H. [A. hippocastanum < A. pavial] DECRIE MDBG VAMP DCSE MDCP x dupontii Sarg. [A. flava x (A. pavia < A. sylvatica)] DEEM DEWG flava Sol. Yellow B. DCCG MDHN NCTE VAKH DCNA MDJH TNFE VAMP DEEM MDNA TNSC VAWP MDBP NCBE VACP MDCP NCKH VACW flava Ait. = A. flava Sol. glabra Willd. Ohio B. ARHS DCWR MDJH TNFE DCNA DEWG MDLS VAMP DCWH MDHN SCWI x glaucescens Sarg. [A. flava x A. sylvatica] DEWG hippocastanum L. H. DECG DEWG MDMM VAPS DCNA MDBG VACW DCSE MDCP VAGW DCWH MDLA VAMP Becc. FLUF ACTINIDIA Lindl. ACER ex J.A.Murr.] = ‘Dissectum Variegatum’ ‘Variegatum’ [A. rufinerve Sieb. & Zucc.] MDBG velutinum Boiss. var. vanvolxemii (Mast.) Rehd. Velvet M. DCNA ‘Versicolor’ [A. palmatum Thunb. ex J.A.Murr.] GACG MDKN ‘Vitifolium’ [A. japonicum Thunb. ex J.A.Murr.] DCNA ‘Waka momiji’ [A. palmatum Thunb. ex J.A.Murr.] MDOF ‘Washi no o’ [A. palmatum Thunb. ex J.A.Murr] = ‘Dissectum Palmatifidum’ ‘Waterfall’ [A. palmatum Thunb. ex J.A.Murr.] SCCE wilsonii Rehd. DCNA ‘Yatsubusa’ [A. palmatum Thunb. ex J.A.Murr.] DCNA GACG MDPJ ‘Yayoigasa’ [A. japonicum Thunb. ex J.A.Murr.] DCNA ‘Yezo nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA GACG ‘Yukigumi’ [A. palmatum Thunb. ex J.A.Murr.] DCNA <x zoeschense Pax ‘Variegatum’ [A. rufinerve Sieb. & Zucc.] MDBG ‘Vitifolium’ [A. japonicum Thunb. ex J.A.Murr.] DCNA ‘Tsukushi gata’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Tsuri nishiki’ [A. palmatum Thunb. ex J.A.Murr.] DCNA ‘Ukon’ [A. palmatum Thunb. ex J.A.Murr.] = ‘Aoyagi’ ) ) nd NS AESCULUS ACOELORRHAPHE AESCULUS L. _ Horsechestnut, Buckeye HIPPOCASTANACEAE Buckeye Family SSG FETT Pe he eae Sh eed ‘Baumanii’ [A. hippocastanum L.] Double-flowered H. DCNA MDBG DCSE MDCP NCBE NCDU ‘Briotii’ [A. x carnea Hayne] [A. hippocastanum x A. pavia] DCNA DEWG MDPJ DCWR MDKN VACW < bushii Schneid. [A. glabra x A. pavia] MDBP californica (Spach) Nutt. California B. DCNA SCWI x carnea Hayne Red H. [A. hippocastanum < A. pavial] DECRIE MDBG VAMP DCSE MDCP x dupontii Sarg. [A. flava x (A. pavia < A. sylvatica)] DEEM DEWG flava Sol. Yellow B. DCCG MDHN NCTE VAKH DCNA MDJH TNFE VAMP DEEM MDNA TNSC VAWP MDBP NCBE VACP MDCP NCKH VACW flava Ait. = A. flava Sol. glabra Willd. Ohio B. ARHS DCWR MDJH TNFE DCNA DEWG MDLS VAMP DCWH MDHN SCWI x glaucescens Sarg. [A. flava x A. sylvatica] DEWG hippocastanum L. H. DECG DEWG MDMM VAPS DCNA MDBG VACW DCSE MDCP VAGW AESCULUS L. _ Horsechestnut, Buckeye HIPPOCASTANACEAE Buckeye Family SSG FETT Pe he eae Sh eed ‘Baumanii’ [A. hippocastanum L.] Double-flowered H. DCNA MDBG DCSE MDCP NCBE NCDU ‘Briotii’ [A. x carnea Hayne] [A. hippocastanum x A. pavia] DCNA DEWG MDPJ DCWR MDKN VACW < bushii Schneid. [A. glabra x A. pavia] MDBP californica (Spach) Nutt. California B. DCNA SCWI x carnea Hayne Red H. [A. hippocastanum < A. pavial] DECRIE MDBG VAMP DCSE MDCP x dupontii Sarg. [A. flava x (A. pavia < A. sylvatica)] DEEM DEWG flava Sol. Yellow B. ACER ACTINIDIACEAE Actinidia Family a Oe ee SE OT oe TC SES Vn Se SS SET] arguta (Sieb. & Zucc.) Planch. ex Miq. Tara Vine DCDO DCNA MDGD VATA ‘Chico’ [A. deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa] VATA chinensis Planch. = A. deliciosa var. deliciosa chinensis Planch. var. deliciosa (A.Chev.) A.Chev. = A. deliciosa var. deliciosa deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa Kiwi Fruit, Chinese Gooseberry GAAB NCDU x fairchildii Rehd. [A. arguta < A. chinensis] MDGD VATA kolomikta (Maxim. & Rupr.) Maxim. VAGS polygama (Sieb. & Zucc.) Maxim. Silver Vine DCNA purpurea Rehd. VATA ADINA Salisb. RUBIACEAE Madder Family Ce ee EE ae EET ee a See SS Se] rubella Hance Chinese Buttonbush ALAU SCCU LASL VACW VAHM 21 Pap I ran rahe att PN \ ACTINIDIA arguta (Sieb. & Zucc.) Planch. ex Miq. [illustrator Lillian Nicholson Meyer] Pap AESCULUS x hybrida DC. [A. flava x A. pavia] DCNA DEWG VAGW DCWH VAAL VAWP < marilandica Booth ex Kirchn. [A. glabra x A. flava] DCSE DEEM maxima Drake = A. flava Sol. ‘Memmingeri’ [A. hippocastanum L.] DCNA x mutabilis (Spach) Scheele [A. sylvatica < A. pavia] GACG GAEC octandra Marsh. = A. flava Sol. parviflora Walt. Bottlebrush Buckeye DCNA LARP MDKN TNNE DCWR MDAM MDLT TNUT GACG MDHN MDRT parviflora Walt. var. serotina Rehd. DCNA pavia L. Red Buckeye ALBG LAAL MDKN TNHT DEWG LALG NCTP TNMB FLUF MDBG SCBR TXMS GACG MDIH SCKG VACW GASM MDJH SCMG VAWR pavia L. var. flavescens (Sarg.) Correll TXLL x plantierensis Andre [A. hippocastanum < A. <carnea] DCNA DEWG sylvatica Bartram DCNA GAEC NCCA SCWI x woerlitzensis Koehne [A. flava x (A. sylvatica < A. pavia)] DEWG ALBIZIA ALBIZIA AGARISTA D.Don ex’G.Don ERICACEAE Heath Family populifolia (Lam.) D.Don ex Judd Pipe Plant, Pipe-stem Wood ALAU MDLT SCBR SCWI LAAL MDPJ SCCU VAGS LALG NCBE SCMP VAPH AGAVE L. Century Plant AGAVACEAE Agave Family americana L. FLUF ‘Marginata’ [A. americana L.] Variegated C. P. FLUF AILANTHUS Desf. SIMAROUBACEAE Quassia Family De ee ee \| altissima (Mill.) Swingle Tree-of-heaven DEEM MDHN MDSJ MDCP MDLT VABP glandulosa Desf. = A. altissima AKEBIA Decne. Akebia LARDIZABALACEAE Lardizabala Family quinata (Thunb. ex Houtt.) Decne. Chocolate Vine DCNA MDFM VASS MDBG NCDU ‘Shirobana’ [A. quinata (Thunb. ex Houtt.) Decne.] MDBG ALBIZIA Durazz. FABACEAE (Mimosoideae) Bean Family aI ee a Oe NTRS BL OR BREEN Ce julibrissin Durazz. ACER Silk Tree, Mimosa Tree DCNA MSHB NCTR FLUF NCDU SCBR MSBN NCOP TNTV AGARISTA D.Don ex’G.Don ERICACEAE Heath Family populifolia (Lam.) D.Don ex Judd Pipe Plant, Pipe-stem Wood ALAU MDLT SCBR SCWI LAAL MDPJ SCCU VAGS LALG NCBE SCMP VAPH AGAVE L. Century Plant AGAVACEAE Agave Family americana L. FLUF ‘Marginata’ [A. americana L.] Variegated C. P. FLUF AILANTHUS Desf. SIMAROUBACEAE Quassia Family De ee ee \| altissima (Mill.) Swingle Tree-of-heaven DEEM MDHN MDSJ MDCP MDLT VABP glandulosa Desf. = A. altissima AKEBIA Decne. Akebia LARDIZABALACEAE Lardizabala Family quinata (Thunb. ex Houtt.) Decne. Chocolate Vine DCNA MDFM VASS MDBG NCDU ‘Shirobana’ [A. quinata (Thunb. ex Houtt.) Decne.] MDBG ALBIZIA Durazz. FABACEAE (Mimosoideae) Bean Family aI ee a Oe NTRS BL OR BREEN Ce julibrissin Durazz. Silk Tree, Mimosa Tree DCNA MSHB NCTR FLUF NCDU SCBR MSBN NCOP TNTV AGARISTA D.Don ex’G.Don ERICACEAE Heath Family populifolia (Lam.) D.Don ex Judd Pipe Plant, Pipe-stem Wood ALAU MDLT SCBR SCWI LAAL MDPJ SCCU VAGS LALG NCBE SCMP VAPH AGAVE L. Century Plant AGAVACEAE Agave Family americana L. FLUF ‘Marginata’ [A. americana L.] Variegated C. P. FLUF AILANTHUS Desf. SIMAROUBACEAE Quassia Family De ee ee \| altissima (Mill.) Swingle Tree-of-heaven DEEM MDHN MDSJ MDCP MDLT VABP glandulosa Desf. = A. altissima AKEBIA Decne. Akebia LARDIZABALACEAE Lardizabala Family quinata (Thunb. ex Houtt.) Decne. Chocolate Vine DCNA MDFM VASS MDBG NCDU ‘Shirobana’ [A. quinata (Thunb. ex Houtt.) Decne.] MDBG ALBIZIA Durazz. FABACEAE (Mimosoideae) Bean Family aI ee a Oe NTRS BL OR BREEN Ce julibrissin Durazz. Silk Tree, Mimosa Tree DCNA MSHB NCTR AGARISTA D.Don ex’G.Don ERICACEAE Heath AGARISTA D.Don ex’G.Don ERICACEAE He 23 €y,) WW yi AKEBIA quinata, (Thunb. ex Houtt.) De [illustrator Susan M. Johnston] AMPELOPSIS ALBIZIA kalkora (Roxb.) Prain DCNA NCBE GAIS NCDU NCTR VATA VABF ALEURITES J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family (er ae re a Pe en ee eS SE \| fordii Hemsl. Tung-oil Tree ALBG FLUF MSMN FLLE GATS NCOP FLMG LAAL SCFW montana (Lour.) Wils. Mu-oil Tree FLUF ALLAMANDA L. APOCYNACEAE Dogbane Family (a OOS ATT DE Se De] cathartica L. ALBG ALNUS Mill. Alder BETULACEAE Birch Family formosana (Burkw.) Makino MDJC TXLL glutinosa (L.) Gaertn. European Alder, Black Alder DCCG DCNA VATA hirsuta (Spach) Rupr. var. sibirica (Spach) Schneid. DCNA japonica (Thunb.) Steud. MDGD maritima (Marsh.) Nutt. Seaside Alder VACW serrulata (Ait.) Willd. Smooth Alder ALAU NCDU TXLL LAHG SCPL ALOYSIA Juss. ACER Strawberry Tree FLUF LASL NCEL SCMP SCWI VANB ARCTOSTAPHYLOS Adans. ERICACEAE Heath Family BSS SO EE I OE Ry a eT \| uva-ursi (L.) Spreng. Bearberry, Kinnikinnick DCNA MDLT ARDISIA O.Swartz MYRSINACEAE Myrsine Family ‘Alba’ [A. crenata Sims] FLUF crenata Sims Coralberry ALBG FLUF GADG TXDC FLMG GACH GATS crispa (Thunb. ex J.A.Murr.) A.DC. Coral A. ALBG FLDG GACG LAAL ARDISIA ARDISIA ACER VERBENACEAE Verbena Family a Rd AMELANCHIER Medik. ROSACEAE Shadbush Rose Family alnifolia (Nutt.) Nutt. MDGD arborea (Michx.f.) Fern. Common S., Downy Serviceberry ALEH DCSG MDBG SCUC DCEL DEMC MDLT VACW DCNA GACG SCBR VAGW asiatica (Sieb. & Zucc.) Endl. DCNA MDKN canadensis (L.) Medik. DCNA SCBR VAGW MDWJ VACW laevis Wieg. Allegheny S., Smooth S. DCNA GAEC MDLM VASK oblongifolia T. & G. = A. canadensis ‘Rosea’ [A. laevis Wie¢g.] DCNA sanguinea (Pursh) DC. VAGS spicata (Lam.) K.Koch DCNA GAUG TNUT stolonifera Wieg. = A. spicata ‘Success’ MDOF AMORPHA iL. FABACEAE (Faboideae) Bean Family fruticosa L. - Indigo Bush DCNA TNDR VAGW MDNA VABF glabra Desf. ex Poir. Mountain Indigo ALTR AMPELOPSIS Michx. VITACEAE Grape Family AMELANCHIER Medik. ROSACEAE Shadbush Rose Family alnifolia (Nutt.) Nutt. MDGD arborea (Michx.f.) Fern. Common S., Downy Serviceberry ALEH DCSG MDBG SCUC DCEL DEMC MDLT VACW DCNA GACG SCBR VAGW asiatica (Sieb. & Zucc.) Endl. DCNA MDKN canadensis (L.) Medik. DCNA SCBR VAGW MDWJ VACW laevis Wieg. Allegheny S., Smooth S. DCNA GAEC MDLM VASK oblongifolia T. & G. = A. canadensis ‘Rosea’ [A. laevis Wie¢g.] DCNA sanguinea (Pursh) DC. VAGS spicata (Lam.) K.Koch DCNA GAUG TNUT stolonifera Wieg. = A. spicata ‘Success’ MDOF AMORPHA iL. FABACEAE (Faboideae) Bean Family fruticosa L. - Indigo Bush DCNA TNDR VAGW MDNA VABF glabra Desf. ex Poir. Mountain Indigo AMELANCHIER Medik. ROSACEAE Shadbush Rose Family alnifolia (Nutt.) Nutt. MDGD arborea (Michx.f.) Fern. Common S., Downy Serviceberry ALEH DCSG MDBG SCUC DCEL DEMC MDLT VACW DCNA GACG SCBR VAGW asiatica (Sieb. & Zucc.) Endl. DCNA MDKN canadensis (L.) Medik. DCNA SCBR VAGW MDWJ VACW laevis Wieg. Allegheny S., Smooth S. DCNA GAEC MDLM VASK oblongifolia T. & G. = A. canadensis ‘Rosea’ [A. laevis Wie¢g.] DCNA sanguinea (Pursh) DC. VAGS spicata (Lam.) K.Koch DCNA GAUG TNUT stolonifera Wieg. = A. spicata ‘Success’ MDOF AMORPHA iL. FABACEAE (Faboideae) Bean Family fruticosa L. - Indigo Bush DCNA TNDR VAGW MDNA VABF glabra Desf. ex Poir. Mountain Indigo ALTR 25 ARDISIA ARALIA L. ARALIACEAE Ginseng Family elata (Miq.) Seemann DCNA DEMC spinosa L. Devil’s Walking Stick ALAU MDLT SCBR VAWP ARAUCARIA Juss. ARAUCARIACEAE Araucaria Family EET EL a TT RE \| SES RE EE \| araucana (Mol.) K.Koch Monkey Puzzle MDJR bidwillii Hook. Bunya-Bunya Tree FLUF LAHS ARBUTUS L. ERICACEAE Heath Family Sa Se ee a es SO SEE SS Te a \| unedo L. AMPELOPSIS brevipedunculata (Maxim.) Traut. = A. glandulosa var. brevipedunculata glandulosa (Wall.) Momiy. var. brevipedunculata (Maxim.) Momiy. Porcelain-berry GAAB MDBG VAWP ‘Elegans’ [A. glandulosa var. brevipedunculata (Maxim.) Momiy.] DCNA brevipedunculata (Maxim.) Traut. = A. glandulosa var. brevipedunculata glandulosa (Wall.) Momiy. var. brevipedunculata (Maxim.) Momiy. Porcelain-berry GAAB MDBG VAWP ‘Elegans’ [A. glandulosa var. brevipedunculata (Maxim.) Momiy.] DCNA ARALIA L. ARALIACEAE Ginseng Family elata (Miq.) Seemann DCNA DEMC spinosa L. Devil’s Walking Stick ALAU MDLT SCBR VAWP ARAUCARIA Juss. ARAUCARIACEAE Araucaria Family EET EL a TT RE \| SES RE EE \| araucana (Mol.) K.Koch Monkey Puzzle MDJR bidwillii Hook. Bunya-Bunya Tree FLUF LAHS ARBUTUS L. ERICACEAE Heath Family Sa Se ee a es SO SEE SS Te a \| unedo L. Strawberry Tree FLUF LASL NCEL SCMP SCWI VANB ARCTOSTAPHYLOS Adans. ERICACEAE Heath Family BSS SO EE I OE Ry a eT \| uva-ursi (L.) Spreng. Bearberry, Kinnikinnick DCNA MDLT ARDISIA O.Swartz MYRSINACEAE Myrsine Family ‘Alba’ [A. crenata Sims] FLUF crenata Sims Coralberry ALBG FLUF GADG TXDC FLMG GACH GATS crispa (Thunb. ex J.A.Murr.) A.DC. Coral A. ALBG FLDG GACG LAAL ‘Elegans’ [A. glandulosa var. brevipedunculata (Maxim.) Momiy.] DCNA AMYRIS P.Br. RUTACEAE Citrus Family CRT OE Ee SE OE OE OO TE) texana (Buckl.) P.Wilson Texas Torchwood ADL, ANDROMEDA L. ERICACEAE Heath Family a PS ST RS ea a EL eS a \| ‘Nana’ [A. polifolia L.] MDLT polifolia L. NCFR ANISACANTHUS Nees ACANTHACEAE Acanthus Family thurberi (Torr.) A.Gr. TXCT ANISOSTICHUS Bur. = BIGNONIA ANTIGONON Endl. POLYGONACEAE Buckwheat Family leptopus Hook. & Arn. Mexican Creeper ALBG FLMG APHANANTHE Planch. ULMACEAE Elm Family PRD aS Sas MS RES EMS STO MEER aspera (Thunb. ex J.A.Murr.) Planch. GAME GANG GATC SCHS texana (Buckl.) P.Wilson Texas Torchwood ADL, ANISACANTHUS Nees ACANTHACEAE Acanthus Family we) o) AUCUBA ARDISIA ASCYRUM L. a HYPERICACEAE st. John’s-wort Family Ea a eT a ee hypericoides L. St. Andrew’s Cross NCEG SCWI stans Michx. = Hypericum stans ASIMINA Adans. ANNONACEAE Custard-apple Family QS NEI triloba (L.) Dunal Pawpaw DEWG NCCA VACM VAGW MDLT TXLL VACW ASTER L. ASTERACEAE Aster Family carolinianus Walt. Carolina Aster SCWI AUCUBA Thunb. CORNACEAE Dogwood Family chinensis Benth. DCNA ‘Goldieana’ [A. japonica Thunb.] VAPH Japonica Thunb. Japanese Aucuba ALEH GACG MDLT SCNS ARWS GAJS MDPJ VACW DCCG LAAL SCMG VAWR FLMG MDHN SCMP japonica Thunb. var. borealis Miyabe & ~ Kudo DCNA ‘Limbata’ [A. japonica Thunb.] DCNA ‘Longifolia’ [A. japonica Thunb.] DCNA VAPH ‘Meigetsw’ [A. AMPELOPSIS japonica Thunb.] MDMG ‘Nana’ [A. japonica Thunb.] japonica (Thunb.) Bl. Japanese A. ALFS LAAL MSMN FLDG LAHP SCLM GACG LAPA SCMG ARECASTRUM (Drude) Becc. romanzoffianum (Cham.) Becc. = Syagrus romanzoffianum ARISTOLOCHIA L. ARISTOLOCHIACEAE Aristolochia Family elegans Mast. Calico Dutchman’s Pipe TXPO ARONIA Medik. Chokeberry ROSACEAE Rose Family arbutifolia (L.) Ell. Red C. DCNA MDMM VADW VAPO GAEC TXSE VAFB MDLT VABP VAGW melanocarpa (Michx.) Fll. Black C. DCNA VACW VAPO MDGD VAGS melanocarpa (Michx.) Ell. var. elata Rehd. = A. melanocarpa prunifolia (Marsh.) Rehd. Purplefruit C. MDGJ ARTEMISIA L. ASTERACEAE Aster Family abrotanum L. Southernwood DCNA ARUNDINARIA Michx. POACEAE Grass Family ‘Variegata’ [A. pygmaea (Miq.) Mitf.] SCBR ARECASTRUM (Drude) Becc. ARECASTRUM (Drude) Becc. romanzoffianum (Cham.) Becc. = Syagrus romanzoffianum ARISTOLOCHIA L. ARISTOLOCHIACEAE Aristolochia Family elegans Mast. Calico Dutchman’s Pipe TXPO ARONIA Medik. Chokeberry ROSACEAE Rose Family arbutifolia (L.) Ell. Red C. DCNA MDMM VADW VAPO GAEC TXSE VAFB MDLT VABP VAGW melanocarpa (Michx.) Fll. Black C. DCNA VACW VAPO MDGD VAGS melanocarpa (Michx.) Ell. var. elata Rehd. = A. melanocarpa prunifolia (Marsh.) Rehd. Purplefruit C. MDGJ ARTEMISIA L. ASTERACEAE Aster Family abrotanum L. Southernwood DCNA ARUNDINARIA Michx. POACEAE Grass Family ‘Variegata’ [A. pygmaea (Miq.) Mitf.] SCBR romanzoffianum (Cham.) Becc. = Syagrus romanzoffianum AUCUBA Thunb. CORNACEAE Dogwood Family chinensis Benth. DCNA ‘Goldieana’ [A. japonica Thunb.] VAPH Japonica Thunb. Japanese Aucuba ALEH GACG MDLT SCNS ARWS GAJS MDPJ VACW DCCG LAAL SCMG VAWR FLMG MDHN SCMP japonica Thunb. var. borealis Miyabe & ~ Kudo DCNA ‘Limbata’ [A. japonica Thunb.] DCNA ‘Longifolia’ [A. japonica Thunb.] DCNA VAPH ‘Meigetsw’ [A. japonica Thunb.] MDMG ‘Nana’ [A. japonica Thunb.] MDTD SCMG 27 BERBERIS ‘Salicifolia’ [A. japonica Thunb.] DCNA ‘Variegata’ [A. japonica Thunb.] Gold-dust Shrub ALBG GAHC MDPJ SCFW ARPT GAJS MSMN TNUT DECE LALG NCOP VACW FLMG MDLT SCBR BACCHARIS L. Groundsel ASTERACEAE Aster Family ‘Dauphin Island’ [B. halimifolia L.] SCWI glomeruliflora Pers. Southern B. SCWI halimifolia L. Sea Myrtle, G. Tree GACG BAMBUSA_ Schreber Bamboo POACEAE Grass Family ‘Alphonse Karr’ [B. multiplex (Lour.) Raeusch.] GAIS ‘Fernleaf’ [B. multiplex (Lour.) Raeusch.] FLDG SCOY TXVO glaucescens (Willd.) Sieb. ex Munro = B. multiplex multiplex (Lour.) Raeusch. Hedge Bamboo FLDG FLMG LAAL NCOP ‘Silverstripe’ [B. multiplex (Lour.) Raeusch.] SCCY tuldoides Munro FLUF ‘Variegata’ [B. multiplex (Lour.) Raeusch.] LAAL BAUHINIA L. FABACEAE (Caesalpinioideae) Bean Family EE ea Cae ee a Sr oS Te aE EST) congesta (Britton & Rose) Lundell = B. lunarioides divaricata L. LASL lunarioides A.Gr. AMPELOPSIS ex S.Wats. Texas B. TXMS TXSE saigonensis Pierre ex Gagnep. = B. yunnanensts yunnanensis Franch. TXPS BEFARIA Mutis ex L. ERICACEAE Heath Family racemosa Vent. Tarflower ALAU BELOPERONE Nees ACANTHACEAE Acanthus Family Se Se Se ee ee ee ee a ae eee guttata Brandegee = Justicia brandegeana BERBERIS L. BERBERIDACEAE Barberry Barberry Family ‘Atropurpurea’ [B. koreana Palib.] Redleaf Korean B. VAGS ‘Atropurpurea’ [B. thunbergii DC.] Redleaf Japanese B. ALBH MDBG TNDR FLMN NCBE VAPH FLRH SCCU VAWR ‘Atropurpurea Nana’ [B. thunbergii DC.] Dwarf Redleaf Japanese B. ‘Salicifolia’ [A. japonica Thunb.] DCNA ‘Variegata’ [A. japonica Thunb.] Gold-dust Shrub ALBG GAHC MDPJ SCFW ARPT GAJS MSMN TNUT DECE LALG NCOP VACW FLMG MDLT SCBR BACCHARIS L. Groundsel ASTERACEAE Aster Family ‘Dauphin Island’ [B. halimifolia L.] SCWI glomeruliflora Pers. Southern B. SCWI halimifolia L. Sea Myrtle, G. Tree GACG BAMBUSA_ Schreber Bamboo POACEAE Grass Family ‘Alphonse Karr’ [B. multiplex (Lour.) Raeusch.] GAIS ‘Fernleaf’ [B. multiplex (Lour.) Raeusch.] FLDG SCOY TXVO glaucescens (Willd.) Sieb. ex Munro = B. multiplex multiplex (Lour.) Raeusch. Hedge Bamboo FLDG FLMG LAAL NCOP ‘Silverstripe’ [B. multiplex (Lour.) Raeusch.] SCCY tuldoides Munro FLUF ‘Variegata’ [B. multiplex (Lour.) Raeusch.] LAAL BAUHINIA L. FABACEAE (Caesalpinioideae) Bean Family EE ea Cae ee a Sr oS Te aE EST) congesta (Britton & Rose) Lundell = B. lunarioides divaricata L. LASL lunarioides A.Gr. ex S.Wats. Texas B. TXMS TXSE saigonensis Pierre ex Gagnep. = B. yunnanensts yunnanensis Franch. TXPS BEFARIA Mutis ex L. ERICACEAE Heath Family racemosa Vent. Tarflower ALAU BELOPERONE Nees ACANTHACEAE Acanthus Family Se Se Se ee ee ee ee a ae eee guttata Brandegee = Justicia brandegeana BERBERIS L. BERBERIDACEAE Barberry Barberry Family ‘Atropurpurea’ [B. koreana Palib.] Redleaf Korean B. VAGS ‘Atropurpurea’ [B. thunbergii DC.] Redleaf Japanese B. ALBH MDBG TNDR FLMN NCBE VAPH FLRH SCCU VAWR ‘Atropurpurea Nana’ [B. thunbergii DC.] Dwarf Redleaf Japanese B. NCBE VACW VAPH ‘Aurea’ [B. thunbergii DC.] Yellowleaf Japanese B. MDBG TNTV VAGS ‘Byers’ [B. julianae Schneid.] we) CO BERBERIS ‘Chenault’ [B. < hybrido-gagnepainii Suring.] [B. gagnepainii < B. verruculosa] VAWR x chenaultii Ahrendt = B. x hybrido-gagnepainii ‘Chenault’ ‘Crimson Pygmy’ [B. thunbergii DC.] DCNA MDBG TNTV GACG TNSN VAGS hookeri Lem. Hooker B. FLUF hookeri Lem. var. viridis Schneid. Hooker B. SCCU julianae Schneid. Wintergreen B. ALAU GACG MDNA SCUC ALBH GAHC NCBE VACW ALIT GAJY NCDU VAMP ALTD GAUG NCEG VATA FLMG LAHG SCCU VAWR ‘Kobold’ [B. AMPELOPSIS thunbergii DC.] GACG x mentorensis H.Schultz & Horvath ex L.M.Ames Mentor B. [B. julianae x B. thunbergiil (Plant Patent No. 99) ALAU ALIT SCCU ALBH NCBE VATA mouillacana Schneid. DCNA ‘Parkjuweel’ [B. < media Grootend.] [B. hybrido-gagnepainii ‘Chenault’ x B. thunbergii\| DCNA sargentiana Schneid. Sargent B. ALAU GABS soulieana Schneid. Soulie B. GAJS ‘Sparkle’ [B. thunbergii DC.] BETULA thunbergii DC. Japanese B. DEWG MDLT SCCU VAWL FLMG MDNA VAGG _= triacanthophora Hort. not Fedde = B. x wisleyensis verruculosa Hemsl. & Wils. Warty B. DCNA TNTV VAMP VATA ‘William Penn’ [B. x gladwynensis Li\| [B. julianae < B. verruculosa] MDBG VATA X wisleyensis Ahrendt (Parentage unknown; sometimes labeled triacanthophora) MDBG NCDU SCCU VAWR MDPJ NCWC VAMP BERCHEMIA Necker ex DC. RHAMNACEAE Buckthorn Family ee TE EEN Pon Te ee] ‘Issai’ [B. racemosa Sieb. & Zucc.] MDBG racemosa Sieb. & Zucc. Japanese Supplejack TNTV scandens (J.Hill) K.Koch Alabama Supplejack SCBR TNSN BETULA L. Birch BETULACEAE Birch Family ES ES A EE 1S SS ES I eee] alleghaniensis Britton Yellow B. VACW costata Traut. MDBG VATA ‘Dalecarlica’ [B. pendula Roth] MDNA TNSN VAJL davurica Pall. DCNA fontinalis Sarg. = B. occidentalis jaquemontii Spach 29 BETULA lenta L. Sweet B. DCNA lutea Michx.f. = B. alleghaniensis maximowicziana Regel VATA nigra L. River B. DCDO NCCA SCBR VACW DCWR NCDU SCCU VAMP FLUF NCTP TXDM occidentalis Hook. DCNA papyrifera Marsh. Paper B. DCNA MDGD NCBE MDBG MDLM papyrifera x B. maximowicziana VAGM pendula Roth European White B. ALBH MDLT INCH VATA DCNA NCDU TNFN VAWR DCWH SECU TNSM pendula x B. nigra VATA platyphylla Sukachev var. japonica (Miq.) Hara Japanese White B. DCNA SCCU populifolia Marsh. Gray B. DCCG MDBG NCWC DCNA MDJS TNBO potaninii Batal. DCNA pubescens Ehrh. Hairy B. DCNA BRUGMANSIA BRUGMANSIA ‘Youngii’ [B. pendula Roth] MDBG BIGNONIA L. BIGNONIACEAE Bignonia Family capreolata L. Cross Vine FLMG GAJI SCWR FLUF MDFM VACW unguis-cati L. = Macfadyena unguis-cati BIOTA (D.Don) Endl. = PLATYCLADUS BREYNIA J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family disticha J.R. & J.G.Forst. Snowbush LAHG nivosa (W.G.Sm.) Small = B. disticha BROUSSONETIA ULHer. ex Vent. MORACEAE Mulberry Family papyrifera (L.) Vent. Paper Mulberry DCNA MSRN TNSG VABF MDJH SCUC TNTV VACW ‘Variegata’ [B. papyrifera (L.) Vent.] SCWI BRUCKENTHALIA Reichenb. ERICACEAE Heath Family spiculifolia (Salisb.) Reichenb. Spike Heath DEWG BRUGMANSIA Pers. SOLANACEAE Nightshade Family _—— —————————— EE suaveolens (Humboldt& Bonpland ex Willd.) Bercht.& J.Presl Angel's Trumpet BRUGMANSIA ‘Youngii’ [B. AMPELOPSIS pendula Roth] MDBG BIGNONIA L. BIGNONIACEAE Bignonia Family capreolata L. Cross Vine FLMG GAJI SCWR FLUF MDFM VACW unguis-cati L. = Macfadyena unguis-cati BIOTA (D.Don) Endl. = PLATYCLADUS BREYNIA J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family disticha J.R. & J.G.Forst. Snowbush LAHG nivosa (W.G.Sm.) Small = B. disticha BROUSSONETIA ULHer. ex Vent. MORACEAE Mulberry Family papyrifera (L.) Vent. Paper Mulberry DCNA MSRN TNSG VABF MDJH SCUC TNTV VACW ‘Variegata’ [B. papyrifera (L.) Vent.] SCWI BRUCKENTHALIA Reichenb. ERICACEAE Heath Family spiculifolia (Salisb.) Reichenb. Spike Heath DEWG BRUGMANSIA Pers. SOLANACEAE Nightshade Family ‘Youngii’ [B. pendula Roth] MDBG BIGNONIA L. BIGNONIACEAE Bignonia Family capreolata L. Cross Vine FLMG GAJI SCWR FLUF MDFM VACW unguis-cati L. = Macfadyena unguis-cati BIOTA (D.Don) Endl. = PLATYCLADUS BREYNIA J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family disticha J.R. & J.G.Forst. Snowbush LAHG nivosa (W.G.Sm.) Small = B. disticha BROUSSONETIA ULHer. ex Vent. MORACEAE Mulberry Family papyrifera (L.) Vent. Paper Mulberry DCNA MSRN TNSG VABF MDJH SCUC TNTV VACW ‘Variegata’ [B. papyrifera (L.) Vent.] SCWI BRUCKENTHALIA Reichenb. ERICACEAE Heath Family spiculifolia (Salisb.) Reichenb. Spike Heath DEWG BRUGMANSIA Pers. SOLANACEAE Nightshade Family _—— —————————— EE suaveolens (Humboldt& Bonpland ex Willd.) ‘Youngii’ [B. pendula Roth] MDBG BIGNONIA L. BIGNONIACEAE Bignonia Family capreolata L. Cross Vine FLMG GAJI SCWR FLUF MDFM VACW unguis-cati L. = Macfadyena unguis-cati BIOTA (D.Don) Endl. = PLATYCLADUS BREYNIA J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family disticha J.R. & J.G.Forst. Snowbush LAHG nivosa (W.G.Sm.) Small = B. disticha BROUSSONETIA ULHer. ex Vent. MORACEAE Mulberry Family papyrifera (L.) Vent. Paper Mulberry DCNA MSRN TNSG VABF MDJH SCUC TNTV VACW ‘Variegata’ [B. papyrifera (L.) Vent.] SCWI BRUCKENTHALIA Reichenb. ERICACEAE Heath Family spiculifolia (Salisb.) Reichenb. Spike Heath DEWG BRUGMANSIA Pers. SOLANACEAE Nightshade Family ‘Youngii’ [B. pendula Roth] MDBG = PLATYCLADUS verrucosa Ehrh. = B. pendula verrucosa Ehrh. = B. pendula BIGNONIA capreolata L. [illustrator Lillian Nicholson Meyer] BIGNONIA capreolata L. [illustrator Lillian Nicholson Meyer] 31 X weyeriana Weyer [B. davidii x B. globosa] SCJF <x whiteana R.J.Moore [B. alternifolia < B. asiatica] DCNA BUMELIA O.Swartz SAPOTACEAE Sapodilla Family (EES SE LE EL SE, A MRED SO I TET \| lacuum Small = B. tenax lanuginosa (Michx.) Pers. Chittamwood SCMG TXMS lycioides (L.) Pers. Southern Buckthorn SCBR VACW tenax (L.) Willd. Ironwood SCWI BUTIA (Becc.) Becc. ARECACEAE Palm Family ES a a CPR SRS ES SFE ET ES] capitata (Mart.) Becc. Jelly Palm FLMG FLUF LAAL BUXUS L. Box BUXACEAE Boxwood Family ‘Abilene’ [B. AMPELOPSIS sempervirens L.] DCNA ‘Agram’ [B. sempervirens L.] DCNA ‘Albo-marginata’ [B. sempervirens L.] = ‘Marginata’ ‘Angustifolia’ [B. sempervirens L.] Willow-leaved B. DCNA MSMN TNLW FLDG NCCA ‘Arborescens’ [B. sempervirens L.] Tree B. DCNA DEMC TNLW VACW ‘Arborescens Decussata’ [B. sempervirens BRUNFELSIA BRUNFELSIA BRUNFELSIA L. SOLANACEAE Nightshade Family australis Benth. Yesterday-today-tomorrow FLCG GATS TXAV FLUF LALG TXLL GAJI LASL TXMK BUCKLEYA Torr. SANTALACEAE Sandalwood Family distichophylla (Nutt.) Torr. Piratebush GAEC NCBE BUDDLEJA L. Butterfly Bush BUDDLEJACEAE Buddleja Family ‘Alba’ [B. davidii Franch.] MDWP alternifolia Maxim. Fountain B. DEWG crispa Benth. var. farreri (Balf.f. & W.W.Sm.)Hand.-Mazz. DCNA davidii Franch. Orange-eye B. MDBG NCBE SCCU MDWP NCOP fallowiana Balf.f. & W.W.Sm. GATS farreri Balf.f. & W.W.Sm. DCNA japonica Hemsl. DCNA lindleyana Fort. ex Lindl. GATN LARP MSHB LAAL MSEN ‘Sungold’ [B. x weyeriana Weyer] [B. davidii x B. globosa] VAGS venenifera Makino X weyeriana Weyer [B. davidii x B. globosa] SCJF <x whiteana R.J.Moore [B. alternifolia < B. asiatica] DCNA BRUNFELSIA L. SOLANACEAE Nightshade Family australis Benth. Yesterday-today-tomorrow FLCG GATS TXAV FLUF LALG TXLL GAJI LASL TXMK BUCKLEYA Torr. SANTALACEAE Sandalwood Family distichophylla (Nutt.) Torr. Piratebush GAEC NCBE BUDDLEJA L. Butterfly Bush BUDDLEJACEAE Buddleja Family ‘Alba’ [B. davidii Franch.] MDWP alternifolia Maxim. Fountain B. DEWG crispa Benth. var. farreri (Balf.f. & W.W.Sm.)Hand.-Mazz. DCNA davidii Franch. Orange-eye B. MDBG NCBE SCCU MDWP NCOP fallowiana Balf.f. & W.W.Sm. GATS farreri Balf.f. & W.W.Sm. DCNA japonica Hemsl. DCNA lindleyana Fort. ex Lindl. GATN LARP MSHB LAAL MSEN ‘Sungold’ [B. x weyeriana Weyer] 32 BRUNFELSIA australis Benth. [Illustrator Lillian Nicholson Meyer] 33 BUXUS BUXUS BUXUS BUXUS * ‘National’ [B. microphylla Sieb. & Zucc. var. japonica (Muell.-Args:) Rehd. & Wils.] DCNA MDBG ‘Nish’ [B. sempervirens L.] DCNA ‘Northern Find’ [B. sempervirens L.] DCNA ‘Northern New York’ [B. sempervirens L.] DCNA ‘Northland’ [B. sempervirens L.] DCNA ‘Pendula’ [B. sempervirens L.] Weeping B. DCCG DCNA ‘Pincushion’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] DCNA ‘Ponteyi’ [B. sempervirens L.] DCNA ‘Prostrata’ [B. sempervirens L.] DCNA ‘Pyramidalis’ [B. sempervirens L.] DCNA TNTV ‘Richard’ [B. harlandii Hance] ARWS DCNA LASL ‘Rotundifolia’ [B. sempervirens L.] DCNA LARP TNLW VAHM ‘Ste. Genevieve’ [B. sempervirens L.] DCNA ‘Salicifolia’ [B. sempervirens L.] DCNA ‘Salicifolia Elata’ [B. sempervirens L.] DCNA sempervirens L. Common B. ALBG GABG NCDU SCMP ARRB GACG NCTP TNCT DCCG GAHC SCBR TNRU DCNA GAKN SCCU TNUT DCWH GAOH SCFW VAGS DEEM GATN SCJM VAGW FLMG MDHN SCMG VAWR sinica (Rehd. & Wils.) M.Cheng var. ‘Joy’ [B. sempervirens L.] DCNA ‘Kingsville Dwarf [B. microphylla Sieb. & Zucc. \| = ‘Compacta’ ‘Latifolia Macrophylla’ [B. sempervirens L.] DCNA ‘Latifolia Nova’ [B. sempervirens L.] DCNA ‘Lynnhaven’ [B. sempervirens L.] DCNA ‘Macrophylla’ [B. sempervirens L.] DCNA ‘Marginata’ [B. sempervirens L.] DCNA MDHN ‘Memorial’ [B. sempervirens L.] DCNA microphylla Sieb. & Zucc. Japanese B. ARHS DEEM MDJS DCCG DEWG TNSN DCNA GAEC TNUT microphylla Sieb. & Zucc. var. insularis Nakai = B. sinica var. insularis microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. Japanese Box ALBG FLCG LASL SCMP ALTD FLMG SECU TNCT ARHS FLUF SCEA DCCG GATS SCJM DCNA LAAL SCMG microphylla Sieb. & Zucc. var. koreana Nakai ex Rehd. = B. sinica var. insularis * ‘Morris Dwarf [B. microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils.] DCNA * ‘Morris Midget’ [B. microphylla Sieb. & BUXUS BUXUS ‘Argenteo-variegata’ [B. sempervirens L.] DCNA LARP VAGS ‘Aristocrat’ [B. sempervirens L.] DCNA ‘Aurea Maculata’ [B. sempervirens L.] = ‘Aureo-variegata’ ‘Aurea Marginata’ [B. sempervirens L.] = ‘Marginata’ ‘Aurea Pendula’ [B. sempervirens L.] DCNA ‘Aureo-variegata’ [B. sempervirens L.] DCNA balearica Lam. Balearic B. DCNA SCMG ‘Belleville’ [B. sempervirens L.] DCNA ‘Bullata’ [B. sempervirens L.] ARRB DCNA GAHC VAAC ‘Butterworth’ [B. sempervirens L.] DCNA ‘Compacta’ [B. microphylla Sieb. & Zucc.] ALHC DCNA NCFR VATA ALTD MDBG VACW VATW ‘Curly Locks’ [B. microphylla Sieb. & Zucc.] DCNA ‘Denmark’ [B. sempervirens L.] DCNA ‘Edgar Anderson’ [B. sempervirens L.] DCNA ‘Elegantissima’ [B. sempervirens L.] DCNA VAGS ‘Fastigiata’ [B. sempervirens L.] Columnar B. DCNA VABF ‘Glauca’ [B. sempervirens L.] DCNA ‘Grace Hendrick Phillips’ [B. microphylla BUXUS ‘Green Gem’ (? Hybrid) DCNA ‘Green Mountain’ (? Hybrid) DCNA ‘Green Pillow’ [B. microphylla Sieb. & Zucc.] DCNA ‘Green Velvet’ (°° Hybrid) DCNA ‘Handsworthiensis’ [B. sempervirens L.] Handsworth B. DCNA TNSN TNTV ‘Hardwickensis’ [B. sempervirens L.] DCNA harlandii Hance Harland B. ALBG FLMG MSEN VAGS ALTD GADG MSEP VATA ARHS LALG NCTP VAWM ARRB LATU SCFW DCNA MDKN SCJM ‘Heinrich Bruns’ [B. sempervirens L.] DCNA ‘Helen Whiting’ [B. microphylla Sieb. & Zucc.] DCNA ‘Henry Hohman’ [B. microphylla Sieb. & Zucc.] DCNA ‘Henry Shaw’ [B. sempervirens L.] DCNA himalayensis Hort. = B. wallichiana ‘Inglis’ [B. sempervirens L.] DCNA ‘Ipek’ [B. sempervirens L.] DCNA ‘Joe Gable’ [B. sempervirens L.] DCNA TNSN TNTV ‘John Baldwin’ [B. microphylla Sieb. & Zucc.] ‘Joy’ [B. sempervirens L.] DCNA DCNA ‘Kingsville Dwarf [B. microphylla Sieb. & Zucc. \| = ‘Compacta’ ‘Latifolia Macrophylla’ [B. sempervirens L.] DCNA ‘Latifolia Nova’ [B. sempervirens L.] DCNA ‘Lynnhaven’ [B. sempervirens L.] DCNA ‘Macrophylla’ [B. sempervirens L.] DCNA ‘Marginata’ [B. sempervirens L.] DCNA MDHN ‘Memorial’ [B. sempervirens L.] DCNA microphylla Sieb. & Zucc. Japanese B. ARHS DEEM MDJS DCCG DEWG TNSN DCNA GAEC TNUT microphylla Sieb. & Zucc. var. insularis Nakai = B. sinica var. insularis microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. Japanese Box ALBG FLCG LASL SCMP ALTD FLMG SECU TNCT ARHS FLUF SCEA DCCG GATS SCJM DCNA LAAL SCMG microphylla Sieb. & Zucc. var. koreana Nakai ex Rehd. ‘Kingsville Dwarf [B. microphylla Sieb. & Zucc. \| = ‘Compacta’ ‘Nish’ [B. sempervirens L.] DCNA ‘Latifolia Macrophylla’ [B. sempervirens L.] DCNA = B. sinica var. insularis microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. Japanese Box ALBG FLCG LASL SCMP ALTD FLMG SECU TNCT ARHS FLUF SCEA DCCG GATS SCJM DCNA LAAL SCMG microphylla Sieb. & Zucc. var. koreana Nakai ex Rehd. = B. sinica var. insularis microphylla Sieb. & Zucc. var. koreana Nakai ex Rehd. = B. sinica var. insularis = B. sinica var. insularis 35 BUXUS stenophylla Hance DCNA ‘Suffruticosa’ [B. sempervirens L.] Dwarf English B. DCNA GAKN NCSM TNCT DCWH GAOH NCTE VACW FLCM MDJS SCBR VAGU FLDG MSEN SCCU VATA GAHC NCDU SCMP VAWR ‘Sunnyside’ [B. microphylla Sieb. & Zucc.] DCNA ‘Tall Boy’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] DCNA ‘Tide Hill’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] VATA ‘Vardar Valley’ [B. sempervirens L.] Jugoslavian B. . DCNA GACG TNPW ‘Varifolia’ [B. sempervirens L.] DCNA wallichiana Baill. DCNA ‘Welleri’ [B. sempervirens L.] DCNA GACG ‘Winter Beauty’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] DCNA ‘Wintergreen’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] SCCU CAESALPINIA L. FABACEAE (Caesalpinioideae) Bean Family LS A a) gilliesii Hook. SCWI CALLIANDRA Benth. BUXUS stenophylla Hance DCNA ‘Suffruticosa’ [B. sempervirens L.] Dwarf English B. DCNA GAKN NCSM TNCT DCWH GAOH NCTE VACW FLCM MDJS SCBR VAGU FLDG MSEN SCCU VATA GAHC NCDU SCMP VAWR ‘Sunnyside’ [B. microphylla Sieb. & Zucc.] DCNA ‘Tall Boy’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] DCNA ‘Tide Hill’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] VATA ‘Vardar Valley’ [B. sempervirens L.] Jugoslavian B. . ‘Joy’ [B. sempervirens L.] DCNA DCNA GACG TNPW ‘Varifolia’ [B. sempervirens L.] DCNA wallichiana Baill. DCNA ‘Welleri’ [B. sempervirens L.] DCNA GACG ‘Winter Beauty’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] DCNA ‘Wintergreen’ [B. sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng] SCCU CAESALPINIA L. FABACEAE (Caesalpinioideae) Bean Family LS A a) gilliesii Hook. SCWI CALLIANDRA Benth. FABACEAE (Mimosoideae) Bean Family [ Ree RLS Rohe aE SMS AAC. AON AR ee LOS OE ee tweediei Benth. Mexican Flamebush GACH LAAL BUXUS CALLISTEMON CALLICARPA L. Beautyberry VERBENACEAE Verbena Family americana L. American B. ALAU GACG NCDU VAGS ALBG LAAL SCBR FLUF NCAG TXRS bodinieri Lev. Bodinier B. MDPJ bodinieri Lev. var. giraldii Rehd. DCBG DCNA SCCU dichotoma (Lour.) K.Koch Purple B. MDBG SCCU VACW japonica Thunb. ex J.A.Murr. Japanese B. DCNA GAEH MDKN SCCU SCWI TNUT ‘Lactea’ [C. americana L.] White-fruited B. GACG SCBR SCWI TXRS ‘Leucocarpa’ [C. dichotoma (Lour.) K.Koch] DCNA macrophylla Vahl FLUF tosaensis Makino VATA CALLISTEMON R.Br. Bottlebrush MYRTACEAE Myrtle Family citrinus (Curtis) Skeels ALFS FLUF LASL FLCS GASM NCOP linearis (Schrad. & J.C.Wendl.) DC. Narrowleaf B. FLUF LASL rigidus R.Br. Stiff B. FLUF rugulosus Miq. SCWI salignus (Sm.) DC. CALLICARPA L. Beautyberry VERBENACEAE Verbena Family americana L. American B. ALAU GACG NCDU VAGS ALBG LAAL SCBR FLUF NCAG TXRS bodinieri Lev. Bodinier B. MDPJ bodinieri Lev. var. giraldii Rehd. DCBG DCNA SCCU dichotoma (Lour.) K.Koch Purple B. MDBG SCCU VACW japonica Thunb. ex J.A.Murr. Japanese B. DCNA GAEH MDKN SCCU SCWI TNUT ‘Lactea’ [C. americana L.] White-fruited B. GACG SCBR SCWI TXRS ‘Leucocarpa’ [C. dichotoma (Lour.) K.Koch] DCNA macrophylla Vahl FLUF tosaensis Makino VATA CALLISTEMON R.Br. Bottlebrush MYRTACEAE Myrtle Family citrinus (Curtis) Skeels ALFS FLUF LASL FLCS GASM NCOP linearis (Schrad. & J.C.Wendl.) DC. Narrowleaf B. FLUF LASL rigidus R.Br. Stiff B. FLUF rugulosus Miq. SCWI CALLICARPA L. Beautyberry VERBENACEAE Verbena Family americana L. American B. ALAU GACG NCDU VAGS ALBG LAAL SCBR FLUF NCAG TXRS bodinieri Lev. Bodinier B. MDPJ bodinieri Lev. var. giraldii Rehd. DCBG DCNA SCCU dichotoma (Lour.) K.Koch Purple B. MDBG SCCU VACW japonica Thunb. ex J.A.Murr. Japanese B. DCNA GAEH MDKN SCCU SCWI TNUT ‘Lactea’ [C. americana L.] White-fruited B. GACG SCBR SCWI TXRS ‘Leucocarpa’ [C. dichotoma (Lour.) K.Koch] DCNA macrophylla Vahl FLUF tosaensis Makino VATA CALLISTEMON R.Br. Bottlebrush MYRTACEAE Myrtle Family citrinus (Curtis) Skeels ALFS FLUF LASL FLCS GASM NCOP linearis (Schrad. & J.C.Wendl.) DC. Narrowleaf B. FLUF LASL rigidus R.Br. ‘Joy’ [B. sempervirens L.] DCNA Stiff B. FLUF 36 CALLISTEMON viminalis (Sol. ex Gaertn.) G.Don FLMG FLUF GACG CALLITRIS Vent. CUPRESSACEAE Cypress Family columellaris F.Muell. FLUF CALLUNA Salisb. Heather ERICACEAE Heath Family ‘August Beauty’ [C. vulgaris (L.) Hull] MDLT ‘Aureafolia’ [C. vulgaris (L.) Hull] MDLT ‘H. E. Beale’ [C. vulgaris (L.) Hull] MDLT ‘Juno’ [C. vulgaris (L.) Hull] MDLT ‘Plena’ [C. vulgaris (L.) Hull] MDLT ‘Silver Queen’ [C. vulgaris (L.) Hull] MDLT ‘Tib’ [C. vulgaris (L.) Hull] MDLT vulgaris (L.) Hull Scotch H. DCNA CALOCEDRUS Kurz CUPRESSACEAE Cypress Family RR cc a a i a ge Ee Ee decurrens (Torr.) Florin Incense Cedar ALFN GAMP NCCA TNUT DCPC GAWJ NCTE VAMP FLUF MDBG SCHS GAHC MDJH TNPC CALLISTEMON viminalis (Sol. ex Gaertn.) G.Don FLMG FLUF GACG CAMELLIA CALLISTEMON viminalis (Sol. ex Gaertn.) G.Don FLMG FLUF GACG CALLITRIS Vent. CUPRESSACEAE Cypress Family columellaris F.Muell. FLUF CALLUNA Salisb. Heather ERICACEAE Heath Family ‘August Beauty’ [C. vulgaris (L.) Hull] MDLT ‘Aureafolia’ [C. vulgaris (L.) Hull] MDLT ‘H. E. Beale’ [C. vulgaris (L.) Hull] MDLT ‘Juno’ [C. vulgaris (L.) Hull] MDLT ‘Plena’ [C. vulgaris (L.) Hull] MDLT ‘Silver Queen’ [C. vulgaris (L.) Hull] MDLT ‘Tib’ [C. vulgaris (L.) Hull] MDLT vulgaris (L.) Hull Scotch H. DCNA CALOCEDRUS Kurz CUPRESSACEAE Cypress Family CAMELLIA floridus L. ~ Sweetshrub, Carolina Allspice ALAU GAVA NCBE TNDG ALBH LASL NCCA VACW DEEM MDBM NCTP VADW DEWG MDJS SCCU VAGW GACG MDLA SCDB VANB GAEC MDNA SCHW VAWP GAFF MDWP SCMP GAUG MSEP SCWC floridus L. var. laevigatus (Willd.) T. & G. = C. floridus ‘Margarita’ [C. floridus L.] GAEC VAPH CAMELLIA LL. THEACEAE Tea Family * ‘Ack-Scent’ [C. japonica ‘Kramer’s Supreme’ < C. ‘Fragrant Pink Improved’] DCNA * ‘Ack-Scent Pink’ [C. japonica ‘Fragrant Star’ x C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Red’ [C. japonica ‘Arabella’ x C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Sno’ [C. japonica ‘Frost Queen’ X C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Spice’ [C. japonica ‘Fragrant Star’ < C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Star’ [C. japonica ‘Fragrant Star’ X C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent White’ [C. japonica ‘Frost Queen’ x C. ‘Fragrant Pink’] DCNA ‘Alice Morrison’ [C. japonica L.] ALBG ‘Anemoniflora’ [C. japonica L.] floridus L. ~ Sweetshrub, Carolina Allspice ALAU GAVA NCBE TNDG ALBH LASL NCCA VACW DEEM MDBM NCTP VADW DEWG MDJS SCCU VAGW GACG MDLA SCDB VANB GAEC MDNA SCHW VAWP GAFF MDWP SCMP GAUG MSEP SCWC floridus L. var. laevigatus (Willd.) T. & G. = C. ‘Joy’ [B. sempervirens L.] DCNA floridus ‘Margarita’ [C. floridus L.] GAEC VAPH CAMELLIA LL. THEACEAE Tea Family * ‘Ack-Scent’ [C. japonica ‘Kramer’s Supreme’ < C. ‘Fragrant Pink Improved’] DCNA * ‘Ack-Scent Pink’ [C. japonica ‘Fragrant Star’ x C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Red’ [C. japonica ‘Arabella’ x C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Sno’ [C. japonica ‘Frost Queen’ X C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Spice’ [C. japonica ‘Fragrant Star’ < C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Star’ [C. japonica ‘Fragrant Star’ X C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent White’ [C. japonica ‘Frost Queen’ x C. ‘Fragrant Pink’] DCNA ‘Alice Morrison’ [C. japonica L.] ALBG ‘Anemoniflora’ [C. japonica L.] Warratah C. SCMP ‘Annette’ [C. sasanqua Thunb. ex J.A.Murr.] DCNA assimilis Champ. ex Benth. MDGD * ‘Ack-Scent Red’ [C. japonica ‘Arabella’ x C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Sno’ [C. japonica ‘Frost Queen’ X C. ‘Fragrant Pink’] DCNA * ‘Ack-Scent Spice’ [C. japonica ‘Fragrant Star’ < C. ‘Fragrant Pink’] DCNA CALOCEDRUS Kurz CUPRESSACEAE Cypress Family RR cc a a i a ge Ee Ee decurrens (Torr.) Florin Incense Cedar ALFN GAMP NCCA TNUT DCPC GAWJ NCTE VAMP FLUF MDBG SCHS GAHC MDJH TNPC GAHS MDJS TNTV CALYCANTHUS L. CALYCANTHACEAE - Calycanthus Family ‘Athens’ [C. floridus L.] GACG GAMD GAPG fertilis Walt. = C. floridus CALOCEDRUS Kurz CUPRESSACEAE Cypress Family RR cc a a i a ge Ee Ee decurrens (Torr.) Florin Incense Cedar ALFN GAMP NCCA TNUT DCPC GAWJ NCTE VAMP FLUF MDBG SCHS GAHC MDJH TNPC GAHS MDJS TNTV CALYCANTHUS L. CALYCANTHACEAE - Calycanthus Family ‘Athens’ [C. floridus L.] GACG GAMD GAPG fertilis Walt. = C. floridus * ‘Ack-Scent Star’ [C. japonica ‘Fragrant Star’ X C. ‘Fragrant Pink’] DCNA 37 CALYCANTAUS floridus L. [illustrator Peggy K. Duke] CAMELLIA ‘Aunt Jetty’ [C. japonica L.] FLMG ‘Barbara Morgan’ [C. japonica L.] DCNA ‘Capt. Rawes’ [C. reticulata Lind].] LAGN ‘Chandleri Elegans’ [C. japonica L.] VACW ‘Cherokee’ [C. sasanqua Thunb. ex J.A.Murr.] DCNA * ‘Cinnamon Cindy’ [C. japonica ‘Kenyo-Tai’ < C. lutchuensis] DCNA ‘Cleopatra’ [C. sasanqua Thunb. ex J.A.Murr.] VATA crapnelliana Tutch. DCNA MDGD ‘Crimson Bride’ [C. sasanqua Thunb. ex J.A.Murr.] DCNA cuspidata (Kochs) J.G.Veitch SCMG +‘Daisy Eagleson’ [C. sasanqua ‘Maiden’s Blush’ + C. japonica] (Graft chimera) DCNA ‘Daitairin’ [C. japonica L.] ALBG ‘Dawn’ [C. vernalis (Makino) Makino] MDGD ‘Doctor Tinsley’ [C. japonica L.] ARRB ‘Donation’ [C. x williamsii W.W.Sm.] [C. japonica < C. saluenensis] ALBG ‘Donation Sport’ [C. x williamsii W.W.Sm.] [C. japonica x C. saluenensis] DCNA CAMELLIA CAMELLIA CAMELLIA * ‘Fragrant Pink’ [C. rusticana ‘Yoshida’ x C. lutchuensis] DCNA * ‘Fragrant Pink Improved’ (Polyploid of C. ‘Fragrant Pink’) DCNA Sfraterna Hance ALTD LASL MDGD * ‘Frost Prince’ [C. hiemalis ‘Shishigashira’ < C. oleifera] DCNA MDLT * ‘Frost Princess’ [C. hiemalis ‘Billy Wylam’ C. oleifera] DCNA * ‘Frost Queen’ [C. japonica L.] DCNA ‘Gov. Mouton Variegated’ [C. japonica L.] SCMP granthamiana Sealy MDGD SCMG VANB ‘Guilio Nuccio’ [C. japonica L.] TNMB ‘Inspiration’ [C. reticulata < C. saluenensis] VAGS Japonica L. Common C. ALSH FLUF SCBR SCMP DCNA MDBG SCMG VACW kissii Wall. MDGD ‘Kumasaka’ [C. japonica L.] ALBG ‘Leucantha’ [C. japonica L.] DCWH ‘Little Princess’ [C. fraterna < C. japonica] ALBG lutchuensis Ito MDGD ‘Marie Wood’ [C. japonica L.] VANB 39 CAMPYLOTROPIS CAMELLIA ‘November Pink’ [C. < williamsii W.W.Sm.] [C. japonica < C. saluenensis] MDGD oleifera Abel Oil C. DCNA GAEC MDLT GACG MDGD VACW * ‘Polar Ice’ [C. ‘Frost Princess’ < C. oleifera] DCNA ‘Professor Sargent’ [C. japonica L.] DCWH ‘Reverend John G. Drayton’ [C. japonica L.] SCMG saluenensis Stapf NCEL ‘Sarah Frost’ [C. japonica L.] ALRH NCTP sasanqua Thunb. ex J.A.Murr. Sasanqua C. ALBG FLUF NCAG SCMG ARRB GAWH NCDU SCWP DCNG MDGD SCKG ‘Sayehime’ [C. vernalis (Makino) Makino] DCNA ‘Sharon Elizabeth’ [C. sasanqua Thunb. ex J.A.Murr.] DCNA ‘Sieboldii’ [C. japonica L.] DCWH sinensis (L.) O.Ktze. Tea Plant DCNA GAJS MSEP SCMP FLIF MDFM NCTE VAGS GACG MDLT SCBR VAMP GAEH MDRP SCKG * ‘Snow Flurry’ [C. ‘Frost Princess’ < C. oleifera] DCNA * ‘Sunworshiper’ [C. hongkongensis < C. rusticana] DCNA CAMPYLOTROPIS ‘Texas Star’ [C. sasanqua Thunb. ex J.A.Murr.] ALCW ‘Tiny Princess’ VAPH tsaii H.H.Hu MDGD * ‘Two Marthas’ [C. sasanqua ‘Onishiki’ < C. kissii] DCNA ‘Ville de Nantes’ [C. japonica L.] FLUF * ‘Winter’s Charm’ [C. oleifera x C. sasanqua ‘Takara-awase’] DCNA * ‘Winter’s Hope’ [C. ‘Frost Princess’ < C. oleifera] DCNA * ‘Winter’s Rose’ [C. hiemalis ‘Otome’ x C. oleifera] DCNA * ‘Winter’s Star’ [C. ‘Frost Prince’ < C. hiemalis] DCNA CAMPSIS Lour. BIGNONIACEAE Bignonia Family grandiflora (Thunb. ex J.A.Murr.) Schum. Chinese Trumpet-creeper MDFM TNSC ‘Mme. Galen’ [C. <tagliabuana (Vis.) Rehd.] [C. grandiflora x C. radicans] DCNA MDFM TNSC DCRS SCCU VAGS radicans (L.) Seemann ex Bur. Trumpet-creeper, Trumpet Vine DCNA MDLT SCBR VACW LAAL NCBE SCCU VAMW ‘Yellow Trumpet’ [C. radicans (L.) Seemann ex Bur.] SCCU VAGS CAMPYLOTROPIS' Bunge CARYA CARYA CARAGANA CARAGANA orientalis Mill. = DCNA ‘Purpurea’ [C. betulus L.] Purple-leaf H. TNTV CARAGANA Fabr. Pea Shrub FABACEAE (Faboideae) Bean Family Pas SS ee eee arborescens Lam. IPS). DEWG chamlagu Lam. = C. sinica ‘Lorbergii’ [C. arborescens Lam.] MDKN ‘Pendula’ [C. arborescens Lam.] DCNA TNSN sinica (Buc’hoz) Rehd. Chinese P.S. DCNA GAEC MDKN ‘Sylvatica’ [C. frutex (L.) K.Koch] DCNA CARISSA L. APOCYNACEAE Dogbane Family EE a ee Ss 7 ena ae eT \| macrocarpa (Eckl. & Zeyh.) A.DC. LALG LASL CARPINUS lL. Hornbeam BETULACEAE Birch Family EF a ee ee a ORES: SES Me TPES EEE S\| ‘Ascendens’ [C. caroliniana Walt.] TNLW betulus L. European H. DCCG DCNA DEMC MDTD caroliniana Walt. American H., Ironwood ALAU FLUF NCCA TXMS DCCG MDHN SCBR VACW DEWG MDWP SCMP cordata BI. Heartleaf H. MDGD TNDG ‘Fastigiata’ [C. CAMPYLOTROPIS betulus L.] Columnar H. DCCG TNEN TNTV DCNA TNSN VACW japonica BI. Japanese H. CARAGANA Fabr. Pea Shrub FABACEAE (Faboideae) Bean Family Pas SS ee eee arborescens Lam. IPS). DEWG chamlagu Lam. = C. sinica ‘Lorbergii’ [C. arborescens Lam.] MDKN ‘Pendula’ [C. arborescens Lam.] DCNA TNSN sinica (Buc’hoz) Rehd. Chinese P.S. DCNA GAEC MDKN ‘Sylvatica’ [C. frutex (L.) K.Koch] DCNA CARISSA L. APOCYNACEAE Dogbane Family EE a ee Ss 7 ena ae eT \| macrocarpa (Eckl. & Zeyh.) A.DC. LALG LASL CARPINUS lL. Hornbeam BETULACEAE Birch Family EF a ee ee a ORES: SES Me TPES EEE S\| ‘Ascendens’ [C. caroliniana Walt.] TNLW betulus L. European H. DCCG DCNA DEMC MDTD caroliniana Walt. American H., Ironwood ALAU FLUF NCCA TXMS DCCG MDHN SCBR VACW DEWG MDWP SCMP cordata BI. Heartleaf H. MDGD TNDG CARYA Nutt. Hickory JUGLANDACEAE Walnut Family aquatica (Michx.f.) Nutt. Bitter Pecan, Water H. SCBR cordiformis (Wang.) K.Koch Bitternut DCWM glabra (Mill.) Sweet Pignut ALAU FLWC TNBM VASC FLUF SCBR VAGG ‘Glover’ [C. ovata (Mill.) K.Koch] MDAB ‘Hines’ [C. ovata (Mill.) K.Koch] MDAB illinoinensis (Wang.) K.Koch Pecan ALCG GAIS MDHN SCWC DCCG LATU SCBR SCWI DCJS MDBR SCMP VAGW illinoinensis < C. sp. Hican TNSN ‘Koon’ [C. illinoinensis x C. sp.] MDGD laciniosa (Michx.f.) Loud. Shellbark H. FLMG NCCP _ ‘Lake’ [C. ovata (Mill.) K.Koch] MDAB ‘Lingenfelter’ [C. ovata (Mill.) K.Koch] MDAB ‘Mehan’ [C. illinoinensis (Wang.) K.Koch] Mehan Pecan ALBH ovalis (Wang.) Sarg. Sweet Pignut ALAU SCBR 4] CARYA CARYA CASTANOPSIS CARYA ovata (Mill.) K.Koch Shagbark H. DEEM VAGG ovata (Mill.) K.Koch var. pubescens Sarg. TNHG pallida (Ashe) Engl. & Graebn. Sand H. ALAU VASC ‘Romig’ [C. ovata (Mill.) K.Koch] MDAB ‘Schaul’ [C. ovata (Mill.) K.Koch] MDAB ‘Schinnerling’ [C. ovata (Mill.) K.Koch] MDAB ‘Stuart’ [C. illinoinensis (Wang.) K.Koch] Stuart Pecan ALBH DCNA FLUF NCCA tomentosa (Lam.) Nutt. Mockernut H. DCCG FLUF SCBR VAMP FLMG NCWK TNBM ‘Vest’ [C. ovata (Mill.) K.Koch] MDAB CARYOPTERIS Bunge VERBENACEAE Verbena Family <x clandonensis Simmonds [C. incana < C. mongholica] MDBG incana (Thunb. ex Houtt.) Miq. DCNA CASIMIROA Liave & Lex. RUTACEAE Citrus Family edulis Llave & Lex. White Sapote FLUF pringlei (S.Wats.) Engelm. TXMS TXPS CASSIA L. Senna FABACEAE (Caesalpinioideae) Bean Family \| ACARI SE a Sw ND a i alata L. Ringworm S. ALBG NCOP coluteoides Coll. ALBG FLMG LAAP FLDJ GATS SCCL corymbosa Lam. FLUF CASTANEA Mill. Chestnut FAGACEAE Beech Family LM a en Lg ‘Argenteo-variegata’ [C. sativa L.] DCNA ‘Crane’ [C. mollissima B1.] MDAB crenata Sieb. & Zucc. Japanese C. DCCT ‘Meiling’ [C. mollissima B1.] MDAB mollissima Bl. Chinese C. DCNA GAIS TNSC VAWR DCWH MDHN TNWF DEMC MDLT VACM ‘Nanking’ [C. mollissima B1.] MDAB ‘Orrin’ [C. mollissima B1.] MDAB sativa Mill. Common C., European C. MDBR vesca Gaertn. = C. sativa CASTANOPSIS (D.Don) Spach CASTANOPSIS cuspidata (Thunb. ex J.A.Murr.) Schottky var. sieboldii (Makino) Nakai FLMG sclerophylla Schottky GAIS CASUARINA Adans. CASUARINACEAE Casuarina Family cunninghamiana Miq. Australian Pine, Beefwood FLDH FLGA CATALPA Scop. BIGNONIACEAE Bignonia Family (a a a BU RE SESE ARE OS SINE eT) bignonioides Walt. Common C., Indian Bean DCSH MDHN GAMC SCGB VAWR bungei C.A.Mey. Manchurian C. TNRW ovata G.Don Chinese C. DCCG DCJJ DCPC SCCU speciosa Warder ex Engelm. Western C. DEWG GABS NCCA TNHG VACM CEANOTHUS L. RHAMNACEAE Buckthorn Family americanus L. New Jersey Tea DCNA VAMP xX delilianus Spach [C. americanus X< C. caeruleus] VAGS ‘Gloire de Versailles’ [C. < delilianus Spach] [C. americanus Xx C. caeruleus] SCWI CASTANOPSIS cuspidata (Thunb. ex J.A.Murr.) Schottky var. sieboldii (Makino) Nakai FLMG sclerophylla Schottky GAIS CASUARINA Adans. CASUARINACEAE Casuarina Family cunninghamiana Miq. Australian Pine, Beefwood FLDH FLGA CATALPA Scop. BIGNONIACEAE Bignonia Family (a a a BU RE SESE ARE OS SINE eT) bignonioides Walt. Common C., Indian Bean DCSH MDHN GAMC SCGB VAWR bungei C.A.Mey. Manchurian C. TNRW ovata G.Don Chinese C. DCCG DCJJ DCPC SCCU speciosa Warder ex Engelm. Western C. CARYA DEWG GABS NCCA TNHG VACM CEANOTHUS L. RHAMNACEAE Buckthorn Family americanus L. New Jersey Tea DCNA VAMP xX delilianus Spach [C. americanus X< C. caeruleus] VAGS ‘Gloire de Versailles’ [C. < delilianus Spach] [C. americanus Xx C. caeruleus] SCWI ‘Marie Simon’ [C. x pallidus Lind1.] [C. ? xdelilianus < C. ovatus] CEDRUS velutinus Doug]. Snowbrush C. SCJF velutinus Doug]. ~ Snowbrush C. SCJF CEDRELA P.Br. MELIACEAE Mahogany Family ER ae Os eS a ER a Re NS sinensis Juss. = Toona sinensis CEDRUS Trew Cedar PINACEAE Pine Family Bas, atlantica (Endl.) Manetti ex Carr. Atlas C. DCNA NCCA VABF GAUG NCTP VACW MDJS TNMB VARH ‘Aurea’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA MDJS ‘Aurea’ [C. deodara (Roxb.) G.Don] DCNA MDBG brevifolia (Hook.f.) A.Henry = C. libani ssp. brevifolia deodara (Roxb.) G.Don Deodar C. ALBG GACG MDHN TNSN ARUM GAJI NCBE VAMP DCCG GAOH NCTE VARH DCNA GAUG SCCU FLMG GAWS SCHG ‘Fastigiata’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA ‘Fastigiata’ [C. deodara (Roxb.) G.Don] DCNA _‘Glauca’ [C. atlantica (Endl.) Manetti ex Carr.] Blue Atlas C. DCNA MDCP TNSG MDBG MDLT ‘Glauca Pendula’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA MDBG MDLT libanensis Juss. ex Mirb. Snowbrush C. SCJF CEDRELA P.Br. MELIACEAE Mahogany Family ER ae Os eS a ER a Re NS sinensis Juss. = Toona sinensis CEDRUS Trew Cedar PINACEAE Pine Family Bas, atlantica (Endl.) Manetti ex Carr. Atlas C. DCNA NCCA VABF GAUG NCTP VACW MDJS TNMB VARH ‘Aurea’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA MDJS ‘Aurea’ [C. deodara (Roxb.) G.Don] DCNA MDBG brevifolia (Hook.f.) A.Henry = C. libani ssp. brevifolia deodara (Roxb.) G.Don Deodar C. ALBG GACG MDHN TNSN ARUM GAJI NCBE VAMP DCCG GAOH NCTE VARH DCNA GAUG SCCU FLMG GAWS SCHG ‘Fastigiata’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA ‘Fastigiata’ [C. deodara (Roxb.) G.Don] DCNA _‘Glauca’ [C. atlantica (Endl.) Manetti ex Carr.] Blue Atlas C. DCNA MDCP TNSG MDBG MDLT ‘Glauca Pendula’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA MDBG MDLT libanensis Juss. ex Mirb. = C. libani CEANOTHUS L. RHAMNACEAE Buckthorn Family americanus L. New Jersey Tea DCNA VAMP xX delilianus Spach [C. americanus X< C. caeruleus] VAGS ‘Gloire de Versailles’ [C. < delilianus Spach] [C. americanus Xx C. caeruleus] SCWI ‘Marie Simon’ [C. x pallidus Lind1.] [C. ? xdelilianus < C. ovatus] DCNA DEWG microphyllus Michx. SCWI 43 CEDRUS libani A.Rich. Cedar-of-Lebanon ALAU ALTD MDBG ALFN GALS MDHN libani A.Rich. ssp. CEDRUS CEPHALOTAXUS CEPHALOTAXUS caucasica Willd. Caucasian H. GAIS laevigata Willd. Sugarberry ALTD FLUF SCMP VAMP DCLP LAAL TXHN occidentalis L. Common H. ALUA GAHC TNFE TNWF DCCG MDLA TNLW VARR DEMC NCWT TNUT VASC pallida Torr. Spiny H. TXPS sinensis Pers. Chinese H. DCGP DCPG TNFE DCNA GAUG sinensis Pers. var. japonica (Planch.) Nakai VATA tala Gillies ex Planch. GAIS CEPHALANTHUS lL. RUBIACEAE Madder Family ilies Sa as ee OR ee a es ee eee occidentalis L. Buttonbush ALAU LAMP SCBR FLUF MDWP CEPHALOTAXUS Sieb. & Zucc. ex Endl. CEPHALOTAXACEAE Plum-yew Family ‘Fastigiata’ [C. harringtonia (J.Knight ex Forbes) K.Koch] ALTD FLUF MDLT VAGS FLCG GAHC NCOP VAMP FLMG GAOH SCMG harringtonia (J.Knight ex Forbes) K.Koch Plum Yew ALEH GAEH MDGD VABR DCNA GAGR NCBE VAGG GABG GAIS NCCA VAMP ‘Nana’ [C. harringtonia (J.Knight caucasica Willd. Caucasian H. GAIS laevigata Willd. Sugarberry ALTD FLUF SCMP VAMP DCLP LAAL TXHN occidentalis L. Common H. ALUA GAHC TNFE TNWF DCCG MDLA TNLW VARR DEMC NCWT TNUT VASC pallida Torr. Spiny H. TXPS sinensis Pers. Chinese H. DCGP DCPG TNFE DCNA GAUG sinensis Pers. var. japonica (Planch.) Nakai VATA tala Gillies ex Planch. GAIS CEPHALANTHUS lL. RUBIACEAE Madder Family ilies Sa as ee OR ee a es ee eee occidentalis L. Buttonbush ALAU LAMP SCBR FLUF MDWP CEPHALOTAXUS Sieb. & Zucc. ex Endl. CEPHALOTAXACEAE Plum-yew Family ‘Fastigiata’ [C. harringtonia (J.Knight ex Forbes) K.Koch] ALTD FLUF MDLT VAGS FLCG GAHC NCOP VAMP caucasica Willd. Caucasian H. GAIS laevigata Willd. Sugarberry ALTD FLUF SCMP VAMP DCLP LAAL TXHN occidentalis L. Common H. ALUA GAHC TNFE TNWF DCCG MDLA TNLW VARR DEMC NCWT TNUT VASC pallida Torr. Spiny H. TXPS sinensis Pers. Chinese H. DCGP DCPG TNFE DCNA GAUG sinensis Pers. var. japonica (Planch.) Nakai VATA tala Gillies ex Planch. GAIS CEPHALANTHUS lL. RUBIACEAE Madder Family ilies Sa as ee OR ee a es ee eee occidentalis L. Buttonbush ALAU LAMP SCBR FLUF MDWP CEPHALOTAXUS Sieb. & Zucc. ex Endl. CEPHALOTAXACEAE Plum-yew Family ‘Fastigiata’ [C. harringtonia (J.Knight ex Forbes) K.Koch] ALTD FLUF MDLT VAGS FLCG GAHC NCOP VAMP FLMG GAOH SCMG harringtonia (J.Knight ex Forbes) K.Koch Plum Yew ALEH GAEH MDGD VABR DCNA GAGR NCBE VAGG GABG GAIS NCCA VAMP ‘Nana’ [C. harringtonia (J.Knight ex Forbes) K.Koch] SCMG CEPHALOTAXUS Sieb. & Zucc. ex Endl. CEPHALOTAXACEAE Plum-yew Family ‘Fastigiata’ [C. CARYA brevifolia (Hook.f.) Meikle Cyprus Cedar DCNA libani A.Rich. var. stenocoma (O.Schwarz) Davis DCNA MDDF libanotica Trew ex Pilger ssp. brevifolia (Hook.f.) O.Schwarz = C. libani ssp. brevifolia ‘Limelight’ [C. deodara (Roxb.) G.Don] DCNA ‘Nana’ [C. libani A.Rich.] DCNA ‘Pendula’ [C. atlantica (Endl.) Manetti ex Carr.] DCNA ‘Pendula’ [C. deodara (Roxb.) G.Don] DCNA ‘Pygmy’ [C. deodara (Roxb.) G.Don] DCNA ‘Repandens’ [C. deodara (Roxb.) G.Don] DCNA CELASTRUS L. Bittersweet CELASTRACEAE Staff-tree Family ER a a angulatus Maxim. TNSN orbiculatus Thunb. ex J.A.Murr. Oriental B. ALAU NCBE TNTV MDGJ SCBR VACW MDPJ Secu VANB rosthornianus Loesn. SCBR CELTIS L. Hackberry ULMACEAE Elm Family australis L. DCNA GAIS CEDRUS harringtonia (J.Knight ex Forbes) K.Koch] ALTD FLUF MDLT VAGS FLCG GAHC NCOP VAMP FLMG GAOH SCMG harringtonia (J.Knight ex Forbes) K.Koch Plum Yew ALEH GAEH MDGD VABR DCNA GAGR NCBE VAGG GABG GAIS NCCA VAMP ‘Nana’ [C. harringtonia (J.Knight ex Forbes) K.Koch] SCMG et CHAENOMELES CERATIOLA CHAENOMELES chinensis Bunge ~ Chinese Redbud ALIT GACG MDBG MDWP DCCG GACW MDDF VACW ‘Forest Pansy’ [C. canadensis L.] DCNA TNCT TNSC TNTV GAUG TNFEN TNSM VACW ‘Oklahoma’ [C. canadensis L. ssp. texensis (S.Wats.) E.Murr.] MDBG NC ‘Plena’ [C. canadensis L.] FLMG SCBR reniformis Engelm. = C. canadensis ssp. texensis ‘Rubrum’ [C. siliquastrum L.] DCNA ‘Ruby Atkinson’ [C. canadensis L.] TNCT ‘Wither’s Pink Charm’ [C. canadensis L.] DEWG MDKN CESTRUM lL. SOLANACEAE Nightshade Family nocturnum L. Night Jessamine FLUF CHAENOMELES Lindl. ROSACEAE Rose Family (Chaenomeles is an orthographic correction of the name published by Lindley in 1821 as Choenomeles.) ‘Apple Blossom’ [C. speciosa (Sweet) Nakai] DEWG GACG - ‘Candida’ [C. speciosa (Sweet) Nakai] ARPT MDHN VAMP DEMC VACW ‘Cardinalis’ [C. speciosa (Sweet) Nakai] GACG ‘Corallina’ [C. < superba (Frahm) Rehd.] [C. japonica < C. speciosa] SCBR CERATIOLA Michx. EMPETRACEAE Crowberry Family fe a eee ee ericoides Michx. Sandheath SCBR CERATOSTIGMA Bunge PLUMBAGINACEAE Leadwort Family SS ES ES A EES ee SS) plumbaginoides Bunge DCNA DEMC willmottianum Stapf Chinese Plumbago SCWI CERCIDIPHYLLUM Sieb. & Zucc. CERCIDIPHYLLACEAE Katsura-tree Family Sa, SE SES Se \| japonicum Sieb. & Zucc. Katsura Tree DCAC DCNA DEMC MDJS DCCG DCWR GAUG TNHT DCDO DEEM MDJH TNMB ‘Pendula’ [C. japonicum Sieb. & Zucc.] MDBG CERCIS L. FABACEAE (Caesalpinioideae) Bean Family SSS a a a a LEE LS ES \| ‘Alba’ [C. canadensis L.] ALTD DCNA LASL ARPT GAEC TNCT VAWR ‘Alba’ [C. canadensis L. ssp. texensis (S.Wats.) E.Murr.] TNTV ‘Alba’ [C. chinensis Bunge] DCNA canadensis L. North American Redbud ALBG DEMC MDBG SCRL ALEH DEWG MDJH SCUC ALTD FLCG MDJS VACW ARRB FLMG MSHB VAGW ARUM FLUF NCTP VAMP IXOSE GAVI SCCU VATA DCWH LASL SCMP 45 CHAENOMELES CHAENOMELES ‘Crimson and Gold’ [C. < superba (Frahm) Rehd.] [C. japonica x C. speciosa] DEWG ‘Hanazono’ [C. speciosa (Sweet) Nakai] GACG japonica (Thunb.) Lindl. ex Spach Japanese Flowering Quince MSHB ‘Knap Hill Scarlet’ [C. x superba (Frahm) Rehd.] [C. japonica < C. speciosa] DEWG lagenaria (Loisel.) Koidz. = C. speciosa ‘Mandarin’ [C. < superba (Frahm) Rehd.] [C. japonica < C. speciosa] DCNA ‘Marmorata’ [C. speciosa (Sweet) Nakai] GACG ‘Mt. Everest’ [C. < vilmoriniana Weber] [C. cathayensis < C. speciosa] GACG ‘Nivalis’ [C. speciosa (Sweet) Nakai] GACG ‘Perfecta’ [C. x superba (Frahm) Rehd.] [C. japonica < C. speciosa] DEWG ‘Phyllis Moore’ [C. speciosa (Sweet) Nakai] DEWG ‘Pink Lady’ [C. < superba (Frahm) Rehd.] [C. japonica = C. speciosa] GACG ‘Rosy Morn’ [C. californica Clarke & Weber] [C. cathayensis < C. < superba] GACG ‘Roxana Foster’ [C. < superba (Frahm) Rehd.] [C. japonica x C. speciosa] CHAMAECYPARIS ‘Simonii’ [C. speciosa (Sweet) Nakai] GACG ‘Snow’ [C. speciosa (Sweet) Nakai] DEWG GACG speciosa (Sweet) Nakai Japanese Quince, Flowering Quince DCWH MDHN SCBR VACW FLDG MDLA SCPR VAMP GAGM MSMN SCSR MDBG NCWC TXAB ‘Stanford Red’ [C. < superba (Frahm) Rehd.] [C. japonica < C. speciosa] GACG x superba (Frahm) Rehd. [C. japonica x C. speciosa] DCNA FLCG FLMN SCFW ‘Toyo nishiki’ [C. speciosa (Sweet) Nakai] GACG ‘Umbilicata’ [C. speciosa (Sweet) Nakai] GACG ‘Simonii’ [C. speciosa (Sweet) Nakai] GACG ‘Snow’ [C. speciosa (Sweet) Nakai] DEWG GACG speciosa (Sweet) Nakai Japanese Quince, Flowering Quince DCWH MDHN SCBR VACW FLDG MDLA SCPR VAMP GAGM MSMN SCSR MDBG NCWC TXAB ‘Stanford Red’ [C. < superba (Frahm) Rehd.] [C. japonica < C. speciosa] GACG x superba (Frahm) Rehd. [C. japonica x C. speciosa] DCNA FLCG FLMN SCFW ‘Toyo nishiki’ [C. speciosa (Sweet) Nakai] GACG ‘Umbilicata’ [C. speciosa (Sweet) Nakai] GACG ‘Knap Hill Scarlet’ [C. x superba (Frahm) Rehd.] [C. japonica < C. speciosa] DEWG ‘Mandarin’ [C. < superba (Frahm) Rehd.] [C. japonica < C. speciosa] DCNA ‘Marmorata’ [C. speciosa (Sweet) Nakai] GACG ‘Mt. Everest’ [C. < vilmoriniana Weber] [C. cathayensis < C. speciosa] GACG ‘Nivalis’ [C. speciosa (Sweet) Nakai] GACG ‘Mt. Everest’ [C. < vilmoriniana Weber] [C. cathayensis < C. speciosa] GACG CHAMAECYPARIS Spach False Cypress CUPRESSACEAE Cypress Family SSS OEE EE TS CE SS TEL ET) ‘Albospica’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Allumii’ [C. lawsoniana (A.Murr.) Parl.] VACR ‘Andelyensis’ [C. thyoides (L.) BSP.] DCNA MDBG ‘Aurea Nana’ [C. pisifera (Sieb. & Zucc.) End1.] MDLT ‘Blom’ [C. CHAENOMELES lawsoniana (A.Murr.) Parl.] DCNA ‘Boulevard’ [C. pisifera (Sieb. & Zucc.) Endl.] ALBH MDBG VATA ‘Breviramea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Compacta’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Nivalis’ [C. speciosa (Sweet) Nakai] GACG ‘Albospica’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Perfecta’ [C. x superba (Frahm) Rehd.] [C. japonica < C. speciosa] DEWG ‘Allumii’ [C. lawsoniana (A.Murr.) Parl.] VACR ‘Phyllis Moore’ [C. speciosa (Sweet) Nakai] DEWG ‘Andelyensis’ [C. thyoides (L.) BSP.] DCNA MDBG ‘Pink Lady’ [C. < superba (Frahm) Rehd.] [C. japonica = C. speciosa] GACG ‘Aurea Nana’ [C. pisifera (Sieb. & Zucc.) End1.] MDLT ‘Rosy Morn’ [C. californica Clarke & Weber] [C. cathayensis < C. < superba] GACG ‘Rosy Morn’ [C. californica Clarke & Weber] [C. cathayensis < C. < superba] ‘Blom’ [C. lawsoniana (A.Murr.) Parl.] DCNA [C. cathayensis < C. < superba] GACG ‘Boulevard’ [C. pisifera (Sieb. & Zucc.) Endl.] ALBH MDBG VATA ‘Roxana Foster’ [C. < superba (Frahm) Rehd.] [C. japonica x C. speciosa] GACG ‘Breviramea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Compacta’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Breviramea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Rubra’ [C. speciosa (Sweet) Nakai] GACG ‘Ruby Glow’ [C. < superba (Frahm) Rehd.] [C. japonica < C. speciosa] GACG 46 CHAMAECYPARIS ‘Contorta’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Coralliformis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Crippsii’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDBG NCDU GAEC MDJS ‘Cyanoviridis’ [C. pisifera (Sieb. & Zucc.) End1.] = ‘Boulevard’ ‘Ericoides’ [C. thyoides (L.) BSP.] ALSM FLUF NCTP VATW DCNA GABC SCFW FLMG GAEC SCRS FLRI MDBG SCTR ‘Filicoides’ [C. obtusa (Sieb. & Zucc.) Endl.] GAWH MDBG MDJS NCDU ‘Filifera’ [C. pisifera (Sieb. & Zucc.) End1.] DCNA MDBG VAOD GAEH VAMP VATA ‘Filifera Aurea’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA MDLT VAMP ‘Filiformis’ [C. lawsoniana (A.Murr.) Parl.] DCNA ‘Filiformis Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] MDLT ‘Filiformis Compacta’ [C. lawsoniana (A.Murr.) Parl.] DCNA funebris (Endl.) Franco Mourning Cypress GAFN LARP SCHG LAAL LATU ‘Gold Drop’ [C. obtusa (Sieb. & Zucc.) Endl.] MDLT ‘Gold Spangle’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Golden Mop’ [C. pisifera (Sieb. & Zucc.) Endl.] MDBG ‘Gracilis’ [C. obtusa (Sieb. & Zucc.) Endl.] CHAMAECYPARIS ‘Contorta’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Coralliformis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Crippsii’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDBG NCDU GAEC MDJS ‘Cyanoviridis’ [C. pisifera (Sieb. CHAENOMELES & Zucc.) End1.] = ‘Boulevard’ ‘Ericoides’ [C. thyoides (L.) BSP.] ALSM FLUF NCTP VATW DCNA GABC SCFW FLMG GAEC SCRS FLRI MDBG SCTR ‘Filicoides’ [C. obtusa (Sieb. & Zucc.) Endl.] GAWH MDBG MDJS NCDU ‘Filifera’ [C. pisifera (Sieb. & Zucc.) End1.] DCNA MDBG VAOD GAEH VAMP VATA ‘Filifera Aurea’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA MDLT VAMP ‘Filiformis’ [C. lawsoniana (A.Murr.) Parl.] DCNA ‘Filiformis Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] MDLT ‘Filiformis Compacta’ [C. lawsoniana (A.Murr.) Parl.] DCNA funebris (Endl.) Franco Mourning Cypress GAFN LARP SCHG LAAL LATU ‘Gold Drop’ [C. obtusa (Sieb. & Zucc.) Endl.] MDLT ‘Gold Spangle’ [C. pisifera (Sieb. & Zucc.) CHAMAECYPARIS CHAMAECYPARIS henryae Li = = C. thyoides ‘Intermedia’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Juniperoides Aurea’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Kosteri’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA lawsoniana (A.Murr.) Parl. Lawson Cypress, Port Orford Cedar FLGS SCCU VAMP VAWM ‘Lutea’ [C. lawsoniana (A.Murr.) Parl.] ALTD ‘Lycopodioides’ [C. lawsoniana (A.Murr.) Parl.] DCNA ‘Lycopodioides’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDBG ‘Mariesii’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Monstrosa’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Nana’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA VATA ‘Nana Argentea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Nana Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Nana Gracilis’ [C. obtusa (Sieb. & Zucc.) - Endl.] DCNA MDBG MDLT NCGC ‘Nana Miko’ [C. pisifera (Sieb. & Zucc.) Endl.] = ‘Snow’ ‘Nana Pyramidalis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Lycopodioides’ [C. lawsoniana (A.Murr.) Parl.] DCNA ‘Lycopodioides’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDBG ‘Mariesii’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Filiformis’ [C. lawsoniana (A.Murr.) Parl.] DCNA ‘Monstrosa’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Filiformis Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] MDLT ‘Nana’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA VATA ‘Nana Argentea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Nana Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Golden Mop’ [C. pisifera (Sieb. & Zucc.) Endl.] MDBG ‘Gracilis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDJS VAGG ‘Gracilis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDJS VAGG 47 CHAMAECYPARIS CHAMAEDAPHNE ‘Snow’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Spiralis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Squarrosa’ [C. pisifera (Sieb. & Zucc.) Endl.] Moss False Cypress DCSH GAEC SCCU VATA DCWR MDHN VAMP FLUF MDJS VASK ‘Squarrosa Intermedia’ [C. pisifera (Sieb. & Zucc.) Endl.) DCNA MDBG VATA ‘Squarrosa Minima’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Stoneham’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Tempelhof [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Tetragona Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDBG MDLT VAMP SCCEC VATA thyoides (L.) BSP. Atlantic White Cedar MDBG SCBR SCWI VACW ‘Tonia’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Verdonii’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA CHAMAECYTISUS Link FABACEAE (Faboideae) Bean Family \| Se i a Se Oe ee supinus (L.) Link MDSC CHAMAEDAPHNE Moench ERICACEAE Heath Family calyculata (L.) Moench Leatherleaf CHAMAEDAPHNE ‘Snow’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Spiralis’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Squarrosa’ [C. pisifera (Sieb. & Zucc.) Endl.] Moss False Cypress DCSH GAEC SCCU VATA DCWR MDHN VAMP FLUF MDJS VASK ‘Squarrosa Intermedia’ [C. pisifera (Sieb. & Zucc.) Endl.) DCNA MDBG VATA ‘Squarrosa Minima’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Stoneham’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Tempelhof [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Tetragona Aurea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA MDBG MDLT VAMP SCCEC VATA thyoides (L.) BSP. Atlantic White Cedar MDBG SCBR SCWI VACW ‘Tonia’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Verdonii’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA CHAMAECYTISUS Link FABACEAE (Faboideae) Bean Family \| Se i a Se Oe ee supinus (L.) Link MDSC CHAMAEDAPHNE Moench ‘Pembury Blue’ [C. lawsoniana (A.Murr.) Parl.] DCNA ‘Plumosa’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Stoneham’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Plumosa Aurea’ [C. pisifera (Sieb. & Zucc.) Endl.] MDJS ‘Tempelhof [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Plumosa Aurea Nana’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA MDBG VATA ‘Plumosa Compacta’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA MDBG VATA ‘Plumosa Compressa’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Pygmaea’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Pygmaea’ [C. pisifera (Sieb. & Zucc.) Endl.] DCNA ‘Pygmaea Aurescens’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Reis Dwarf [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Repens’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Rigid Dwarf’ [C. obtusa (Sieb. & Zucc.) Endl.] DCNA ‘Sanderi’ [C. obtusa (Sieb. CHAMAECYPARIS & Zucc.) Endl.] ALHC DCNA aN (ee) CISTUS CHAMAEROPS Palm Family SALICACEAE Willow Family arbutifolia (Pall.) Skvortz. DCNA CHRYSANTHEMUM lL. ASTERACEAE Aster Family nipponicum (Franch. ex Maxim.) Matsum. Nippon Daisy DCNA MDFM CHRYSOLARIX H.E.Moore = PSEUDOLARIX CINNAMOMUM. § Schaeffer LAURACEAE Laurel Family camphora (L.) T.Nees & Eberm. Camphor Tree ALTD FLUF MSEN SCCL FLCG GAIS MSHB SCMP FLPM GAJI NCOP SCUC daphnoides Sieb. & Zucc. LASL CISSUS L. VITACEAE Grape Family incisa (Nutt.) Desmoul. Marine Ivy, Marine Vine LAIS CISTUS L. CISTACEAE Rock-rose Family ~corbariensis Pourr. = C. < hybridus < hybridus Pourr. [C. populifolius < C. salvifolius] MDFM SCWI VACW ladanifer L. Laudanum SCWI VACW x purpureus Lam. [C. ladanifer < C. villosus] SCWI VACW arbutifolia (Pall.) Skvortz. DCNA DCNA CHRYSANTHEMUM lL. ASTERACEAE Aster Family nipponicum (Franch. ex Maxim.) Matsum. Nippon Daisy DCNA MDFM CHRYSOLARIX H.E.Moore = PSEUDOLARIX CINNAMOMUM. § Schaeffer LAURACEAE Laurel Family camphora (L.) T.Nees & Eberm. Camphor Tree ALTD FLUF MSEN SCCL FLCG GAIS MSHB SCMP FLPM GAJI NCOP SCUC daphnoides Sieb. & Zucc. LASL CISSUS L. VITACEAE Grape Family linearis (Cav.) Sweet Desert Willow LASL CHIMONANTHUS Lindl. CALYCANTHACEAE Calycanthus Family CT a a aS ae ee Pee a ee ee) ‘Luteus’ [C. praecox (L.) Link] DCNA ‘Mangetsw’ [C. praecox (L.) Link] DCNA nitens Oliv. MDBG praecox (L.) Link Wintersweet DCAC GAUG SCHW VAGS DCNA LAHG SCUC VAGW GAHC MDBG TXPS VATW GAOH MSMN VACW CHIONANTAUS L. OLEACEAE Fringe Tree Olive Family retusus Lindl. & Paxt. Japanese F. DCEL DCNA MDDF retusus Lindl. & Paxt. var. serrulatus (Hayata) Koidz. Taiwan F. ALAU FLUF TNSN TXDC DCNA GAGB TNUT VACW DEWG TNBM TXBA virginicus L. American F.T., Old-man’s Beard DCNA GACG MDJS VAGW DEWG LAAL MSMN VANB FLMG LASL SCMG FLUF MDBG VACP GACC MDJH VACW 49 CHIMONANTHUS praecox (L.) Link [illustrator Lillian Nicholson Meyer] CLEMATIS CLEMATIS CITHAREXYLUM CLADRASTIS kat. FABACEAE (Faboideae) Bean Family a eS ee SE ey a a a SE AE kentukea (Dum.-Cours.) Rudd Yellow-wood DCNA DEWG MDHC VABF DCWH MDCR TNHG VACW DEMC MDDF TNUT VAMP lutea (Michx.f.) K.Koch = C. kentukea tinctoria Raf. = C. kentukea CLEMATIS L. RANUNCULACEAE Buttercup Family ee SS ee a eS armandii Franch. Evergreen C. ALBG DCNA MDWP * ‘Betty Corning’ [C. viticella L.] DCNA MDFM MDTD chrysocoma Franch. var. sericea (Franch.) Schneid. Goldwool C. DCNA crispa L. Blue Jasmine SCBR dioscoreifolia Lev. & Van. = C. terniflora ‘Farquhariana’ [C. armandii Franch.] Pink-flowered Evergreen C. CHAMAECYPARIS ALBG ‘Grandiflora’ [C. montana Buch.-Ham.] VAGS _xjackmanii T.Moore [C. lanuginosa C. viticella] ALHC MDJS VAGS maximowicziana Franch. & Sav. = C. terniflora CITHAREXYLUM L. VERBENACEAE Verbena Family berlandieri Robinson Fiddlewood TXLL DCWH MDCR TNHG VACW DEMC MDDF TNUT VAMP lutea (Michx.f.) K.Koch = C. kentukea tinctoria Raf. = C. kentukea CLEMATIS L. RANUNCULACEAE Buttercup Family ee SS ee a eS armandii Franch. Evergreen C. ALBG DCNA MDWP * ‘Betty Corning’ [C. viticella L.] DCNA MDFM MDTD chrysocoma Franch. var. sericea (Franch.) Schneid. Goldwool C. DCNA crispa L. Blue Jasmine SCBR dioscoreifolia Lev. & Van. = C. terniflora ‘Farquhariana’ [C. armandii Franch.] Pink-flowered Evergreen C. ALBG ‘Grandiflora’ [C. montana Buch.-Ham.] VAGS _xjackmanii T.Moore [C. lanuginosa C. viticella] ALHC MDJS VAGS maximowicziana Franch. & Sav. = C. terniflora paniculata Thunb. <CITROFORTUNELLA J.Ingram & H.E.Moore RUTACEAE [Citrus < Fortunella] Citrus Family mitis (Blanco) J.Ingram & H.E.Moore Calamondin [Citrus reticulata < Fortunella ?margarita] ALTD FLMG LALG FLCG FLUF XCITRONCIRUS J.Ingram & H.E.Moore RUTACEAE Citrus Family [Citrus < Poncirus] webberi J.Ingram & H.E.Moore Citrange [Citrus sinensis < Poncirus trifoliata] SCRP CITRUS L. RUTACEAE Citrus Family aurantium L. Sour Orange, Seville Orange PLUF ‘Etrog’ [C. medica L.] Citron FLUF hystrix DC. FLUF mitis Blanco = X Citrofortunella mitis xX paradisi Macf. Grapefruit [C. maxima <x C. sinensis] FLUF TXJS reticulata Blanco Mandarin, Tangerine, Satsuma Orange FLUF ‘Thomasville Citrangequat’ [(Citrus sinensis < Poncirus trifoliata) < Fortunella sp.] GAIS <CITROFORTUNELLA J.Ingram & H.E.Moore RUTACEAE [Citrus < Fortunella] Citrus Family mitis (Blanco) J.Ingram & H.E.Moore Calamondin [Citrus reticulata < Fortunella ?margarita] ALTD FLMG LALG FLCG FLUF XCITRONCIRUS J.Ingram & H.E.Moore RUTACEAE Citrus Family [Citrus < Poncirus] webberi J.Ingram & H.E.Moore Citrange [Citrus sinensis < Poncirus trifoliata] SCRP CITRUS L. RUTACEAE Citrus Family aurantium L. Sour Orange, Seville Orange PLUF ‘Etrog’ [C. medica L.] Citron FLUF hystrix DC. FLUF mitis Blanco = X Citrofortunella mitis xX paradisi Macf. Grapefruit [C. maxima <x C. sinensis] FLUF TXJS reticulata Blanco Mandarin, Tangerine, Satsuma Orange FLUF ‘Thomasville Citrangequat’ [(Citrus sinensis < Poncirus trifoliata) < Fortunella sp.] lutea (Michx.f.) K.Koch = C. kentukea tinctoria Raf. = C. kentukea D1 CLIFTONIA CLEMATIS spooneri Rehd. & Wils. = C. chrysocoma var. sericea stans Sieb. & Zucc. MDTD ‘Superba’ [C. <jackmanii T.Moore] [C. lanuginosa C. viticella] MDBG MDJS terniflora DC. Virgin’s Bower ALBG DCNA NCOP SCBR MDGD SCCU MSHB texensis Buckl. Scarlet C. SCWI versicolor Britton Leatherflower SCWI viorna L. Vase Vine DCNA SCBR CLETHRA L. CLETHRACEAE White Alder White-alder Family acuminata Michx. DCNA GACG MDLT alnifolia L. Sweet Pepperbush DCNA MDLT MDBG NCDB NCOP SCBR VATA barbinervis Sieb. & Zucc. Japanese Summersweet TNTV fargesii Franch. DCNA ‘Rosea’ [C. alnifolia L.] Pink Summersweet DEMC MDLT VACW tomentosa Lam. Woolly Summersweet ALAU MDLT ALBG NCBE SCBR CLERODENDRUM L. VERBENACEAE Verbena Family SE ee ee eee ee ES Se ee eee bungei Steud. LAAL LASL VACW fragrans (Vent.) R.Br. = C. philippinum indicum (L.) O.Ktze. Tubeflower, Turk’s Turban ALBG SCBR philippinum Schauer Fragrant Glorybower FLUF thomsonae Balf.f. Bleedingheart Glorybower FLUF LALG trichotomum Thunb. Harlequin Glorybower DCNA MDBG GAJS MDLT VATW THEACEAE gymnanthera Wight & Arn. = Ternstroemia gymnanthera japonica Thunb. Japanese C. (Thunberg originally included material from another plant, now known as Ternstroemia gymnanthera(Wight & Arn.) Sprague, in his description of Cleyera japonica. As a result, these names have been confused in the nursery trade.) ALEH LAAL GAIS SCCU VANB VAWM ochnacea DC. = C. japonica ‘Tricolor’ [C. japonica Thunb.] Variegated C. GACG CLIFTONIA Banks ex C.F.Gaertn. CYRILLACEAE Cyrilla Family SSL a a ID) monophylla (Lam.) Britton ex Sarg. Buckwheat Tree, Titi ALAU GAEC ALBG LALG SCWI ES a) as CORNUS CLINOPODIUM CLINOPODIUM CLINOPODIUM lL. LAMIACEAE Mint Family =a ee ES Fe Ee ES SS ashei (Weatherby) Small SCWI coccineum (Nutt.) O.Ktze. SCWI dentatum (Chapm.) O.Ktze. SCWI CLYTOSTOMA Miliers ex Bur. BIGNONIACEAE Bignonia Family DR a ee 2 ee callistegioides (Cham.) Bur. Argentine Trumpetvine FLCG FLUF GACH TXMK CNEORUM lL. CNEORACEAE Spurge-olive Family SSS EE SEE EEE ANG AL REET \| tricoccon L. Spurge-olive SCWI COCCULUS DC. MENISPERMACEAE Moonseed Family EEE ET DE A AD APES ADOT] laurifolius DC. Laurel-leaf Snailseed FLUF LAAL LALG LATU trilobus (Thunb.) DC. Coralbeads GAGS COLUTEA L. FABACEAE (Faboideae) Bean Family arborescens L. Bladder Senna VABF COMPTONIA UHer. ex Ait. MYRICACEAE Wax-myrtle Family peregrina (L.) Coult. Sweetfern MDWP VAHB CORNUS by CONDALIA Cav. RHAMNACEAE Buckthorn Family hookeri M.C.Johnst. TXSC CONRADINA A.Gr. LAMIACEAE Mint Family eas A ee EE Se a SAO TT] canescens (T. & G.) A.Gr. Bluesage C. ALTD GAJI VAPH GACG SCWI glabra Shinners SCWI grandiflora Small SCWI verticillata Jennison Whorled C. GAFG SCWI CORDIA lL. BORAGINACEAE Borage Family a Ee EE ee eee aS eS 2 ES eee ee] boissieri A.DC. Anacahuita FLJH TXRE CORIARIA L. CORIARIACEAE Coriaria Family Japonica A.Gr. Japanese C. DCNA GAEC MDKN CORNUS L. Dogwood CORNACEAE Dogwood Family alba L. MDGD alternifolia Lf. Green Osier, Alternate-leaved D. MDJS TNUT VAHR amomum Mill. Silky D. ALUA MDLT SCCU VAPO DCNA NCDU VAGW CORNUS by CONDALIA Cav. RHAMNACEAE Buckthorn Family hookeri M.C.Johnst. TXSC CONRADINA A.Gr. LAMIACEAE Mint Family eas A ee EE Se a SAO TT] canescens (T. & G.) A.Gr. Bluesage C. ALTD GAJI VAPH GACG SCWI glabra Shinners SCWI grandiflora Small SCWI verticillata Jennison Whorled C. GAFG SCWI CORDIA lL. BORAGINACEAE Borage Family a Ee EE ee eee aS eS 2 ES eee ee] boissieri A.DC. Anacahuita FLJH TXRE CORIARIA L. CORIARIACEAE Coriaria Family Japonica A.Gr. Japanese C. DCNA GAEC MDKN CORNUS L. Dogwood CORNACEAE Dogwood Family alba L. MDGD alternifolia Lf. Green Osier, Alternate-leaved D. gymnanthera Wight & Arn. = Ternstroemia gymnanthera MDJS TNUT VAHR amomum Mill. Silky D. CLINOPODIUM lL. LAMIACEAE Mint Family =a ee ES Fe Ee ES SS ashei (Weatherby) Small SCWI coccineum (Nutt.) O.Ktze. SCWI dentatum (Chapm.) O.Ktze. SCWI CLYTOSTOMA Miliers ex Bur. BIGNONIACEAE Bignonia Family DR a ee 2 ee callistegioides (Cham.) Bur. Argentine Trumpetvine FLCG FLUF GACH TXMK CNEORUM lL. CNEORACEAE Spurge-olive Family SSS EE SEE EEE ANG AL REET \| tricoccon L. Spurge-olive SCWI COCCULUS DC. MENISPERMACEAE Moonseed Family EEE ET DE A AD APES ADOT] laurifolius DC. Laurel-leaf Snailseed FLUF LAAL LALG LATU trilobus (Thunb.) DC. Coralbeads GAGS COLUTEA L. FABACEAE (Faboideae) Bean Family arborescens L. Bladder Senna VABF COMPTONIA UHer. ex Ait. MYRICACEAE Wax-myrtle Family CLINOPODIUM lL. LAMIACEAE Mint Family ashei (Weatherby) Small SCWI coccineum (Nutt.) O.Ktze. SCWI 53 CORNUS CORNUS CORNUS ‘Argenteo-marginata’ [C. alba L.] Cream-edged D. MDBG VAGS asperifolia Michx. DCNA LAAL SCBR ‘Aurea’ [C. mas L.] DCNA australis C.A.Mey. var. koenigii (Schneid.) Wang. DCNA MDGD ‘Bay Beauty’ [C. florida L.] Double-flowered D. TXLL ‘Cherokee Chief [C. florida L.] DCNA ‘Cherokee Princess’ [C. florida L.] DCNA ‘Cherokee Sunset’ [C. florida L.] TNSN ‘Cloud Nine’ [C. florida L.] DCNA controversa Hemsl. Giant D. DEEM MDGJ TNPW GARW MDMG VAGS MDBG TNDG VANB coreana Wang. DCNA drummondtii C.A.Mey. AD COAL ‘Dwarf’ [C. florida L.] DCNA ‘First Lady’ [C. florida L.] DCNA TNBO ‘Flaviramea’ [C. sericea L.] MDBG MDLT VATA florida L. Flowering D. ALBG FLUF MDLT SCUC ARRB GAdJI MDWP SCWI DCCG GAKN NCCS TNMB DCFM LAAL NCPR VACW florida L. f. pluribracteata Rehd. Double-flowered D. DCCG MDLT NCGC SCMG FLMG MDMM NCPL GAEC NCBE SCDB florida L. f. rubra (Weston) Schelle Pink Flowering D. ARSN DCWH SCPR DCCG FLMG VAGG VAMP florida L. f. xanthocarpa Rehd. Yellow-fruited Flowering D. MDCG foemina Mill. Stiff D. GAUG TNHS ‘Fragrant Cloud’ [C. florida L.] DCNA ‘Gold Star’ [C. kousa Hance] DCNA ‘Kesselringii’ [C. alba L.] DCNA kousa (Buerger ex Miq.) Hance Kousa D. DCAC GAEC MDMG TNSN DCNA GAUG MDPJ TNUT IDYCIRE MDBG MDSJ VACW DCWH MDJH NCBE VAPH DEWG MDLT TNSC ‘Lustgarten Weeping’ [C. kousa (Buerger ex Miq.) Hance] MDBG TNSN ‘Macrocarpa’ [C. mas L.] DCNA mas L. Cornelian Cherry DCCG MDBG TNCT VAGW DCNA MDJH TNTV DEWG MDLT VACW ‘Milky Way’ [C. kousa (Buerger ex Miq.) Hance] DCNA DEWG ‘Nana’ [C. mas L.] VAGS florida L. f. pluribracteata Rehd. Double-flowered D. asperifolia Michx. DCNA LAAL SCBR ‘Aurea’ [C. mas L.] DCNA australis C.A.Mey. var. koenigii (Schneid.) Wang. DCNA MDGD ‘Bay Beauty’ [C. florida L.] Double-flowered D. TXLL ‘Cherokee Chief [C. florida L.] DCNA ‘Cherokee Princess’ [C. florida L.] DCNA ‘Cherokee Sunset’ [C. florida L.] TNSN ‘Cloud Nine’ [C. florida L.] DCNA controversa Hemsl. Giant D. DEEM MDGJ TNPW GARW MDMG VAGS MDBG TNDG VANB coreana Wang. DCNA drummondtii C.A.Mey. AD COAL ‘Dwarf’ [C. florida L.] DCNA ‘First Lady’ [C. florida L.] DCNA TNBO ‘Flaviramea’ [C. sericea L.] MDBG MDLT VATA florida L. Flowering D. ALBG FLUF MDLT SCUC ARRB GAdJI MDWP SCWI DCCG GAKN NCCS TNMB DCFM LAAL NCPR VACW DCNA LAHG NCTE VAMP DCWH LASL SCBR VATA FLMG MDBG SCFW FLRD MDJS SCMP australis C.A.Mey. var. koenigii (Schneid.) Wang. DCNA MDGD CORYLOPSIS CORNUS CORNUS officinalis Sieb. & Zucc. Japanese Cornelian Cherry DCNA MDBG TNTV DEWG MDLT paucinervis Hance DCNA ‘Prosser Red’ [C. florida L.] DCNA racemosa Lam. Gray D. DEWG SCBR VAHB NCAS VAGW ‘Rainbow’ [C. florida L.] TNAB ‘Royal Red’ [C. florida L.] DCNA ‘Rubra’ [C. kousa (Buerger ex Miq.) Hance] DCNA ‘Salicifolia’ [C. florida L.] TNBO TNTV sanguinea L. DCNA sericea L. Red Osier LARP MSMN TNTV TXCT MDLT TNAB TNUT VACW ‘Sibirica’ [C. alba L.] Siberian D. ALBH ‘Springtime’ [C. florida L.] DCNA ‘Steele’s Fastigiata’ [C. florida L.] DCNA ‘Stokes’ Pink’ [C. florida L.] LAAU stolonifera Michx. = C. sericea stricta Lam. = C. foemina ‘Sweetwater’ [C. florida L.] DCNA ‘Variegata’ [C. kousa (Buerger ex Miq.) CORNUS officinalis Sieb. & Zucc. Japanese Cornelian Cherry DCNA MDBG TNTV DEWG MDLT paucinervis Hance DCNA ‘Prosser Red’ [C. florida L.] DCNA racemosa Lam. Gray D. DEWG SCBR VAHB NCAS VAGW ‘Rainbow’ [C. florida L.] TNAB ‘Royal Red’ [C. florida L.] DCNA ‘Rubra’ [C. kousa (Buerger ex Miq.) Hance] DCNA ‘Salicifolia’ [C. florida L.] TNBO TNTV sanguinea L. DCNA sericea L. Red Osier LARP MSMN TNTV TXCT MDLT TNAB TNUT VACW ‘Sibirica’ [C. alba L.] Siberian D. ALBH ‘Springtime’ [C. florida L.] DCNA ‘Steele’s Fastigiata’ [C. florida L.] DCNA ‘Stokes’ Pink’ [C. florida L.] LAAU stolonifera Michx. = C. sericea stricta Lam. = C. foemina ‘Sweetwater’ [C. florida L.] DCNA ‘Variegata’ [C. kousa (Buerger ex Miq.) Hance] DEWG ‘Variegata’ [C. mas L.] VAMP = walteri Wang. DCNA ‘Welch’s Junior Miss’ [C. florida L.] ALCW DCNA FLJU ‘Welchii’ [C. florida L.] DCNA GARW MDBG CORYLOPSIS Sieb. & Zucc. Winter Hazel HAMAMELIDACEAE Witch-hazel Family coreana Uyeki = C. glabrescens glabrescens Franch. & Sav. Fragrant W. DCNA MDBG MDKN gotoana Makino = C. glabrescens pauciflora Sieb. & Zucc. Buttercup W. DCNA MDBG VACW DEWG MDKN VAGS platypetala Rehd. & Wils. = C. sinensis var. calvescens sinensis Hemsl. var. calvescens Rehd. & Wils. DCNA DCPP MDFM VAGS DCNC MDBG MDKN sinensis Hemsl. var. calvescens Rehd. & Wils. f. veitchiana (Bean) Morley & Chao MDKN sinensis Hemsl. var. sinensis Chinese W. DCNA DEWG MDFM MDLT DCNC GACG MDGD VAGS spicata Sieb. & Zucc. DCNA MDBG NCBE VAPH DCNC MDKN VACW DEWG MDLT VAGS ‘Spring Purple’ [C. sinensis Hemsl. var. sinensis] DCNA veitchiana Bean = C. sinensis var. calvescens f. veitchiana ‘Variegata’ [C. mas L.] VAMP = walteri Wang. DCNA ‘Welch’s Junior Miss’ [C. MDGD florida L.] ALCW DCNA FLJU ‘Welchii’ [C. florida L.] DCNA GARW MDBG CORYLOPSIS Sieb. & Zucc. Winter Hazel HAMAMELIDACEAE Witch-hazel Family coreana Uyeki = C. glabrescens glabrescens Franch. & Sav. Fragrant W. DCNA MDBG MDKN gotoana Makino = C. glabrescens pauciflora Sieb. & Zucc. Buttercup W. DCNA MDBG VACW DEWG MDKN VAGS platypetala Rehd. & Wils. = C. sinensis var. calvescens sinensis Hemsl. var. calvescens Rehd. & Wils. DCNA DCPP MDFM VAGS DCNC MDBG MDKN sinensis Hemsl. var. calvescens Rehd. & Wils. f. veitchiana (Bean) Morley & Chao MDKN sinensis Hemsl. var. sinensis Chinese W. DCNA DEWG MDFM MDLT DCNC GACG MDGD VAGS spicata Sieb. & Zucc. DCNA MDBG NCBE VAPH DCNC MDKN VACW DEWG MDLT VAGS ‘Spring Purple’ [C. sinensis Hemsl. var. sinensis] DCNA veitchiana Bean = C. sinensis var. calvescens f. veitchiana walteri Wang. DCNA ‘Welch’s Junior Miss’ [C. florida L.] ALCW DCNA FLJU ‘Welchii’ [C. florida L.] DCNA GARW MDBG 55 CORYLOPSIS wilmottiae Rehd. = C. sinensis var. sinensis ‘Winterthur’ [C. sinensis Hems].] DEWG CORYLUS L. Hazelnut, Filbert BETULACEAE Birch Family [isi ae ES ae ee eT oe ee a Se americana Marsh. American H. GAFN MDBG SCCU ‘Atropurpurea’ [C. avellana L.] = ‘Fusco-rubra’ avellana L. European H. DCCG DCNA MDLA SNA DCDO MDGJ MDPJ chinensis Franch. Chinese F. MDGD colurna L. Turkish F. DCWR MDAB MDGD ‘Contorta’ [C. avellana L.] Harry Lauder’s Walking-stick GAEC MDBG VAGS GAEH TNUT VATA cornuta Marsh. Beaked F. VAHR ‘Cosford’ [C. avellana L.] MDAB ‘Fusco-rubra’ [C. avellana L.] Purple-leaved H. DCAE DCNA DCCG DEWG MDHN TNSN ‘Potomac’ [C. americana < C. avellana ]} MDAB ‘Purpurea’ [C. maxima Mill.] Purple-leaved F. TNUT ‘Reed’ [C. americana < C. avellana ] COTONEASTER COTONEASTER sieboldiana BI. var. mandschurica (Maxim. & Rupr.) Schneid. DCNA COTINUS Mill. Smoke Tree ANACARDIACEAE Cashew Family americanus Nutt. = C. obovatus ‘Atropurpurea’ [C. coggygria Scop.] = ‘Purpureus’ ‘Baby Doll’ [C. coggygria Scop.] TNTV coggygria Scop. Smoke Tree DCCG MDHN VABF VAGW DCNA SCCU VACP VAMF DEMC INS VACW VAMP cotinoides (Nutt. ex Chapm.) Britton = C. obovatus ‘Flame’ [C. coggygria Scop.] DCNA ‘Nordine Red’ [C. coggygria Scop.] DCNA TNSC obovatus Raf. American Smoke Tree ALAU GAEC SCWI TNSN ALBH MDDS TNSC ‘Purpureus’ [C. coggygria Scop.] DCNA MDJS TNSC VACW GAUG MDKN TNUT ‘Royal Purple’ [C. coggygria Scop.] TNSC ‘Rubrifolius’ [C. coggygria Scop.] DCNA ‘Velvet Cloak’ [C. coggygria Scop.] DCNA COTONEASTER Medik. ROSACEAE Rose Family acutifolius Turcz. sieboldiana BI. var. mandschurica (Maxim. MDGD & Rupr.) Schneid. DCNA COTINUS Mill. Smoke Tree ANACARDIACEAE Cashew Family americanus Nutt. = C. obovatus ‘Atropurpurea’ [C. coggygria Scop.] = ‘Purpureus’ ‘Baby Doll’ [C. coggygria Scop.] TNTV coggygria Scop. Smoke Tree DCCG MDHN VABF VAGW DCNA SCCU VACP VAMF DEMC INS VACW VAMP cotinoides (Nutt. ex Chapm.) Britton = C. obovatus ‘Flame’ [C. coggygria Scop.] DCNA ‘Nordine Red’ [C. coggygria Scop.] DCNA TNSC obovatus Raf. American Smoke Tree ALAU GAEC SCWI TNSN ALBH MDDS TNSC ‘Purpureus’ [C. coggygria Scop.] DCNA MDJS TNSC VACW GAUG MDKN TNUT ‘Royal Purple’ [C. coggygria Scop.] TNSC ‘Rubrifolius’ [C. coggygria Scop.] DCNA ‘Velvet Cloak’ [C. coggygria Scop.] DCNA COTONEASTER Medik. ROSACEAE Rose Family acutifolius Turcz. COTONEASTER COTONEASTER dammeri Schneid.] GAUG splendens Flinck & Hylmo ALBH sternianus (Turrill) Boom DCNA MDTD ‘Variegata’ [C. horizontalis Decne.] MDBG MDLT <x watereri Exell [C. frigidus x C. salicifolius] DCNA wilsonti Nakai TNSN zabelii Schneid. GAWH X CRATAEGOSORBUS Makino ex Pojark. ROSACEAE [Crataegus < Sorbus] Rose Family miczurinii Pojark. [Crataegus sanguinea < Sorbus aucuparia] DCNA CRATAEGUS lL. Hawthorn ROSACEAE Rose Family aestivalis (Walt.) T. & G. May H. DCNA GAEC VACW FLUF MSMN arnoldiana Sarg. DEWG ‘Autumn Glory’ [C. laevigata (Poir.) DC.] DCNA brachyacantha Sarg. & Engelm. COTONEASTER COTONEASTER ignavus E.Wolf MDTD integerrimus Medik. European C. MDJH lacteus W.W.Sm. GATS GAUG GAWH ‘Lowfast’ [C. dammeri Schneid.] VATA lucidus Schlechtend. TNSN microphyllus Wall. ex Lindl. DCNA MDLT VATA microphyllus Wall. ex Lindl. f. thymifolius (Lindl.) Koehne DCNA var. cochleatus (Franch.) Rehd. & Wils. microphyllus Wall. ex Lindl. VACW TNSN microphyllus Wall. ex Lindl. var. glacialis Hook.f. = C. congestus nanshan Mottet MDLT nitens Rehd. & Wils. DEMC nummularius Fischer & C.A.Mey. DCNA obscurus Rehd. & Wils. Bloodberry C. SCCU VABF ‘Parkteppich’ [C. salicifolius Franch.] DCNA racemiflorus (Desf.) K.Koch MDGD racemiflorus (Desf.) K.Koch var. songoricus (Regel & Herd.) Schneid. DEMC roseus Edgew. DEMC MDTD ‘Royal Beauty’ [C. dammeri Schneid.] GAAB VATA salicifolius Franch. ambiguus Rehd. & Wils. VABF apiculatus Rehd. & Wils. Cranberry C. ALBH VATA cochleatus (Franch.) Klotz. = C. microphyllus var. cochleatus congestus Baker VAGS conspicuus Marq. Wintergreen C. DCNA GACG ‘Cornubia’ [C. < watereri Exell] [C. frigidus x C. salicifolius] DCNA dammeri Schneid. MDBG SCWI ‘Decorus’ [C. conspicuus Marq.] GACG SCCU dielsianus Pritz. ex Diels Diels C. MDBG VABF divaricatus Rehd. & Wils. Spreading C. MDNA floccosus (Rehd. & Wils.) Flinck & Hylmo DCNA NCTE foveolatus Rehd. & Wils. DEMC franchetii Bois DCNA MDLT MDNA SCCU henryanus Hort. not (Schneid.) Rehd. & Wils. = C. salicifolius ‘Hessei’ [C. adpressus Bois] DCNA horizontalis Decne. Rock C. DCLC GAUG MDLT VATA DCNA MDBG SCCU horizontalis Decne. var. perpusillus Schneid. DCNA hupehensis Rehd. & Wils. = C. silvestri microphyllus Wall. ex Lindl. f. thymifolius (Lindl.) Koehne DCNA microphyllus Wall. ex Lindl. f. thymifolius (Lindl.) Koehne DCNA 57 COTONEASTER salicifolius Franch. var. floccosus Rehd. & Wils. = C. floccosus ‘Scarlet Leader’ [C. salicifolius Franch.] MDBG silvestrii Pamp. DEWG MDTD ‘Skogsholmen’ [C. dammeri Schneid.] GAUG splendens Flinck & Hylmo ALBH sternianus (Turrill) Boom DCNA MDTD ‘Variegata’ [C. horizontalis Decne.] MDBG MDLT <x watereri Exell [C. frigidus x C. salicifolius] DCNA wilsonti Nakai TNSN zabelii Schneid. GAWH X CRATAEGOSORBUS Makino ex Pojark. ROSACEAE [Crataegus < Sorbus] Rose Family miczurinii Pojark. [Crataegus sanguinea < Sorbus aucuparia] DCNA CRATAEGUS lL. Hawthorn ROSACEAE Rose Family aestivalis (Walt.) T. & G. May H. DCNA GAEC VACW FLUF MSMN arnoldiana Sarg. DEWG ‘Autumn Glory’ [C. laevigata (Poir.) DC.] DCNA brachyacantha Sarg. & Engelm. COTONEASTER salicifolius Franch. var. floccosus Rehd. & Wils. = C. floccosus ‘Scarlet Leader’ [C. salicifolius Franch.] MDBG silvestrii Pamp. DEWG MDTD ‘Skogsholmen’ [C. COTONEASTER CRATAEGUS CRATAEGUS calpodendron (Ehrh.) Medik. Pear H. TNGN ‘Carrierei’ [C. x lavallei Herincq ex Lav.] [C. stipulacea x C. crus-galli] DCSH VAMP coccinioides Ashe TNGN compta Sarg. MDAC concinna Beadle GACG cordata Ait. = C. phaenopyrum crus-galli L. Cockspur H. DECG DCNG GANG TNLW flava Ait. Yellow H. FLUF floridana Sarg. FLUF fulleriana Sarg. DCRC DEEM DEWG ‘Inermis’ [C. monogyna Jacq.] DCNA ‘Inermis’ [C. punctata Jacq.] DCNA intricata J.Lange Thicket H. VACW lacrimata Small GAEC SCWI laevigata (Poir.) DC. English H. DCNA MDKN < lavallei Herincq ex Lav. [C. stipulacea x C. crus-galli} DCSH VAMP macrosperma Ashe DCWH TNBO calpodendron (Ehrh.) Medik. Pear H. TNGN ‘Carrierei’ [C. x lavallei Herincq ex Lav.] [C. stipulacea x C. crus-galli] DCSH VAMP coccinioides Ashe TNGN compta Sarg. MDAC concinna Beadle GACG cordata Ait. = C. phaenopyrum crus-galli L. Cockspur H. DECG DCNG GANG TNLW flava Ait. Yellow H. FLUF floridana Sarg. FLUF fulleriana Sarg. DCRC DEEM DEWG ‘Inermis’ [C. monogyna Jacq.] DCNA ‘Inermis’ [C. punctata Jacq.] DCNA intricata J.Lange Thicket H. VACW lacrimata Small GAEC SCWI laevigata (Poir.) DC. English H. DCNA MDKN < lavallei Herincq ex Lav. [C. stipulacea x C. crus-galli} DCSH VAMP macrosperma Ashe DCWH TNBO marshallii Egglest. Parsley H. ARRB LALG TXSE X CRATAEGOSORBUS Makino ex Pojark. ROSACEAE [Crataegus < Sorbus] Rose Family miczurinii Pojark. [Crataegus sanguinea < Sorbus aucuparia] DCNA CRATAEGUS lL. Hawthorn ROSACEAE Rose Family aestivalis (Walt.) T. & G. May H. DCNA GAEC VACW FLUF MSMN arnoldiana Sarg. DEWG ‘Autumn Glory’ [C. laevigata (Poir.) DC.] DCNA brachyacantha Sarg. & Engelm. Blueberry H. TXDM X CRATAEGOSORBUS Makino ex Pojark. ROSACEAE [Crataegus < Sorbus] Rose Family miczurinii Pojark. [Crataegus sanguinea < Sorbus aucuparia] DCNA CRATAEGUS lL. Hawthorn ROSACEAE Rose Family aestivalis (Walt.) T. & G. May H. DCNA GAEC VACW FLUF MSMN X CRATAEGOSORBUS Makino ex Pojark. ROSACEAE [Crataegus < Sorbus] Rose Family miczurinii Pojark. [Crataegus sanguinea < Sorbus aucuparia] DCNA CRATAEGUS lL. Hawthorn ROSACEAE Rose Family aestivalis (Walt.) T. & G. May H. DCNA GAEC VACW FLUF MSMN arnoldiana Sarg. DEWG ‘Autumn Glory’ [C. laevigata (Poir.) DC.] DCNA brachyacantha Sarg. & Engelm. Blueberry H. TXDM 58 CRATAEGUS mollis (T. & G.) Scheele Downy H. DEMC TNGN monogyna Jacq. Common H. DCGW MDBG MDGB VAGW DCNA MDCD TNGN VAMP GAUG MDCP VACW opaca Hook. & Arn. DCNA VATA oxyacantha L. = C. laevigata ‘Paul’s Scarlet’ [C. laevigata (Poir.) DC.] DEMC MDKN NCBE pennsylvanica Ashe DEWG phaenopyrum (L.f.) Medik. CRYPTOMERIA CRYPTOMERIA ‘Enko sugi’ [C. japonica (Thunb. ex L.f.) D.Don] DCNA ‘Globosa’ [C. japonica (Thunb. ex L.f.) D.Don] LASL ‘Globosa Nana’ [C. japonica (Thunb. ex Lf.) D.Don] DCNA japonica (Thunb. ex L.f.) D.Don Japanese Cedar ALBG GAHC MDHN SCMP ALEH GAJI MDJH SCPP ALFN GATN MDJS TNMB DCCG LAAL MSHB TXAB DCNA LARP NCDU VAMP FLMG MDBG SCBR FLUF MDEF SCDB GAAB MDFM SCMG ‘Jindai sugi’ [C. japonica (Thunb. ex L.f.) D.Don] ALFN DCNA ‘Knaptonensis’ [C. japonica (Thunb. ex L.f.) D.Don] MDBA ‘Lobbii’ [C. japonica (Thunb. ex L.f.) D.Don] SECU ‘Lycopodioides’ [C. japonica (Thunb. ex L.f.) D.Don] MDJS ‘Pygmaea’ [C. japonica (Thunb. ex L.f.) D.Don] DCNA ‘Vilmoriniana’ [C. japonica (Thunb. ex L.f.) D.Don] DCNA FLUF MDBG VAGS ‘Yoshino’ [C. japonica (Thunb. ex L.f.) D.Don] DCNA CUDRANIA Trecul MORACEAE Mulberry Family tricuspidata (Carr.) Bur. ex Lav. CUNNINGHAMIA R.Br. China Fir TAXODIACEAE Taxodium Family ‘Glauca’ [C. lanceolata (Lamb.) Hook.f.] Blue C.F. GAAB MDLT MDPJ VATW MDJS MDMG TNHD VAWR lanceolata (Lamb.) Hook.f. China F. ALTD GACG MSEN TNMB FLDG GAHC NCBE TNSN FLIF GAIS NCKH TNTV FLMG GAVI NCTE VAMP BERS LAAL NCWK FLUF LASL SCCU GABG MDBG SCHA CUPHEA P.Br. LYTHRACEAE Loosestrife Family a I ST ES Ok SEE TE ET hyssopifolia HBK. False Heather, Elfin Herb FLUF GACG LAHG LALG micropetala HBK. LAHG LASL x CUPRESSOCYPARIS Dallim. CUPRESSACEAE Cypress Family [Chamaecyparis < Cupressus] ‘Leighton Green’ [<C. leylandii (Dallim. & A.B.Jacks.) Dallim.] [Chamaecyparis nootkatensis < Cupressus macrocarpa] DCNA leylandii (Dallim. & A.B.Jacks.) Dallim. Leyland Cypress [Chamaecyparis nootkatensis < Cupressus macrocarpa] ALAU MDLT SCBR VATA MDBG NCDU SCCU * Silver Dust [<C. leylandii (Dallim. & A.B.Jacks.) Dallim.] [Chamaecyparis nootkatensis « Cupressus CUNNINGHAMIA R.Br. China Fir TAXODIACEAE Taxodium Family ‘Glauca’ [C. lanceolata (Lamb.) Hook.f.] Blue C.F. GAAB MDLT MDPJ VATW MDJS MDMG TNHD VAWR lanceolata (Lamb.) Hook.f. China F. ALTD GACG MSEN TNMB FLDG GAHC NCBE TNSN FLIF GAIS NCKH TNTV FLMG GAVI NCTE VAMP BERS LAAL NCWK FLUF LASL SCCU GABG MDBG SCHA CUPHEA P.Br. LYTHRACEAE Loosestrife Family a I ST ES Ok SEE TE ET hyssopifolia HBK. False Heather, Elfin Herb FLUF GACG LAHG LALG micropetala HBK. LAHG LASL x CUPRESSOCYPARIS Dallim. CUPRESSACEAE Cypress Family [Chamaecyparis < Cupressus] ‘Leighton Green’ [<C. leylandii (Dallim. & A.B.Jacks.) Dallim.] [Chamaecyparis nootkatensis < Cupressus macrocarpa] DCNA leylandii (Dallim. & A.B.Jacks.) Dallim. Leyland Cypress [Chamaecyparis nootkatensis < Cupressus macrocarpa] ALAU MDLT SCBR VATA MDBG NCDU SCCU * Silver Dust [<C. leylandii (Dallim. COTONEASTER Washington Thorn DCBG DEMC MDAC VAGW DCNA GAUG MDLT VATA pinnatifida Bunge MDGD ‘Plena’ [C. laevigata (Poir.) DC.] MDKN populifolia Walt. FLEM ‘Praecox’ [C. monogyna Jacq.] DCNC pringlei Sarg. Pringle H. MDAC pruinosa (H.Wendl.) K.Koch var. leiophylla (Sarg.) Phipps DECK DCNA punctata Jacq. Dotted H. TNGN VAAH schuettei Ashe DCCK spathulata Michx. Littlehip H. CRATAEGUS mollis (T. & G.) Scheele Downy H. DEMC TNGN monogyna Jacq. Common H. DCGW MDBG MDGB VAGW DCNA MDCD TNGN VAMP GAUG MDCP VACW opaca Hook. & Arn. DCNA VATA oxyacantha L. = C. laevigata ‘Paul’s Scarlet’ [C. laevigata (Poir.) DC.] DEMC MDKN NCBE pennsylvanica Ashe DEWG phaenopyrum (L.f.) Medik. Washington Thorn DCBG DEMC MDAC VAGW DCNA GAUG MDLT VATA pinnatifida Bunge MDGD ‘Plena’ [C. laevigata (Poir.) DC.] MDKN populifolia Walt. FLEM ‘Praecox’ [C. monogyna Jacq.] DCNC pringlei Sarg. Pringle H. MDAC pruinosa (H.Wendl.) K.Koch var. leiophylla (Sarg.) Phipps DECK DCNA punctata Jacq. Dotted H. TNGN VAAH schuettei Ashe DCCK spathulata Michx. Littlehip H. CRYPTOMERIA CRYPTOMERIA ‘Superba’ [C. laevigata (Roir.) DC.] DCNA ‘Toba’ [C. X mordenensis Boom] [C. laevigata ‘Paul’s Scarlet’ «C. succulenta] DCNA uniflora Moench ALAU FLUF vaileae Britton MDCD MDGB viridis L. Green H. ARPT GACG MDUM SCWP DEWG MDBP SCBR TNMB ‘Winter King’ [C. viridis L.] DCNA CROOKEA Small = HYPERICUM microsepalum CROTON L. EUPHORBIACEAE Spurge Family alabamensis E.A.Sm. Alabama C. ALTD GACG MDLT VACW ALUA GAEC TNCT DCNA MDJW VACG CRYPTOMERIA D.Don TAXODIACEAE Taxodium Family ESE ee ae ee ee ee ‘Bandai sugi’ [C. japonica (Thunb. ex L.f.) D.Don] MDPJ ‘Cristata’ [C. japonica (Thunb. ex L.f.) D.Don] MDBG ‘Dacrydioides’ [C. japonica (Thunb. ex L.f.) ‘Elegans’ [C. japonica (Thunb. ex L.f.) D.Don] MDJW MDWP VAGS x CUPRESSOCYPARIS x CUPRESSOCYPARIS CRYPTOMERIA CRYPTOMERIA & A.B.Jacks.) Dallim.] [Chamaecyparis nootkatensis « Cupressus macrocarpa] (Sport of <C. leylandii ‘Leighton Green’) DCNA GAEC MDBG GACG LASL VATA CUPHEA P.Br. LYTHRACEAE Loosestrife Family a I ST ES Ok SEE TE ET hyssopifolia HBK. False Heather, Elfin Herb FLUF GACG LAHG LALG micropetala HBK. LAHG LASL x CUPRESSOCYPARIS Dallim. CUPRESSACEAE Cypress Family [Chamaecyparis < Cupressus] ‘Leighton Green’ [<C. leylandii (Dallim. & A.B.Jacks.) Dallim.] [Chamaecyparis nootkatensis < Cupressus macrocarpa] DCNA leylandii (Dallim. & A.B.Jacks.) Dallim. Leyland Cypress [Chamaecyparis nootkatensis < Cupressus macrocarpa] ALAU MDLT SCBR VATA MDBG NCDU SCCU * Silver Dust [<C. leylandii (Dallim. & A.B.Jacks.) Dallim.] [Chamaecyparis nootkatensis « Cupressus macrocarpa] (Sport of <C. leylandii ‘Leighton Green’) DCNA GAEC MDBG GACG LASL VATA ‘Pygmaea’ [C. japonica (Thunb. ex L.f.) D.Don] DCNA (e)) © DALBERGIA CUPRESSUS CUPRESSUS lL. Cypress CUPRESSACEAE Cypress Family arizonica Greene ALBG GAAB GAUG ARHS GAEC SCCU FLUF GAPM VATA funebris Endl. = Chamaecyparis funebris ‘Gareei’ [C. arizonica Greene] FLUF lusitanica Mill. Portuguese C. FLMG FLUF macrocarpa Hartw. Monterey C. SCHA sempervirens L. Italian C. ALSH GAJI MSHB VAGW FLMG GANG SCMG FLUF GASU VACW CYCAS L. CYCADACEAE Cycad Family circinalis L. Queen Sago FLUF revoluta Thunb. Sago Palm ALBG FLMG SCNS ALSM FLUF TXJIS CYDONIA Mill. Quince ROSACEAE Rose Family Se Oe EE Ne "Sai PON ne MPa TE TT] oblonga Mill. Common Q. DCNA MDGD NCTP VACW MDAB MSHB SCUC VASK sinensis Thouin = Pseudocydonia sinensis by CYRILLA Gard. ex L. CYRILLACEAE Cyrilla Family racemiflora L. Leatherwood, Titi LALG LASL MDLT NCBE NCGP SCBR CYTISUS Desf. Broom FABACEAE (Faboideae) Bean Family a A RO ENED YEO RLS UA Pe] ‘Andreanus’ [C. scoparius (L.) Link] DEMC VAGS battandieri Maire DCNA commutatus (Willd.) Briq. MDSC decumbens (Durande) Spach MDSC ‘Hollandia’ [C. x praecox Bean] [C. multiflorus < C. purgans] DCNA ‘Moonlight’ [C. x praecox Bean] [C. multiflorus < C. purgans] MDBG X praecox Bean Warminster B. [C. multiflorus < C. purgans] DEWG MDKN VAGS GACG MDSC scoparius (L.) Link Scotch B. DEMC NCGR VAMP GACG VAGS VANB DALBERGIA Lf. FABACEAE (Faboideae) Bean Family CYTISUS Desf. Broom FABACEAE (Faboideae) Bean Family a A RO ENED YEO RLS UA Pe] ‘Andreanus’ [C. scoparius (L.) Link] DEMC VAGS battandieri Maire DCNA commutatus (Willd.) Briq. MDSC decumbens (Durande) Spach MDSC ‘Hollandia’ [C. x praecox Bean] [C. multiflorus < C. purgans] DCNA ‘Moonlight’ [C. x praecox Bean] [C. multiflorus < C. purgans] MDBG X praecox Bean Warminster B. [C. CRYPTOMERIA multiflorus < C. purgans] DEWG MDKN VAGS GACG MDSC scoparius (L.) Link Scotch B. DEMC NCGR VAMP GACG VAGS VANB DALBERGIA Lf. FABACEAE (Faboideae) Bean Family hupeana Hance DCNA sissoo Roxb. ex DC. Sissoo FLUF 61 DANAE DANAE Medik. LILIACEAE Lily Family racemosa (L.) Moench Alexandrian Laurel GACG MDMG NCWM GAJS NCDU VAGW GAJY NCOP VANB DAPHNE L. THYMELAEACEAE Mezereum Family ‘Alba’ [D. odora Thunb. ex J.A.Murr.] VAPH ‘Albo-marginata’ [D. odora Thunb. ex J.A.Murr.] DCNA GAHC NCEG FLDG MDLT VANB ‘Aureo-marginata’ [D. odora Thunb. ex J.A.Murr.] SCMP ‘Carol Mackie’ [D. < burkwoodii Turrill] [D. caucasica < D. cneorum] VAPH caucasica Pall. MDLT cneorum L. Garland Flower, Rose D. MDLT VAPH genkwa Sieb. & Zucc. Lilac D. DCNA MDLT VAGS kamtschatica Maxim. var. jezoensis (Maxim.) Ohwi DCNA ‘Manten’ [D. x mantensiana T.M.C.Taylor & F. Vrugtman] [D. Xburkwoodii < D. retusa] DCNA MDLT mezereum L. February Daphne, Mezereon MDLT odora Thunb. ex J.A.Murr. DANAE DANAE Medik. LILIACEAE Lily Family racemosa (L.) Moench Alexandrian Laurel GACG MDMG NCWM GAJS NCDU VAGW GAJY NCOP VANB DAPHNE L. THYMELAEACEAE Mezereum Family ‘Alba’ [D. odora Thunb. ex J.A.Murr.] VAPH ‘Albo-marginata’ [D. odora Thunb. ex J.A.Murr.] DCNA GAHC NCEG FLDG MDLT VANB ‘Aureo-marginata’ [D. odora Thunb. ex J.A.Murr.] SCMP ‘Carol Mackie’ [D. < burkwoodii Turrill] [D. caucasica < D. cneorum] VAPH caucasica Pall. MDLT cneorum L. Garland Flower, Rose D. MDLT VAPH genkwa Sieb. & Zucc. Lilac D. DCNA MDLT VAGS kamtschatica Maxim. var. jezoensis (Maxim.) Ohwi DCNA ‘Manten’ [D. x mantensiana T.M.C.Taylor & F. Vrugtman] [D. Xburkwoodii < D. retusa] DCNA MDLT mezereum L. February Daphne, Mezereon MDLT odora Thunb. ex J.A.Murr. Winter D. GACG NCEG SCMP DENDROPANAX DANAE DANAE Medik. LILIACEAE Lily Family racemosa (L.) Moench Alexandrian Laurel GACG MDMG NCWM GAJS NCDU VAGW GAJY NCOP VANB DAPHNE L. THYMELAEACEAE Mezereum Family ‘Alba’ [D. odora Thunb. ex J.A.Murr.] VAPH ‘Albo-marginata’ [D. odora Thunb. ex J.A.Murr.] DCNA GAHC NCEG FLDG MDLT VANB ‘Aureo-marginata’ [D. odora Thunb. ex J.A.Murr.] SCMP ‘Carol Mackie’ [D. < burkwoodii Turrill] [D. caucasica < D. cneorum] VAPH caucasica Pall. MDLT cneorum L. Garland Flower, Rose D. MDLT VAPH genkwa Sieb. & Zucc. Lilac D. DCNA MDLT VAGS kamtschatica Maxim. var. jezoensis (Maxim.) Ohwi DCNA ‘Manten’ [D. x mantensiana T.M.C.Taylor & F. Vrugtman] [D. Xburkwoodii < D. retusa] DCNA MDLT mezereum L. February Daphne, Mezereon MDLT odora Thunb. ex J.A.Murr. Winter D. GACG NCEG SCMP GATS SCBR ‘Ringmaster’ [D. odora Thunb. CRYPTOMERIA ex DENDROPANAX ‘Ruby Glow’ [D. cneorum L.] DEMC ‘Somerset’ [D. X burkwoodii Turrill] [D. caucasica < D. cneorum] DEWG GACG VAPH tangutica Maxim. MDLT ‘Zuiko nishiki’ [D. odora Thunb. ex J.A.Murr.] MDBG DAPHNIPHYLLUM Bl. EUPHORBIACEAE Spurge Family macropodum Miq. ALEH DCNA FLRP LAAL DATURA L SOLANACEAE Nightshade Family suaveolens Humboldt & Bonpland ex Willd. = Brugmansia suaveolens DAUBENTONIA_ DC. = SESBANIA DAVIDIA Baill. NYSSACEAE Sour-gum Family involucrata Baill. var. vilmoriniana (Dode) Wang. Dove Tree DCNA MDHA VAAH MDDF MDHW VAWR DECUMARIA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family barbara L. Wood-vamp SCBR TNTV DENDROPANAX Decne. & Planch. ARALIACEAE Ginseng Family trifidus (Thunb. ex J.A.Murr.) Makino ex DECUMARIA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family barbara L. Wood-vamp SCBR TNTV DENDROPANAX Decne. & Planch. ARALIACEAE Ginseng Family trifidus (Thunb. ex J.A.Murr.) Makino ex Hara SCWI ‘@) VJ DAVIDIA involucrata var. vilmoriniana (Dode) Wang. [illustrator Susan M. Johnston] 63 DESMOTHAMNUS DESMOTHAMNUS Small = LYONIA [RNS MES \| AO OE ERIN BLA A RT! a ee ee DEUTZIA Thunb. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family RRS 7d ORE ORE TSP EIS OE SIO NE RS LR oh LO SDS a Ee ae a Bil <candelabrum (Lem.) Rehd. [D. gracilis x D. sieboldiana] DEWG <x candida (Lem.) Rehd. [D. <lemoinei < D. sieboldiana] MDGD ‘Candidissima’ [D. scabra Thunb.] DCSH GAOH LATU MSHB DEWG GARW MDGD NCCA FLDJ GAUG MDPJ VABF GACD LAHG MDSJ ‘Carminea’ [D. < rosea (Lem.) Rehd.] [D. gracilis <x D. purpurascens] DEWG MDSG chunti H.H.Hu DEWG ‘Conspicua’ [D. x elegantissima (Lem.) Rehd.] [D. purpurascens < D. sieboldiana] MDGD ‘Contraste’ [D. x hybrida E.Lemoine] [D. discolor < D. longifolia] MDGD discolor Hemsl. MDGD ‘Eburnea’ [D. < magnifica (Lem.) Rehd.] [D. scabra < D. vilmoriniae] MDGD x elegantissima (Lem.) Rehd. [D. purpurascens < D. sieboldiana] DEMC MDGD ‘Eminens’ [D. x magnifica (Lem.) Rehd.] [D. scabra < D. vilmoriniae] MDGD ‘Erecta’ [D. < magnifica (Lem.) Rehd.] [D. scabra < D. vilmoriniae] DEUTZIA glabrata Komar. DCNA gracilis Sieb. & Zucc. DCWR GAOH MDJS VADW DEEM GAUG MDLT VAGS DEWG LAHG SCFR GAFG MDCP VACW ‘Latiflora’ [D. x magnifica (Lem.) Rehd.] [D. scabra x D. vilmoriniae] MDGD <x lemoinei E.Lemoine ex Bois [D. gracilis < D. parviflora] DCDO MSHB ‘Magicien’ [D. < hybrida E.Lemoine] [D. discolor < D. longifolia] DEWG MDGD xX magnifica (Lem.) Rehd. [D. scabra < D. vilmoriniae] DEWG MDGD MDPJ ‘Mohican’ [D. gracilis Sieb. & Zucc.] MDBG ‘Nikko’ [D. gracilis Sieb. & Zucc.] DCNA ‘Plena’ [D. CRYPTOMERIA scabra Thunb.] DCSH MDPJ NCCW GABS MDSJ ‘Pride of Rochester’ [D. scabra Thunb.] VAAC < rosea (Lem.) Rehd. [D. gracilis <x D. purpurascens] MDKN MDPJ scabra Thunb. Rough-leaved Deutzia DCSH GAOH SCMG VAWP DEEM LAAL SCMP DEWG NCBE VAGW schneideriana Rehd. DEWG sieboldiana Maxim. DCNA LALG ‘Summer Snow’ [D. scabra Thunb.] MDBG MDMG ‘Suspensa’ [D. scabra Thunb.] MDGD ‘Watereri’ [D. scabra Thunb.] GAOH glabrata Komar. DCNA gracilis Sieb. & Zucc. DCWR GAOH MDJS VADW DEEM GAUG MDLT VAGS DEWG LAHG SCFR GAFG MDCP VACW ‘Latiflora’ [D. x magnifica (Lem.) Rehd.] [D. scabra x D. vilmoriniae] MDGD <x lemoinei E.Lemoine ex Bois [D. gracilis < D. parviflora] DCDO MSHB ‘Magicien’ [D. < hybrida E.Lemoine] [D. discolor < D. longifolia] DEWG MDGD xX magnifica (Lem.) Rehd. [D. scabra < D. vilmoriniae] DEWG MDGD MDPJ ‘Mohican’ [D. gracilis Sieb. & Zucc.] MDBG ‘Nikko’ [D. gracilis Sieb. & Zucc.] DCNA ‘Plena’ [D. scabra Thunb.] DCSH MDPJ NCCW GABS MDSJ ‘Pride of Rochester’ [D. scabra Thunb.] VAAC < rosea (Lem.) Rehd. [D. gracilis <x D. purpurascens] MDKN MDPJ scabra Thunb. Rough-leaved Deutzia DCSH GAOH SCMG VAWP DEEM LAAL SCMP DEWG NCBE VAGW schneideriana Rehd. DEWG sieboldiana Maxim. DCNA LALG ‘Summer Snow’ [D. scabra Thunb.] glabrata Komar. DCNA gracilis Sieb. & Zucc. DCWR GAOH MDJS VADW DEEM GAUG MDLT VAGS DEWG LAHG SCFR GAFG MDCP VACW ‘Latiflora’ [D. x magnifica (Lem.) Rehd.] [D. scabra x D. vilmoriniae] MDGD <x lemoinei E.Lemoine ex Bois [D. gracilis < D. parviflora] DCDO MSHB ‘Magicien’ [D. < hybrida E.Lemoine] [D. discolor < D. longifolia] DEWG MDGD xX magnifica (Lem.) Rehd. [D. scabra < D. vilmoriniae] DEWG MDGD MDPJ ‘Mohican’ [D. gracilis Sieb. & Zucc.] MDBG ‘Nikko’ [D. gracilis Sieb. & Zucc.] DCNA ‘Plena’ [D. scabra Thunb.] DCSH MDPJ NCCW GABS MDSJ ‘Pride of Rochester’ [D. scabra Thunb.] VAAC < rosea (Lem.) Rehd. [D. gracilis <x D. purpurascens] MDKN MDPJ scabra Thunb. Rough-leaved Deutzia DCSH GAOH SCMG VAWP DEEM LAAL SCMP DEWG NCBE VAGW schneideriana Rehd. DEWG sieboldiana Maxim. DCNA LALG ‘Summer Snow’ [D. scabra Thunb.] glabrata Komar. DCNA ©) es EDGEWORTHIA DIERVILLA DIERVILLA DIERVILLA Mill. Bush Honeysuckle CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) = es ee ee lonicera Mill. Dwarf Bush H. MDWP NCDB VAGS rivularis Gatt. Georgia Bush H. SCCU X splendens (Carr.) Kirchn. [D. lonicera X< D. sessilifolia] DEWG DIOON Lindl. CYCADACEAE Cycad Family Ge Ey ee ee SM RE er edule Lindl. Chestnut D. CRYPTOMERIA TXPS DIOSPYROS L. Persimmon EBENACEAE Ebony Family EES a PIS FS PO ORO Mer REE FEE Tee) ‘Hagabushi’ [D. kaki L.f.] MDAB kaki L.f. Kaki, Japanese P. DCCA DCHM TXHL DCCL SCCC VANB ‘Kyungsun bansi’ [D. kaki L.f.] MDAB sinensis Hemsl. Chinese P. DCNA GAIS texana Scheele Texas P. TXDC TXDM TXPS virginiana L. Common P. ALAU DEWG MDLT TNSN DCCG MDAB MSEP VAMP DCSH MDBP SCBR DIPELTA Maxim. = CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) floribunda Maxim. MDGD yunnanensis Franch. DCNA Mezereum Family HAMAMELIDACEAE Witch-hazel Family cercidifolius Maxim. MDBG MDFM MDJS MDKN DISTYLIUM Sieb. & Zucc. HAMAMELIDACEAE Witch-hazel Family ‘Akebono’ [D. racemosum Sieb. & Zucc.] MDBG racemosum Sieb. & Zucc. ALTD FLRH GAIS TXPS DCNA FLUF LASL VAWM DOXANTHA Miers = MACFADYENA DURANTA L. VERBENACEAE Verbena Family repens L. Creeping Skyflower FLMG GACG LAAL FLUF GAJI LASL EDGEWORTHIA Meisn. THYMELAEACEAE Mezereum Family chrysantha Lindl. Paperbush GACG GAEC MDFM = MACFADYENA 65 va naeee \\\\ ‘ wee a yi! DISANTHUS cercidifolius Maxim. [illustrator Susan M. Johnston] ep) oO) ERICA EHRETIA ERICA umbellata Thunb. ex J.A.Murr. Autumn Olive ALON LASL SCBR VAGW ALTD MDLT SCCU VAWR DCNA MDPJ SCDB DEMC MSHB SCMM LAHG NCBE VACW ‘Variegata’ [E. pungens Thunb. ex J.A.Murr.] VATA ELEUTHEROCOCCUS Maxim. ARALIACEAE Ginseng Family gracilistylus (W.W.Sm.) S.Y.Hu MDBG sieboldianus (Makino) Koidz. VATA trifoliatus (L.) S.Y.Hu FLUF GATS ELLIOTTIA Muh. ex Ell. ERICACEAE Heath Family bracteata (Maxim.) Hook.f. MDLT racemosa Muhl. ex El. Southern Plume GATS MDFM NCBE ENKIANTHUS Lour. ERICACEAE Heath Family ‘Ambassador’ [E. campanulatus (Miq.) Nichols.] MDKN campanulatus (Miq.) Nichols. DEWG MDBG MDLT NCBE GAEC MDJS MDMG perulatus (Miq.) Schneid. DCNA DEWG MDBG VAMP ERICA L. Heath ERICACEAE Heath Family CSRS PE a EE CP A Ie OS ST 7] carnea L. Spring H. umbellata Thunb. ex J.A.Murr. Autumn Olive ALON LASL SCBR VAGW ALTD MDLT SCCU VAWR DCNA MDPJ SCDB DEMC MSHB SCMM LAHG NCBE VACW ‘Variegata’ [E. pungens Thunb. ex J.A.Murr.] VATA ELEUTHEROCOCCUS Maxim. ARALIACEAE Ginseng Family gracilistylus (W.W.Sm.) S.Y.Hu MDBG sieboldianus (Makino) Koidz. VATA trifoliatus (L.) S.Y.Hu FLUF GATS ELLIOTTIA Muh. ex Ell. ERICACEAE Heath Family bracteata (Maxim.) Hook.f. MDLT racemosa Muhl. ex El. Southern Plume GATS MDFM NCBE ENKIANTHUS Lour. ERICACEAE Heath Family ‘Ambassador’ [E. campanulatus (Miq.) Nichols.] MDKN campanulatus (Miq.) Nichols. DEWG MDBG MDLT NCBE GAEC MDJS MDMG perulatus (Miq.) Schneid. DCNA DEWG MDBG VAMP ERICA L. CRYPTOMERIA Heath ERICACEAE Heath Family CSRS PE a EE CP A Ie OS ST 7] EHRETIA P.Br. BORAGINACEAE Borage Family —_ a EIS ME SILT ey ee ee) acuminata R.Br. var. serrata (Roxb.) I.M.Johnst. FLUF anacua (Teran & Berl.) I.M.Johnst. LAAL TXAB TXGM TXPS thyrsiflora (Sieb. & Zucc.) Nakai = E. acuminata var. serrata ELAEAGNUS lL. ELAEAGNACEAE Oleaster Family ST aE OAS TRE TSS RS TS ES) angustifolia L. Russian Olive MDJS MDSJ ‘Aurea’ [E. pungens Thunb. ex J.A.Murr.] SCMA ‘Aureo-variegata’ [E. pungens Thunb. ex J.A.Murr.] = ‘Maculata’ x ebbingei Doorenbos [E. macrophylla x E. pungens] VATA ‘Ebbingei’ = E. x ebbingei ‘Fruitlandii’ [E. pungens Thunb. ex J.A.Murr.] GABS GAFN SCMP glabra Thunb. ex J.A.Murr. SCBR macrophylla Thunb. MDBG ‘Maculata’ [E. pungens Thunb. ex J.A.Murr.] ALAU FLMG GACG multiflora Thunb. ex J.A.Murr. Cherry E., Gumi MDKN SCCU pungens Thunb. ex J.A.Murr. Thorny E. ALAU GAUG SCCL VANB ALIT MDLT SCDB ARHS NCBE TNDG umbellata Thunb. ex J.A.Murr. DCNA MDPJ SCDB DEMC MSHB SCMM LAHG NCBE VACW ‘Variegata’ [E. pungens Thunb. ex J.A.Murr.] VATA ELEUTHEROCOCCUS Maxim. ARALIACEAE Ginseng Family gracilistylus (W.W.Sm.) S.Y.Hu MDBG sieboldianus (Makino) Koidz. VATA trifoliatus (L.) S.Y.Hu FLUF GATS ELLIOTTIA Muh. ex Ell. ERICACEAE Heath Family bracteata (Maxim.) Hook.f. MDLT racemosa Muhl. ex El. Southern Plume GATS MDFM NCBE ENKIANTHUS Lour. ERICACEAE Heath Family ‘Ambassador’ [E. campanulatus (Miq.) Heath Family 67 EUCOMMIA ERICA ‘Silberschmelze’ [E. x darleyensis Bean] [E. carnea X< E. mediterranea] MDWP ‘Springwood Pink’ [E. carnea L.] DCNA MDWP ‘Springwood White’ [E. carnea L.] DCNA MDBG vagans L. Cornish H. NCBE VAGS ERIOBOTRYA Lindl. ROSACEAE Loquat Rose Family deflexa (Hemsl.) Nakai FLUF LASL japonica (Thunb.) Lindl. Loquat ALBG FLUP NCEL VACW ALTM GADG SCBH VANB DCNA GAJI SCEM FLMG GATS SCNS ERYTHRINA L. FABACEAE (Faboideae) Bean Family ES SPT eae a ee A ee ee kee eS ee ee a) arborea (Chapm.) Small = E. herbacea x bidwillii Lindl. [E. crista-galli x E. herbacea] LAMP crista-galli L. Cockspur Coral Tree ALTD LAAL LANR FLUF LAMP MSHB herbacea L. Coral Bean DCNA MSEP SCBR herbacea L. var. arborea Chapm. = E. herbacea ESCALLONIA Mutis ex L.f. SAXIFRAGACEAE (Escallonioideae) Saxifrage Family bifida Link & Otto FLUF SCBG VANB GACH SCBR montevidensis (Cham. & Schlechtend.) DC. = E. bifida rosea Griseb. SCJF rubra (Ruiz & Pavon) Pers. LASL MSHB EUCALYPTUS MYRTACEAE Myrtle Family eT Re EE EIS CE TS EE) camaldulensis Dehnh. FLIF LHer. cocctifera Hook.f. CRYPTOMERIA Tasmanian Snow Gum SCJUF dalrympleana Maiden Mountain Gum SCJUF gunnii Hook.f. Cider Gum SCJUF robusta Sm. Swamp Mahogany FLUF LAAP TXZH viminalis Labill. Manna Gum ALEH EUCOMMIA Oliv. EUCOMMIACEAE Eucommia Family a eCeiiS On Si n oe Ee eo a ey] ulmoides Oliv. Chinese Rubber Tree MDGD TNSN VABF VATA ESCALLONIA Mutis ex L.f. SAXIFRAGACEAE (Escallonioideae) Saxifrage Family bifida Link & Otto FLUF SCBG VANB GACH SCBR montevidensis (Cham. & Schlechtend.) DC. = E. bifida rosea Griseb. SCJF rubra (Ruiz & Pavon) Pers. LASL MSHB EUCALYPTUS MYRTACEAE Myrtle Family eT Re EE EIS CE TS EE) camaldulensis Dehnh. FLIF LHer. cocctifera Hook.f. Tasmanian Snow Gum SCJUF dalrympleana Maiden Mountain Gum SCJUF gunnii Hook.f. Cider Gum SCJUF robusta Sm. Swamp Mahogany FLUF LAAP TXZH viminalis Labill. Manna Gum ALEH EUCOMMIA Oliv. EUCOMMIACEAE Eucommia Family a eCeiiS On Si n oe Ee eo a ey] ulmoides Oliv. Chinese Rubber Tree MDGD TNSN VABF VATA ESCALLONIA Mutis ex L.f. SAXIFRAGACEAE (Escallonioideae) Saxifrage Family ERYTHRINA L. FABACEAE (Faboideae) Bean F ES SPT eae a ee A ee ee kee eS arborea (Chapm.) Small = E. herbacea x bidwillii Lindl. [E. crista-galli x E. herbacea] LAMP crista-galli L. Cockspur Coral Tree ALTD LAAL LANR FLUF LAMP MSHB herbacea L. Coral Bean DCNA MSEP SCBR herbacea L. var. arborea Chapm. = E. herbacea herbacea L. var. arborea Chapm. = E. herbacea EUONYMUS EUODIA EUODIA J.R. &J.G. Forst. RUTACEAE Citrus Family a RE ET ean a Te aa daniellii (Benn.) Hemsl. DCNA MDGD NCWM VACW GAEH NCJP VABF VAPC EUONYMUS UL. Spindle Tree CELASTRACEAE Staff-tree Family alatus (Thunb. ex J.A.Murr.) Sieb. Winged Spindle T. DCDO MDGJ MDMG VACP DEWG MDJH NCBE VACW GACG MDJS NCWC VADW LAHG MDLA SCCU VAGW MDBG MDLT TNMB VASK alatus (Thunb. ex J.A.Murr.) Sieb. f. apterus (Regel) Rehd. MDDF ‘Albo-marginatus’ [E. japonicus Thunb.] ALHC VATW ‘Aldenhamensis’ [E. europaeus L.] TNSN TNTV americanus L. Strawberry Bush GABQ MDPL SCWI TXSE GAEC NCDU TXLL MDLT SCBR TXPS ‘Argenteo-marginatus’ [E. fortunei (Turcz.) Hand.-Mazz.] = ‘Variegatus’ ‘Argenteo-variegatus’ [E. japonicus Thunb.] LALG NCWM ‘Aureo-marginatus’ [E. japonicus Thunb.] FLMG LALG LASL TNCT ‘Aureus’ [E. japonicus Thunb.] ALBG GAIS MSRN FLMG LALG VADW bungeanus Maxim. Winterberry Spindle T. DCNA GAPN MDHN TXBA FLUF LASL NCDU VASK bungeanus Maxim. var. semipersistans ‘Compactus’ [E. alatus (Thunb. ex J.A.Murr.) Sieb.] DCNA GAUG SCCU GACG MDLT VATA ‘Duc d’Anjow’ [E. japonicus Thunb.] FLUF ‘Emerald Gaiety’ [E. fortunei (Turcz.) Hand.-Mazz.] (Plant Patent No. CRYPTOMERIA ROSACEAE Rose Family (EST a ES Gy aT ae SOT) giraldii Hesse = E. racemosa racemosa (Lindl.) Rehd. Pearlbush ALBH MDAB SCCU VAGS ALIT MDAF SCDB VAGW DCNA MDJH SCEI VAMP DEWG MDLA SCMM VATA GAHC MDSS SCMP LAAL MSEP TNRM LASL NCOP VACW ‘The Bride’ [E. « macrantha (V.Lemoine) Schneid.] [E. korolkowii < E. racemosa] DCNA DEWG MDLT VAPH FAGUS L. Beech FAGACEAE Beech Family ‘Asplenifolia’ [F. sylvatica L.] DCFP DCWH DEMC NCKH CRYPTOMERIA 1,960) GACG TNTV europaeus L. European Spindle T. GAUG NCBE fortunei (Turcz.) Hand.-Mazz. Wintercreeper Spindlebush ARWS GAJY NCEL VACP DEWG NCDU NCWC VATA Jfortunei (Turcz.) Hand.-Mazz. var. radicans (Miq.) Rehd. DCNA FLPM ‘Gracilis’ [E. fortunei (Turcz.) Hand.-Mazz.] ARSN DCNA GACD LALG ‘Harlequin’ [E. fortunei (Turcz.) Hand.-Mazz.] DCNA MDBG japonicus Thunb. Japanese Spindle T. DCNA MDNA NCWC GAIS MDRT SCCG MDJH MSHB SCUC kiautschovicus Loesn. GAEH GAUG SCCU VABF GAMM MDPJ SCUC VAWR lanceolatus Yatabe DCNA ‘Longwood’ [E. fortunei (Turcz.) Hand.-Mazz.] MDMG maackii Rupr. MDGD TXGS ‘Macrophyllus’ [E. japonicus Thunb.] DCNA EUODIA J.R. &J.G. Forst. RUTACEAE Citrus Family a RE ET ean a Te aa daniellii (Benn.) Hemsl. DCNA MDGD NCWM VACW GAEH NCJP VABF VAPC EUONYMUS UL. Spindle Tree CELASTRACEAE Staff-tree Family alatus (Thunb. ex J.A.Murr.) Sieb. Winged Spindle T. DCDO MDGJ MDMG VACP DEWG MDJH NCBE VACW GACG MDJS NCWC VADW LAHG MDLA SCCU VAGW MDBG MDLT TNMB VASK alatus (Thunb. ex J.A.Murr.) Sieb. f. apterus (Regel) Rehd. MDDF ‘Albo-marginatus’ [E. japonicus Thunb.] ALHC VATW ‘Aldenhamensis’ [E. europaeus L.] TNSN TNTV americanus L. Strawberry Bush GABQ MDPL SCWI TXSE GAEC NCDU TXLL MDLT SCBR TXPS ‘Argenteo-marginatus’ [E. fortunei (Turcz.) Hand.-Mazz.] = ‘Variegatus’ ‘Argenteo-variegatus’ [E. japonicus Thunb.] LALG NCWM ‘Aureo-marginatus’ [E. japonicus Thunb.] FLMG LALG LASL TNCT ‘Aureus’ [E. japonicus Thunb.] ALBG GAIS MSRN FLMG LALG VADW bungeanus Maxim. alatus (Thunb. ex J.A.Murr.) Sieb. 69 EUONYMUS ‘Minima’ [E. fortunei (Turcz.) Hand.-Mazz.] NCDU nanus Bieb. VAGS patens Rehd. = E. kiautschovicus ‘Sarcoxie’ [E. fortunei (Turcz.) Hand.- Mazz.] DCNA sieboldianus BI. DCNA TNTV VABF ‘Silver King’ [E. japonicus Thunb.] FLUF ‘Silver Queen’ [E. fortunei (Turcz.) Hand.- Mazz.] LALG MDET VATA ‘Variegatus’ [E. fortunei (Turcz.) Hand.- Mazz.] GACB VAHF wilsonii Sprague GACG EUPHORBIA L. Spurge EUPHORBIACEAE Spurge Family SE eS NES SE ES eS a oe characias L. ssp. wulfenii (Hoppe ex K.Koch) A.R.Sm. SCWI leucocephala Lotsy Pascuita FLUF pulcherrima Willd. Poinsettia FLUF EUPTELEA Sieb. & Zucc. EUPTELEACEAE Euptelea Family polyandra Sieb. & Zucc. MDMG EURYA Thunb. THEACEAE Tea Family acuminata DC. GAIS FAGUS ‘Confetti’ [E. japonica Thunb.] MDBG emarginata (Thunb. ex J.A.Murr.) Makino ALEH FLMG LASL SCBY ALTD FLUF SCBP FLLE LAAL SCBR ‘Harmony’ [E. japonica Thunb.] MDBG japonica Thunb. ALEH ALTD DCNA LAAL ochnacea (DC.) Szysz. = Cleyera japonica * ‘Winter Wine’ [E. japonica Thunb.] DCNA EUSCAPHIS Sieb. & Zucc. STAPHYLEACEAE Bladdernut Family japonica (Thunb. ex J.A.Murr.) Kanitz DCNA EVODIA Lam. = EUODIA EXOCHORDA. Lindl. EUONYMUS FAGUS ‘Confetti’ [E. japonica Thunb.] MDBG emarginata (Thunb. ex J.A.Murr.) Makino ALEH FLMG LASL SCBY ALTD FLUF SCBP FLLE LAAL SCBR ‘Harmony’ [E. japonica Thunb.] MDBG japonica Thunb. ALEH ALTD DCNA LAAL ochnacea (DC.) Szysz. = Cleyera japonica * ‘Winter Wine’ [E. japonica Thunb.] DCNA EUSCAPHIS Sieb. & Zucc. STAPHYLEACEAE Bladdernut Family japonica (Thunb. ex J.A.Murr.) Kanitz DCNA EVODIA Lam. = EUODIA EXOCHORDA. Lindl. ROSACEAE Rose Family (EST a ES Gy aT ae SOT) giraldii Hesse = E. racemosa racemosa (Lindl.) Rehd. Pearlbush ALBH MDAB SCCU VAGS ALIT MDAF SCDB VAGW DCNA MDJH SCEI VAMP DEWG MDLA SCMM VATA GAHC MDSS SCMP LAAL MSEP TNRM LASL NCOP VACW ‘The Bride’ [E. « macrantha (V.Lemoine) Schneid.] [E. korolkowii < E. racemosa] DCNA DEWG MDLT VAPH FAGUS L. Beech EUPHORBIA L. Spurge EUPHORBIACEAE Spurge Family SE eS NES SE ES eS a oe characias L. ssp. wulfenii (Hoppe ex K.Koch) A.R.Sm. SCWI leucocephala Lotsy Pascuita FLUF pulcherrima Willd. Poinsettia FLUF EUPTELEA Sieb. & Zucc. EUPTELEACEAE Euptelea Family polyandra Sieb. & Zucc. MDMG EURYA Thunb. THEACEAE Tea Family acuminata DC. GAIS EUPTELEA Sieb. & Zucc. EUPTELEACEAE Euptelea Family polyandra Sieb. & Zucc. MDMG EURYA Thunb. THEACEAE Tea Family acuminata DC. GAIS FICUS FAGUS FAGUS ‘Atropunicea’ [F. sylvatica L.] = F. sylvatica f. purpurea ‘Cristata’ [F. sylvatica L.] MDAN ‘Dawyck’ [F. sylvatica L.] Columnar Beech DEWG MDBG MDHC TNSN ‘Fastigiata’ [F. sylvatica L.] = ‘Dawyck’ grandifolia Ehrh. American B. ALBG DEMC MDWP £TNHD ALMC DEWG NCBE TNHG ALSH GAEH NCTE TNSC DCCG GAUG NCTP VAGG DCDO MDBG SCBR VAMP DCNA MDHN SCKG VAWR DCWH MDLA SCMP ‘Laciniata’ [F. sylvatica L.] Fernleaf Beech TNSM ‘Pendula’ [F. sylvatica L.] Weeping Beech DCAC DEWG VABR VAHC ‘Purpurea Tricolor’ [F. sylvatica L.] MDKN ‘Quercifolia’ [F. sylvatica L.] DCNA NCBE ‘Rohanii’ [F. sylvatica L.] DCNA MDAN TNSN TNTV ‘Rotundifolia’ [F. sylvatica L.] DCNA MDAN MDBG sylvatica L. European B. DCCG DCWH MDCP VASS sylvatica L. f. purpurea (Ait.) Schneid. Purple B. DCCG DCWH MDHC MDLA DCDO DEMC MDHN NCBE DCMS MDBR MDJS ‘Tricolor [F. sylvatica L.] = ‘Purpurea Tricolor’ ‘Zlatia’ [F. sylvatica L.] paradoxa (D.Don) Endl. Apache Plume VACW FARGESIA Franch. POACEAE Grass Family spathacea Franch. = Thamnocalamus spathaceus XFATSHEDERA Guill. ARALIACEAE [Fatsia < Hedera] Ginseng Family lizei (Cochet) Guill. Tree Ivy [Fatsia japonica ‘Moseri’ < Hedera helix var. hibernica] ALBG DCNA GACH LALG FATSIA Decne. & Planch. ARALIACEAE Ginseng Family EE ae Sa ae ee japonica (Thunb.) Decne. & Planch. Japanese Fatsia ALBG FLMG GAWH VANB DCNA FLUF MSHB FLDG GACH SCBR FEIJOA O.Berg = ACCA sellowiana aa es tet ee a a eT) FICUS L. Fig MORACEAE Mulberry Family -carica L. Common F. DCCG GAJS MSEN VAWP DCNA MDNA VACW FLUF MDRP VASC carica X F. pumila SCWI ‘Minima’ [F. pumila L.] SCBR palmata Forssk. FLUF FARGESIA Franch. POACEAE Grass Family spathacea Franch. = Thamnocalamus spathaceus XFATSHEDERA Guill. ARALIACEAE [Fatsia < Hedera] Ginseng Family lizei (Cochet) Guill. Tree Ivy [Fatsia japonica ‘Moseri’ < Hedera helix var. hibernica] ALBG DCNA GACH LALG FATSIA Decne. & Planch. ARALIACEAE Ginseng Family EE ae Sa ae ee japonica (Thunb.) Decne. & Planch. Japanese Fatsia ALBG FLMG GAWH VANB DCNA FLUF MSHB FLDG GACH SCBR FEIJOA O.Berg = ACCA sellowiana aa es tet ee a a eT) FICUS L. Fig MORACEAE Mulberry Family -carica L. Common F. DCCG GAJS MSEN VAWP DCNA MDNA VACW FLUF MDRP VASC carica X F. pumila SCWI ‘Minima’ [F. pumila L.] SCBR palmata Forssk. FLUF FARGESIA Franch. POACEAE Grass Family spathacea Franch. = Thamnocalamus spathaceus XFATSHEDERA Guill. ARALIACEAE [Fatsia < Hedera] Ginseng Family lizei (Cochet) Guill. Tree Ivy [Fatsia japonica ‘Moseri’ < Hedera helix var. FAGUS hibernica] ALBG DCNA GACH LALG FORSYTHIA FICUS pumila L. Creeping F. ALBG GAHC LAHG TXJS FLDG GAJI MSRN VANB FLMM GAJY SCBR FLUF GAUG SCMP tikoua Bur. LASL FIRMIANA Marsili STERCULIACEAE Sterculia Family {sine LAO Uae MNT RE SRE NA EP oe ae A Re RE a] simplex (L.) W.F. Wight Chinese Parasol Tree ALAU FLUF LAAL MSHB DCNA GAAB LASL SCMP platanifolia (L.f.) Marsili = F. simplex FONTANESIA Labill. OLEACEAE Olive Family fortunei Carr. DCNA MDGJ NCCA TNSN DCSH NCBE TNRG VATA FORESTIERA Poir. OLEACEAE Olive Family acuminata (Michx.) Poir. Swamp Privet TXRS FORSYTHIA Vahl Golden Bells OLEACEAE Olive Family fe 2. ROT ee ER SO NS CY RM ‘Beatrix Farrand’ [F. intermedia Zab.]\| = ‘Karl Sax’ ‘Bronxensis’ [F. viridissima Lind1.] DCNA ‘Ilgwang’ [F. koreana Nakail] MDBG xX intermedia Zab. [F. suspensa x F. viridissima] ALIT GACG VACW DEWG SCUC FORSYTHIA ‘Karl Sax’ [F. < intermedia Zab.] [F. suspensa X F. viridissima] ALEH DEWG ‘Lynwood’ [F. < intermedia Zab.] [F. suspensa xX F. viridissimal] DCNA DEWG MDLT ‘Lynwood Gold’ [F. < intermedia Zab.] = ‘Lynwood’ ‘New Hampshire Gold’ [F. X intermedia Zab.] [F. suspensa X F. viridissima] DCNA ‘Ottawa’ [F. ovata (? hybrid)] DEWG ovata Nakai Korean G.B. DCNA SCCU ‘Pallida’ [F. suspensa (Thunb.) Vahl] DEWG ‘Spectabilis’ [F. < intermedia Zab.] [F. suspensa < F. viridissima] DEWG ‘Spring Glory’ [F. < intermedia Zab.] [F. suspensa X F. viridissima] DCNA DCWH DEWG MDHN suspensa (Thunb.) Vahl Weeping G.B. DCDO MDLT NCDU VAPH DEWG MDPJ VACW suspensa (Thunb.) Vahl var. fortunei (Lindl.) Rehd. DEWG ‘Variegata’ [F. < intermedia Zab.] [F. suspensa < F. viridissima] VATA ‘Vermont Sun’ [F. mandshurica Nakai] DCNA viridissima Lindl. ALEH NCEG SCMP NCBE NCSM VAMP ‘Winterthur’ [F. ovata < F. <intermedia ‘Spring Glory’] DEWG TXRS FORSYTHIA Vahl Golden Bells OLEACEAE Olive Family fe 2. ROT ee ER SO NS CY RM ‘Beatrix Farrand’ [F. intermedia Zab.]\| = ‘Karl Sax’ ‘Bronxensis’ [F. viridissima Lind1.] DCNA ‘Ilgwang’ [F. koreana Nakail] MDBG xX intermedia Zab. [F. suspensa x F. viridissima] ALIT GACG VACW DEWG SCUC FORTUNEARIA FORTUNEARIA kehd. & Wils. HAMAMELIDACEAE Witch-hazel Family [EE a LE Se a) sinensis Rehd. & Wils. MDGD FORTUNELLA Swingle RUTACEAE Kumquat Citrus Family x crassifolia Swingle Meiwa K. [F. margarita < F. japonica] LAGN japonica (Thunb.) Swingle FOTHERGILLA L. HAMAMELIDACEAE Witch-hazel Family gardenii J.A.Murr. Witch Alder GAEC NCBE SCBR VAPH MDWP NCFR SCWI major (Sims) Lodd. FAGUS DCEL GAEC MDLT SCWI DEMC MDBG MDPJ VABF DEWG MDFM MDWP VAGS GACG MDKN NCBE major < F. gardenii DEWG VAPH monticola Ashe Buckthorn Family alnus Mill. DEMC VATA FRANKLINIA Marsh. THEACEAE Tea Family alatamaha Marsh. Franklin Tree DCCG MDBD MDLT SCCU DEMC MDBG NCBE VACW GARW MDGD NCDB VAMP FORTUNEARIA FORTUNEARIA kehd. & Wils. HAMAMELIDACEAE Witch-hazel Family [EE a LE Se a) sinensis Rehd. & Wils. MDGD FORTUNELLA Swingle RUTACEAE Kumquat Citrus Family x crassifolia Swingle Meiwa K. [F. margarita < F. japonica] LAGN japonica (Thunb.) Swingle FOTHERGILLA L. HAMAMELIDACEAE Witch-hazel Family gardenii J.A.Murr. Witch Alder GAEC NCBE SCBR VAPH MDWP NCFR SCWI major (Sims) Lodd. DCEL GAEC MDLT SCWI DEMC MDBG MDPJ VABF DEWG MDFM MDWP VAGS GACG MDKN NCBE major < F. gardenii DEWG VAPH monticola Ashe Buckthorn Family alnus Mill. DEMC VATA FRANKLINIA Marsh. THEACEAE Tea Family FRAXINUS FRAXINUS FORTUNEARIA Flowering A. FRAXINUS L. Ash OLEACEAE Olive Family RS LE americana L. American A., White A. DCCG MDJS TNFL VAWR FLUF NCCA TNSC MDBP SCBR VAMP MDHN TNDR VAPO americana L. var. biltmoreana (Beadle) J.Wright Biltmore A. MDCP MDHN ‘Aurea’ [F. excelsior L.] DCNA berlandieriana A.DC. Berlandier A. TXBA chinensis Roxb. var. rhynchophylla (Hance) Hemsl. TNDR ‘Doorenbos #5’ [F. excelsior L.] DCNA excelsior L. European A. MDSJ holotricha Koehne DCNA lanceolata Borkh. = F. pennsylvanica var. subintegerrima longicuspis Sieb. & Zucc. VATA nigra Marsh. Black A. TNLW ornus L. GACG MDLT ornus X F. excelsior DCNA pennsylvanica Marsh. Red A. VABF pennsylvanica Marsh. var. subintegerrima (Vahl) Fern. Green A. Flowering A. FRAXINUS L. Ash OLEACEAE Olive Family RS LE americana L. American A., White A. DCCG MDJS TNFL VAWR FLUF NCCA TNSC MDBP SCBR VAMP MDHN TNDR VAPO americana L. var. biltmoreana (Beadle) J.Wright Biltmore A. MDCP MDHN ‘Aurea’ [F. excelsior L.] DCNA berlandieriana A.DC. Berlandier A. TXBA chinensis Roxb. var. rhynchophylla (Hance) Hemsl. TNDR ‘Doorenbos #5’ [F. excelsior L.] DCNA excelsior L. European A. MDSJ holotricha Koehne DCNA lanceolata Borkh. = F. pennsylvanica var. subintegerrima longicuspis Sieb. & Zucc. VATA nigra Marsh. Black A. TNLW ornus L. GACG MDLT ornus X F. excelsior DCNA pennsylvanica Marsh. FORTUNEARIA kehd. & Wils. HAMAMELIDACEAE Witch-hazel Family FORTUNELLA Swingle RUTACEAE Kumquat Citrus Family Buckthorn Family alnus Mill. DEMC VATA FRANKLINIA Marsh. THEACEAE Tea Family alatamaha Marsh. Franklin Tree DCCG MDBD MDLT SCCU DEMC MDBG NCBE VACW GARW MDGD NCDB VAMP 73 . FAGUS ik K iff , 0 FOTHERGILLA major (Sims) Lodd. [illustrator Susan M. Johnston] ss] — FRAXINUS pubescens Lam. = F. pennsylvanica sieboldiana BI. DCNA VATA toumeyi Britton = F. velutina velutina Torr. var. coriacea (S.Wats.) Rehd. Leatherleaf A. FLUF SCCU velutina Torr. var. glabra Rehd. Modesto A. FLMM FLUF FLWG GALPHIMIA Cav. MALPIGHIACEAE Malpighia Family Se aa a: SaaS ST Se See glauca Cav. ALBG FLUF GACG SCWI GARDENIA Ellis RUBIACEAE Madder Family ee ee ee ee CS \| es ee ee jasminoides Ellis Cape Jasmine FLMG GATS LAAL LARP LAHG MSMN NCWC VACM jasminoides Ellis var. radicans (Thunb.) Makino = ‘Radicans’ ‘Radicans’ [G. jasminoides Ellis] LAHG LARP MSMN VANB GAULTHERIA lL. ERICACEAE Heath Family procumbens L. Wintergreen, Tea Berry DCNA GAYLUSSACIA HBK. ERICACEAE Huckleberry Heath Family baccata (Wang.) K.Koch Black H. VATG brachycera (Michx.) A.Gr. GINKGO GINKGO GELSEMIUM Juss. Jessamine LOGANIACEAE Logania Family Py wR, a ‘Plena’ [G. sempervirens (L.) Ait.f.] = ‘Pride of Augusta’ ‘Pride of Augusta’ [G. sempervirens (L.) Ait.f.] Double-flowered Jessamine ALTD VACW VAPH LASL VAGS rankinii Small SCWI VACW sempervirens (L.) Ait.f. Yellow J., Carolina J. ALON LAHG TNCG VAWM ARPT LASL VACW VAWR ARSN NCOP VAGG GAEC SCBR VAGS GENISTA L. Broom FABACEAE (Faboideae) Bean Family lydia Boiss. VACW ‘Prostrata’ [G. germanica L.] DCNA GINKGO lL. GINKGOACEAE Ginkgo Family eae SOS Se See ee biloba L. Maidenhair Tree, Ginkgo DCCG LASL NCDU VABF DCWH MDHN NCTE VAMP FLCG MDJS TNCT VATA GAGN MSMN TNFE VAWR GAMW NCCA TNPW (The specimen at GAGN develops pendent mammillate projections (called “chi” in Japan) _from the lower side of the main branches; this is the first specimen of its kind recorded in the United States.) ‘Fastigiata’ [G. biloba L.] GACD ‘Lakeview’ [G. biloba L.] TNPW (8) GLEDITSIA L. Honeylocust FABACEAE (Caesalpinioideae) Bean Family LE IE A FRET OE ETO OR PEE PO SY GET LUE RS SS SES ETN aquatica Marsh. Water Locust DCLC japonica Miq. Japanese H. SCCU ‘Shademaster’ [G. triacanthos L. f. inermis (Pursh) Schneid.] NCDU ‘Sunburst’ [G. triacanthos L. f. inermis (Pursh) Schneid.] NCCA TNSC VACW triacanthos L. Common H. MDHN MDJH VACW triacanthos L. f. inermis (Pursh) Schneid. Spineless H. ALBH NCCA TNBO GLOCHIDION J.R. &J.G.Forst. EUPHORBIACEAE Spurge Family 2 SS aE NORE NEES SEES a ie puberum (L.) Hutch. FLUF GLYCOSMIS_ Correa RUTACEAE citrifolia (Willd.) Lindl. FLUF Citrus Family parviflora (Sims) Little = G. citrifolia GLYPTOSTROBUS Endl. TAXODIACEAE Taxodium Family heterophyllus (Brongn.) Endl. FAGUS = G. lineatus lineatus (Poir.) Druce Chinese Water-pine DCNA MDBG pensilis (Staunt.) K.Koch = G. lineatus GORDONIA Ellis THEACEAE Tea Family CE a SS LT alatamaha Sarg. = Franklinia alatamaha axillaris (Roxb. ex Ker-Gawl.) D.Dietr. FLUF GACG chrysandra Cowan FLUF lasianthus (L.) Ellis Loblolly Bay ALAU SCBR VATA DCNA VANB CE a SS LT alatamaha Sarg. = Franklinia alatamaha axillaris (Roxb. ex Ker-Gawl.) D.Dietr. FLUF GACG chrysandra Cowan FLUF lasianthus (L.) Ellis Loblolly Bay ALAU SCBR VATA DCNA VANB GREWIA lL. TILIACEAE Linden Family EEE a aS a a aS \| biloba G.Don GAIS VABF GYMNOCLADUS Lam. FABACEAE (Caesalpinioideae) Bean Family EE a a a a a SA 2 a \| dioica (L.) K.Koch Kentucky Coffee-tree DCCG DEMC VACW VAMF DCNA MDHN VAGW IDXOJ212 MDJS VAHC HALESIA Ellis ex L. Silverbell Tree STYRACACEAE Storax Family carolina L. ALEH MDCA NCTE VACP DCCG MDGJ NCTP VAMP GARW MDJH SCBR VATA GASM MDJS SCWI VAWR GATS MDWP TNMB LALG MSMN TNSC MDBG NCAK TNSM diptera Ellis DCNA LAAL LASL NCTE FLMG LAHG MSMN TXLL GAEC LALG NCBE diptera Ellis var. magniflora Godfrey DCNA FLMG NCBE monticola Rehd. & Sarg. = H. carolina parviflora Michx. ALEH GLOCHIDION J.R. &J.G.Forst. EUPHORBIACEAE Spurge Family 2 SS aE NORE NEES SEES a ie puberum (L.) Hutch. FLUF GLYCOSMIS_ Correa RUTACEAE citrifolia (Willd.) Lindl. FLUF Citrus Family parviflora (Sims) Little = G. citrifolia GLYPTOSTROBUS Endl. TAXODIACEAE Taxodium Family heterophyllus (Brongn.) Endl. = G. lineatus lineatus (Poir.) Druce Chinese Water-pine DCNA MDBG pensilis (Staunt.) K.Koch = G. lineatus HALESIA carolina L. [illustrator Susan M. Johnston] 77 HALESIA ‘Rosea’ [H. carolina L.] DCNA tetraptera Ellis = H. carolina tetraptera Ellis var. monticola (Rehd.) Reveal & Seldin = H. carolina HALIMODENDRON Fischer ex DC. FABACEAE (Faboideae) halodendron (L.) Voss DCNA HAMAMELIS L. HAMAMELIDACEAE arborea Ottol. = H. japonica ‘Arnold Promise’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG ‘Brevipetala’ [H. mollis Oliv.] DCNA MDBG MDKN ‘Carnea’ [H. vernalis Sarg.] MDBG ‘Christmas Cheer’ [H. vernalis Sarg.] MDBG ‘Copper’ [H. vernalis Sarg.] MDBG ‘Copper Beauty’ [H. X intermedia Rehd.] = ‘Jelena’ ‘Feuerzauber’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA ‘Fire Charm’ [H. < intermedia Rehd.] = ‘Feuerzauber’ X intermedia Rehd. [H. japonica < H. mollis] Bean Family EAT IE NE RI A AITO PEN, MORN TROT SR SEE SRO A CTR Oe ROE Witch Hazel Witch-hazel Family HALESIA ‘Rosea’ [H. HAMAMELIS HALESIA ‘Rosea’ [H. carolina L.] DCNA tetraptera Ellis = H. carolina tetraptera Ellis var. monticola (Rehd.) Reveal & Seldin = H. carolina HALIMODENDRON Fischer ex DC. FABACEAE (Faboideae) halodendron (L.) Voss DCNA HAMAMELIS L. HAMAMELIDACEAE arborea Ottol. = H. japonica ‘Arnold Promise’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG ‘Brevipetala’ [H. mollis Oliv.] DCNA MDBG MDKN ‘Carnea’ [H. vernalis Sarg.] MDBG ‘Christmas Cheer’ [H. vernalis Sarg.] MDBG ‘Copper’ [H. vernalis Sarg.] MDBG ‘Copper Beauty’ [H. X intermedia Rehd.] = ‘Jelena’ ‘Feuerzauber’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA ‘Fire Charm’ [H. < intermedia Rehd.] = ‘Feuerzauber’ X intermedia Rehd. [H. japonica < H. mollis] DCNA MDGC MDKN xjapollis J.Lange = H. Xintermedia japonica Sieb. & Zucc. DCNA MDBG MDFM MDKN Bean Family EAT IE NE RI A AITO PEN, MORN TROT SR SEE SRO A CTR Oe ROE Witch Hazel Witch-hazel Family japonica Sieb. & Zucc. f. flavopurpurascens (Makino) Rehd. DCNA MDBG MDKN ‘Jelena’ [H. Xintermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG ‘Lombarts Weeping’ [H. vernalis Sarg.] DCNA MDBG MDFM ‘Luna’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG macrophylla Pursh = H. virginiana ‘Magic Fire’ [H. < intermedia Rehd.] = ‘Feuerzauber’ mollis Oliv. Chinese W.H. ALBG DEMC MDBG DCNA GAUG MDKN ‘Orange Beauty’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA ‘Pallida’ [H. X intermedia Rehd.] [H. japonica x H. mollis] MDBG ‘Primavera’ [H. < intermedia Rehd.] [H. japonica x H. mollis] DCNA ‘Rubra’ [H. < intermedia Rehd.] [H. japonica = H. mollis] DCNA ‘Ruby Glow’ [H. < intermedia Rehd.] [H. japonica < H. mollis] (Sometimes listed as ‘Adonis’ DCNA MDBG ‘Sandra’ [H. vernalis Sarg.] MDBG vernalis Sarg. DCNA MDKN MDWP vernalis Sarg. f. tomentella Rehd. = H. vernalis vernalis Sarg. var. tomentella (Rehd.) Palm. = H. vernalis halodendron (L.) Voss DCNA HAMAMELIS L. HAMAMELIDACEAE arborea Ottol. = H. japonica ‘Arnold Promise’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG ‘Brevipetala’ [H. mollis Oliv.] DCNA MDBG MDKN ‘Carnea’ [H. vernalis Sarg.] MDBG ‘Christmas Cheer’ [H. vernalis Sarg.] MDBG ‘Copper’ [H. vernalis Sarg.] MDBG ‘Copper Beauty’ [H. X intermedia Rehd.] = ‘Jelena’ ‘Feuerzauber’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA ‘Fire Charm’ [H. < intermedia Rehd.] = ‘Feuerzauber’ X intermedia Rehd. [H. japonica < H. mollis] DCNA MDGC MDKN xjapollis J.Lange = H. Xintermedia japonica Sieb. & Zucc. FAGUS carolina L.] DCNA tetraptera Ellis = H. carolina tetraptera Ellis var. monticola (Rehd.) Reveal & Seldin = H. carolina HALIMODENDRON Fischer ex DC. FABACEAE (Faboideae) halodendron (L.) Voss DCNA HAMAMELIS L. HAMAMELIDACEAE arborea Ottol. = H. japonica ‘Arnold Promise’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG ‘Brevipetala’ [H. mollis Oliv.] DCNA MDBG MDKN ‘Carnea’ [H. vernalis Sarg.] MDBG ‘Christmas Cheer’ [H. vernalis Sarg.] MDBG ‘Copper’ [H. vernalis Sarg.] MDBG ‘Copper Beauty’ [H. X intermedia Rehd.] = ‘Jelena’ ‘Feuerzauber’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA ‘Fire Charm’ [H. < intermedia Rehd.] = ‘Feuerzauber’ X intermedia Rehd. [H. japonica < H. mollis] DCNA MDGC MDKN xjapollis J.Lange = H. Xintermedia Bean Family EAT IE NE RI A AITO PEN, MORN TROT SR SEE SRO A CTR Oe ROE Witch Hazel Witch-hazel Family HAMAMELIS HAMAMELIS DCNA MDBG MDFM MDKN Witch Hazel Witch-hazel Family HAMAMELIS L. HAMAMELIDACEAE arborea Ottol. = H. japonica ‘Arnold Promise’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA MDBG ‘Brevipetala’ [H. mollis Oliv.] DCNA MDBG MDKN ‘Carnea’ [H. vernalis Sarg.] MDBG ‘Christmas Cheer’ [H. vernalis Sarg.] MDBG ‘Copper’ [H. vernalis Sarg.] MDBG ‘Copper Beauty’ [H. X intermedia Rehd.] = ‘Jelena’ ‘Feuerzauber’ [H. < intermedia Rehd.] [H. japonica < H. mollis] DCNA ‘Fire Charm’ [H. < intermedia Rehd.] = ‘Feuerzauber’ X intermedia Rehd. [H. japonica < H. mollis] DCNA MDGC MDKN xjapollis J.Lange = H. Xintermedia japonica Sieb. & Zucc. DCNA MDBG MDFM MDKN Witch Hazel Witch-hazel Family HAMAMELIS mollis Oliv. [illustrator Susan M. Johnston] 79 HAMAMELIS virginiana L. Common W. H. ALBG GAEH DCNA LALG MDBG TXDC TXSE VACW virginiana L. var. parvifolia Nutt. = H. virginiana ‘Zuccariniana’ [H. japonica Sieb. & Zucc.] DCNA MDKN HAMELIA Jacq. RUBIACEAE Madder Family (RAS TES TE TPS RCT EW ES OT PN] patens Jacq. Scarlet Bush TXRS HEDERA L. Ivy ARALIACEAE Ginseng Family RES Ce ES ee AS Ta te TE BES Fe a ks algeriensis Hibb. = H. canariensis ‘Alpha’ [H. helix L.] SCWF ‘Anchor’ [H. helix L.] SCWF ‘Angularis Aurea’ [H. helix L.] SCWF Arborescens group [H. helix L.] Shrub Ivy (This group name applies to the adult flowering form of the species.) ALAU GAJY MDNA VACM ALON LASL MDPJ VACW DCCG MDBG NCBE DCSE MDFM TNUT ‘Arrowhead’ [H. helix L.] SCWF ‘Aurea Spectabilis’ [H. helix L.] SCWF ‘Baby Merion’ [H. helix L.] GAAB HEDERA HAMAMELIS virginiana L. Common W. H. ALBG GAEH DCNA LALG MDBG TXDC TXSE VACW virginiana L. var. parvifolia Nutt. = H. virginiana ‘Zuccariniana’ [H. japonica Sieb. & Zucc.] DCNA MDKN HAMELIA Jacq. RUBIACEAE Madder Family (RAS TES TE TPS RCT EW ES OT PN] patens Jacq. Scarlet Bush TXRS HEDERA L. Ivy ARALIACEAE Ginseng Family RES Ce ES ee AS Ta te TE BES Fe a ks algeriensis Hibb. = H. canariensis ‘Alpha’ [H. helix L.] SCWF ‘Anchor’ [H. helix L.] SCWF ‘Angularis Aurea’ [H. helix L.] SCWF Arborescens group [H. helix L.] Shrub Ivy (This group name applies to the adult flowering form of the species.) ALAU GAJY MDNA VACM ALON LASL MDPJ VACW DCCG MDBG NCBE DCSE MDFM TNUT ‘Arrowhead’ [H. helix L.] SCWF ‘Aurea Spectabilis’ [H. helix L.] SCWF ‘Baby Merion’ [H. helix L.] GAAB ‘Baccifer’ [H. helix L.] SCWF ‘Big Deal’ [H. HAMAMELIS helix L.] SCWF HEDERA ‘Bulgaria’ [H. helix L.] MDBG ‘California’ [H. helix L.] SCWF ‘California Gold’ [H. helix L.] SCWF canariensis Willd. Algerian I. DEWG GAJI SCCL SCNS FLCG LAHG SCFW VADW FLMG MDMG SCMI FLUF SCBR SCMP ‘Canary Cream’ [H. canariensis Willd.] MDMG ‘Carolina Crinkle’ [H. helix L.] GAHC MDBG SCBR SCWF ‘Cascade’ [H. helix L.] SCWF ‘Cathedral Wall’ [H. helix L.] SCWF ‘Cavendishii’ [H. helix L.] ALHC FLCG ‘Christian’ [H. helix L.] GAAB ‘Chrysantha’ [H. helix L.] DCNA MDBG ‘Chrysocarpa’ [H. helix L.] = H. helix var. poetica ‘Cockle Shell’ [H. helix L.] GAAB SCWF colchica K.Koch Caucasus Ivy DCCC NCEL SCMP VAGS MDBG SCBR SCWF ‘Conglomerata’ [H. helix L.] GACG SCWF VAGS ‘Crenata’ [H. helix L.] DCDO ‘Deltoidea’ [H. helix L.] MDBG SCWF ‘Dentata’ [H. helix L.] SCWF ‘Denticulata’ [H. helix L.] SCWF HEDERA ‘Bulgaria’ [H. helix L.] MDBG ‘California’ [H. helix L.] SCWF ‘California Gold’ [H. helix L.] SCWF canariensis Willd. Algerian I. DEWG GAJI SCCL SCNS FLCG LAHG SCFW VADW FLMG MDMG SCMI FLUF SCBR SCMP ‘Canary Cream’ [H. canariensis Willd.] MDMG ‘Carolina Crinkle’ [H. helix L.] GAHC MDBG SCBR SCWF ‘Cascade’ [H. helix L.] SCWF ‘Cathedral Wall’ [H. helix L.] SCWF ‘Cavendishii’ [H. helix L.] ALHC FLCG ‘Christian’ [H. helix L.] GAAB ‘Chrysantha’ [H. helix L.] DCNA MDBG ‘Chrysocarpa’ [H. helix L.] = H. helix var. poetica ‘Cockle Shell’ [H. helix L.] GAAB SCWF colchica K.Koch Caucasus Ivy DCCC NCEL SCMP VAGS MDBG SCBR SCWF ‘Conglomerata’ [H. helix L.] GACG SCWF VAGS ‘Crenata’ [H. helix L.] DCDO ‘Deltoidea’ [H. helix L.] MDBG SCWF HEDERA HEDERA HEDERA ‘Digitata’ [H. helix L.] ALHC SCWF ‘Discolor’ [H. helix L.] SCWF ‘Dragon Claw’ [H. helix L.] SCWF ‘Edison’ [H. helix L.] SCWF ‘Emerald Beauty’ [H. helix L.] GAFN ‘Emerald Gem’ [H. helix L.] SCWF ‘Emerald Jewel’ [H. helix L.] DCNA ‘Erecta’ [H. helix L.] VATA ‘Erin’ [H. helix L.] SCWF ‘Eva’ [H. helix L.] SCWF ‘Fan’ [H. helix L.] DCNA MDBG ‘Fantasia’ [H. helix L.] SCWF ‘Ferney’ [H. helix L.] SCWF ‘Fleur’ [H. helix L.] SCBR SCWF ‘Fleur de Lis’ [H. helix L.] SCWF ‘Four Square’ [H. helix L.] SCWF ‘Garland’ [H. helix L.] SCWF ‘Gavotte’ [H. helix L.] SCWF ‘Ginkgo’ [H. helix L.] SCWF ‘Glacier’ [H. helix L.] SCWF HEDERA ‘Glymii’ [H. helix L.] as SCWF ‘Goldcraft’ [H. helix L.] SCWF ‘Gold Dust’ [H. helix L.] SCWF ‘Gold Heart’ [H. helix L.] MDBG SCWF ‘Goods Selfbranching’ [H. HAMAMELIS helix L.] SCWF ‘Gracilis’ [H. helix L.] SCWF ‘Green Crown’ [H. helix L.] SCWF ‘Green Feather [H. helix L.] = ‘Meagheri’ ‘Green Finger’ [H. helix L.] SCWF ‘Green Quartz’ [H. helix L.] SCWF ‘Green Ripples’ [H. helix L.] GAFN SCWF ‘Green Spear’ [H. helix L.] SCWF ‘Green Velvet’ [H. helix L.] GAFN ‘Hahn Selfbranching’ [H. helix L.] SCWF ‘Hahn Variegated’ [H. helix L.] SCWF ‘Harold’ [H. helix L.] SCWF ‘Harrison’ [H. helix L.] SCWF ‘Hebron’ [H. helix L.] SCWF helix L. English I. ALBG GABW NCEG TXSH FLBF LAHG NCOP FLCG LASL NCTP ‘Green Crown’ [H. helix L.] SCWF ‘Green Feather [H. helix L.] = ‘Meagheri’ ‘Green Finger’ [H. helix L.] SCWF ‘Green Quartz’ [H. helix L.] SCWF ‘Green Ripples’ [H. helix L.] GAFN SCWF ‘Green Spear’ [H. helix L.] SCWF 81 HEDERA HEDERA HEDERA helix L. var. poetica Weston Italian I. SCWF ‘Helvetica’ [H. helix L.] SCWF ‘Heterophylla’ [H. helix L.] SCWF ‘Hibernica’ [H. helix L.] SCWF ‘Hite Miniature’ [H. helix L.] SCWF ‘Holly’ [H. helix L.] SCWF ‘Ideal’ [H. helix L.] SCWF ‘Imp’ [H. helix L.] SCBR SCWF ‘Itsy Bitsy’ [H. helix L.] SCWF ‘Iva Lace’ [H. helix L.] MDBG SCWEF ‘Jack Frost’ [H. helix L.] SCWF japonica Tobl. = H. rhombea ‘Lady Kay’ [H. helix L.] SCWF ‘La Platta’ [H. helix L.] SCWF ‘Lobata Major’ [H. helix L.] SCWF ‘Lolla Rookh’ [H. helix L.] MDBG ‘Long Point’ [H. helix L.] SCWF ‘Lucida Aurea’ [H. helix L.] SCWF ‘Luzzi’ [H. helix L.] SCWF ‘Manda Crested’ [H. helix L.] MDBG SCWF HEDERA ‘Manda’s Star’ [H. helix L.] SCWF ‘Maple Queen’ [H. helix L.] SCWF ‘Marbled Dragon’ [H. helix L.] SCWF ‘Marginata’ [H. helix L.] SCWF ‘Meagheri’ [H. helix L.] SCWF VATA ‘Merion Beauty’ [H. helix L.] GAFN VATA ‘Merrie’s Albany’ [H. helix L.] SCWF ‘Microphylla Variegata’ [H. helix L.] SCWF ‘Midget’ [H. helix L.] SCWF ‘Miniature Needlepoint’ [H. helix L.] VATA ‘Minima’ [H. helix L.] SCWF ‘Minor Marmorata’ [H. helix L.] MDBG ‘Mount Vernon’ [H. helix L.] SCWF ‘My Variegated’ [H. helix L.] SCWF ‘Needlepoint’ [H. helix L.] SCBR SCWF nepalensis K.Koch var. sinensis Tobl. SCWF ‘Obscura’ [H. helix L.] SCWF ‘Palmata’ [H. helix L.] = ‘Digitata’ ‘Paper Doll’ [H. helix L.] SCWF ‘La Platta’ [H. helix L.] SCWF ‘Lobata Major’ [H. helix L.] SCWF ‘Lolla Rookh’ [H. helix L.] MDBG ‘Long Point’ [H. helix L.] SCWF ‘Luzzi’ [H. helix L.] SCWF ‘Manda Crested’ [H. helix L.] MDBG SCWF HEDERA HEDERA ‘Perfection’ [H. helix L.] SCWF ‘Permanent Wave’ [H. helix L.] SCWF ‘Pin Oak’ [H. helix L.] SCWF ‘Pin Oak Improved’ [H. helix L.] SCWF ‘Pittsburgh’ [H. helix L.] MDBG SCWF ‘Pittsburgh Variegated’ [H. helix L.] SCWF ‘Pixie’ [H. helix L.] SCWF ‘Plume de Or’ [H. helix L.] SCWF ‘Poetica’ [H. helix L.] = H. helix var. poetica ‘Preston Tiny’ [H. helix L.] SCWF ‘Purpurea’ [H. helix L.] SCWF ‘Ralf’ [H. helix L.] GAAB ‘Rambler’ [H. helix L.] SCWF ‘Ray’s Supreme’ [H. helix L.] FLMG SCWF rhombea (Miq.) Bean Japanese I. DCNA ‘Ripples’ [H. helix L.] SCWF ‘Rochester’ [H. helix L.] SCWF ‘Roehr’s Minor’ [H. helix L.] SCWF ‘Rubaiyat’ [H. helix L.] HED ‘Sagittaefolia’ [H. helix L.] SCWF ‘Sea Foam’ [H. helix L.] SCMP ‘Shamrock’ [H. helix L.] = ‘Meagheri’ ‘Shannon’ [H. HEDERA helix L.] SCWF ‘Silver Queen’ [H. helix L.] SCWF ‘Sinclair Silverleaf’ [H. helix L.] GAAB ‘Small Deal’ [H. helix L.] SCWF ‘Spearpoint’ [H. helix L.] SCWF ‘Springtime Snow’ [H. helix L.] SCWF ‘Staghorn’ [H. helix L.] SCWF ‘Stardust’ [H. helix L.] SCWF ‘Stare’ [H. helix L.] SCWF ‘Sulphurea’ [H. helix L.] SCWF ‘Sulfur Heart’ [H. colchica K.Koch var. dentata Hibb.] MDBG ‘Susan Gibles’ [H. helix L.] SCBR ‘Suzanne’ [H. helix L.] SCWEF ‘Sylvanian’ [H. helix L.] SCWF ‘Teardrop’ [H. helix L.] SCWF ‘Teena’ [H. helix L.] SCWF ‘Telecurl’ [H. helix L.] MDBG SCWF ‘Tesselata’ [H. helix L.] 83 HEDERA HEDERA HIBISCUS HIBISCUS nummularium (L.) Mill. MDSC HELICHRYSUM Miill. Strawflower ASTERACEAE Aster Family italicum (Roth) G.Don VAGS HEMIPTELEA Planch. ULMACEAE Elm Family ee ae ES ES a Sa ee ET davidii (Hance) Planch. VABF HEPTACODIUM Rehd. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) EE a a aa aE EE TE TEE] jJasminoides Airy-Shaw = H. miconioides miconioides Rehd. Seven-sun Flower DCNA HETEROPTERIS' HBK. MALPIGHIACEAE Malpighia Family angustifolia Griseb. FLJH syringifolia Griseb. LAAL LASL TXSC HIBISCUS L. MALVACEAE Mallow Family LS a TS a a) ‘Admiral Dewey’ [H. syriacus L.] DCNA ‘Aka yae’ [H. syriacus L.] DCNA ‘Albo-laciniata’ [H. rosa-sinensis L.] FLUF ‘Albus Plenus’ [H. syriacus L.] DCNA ‘Amarantus’ [H. syriacus L.] DCNA HEDERA ‘Thorndale’ [H. helix L.] SCWF ‘Tidal Wave’ [H. helix L.] SCWF ‘Tribairn’ [H. helix L.] SCWE ‘Triloba’ [H. helix L.] SCWF ‘Triton’ [H. helix L.] MDTD SCWF ‘Trustee’ [H. helix L.] SCWF ‘238th Street’ [H. helix L.] SCWF ‘Ustlers’ [H. helix L.] SCWF ‘Vanderhof’ [H. helix L.] SCWF ‘Variegata’ [H. canariensis Willd.] Gloire de Marengo I. GACG VANB ‘Walthamensis’ [H. helix L.] SCWF ‘Weber’s California’ [H. helix L.] SCWF ‘Wilson’ [H. helix L.] LASL SCWF ‘Woodsii’ [H. helix L.] SCWF ‘Yalta’ [H. helix L. var. taurica Rehd.] SCWF HELIANTHEMUM Mill. CISTACEAE Rock-rose apenninum (L.) Mill. MDSC apenninum (L.) Mill. var. roseum (Jac Schneid. MDSC nummularium (L.) Mill. MDSC nummularium (L.) Mill. MDSC ‘Yalta’ [H. helix L. var. taurica Rehd.] SCWF HELIANTHEMUM Mill. CISTACEAE Rock-rose Family apenninum (L.) Mill. MDSC apenninum (L.) Mill. var. roseum (Jacq.) Schneid. MDSC ‘Buttercup’ [H. nummularium (L.) Mill.] DEMC ‘Fireball’ [H. nummularium (L.) Mill.] MDSC 84 Orx HIBISCUS HIBISCUS ‘Effie Riegel’ [H. syriacus L.] SCCU ‘Elegantissimus’ [H. syriacus L.] DCNA ‘Gion mamori’ [H. syriacus L.] DCNA ‘Grandiflorus Superbus’ [H. syriacus L.] DCNA ‘Hagan Hybrid #2’ [H. syriacus L.] DCNA ‘Hamabo’ [H. syriacus L.] DCNA ‘Hanagasa’ [H. syriacus L.] = ‘Shiro hanagasa’ * ‘Helene’ [H. syriacus L.] DCNA ‘Hinomarw’ [H. syriacus L.] DCNA ‘Hitoe’ [H. syriacus L.] DCNA ‘Jeanne d’Arc’ [H. syriacus L.] DCNA ‘Koki yae’ [H. syriacus L.] DCNA ‘Kreider Blue’ [H. syriacus L.] DCNA SCCU ‘Lady Stanley’ [H. syriacus L.] DCNA ‘La Fleur’ [H. syriacus L.] DCNA ‘Leopoldii’ [H. syriacus L.] DCNA ‘Leopoldii Plenus’ [H. syriacus L.] DCNA ‘Lovely Pink’ [H. syriacus L.] DCNA ‘Lucy’ [H. syriacus L.] DCNA ‘Luteus Plenus’ [H. syriacus L.] DCNA ‘Martha Jane’ [H. syriacus L.] HIBISCUS HIBISCUS * ‘Aphrodite’ [H. syriacus L.] [(H. syriacus ‘Suminokura’ /diploid/ x H. syriacus ‘William R. Smith’) <tetraploid seedling of H. syriacus ‘William R. Smith’] DCNA ‘Ardens’ [H. syriacus L.] DCNA ‘Ardens Plena’ [H. syriacus L.] DCNA ‘Bicolor’ [H. syriacus L.] DCNA ‘Blue Bird’ [H. syriacus L.] DCNA ‘Blue Rouge’ [H. syriacus L.] DCNA ‘Boule de Feu’ [H. syriacus L.] DCNA ‘Brilliant’ [H. rosa-sinensis L.] FLUF ‘Caeruleus Plenus’ [H. syriacus L.] DCNA ‘Campanha’ [H. syriacus L.] DCNA ‘Celestial Blue’ [H. syriacus L.] DCNA ‘Colie Mullins’ [H. syriacus L.] DCNA SCCU ‘Comte de Flandre’ [H. syriacus L.] DCNA ‘Comte de Haimout’ [H. syriacus L.] DCNA ‘Dela Vaux’ [H. syriacus L.] DCNA ‘De La Veuve’ [H. syriacus L.] DCNA * ‘Diana’ [H. syriacus L.] (Tetraploid sdlg. x diploid sdlg.) DCNA MDBG ‘Double Light Pink’ [H. syriacus L.] DCNA ‘Brilliant’ [H. rosa-sinensis L.] FLUF ‘Caeruleus Plenus’ [H. syriacus L.] DCNA ‘Campanha’ [H. syriacus L.] DCNA ‘Celestial Blue’ [H. syriacus L.] DCNA ‘Koki yae’ [H. syriacus L.] DCNA ‘Kreider Blue’ [H. syriacus L.] DCNA SCCU ‘Colie Mullins’ [H. syriacus L.] DCNA SCCU ‘Comte de Flandre’ [H. syriacus L.] DCNA 85 HIBISCUS HIBISCUS HIBISCUS HIBISCUS ‘Mauve Queen’ [H. syriacus L.] DCNA ‘Meehanii’ [H. syriacus L.] DCNA ‘Mimihara’ [H. syriacus L.] DCNA * ‘Minerva’ [H. syriacus L.] [H. syriacus ‘Blue Bird’ < H. syriacus ‘Hanagasa’] DCNA ‘Monstrosus’ [H. syriacus L.] DCNA ‘Monstrosus Plenus’ [H. syriacus L.] DCNA ‘Monstrosus Simple’ [H. syriacus L.] DCNA mutabilis L. Confederate Rose FLUF ‘Oiseau Blew’ [H. syriacus L.] DCNA paramutabilis Bailey VABF ‘Perry’s Purple’ [H. syriacus L.] DCNA ‘Pheasant Eye’ [H. syriacus L.] DCNA ‘Pink Delight’ [H. syriacus L.] DCNA SeéCU ‘Plume’ [H. syriacus L.] DCNA ‘Pom Pom Rouge’ [H. syriacus L.] DCNA ‘Pompon’ [H. syriacus L.] DCNA ‘Pulcherrimus’ [H. syriacus L.] DCNA ‘Puniceus Plenus’ [H. syriacus L.] DCNA ‘Purpurea Semiplena’ [H. syriacus L.] HIBISCUS ‘Ranunculiflorus’ [H. syriacus L.] DCNA ‘Ranunculiflorus Plenus’ [H. syriacus L.] DCNA ‘Rosalinda’ [H. syriacus L.] DCNA ‘Roseus Plenus’ [H. syriacus L.] DCNA ‘Roxanus’ [H. syriacus L.] DCNA ‘Rubis’ [H. syriacus L.] DCNA ‘Rubra Grandiflora’ [H. syriacus L.] DCNA ‘Rubra Plena’ [H. syriacus L.] DCNA ‘Shiro hanagasa’ [H. syriacus L.] DCNA ‘Shiro midare’ [H. syriacus L.] DCNA ‘Sir de Charles Breton’ [H. syriacus L.] DCNA ‘Snowdrift’ [H. syriacus L.] DCNA ‘Soft Pink’ [H. syriacus L.] DCNA ‘Sokobeni yae’ [H. syriacus L.] DCNA ‘Sonde’ [H. syriacus L.] DCNA ‘Souvenir de Charles Breton’ [H. syriacus L.] DCNA ‘Speciosus’ [H. syriacus L.] LAHP ‘Spectabilis Plena’ [H. syriacus L.] DCNA ‘Shiro hanagasa’ [H. syriacus L.] DCNA ‘Shiro midare’ [H. syriacus L.] DCNA ‘Sir de Charles Breton’ [H. syriacus L.] DCNA ‘Purpurea Semiplena’ [H. syriacus L.] DCNA ‘Purpureus Plenus’ [H. syriacus L.] DCNA QO (ep) HYDRANGEA HYDRANGEA HYDRANGEA anomala D.Don ssp. petiolaris (Sieb. & Zucc.) McClint. Climbing H. DCNA MDBG GAEC MDLT MDMG VAPH TNRW VARC ‘Grandiflora’ [H. arborescens L.]\| = ‘Annabelle’ ‘Grandiflora’ [H. paniculata Sieb.] Peegee H. DCCG SCBR VARC ‘Grayswood’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser.] (Lacecap group) MDMG ‘Harmony’ [H. quercifolia Bartram] DCNA MDFM TNTV macrophylla (Thunb. ex J.A.Murr.) Ser. Lacecap (Lacecap group) DCNA LASL MSPR LALG MDBG VAGS LARP MDBR VAMP macrophylla (Thunb. ex J.A.Murr.) Ser. Hortensia, Garden Hydrangea (Hortensia group) ALBG LARP MSKR VATA DCNA LASL VAGS LAAL MSEP VAPH macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino DCNA MDBG MDJS MDPJ DEWG MDGD MDMG ‘Maculata’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser.] (Lacecap group) DCNA LASL ‘Otaksa’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser.] = H. macrophylla (Hortensia group) paniculata Sieb. Peegee TH: DCNA MDGJ NCAS VABK MDBG MDLT NCSM VAMP ‘Prolifera’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino] DCNA HIBISCUS ‘Suminokura yae’ [H. syriacus L.] DCNA syriacus L. Rose-of-Sharon, Shrub Althea ALBG FLUF NCAS DCCG LAAL SCSR VACW ‘Tama usagi’ [H. syriacus L.] DCNA ‘Totus Albus’ [H. syriacus L.] DCNA ‘Usu hitoe’ [H. syriacus L.] DCNA ‘Variegatus’ [H. syriacus L.] DCNA ‘Violaceus Plenus’ [H. syriacus L.] DCNA ‘Violet Clair’ [H. syriacus L.] DCNA ‘White Red Eye’ [H. syriacus L.] DCNA ‘White Supreme’ [H. syriacus L.] DCNA ‘William P. Smith’ [H. syriacus L.] DCNA ‘Woodbridge’ [H. syriacus L.] DCNA ‘Zulauf [H. syriacus L.] DCNA HOVENIA ‘Thunb. RHAMNACEAE Buckthorn Family ES SS ee ee ee SE ae TS ee dulcis Thunb. Japanese Raisin-tree DCNA FLFB GAGR DCTB FLUF GAWM HYDRANGEA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family acuminata Sieb. & Zucc. = H. macrophylla ssp. serrata HIBISCUS ‘Suminokura yae’ [H. syriacus L.] DCNA syriacus L. Rose-of-Sharon, Shrub Althea ALBG FLUF NCAS DCCG LAAL SCSR VACW ‘Tama usagi’ [H. syriacus L.] DCNA ‘Totus Albus’ [H. syriacus L.] DCNA ‘Usu hitoe’ [H. syriacus L.] DCNA ‘Variegatus’ [H. syriacus L.] DCNA ‘Violaceus Plenus’ [H. syriacus L.] DCNA ‘Violet Clair’ [H. syriacus L.] DCNA ‘White Red Eye’ [H. syriacus L.] DCNA ‘White Supreme’ [H. syriacus L.] DCNA ‘William P. Smith’ [H. syriacus L.] DCNA ‘Woodbridge’ [H. syriacus L.] DCNA ‘Zulauf [H. syriacus L.] DCNA HOVENIA ‘Thunb. RHAMNACEAE Buckthorn Family ES SS ee ee ee SE ae TS ee dulcis Thunb. Japanese Raisin-tree DCNA FLFB GAGR DCTB FLUF GAWM HYDRANGEA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family HYDRANGEA anomala D.Don ssp. petiolaris (Sieb. HYDRANGEA & Zucc.) McClint. Climbing H. DCNA MDBG GAEC MDLT MDMG VAPH TNRW VARC ‘Grandiflora’ [H. arborescens L.]\| = ‘Annabelle’ ‘Grandiflora’ [H. paniculata Sieb.] Peegee H. DCCG SCBR VARC ‘Grayswood’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser.] (Lacecap group) MDMG ‘Harmony’ [H. quercifolia Bartram] DCNA MDFM TNTV macrophylla (Thunb. ex J.A.Murr.) Ser. Lacecap (Lacecap group) DCNA LASL MSPR LALG MDBG VAGS LARP MDBR VAMP macrophylla (Thunb. ex J.A.Murr.) Ser. Hortensia, Garden Hydrangea (Hortensia group) ALBG LARP MSKR VATA DCNA LASL VAGS LAAL MSEP VAPH macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino DCNA MDBG MDJS MDPJ DEWG MDGD MDMG ‘Maculata’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser.] (Lacecap group) DCNA LASL ‘Otaksa’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser.] = H. macrophylla (Hortensia group) paniculata Sieb. Peegee TH: DCNA MDGJ NCAS VABK MDBG MDLT NCSM VAMP ‘Prolifera’ [H. macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino] 87 [illustrator Lillian Nicholson Meyer] HOVENIA dulcis Thunb. e@) ee) HYDRANGEA anomala ssp. petiolaris (Sieb. & Zucc.) McClint. 89 HYDRANGEA quercifolia Bartram Oakleaf H. ALAU LALG MSRN TNSC ALHC MDJH NCBE TNUT DCNA MDKN NCSM VAPH FLUF MDLT SCBR GAUG MDPJ SCMP LAHG MSMN SCWI scandens (L.f.) Ser. ssp. liukiuensis (Nakai) McClint. GALH ‘Snowflake’ [H. quercifolia Bartram] MDBG MDMG VAPH MDFM SCWI ‘Tardiva’ [H. paniculata Sieb.] SCCU HYPERICUM L. St. John’s-wort HYPERICACEAE St. John’s-wort Family Se ES ES SET SE EE ES SPO SE brachyphyllum (Spach) Steud. SCWI buckleyi M.A.Curtis MDJW calycinum L. Aaron’s Beard, Goldflower MDBG fasciculatum Lam. Sandweed SCWI frondosum Michx. DCNA galioides Lam. LALG SCBR SCWI ‘Hidcote’ [H. ?calycinum H. forrestii] MDLT NCBE TNUT VAWP hircinum L. VAGS hookerianum Wight & Arn. ILEX ILEX HYDRANGEA HYDRANGEA quercifolia Bartram Oakleaf H. ALAU LALG MSRN TNSC ALHC MDJH NCBE TNUT DCNA MDKN NCSM VAPH FLUF MDLT SCBR GAUG MDPJ SCMP LAHG MSMN SCWI scandens (L.f.) Ser. ssp. liukiuensis (Nakai) McClint. GALH ‘Snowflake’ [H. quercifolia Bartram] MDBG MDMG VAPH MDFM SCWI ‘Tardiva’ [H. paniculata Sieb.] SCCU HYPERICUM L. St. John’s-wort HYPERICACEAE St. John’s-wort Family Se ES ES SET SE EE ES SPO SE brachyphyllum (Spach) Steud. SCWI buckleyi M.A.Curtis MDJW calycinum L. Aaron’s Beard, Goldflower MDBG fasciculatum Lam. Sandweed SCWI frondosum Michx. DCNA galioides Lam. LALG SCBR SCWI ‘Hidcote’ [H. ?calycinum H. forrestii] MDLT NCBE TNUT VAWP hircinum L. VAGS hookerianum Wight & Arn. DCNA hypericoides (L.) Crantz = Ascyrum hypericoides kalmianum L. ILEX lloydii (Svenson) Adams MDJW microsepalum (T. HYDRANGEA & G.) A. Gr. SCWI patulum Thunb. ex J.A.Murr. LARP prolificum L. NCBE SCBR SCWI reductum (Svenson) Adams SCBR SCWI stans (Michx.) Adams & N.Robs. St. Peter’s-wort SCBR SCWI ‘Sunburst’ [H. frondosum Michx.] DCNA ‘Van Fleetii’ DCNA HYSSOPUS L. LAMIACEAE Mint Family officinalis L. DCNA IDESIA Maxim. FLACOURTIACEAE Flacourtia Family polycarpa Maxim. ALON ALTD DCNA ILEX L. AQUIFOLIACEAE (Contributed by T. R. Dudley) Holly Holly Family (Because of a large number of cultivars in the hybrid species of Ilex, parentage is not shown for each cultivar. Parentage is listed under the respective hybrid-species names.) ‘Aalto’ [I. opaca Ait.] NCSH ‘Aalto #2’ [I. opaca Ait.] = ‘Harriet’ ‘Aalto #3’ [I. opaca Ait.] = ‘Kate’ scandens (L.f.) Ser. ssp. liukiuensis (Nakai) McClint. GALH ‘Snowflake’ [H. quercifolia Bartram] MDBG MDMG VAPH MDFM SCWI ‘Tardiva’ [H. paniculata Sieb.] SCCU HYPERICUM L. St. John’s-wort HYPERICACEAE St. John’s-wort Family Se ES ES SET SE EE ES SPO SE brachyphyllum (Spach) Steud. SCWI buckleyi M.A.Curtis MDJW calycinum L. Aaron’s Beard, Goldflower MDBG fasciculatum Lam. Sandweed SCWI frondosum Michx. DCNA galioides Lam. LALG SCBR SCWI ‘Hidcote’ [H. ?calycinum H. forrestii] MDLT NCBE TNUT VAWP hircinum L. VAGS hookerianum Wight & Arn. DCNA hypericoides (L.) Crantz = Ascyrum hypericoides kalmianum L. MDWP lissophloeus Adams SCWI = ILEX ILEX ILEX ‘Aalto #5A’ [I. opaca Ait.] DCNA * ‘Accent’ [I. integra x I. pernyi] DCNA ‘Afterglow’ [I. verticillata (L.) A.Gr.] DCNA ‘Aglo’ [I. cornuta Lindl. & Paxt.] DCCB ‘Aka tsuge’ [I. sugerokii Maxim.] DCNA ‘Alagold’ [I. x attenuata Ashe] VATA ‘Alice’ [I. X altaclerensis (Loud.) Dallim.] DCNA ‘Alice’ [I. aquifolium] = ‘Alice’ [I. < altaclerensis] x altaclerensis (Loud.) Dallim. Highclere H. [I. aquifolium x I. perado] DCNA MDLT MDWP VATA ambigua (Michx.) Torr. Carolina H., Ambiguous Winterberry VAGW amelanchier M.A.Curtis Sarvis H. ALJG GACG TNFB DCNA LASL VACW ‘Andorra’ [I. opaca Ait.] NCSH ‘Angustifolium’ [I. aquifolium L.] (Female and male clones of this cultivar exist) ALTD GACG MDLT DCNA MDBG TNUT ‘Angyo’ [I. crenata Thunb. ex J.A.Murr.] DCNA MDGD ‘Anicet Delcambrie’ [I. cornuta Lindl. & Paxt.] DCNA LAMP ‘Anna Mae’ [I. cornuta Lindl. & Paxt.] GACG LASL ILEX ILEX ‘Aalto #5A’ [I. opaca Ait.] DCNA * ‘Accent’ [I. integra x I. pernyi] DCNA ‘Afterglow’ [I. verticillata (L.) A.Gr.] DCNA ‘Aglo’ [I. cornuta Lindl. & Paxt.] DCCB ‘Aka tsuge’ [I. sugerokii Maxim.] DCNA ‘Alagold’ [I. x attenuata Ashe] VATA ‘Alice’ [I. X altaclerensis (Loud.) Dallim.] DCNA ‘Alice’ [I. aquifolium] = ‘Alice’ [I. < altaclerensis] x altaclerensis (Loud.) Dallim. Highclere H. [I. aquifolium x I. perado] DCNA MDLT MDWP VATA ambigua (Michx.) Torr. Carolina H., Ambiguous Winterberry VAGW amelanchier M.A.Curtis Sarvis H. ALJG GACG TNFB DCNA LASL VACW ‘Andorra’ [I. opaca Ait.] NCSH ‘Angustifolium’ [I. aquifolium L.] (Female and male clones of this cultivar exist) ALTD GACG MDLT DCNA MDBG TNUT ‘Angyo’ [I. crenata Thunb. ex J.A.Murr.] DCNA MDGD ‘Anicet Delcambrie’ [I. cornuta Lindl. & Paxt.] DCNA LAMP ‘Anna Mae’ [I. cornuta Lindl. & Paxt.] GACG LASL ‘Anne Arundel’ [I. opaca Ait.] ILEX ‘Apricot’ [I. aquifolium L.] NCSH aquifolium L. English H. DCCG DCWH TXHN VATW DCLC GACG VAGW DCNA MDLT VATA aquifolium x I. spinigera DCNA ‘Aquipern’ [I. X aquipernyi Gable] DCNA DCWH GAIS MDTD X aquipernyi Gable [I. aquifolium x I. pernyi] (Sometimes confused with I. bioritensis) DCNA GACG MDBG MDLT ‘Arden’ [I. opaca Ait.] DCNA ‘Argentea Marginata’ [I. aquifolium L.] DCNA MDLT NCSH ‘Arthur Bruner’ [I. cornuta ‘Burfordii’ < I. latifolia] TNHH asprella (Hook. & Arn.) Champ. ex Benth. DCNA ‘Astoria’ [I. aquifolium L.] = ‘Beacon’ ‘Atlas’ [I. aquifolium L.] DCNA ‘Atlas’ [I. cornuta x I. pernyji] GACG < attenuata Ashe Topel H., Attenuate-leaved H. [I. cassine x I. opaca] ANCADD) MDGD NCEG SCMP DCNA MDLT SCBR GAOH MSHB SCFR ‘Audry’ [I. cornuta X I. pernyi] MDPF ‘Aurantiaca’ [I. verticillata (L.) A.Gr.] DCNA TNFB ‘Aurea Regina’ [I. aquifolium L.] = ‘Golden Queen’ ‘Apricot’ [I. aquifolium L.] NCSH aquifolium L. English H. DCCG DCWH TXHN VATW DCLC GACG VAGW DCNA MDLT VATA aquifolium x I. spinigera DCNA ‘Aquipern’ [I. X aquipernyi Gable] DCNA DCWH GAIS MDTD X aquipernyi Gable [I. aquifolium x I. pernyi] (Sometimes confused with I. bioritensis) DCNA GACG MDBG MDLT ‘Arden’ [I. opaca Ait.] DCNA ‘Argentea Marginata’ [I. aquifolium L.] DCNA MDLT NCSH ‘Arthur Bruner’ [I. cornuta ‘Burfordii’ < I. latifolia] TNHH asprella (Hook. & Arn.) Champ. ex Benth. DCNA ‘Astoria’ [I. aquifolium L.] = ‘Beacon’ ‘Atlas’ [I. aquifolium L.] DCNA ‘Atlas’ [I. cornuta x I. pernyji] GACG < attenuata Ashe Topel H., Attenuate-leaved H. [I. cassine x I. ILEX ARRB FLUF NCBE TNSB DCCG GACG NCEG TXSH DCNA GAWH NCGP VACW FLCG MDLT SCFW VATA FLMG MSHB SCUC buswellii Small Sand H., Buswell’s H. DCNA TNFB ‘Butler’ [I. aquifolium L.] MDWA ‘Buxifolia’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of different origins) DCNA ‘Byers Golden’ [I. decidua Walt.] ALBH DCNA GACG ‘Cacapon’ [I. verticillata (L.) A.Gr.] DCNA ‘Cajun Gold’ [I. cornuta Lindl. & Paxt.] LAGN ‘Callina’ [I. cornuta x I. aquifolium] DCNA NCNB ‘Calloway’ [I. opaca Ait. f. xanthocarpa ILEX ‘Camelliifolia’ [I. x altaclerensis (Loud.) Dallim.] DCLC DCNA MDBG MDTD ‘Campus Variegated’ [I. aquifolium L.] DCNA canariensis Poir. Canary Islands H. DCNA ‘Canary’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA DEMC MDLT ‘Cape Christmas’ [I. opaca Ait.] MDTN ‘Captain Bonneville’ [I. aquifolium L.] GACG ‘Carefree’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Carissa’ [I. cornuta Lindl. & Paxt.] MDTD ‘Casey’s Dwarf [I. cornuta Lindl. & Paxt.] GACG SCMG cassine L. Dahoon, Cassine DCNA GACG LASL FLIF GASM SCBR FLUF LAHG VANB cassine L. var. angustifolia Ait. Narrow-leaved Dahoon ALAU FLIF TNUT DCNA NCDU cassine L. var. angustifolia f. aurea-baccata Tarbox ex S.F.Blake Yellow-berried Dahoon FLUF SCBR cassine L. var. bryanii Tarbox ex S.F.Blake DCNA SCBR cassine L. var. myrtifolia (Walt.) Sarg. = I. myrtifolia ‘Cetus’ [I. cornuta x I. pernyi] GACG ‘Changsha’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Charles’ [I. opaca Ait.] ILEX ‘Brighter Shine’ [I. cornuta x I. pernyi] GACG ‘Bright Horizon’ [I. verticillata (L.) A.Gr.] DCNA ‘Brilliant’ [I. aquifolium x I. ciliospinosa] ALBG MDBG VAGS DCNA SCWI VATA ‘Bronze’ [I. aquifolium L.] DCNA ‘Brown #3’ [I. opaca Ait.] = ‘Judge Brown’ ‘Brown #5’ [I. opaca Ait.] NCSH ‘Brown #16’ [I. opaca Ait.] DCNA ‘Brownell’ [I. aquifolium L.] GACG buergeri Miq. ALTD DCNA ‘Burfordii’ [I. cornuta Lindl. & Paxt.] Burford H. ARRB FLUF NCBE TNSB DCCG GACG NCEG TXSH DCNA GAWH NCGP VACW FLCG MDLT SCFW VATA FLMG MSHB SCUC buswellii Small Sand H., Buswell’s H. DCNA TNFB ‘Butler’ [I. aquifolium L.] MDWA ‘Buxifolia’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of different origins) DCNA ‘Byers Golden’ [I. decidua Walt.] ALBH DCNA GACG ‘Cacapon’ [I. verticillata (L.) A.Gr.] DCNA ‘Cajun Gold’ [I. cornuta Lindl. & Paxt.] LAGN ‘Camelliifolia’ [I. x altaclerensis (Loud.) Dallim.] DCLC DCNA MDBG MDTD ‘Campus Variegated’ [I. aquifolium L.] DCNA canariensis Poir. Canary Islands H. DCNA ‘Canary’ [I. opaca Ait. f. ILEX opaca] ANCADD) MDGD NCEG SCMP DCNA MDLT SCBR GAOH MSHB SCFR ‘Audry’ [I. cornuta X I. pernyi] MDPF ‘Aurantiaca’ [I. verticillata (L.) A.Gr.] DCNA TNFB ‘Aurea Regina’ [I. aquifolium L.] = ‘Golden Queen’ ‘Aureo-marginata’ [I. aquifolium L.] DCNA VATA 91 ILEX ILEX ILE ‘Big Bull’ [I. aquifolium L.] DCNA GACG bioritensis Hayata (Sometimes confused with I. pernyi and I. X aquipernyji) DCNA FLMG DCWH FLUF GACG NCSH ‘Bleeg’ [I. aquifolium L.] DCNA ‘Blue Angel’ [I. < meserveae S.Y.Hu] MDBG VATA ‘Blue Boy’ [I. < meserveae S.Y.Hu] DCNA MDBG ‘Blue Girl’ [I. meserveae S.Y.Hu] DCNA ‘Blue Maid’ [I. X meserveae S.Y.Hu] MDLT ‘Blue Prince’ [I. <X meserveae S.Y.Hu] MDTD VATA ‘Blue Princess’ [I. <X meserveae S.Y.Hu] MDBG VATA ‘Blue Stallion’ [I. < meserveae S.Y.Hu] MDLT VATA ‘Bob Bruner’ [I. cornuta ‘Burfordii’ < I. latifolia] TNAB TNHH ‘Bodley’s Bleeg’ [I. aquifolium L.] DCNA ‘Bonanza’ [I. aquifolium L.] GACG ‘Bostic’ [I. cornuta Lindl. & Paxt.] GACG ‘Boyce Thompson’ [I. opaca Ait.] MDLT ‘Boyce Thompson #3’ [I. opaca Ait.] DCNA ‘Boyce Thompson Xanthocarpa’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA MDBG ‘Braddock Heights’ [I. crenata Thunb. ex J.A.Murr.] ILEX ‘Aurifodina’ [I. aquifolium L.] GACG ‘Autumn Glow’ [I. serrata < I. verticillata] DCNA MDLT ‘Avery Island’ [I. cornuta Lindl. & Paxt.] LAAL SCBR ‘B. & O.’ [I. opaca Ait.] DCNA MDBO ‘Bacciflava’ [I. aquifolium L.] DCNA GACG MDMG VATW ‘Baldwin’ [I. cassine L.] GACG ‘Balearica’ [I. < altaclerensis (Loud.) Dallim.] ARSN DCCG DCNA ‘Balkans’ [I. aquifolium L.] DCNA ‘Barclay’ [I. opaca Ait.] DCNA ‘Barnes’ [I. aquifolium L.] DCNA ‘Beacon’ [I. aquifolium L.] DCNA GACG beadlei Ashe Beadle’s H. TNFB xX beanii Rehd. [I. aquifolium x I. dipyrena] DCNA MDTD ‘Beauty Spra’ [I. aquifolium L.] DCNA GACG ‘Beauty Spra Espalier’ [I. aquifolium L.] DCNA ‘Belgica’ [I. < altaclerensis (Loud.) Dallim.] DCNA LASL ‘Belin’s Weeping’ [I. x altaclerensis (Loud.) Dallim.] DCNA ‘Berigold’ [I. aquifolium L.] DCNA GACG ‘Boyce Thompson #3’ [I. opaca Ait.] DCNA ILEX ILEX ILEX ‘Brighter Shine’ [I. cornuta x I. pernyi] GACG ‘Bright Horizon’ [I. verticillata (L.) A.Gr.] DCNA ‘Brilliant’ [I. aquifolium x I. ciliospinosa] ALBG MDBG VAGS DCNA SCWI VATA ‘Bronze’ [I. aquifolium L.] DCNA ‘Brown #3’ [I. opaca Ait.] = ‘Judge Brown’ ‘Brown #5’ [I. opaca Ait.] NCSH ‘Brown #16’ [I. opaca Ait.] DCNA ‘Brownell’ [I. aquifolium L.] GACG buergeri Miq. ALTD DCNA ‘Burfordii’ [I. cornuta Lindl. & Paxt.] Burford H. ILEX xanthocarpa Rehd.] DCNA DEMC MDLT ‘Cape Christmas’ [I. opaca Ait.] MDTN ‘Captain Bonneville’ [I. aquifolium L.] GACG ‘Carefree’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Carissa’ [I. cornuta Lindl. & Paxt.] MDTD ‘Casey’s Dwarf [I. cornuta Lindl. & Paxt.] GACG SCMG cassine L. Dahoon, Cassine DCNA GACG LASL FLIF GASM SCBR FLUF LAHG VANB cassine L. var. angustifolia Ait. Narrow-leaved Dahoon ALAU FLIF TNUT DCNA NCDU cassine L. var. angustifolia f. aurea-baccata Tarbox ex S.F.Blake Yellow-berried Dahoon FLUF SCBR cassine L. var. bryanii Tarbox ex S.F.Blake DCNA SCBR cassine L. var. myrtifolia (Walt.) Sarg. = I. myrtifolia ‘Cetus’ [I. cornuta x I. pernyi] GACG ‘Changsha’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Camelliifolia’ [I. x altaclerensis (Loud.) Dallim.] DCLC DCNA MDBG MDTD ‘Campus Variegated’ [I. aquifolium L.] DCNA canariensis Poir. Canary Islands H. DCNA ‘Canary’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA DEMC MDLT ‘Cape Christmas’ [I. opaca Ait.] MDTN ‘Captain Bonneville’ [I. aquifolium L.] GACG ‘Carefree’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Carissa’ [I. cornuta Lindl. & Paxt.] MDTD ‘Casey’s Dwarf [I. cornuta Lindl. & Paxt.] GACG SCMG cassine L. Dahoon, Cassine DCNA GACG LASL FLIF GASM SCBR FLUF LAHG VANB cassine L. var. angustifolia Ait. Narrow-leaved Dahoon ALAU FLIF TNUT DCNA NCDU cassine L. var. angustifolia f. aurea-baccata Tarbox ex S.F.Blake Yellow-berried Dahoon FLUF SCBR cassine L. var. bryanii Tarbox ex S.F.Blake DCNA SCBR cassine L. var. myrtifolia (Walt.) Sarg. = I. myrtifolia ‘Cetus’ [I. cornuta x I. pernyi] GACG ‘Changsha’ [I. crenata Thunb. ex J.A.Murr.] GACG 93 ILEX ILEX ILEX ‘Cherry Berry’ [I. < altaclerensis (Loud.) Dallim.] DCNA ‘Chief’ [I. aquifolium L.] NCSH ‘Chieftan’ [I. < koehneana Loesn.] DCNA MDBO ‘China Boy’ [I. cornuta x I. rugosa] VATA ‘China Girl’ [I. cornuta < I. rugosa] VATA chinensis (sensu S.Y.Hu & nurseries, non Sims) = I. purpurea ‘Christmas Carol’ [I. opaca Ait.] DCNA ‘Christmas Cheer’ [I. verticillata (L.) A.Gr.] DCNA ‘Christmas Gem)’ [I. verticillata (L.) A.Gr.] MDJN ‘Christmas Hedge’ [I. opaca Ait.] DCNA ‘Christmas Queen’ [I. opaca Ait.] = ‘Cape Christmas’ ciliospinosa Loesn. (Often confused with ‘Brilliant’) DCNA MDBG MDTD MDBA MDHS ciliospinosa x I. fargesii DCNA DEFC MDMN cinerea Champ. DCNA ‘Clarendon’ [I. opaca Ait.] GACG ‘Clarendon Batwing’ [I. cornuta Lindl. & Paxt.] NCCG ‘Clarendon Small Leaf’ [I. cornuta Lindl. & Paxt.] DCNA GACG ‘Clarendon Spreading’ [I. opaca Ait.] DCNA NCCG TNTV ‘Clarissa’ [I. opaca Ait.] ‘Clark’ [I. ILEX opaca Ait.] DCNA ‘Clark’ [I. opaca Ait.] DCNA ‘Clouded Gold’ [I. aquifolium L.] NCSH * ‘Clusterberry’ (I. cornuta X< I. aquifolium) ‘Nellie R. Stevens’ <I. leucoclada] DENAW VAD ‘Colburn’ [I. < altaclerensis (Loud.) Dallim.] DCNA colchica Pojark. Pontic H., Caucasian H. DCNA ‘Coleman’ [I. aquifolium L.] DCNA collina Alex. Appalachian H. DCNA MDPJ TNFB ‘Compacta’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of different origins) DCNA GACG ‘Compacta’ [I. vomitoria Ait.] (Name applied to numerous clones of different origins) VATA ‘Conners’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Convexa’ [I. crenata Thunb. ex J.A.Murr.] DCNA FLMG MDMG SCMG FLCG GACG NCEG VATA corallina Franch. DCNA MDGD coriacea (Pursh) Chapm. Large Gallberry DCNA SCBR cornuta Lindl. & Paxt. Chinese H., Horned H. ALBG DCWH LAMP TNDR ALTD FLMG MDTD VAGF ARRB FLUF NCDU VATA DCCG GACG SCBR DCNA GAIS SCEG ‘Christmas Carol’ [I. opaca Ait.] DCNA ‘Compacta’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of different origins) DCNA GACG ‘Compacta’ [I. vomitoria Ait.] (Name applied to numerous clones of different origins) VATA ‘Conners’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Convexa’ [I. crenata Thunb. ex J.A.Murr.] DCNA FLMG MDMG SCMG FLCG GACG NCEG VATA corallina Franch. DCNA MDGD coriacea (Pursh) Chapm. Large Gallberry DCNA SCBR cornuta Lindl. & Paxt. Chinese H., Horned H. ALBG DCWH LAMP TNDR ALTD FLMG MDTD VAGF ARRB FLUF NCDU VATA DCCG GACG SCBR DCNA GAIS SCEG ‘Clarendon Small Leaf’ [I. cornuta Lindl. & Paxt.] DCNA GACG ‘Clarendon Spreading’ [I. opaca Ait.] DCNA NCCG TNTV ‘Clarissa’ [I. opaca Ait.] DCNA 94 ILEX ILEX ILEX ILEX ILEX curtissii (Fern.) Small Suwanee H. DCNA GACG TNFB TXDM cyrtura Merr. DCNA ‘D’Or’ [I. cornuta Lindl. & Paxt.] DCNA GAIS VACW GACG LASL ‘Daddyo’ [I. aquifolium L.] DCNA ‘Dan Fenton’ [I. opaca Ait.] DCNA ‘Dare County’ [I. vomitoria Ait.] NCMH decidua Walt. Possum Haw DCNA LAAP NCTE VACW FLIF LAMP SCBR GACG MDLT TNUT GAEC NCDU TXPS decidua Walt. var. curtissii Fern. = I. curtissti ‘Delaware Diamond’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Deluxe’ [I. aquifolium L.] DCNA ‘Dengle Belles’ [I. opaca Ait. f. xanthocarpa Rehd.] MDBG ‘Dewerth’ [I. vomitoria Ait.] GACG ‘Dick’ [I. opaca Ait.] (Originally ‘Wheeler #1’) NCSH _dimorphophylla Koidz. Okinawa H. DCNA dipyrena Wall. DCNA ‘Divaricata’ [I. crenata Thunb. ex J.A.Murr.] cornuta X I. pernyi (CB hybrids originated at Kingsville Nursery, Kingsville, MD; CB is an acronym for I. cornuta x I. cornuta ‘Burfordii’) DCCG DCNA GACG ‘Corpening #1’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA ‘Cover Girl’ [I. aquifolium L.] DCNA crenata Thunb. ex J.A.Murr. Japanese H., Box-leaved H. DCCG FLCG GACG DCNA FLMG MDLT NCBE SCMG crenata Thunb. ex J.A.Murr. ssp. fukasawana (Makino) Murata DCNA crenata Thunb. ex J.A.Murr. var. mutchagara Makino = J. maximowicziana var. kanahirae crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara DCNA GACG crenata Thunb. ex J.A.Murr. var. thomsonii (Hook.f.) Loesn. DCNA crenata Thunb. ex J.A.Murr. f. watanabeana Makino DCNA GACG MDTD ‘Crescent’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Crinkle Variegated’ [I. aquifolium L.] DCNA ‘Crispa’ [I. aquifolium L.] Green Screw DCNA GACG ‘Crispa Aureo-picta’ [I. aquifolium L.] GACG ‘Croonenberg’ [I. opaca Ait.] VAGF ‘Cumberland’ [I. opaca Ait.] DCNA 95 ILEX ILEX ‘Edna Jean’ [I. < attenuata Ashe] TNML ‘Edward J. Stevens’ [I. cornuta < I. aquifolium] DCNA ‘Edwin Dozier’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Eldridge’ [I. x altaclerensis (Loud.) Dallim.] DCNA MDHA NCSH * ‘Elegance’ [I. integra x I. pernyi] DCNA FLUF ‘Elegantissima’ [I. aquifolium L.] (Commercially known as ‘Mistigold’) DCNA GACG ‘Elfin’ [I. crenata Thunb.] = ‘Delaware Diamond’ ‘Elizabeth’ [I. opaca Ait.] DCNA ‘Emily’ [I. opaca Ait.] DCNA ‘Emily Bruner’ [I. cornuta ‘Burfordii’ < I. latifolia] DCNA TNAB VATA GACG TNHH ‘Erma Byrd’ [I. < attenuata Ashe] DCNA MDTD ‘Escort’ [I. aquifolium L.] DCNA ‘Evangeline’ [I. x altaclerensis (Loud.) Dallim.] = ‘Hazel’ ‘Fairfax’ [I. verticillata (L.) A.Gr.] DCNA ‘Farage’ [I. opaca Ait.] DCNA fargesii Franch. Farges’ H. DCNA GAEC fargesii Franch. ssp. melanotricha (Merr.) S.Andrews ‘Dodd Special’ [I. cornuta Lindl. ILEX & Paxt.] GACG ‘Dorsey’ [I. opaca Ait.] MDWA ‘Drace’ [I. cornuta < I. pernyji] GACG ‘Dragon Lady’ [I. x aquipernyi Gable] MDLT VATA ‘Dr. James Foret’ [I. cornuta Lindl. & Paxt.] LAAL ‘Dr. John Creech’ [I. cornuta Lindl. & Paxt.] ALOS LAAL ‘Dr. T. B. Symons’ [I. opaca Ait.] DCNA ‘Dude’ [I. aquifolium L.] DCNA GACG ‘Dumbarton Oaks’ [I. aquifolium L.] DCNA ‘Dwarf Burford’ [I. cornuta Lindl. & Paxt.] DCNA LAHG NCDU GACG MDBG ‘Dwarf Cone’ [I. crenata Thunb. ex J.A.Murr.] DCNA DECN ‘Dwarf Pagoda’ [I. crenata Thunb. ex J.A.Murr.] DCNA MDMN ‘Eagleson’ [I. x attenuata Ashe] TXEN ‘E. A. McIlhenny’ [I. cornuta Lindl. & Paxt.] LAAL ‘Earlibright’ [I. verticillata (L.) A.Gr.] DCNA ‘Early Cluster’ [I. < altaclerensis (Loud.) Dallim.] DCNA ‘East Palatka’ [I. x attenuata Ashe] ALFN DCWH LASL VACW ARRB FLUF NCEG VATA DCCG GACG SCMG DCNA GAUG TNSB 96 ILEX ILEX ‘Fort McCoy’ [I. vomitoria Ait.] (Cultivar name illegitimate) FLUF ‘Foster #1’ [I. x attenuata Ashe] ALAU ALTD ‘Foster #2’ [I. x attenuata Ashe] ALAU GABS MDLT NCDU ALFN MDBG MDTD VATA ‘Foster #3’ [I. x attenuata Ashe] ALFN GACG ‘Foster #4’ [I. xX attenuata Ashe] DCNA ‘Foster No. 1’ [I. crenata Thunb. ex J.A.Murr.] (Cultivar name illegitimate) GACG ‘Foster No. 2’ [I. crenata Thunb. ex J.A.Murr.] (Cultivar name illegitimate) GACG ‘Foxii’ [I. aquifolium L.] DCNA GACG ‘Francis Lewis’ [I. opaca Ait.] DEFL ‘Freeman’ [I. opaca Ait.] DCNA ‘Frierson’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Fructo-lutea’ [I. aquifolium L.] DCNA GACG ‘Fruitland Nursery’ [I. opaca Ait. f. xanthocarpa Rehd.] GACG NCSH ‘Gable’ [I. < aquipernyi Gable] DCNA ‘Gayle’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Gee’ [I. opaca Ait.] DCNA geniculata Maxim. Furin H. DCNA ILEX ‘Father Charles’ [I. < altaclerensis (Loud.) Dallim. ex Rehd.] DCNA GACG ‘Faulkner’ [I. opaca Ait.] MDHH ‘Favorite’ [I. aquifolium L.] DCNA GACG ‘Felten’s Selection’ [I. opaca Ait.] DCNA ‘Ferox’ [I. aquifolium L.] DCNA GACG MDBG VADW ‘Ferox Argentea’ [I. aquifolium L.] DCNA ‘Ferox Aurea Marginata’ [I. aquifolium L.] VATA ficoidea Hemsl. Fig-leaved H. DCNA ‘Fine Line’ [I. cornuta Lindl. & Paxt.] ALTD ‘Fire Chief [I. opaca Ait.] DCNA ‘Firecracker’ [I. aquifolium L.] GACG ‘Firefly’ [I. crenata Thunb. ex J.A.Murr.] DECN ‘Firelight’ [I. x altaclerensis (Loud.) Dallim.] DCNA ‘Flavescens’ [I. aquifolium L.] GACG ‘Flushing’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Folsom’s Weeping’ [I. vomitoria Ait. f. pendula Foret & Solym.] GACG GAUG TNCT TXSE ‘Formal’ [I. opaca Ait.] NCSH ‘Formax’ [I. cornuta x I. pernyji] GACG ‘Fort McCoy’ [i. cumulicola Small] ILEX ‘Fort McCoy’ [I. vomitoria Ait.] (Cultivar name illegitimate) FLUF ‘Foster #1’ [I. x attenuata Ashe] ALAU ALTD ‘Foster #2’ [I. x attenuata Ashe] ALAU GABS MDLT NCDU ALFN MDBG MDTD VATA ‘Foster #3’ [I. x attenuata Ashe] ALFN GACG ‘Foster #4’ [I. xX attenuata Ashe] DCNA ‘Foster No. 1’ [I. crenata Thunb. ex J.A.Murr.] (Cultivar name illegitimate) GACG ‘Foster No. 2’ [I. crenata Thunb. ex J.A.Murr.] (Cultivar name illegitimate) GACG ‘Foxii’ [I. aquifolium L.] DCNA GACG ‘Francis Lewis’ [I. opaca Ait.] DEFL ‘Freeman’ [I. opaca Ait.] DCNA ‘Frierson’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Fructo-lutea’ [I. aquifolium L.] DCNA GACG ‘Fruitland Nursery’ [I. opaca Ait. f. xanthocarpa Rehd.] GACG NCSH ‘Gable’ [I. < aquipernyi Gable] DCNA ‘Gayle’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Gee’ [I. ILEX opaca Ait.] DCNA geniculata Maxim. 97 ILEX ILEX ILEX glabra (L.) A.Gr. Inkberry ALBG GACG MDLT ALUA GAUG MDTD DCNA MDBG VAWP glabra (L.) A.Gr. f. leucocarpa F.W.Woods White-fruited Inkberry DCNA GACG SCBR ‘Glass’ [I. crenata Thunb. ex J.A.Murr.] GACG VATA ‘Glenwood’ [I. cornuta Lindl. & Paxt.] VAIG ‘Globe’ [I. aquifolium L.] DCNA ‘Glory’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Golden Beauty’ [I. aquifolium L.] = ‘Golden Milkmaid’ ‘Golden Butterfly’ [I. aquifolium L.] DCNA GACG ‘Golden Fleece’ [I. opaca Ait.] DCNA ‘Golden Gate’ [I. aquifolium] = ‘Rubricaulis Aurea’ ‘Golden Gem’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Golden Girl’ [I. X meserveae S.Y.Hul] (Commercially known as ‘Mesglog’) DCNA ‘Golden Heller’ [I. crenata Thunb. ex J.A.Murr.] VATA ‘Golden Milkboy’ [I. aquifolium L.] DCNA ‘Golden Milkmaid’ [I. aquifolium L.] DCNA ‘Golden Queen’ [I. aquifolium L.] NCSH ‘Goldie’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA ‘Good Taste’ [I. cornuta < I. pernyji] DCNA GACG ILEX ‘Good Will Park’ [I. opaca Ait.] DCNA ‘Governor William Paca’ [I. opaca Ait.] MDWP ‘Gracean’ [I. aquifolium L.] DCNA ‘Grandpappy’ [I. opaca Ait.] NCBW ‘Grandview’ [I. cornuta Lindl. & Paxt.] DCNA ‘Gray’s Bigleaf’ [I. vomitoria Ait.] NCMH ‘Gray’s Little Leaf’ [I. vomitoria Ait.] DCNA TXDM ‘Green Dragon’ [I. crenata Thunb. ex J.A.Murr.] DCNA MDLT ‘Green Knight’ [I. aquifolium L.] DCNA GACG ‘Green Lustre’ [I. crenata Thunb. ex J.A.Murr.] DCNA GACG VATA ‘Green Maid’ [I. aquifolium L.] DCNA GACG ‘Green Screw’ [I. aquifolium L.] = ‘Crispa’ ‘Green Shadow’ [I. integra Thunb. ex J.A.Murr.] DCNA ‘Green Thumb’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Grier’ [I. crenata Thunb. ex J.A.Murr.] VATA ‘Griscom’ [I. opaca Ait.] DCNA ‘Hamlet’ [I. opaca Ait.] DCNA ‘Handsworthensis’ [I. aquifolium L.] DCNA ‘Golden Gem’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Golden Girl’ [I. X meserveae S.Y.Hul] (Commercially known as ‘Mesglog’) DCNA ‘Golden Heller’ [I. crenata Thunb. ex J.A.Murr.] VATA ‘Golden Milkmaid’ [I. aquifolium L.] DCNA ILEX ILEX ILEX ILEX ILEX ‘Harry Gunning’ [I. ciliospinosa x I. leucoclada] DCNA ‘Harvest Red’ [I. serrata < I. verticillata] DCNA MDBG ‘Hastata’ [I. aquifolium L.] GACG ‘Hatfield’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Hazel’ [I. < altaclerensis (Loud.) Dallim.] DCNA ‘Hazel [I. aquifolium L.] = ‘Hazel’ [I. x altaclerensis] ‘H. B. Red’ [I. decidua Walt.] = ‘Pocahontas’ ‘Hedgeholly’ [I. opaca Ait.] DCNA ‘Helen Makepeace’ [I. opaca Ait.] DCNA ‘Helleri’ [I. crenata Thunb. ex J.A.Murr.] DCNA MDBG NCEG ‘Hendersonii’ [I. < altaclerensis (Loud.) Dallim.] DCNA ‘Hendersonii Aurea’ [I. < altaclerensis (Loud.) Dallim.] GACG ‘Heterophylla Aureomarginata’ [I. aquifolium L.] = ‘Elegantissima’ ‘Hetzii’ [I. crenata Thunb. ex J.A.Murr.] GACG * ‘Highlight’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Hodginsii’ [I. x altaclerensis (Loud.) Dallim.] DCNA GACG VAWM ‘Hohman’ [I. < koehneana Loesn.] MDBG ILEX ‘Hollycroft Jack’ [I. x altaclerensis (Loud.) Dallim.] = = ‘Hollycroft Jack’ [I. aquifolium] ‘Hollycroft Jack’ [I. aquifolium L.] DCNA ‘Homer’ [I. opaca Ait.] DCNA ‘Honeycomb’ [I. crenata Thunb. ex J.A.Murr.] MDMN ‘Hopkins’ [I. opaca Ait.] DCNA ‘Howard’ [I. <x attenuata Ashe] DCNA GABS GAFN FLDG GACG LASL ‘Howard’ [I. opaca Ait.] = ‘Howard’ [I. x attenuata] ‘Huber’s Compact’ [I. vomitoria Ait.] LASL ‘Hume’ [I. cornuta Lindl. & Paxt.] MDMN VATA ‘Hume #1’ [I. X attenuata Ashe] DCNA ‘Hume #2’ [I. <x attenuata Ashe] ALFN DCNA GACG ‘Hume’s Choice’ [I. opaca Ait.] MDMN ‘Hutchinson’ [I. x attenuata Ashe] MSWB ‘Indian Bayou #3’ [I. decidua Walt.] LASL ‘Indian Chief [I. cornuta x I. pernyji] GACG ‘Ingramii’ [I. aquifolium L.] MDTN insignis Hook. = I. kingiana integra Thunb. ex J.A.Murr. Mochi H. ALBG DCNA GAIS ALTD FLMG LAAL DCCG FLUF VAWR ‘Hume’s Choice’ [I. opaca Ait.] MDMN ‘Hutchinson’ [I. x attenuata Ashe] MSWB 99 ILEX integra xX I. aquifolium (Plants of this cross from different origins are widely distributed) DCNA MDTD integra X< I. cornuta GACG integra x I. pernyi (Plants of this cross from different origins are widely distributed) DCNA ‘Integrifolium’ [I. aquifolium L.] NCSH ‘Ira Nelson’ [I. cornuta Lindl. & Paxt.] DCNA LAAL LASL VACW ‘Iso’ [I. opaca Ait.] DCNA ‘Ivory’ [I. aquifolium L.] DCNA ‘Ivory Hall’ [I. crenata Thunb. ex J.A.Murr.] DECN VATA ‘Ivory Queen’ [I. glabra (L.) A.Gr. f. leucocarpa F.W.Woods] DCNA MDMN ‘Ivory Tower’ [I. crenata Thunb. ex J.A.Murr.] DECN VATA ‘Jackson’ [I. verticillata (L.) A.Gr.] DCNA * ‘Jade’ [I. x koehneana Loesn.] DCNA ‘James G. Esson’ [I. < altaclerensis (Loud.) Dallim.] DCNA GACG MDTD DEMC MDMN ‘James Swan’ [I. ILEX cornuta ‘Burfordii’ < I. latifolia] TNAB TNHH ‘J. C. van Tol’ [I. aquifolium L.] GACG ‘Jeannette Adamson’ [I. opaca Ait.] MDPJ ‘Jersey Pinnacle’ [I. crenata Thunb. ex J.A.Murr.] DCNA ILEX integra xX I. aquifolium (Plants of this cross from different origins are widely distributed) DCNA MDTD integra X< I. cornuta GACG integra x I. pernyi (Plants of this cross from different origins are widely distributed) DCNA ‘Integrifolium’ [I. aquifolium L.] NCSH ‘Ira Nelson’ [I. cornuta Lindl. & Paxt.] DCNA LAAL LASL VACW ‘Iso’ [I. opaca Ait.] DCNA ‘Ivory’ [I. aquifolium L.] DCNA ‘Ivory Hall’ [I. crenata Thunb. ex J.A.Murr.] DECN VATA ‘Ivory Queen’ [I. glabra (L.) A.Gr. f. leucocarpa F.W.Woods] DCNA MDMN ‘Ivory Tower’ [I. crenata Thunb. ex J.A.Murr.] DECN VATA ‘Jackson’ [I. verticillata (L.) A.Gr.] DCNA * ‘Jade’ [I. x koehneana Loesn.] DCNA ‘James G. Esson’ [I. < altaclerensis (Loud.) Dallim.] DCNA GACG MDTD DEMC MDMN ‘James Swan’ [I. cornuta ‘Burfordii’ < I. latifolia] TNAB TNHH ‘J. C. van Tol’ [I. aquifolium L.] GACG ‘Jeannette Adamson’ [I. opaca Ait.] MDPJ ‘Jersey Pinnacle’ [I. crenata Thunb. ex J.A.Murr.] DCNA ILEX ILEX ILEX ILEX ‘Jersey Princess’ [I. opaca Ait.] DCNA MDLT VATA ‘Jinny Bruner’ [I. cornuta ‘Burfordii’ x I. latifolia] TNAB TNHH ‘John Higgins’ [I. opaca Ait.] DCNA ‘John Nosal’ [I. crenata Thunb. ex J.A.Murr.] DCNA * ‘John T. Morris’ [I. cornuta x I. pernyi] DCNA GACG VATA ‘Joyce’ [I. opaca Ait.] DCNA ‘Judge Brown’ [I. opaca Ait.] DCNA NCSH ‘Jungle Garden’ [I. cornuta Lindl. & Paxt.] LAAL NCDU ‘Kate’ [I. opaca Ait.] DCNA kingiana Cockerell DCNA ‘Kingsville Dwarf [I. crenata Thunb. ex J.A.Murr.] DCNA MDBG SCMG GACG NCEG VATA ‘Kingsville Special’ [I. cornuta Lindl. & Paxt.] GACG ‘Kirofukurin’ [I. crenata Thunb. ex J.A.Murr.] = ‘Angyo’ < kiusiana Hatusima [I. buergeri x I. integra] DCNA ‘Knight’ [I. opaca Ait.] DCNA MDWA < koehneana Loesn. Koehne’s H. [I. aquifolium ~ I. latifolia] DCNA MDBG MDMG- VATA DCWH MDLT VACW VAWR ‘Kunming’ [I. crenata Thunb. ex J.A.Murr.] 100 ILEX ILEX ILEX ILEX ‘La Bar’ [I. opaca Ait.] DCNA NCSH ‘Lacerta’ [I. cornuta x I. pernyi] GACG ‘Lady Baltimore’ [I. aquifolium L.] DCNA laevigata (Dum.-Cours.) A.Gr. Smooth Winterberry DCNA GACG NCBE ‘Lagniappe’ [I. longipes Chapm. ex Trelease] ALJG ‘Lake City’ [I. opaca Ait.] DCNA GACG LAHG LASL ‘Lassie’ [I. x koehneana Loesn.] MDMN latifolia Thunb. ex J.A.Murr. Luster-leaved H., Broad-leaved H. ILEX ALAU GACG MDLT TXDC ALBG GADG NCDU VACW ALTD GAEC NCEL VAGS DCCG GAIS SCMG VASC DCNA GAWH SCMP DCWH LASL TNSB FLUF MDBG TNUT ‘Latifolia’ [I. crenata Thunb. ex J.A.Murr.] (Many cultivars of different origins) DCNA GACG MDLT FLMG GAFN ‘Latispina’ [I. aquifolium L.] VADW ‘Laura’ [I. opaca Ait.] DCNA NCSH ‘Laurifolia’ [I. <x altaclarensis (Loud.) Dallim. ex Rehd.] DCNA ‘Laurifolia’ [I. aquifolium L.] = ‘Laurifolia’ [I. <x altaclerensis] ‘Lenape Moon’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA ‘Lepux’ [I. cornuta x I. pernyji] GACG ‘Leucocarpa’ [I. serrata Thunb. ex ILEX leucoclada Makino Yellow-stemmed H. DCNA ‘Lewis’ [I. aquifolium L.] MDHA ‘Libby’s Favorite’ [I. integra < I. cornuta] ALTD ‘Lilliput’ [I. aquifolium L.] DCNA ‘Lilygold’ [I. aquifolium L.] DCNA ‘Lindleyana’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Little Bull’ [I. aquifolium L.] DCNA liukiuensis Loesn. Liukiu H. GACG ‘Lock Raven’ [I. x koehneana Loesn.] DCNA lohfauensis Merr. DCNA ‘Longfellow’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Longifolia’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of diverse origins.) DCWH longipes Chapm. ex Trelease Long-stalked H., Georgian H. ALTD LAHG NCDU TXLL DCNA MDTD TNFB _‘Longspra’ [I. aquifolium L.] NCSH ‘Lord’ [I. rotunda Thunb. ex J.A.Murr.] DCNA ‘Louise’ [I. aquifolium L.] DCNA ‘Louise Holmes’ [I. x attenuata Ashe] DCNA VACW ‘Lowei’ [I. myrtifolia Walt.] DCNA FLUF ILEX ‘La Bar’ [I. opaca Ait.] DCNA NCSH ‘Lacerta’ [I. cornuta x I. pernyi] GACG ‘Lady Baltimore’ [I. aquifolium L.] DCNA laevigata (Dum.-Cours.) A.Gr. Smooth Winterberry DCNA GACG NCBE ‘Lagniappe’ [I. longipes Chapm. ex Trelease] ALJG ‘Lake City’ [I. opaca Ait.] DCNA GACG LAHG LASL ‘Lassie’ [I. x koehneana Loesn.] MDMN latifolia Thunb. ex J.A.Murr. Luster-leaved H., Broad-leaved H. ALAU GACG MDLT TXDC ALBG GADG NCDU VACW ALTD GAEC NCEL VAGS DCCG GAIS SCMG VASC DCNA GAWH SCMP DCWH LASL TNSB FLUF MDBG TNUT ‘Latifolia’ [I. crenata Thunb. ex J.A.Murr.] (Many cultivars of different origins) DCNA GACG MDLT FLMG GAFN ‘Latispina’ [I. aquifolium L.] VADW ‘Laura’ [I. opaca Ait.] DCNA NCSH ‘Laurifolia’ [I. <x altaclarensis (Loud.) Dallim. ex Rehd.] DCNA ‘Laurifolia’ [I. aquifolium L.] = ‘Laurifolia’ [I. <x altaclerensis] ‘Lenape Moon’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA ‘Lepux’ [I. cornuta x I. pernyji] GACG ‘Leucocarpa’ [I. serrata Thunb. ex J.A.Murr.] Yellow-fruited Japanese Winterberry DCNA ILEX leucoclada Makino Yellow-stemmed H. DCNA ‘Lewis’ [I. aquifolium L.] MDHA ‘Libby’s Favorite’ [I. integra < I. cornuta] ALTD ‘Lilliput’ [I. aquifolium L.] DCNA ‘Lilygold’ [I. ILEX aquifolium L.] DCNA ‘Lindleyana’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Little Bull’ [I. aquifolium L.] DCNA liukiuensis Loesn. Liukiu H. GACG ‘Lock Raven’ [I. x koehneana Loesn.] DCNA lohfauensis Merr. DCNA ‘Longfellow’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Longifolia’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of diverse origins.) DCWH longipes Chapm. ex Trelease Long-stalked H., Georgian H. ALTD LAHG NCDU TXLL DCNA MDTD TNFB _‘Longspra’ [I. aquifolium L.] NCSH ‘Lord’ [I. rotunda Thunb. ex J.A.Murr.] DCNA ‘Louise’ [I. aquifolium L.] DCNA ‘Louise Holmes’ [I. x attenuata Ashe] DCNA VACW ‘Lowei’ [I. myrtifolia Walt.] leucoclada Makino Yellow-stemmed H. DCNA ‘Lewis’ [I. aquifolium L.] MDHA ‘Libby’s Favorite’ [I. integra < I. cornuta] ALTD ‘Lilliput’ [I. aquifolium L.] DCNA ‘Lilygold’ [I. aquifolium L.] DCNA ‘Lindleyana’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Little Bull’ [I. aquifolium L.] DCNA liukiuensis Loesn. Liukiu H. GACG ‘Lock Raven’ [I. x koehneana Loesn.] DCNA lohfauensis Merr. DCNA ‘Longfellow’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Longifolia’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of diverse origins.) DCWH longipes Chapm. ex Trelease Long-stalked H., Georgian H. ALTD LAHG NCDU TXLL DCNA MDTD TNFB _‘Longspra’ [I. aquifolium L.] NCSH ‘Lord’ [I. rotunda Thunb. ex J.A.Murr.] DCNA ‘Louise’ [I. aquifolium L.] DCNA ‘Louise Holmes’ [I. x attenuata Ashe] DCNA VACW 101 ILEX ILEX ILEX ‘Marnockii’ [I. <x altaclerensis (Loud.) Dallim.] GACG ‘Marshal Tito’ [I. aquifolium L.] DCNA ‘Mary Nell’ [(U. cornuta ‘Burfordii’ <I. pernyi) ‘Red Delight’ <I. latifolia] ALTD ‘Maryland’ [I. opaca Ait.] DCNA ‘Maryland Beauty’ [I. verticillata (L.) A.Gr.] MDJN ‘Maryland Dwarf [I. opaca Ait.] DCNA MDBG VATA maximowicziana Loesn. var. kanehirae (Yamamoto) Yamazaki DCNA GACG VATA ‘Maxwell’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Medallion’ [I. cornuta Lindl. & Paxt.] DCNA ‘Menantico’ [I. opaca Ait.] DCNA ‘Mentor Dense’ [I. crenata Thunb. ex J.A.Murr.] DCNA GACG ‘Mentor Glossy’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Merry Christmas’ [I. opaca Ait.] MDAL x meserveae S.Y.Hu [I. rugosa X I. aquifolium] (The “blue” hollies; see cultivar names beginning with Blue) ‘Mesglog [I. x meserveae S.Y.Hul] = ‘Golden Girl’ ‘Microphylla’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of different origins) ALFN DCNA GACG macropoda Miq. DCNA VABF ‘Maderensis Variegata’ [I. aquifolium L.] DCNA ‘Mae’ [I. opaca Ait.] DCNA ‘Mae’ [I. opaca Ait.] DCNA ‘Magna Semen’ [I. opaca Ait.] DCNA ‘Magna Semen’ [I. opaca Ait.] DCNA ‘Major’ [I. crenata Thunb. ex J.A.Murr.] (Name applied to numerous clones of different origins) DCNA GACG ‘Malcolm S. Whipple’ [I. aquifolium L.] MDBG ‘Mamie Eisenhower’ [I. opaca Ait.] DCNA GACG ‘Manig’ [I. opaca Ait.] DCNA MDBG ‘Manig’ [I. opaca Ait.] DCNA MDBG ‘Maplehurst’ [I. cornuta x I. aquifolium] DCNA ILEX ILEX ‘Nellie R. Stevens’ [I. cornuta < mutchagara Makino = I. maximowicziana var. kanahirae myrtifolia Walt. Myrtle-leaved H. DCNA LAHG SCJF GACG MSFG SCRH GAFN SCBR VATA myrtifolia Walt. (Yellow-fruited; not equivalent to ‘Lowei’) GAEC ‘Myrtifolia’ [I. aquifolium L.] GACG ‘Nakada’ [I. crenata Thunb. ex J.A.Murr.] DCNA MDTD VATA ‘Nakaharae’ [I. sugerokii Maxim.] DCNA ‘Nana’ [I. glabra (L.) A.Gr.] (Several clones of different origins) DCNA ‘Nana’ [I. verticillata (L.) A.Gr.] = ‘Red Sprite’ ‘Nana’ [I. vomitoria Ait.] (Several clones of different origins) ALAU FLMG GACG LAHG ‘Nanking’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘NASA’ [I. <x attenuata Ashe] ALTD ‘Natchez Belle’ [I. longipes Chapm. ex Trelease] MSBH ‘National’ [I. cornuta Lindl. & Paxt.] ALTD ‘Needle Point’ [I. cornuta Lindl. & Paxt.] LASL I. aquifolium] ALTD DCWH MDLT TNUT DCCG GACG NCTP VATA DCNA MDBG SCKG ‘Nelson West’ [I. opaca Ait.] DCNA ‘N. F. Barnes’ [I. aquifolium L.] GACG mutchagara Makino = I. maximowicziana var. kanahirae ‘Morgan Gold’ [I. opaca Ait. f. xanthocarpa Rehd.] DCIW ‘Morrell No. 1’ [I. cornuta Lindl. & Paxt.] GACG ‘Morrell No. 3’ [I. cornuta Lindl. & Paxt.] GACG 103 ILEX ILEX ILEX ILEX ‘Pale Moon’ [I. aquifolium L.] GACG ‘Palmetto’ [I. opaca Ait.] ALTD paraguariensis St.Hil. Paraguay Tea, Yerba Mate DCNA FLUF ‘Peconic’ [I. crenata Thunb. ex J.A.Murr.] DCNA pedunculosa Miq. Evergreen Long-stalked H. ALTD DEWG MDLT MDTD DCCG GACG MDMG SCWI DCNA GAEC MDMN pedunculosa Miq. var. continentalis Loesn. = I. pedunculosa ‘Pendula’ [I. aquifolium L.] DCNA GACG perado Ait. ssp. platyphylla (P.B.Webb & Berth.) S.Andrews DCNA ‘Perkins #1’ [I. aquifolium L.] DCNA ‘Perkins-de-Wilde #1’ [I. opaca Ait.] DCNA ‘Perkins-de-Wilde #2’ [I. opaca Ait. ] DCNA ‘Perkins-de-Wilde #3’ [I. opaca Ait.] DCNA ‘Perle LeClair’ [I. opaca Ait. f. subintegra Weatherby] DCNA NCDU pernyi Franch. ALFN FLMG GALA VABF ALTD GACG MDMG VAWR DCNA GAFN TNUT pernyi Franch. var. veitchii (J.G.Veitch) Bean = I. bioritensis ‘Perrine’ [I. opaca Ait.] DCNA ‘Petite’ [I. aquifolium L.] DCNA GACG ‘Phantom Gold’ [I. aquifolium L.] ILEX ‘Pale Moon’ [I. aquifolium L.] GACG ‘Palmetto’ [I. opaca Ait.] ALTD paraguariensis St.Hil. ‘Magna Semen’ [I. opaca Ait.] DCNA Paraguay Tea, Yerba Mate DCNA FLUF ‘Peconic’ [I. crenata Thunb. ex J.A.Murr.] DCNA pedunculosa Miq. Evergreen Long-stalked H. ALTD DEWG MDLT MDTD DCCG GACG MDMG SCWI DCNA GAEC MDMN pedunculosa Miq. var. continentalis Loesn. = I. pedunculosa ‘Pendula’ [I. aquifolium L.] DCNA GACG perado Ait. ssp. platyphylla (P.B.Webb & Berth.) S.Andrews DCNA ‘Perkins #1’ [I. aquifolium L.] DCNA ‘Perkins-de-Wilde #1’ [I. opaca Ait.] DCNA ‘Perkins-de-Wilde #2’ [I. opaca Ait. ] DCNA ‘Perkins-de-Wilde #3’ [I. opaca Ait.] DCNA ‘Perle LeClair’ [I. opaca Ait. f. subintegra Weatherby] DCNA NCDU pernyi Franch. ALFN FLMG GALA VABF ALTD GACG MDMG VAWR DCNA GAFN TNUT pernyi Franch. var. veitchii (J.G.Veitch) Bean = I. bioritensis ‘Perrine’ [I. opaca Ait.] DCNA ILEX ‘Nigrescens’ [I. <x altaclerensis (Loud.) Dallim.] DCNA nobilis Gumbleton = I. kingiana ‘NYBG #2’ [I. aquifolium L.] DCNA ‘Oconee River’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Old Gold’ [I. opaca Ait. f. xanthocarpa Rehd.] DCNA ‘Old Heavy Berry’ [I. opaca Ait.] DCNA GACG VACW ‘Oleafera’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Olga’ [I. cornuta Lindl. & Paxt.] DCCB opaca Ait. American H. ALSH FLCG MDLT SCNS DCCG FLMG MDMG VACW DCNA GAOH SCBR DCWH MDBG SCMP opaca Ait. var. arenicola (Ashe) Ashe = I. cumulicola opaca Ait. f. subintegra Weatherby DCNA MDAB MDSJ SCMP FLPW MDJS NCBE LASL MDMN SCBR opaca Ait. f. xanthocarpa Rehd. Yellow-fruited American H. DCNA GACG NCEG VACW * ‘Oriole’ [(1. myrtifolia < I. opaca) x I. myrtifolia] DCNA ‘Osa’ [I. opaca Ait.] DCNA ‘O. Spring’ [I. cornuta Lindl. & Paxt.] DCNA ‘Otis Miley’ [I. vomitoria Ait.] GACG ILEX ILEX ILEX ‘Piccolo’ [I. crenata Thunb. ex J.A.Murr.] DECN ‘Pin Cushion’ [I. opaca Ait.] DCNA ‘Pinto’ [I. aquifolium L.] DCNA GACG ‘Planifolia’ [I. aquifolium L.] DCNA GACG ‘Pocahontas’ [I. decidua Walt.] (Originally known as ‘H. B. Red’, an invalid name) DCNA ‘Polly’ [I. opaca Ait.] DCNA ‘Pomona’ [I. opaca Ait.] DCNA poneantha Koidz. DCNA ‘Pot-O-Gold’ [I. aquifolium L.] GACG ‘Pride Dwarf’ [I. crenata Thunb. ex J.A.Murr.] DCNA ‘Pride’s Tiny’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Princess Pat’ [I. aquifolium L.] DCNA pubescens Hook. & Arn. ALTD DCNA FLUF purpurea Hassk. Peach-leaved H., Kachi H. DCNA GAIS TNSN GACG GAUG TNUT ‘Pyramidalis’ [I. aquifolium L.] DCNA ‘Pyramidalis’ [I. crenata Thunb. ex J.A.Murr.] MDAL ‘Pyramidalis Compacta’ [I. aquifolium L.] DCNA ‘Red Delight’ [I. cornuta ‘Burfordii’ < I. pernyjij DCNA GACG ‘Rederly’ [I. aquifolium L.] DCNA GACG ‘Red Robe’ [I. cornuta x I. ‘Magna Semen’ [I. opaca Ait.] DCNA pernyi] GACG ‘Red Sprite’ [I. verticillata (L.) A.Gr.] DCNA ‘Repandens’ [I. crenata Thunb. ex J.A.Murr.] DCNA GACG ‘Reynolds’ [I. opaca Ait.]} DCNA TNTV ‘Richard E. Lincoln’ [I. verticillata (L.) A.Gr.] MDES ‘Richards’ [I. opaca Ait.] MDHR ‘Ricker’ [I. aquifolium L.] DCNA MDHA ‘Riddle Farm’ [I. aquifolium L.] DCNA ‘Rock Garden’ [I. Xaquipernyi < (I. integra x I. pernyji) ‘Accent’] DCNA ‘Rocky Creek’ [I. crenata Thunb. ex J.A.Murr.] DCNA rotunda Thunb. ex J.A.Murr. Kurogane H. ALTD FLFS GACH VANB DCNA FLUF GAIS FLFB GACG LAAL ‘Rotunda’ [I. cornuta Lindl. & Paxt.] ALTD FLUF MDLT TXHA DCLC GACG MDTD DCNA GAFN SCMG ‘Royal Red’ [I. < altaclerensis (Loud.) Dallim.] ‘Pocahontas’ [I. decidua Walt.] (Originally known as ‘H. B. Red’, an invalid name) DCNA 105 ILEX ‘Sentinel’ [I. crenata Thunb. ex J.A.Murr.] VATA * ‘September Gem’ [I. ciliospinosa x I. X aquipernyi] DCNA MDWP serrata Thunb. ex J.A.Murr. Japanese Winterberry DCNA GACG MDPJ NCBE DEEM MDBG MDTD serrata xX I. verticillata SCCU serrata Thunb. ex J.A.Murr. var. argutidens (Miq.) Rehd. = I. serrata serrata Thunb. ex J.A.Murr. var. sieboldii (Miq.) Rehd. = I. serrata ‘Shanghai’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Shangri-La’ [I. cornuta Lindl. & Paxt.] DCNA GACG ‘Shaver’ [I. verticillata (L.) A.Gr.] DCNA shennongjiaensis T.R.Dudley & Sun DCNA ‘Shilling’s’ [I. vomitoria Ait.] GACL NCTP ‘Shilling’s Dwarf’ [I. vomitoria Ait.] (This cultivar and ‘Schilling’s’ are confused) GAJI ‘Shiu-ying’ [I. cornuta Lindl. & Paxt.] MDMN ‘Shortspra’ [I. aquifolium L.] DCNA GACG sikkimensis Hook. Sikkim H. DCNA ‘Silver Edge’ [I. aquifolium L.] = ‘Argentea Marginata’ ‘Silver Milkboy’ [I. aquifolium L.] GACG ‘Silver Milkmaid’ [I. aquifolium L.] DCNA ILEX ILEX ILEX ILEX ‘Rubricaulis Aurea’ [I. aquifolium L.] DCNA GACG * ‘Ruby’ [I. < koehneana Loesn.] DCNA MDTD VATA ‘Ruby Red’ [I. opaca Ait.] TNTV rugosa F.Schmidt Tsuru H., Creeping H. DCNA ‘Rushton’ [I. opaca Ait.] DCNA ‘R. V. P. Special’ [I. cornuta Lindl. & Paxt.] MDHD ‘St. Ann’ [I. opaca Ait.} DCNA ‘St. John’s’ [I. opaca Ait.] MDSJ ‘St. Mary’ [I. opaca Ait.] DCNA GACG ‘San Jose’ [I. < aquipernyi Gable] GACG MDTD ‘San Jose’ [I. koehneana Loesn.] DCNA GACG ‘Sandy Hook #5’ [I. opaca Ait.] DCNA ‘Sara Higgins’ [I. opaca Ait.] NCSH ‘Satyr Hill’ [I. opaca Ait.] MDMN VATA ‘Savannah’ [I. x attenuata Ashe] ALBG GACG LASL SCKG DCNA GAET SCBR FLUF LAHG SCFW ‘Schlupp’ [I. opaca Ait.] VACW ‘Scotica’ [I. aquifolium L.] MDBG ‘Scram’s Dwarf [I. aquifolium L.] DCNA ‘Secrest’ [I. opaca Ait.] ‘Shanghai’ [I. crenata Thunb. ex J.A.Murr.] GACG ‘Shangri-La’ [I. cornuta Lindl. & Paxt.] DCNA GACG ‘Shaver’ [I. verticillata (L.) A.Gr.] DCNA shennongjiaensis T.R.Dudley & Sun DCNA ‘Shilling’s’ [I. vomitoria Ait.] GACL NCTP ILEX ILEX ILEX ILEX ‘Tankard’s Compact’ [I. glabra (L.) A.Gr.] VATA ‘Tennyson’ [I. crenata Thunb. ex J.A.Murr.] DCNA GACG ‘Teufel’s Hybrid’ [I. aquifolium L.] DCNA GACG ‘Teufel’s Variegated’ [I. aquifolium L.] GACG thomsonii (Hook.f.) Hara = I. crenata var. thomsonii ‘Thornton’ [I. aquifolium L.] DCNA ‘Tiny [I. crenata Thunb. ex J.A.Murr.] = ‘Pride’s Tiny’ ‘Tiny’ [I. opaca Ait.] DCNA ‘Tiny Tim’ [I. crenata Thunb. ex J.A.Murr.] = ‘Pride’s Tiny’ ‘Titan’ [I. cornuta x I. pernyji] DCNA GACG MDBG ‘Tom Everett’ [I. aquifolium L.] GACG ‘T-one’ [I. crenata Thunb. ex J.A.Murr.] VATA ‘Toner’ [I. opaca Ait.] DCSE MDHD MDKN ‘Topeli’ [I. x attenuata Ashe] DCNA ‘Trisco’ [I. opaca Ait.] DCNA ‘24 Karat’ [I. opaca Ait.] MDPM ‘Tyke’ [I. crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara] SCCU ‘Variegata’ [I. cornuta Lindl. & Paxt.] GACG ‘Vaseyi’ [I. crenata Thunb. ex J.A.Murr.] GACG ILEX ‘Silver Princess’ [I. aquifolium L.] = ‘Argentea Marginata’ ‘Sirofukurin’ [I. crenata Thunb. ex J.A.Murr.] = ‘Snowflake’ ‘Slim Jim’ [I. opaca Ait.] DCNA ‘Snowflake’ [I. crenata Thunb. ex J.A.Murr.] DCNA * ‘Sparkleberry’ [I. serrata x I. verticillata] DCNA VATA ‘Sparkler’ [I. aquifolium L.] GACG spinigera (Loesn.) Loesn. DCNA ‘Star’ [I. opaca Ait.] DCNA ‘Starker’s Silver’ [I. aquifolium L.] = ‘Elegantissima’ ‘Stokes’ [I. crenata Thunb. ex J.A.Murr.] DCNA GACG ‘Stokes Dwarf’ [I. vomitoria Ait.] LASL sugerokii Maxim. DCNA VATA ‘Sunnybrooke’ [I. aquifolium L.] DCNA GACG * ‘Sunny Foster’ [I. x attenuata Ashe] DCNA MDBG ‘Sunnyside’ [I. aquifolium L.] DCNA ‘Sunset’ [I. verticillata (L.) A.Gr.] DCNA ‘Taber’ [I. opaca Ait.] GACG ‘Taber #2’ [I. opaca Ait.] ALFN * ‘Tanager’ [(I. myrtifolia < I. opaca) x I. myrtifolia] 107 ILEX ‘Vera’ [I. opaca Ait.] MDBG verticillata (L.) A.Gr. Winterberry, Black Alder ALMG MDBG SCCU VAPH ALTD MDLT TNFB VATA ALUA NCAS VAAC DCNA NCBE VAGW GACG SCBR VAHR verticillata (L.) A.Gr. f. chrysocarpa Robinson Yellow-berried Winterberry DCNA ‘Virginia Dare’ [I. vomitoria Ait.] = ‘Dare County’ ‘Virgo’ [I. cornuta x I. pernyi] DCNA ‘Viridis’ [I. glabra (L.) A.Gr.] DCNA vomitoria Ait. Yaupon ALBG FLMG NCDU SCUC ARSN FLUF NCEG TXDM DCNA GACG SCBR VACW FLAL GAJI SCMP VAGW FLCG GAJY SCNS vomitoria Ait. var. chiapiensis Sharp Mexican Yaupon DCNA vomitoria Ait. f. pendula Foret & Solym. Pendulous Yaupon DCNA LAHG LASL GACG LAHP ‘Walker’ [I. cornuta Lindl. & Paxt.] GACG ‘Watereriana’ [I. aquifolium L.] Waterer’s Gold H. DCNA ‘Wayne’ [I. crenata Thunb. ILEX ex J.A.Murr.] MDAL ILEX ILEX ILEX ALTD MDLT TNFB VATA ALUA NCAS VAAC DCNA NCBE VAGW GACG SCBR VAHR verticillata (L.) A.Gr. f. chrysocarpa Robinson Yellow-berried Winterberry DCNA ‘Virginia Dare’ [I. vomitoria Ait.] = ‘Dare County’ ‘Virgo’ [I. cornuta x I. pernyi] DCNA ‘Viridis’ [I. glabra (L.) A.Gr.] DCNA vomitoria Ait. Yaupon ALBG FLMG NCDU SCUC ARSN FLUF NCEG TXDM DCNA GACG SCBR VACW FLAL GAJI SCMP VAGW FLCG GAJY SCNS vomitoria Ait. var. chiapiensis Sharp Mexican Yaupon DCNA vomitoria Ait. f. pendula Foret & Solym. Pendulous Yaupon DCNA LAHG LASL GACG LAHP ‘Walker’ [I. cornuta Lindl. & Paxt.] GACG ‘Watereriana’ [I. aquifolium L.] Waterer’s Gold H. DCNA ‘Wayne’ [I. crenata Thunb. ex J.A.Murr.] MDAL ‘Wheeler #1’ [I. opaca] = ‘Dick’ ‘Wheeler #4’ [I. aquifolium L.] DCNA NCSH ‘Wheeler #4 [I. opaca Ait.] (Cultivar name illegitimate) NCSH ILEX ‘Yule Glow’ [I. aquifolium L.] GACG ‘Yunnan’ [I. crenata Thunb. ex J.A.Murr.] GACG yunnanensis Franch. Yunnan H. DCNA yunnanensis Franch. var. gentilis Loesn. DCNA ‘Zero’ [I. aquifolium L.] DCNA GACG zhejiangensis C.J.Tseng DCNA ILLICIUM L. ILLICIACEAE Ilicium Family anisatum L. Japanese Anise Tree ALEH GACG TXLL ALTD GAEC VATA floridanum Ellis Florida Anise ALAU FLGS GAEC SCWI ALBG FLMG GAWH TNDG ALEH FLUF LALG TXLL ALSH GACE MDLT VACW DCNA GACG NCBE VAPH FLAL GACH SCMP VAZC floridanum Ellis f. album F.G.Mey. & Mazzeo ALTD DCNA SCWI henryi Diels Chinese Anise Tree ALTD mexicanum A.C.Sm. SCWI parviflorum Michx. ex Vent. FLLE LAGN SCMG VATA FLMG NCAG SCMP FLUF NCDU TNFR GAJC SCBR VACW ‘Semmes’ [I. floridanum Ellis f. album F.G.Mey. & Mazzeo] ALTD ALWR DCNA JACARANDA INDIGOFERA L. Indigo FABACEAE (Faboideae) Bean Family ‘Alba’ [I. decora Lindl.] DCNA DEWG MDGD decora Lindl. LAAL LARP SCJA LAHP LASL incarnata (Willd.) Nakai = I. decora kirilowii Maxim. DEWG VAGS potaninii Craib DCNA suffruticosa Mill. SCBR SCCL ITEA L. SAXIFRAGACEAE (Iteoideae) Saxifrage Family ilicifolia Oliv. NCEL virginica L. Virginia Willow DCNA LASL SCBR VACW GAGB MDBG TNRT LALG MDLT TXRS JACARANDA Juss. BIGNONIACEAE Bignonia Family acutifolia HBK. Jacaranda, Green Ebony LALG INDIGOFERA L. Indigo FABACEAE (Faboideae) Bean Family ‘Alba’ [I. decora Lindl.] DCNA DEWG MDGD decora Lindl. LAAL LARP SCJA LAHP LASL incarnata (Willd.) Nakai = I. decora kirilowii Maxim. DEWG VAGS potaninii Craib DCNA suffruticosa Mill. SCBR SCCL ITEA L. SAXIFRAGACEAE (Iteoideae) Saxifrage Family ilicifolia Oliv. NCEL virginica L. Virginia Willow DCNA LASL SCBR VACW GAGB MDBG TNRT LALG MDLT TXRS JACARANDA Juss. ILEX BIGNONIACEAE Bignonia Family acutifolia HBK. Jacaranda, Green Ebony LALG ITEA L. SAXIFRAGACEAE (Iteoideae) Saxifrage Family ilicifolia Oliv. NCEL virginica L. Virginia Willow DCNA LASL SCBR VACW GAGB MDBG TNRT LALG MDLT TXRS JACARANDA Juss. BIGNONIACEAE Bignonia Family acutifolia HBK. Jacaranda, Green Ebony LALG 109 ILLICIUM floridanum Ellis [illustrator Susan M. Johnston] 110 110 ILLICIUM parviflorum Michx. ex Vent. [illustrator Susan M. Johnston] 111 111 JASMINUM JASMINUM L. OLEACEAE Jasmine Olive Family beesianum Forrest & Diels MDGD floridum Bunge ALBH NCDU TNRM VANB GABS SCBR TNUT VAPH LAHG SCCU TXLL MDGD SCWI VAGW Sruticans L. SCWI humile L. LAMP LASL mesnyi Hance Primrose J. ALSH GAJI LASL sSCuUCc FLDG LAAP SCBR VACW multiflorum (Burm.f.) Andr. Star J. FLUF LALG nitidum Skan Angel-wing J. FLUF GACG LALG nudiflorum Lindl. Winter J. MDBG MDLA MDLT parkeri S.T.Dunn VACW ‘Revolutum’ [J. humile L.] Italian J. SCWI x stephanense E.Lemoine [J. beesianum < J. officinale] SCWI JUGLANS L. Walnut JUGLANDACEAE Walnut Family ailantifolia Carr. DCNA TNFE TNSC JUNIPERUS JASMINUM JASMINUM L. OLEACEAE Jasmine Olive Family beesianum Forrest & Diels MDGD floridum Bunge ALBH NCDU TNRM VANB GABS SCBR TNUT VAPH LAHG SCCU TXLL MDGD SCWI VAGW Sruticans L. SCWI humile L. LAMP LASL mesnyi Hance Primrose J. ALSH GAJI LASL sSCuUCc FLDG LAAP SCBR VACW multiflorum (Burm.f.) Andr. Star J. FLUF LALG nitidum Skan Angel-wing J. FLUF GACG LALG nudiflorum Lindl. Winter J. MDBG MDLA MDLT parkeri S.T.Dunn VACW ‘Revolutum’ [J. humile L.] Italian J. SCWI x stephanense E.Lemoine [J. beesianum < J. officinale] SCWI JUGLANS L. Walnut JUGLANDACEAE Walnut Family ailantifolia Carr. DCNA TNFE TNSC DCWR TNLW ailantifolia Carr. var. cordiformis (Maxim.) Rehd. Japanese W. JUNIPERUS ‘Broadview’ [J. regia L.] MDGD cinerea L. Butternut DCCG ‘Hansen’ [J. regia L.] MDGD ‘Laciniata’ [J. nigra L.] TNNE major (Torr.) Heller Arizona W. DCNA ‘McDermid’ [J. regia L.] MDGD nigra L. Black W. DECCE GAJS MDNA VAWP DEMC MDCP SCBR FLUF MDHN TNUT GAIS MDLT VACW ‘Paradox’ [J. hindsii < J. regia] DCNA regia L. English W., Persian W. DCCG VACW JUNIPERUS L. Juniper CUPRESSACEAE Cypress Family ‘Admirabilis’ [J. horizontalis Moench] DCNA ‘Alba’ [J. scopulorum Sarg.] DCNA ‘Arcadia’ [J. sabina L.] DCNA ‘Aurea’ [J. chinensis L.] VAMP ‘Aurea’ [J. virginiana L.] DCNA ‘Bar Harbor’ [J. horizontalis Moench] DCNA FLUF MDBG ‘Berkshire’ [J. communis L.] — — i) JASMINUM nudiflorum Lindl. [illustrator Lillian Nicholson Meyer] 113 ia L. [illustrator Lillian Nicholson Meyer] JUGLANS reg ia L. ILEX [illustrator Lillian Nicholson Meyer] JUGLANS reg 114 JUNIPERUS communis L. ssp. depressg (Pursh) Franco Ground J. DCNA communis L. var. montana Ait. DCNA GAEC conferta Parl. Shore J. ALAU FLLE LAAL SCBR ALBG FLMG LAHG SCMG ALFN FLUF LATU VACW DCNA GAFN MDLT VANB DEMC GAJI MSHB VAPH FLFS GAUG NCAS ‘Den Boer’ [J. < media Van Melle] [J. sabina < J. chinensis] DCNA deppeana Steud. var. pachyphlaea (Torr.) Martinez Alligator J. VABF ‘Douglasii’ [J. horizontalis Moench] DCNA ‘Echiniformis’ [J. chinensis L.] DCNA ‘Emerald Sea’ [J. conferta Parl.] DCNA MDBG VATA ‘Expansa’ [J. davurica Pall.] DCNA FLUF ‘Expansa Aureo-spicata’ [J. davurica Pall.] ALFN DCNA LASL SCMG ‘Expansa Variegata’ [J. davurica Pall.] DCNA MDBG ‘Fastigiata’ [J. sabina L.] DCNA ‘Fastigiata’ [J. virginiana L.] DCNA ‘Filicinus’ [J. horizontalis Moench] DCNA ‘Filicinus Minimus’ [J. horizontalis Moench] MDBG formosana Hayata NCWT ‘Fruitlandii’ [J. < media Van Melle] [J. sabina < J. chinensis] GAFN JUNIPERUS JUNIPERUS JUNIPERUS ‘Blue Chip’ [J. horizontalis Moench] DCNA ‘Blue Cloud’ [J. x media Van Melle] [J. sabina < J. chinensis] DCNA FLMG ‘Blue Forest’ [J. horizontalis Moench] DCNA MDBG ‘Blue Heaven’ [J. scopulorum Sarg.] MDBG ‘Blue Horizon’ [J. horizontalis Moench] DCNA ‘Blue Muffet’ [J. horizontalis Moench] MDLT ‘Blue Pacific’ [J. conferta Parl.] VATA ‘Blue Rug’ [J. horizontalis Moench] GAEC ‘Blue Star’ [J. squamata Buch.-Ham. ex Lamb.] DCNA MDBG MDLT ‘Blue Vase’ [J. < media Van Melle] [J. sabina < J. chinensis] ALBH DCNA FLUF LASL ‘Broadmoor’ [J. sabina L.] DCNA ‘Buffalo’ [J. sabina L.] DCNA ‘Canaertii’ [J. virginiana L.] LASL VACW ‘Chandler Blue’ [J. scopulorum Sarg.] DCNA chinensis L. Chinese J. ALAU FLMG MDMR VATA ALBG FLUF SCMG ARUM GANG TNBC FLLE LATU VABF chinensis L. var. sargentii A.Henry Sargent J. DCNA GAUG MDBG GAFN LAAL JUNIPERUS communis L. ssp. depressg (Pursh) Franco Ground J. DCNA communis L. var. montana Ait. DCNA GAEC conferta Parl. Shore J. ALAU FLLE LAAL SCBR ALBG FLMG LAHG SCMG ALFN FLUF LATU VACW DCNA GAFN MDLT VANB DEMC GAJI MSHB VAPH FLFS GAUG NCAS ‘Den Boer’ [J. < media Van Melle] [J. sabina < J. chinensis] DCNA deppeana Steud. var. pachyphlaea (Torr.) Martinez Alligator J. VABF ‘Douglasii’ [J. horizontalis Moench] DCNA ‘Echiniformis’ [J. chinensis L.] DCNA ‘Emerald Sea’ [J. conferta Parl.] DCNA MDBG VATA ‘Expansa’ [J. davurica Pall.] DCNA FLUF ‘Expansa Aureo-spicata’ [J. davurica Pall.] ALFN DCNA LASL SCMG ‘Expansa Variegata’ [J. davurica Pall.] DCNA MDBG ‘Fastigiata’ [J. sabina L.] DCNA ‘Fastigiata’ [J. virginiana L.] DCNA ‘Filicinus’ [J. horizontalis Moench] DCNA ‘Filicinus Minimus’ [J. horizontalis Moench] MDBG formosana Hayata LS JUNIPERUS JUNIPERUS JUNIPERUS ‘Gareei’ [J. scopulorum Sarg.] DCNA ‘Glauca’ [J. horizontalis Moench] ALBH ‘Glauca’ [J. virginiana L.] ALTD ‘Glenmore’ [J. horizontalis Moench] DCNA ‘Globosa Cinerea’ [J. chinensis L.] DCNA ‘Glomerata’ [J. horizontalis Moench] DCNA ‘Gold Star’ [J. chinensis L.] MDBG ‘Gray Gleam’ [J. scopulorum Sarg.] DCNA ‘Grey Owl’ [J. virginiana L.] SCJUF VATA ‘Gulf Tide’ [J. conferta Parl.] SCMG ‘Hetzii’ [J. chinensis L.] Hetz Blue J. MDBG MDJW NCDU SCCU ‘Hibernica’ [J. communis L.] Irish J. ALAU DCNA GAGM VAMP ALBH GAEC NCBE ‘Hill‘s Silver’ [J. scopulorum Sarg.] DCNA horizontalis Moench Creeping J. DCCG NCDU VATA DCNA NCWC ‘Hornibrookii’ [J. communis L. var. montana Ait.] DCNA ‘Hughes’ [J. horizontalis Moench] DCNA ‘Kaizuka’ [J. chinensis L.] Hollywood J. ALBG FLUF SCCU DCNA LASL VABF FLFS MDBG VATA JUNIPERUS ‘Kaizuka Variegated’ [J. chinensis L.] MDBG VATA ‘Keteleeri’ [J. virginiana L.] ALFN VACW ‘Lakewood’ [J. scopulorum Sarg.] DCNA ‘Lakewood Globe’ [J. scopulorum Sarg.] DCNA ‘Livida’ [J. horizontalis Moench] DCNA ‘Loderi’ [J. squamata Buch.-Ham. ex Lamb.] DCNA ‘Marcella’ [J. horizontalis Moench] DCNA ‘Meyeri’ [J. squamata Buch.-Ham. ex Lamb.] GAEC LAAL ‘Mint Julep’ [J. x media Van Melle] [J. sabina < J. chinensis] FLUF ‘Nana’ [J. procumbens (Endl.) Miq.] DCNA MDBG ‘Nova’ [J. virginiana L.] DCNA ‘Oblonga Pendula’ [J. communis L.] MDEM NCSM VACW VAPH ‘Old Gold’ [J. « media Van Melle] [J. sabina < J. chinensis] DCNA pachyphlaea Torr. = J. deppeana var. pachyphlaea ‘Parsonii’ [J. chinensis L.] VATA ‘Pathfinder’ [J. scopulorum Sarg.] DCCG DCNA ‘Pencil Point’ [J. communis L.] DCNA ‘Petraea’ [J. horizontalis Moench] DCNA JUNIPERUS _ JUNIPERUS - ‘Pfitzeriana’ [J. x media Van Melle] Pitzer J. [J. sabina X< J. chinensis] ALIT DCNA MDAB ALSM FLMG NCWC \| ARRB FLPM SCFW ‘Pfitzeriana Aurea’ [J. < media Van Melle] Golden Pfitzer J. [J. sabina < J. chinensis] GAEC MDJS TNMB ‘Pfitzeriana Compacta’ [J. x media Van Melle] [J. sabina x J. chinensis] DCNA ‘Platinum’ [J. scopulorum Sarg.] DCNA ‘Plumosa’ [J. horizontalis Moench] Andorra J. GAUG SCMP ‘Plumosa Aurea’ [J. < media Van Melle] [J. sabina < J. chinensis] DCNA ‘Prince of Wales’ [J. horizontalis Moench] DCNA procumbens (Endl.) Miq. DCNA FLUF LAHG VAMP FLDG GAAB LASL VATA FLLE GAEC SCMG ‘Procumbens’ [J. horizontalis Moench] DCNA ‘Pulchella’ [J. horizontalis Moench] DCNA ‘Reptans’ [J. virginiana L.] DCNA rigida Sieb. & Zucc. Needle J. DCNA GAEC LAAL ‘Robusta Green’ [J. chinensis L.] DCNA VATA ‘San Jose’ [J. chinensis L.] ARSN GAUG scopulorum Sarg. Rocky Mountain J. JUNIPERUS DCNA JUNIPERUS JUNIPERUS ‘Sentinel’ [J. communis L.]_ DCNA ‘Sheppardii’ [J. chinensis L.] DCNA silicicola (Small) Bailey Southern Red Cedar ALBG FLCG GATS SCMP ALTR FLMG NCRM TXMS ALUA FLUF SCMG ‘Skandia’ [J. sabina L.] DCNA ‘Skyrocket’ [J. virginiana L.] MDBG VATA ‘Spartan’ [J. chinensis L.] DCNA squamata Buch.-Ham. ex Lamb. Single-seed J. FLMG NCGM ‘Stricta’ [J. excelsa Bieb.] DCNA VATA ‘Tolleson‘s Blue Weeping’ [J. scopulorum Sarg.] DCNA MDMR VAMD ‘Torulosa’ [J. chinensis L.] = ‘Kaizuka’ ‘Variegata’ [J. chinensis L.] GAFN TNUT virginiana L. Red Cedar DCNA MDHN SCFS VACW FLCG MDLT SCMG VAMP FLUF NCDU SCWP GAUG NCWT TNHG MDBG SCCU TNSG ‘Viridis’ [J. chinensis L. var. sargentii A.Henry] DCNA ‘Von Ehren’ [J. sabina L.] LAAL ‘Wichita Blue’ [J. scopulorum Sarg.] DCNA ‘Wiltonii’ [J. horizontalis Moench] DCNA GAUG MDBG VATA P17 KOELREUTERIA JUSTICIA KOELREUTERIA ‘Ostbo Red’ [K. latifolia L.] MDLT ‘Sharon Rose’ [K. latifolia L.] MDBG ‘Shooting Star’ [K. latifolia L.] GAEC KALOPANAX Miq. ARALIACEAE Ginseng Family pictus (Thunb.) Nakai = K. septemlobus ricinifolius (Sieb. & Zucc.) Miq. = K. septemlobus septemlobus (Thunb. ex J.A.Murr.) Koidz. DCNA MDBG MDGJ KERRIA DC. ROSACEAE Rose Family TE a TE TE TL RE ET) ‘Albescens’ [K. japonica (L.) DC.] DCNA japonica (L.) DC. Japanese Rose DEMC MSMN VAPH GAEC SCWI VAWR ‘Picta’ [K. japonica (L.) DC.] MDBG MDFM MDLT ‘Pleniflora’ [K. japonica (L.) DC.] ARHS SCCuU VADW VAWL MDJS TNCT VAGW KOELREUTERIA Laxm. Golden-rain Tree SAPINDACEAE Soapberry Family bipinnata Franch. DCNA DCVK VATA elegans (Seemann) A.C.Sm. ssp. formosana (Hayata) F.G.Mey. ALTD FLUF FLPE LASL MSPR TXMA JUSTICIA JUSTICIA L. ACANTHACEAE Acanthus Family AR DL DPE N PIRI ET RoE MTD ORE ES PTL OS brandegeana Wassh. & L.B.Sm. Shrimp Plant FLCG FLDG FLUF californica (Benth.) D.Gibson Shrimp Plant TXWE ‘Yellow Queen’ [J. brandegeana Wassh. & L.B.Sm.] LALG KADSURA Juss. SCHISANDRACEAE Schisandra Family (CRS IE ORES SD OE EE SN EE es \| japonica (Thunb.) Dunal Kadsura Vine NCEL SCAC SCCU KALMIA L. ERICACEAE Heath Family 2 a Se a a OE ee Ee PSST SEES SS, angustifolia L. Sheep Laurel DEWG MDLT VAZC angustifolia L. var. carolina (Small) Fern. NCDU * ‘Bettina’ [K. latifolia L.] DCNA cuneata Michx. White Wicky SCWI ‘Dexter Pink’ [K. latifolia L.] MDBG ‘Fuscata’ [K. latifolia L.] MDLT hirsuta < K. latifolia GAEC SCWI latifolia L. Mountain Laurel Acanthus Family ‘Yellow Queen’ [J. brandegeana Wassh. & L.B.Sm.] LALG ‘Yellow Queen’ [J. brandegeana Wassh. & L.B.Sm.] LALG KADSURA Juss. KOELREUTERIA LAGERSTROEMIA KOELREUTERIA integrifoliola Merr. = K. bipinnata paniculata Laxm. DCCG GAUG MDWP TNUT DCJM LAHG MSMN VACP DCNA MDBG NCBE VACW DCSE MDJB SCCU VAMP DCWH MDLT TNSC VATA DEMC MDNA TNSN paniculata Laxm. var. apiculata (Rehd. & Wils.) Rehd. = K. paniculata ‘September’ [K. paniculata Laxm.] DCNA KOLKWITZIA Graebn. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) amabilis Graebn. Beauty Bush DEEP MDBE TNSC VACM DEWG MDDF TNSN VASK GABS NCBE TNTV ‘Rosea’ [K. amabilis Graebn.] DCNA +LABURNOCYTISUS Schneid. FABACEAE (Faboideae) Bean Family [Laburnum + Cytisus] adamii (Poit.) Schneid. [Laburnum anagyroides + Cytisus purpureus] MDBG LABURNUM Medik. Golden-chain Tree FABACEAE (Faboideae) Bean Family a a a RE ee WE ETI em} anagyroides Medik. Golden-chain Tree DCNA DEEM MDLA VACW ‘Vossii’ [L. X watereri (Kirchn.) Dipp.] = L. anagyroides < watereri (Kirchn.) Dipp. [L. anagyroides < L. alpinum] DCNA MDLT VAGS KOELREUTERIA integrifoliola Merr. = K. bipinnata paniculata Laxm. DCCG GAUG MDWP TNUT DCJM LAHG MSMN VACP DCNA MDBG NCBE VACW DCSE MDJB SCCU VAMP DCWH MDLT TNSC VATA DEMC MDNA TNSN paniculata Laxm. var. apiculata (Rehd. & Wils.) Rehd. = K. paniculata ‘September’ [K. paniculata Laxm.] DCNA KOLKWITZIA Graebn. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) amabilis Graebn. Beauty Bush DEEP MDBE TNSC VACM DEWG MDDF TNSN VASK GABS NCBE TNTV ‘Rosea’ [K. amabilis Graebn.] DCNA +LABURNOCYTISUS Schneid. FABACEAE (Faboideae) Bean Family [Laburnum + Cytisus] adamii (Poit.) Schneid. [Laburnum anagyroides + Cytisus purpureus] MDBG LABURNUM Medik. Golden-chain Tree FABACEAE (Faboideae) Bean Family a a a RE ee WE ETI em} anagyroides Medik. Golden-chain Tree DCNA DEEM MDLA VACW ‘Vossii’ [L. X watereri (Kirchn.) Dipp.] KOELREUTERIA integrifoliola Merr. = K. bipinnata paniculata Laxm. DCCG GAUG MDWP TNUT DCJM LAHG MSMN VACP DCNA MDBG NCBE VACW DCSE MDJB SCCU VAMP DCWH MDLT TNSC VATA DEMC MDNA TNSN paniculata Laxm. var. apiculata (Rehd. & Wils.) Rehd. = K. paniculata ‘September’ [K. paniculata Laxm.] DCNA KOLKWITZIA Graebn. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) amabilis Graebn. Beauty Bush DEEP MDBE TNSC VACM DEWG MDDF TNSN VASK GABS NCBE TNTV ‘Rosea’ [K. amabilis Graebn.] DCNA +LABURNOCYTISUS Schneid. FABACEAE (Faboideae) Bean Family [Laburnum + Cytisus] adamii (Poit.) Schneid. [Laburnum anagyroides + Cytisus purpureus] MDBG LABURNUM Medik. Golden-chain Tree FABACEAE (Faboideae) Bean Family a a a RE ee WE ETI em} anagyroides Medik. Golden-chain Tree DCNA DEEM MDLA VACW ‘Vossii’ [L. X watereri (Kirchn.) Dipp.] = L. anagyroides LAGERSTROEMIA lL. JUNIPERUS SCHISANDRACEAE Schisandra Family (CRS IE ORES SD OE EE SN EE es \| japonica (Thunb.) Dunal Kadsura Vine NCEL SCAC SCCU KALMIA L. ERICACEAE Heath Family 2 a Se a a OE ee Ee PSST SEES SS, angustifolia L. Sheep Laurel DEWG MDLT VAZC angustifolia L. var. carolina (Small) Fern. NCDU * ‘Bettina’ [K. latifolia L.] DCNA cuneata Michx. White Wicky SCWI ‘Dexter Pink’ [K. latifolia L.] MDBG ‘Fuscata’ [K. latifolia L.] MDLT hirsuta < K. latifolia GAEC SCWI latifolia L. Mountain Laurel ALAU FLMG LALG NCOP ALBG FLUF MDBG VACW ALSH GAEC MDLT VAGW DEWG GAHC NCBE VAHB latifolia L. f. angustata Rehd. Ss io Ags 4 Peamehertegar: Wea y, ta Franch. ipinna ip KOELREUTERIA b ta Franch. ion from F.G. Meyer Journal of the Arnold Arboretum. ©1976 by The Arnold Arboretum. [illustrator Lillian Nicholson Meyer] ipinna ip 1Ss ted by perm ’ KOELREUTERIA b rin Rep (1976) 9 9 KOELREUTERIA indica ‘Pink Lace’ < L. fauriei) x ((L. amabilis x L. indica /hardy light pink/) < L. indica /very dwarf red/)] DCNA * ‘Zuni’ [(L. indica /dwarf red/ < L. fauriei) < L. indica ‘Low Flame’ \|} DCNA LANTANA L. VERBENACEAE Verbena Family SSSR A EOE a OS A eR OE ee camara L. ALBG GAAG LAAL LALG FLUF GATS LAHG VACW montevidensis (Spreng.) Briq. Trailing Lantana ALBG FLCG SCHW TXRS sellowiana Link & Otto = L. montevidensis LARIX $ Mill. Larch PINACEAE Pine Family decidua Mill. European L. DCCG DCNA MDBG MDHN MDNA NCBE VABF gmelinii (Rupr.) Rupr. Dahurian L. MDPJ LAGERSTROEMIA * ‘Hopi’ [((L. indica ‘Pink Lace’ < L. fauriei) < L. indica ‘Alba-Nana’] DCNA indica L. ALBG FLUF NCSM VACW ALCM LARP NCTE VAHC DCCG LASL SCMP VAMP FLMG NCDU VACM * ‘Lipan’ [(L. indica ‘Pink Lace’ x L. fauriei) x ((L. indica /red/ X ‘Carolina Beauty’) x ‘Basham’s Party Pink’)] DCNA * ‘Miami’ [((L. indica ‘Pink Lace’ x L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Muskogee’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Natchez’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Osage’ [(L. indica /dwarf red/ < L. fauriei) < (L. indica ‘Pink Lace’ x L. fauriei)] DCNA * ‘Pecos’ [((L. indica /dwarf red/ < L. fauriei) x (L. indica /dark red/ X L. fauriei)] DCNA * ‘Potomac’ [L. indica L.] (Seed was colchicine treated) DCNA SCCU * ‘Powhatan’ [L. indica /lilac/ < L. indica /lavender/] DCNA * ‘Seminole’ [L. indica /pink/ < L. indica ‘Low Flame’] DCNA * ‘Sioux’ LAGERSTROEMIA * ‘Hopi’ [((L. indica ‘Pink Lace’ < L. fauriei) < L. indica ‘Alba-Nana’] DCNA indica L. ALBG FLUF NCSM VACW ALCM LARP NCTE VAHC DCCG LASL SCMP VAMP FLMG NCDU VACM * ‘Lipan’ [(L. indica ‘Pink Lace’ x L. fauriei) x ((L. indica /red/ X ‘Carolina Beauty’) x ‘Basham’s Party Pink’)] DCNA * ‘Miami’ [((L. indica ‘Pink Lace’ x L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Muskogee’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Natchez’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Osage’ [(L. indica /dwarf red/ < L. fauriei) < (L. indica ‘Pink Lace’ x L. fauriei)] DCNA * ‘Pecos’ [((L. indica /dwarf red/ < L. fauriei) x (L. indica /dark red/ X L. fauriei)] DCNA * ‘Potomac’ [L. KOELREUTERIA Crape Myrtle LYTHRACEAE Loosestrife Family * ‘Acoma’ [L. indica ‘Pink Ruffles’ < (L. indica x L. fauriei sdlg.)] DCNA ‘Alba’ [L. indica L.] DCCG GAMM VAMP FLUF VACM * ‘Apalachee’ [L. indica /Asuka dwarf hybrid/ xX L. fauriei] DCNA ‘Basham’s Party Pink’ [L. indica x L. fauriei] TXSB * ‘Biloxi’ [(L. indica /dwarf red/ x L. fauriei) x (L. indica ‘Low Flame’ < L. fauriei)] DCNA * ‘Caddo’ [(F., sdlg. from ‘Basham’s Party Pink’ < ‘Cherokee’) <x unknown pollen parent] DCNA ‘Carolina Beauty’ [L. indica L.] SCCU * ‘Catawba’ [L. indica /lilac/ < L. indica /lavender/] DCNA SCCU * ‘Cherokee’ [L. indica ‘Hardy Red’ L. indica ‘Low Flame’\|] DCNA MDLT * ‘Choctaw’ [(L. indica < L. fauriei) x ‘Potomac’] DCNA * ‘Comanche’ [L. indica /dark red/ x (L. indica x L. fauriei sdlg.)] DCNA * ‘Conestoga’ [L. indica /white/ < L. indica ‘Low Flame’] DCNA fauriei Koehne DCNA FLUF LASL TXPH 120 LAGERSTROEMIA * ‘Hopi’ [((L. indica ‘Pink Lace’ < L. fauriei) < L. indica ‘Alba-Nana’] DCNA indica L. ALBG FLUF NCSM VACW ALCM LARP NCTE VAHC DCCG LASL SCMP VAMP FLMG NCDU VACM * ‘Lipan’ [(L. indica ‘Pink Lace’ x L. fauriei) x ((L. indica /red/ X ‘Carolina Beauty’) x ‘Basham’s Party Pink’)] DCNA * ‘Miami’ [((L. indica ‘Pink Lace’ x L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Muskogee’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Natchez’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Osage’ [(L. indica /dwarf red/ < L. fauriei) < (L. indica ‘Pink Lace’ x L. fauriei)] DCNA * ‘Pecos’ [((L. indica /dwarf red/ < L. fauriei) x (L. indica /dark red/ X L. fauriei)] DCNA * ‘Potomac’ [L. indica L.] (Seed was colchicine treated) DCNA SCCU * ‘Powhatan’ [L. indica /lilac/ < L. indica /lavender/] DCNA * ‘Seminole’ [L. indica /pink/ < L. indica ‘Low Flame’] DCNA * ‘Sioux’ [(L. indica ‘Tiny Fire’ < (L. indica x L. fauriei sdlg.)) x ((L. indica ‘Pink Lace’ < L. fauriei) < ‘Catawba’)\| DCNA subcostata Koehne FLUF LASL SCCU * ‘Tonto’ [(((L. indica ‘Pink Lace’ x L. fauriei) < ‘Catawba’) < ‘Tuscarora’\| DCNA * ‘Tuscarora’ [‘Basham’s Party Pink’ < ‘Cherokee’] DCNA * ‘Tuskegee’ [L. indica ‘Dallas Red’ x ‘Basham’s Party Pink’\| DCNA * ‘Wichita’ [((L. indica ‘Pink Lace’ < L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Yuma’ [(L. KOELREUTERIA indica L.] (Seed was colchicine treated) DCNA SCCU * ‘Powhatan’ [L. indica /lilac/ < L. indica /lavender/] DCNA * ‘Seminole’ [L. indica /pink/ < L. indica ‘Low Flame’] DCNA * ‘Sioux’ [(L. indica ‘Tiny Fire’ < (L. indica x L. fauriei sdlg.)) x ((L. indica ‘Pink Lace’ < L. fauriei) < ‘Catawba’)\| DCNA subcostata Koehne FLUF LASL SCCU LAGERSTROEMIA LAGERSTROEMIA * ‘Hopi’ [((L. indica ‘Pink Lace’ < L. fauriei) < L. indica ‘Alba-Nana’] DCNA indica L. ALBG FLUF NCSM VACW ALCM LARP NCTE VAHC DCCG LASL SCMP VAMP FLMG NCDU VACM * ‘Lipan’ [(L. indica ‘Pink Lace’ x L. fauriei) x ((L. indica /red/ X ‘Carolina Beauty’) x ‘Basham’s Party Pink’)] DCNA * ‘Miami’ [((L. indica ‘Pink Lace’ x L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Muskogee’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Natchez’ [L. indica ‘Pink Lace’ x L. fauriei] DCNA * ‘Osage’ [(L. indica /dwarf red/ < L. fauriei) < (L. indica ‘Pink Lace’ x L. fauriei)] DCNA * ‘Pecos’ [((L. indica /dwarf red/ < L. fauriei) x (L. indica /dark red/ X L. fauriei)] DCNA * ‘Potomac’ [L. indica L.] (Seed was colchicine treated) DCNA SCCU * ‘Powhatan’ [L. indica /lilac/ < L. indica /lavender/] DCNA * ‘Seminole’ [L. indica /pink/ < L. indica ‘Low Flame’] DCNA * ‘Sioux’ [(L. indica ‘Tiny Fire’ < (L. indica x L. fauriei sdlg.)) x ((L. indica ‘Pink Lace’ < L. fauriei) < ‘Catawba’)\| DCNA subcostata Koehne FLUF LASL SCCU * ‘Tonto’ [(((L. indica ‘Pink Lace’ x L. fauriei) < ‘Catawba’) < ‘Tuscarora’\| DCNA * ‘Tuscarora’ [‘Basham’s Party Pink’ < ‘Cherokee’] DCNA * ‘Tuskegee’ [L. indica ‘Dallas Red’ x ‘Basham’s Party Pink’\| DCNA * ‘Wichita’ [((L. indica ‘Pink Lace’ < L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Yuma’ [(L. indica ‘Pink Lace’ < L. fauriei) x ((L. amabilis x L. indica /hardy light pink/) < L. indica /very dwarf red/)] DCNA * ‘Zuni’ [(L. indica /dwarf red/ < L. fauriei) < L. indica ‘Low Flame’ \|} DCNA LANTANA L. VERBENACEAE Verbena Family SSSR A EOE a OS A eR OE ee \| camara L. ALBG GAAG LAAL LALG FLUF GATS LAHG VACW montevidensis (Spreng.) Briq. Trailing Lantana ALBG FLCG SCHW TXRS sellowiana Link & Otto = L. montevidensis LARIX $ Mill. Larch PINACEAE Pine Family decidua Mill. European L. KOELREUTERIA thunbergii (DC.) Nakai] DCNA GACG NCSM bicolor Turcz. MDGD MDSC bicolor Turcz. f. acutifolia Matsum. = L. bicolor var. bicolor cyrtobotrya Miq. MDSC maximowiczii Schneid. MDSC LARIX LARIX LARIX kaempferi (Lamb.) Carr. Japanese L. DCCG DCNA MDGD NCBE leptolepis (Sieb. & Zucc.) Gord. = L. kaempferi LAURUS lL. Laurel LAURACEAE Laurel Family ee EE SEE Te errs ERS nobilis L. Grecian L. ALSH MSRN SCHW VACS FLCG NCTP SCUC LARP SCBR TXSH LAVANDULA L. Lavender LAMIACEAE Mint Family angustifolia Mill. DCNA MDBG MDLT VACS ‘Hidcote’ [L. angustifolia Mill. ssp. angustifolia] DCNA MDBG VAGS x intermedia Emeric ex Loisel. Lavandin [L. angustifolia < L. latifolia} DCNA officinalis L. = L. angustifolia ‘Provence’ [L. angustifolia Mill.] DCNA stoechas L. French L. SCWI LEDUM L. ERICACEAE Heath Family palustre L. var. diversipilosum Nakai Wild Rosemary VAGS buxifolium (Bergius) Ell. var. hugeri (Small) Schneid. DEMC GAEC LEITNERIA Chapm. LEITNERIACEAE Leitneria Family floridana Chapm. Florida Corkwood DCNA LEPTODERMIS Wall. RUBIACEAE Madder Family oblonga Bunge DEWG TNSN TNTV LEPTOSPERMUM J.R. &J.G. Forst. MYRTACEAE Myrtle Family ‘Plenum’ [L. scoparium J.R. & J.G. Forst.] New Zealand Tea Tree LASL LESPEDEZA Michx. Bush Clover FABACEAE (Faboideae) Bean Family ‘Albiflora’ [L. thunbergii (DC.) Nakai] DCNA GACG NCSM bicolor Turcz. MDGD MDSC bicolor Turcz. f. acutifolia Matsum. = L. bicolor var. bicolor cyrtobotrya Miq. MDSC maximowiczii Schneid. MDSC penduliflora (OQudemans) Nakai = L. thunbergii thunbergii (DC.) Nakai Japanese Bush C. MDSC MSHB NCWK floridana Chapm. Florida Corkwood DCNA floridana Chapm. Florida Corkwood DCNA LEPTODERMIS Wall. RUBIACEAE Madder Family oblonga Bunge DEWG TNSN TNTV LEPTOSPERMUM J.R. &J.G. Forst. MYRTACEAE Myrtle Family ‘Plenum’ [L. scoparium J.R. & J.G. Forst.] New Zealand Tea Tree LASL LESPEDEZA Michx. Bush Clover FABACEAE (Faboideae) Bean Family ‘Albiflora’ [L. thunbergii (DC.) Nakai] DCNA GACG NCSM bicolor Turcz. MDGD MDSC bicolor Turcz. f. acutifolia Matsum. = L. bicolor var. bicolor cyrtobotrya Miq. MDSC maximowiczii Schneid. MDSC penduliflora (OQudemans) Nakai = L. thunbergii thunbergii (DC.) Nakai Japanese Bush C. MDSC MSHB NCWK LAVANDULA L. Lavender LAMIACEAE Mint Family angustifolia Mill. DCNA MDBG MDLT VACS ‘Hidcote’ [L. angustifolia Mill. ssp. angustifolia] DCNA MDBG VAGS x intermedia Emeric ex Loisel. Lavandin [L. angustifolia < L. latifolia} DCNA officinalis L. = L. angustifolia ‘Provence’ [L. angustifolia Mill.] DCNA stoechas L. French L. SCWI LESPEDEZA Michx. Bush Clover FABACEAE (Faboideae) Bean Family ‘Albiflora’ [L. thunbergii (DC.) Nakai] DCNA GACG NCSM bicolor Turcz. MDGD MDSC bicolor Turcz. f. acutifolia Matsum. = L. bicolor var. bicolor cyrtobotrya Miq. MDSC maximowiczii Schneid. MDSC penduliflora (OQudemans) Nakai = L. thunbergii thunbergii (DC.) Nakai Japanese Bush C. MDSC MSHB NCWK palustre L. var. KOELREUTERIA DCCG DCNA MDBG MDHN MDNA NCBE VABF gmelinii (Rupr.) Rupr. Dahurian L. MDPJ * ‘Tonto’ [(((L. indica ‘Pink Lace’ x L. fauriei) < ‘Catawba’) < ‘Tuscarora’\| DCNA * ‘Tuscarora’ [‘Basham’s Party Pink’ < ‘Cherokee’] DCNA * ‘Tuskegee’ [L. indica ‘Dallas Red’ x ‘Basham’s Party Pink’\| DCNA * ‘Wichita’ [((L. indica ‘Pink Lace’ < L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Yuma’ [(L. indica ‘Pink Lace’ < L. fauriei) x ((L. amabilis x L. indica /hardy light pink/) < L. indica /very dwarf red/)] DCNA * ‘Zuni’ [(L. indica /dwarf red/ < L. fauriei) < L. indica ‘Low Flame’ \|} DCNA LANTANA L. VERBENACEAE Verbena Family SSSR A EOE a OS A eR OE ee \| camara L. ALBG GAAG LAAL LALG FLUF GATS LAHG VACW montevidensis (Spreng.) Briq. Trailing Lantana ALBG FLCG SCHW TXRS sellowiana Link & Otto = L. montevidensis LARIX $ Mill. Larch PINACEAE Pine Family decidua Mill. European L. DCCG DCNA MDBG MDHN MDNA NCBE VABF gmelinii (Rupr.) Rupr. Dahurian L. MDPJ * ‘Tonto’ [(((L. indica ‘Pink Lace’ x L. fauriei) < ‘Catawba’) < ‘Tuscarora’\| DCNA * ‘Tuscarora’ [‘Basham’s Party Pink’ < ‘Cherokee’] DCNA * ‘Tuskegee’ [L. indica ‘Dallas Red’ x ‘Basham’s Party Pink’\| DCNA * ‘Wichita’ [((L. indica ‘Pink Lace’ < L. fauriei) < (L. indica ‘Firebird’ < (L. indica < L. fauriei sdlg.))] DCNA * ‘Yuma’ [(L. indica ‘Pink Lace’ < L. fauriei) x ((L. amabilis x L. indica /hardy light pink/) < L. indica /very dwarf red/)] DCNA * ‘Zuni’ [(L. indica /dwarf red/ < L. fauriei) < L. indica ‘Low Flame’ \|} DCNA LANTANA L. VERBENACEAE Verbena Family SSSR A EOE a OS A eR OE ee \| camara L. ALBG GAAG LAAL LALG FLUF GATS LAHG VACW montevidensis (Spreng.) Briq. Trailing Lantana ALBG FLCG SCHW TXRS sellowiana Link & Otto = L. montevidensis LARIX $ Mill. Larch PINACEAE Pine Family decidua Mill. European L. DCCG DCNA MDBG MDHN MDNA NCBE VABF gmelinii (Rupr.) Rupr. Dahurian L. MDPJ PAI LESPEDEZA buxifolium (Bergius) Ell. var. hugeri (Small) Schneid. DEMC GAEC LEITNERIA Chapm. LEITNERIACEAE Leitneria Family floridana Chapm. Florida Corkwood DCNA LEPTODERMIS Wall. RUBIACEAE Madder Family oblonga Bunge DEWG TNSN TNTV LEPTOSPERMUM J.R. &J.G. Forst. MYRTACEAE Myrtle Family ‘Plenum’ [L. scoparium J.R. & J.G. Forst.] New Zealand Tea Tree LASL LESPEDEZA Michx. Bush Clover FABACEAE (Faboideae) Bean Family ‘Albiflora’ [L. LARIX diversipilosum Nakai Wild Rosemary VAGS Sand Myrtle Heath Family IQA 1 °)) LA4a LAURUS nobilis L. (staminate) [illustrator Lillian Nicholson Meyer] 123 LIGUSTRUM LEUCAENA LIGUSTRUM racemosa (L.) A.Gr. Sweetbells GAEC LALG NCDU VACW GAJS MDLT SCBR ‘Rollissonii’ [L. fontanesiana (Steud.) Sleumer] MDBG ‘Zebekot’ [L. fontanesiana L. ?axillaris] (Plant Patent No. 5,224; patented name for Carinella(TM)) MDFM ‘Zebonard’ [L. fontanesiana L. ?axillaris] (Plant Patent No. 5,229; patented name for Lovita(TM)) MDFM LIBOCEDRUS Endl. CUPRESSACEAE Cypress Family [Ree RO ee a ee ee ee eee decurrens Torr. = Calocedrus decurrens LIGUSTRUM L. Privet OLEACEAE Olive Family ‘Aureo-marginatum’ [L. ovalifolium Hassk.] SCCU compactum Hook. f. & Thoms. DCNA delavayanum Hariot SCCU ‘Erecta’ [L. japonicum Thunb.] FLGS ‘Fraseri’ [L. japonicum Thunb.] FLGS ‘Fraseri’ [L. sinense Lour.] ALBG FLMG GAIS TNTV ALFN FLUF NCWM ALPH GAAB SCCU FLCG GACG SCLM ioandrum Diels = L. delavayanum ‘Iwata’ [L. japonicum Thunb.] racemosa (L.) A.Gr. Sweetbells LEUCAENA Benth. FABACEAE (Mimosoideae) Bean Family glauca (L.) Benth. = L. leucocephala leucocephala (Lam.) de Wit White Popinac, Ipil-ipil FLUF leucocephala (Lam.) de Wit White Popinac, Ipil-ipil FLUF FLUF LEUCOPHYLLUM Humboldt & Bonpland SCROPHULARIACEAE Figwort Family se soa raer eerie ee re frutescens (Berl.) 1.M.Johnst. Ceniza, Barometer Bush FLUF LASL TXLL TXSE LEUCOTHOE D.Don ERICACEAE Heath Family (SSS ee Se ST) axillaris (Lam.) D.Don GAEC SCDB SCWI VACW Carinella (TM) [L. fontanesiana x L. ?axillaris] = Trademark name for ‘Zebekot’ catesbaei (Walt.) A.Gr. = L. fontanesiana editorum Fern. & Schub. = L. fontanesiana fontanesiana (Steud.) Sleumer Doghobble DCNA MDWP TNDG VAPO DEWG NCAS VACW VATA GACG NCBE VADW GAUG SCCU VAGS MDLT SCWI VAGW ‘Girard’s Rainbow’ [L. fontanesiana (Steud.) Sleumer] GACG GAJS MDBG Lovita (TM) [L. fontanesiana x L. ?axillaris] = Trademark name for ‘Zebonard’ ‘Nana’ [L. fontanesiana (Steud.) Sleumer] GACG MDLT populifolia (Lam.) Dipp. = Agarista populifolia LEUCOPHYLLUM Humboldt & Bonpland SCROPHULARIACEAE Figwort Family se soa raer eerie ee re frutescens (Berl.) 1.M.Johnst. Ceniza, Barometer Bush FLUF LASL TXLL TXSE LEUCOTHOE D.Don ERICACEAE Heath Family (SSS ee Se ST) axillaris (Lam.) D.Don GAEC SCDB SCWI VACW Carinella (TM) [L. fontanesiana x L. ?axillaris] = Trademark name for ‘Zebekot’ catesbaei (Walt.) A.Gr. = L. fontanesiana editorum Fern. & Schub. = L. fontanesiana fontanesiana (Steud.) Sleumer Doghobble DCNA MDWP TNDG VAPO DEWG NCAS VACW VATA GACG NCBE VADW GAUG SCCU VAGS MDLT SCWI VAGW ‘Girard’s Rainbow’ [L. fontanesiana (Steud.) Sleumer] GACG GAJS MDBG Lovita (TM) [L. fontanesiana x L. LARIX ?axillaris] = Trademark name for ‘Zebonard’ ‘Nana’ [L. fontanesiana (Steud.) Sleumer] GACG MDLT populifolia (Lam.) Dipp. = Agarista populifolia LIQUIDAMBAR tschonoskii Decne. DCNA ‘Variegatum’ [L. japonicum Thunb.] LASL <vicaryi Rehd. [L. ovalifolium {. aureum L. vulgare] FLUF SCceu vulgare L. Common P. DEWG TXRS ‘Wimbish’ [L. sinense Lour.] DCNA GAEC VACW LINDERA Thunb. LAURACEAE Spicebush Laurel Family angustifolia W.C.Cheng MDBG benzoin (L.) Bl. DCNA MDBG NCBE DEWG MDWP VACW benzoin (L.) Bl. var. pubescens (Palm. & Steyerm.) Rehd. TXLL obtusiloba Bl. DCNA VACW strychnifolia (Sieb. & Zucc.) F.Vill. DCNA LIPPIA L. VERBENACEAE Verbena Family a SE Se EE ee ee citriodora = Aloysia gratissima Sweet Gum Witch-hazel Family LIQUIDAMBAR L. HAMAMELIDACEAE formosana Hance Formosan S.G. ALTD GAIS MDGJ TXMS DCUR GAUG MDPJ VABF FLUF LAAL SCCU ‘Gum Ball’ [L. styraciflua L.] GAEC TNSM TNSN TNTV LIQUIDAMBAR LIGUSTRUM LIGUSTRUM japonicum Thunb. Japanese P. ALBG DCWH NCDU SCUC ALCW FLMG NCWC TNUT ALFN GAUG SCBR TXPO DCCG MDJH SCRH ‘Lake Treska’' [L. japonicum Thunb.] FLUF lucidum Ait.f. Wax-leaf P. ALAU NCDU SCUC VAWR FLUF SCCU TXPO LAAL SCMP VACM ‘Nobilis’ [L. japonicum Thunb.] FLGS SECU obtusifolium Sieb. & Zucc. Border P. ALBH MDLT VARC DEWG NCCA ovalifolium Hassk. California P. DCCG LAHG MDCP VARO ‘Pendulum’ [L. sinense Lour.] Weeping P. DCNA DCNE quihoui Carr. FLIF MDKN MDNA TXMS LAAP MDKS SCAK ‘Repandens’ [L. japonicum Thunb.] ALFN ‘Rotundifolium’ [L. japonicum Thunb.] ALBG FLMG LAHG FLDG FLUF SCCU FLGS GAHC SCRH sempervirens (Franch.) Mansf. LAGN ‘Silver Star’ [L. japonicum Thunb.] SCCU sinense Lour. Chinese P. ALBH GAUG NCBE TNUT DCRR LAAL NCDU VACM LIQUIDAMBAR tschonoskii Decne. DCNA ‘Variegatum’ [L. japonicum Thunb.] LASL <vicaryi Rehd. [L. ovalifolium {. aureum L. vulgare] FLUF SCceu vulgare L. Common P. DEWG TXRS ‘Wimbish’ [L. sinense Lour.] DCNA GAEC VACW LINDERA Thunb. LAURACEAE Spicebush Laurel Family angustifolia W.C.Cheng MDBG benzoin (L.) Bl. DCNA MDBG NCBE DEWG MDWP VACW benzoin (L.) Bl. var. pubescens (Palm. & Steyerm.) Rehd. TXLL obtusiloba Bl. DCNA VACW strychnifolia (Sieb. & Zucc.) F.Vill. DCNA LIPPIA L. VERBENACEAE Verbena Family a SE Se EE ee ee citriodora = Aloysia gratissima Sweet Gum Witch-hazel Family LIQUIDAMBAR L. HAMAMELIDACEAE formosana Hance Formosan S.G. ALTD GAIS MDGJ TXMS DCUR GAUG MDPJ VABF FLUF LAAL SCCU ‘Gum Ball’ [L. styraciflua L.] japonicum Thunb. Japanese P. DCNA LIPPIA L. VERBENACEAE Verbena Family a SE Se EE ee ee citriodora = Aloysia gratissima Sweet Gum Witch-hazel Family LIQUIDAMBAR L. LARIX HAMAMELIDACEAE formosana Hance Formosan S.G. ALTD GAIS MDGJ TXMS DCUR GAUG MDPJ VABF FLUF LAAL SCCU ‘Gum Ball’ [L. styraciflua L.] GAEC TNSM TNSN TNTV LIQUIDAMBAR ‘Obtusiloba’ = L. styraciflua f. rotundiloba orientalis Mill. DCNA GAIS styraciflua L. S.G. ALBG LAAL SCBR TNGN DCCG MDPJ SCCU VAHC FLUF NCBE SCMP VAMP styraciflua L. f. rotundiloba Rehd. NCCA ‘Variegata’ [L. styraciflua L.] VAGS LIRIODENDRON L. MAGNOLIACEAE Tulip Tree Magnolia Family ‘Aureo-marginatum’ [L. tulipifera L.] DCSH chinense (Hemsl.) Sarg. Chinese Tulip T. NCCA VABF tulipifera L. Tulip T., Yellow Poplar ALBH LASL MSEP VACM DCCG MDBP NCSM VACW DCWH MDHN SCBR VAGW DEWG MDLT TNSC VATA GAES MDSJ VABF LITHOCARPUS Bl. Tanbark Oak FAGACEAE Beech Family corneus (Lour.) Rehd. GAIS edulis (Makino) Nakai GAIS glaber (Thunb.) Nakai Tanbark Oak GAIS LONICERA LONICERA ‘Obtusiloba’ = L. styraciflua f. rotundiloba orientalis Mill. DCNA GAIS styraciflua L. S.G. ALBG LAAL SCBR TNGN DCCG MDPJ SCCU VAHC FLUF NCBE SCMP VAMP styraciflua L. f. rotundiloba Rehd. NCCA ‘Variegata’ [L. styraciflua L.] VAGS LIRIODENDRON L. MAGNOLIACEAE Tulip Tree Magnolia Family ‘Aureo-marginatum’ [L. tulipifera L.] DCSH chinense (Hemsl.) Sarg. Chinese Tulip T. NCCA VABF tulipifera L. Tulip T., Yellow Poplar ALBH LASL MSEP VACM DCCG MDBP NCSM VACW DCWH MDHN SCBR VAGW DEWG MDLT TNSC VATA GAES MDSJ VABF orientalis Mill. DCNA GAIS styraciflua L. S.G. ALBG LAAL SCBR TNGN DCCG MDPJ SCCU VAHC FLUF NCBE SCMP VAMP styraciflua L. f. rotundiloba Rehd. NCCA ‘Variegata’ [L. styraciflua L.] VAGS LIRIODENDRON L. MAGNOLIACEAE Tulip Tree Magnolia Family ‘Aureo-marginatum’ [L. tulipifera L.] DCSH chinense (Hemsl.) Sarg. Chinese Tulip T. NCCA VABF tulipifera L. Tulip T., Yellow Poplar ALBH LASL MSEP VACM DCCG MDBP NCSM VACW DCWH MDHN SCBR VAGW DEWG MDLT TNSC VATA GAES MDSJ VABF LITHOCARPUS Bl. Tanbark Oak FAGACEAE Beech Family corneus (Lour.) Rehd. GAIS edulis (Makino) Nakai GAIS glaber (Thunb.) Nakai Tanbark Oak GAIS henryi (Seemann) Rehd. & Wils. ALTD DCNA GAIS VACW CE TS A, CN ROE TESTES OE chinensis (Jacq.) R.Br. ex Mart. Chinese Fan Palm FLUF LONICERA L. Honeysuckle CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) ES aS EE SES EET S ‘Arnold Red’ [L. tatarica L.] MDPJ ‘Baggesen’s Gold’ [L. nitida Wils.] MDBG x bella Zab. [L. morrowiti < L. tatarica] VACW < brownii (Regel) Carr. [L. sempervirens < L. hirsuta] GAFN LARS chinensis P.W.Wats. = L. japonica f. chinensis ‘Ernest Wilson’ [L. nitida Wils.] GACG flava Sims Yellow H. LARIX SCWI ‘Floribunda’ [L. korolkowii Stapf] MDTD Sragrantissima Lindl. & Paxt. Winter H. ALIT FLMG MDLT SCUC ALSH GAOH MDTD TNCT ARPT GATS MSHB TNDG DCCG GAWP NCBE VACW DCHU LARP SCBR VADW DEWG MDDP SCDB VAMP gracilipes Miq. MDKN LONICERA L. Honeysuckle CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) ES aS EE SES EET S\| ‘Arnold Red’ [L. tatarica L.] MDPJ ‘Baggesen’s Gold’ [L. nitida Wils.] MDBG x bella Zab. [L. morrowiti < L. tatarica] VACW < brownii (Regel) Carr. [L. sempervirens < L. hirsuta] GAFN LARS chinensis P.W.Wats. = L. japonica f. chinensis ‘Ernest Wilson’ [L. nitida Wils.] GACG flava Sims Yellow H. SCWI ‘Floribunda’ [L. korolkowii Stapf] MDTD Sragrantissima Lindl. & Paxt. Winter H. ALIT FLMG MDLT SCUC ALSH GAOH MDTD TNCT ARPT GATS MSHB TNDG DCCG GAWP NCBE VACW DCHU LARP SCBR VADW DEWG MDDP SCDB VAMP gracilipes Miq. MDKN LONICERA L. Honeysuckle CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) ES aS EE SES EET S\| ‘Arnold Red’ [L. tatarica L.] MDPJ ‘Baggesen’s Gold’ [L. nitida Wils.] MDBG x bella Zab. [L. morrowiti < L. tatarica] VACW < brownii (Regel) Carr. [L. sempervirens < L. hirsuta] GAFN LARS chinensis P.W.Wats. = L. japonica f. chinensis ‘Ernest Wilson’ [L. nitida Wils.] GACG flava Sims Yellow H. SCWI ‘Floribunda’ [L. korolkowii Stapf] MDTD Sragrantissima Lindl. & Paxt. Winter H. ALIT FLMG MDLT SCUC ALSH GAOH MDTD TNCT ARPT GATS MSHB TNDG DCCG GAWP NCBE VACW DCHU LARP SCBR VADW DEWG MDDP SCDB VAMP gracilipes Miq. MDKN LITHOCARPUS Bl. Tanbark Oak FAGACEAE Beech Family corneus (Lour.) Rehd. GAIS edulis (Makino) Nakai GAIS glaber (Thunb.) Nakai Tanbark Oak GAIS henryi (Seemann) Rehd. & Wils. ALTD DCNA GAIS VACW LONICERA gracilipes Miq. var. glandulosa Maxim. MDAB ‘Hack’s Red’ [L. tatarica L.] DCNA x heckrottii Rehd. Everblooming H. [L. Xamericana X< L. sempervirens] ARPT GAFN SCCU VATA japonica Thunb. ex J.A.Murr. Japanese H. ALBG LAAL MSHB ALBH MDBG TNSC japonica Thunb. ex J.A.Murr. f. chinensis (P.W.Wats.) Hara ALBH VAGW maackii Maxim. Amur H. DCAG LAHP MDTD VACW DEEM MDAB NCDU VAMP GANA MDBW TNRM VARG GAUG MDDF VABF maackii Maxim. var. podocarpa Franch. MDAB MDGD ‘Magnifica’ [L. sempervirens L.] = ‘Superba’ morrowiti A.Gr. Morrow’s H. DEEM MDTD TNNE VAWD MDLT NCBE TNRM ‘Nana’ [L. xylosteum L.] VAGS nitida Wils. MDMG pileata Oliv. Privet H. DCNA GAGB TNCT VANB FLUF GAUG TNDR GACG MDLT VADW ‘Punicea’ [L. LARIX tatarica L.] MDAB MDGD sempervirens L. LYONIA LYONIA standishii Jacq. Standish’s H. 3 DEWG MDLT subsessilis Rehd. DCNA ‘Sulphurea’ [L. sempervirens L.] DCNA SCWI VACW ‘Superba’ [L. sempervirens L.] SCWI VAGS VAPH tatarica L. Tatarian H. MDFF MDLA MDMG MDJH MDMC NCBE xylosteum L. Fly H. MDHN VAGS xylosteum L. f. glabrescens Zab. VALU LOROPETALUM R.Br. ex Reichenb. HAMAMELIDACEAE Witch-hazel Family eS Se es OE ey Ca I es EET, ORE OE SS 3 chinense (R.Br.) Oliv. ALEH GACE GAUG SCLM DCNA GACG MDLT SCRH FLGS GACH NCWD SCWI FLMG GACL SCBR VACW FLUF GAIS SCCU GAAB GAJI SCDB GABS GAJY SCFW LYCIUM L. Matrimony Vine SOLANACEAE Nightshade Family aT a ES Pe Es SE Oe DE nC carolinianum Walt. Carolina M. V. SCWI chinense Mill. Chinese M. V. MDSC LYONIA Nutt. ERICACEAE Heath Family standishii Jacq. Standish’s H. 3 DEWG MDLT subsessilis Rehd. DCNA ‘Sulphurea’ [L. sempervirens L.] DCNA SCWI VACW ‘Superba’ [L. sempervirens L.] SCWI VAGS VAPH tatarica L. Tatarian H. MDFF MDLA MDMG MDJH MDMC NCBE xylosteum L. Fly H. MDHN VAGS xylosteum L. f. glabrescens Zab. VALU LYCIUM L. Matrimony Vine SOLANACEAE Nightshade Family aT a ES Pe Es SE Oe DE nC carolinianum Walt. Carolina M. V. SCWI chinense Mill. Chinese M. V. MDSC LYONIA Nutt. ERICACEAE Heath Family ferruginea (Walt.) Nutt. GAEC ligustrina (L.) DC. GAEC SCBR VAPO LYCIUM L. Matrimony Vine SOLANACEAE Nightshade Family aT a ES Pe Es SE Oe DE nC carolinianum Walt. Carolina M. V. SCWI chinense Mill. Chinese M. V. MDSC LYONIA Nutt. ERICACEAE Heath Family ferruginea (Walt.) Nutt. GAEC ligustrina (L.) DC. GAEC SCBR VAPO WATE MAGNOLIA MAGNOLIA LYONIA LYONIA lucida (Lam.) K.Koch Fetterbush SCBR SCWI TXLL VAPH mariana (L.) D.Don Staggerbush SCWI MAACKIA Rupr. FABACEAE (Faboideae) Bean Family TE LE Pe TS SS EE DT OE TE CE SET RE \| amurensis Rupr. & Maxim. MDJS SCCU VAIS NCBE TNTV tenuifolia (Hemsl.) Hand.-Mazz. DCNA MACFADYENA A.DC. BIGNONIACEAE Bignonia Family unguis-cati (L.) A.Gentry Cat’s-claw ALBG FLUF LAHG SCHW FLCG LAAL SCBH MACHILUS Nees = PERSEA MACLURA Nutt. MORACEAE Mulberry Family pomifera (Raf.) Schneid. Osage Orange DCCG MDLT NCTE VAWR DCSH MDSJ NCWT GAEH MSMN = TNLW MDHN NCSM ~~ VACW MAGNOLIA lL. Magnolia MAGNOLIACEAE Magnolia Family acuminata L. Cucumber Tree DCCG GAGC TNHG VAMP DCNA GASC TNMB VAWR DEEM NCBE VACM GACG SCBR VACW MAGNOLIA ‘Alexandrina’ [M. x soulangeana Soul.- Bod.] [M. denudata < M. liliiflora] DCNA ‘Ann’ [M. liliiflora ‘Nigra’ < M. stellata ‘Rosea’] DCNA ashei Weatherby Florida Bigleaf M. DCNA MDFM MSFG TNSN GAEC MDLT NCBE TNTV ‘Baldwin’ [M. grandiflora L.] DCNA * ‘Betty’ [M. liliiflora ‘Nigra’ < M. stellata ‘Rosea’] DCNA ‘Biloba’ [M. officinalis Rehd. & Wils.] = M. officinalis var. biloba ‘Charles Dickens’ [M. grandiflora L.] TNSN TNTV ‘C. H. Kern’ [M. <soulangeana Soul.-Bod.] [M. denudata < M. liliiflora] DCNA ‘Claudia Wanamaker’ [M. grandiflora L.] SCSG cordata Michx. = M. acuminata cylindrica Wils. MDLT dealbata Zucc. Mexican Bigleaf M. MSFG denudata Dest. Yulan, Lily Tree DCNA FLMG MDLT ‘Diva’ [M. sprengeri Pamp.] Goddess M. DCNA MDLT ‘Elizabeth’ [M. acuminata < M. denudata] DCNA Sraseri Walt. Fraser M. NCAS TNTV VAMP * ‘Freeman’ [M. virginiana x M. grandi- flora] ‘Alexandrina’ [M. x soulangeana Soul.- Bod.] [M. denudata < M. liliiflora] DCNA * ‘Ann’ [M. liliiflora ‘Nigra’ < M. stellata ‘Rosea’] DCNA ashei Weatherby Florida Bigleaf M. DCNA MDFM MSFG TNSN GAEC MDLT NCBE TNTV ‘Baldwin’ [M. grandiflora L.] DCNA * ‘Betty’ [M. liliiflora ‘Nigra’ < M. stellata ‘Rosea’] DCNA ‘Biloba’ [M. officinalis Rehd. & Wils.] = M. officinalis var. biloba ‘Charles Dickens’ [M. grandiflora L.] TNSN TNTV ‘C. H. Kern’ [M. <soulangeana Soul.-Bod.] [M. denudata < M. liliiflora] DCNA ‘Claudia Wanamaker’ [M. grandiflora L.] SCSG cordata Michx. = M. acuminata cylindrica Wils. MDLT dealbata Zucc. Mexican Bigleaf M. MSFG denudata Dest. Yulan, Lily Tree DCNA FLMG MDLT ‘Diva’ [M. sprengeri Pamp.] Goddess M. DCNA MDLT ‘Elizabeth’ [M. acuminata < M. denudata] DCNA Sraseri Walt. MAGNOLIA macrophylla Michx. (fruit) [illustrator Susan M. Johnston] MAGNOLIA ashei Weatherby (fruit) [ilustrator Susan M. Johnston] 129 MAGNOLIA macrophylla Michx. (fruit) [illustrator Susan M. Johnston] MAGNOLIA ashei Weatherby (fruit) [ilustrator Susan M. Johnston] 129 129 MAGNOLIA x loebneri Kache [M. kobus < M. stellata] DCNA DCWH DCWR DEWG macrophylla Michx. Big-leaf M. DCCG GAEC MSFG TNSC DERE LASL NCBE TNSN DEEM MDAM NCTE VAWH DEWG MDHC SCBR GACB MDPJ SCMP ‘Majestic Beauty’ [M. grandiflora L.] DCNA ‘Margaret Davis’ [M. grandiflora L.] SCSG * ‘Maryland’ [M. virginiana x M. grandiflora] DCNA ‘Merrill’ [M. < loebneri Kache] [M. kobus x M. stellata] DCNA ‘Mrs. Jack’ [M. salicifolia (Sieb. & Zucc.) Maxim.] MDLT ‘Neil McEacharn’ [M. < loebneri Kache] [M. kobus < M. stellata] DCNA ‘Nigra’ [M. liliiflora Desr.] DCNA * ‘Nimbus’ [M. obovata < M. virginiana] DCNA ‘O’Neill’ [M. liliiflora Desr.] MDLT obovata Thunb. DCNA GAFN MDLT officinalis Rehd. & Wils. GAEH MDJS officinalis Rehd. & Wils. var. biloba Rehd. & Wils. DCNA GAWH MDJS MAGNOLIA x loebneri Kache [M. kobus < M. stellata] DCNA DCWH DCWR DEWG macrophylla Michx. Big-leaf M. DCCG GAEC MSFG TNSC DERE LASL NCBE TNSN DEEM MDAM NCTE VAWH DEWG MDHC SCBR GACB MDPJ SCMP ‘Majestic Beauty’ [M. grandiflora L.] DCNA ‘Margaret Davis’ [M. grandiflora L.] SCSG * ‘Maryland’ [M. virginiana x M. grandiflora] DCNA ‘Merrill’ [M. < loebneri Kache] [M. kobus x M. stellata] DCNA ‘Mrs. Jack’ [M. salicifolia (Sieb. & Zucc.) Maxim.] MDLT ‘Neil McEacharn’ [M. < loebneri Kache] [M. kobus < M. stellata] DCNA ‘Nigra’ [M. liliiflora Desr.] DCNA * ‘Nimbus’ [M. obovata < M. virginiana] DCNA ‘O’Neill’ [M. liliiflora Desr.] MDLT obovata Thunb. DCNA GAFN MDLT officinalis Rehd. & Wils. GAEH MDJS officinalis Rehd. & Wils. var. biloba Rehd. & Wils. DCNA GAWH MDJS parviflora Sieb. & Zucc. = M. sieboldii ‘Picture’ [M. x soulangeana Soul.-Bod.] [M. denudata ~ M. liliiflora] MDLT ‘Pink Stardust’ [M. stellata Sieb. & Zucc.] MAGNOLIA * ‘Galaxy’ [M. liliiflora ‘Nigra’ < M. sprengeri ‘Diva’] DCNA MDLT glauca L. = M. virginiana ‘Grace McDade’ [M. <soulangeana Soul.- Bod.] [M. denudata ~ M. liliiflora] DCNA grandiflora L. Southern M. ALBG FLMG MDCP SCMP DCCG FLUF MDFM SCRP DCE GAHC MDHN TNHG DCSH GATN MSEP TNUT DCWA LAAL NCDU VAGW DCWH LAHG NCTE VAMP DEWG LASL SCBR VATA ‘Hasse’ [M. grandiflora L.] SCSG ‘Henry Hicks’ [M. virginiana L.] DCNA TNSN TNTV heptapeta (Buc’hoz) Dandy = M. denudata hypoleuca Sieb. & Zucc. = M. obovata * ‘Jane’ [M. liliiflora ‘Reflorescens’ < M. stellata ‘Waterlily’] DCNA * ‘Judy’ [M. MAGNOLIA macrophylla Michx. (fruit) [illustrator Susan M. Johnston] MAGNOLIA ashei Weatherby (fruit) [ilustrator Susan M. Johnston] 129 liliiflora ‘Nigra’ < M. stellata ‘Rosea’]} DCNA kobus DC. Kobus M. DCNA DCWR MDLT ‘Lennei’ [M. < soulangeana Soul.-Bod.] [M. denudata x M. liliiflora] ALON DCNA LAAL DCCG FLMG VACG liliiflora Desr. Lily-flowered M. LAAL VAGW ‘Lilliputian’ [M. x soulangeana Soul.-Bod.] [M. denudata x M. liliiflora] DCNA MAGNOLIA MAGNOLIA * ‘Pinkie’ [M. liliiflora ‘Reflorescens’ < M. stellata ‘Rosea’] DCNA ‘Pioneer’ [M. grandiflora L.] DCNA praecocissima Koidz. = M. kobus ‘Praecox’ [M. grandiflora L.] DCNA ‘Praecox Fastigiata’ [M. grandiflora L.] DCNA < proctoriana Rehd. [M. kobus X M. salicifolia] GAFN ‘Purpliana’ [M. x soulangeana Soul.-Bod.] [M. denudata x M. liliiflora] DCNA pyramidata Bartram ex Pursh ALBG DCNA MDLT TNTV quinquepeta (Buc’hoz) Dandy = M. liliiflora * ‘Randy’ [M. liliiflora ‘Nigra’ <x M. stellata ‘Rosea’] DCNA ‘Reflorescens’ [M. liliiflora Desr.] DCNA * ‘Ricki’ [M. liliiflora ‘Nigra’ < M. stellata ‘Rosea’]} DCNA ‘Rohrbach’ [M. stellata (Sieb. & Zucc.) Maxim.] DCNA ‘Rosea’ [M. stellata (Sieb. & Zucc.) Maxim.] ALOS DEWG LAHP DCNA FLMG MDLT ‘Royal Star’ [M. stellata (Sieb. & Zucc.) Maxim.] DCNA MDLT ‘Rubra’ [M. < soulangeana Soul.-Bod.] [M. denudata x M. liliiflora] SCBR ‘Rubra’ [M. stellata (Sieb. & Zucc.) Maxim.] DCNA ‘Rustica’ [M. x soulangeana Soul.-Bod.] [M. denudata x M. liliifloraj~ DCNA ‘St. Mary’ [M. grandiflora L.] DCNA FLUF ‘Samuel Sommer’ [M. grandiflora L.] (Plant Patent No. 2,015) MDLT ‘San Jose’ [M. <soulangeana Soul.-Bod.] [M. denudata x M. liliiflora] DCNA DEWG * ‘Satellite’ [M. virginiana L.] DCNA ‘Shady Grove No. 4’ [M. grandiflora L.] SCSG ‘Shady Grove No. 5’ [M. grandiflora L.] SCSG ‘Shady Grove No. 6’ [M. grandiflora L.] SCSG sieboldii K.Koch Otama M. DEWG MDBG TNSN VAGS GAFN MDLT TNTV x soulangeana Soul.-Bod. Saucer M. [M. denudata x M. liliiflora] DCCG FLDG LASL MDLT DCNA GAAG MDHN SCBR DCWH GATS MDJS VAGG * ‘Spectrum’ [M. liliiflora ‘Nigra’ < M. sprengeri ‘Diva’] DCNA ‘Spring Snow’ [M. <loebneri Kache] [M. kobus < M. stellata] MDLT stellata (Sieb. & Zucc.) Maxim. Star Magnolia DCCG DCWH DEWG MDLT ‘Sundance’ [M. acuminata x M. ?denudatal] DCNA ‘Sundew’ [M. <x soulangeana Soul.-Bod.] [M. denudata < M. liliiflora] 131 MAGNOLIA MAHONIA ‘Superba Rosea’ [M. <soulangeana Soul.- Bod.] [M. denudata x M. liliiflora] DCNA * ‘Susan’ [M. liliiflora ‘Nigra’ < M. stellata ‘Rosea’] DCNA MDLT tripetala L. Umbrella M. ALBG DEWG MDLT NCCA DCCG GARW MDMS SCMP DCNA GAUG MDVV TNSC DEEM MDJS NCBE VACW ‘Variegata’ [M. grandiflora L.] LASL x veitchii Bean [M. campbellii x M. denudata] DCNA NCEL ‘Verbanica’ [M. x soulangeana Soul.-Bod.] [M. denudata < M. liliiflora] DCNA ‘Victoria’ [M. grandiflora L.] DCNA virginiana L. Sweet Bay ALAU FLUF MDBG NCCA ALBG GAEC MDHN SCCU ALBH GAUG MDJH TNPW DCCG LAAL MDLT TNUT DCNA LAHG MSMN VAGW DCWA LALG NCBE VAHC virginiana L. var. australis Sarg. = M. virginiana ‘Wada’s Memory’ [M. < kewensis Pearce] [M. kobus < M. salicifolia] DCNA DEWG MDLT ‘Water Lily’ [M. stellata Sieb. & Zucc.] DCNA x watsonii Hook.f. = M. X wieseneri < wieseneri Carr. [M. obovata < M. sieboldii] DCNA VAGS <MAHOBERBERIS _ Schneid. BERBERIDACEAE Barberry Family [Berberis < Mahonia] CE aa, a a a LE PINT RITE MST \| aquisargentii Kruessm. [Berberis sargentiana < Mahonia aquifolium] TNTV miethkeana Melander & Eade [Berberis ‘Renton’ < Mahonia aquifolium] DCNA SCCU MAHONIA Nutt. BERBERIDACEAE Barberry Family SS EE ES IE ET ES ST) aquifolium (Pursh) Nutt. Oregon Hollygrape DCNA GAEC MDLT DEMC GAUG NCDU GACG MDKN VACW bealei (Fort.) Carr. Leatherleaf Hollygrape ALBG FLDG MDHN TXGM ALRH FLMG MDLA VACW ARSN FLUF MDLT VAMP DCCG GACH MSMN DCNA GAEC NCCA DCTF GAOH SCRE ‘Charity’ [M. < media Brickell] [M. napaulensis < M. oiwakensis] DCNA chochoca Fedde ID ALL, fortunei (Lindl.) Fedde Chinese M. ALBG FLUF MSEP TXHA FLDG LAAL SCBR IDAUL, FLMG LASL SCJM japonica (Thunb. ex J.A.Murr.) DC. Japanese M. GACG oiwakensis Hayata ALEH FLUF LAMP VACW pinnata (Lag.) Fedde Cluster M. ALBH TNHS TNPW VAGG MDBG TNMB TNUT <MAHOBERBERIS _ Schneid. 132 CAE ae MAGNOLIA tripetala L. MATTIONIA trifoliolata (Moric.) Fedde Laredo M, hLUT TXDC TALL VACW MALLOTUS § Lour. EUPHORBIACEAE Spurge Family \| SERRE et ee RR RE RESP EPR RI: OE EES FT\| japonicus (TMiunb. ex Lt.) Muell.-Arg, Japanese M, LUT GAIS <MALOSORBUS — Browicz ROSACEAE Rose Family {Malus * Sorbus] \|S a ve ep aN BT REE ea oe aa SS Sd florentina (Zuccagni) Browicz \|Malus domestica « Sorbus torminalis] DCUNA MALPIGHIA LL. MALPIGHIACEAE Malpighia Family LI SAT EEL SE ETE SS TS LIT LILO OIE IE IPE LS SE \| glabra \.. MAGNOLIA Barbados Cherry LUT LASL MALUS Mill. ROSACEAE Rose Family * ‘Adirondack’ [M. halliana hybrid] DCONA ‘Aldenhamensis’ [M. * purpurea (Barbicr) Rehad.] [M. < atrosanguinea » ‘Niedzwetzkeyana’] M. domestica DCAR DCNA GAIN ‘Almey’' DCNA GAIN MDGD angustifolia (Ail.) Michx, Southern Crabapple ALE RLU FLMG SCILIW TNSC <arnoldiana (Rehd.) Sarg. [M. baccata < M. floribunda] DCAR DCNA ‘Athabasca’ [M. < adstringens Zab.] \|M. baccata < M. domestica] MDGbD MALUS MATTIONIA MATTIONIA < atrosanguinea (Spaeth) Schneid. [M. halliana * M. sieboldii] DCAR MSMN VACW baccata (L.) Borkh. Siberian Crabapple DONA MDGD baccata (L.) Borkh. var. mandschurica (Maxim.) Schneid. DCAR DCNA ‘Centennial’ DCNA coronaria (L.) Mill. Wild Sweet Crabapple GACG NCOP VAGG coronaria (L.) Mill. var. dasycalyx Rehd. MDGD coronaria (L.) Mill. var. elongata Rehd. DCNA cralaegifolia Koehne <Malosorbus florentina ‘Crimson Brilliant’ DCNA ‘David’ DCNA domestica Borkh. Common Apple (Culligen of mixed parentage) MDLYT VACW VATG ‘Dorothea’ [M. « scheideckeri (Spacth) Zab.) \|[M. floribunda » DCNA M. prunifolial\| DIEMC ‘East Malling’ DCNA ‘Eleyi’ [M. < purpurea (Barbier) Rehd.] \|[M. <atrosanguinea < M. domestica ‘Niedzwetzkyana’' \| GAFFE ‘Evelyn’ DCNA ‘Flame’ DCNA japonicus (TMiunb. ex Lt.) Muell.-Arg, Japanese M, LUT GAIS MALUS Mill. ROSACEAE Rose Family * ‘Adirondack’ [M. halliana hybrid] DCONA ‘Aldenhamensis’ [M. * purpurea (Barbicr) Rehad.] [M. < atrosanguinea » ‘Niedzwetzkeyana’] M. domestica DCAR DCNA GAIN ‘Almey’' DCNA GAIN MDGD angustifolia (Ail.) Michx, Southern Crabapple ALE RLU FLMG SCILIW TNSC <arnoldiana (Rehd.) Sarg. [M. baccata < M. floribunda] DCAR DCNA ‘Athabasca’ [M. < adstringens Zab.] \|M. baccata < M. domestica] MDGbD MALUS Mill. ROSACEAE Rose Family * ‘Adirondack’ [M. halliana hybrid] DCONA ‘Aldenhamensis’ [M. * purpurea (Barbicr) Rehad.] [M. < atrosanguinea » ‘Niedzwetzkeyana’] M. domestica DCAR DCNA GAIN ‘Almey’' DCNA GAIN MDGD angustifolia (Ail.) Michx, Southern Crabapple ALE RLU FLMG SCILIW TNSC <arnoldiana (Rehd.) Sarg. [M. baccata < M. floribunda] DCAR DCNA ‘Athabasca’ [M. < adstringens Zab.] \|M. baccata < M. domestica] MDGbD MALUS Mill. ROSACEAE Rose Family * ‘Adirondack’ [M. halliana hybrid] DCONA ‘Aldenhamensis’ [M. * purpurea (Barbicr) Rehad.] [M. < atrosanguinea » ‘Niedzwetzkeyana’] M. domestica DCAR DCNA GAIN ‘Almey’' DCNA GAIN MDGD angustifolia (Ail.) Michx, Southern Crabapple ALE RLU FLMG SCILIW TNSC <arnoldiana (Rehd.) Sarg. [M. baccata < M. floribunda] DCAR DCNA ‘Athabasca’ [M. < adstringens Zab.] \|M. baccata < M. domestica] MDGbD MALUS MALUS MALUS MALUS floribunda Sieb. Showy Crabapple DCAR DCWH DCCG DEMC DCNA FLMG GACG MDHP VACW VASK * ‘Fuji’ [M. sieboldii (Regel) Rehd.] DCNA MDGD ‘Golden Anniversary’ MDGD ‘Golden Hornet’ [M. robusta (Carr.) Rehd.] [M. baccata < M. prunifolia\| DEMC ‘Guiding Star’ DCNA halliana Koehne DCNA ‘Henrietta Crosby’ [M. <arnoldiana * M. domestica ‘Niedzwetzkyana’] DCNA ‘Henry F. Dupont’ DCNA ‘Hillier’ DCNA ‘Hopa’ [M. adstringens Zab.] [M. baccata * M. domestica] DECC DCNA LATU DCCG LAGN NCTP hupehensis (Pamp.) Rehd. DCWH DEMC SCCU ioensis (A.Wood) Britton var. palmeri Rehd. DCNA ‘Katherine’ [M. * hartwigii Koehne] [M. MATTIONIA baccata * M. halliana] DCWH VACW VATA lancifolia Rehd. DCNA ‘Lemoinei’ [M. * purpurea (Barbier) Rehd.] [M. x atrosanguinea * M. domestica ‘Niedzwetzkyana \| MDBG MDGD ‘Marshall Oyama’ MALUS ‘Mathews’ DCNA = Xmicromalus Makino [M. baccata M. spectabilis] DCNA ‘Mrs. Bayard Thayer’ DCNA * ‘Naragansett’ [M. /hybrid #28/ x M. ‘Wintergold’] DCNA ‘Nieuwlandiana’ [M. coronaria (L.) Mill.] GACG ‘Normand’ DCNA ‘Nova’ [M. ioensis (A.Wood) Britton] DCNA ‘Oporto’ DCNA ‘Parkmanii’ [M. halliana Koehne] DCNA DEWG VAMP DEMC MDHN ‘Pink Pearl’ DCNA ‘Pink Spires’ DCNA x platycarpa Rehd. [M. coronaria ~ M. domestica] DCNA ‘Plena’ [M. toensis (A.Wood) Britton] Bechtel’s Crabapple DCNA MDBG NCOP ‘Prairie Rose’ DCNA ‘Prince George’s’ DCNA ‘Profusion’ DCNA prunifolia (Willd.) Borkh. DCNA FLBW floribunda Sieb. Showy Crabapple DCAR DCWH DCCG DEMC DCNA FLMG GACG MDHP VACW VASK ‘Fuji’ [M. sieboldii (Regel) Rehd.] DCNA MDGD ‘Golden Anniversary’ MDGD ‘Golden Hornet’ [M. robusta (Carr.) Rehd.] [M. baccata < M. prunifolia\| DEMC ‘Guiding Star’ DCNA halliana Koehne DCNA ‘Henrietta Crosby’ [M. <arnoldiana * M. domestica ‘Niedzwetzkyana’] DCNA ‘Henry F. Dupont’ DCNA ‘Hillier’ DCNA ‘Hopa’ [M. adstringens Zab.] [M. baccata * M. domestica] DECC DCNA LATU DCCG LAGN NCTP hupehensis (Pamp.) Rehd. DCWH DEMC SCCU ioensis (A.Wood) Britton var. palmeri Rehd. DCNA ‘Katherine’ [M. * hartwigii Koehne] [M. baccata * M. halliana] DCWH VACW VATA lancifolia Rehd. DCNA ‘Lemoinei’ [M. * purpurea (Barbier) Rehd.] [M. x atrosanguinea * M. domestica ‘Niedzwetzkyana \| MDBG MDGD ‘Marshall Oyama’ DCNA MELALEUCA MALUS X purpurea (Barbier) Rehd. [M. x atrosanguinea < M. domestica ‘Niedzwetzkeyana’] DCCG DEMC FLMG ‘Pygmy’ DCNA ‘Red Jade’ DCNA TNSC ‘Red Jewel’ DCNA ‘Red Silver’ DCNA ‘Red Tip’ [M. <soulardii (Bailey) Britton] [M. ioensis x M. domestica] MDGD < robusta (Carr.) Rehd. [M. baccata < M. prunifolia] DCAR DCNA NCDU ‘Rondo’ DCNA ‘Rosea’ [M. sargentii Rehd.] DCNA sargentii Rehd. Sargent’s Crabapple DCAR GARW VACW DCNA SCCU x scheideckeri (Spaeth) Zab. [M. floribunda x M. prunifolia] DCCG DCNA SCCU ‘Shakespeare’ DCNA sieboldii (Regel) Rehd. Toringo Crabapple DCAR DCNA NCDU VATA sieboldii var. zumi (Matsum.) Asami = M. zumi ‘Silver Moon’ DCNA ‘Sissipuk’ DCNA spectabilis (Ait.) Borkh. DCAR ‘Thomas Roland’ DCNA MELALEUCA ‘Timiskaming’ DCNA ‘Tops-in-Bloom’ DCNA ‘Van Eseltine’ [M. <x arnoldiana x M. spectabilis] DCNA MDRP ‘Veitch’s Scarlet’ [M. domestica L.] DCNA ‘Wintergold’ DCNA ‘Wynema’ DCNA MDGD <zumi (Matsum.) Rehd. [M. baccata var. mandshurica < M. sieboldii] DCNA DEMC MALVAVISCUS Fabr. non Adans. MALVACEAE Mallow Family arboreus Cav. var. MATTIONIA drummondii Schery Turk’s Cap VACW arboreus Cav. var. penduliflorus (Sesse & Moc. ex DC.) Schery Turk’s Cap ALDM FLUF drummondii Torr. & A.Gr. = M. arboreus var. drummondii MANIHOT Miill. Cassava EUPHORBIACEAE Spurge Family grahamii Hook. FLUF LAAL MSBN MELALEUCA L. MYRTACEAE Myrtle Family leucadendron (L.) L. = M. quinquenervia quinquenervia (Cav.) S.T.Blake Paperbark Tree, Punk Tree ‘Timiskaming’ DCNA ‘Tops-in-Bloom’ DCNA ‘Van Eseltine’ [M. <x arnoldiana x M. spectabilis] DCNA MDRP ‘Veitch’s Scarlet’ [M. domestica L.] DCNA ‘Wintergold’ DCNA ‘Wynema’ DCNA MDGD <zumi (Matsum.) Rehd. [M. baccata var. mandshurica < M. sieboldii] DCNA DEMC 136 MELIA MELIA lL. MELIACEAE Mahogany Family eS ee ae Sy Se) azedarach L. Chinaberry ALCG FLUF NCDU VAWR ARUM MDPJ SCMG DCNA NCCA VACW MENZIESIA Sm. ERICACEAE Heath Family Ea a SE SRE SEE AE LITRES LEST TT ETT TT) pilosa (Michx.) Juss. Minniebush VAPO MESPILUS L. ROSACEAE Rose Family CE a a a EEE OE CLL Te SS ES ETS] germanica L. Medlar DCHU VABF VACW METASEQUOIA Miki ex H.H.Hu & W.C.Cheng TAXODIACEAE Taxodium Family glyptostroboides H.H.Hu & W.C.Cheng Dawn Redwood figo (Lour.) Spreng. Banana Shrub = ALBG GAFF LALG SCMG ALSH GAHC LASL SCMP FLDG GAJI MDFM SCWI FLMG GATS MSMN TXHE FLRM LAAL SCFW FLUF LAHG SCJM Jfuscata (Andr.) Bl. ex Wall. = M. figo MICROBIOTA Komar. CUPRESSACEAE Cypress Family decussata Komar. DCNA GAEC MDBG MICROCITRUS' Swingle RUTACEAE Citrus Family SSS EN SP SE Se RE RE BEE Phe ae SESS] australasica (F.Muell.) Swingle Australian Finger Lime FLUF MILLETTIA Wight & Arn. FABACEAE (Faboideae) Bean Family SE SE RS ES Sp EES TW SET TEN ae ER reticulata Benth. Leatherleaf M. FLUF TXCT MIMOSA L. FABACEAE (Mimosoideae) Bean Family biuncifera Benth. Catclaw M. LAHG LASL TXCT pigra L. var. berlandieri (A.Gr.) B.L.Turner DCNA TXPS MITCHELLA L. RUBIACEAE Madder Family A Te a Se SOE EE a ee I eee repens L. Partridgeberry GAEC MDLT NCEG SCBR 137 MESPILUS L. ROSACEAE Rose Family CE a a a EEE OE CLL Te SS ES ETS] germanica L. Medlar DCHU VABF VACW METASEQUOIA Miki ex H.H.Hu & W.C.Cheng TAXODIACEAE Taxodium Family glyptostroboides H.H.Hu & W.C.Cheng Dawn Redwood ALAU GAUG NCDU VATA DCNA MDBG SCMG VAWM DEMC MDJS TNHD GAFN MDPJ VACW * ‘National’ [M. glyptostroboides H.H.Hu & W.C.Cheng] DCNA MICHELIA L. MAGNOLIACEAE Magnolia Family haar mea iS Sa compressa (Maxim.) Sarg. NCOP METASEQUOIA Miki ex H.H.Hu & W.C.Cheng MIMOSA L. MATTIONIA FABACEAE (Mimosoideae) Bean Family biuncifera Benth. Catclaw M. LAHG LASL TXCT pigra L. var. berlandieri (A.Gr.) B.L.Turner DCNA TXPS MITCHELLA L. RUBIACEAE Madder Family A Te a Se SOE EE a ee I eee repens L. Partridgeberry GAEC MDLT NCEG SCBR 137 MICHELIA figo (Lour.) Spreng. [illustrator Susan M. Johnston] 138 MORUS MORUS L. Mulberry MORACEAE Mulberry Family ETC Fase wa SSS a alba L. White M. DCSH MDHN SCCL VACW DEMC MDLT TNSG VAGG FLLT NCCA TNTV VAMP ‘Hicks’ [M. alba L.] TNNE ‘Pendula’ [M. alba L.] Weeping M. DCNC MSMN TNTV DCSH SCUC rubra L. Red M. ALHC GAUG TXDC ALTR NCBE VACW FLUF TNSC VAMP ‘Teas Weeping’ [M. alba L.] TNSN MUEHLENBECKIA Meisn. POLYGONACEAE Buckwheat Family Cee Se a Te TE = ele \| axillaris (Hook.f.) Walp. Matbrush Wire Vine ALHC MURRAYA Koenig ex L. RUTACEAE Citrus Family ES Se ee Se Se er ee \| exotica L. = M. paniculata paniculata (L.) Jack Orange Jasmine, Chinese Box FLUF MUSA L. Banana MUSACEAE Banana Family basjoo Sieb. & Zucc. DCNA ornata Roxb. MYRTUS MYRTUS rosacea Jacq. LASL VACW cay ‘Sapientum’ [M. x paradisiaca L.] [M. acuminata < M. balbisiana\| FLUF velutina H.Wendl. & Drude GACG LASL MYRICA L. MYRICACEAE Bayberry Family A RS ROS Re EE ES Ee ET SEER EAS] cerifera L. Wax-myrtle, Waxberry ALBG GACG LASL SCCU FLMG GAIS MDJS TXHA FLPW GAJY MDLT VAMP FLUF LAHG NCE heterophylla Raf. Wax-myrtle GAEC TXSE inodora Bartram SCWI pensylvanica Loisel. Bayberry DCNA MDBG TNBO DEMC MDTD pumila Michx. = M. pusilla pusilla Raf. Dwarf Wax-myrtle SCWI TXSE rubra Sieb. & Zucc. FLUF GAIS VAWL MYRTUS L. Myrtle MYRTACEAE Myrtle Family communis L. Common M. FLMG GADR SCKS SCNR FLUF GAJI SCMG TXLL ‘Microphylla’ [M. communis L.] Dwarf Myrtle ALFN FLMG SCBR MORUS L. Mulberry MORACEAE Mulberry Family ETC Fase wa SSS a alba L. White M. DCSH MDHN SCCL VACW DEMC MDLT TNSG VAGG FLLT NCCA TNTV VAMP ‘Hicks’ [M. alba L.] TNNE ‘Pendula’ [M. alba L.] Weeping M. DCNC MSMN TNTV DCSH SCUC rubra L. Red M. ALHC GAUG TXDC ALTR NCBE VACW FLUF TNSC VAMP ‘Teas Weeping’ [M. alba L.] TNSN MUEHLENBECKIA Meisn. POLYGONACEAE Buckwheat Family Cee Se a Te TE = ele \| axillaris (Hook.f.) Walp. Matbrush Wire Vine ALHC MURRAYA Koenig ex L. RUTACEAE Citrus Family ES Se ee Se Se er ee \| exotica L. = M. MATTIONIA paniculata paniculata (L.) Jack Orange Jasmine, Chinese Box FLUF MUSA L. Banana MUSACEAE Banana Family basjoo Sieb. & Zucc. DCNA rosacea Jacq. LASL VACW cay ‘Sapientum’ [M. x paradisiaca L.] [M. acuminata < M. balbisiana\| FLUF velutina H.Wendl. & Drude GACG LASL MYRICA L. MYRICACEAE Bayberry Family A RS ROS Re EE ES Ee ET SEER EAS] cerifera L. Wax-myrtle, Waxberry ALBG GACG LASL SCCU FLMG GAIS MDJS TXHA FLPW GAJY MDLT VAMP FLUF LAHG NCE heterophylla Raf. Wax-myrtle GAEC TXSE inodora Bartram SCWI pensylvanica Loisel. Bayberry DCNA MDBG TNBO DEMC MDTD pumila Michx. = M. pusilla pusilla Raf. Dwarf Wax-myrtle SCWI TXSE rubra Sieb. & Zucc. FLUF GAIS VAWL MYRTUS L. Myrtle MYRTACEAE Myrtle Family communis L. Common M. FLMG GADR SCKS SCNR FLUF GAJI SCMG TXLL MORUS L. Mulberry MORACEAE Mulberry Family ETC Fase wa SSS a alba L. White M. DCSH MDHN SCCL VACW DEMC MDLT TNSG VAGG FLLT NCCA TNTV VAMP ‘Hicks’ [M. alba L.] TNNE ‘Pendula’ [M. alba L.] Weeping M. DCNC MSMN TNTV DCSH SCUC rubra L. Red M. ALHC GAUG TXDC ALTR NCBE VACW FLUF TNSC VAMP ‘Teas Weeping’ [M. alba L.] TNSN MUEHLENBECKIA Meisn. POLYGONACEAE Buckwheat Family Cee Se a Te TE = ele \| axillaris (Hook.f.) Walp. Matbrush Wire Vine ALHC MURRAYA Koenig ex L. RUTACEAE Citrus Family ES Se ee Se Se er ee \| exotica L. = M. paniculata paniculata (L.) Jack Orange Jasmine, Chinese Box FLUF MUSA L. Banana MUSACEAE Banana Family basjoo Sieb. & Zucc. DCNA MYRICA L. MYRICACEAE Bayberry Family A RS ROS Re EE ES Ee ET SEER EAS] cerifera L. Wax-myrtle, Waxberry ALBG GACG LASL SCCU FLMG GAIS MDJS TXHA FLPW GAJY MDLT VAMP FLUF LAHG NCE heterophylla Raf. Wax-myrtle GAEC TXSE inodora Bartram SCWI pensylvanica Loisel. Bayberry DCNA MDBG TNBO DEMC MDTD pumila Michx. = M. pusilla pusilla Raf. Dwarf Wax-myrtle SCWI TXSE rubra Sieb. & Zucc. FLUF GAIS VAWL MYRTUS L. Myrtle MYRTACEAE Myrtle Family communis L. Common M. FLMG GADR SCKS SCNR FLUF GAJI SCMG TXLL ‘Microphylla’ [M. communis L.] Dwarf Myrtle ALFN FLMG SCBR MUEHLENBECKIA Meisn. POLYGONACEAE Buckwheat Family Cee Se a Te TE = ele \| axillaris (Hook.f.) Walp. Matbrush Wire Vine ALHC MURRAYA Koenig ex L. RUTACEAE Citrus Family ES Se ee Se Se er ee \| exotica L. = M. paniculata paniculata (L.) Jack Orange Jasmine, Chinese Box FLUF MUSA L. Banana MUSACEAE Banana Family basjoo Sieb. MATTIONIA & Zucc. DCNA ornata Roxb. = M. rosacea 139 NANDINA oleander L. Oleander ALBG LAHG MSBN VANB FLUF LASL SCWC ‘Variegata’ [N. oleander L.] Variegated Oleander DCBG NANDINA Thunb. BERBERIDACEAE Barberry Family ‘Alba’ [N. domestica Thunb.] ARRB FLMG MDBG domestica Thunb. Heavenly Bamboo ALBG FLMG MDBG VACW ALRH FLUF MDLT VANB ARRB GAHC MSBN DCNA LAAL MSEN FLDG LASL TNSG ‘Harbor Dwarf’ [N. domestica Thunb.] = ‘Harbour Dwarf’ ‘Harbour Dwarf’ [N. domestica Thunb.] GACG ‘Purpurea Nana’ [N. domestica Thunb.] NCNB VATA NEILLIA D.Don ROSACEAE Rose Family EE ETS NT EVEREST ESS FRE TTS SE Ee) sinensis Oliv. Chinese Neillia NCCA TNSN NEMOPANTHUS Raf. AQUIFOLIACEAE Holly Family mucronatus (L.) Trelease Catberry, Northern Mountain Holly DCNA NEOPIERIS Britton ERICACEAE = Lyonia mariana TE Re SDN aaa Ro Heath Family NERIUM L. APOCYNACEAE Dogbane Family TR eS a indicum Mill. = N. oleander odorum Ait. NEVIUSIA A.Gr. ROSACEAE Rose Family EE aE FS a AS TE IY OMNES EL ED) alabamensis A.Gr. Snow-wreath ALUA NCCA ING VAPH DCNA SCBR TNTV DEMC SCWI TNUT GAEC TNAB VACW NICODEMIA Tenore BUDDLEJACEAE Buddleja Family diversifolia Tenore FLUF NICOTIANA L. SOLANACEAE Nightshade Family EET a RY eS RET RE We EE ET ES glauca R.Graham Tree Tobacco LASL NIEREMBERGIA Ruiz & Pavon SOLANACEAE Nightshade Family frutescens Durieu = N. scoparia scoparia Sendtn. Tall Cup-flower GACE GAUG NYSSA L. NYSSACEAE Sour-gum Family CE a \| ogeche Marsh. Ogeechee Tupelo DCNA SCBR VAWM NIEREMBERGIA Ruiz & Pavon SOLANACEAE Nightshade Family frutescens Durieu = N. scoparia scoparia Sendtn. Tall Cup-flower GACE GAUG NYSSA L. NYSSACEAE Sour-gum Family CE a \| ogeche Marsh. Ogeechee Tupelo DCNA SCBR VAWM ws @ NYSSA sylvatica Marsh. Sour Gum, Black Gum ALAU FLMG NCAS VAGW DCCG LAHG TNFN DCWH MDBP TNHG DEMC MDJH VACW sylvatica Marsh. var. biflora (Walt.) Sarg. SCBR OCHNA L. OCHNACEAE Ochna Family atropurpurea DC. LASL multiflora Hort. = O. atropurpurea OLEA L. OLEACEAE Olive Family EE CS I RE ES SE REE, TEE SE) europaea L. Olive FLUF ORIGANUM L. LAMIACEAE onites L. Pot Marjoram DCNA ORIXA Thunb. RUTACEAE Citrus Family japonica Thunb. MDKN OSMANTHUS Lour. OLEACEAE Olive Family aS eT ET ERT SS OSES SST) SS) americanus (L.) A.Gr. Devilwood ALAU GACG NCTP VACW ALBG GATS SCBR VAWR ALIT GAUG SCMP FLMG NCBE TNDG aquifolium Sieb. = O. heterophyllus var. heterophyllus OSMANTHUS NYSSA NYSSA armatus Diels ALTD GAEH MDMG_ VAGS GACG MDBG NCDU ‘Aurantiacus’ [O. MATTIONIA fragrans Lour.] DCNA SCHS SCMG <fortunei Carr. [O. fragrans < O. heterophyllus var. heterophyllus } ALAU FLMG MDMG NCTP ALBG GAUG NCCA VAMP ALTD LARP NCDU DCCG MDCO NCGP Sfragrans Lour. Sweet Olive, Tea Olive ALBG LAAL MDRP SCMG DCNA LAGN NCDU SCMP FLDG LARP NCEL SCUC FLMG LASL NCRC TXMK FLUF LATU SCEA VANB GAAB MDFM SCFW ‘Gulf Tide’ [O. heterophyllus (G.Don) P.S.Green var. heterophyllus] MDGD TNSN VATA heterophyllus (G.Don) P.S.Green var. heterophyllus Holly Olive ALTD GATS MDSJ NCWC DCCG MDGD NCCA VAGS FLMG MDLT NCDU ilicifolius (Hassk.) Hort. ex Carr. = O. heterophyllus var. heterophyllus ‘Purpureus’ [O. heterophyllus (G.Don) P.S.Green var. heterophyllus] VAGS ‘Rotundifolius’ [O. heterophyllus (G.Don) P.S.Green var. heterophyllus] ALTD LALG SCCU GAUG MDMG VANB ‘San Jose’ [O. <fortunei Carr.] -[O. fragrans x O. heterophyllus var. heterophyllus ] DCCG DCNA GABS NCEL ‘Variegatus’ [O. heterophyllus (G.Don) P.S.Green var. heterophyllus] ALBG FLUF LALG FLMG GACG VADW ilicifolius (Hassk.) Hort. ex Carr. = O. heterophyllus var. heterop 141 OSTEOMELES PASANIA PASANIA PALAFOXIA Lag. ASTERACEAE Aster Family EE ES PELE Ee TES ETE EOE \| feayi A.Gr. SCWI PARKINSONIA L. FABACEAE (Caesalpinioideae) Bean Family EES ET EE TN OE BERS EEE SEER RITE] aculeata L. Jerusalem Thorn FLUF LASL LALG MSMN SCSB PARROTIA C.A.Mey. HAMAMELIDACEAE Witch-hazel Family GS Ee ee Sees Soa ee S\| persica (DC.) C.A.Mey. Persian Ironwood DCEL GAEH VABF DCWH MDBG VACW DEEM MDSJ VAMP PARROTIOPSIS (Niedenzu) Schneid. HAMAMELIDACEAE Witch-hazel Family jacquemontiana (Decne.) Rehd. DCNA PARTHENOCISSUS Planch. VITACEAE Grape Family henryana (Hemsl.) Diels & Gilg DCNA MDMG VAPH quinquefolia (L.) Planch. Virginia Creeper, Woodbine MDLT tricuspidata (Sieb. & Zucc.) Planch. Boston Ivy DECE SCRH PASANIA_ (Miq.) Oerst. = LITHOCARPUS EE ES PELE Ee TES ETE EOE \| feayi A.Gr. SCWI PARKINSONIA L. FABACEAE (Caesalpinioideae) Bean Family EES ET EE TN OE BERS EEE SEER RITE] aculeata L. Jerusalem Thorn FLUF LASL LALG MSMN SCSB PARROTIA C.A.Mey. HAMAMELIDACEAE Witch-hazel Family GS Ee ee Sees Soa ee S\| persica (DC.) C.A.Mey. Persian Ironwood DCEL GAEH VABF DCWH MDBG VACW DEEM MDSJ VAMP PARROTIOPSIS (Niedenzu) Schneid. HAMAMELIDACEAE Witch-hazel Family jacquemontiana (Decne.) Rehd. DCNA PARTHENOCISSUS Planch. VITACEAE Grape Family henryana (Hemsl.) Diels & Gilg DCNA MDMG VAPH quinquefolia (L.) Planch. Virginia Creeper, Woodbine MDLT tricuspidata (Sieb. & Zucc.) Planch. Boston Ivy DECE SCRH PASANIA_ (Miq.) Oerst. = LITHOCARPUS schwerinae Schneid. Chinese Boneberry DCNA OSTRYA Scop. MATTIONIA BETULACEAE Birch Family eT AE a RT ES SET A TG virginiana (Mill.) K.Koch Hop Hornbeam ALAU DCCG FLUF TNFN VAMP OXYDENDRUM DC. ERICACEAE Heath Family arboreum (L.) DC. Sourwood, Sorrel Tree ALAU MDJH SCBR VANB DCNA MDKN TNSC VAPO DCWR MDLT VACP PACHYSANDRA Michx. BUXACEAE Boxwood Family SE EE PE SE SS TSS SS axillaris Franch. NCWD procumbens Michx. Allegheny P., Allegheny Spurge MDBG SCWI VAPH ‘Silveredge’ [P. terminalis Sieb. & Zucc.] DCNA terminalis Sieb. & Zucc. Japanese P., Japanese Spurge DCNA DEWG LALG VACW PACHYSTIMA Endl. = PAXISTIMA PAEONIA lL. Peony PAEONIACEAE Peony Family suffruticosa Andr. Abtae le. DCWH VACW PACHYSANDRA Michx. BUXACEAE Boxwood Family SE EE PE SE SS TSS SS axillaris Franch. NCWD procumbens Michx. Allegheny P., Allegheny Spurge MDBG SCWI VAPH ‘Silveredge’ [P. terminalis Sieb. & Zucc.] DCNA terminalis Sieb. & Zucc. Japanese P., Japanese Spurge DCNA DEWG LALG VACW PACHYSTIMA Endl. = PAXISTIMA PAEONIA lL. Peony PAEONIACEAE Peony Family suffruticosa Andr. Abtae le. DCWH VACW jacquemontiana (Decne.) Rehd. DCNA PARTHENOCISSUS Planch. VITACEAE Grape Family henryana (Hemsl.) Diels & Gilg DCNA MDMG VAPH quinquefolia (L.) Planch. Virginia Creeper, Woodbine MDLT tricuspidata (Sieb. & Zucc.) Planch. Boston Ivy DECE SCRH PASANIA_ (Miq.) Oerst. = LITHOCARPUS PARROTIOPSIS jacquemontiana (Decne.) Rehd. [illustrator Susan M. Johnston] PARROTIOPSIS jacquemontiana (Decne.) Rehd. [illustrator Susan M. Johnston] 143 PASSIFLORA PHELLODENDRON PEROVSKIA XKarelin LAMIACEAE Mint Family EEE SS a a aS IEE TE BIT atriplicifolia Benth. Russian Sage MDBG VAGS PERSEA Mill. LAURACEAE Laurel Family americana Mill. Avocado, Alligator Pear FLUF borbonia (L.) Spreng. Red Bay ALAU LAMP SCBR VACW FLUF NCGP SCMP VANB humilis Nash SCWI thunbergii (Sieb. & Zucc.) Kosterm. LASL PETTERIA Presl FABACEAE (Faboideae) Bean Family EEE ee aes TS ae ee ee a eS \| ramentacea Presl MDGD PHELLODENDRON Rupr. RUTACEAE Citrus Family a el amurense Rupr. Amur Cork-tree DCTB DCWH GARW MDCP MDGJ TNSN VABF VABP amurense Rupr. var. japonicum (Maxim.) Ohwi DCLC MDWG TNWF amurense Rupr. var. lavallei (Dode) Sprague TNUT PEROVSKIA XKarelin LAMIACEAE Mint Family EEE SS a a aS IEE TE BIT atriplicifolia Benth. Russian Sage MDBG VAGS PERSEA Mill. LAURACEAE Laurel Family americana Mill. Avocado, Alligator Pear FLUF borbonia (L.) Spreng. Red Bay ALAU LAMP SCBR VACW FLUF NCGP SCMP VANB humilis Nash SCWI thunbergii (Sieb. & Zucc.) Kosterm. LASL PETTERIA Presl FABACEAE (Faboideae) Bean Family EEE ee aes TS ae ee ee a eS \| ramentacea Presl MDGD PHELLODENDRON Rupr. MATTIONIA RUTACEAE Citrus Family a el amurense Rupr. Amur Cork-tree DCTB DCWH GARW MDCP MDGJ TNSN VABF VABP amurense Rupr. var. japonicum (Maxim.) Ohwi DCLC MDWG TNWF amurense Rupr. var. lavallei (Dode) Sprague TNUT PASSIFLORA L. PASSIFLORACEAE _ Passion-flower Family PASSIFLORA L. PASSIFLORACEAE _ Passion-flower Family PAULOWNIA Sieb. & Zucc. SCROPHULARIACEAE Figwort Family AE ae EE RL ST OR ee tee SV) kawakamii Ito SCWI tomentosa (Thunb. ex J.A.Murr.) Steud. Princess Tree DCWH MDHN MSRN VAWR GAAB MDLT TNHG PAUROTIS Cook ARECACEAE Palm Family TT ES Te a ee eee eee wrightii (Griseb.) Britton = Acoelorrhaphe wrightii PAVONIA Cav. MALVACEAE Mallow Family hastata Cav. Spearleaf P. LASL PAXISTIMA Raf. CELASTRACEAE Staff-tree Family canbyi A.Gr. Cliff-green DCNA MDLT PERIPLOCA L. ASCLEPIADACEAE Asclepias Family (ES ES a a aT graeca L. Silk Vine TNSN PAULOWNIA Sieb. & Zucc. SCROPHULARIACEAE Fig PHELLODENDRON Rupr. RUTACEAE Citrus Family a el amurense Rupr. Amur Cork-tree DCTB DCWH GARW MDCP MDGJ TNSN VABF VABP amurense Rupr. var. japonicum (Maxim.) Ohwi DCLC MDWG TNWF amurense Rupr. var. lavallei (Dode) Sprague TNUT graeca L. Silk Vine TNSN 144 PHOENIX PHILADELPHUS PHOENIX schrenkii Rupr. DCNA ‘Silberregen’ DCNA triflorus Wall. VABF verrucosus Schrad. ex DC. = P. pubescens var. verrucosus ‘Virginal’ [P. <virginalis Rehd.] [P. Xlemoinei < P. ?nivalis ‘Plenus’] ALBH DEWG MDCC TNSN DCDO GAPG MDJS ‘Voie Lactee’ MDGD PHILLYREA L. OLEACEAE Olive Family angustifolia L. Narrow-leaf P. ALTD SCPP latifolia L. var. media (L.) Schneid. Tree P. LASL media L. = P. latifolia var. media ‘Spinosa’ [P. latifolia L. var. media (L.) Schneid.] GAUG PHOENIX L. ARECACEAE Palm Family SE PE eS en ee Soe a) canariensis Hort. ex Chabaud Canary Island Date Palm FLUF reclinata Jacq. Senegal Date Palm FLUF sylvestris < P. canariensis FLUF PHOENIX schrenkii Rupr. DCNA ‘Silberregen’ DCNA triflorus Wall. VABF verrucosus Schrad. ex DC. = P. pubescens var. verrucosus ‘Virginal’ [P. <virginalis Rehd.] [P. Xlemoinei < P. ?nivalis ‘Plenus’] ALBH DEWG MDCC TNSN DCDO GAPG MDJS ‘Voie Lactee’ MDGD PHILLYREA L. OLEACEAE Olive Family angustifolia L. Narrow-leaf P. ALTD SCPP latifolia L. var. media (L.) Schneid. Tree P. LASL media L. = P. latifolia var. media ‘Spinosa’ [P. latifolia L. var. media (L.) Schneid.] GAUG PHOENIX L. ARECACEAE Palm Family SE PE eS en ee Soe a) canariensis Hort. ex Chabaud Canary Island Date Palm FLUF reclinata Jacq. Senegal Date Palm FLUF sylvestris < P. canariensis FLUF PHILADELPHUS L. MATTIONIA Mock Orange SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family == nS eS a ES ae ‘Belle Etoile’ [P. coronarius L.] VAPH coronarius L. European Mock O. GAOH MDCP MDPJ VAGW GAVA MDHN TNSN VANS MDBP MDLT VACW ‘Duplex’ [P. coronarius L.] MDJS VABF <falconeri Sarg. [P. coronarius X< P. laxus] MSMN NCCA gordonianus Lindl. = P. lewisii hirsutus Nutt. GAEC inodorus L. FLDJ LALG NCCA TXMA FLMG MSHB SCCU VACW GAOH NCBE SCRH VAGG x lemoinei V.Lemoine [P. microphyllus < P. coronarius] VAPH lewisii Pursh MDJS ‘Minnesota Snowflake’ [P. < virginalis Rehd.] [P. Xlemoinei < P. ?nivalis ‘Plenus’] DEWG ‘Natchez’ [P. coronarius L.] DCNA ‘Nymans’ [P. delavayi L.Henry] DCNA pubescens Loisel. SCBR pubescens Loisel. var. verrucosus (Schrad. ex DC.) S.Y.Hu DCDO MDSJ VAHS satsumanus Sieb. ex Miq. var. nikoensis Rehd. DEWG PHILADELPHUS L. Mock Orange SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family 145 PHOTINIA PHYLLOSTACHYS PHOTINIA Lindl. ROSACEAE Rose Family ‘Birmingham’ [P. <fraseri W.J.Dress] [P. serratifolia < P. glabra] ALBG FLUF SCWE VAWR ALFN GAWH TXSE ALTD MSHB VACW DCSD SCFW VAMP glabra (Thunb. ex J.A.Murr.) Maxim. Japanese P. FLGS GAJY SCCL VAMP FLMG MSHB SCES GACG NCCA SCMG serratifolia (Desf.) Kalk Chinese P. ALBG GACG NCTE SCUC ALTD GAUG SCBR TNMB ARRB LASL SCDB VANB DCCG MSEN SCFW VAOD FLMG NCDU SCMG VAWP FLUF NCOP SCMP serrulata Lindl. = P. serratifolia villosa (Thunb. ex J.A.Murr.) DC. DCWR MDBG NCBE VAWR DEWG MDGJ VABF GAEH MDPJ VAGH villosa (Thunb. ex J.A.Murr.) DC. var. laevis (Thunb. ex J.A.Murr.) Dipp. MDGJ villosa (Thunb. ex J.A.Murr.) DC. var. maximowicziana (Lev.) Rehd. DCCG DEMC NCBE villosa (Thunb. ex J.A.Murr.) DC. var. sinica Rehd. & Wils. VACW PHYGELIUS' E.Mey. ex Benth. SCROPHULARIACEAE Figwort Family capensis E.Mey. Cape Fuchsia VACW PHYLLANTHUS L. EUPHORBIACEAE Spurge Family nivosa W.G.Sm. = Breynia nivosa PHYLLOSTACHYS Sieb. & Zucc. Bamboo POACEAE Grass Family = aS OS EE SER ET a ee \| angusta McClure MDST arcana McClure GAIS aurea A.& C.Riv. Fishpole B., Golden B. FLDG GAIS VAMP aureosulcata McClure Yellow-groove B. TNFE bambusoides Sieb. & Zucc. GAIS NCOP ‘Castillon’ [P. bambusoides Sieb. & Zucc.] MDFM congesta McClure GAIS dulcis McClure Sweetshoot B. GAIS MDFM elegans McClure GAIS flexuosa A.& C.Riv. GAIS ‘Henon’ [P. nigra (Lodd.) Munro] GAIS VABB heterocycla (Carr.) Mitf. Giant Timber Bamboo ALBG GAIS SCCU SCWL meyeri McClure FLUF GAIS PHYLLANTHUS L. EUPHORBIACEAE Rose Family = Breynia nivosa PHYLLOSTACHYS Sieb. & Zucc. MATTIONIA Bamboo POACEAE Grass Family = aS OS EE SER ET a ee \| angusta McClure MDST arcana McClure GAIS aurea A.& C.Riv. Fishpole B., Golden B. FLDG GAIS VAMP aureosulcata McClure Yellow-groove B. TNFE bambusoides Sieb. & Zucc. GAIS NCOP ‘Castillon’ [P. bambusoides Sieb. & Zucc.] MDFM congesta McClure GAIS dulcis McClure Sweetshoot B. GAIS MDFM elegans McClure GAIS flexuosa A.& C.Riv. GAIS ‘Henon’ [P. nigra (Lodd.) Munro] GAIS VABB heterocycla (Carr.) Mitf. Giant Timber Bamboo ALBG GAIS SCCU SCWL meyeri McClure FLUF GAIS nidularia Munro GAIS nigra (Loud.) Munro PHYGELIUS' E.Mey. ex Benth. SCROPHULARIACEAE Figwort Family 146 PHYLLOSTACHYS nuda McClure MDGD MDPJ propinqua McClure GAIS pubescens Mazel ex Houz.de Leh. = P. heterocycla rubromarginata McClure DCNA ‘Smoothsheath’ [P. nidularia Munro] GAIS ‘Solidstem’ [P. purpurata McClure] GAIS ‘Straightstem’ [P. purpurata McClure] GAIS viridiglaucescens A.& C.Riv. GAIS viridis (Young) McClure GAIS NCSL vivax McClure GAIS PHYLLOSTACHYS nuda McClure MDGD MDPJ propinqua McClure GAIS pubescens Mazel ex Houz.de Leh. = P. heterocycla rubromarginata McClure DCNA ‘Smoothsheath’ [P. nidularia Munro] GAIS ‘Solidstem’ [P. purpurata McClure] GAIS ‘Straightstem’ [P. purpurata McClure] GAIS viridiglaucescens A.& C.Riv. GAIS viridis (Young) McClure GAIS NCSL vivax McClure GAIS PHYSOCARPUS (Cambess.) Maxim. Ninebark ROSACEAE Rose Family ES a EE OL EA Sa ee \| amurensis (Maxim.) Maxim. DCNA intermedius (Rydb.) Schneid. TNBM ‘Luteus’ [P. opulifolius (L.) Maxim.] VACW malvaceous (Greene) O.Ktze. TNUT opulifolius (L.) Maxim. GAEC MDGJ TNFN PICEA A.Dietr. Spruce PINACEAE Pine Family abies (L.) Karst. Norway S. DCNA MDBG MDJS TNWF GAHC MDHN NCBE VASP PICEA = P. glauca = alcoquiana (J.G.Veitch ex Lindl.) Carr. Alcock’s S. DCNA asperata Mast. Dragon S. DCNA NCBE asperata Mast. var. heterolepis (Rehd. & Wils.) W.C.Cheng ex Rehd. DCNA ‘Aurea Compacta’ [P. orientalis (L.) Link] DCNA ‘Barryi’ [P. abies (L.) Karst.] DCNA ‘Beissneri Compacta’ [P. mariana (Mill.) BSP.] DCNA ‘Brevifolia’ [P. abies (L.) Karst.] DCNA ‘Capitata’ [P. abies (L.) Karst.] DCNA ‘Clanbrassiliana’ [P. abies (L.) Karst.] DCNA ‘Compacta’ [P. pungens Engelm.] DCNA ‘Compacta Asselyn’ [P. abies (L.) Karst.] DCNA ‘Conica’ [P. glauca (Moench) Voss] Dwarf Alberta S. DCNA MDBG MDLT GAEC MDJS VATW ‘Doumetii’ [P. mariana (Mill.) BSP.] Black S. NCBE -‘Echiniformis’ [P. glauca (Moench) Voss] MDLT ‘Foxtail’ [P. pungens Engelm.] DCNA gemmata Rehd. & Wils. DCNA glauca (Moench) Voss ‘Aurea Compacta’ [P. orientalis (L.) Link] DCNA PHYSOCARPUS (Cambess.) Maxim. Ninebark ROSACEAE Rose Family ES a EE OL EA Sa ee \| amurensis (Maxim.) Maxim. DCNA intermedius (Rydb.) Schneid. MATTIONIA TNBM ‘Luteus’ [P. opulifolius (L.) Maxim.] VACW malvaceous (Greene) O.Ktze. TNUT opulifolius (L.) Maxim. GAEC MDGJ TNFN PICEA A.Dietr. Spruce PINACEAE Pine Family abies (L.) Karst. Norway S. DCNA MDBG MDJS TNWF GAHC MDHN NCBE VASP 147 PICEA PICEA PICEA ‘Glauca’ [P. asperata Mast.] DCNA ‘Glauca Pendula’ [P. pungens Engelm. f. glauca (Regel) Beissn.] DCNA ‘Glauca Procumbens’ [P. pungens Engelm. f. glauca (Regel) Beissn.] DCNA ‘Glauca Prostrata’ [P. pungens Engelm. f. glauca (Regel) Beissn.] DCNA glehnii (F.Schmidt) Mast. DCNA NCBE ‘Globosa’ [P. abies (L.) Karst.] DCNA ‘Globosa’ [P. pungens Engelm.] DCNA ‘Gracilis’ [P. orientalis (L.) Link] DCNA ‘Gregoryana Parsonii’ [P. abies (L.) Karst.] DCNA ‘Highlandia’ [P. abies (L.) Karst.] DCNA ‘Hoopsii’ [P. pungens Engelm.] DCNA ‘Howell’s Dwarf’ [P. jezoensis (Sieb. & Zucc.) Carr.] MDBG ‘Humilis’ [P. abies (L.) Karst.] DCNA ‘Hunnewelliana’ [P. pungens Engelm.] DCNA MDBG ‘Hystrix’ [P. abies (L.) Karst.] DCNA ‘Inversa’ [P. abies (L.) Karst.] DCNA MDBG NCBE ‘Iseli Fastigiata’ [P. pungens Engelm.] DCNA ‘Kingsville’ [P. abies (L.) Karst.] DCNA ‘Koster’ [P. pungens Engelm.] PIC ‘Maxwellii’ [P. abies (L.) Karst.] DCNA MDBG ‘Merhii’ [P. abies (L.) Karst.] DCNA ‘Microphylla’ [P. abies (L.) Karst.] DCNA ‘Microsperma’ [P. abies (L.) Karst.] DCNA ‘Montgomery’ [P. pungens Engelm.] DCNA ‘Montigena’ [P. abies (L.) Karst.] DCNA ‘Mucronata’ [P. abies (L.) Karst.] DCNA MDBG ‘Nidiformis’ [P. abies (L.) Karst.] DCNA MDBG MDLT obovata Ledeb. DCNA ‘Ohlendorffii’ [P. abies (L.) Karst.] DCNA ‘Oldhamiana’ [P. abies (L.) Karst.] DCNA omorika (Pancic) Purk. Serbian S. DCNA MDBG MDLT orientalis (L.) Link Oriental S. DCNA DEWG NCBE VAAC DCWH MDJS NCKH VABF ‘Parsonii’ [P. abies (L.) Karst.] DCNA ‘Pendula’ [P. abies (L.) Karst.] DCNA MDJS MDLT ‘Pendula’ [P. omorika (Pancic) Purk.] DCNA polita (Sieb. & Zucc.) Carr. = P. torano ‘Procumbens’ [P. abies (L.) Karst.] DCNA ‘Prostrata’ [P. abies (L.) Karst.] DCNA 148 PICEA ‘Pumila’ [P. abies (L.) Karst.] DCNA ‘Pumila Glauca’ [P. abies (L.) Karst.] DCNA pungens Engelm. Colorado S. DCNA MDBG VASP pungens Engelm. f. glauca (Regel) Beissn. Colorado Blue S. DCNA ‘Pygmaea’ [P. abies (L.) Karst.] DCNA MDLT ‘Pyramidata’ [P. abies (L.) Karst.] DCNA ‘Remonte’ [P. abies (L.) Karst.] DCNA ‘Repens’ [P. abies (L.) Karst.] DCNA ‘Tabuliformis’ [P. abies (L.) Karst.] DCNA torano (K.Koch) Koehne Tiger-tail S. DCNA NCBE VAMP ‘Wild Acres’ [P. glauca (Moench) Voss] DCNA PIERIS D.Don ERICACEAE Heath Family ‘Bert Chandler’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] MDLT MDMG ‘Brower’s Beauty’ [P. floribunda x P. japonica] DCNA MDBG MDLT ‘Compacta’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA ‘Crispa’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA MDBG ‘Daisen’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] PIERIS PIERIS ‘Dorothy Wyckoff [P. japonica (Thunb. PICEA ex J.A.Murr.) D.Don ex G.Donk. DEWG MDLT VAPH ‘Flamingo’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA MDBG floribunda (Pursh) Benth. & Hook. Fetterbush DEWG MDLT MDWP NCDB ‘Forest Flame’ [P. japonica x P. formosa] DEWG MDFM MDJW VAPH formosa (Wall.) D.Don MDKN forrestii R.Harrow ex W.W.Sm. = P. formosa japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don Japanese Andromeda ALBG DEWG MDLT VACW ALBH GACG TNCT DCNA GASM TNDG ‘Mountain Fire’ [P. formosa x P. japonica] MDLT phillyreifolia (Hook.) DC. ALAU MDKN VAPH GAEC SCWI ‘Purity’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA ‘Pygmaea’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA GAEC ‘Pygmy Variegata’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] MDKN ‘Red Mill’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] MDBG MDLT ‘Scarlet O’Hara’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA MDBG ‘Shojo’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] 149 PINUS PIERIS PIERIS ‘Variegata’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA MDBG VATW DEWG MDLT ‘Wada’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] VAPH ‘Wakehurst’ [P. formosa (Wall.) D.Don] DCNA ‘Whitecaps’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA ‘White Cascade’ [P. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don] DCNA MDBG PINCKNEYA Michx. RUBIACEAE Madder Family pubens Michx. VAPH PINUS L. Pine PINACEAE Pine Family ‘Adcock’s Dwarf’ [P. parviflora Sieb. & Zucc.] DCNA ‘Albyns’ [P. sylvestris L.] DCNA ‘Amelia Dwarf’ [P. strobus L.] DCNA ‘Argentea Compacta’ [P. sylvestris L.] DCNA armandii Franch. DCNA ‘Arnold Dwarf’ [P. peuce Griseb.] DCNA attenuata J.G.Lemmon Knob-cone P. VABF ‘Aurea’ [P. sylvestris L.] DCNA ayacahuite K.Ehrenb. GAEC DCNA banksiana Lamb. JackoR: TNMB TNUT ‘Beuvronensis’ [P. sylvestris L.] DCNA brutia Tenore DCNA bungeana Zucc. ex Endl. Lacebark P. DCNA MDBA VABF GAUG TNRM cembra L. Swiss Stone P. DCNA MDBG MDPJ cembra L. var. sibirica Loud. DCNA cembroides Zucc. Mexican Stone P. DCNA VABF clausa (Chapm. ex Engelm.) Vasey ex Sarg. Sand P. ALAU FLFB FLPL FLUF ‘Compact Gem’ [P. leucodermis Ant.] DCNA ‘Compacta Glauca’ [P. cembra L.] DCNA ‘Contorta’ [P. strobus L.] MDBG ‘Corticosa’ [P. thunbergiana Franco] DCNA densiflora Sieb. & Zucc. Japanese Red P. ALTD FLHJ GAFN DCNA FLUF VANB ‘Dwarf’ [P. strobus L.] GAJS ‘Dwarf Blue’ [P. pumila (Pall.) Regel] DCNA echinata Mill. Shortleaf P. DCCG FLMG GAIS SCFW DCNC GACE NCBM TNFL DCNA banksiana Lamb. JackoR: TNMB TNUT ‘Beuvronensis’ [P. sylvestris L.] DCNA brutia Tenore DCNA bungeana Zucc. ex Endl. Lacebark P. DCNA MDBA VABF GAUG TNRM cembra L. Swiss Stone P. DCNA MDBG MDPJ cembra L. var. sibirica Loud. DCNA cembroides Zucc. Mexican Stone P. DCNA VABF clausa (Chapm. ex Engelm.) Vasey ex Sarg. Sand P. ALAU FLFB FLPL FLUF ‘Compact Gem’ [P. leucodermis Ant.] DCNA ‘Compacta Glauca’ [P. cembra L.] DCNA ‘Contorta’ [P. strobus L.] MDBG ‘Corticosa’ [P. thunbergiana Franco] DCNA densiflora Sieb. & Zucc. Japanese Red P. ALTD FLHJ GAFN DCNA FLUF VANB ‘Dwarf’ [P. strobus L.] GAJS ‘Dwarf Blue’ [P. pumila (Pall.) Regel] DCNA echinata Mill. Shortleaf P. DCCG FLMG GAIS SCFW DCNC GACE NCBM TNFL PINUS PINUS PINUS elliottiti Engelm. Slash P. ALAU FLFB excelsa Wall. ex D.Don = P. wallichiana ‘Fastigiata’ [P. strobus L.] GAEC MDBG MDJS VABF ‘Fastigiata’ [P. sylvestris L.] DCNA MDBG flexilis James Limber P. DCCG DCNA MDBG glabra Walt. Spruce P. PIERIS ALEH LAMP SCFW FLMG LASL SCMP FLUF NCCA TXPS ‘Glauca’ [P. parviflora Sieb. & Zucc.] DCNA ‘Glauca Nana’ [P. parviflora Sieb. & Zucc.] DCNA ‘Glenmore’ [P. flexilis James] DCNA ‘Globosa’ [P. nigra Arn.] DCNA ‘Globosa Viridis’ [P. sylvestris L.] DCNA ‘Gnom’ [P. mugo Turra var. mughus (Scop.) Zenari] DCNA greggii Engelm. & Parl. TXMA griffithii McClelland = P. wallichiana halepensis Mill. Aleppo P. TXAO heldreichii Christ var. leucodermis (Ant.) Markgr. ex Fitschen = P. leucodermis PINUS koraiensis Sieb. & Zucc. Korean P. aE DCNA MDDF MDJS leucodermis Ant. DCNA VABF longifolia Roxb. = P. roxburghii ‘Merrimack’ [P. strobus L.] DCNA ‘Monstrosa’ [P. nigra Arn.] GAEC ‘Mops’ [P. mugo Turra var. mughus (Scop.) Zenari] DCNA ‘Moseri’ [P. sylvestris L.] DCNA mugo Turra var. mugo Swiss Mountain P. ALTD DEWG MDLT DCLC GAFN TNHD DCNA MDBG VABF mugo Tutra var. pumilio (Haenke) Zenari MDBG ‘Nana’ [P. nigra Arn.] (Nana group) DCNA GAEC MDBG MDLT ‘Nana’ [P. strobus L.] DCNA MDBG MDLT nepalensis Chambr. = P. wallichiana nigra Arn. ssp. nigra Austrian P. DCLC MDHN SCCU VAMP MDAF MDLA VABF VATW MDCP MDSJ VAHN VAWR ‘Oculus-draconis’ [P. densiflora Sieb. & Zucc.] DCNA MDBG TNTV GAJS MDTD palustris Mill. Longleaf P. ALSM NCGP TNBM NCBE NCWK parviflora Sieb. & Zucc. 151 PINUS PINUS patula Schiede & Deppe Jelecote P. SCCU ‘Pendula’ [P. densiflora Sieb. & Zucc.] Weeping Japanese Red P. DCNA MDJS TNTV MDBG MDLT ‘Pendula’ [P. ponderosa Dougl. ex P.& C.Laws.] DCNA ‘Pendula’ [P. strobus L.] DCNA MDBG MDLT pinea L. Italian Stone P., Umbrella P. LAMP ponderosa Dougl. ex P.& C.Laws Western Yellow P., Ponderosa P. VABF pumila (Pall.) Regel DCNA ‘Pumila’ [P. strobus L.] DCNA pungens Lamb. Table-mountain P. DCNA Sccu VABF ‘Radiata’ [P. strobus L.] DCNA ‘Repens’ [P. sylvestris L.] DCNA resinosa Ait. Red P. DCCG DCNA TNSC VATG rigida Mill. Pitehir: MDDF roxburghii Sarg. Long-leaved Indian P., Chir P. TXHJ sabiniana Dougl. ex D.Don Digger P. VABF ‘Seacrest’ [P. strobus L.] PINU serotina Michx. Pond P. ALAU GACL ‘Spaan’s Dwarf [P. contorta Doug. ex Loud.] DCNA strobus L. Eastern White P. ALIT MDHN NCOP VAGW DCNA MDJS SCAK VAMP GAOH MDLT SCCU GAUG NCCA TNBM MDBG NCKH TNHG strobus X< P. wallichiana DCNA sylvestris L. Scotch P. DCNA NCCM TNSC GAUG SCCU VABF taeda L. Loblolly P. DCWH GAIS MDBG SCMG FLMG LAAL NCGP TNRM ‘Tanoyosho’ [P. densiflora Sieb. & Zucc.] = P. densiflora ‘Umbraculifera’ thunbergiana Franco Japanese Black P. ALTD DCNA MDKN TNCT DCCG LAMP MDLT VABF DCFP MDBG NCBE VATA thunbergii Parl. = P. thunbergiana ‘Torulosa’ [P. strobus L.] DCNA ‘Umbraculifera’ [P. densiflora Sieb. & Zucc.] Japanese Umbrella P. ALOS DCNA MDJS TNSC DCCG MDBG MDLT VATS ‘Venus’ [P. parviflora Sieb. & Zucc.] DCNA virginiana Mill. Virginia P., Scrub P. ALAU GAJS NCWK VAGW DCNA GAUG SCCU GACE MDBG TNSC ponderosa Dougl. ex P.& C.Laws Western Yellow P., Ponderosa P. VABF pumila (Pall.) Regel DCNA ‘Pumila’ [P. strobus L.] DCNA PLATYCLADUS ‘Variegata’ [P. tobira (Willd.) Ait.] FLMG LALG SCBR SCJM GACH MSEN SCCU TXSH ‘Wheeler’s Dwarf’ [P. tobira (Willd.) Ait.] FLMG FLUF PLANERA J.F.Gmel. ULMACEAE Elm Family (RE SE Ne ae ee eM eR Re Ee Se) aquatica (Walt.) J.F.Gmel. Water Elm SCMP PLATANUS L. PLATANACEAE Sycamore Plane-tree Family x acerifolia (Ait.) Willd. [P. occidentalis < P. orientalis] London Plane DCCG DEMC MDHN NCBE VASC MDJH TNUT * ‘Columbia’ [P. < acerifolia (Ait.) Willd.] [P. occidentalis < P. orientalis] DCNA x hispanica Muenchh. = P. Xacerifolia * ‘Liberty’ [P. < acerifolia (Ait.) Willd.] [P. occidentalis < P. orientalis] DCNA FLLT occidentalis L. American S. DCCG FLUF MDHN TNUT orientalis L. Oriental Plane DCNA FLUF PLATYCARYA Sieb. & Zucc. PINUS JUGLANDACEAE Walnut Family LRAT AE EM SE CIS LN To ODOM RIT SPE) strobilacea Sieb. & Zucc. TNLW VABF PLATYCLADUS Spach ‘Yatsubusa’ [P. thunbergiana Franco] DCNA ‘Zebrina’ [P. wallichiana A.B.Jacks.] DCNA PISTACIA L. Pistache ANACARDIACEAE Cashew Family chinensis Bunge Chinese P. ALBT GAIS SCEM VAHM ALTD LAAL SCRP VAMP DCNA LASL TNSG FLJH MDGD TXMS FLUF MSMN VABF texana Swingle Texas P. TXLL TXMS PISTACIA L. Pistache ANACARDIACEAE Cashew Family chinensis Bunge Chinese P. ALBT GAIS SCEM VAHM ALTD LAAL SCRP VAMP DCNA LASL TNSG FLJH MDGD TXMS FLUF MSMN VABF texana Swingle Texas P. TXLL TXMS PITHECELLOBIUM Mart. FABACEAE (Mimosoideae) Bean Family ET TLS aT aN SAT eT] ebano (Berl.) Muller Texas Ebony TXRE flexicaule (Benth.) Coult. = P. ebano PITHECOLOBIUM Benth. = PITHECELLOBIUM PITTOSPORUM Banks ex Sol. PITTOSPORACEAE Pittosporum Family glabratum Lindl. LAAL tobira (Willd.) Ait Japanese P. DCNA LAAL SCHW VANB FLMG LARP SCMP VARR FLUF LASL SCUC GAHC NCGP TXSH GAJI SCBR VAMF undulatum Vent. Victorian Box FLUF LAAL PLATYCARYA Sieb. & Zucc. JUGLANDACEAE Walnut Family LRAT AE EM SE CIS LN To ODOM RIT SPE) strobilacea Sieb. & Zucc. TNLW VABF PLATYCLADUS Spach CUPRESSACEAE Cypress Family LA Ey ID PNR PE CCN A EO a Cay HS ‘Aurea Nana’ [P. orientalis (L.) Franco] ALFN FLCG GABC LASL DCNA FLEM GAFN SCMG PLATYCLADUS ‘Bakeri’ [P. orientalis (L.) Franco] ALAU ALFN SCMG ‘Berckman’s Golden’ [P. orientalis (L.) Franco] ALAU ‘Beverleyensis’ [P. orientalis (L.) Franco] DCNA LAAL ‘Conspicua’ [P. orientalis (L.) Franco] DCNA SCCU ‘Flagelliformis’ [P. orientalis (L.) Franco] GAGC ‘Fruitlandii’ [P. orientalis (L.) Franco] ALFN ‘Hohman’ [P. orientalis (L.) Franco] ALBH ‘Juniperoides’ [P. orientalis (L.) Franco] DCNA orientalis (L.) Franco Oriental Arborvitae ALSH FLUF MDJS SCRH ALUA GAJI MDLA TNUT DCFP GASU NCCA FLDG LAAL SCBS FLMG MDCP SCMG ‘Rosedalis’ [P. orientalis (L.) Franco] MDBG ‘Semperaurea’ [P. orientalis (L.) Franco] GAJI PLUMBAGO lL. PLUMBAGINACEAE Leadwort Family Ee EO EE ES SR a er a auriculata Lam. Cape Leadwort ALBG FLUF GACG LAAL LALG SCHW capensis Thunb. = P. auriculata PODOCARPUS WdHer. ex Pers. PODOCARPACEAE Podocarpus Family gracilior Pilger African Fern Pine FLUF PONCIRUS PLATYCLADUS PONCIRUS macrophyllus (Thunb. ex J.A.Murr.) Sweet Wewal: ALBG LAAL MSMN FLMG LASL NCCA FLUF MSHB SCMG macrophyllus (Thunb. ex J.A.Murr.) Sweet var. maki Sieb. ALBG FLUF LALG NCDU ALON GAHC LARP SCBR FLDC GAIS LASL SCMG FLDG GAdJI MSMN SCWI FLMG GATS NCCA nagi (Thunb. ex J.A.Murr.) Makino Broadleaf P. FLRP FLUF GACH LAAL LAGN TXHA totara D.Don Totara Pine ALEH POINCIANA L. = CAESALPINIA POLIOTHYRSIS Oliv. FLACOURTIACEAE Flacourtia Family sinensis Oliv. DCNA VAGS POLYGONELLA Michx. POLYGONACEAE Buckwheat Family a a eS Se ee a ee SEE ORE a) americana (Fischer & C.A.Mey.) Small SCBR SCWI myriophylla (Small) Horton SCWI polygama (Vent.) Engelm. & A.Gr. SCWI PONCIRUS Raf. RUTACEAE Citrus Family trifoliata (L.) Raf. Hardy Orange ALTD FLUF MDLA VAMP DCCG GAHC SCCU VAPH DCWH GAIS VACW VATA FLMG MDHN VAGW macrophyllus (Thunb. ex J.A.Murr.) Sweet Wewal: ALBG LAAL MSMN FLMG LASL NCCA FLUF MSHB SCMG macrophyllus (Thunb. ex J.A.Murr.) Sweet var. maki Sieb. ALBG FLUF LALG NCDU ALON GAHC LARP SCBR FLDC GAIS LASL SCMG FLDG GAdJI MSMN SCWI FLMG GATS NCCA nagi (Thunb. ex J.A.Murr.) Makino Broadleaf P. FLRP FLUF GACH LAAL LAGN TXHA totara D.Don Totara Pine ALEH POINCIANA L. = CAESALPINIA POLIOTHYRSIS Oliv. FLACOURTIACEAE Flacourtia Family sinensis Oliv. DCNA VAGS POLYGONELLA Michx. POLYGONACEAE Buckwheat Family a a eS Se ee a ee SEE ORE a) americana (Fischer & C.A.Mey.) Small SCBR SCWI myriophylla (Small) Horton SCWI polygama (Vent.) Engelm. & A.Gr. SCWI PONCIRUS Raf. RUTACEAE Citrus Family trifoliata (L.) Raf. ‘Bakeri’ [P. orientalis (L.) Franco] ALAU ALFN SCMG ‘Bakeri’ [P. orientalis (L.) Franco] ALAU ALFN SCMG macrophyllus (Thunb. ex J.A.Murr.) Sweet Wewal: POINCIANA L. = CAESALPINIA POLIOTHYRSIS Oliv. FLACOURTIACEAE Flacourtia Family sinensis Oliv. DCNA VAGS POLYGONELLA Michx. POLYGONACEAE Buckwheat Family a a eS Se ee a ee SEE ORE a) americana (Fischer & C.A.Mey.) Small SCBR SCWI myriophylla (Small) Horton SCWI polygama (Vent.) Engelm. & A.Gr. SCWI PONCIRUS Raf. RUTACEAE Citrus Family trifoliata (L.) Raf. Hardy Orange ALTD FLUF MDLA VAMP DCCG GAHC SCCU VAPH DCWH GAIS VACW VATA FLMG MDHN VAGW POTENTILLA POPULUS POPULUS POPULUS L. SALICACEAE alba L. White P. ALTC NCDU VACW MDHN TNFR VASP alba L. var. bolleana (Lauche) Otto = ‘Pyramidalis’ balsamifera L. var. candicans (Ait.) A.Gr. = P, candicans balsamifera L. var. subcordata Hylander = P, candicans bolleana Lauche = ‘Pyramidalis’ <x canadensis Moench Carolina P. [P. deltoides < P. nigra] VACP candicans Ait. Balm-of-Gilead NCCA VAGW x canescens (Ait.) Sm. Gray P. [P. alba < P. tremula] MDPJ VAGW VASP MDWP VARB deltoides Marsh. Cottonwood DCCG GAUG TXWH FLUF MSMN VASC ‘Fastigiata’ [P. simonii Carr.] SCCU x gileadensis Rouleau = P. candicans grandidentata Michx. Large-toothed Aspen VAWP heterophylla L. Swamp Cottonwood SCBR VACW ‘Italica’ [P. nigra L.] Lombardy P. ALBH GAVI TNBO Poplar Willow Family Pa Ee VASP POTENTILLA maximowiczii A.Henry Japanese P. VAGG maximowiczii < P. trichocarpa VATA ‘Pyramidalis’ [P. alba L.] GABS MDNA TNSC GAUG NCWT tremuloides Michx. Quaking Aspen DCNA VASP PORLIERIA Ruiz & Pavon ZYGOPHYLLACEAE Caltrop Family angustifolia (Engelm.) A.Gr. diexas RP: LASL POTENTILLA L. Cinquefoil ROSACEAE Rose Family ‘Abbotswood’ [P. fruticosa L.] VAGS ‘Everest’ [P. fruticosa L.] MDWP fruticosa L. Shrubby C. MDBG MDJS VAGS ‘Grandiflora’ [P. fruticosa L.] MDKN ‘Jackman’s’ [P. fruticosa L. VATA ‘Katherine Dykes’ [P. fruticosa L.] MDKN ‘Maanelys’ [P. fruticosa L.] MDKN VATA ‘Moonlight’ [P. fruticosa L.] = ‘Maanelys’ ‘Primrose Beauty’ [P. fruticosa L.] MDKN ‘Purdomii’ [P. fruticosa L.] = ‘William Purdom’ ‘Sophie Blush’ maximowiczii A.Henry Japanese P. VAGG maximowiczii < P. trichocarpa VATA ‘Pyramidalis’ [P. alba L.] GABS MDNA TNSC GAUG NCWT tremuloides Michx. Quaking Aspen DCNA VASP 155 POTENTILLA tridentata Ait. DCNA ‘William Purdom’ [P. fruticosa L.] DEMC MDKN PRINSEPIA Royle ROSACEAE Rose Family sinensis (Oliv.) Oliv. DEWG PROSOPIS L. FABACEAE (Mimosoideae) Bean Family glandulosa Torr. var. torreyana (L.Benson) M.C.Johnst. Western Honey Mesquite TXGS PRUNUS L. ROSACEAE Rose Family ‘Accolade’ [P. sargentii < P. < subhirtella] DEWG ‘Akebono’ [P. x yedoensis Matsum.] [Parentage unknown] DCNA DCTB DCWH DCWM ‘Alba’ [P. ‘Bakeri’ [P. orientalis (L.) Franco] ALAU ALFN SCMG mume Sieb. & Zucc.] MDBG ‘Alba’ [P. persica (L.) Batsch] NCOP ‘Albo-plena’ [P. glandulosa Thunb.] ALIT NCEG TNDG VAPH ‘Albo-plena’ [P. persica (L.) Batsch] DCNA NCEG VACW LAAP SCLM ‘Amanogawa’ [P. serrulata Lindl.] DCNA americana Marsh. Wild Plum VASK PRUNUS angustifolia Marsh. Chickasaw Plum LATU NCDF apetala (Sieb. & Zucc.) Franch. & Sav. DCNA ‘Ariake’ (Sato zakura group) DCPP armeniaca L. Apricot MDPD NCBE VACW VAJL ‘Asagi’ (Sato zakura group) MDGD ‘Atropurpurea’ [P. cerasifera Ehrh.] Purple-leaf Plum ALBG FLHP ALSH LAMP SCUC VACW ‘Autumnalis’ [P. < subhirtella Lindl.] [Parentage unknown] DCCG GAEC MDLT DCNA MDBG NCEL DCWH MDKN VANB avium L. Sweet Cherry DECE MDHN VACW MDHC MDJH VASP ‘Benden’ (Sato zakura group) MDGD ‘Beni fugen’ = ‘Fugenzo’ < blireiana Andre [P. cerasifera ’Atropurpurea’ < P. mumel] DCDO LATU ‘Botan zakura’ (Sato zakura group) MDKN ‘Camelliifolia’ [P. laurocerasus L.] ALFN ‘Camelliifolia’ [P. persica (L.) Batsch] DCCG campanulata Maxim. Taiwan Cherry ALCW ALEH FLMG GATS glandulosa Torr. var. torreyana (L.Benson) M.C.Johnst. Western Honey Mesquite TXGS PRUNUS L. ROSACEAE Rose Family ‘Accolade’ [P. sargentii < P. < subhirtella] DEWG ‘Akebono’ [P. x yedoensis Matsum.] [Parentage unknown] DCNA DCTB DCWH DCWM ‘Alba’ [P. mume Sieb. & Zucc.] MDBG ‘Alba’ [P. persica (L.) Batsch] NCOP ‘Albo-plena’ [P. glandulosa Thunb.] ALIT NCEG TNDG VAPH ‘Albo-plena’ [P. persica (L.) Batsch] DCNA NCEG VACW LAAP SCLM ‘Amanogawa’ [P. serrulata Lindl.] DCNA americana Marsh. Wild Plum VASK amygdalus Batsch = P. dulcis ol Oo) PRUNUS PRUNUS PRUNUS PRUNUS ‘Forest Green’ [P. laurocerasus L.\|] DCNA = ‘Fudan sakura’ [P. serrulata Lindl.] DCNA ‘Fugenzo’ (Sato zakura group) MDGD MDKN ‘Fuku rokujw’ [P. serrulata Lindl.] MDKN glandulosa Thunb. ex J.A.Murr. Flowering Almond LAHP LASL MSMV SKOCMW) ‘Gozanoma nioi’ [P. serrulata Lindl.] MDKN ‘Grandiflorus’ [P. padus L.] DCNA ‘Gyoiko’ (Sato zakura group) MDGD ‘Hally Jolivette’ [(P. <subhirtella < P. x yedoensis) < P. < subhirtella] DCNA DEWG VAPH DCWH MDKN ‘Hatazakura’ (Sato zakura group) MDGD MDKN x hillieri (listed name with no standing) = ‘Spire’ ‘Hizakura’ (Sato zakura group) MDKN ‘Hosokawa’ [P. serrulata Lindl.] DCTB MDGD humilis Bunge NCEL ‘Imose’ [P. serrulata Lindl.] MDGD incisa Thunb. ex J.A.Murr. DCNA VAPH incisa Thunb. ex J.A.Murr. f. serrata Koidz. ex Wils. MDGD incisa Thunb. ex J.A.Murr. var. tomentosa Koidz. PRUNUS ‘Forest Green’ [P. laurocerasus L.\|] DCNA = ‘Fudan sakura’ [P. serrulata Lindl.] DCNA ‘Fugenzo’ (Sato zakura group) MDGD MDKN ‘Fuku rokujw’ [P. serrulata Lindl.] MDKN glandulosa Thunb. ex J.A.Murr. ‘Bakeri’ [P. orientalis (L.) Franco] ALAU ALFN SCMG Flowering Almond LAHP LASL MSMV SKOCMW) ‘Gozanoma nioi’ [P. serrulata Lindl.] MDKN ‘Grandiflorus’ [P. padus L.] DCNA ‘Gyoiko’ (Sato zakura group) MDGD ‘Hally Jolivette’ [(P. <subhirtella < P. x yedoensis) < P. < subhirtella] DCNA DEWG VAPH DCWH MDKN ‘Hatazakura’ (Sato zakura group) MDGD MDKN x hillieri (listed name with no standing) = ‘Spire’ ‘Hizakura’ (Sato zakura group) MDKN ‘Hosokawa’ [P. serrulata Lindl.] DCTB MDGD humilis Bunge NCEL ‘Imose’ [P. serrulata Lindl.] MDGD incisa Thunb. ex J.A.Murr. DCNA VAPH incisa Thunb. ex J.A.Murr. f. serrata Koidz. ex Wils. MDGD incisa Thunb. ex J.A.Murr. var. tomentosa Koidz. caroliniana Ait. Carolina Cherry Laurel ALAU FLLE MSBN TNDR ALBG FLPM NCDU TNRM ALEH FLUF NCTP TNUT ALIT GAHC SCCU VACW ARRB GARW SCMP VASC DCWH LAHG SCUC VAWR FLCG LASL SCWI cerasifera Ehrh. MDBG MDGD caroliniana Ait. Carolina Cherry Lau ALIT GAHC SCCU VACW ARRB GARW SCMP VASC DCWH LAHG SCUC VAWR FLCG LASL SCWI cerasifera Ehrh. MDBG MDGD cerasoides D.Don var. campanulata (Maxim.) Koidz. = P. campanulata x cistena N.E.Hansen [P. cerasifera ‘Atropurpurea’ < P. pumila] MDBG conradinae Koehne MDGD cyclamina Koehne DCNA ‘Daikakuw’ [P. serrulata Lindl.] DCNA ‘Dianthiflora’ [P. persica (L.) Batsch] FLHP GADG SCHW domestica L. ssp. insititia (L.) Schneid. Damson Plum VACW ‘Dr. S. Edwin Mueller’ [P. sargentii Rehd.] MDKN dulcis (Mill.) D.A.Webb Almond VACW ‘Duplex’ [P. persica (L.) Batsch] NCOP ‘Elizabeth’ [P. <subhirtella Miq.] [Parentage unknown] MDKN ‘Fastigiata’ [P. persica (L.) Batsch] VACW ‘February Pink’ [P. incisa Thunb. ex J.A.Murr.] MDGD ‘Flore-plena’ [P. < subhirtella Miq. var. ascendens (Makino) Wils.] [Parentage unknown] MDGD ‘Flore-plena’ [P. < subhirtella Miq. var. ascendens (Makino) Wils.] [Parentage unknown] MDGD 157 PRUNUS PRUNUS Japonica Thunb. ex J.A.Murr. Oriental Bushcherry ALEH ‘Jugatsu zakura’ [P. x subhirtella Miq.] = ‘Autumnalis’ ‘Kiku shidare’ [P. serrulata Lindl.] MDKN ‘Kursar’ (P. nipponica var. kurilensis (Miyabe) Wils. unknown pollen parent) MDGD ‘Kwanzan’ [P. serrulata Lindl.] Kwanzan Cherry DCCG GACG MDBG TNCT FLMG GARW NCTP laurocerasus L. Cherry Laurel, English Laurel DCCG GAUG NCWC VACM FLMG MDWP SCDB VAHEC GACC NCBM SCFW VAMP GAHC NCOP TNAB laurocerasus L. var. schipkaensis Spaeth Carpathian Cherry Laurel DECG GAUG NCWT VAGS DEEM MDMM TNCT VATA GACG MDWP TNUT VATW lusitanica L. Portuguese Cherry Laurel DCNA GARW mahaleb L. Mahaleb Cherry ALBH maritima Marsh. Beach Plum MDGD VAGW ‘Matsubara Red’ [P. mume Sieb. & Zucc.] MDBG ‘Meigetsu’ (Sato zakura group) MDKN ‘Methley’ [P. maritima Marsh.] ALEH mexicana S.Wats. Mexican Plum FLUF LAHG MSEN TXMA ‘Moseri’ [P. cerasifera Ehrh.] GAFN ‘Mt. Fuji’ [P. serrulata Lindl.] DERE MDLT ‘Multiplex’ [P. triloba Lindl.] Double-flowered Flowering Almond DEWG NCGM VAGW mume Sieb. & Zucc. Japanese Apricot DCNA GAIS MDMW #£NCOP GAHC MDGD NCDU SCDB munsoniana W.F.Wight & Hedr. Wild Goose Plum FLUF NCCA TNBO ‘Nishiki ume’ [P. mume Sieb. & Zucc.] MDLT ‘Ojochin’ (Sato zakura group) MDKN ‘Okami’ [P. <incam Fletcher] [P. incisa x P. campanulata] DCNA ‘Oshima zakura’ [P. serrulata Lindl.] MDGD ‘Otto Luyken’ [P. laurocerasus L.] GACG MDBG MDLT padus L. Bird Cherry GARW ‘Pandora’ [P. < subhirtella < P. x yedoensis] MDGD ‘Peggy Clarke’ [P. mume Sieb. & Zucc.] DCNA ‘Pendula’ [P. x subhirtella Miq.] = ‘Shidare higan’ persica (L.) Batsch Peach DCCG DCNA GAGM VACW persica (L.) Batsch var. nectarina (Ait.) Maxim. = P. persica var. nucipersica ‘Pink Perfection’ [P. serrulata Lindl.] MDGD pS Ol QO PRUNUS PRUNUS ‘Plena’ [P. avium L.] DCNA ‘Purpusii’ [P. cerasifera Ehrh.] MDGD ‘Rancho’ [P. sargentii Rehd.] DCNA rivularis Scheele Creek Plum TXLL ‘Rosea’ [P. serrulata Lindl.] MDGD ‘Rosea’ [P. < subhirtella Miq.] [Parentage unknown] MDGD ‘Rosea Plena’ [P. glandulosa] = ‘Sinensis’ ‘Rosemary Clarke’ [P. mume Sieb. & Zucc.] DCNA ‘Rubro-plena’ [P. persica (L.) Batsch] NCOP sargentii Rehd. Sargent Cherry DCNA MDKN MDLT NCDU ‘Schubert’ [P. virginiana L.] DCNA ‘Sekiyama’ [P. serrulata Lindl.] = ‘Kwanzan’ ‘Senriko’ (Sato zakura group) MDGD MDKN serotina Ehrh. Black Cherry ALON LAAL MSBN TNMB DCNA MDCP TNHG VAPO FLLE MDLT TNHS VASJ serrula Franch. MDKA serrulata Lindl. Japanese Flowering Cherry FLMG MDGD serrulata Lindl. var. spontanea (Maxim.) Wils. DCNA PRUNUS ‘Shidare higan’ [P. PRUNUS < subhirtella Lindl.] Weeping Higan Cherry a [Parentage unknown] DCCG DEMC MDGD MDPJ DCNA MDBG MDKN NCWC DCPP MDDF MDLT ‘Shidare yoshino’ [P. x yedoensis Matsum.] [Parentage unknown] MDGD ‘Shirofugen’ [P. serrulata Lindl.] (Sato zakura group) DCNA MDGD NCOP ‘Shirotae’ [P. serrulata Lindl.] (Sato zakura group) DCMA MDKN NCRW ‘Shogetsuw’ (Sato zakura group) MDGD x sieboldii (Carr.) Wittmack Takasago Cherry [P. apetala < P. serrulata] DCNA ‘Sinensis’ [P. glandulosa Thunb.] ALSM DEWG NCOP VAPH ‘Snow Fountain’ DCNA ‘Somei yoshino’ [P. x yedoensis Matsum.] Yoshino Cherry [Parentage unknown] DCCG MDDF MDLT DCTB MDGD ‘Spire’ [P. sargentii < P. ?incisa] MDGD x subhirtella Miq. Higan Cherry (Alleged hybrid of unknown origin) DCCG DCNA SCCU syodoi Nakai DCNA ‘Tai hakw’ [P. serrulata Lindl.] MDGD ‘Takasago’ = P. sieboldii ‘Taki nioi’ (Sato zakura group) PRUNUS ‘Plena’ [P. avium L.] DCNA ‘Purpusii’ [P. cerasifera Ehrh.] MDGD ‘Rancho’ [P. sargentii Rehd.] DCNA rivularis Scheele Creek Plum TXLL ‘Rosea’ [P. serrulata Lindl.] MDGD ‘Rosea’ [P. < subhirtella Miq.] [Parentage unknown] MDGD ‘Rosea Plena’ [P. glandulosa] = ‘Sinensis’ ‘Rosemary Clarke’ [P. mume Sieb. & Zucc DCNA ‘Rubro-plena’ [P. persica (L.) Batsch] NCOP sargentii Rehd. Sargent Cherry DCNA MDKN MDLT NCDU ‘Schubert’ [P. virginiana L.] DCNA ‘Sekiyama’ [P. serrulata Lindl.] = ‘Kwanzan’ ‘Senriko’ (Sato zakura group) MDGD MDKN serotina Ehrh. Black Cherry ALON LAAL MSBN TNMB DCNA MDCP TNHG VAPO FLLE MDLT TNHS VASJ serrula Franch. MDKA serrulata Lindl. Japanese Flowering Cherry FLMG MDGD serrulata Lindl. var. spontanea (Maxim.) Wils. DCNA ‘Shidare higan’ [P. < subhirtella Lindl.] Weeping Higan Cherry a [Parentage unknown] DCCG DEMC MDGD MDPJ DCNA MDBG MDKN NCWC DCPP MDDF MDLT ‘Shidare yoshino’ [P. x yedoensis Matsum.] [Parentage unknown] MDGD ‘Shirofugen’ [P. serrulata Lindl.] (Sato zakura group) DCNA MDGD NCOP ‘Shirotae’ [P. serrulata Lindl.] (Sato zakura group) DCMA MDKN NCRW ‘Shogetsuw’ (Sato zakura group) MDGD x sieboldii (Carr.) Wittmack Takasago Cherry [P. apetala < P. serrulata] DCNA ‘Sinensis’ [P. glandulosa Thunb.] ALSM DEWG NCOP VAPH ‘Snow Fountain’ DCNA ‘Somei yoshino’ [P. x yedoensis Matsum.] Yoshino Cherry [Parentage unknown] DCCG MDDF MDLT DCTB MDGD ‘Spire’ [P. sargentii < P. ?incisa] MDGD x subhirtella Miq. Higan Cherry (Alleged hybrid of unknown origin) DCCG DCNA SCCU syodoi Nakai DCNA ‘Tai hakw’ [P. serrulata Lindl.] MDGD ‘Takasago’ = P. sieboldii ‘Taki nioi’ (Sato zakura group) 159 PSEUDOTSUGA PRUNUS ‘Thundercloud’ [P. cerasifera Ehrh.] MDBG SCCU VATA tomentosa Thunb. ex J.A.Murr. Nanking Cherry DEMC DEWG MDLT MDOF VACW triloba Lindl. PRUNUS Flowering Almond DEWG umbellata Ell. Black-sloe Plum FLMG GAGM ‘Umineko’ [P. incisa < P. serrulata] MDGD verecunda (Koidz.) Koehne DCNA ‘Versicolor’ [P. persica (L.) Batsch] NCOP ‘Wase mikayo’ [P. serrulata Lindl.] MDKN ‘W. B. Clarke’ [P. mume Sieb. & Zucc.] MDBG ‘White Glory’ [P. persica (L.) Batsch var. nucipersica (Borkh.) Schneid.] Nectarine NCNS ‘Yae akebono’ (Sato zakura group) MDKN ‘Yae murasaki zakura’ (Yato zakura group) MDKN ‘Yae shidare higan’ [P. < subhirtella Lind1 [Parentage unknown] DECE MDGD MDLT MDNH ‘Yama zakura’ [P. serrulata Lindl.] MDGD x yedoensis Matsum. Tokyo Cherry (Alleged hybrid of unknown origin) ALON DCPP GAIH ‘Yoshino’ = ‘Somei yoshino’ PSEUDOCYDONIA (Schneid.) Schneid. ROSACEAE Rose Family a a RS \| sinensis (Dum.-Cours.) Thouin Chinese Quince DCNA LASL NCEL FLDG MDBG VAGG LAAL MDJH VATA PSEUDOLARIX Gord. PINACEAE Pine Family amabilis (J.Nelson) Rehd. Golden Larch DCNA MDPJ VAMP PSEUDOSASA_ Makino ex Nakai POACEAE Grass Family Japonica Sieb. & Zucc. ex Steud. Metake ALON DCMC LALG MDFM PSEUDOTSUGA Carr. Douglas Fir PINACEAE Pine Family ‘Densa’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA ‘Fastigiata’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA ‘Glauca Pendula’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA VAAC menziesii (Mirb.) Franco var. glauca (Beissn.) Franco Rocky Mountain Douglas Fir DECGE GAEC MDBG SCUC DCNA GATN MDNA VAAC DEMC GAUG NCKH VAMP ‘Oudemansii’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA PSEUDOCYDONIA (Schneid.) Schneid. ROSACEAE Rose Family a a RS \| sinensis (Dum.-Cours.) Thouin Chinese Quince DCNA LASL NCEL FLDG MDBG VAGG LAAL MDJH VATA PSEUDOLARIX Gord. PINACEAE Pine Family amabilis (J.Nelson) Rehd. Golden Larch DCNA MDPJ VAMP PSEUDOSASA_ Makino ex Nakai POACEAE Grass Family Japonica Sieb. & Zucc. ex Steud. Metake ALON DCMC LALG MDFM PSEUDOTSUGA Carr. Douglas Fir PINACEAE Pine Family ‘Densa’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA ‘Fastigiata’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA ‘Glauca Pendula’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA VAAC menziesii (Mirb.) Franco var. glauca (Beissn.) Franco Rocky Mountain Douglas Fir DECGE GAEC MDBG SCUC DCNA GATN MDNA VAAC DEMC GAUG NCKH VAMP ‘Oudemansii’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA LAAL MDJH VATA PSEUDOLARIX Gord. PINACEAE Pine Family amabilis (J.Nelson) Rehd. Golden Larch DCNA MDPJ VAMP PSEUDOSASA_ Makino ex Nakai POACEAE Grass Family Japonica Sieb. & Zucc. ex Steud. Metake ALON DCMC LALG MDFM PSEUDOTSUGA Carr. Douglas Fir PINACEAE Pine Family ‘Densa’ [P. PRUNUS menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA ‘Fastigiata’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA ‘Glauca Pendula’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA VAAC menziesii (Mirb.) Franco var. glauca (Beissn.) Franco Rocky Mountain Douglas Fir DECGE GAEC MDBG SCUC DCNA GATN MDNA VAAC DEMC GAUG NCKH VAMP ‘Oudemansii’ [P. menziesii (Mirb.) Franco var. glauca (Beissn.) Franco] DCNA DCNA MDPJ VAMP 160 ‘ ee ~ a PSEUDOLARIX amabilis (J. Nelson) Rehd. [illustrator Lillian Nicholson Meyer] PSEUDOLARIX amabilis (J. Nelson) Rehd. [illustrator Lillian Nicholson Meyer] 161 PYRACANTHA ‘Nana’ [P. granatum L.] Dwarf Pomegranate GAUG LASL SCCU VABF ‘Plena’ [P. granatum L.] GAUG LAMP VAGS PYRACANTHA M.J.Roem. Firethorn ROSACEAE Rose Family : angustifolia (Franch.) Schneid. DCNA GAIS NCWK * ‘Apache’ [P. koidzumii ‘Victory’ < (P. koidzumii ‘Rosedale’ < P. fortuneana ‘Orange Glow’)] DCNA atalantioides (Hance) Stapf DCNA DCSH GAIS ‘Aurea’ [P. atalantioides (Hance) Stapf] VATA ‘Bad Zwischenahn’ DCNA ‘Baker’ DCNA ‘Belli’ [P. koidzumii (Hayata) Rehd.] DCNA ‘Bloss’ DCNA ‘Bound’ [P. coccinea Roem.] DCNA ‘Brilliant’ DCNA ‘Cal Poly’ DCNA ‘Chadwick’ [P. coccinea Roem.] DCNA ‘Chinese Brocade’ DCNA coccinea Roem. PYRACANTHA PYRACANTHA ‘Nana’ [P. granatum L.] Dwarf Pomegranate GAUG LASL SCCU VABF ‘Plena’ [P. granatum L.] GAUG LAMP VAGS PYRACANTHA M.J.Roem. Firethorn ROSACEAE Rose Family : angustifolia (Franch.) Schneid. DCNA GAIS NCWK * ‘Apache’ [P. koidzumii ‘Victory’ < (P. koidzumii ‘Rosedale’ < P. fortuneana ‘Orange Glow’)] DCNA atalantioides (Hance) Stapf DCNA DCSH GAIS ‘Aurea’ [P. atalantioides (Hance) Stapf] VATA ‘Bad Zwischenahn’ DCNA ‘Baker’ DCNA ‘Belli’ [P. koidzumii (Hayata) Rehd.] DCNA ‘Bloss’ DCNA ‘Bound’ [P. coccinea Roem.] DCNA ‘Brilliant’ DCNA ‘Cal Poly’ DCNA ‘Chadwick’ [P. coccinea Roem.] DCNA ‘Chinese Brocade’ DCNA coccinea Roem. SECU VAGW ‘Cole’s Erect’ [P. coccinea Roem.] DCNA PSIDIUM L. Purple Guava MYRTACEAE Myrtle Family littorale Raddi var. longipes (O. Berg) Fosberg Purple Guava FLUF LAAL PSIDIUM L. Purple Guava MYRTACEAE Myrtle Family littorale Raddi var. longipes (O. Berg) Fosberg Purple Guava FLUF LAAL littorale Raddi var. longipes (O. Berg) Fosberg Purple Guava FLUF LAAL PTELEA lL. Hop [ree RUTACEAE Citrus Family trifoliata L. Wafer Ash DCWH NCBE VACW VAGG PTEROCARYA Kunth Wingnut JUGLANDACEAE Walnut Family stenoptera DC. Chinese W. DCNA GANG TNSG VACW GAIS NCDS TXMA PTEROSTYRAX Sieb. & Zucc. Epaulette Tree STYRACACEAE Storax Family corymbosum Sieb. & Zucc. MDGJ hispidum Sieb. & Zucc. DCNA DEWG TNTV VAGS PUNICA L. PUNICACEAE Pomegranate Family granatum L. Pomegranate DCWA FLCG LAMP MSMN SCKG VAGS granatum L. var. PRUNUS nana (L.) Pers. = ‘Nana’ ‘Legrellei’ [P. granatum L.] LAHG SCWC VAFN MSEP VABR ‘Mme. Legrelle’ [P. granatum L.] = ‘Legrellei’ 162 PYRACANTHA PYRACANTHA crenatoserrata (Hance) Rehd. = P. fortuneana crenulata (D.Don) Roem. DCNA GAIS NCBE FLUF MSHB SCMG SCMP crenulata (D.Don) Roem. f. flava Meunissier DCNA crenulata (D.Don) Roem. var. kansuensis Rehd. DCNA crenulata (D.Don) Roem. var. rogersiana A.B.Jacks. DCNA ‘Crimson Tide’ [P. koidzumii (Hayata) Rehd.] DCNA ‘Dauerbrand’ [P. coccinea Roem.] DCNA ‘Dr. Hook’ DCNA ‘Early Red’ DCNA ‘Eddie’s Coral’ [P. coccinea < P. crenulata var. rogersiana] DCNA ‘Flava’ [P. crenulata var. rogersiana] DCNA fortuneana (Maxim.) Li DCNA GAIS FLJU MSHB SCMG SCMP VACW gibbsii Rehd. non A.B.Jacks. var. yunnanensis Osborn = P. fortuneana ‘Gnome’ [P. angustifolia (Franch.) Schneid.] (Plant Patent No. 1,816) DCNA ‘Gnozam’ [P. angustifolia (Franch.) Schneid.] = ‘Gnome’ ‘Gold Nugget’ [P. coccinea Roem.] DCNA ‘Golden Queen’ DCNA ‘Government Red’ [P. koidzumii (Hayata) Rehd.] ALBH ‘Graberi’ [P. fortuneana (Maxim.) Li] DCNA VANB ‘Heyden’s Bright Yellow’ DCNA ‘Heyden’s Hi Yellow’ DCNA ‘Ingleside Crimson’ DCNA ‘Kasan’ [P. coccinea Roem.] DCNA ‘Keessen’ [P. coccinea Roem.] DCNA ‘Knap Hill Buttercup’ DCNA koidzumii (Hayata) Rehd. DCNA FLUF NCOP SCKG FLCG GATS SCBR FLMA NCEL SCFW ‘Lalandei’ [P. coccinea Roem.] DCNA ‘Low Boy’ [P. coccinea Roem.] DCNA GACG ‘Low Dense’ [P. koidzumii (Hayata) Rehd.] DCNA FLUF ‘Miller’ [P. koidzumii (Hayata) Rehd.] DCNA ‘Minute Man’ [P. coccinea Roem.] DCNA ‘Mioun’ DCNA * ‘Mohave’ [P. koidzumti < P. coccinea ‘Wyattii’] DCNA NCDU ‘Monon’ [P. angustifolia (Franch.) Schneid.] = ‘Gnome’ ‘Monrovia’ [P. coccinea Roem.] 163 PYRACANTHA PYRACANTHA PYRACANTHA ‘Moonbeam’ DCNA FLUF VANB * ‘Navaho’ [P. angustifolia < P. ‘Watereri’] DCNA ‘Orange Giant’ [P. coccinea Roem.] DCNA ‘Orange Glow’ [P. fortuneana (Maxim.) Li] DCNA VATA ‘Orange King’ DCNA ‘Pauciflora’ [P. coccinea Roem.] DCNA ‘Pine Cone’ DCNA ‘Pinkie’ DCNA ‘Praecox’ [P. coccinea Roem.] DCNA ‘Pride of Portsmouth’ DCNA * ‘Pueblo’ [P. koidzumii ‘Belli’ < P. coccinea var. pauciflora] DCNA ‘Pyrabox’ DCNA ‘Red Berry’ DCNA rogersiana (A.B.Jacks.) Chittenden = P. crenulata var. rogersiana ‘Rosedale’ [P. koidzumii (Hayata) Rehd.] DCNA ‘Runyan’ [P. coccinea Roem.] DCNA ‘San Jose’ [P. koidzumii < P. fortuneana] DCNA ‘Santa Cruz’ DCNA ‘Schwartz’ PYRACANTHA ‘Sensation’ DCNA ‘Sepers’ [P. coccinea Roem.] DCNA * ‘Shawnee’ [P. koidzumii < P. fortuneana] DCNA ‘Spring Hill’ DCNA ‘Stribling’ DCNA ‘Taliensis’ DCNA * ‘Teton’ [P. ‘Orangeglow’ x P. ‘Flava’] DCNA ‘Thornless’ [P. coccinea Roem.] DCNA ‘Tiny Tim’ DCNA ‘True Yellow’ DCNA ‘Variegated’ GACG LALG LASL ‘Vincent’ [P. coccinea Roem.] DCNA ‘Walder’ DCNA ‘Walder Prostrate’ DCNA ‘Watereri’ DCNA ‘Waterer’s Dwarf’ DCNA ‘Waterer’s Orange’ DCNA ‘Waterer’s Yellow’ DCNA ‘Wayside’s Compact’ [P. coccinea Roem.] 164 PYRACANTHA ‘Wight Early’ DCNA ‘Wonderberry’ DCNA ‘Wyattii’ [P. coccinea Roem.] DCNA ‘Yella Berry’ DCNA ‘Yokohama’ DCNA ‘Yukon Belle’ [P. angustifolia (Franch.) Schneid.] = ‘Gnome’ ‘Yukon Jack’ [P. angustifolia (Franch.) Schneid.] = ‘Gnome’ yunnanensis (M.L.Vilm. ex Mottet) Chittenden = P. fortuneana XPYRACOMELES Rehd. ex Guill. ROSACEAE Rose Family [Osteomeles <X Pyracantha] vilmorinii Rehd. ex Guill. [Pyracantha fortuneana < Osteomeles subrotunda] DCNA FLUF PYRUS L. Pear ROSACEAE Rose Family amygdaliformis Villars MDAB ‘Aristocrat’ [P. calleryana Decne.] DCNA VATA betulifolia Bunge Birchleaf P. ALCW MDAB ‘Bradford’ [P. calleryana Decne.] Bradford P. ALEH DCNA MDAB VACW DCCG GAAB MDLT VATA PYRU calleryana Decne. Callery P. = ALCW ARPT LATU SCEM ALTD LASL MDAB TNUT calleryana Decne. var. dimorphophylla (Makino) Koidz. MDAB xX canescens Spach [P. nivalis < P. salicifolia] MDAB * ‘Capital’ [P. calleryana Decne.] DCNA ‘Chanticleer’ [P. calleryana Decne.] DCNA ‘Chojure’ [P. pyrifolia (Burm.) Nakai] MDKN communis L. Common P. MDAB VACW VAMP VASK elaeagrifolia Pall. MDAB fauriei Schneid. MDAB kawakamii Hayata Evergreen P. DCNA FLMG TXSH x lecontei Rehd. Leconte P. [P. communis < P. pyrifolia] ALSM LAAP SCMG FLDG MDWP SCMP < michauxii Bosc ex Poir. [P. ?amygdaliformis < P. nivalis] MDAB nivalis Jacq. Snow P. MDAB MDGD MDHN pashia Buch.-Ham. ex D.Don MDCH ‘Pendula’ [P. salicifolia Pall.] DCNA MDAB phaeocarpa Rehd. MDAB 165 PYRUS pyrifolia (Burm.f.) Nakai Sand P., Asian P. PYRACANTHA LATU MDCH TXMS MDAB MDGD VATG pyrifolia (Burm.f.) Nakai var. culta (Makino) Nakai Nashi P. MDAB MDPJ regelii Rehd. MDAB salicifolia Pall. MDAB VAPH ussuriensis Maxim. DCCI MDAB ussuriensis Maxim. var. hondoensis (Kikuchi & Nakai) Rehd. IDXCLE) MDAB ussuriensis Maxim. var. ovoidea Rehd. MDAB * ‘Whitehouse’ [P. calleryana Decne.] DCNA VATA QUERCUS L. Oak FAGACEAE Beech Family acutissima Carruthers Oriental Sawtooth O. ARUM GAFN MDTH TXMS DCAE GAIS NCBE VABP DCNA LAAL NCGR FLUF MDGD TNSG GACG MDLA TXHG alba L. White O. ALAU DEMC MDWM ARUM GAUG SCBR DCCG MDHN SCMP alba < Q. virginiana SCRH arkansana Sarg. Arkansas Oak ARCA x asheana Little [Q. incana x Q. laevis] FLUF austrina Small FLUF QUERCUS QUERCUS PYRUS PYRUS PYRUS pyrifolia (Burm.f.) Nakai Sand P., Asian P. LATU MDCH TXMS MDAB MDGD VATG pyrifolia (Burm.f.) Nakai var. culta (Makino) Nakai Nashi P. MDAB MDPJ regelii Rehd. MDAB salicifolia Pall. MDAB VAPH ussuriensis Maxim. DCCI MDAB ussuriensis Maxim. var. hondoensis (Kikuchi & Nakai) Rehd. IDXCLE) MDAB ussuriensis Maxim. var. ovoidea Rehd. MDAB * ‘Whitehouse’ [P. calleryana Decne.] DCNA VATA QUERCUS L. Oak FAGACEAE Beech Family acutissima Carruthers Oriental Sawtooth O. ARUM GAFN MDTH TXMS DCAE GAIS NCBE VABP DCNA LAAL NCGR FLUF MDGD TNSG GACG MDLA TXHG alba L. White O. ALAU DEMC MDWM ARUM GAUG SCBR DCCG MDHN SCMP alba < Q. virginiana SCRH arkansana Sarg. Arkansas Oak ARCA x asheana Little [Q. incana x Q. laevis] FLUF bicolor Willd. Swamp White O. DCCG SCMG < bimundorum Palm. [Q. alba x Q. robur] DCCH borealis Michx.f. = Q. rubra catesbaei Michx. = Q. laevis cerris L. Turkey O. NCTP TNWF cheniti Nakai DCNA coccinea Muenchh. Scarlet O. DCNG DEMC MDAF MDHN ‘Columna’ [Q. robur ‘Fastigiata’ < Q. petraea ‘Muscaviensis’] DCNA GAUG <comptonae Sarg. Compton O. [Q. lyrata < Q. virginiana] DCHP LASL VACW LAMB VACG ‘Cristata’ [Q. robur L.] DCNA ‘Cucullata’ [Q. robur L.] DCNA dentata Thunb. ex J.A.Murr. Daimyo O. DCNA GAIS GAUG LAAL fabri Hance DCNA falcata Michx. Southern Red O., Spanish O. ALBG GAHC SCBR falcata Michx. var. pagodifolia Ell. SCMP ‘Falkenbergensis’ [Q. petraea (Mattusch.) Liebl.] DCNA farnetto Tenore = Q. frainetto 166 QUERCUS frainetto Tenore Italian Oak DCNA ‘Geisleri’ [Q. petraea (Mattusch.) Liebl.] DCNA georgiana M.A.Curtis Georgia O. GAWD gilva Bl. LAAL glandulifera Bl. Konara Oak DCNA glauca Thunb. ex J.A.Murray Ring-cupped O. ALAU GACD NCOP TXHG ALON GAHC SCBR VANB ALTD GAIS SCKG VAWR DCNA GAJI SCRP FLMG LAAL TNMB FLUF MSMN TNUT haas Kotschy DCNA hemisphaerica Bartram ex Willd. Darlington O. DCNA GAWM SCSG SCWP x heterophylla Michx.f. Bartram O. [Q. phellos < Q. rubra] DCCG imbricaria Michx. Shingle O. DCNA MDBG incana Bartram Bluejack O. FLUF laevis Walt. Turkey O. FLUF NCGP laurifolia Michx. Laurel O. MDKB SCBR NCAG SCKG ALBG FLUF NCTP SCSG QUERCUS QUERCUS macrocarpa Michx. Bur O. 2S DCCG DCNA GARC DCMS GACD NCWM margaretta Ashe Sand Post O. GAGR maxima Marsh. = Q. rubra michauxii Nutt. Basket O. DCCG FLMG SCBR minima (Sarg.) Small SCWI mongolica Fischer var. grosseserrata (Bl.) Rehd. & Wils. DCNA ‘Muscaviensis’ [Q. petraea (Mattusch.) Liebl.] DCNA myrsinifolia Bl. Japanese Evergreen O. PYRUS ALON FLMG LAAL ALTD FLUF MSHB DCNA GAUG NCEL nigra L. Water O. ALBG PEUE SCBR DCHP GAJS SCDH FLLE NCTE VAMP nuttallii Palm. Nuttall O. LAAP pagoda Raf. = Q. falcata var. pagodifolia palustris Muenchh. Pin O. DCCG DCNA SCBR petraea (Mattusch.) Liebl. Durmast Oak DCNA phellos L. Willow O. DCCG SCBR SCWP VAGG NCDU SCMG VACM VALC macrocarpa Michx. Bur O. 2S DCCG DCNA GARC DCMS GACD NCWM margaretta Ashe Sand Post O. GAGR maxima Marsh. = Q. rubra michauxii Nutt. Basket O. DCCG FLMG SCBR minima (Sarg.) Small SCWI margaretta Ashe Sand Post O. GAGR maxima Marsh. = Q. rubra michauxii Nutt. Basket O. DCCG FLMG SCBR minima (Sarg.) Small SCWI mongolica Fischer var. grosseserrata (Bl.) Rehd. & Wils. DCNA ‘Muscaviensis’ [Q. petraea (Mattusch.) Liebl.] DCNA myrsinifolia Bl. Japanese Evergreen O. ALON FLMG LAAL ALTD FLUF MSHB DCNA GAUG NCEL nigra L. Water O. ALBG PEUE SCBR DCHP GAJS SCDH FLLE NCTE VAMP nuttallii Palm. Nuttall O. LAAP pagoda Raf. = Q. falcata var. pagodifolia palustris Muenchh. Pin O. DCCG DCNA SCBR petraea (Mattusch.) Liebl. Durmast Oak DCNA phellos L. Willow O. DCCG SCBR SCWP VAGG NCDU SCMG VACM VALC phillyraeoides A.Gr. Ubame O. DCNA GAIS SCCU TXPS 167 QUERCUS prinus L. Chestnut O. DCCG MDHN VAGF pubescens Willd. DCCG DCLC robur L. English O. DCCG DCNA MDCP NCTP robur L. f. fastigiata (Lam.) O.Schwarz Columnar English O. DCNA MDAB VAMP GAUG TNSN < rosacea Bechst. [Q. petraea < Q. robur] DCNA rubra L. Red O. DCCG DEMC GAUG MDHN ‘Salicifolia’ [Q. robur L.] DCNA xX sargentii Rehd. Sargent O. [Q. prinus < Q. robur] IDXC}sle serrata Thunb. ex J.A.Murr. = Q. glandulifera shumardii Buckl. Shumard O. ALAU FLUF SCMP stellata Wang. Post O. DCCG GAJS stellata Wang. var. margaretta (Ashe) Sarg. = Q. margaretta suber L. Cork O. ALUA NCTE SCRP variabilis Bl. Chinese Cork O. DCDO DCNA FLGS MDDF velutina Lam. Black O. MDHN SCBR RHAPHIOLEPIS RHAPHIOLEPIS virginiana Mill. Southern Live O. ALBB GAAB SCKG VARO ALCM LAAP SCMG VASC ALEC NCAG SCMP VATA ALTD SCBP VACM VAWR FLCG SCBR VAGW FLUF SCDH VANB RAPHIOLEPIS Lindl. = RHAPHIOLEPIS REHSONIA Stritch = WISTERIA Buckthorn Buckthorn Family alaternus L. Italian B. SCCU carolinianus L. Carolina B. GAUG MSMN TNSC catharticus L. Common B. DEEM MDGJ TNRG MDBP TNLW davuricus Pall. MDGJ TNLW VACW TNDR TNUT frangula L. = Frangula alnus japonicus Maxim. DCNA utilis Decne. PYRUS LAAL MDBP RHAPHIOLEPIS Lindl. ROSACEAE Rose Family ‘Enchantress’ [R. indica (L.) Lindl.] serrata Thunb. ex J.A.Murr. = Q. glandulifera shumardii Buckl. Shumard O. ALAU FLUF SCMP stellata Wang. Post O. DCCG GAJS stellata Wang. var. margaretta (Ashe) Sarg. = Q. margaretta suber L. Cork O. ALUA NCTE SCRP variabilis Bl. Chinese Cork O. DCDO DCNA FLGS MDDF velutina Lam. Black O. MDHN SCBR serrata Thunb. ex J.A.Murr. = Q. glandulifera shumardii Buckl. Shumard O. ALAU FLUF SCMP stellata Wang. Post O. DCCG GAJS stellata Wang. var. margaretta (Ashe) Sarg. = Q. margaretta suber L. Cork O. ALUA NCTE SCRP variabilis Bl. Chinese Cork O. DCDO DCNA FLGS MDDF velutina Lam. Black O. MDHN SCBR RHODODENDRON RHAPHIOLEPIS RHAPHIOLEPIS indica (L.) Lindl. Indian Hawthorn ALTD GAUG SCBA SCPR FLMG GAWH SCBH SCRH FLRH MSHB SCCU GACL NCEG SCFW GATS NCOP SCMP indica var. integerrima (Hook.) Kitam. = R. umbellata var. integerrima ‘Majestic Beauty’ TXSN ‘Pink Cloud’ TXSN ‘Snow’ [R. indica (L.) Lindl.] TXSE ‘Springtime’ TXSN umbellata (Thunb. ex J.A.Murr.) Makino Yeddo Hawthorn ALBG GAIS GACH NCEG VATA umbellata (Thunb. ex J.A.Murr.) Makino var. integerrima (Hook. & Arn.) Rehd. DCNA LAHG SCLM FLUF NCAG VANB GATS SCBR VATA RHAPIDOPHYLLUM H.Wendl. & Drude ARECACEAE Palm Family Se ESET He) hystrix (Pursh) H.Wendl. & Drude Needle Palm ALAU FLUF LAMP SCWI FLDG GAHC NCOP TXSE RHAPIS L.f. ex Ait. ARECACEAE Palm Family excelsa (Thunb. ex J.A.Murr.) A.Henry FLUF RHODODENDRON L. ERICACEAE Heath Family aan RNR RET PTE PERRIS eo ET] (The plants recognized by landscapers and horticulturists as “rhododendrons’” fall into two main categories: lepidote (scaly) and elepidote (nonscaly). Botanically, the lepidotes are found in subgenus Rhododendron and the elepidotes indica (L.) Lindl. Indian Hawthorn ALTD GAUG SCBA SCPR FLMG GAWH SCBH SCRH FLRH MSHB SCCU GACL NCEG SCFW GATS NCOP SCMP in subgenus Hymenanthes. The leaves of the lepidotes have minute surface scales and are generally smaller than those of the elepidotes. The lepidotes include, among others, species native to the southeastern United States (for example, R. minus), to subarctic regions (for example, R. lapponicum), and to the Alps and Pyrenees (for example, R. ferrugineum). Many of the subarctic and alpine species and their hybrids do not thrive in the summer heat of the southeastern United States. in subgenus Hymenanthes. The leaves of the lepidotes have minute surface scales and are generally smaller than those of the elepidotes. PYRUS The lepidotes include, among others, species native to the southeastern United States (for example, R. minus), to subarctic regions (for example, R. lapponicum), and to the Alps and Pyrenees (for example, R. ferrugineum). Many of the subarctic and alpine species and their hybrids do not thrive in the summer heat of the southeastern United States. The elepidotes (R. maximum and R. catawbiense, for example) are species generally recognized by the layperson as rhododendrons. Some of the elepidotes have leaves that are glabrous (without hairs), while others have leaves with indumentum (matted branched hairs) on the lower surface. Azaleas are also members of the genus Rhododendron. Approximately 15 deciduous species are native in the eastern United States, and a number of these have been crossed with Japanese, Chinese, and east- European species to develop showy hybrids (for example, the Ghent, Knap Hill, Exbury, and Ilam hybrids). The evergreen azaleas are of east-Asian origin. Their degree of leaf retention may vary with severity of the climate.) ‘A. Bedford’ (Elepidote rhododenron) VAZC ‘Addy Wery’ (Evergreen azalea) DEMC alabamense Rehd. Alabama Azalea (Deciduous azalea) DCNA FLMG DEWG GACG MDLT NCBE SCBR ‘Albiflorum’ [R. mucronulatum Turcz.] (Lepidote rhododendron) DCNA ‘Album’ [R. carolinianum Rehd.] (Lepidote rhododendron) DEWG MDLT ‘Album Elegans’ [R. catawbiense hybrid] (Elepidote rhododendron) DCWH amagianum Makino Mt. Amagi Azalea (Deciduous azalea) MDMG ‘Amethystinum’ (Evergreen azalea; Mucronatum group) DEMC ‘A. Bedford’ (Elepidote rhododenron) VAZC ‘Addy Wery’ (Evergreen azalea) DEMC alabamense Rehd. Alabama Azalea (Deciduous azalea) DCNA FLMG DEWG GACG MDLT NCBE SCBR ‘Albiflorum’ [R. mucronulatum Turcz.] (Lepidote rhododendron) DCNA ‘Album’ [R. carolinianum Rehd.] (Lepidote rhododendron) DEWG MDLT ‘Album Elegans’ [R. catawbiense hybrid] (Elepidote rhododendron) DCWH amagianum Makino Mt. Amagi Azalea (Deciduous azalea) MDMG ‘Amethystinum’ (Evergreen azalea; Mucronatum group) DEMC excelsa (Thunb. ex J.A.Murr.) A.Henry FLUF (The plants recognized by landscapers and horticulturists as “rhododendrons’” fall into two main categories: lepidote (scaly) and elepidote (nonscaly). Botanically, the lepidotes are found in subgenus Rhododendron and the elepidotes 169 RHODODENDRON RHODODENDRON ‘Amoenum’ (Evergreen azalea; Obtusum group) DEWG NCBE ‘Anah Kruschke’ [R. ponticum seedling] (Elepidote rhododendron) VATA ‘Annie E. Endtz’ (Elepidote rhododendron) VANB ‘April Rose’ (Evergreen azalea) SCMG arborescens (Pursh) Torr. Sweet Azalea (Deciduous azalea) DEMC DEWG NCBE TNEFN atlanticum (Ashe) Rehd. Coastal Azalea (Deciduous azalea) DCNA MDLT SCBR DEWG NCBE augustinii Hemsl. (Lepidote rhododendron) MDMG VANB austrinum (Small) Rehd. Florida Azalea (Deciduous azalea) ALEH FLAL MDLT IDAIL, ALFS FLMG NCBE VAGS ARHS GACG SCBR VATW DCNA GATS SCMG DEWG MDKN SCWI ‘Autumn Glory’ (Elepidote rhododendron) VAZC ‘Azma’ (Elepidote rhododendron) VANB ‘Azor’ (Elepidote rhododendron) VANB bakeri (W.P.Lemmon & McKay) Hume Cumberland Azalea (Deciduous azalea) DEWG MDLT TNAB RHODODENDRON RHODODENDRON ‘Amoenum’ (Evergreen azalea; Obtusum group) DEWG NCBE ‘Anah Kruschke’ [R. ponticum seedling] (Elepidote rhododendron) VATA ‘Annie E. Endtz’ (Elepidote rhododendron) VANB ‘April Rose’ (Evergreen azalea) SCMG arborescens (Pursh) Torr. Sweet Azalea (Deciduous azalea) DEMC DEWG NCBE TNEFN atlanticum (Ashe) Rehd. Coastal Azalea (Deciduous azalea) DCNA MDLT SCBR DEWG NCBE augustinii Hemsl. (Lepidote rhododendron) MDMG VANB austrinum (Small) Rehd. Florida Azalea (Deciduous azalea) ALEH FLAL MDLT IDAIL, ALFS FLMG NCBE VAGS ARHS GACG SCBR VATW DCNA GATS SCMG DEWG MDKN SCWI ‘Autumn Glory’ (Elepidote rhododendron) VAZC ‘Azma’ (Elepidote rhododendron) VANB ‘Azor’ (Elepidote rhododendron) VANB bakeri (W.P.Lemmon & McKay) Hume Cumberland Azalea (Deciduous azalea) DEWG MDLT TNAB GAEC MDMG ‘Ballerina’ (Deciduous azalea) DEMC ‘Beauty of Littleworth’ (Elepidote rhododendron) DCNA ‘Beni kirishima’ (Evergreen azalea) MDMG ‘Ben Morrison’ (Evergreen azalea) MDMG ‘Betty Wormald’ (Elepidote rhododendron) DCNA ‘Bibiani’ (Elepidote rhododendron) ALEH ‘Blaauw’s Pink’ (Evergreen azalea) DCWH ‘Blue Tit’ (Lepidote rhododendron) DCNA ‘Bosley 1020’ (Elepidote rhododendron) VATA * ‘Bowie’ [R. chapmanii < R. minus] (Lepidote rhododendron) DCNA brachycarpum D.Don ex G.Don Fujiama R. (Elepidote rhododendron) VANB ‘Brandywine’ (Lepidote rhododendron) DCNA ‘Brick-dust Red’ (Evergreen azalea) SCMG ‘Cadis’ (Elepidote rhododendron) DEMC calendulaceum (Michx.) Torr. Flame Azalea (Deciduous azalea) DEWG MDMG VADF VAZC FLMG NCBE VAGW GAEC SCBR VASP 170 RHODODENDRON RHODODENDRON RHODODENDRON * ‘Camp’s Red’ [R. bakeri (W.P.Lemmon & McKay) Hume] (Deciduous azalea) DCNA MDKN canescens (Michx.) Sweet Piedmont Azalea (Deciduous azalea) DEWG GASM NCBE SCMG FLMG LAAL NCOP SCWI FLUF LASL SCBR TNDG GACG MDLT SCFW ADL, ‘Carmen’ (Evergreen azalea) DEMC ‘Carminata Splendens’ (Evergreen azalea; Amoenum hybrid) DCNA carolinianum Rehd. Carolina R. (Lepidote rhododendron) DCWH MDKN VANB DEWG MDLA VATW carolinianum X< R. mucronulatum (Lepidote rhododendron) DCNA carolinianum X< R. racemosum (Lepidote rhododendron) DCNA catawbiense Michx. RHODODENDRON Mountain Rosebay (Elepidote rhododendron) DCCG MDOM VAHB VAPO ‘Cerise’ (Evergreen azalea) SCMG chapmanii A.Cr. Chapman R. (Lepidote rhododendron) DCNA FLUF NCBE IDOE, FLMG GAEC SCWI VANB ‘Cherry Red’ (Elepidote rhododendron) MDLT ‘Christmas Cheer’ (Evergreen azalea) ALEH ARPT FLMG ‘Christopher Wren’ (Deciduous azalea) DEMC MDBG RHODODENDRON * ‘Camp’s Red’ [R. bakeri (W.P.Lemmon & McKay) Hume] (Deciduous azalea) DCNA MDKN canescens (Michx.) Sweet Piedmont Azalea (Deciduous azalea) DEWG GASM NCBE SCMG FLMG LAAL NCOP SCWI FLUF LASL SCBR TNDG GACG MDLT SCFW ADL, ‘Carmen’ (Evergreen azalea) DEMC ‘Carminata Splendens’ (Evergreen azalea; Amoenum hybrid) DCNA carolinianum Rehd. Carolina R. (Lepidote rhododendron) DCWH MDKN VANB DEWG MDLA VATW carolinianum X< R. mucronulatum (Lepidote rhododendron) DCNA carolinianum X< R. racemosum (Lepidote rhododendron) DCNA catawbiense Michx. Mountain Rosebay (Elepidote rhododendron) DCCG MDOM VAHB VAPO ‘Cerise’ (Evergreen azalea) SCMG chapmanii A.Cr. Chapman R. (Lepidote rhododendron) DCNA FLUF NCBE IDOE, FLMG GAEC SCWI VANB ‘Cherry Red’ (Elepidote rhododendron) MDLT ‘Christmas Cheer’ (Evergreen azalea) RHODODENDRON ‘Coccinea Major’ (Evergreen azalea) FLMG SCMG i ‘Coccinea Speciosa’ (Deciduous azalea) DEMC ‘Comte de Newport’ (Evergreen azalea) SCMG ‘Conemaugh’ [R. racemosum R. mucronulatum] (Lepidote rhododendron) DEWG ‘Conestoga’ [R. carolinianum x R. racemosum\| (Lepidote rhododendron) DCNA DEWG ‘Conewago’ [R. carolinianum x R. mucronulatum] (Lepidote rhododendron) DEWG ‘Coral Bells’ (Evergreen azalea) ALEH ARPT ‘Coral Cluster’ (Evergreen azalea) DCNA ‘Corneille’ (Deciduous azalea) MDLT ‘Cornell Pink’ [R. mucronulatum Turcz.] (Lepidote rhododendron) DCNA DEWG MDBG ‘Crater Lake’ [R. augustinii Hemsl.] (Lepidote rhododendron) MDBG cumberlandense E.Braun = R. bakeri ‘Cunningham’s White’ [R. caucasicum R. ponticum var. album] (Elepidote rhododendron) VANB ‘Cynthia’ [R. catawbiense R. griffithianum] (Elepidote rhododendron) VANB dauricum L. (Lepidote rhododendron) DCNA 171 RHODODENDRON RHODODENDRON RHODODENDRON RHODODENDRON ‘David Gable’ [R. catawbiense ‘Atrosanguineum’ X R. fortunei] (Elepidote rhododendron) DEWG MDLT decorum Franch. (Elepidote rhododendron) MDFM VANB degronianum Carr. ssp. heptamerum (Maxim.) Hara (Elepidote rhododendron) DCNA MDKN MDMG VANB DEWG MDLT SCWI ‘Dexter Purple’ (Elepidote rhododendron) MDLT ‘Dexter’s Champagne’ (Elepidote rhododendron) MDLT discolor Franch. = R. fortunei subsp. discolor ‘Dora Amateis’ [R. carolinianum R. ciliatum] (Lepidote rhododendron) DEMC GAEC SCWI ‘Dorsett’ [R. kaempferi Planch.] (Evergreen azalea) DCNA ‘Duc de Rohan’ (Evergreen azalea) FLMG SCMG ‘Early Lavender’ (Evergreen azalea) SCMG ‘Early Salmon’ (Evergreen azalea) SCMG ‘Evening Glow’ (Elepidote rhododendron) MDLT ‘Everestianum’ [R. catawbiense hybrid] (Elepidote rhododendron) VANB fargesii Franch. = R. oreodoxa var. fargesii ‘Fedora’ (Evergreen azalea) DEMC ‘Flamingo’ (Evergreen azalea) VATA flammeum (Michx.) Sarg. Oconee Azalea (Deciduous azalea) DCNA FLMG MDLT DEWG GACG NCBE flavum (Hoffmgg.) G.Don = R. RHODODENDRON luteum ‘Flowerdale Pink’ (Evergreen azalea) SCMG ‘Flowerdale Red’ (Evergreen azalea) SCMG ‘Formosa’ (Evergreen azalea) ALBG FLUF SCMG ‘Forsterianum’ [R. veitchianum << R. edgeworthii] (Lepidote rhododendron) DCNA fortunei Lindl. (Elepidote rhododendron) DCWH NCBE VATW MDMG VANB fortunei Lindl. ssp. discolor (Franch.) Chamb. (Elepidote rhododendron) MDKN MDMG VATW ‘George Lindley Taber’ (Evergreen azalea; sport of ‘Phoeniceum’) FLGS FLMG MDLG ‘Georgiana Maclay’ (Evergreen azalea) FLMG ‘Giant Elegans’ (Evergreen azalea) SCMG ‘Giant White’ (Evergreen azalea) ‘Fedora’ (Evergreen azalea) DEMC ‘Flamingo’ (Evergreen azalea) VATA flammeum (Michx.) Sarg. Oconee Azalea (Deciduous azalea) DCNA FLMG MDLT DEWG GACG NCBE flavum (Hoffmgg.) G.Don = R. luteum ‘Flowerdale Pink’ (Evergreen azalea) SCMG ‘Flowerdale Red’ (Evergreen azalea) SCMG ‘Formosa’ (Evergreen azalea) ALBG FLUF SCMG ‘Forsterianum’ [R. veitchianum << R. edgeworthii] (Lepidote rhododendron) DCNA fortunei Lindl. (Elepidote rhododendron) DCWH NCBE VATW MDMG VANB fortunei Lindl. ssp. discolor (Franch.) Chamb. (Elepidote rhododendron) MDKN MDMG VATW ‘George Lindley Taber’ (Evergreen azalea; sport of ‘Phoeniceum’) FLGS FLMG MDLG ‘Georgiana Maclay’ (Evergreen azalea) FLMG ‘Giant Elegans’ (Evergreen azalea) SCMG ‘Giant White’ (Evergreen azalea) SCMG degronianum Carr. ssp. heptamerum (Maxim.) Hara (Elepidote rhododendron) DCNA MDKN MDMG VANB DEWG MDLT SCWI ‘Everestianum’ [R. catawbiense hybrid] (Elepidote rhododendron) VANB fargesii Franch. = R. oreodoxa var. fargesii ‘Fawley’ (Deciduous azalea) DEMC 172 RHODODENDRON RHODODENDRON indicum (L.) Sweet var. tamurae Makino = R. tamurae =~ ‘Iveryana’ (Evergreen azalea) FLMG SCMG japonicum (A.Gr.) Suring. DCNA ‘Jean Marie de Montague’ = ‘The Hon. Jean Marie de Montague’ ‘John Walter’ [R. catawbiense < R. arboreum ssp. arboreum] (Elepidote rhododendron) VANB kaempferi Planch. Kaempfer Azalea (Evergreen azalea) DEWG MDGD kaempferi x R. komiyamae (Evergreen azalea; natural hybrid) MDGD kanahirae Wibls. Taibei Azalea (Evergreen azalea) MDGD keiskei Miq. (Lepidote rhododendron) DCNA DEMC keiskei x R. racemosum (Lepidote rhododendron) DEWG MDKN ‘Ken Janeck’ [R. yakushimanum Nakai ssp. yakushimanum] (Elepidote rhododendron) MDLT kiusianum Makino Kyushu Azalea (Evergreen azalea) DEWG MDGD VANB ‘Koran yuki’ (Evergreen azalea) DCNA ‘Koromo shikibu’ (Evergreen azalea) Glenn Dale Azaleas This series of 454 named Glenn Dale Azaleas This series of 454 named cultivars was created at the U.S. Plant Introduction Garden, Glenn Dale, MD, by B.Y. Morrison, who became the first director of the U.S. National Arboretum. The project began around 1929, with the objective of creating azaleas with flowers as large as those of the Southern Indian hybrids, but hardy in the Washington, DC, area. Many of the selections met this criterion, and others equally hardy were selected for different ornamental values (see B.Y. Morrison. 1953. The Glenn Dale azaleas. U.S. Department of Agriculture, Agriculture Mono- graph No. 20, Washington, DC.) A list of the Glenn Dale azalea cultivars documented in the U.S. National Arboretum herbarium appears in Appendix C. ‘Glory of Sunninghill’ (Evergreen azalea) FLMG ‘Glory of Sunninghill’ (Evergreen azalea) FLMG ‘Gomer Waterer’ [R. catawbiense hybrid] (Elepidote rhododendron) SCWI ‘Gumpo’ [R. eriocarpum (Hayata) Nakail] (Evergreen azalea) LAHG ‘Helen’ (Evergreen azalea) VATA ‘Hinode giri’ (often ‘Hinodegiri’) (Evergreen azalea) * ‘Hohman’ [R. prunifolium (Small) Millais] (Deciduous azalea) DCNA houlstonii Hemsl. & Wils. = R. fortunei subsp. discolor ‘Hugh Koster’ (Elepidote rhododendron) VANB ‘Ima shojo’ = ‘Christmas Cheer’ indicum (L.) Sweet Indica Azalea (Evergreen azalea) DCNA LAAL LAHG TNSG ‘Gumpo’ [R. eriocarpum (Hayata) Nakail] (Evergreen azalea) LAHG ‘Helen’ (Evergreen azalea) VATA * ‘Hohman’ [R. prunifolium (Small) Millais] (Deciduous azalea) DCNA houlstonii Hemsl. & Wils. = R. fortunei subsp. discolor ‘Hugh Koster’ (Elepidote rhododendron) VANB ‘Ima shojo’ = ‘Christmas Cheer’ indicum (L.) Sweet Indica Azalea (Evergreen azalea) DCNA LAAL LAHG TNSG WS RHODODENDRON ‘La Roche’ (Evergreen azalea) TNCT ‘Lady Mulberry’ (Evergreen azalea) SCMG lasiostylum Hayata (Evergreen azalea) MDGD ‘Late Orchid’ (Evergreen azalea) SCMG ‘Lawsal’ (Evergreen azalea) SCMG ledifolium (Hook.) G.Don = ‘Mucronatum’ ‘Linearifolium’ [R. macrosepalum Maxim.] Spider Azalea (Evergreen azalea) MDGD ‘Lizette’ (Evergreen azalea) DCNA ‘Lucinda’ (Evergreen azalea) DCNA luteum Sweet Pontic Azalea (Deciduous azalea) (The early synonymy of this plant is confused. It is possible that R. flavum may have priority over R. luteum under the rules governing botanical nomenclature. The International Registrar for Rhododendron, however, lists the subject plant as R. luteum, and that name is almost univer- sally used in horticulture and the nursery trade.) DCNA DEWG MDGD MDKN ‘Luteum’ [R. carolinianum Rehd.] (Lepidote rhododendron) DCNA macranthum (Bunge) G.Don = R. indicum macrosepalum Maxim. RHODODENDRON RHODODENDRON ‘Magnolia Alba’ (Evergreen azalea) SCMG makinoi Tagg = R. yakushimanum ssp. makinoi ‘Maria Derby’ (Evergreen azalea) MDMG ‘Mary Fleming’ [(R. keiskei < R. racemosum) X R. keiskei] (Lepidote rhododendron) MDLT maximum L. Rosebay (Elepidote rhododendron) DCNA MDET MDLT NCDR MDBG MDHN MDMG £TNHD MDDF MDJH NCBE VASP maximum L. (Elepidote rhododendron; red-flowered form) VATW metternichii Sieb. & Zucc. = R. degronianum ssp. heptamerum micranthum Turcz. (Lepidote rhododendron) MDLT SCWI VATW minus Michx. Piedmont R. (Lepidote rhododendron) DCSH MDCH SCWI DEWG NCBE TNAB GAEC SCBR VAPO ‘Mrs. Betty Robertson’ (Elepidote rhododendron) VANB ‘Mrs. Charles S. Sargent’ [R. catawbiense hybrid] (Elepidote rhododendron) VANB ‘Mrs. E. C. Stirling’ [R. griffithianum hybrid] (Elepidote rhododendron) VANB ‘Mrs. G. G. Gerbing’ (Evergreen azalea) FLMG ‘Mrs. LBJ’ (Evergreen azalea) MDGD RHODODENDRON ‘Magnolia Alba’ (Evergreen azalea) SCMG makinoi Tagg = R. yakushimanum ssp. makinoi ‘Maria Derby’ (Evergreen azalea) MDMG ‘Mary Fleming’ [(R. keiskei < R. racemosum) X R. RHODODENDRON RHODODENDRON ‘Obtusum Album’ (Evergreen azalea; Obtusum group) DCHU a3 occidentale (Torr. & A.Gr.) A. Gr. Western Azalea (Deciduous azalea) DCNA oldhamii Maxim. Oldham Azalea (Evergreen azalea) DCNA MDGD GADG SCWI VAZC oreodoxa Franch. var. fargesii (Franch.) Chamb. (Elepidote rhododendron) DEWG ‘Oritani’ (Lepidote rhododendron) MDLT ovatum (Lindl.) Planch. ex Maxim. (Azaleastrum) DCNA GACG ‘Palestrina’ (Evergreen azalea) DEMC periclymenoides (Michx.) Shinners Pinxterbloom (Deciduous azalea) DCNA MDLT VAAC VAGW DEWG NCBE VADW VATW phoeniceum (Sweet) G.Don = ‘Phoeniceum’ ‘Phoeniceum’ [R. scabrum selection or hybrid] (Evergreen azalea; Phoeniceum group) ARGD MDGD SCMG ‘Pink Diamond’ [R. griffithianum x R. fortunei ssp. fortunei] (Elepidote rhododendron; Loderi group) VANB ‘Pioneer’ (Lepidote rhododendron; Gable hybrid) DCNA MDBG MDLT ‘P. J. M.’ [R. carolinianum x R. dauricum] (Lepidote rhododendron; ‘P.J.M.’ originally ‘Obtusum Album’ (Evergreen azalea; Obtusum group) DCHU a3 occidentale (Torr. & A.Gr.) A. Gr. Western Azalea (Deciduous azalea) DCNA oldhamii Maxim. Oldham Azalea (Evergreen azalea) DCNA MDGD GADG SCWI VAZC oreodoxa Franch. var. fargesii (Franch.) Chamb. (Elepidote rhododendron) DEWG ‘Oritani’ (Lepidote rhododendron) MDLT ovatum (Lindl.) Planch. ex Maxim. (Azaleastrum) DCNA GACG ‘Palestrina’ (Evergreen azalea) DEMC periclymenoides (Michx.) Shinners Pinxterbloom (Deciduous azalea) DCNA MDLT VAAC VAGW DEWG NCBE VADW VATW phoeniceum (Sweet) G.Don = ‘Phoeniceum’ ‘Phoeniceum’ [R. scabrum selection or hybrid] (Evergreen azalea; Phoeniceum group) ARGD MDGD SCMG ‘Pink Diamond’ [R. griffithianum x R. fortunei ssp. fortunei] (Elepidote rhododendron; Loderi group) VANB ‘Pioneer’ (Lepidote rhododendron; Gable hybrid) DCNA MDBG MDLT ‘P. J. M.’ [R. carolinianum x R. dauricum] (Lepidote rhododendron; ‘P.J.M.’ originally applied to various seedlings from the cross; other cultivar names are being given to clonal selec- tions, e.g. ‘P.J. Mezitt’) ‘Mrs. R. S. Holford’ (Elepidote rhododendron) VATA (Elepidote rhododendron) VATA mucronatum (Bl.) G.Don = Mucronatum group ‘Mucronatum’ Snow Azalea (Evergreen azalea; Mucronatum group) (Although Chamberlain and Rae (1990) retain R. mucronatum (Bl.) G. Don var. mucronatum as a botanical taxon, they note that the plant is “only known in cultivation.” They also note that var. mucronatum “may occur in the wild as the albino form of [R. mucronatum\| var. ripense.” Because of the amibiguity surrounding its origin, the Snow Azalea is accorded cultivar status here as ‘Mucronatum’, and its closely related variants are identified as being in the “Mucronatum group.”) ARPT DEWG MDGD DCDO LASL MSHB mucronulatum Turcz. (Lepidote rhododendron) DEMC DEWG MDBG MDLT ‘Myrtifolium’ [R. minus < R. hirsutum] (Lepidote rhododendron; this cultivar name sometimes leads to confusion with the quite different R. RHODODENDRON myrtifolium) MDLT MDMG ‘Nadine’ (Evergreen azalea) DCNA ‘Narcissiflora’ (Deciduous azalea) MDLT ‘Nodding Bells’ (Elepidote rhododendron) VADF nudiflorum (L.) Torr. = R. periclymenoides oblongifolium (Small) Millais Texas Azalea (Deciduous azalea) MDLT NCBE SCWI obtusum (Lindl.) Planch. = Obtusum group ‘Obtusum’ Kirishima Azalea (Evergreen azalea; Obtusum group) FLMG mucronatum (Bl.) G.Don = Mucronatum group ‘Glory of Sunninghill’ (Evergreen azalea) FLMG keiskei] (Lepidote rhododendron) MDLT maximum L. Rosebay (Elepidote rhododendron) DCNA MDET MDLT NCDR MDBG MDHN MDMG £TNHD MDDF MDJH NCBE VASP maximum L. (Elepidote rhododendron; red-flowered form) VATW metternichii Sieb. & Zucc. = R. degronianum ssp. heptamerum micranthum Turcz. (Lepidote rhododendron) MDLT SCWI VATW minus Michx. Piedmont R. (Lepidote rhododendron) DCSH MDCH SCWI DEWG NCBE TNAB GAEC SCBR VAPO ‘Mrs. Betty Robertson’ (Elepidote rhododendron) VANB ‘Mrs. Charles S. Sargent’ [R. catawbiense hybrid] (Elepidote rhododendron) VANB ‘Mrs. E. C. Stirling’ [R. griffithianum hybrid] (Elepidote rhododendron) VANB ‘Mrs. G. G. Gerbing’ (Evergreen azalea) FLMG ‘Mrs. LBJ’ (Evergreen azalea) RHODODENDRON ‘Magnolia Alba’ (Evergreen azalea) SCMG makinoi Tagg = R. yakushimanum ssp. makinoi ‘Maria Derby’ (Evergreen azalea) MDMG ‘Mary Fleming’ [(R. keiskei < R. racemosum) X R. keiskei] (Lepidote rhododendron) MDLT maximum L. Rosebay (Elepidote rhododendron) DCNA MDET MDLT NCDR MDBG MDHN MDMG £TNHD MDDF MDJH NCBE VASP maximum L. (Elepidote rhododendron; red-flowered form) VATW metternichii Sieb. & Zucc. = R. degronianum ssp. heptamerum micranthum Turcz. (Lepidote rhododendron) MDLT SCWI VATW minus Michx. Piedmont R. (Lepidote rhododendron) DCSH MDCH SCWI DEWG NCBE TNAB GAEC SCBR VAPO ‘Mrs. Betty Robertson’ (Elepidote rhododendron) VANB ‘Mrs. Charles S. Sargent’ [R. catawbiense hybrid] (Elepidote rhododendron) VANB ‘Mrs. E. C. Stirling’ [R. griffithianum hybrid] (Elepidote rhododendron) VANB ‘Mrs. G. G. Gerbing’ ‘Lawsal’ (Evergreen azalea) SCMG ledifolium (Hook.) G.Don = ‘Mucronatum’ ‘Linearifolium’ [R. macrosepalum Maxim.] Spider Azalea (Evergreen azalea) MDGD ‘Lizette’ (Evergreen azalea) DCNA ‘Lucinda’ (Evergreen azalea) DCNA luteum Sweet Pontic Azalea (Deciduous azalea) (The early synonymy of this plant is confused. It is possible that R. flavum may have priority over R. luteum under the rules governing botanical nomenclature. The International Registrar for Rhododendron, however, lists the subject plant as R. luteum, and that name is almost univer- sally used in horticulture and the nursery trade.) DCNA DEWG MDGD MDKN ‘Luteum’ [R. carolinianum Rehd.] (Lepidote rhododendron) DCNA macranthum (Bunge) G.Don = R. indicum macrosepalum Maxim. Large-sepal Azalea (Evergreen azalea) VAPH ‘Madame de Bruin’ (Elepidote rhododendron) VANB 174 Snow Azalea (Evergreen aza 175 175 RHODODENDRON RHODODENDRON RHODODENDRON RHODODEND ‘Ramapo’ [R. fastigiatum < R. carolinianum] (Lepidote rhododendron) NCAS ‘Red Flare’ [R. keiskei Miq.] (Lepidote rhododendron) MDLT ‘Red Formosa’ (Evergreen azalea) FLMG ‘Red Head’ (Elepidote rhododendron) VANB reticulatum D.Don ex G.Don Rose Azalea (Deciduous azalea) DCNA MDGD ‘Robert Allison’ (Elepidote rhododendron) DEWG roseum (Loisel.) Rehd. = R. prinophyllum ‘Roseum Elegans’ [R. catawbiense hybrid] (Elepidote rhododendron) DCCG DCWH VAMP ‘Roseum Magnificum’ (Evergreen azalea) SCMG ‘Royal Red’ (Evergreen azalea) FLMG ‘Royal Splendor’ (Evergreen azalea) SCMG rubropilosum Hayata (Evergreen azalea) MDGD MSBA ‘Salmon King’ (Evergreen azalea) SCMG schlippenbachii Maxim. Royal Azalea (Deciduous azalea) DCNA DEWG MDPB DEMC MDBG TNDG RHODODENDRON ‘Ramapo’ [R. fastigiatum < R. carolinianum] (Lepidote rhododendron) NCAS ‘Red Flare’ [R. keiskei Miq.] (Lepidote rhododendron) MDLT ‘Red Formosa’ (Evergreen azalea) FLMG ‘Red Head’ (Elepidote rhododendron) VANB reticulatum D.Don ex G.Don Rose Azalea (Deciduous azalea) DCNA MDGD ‘Robert Allison’ (Elepidote rhododendron) DEWG roseum (Loisel.) Rehd. = R. prinophyllum ‘Roseum Elegans’ [R. catawbiense hybrid] (Elepidote rhododendron) DCCG DCWH VAMP ‘Roseum Magnificum’ (Evergreen azalea) SCMG ‘Royal Red’ (Evergreen azalea) FLMG ‘Royal Splendor’ (Evergreen azalea) SCMG rubropilosum Hayata (Evergreen azalea) MDGD MSBA ‘Salmon King’ (Evergreen azalea) SCMG schlippenbachii Maxim. Royal Azalea ‘P. J. Mezitt’ [R. carolinianum x R. dauricum] (Lepidote rhododendron; P.J.M. group) GACG MDLT ‘Plenum’ (Evergreen azalea; Mucronatum group) MDMG ‘Ponticum Roseum’ [R. ponticum x R. maximum] (Elepidote rhododendron) VATA (Elepidote rhododendron) VATA ‘Ponticum Variegatum’ [R. ponticu sport] (Elepidote rhododendron) DCNA poulhanense Lev. = R. yedoense var. poukhanense ‘President Claeys’ (Evergreen azalea) SCMG ‘Pride of Dorking’ (Evergreen azalea) FLMG ‘Pride of Mobile’ (Evergreen azalea) ALCW SCMG ‘Pride of Summerville’ = ‘Lawsal’ prinophyllum (Small) Millais Roseshell Azalea (Deciduous azalea) DEWG MDTD SCBR MDLT NCBE prunifolium (Small) Millais Plumleaf Azalea (Deciduous azalea) DCNA MDLT NCBE punctatum Andr. (Lepidote rhododendron) SCBR ‘Purple Splendor’ (Evergreen azalea) DCNA ‘Queen of Orange’ (Evergreen azalea) SCMG racemosum X< R. keiskei (Lepidote rhododendron) DCNA 176 ‘Ponticum Variegatum’ [R. ponticum sport] (Elepidote rhododendron) DCNA 176 RHODODENDRON schlippenbachii Maxim. Royal Azalea (Deciduous azalea; white-flowered form) DCNA DEWG MDBG MDKN serpyllifolium (A.Gr.) Miq. Wild-thyme Azalea (Evergreen azalea) MDJW serrulatum (Small) Millais Hammock Sweet Azalea (Deciduous azalea) LASL NCBE ‘Shrimp Pink’ (Lepidote rhododendron) MDBG simiarum Hance (Elepidote rhododendron) DCNA MDGD simsii Planch. Sims Azalea (Evergreen azalea) VANB simsii Planch. var. tamurae (Makino) Kaneh. & Hatusima = R. tamurae ‘Small Elegans’ (Evergreen azalea) SCMG smirnowii Traut. (Elepidote rhododendron) DEWG MDKN MDLT speciosum (Willd.) Sweet = R. RHODODENDRON flammeum ‘Spring Glory’ (Elepidote rhododendron) DCNA ‘Stewartstonian’ (Evergreen azalea) DEMC ‘Suetsuma’ (Evergreen azalea) DCNA ‘Sunset’ (Evergreen azalea) DCNA ‘Susugonoito’ (Evergreen azalea) RHODODENDRON 177 177 RHODODENDRON RHODODENDRON RIBES RIBES ‘William Bull’ (Evergreen azalea) FLMG williamsianum Rehd. & Wils. (Elepidote rhododendron) DCNA ‘Windbeam’ (Lepidote rhododendron) DEMC GAEC MDLT ‘Winterthur’ (Evergreen azalea; Mucronatum group) DEWG ‘Wissahickon’ (Elepidote rhododendron) MDLT ‘Wyanokie’ (Lepidote rhododendron) DCNA ‘Yae shojo’ (Evergreen azalea) DCNA yakushimanum Nakai ssp. makinoi (Tagg) Chamb. (Elepidote rhododendron) VAZC yakushimanum Nakai ssp. yakushimanum (Elepidote rhododendron) DEWG MDBG MDLT yedoense Maxim. ex Regel var. poukhanense (Lev.) Nakai Korean Azalea (Evergreen azalea) DEWG GAEC NCBE yedoense Maxim. ex Regel var. poukhanense (Lev.) Nakai (Evergreen azalea; white-flowered form) DCNA RHODOLEIA Champ. ex Hook. HAMAMELIDACEAE Witch-hazel Family a NR championii Hook. FLUF ‘William Bull’ (Evergreen azalea) FLMG ‘William Bull’ (Evergreen azalea) FLMG RHODOTYPOS Sieb. & Zucc. Jetbead ROSACEAE Rose Family ES RS SS aa OE RE SET scandens (Thunb.) Makino DEMC MDGD NCBE MDBG MDJH NCCA VACW VAGG RHUS UL. Sumac ANACARDIACEAE Cashew Family EE Sea eS SEE OE Ee Re EERE CE aromatica Ait. Fragrant S. DCNA DEMC VACW chinensis Mill. VABF copallina L. Shining S., Winged S. ALBG SCCU VAPO glabra L. Smooth S. DEMC ‘Laciniata’ [R. typhina L.] MDBG MDLT TNSC michauxii Sarg. GAWD punjabensis J.L.Stewart var. sinica (Diels) Rehd. & Wils. DCNA radicans L. = Toxicodendron radicans typhina L. Staghorn S. DCNA VACW verniciflua Stokes = Toxicodendron vernicifluum RIBES L. Currant, Gooseberry SAXIFRAGACEAE (Ribesioideae) Saxifrage Family alpinum L. Mountain C. MDWP curvatum Small RHODOTYPOS Sieb. & Zucc. Jetbead ROSACEAE Rose Family ES RS SS aa OE RE SET scandens (Thunb.) Makino DEMC MDGD NCBE MDBG MDJH NCCA VACW VAGG RHUS UL. Sumac ANACARDIACEAE Cashew Family EE Sea eS SEE OE Ee Re EERE CE aromatica Ait. Fragrant S. DCNA DEMC VACW chinensis Mill. VABF copallina L. Shining S., Winged S. ALBG SCCU VAPO glabra L. Smooth S. DEMC ‘Laciniata’ [R. typhina L.] MDBG MDLT TNSC michauxii Sarg. GAWD punjabensis J.L.Stewart var. sinica (Diels) Rehd. & Wils. DCNA radicans L. = Toxicodendron radicans typhina L. Staghorn S. DCNA VACW verniciflua Stokes = Toxicodendron vernicifluum RIBES L. Currant, Gooseberry SAXIFRAGACEAE (Ribesioideae) Saxifrage Family alpinum L. Mountain C. MDWP RHODOLEIA Champ. ex Hook. HAMAMELIDACEAE Witch-hazel Family ROSA RIBES pseudoacacia L. var. rectissima Raber Ship-mast Locust ~ DCNA ‘Purple Rose’ [R. pseudoacacia L.] DCNA ‘Pyramidalis’ [R. pseudoacacia L.] MDGJ VAJL ‘Rectissima’ [R. pseudoacacia L.] = R. pseudoacacia var. rectissima ‘Sandraudiga’ [R. pseudoacacia L.] DCNA ‘Tortuosa’ [R. pseudoacacia L.] DCNA ‘Unifoliola’ [R. pseudoacacia L.] DCNA viscosa Vent. Clammy L. NCCA ROSA L. RHODODENDRON Rose ROSACEAE Rose Family CE TS a SE ID OP, TRE PTET RI SE SS (The list includes cultivars introduced mainly before 1900; dates indicate year of introduction to gardens.) acicularis Lindl. Prickly R. VABF ‘Agathe Incarnata’ (Damask; 1866) DCNA NCCB ‘Aglaia’ [R. multiflora < R. ‘Reve d’Or’] (Yellow Rambler; 1896) VACW <x alba L. White Rose-of-York [R. ?canina < R. xdamascena] (Alba; before 1597) NCCB VAGW ‘Alba’ [R. rugosa Thunb. ex J.A.Murr.] DCNA NCCB VANB VAPH ‘Alba Plena’ [R. banksiae Ait.f. var. banksiae] echinellum (Cov.) Rehd. Florida G. MDJW SCWI grossularia L. = R. uva-crispa odoratum H.Wendl. Clove C. ARWS MDBG TNSM GAOH MDGL ‘Pixwell’ [R. uva-crispa L.] MDFM ‘Pulborough Scarlet’ [R. sanguineum Pursh] MDBG uva-crispa L. English G. MDFM ROBINIA L. Locust FABACEAE (Faboideae) © Bean Family ER Ee a EE a x ambigua Poir. [R. pseudoacacia < R. viscosa] DCNA ‘Bicolor’ [R. pseudoacacia L.] DCNA boyntonii Ashe NCCA ‘Burgundy’ [R. pseudoacacia L.] DCNA ‘Decaisneana’ [R. x ambigua Poir.] [R. pseudoacacia < R. viscosa] DCNA ‘Fastigiata’ [R. pseudoacacia L.] = ‘Pyramidalis’ ‘Frisia’ [R. pseudoacacia L.] DCNA hispida L. Rose Acacia, Bristly Locust NCCA VACW VAGC pseudoacacia L. Black L. DCNA GAJA MDHN SCMP DCWR MDBP MDLT TNFR FLWJ MDCP MSBN TXHA GAES MDGJ SCFW VACW echinellum (Cov.) Rehd. Florida G. MDJW SCWI grossularia L. = R. uva-crispa odoratum H.Wendl. Clove C. ARWS MDBG TNSM GAOH MDGL ‘Pixwell’ [R. uva-crispa L.] MDFM ‘Pulborough Scarlet’ [R. sanguineum Pursh] MDBG uva-crispa L. English G. MDFM ROBINIA L. Locust FABACEAE (Faboideae) © Bean Family ER Ee a EE a x ambigua Poir. [R. pseudoacacia < R. viscosa] DCNA ‘Bicolor’ [R. pseudoacacia L.] DCNA boyntonii Ashe NCCA ‘Burgundy’ [R. pseudoacacia L.] DCNA ‘Decaisneana’ [R. x ambigua Poir.] [R. pseudoacacia < R. viscosa] DCNA ‘Fastigiata’ [R. pseudoacacia L.] = ‘Pyramidalis’ ‘Frisia’ [R. pseudoacacia L.] DCNA hispida L. Rose Acacia, Bristly Locust NCCA VACW VAGC pseudoacacia L. Black L. DCNA GAJA MDHN SCMP (The list includes cultivars introduced mainly before 1900; dates indicate year of introduction to gardens.) acicularis Lindl. Prickly R. VABF ‘Agathe Incarnata’ (Damask; 1866) DCNA NCCB ‘Aglaia’ [R. multiflora < R. ‘Reve d’Or’] (Yellow Rambler; 1896) VACW <x alba L. White Rose-of-York [R. ?canina < R. xdamascena] (Alba; before 1597) NCCB VAGW ‘Alba’ [R. rugosa Thunb. ex J.A.Murr.] DCNA NCCB VANB VAPH ‘Alba Plena’ [R. banksiae Ait.f. var. banksiae] Banks R. (double white) (1807) GATS SCMG SCNS LAGN SCMP VAMW 79 ROSA ROSA ‘Baltimore Belle’ [R. setigera < R. gallica hybrid] (1843) DCNA banksiae Ait. (See cultivars ‘Alba Plena’, ‘Lutea’, and ‘Lutescens’) ‘Baronne Prevost’ (Hybrid Perpetual; 1842) NCCB ‘Beauty of Glazenwood’ = ‘Fortune’s Double Yellow’ ‘Belinda’ (Hybrid Musk; 1936) NCCB ‘Belle Amour’ (Alba; before 1950) NCCB ‘Belle de Crecy’ (Gallica; before 1848) DCNA NCCB 180 ‘Baltimore Belle’ [R. setigera < R. gallica hybrid] (1843) DCNA banksiae Ait. (See cultivars ‘Alba Plena’, ‘Lutea’, and ‘Lutescens’) ‘Baronne Prevost’ (Hybrid Perpetual; 1842) NCCB ‘Beauty of Glazenwood’ = ‘Fortune’s Double Yellow’ ‘Belinda’ (Hybrid Musk; 1936) NCCB ‘Belle Amour’ (Alba; before 1950) NCCB ‘Belle de Crecy’ (Gallica; before 1848) DCNA NCCB 180 180 ROSA ‘Cardinal de Richelieu’ (Gallica; 1840) DCNA ‘Carnea’ [R. multiflora Thunb.] (1804) NCCB carolina L. Pasture R. (1826) TNKS ‘Catherine Mermet’ (Tea; 1869) DCNA ‘Celeste’ [R. xalba L.] [R. ?canina < R. x damascena] (Alba; before 1759) DCNA NCCB ‘Celestial’ [R. <x alba L.] = ‘Celeste’ ‘Celsiana’ (Damask; before 1750) NCCB centifolia L. Cabbage R., Provence R. (1596) DCNA NCCB VAGW ‘Champney’s Pink Cluster’ Champney R. (Noisette; 1811) DCNA ‘Charles de Mills’ (Gallica) NCCB chinensis Jacq. China R. (1759) MSHB VAGW chinensis Jacq. f. mutabilis (Corr.) = ‘Mutabilis’ ‘Commandant Beaurepaire’ (Bourbon; 1874) NCCB NCMM ‘Comte de Chambord’ (Portland; 1860) DCNA NCCB ‘Conditorum’ [R. gallica L.] Tidbit R., Hungarian R. DESC ROSA ‘Conrad Ferdinand Meyer’ [R. rugosa hybrid X ‘Gloire de Dijon’]__ (Hybrid Rugosa; 1899) NCCB ‘Cramoisi Superieur’ (China; 1832) NCMM VACW ‘Crested Jewel’ (Moss; 1971) NCCB ‘Cristata’ [R. centifolia L.] Crested Moss R. (Crested Moss; 1827) NCCA NCMM x damascena Mill. Damask R. [R. gallica x R. moschata] (16th century; before Miller's use of the name R. Xdamascena, J. Herrmann applied the epithet damascena, apparently to a different rose. Miller's name thus may be a later hom- onym and therefore illegitimate, but there is no generally accepted replacement.) DCNA LASL NCCB VAGW <x damascena Mill. var. semperflorens (Loisel.) Rowley Four Seasons R., Monthly R. [R. gallica x R. moschata] (Ancient) DCNA GASH NCCB ‘De la Grifferaie’ (Shrub; 1845) NCCB ‘Delicata’ (Hybrid Rugosa; 1898) NCCB ‘Duc de Fitzjames’ (Gallica) NCCB ‘Duc de Guiche’ (Gallica; before 1829) NCCB ‘Duchesse de Brabant’ (Tea; 1857) DCNA SCHW ‘Duchesse de Montebello’ (Gallica; before 1829) DCNA NCCB ROSA ROSA ROSA 181 ROSA ‘Frau Dagmar Hartopp’ (Hybrid Rugosa) NCCB ‘Frau Dagmar Hastrup’ = ‘Frau Dagmar Hartopp’ ‘Frau Karl Druschki’ (Hybrid Perpetual; 1906) NCCB ‘Fun Jwan Lo’ [R. < odorata (Andr.) Sweet] [R. ROSA chinensis < R. gigantea] SCMG ‘Gardenia’ [R. wichuraiana X ‘Perle des Jardins’] (Rambler; 1899) NCCB ‘General Jacqueminot’ (Hybrid Perpetual; 1853) DCNA ‘General Kleber’ (Moss; 1856) NCCB glauca Pourr. (Before 1830) DCNA ‘Gloire de Dijon’ [? Tea X ‘Souvenir de la Malmaison’\| (1853) DCNA ‘Gloire des Mosseux’ (Moss; 1853) DCNA GASH NCCB ‘Gold of Ophir’ = ‘Fortune’s Double Yellow’ ‘Gracilis’ [R. < lheritieranea Thory] Boursault R. [R. chinensis < R. ?pendulina] NCCB ‘Hanosa’ [R. rugosa Thunb.] DCDO < harisonii Rivers Yellow Rose-of-Texas [R. foetida < R. pimpinellifolia] (ca. 1830) DCNA MDFM MDHN VABF NCMM VAGW NCCB ‘Felicite et Perpetue’ [R. sempervirens hybrid] (1827) NCCB ‘Felicite Parmentier’ (Alba; 1834) NCMM ‘i —) ee) NS) ROSA ROSA ‘Hermosa’ (China; 1840) DCNA NCMM ‘Honorine de Brabant’ (Bourbon) DCNA NCCB hugonis Hemsl. = R. xanthina f. hugonis ‘Ipsilante’ (Gallica; 1821) NCCB ‘Isabella Sprunt’ (Tea; 1865) DCNA ‘Jacques Cartier’ (Portland; 1868) GASH NCCB ‘Jeanne d’Arc’ (Alba; 1818) NCCB ‘Jeanne de Montfort’ (Moss; 1851 or 1854) MDHN ‘Juno’ (Centifolia; before 1832) DCNA ‘Koenigin von Daenemark’ [R. ?<alba < Damask Hybrid] (Alba; 1826) DCNA MDJS NCMM laevigata Michx. Cherokee R. (1759) FLMG SCBR SCMP GAIS SCCU SCWI GAJI SCMG VACW ‘La France’ (Hybrid Tea; 1867) DCNA ‘Leda’ (Damask; before 1827) DCNA NCCB ‘Louis Philippe’ (China; 1834) FLMS ‘Louise Odier’ (Bourbon; 1851) DCNA NCCB ROSA ‘Lutea’ [R. banksiae Ait.f. var. banksiae] Banks R. (double yellow), LadyBanks' R. (1824) DCNA LAGN SCHW SCWI GACG MSMN SCMG TXMK GATS NCOP SCUC ‘Lutescens’ [R. banksiae Ait.f. var. normalis Regel] Banks R. (single yellow) (ca. 1870) FLCG ‘Mabel Morrison’ (Hybrid Perpetual; 1878) NCCB ‘Macrantha’ [Hort., not Desp.] DCNA ‘Macrantha’ [R. < waitziana Tratt.] (Early 18th Century) [R. canina < R. gallica] NCCB ‘Maiden’s Blush’ [R. <x alba x R. centifolia] (1797) DCNA GASH MDHN NCMM ‘Marcel Bourgouin’ (Gallica; 1899) NCCB ‘Marie Louise’ (Damask; 1813) NCMM ‘May Queen’ [R. wichuraiana < ‘Cham- pion of the World’] (Rambler; 1898) NCCB ‘Minima’ [R. chinensis Jacq.] Fairy Rose (1815) DCNA “Mme. Alfred Carriere’ (Noisette; 1879) DCNA ‘Mme. Hardy’ (Damask; 1832) ROSA ROSA ROSA ‘Nuits de Young’ (Moss; 1845) NCCB <x odorata (Andr.) Sweet Meauke [R. chinensis < R. gigantea] (Cult. 1752) NCCB ‘Oeillet de Saint Arquey’ = ‘Serratipetala’ ‘Oeillet Panachee’ Striped Moss (Moss; 1888) DCNA NCMM ‘Officinalis’ Apothecary R. (Gallica; 13th Century) DCNA NCCB ‘Old Blush’ [R. x odorata (Andr.) Sweet] Monthly R. [R. chinensis < R. gigantea] (1752) DCNA NCCB VAGW ‘Omar Khayyam’ (Damask; 1893) NCCB palustris Marsh. Swamp R. (1726) FLUF SCBR < paulii Rehd. [R. arvensis < R. rugosa] (Before 1903) DEMC ‘Paul Neyron’ (Hybrid Perpetual; 1869) DCNA NCCB ‘Perle d’Or’ (Polyantha; 1884) NCCB ‘Perseana’ [R. foetida J.Herrm.] Persian Yellow R. (1837) DCNA VAGW ‘Petite de Hollande’ (Centifolia; before 1802) ‘Mme. Legras de Saint Germain’ (Hybrid Alba; 1846) NCCB ‘Mme. Louis Leveque’ (Moss; 1898) NCMM ‘Mme. Pierre Oger’ (Bourbon; 1878) NCMM ‘Mme. Plantier’ (Hybrid Alba; 1835) DCNA NCCB moschata J.Herrm. Musk R. (1540) DCNA moyesii Hemsl. & Wils. (1894) DCNA multibracteata Hemsl. & Wils. (1910) DCNA multiflora Thunb. ex J.A.Murr. Japanese R., Baby R. (Before 1868) ALBH NCCA VACW DEMC NCWC VAHI MSHB SCBR VASK ‘Muscosa’ [R. centifolia L.] Moss R. (Before 1750) DCNA NCCB VAGW mutabilis Corr. = ‘Mutabilis’ ‘Mutabilis’ [R. chinensis Jacq.] (Cult. 1932 or earlier) GAUG LASL NCCB ‘Niphetos’ (Tea; 1889) DCNA xX noisettiana Thory Noisette R. [R. chinensis < R. moschata] (ca. 1812) NCCB VABR VAGW 184 ROSA ROSA ROSA ROSA pimpinellifolia L. Scotch R. (Before 1600) VAGW pimpinellifolia L. var. altaica (Willd.) Thory (Ca. 1820) DCDO ‘Platyphylla’ [R. multiflora Thunb. ex J.A.Murr.] Seven Sisters R. (1817) NCCB ‘Plena’ [R. moschata J.Herrm.] (Before 1596) DCNA ‘Pompon Elegant’ (Hybrid China) NCCB ‘President de Seze’ (Gallica; 1836) NCCB ‘Prince Camille de Rohan’ (Hybrid Perpetual; 1861) DCNA ‘Ramona’ [R. X anemonoides Rehd.] Red Cherokee R. [R. laevigata < R. ?odorata] (1913) SCLS VACW ‘Rampant’ [R. sempervirens L.] (1830) NCCB ‘Reine des Violettes’ (Hybrid Perpetual; 1860) DCNA NCCB ‘Reine Victoria’ (Bourbon; 1872) DCNA MDJS ‘Reve d’Or’ (Noisette; 1869) FLCG ‘Rivers’ George IV’ (Hybrid China; 1820) ROSA ‘Rose des Peintres’ [R. centifolia L.] (Centifolia) = DCNA NCMM roxburghii Tratt. f. normalis Rehd. & Wils. Chestnut R., Bur R. (Flowers single; 1908) DCNA GASH MDHN MSMN ‘Roxburghii’ [R. roxburghii Tratt.] (Flowers double; before 1814) GAJS MDHN NCCB VAWP LARP MSMV SCBR rubiginosa L. (Before 1551) DCNA MDHN NCCB GASH MDJS VAGW rubrifolia Villars = R. glauca rugosa Thunb. ex J.A.Murr. Rugosa R. (Ca. ROSA 1845) DCNA VACW VANB ‘Safrano’ (Tea; 1839) DCNA ‘Salet’ (Moss; 1854) DCNA ‘Schneezwerg’ (Hybrid Rugosa) MDJS ‘Semiplena’ [R. <x alba L.] (Alba) [R. canina < R. xdamascena] DCNA ‘Serratipetala’ (China; 1912) NCCB setigera Michx. Prairie R. (1810) DCNA SCBR VABF NCCB SCMP ‘Shailer’s Provence’ (Centifolia) NCCB ‘Rose des Peintres’ [R. centifolia L.] (Centifolia) = 185 ROSA ROSA ROSA spinosissima L. = R. pimpinellifolia ‘Stanwell Perpetual’ [R. xdamascena var. semperflorens < R. pimpinellifolia] (Hybrid Pimpinellifolia; 1838) DCNA MDHN ‘Suzanne’ [R. laxa < R. pimpinellifolia] (Shrub; 1950) MDJS NCCB ‘The Fairy’ (Polyantha; 1932) VANB ‘Tour de Malakoff (Centifolia; 1856) NCCB ‘Trigintipetala’ [R. x damascena Mill.] Kazanlik Rose [R. gallica x R. moschata] (Before 1850) DCNA ‘Tuscany’ (Gallica; 1596) DCNA ‘Tuscany Superb’ (Gallica) DCNA NCCB ‘Variegata’ [R. centifolia L.] (1845) NCMM ‘Variegata di Bologna’ (Bourbon; 1909) NCMM ‘Versicolor’ [R. x damascena Mill.] York and Lancaster R. [R. gallica x R. moschata] (Before 1700) DCNA NCCB ‘Versicolor’ [R. gallica L.] (Before 1581) DCNA MDFM VAGW DESC NCCB virginiana Mill. Virginia R. (Before 1807) wichuraiana Crepin Memorial R. (1891) NCWC TNBO VAWE wichuraiana Crepin var. poteriifolia Koidz. DCNA woodsii Lindl. (1820) VABF xanthina Lindl. f. hugonis (Hemsl.) Roberts Father Hugo R. (1899) DCDO MDJS NCBE DEWG MDLA wichuraiana Crepin Memorial R. (1891) NCWC TNBO VAWE wichuraiana Crepin var. poteriifolia Koidz. DCNA woodsii Lindl. (1820) VABF xanthina Lindl. f. hugonis (Hemsl.) Roberts Father Hugo R. (1899) DCDO MDJS NCBE DEWG MDLA xanthina Lindl. f. hugonis (Hemsl.) Roberts Father Hugo R. (1899) DCDO MDJS NCBE DEWG MDLA NCCB ‘Trigintipetala’ [R. x damascena Mill.] Kazanlik Rose [R. gallica x R. moschata] (Before 1850) DCNA ‘Tuscany’ (Gallica; 1596) DCNA ‘Tuscany Superb’ (Gallica) DCNA NCCB ‘Variegata’ [R. centifolia L.] (1845) NCMM ‘Variegata di Bologna’ (Bourbon; 1909) NCMM ‘Versicolor’ [R. x damascena Mill.] York and Lancaster R. [R. gallica x R. moschata] (Before 1700) DCNA NCCB ‘Versicolor’ [R. gallica L.] (Before 1581) DCNA MDFM VAGW DESC NCCB virginiana Mill. Virginia R. (Before 1807) DCNA NCCB VACW VAPO MDRL NCDU VAGS ‘Viridiflora’ [R. chinensis Jacq.] Green R. (1855) LASL ROSMARINUS L. LAMIACEAE Mint Family officinalis L. Rosemary FLUF SCCL VANB LAHG VACW VATA ‘Prostratus’ [R. officinalis L.] GAJI RUBUS L. ROSACEAE Rose Family calycinoides Hayata & Koidz. DCNA VAGS VAPH cockburnianus Hemsl. MDBG ‘Coronarius’ [R. rosifolius Sm.] Blackberry Rose GAIH MDFM SCWI odoratus L. Thimbleberry MDJH RUSCUS L. LILIACEAE Lily Family CE I SD SE SE RS aculeatus L. Butcher’s Broom DCNA LAAL NCEL SCBR GACC LASL NCTP TXHN GAUG MSEN NCWM ROSMARINUS L. LAMIACEAE Mint Family officinalis L. Rosemary FLUF SCCL VANB LAHG VACW VATA ‘Prostratus’ [R. officinalis L.] GAJI RUBUS L. ROSACEAE Rose Family calycinoides Hayata & Koidz. DCNA VAGS VAPH cockburnianus Hemsl. MDBG ‘Coronarius’ [R. rosifolius Sm.] Blackberry Rose GAIH MDFM SCWI odoratus L. ROSA Thimbleberry MDJH RUSCUS L. LILIACEAE Lily Family CE I SD SE SE RS aculeatus L. Butcher’s Broom DCNA LAAL NCEL SCBR GACC LASL NCTP TXHN GAUG MSEN NCWM ROSMARINUS L. LAMIACEAE Mint Family officinalis L. Rosemary FLUF SCCL VANB LAHG VACW VATA ‘Prostratus’ [R. officinalis L.] GAJI RUBUS L. ROSACEAE Rose Family calycinoides Hayata & Koidz. DCNA VAGS VAPH cockburnianus Hemsl. MDBG ‘Coronarius’ [R. rosifolius Sm.] Blackberry Rose GAIH MDFM SCWI odoratus L. Thimbleberry MDJH RUSCUS L. LILIACEAE Lily Family CE I SD SE SE RS aculeatus L. Butcher’s Broom DCNA LAAL NCEL SCBR GACC LASL NCTP TXHN GAUG MSEN NCWM ROSMARINUS L. LAMIACEAE Mint Family officinalis L. Rosemary FLUF SCCL VANB LAHG VACW VATA ‘Prostratus’ [R. officinalis L.] GAJI RUBUS L. ROSACEAE Rose Family calycinoides Hayata & Koidz. DCNA VAGS VAPH cockburnianus Hemsl. MDBG ‘Coronarius’ [R. rosifolius Sm.] Blackberry Rose GAIH MDFM SCWI odoratus L. Thimbleberry MDJH RUSCUS L. LILIACEAE Lily Family CE I SD SE SE RS aculeatus L. Butcher’s Broom DCNA LAAL NCEL SCBR GACC LASL NCTP TXHN GAUG MSEN NCWM SALIX RUSCUS hypoglossum L. TXDM SALIX RUSCUS hypoglossum L. TXDM RUSSELIA Jacq. SCROPHULARIACEAE Figwort Family equisetiformis Schlechtend. & Cham. Coral Plant ALBG LAAL LASL SABAL Adans. ARECACEAE minor (Jacq.f.) Pers. Dwarf Palmetto LASL NCTE SCBR SCWI palmetto (Walt.) Lodd. ex J.A. & J.H. Schultes Cabbage Palm ALBG FLUF SCBR SAGERETIA Brongn. RHAMNACEAE Buckthorn Family thea (Osbeck) M.C.Johnst. LAAL TXMS theezans (L.) Brongn. = S. thea SALIX L. Willow SALICACEAE Willow Family ee ES SS TS a Se \| aegyptiaca L. DCNA MDSC alba L. White W. NCDU TNLW VAHI TNBM VAGG alba X S. fragilis DCSH DEMC MDSC babylonica L. Weeping W. ALEH GANG NCTP VACW ARRR GAUG NCWD VALC DCDO LAHG TNCT VARG DCNA MDGD TXWW GAAB MDSJ VACM hypoglossum L. TXDM RUSSELIA Jacq. SCROPHULARIACEAE Figwort Family equisetiformis Schlechtend. & Cham. Coral Plant ALBG LAAL LASL SABAL Adans. ARECACEAE minor (Jacq.f.) Pers. Dwarf Palmetto LASL NCTE SCBR SCWI palmetto (Walt.) Lodd. ex J.A. & J.H. Schultes Cabbage Palm ALBG FLUF SCBR SAGERETIA Brongn. RHAMNACEAE Buckthorn Family thea (Osbeck) M.C.Johnst. LAAL TXMS theezans (L.) Brongn. = S. thea SALIX L. Willow SALICACEAE Willow Family ee ES SS TS a Se \| aegyptiaca L. DCNA MDSC alba L. White W. NCDU TNLW VAHI TNBM VAGG alba X S. fragilis DCSH DEMC MDSC babylonica L. Weeping W. ROSA ALEH GANG NCTP VACW ARRR GAUG NCWD VALC DCDO LAHG TNCT VARG x bicolor Ehrh. [S. arbuscula < S. nigricans] MDSC x blanda Anderss. Niobe W. [S. babylonica x S. fragilis] DCNA caprea L. Goat W. DCNA DCRP caprea X< S. elaeagnos DCNA caprea X< S. purpurea DCNA chaenomeles Kimura DCNA ‘Chrysocoma’ [S. alba var. vitellina x S. babylonica] ALBG DCNA VACW VAHI cinerea L. Gray W. DCNA MDPJ SCCU GACG MDSC TNTV cordata Michx. MDSC ‘Crispa’ [S. babylonica L.] TNTV x dasyclados Wimmer [S. caprea < S. cinerea < S. viminalis] MDSC discolor Muhl. DCNA elaeagnos Scop. DEMC MDSC ‘Eugenei’ [S. purpurea L.] DCNA fragilis L. DCAG ‘Ginme’ [S. < tsugaluensis Koidz.] [S. integra < S.vulpina] MDSC ‘Ginryu’ [S. subopposita Miq.] MDBG gracilistyla Miq. equisetiformis Schlechtend. & Cham. Coral Plant ALBG LAAL LASL 187 SALIX SALIX SALIX SALIX purpurea L. var. amplexicaulis (Bory & Chaub.) Boiss. GACG purpurea L. f. gracilis (Gren. & Godr.) Schneid. DCNA MDSC repens L. ssp. argentea (Sm.) A.A. & E.G. Camus DCNA MDSC rigida Muhl. MDSC < rubra Huds. [S. purpurea xX S. viminalis] MDSC ‘Sekka’ [S. sachalinensis F.Schmidt] DCNA MDBG VANB GACG MDGD x sepulcralis Simonk. [S. alba < S. babylonica] DCNA DEMC MDKN MDSJ sericans Tausch ex A. Kerner MDSC sericea Marsh. DCNA X seringeana Gaudin [S. caprea x S. elaeagnos] MDSC <x smithiana Willd. [S. caprea x S. viminalis] MDSC syrticola Fern. MDSC ‘Tortuosa’ [S. babylonica L.] Contorted Hankow W. ALBH GACD MDLA VAWR DEWG GAUG VACW FLUF MDBG VATA ‘Tortuosa’ [S. matsudana Koidz.] = ‘Tortuosa’ [S. babylonica] udensis Traut. DCNA variegata Franch. MDSC vulpina Anderss. DCNA hookeriana Barratt var. tomentosa J.K.Henry DCNA humilis Marsh. DCNA integra Thunb. ex J.A.Murr. DCNA interior Rowlee MDSC irrorata Anderss. MDSC japonica Thunb. ex J.A.Murr. DCNA x laestadiana Hartm. [S. cineria <x S. lapponum] MDSC ‘Melanostachys’ [S. gracilistyla Miq.] DCNA miyabeana Seemen DCNA muscina Dode DCNA MDSC x myricoides Muhl. [S. rigida x S. sericea] MDSC myrsinifolia Salisb. DCNA MDSC nigra L. Black W. ALAU DEWG LAAL MSMV ALTD GAFN MSBN NCDU ‘Pendula’ [S. alba L. var. vitellina (L.) Stokes] DEWG ‘Pendula’ [S. purpurea L.] MDSC pentandra L. DCNA x pontederana Willd. [S. cineria < S. purpurea] MDSC purpurea L. SASSAFRAS SALVIA SASSAFRAS marginatus Willd. Florida S. = GACB mukorossi Gaertn. Chinese S. ALCS GACR GASM FLUF GAIS SAPIUM P.Br. - Tallow Tree EUPHORBIACEAE Spurge Family japonicum (Sieb. & Zucc.) Pax & K.Hoffm. MDBG sebiferum (L.) Roxb. Chinese Tallow T. ALAU GAIS SCCL VACW ALBG LAAL SCJC ALCG LASL SCMG FLWG LATU TXGS SARCOCOCCA Lindl. Sweet Box BUXACEAE Boxwood Family Ee eS EL Se ee ee] hookeriana Baill. var. digyna Franch. GACG hookeriana Baill. var. humilis Rehd. & Wils. DCNA GAWH MDSJ DEMC MDBG NCGC DEWG MDLT SCWI ruscifolia Stapf Fragrant Sweet B. NCWM VACW VANB SASA Makino & Shibata POACEAE Grass Family veitchii (Carr.) Rehd. ‘Kuma Bamboo LAAL TXLL SASSAFRAS T.Nees & Eberm. LAURACEAE Laurel Family albidum (Nutt.) Nees Sassafras MDJS VACW VAWR MDLA VAPH marginatus Willd. LAHG SCWI VACW VAPH leucantha Cav. Mexican Bush S. SCWI SAMBUCUS L. Elder CAPRIFOLIACEAE Honeysuckle Famil (Contributed by T. R. Dudley) - (Cea EE ETT ER A RE] ‘Aurea’ [S. nigra L.] MDBG canadensis L. American E. SALIX ALBG LAAL TNSC VAHR DCNA MDTD VAGW ebulus L. Dwarf E. DCNA racemosa L. ssp. pubens (Michx.) H.House American Red E. MDWP racemosa ssp. sieboldiana (Miq.) Hara Asiatic Red E. DCNA sieboldiana Bl. ex Graebn. = S. racemosa ssp. sieboldiana SANTOLINA L. ASTERACEAE Aster Family LT TET BT II BE OTN CAIVE RE RATED OD RR PRES EID chamaecyparissus L. Lavender Cotton MDBG MDLT TNUT SAPINDUS L. Soapberry SAPINDACEAE Soapberry Family a SS EN RA CEST ORE En TE CRE] drummondii Hook. & Arn. Texas S. ALUA GATG MSMN VABF DCNA LAAL TNSC GAMP LAHP TXZH SAPIUM P.Br. - Tallow Tree EUPHORBIACEAE Spurge Family japonicum (Sieb. & Zucc.) Pax & K.Hoffm. MDBG sebiferum (L.) Roxb. Chinese Tallow T. ALAU GAIS SCCL VACW ALBG LAAL SCJC ALCG LASL SCMG FLWG LATU TXGS leucantha Cav. Mexican Bush S. SCWI SAMBUCUS L. Elder CAPRIFOLIACEAE Honeysuckle Famil (Contributed by T. R. Dudley) - (Cea EE ETT ER A RE] ‘Aurea’ [S. nigra L.] MDBG canadensis L. American E. ALBG LAAL TNSC VAHR DCNA MDTD VAGW ebulus L. Dwarf E. DCNA Mexican Bush S. SCWI SAMBUCUS L. Elder CAPRIFOLIACEAE Honeysuckle Famil (Contributed by T. R. Dudley) - (Cea EE ETT ER A RE] ‘Aurea’ [S. nigra L.] MDBG canadensis L. American E. ALBG LAAL TNSC VAHR DCNA MDTD VAGW ebulus L. Dwarf E. DCNA racemosa L. ssp. pubens (Michx.) H.House American Red E. MDWP racemosa ssp. sieboldiana (Miq.) Hara Asiatic Red E. DCNA sieboldiana Bl. ex Graebn. = S. racemosa ssp. sieboldiana SANTOLINA L. ASTERACEAE Aster Family LT TET BT II BE OTN CAIVE RE RATED OD RR PRES EID chamaecyparissus L. Lavender Cotton MDBG MDLT TNUT SAPINDUS L. Soapberry SAPINDACEAE Soapberry Family a SS EN RA CEST ORE En TE CRE] drummondii Hook. & Arn. Texas S. ALUA GATG MSMN VABF DCNA LAAL TNSC GAMP LAHP TXZH 189 SERISSA SERISSA SATUREJA SATUREJA SENECIO L. ASTERACEAE Aster Family salignus DC. FLUF SEQUOIA Endl. California Redwood TAXODIACEAE Taxodium Family ‘Adpressa’ [S. sempervirens (D.Don) Endl.] DCNA sempervirens (D.Don) Endl. Coast Redwood ALAU SCMG VACW VAOD MDPL TNMB VAMR VAWM SCHA VABR VANB SEQUOIADENDRON J.Buchh. Giant Sequoia TAXODIACEAE Taxodium Family giganteum (Lindl.) J.Buchh. Gai Bis direc DECG NCBR SERENOA_ Hook.f. ARECACEAE Palm Family SSS a SE EE EE ES. a Sa SE aS) repens (Bartram) Small Saw Palmetto FLUF SERISSA Comm. ex Juss. RUBIACEAE Madder Family foetida (L.f.) Lam. = S. japonica japonica (Thunb.) Thunb. SALIX ALFS FLUF GAUG VANB DCNA GAJI LALG FLCG GAJY SCCU FLLE GATS VACW Mint Family DCNA SCHINUS L. Peppertree ANACARDIACEAE Cashew Family (SE ea RTE IPS. IE RET RAF DEPEND a ES terebinthifolius Raddi Brazilian P. FLUF SCHIZOPHRAGMA Sieb. & Zucc. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family [REE ONE ti SSS ee hE it EE hydrangeoides Sieb. & Zucc. Japanese Hydrangea Vine ALUA MDGD DCNA MDLT MDMG SCIADOPITYS \| Sieb. & Zucc. TAXODIACEAE Taxodium Family verticillata (Thunb. ex J.A.Murr.) Sieb. & Zucc. Umbrella Pine DCNA DEWG MDBG MDJS MDLT VABF VANP SEBASTIANA Spreng. EUPHORBIACEAE Spurge Family ligustrina (Michx.) Muell.-Arg. GAEC SEMIARUNDINARIA Makino ex Nakai POACEAE Grass Family fastuosa (Latour-Marl. ex Mitf.) Makino ex Nakai SCJIC sempervirens (D.Don) Endl. Coast Redwood SEQUOIADENDRON J.Buchh. Giant Sequoia TAXODIACEAE Taxodium Family giganteum (Lindl.) J.Buchh. Gai Bis direc DECG NCBR SERENOA_ Hook.f. ARECACEAE Palm Family SSS a SE EE EE ES. a Sa SE aS) repens (Bartram) Small Saw Palmetto FLUF SERISSA Comm. ex Juss. RUBIACEAE Madder Family foetida (L.f.) Lam. = S. japonica japonica (Thunb.) Thunb. ALFS FLUF GAUG VANB DCNA GAJI LALG FLCG GAJY SCCU FLLE GATS VACW SEQUOIADENDRON J.Buchh. Giant TON BI JU f NX y AN IZAYY\| J , =! Vis L7SF SCHIZOPHRAGMA hydrangeoides Sieb. Zucc. [illustrator Susan M. Johnston] SCHIZOPHRAGMA hydrangeoides Sieb. & Zucc. [illustrator Susan M. Johnston] SOLANUM SESBANIA SESBANIA Scop. FABACEAE (Faboideae) Bean Family drummondii (Rydb.) Cory SCWI punicea (Cav.) Benth. LAAL NCEG SCMP LASL SCBR SCWI tripetii (Poit.) F.T.Hubb. = S. punicea SEVERINIA ‘Tenore ex Endl. RUTACEAE Citrus Family TEE EY DTS AT ST ALS MP PO ST buxifolia (Poir.) Tenore Chinese Box Orange FLCG FLUF FLMG GAJI LAAL VACW SHIBATAEA Makino ex Nakai POACEAE Grass Family kumasaca (Zoll.) Makino NCBE SINARUNDINARIA Nakai POACEAE Grass Family murielae (Gamble) Nakai = Thamnocalamus spathaceus nitida (Mitf.) Nakai DCNA SIPHONOSMANTHAUS Stapf OLEACEAE Olive Family [ RARE cM TE NE IGN ES ae ERLE US ok ea \| delavayi (Franch.) Stapf ALTD MDJW SKIMMIA Thunb. Skimmia RUTACEAE Citrus Family RE I ‘Fisheri’ [S. japonica Thunb.] DCNA SESBANIA Scop. FABACEAE (Faboideae) Bean Family drummondii (Rydb.) Cory SCWI punicea (Cav.) Benth. LAAL NCEG SCMP LASL SCBR SCWI tripetii (Poit.) F.T.Hubb. = S. punicea SEVERINIA ‘Tenore ex Endl. SALIX RUTACEAE Citrus Family TEE EY DTS AT ST ALS MP PO ST buxifolia (Poir.) Tenore Chinese Box Orange FLCG FLUF FLMG GAJI LAAL VACW SHIBATAEA Makino ex Nakai POACEAE Grass Family kumasaca (Zoll.) Makino NCBE SINARUNDINARIA Nakai POACEAE Grass Family murielae (Gamble) Nakai = Thamnocalamus spathaceus nitida (Mitf.) Nakai DCNA SIPHONOSMANTHAUS Stapf OLEACEAE Olive Family [ RARE cM TE NE IGN ES ae ERLE US ok ea \| delavayi (Franch.) Stapf ALTD MDJW SKIMMIA Thunb. Skimmia RUTACEAE Citrus Family SMILAX L. SMILACACEAE Greenbrier Cat-brier Family biflora Sieb. ex Miq. SCWI laurifolia L. SCWI pumila Walt. SCBR SCWI smallii Morong ALON GAUG TNHD SMILAX L. SMILACACEAE Greenbrier Cat-brier Family biflora Sieb. ex Miq. SCWI laurifolia L. SCWI pumila Walt. SCBR SCWI smallii Morong ALON GAUG TNHD SOLANUM L. SOLANACEAE Nightshade Family diphyllum L. FLUF TXGS jasminoides Paxt. Potato Vine GACL MSMN TXGM rantonnetii Carr. Blue Potato Bush TXPS TXRS = ce NO SORBUS SOLANUM seaforthianum Andr. Brazilian Nightshade ALBG wendlandii Hook.f. Paradise Flower LALG fallax Schneid. [Aronia melanocarpa < Sorbus aucuparia] MDAB sorbifolia (Poir.) Schneid. [Aronia melanocarpa < Sorbus americana] DCNA xSORBOCOTONEASTER Pojark. ROSACEAE Rose Family [Cotoneaster < Sorbus] pozdnjakovii Pojark. [Cotoneaster niger < Sorbus sibirica] DCNA x SORBOPYRUS ROSACEAE [Pyrus < Sorbus] Schneid. Rose Family ‘Bulbiformis’ [x S. auricularis (Knoop) Schneid.] [Pyrus communis < Sorbus aria] MDSC SORBUS L. Mountain Ash ROSACEAE Rose Family alnifolia (Sieb. & Zucc.) K.Koch DCNA MDGD VABF americana Marsh. American Mountain A. DCWH DEWG NCAS ‘Apricot Queen’ DCNA aucuparia L. Rowan DCMS DEMC TNBO DCNA NCAS ‘Cardinal’ [S. aucuparia L.] DCNA domestica L. Service Tree NCMS ‘Fastigiata’ [S. aucuparia L.] MDKN hybrida L. SOPHORA lL. FABACEAE (Faboideae) Bean Family affinis T. & G. LASL TXDM davidii (Franch.) Skeels SCWI TNLW VABF ‘Dot’ [S. japonica L.] ALTD japonica L. Pagoda Tree, Chinese Scholar Tree DCCG DEMC VAMP DCNA TNLW VASC ‘Pendula’ [S. japonica L.] MDBG MDCP MDHN VAMP ‘Regent’ [S. japonica L.] DCNA secundiflora (Ort.) Lag. ex DC. Mescal Bean FLJH LAHG TXJS TXSH FLMG MSMN TXPS VACW viciifolia Hance = S. davidii SORBARIA (Ser. ex DC.) A.Braun False Spirea ROSACEAE Rose Family ea ERR NES FST TR OS Is a Se kirilowii (Regel) Maxim. VAGO tomentosa (Lindl.) Rehd. var. tomentosa DCBG DCNA NCBE <SORBARONIA Schneid. ROSACEAE [Aronia < Sorbus] a a I a ER I A \| ‘Brilliantissima’ DCNA Rose Family 193 SORBUS SPIRAEA SPIRAEA SORBUS ‘Magnifica’ [S. aria (L.) Crantz] DCNA ‘Pendula’ [S. aucuparia L.] MDKN pohuashanensis (Hance) Hed. MDKN ‘Red Copper Glow’ DCNA torminalis Crantz DCNA SPARTIUM L. FABACEAE (Faboideae) Bean Family junceum L. Spanish Broom SCWI VACW SPIRAEA L. Spirea ROSACEAE Rose Family alba Du Roi DCNA albiflora (Miq.) Zab. DCSH ‘Alpestris’ [S. < lemoinei Zab.] [S. bullata « S. x bumalda] DEWG ‘Alpina’ [S. japonica L.f.] DCNA GAEC VAGS VATA GAAM SECU VAPH ‘Anthony Waterer’ [S. x bumalda Burv.] [S. albiflora < S. japonica] MDBN MDJS NCAS SCBR arcuata Hook.f. DCNA MDDF <arguta Zab. [S. multiflora < S. thunbergii] DEWG betulifolia Pall. DCNA < billiardii Herincq [S. douglasii < S. salicifolia] DEWG VACW SORBUS ‘Magnifica’ [S. aria (L.) Crantz] DCNA ‘Pendula’ [S. aucuparia L.] MDKN pohuashanensis (Hance) Hed. MDKN ‘Red Copper Glow’ DCNA torminalis Crantz DCNA SPARTIUM L. FABACEAE (Faboideae) Bean Family junceum L. Spanish Broom SCWI VACW SPIRAEA L. Spirea ROSACEAE Rose Family alba Du Roi DCNA albiflora (Miq.) Zab. DCSH ‘Alpestris’ [S. < lemoinei Zab.] [S. bullata « S. x bumalda] DEWG ‘Alpina’ [S. japonica L.f.] DCNA GAEC VAGS VATA GAAM SECU VAPH ‘Anthony Waterer’ [S. x bumalda Burv.] [S. albiflora < S. japonica] MDBN MDJS NCAS SCBR arcuata Hook.f. DCNA MDDF <arguta Zab. [S. multiflora < S. thunbergii] DEWG betulifolia Pall. blumei G.Don DCNA bullata Maxim. DEWG MDJW <x bumalda Burv. [S. albiflora < S. japonica] TNBM TNFE VAPH cantoniensis Lour. Reeves S. DCWR FLUF MDPJ SCPP DEWG GACG NCTE TNDG FLJU LAAP SCMG FLMG LATU SCMM chinensis Maxim. GAIS SCPP ‘Compacta’ [S. thunbergii Sieb. ex. Bl.] DCNA ‘Crispa’ [S. <x bumalda Burv.] [S. albiflora < S. japonica] ALBH VAPH ‘Goldflame’ [S. < bumalda Burv.] [S. albiflora < S. japonica] DCNA japonica L.f. Japanese S. DCNA LAHG NCBE VATA DEWG MDBG SCCU ‘Lanceata’ [S. cantoniensis Lour.] ALBG JAG AD LAAP NCSM ALBH FLMG LALG NCTE ALEH FLUF LASL SCLM DEWG GAJI MDGJ TNDG FLJU GAVI MDLA < margaritae Zab. [S. japonica < S. superba] DEWG MDJH miyabei Koidz. DCNA nipponica Maxim. var. tosaensis (Yatabe) Makino DCWH VATA prunifolia Sieb. & Zucc. Bridal Wreath ALIT FLDG MDMM VAPH ARRB FLMG SCMP VATA DCDO GACG TNCT VAWR DEWG MDHN VACW STEWARTIA SPIRAEA reevesiana Lindl. = S. cantoniensts salicifolia L. Willowleaf S. SCCU < sanssouciana K.Koch [S. douglasii < S. japonica] DCNA DEWG ‘Snowmound’ [S. nipponica Maxim. var. tosaensis (Yatabe) Makino] MDBG SCCU TNTV MDBN TNSN ‘Swan Lake’ [S. SORBUS trilobata L.] DEWG thunbergii Sieb. ex Bl. ALBG FLUF SCLM TNDG DEWG GAGM SCMP FLMG NCBE SCUC tomentosa L. Hardhack NCDB x vanhouttei (C. Briot) Zab. Vanhoutte S., Bridal Wreath [S. cantoniensis x S. trilobata] ARRB GACG NCGR VAGJ DCDO MDHF SCLM VAMP DCWR MDLT SCMP DEWG NCBE VACW STACHYURUS Sieb. & Zucc. STACHYURACEAE Stachyurus Family (SS ER chinensis Franch. DCNA ‘Issai’ [S. praecox Sieb. & Zucc.] MDMG praecox Sieb. & Zucc. DCNA MDLT VAGS MDKN SCCU STAPHYLEA L. STAPHYLEACEAE Bladdernut Family STEWARTIA trifolia L. ALAU DCNA VACW VAGW STEPHANANDRA Sieb. & Zucc. ROSACEAE Rose Family ‘Crispa’ [S. incisa (Thunb. ex J.A.Murr.) Zab.] MDBG incisa (Thunb. ex J.A.Murr.) Zab. Lace Shrub DEMC MDLN NCBE DEWG MDLT tanakae (Franch. & Sav.) Franch. & Sav. DCNA STEWARTIA L. THEACEAE Tea Family malacodendron L. Silky Camellia GAEC MDKN MDLT MDMG monadelpha Sieb. & Zucc. ALBG MDBG SCWI DCNA MDLT TNAB GARW NCBE VAGS ovata (Cav.) Weatherby Mountain Camellia DCNA TNPW pentagyna L’Her. = S. ovata pseudocamellia Maxim. Japanese S. DCCG MDFA NCDB TNAB DCNA MDLT NCDU VAGS DEMC MDMG NCEL VATA MDBG NCBE SCBR rostrata Spongberg DEWG VAGS serrata Maxim. VAGS sinensis Rehd. & Wils. DCNA GAEC TNSN STEWARTIA trifolia L. ALAU DCNA VACW VAGW STEPHANANDRA Sieb. & Zucc. ROSACEAE Rose Family ‘Crispa’ [S. incisa (Thunb. ex J.A.Murr.) Zab.] MDBG incisa (Thunb. ex J.A.Murr.) Zab. Lace Shrub DEMC MDLN NCBE DEWG MDLT tanakae (Franch. & Sav.) Franch. & Sav. DCNA STEWARTIA L. THEACEAE Tea Family malacodendron L. Silky Camellia GAEC MDKN MDLT MDMG monadelpha Sieb. & Zucc. ALBG MDBG SCWI DCNA MDLT TNAB GARW NCBE VAGS ovata (Cav.) Weatherby Mountain Camellia DCNA TNPW pentagyna L’Her. = S. ovata pseudocamellia Maxim. Japanese S. DCCG MDFA NCDB TNAB DCNA MDLT NCDU VAGS DEMC MDMG NCEL VATA MDBG NCBE SCBR rostrata Spongberg DEWG VAGS serrata Maxim. VAGS sinensis Rehd. & Wils. DCNA GAEC TNSN STACHYURUS Sieb. & Zucc. STACHYURACEAE Stachyurus Family (SS ER chinensis Franch. DCNA ‘Issai’ [S. praecox Sieb. & Zucc.] MDMG praecox Sieb. & Zucc. DCNA MDLT VAGS MDKN SCCU STAPHYLEA L. STAPHYLEACEAE Bladdernut Family Sa RRR IS FIT YS ES VERE FRE TIO] colchica Steven DCNA DEWG pinnata L. NCBE STACHYURUS Sieb. & Zucc. 195 STIGMAPHYLLON SYMPLOCOS SYAGRUS Mart. SORBUS ARECACEAE Palm Family romanzoffianum (Cham.) Glassman Queen Palm FLUF romanzoffianum xX Butia capitata FLUF <SYCOPARROTIA P.Endress & J.Anliker HAMAMELIDACEAE Witch-hazel Family [Parrotia <x Sycopsis] semidecidua P.Endress & J.Anliker [Parrotia persica < Sycopsis sinensis] DCNA SYCOPSIS Oliv. HAMAMELIDACEAE Witch-hazel Family sinensis Oliv. DCNA MDBG SYMPHORICARPOS Duham. Snowberry CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) albus (L.) S.F.Blake MDLG MDPJ albus (L.) S.F.Blake var. laevigatus (Fern.) S.F.Blake MDJS <x chenaultiti Rehd. [S. microphyllus < S. orbiculatus] VARE orbiculatus Moench Coralberry, Indian Currant ALAU NCBE VAHB VASP DCCG TXDC VAPO SYMPLOCOS Jacq. SYMPLOCACEAE Sweetleaf Family chinensis (Lour.) Druce f. pilosa (Nakai) Ohwi = S. paniculata STIGMAPHYLLON Juss. MALPIGHIACEAE Malpighia Family ledifolium (HBK.) Small TXCT STRANVAESIA Lindl. ROSACEAE Rose Family LFA TA EP a MN ERO YT TO A) davidiana Decne. Chinese S. DCCG DCWA MDLT TNUT DCNA MDBG TNLW ‘Lutea’ [S. davidiana Decne.] Yellow-fruited Chinese S. DCCG ntitakayamensis (Hayata) Hayata VAPH STYRAX L Snowbell, Storax STYRACACEAE Storax Family ih ea Ee CE ET ae a a a americanus Lam. American Snowbell TNSN TXMA ‘Carillon’ [S. japonica Sieb. & Zucc.] DCNA dasyanthus Perk. MDKN grandifolius Ait. Bigleaf Snowbell LAHG NCDU VACW NCAG SCBR japonicus Sieb. & Zucc. Japanese Snowbell DCSH MDGJ TNSG VACW DEWG MDLT TNSN VAGS MDBG TNSC TNUT obassia Sieb. & Zucc. Fragrant Snowbell DCDO MDBG MDPJ VANB DEEM MDGJ TNTV VATA MDAB MDLT VAGS ‘Pink Chimes’ [S. japonica Sieb. & Zucc.] SYAGRUS Mart. ARECACEAE 196 Sieb. & Zucc. japonicus STYRAX [i/lustrator Lillian Nicholson Meyer] KO, KO, SYCOPSIS sinensis Oliv. [illustrator Susan M. Johnston] SYCOPSIS sinensis Oliv. [illustrator Susan M. Johnston] 198 SYRINGA ‘Decaisne’ [S. vulgaris L.] DCNA = ‘Esther Staley’ [S. vulgaris L.] DCNA ‘Ethel M. Webster’ [S. < prestoniae McKelvey] [S. reflexa x S. villosa] DEWG ‘Henri Robert’ [S. vulgaris L.] DCNA x henryi Schneid. [S. josikaea x S. villosa] DEWG ‘Jessica’ DCNA josikaea Jacq.f. ex Reichenb. Hungarian L. NCBE ‘Jules Ferry’ [S. vulgaris L.] DCNA ‘Katherine Havemeyer’ [S. vulgaris L.] DCNA laciniata Mill. Cutleaf L. DCNA GAFN MDWP SCCU DEWG GAJS NCBE ‘Laciniata’ [S. persica L.] MDWP NCBE SCCU ‘Lamartine’ [S. x hyacinthiflora (Lem.) Rehd.] [S. oblata x S. vulgaris] DCNA DEWG ‘Laurentian’ [S. oblata Lindl. var. dilatata (Nakai) Rehd.] DCNA ‘Leon Gambetta’ [S. vulgaris L.] DCNA ‘Marechal Lannes’ [S. vulgaris L.] DCNA ‘Maurice Barres’ [S. vulgaris L.] DCNA meyeri Schneid. DCNA DEWG VAGS microphylla Diels SYRINGA L. SORBUS Lilac OLEACEAE Olive Family a ae ee PR ee ‘Alba’ [S. vulgaris L.] ARRB MDMM VAGW VAWR MDLA VACW VASP ‘Alba Plena’ [S. vulgaris L.] Double White L. MDLA ‘Alexander’s Perfection’ DCNA amurensis var. japonica (Maxim.) Franch. & Sav. = S. reticulata ‘Annabelle’ [S. oblata Lindl. var. dilatata (Nakai) Rehd.] DCNA ‘Buffon’ [S. < hyacinthiflora (V.Lemoine) Rehd.] [S. oblata x S. vulgaris] DCNA ‘Charles Giant’ [S. x hyacinthiflora (V.Lemoine) Rehd.] [S. oblata < S. vulgaris] DEWG ‘Charles Joly’ [S. vulgaris L.] DCNA ‘Charles Nordine’ [S. vulgaris L.] DCNA x chinensis Willd. Chinese L. [S. x persica x S. vulgaris] DCNA ‘Crayton’ [S. villosa Vahl] Late Lilac DEWG 199 SYRINGA SYRINGA ‘Miss Ellen Willmott’ [S. vulgaris L.] DCNA ‘Miss Kim’ [S. patula (Palib.) Nakai] MDJS ‘Mme. Charles Souchet’ [S. vulgaris L.] DCNA ‘Monge’ [S. vulgaris L.] DCNA ‘Mrs. Edward Harding’ [S. vulgaris L.] DCNA oblata Lindl. var. dilatata (Nakai) Rehd. VATA patula (Palib.) Nakai DCNA VACW persica L. Persian L. DEWG MDWP VACW VAWR MDPJ SCCU VAGW ‘Pocohantas’ [S. < hyacinthiflora (Lem.) Rehd.] [S. oblata x S. vulgaris] DEWG ‘President Grevy’ [S. vulgaris L.] DCNA ‘Primrose’ [S. vulgaris L.] DEWG ‘Priscella’ [S. vulgaris L.] DCNA pubescens Turcz. DCNA DEWG ‘Purpurea’ [S. vulgaris L.] Common Lilac ARRB MDLA TNCT DCWH MDMM VAGW GAAM MDSM VASP ‘Rene Jarry-Desloges’ [S. vulgaris L.] DCNA reticulata (Bl.) Hara Japanese Tree L. DCNA DEWG MDHC VAMP ‘Superba’ [S. microphylla Diels] DEWG ‘Vaubon’ [S. < hyacinthiflora (Lem.) Rehd.] [S. oblata x S. vulgaris] DCNA velutina Komar. = S. patula villosa Vahl Late L: MDBG vulgaris L. var. alba Weston = ‘Alba’ vulgaris L. var. purpurea Weston = ‘Purpurea’ yunnanensis Franch. DCNA ‘Zulu’ [S. vulgaris L.] DCNA TABERNAEMONTANA L. APOCYNACEAE Dogbane Family aS ee re ES a SS OE PREY a TS ae el RE) coronaria (Jacq.) Willd. = T. divaricata divaricata (L.) R.Br. ex Roem. & J.A.Schultes Crape Jasmine LALG TAIWANIA Hayata TAXODIACEAE Taxodium Family cryptomerioides Hayata ALTD DCNA GACG TAMARIX L. TAMARICACEAE Tamarisk Family parviflora DC. DEWG VACW pentandra Pall. = T. ramosissima ramosissima Ledeb. DCNA TXHN VAGS TAXUS baccata L. English Y. ~ DCCG MDFM NCTE VASJ DCNA MDRT VACS VAWR baccata L. f. fastigiata (Lindl.) Pilger FLMG MDHN VABA VAMP GABG MDJH VAGS canadensis Marsh. Canadian Y., Ground Hemlock SCBR VADW ‘Citation’ [T. < media Rehd.] [T. baccata < T. cuspidata] DCNA cuspidata Sieb. & Zucc. Japanese Y. ALTD FLMG VASP DCCG MDDF ‘Elegantissima’ [T. baccata L.] VAFN ‘Expansa’ [T. cuspidata Sieb. & Zucc.] MDLT ‘Fastigiata Robusta’ [T. baccata L.] DCNA floridana Nutt. Florida Y. FLUF TNAB ‘Flushing’ [T. x media Rehd.] [T. baccata < T. cuspidata] DCNA ‘Fowle’ [T. baccata L.] = ‘Adpressa Fowle’ ‘Hicksii’ [T. < media Rehd.] [T. baccata < T. cuspidata] DCNA FLMG NCDU ‘Luteobaccata’ [T. cuspidata Sieb. & Zucc.] DCNA < media Rehd. [T. baccata < T. cuspidata] DCFP DCNA DCSI MDAB ‘Minima’ [T. cuspidata Sieb. & Zucc.] DCNA ‘Nana’ [T. cuspidata Sieb. & Zucc.] TAXUS baccata L. English Y. ~ DCCG MDFM NCTE VASJ DCNA MDRT VACS VAWR baccata L. f. SYRINGA fastigiata (Lindl.) Pilger FLMG MDHN VABA VAMP GABG MDJH VAGS canadensis Marsh. Canadian Y., Ground Hemlock SCBR VADW ‘Citation’ [T. < media Rehd.] [T. baccata < T. cuspidata] DCNA cuspidata Sieb. & Zucc. Japanese Y. ALTD FLMG VASP DCCG MDDF ‘Elegantissima’ [T. baccata L.] VAFN ‘Expansa’ [T. cuspidata Sieb. & Zucc.] MDLT ‘Fastigiata Robusta’ [T. baccata L.] DCNA floridana Nutt. Florida Y. FLUF TNAB ‘Flushing’ [T. x media Rehd.] [T. baccata < T. cuspidata] DCNA ‘Fowle’ [T. baccata L.] = ‘Adpressa Fowle’ ‘Hicksii’ [T. < media Rehd.] [T. baccata < T. cuspidata] DCNA FLMG NCDU ‘Luteobaccata’ [T. cuspidata Sieb. & Zucc.] DCNA < media Rehd. [T. baccata < T. cuspidata] DCFP DCNA DCSI MDAB ‘Minima’ [T. cuspidata Sieb. & Zucc.] DCNA ‘Nana’ [T. cuspidata Sieb. & Zucc.] MDTD ‘Rubra’ [T. ramosissima Ledeb.] DCNA ‘Summer Glow’ [T. ramosissima Ledeb.] DCNA TAXODIUM L.C.Rich. Bald Cypress TAXODIACEAE Taxodium Family POA a ascendens Brongn. = T. distichum var. nutans distichum (L.) L.C.Rich. var. distichum Bald Cypress ALAU DCWH NCCA VAMC ALBG DEMC SCBR VAMP ARUM GAMW SCMG DCCG MDBG TNFE DCNA MSEP TNSC distichum (L.) L.C.Rich. var. imbricarium (Nutt.) Croom = T. distichum var. nutans distichum (L.) L.C.Rich. var. nutans (Ait.) Sweet Pond C. ALAU DEMC MDCP SCBR ALBG FLMG NCCA VAMP DCNA MDBG NCOP mucronatum Tenore Montezuma Cypress LASL TXDM TXPS ‘Nutans’ [T. distichum (L.) L.C.Rich.] = T. distichum var. nutans ‘Pendens’ [T. distichum (L.) L.C.Rich.] DCNA TAXUS L. Yew TAXACEAE Yew Family ‘Adpressa’ [T. baccata L.] NCBE ‘Adpressa Fowle’ [T. baccata L.] DCNA ‘Amersfoort’ [T. baccata L.] DCNA MDTD ‘Aurea’ [T. baccata L.] Golden Y. DCSH ‘Aurea Marginata’ [T. baccata L.] VABR VAMP TAXODIUM L.C.Rich. Bald Cypress TAXODIACEAE Taxodium Family POA a ascendens Brongn. = T. distichum var. nutans distichum (L.) L.C.Rich. var. distichum Bald Cypress ALAU DCWH NCCA VAMC ALBG DEMC SCBR VAMP ARUM GAMW SCMG DCCG MDBG TNFE DCNA MSEP TNSC distichum (L.) L.C.Rich. var. imbricarium (Nutt.) Croom = T. distichum var. nutans distichum (L.) L.C.Rich. var. nutans (Ait.) Sweet Pond C. ALAU DEMC MDCP SCBR ALBG FLMG NCCA VAMP DCNA MDBG NCOP mucronatum Tenore Montezuma Cypress LASL TXDM TXPS ‘Nutans’ [T. distichum (L.) L.C.Rich.] = T. distichum var. nutans ‘Pendens’ [T. distichum (L.) L.C.Rich.] DCNA TAXUS L. Yew TAXACEAE Yew Family ‘Adpressa’ [T. baccata L.] NCBE ‘Adpressa Fowle’ [T. baccata L.] DCNA ‘Amersfoort’ [T. baccata L.] DCNA MDTD ‘Aurea’ [T. baccata L.] Golden Y. DCSH ‘Aurea Marginata’ [T. SYRINGA baccata L.] VABR VAMP ascendens Brongn. = T. distichum var. nutans 201 TAXUS TAXUS THUJA THUJA ‘Overeynderi’ [T. baccata L.] DCCG ‘Repandens’ [T. baccata L.] DCNA MDBG MDHN ‘Richard Horsey’ [T. <x hunnewelliana Rehd.] [T. canadensis < T. cuspidata] DCNA ‘Stricta’ [T. baccata] = T. baccata f. fastigiata ‘Thompson’ [T. cuspidata Sieb. & Zucc.] DCNA TECOMA Juss. BIGNONIACEAE Bignonia Family stans (L.) HBK. var. angustata Rehd. TXILL TECOMARIA (Endl.) Spach BIGNONIACEAE Bignonia Family aE Re Oe a a Se A eee, capensis (Thunb.) Spach Cape Honeysuckle FLUF TETRAPANAX (K.Koch) K.Koch papyriferus (Hook.) K.Koch Rice-paper Plant MSHB Rice-paper Plant MSHB TEUCRIUM lL. Germander LAMIACEAE Mint Family (TS RT aa a DT SS SCT TE OT PEE) chamaedrys L. European Germander DCNA x lucidrys Boom [T. chamaedrys < T. lucidum] DCNA lucidum L. MDHN THAMNOCALAMUS Munro POACEAE Grass Family spathaceus (Franch.) Soderstrom Green Fountain Bamboo DCNA MDFM THRYALLIS Mart. MALPIGHIACEAE Malpighia Family SS ae eS ae ee ee eee ee ee ee ee a} glauca (Cav.) O.Ktze. = Galphimia glauca THUJA L. CUPRESSACEAE Arborvitae Cypress Family ‘Alba’ [T. occidentalis L.] DCNA ‘Aurea’ [T. occidentalis L.] VAMP ‘Beaufort’ [T. occidentalis L.] DCNA ‘Buchananii’ [T. occidentalis L.] DCNA ‘Columna’ [T. occidentalis L.] MDJS LAMIACEAE Mint Family (TS RT aa a DT SS SCT TE OT PEE) chamaedrys L. European Germander DCNA x lucidrys Boom [T. chamaedrys < T. lucidum] DCNA lucidum L. MDHN THAMNOCALAMUS Munro POACEAE Grass Family spathaceus (Franch.) Soderstrom Green Fountain Bamboo DCNA MDFM THRYALLIS Mart. MALPIGHIACEAE Malpighia Family SS ae eS ae ee ee eee ee ee ee ee a} glauca (Cav.) O.Ktze. = Galphimia glauca THUJA L. CUPRESSACEAE Arborvitae Cypress Family ‘Alba’ [T. occidentalis L.] DCNA ‘Aurea’ [T. occidentalis L.] VAMP ‘Beaufort’ [T. occidentalis L.] DCNA ‘Buchananii’ [T. occidentalis L.] DCNA ‘Columna’ [T. occidentalis L.] MDJS TECOMARIA (Endl.) Spach BIGNONIACEAE Bignonia Family aE Re Oe a a Se A eee, TECOMARIA (Endl.) Spach BIGNONIACEAE Bignonia Family aE Re Oe a a Se A eee, capensis (Thunb.) Spach Cape Honeysuckle FLUF TERNSTROEMIA Mutis ex Lf. TERNSTROEMIA Mutis ex Lf. TERNSTROEMIA Mutis ex Lf. THEACEAE Tea Family gymnanthera (Wight & Arn.) Sprague (Distinct from Cleyera japonica, this plant is often grown under that name because of its confused nomenclatural history; referral to Cleyera japonica Thunb., pro parte, merely continues the chances for misidentification of the plant.) ALAU FLMG LAMP SCKG ALBG FLUF LASL VAMP ALTD GAHC MSHB VANB DEEG GAMM NCAG VAWM DCDO GAWP NCDU DCNA LAHG NCOP FLCG LALG NCTE japonica Thunb. = T. gymnanthera THEACEAE Tea Family gymnanthera (Wight & Arn.) Sprague (Distinct from Cleyera japonica, this plant is often grown under that name because of its confused nomenclatural history; referral to Cleyera japonica Thunb., pro parte, merely continues the chances for misidentification of the plant.) ALAU FLMG LAMP SCKG ALBG FLUF LASL VAMP ALTD GAHC MSHB VANB DEEG GAMM NCAG VAWM DCDO GAWP NCDU DCNA LAHG NCOP FLCG LALG NCTE japonica Thunb. = T. gymnanthera THUJOPSIS THUJA THUJA THUJA ‘Compacta’ [T. occidentalis L.] DCNA ‘Cuprea’ [T. plicata D.Don] DCNA ‘Endean’ [T. occidentalis L.] DCNA ‘Fastigiata’ [T. occidentalis L.] NCBE ‘Filifera’ [T. occidentalis L.] NCBE ‘Filiformis’ [T. occidentalis L.] DCNA ‘Froebelii’ [T. occidentalis L.] DCNA ‘Globosa’ [T. occidentalis L.] DCNA SCCU ‘Globosa Rheindiana’ [T. occidentalis L.] DCNA ‘Hetz Midget’ [T. occidentalis L.] DCNA ‘Holmstrup’ [T. occidentalis L.] DCNA ‘Hudsonica’ [T. occidentalis L.] DCNA ‘Little Gem’ [T. occidentalis L.] VATA ‘Lutea’ [T. occidentalis L.] VABF ‘Malonyana’ [T. occidentalis L.] DCNA occidentalis L. American A. ALSH MDHN NCCA TNBV DCNA MDJH NCSM ‘Ohlendorfii’ [T. occidentalis L.] MDBG orientalis L. = Platycladus orientalis ‘Pendula’ [T. occidentalis L.] TNTV THUJOPSIS ‘Pygmaea’ [T. occidentalis L.] DCNA MDBG = ‘Pyramidalis’ [T. occidentalis L.] MDBG MDHN ‘Pyramidalis Nigra’ [T. occidentalis L.] VATA ‘Recurva Nana’ [T. occidentalis L.] DCNA ‘Rheingold’ [T. occidentalis L.] DCNA ‘Rogersii’ [T. plicata D.Don] DCNA ‘Semperaurea’ [T. occidentalis L.] DCNA ‘Spiralis’ [T. occidentalis L.] NCBE standishii (Gord.) Carr. DCNA ‘Stricta’ [T. occidentalis L.] DCNA ‘Sunkist’ [T. occidentalis L.] DCNA MDBG ‘Techny’ [T. occidentalis L.] GACG ‘Umbraculifera’ [T. occidentalis L.] DCNA ‘Wareana’ [T. occidentalis L.] DCNA ‘Wareana Lutescens’ [T. occidentalis L.] DCNA ‘Woodwardii’ [T. occidentalis L.] DCNA ‘Zebrina’ [T. plicata D.Don] DCNA THUJOPSIS Sieb. & Zucc. ex Endl. False Arborvitae CUPRESSACEAE Cypress Family dolabrata (Thunb. ex L.f.) Sieb. & Zucc. THUJOPSIS dolabrata (Thunb. ex L.f.) Sieb. & Zucc. var. hondae Makino DCNA MDGD ‘Nana’ [T. dolabrata (Thunb. ex L.f.) Sieb. & Zucc.] LASL THUNBERGIA ketz. ACANTHACEAE Acanthus Family grandiflora (Roxb. ex Rottl.) Roxb. Blue Trumpet Vine TXAB THYMUS L. LAMIACEAE Mint Family vulgaris L. Common Thyme DCNA MDBG TIBOUCHINA Aubl. MELASTOMATACEAE Melastoma Family (aS semidecandra Hort. non (DC.) Cogn. = T. urvilleana urvilleana (DC.) Cogn. Glory Bush ALBG FLUF TILIA L. Basswood, Linden TILIACEAE Linden Family ER A GeO Se ETN AB eal americana L. American L. ALUA DCMS MDHN VABR DCBG DCNA MDNA VACM DCEE DESI NCBE VACW DCCG DCTB SCBR VAMP DCJJ FLUF TNFE VARO DCMH MDAB TNSG caroliniana Mill. = T. americana cordata Mill. Littleleaf L. THUJOPSIS pubescens Makino LAHG MSMN TOONA TOONA (Endl.) M.J.Roem. MELIACEAE Mahogany Family ou EE TS SES ST sinensis (Juss.) M.J.Roem. Chinese T. DCCG DCNA MDBG VABF DCGD DCTN TNRT TORREYA Arn. TAXACEAE Yew Family (Te a a eae ME EA eS a ee] ‘Gold Strike’ [T. nucifera (L.) Sieb. & Zucc.] DCNA nucifera (L.) Sieb. & Zucc. Japanese T. DCDO DCNA TNTV taxifolia Arn. Stinking Cedar, Florida T. ALEH FLDG FLUF SCBR DCNA FLMG NCBE SCMG FLAL FLIP NCHS TOXICODENDRON Mill. ANACARDIACEAE Cashew Family radicans (L.) O.Ktze. Poison Ivy MDBG vernicifluum (Stokes) F.A.Barkley Varnish Tree DCNA TRACHELOSPERMUM Lem. APOCYNACEAE Dogbane Family asiaticum (Sieb. & Zucc.) Nakai GAEC GAWH difforme (Walt.) A.Gr. MSEP SCBR jasminoides (Lindl.) Lem. Confederate Jasmine, Star Jasmine FLPM GAUG MSEN SCCM FLUF LALG NCDU SCWI GAOH LASL NCWM jasminoides (Lindl.) Lem. var. pubescens Makino TSUGA ‘Variegatum’ [T. jasminoides (Lindl.) Lem.] ALBG LASL SCWC TRACHYCARPUS' H.Wend1. ARECACEAE Palm Family fortunei (Hook.) H.Wendl. Windmill! Palm ALBG FLMG SCFW TXDM VAGS TRIPETALEIA Sieb. & Zucc. = ELLIOTIA TRIPTERYGIUM Hook.f. CELASTRACEAE Staff-tree Family regelii Sprague & Takeda Three-wing Wingnut DCNA TNTV TRITHRINAX Mart. ARECACEAE Palm Family acanthocoma Drude FLUF TROCHODENDRON Sieb. & Zucc. TROCHODENDRACEAE Trochodendron Family aralioides Sieb. & Zucc. MDLT TSUGA Carr. Hemlock PINACEAE Pine Family ‘Abbott’s Dwarf’ [T. canadensis (L.) Carr.] MDMG ‘Angustifolia’ [T. canadensis (L.) Carr.] DCNA ‘Armistice’ [T. canadensis (L.) Carr.] ‘Variegatum’ [T. jasminoides (Lindl.) Lem.] ALBG LASL SCWC ‘Variegatum’ [T. jasminoides (Lindl.) Lem.] ALBG LASL SCWC TRACHYCARPUS' H.Wend1. ARECACEAE Palm Family fortunei (Hook.) H.Wendl. Windmill! Palm ALBG FLMG SCFW TXDM VAGS TRIPETALEIA Sieb. & Zucc. = ELLIOTIA TRIPTERYGIUM Hook.f. CELASTRACEAE Staff-tree Family regelii Sprague & Takeda Three-wing Wingnut DCNA TNTV TRITHRINAX Mart. ARECACEAE Palm Family acanthocoma Drude FLUF TROCHODENDRON Sieb. & Zucc. TROCHODENDRACEAE Trochodendron Family aralioides Sieb. & Zucc. MDLT TSUGA Carr. Hemlock PINACEAE Pine Family ‘Abbott’s Dwarf’ [T. canadensis (L.) Carr.] MDMG ‘Angustifolia’ [T. canadensis (L.) Carr.] DCNA ‘Armistice’ [T. canadensis (L.) Carr.] DCNA ‘Variegatum’ [T. jasminoides (Lindl.) Lem.] ALBG LASL SCWC TRACHYCARPUS' H.Wend1. ARECACEAE Palm Family fortunei (Hook.) H.Wendl. Windmill! Palm ALBG FLMG SCFW TXDM VAGS TRIPETALEIA Sieb. & Zucc. = ELLIOTIA TRIPTERYGIUM Hook.f. CELASTRACEAE Staff-tree Family regelii Sprague & Takeda Three-wing Wingnut DCNA TNTV TRITHRINAX Mart. ARECACEAE Palm Family acanthocoma Drude FLUF TROCHODENDRON Sieb. & Zucc. TROCHODENDRACEAE Trochodendron Family aralioides Sieb. & Zucc. MDLT TSUGA Carr. Hemlock PINACEAE Pine Family ‘Abbott’s Dwarf’ [T. canadensis (L.) Carr.] MDMG ‘Angustifolia’ [T. THUJOPSIS TIPUANA x euchlora K.Koch [T. ?cordata < T. dasystyla] DCBG DCML MDNA DCJM DCNA < europaea L. [T. cordata < T. platyphyllos] DCGP GAAC MDSJ VASC DCJJ GALE NCBE VAWR DCML MDHC NCCP DCMN MDHN VACM heterophylla Vent. = T. americana ‘Laciniata’ [T. platyphyllos Scop.] DCBG MDCP VALC DCML TNTV x moltkei Spaeth [T. americana x T. tomentosa ‘Pendula’] DCML MDNA mongolica Maxim. Mongolian L. DCNA ‘Pendula’ [T. tomentosa Moench] DCGP DCPA DCWH petiolaris DC. = T. tomentosa ‘Pendula’ platyphyllos Scop. Bigleaf L. DCBG MDDF NCBE VAMP DCCG MDEC TNFE VARB DCML MDHN TNHG DCWH MDJH TNSG MDCP MDPJ VAMF tomentosa Moench Silver L. DCAC DCWH MDCP TNUT DCCG GAEH MDJH VALC DCGP MDCL NCBE ‘Vitifolia’ [T. platyphyllos Scop.] DCSI x vulgaris Hayne = T. Xeuropaea TIPUANA (Benth.) Benth. FABACEAE (Faboideae) Bean Family CE ST IP eT TS tipu (Benth.) O.Ktze. Tipu Tree, Rosewood FLUF ‘Nana’ [T. dolabrata (Thunb. ex L.f.) Sieb. & Zucc.] LASL THUNBERGIA ketz. ACANTHACEAE Acanthus Family grandiflora (Roxb. ex Rottl.) Roxb. Blue Trumpet Vine TXAB THYMUS L. LAMIACEAE Mint Family vulgaris L. Common Thyme DCNA MDBG TIBOUCHINA Aubl. MELASTOMATACEAE Melastoma Family (aS semidecandra Hort. non (DC.) Cogn. = T. urvilleana urvilleana (DC.) Cogn. Glory Bush ALBG FLUF TILIA L. Basswood, Linden TILIACEAE Linden Family ER A GeO Se ETN AB eal americana L. American L. ALUA DCMS MDHN VABR DCBG DCNA MDNA VACM DCEE DESI NCBE VACW DCCG DCTB SCBR VAMP DCJJ FLUF TNFE VARO DCMH MDAB TNSG caroliniana Mill. = T. americana cordata Mill. Littleleaf L. DCCG DCNY MDBG SCCU DCJM DCSH MDHN TNSC DCMH DCWH MDJH TNUT DCMN DESR MDLT VACM DCNA GARW NCDU TOONA TOONA (Endl.) M.J.Roem. MELIACEAE Mahogany Family ou EE TS SES ST sinensis (Juss.) M.J.Roem. Chinese T. DCCG DCNA MDBG VABF DCGD DCTN TNRT TORREYA Arn. TAXACEAE Yew Family (Te a a eae ME EA eS a ee] ‘Gold Strike’ [T. nucifera (L.) Sieb. & Zucc.] DCNA nucifera (L.) Sieb. & Zucc. Japanese T. DCDO DCNA TNTV taxifolia Arn. Stinking Cedar, Florida T. ALEH FLDG FLUF SCBR DCNA FLMG NCBE SCMG FLAL FLIP NCHS TOXICODENDRON Mill. ANACARDIACEAE Cashew Family radicans (L.) O.Ktze. Poison Ivy MDBG vernicifluum (Stokes) F.A.Barkley Varnish Tree DCNA TRACHELOSPERMUM Lem. APOCYNACEAE Dogbane Family asiaticum (Sieb. & Zucc.) Nakai GAEC GAWH difforme (Walt.) A.Gr. MSEP SCBR jasminoides (Lindl.) Lem. Confederate Jasmine, Star Jasmine FLPM GAUG MSEN SCCM FLUF LALG NCDU SCWI GAOH LASL NCWM jasminoides (Lindl.) Lem. var. ULMUS ULMUS ‘Belgica’ [U. <hollandica Mill.] [U. minor < U. glabra] DCCG DCWP ‘Camperdownii’ [U. glabra Huds.] DCWH TNDR VACW carpinifolia Gleditsch = U. minor ‘Christine Buisman’ [U. minor Mill.] DCPP crassifolia Nuit. Cedar E. TXGS ‘Dauvessei’ [U. < hollandica Mill.] [U. minor < U. glabra] DCWP ‘Drake’ [U. parvifolia Jacq.] SCCU SCSG VATA * ‘Dynasty‘ [U. parvifolia Jacq.] DCNA elliptica K.Koch VABF ‘Frosty’ [U. parvifolia Jacq.] GAFG fulva Michx. = U. rubra glabra Huds. Wych E., Scotch E. DCCG ‘Hokkaido’ [U. parvifolia Jacq.] GAFG <hollandica Mill. [U. minor < U. glabra] VAMW * ‘Homestead’ [U. pumila x ((U. xhollandica ‘Vegeta’ < U. minor) < (U. pumila var. arborea < U. minor ‘Hoersholm’))] DCNA japonica (Rehd.) Sarg. DCNA ULMUS macrocarpa Hance DCNA MDGD ‘Major’ [U. <x hollandica Mill.] [U. minor x U. glabra] DCCG ‘Marginata’ [U. procera Salisb.] DCSE minor Mill. DCCG SCCU TNRM ‘Moline’ [U. americana L.] Moline Elm MDGD parvifolia Jacq. Chinese E. ALAU DCLC GAJY SCCU ALFN FLMN MDAF DCAC FLUF NCEL * ‘Pioneer’ [U. < hollandica] [U. minor x U. glabra] DCNA procera Salisb. English E. DCCG DCWH MDHN DCSE MDCP pumila L. Siberian E. ALBH DCWR MDGG SCNS ARPT GACL MDMM VAWD DCCG LATU NCCC rubra Muhl. Slippery E. DCCG NCKH VACW MDHN SCMP VAMP ‘Sarniensis’ [U. minor Mill.] Jersey E. DCLP serotina Sarg. September E. NCBE VASC ‘Superba’ [U. < hollandica Mill.] [U. minor < U. glabra] DCGP thomasii Sarg. ULMUS ‘Belgica’ [U. <hollandica Mill.] [U. minor < U. glabra] DCCG DCWP ‘Camperdownii’ [U. glabra Huds.] DCWH TNDR VACW carpinifolia Gleditsch = U. minor ‘Christine Buisman’ [U. minor Mill.] DCPP crassifolia Nuit. Cedar E. TXGS ‘Dauvessei’ [U. < hollandica Mill.] [U. minor < U. glabra] DCWP ‘Drake’ [U. parvifolia Jacq.] SCCU SCSG VATA * ‘Dynasty‘ [U. parvifolia Jacq.] DCNA elliptica K.Koch VABF ‘Frosty’ [U. parvifolia Jacq.] GAFG fulva Michx. = U. rubra glabra Huds. Wych E., Scotch E. DCCG ‘Hokkaido’ [U. parvifolia Jacq.] GAFG <hollandica Mill. [U. minor < U. glabra] VAMW * ‘Homestead’ [U. pumila x ((U. xhollandica ‘Vegeta’ < U. minor) < (U. pumila var. arborea < U. minor ‘Hoersholm’))] DCNA japonica (Rehd.) Sarg. DCNA macrocarpa Hance DCNA MDGD ‘Major’ [U. <x hollandica Mill.] [U. minor x U. glabra] DCCG ‘Marginata’ [U. procera Salisb.] DCSE minor Mill. DCCG SCCU TNRM ‘Moline’ [U. americana L.] Moline Elm MDGD parvifolia Jacq. Chinese E. THUJOPSIS canadensis (L.) Carr.] DCNA ‘Armistice’ [T. canadensis (L.) Carr.] DCNA ‘Beaujean’ [T. canadensis (L.) Carr.] DCNA 205 (Juss.) M.J. Roem. inensis TOONA s [#Mustrator Lillian Nicholson Meyer] (Juss.) M.J. Roem. inensis TOONA s [#Mustrator Lillian Nicholson Meyer] ULMUS TSUGA ‘Bennett’ [T. canadensis (L.) Carr.] DCNA ‘Boulevard’ [T. canadensis (L.) Carr.] DCNA ‘Brandley’ [T. canadensis (L.) Carr.] DCNA canadensis (L.) Carr. Canadian H. ALEH GAAB NCBE TNWF DCCG MDHN NCTE VAMP DCE MDJS SCCU VAPO DCNA MDLA SCPE VAWP DCWH MDLT TNDG VAWR FLUF MDWP TNSM caroliniana Engelm. Carolina H. DCWH MDLT NCBE VAPO MDBG MDNA NCCA MDDF MDWP NCDU MDLA NCAS VAML chinensis (Franch.) Pritz. DCNA ‘Cinnamomea’ [T. canadensis (L.) Carr.] DCNA ‘Curtis Ideal’ [T. canadensis (L.) Carr.] DCNA ‘Curtis Spreader’ [T. canadensis (L.) Carr.] DCNA diversifolia (Maxim.) Mast. DCNA GAEC ‘Doc’s Choice’ [T. canadensis (L.) Carr.] DCNA ‘Doran’ [T. canadensis (L.) Carr.] DCNA ‘Fastigiata’ [T. canadensis (L.) Carr.] DCNA ‘Gensch White’ [T. canadensis (L.) Carr.] MDLT ‘Globosa’ [T. canadensis (L.) Carr.] DCNA ‘Hawkersmith Weeping’ [T. canadensis (L.) Carr.] ULMUS ‘Jacqueline Verkade’ [T. canadensis (L.) Carr.] — DCNA ‘Jervis’ [T. canadensis (L.) Carr.] DCNA ‘Kelsey’s Weeping’ [T. canadensis (L.) Carr.] DCNA ‘Macrophylla’ [T. canadensis (L.) Carr.] DCNA ‘Minima’ [T. canadensis (L.) Carr.] DCNA ‘Minuta’ [T. canadensis (L.) Carr.] DCNA ‘Pendula’ [T. canadensis (L.) Carr.] Weeping H. DCNA MDLT MDNA NCBE ‘Sargentii’ [T. canadensis (L.) Carr.] Sargent’s Weeping H. DCNA MDBG sieboldii Carr. MDKN MDLT ‘Verkade Recurved’ [T. canadensis (L.) Carr.] DCNA ‘Von Helms’ [T. canadensis (L.) Carr.] DCNA ‘Youngcone’ [T. canadensis (L.) Carr.] DCNA ULMUS L. Elm ULMACEAE Elm Family alata Michx. Winged E. ARSN GAUG SCCU VAMP FLUF LAAP SCMG VASC GAAB NCDU VACW VAWR GATN NCWT VAGG americana L. American E. DCCG MDCP SCUC TNSC DCNA MDHN TNDR VACW DCWH SCCU TNLW VASC 207 ULMUS VIBURNUM fuscatum Ait. = V. corymbosum, in part myrsinites Lam. Ground B. ALTD FLUF GAJI SCWI VAPH simulatum Small VACW stamineum L. Deerberry ALBG GACG MDLT VACW tenellum Ait. SCBR vacillans Torr. MDLT viurgatum Ait. = V. corymbosum, in part vitis-idaea L. Cowberry, Mountain Cranberry DCNA DEMC ‘Wells Delight’ [V. crassifolium Andr. ssp. crassifolium] NCNS VIBURNUM lL. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) SSS FS SE CY PIS CE, EE Se ET) acertfolium L. Maple-leaved V. GAUG MDLT VAAC * ‘Alleghany’ [V. x rhytidophylloides Suring.] [V. lantana x V. rhytidophyllum] DCNA MDLT alnifolium Marsh. = V. lantanoides ‘Aurantiacum’ [V. setigerum Hance] DCNA MDBG MDGD awabuki K.Koch FLJU FLUF LASL SCCU FLMG LAAL NCDU SCMG ‘Ben Blackburn’ [V. rhytidophyllum Hemsl.] MDKN VIBURNUM ‘Wredei’ [U. minor Mill.] DCPL ‘Wredei’ [U. minor Mill.] DCPL UNGNADIA Endl. SAPINDACEAE Soapberry Family LETT SEE DOT TE Mee ORLY AR, RRR DE EE Pay PRT NG IT MEY AP EE Be FY speciosa Endl. Mexican Buckeye LASL SCWI TXDC TXLL VACCINIUM lL. Blueberry ERICACEAE Heath Family LP ST ANNES TGS ORL E TERN RE SEE ST a amoenum Ait. GASM arboreum Marsh. Farkleberry, Sparkleberry ALBG FLUF FLMG LAHG LALG MSMN SCHS ashei Reade = V. corymbosum, in part atrococcum (A.Gr.) Heller = V. corymbosum, in part ‘Bloodstone’ [V. crassifolium Andr. ssp. sempervirens (Rayner & Henderson) Kirkman & Bal.] NCNS bracteatum Thunb. ex J.A.Murr. DCNA constablaei A.Gr. = V. corymbosum, in part corymbosum L. Highbush B. ALON LAHG SCCU VACW DCNA LALG SCKG VAPH FLDG MDWJ SCWI VAPO GACG SCBR TNMB VATW crassifolium Andr. Creeping B. SCWI cylindraceum Sm. DCNA UNGNADIA Endl. SAPINDACEAE Soapberry Family LETT SEE DOT TE Mee ORLY AR, RRR DE EE Pay PRT NG IT MEY AP EE Be FY speciosa Endl. Mexican Buckeye LASL SCWI TXDC TXLL VACCINIUM lL. Blueberry ERICACEAE Heath Family LP ST ANNES TGS ORL E TERN RE SEE ST a amoenum Ait. GASM arboreum Marsh. Farkleberry, Sparkleberry ALBG FLUF FLMG LAHG LALG MSMN SCHS ashei Reade = V. corymbosum, in part atrococcum (A.Gr.) Heller = V. corymbosum, in part ‘Bloodstone’ [V. crassifolium Andr. ssp. sempervirens (Rayner & Henderson) Kirkman & Bal.] NCNS bracteatum Thunb. ex J.A.Murr. DCNA constablaei A.Gr. = V. corymbosum, in part corymbosum L. Highbush B. ALON LAHG SCCU VACW DCNA LALG SCKG VAPH FLDG MDWJ SCWI VAPO GACG SCBR TNMB VATW crassifolium Andr. Creeping B. SCWI cylindraceum Sm. DCNA elliottii Chapm. = V. ULMUS ALAU DCLC GAJY SCCU ALFN FLMN MDAF DCAC FLUF NCEL * ‘Pioneer’ [U. < hollandica] [U. minor x U. glabra] DCNA procera Salisb. English E. DCCG DCWH MDHN DCSE MDCP pumila L. Siberian E. ALBH DCWR MDGG SCNS ARPT GACL MDMM VAWD DCCG LATU NCCC rubra Muhl. Slippery E. DCCG NCKH VACW MDHN SCMP VAMP ‘Sarniensis’ [U. minor Mill.] Jersey E. DCLP serotina Sarg. September E. NCBE VASC ‘Superba’ [U. < hollandica Mill.] [U. minor < U. glabra] DCGP thomasii Sarg. Rock E. SCCU 208 ULMUS parvifolia Jacq. [illustrator Susan M. Johnston] 209 ULMUS ULMUS corymbosum, in part floridanum (Nutt.) Sleumer = V. stamineum, in part VIBURNUM VIBURNUM betulifolium Batal. Birch-leaved V. GAUG bitchiuense Makino Yeddo V. DCWH GAEC VAPH VARG brachybotryum Hemsl. GACG buddleifolium Wright Buddleja-leaved V. DEWG MDKN burejaeticum Regel & Herd. Manchurian V. MDLT MDTD NU x burkwoodii Burkw. & Skipwith Burkwood’s V. [V. carlesii < V. utile] DCNA MDDF TNMB VAPH DEWG SCCU TNUT GAFN TNDG VACW calvum Rehd. LASL xcarlcephalum Burkw. ex Pike [V. carlesii x V. macrocephalum] ARWS MDLT TNCT VACW DEWG MDME TNMB VAWM carlesii Hemsl. Korean-spice V. DCNA MDMM VANB GAWH TNCT VAPH MDBG TNDG VAWM carlesii Hemsl. var. bitchiuense (Makino) Nakai = V. bitchiuense cassinoides L. Withe-rod V. NCBE TXHN * ‘Catskill’ [V. dilatatum Thunb. ex J.A.Murr.] DCNA MDBG * ‘Cayuga’ [V. xcarlcephalum Burkw. ex Pike] [V. carlesii x V. macrocephalum] DCNA VIBURNUM * ‘Chesapeake’ [V. xcarlcephalum ‘Cayuga’ x V. utile] DCNA GAEC * ‘Chippewa’ [V. japonicum x V. dilatatum] DCNA cinnamomifolium Rehd. Cinnamon-leaved V. LASL ‘Compacta’ [V. carlesii Hemsl.] DEWG * ’Conoy’ [V. x burkwoodii Burkw. & Skipwith] [V. carlesii < V. utile] DCNA cylindricum D.Don GAIS dasyanthum Rehd. MDKN ‘Dawn’ [V. < bodnantense Aberc.] [V. farreri < V. grandiflorum] DEWG MDLT ‘Deben’ [V. x bodnantense Aberc.] [V. farreri < V. grandiflorum] DEWG dentatum L. Arrow-wood MDBG NCBE SCWI MDLT SCBR TNSN dilatatum Thunb. ex J.A.Murr. Linden V. DEWG MDGD SCCU VANB GAEC MDLT VABF VATA MDBG NCBE VAFC * ‘Erie’ [V. dilatatum Thunb. ex J.A.Murr.] DCNA MDBG -erosum Thunb. ex J.A.Murr. GACG MDGD NCSW GAEC MDPJ * ‘Eskimo’ [V. <carlcephalum ‘Cayuga’ x V. utile] DCNA farreri Stearn DEWG foetidum Wall. var. rectangulatum (Graebn.) Rehd. 211 VIBURNUM VIBURNUM macrocephalum Fort. f. macrocephalum Chinese Snowball DCWH GACG MDKN DEWG MDBG VAGS ‘Mariesii’ [V. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.] DCWR MDLA TNDG VATW MDJS MDLT VANB * ‘Mohawk’ [V. <x burkwoodii < V. carlesii] DCNA MDBG * ‘Mohican’ [V. lantana L.] DCNA molle Michx. Poison Haw DEWG ‘Mt. Fuji’ [V. plicatum Fort. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.] DCNA mullaha Buch.-Ham. ex D.Don DCNA ‘Newport’ [V. plicatum Thunb. f. plicatum] GACG MDBG nudum L. Nannyberry Haw, Possum Haw ALBG LAHG SCBR VACW ALUA LALG TNUT obovatum Walt. Walter's V. ALEH DCNA TXPS ALTD SCBR odoratissimum Ker-Gawl. Sweet V. FLEM LAAL MSBN MSMN FLMN LAHG MSEP TXJS odoratissimum Ker-Gawl. var. awabuki (K.Koch) K.Koch ex Ruempler = V. awabuki * ‘Oneida’ [V. dilatatum x V. ULMUS lobophyllum] DCNA * ‘Onondaga’ [V. sargentii Koehne] DCNA opulus L. European Cranberry-bush fordiae Hemsl. TNTV fragrans Bunge = V. farreri x hillieri Stearn [V. erubescens x V. henryi] MDBG hirtulum Rehd. SCCU hupehense Rehd. MDGD TNTV * ‘Huron’ [V. lobophyllum x V. japonicum] DCNA ichangense (Hemsl.) Rehd. MDKN * ‘Iroquois’ [V. dilatatum Thunb. ex J.A.Murr.] DCNA MDBG japonicum (Thunb. ex J.A.Murr.) Spreng. Japanese V. DCNA GAJI MDGD FLCG LALG MDLT FLUF MDBG SCBH x juddii Rehd. [V. bitchiuense < V. carlesii] DEWG MDBG VAPH lantana L. Wayfaring Tree MDJS MDLT NCBE lantanoides Michx. Hobblebush NCDB lentago L. Nannyberry, Sheepberry VACW VAGW lentago L. f. sphaerocarpum (Fern.) Rehd. MDBG lobophyllum Graebn. fordiae Hemsl. TNTV fragrans Bunge = V. farreri x hillieri Stearn [V. erubescens x V. henryi] MDBG hirtulum Rehd. SCCU hupehense Rehd. MDGD TNTV * ‘Huron’ [V. lobophyllum x V. japonicum] DCNA ichangense (Hemsl.) Rehd. MDKN * ‘Iroquois’ [V. dilatatum Thunb. ex J.A.Murr.] DCNA MDBG japonicum (Thunb. ex J.A.Murr.) Spreng. Japanese V. DCNA GAJI MDGD FLCG LALG MDLT FLUF MDBG SCBH x juddii Rehd. [V. bitchiuense < V. carlesii] DEWG MDBG VAPH lantana L. Wayfaring Tree MDJS MDLT NCBE lantanoides Michx. Hobblebush NCDB lentago L. Nannyberry, Sheepberry VACW VAGW lentago L. f. sphaerocarpum (Fern.) Rehd. MDBG lobophyllum Graebn. DEWG MDBG luzonicum Rolfe Philippine V. TXLL TXSE macrocephalum Fort. f. keteleeri (Carr.) Rehd. NOD —_ iS) VIBURNUM ovatifolium Rehd. DEWG plicatum Thunb. f. lanceolatum (Rehd.) Rehd. TNUT plicatum Thunb. f. parvifolium (Miq.) Rehd. MDKN VATW plicatum Thunb. f. plicatum Japanese Snowball ALBH MDJS NCCA VAMP DCDO MDKN NCSM VASK GACG MDLA VAAC MDBG NCBE VACW plicatum Thunb. var. rotundifolium Rehd. Round-leaved double-file V. ARPT MDDF plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. Double-file V. ALBH GACG MDKN VACW DCWH GAFN NCGC VAPH DEWG MDBG VAAC VASK ‘Pragense’ [V. rhytidophyllum x V. utile] TNTV VAGS VATA prunifolium L. Black Haw, Sheepberry DCNA MDLA TNMB VAMP GARW MDLT TNRG VAPO MDBA NCBE VACW VATA MDBG SCCU VAGS MDKN TNDG VAGW recognitum Fern. Northern Arrow-wood DECG LASL VACW VAGW ‘Reticulatum’ [V. sieboldii Miq.] MDPJ VADW x rhytidocarpum E.Lemoine [V. buddleifolium < V. rhytidophyllum] GACG MDBG TNPW VATW x rhytidophylloides Suring. [V. lantana x V. rhytidophyllum] GACG MDPJ VATA MDKN VAMP rhytidophyllum Hemsl. VIBURNUM ‘Roseum’ [V. opulus L.] Common Snowball, European Snowball GAOH VACW VAGW MDTD VADW VASP ‘Roseum’ [V. rhytidophyllum Hemsl.] MDKN rufidulum Raf. ULMUS Rusty Black Haw, Southern Black Haw ALTD LARP TNSG GACG MDGJ TXMK LAAL SCBR TXRS ‘St. Keverne’ [V. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.] TNTV sargentii Koehne MDBG MDGD VACW sargentii Koehne var. calvescens Rehd. SCCU * ‘Seneca’ [V. sieboldii Miq.] DCNA MDBG setigerum Hance Tea-leaved V. GAAB MDBG MDPJ VABF GACG MDDF NCBE VATW GAEC MDJS SCCU VAWR GAEH MDKN TNMB GAIS MDLT TNSN * ‘Shasta’ [V. plicatum f. tomentosum < V. plicatum f. tomentosum ‘Mariesii’] DCNA * ‘Shoshoni’ [V. plicatum Fort. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.] DCNA sieboldii Miq. ree We DCCG MDJH MDLT VATA DEMC MDKN MDWS DEWG MDLA TNUT stellulatum Wall. = V. mullaha suspensum Lindl. ALEH GAFN LAGN FLMG GAJI SCBR FLUF LAAL TXSH * ‘Susquehanna’ [V. sargentii Koehne] DCNA theiferum Rehd. = V. setigerum VIBURNUM ‘Roseum’ [V. opulus L.] Common Snowball, European Snowball GAOH VACW VAGW MDTD VADW VASP ‘Roseum’ [V. rhytidophyllum Hemsl.] MDKN rufidulum Raf. Rusty Black Haw, Southern Black Haw ALTD LARP TNSG GACG MDGJ TXMK LAAL SCBR TXRS ‘St. Keverne’ [V. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.] TNTV sargentii Koehne MDBG MDGD VACW sargentii Koehne var. calvescens Rehd. SCCU * ‘Seneca’ [V. sieboldii Miq.] DCNA MDBG setigerum Hance Tea-leaved V. GAAB MDBG MDPJ VABF GACG MDDF NCBE VATW GAEC MDJS SCCU VAWR GAEH MDKN TNMB GAIS MDLT TNSN * ‘Shasta’ [V. plicatum f. tomentosum < V. plicatum f. tomentosum ‘Mariesii’] DCNA * ‘Shoshoni’ [V. plicatum Fort. f. tomentosum (Thunb. ex J.A.Murr.) Rehd.] DCNA sieboldii Miq. ree We DCCG MDJH MDLT VATA DEMC MDKN MDWS DEWG MDLA TNUT stellulatum Wall. = V. mullaha suspensum Lindl. ALEH GAFN LAGN 213 VIBURNUM VIBURNUM WASHINGTONIA VIBURNUM tinus L. Laurustinus FLUF MSMN SCFW VACW GACG NCEG SCHW LALG NCTP SCMG LASL SCBR SCMP trilobum Marsh. Highbush Cranberry DCDO NCAS VARG MDBG NCCA urceolatum Sieb. & Zucc. DCNA utile Hemsl. GAEC MDBG NCEL SCWI ‘Variegatum’ [V. rhytidophyllum Hemsl.] MDBG ‘Willow Leaf [V. x rhytidophylloides Suring.] [V. lantana < V. rhytidophyllum] DEWG GACG ‘Willowwood’ [V. < rhytidophylloides Suring.] [V. lantana < V. rhytidophyllum] TNPW wrightii Miq. MDGD MDJS MDKN VABF VINCA L. Periwinkle APOCYNACEAE Dogbane Family ‘Alba’ [V. minor L.] MDLT MDSM ‘Atropurpurea’ [V. minor L.] VAPH major L. Greater P. FLDG FLMG MDWP VAWR minor L. Common P. MDLT VACW ‘Multiplex’ [V. minor L.] Double-flowered P. DEWG ‘Oxyloba’ [V. major L.] MDFM ‘Variegata’ [V. major L.] tinus L. ULMUS Laurustinus FLUF MSMN SCFW VACW GACG NCEG SCHW LALG NCTP SCMG LASL SCBR SCMP trilobum Marsh. Highbush Cranberry DCDO NCAS VARG MDBG NCCA urceolatum Sieb. & Zucc. DCNA utile Hemsl. GAEC MDBG NCEL SCWI ‘Variegatum’ [V. rhytidophyllum Hemsl.] MDBG ‘Willow Leaf [V. x rhytidophylloides Suring.] [V. lantana < V. rhytidophyllum] DEWG GACG ‘Willowwood’ [V. < rhytidophylloides Suring.] [V. lantana < V. rhytidophyllum] TNPW wrightii Miq. MDGD MDJS MDKN VABF VINCA L. Periwinkle APOCYNACEAE Dogbane Family ‘Alba’ [V. minor L.] MDLT MDSM ‘Atropurpurea’ [V. minor L.] VAPH major L. Greater P. FLDG FLMG MDWP VAWR minor L. Common P. MDLT VACW ‘Multiplex’ [V. minor L.] Double-flowered P. DEWG VITEX L. VERBENACEAE Verbena Family ae ae aa a a a TE agnus-castus L. Chaste Tree ALAU LAMP NCBE VACP ALBG MDBG NCSM VACW DCCG MDBR SCBR GAAB MDFD SCCU LAAL MDWP SCMP ‘Incisa’ [V. negundo L.] FLUF VACW negundo L. FLUF GAIS rotundifolia L.f. MDFM ‘Silver Spire’ [V. agnus-castus L.] DCNA ‘Variegata’ [V. trifolia L.] FLUF VITIS L. Grape VITACEAE Grape Family EE SS SE EE EE ERE \| candicans Engelm. = V. mustangensis labrusca L. Fox G. NCDU mustangensis Buckl. Mustang G. DCNA rotundifolia Michx. Muscadine G., Scuppernong NCMO SCPL WASHINGTONIA H.Wendl. Washington Palm ARECACEAE Palm Family \| US a NT robusta H.Wendl. LAAL WISTERIA WIKSTROEMIA Endl. > THYMELAEACEAE Mezereum Family trichotoma (Thunb.) Makino DCNA WISTERIA Nutt. FABACEAE (Faboideae) Wisteria Bean Family ‘Alba’ [W. brachybotrys Sieb. & Zucc.] DCNA ‘Alba’ [W. floribunda (Willd.) DC.] DCDO MDBG NCDU VAGS DEEM MDPJ TNCT VANB ‘Alba’ [W. sinensis (Sims) Sweet] FLMN SCLO SCWI VAGS GASM SCMG TXLL brachybotrys Sieb. & Zucc. DCNA floribunda (Willd.) DC. Japanese W. ALBG DEEM MDBG SCMG DCCC FLUF MDJH TNSC DCDO GAHC MSHB VAGS DCNA GANG NCCB VANB DCWH GASS SCDB < formosa Rehd. [W. floribunda x W. sinensis] ALEH FLUF MSEN SCFW DCCC GAJI MSHB VATW FLCG LAAL SCBR frutescens (L.) Poir. American W. DCNA GALF SCWI VACW GAFN SCBR TXWE ‘Honbeni’ [W. floribunda (Willd.) DC.] DCNA ‘Itoe kokuryw’ [W. floribunda (Willd.) DC.] DCNA ‘Jabo’ [W. floribunda (Willd.) DC.] DCNA ‘Koshigaya’ [W. floribunda (Willd.) DC.] MDGD ‘Macrobotrys’ [W. floribunda (Willd.) DC.] MDBG macrostachya Nutt. = W. frutescens WISTERIA WEIGELA WEIGELA WEIGELA WEIGELA Thunb. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) ‘Bristol Ruby’ [W. florida x W. ‘Eva Rathke’] VAMP decora (Nakai) Nakai DCDO NCBE ‘Eva Rathke’ [W. floribunda x W. ULMUS coraeensis] DEWG ‘Feerie’ (Hybrid of unknown parentage) DEWG florida (Bunge) A.DC. ALBH MDLA LAHG SCMG VACW hortensis (Sieb. & Zucc.) K.Koch FLMG NCBE SCCU VALU x hybrida Jaeger = Untenable name for unassigned hybrids ‘Mont Blanc’ (Hybrid of unknown parentage) DEWG ‘Newport Red’ (Hybrid of unknown parentage) DEWG ‘Rosea’ [W. praecox (V.Lemoine) Bailey] DCNA subsessilis (Nakai) Bailey DCNA ‘Vanicek’ = ‘Newport Red’ ‘Variegata’ [W. florida (Bunge) A.DC.] MDPJ VAGS WESTRINGIA Sm. LAMIACEAE Mint Family fruticosa (Willd.) Druce = W. rosmariniformis rosmariniformis Sm. FLUF WEIGELA Thunb. CAPRIFOLIACEAE Honeysuckle Family (Contributed by T. R. Dudley) ‘Bristol Ruby’ [W. florida x W. ‘Eva Rathke’] VAMP decora (Nakai) Nakai DCDO NCBE ‘Eva Rathke’ [W. floribunda x W. coraeensis] DEWG ‘Feerie’ (Hybrid of unknown parentage) DEWG florida (Bunge) A.DC. ALBH MDLA LAHG SCMG VACW hortensis (Sieb. & Zucc.) K.Koch FLMG NCBE SCCU VALU x hybrida Jaeger = Untenable name for unassigned hybrids ‘Mont Blanc’ (Hybrid of unknown parentage) DEWG ‘Newport Red’ (Hybrid of unknown parentage) DEWG ‘Rosea’ [W. praecox (V.Lemoine) Bailey] DCNA subsessilis (Nakai) Bailey DCNA ‘Vanicek’ = ‘Newport Red’ ‘Variegata’ [W. florida (Bunge) A.DC.] MDPJ VAGS WESTRINGIA Sm. LAMIACEAE Mint Family fruticosa (Willd.) Druce = W. rosmariniformis WISTERIA Nutt. FABACEAE (Faboideae) Wisteria Bean Family ‘Alba’ [W. brachybotrys Sieb. & Zucc.] DCNA ‘Alba’ [W. floribunda (Willd.) DC.] DCDO MDBG NCDU VAGS DEEM MDPJ TNCT VANB ‘Alba’ [W. sinensis (Sims) Sweet] FLMN SCLO SCWI VAGS GASM SCMG TXLL brachybotrys Sieb. & Zucc. DCNA floribunda (Willd.) DC. Japanese W. ALBG DEEM MDBG SCMG DCCC FLUF MDJH TNSC DCDO GAHC MSHB VAGS DCNA GANG NCCB VANB DCWH GASS SCDB < formosa Rehd. [W. floribunda x W. sinensis] ALEH FLUF MSEN SCFW DCCC GAJI MSHB VATW FLCG LAAL SCBR frutescens (L.) Poir. American W. DCNA GALF SCWI VACW GAFN SCBR TXWE ‘Honbeni’ [W. floribunda (Willd.) DC.] DCNA ‘Itoe kokuryw’ [W. floribunda (Willd.) DC.] DCNA ‘Jabo’ [W. floribunda (Willd.) DC.] DCNA ‘Koshigaya’ [W. floribunda (Willd.) DC.] MDGD ‘Macrobotrys’ [W. floribunda (Willd.) DC.] MDBG macrostachya Nutt. = W. frutescens 215 WISTERIA ZAMIA ‘Murasaki kapitan’ [W. brachybotrys Sieb. & Zucc.] DCNA ‘Nivea’ [W. frutescens (L.) Poir.] SCWI ‘Noda’ [W. floribunda (Willd.) DC.] DCNA ‘Ossai’ [W. floribunda (Willd.) DC.] DCNA ‘Rosea’ [W. floribunda (Willd.) DC.] DCNA GASM VAGS DEEM MDBG ‘Shino kapitan’ [W. floribunda (Willd.) DC.] DCNA sinensis (Sims) Sweet Chinese W. DCDO FLMG MSHB SCWI DEWG GAVI NCGP VAGS FLDG LASL SCPP ‘Siro kapitan’ [W. venusta Rehd. & Wils.] DCNA villosa Rehd. ARRB DCDO DCNA ‘Violacea Plena’ [W. floribunda (Willd.) DC.] DCNA GAWP NCOP GASM MDBG SCMP XANTHOCERAS Bunge SAPINDACEAE Soapberry Family sorbifolium Bunge Yellowhorn DEWG VAGS VAPH XANTHORHIZA Marsh. RANUNCULACEAE Buttercup Family simplicissima Marsh. Shrub Yellowroot SCWI VAPO XYLOSMA J.G.Forst. FLACOURTIACEAE Flacourtia Family congestum (Lour.) Merr. FLUF LASL TXSH LAAL NCOP Sflexuosum (HBK.) Hemsl. FLUF senticosum Hance = X. congestum YUCCA L. Adam’s Needle, Spanish Dagger AGAVACEAE Agave Family ‘Bright Edge’ [Y. filamentosa L.] VAGS filamentosa L. Adam's Needle TNHS filamentosa L. var. smalliana (Fern.) Ahles = Y. smalliana gloriosa L. Spanish Dagger DCCG FLMG NCRM DCNA LAAL ‘Golden Sword’ [Y. filamentosa L.] VAGS ‘Marginata’ [Y. aloifolia L.] Spanish Bayonet ALBG LAAL recurvifolia Salisb. VAGS VATA smalliana Fern. VATA whipplei Torr. Our-Lord’s-Candle ALBG ZAMIA L. CYCADACEAE Cycad Family floridana A.DC. = Z. pumila integrifolia Ait. = Z. pumila XYLOSMA J.G.Forst. FLACOURTIACEAE Flacourtia Family congestum (Lour.) Merr. FLUF LASL TXSH LAAL NCOP Sflexuosum (HBK.) Hemsl. FLUF senticosum Hance = X. congestum YUCCA L. Adam’s Needle, Spanish Dagger AGAVACEAE Agave Family ‘Bright Edge’ [Y. filamentosa L.] VAGS filamentosa L. Adam's Needle TNHS filamentosa L. var. smalliana (Fern.) Ahles = Y. smalliana gloriosa L. Spanish Dagger DCCG FLMG NCRM DCNA LAAL ‘Golden Sword’ [Y. filamentosa L.] VAGS ‘Marginata’ [Y. aloifolia L.] Spanish Bayonet ALBG LAAL recurvifolia Salisb. VAGS VATA smalliana Fern. VATA whipplei Torr. Our-Lord’s-Candle ALBG ZAMIA L. CYCADACEAE Cycad Family ZAMIA pumila L. Coontie FPLCG FLMG GACG GASM FLDG FLUF GAJI ZANTHOXYLUM lL. Prickly Ash RUTACEAE Citrus Family CE a PS EEE ee RS NE] bungei Planch. = Z. simulans clava-herculis L. Southern P. A. FLUF LAAL VACW FLWC MSMN fagara (L.) Sarg. Wild Lime TXPH piperitum DC. DCNA simulans Hance GAEC GAIS TNSG GAEH MDPL VABF ZELKOVA Spach ULMACEAE Elm Family acuminata (Lindl.) Planch. = Z. serrata carpinifolia (Pall.) K.Koch Caucasian Z. DCLC DCUS crenata Spach = Z. carpinifolia schneideriana Hand.-Mazz. WISTERIA DCAC DCCG MDBG MDGD serrata (Thunb.) Makino Japanese Z., Keaki ALIT DCGC MDBG MDJH DCCG DCGP MDGG TNDR DCEL GAIS MDIH x verschaffeltii Nichols. [Z. carpinifolia < Z. serrata] MDGD ZIZIPHUS ZIZIPHUS Miill. RHAMNACEAE Buckthorn Family ee eee Ro eee) ‘Contorta’ [Z. jujuba Mill.] TNSN Jujuba Mill. Jujube DCCG GAIS MSEP TNNE DCNA GALE MSMN TNRM GAHC LAAL SCWI VABF Ant.: Franz Antoine, 1815-1886, Austria (Coniferae) Arn.: George Arnott (Arnold) Walker Arnott, 1799-1868, United Kingdom Asami: Yoshichi Asami, 1894— , Japan (Malus) Ashe: William Willard Ashe, 1872-1932, United States Aubl.: Jean Baptiste Christophore Fusée Aublet, 1720-1778, France Audubon: John James Audubon, 1785-1851, United States BSP.: See Britton; Sterns; Poggenburg Bailey: Liberty Hyde Bailey, 1858-1954, United States (cultivated plants) Baill.: Henri Ernest Baillon, 1827-1895, France Baker: John Gilbert Baker, 1834-1920, United Kingdom Bal.: Benedict Balansa, 1825-1892, France Balf.f.: Isaac Bayley Balfour, 1853-1922, United Kingdom Ballington: James R. Ballington, contemporary, United States (horticultural plants) Banks: Joseph Banks, 1743-1820, United Kingdom Barbier: Barbier, fl. 1911, France (nurseryman) Barkley, F.A.: Fred Alexander Barkley, 1908-1989, United States Barratt: Joseph Barratt, 1796-1882, United States (Salix) Appendix A: Authors Cited Appendix A: Authors Cited Abel: Clarke Abel, 1789-1826, United Kingdom Aberc.: Henry Duncan McLaren, 2nd Baron Aberconway, 1879-1953, United Kingdom Adams: William Preston Adams, 1930- , United Kingdom (Hypericum) Adans.: Michel Adanson, 1727-1806, France Ahles: Harry E. Ahles, 1924-1981, United States Ahrendt: Leslie Walter Allen Ahrendt, 1903-1969, United Kingdom (Berberis, Mahonia) Airy-Shaw: Herbert Kenneth Airy-Shaw, 1902-1985, United Kingdom Ait.: William Aiton, 1731-1793, United Kingdom (cultivated plants) Ait.f.: William Townsend Aiton, 1766-1849, United Kingdom (cultivated plants) Alex.: Edward Johnston Alexander, 1901-1985, United States Ames, L.M.: Lawrence M. Ames, 1900-1966, United States Anderss.: Nils Johan Andersson, 1821-1880, Sweden (Salix) Andr.: Henry Charles Andrews, 1752?-1830, United Kingdom Andre: Edouard-Francois André, 1840-1911, France Andrews, S.: Susyn Andrews, 1953— , United Kingdom (Ilex) Anliker, J.: Johann Anliker, fl. 1931, Switzerland Andre: Edouard-Francois André, 1840-1911, France Andrews, S.: Susyn Andrews, 1953— , United Kingdom (Ilex) Anliker, J.: Johann Anliker, fl. 1931, Switzerland 2S Batal.: Alexander Theodorowicz Batalin, 1847-1896, Russia Batsch: August Johann Georg Karl Batsch, 1761-1802, Germany (horticulture) Beadle: Chauncey Delos Beadle, 1866-1950, United States Bean: William Jackson Bean, 1863-1947, United Kingdom (dendrology) Becc.: Odoardo Beccari, 1843-1920, Italy (palms) Bechst.: Johann Matthaeus Bechstein, 1757-1822, Germany Beissn.: Ludwig Beissner, 1843-1927, Germany (Coniferae) Benn.: John Joseph Bennett, 1801-1876, United Kingdom Benson, L.: Lyman David Benson, 1909-1993, United States Benth.: George Bentham, 1800-1884, United Kingdom Bercht.: Friedrich von Berchtold, 1781-1876, Czechoslovakia Berg, O.: Otto Karl Berg, 1815-1866, Germany Bergius: Peter Jonas Bergius, 1730-1790, Sweden Berl.: Jean Louis Berlandier, 1805-1851, France Berth.: Sabin Berthelot, 1794-1880, France Bieb.: Friedrich August Marschall von Bieberstei 1768-1826, Germany, Russia Bl.: Karl Ludwig von Blume, 1796-1862, Netherlands Blake, S.F.: Sydney Fay Blake, 1892-1959, United States Blanco: Francisco Manuel Blanco, 1780-1845, Spain, Philippines. Bois: Désiré Georges Jean Marie Bois, 1856-1946, France Boiss.: Pierre Edmond Boissier, 1810-1885, Switzerland Bonpland: Aimé Jacques Alexandre Bonpland, 1773-1858, France, South America Boom: Boudewijn Karel Boom, 1903-1980, Netherlands (cultivated plants) Booth: J.B. Booth, 1836-1908, Germany (nurseryman) Bord.-Rey: O. Bordéres-Rey, fl. Appendix A: Authors Cited 1939-68, France (Coniferae) Borkh.: Moritz Balthasar Borkhausen, 1760-1806, Germany Bory: Jean Baptiste Genevieve Marcellin Bory de Saint-Vincent, 1778-1846, France Bosc: Louis Augustin Guillaume Bosc, 1759-1828, France Boulenger: George Albert Boulenger, 1858-1937, Belgium (Rosa) Br., N.E.: Nicholas Edward Brown, 1849-1934, United Kingdom Br., P.: Patrick Browne, 1720-1790, Ireland Br., R.: Robert Brown, 1773-1858, United Kingdom Brandegee: Townshend Stith Brandegee, 1843-1925, United States Braun, A.: Alexander Karl Heinrich Braun, 1805-1877, Germany Braun, E.: Emma Lucy Braun, 1889-1971, United States Brickell: Christopher David Brickell, 1932-— , United Kingdom (cultivated plants) 219 Camus, E.G.: Edmond Gustav Camus, 1852-1915, France Candolle: See DC. Carr.: Elie Abel Carriére, 1818-1896, France (dendrology) Carruthers: William Carruthers, 1830-1922, United Kingdom Cav.: Antonio José Cavanilles, 1745-1804, Spain Chabaud: J. Benjamin Chabaud, 1833-1915, France Cham.: Ludolf Karl Adalbert von Chamisso, 1781-1838, Germany Chamb.: Charles Joseph Chamberlain, 1863-1943, United States Chambr.: Georges de Chambray, 1783-1849, France Champ.: John George Champion, 1815-1854, United Kingdom Chao: Jew Ming Chao, fl. 1977, China Chapm.: Alvan Wentworth Chapman, 1809-1899, United States Chaub.: Louis Athanase (Anastase) Chaubard, 1785-1854, France Cheng, M.: Mien Cheng, fl. Appendix A: Authors Cited 1956-1980, China Cheng, W.C.: Wan Chun Cheng, 1904-1983, China (dendrology) Chev., A.: Auguste Jean Baptiste Chevalier, 1873-1956, France Chittenden: Frederick James Chittenden, 1873-1950, United Kingdom (cultivated plants) Christ: Konrad Hermann Heinrich Christ, Briot, C.: Pierre Louis (Charles, in error) Briot, 1804-1888, France Briq.: John Isaac Briquet, 1870-1931, Switzerland Britton: Nathaniel Lord Britton, 1859-1934, United States Brongn.: Adolphe Théodore Brongniart, 1801-1876, France Browicz: Kazimierz Browicz, 1925- , Poland (dendrology) Buch.-Ham.: Francis Buchanan-Hamilton, 1762-1829, United Kingdom Buchh., J.: John Theodore Buchholz, 1888-1951, United States Buc’hoz: Pierre Joseph Buc’hoz, 1731-1807, France Buckl.: Samuel Botsford Buckley, 1809-1884, United States Buerger: Heinrich Burger, 1806-1858, Germany, Netherlands Bunge: Alexander Andrejewitsch von Bunge, 1803-1890, Russia Bur.: Louis Edouard Bureau, 1830-1918, France Burkw.: Arthur Burkwood, 1888-1951, United Kingdom (nurseryman) Burm.: Johannes Burman, 1707-1779, Netherlands Burm.f.: Nikolaas Laurens Burman, 1734-1793, Netherlands Burret: (Maximilian) Karl Ewald Burret, 1883-1964, Germany Burv.: Fréderic Burvenich, 1857-1917, Belgium Cambess.: Jacques Cambessédes, 1799-1863, France Camus, A.A.: Aimée Antoinette Camus, 1879-1965, France Curtis: William Curtis, 1746-1799, United Kingdom > Curtis, M.A.: Moses Ashley Curtis, 1808-1872, United States DC.: Augustin Pyramus de Candolle, 1778-1841, Switzerland DC., A.: Alphonse Louis Pierre Pyramus de Candolle, 1806-1893, Switzerland Dallim.: William Dallimore, 1871-1959, United Kingdom (horticulture, dendrology) Dandy: James Edgar Dandy, 1903-1976, United Kingdom (Magnolia) Davis: Peter Hadland Davis, 1918-1992, United Kingdom Decne.: Joseph Decaisne, 1807-1882, France Dehnh.: Friedrich Dehnhardt, 1787-1870, Germany de Lannoy: de Lannoy, fl. 1863, France Deppe: Ferdinand Deppe, 1794-1861, Germany Deseglise: Pierre Alfred Déséglise, 1823-1883, France Desf.: René Louiche Desfontaines, 1750-1833, France Desm.: Yves Desmarais, 1918- , Canada ‘Desmoul.: Charles Robert des Moulins, 1798-1875, France Desp.: Narcisse Henri Francois Desportes, 1776-1856, France Desr.: Louis Auguste Joseph Desrousseaux, 1753-1838, France Curtis: William Curtis, 1746-1799, United Kingdom > Curtis, M.A.: Moses Ashley Curtis, 1808-1872, United States DC.: Augustin Pyramus de Candolle, 1778-1841, Switzerland DC., A.: Alphonse Louis Pierre Pyramus de Candolle, 1806-1893, Switzerland Dallim.: William Dallimore, 1871-1959, United Kingdom (horticulture, dendrology) Dandy: James Edgar Dandy, 1903-1976, United Kingdom (Magnolia) Davis: Peter Hadland Davis, 1918-1992, United Kingdom Decne.: Joseph Decaisne, 1807-1882, France Dehnh.: Friedrich Dehnhardt, 1787-1870, Germany de Lannoy: de Lannoy, fl. Appendix A: Authors Cited 1863, France Deppe: Ferdinand Deppe, 1794-1861, Germany Deseglise: Pierre Alfred Déséglise, 1823-1883, France Desf.: René Louiche Desfontaines, 1750-1833, France Desm.: Yves Desmarais, 1918- , Canada ‘Desmoul.: Charles Robert des Moulins, 1798-1875, France Desp.: Narcisse Henri Francois Desportes, 1776-1856, France , tuart, Curtis: William Curtis, 1746-1799, United Ki Curtis, M.A.: Moses As 1808-1872, United Sta DC.: Augustin Pyramus 1778-1841, Switzerlan DC., A.: Alphonse Loui Pyramus de Candolle, 1806-1893, Switzerlan Dallim.: William Dallim Cochet: Pierre Charles Marie Cochet, 1866-1936, France Cockerell: Theodore Dru Alison Cockerell, 1866-1948, United States Cogn.: Célestin Alfred Cogniaux, 1841-1916, Belgium Cohen-Stuart: Combertus Pieter Cohen-Stuart, 1889-1945, Netherlands, Java Coll.: Henry Collett, 1836-1901, United Kingdom Comber: Harold Frederick Comber, 1897-1969, United Kingdom Comm.: Philibert Commerson, 1727-1773, France Cook: Orator Fuller Cook, 1867-1949, United States Corr.: Louis Henry Correvon, 1854-1939, Switzerland Correa: José Francisco Corréa da Serra, 1751-1823, Portugal Correll: Donovan Stewart Correll, 1908-1983, United States Cory: Victor Louis Cory, 1880-1964, United States Coult.: John Merle Coulter, 1851-1928, United States Cov.: Frederick Vernon Coville, 1867-1937, United States Cowan: John Macqueen Cowan, 1891-1960, United Kingdom Craib: William Grant Craib, 1882-1933, United Kingdom Crantz: Heinrich Johann Nepomuk von Crantz, 1722-1797, Austria Crepin: Francois Crépin, 1830-1903, Belgium Croom: Hardy Bryan Croom, 1797-1837, United States Za Dum.-Cours.: George Louis Marie Dumont de Courset, 1746-1824, France Dunal: Michel Felix Dunal, 1789-1856, France Dunn, S.T.: Stephen Troyte Dunn, 1868-1938, United Kingdom Durande: Jean Francois Durande, 1732-1794, France Durazz.: Antonio Durazzini, ii 2— tally: Durieu: Michel Charles Durieu de Maisonneuve, 1796-1878, France Du Roi: Johann Philipp Du Roi, 1741-1785, Germany Eade: George William Eade, 1905-— , United States Eberm.: Carl Heinrich Ebermaier, 1802-1870, Germany Eckl.: Christian Friedrich Ecklon, 1795-1868, Germany Edgew.: Michael Pakenham Edgeworth, 1812-1881, United Kingdom Egglest.: Willard Webster Eggleston, 1863-1935, United States Ehrenb., K.: Karl August Ehrenberg, 1801-1849, Germany Ehrh.: Jakob Friedrich Ehrhart, 1742-1795, Germany Ell.: Stephen Elliot, 1771-1830, United States Ellis: John Ellis, 1710-1776, United Kingdom Emeric: D. Emeric, fl. ca. 1828, France Dicks.: George Frederick Dickson, fl. 1839, United Kingdom Dieck: Georg Dieck, 1847-1925, Germany Diels: Friedrich Ludwig Emil Diels, 1874-1945, Germany Dietr., A.: Albert Gottfried Dietrich, 1795-1856, Germany Dietr., D.: David Nathaniel Friedrich Dietrich, 1799-1888, Germany Dipp.: Leopold Dippel, 1827-1914, Germany (dendrology) Dode: Louis-Albert Dode, 1875-1943, France Don, D.: David Don, 1799-1841, United Kingdom Don, G.: George Don, 1798-1856, United Kingdom Donn, J.: James Donn, 1758-1813, United Kingdom Doorenbos: J. Appendix A: Authors Cited Doorenbos, 1921- , Netherlands (dendrology) Dougl.: David Douglas, 1798-1834, United Kingdom Drake: Emmanuel Drake del Castillo, 1855-1904, France Dress, W.J.: William John Dress, 1918- , United States (cultivated plants) Druce: George Claridge Druce, 1850-1932, United Kingdom Drude: Carl Georg Oscar Drude, 1852-1933, Germany Dudley, T.R.: Theodore Robert Dudley, 1936-— , United States (Ilex, Viburnum, cultivated plants) Duham.: Henri Louis Duhamel du Monceau, 1700-1782, France Dudley, T.R.: Theodore Robert Dudley, 1936-— , United States (Ilex, Viburnum, cultivated plants) Endl.: Stephan Friedrich Ladislaus Endlicher, 1804-1849, Austria Endress, P.: Peter Karl Endress, 1942- , Switzerland (Hamamelidaceae) 222 Fosberg: Francis R 1908— , United Sta Frahm: G. Frahm, fl. 1898-— , German Franch.: Adrien Re 1834-1900, France Franco: Joao Manue do Amaral Franco, 1921—, Portugal Engelm.: George Engelmann, 1809-1884, United States Engl.: Heinrich Gustav Adolf Engler, 1844-1930, Germany Exell: Arthur Wallis Exell, 1901- , United Kingdom Fabr.: Philipp Conrad Fabricius, 1714-1774, Germany Fedde: Friedrich Karl Georg Fedde, 1873-1942, Germany Ferguson, A.R.: Allan Ross Ferguson, 1943- , New Zealand (Actinidia) Fern.: Merritt Lyndon Fernald, 1873-1950, United States Fischer: Friedrich Ernst Ludwig von Fischer, 1782-1854, Russia Fitschen: Jost Fitschen, 1869-1947, Germany (Coniferae) Fletcher: Fletcher & Sons Nursery, fl. 1913, United Kingdom Flinck: Karl Evert Flinck, 1915-— , Sweden Florin: Carl Rudolph Florin, 1894-1965, Sweden Forbes: James Forbes, 1773-1861, United Kingdom Foret: James Aloysius Foret, 1921- , United States Forrest: George Forrest, 1873-1932, United Kingdom (collector in China) Forssk.: Pehr Forsskal, 1732-1763, Finland Forst., J.G.: Johann Georg Adam Forster, 1754-1794, Germany Forst., J.R.: Johann Reinhold Forster, 1729-1798, Germany Fort.: Robert Fortune, 1812-1880, United Kingdom (collector in China) 223 Gleditsch: Johann Gottlieb Gleditsch, 1714-1786, Germany Glend.: Robert Glendinning, 1805-1862, United Kingdom Gmel., J.F.: Johann Friedrich Gmelin, 1748-1804, Germany Godfrey: Robert Kenneth Godfrey, 1911- , United States Godr.: Dominique Alexandre Godron, 1807-1880, France Gord.: George Gordon, 1806-1879, Ireland (Coniferae) Gr., A.: Asa Gray, 1810-1888, United States Graebn.: Karl Otto Robert Peter Paul Graebner, 1871-1933, Germany Graham, R.: Robert C. Graham, 1786-1845, United Kingdom Green, P.S.: Peter Shaw Green, 1920-— , United Kingdom Greene: Edward Lee Greene, 1843-1915, United States Gren.: Jean Charles Marie Grenier, 1808-1875, France Griseb.: August Heinrich Rudolph Grisebach, 1814-1879, Germany Grootend.: Herman Johannes Grootendorst, 1911- , Netherlands (dendrology, Rhododendron) Guill.: André Guillaumin, 1885-1974, France Gumbleton: William Edward Gumbleton, 1840-1911, Ireland HBK.: See Humboldt; Bonpland; Kunth Haenke: Thaddaus Peregrinus Xaverius Haenke, 1761-1817, Czechoslovakia Hance: Henry Fletcher Hance, 1827-1886, United Kingdom, China Hand.-Mazz.: Heinrich R. E. Appendix A: Authors Cited Handel-Mazze 1882-1940, Austria Hansen, N.E.: Niels Ebbesen Hansen, 1866-1950, Denmark Hara: Hiroshi Hara, 1911-1986, Japan Hariot: Paul Auguste Hariot, 1854-1917, France Harrow, R.: Robert Lewis Harrow, 1867-1954, United Kingdom Hartm.: Carl Johan Hartman, 1790-1849, Sweden Hartw.: Carl Theodor Hartweg, 1812-1871, Germany Harv.: William Henry Harvey, 1811-1866, Ireland Hassk.: Justus Karl Hasskarl, 1811-1894, Germany, Java Hatusima: Sumihiko Hatusima, 1906— , Japan (dendrology) Hayata: Bunzo Hayata, 1874-1934, Japan, Taiwan Hayne: Friedrich Gottlob Hayne, 1763-1832, Germany Hedl.: Johan Theodor Hedlund, 1861-1953, Sweden Hedr.: Ulysses Prentiss Hedrick, 1870-1951, United States Hedwig, R.: Romanus Adolf Hedwig, 1772-1806, Germany Heller: Amos Arthur Heller, 1867-1944, United States Hemsl.: William Botting Hemsley, 1843-1924, United Kingdom Henderson: James Henderson, fl. 1980, United States Henry, A.: Augustine Henry, 224 Horton: James H. Horton, 1931- , United States = Horvath: Michael Henry Horvath, fl. 1934, United States (Berberis) House, H.: Homer Doliver House, 1878-1949, United States Houtt.: Maarten Houttuyn, 1720-1798, Netherlands Houz. de Leh.: Jean Auguste-Hippolyte Houzeau de Lehaie, 1867-1959, Belgium (bamboo) Hu, H.H.: Hsen Hsu Hu (Xian Su Hu), 1894-1968, China (dendrology) Hu, S.Y.: Shiu-Ying Hu, 1910— , United States, China Hubb., F.T.: Frederic Tracy Hubbard, 1875-1962, United States Huds.: William Hudson, 1730-1793, United Kingdom Hull: John Hull, 1761-1843, United Kingdom Humboldt: Friedrich Wilhelm Heinrich Alexander von Humboldt, 1769-1859, Germany, South America Hume: Hardrada Harold Hume, 1875-1965, United States, Canada (Ilex, Camellia) Hutch.: John Hutchinson, 1884-1972, United Kingdom Hylander: Nils Hylander, 1904-1970, Sweden Hylmo: Bertil Hylmé, 1915—... Sweden Ingram, J.: John William Ingram, 1924— , United States (cultivated plants) Ito: Tokutaro Ito, 1868-1941, Japan Henry, J.K.: Joseph Kaye Henry, 1866-1930, Canada Henry, L.: Louis Henry, 1853-1908, France (horticulture) Herd.: Ferdinand Gottfried Maximilian Theobold von Herder, 1828-1896, Germany, Russia Herineq: Francois Hérincq, 1820-1891, France Herrm., J.: J. Herrmann, 1738-1800, France Hesse: Hermann Albrecht Hesse, 1852-1937, Germany (dendrology) Hibb.: James Shirley Hibberd, 1825-1890, United Kingdom (horticulture) Hiern: William Philip Hiern, 1839-1925, United Kingdom Hildebr.: F.H.G. Appendix A: Authors Cited Hildebrand, 1835-1915, Germany Hill, J.: John Hill, 1716-1775, United Kingdom Hochst.: Christian Ferdinand Friedrich Hochstetter, 1787-1860, Germany Hoffm., K.: Kathe Hoffmann, 1883-ca.1931, Germany Hoffmgg.: Johann Centurius von Hoffmannsegg, 1766-1849, Germany Holtt.: Richard Eric Holttum, 1895-1990, United Kingdom Hook.: William Jackson Hooker, 1785-1865, United Kingdom Hook.f.: Joseph Dalton Hooker, 1817-1911, United Kingdom Hoppe: David Heinrich Hoppe, 1760-1846, Germany Hort.: Hortorum (of gardens) or hortulanorum Jack: William Jack, 1795-1822, United Kingdom 229 Ker-Gawl.: John Bellenden Ker Gawler, 1764-1842, United Kingdom Kerner, A.: Anton Joseph Kerner von Marilaun, 1831-1898, Austria Kikuchi: Akio Kikuchi, 1883-1951, Japan Kimura: Arika Kimura, 1900-— , Japan Kirchn.: George Kirchner, 1837-1885, Germany Kirkman: W. Benson Kirkman, contemporary, United States Kitam.: Siro Kitamura, 1906-— , Japan Klotz.: Johann Friedrich Klotzsch, 1805-1860, Germany Knight, H.: Henry Knight, 1834-1896, United Kingdom (nurseryman) Knight, J.: Joseph Knight, 1777?-1855, United Kingdom (nurseryman) Knoop: Johann Hermann Knoop, ca. 1700-1769, Germany (horticulture) Koch, K.: Karl Heinrich Emil Koch, 1809-1879, Germany (dendrology) Kochs: Julius Kochs, 1900-— , Germany Koehne: Bernhard Adalbert Emil Koehne, 1848-1918, Germany (dendrology) Koenig: Johann Gerhard K6nig, 1728-1785, Germany Koidz.: Gen’ichi Koidzumi, 1883-1953, Japan Komar.: Vladimir Leontjevich Komarov, 1869-1945, Russia Kosterm.: André Joseph Guillaume Henri Kostermans, 1907-— , Netherlands, Indonesia Kotschy: Carl Georg Theodor Kotschy, Koehne: Bernhard Adalbert Emil Koehne, 1848-1918, Germany (dendrology) Koenig: Johann Gerhard K6nig, 1728-1785, Germany Koidz.: Gen’ichi Koidzumi, 1883-1953, Japan Komar.: Vladimir Leontjevich Komarov, 1869-1945, Russia Kosterm.: André Joseph Guillaume Henri Kostermans, 1907-— , Netherlands, Indonesia Kotschy: Carl Georg Theodor Kotschy, 1813-1866, Austria 226 Laxm.: Erich Laxmann, 1737-1796, Finland, Russia > Ledeb.: Karl Friedrich von Ledebour, 1785-1851, Germany Lem.: (Antoine) Charles Lemaire, 1801-1871, France Lemmon, J.G.: John Gill Lemmon, 1832-1908, United States Lemmon, W.P.: W.P. Lemmon, fl. 1938, United States (Rhododendron) Lemoine, E.: Emile Lemoine, 1862-1943, France Lemoine, V.: Pierre Louis Victor Lemoine, 1823-1911, France Lev.: Augustin Abel Hector Léveillé, 1863-1918, France Lex.: Juan José Martinez de Lexarza, 1785-1824, Mexico Li: Hui Lin Li, 1911- , United States Liang, C.F.: Chou Fen Liang, 1921- , China (Actinidia) Liebl.: Franz Kaspar Lieblein, 1744-1810, Germany Lindl.: John Lindley, 1799-1865, United Kingdom Link: Johann Heinrich Friedrich Link, 1767-1851, Germany Little: Elbert Luther Little, Jr., ‘1907-— , United States Litvin.: Dmitri Ivanovich Litvinov, 1854-1929, Russia Liu: Tang Shui Liu, fl. Appendix A: Authors Cited 1966-1982, Taiwan (Coniferae, Abies) Llave: Pablo de La Llave, 1773-1833, Mexico Lodd.: Conrad Loddiges, 1738-1826, Netherlands, United Kingdom Kruessm.: Gerd Kriissmann, 1910-1980, Germany (dendrology) Ktze., O.: Karl Ernst Otto Kuntze, 1843-1907, Germany Kudo: Yushun Kudo, 1887-1932, Japan Kunth: Carl Sigismund Kunth, 1788-1850, Germany Kurz: Wilhelm Sulpiz Kurz, 1834-1878, Germany L’Her.: Charles Louis L’Héritier de Brutelle, 1746-1800, France L.: Carl Linnaeus, 1707-1778, Sweden L.f.: Carl Linnaeus, filius, 1741-1783, Sweden Labill.: Jacques Julien Houtton de Labillardiére, 1755-1834, France Lag.: Mariano Lagasca y Segura, 1776-1839, Spain Lam.: Jean Baptiste Antoine Pierre de Monnet de Lamarck, 1744-1829, France Lamb.: Alymer Bourke Lambert, 1761-1842, United Kingdom Lange, J.: Johan Martin Christian Lange, 1818-1898, Denmark Latour-Marl.: Joseph (Bory) Latour-Marliac, 1830-1911, France Lauche: Wilhelm Lauche, 1827-1883, Germany (dendrology) Lauener: Lucien André Lauener, 1918-1991, United Kingdom Lav.: Pierre Alphonse Martin Lavallée, 1836-1884, France Laws., C.: Charles Lawson, 1794-1873, United Kingdom (Coniferae) Laws., P.: Peter Lawson, 227 Loesn.: Ludwig Eduard Theodor Loesener, 1865-1941, Germany Loisel.: Jean Louis Auguste Loiseleur- Deslongchamps, 1774-1849, France Lotsy: Johannes Paulus Lotsy, 1867-1931, Netherlands Loud.: John Claudius Loudon, 1783-1843, United Kingdom Lour.: Joao de Loureiro, 1717-1791, Portugal Lundell: Cyrus Longworth Lundell, 1907-— , United States Macf.: James Macfadyen, 1798-1850, United Kingdom Maiden: Joseph Henry Maiden, 1859-1925, United Kingdom, Australia Maire: Réne Charles Joseph Ernest Maire, 1878-1949, France, Algeria Makino: Tomitaro Makino, 1862-1957, Japan Manetti: Guiseppe Manetti, 1831-1858, Italy Mansf.: Rudolph Mansfeld, 1901-1960, Germany Markgr.: Friedrich Markgraf, 1897-1987, Germany, Switzerland Marq.: Cecil Victor Boley Marquand, 1897-1943, United Kingdom Marsh.: Humphrey Marshall, 1722-1801, United States Marsili: Giovanni Marsili, 1727-1794, Italy Mart.: Carl Friedrich Philipp von Martius, 1794-1868, Germany Martinez: Maximino Martinez, Mast.: Maxwell Tylden Masters, 1833-1907, United Kingdom Matsum.: Jinzo Matsumura, 1856-1928, Japan Mattusch.: Heinrich Gottfried von Mattuschka, 1734-1779, Germany Maxim.: Carl Johann Maximowicz, 1827-1891, Russia Mayr: Heinrich Mayr, 1856-1911, Germany (dendrology) Mazel: Eugene Mazel, fl. 1872, France (cultivated plants) Mazzeo: Peter M. Mazzeo, 1940-— , United States (dendrology) McClelland: John McClelland, 1805-1883, United Kingdom McClint.: Elizabeth May McClintock, 1912-— , United States (cultivated plants, Hydrangea) McClure: Floyd Alonso McClure, 1897-1970, United States (bamboo) McKay: John A. McKay, fl. Appendix A: Authors Cited 1938, United States (Rhododendron) McKelvey: Susan Adams (nee Delano) McKelvey, 1888-1964, United States Medik.: Friedrich Kasimir Medikus, 1736-1808, Germany Meikle: Robert Desmond Meikle, 1923-— , United Kingdom Meisn.: Carl Daniel Friedrich Meisner, 1800-1874, Switzerland Melander: Leonard William Melander, 1893-— , United States Merr.: Elmer Drew Merrill, 1876-1956, United States Meunissier: Auguste Alexandre Meunissier, 1876-1947, France A) No @0) Moric.: Moise Etienne (Stefano) Moricand, 1779-1854, Switzerland = Morley: Brian D. Morley, 1943- , Australia Morong: Thomas Morong, 1827-1894, United States Mottet: Séraphin Joseph Mottet, 1861-1930, France (horticulture) Muell., F.: Ferdinand Jacob Heinrich von Mueller, 1825-1896, Germany, Australia Muell.-Arg.: Johannes Muller Argoviensis [of Aargau], 1828-1896, Switzerland Muenchh.: Otto von Mtinchhausen, 1716-1774, Germany Muhl.: Gotthilf Henry Ernest Muhlenberg, 1753-1815, United States Muller: Cornelius Herman Muller, 1909- , United States Munro: William Munro, 1818-1880, United Kingdom (bamboo) Murata: Gen Murata (Gen Nakai), 1927- , Japan Murr., A.: Andrew Murray, 1812-1878, United Kingdom (Coniferae) Murr., E.: Albert Edward Murray, 1935— , United States Murr., J.A.: Johan Andreas Murray, 1740-1791, Sweden Mutis: José Celestino Bruno Mutis, 1732-1808, Spain, Colombia Nakai: Takenoshin Nakai, 1882-1952, Japan Nash: George Valentine Nash, 1864-1921, United States Mey., E.: Ernst Heinrich Friedrich Meyer, 1791-1858, Germany Mey., F.G.: Frederick Gustav Meyer, 1917- , United States Michx.: André Michaux, 1746-1803, France, United States Michx.f.: Francois André Michaux, 1770-1855, France, United States Miers: John Miers, 1789-1879, United Kingdom Miki: Shigeru Miki, 1901-1974, Japan Mill.: Philip Miller, 1691-1771, United Kingdom Millais: John Guille Millais, 1865-1931, United Kingdom, (Magnolia, Rhododendron) Miq.: Friedrich Anton Wilhelm Miquel, 1811-1871, Netherlands Mirb.: Charles Francois Brisseau de Mirbel, 1776-1854, France Mitf.: Algernon Bertram Freeman Mitford, 1837-1916, United Kingdom (bamboo) Miyabe: Kingo Miyabe, 1860-1951, Japan Moc.: José Mariano Mocino, 1757-1820, Mexico Moench: Conrad Moench, 1744-1805, Germany Mol.: Juan Ignacio Molina, 1737-1829, Chile Momiy.: Yasuichi Momiyama, 1904— , Japan Moore, H.E.: Harold Emery Moore, Jr., 1917-1980, United States Moore, R.J.: Raymond John Moore, 1918- , Canada Nees: Christian Gottfried Nees von Esenbeck, 1776-1858, Germany Nees, T.: Theodor Friedrich Ludwig Nees von Esenbeck, 1787-1837, Germany Nelson, J.: John Nelson (pseudonym “Johannes Senilis”), fl. Appendix A: Authors Cited 1860’s, United Kingdom Nemoto: Kwanji Nemoto, 1860-1936, Japan Nichols.: George Nicholson, 1847-1908, United Kingdom (horticulture) Niedenzu: Franz Joseph Niedenzu, 1857-1937, Germany Nutt.: Thomas Nuttall, 1786-1859, United Kingdom, United States Oerst.: Anders Sandoe Oersted, 1816-1872, Denmark, Central America Ohwi: Jisaburo Ohwi, 1905-1977, Japan Oliv.: Daniel Oliver, 1830-1916, United Kingdom Opiz: Philipp Maximilian Opiz, 1787-1858, Czechoslovakia Ort.: Casimiro Gomez Ortega, 1740-1818, Spain Osbeck: Pehr Osbeck, 1723-1805, Sweden (collector in China) Osborn: Arthur Osborn, 1878-1964, United Kingdom Otto: Christoph Friedrich Otto, 1783-1856, Germany Ottol.: Kornelius Johannes Willem Ottolander, 1822-1887, Netherlands Oudemans: Cornelius Anton Jan Abraham Oudemans, 1825-1906, Netherlands (dendrology) Palib.: Ivan Vladimirovich Palibin, 1872-1949, Russia Pall.: Peter Simon Pallas, 1741-1811, Germany, Russia Palm.: Ernest Jesse Palmer, 1875-1962, United Kingdom, United States Pamp.: Renato Pampanini, 1875-1949, Italy Pancic.: Josef Panéic, 1814-1888, Yugoslavia Parl.: Filippo Parlatore, 1816-1877, Italy (Coniferae) Pavon: José Antonio Pavon, 1754-1844, Spain, Peru, Chile Pax: Ferdinand Albin Pax, 1858-1942, Germany Paxt.: Joseph Paxton, 1803-1865, United Kingdom Pearce: Sydney Albert Pearce, 1906-1972, United Kingdom (cultivated plants) Perk.: Janet Russell Perkins, 1853-1933, United States Pers.: Christiaan Hendrik Persoon, 1761-1836, So. Africa, Germany, France Phipps: James Bird Phipps, 1934 , Canada (Crataegus) Pierre: Jean Baptiste Louis Pierre, 1833-1905, France Pike: Arnold Pike, fl. 1890's, United Kingdom Pilger: Robert Knud Friedrich Pilger, 1876-1953, Germany Planch.: Jules Emile Planchon, 1823-1888, France Poggenburg: Justus Ferdinand Poggenburg, 1840-1893, Germany, United States Poir.: Jean Louis Marie Poiret, 1755-1834, France Poit.: Pierre Antoine Poiteau, Palib.: Ivan Vladimirovich Palibin, 1872-1949, Russia 230 Reveal: James Lauritz Reveal, 1941- , United States = Rich., A.: Achille Richard, 1794-1852, France Rich., L.C.: Louis Claude Marie Richard, 1754-1821, France Riv., A.: Marie Auguste Riviere, 1821-1877, France (bamboo) Riv., C.: Charles Marie Riviére, 1845-?, France (bamboo) Rivers: Thomas Rivers, 1798-1877, United Kingdom (nurseryman) Robinson: Benjamin Lincoln Robinson, 1864-1935, United States Robs., N.: Norman K. B. Robson, 1928— , United Kingdom (Hypericum) Roberts: A.V. Roberts, United Kingdom Roem.: Johann Jacob Roemer, 1763-1819, Germany Roem., M.J.: Max J. Appendix A: Authors Cited Roemer, 1791-1849, Germany Rolfe: Robert Allen Rolfe, 1855-1921, United Kingdom Rose: Joseph Nelson Rose, 1862-1928, United States Roth: Albrecht Wilhelm Roth, 1757-1834, Germany Rottl.: Johann Peter Rottler, - 1749-1836, France, India Rouleau: Joseph Albert Ernest Rouleau, 1916-1991, Canada Rowlee: Willard Winfield Rowlee, 1861-1923, United States Pojark.: Antonina Ivanovna Pojarkova, 1897-1980, Russia Pollard: Charles Louis Pollard, 1872-1945, United States Pourr.: Pierre André Pourret, 1754-1818, France Prain: David Prain, 1857-1944, United Kingdom Presl: Karel Boriwag Pres], 1794-1852, Czechoslovakia Pres], J.: Jan Svatopluk Presl, 1791-1849, Czechoslovakia Pritz.: Ernst Georg Pritzel, 1875-1946, Germany Purk.: Emanuel von Purkyné, 1832-1882, Czechoslovakia Pursh: Frederick Traugott Pursh, 1774-1820, Germany, United States Raber: Oran Lee Raber, 1893-1940, United States Raddi: Giuseppe Raddi, 1770-1829, Italy Raeusch.: Ernst Adolf Raeuschel, fl. 1772-1797, Germany Raf.: Constantine Samuel Rafinesque, 1783-1840, United States Rayner: Douglas A. Rayner, 1949- , United States Reade: John Moore Reade, 1876-1937, Canada, United States Regel: Eduard August von Regel, 1815-1892, Germany, Russia Rehd.: Alfred Rehder, 1863-1949, Germany, United States (dendrolog Reichenb.: Heinrich Gottlieb Ludwig Reichenbach, 1793-1879, Germany 231 Royle: John Forbes Royle, 1798-1858, United Kingdom Rudd: Velva Elaine Rudd, 1910- , United States Ruempler: Karl Theodor Rtimpler, 1817-1891, Germany Ruiz: Hipolito Ruiz Lopez, 1754-1815, Spain, Peru, Chile Rupr.: Franz Joseph Ruprecht, 1814-1870, Czechoslovakia, Russia Rushforth: K.D. Rushforth, fl. 1983, United Kingdom (Coniferae) Rydb.: Per Axel Rydberg, 1860-1931, United States Salisb.: Richard Anthony Salisbury, 1761-1829, United Kingdom Sarg.: Charles Sprague Sargent, 1841-1927, United States (dendrology) Sav.: Paul Amedée Ludovic Savatier, 1830-1891, France (flora of Japan) Schaeffer: Jacob Christian Schaeffer, 1718-1790, Germany Schauer: Johannes Conrad Schauer, 1813-1848, Germany Scheele: Georg Heinrich Adolph Scheele, 1808-1864, Germany Schelle: Ernst Schelle, 1864-1945, Germany (horticulture) Schery: Robert Walter Schery, 1917-1987, United States Schiede: Christian Julius Wilhelm Schiede, 1798-1836, Germany Schlechtend.: Diederich Franz Leonhard von Schlechtendal, 1794-1866, Germany Schmidt, F.: Friedrich Karl Schmidt, 1832-1908, Russia Royle: John Forbes Royle, 1798-1858, United Kingdom Royle: John Forbes Royle, 1798-1858, United Kingdo Schottky: Ernst Max Schottky, 1888-1915, Germany Schrad.: Heinrich Adolph Schrader, 1767-1836, Germany Schreber: Johann Christian Daniel von Schreber, 1739-1810, Germany Schub.: Bernice Giduz Schubert, 1913- , United States Schultes, J.A.: Josef August Schultes, 1773-1831, Austria Schultes, J.H.: Julius Hermann Schultes, 1804-1840, Austria Schultz, H.: Elmer H. Schultz, fl. Appendix A: Authors Cited Hil.: Auguste Francois César Prouvencal de Saint-Hilaire, 1779-1853, France Stapf: Otto Stapf, 1857-1933, Austria, United Kingdom Staunt.: George Leonard Staunton, 1737-1801, United Kingdom Stearn: William Thomas Stearn, 1911- , United Kingdom Sterns: Emerson Ellick Sterns, 1846-1926, United States Steud.: Ernst Gottlieb von Steudel, 1783-1856, Germany Steven: Christian von Steven, 1781-1863, France, United States Stewart, J.L.: John Lindsay Stewart, 1832-1873, United Kingdom, India Shibata: Keita Shibata, 1877-1949, Japan 233 Thoms.: Thomas Thomson, 1817-1878, United Kingdom Thory: Claude Antoine Thory, 1759-1827, France Thouin: André Thouin, 1747-1824, France Thunb.: Carl Peter Thunberg, 1743-1828, Sweden, Japan, South Africa Tieghem: Philippe Edouard Léon van Tieghem, 1839-1914, France Tobl.: Friedrich Tobler, 1879-1957, Switzerland (Hedera) Torr.: John Torrey, 1796-1873, United States Tratt.: Leopold Trattinnick, 1764-1849, Austria Traut.: Ernst Rudolph von Trautvetter, 1809-1889, Russia Trecul: Auguste Adolfe Lucien Trécul, 1818-1896, France Trelease: William Trelease, 1857-1945, United States Trew: Christoph Jakob Trew, 1695-1769, Germany Troncoso: Nélida Sara Troncoso, 1914-1988, Argentina Tseng, C.J.: Chang Jiang Tseng, 1896-1980, China (Ilex) Turcz.: Nikolai Stepanovich Turczaninow, 1796-1863, Russia Turner, B.L.: Billie Lee Turner, 1925- , United States Turra: Antonio Turra, 1730-1796, Italy Turrill: William Bertram Turrill, 1890-1961, United Kingdom Tutch.: William James Tutcher, 1867-1920, United Kingdom Thoms.: Thomas Thomson, 1817-1878, United Kingdom Thory: Claude Antoine Thory, 1759-1827, France Thouin: André Thouin, 1747-1824, France Thunb.: Carl Peter Thunberg, 1743-1828, Sweden, Japan, South Africa Tieghem: Philippe Edouard Léon van Tieghem, 1839-1914, France Tobl.: Friedrich Tobler, 1879-1957, Switzerland (Hedera) Torr.: John Torrey, 1796-1873, United States Tratt.: Leopold Trattinnick, 1764-1849, Austria Traut.: Ernst Rudolph von Trautvetter, 1809-1889, Russia Trecul: Auguste Adolfe Lucien Trécul, 1818-1896, France Trelease: William Trelease, 1857-1945, United States Trew: Christoph Jakob Trew, 1695-1769, Germany Troncoso: Nélida Sara Troncoso, 1914-1988, Argentina Tseng, C.J.: Chang Jiang Tseng, 1896-1980, China (Ilex) Turcz.: Nikolai Stepanovich Turczaninow, 1796-1863, Russia Turner, B.L.: Billie Lee Turner, 1925- , United States Turra: Antonio Turra, 1730-1796, Italy Turrill: William Bertram Turrill, 1890-1961, United Kingdom Stokes: Jonathan Stokes, 1755-1831, United Kingdom Stritch: Lawrence R. Stritch, 1953- , United States Sukachev: Vladimir Nikolaevich Sukachev, 1880-1967, Russia Sun: Siang Chung Sun, 1908- , China Suring.: Willem Frederik Reinier Suringar, 1832-1898, Netherlands Svenson: Henry Knute Svenson, 1897-1986, United States Swartz, O.: Olof Peter Swartz, 1760-1818, Sweden Sweet: Robert Sweet, 1783-1835, United Kingdom (cultivated plants) Swingle: Walter Tennyson Swingle, 1871-1952, United States (Citrus) Szysz.: Ignaz von Szyszylowicz, 1857-1910, Poland TT. & G.: See Torr.: A. Gr. Appendix A: Authors Cited 1934, United States (Berberis) Schum.: Karl Moritz Schumann, 1851-1904, Germany Schwarz, O.: Otto Karl Anton Schwarz, 1900-1983, Germany Schwerin: Fritz Kurt Alexander von Schwerin, 1856-1934, Germany (dendrology) Scop.: Giovanni Antonio Scopoli, 1723-1788, Austria, Italy Sealy: Joseph Robert Sealy, 1907-— , United Kingdom Seemann: Berthold Carl Seemann, 1825-1871, Germany Seemen: Karl Otto von Seemen, 1838-1910, Germany Seldin: Margaret J. Seldin, fl. 1976, United States Sendtn.: Otto Sendtner, 1813-1859, Germany Ser.: Nicolas Charles Seringe, 1776-1858, France Sesse: Martin de Sessé y Lacasta, 1751-1808, Spain, Mexico Sharp: Aaron John Sharp, 1798-1858, United Kingdom 1798-1858, United Kingdom Rudd: Velva Elaine Rudd, 1910- , United States Rushforth: K.D. Rushforth, fl. 1983, United Kingdom (Coniferae) 22 Sharp, W.: Ward McClintic Sharp, 1904— , United States Shibata: Keita Shibata, 1877-1949, Japan Shinners: Lloyd Herbert Shinners, 1918-1971, Canada, United States Sieb.: Philipp Franz von Siebold, 1796-1866, Germany Simmonds: Arthur Simmonds, 1892-1968, United Kingdom (horticulture) Simonk.: Lajos Simonkai, 1851-1910, Hungary Sims: John Sims, 1749-1831, United Kingdom Skan: Sidney Alfred Skan, 1870-1939, United Kingdom (horticulture) Skeels: Homer Collar Skeels, 1873-1934, United States Skipwith: Geoffrey Skipwith, fl. 1924-29, United Kingdom (nurseryman) Skvortz.: Boris Vassilievich Skvortzov, 1890-1980, Poland, Brazil Sleumer: Hermann Otto Sleumer, 1906- , Germany, Netherlands Sm.: James Edward Smith, 1759-1828, United Kingdom Sm., A.C.: Albert Charles Smith, 1906- , United States Sm., A.R.: Alan Reed Smith, 1938- , United Kingdom Sm., E.A.: Eugene Allen Smith, 1841-1927, United States Sm., L.B.: Lyman Bradford Smith, 1904— , United States Sm., W.G.: William Gardner Smith, 1866-1928, United Kingdom Small: John Kunkel Small, 1869-1938, United States cae Soderstrom: Thomas Robert Soderstrom, 1936-1987, United States Sol.: Daniel Carl Solander, 1733-1782, Sweden, United Kingdom Solym.: Sigmond L. Solymosy, 1906-1974, United States Soul.-Bod.: Etienne Soulange-Bodin, 1774-1846, France (horticulture) Spach: Edouard Spach, 1801-1879, France Spaeth: Franz Ludwig Spath, 1838-1913, Germany (horticulture) Spongberg: Stephen A. Spongberg, 1942-— , United States Sprague: Thomas Archibald Sprague, 1877-1958, United Kingdom Spreng.: Kurt Polykarp Joachim Sprengel, 1766-1833, Germany St. Appendix A: Authors Cited Tagg: Henry Frank Tagg, 1874-1933, United Kingdom (Rhododendron) Takeda: Hisayoshi Takeda, 1883-1972, Japan Tarbox: Gurdon Lucius Tarbox, Jr., 1927- , United States (horticulture) Tausch: Ignaz Friedrich Tausch, 1793-1848, Czechoslovakia Taylor, T.M.C.: Thomas Mayne Cunninghame Taylor, 1904-1983, Canada, South Africa femple; h:L; vemple; fl. 1885-1893, United States (nurseryman) Stritch: Lawrence R. Stritch, 1953- , United States Sukachev: Vladimir Nikolaevich Sukachev, 1880-1967, Russia Sun: Siang Chung Sun, 1908- , China Suring.: Willem Frederik Reinier Suringar, 1832-1898, Netherlands Svenson: Henry Knute Svenson, 1897-1986, United States Swartz, O.: Olof Peter Swartz, 1760-1818, Sweden Sweet: Robert Sweet, 1783-1835, United Kingdom (cultivated plants) Swingle: Walter Tennyson Swingle, 1871-1952, United States (Citrus) Szysz.: Ignaz von Szyszylowicz, 1857-1910, Poland TT. & G.: See Torr.: A. Gr. Tagg: Henry Frank Tagg, 1874-1933, United Kingdom (Rhododendron) Takeda: Hisayoshi Takeda, 1883-1972, Japan Tarbox: Gurdon Lucius Tarbox, Jr., 1927- , United States (horticulture) Tausch: Ignaz Friedrich Tausch, 1793-1848, Czechoslovakia Taylor, T.M.C.: Thomas Mayne Cunninghame Taylor, 1904-1983, Canada, South Africa femple; h:L; vemple; fl. 1885-1893, United States (nurseryman) Tenore: Michele Tenore, 1780-1861, Italy Teran: Manuel de Mier y Teran, 1789?-1852, Mexico Warder: John Aston Warder, 1812-1883, United States (horticulture) Wassh.: Dieter Carl Wasshausen, 1938- , United States (Acanthaceae) Wats., P.W.: Peter William Watson, 1761-1830, United Kingdom Wats., S.: Sereno Watson, 1826-1892, United States Weatherby: Charles Alfred Weatherby, 1875-1949, United States Webb, D.A.: David Allardice Webb, 1912- , Ireland Webb, P.B.: Philip Barker Webb, 1793-1854, United Kingdom Weber: Georg Heinrich Weber, 1752-1828, Germany Wendl., H.: Hermann Wendland, 1825-1903, Germany Wendl., J.C.: Johann Christoph Wendland, 1755-1828, Germany Wesmael: Alfred Wesmael, 1832-1905, Belgium (dendrology) Weston: Richard Weston, 1733-1806, United Kingdom Weyer: W. van de Weyer, fl. Appendix A: Authors Cited 1914-20, United Kingdom Wieg.: Karl McKay Wiegand, 1873-1942, United States Wight: Robert Wight, 1796-1872, United Kingdom, India Wight, W.F.: William Franklin Wight, 1874-1954, United States Willd.: Karl Ludwig von Willdenow, 1765-1812, Germany Wils.: Ernest Henry Wilson, 1876-1930, United Kingdom, United States Uyeki: Homiki Uyeki, 1882-— , Japan Vahl: Martin Henrichsen Vahl, 1749-1804, Denmark Van Melle: Peter Jacobus van Melle, 1891-1953, Netherlands, United States (Juniperus) Van.: Eugene Vaniot, 2-1913, France Vasey: George Vasey, 1822-1893, United States Veitch, J.G.: John Gould Veitch, 1839-1870, United Kingdom Vent.: Etienne Pierre Ventenat, 1757-1808, France Vill.: Celestino Fernandez-Villar, 1838-1907, Spain, Philippines Villars: Dominique Villars, 1745-1814, France Vilm., M.L.: Auguste Louis Maurice Levéque de Vilmorin, 1849-1918, France Vis.: Roberto de Visiani, 1800-1878, Italy Voss: Andreas Voss, 1857-1924, Germany (cultivated plants) Vrughtman, F.: Freek Vrughtman, 1927-— , Canada (horticulture) Wall.: Nathaniel Wallich (Nathan Wolff), 1786-1854, Denmark, India, Nepal Wallroth: Carl Friedrich Wilhelm Wallroth, 1792-1857, Germany Walp.: Wilhelm Gerhard Walpers, 1816-1853, Germany Walt.: Thomas Walter, 1740-1789, United States Wang.: Friedrich Adam Julius von Wangenheim, Van Melle: Peter Jacobus van Melle, 1891-1953, Netherlands, United States (Juniperus) 235 Wimmer: Christian Friedrich Heinrich Wim 1803-1868, Germany Wittmack: Max Carl Ludwig Wittmack, 1839-1929, Germany Wolf, E.: Egbert Ludwigowitsch Wolf, 1860-1931, Germany (dendrology) Wood, A.: Alphonso Wood, 1810-1881, United States Woods, F.W.: Frank W. Woods, contemporary, United States Wright: Charles Henry Wright, 1864-1941, United Kingdom Wright, J.: John Wright, 1811-1846, United States Yamamoto: Yoshimatsu Yamamoto, 1893-1947, Japan, Taiwan Yamazaki: Takasi Yamazaki, LO2 Japan Yatabe: Ryokichi Yatabe, 1851-1899, Japan Young: Robert Armstrong Young, 1876-1963, United States Zab.: Hermann Zabel, 1832-1912, Germany (dendrology) Zenari: Silvia Zenari, 1896-1956, Italy Zeyh.: Carl Ludwig Philipp Zeyher, 1799-1858, Germany, So. Africa Zoll.: Heinrich Zollinger, 1818-1859, Switzerland, Java Zucc.: Joseph Gerhard Zuccarini, 1797-1848, Germany Zuccagni: Attilio Zuccagni, 1754-1807, Italy iw) ee) ©) Appendix B: Plant Collection Sites, Cited by Acronym The acronyms cited here identify the 677 sites where plant specimens were collected. Arranged alphabetically by state, the acronyms consist of two elements: (1) the first two letters are the postal code for the state or the District of Colum- bia, and (2) the second two letters identify a precise locale in the state where the plant was collected. ALAU thus stands for Alabama, Au- burn University; and DCNA, for the District of Columbia, the U.S. National Arboretum. Appendix A: Authors Cited ALAU ALBB ALBG ALBH ALBT ALCG ALCM ALCS ALCW ALDM ALEC ALEH ALFN ALFS ALHC ALIT ALJG ALMC ALMG ALON ALOS ALPH ALRH ALSH Auburn University, Auburn, Lee Co., AL Bank in downtown area, Birmingham, Jefferson Co., AL Bellingrath Gardens, Theodore, Mobile Co., AL Byers Nursery, Huntsville, Madison Co., AL Bryce State Mental Hospital, Tuscaloosa, Tuscaloosa Co., AL Vacant corner lot on Church Street near Government Street, Mobile, Mobile Co., AL Church Street Graveyard, Mobile, Mobile Co., AL Chamberlain Street, Mobile, Mobile Co., AL Nursery of Clarence H. Welch, US Highway 98, Wilmer, Mobile Co., AL House on Dauphin Street, Mobile, Mobile Co., AL Elmwood Cemetery, Birmingham, Jefferson Co., AL Private garden, 4558 Brookmoor Drive, Mobile, Mobile Co., AL Fraser Nursery, Birmingham, Jefferson Co., AL Horticulture Field Station Mobile (Spring Hill School), Mobile, Mobile Co., AL Private garden, 168 Peachtree Circle, Birming- ham, Jefferson Co., AL Milepost 36.5 on Interstate 85 near Tuskegee exit, Macon Co., AL Private garden, Eight Mile, Mobile Co., AL Beech Grove United Methodist Church, Francisco, Jackson Co., AL Magnolia Gardens, Chunchulla, Mobile Co., AL Overlook Nurseries, Route 98, Mobile, Mobile Co., AL Overlook Nursery, Schillinger Road, West Mobile, Mobile Co., AL Albert Pick Motel, US Route 72, Huntsville, Madison Co., AL Private garden, 4058 Old Shell Road, Spring Hill, Mobile, Mobile Co., AL Spring Hill College, Spring Hill, Mobile, Mobile Co., AL Scenic Motel, Highway 98, Mobile, Mobile Co., AL ae Roadside, US Route 11, 3 mi. Appendix A: Authors Cited south of Tuscaloosa, Tuscaloosa Co., AL Tom Dodd Nursery, Semmes, Mobile Co., AL Travelodge Motel, Government Street, Mobile, Mobile Co., AL Roadside, US Route 11 at Tombigbee River (west side), Sumter Co., AL University of Alabama, Tuscaloosa, Tuscaloosa Co., AL Private garden, Wulff Road, Semmes, Mobile Co., AL Private garden, Calion, Union Co., AR Private garden, Glendale, Lincoln Co., AR Private garden, 34 Edgehill Road, Little Rock, Pulaski Co., AR Private garden, 4 Longfellow Lane, Little Rock, Pulaski Co., AR Private garden, 36 River Ridge Road, Little Rock, Pulaski Co., AR Roadside, Route 130, Scott, Pulaski Co., AR Private garden, 12 Sherrill Road, Little Rock, Pulaski Co., AR University of Arkansas, Monticello, Drew Co., AR Private garden, 8 Longfellow Place, Little Rock, Pulaski Co., AR National Academy of Sciences, Wisconsin Avenue NW, Washington, DC Vacant lot at corner of Anacostia Avenue and Douglas Street NE, Washington, DC Arnold Estate Garden (now abandoned), 4000 block, Massachusetts Avenue NW, Washington, DC Kenilworth Aquatic Gardens, Douglas Street NE (off Kenilworth Avenue), Washington, DC Anacostia Park, near Sousa Bridge (Pennsylvania Avenue SE), Washington, DC US Botanic Garden grounds, 3d Street SW, Washington, DC Center for the Aging, 2601 18th Street NE, Washington, DC Private garden, 6010 31st Street NW, Washington, DC Cosmos Club, 2121 Massachusetts Avenue NW, Washington, DC US Capitol grounds, Washington DC Sidewalk along Delaware Avenue NE, by Senate Office Building, Washington, DC Columbia Island, Washington, DC Small park at Columbia and Kalorama Roads NW, Washington, DC Private garden, 3815 Alton Place NW, Washington, DC Connecticut Avenue NW, between Kanawha and Military Roads, Washington, DC Private garden, Dahlia Street and Georgia Avenue NW, Washington, DC Dumbarton Oaks Garden, 31st and R Streets NW, Washington, DC Vacant lot near Eastern Avenue and Laurel Appendix B: Plant Collection Sites, Cited by Acronym The acronyms cited here identify the 677 sites where plant specimens were collected. Arranged alphabetically by state, the acronyms consist of two elements: (1) the first two letters are the postal code for the state or the District of Colum- bia, and (2) the second two letters identify a precise locale in the state where the plant was collected. ALAU thus stands for Alabama, Au- burn University; and DCNA, for the District of Columbia, the U.S. National Arboretum. Appendix A: Authors Cited ALAU ALBB ALBG ALBH ALBT ALCG ALCM ALCS ALCW ALDM ALEC ALEH ALFN ALFS ALHC ALIT ALJG ALMC ALMG ALON ALOS Auburn University, Auburn, Lee Co., AL Bank in downtown area, Birmingham, Jefferson Co., AL Bellingrath Gardens, Theodore, Mobile Co., AL Byers Nursery, Huntsville, Madison Co., AL Bryce State Mental Hospital, Tuscaloosa, Tuscaloosa Co., AL Vacant corner lot on Church Street near Government Street, Mobile, Mobile Co., AL Church Street Graveyard, Mobile, Mobile Co., AL Chamberlain Street, Mobile, Mobile Co., AL Nursery of Clarence H. Welch, US Highway 98, Wilmer, Mobile Co., AL House on Dauphin Street, Mobile, Mobile Co., AL Elmwood Cemetery, Birmingham, Jefferson Co., AL Private garden, 4558 Brookmoor Drive, Mobile, Mobile Co., AL Fraser Nursery, Birmingham, Jefferson Co., AL Horticulture Field Station Mobile (Spring Hill School), Mobile, Mobile Co., AL Private garden, 168 Peachtree Circle, Birming- ham, Jefferson Co., AL Milepost 36.5 on Interstate 85 near Tuskegee exit, Macon Co., AL Private garden, Eight Mile, Mobile Co., AL Beech Grove United Methodist Church, Francisco, Jackson Co., AL Magnolia Gardens, Chunchulla, Mobile Co., AL Overlook Nurseries, Route 98, Mobile, Mobile Co., AL Overlook Nursery, Schillinger Road, West Scenic Motel, Highway 98, Mobile, Mobile Co., AL ae Roadside, US Route 11, 3 mi. Appendix A: Authors Cited south of Tuscaloosa, Tuscaloosa Co., AL Tom Dodd Nursery, Semmes, Mobile Co., AL Travelodge Motel, Government Street, Mobile, Mobile Co., AL Roadside, US Route 11 at Tombigbee River (west side), Sumter Co., AL University of Alabama, Tuscaloosa, Tuscaloosa Co., AL Private garden, Wulff Road, Semmes, Mobile Co., AL Private garden, Calion, Union Co., AR Private garden, Glendale, Lincoln Co., AR Private garden, 34 Edgehill Road, Little Rock, Pulaski Co., AR Private garden, 4 Longfellow Lane, Little Rock, Pulaski Co., AR Private garden, 36 River Ridge Road, Little Rock, Pulaski Co., AR Roadside, Route 130, Scott, Pulaski Co., AR Private garden, 12 Sherrill Road, Little Rock, Pulaski Co., AR University of Arkansas, Monticello, Drew Co., AR Private garden, 8 Longfellow Place, Little Rock, Pulaski Co., AR National Academy of Sciences, Wisconsin Avenue NW, Washington, DC Vacant lot at corner of Anacostia Avenue and Douglas Street NE, Washington, DC Arnold Estate Garden (now abandoned), 4000 block, Massachusetts Avenue NW, Washington, DC Kenilworth Aquatic Gardens, Douglas Street NE (off Kenilworth Avenue), Washington, DC Anacostia Park, near Sousa Bridge (Pennsylvania Avenue SE), Washington, DC US Botanic Garden grounds, 3d Street SW, Washington, DC Center for the Aging, 2601 18th Street NE, Washington, DC Private garden, 6010 31st Street NW, Washington, DC Cosmos Club, 2121 Massachusetts Avenue NW, Washington, DC US Capitol grounds, Washington DC Sidewalk along Delaware Avenue NE, by Senate Office Building, Washington, DC Columbia Island, Washington, DC Small park at Columbia and Kalorama Roads NW, Washington, DC Private garden, 3815 Alton Place NW, Washington, DC ALFN Fraser Nursery, Birmingham, Jefferson Co., AL Jefferson Co., AL Horticulture Field ALFS Jefferson Co., AL Horticulture Field Station Mobile (Spring Hill Wa Ce ALFS Jefferson Co., AL Horticulture Field Station Mobile (Spring Hil ALFS Horticulture Field Station Mobile (Spring Hill School), Mobile, Mobile Co., AL ALHC School), Mobile, Mobile Co., AL Private garden, 168 Peachtree Cir ALHC Private garden, 168 Peachtree Circle, Birming- ham, Jefferson Co., AL ham, Jefferson Co., AL ALIT Milepost 36.5 on Interstate 85 near Tuskegee exit, Macon Co., AL ALIT Milepost 36.5 on Interstate 85 near Tuskegee ALJG ALMC Private garden, Eight Mile, Mobile Co., AL Beech Grove United Methodist Church, US Capitol grounds, Washington DC Sidewalk along Delaware Avenue NE, b Sidewalk along Delaware Avenue NE, by Senate Office Building, Washington, DC Columbia Island, Washington, DC ALMC Beech Grove United Methodist Church, Francisco, Jackson Co., AL ALMG Francisco, Jackson Co., AL Magnolia Gardens, Chunchull ALMG Francisco, Jackson Co., AL Magnolia Gardens, Chunchulla, Columbia Island, Washington, DC Small park at Columbia and Kalora ALMG Magnolia Gardens, Chunchulla, Mobile Co., AL ALON Mobile Co., AL Overlook Nurseries, Route 98, Mobile, Mobile Co., AL Overlook Nurseri Small park at Columbia and Kalorama Roads NW, Washington, DC ALON Overlook Nurseries, Route 98, Mobile, Mobile Co., AL Private garden, 3815 Alton Place NW, Washington, DC ALOS Mobile Co., AL Overlook Nursery, Schillinger Road, West ALOS Mobile Co., AL Overlook Nurser ALOS Overlook Nursery, Schillinger Road, West Mobile, Mobile Co., AL Washington, DC Connecticut Avenue NW, between Kanawha Connecticut Avenue NW, between Kanawha and Military Roads, Washington, DC ALPH Mobile, Mobile Co., AL Albert Pick Motel, US Ro ALPH Mobile, Mobile Co., AL Albert Pick Motel, US Route 72, Huntsv Connecticut Avenue NW, between Kanawha and Military Roads, Washington, DC Private garden, Dahlia Street and Georgia ALPH Albert Pick Motel, US Route 72, Huntsville, Madison Co., AL and Military Roads, Washington, DC Private garden, Dahlia Street and Georgia Private garden, Dahlia Street and Georgia Avenue NW, Washington, DC Madison Co., AL Private garden, 4058 Old Shell Road, Spring ALRH ALSH Private garden, 4058 Old Shell Road, Spring Hill, Mobile, Mobile Co., AL Spring Hill College, Spring Hill, Mobile, Mobile Co., AL ALRH ALSH Private garden, 4058 Old Shell Road, Spring Hill, Mobile, Mobile Co., AL Spring Hill College, Spring Hill, Mobile, Avenue NW, Washington, DC Dumbarton Oaks Garden, 31st 237 DCSE DCSG DCSH DCSI Saint Elizabeth’s Hospital grounds, Martin Luther King Jr Avenue SE, Washington, DC Edge of woods at corner of Sargent Road and Galloway Road NE, Washington, DC Soldiers Home (near police station), Washington, DC Old Smithsonian Institution building (the “Castle”), Washington, DC DCSE DCSG Saint Elizabeth’s Hospital grounds, Martin Luther King Jr Avenue SE, Washington, DC Edge of woods at corner of Sargent Road and DCFM Vacant lot across from Franciscan Monastery, 18th Street NE, Washington, DC Folger Park, 2d and D Streets SE, DCFP Folger Park, 2d and D Streets SE, Washington, DC DCGC Near greenhouse, Independence Avenue at Ist Street SW, Washington, DC DCSH Soldiers Home (near police station), Washington, DC Street SW, Washington, DC DCGD Private garden at 1617 29th Street (Georgetown), Washington, DC DCGP (Georgetown), Washington, DC Garfield Park, F Street between Ist and 3rd DCGP Garfield Park, F Street between Ist and 3rd (the “Castle”), Washington, DC DCGP Garfield Park, F Street between Ist DCTB Tidal Basin, Washington, DC Private garden, 1700 24th Stree DCGW Streets SE, Washington, DC DCTF Private garden, 1700 24th Street NE, Washington, DC DCGW George Washington University campus, Washington, DC Washington, DC DCTN Private garden, 4701 Fessenden Street NW, Washington, DC DCTN Private garden, 4701 Fessenden Street NW, Washington, DC DCHM Hillwood Museum, 4115 Linnean Avenue NW, Washington, DC Mount Alto, site for new Russian Federation DCUR Mount Alto, site for new Russian Federation Embassy, Wisconsin Avenue NW, DCHP East Potomac Golf Course, Hains Point, Washington, DC HP East Potomac Golf Course, Hains Point, DCUR DCUS Mount Alto, site for new Russian Federation Embassy, Wisconsin Avenue NW, Washington, DC Union Square, Ist Street and The Mall, DCHU DCIW DCJJ DCJM DCJS DELE DCLP DCMA DCMC DCMH DCML DCMN DCMS DCNA DCNC DCNE DCNG DCNY DCPA DERC DCPG DCPL Washington, DC Howard University campus, Washington, DC Private garden, 7433 Berkeley Terrace NW, Washington, DC Near John Paul Jones statue, 17th Street and Independence Avenue SW, Washington, DC Jefferson Memorial grounds, Washington, DC Private garden, corner of Jackson Street and 14th Street NE, Washington, DC Library of Congress, Washington, DC Lafayette Park, Washington, DC Private garden, MacArthur Boulevard near Arizona Avenue NW, Washington, DC Private garden, 1616 31st Street NW (Georgetown), Washington, DC Meridian Hill Park, 16th Street NW, Washington, DC The Mall, Washington, DC Fort McNair, Washington, DC US Navy, Bureau of Medicine and Surgery, 23d and E Streets NW, Washington, DC US National Arboretum, Washington, DC National Cathedral, Washington, DC Private garden, 3910 18th Street NE, Washington, DC National Gallery of Art, Washington, DC New York Avenue and 7th Street NW, Washington, DC Pan American Building, 17th and C Streets NW, Washington, DC Rock Creek Park Cemetery, Rock Creek Church Road NW, Washington, DC National Portrait Gallery, F and 7th Streets NW, Washington, DC Old District of Columbia Public Library (now UDC), New York Avenue and 7th Street NW, Washington, DC East Potomac Park, Washington, DC Rock Creek Park, Washington, DC Private garden, 2122 Rand Place NE, Washington, DC Private garden, 3051 Porter Street NW, Washington, DC Private garden, 3224 R Street NW, Washington, DC Department of State, C and 22d Streets NW, Washington, DC Embassy, Wisconsin Avenue NW, Washington, DC Washington, DC DCUS Embassy, Wisconsin Avenue NW, Washington, DC Union Square, Ist Street and The Mall, Washington, DC DCHU DCIW Howard University campus, Washington, DC Private garden, 7433 Berkeley Terrace NW, DCUS Union Square, Ist Street and The Mall, Washington, DC DCIW Private garden, 7433 Berkeley Terrace NW, Washington, DC DCIW Private garden, 7433 Berkeley Terrace NW, Washington, DC Washington, DC Private garden, 15 Washington, DC Private garden, 152 North Carolina Avenue Washington, DC Near John Paul Jo DCVK Private garden, 152 North Carolina Avenue SE, Washington, DC DCJJ Near John Paul Jones statue, 17th Street and Independence Avenue SW, Washington, DC DCJJ Near John Paul Jones statue, 17th Street and Independence Avenue SW, Washington, DC Jefferson Memorial grounds, Washington, DC DCWA Westchester Apartments, 4000 Cathedral Avenue NW, Washington, DC DCJM Jefferson Memorial grounds, Washington, DC Private garden, corner of Jackson Street and DCWH Avenue NW, Washington, DC DCJS Private garden, corner of Jackson Street and 14th Street NE, Washington, DC DCWH The White House, 1600 Pennsylvania Avenue NW, Washington, DC Washington Monument grounds, 14th Street NE, Washington, DC Library of Congress, Washington, DC NW, Washington, DC DELE Library of Congress, Washington, DC Lafayette Park, Washington, DC DCWM Washington Monument grounds, Washington, DC DCLP Lafayette Park, Washington, DC Washington, DC DCMA Private garden, MacArthur Boulevard near DCWP DCWR West Potomac Park, Washington, DC Arizona Avenue NW, Washington, DC DCWR Grounds of Walter Reed Hospital, 16t DCWR Grounds of Walter Reed Hospital, 16t DCWR Grounds of Walter Reed Hospital, 16th Street NW, Washington, DC DCMC Private garden, 1616 31st Street (Georgetown), Washington, DC DCWR Grounds of Walter Reed Hospital, 16th Street NW, Washington, DC DCWR Grounds of Walter Reed Hospital, 16th Street NW, Washington, DC NW, Washington, DC (Georgetown), Washington, DC Meridian Hill Park, 16th Street NW, DECN DEEM Cannon Nursery, Greenwood, Sussex Co., DE Eleutherian Mills, Greenville, DCMH Meridian Hill Park, 16th Street NW, Washington, DC Washington, DC south of state line, near Snuffmill Road, Centerville, near Snuffmill Road, Centerville, New Castle Co., DE DCNY New York Avenue and 7th Street NW, Washington, DC New Castle Co., DE Winterthur Gardens, Washington, DC DEWG Winterthur Gardens, Winterthur, New Castle Co., DE New Castle Co., DE FLAL Private garden, Northwest 45th Avenue, Gainesville, Alachua Co., FL FLBF Gainesville, Alachua Co., FL Briar Farm, 15th Street NW, Church Road NW, Washington, DC FLBF Briar Farm, 15th Street NW, Gainesville, Alachua Co., FL DCPG National Portrait Gallery, F and 7th FLBW Private garden, Lakeview Drive, DeFuniak Springs, Walton Co., NW, Washington, DC FLCG DeFuniak Springs, Walton Co., FL Cummer Gallery of Art, 829 Riversid FLCG Cummer Gallery of Art, 829 Riverside Avenue, Jacksonville, Duval Co., FL FLCM Cemetery adjacent to Monticello Nursery, Monticello, Jefferson Co., FL Rock Creek Park, Washington, DC Monticello, Jefferson Co., FL FLCS Apartment building, Charles Street, Tallahassee, Leon Co., FL Apartment building, Charles Street, Tallahassee, Leon Co., FL FLDC Tallahassee, Leon Co., FL Doyle Conner Building, 2010 Southwest 34th Private garden, 1313 Southwest 23d Drive, Gainesville, Alachua Co., FL FLWC Women’s Club of Jacksonville, Park Street, FLDJ Private garden, 335 Cypress Street, DeFuniak Springs, Walton Co., FL FLWC Jacksonville, Duval Co., FL FLWG West Gate Shopping Centre, University FLEM Lake Ella Motel, Tallahassee, Leon Co., FL FLWG FLFB Private garden, 2356 West University Avenue, Avenue, Gainesville, Alachua Co., FL FLWJ Avenue, Gainesville, Alachua Co., FL Private garden, 2243 University Boulevard, FLWJ Private garden, 2243 University Boul Gainesville, Alachua Co., FL FLFS Florida State University, Tallahassee, North Jacksonville, Duval Co., FL GAAB Atlanta Botanical Garden, Piedmont Park, Prado Street S, Atlanta, Fulton Co., GA FLGA Apartment house, 13th Street and 13th Avenue SW, Gainesville, Alachua Co., FL GAAC GAAC Camak House, Finley and Meigs Streets, FLGS Glen Saint Mary Nurseries, Glen Street, Athens, Clarke Co., GA Glen Saint Mary, Baker Co., FL GAAG Private garden, Augusta, Richmond Co., GA Glen Saint Mary, Baker Co., FL FLHJ Howard Johnson Restaurant, 13th Street NW GAA Carr’s Hill, Athens, Clarke Co., GA and 29th Road, Gainesville, Alachua Co., FL GAA GAAT GABC Private garden, Athens, Clarke Co., GA Bonaventure Cemetery, Savannah, FLHP Private garden, Highland Park area, GABC Bonaventure Cemetery, Savannah, Chatham Co., GA Jacksonville, Duval Co., FL GABG Barnsley Gardens, Route 3, Adairsville, FLIF IFAS Horticultural Unit (University of Florida), GABP Bartow Co., GA Millhopper Road, Gainesville, Alachua Co., FL FLJH Private garden, 2 Northwest 29th Street, GABP GABQ Buttner Park, Savannah, Chatham Co., GA FLJH Private garden, 2 Northwest 29 Private garden, 2 Northwest 29th Street, GABQ Private garden, 209 Sherwood Road, Rome, Gainesville, Alachua Co., FL GABQ Private garden, 209 Sherwood Road, Rome, Floyd Co., GA FLJU Jacksonville University, Jacksonville, FLJU Jacksonville University, Jacksonville, GABS Berry School, Rome, Floyd Co., GA Benjamin Wilson House, Calhoun S Duval Co., FL American Legion Building, Lake Ella, Duval Co., FL FLLE American Legion Building, Lake Ella, GABW Benjamin Wilson House, Calhoun Square, Savannah, Chatham Co., GA Tallahassee, Leon Co., FL Savannah, Chatham Co., GA Along Canton bypass, Route 5 Tallahassee, Leon Co., FL FLLT Building along Route 27 near Lake Ella, GACB Along Canton bypass, Route 5, Cherokee Co., GA Tallahassee, Leon Co., FL Tallahassee, Leon Co., FL Cherokee Co., GA GACC Christ Church, near Fort Frederica, Saint Simon’s Island, Glynn Co., GA FLMA Marcos de Apalache Museum, Saint Marks, Wakulla Co., FL GACD Simon’s Island, Glynn Co., GA Private garden, 5 Club Drive, R Wakulla Co., FL GACD Private garden, 5 Club Drive, Rome, Floyd Co., GA FLMG Alfred B. Washington, DC Maclay Gardens State Park, Floyd Co., GA Floyd Co., GA US Route 319, Leon Co., FL GA US Route 319, Leon Co., FL GACE Private garden, 4130 Amsterdam Circle, Savannah, Chatham Co., GA FLMM Mason Manor, 1827 Stockton Street, G FLMN Jacksonville, Duval Co., FL Monticello Nursery, Monticello, Jacksonville, Duval Co., FL GACG Savannah, Chatham Co., GA Callaway Gardens, Pine Mounta FLMN Monticello Nursery, Monticello, FLMN Monticello Nursery, Monticello, GACG Callaway Gardens, Pine Mountain, Harris Co., GA FLMS Jefferson Co., FL Along Highway 441, south side of Gainesville, Harris Co., GA Jefferson Co., FL GACH Cloisters Hotel, Sea Island, Saint Simon’s Island, Glynn FLMS Along Highway 441, south side of Gainesville, Alachua Co., FL GACL Saint Simon’s Island, Glynn Co., GA Private garden, 308 East 5th Street, Alachua Co., FL Alachua Co., FL FLMT Vacant lot on Monroe Street, Tallahassee, GACL Private garden, 308 East 5th Street, Savannah, Chatham Co., GA GACM Savannah, Chatham Co., GA Maxwelton Plantation, Colonel’s Leon Co. FL FLPE Private garden, 4859 Empire Avenue, Jacksonville, Duval Co. FL GACM Maxwelton Plantation, Colonel’s Island, Liberty Co., GA Liberty Co., GA Jacksonville, Duval Co. FL GACR Liberty Co., GA Old Knight Planta Jacksonville, Duval Co. Washington, DC DEEM Eleutherian Mills, Greenville, DEEM Eleutherian Mills, Greenville, New Castle Co., DE DCML The Mall, Washington, DC New Castle Co., DE DEFC Frorer Collection, Wilmington, New Castle Co., DE DCMN Fort McNair, Washington, DC DCMS US Navy, Bureau of Medicine and New Castle Co., DE 23d and E Streets NW, Washington, DC DEFL DEMC Private garden, Bridgeville, Sussex Co., DE Mount Cuba Botanical Park, Greenville, DCNA US National Arboretum, Washington, DC National Cathedral, Washington, DC DEMC Mount Cuba Botanical Park, Greenville, New Castle Co., DE DCNC National Cathedral, Washington, DC New Castle Co., DE DESC DESR Delaware State College, Dover, Kent Co., DE DCNE Private garden, 3910 18th Street NE, Washington, DC Washington, DC DESR Along Route 52, 1/2 mi. Washington, DC south of Altamaha River bridge, near Jesup, Wayne Co., GA south of Altamaha River bridge, near Jesup, GAVA GAVI DeSoto Hotel, Kingston Valley Inn Motel, Rout GAVA GAVI DeSoto Hotel, Kingston, Floyd Co., GA Valley Inn Motel, Route 27, Harris Co., GA GAVI GAWD Valley Inn Motel, Route 27, Harris Co., GA Private garden, 2600 Lexington Road, GAJC Private garden, 3820 Northside Drive, Atlanta, Fulton Co., GA GAWD Private garden, 2600 Lexington Road, Athens, Clarke Co., GA Athens, Clarke Co., GA Private garden, Harden B Fulton Co., GA GAJI Private garden, Jekyll Island, Glynn Co., GA GA GAWH Private garden, Harden Bridge Road, Banks Co., GA GAJS Cedar Lane Farms Nursery, Madison, Banks Co., GA GAJS Cedar Lane Farms Nursery, Madison, GAWJ Private garden along Route 83, near West Jefferson and Washington Streets, Madison, Morgan Co., GA GAJY Private garden, 881 Conway Drive W, Atlanta, Fulton Co., GA GAKN Fulton Co., GA Private garden, 357 Academy Street, Madison, GAKN Private garden, 357 Academy Street, Madison, Morgan Co., GA Morgan Co., GA Monte Morgan Co., GA Morgan Co., GA GALA Private garden, 3700 Northside Drive NW, Richmond GALA Private garden, 3700 Northside Drive NW, Atlanta, Fulton Co., GA Wormsloe Plantation, RR 6 (Skidaway Road), Savannah, Chatham Co., GA GALE Atlanta, Fulton Co., GA Lawton B. Evans School grounds, Walton Way, Wormsloe Plant Savannah, Cha Atlanta, Fulton Co., GA Atlanta, Fulton Co., GA ton B. Washington, DC FL GACR Old Knight Plantation, Quitman, Brooks Co., GA FLPL Park Street and Laclede (western section), Jacksonville, Duval Co., FL FLPL Park Street and Laclede (west FLPM Jacksonville, Duval Co., FL GACW Brooks Co., GA GACW Clark and Waddell Streets, Athens, Clarke Co., GA FLPM Prince Murat Motel, Monroe Street, FLPS Tallahassee, Leon Co., FL Private garden, Park Street extension, Clarke Co., GA Tallahassee, Leon Co., FL GADG Clarke Co., GA Diamond Garden FLPS Private garden, Park Street extension, GADG Diamond Garden, South Rockwell Avenue, Vernonburg, Chatham Co., GA FLPS FLPU Private garden, Park Street extension, Riverside area (western section), Jacksonville Duval Co., FL Private garden, Providence, Union Co., FL Riverside area (western section), Jacksonville, Duval Co., FL Private garden, Providence, Union Co., FL GADR Vernonburg, Chatham Co., GA Private garden, corner of Drayton GADR Vernonburg, Chatham Co., GA Private garden, corner of Drayton and Gaston GADR Private garden, corner of Drayton and Gaston Streets, Savannah, Chatham Co., GA FLPU FLPW Private garden, Providence, Union Co., FL Welcome station, Florida state line, Route 1, GAEC Streets, Savannah, Chatham Co., GA Private garden, Highway 140, Canton, Streets, Savannah, Chatham Co., GA Private garden, Highway 140, Canton, GAEC Private garden, Highway 140, Canton, Cherokee Co., GA FLPW Welcome station, Florida state line, Route 1, Nassau Co., FL Private garden, Randall and Dancy Streets, GAEH Cherokee Co., GA Mimosa Hall, 127 B FLRD Private garden, Randall and Dancy Streets, Jacksonville, Duval Co., FL GAEH Mimosa Hall, 127 Bullock Avenue, Roswell, Fulton Co., GA FLRH Jacksonville, Duval Co., FL North Florida Landscaping Co., 1310 Glen GAES Private garden, 316 55th Street, East Savannah, Chatham Co., GA North Florida Landscaping Co., 1310 Gle GAET East Savannah, Chatham Co., GA Private garden, 2021 East 37th Stree FLRI Laura Road, Jacksonville, Duval Co., FL FLRI Riverside Motel, Route 17, near Florida state line, Nassau Co., FL GAET Private garden, 2021 East 37th Street, Savannah, Chatham Co., GA GAFF Savannah, Chatham Co., GA Private garden, front of 318 55 FLRM Private garden, San Jose Boulevard, GAFF Private garden, front of 318 55th Street E, Savannah, Chatham Co., GA GAFG Savannah, Chatham Co., GA Private garden, Hamilton, Harr Jacksonville, Duval Co., FL FLRP Private garden, 4806 River Basin Drive and GAFG GAFN Private garden, Hamilton, Harris Co., GA Fruitland Nursery, Augusta, iver Point Road, Jacksonville, Duval Co., FL orreya State Park, Liberty Co., FL GAFN Fruitland Nursery, Augusta, Richmond Co., GA GAGB Richmond Co., GA University of Georgi FLTP FLUF Torreya State Park, Liberty Co., FL University of Florida, Gainesville, FLTP FLUF Torreya State Park, Liberty Co., FL University of Florida, Gainesville, GAGB University of Georgia Botanical Garden, Athens, Clarke Co., GA FLUF University of Florida, Gainesville, Alachua Co., FL 239 GARW GASC Reinhardt College, Waleska, Cherokee Co., GA Sadis Church, Route 140, Cherokee Co., GA GAGC Gould’s Corner, Walton Way and Milledge Road, Augusta, Richmond Co., GA Road, Augusta, Richmond Co., GA Abandoned garden along Route 27 GASC GASH Sadis Church, Route 140, Cherokee Co., GA Tullie Smith House Restoration, 3099 Andrews GAGM Road, Augusta, Richmond Co., GA Abandoned garden along Route 27 Alt., 10 mi. Washington, DC GAGM Abandoned garden along Route 27 Alt., 10 mi. north of Greenville, Meriwether Co., GA GASH GAGM Abandoned garden along Route 27 Alt., 10 mi. GASH GASH Tullie Smith House Restoration, 3099 Andrews Drive NW, Atlanta, Fulton Co., GA north of Greenville, Meriwether Co., GA GASM Drive NW, Atlanta, Fulton Co., GA Private garden, 4637 Oakview Drive GAGN Private garden, front of 415 Green Street, GASM Private garden, 4637 Oakview Drive, Savannah, Chatham Co., GA Augusta, Richmond Co., GA GASS Savannah, Chatham Co., GA Augusta, Richmond Co., GA Augusta, Richmond Co., GA GAS Private garden, 2111 Gardner Street, Augusta, Richmond Co., GA Queens Court Motel, Saint Simon’s Island, Glynn Co., GA GASS GASU Queens Court Motel, Saint Simon’s Island, Glynn Co., GA Summerville Cemetery, Cuming and Johns GASS Queens Court Motel, Saint Simon’s Island, Glynn Co., GA Richmond Co., GA Richmond Co., GA GAGS Private garden, adjacent to 2111 Gardner Street, Augusta, Richmond Co., GA GAS GASU Summerville Cemetery, Cuming and Johns Roads, Augusta, Richmond Co., GA Street, Augusta, Richmond Co., GA Private garden, 933 Milledge Road, Augusta, GATC GATC Roads, Augusta, Richmond Co., GA Theta Chi Fraternity House, 645 Mille Street, Augusta, Richmond Co., GA Private garden, 933 Milledge Road, Augu GAHC Private garden, 933 Milledge Road, Augusta, Richmond Co., GA GAT GATC Theta Chi Fraternity House, 645 Milledge Avenue, Athens, Clarke Co., GA Richmond Co., GA GATG Avenue, Athens, Clarke Co., GA Richmond Co., GA HS Private garden, 869 Hill Street, Athens, Clark Co., GA GAHS Private garden, 869 Hill Street, Athens, Clark Co., GA GATG Taylor-Grady House, Prince Avenue, Athens, Clarke Co., GA GAHS Private garden, 869 Hill Street, Athens, Clarke Co., GA GATN Bonar Hall, Dixy Avenue, Madison, Morgan Co., GA GAIH Private garden, Isle of Hope, La Roche Avenue, Savannah, Chatham Co., GA Morgan Co., GA Savannah, Chatham Co., GA Plant Introduction Station, Rou Savannah, Chatham Co., GA G GATS Private garden, Route 6, Turner’s Rock, Savannah, Chatham Co., GA GAIS Plant Introduction Station, Route 17, 10 mi. GATS Savannah, Chatham Co., GA University of Georgia, Athens, south of Savannah, Chatham Co., GA GAUG GAVA University of Georgia, Athens, Clark Co., GA DeSoto Hotel, Kingston, Floyd Co., GA GAJA GAJC Edge of southbound Route 301, ca. 2 mi. south of Altamaha River bridge, near Jesup, Wayne Co., GA Private garden, 3820 Northside Drive, Atlanta, GAJA Edge of southbound Route 301, ca. 2 mi. Montgomery Co., MD Private garden, 24 Acac MDHS Private garden, 24 Acacia, Scientists’ Cliffs, Port Republic, Calvert Co., MD MDCH Private garden, 6510 River Road, Bethesda, Montgomery Co., MD Montgomery Co., MD Carson Circle and LaSa Kent Co., MD MDGB Small park near shopping area, Greenbelt, Prince George’s Co., MD Kent Co., MD MDBA Bond Arboretum, Easton, Talbot Co., MD MDGC Prince George’s Co., MD MDGC Goucher College (Science Building), Towson, Baltimore Co., MD MDBD Brighton Dam, Brighton Dam Road, Ashton, Montgomery Co., MD Montgomery Co., MD Calvert Co., MD MDCD Crescent Drive, Greenbelt, Prince George’s Co., MD MDCD Crescent Drive, Greenbelt, Prince George’s Co., MD MDHP Lane, Towson, Baltimore Co., MD Hilltop and Park Valley Roads, MDHP Hilltop and Park Valley Roads, near Piney Branch Road, Takoma MDCG Prince George’s Co., MD Private garden, 4327 Kent MDCG Prince George’s Co., MD Private garden, 4327 Kentbury Drive, near Piney Branch Road, Takoma Park, Montgomery Co., MD MDCG Private garden, 4327 Kentbury Drive, Bethesda, Montgomery Co., MD MDCH Bethesda, Montgomery Co., MD Private garden, 6510 River Road, Faulkner, Charles Co., MD Faulkner, Charles Co., MD MDHN Hampton National Historic Site, 535 Hampton Lane, Towson, Baltimore Co., MD Calvert Co., MD Montgomery Co., MD Carson Circle and LaSa Port Republic, Calvert Co., MD Private garden, 7001 Glenn Broo MDHW Private garden, 7001 Glenn Brook, Bethesda, Montgomery Co., MD MDCL Carson Circle and LaSalle Drive, Avondale, Prince George’s Co., MD MDIH Montgomery Co., MD National Institutes of H Montgomery Co., MD Montgomery Co., MD Cliffs, Port Republic, Calvert Co., MD Hood College, Frederick, Frederick Co., MD MDHC MDHD Hood College, Frederick, Frederick Co., MD Private garden, 3108 Lancer Drive, Hyattsville, MDBR Bordley-Randall House, Randall Court, Annapolis, Anne Arundel Co., MD MDBR Bordley-Randall House, Randall Court, Annapolis, Anne Arundel Co., MD MDHD Private garden, 3108 Lancer Drive, Prince George’s Co., MD rivate garden, 3108 Lancer Drive, Hyattsville, rince George’s Co., MD MDHF Prince George’s Co., MD Memorial Hospital grounds MDBW MDCA Blackwater Wildlife Refuge, Dorcester Co., MD Private garden across from St. John’s College, MDHF Memorial Hospital grounds, Frederick, Frederick Co., MD MDCA MDCC Private garden across from St. John’s Colleg College Avenue, Annapolis, Anne Arundel Co., MD Along Route 4 at Calvert County line, MDHH Frederick Co., MD Hardy Farm, 7 mi. N College Avenue, Annapolis, Anne Arundel Co., MD Washington, DC Evans School grounds, Walton Way, usta, Richmond Co., GA Augusta, Richmond Co., GA Private garden, 1 Beech Knoll Augusta, Richmond Co., GA Private garden, 575 Waddell Street, Athens, Clarke Co., GA Augusta, Richmond Co., GA Private garden, 575 Waddell Street, Athens, GALF Private garden, 1 Beech Knoll Drive, Canton, Cherokee Co., GA Clarke Co., GA Jungle Gardens, Avery Island, Iberia Parish, LA GALH Cherokee Co., GA Private garden, Thomson, McDuffie Co., GA Jungle Gardens, Iberia Parish, LA Cherokee Co., GA GALH Private garden, Thomson, McDuffie Co., GA Private garden, Lumpkin Street, Athens, Iberia Parish, LA GALS Private garden, Lumpkin Street, Athens, Audubon Par Clarke Co., GA Clarke Co., GA GAMC Private garden, 720 Milledge Circle, Athens, Clarke Co., GA GAMD Clarke Co., GA Private garden, Hallowcreek Lane, Athens, GAMD GAME Private garden, Hallowcreek Lane, Athens, Clarke Co., GA Private garden, 708 Milledge Road, Augusta, LAGN Lafayette Parish Grandview Nurs Lafayette Parish GAME Private garden, 708 Milledge Road, Augusta, Richmond Co., GA GAME Private garden, 708 Milledge Road, Augusta, Richmond Co., GA La Ho GAMM City Hall, Madison, Morgan Co., GA Georgia May Park, 4th Avenue at Fen GAMP Georgia May Park, 4th Avenue at Fenwick, Augusta, Richmond Co., GA GAMP Georgia May Park, 4th Avenue at Fen Augusta, Richmond Co., GA GAMP GAMW Georgia May Park, 4th Avenue at Fenwick, Augusta, Richmond Co., GA Private garden, 126 Dearing Street, Athens, Augusta, Richmond Co., GA Private garden, 126 Dearing S GAMW Augusta, Richmond Co., GA Private garden, 126 Dearing Street, Athens, GAMW Private garden, 126 Dearing Street, Athens, Clark Co., GA GANA Private garden, 530 Academy Street, Madison, Morgan Co., GA GANG Augusta National Golf Course, Augusta, Richmond Co., GA Richmond Co., GA GAOH Richmond Co., GA Stre GAOH Private garden, Oak Hill, Rome, Floyd Co., GA Private garden, Athens, Clarke Co., GA Private garden, Athens, Clarke Co., GA Corner of State and Main Streets, Private garden, Athens, Clarke Co., GA Corner of State and Main Streets, Parish, LA Private garden, Athens, Clarke Co., GA Corner of State and Main Streets, Corner of State and Main Streets, Pine Mountain, Harris Co., GA LA Corner of State and Main Streets, Pine Mountain, Harris Co., GA Pine Mountain, Harris Co., GA Private garden, 3036 Pine Needle Street, Private garden, 3036 Pine Needle Str Augusta, Richmond Co., GA Augusta, Richmond Co., GA Private garden, 188 South Mil Private garden, 188 South Milledge Avenue, Athens, Clarke Co., GA LARP LARS LASL LATU MDAB MDAC MDAF MDAG MDAL MDAM MDAN MDBA MDBD MDBE Rosedown Plantation, Saint Francisville, West Feliciana Parish, LA Private garden, Jonesville, Concordia Parish, LA Southwestern Louisiana University, Lafayette, Lafayette Parish, LA Tulane University, New Orleans, Orleans Parish, LA USDA Agricultural Research Center (formerly Plant Industry Sta.), Beltsville, Prince George’s Co., MD Adventist Church, Laurel and Eastern Avenues, Takoma Park, Montgomery Co., MD Society of American Foresters, 5400 Grosvenor Lane, Bethesda, Montgomery Co., MD Private garden, Bethesda, Montgomery Co., MD Private garden, Lutherville, Baltimore Co., MD Private garden at house across from US Post Office, Ashton, Montgomery Co., MD Angelica Nurseries, RR 1, Kennedyville, Kent Co., MD Bond Arboretum, Easton, Talbot Co., MD Brighton Dam, Brighton Dam Road, Ashton, Montgomery Co., MD Private garden, 1600 Bishop Road, Edgewater, Anne Arundel Co., MD MDDP MDDS MDEC MDEF MDEM MDES MDET MDFA MDFD MDFF MDFM MDGB Private garden, corner of Dale and Pershing Drives, Silver Spring, Montgomery Co., MD Private garden, 727 Dartmouth Street, Silver Spring, Montgomery Co., MD Episcopal Church, Prince Frederick, Calvert Co., MD Private garden, near Elm Avenue, Frederick, Frederick Co., MD Private garden, 400 East Main Street, Middletown, Frederick Co., MD Private garden, Haller Vista Avenue, Frederick, Frederick Co., MD Private garden, 7100 Armat Drive, Bethesda, Montgomery Co., MD Private garden, Camden Street, Salisbury, Wicomico Co., MD Private garden, 730 Dartmouth Avenue, Silver Spring, Montgomery Co., MD Along Route 15 at Frederick, Frederick Co., MD Private garden, 7417 Buffalo Avenue, Takoma Park, Montgomery Co., MD Small park near shopping area, Greenbelt, Prince George’s Co., MD MDDP Private garden, corner of Dale and Pershing Drives, Silver Spring, Montgomery Co., MD LARS LASL LATU MDAB MDAC MDAF West Feliciana Parish, LA Private garden, Jonesville, Concordia Parish, LA Southwestern Louisiana University, Lafayette, Lafayette Parish, LA Tulane University, New Orleans, Orleans Parish, LA USDA Agricultural Research Center (formerly Plant Industry Sta.), Beltsville, Prince George’s Co., MD Adventist Church, Laurel and Eastern Avenues, Takoma Park, Montgomery Co., MD Society of American Foresters, 5400 Grosvenor Lane, Bethesda, Montgomery Co., MD MDDS Private garden, 727 Dartmouth Street, Silver Spring, Montgomery Co., MD MDEC Episcopal Church, Prince Frederick, MDEF Private garden, near Elm Avenue, Frederick, Frederick Co., MD MDEM Private garden, 400 East Main Street, MDES Private garden, Haller Vista Avenue, Frederick, Frederick Co., MD Dale, Prince George’s Co., MD Dale, Prince George’s Co., MD Private garden, 7112 Cedar Avenue, Takoma Park, Montgomery Co., MD MDBN J. H. Burton Nursery, 5950 Ager Road Takoma Park, Montgomery Co., MD Private garden, 22 Orchard Way, Hyattsville, Prince George’s Co., MD Field along B & O Railroad, Jackson, MDGR Private garden, 22 Orchard Way, MDBO Field along B & O Railroad, Jackson, Cecil Co., MD North Rockville, Montgomery Co., MD Cecil Co., MD MDHA Henry Allanson Memorial Garden, Scientists’ Cliffs, Port Republic, Calvert Co., MD MDHA Henry Allanson Memorial Garden, Scientists’ Cliffs, Port Republic, Calvert Co., MD MDBP Jesup Blair Park, Silver Spring, Montgomery Co., MD M MDBP Jesup Blair Park, Silver Spring, Montgomery Co., MD MDCO Prince George’s Co., MD Corner of Colesville Road MDCO Prince George’s Co., MD Corner of Colesville Road MDCO Corner of Colesville Road and Dale Drive, Silver Spring, Montgomery Co., MD MDIH National Institutes of Health, Bethesda, Montgomery Co., MD Anne Arundel Co., MD Along Route 4 at Calvert MDHH Hardy Farm, 7 mi. N of Potomac River bridge, Faulkner, Charles Co., MD Anne Arundel Co., MD Along Route 4 at Calvert MDCC Along Route 4 at Calvert County line, Calvert Co., MD Baltimore Co., MD MDBE Private garden, 1600 Bishop Road, Edgewater, Anne Arundel Co., MD MDGD USDA Plant Introduction Station (now National Plant Quarantine Center), Glenn Dale, Prince George’s Co., MD Anne Arundel Co., MD Brookside Gardens, 150 MDBG Brookside Gardens, 1500 Glenall MDBG Brookside Gardens, 1500 Glenallen Road, Wheaton, Montgomery Co., MD MDGG Private garden, 11002 Kenilworth Avenue, Garrett Park, Montgomery Co., MD Wheaton, Montgomery Co., MD MDBM Private garden, 6704 Glenn Avenue, Glenn Dale, Prince George’s Co., MD Garrett Park, Montgomery Co., MD Private garden, Bryans Road, Charle MDGJ MDGL Private garden, Bryans Road, Charles Co., MD Private garden, 7112 Cedar Avenue, Prince George’s Co., MD Adventist Church, Laurel and Eastern MDET Private garden, 7100 Armat Drive, Bethesda, Montgomery Co., MD Avenues, Takoma Park, Montgomery Co., MD MDAF Society of American Foresters, MDFA Private garden, Camden Street, Salisbury, Wicomico Co., MD MDFD Private garden, 730 Dartmouth Avenue, Silver Spring, Montgomery Co., MD MDAG Private garden, Bethesda, Montgomery Co., MD Silver Spring, Montgomery Co., MD Montgomery Co., MD MDFF Along Route 15 at Frederick, Frederick Co., MD MDAM Private garden at house across from US Post Office, Ashton, Montgomery Co., MD Frederick Co., MD MDAM Private garden at house across from US Post Office, Ashton, Montgomery Co., MD Angelica Nurseries, RR 1, Kennedyville, MDAM MDAN Office, Ashton, Montgomery Co., MD Angelica Nurseries, RR 1, Kennedyville, MDFM Private garden, 7417 Buffalo Avenue, Takoma Park, Montgomery Co., MD MDAN Angelica Nurseries, RR 1, Kennedyville, Kent Co., MD MDGB Takoma Park, Montgomery Co., MD Small park near shopping area, Greenb MDCP Silver Spring, Montgomery Co., MD Mount Clare, Carroll Park, Baltimore, MDCP Silver Spring, Montgomery Co., MD Mount Clare, Carroll Park, Baltimore, MD MDJB MDJC MDJH Montgomery Co., MD Jones Mill and Brookville Roads, adjacent to Route 410 at Rock Creek, Montgomery Co., MD Private garden, New Carrollton, Prince George’s Co., MD Johns Hopkins University campus, Baltimore, MD MDCP MDCR Mount Clare, Carroll Park, Baltimore, MD Private garden, 1634 Chesapeake Road, adjacent to Route 410 at Rock Creek, Montgomery Co., MD MDCR Private garden, 1634 Chesapeake Road, Edgewater, Anne Arundel Co., MD MDJC MDJH adjacent to Route 410 at Rock Creek, Montgomery Co., MD Private garden, New Carrollton, Prince George’s Co., MD Johns Hopkins University campus, Baltimore, MD DDF Edgewater, Anne Arundel Co., MD Private garden, “In the Woods,” Montgomery Co., MD Montpelier Mansion, 12828 Laurel-Bowie Road, Laurel, Prince George’s Co., MD McLean Nursery, Satyr Hill Road, Towson, MDTW MDUM Towson Nursery, Towson, Baltimore Co., MD North Administration Building, MDUM North Administration Building, University of Maryland, College McLean Nursery, Satyr Hill Road, Towson, Baltimore Co., MD Restaurant on Mayo Road University of Maryland, College Park, Prince George’s Co., MD Prince George’s Co., MD Corner of Valley View and MDVV Corner of Valley View and Maple Avenues, Takoma Park, Montgomery Co., MD Restaurant on Mayo Road (near Londontown Road), Edgewater, Anne Arundel Co., MD Private garden, 1 Valley View, Takoma Park, Takoma Park, Montgomery Co., MD MDWA Ten Oaks Nursery, Clarkesville, Howard Co., MD Private garden, 1 Valley View, Takoma Park, Montgomery Co., MD MDW Howard Co., MD Private garden, Bu Montgomery Co., MD Private garden, 2212 Merzerott Road, Adelphi, Prince George’s Co., MD US Naval Academy, Annapolis, Anne Arundel Co., MD Bethesda Naval Hospital, Bethesda, Montgomery Co., MD Olallie Farm, Glenn Dale, Prince George’s Co., MD Chatolanee Hill, Owings Mills, Baltimore Co., MD Private garden, 7432 Piney Branch Road, Takoma Park, Montgomery Co., MD Private garden, 420 Pershing Drive, Silver Spring, Montgomery Co., MD Private garden, Fox Chapel Drive, Lutherville, Baltimore Co., MD Private garden, Scientists’ Cliffs, Prince Frederick, Calvert Co., MD MDWG Private garden, Burnt Mills, 10705 Blossom Private garden, 2212 Merzerott Road, Adelphi, Prince George’s Co., MD MDWJ Lane, Silver Spring, Montgomery Co., MD Roadside at intersection of Wisconsin Avenue MDWJ Roadside at intersection of Wisconsin Avenue US Naval Academy, Annapolis, Anne Arundel Co., MD MDW US Naval Academy, Annapolis, MDWK and Jones Bridge Road, Montgomery Co., MD Private garden, 415 Pershing Drive, MDWK Private garden, 415 Pershing Drive, Silver Spring, Montgomery Co., MD Anne Arundel Co., MD Bethesda Naval Hospital, Bethesda, Silver Spring, Montgomery Co., MD MDWM Wye Oak State Park, Wye Mills, MDWM Wye Oak State Park, Wye Mills, Olallie Farm, Glenn Dale, MDDF Edgewater, Anne Arundel Co., MD Private garden, “In the Woods,” MDDF Private garden, “In the Woods,” 8922 Spring Valley Road, Chevy Chase, Montgomery Co., MD MDJC Private garden, New Carrollton, Prince George’s Co., MD MDDF Private garden, “In the Woods,” 8922 Spring Valley Road, Chevy 8922 Spring Valley Road, Chevy Chase, Montgomery Co., MD 241 Jenkins Nursery, Mitchelville, Jenkins Nursery, Mitchelville, MD Prince George’s Co., MD Private garden, 6200 Kennedy Drive, MDP Kenwood, Chevy Chase, Montgomery Co., MD MDPM Private garden, 1304 West Seminary Road, MDRL MDRL Private garden, 11954 Simpson Road, Clarksville, Howard Co., MD Lutherville, Baltimore Co., MD Private garden, 4400 Samar Street, Beltsville, MDRP Prince George’s Co., MD MDRP Private garden, 4502 Brandon Lane, Beltsville, Private garden, 1138 Kenilworth Avenue, Garrett Park, Montgomery Co., MD MDRT MDRT Rideout Tenements, 114 Duke of Gloucester Street, Annapolis, Garrett Park, Montgomery Co., MD Gloucester Street, Annapolis, Private garden, 6102 Kirby Street, Bethesda, MDSC Anne Arundel Co., MD Plant Materials Center, Montgomery Co., MD MDSC Plant Materials Center, Soil Conservation Service Kingsville Nursery, Kingsville, Kingsville Nursery, Kingsville, Baltimore Co., MD MDSC Soil Conservation Service, Beltsville, Prince George’s Co., MD Baltimore Co., MD Prince George’s Co., MD Private garden, Kennett Street, Silver Spring, MDSG Private garden, 4102 Chelmont Lane, Bowie, Prince George’s Co., MD Montgomery Co., MD Montgomery Co., MD Tulip Hill, Harwood, Anne Arundel Co., MD MDSG Prince George’s Co., MD Tulip Hill, Harwood, Anne Arundel Co., MD Landon School Gardens, Bethesda, MDSH MDSH MDSJ Satyr Hill Nursery, Baltimore, MD Saint John’s College, Annapolis, Landon School Gardens, Bethesda, Montgomery Co., MD MDSH MDSJ MDSJ Saint John’s College, Annapolis, Anne Arundel Co., MD Montgomery Co., MD Across from National L Across from National Library of Medicine, Anne Arundel Co., MD MDSM Private garden, 10926 Montrose Street Lyon Nursery, 13520 New Hampshire Avenue, MDSS Garrett Park, Montgomery Co., MD Private garden at corner of Georgia MDSS Private garden at corner of Georgia Avenue Silver Spring, Montgomery Co., MD MDS Silver Spring, Montgomery Co., MD and Highland Drive, Silver Spring, Private garden, 415 Ethan Allen, Private garden, 415 Ethan Allen, Takoma Park, Prince George’s Co., MD MDST Private garden, 5520 Spruce Tree Avenue, London Town Publik House and Gardens, 839 Londontown Road, Edgewater, Anne Arundel Co., MD MDS MDT Bethesda, Montgomery Co., MD Private garden, 10041 Worrell Av MDTD Private garden, 10041 Worrell Avenu Glenn Dale, Prince George’s Co., MD Montgomery College, Fenton Street, Glenn Dale, Prince George’s Co., MD Tyler Heights Elementary School, Janw Takoma Park, Montgomery Co., MD MDT MDTH Tyler Heights Elementary School, Janwal Street, Annapolis, Anne Arundel Co., MD Montgomery Co., MD METRO Station, Colesville Road, Silver Spring, MDT Street, Annapolis, Anne Arundel Co., MD Tingle Nursery, Pittsville, Wicomico Co., MD MDTN MDTP Tingle Nursery, Pittsville, Wicomico Co., MD Gymnasium, Chicago Avenue MDTP Gymnasium, Chicago Avenue McCrillis Gardens and Gallery, 6910 Greentree Road, Bethesda, Montgomery Co., MD Montpelier Mansion, 12828 Laurel-Bowie near Philadelphia Avenue, Takoma Park, Montgomery Co., MD Talbot Co., MD south of Wilmington, Brunswick Co., NC NCBR YMCA Blue Ridge Assembly, near Black Mountain, Buncombe Co., NC of Wilmington, Brunswick Co., NC NCPL Planters National Bank, Rocky Mount, Edgecombe Co., NC NCBW Private garden, Willard, Pender Co., NC Coker Arboretum, University of North Edgecombe Co., NC NCCA Coker Arboretum, University of North Carolina, Chapel Hill, Orange Co., NC Carolina, Chapel Hill, Orange Co., NC NCPR Prudent National Bank, Rocky Mount, Edgecombe Co., NC Carolina, Chapel Hill, Orange Co., NC Edgecombe Co., NC NCCB Chatwood, Faucette Mill Road, Hillsborough, Orange Co., NC NCRC Private garden, 2800 Saint Andrews Lane, Charlotte, Mecklenburg Co., NC NCCC Orange Co., NC Calico Cottage Restaurant, Saluda, Orange Co., NC NCCC Calico Cottage Restaurant, Saluda, Polk Co., NC Charlotte, Mecklenburg Co., NC NCRM NCRW Riverside Motel, Southport, Brunswick Co., NC Private garden, 342 Ridgewood Avenue, NCCG NCCM Clarendon Gardens, Pinehurst, Moore Co., NC Carleton Motel, Rocky Mount, NCRW Private garden, 342 Ridgewood Avenue, Charlotte, Mecklenburg Co., NC Charlotte, Mecklenburg Co., NC NCCM Carleton Motel, Rocky Mount, Edgecombe Co., NC NCSH Ebersole Holly Garden, Sand Hills Community College, Southern Pines, Moore Co., NC Edgecombe Co., NC NCSH Ebersole Holly Garden, Sand Hills College, Southern Pines, Moore Co. Talbot Co., MD MDWP The William Paca Garden, 1 Martin Street, Annapolis, Anne Arundel Co., MD Annapolis, Anne Arundel Co., MD Private garden, 40 Cornhill Street, A MDWS Private garden, 40 Cornhill Street, Annapolis, Anne Arundel Co., MD MSBA Private garden, Pass Christian, Harrison Co., MS Takoma Park, Montgomery Co., MD MSBA Private garden, Pass Christian, Harrison Co., MS Private garden, 420 Pershing Drive, Silver Spring, Montgomery Co., MD Private garden, Fox Chapel Drive, Lut MSBH Private garden, Crosby, Wilkinson Co., MS Bellemont Motel, US Route 61 near southern MSBN Bellemont Motel, US Route 61 near southern boundary of Natchez, Adams Co., MS i) NCHO Private garden, Hillsborough, Orange Co., NC MSEP D’Evereux Plantation, Natchez, Adams Co., MS NCHS Private garden, Norlina, Warren Co., NC MSFG Gloster Arboretum, Highway 33, Gloster, Amite Co., MS NCJP Private garden, 2260 Crawford, Durham, Durham Co., NC NCJP Private garden, 2260 Crawford, Durham, Durham Co., NC MSHB Private garden, Hattiesburg, Forrest Co., MS NCKH Private garden, West King and South Hasel MSKR Private garden, Perry Co., MS MSKR Private garden, Perry Co., MS Streets, Hillsborough, Orange Co., NC MSMN Mistletoe House, Airport Road, Natchez, Adams Co., MS MSMN Mistletoe House, Airport Road, Natchez, NCKK Private garden, North Rugby Road, Adams Co., MS NCKK Private garden, North Rugby Ro RR 8 Box 48, Hendersonville, Henderson Co., NC RR 8 Box 48, Hendersonville, MSMV Magnolia Vale, Natchez, Adams Co., MS Henderson Co., NC MSPR Private garden, Poplarville, Pearl River Co., MS Rosalie House garden, Natchez, NCKN Kitty’s Nursery, 106 Airlie Road, Wilmington, MSRN Rosalie House garden, Natchez, Adams Co., MS MSRN Rosalie House garden, Natchez, Adams Co., MS MSRN Rosalie House garden, Natchez, Adams Co., MS New Hanover Co., NC NCMH Private garden, Manns Harbor, Dare Co., NC MSWB Private garden, Port Gibson, Claiborne Co., MS NCAG Cemetery of Airlie Gardens, Wrightsville Beach NCMM Private garden, Elon College, NCMM Private garden, Elon College, Alamance Co., NC Hwy., near Wilmington, New Hanover Co., NC NCMO Private garden (Mother Vine), Manteo, NCAK Private garden, 240 Tranquility Place, Dare Co., NC Dare Co., NC Hendersonville, Henderson Co., NC NCMS Private garden, Ravenswood Road, Flatrock, Henderson Co., NC Appalachian State University, Boone, NCAS Appalachian State University, Boone, Watauga Co., NC Henderson Co., NC Henderson Co., NC Watauga Co., NC NCNB Private garden, Pollock Street, New Bern, Craven Co., NC NCBE Biltmore Estate Gardens, Biltmore, Buncombe Co., NC Craven Co., NC NCNS North Carolina State University Arboretum, Raleigh, Wake Co., NC NCBM Private garden, 410 Buchanan and Monmouth Avenues, Durham, Durham Co., NC Raleigh, Wake Co., NC Avenues, Durham, Durham Co., NC YMCA Blue Ridge Assembly, NCOP Orton Plantation, off Rout of Wilmington, Brunswick NCOP Orton Plantation, off Route 133, 13 mi. Talbot Co., MD Edgecombe Co., NC NCCP Cameron Park School, Hillsborough, Orange Co., NC NCSL College, Southern Pines, Moore Co., NC NCSL Near house of Sidney Lanier, along stream 300 yards from Route 176, Tryon, Polk Co., NC Orange Co., NC Orange Co., NC NCCS Church Street, Rocky Mount, Edgecombe Co., NC NCCS Church Street, Rocky Mount, Edgecombe Co., NC NCSM yards from Route 176, Tryon, Polk Co., NC Saint Matthew's Episcopal Church, Edgecombe Co., NC NCCW Edgecombe Co., NC Private garden, Weaver Dairy Road, NCSM Saint Matthew's Episcopal Church, Hillsborough, Orange Co., NC NCCW Private garden, Weaver Dairy Chapel Hill, Orange Co., NC NCCW Private garden, Weaver Dairy Road, Chapel Hill, Orange Co., NC Hillsborough, Orange Co., NC NCSW Parking strip on South Wake Street, Hillsborough, Orange Co., NC Chapel Hill, Orange Co., NC NCDB Daniel Boone Native Gardens, Boone, Watauga Co., NC NCTE Hillsborough, Orange Co., NC Calvary Episcopal Church, Chu NCTE Calvary Episcopal Church, Church Street, Tarboro, Edgecombe Co., NC Watauga Co., NC NCTE Calvary Episcopal Church, Chu Tarboro, Edgecombe Co., NC NCDF Watauga Co., NC Private garden, Dixie Lane, Hendersonville, NCDF Private garden, Dixie Lane, Hendersonville, Henderson Co., NC NCTP Tarboro, Edgecombe Co., NC Tryon Palace, New Bern, Crave NCTP NCTR Tryon Palace, New Bern, Craven Co., NC Intersection of Towerview Road and Edwin Henderson Co., NC Bluff on Dan River, below Henderson Co., NC NCDR Henderson Co., NC Bluff on Dan River, below Leaksville, NCDR Bluff on Dan River, below Leaksville, Rockingham Co., NC NCTR Intersection of Towerview Road and Edwin Avenue, Durham, Durham Co., NC Rockingham Co., NC NCWC Avenue, Durham, Durham Co., NC NCDS Rockingham Co., NC Private garden, 2706 Spencer Street, Durham, NCDS Private garden, 2706 Spencer Street, Durham, Durham Co., NC NCWC Western Carolina University, Cullowhee, Jackson Co., NC NCWD Jackson Co., NC NCDU Durham Co., NC Duke University, Durham, Durham Co., NC NCDU Durham Co., NC Duke University, NCWD Jackson Co., NC NCWD NCWK We-Do Nursery, RR 5 Box 724, Marion, McDowell Co., NC Private garden, Norlina, Warren Co., NC NCDU NCEG Duke University, Durham, Durham Co., NC Elizabethan Gardens, Manteo, Dare Co., NC NCWD We-Do Nursery, RR 5 Box 724, Marion, McDowell Co., NC NCEG NCEL Elizabethan Gardens, Manteo, Dare Co., NC Private garden, 384 Ridgewood Road, NCWK NCWM Private garden, Norlina, Warren Co., NC Private garden, 2828 Saint Andrews Lane, NCEL Private garden, 384 Ridgewood Road, Charlotte, Mecklenburg Co., NC NCFR Charlotte, Mecklenburg Co., NC Fred Rees Nursery, Lake Hosea, NCWM Private garden, 2828 Saint Andrews Lane, Charlotte, Mecklenburg Co., NC NCFR Fred Rees Nursery, Lake Hosea, Saluda, Henderson Co., NC NCWT Charlotte, Mecklenburg Co., NC Episcopal Cemetery, West Tryon NCWT Episcopal Cemetery, West Tryon Str Hillsborough, Orange Co., NC NCGC Henderson Co., NC NCGC Private garden, Chisholm Street, Saluda, Polk Co., NC SCAB Hillsborough, Orange Co., NC Private garden, 933 Abbeville S SCAB Hillsborough, Orange Co., NC Private garden, 933 Abbeville Street, Aiken, SCAB SCAC Private garden, 933 Abbeville Street, Aiken, Aiken Co., SC Private garden, 127 Greenville Street, Aiken, NCGM Polk Co., NC Gra-Mar Lodge, Box 122, Fayetteville, NCGM Gra-Mar Lodge, Box 122, Fayetteville, Cumberland Co., NC SCAC Private garden, 127 Greenville Street, Aiken, Aiken Co., SC SCAC Private garden, 127 Greenville Street, Aiken, Aiken Co., SC NCGP Cumberland Co., NC Greenfield Park, Wilmi NCGP Cumberland Co., NC Greenfield Park, Wilmington, NCGP Greenfield Park, Wilmington, New Hanover Co., NC NCGP Greenfield Park, Wilmington, New Hanover Co., NC New Hanover Co., NC Private garden, Aiken, Aiken Co., SC Private garden, Barre and Calhoun St NCGR Park near Guess Road and Interstate 85, Durham, Durham Co., NC Private garden, Barre and Calhoun Stree Charleston, Charleston Co., SC Durham, Durham Co., NC Charleston, Charleston Co., SC 243 SCBG Bailston Garden, Richland Street, Columbia, SCJM Private garden, 42 Society Street, Charleston, Charleston Co., SC bia, SCJM Private garden, 42 Society Street, Charleston, Charleston Co., SC Richland Co., SC SCBH Private garden, 68 South Battery, Charleston, Charleston Co., SC SCKG Kalmia Gardens, Coker College, Hartsville, Darlington Co., SC Darlington Co., SC Private garden, King ation, Route 17, SC SCKS Private garden, King Street, Charleston, Charleston Co., SC SCBP Boone Hall Plantation, Route 17, Charleston Co., SC SCKS Private g Charlesto SCBP Boone Hall Plantation, Route 17, Charleston Co., SC SCKS Private garden, King Street, Charleston, Charleston Co., SC SC Charleston Co., SC Charleston Co., SC SCBR Charleston Co., SC Brookgreen Gardens, Route 17, SCLM Charleston Co., S Private garden, 74 SCBR Brookgreen Gardens, Route 17, Georgetown Co., SC SCLM Private garden, 74 Charleston Co., S Charleston Co., SC Georgetown Co., SC SCLO Charleston Co., S Live Oak Memorial Georgetown Co., SC Charleston Co., SC O Live Oak Memorial Gardens, Charleston, Charleston Co., SC Streets, Abbeville, Abbeville Co., SC C Streets, Abbeville, Abbeville Co., SC Charleston Co., SC Charleston Co., SC Streets, Abbeville, Abbeville Co., SC Private garden, 531 Berrie Road, Aiken, 531 Berrie Road, Aiken, SCLS Garden by entrance gate, 22 Lagare Street, Private garden, 531 Berrie Road, Aiken, SCLS Garden by entrance gate, Charleston, Charleston Co. Talbot Co., MD SCBY Private garden, 531 Berrie Road, Aiken, SCLS Gard Charl Aiken Co., SC SCMA Charleston, Charl Private garden, 20 Charleston, Charleston Co., SC MA Charleston, Charleston Co., SC SECE Private garden, 7 Church Street, Charleston, SCMA Private garden, 206 South Main Street, SECE Private garden, 7 Church Street, Charleston, SCMA Private garden, 20 Charleston Co., SC SCMG Abbeville, Abbevill Magnolia Gardens, SCCF Private garden, Carolina and Forest Avenues, SCMG Magnolia Gardens, north of Charleston, Charleston Co., SC s, SCMG Magnolia Gardens, north of Charleston, Charleston Co., SC SCCF Private garden, Carolina and Forest Avenues, Aiken, Aiken Co., SC SCMG Magnolia Gardens, Charleston Co., S Aiken, Aiken Co., SC George, Winyah, getown Co., SC SCMI Private garden, 204 South Main Street, Abbeville, Abbeville Co., SC SCMI Private garden, 204 South Main Street, Abbeville, Abbeville Co., SC SCCG Church of Prince George, Winyah, Georgetown, Georgetown Co., SC SCMI SCCG Church of Prince George, Winyah, SCMI Private garden, 204 South Main Street, Georgetown, Georgetown Co., SC Abbeville, Abbeville Motel near southern Georgetown, Georgetown Co., SC SCMM Abbeville, Abbeville Co., SC Motel near southern boundar ding, Charleston, SCMM Motel near southern boundary of Manning, Clarendon Co., SC Motel near southern boundary of Manning, Clarendon Co., SC SCCL Charlestowne Landing, Charleston, SCMM Motel near so Clarendon Co. SCMM Motel near southern boundary of Manning, Clarendon Co., SC Charleston Co., SC SCMP Clarendo Middleton Charleston Co., SC Clarendon Co., SC Clarendon Co., SC SCCM Charleston Motor Hotel, Meeting Street, SCMP SCMP Middleton Place Gardens, Dorcester Co., SC Charleston, Charleston Co., SC Charleston, Charleston Co., SC SCNR Nathaniel Russell House, Meeting SCNR Nathaniel Russell House, Meeting Street, Charleston, Charleston Co., SC SCCS Private garden at house across from 7 Church SCNS SCNS Charleston, Charleston Co., SC Private garden, 14 Legare Street, Street, Charleston, Charleston Co., SC SCNS Private garden, Charleston Co., SCNS Private garden, 14 Legare Street, Charleston, Charleston Co., SC Street, Charleston, Charleston Co., SC SCCU Clemson University, Clemson, Pickens Co., SC SCPL Charleston Co. Talbot Co., MD SCPL Palmetto Landscaping and Nursery Co., 247 Springville Road, Darlington, SECY, Cypress Gardens, off US Route 52, SCPL Palmetto Land ECY, Cypress Gardens, off US Route 52, Berkeley Co., SC Berkeley Co., SC Berkeley Co., SC 247 Springville Road, Darlington, Darlington Co., SC SCDB Private garden, Dibble and Bissel Roads, Aiken, Aiken Co., SC SCPP D P SCDB Private garden, Dibble and Bissel Roads, Aiken, Aiken Co., SC SCPP Darlington Pomaria Pla Aiken, Aiken Co., SC SCPP Pomaria Plantation, US Route 176, Pomaria, Pomaria Plantation, US Route 176, Pomaria, Newberry Co., SC Aiken, Aiken Co., SC SCDH Drayton Hall, north of Charleston, Charleston Co., SC SCPR SCPR Newberry Co., SC Median strip, Pendl SCEA Charleston Co., SC Private garden, 120 Edwards Avenue, S Charleston Co., SC SCPR Median strip, Pendleton and Richland Avenues, Aiken, Aiken Co., SC SCEA Private garden, 120 Edwards Avenue, Darlington, Darlington Co., SC SCRH Avenues, Aiken, Aiken Co., SC SCRH Rosehill, 200 block of Greenville Street, Aiken, Aiken Co., SC SCEG Darlington, Darlington Co., SC Edisto Memorial Garden, Orangeburg, SCRH SCEG Edisto Memorial Garden, Orangeburg, Aiken, Aiken Co., SC SCRL Median strip, Richland and Lancaster Avenues, Aiken, Aiken Co., SC Orangeburg Co., SC SCEI Private garden (formerly Baynard’s Bluff), Bluffton, Beaufort Co., SC SCRP Avenues, Redcliffe SCEI Private garden (formerly Baynard’s Bluff), EI Private garden (formerly Baynard’s Bluff), Bluffton, Beaufort Co., SC SCRP Avenues, Aiken, Aiken Co., SC Redcliffe State Park, Beech Island, Route 125, SCRP Avenues, Aiken, Aiken Co., SC Redcliffe State Park, Beech Island, (formerly Baynard’s Bluff), ort Co., SC Avenues, Aiken, Aiken Co., SC Redcliffe State Park, Beech Island, Route 125, Bluffton, Beaufort Co., SC SCRP Redcliffe State Pa Bluffton, Beaufort Co., SC SCRP Redcliffe State Park, Beech Island, Route 125, Aiken Co., SC SCEM Private garden, 27 King Street, Charleston, Aiken Co., SC Charleston Co., SC SCRS Roadside, Route SCRS Roadside, Route 17 Alt. north of Summerville, Dorchester Co., SC SCFR Private garden, 100 Tradd Street, Charleston, Dorchester Charleston Co., SC SCFS Charleston Co., SC SCSB Private garden, along South Battery Street, Charleston, Charleston Co., SC SCSG Charleston, Charleston Co., SC SCSG Shady Grove Nursery, 3030 Charleston Road SW, Orangeburg, Orangeburg Co., SC Charleston Co., SC SCSR SW, Orangeburg, Orangeburg Co., SC Private garden, 1115 Glen Oaks Road, SCSR Private garden, 1115 Glen Oaks Road, Columbia, Richland Co., SC SCTR Columbia, Richland Co., SC Private garden, near Table Ro SCTR Private garden, near Table Rock State Park, Highway 11, Greenville Co., SC Aiken, Aiken Co., SC Highway 11, Greenville Co., SC UC University of South Carolina Campus, South Caroliniana Library, Columbia, Abbeville, Abbeville Co., SC South Caroliniana Library, Columbia, Richland Co., SC SCWE Wendy’s restaurant, US Route 1, Aiken, Aiken Co., SC Dorchester Co., SC SCWF Private garden, 4032 Rosewood Drive, Columbia, Richland Co., SC SCWI Woodlanders, Inc. Aiken, Aiken Co., SC (nursery), 1128 Colleton Avenue, Aiken, Aiken Co., SC SCWL Woodland, southwest corner of Colleton and Laurens Streets, Aiken, Aiken Co., SC SCWL Woodland, southwest corner of Colleton and Laurens Streets, Aiken, Aiken Co., SC 244 SCWP SCWR TNAB TNBC TNBM TNBO TNBV TNCG TNCT TNDG TNDR TNFB TNFE TNFL TNFEN TNFR TNGN TNHD TNHG TNHH TNHS TNHT TNKS TNLW TNMB TNML TNNE TNPC Private garden, Mechanicsville Highway, Darlington Co., SC Williamsburg and Richland Streets, Aiken, Aiken Co., SC Private garden, 709 Forest Hills Boulevard, Knoxville, Knox Co., TN Bay View Motel, US Route 27 (north of tunnel), Chattanooga, Hamilton Co., TN Private garden, 3359 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Boyd Nursery, McMinnville, Warren Co., TN Private garden, Route 261, 2 mi. north of Bakersville, Mitchell Co., TN Chickasaw Golf Course, Central Avenue and Lafayette Street, Memphis, Shelby Co., TN Cheekwood, Tennessee Botanical Gardens, Cheek Road, Nashville, Davidson Co., TN Dixon Gallery and Gardens, 4339 Park Avenue, Memphis, Shelby Co., TN Private garden, 3417 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, Alcoa, Blount Co., TN Private garden, 645 Cheowa Circle (formerly Sanford Arboretum), Knoxville, Knox Co., TN Fort Loudoun Dam (near powerhouse), Loudoun Co., TN Forest Nursery Company, McMinnville, Warren Co., TN Private garden, 3400 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, 3410 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, 1079 Scenic Drive, Knoxville, Knox Co., TN The Hermitage (home of Andrew Jackson), Hermitage, Davidson Co., TN Holly Haven Nursery, Knoxville, Knox Co., TN Private garden, 3350 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, 600 High Street, Winchester, Franklin Co., TN Keith Spring Mountain (1,200 ft. Aiken, Aiken Co., SC alt.), Franklin Co., TN Private garden, 3428 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Memphis Botanic Garden, Audubon Park, 750 Cherry Street, Memphis, Shelby Co., TN Private garden, Montreal Lane, Oak Ridge, Anderson Co., TN Norris Experimental Area, Tennessee Valley Authority, Norris, Anderson Co., TN Corner of Parkway E and Central Avenue, TNRG TNRM TNRT TNRU TNRW TNSB TNSC TNSG TNSM TNSN TNTV TNUT TNWF TXAB TXAO TXAV TXBA TXCC TXCT TXDC TXDM TXEN TXGM TXGS TXHA TXHE TXHG Private garden, 3351 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, 3403 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, 4805 East Summit, Knoxville, Knox Co., TN Gravesite of Ferdinand Rugel (botanist), Old Westminster Cemetery, ca. 2 mi. Aiken, Aiken Co., SC from White Pine, Jefferson Co., TN Private garden, 3358 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Swan Bakery, 1801 Magnolia Avenue NE, Knoxville, Knox Co., TN Private garden, Holt Road, Nashville, Davidson Co., TN Private garden, 3418 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, McMinnville, Warren Co., TN Shadow Nursery, Route 64, Winchester, Franklin Co., TN Tennessee Valley Nursery, Winchester, Franklin Co., TN University of Tennessee, Agriculture Campus, Knoxville, Knox Co., TN Private garden, 3425 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN Private garden, 2 Tealway, River Oaks, Houston, Harris Co., TX Private garden, 4019 Avenue O, Galveston, Galveston Co., TX Private garden, 4318 Avenue O, Galveston, Galveston Co., TX Private garden, 2030 Banks Avenue, Houston, Harris Co., TX Private garden, 3325 Chevy Chase Drive, Houston, Harris Co., TX Private garden, Spring Branch, Holidan Street Houston, Harris Co., TX Private garden, 3408 Ella Lee Lane, Houston, Harris Co., TX Private garden, 3453 Inwood Street, Houston, Harris Co., TX Eagleson Nursery, Port Arthur, Jefferson Co., TX Private garden, 2515 Reba Street, Houston, Harris Co., TX Private garden, Bel Air Boulevard, Sharpstown, Houston, Harris Co., TX Houston Arboretum and Botanical Gardens, Woodway Lane, Houston, Harris Co., TX Hogg Estate, Bayou Bend, 2940 Lazy Lane Boulevard, Houston, Harris Co., TX Private garden, 406 Hedwig Green Street, Houston, Harris Co., TX NDG Dixon Gallery and Gardens, 4339 Park TNWF Private garden, 3425 Lakeview Drive (formerly Sanford Arboretum), Knoxville, Knox Co., TN TXAB Private garden, 2 Tealway, River Oaks Houston, Harris Co., TX TXAO Houston, Harris Co., TX Private garden, 4019 Aven TXAO Private garden, 4019 Avenue O, Galveston, Galveston Co., TX TXAV Private garden, 4318 Avenue O, Galveston, Galveston Co., TX TXBA Galveston Co., TX Private garden, 2030 Banks Avenue, Houston, TXBA Private garden, 2030 Banks Avenue, Houston, Harris Co., TX TXCC Private garden, 3325 Chevy Chase Drive, Houston, Harris Co., TX TXCT Houston, Harris Co., TX Private garden, Spring Bra TXCT Private garden, Spring Branch, Holidan Street, Houston, Harris Co., TX TXDC Houston, Harris Co., TX Private garden, 3408 Ella TXDC Houston, Harris Co., TX Private garden, 3408 Ella Lee Lane, Housto TXDC Private garden, 3408 Ella Lee Lane, Houston, Harris Co., TX TXDM Harris Co., TX Private garden, 3453 Inwood Street, Houston, TXDM Private garden, 3453 Inwood Street, Houston, Harris Co., TX TXEN Eagleson Nursery, Port Arthur, Jefferson Co., TX TXEN Eagleson Nursery, Port Arthur, Jefferson Co., TX TXGM Jefferson Co., TX Private garden, 251 TNLW Franklin Co., TN Private garden, 34 TXGM Private garden, 2515 Reba Street, Houston, Harris Co., TX TXGS Harris Co., TX Private garden, TXGS Private garden, Bel Air Boulevard, Sharpstown, Houston, Harris Co., T TNMB Memphis Botanic Garden, Audubon Park, TXHA Sharpstown, Houston, Harris Co., TX Houston Arboretum and Botanical Gard TXHA Houston Arboretum and Botanical Gardens, Woodway Lane, Houston, Harris Co., TX TXHE Woodway Lane, Houston, Harris Co., TX Hogg Estate, Bayou Bend, 2940 Lazy Lane TXHE Hogg Estate, Bayou Bend, 2940 Lazy Lane Boulevard, Houston, Harris Co., TX TXHG Boulevard, Houston, Harris Co., TX Private garden, 406 Hedwig Green St TXHG Private garden, 406 Hedwig Green Street, Houston, Harris Co., TX Houston, Harris Co., TX Howard Johnson Motel, I TXHJ TXHL Howard Johnson Motel, Interstate 10, west of Houston, Harris Co., TX Old nursery of Lynn Lowrey, 1404 Upland, Houston, Harris Co., TX TNPW Memphis, Shelby Co., TN Phytotektor, Inc., Wincheste 245 VACP VACR VACS VACW VADC VADF VADW VAFB VAFC VAFN Chippokes Plantation State Park, Surry Co., VA Private garden, 1306 Claremont Avenue, Richmond, VA Private garden, Front Street, Accomac, Accomack Co., VA Colonial Williamsburg, Williamsburg, James City Co., VA Cultivated area along Dickey Creek, Dickey Knob, Highway 16, Sugar Grove, Smythe Co., VA Private garden, 116 Woodland Drive, Newport News, VA Private garden, 8709 Cherokee Road, Richmond, VA Along Interstate 95 near Fredericksburg, Spotsylvania Co., VA Falls Church City Hall, Falls Church, Fairfax Co.,VA Private garden, Back Street, Accomac, VACP Chippokes Plantation State Park, Surry Co., VA TXHN Hester Nursery, 3029 Todville Road, PO Box 413, Seabrook, Harris Co., TX TXHN Hester Nursery, 3029 Todville Road, PO Box 413, Seabrook, Harris Co., TX VACR Surry Co., VA Private garden, TXJS Private garden, 1702 Broadway Street, Galveston, Galveston Co., TX Galveston, Galveston Co., TX TXLL Nursery of Lynn Lowrey, 2323 Sleepy Hollow Road, Conroe, Montgomery Co., TX VACS Private garden, Front Street, Accomac, Accomack Co., VA Road, Conroe, Montgomery Co., TX VACW Colonial Williamsburg, Williamsburg, James City Co., VA TXMA Private garden, 3376 Inwood Street, Houston, Harris Co., TX James City Co., VA TXMK Private garden, 2 Longbow Lane, Houston, Harris Co., TX VADF Private garden, 116 Woodland Drive, Newport News, VA VADW Private garden, 8709 Cherokee Road, Richmond, VA Boulevard, Houston, Harris Co., TX TXPO Private garden, 10656 Memorial Drive, Houston, Harris Co., TX VAFB Richmond, VA Along Interstate Private garden, 10656 Memorial Drive, Houston, Harris Co., TX VAFB Along Interstate 95 near Fredericksburg, Spotsylvania Co., VA Houston, Harris Co., TX Houston, Harris Co., TX Private garden, 6810 Hendon, Sharpstown, Spotsylvania Co., VA TXPS Private garden, 6810 Hendon, Sharpstown, Houston, Harris Co., TX VAFC TX 8 Meadowick, Houston, VAFC Falls Church City Hall, Falls Church, Fairfax Co.,VA VAFC Falls Church City Hall, Falls Church, Fairfax Co.,VA TXRE Houston, Harris Co., TX Private garden, 11108 Meadowick, Houston, VAFC Falls Church City Ha Fairfax Co.,VA Harris Co., TX VAFN Private garden, Back Street, Accomac, TXRS TXSB Private garden, Woodland Garden, Houston, Harris Co., TX Private garden, Spring Branch area, Houston, VAFR Accomack Co., VA Old house on Flat Ri Smythe Co., VA TXSB Harris Co., TX Private garden, Spring Branch area, Houston, VAFR Old house on Flat Ridge Road, Sugar Grove, Smythe Co., VA Private garden, Spring Branch area, Houston, Smythe Co., VA Harris Co., TX Private garden, 7107 Sharp Crest, VAGC Roadbank along Interstate Groseclose, Wythe Co., VA TXSC Private garden, 7107 Sharp Crest, VAGF Groseclose, Wythe Greenbrier Farms Sharpstown, Houston, Harris Co., TX Private garden, 700 Cavalcade Street W, VAGF Greenbrier Farms Nursery, Chesapeake, VA TXSE Sharpstown, Houston, Harris Co., TX Private garden, 700 Cavalcade Street W, VAGF Greenbrier Farms Nursery, 201 Hickory Road, Chesapeake, VA TXSE Private garden, 700 Cavalcade Street W, Chesapeake, VA TXSH Houston, Harris Co., TX Private garden, Avenue N 1/2, Galveston, VAGG The Lewis Ginter Avenue, Henrico TXSH Private garden, Avenue N 1/2, Galveston, Avenue, Henrico TXSH Private garden, Avenue N 1/2, Galveston, Avenue, Henrico Co., VA TXSN Galveston Co., TX VAGH Glass-blower hous James City Co., V TXSN Seaside Nursery, 2315 45th Street, Galveston, James City Co., V TXVO Galveston Co., TX Private garden, 3 mi. Aiken, Aiken Co., SC north of Vidor VAGJ House at junction of Routes 16 and 741, Grayson Co., VA TXVO TXWE TXWH TXWW TXZH VAAC VAAH VAAL VABA VABB VABF Private garden, 3 mi. north of Vidor (1 mi. north of Interstate 10), Orange Co., TX West End Market, 2203 45th Street, Galveston, Galveston Co., TX Whataburgers’ Hamburger Place, 3639 Westheimer Road, Houston, Harris Co., TX Private garden, 4 Woods Edge Lane, Houston, Harris Co., TX Zoo grounds, Hermann Park, Houston, Harris Co., TX Arlington National Cemetery, Arlington, Arlington Co., VA American Horticultural Society Headquarters, River Farm, Mount Vernon, Fairfax Co., VA Private garden, Aldie, Loudon Co., VA Private garden, Front Street, Accomac, Accomack Co., VA Bamboo grove along Richmond Road, about 2 mi. west of Williamsburg, James City Co., VA Blandy Experimental Farm (O. E. White Arboretum), Boyce, Clarke Co., VA Private garden, Henrico Co., VA Bryan Park, Richmond, VA Private garden, Roseland, Route 605, Accomac, Accomack Co., VA Carter’s Grove Plantation, James City Co., VA Claremont Manor, Claremont, Surry Co., VA VAGM VAGO VAGS VAGU VAGW VAHB VAHC VAHF VAHI VAHM VAHN VAHR VAHS VAIG VAIS VAJL VAKH Grayson Co., VA Private garden, 8008 Park Street N, Dunn Loring, Fairfax Co., VA 4602 17th Street N, Arlington, Arlington Co., VA Gulf Stream Nursery (Jacques Legendre), Wachapreague, Accomack Co., VA Gunston Hall, Fairfax Co., VA George Washington Estate, Mount Vernon, Fairfax Co., VA Harry F. Byrd, Sr., Visitors’ Center, Shenandoah National Park, Madison Co., VA Hollywood Cemetery, Richmond, VA Private garden, Route 601, Flat Ridge Road, Sugar Grove, Smythe Co., VA Holiday Inn Motel, Marion, Smythe Co., VA Hermitage Methodist Home, Hermitage Avenue, Richmond, VA Hill’s Nursery and Camellia Gardens, Arlington, Arlington Co., VA Humpback Rocks Visitors’ Center, Blue Ridge Parkway, Augusta Co., VA Private garden, across from store in downtown area, Sugar Grove, Smythe Co., VA Glenwood Garden Center, Richmond, VA Private garden, Idlewood Street, Richmond, VA Private garden, 2822 Avenham Avenue SW, Roanoke, Roanoke Co., VA Garden at Kenmore House, Fredericksburg, Spotsylvania Co., VA (1 mi. north of Interstate 10), Orange Co., TX VAGM Private garden, 8008 Park Street N, (1 mi. north of Interstate 10), Orange Co., TX VAGM VAGO Dunn Loring, Fairfax Co., VA 4602 17th Street N, Arlington, A AGW George Washington Estate, Mount Vernon, Fairfax Co., VA VAHB Harry F. Aiken, Aiken Co., SC Byrd, Sr., Visitors’ Center, Shenandoah National Park, Madison VAHC Shenandoah National Park, Madison Co., VA Hollywood Cemetery, Richmond, VA Shenandoah National Park, Madison C VAHC VAHF Hollywood Cemetery, Richmond, VA Private garden, Route 601, Flat Ridge VAHF Private garden, Route 601, Flat Ridge Road, Sugar Grove, Smythe Co., VA Sugar Grove, Smythe Co., VA VAHI VAHM Holiday Inn Motel, Marion, Smythe Co., VA Hermitage Methodist Home, Private garden, Aldie, Loudon Co., VA Private garden, Front Street, Accomac, VAHI Holiday Inn Motel, Marion, Smy VAHM Hermitage Methodist Home, Hermitage Avenue, Richmond, Hermitage Avenue, Richmond, VA VAHN Hill’s Nursery and Camellia Gardens, Arlington, Arlington Co., VA Arlington, Arlington Co., VA VAHR Humpback Rocks Visitors’ Center, Blue Ridge Parkway, Augusta Co., V downtown area, Sugar Grove, Smythe Co., VA Glenwood Garden Center, Richmond, VA VAWE Wytheville exit, Interstate 81, Wythe Co., VA VAWH._ Westbrook Psychiatric Hagspital, 1500 Westbrook Avenue, Richmond, VA VAWI Private garden, 610 North Cameron Street, Winchester, VA VAWL White Lion Motel, Williamsburg, James City Co., VA VAWM. College of William and Mary, Williamsburg, James City Co., VA VAWP Woodlawn Plantation gardens, Fairfax Co., VA VAWR Private garden, Highway 6, 3 mi. Aiken, Aiken Co., SC west of Manakin, Goochland Co., VA VAZC_ Private garden, 5236 Shenstone Circle, Virginia Beach, VA VAWE Wytheville exit, Interstate 81, Wythe Co., VA VALC Jefferson Lakeside Country Club, VAWH._ Westbrook Psychiatric Hagspital, Lakeside Avenue, Henrico Co., VA VAMC Medical College of Virginia, College and Marshall Streets, Richmond, VA VAWL White Lion Motel, Williamsburg, Marshall Streets, Richmond, VA VAMD McDonald’s Garden Center, Hampton, VA VAMF Private garden, near East and Market Streets, Onancock, Accomack Co., VA Onancock, Accomack Co., VA VAML Private garden, Mountain Lake, Giles Co., VA VAWP Woodlawn Pl VAML Private garden, Mountain Lake, Giles Co., VA VAMP Maymont Park, Richmond, VA VAMR Private garden, 1027 Magnolia Roa VAMR Private garden, 1027 Magnolia Road (along street), Norfolk, VA VAMR Private garden, 1027 Magnolia Road (along street), Norfolk, VA (along street), Norfolk, VA VAMW Mews across from Saint John’s Church, off 24th Street, Richmond, VA off 24th Street, Richmond, VA VANB Norfolk Botanical Gardens, Airport Way, VANB Norfolk Botanical Gardens, Airport Way, Norfolk, VA Norfolk, VA VANP Heronwood, Route 623 near Upperville, Loudon Co., VA VANS Roadside near settlement along Route 16 near Marion, Smythe Co., VA VAOD Old Dominion University, Norfolk, VA Private garden, corner of Pope Avenue VAPC Private garden, corner of Pope Avenue and Crestwood Road, Richmond, VA Crestwood Road, Richmond, VA VAPH Private garden, 219 Robanna Shores, Seaford, York Co., VA VAPO Peaks of Otter Lodge, Blue Ridge Parkway, Bedford Co., VA Bedford Co., VA Bedford Co., VA Private garden, Pitt Street, Fredericksburg, VAPS Private garden, Pitt Street, Fredericksburg, Spotsylvania Co., VA VAPS Private garden, Pitt Street, Fredericksburg, Spotsylvania Co., VA VAPS Private garden, Pitt Street, Fredericksburg, Spotsylvania Co., VA Anly Farm, Onancock, Accomack Co., VA Spotsylvania Co., VA VARB VARC Anly Farm, Onancock, Accomack Co., VA Near milepost 155, Interstate 81, VARC Near milepost 155, Interstate 81, Roanoke Co., VA Roanoke Co., VA VARD VARE Private garden, Richmond, VA Private garden, 6611 Brauwner St, McLean, VARD VARE Private garden, Richmond, VA Private garden, 6611 Brauwner VARE Private garden, 6611 Brauwner St, McLean, Fairfax Co, VA Fairfax Co, VA VARG Russell’s Grocery, near stream, along Route 16, Sugar Grove, Sm VARH along Route 16, Sugar Grove, Smythe Co., VA Private garden, Nock’s Pasture, Back Street, VARH Private garden, Nock’s Pasture, Back Street, Accomac, Accomack Co., VA Accomac, Accomack Co., VA Hotel Roanoke grounds, Roano VARO Hotel Roanoke grounds, Roanoke, Roanoke Co., VA Private garden, Quinby, Accomack Co., VA VARR VASC Private garden, Quinby, Accomack Co., VA State Capitol Building grounds, Richmond, VA VASC VASG State Capitol Building grounds, Richmond, VA Abandoned house, Route 601, Sugar Grove, VASG Abandoned house, Route 601, Sugar Grove, Smythe Co., VA Saint John’s Churchyard, Richmond, VA Skyland, Shenandoah National Park, VASK Skyland, Shenandoah National Park, Page Co., VA VASK VASP Skyland, Shenandoah National Park, Page Co., VA Shenandoah National Park, VA VASP Page Co., VA Shenandoah National Park, VA VASP Page Co., VA Shenandoah N VASP Shenandoah National Park, VA Stony Point School, Richmond, V VASP VASS Shenandoah National Park, VA Stony Point School, Richmond, VA VASS VATA Stony Point School, Richmond, VA Virginia Truck and Ornamental Rese VATA Virginia Truck and Ornamental Research Station (Tidewater Arboretum), 1444 Diamond Springs Road, Virginia Beach, VA Springs Road, Virginia Beach, VA VATG VATS Thornton Gap, Shenandoah National Park, VA Todd Shopping Center, 220 Mercury VATS Todd Shopping Center, 220 Mercury Boulevard, Hampton, VA VATW Boulevard, Hampton, VA Private garden, 6311 Three VATW Boulevard, Hampton, VA Private garden, 6311 Three VATW Private garden, 6311 Three Chopt Road, Richmond, VA VAWD Richmond, VA VAWD Richmond, VA VAWD Private garden, Wassona Drive, Marion, Smythe Co., VA mythe Co., VA 247 Appendix C: Glenn Dale Azalea Cultivars Documented in the U.S. National Arboretum Herbarium Abbott Acme Acrobat Adorable Advance Alight Allegory Allure Altair Ambrosia Anchorite Andros Angela Place Anthem Antique Aphrodite Arcadia Argosy Astarte Ave Maria Aviator Bacchante Bagatelle Bagdad Beacon Bettina Bishop Blushing Maid Bohemian Boldface Bolivar Bopeep Bountiful Bowman Brangaene Bravo Buccaneer Burgundy Cadenza Camelot Cantabile Capella Captivation Carbineer Carnival Carrara Cascade Cavalier Cavatina Challenger Chameleon Chanticleer Cocktail Colleen Commando Commodore Con Amore Concordia Consuela Content Copperman Coquette Cordial Corsair Crinoline Cygnet Cytherea Damaris Damask Damozel Daphnis Darkness Dawning Dayspring Dazzler Delight Delos Demure Desire Dimity Dowager Dragon Dream Driven Snow Duenna Echo Egoist Ember Enchantment Eros Eucharis Evangeline Evensong Everest F. C. Bradford Fairy Bells Fakir Fandango Fantasy Fashion Fawn Felicity Folly Frivolity Ganymede Gawain Geisha Glacier Gladiator Glamour Glee Gracious Granat Grandam Grandee Greeting Grenadier Guerdon Harbinger Harlequin Helen Fox Helen Gunning Hopeful Illusion Isolde Jamboree Janet Noyes Jessica Jubilant Juneglow Kashmir Kathleen Killarney Kobold Lacquer Leonore Lillie Maude Limerick Litany Louise Dowdle Lucette Luminary Lustre Madcap Madeira Madrigal Mandarin Manhattan Marionette Marjorie Marmora Martha Hitchcock Mary Margaret Mascot Masquerade Masterpiece Matins Mavis Mavourneen Mayflower Medea Melanie Memento Moira Morgana Mother of Pearl Motley Muscadine Nerissa Niagara Niphetos Nocturne Noreen Novelty Oriflamme Orison Paladin Parade Pastel Patriot Peerless Peter Pan Phoebe Pied Piper Pilgrim Pinkie Pinocchio Pippin Pirate Pixie Polonaise Portent Prelate Presto Progress Prosperity Prudence Puck Punchinello Quakeress Ranger Red Bird Red Hussar Refrain Remembrance Revery Reward Robinhood Rogue Rosalie Roselight Rosette Roundelay Safrano Saga Sambo Samite Samson Sarabande Satrap Satyr Scherzo Seneca Serenade Serenity Signal Silver Cup Silver Lace Silver Mist Silver Moon Simplicity Sligo Snowclad Snowscape Sonata Souvenir Spangles Sprite Stampede Stardust Sterling Surprise Suwanee Swansong Swashbuckler Taffeta Tanager Tango Templar Temptation Thisbe Touchstone Treasure Trilby Trinket Tristan Trophy Valentine Vanity Vespers Vestal Viking Violetta Vision Volcan Warrior Wavelet Welcome Whimsical Whirlwind Wildfire Winedrop Winner Wisdom Witchery Zephyr Zulu Appendix D: Plants Listed by Botanical Names and Cultivar Names Appendix D: Plants Listed by Botanical Names and Cultivar Names Appendix D: Plants Listed by Botanical Names and Cultivar Names nordmanniana (Steven) Spach numidica de Lannoy exLarr. pinsapo Boiss. pinsapo Boiss. ‘Glauca’ procera Rehd. xumbellata (Mayr) Liu x vilmorinii Mast. nordmanniana (Steven) Spach numidica de Lannoy exLarr. pinsapo Boiss. pinsapo Boiss. ‘Glauca’ procera Rehd. xumbellata (Mayr) Liu x vilmorinii Mast. ABUTILON Mill. MALVACEAE Flowering Maple Mallow Family pictum (Gillies ex Hook. & Arn.) Walp. pictum (Gillies ex Hook. & Arn.) Walp. ‘Thompsonii’ ACACIA Mill. FABACEAE (Mimosoideae) Wattle, Mimosa Bean Family berlandieri Benth. farnesiana (L.) Willd. rigidula Benth. roemeriana Scheele wrightii Benth. ACALYPHA L. EUPHORBIACEAE Spurge Family hispida Burm.f. ACCA O.Berg MYRTACEAE Myrtle Family sellowiana (O.Berg) Burret ACER L. ACERACEAE Maple Maple Family buergerianum Miq. buergerianum Miq. ‘Mino yatsufusa’ buergerianum Miq. ‘Nauto kaede’ buergerianum Miq. ssp. formosanum (Hayata) E.Murr. & Lauener ‘Miyasama’ campbellii Hiern ssp. flabellatum (Rehd.) E.Murr. campestre L. campestre L. ‘Nanum’ campestre L. var. leiocarpum (Opiz) Wallroth capillipes Maxim. cappadocicum Gleditsch cappadocicum Gleditsch ‘Aureum’ carpinifolium Sieb. & Zucc. cissifolium (Sieb. & Zucc.) K.Koch davidii Franch. diabolicum Bl. ex K.Koch xfreemanii E.Murr. This appendix provides a cross-reference to the legitimate botanical names and, where appli- cable, the cultivar names for the plants included in the catalog. Genera are arranged alphabeti- cally. The catalog presents the specific epithets and cultivar names in alphabetical order follow- ing each genus. This list is organized as follows: Genus name and family name Specific epithet or hybrid specific epithet Cultivar name for selected species material Forma (minor variant of species) Infraspecific epithet for subspecies or varietas Cultivar name at infraspecific rank Hybrid without cultivar name Hybrid with cultivar name Cultivar name not otherwise identified. This appendix provides a cross-reference to the legitimate botanical names and, where appli- cable, the cultivar names for the plants included in the catalog. Genera are arranged alphabeti- cally. The catalog presents the specific epithets and cultivar names in alphabetical order follow- ing each genus. This list is organized as follows: Genus name and family name legitimate botanical names and, where appli- cable, the cultivar names for the plants included in the catalog. Genera are arranged alphabeti- cally. The catalog presents the specific epithets and cultivar names in alphabetical order follow- ing each genus. Appendix D: Plants Listed by Botanical Names and Cultivar Names This list is organized as follows: Genus name and family name Specific epithet or hybrid specific epithet Cultivar name for selected species material Forma (minor variant of species) Infraspecific epithet for subspecies or varietas Cultivar name at infraspecific rank Hybrid without cultivar name Hybrid with cultivar name Cultivar name not otherwise identified. Genus name and family name Specific epithet or hybrid specific epithet Cultivar name for selected species material Forma (minor variant of species) Infraspecific epithet for subspecies or varietas Cultivar name at infraspecific rank Hybrid without cultivar name Hybrid with cultivar name Cultivar name not otherwise identified. ABELIA R.Br. CAPRIFOLIACEAE Honeysuckle Family chinensis R.Br. x grandiflora (Andre) Rehd. <x grandiflora (Andre) Rehd. ‘Prostrata’ < grandiflora (Andre) Rehd. ‘Sherwoodii’ (x grandiflora x A. schumannii) ‘Edward Goucher’ ABELIOPHYLLUM Nakai OLEACEAE Olive Family distichum Nakai ABIES Mill. Fir PINACEAE alba Mill. balsamea (L.) Mill. cephalonica Loud. chensiensis Tieghem ssp. salouenensis (Bord.-Rey. & Gaussen) Rushforth cilicica (Ant. & Kotschy) Carr. concolor (Gord. & Glend.) Lindl. ex Hildebr. firma Sieb. & Zucc. fraseri (Pursh) Poir. holophylla Maxim. homolepis Sieb. & Zucc. koreana Wils. Pine Family ABELIA R.Br. CAPRIFOLIACEAE balsamea (L.) Mill. cephalonica Loud. cephalonica Loud. chensiensis Tieghem firma Sieb. & Zucc. fraseri (Pursh) Poir. fraseri (Pursh) Poir. holophylla Maxim. holophylla Maxim. homolepis Sieb. & Z homolepis Sieb. & Zucc. koreana Wils. koreana Wils. xfreemanii E.Murr. 249 palmatum Thunb. ‘Chitoseyama’ palmatum Thunb. ‘Corallinum’ palmatum Thunb. ‘Crimson Queen’ palmatum Thunb. ‘Dissectum’ palmatum Thunb. palmatum Thunb. palmatum Thunb. palmatum Thunb. palmatum Thunb. palmatum Thunb. palmatum Thunb. ‘Hagoromo’ palmatum Thunb. palmatum Thunb. ‘Higasayama’ palmatum Thunb. ‘Hogyoku’ palmatum Thunb. ‘Ichigyo ji’ palmatum Thunb. ‘lijima sunago’ palmatum Thunb. ‘Inaba shidare’ palmatum Thunb. ‘Jiro shidare’ palmatum Thunb. ‘Kagiri nishiki’ palmatum Thunb. ‘Killarney’ palmatum Thunb. ‘Kingsville Red’ palmatum Thunb. ‘Koshimino’ palmatum Thunb. ‘Kurabeyama’ palmatum Thunb. ‘Linearilobum’ palmatum Thunb. ‘Lutescens’ ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ‘Dissectum Flavescens’ ex J.A.Murr. ‘Dissectum Palmatifidum’ ex J.A.Murr. ‘Dissectum Rubrifolium’ ex J.A.Murr. ‘Dissectum Variegatum’ ex J.A.Murr ex J.A.Murr . ‘Filigree’ . ‘Garnet’ ex J.A.Murr. ex J.A.Murr . ‘Hessei’ ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ginnala Maxim. griseum (Franch.) ginnala Maxim. griseum (Franch.) Pax japonicum Thunb japonicum Thunb japonicum Thunb ex J.A.Murr. japonicum Thunb ‘Aconitifolium’ ex J.A.Murr. japonicum Thunb. ‘Aureum’ ex J.A.Murr. japonicum Thunb. ‘Aureum’ japonicum Thunb. ex J.A.Murr. japonicum Thunb. ‘Itaya’ japonicum Thunb. ex J.A.Murr. palmatum Thunb. ‘Dissectum Flavescens’ ex J.A.Murr. ‘Dissectum Palmatifidum’ japonicum Thunb. ‘Kinugasayama’ ex J.A.Murr. palmatum Thunb. ‘Dissectum Palmatifidum’ ex J.A.Mur palmatum Thunb. ‘Dissectum Palmatifidum’ ex J.A.Murr. ‘Dissectum Rubrifolium’ palmatum Thunb. ex J.A.Murr. ‘Dissectum Rubrifolium’ palmatum Thunb. ‘Dissectum Rubrifolium’ palmatum Thunb. ‘Dissectum Rubrifolium’ ex J.A.Murr. ‘Dissectum Variegatum’ palmatum Thunb. ex J.A.Murr. ‘Dissectum Variegatum’ palmatum Thunb. palmatum Thunb. palmatum Thunb. palmatum Thunb. ‘Hagoromo’ palmatum Thunb. palmatum Thunb. ‘Higasayama’ palmatum Thunb. ‘Hogyoku’ palmatum Thunb. ‘Ichigyo ji’ palmatum Thunb. ‘lijima sunago’ palmatum Thunb. ‘Inaba shidare’ palmatum Thunb. ‘Jiro shidare’ palmatum Thunb. ‘Kagiri nishiki’ palmatum Thunb. ‘Killarney’ palmatum Thunb. ‘Kingsville Red’ palmatum Thunb. ‘Koshimino’ palmatum Thunb. ‘Kurabeyama’ palmatum Thunb. ‘Linearilobum’ palmatum Thunb. ‘Lutescens’ palmatum Thunb. palmatum Thunb. ‘Masukagami’ palmatum Thunb. ‘Matsukaze’ palmatum Thunb. palmatum Thunb. ‘Mizu kuguri’ palmatum Thunb. ex J.A.Murr. ‘Dissectum Variegatum’ ex J.A.Murr ex J.A.Murr . ‘Filigree’ . ‘Garnet’ ex J.A.Murr. ex J.A.Murr . ‘Hessei’ ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr . ‘Maiko’ ex J.A.Murr. ex J.A.Murr. ex J.A.Murr . ‘Mioun’ ex J.A.Murr. ABELIA R.Br. CAPRIFOLIACEAE ex J.A.Murr. palmatum Thunb. ‘Dissectum Variegatum’ japonicum Thunb. ex J.A.Murr. ‘Yayoigasa’ maximowiczianum Miq. miyabei Maxim. negundo L. ‘Variegatum’ negundo L. ssp. mexicanum (DC.) palmatum Thunb. ‘lijima sunago’ ex J.A.Murr. negundo L. ssp. mexicanum (DC.) Wesmael palmatum Thunb. ‘Inaba shidare’ ex J.A.Murr. negundo L. ssp. negundo var. texanum Pax oblongum Wall. ex DC. oliverianum Pax palmatum Thunb. ex J.A.Murr. ‘Akaji nishiki’ palmatum Thunb. ex J.A.Murr. ‘Aoyagi’ palmatum Thunb. ex J.A.Murr. ‘Asahi zuru’ palmatum Thunb. ex J.A.Murr. ‘Atropurpureum’ palmatum Thunb. ex J.A.Murr. ‘Atropurpureum Superbum’ palmatum Thunb. ex J.A.Murr. ‘Aureo-variegatum’ palmatum Thunb. ex J.A.Murr. ‘Aureum’ palmatum Thunb. ex J.A.Murr. ‘Autumn Glory’ palmatum Thunb. ex J.A.Murr. ‘Bloodgood’ palmatum Thunb. ex J.A.Murr. ‘Burgundy Lace’ palmatum Thunb. ex J.A.Murr. ‘Butterfly’ palmatum Thunb. ex J.A.Murr. ‘Chirimen nishiki’ negundo L. ssp. negundo var. texanum Pax oblongum Wall. ex DC. oliverianum Pax palmatum Thunb. ex J.A.Murr. ‘Akaji nishiki’ palmatum Thunb. ex J.A.Murr. ‘Aoyagi’ palmatum Thunb. ex J.A.Murr. ‘Asahi zuru’ palmatum Thunb. ex J.A.Murr. ‘Atropurpureum’ palmatum Thunb. ex J.A.Murr. ‘Atropurpureum Superbum’ palmatum Thunb. ex J.A.Murr. ‘Aureo-variegatum’ palmatum Thunb. ex J.A.Murr. ‘Aureum’ palmatum Thunb. ex J.A.Murr. ‘Autumn Glory’ palmatum Thunb. ex J.A.Murr. ‘Bloodgood’ palmatum Thunb. ex J.A.Murr. ‘Burgundy Lace’ palmatum Thunb. ex J.A.Murr. ‘Butterfly’ palmatum Thunb. ex J.A.Murr. ‘Chirimen nishiki’ palmatum Thunb. ‘Jiro shidare’ ex J.A.Murr. palmatum Thunb. ‘Kagiri nishiki’ ex J.A.Murr. palmatum Thunb. ex J.A.Murr. ‘Akaji nishiki’ palmatum Thunb. ‘Kingsville Red’ ex J.A.Murr. ‘Kingsville Red’ palmatum Thunb. palmatum Thunb. ‘Koshimino’ ex J.A.Murr. ‘Atropurpureum’ palmatum Thunb. palmatum Thunb. ex J.A.Murr. ‘Atropurpureum Superbum’ palmatum Thunb. ‘Kurabeyama’ ex J.A.Murr. ‘Atropurpureum Superbum’ palmatum Thunb. ex J.A.Murr. palmatum Thunb. ‘Linearilobum’ ex J.A.Murr. palmatum Thunb. ‘Monzukushi’ ex J.A.Murr. 250 palmatum Thunb. ‘Moonfire’ palmatum Thunb. ‘Mure hibari’ palmatum Thunb. ‘Naruo nishiki’ palmatum Thunb. palmatum Thunb. nishiki’ palmatum Thunb. nagare’ palmatum Thunb. sarasa’ palmatum Thunb. ‘O kagami’ palmatum Thunb. ‘Okushimo’ palmatum Thunb. palmatum Thunb. ‘Oo shi rini’ palmatum Thunb. Sunset’ palmatum Thunb. nishiki’ palmatum Thunb. ‘Ornatum’ palmatum Thunb. ‘Osakazuki’ palmatum Thunb. ‘Oshio beni’ palmatum Thunb. ‘Oshu shidare’ palmatum Thunb. palmatum Thunb. Leaf’ palmatum Thunb. nishiki’ palmatum Thunb. kakuw’ palmatum Thunb. ‘Sazanami’ palmatum Thunb. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ‘Nomura’ ‘Nomura ‘Ogino ‘Ogon ‘Omato’ ‘Oregon ‘Orido ‘Pixie’ ‘Ribbon ‘Sagara ‘Sango ‘Seigen’ palmatum Thunb. ‘Tamukeyama’ palmatum Thunb. palmatum Thunb. Bishop’ palmatum Thunb. nishiki’ palmatum Thunb. ‘Trompenburg’ palmatum Thunb. ‘Tsuchi uno’ palmatum Thunb. ‘Tsuku bane’ palmatum Thunb. ‘Tsukushi gata’ palmatum Thunb. ‘Tsuri nishiki’ palmatum Thunb. ‘Utsu semi’ palmatum Thunb. ‘Versicolor’ palmatum Thunb. ‘Waka momiji’ palmatum Thunb. ‘Waterfall’ palmatum Thunb. ‘Yatsubusa’ palmatum Thunb. ‘Yezo nishiki’ palmatum Thunb. ‘Yukigumi’ palmatum Thunb palmatum Thunb. var. palmatum pensylvanicum L. platanoides L. ex J.A.Murr. ex J-A.Murr ex J.A.Murr ex J.A.Murr . ‘Tatsuta’ . ‘The . ‘Toyama ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. ex J.A.Murr. var. amoenum (Carr.) Ohwi ex J.A.Murr. var. matsumurae (Koidz.) Makino palmatum Thunb. ex J.A.Murr. platanoides L. ‘Almira’ platanoides L. ‘Chas. F. Irish’ platanoides L. ‘Crimson King’ platanoides L. ‘Drummondii’ platanoides L. ‘Faassen’s Black’ platanoides L. ‘Oekonomierat Stoll’ platanoides L. ‘Schwedleri’ pseudoplatanus L. pseudoplatanus L. ‘Atropurpureum’ pseudoplatanus L. ‘Brilliantissimum’ pseudosieboldianum (Pax) Komar. rubrum L. rubrum L. ‘Gerling’ rubrum L. ‘October Glory’ rubrum L. ‘Pyramidale’ palmatum Thunb. ‘Naruo nishiki’ ex J.A.Murr. ‘O kagami’ palmatum Thunb. ‘Okushimo’ ex J.A.Murr. ‘Okushimo’ palmatum Thunb. palmatum Thunb. ‘Oo shi rini’ ex J.A.Murr. ex J.A.Murr. ‘Omato’ ‘Oo shi rini’ palmatum Thunb. Sunset’ ex J.A.Murr. ‘Oregon ‘Orido palmatum Thunb. nishiki’ ex J.A.Murr. ‘Orido palmatum Thunb. ‘Ornatum’ ex J.A.Murr. palmatum Thunb. ‘Yatsubusa’ ex J.A.Murr. palmatum Thunb. ‘Osakazuki’ ex J.A.Murr. palmatum Thunb. ‘Oshu shidare’ ex J.A.Murr. palmatum Thunb. Leaf’ palmatum Thunb. ex J.A.Murr. ex J.A.Murr. ‘Shigitatsu sawa’ palmatum Thunb. ‘Shigitatsu sawa’ palmatum Thunb. palmatum Thunb. ‘Shigurezome’ ex J.A.Murr. ‘Atropurpureum’ palmatum Thunb. ‘Ribbon ‘Sagara palmatum Thunb. nishiki’ palmatum Thunb. ex J.A.Murr. ‘Sagara ‘Sango palmatum Thunb. kakuw’ palmatum Thunb. ex J.A.Murr. ex J.A.Murr. ‘Sango palmatum Thunb. ‘Sazanami’ ex J.A.Murr. ‘Sazanami’ palmatum Thunb. ex J.A.Murr. ‘Seigen’ palmatum Thunb. palmatum Thunb. ex J.A.Murr. ex J.A.Murr. ‘Seigen’ ‘Seiryu’ palmatum Thunb. palmatum Thunb. ex J.A.Murr. ‘Seiryu’ palmatum Thunb. ex J.A.Murr. ‘Sherwood Flame’ ‘Sherwood Flame’ palmatum Thunb. palmatum Thunb. ‘Shigitatsu sawa’ ex J.A.Murr. ‘Shigurezome’ palmatum Thunb ‘“Variegatum’ saccharinum L. saccharinum L. ‘Palmatum’ rufinerve Sieb. & Zucc. ‘“Variegatum’ saccharinum L. saccharinum L. ‘Palmatum’ saccharum Marsh. ‘Newton Sentry’ saccharum Marsh. ‘Sweet Shadow Cut- saccharinum L. saccharinum L. ‘Palmatum’ saccharinum L. ‘Palmatum’ saccharum Marsh. ‘Newton Sentry’ saccharum Marsh. ‘Newton Sentry’ saccharum Marsh. ‘Sweet Shadow Cu saccharum Marsh. ‘Sweet Shadow Cut- Leaf’ saccharum Marsh. ‘Temple’s Upright’ ‘Shigurezome’ palmatum Thunb 251 ADINA Salisb. RUBIACEAE Madder Family rubella Hance AESCULUS L._ Horsechestnut, Buckeye HIPPOCASTANACEAE Buckeye Family x<bushii Schneid. californica (Spach) Nutt. <x carnea Hayne x carnea Hayne ‘Briotii’ x dupontii Sarg. flava Sol. glabra Willd. x glaucescens Sarg. hippocastanum L. hippocastanum L. ‘Baumanii’ hippocastanum L. ‘Memmingeri’ < hybrida DC. < marilandica Booth ex Kirchn. < mutabilis (Spach) Scheele parviflora Walt. parviflora Walt. var. serotina Rehd. pavia L. pavia L. var. flavescens (Sarg.) Correll x plantierensis Andre sylvatica Bartram x woerlitzensis Koehne AGARISTA D.Don ex G.Don ERICACEAE Heath Family populifolia (Lam.) D.Don ex Judd AGAVE L. AGAVACEAE Century Plant Agave Family americana L. americana L. ‘Marginata’ AILANTHUS Dest. SIMAROUBACEAE Quassia Family altissima (Mill.) Swingle AKEBIA Decne. LARDIZABALACEAE Akebia Lardizabala Family quinata (Thunb. ex Houtt.) Decne. quinata (Thunb. ex Houtt.) Decne. ‘Shirobana’ ADINA Salisb. RUBIACEAE Madder Family rubella Hance AESCULUS L._ Horsechestnut, Buckeye HIPPOCASTANACEAE Buckeye Family x<bushii Schneid. californica (Spach) Nutt. <x carnea Hayne x carnea Hayne ‘Briotii’ x dupontii Sarg. flava Sol. glabra Willd. x glaucescens Sarg. hippocastanum L. hippocastanum L. ‘Baumanii’ hippocastanum L. ‘Memmingeri’ < hybrida DC. < marilandica Booth ex Kirchn. < mutabilis (Spach) Scheele parviflora Walt. parviflora Walt. var. serotina Rehd. pavia L. pavia L. var. flavescens (Sarg.) Correll x plantierensis Andre sylvatica Bartram x woerlitzensis Koehne AGARISTA D.Don ex G.Don ERICACEAE Heath Family populifolia (Lam.) D.Don ex Judd AGAVE L. AGAVACEAE Century Plant Agave Family americana L. americana L. ‘Marginata’ AILANTHUS Dest. SIMAROUBACEAE Quassia Family altissima (Mill.) Swingle AKEBIA Decne. LARDIZABALACEAE Akebia Lardizabala Family quinata (Thunb. ex Houtt.) Decne. quinata (Thunb. ex Houtt.) Decne. ‘Shirobana’ rubrum L. var. trilobum T. & G. ex K.Koch rufinerve Sieb. & Zucc. Madder Family rufinerve Sieb. & Zucc. rufinerve Sieb. & Zucc. & Zucc. & Zucc. ‘Hatsuyuki’ & Zucc. ‘“Variegatum’ . . ‘Palmatum’ sh. ‘Newton Sentry’ sh. ‘Sweet Shadow Cut- sh. ‘Temple’s Upright’ sh. ssp. floridanum . sh. ssp. leucoderme sh. ssp. nigrum (Michx.f.) sh. var. rugelii (Pax) sh. ssp. saccharum Koidz. Miq. Miq. ‘Kohauchina kaido’ Miq. ‘Mikasayama’ Maxim. ge ge ‘Akikaze nishiki’ ge ‘Tokiwa nishiki’ ge ‘Usugumo’ ge f. dissectum Wesmael . var. vanvolxemii ax E H.Wendl. Palm Family . & H.Wendl.) H.Wendl. ex l. Actinidia Family Zucc.) Planch. ex Miq. v.) C.F.Liang & ar. deliciosa v.) C.F.Liang & ar. deliciosa ‘Chico’ hd. im. & Rupr.) Maxim. & Zucc.) Maxim. rufinerve Sieb. & Zucc. ‘Hatsuyuki’ rufinerve Sieb. & Zucc. ‘“Variegatum’ rufinerve Sieb. & Zucc. ‘“Variegatum’ saccharinum L. rufinerve Sieb. & Zucc. Leaf’ saccha saccharum Marsh. ‘Temple’s Upright’ saccharum Marsh. ssp. floridanum (Chapm.) Desm. saccharum Marsh. ssp. leucoderme saccharum Marsh. ‘Temple’s Upright’ saccharum Marsh. ssp. floridanum (Chapm.) Desm. saccharum Marsh. ssp. leucoderme (Small) Desm. saccharum Marsh. ssp. nigrum (Michx.f.) Desm. saccharum Marsh. var. rugelii (Pax) Rehd. saccharum Marsh. ssp. saccharum shirasawanum Koidz. sieboldianum Miq. sieboldianum Miq. ‘Kohauchina kaido’ _ sieboldianum Miq. ‘Mikasayama’ spicatum Lam. tataricum L. tegmentosum Maxim. truncatum Bunge truncatum Bunge ‘Akikaze nishiki’ truncatum Bunge ‘Tokiwa nishiki’ truncatum Bunge ‘Usugumo’ truncatum Bunge f. dissectum Wesmael velutinum Boiss. var. vanvolxemii (Mast.) Rehd. wilsonii Rehd. x zoeschense Pax ACOELORRHAPHE H.Wendl. RECACEAE Palm Family wrightii (Griseb. & H.Wendl.) H.Wendl. ex BEeEce: CTINIDIA Lindl. CTINIDIACEAE Actinidia Family arguta (Sieb. & Zucc.) Planch. ex Miq. deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa deliciosa (A.Chev.) C.F.Liang & x carnea Hayne ‘Briotii’ x dupontii Sarg. x dupontii Sarg. flava Sol. saccharum Marsh. ssp. leucoderme (Small) Desm. saccharum Marsh. ssp. nigrum (Michx.f.) Desm. saccharum Marsh. var. rugelii (Pax) saccharum Marsh. var. rugelii (Pax) Rehd. saccharum Marsh. ssp. saccharum Rehd. saccharum Marsh. ssp. saccharum shirasawanum Koidz. sieboldianum Miq. sieboldianum Miq. ‘Kohauchina kaido’ _ sieboldianum Miq. ‘Mikasayama’ spicatum Lam. tataricum L. tegmentosum Maxim. truncatum Bunge truncatum Bunge ‘Akikaze nishiki’ truncatum Bunge ‘Tokiwa nishiki’ truncatum Bunge ‘Usugumo’ truncatum Bunge f. dissectum Wesmael velutinum Boiss. var. vanvolxemii (Mast.) Rehd. wilsonii Rehd. x zoeschense Pax ACOELORRHAPHE H.Wendl. ARECACEAE Palm Family parviflora Walt. parviflora Walt. tataricum L. tegmentosum tegmentosum Maxim. truncatum Bunge truncatum Bunge truncatum Bunge (Mast.) Rehd. wilsonii Rehd. ACOELORRHAPHE H.Wendl. ARECACEAE Palm Family wrightii (Griseb. & H.Wendl.) H.Wendl. ex BEeEce: ACTINIDIA Lindl. ACTINIDIACEAE Actinidia Family arguta (Sieb. & Zucc.) Planch. ex Miq. deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa deliciosa (A.Chev.) C.F.Liang & A.R.Ferguson var. deliciosa ‘Chico’ <fairchildii Rehd. kolomikta (Maxim. & Rupr.) Maxim. polygama (Sieb. & Zucc.) Maxim. purpurea Rehd. NO Oi NS) AMPELOPSIS Michx. VITACEAE > Grape Family arborea (L.) Koehne glandulosa var. brevipedunculata (Maxim.) Momiy. glandulosa var. brevipedunculata (Maxim.) Momiy. ‘Elegans’ AMYRIS P.Br. RUTACEAE Citrus Family texana (Buckl.) P.Wilson ANDROMEDA L. ERICACEAE Heath Family polifolia L. polifolia L. ‘Nana’ ANISACANTHUS Nees ACANTHACEAE Acanthus Family thurberi (Torr.) A.Gr. ANTIGONON Endl. POLYGONACEAE Buckwheat Family leptopus Hook. & Arn. APHANANTHE Planch. ULMACEAE Elm Family aspera (Thunb. ex J.A.Murr.) Planch. ARALIA L. ARALIACEAE Ginseng Family elata (Miq.) Seemann spinosa L. ARAUCARIA Juss. ARAUCARIACEAE Araucaria Family araucana (Mol.) K.Koch bidwillii Hook. ARBUTUS L. ERICACEAE Heath Family unedo L. ARCTOSTAPHYLOS Adans. ERICACEAE Heath Family ALBIZIA Durazz. Leaf’ saccha FABACEAE (Mimosoideae) Bean Family julibrissin Durazz. kalkora (Roxb.) Prain ALEURITES J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family fordii Hemsl. montana (Lour.) Wils. ALLAMANDA L. APOCYNACEAE Dogbane Family cathartica L. ALNUS Mill. BETULACEAE Alder Birch Family formosana (Burkw.) Makino glutinosa (L.) Gaertn. hirsuta (Spach) Rupr. var. sibirica (Spach) Schneid. japonica (Thunb.) Steud. maritima (Marsh.) Nutt. serrulata (Ait.) Willd. ALOYSIA Juss. VERBENACEAE Verbena Family gratissima (Gillies & Hook.) Troncoso AMELANCHIER Medik. ROSACEAE Shadbush Rose Family alnifolia (Nutt.) Nutt. arborea (Michx.f.) Fern. asiatica (Sieb. & Zucc.) Endl. canadensis (L.) Medik. laevis Wieg. laevis Wieg. ‘Rosea’ sanguinea (Pursh) DC. spicata (Lam.) K.Koch ‘Success’ AMORPHA lL. FABACEAE (Faboideae) Bean Family Sfruticosa L. glabra Desf. ex Poir. Grape Family 253 ARDISIA O.Swartz MYRSINACEAE Myrsine Family crenata Sims crenata Sims ‘Alba’ crispa (Thunb. ex J.A.Murr.) A.DC. japonica (Thunb.) BI. ARISTOLOCHIA L. ARISTOLOCHIACEAE Aristolochia Family elegans Mast. ARONIA Medik. ROSACEAE Chokeberry Rose Family arbutifolia (L.) Ell. melanocarpa (Michx.) EIl. prunifolia (Marsh.) Rehd. ARTEMISIA L. ASTERACEAE Aster Family abrotanum L. ARUNDINARIA Michx. POACEAE Grass Family pygmaea (Miq.) Mitf. “Variegata’ ASCYRUM L. HYPERICACEAE St. John’s-wort Family hypericoides L. ASIMINA Adans. ANNONACEAE Custard-apple Family triloba (L.) Dunal ASTER L. ASTERACEAE Aster Family carolinianus Walt. AUCUBA Thunb. CORNACEAE Dogwood Family chinensis Benth. japonica Thunb. japonica Thunb. ‘Goldieana’ japonica Thunb. ‘Limbata’ japonica Thunb. ‘Longifolia’ japonica Thunb. ‘Meigetsu’ BACCHARIS L. ASTERACEAE Groundsel Aster Family glomeruliflora Pers. halimifolia L. halimifolia L. ‘Dauphin Island’ BAMBUSA_ Schreber POACEAE Bamboo Grass Family multiplex (Lour.) Raeusch. multiplex (Lour.) Raeusch. ‘Alphonse Karr’ multiplex (Lour.) Raeusch. ‘Fernleaf’ multiplex (Lour.) Raeusch. ‘Silverstripe’ multiplex (Lour.) Raeusch. ‘Variegata’ tuldoides Munro BAUHINIA lL. FABACEAE (Caesalpinioideae) Bean Family divaricata L. lunarioides A.Gr. ex S.Wats. yunnanensis Franch. BEFARIA Mutis ex L. ERICACEAE Heath Family racemosa Vent. BERBERIS L. BERBERIDACEAE Barberry Barberry Family x gladwynensis Li ‘William Penn’ hookeri Lem. hookeri Lem. var. viridis Schneid. < hybrido-gagnepainii Suring. ‘Chenault’ julianae Schneid. julianae Schneid. ‘Byers’ koreana Palib. ‘Atropurpurea’ < media Grootend. ‘Parkjuweel’ x mentorensis H.Schultz & Horvath ex L.M.Ames mouillacana Schneid. sargentiana Schneid. soulieana Schneid. thunbergii DC. thunbergii DC. ‘Atropurpurea’ thunbergii DC. ‘Atropurpurea Nana’ thunbergii DC. ‘Aurea’ thunbergii DC. ‘Crimson Pygmy’ thunbergii DC. ‘Kobold’ BERCHEMIA Necker ex DC. RHAMNACEAE Buckthorn Family BERCHEMIA Necker ex DC. RHAMNACEAE Buckthorn Family BERCHEMIA Necker ex DC. RHAMNACEAE Buckthorn Family racemosa Sieb. & Zucc. racemosa Sieb. & Zucc. ‘Issai’ scandens (J.Hill) K.Koch racemosa Sieb. & Zucc. racemosa Sieb. & Zucc. ‘Issai’ scandens (J.Hill) K.Koch Birch Birch Family BACCHARIS L. ASTERACEAE Groundsel Aster Family Myrsine Family Chokeberry Rose Family BRUNFELSIA L. SOLANACEAE Nightshade Family australis Benth. BUCKLEYA Torr. SANTALACEAE Sandalwood Family distichophylla (Nutt.) Torr. BUDDLEJA L. BUDDLEJACEAE Butterfly Bush Buddleja Family alternifolia Maxim. crispa Benth. var. farreri (Balf.f. & W.W.Sm.)Hand.-Mazz. davidii Franch. davidii Franch. ‘Alba’ faltlowiana Balf.f. & W.W.Sm. farreri Balf.f. & W.W.Sm. japonica Hemsl. lindleyana Fort. ex Lindl. venenifera Makino xX weyeriana Weyer xX weyeriana Weyer ‘Sungold’ <x whiteana R.J.Moore BUMELIA O.Swartz SAPOTACEAE Sapodilla Family lanuginosa (Michx.) Pers. lycioides (L.) Pers. tenax (L.) Willd. BUTIA (Becc.) Becc. ARECACEAE Palm Family capitata (Mart.) Becc. BUXUS L. Box BUXACEAE Boxwood Family balearica Lam. harlandii Hance harlandii Hance ‘Richard’ microphylla Sieb. & Zucc. microphylla Sieb. & Zucc. ‘Compacta’ microphylla Sieb. & Zucc. ‘Curly Locks’ microphylla Sieb. & Zucc. ‘Grace Hendrick Phillips’ microphylla Sieb. & Zucc. ‘Green Pillow’ microphylla Sieb. & Zucc. ‘Helen Whiting’ microphylla Sieb. & Zucc. ‘Henry Hohman’ microphylla Sieb. & Zucc. ‘John BETULA L. BETULACEAE sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. Macrophylla’ sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. ‘Abilene’ ‘Agram’ ‘Angustifolia’ ‘Arborescens’ ‘Arborescens ‘Argenteo-variegata’ ‘Aristocrat’ ‘Aurea Pendula’ ‘Aureo-variegata’ ‘Belleville’ ‘Bullata’ ‘Butterworth’ ‘Denmark’ ‘Edgar Anderson’ ‘Elegantissima’ ‘Fastigiata’ ‘Glauca’ ‘Graham Blandy’ ‘Handsworthiensis’ ‘Hardwickensis’ ‘Heinrich Bruns’ ‘Henry Shaw’ ‘Inglis’ ‘Ipek’ ‘Joe Gable’ ‘Joy’ ‘Latifolia ‘Latifolia Nova’ ‘Lynnhaven’ ‘Macrophylla’ ‘Marginata’ ‘Memorial’ ‘Myrtifolia’ ‘Nish’ ‘Northern Find’ ‘Northern New York’ ‘Northland’ ‘Pendula’ ‘Ponteyi’ ‘Prostrata’ ‘Pyramidalis’ ‘Rotundifolia’ sempervirens L. ‘Salicifolia’ sempervirens L. ‘Salicifolia Elata’ sempervirens L. ‘Suffruticosa’ sempervirens L. ‘Vardar Valley’ sempervirens L. ‘Varifolia’ sempervirens L. ‘Welleri’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Pincushion’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Tall Boy’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Tide Hill’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Winter Beauty’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Wintergreen’ stenophylla Hance wallichiana Baill. ‘Green Gem’ ‘Green Mountain’ ‘Green Velvet’ CAESALPINIA L. FABACEAE (Caesalpinioideae) Bean Family gilliesii Hook. CALLIANDRA Benth. FABACEAE (Mimosoideae) Bean Family tweediei Benth. CALLICARPA lL. Beautyberry VERBENACEAE Verbena Family americana L. americana L. ‘Lactea’ bodinieri Lev. bodinieri Lev. var. giraldii Rehd. dichotoma (Lour.) K.Koch dichotoma (Lour.) K.Koch ‘Leucocarpa’ japonica Thunb. macrophylla Vahl tosaensis Makino CALLISTEMON R.Br. Bottlebrush MYRTACEAE Myrtle Family citrinus (Curtis) Skeels microphylla Sieb. & Zucc. ‘Sunnyside’ microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. * microphylla Sieb. & Zucc. var. japonica microphylla Sieb. & Zucc. ‘Sunnyside’ microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. * microphylla Sieb. & Zucc. var. japonica * microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. ‘Morris Dwarf’ * microphylla Sieb. & Zucc. var. japonica * microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. ‘Morris Midget’ * microphylla Sieb. & Zucc. var. japonica Midget’ * microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. ‘National’ sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. Decussata’ sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. Macrophylla’ sempervirens L. BETULA L. BETULACEAE alleghaniensis Britton costata Traut. davurica Pall. jaquemontii Spach lenta L. maximowicziana Regel nigra L. occidentalis Hook. papyrifera Marsh. papyrifera < B. maximowicziana pendula Roth pendula x B. nigra pendula Roth ‘Dalecarlica’ pendula Roth ‘Purpurea’ pendula Roth ‘Youngii’ platyphylla Sukachev var. japonica (Miq.) Hara populifolia Marsh. potaninii Batal. pubescens Ehrh. uber (Ashe) Fern. BIGNONIA L. BIGNONIACEAE Bignonia Family capreolata L. BREYNIA J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family disticha J.R. & J.G.Forst. BROUSSONETIA UWLdHer. ex Vent. MORACEAE papyrifera (L.) Vent. papyrifera (L.) Vent. ‘Variegata’ BRUCKENTHALIA Reichenb. ERICACEAE Heath Family spiculifolia (Salisb.) Reichenb. BRUGMANSIA Pers. SOLANACEAE Nightshade Family suaveolens (Humboldt & Bonpland ex Willd.) Bercht. & J.Presl Mulberry Family BIGNONIA L. BIGNONIACEAE Bignonia Family capreolata L. BREYNIA J.R. &J.G. Forst. EUPHORBIACEAE Spurge Family disticha J.R. & J.G.Forst. BROUSSONETIA UWLdHer. ex Vent. MORACEAE papyrifera (L.) Vent. papyrifera (L.) Vent. ‘Variegata’ BRUCKENTHALIA Reichenb. ERICACEAE Heath Family spiculifolia (Salisb.) Reichenb. BRUGMANSIA Pers. SOLANACEAE Nightshade Family suaveolens (Humboldt & Bonpland ex Willd.) Bercht. & J.Presl Mulberry Family \| 255 sempervirens L. ‘Salicifolia’ sempervirens L. ‘Salicifolia Elata’ sempervirens L. ‘Suffruticosa’ sempervirens L. ‘Vardar Valley’ sempervirens L. ‘Varifolia’ sempervirens L. ‘Welleri’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Pincushion’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Tall Boy’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Tide Hill’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Winter Beauty’ sinica (Rehd. & Wils.) M.Cheng var. insularis (Nakai) M.Cheng ‘Wintergreen’ stenophylla Hance wallichiana Baill. ‘Green Gem’ ‘Green Mountain’ ‘Green Velvet’ CAESALPINIA L. FABACEAE (Caesalpinioideae) Bean Family gilliesii Hook. CALLIANDRA Benth. FABACEAE (Mimosoideae) Bean Family tweediei Benth. CALLICARPA lL. Beautyberry VERBENACEAE Verbena Family americana L. americana L. ‘Lactea’ bodinieri Lev. bodinieri Lev. var. giraldii Rehd. dichotoma (Lour.) K.Koch dichotoma (Lour.) K.Koch ‘Leucocarpa’ japonica Thunb. macrophylla Vahl tosaensis Makino microphylla Sieb. & Zucc. ‘Sunnyside’ microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. * microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. ‘Morris Dwarf’ * microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. ‘Morris Midget’ * microphylla Sieb. & Zucc. var. japonica (Muell.-Arg.) Rehd. & Wils. ‘National’ sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. Decussata’ sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. BETULA L. BETULACEAE sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. sempervirens L. ‘Abilene’ ‘Agram’ ‘Angustifolia’ ‘Arborescens’ ‘Arborescens ‘Argenteo-variegata’ ‘Aristocrat’ ‘Aurea Pendula’ ‘Aureo-variegata’ ‘Belleville’ ‘Bullata’ ‘Butterworth’ ‘Denmark’ ‘Edgar Anderson’ ‘Elegantissima’ ‘Fastigiata’ ‘Glauca’ ‘Graham Blandy’ ‘Handsworthiensis’ ‘Hardwickensis’ ‘Heinrich Bruns’ ‘Henry Shaw’ ‘Inglis’ ‘Ipek’ ‘Joe Gable’ ‘Joy’ ‘Latifolia ‘Latifolia Nova’ ‘Lynnhaven’ ‘Macrophylla’ ‘Marginata’ ‘Memorial’ ‘Myrtifolia’ ‘Nish’ ‘Northern Find’ ‘Northern New York’ ‘Northland’ ‘Pendula’ ‘Ponteyi’ ‘Prostrata’ ‘Pyramidalis’ ‘Rotundifolia’ ‘Ste. Genevieve’ rugulosus Miq. salignus (Sm.) DC. viminalis (Sol. ex Gaertn.) G.Don CALLITRIS Vent. CUPRESSACEAE Cypress Family columellaris F.Muell. CALLUNA Salisb. ERICACEAE Heather Heath Family vulgaris (L.) Hull vulgaris (L.) Hull ‘August Beauty’ vulgaris (L.) Hull ‘Aureafolia’ vulgaris (L.) Hull ‘H. E. Beale’ vulgaris (L.) Hull ‘Juno’ vulgaris (L.) Hull ‘Plena’ vulgaris (L.) Hull ‘Silver Queen’ vulgaris (L.) Hull ‘Tib’ CALOCEDRUS Kurz CUPRESSACEAE Cypress Family decurrens (Torr.) Florin CALYCANTHUS L. CALYCANTHACEAE Calycanthus Family floridus L. floridus L. ‘Athens’ floridus L. ‘Margarita’ CAMELLIA L. THEACEAE Tea Family assimilis Champ. ex Benth. crapnelliana Tutch. cuspidata (Kochs) J.G.Veitch drupifera (Lour.) Pierre Sfraterna Hance (fraterna X C. japonica) ‘Little Princess’ granthamiana Sealy * (hiemalis ‘Billy Wylam’ x C. oleifera) ‘Frost Princess’ * (‘Frost Princess’ x C. oleifera) ‘Polar Ice’ * (‘Frost Princess’ x C. oleifera) ‘Snow Flurry’ * (‘Frost Princess’ x C. oleifera) ‘Winter’s Hope’ * (hiemalis ‘Otome’ x C. oleifera) ‘Winter’s Rose’ * (hiemalis ‘Shishigashira’ < C. oleifera) ‘Frost Prince’ * (‘Frost Prince’ < C. hiemalis) ‘Winter’s Star’ = * (hongkongensis < C. rusticana) ‘Sunworshiper’ Japonica L. japonica L. ‘Alice Morrison’ Japonica L. ‘Anemoniflora’ Japonica L. ‘Aunt Jetty’ japonica L. ‘Barbara Morgan’ japonica L. ‘Chandleri Elegans’ japonica L. ‘Daitairin’ Japonica L. ‘Doctor Tinsley’ * japonica L. ‘Frost Queen’ japonica L. ‘Gov. Mouton Variegated’ japonica L. ‘Guilio Nuccio’ japonica L. ‘Kumasaka’ Japonica L. ‘Leucantha’ japonica L. ‘Marie Wood’ japonica L. ‘Professor Sargent’ japonica L. ‘Reverend John G. Drayton’ japonica L. ‘Sarah Frost’ japonica L. ‘Sieboldii’ japonica L. ‘Tama no ura’ japonica L. ‘Ville de Nantes’ * (japonica ‘Arabella’ x C. ‘Fragrant Pink’) ‘Ack-Scent Red’ * (japonica ‘Fragrant Star’ x C. ‘Fragrant Pink’) ‘Ack-Scent Pink’ * (japonica ‘Fragrant Star’ x C. ‘Fragrant Pink’) ‘Ack-Scent Spice’ * (japonica ‘Fragrant Star’ x C. ‘Fragrant Pink’) ‘Ack-Scent Star’ * (japonica ‘Frost Queen’ * C. ‘Fragrant Pink’) ‘Ack-Scent Sno’ * (japonica ‘Frost Queen’ x C. ‘Fragrant Pink’) ‘Ack-Scent White’ * (japonica ‘Kenyo-Tai’ < C. BETULA L. BETULACEAE lutchuensis) ‘Cinnamon Cindy’ * (japonica ‘Kramer’s Supreme’ C. ‘Fragrant Pink Improved’) ‘Ack-Scent’ kissii Wall. lutchuensis Ito oleifera Abel * (oleifera <x C. sasanqua ‘Takara-awase’) ‘Winter’s Charm’ reticulata Lindl. ‘Capt. Rawes’ (reticulata < C. saluenensis) ‘Inspiration’ * (rusticana ‘Yoshida’ < C. lutchuensis) ‘Fragrant Pink’ * (rusticana x C. lutchuensis) ‘Fragrant Joy’ saluenensis Stapf sasanqua Thunb. ex J.A.Murr. sasanqua Thunb. ex J.A.Murr. ‘Annette’ sasanqua Thunb. ex J.A.Murr. ‘Cherokee’ rugulosus Miq. salignus (Sm.) DC. viminalis (Sol. ex Gaertn.) G.Don rugulosus Miq. salignus (Sm.) DC. viminalis (Sol. ex Gaertn.) G.Don rugulosus Miq. salignus (Sm.) DC. viminalis (Sol. ex Gaertn.) G.Don salignus (Sm.) DC. viminalis (Sol. ex Gaertn.) G.Don CALLITRIS Vent. CUPRESSACEAE Cypress Family columellaris F.Muell. CALLUNA Salisb. ERICACEAE Heather Heath Family vulgaris (L.) Hull vulgaris (L.) Hull ‘August Beauty’ vulgaris (L.) Hull ‘Aureafolia’ vulgaris (L.) Hull ‘H. E. Beale’ vulgaris (L.) Hull ‘Juno’ vulgaris (L.) Hull ‘Plena’ vulgaris (L.) Hull ‘Silver Queen’ vulgaris (L.) Hull ‘Tib’ CALOCEDRUS Kurz CUPRESSACEAE Cypress Family decurrens (Torr.) Florin CALYCANTHUS L. CALYCANTHACEAE Calycanthus Family floridus L. floridus L. ‘Athens’ floridus L. ‘Margarita’ CAMELLIA L. THEACEAE Tea Family assimilis Champ. ex Benth. crapnelliana Tutch. cuspidata (Kochs) J.G.Veitch drupifera (Lour.) Pierre Sfraterna Hance (fraterna X C. japonica) ‘Little Princess’ granthamiana Sealy * (hiemalis ‘Billy Wylam’ x C. oleifera) ‘Frost Princess’ * (‘Frost Princess’ x C. oleifera) ‘Polar Ice’ * (‘Frost Princess’ x C. oleifera) ‘Snow Flurry’ * (‘Frost Princess’ x C. oleifera) ‘Winter’s Hope’ * (hiemalis ‘Otome’ x C. oleifera) ‘Winter’s Rose’ * (hiemalis ‘Shishigashira’ < C. oleifera) ‘Frost Prince’ Tea Family ‘Cherokee’ 257 sasanqua Thunb. ex J.A.Murr. ‘Cleopatra’ sasanqua Thunb. ex J.A.Murr. ‘Crimson Bride’ sasanqua Thunb. ex J.A.Murr. ‘Sharon Elizabeth’ sasanqua Thunb. ex J.A.Murr. ‘Texas Star’ (sasanqua ‘Maiden’s Blush’ + C. japonica) +‘Daisy Eagleson’ * (sasanqua ‘Onishiki’ x C. kissii) ‘Two Marthas’ sinensis (L.) O.Ktze. tenuifolia (Hayata) Cohen-Stuart tsaii H.H.Hu vernalis (Makino) Makino ‘Dawn’ vernalis (Makino) Makino ‘Sayehime’ x williamsii W.W.Sm. ‘Donation’ x williamsii W.W.Sm. ‘Donation Sport’ <williamsii W.W.Sm. ‘November Pink’ * ‘Fragrant Pink Improved’ (Polyploid ‘Fragrant Pink’) ‘Tiny Princess’ CAMPSIS Lour. BIGNONIACEAE Bignonia Family grandiflora (Thunb. ex J.A.Murr.) Schum. radicans (L.) Seemann ex Bur. radicans (L.) Seemann ex Bur. ‘Yellow Trumpet’ x tagliabuana (Vis.) Rehd. ‘Mme. Galen’ CAMPYLOTROPIS Bunge FABACEAE (Papilionoideae) Bean Family macrocarpa Bunge CARAGANA Fabr. FABACEAE (Faboideae) Pea Shrub Bean Family arborescens Lam. arborescens Lam. ‘Lorbergii’ arborescens Lam. ‘Pendula’ Srutex (L.) K.Koch ‘Sylvatica’ sinica (Buc’hoz) Rehd. CARISSA L. APOCYNACEAE Dogbane Family macrocarpa (Eckl. & Zeyh.) A.DC. CARPINUS lL. BETULACEAE betulus L. betulus L. ‘Fastigiata’ betulus L. ‘Purpurea’ caroliniana Walt. caroliniana Walt. ‘Ascendens’ cordata BI. Japonica Bl. orientalis Mill. CARYA Nutt. JUGLANDACEAE Hickory Walnut Family aquatica (Michx.f.) Nutt. cordiformis (Wang.) K.Koch glabra (Mill.) Sweet illinoinensis (Wang.) K.Koch illinoinensis (Wang.) K.Koch ‘Mehan’ illinoinensis (Wang.) K.Koch ‘Stuart’ illinoinensis X C. sp. (illinoinensis < C. sp.) ‘Koon’ laciniosa (Michx.f.) Loud. ovalis (Wang.) Sarg. ovata (Mill.) K.Koch ovata (Mill.) K.Koch ‘Glover’ ovata (Mill.) K.Koch ‘Hines’ ovata (Mill.) K.Koch ‘Lake’ ovata (Mill.) K.Koch ‘Lingenfelter’ ovata (Mill.) K.Koch ‘Romig’ ovata (Mill.) K.Koch ‘Schaul’ ovata (Mill.) K.Koch ‘Schinnerling’ ovata (Mill.) K.Koch ‘Vest’ ovata (Mill.) K.Koch var. pubescens Sarg. pallida (Ashe) Engl. & Graebn. tomentosa (Lam.) Nutt. CARYOPTERIS Bunge VERBENACEAE Verbena Family x clandonensis Simmonds incana (Thunb. ex Houtt.) Miq. CASIMIROA Liave & Lex. RUTACEAE Citrus Family edulis Llave & Lex. pringlei (S.Wats.) Engelm. CASSIA L. Senna FABACEAE (Caesalpinioideae) Bean Family alata L. coluteoides Coll. corymbosa Lam. CARYA Nutt. JUGLANDACEAE Hickory Walnut Family aquatica (Michx.f.) Nutt. cordiformis (Wang.) K.Koch glabra (Mill.) Sweet illinoinensis (Wang.) K.Koch illinoinensis (Wang.) K.Koch ‘Mehan’ illinoinensis (Wang.) K.Koch ‘Stuart’ illinoinensis X C. sp. (illinoinensis < C. sp.) ‘Koon’ laciniosa (Michx.f.) Loud. ovalis (Wang.) Sarg. ovata (Mill.) K.Koch ovata (Mill.) K.Koch ‘Glover’ ovata (Mill.) K.Koch ‘Hines’ ovata (Mill.) K.Koch ‘Lake’ ovata (Mill.) K.Koch ‘Lingenfelter’ ovata (Mill.) K.Koch ‘Romig’ ovata (Mill.) K.Koch ‘Schaul’ ovata (Mill.) K.Koch ‘Schinnerling’ ovata (Mill.) K.Koch ‘Vest’ ovata (Mill.) K.Koch var. pubescens Sarg. pallida (Ashe) Engl. & Graebn. tomentosa (Lam.) Nutt. CARYOPTERIS Bunge VERBENACEAE Verbena Family x clandonensis Simmonds incana (Thunb. ex Houtt.) Miq. CASIMIROA Liave & Lex. RUTACEAE Citrus Family edulis Llave & Lex. pringlei (S.Wats.) Engelm. CASSIA L. Senna FABACEAE (Caesalpinioideae) Bean Family alata L. coluteoides Coll. corymbosa Lam. 0 atlantica (Endl.) Manetti ex Carr. ‘Pendula’ = deodara (Roxb.) G.Don deodara (Roxb.) G.Don ‘Aurea’ deodara (Roxb.) G.Don ‘Fastigiata’ deodara (Roxb.) G.Don ‘Limelight’ deodara (Roxb.) G.Don ‘Pendula’ deodara (Roxb.) G.Don ‘Pygmy’ deodara (Roxb.) G.Don ‘Repandens’ libani A.Rich. libani A.Rich. ‘Nana’ libani A.Rich. ssp. brevifolia (Hook.f.) Meikle libani A.Rich. var. stenocoma (O.Schwarz) Davis CELASTRUS lL. CELASTRACEAE Bittersweet Staff-tree Family angulatus Maxim. orbiculatus Thunb. ex J.A.Murr. rosthornianus Loesn. CELTIS L. ULMACEAE Hackberry Elm Family australis L. caucasica Willd. laevigata Willd. occidentalis L. pallida Torr. sinensis Pers. sinensis Pers. var. japonica (Planch.) Nakai tala Gillies ex Planch. CEPHALANTHUS lL. CASTANEA Mill. FAGACEAE crenata Sieb. & Zucc. mollissima BI. mollissima BI. ‘Crane’ mollissima Bl. ‘Meiling’ mollissima Bl. ‘Nanking’ mollissima Bl. ‘Orrin’ sativa Mill. sativa L. ‘Argenteo-variegata’ CASTANOPSIS (D.Don) Spach Chinquapin FAGACEAE Beech Family cuspidata (Thunb. ex J.A.Murray) Schottky cuspidata (Thunb.) Schottky var. sieboldii (Makino) Nakai sclerophylla Schottky CASUARINA Adans. CASUARINACEAE Casuarina Family cunninghamiana Miq. CATALPA Scop. BIGNONIACEAE Bignonia Family bignonioides Walt. bungei C.A.Mey. ovata G.Don speciosa Warder ex Engelm. CEANOTHUS lL. RHAMNACEAE Buckthorn Family americanus L. x delilianus Spach <x delilianus Spach ‘Gloire de Versailles’ microphyllus Michx. x pallidus Lindl. ‘Marie Simon’ velutinus Dougl. CEDRUS ‘Trew PINACEAE Cedar Pine Family atlantica (Endl.) Manetti ex Carr. atlantica (Endl.) Manetti ex Carr. ‘Aurea’ atlantica (Endl.) Manetti ex Carr. deodara (Roxb.) G.Don ‘Limelight’ deodara (Roxb.) G.Don ‘Pendula’ deodara (Roxb.) G.Don ‘Pygmy’ deodara (Roxb.) G.Don ‘Repandens’ libani A.Rich. libani A.Rich. ‘Nana’ libani A.Rich. ssp. brevifolia (Hook.f.) Meikle libani A.Rich. var. stenocoma (O.Schwarz) Davis CELASTRUS lL. CELASTRACEAE Bittersweet Staff-tree Family angulatus Maxim. orbiculatus Thunb. ex J.A.Murr. rosthornianus Loesn. CELTIS L. ULMACEAE Hackberry Elm Family australis L. caucasica Willd. laevigata Willd. occidentalis L. pallida Torr. sinensis Pers. sinensis Pers. var. japonica (Planch.) Nakai tala Gillies ex Planch. CEPHALANTHUS lL. RUBIACEAE Madder Family occidentalis L. CEPHALOTAXUS Sieb. & Zucc. ex Endl. CEPHALOTAXACEAE Plum-yew Family harringtonia (J.Knight ex Forbes) K.Koch harringtonia (J.Knight ex Forbes) K.Koch ‘Fastigiata’ harringtonia (J.Knight ex Forbes) K.Koch ‘Nana’ CERATIOLA Michx. EMPETRACEAE Crowberry Family ericoides Michx. sativa Mill. sativa L. ‘Argenteo-variegata’ CASTANOPSIS (D.Don) Spach Chinquapin FAGACEAE Beech Family cuspidata (Thunb. ex J.A.Murray) Schottky cuspidata (Thunb.) Schottky var. sieboldii (Makino) Nakai sclerophylla Schottky CASUARINA Adans. CASUARINACEAE Casuarina Family cunninghamiana Miq. CATALPA Scop. BIGNONIACEAE Bignonia Family bignonioides Walt. bungei C.A.Mey. ovata G.Don speciosa Warder ex Engelm. CEANOTHUS lL. CEANOTHUS lL. RHAMNACEAE Buckthorn Family americanus L. x delilianus Spach <x delilianus Spach ‘Gloire de Versailles’ microphyllus Michx. x pallidus Lindl. ‘Marie Simon’ velutinus Dougl. CEDRUS ‘Trew PINACEAE Cedar Pine Family atlantica (Endl.) Manetti ex Carr. atlantica (Endl.) Manetti ex Carr. ‘Aurea’ atlantica (Endl.) Manetti ex Carr. ‘Fastigiata’ atlantica (Endl.) Manetti ex Carr. ‘Glauca’ atlantica (Endl.) Manetti ex Carr. ‘Glauca Pendula’ CEPHALANTHUS lL. RUBIACEAE Madder Family occidentalis L. CEPHALOTAXUS Sieb. & Zucc. ex Endl. CEPHALOTAXACEAE Plum-yew Family harringtonia (J.Knight ex Forbes) K.Koch harringtonia (J.Knight ex Forbes) K.Koch ‘Fastigiata’ harringtonia (J.Knight ex Forbes) K.Koch ‘Nana’ CERATIOLA Michx. EMPETRACEAE Crowberry Family ericoides Michx. AO) x superba (Frahm) Rehd. ‘Knap Hill Scarlet’ x superba (Frahm) Rehd. ‘Mandarin’ x superba (Frahm) Rehd. ‘Pendula Vera’ nootkatensis (D. ‘Pendula Vera’ nootkatensis (D.Don) Spach obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. ‘Albospica’ ‘Breviramea’ ‘Contorta’ obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. ‘Breviramea’ ‘Contorta’ ‘Coralliformis’ obtusa (Sieb. & Z ‘Coralliformis’ obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. Aurea’ obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. ‘Crippsii’ ‘Filicoides’ ‘Filiformis ‘Gold Drop’ ‘Gracilis’ ‘Intermedia’ ‘Kosteri’ & Zucc.) Endl. ‘Lycopodioides’ CERCIS L. FABACEAE ( awsoniana (A.Murr.) Parl. awsoniana (A.Murr.) Parl. ‘Filiformis ‘Filiformis lawsoniana (A.Murr.) Parl. ‘Lycopodioides’ ‘Pembury Blue’ lawsoniana (A.Murr.) Parl. ‘Pendula Vera’ CASTANEA Mill. FAGACEAE ‘Perfecta’ x superba (Frahm) Rehd. ‘Pink Lady’ x superba (Frahm) Rehd. ‘Roxana Foster’ x superba (Frahm) Rehd. ‘Ruby Glow’ x superba (Frahm) Rehd. ‘Stanford Red’ x vilmoriniana Weber ‘Mt. Everest’ PLUMBAGINACEAE Leadwort Family plumbaginoides Bunge willmottianum Stapf CERCIDIPHYLLUM Sieb. & Zucc. CERCIDIPHYLLACEAE Katsura-tree Family japonicum Sieb. & Zucc. japonicum Sieb. & Zucc. ‘Pendula’ CERCIS L. FABACEAE (Caesalpinioideae) Bean Family canadensis L. canadensis L. canadensis L. canadensis L. canadensis L. canadensis L. canadensis L. ‘Alba’ ‘Forest Pansy’ ‘Plena’ ‘Ruby Atkinson’ ‘Wither’s Pink Charm’ ssp. texensis (S.Wats.) E.Murr. ‘Alba’ canadensis L. ssp. texensis (S.Wats.) E.Murr. ‘Oklahoma’ chinensis Bunge chinensis Bunge ‘Alba’ siliquastrum L. ‘Rubrum’ CESTRUM lL. SOLANACEAE Nightshade Family nocturnum L. CHAENOMELES Lindl. ROSACEAE Rose Family x californica Clarke & Weber ‘Rosy Morn’ japonica (Thunb.) Lindl. ex Spach speciosa (Sweet) Nakai speciosa (Sweet) Nakai ‘Apple Blossom’ speciosa (Sweet) Nakai ‘Candida’ speciosa (Sweet) Nakai ‘Cardinalis’ speciosa (Sweet) Nakai ‘Hanazono’ speciosa (Sweet) Nakai ‘Marmorata’ speciosa (Sweet) Nakai ‘Nivalis’ speciosa (Sweet) Nakai ‘Phyllis Moore’ speciosa (Sweet) Nakai ‘Rubra’ speciosa (Sweet) Nakai ‘Simonii’ speciosa (Sweet) Nakai ‘Snow’ speciosa (Sweet) Nakai ‘Toyo nishiki’ speciosa (Sweet) Nakai ‘Umbilicata’ < superba (Frahm) Rehd. < superba (Frahm) Rehd. ‘Corallina’ x superba (Frahm) Rehd. ‘Crimson and Gold’ CHAMAECYPARIS Spach False Cypress CUPRESSACEAE Cypress Family Jfunebris (Endl.) Franco lawsoniana (A.Murr.) Parl. lawsoniana (A.Murr.) Parl. lawsoniana (A.Murr.) Parl. lawsoniana (A.Murr.) Parl. lawsoniana (A.Murr.) Parl. Compacta’ lawsoniana (A.Murr.) Parl. lawsoniana (A.Murr.) Parl. ‘Lycopodioides’ lawsoniana (A.Murr.) Parl. ‘Pembury Blue’ lawsoniana (A.Murr.) Parl. ‘Pendula Vera’ ‘Allumii’ ‘Blom’ ‘Filiformis’ ‘Filiformis ‘Lutea’ nootkatensis (D.Don) Spach obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. ‘Albospica’ ‘Breviramea’ pisifera (Sieb. ‘Juniperoides Aurea’ & Zucc.) pisifera (Sieb. pistfera (Sieb. & Zucc.) Endl. & Zucc.) Endl. ‘Monstrosa’ ‘Plumosa’ pistfera (Sieb. pistfera (Sieb. & Zucc.) Endl. & Zucc.) Endl. ‘Plumosa’ ‘Plumosa obtusa (Sieb. & Zucc.) Endl. ‘Lycopodioides’ ‘Ericoides’ CHAMAECYTISUS Link FABACEAE (Faboideae) supinus (L.) Link CHAMAEDAPHNE ERICACEAE Moench calyculata (L.) Moench ‘Monstrosa’ ‘Plumosa’ ‘Plumosa ‘Plumosa ‘Plumosa ‘Plumosa ‘Pygmaea’ ‘Snow’ ‘Squarrosa’ ‘Squarrosa ‘Squarrosa Bean Family Heath Family CHAMAEROPS L. ARECACEAE = Palm Family humilis L. CHILOPSIS D.Don BIGNONIACEAE Bignonia Family linearis (Cav.) Sweet CHIMONANTAUS Lindl. CALYCANTHACEAE Calycanthus Family nitens Oliv. praecox (L.) Link praecox (L.) Link ‘Luteus’ praecox (L.) Link ‘Mangetsuw’ CHIONANTHUS L. OLEACEAE Fringe Tree Olive Family retusus Lindl. & Paxt. retusus Lindl. & Paxt. var. serrulatus (Hayata) Koidz. virginicus L. CHOSENIA Nakai SALICACEAE Willow Family arbutifolia (Pall.) Skvortz. CHRYSANTHEMUM L. ASTERACEAE Aster Family nipponicum (Franch. ex Maxim.) Matsum. CINNAMOMUM $= Schaeffer LAURACEAE Laurel Family camphorea (L.) T. Nees & Eberm. daphnoides Sieb. & Zucc. CISSUS L. VITACEAE Grape Family incisa (Nutt.) Desmoul. CISTUS L. CISTACEAE Rock-rose Family < hybridus Pourr. ladanifer L. < purpureus Lam. obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb Aurea’ obtusa (Sieb obtusa (Sieb . & Zuce. . & Zuce. . & Zucc.) Endl. . & Zucc.) Endl. ) Endl. . & Zucc.) Endl. ) Endl. . & Zucc.) Endl. . & Zucc.) Endl. . & Zucc.) Endl. . & Zucc.) Endl. pisifera (Sieb. pisifera (Sieb. Nana’ pistfera (Sieb. pistfera (Sieb. pisifera (Sieb. pistfera (Sieb. Aurea’ pisifera (Sieb. Spangle’ pistfera (Sieb. Mop’ pisifera (Sieb. pisifera (Sieb. pistfera (Sieb. pistfera (Sieb. Aurea’ pisifera (Sieb. Aurea Nana’ pisifera (Sieb. Compacta’ pisifera (Sieb. Compressa’ pisifera (Sieb. pisifera (Sieb. pisifera (Sieb. pisifera (Sieb. Intermedia’ pisifera (Sieb. Minima’ ‘Repens’ ‘Rigid Dwarf’ ‘Sanderi’ ‘Spiralis’ ‘Stoneham’ ‘Tempelhof ‘Tetragona ‘Tonia’ ‘Verdonii’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. ‘Aurea ‘Boulevard’ ‘Compacta’ ‘Filifera’ ‘Filifera ‘Gold ‘Golden & Zucc.) Endl. ‘Juniperoides Aurea’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. thyoides (L.) BSP. thyoides (L.) BSP. ‘Andelyensis’ thyoides (L.) BSP. ‘Ericoides’ CHAMAECYTISUS Link FABACEAE (Faboideae) supinus (L.) Link CHAMAEDAPHNE ERICACEAE Moench ‘Monstrosa’ ‘Plumosa’ ‘Plumosa ‘Plumosa ‘Plumosa ‘Plumosa ‘Pygmaea’ ‘Snow’ ‘Squarrosa’ ‘Squarrosa ‘Squarrosa Bean Family Heath Family btusa (Sieb . & Zucc.) Endl. ‘Tonia’ pisifera (Sieb. pisifera (Sieb. & Zucc.) Endl. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. ‘Lycopodioides’ obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. Argentea’ obtusa (Sieb. obtusa (Sieb. Gracilis’ obtusa (Sieb. & Zucc.) Endl. ‘Lycopodioides’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. Pyramidalis’ obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. Aurescens’ obtusa (Sieb. & Zucc.) Endl. ‘Mariesii’ ‘Nana’ ‘Nana ‘Nana Aurea’ ‘Nana ‘Nana ‘Pygmaea’ ‘Pygmaea ‘Reis Dwarf’ obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. Argentea’ obtusa (Sieb. obtusa (Sieb. Gracilis’ obtusa (Sieb. ‘Lycopodioides’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. Pyramidalis’ obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. Aurescens’ obtusa (Sieb. & Zucc.) Endl. ‘Mariesii’ ‘Nana’ ‘Nana ‘Nana Aurea’ ‘Nana ‘Nana ‘Pygmaea’ ‘Pygmaea ‘Reis Dwarf’ obtusa (Sieb. obtusa (Sieb. obtusa (Sieb. Argentea’ obtusa (Sieb. obtusa (Sieb. Gracilis’ obtusa (Sieb. ‘Lycopodioides’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. Pyramidalis’ obtusa (Sieb. & Zucc.) Endl. obtusa (Sieb. & Zucc.) Endl. Aurescens’ obtusa (Sieb. & Zucc.) Endl. ‘Mariesii’ ‘Nana’ ‘Nana ‘Nana Aurea’ ‘Nana ‘Nana ‘Pygmaea’ ‘Pygmaea ‘Reis Dwarf’ obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb obtusa (Sieb Aurea’ obtusa (Sieb obtusa (Sieb . & Zuce. . & Zuce. . & Zucc.) Endl. . & Zucc.) Endl. ) Endl. . & Zucc.) Endl. ) Endl. . & Zucc.) Endl. . & Zucc.) Endl. . & Zucc.) Endl. . & Zucc.) Endl. pisifera (Sieb. pisifera (Sieb. Nana’ pistfera (Sieb. pistfera (Sieb. pisifera (Sieb. pistfera (Sieb. Aurea’ pisifera (Sieb. Spangle’ pistfera (Sieb. Mop’ pisifera (Sieb. pisifera (Sieb. pistfera (Sieb. pistfera (Sieb. Aurea’ pisifera (Sieb. Aurea Nana’ pisifera (Sieb. Compacta’ pisifera (Sieb. Compressa’ pisifera (Sieb. pisifera (Sieb. pisifera (Sieb. pisifera (Sieb. Intermedia’ pisifera (Sieb. Minima’ ‘Repens’ ‘Rigid Dwarf’ ‘Sanderi’ ‘Spiralis’ ‘Stoneham’ ‘Tempelhof ‘Tetragona ‘Tonia’ ‘Verdonii’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. ‘Aurea ‘Boulevard’ ‘Compacta’ ‘Filifera’ ‘Filifera ‘Gold ‘Golden & Zucc.) Endl. ‘Juniperoides Aurea’ & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. & Zucc.) Endl. thyoides (L.) BSP. thyoides (L.) BSP. ‘Andelyensis’ thyoides (L.) BSP. Nana’ pistfera pistfera (Sieb. pistfera (Sieb. & Zucc.) Endl. & Zucc.) Endl. ‘Boulevard’ ‘Compacta’ pistfera (Sieb. pisifera (Sieb. & Zucc.) Endl. & Zucc.) Endl. ‘Compacta’ ‘Filifera’ pisifera (Sieb. pistfera (Sieb. & Zucc.) Endl. & Zucc.) Endl. ‘Filifera’ ‘Filifera pisifera (Sieb. & Zucc.) Endl. ‘Juniperoides Aurea’ pisifera (Sieb. ‘Juniperoides Aurea’ & Zucc.) Intermedia’ pisifera (Sieb. 261 CITHAREXYLUM L. VERBENACEAE Verbena Family berlandieri Robinson x CITROFORTUNELLA J.Ingram & H.E.Moore RUTACEAE Citrus Family mitis (Blanco) J.Ingram & H.E.Moore XCITRONCIRUS J.Ingram & H.E.Moore RUTACEAE Citrus Family webberi J.Ingram & H.E.Moore CITRUS L. RUTACEAE Citrus Family aurantium L. hystrix DC. medica L. ‘Etrog’ x paradisi Macf. reticulata Blanco ‘Thomasville Citrangequat’ CLADRASTIS kaf. FABACEAE (Faboideae) Bean Family kentukea (Dum.-Cours.) Rudd CLEMATIS L. RANUNCULACEAE Buttercup Family armandii Franch. armandii Franch. ‘Farquhariana’ chrysocoma Franch. var. sericea (Franch.) Schneid. crispa L. <jackmanii T.Moore xjackmaniti T.Moore ‘Superba’ montana Buch.-Ham. ‘Grandiflora’ pitcheriT. & G. stans Sieb. & Zucc. terniflora DC. texensis Buckl. < triternata DC. ‘Rubro-marginata’ versicolor Britton viorna L. * viticella L. ‘Betty Corning’ microphyllus Wall. ex Lindl. var. cochleatus (Franch.) Rehd. & Wils. nanshan Mottet nitens Rehd. & Wils. nummularius Fischer & C.A.Mey. obscurus Rehd. & Wils. racemiflorus (Desf.) K.Koch racemiflorus (Desf.) K.Koch var. soongoricus (Regel & Herd.) Schneid. roseus Edgew. salicifolius Franch. salicifolius Franch. ‘Parkteppich’ salicifolius Franch. ‘Scarlet Leader’ silvestrii Pamp. splendens Flinck & Hylmo sternianus (Turrill) Boom <x watereri Exell <x watereri Exell ‘Cornubia’ wilsonii Nakai zabelti Schneid. microphyllus Wall. ex Lindl. var. cochleatus (Franch.) Rehd. & Wils. nanshan Mottet nitens Rehd. & Wils. nummularius Fischer & C.A.Mey. obscurus Rehd. & Wils. racemiflorus (Desf.) K.Koch racemiflorus (Desf.) K.Koch var. soongoricus (Regel & Herd.) Schneid. roseus Edgew. salicifolius Franch. salicifolius Franch. ‘Parkteppich’ salicifolius Franch. ‘Scarlet Leader’ silvestrii Pamp. splendens Flinck & Hylmo sternianus (Turrill) Boom <x watereri Exell <x watereri Exell ‘Cornubia’ wilsonii Nakai zabelti Schneid. avellana L. avellana L. ‘Contorta’ avellana L. ‘Cosford’ avellana L. ‘Fusco-rubra’ chinensis Franch. colurna L. cornuta Marsh. maxima Mill. ‘Purpurea’ sieboldiana BI. sieboldiana Bl. var. mandschurica (Maxim. & Rupr.) Schneid. avellana L. avellana L. ‘Contorta’ avellana L. ‘Cosford’ avellana L. ‘Fusco-rubra’ chinensis Franch. colurna L. cornuta Marsh. maxima Mill. ‘Purpurea’ sieboldiana BI. sieboldiana Bl. var. mandschurica (Maxim. & Rupr.) Schneid. COTINUS Mill. Smoke Tree ANACARDIACEAE Cashew Family coggygria Scop. coggygria Scop. ‘Baby Doll’ coggygria Scop. ‘Flame’ coggygria Scop. ‘Nordine Red’ coggygria Scop. ‘Purpureus’ coggygria Scop. ‘Royal Purple’ coggygria Scop. ‘Rubrifolius’ coggygria Scop. ‘Velvet Cloak’ -obovatus Raf. Rose Family CLERODENDRUM lL. VERBENACEAE & Wils.) Flinck & Hylmo foveolatus Rehd. & Wils. franchetii Bois horizontalis Decne. horizontalis Decne. ‘Variegata’ horizontalis Decne. var. perpusillus Schneid. ignavus E.Wolf integerrimus Medik. lacteus W.W.Sm. lucidus Schlechtend. microphyllus Wall. ex Lindl. microphyllus Wall. ex Lindl. f. thymifolius (Lindl.) Koehne CLERODENDRUM lL. VERBENACEAE Verbena Family White Alder White-alder Family CLETHRA lL. CLETHRACEAE White Alder White-alder Family acuminata Michx. alnifolia L. alnifolia L. ‘Rosea’ barbinervis Sieb. & Zucc. fargesii Franch. tomentosa Lam. CLEYERA Thunb. THEACEAE Tea Family japonica Thunb. japonica Thunb. ‘Tricolor’ stans Sieb. & Zucc. terniflora DC. terniflora DC. texensis Buckl. we) COMPTONIA Liter. ex Ait. MYRICACEAE Wax-myrtle Family peregrina (L.) Coult. CONDALIA Cav. RHAMNACEAE Buckthorn Family hookeri M.C.Johnst. CONRADINA A.Gr. LAMIACEAE Mint Family canescens (T. & G.) A.Gr. glabra Shinners grandiflora Small verticillata Jennison CORDIA L. BORAGINACEAE Borage Family boissieri A.DC. CORIARIA L. CORIARIACEAE Coriaria Family japonica A.Gr. CORNUS L. Dogwood CORNACEAE Dogwood Family alba L. alba L. ‘Argenteo-marginata’ alba L. ‘Kesselringii’ alba L. ‘Sibirica’ alternifolia L.f. amomum Mill. asperifolia Michx. australis C.A.Mey. var. koenigii (Schneid.) Wang. controversa Hemsl. coreana Wang. drummondii C.A.Mey. florida L. florida L. ‘Bay Beauty’ florida L. ‘Cherokee Chief’ florida L. ‘Cherokee Princess’ florida L. ‘Cherokee Sunset’ florida L. ‘Cloud Nine’ florida L. ‘Dwarf florida L. ‘First Lady’ florida L. ‘Fragrant Cloud’ florida L. ‘Prosser Red’ florida L. ‘Rainbow’ mas L. ‘Macrocarpa’ mas L. ‘Nana’ mas L. ‘Nana’ mas L. ‘Variega mas L. ‘Variegata’ nuttallii Audubon nuttallii Audubon obliqua Raf. obliqua Raf. officinalis Si glabrescens Franch. & Sav. pauciflora Sieb. & Zucc. sinensis Hemsl. ‘Winterthur’ sinensis Hemsl. var. calvescens Rehd. & Wils. sinensis Hemsl. var. calvescens Rehd. & Wils. f. veitchiana (Bean) Morley & Chao sinensis Hemsl. var. sinensis sinensis Hemsl. var. sinensis ‘Spring Purple’ spicata Sieb. & Zucc. Hazelnut, Filbert Birch Family CORYLUS L. BETULACEAE americana Marsh. americana Walt. ‘Rush’ (americana < C. avellana) ‘Potomac’ (americana X C. avellana) ‘Reed’ florida L. ‘Royal Red’ florida L. ‘Salicifolia’ florida L. ‘Salicifolia’ florida L. ‘Springtime’ florida L. ‘Springtime’ 263 avellana L. avellana L. ‘Contorta’ avellana L. ‘Cosford’ avellana L. ‘Fusco-rubra’ chinensis Franch. colurna L. cornuta Marsh. maxima Mill. ‘Purpurea’ sieboldiana BI. sieboldiana Bl. var. mandschurica (Maxim. & Rupr.) Schneid. COTINUS Mill. Smoke Tree ANACARDIACEAE Cashew Family coggygria Scop. coggygria Scop. ‘Baby Doll’ coggygria Scop. ‘Flame’ coggygria Scop. ‘Nordine Red’ coggygria Scop. ‘Purpureus’ coggygria Scop. ‘Royal Purple’ coggygria Scop. ‘Rubrifolius’ coggygria Scop. ‘Velvet Cloak’ -obovatus Raf. COTONEASTER Medik. ROSACEAE Rose Family acutifolius Turcz. adpressus Bois adpressus Bois ‘Hessei’ ambiguus Rehd. & Wils. apiculatus Rehd. & Wils. congestus Baker conspicuus Marq. conspicuus Marq. ‘Decorus’ dammeri Schneid. dammeri Schneid. ‘Lowfast’ dammeri Schneid. ‘Royal Beauty’ dammeri Schneid. ‘Skogsholmen’ dielsianus Pritz. ex Diels divaricatus Rehd. & Wils. floccosus (Rehd. CRATAEGUS L. ROSACEAE & Zucc. gracilis Sieb. & Zucc. ‘Mohican’ gracilis Sieb. & Zucc. ‘Nikko’ < hybrida E.Lemoine ‘Contraste’ < hybrida E.Lemoine ‘Magicien’ x lemoinei E.Lemoine ex Bois x magnifica (Lem.) Rehd. < magnifica (Lem.) Rehd. ‘Eburnea’ x magnifica (Lem.) Rehd. ‘Eminens < magnifica (Lem.) Rehd. ‘Erecta’ <x magnifica (Lem.) Rehd. ‘Formosa’ x magnifica (Lem.) Rehd. ‘Latiflora’ < rosea (Lem.) Rehd. < rosea (Lem.) Rehd. ‘Carminea’ scabra Thunb. scabra Thunb. ‘Candidissima’ scabra Thunb. ‘Plena’ scabra Thunb. ‘Pride of Rochester’ scabra Thunb. ‘Summer Snow’ scabra Thunb. ‘Suspensa’ scabra Thunb. ‘Watereri’ schneideriana Rehd. sieboldiana Maxim. DIERVILLA Mill. CAPRIFOLIACEAE Bush Honeysuckle Honeysuckle Family lonicera Mill. rivularis Gatt. x splendens (Carr.) Kirchn. DIOON Lindl. CYCADACEAE Cycad Family edule Lindl. DENDROPANAX Decne. & Planch. ARALIACEAE Ginseng Family trifidus (Thunb. ex J.A.Murr.) Makino ex Hara DEUTZIA Thunb. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family <x candelabrum (Lem.) Rehd. <x candida (Lem.) Rehd. chunti H.H.Hu discolor Hemsl. x elegantissima (Lem.) Rehd. x elegantissima (Lem.) Rehd. ‘Conspicua’ glabrata Komar. gracilis Sieb. & Zucc. gracilis Sieb. & Zucc. ‘Mohican’ gracilis Sieb. & Zucc. ‘Nikko’ < hybrida E.Lemoine ‘Contraste’ < hybrida E.Lemoine ‘Magicien’ x lemoinei E.Lemoine ex Bois x magnifica (Lem.) Rehd. < magnifica (Lem.) Rehd. ‘Eburnea’ x magnifica (Lem.) Rehd. ‘Eminens < magnifica (Lem.) Rehd. ‘Erecta’ <x magnifica (Lem.) Rehd. ‘Formosa’ x magnifica (Lem.) Rehd. ‘Latiflora’ < rosea (Lem.) Rehd. < rosea (Lem.) Rehd. ‘Carminea’ scabra Thunb. scabra Thunb. ‘Candidissima’ scabra Thunb. ‘Plena’ scabra Thunb. ‘Pride of Rochester’ scabra Thunb. ‘Summer Snow’ scabra Thunb. ‘Suspensa’ scabra Thunb. ‘Watereri’ schneideriana Rehd. sieboldiana Maxim. DIERVILLA Mill. CAPRIFOLIACEAE Bush Honeysuckle Honeysuckle Family lonicera Mill. rivularis Gatt. x splendens (Carr.) Kirchn. DALBERGIA L.f. FABACEAE (Faboideae) Bean Family hupeana Hance sissoo Roxb. ex DC. DANAE Medik. LILIACEAE Lily Family racemosa (L.) Moench DAPHNE lL. THYMELAEACEAE Mezereum Family < burkwoodii Turrill ‘Carol Mackie’ < burkwoodii Turrill ‘Somerset’ caucasica Pall. cneorum L. cneorum L. ‘Ruby Glow’ genkwa Sieb. & Zucc. kamtschatica Maxim. var. jezoensis (Maxim.) Ohwi <x mantensiana T.M.C.Taylor & F. Vrugtman ‘Manten’ mezereum L. odora Thunb. ex J.A.Murr. odora Thunb. ex J.A.Murr. ‘Alba’ odora Thunb. ex J.A.Murr. ‘Albo-marginata’ odora Thunb. ex J.A.Murr. ‘Aureo-marginata’ odora Thunb. ex J.A.Murr. ‘Ringmaster’ odora Thunb. ex J.A.Murr. ‘Zuiko nishiki’ tangutica Maxim. DAPHNIPHYLLUM Bl. EUPHORBIACEAE Spurge Family macropodum Miq. DALBERGIA L.f. FABACEAE (Faboideae) Bean Family hupeana Hance sissoo Roxb. ex DC. DANAE Medik. LILIACEAE Lily Family racemosa (L.) Moench DAPHNE lL. THYMELAEACEAE Mezereum Family < burkwoodii Turrill ‘Carol Mackie’ < burkwoodii Turrill ‘Somerset’ caucasica Pall. cneorum L. CRATAEGUS L. ROSACEAE aestivalis (Walt.) T. & G. arnoldiana Sarg. arnoldiana Sarg. brachyacantha Sa calpodendron (Ehrh.) Medik. coccinioides Ashe coccinioides Ashe compta Sarg. floridana Sarg. fulleriana Sarg. fulleriana Sarg. intricata J.Lange floccosus (Rehd. & Wils.) Flinck & Hylmo foveolatus Rehd. & Wils. aevigata (Poir.) DC. ‘Autumn Glory’ aevigata (Poir.) DC. ‘Paul’s Scarlet’ aevigata (Poir.) DC. ‘Paul’s Scarlet’ aevigata (Poir.) DC. ‘Plena’ laevigata (Poir.) DC. ‘Plena’ laevigata (Poir.) DC. ‘Superba x lavallei Herincq ex Lav. < lavallei Herincq ex Lav. < lavallei Herincq ex Lav. ‘Carrierei’ macrosperma Ashe macrosperma Ashe marshallii Egglest. marshallii Egglest. mollis (T. & G.) Sche mollis (T. & G.) Scheele monogyna Jacq. monogyna Jacq. monogyna Jacq. monogyna Jacq. ‘Inermis’ monogyna Jacq. ‘Praecox’ monogyna Jacq. ‘ monogyna Jacq. ‘ o os CUNNINGHAMIA R.Br. TAXODIACEAE China Fir ~laxodium Family lanceolata (Lamb.) Hook.f. lanceolata (Lamb.) Hook.f. ‘Glauca’ CUPHEA P.Br. LYTHRACEAE Loosestrife Family hyssoptfolia HBK. micropetala HBK. Xx CUPRESSOCYPARIS Dallim. CUPRESSACEAE Cypress Family leylandii (Dallim. & A.B.Jacks.) Dallim. leylandii (Dallim. & A.B.Jacks.) Dallim. ‘Leighton Green’ leylandii (Dallim. & A.B.Jacks.) Dallim. ‘Silver Dust’ CUPRESSUS L. CUPRESSACEAE Cypress Cypress Family arizonica Greene arizonica Greene ‘Gareei’ lusitanica Mill. macrocarpa Hartw. sempervirens L. CYCAS L. CYCADACEAE Cycad Family circinalis L. revoluta Thunb. CYDONIA Miill. Quince ROSACEAE Rose Family oblonga Mill. CYRILLA Gard. ex L. CYRILLACEAE Cyrilla Family racemiflora L. CYTISUS Desf. Broom FABACEAE (Faboideae) Bean Family battandieri Maire commutatus (Willd.) Brig. decumbens (Durande) Spach X praecox Bean X praecox Bean ‘Hollandia’ CUNNINGHAMIA R.Br. TAXODIACEAE China Fir ~laxodium Family lanceolata (Lamb.) Hook.f. lanceolata (Lamb.) Hook.f. ‘Glauca’ monogyna Jacq. ‘Stricta’ x mordenensis Boom ‘Toba’ opaca Hook. & Arn. pennsylvanica Ashe phaenopyrum (L.f.) Medik. pinnatifida Bunge populifolia Walt. pringlei Sarg. pruinosa (H.Wendl.) K.Koch var. leiophylla (Sarg.) Phipps punctata Jacq. punctata Jacq. ‘Inermis’ schuettei Ashe spathulata Michx. uniflora Moench vaileae Britton viridis L. viridis L. ‘Winter King’ monogyna Jacq. ‘Stricta’ x mordenensis Boom ‘Toba’ opaca Hook. & Arn. pennsylvanica Ashe phaenopyrum (L.f.) Medik. pinnatifida Bunge populifolia Walt. pringlei Sarg. pruinosa (H.Wendl.) K.Koch var. leiophylla (Sarg.) Phipps punctata Jacq. punctata Jacq. ‘Inermis’ schuettei Ashe spathulata Michx. uniflora Moench vaileae Britton viridis L. viridis L. ‘Winter King’ CUDRANIA Trecul MORACEAE Mulberry Family tricuspidata (Carr.) Bur. ex Lav. 265 DENDROPANAX Decne. & Planch. ARALIACEAE Ginseng Family trifidus (Thunb. ex J.A.Murr.) Makino ex Hara DEUTZIA Thunb. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family <x candelabrum (Lem.) Rehd. <x candida (Lem.) Rehd. chunti H.H.Hu discolor Hemsl. x elegantissima (Lem.) Rehd. x elegantissima (Lem.) Rehd. ‘Conspicua’ glabrata Komar. gracilis Sieb. CRATAEGUS L. ROSACEAE cneorum L. ‘Ruby Glow’ genkwa Sieb. & Zucc. kamtschatica Maxim. var. jezoensis (Maxim.) Ohwi <x mantensiana T.M.C.Taylor & F. Vrugtman ‘Manten’ mezereum L. odora Thunb. ex J.A.Murr. odora Thunb. ex J.A.Murr. ‘Alba’ odora Thunb. ex J.A.Murr. ‘Albo-marginata’ odora Thunb. ex J.A.Murr. ‘Aureo-marginata’ odora Thunb. ex J.A.Murr. ‘Ringmaster’ odora Thunb. ex J.A.Murr. ‘Zuiko nishiki’ tangutica Maxim. DAPHNIPHYLLUM Bl. EUPHORBIACEAE Spurge Family macropodum Miq. DAVIDIA Baill. NYSSACEAE Sour-gum Family Bean Family Lily Family Bean Family hupeana Hance sissoo Roxb. ex DC. DANAE Medik. LILIACEAE Lily Family racemosa (L.) Moench DAPHNE lL. THYMELAEACEAE Mezereum Family < burkwoodii Turrill ‘Carol Mackie’ < burkwoodii Turrill ‘Somerset’ caucasica Pall. cneorum L. cneorum L. ‘Ruby Glow’ genkwa Sieb. & Zucc. kamtschatica Maxim. var. jezoensis (Maxim.) Ohwi <x mantensiana T.M.C.Taylor & F. Vrugtman ‘Manten’ mezereum L. odora Thunb. ex J.A.Murr. odora Thunb. ex J.A.Murr. ‘Alba’ odora Thunb. ex J.A.Murr. ‘Albo-marginata’ odora Thunb. ex J.A.Murr. ‘Aureo-marginata’ odora Thunb. ex J.A.Murr. ‘Ringmaster’ odora Thunb. ex J.A.Murr. ‘Zuiko nishiki’ tangutica Maxim. DAPHNIPHYLLUM Bl. EUPHORBIACEAE Spurge Family macropodum Miq. DAVIDIA Baill. NYSSACEAE Sour-gum Family involucrata Baill. var. vilmoriniana (Dode) Wang. DECUMARIA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family barbara L. <x candida (Lem.) Rehd. chunti H.H.Hu <x magnifica (Lem.) Rehd. ‘Formosa’ x magnifica (Lem.) Rehd. ‘Latiflora’ x magnifica (Lem.) Rehd. ‘Latiflora’ < rosea (Lem.) Rehd. < rosea (Lem.) Rehd. < rosea (Lem.) Rehd. Cycad Family pungens Thunb. ex J.A.Murr. pungens Thunb. ex J.A.Murr. ‘Aurea’ pungens Thunb. ex J.A.Murr. ‘Fruitlandii’ pungens Thunb. ex J.A.Murr. ‘Maculata’ pungens Thunb. ex J.A.Murr. ‘Variegata’ umbellata Thunb. ex J.A.Murr. ELEUTHEROCOCCUS Maxim. ARALIACEAE Ginseng Family gracilistylus (W.W.Sm.) S.Y.Hu sieboldianus (Makino) Koidz. trifoliatus (L.) S.Y.Hu ELLIOTTIA Muhl. ex Ell. ERICACEAE Heath Family bracteata (Maxim.) Hook.f. racemosa Muhl. ex Ell. ENKIANTHUS Lour. ERICACEAE Heath Family campanulatus (Miq.) Nichols. campanulatus (Miq.) Nichols. ‘Ambassador’ perulatus (Miq.) Schneid. ERICA L. Heath ERICACEAE Heath Family carnea L. carnea L. ‘Springwood Pink’ carnea L. ‘Springwood White’ x darleyensis Bean ‘Silberschmelze’ vagans L. ERIOBOTRYA Lindl. ROSACEAE Loquat Rose Family deflexa (Hemsl.) Nakai japonica (Thunb.) Lindl. ERYTHRINA L. FABACEAE (Faboideae) Bean Family x bidwillii Lindl. crista-galli L. herbacea L. ESCALLONIA Mutis ex Lf. SAXIFRAGACEAE (Escallonioideae) Saxifrage Family Persimmon Ebony Family DIOSPYROS L. EBENACEAE kaki L.f. kaki L.f. ‘Hagabushi’ kaki L.f. ‘Kyungsun bansi’ sinensis Hemsl. texana Scheele virginiana L. DIPELTA Maxim. CAPRIFOLIACEAE Honeysuckle Family floribunda Maxim. yunnanensis Franch. DIRCA L. THYMELAEACEAE Mezereum Family palustris L. DISANTHUS Maxim. HAMAMELIDACEAE Witch-hazel Family cercidifolius Maxim. DISTYLIUM Sieb. & Zucc. HAMAMELIDACEAE Witch-hazel Family racemosum Sieb. CRATAEGUS L. ROSACEAE & Zucc. racemosum Sieb. & Zucc. ‘Akebono’ DURANTA L. VERBENACEAE Verbena Family repens L. EDGEWORTHIA Meisn. THYMELAEACEAE Mezereum Family chrysantha Lindl. EHRETIA P.Br. BORAGINACEAE Borage Family acuminata R.Br. var. serrata (Roxb.) I.M.Johnst. anacua (Teran & Berl.) I.M.Johnst. ELAEAGNUS L. ELAEAGNACEAE Oleaster Family angustifolia L. x ebbingei Doorenbos glabra Thunb. ex J.A.Murr. macrophylla Thunb. Persimmon Ebony Family DIOSPYROS L. EBENACEAE kaki L.f. kaki L.f. ‘Hagabushi’ kaki L.f. ‘Kyungsun bansi’ sinensis Hemsl. texana Scheele virginiana L. 267 kiautschovicus Loesn. kiautschovicus Loesn. ‘Manhattan’ lanceolatus Yatabe maackii Rupr. nanus Bieb. sieboldianus Bl. wilsonii Sprague EUPHORBIA lL. EUPHORBIACEAE Spurge Spurge Family characias L. ssp. wulfenii (Hoppe ex K.Koch) A.R.Sm. leucocephala Lotsy pulcherrima Willd. EUPTELEA Sieb. & Zucc. EUPTELEACEAE Euptelea Family polyandra Sieb. & Zucc. EURYA Thunb. THEACEAE Tea Family acuminata DC. emarginata (Thunb. ex J.A.Murr.) Makino japonica Thunb. japonica Thunb. ‘Confetti’ japonica Thunb. ‘Harmony’ * japonica Thunb. ‘Winter Wine’ EUSCAPHIS Sieb. & Zucc. STAPHYLEACEAE Bladdernut Family japonica (Thunb. ex J.A.Murr.) Kanitz EXOCHORDA Lindl. ROSACEAE Rose Family <x macrantha (V.Lemoine) Schneid. ‘The Bride’ racemosa (Lindl.) Rehd. FAGUS lL. Beech FAGACEAE Beech Family grandifolia Ehrh. sylvatica L. kiautschovicus Loesn. kiautschovicus Loesn. ‘Manhattan’ lanceolatus Yatabe maackii Rupr. nanus Bieb. sieboldianus Bl. wilsonii Sprague EUONYMUS L. CELASTRACEAE alatus (Thunb. ex J.A.Murr.) Sieb. alatus (Thunb. ex J.A.Murr.) Sieb. ‘Compactus’ EUCALYPTUS ULHer. MYRTACEAE Myrtle Family Myrtle Family camaldulensis Dehnh. coccifera Hook.f. dalrympleana Maiden gunnii Hook.f. robusta Sm. viminalis Labill. wilsonii Sprague EUPHORBIA lL. EUPHORBIACEAE Spurge Spurge Family characias L. ssp. wulfenii (Hoppe ex K.Koch) A.R.Sm. leucocephala Lotsy pulcherrima Willd. EUPTELEA Sieb. & Zucc. EUPTELEACEAE Euptelea Family polyandra Sieb. & Zucc. EURYA Thunb. THEACEAE Tea Family acuminata DC. emarginata (Thunb. ex J.A.Murr.) Makino japonica Thunb. japonica Thunb. ‘Confetti’ japonica Thunb. ‘Harmony’ * japonica Thunb. ‘Winter Wine’ EUSCAPHIS Sieb. & Zucc. STAPHYLEACEAE Bladdernut Family japonica (Thunb. ex J.A.Murr.) Kanitz EXOCHORDA Lindl. ROSACEAE Rose Family <x macrantha (V.Lemoine) Schneid. ‘The Bride’ racemosa (Lindl.) Rehd. FAGUS lL. Beech FAGACEAE Beech Family grandifolia Ehrh. sylvatica L. sylvatica L. ‘Asplenifolia’ sylvatica L. ‘Cristata’ sylvatica L. ‘Dawyck’ sylvatica L. ‘Laciniata’ sylvatica L. ‘Pendula’ sylvatica L. ‘Purpurea Tricolor’ sylvatica L. ‘Quercifolia’ sylvatica L. ‘Rohanii’ ‘Compactus’ fortunei (Turcz.) Hand.-Mazz. fortunei (Turcz.) Hand.-Mazz. EUCOMMIA Oliv. EUCOMMIACEAE EUODIA J.R. &J.G. Forst. RUTACEAE Citrus Family daniellii (Benn.) Hemsl. EUONYMUS L. CELASTRACEAE Spindle Tree Staff-tree Family alatus (Thunb. ex J.A.Murr.) Sieb. alatus (Thunb. ex J.A.Murr.) Sieb. ‘Compactus’ alatus (Thunb. ex J.A.Murr.) Sieb. f. apterus (Regel) Rehd. americanus L. bungeanus Maxim. bungeanus Maxim. var. semipersistans (Rehd.) Schneid. europaeus L. europaeus L. ‘Aldenhamensis’ fortunei (Turcz.) Hand.-Mazz. fortunei (Turcz.) Hand.-Mazz. ‘Emerald Gaiety’ fortunei (Turcz.) Hand.-Mazz. ‘Gracilis’ fortunei (Turcz.) Hand.-Mazz. ‘Harlequin’ fortunei (Turcz.) Hand.-Mazz. ‘Longwood’ fortunei (Turcz.) Hand.-Mazz. ‘Minima’ fortunei (Turcz.) Hand.-Mazz. ‘Sarcoxie’ fortunei (Turcz.) Hand.-Mazz. ‘Silver Queen’ fortunei (Turcz.) Hand.-Mazz. ‘Variegatus’ fortunei (Turcz.) Hand.-Mazz. var. radicans (Miq.) Rehd. japonicus Thunb. japonicus Thunb. ‘Albo-marginatus’ japonicus Thunb. ‘Argenteo-variegatus’ japonicus Thunb. ‘Aureo-marginatus’ japonicus Thunb. ‘Aureus’ japonicus Thunb. ‘Duc d’Anjow’ japonicus Thunb. ‘Macrophyllus’ japonicus Thunb. ‘Microphyllus’ japonicus Thunb. ‘Silver King’ daniellii (Benn.) Hemsl. ‘Emerald Gaiety’ fortunei (Turcz.) Ha 268 sylvatica L. ‘Rotundifolia’ sylvatica L. ‘Zlatia’ sylvatica L. f. purpurea (Ait.) Schneid. FALLUGIA Endl. ROSACEAE Rose Family paradoxa (D.Don) Endl. XFATSHEDERA Guill. ARALIACEAE Ginseng Family lizei (Cochet) Guill. FATSIA Decne. & Planch. ARALIACEAE Ginseng Family japonica (Thunb.) Decne. & Planch. FICUS L. Fig MORACEAE Mulberry Family carica L. carica X F. pumila palmata Forssk. pumila L. pumila L. ‘Minima’ tikoua Bur. FIRMIANA Marsili STERCULIACEAE Sterculia Family simplex (L.) W.F.Wight FONTANESIA Labill. OLEACEAE Olive Family fortunei Carr. FORESTIERA Poir. OLEACEAE Olive Family acuminata (Michx.) Poir. FORSYTHIA Vahl OLEACEAE Golden Bells Olive Family x intermedia Zab. x intermedia Zab. ‘Karl Sax’ X intermedia Zab. ‘Lynwood’ Xintermedia Zab. ‘New Hampshire Gold’ x intermedia Zab. ‘Spectabilis’ x intermedia Zab. ‘Spring Glory’ x intermedia Zab. ‘Variegata’ koreana Nakai ‘Ilgwang’ ovata (hybrid) ‘Ottawa’ (ovata x F. <intermedia ‘Spring Glory’) ‘Winterthur’ suspensa (Thunb.) Vahl suspensa (Thunb.) Vahl ‘Pallida’ suspensa (Thunb.) Vahl var. fortunei (Lindl.) Rehd. viridissima Lindl. viridissima Lindl. ‘Bronxensis’ FORTUNEARIA Rehd. & Wils. HAMAMELIDACEAE Witch-hazel Family sinensis Rehd. & Wils. FORTUNELLA Swingle RUTACEAE Kumquat Citrus Family x crassifolia Swingle japonica (Thunb.) Swingle FOTHERGILLA L. HAMAMELIDACEAE Witch-hazel Family gardenii J.A.Murr. major (Sims) Lodd. major < F. gardenii FRANGULA Mill. RHAMNACEAE Buckthorn Family alnus Mill. FRANKLINIA Marsh. THEACEAE Tea Family alatamaha Marsh. FRAXINUS L. Ash OLEACEAE Olive Family americana L. americana L. var. biltmoreana (Beadle) J.Wright berlandieriana A.DC. chinensis Roxb. var. rhynchophylla (Hance) Hemsl. excelsior L. excelsior L. ‘Aurea’ excelsior L. ‘Doorenbos #5’ holotricha Koehne longicuspis Sieb. & Zucc. nigra Marsh. ornus L. ornus L. < F. excelsior L. sylvatica L. ‘Rotundifolia’ sylvatica L. ‘Zlatia’ sylvatica L. f. purpurea (Ait.) Schneid. FALLUGIA Endl. ROSACEAE Rose Family paradoxa (D.Don) Endl. XFATSHEDERA Guill. ARALIACEAE Ginseng Family lizei (Cochet) Guill. FATSIA Decne. & Planch. ARALIACEAE Ginseng Family japonica (Thunb.) Decne. & Planch. FICUS L. Fig MORACEAE Mulberry Family carica L. carica X F. pumila palmata Forssk. pumila L. pumila L. ‘Minima’ tikoua Bur. FIRMIANA Marsili STERCULIACEAE Sterculia Family simplex (L.) W.F.Wight FONTANESIA Labill. OLEACEAE Olive Family fortunei Carr. FORESTIERA Poir. OLEACEAE Olive Family acuminata (Michx.) Poir. FORSYTHIA Vahl OLEACEAE Golden Bells Olive Family x intermedia Zab. x intermedia Zab. ‘Karl Sax’ X intermedia Zab. ‘Lynwood’ Xintermedia Zab. ‘New Hampshire Gold’ x intermedia Zab. ‘Spectabilis’ x intermedia Zab. ‘Spring Glory’ x intermedia Zab. ‘Variegata’ ovata (hybrid) ‘Ottawa’ (ovata x F. <intermedia ‘Spring Glory’) ‘Winterthur’ suspensa (Thunb.) Vahl suspensa (Thunb.) Vahl ‘Pallida’ suspensa (Thunb.) Vahl var. fortunei (Lindl.) Rehd. viridissima Lindl. ‘Emerald Gaiety’ fortunei (Turcz.) Ha viridissima Lindl. ‘Bronxensis’ ovata (hybrid) ‘Ottawa’ (ovata x F. <intermedia ‘Spring Glory’) ‘Winterthur’ suspensa (Thunb.) Vahl suspensa (Thunb.) Vahl ‘Pallida’ suspensa (Thunb.) Vahl var. fortunei (Lindl.) Rehd. viridissima Lindl. viridissima Lindl. ‘Bronxensis’ FORTUNEARIA Rehd. & Wils. HAMAMELIDACEAE Witch-hazel Family sinensis Rehd. & Wils. FORTUNELLA Swingle RUTACEAE Kumquat Citrus Family x crassifolia Swingle japonica (Thunb.) Swingle FOTHERGILLA L. HAMAMELIDACEAE Witch-hazel Family gardenii J.A.Murr. major (Sims) Lodd. major < F. gardenii FRANGULA Mill. RHAMNACEAE Buckthorn Family alnus Mill. FRANKLINIA Marsh. THEACEAE Tea Family alatamaha Marsh. FRAXINUS L. Ash OLEACEAE Olive Family americana L. americana L. var. biltmoreana (Beadle) J.Wright berlandieriana A.DC. chinensis Roxb. var. rhynchophylla (Hance) Hemsl. excelsior L. excelsior L. ‘Aurea’ excelsior L. ‘Doorenbos #5’ holotricha Koehne longicuspis Sieb. & Zucc. nigra Marsh. x intermedia Zab. ‘Spring Glory’ x intermedia Zab. ‘Variegata’ x intermedia Zab. ‘Variegata’ koreana Nakai ‘Ilgwang’ 269 pennsylvanica Marsh. var. subintegerrima (Vahl) Fern. sieboldiana Bl. velutina Torr. var. coriacea (S.Wats.) Rehd. velutina Torr. var. glabra Rehd. GALPHIMIA Cav. MALPIGHIACEAE Malpighia Family glauca Cav. GARDENIA Ellis RUBIACEAE Madder Family jasminoides Ellis jasminoides Ellis ‘Radicans’ GAULTHERIA lL. ERICACEAE Heath Family procumbens L. GAYLUSSACIA HBK. ERICACEAE Huckleberry Heath Family baccata (Wang.) K.Koch brachycera (Michx.) A.Gr. GELSEMIUM Juss. LOGANIACEAE Logania Family rankinii Small sempervirens (L.) Ait.f. sempervirens (L.) Ait.f. ‘Pride of Augusta’ GENISTA L. FABACEAE (Faboideae) Broom Bean Family germanica L. ‘Prostrata’ lydia Boiss. GINKGO L. GINKGOACEAE Ginkgo Family biloba L. biloba L. ‘Fastigiata’ biloba L. ‘Lakeview’ biloba L. ‘Mayfield’ GLEDITSIA lL. Honeylocust FABACEAE (Caesalpinioideae) Bean Family aquatica Marsh. japonica Miq. triacanthos L. triacanthos L. f. inermts (Pursh) Schneid. triacanthos L. f. inermis (Pursh) Schneid. ‘Shademaster’ triacanthos L. f. inermis (Pursh) Schneid. ‘Sunburst’ GLOCHIDION J.R. &J.G.Forst. EUPHORBIACEAE Spurge Family puberum (L.) Hutch. GLYCOSMIS Correa RUTACEAE Citrus Family citrifolia (Willd.) Lindl. GLYPTOSTROBUS Endl. TAXODIACEAE Taxodium Family lineatus (Poir.) Druce GORDONIA Ellis THEACEAE Tea Family axillaris (Roxb. ex Ker-Gawl.) D.Dietr. chrysandra Cowan lasianthus (L.) Ellis GREWIA L. TILIACEAE Linden Family biloba G.Don GYMNOCLADUS Lam. FABACEAE (Caesalpinioideae) Bean Family dioica (L.) K.Koch HALESIA FE llis ex L. STYRACACEAE Silverbell Tree Storax Family carolina L. carolina L. ‘Rosea’ diptera Ellis diptera Ellis var. magniflora Godfrey parviflora Michx. HALIMODENDRON Fischer ex DC. FABACEAE (Faboideae) Bean Family halodendron (L.) Voss triacanthos L. f. inermts (Pursh) Schneid. triacanthos L. f. inermis (Pursh) Schneid. ‘Shademaster’ triacanthos L. f. inermis (Pursh) Schneid. ‘Sunburst’ GLOCHIDION J.R. &J.G.Forst. EUPHORBIACEAE Spurge Family puberum (L.) Hutch. GLYCOSMIS Correa RUTACEAE Citrus Family citrifolia (Willd.) Lindl. ‘Emerald Gaiety’ fortunei (Turcz.) Ha helix L. helix L. ‘Cascade’ ‘Cathedral Wall’ ‘Cavendishii’ ‘Christian’ ‘Chrysantha’ ‘Cockle Shell’ ‘Conglomerata’ ‘Crenata’ ‘Deltoidea’ ‘Dentata’ ‘Denticulata’ ‘Digitata’ ‘Discolor’ ‘Dragon Claw’ ‘Edison’ ‘Emerald Beauty ‘Emerald Gem’ ‘Emerald Jewel’ ‘Erecta’ ‘Erin’ ‘Eva’ ‘Fan’ ‘Fantasia’ ‘Ferney’ ‘Fleur’ ‘Fleur de Lis’ ‘Four Square’ ‘Garland’ ‘Gavotte’ ‘Ginkgo’ ‘Glacier’ ‘Gladiator’ ‘Glymii’ ‘Goldcraft’ ‘Gold Dust’ ‘Gold Heart’ ‘Goods Selfbranching’ ‘Gracilis’ ‘Green Crown’ ‘Green Finger’ ‘Green Quartz’ ‘Green Ripples’ ‘Green Spear’ ‘Green Velvet’ ‘Hahn Selfbranching’ ‘Hahn Variegated’ ‘Harold’ ‘Harrison’ ‘Hebron’ ‘Helvetica’ ‘Heterophylla’ ‘Hibernica’ ‘Hite Miniature’ ‘Holly’ ‘Ideal’ ‘Imp’ ‘Itsy Bitsy’ ‘Emerald Gaiety’ fortunei (Turcz.) Ha GLYPTOSTROBUS Endl. TAXODIACEAE Taxodium Family lineatus (Poir.) Druce GORDONIA Ellis THEACEAE Tea Family axillaris (Roxb. ex Ker-Gawl.) D.Dietr. chrysandra Cowan lasianthus (L.) Ellis GREWIA L. TILIACEAE Linden Family biloba G.Don GYMNOCLADUS Lam. FABACEAE (Caesalpinioideae) Bean Family dioica (L.) K.Koch HALESIA FE llis ex L. STYRACACEAE Silverbell Tree Storax Family carolina L. carolina L. ‘Rosea’ diptera Ellis diptera Ellis var. magniflora Godfrey parviflora Michx. HALIMODENDRON Fischer ex DC. FABACEAE (Faboideae) Bean Family halodendron (L.) Voss pennsylvanica Marsh. var. subintegerrima (Vahl) Fern. sieboldiana Bl. velutina Torr. var. coriacea (S.Wats.) Rehd. velutina Torr. var. glabra Rehd. pennsylvanica Marsh. var. subintegerrima (Vahl) Fern. sieboldiana Bl. velutina Torr. var. coriacea (S.Wats.) Rehd. velutina Torr. var. glabra Rehd. HAMAMELIS L. Witch Hazel HAMAMELIDACEAE Witch-hazel Family x intermedia Rehd. x intermedia Rehd. ‘Arnold Promise’ x intermedia Rehd. ‘Feuerzauber’ < intermedia Rehd. ‘Jelena’ x intermedia Rehd. ‘Luna’ Xintermedia Rehd. ‘Orange Beauty’ x intermedia Rehd. ‘Pallida’ x intermedia Rehd. ‘Primavera’ x intermedia Rehd. ‘Rubra’ x intermedia Rehd. ‘Ruby Glow’ x intermedia Rehd. ‘Vesna’ japonica Sieb. & Zucc. japonica Sieb. & Zucc. ‘Zuccariniana japonica Sieb. & Zucc. f. flavopurpurascens (Makino) Rehd. mollis Oliv. mollis Oliv. ‘Brevipetala’ vernalis Sarg. vernalis Sarg. ‘Carnea’ vernalis Sarg. ‘Christmas Cheer’ vernalis Sarg. ‘Copper’ vernalis Sarg. ‘Lombart’s Weeping’ vernalis Sarg. ‘Sandra’ virginiana L. HAMELIA Jacq. RUBIACEAE Madder Family patens Jacq. HEDERA L. Ivy ARALIACEAE Ginseng Family canariensis Willd. canariensis Willd. ‘Canary Cream’ canariensis Willd. ‘Variegata’ colchica K.Koch colchica K.Koch var. dentata Hibb. ‘Sulfur Heart’ helix L. helix L. ‘Alpha’ helix L. ‘Anchor’ helix L. ‘Angularis Aurea’ helix L. Arborescens group helix L. ‘Arrowhead’ helix L. ‘Aurea Spectabilis’ helix L. ‘Baby Merion’ helix L. ‘Baccifer’ helix L. ‘Big Deal’ helix L. ‘Boskoop’ helix L. ‘Bulgaria’ helix L. ‘California’ helix L. ‘California Gold’ helix L. ‘Carolina Crinkle’ helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. helix L. HAMAMELIS L. HAMAMELIDACEAE Strawflower Aster Family Elm Family Honeysuckle Family ‘Iva Lace’ ‘Jack Frost’ ‘La Platta’ ‘Lady Kay’ ‘Lobata Major’ ‘Lolla Rookh’ ‘Long Point’ ‘Lucida Aurea’ ‘Luzzi’ ‘Manda Crested’ ‘Manda Fringette’ ‘Manda’s Star’ ‘Maple Queen’ ‘Marbled Dragon’ ‘Marginata’ ‘Meagheri’ ‘Merion Beauty’ ‘Merrie’s Albany’ ‘Microphylla Variegata’ ‘Midget’ ‘Miniature Needlepoint’ ‘Minima’ ‘Minor Marmorata’ ‘Mount Vernon’ ‘My Variegated’ ‘Needlepoint’ ‘Obscura’ ‘Paper Doll’ ‘Parsley Crested’ ‘Pedata’ ‘Perfection’ ‘Permanent Wave’ ‘Pin Oak’ ‘Pin Oak Improved’ ‘Pittsburgh’ ‘Pittsburgh Variegated’ ‘Pixie’ ‘Plume de Or’ ‘Preston Tiny’ ‘Purpurea’ ‘Ralf’ ‘Rambler’ ‘Ray’s Supreme’ ‘Ripples’ ‘Rochester’ ‘Roehr’s Minor’ ‘Rubaiyat’ ‘Russell Gold’ ‘Ruth’ ‘Sagittaefolia’ ‘Sea Foam’ ‘Shannon’ ‘Silver Queen’ ‘Sinclair Silverleaf ‘Small Deal’ ‘Spearpoint’ ‘Springtime Snow’ helix L. ‘Staghorn’ helix L. ‘Stardust’ helix L. ‘Stare’ helix L. ‘Sulphurea’ helix L. ‘Susan Gibles’ helix L. ‘Suzanne’ helix L. ‘Sylvanian’ helix L. ‘Teardrop’ helix L. ‘Teena’ helix L. ‘Telecurl’ helix L. ‘Tesselata’ helix L. ‘Thorndale’ helix L. ‘Tidal Wave’ helix L. ‘Tribairn’ helix L. ‘Triloba’ helix L. ‘Triton’ helix L. ‘Trustee’ helix L. ‘238th Street’ helix L. ‘Ustlers’ helix L. ‘Vanderhof’ helix L. ‘Walthamensis’ helix L. ‘Weber’s California’ helix L. ‘Wilson’ helix L. ‘Woodsii’ helix L. var. poetica Weston helix L. var. taurica Rehd. ‘Yalta’ nepalensis K.Koch var. sinensis Tobl. rhombea (Miq.) Bean HELIANTHEMUM Mill. CISTACEAE apenninum (L.) Mill. Rock-rose Family apenninum (L.) Mill. var. roseum (Jacq.) Schneid. nummularium (L.) Mill. nummularium (L.) Mill. ‘Buttercup’ nummularium (L.) Mill. ‘Fireball’ HELICHRYSUM Mill. ASTERACEAE italicum (Roth) G.Don HEMIPTELEA Planch. ULMACEAE davidii (Hance) Planch. HEPTACODIUM Rehd. CAPRIFOLIACEAE miconioides Rehd. Strawflower Aster Family Elm Family Honeysuckle Family ’ iegata’ epoint’ a’ ’ ed’ egated’ eaf w’ helix L. ‘Staghorn’ helix L. ‘Stardust’ helix L. ‘Stare’ helix L. ‘Sulphurea’ helix L. ‘Susan Gibles’ helix L. ‘Suzanne’ helix L. ‘Sylvanian’ helix L. ‘Teardrop’ helix L. ‘Teena’ helix L. ‘Telecurl’ helix L. ‘Tesselata’ helix L. ‘Thorndale’ helix L. ‘Tidal Wave’ helix L. ‘Tribairn’ helix L. ‘Triloba’ helix L. ‘Triton’ helix L. ‘Trustee’ helix L. ‘238th Street’ helix L. ‘Ustlers’ helix L. ‘Vanderhof’ helix L. ‘Walthamensis’ helix L. ‘Weber’s California’ helix L. ‘Wilson’ helix L. ‘Woodsii’ helix L. var. poetica Weston helix L. var. taurica Rehd. ‘Yalta’ nepalensis K.Koch var. sinensis Tobl. rhombea (Miq.) Bean HELIANTHEMUM Mill. CISTACEAE apenninum (L.) Mill. Rock-rose Family apenninum (L.) Mill. var. roseum (Jacq.) Schneid. nummularium (L.) Mill. nummularium (L.) Mill. ‘Buttercup’ nummularium (L.) Mill. ‘Fireball’ HELICHRYSUM Mill. ASTERACEAE italicum (Roth) G.Don HEMIPTELEA Planch. ULMACEAE davidii (Hance) Planch. HEPTACODIUM Rehd. CAPRIFOLIACEAE miconioides Rehd. HAMAMELIS L. HAMAMELIDACEAE helix L. helix L. ‘Gold Heart’ ‘Goods Selfb helix L. helix L. ‘Goods Selfbranching’ ‘Gracilis’ helix L. helix L. ‘Gracilis’ ‘Green Cr helix L. helix L. ‘Green Crown’ ‘Green Finger’ helix L. helix L. ‘Green Finger’ ‘Green Quartz’ helix L. ‘Alpha’ helix L. ‘Anchor’ helix L. ‘Anchor’ helix L. ‘Angularis helix L. ‘Angularis Aurea’ helix L. Arborescens group helix L. helix L. ‘Harrison’ ‘Hebron’ helix L. helix L. ‘Hebron’ ‘Helvetica’ helix L. helix L. ‘Helvetica’ ‘Heterophyll helix L. helix L. ‘Heterophylla’ ‘Hibernica’ helix L. ‘Baccifer’ helix L. ‘Big Deal’ helix L. helix L. ‘Hibernica’ ‘Hite Miniat helix L. ‘Big Deal’ helix L. ‘Boskoop’ helix L. helix L. ‘Hite Miniature’ ‘Holly’ helix L. ‘Boskoop’ helix L. ‘Bulgaria’ helix L. helix L. ‘Holly’ ‘Ideal’ helix L. ‘Bulgaria’ helix L. ‘California’ helix L. ‘California’ helix L. ‘California G helix L. helix L. ‘Ideal’ ‘Imp’ helix L. ‘California Gold’ helix L. ‘Carolina Crinkle’ helix L. helix L. ‘Imp’ ‘Itsy B helix L. ‘Itsy Bitsy’ helix L. ‘Carolina Crinkle’ 271 helix L. ‘Staghorn’ helix L. ‘Stardust’ helix L. ‘Stare’ helix L. ‘Sulphurea’ helix L. ‘Susan Gibles’ helix L. ‘Suzanne’ helix L. ‘Sylvanian’ helix L. ‘Teardrop’ helix L. ‘Teena’ helix L. ‘Telecurl’ helix L. ‘Tesselata’ helix L. ‘Thorndale’ helix L. ‘Tidal Wave’ helix L. ‘Tribairn’ helix L. ‘Triloba’ helix L. ‘Triton’ helix L. ‘Trustee’ helix L. ‘238th Street’ helix L. ‘Ustlers’ helix L. ‘Vanderhof’ helix L. ‘Walthamensis’ helix L. ‘Weber’s California’ helix L. ‘Wilson’ helix L. ‘Woodsii’ helix L. var. poetica Weston helix L. var. taurica Rehd. ‘Yalta’ nepalensis K.Koch var. sinensis Tobl. rhombea (Miq.) Bean HELIANTHEMUM Mill. CISTACEAE apenninum (L.) Mill. Rock-rose Family apenninum (L.) Mill. var. roseum (Jacq.) Schneid. nummularium (L.) Mill. nummularium (L.) Mill. ‘Buttercup’ nummularium (L.) Mill. ‘Fireball’ HELICHRYSUM Mill. ASTERACEAE italicum (Roth) G.Don HEMIPTELEA Planch. ULMACEAE davidii (Hance) Planch. HEPTACODIUM Rehd. CAPRIFOLIACEAE miconioides Rehd. HAMAMELIS L. HAMAMELIDACEAE ‘Comte de Haimout’ syriacus L. ‘Sokobeni yae’ syriacus L. ‘Dela Vaux’ syriacus L. ‘Sonde’ syriacus L. ‘De La Veuve’ syriacus L. ‘Souvenir de Charles Breton’ * syriacus L. ‘Diana’ syriacus L. ‘Speciosus’ syriacus L. ‘Double Light Pink’ syriacus L. ‘Spectabilis Plena’ syriacus L. ‘Duc de Brabant’ syriacus L. ‘Suminokura’ syriacus L. ‘Effie Riegel’ syriacus L. ‘Suminokura hanagasa’ syriacus L. ‘Elegantissimus’ syriacus L. ‘Suminokura yae’ syriacus L. ‘Gion mamori’ syriacus L. ‘Tamausagi’ syriacus L. ‘Grandiflorus Superbus’ syriacus L. ‘Totus Albus’ syriacus L. ‘Hagan Hybrid #2’ syriacus L. ‘Usu hitoe’ syriacus L. ‘Hamabo’ syriacus L. ‘Variegatus’ * syriacus L. ‘Helene’ syriacus L. ‘Violaceus Plenus’ syriacus L. ‘Hinomaruw’ syriacus L. ‘Violet Clair’ syriacus L. ‘Hitoe’ syriacus L. ‘White Red Eye’ syriacus L. ‘Jeanne d’Arc’ syriacus L. ‘White Supreme’ syriacus L. ‘Koki yae’ syriacus L. ‘William P. Smith’ syriacus L. ‘Kreider Blue’ syriacus L. ‘Woodbridge’ syriacus L. ‘La Fleur’ syriacus L. ‘Zulauf’ syriacus L. ‘Lady Stanley’ syriacus L. ‘Leopoldii’ HOVENIA Thunb. syriacus L. ‘Leopoldii Plenus’ RHAMNACEAE Buckthorn Family syriacus L. ‘Lovely Pink’ syriacus L. ‘Lucy’ dulcis Thunb. syriacus L. ‘Luteus Plenus’ syriacus L. ‘Martha Jane’ HAMAMELIS L. HAMAMELIDACEAE Strawflower Aster Family Elm Family Honeysuckle Family ‘Iva Lace’ ‘Jack Frost’ ‘La Platta’ ‘Lady Kay’ ‘Lobata Major’ ‘Lolla Rookh’ ‘Long Point’ ‘Lucida Aurea’ ‘Luzzi’ ‘Manda Crested’ ‘Manda Fringette’ ‘Manda’s Star’ ‘Maple Queen’ ‘Marbled Dragon’ ‘Marginata’ ‘Meagheri’ ‘Merion Beauty’ ‘Merrie’s Albany’ ‘Microphylla Variegata’ ‘Midget’ ‘Miniature Needlepoint’ ‘Minima’ ‘Minor Marmorata’ ‘Mount Vernon’ ‘My Variegated’ ‘Needlepoint’ ‘Obscura’ ‘Paper Doll’ ‘Parsley Crested’ ‘Pedata’ ‘Perfection’ ‘Permanent Wave’ ‘Pin Oak’ ‘Pin Oak Improved’ ‘Pittsburgh’ ‘Pittsburgh Variegated’ ‘Pixie’ ‘Plume de Or’ ‘Preston Tiny’ ‘Purpurea’ ‘Ralf’ ‘Rambler’ ‘Ray’s Supreme’ ‘Ripples’ ‘Rochester’ ‘Roehr’s Minor’ ‘Rubaiyat’ ‘Russell Gold’ ‘Ruth’ ‘Sagittaefolia’ ‘Sea Foam’ ‘Shannon’ ‘Silver Queen’ ‘Sinclair Silverleaf ‘Small Deal’ ‘Spearpoint’ ‘Iva Lace’ ‘Jack Frost’ ‘Jack Frost’ ‘La Platta’ ‘La Platta’ ‘Lady Kay’ ‘Lady Kay’ ‘Lobata Maj ‘Lolla Rookh’ ‘Long Point’ ‘Long Point’ ‘Lucida Aurea ‘Lucida Aurea’ ‘Luzzi’ helix L. ‘Teena’ helix L. ‘Telecurl’ helix L. ‘Telecurl’ helix L. ‘Tesselata’ helix L. ‘Tesselata’ helix L. ‘Thorndale’ helix L. ‘Thorndale’ helix L. ‘Tidal Wave’ helix L. ‘Tidal Wave’ helix L. ‘Tribairn’ helix L. ‘Tribairn’ helix L. ‘Triloba’ helix L. ‘Triloba’ helix L. ‘Triton’ helix L. ‘Triton’ helix L. ‘Trustee’ helix L. ‘Trustee’ helix L. ‘238th Str helix L. ‘238th Street’ helix L. ‘Ustlers’ helix L. ‘Vanderhof’ helix L. ‘Walthamensi HETEROPTERIS HBK. syriacus L. ‘Meehanii’ MALPIGHIACEAE Malpighia Family syriacus L. ‘Mimihara’— * syriacus L. ‘Minerva’ angustifolia Griseb. syriacus L. ‘Monstrosus’ syringifolia Griseb. syriacus L. ‘Monstrosus Plenus’ syriacus L. ‘Monstrosus Simple’ HIBISCUS L. syriacus L. ‘Oiseau Blew’ MALVACEAE Mallow Family syriacus L. ‘Perry’s Purple’ syriacus L. ‘Pheasant Eye’ mutabilis L. syriacus L. ‘Pink Delight’ paramutabilis Bailey syriacus L. ‘Plume’ rosa-sinensis L. ‘Albo-laciniata’ syriacus L. ‘Pom Pom Rouge’ rosa-sinensis L. ‘Brilliant’ syriacus L. ‘Pompon’ syriacus L. syriacus L. ‘Pulcherrimus’ syriacus L. ‘Admiral Dewey’ syriacus L. ‘Puniceus Plenus’ syriacus L. ‘Aka-Yae’ syriacus L. ‘Purpurea Semiplena’ syriacus L. ‘Albus Plenus’ syriacus L. ‘Purpureus Plenus’ syriacus L. ‘Amarantus’ syriacus L. ‘Ranunculiflorus’ * syriacus L. ‘Aphrodite’ syriacus L. ‘Ranunculiflorus Plenus’ syriacus L. ‘Ardens’ syriacus L. ‘Rosalinda’ syriacus L. ‘Ardens Plena’ syriacus L. ‘Roseus Plenus’ syriacus L. ‘Bicolor’ syriacus L. ‘Roxanus’ syriacus L. ‘Blue Bird’ syriacus L. ‘Rubis’ syriacus L. ‘Blue Rouge’ syriacus L. ‘Rubra Grandiflora’ syriacus L. ‘Boule de Few’ syriacus L. ‘Rubra Plena’ syriacus L. ‘Caeruleus Plenus’ syriacus L. ‘Shiro hanagasa’ syriacus L. ‘Campanha’ syriacus L. ‘Shiro midare’ syriacus L. ‘Celestial Blue’ syriacus L. ‘Sir de Charles Breton’ syriacus L. ‘Colie Mullins’ syriacus L. ‘Snowdrift’ syriacus L. ‘Comte de Flandre’ syriacus L. ‘Soft Pink’ syriacus L. HIBISCUS L. MALVACEAE Mallow Family MALVACEAE Mallow Fami mutabilis L. paramutabilis Bailey rosa-sinensis L. ‘Albo-laciniata’ rosa-sinensis L. ‘Brilliant’ syriacus L. syriacus L. ‘Admiral Dewey’ syriacus L. ‘Aka-Yae’ syriacus L. ‘Albus Plenus’ syriacus L. ‘Amarantus’ * syriacus L. ‘Aphrodite’ syriacus L. ‘Ardens’ syriacus L. ‘Ardens Plena’ syriacus L. ‘Bicolor’ syriacus L. ‘Blue Bird’ syriacus L. ‘Blue Rouge’ syriacus L. ‘Boule de Few’ syriacus L. ‘Caeruleus Plenus’ syriacus L. ‘Campanha’ syriacus L. ‘Celestial Blue’ syriacus L. ‘Colie Mullins’ syriacus L. ‘Comte de Flandre’ syriacus L. ‘Comte de Haimout’ syriacus L. ‘Dela Vaux’ syriacus L. ‘De La Veuve’ * syriacus L. ‘Diana’ syriacus L. ‘Double Light Pink’ syriacus L. ‘Duc de Brabant’ syriacus L. ‘Effie Riegel’ syriacus L. ‘Elegantissimus’ syriacus L. ‘Gion mamori’ syriacus L. ‘Grandiflorus Superbus’ syriacus L. ‘Hagan Hybrid #2’ syriacus L. ‘Hamabo’ * syriacus L. ‘Helene’ syriacus L. ‘Hinomaruw’ syriacus L. ‘Hitoe’ syriacus L. ‘Jeanne d’Arc’ syriacus L. ‘Koki yae’ syriacus L. ‘Kreider Blue’ syriacus L. ‘La Fleur’ syriacus L. ‘Lady Stanley’ syriacus L. ‘Leopoldii’ syriacus L. ‘Leopoldii Plenus’ syriacus L. ‘Lovely Pink’ syriacus L. ‘Lucy’ syriacus L. ‘Luteus Plenus’ syriacus L. ‘Martha Jane’ syriacus L. ‘Mauve Queen’ syriacus L. ‘Jeanne d’Arc’ syriacus L. ‘Koki yae’ syriacus L. ‘Koki yae’ syriacus L. ‘Kreider Blu syriacus L. ‘Kreider Blue’ syriacus L. ‘La Fleur’ syriacus L. ‘La Fleur’ syriacus L. ‘Lady Stan syriacus L. ‘Lady Stanley’ syriacus L. ‘Leopoldii’ 273 IDESIA Maxim. FLACOURTIACEAE Flacourtia Family polycarpa Maxim. ILEX L. Holly AQUIFOLIACEAE Holly Family x altaclerensis (Loud.) Dallim. x altaclerensis (Loud.) Dallim. ‘Alice’ x altaclerensis (Loud.) Dallim. ‘Balearica’ < altaclerensis (Loud.) Dallim. ‘Belgica’ x altaclerensis (Loud.) Dallim. ‘Belin’s Weeping’ x altaclerensis (Loud.) Dallim. ‘Camelliifolia’ <x altaclerensis (Loud.) Dallim. ‘Cherry Berry’ x altaclerensis (Loud.) Dallim. ‘Colburn’ < altaclerensis (Loud.) Dallim. ‘Early Cluster’ < altaclerensis (Loud.) Dallim. ‘Eldridge’ <x altaclerensis (Loud.) Dallim. ‘Father Charles’ < altaclerensis (Loud.) Dallim. ‘Firelight’ <x altaclerensis (Loud.) Dallim. ‘Hazel’ <x altaclerensis (Loud.) Dallim. ‘Hendersonii’ x altaclerensis (Loud.) Dallim. ‘Hendersonii Aurea’ x altaclerensis (Loud.) Dallim. ‘Hodginsii’ x altaclerensis (Loud.) Dallim. ‘James G. Esson’ x altaclerensis (Loud.) Dallim. ‘Laurifolia’ x altaclerensis (Loud.) Dallim. ‘Marnockii’ < altaclerensis (Loud.) Dallim. ‘Mundyi’ x altaclerensis (Loud.) Dallim. ‘Nigrescens’ x altaclerensis (Loud.) Dallim. ‘Royal Red’ x altaclerensis (Loud.) Dallim. ‘Wilsonii’ ambigua (Michx.) Torr. amelanchier M.A.Curtis aquifolium L. aquifolium L. ‘Angustifolium’ aquifolium L. ‘Apricot’ aquifolium L. ‘Argentea Marginata’ IDESIA Maxim. FLACOURTIACEAE polycarpa Maxim. HYDRANGEA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage F HYDRANGEA L. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family anomala D.Don ssp. petiolaris (Sieb. & Zucc.) McClint. arborescens L. ‘Annabelle’ macrophylla (Thunb. ex J.A.Murr.) Ser. macrophylla (Thunb. ex J.A.Murr.) Ser. ‘Grayswood’ macrophylla (Thunb. ex J.A.Murr.) Ser. ‘Maculata’ macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino ‘Prolifera’ paniculata Sieb. paniculata Sieb. ‘Grandiflora’ paniculata Sieb. ‘Tardiva’ quercifolia Bartram quercifolia Bartram ‘Harmony’ quercifolia Bartram ‘Snowflake’ scandens (L.f.) Ser. ssp. liukiuensis (Nakai) McClint. HYPERICUM L. St. John’s-Wort HYPERICACEAE St. John’s-wort Family brachyphyllum (Spach) Steud. buckleyi M.A.Curtis calycinum L. (?calycinum < H. forrestii) ‘Hidcote’ fasciculatum Lam. frondosum Michx. Sfrondosum Michx. ‘Sunburst’ galioides Lam. hircinum L. hookerianum Wight & Arn. kalmianum L. lissophloeus Adams lloydii (Svenson) Adams microsepalum (T. & G.) A.Gr. patulum Thunb. ex J.A.Murr. prolificum L. reductum (Svenson) Adams stans (Michx.) Adams & N.Robs. ‘Van Fleetii’ HYSSOPUS L. LAMIACEAE Mint Family officinalis L. Saxifrage Family anomala D.Don ssp. petiolaris (Sieb. & Zucc.) McClint. arborescens L. ‘Annabelle’ macrophylla (Thunb. ex J.A.Murr.) Ser. macrophylla (Thunb. ex J.A.Murr.) Ser. ‘Grayswood’ macrophylla (Thunb. ex J.A.Murr.) Ser. ‘Maculata’ macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino macrophylla (Thunb. ex J.A.Murr.) Ser. ssp. serrata (Thunb. ex J.A.Murr.) Makino ‘Prolifera’ paniculata Sieb. paniculata Sieb. ‘Grandiflora’ paniculata Sieb. ‘Tardiva’ quercifolia Bartram quercifolia Bartram ‘Harmony’ quercifolia Bartram ‘Snowflake’ scandens (L.f.) Ser. ssp. liukiuensis (Nakai) McClint. AQUIFOLIACEAE arborescens L. ‘Annabelle’ macrophylla (Thunb. ex J.A macrophylla (Thunb. ex J.A.Murr.) Ser. macrophylla (Thunb. ex J.A.Murr.) Ser. brachyphyllum (Spach) Steud. buckleyi M.A.Curtis calycinum L. (?calycinum < H. forrestii) ‘Hidcote’ fasciculatum Lam. frondosum Michx. Sfrondosum Michx. ‘Sunburst’ galioides Lam. hircinum L. hookerianum Wight & Arn. kalmianum L. lissophloeus Adams lloydii (Svenson) Adams microsepalum (T. & G.) A.Gr. patulum Thunb. ex J.A.Murr. prolificum L. reductum (Svenson) Adams stans (Michx.) Adams & N.Robs. ‘Van Fleetii’ HYSSOPUS L. LAMIACEAE Mint Family officinalis L. aquifolium L. ‘Aurifodina’ aquifolium L. ‘Bacciflava’ aquifolium L. ‘Lutescens’ aquifolium L. ‘Maderensis Variegata’ aquifolium L. ‘Malcolm S. Whipple’ aquifolium L. ‘Marshal Tito’ aquifolium L. ‘Monstrosa’ aquifolium L. ‘Myrtifolia’ aquifolium L. ‘N. F. Barnes’ aquifolium L. ‘NYBG #2’ aquifolium L. ‘Painted Lady’ aquifolium L. ‘Pale Moon’ aquifolium L. ‘Pendula’ aquifolium L. ‘Perkins #1’ aquifolium L. ‘Petite’ aquifolium L. ‘Phantom Gold’ aquifolium L. ‘Pinto’ aquifolium L. ‘Planifolia’ aquifolium L. ‘Pot-O-Gold’ aquifolium L. ‘Princess Pat’ aquifolium L. ‘Pyramidalis’ aquifolium L. ‘Pyramidalis Compacta’ aquifolium L. ‘Recurva’ aquifolium L. ‘Rederly’ aquifolium L. ‘Ricker’ aquifolium L. ‘Riddle Farm’ aquifolium L. ‘Rubricaulis Aurea’ aquifolium L. ‘Scotia’ aquifolium L. ‘Scram’s Dwarf’ aquifolium L. ‘Shortspra’ aquifolium L. ‘Silver Milkboy’ aquifolium L. ‘Silver Milkmaid’ aquifolium L. ‘Sparkler’ aquifolium L. ‘Sunnybrooke’ aquifolium L. ‘Sunnyside’ aquifolium L. ‘Teufel’s Hybrid’ aquifolium L. ‘Teufel’s Variegated’ aquifolium L. ‘Thornton’ aquifolium L. ‘Tom Everett’ aquifolium L. ‘Watereriana’ aquifolium L. ‘Wheeler #4’ aquifolium L. ‘Whittingtonensis’ aquifolium L. ‘Winter King’ aquifolium L. ‘Winter Queen’ aquifolium L. ‘Wintergreen’ aquifolium L. ‘Yellow Beam’ aquifolium L. ‘Yonkers’ aquifolium L. ‘Yule Glow’ aquifolium L. ‘Zero’ (aquifolium x I. ciliospinosa) ‘Brilliant’ aquifolium x I. spinigera x aquipernyi Gable < aquipernyi Gable ‘Aquipern’ x aquipernyi Gable ‘Dragon Lady’ xX aquipernyi Gable ‘Gable’ x aquipernyi Gable ‘San Jose’ (< aquipernyi X (I. integra x I. pernyji) ‘Accent’) ‘Rock Garden’ asprella (Hook. & Arn.) Champ. ex Benth. aquifolium L. ‘Lutescens’ aquifolium L. ‘Maderensis Variegata’ aquifolium L. ‘Malcolm S. Whipple’ aquifolium L. ‘Marshal Tito’ aquifolium L. ‘Monstrosa’ aquifolium L. ‘Myrtifolia’ aquifolium L. ‘N. F. Barnes’ aquifolium L. ‘NYBG #2’ aquifolium L. ‘Painted Lady’ aquifolium L. ‘Pale Moon’ aquifolium L. ‘Pendula’ aquifolium L. ‘Perkins #1’ aquifolium L. ‘Petite’ aquifolium L. ‘Phantom Gold’ aquifolium L. ‘Pinto’ aquifolium L. ‘Planifolia’ aquifolium L. ‘Pot-O-Gold’ aquifolium L. ‘Princess Pat’ aquifolium L. ‘Pyramidalis’ aquifolium L. ‘Pyramidalis Compacta’ aquifolium L. ‘Recurva’ aquifolium L. ‘Rederly’ aquifolium L. ‘Ricker’ aquifolium L. ‘Riddle Farm’ aquifolium L. ‘Rubricaulis Aurea’ aquifolium L. ‘Scotia’ aquifolium L. ‘Scram’s Dwarf’ aquifolium L. AQUIFOLIACEAE ‘Shortspra’ aquifolium L. ‘Silver Milkboy’ aquifolium L. ‘Silver Milkmaid’ aquifolium L. ‘Sparkler’ aquifolium L. ‘Sunnybrooke’ aquifolium L. ‘Sunnyside’ aquifolium L. ‘Teufel’s Hybrid’ aquifolium L. ‘Teufel’s Variegated’ aquifolium L. ‘Thornton’ aquifolium L. ‘Tom Everett’ aquifolium L. ‘Watereriana’ aquifolium L. ‘Wheeler #4’ aquifolium L. ‘Whittingtonensis’ aquifolium L. ‘Winter King’ aquifolium L. ‘Winter Queen’ aquifolium L. ‘Wintergreen’ aquifolium L. ‘Yellow Beam’ aquifolium L. ‘Yonkers’ aquifolium L. ‘Yule Glow’ aquifolium L. ‘Zero’ (aquifolium x I. ciliospinosa) ‘Brilliant’ aquifolium x I. spinigera x aquipernyi Gable < aquipernyi Gable ‘Aquipern’ x aquipernyi Gable ‘Dragon Lady’ aquifolium L. ‘Beacon’ aquifolium L. ‘Beauty Spra’ aquifolium L. ‘Beauty Spra Espalier’ aquifolium L. ‘Berigold’ aquifolium L. ‘Big Bull’ aquifolium L. ‘Bleeg’ aquifolium L. ‘Bodley’s Bleeg’ aquifolium L. ‘Bonanza’ aquifolium L. ‘Bronze’ aquifolium L. ‘Brownell’ aquifolium L. ‘Butler’ aquifolium L. ‘Campus Variegated’ aquifolium L. ‘Captain Bonneville’ aquifolium L. ‘Chief’ aquifolium L. ‘Clouded Gold’ aquifolium L. ‘Coleman’ aquifolium L. ‘Cover Girl’ aquifolium L. ‘Crinkle Variegated’ aquifolium L. ‘Crispa’ aquifolium L. ‘Crispa Aureo-picta’ aquifolium L. ‘Daddyo’ aquifolium L. ‘Deluxe’ aquifolium L. ‘Dude’ aquifolium L. ‘Dumbarton Oaks’ aquifolium L. ‘Elegantissima’ aquifolium L. ‘Escort’ aquifolium L. ‘Favorite’ aquifolium L. ‘Ferox’ aquifolium L. ‘Ferox Argentea’ aquifolium L. ‘Ferox Aurea Marginata’ aquifolium L. ‘Firecracker’ aquifolium L. ‘Flavescens’ aquifolium L. ‘Foxii’ aquifolium L. ‘Fructo-lutea’ aquifolium L. ‘Globe’ aquifolium L. ‘Golden Butterfly’ aquifolium L. ‘Golden Milkboy’ aquifolium L. ‘Golden Milkmaid’ aquifolium L. ‘Golden Queen’ aquifolium L. ‘Gracean’ aquifolium L. ‘Green Knight’ aquifolium L. ‘Green Maid’ aquifolium L. ‘Handsworthensis’ aquifolium L. ‘Hastata’ aquifolium L. ‘Hollycroft Jack’ aquifolium L. ‘Ingramii’ aquifolium L. ‘Integrifolium’ aquifolium L. ‘Ivory’ aquifolium L. ‘J. C. van Tol’ aquifolium L. ‘Lady Baltimore’ aquifolium L. ‘Latispina’ aquifolium L. ‘Lewis’ aquifolium L. ‘Lilliput’ aquifolium L. ‘Lilygold’ aquifolium L. ‘Little Bull’ aquifolium L. ‘Longspra’ aquifolium L. ‘Louise’ aquifolium L. ‘Whittingtonensis’ aquifolium L. ‘Winter King’ aquifolium L. ‘Wintergreen’ aquifolium L. ‘Yellow Beam’ aquifolium L. ‘Yonkers’ aquifolium L. ‘Yule Glow’ aquifolium L. ‘Yule Glow’ aquifolium L. ‘Zero’ ‘Accent’) ‘Rock Garden’ asprella (Hook. & Arn.) Ch asprella (Hook. & Arn.) Champ. ex Benth. aquifolium L. ‘Louise’ 275 x attenuata Ashe < attenuata Ashe ‘Alagold’ x attenuata Ashe ‘Eagleson’ x attenuata Ashe ‘East Palatka’ < attenuata Ashe ‘Edna Jean’ x attenuata Ashe ‘Erma Byrd’ <x attenuata Ashe ‘Foster #1’ <x attenuata Ashe ‘Foster #2’ <x attenuata Ashe ‘Foster #3’ x attenuata Ashe ‘Foster #4’ <x attenuata Ashe ‘Howard’ <x attenuata Ashe ‘Hume #1’ <x attenuata Ashe ‘Hume #2’ <x attenuata Ashe ‘Hutchinson’ <x attenuata Ashe ‘Louise Holmes’ x attenuata Ashe ‘NASA’ <x attenuata Ashe ‘Savannah’ * x<attenuata Ashe ‘Sunny Foster’ < attenuata Ashe ‘Topeli’ beadlei Ashe x beanii Rehd. bioritensis Hayata buergeri Miq. buswellii Small canariensis Poir. cassine L. cassine L. ‘Baldwin’ cassine L. var. angustifolia Ait. cassine L. var. angustifolia f. aurea-baccata Tarbox ex S.F.Blake cassine L. var. bryanii Tarbox * ex S.F.Blake ciliospinosa Loesn. (ciliospinosa x I. x aquipernyji) ‘September Gem’ ciliospinosa < I. fargesii (ciliospinosa x I. leucoclada) ‘Harry Gunning’ (ciliospinosa x I. leucoclada) ‘William Cowsgill’ cinerea Champ. colchica Pojark. collina Alex. corallina Franch. coriacea (Pursh) Chapm. cornuta Lindl. & Paxt. cornuta Lindl. & Paxt. ‘Aglo’ cornuta Lindl. & Paxt. ‘Anicet Delcambrie’ cornuta Lindl. & Paxt. ‘Anna Mae’ cornuta Lindl. & Paxt. ‘Avery Island’ cornuta Lindl. & Paxt. ‘Bostic’ cornuta Lindl. & Paxt. ‘Brawley’ cornuta Lindl. & Paxt. ‘Burfordii’ cornuta Lindl. & Paxt. ‘Cajun Gold’ cornuta Lindl. & Paxt. ‘Carissa’ cornuta Lindl. & Paxt. ‘Clarendon Batwing’ cornuta Lindl. & Paxt. ‘Clarendon Small Leaf’ cornuta Lindl. & Paxt. ‘Dodd Special’ cornuta Lindl. & Paxt. ‘D’Or’ cornuta Lindl. & Paxt. ‘Dr. James Foret’ cornuta Lindl. & Paxt. ‘Dr. John Creech’ cornuta Lindl. & Paxt. ‘Dwarf Burford’ cornuta Lindl. & Paxt. ‘E. A. McIlIhenny’ cornuta Lindl. & Paxt. ‘Fine Line’ cornuta Lindl. & Paxt. ‘Glenwood’ cornuta Lindl. & Paxt. ‘Grandview’ cornuta Lindl. & Paxt. ‘Hume’ cornuta Lindl. & Paxt. ‘Ira Nelson’ cornuta Lindl. & Paxt. ‘Jungle Garden’ cornuta Lindl Toa axt ‘Kingsville Special’ cornuta Lindl. & Paxt. ‘Medallion’ cornuta Lindl. & Paxt. ‘Morrell No. 1’ cornuta Lindl. & Paxt. ‘Morrell No. 3’ cornuta Lindl. & Paxt. ‘National’ cornuta Lindl. & Paxt. ‘Needle Point’ cornuta Lindl. & Paxt. ‘Olga’ cornuta Lindl. & Paxt. ‘O. Spring’ cornuta Lindl. & Paxt. ‘Rotunda’ cornuta Lindl. & Paxt. ‘R. V. P. Special’ cornuta Lindl. & Paxt. ‘Shangri-La’ cornuta Lindl. & Paxt. ‘Shiu-Ying’ cornuta Lindl. & Paxt. ‘Variegata’ cornuta Lindl. & Paxt. ‘Walker’ (cornuta < I. aquifolium) ‘Callina’ (cornuta x I. ‘Edwin Dozier’ crenata Thunb. crenata Thunb. ex J.A.Murr. ‘Firefly’ crenata Thunb. ex J.A.Murr. ‘Flushing’ crenata Thunb. ex J.A.Murr. ‘Flushing’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Foster No. 1’ crenata Thunb. ex J.A.Murr. ‘Foster No. 1’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Foster No. 2’ crenata Thunb. ex J.A.Murr. ‘Foster No. 2’ crenata Thunb. ex J.A.Murr. ‘ crenata Thunb. ex J.A.Murr. ‘Frierson’ crenata Thunb. ex J.A.Murr. ‘Glass’ crenata Thunb. ex J.A.Murr. ‘Glass’ crenata Thunb. ex J.A.Murr. ‘Glory’ crenata Thunb. ex J.A.Murr. ‘Glory’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Golden Gem’ ‘Loyce Nelson’ crenata Thunb. crenata Thunb. ex J.A.Murr. ‘Buxifolia’ crenata Thunb. ex J.A.Murr. ‘Changsha’ crenata Thunb. ex J.A.Murr. ‘Luteo-variegata’ crenata Thunb. ex J.A.Murr. ‘Changsha’ crenata Thunb. ex J.A.Murr. ‘Compacta’ crenata Thunb. ex J.A.Murr. ‘Compacta’ crenata Thunb. ex J.A.Murr. ‘Conners’ crenata Thunb. ex J.A.Murr. ‘Conners’ crenata Thunb. ex J.A.Murr. ‘Convexa’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Mariesii’ crenata Thunb. ex J.A.Murr. ‘Maxwell’ crenata Thunb. ex J.A.Murr. ‘Delaware Diamond’ crenata Thunb. ex J.A.Murr. ‘Maxwell’ crenata Thunb. ex J.A.Murr. ‘Oconee River’ crenata Thunb. crenata Thunb. ex J.A.Murr. ‘Green Dragon’ ‘Accent’) ‘Rock Garden’ asprella (Hook. & Arn.) Ch aquifolium) ‘Edward J. Stevens’ (cornuta < I. aquifolium) ‘Hollowell’ (cornuta x I. aquifolium) ‘Maplehurst’ (cornuta x I. aquifolium) ‘Nellie R. Stevens’ * ((cornuta x I. aquifolium) ‘Nellie R. Stevens’ x I. leucoclada) ‘Clusterberry’ cornuta X I. pernyi (cornuta < I. pernyi) ‘Atlas’ (cornuta X< I. pernyi) ‘Audry’ (cornuta x I. pernyi) ‘Brighter Shine’ (cornuta < I. pernyi) ‘Cetus’ (cornuta < I. pernyi) ‘Doctor Kassab’ (cornuta < I. pernyi) ‘Drace’ (cornuta < I. pernyi) ‘Formax’ (cornuta < I. pernyi) ‘Good Taste’ (cornuta < I. pernyji) ‘Indian Chief’ * (cornuta x I. pernyi) ‘John T. Morris’ (cornuta < I. pernyi) ‘Lacerta’ (cornuta < I. pernyi) ‘Lepux’ * (cornuta < I. pernyi) ‘Lydia Morris’ (cornuta < I. pernyji) ‘Lyra’ (cornuta x I. pernyi) ‘Moonglow’ cassine L. ‘Baldwin’ cassine L. var. angust cassine L. var. angustifolia Ait. cassine L. var. angustifolia corallina Franch. coriacea (Pursh) C ‘Anicet Delcambrie’ cornuta Lindl. & Paxt. ‘Anna Mae’ cornuta Lindl. & Paxt. ‘Avery Island’ cornuta Lindl. & Paxt. ‘Bostic’ cornuta Lindl. & Paxt. ‘Brawley’ cornuta Lindl. & Paxt. ‘Burfordii’ cornuta Lindl. & Paxt. ‘Cajun Gold’ cornuta Lindl. & Paxt. ‘Carissa’ cornuta Lindl. & Paxt. ‘Casey’s Dwarf’ crenata Thunb. ex J.A.Murr. ‘Hatf crenata Thunb. ex J.A-Murr. ‘Helle crenata Thunb. ex J.A.Murr. ‘Hetzi crenata Thunb. ex J.A.Murr. ‘Highlight’ crenata Thunb. ex J.A.Murr. ‘Honeycomb’ crenata Thunb. ex J.A.Murr. ‘Ivor crenata Thunb. ex J.A.Murr. ‘Ivory Tower’ crenata Thunb. ex J.A.Murr. ‘Jers Pinnacle’ crenata Thunb. ex J.A.Murr. ‘John Nosal’ crenata Thunb. ex J.A.Murr. ‘Kingsville Dwarf crenata Thunb. ex J.A.Murr. ‘Kun crenata Thunb. ex J.A.Murr. ‘Latif crenata Thunb. ex J.A.Murr. ‘Lindleyana’ crenata Thunb. ex J.A.Murr. ‘Long crenata Thunb. ex J.A.Murr. ‘Long crenata Thunb. ex J.A.Murr. ‘Loyce Nelson’ crenata Thunb. ex J.A.Murr. ‘Luteo-variegata’ crenata Thunb. ex J.A.Murr. ‘Majo crenata Thunb. ex J.A.Murr. ‘Mari crenata Thunb. ex J.A.Murr. ‘Maxw crenata Thunb. ex J.A.Murr. ‘Mentor Dense’ crenata Thunb. ex J.A.Murr. ‘Mentor Glossy’ crenata Thunb. ex J.A.Murr. ‘Microphylla’ crenata Thunb. ex J.A.Murr. ‘Microphylla Supreme’ crenata Thunb. ex J.A.Murr. ‘Midas Touch’ crenata Thunb. ex J.A.Murr. ‘Miss Muffet’ crenata Thunb. ex J.A.Murr. ‘Mobjack Supreme’ crenata Thunb. ex J.A.Murr. ‘Morris Dwarf’ crenata Thunb. ex J.A.Murr. ‘Mount Halla’ crenata Thunb. ex J.A.Murr. ‘Naka crenata Thunb. ex J.A.Murr. ‘Nank crenata Thunb. ex J.A.Murr. ‘Oconee River’ crenata Thunb. ex J.A.Murr. ‘Olea crenata Thunb. ex J.A.Murr. ‘Peco crenata Thunb. ex J.A.Mutrr. ‘Picc crenata Thunb. ex J.A.Murr. ‘Pride Dwarf’ (cornuta < I. pernyji) ‘Titan’ (cornuta x I. pernyi) ‘Virgo’ (cornuta X< I. ‘Golden Gem’ crenata Thunb. crenata Thunb. ex J.A.Murr. ‘Golden Heller’ ‘Golden Heller’ crenata Thunb. e ‘Accent’) ‘Rock Garden’ asprella (Hook. & Arn.) Ch rugosa) ‘China Boy’ (cornuta X< I. rugosa) ‘China Girl’ (cornuta ‘Burfordii’ < I. latifolia) ‘Arthur Bruner’ (cornuta ‘Burfordii’ < I. latifolia) ‘Bob Bruner’ (cornuta ‘Burfordii’ < I. latifolia) ‘Emily Bruner’ (cornuta ‘Burfordii’ < I. latifolia) ‘James Swan’ (cornuta ‘Burfordii’ < I. latifolia) ‘Jinny Bruner’ (cornuta ‘Burfordii’ < I. pernyi) ‘Red Delight’ ((cornuta ‘Burfordii’ < I. pernyi) ‘Red Delight’ x I. latifolia) ‘Mary Nell’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Angyo’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Longifolia’ crenata Thunb. ex J.A.Murr. ‘Delaware Diamond’ crenata Thunb. ex J.A. crenata Thunb. ex J.A.Murr. ‘Mentor Dense’ ‘Delaware Diamond’ crenata Thunb. ex J.A.Murr. ‘Divaricata’ crenata Thunb. ex J.A.Murr. ‘Dwarf Cone’ crenata Thunb. ex J.A.Murr. ‘Divaricata’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘Mentor Glossy’ crenata Thunb. ex J.A.Murr. ‘Dwarf Cone’ ‘Dwarf Cone’ crenata Thunb. ex J.A.Murr. ‘Dwarf Pagoda’ crenata Thunb. ex J.A.Murr. ‘Dwarf Pagoda’ crenata Thunb. ex J.A.Murr. ‘Microphylla’ crenata Thunb. ex J.A.Murr. ‘Edwin Dozier’ crenata Thunb. ex J.A.Murr. ‘Microphylla Supreme’ ‘Braddock Heights’ crenata Thunb. ex J. crenata Thunb. ex J.A.Murr. ‘Loyce Nelson’ ‘Rocky Creek’ crenata Thunb. ‘Rocky Creek’ crenata Thunb. crenata Thunb. ex J.A.Murr. ‘Sentinel’ crenata Thunb. ex J.A.Murr. ‘Shanghai’ crenata Thunb. ex J.A.Murr. ‘Shanghai’ crenata Thunb. ex J.A.Murr. ‘Snowflake’ crenata Thunb. ex J.A.Murr. ‘Snowflake’ crenata Thunb. ex J.A.Murr. ‘Stokes’ crenata Thunb. ex J.A.Murr. ‘Stokes’ crenata Thunb. ex J.A.Murr. ‘Tennyson crenata Thunb. ex J.A.Murr. ‘Tennyson’ crenata Thunb. ex J.A.Murr. ‘T-one’ crenata Thunb. ex J.A.Murr. ‘T-one’ crenata Thunb. ex J.A.Murr. ‘Vaseyi’ crenata Thunb. ex J.A.Murr. ‘Vaseyi’ crenata Thunb. ex J.A.Murr. ‘Wayne’ crenata Thunb. ex J.A.Murr. ‘Wayne’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. ‘William Jackson’ ‘William Jackson’ crenata Thunb. ex J.A.Murr. ‘Willow Leaf’ ‘Green Dragon’ crenata Thunb. e ‘Green Dragon’ crenata Thunb. e crenata Thunb. ex J.A.Murr. ‘Green Lustre’ crenata Thunb. ex J.A.Murr. ‘Green Zila glabra (L.) A.Gr. f. leucocarpa F.W.Woods glabra (L.) A.Gr. f. leucocarpa F.W.Woods ‘Ivory Queen’ integra Thunb. ex J.A.Murr. integra Thunb. ex J.A.Murr. ‘Green Shadow’ integra < I. aquifolium integra < I. cornuta (integra x I. cornuta) ‘Libby’s Favorite’ (integra < I. cornuta) ‘Semala’ integra X I. pernyi * (integra x I. pernyji) ‘Accent’ * (integra x I. pernyi) ‘Elegance’ kingiana Cockerell < kiusiana Hatusima <x koehneana Loesn. <x koehneana Loesn. ‘Chieftan’ <x koehneana Loesn. ‘Hohman’ * <koehneana Loesn. ‘Jade’ <x koehneana Loesn. ‘Lassie’ <x koehneana Loesn. ‘Lock Raven’ * <koehneana Loesn. ‘Ruby’ < koehneana Loesn. ‘San Jose’ < koehneana Loesn. ‘Wirt L. Winn’ laevigata (Dum.-Cours.) A.Gr. latifolia Thunb. ex J.A.Murr. leucoclada Makino liukiuensis Loesn. lohfauensis Merr. longipes Chapm. ex Trelease longipes Chapm. ex Trelease ‘Lagniappe’ longipes Chapm. ex Trelease ‘Natchez Belle’ macrocarpa Oliv. macropoda Miq. maximowicziana Loesn. var. kanehirae (Yamamoto) Yamazaki <x meserveae S.Y.Hu ‘Blue Angel’ x meserveae S.Y.Hu ‘Blue Boy’ x meserveae S.Y.Hu ‘Blue Girl’ x meserveae S.Y.Hu ‘Blue Maid’ x meserveae S.Y.Hu ‘Blue Prince’ <x meserveae S.Y.Hu ‘Blue Princess’ x meserveae S.Y.Hu ‘Blue Stallion’ x meserveae S.Y.Hu ‘Golden Girl’ monticola A. Gr. myrtifolia Walt. myrtifolia Walt. ‘Lowei’ * ((myrtifolia < I. opaca) < I. myrtifolia) ‘Oriole’ * ((myrtifolia < I. opaca) < I. myrtifolia) ‘Tanager’ opaca Ait. opaca Ait. ‘Aalto’ opaca Ait. ‘Aalto #5’ glabra (L.) A.Gr. f. leucocarpa F.W.Woods glabra (L.) A.Gr. f. leucocarpa F.W.Woods ‘Ivory Queen’ integra Thunb. ex J.A.Murr. integra Thunb. ex J.A.Murr. ‘Green Shadow’ integra < I. aquifolium integra < I. cornuta (integra x I. cornuta) ‘Libby’s Favorite’ (integra < I. cornuta) ‘Semala’ integra X I. pernyi * (integra x I. pernyji) ‘Accent’ * (integra x I. pernyi) ‘Elegance’ kingiana Cockerell < kiusiana Hatusima <x koehneana Loesn. <x koehneana Loesn. ‘Chieftan’ <x koehneana Loesn. ‘Hohman’ * <koehneana Loesn. ‘Jade’ <x koehneana Loesn. ‘Lassie’ <x koehneana Loesn. ‘Lock Raven’ * <koehneana Loesn. ‘Ruby’ < koehneana Loesn. ‘San Jose’ < koehneana Loesn. ‘Wirt L. Winn’ laevigata (Dum.-Cours.) A.Gr. latifolia Thunb. ex J.A.Murr. leucoclada Makino crenata Thunb. ex J.A.Murr. ‘Pride’s Tiny’ ‘Pride’s Tiny’ crenata Thunb. ex J.A.Murr. ‘Pyramidalis’ crenata Thunb. ex J.A.Murr. ‘Repandens’ ‘Repandens’ crenata Thunb. ex J.A.Murr. ‘Rocky Creek’ ‘Willow Leaf’ crenata Thunb. & O.’ ‘Barclay’ ‘Beulah’ ‘Boyce Thompson’ ‘Boyce Thompson #3’ ‘Brown #5’ ‘Brown #16’ ‘Cape Christmas’ ‘Charles’ ‘Christmas Carol’ ‘Christmas Hedge’ ‘Clarendon’ ‘Clarendon Spreading ‘Clarissa’ ‘Clark’ ‘Croonenberg’ ‘Cumberland’ ‘Dan Fenton’ ‘Dick’ ‘Dorsey’ ‘Dr. T. B. Symons’ ‘Elizabeth’ ‘Emily’ ‘Farage’ ‘Faulkner’ ‘Felten’s Selection’ ‘Fire Chief’ ‘Formal’ ‘Francis Lewis’ ‘Freeman’ ‘Gee’ ‘Golden Fleece’ ‘Good Will Park’ ‘Governor William Paca’ ‘Grandpappy’ ‘Griscom’ ‘Hamlet’ ‘Harriet’ ‘Hedgeholly’ ‘Helen Makepeace’ ‘Homer’ ‘Hopkins’ ‘Hume’s Choice’ ‘Iso’ ‘Jeannette Adamson ‘Jersey Princess’ ‘John Higgins’ ‘Joyce’ ‘Judge Brown’ ‘Kate’ ‘Knight’ ‘La Bar’ ‘Lake City’ 9 opaca Ait. opaca Ait. ‘Pomona’ ‘Reynolds’ opaca Ait. opaca Ait. ‘Dr. T. B. Symons’ ‘Elizabeth’ opaca Ait. opaca Ait. ‘Reynolds’ ‘Richards’ opaca Ait. opaca Ait. ‘Elizabeth’ ‘Emily’ opaca Ait. opaca Ait. ‘Richards’ ‘Ruby Red’ opaca Ait. opaca Ait. ‘St. Ann’ ‘St. John’s opaca Ait. opaca Ait. ‘St. John’s’ ‘St. Mary’ opaca Ait. opaca Ait. ‘St. Mary’ ‘Sandy Hoo opaca Ait. opaca Ait. ‘Sara Higgins’ ‘Satyr Hill’ opaca Ait. opaca Ait. ‘Satyr Hill’ ‘Schlupp’ opaca Ait. opaca Ait. ‘Schlupp’ ‘Secrest’ opaca Ait. opaca Ait. ‘Secrest’ ‘Slim Jim’ opaca Ait. opaca Ait. ‘Good Will Park’ ‘Governor Willia opaca Ait. opaca Ait. ‘Slim Jim’ ‘Star’ opaca Ait. opaca Ait. ‘Star’ ‘Taber’ opaca Ait. opaca Ait. ‘Taber’ ‘Taber # opaca Ait. opaca Ait. ‘Taber #2’ ‘Tiny’ opaca Ait. opaca Ait. ‘Hamlet’ opaca Ait. opaca Ait. ‘Tiny’ ‘Toner’ opaca Ait. opaca Ait. ‘Harriet’ ‘Hedgehol opaca Ait. opaca Ait. ‘Toner’ ‘Trisco’ opaca Ait. opaca Ait. ‘Hedgeholly’ ‘Helen Makepe opaca Ait. opaca Ait. ‘Trisco’ ‘24 Karat opaca Ait. opaca Ait. ‘Helen Makepeace’ ‘Homer’ opaca Ait. opaca Ait. ‘24 Karat’ ‘Vera’ opaca Ait. opaca Ait. ‘Homer’ ‘Hopkins’ opaca Ait. opaca Ait. ‘Vera’ ‘Wheele opaca Ait. opaca Ait. ‘Hopkins’ ‘Hume’s Ch opaca Ait. opaca Ait. ‘Wheeler #4’ ‘William Haw opaca Ait. opaca Ait. ‘Hume’s Choice’ ‘Iso’ opaca Ait. opaca Ait. ‘William Hawkins’ ‘Yule’ opaca Ait. opaca Ait. ‘Iso’ opaca Ait. opaca Ait ‘Yule’ f. subin opaca Ait. opaca Ait. ‘Jeannette Adamson ‘Jersey Princess’ 9 opaca Ait opaca Ait f. subintegra Weatherby f. subintegra Weatherby opaca Ait. opaca Ait. ‘Jersey Princess’ ‘John Higgins’ opaca Ait f. subintegra Weatherby ‘Perle LeClair’ opaca Ait. opaca Ait. ‘John Higgins’ ‘Joyce’ ‘Willow Leaf’ crenata Thunb. opaca Ait. ‘Aalto #5A’ ‘Andorra’ ‘Anne Arundel’ ‘Arden’ ‘B. & O.’ ‘Barclay’ ‘Beulah’ ‘Boyce Thompson’ ‘Boyce Thompson #3’ ‘Brown #5’ ‘Brown #16’ ‘Cape Christmas’ ‘Charles’ ‘Christmas Carol’ ‘Christmas Hedge’ ‘Clarendon’ ‘Clarendon Spreading ‘Clarissa’ ‘Clark’ ‘Croonenberg’ ‘Cumberland’ ‘Dan Fenton’ ‘Dick’ ‘Dorsey’ ‘Dr. T. B. Symons’ ‘Elizabeth’ ‘Emily’ ‘Farage’ ‘Faulkner’ ‘Felten’s Selection’ ‘Fire Chief’ ‘Formal’ ‘Francis Lewis’ ‘Freeman’ ‘Gee’ ‘Golden Fleece’ ‘Good Will Park’ ‘Governor William Paca’ ‘Grandpappy’ ‘Griscom’ ‘Hamlet’ ‘Harriet’ ‘Hedgeholly’ ‘Helen Makepeace’ ‘Homer’ ‘Hopkins’ ‘Hume’s Choice’ ‘Iso’ ‘Jeannette Adamson ‘Jersey Princess’ ‘John Higgins’ ‘Joyce’ 9 opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait opaca Ait ‘Laura’ ‘Lowell’ ‘Mae’ ‘Magna Semen’ ‘Mamie Eisenhower’ ‘Manig’ ‘Maryland’ ‘Maryland Dwarf ‘Menantico’ ‘Merry Christmas’ ‘Miss Helen’ ‘Miss Liberty’ ‘Mrs. Santa’ ‘Nelson West’ ‘Old Heavy Berry’ ‘Osa’ ‘Palmetto’ ‘Perkins-de-Wilde #1’ ‘Perkins-de-Wilde #2’ ‘Perkins-de-Wilde #3’ ‘Perrine’ ‘Pin Cushion’ ‘Polly’ ‘Pomona’ ‘Reynolds’ ‘Richards’ ‘Ruby Red’ ‘Rushton’ ‘St. Ann’ ‘St. John’s’ ‘St. Mary’ ‘Sandy Hook #5’ ‘Sara Higgins’ ‘Satyr Hill’ ‘Schlupp’ ‘Secrest’ ‘Slim Jim’ ‘Star’ ‘Taber’ ‘Taber #2’ ‘Tiny’ ‘Toner’ ‘Trisco’ ‘24 Karat’ ‘Vera’ ‘Wheeler #4’ ‘William Hawkins’ ‘Yule’ f. subintegra Weatherby f. subintegra Weatherby ‘Perle LeClair’ opaca Ait. f. xanthocarpa Rehd. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. ‘Aalto #5A’ ‘Andorra’ ‘Anne Arundel’ ‘Arden’ ‘B. ‘Willow Leaf’ crenata Thunb. crenata Thunb. ex J.A.Murr. ‘Yunnan’ crenata Thunb. ex J.A.Murr. crenata Thunb. ex J.A.Murr. f. watanabeana Makino crenata Thunb. ex J.A.Murr. f. watanabeana Makino crenata Thunb. ex J.A.Murr. ssp. fukasawana (Makino) Murata crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara ‘Carefree’ crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara ‘Crescent’ crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara ‘Gayle’ crenata Thunb. ex J.A.Murr. var. paludosa (Nakai) Hara ‘Tyke’ crenata Thunb. ex J.A.Murr. var. thomsonii (Hook.f.) Loesn. cumulicola Small cumulicola Small ‘Fort McCoy’ curtissii (Fern.) Small cyrtura Merr. decidua Walt. decidua Walt. ‘Byers Golden’ decidua Walt. ‘Indian Bayou #3’ decidua Walt. ‘Pocahontas’ dimorphophylla Koidz. dipyrena Wall. fargesii Franch. fargesii Franch. ssp. melanotricha (Merr.) S.Andrews ficoidea Hemsl. geniculata Maxim. georgei Comber glabra (L.) A.Gr. glabra (L.) A.Gr. ‘Nana’ glabra (L.) A.Gr. ‘Tankard’s Compact’ glabra (L.) A.Gr. ‘Viridis’ latifolia Thunb. ex J.A.Murr. leucoclada Makino latifolia Thunb. ex J.A.Murr. leucoclada Makino leucoclada Makino liukiuensis Loesn. liukiuensis Loesn. lohfauensis Merr. lohfauensis Merr. longipes Chapm. ex longipes Chapm. ex Trelease longipes Chapm. ex Trelease longipes Chapm. ex Trelease ‘Lagniappe’ longipes Chapm. ex Trelease ‘Natchez Belle’ macrocarpa Oliv. macrocarpa Oliv. macropoda Miq. macropoda Miq. maximowicziana crenata Thunb. ex J.A.Murr. var. thomsonii (Hook.f.) Loes var. thomsonii (Hook.f.) Loesn. cumulicola Small decidua Walt. decidua Walt. decidua Walt. ‘Indian Bayou #3’ decidua Walt. ‘Pocahontas’ decidua Walt. ‘Pocahontas’ dimorphophylla Koidz. dimorphophylla Koidz. dipyrena Wall. dipyrena Wall. fargesii Franch. monticola A. Gr. myrtifolia Walt. myrtifolia Walt. myrtifolia Walt. fargesii Franch. fargesii Franch. myrtifolia Walt. ‘Lowei’ ((myrtifolia < I. opaca) < (Merr.) S.Andrews ficoidea Hemsl. glabra (L.) A.Gr. glabra (L.) A.Gr. opaca Ait. ‘Aalto’ opaca Ait. ‘Aalto # glabra (L.) A.Gr. ‘Nana’ glabra (L.) A.Gr. ‘Tankar opaca Ait. ‘Aalto #5’ glabra (L.) A.Gr. ‘Tankard’s Compact’ glabra (L.) A.Gr. ‘Viridis’ glabra (L.) A.Gr. ‘Viridis’ 278 opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. opaca Ait. ‘Perle LeClair’ opaca Ait. f. xan opaca Ait. opaca Ait. f. xanthocarpa Rehd. f. xanthocarpa Rehd. opaca Ait. opaca Ait. ‘Joyce’ ‘Judge opaca Ait. opaca Ait. f. xanthocarpa Rehd. f. xanthocarpa Rehd. opaca Ait. opaca Ait. ‘Judge Brown’ ‘Kate’ opaca Ait. f. xanthocarpa Rehd. ‘Boyce Thompson Xanthocarpa’ opaca Ait. opaca Ait. ‘Kate’ ‘Knight ‘Boyce Thompson Xanthocarpa’ opaca Ait. f. xanthocarpa Rehd. ‘Calloway’ ‘Boyce Thompson Xanthocarpa’ opaca Ait. f. xanthocarpa Rehd. ‘Calloway’ ‘Boyce Thompson Xanthocarpa’ opaca Ait. f. xanthocarpa Rehd. opaca Ait. opaca Ait. ‘Knight’ ‘La Bar’ ‘Calloway’ opaca Ait. opaca Ait. ‘La Bar’ ‘Lake Ci opaca Ait. ‘Lake City’ 29 opaca Ait. f. xanthocarpa Rehd. ‘Canary’ opaca Ait. f. xanthocarpa Rehd. ‘Corpening #1’ opaca Ait. f. xanthocarpa Rehd. ‘Dengle Belles’ opaca Ait. f. xanthocarpa Rehd. ‘Fruitland Nursery’ opaca Ait. f. xanthocarpa Rehd. ‘Goldie’ opaca Ait. f. xanthocarpa Rehd. ‘Lenape Moon’ opaca Ait. f. xanthocarpa Rehd. ‘Margaret Moran’ opaca Ait. f. xanthocarpa Rehd. ‘Morgan Gold’ opaca Ait. f. xanthocarpa Rehd. ‘Old Gold’ opaca Ait. f. xanthocarpa Rehd. ‘Wilmat Yellow’ opaca Ait. f. xanthocarpa Rehd. ‘Yellow Jacquet’ paraguariensis St.Hil. pedunculosa Miq. perado Ait. ssp. platyphylla (P.B.Webb & Berth.) S.Andrews pernyi Franch. pernyi Franch. ‘Recurva’ poneantha Koidz. pubescens Hook. & Arn. purpurea Hassk. rotunda Thunb. ex J.A.Murr. rotunda Thunb. ex J.A.Murr. ‘Lord’ rugosa F.Schmidt serrata Thunb. ex J.A.Murr. serrata Thunb. ex J.A.Murr. ‘Leucocarpa’ serrata ~ I. verticillata (serrata < I. verticillata) ‘Apollo’ (serrata < I. verticillata) ‘Autumn Glow’ (serrata < I. verticillata) ‘Harvest Red’ (serrata x I. verticillata) ‘Sparkleberry’ shennongjiaensis T.R.Dudley & Sun sikkimensis Hook. spinigera (Loesn.) Loesn. sugerokii Maxim. sugerokii Maxim. ‘Aka tsuge’ sugerokii Maxim. ‘Nakaharae’ verticillata (L.) A.Gr. verticillata (L.) A.Gr. ‘Afterglow’ verticillata (L.) A.Gr. ‘Aurantiaca’ verticillata (L.) A.Gr. ‘Bright Horizon’ verticillata (L.) A.Gr. ‘Cacapon’ verticillata (L.) A.Gr. ‘Christmas Cheer’ verticillata (L.) A.Gr. ‘Christmas Gem’ verticillata (L.) A.Gr. ‘Earlibright’ verticillata (L.) A.Gr. ‘Fairfax’ verticillata (L.) A.Gr. ‘Jackson’ verticillata (L.) A.Gr. ‘Maryland Beauty’ INDIGOFERA LL. FABACEAE (Faboideae) verticillata (L.) A.Gr. ‘Red Sprite’ verticillata (L.) A.Gr. ‘Richard E. Lincoln’ verticillata (L.) A.Gr. ‘Shaver’ verticillata (L.) A.Gr. ‘Sunset’ verticillata (L.) A.Gr. ‘Winter Red’ verticillata (L.) A.Gr. f. chrysocarpa Robinson vomitoria Ait. vomitoria Ait. ‘Compacta’ vomitoria Ait. ‘Dare County’ vomitoria Ait. ‘Dewerth’ vomitoria Ait. ‘Fort McCoy’ vomitoria Ait. ‘Gray’s Bigleaf vomitoria Ait. ‘Gray’s Little Leaf’ vomitoria Ait. ‘Huber’s Compact’ vomitoria Ait. ‘Nana’ vomitoria Ait. ‘Otis Miley’ vomitoria Ait. ‘Shilling’s’ vomitoria Ait. ‘Shilling’s Dwarf’ vomitoria Ait. ‘Stokes Dwarf’ vomitoria Ait. ‘Yawkeyii’ vomitoria Ait. f. pendula Foret & Solym. vomitoria Ait. f. ‘Perle LeClair’ opaca Ait. f. xan pendula Foret & Solym. ‘Folsom’s Weeping’ vomitoria Ait. var. chiapiensis Sharp wilsonii Loesn. yunnanensis Franch. yunnanensis Franch. var. gentilis Loesn. zhejiangensis C.J.Tseng ILLICIUM L. ILLICIACEAE Mlicitum Family anisatum L. floridanum Ellis floridanum Ellis f. album F.G.Mey. & Mazzeo floridanum Ellis f. album F.G.Mey. & Mazzeo ‘Semmes’ henryi Diels mexicanum A.C.Sm. parviflorum Michx. ex Vent. Indigo Bean Family decora Lindl. decora Lindl. ‘Alba’ kirilowii Maxim. potaninii Craib suffruticosa Mill. opaca Ait. f. xanthocarpa Rehd. ‘Corpening #1’ opaca Ait. f. xanthocarpa Rehd. opaca Ait. f. xanthocarpa Rehd. ‘Dengle Belles’ ‘Dengle Belles’ opaca Ait. f. xanthocarpa Rehd. ‘Fruitland Nursery’ opaca Ait. f. xanthocarpa Rehd. ‘Fruitland Nursery’ ‘Fruitland Nursery’ opaca Ait. f. xanthoc ‘Fruitland Nursery’ opaca Ait. f. xanthoca ‘Fruitland Nursery’ opaca Ait. f. xanthocarpa Rehd. ‘Goldie’ opaca Ait. f. xanthocarpa Rehd. opaca Ait. f. xanthocarpa Rehd. ‘Lenape Moon’ ‘Lenape Moon’ opaca Ait. f. xan ‘Lenape Moon’ opaca Ait. f. xan opaca Ait. f. xanthocarpa Rehd. ‘Margaret Moran’ ‘Margaret Moran’ opaca Ait. f. xantho opaca Ait. f. xanthocarpa Rehd. ‘Yellow Jacquet’ pernyi Franch. ‘Recurva’ poneantha Koidz. INDIGOFERA LL. FABACEAE (Faboideae) ILLICIUM L. ILLICIACEAE Mlicitum Family anisatum L. floridanum Ellis floridanum Ellis f. album F.G.Mey. & Mazzeo floridanum Ellis f. album F.G.Mey. & Mazzeo ‘Semmes’ henryi Diels mexicanum A.C.Sm. parviflorum Michx. ex Vent. Indigo Bean Family decora Lindl. decora Lindl. ‘Alba’ kirilowii Maxim. potaninii Craib suffruticosa Mill. 280 chinensis L. ‘Robusta Green’ chinensis L. ‘San Jose... chinensis L. ‘Seagreen’ chinensis L. ‘Sheppardii’ chinensis L. ‘Spartan’ chinensis L. ‘Variegata’ chinensis L. var. sargentii A.Henry chinensis L. var. sargentii A.Henry ‘Viridis’ communis L. communis L. ‘Berkshire’ communis L. ‘Hibernica’ communis L. ‘Oblonga Pendula’ communis L. ‘Pencil Point’ communis L. ‘Sentinel’ communis L. ssp. depressa (Pursh) Franco communis L. var. montana Ait. communis L. var. montana Ait. ‘Hornibrookii’ conferta Parl. conferta Parl. ‘Blue Pacific’ conferta Parl. ‘Emerald Sea’ conferta Parl. ‘Gulf Tide’ davurica Pall. ‘Expansa’ davurica Pall. ‘Expansa Aureo-spicata’ davurica Pall. ‘Expansa Variegata’ deppeana Steud. var. pachyphlaea (Torr.) Martinez excelsa Bieb. ‘Stricta’ formosana Hayata horizontalis Moench horizontalis Moench ‘Admirabilis’ horizontalis Moench ‘Bar Harbor’ horizontalis Moench ‘Blue Chip’ horizontalis Moench ‘Blue Forest’ horizontalis Moench ‘Blue Horizon’ horizontalis Moench ‘Blue Muffet’ horizontalis Moench ‘Blue Rug’ horizontalis Moench ‘Douglasii’ horizontalis Moench ‘Filicinus’ horizontalis Moench ‘Filicinus Minimus’ horizontalis Moench ‘Glauca’ horizontalis Moench ‘Glenmore’ horizontalis Moench ‘Glomerata’ horizontalis Moench ‘Hughes’ horizontalis Moench ‘Livida’ horizontalis Moench ‘Marcella’ horizontalis Moench ‘Petraea’ horizontalis Moench ‘Plumosa’ horizontalis Moench ‘Prince of Wales’ horizontalis Moench ‘Procumbens’ horizontalis Moench ‘Pulchella’ horizontalis Moench ‘Wiltonii’ <x media Van Melle ‘Blue Cloud’ <x media Van Melle ‘Blue Vase’ chinensis L. ‘Robusta Green’ chinensis L. ‘San Jose... chinensis L. ‘Seagreen’ chinensis L. ‘Sheppardii’ chinensis L. ‘Spartan’ chinensis L. ‘Variegata’ chinensis L. var. sargentii A.Henry chinensis L. var. sargentii A.Henry ‘Viridis’ communis L. communis L. ‘Berkshire’ communis L. ‘Hibernica’ communis L. ‘Oblonga Pendula’ communis L. ‘Pencil Point’ communis L. ‘Sentinel’ communis L. ssp. depressa (Pursh) Franco communis L. var. montana Ait. communis L. var. montana Ait. ‘Hornibrookii’ conferta Parl. conferta Parl. ‘Blue Pacific’ conferta Parl. ‘Emerald Sea’ conferta Parl. ‘Gulf Tide’ davurica Pall. ‘Expansa’ davurica Pall. ‘Expansa Aureo-spicata’ davurica Pall. ‘Expansa Variegata’ deppeana Steud. var. pachyphlaea (Torr.) Martinez excelsa Bieb. ‘Lenape Moon’ opaca Ait. f. xan < media Van Melle ‘Fruitlandii’ < media Van Melle ‘Mint Julep’ <x media Van Melle ‘Old Gold’ < media Van Melle ‘Pfitzeriana’ < media Van Melle ‘Pfitzeriana Aurea’ < media Van Melle ‘Pfitzeriana Compacta’ < media Van Melle ‘Plumosa Aurea’ procumbens (Endl.) Miq. procumbens (Endl.) Miq. ‘Nana’ rigida Sieb. & Zucc. sabina L. ‘Arcadia’ sabina L. ‘Broadmoor’ sabina L. ‘Buffalo’ sabina L. ‘Fastigiata’ sabina L. ‘Skandia’ sabina L. ‘Von Ehren’ scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. scopulorum Sarg. Weeping’ scopulorum Sarg ‘Alba’ ‘Blue Heaven’ ‘Chandler Blue’ ‘Gareei’ ‘Gray Gleam’ ‘Hill’s Silver’ ‘Lakewood’ ‘Lakewood Globe’ ‘Pathfinder’ ‘Platinum’ ‘Tolleson’s Blue ‘Wichita Blue’ silicicola (Small) Bailey squamata Buch.-Ham. ex Lamb. squamata Buch.-Ham. ex Lamb. ‘Blue Star’ squamata Buch.-Ham. ex Lamb. ‘Loderi’ squamata Buch.-Ham. ex Lamb. ‘Meyeri’ virginiana L. virginiana L. ‘Aurea’ virginiana L. ‘Canaertii’ virginiana L. ‘Fastigiata’ virginiana L. ‘Glauca’ virginiana L. ‘Grey Owl’ virginiana L. ‘Keteleeri’ virginiana L. ‘Nova’ virginiana L. ‘Reptans’ virginiana L. ‘Skyrocket’ JUSTICIA L. ACANTHACEAE Acanthus Family brandegeana Wassh. & L.B.Sm. brandegeana Wassh. & L.B.Sm. ‘Yellow Queen’ californica (Benth.) D.Gibson KADSURA Juss. SCHISANDRACEAE Schisandra Family Japonica (Thunb.) Dunal KALMIA lL. ERICACEAE Heath Family angustifolia L. angustifolia L. var. carolina (Small) Fern. cuneata Michx. hirsuta < K. latifolia latifolia L. * latifolia L. ‘Bettina’ latifolia L. ‘Dexter Pink’ latifolia L. ‘Fuscata’ latifolia L. ‘Ostbo Red’ latifolia L. ‘Sharon Rose’ latifolia L. ‘Shooting Star’ latifolia L. f. angustata Rehd. KALOPANAX Miq. ARALIACEAE Ginseng Family septemlobus (Thunb. ex J.A.Murr.) Koidz. KERRIA DC. ROSACEAE Rose Family japonica (L.) DC. japonica (L.) DC. ‘Albescens’ japonica (L.) DC. ‘Picta’ japonica (L.) DC. ‘Pleniflora’ KOELREUTERIA Laxm. Golden-rain Tree SAPINDACEAE Soapberry Family bipinnata Franch. elegans (Seemann) A.C.Sm. ssp. formosana (Hayata) F.G.Mey. paniculata Laxm. paniculata Laxm. ‘Fastigiata’ paniculata Laxm. ‘September’ KOLKWITZIA_ Graebn. CAPRIFOLIACEAE Honeysuckle Family amabilis Graebn. amabilis Graebn. ‘Rosea’ +LABURNOCYTISUS Schneid. FABACEAE (Faboideae) Bean Family adamii (Poit.) Schneid. sabina L. ‘Arcadia’ sabina L. ‘Broadmoo scopulorum Sarg. scopulorum Sarg. ‘Hill’s Silver’ ‘Lakewood’ scopulorum Sarg. scopulorum Sarg. ‘Lakewood’ ‘Lakewood G scopulorum Sarg. scopulorum Sarg. ‘Lakewood Globe’ ‘Pathfinder’ scopulorum Sarg. scopulorum Sarg. ‘Pathfinder’ ‘Platinum’ scopulorum Sarg. scopulorum Sarg. ‘Platinum’ ‘Tolleson’s scopulorum Sarg. Weeping’ ‘Tolleson’s Blue ‘Lenape Moon’ opaca Ait. f. xan ‘Stricta’ formosana Hayata horizontalis Moench horizontalis Moench ‘Admirabilis’ horizontalis Moench ‘Bar Harbor’ horizontalis Moench ‘Blue Chip’ horizontalis Moench ‘Blue Forest’ horizontalis Moench ‘Blue Horizon’ horizontalis Moench ‘Blue Muffet’ horizontalis Moench ‘Blue Rug’ horizontalis Moench ‘Douglasii’ horizontalis Moench ‘Filicinus’ horizontalis Moench ‘Filicinus Minimus’ horizontalis Moench ‘Glauca’ horizontalis Moench ‘Glenmore’ horizontalis Moench ‘Glomerata’ horizontalis Moench ‘Hughes’ horizontalis Moench ‘Livida’ horizontalis Moench ‘Marcella’ horizontalis Moench ‘Petraea’ horizontalis Moench ‘Plumosa’ horizontalis Moench ‘Prince of Wales’ horizontalis Moench ‘Procumbens’ horizontalis Moench ‘Pulchella’ horizontalis Moench ‘Wiltonii’ JASMINUM L. OLEACEAE JUGLANS lL. JUGLANDACEAE JUNIPERUS L. CUPRESSACEAE ITEA L. SAXIFRAGACEAE (Iteoideae) Saxifrage Family ilicifolia Oliv. virginica L. JACARANDA Juss. BIGNONIACEAE Bignonia Family acutifolia HBK. Jasmine Olive Family beesianum Forrest & Diels floridum Bunge fruticans L. humile L. humile L. ‘Revolutum’ mesnyi Hance multiflorum (Burm.f.) Andr. nitidum Skan nudiflorum Lindl. parkeri S.T.Dunn x stephanense E.Lemoine Walnut Walnut Family ailantifolia Carr. ailantifolia Carr. var. cordiformis (Maxim.) Rehd. cinerea L. (hindsii < J. regia) ‘Paradox’ major (Torr.) Heller nigra L. nigra L. ‘Laciniata’ regia L. regia L. ‘Broadview’ regia L. ‘Hansen’ regia L. ‘McDermid’ Juniper Cypress Family chinensis L. chinensis L. ‘Aurea’ chinensis L. ‘Blaauw’ chinensis L. ‘Columnaris’ chinensis L. ‘Echiniformis’ chinensis L. ‘Globosa Cinerea’ chinensis L. ‘Gold Star’ chinensis L. ‘Hetzii’ chinensis L. ‘Kaizuka’ chinensis L. ‘Kaizuka Variegated’ chinensis L. ‘Parsonii’ chinensis L. ‘Blaauw’ chinensis L. ‘Columnar chinensis L. ‘Columnaris’ chinensis L. ‘Echiniformis’ chinensis L. ‘Echiniformis’ chinensis L. ‘Globosa Cinerea chinensis L. ‘Globosa Cinerea’ chinensis L. ‘Gold Star’ chinensis L. ‘Gold Star’ chinensis L. ‘Hetzii’ chinensis L. ‘Hetzii’ chinensis L. ‘Kaizuka chinensis L. ‘Kaizuka’ chinensis L. ‘Kaizuka V chinensis L. ‘Kaizuka Variegated’ chinensis L. ‘Parsonii’ chinensis L. ‘Parsonii’ 281 KADSURA Juss. SCHISANDRACEAE Schisandra Family Japonica (Thunb.) Dunal KALMIA lL. ERICACEAE Heath Family angustifolia L. angustifolia L. var. carolina (Small) Fern. cuneata Michx. hirsuta < K. latifolia latifolia L. * latifolia L. ‘Bettina’ latifolia L. ‘Dexter Pink’ latifolia L. ‘Fuscata’ latifolia L. ‘Ostbo Red’ latifolia L. ‘Sharon Rose’ latifolia L. ‘Shooting Star’ latifolia L. f. angustata Rehd. KALOPANAX Miq. ARALIACEAE Ginseng Family septemlobus (Thunb. ex J.A.Murr.) Koidz. KERRIA DC. ROSACEAE Rose Family japonica (L.) DC. japonica (L.) DC. ‘Albescens’ japonica (L.) DC. ‘Picta’ japonica (L.) DC. ‘Pleniflora’ KOELREUTERIA Laxm. Golden-rain Tree SAPINDACEAE Soapberry Family bipinnata Franch. elegans (Seemann) A.C.Sm. ssp. formosana (Hayata) F.G.Mey. paniculata Laxm. paniculata Laxm. ‘Fastigiata’ paniculata Laxm. ‘September’ KOLKWITZIA_ Graebn. CAPRIFOLIACEAE Honeysuckle Family amabilis Graebn. amabilis Graebn. ‘Rosea’ +LABURNOCYTISUS Schneid. FABACEAE (Faboideae) Bean Family adamii (Poit.) Schneid. Weeping’ scopulorum japonica (L.) DC. ‘Picta’ japonica (L.) DC. ‘Pleniflora squamata Buch.-Ham. ex Lamb. squamata Buch.-Ham. ex Lamb. japonica (L.) DC. ‘Pleniflora’ 282 Golden-chain Tree Bean Family LABURNUM Medik. FABACEAE (Faboideae) anagyroides Medik. xX watereri (Kirchn.) Dipp. LAGERSTROEMIA lL. LYTHRACEAE Crape Myrtle Loosestrife Family fauriei Koehne _ indica L. indica L. ‘Alba’ indica L. ‘Carolina Beauty’ * indica L. ‘Potomac’ subcostata Koehne Note: The parentage of the following cultivars is complex; see catalog entries for details. * ‘Acoma’ * ‘Apalachee’ ‘Basham’s Party Pink’ * ‘Biloxi’ * ‘Caddo’ * ‘Catawba’ * ‘Cherokee’ * ‘Choctaw’ * ‘Comanche’ * ‘Conestoga’ * ‘Hopi’ * ‘Lipan’ * ‘Miami’ * ‘Muskogee’ * ‘Natchez’ * ‘Osage’ * ‘Pecos’ * ‘Powhatan’ * ‘Seminole’ * ‘Sioux’ * ‘Tonto’ * ‘Tuscarora’ * ‘Tuskegee’ * ‘Wichita’ * ‘Yuma’ * ‘Zuni’ LANTANA L. VERBENACEAE Verbena Family camara L. montevidensis (Spreng.) Briq. LARIX Mill. Larch PINACEAE Pine Family LAURUS lL. Laurel LAURACEAE ~ Laurel Family nobilis L. LAVANDULA L. LAMIACEAE Lavender Mint Family angustifolia Mill. angustifolia Mill. ‘Provence’ angustifolia Mill. ssp. angustifolia ‘Hidcote’ x intermedia Emeric ex Loisel. stoechas L. LEDUM L. ERICACEAE Heath Family palustre L. var. diversipilosum Nakai LEIOPHYLLUM R.Hedwig ERICACEAE Sand Myrtle Heath Family buxifolium (Bergius) Ell. buxifolium (Bergius) Ell. var. hugeri (Small) Schneid. LEITNERIA Chapm. LEITNERIACEAE Leitneria Family floridana Chapm. LEPTODERMIS Wall. RUBIACEAE Madder Family oblonga Bunge LEPTOSPERMUM J.R. &J.G. Forst. MYRTACEAE Myrtle Family scoparium J.R. & J.G. Forst. ‘Plenum’ LESPEDEZA Michx. FABACEAE (Faboideae) Bush Clover Bean Family bicolor Turcz. cyrtobotrya Miq. maximowiczii Schneid. thunbergii (DC.) Nakai thunbergii (DC.) Nakai ‘Albiflora’ Crape Myrtle Loosestrife Family * indica L. ‘Potomac’ * ‘Acoma’ * ‘Apalachee * ‘Apalachee’ ‘Basham’s Pa ‘Basham’s Party Pink’ * ‘Biloxi’ * ‘Biloxi’ * ‘Caddo’ * ‘Biloxi’ * ‘Caddo’ * ‘Caddo’ * ‘Catawba’ * ‘Catawba’ * ‘Cherokee’ * ‘Cherokee’ * ‘Choctaw’ * ‘Choctaw’ * ‘Comanche’ * ‘Comanche’ * ‘Conestoga’ * ‘Conestoga’ * ‘Hopi’ * ‘Hopi’ * ‘Lipan’ * ‘Lipan’ * ‘Miami’ * ‘Miami’ * ‘Muskoge 283 benzoin (L.) Bl. var. pubescens (Palm. & Steyerm.) Rehd. obtusiloba BI. strychnifolia (Sieb. & Zucc.) F.Vill. LIQUIDAMBAR L. HAMAMELIDACEAE Sweet Gum Witch-hazel Family formosana Hance orientalis Mill. styraciflua L. styraciflua L. ‘Gum Ball’ styraciflua L. ‘Variegata’ styraciflua L. f. rotundiloba Rehd. LIRIODENDRON L. MAGNOLIACEAE Tulip Tree Magnolia Family chinense (Hemsl.) Sarg. tulipifera L. tulipifera L. ‘Aureo-marginatum’ LITHOCARPUS Bl. FAGACEAE Tanbark Oak Beech Family corneus (Lour.) Rehd. edulis (Makino) Nakai glaber (Thunb.) Nakai henryi (Seemann) Rehd. & Wils. LITSEA Lam. Weeping’ scopulorum ‘Shady Grove No. 5’ grandiflora L. ‘Shady Grove No. 6’ grandiflora L. ‘Variegata’ grandiflora L. ‘Victoria’ x kewensis Pearce ‘Wada’s Memory’ kobus DC. liliiflora Desr. liliiflora Desr. ‘Nigra’ liliiflora Desr. ‘O’Neill’ liliiflora Desr. ‘Reflorescens’ * (liliiflora ‘Nigra’ < M. sprengeri ‘Diva’) ‘Galaxy’ * (liliiflora ‘Nigra’ < M. sprengeri ‘Diva’) ‘Spectrum’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) aay’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Betty’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Judy’ x (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Randy’ x (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Ricki’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Susan’ * (liliiflora ‘Reflorescens’ < M. stellata ‘Rosea’) ‘Pinkie’ * (liliiflora ‘Reflorescens’ < M. stellata ‘Waterlily’) ‘Jane’ <x loebneri Kache < loebneri Kache ‘Merrill’ x loebneri Kache ‘Neil McEacharn’ < loebneri Kache ‘Spring Snow’ macrophylla Michx. 9 morrowii A.Gr. nitida Wils. nitida Wils. ‘Baggesen’s Gold’ nitida Wils. ‘Ernest Wilson’ pileata Oliv. sempervirens L. sempervirens L. ‘Sulphurea’ sempervirens L. ‘Superba’ standishii Jacq. subsessilis Rehd. tatarica L. tatarica L. ‘Arnold Red’ tatarica L. ‘Hack’s Red’ tatarica L. ‘Punicea’ xylosteum L. xylosteum L. ‘Nana’ xylosteum L. f. glabrescens Zab. LOROPETALUM R.Br. ex Reichenb. HAMAMELIDACEAE Witch-hazel Family chinense (R.Br.) Oliv. LYCIUM L. Matrimony Vine SOLANACEAE Nightshade Family carolinianum Walt. chinense Mill. LYONIA Nutt. ERICACEAE Heath Family ferruginea (Walt.) Nutt. ligustrina (L.) DC. lucida (Lam.) K.Koch mariana (L.) D.Don MAACKIA Rupr. FABACEAE (Faboideae) Bean Family amurensis Rupr. & Maxim. tenuifolia (Hemsl.) Hand.-Mazz. MACFADYENA A.DC. BIGNONIACEAE Bignonia Family unguis-cati (L.) A.Gentry MACLURA Nuit. MORACEAE Mulberry Family pomifera (Raf.) Schneid. MAGNOLIA L. MAGNOLIACEAE Magnolia Magnolia Family (?acuminata <x M. denudata) ‘Sundance’ > ashei Weatherby x brooklynensis Kalmb. ‘Woodsman’ cylindrica Wils. dealbata Zucc. denudata Desr. Sfraseri Walt. grandiflora L. grandiflora L. ‘Baldwin’ grandiflora L. ‘Charles Dickens’ grandiflora L. ‘Claudia Wanamaker grandiflora L. ‘Hasse’ grandiflora L. ‘Little Gem’ grandiflora L. ‘Majestic Beauty’ grandiflora L. ‘Margaret Davis’ grandiflora L. ‘Pioneer’ grandiflora L. ‘Praecox’ grandiflora L. ‘Praecox Fastigiata’ grandiflora L. ‘St. Mary’ grandiflora L. ‘Samuel Sommer’ grandiflora L. ‘Shady Grove No. 4’ grandiflora L. ‘Shady Grove No. 5’ grandiflora L. ‘Shady Grove No. 6’ grandiflora L. ‘Variegata’ grandiflora L. ‘Victoria’ x kewensis Pearce ‘Wada’s Memory’ kobus DC. liliiflora Desr. liliiflora Desr. ‘Nigra’ liliiflora Desr. ‘O’Neill’ liliiflora Desr. ‘Reflorescens’ * (liliiflora ‘Nigra’ < M. sprengeri ‘Diva’) ‘Galaxy’ * (liliiflora ‘Nigra’ < M. sprengeri ‘Diva’) ‘Spectrum’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) aay’ * (liliiflora ‘Nigra’ < M. Weeping’ scopulorum LAURACEAE Laurel Family aestivalis (L.) Fern. LIVISTONA R.Br. ARECACEAE Palm Family chinensis (Jacq.) R.Br. ex Mart. LONICERA lL. Honeysuckle CAPRIFOLIACEAE Honeysuckle Family x bella Zab. < brownii (Regel) Carr. flava Sims fragrantissima Lindl. & Paxt. gracilipes Miq. gracilipes Miq. var. glandulosa Maxim. x heckrottii Rehd. LEUCOPHYLLUM Humboldt & Bonpland SCROPHULARIACEAE Figwort Family Srutescens (Berl.) I.M.Johnst. LEUCOTHOE D.Don ERICACEAE Heath Family axillaris (Lam.) D.Don fontanesiana (Steud.) Sleumer fontanesiana (Steud.) Sleumer ‘Girard’s Rainbow’ fontanesiana (Steud.) Sleumer ‘Nana’ fontanesiana (Steud.) Sleumer ‘Rollissonii’ fontanesiana (Steud.) Sleumer ‘Zebekot’ fontanesiana (Steud.) Sleumer ‘Zebonard’ racemosa (L.) A.Gr. LEUCOTHOE D.Don ERICACEAE Heath Family axillaris (Lam.) D.Don fontanesiana (Steud.) Sleumer fontanesiana (Steud.) Sleumer ‘Girard’s Rainbow’ fontanesiana (Steud.) Sleumer ‘Nana’ fontanesiana (Steud.) Sleumer ‘Rollissonii’ fontanesiana (Steud.) Sleumer ‘Zebekot’ fontanesiana (Steud.) Sleumer ‘Zebonard’ racemosa (L.) A.Gr. compactum Hook.f. & Thoms. delavayanum Hariot japonicum Thunb. japonicum Thunb. ‘Erecta’ japonicum Thunb. ‘Fraseri’ japonicum Thunb. ‘Iwata’ japonicum Thunb. ‘Lake Treska’ japonicum Thunb. ‘Nobilis’ japonicum Thunb. ‘Repandens’ japonicum Thunb. ‘Rotundifolium’ japonicum Thunb. ‘Silver Star’ japonicum Thunb. ‘Suwanee River’ japonicum Thunb. ‘Variegatum’ lucidum Ait.f. obtusifolium Sieb. & Zucc. ovalifolium Hassk. ovalifolium Hassk. ‘Aureo-marginatum’ quihoui Carr. sempervirens (Franch.) Mansf. sinense Lour. sinense Lour. ‘Fraseri’ sinense Lour. ‘Pendulum’ sinense Lour. ‘Wimbish’ tschonoskii Decne. X< vicaryi Rehd. vulgare L. LINDERA Thunb. LAURACEAE Spicebush Laurel Family angustifolia W.C.Cheng benzoin (L.) Bl. compactum Hook.f. & Thoms. delavayanum Hariot japonicum Thunb. japonicum Thunb. ‘Erecta’ japonicum Thunb. ‘Fraseri’ japonicum Thunb. ‘Iwata’ japonicum Thunb. ‘Lake Treska’ japonicum Thunb. ‘Nobilis’ japonicum Thunb. ‘Repandens’ japonicum Thunb. ‘Rotundifolium’ japonicum Thunb. ‘Silver Star’ japonicum Thunb. ‘Suwanee River’ japonicum Thunb. ‘Variegatum’ lucidum Ait.f. obtusifolium Sieb. & Zucc. ovalifolium Hassk. ovalifolium Hassk. ‘Aureo-marginatum’ quihoui Carr. sempervirens (Franch.) Mansf. sinense Lour. sinense Lour. ‘Fraseri’ sinense Lour. ‘Pendulum’ sinense Lour. ‘Wimbish’ tschonoskii Decne. X< vicaryi Rehd. vulgare L. LINDERA Thunb. LAURACEAE Spicebush Laurel Family angustifolia W.C.Cheng benzoin (L.) Bl. obtusifolium Sieb. & Zucc. ovalifolium Hassk. ovalifolium Hassk. ovalifolium Hassk. quihoui Carr. sempervirens angustifolia W.C.Cheng benzoin (L.) Bl. angustifolia W.C.Cheng benzoin (L.) Bl. maackii Maxim. maackii Maxim. 284 (?acuminata <x M. denudata) ‘Sundance’ > ashei Weatherby x brooklynensis Kalmb. ‘Woodsman’ cylindrica Wils. dealbata Zucc. denudata Desr. Sfraseri Walt. grandiflora L. grandiflora L. ‘Baldwin’ grandiflora L. ‘Charles Dickens’ grandiflora L. ‘Claudia Wanamaker grandiflora L. ‘Hasse’ grandiflora L. ‘Little Gem’ grandiflora L. ‘Majestic Beauty’ grandiflora L. ‘Margaret Davis’ grandiflora L. ‘Pioneer’ grandiflora L. ‘Praecox’ grandiflora L. ‘Praecox Fastigiata’ grandiflora L. ‘St. Mary’ grandiflora L. ‘Samuel Sommer’ grandiflora L. ‘Shady Grove No. 4’ grandiflora L. ‘Superba Rosea’ x soulangeana So x soulangeana Soul.-Bod. ‘Verbanica’ sprengeri Pamp. ‘Diva’ sprengeri Pamp. ‘Diva’ stellata (Sieb. & Zucc.) stellata (Sieb. & Zucc.) Maxim. stellata (Sieb. & Zucc.) Maxim. stellata (Sieb. & Zucc.) Maxim. stellata (Sieb. & Zucc.) Maxim. stellata (Sieb. & Zucc.) Maxim. ‘Pink Stardust’ stellata (Sieb. & Zucc.) Maxim. ‘Pink Stardust’ ‘Grace McDade’ < soulangeana So < soulangeana Soul.-Bod. ‘Lennei’ x soulangeana Soul.-Bod. ‘Lilliputian’ x soulangeana Soul.-Bod. ‘Lilliputian’ < soulangeana Soul.-Bod. ‘Picture’ < soulangeana Soul.-Bod. ‘Picture’ x soulangeana Soul.-Bod. ‘Purpliana’ x soulangeana Soul.-Bod. ‘Purpliana’ < soulangeana Soul.-Bod. ‘Rubra’ < soulangeana Soul.-Bod. ‘Rubra’ x soulangeana Soul.-Bod. ‘Rustica’ x soulangeana Soul.-Bod. ‘Rustica’ x soulangeana Soul.-Bod. ‘San Jose’ x soulangeana Soul.-Bod. ‘San Jose’ x soulangeana Soul.-Bod. ‘Sundew’ x soulangeana Soul.-Bod. ‘Sundew’ x soulangeana Soul.-Bod. x soulangeana Soul.-Bod. ‘Superba Rosea’ Weeping’ scopulorum ‘Alexan x soulangeana Soul.-Bod. ‘Alexandrina’ < soulangeana Soul.-Bod. ‘C. H. Kern’ < soulangeana Soul.-Bod. ‘C. H. Kern’ x soulangeana Soul.-Bod. soulangeana Soul.-Bod. ‘Grace McDade’ Weeping’ scopulorum stellata ‘Rosea’) ‘Betty’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Judy’ x (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Randy’ x (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Ricki’ * (liliiflora ‘Nigra’ < M. stellata ‘Rosea’) ‘Susan’ * (liliiflora ‘Reflorescens’ < M. stellata ‘Rosea’) ‘Pinkie’ * (liliiflora ‘Reflorescens’ < M. stellata ‘Waterlily’) ‘Jane’ <x loebneri Kache < loebneri Kache ‘Merrill’ 9 morrowii A.Gr. nitida Wils. nitida Wils. ‘Baggesen’s Gold’ nitida Wils. ‘Ernest Wilson’ pileata Oliv. sempervirens L. sempervirens L. ‘Sulphurea’ sempervirens L. ‘Superba’ standishii Jacq. subsessilis Rehd. tatarica L. tatarica L. ‘Arnold Red’ tatarica L. ‘Hack’s Red’ tatarica L. ‘Punicea’ xylosteum L. xylosteum L. ‘Nana’ xylosteum L. f. glabrescens Zab. LOROPETALUM R.Br. ex Reichenb. HAMAMELIDACEAE Witch-hazel Family chinense (R.Br.) Oliv. LYCIUM L. Matrimony Vine SOLANACEAE Nightshade Family carolinianum Walt. chinense Mill. LYONIA Nutt. ERICACEAE Heath Family ferruginea (Walt.) Nutt. ligustrina (L.) DC. lucida (Lam.) K.Koch mariana (L.) D.Don MAACKIA Rupr. FABACEAE (Faboideae) Bean Family amurensis Rupr. & Maxim. tenuifolia (Hemsl.) Hand.-Mazz. MACFADYENA A.DC. BIGNONIACEAE Bignonia Family unguis-cati (L.) A.Gentry MACLURA Nuit. MORACEAE Mulberry Family pomifera (Raf.) Schneid. (?acuminata <x M. denudata) ‘Sundance’ > denudata Desr. Sfraseri Walt. Sfraseri Walt. grandiflora L grandiflora L. grandiflora L. standishii Jacq. subsessilis Rehd. subsessilis Rehd. tatarica L. tatarica L. tatarica L. 285 MAHONIA Nutt. BERBERIDACEAE Barberry Family aquifolium (Pursh) Nutt. bealei (Fort.) Carr. chochoca Fedde fortunei (Lindl.) Fedde japonica (Thunb. ex J.A.Murr.) DC. oiwakensis Hayata < media Brickell ‘Charity’ pinnata (Lag.) Fedde swazeyi Buckl. trifoliolata (Moric.) Fedde Barberry Family bealei (Fort.) Carr. chochoca Fedde fortunei (Lindl.) Fedde japonica (Thunb. ex J.A.Murr.) DC. oiwakensis Hayata < media Brickell ‘Charity’ pinnata (Lag.) Fedde swazeyi Buckl. trifoliolata (Moric.) Fedde MALLOTUS Lour. EUPHORBIACEAE Spurge Family japonicus (Thunb. ex L.f.) Muell.-Arg. <xMALOSORBUS _ Browicz ROSACEAE Rose Family florentina (Zuccagni) Browicz MALPIGHIA lL. MALPIGHIACEAE Malpighia Family glabra L. MALUS UMiill. ROSACEAE Rose Family x adstringens Zab. ‘Athabasca’ < adstringens Zab. ‘Hopa’ angustifolia (Ait.) Michx. < arnoldiana (Rehd.) Sarg. (<x arnoldiana < M. domestica ‘Niedzwetzkyana’) ‘Henrietta Crosby’ (<arnoldiana < M. spectabilis) ‘Van Eseltine’ < atrosanguinea (Spaeth) Schneid. (<x atrosanguinea < M. sargentii ‘Rosea’) ‘Mary Potter’ baccata (L.) Borkh. baccata (L.) Borkh. var. mandschurica (Maxim.) Schneid. coronaria (L.) Mill. coronaria (L.) Mill. ‘Nieuwlandiana’ coronaria (L.) Mill. var. dasycalyx Rehd. coronaria (L.) Mill. var. elongata Rehd. domestica Borkh. domestica Borkh. ‘Veitch’s Scarlet’ floribunda Sieb. halliana Koehne halliana Koehne ‘Parkmanii’ * halliana Koehne hybrid ‘Adirondack’ < hartwigii Koehne ‘Katherine’ < soulangeana Soul.-Bod. ‘Alba’ x soulangeana Soul.-Bod. ‘Pink Stardust’ stellata (Sieb. & stellata (Sieb. & Zucc.) Maxim. ‘Rohrbach’ stellata (Sieb. & Zucc.) Maxim. ‘Rohrbach’ stellata (Sieb. & Zucc.) Maxim. ‘Rohrbach’ stellata (Sieb. & Zucc.) Maxim. ‘Rosea’ stellata (Sieb. & Zucc.) Maxim. ‘Royal Star’ stellata (Sieb. & Zucc.) Maxim. ‘Rubra’ stellata (Sieb. & Zucc.) Maxim. ‘Water Lily’ tripetala L. x veitchii Bean virginiana L. virginiana L. ‘Henry Hicks’ * virginiana L. ‘Satellite’ * (virginiana < M. grandiflora) ‘Freeman’ * (virginiana xX M. grandiflora) ‘Maryland’ <wieseneri Carr. <MAHOBERBERIS Schneid. BERBERIDACEAE Barberry Family ‘Rohrbach’ stellata (Sieb. & Zucc.) Maxim. ‘Rosea’ stellata (Sieb. & Zucc.) Maxim. ‘Royal Star’ ‘Water Lily’ tripetala L. 286 hupehensis (Pamp.) Rehd. ‘Sissipuk’ ioensis (A.Wood) Britton ‘Nova’ ‘Thomas Roland’ = ioensis (A.Wood) Britton ‘Plena’ ‘Timiskaming’ ioensis (A.Wood) Britton var. palmeri ‘Tops-in-Bloom’ Rehd. ‘Wintergold’ lancifolia Rehd. ‘Wynema’ x micromalus Makino x platycarpa Rehd. MALVAVISCUS Fabr. non Adans. prunifolia (Willd.) Borkh. MALVACEAE Mallow Family X purpurea (Barbier) Rehd. X purpurea (Barbier) Rehd. arboreus Cav. var. drummondii Schery ‘Centennial’ ‘Crimson Brilliant’ ‘David’ ‘East Malling’ ‘Evelyn’ ‘Flame’ ‘Golden Anniversary’ ‘Guiding Star’ ‘Henry F. Dupont’ ‘Hillier’ ‘Marshall Oyama’ ‘Mathews’ ‘Mrs. Bayard Thayer’ ‘Normand’ ‘Aldenhamensis’ arboreus Cav. var. penduliflorus (Sesse X purpurea (Barbier) Rehd. ‘Eleyi’ & Moc. ex DC.) Schery X purpurea (Barbier) Rehd. ‘Lemoinei’ x robusta (Carr.) Rehd. MANIHOT Mill. Cassava x robusta (Carr.) Rehd. ‘Golden EUPHORBIACEAE Spurge Family Hornet’ sargentii Rehd. grahamii Hook. sargentii Rehd. ‘Rosea’ x scheideckeri (Spaeth) Zab. MELALEUCA lL. x scheideckeri (Spaeth) Zab. MYRTACEAE Myrtle Family ‘Dorothea’ sieboldii (Regel) Rehd. quinquenervia (Cav.) S.T.Blake * sieboldii (Regel) Rehd. ‘Fuji’ x soulardii (Bailey) Britton ‘Red Tip’ MELIA L. spectabilis (Ait.) Borkh. MELIACEAE Mahogany Family x zumi (Matsum.) Rehd. * (/hybrid #28/ x M. ‘Wintergold’) azedarach L. ‘Narragansett’ ‘Almey’ MENZIESIA Sm. ERICACEAE Heath Family pilosa (Michx.) Juss. MESPILUS L. ROSACEAE Rose Family germanica L. METASEQUOIA Miki ex H.H.Hu & W.C.Cheng TAXODIACEAE Taxodium Family glyptostroboides H.H.Hu & W.C.Cheng ‘Oporto’ * glyptostroboides H.H.Hu & W.C.Cheng ‘Pink Pearl’ ‘National’ ‘Pink Spires’ ‘Prairie Rose’ MICHELIA UL. ‘Prince George’s’ MAGNOLIACEAE Magnolia Family ‘Profusion’ ‘Pygmy’ compressa (Maxim.) Sarg. ‘Red Jade’ figo (Lour.) Spreng. ‘Red Jewel’ ‘Red Silver’ MICROBIOTA Komar. hupehensis (Pamp.) Rehd. ioensis (A.Wood) Britton ‘Nova’ ioensis (A.Wood) Britton ‘Plena’ ioensis (A.Wood) Britton var. palmeri Rehd. lancifolia Rehd. x micromalus Makino x platycarpa Rehd. prunifolia (Willd.) Borkh. X purpurea (Barbier) Rehd. X purpurea (Barbier) Rehd. ‘Centennial’ ‘Crimson Brilliant’ ‘David’ ‘East Malling’ ‘Evelyn’ ‘Flame’ ‘Golden Anniversary’ ‘Guiding Star’ ‘Henry F. Dupont’ ‘Hillier’ ‘Marshall Oyama’ ‘Mathews’ ‘Mrs. ‘Pink Stardust’ stellata (Sieb. & Bayard Thayer’ ‘Normand’ ‘Aldenhamensis’ X purpurea (Barbier) Rehd. ‘Eleyi’ X purpurea (Barbier) Rehd. ‘Lemoinei’ x robusta (Carr.) Rehd. x robusta (Carr.) Rehd. ‘Golden Hornet’ sargentii Rehd. sargentii Rehd. ‘Rosea’ x scheideckeri (Spaeth) Zab. x scheideckeri (Spaeth) Zab. ‘Dorothea’ sieboldii (Regel) Rehd. * sieboldii (Regel) Rehd. ‘Fuji’ x soulardii (Bailey) Britton ‘Red Tip’ spectabilis (Ait.) Borkh. x zumi (Matsum.) Rehd. * (/hybrid #28/ x M. ‘Wintergold’) ‘Narragansett’ ‘Almey’ ‘Oporto’ ‘Pink Pearl’ ‘Pink Spires’ ‘Prairie Rose’ ‘Prince George’s’ ‘Profusion’ ‘Pygmy’ ‘Red Jade’ ‘Red Jewel’ ‘Red Silver’ ‘Golden Anniversary’ ‘Guiding Star’ ‘Guiding Star’ ‘Henry F. Dupon ‘Henry F. Dupont’ ‘Hillier’ ‘Pink Pearl’ ‘Pink Spires’ ‘Pink Spires’ ‘Prairie Rose’ ‘Prairie Rose’ ‘Prince George’ ‘Prince George’s’ ‘Profusion’ ‘Red Jade’ ‘Red Jewel’ ‘Red Jewel’ ‘Red Silver’ 287 MICROCITRUS Swingle RUTACEAE Citrus Family australasica (F.Muell.) Swingle MILLETTIA Wight & Arn. FABACEAE (Faboideae) Bean Family reticulata Benth. MIMOSA L. FABACEAE (Mimosoideae) Bean Family biuncifera Benth. pigra L. var. berlandieri (A.Gr.) B.L.Tumer MITCHELLA L. RUBIACEAE Madder Family repens L. MORUS L. Mulberry MORACEAE Mulberry Family alba L. alba L. ‘Hicks’ alba L. ‘Pendula’ alba L. ‘Teas Weeping’ rubra L. MUEHLENBECKIA Meisn. POLYGONACEAE Buckwheat Family axillaris (Hook.f.) Walp. MURRAYA Koenig ex L. RUTACEAE Citrus Family paniculata (L.) Jack MUSA L. MUSACEAE Banana Banana Family basjoo Sieb. & Zucc. x paradisiaca L. ‘Sapientum’ rosacea Jacq. velutina H.Wendl. & Drude MYRICA L. MYRICACEAE Bayberry Family cerifera L. heterophylla Raf. inodora Bartram pensylvanica Loisel. pusilla Raf. rubra Sieb. & Zucc. MYRTUS L. Myrtle MYRTACEAE Myrtle Family communis L. communis L. ‘Microphylla’ NANDINA Thunb. BERBERIDACEAE Barberry Family domestica Thunb. domestica Thunb. ‘Alba’ domestica Thunb. ‘Harbour Dwarf’ domestica Thunb. ‘Purpurea Nana’ NEILLIA D.Don ROSACEAE Rose Family sinensis Oliv. NEMOPANTAUS Raf. AQUIFOLIACEAE Holly Family mucronatus (L.) Trelease NERIUM L. APOCYNACEAE Dogbane Family oleander L. oleander L. ‘Variegata’ NEVIUSIA A.Gr. ROSACEAE Rose Family alabamensis A.Gr. NICODEMIA ‘Tenore BUDDLEJACEAE Buddleja Family diversifolia Tenore NICOTIANA lL. SOLANACEAE Nightshade Family glauca R.Graham NIEREMBERGIA Ruiz & Pavon SOLANACEAE Nightshade Family scoparia Sendtn. inodora Bartram pensylvanica Loisel. pusilla Raf. rubra Sieb. & Zucc. inodora Bartram pensylvanica Loisel. pusilla Raf. rubra Sieb. & Zucc. MICROCITRUS Swingle RUTACEAE Citrus Family australasica (F.Muell.) Swingle MILLETTIA Wight & Arn. FABACEAE (Faboideae) Bean Family reticulata Benth. MIMOSA L. FABACEAE (Mimosoideae) Bean Family biuncifera Benth. pigra L. var. berlandieri (A.Gr.) B.L.Tumer MITCHELLA L. RUBIACEAE Madder Family repens L. MORUS L. Mulberry MORACEAE Mulberry Family alba L. alba L. ‘Hicks’ alba L. ‘Pendula’ alba L. ‘Teas Weeping’ rubra L. ‘Pink Stardust’ stellata (Sieb. & MUEHLENBECKIA Meisn. POLYGONACEAE Buckwheat Family axillaris (Hook.f.) Walp. MURRAYA Koenig ex L. RUTACEAE Citrus Family paniculata (L.) Jack MUSA L. MUSACEAE Banana Banana Family basjoo Sieb. & Zucc. x paradisiaca L. ‘Sapientum’ rosacea Jacq. velutina H.Wendl. & Drude MYRICA L. MYRICACEAE Bayberry Family cerifera L. heterophylla Raf. Citrus Family 288 OXYDENDRUM DC. ERICACEAE ~ Heath Family arboreum (L.) DC. PACHYSANDRA Michx. BUXACEAE Boxwood Family axillaris Franch. procumbens Michx. terminalis Sieb. & Zucc. terminalis Sieb. & Zucc. ‘Silveredge’ PAEONIA lL. Peony PAEONIACEAE Peony Family suffruticosa Andr. PALAFOXIA Lag. ASTERACEAE Aster Family feayi A.Gr. PARKINSONIA lL. FABACEAE (Caesalpinioideae) Bean Family aculeata L. PARROTIA C.A.Mey. HAMAMELIDACEAE Witch-hazel Family persica (DC.) C.A.Mey. PARROTIOPSIS (Niedenzu) Schneid. HAMAMELIDACEAE Witch-hazel Family Jjacquemontiana (Decne.) Rehd. PARTHENOCISSUS Planch. VITACEAE Grape Family henryana (Hemsl.) Diels & Gilg quinquefolia (L.) Planch. tricuspidata (Sieb. & Zucc.) Planch. PASSIFLORA L. PASSIFLORACEAE __ Passion-flower Family coccinea Aubl. PAULOWNIA Sieb. & Zucc. NYSSA L. NYSSACEAE Sour-gum Family ogeche Marsh. sylvatica Marsh. sylvatica Marsh. var. biflora (Walt.) Sarg. OCHNA L. OCHNACEAE Ochna Family atropurpurea DC. OLEA L. OLEACEAE Olive Family europaea L. ORIGANUM lL. LAMIACEAE Mint Family onites L. ORIXA Thunb. RUTACEAE Citrus Family japonica Thunb. OSMANTHAUS Lour. OLEACEAE Olive Family americanus (L.) A.Gr. armatus Diels x fortunei Carr. x fortunei Carr. ‘San Jose’ fragrans Lour. fragrans Lour. ‘Aurantiacus’ heterophyllus (G.Don) P.S.Green var. heterophyllus heterophyllus (G.Don) P.S.Green var. heterophyllus ‘Gulf Tide’ heterophyllus (G.Don) P.S.Green var. heterophyllus ‘Purpureus’ heterophyllus (G.Don) P.S.Green var. heterophyllus ‘Rotundifolius’ heterophyllus (G.Don) P.S.Green var. heterophyllus ‘Variegatus’ OSTEOMELES Lindl. ROSACEAE Rose Family schwerinae Schneid. OSTRYA Scop. BETULACEAE Birch Family virginiana (Mill.) K.Koch PAVONIA Cav. MALVACEAE Rose Family NYSSA L. NYSSACEAE Sour-gum Family 289 PAVONIA Cav. MALVACEAE Mallow Family hastata Cav. PAXISTIMA Raf. CELASTRACEAE Staff-tree Family canbyi A.Gr. PERIPLOCA UL. ASCLEPIADACEAE Asclepias Family graeca L. PEROVSKIA Karelin LAMIACEAE Mint Family atriplicifolia Benth. PERSEA Mill. LAURACEAE Laurel Family americana Mill. borbonia (L.) Spreng. humilis Nash thunbergii (Sieb. & Zucc.) Kosterm. PETTERIA Presl FABACEAE (Faboideae) Bean Family ramentacea Presl PHELLODENDRON Rupr. RUTACEAE Citrus Family amurense Rupr. amurense Rupr. var. japonicum (Maxim.) Ohwi amurense Rupr. var. lavallei (Dode) Sprague PHILADELPHUS L. Mock Orange SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family coronarius L. coronarius L. ‘Belle Etoile’ coronarius L. ‘Duplex’ coronarius L. ‘Natchez’ delavayi L.Henry ‘Nymans’ <falconeri Sarg. hirsutus Nutt. inodorus L. x lemoinei V.Lemoine lewisii Pursh pubescens Loisel. var. verrucosus (Schrad,ex DC,)\|S{Y Hu satsumanus Sieb. ex Miq. var. nikoensis Rehd. schrenkii Rupr. triflorus Wall. x virginalis Rehd. ‘Minnesota Snowflake’ < virginalis Rehd. ‘Virginal’ ‘Silberregen’ ‘Voie Lactee’ PHILLYREA lL. OLEACEAE Olive Family angustifolia L. latifolia L. var. media (L.) Schneid. latifolia L. var. media (L.) Schneid. ‘Spinosa’ PHOENIX lL. ARECACEAE Palm Family canariensis Hort. ex Chabaud reclinata Jacq. sylvestris < P. canariensis PHOTINIA Lindl. ROSACEAE Rose Family <fraseri W.J.Dress ‘Birmingham’ glabra (Thunb. ex J.A.Murr.) Maxim. serratifolia (Desf.) Kalk. villosa (Thunb. ex J.A.Murr.) DC. villosa (Thunb. ex J.A.Murr.) DC. var. laevis (Thunb. ex J.A.Murr.) Dipp. villosa (Thunb. ex J.A.Murr.) DC. var. maximowicziana (Lev.) Rehd. villosa (Thunb. ex J.A.Murr.) DC. var. sinica Rehd. & Wils. PHYGELIUS E.Mey. ex Benth. SCROPHULARIACEAE Figwort Family capensis E.Mey. PHYLLOSTACHYS Sieb. & Zucc. Bamboo POACEAE Grass Family angusta McClure arcana McClure aurea A.& C.Riv. aureosulcata McClure bambusoides Sieb. & Zucc. bambusoides Sieb. & Zucc. ‘Castillon’ PAVONIA Cav. MALVACEAE Mallow Family hastata Cav. PAXISTIMA Raf. CELASTRACEAE Staff-tree Family canbyi A.Gr. PERIPLOCA UL. ASCLEPIADACEAE Asclepias Family graeca L. PEROVSKIA Karelin LAMIACEAE Mint Family atriplicifolia Benth. PERSEA Mill. LAURACEAE Laurel Family americana Mill. borbonia (L.) Spreng. humilis Nash thunbergii (Sieb. & Zucc.) Kosterm. PETTERIA Presl FABACEAE (Faboideae) Bean Family ramentacea Presl PHELLODENDRON Rupr. RUTACEAE Citrus Family amurense Rupr. amurense Rupr. var. japonicum (Maxim.) Ohwi amurense Rupr. var. lavallei (Dode) Sprague PHILADELPHUS L. Mock Orange SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family coronarius L. coronarius L. ‘Belle Etoile’ coronarius L. ‘Duplex’ coronarius L. ‘Natchez’ delavayi L.Henry ‘Nymans’ <falconeri Sarg. hirsutus Nutt. inodorus L. x lemoinei V.Lemoine lewisii Pursh ROSACEAE ‘Glauca’ asperata Mast. var. heterolepis (Rehd. & Wils.) W.C.Cheng ex Rehd. gemmata Rehd. & Wils. glauca (Moench) Voss glauca (Moench) Voss ‘Conica’ glauca (Moench) Voss ‘Echiniformis’ glauca (Moench) Voss ‘Wild Acres’ glehnii (F.Schmidt) Mast. jezoensis (Sieb. & Zucc.) Carr. ‘Howell’s Dwarf’ mariana (Mill.) BSP. dulcis McClure elegans McClure flexuosa A.& C.Riv. heterocycla (Carr.) Mitf. meyeri McClure nidularia Munro nidularia Munro ‘Smoothsheath’ nigra (Loud.) Munro nigra (Lodd.) Munro ‘Henon’ nuda McClure propinqua McClure purpurata McClure ‘Solidstem’ purpurata McClure ‘Straightstem’ rubromarginata McClure viridiglaucescens A.& C.Riv. viridis (Young) McClure vivax McClure dulcis McClure elegans McClure flexuosa A.& C.Riv. heterocycla (Carr.) Mitf. meyeri McClure nidularia Munro nidularia Munro ‘Smoothsheath’ nigra (Loud.) Munro nigra (Lodd.) Munro ‘Henon’ nuda McClure propinqua McClure purpurata McClure ‘Solidstem’ purpurata McClure ‘Straightstem’ rubromarginata McClure viridiglaucescens A.& C.Riv. viridis (Young) McClure vivax McClure dulcis McClure elegans McClure flexuosa A.& C.Riv. heterocycla (Carr.) Mitf. meyeri McClure nidularia Munro nidularia Munro ‘Smoothsheath’ nigra (Loud.) Munro nigra (Lodd.) Munro ‘Henon’ nuda McClure propinqua McClure purpurata McClure ‘Solidstem’ purpurata McClure ‘Straightstem’ rubromarginata McClure viridiglaucescens A.& C.Riv. viridis (Young) McClure vivax McClure abies (L.) Karst. ‘Tabuliformis’ nigra (Lodd.) Munro ‘Henon’ nuda McClure propinqua McClure purpurata McClure ‘Solidstem’ purpurata McClure ‘Straightstem’ rubromarginata McClure viridiglaucescens A.& C.Riv. viridis (Young) McClure vivax McClure PHYSOCARPUS (Cambess.) Maxim. Ninebark ROSACEAE Rose Family amurensis (Maxim.) Maxim. intermedius (Rydb.) Schneid. malvaceous (Greene) O.Ktze. opulifolius (L.) Maxim. opulifolius (L.) Maxim. ‘Luteus’ PICEA A.Dietr. PINACEAE Spruce Pine Family abies (L.) Karst. abies (L.) Karst. ‘Barryi’ abies (L.) Karst. ‘Brevifolia’ abies (L.) Karst. ‘Capitata’ abies (L.) Karst. ‘Clanbrassiliana’ abies (L.) Karst. ‘Compacta Asselyn’ abies (L.) Karst. ‘Globosa’ abies (L.) Karst. ‘Gregoryana Parsonii’ abies (L.) Karst. ‘Highlandia’ abies (L.) Karst. ‘Humilis’ abies (L.) Karst. ‘Hystrix’ abies (L.) Karst. ‘Inversa’ abies (L.) Karst. ‘Kingsville’ abies (L.) Karst. ‘Little Gem’ abies (L.) Karst. ‘Maxwellii’ abies (L.) Karst. ‘Merhii’ abies (L.) Karst. ‘Microphylla’ abies (L.) Karst. ‘Microsperma’ abies (L.) Karst. ‘Montigena’ abies (L.) Karst. ‘Mucronata’ abies (L.) Karst. ‘Nidiformis’ abies (L.) Karst. ‘Ohlendorffii’ abies (L.) Karst. ‘Oldhamiana’ abies (L.) Karst. ‘Parsonii’ abies (L.) Karst. ‘Pendula’ abies (L.) Karst. ‘Procumbens’ viridiglaucescens A.& C.Riv. viridis (Young) McClure vivax McClure PHYSOCARPUS (Cambess.) Maxim. Ninebark ROSACEAE Rose Family amurensis (Maxim.) Maxim. intermedius (Rydb.) Schneid. malvaceous (Greene) O.Ktze. opulifolius (L.) Maxim. opulifolius (L.) Maxim. ‘Luteus’ PICEA A.Dietr. PINACEAE Spruce Pine Family abies (L.) Karst. abies (L.) Karst. ‘Barryi’ abies (L.) Karst. ‘Brevifolia’ abies (L.) Karst. ‘Capitata’ abies (L.) Karst. ‘Clanbrassiliana’ abies (L.) Karst. ROSACEAE <fraseri W.J.Dress ‘Birmingham’ glabra (Thunb. ex J.A.Murr.) Maxim. serratifolia (Desf.) Kalk. villosa (Thunb. ex J.A.Murr.) DC. villosa (Thunb. ex J.A.Murr.) DC. var. laevis (Thunb. ex J.A.Murr.) Dipp. villosa (Thunb. ex J.A.Murr.) DC. var. maximowicziana (Lev.) Rehd. villosa (Thunb. ex J.A.Murr.) DC. var. sinica Rehd. & Wils. PHYGELIUS E.Mey. ex Benth. SCROPHULARIACEAE Figwort Family capensis E.Mey. PHYLLOSTACHYS Sieb. & Zucc. Bamboo POACEAE Grass Family angusta McClure arcana McClure aurea A.& C.Riv. aureosulcata McClure bambusoides Sieb. & Zucc. bambusoides Sieb. & Zucc. ‘Castillon’ congesta McClure 290 abies (L.) Karst. ‘Prostrata’ abies (L.) Karst. ‘Pseutoprostrata’ abies (L.) Karst. ‘Pumila’ abies (L.) Karst. ‘Pumila Glauca’ abies (L.) Karst. ‘Pygmaea’ abies (L.) Karst. ‘Pyramidata’ abies (L.) Karst. ‘Remonte’ abies (L.) Karst. ‘Repens’ abies (L.) Karst. ‘Tabuliformis’ alcoquiana (J.G.Veitch ex Lindl.) Carr. asperata Mast. asperata Mast. ‘Glauca’ asperata Mast. var. heterolepis (Rehd. & Wils.) W.C.Cheng ex Rehd. gemmata Rehd. & Wils. glauca (Moench) Voss glauca (Moench) Voss ‘Conica’ glauca (Moench) Voss ‘Echiniformis’ glauca (Moench) Voss ‘Wild Acres’ glehnii (F.Schmidt) Mast. jezoensis (Sieb. & Zucc.) Carr. ‘Howell’s Dwarf’ mariana (Mill.) BSP. ‘Beissneri Compacta’ mariana (Mill.) BSP. ‘Doumetii’ obovata Ledeb. omorika (Pancic) Purk. omorika (Pancic) Purk. ‘Pendula’ orientalis (L.) Link orientalis (L.) Link ‘Aurea Compacta’ orientalis (L.) Link ‘Gracilis’ pungens Engelm. pungens Engelm. ‘Compacta’ pungens Engelm. ‘Foxtail’ pungens Engelm. f. glauca (Regel) Beissn. ‘Glauca Pendula’ pungens Engelm. f. glauca (Regel) Beissn. ‘Glauca Procumbens’ pungens Engelm. f. glauca (Regel) Beissn.‘Glauca Prostrata’ pungens Engelm. ‘Globosa’ pungens Engelm. ‘Hoopsii’ pungens Engelm. ‘Hunnewelliana’ pungens Engelm. ‘Iseli Fastigiata’ pungens Engelm. ‘Koster’ pungens Engelm. ‘Montgomery’ pungens Engelm. f. glauca (Regel) Beissn. torano (K.Koch) Koehne IERIS' D.Don RICACEAE Heath Family abies (L.) Karst. ‘Prostrata’ abies (L.) Karst. ‘Pseutoprostrata’ abies (L.) Karst. ‘Pumila’ abies (L.) Karst. ‘Pumila Glauca’ abies (L.) Karst. ‘Pygmaea’ abies (L.) Karst. ‘Pyramidata’ abies (L.) Karst. ‘Remonte’ abies (L.) Karst. ‘Repens’ abies (L.) Karst. ‘Tabuliformis’ alcoquiana (J.G.Veitch ex Lindl.) Carr. asperata Mast. asperata Mast. ‘Glauca’ asperata Mast. var. heterolepis (Rehd. & Wils.) W.C.Cheng ex Rehd. gemmata Rehd. & Wils. glauca (Moench) Voss glauca (Moench) Voss ‘Conica’ glauca (Moench) Voss ‘Echiniformis’ glauca (Moench) Voss ‘Wild Acres’ glehnii (F.Schmidt) Mast. jezoensis (Sieb. & Zucc.) Carr. ‘Howell’s Dwarf’ abies (L.) Karst. ‘Prostrata’ abies (L.) Karst. ‘Pseutoprostrata’ abies (L.) Karst. ‘Pumila’ abies (L.) Karst. ‘Pumila Glauca’ abies (L.) Karst. ‘Pygmaea’ abies (L.) Karst. ‘Pyramidata’ abies (L.) Karst. ‘Remonte’ abies (L.) Karst. ‘Repens’ abies (L.) Karst. ‘Tabuliformis’ alcoquiana (J.G.Veitch ex Lindl.) Carr. asperata Mast. asperata Mast. ex G.Don ‘Pygmaea’ Japonica (Thunb. ex J halepensis Mill. koraiensis Sieb. Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Pygmy Variegata’ japonica (Thunb. ex J.A.Murr.) D.Don koraiensis Sieb. & Zucc. leucodermis Ant. leucodermis Ant. leucodermis Ant. leucodermis Ant. ‘Compact Gem’ mugo Turra var. mugo japonica (Thunb. ex J.A.Murr.) D.Don ~ ex G.Don ‘Red Mill’ ex G.Don ‘Red Mill’ japonica (Thunb. ex J ~ ex G.Don ‘Red Mill’ japonica (Thunb. ex J mugo Turra var. mugo mugo Turra var. mughus japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Scarlet O’Hara’ ROSACEAE ‘Compacta Asselyn’ abies (L.) Karst. ‘Globosa’ abies (L.) Karst. ‘Gregoryana Parsonii’ abies (L.) Karst. ‘Highlandia’ abies (L.) Karst. ‘Humilis’ abies (L.) Karst. ‘Hystrix’ abies (L.) Karst. ‘Inversa’ abies (L.) Karst. ‘Kingsville’ abies (L.) Karst. ‘Little Gem’ abies (L.) Karst. ‘Maxwellii’ abies (L.) Karst. ‘Merhii’ abies (L.) Karst. ‘Microphylla’ abies (L.) Karst. ‘Microsperma’ abies (L.) Karst. ‘Montigena’ abies (L.) Karst. ‘Mucronata’ abies (L.) Karst. ‘Nidiformis’ abies (L.) Karst. ‘Ohlendorffii’ abies (L.) Karst. ‘Oldhamiana’ abies (L.) Karst. ‘Parsonii’ abies (L.) Karst. ‘Pendula’ abies (L.) Karst. ‘Procumbens’ PHYSOCARPUS (Cambess.) Maxim. Ninebark ROSACEAE Rose Family amurensis (Maxim.) Maxim. intermedius (Rydb.) Schneid. malvaceous (Greene) O.Ktze. opulifolius (L.) Maxim. opulifolius (L.) Maxim. ‘Luteus’ jezoensis (Sieb. & Zucc.) Carr. ‘Howell’s Dwarf’ ‘Beissneri Compacta’ mariana (Mill.) BSP. ‘Doumetii’ obovata Ledeb. omorika (Pancic) Purk. omorika (Pancic) Purk. ‘Pendula’ orientalis (L.) Link orientalis (L.) Link ‘Aurea Compacta’ orientalis (L.) Link ‘Gracilis’ pungens Engelm. pungens Engelm. ‘Compacta’ pungens Engelm. ‘Foxtail’ pungens Engelm. f. glauca (Regel) Beissn. ‘Glauca Pendula’ pungens Engelm. f. glauca (Regel) Beissn. ‘Glauca Procumbens’ pungens Engelm. f. glauca (Regel) Beissn.‘Glauca Prostrata’ pungens Engelm. ‘Globosa’ pungens Engelm. ‘Hoopsii’ pungens Engelm. ‘Hunnewelliana’ pungens Engelm. ‘Iseli Fastigiata’ pungens Engelm. ‘Koster’ pungens Engelm. ‘Montgomery’ pungens Engelm. f. glauca (Regel) Beissn. torano (K.Koch) Koehne PIERIS' D.Don ERICACEAE Heath Family floribunda (Pursh) Benth. & Hook. (floribunda x P. japonica) ‘Brower’s Beauty’ formosa (Wall.) D.Don 291 mariana (Mill.) BSP. ‘Doumetii’ obovata Ledeb. abies (L.) Karst. ‘Ohlendorffii’ abies (L.) Karst. ‘Oldhamiana’ abies (L.) Karst. ‘Oldhamiana’ abies (L.) Karst. ‘Parsonii’ 291 formosa (Wall.) D.Don ‘Wakehurst’ (formosa X< P. japonica) ‘Mountain Fire’ Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Bert Chandler’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Compacta’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Crispa’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Daisen’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Dorothy Wyckoff japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Flamingo’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Purity’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Pygmaea’ Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Pygmy Variegata’ japonica (Thunb. ex J.A.Murr.) D.Don ~ ex G.Don ‘Red Mill’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Scarlet O’Hara’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Shojo’ Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Variegata’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Wada’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘White Cascade’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Whitecaps’ (japonica < P. ‘Gnom’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Shojo’ Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Purity’ japonica (Thunb. e japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Pygmaea’ greggii Engelm. & Parl. halepensis Mill. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Pygmaea’ Japonica (Thunb. ex J.A.Murr.) D.Don japonica (Thunb. ex J ex G.Don ‘Pygmaea’ Japonica (Thunb. ex J ex G.Don ‘Daisen’ japonica (Thunb. ex japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Dorothy Wyckoff ex G.Don ‘Dorothy Wyckoff japonica (Thunb. ex J.A.Murr.) elliottii Engelm. flexilis James japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Flamingo’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Flamingo’ japonica (Thunb. ex J.A.Murr.) D.Don ROSACEAE & Parl. halepensis Mill. koraiensis Sieb. & Zucc. leucodermis Ant. leucodermis Ant. ‘Compact Gem’ mugo Turra var. mugo mugo Turra var. mughus (Scop.) Zenari ‘Gnom’ mugo Turra var. mughus (Scop.) Zenari ‘Mops’ mugo Turra var. pumilio (Haenke) Zenari nigra Arn. ‘Globosa’ nigra Arn. ‘Hornibrookiana’ nigra Arn. ‘Monstrosa’ nigra Arn. ‘Nana’ nigra Arn. ssp. nigra palustris Mill. parviflora Sieb. & Zucc. parviflora Sieb. & Zucc. ‘Adcock’s Dwarf’ parviflora Sieb. & Zucc. ‘Baldwin’ parviflora Sieb. & Zucc. ‘Glauca’ parviflora Sieb. & Zucc. ‘Glauca Nana’ parviflora Sieb. & Zucc. ‘Kokonoe’ parviflora Sieb. & Zucc. ‘Venus’ parviflora Sieb. & Zucc. ‘Watnong’ patula Schiede & Deppe peuce Griseb. ‘Arnold Dwarf’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Daisen’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Flamingo’ japonica (Thunb. ex J. flexilis James flexilis James ‘ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Purity’ japonica (Thunb. ex J.A.Murr.) D.Don flexilis James ‘Glenmore’ glabra Walt. ROSACEAE formosa) ‘Forest Flame’ phillyreifolia (Hook.) DC. taiwanensis Hayata PINCKNEYA Michx. RUBIACEAE Madder Family pubens Michx. PINUS L. Pine PINACEAE Pine Family armandii Franch. attenuata J.G.Lemmon ayacahuite K.Ehrenb. banksiana Lamb. brutia Tenore bungeana Zucc. ex Endl. cembra L. formosa (Wall.) D.Don ‘Wakehurst’ (formosa X< P. japonica) ‘Mountain F cembra L. cembra L. (formosa X< P. japonica) ‘Mountain Fire’ Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don japonica (Thunb. ex J.A.Murr.) D.Don cembra L. cembra L. ‘Compacta Glauca’ cembra L. var. sibirica Loud. cembroides Zucc. clausa (Chapm. ex Engelm.) Vasey ex Sarg. contorta Doug]. ex Loud. ‘Spaan’s Dwarf densiflora Sieb. & Zucc. densiflora Sieb. & Zucc. ‘Oculus-draconis’ densiflora Sieb. & Zucc. ‘Pendula’ densiflora Sieb. & Zucc. ‘Umbraculifera’ echinata Mill. elliottii Engelm. flexilis James flexilis James ‘Glenmore’ glabra Walt. greggii Engelm. & Parl. halepensis Mill. koraiensis Sieb. & Zucc. leucodermis Ant. leucodermis Ant. ‘Compact Gem’ mugo Turra var. mugo mugo Turra var. mughus (Scop.) Zenari ‘Gnom’ mugo Turra var. mughus (Scop.) Zenari ‘Mops’ mugo Turra var. pumilio (Haenke) Zenari nigra Arn. ‘Globosa’ nigra Arn. ‘Hornibrookiana’ nigra Arn. ‘Monstrosa’ nigra Arn. ‘Nana’ nigra Arn. ssp. nigra palustris Mill. parviflora Sieb. & Zucc. parviflora Sieb. & Zucc. ‘Adcock’s Dwarf’ parviflora Sieb. & Zucc. ‘Baldwin’ parviflora Sieb. & Zucc. ‘Glauca’ parviflora Sieb. & Zucc. ‘Glauca Nana’ parviflora Sieb. & Zucc. ‘Kokonoe’ parviflora Sieb. & Zucc. ‘Venus’ parviflora Sieb. & Zucc. ‘Watnong’ patula Schiede & Deppe peuce Griseb. ‘Arnold Dwarf’ pinea L. ponderosa Dougl. ex P.& C.Laws ponderosa Doug. ex P.& C.Laws. ‘Pendula’ pumila (Pall.) Regel pumila (Pall.) Regel ‘Dwarf Blue’ pungens Lamb. resinosa Ait. rigida Mill. roxburghii Sarg. sabiniana Doug]. ex D.Don Japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don japonica (Thunb. ex J.A.Murr.) D.Don japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Bert Chandler’ japonica (Thunb. ex J.A.Murr.) D.Don cembroides Zucc. clausa (Chapm. ex Engelm.) Vasey ex Sarg. contorta Doug]. ex Loud. ‘Spaan’s Dwa ex G.Don ‘Bert Chandler’ japonica (Thunb. ex J.A.Mur japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Compacta’ japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Compacta’ japonica (Thunb. ex J.A.Murr.) D.Don contorta Doug]. ex Loud. ‘Spaan’s Dwarf densiflora Sieb. & Zucc. densiflora Sieb. & Zucc. densiflora Sieb. & Zucc. japonica (Thunb. ex J.A.Murr.) D.Don ex G.Don ‘Crispa’ japonica (Thunb. ex J.A.Murr.) D.Don densiflora Sieb. & Zucc. ‘Oculus-draconis’ densiflora Sieb. & Zucc. ‘Oculus-draconis’ densiflora Sieb. & Zucc. ‘Pendula’ densiflora Sieb. & Zucc. ‘Umbraculifera’ echinata Mill. elliottii Engelm. flexilis James flexilis James ‘Glenmore’ glabra Walt. greggii Engelm. ex G.Don ‘Shojo’ Japonica (Thunb. nigra Arn. ‘Globosa’ nigra Arn. ‘Hornibroo nigra Arn. ‘Hornibrookiana’ nigra Arn. ‘Monstrosa’ nigra Arn. ‘Monstrosa’ nigra Arn. ‘Nana’ nigra Arn. ‘Nana’ nigra Arn. ssp. nig nigra Arn. ssp. nigra palustris Mill. parviflora Sieb. & Zucc. ‘Adcock’s Dwarf’ parviflora Sieb. & Zucc. ‘Venus’ parviflora Sieb. & Zucc. ‘Watnong’ parviflora Sieb. & Zucc. ‘Watnong’ patula Schiede & Deppe patula Schiede & Deppe peuce Griseb. ‘Arnold Dwa peuce Griseb. ‘Arnold Dwarf’ pinea L. 292 PLANERA J.F.Gmel. ULMACEAE = Elm Family aquatica (Walt.) J.F.Gmel. PLATANUS L. Sycamore PLATANACEAE Plane-tree Family x acertfolia (Ait.) Willd. * Xacerifolia (Ait.) Willd. ‘Columbia’ * Xacerifolia (Ait.) Willd. ‘Liberty’ occidentalis L. orientalis L. PLATYCARYA Sieb. & Zucc. JUGLANDACEAE Walnut Family strobilacea Sieb. & Zucc. PLATYCLADUS Spach CUPRESSACEAE Cypress Family orientalis (L.) Franco orientalis (L.) Franco ‘Aurea Nana’ orientalis (L.) Franco ‘Bakeri’ orientalis (L.) Franco ‘Berckman’s Golden’ orientalis (L.) Franco ‘Beverleyensis’ orientalis (L.) Franco ‘Conspicua’ orientalis (L.) Franco ‘Flagelliformis’ orientalis (L.) Franco ‘Fruitlandii’ orientalis (L.) Franco ‘Hohman’ orientalis (L.) Franco ‘Juniperoides’ orientalis (L.) Franco ‘Rosedalis’ orientalis (L.) Franco ‘Semperaurea’ PLUMBAGO lL. PLUMBAGINACEAE Leadwort Family auriculata Lam. PODOCARPUS. VL Her. ex Pers. PODOCARPACEAE Podocarpus Family gracilior Pilger macrophyllus (Thunb. ex J.A.Murr.) Sweet macrophyllus (Thunb. ex J.A.Murr.) Sweet var. maki Sieb. nagi (Thunb. ex J.A.Murr.) Makino totara D.Don POLIOTHYRSIS Oliv. FLACOURTIACEAE Flacourtia Family serotina Michx. strobus L. strobus L. ‘Amelia Dwarf strobus L. ‘Contorta’ strobus L. ‘Dwarf strobus L. ‘Elf strobus L. ‘Fastigiata’ strobus L. ‘Merrimack’ strobus L. ‘Nana’ strobus L. ‘Pendula’ strobus L. ‘Pumila’ strobus L. ‘Radiata’ strobus L. ‘Seacrest’ strobus L. ‘Torulosa’ strobus < P. sylvestris L. sylvestris L. sylvestris L. sylvestris L. sylvestris L. sylvestris L. sylvestris L. sylvestris L. wallichiana ‘Albyns’ ‘Argentea Compacta’ ‘Aurea’ ‘Beuvronensis’ ‘Fastigiata’ ‘Globosa Viridis’ ‘Moseri’ sylvestris L. ‘Repens’ sylvestris L. ‘Sentinel’ taeda L. thunbergiana Franco thunbergiana Franco ‘Corticosa’ thunbergiana Franco ‘Yatsubusa’ virginiana Mill. wallichiana A.B.Jacks. wallichiana A.B.Jacks. ‘Zebrina’ PISTACIA L. Pistache ANACARDIACEAE Cashew Family chinensis Bunge texana Swingle PITHECELLOBIUM Mart. FABACEAE (Mimosoideae) Bean Family ebano (Berl.) Muller PITTOSPORUM Banks ex Sol. PITTOSPORACEAE Pittosporum Family glabratum Lindl. tobira (Willd.) Ait tobira (Willd.) Ait. ‘Variegata’ tobira (Willd.) Ait. ‘Wheeler’s Dwarf’ undulatum Vent. serotina Michx. strobus L. strobus L. ‘Amelia Dwarf strobus L. ‘Contorta’ strobus L. ‘Dwarf strobus L. ‘Elf strobus L. ‘Fastigiata’ strobus L. ‘Merrimack’ strobus L. ‘Nana’ strobus L. ‘Pendula’ strobus L. ‘Pumila’ strobus L. ‘Radiata’ strobus L. ‘Seacrest’ strobus L. ‘Torulosa’ strobus < P. sylvestris L. sylvestris L. sylvestris L. sylvestris L. sylvestris L. PROSOPIS L. FABACEAE (Mimosoideae) Bean Family FABACEAE (Mimosoideae) Bean Family glandulosa Torr. var. torreyana (L.Benson) M.C.Johnst. PRUNUS L. ROSACEAE Rose Family americana Marsh. angustifolia Marsh. apetala (Sieb. & Zucc.) Franch. & Sav. armeniaca L. avium L. avium L. ‘Plena’ < blireiana Andre campanulata Maxim. caroliniana Ait. cerasifera Ehrh. cerasifera Ehrh. ‘Atropurpurea’ cerasifera Ehrh. ‘Moseri’ cerasifera Ehrh. ‘Purpusii’ cerasifera Ehrh. ‘Thundercloud’ cerasus L. ‘Montmorency’ < cistena N.E.Hansen conradinae Koehne cyclamina Koehne domestica L. ssp. insititia (L.) Schneid. dulcis (Mill.) D.A.Webb glandulosa Thunb. ex J.A.Murr. glandulosa Thunb. ex J.A.Murr. ‘Albo-plena’ glandulosa Thunb. ex J.A.Murr. ‘Sinensis’ humilis Bunge <incam Fletcher ‘Okami’ incisa Thunb. ex J.A.Murr. incisa Thunb. ex J.A.Murr. ‘February Pink’ FABACEAE (Mimosoideae) Bean Family glandulosa Torr. var. torreyana (L.Benson) M.C.Johnst. PRUNUS L. ROSACEAE Rose Family americana Marsh. angustifolia Marsh. apetala (Sieb. & Zucc.) Franch. & Sav. armeniaca L. avium L. avium L. ‘Plena’ < blireiana Andre campanulata Maxim. caroliniana Ait. cerasifera Ehrh. cerasifera Ehrh. ‘Atropurpurea’ cerasifera Ehrh. ‘Moseri’ cerasifera Ehrh. ‘Purpusii’ cerasifera Ehrh. ‘Thundercloud’ cerasus L. ‘Montmorency’ < cistena N.E.Hansen conradinae Koehne cyclamina Koehne domestica L. ssp. insititia (L.) Schneid. dulcis (Mill.) D.A.Webb glandulosa Thunb. ex J.A.Murr. glandulosa Thunb. ex J.A.Murr. ‘Albo-plena’ glandulosa Thunb. ex J.A.Murr. ‘Sinensis’ humilis Bunge <incam Fletcher ‘Okami’ incisa Thunb. ex J.A.Murr. incisa Thunb. ex J.A.Murr. ‘February Pink’ incisa Thunb. ex J.A.Murr. f. serrata Koidz. ex Wils. incisa Thunb. ex J.A.Murr. var. tomentosa Koidz. (incisa < P. serrulata) ‘Umineko’ Japonica Thunb. ex J.A.Murr. laurocerasus L. laurocerasus L. ‘Camelliifolia’ laurocerasus L. ‘Forest Green’ glandulosa Torr. var. torreyana (L.Benson) M.C.Johnst. ex G.Don ‘Shojo’ Japonica (Thunb. sylvestris L. sylvestris L. sylvestris L. wallichiana ‘Albyns’ ‘Argentea Compacta’ ‘Aurea’ ‘Beuvronensis’ ‘Fastigiata’ ‘Globosa Viridis’ ‘Moseri’ sylvestris L. ‘Repens’ sylvestris L. ‘Sentinel’ taeda L. thunbergiana Franco thunbergiana Franco ‘Corticosa’ thunbergiana Franco ‘Yatsubusa’ virginiana Mill. wallichiana A.B.Jacks. wallichiana A.B.Jacks. ‘Zebrina’ PISTACIA L. Pistache ANACARDIACEAE Cashew Family chinensis Bunge texana Swingle PITHECELLOBIUM Mart. FABACEAE (Mimosoideae) Bean Family ebano (Berl.) Muller PITTOSPORUM Banks ex Sol. PITTOSPORACEAE Pittosporum Family glabratum Lindl. tobira (Willd.) Ait tobira (Willd.) Ait. ‘Variegata’ tobira (Willd.) Ait. ‘Wheeler’s Dwarf’ undulatum Vent. tobira (Willd.) Ait. ‘Variegata’ tobira (Willd.) Ait. ‘Wheeler’s Dwa TASKS) POLYGONELLA Michx. POLYGONACEAE Buckwheat Family americana (Fischer & C.A.Mey.) Small myriophylla (Small) Horton polygama (Vent.) Engelm. & A.Gr. PONCIRUS Raf. RUTACEAE Citrus Family trifoliata (L.) Raf. POPULUS L. Poplar SALICACEAE Willow Family alba L. alba L. ‘Pyramidalis’ <x canadensis Moench candicans Ait. <x canescens (Ait.) Sm. deltoides Marsh. grandidentata Michx. heterophylla L. -maximowiczii A.Henry maximowiczii < P. trichocarpa nigra L. ‘Italica’ simonii Carr. ‘Fastigiata’ tremuloides Michx. PORLIERIA Ruiz & Pavon ZYGOPHYLLACEAE Caltrop Family angustifolia (Engelm.) A.Gr. POTENTILLA L. Cinquefoil ROSACEAE Rose Family fruticosa L. fruticosa L. ‘Abbotswood’ fruticosa L. ‘Everest’ fruticosa L. ‘Grandiflora’ fruticosa L. ‘Jackman’s’ JSruticosa L. ‘Katherine Dykes’ JSruticosa L. ‘Maanelys’ JSruticosa L. ‘Primrose Beauty’ fruticosa L. ‘William Purdom’ tridentata Ait. ‘Sophie Blush’ PRINSEPIA Royle ROSACEAE Rose Family sinensis (Oliv.) Oliv. PROSOPIS L. FABACEAE (Mimosoideae) Bean F glandulosa Torr. var. torreyana (L.Benson) M.C.Johnst. PRUNUS L. ROSACEAE Rose Family ROSACEAE Rose Family americana Marsh. angustifolia Marsh. apetala (Sieb. & Zucc.) Franch. & Sav. armeniaca L. avium L. avium L. ‘Plena’ < blireiana Andre campanulata Maxim. caroliniana Ait. cerasifera Ehrh. cerasifera Ehrh. ‘Atropurpurea’ cerasifera Ehrh. ‘Moseri’ cerasifera Ehrh. ‘Purpusii’ cerasifera Ehrh. ‘Thundercloud’ cerasus L. ‘Montmorency’ < cistena N.E.Hansen conradinae Koehne cyclamina Koehne domestica L. ssp. insititia (L.) Schneid. dulcis (Mill.) D.A.Webb glandulosa Thunb. ex J.A.Murr. glandulosa Thunb. ex J.A.Murr. ‘Albo-plena’ glandulosa Thunb. ex J.A.Murr. ‘Sinensis’ humilis Bunge <incam Fletcher ‘Okami’ incisa Thunb. ex J.A.Murr. incisa Thunb. ex J.A.Murr. ‘February Pink’ Citrus Family avium L. avium L. avium L. ‘Plena’ < blireiana Andre < blireiana Andre campanulata Maxi campanulata Maxim. caroliniana Ait. caroliniana Ait. cerasifera Ehrh. glandulosa Thunb. ex J.A.Murr. ‘Albo-plena’ incisa Thunb. ex J.A.Murr. var. tomentosa Koidz. var. tomentosa Koidz. (incisa < P. serrulata) var. tomentosa Koidz. (incisa < P. serrulata) ‘Umineko’ Japonica Thunb. ex J.A.Murr. (incisa < P. serrulata) ‘Umineko’ Japonica Thunb. ex J.A.Murr. Japonica Thunb. ex J.A.Murr. laurocerasus L. laurocerasus L. laurocerasus L. laurocerasus L. ‘Forest Green’ laurocerasus L. ‘Otto Luyken’ laurocerasus L. ‘Otto Luyken’ laurocerasus L. ‘Zabeliana’ laurocerasus L. ‘Zabeliana’ laurocerasus L. var. schipka lusitanica L. mahaleb L. mahaleb L. maritima Ma maritima Marsh. maritima Marsh. maritima Marsh. ‘Methley’ mexicana S.Wats. mexicana S.Wats. 294 x subhirtella Migq. ‘Elizabeth’ x subhirtella Mig. ‘Rosea’ x subhirtella Miq. ‘Shidare higan’ x subhirtella Miq. ‘Yae shidare higan’ x subhirtella Mig. var. ascendens (Makino) Wils. ‘Flore-plena’ (<subhirtella < P. x yedoensis) ‘Pandora’ ((<xsubhirtella x P. x yedoensis) < P. <subhirtella) ‘Hally Jolivette’ syodoi Nakai tomentosa Thunb. ex J.A.Murr. triloba Lindl. triloba Lindl. ‘Multiplex’ umbellata Ell. verecunda (Koidz.) Koehne virginiana L. ‘Schubert’ x yedoensis Matsum. x yedoensis Matsum x yedoensis Matsum x yedoensis Matsum ‘Ariake’ ‘Asagi’ ‘Benden’ ‘Botan zakura’ ‘Fugenzo’ ‘Gyoiko’ ‘Hatazakura’ ‘Hizakura’ ‘Meigetsu’ ‘Ojochin’ ‘Senriko’ ‘Shogetsu’ ‘Snow Fountain’ ‘Taki nioi’ ‘Yae akebono’ . ‘Akebono’ . ‘Shidare yoshino’ . ‘Somei yoshino’ ‘Yae murasaki zakura’ PSEUDOCYDONIA ROSACEAE (Schneid.) Schneid. Rose Family sinensis (Dum.-Cours.) Thouin PSEUDOLARIX Gord. PINACEAE Pine Family amabilis (J.Nelson) Rehd. PSEUDOSASA Makino ex Nakai POACEAE Grass Family japonica Sieb. & Zucc. ex Steud. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. ‘Rosemary Clarke’ mume Sieb. & Zucc. “‘W. B. Clarke’ munsoniana W.F.Wight & Hedr. (nipponica Matsum. var. kurilensis (Miyabe) Wils. x unknown pollen parent) ‘Kursar’ padus L. padus L. PRUNUS L. ROSACEAE ‘Grandiflorus’ persica (L.) Batsch persica (L.) Batsch ‘Alba’ persica (L.) Batsch ‘Albo-plena’ persica (L.) Batsch ‘Camelliifolia’ persica (L.) Batsch ‘Dianthiflora’ ‘Alba’ ‘Matsubara Red’ ‘Nishiki ume’ ‘Peggy Clarke’ persica (L.) Batsch ‘Duplex’ persica (L.) Batsch ‘Fastigiata’ persica (L.) Batsch ‘Rubro-plena’ persica (L.) Batsch ‘Versicolor’ persica (L.) Batsch var. nucipersica (Borkh.) Schneid. ‘White Glory’ rivularis Scheele sargentii Rehd. sargentii Rehd. ‘Dr. S. Edwin Mueller’ sargentii Rehd. ‘Rancho’ (sargentii < P. ?incisa) ‘Spire’ (sargentii < P. subhirtella) ‘Accolade’ serotina Ehrh. serrula Franch. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. (Maxim.) Wils. ‘Amanogawa’ ‘Daikaku’ ‘Fudan sakura’ ‘Fuku rokuju’ ‘Gozanoma nioi’ ‘Hosokawa’ ‘Imose’ ‘Kiku shidare’ ‘Kwanzan’ ‘Mt. Fuji’ ‘Oshima zakura’ ‘Pink Perfection’ ‘Rosea’ ‘Shirofugen’ ‘Shirotae’ ‘Tai haku’ ‘Wase mikayo’ ‘Yama zakura’ var. spontanea mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. mume Sieb. & Zucc. ‘Rosemary Clarke’ mume Sieb. & Zucc. “‘W. B. Clarke’ munsoniana W.F.Wight & Hedr. (nipponica Matsum. var. kurilensis (Miyabe) Wils. x unknown pollen parent) ‘Kursar’ padus L. padus L. ‘Grandiflorus’ persica (L.) Batsch persica (L.) Batsch ‘Alba’ persica (L.) Batsch ‘Albo-plena’ persica (L.) Batsch ‘Camelliifolia’ persica (L.) Batsch ‘Dianthiflora’ ‘Alba’ ‘Matsubara Red’ ‘Nishiki ume’ ‘Peggy Clarke’ persica (L.) Batsch ‘Duplex’ persica (L.) Batsch ‘Fastigiata’ persica (L.) Batsch ‘Rubro-plena’ persica (L.) Batsch ‘Versicolor’ persica (L.) Batsch var. nucipersica (Borkh.) Schneid. ‘White Glory’ rivularis Scheele sargentii Rehd. sargentii Rehd. ‘Dr. S. Edwin Mueller’ sargentii Rehd. ‘Rancho’ (sargentii < P. ?incisa) ‘Spire’ (sargentii < P. subhirtella) ‘Accolade’ serotina Ehrh. serrula Franch. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. (Maxim.) Wils. ‘Amanogawa’ ‘Daikaku’ ‘Fudan sakura’ ‘Fuku rokuju’ ‘Gozanoma nioi’ ‘Hosokawa’ ‘Imose’ ‘Kiku shidare’ ‘Kwanzan’ ‘Mt. Fuji’ ‘Oshima zakura’ ‘Pink Perfection’ ‘Rosea’ ‘Shirofugen’ ‘Shirotae’ ‘Tai haku’ ‘Wase mikayo’ ‘Yama zakura’ var. spontanea x sieboldii (Carr.) Wittmack triloba Lindl. ‘Multiplex’ umbellata Ell. ‘Asagi’ ‘Benden’ ‘Benden’ ‘Botan zak rivularis Scheele sargentii Rehd. ‘Botan zakura’ ‘Fugenzo’ sargentii Rehd. sargentii Rehd. ‘Fugenzo’ ‘Gyoiko’ ‘Hatazakura’ ‘Hizakura’ ‘Hizakura’ ‘Meigetsu’ (sargentii < P. ?incisa) ‘Spire’ (sargentii < P. subhirtella) ‘Ac serotina Ehrh. serrula Franch. PRUNUS L. ROSACEAE serrula Franch. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. serrulata Lindl. ‘Hosokawa’ ‘Imose’ serrulata Lindl. serrulata Lindl. ‘Pink Perfection’ ‘Rosea’ serrulata Lindl. serrulata Lindl. ‘Rosea’ ‘Shirofu serrulata Lindl. serrulata Lindl. ‘Shirofugen’ ‘Shirotae’ serrulata Lindl. serrulata Lindl. ‘Shirotae’ ‘Tai haku’ serrulata Lindl. serrulata Lindl. ‘Tai haku’ ‘Wase mika serrulata Lindl. serrulata Lindl. ‘Wase mikayo’ ‘Yama zakura’ serrulata Lindl. serrulata Lindl. ‘Yama zakura’ var. spontanea x sieboldii (Carr.) Wittmack x subhirtella Miq. x subhirtella Miq. x subhirtella Miq. x subhirtella Miq. ‘Autumnalis’ 295 coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. coccinea Roem. ‘Dauerbrand’ ‘Gold Nugget’ ‘Kasan’ ‘Keessen’ ‘Lalandei’ ‘Low Boy’ ‘Minute Man’ ‘Monrovia’ ‘Orange Giant’ ‘Pauciflora’ ‘Praecox’ ‘Runyan’ ‘Sepers’ ‘Thornless’ ‘Vincent’ ‘Wayside’s Compact’ ‘Wyattii’ (coccinea < P. crenulata var. rogersiana) ‘Eddie’s Coral’ crenulata (D.Don) Roem. crenulata (D.Don) Roem. f. flava Meunissier crenulata (D.Don) Roem. var. kansuensis Rehd. crenulata (D.Don) Roem. var. rogersiana A.B.Jacks. crenulata (D.Don) Roem. var. rogersiana A.B.Jacks. ‘Flava’ fortuneana (Maxim.) Li PSEUDOTSUGA Carr. PINACEAE PSEUDOTSUGA Carr. PINACEAE Douglas Fir Pine Family menziesii (Mirb.) Franco var. glauca (Beissn.) Franco menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Densa’ menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Fastigiata’ menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Glauca Pendula’ menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Oudemansii’ (Beissn.) Franco ‘Densa’ menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Fastigiata’ menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Glauca Pendula’ menziesii (Mirb.) Franco var. glauca (Beissn.) Franco ‘Oudemansii’ PSIDIUM L. Purple Guava MYRTACEAE Myrtle Family littorale Raddi var. longipes (O. Berg) Fosberg PTELEA L. Hop Tree RUTACEAE Citrus Family trifoliata L. PTEROCARYA Kunth JUGLANDACEAE Wingnut Walnut Family stenoptera DC. PTEROSTYRAX Sieb. & Zucc. Epaulette Tree STYRACACEAE Storax Family corymbosum Sieb. & Zucc. hispidum Sieb. & Zucc. PUNICA L. PUNICACEAE Pomegranate Family granatum L. granatum L. ‘Legrellei’ granatum L. ‘Nana’ granatum L. ‘Plena’ PYRACANTHA M.J.Roem. ROSACEAE Firethorn Rose Family angustifolia (Franch.) Schneid. angustifolia (Franch.) Schneid. ‘Gnome’ * (angustifolia < P. ‘Watereri’) ‘Navaho’ atalantioides (Hance) Stapf atalantioides (Hance) Stapf ‘Aurea’ coccinea Roem. coccinea Roem. ‘Bound’ coccinea Roem. ‘Chadwick’ coccinea Roem. ‘Cole’s Erect’ crenulata (D.Don) Roem. var. rogersiana A.B.Jacks. crenulata (D.Don) Roem. crenulata (D.Don) Roem. var. rogersiana A.B.Jacks crenulata (D.Don) Roem. var. rogersiana A.B.Jacks. ‘Fla crenulata (D.Don) Roem. var. rogersiana A.B.Jacks. ‘Flava’ fortuneana (Maxim.) Li var. rogersiana A.B.Jacks. ‘Flava’ fortuneana (Maxim.) Li fortuneana (Maxim.) Li Jfortuneana (Maxim.) Li Jfortuneana (Maxim.) Li ‘Graberi’ Jfortuneana (Maxim.) Li ‘Orange G Jfortuneana (Maxim.) Li ‘Orange Glow’ (fortuneana ‘Orange Glow’ x (fortuneana ‘Orange Glow’ x P. crenulata var. rogersiana ‘Flava’) ‘Teton’ P. crenulata var. rogersiana ‘Flava’) ‘Teton’ P. crenulata var. rogersiana ‘Flava’ koidzumii (Hayata) Rehd. koidzumii (Hayata) Rehd. koidzumii (Hayata) Rehd. koidzumii (Hayata) Rehd. ‘Belli’ ‘Crimso koidzumii (Hayata) Rehd. koidzumii (Hayata) Rehd. ‘Crimson Tide’ ‘Government koidzumii (Hayata) Rehd. Red’ koidzumii (Hayata) Rehd. ‘Government koidzumii (Hayata) Rehd. koidzumii (Hayata) Rehd. ‘Low Dense’ koidzumii (Hayata) Rehd. koidzumii (Hayata) Rehd. ‘Miller’ ‘Rosedal koidzumii (Hayata) Rehd. ‘Rosedale’ (koidzumii < P. coccinea ‘Wyattii’) granatum L. ‘Nana’ granatum L. ‘Plena’ granatum L. ‘Plena’ (koidzumii < P. fortuneana) ‘San Jose’ * (koidzumii < P. fortuneana) ‘Shawnee’ (koidzumii < P. fortuneana) ‘San Jose’ * (koidzumii < P. fortuneana) ‘Shawnee’ * (koidzumii < P. fortuneana) ‘Shawnee’ * (koidzumii ‘Belli’ x P. coccinea * (koidzumii < P. fortuneana) ‘Shawnee’ * (koidzumii ‘Belli’ x P. coccinea ‘Baker’ ‘Bloss’ ‘Bloss’ ‘Brillian ‘Brilliant’ ‘Cal Poly’ ‘Cal Poly’ ‘Chinese B ‘Chinese Brocade’ 296 ‘Coplen’s Royal’ ‘Dr. PSEUDOTSUGA Carr. PINACEAE & Drude RHAPIS L.f. ex Ait. ARECACEAE Palm Family excelsa (Thunb. ex J.A.Murr.) A.Henry RHODODENDRON L. ERICACEAE Heath Family alabamense Rehd. amagianum Makino arborescens (Pursh) Torr. atlanticum (Ashe) Rehd. augustinii Hemsl. augustinii Hemsl. ‘Crater Lake’ austrinum (Small) Rehd. bakeri (W.P.Lemmon & McKay) Hume * bakeri (W.P.Lemmon & McKay) Hume ‘Camp’s Red’ brachycarpum D.Don ex G.Don calendulaceum (Michx.) Torr. canescens (Michx.) Sweet carolinianum Rehd. carolinianum Rehd. ‘Album’ carolinianum Rehd. ‘Luteum’ (carolinianum X R. ciliatum) ‘Dora Amateis’ (carolinianum < R. dauricum) ‘P. J. M.’ (carolinianum < R. dauricum) ‘P. J. Mezitt’ carolinianum < R. mucronulatum carolinianum < R. mucronulatum ‘Conewago’ carolinianum < R. racemosum (carolinianum < R. racemosum) ‘Conestoga’ catawbiense Michx. (catawbiense < R. arboreum ssp. arboreum) ‘John Walter’ (catawbiense < R. griffithianum) ‘Cynthia’ (catawbiense ‘Atrosanguineum’ margaretta Ashe michauxti Nutt. minima (Sarg.) Small mongolica Fischer var. grosseserrata (Bl.) Rehd. & Wils. myrsinifolia Bl. nigra L. nuttallii Palm. palustris Muenchh. petraea (Mattusch.) Liebl. petraea (Mattusch.) Liebl. ‘Falkenbergensis’ petraea (Mattusch.) Liebl. ‘Geisleri’ petraea (Mattusch.) Liebl. ‘Muscaviensis’ phellos L. phillyraeoides A.Gr. prinus L. pubescens Willd. robur L. robur L. ‘Cristata’ robur L. ‘Cucullata’ robur L. ‘Salicifolia’ robur L. f. fastigiata (Lam.) O.Schwarz (robur f. fastigiata <x Q. petraea ‘Muscaviensis’) ‘Columna’ < rosacea Bechst. rubra L. x sargentii Rehd. shumardii Buckl. stellata Wang. suber L. variabilis Bl. velutina Lam. virginiana Mill. RHAMNUS L. Buckthorn RHAMNACEAE Buckthorn Family alaternus L. carolinianus L. catharticus L. davuricus Pall. japonicus Maxim. utilis Decne. RHAPHIOLEPIS Lind. ROSACEAE Rose Family indica (L.) Lindl. indica (L.) Lindl. ‘Enchantress’ indica (L.) Lindl. ‘Snow’ umbellata (Thunb. ex J.A.Murr.) Makino umbellata (Thunb. ex J.A.Murr.) Makino var. integerrima (Hook. & Arn.) Rehd. ‘Majestic Beauty’ margaretta Ashe michauxti Nutt. minima (Sarg.) Small mongolica Fischer var. grosseserrata (Bl.) Rehd. & Wils. myrsinifolia Bl. nigra L. nuttallii Palm. palustris Muenchh. petraea (Mattusch.) Liebl. petraea (Mattusch.) Liebl. ‘Falkenbergensis’ petraea (Mattusch.) Liebl. ‘Geisleri’ petraea (Mattusch.) Liebl. ‘Muscaviensis’ phellos L. phillyraeoides A.Gr. prinus L. pubescens Willd. robur L. robur L. ‘Cristata’ robur L. ‘Cucullata’ robur L. ‘Salicifolia’ robur L. f. fastigiata (Lam.) O.Schwarz (robur f. fastigiata <x Q. petraea ‘Muscaviensis’) ‘Columna’ < rosacea Bechst. rubra L. x sargentii Rehd. shumardii Buckl. stellata Wang. suber L. variabilis Bl. velutina Lam. virginiana Mill. RHAMNUS L. Buckthorn RHAMNACEAE Buckthorn Family alaternus L. carolinianus L. catharticus L. davuricus Pall. japonicus Maxim. utilis Decne. RHAPHIOLEPIS Lind. ROSACEAE Rose Family indica (L.) Lindl. indica (L.) Lindl. ‘Enchantress’ indica (L.) Lindl. ‘Snow’ umbellata (Thunb. ex J.A.Murr.) Makino robur L. PSEUDOTSUGA Carr. PINACEAE Hook’ ‘Early Red’ ‘Golden Charmer’ ‘Golden Queen’ ‘Heyden’s Bright Yellow’ ‘Heyden’s Hi Yellow’ ‘Ingleside Crimson’ ‘Knap Hill Buttercup’ ‘Mioun’ ‘Moonbeam’ ‘Orange King’ ‘Pine Cone’ ‘Pinkie’ ‘Pride of Portsmouth’ ‘Pyrabox’ ‘Red Berry’ ‘Santa Cruz’ ‘Schwartz’ ‘Select Yellow’ ‘Sensation’ ‘Spring Hill’ ‘Stribling’ ‘Taliensis’ ‘Tiny Tim’ ‘True Yellow’ ‘Variegated’ ‘Walder’ ‘Walder Prostrate’ ‘Waterer’s Dwarf’ ‘Waterer’s Orange’ ‘Waterer’s Yellow’ ‘Watereri’ ‘Weaver’s Superb’ ‘Wheeler’ ‘Wight Early’ ‘Wonderberry’ ‘Yella Berry’ ‘Yokohama’ xPYRACOMELES Rehd. ex Guill. ROSACEAE Rose Family vilmorinii Rehd. ex Guill. PYRUS L. Pear ROSACEAE Rose Family amygdaliformis Villars betulifolia Bunge bretschneideri Rehd. calleryana Decne. calleryana Decne. ‘Aristocrat’ calleryana Decne. ‘Bradford’ calleryana Decne. var. dimorphophylla (Makino) Koidz. = xX canescens Spach communis L. elaeagrifolia Pall. fauriei Schneid. kawakamii Hayata x lecontei Rehd. <x michauxii Bosc ex Poir. nivalis Jacq. pashia Buch.-Ham. ex D.Don phaeocarpa Rehd. pyrifolia (Burm.f.) Nakai pyrifolia (Burm.) Nakai ‘Chojure’ pyrifolia (Burm.f.) Nakai var. culta (Makino) Nakai regelii Rehd. salicifolia Pall. salicifolia Pall. ‘Pendula’ ussuriensis Maxim. ussuriensis Maxim. var. hondoensis (Kikuchi & Nakai) Rehd. ussuriensis Maxim. var. ovoidea Rehd. QUERCUS L. FAGACEAE acutissima Carruthers alba L. alba < Q. virginiana arkansana Sarg. asheana Little austrina Small bicolor Willd. <x bimundorum Palm. cerris L. chenti Nakai coccinea Muenchh. <x comptonae Sarg. Oak Beech Family dentata Thunb. ex J.A.Murr. fabri Hance falcata Michx. falcata Michx. var. pagodifolia Ell. frainetto Tenore georgiana M.A.Curtis gilva Bl. glandulifera Bi. glauca Thunb. ex J.A.Murray haas Kotschy hemisphaerica Bartram ex Willd. < heterophylla Michx.f. imbricaria Michx. incana Bartram laevis Walt. calleryana Decne. var. dimorphophylla (Makino) Koidz. = xX canescens Spach communis L. elaeagrifolia Pall. fauriei Schneid. kawakamii Hayata x lecontei Rehd. <x michauxii Bosc ex Poir. nivalis Jacq. pashia Buch.-Ham. ex D.Don phaeocarpa Rehd. pyrifolia (Burm.f.) Nakai pyrifolia (Burm.) Nakai ‘Chojure’ pyrifolia (Burm.f.) Nakai var. culta (Makino) Nakai regelii Rehd. salicifolia Pall. salicifolia Pall. ‘Pendula’ ussuriensis Maxim. ussuriensis Maxim. var. hondoensis (Kikuchi & Nakai) Rehd. ussuriensis Maxim. var. ovoidea Rehd. QUERCUS L. FAGACEAE acutissima Carruthers alba L. alba < Q. virginiana arkansana Sarg. asheana Little austrina Small bicolor Willd. <x bimundorum Palm. cerris L. chenti Nakai coccinea Muenchh. <x comptonae Sarg. Oak Beech Family dentata Thunb. ex J.A.Murr. fabri Hance falcata Michx. falcata Michx. var. pagodifolia Ell. frainetto Tenore georgiana M.A.Curtis gilva Bl. glandulifera Bi. glauca Thunb. ex J.A.Murray haas Kotschy hemisphaerica Bartram ex Willd. < heterophylla Michx.f. imbricaria Michx. incana Bartram laevis Walt. laurifolia Michx. Pear Rose Family ZOOM RHAPIDOPHYLLUM H.Wendl. & Drude ARECACEAE Palm Family hystrix (Pursh) H.Wendl. PSEUDOTSUGA Carr. PINACEAE ‘Cristata’ robur L. ‘Cucullata’ robur L. ‘Cucullata’ robur L. ‘Salicifolia’ robur L. ‘Salicifolia’ robur L. f. fastigiata ‘Camp’s Red’ brachycarpum ‘Muscaviensis’) ‘Columna’ < rosacea Bechst. < rosacea Bechst. rubra L. x sargenti x sargentii Rehd. shumardii Buckl. shumardii Buckl. stellata Wang. (carolinianum X R. ciliatum) ‘Dora Amateis’ stellata Wang. suber L. japonicus Maxim. utilis Decne. utilis Decne. ROSACEAE indica (L.) Lindl. indica (L.) Lindl. ‘Enchantress’ indica (L.) Lindl. ‘Snow’ umbellata (Thunb. ex J.A.Murr.) Makino umbellata (Thunb. ex J.A.Murr.) Makino var. integerrima (Hook. & Arn.) Rehd. ‘Majestic Beauty’ ‘Pink Cloud’ ‘Springtime’ R. fortunei) ‘David Gable’ catawbiense hybrid ‘Album Elegans’ catawbiense hybrid ‘Everestianum’ catawbiense hybrid ‘Gomer Waterer’ catawbiense hybrid ‘Mrs. Charles S. Sargent’ ‘Mrs. Charles S. Sargent’ catawbiense hybrid ‘Roseum Elegans’ (caucasicum < R. ponticum var. album) ‘Cunningham’s White’ chapmanii A.Gr. var. album) ‘Cunningham’s White’ chapmanii A.Gr. 298 racemosum X R. keiskei (racemosum < R. mucronulatum) ‘Conemaugh’ reticulatum D.Don ex G.Don rubropilosum Hayata scabrum (selection or hybrid) ‘Phoeniceum’ schlippenbachii Maxim. serpyllifolium (A.Gr.) Miq. serrulatum (Small) Millais simiarum Hance simsii Planch. smirnowii Traut. tamurae (Makino) Masam. tosaense Makino vaseyi A.Gr. vaseyi A.Gr. f. album (Bean) Rehd. (veitchianum x R. edgeworthii) ‘Forsterianum’ vernicosum Franch. viscosum (L.) Torr. weyrichii Maxim. williamsianum Rehd. & Wils. yakushimanum Nakai ssp. makinoi (Tagg) Chamb. yakushimanum Nakai ssp. yakushimanum yakushimanum Nakai ssp. yakushimanum ‘Ken Janeck’ yedoense Maxim. ex Regel var. poukhanense (Lev.) Nakai ‘A. Bedford’ ‘Addy Wery’ ‘Amethystinum’ ‘Amoenum’ ‘Annie E. Endtz’ ‘April Rose’ ‘Autumn Glory’ ‘Azma’ ‘Azor’ ‘Ballerina’ ‘Beauty of Littleworth’ ‘Ben Morrison’ ‘Beni kirishima’ ‘Betty Wormald’ ‘Bibiani’ ‘Blaauw’s Pink’ ‘Blue Tit’ ‘Bosley 1020’ ‘Brandywine’ racemosum X R. keiskei (racemosum < R. mucronulatum) ‘Conemaugh’ reticulatum D.Don ex G.Don rubropilosum Hayata scabrum (selection or hybrid) ‘Phoeniceum’ schlippenbachii Maxim. serpyllifolium (A.Gr.) Miq. serrulatum (Small) Millais simiarum Hance simsii Planch. smirnowii Traut. tamurae (Makino) Masam. tosaense Makino vaseyi A.Gr. vaseyi A.Gr. f. album (Bean) Rehd. (veitchianum x R. edgeworthii) ‘Forsterianum’ * (chapmanii < R. minus) ‘Bowie’ dauricum L. decorum Franch. degronianum Carr. ssp. heptamerum (Maxim.) Hara eriocarpum (Hayata) Nakai ‘Gumpo’ (fastigiatum < R. carolinianum) ‘Ramapo’ flammeum (Michx.) Sarg. fortunei Lindl. fortunei Lindl. ssp. discolor (Franch.) Chamb. (griffithianum < R. fortunei ssp. fortunei) ‘Pink Diamond’ griffithianum hybrid ‘Mrs. E. C. Stirling’ griffithianum hybrid ‘The Hon. Jean Marie de Montague’ indicum (L.) Sweet japonicum (A.Gr.) Suring. kaempferi Planch. kaempferi Planch. ‘Dorsett’ kaempferi x R. komiyamae kanahirae Wils. keiskei Miq. keiskei Miq. ‘Red Flare’ keiskei < R. racemosum ((keiskei xX R. racemosum) < R. keiskei) ‘Mary Fleming’ kiusianum Makino lasiostylum Hayata luteum Sweet macrosepalum Maxim. macrosepalum Maxim. ‘Linearifolium’ maximum L. micranthum Turcz. minus Michx. (minus X R. hirsutum) ‘Myrtifolium’ mucronulatum Turcz. mucronulatum Turcz. ‘Albiflorum’ mucronulatum Turcz. ‘Cornell Pink’ mucronulatum Turcz. ‘Wheeldon’s Pink’ oblongifolium (Small) Millais occidentale (Torr. & A.Gr.) A. Gr. oldhamii Maxim. oreodoxa Franch. var. fargesii (Franch.) Chamb. ovatum (Lindl.) Planch. ROSACEAE ex Maxim. periclymenoides (Michx.) Shinners fortunei Lindl. ssp. discolor (Franch.) Chamb. (griffithianum < R. fortunei vaseyi A.Gr. f. album (Bean) Rehd. (veitchianum x R. edgeworthii) ‘Forsterianum’ vernicosum Franch. viscosum (L.) Torr. weyrichii Maxim. williamsianum Rehd. & Wils. yakushimanum Nakai ssp. makinoi (Tagg) Chamb. yakushimanum Nakai ssp. yakushimanum yakushimanum Nakai ssp. yakushimanum ‘Ken Janeck’ yedoense Maxim. ex Regel var. poukhanense (Lev.) Nakai ‘A. Bedford’ ‘Addy Wery’ ‘Amethystinum’ ‘Amoenum’ ‘Annie E. Endtz’ ‘April Rose’ ‘Autumn Glory’ ‘Azma’ ‘Azor’ ‘Ballerina’ ‘Beauty of Littleworth’ ‘Ben Morrison’ ‘Beni kirishima’ ‘Betty Wormald’ ‘Bibiani’ ‘Blaauw’s Pink’ ‘Blue Tit’ ‘Bosley 1020’ ‘Brandywine’ ‘Brick-dust Red’ ‘Cadis’ ‘Carmen’ ‘Carminata Splendens’ ‘Cerise’ ‘Cherry Red’ ‘Christmas Cheer’ kaempferi Planch. ‘Dorsett’ kaempferi x R. komiyamae kaempferi x R. komiyamae kanahirae Wils. ‘Ponticum Roseum’ ponticum seedling ‘Anah Kruschke’ prinophyllum (Small) Millais prunifolium (Small) Millais prunifolium (Small) Millais ‘Hohman’ punctatum Andr. ‘Ponticum Roseum’ ponticum seedling ‘Anah Kruschke’ prinophyllum (Small) Millais prunifolium (Small) Millais prunifolium (Small) Millais ‘Hohman’ punctatum Andr. 299 ‘Christopher Wren’ ‘Coccinea Major’ ‘Coccinea Speciosa’ ‘Comte de Newport’ ‘Coral Bells’ ‘Coral Cluster’ ‘Corneille’ ‘Dexter Purple’ ‘Dexter’s Champagne’ ‘Duc de Rohan’ ‘Early Lavender’ ‘Early Salmon’ ‘Evening Glow’ ‘Fawley’ ‘Fedora’ ‘Flamingo’ ‘Flowerdale Pink’ ‘Flowerdale Red’ ‘Formosa’ ‘George Lindley Taber’ ‘Georgiana Maclay’ ‘Giant Elegans’ ‘Giant White’ ‘Gibraltar’ Glenn Dale Azaleas (see App. ‘Glory of Sunninghill’ ‘Golden Dream’ ‘Helen’ ‘Hinode giri’ ‘Hugh Koster’ ‘Iveryana’ ‘Koran yuki’ ‘Koromo shikibw’ ‘Kristin’ ‘La Roche’ ‘Lady Mulberry’ ‘Late Orchid’ ‘Lawsal’ ‘Lizette’ ‘Lucinda’ ‘Madame de Bruin’ ‘Magnolia Alba’ ‘Maria Derby’ ‘Mrs. Betty Robertson’ ‘Mrs. G. G. Gerbing’ ‘Mrs. LBJ’ ‘Mrs. R. S. Holford’ ‘Mucronatum’ ‘Nadine’ ‘Narcissiflora’ ‘Nodding Bells’ ‘Obtusum’ ‘Obtusum Album’ ‘Oritani’ C) ‘President Claeys’ ‘Pride of Dorking’ ‘Pride of Mobile’ ‘Purple Splendor’ ‘Queen of Orange’ ‘Red Formosa’ ‘Red Head’ ‘Robert Allison’ ‘Roseum Magnificum’ ‘Royal Red’ ‘Royal Splendor’ ‘Salmon King’ ‘Shrimp Pink’ ‘Small Elegans’ ‘Spring Glory’ ‘Stewartstonian’ ‘Suetsuma’ ‘Sunset’ ‘Susugonoito’ ‘Tiffany’ ‘Trilby’ ‘Viscosepala’ ‘Wheatley’ ‘William Bull’ ‘Windbeam’ ‘Winterthur’ ‘Wissahickon’ ‘Wyanokie’ ‘Yae shojo’ RHODOLEIA Champ. ex Hook. HAMAMELIDACEAE Witch-hazel Family championii Hook. RHODOTYPOS. Sieb. & Zucc. ROSACEAE Jetbead Rose Family scandens (Thunb.) Makino RHUS UL. Sumac ANACARDIACEAE Cashew Family aromatica Ait. chinensis Mill. copallina L. glabra L. michauxii Sarg. punjabensis J.L.Stewart var. sinica (Diels) Rehd. & Wils. typhina L. typhina L. ‘Laciniata’ ‘Christopher Wren’ ‘Coccinea Major’ ‘Coccinea Speciosa’ ‘Comte de Newport’ ‘Coral Bells’ ‘Coral Cluster’ ‘Corneille’ ‘Dexter Purple’ ‘Dexter’s Champagne’ ‘Duc de Rohan’ ‘Early Lavender’ ‘Early Salmon’ ‘Evening Glow’ ‘Fawley’ ‘Fedora’ ‘Flamingo’ ‘Flowerdale Pink’ ‘Flowerdale Red’ ‘Formosa’ ‘George Lindley Taber’ ‘Georgiana Maclay’ ‘Giant Elegans’ ‘Giant White’ ‘Gibraltar’ Glenn Dale Azaleas (see App. ‘Glory of Sunninghill’ ‘Golden Dream’ ‘Helen’ ‘Hinode giri’ ‘Hugh Koster’ ‘Iveryana’ ‘Koran yuki’ ‘Koromo shikibw’ ‘Kristin’ ‘La Roche’ ‘Lady Mulberry’ ‘Late Orchid’ ‘Lawsal’ ‘Lizette’ ‘Lucinda’ ‘Madame de Bruin’ ‘Magnolia Alba’ ‘Maria Derby’ ‘Mrs. Betty Robertson’ ‘Mrs. G. G. Gerbing’ ‘Mrs. LBJ’ ‘Mrs. R. S. Holford’ ‘Mucronatum’ ‘Nadine’ ‘Narcissiflora’ ‘Nodding Bells’ ‘Obtusum’ ‘Obtusum Album’ C) ‘President Claeys’ ‘Pride of Dorking’ ‘Pride of Mobile’ ‘Purple Splendor’ ‘Queen of Orange’ ‘Red Formosa’ ‘Red Head’ ‘Robert Allison’ ‘Roseum Magnificum’ ‘Royal Red’ ‘Royal Splendor’ ‘Salmon King’ ‘Shrimp Pink’ ‘Small Elegans’ ‘Spring Glory’ ‘Stewartstonian’ ‘Suetsuma’ ‘Sunset’ ‘Susugonoito’ ‘Tiffany’ ‘Trilby’ ‘Viscosepala’ ‘Wheatley’ ‘William Bull’ ‘Windbeam’ ‘Winterthur’ ‘Wissahickon’ ‘Wyanokie’ ‘Yae shojo’ RHODOLEIA Champ. ex Hook. HAMAMELIDACEAE Witch-hazel Family championii Hook. RHODOTYPOS. Sieb. & Zucc. ROSACEAE Jetbead Rose Family scandens (Thunb.) Makino RHUS UL. Sumac ANACARDIACEAE Cashew Family aromatica Ait. chinensis Mill. copallina L. glabra L. michauxii Sarg. punjabensis J.L.Stewart var. sinica (Diels) Rehd. & Wils. typhina L. typhina L. ‘Laciniata’ ‘Corneille’ ‘Dexter Pur ‘Duc de Rohan’ ‘Early Lavender’ ‘Early Lavender’ ‘Early Salmon’ ‘Early Salmon’ ‘Evening Glow’ ‘Evening Glow’ ‘Fawley’ ‘Fawley’ ‘Fedora’ ‘Fedora’ ‘Flamingo’ ‘Flamingo’ ‘Flowerdale ‘Flowerdale Pink’ ‘Flowerdale Red’ ‘Gibraltar’ Glenn Dale ‘Golden Dream’ ‘Helen’ ‘Helen’ ‘Hinode ‘Hinode giri’ ‘Hugh Koster’ ‘Hugh Koster’ ‘Iveryana’ ‘Iveryana’ ‘Koran yuki ‘Koran yuki’ ‘Koromo shik ‘Koromo shikibw’ ‘Kristin’ ‘La Roche’ ‘Lady Mulbe ‘Late Orchid’ ‘Lawsal’ ‘Lawsal’ ‘Lizette’ ‘Lizette’ ‘Lucinda’ ‘Lucinda’ ‘Madame typhina L. ‘Laciniata’ 300 centifolia L. ‘Muscosa’ centifolia L. ‘Rose des_Peintres’ centifolia L. ‘Variegata’ chinensis Jacq. chinensis Jacq. ‘Minima’ chinensis Jacq. ‘Mutabilis’ chinensis Jacq. ‘Viridiflora’ (chinensis < R. gigantea) ‘Old Blush’ x damascena Mill. <x damascena Mill. var. semperflorens (Loisel.) Rowley x damascena Mill. ‘Trigintipetala’ x damascena Mill. ‘Versicolor’ (xdamascena var. semperflorens < R. pimpinellifolia) ‘Stanwell Perpetual’ x dupontii Deseglise foetida J.Herrm. foetida J.Herrm. ‘Bicolor’ foetida J.Herrm. ‘Perseana’ forrestiana Boulenger <fortuneana Lindl. <francofurtana Muenchh. ‘Empress Josephine’ gallica L. ‘Conditorum’ gallica L. ‘Versicolor’ glauca Pourr. < harisonii Rivers laevigata Michx. (laxa < R. pimpinellifolia) ‘Suzanne’ x lheritieranea Thory ‘Gracilis’ moschata J.Herrm. moschata J.Herrm. ‘Plena’ moyesii Hemsl. & Wils. multibracteata Hemsl. & Wils. multiflora Thunb. ex J.A.Murr. multiflora Thunb. ex J.A.Murr. ‘Carnea’ multiflora Thunb. ex J.A.Murr. ‘Platyphylla’ (multiflora < R. ‘Reve d’Or’) ‘Aglaia’ xX noisettiana Thory odorata (Andr.) Sweet odorata (Andr.) Sweet ‘Fun Jwan Lo’ odorata (Andr.) Sweet var. pseudindica (Lindl.) Rehd. ‘Fortune’s Double Yellow’ palustris Marsh. <x paulii Rehd. pimpinellifolia L. pimpinellifolia L. var. altaica (Willd.) Thory roxburghii Tratt. ‘Roxburghii’ roxburghii Tratt. f. normalis Rehd. & Wils. rubiginosa L. rugosa Thunb. ex J.A.Murr. rugosa Thunb. ex J.A.Murr. ‘Alba’ rugosa Thunb. ex J.A.Murr. ‘Hanosa’ alpinum L. curvatum Small echinellum (Cov.) Rehd. odoratum H.Wend1. sanguineum Pursh ‘Pulborough Scarlet’ uva-crispa L. uva-crispa L. ‘Pixwell’ ROBINIA L. Locust FABACEAE (Faboideae) Bean Family x ambigua Poir. <x ambigua Poir. ‘Decaisneana’ boyntonii Ashe hispida L. pseudoacacia L. pseudoacacia L. ‘Bicolor’ pseudoacacia L. ‘Christopher Wren’ ‘Coccinea Major’ ‘Coccinea Speciosa’ ‘Comte de Newport’ ‘Coral Bells’ ‘Coral Cluster’ ‘Corneille’ ‘Dexter Purple’ ‘Dexter’s Champagne’ ‘Duc de Rohan’ ‘Early Lavender’ ‘Early Salmon’ ‘Evening Glow’ ‘Fawley’ ‘Fedora’ ‘Flamingo’ ‘Flowerdale Pink’ ‘Flowerdale Red’ ‘Formosa’ ‘George Lindley Taber’ ‘Georgiana Maclay’ ‘Giant Elegans’ ‘Giant White’ ‘Gibraltar’ Glenn Dale Azaleas (see App. ‘Glory of Sunninghill’ ‘Golden Dream’ ‘Helen’ ‘Hinode giri’ ‘Hugh Koster’ ‘Iveryana’ ‘Koran yuki’ ‘Koromo shikibw’ ‘Kristin’ ‘La Roche’ ‘Lady Mulberry’ ‘Late Orchid’ ‘Lawsal’ ‘Lizette’ ‘Lucinda’ ‘Madame de Bruin’ ‘Magnolia Alba’ ‘Maria Derby’ ‘Mrs. Betty Robertson’ ‘Mrs. G. G. Gerbing’ ‘Mrs. LBJ’ ‘Mrs. R. S. Holford’ ‘Mucronatum’ ‘Nadine’ ‘Narcissiflora’ ‘Nodding Bells’ ‘Obtusum’ ‘Obtusum Album’ C) ‘Burgundy’ pseudoacacia L. ‘Frisia’ pseudoacacia L. ‘Purple Rose’ pseudoacacia L. ‘Pyramidalis’ pseudoacacia L. ‘Sandraudiga pseudoacacia L. ‘Tortuosa’ pseudoacacia L. ‘Unifoliola’ pseudoacacia L. var. rectissima Raber viscosa Vent. 9 ROSA L. Rose ROSACEAE Rose Family acicularis Lindl. x alba L. x alba L. ‘Celeste’ x alba L. ‘Semiplena’ (xalba X R. centifolia) ‘Maiden’s Blush’ (? xalba x Damask Hybrid) ‘Koenigin von Daenemark’ <x anemonoides Rehd. ‘Ramona’ banksiae Ait.f. var. banksiae ‘Alba Plena’ banksiae Ait.f. var. banksiae ‘Lutea’ banksiae Ait.f. var. normalis Regel ‘Lutescens’ blanda Ait. bracteata J.C.Wendl. brunonii Lindl. canina L. carolina L. centifolia L. centifolia L. ‘Bullata’ centifolia L. ‘Cristata’ foetida J.Herrm. foetida J.Herrm. foetida J.Herrm. ‘Bicolor’ foetida J.Herrm. ‘Perseana’ foetida J.Herrm. ‘Perseana’ forrestiana Boulenger forrestiana Boulenger <fortuneana Lindl. gallica L. ‘Conditorum’ gallica L. ‘Versicolor’ gallica L. ‘Versicolor’ glauca Pourr. glauca Pourr. < harisonii Riv < harisonii Rivers laevigata Michx. laevigata Michx. (laxa < R. pimpine (laxa < R. pimpinellifolia) ‘Suzanne’ x lheritieranea Thory ‘Gracilis’ x lheritieranea Thory ‘Gracilis’ moschata J.Herrm. moschata J.Herrm. moschata J.Herrm. moschata J.Herrm. ‘Plena’ moyesii Hemsl. & Wils. moyesii Hemsl. & Wils. multibracteata Hemsl. multibracteata Hemsl. & Wils. multiflora Thunb. ex J.A.Murr. multiflora Thunb. ex J.A.Murr. multiflora Thunb. ex J.A.Murr. multiflora Thunb. ex J.A.Murr. ‘Carnea’ multiflora Thunb. ex J.A.Murr. multiflora Thunb. ex J.A.Murr. ‘Platyphylla’ ‘Platyphylla’ (multiflora < (xalba X R. centifolia) ‘Maiden’s Blush’ bracteata J.C.Wendl. brunonii Lindl. brunonii Lindl. canina L. canina L. carolina L carolina L. centifolia L centifolia L. centifolia L. centifolia L. ‘Bullata’ centifolia L. ‘Cristata’ centifolia L. ‘Cristata’ ‘Conrad Ferdinand Meyer’ 301 sempervirens L. ‘Rampant’ sempervirens hybrid ‘Felicite et Perpetue’ setigera Michx. (setigera < R. gallica hybrid) ‘Baltimore Belle’ virginiana Mill. < waitziana Tratt. ‘Macrantha’ wichuraiana Crepin wichuraiana Crepin var. poteriifolia Koidz. (wichuraiana < R. multiflora) ‘America’ (wichuraiana < ‘Champion of the World’) ‘May Queen’ (wichuraiana < ‘Perle des Jardins’) ‘Gardenia’ (wichuraiana x ‘Shirley Hibberd’) ‘Alberic Barbier’ woodsii Lindl. xanthina f. hugonis (Hemsl.) Roberts ‘Agathe Incarnata’ ‘Alfred de Dalmas’ ‘Alika’ ‘Anais Segalas’ ‘Angelica Minor’ ‘Arielle’ ‘Aurora’ ‘Baronne Prevost’ ‘Belinda’ ‘Belle Amour’ ‘Belle de Crecy’ ‘Belle Isador’ ‘Belle Vichysoise’ ‘Blanc Double de Coubert’ ‘Blanche Moreau’ ‘Blanchefleur’ ‘Blush Boursault’ ‘Camaieux’ ‘Captain Christy’ ‘Cardinal de Richelieu’ ‘Catherine Mermet’ ‘Celsiana’ ‘Champney’s Pink Cluster’ ‘Charles de Mills’ ‘Commandant Beaurepaire’ ‘Comte de Chambord’ ‘Cramoisi Superieur’ ‘Crested Jewel’ ‘De la Grifferaie’ ‘Delicata’ ‘Duc de Fitzjames’ ‘Duc de Guiche’ ‘Duchesse de Brabant’ ‘F. J. Grootendorst’ ‘Fraser’s Pink Musk’ ‘Frau Dagmar Hartopp’ ‘Frau Karl Druschki’ ‘General Jacqueminot’ ‘General Kleber’ ‘Gloire de Dijon’ ‘Gloire des Mosseux’ ‘Henri Martin’ ‘Hermosa’ ‘Honorine de Brabant’ ‘Ipsilante’ ‘Isabella Sprunt’ ‘Jacques Cartier’ ‘Jeanne d’Arc’ ‘Jeanne de Montfort’ ‘Juno’ ‘La France’ ‘Leda’ ‘Louis Philippe’ ‘Louise Odier’ ‘Mabel Morrison’ ‘Macrantha’ ‘Marcel Bourgouin’ ‘Marie Louise’ ‘Mme. Alfred Carriere’ ‘Mme. Hardy’ ‘Mme. Isaac Pereire’ ‘Mme. Legras de Saint Germain’ ‘Mme. Louis Leveque’ ‘Mme. Pierre Oger’ ‘Mme. Plantier’ ‘Niphetos’ ‘Nuits de Young’ ‘Oeillet Panachee’ ‘Officinalis’ ‘Omar Khayyam’ ‘Paul Neyron’ ‘Perle d’Or’ ‘Petite de Hollande’ ‘Pompon Elegant’ ‘President de Seze’ ‘Prince Camille de Rohan’ ‘Reine des Violettes’ ‘Reine Victoria’ ‘Reve d’Or’ ‘Rivers’ George IV’ ‘Rose de Meaux’ ‘Safrano’ ‘Salet’ ‘Schneezwerg’ ‘Serratipetala’ ‘Shailer’s Provence’ ‘Sombreuil’ sempervirens L. ‘Rampant’ sempervirens hybrid ‘Felicite et Perpetue’ setigera Michx. (setigera < R. gallica hybrid) ‘Baltimore Belle’ virginiana Mill. < waitziana Tratt. ‘Macrantha’ wichuraiana Crepin wichuraiana Crepin var. poteriifolia Koidz. (wichuraiana < R. multiflora) ‘America’ (wichuraiana < ‘Champion of the World’) ‘May Queen’ (wichuraiana < ‘Perle des Jardins’) ‘Gardenia’ (wichuraiana x ‘Shirley Hibberd’) ‘Alberic Barbier’ woodsii Lindl. xanthina f. ‘Platyphylla’ (multiflora < hugonis (Hemsl.) Roberts ‘Agathe Incarnata’ ‘Alfred de Dalmas’ ‘Alika’ ‘Anais Segalas’ ‘Angelica Minor’ ‘Arielle’ ‘Aurora’ ‘Baronne Prevost’ ‘Belinda’ ‘Belle Amour’ ‘Belle de Crecy’ ‘Belle Isador’ ‘Belle Vichysoise’ ‘Blanc Double de Coubert’ ‘Blanche Moreau’ ‘Blanchefleur’ ‘Blush Boursault’ ‘Camaieux’ ‘Captain Christy’ ‘Cardinal de Richelieu’ ‘Catherine Mermet’ ‘Celsiana’ ‘Champney’s Pink Cluster’ ‘Charles de Mills’ ‘Commandant Beaurepaire’ ‘Comte de Chambord’ ‘Cramoisi Superieur’ ‘Crested Jewel’ ‘De la Grifferaie’ ‘Delicata’ ‘Duc de Fitzjames’ ‘Duc de Guiche’ ‘Hermosa’ ‘Honorine ‘La France’ ‘Leda’ ‘Leda’ ‘Louis ‘Louise Odier’ ‘Mabel Morrison ‘Mabel Morrison’ ‘Macrantha’ ‘Macrantha’ ‘Marcel Bourg ‘Marcel Bourgouin’ ‘Marie Louise’ ‘Marie Louise’ ‘Mme. Alfred C ‘Mme. Alfred Carriere’ ‘Mme. Hardy’ ‘Mme. Hardy’ ‘Mme. Isaac Pe ‘Aurora’ ‘Baronne ‘Mme. Isaac Pereire’ ‘Mme. Legras de Sain ‘Baronne Prevost’ ‘Belinda’ ‘Belinda’ ‘Belle Amo ‘Belle Amour’ ‘Belle de Crecy ‘Mme. Pierre Oger’ ‘Mme. Plantier’ ‘Belle de Crecy’ ‘Belle Isador’ ‘Mme. Plantier’ ‘Niphetos’ ‘Niphetos’ ‘Nuits de Y ‘Belle Isador’ ‘Belle Vichysoi ‘Nuits de Young’ ‘Oeillet Panachee’ ‘Belle Vichysoise’ ‘Blanc Double de C ‘Blanc Double de Coubert’ ‘Blanche Moreau’ ‘Oeillet Panachee’ ‘Officinalis’ ‘Officinalis’ ‘Omar Khayy ‘Omar Khayyam’ ‘Paul Neyron’ ‘Paul Neyron’ ‘Perle d’Or’ ‘Perle d’Or’ ‘Petite de H ‘Camaieux’ ‘Captain Chr ‘Tuscany’ ‘Tuscany ‘Tuscany Superb’ ‘Variegata di Bolog ‘Variegata di Bologna’ 302 ROSMARINUS L. LAMIACEAE Mint Family officinalis L. officinalis L. ‘Prostratus’ RUBUS L. ROSACEAE Rose Family calycinoides Hayata & Koidz. cockburnianus Hemsl. odoratus L. rosifolius Sm. ‘Coronarius’ RUSCUS L. LILIACEAE Lily Family aculeatus L. hypoglossum L. RUSSELIA Jacq. SCROPHULARIACEAE Figwort Family equisetiformis Schlechtend. & Cham. SABAL Adans. ARECACEAE Palm Family minor (Jacq.f.) Pers. palmetto (Walt.) Lodd. ex J.A. & J.H. Schultes SAGERETIA Brongn. RHAMNACEAE Buckthorn Family thea (Osbeck) M.C.Johnst. Willow Willow Family SALIX L. SALICACEAE aegyptiaca L. alba L. alba L. var. vitellina (L.) Stokes ‘Pendula’ alba x S. fragilis (alba var. vitellina <x S. babylonica) ‘Chrysocoma’ babylonica L. babylonica L. ‘Crispa’ babylonica L. ‘Tortuosa’ x bicolor Ehrh. x blanda Anderss. caprea L. caprea X< S. elaeagnos caprea X S. purpurea chaenomeles Kimura cinerea L. Mint Family Mint Family officinalis L. officinalis L. ‘Prostratus’ RUBUS L. ROSACEAE Rose Family calycinoides Hayata & Koidz. cockburnianus Hemsl. odoratus L. rosifolius Sm. ‘Coronarius’ RUSCUS L. LILIACEAE Lily Family aculeatus L. hypoglossum L. RUSSELIA Jacq. SCROPHULARIACEAE Figwort Family equisetiformis Schlechtend. & Cham. SABAL Adans. ARECACEAE Palm Family minor (Jacq.f.) Pers. palmetto (Walt.) Lodd. ex J.A. & J.H. Schultes SAGERETIA Brongn. RHAMNACEAE Buckthorn Family thea (Osbeck) M.C.Johnst. Willow Willow Family SALIX L. SALICACEAE aegyptiaca L. alba L. alba L. ‘Platyphylla’ (multiflora < var. vitellina (L.) Stokes ‘Pendula’ alba x S. fragilis (alba var. vitellina <x S. babylonica) ‘Chrysocoma’ babylonica L. babylonica L. ‘Crispa’ babylonica L. ‘Tortuosa’ x bicolor Ehrh. x blanda Anderss. caprea L. caprea X< S. elaeagnos caprea X S. purpurea chaenomeles Kimura cinerea L. x pontederana Willd. purpurea L. x pontederana Willd. purpurea L. purpurea L. purpurea L. purpurea L. ‘Eugenei’ purpurea L. ‘Pendula’ purpurea L. ‘Pendula’ purpurea L. f. gracilis purpurea L. f. gracilis (Gren. & Godr.) Schneid. rigida Muhl. x rubra Huds. SALVIA L. LAMIACEAE greggii A.Gr. leucantha Cav. babylonica L. ‘Crispa’ babylonica L. ‘Tortuosa babylonica L. ‘Tortuosa’ x bicolor Ehrh. x blanda Anderss. caprea L. caprea L. caprea X< caprea X< S. elaeagnos caprea X S. purpurea caprea X S. purpurea chaenomeles Kimura chaenomeles Kimura cinerea L. 303 SCHIZOPHRAGMA Sieb. & Zucc. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family hydrangeoides Sieb. & Zucc. SCIADOPITYS Sieb. & Zucc. TAXODIACEAE Taxodium Family verticillata (Thunb. ex J.A.Murr.) Sieb. & ZUcc. SEBASTIANA Spreng. EUPHORBIACEAE Spurge Family ligustrina (Michx.) Muell.-Arg. SEMIARUNDINARIA Makino ex Nakai POACEAE Grass Family fastuosa (Latour-Marl. ex Mitf.) Makino ex Nakai SENECIO L. ASTERACEAE Aster Family salignus DC. SEQUOIA Endl. TAXODIACEAE California Redwood Taxodium Family sempervirens (D.Don) Endl. sempervirens (D.Don) Endl. ‘Adpressa’ SEQUOIADENDRON J. Buchh. TAXODIACEAE Taxodium Family giganteum (Lindl.) J.Buchh. SERENOA Hook.f. ARECACEAE Palm Family repens (Bartram) Small SERISSA Comm. ex Juss. RUBIACEAE Madder Family japonica (Thunb.) Thunb. SESBANIA Scop. FABACEAE (Faboideae) Bean Family drummondii (Rydb.) Cory punicea (Cav.) Benth. SCHIZOPHRAGMA Sieb. & Zucc. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family hydrangeoides Sieb. & Zucc. SCIADOPITYS Sieb. & Zucc. TAXODIACEAE Taxodium Family verticillata (Thunb. ex J.A.Murr.) Sieb. & ZUcc. SEBASTIANA Spreng. EUPHORBIACEAE Spurge Family ligustrina (Michx.) Muell.-Arg. SEMIARUNDINARIA Makino ex Nakai POACEAE Grass Family fastuosa (Latour-Marl. ex Mitf.) Makino ex Nakai SENECIO L. ASTERACEAE Aster Family salignus DC. SEQUOIA Endl. TAXODIACEAE California Redwood Taxodium Family sempervirens (D.Don) Endl. sempervirens (D.Don) Endl. ‘Adpressa’ SEQUOIADENDRON J. Buchh. TAXODIACEAE Taxodium Family giganteum (Lindl.) J.Buchh. SERENOA Hook.f. ARECACEAE Palm Family repens (Bartram) Small SERISSA Comm. ex Juss. RUBIACEAE Madder Family japonica (Thunb.) Thunb. SESBANIA Scop. FABACEAE (Faboideae) Bean Family drummondii (Rydb.) Cory punicea (Cav.) Benth. SAMBUCUS L. CAPRIFOLIACEAE Elder Honeysuckle Family canadensis L. ebulus L. nigra L. ‘Aurea’ racemosa L. ssp. pubens (Michx.) H.House racemosa L. ssp. sieboldiana (Miq.) Hara SANTOLINA L. ASTERACEAE Aster Family chamaecyparissus L. SAPINDUS lL. SAPINDACEAE Soapberry Soapberry Family drummondii Hook. & Arn. marginatus Willd. mukorossi Gaertn. SAPIUM 1 P.Br. ‘Platyphylla’ (multiflora < EUPHORBIACEAE Tallow Tree Spurge Family japonicum (Sieb. & Zucc.) Pax & K.Hoffm. sebiferum (L.) Roxb. SARCOCOCCA Lindl. BUXACEAE Sweet Box Boxwood Family hookeriana Baill. var. digyna Franch. hookeriana Baill. var. humilis Rehd. & Wils. ruscifolia Stapf SASA Makino & Shibata POACEAE Grass Family veitchii (Carr.) Rehd. SASSAFRAS T. Nees & Eberm. LAURACEAE Laurel Family albidum (Nutt.) Nees SATUREJA L. LAMIACEAE Mint Family montana L. SCHINUS lL. Peppertree SCHIZOPHRAGMA Sieb. & Zucc. SAXIFRAGACEAE (Hydrangeoideae) Saxifrage Family hydrangeoides Sieb. & Zucc. SCIADOPITYS Sieb. & Zucc. TAXODIACEAE Taxodium Family verticillata (Thunb. ex J.A.Murr.) Sieb. & ZUcc. SEBASTIANA Spreng. EUPHORBIACEAE Spurge Family ligustrina (Michx.) Muell.-Arg. SEMIARUNDINARIA Makino ex Nakai POACEAE Grass Family fastuosa (Latour-Marl. ex Mitf.) Makino ex Nakai SENECIO L. ASTERACEAE Aster Family salignus DC. SEQUOIA Endl. TAXODIACEAE California Redwood Taxodium Family sempervirens (D.Don) Endl. sempervirens (D.Don) Endl. ‘Adpressa’ SEQUOIADENDRON J. Buchh. TAXODIACEAE Taxodium Family giganteum (Lindl.) J.Buchh. SERENOA Hook.f. ARECACEAE Palm Family repens (Bartram) Small SERISSA Comm. ex Juss. RUBIACEAE Madder Family japonica (Thunb.) Thunb. SESBANIA Scop. FABACEAE (Faboideae) Bean Family japonica L. ‘Dot’ japonica L. ‘Pendula’~— japonica L. ‘Regent’ secundiflora (Ort.) Lag. ex DC. SORBARIA (Ser. ex DC.) A.Braun False Spirea ROSACEAE Rose Family kirilowii (Regel) Maxim. tomentosa (Lindl.) Rehd. var. tomentosa <XSORBARONIA Schneid. ROSACEAE Rose Family fallax Schneid. sorbifolia (Poir.) Schneid. ‘Brilliantissima’ xSORBOCOTONEASTER Pojark. ROSACEAE Rose Family pozdnjakovii Pojark. <SORBOPYRUS Schneid. ROSACEAE Rose Family auricularis (Knoop) Schneid. ‘Bulbiformis’ SORBUS L. Mountain Ash ROSACEAE Rose Family alnifolia (Sieb. & Zucc.) K.Koch americana Marsh. aria (L.) Crantz ‘Magnifica’ aucuparia L. aucuparia L. ‘Cardinal’ aucuparia L. ‘Fastigiata’ aucuparia L. ‘Pendula’ domestica L. hybrida L. pohuashanensis (Hance) Hed. torminalis Crantz ‘Apricot Queen’ ‘Red Copper Glow’ SPARTIUM L. FABACEAE (Faboideae) Bean Family junceum L. SPIRAEA L. Spirea ROSACEAE Rose Family alba Du Roi SEVERINIA Tenore ex Endl. RUTACEAE Citrus Family buxifolia (Poir.) Tenore SHIBATAEA Makino ex Nakai POACEAE Grass Family kumasaca (Zoll.) Makino SINARUNDINARIA_ Nakai POACEAE Grass Family nitida (Mitf.) Nakai SIPHONOSMANTHUS Stapf OLEACEAE Olive Family delavayi (Franch.) Stapf SKIMMIA Thunb. RUTACEAE Skimmia Citrus Family <foremanii H.Knight japonica Thunb. japonica Thunb. ‘Fisheri’ japonica Thunb. ‘Fructo-albo’ japonica Thunb. ‘Nana’ japonica Thunb. ‘Veitchii’ japonica Thunb. ‘Wisley Red’ japonica Thunb. var. repens (Nakai) Ohwi laureola Sieb. & Zucc. reevesiana Fort. SMILAX L. SMILACACEAE Greenbrier Greenbrier Family biflora Sieb. ex Miq. laurifolia L. pumila Walt. smallii Morong SOLANUM L. SOLANACEAE Nightshade Family diphyllum L. jasminoides Paxt. rantonnetii Carr. seaforthianum Andr. wendlandii Hook.f. SOPHORA lL. ‘Platyphylla’ (multiflora < FABACEAE (Faboideae) Bean Family Greenbrier Greenbrier Family Nightshade Family Greenbrier Greenbrier Family Nightshade Family 305 < arguta Zab. betulifolia Pall. < billiardii Herincq blumei G.Don bullata Maxim. <x bumalda Burv. <x bumalda Burv. ‘Anthony Waterer’ x bumalda Burv. ‘Crispa’ x bumalda Burv. ‘Goldflame’ cantoniensis Lour. cantoniensis Lour. ‘Lanceata’ chinensis Maxim. japonica L.f. japonica L.f. ‘Alpina’ x lemoinei Zab. ‘Alpestris’ x margaritae Zab. miyabei Koidz. nipponica Maxim. var. tosaensis (Yatabe) Makino nipponica Maxim. var. tosaensis (Yatabe) Makino ‘Snowmound’ prunifolia Sieb. & Zucc. salicifolia L. 'X sanssouciana K.Koch thunbergii Sieb. ex BI. thunbergii Sieb. ex Bl. ‘Compacta’ tomentosa L. trilobata L. ‘Swan Lake’ x vanhouttei (C. Briot) Zab. STACHYURUS Sieb. & Zucc. STACHYURACEAE Stachyurus Family chinensis Franch. praecox Sieb. & Zucc. praecox Sieb. & Zucc. ‘Issai’ STAPHYLEA L. STAPHYLEACEAE Bladdernut Family colchica Steven pinnata L. trifolia L. STEPHANANDRA Sieb. & Zucc. ROSACEAE Rose Family incisa (Thunb. ex J.A.Murr.) Zab. incisa (Thunb. ex J.A.Murr.) Zab. ‘Crispa’ tanakae (Franch. & Sav.) Franch. & Sav. STEWARTIA L. THEACEAE Tea Family malacodendron L. monadelpha Sieb. & Zucc. ovata (Cav.) Weatherby pseudocamellia Maxim. rostrata Spongberg serrata Maxim. sinensis Rehd. & Wils. ovata (Cav.) Weatherby pseudocamellia Maxim. rostrata Spongberg serrata Maxim. sinensis Rehd. & Wils. betulifolia Pall. < billiardii Herin < billiardii Herincq blumei G.Don blumei G.Don bullata Maxim. bullata Maxim. <x bumalda Burv. sinensis Rehd. & Wils. STIGMAPHYLLON Juss. MALPIGHIACEAE Malpighia Family ledifolium (HBK.) Small STRANVAESIA Lindl. ROSACEAE Rose Family davidiana Decne. davidiana Decne. ‘Lutea’ niitakayamensis (Hayata) Hayata STYRAX L. STYRACACEAE Storax, Snowbell Storax Family americanus Lam. dasyanthus Perk. grandifolius Ait. japonicus Sieb. & Zucc. japonicus Sieb. & Zucc. ‘Carillon’ japonicus Sieb. & Zucc. ‘Pink Chimes’ obassia Sieb. & Zucc. platanifolius Engelm. SYAGRUS Mart. ARECACEAE Palm Family romanzoffianum (Cham.) Glassman romanzoffianum < Butia capitata <SYCOPARROTIA P.Endress & J.Anliker HAMAMELIDACEAE Witch-hazel Family semidecidua P.Endress & J.Anliker SYCOPSIS Oliv. HAMAMELIDACEAE Witch-hazel Family sinensis Oliv. SYMPHORICARPOS Duham. Snowberry CAPRIFOLIACEAE Honeysuckle Family albus (L.) S.F.Blake albus (L.) S.F.Blake var. laevigatus (Fern.) S.F.Blake < chenaultii Rehd. orbiculatus Moench <x bumalda Burv. <x bumalda Burv. <x bumalda Burv. ‘Anthony Waterer’ x bumalda Burv. ‘Crispa’ x bumalda Burv. ‘Crispa’ x bumalda Burv. ‘Goldflam x bumalda Burv. ‘Goldflame’ cantoniensis Lour. cantoniensis Lour. cantoniensis Lour. cantoniensis Lour. ‘Lanceata’ chinensis Maxim. chinensis Maxim. japonica L.f. japonica L.f. japonica L.f. x lemoinei Zab. ‘Alpestris’ x margaritae Zab. x margaritae Zab. miyabei Koidz. Makino ‘Snowmound’ prunifolia Sieb. & Zucc. prunifolia Sieb. & Zucc. salicifolia L. thunbergii Sieb. ex BI. SYMPLOCOS Jacq. SYMPLOCACEAE vulgaris L. ‘Mrs. Edward Harding’ vulgaris L. ‘President-Grevy’ vulgaris L. ‘Primrose’ vulgaris L. ‘Priscella’ vulgaris L. ‘Purpurea’ vulgaris L. ‘Rene Jarry-Desloges’ vulgaris L. ‘Sarah Sands’ vulgaris L. ‘Zulu’ yunnanensis Franch. ‘Alexander’s Perfection’ ‘Jessica’ SYMPLOCOS Jacq. SYMPLOCACEAE Sweetleaf Family lucida Sieb. & Zucc. paniculata (Thunb. ex J.A.Murr.) Miq. tinctoria (L.) L’Her. SYRINGA L. Lilac OLEACEAE Olive Family x chinensis Willd. x henryi Schneid. < hyacinthiflora (V.Lemoine) Rehd. ‘Buffon’ < hyacinthiflora (V.Lemoine) Rehd. ‘Charles Giant’ x hyacinthiflora (V.Lemoine) Rehd. ‘Lamartine’ x hyacinthiflora (V.Lemoine) Rehd. ‘Pocohantas’ x hyacinthiflora (V.Lemoine) Rehd. ‘Vaubon’ josikaea Jacq.f. ex Reichenb. laciniata Mill. meyeri Schneid. microphylla Diels microphylla Diels ‘Superba’ oblata Lindl. var. dilatata (Nakai) Rehd. oblata Lindl. var. dilatata (Nakai) Rehd. ‘Annabelle’ oblata Lindl. var. dilatata (Nakai) Rehd. ‘Laurentian’ patula (Palib.) Nakai patula (Palib.) Nakai ‘Miss Kim’ persica L. persica L. ‘Laciniata’ xX prestoniae McKelvey ‘Ethel M. Webster’ pubescens Turcz. reticulata (Bl.) Hara villosa Vahl villosa Vahl ‘Crayton’ vulgaris L. ‘Alba’ vulgaris L. ‘Alba Plena’ vulgaris L. ‘Charles Joly’ vulgaris L. ‘Charles Nordine’ vulgaris L. ‘Decaisne’ vulgaris L. ‘Esther Staley’ vulgaris L. ‘Henri Robert’ vulgaris L. ‘Jules Ferry’ vulgaris L. ‘Katherine Havemeyer’ vulgaris L. ‘Leon Gambetta’ vulgaris L. ‘Marechal Lannes’ vulgaris L. ‘Maurice Barres’ vulgaris L. ‘Miss Ellen Willmott’ vulgaris L. ‘Mme. Charles Souchet’ vulgaris L. ‘Monge’ lucida Sieb. & Zucc. paniculata (Thunb. ex J.A.Murr.) Miq. tinctoria (L.) L’Her. ‘Platyphylla’ (multiflora < thunbergii Sieb. ex Bl. 306 SYMPLOCOS Jacq. SYMPLOCACEAE Sweetleaf Family lucida Sieb. & Zucc. paniculata (Thunb. ex J.A.Murr.) Miq. tinctoria (L.) L’Her. SYRINGA L. Lilac OLEACEAE Olive Family x chinensis Willd. x henryi Schneid. < hyacinthiflora (V.Lemoine) Rehd. ‘Buffon’ < hyacinthiflora (V.Lemoine) Rehd. ‘Charles Giant’ x hyacinthiflora (V.Lemoine) Rehd. ‘Lamartine’ x hyacinthiflora (V.Lemoine) Rehd. ‘Pocohantas’ x hyacinthiflora (V.Lemoine) Rehd. ‘Vaubon’ josikaea Jacq.f. ex Reichenb. laciniata Mill. meyeri Schneid. microphylla Diels microphylla Diels ‘Superba’ oblata Lindl. var. dilatata (Nakai) Rehd. oblata Lindl. var. dilatata (Nakai) Rehd. ‘Annabelle’ oblata Lindl. var. dilatata (Nakai) Rehd. ‘Laurentian’ patula (Palib.) Nakai patula (Palib.) Nakai ‘Miss Kim’ persica L. persica L. ‘Laciniata’ xX prestoniae McKelvey ‘Ethel M. Webster’ pubescens Turcz. reticulata (Bl.) Hara villosa Vahl villosa Vahl ‘Crayton’ vulgaris L. ‘Alba’ vulgaris L. ‘Alba Plena’ vulgaris L. ‘Charles Joly’ vulgaris L. ‘Charles Nordine’ vulgaris L. ‘Decaisne’ vulgaris L. ‘Esther Staley’ vulgaris L. ‘Henri Robert’ vulgaris L. ‘Jules Ferry’ vulgaris L. ‘Katherine Havemeyer’ vulgaris L. ‘Leon Gambetta’ vulgaris L. ‘Marechal Lannes’ vulgaris L. ‘Maurice Barres’ vulgaris L. ‘Mrs. Edward Harding’ vulgaris L. ‘President-Grevy’ vulgaris L. ‘Primrose’ vulgaris L. ‘Priscella’ vulgaris L. ‘Purpurea’ vulgaris L. ‘Rene Jarry-Desloges’ vulgaris L. ‘Sarah Sands’ vulgaris L. ‘Zulu’ yunnanensis Franch. ‘Alexander’s Perfection’ ‘Jessica’ TABERNAEMONTANA L. APOCYNACEAE Dogbane Family divaricata (L.) R.Br. ex Roem. & J.A. Schultes TAIWANIA Hayata TAXODIACEAE Taxodium Family cryptomerioides Hayata TAMARIX L. TAMARICACEAE Tamarisk Family parviflora DC. ramosissima Ledeb. ramosissima Ledeb. ‘Rubra’ ramosissima Ledeb. ‘Summer Glow’ TAXODIUM L.C.Rich. TAXODIACEAE Bald Cypress Taxodium Family distichum (L.) L.C.Rich. ‘Pendens’ distichum (L.) L.C.Rich. var. distichum distichum (L.) L.C.Rich. var. nutans (Ait.) Sweet mucronatum Tenore TAXUS L. Yew TAXACEAE Yew Family baccata L. baccata L. ‘Adpressa’ baccata L. ‘Adpressa Fowle’ baccata L. ‘Amersfoort’ baccata L. ‘Aurea’ baccata L. ‘Aurea Marginata’ baccata L. ‘Elegantissima’ baccata L. ‘Fastigiata Robusta’ baccata L. ‘Overeynderi’ baccata L. ‘Repandens’ baccata L. f. fastigiata (Lindl.) Pilger vulgaris L. ‘Mrs. Edward Harding’ vulgaris L. ‘President-Grevy’ vulgaris L. ‘Primrose’ vulgaris L. ‘Priscella’ vulgaris L. ‘Purpurea’ vulgaris L. ‘Rene Jarry-Desloges’ vulgaris L. ‘Sarah Sands’ vulgaris L. ‘Zulu’ yunnanensis Franch. ‘Alexander’s Perfection’ ‘Jessica’ SYRINGA L. OLEACEAE ‘Filiformis’ ‘Froebelii’ ‘Globosa’ ‘Globosa Rheindiana’ ‘Hetz Midget’ ‘Holmstrup’ ‘Hudsonica’ ‘Little Gem’ ‘Lutea’ ‘Malonyana’ ‘Ohlendorfii’ ‘Pendula’ ‘Pygmaea’ ‘Pyramidalis’ ‘Pyramidalis Nigra’ ‘Recurva Nana’ ‘Rheingold’ ‘Semperaurea’ ‘Spiralis’ ‘Stricta’ ‘Sunkist’ ‘Techny’ ‘Umbraculifera’ occidentalis L. ‘Wareana’ occidentalis L. ‘Wareana Lutescens’ occidentalis L. ‘Woodwardii’ plicata J.Donn ex D.Don plicata J.Donn ex D.Don ‘Cuprea’ plicata J.Donn ex D.Don ‘Rogersii’ plicata J.Donn ex D.Don ‘Zebrina’ standishii (Gord.) Carr. cuspidata Sieb. & Zucc. ‘Luteobaccata’ cuspidata Sieb. & Zucc. ‘Minima’ cuspidata Sieb. & Zucc. ‘Nana’ cuspidata Sieb. & Zucc. ‘Nana Variegata’ cuspidata Sieb. & Zucc. ‘Thompson’ floridana Nutt. < hunnewelliana Rehd. ‘Richard Horsey’ < media Rehd. < media Rehd. ‘Citation’ x media Rehd. ‘Flushing’ < media Rehd. ‘Hicksii’ cuspidata Sieb. & Zucc. ‘Luteobaccata’ cuspidata Sieb. & Zucc. ‘Minima’ cuspidata Sieb. & Zucc. ‘Nana’ cuspidata Sieb. & Zucc. ‘Nana Variegata’ cuspidata Sieb. & Zucc. ‘Thompson’ floridana Nutt. < hunnewelliana Rehd. ‘Richard Horsey’ < media Rehd. < media Rehd. ‘Citation’ x media Rehd. ‘Flushing’ < media Rehd. ‘Hicksii’ occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. ‘Filiformis’ ‘Froebelii’ ‘Globosa’ ‘Globosa Rheindiana’ ‘Hetz Midget’ ‘Holmstrup’ ‘Hudsonica’ ‘Little Gem’ ‘Lutea’ ‘Malonyana’ ‘Ohlendorfii’ ‘Pendula’ ‘Pygmaea’ ‘Pyramidalis’ ‘Pyramidalis Nigra’ ‘Recurva Nana’ ‘Rheingold’ ‘Semperaurea’ ‘Spiralis’ ‘Stricta’ ‘Sunkist’ ‘Techny’ ‘Umbraculifera’ occidentalis L. ‘Wareana’ occidentalis L. ‘Wareana Lutescens’ occidentalis L. ‘Woodwardii’ plicata J.Donn ex D.Don plicata J.Donn ex D.Don ‘Cuprea’ plicata J.Donn ex D.Don ‘Rogersii’ plicata J.Donn ex D.Don ‘Zebrina’ standishii (Gord.) Carr. THUJOPSIS Sieb. & Zucc. ex Endl. False or Hiba Arborvitae CUPRESSACEAE Cypress Family dolabrata (Thunb. ex L.f.) Sieb. & Zucc. dolabrata (Thunb. ex L.f.) Sieb. & Zucc. ‘Nana’ dolabrata (Thunb. ex L.f.) Sieb. & Zucc. var. hondae Makino THUNBERGIA Retz. ACANTHACEAE Acanthus Family grandiflora (Roxb. ex Rottl.) Roxb. THYMUS L. LAMIACEAE Mint Family vulgaris L. TIBOUCHINA Aubl. MELASTOMATACEAE Melastoma Family urvilleana (DC.) Cogn. x media Rehd. ‘Flushing’ < media Rehd. ‘Hicksii’ TECOMA Juss. BIGNONIACEAE Bignonia Family stans (L.) HBK. var. angustata Rehd. TECOMARIA (Endl.) Spach BIGNONIACEAE Bignonia Family capensis (Thunb.) Spach TERNSTROEMIA Mutis ex Lf. THEACEAE Tea Family gymnanthera (Wight & Arn.) Sprague TETRAPANAX (K.Koch) K.Koch ARALIACEAE Ginseng Family papyriferus (Hook.) K.Koch TEUCRIUM L. LAMIACEAE Germander Mint Family chamaedrys L. x lucidrys Boom lucidum L. THAMNOCALAMUS Munro POACEAE Grass Family spathaceus (Franch.) Soderstrom THUJA L. SYRINGA L. OLEACEAE ‘Jessica’ TABERNAEMONTANA L. APOCYNACEAE Dogbane Family divaricata (L.) R.Br. ex Roem. & J.A. Schultes TAIWANIA Hayata TAXODIACEAE Taxodium Family cryptomerioides Hayata TAMARIX L. TAMARICACEAE Tamarisk Family parviflora DC. ramosissima Ledeb. ramosissima Ledeb. ‘Rubra’ ramosissima Ledeb. ‘Summer Glow’ TAXODIUM L.C.Rich. TAXODIACEAE Bald Cypress Taxodium Family distichum (L.) L.C.Rich. ‘Pendens’ distichum (L.) L.C.Rich. var. distichum distichum (L.) L.C.Rich. var. nutans (Ait.) Sweet mucronatum Tenore TAXUS L. Yew TAXACEAE Yew Family baccata L. baccata L. ‘Adpressa’ baccata L. ‘Adpressa Fowle’ baccata L. ‘Amersfoort’ baccata L. ‘Aurea’ baccata L. ‘Aurea Marginata’ baccata L. ‘Elegantissima’ baccata L. ‘Fastigiata Robusta’ baccata L. ‘Overeynderi’ baccata L. ‘Repandens’ baccata L. f. fastigiata (Lindl.) Pilger canadensis Marsh. cuspidata Sieb. & Zucc. cuspidata Sieb. & Zucc. ‘Expansa’ 307 cuspidata Sieb. & Zucc. ‘Luteobaccata’ cuspidata Sieb. & Zucc. ‘Minima’ cuspidata Sieb. & Zucc. ‘Nana’ cuspidata Sieb. & Zucc. ‘Nana Variegata’ cuspidata Sieb. & Zucc. ‘Thompson’ floridana Nutt. < hunnewelliana Rehd. ‘Richard Horsey’ < media Rehd. < media Rehd. ‘Citation’ x media Rehd. ‘Flushing’ < media Rehd. ‘Hicksii’ TECOMA Juss. BIGNONIACEAE Bignonia Family stans (L.) HBK. var. angustata Rehd. TECOMARIA (Endl.) Spach BIGNONIACEAE Bignonia Family capensis (Thunb.) Spach TERNSTROEMIA Mutis ex Lf. THEACEAE Tea Family gymnanthera (Wight & Arn.) Sprague TETRAPANAX (K.Koch) K.Koch ARALIACEAE Ginseng Family papyriferus (Hook.) K.Koch TEUCRIUM L. LAMIACEAE Germander Mint Family chamaedrys L. x lucidrys Boom lucidum L. THAMNOCALAMUS Munro POACEAE Grass Family spathaceus (Franch.) Soderstrom THUJA L. CUPRESSACEAE Arborvitae Cypress Family occidentalis L. occidentalis L. ‘Alba’ occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. ‘Filiformis’ ‘Froebelii’ ‘Globosa’ ‘Globosa Rheindiana’ ‘Hetz Midget’ ‘Holmstrup’ ‘Hudsonica’ ‘Little Gem’ ‘Lutea’ ‘Malonyana’ ‘Ohlendorfii’ ‘Pendula’ ‘Pygmaea’ ‘Pyramidalis’ ‘Pyramidalis Nigra’ ‘Recurva Nana’ ‘Rheingold’ ‘Semperaurea’ ‘Spiralis’ ‘Stricta’ ‘Sunkist’ ‘Techny’ ‘Umbraculifera’ occidentalis L. ‘Wareana’ occidentalis L. ‘Wareana Lutescens’ occidentalis L. ‘Woodwardii’ plicata J.Donn ex D.Don plicata J.Donn ex D.Don ‘Cuprea’ plicata J.Donn ex D.Don ‘Rogersii’ plicata J.Donn ex D.Don ‘Zebrina’ standishii (Gord.) Carr. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. occidentalis L. SYRINGA L. OLEACEAE ‘Minuta’ canadensis (L.) Carr. ‘Pendula’ canadensis (L.) Carr. ‘Sargentii’ canadensis (L.) Carr. ‘Verkade Recurved’ canadensis (L.) Carr. ‘Von Helms’ canadensis (L.) Carr. ‘Youngcone’ caroliniana Engelm. TILIA L. TILIACEAE Linden, Basswood Linden Family TILIA L. TILIACEAE Linden, Basswood Linden Family americana L. cordata Mill. x euchlora K.Koch x europaea L. x moltkei Spaeth mongolica Maxim. platyphyllos Scop. platyphyllos Scop. ‘Laciniata’ platyphyllos Scop. ‘Vitifolia’ tomentosa Moench tomentosa Moench ‘Pendula’ TIPUANA (Benth.) Benth. FABACEAE (Faboideae) Bean Family tipu (Benth.) O.Ktze. TOONA (Endl.) M.J.Roem. MELIACEAE Mahogany Family sinensis (Juss.) M.J.Roem. TORREYA Arn. TAXACEAE Yew Family nucifera (L.) Sieb. & Zucc. nucifera (L.) Sieb. & Zucc. ‘Gold Strike’ taxifolia Arn. TOXICODENDRON UMiili. ANACARDIACEAE Cashew Family radicans (L.) O.Ktze. vernicifluum (Stokes) F.A.Barkley TRACHELOSPERMUM Lem. APOCYNACEAE Dogbane Family asiaticum (Sieb. & Zucc.) Nakai difforme (Walt.) A.Gr. jasminoides (Lindl.) Lem. jasminoides (Lindl.) Lem. ‘Variegatum’ Jasminoides (Lindl.) Lem. var. pubescens Makino TRACHYCARPUS' H.Wendl. ARECACEAE Palm Family fortunei (Hook.) H.Wendl. TRIPTERYGIUM Hook.f. CELASTRACEAE Staff-tree Family acanthocoma Drude TROCHODENDRON Sieb. & Zucc. TROCHODENDRACEAE Trochodendron Family aralioides Sieb. & Zucc. TSUGA Carr. Hemlock PINACEAE Pine Family canadensis (L.) Carr. canadensis (L.) Carr. ‘Abbott’s Dwarf’ canadensis (L.) Carr. ‘Angustifolia’ canadensis (L.) Carr. ‘Armistice’ canadensis (L.) Carr. ‘Beaujean’ canadensis (L.) Carr. ‘Bennett’ canadensis (L.) Carr. ‘Boulevard’ canadensis (L.) Carr. ‘Brandley’ canadensis (L.) Carr. ‘Cinnamomea’ canadensis (L.) Carr. ‘Curtis Ideal’ canadensis (L.) Carr. ‘Curtis Spreader’ canadensis (L.) Carr. ‘Doc’s Choice’ canadensis (L.) Carr. ‘Doran’ canadensis (L.) Carr. ‘Fastigiata’ canadensis (L.) Carr. ‘Gensch White’ canadensis (L.) Carr. ‘Globosa’ canadensis (L.) Carr. ‘Hawkersmith Weeping’ canadensis (L.) Carr. ‘Henry Hohman’ canadensis (L.) Carr. ‘Jacqueline Verkade’ canadensis (L.) Carr. ‘Jervis’ canadensis (L.) Carr. ‘Kelsey’s Weeping’ canadensis (L.) Carr. ‘Macrophylla’ canadensis (L.) Carr. ‘Minima’ canadensis (L.) Carr. ‘Minuta’ canadensis (L.) Carr. ‘Pendula’ canadensis (L.) Carr. ‘Sargentii’ canadensis (L.) Carr. ‘Verkade Recurved’ canadensis (L.) Carr. ‘Von Helms’ canadensis (L.) Carr. ‘Youngcone’ caroliniana Engelm. chinensis (Franch.) Pritz. diversifolia (Maxim.) Mast. sieboldii Carr. ULMUS L. Elm SYRINGA L. OLEACEAE CUPRESSACEAE Arborvitae Cypress Family occidentalis L. occidentalis L. ‘Alba’ occidentalis L. ‘Aurea’ occidentalis L. ‘Beaufort’ occidentalis L. ‘Buchananii’ occidentalis L. ‘Columna’ occidentalis L. ‘Compacta’ occidentalis L. ‘Endean’ occidentalis L. ‘Fastigiata’ occidentalis L. ‘Filifera’ THUJOPSIS Sieb. & Zucc. ex Endl. False or Hiba Arborvitae CUPRESSACEAE Cypress Family dolabrata (Thunb. ex L.f.) Sieb. & Zucc. dolabrata (Thunb. ex L.f.) Sieb. & Zucc. ‘Nana’ dolabrata (Thunb. ex L.f.) Sieb. & Zucc. var. hondae Makino THUNBERGIA Retz. ACANTHACEAE Acanthus Family grandiflora (Roxb. ex Rottl.) Roxb. THYMUS L. LAMIACEAE Mint Family vulgaris L. TIBOUCHINA Aubl. MELASTOMATACEAE Melastoma Family urvilleana (DC.) Cogn. THUJOPSIS Sieb. & Zucc. ex Endl. False or Hiba Arborvitae CUPRESSACEAE Cypress Family dolabrata (Thunb. ex L.f.) Sieb. & Zucc. dolabrata (Thunb. ex L.f.) Sieb. & Zucc. ‘Nana’ dolabrata (Thunb. ex L.f.) Sieb. & Zucc. var. hondae Makino THUNBERGIA Retz. ACANTHACEAE Acanthus Family grandiflora (Roxb. ex Rottl.) Roxb. THYMUS L. LAMIACEAE Mint Family vulgaris L. TIBOUCHINA Aubl. MELASTOMATACEAE Melastoma Family urvilleana (DC.) Cogn. 308 TILIA L. TILIACEAE Linden, Basswood Linden Family americana L. cordata Mill. x euchlora K.Koch x europaea L. x moltkei Spaeth mongolica Maxim. platyphyllos Scop. platyphyllos Scop. ‘Laciniata’ platyphyllos Scop. ‘Vitifolia’ tomentosa Moench tomentosa Moench ‘Pendula’ TIPUANA (Benth.) Benth. FABACEAE (Faboideae) Bean Family tipu (Benth.) O.Ktze. TOONA (Endl.) M.J.Roem. MELIACEAE Mahogany Family sinensis (Juss.) M.J.Roem. TORREYA Arn. TAXACEAE Yew Family nucifera (L.) Sieb. & Zucc. nucifera (L.) Sieb. & Zucc. ‘Gold Strike’ taxifolia Arn. TOXICODENDRON UMiili. ANACARDIACEAE Cashew Family radicans (L.) O.Ktze. vernicifluum (Stokes) F.A.Barkley TRACHELOSPERMUM Lem. APOCYNACEAE Dogbane Family asiaticum (Sieb. & Zucc.) Nakai difforme (Walt.) A.Gr. jasminoides (Lindl.) Lem. jasminoides (Lindl.) Lem. ‘Variegatum’ Jasminoides (Lindl.) Lem. var. pubescens Makino TRITHRINAX M<art. ARECACEAE = Palm Family acanthocoma Drude TROCHODENDRON Sieb. & Zucc. TROCHODENDRACEAE Trochodendron Family aralioides Sieb. & Zucc. TSUGA Carr. Hemlock PINACEAE Pine Family canadensis (L.) Carr. canadensis (L.) Carr. ‘Abbott’s Dwarf’ canadensis (L.) Carr. ‘Angustifolia’ canadensis (L.) Carr. ‘Armistice’ canadensis (L.) Carr. ‘Beaujean’ canadensis (L.) Carr. ‘Bennett’ canadensis (L.) Carr. ‘Boulevard’ canadensis (L.) Carr. ‘Brandley’ canadensis (L.) Carr. ‘Cinnamomea’ canadensis (L.) Carr. ‘Curtis Ideal’ canadensis (L.) Carr. ‘Curtis Spreader’ canadensis (L.) Carr. ‘Doc’s Choice’ canadensis (L.) Carr. ‘Doran’ canadensis (L.) Carr. ‘Fastigiata’ canadensis (L.) Carr. ‘Gensch White’ canadensis (L.) Carr. ‘Globosa’ canadensis (L.) Carr. ‘Hawkersmith Weeping’ canadensis (L.) Carr. ‘Henry Hohman’ canadensis (L.) Carr. ‘Jacqueline Verkade’ canadensis (L.) Carr. ‘Jervis’ canadensis (L.) Carr. ‘Kelsey’s Weeping’ canadensis (L.) Carr. ‘Macrophylla’ canadensis (L.) Carr. ‘Minima’ canadensis (L.) Carr. TSUGA Carr. PINACEAE ‘Frosty’ parvifolia Jacq. ‘Hokkaido’ procera Salisb. procera Salisb. ‘Marginata’ pumila L. * (pumila x ((U. x hollandica ‘Vegeta’ < U. minor) < (U. pumila var. arborea < U. minor ‘Hoersholm’))) ‘Homestead’ rubra Muhl. serotina Sarg. thomasii Sarg. UNGNADIA Endl. SAPINDACEAE Soapberry Family speciosa End. VACCINIUM L. Blueberry ERICACEAE Heath Family amoenum Ait. arboreum Marsh. bracteatum Thunb. ex J.A.Murr. corymbosum L. crassifolium Andr. crassifolium Andr. ssp. crassifolium ‘Wells Delight’ crassifolium Andr. ssp. sempervirens (Rayner&Henderson) Kirkman&Bal. ‘Bloodstone’ cylindraceum Sm. myrsinites Lam. vacillans Torr. vitis-idaea L. glabra Huds. glabra Huds. ‘Camperdownii’ <hollandica Mill. <hollandica Mill. ‘Belgica’ <hollandica Mill. ‘Dauvessei’ < hollandica Mill. ‘Major’ <hollandica Mill. ‘Pioneer’ <hollandica Mill. ‘Superba’ japonica (Rehd.) Sarg. japonica < U. wilsoniana laevis Pall. macrocarpa Hance minor Mill. minor Mill. ‘Christine Buisman’ miner Mill. ‘Sarniensis’ minor Mill. ‘Umbraculifera’ minor Mill. ‘Wredei’ parvifolia Jacq. parvifolia Jacq. ‘Drake’ parvifolia Jacq. ‘Dynasty’ parvifolia Jacq. ‘Frosty’ parvifolia Jacq. ‘Hokkaido’ procera Salisb. procera Salisb. ‘Marginata’ pumila L. (pumila x ((U. x hollandica ‘Vegeta’ < U. minor) < (U. pumila var. arborea < U. minor ‘Hoersholm’))) ‘Homestead’ rubra Muhl. serotina Sarg. thomasii Sarg. NGNADIA Endl. APINDACEAE Soapberry Family speciosa End. ACCINIUM L. Blueberry RICACEAE Heath Family amoenum Ait. arboreum Marsh. bracteatum Thunb. ex J.A.Murr. corymbosum L. crassifolium Andr. crassifolium Andr. ssp. crassifolium ‘Wells Delight’ crassifolium Andr. ssp. sempervirens (Rayner&Henderson) Kirkman&Bal. ‘Bloodstone’ cylindraceum Sm. myrsinites Lam. simulatum Small stamineum L. tenellum Ait. VIBURNUM lL. CAPRIFOLIACEAE Honeysuckle Family acertfolium L. awabuki K.Koch betulifolium Batal. bitchiuense Makino x bodnantense Aberc. ‘Dawn’ x bodnantense Aberc. ‘Deben’ brachybotryum Hemsl. buddleifolium Wright burejaeticum Regel & Herd. < burkwoodii Burkw. & Skipwith < burkwoodii Burkw. & Skipwith ‘Chenault’ * <burkwoodii Burkw. & Skipwith ‘Conoy’ * (<burkwoodii < V. carlesii) ‘Mohawk’ calvum Rehd. <carlcephalum Burkw. ex Pike * <carlcephalum Burkw. ex Pike ‘Cayuga’ * (xcarlcephalum ‘Cayuga’ x V. utile) ‘Chesapeake’ * (xcarlcephalum ‘Cayuga’ x V. utile) ‘Eskimo’ carlesii Hemsl. carlesii Hemsl. ‘Compacta’ cassinoides L. cinnamomifolium Rehd. cylindricum D.Don dasyanthum Rehd. dentatum L. dilatatum Thunb. ex J.A.Murr. * dilatatum Thunb. ex J.A.Murr. ‘Catskill’ * dilatatum Thunb. ex J.A.Murr. ‘Erie’ * dilatatum Thunb. ex J.A.Murr. ‘Iroquois’ * (dilatatum x V. lobophyllum) ‘Oneida’ erosum Thunb. ex J.A.Murr. farreri Stearn foetidum Wall. var. rectangulatum (Graebn.) Rehd. fordiae Hemsl. < hillieri Stearn hirtulum Rehd. hupehense Rehd. ichangense (Hemsl.) Rehd. japonicum (Thunb. ex J.A.Murr.) Spreng. * (japonicum x V. dilatatum) ‘Chippewa’ <juddii Rehd. lantana L. * lantana L. ‘Mohican’ lantanoides Michx. lentago L. lentago L. f. sphaerocarpum (Fern.) Rehd. TSUGA Carr. PINACEAE canadensis (L.) Carr. canadensis (L.) Carr. ‘Abbott’s Dwarf’ canadensis (L.) Carr. ‘Angustifolia’ canadensis (L.) Carr. ‘Armistice’ canadensis (L.) Carr. ‘Beaujean’ canadensis (L.) Carr. ‘Bennett’ canadensis (L.) Carr. ‘Boulevard’ canadensis (L.) Carr. ‘Brandley’ canadensis (L.) Carr. ‘Cinnamomea’ canadensis (L.) Carr. ‘Curtis Ideal’ canadensis (L.) Carr. ‘Curtis Spreader’ canadensis (L.) Carr. ‘Doc’s Choice’ canadensis (L.) Carr. ‘Doran’ canadensis (L.) Carr. ‘Fastigiata’ canadensis (L.) Carr. ‘Gensch White’ canadensis (L.) Carr. ‘Globosa’ canadensis (L.) Carr. ‘Hawkersmith Weeping’ canadensis (L.) Carr. ‘Henry Hohman’ canadensis (L.) Carr. ‘Jacqueline Verkade’ canadensis (L.) Carr. ‘Jervis’ canadensis (L.) Carr. ‘Kelsey’s Weeping’ canadensis (L.) Carr. ‘Macrophylla’ canadensis (L.) Carr. ‘Minima’ canadensis (L.) Carr. ‘Minuta’ canadensis (L.) Carr. ‘Pendula’ canadensis (L.) Carr. ‘Sargentii’ canadensis (L.) Carr. ‘Verkade Recurved’ canadensis (L.) Carr. ‘Von Helms’ canadensis (L.) Carr. ‘Youngcone’ caroliniana Engelm. chinensis (Franch.) Pritz. diversifolia (Maxim.) Mast. sieboldii Carr. ULMUS L. Elm ULMACEAE Elm Family alata Michx. americana L. americana L. ‘Augustine Ascending’ americana L. ‘Moline’ 309 ULMUS L. Elm ULMACEAE Elm Family alata Michx. americana L. americana L. ‘Augustine Ascending’ americana L. ‘Moline’ alata Michx. americana L. americana L. ‘Augustine Ascending’ americana L. ‘Moline’ 309 crassifolia Nutt. elliptica K.Koch glabra Huds. glabra Huds. ‘Camperdownii’ <hollandica Mill. <hollandica Mill. ‘Belgica’ <hollandica Mill. ‘Dauvessei’ < hollandica Mill. ‘Major’ * <hollandica Mill. ‘Pioneer’ <hollandica Mill. ‘Superba’ japonica (Rehd.) Sarg. japonica < U. wilsoniana laevis Pall. macrocarpa Hance minor Mill. minor Mill. ‘Christine Buisman’ miner Mill. ‘Sarniensis’ minor Mill. ‘Umbraculifera’ minor Mill. ‘Wredei’ parvifolia Jacq. parvifolia Jacq. ‘Drake’ * parvifolia Jacq. ‘Dynasty’ parvifolia Jacq. ‘Frosty’ parvifolia Jacq. ‘Hokkaido’ procera Salisb. procera Salisb. ‘Marginata’ pumila L. * (pumila x ((U. x hollandica ‘Vegeta’ < U. minor) < (U. pumila var. arborea < U. minor ‘Hoersholm’))) ‘Homestead’ rubra Muhl. serotina Sarg. thomasii Sarg. UNGNADIA Endl. SAPINDACEAE Soapberry Family speciosa End. VACCINIUM L. Blueberry ERICACEAE Heath Family amoenum Ait. arboreum Marsh. bracteatum Thunb. ex J.A.Murr. corymbosum L. crassifolium Andr. crassifolium Andr. ssp. crassifolium ‘Wells Delight’ crassifolium Andr. ssp. sempervirens (Rayner&Henderson) Kirkman&Bal. ‘Bloodstone’ cylindraceum Sm. myrsinites Lam. simulatum Small stamineum L. tenellum Ait. crassifolia Nutt. elliptica K.Koch glabra Huds. glabra Huds. ‘Camperdownii’ <hollandica Mill. <hollandica Mill. ‘Belgica’ <hollandica Mill. ‘Dauvessei’ < hollandica Mill. ‘Major’ * <hollandica Mill. ‘Pioneer’ <hollandica Mill. ‘Superba’ japonica (Rehd.) Sarg. japonica < U. wilsoniana laevis Pall. macrocarpa Hance minor Mill. minor Mill. ‘Christine Buisman’ miner Mill. ‘Sarniensis’ minor Mill. ‘Umbraculifera’ minor Mill. ‘Wredei’ parvifolia Jacq. parvifolia Jacq. ‘Drake’ * parvifolia Jacq. ‘Dynasty’ parvifolia Jacq. VIBURNUM lL. CAPRIFOLIACEAE ‘Shoshoni’ * (plicatum Thunb. f. tomentosum (Thunb. ex J.A. Murr.) Rehd. x V. plicatum f. tomentosum ‘Mariesii’) ‘Shasta’ plicatum Thunb. var. rotundifolium Rehd. prunifolium L. recognitum Fern. xrhytidocarpum E.Lemoine x rhytidophylloides Suring. * x<rhytidophylloides Suring. ‘Alleghany’ xrhytidophylloides Suring. ‘Willow Leaf’ x rhytidophylloides Suring. ‘Willowwood’ rhytidophyllum Hemsl. rhytidophyllum Hemsl. ‘Ben Blackburn’ rhytidophyllum Hemsl. ‘Roseum’ rhytidophyllum Hemsl. ‘Variegatum’ (rhytidophyllum V. utile) ‘Pragense’ setigerum Hance setigerum Hance ‘Aurantiacum’ sieboldii Miq. sieboldii Miq. ‘Reticulatum’ * sieboldii Miq. ‘Seneca’ suspensum Lindl. tinus L. trilobum Marsh. urceolatum Sieb. & Zucc. utile Hemsl. wrightii Miq. VINCA L. Periwinkle APOCYNACEAE Dogbane Family major L. major L. ‘Oxyloba’ major L. ‘Variegata’ minor L. minor L. ‘Alba’ minor L. ‘Atropurpurea’ minor L. ‘Multiplex’ VITEX L. VERBENACEAE Verbena Family agnus-castus L. agnus-castus L. ‘Silver Spire’ negundo L. negundo L. ‘Incisa’ rotundifolia L.f. trifolia L. ‘Variegata’ VITIS L. Grape VITACEAE Grape Family labrusca L. mustangensis Buckl. rotundifolia Michx. WASHINGTONIA H.Wendl. Washington Palm ARECACEAE Palm Family robusta H.Wendl. WEIGELA Thunb. CAPRIFOLIACEAE Honeysuckle Family decora (Nakai) Nakai lobophyllum Graebn. * (lobophyllum x V. jap opulus L. ‘Roseum’ ovatifolium Rehd. ovatifolium Rehd. plicatum Thunb. f. plicatum Thunb. f. lanceolatum (Rehd.) Rehd. plicatum Thunb. f. parvifolium (Miq.) plicatum Thunb. f. parvifolium (Miq.) Rehd. plicatum Thunb. f. plicatum plicatum Thunb. f. parvifolium (Miq.) Rehd. plicatum Thunb. f. plicatum plicatum Thunb. f. plicatum plicatum Thunb. f. plicatum ‘ minor L. minor L. plicatum Thunb. f. plicatum ‘Newport’ plicatum Thunb. f. tomentosum (Thunb. plicatum Thunb. f. plicatum ‘Newport’ plicatum Thunb. f. tomentosum (Thunb. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. plicatum Thunb. f. tomentosum (Thunb. minor L. ‘Multiplex’ plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘Mariesii’ plicatum Thunb. f. tomentosum (Thunb. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘Mariesii’ plicatum Thunb. f. tomentosum ex J.A.Murr.) Rehd. ‘Mariesii’ plicatum Thunb. f. tomentosum plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘Mt. Fuji’ plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘St. Keverne’ plicatum Thunb. f. tomentosum (Thunb ex J.A.Murr.) Rehd. ‘St. Keverne’ * plicatum Thunb. f. tomentosum (Thunb ex J.A.Murr.) Rehd. ‘St. Keverne’ plicatum Thunb. f. tomentosum (T * plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘Shoshoni’ * (plicatum Thunb. f. tomentosum ex J.A.Murr.) Rehd. ‘Shoshoni’ * (plicatum Thunb. f. tomentosum (Thunb. ex J.A. Murr.) Rehd. x V. plicatum f. tomentosum ‘Mariesii’) ‘Shasta’ plicatum Thunb. var. rotundifolium Rehd. prunifolium L. recognitum Fern. xrhytidocarpum E.Lemoine x rhytidophylloides Suring. * x<rhytidophylloides Suring. ‘Alleghany’ xrhytidophylloides Suring. VIBURNUM lL. CAPRIFOLIACEAE < < ’ mily erry mily s CAPRIFOLIACEAE Honeysuckle Family acertfolium L. awabuki K.Koch betulifolium Batal. bitchiuense Makino x bodnantense Aberc. ‘Dawn’ x bodnantense Aberc. ‘Deben’ brachybotryum Hemsl. buddleifolium Wright burejaeticum Regel & Herd. < burkwoodii Burkw. & Skipwith < burkwoodii Burkw. & Skipwith ‘Chenault’ * <burkwoodii Burkw. & Skipwith ‘Conoy’ * (<burkwoodii < V. carlesii) ‘Mohawk’ calvum Rehd. <carlcephalum Burkw. ex Pike * <carlcephalum Burkw. ex Pike ‘Cayuga’ * (xcarlcephalum ‘Cayuga’ x V. utile) ‘Chesapeake’ * (xcarlcephalum ‘Cayuga’ x V. utile) ‘Eskimo’ carlesii Hemsl. carlesii Hemsl. ‘Compacta’ cassinoides L. cinnamomifolium Rehd. cylindricum D.Don dasyanthum Rehd. dentatum L. dilatatum Thunb. ex J.A.Murr. * dilatatum Thunb. ex J.A.Murr. ‘Catskill’ * dilatatum Thunb. ex J.A.Murr. ‘Erie’ * dilatatum Thunb. ex J.A.Murr. ‘Iroquois’ * (dilatatum x V. lobophyllum) ‘Oneida’ erosum Thunb. ex J.A.Murr. farreri Stearn foetidum Wall. var. rectangulatum (Graebn.) Rehd. fordiae Hemsl. < hillieri Stearn hirtulum Rehd. hupehense Rehd. ichangense (Hemsl.) Rehd. japonicum (Thunb. ex J.A.Murr.) Spreng. * (japonicum x V. dilatatum) ‘Chippewa’ <juddii Rehd. lantana L. * lantana L. ‘Mohican’ lantanoides Michx. lentago L. lentago L. f. sphaerocarpum (Fern.) Rehd. parvifolia Jacq. ‘Hokkaido’ procera Salisb. procera Salisb. procera Salisb. procera Salisb. ‘Marginata’ pumila L. serotina Sarg. thomasii Sarg. thomasii Sarg. UNGNADIA Endl. SAPINDACEAE * dilatatum Thunb. ex J.A.Murr. ‘Catskill’ * dilatatum Thunb. ex J.A.Murr. ‘Erie’ * dilatatum Thunb. ex J.A.Murr. ‘Erie’ * dilatatum Thunb. ex J.A.Murr. ‘Iroquois * dilatatum Thunb. ex J.A.Murr. ‘Iroquois’ * (dilatatum x V. lobophyllum) ‘Oneida’ amoenum Ait. arboreum Mars arboreum Marsh. bracteatum Thunb. fordiae Hemsl. < hillieri Stearn < hillieri Stearn hirtulum Rehd. hirtulum Rehd. hupehense Rehd. corymbosum L. crassifolium And crassifolium Andr. crassifolium Andr. lantana L. lantana L. * lantana L. ‘Mohican’ lantanoides Michx. lantanoides Michx. lentago L. lentago L. lentago L. lentago L. f. sphaerocarpum (Fern.) Rehd. 310 lobophyllum Graebn. * (lobophyllum x V. japonicum) ‘Huron’ luzonicum Rolfe macrocephalum Fort. f. keteleeri (Carr.) Rehd. macrocephalum Fort. f. macrocephalum molle Michx. mullaha Buch.-Ham. ex D.Don nudum L. obovatum Walt. odoratissimum Ker-Gawl. opulus L. opulus L. ‘Roseum’ ovatifolium Rehd. plicatum Thunb. f. lanceolatum (Rehd.) Rehd. plicatum Thunb. f. parvifolium (Miq.) Rehd. plicatum Thunb. f. plicatum plicatum Thunb. f. plicatum ‘Newport’ plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘Mariesii’ plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘Mt. Fuji’ plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. ‘St. Keverne’ * plicatum Thunb. f. tomentosum (Thunb. ex J.A.Murr.) Rehd. Appendix E: Vernacular Names This alphabetical list of the vernacular (or com- mon) names of plants in the catalog has two sections: one that identifies the genera and another that identifies the species and cultivars. The list provides common names for about 40 percent of the catalog entries. Only a few com- mon-name synonyms are given. In the list of species and cultivars, the common names are alphabetized under group names, such as alder, box, cypress, gum. VIBURNUM lL. CAPRIFOLIACEAE Fig: FICUS Filbert: CORYLUS Fir: ABIES Firethorn: PYRACANTHA Flowering Maple: ABUTILON Fringe Tree: CHIONANTHUS Germander: TEUCRIUM Giant Sequoia: SEQUOIADENDRON Golden Bells: FORSYTHIA Golden-rain Tree: KOELREUTERIA Golden-chain Tree: LABURNUM Gooseberry: RIBES Grape: VITIS Greenbrier: SMILAX Groundsel: BACCHARIS Hackberry: CELTIS Hawthorn: CRATAEGUS Hazelnut: CORYLUS Heath: ERICA Heather: CALLUNA Hemlock: TSUGA Hickory: CARYA Holly: ILEX Honeylocust: GLEDITSIA Honeysuckle: LONICERA Hop Tree: PTELEA Hornbeam: CARPINUS Horsechestnut: AESCULUS Huckleberry: GAYLUSSACIA Indigo: INDIGOFERA Ivy: HEDERA Jasmine: JASMINUM Jessamine: GELSEMIUM Jetbead: RHODOTYPOS Juniper: JUNIPERUS Kumquat: FORTUNELLA Larch: LARIX Laurel: LAURUS Lavender: LAVANDULA Lilac: SYRINGA Linden: TILIA Locust: ROBINIA Loquat: ERIOBOTRYA Magnolia: MAGNOLIA Maple: ACER Matrimony Vine: LYCIUM Mimosa: ACACIA Mock Orange: PHILADELPHUS Mountain Ash: SORBUS Mulberry: MORUS Myrtle: MYRTUS Ninebark: PHYSOCARPUS Oak: QUERCUS Pea Shrub: CARAGANA Pear: PYRUS Peony: PAEONIA Peppertree: SCHINUS Periwinkle: VINCA Persimmon: DIOSPYROS Pine: PINUS VIBURNUM lL. CAPRIFOLIACEAE ‘Willow Leaf’ x rhytidophylloides Suring. ‘Willowwood’ rhytidophyllum Hemsl. rhytidophyllum Hemsl. ‘Ben Blackburn’ rhytidophyllum Hemsl. ‘Roseum’ rhytidophyllum Hemsl. ‘Variegatum’ (rhytidophyllum V. utile) ‘Pragense’ rufidulum Raf. sargentii Koehne * sargentii Koehne ‘Onondaga’ * sargentii Koechne ‘Susquehanna’ sargentii Koehne var. calvescens Rehd. scabrellum Chapm. 311 XYLOSMA J.G.Forst. FLACOURTIACEAE Flacourtia Family congestum (Lour.) Merr. Sflexuosum (HBK.) Hemsl. YUCCA L. Adam’s Needle, Spanish Dagger AGAVACEAE Agave Family aloifolia L. ‘Marginata’ filamentosa L. Silamentosa L. ‘Bright Edge’ filamentosa L. ‘Golden Sword’ gloriosa L. recurvifolia Salisb. smalliana Fern. whipplei Torr. ZAMIA lL. CYCADACEAE Cycad Family pumila L. ZANTHOXYLUM L. Prickly Ash RUTACEAE Citrus Family clava-herculis L. fagara (L.) Sarg. piperitum DC. simulans Hance ZELKOVA Spach ULMACEAE Elm Family carpinifolia (Pall.) K.Koch schneideriana Hand.-Mazz. serrata (Thunb.) Makino x verschaffeltii Nichols. ZENOBIA D.Don ERICACEAE Heath Family pulverulenta (Bartram) Pollard pulverulenta (Bartram) Pollard f. nuda (Vent.) Fern. ZIZIPHUS Mill. RHAMNACEAE Buckthorn Family jujuba Mill. jujuba Mill. ‘Contorta’ hortensis (Sieb. & Zucc.) K.Koch praecox (V.Lemoine) Bailey ‘Rosea’ subsessilis (Nakai) Bailey ‘Feerie’ ‘Mont Blanc’ ‘Newport Red’ WESTRINGIA Sim. LAMIACEAE Mint Family rosmariniformis Sm. WIKSTROEMIA Endl. THYMELAEACEAE Mezereum Family trichotoma (Thunb.) Makino WISTERIA Nuit. FABACEAE (Faboideae) Wisteria Bean Family brachybotrys Sieb. & Zucc. brachybotrys Sieb. & Zucc. ‘Alba’ brachybotrys Sieb. & Zucc. ‘Murasaki kapitan’ floribunda (Willd.) DC. floribunda (Willd.) DC. ‘Alba’ floribunda (Willd.) DC. ‘Honbeni’ floribunda (Willd.) DC. ‘Itoe kokuryw’ floribunda (Willd.) DC. ‘Jabo’ floribunda (Willd.) DC. ‘Koshigaya’ floribunda (Willd.) DC. ‘Macrobotrys’ floribunda (Willd.) DC. ‘Noda’ floribunda (Willd.) DC. ‘Ossai’ floribunda (Willd.) DC. ‘Rosea’ floribunda (Willd.) DC. ‘Shino kapitan’ floribunda (Willd.) DC. ‘Violacea Plena’ < formosa Rehd. Sfrutescens (L.) Poir. Sfrutescens (L.) Poir. ‘Nivea’ sinensis (Sims) Sweet sinensis (Sims) Sweet ‘Alba’ venusta Rehd. & Wils. ‘Siro kapitan’ villosa Rehd. XANTHOCERAS Bunge SAPINDACEAE Soapberry Family sorbifolium Bunge XANTHORHIZA Marsh. RANUNCULACEAE Buttercup Family Mint Family XANTHOCERAS Bunge SAPINDACEAE Soapberry Family sorbifolium Bunge XANTHORHIZA Marsh. RANUNCULACEAE Buttercup Family simplicissima Marsh. 312 False Spirea: SORBARIA False Arborvitae: THUJOPSIS~. Genera glabra Red: FRAXINUS pennsylvanica Wafer: PTELEA trifoliata White: FRAXINUS americana Aspen, Large-toothed: POPULUS grandidentata Quaking: POPULUS tremuloides Aster, Carolina: ASTER carolinianus Aucuba, Japanese: AUCUBA japonica Avocado: PERSEA americana Azalea, Alabama: RHODODENDRON alabamense Coastal: RHODODENDRON atlanticum Cumberland: RHODODENDRON bakeri Dwarf Indica: RHODODENDRON tamurae Flame: RHODODENDRON calendulaceum Florida: RHODODENDRON austrinum Hammock Sweet: RHODODENDRON serrulatum Indica: RHODODENDRON indicum Kaempfer: RHODODENDRON kaempferi Kirishima: RHODODENDRON ‘Obtusum’ Korean: RHODODENDRON yedoense var. poukhanense Kyushu: RHODODENDRON kiusianum Large-sepal: RHODODENDRON macrosepalum Mt. Amagi: RHODODENDRON amagianum Oconee: RHODODENDRON flammeum Smoke Tree: COTINUS Spanish Dagger: YUCCA Spicebush: LINDERA Spindle Tree: EUONYMUS Spirea: SPIRAEA Spruce: PICEA Spurge: EUPHORBIA Storax: STYRAX Strawflower: HELICHRYSUM Strawflower: HELICHRYSUM Genera Adam’s Needle: YUCCA Akebia: AKEBIA Alder: ALNUS Arborvitae: THUJA Ash: FRAXINUS Bald Cypress: TAXODIUM Bamboo: BAMBUSA, PHYLLOSTACHYS Banana: MUSA Barberry: BERBERIS Basswood: TILIA Beautyberry: CALLICARPA Beech: FAGUS Birch: BETULA Bittersweet: CELASTRUS Blueberry: VACCINIUM Bottlebrush: CALLISTEMON Box: BUXUS Broom: CYTISUS, GENISTA Buckeye: AESCULUS Buckthorn: RHAMNUS Bush Clover: LESPEDEZA Bush Honeysuckle: DIERVILLA Butterfly Bush: BUDDLEJA California Redwood: SEQUOIA Cassava: MANIHOT Cedar: CEDRUS Century Plant: AGAVE Chestnut: CASTANEA China Fir: CUNNINGHAMIA Chinquapin: CASTANOPSIS Chokeberry: ARONIA Cinquefoil: POTENTILLA Coral Tree: ERYTHRINA Crape Myrtle: LAGERSTROEMIA Currant: RIBES Cypress: CUPRESSUS Dogwood: CORNUS Douglas Fir: PSEUDOTSUGA Elder: SAMBUCUS Elm: ULMUS Epaulette Tree: PTEROSTYRAX False Cypress: CHAMAECYPARIS Crape Myrtle: LAGERSTROEMIA Currant: RIBES 313 Poplar: POPULUS Prickly Ash: ZANTHOXYLUM Privet: LIGUSTRUM Purple Guava: PSIDIUM Quince: CYDONIA Rose: ROSA Sage: SALVIA St. John’s-wort: HYPERICUM Sand Myrtle: LEIOPHYLLUM Senna: CASSIA Shadbush: AMELANCHIER Silverbell Tree: HALESIA Skimmia: SKIMMIA Smoke Tree: COTINUS Snowbell: STYRAX Snowberry: SYMPHORICARPOS Soapberry: SAPINDUS Spanish Dagger: YUCCA Spicebush: LINDERA Spindle Tree: EUONYMUS Spirea: SPIRAEA Spruce: PICEA Spurge: EUPHORBIA Storax: STYRAX Strawflower: HELICHRYSUM Sumac: RHUS Sweet Box: SARCOCOCCA Sweet Gum: LIQUIDAMBAR Sycamore: PLATANUS Tallow Tree: SAPIUM Tanbark Oak: LITHOCARPUS Tulip Tree: LIRIODENDRON Walnut: JUGLANS Washington Palm: WASHINGTONIA Wattle: ACACIA White Alder: CLETHRA Willow: SALIX Wingnut: PTEROCARYA Winter Hazel: CORYLOPSIS Wisteria: WISTERIA Witch Hazel: HAMAMELIS Yew: TAXUS Species and Cultivars Aaron’s Beard: HYPERICUM calycinum Abelia, Chinese: ABELIA chinensis Glossy: ABELIA x grandiflora Acacia, Rose: ROBINIA hispida Sweet: ACACIA farnesiana Adam’s Needle: YUCCA filamentosa Alder, Black: ALNUS glutinosa, ILEX verticillata European: ALNUS glutinosa Seaside: ALNUS maritima Smooth: ALNUS serrulata Witch: FOTHERGILLA gardenii Allspice, Carolina: CALYCANTHUS floridus Almond: PRUNUS dulcis Double-flowered Flowering: PRUNUS triloba ‘Multiplex’ Flowering: PRUNUS glandulosa, P. triloba, Althea, Shrub: HIBISCUS syriacus Anacahuita: CORDIA boisseri Andromeda, Japanese: PIERIS japonica Angel's Trumpet: BRUGMANSIA suaveolens Anise, Florida: ILLICIUM floridanum Anise Tree, Chinese: ILLICIUM henryi Japanese: ILLICIUM anisatum Apache Plume: FALLUGIA paradoxa Apple, Common: MALUS domestica Apricot: PRUNUS armeniaca Japanese: PRUNUS mume Arborvitae, American: THUJA occidentalis Hiba: THUJOPSIS dolabrata Oriental: PLATYCLADUS orientalis Ardisia, Coral: ARDISIA crispa Japanese: ARDISIA japonica Arrow-wood: VIBURNUM dentatum Northern: VIBURNUM recognitum Ash, American: FRAXINUS americana American Mountain: SORBUS americana Berlandier: FRAXINUS berlandieriana Biltmore: FRAXINUS americana var. biltmoreana Black: FRAXINUS nigra European: FRAXINUS excelsior Flowering: FRAXINUS ornus Green: FRAXINUS pennsylanica var. subintegerrima Leatherleaf: FRAXINUS velutina var. coriacea Modesto: FRAXINUS velutina var. Sumac: RHUS Sumac: RHUS Sweet Box: SARCOCOCCA Sweet Box: SARCOCOCCA Sweet Gum: LIQUIDAMBAR Sycamore: PLATANUS Tallow Tree: SAPIUM Tanbark Oak: LITHOCARPUS Tulip Tree: LIRIODENDRON Tanbark Oak: LITHOCARPUS Tulip Tree: LIRIODENDRON Tulip Tree: LIRIODENDRON Walnut: JUGLANS White: FRAXINUS americana Aspen, Large-toothed: POPULUS g Washington Palm: WASHINGTONIA Aspen, Large-toothed: POPULUS grandidentata Quaking: POPULUS tremuloides Wattle: ACACIA Aster, Carolina: ASTER carolinianus Aucuba, Japanese: AUCUBA japonica Aucuba, Japanese: AUCUBA japonica Avocado: PERSEA americana Species and Cultivars Aaron’s Beard: HYPERICUM calycinum Abelia, Chinese: ABELIA chinensis Glossy: ABELIA x grandiflora Acacia, Rose: ROBINIA hispida Sweet: ACACIA farnesiana Adam’s Needle: YUCCA filamentosa Alder, Black: ALNUS glutinosa, ILEX verticillata European: ALNUS glutinosa Seaside: ALNUS maritima Smooth: ALNUS serrulata Witch: FOTHERGILLA gardenii Allspice, Carolina: CALYCANTHUS floridus Almond: PRUNUS dulcis Double-flowered Flowering: PRUNUS triloba ‘Multiplex’ Species and Cultivars Aaron’s Beard: HYPERICUM calycinum Abelia, Chinese: ABELIA chinensis Glossy: ABELIA x grandiflora Acacia, Rose: ROBINIA hispida Sweet: ACACIA farnesiana Adam’s Needle: YUCCA filamentosa Alder, Black: ALNUS glutinosa, ILEX verticillata European: ALNUS glutinosa Seaside: ALNUS maritima Smooth: ALNUS serrulata Witch: FOTHERGILLA gardenii Allspice, Carolina: CALYCANTHUS floridus Almond: PRUNUS dulcis Double-flowered Flowering: PRUNUS triloba ‘Multiplex’ var. poukhanense Kyushu: RHODODENDRON kiusianum Large-sepal: RHODODENDRON macrosepalum Mt. Amagi: RHODODENDRON amagianum Oconee: RHODODENDRON flammeum Oldham: RHODODENDRON oldhamii Piedmont: RHODODENDRON canescens Pinkshell: RHODODENDRON vaseyi Plumleaf: RHODODENDRON prunifolium Pontic: RHODODENDRON luteum Rose: RHODODENDRON reticulatum Roseshell: RHODODENDRON prinophyllum 314 Beefwood: CASUARINA cunninghamiana Big Tree: SEQUOIADENDRON-giganteum Birch, European White: BETULA pendula Gray: BETULA populifolia Hairy: BETULA pubescens Japanese White: BETULA platyphylla var. japonica Paper: BETULA papyrifera River: BETULA nigra Sweet: BETULA lenta Virginia Roundleaf: BETULA uber Yellow: BETULA alleghaniensis Bitternut: CARYA cordiformis Bittersweet, Oriental: CELASTRUS orbiculatus Blueberry, Creeping: VACCINIUM crassifolium Ground: VACCINIUM myrsinites Highbush: VACCINIUM corymbosum Boneberry, Chinese: OSTEOMELES schwerinae Bottlebrush, Narrowleaf: CALLISTEMON linearis Royal: RHODODENDRON schlippenbachii Sims: RHODODENDRON simsii Snow: RHODODENDRON ‘Mucronatum’ Spider: RHODODENDRON macrosepalum Spider: RHODODENDRON macrosepalum ‘Linearifolium’ Swamp: RHODODENDRON viscosum Sweet: RHODODENDRON arborescens Taibei: RHODODENDRON kanahirae Texas: RHODODENDRON oblongifolium Western: RHODODENDRON occidentale Wild-thyme: RHODODENDRON serpyllifolium Baccharis, Southern: BACCHARIS glomerulifera Balm-of-Gilead: POPULUS candicans Bamboo, Fishpole: PHYLLOSTACHYS aurea Giant Timber: PHYLLOSTACHYS heterocycla Golden: PHYLLOSTACHYS aurea Green Fountain: THAMNOCALAMUS spathaceus Heavenly: NANDINA domestica Hedge: BAMBUSA multiplex Kuma: SASA veitchii Sweetshoot: PHYLLOSTACHYS dulcis Yellow-groove: PHYLLOSTACHYS aureosulcata Banana Shrub: MICHELIA figo Barberry, Byers Wintergreen: BERBERIS julianae ‘Byers’ Dwarf Redleaf Japanese: BERBERIS thunbergii ‘Atropurpurea Nana’ Hooker: BERBERIS hookeri, BERBERIS hookeri var. orbiculatus l Plant: RUSS orbiculatus Coral Plant: RUSSELIA equisetiformis Coral Tree, Cockspur: ERYTHRINA crista-galli Coriaria, Japanese: CORIARIA japonica Cork-tree, Amur: PHELLODENDRON amurense Corkwood, Florida: LEITNERIA floridana Cotoneaster, Bloodberry: COTONEASTER obscurus Cranberry: COTONEASTER apiculatus Creeping: COTONEASTER adpressus Diels: COTONEASTER dielsianus European: COTONEASTER integerrimus Rock: COTONEASTER horizontalis Spreading: COTONEASTER divaricatus Wintergreen: COTONEASTER conspicuus Cottonwood: POPULUS deltoides Swamp: POPULUS heterophylla Cowberry: VACCINIUM vitis-idaea Crabapple, Bechtel’s: MALUS ioensis ‘Plena’ Sargent’s: MALUS sargentii Showy: MALUS floribunda Siberian: MALUS baccata Southern: MALUS angustifolia Toringo: MALUS sieboldii orbiculatus Coral Plant: RUSSELIA equisetiformis Coral Tree, Cockspur: ERYTHRINA crista-galli Coriaria, Japanese: CORIARIA japonica Cork-tree, Amur: PHELLODENDRON amurense Corkwood, Florida: LEITNERIA floridana Cotoneaster, Bloodberry: COTONEASTER obscurus Cranberry: COTONEASTER apiculatus Creeping: COTONEASTER adpressus Diels: COTONEASTER dielsianus European: COTONEASTER integerrimus Rock: COTONEASTER horizontalis Spreading: COTONEASTER divaricatus Wintergreen: COTONEASTER conspicuus Cottonwood: POPULUS deltoides Swamp: POPULUS heterophylla Cowberry: VACCINIUM vitis-idaea Crabapple, Bechtel’s: MALUS ioensis ‘Plena’ Sargent’s: MALUS sargentii Showy: MALUS floribunda Siberian: MALUS baccata Southern: MALUS angustifolia Toringo: MALUS sieboldii Wild Sweet: MALUS coronaria Cranberry, Highbush: VIBURNUM trilobum Mountain: VACCINIUM vitis-idaea Cranberry-bush, European: VIBURNUM opulus Creeper, Mexican: ANTIGONON leptopus Virginia: PARTHENOCISSUS quinquefolia Cross Vine: BIGNONIA capreolata Croton, Alabama: CROTON alabamensis Cucumber Tree: MAGNOLIA acuminata Cup-flower, Tall: NIEREMBERGIA scoparia Currant, Clove: RIBES odoratum Indian: SYMPHORICARPOS orbiculatus Mountain: RIBES alpinum Cypress, Bald: TAXODIUM distichum var. distichum Italian: CUPRESSUS sempervirens Lawson: CHAMAECYPARIS lawsoniana Leyland: x CUPRESSOCYPARIS leylandii ‘Marginata’ Chenille Plant: ACALYPHA hispida Cherry, Barbados: MALPIGHIA glabra Bird: PRUNUS padus Black: PRUNUS serotina Cornelian: CORNUS mas Higan: PRUNUS xsubhirtella Japanese Cornelian: CORNUS officinalis Japanese Flowering: PRUNUS serrulata Kwanzan: PRUNUS serrulata ‘Kwanzan’ Mahaleb: PRUNUS mahaleb Nanking: PRUNUS tomentosa Sargent: PRUNUS sargentii Sour: PRUNUS cerasus Sweet: PRUNUS avium Taiwan: PRUNUS campanulata Takasago: PRUNUS sieboldii Tokyo: PRUNUS yedoensis Weeping Higan: PRUNUS subhirtella Croton, Alabama: CROTON alabamensis Cucumber Tree: MAGNOLIA acuminata ‘Shidare Higan’ Yoshino: PRUNUS x yedoensis ‘Somei Yoshino’ Chestnut, Chinese: CASTANEA mollissima Common: CASTANEA sativa European: CASTANEA sativa Japanese: CASTANEA crenata Chinaberry: MELIA azedarach Chinquapin, Japanese: CASTANOPSIS cuspidata Coralberry: ARDISIA crenata, SY orbiculatus Coralberry: ARDISIA crenata, SYMPHORICARPOS orbiculatus Coral Plant: RUSSELIA equisetiformis Coral Tree, Cockspur: ERYTHRINA crista-galli Coriaria, Japanese: CORIARIA japonica Cork-tree, Amur: PHELLODENDRON amurense Corkwood, Florida: LEITNERIA floridana Cotoneaster, Bloodberry: COTONEASTER obscurus Cranberry: COTONEASTER apiculatus Creeping: COTONEASTER adpressus Diels: COTONEASTER dielsianus European: COTONEASTER integerrimus Rock: COTONEASTER horizontalis Spreading: COTONEASTER divaricatus Wintergreen: COTONEASTER conspicuus Cottonwood: POPULUS deltoides Swamp: POPULUS heterophylla Cowberry: VACCINIUM vitis-idaea Crabapple, Bechtel’s: MALUS ioensis ‘Plena’ Sargent’s: MALUS sargentii Showy: MALUS floribunda Siberian: MALUS baccata Southern: MALUS angustifolia Toringo: MALUS sieboldii Wild Sweet: MALUS coronaria Cranberry, Highbush: VIBURNUM trilobum Mountain: VACCINIUM vitis-idaea Cranberry-bush, European: VIBURNUM opulus Creeper, Mexican: ANTIGONON leptopus Virginia: PARTHENOCISSUS quinquefolia Cross Vine: BIGNONIA capreolata Croton, Alabama: CROTON alabamensis Cucumber Tree: MAGNOLIA acuminata Cup-flower, Tall: NIEREMBERGIA scoparia Currant, Clove: RIBES odoratum Indian: SYMPHORICARPOS orbiculatus Mountain: RIBES alpinum Cypress, Bald: TAXODIUM distichum var. distichum Italian: CUPRESSUS sempervirens Lawson: CHAMAECYPARIS lawsoniana Leyland: x CUPRESSOCYPARIS leylandii Sumac: RHUS brevifolia Deodar: CEDRUS deodara Incense: CALOCEDRUS decurrens Japanese: CRYPTOMERIA japonica -of-Lebanon: CEDRUS libani Port Orford: CHAMAECYPARIS lawsoniana Red: JUNIPERUS virginiana Southern Red: JUNIPERUS silicicola Stinking: TORREYA taxifolia Western Red: THUJA plicata Ceniza: LEUCOPHYLLUM frutescens ‘Farquhariana’ Scarlet: CLEMATIS texensis Cleyera, Japanese: CLEYERA japonica Variegated: CLEYERA japonica ‘Tricolor’ Cliff-green: PAXISTIMA canbyi Coffee-tree, Kentucky: GYMNOCLADUS dioica Conradina, Bluesage: CONRADINA canescens Whorled: CONRADINA verticillata Coontie: ZAMIA pumila Coralbeads: COCCULUS triloba Coralberry: ARDISIA crenata, SYMPHORICARPOS orbiculatus Sumac: RHUS viridis Japanese: BERBERIS thunbergii Mentor: BERBERIS < mentorensis Redleaf Japanese: BERBERIS thunbergii ‘Atropurpurea’ Redleaf Korean: BERBERIS koreana ‘Atropurpurea’ Sargent: BERBERIS sargentiana Soulie: BERBERIS soulieana Warty: BERBERIS verruculosa Wintergreen: BERBERIS julianae Yellowleaf Japanese: BERBERIS thunbergii ‘Aurea’ Barometer Bush: LEUCOPHYLLUM frutescens Bauhinia, Texas: BAUHINIA lunarioides Bay, Loblolly: GORDONIA lasianthus Red: PERSEA borbonia Sweet: MAGNOLIA virginiana Bayberry: MYRICA pensylvanica Bean, Coral: ERYTHRINA herbacea Indian: CATALPA bignonioides Mescal: SOPHORA secundiflora Bearberry: ARCTOSTAPHYLOS uva-ursi Beauty Bush: KOLKWITZIA amabilis Beautyberry, American: CALLICARPA americana Bodinier: CALLICARPA bodinieri Japanese: CALLICARPA japonica Purple: CALLICARPA dichotoma White-fruited: CALLICARPA americana ‘Lactea’ Beech, American: FAGUS grandifolia Columnar: FAGUS sylvatica ‘Dawyck’ European: FAGUS sylvatica Fernleaf: FAGUS sylvatica ‘Laciniata’ Purple: FAGUS sylvatica f. purpurea Weeping: FAGUS sylvatica ‘Pendula’ Stiff: CALLISTEMON rigidus Willow: CALLISTEMON salignus Box, Balearic: BUXUS balearica Chinese: MURRAYA paniculata Columnar: BUXUS sempervirens ‘Fastigiata’ Common: BUXUS sempervirens Dwarf English: BUXUS sempervirens ‘Suffruticosa’ Handsworth: BUXUS sempervirens ‘Handsworthiensis’ ‘Handsworthiensis’ Harland: BUXUS harlandii Japanese: BUXUS microphylla, B. microphylla var. japonica Korean: BUXUS sinica var. insularis Tree: BUXUS sempervirens ‘Arborescens’ Victorian: PITTOSPORUM undulatum Weeping: BUXUS sempervirens ‘Pendula’ Willow-leaved: BUXUS sempervirens ‘Angustifolia’ Yugoslavian: BUXUS sempervirens ‘Vardar Valley’ Boxelder: ACER negundo ‘Handsworthiensis’ Harland: BUXUS harlandii Japanese: BUXUS microphylla, B. microphylla var. japonica Broom, Butcher’s: RUSCUS aculeatus Scotch: CYTISUS scoparius Scotch: CYTISUS scoparius Spanish: SPARTIUM junceum Warminster: CYTISUS < praecox Buckthorn, Carolina: RHAMNUS carolinianus Common: RHAMNUS catharticus Common: RHAMNUS catharticus Italian: RHAMNUS alaternus Southern: BUMELIA lycioides Common: RHAMNUS catharticus Italian: RHAMNUS alaternus 315 Butternut: JUGLANS cinerea Buttonbush: CEPHALANTHUS occidentalis Chinese: ADINA rubella Calamondin: < CITROFORTUNELLA mitis Camellia, Common: CAMELLIA japonica Mountain: STEWARTIA ovata Oil: CAMELLIA oleifera Sasanqua: CAMELLIA sasanqua Silky: STEWARTIA malacodendron Warratah: CAMELLIA japonica ‘Anemoniflora’ Camphor Tree: CINNAMOMUM camphora Cassine: ILEX cassine Cat’s-claw: MACFADYENA unguis-cati Catalpa, Chinese: CATALPA ovata Common: CATALPA bignonioides Manchurian: CATALPA bungei Western: CATALPA speciosa Catberry: NEMOPANTHUS mucronatus Ceanothus, Snowbrush: CEANOTHUS velutinus Cedar, Alaska: CHAMAECYPARIS nootkatensis Atlantic White: CHAMAECYPARIS thyoides Atlas: CEDRUS atlantica Blue Atlas: CEDRUS atlantica ‘Glauca’ Cyprus: CEDRUS libani ssp. Sumac: RHUS brevifolia Deodar: CEDRUS deodara Incense: CALOCEDRUS decurrens Japanese: CRYPTOMERIA japonica -of-Lebanon: CEDRUS libani Port Orford: CHAMAECYPARIS lawsoniana Red: JUNIPERUS virginiana Southern Red: JUNIPERUS silicicola Stinking: TORREYA taxifolia Western Red: THUJA plicata Ceniza: LEUCOPHYLLUM frutescens Century Plant, Variegated: AGAVE americana ‘Marginata’ Chaste Tree: VITEX agnus-castus Chenille Plant: ACALYPHA hispida Cherry, Barbados: MALPIGHIA glabra Bird: PRUNUS padus Black: PRUNUS serotina Cornelian: CORNUS mas Higan: PRUNUS xsubhirtella Japanese Cornelian: CORNUS officinalis Japanese Flowering: PRUNUS serrulata Kwanzan: PRUNUS serrulata ‘Kwanzan’ Mahaleb: PRUNUS mahaleb Nanking: PRUNUS tomentosa Sargent: PRUNUS sargentii Sour: PRUNUS cerasus Sweet: PRUNUS avium Taiwan: PRUNUS campanulata Takasago: PRUNUS sieboldii Tokyo: PRUNUS yedoensis Weeping Higan: PRUNUS subhirtella ‘Shidare Higan’ Yoshino: PRUNUS x yedoensis ‘Somei Yoshino’ Chestnut, Chinese: CASTANEA mollissima Common: CASTANEA sativa European: CASTANEA sativa Japanese: CASTANEA crenata Chinaberry: MELIA azedarach Chinquapin, Japanese: CASTANOPSIS cuspidata 316 Chittamwood: BUMELIA lanuginosa Chocolate Vine: AKEBIA quinata Chokeberry, Black: ARONIA melanocarpa Purplefruit: ARONIA prunifolia Red: ARONIA arbutifolia Cinquefoil, Shrubby: POTENTILLA fruticosa Citrange: x CITRONCIRUS webberi Citron: CITRUS medica ‘Etrog’ Clematis, Evergreen: CLEMATIS armandii Goldwool: CLEMATIS chrysocoma var. sericea Pink-flowered Evergreen: CLEMATIS armandii ‘Farquhariana’ Butternut: JUGLANS cinerea Buttonbush: CEPHALANTHUS occidentalis Chinese: ADINA rubella Buttonbush: CEPHALANTHUS occidentalis Chinese: ADINA rubella Calamondin: < CITROFORTUNELLA mitis Camellia, Common: CAMELLIA japonica Mountain: STEWARTIA ovata Oil: CAMELLIA oleifera Sasanqua: CAMELLIA sasanqua Silky: STEWARTIA malacodendron Warratah: CAMELLIA japonica ‘Anemoniflora’ Camphor Tree: CINNAMOMUM camphora Cassine: ILEX cassine Cat’s-claw: MACFADYENA unguis-cati Catalpa, Chinese: CATALPA ovata Common: CATALPA bignonioides Manchurian: CATALPA bungei Western: CATALPA speciosa Catberry: NEMOPANTHUS mucronatus Ceanothus, Snowbrush: CEANOTHUS velutinus Cedar, Alaska: CHAMAECYPARIS nootkatensis Atlantic White: CHAMAECYPARIS thyoides Atlas: CEDRUS atlantica Blue Atlas: CEDRUS atlantica ‘Glauca’ Cyprus: CEDRUS libani ssp. brevifolia Deodar: CEDRUS deodara Incense: CALOCEDRUS decurrens Japanese: CRYPTOMERIA japonica -of-Lebanon: CEDRUS libani Port Orford: CHAMAECYPARIS lawsoniana Red: JUNIPERUS virginiana Southern Red: JUNIPERUS silicicola Stinking: TORREYA taxifolia Western Red: THUJA plicata Ceniza: LEUCOPHYLLUM frutescens Buttonbush: CEPHALANTHUS occidentalis Chinese: ADINA rubella Calamondin: < CITROFORTUNELLA mitis Camellia, Common: CAMELLIA japonica Mountain: STEWARTIA ovata Oil: CAMELLIA oleifera Sasanqua: CAMELLIA sasanqua Silky: STEWARTIA malacodendron Warratah: CAMELLIA japonica ‘Anemoniflora’ Camphor Tree: CINNAMOMUM camphora Cassine: ILEX cassine Cat’s-claw: MACFADYENA unguis-cati Catalpa, Chinese: CATALPA ovata Common: CATALPA bignonioides Manchurian: CATALPA bungei Western: CATALPA speciosa Catberry: NEMOPANTHUS mucronatus Ceanothus, Snowbrush: CEANOTHUS velutinus Cedar, Alaska: CHAMAECYPARIS nootkatensis Atlantic White: CHAMAECYPARIS thyoides Atlas: CEDRUS atlantica Blue Atlas: CEDRUS atlantica ‘Glauca’ Cyprus: CEDRUS libani ssp. ‘Shidare Higan’ ‘Shidare Higan’ Yoshino: PRUNUS x yedoensis ‘Somei Yoshino’ Chestnut, Chinese: CASTANEA mollissima Common: CASTANEA sativa European: CASTANEA sativa Japanese: CASTANEA crenata Chinaberry: MELIA azedarach Chinquapin, Japanese: CASTANOPSIS cuspidata 316 Monterey: CUPRESSUS macrocarpa Montezuma: TAXODIUM mucronatum Mourning: CHAMAECYPARIS funebris Pond: TAXODIUM distichum var. nutans Portuguese: CUPRESSUS lusitanica Dahoon: ILEX cassine Narrow-leaved: ILEX cassine var. angustifolia Yellow-berried: ILEX cassine var. angustifolia f. aurea-baccata Daisy, Nippon: CHRYSANTHEMUM nipponicum Daphne, February: DAPHNE mezereum Lilac: DAPHNE genkwa Rose: DAPHNE cneorum Winter: DAPHNE odora Deerberry: VACCINIUM stamineum Deutzia, Rough-leaved: DEUTZIA scabra Devil's Walking Stick: ARALIA spinosa Devilwood: OSMANTHUS americanus Dioon, Chestnut: DIOON edule Doghobble: LEUCOTHOE fontanesiana Dogwood, Alternate-leaved: CORNUS alternifolia Cream-edged: CORNUS alba ‘Argenteo Marginata’ Double-flowered: CORNUS florida ‘Bay Beauty’ Double-flowered: CORNUS florida f. pluribracteata Flowering: CORNUS florida Giant: CORNUS controversa Gray: CORNUS racemosa Kousa: CORNUS kousa Pale: CORNUS obliqua Pink-flowering: CORNUS florida f. rubra Siberian: CORNUS alba ‘Sibirica’ Silky: CORNUS amomum Stiff: CORNUS foemina Western: CORNUS nuttallii Yellow-fruited flowering: CORNUS florida f. xanthocarpa Monterey: CUPRESSUS macrocarpa Montezuma: TAXODIUM mucronatum Mourning: CHAMAECYPARIS funebris Pond: TAXODIUM distichum var. nutans Portuguese: CUPRESSUS lusitanica Dahoon: ILEX cassine Narrow-leaved: ILEX cassine var. angustifolia Yellow-berried: ILEX cassine var. angustifolia f. aurea-baccata Wych: ULMUS glabra ue False Cypress, Hinoki: CHAMAECYPARIS obtusa Moss: CHAMAECYPARIS pisifera ‘Squarrosa’ Nootka: CHAMAECYPARIS nootkatensis Sawara: CHAMAECYPARIS pisifera Farkleberry: VACCINIUM arboreum Fatsia, Japanese: FATSIA japonica Fetterbush: LYONIA lucida, PIERIS floribunda Fiddlewood: CITHAREXYLUM berlandieri Fig, Common: FICUS carica Creeping: FICUS pumila Filbert, Beaked: CORYLUS cornuta Chinese: CORYLUS chinensis Purple-leaved: CORYLUS maxima ‘Purpurea’ Turkish: CORYLUS colurna Fir, Algerian: ABIES numidica Balsam: ABIES balsamea Blue China: CUNNINGHAMIA lanceolata ‘Glauca’ China: CUNNINGHAMIA lanceolata Cilician: ABIES cilicica Douglas: See Rocky Mountain Douglas Fraser: ABIES fraseri Greek: ABIES cephalonica Korean: ABIES koreana Manchurian: ABIES holophylla Momi: ABIES firma Nikko: ABIES homolepis Noble: ABIES procera Nordmann: ABIES nordmanniana Rocky Mountain Douglas: PSEUDOTSUGA Wych: ULMUS glabra ue False Cypress, Hinoki: CHAMAECYPARIS obtusa Moss: CHAMAECYPARIS pisifera ‘Squarrosa’ Nootka: CHAMAECYPARIS nootkatensis Sawara: CHAMAECYPARIS pisifera Dahoon: ILEX cassine Narrow-leaved: ILEX cassine var. angustifolia Yellow-berried: ILEX cassine var. angustifolia f. aurea-baccata Daisy, Nippon: CHRYSANTHEMUM nipponicum Daphne, February: DAPHNE mezereum Rose: DAPHNE cneorum Douglas: See Rocky Mountain Douglas Greek: ABIES cephalonica Korean: ABIES koreana Gray: CORNUS racemosa Nikko: ABIES homolepis Rocky Mountain Douglas: PSEUDOTSUGA menziesii var. glauca Shensi: ABIES chensiensis ssp. salouenensis Silver: ABIES alba Spanish: ABIES pins Spanish: ABIES pinsapo White: ABIES concolor f. xanthocarpa Dove Tree: DAVIDIA involucrata var. ‘Shidare Higan’ longipes Gum, Black: NYSSA sylvatica Cider: EUCALYPTUS gunnii Formosan Sweet: LIQUIDAMBAR formosana Manna: EUCALYPTUS viminalis Mountain: EUCALYPTUS dalrympleana Sour: NYSSA sylvatica Sweet: LIQUIDAMBAR styraciflua Tasmanian Snow: EUCALYPTUS coccifera Gumi: ELAEAGNUS multiflora Hackberry: Caucasian: CELTIS caucasica Chinese: CELTIS sinensis Common: CELTIS occidentalis Spiny: CELTIS pallida Hardhack: SPIRAEA tomentosa Harry Lauder’s Walking-stick: CORYLUS avellana ‘Contorta’ Haw, Black: VIBURNUM prunifolium Nannyberry: VIBURNUM nudum Poison: VIBURNUM molle Possum: ILEX decidua, VIBURNUM nudum Rusty Black: VIBURNUM rufidulum Southern Black: VIBURNUM rufidulum Hawthorn: Blueberry: CRATAEGUS brachyacantha Cockspur: CRATAEGUS crus-galli Common: CRATAEGUS monogyna Dotted: CRATAEGUS punctata Downy: CRATAEGUS mollis English: CRATAEGUS laevigata Green: CRATAEGUS viridis Indian: RHAPHIOLEPIS indica Littlehip: CRATAEGUS spathulata May: CRATAEGUS aestivalis Parsley: CRATAEGUS marshallii Pear: CRATAEGUS calpodendron Pringle: CRATAEGUS pringlei Thicket: CRATAEGUS intricata Yeddo: RHAPHIOLEPIS umbellata Yellow: CRATAEGUS flava Hazelnut, American: CORYLUS americana European: CORYLUS avellana Purple-leaved: CORYLUS avellana ‘Fusco-rubra’ Heath, Cornish: ERICA vagans Spike: BRUCKENTHALIA spiculifolia Spring: ERICA carnea Heather, False: CUPHEA hyssopifolia Scotch: CALLUNA vulgaris Hemlock, Canadian: TSUGA canadensis Carolina: TSUGA caroliniana Ground: TAXUS canadensis Sand: CARYA pallida Shagbark: CARYA ovata Shellbark: CARYA laciniosa Water: CARYA aquatica Hobblebush: VIBURNUM lantanoides Holly, American: ILEX opaca Appalachian: ILEX collina Attenuate-leaved: ILEX x attenuata Beadle’s: ILEX beadlei Box-leaved: ILEX crenata Broad-leaved: ILEX latifolia Burford: ILEX cornuta ‘Burfordii’ Buswell’s: ILEX buswellii Canary Islands: ILEX canariensis Carolina: ILEX ambigua Caucasian: ILEX colchica Chinese: ILEX cornuta Coin-leaved: ILEX crenata ‘Mariesii’ Creeping: ILEX rugosa Dune: ILEX cumulicola English: ILEX aquifolium Evergreen Long-stalked: ILEX pedunculosa Farges’: ILEX fargesii Fig-leaved: ILEX ficoidea Furin: ILEX geniculata Georgian: ILEX longipes Highclere: ILEX = altaclerensis Horned: ILEX cornuta Hummock: ILEX cumulicola Japanese: ILEX crenata Kachi: ILEX purpurea Koehne’s: ILEX x koehneana Kurogane: ILEX rotunda Liukiu: ILEX liukiuensis Long-stalked: ILEX longipes Luster-leaved: ILEX latifolia Mochi: ILEX integra Mountain: ILEX monticola Myrtle-leaved: ILEX myrtifolia Northern Mountain: NEMOPANTHUS mucronatus Okinawa: ILEX dimorphophylla Peach-leaved: ILEX purpurea Pontic: ILEX colchica Sand: ILEX buswellii Sarvis: ILEX amelanchier Sikkim: ILEX sikkimensis Suwanee: ILEX curtissii Topel: ILEX x attenuata Tsuru: ILEX rugosa Waterer’s Gold: ILEX aquifolium ‘Watereriana’ Wilson’s: ILEX wilsonii Yellow-fruited American: ILEX opaca f. xanthocarpa Yellow-stemmed: ILEX leucoclada Yunnan: ILEX yunnanensis Hollygrape, Leatherleaf: MAHONIA bealei Oregon: MAHONIA aquifolium Texas: MAHONIA swazeyi Grape, Fox: VITIS labrusca Muscadine: VITIS rotundifolia Mustang: VITIS mustangensis Grapefruit: CITRUS x paradisi Green Screw: ILEX aquifolium ‘Crispa’ Groundsel Tree: BACCHARIS halimifolia Guava, Pineapple: ACCA sellowiana Purple: PSIDIUM littorale var. ‘Shidare Higan’ vilmoriniana Dutchman’s Pipe, Calico: ARISTOLOCHIA elegans Ebony, Green: JACARANDA acutifolia Texas: PITHECELLOBIUM flexicaule Elaeagnus, Cherry: ELAEAGNUS multiflora Thorny: ELAEAGNUS pungens Elder, American: SAMBUCUS canadensis American Red: SAMBUCUS racemosa ssp. pubens Asiatic Red: SAMBUCUS racemosa ssp. sieboldiana Dwarf: SAMBUCUS ebulus Elfin Herb: CUPHEA hyssopifolia Elm, American: ULMUS americana Cedar: ULMUS crassifolia Chinese: ULMUS parvifolia English: ULMUS procera European White: ULMUS laevis Globe: ULMUS minor ‘Umbraculifera’ Jersey: ULMUS minor ‘Sarniensis’ Moline: ULMUS americana ‘Moline’ Rock: ULMUS thomasii Scotch: ULMUS glabra September: ULMUS serotina Siberian: ULMUS pumila Slippery: ULMUS rubra Water: PLANERA aquatica White: ABIES concolor Flamebush, Mexican: CALLIANDRA tweediei Forsythia, White: ABELIOPHYLLUM distichum Franklin Tree: FRANKLINIA alatamaha Fringe Tree, American: CHIONANTHUS virginicus Japanese: CHIONANTHUS retusus Taiwan: CHIONANTHUS retusus var. serrulatus Fuchsia, Cape: PHYGELIUS capensis Garland Flower: DAPHNE cneorum Gallberry, Large: ILEX coriacea Ginkgo: GINKGO biloba Glorybower, Bleedingheart: CLERODENDRUM thomsonae thomsonae Fragrant: CLERODENDRUM philippinum Harlequin: CLERODENDRUM trichotomum Glory Bush: TIBOUCHINA urvilleana Gold-dust Shrub: AUCUBA japonica ‘Variegata’ Golden Bells, Korean: FORSYTHIA ovata Weeping: FORSYTHIA suspensa Golden-chain Tree: LABURNUM anagyroides Golden-rain Tree: KOELREUTERIA bipinnata, K. elegans ssp. formosana, K. paniculata Weeping: FORSYTHIA suspensa Golden-chain Tree: LABURNUM anagyroides Golden-rain Tree: KOELREUTERIA bipinnata, K. elegans ssp. formosana, K. paniculata K. elegans ssp. formosana, K. paniculata Goldflower: HYPERICUM calycinum Gooseberry, Chinese: ACTINIDIA deliciosa var. deliciosa English: RIBES uva-crispa Florida: RIBES echinellum Granite: RIBES curvatum Siberian: ULMUS pumila Slippery: ULMUS rubra Slippery: ULMUS rubra Water: PLANERA aquati Water: PLANERA aquatica 317 Grape, Fox: VITIS labrusca Muscadine: VITIS rotundifolia Mustang: VITIS mustangensis Grapefruit: CITRUS x paradisi Green Screw: ILEX aquifolium ‘Crispa’ Groundsel Tree: BACCHARIS halimifolia Guava, Pineapple: ACCA sellowiana Purple: PSIDIUM littorale var. ‘Shidare Higan’ longipes Gum, Black: NYSSA sylvatica Cider: EUCALYPTUS gunnii Formosan Sweet: LIQUIDAMBAR formosana Manna: EUCALYPTUS viminalis Mountain: EUCALYPTUS dalrympleana Sour: NYSSA sylvatica Sweet: LIQUIDAMBAR styraciflua Tasmanian Snow: EUCALYPTUS coccifera Gumi: ELAEAGNUS multiflora Hackberry: Caucasian: CELTIS caucasica Chinese: CELTIS sinensis Common: CELTIS occidentalis Spiny: CELTIS pallida Hardhack: SPIRAEA tomentosa Harry Lauder’s Walking-stick: CORYLUS avellana Spiny: CELTIS pallida poetica Japanese: HEDERA rhombea Marine: CISSUS incisa Poison: TOXICODENDRON radicans Shrub: HEDERA helix (Arborescens group) Tree: X FATSHEDERA lizei Jacaranda: JACARANDA acutifolia Jasmine, Angel-wing: JASMINUM nitidum Blue: CLEMATIS crispa Cape: GARDENIA jasminoides Confederate: TRACHELOSPERMUM jasminioides Crape: TABERNAEMONTANA divaricata Italian: JASMINUM humile ‘Revolutum’ Orange: MURRAYA paniculata Spiny: CELTIS pallida Hardhack: SPIRAEA tomentosa Harry Lauder’s Walking-stick: CORYLUS avellana Nannyberry: VIBURNUM nudum Poison: VIBURNUM molle Thicket: CRATAEGUS intricata Yeddo: RHAPHIOLEPIS umbellata Yeddo: RHAPHIOLEPIS umbellata Yellow: CRATAEGUS flava Yellow: CRATAEGUS flava Hazelnut, American: CORYLUS Hazelnut, American: CORYLUS americana European: CORYLUS avellana xanthocarpa Yellow-stemmed: ILEX leucoclada Yunnan: ILEX yunnanensis Hollygrape, Leatherleaf: MAHONIA bealei Oregon: MAHONIA aquifolium Texas: MAHONIA swazeyi Honeylocust, Common: GLEDITSIA triacanthos Japanese: GLEDITSIA japonica Spineless: GLEDITSIA triacanthos f. inermis Honeysuckle, Amur: LONICERA maackii 318 Primrose: JASMINUM mesyni Star: JASMINUM multiflorym, Cape: TECOMARIA capensis Everblooming: LONICERA x heckrottii Fly: LONICERA xylosteum Japanese: LONICERA japonica Morrow’s: LONICERA morrowii Privet: LONICERA pileata Standish’s: LONICERA standishii Tatarian: LONICERA tatarica Trumpet: LONICERA sempervirens Winter: LONICERA fragrantissima Yellow: LONICERA flava Star: JASMINUM multiflorym, TRACHELOSPERMUM jasminioides Winter: JASMINUM nudiflorum Jessamine, Carolina: GELSEMIUM sempervirens Double-flowered: GELSEMIUM sempervirens ‘Pride of Augusta’ Night: CESTRUM nocturnum Yellow: GELSEMIUM sempervirens Jetbead: RHODOTYPOS scandens Jujube: ZIZIPHUS jujuba Juniper, Alligator: JUNIPERUS deppeana var. pachyphlaea Andorra: JUNIPERUS horizontalis ‘Plumosa’ Chinese: JUNIPERUS chinensis Creeping: JUNIPERUS horizontalis Golden Pfitzer: JUNIPERUS < media Winter: LONICERA fragrantissima Winter: LONICERA fragrantissima Yellow: LONICERA flava Hornbeam, American: CARPINUS caroliniana Columnar: CARPINUS betulus ‘Fastigiata’ European: CARPINUS betulus Heartleaf: CARPINUS cordata Hop: OSTRYA virginiana Japanese: CARPINUS japonica Purple-leaf: CARPINUS betulus ‘Purpurea’ Horse Sugar: SYMPLOCOS tinctoria Horsechestnut: AESCULUS hippocastanum Double-flowered: AESCULUS hippocastanum ‘Pfitzeriana Aurea’ Ground: JUNIPERUS communis ssp. depressa Hetz Blue: JUNIPERUS chinensis ‘Hetzii’ Hollywood: JUNIPERUS chinensis ‘Kaizuka’ Irish: JUNIPERUS communis ‘Hibernica’ Needle: JUNIPERUS rigida Pfitzer: JUNIPERUS x media ‘Pfitzeriana’ Rocky Mountain: JUNIPERUS scopulorum Sargent: JUNIPERUS chinensis var. sargentii Shore: JUNIPERUS conferta Single-seed: JUNIPERUS squamata Kadsura Vine: KADSURA japonica Kaki: DIOSPYROS kaki hydrangeoides Indigo, Mountain: AMORPHA giabra Indigo Bush: AMORPHA fruticosa Inkberry: ILEX glabra White-fruited: ILEX glabra f. leucocarpa Ipil-ipil: LEUCAENA leucocephala Ironwood: BUMELIA tenax, CARPINUS caroliniana Persian: PARROTIA persica Ivy, Algerian: HEDERA canariensis Boston: PARTHENOCISSUS tricuspidata Caucasus: HEDERA colchica English: HEDERA helix Gloire de Marengo: HEDERA canariensis ‘Variegata’ hydrangeoides Indigo, Mountain: AMORPHA giabra Indigo Bush: AMORPHA fruticosa Inkberry: ILEX glabra White-fruited: ILEX glabra f. leucocarpa Ipil-ipil: LEUCAENA leucocephala Ironwood: BUMELIA tenax, CARPINUS caroliniana Persian: PARROTIA persica Ivy, Algerian: HEDERA canariensis Boston: PARTHENOCISSUS tricuspidata Caucasus: HEDERA colchica English: HEDERA helix Gloire de Marengo: HEDERA canariensis ‘Variegata’ Kumquat, Meiwa: FORTUNELLA crassifolia Round: FORTUNELLA japonica Lace Shrub: STEPHANANDRA incisa Lacecap: HYDRANGEA macrophylla ( Gloire de Marengo: HEDERA canariensis ‘Variegata’ Italian: HEDERA helix var. Chinese: LYCIUM chinense Kobus: MAGNOLIA kobus Kobus: MAGNOLIA kobus Medlar: MESPILUS germanica Mesquite, Western Honey: PROSO Medlar: MESPILUS germanica Mesquite, Western Honey: PROSO Mesquite, Western Honey: PROSOPIS glandulosa var. torreyana Metake: PSEUDOSASA japonica Mezereon: DAPHNE mezereum Milletia, Leatherleaf: MILLETTIA reticulata Mimosa, Catclaw: MIMOSA biuncifera Mimosa Tree: ALBIZIA julibrissin Minniebush: MENZIESIA pilosa Mock Orange, European: PHILADELPHUS coronarius Monkey Puzzle: ARAUCARIA araucana Mu-oil Tree: ALEURITES montana Mulberry, Paper: BROUSSONETIA papyrifera Red: MORUS rubra Weeping: MORUS alba ‘Pendula’ White: MORUS alba Myrtle, Common: MYRTUS communis Dwarf: MYRTUS communis ‘Microphylla’ Sea: BACCHARIS halimifolia Nannyberry: VIBURNUM lentago Nectarine: PRUNUS persica var. nucipersica ‘White Glory’ Otama: MAGNOLIA sieboldii Saucer: MAGNOLIA X< soulangeana Southern: MAGNOLIA grandiflora Southern: MAGNOLIA grandiflora Star: MAGNOLIA stellata ‘White Glory’ Neillia, Chinese: NEILLIA sinensis Nightshade, Brazilian: SOLANUM seaforthianum Oak, Arkansas: QUERCUS arkansana Bartram: QUERCUS heterophylla Basket: QUERCUS michauxii Black: QUERCUS velutina Bluejack: QUERCUS incana Bur: QUERCUS macrocarpa Chestnut: QUERCUS prinus Chinese Cork: QUERCUS variabilis Columnar English: QUERCUS robur f. fastigiata Compton: QUERCUS x comptonae Cork: QUERCUS suber Daimyo: QUERCUS dentata Golden Full-moon: ACER japonicum ‘Aureum’ Hedge: ACER campestre, ACER campestre Blue: CLEMATIS crispa Cape: GARDENIA jasmino Orange: MURRAYA paniculata DIRCA palustris 319 Lilac, Chinese: SYRINGA X chinensis Common: SYRINGA vulgaris ‘Purpurea’ Cutleaf: SYRINGA laciniata Double White: SYRINGA vulgaris ‘Alba Plena’ Hungarian: SYRINGA josikaea Japanese Tree: SYRINGA reticulata Late: SYRINGA villosa, S. villosa ‘Crayton’ Persian: SYRINGA x persica Lily Tree: MAGNOLIA denudata Lime, Australian Finger: MICROCITRUS australasica Wild: ZANTHOXYLUM fagara Linden, American: TILIA americana Bigleaf: TILIA platyphyllos Littleleaf: TILIA cordata Mongolian: TILIA mongolica Silver: TILIA tomentosa Locust, Black: ROBINIA pseudoacacia Bristly: ROBINIA hispida Clammy: ROBINIA viscosa Ship-mast: ROBINIA pseudoacacia var. rectissima Water: GLEDITSIA aquatica Loquat: ERIOBOTRYA japonica Magnolia, Bigleaf: MAGNOLIA macrophylla Florida Bigleaf: MAGNOLIA ashei Fraser: MAGNOLIA fraseri Goddess: MAGNOLIA sprengeri ‘Diva’ Kobus: MAGNOLIA kobus Lily-flowered: MAGNOLIA liliiflora Mexican Bigleaf: MAGNOLIA dealbata Otama: MAGNOLIA sieboldii Saucer: MAGNOLIA X< soulangeana Southern: MAGNOLIA grandiflora Star: MAGNOLIA stellata Umbrella: MAGNOLIA tripetala Mahogany, Swamp: EUCALYPTUS robusta Mahonia, Chinese: MAHONIA fortunei Cluster: MAHONIA pinnata Japanese: MAHONIA japonica Laredo: MAHONIA trifoliata Maidenhair Tree: GINKGO biloba Mallotus, Japanese: MALLOTUS japonicus Mandarin: CITRUS reticulata Maple, Amur: ACER ginnala Black: ACER saccharum ssp. nigrum Bloodleaf: ACER palmatum ‘Atropurpureum’ Chalk: ACER saccharum ssp. leucoderme Colosseum: ACER cappadocicum David's: ACER davidii Devil: ACER diabolicum Drummond: ACER rubrum ssp. drummondii Dwarf Hedge: ACER campestre ‘Nanum’ Evergreen: ACER oblongum Florida: ACER saccharum ssp. floridanum Manchurian Striped: ACER tegmentosum Miyabe: ACER miyabei Montpellier: ACER monspessulanum Mountain: ACER spicatum Nikko: ACER maximowiczianum Norway: ACER platanoides Paperbark: ACER griseum Purpleleaf Sycamore: ACER pseudoplatanus ‘Atropurpureum’ ‘Atropurpureum’ Purplebloom: ACER pseudosieboldianum Red: ACER rubrum Redvein: ACER rufinerve Schwedler: ACER platanoides ‘Schwedleri’ Shantung: ACER truncatum Siebold: ACER sieboldianum Silver: ACER saccharinum Striped: ACER pensylvanicum Sugar: ACER saccharum ssp. saccharum Sycamore: ACER pseudoplatanus Tatarian: ACER tataricum Trident: ACER buergerianum Velvet: ACER velutinum var. vanvolxemii Ship-mast: ROBINIA pseudoacacia var. rectissima Water: GLEDITSIA aquatica Water: GLEDITSIA aquatica Loquat: ERIOBOTRYA japonica Magnolia, Bigleaf: MAGNOLIA mac Magnolia, Bigleaf: MAGNOLIA macrophylla Florida Bigleaf: MAGNOLIA ashei Magnolia, Bigleaf: MAGNOLIA macrophylla Florida Bigleaf: MAGNOLIA ashei Florida Bigleaf: MAGNOLIA ashei Fraser: MAGNOLIA fraseri Florida Bigleaf: MAGNOLIA ashei Fraser: MAGNOLIA fraseri Matrimony Vine, Carolina: LYCIUM carolinianum Chinese: LYCIUM chinense Matrimony Vine, Carolina: LYCIUM carolinianum Chinese: LYCIUM chinense Fraser: MAGNOLIA fraseri Goddess: MAGNOLIA spreng Goddess: MAGNOLIA sprengeri ‘Diva’ Kobus: MAGNOLIA kobus Goddess: MAGNOLIA sprengeri ‘Diva’ var. leiocarpum 320 Darlington: QUERCUS hemisphaerica Durmast: QUERCUS petraea English: QUERCUS robur Georgia: QUERCUS georgiana Italian: QUERCUS frainetto Japanese Evergreen: QUERCUS myrsinifolia Konara: QUERCUS glandulifera Laurel: QUERCUS laurifolia Nuttall: QUERCUS nuttallii Oriental Sawtooth: QUERCUS acutissima Overcup: QUERCUS lyrata Pin: QUERCUS palustris Post: QUERCUS stellata Red: QUERCUS rubra Ring-cupped: QUERCUS glauca Sand Post: QUERCUS margaretta Sargent: QUERCUS x sargentii Scarlet: QUERCUS coccinea Shingle: QUERCUS imbricaria Shumard: QUERCUS shumardii Southern Live: QUERCUS virginiana Southern Red: QUERCUS falcata Spanish: QUERCUS falcata Swamp White: QUERCUS bicolor Tanbark: LITHOCARPUS glaber Turkey: QUERCUS cerris; QUERCUS laevis Ubame: QUERCUS phillyraeoides Water: QUERCUS nigra White: QUERCUS alba Willow: QUERCUS phellos Old-man’s Beard: CHIONANTHUS virginicus Oleander: NERIUM oleander Variegated: NERIUM oleander ‘Variegata’ Olive: OLEA europaea Autumn: ELAEAGNUS umbellata Holly: OSMANTHUS heterophyllus var. heterophyllus Russian: ELAEAGNUS angustifolia Sweet: OSMANTHUS fragrans Tea: OSMANTHUS fragrans Orange, Chinese Box: SEVERINIA buxifolia Hardy: PONCIRUS trifoliata Osage: MACLURA pomifera Satsuma: CITRUS reticulata Seville: CITRUS aurantium Sour: CITRUS aurantium Osier, Green: CORNUS alternifolia Red: CORNUS sericea Our-Lord’s-Candle: YUCCA whipplei Pachysandra, Allegheny: PACHYSANDRA procumben Japanese: PACHYSANDRA terminalis Pagoda Tree: SOPHORA japonica Palm, Cabbage: SABAL palmetto Canary Island Date: PHOENIX canariensis Chinese Fan: LIVISTONA chinensis European Fan: CHAMAEROPS humilis Jelly: BUTIA capitata Needle: RHAPIDOPHYLLUM hystrix Queen: SYAGRUS romanzoffianum Palmetto, Dwarf: SABAL minor Saw: SERENOA repens ~ Palmetto, Dwarf: SABAL minor Saw: SERENOA repens ~ Paperbark Tree: MELALEUCA quinquenervis Paperbush: EDGEWORTHIA chrysantha Paradise Flower: SOLANUM wendlandii Paraguay Tea: ILEX paraguariensis Parasol Tree, Chinese: FIRMIANA simplex Partridgeberry: MITCHELLA repens Pascuita: EUPHORBIA leucocephala Passionflower, Red: PASSIFLORA coccinea Pavonia, Spearleaf: PAVONIA hastata Pawpaw: ASIMINA triloba Pea Shrub: CARAGANA arborescens Chinese: CARAGANA sinica Peach: PRUNUS persica Pear, Alligator: PERSEA americana Asian: PYRUS pyrifolia Birchleaf: PYRUS betulifolia Bradford: PYRUS calleryana ‘Bradford’ Callery: PYRUS calleryana Common: PYRUS communis Evergreen: PYRUS kawakamii Leconte: PYRUS x lecontei Nashi: PYRUS pyrifolia var. culta Sand: PYRUS pyrifolia Snow: PYRUS nivalis Pearlbush: EXOCHORDA racemosa Pecan: CARYA illinoinensis Bitter: CARYA aquatica Mehan: CARYA illinoinensis ‘Mehan’ Stuart: CARYA illinoinensis ‘Stuart’ Peony, Tree: PAEONIA suffruticosa Pepperbush, Sweet: CLETHRA alnifolia Peppertree, Brazilian: SCHINUS terebinthifolius Pepper-vine: AMPELOPSIS arborea Periwinkle, Common: VINCA minor Double-flowered: VINCA minor ‘Multiplex’ Greater: VINCA major Persimmon, Common: DIOSPYROS virginiana Chinese: DIOSPYROS sinensis Japanese: DIOSPYROS kaki Texas: DIOSPYROS texana Phillyrea, Narrow-leaf: PHILLYREA angustifolia Tree: PHILLYREA latifolia var. media Photinia, Chinese: PHOTINIA serratifolia Japanese: PHOTINIA glabra Pignut: CARYA glabra Sweet: CARYA ovalis Pine, African Fern: PODOCARPUS gracilior Aleppo: PINUS halepensis Australian: CASUARINA cunninghamiana Austrian: PINUS nigra ssp. var. leiocarpum mugo Swiss Stone: PINUS cembra Table-mountain: PINUS pungens Totara: PODOCARPUS totara Umbrella: PINUS pinea; SCIADOPITYS verti Virginia: PINUS virginiana Weeping Japanese Red: PINUS densiflora ‘P Japanese White: PINUS parviflora Jelecote: PINUS patula Knob-cone: PINUS attenuata Korean: PINUS koraiensis Lacebark: PINUS bungeana Limber: PINUS flexilis Loblolly: PINUS taeda Long-leaved Indian: PINUS roxburghii Longleaf: PINUS palustris Mexican Stone: PINUS cembroides Pitch: PINUS rigida Pond: PINUS serotina Ponderosa: PINUS ponderosa Red: PINUS resinosa Sand: PINUS clausa Scotch: PINUS sylvestris Scrub: PINUS virginiana Shortleaf: PINUS echinata Slash: PINUS elliottii Spruce: PINUS glabra Swiss Mountain: PINUS mugo var. mugo Swiss Stone: PINUS cembra Table-mountain: PINUS pungens Totara: PODOCARPUS totara Umbrella: PINUS pinea; SCIADOPITYS verticillata Virginia: PINUS virginiana Weeping Japanese Red: PINUS densiflora ‘Pendula’ Western Yellow: PINUS ponderosa Yew: PODOCARPUS macrophyllus Dawn: METASEQUOIA glyptostroboides Rhododendron, Carolina: RHODODENDRON carolinianum carolinianum Chapman: RHODODENDRON chapmanii Fujiama: RHODODENDRON brachycarpum Piedmont: RHODODENDRON minus Rice-paper Plant: TETRAPANAX papyriferum Rose, Apothecary: ROSA ‘Officinalis’ Baby: ROSA multiflora Banks (double white): ROSA banksiae var. banksiae ‘Alba Plena’ Banks (double yellow): ROSA banksiae var. banksiae ‘Lutea’ Yew: PODOCARPUS macrophyllus Pinxterbloom: RHODODENDRON periclymenoides Pipe Plant: AGARISTA populifolia Pipe-stem Wood: AGARISTA populifolia Piratebush: BUCKLEYA distichophylla Pistache, Chinese: PISTACIA chinensis Texas: PISTACIA texana Texas: PISTACIA texana Pittosporum, Japanese: PITTOSPORUM tobira Plane, American: PLATANUS occidentalis London: PLATANUS acerifolia Oriental: PLATANUS orientalis Plum, Beach: PRUNUS maritima Black-sloe: PRUNUS umbellata Chickasaw: PRUNUS angustifolia Creek: PRUNUS rivularis Damson: PRUNUS domestica ssp. insititia Mexican: PRUNUS mexicana Purple-leaf: PRUNUS cerasifera ‘Atropurpurea’ Wild Goose: PRUNUS munsoniana var. banksiae ‘Alba Plena’ Banks (double yellow): ROSA banksiae var. banksiae ‘Lutea’ var. banksiae ‘Lutea’ Banks (single yellow): ROSA banksiae Banks (single yellow): ROSA banksiae var. normalis ‘Lutescens’ Blackberry: RUBUS rosifolius ‘Coronarius’ Boursault: ROSA x lheritierana ‘Gracilis’ Bur: ROSA roxburghii f. normalis Cabbage: ROSA centifolia Champney: ROSA ‘Champney’s Pink Cluster’ Cherokee: ROSA laevigata Chestnut: ROSA roxburghii f. normalis China: ROSA chinensis Confederate: HIBISCUS mutabilis Crested Moss: ROSA centifolia ‘Cristata’ Damask: ROSA x damascena Dog: ROSA canina Fairy: ROSA chinensis ‘Minima’ Father Hugo: ROSA xanthina f. hugonis Four Seasons: ROSA < damascena var. var. normalis ‘Lutescens’ Blackberry: RUBUS rosifolius ‘Coronarius’ Poinsettia: EUPHORBIA pulcherri Pomegranate: PUNICA granatum Dwarf: PUNICA granatum ‘Nana’ Dwarf: PUNICA granatum ‘Nana’ Pond-spice: LITSEA aestivalis Popinac, White: LEUCAENA leucocephala Poplar, Carolina: POPULUS x canadensis Gray: POPULUS x canescens Japanese: POPULUS maximowiczii Lombardy: POPULUS nigra ‘Italica’ White: POPULUS alba Yellow: LIRIODENDRON tulipifera Porcelain-berry: AMPELOPSIS glandulosa var. var. leiocarpum nigra Chir: PINUS roxburghii Digger: PINUS sabiniana Eastern White: PINUS strobus Nashi: PYRUS pyrifolia var. culta Sand: PYRUS pyrifolia Texas: DIOSPYROS texana Phillyrea, Narrow-leaf: PHILLYREA angustifolia Tree: PHILLYREA latifolia var. media Seville: CITRUS aurantium Sour: CITRUS aurantium Pine, African Fern: PODOCARPUS gracilior Aleppo: PINUS halepensis Australian: CASUARINA cunninghamiana Austrian: PINUS nigra ssp. nigra Chir: PINUS roxburghii Digger: PINUS sabiniana Eastern White: PINUS strobus Himalayan: PINUS wallichiana Italian Stone: PINUS pinea Jack: PINUS banksiana Japanese Black: PINUS thunbergiana Japanese Red: PINUS densiflora Japanese Umbrella: PINUS densiflora ‘Umbraculifera’ ‘Umbraculifera’ 321 Porlieria, Texas: PORLIERIA angustifolia Potato Bush, Blue: SOLANUM rantonnetii Potato Vine: SOLANUM jasminoides Prickly Ash, Southern: ZANTHOXYLUM clava-herculis Princess Tree: PAULOWNIA tomentosa Privet, Border: LIGUSTRUM obtusifolium California: LIGUSTRUM ovalifolium Chinese: LIGUSTRUM sinense Common: LIGUSTRUM vulgare Japanese: LIGUSTRUM japonicum Swamp: FORESTIERA acuminata Wax-leaf: LIGUSTRUM lucidum Weeping: LIGUSTRUM sinense ‘Pendulum’ Punk Tree: MELALEUCA quinquenervis Quince, Chinese: PSEUDOCYDONIA sinensis Common: CYDONIA oblonga Flowering: CHAENOMELES speciosa Japanese: CHAENOMELES speciosa Japanese Flowering: CHOENOMELES japonica Raisin-tree, Japanese: HOVENIA dulcis Redbud, Chinese: CERCIS chinensis North American: CERCIS canadensis Redwood, Coast: SEQUOIA sempervirens Dawn: METASEQUOIA glyptostroboides Porlieria, Texas: PORLIERIA angustifolia Potato Bush, Blue: SOLANUM rantonnetii Potato Vine: SOLANUM jasminoides Prickly Ash, Southern: ZANTHOXYLUM clava-herculis Princess Tree: PAULOWNIA tomentosa Privet, Border: LIGUSTRUM obtusifolium California: LIGUSTRUM ovalifolium Chinese: LIGUSTRUM sinense Common: LIGUSTRUM vulgare Japanese: LIGUSTRUM japonicum Swamp: FORESTIERA acuminata Wax-leaf: LIGUSTRUM lucidum Weeping: LIGUSTRUM sinense ‘Pendulum’ Punk Tree: MELALEUCA quinquenervis Quince, Chinese: PSEUDOCYDONIA sinensis Common: CYDONIA oblonga Flowering: CHAENOMELES speciosa Japanese: CHAENOMELES speciosa Japanese Flowering: CHOENOMELES japonica Raisin-tree, Japanese: HOVENIA dulcis Redbud, Chinese: CERCIS chinensis North American: CERCIS canadensis Redwood, Coast: SEQUOIA sempervirens Dawn: METASEQUOIA glyptostroboides Japanese White: PINUS parviflora Jelecote: PINUS patula Knob-cone: PINUS attenuata Korean: PINUS koraiensis Lacebark: PINUS bungeana Limber: PINUS flexilis Loblolly: PINUS taeda Long-leaved Indian: PINUS roxburghii Longleaf: PINUS palustris Mexican Stone: PINUS cembroides Pitch: PINUS rigida Pond: PINUS serotina Ponderosa: PINUS ponderosa Red: PINUS resinosa Sand: PINUS clausa Scotch: PINUS sylvestris Scrub: PINUS virginiana Shortleaf: PINUS echinata Slash: PINUS elliottii Spruce: PINUS glabra Swiss Mountain: PINUS mugo var. Ringworm: CASSIA alata Sequoia, Giant: SEQUOIADEN Serbian: PICEA omorika Tiger-tail: PICEA torano var. leiocarpum gigantea ‘Old Blush’, ROSA x damascena var. semperflorens Moss: ROSA centifolia ‘Muscosa’ Musk: ROSA moschata Noisette: ROSA Xnoisettiana -of-Sharon: HIBISCUS syriacus Pasture: ROSA carolina Persian Yellow: ROSA foetida ‘Perseana’ Prairie: ROSA setigera Prickly: ROSA acicularis Provence: ROSA centifolia Red Cherokee: ROSA < anemonoides ‘Ramona Rugosa: ROSA rugosa Scotch: ROSA pimpinellifolia Seven Sisters: ROSA multiflora ‘Platyphylla’ Striped Moss: ROSA ‘Oeillet Panachee’ Swamp: ROSA palustris Tea: ROSA x odorata Tidbit: ROSA gallica ‘Conditorum’ Virginia: ROSA virginiana White Rose-of-York: ROSA x alba Yellow Rose-of-Texas: ROSA x harisonii York and Lancaster Rose: ROSA damascena ‘Versicolor’ Memorial: ROSA wichuraiana Sissoo: DALBERGIA sissoo Skimmia, White-fruited: SKIMMIA japonica ‘Fructo-albo’ Skyflower, Creeping: DURANTA repens Smoke Tree: COTINUS coggygria American: COTINUS obovatus Snailseed, Laurel-leaf: COCCULUS laurifolius Snowball, Chinese: VIBURNUM macrocephalum American: COTINUS obovatus Snailseed, Laurel-leaf: COCCULUS laurifolius Snowball, Chinese: VIBURNUM macrocephalum Common: VIBURNUM opulus ‘Roseum’ European: VIBURNUM opulus ‘Roseum’ Japanese: VIBURNUM plicatum f. plicatum Snowbell, American: STYRAX americanus Bigleaf: STYRAX grandifolius Fragrant: STYRAX obassia Japanese: STYRAX japonicus Texas: STYRAX platanifolius Snowbrush Ceanothus: CEANOTHUS velutinus Snowbush: BREYNIA disticha Snow-wreath: NEVIUSIA alabamensis Soapberry, Chinese: SAPINDUS mukorossi Florida: SAPINDUS marginatus Texas: SAPINDUS drummondii Sorrel Tree: OXYDENDRUM arboreum Sourwood: OXYDENDRUM arboreum Southern Plume: ELLIOTTIA racemosa Southernwood: ARTEMESIA abrotanum Spanish Bayonet: YUCCA aloifolia ‘Marginata’ Spanish Dagger: YUCCA gloriosa Sparkleberry: VACCINIUM arboreum Spindle Tree, European: EUONYMUS europaeus Japanese: EUONYMUS japonicus Winged: EUONYMUS alatus Winterberry: EUONYMUS bungeanus Spindlebush, Wintercreeper: EUONYMUS fortunei Spirea, Japanese: SPIRAEA japonica Reeves: SPIRAEA cantoniensis Vanhoutte: SPIRAEA <vanhouttei Willowleaf: SPIRAEA salicifolia Spruce, Alcock’s: PICEA alcoquiana Black: PICEA mariana ‘Doumetii’ Colorado: PICEA pungens Colorado Blue: PICEA pungens f. glauca Dragon: PICEA asperata Dwarf Alberta: PICEA glauca ‘Conica’ Norway: PICEA abies Oriental: PICEA orientalis Serbian: PICEA omorika Tiger-tail: PICEA torano White: PICEA glauca Spurge, Allegheny: PACHYSANDRA procumbens Japanese: PACHYSANDRA terminalis Spurge-olive: CNEORUM tricoccon Staggerbush: LYONIA mariana Stewartia, Japanese: STEWARTIA pseudocamellia Stranvaesia, Chinese: STRANVAESIA davidiana Yellow-fruited Chinese: STRANVAESIA davidiana ‘Lutea’ var. leiocarpum brevipedunculata semperflorens Green: ROSA chinensis ‘Viridiflora’ Himalayan Musk: ROSA brunonii Hungarian: ROSA gallica ‘Conditorum’ Japanese: KERRIA japonica, ROSA multiflora Kazanlik: ROSA < damascena ‘Trigintipetala’ Lady Bank’s: ROSA banksiae var. banksiae ‘Lutea’ M‘Cartney: ROSA bracteata 322 Meadow: ROSA blanda Memorial: ROSA wichuraiana Monthly: ROSA chinensis < R. gigantea ‘Old Blush’, ROSA x damascena var. semperflorens Moss: ROSA centifolia ‘Muscosa’ Musk: ROSA moschata Noisette: ROSA Xnoisettiana -of-Sharon: HIBISCUS syriacus Pasture: ROSA carolina Persian Yellow: ROSA foetida ‘Perseana’ Prairie: ROSA setigera Prickly: ROSA acicularis Provence: ROSA centifolia Red Cherokee: ROSA < anemonoides ‘Ramona Rugosa: ROSA rugosa Scotch: ROSA pimpinellifolia Seven Sisters: ROSA multiflora ‘Platyphylla’ Striped Moss: ROSA ‘Oeillet Panachee’ Swamp: ROSA palustris Tea: ROSA x odorata Tidbit: ROSA gallica ‘Conditorum’ Virginia: ROSA virginiana White Rose-of-York: ROSA x alba Yellow Rose-of-Texas: ROSA x harisonii York and Lancaster Rose: ROSA damascena ‘Versicolor’ Rosebay: RHODODENDRON maximum Mountain: RHODODENDRON catawbiense Rosemary: ROSMARINUS officinalis Wild: LEDUM palustre var. diversipilosum Rosewood: TIPUANA tipu Rowan: SORBUS aucuparia Rubber Tree, Chinese: EUCOMMIA ulmoides Sage, Autumn: SALVIA greggii Mexican Bush: SALVIA leucantha Russian: PEROVSKIA atriplicifolia Sago, Queen: CYCAS circinalis St. Andrew’s Cross: ASCYRUM hypericoides St. Peter’s-wort: HYPERICUM stans Sandheath: CERATIOLA ericoides Sand Myrtle, Box: LEIOPHYLLUM buxifolium Sandweed: HYPERICUM fasciculatum Sapote, White: CASIMIROA edulis Sassafras: SASSAFRAS albidum Satureja, Winter: SATUREJA montana Scarlet Bush: HAMELIA patens Scholar Tree, Chinese: SOPHORA japonica Scuppernong: VITIS rotundifolia Senna, Bladder: COLUTEA arborescens Ringworm: CASSIA alata Sequoia, Giant: SEQUOIADENDRON giganteum Serviceberry, Downy: AMELANCHIER arborea Service Tree: SORBUS domestica Seven-sun Flower: HEPTACODIUM miconioides Shadbush, Allegheny: AMELANCHIER laevis Common: AMELANCHIER arborea Smooth: AMELANCHIER laevis Sheepberry: VIBURNUM lentago, VIBURNUM prunifolium Shrimp Plant: JUSTICIA brandegeana, Silk Tree: ALBIZIA julibrissin Silk Vine: PERIPLOCA graece. Silk-worm Tree: CUDRANIA tricuspidata Silver Vine: ACTINIDIA polygama Sissoo: DALBERGIA sissoo Skimmia, White-fruited: SKIMMIA japonica ‘Fructo-albo’ Meadow: ROSA blanda Memorial: ROSA wichuraiana Monthly: ROSA chinensis < R. gigantea ‘Old Blush’, ROSA x damascena var. semperflorens Moss: ROSA centifolia ‘Muscosa’ Musk: ROSA moschata Noisette: ROSA Xnoisettiana -of-Sharon: HIBISCUS syriacus Pasture: ROSA carolina Persian Yellow: ROSA foetida ‘Perseana’ Prairie: ROSA setigera Prickly: ROSA acicularis Provence: ROSA centifolia Red Cherokee: ROSA < anemonoides ‘Ramona Rugosa: ROSA rugosa Scotch: ROSA pimpinellifolia Seven Sisters: ROSA multiflora ‘Platyphylla’ Striped Moss: ROSA ‘Oeillet Panachee’ Swamp: ROSA palustris Tea: ROSA x odorata Tidbit: ROSA gallica ‘Conditorum’ Virginia: ROSA virginiana White Rose-of-York: ROSA x alba Yellow Rose-of-Texas: ROSA x harisonii York and Lancaster Rose: ROSA damascena ‘Versicolor’ Meadow: ROSA blanda Memorial: ROSA wichuraiana Monthly: ROSA chinensis < R. ‘Versicolor’ ‘Versicolor’ Rosebay: RHODODENDRON maximum Mountain: RHODODENDRON catawbiense Rosemary: ROSMARINUS officinalis Wild: LEDUM palustre var. diversipilosum Rosewood: TIPUANA tipu Rowan: SORBUS aucuparia Rubber Tree, Chinese: EUCOMMIA ulmoides Sage, Autumn: SALVIA greggii Mexican Bush: SALVIA leucantha Russian: PEROVSKIA atriplicifolia Sago, Queen: CYCAS circinalis St. Andrew’s Cross: ASCYRUM hypericoides St. Peter’s-wort: HYPERICUM stans Sandheath: CERATIOLA ericoides Sand Myrtle, Box: LEIOPHYLLUM buxifolium Sandweed: HYPERICUM fasciculatum Sapote, White: CASIMIROA edulis Sassafras: SASSAFRAS albidum Satureja, Winter: SATUREJA montana Scarlet Bush: HAMELIA patens Scholar Tree, Chinese: SOPHORA japonica Scuppernong: VITIS rotundifolia Senna, Bladder: COLUTEA arborescens Ringworm: CASSIA alata Sequoia, Giant: SEQUOIADENDRON giganteum Serviceberry, Downy: AMELANCHIER arborea Service Tree: SORBUS domestica Seven-sun Flower: HEPTACODIUM miconioides Shadbush, Allegheny: AMELANCHIER laevis Common: AMELANCHIER arborea Smooth: AMELANCHIER laevis Sheepberry: VIBURNUM lentago, VIBURNUM prunifolium Shrimp Plant: JUSTICIA brandegeana, J. californica ‘Versicolor’ Rosebay: RHODODENDRON maximum Mountain: RHODODENDRON catawbiense Rosemary: ROSMARINUS officinalis Wild: LEDUM palustre var. diversipilosum Rosewood: TIPUANA tipu Rowan: SORBUS aucuparia Rubber Tree, Chinese: EUCOMMIA ulmoides Sage, Autumn: SALVIA greggii Mexican Bush: SALVIA leucantha Russian: PEROVSKIA atriplicifolia Sago, Queen: CYCAS circinalis St. Andrew’s Cross: ASCYRUM hypericoides St. Peter’s-wort: HYPERICUM stans Sandheath: CERATIOLA ericoides Sand Myrtle, Box: LEIOPHYLLUM buxifolium Sandweed: HYPERICUM fasciculatum Sapote, White: CASIMIROA edulis Sassafras: SASSAFRAS albidum Satureja, Winter: SATUREJA montana Scarlet Bush: HAMELIA patens Scholar Tree, Chinese: SOPHORA japonica Scuppernong: VITIS rotundifolia Senna, Bladder: COLUTEA arborescens Spruce, Alcock’s: PICEA alcoquiana Black: PICEA mariana ‘Doumetii’ Black: PICEA mariana ‘Doumetii’ Colorado: PICEA pungens Colorado: PICEA pungens Colorado Blue: PICEA pung Scarlet Bush: HAMELIA patens Scholar Tree, Chinese: SOPHORA Scholar Tree, Chinese: SOPHORA japonica Scuppernong: VITIS rotundifolia Scuppernong: VITIS rotundifolia Senna, Bladder: COLUTEA arbores Dwarf Alberta: PICEA glauca ‘Conica’ Norway: PICEA abies Oriental: PICEA orientalis Serbian: PICEA omorika Spurge-olive: CNEORUM tricoccon Staggerbush: LYONIA mariana sinensis Fragrant: CORYLOPSIS glabrescens Winterberry: ILEX verticillata Ambiguous: ILEX ambigua Japanese: ILEX serrata Smooth: ILEX laevigata Yellow-berried: ILEX verticillata f. chrysocarpa Yellow-fruited Japanese: ILEX serrata ‘Leucocarpa’ Wintergreen: GAULTHERIA procumbens Wintersweet: CHIMONANTHUS praecox Wire Vine, Matbrush: MUEHLENBECKIA axillaris Wisteria, American: WISTERIA frutescens Chinese: WISTERIA sinensis Japanese: WISTERIA floribunda Witch Hazel, Common: HAMAMELIS virginiana var. rotundifolium Sweet: VIBURNUM odoratissimum Tea-leaved: VIBURNUM setigerum Tree: VIBURNUM sieboldii Walter's: VIBURNUM obovatum Withe-rod: VIBURNUM cassinoides Yeddo: VIBURNUM bitchiuense Virgin’s Bower: CLEMATIS terniflora Walnut, Arizona: JUGLANS major Black: JUGLANS nigra English: JUGLANS regia Japanese: JUGLANS ailantifolia var. cordiformis Persian: JUGLANS regia Water-pine, Chinese: GLYPTOSTROBUS lineatus Waxberry: MYRICA cerifera Wax-myrtle: MYRICA cerifera, M. heterophylla Wayfaring Tree: VIBURNUM lantana White Wicky: KALMIA cuneata Tea Berry: GAULTHERIA procumbens Tea Plant: CAMELLIA sinensis Tea Berry: GAULTHERIA procumbens Tea Plant: CAMELLIA sinensis Tea Plant: CAMELLIA sinensis Tea Tree, New Zealand: LEPTOSPERMUM scoparium Thimbleberry: RUBUS odoratus Thimbleberry: RUBUS odoratus Thorn, Jerusalem: PARKINSONIA Black: SALIX nigra Contorted Hankow: SALIX babylonica ‘Tortuosa’ Desert: CHILOPSIS linearis Goat: SALIX caprea Thorn, Jerusalem: PARKINSONIA aculeata Washington: CRATAEGUS phaenopyrum Weeping: SALIX babylonica Spurge-olive: CNEORUM tricoccon Staggerbush: LYONIA mariana Smooth: AMELANCHIER laevis heepberry: VIBURNUM lentago, J. californica ‘Lutea’ 323 Strawberry Bush: EUONYMUS americanus Strawberry Tree: ARBUTUS unedo Sugarberry: CELTIS laevigata Sumac, Fragrant: RHUS aromatica Shining: RHUS copallina Smooth: RHUS glabra Staghorn: RHUS typhina Winged: RHUS copallina Summersweet, Japanese: CLETHRA barbinervis Pink: CLETHRA alnifolia ‘Rosea’ Woolly: CLETHRA tomentosa Supplejack, Alabama: BERCHEMIA scandens Japanese: BERCHEMIA racemosa Sweetbells: LEUCOTHOE racemosa Sweet Box, Fragrant: SARCOCOCCA ruscifolia Sweetfern: COMPTONIA peregrina Sweetshrub: CALYCANTHUS floridus Sycamore, American: PLATANUS occidentalis Tallow Tree, Chinese: SAPIUM sebiferum Tangerine: CITRUS reticulata Tara Vine: ACTINIDIA arguta Tarflower: BEFARIA racemosa Tea, New Jersey: CEANOTHUS americanus Tea Berry: GAULTHERIA procumbens Tea Plant: CAMELLIA sinensis Tea Tree, New Zealand: LEPTOSPERMUM scoparium Thimbleberry: RUBUS odoratus Thorn, Jerusalem: PARKINSONIA aculeata Washington: CRATAEGUS phaenopyrum Thyme, Common: THYMUS vulgaris Tipu Tree: TIPUANA tipu Titi: CLIFTONIA monophylla, CYRILLA racemiflora Tobacco, Tree: NICOTIANA glauca Toona, Chinese: TOONA sinensis Torchwood, Texas: AMYRIS texana Torreya, Florida: TORREYA taxifolia Japanese: TORREYA nucifera Tree-of-heaven: AILANTHUS altissima Trumpet-creeper: CAMPSIS radicans Chinese: CAMPSIS grandiflora Trumpet Vine: CAMPSIS radicans Argentine: CLYTOSTOMA callistegioides Blue: THUNBERGIA grandiflora Tubeflower: CLERODENDRUM indicum Tulip Tree: LIRIODENDRON tulipifera Chinese: LIRIODENDRON chinense Tung-oil Tree: ALEURITES fordii Tupelo, Ogeechee: NYSSA ogeche Turk’s Cap: MALVAVISCUS arboreus var. drummondii, M. arboreus var. penduliflorus Turk’s Turban: CLERODENDRUM indicum Varnish Tree: TOXICODENDRON vernicifluum Vase Vine: CLEMATIS viorna Verbena, Lemon: ALOYSIA gratissima Viburnum, Birch-leaved: VIBURNUM betulifolium Buddleja-leaved: VIBURNUM buddleifolium Burkwood’s: VIBURNUM X burkwoodii Cinnamon-leaved: VIBURNUM cinnamomifolium Double-file: VIBURNUM plicatum f. tomentosum vis Leatherleaf: VIBURNUM r Linden: VIBURNUM dila Manchurian: VIBURNUM Maple-leaved: VIBURNUM Philippine: VIBURNUM l Round-leaved double-file: var. rotundifolium Sweet: VIBURNUM odora Tea-leaved: VIBURNUM s Tree: VIBURNUM sieboldi Leatherleaf: VIBURNUM rhytidophyllum Linden: VIBURNUM dilatatum Manchurian: VIBURNUM burejaeticum Maple-leaved: VIBURNUM acerifolium Philippine: VIBURNUM luzonicum Round-leaved double-file: VIBURNUM plicatum var. rotundifolium Sweet: VIBURNUM odoratissimum Tea-leaved: VIBURNUM setigerum Tree: VIBURNUM sieboldii Walter's: VIBURNUM obovatum Withe-rod: VIBURNUM cassinoides Yeddo: VIBURNUM bitchiuense Virgin’s Bower: CLEMATIS terniflora Walnut, Arizona: JUGLANS major Black: JUGLANS nigra English: JUGLANS regia Japanese: JUGLANS ailantifolia var. cordiformis Persian: JUGLANS regia Water-pine, Chinese: GLYPTOSTROBUS lineatus Waxberry: MYRICA cerifera Wax-myrtle: MYRICA cerifera, M. heterophylla Dwarf: MYRICA pusilla Wayfaring Tree: VIBURNUM lantana White Wicky: KALMIA cuneata Willow, Basket: SALIX purpurea Black: SALIX nigra Contorted Hankow: SALIX babylonica ‘Tortuosa’ Desert: CHILOPSIS linearis Goat: SALIX caprea Gray: SALIX cinerea Niobe: SALIX x blanda Virginia: ITEA virginica Weeping: SALIX babylonica White: SALIX alba Wingnut, Chinese: PTEROCARYA stenoptera Three-wing: TRIPTERYGIUM regelii Winter Hazel, Buttercup: CORYLOPSIS pauciflora Chinese: CORYLOPSIS sinensis var. Turk’s Turban: CLERODENDRUM indicum Verbena, Lemon: ALOYSIA gratissima Viburnum, Birch-leaved: VIBURNUM betulifolium Buddleja-leaved: VIBURNUM buddleifolium Burkwood’s: VIBURNUM X burkwoodii Cinnamon-leaved: VIBURNUM cinnamomifolium Double-file: VIBURNUM plicatum f. tomentosum Japanese: VIBURNUM japonicum Korean-spice: VIBURNUM carlesii Yaupon: ILEX vomitoria Mexican: ILEX vomitoria var. chiapiensis Pendulous: ILEX vomitoria f. pendula Yellowhorn: XANTHOCERAS sorbifolium Yellowroot, Shrub: XANTHORHIZA simplicissima Yellow-wood: CLADRASTIS kentukea, SYMPLOCOS tinctoria 324 Yerba Maté: ILEX paraguariensis Yesterday-today-tomorrow: BRUNFELSIA australis Yew, Canadian: TAXUS canadensis English: TAXUS baccata Florida: TAXUS floridana Golden: TAXUS baccata ‘Aurea’ Japanese: TAXUS cuspidata Plum: CEPHALOTAXUS harringtonia Yulan: MAGNOLIA denudata Zelkova, Caucasian: ZELKOVA carpinifolia Japanese: ZELKOVA serrata Florida: TAXUS floridana Golden: TAXUS baccata ‘Aurea’ Japanese: TAXUS cuspidata Plum: CEPHALOTAXUS harringtonia Yulan: MAGNOLIA denudata Zelkova, Caucasian: ZELKOVA carpinifolia Japanese: ZELKOVA serrata 329 Selected Bibliography Brown, R.G., and M.L. 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https://openalex.org/W3102426320	https://hal.science/hal-02960787/file/aa38456-20.pdf	English	null	A wide field-of-view low-resolution spectrometer at APEX: Instrument design and scientific forecast	Astronomy & astrophysics	2,020	cc-by	15,543	To cite this version: P. Ade, M. Aravena, E. Barria, A. Beelen, A. Benoit, et al.. A wide field-of-view low-resolution spectrometer at APEX: Instrument design and scientific forecast. Astronomy and Astrophysics - A&A, 2020, 642, pp.A60. 10.1051/0004-6361/202038456. hal-02960787 A wide field-of-view low-resolution spectrometer at APEX: Instrument design and scientific forecast P. Ade, M. Aravena, E. Barria, A. Beelen, A. Benoit, M. Béthermin, J. Bounmy, O. Bourrion, G. Bres, C. de Breuck, et al. Distributed under a Creative Commons Attribution 4.0 International License A wide ﬁeld-of-view low-resolution spectrometer at APEX: Instrument design and scientiﬁc forecast The CONCERTO Collaboration: P. Ade1, M. Aravena2, E. Barria3,4, A. Beelen5, A. Benoit3,4, M. Béthermin5, J. Bounmy6,4, O. Bourrion6,4, G. Bres3,4, C. De Breuck7, M. Calvo3,4, Y. Cao5, A. Catalano6,4, F.-X. Désert8,4, C.A. Durán9, A. Fasano3,4, T. Fenouillet5, J. Garcia5, G. Garde3,4, J. Goupy3,4, C. Groppi10, C. Hoarau6,4, G. Lagache5, J.-C. Lambert5, J.-P. Leggeri3,4, F. Levy-Bertrand3,4, J. Macías-Pérez6,4, H. Mani10, J. Marpaud6,4, P. Mauskopf10, A. Monfardini3,4, G. Pisano1, N. Ponthieu8,4, L. Prieur5, S. Roni6, S. Roudier6, D. Tourres6,4, and C. Tucker1 1 Astronomy Instrumentation Group, University of Cardiﬀ, The Parade CF24 3AA, UK 2 1 Astronomy Instrumentation Group, University of Cardiﬀ, The Parade CF24 3AA, UK 2 2 Núcleo de Astronomía, Facultad de Ingeniería y Ciencias, Universidad Diego Portales, Av. Ejército 441, Santiago, C U i G bl Al CNRS G bl INP I i Né l 38000 G bl F 2 Núcleo de Astronomía, Facultad de Ingeniería y Ciencias, Universidad Diego Portales, 3 3 Univ. Grenoble Alpes, CNRS, Grenoble INP, Institut Néel, 38000 Grenoble, France e-mail: monfardini@neel.cnrs.fr 4 Groupement d’Interet Scientiﬁque KID, 38000 Grenoble and 38400 Saint Martin d’Hères, France 5 4 Groupement d’Interet Scientiﬁque KID, 38000 Grenoble and 38400 Saint Martin d’Hères, France 5 5 Aix Marseille Université, CNRS, LAM (Laboratoire d’Astrophysique de Marseille), 13388 Marseille, Franc e-mail: guilaine.lagache@lam.fr g g 6 Univ. Grenoble Alpes, CNRS, LPSC/IN2P3, 38000 Grenoble, France 7 6 Univ. Grenoble Alpes, CNRS, LPSC/IN2P3, 38000 Grenoble, France 7 European Southern Observatory, Karl Schwarzschild Straße 2, 85748 Garching, Germany 7 European Southern Observatory, Karl Schwarzschild Straße 2, 85748 Garching, Germany 8 8 Univ. Grenoble Alpes, CNRS, IPAG, 38400 Saint Martin d’Hères, France 8 Univ. Grenoble Alpes, CNRS, IPAG, 38400 Saint Martin d’Hères, France 9 European Southern Observatory, Alonso de Cordova 3107, Vitacura, Santiago, Chile 10 S h l f E h d S E l i d D f Ph i A i S U i 9 European Southern Observatory, Alonso de Cordova 3107, Vitacura, Santiago, Chile 10 School of Earth and Space Exploration and Department of Physics, Arizona State Uni p y g 0 School of Earth and Space Exploration and Department of Physics, Arizona State University, Tempe, AZ 85287, US Received 20 May 2020 / Accepted 24 July 2020 ABSTRACT Context. Characterising the large-scale structure in the Universe from present times to the high redshift epoch of reionisation is essen- tial to constraining the cosmology, the history of star formation, and reionisation, to measuring the gas content of the Universe, and to obtaining a better understanding of the physical processes that drive galaxy formation and evolution. Using the integrated emission from unresolved galaxies or gas clouds, line intensity mapping (LIM) provides a new observational window to measure the larger properties of structures. This very promising technique motivates the community to plan for LIM experiments. Aims. We describe the development of a large ﬁeld-of-view instrument, named CONCERTO (for CarbON CII line in post-rEionisation and ReionisaTiOn epoch), operating in the range 130–310 GHz from the APEX 12-m telescope (5100 m above sea level). CONCERTO is a low-resolution spectrometer based on the lumped element kinetic inductance detectors (LEKID) technology. Spectra are obtained using a fast Fourier transform spectrometer (FTS), coupled to a dilution cryostat with a base temperature of 0.1 K. Two two kilo-pixel arrays of LEKID are mounted inside the cryostat that also contains the cold optics and the front-end electronics. y y p Methods. We present, in detail, the technological choices leading to the instrumental concept, together with the design and fabrication of the instrument and preliminary laboratory tests on the detectors. We also give our best estimates for CONCERTO sensitivity and give predictions for two of the main scientiﬁc goals of CONCERTO, that is, a [CII]-intensity mapping survey and observations of galaxy clusters. g y Results. We provide a detailed description of the instrument design. Based on realistic comparisons with existing instruments devel- oped by our group (NIKA, NIKA2, and KISS), and on the laboratory characterisation of our detectors, we provide an estimate for CONCERTO sensitivity on the sky. Finally, we describe, in detail, two of the main scientiﬁc goals oﬀered by CONCERTO at APEX. Key words. instrumentation: detectors – instrumentation: spectrographs – telescopes – cosmology: observations Open Access article, published by EDP Sciences, under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. HAL Id: hal-02960787 https://hal.science/hal-02960787v1 Submitted on 7 Oct 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Astronomy & Astrophysics Astronomy & Astrophysics A&A 642, A60 (2020) https://doi.org/10.1051/0004-6361/202038456 c⃝The CONCERTO collaboration 2020 1. Introduction A pathﬁnder instrument, named KISS that is based on the same concept, has been built by our collabora- tion and deployed in November 2018, at the Teide Observatory (Fasano et al. 2020a). sky, the spectral dimension has to be added, without sacriﬁcing the instantaneous ﬁeld-of-view. The large ﬁeld-of-view and the mapping speed are, actually, the main asset of single dish tele- scopes when compared to variable baseline interferometers, such as ALMA1 or NOEMA (Lefèvre et al. 2020). For this reason, we are developing a millimetre-wave low spectral resolution (R = ν/∆ν ≤300) spectrometer with an instantaneous ﬁeld-of-view of 20 arcmin. In order to preserve the angular resolution at frequen- cies of around 300 GHz and assuming a 10-m class telescope, a focal-plane containing around 2000 spatial pixels is needed. Toachieve these ﬁgures, we adopt a room-temperature Martin- Puplett Interferometer (MpI) (Martin & Puplett 1970) coupled to a large ﬁeld millimetre-wave LEKID camera. The instrument, named CONCERTO (for CarbON CII line in post-rEionisation and ReionisaTiOn epoch), has been designed to interface with the Atacama Pathﬁnder EXperiment (APEX) 12-m telescope (Güsten et al. 2006). A pathﬁnder instrument, named KISS that is based on the same concept, has been built by our collabora- tion and deployed in November 2018, at the Teide Observatory (Fasano et al. 2020a). Telescope primary mirror diameter [m] 12 Field-of-view diameter [arcmin] 20 Absolute spectral resolution [GHz] ≥1 Relative spectral resolution R [#] 1–300 Frequency range HF \| LF [GHz] 195–310 \| 130–270 Pixels on Sky HF \| LF [#] 2152 \| 2152 Angular resolution HF \| LF [arcsec] 20–32 \| 23–45 Average angular resolution HF \| LF [arcsec] 26 \| 34 Instrument geometrical throughput [sr m2] 2.5 × 10−3 Single Pixel geometrical throughput [sr m2] 1.16 × 10−6 Data rate [MBytes s−1] 128 Notes. For comparison, the NIKA2 instrument (6.5′ ﬁeld-of-view, 30- metre telescope) geometrical throughput (A×Ω), characterising the camera collecting power, is 1.7×10−3 sr m2. Concerning the overall opti- cal transmission of CONCERTO compared to NIKA2, we refer the reader to Sect. 4.1. ( ) One important scientiﬁc driver that has motivated our devel- opments is the study of the [CII] emission line at high redshift. The [CII] line is among the brightest lines originating from star- forming galaxies and it is a reliable tracer of star formation on global scales. 1. Introduction With CONCERTO at APEX, we will map, in three dimensions, the ﬂuctuations of the [CII] line intensity in the reionisation and post-reionisation epoch (z ≳5). This tech- nique, known as “intensity mapping”, will allow us to answer the questions of whether dusty star-formation contributes to early galaxy evolution, and whether [CII]-emitters play an important role in shaping cosmic reionisation. The dedicated [CII] survey will provide a (spatial-spectral) data cube in which intensity is mapped as a function of the sky position and frequency. The 3D ﬂuctuations will then be studied in Fourier space with the power spectrum. The [CII] survey will also be sensitive to the CO intensity ﬂuctuations arising from 0.3 < z < 2 galaxies, giving the spatial distribution and abundance of molecular gas over a broad range of cosmic time. The [CII] intensity mapping is also one of the main goals of CCAT-prime (Choi et al. 2020) and TIME (Crites et al. 2014), which are two experiments that are based on diﬀerent technologies than CONCERTO: Gratings and TES bolometers are used for TIME, and KID and Fabry- Perot interferometers are used for CCAT-p. In addition to the main [CII] survey, we expect CONCERTO to bring a signiﬁ- cant contribution in a number of areas, including the study of galaxy clusters (via the thermal and kinetic SZ eﬀect), the obser- vation of local and intermediate-redshift galaxies, and the study of Galactic star-forming clouds. In this paper, we detail the main goals of the [CII] intensity mapping survey and galaxy cluster observations. To RTA & DISKS Fig. 1. Location of the CONCERTO sub-systems: the chassis and optics in the C-cabin, the Data AcQuisition computers (DAQ) in the middle container (“instrumentation container”), and the gas handling system and the pulse-tube compressor in the bottom container (“compressors room”). Real time analysis (RTA) computers and hard disks are not in the telescope tower. Fig. 1. Location of the CONCERTO sub-systems: the chassis and optics in the C-cabin, the Data AcQuisition computers (DAQ) in the middle container (“instrumentation container”), and the gas handling system and the pulse-tube compressor in the bottom container (“compressors room”). Real time analysis (RTA) computers and hard disks are not in the telescope tower. optics box. The “chassis” includes the camera (cryostat), the MpI interferometer, the readout, and control electronics. The “optics box” includes a number of mirrors and polarisers as well as a cold reference for the MpI. 1. Introduction In Table 1 we summarise the main instrument characteristics. The location of the CONCERTO sub- systems is shown in Fig. 1. In this section, we describe, in detail, the camera and its con- tent (Sect. 2.1), the MpI (Sect. 2.2), and the chassis and related electronics (Sect. 2.3). The room temperature optics, including the cold reference source, is presented in Sect. 2.4. Section 2.5 is devoted to a brief description of the CONCERTO hardware components, which are located elsewhere than in the telescope tower. Information about the installation at the telescope is found in Sect. 2.6. The paper is organised as follows. We present the instru- mental concept, design, and preliminary results in Sect. 2. The discussion pertains to KID detectors, cryogenics, and optics. In Sect. 3, we describe the ﬁrst laboratory tests. In Sect. 4, we present the sensitivity estimates, while Sect. 5 is dedicated to the [CII] intensity mapping and SZ surveys. 1 https://almascience.eso.org 1. Introduction et al. 2018; Perotto et al. 2020). The particular type of KID used for NIKA2 are front-illuminated lumped element KID (LEKID; Doyle et al. 2010). They consist in inductor-capacitor (LC) superconducting planar resonators made by a long meandered inductor (wire), which is terminated at both ends by an inter- digitated capacitor. NIKA2 supersedes previous cameras based on transition edge sensors (referred to as TES bolometers) in the frequency range of 150–360 GHz, such as MAMBO2 at IRAM (Kreysa et al. 1998), LABOCA at APEX (Siringo et al. 2009), and SCUBA-2 at the JCMT (Holland et al. 2013). Modern imaging and polarimetry cameras, at millimetre and sub-millimetre wavelengths, are currently operating on large (e.g. D > 10 m) single-dish telescopes. The main goal of these instruments is to map, at relatively high angular resolution (e.g. 5–30 arcsec), large portions of the sky (e.g. several deg2) with a high sensitivity (e.g. RMSMAP ≲1 mJy). Polarised emissions are also measured with similar speciﬁcations. In this context, the dual-band NIKA2 camera represents the ﬁrst kilo-pixel instrument operating at these wavelengths based on the kinetic inductance detectors (KID) technology (Adam In order to extend the capabilities of the existing instru- ments and to open new observational windows of the millimetre A60, page 1 of 13 A&A 642, A60 (2020) Table 1. Main characteristics of CONCERTO. sky, the spectral dimension has to be added, without sacriﬁcing the instantaneous ﬁeld-of-view. The large ﬁeld-of-view and the mapping speed are, actually, the main asset of single dish tele- scopes when compared to variable baseline interferometers, such as ALMA1 or NOEMA (Lefèvre et al. 2020). For this reason, we are developing a millimetre-wave low spectral resolution (R = ν/∆ν ≤300) spectrometer with an instantaneous ﬁeld-of-view of 20 arcmin. In order to preserve the angular resolution at frequen- cies of around 300 GHz and assuming a 10-m class telescope, a focal-plane containing around 2000 spatial pixels is needed. Toachieve these ﬁgures, we adopt a room-temperature Martin- Puplett Interferometer (MpI) (Martin & Puplett 1970) coupled to a large ﬁeld millimetre-wave LEKID camera. The instrument, named CONCERTO (for CarbON CII line in post-rEionisation and ReionisaTiOn epoch), has been designed to interface with the Atacama Pathﬁnder EXperiment (APEX) 12-m telescope (Güsten et al. 2006). 2.1. The camera 2, are the image stop (IS) at a temperature of 4 K and the cold pupil (CP) at the base temperature of around 0.1 K. Three HDPE (High Density PolyEthilene) lenses are used in the camera: L1 (room temperature), L2 (4 K), and L3 (0.1 K). In order to analyse the polarised signal, the last polariser of the MpI (P3) is placed just in front of the LEKID arrays (FP) at the base temperature. P3 is a custom wire-grid polariser. It is realised on a 12 µm-thick Polyimide membrane, and with Copper wires with a pitch of 50 µm. A number of infrared (IR)-blocking (thermal) and metal- lic multi-mesh ﬁlters (Ade et al. 2006) are mounted at diﬀer- ent stages. In particular, we have thermal ﬁlters on the warmest stages (room temperature to 50 K), low-pass multi-mesh ﬁlters at the intermediate temperatures (50 K–1 K), and band-deﬁning ﬁl- ters at the base temperature (i.e. just in front of each focal-plane array). A specially blackened baﬄe is installed at 4 K, between IS and L2, in order to suppress the stray light. chamber by soft ion milling. The superconducting ﬁlm depo- sition, which is made of Aluminium with a thickness of 20 nm, is achieved by e-beam evaporation and under a residual cham- ber pressure of 5 × 10−8 mbars. The deposition rates is ﬁxed at 0.1 nm s−1. The ultraviolet (UV) photo-lithography step is based on a positive resist and is followed by wet etching. The etch- ing is done using a standard aluminium etching solution based on phosphoric acid. The diced detectors arrays are packaged in custom holders and bonded, via 17 µm Aluminium wires, to the 50-Ohms micro-strip launchers. Those are then tin-soldered to the inner pin of the SMA3 feed-throughs. p g The front-end electronics stage is installed in the cryostat, at a temperature of 4 K. It is made of a series of twelve low- noise ampliﬁers (LNA) operating at the resonance frequencies4, that is, in the range of 1.5–2.5 GHz. A second stage of cryogenic ampliﬁcation, made of twelve commercial LNA, has been added on the 50 K cryogenic stage to simplify the room-temperature electronics and reduce its power consumption. The connections between the cold electronics stages, the arrays, and the SMA vacuum feed-throughs plate (see Fig. 2), are ensured by commer- cial semi-rigid cryogenic coaxial cables. 2.1. The camera CONCERTO has been speciﬁcally designed to ﬁt into the Cassegrain cabin (C-cabin) of the APEX telescope. It is com- posed of two main components: the so-called chassis and the The CONCERTO camera is based on a cryogenic-liquid-free custom dilution cryostat. The dilution insert and the pulse- tube orientation, in particular, have been speciﬁcally designed to allow the rotation of the cryostat axis following telescope A60, page 2 of 13 The CONCERTO collaboration: A wide ﬁeld-of-view low-resolution spectrometer at APEX Polariser P3 Cold Pupil CP Lens L3 Focal-Plane LF Focal-Plane HF Fig. 3. Picture of the ﬁrst “100 mK block” including the cold pupil, the L3 lens, the P3 polariser, and the two arrays (HF and LF) containing 2152 pixels each. The array holders are, in this case, realised in Alu- minium. A version of the block with Copper holders is also available. L1 285K L2 4K L3 0.1K P3 0.1K FP 0.1K IS 4K CP 0.1K Fig. 2. CONCERTO camera cross-section and 3D view (inset). The positions of the three HDPE lenses are shown (L1, L2, L3), together with the image stop (IS), the cold pupil (CP), the cold polariser (P3), and the two focal planes (FP). The diameter of L1 is around 250 mm. L1 285K L2 4K L3 0.1K P3 0.1K FP 0.1K IS 4K CP 0.1K L1 285K L2 4K L3 0.1K P3 0.1K FP 0.1K IS 4K CP 0.1K Cold Pupil CP Fig. 2. CONCERTO camera cross-section and 3D view (inset). The positions of the three HDPE lenses are shown (L1, L2, L3), together with the image stop (IS), the cold pupil (CP), the cold polariser (P3), and the two focal planes (FP). The diameter of L1 is around 250 mm. Focal-Plane LF Fig. 3. Picture of the ﬁrst “100 mK block” including the cold pupil, the L3 lens, the P3 polariser, and the two arrays (HF and LF) containing 2152 pixels each. The array holders are, in this case, realised in Alu- minium. A version of the block with Copper holders is also available. movements. The cryostat is optimised for the range of telescope elevations (EL) comprised between 30 and 90 degrees. The best working point is achieved for EL = 60 degrees. The main camera optical features, shown in Fig. 3 SubMiniature version A. 4 http://thz.asu.edu/products.html 2.1. The camera In particular, we adopt NbTi superconducting coaxial cables for the portion connect- ing the output of the LEKID arrays to the input of the front-end ampliﬁers. Fixed attenuators are mounted on each input line at the 4 K stage. The overall electrical gain of each radio-frequency line to and from the room temperature electronics has been mea- sured and is about +25 dB. Since LEKID are sensitive to variations of magnetic ﬁelds, they have to be protected by a multi-stage B ﬁeld screen. Four concentric high-permittivity alloy (i.e. mu-metal and cryogenics variations) cylinders are installed at 300 K, 50 K (double screen), and 4 K. An additional superconducting screen will be wrapped around the focal planes section. The focal plane arrays are microstrip-coupled LEKID simi- lar to those used for NIKA2 (Adam et al. 2018). Six feed-lines (i.e. excitation and readout lines) are needed to read out each of the 2152 pixel arrays. A total of twelve pairs of coaxial cables are thus running into the cryostat. The pixels design itself is derived from NIKA (Monfardini et al. 2011). The LEKID details have been optimised to meet the CONCERTO speciﬁcations. In par- ticular, the shape of the meander and its coupling quality fac- tor have been adjusted to the target range of frequencies and expected background. The coupling quality factor is designed to be Qc ≈∆f−3 dB/ f0 ≈2.5 × 104, where ∆f−3 dB is the typi- cal width of the resonance under dark conditions. The thickness of the dielectric substrate is calibrated in order to maximise the quantum eﬃciency, and it is in the range of 100–120 µm. 2 https://www.sil-tronix-st.com/en/ 2.2. The Martin-Puplett interferometer Two identical linear motors, which develop a force of ≥1000 N each, are acted on in counter-phase to null the total momentum. Fig. 4. Schematics of the MpI concept. Two options are shown for the reference source: (a) a de-focused image of the instantaneous ﬁeld-of- view; and (b) a cold reference. The polariser P1 provides the needed polarised input to the MpI. P2 is the beam splitter deﬁning the two arms, while P3, in the cryostat, dispatches the two projections of the polarised signal to the focal-plane arrays. The incoming beam is represented by a spectral distribution S(ν). include the camera itself, the MpI motors and moving mirror, and the electronics boards, along with a large number of modules devoted to monitoring and controlling the instrument. The chas- sis was designed and fabricated to match the constraints related to the limited space available in the C-cabin. It allows for the installation of multiple sub-systems of CONCERTO as a single element inside the APEX C-cabin. The chassis is pre-mounted in the laboratory and it can slide through the C-cabin door. mostly for laboratory characterisations, but also for narrow5 ﬁeld-of-view observations from space in the past (Mather 1999; Griﬃn et al. 2010). The ﬁrst examples of wide ﬁeld instruments making use of an MpI to obtain spectral information are the stratospheric balloon OLIMPO (Schillaci et al. 2014) and the previously mentioned KISS ground-based spectro-photometer (Fasano et al. 2010b). y g Five microTCA6 racks mounted on the side of the chassis host the 12 advanced mezzanine cards (AMC) used to read out the two arrays. The cards have an architecture similar to those used for NIKA2 (Bourrion et al. 2012), but they have been improved to be able to generate up to 400 excitation tones span- ning 1 GHz in bandwidth. The data acquisition rate has been increased from less than 100 Hz to up to 4 kHz in order to prop- erly sample the interferograms generated by the MpI. The cali- bration strategy has been inherited from the NIKA and NIKA2 instruments (Calvo et al. 2014), but the continuous frequency modulation used there is no longer viable because of the high- sampling rate. As a consequence, in CONCERTO, the position and shape of each resonance circle, which is used to calibrate the data, is reconstructed by sampling three points around the resonance: f0, f0 + δ f, and f0 −δ f. 2.2. The Martin-Puplett interferometer The δ f is much smaller than the resonance width, and it is of the order of a few kHz. The calibration step is performed at the beginning of each interfero- gram, while the direction of motion of the MpI rooftop mirror is changing (Fasano et al. 2020a). In our opinion, this approach represents the best trade-oﬀbetween optimal calibration and observing eﬃciency, that is, the fraction of time that is devoted to the science data stream. To achieve a spectral resolution better than 1 GHz, the maxi- mum range ∆lmax of the moving rooftop mirror has to be larger than 75 mm. In CONCERTO, the motors can move by up to 90 mm. The range ∆l spanned by the interferogram can be adjusted, on a scan-by-scan basis and depending on the science target, from zero to the maximum. The spectral resolution will then be ∆ν = c 4 × ∆l· The optical path diﬀerence is thus OPD = 2∆l. Leaving the rooftop mirror immobile at the zero-path diﬀerence position results in using CONCERTO as a broad- and dual-band large ﬁeld-of-view imager. The optical path diﬀerence is thus OPD = 2∆l. Leaving the rooftop mirror immobile at the zero-path diﬀerence position results in using CONCERTO as a broad- and dual-band large ﬁeld-of-view imager. g The distinctive feature of the CONCERTO (and KISS) MpI is the combination of the speed of movement of the rooftop mir- ror and its size and mass. In order to avoid atmospheric drifts during a single interferogram, the mechanical frequency of the motors is set to around 4 Hz, that is, eight full interferograms (and spectra) per second are produced by each of the pixels. The lateral size of the mirror that is to be moved exceeds 0.5 m for a mass exceeding 3 kg. In order to counterbalance the linear momentum associated with this moving mass, a second motor, with an equivalent mass, oscillates with an opposite instanta- neous velocity. With a maximum force around 1000 N, the accel- eration that can be imposed to the moving mass, including the motor piston, exceeds 100 m s−2. The theoretical curve that is commanded to the motor is a square wave (i.e. a constant speed for both directions and a maximum acceleration at the turn- backs).The real curve is of course smoothed out by the ﬁnite acceleration near the extremes. 2.2. The Martin-Puplett interferometer The MpI is a particular kind of Fourier transform spectrometer (FTS). It is capable of measuring the diﬀerential spectrum of a source, with respect to a given reference. The key elements are three polarisers (P1-beam divider, P2-splitter, and P3-analyser), two (one ﬁxed and one variable) arms, and two (one ﬁxed and one moving) rooftop mirrors (see Fig. 4). This technique is widely adopted in the millimetre and sub-millimetre domains, The fabrication process (Goupy et al. 2016) looks straight- forward when compared to competing detectors that have sim- ilar performances. The substrate, a 100-millimetre high-purity mono-crystalline silicon wafer2, is prepared in the deposition 2 https://www.sil-tronix-st.com/en/ A60, page 3 of 13 A&A 642, A60 (2020) a) b) Fig. 4. Schematics of the MpI concept. Two options are shown for the reference source: (a) a de-focused image of the instantaneous ﬁeld-of- view; and (b) a cold reference. The polariser P1 provides the needed polarised input to the MpI. P2 is the beam splitter deﬁning the two arms, while P3, in the cryostat, dispatches the two projections of the polarised signal to the focal-plane arrays. The incoming beam is represented by a t l di t ib ti S( ) COUNTER-BALANCE MOTOR MAIN MOTOR COUNTER-WEIGHT POSITION MPI MOVING ROOFTOP MIRROR Fig. 5. CONCERTO double-motors MpI. Two identical linear motors, which develop a force of ≥1000 N each, are acted on in counter-phase to null the total momentum. include the camera itself, the MpI motors and moving mirror, and the electronics boards, along with a large number of modules COUNTER-BALANCE MOTOR MAIN MOTOR COUNTER-WEIGHT POSITION MPI MOVING ROOFTOP MIRROR Fig. 5. CONCERTO double-motors MpI. Two identical linear motors, which develop a force of ≥1000 N each, are acted on in counter-phase to null the total momentum. a) b) Fig. 4. Schematics of the MpI concept. Two options are shown for the reference source: (a) a de-focused image of the instantaneous ﬁeld-of- view; and (b) a cold reference. The polariser P1 provides the needed polarised input to the MpI. P2 is the beam splitter deﬁning the two arms, while P3, in the cryostat, dispatches the two projections of the polarised signal to the focal-plane arrays. The incoming beam is represented by a spectral distribution S(ν). COUNTER-BALANCE MOTOR MAIN MOTOR b) a) b) a) MPI MOVING ROOFTOP MIRROR Fig. 5. CONCERTO double-motors MpI. 2.2. The Martin-Puplett interferometer We present a picture of the sys- tem that was built for CONCERTO in Fig. 5. The moving elements inside the chassis, in particular the MpI motors and the gas ﬂowing in the pulse-tube head, gener- ate vibrations which could aﬀect the detector performances. In order to suppress their propagation to the focal plane, the camera is ﬁxed to the chassis only via a series of soft rubber pneumatic actuators, which strongly dampen the vibrations. Furthermore, the pressure inside the actuators (eight in total, with diﬀerent orientations, as shown in Fig. 6) is constantly adjusted by a ded- icated software, so that the cryostat position and axis, which are monitored by means of linear position transducers, are kept con- stant independently of the telescope elevation. The cryostat posi- tion adjustment, requiring a few seconds to complete, is done automatically after each re-pointing and, upon request, between two subsequent observing blocks (scans). The position is, on the other hand, monitored in real time even during scans. 5 In this context, the ﬁeld-of-view has to be expressed in the number of beams. The number of beams is around 2000 for CONCERTO and 300 for KISS, which can be compared to few tens, at most, for the previous instruments. 2.3. The chassis and electronics The chassis is a single, compact support structure to which many of the core components of CONCERTO are attached. These 6 https://en.wikipedia.org/wiki/MicroTCA 2.4. The cabin optics and the cold reference (optics box) A large number of mirrors (M5 to M11), the two polarisers P1 and P2, and the cold reference optics complete the optical chain. A remotely controllable three-position mirror is inserted in the optics chain in order to select the type of reference input for the MpI. The three options are as follows. the largest among the CONCERTO mirrors. The next mirror, M4, which is attached to C-cabin upper ring, reﬂects the beam towards the C-cabin ﬂoor and directly into the so-called optics box. A ﬁrst virtual image is generated, by the combination M1- M2-M3-M4, before M5. This virtual image plane will be used for some of the CONCERTO qualiﬁcation tests. – The ﬁrst option is sky, which is a de-focused image of the full 20 arcmin instantaneous ﬁeld-of-view. The distinctive advantage is that the atmospheric common-mode spectrum is optically subtracted, providing a diﬀerential measurement of the astrophysics source spectrum with respect to the atmosphere along the line-of-sight. This means that, when targeting a ﬁeld populated by weak sources, and at the ﬁrst order, we obtain a null interferogram. This conﬁguration is ideally suited for a com- pact object, that is, one with an angular extension smaller than 20 arcmin. The “optics box” includes a large number of mirrors (M5 to M11), the two polarisers P1 and P2 (Fig. 4) and the part of the optics providing the cold reference for the MpI. It also includes the ﬁxed rooftop mirror of the interferometer. A general 3D view is shown in Fig. 7. – The second option is an external cold black-body, which is a highly-emissive (ϵ ≥0.98) cold disk that is cooled down by an independent pulse-tube cryostat (TBB ≈8 K). This conﬁgura- tion is mostly adapted to extended emission observations, that is, when the spectral and photometric gradients extend, on average, more than 20 arcmin. The mirrors are held at the C-cabin temperature, which is regulated at 11 ◦C = 284 K. We expect an emissivity of the order of 1% per mirror, which is equivalent to an additional back- ground of about 3 K per surface, so not smaller than 30 K in total. The stability of the temperature in the cabin is ±1 K. This means that, for an emissivity of 1%, the eﬀective background tempera- ture variation induced per mirror is around 10 mK. 2.4. The cabin optics and the cold reference (optics box) It is clear that the “walking” of the beam is well contained in the diﬀraction disk. lateral size of the MpI. In order to obtain a diﬀraction-limited combined beam for each position of the movable roof mirror (in the range of 0–90 mm), we imposed the criterion, for each ﬁeld on the sky, producing a quasi-parallel beam inside the MpI. According to the geometrical throughput conservation rule, the ﬁeld-to-ﬁeld divergence is thus ﬁxed by the diameter of the beam. For the 20 arcmin ﬁeld-of-view, and considering a 12 m primary mirror, we obtained an overall beam diameter of about 420 mm inside the interferometer. A consequence of this method is that, in requiring that the combined beam does not “walk” in the focal plane (see Fig. 8), we must accept a jitter on the entrance pupil of the optical system, that is, the “active” portion of the primary mirror. This is, in the end, the main reason why we have decided to under-sample the size of the illuminated pri- mary mirror, to about 11 m. lateral size of the MpI. In order to obtain a diﬀraction-limited combined beam for each position of the movable roof mirror (in the range of 0–90 mm), we imposed the criterion, for each ﬁeld on the sky, producing a quasi-parallel beam inside the MpI. According to the geometrical throughput conservation rule, the ﬁeld-to-ﬁeld divergence is thus ﬁxed by the diameter of the beam. For the 20 arcmin ﬁeld-of-view, and considering a 12 m primary mirror, we obtained an overall beam diameter of about 420 mm inside the interferometer. A consequence of this method is that, in requiring that the combined beam does not “walk” in the focal plane (see Fig. 8), we must accept a jitter on the entrance pupil of the optical system, that is, the “active” portion of the primary mirror. This is, in the end, the main reason why we have decided to under-sample the size of the illuminated pri- mary mirror, to about 11 m. Camera (refractive) Fig. 7. 3D view of the CONCERTO optics, evidencing the M3 mirror interfaced to the APEX sub-reﬂector (M2). M4 is attached to the ceiling of the C-cabin and represents the only reﬂective optics component, with M3 and the MpI rooftop moving mirror, outside of the optics box. 2.4. The cabin optics and the cold reference (optics box) The ﬁrst CONCERTO element along the optical axis, after the telescope mirrors (M1 and M2), is the M3 foldable mir- ror mounted on the chassis. With a diameter of 900 mm, it is 5 In this context, the ﬁeld-of-view has to be expressed in the number of beams. The number of beams is around 2000 for CONCERTO and 300 for KISS, which can be compared to few tens, at most, for the previous instruments. 6 https://en.wikipedia.org/wiki/MicroTCA A60, page 4 of 13 The CONCERTO collaboration: A wide ﬁeld-of-view low-resolution spectrometer at APEX Fig. 8. Simulated focal plane image for the full, 20 arcmin, ﬁeld-of- view. Each spot is shown for the two extreme positions of the roof mir- ror, i.e. green (0 mm) and blue (90 mm). The black circles represent the airy disks. It is clear that the “walking” of the beam is well contained in the diﬀraction disk. Fig. 6. Rotation of the camera (and chassis) following the telescope elevation (EL). The position of the eight soft rubber pneumatic actuators is shown. Two of them are dedicated to the pulse-tube head. For the six remaining actuators, we indicate (with black arrows) those that are in action for three representative elevation cases. Fig. 6. Rotation of the camera (and chassis) following the telescope elevation (EL). The position of the eight soft rubber pneumatic actuators is shown. Two of them are dedicated to the pulse-tube head. For the six remaining actuators, we indicate (with black arrows) those that are in action for three representative elevation cases. Optics Box Virtual Image X Y Z Camera (refractive) M4 M3 to M2 Optics Box Virtual Image X Y Z Camera (refractive) M4 M3 to M2 Fig. 7. 3D view of the CONCERTO optics, evidencing the M3 mirror interfaced to the APEX sub-reﬂector (M2). M4 is attached to the ceiling of the C-cabin and represents the only reﬂective optics component, with M3 and the MpI rooftop moving mirror, outside of the optics box. A large number of mirrors (M5 to M11), the two polarisers P1 and P2, and the cold reference optics complete the optical chain. Fig. 8. Simulated focal plane image for the full, 20 arcmin, ﬁeld-of- view. Each spot is shown for the two extreme positions of the roof mir- ror, i.e. green (0 mm) and blue (90 mm). The black circles represent the airy disks. 2.5. CONCERTO hardware outside the C-cabin In addition to the elements described above and located in the C-cabin, CONCERTO is also made by modules elsewhere in the telescope tower and beyond, in particular, the commercial pulse- tube compressor (Cryomech CPA289C) and, more interestingly, the dilution cryostat gas handling system (GHS) and the Data AcQuisition (DAQ) and real time analysis (RTA) computers. The Atacama Pathﬁnder EXperiment (APEX) telescope is a modiﬁed prototype ALMA antenna with a primary mirror diam- eter of 12 m and a usable ﬁeld-of-view of about 20 arcminutes. The location at around 5100 m a.s.l. on the Chajnantor plateau ensures optimal observing conditions (see Fig. 10). In particular, the fraction of time showing a precipitable water vapour (PWV) column that is lower than 2 mm is of the order of 70% or more. The GHS is composed of: (a) a series of pumps and com- pressors used to circulate the 3He-4He mixture and to provide compressed air to CONCERTO, and, (b) an electronic cabinet hosting a National Instrument CompactRIO7 real-time controller and multiple analogue and digital input-output modules. Ded- icated Labview-based software is loaded on the CompactRIO. The software continuously monitors the state of the cryostat and controls all of the pumps, compressors, valves, and actuators of the dilution circuit. It can perform many tasks automatically, such as pre-cooling the system or putting it in a safe mode if the security thresholds are exceeded. It also acts as a server-side programme for the client graphical user interface (GUI) that is used on remote computers. The GUI allows one to easily see the state of the system and control its components (Fig. 9). The structure of the Cassegrain cabin, and in general the telescope infrastructure, had been designed to host large ﬁeld- of-view instrumentation. The primary mirror surface has been recently refurbished, and it achieves in some conditions a precision of the order of 10 µm RMS. To date, APEX is thus a state-of-the-art installation for millimetre and sub-millimetre astronomy. Since 2007, the telescope has hosted, in the same place that will be occupied by CONCERTO, the Large APEX BOlometer CAmera (LABOCA) operating at 360 GHz (Siringo et al. 2009). The CONCERTO optics box and chassis are slid separately through the C-cabin door and then ﬁxed to the ﬂoor by a suf- ﬁcient number of 12 mm metric screws. 2.4. The cabin optics and the cold reference (optics box) Considering roughly ten mirrors, this translates to a total eﬀective background temperature variation of 0.1 K. The KID detectors, with a NET of the order of ≈mK· √s per pixel, are sensitive to this drift that will produce a correlated signal on all of the pixels. However, these instabilities are harmless since they are slower and smaller than the atmospheric ﬂuctuations sitting on top. – The third option is cold cryostat, which is a de-focused image of the CONCERTO cryostat cold (inner) parts. In other words, CONCERTO “looks” into itself to ﬁnd a cold auto- reference. Considering the number of optical elements (six mir- rors and three lenses) lying between the three-position mirror and the coldest stage of the cryostat, we expect an equivalent eﬀective temperature of the order of 20 K. This is, in any case, lower than the loading of the sky plus the whole optics train between M1 and the focal plane, which is not lower than 50 K. We performed a trade-oﬀbetween the requirements related to the image quality (and the interferometry eﬃciency) and the A60, page 5 of 13 A60 (2020) Fig. 10. Atmosphere transmission at APEX under 1 mm PWV (blue) and 2 mm PWV (orange) conditions. The CONCERTO bands for the HF and LF arrays are shown. A&A 642, A60 (2020) Fig. 9. GUI used to control the dilution refrigerator circuit. Fig. 9. GUI used to control the dilution refrigerator circuit. Fig. 9. GUI used to control the dilution refrigerator circuit. This innovative conﬁguration will be investigated as a simpler alternative to the external cold black body. Fig. 10. Atmosphere transmission at APEX under 1 mm PWV (blue) and 2 mm PWV (orange) conditions. The CONCERTO bands for the HF and LF arrays are shown. We stress the fact that the external cold black-body, or its alternative “old cryostat”, are not used as spectral calibrators. They simply represent cold references, that is to say colder than the combined thermal emission of the atmosphere and the optical chain. In contrast to the “sky” reference case, the interferogram is not expected to be null at ﬁrst order. In this case, we expect potential systematic eﬀects to be minimised by the fact that both the reference and the dominant target (mostly the atmosphere in the common case of weak astrophysical sources) exhibit thermal black-body spectra. 2.4. The cabin optics and the cold reference (optics box) the environment and guarantee the ﬂexibility required by the movement of the telescope. Inside the C-cabin, all of the con- nections are centralised on a dedicated panel located on the front side of the chassis, thus easing the procedures of plugging and unplugging. The DAQ system is located in the middle container and is connected to CONCERTO in the C-cabin, producing 128 MBytes per second, through ﬁve dedicated ethernet cables. The DAQ consists in two commercial computers with 48 GB of RAM and 24 cores each. The disk storage (432 TB) and the RTA systems are installed in the so-called servers room, located a few tens of metres away from the telescope tower. The RTA com- puter has 32 cores and 512 GB of RAM. The network connection between the DAQ and the disk storage or RTA is ensured by two 10 gigabit switches and underground cables. The best choice between the three references will depend on the particular science target, the observing conditions and the unknown systematic eﬀects as of yet aﬀecting this new kind of large ﬁeld-of-view spectrophotometer. A crucial phase of the on- sky commissioning will be dedicated to investigating this item. We will report in further publications the results of this study, as well as a more detailed description of the CONCERTO MpI spectral reference system that is beyond the scope of the present paper. 7 https://en.wikipedia.org/wiki/CompactRIO 3. Detectors laboratory characterisation In this section, we describe the ﬁrst tests performed on CON- CERTO detectors. Some of the electrical tests on the resonances occurred in the CONCERTO cryostat itself. On the other hand, the optical characterisation of the detectors has been achieved in the former NIKA2 test-bench. The so-called NIKA1.5 camera is an easily re-conﬁgurable optical dilution cryostat with a base temperature of 60 mK. It has been recently modiﬁed to host one CONCERTO array at a time. In particular, the optical ﬁlters can be easily replaced, and NIKA1.5 can be interfaced to a custom MpI for spectral characterisation, or alternatively to a sky simu- lator (described in detail in Monfardini et al. 2011) for sensitivity and beams’ geometry measurements. p The spectral response of two HF arrays, with slightly diﬀer- ent substrate thicknesses, was measured. The results are reported in Fig. 14. The sensitivity was measured in terms of NET (noise equiva- lent temperature), with the NIKA1.5 optics system. The average NET per pixel of the CONCERTO arrays, in NIKA1.5, is around 2 mK √ Hz−1. This results in an NET of about 45 µK √ Hz−1 per array (polarisation), or 32 µK √ Hz−18 when combining both polarisations. Since the sensitivities per pixel are in accordance to what had been measured for the very similar NIKA2 detectors (Adam et al. 2018), we base our sensitivity estimate in Sect. 4 on NIKA2 values measured on-sky. We actually believe that the sensitivities measured on the maps on the sky for similar detec- tors are a more realistic prediction compared to somewhat ideal values estimated in laboratory. Since the twelve readout lines of the HF and LF arrays share a common local oscillator (LO, frequency reference for the read- out electronics), it is of vital importance to accommodate all of the blocks of resonances in a common ≤1 GHz band. This is nicely achieved, for example, in the case of the HF array shown in Fig. 12. The spread between blocks of resonances belonging to the same array, and between diﬀerent arrays, is mainly due to inhomogeneities and uncertainties in the thickness of the Alu- minium ﬁlm. The good imaging characteristics of the CONCERTO arrays are demonstrated by the Sky Simulator tests. An example is shown in Fig. 15. 2.5. CONCERTO hardware outside the C-cabin They have been acquired under dark conditions, in the CONCERTO cryostat, at T = 70 mK. 1·104 2·104 3·104 Coupling factor - QC Number of resonators 10 20 30 Fig. 13. Quality factors distribution for one representative block of resonances in the CONCERTO HF array. For this particular block Qc = 23k ± 12k, which is in line with all of the other blocks and with the designed Qc = 25k. These quality factors were measured in the CON- CERTO cryostat at T = 70 mK. 1·104 2·104 3·104 Coupling factor - QC Number of resonators 10 20 30 Fig. 11. Deformations of a wire sensor running between the ﬂoor and the top of the APEX Cassegrain cabin recorded for 48 h. The average absolute length of the wire is 2675.3 mm. Inset: picture of the wire sen- sor. standard APEX observations, is smaller than 0.2 mm and thus negligible for our purposes. The alignment of the mirrors in the optics box will be achieved in laboratory. The position of each mirror is adjusted with three micro-metric screws. No tuning will be possible at the telescope. The alignment of the optics box with respect to the chassis is ensured by the mechanical ﬁxations. A set of spe- ciﬁc lasers will be mounted to achieve the internal alignments in laboratory. At the telescope, we will use these lasers to align the optics box with respect to M4, M4 with respect to M3, and M3 with respect to M2. The alignment procedures will represent a critical step in the installation. Fig. 13. Quality factors distribution for one representative block of resonances in the CONCERTO HF array. For this particular block Qc = 23k ± 12k, which is in line with all of the other blocks and with the designed Qc = 25k. These quality factors were measured in the CON- CERTO cryostat at T = 70 mK. is achieved without requiring complicated and risky additional technology steps, such as cross-the-line micro-bondings or sus- pended micro-bridges. Figure 13 shows an example of the statis- tics obtained for one readout line of the CONCERTO HF array. All of the lines, as well as the LF arrays tested so far, exhibit sim- ilar behaviours. The quality factor distribution does, as designed, peak around 25k. 8 We note that 32 µK √ Hz−1 is equivalent to 22.6 µK s1/2. 2.5. CONCERTO hardware outside the C-cabin Since the beam will bounce between the ﬂoor and the top of the cabin (M4), we mea- sured the deformations of the C-cabin itself under typical APEX observing conditions. This measurement was achieved using two linear wire sensors with a range of 3 m and a single measure- ment precision of 0.1 mm. As is shown in Fig. 11, the root mean square (RMS) of both sensors, over 48 hours of data taken during All of the pipes and cables that are needed to interconnect the elements of CONCERTO situated in diﬀerent rooms (C-cabin, instrumentation, and compressors containers) are routed through ﬂexible hoses. The hoses protect the CONCERTO cabling from A60, page 6 of 13 The CONCERTO collaboration: A wide ﬁeld-of-view low-resolution spectrometer at APEX Fig. 11. Deformations of a wire sensor running between the ﬂoor and the top of the APEX Cassegrain cabin recorded for 48 h. The average absolute length of the wire is 2675.3 mm. Inset: picture of the wire sen- sor. standard APEX observations, is smaller than 0.2 mm and thus LO 1 GHz bandwidth Fig. 12. Frequency sweep (transmission of the feed-line between port 1 and 2, i.e. mod(S21)) of ﬁve blocks of resonances out of the base- line HF array. They have been acquired under dark conditions, in the CONCERTO cryostat, at T = 70 mK. Number of resonators 10 20 30 LO 1 GHz bandwidth Fig. 12. Frequency sweep (transmission of the feed-line between port 1 and 2, i.e. mod(S21)) of ﬁve blocks of resonances out of the base- line HF array. They have been acquired under dark conditions, in the CONCERTO cryostat, at T = 70 mK. 1·104 2·104 3·104 Coupling factor - QC Number of resonators 10 20 30 Fig. 13. Quality factors distribution for one representative block of resonances in the CONCERTO HF array. For this particular block Qc = 23k ± 12k, which is in line with all of the other blocks and with the designed Qc = 25k. These quality factors were measured in the CON- CERTO cryostat at T = 70 mK. Fig. 11. Deformations of a wire sensor running between the ﬂoor and the top of the APEX Cassegrain cabin recorded for 48 h. The average absolute length of the wire is 2675.3 mm. Inset: picture of the wire sen- sor. standard APEX observations, is smaller than 0.2 mm and thus negligible for our purposes. 2.5. CONCERTO hardware outside the C-cabin The alignment of the mirrors in the optics box will be achieved in laboratory. The position of each mirror is adjusted with three micro-metric screws. No tuning will be possible at the telescope. The alignment of the optics box with respect to the chassis is ensured by the mechanical ﬁxations. A set of spe- ciﬁc lasers will be mounted to achieve the internal alignments in laboratory. At the telescope, we will use these lasers to align the optics box with respect to M4, M4 with respect to M3, and M3 with respect to M2. The alignment procedures will represent a critical step in the installation. LO 1 GHz bandwidth Fig. 12. Frequency sweep (transmission of the feed-line between port 1 and 2, i.e. mod(S21)) of ﬁve blocks of resonances out of the base- line HF array. They have been acquired under dark conditions, in the CONCERTO cryostat, at T = 70 mK. 1·104 2·104 3·104 Coupling factor - QC Number of resonators 10 20 30 Fig. 13. Quality factors distribution for one representative block of resonances in the CONCERTO HF array. For this particular block Qc = 23k ± 12k, which is in line with all of the other blocks and with the designed Qc = 25k. These quality factors were measured in the CON- CERTO cryostat at T = 70 mK. is achieved without requiring complicated and risky additional technology steps, such as cross-the-line micro-bondings or sus- LO 1 GHz bandwidth Fig. 12. Frequency sweep (transmission of the feed-line between port 1 and 2, i.e. mod(S21)) of ﬁve blocks of resonances out of the base- line HF array. They have been acquired under dark conditions, in the CONCERTO cryostat, at T = 70 mK. Fig. 11. Deformations of a wire sensor running between the ﬂoor and the top of the APEX Cassegrain cabin recorded for 48 h. The average absolute length of the wire is 2675.3 mm. Inset: picture of the wire sen- sor. Fig. 12. Frequency sweep (transmission of the feed-line between port 1 and 2, i.e. mod(S21)) of ﬁve blocks of resonances out of the base- line HF array. They have been acquired under dark conditions, in the CONCERTO cryostat, at T = 70 mK. Fig. 12. Frequency sweep (transmission of the feed-line between port 1 and 2, i.e. mod(S21)) of ﬁve blocks of resonances out of the base- line HF array. 3. Detectors laboratory characterisation A deeper geometrical characterisation of the thousands of beams is beyond the scope of this paper and will only be performed on the ﬁnal arrays. Another important electrical parameter to be studied for large arrays of LEKID is the coupling quality factor Qc. The micro- strip conﬁguration that has been chosen has the advantage of guaranteeing a relatively well-peaked distribution of Qc. This A60, page 7 of 13 A&A 642, A60 (2020) A60 (2020) TIME SINCE SCAN START (s) RESONANCE FREQUENCY SHIFT (kHz) 75 150 225 300 375 0 -1 -2 -3 Fig. 15. Sky simulator trace. A fake “planet” (point-like source) is cross- ing the ﬁeld-of-view of the considered pixel. A raster scan with sub- scans at a ﬁxed elevation is simulated. The “elevation” steps are 4 mm long each. This measurement was obtained using the NIKA1.5 cryostat at T = 120 mK and under a background temperature around 50 K. Fig. 14. Spectral response of two CONCERTO HF arrays (solid lines) and one NIKA2 260 GHz detector (dashed line). Red: HR Silicon sub- strate thickness of 110 ± 5 µm; blue: thickness of 100 ± 5 µm. Spectral responses were measured in the NIKA1.5 cryostat with low-pass ﬁlters deﬁning an open band up to 300 GHz. TIME SINCE SCAN START (s) RESONANCE FREQUENCY SHIFT (kHz) 75 150 225 300 375 0 -1 -2 -3 Fig. 15. Sky simulator trace. A fake “planet” (point-like source) is cross- ing the ﬁeld-of-view of the considered pixel. A raster scan with sub- scans at a ﬁxed elevation is simulated. The “elevation” steps are 4 mm long each. This measurement was obtained using the NIKA1.5 cryostat at T = 120 mK and under a background temperature around 50 K Fig. 14. Spectral response of two CONCERTO HF arrays (solid lines) and one NIKA2 260 GHz detector (dashed line). Red: HR Silicon sub- strate thickness of 110 ± 5 µm; blue: thickness of 100 ± 5 µm. Spectral responses were measured in the NIKA1.5 cryostat with low-pass ﬁlters deﬁning an open band up to 300 GHz. TIME SINCE SCAN START (s) RESONANCE FREQUENCY SHIFT (kHz) 75 150 225 300 375 0 -1 -2 -3 RESONANCE FREQUENCY SHIFT (kH Fig. 14. Spectral response of two CONCERTO HF arrays (solid lines) and one NIKA2 260 GHz detector (dashed line). 4.1. CONCERTO as an photometer: Dual-band sensitivity where 27.5 and 11 m are the IRAM and APEX telescopes eﬀec- tive sizes, respectively, that is to say the portion of the primary mirrors that are optically conjugated to the cold pupils of the instruments (aperture stops). We ﬁrst computed the sensitivity for CONCERTO as if it was a dual-band imager (LF and HF). For that, we relied on NIKA2 sensitivity measurements on the IRAM 30-m telescope. Aver- age NEFDs for NIKA2 (NEFDNIKA2) are equal to 9.8 and 36.1 mJy s1/2, at 150 and 260 GHz, respectively, for pwv = 2 and an elevation of 60 degrees (Perotto et al. 2020). These numbers already suﬀer from the transmission of the whole experiment. Then we assumed a frequency window of ∆ν = 115 GHz, making the assumption that the two arrays cover the frequency range of 195–310 GHz (HF) and 130–270 GHz (LF), with a notch ﬁlter removing 25 GHz of the low-frequency bandpass (around 183 GHz). Finally, we also had to take the decrease in transmission into account due to the increased optics complexity of CONCERTO compared to NIKA2 (in particular FTS optics will have some transmission loss and additional loading), which we estimate to be T = 0.8 for an unpolarised source. It is impor- tant to note that this is only an additional loss of transmission compared to NIKA2+P1 and not the overall transmission. y p We observe a large diﬀerence between the NIKA2 260 and 150 GHz channel performances. A combination of known eﬀects explains the gap in sensitivity. Indeed, at 260 GHz, (i) the beam eﬃciency of the 30-m telescope is about 55%, (ii) the sensitivity is strongly aﬀected (by 35%) by a known defect of the NIKA2 dichroïc, and (iii) the contribution of residual sky noise to the average NEFDs is important. Therefore, as a realistic starting point for CONCERTO, we assume that the sensitivity for the LF array equals that of NIKA2 at 150 GHz. For the HF array, we assume a sensitivity better than that of the NIKA2 260 GHz channel, thanks to a gain in beam eﬃciency at APEX (which is of the order of 80% at the CONCERTO wavelengths), the lack of dichroïc in CONCERTO, and better atmospheric conditions. 3. Detectors laboratory characterisation Red: HR Silicon sub- strate thickness of 110 ± 5 µm; blue: thickness of 100 ± 5 µm. Spectral responses were measured in the NIKA1.5 cryostat with low-pass ﬁlters deﬁning an open band up to 300 GHz. Fig. 15. Sky simulator trace. A fake “planet” (point-like source) is cross- ing the ﬁeld-of-view of the considered pixel. A raster scan with sub- scans at a ﬁxed elevation is simulated. The “elevation” steps are 4 mm long each. This measurement was obtained using the NIKA1.5 cryostat at T = 120 mK and under a background temperature around 50 K. The typical response time of the LEKID used for CON- CERTO ranges between 30 µs and 100 µs, depending on the background. Even at the chosen sampling rate of 4 kHz, a cosmic-ray hit will thus represent a single-point glitch in the CONCERTO raw-time traces. The order of magnitude of the expected rate is 0.1 Hz per pixel. NEFDHF NIKA2 = 15 [10−20] mJy s1/2. (2) (2) The numbers in brackets give the uncertainties on our assump- tion. As it was the case for NIKA2, we make the hypothesis that no excess noise will appear in CONCERTO at APEX compared to CONCERTO in laboratory. Of course, this cannot be veriﬁed until installation. The numbers in brackets give the uncertainties on our assump- tion. As it was the case for NIKA2, we make the hypothesis that no excess noise will appear in CONCERTO at APEX compared to CONCERTO in laboratory. Of course, this cannot be veriﬁed until installation. 4. Sensitivity estimates Compared to NIKA2, for CONCERTO, we have to scale the sensitivities to match the APEX telescope size and add two polarisers in the optical path (P1 and P2, see Fig. 4). Sensitivity loss is only due to P1, by a factor between √ 2 (if photon noise dominates) and 2. To be conservative, we considered a factor of 2. Thus, for a single array of CONCERTO, the NEFD becomes: Due to the similarities between the NIKA2 and CONCERTO detectors, we used the NIKA2 sensitivity measured on sky and on the reduced maps as a base to estimate the sensitivity for CONCERTO. As already advocated, we think that this approach, coupled with our NET laboratory measurements, provides quite realistic predictions. NEFDLF,HF = NEFDLF,HF NIKA2 × 2 × 27.5 11 !2 (3) (3) 9 A voxel represents a value on a regular grid in three-dimensional space. 4.2. Sensitivity in spectroscopy For the spectroscopic mode, we consider a ﬁx value for spectral resolution of δν = 1.5 GHz. The number of spectral elements in the frequency range is Nse = ∆ν/δν. The sensitivity per spectral element (in mJy s1/2) for a single spectrometer (we note that in our case, with an FTS, the number of pixel equals the number of spectrometers) is given by (9) where FOV is the ﬁeld of view area (with a diameter of 20 arcmin). The numbers are given in Table 2. We note that we ignored the frequency overlap between the two arrays (and thus a gain of ∼ √ 2 on the sensitivity in the frequency overlap region). We considered NEFDLF DB for ν ≤150 GHz, NEFDHF DB for ν ≥260 GHz, and a linear interpolation between the two NEFDs for 150 < ν < 260 GHz. where FOV is the ﬁeld of view area (with a diameter of 20 arcmin). The numbers are given in Table 2. We note that we ignored the frequency overlap between the two arrays (and 4.1. CONCERTO as an photometer: Dual-band sensitivity σarray = NEIFTS/ p NKIDS, (10) (10) where NKIDS is the number of pixels (KIDS) of each array; we use 1720 KIDS, which correspond to 80% of valid KIDS in each array. This would be the sensitivity of each voxel9 of large maps, assuming a RA-Dec (or AZ-EL) raster scan-like scanning strat- egy and also assuming that pixel sizes of the map are equal to beam sizes. Each voxel of the observed map would then be observed by each KIDS. We checked these numbers using a scanning strategy similar to NIKA2 raster scans, with three inter- ferograms per beam. 5. Low spectral-resolution spectroscopic surveys Thus we expect CONCERTO to bring a signiﬁcant contribution in a number of areas, including the study of galaxy clusters (via the thermal and kinetic SZ eﬀect), the follow-up of cosmological deep surveys, the observation of local and intermediate-redshift galaxies, and the study of Galac- tic star-forming clouds. In this section we provide a forecast on the expected signal-to-noise ratio (S/N) that can be obtained on the [CII]-emission power spectrum (Sect. 5.1). In addition, we give some predictions for observing the SZ signal of galaxy clus- ters (Sect. 5.2). with a FWHM that is determined by the Rayleigh criterion for a D = 11 m antenna (our illumination of the APEX 12 m antenna) at a given frequency, (which corresponds to a given redshift for the [CII] line), θbeam = 1.22λobs/D. (7) (7) θbeam = 1.22λobs/D. θbeam = 1.22λobs/D. We can then convert the sensitivity per spectral element from point-source (Eq. (5)) to diﬀuse emission (in MJy sr−1 s1/2) following NEIFTS = NEFDFTS × 10−9/Ωbeam. (8) (8) This is the noise equivalent intensity, on sky, per KIDS, per spec- tral bin (taken as δν = 1.5 GHz). We can ﬁnally compute the mapping speed MS (per spectral element) following, 4.1. CONCERTO as an photometer: Dual-band sensitivity The values are thus (for pwv=2 and an elevation of 60 degrees): p The sensitivity of CONCERTO as a dual-band photometer that is set when the optical path diﬀerence in the FTS is null is thus: NEFDLF,HF phot = NEFDLF,HF × s ∆νLF,HF NIKA2 ∆ν × 1 √ T . (4) NEFDLF,HF phot = NEFDLF,HF × s ∆νLF,HF NIKA2 ∆ν × 1 √ T . (4) (4) We have ∆νHF NIKA2 = 48 GHz and ∆νLH NIKA2 = 39.2 GHz (Perotto et al. 2020) and thus: NEFDLF NIKA2 = 10 [7.5−15] mJy s1/2 and (1) A60, page 8 of 13 he CONCERTO collaboration: A wide ﬁeld-of-view low-resolution spectrometer at APEX The CONCERTO collaboration: A wide ﬁeld-of-view low-resolution spectrometer at APEX p Table 2. Key parameters for CONCERTO instrument. ν [GHz] 131 156 211 238 272 302 Redshift of the [CII] line 13.5 11.2 8.0 7.0 6.0 5.3 Beam size [arcsec] 52.4 44.0 32.5 28.8 25.2 22.7 Beam solid angle [×10−8 sr] 7.30 5.15 2.81 2.21 1.70 1.37 Mapping speed [×10−3 deg2/(MJy sr−1)2 h−1] 42.8 [19.0–76.0] 19.8 [9.0–35.9] 3.4 [1.8–7.1] 1.7 [0.9–3.7] 0.8 [0.5–1.9] 0.5 [0.3–1.2] On sky map sensitivity σarray 2.1 [1.5–3.1] 3.0 [2.3–4.5] 7.3 [5.1–10.2] 10.4 [7.1–14.1] 14.8 [9.9–19.7] 18.2 [12.2–24.3] [(MJy sr−1) s1/2] Notes. Sensitivities and mapping speeds are given for one spectral element (with δν = 1.5 GHz), assuming 80% of valid KIDS, a precipitable water vapour of 2 mm, and an elevation of 60 degrees. Table 2. Key parameters for CONCERTO instrument. Notes. Sensitivities and mapping speeds are given for one spectral element (with δν = 1.5 GHz), assuming 80% of valid KIDS, a precipitable water vapour of 2 mm, and an elevation of 60 degrees. In Table 2, we also provide the sensitivity for the whole array, per spectral element, which is: NEFDLF phot = 81.6 [61.2–122.4] mJy s1/2 and NEFDHF phot = 135.4 [90.3–180.6] mJy s1/2. NEFDLF phot = 81.6 [61.2–122.4] mJy s1/2 and NEFDHF phot = 135.4 [90.3–180.6] mJy s1/2. We note that due to the FTS in front of the cryostat, CON- CERTO is a non-optimal instrument for imaging. However, the option of removing the ﬁrst polariser for purely photomet- ric campaigns could be studied. In that case, the sensitivity is expected to be two times better for each individual array (as we took a factor 2 of penalty for P1). 5. Low spectral-resolution spectroscopic surveys CONCERTO will oﬀer generic access to a large FoV and a low- frequency resolution spectroscopic instrument. This opening of 3D large-scale surveys is the next step after the broad-band pho- tometric experiments, either from the ground (e.g. LABOCA, SCUBA2, NIKA2) or from space (e.g. Herschel and Planck). NEFDFTS = NEFDphot × Nse. (5) (5) NEFDFTS = NEFDphot × Nse. The beam area is computed assuming a Gaussian beam, Ωbeam = 2π  θbeam 2 p 2 log 2  2 , (6 SCUBA2, NIKA2) or from space (e.g. Herschel and Planck). The ﬁrst scientiﬁc aim of CONCERTO is to map, in three dimen- sions, the ﬂuctuations of the [CII] line intensity in the reion- isation and post-reionisation epoch (z ≥5.3). This technique, known as intensity mapping, will measure the clustering of [CII] emissivity and allow for questions to be answered on how and when galaxies and quasars formed and on the history and topol- ogy of reionisation. Even if [CII] intensity mapping has been the basis of instrument deﬁnition, we extended the instrument capa- bilities to make CONCERTO a multi-purpose instrument (e.g. extending the frequency range down to 130 GHz for observa- tions of galaxies clusters). Thus we expect CONCERTO to bring a signiﬁcant contribution in a number of areas, including the study of galaxy clusters (via the thermal and kinetic SZ eﬀect), the follow-up of cosmological deep surveys, the observation of local and intermediate-redshift galaxies, and the study of Galac- tic star-forming clouds. In this section we provide a forecast on the expected signal-to-noise ratio (S/N) that can be obtained on the [CII]-emission power spectrum (Sect. 5.1). In addition, we give some predictions for observing the SZ signal of galaxy clus- ters (Sect. 5.2). (6) The ﬁrst scientiﬁc aim of CONCERTO is to map, in three dimen- sions, the ﬂuctuations of the [CII] line intensity in the reion- isation and post-reionisation epoch (z ≥5.3). This technique, known as intensity mapping, will measure the clustering of [CII] emissivity and allow for questions to be answered on how and when galaxies and quasars formed and on the history and topol- ogy of reionisation. Even if [CII] intensity mapping has been the basis of instrument deﬁnition, we extended the instrument capa- bilities to make CONCERTO a multi-purpose instrument (e.g. extending the frequency range down to 130 GHz for observa- tions of galaxies clusters). MS = FOV/NEI2 FTS, (9) MS = FOV/NEI2 FTS, (9) 5.1. [CII] intensity mapping with CONCERTO (2012) to link the intensity of emission lines to the galaxy infrared luminosity, they computed 3D emission line power spectra for all relevant lines, including [CII]. They computed the expected S/N of cross-power spectra between [CII] and other emission lines, which will constrain the mean amplitude of each signal and thereby help to gain insight into the mean properties of the ISM of high-z galaxies. It is important to note that in their paper, they use for CONCERTO a constant sensitivity for all redshifts of σarray = 155 mJy s1/2, while we have here σarray = [156, 206, 230, 250, 250] mJy s1/2 at z = [11.2, 8.0, 7.0, 6.0, 5.3]. We consider measurements spanning a redshift range of ∆z ∼0.6, which corresponds to a frequency range of Bν ∼ 20 GHz at z = 6.1 for the [CII] line. We note that Gong et al. (2012) give useful relations for computing Vs and Vvoxel for the [CII] line. Table 3 gives the numbers derived from the above compu- tations and Fig. 16 shows the predicted [CII] power spectrum with its error bars. Because these types of predictions are very uncertain, we assumed two extreme models, giving respectively low and high SFRD at z > 3 (see Fig. 3 of Lagache et al. 2018). Low SFRD is the pessimistic prediction as it corresponds to the lowest UV-driven SFRD; high SFRD is the optimistic prediction as it corresponds to the CIB-driven SFRD derived from the halo modelling of Planck CIB measurements (Planck Collaboration XXX 2014). emission lines to the galaxy infrared luminosity, they computed 3D emission line power spectra for all relevant lines, including [CII]. They computed the expected S/N of cross-power spectra between [CII] and other emission lines, which will constrain the mean amplitude of each signal and thereby help to gain insight into the mean properties of the ISM of high-z galaxies. It is important to note that in their paper, they use for CONCERTO a constant sensitivity for all redshifts of σarray = 155 mJy s1/2, while we have here σarray = [156, 206, 230, 250, 250] mJy s1/2 at z = [11.2, 8.0, 7.0, 6.0, 5.3]. 5.1. [CII] intensity mapping with CONCERTO 2019), providing an unbiased view of the dis- tribution of the gas that is diﬃcult to assemble from targeted measurements of individual galaxies, and probing cosmologi- cal volumes, with maps on several-degree scale and large fre- quency (and thus redshift) coverage. Intensity mapping exploits the confusion-limited regime and measures the integrated light emission from all sources, including unresolved faint galaxies. (14) where ∆k is the Fourier bin size and Vs(z) is the survey volume, expressed as Vs(z) = χ(z)2yCIIBνA, (15) Vs(z) = χ(z)2yCIIBνA, (15) with Vs(z) = χ(z)2yCIIBνA, (15) with g g We will conduct with CONCERTO a major survey of about one square degree with 1200 h of APEX telescope time. The sur- vey will provide a data cube in which intensity is mapped as a function of sky position and redshift. Our main target is the [CII] line emission at z ≥5.3. But CONCERTO will also observe the CO intensity ﬂuctuations arising from 0.3 < z < 2 galaxies, giv- ing the spatial distribution and abundance of molecular gas over a broad range of cosmic time. The 3D ﬂuctuations will be stud- ied in Fourier space with the power spectrum. yCII(z) = λCII(1 + z)2/H(z), (16) (16) being the factor to convert the frequency intervals to the comov- ing distance at the wavelength λCII (rest frame wavelength of the [CII] line). In Eq. (15), Bν is the bandwidth considered for the measurement. The volume surveyed by each voxel (Vvoxel in Eq. (13)) is being the factor to convert the frequency intervals to the comov- ing distance at the wavelength λCII (rest frame wavelength of the [CII] line). In Eq. (15), Bν is the bandwidth considered for the measurement. The volume surveyed by each voxel (Vvoxel in Eq. (13)) is (17) Vvoxel = χ(z)2yCII(z)Ωbeamδν. (17) Vvoxel = χ(z)2yCII(z)Ωbeamδν. To compute the expected S/N on the [CII] power spectrum at high z, we used the [CII] model presented in Serra et al. (2016). Using measurements of the cosmic infrared background (CIB) angular power spectra from Herschel/SPIRE together with star formation rate density (SFRD) measurements, they constrain the galaxy FIR luminosity as a function of dark-matter halo mass at all relevant redshifts in the halo model framework. By using scal- ing relations from Spinoglio et al. 5.1. [CII] intensity mapping with CONCERTO The [CII] line is one of the brightest emission lines in the spec- tra of galaxies. It is an excellent coolant for neutral gas in thus a gain of ∼ √ 2 on the sensitivity in the frequency overlap region). We considered NEFDLF DB for ν ≤150 GHz, NEFDHF DB for ν ≥260 GHz, and a linear interpolation between the two NEFDs for 150 < ν < 260 GHz. A60, page 9 of 13 A&A 642, A60 (2020) Assuming a spherically averaged power spectrum measure- ment and a directionally independent on sky sensitivity, the vari- ance of the power spectrum is: photo-dominated regions and an extinction-free tracer of star for- mation at high z. Being redshifted into the sub-millimetre and millimetre atmospheric windows for z > 4.5, it has become one of the most popular lines at high z. Pointing on known objects, with, for example, ALMA, NOEMA, and APEX/FLASH, [CII] is now detected in a large number of galaxies at high z (>150 at z > 4.5, with a large contribution from the ALMA ALPINE survey, e.g. Bethermin et al. 2020). These types of observations are a tremendous step forward but we also need to look at the overall population, that is, observe large volume and unbiased surveys. First observations with ground-based interferometers, for example, with ALMA (‘ALMA Spectroscopic Survey in the Hubble Ultra Deep Field’ large programme – ASPECS, Walter et al. 2016) or JVLA (CO Luminosity Density at High Redshift survey – COLDz, Riechers et al. 2019) oﬀer a three-dimensional view of the molecular gas content of galaxies. The covered areas are about 5–60 square arcmin. var[ ¯PCII(k)] = [PCII(k) + ¯PN CII(k)]2 Nm(k, z) , (12) (12) where Nm(k, z) is the number of modes that leads to the power spectrum measurement at each k and ¯PN CII(k) = Vvoxel σ2 voxel tvoxel , (13) (13) with Vvoxel the volume surveyed by each voxel. In the case of CONCERTO, each KIDS gets a spectrum, and considering one KID per beam, we have σvoxel = σarray. p , voxel array The number of modes at each k is given by p y The number of modes at each k is given by Nm(k, z) = 2π k2∆k Vs(z) (2π)3 , (14) q Intensity mapping complements these eﬀorts beautifully (e.g Kovetz et al. 5.2. Observing galaxy clusters with CONCERTO 2014, 2015, 2016, 2017a; Ruppin et al. 2017, 2018). For the kSZ eﬀect, which is also observed with, for example, NIKA2 (Adam et al. 2017b), a multi-wavelength spec- trometer such as CONCERTO would be a unique tool to separate the tSZ and kSZ as well as the diﬀerent foreground components (CIB, CMB, and the Galactic emission) and to extract informa- tion about the cluster physics. Indeed, with suﬃciently precise spectroscopy measurements (Birkinshaw 1999), we can measure the cluster mass (from the tSZ eﬀect), proper motion along the line-of-sight (from the kSZ), and temperature (from the relativis- tic corrections to the tSZ). or observed with the ALMA ALPINE survey (Schaerer et al. 2020). Our power spectra are thus very likely to be underes- timated. In terms of point source sensitivities, our survey will reach 1σ = [14.4, 14.9, 19.7, 22.0, 23.9, 23.9] mJy, for a spectral element δν = 1.5 GHz, at ν = [131.0, 156.0, 211.0, 238.0, 272.0, 302] GHz, respectively. Finally, one major diﬃculty in inten- sity mapping surveys is the problem of foregrounds. For [CII], the main foregrounds will be the contamination from emission lines from lower redshifts, in particular emission from CO rota- tion transitions (e.g. Yue et al. 2015). Silva et al. (2015) and Breysse et al. (2015) show that this contamination can be par- tially removed by masking out the brightest pixels in the sur- vey or the low-redshift galaxies selected from other surveys. To that end, CONCERTO will highly beneﬁt from the exten- sive visible-IR photometry and spectroscopy galaxy survey data that are available in the chosen ﬁeld (i.e. the COSMOS ﬁeld). In addition, one of the strengths of CONCERTO is in its wide frequency range: Several CO lines are simultaneously observed at the same redshift for all redshifts of z > 0.35. The cross- correlation between these lines will be a very powerful method Particularly, considering the angular resolution and the map- ping speed of CONCERTO, we expect to perform a precise esti- mate of the shape of the SZ spectrum for clusters of galaxies for redshifts between 0.2 and 0.8. As an illustration, we present in Fig. 17 a simulation of a typical cluster that could be observed with CONCERTO. In this simulation, the mass of the cluster is equal to 1015 M⊙and the cluster is located at a redshift of z = 0.4. 5.2. Observing galaxy clusters with CONCERTO Clusters of galaxies are the largest gravitationally bound objects in the Universe and, as such, they are key to understanding the hierarchical large scale structure (Kravtsov & Borgani 2012). The study of galaxy clusters and, in particular, their num- ber as a function of mass and redshift allows us to constrain cosmological parameters (Allen et al. 2011). In the frequency range covered by CONCERTO, a cluster of galaxies will be mainly detected via the thermal and kinetic Sunyaev-Zel’dovitch (SZ) eﬀect (Sunyaev & Zel’dovich 1972; Sunyaev & Zeldovich 1980). The thermal SZ (tSZ) eﬀect (Sunyaev & Zel’dovich 1972) refers to the interaction of the hot electrons in clusters with the CMB photons. It results in a distortion in the CMB spectrum at the position of the cluster. In the case of hot clusters, it will also be aﬀected by relativistic corrections named rSZ (see Itoh & Nozawa 2004, for details on their computation). The kinetic SZ (kSZ) (Sunyaev & Zeldovich 1980) is a Doppler shift of the CMB photons induced by the proper motion of clusters of galax- ies along the line-of-sight. Fig. 16. Predicted [CII] power spectrum at z = 6. Three cases are shown, corresponding to three scenarios of SFRD at high z (see Lagache et al. 2018): high SFRD (dark blue), low SFRD (light blue), and the geo- metrical mean of the two (red). Only points with S/N > 1 are shown. S/Ns were computed considering A = 1.4 square degrees, tsurvey = 840 h, which corresponds to a total observation time of 1200 hours taking the overheads into account, and sensitivities as estimated in Sect. 4. The [CII] power spectra were derived from the modelling of CIB power spectra (Serra et al. 2016), using a conversion from SFR to [CII] that conservatively underestimates the [CII] luminosity by a factor of 6 at z = 5 compared to recent semi-analytical models (e.g. Lagache et al. 2018) or ALMA ALPINE measurements (Schaerer et al. 2020). Our estimates of [CII] power spectra are thus likely to be underestimated. The predicted [CII] power spectra from Yue & Ferrara 2019, using the local SFR-[CII] relation (black line) and Chung et al. 2020 (dashed black line) are also shown. Observations of the tSZ eﬀect have been successfully per- formed at high angular resolution using continuum cameras based on KIDs, such as NIKA and NIKA2 at the IRAM 30-m telescope (Adam et al. 5.1. [CII] intensity mapping with CONCERTO Redshift z 5.5 6.2 7 8 S/N PCII mean SFRD 23 [14–44] 12 [6.9–24] 5.7 [3.1–12] 2.0 [1.0–4.1] S/N PCII low SFRD 4.5 [2.6–9.8] 1.9 [1.1–4.3] 0.78 [0.48–1.7] 0.23 [0.12–0.48] S/N PCII high SFRD 79 [57–112] 55 [36–87] 34 [21–60] 16 [8.5–29] Notes. PCII was computed for ∆z = 0.6 and given for k = [0.1, 1] h Mpc−1. See the caption of Fig. 16 for more details. Numbers in brackets reﬂect the range of sensitivities (as given in Table 2). Table 3. S/N on the [CII] power spectrum. Notes. PCII was computed for ∆z = 0.6 and given for k = [0.1, 1] h Mpc−1. See the caption of Fig. 16 for more details. Numbers in brackets reﬂect the range of sensitivities (as given in Table 2). Fig. 16. Predicted [CII] power spectrum at z = 6. Three cases are shown, corresponding to three scenarios of SFRD at high z (see Lagache et al. 2018): high SFRD (dark blue), low SFRD (light blue), and the geo- metrical mean of the two (red). Only points with S/N > 1 are shown. S/Ns were computed considering A = 1.4 square degrees, tsurvey = 840 h, which corresponds to a total observation time of 1200 hours taking the overheads into account, and sensitivities as estimated in Sect. 4. The [CII] power spectra were derived from the modelling of CIB power spectra (Serra et al. 2016), using a conversion from SFR to [CII] that conservatively underestimates the [CII] luminosity by a factor of 6 at z = 5 compared to recent semi-analytical models (e.g. Lagache et al. 2018) or ALMA ALPINE measurements (Schaerer et al. 2020). Our estimates of [CII] power spectra are thus likely to be underestimated. The predicted [CII] power spectra from Yue & Ferrara 2019, using the local SFR-[CII] relation (black line) and Chung et al. 2020 (dashed black line) are also shown. to remove the contamination. This will be speciﬁcally addressed for CONCERTO in a future paper. 5.1. [CII] intensity mapping with CONCERTO Covering an area of 1.4 square degrees, our survey will pro- vide the ﬁrst measurements of the [CII] power spectrum up to z ∼7, considering the mean SFRD and average sensitivity esti- mate, and up to z ∼8 in the best case for the sensitivity esti- mate. We note that the low SFRD case is unlikely as it gives low shot-noise levels for the CIB, which are not compatible with the Planck and Herschel measurements nor with current SFRD measurements at high-z based on [CII] or far-infrared measure- ments. Moreover, on top of the exact level of the SFRD at high z, the relation used to convert SFR to [CII] luminosity is another capital ingredient of this type of model. Serra et al. (2016) used the relation from Spinoglio et al. (2012), which provides, for a given SFR, a [CII] luminosity that is six times lower than that obtained with the [CII]-SFR relations of Lagache et al. (2018) We followed Gong et al. (2012) to compute uncertainties on the power spectra. The observing time per map voxel, consider- ing one pixel equals one beam, is given by tvoxel = tsurvey ΩbeamNKIDS A , (11) tvoxel = tsurvey ΩbeamNKIDS A , (11) with A the survey area, tsurvey the on-sky survey time (i.e. 1200 × 0.7 = 840 h in our case, considering 30% of overheads), Ωbeam the solid angle of the beam (Table 2), and NKIDS, the number of pixels (we consider 80% of valid KIDS, thus NKIDS = 1720). A60, page 10 of 13 The CONCERTO collaboration: A wide ﬁeld-of-view low-resolution spectrometer at APEX Table 3. S/N on the [CII] power spectrum. Redshift z 5.5 6.2 7 8 S/N PCII mean SFRD 23 [14–44] 12 [6.9–24] 5.7 [3.1–12] 2.0 [1.0–4.1] S/N PCII low SFRD 4.5 [2.6–9.8] 1.9 [1.1–4.3] 0.78 [0.48–1.7] 0.23 [0.12–0.48] S/N PCII high SFRD 79 [57–112] 55 [36–87] 34 [21–60] 16 [8.5–29] Notes. PCII was computed for ∆z = 0.6 and given for k = [0.1, 1] h Mpc−1. See the caption of Fig. 16 for more details. Numbers in brackets reﬂect the range of sensitivities (as given in Table 2). Table 3. S/N on the [CII] power spectrum. 5.2. Observing galaxy clusters with CONCERTO We assumed a universal pressure proﬁle model from Arnaud et al. (2010) to compute the cluster’s Compton param- eter map. From left to right, we present the cluster’s SED as expected to be measured by CONCERTO at diﬀerent radial dis- tances from the centre of the cluster. We considered six bands in frequency with typical bandwidths of 10–25 GHz. We explored both the sensitivity to the relativistic SZ eﬀect (blue points), assuming a cluster temperature of 20 keV, and to the kinetic SZ (red points), assuming the cluster is moving towards the observer A60, page 11 of 13 A&A 642, A60 (2020) Fig. 17. Thermal (green), kinetic (orange), and relativistic (light blue) SZ eﬀect for a simulated cluster at redshift z = 0.4, with a mass of 1015 M⊙ and a temperature of 20 keV, which is moving at 1000 km h−1 towards the observer. From left to right, we present the measured CONCERTO SEDs for tSZ+kSZ and rSZ+tSZ, including uncertainties (red and blue dots, respectively) for diﬀerent radial bins with respect to the centre of the cluster (θ in arcmin) up to the cluster’s characteristic radius, θ500, which is the radial angular distance at which the mean cluster density is 500 times the critical density at the cluster redshift. Fig. 17. Thermal (green), kinetic (orange), and relativistic (light blue) SZ eﬀect for a simulated cluster at redshift z = 0.4, with a mass of 1015 M⊙ and a temperature of 20 keV, which is moving at 1000 km h−1 towards the observer. From left to right, we present the measured CONCERTO SEDs for tSZ+kSZ and rSZ+tSZ, including uncertainties (red and blue dots, respectively) for diﬀerent radial bins with respect to the centre of the cluster (θ in arcmin) up to the cluster’s characteristic radius, θ500, which is the radial angular distance at which the mean cluster density is 500 times the critical density at the cluster redshift. CONCERTO is currently in an advanced stage of fabrica- tion. The installation and technical commissioning at the APEX telescope is scheduled for the ﬁrst semester of 2021. The com- missioning, scientiﬁc veriﬁcation, and observations are foreseen by the end of 2022. CONCERTO is currently in an advanced stage of fabrica- tion. The installation and technical commissioning at the APEX telescope is scheduled for the ﬁrst semester of 2021. The com- missioning, scientiﬁc veriﬁcation, and observations are foreseen by the end of 2022. 5.2. Observing galaxy clusters with CONCERTO with a velocity of 1000 km h−1. Uncertainties were computed from the sensitivity estimates given in Sect. 4, assuming a map- ping area of 310 arcmin2 and a total integration time of 30 hours. In the Fig. 17, we also show the individual tSZ, kSZ, and rSZ eﬀect contributions. We ﬁnd that for reasonable observation times (tens of hours), CONCERTO would provide ﬁrst spectral 2D mapping of the intracluster medium of high redshift clusters and should be able to measure cluster velocities via the kSZ eﬀect. CONCERTO should also be able to detect the relativistic SZ eﬀect and mea- sure the cluster temperature. A more detailed mapping of the cluster temperature would require observation times of about hundreds of hours. Acknowledgements. Besides the authors, the technicians and engineers more involved in the experimental setup development have been Maurice Grol- lier, Olivier Exshaw, Anne Gerardin, Gilles Pont, Guillaume Donnier-Valentin, Philippe Jeantet, Mathilde Heigeas, Christophe Vescovi, and Marc Marton. We acknowledge the crucial contributions of the whole Cryogenics and Electron- ics groups at Institut Néel and LPSC. The arrays described in this paper have been produced at the PTA Grenoble microfabrication facility. We warmly thank the support from the APEX staﬀfor their help in CONCERTO pre-installations and design. The ﬂexible pipes, in particular, have been routed under the compe- tent coordination of Jorge Santana and Marcelo Navarro. We acknowledge sup- port from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (project CONCERTO, grant agreement No 788212) and from the Excellence Initiative of Aix-Marseille University-A*Midex, a French “Investissements d’Avenir” programme. GL warmly thanks Matt Bradford, Jamie Bock and Tzu-Ching Chang for insight- ful discussions on CONCERTO sensitivity computation and J.-G. Cuby for his help and support for the ERC proposal. We are grateful to our administrative staﬀin Grenoble and Marseille, in particular Patricia Poirier, Mathilde Berard, Lilia Todorov and Valérie Favre, and the Protisvalor team. We acknowledge the crucial help of the Institut Néel and MCBT Heads (Etienne Bustarret, Klaus Has- selbach, Thierry Fournier, Laurence Magaud) during the COVID-19 restriction period. 6. Conclusions We have presented the design of the CONCERTO instrument, a novel spectrometer that is set to be installed on the APEX tele- scope. CONCERTO is based on the development of new arrays in the millimetre using kinetic inductance detectors. It will con- tain two arrays of 2152 KIDS, mounted in a dilution cryostat that has a base temperature of 0.1 K. Spectra are obtained by a fast Martin-Puplett interferometer located in front of the cryo- stat. Frequency resolution can be up to δν = 1 GHz. The techno- logical choices leading to the ﬁnal instrument design have been explained in detail. The characterisation of our current detectors is promising. Estimates of expected sensitivity are given, which are mostly based on the NIKA2 experience, that is, an instru- ment on sky that is subject to similar constraints as CONCERTO. The expected sensitivity, combined with the large ﬁeld-of-view (20 arcmin diameter), will provide an unprecedented mapping speed for this kind of instrument. 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https://openalex.org/W3008209398	https://europepmc.org/articles/pmc6990566?pdf=render	English	null	The effects of common structural variants on 3D chromatin structure	BMC genomics	2,020	cc-by	8,055	© The Author(s). 2020 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Shanta et al. BMC Genomics (2020) 21:95 https://doi.org/10.1186/s12864-020-6516-1 Shanta et al. BMC Genomics (2020) 21:95 https://doi.org/10.1186/s12864-020-6516-1 Open Access The effects of common structural variants on 3D chromatin structure mar Shanta1, Amina Noor2, Human Genome Structural Variation Consortium (HGSVC) and Jonat mina Noor2, Human Genome Structural Variation Consortium (HGSVC) and Jonathan Sebat2,3,4* Abstract Background: Three-dimensional spatial organization of chromosomes is defined by highly self-interacting regions 0.1–1 Mb in size termed Topological Associating Domains (TADs). Genetic factors that explain dynamic variation in TAD structure are not understood. We hypothesize that common structural variation (SV) in the human population can disrupt regulatory sequences and thereby influence TAD formation. To determine the effects of SVs on 3D chromatin organization, we performed chromosome conformation capture sequencing (Hi-C) of lymphoblastoid cell lines from 19 subjects for which SVs had been previously characterized in the 1000 genomes project. We tested the effects of common deletion polymorphisms on TAD structure by linear regression analysis of nearby quantitative chromatin interactions (contacts) within 240 kb of the deletion, and we specifically tested the hypothesis that deletions at TAD boundaries (TBs) could result in large-scale alterations in chromatin conformation. Results: Large (> 10 kb) deletions had significant effects on long-range chromatin interactions. Deletions were associated with increased contacts that span the deleted region and this effect was driven by large deletions that were not located within a TAD boundary (nonTB). Some deletions at TBs, including a 80 kb deletion of the genes CFHR1 and CFHR3, had detectable effects on chromatin contacts. However for TB deletions overall, we did not detect a pattern of effects that was consistent in magnitude or direction. Large inversions in the population had a distinguishable signature characterized by a rearrangement of contacts that span its breakpoints. Conclusions: Our study demonstrates that common SVs in the population impact long-range chromatin structure, and deletions and inversions have distinct signatures. However, the effects that we observe are subtle and variable between loci. Genome-wide analysis of chromatin conformation in large cohorts will be needed to quantify the influence of common SVs on chromatin structure. Keywords: Hi-C, Structural variation, Deletion, Inversion, TAD, TAD fusion, Chromatin little is known about patterns of topological variation in the population and the underlying genetic mechanisms. Background h 3D chromatin structure is characterized by Topologically Associated Domains (TADs) and chromatin loops, which create physical interactions between genes and distant regulatory sequences [1]. CTCF and the protein complex cohesin are localized to the boundaries of TADs [2–4], where they serve as barriers to the spread of chromatin. Genetic variation in these sequences has the potential to influence the binding of these factors and contribute to variability in chromatin structure in humans. However, Structural Variants (SVs) are a major source of genetic variability, and SVs have significant functional impact on the genome through the deletion or rearrangement of coding and regulatory sequences. Notably, large SVs that disrupt or re-establish chromatin contacts are associated with two rare monogenic disorders including human limb malformations [5–7] and female-to-male sex rever- sal [5]. Multiple recent studies have begun to examine the potential of SVs to influence chromatin conform- ation by theoretical modeling of ChIA-PET [8] or Hi-C [9] data from a single cell line (GM12878). However, these studies have not directly investigated how genetic variation between individuals contributes to variation in large-scale chromatin structure. * Correspondence: jsebat@ucsd.edu 2Beyster Center for Genomics of Psychiatric Diseases, Department of Psychiatry, UCSD, San Diego, CA, USA 3Department of Cellular and Molecular Medicine, UCSD, San Diego, CA, USA Full list of author information is available at the end of the article Page 2 of 10 Page 2 of 10 Shanta et al. BMC Genomics (2020) 21:95 Shanta et al. BMC Genomics (2020) 21:95 an example in Fig. 1; a large deletion of ~ 80 kb that disrupts the complement factor H-related genes CFHR3 and CFHR1. This deletion has been associated with decreased risk of age-related macular degeneration (AMD), an increased risk of atypical hemolytic uremic syndrome (aHUS), and systemic lupus erythematosus (SLE) [12–15]. A map of chromatin contacts for the de- leted region and two adjacent TADs (spanning 1.24 Mb) is illustrated in Fig. 1 at a 40 kb resolution. The average number of contacts is shown for subjects who were homozygous for the deletion (Fig. 1 a) and for subjects who were homozygous for the reference allele (Fig. 1 b). As expected, the deletion results in loss of contacts in bins that overlap with the deleted region, and as adjacent regions are brought closer together, we observe an in- crease in contacts that span the deletion. In this study, we investigated the effect of common SV polymorphism on 3D chromatin structure in a sample of individuals from the 1000 genomes project [10]. Specif- ically we sought to test the hypothesis that deletions of the boundary regions between adjacent TADs could re- sult in large scale alterations in chromatin conformation. We performed Chromatin Conformation Capture (Hi-C) sequencing of lymphoblastoid cell lines (LCLs) of 19 in- dividuals from the 1000 genomes project, and we tested the effects of common SVs on the numbers of nearby chromatin contacts. Results We hypothesize that SVs could influence TAD structure indirectly by disrupting regulatory sequences that con- trol formation of TADs in adjacent genomic regions. In addition, we anticipate that SVs will have direct effects on the coverage and spacing of paired-end reads similar to the effects that are ordinarily observed for SVs in whole genome sequence data [11]. We sought to distin- guish these two types of effects by separately quantifying the direct effects on chromatin interactions that span a deletion breakpoint and indirect effects on chromatin in- teractions adjacent to a deletion. We illustrate this with The regional effects of the CFHR3/1 deletion on TAD structure was examined in more detail by correlating counts with genotype for all elements of the contact matrix using linear regression controlling for ancestry and sex. The resulting correlation matrix is visualized as a heatmap of the regression coefficients (Fig. 1 c, see methods). The correlation matrix reveals a pattern con- sistent with an increase in interactions between the Fig. 1 Deletion of CFHR3 and CFHR1 is associated with variation in chromatin conformation. Maps of chromatin interaction surrounding an 80 kb deletion of the CFHR3 and CFHR1 genes (hg19 position chr1:196,728,877–196,808,865) are depicted by averaging the counts within the contact matrices of subjects homozygous for the deletion haplotype (N = 3, Panel a) and subjects homozygous for the reference haplotype (N = 12, Panel b). Normalized counts were plotted as a heatmap with red tone representing the number of chromatin interactions in 40 kb bins. To better visualize the effects for this example, the correlation of counts with the deletion haplotype was tested for all bins across a 1.24 Mb region by linear regression, and regression coefficients were displayed as a blue-red heatmap (Panel c) Fig. 1 Deletion of CFHR3 and CFHR1 is associated with variation in chromatin conformation. Maps of chromatin interaction surrounding an 80 kb deletion of the CFHR3 and CFHR1 genes (hg19 position chr1:196,728,877–196,808,865) are depicted by averaging the counts within the contact matrices of subjects homozygous for the deletion haplotype (N = 3, Panel a) and subjects homozygous for the reference haplotype (N = 12, Panel b). Normalized counts were plotted as a heatmap with red tone representing the number of chromatin interactions in 40 kb bins. Results To better visualize the effects for this example, the correlation of counts with the deletion haplotype was tested for all bins across a 1.24 Mb region by linear regression, and regression coefficients were displayed as a blue-red heatmap (Panel c) Fig. 1 Deletion of CFHR3 and CFHR1 is associated with variation in chromatin conformation. Maps of chromatin interaction surrounding an 80 kb deletion of the CFHR3 and CFHR1 genes (hg19 position chr1:196,728,877–196,808,865) are depicted by averaging the counts within the contact matrices of subjects homozygous for the deletion haplotype (N = 3, Panel a) and subjects homozygous for the reference haplotype (N = 12, Panel b). Normalized counts were plotted as a heatmap with red tone representing the number of chromatin interactions in 40 kb bins. To better visualize the effects for this example, the correlation of counts with the deletion haplotype was tested for all bins across a 1.24 Mb region by linear regression, and regression coefficients were displayed as a blue-red heatmap (Panel c) Page 3 of 10 Shanta et al. BMC Genomics (2020) 21:95 Page 3 of 10 positive effects that span the deletion primarily involve contacts between heterologous sequences. proximal TAD (involving the CFH gene) and the distal TAD (involving a broad region between the genes CFHR2 and CRB1). A portion of the CFHR3/1 deletion overlaps with multiple annotated segmental duplications (SDs) which could potentially confound the mapping of Hi-C read pairs. A similar analysis was conducted after masking segmental duplications and the observed effects were unchanged. Therefore, the effects we observe are not explained by the segmental duplications or by con- tacts between paralogous sequences. Furthermore, a map of SDs across the region (Fig. 1 c) shows that the To more rigorously determine the association of dele- tions with chromatin conformation, we used a linear re- gression model to test for the effects of deletions on chromatin contacts. We again use the CFHR3/1 example to illustrate (Fig. 2). Counts were averaged for elements that span the deletion and for flanking regions within 240 kb (Fig. 2 a), a region chosen as the optimal distance by a parameter sweep (see methods). The effects of dele- tions on chromatin conformation were then tested for Fig. 2 Testing the effect of common deletions on chromatin conformation. Results These results suggest that TB dele- tions have effects that are relatively subtle or that are quite variable between loci, but studies of larger samples would be needed to determine if effects differ consist- ently between TB and nonTB deletions. Analysis was re- peated after masking segmental duplications and results were unchanged (Additional file 3: Fig. S2). “span” and “flank” separately by linear regression con- trolling for ancestry principal components (PCs) and sex. Other potential confounders were evaluated, includ- ing surrogate variables, to account for unknown sources of noise (see methods), however including these add- itional covariates did not reduce the overall inflation of the test statistic (Additional file 1: Fig. S1). The effect of the CFHR3/1 deletion on spanning contacts was statisti- cally significant (Fig. 2 b, p-value: 0.002), but the dele- tion did not have a significant effect on the number of contacts in the flanking regions that overlap with the ad- jacent TADs (Fig. 2 c). j g We next sought to extend the analysis of Hi-C data to all common deletions in the phase 3 release of the 1000 genomes project [10]. Analysis was restricted to all dele- tions that were present in ≥3/19 samples (N = 2180 de- letions). The deletions ranged in size from 51 bp to 125 kb, with an average size of 2622 bp. The magnitude of the genetic effects was assessed based on genomic infla- tion of the test statistic (λ). A Quantile-Quantile (QQ) plot of observed regression p-values relative to an empir- ical null distribution based on permutation of genotypes shows very modest effects for deletions overall, λ = 1.10 and 1.04 for span (Fig. 2 d) and flank (Fig. 2 e) respect- ively, but the effects were stronger for large (> 10 kb) de- letions (λ = 3.30 and 1.20 for span and flank respectively). The magnitude of the effect of large dele- tions on the spanning contacts was greater than for small deletions (Kolmogorov-Smirnov test, p-value: 7.63 × 10−6), but was not significantly different for the flank region (p-value: 0.132). Summary statistics for all deletions that were tested are included in Add- itional file 2: Table S1. Given that the effects of com- mon deletions on chromatin conformation are driven by large deletions, our subsequent analyses focused on this subset of SVs. Results A recent paper has described a method to predict the potential of deletions to cause the fusion of two adjacent TADs [9], a potential mechanism described in [16]. This study reported that deletions at TAD boundaries are under negative selection and deletions with a high “fu- sion score” were skewed toward a low frequency. Using the deletion-spanning contacts for 80 large common de- letions as a measure of TAD fusion, we examined whether there was a correlation between the fusion score of the deletion and the coefficient from the regression. We found no correlation of the predicted fusion scores with the observed effects of these deletions on spanning contacts (Additional file 4: Fig. S3). Our results suggest that large SVs have detectable ef- fects on chromatin conformation. Since the above ana- lysis focused on deletions, it did not assess the largest common SVs known to exist in the population, which include large inversions of 8p23.1 (3.87 Mb) and 7q11.1 (2.45 Mb). To characterize the effects of large inversions on chromatin conformation, inversion genotypes were obtained from single-cell strand sequencing (Strand-seq) of a subset of 9 subjects in the 1000 genomes project [17], and the correlation of chromatin contacts across the region was visualized (Fig. 4 a). The most dramatic effects of the inversion involve contacts that span the in- version breakpoints, denoted by the black triangle, and these effects span distances > 2 Mb from the breakpoint. TAD boundaries correlate with insulator and barrier elements that control chromatin conformation and gene regulation [2]. We therefore hypothesized that deletions could have more dramatic effects on chromatin con- formation when they occur in TAD boundaries. Com- mon large deletions (N = 80 deletions) were separated into deletions at TAD boundaries (TB, N = 16 deletions) and those not at a TAD boundary (NonTB, N = 64 dele- tions). The distribution of regression coefficients for common large deletions in TB/NonTB categories was compared against an empirical null distribution based on permutation of genotypes. These results show a sta- tistically significant positive effect for the span region of NonTB deletions (Wilcoxon rank-sum test, p-value: 0.002) (Fig. 3 a). A visualization of the change in chro- matin structure is illustrated by averaging each element of the contact matrix within 240 kb of a deletion across loci in TB/NonTB categories separately (Fig. 3b, c). Results The Hi-C map of chromatin interactions for the 80 kb CFHR3/1 deletion was separated into regions that interact across the deletion (span) and regions that do not cross the deletion (flank) as they can exhibit different behavior with the removal of the deletion bins (Panel a). The effect of the deletion on chromatin conformation was investigated by linear regression, showing a significant effect in the span region (p-value: 0.002, Panel b) and no effect in the flank region (Panel c). The same analysis was run for all common deletions and p-values stratified by size at a 10 kb threshold were displayed in a QQ plot. Large deletions have the strongest effect in the span region while the contribution from small deletions is non-existent (Panel d). Large deletions show a smaller effect in the flank region (Panel e) Fig. 2 Testing the effect of common deletions on chromatin conformation. The Hi-C map of chromatin interactions for the 80 kb CFHR3/1 deletion was separated into regions that interact across the deletion (span) and regions that do not cross the deletion (flank) as they can exhibit different behavior with the removal of the deletion bins (Panel a). The effect of the deletion on chromatin conformation was investigated by linear regression, showing a significant effect in the span region (p-value: 0.002, Panel b) and no effect in the flank region (Panel c). The same analysis was run for all common deletions and p-values stratified by size at a 10 kb threshold were displayed in a QQ plot. Large deletions have the strongest effect in the span region while the contribution from small deletions is non-existent (Panel d). Large deletions show a smaller effect in the flank region (Panel e) Page 4 of 10 Page 4 of 10 Shanta et al. BMC Genomics (2020) 21:95 Shanta et al. BMC Genomics (2020) 21:95 NonTB deletions we observe an increase in the number of deletion spanning contacts (Fig. 3a) that is concentrated within a narrow region around the deletion (Fig. 3b). This pattern is consistent with the “direct” ef- fects of deletion on the number of breakpoint-spanning read pairs. We do not see a significant effect of NonTB deletions on the number of contacts within the adjacent flanking regions. For TB deletions, we did not detect sig- nificant effects on the number of spanning or flanking contacts (Fig. 3a). Results For The availability of a full assembly of the 8p23.1 inversion haplotype [18] enabled us to map TAD structure of the in- version haplotype by directly mapping Hi-C data of sub- jects that were homozygous for the 8p23.1 inversion to the inversion haplotype. The average number of contacts is shown for subjects with homozygous genotypes for the inversion (Fig. 4 b, bottom) and the reference haplotype (Fig. 4 b, top). TAD structures of the reference and inver- sion haplotypes were similar, and the same 5 TADs were defined. Patterns of long-range contacts for the inversion of 7q11.1 were similar (Additional file 5: Fig. S4). Shanta et al. BMC Genomics (2020) 21:95 Page 5 of 10 Shanta et al. BMC Genomics Fig. 3 Large deletions that do not intersect a TAD boundary have a significant positive effect on the number of contacts that span the deletion region. To determine if the strength or direction of effects differed for deletions located at the boundaries of TADs, regression coefficients from our genome wide analysis were compared between groups of deletions located at TAD boundaries (TB) and those not at TAD boundaries (NonTB) (Panel a). A Wilcoxon rank-sum test was performed for each group against a null distribution, resulting in a significant positive effect for the span region of NonTB deletions (p-value: 0.002). To visualize the topological changes of these effects, a blue-red heatmap of regression coefficients was constructed for NonTB and TB deletions separately. A linear regression was performed for each pairwise bin interaction and coefficients were averaged across deletions. Deletions not present at TAD boundaries have positive values in the span region (Panel b). Deletions that intersect TAD boundaries do not have a unique trend in the span or flank region (Panel c) We hypothesize that the genetic variants that influence chromatin conformation could thereby influence gene regulation [19]. However, the effects detectable in our current dataset are restricted to large SVs, relatively few of which represent lead variants for expression quantita- tive trait loci (eQTLs). Of the 2180 common deletions from our analysis and 5128 SV-eQTLs that were previ- ously identified in another study [20], 75 common dele- tions tested in this study correspond to SV-eQTLs, and these were larger on average with an average length of 5.98 kb compared to the rest of the 2105 deletions which had an average length of 2.5 kb. Results A Wilcoxon rank sum test was performed between these two groups to deter- mine if there was a significant difference between the regression p-value distribution of the deletions with SV- eQTLs and the regression p-value distribution of deletions without SV-eQTLs in the span region. How- ever, SVs that were driving eQTLs did not have stronger effects on chromatin contacts (p-value: 0.45). Summary statistics for all deletions are annotated with SV-eQTLs in Additional file 2: Table S1. Discussion Hi-C has enabled discoveries related to understanding the structural and functional basis of the genome. We show that large common deletions have significant ef- fects on patterns of chromatin conformation with effects that are sufficiently large to be detectable in our small sample of 19 subjects. Large common deletions have a distinctive signature characterized by positive effects on contacts that span the deletion. The most dramatic example was a common deletion polymorphism at CFHR3/1, which results in the gain of contacts that span a broad region betweem two adjacent TADs. An increase in the number of contacts between two distinct TADs is an effect reminiscent of “TAD fusion” [21] (Fig. 1). However, for most large common deletions, their effects on the number of deletion-spanning contacts were more subtle and were concentrated within a narrow region around the deletion (Fig. 3 b). The effect of common SVs on 3D chromatin conform- ation has potential significance for gene regulation. Page 6 of 10 Shanta et al. BMC Genomics (2020) 21:95 (2020) 21:95 Shanta et al. BMC Genomics Fig. 4 Long range effects of a large 8p23 inversion on chromatin conformation. A correlation heatmap shows chromatin interactions that are gained (red) and lost (blue) on the inversion haplotype relative to the reference (Panel a). The gray region corresponds to missing values that could not be normalized. The inversion region is depicted by the black triangle. Hi-C matrices for samples that were homozygous for the absence of an inversion and homozygous for the inversion at 8p23.1 were averaged separately and annotated (Panel b). The TAD structure is preserved in a mirrored fashion along with their associated genes. Chromatin interactions for the inversion were mirrored to aid visual comparison with the reference Fig. 4 Long range effects of a large 8p23 inversion on chromatin conformation. A correlation heatmap shows chromatin interactions that are gained (red) and lost (blue) on the inversion haplotype relative to the reference (Panel a). The gray region corresponds to missing values that could not be normalized. The inversion region is depicted by the black triangle. Hi-C matrices for samples that were homozygous for the absence of an inversion and homozygous for the inversion at 8p23.1 were averaged separately and annotated (Panel b). The TAD structure is preserved in a mirrored fashion along with their associated genes. Discussion Chromatin interactions for the inversion were mirrored to aid visual comparison with the reference Large common inversions have distinct effects on chromatin interactions that span the inversion break- points, and these effects can extend for distances > 2 Mb. TAD structures within the large inverted segments of two common inversions appear to be well preserved, suggesting that the sequences within the inverted re- gions are sufficient to determine their 3D structures. However, in our current sample size, we are only able to capture effects from the largest and most common SVs, few of which are associated with expression QTLs. Our results are consistent with common SVs having signatures in Hi-C data that are distinguishable but sub- tle. We reason that common SVs might tend to have relatively small effects on TAD structure as compared to rare pathogenic variants that have been described previ- ously [5–7]. Deletions that remove TAD boundaries and cause TAD fusion may be under negative selection in the population and would therefore tend to be rare. Well-powered characterization of the effects of SVs on chromatin structure and gene regulation would therefore require Hi-C characterization of common variants in lar- ger samples combined with targeted Hi-C and RNA se- quencing of patient samples with specific rare disease associated variants. Generation of hi-C data for 19 subjects Generation of hi-C data for 19 subjects Generation of hi C data for 19 subjects Hi-C data was generated for 19 subjects from the 1000 Genomes Project (Additional file 2: Table S1) using a “dilution” HindIII protocol as previously described [1]. Data collection is described in detail within a companion manuscript [22]. Hi-C allows for unbiased identification of chromatin interactions by using the following process: cells are cross-linked with formaldehyde, DNA is digested using the HindIII restriction enzyme that leaves a five-prime overhang, the five-prime overhang is filled with nucleotides, the resulting fragments are ligated under dilute conditions, DNA is sheared and fragments containing biotin are identified by paired-end sequen- cing [1]. Read ends were aligned to hg19 with BWA- MEM v0.7.8 [23] and in the case of split alignments, the five-prime-most alignment was used as the primary alignment. Reads without a five-prime end alignment and alignments with low mapping quality were fil- tered out. WASP was used to generate alternative reads and realigned using the BWA-MEM [24, 25]. Reads that did not have all alternative reads aligned to the same location were removed. Reads were re- paired and valid read pairs were pairs in which both reads passed this filtering. Defining TAD boundaries TADs were defined as follows. Directionality Index (DI) was computed for each 40 kb bin and used in a Hidden Markov Model to predict the probability of a bin being upstream bias, no bias, or downstream bias [2]. TAD boundaries were called as regions switching from up- stream bias to downstream bias. Selection of covariates used in regression model g The genomic inflation factor (λ) was used to determine how much of the effect could be attributable to con- founding variables such as ethnicity or other unobserved noise in the data that could be captured with surrogate variables. Covariate terms were added one at a time and λ was calculated for the span and flank regions after each addition (Additional file 1: Fig. S1A). The possible confounding variables tested include ancestry PCs to control for population stratification, sex, and surrogate variable PCs to control for variation within each chromosome. Given the sample size of 19, the model be- comes saturated with more than two variables [29]. Co- variates were chosen, according to the combination that minimized λ. The lowest inflation included two ancestry PCs and sex as covariates. The proportion of variance explained by the first two ancestry PCs was calculated to be 47%. The ancestry PC and sex model was used for Conclusions Our analysis has shown that large common SVs can in- fluence local 3D chromatin structure, and the strength and direction of the observed effect varies by locus. De- letions and inversions have distinct signatures. Deletions increase the amount of chromatin interaction between adjacent regions while inversions rearrange the contacts that span its breakpoints. Page 7 of 10 Page 7 of 10 Shanta et al. BMC Genomics (2020) 21:95 Quantifying effects of common deletions on TAD structure Quantitative effects of deletions on chromatin conform- ation were tested by Ordinary Least Squares Regression (OLSR) using Python. First, bins that overlapped with SVs were masked and specific deletion-flanking and de- letion-spanning target regions were defined within 240 kb (six 40 kb bins) on either side of the deletion (Fig. 2 a). For each sample, contacts were averaged across the flanking and spanning target regions respectively. Re- gression was performed for each deletion on the span and flank regions separately, controlling for ancestry PCs ob- tained from SNP genotypes using PLINK1.9 software [28] and sex. The regression was constructed with normalized chromatin interaction counts between regions near the deletion as the independent variable and copy number as the dependent variable (0: Homozygous reference, 1: Het- erozygous deletion, 2: Homozygous deletion). Contact matrices were generated and normalized by dividing read pairs into 40 kb bin pairs and normalizing raw counts using HiCNorm [26, 27]. To compare matri- ces across samples, we needed to remove unwanted variation between matrix elements due to date of pro- cessing as well as remove any other batch effects. This was corrected for by using Bandwise Normalization and Batch effect Correction (BNBC, preprint on bioRxiv https://www.biorxiv.org/content/10.1101/214361v1). This method involves performing quantile normalization on a matrix that contains all contacts between loci at a fixed genomic distance. Methods were present at least once in our sample of 19 subjects. Given that deletions vastly outnumber all other classes of variants, we focused our primary analysis on these. Only deletion alleles that were present in ≥3/19 subjects (N = 2180 deletions, Additional file 2: Table S1) were in- cluded in our analysis. Deletions were then mapped to 40 kb bins within the chromosome Hi-C contact matrices. The bins of the contact matrix that “span” or “flank” each deletion were then defined as illus- trated in Fig. 2. To determine the flanking distance that optimally captures the effect of deletions on flanking regions, multiple bin sizes were tested by a parameter sweep. Effects weakened as the distance in- creased from the deletion and 6 flank bins displayed the largest effect. Extracting structural variant regions from the hi-C contact matrix Genotypes for 68,818 SVs were obtained on the same subjects from the phase 3 SV calls from the 1000 ge- nomes project [10]. The phase 3 SV call set includes 42, 279 deletions, 6,025 duplications and 20,514 inversion/ insertion/complex SVs, of which 5,517 deletions, 101 du- plications, and 227 inversion/insertion/complex SVs Page 8 of 10 Shanta et al. BMC Genomics (2020) 21:95 Shanta et al. BMC Genomics (2020) 21:95 Shanta et al. BMC Genomics the rest of the study and regression coefficients for all loci were displayed in a boxplot (Fig. 3 a). the rest of the study and regression coefficients for all loci were displayed in a boxplot (Fig. 3 a). genomic inflation factor was the metric used to determine the model with the least bias for possible confounding variables: ancestry principal components (PCs), sex, and surrogate variable PCs. The model that used ancestry PCs and sex as covariates had the least bias (λ = 1.10,1.04) and was chosen as the optimal model. P-values of the regression for each deletion in the span (Panel B) and flank (Panel C) region display how the chosen model still has inflation despite the low genomic inflation factor that can be attributed to real effects Additional file 4: Figure S3. Li Additional file 4: Figure S3. Linear regression coefficients in the span region do not correlate with TAD fusion score. We generated the TAD fusion score for our 80 large common deletions and compared the result with the linear regression coefficients in the span region. There was no significant correlation between the two different methods; suggesting that the fusion score is not predictive of patterns of chromatin conformation for common deletions in this study Additional file 5: Figure S4. Long range effects of 7q11.1 inversion on chromatin conformation. A correlation heatmap shows chromatin interactions that are gained (red) and lost (blue) on the 7q11.1 inversion haplotype relative to the reference. The effect of the 7q11.1 inversion on chromatin conformation is similar to the effects of the 8p23.1 inversion, where the most dramatic effects involve contacts that span the inversion breakpoints. The inversion region is depicted by the black triangle Abbreviations AHUS: atypical Hemolytic Uremic Syndrome; DI: Directionality Index; EQTL: Expression Quantitative Trait Loci; LCLs: Lymphoblastoid Cell Lines; Non-TB: Not a TAD Boundary; OLSR: Ordinary Least Squares Regression; PCs: Principal Components; QQ: Quantile-Quantile; SDs: Segmental Duplications; SLE: Systemic Lupus Erythematosus; SV: Structural Variation; TAD: Topological Associating Domains; TB: TAD Boundary; λ: Genomic Inflation Factor Visualization of topological effects for CFHR3/1 and across multiple loci Effects were visualized for select loci as heatmaps of re- gression coefficients. Each heatmap is constructed by ap- plying the regression model for all bins separately across a target genomic region. To visualize the topological ef- fect for CFHR3/1, the regression coefficients for each bin were then plotted as a heatmap with red indicating positive correlation, blue indicating negative correlation, and bins that overlapped the deletion were indicated in gray (Fig. 1 c). Additional file 2: Table S1. Summary statistics for all common deletions. 2180 common deletions from 19 individuals in the 1000 Genomes Project were annotated with TAD boundaries, eQTLs, and GWAS hits. To investigate the effect of these deletions on chromatin conformation, a linear regression was performed between genotype and the median number of chromatin interactions within the flank and span region of each deletion. Ancestry principal components and sex were used as covariates in the regression model Additional file 3: Figure S2. Masking segmental duplications does not change the effects of deletions on chromatin conformation. To determine if the effects on chromatin conformation are driven by segmental duplications (SD), a separate analysis was conducted for all large common deletions after masking every SD found within the deletion or in the flank regions. Deletions were stratified into groups of those that overlap with TAD boundaries (TB) and those that do not overlap with TAD boundaries (NonTB). A Wilcoxon rank-sum test was per- formed for each group against a null distribution and the results are con- sistent with the analysis that did not involve SD masking, showing that the effects of deletions on chromatin contacts are not driven by segmen- tal duplications In addition, to visualize “average” effects across mul- tiple loci, matrices were centered on the left and right deletion boundaries, and the median regression coeffi- cient for each bin across multiple loci was displayed as a heatmap (Fig. 3 b and c). Annotation of structural variants with summary statistics and eQTLs All 2180 common deletions were first annotated with summary statistics from the regression analysis by reporting a p-value and regression coefficient describing the effect of the variant on both the flank region and span region. The SVs were then intersected with the TAD boundaries previously defined in the methods and defined as overlapping that TAD boundary if the inter- section was at least 1 bp. An empty element in the table represents no overlap with a TAD boundary. All dele- tions were intersected with SV-eQTLs previously identi- fied in another study [20]. If these SV-eQTLs were also present within the GWAS Catalog [19], then the table was further annotated with gene information like gene name, gene ID, etc. Acknowledgements We thank Bing Ren and David Gorkin for the generation of the Hi-C data and Yunjiang Qiu for pre-processing the Hi-C contact matrices. We thank the HGSVC for providing SV calls and the patched genome for the 8p23.1 inver- sion. We thank the San Diego Supercomputer Center for the availability of resources. Collaborating authors of the Human Genome Structural Variation Consortium (HGSVC): Analysis of large inversions Hi-C chromatin interactions for the bins that overlap the inversion and 62 bins on each side of the inversion were extracted. A Pearson correlation between number of chromatin interactions and genotype was applied for each bin across the 9 samples that had both Hi-C data and inversion calls available. The Pearson correlation for each bin was displayed as a heatmap (Fig. 4 a). Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-020-6516-1. y Supplementary information accompanies this paper at https://doi.org/10. 1186/s12864-020-6516-1. Mark J.P. Chaisson1,2, Ashley D. Sanders3, Xuefang Zhao4,5, Ankit Malhotra6, David Porubsky1,7,8, Tobias Rausch3, Eugene J. Gardner9, Oscar L. Rodriguez10, Li Guo11,12,13, Ryan L. Collins5,14, Xian Fan15, Jia Wen16, Robert E. Handsaker17,18,19, Susan Fairley20, Zev N. Kronenberg1, Xiangmeng Kong21,22, Fereydoun Hormozdiari23,24, Dillon Lee25, Aaron M. Wenger26, Alex R. Hastie27, Danny Antaki28, Thomas Anantharaman27, Peter A. Audano1, Harrison Brand5, Stuart Cantsilieris1, Han Cao27, Eliza Cerveira6, Chong Chen15, Xintong Chen9, Chen-Shan Chin26, Zechen Chong15, Nelson T. Chuang9, Christine C. Lambert26, Deanna M. Church29, Laura Clarke20, Andrew Farrell25, Additional file 1: Figure S1. Ancestry principal components and sex need to be used as covariates in linear regression. To determine which covariates reduce the bias in the linear regression model, the effect of common deletions on chromatin conformation was tested for 6 different models, with each model adding an extra covariate term (Panel A). The Page 9 of 10 Page 9 of 10 Shanta et al. BMC Genomics (2020) 21:95 Shanta et al. BMC Genomics (2020) 21:95 Joey Flores30, Timur Galeey21,22, Madhusudan Gujral28, Victor Guryev7, William Haynes Heaton29, Jonas Korlach26, Sushant Kumar21,22, Jee Young Kwon6,33, Ernest T. Lam27, Jong Eun Lee34, Joyce Lee27, Wan-Ping Lee6, Sau Peng Lee35, Shantao Li21,22, Patrick Marks29, Karine Viaud-Martinez30, Sascha Meiers3, Kath- erine M. Munson1, Fabio C.P. Navarro21,22, Bradley J. Nelson1, Conor Nodzak16, Amina Noor28, Sofia Kyriazopoulou-Panagiotopoulou29, Andy W.C. Pang27, Gabriel Rosanio28, Mallory Ryan6, Adrian Stütz3, Diana C.J. Spierings7, Alistair Ward25, AnneMarie E. Welch1, Ming Xiao37, Wei Xu29, Chengsheng Zhang6, Qihui Zhu6, Xiangqun Zheng-Bradley20, Ernesto Lowy20, Sergei Yakneen3, Ste- ven McCarroll17,18,38, Goo Jun39, Li Ding40, Chong Lek Koh41, Paul Flicek20, Ken Chen15, Mark B. Gerstein21,22,42,43, Pui-Yan Kwok44, Peter M. Lans- dorp7,45,46, Gabor T. Marth25, Jonathan Sebat28,31,47, Xinghua Shi16, Ali Bashir10, Kai Ye12,13,48, Scott E. Devine9, Michael E. Talkowski5,19,49, Ryan E. Mills4,50, To- bias Marschall8, Jan O. Korbel3,20, Evan E. Eichler1,51 & Charles Lee6,33. 1Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA. 2Quantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA. 3European Molecular Biology Laboratory, Genome Biology Unit, 69117 Heidelberg, Germany. 4Department of Computational Medicine and Bioinformatics, University of Michigan, Ann Arbor, MI 48109, USA. 5Center for Genomic Medicine, Massachusetts General Hospital, Department of Neurology, Harvard Medical School, Boston, MA 02114, USA. Supplementary information 6The Jackson Laboratory for Genomic Medicine, Farmington, CT 06032, USA. 7European Research Institute for the Biology of Ageing, University of Groningen, University Medical Centre Groningen, Groningen, AV NL-9713, The Netherlands. 8Center for Bioinfor- matics, Saarland University and the Max Planck Institute for Informatics, 66123 Saarbrücken, Germany. 9Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, USA. 10Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. 11The School of Life Science and Technology of Xi’an Jiaotong University, 710049 Xi’an, China. 12MOE Key Lab for Intelligent Networks & Networks Security, School of Electronics and Information Engin- eering, Xi’an Jiaotong University, 710049 Xi’an, China. 13Ye-Lab For Omics and Omics Informatics, Xi’an Jiaotong University, 710049 Xi’an, China. 14Pro- gram in Bioinformatics and Integrative Genomics, Harvard Medical School, Boston, MA 02115, USA. 15Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX 77030, USA. 16Department of Bioinformatics and Genomics, College of Com- puting and Informatics, The University of North Carolina at Charlotte, Char- lotte, NC 28223, USA. 17Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. 18The Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 19Program in Med- ical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 20European Molecular Biology Laboratory, European Bioinfor- matics Institute, Wellcome Genome Campus, Hinxton, Cambridge CB10 1SD, United Kingdom. 21Yale University Medical School, Computational Biology and Bioinformatics Program, New Haven, CT 06520, USA. 22Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA. 23Biochemistry and Molecular Medicine, Univer- sity of California Davis, Davis, CA 95616, USA. 24UC Davis Genome Center, University of California, Davis, Davis, CA 95616, USA. 25USTAR Center for Gen- etic Discovery and Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, UT 84112, USA. 26Pacific Biosciences, Menlo Park, CA 94025, USA. 27Bionano Genomics, San Diego, CA 92121, USA. 28Beyster Center for Genomics of Psychiatric Diseases, Department of Psychiatry Uni- versity of California San Diego, La Jolla, CA 92093, USA. 2910X Genomics, Pleasanton, CA 94566, USA. 30Illumina Clinical Services Laboratory, Illumina, Inc., 5200 Illumina Way, San Diego, CA 92122, USA. 31Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA. 32Ludwig Institute for Cancer Research, La Jolla, CA 92093, USA. Funding g This study was supported by a grant to J.S. from the National Human Genome Research Institute (NHGRI #HG007497), which provided support for O.S., A.N. and J.S. and supported the sequencing of the 9 samples (GM19238, GM19239, GM19240, HG00512, HG00513, HG00514, HG00731, HG00732 and HG00733). NHGRI played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. Authors’ contributions JS designed the study. HGSVC provided SV calls on the study cohort of 19 subjects and provided a patched version of the genome containing the inversion haplotype of the 8p23.1 inversion. OS, AN, and JS developed the statistical analysis methodology. OS, AN and JS created the visualization. OS and JS wrote the manuscript. All authors approved the final manuscript. Supplementary information 33Department of Graduate Studies – Life Sciences, Ewha Womans University, 52, Ewhayeodae-gil, Seodaemun- gu, Seoul 03760, South Korea. 34DNA Link, Seodaemun-gu, Seoul, South Korea. 35TreeCode Sdn Bhd, Bandar Botanic, 41200 Klang, Malaysia. 36Bioinformatics and Systems Biology Graduate Pro- gram, University of California, San Diego, La Jolla, CA 92093, USA. 37School of Biomedical Engineering, Drexel University, Philadelphia, PA 19104, USA. 38Pro- 63108, USA. 41High Impact Research, University of Malaya, 50603 Kuala Lum- pur, Malaysia. 42Department of Computer Science, Yale University, 266 Whit- ney Avenue, New Haven, CT 06520, USA. 43Department of Statistics and Data Science, Yale University, 266 Whitney Avenue, New Haven, CT 06520, USA. 44Institute for Human Genetics, University of California–San Francisco, San Francisco, CA 94143, USA. 45Terry Fox Laboratory, BC Cancer Agency, Vancou- ver, BC V5Z 1 L3, Canada. 46Department of Medical Genetics, University of British Columbia, Vancouver, BC V6T 1Z4, Canada. 47Department of Pediatrics, University of California San Diego, La Jolla, CA 92093, USA. 48The First Affili- ated Hospital of Xi’an Jiaotong University, 710061 Xi’an, China. 49Center for Mendelian Genomics, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. 50Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA. 51Howard Hughes Medical Institute, University of Washington, Seattle, WA 98195, USA. Received: 13 September 2019 Accepted: 20 January 2020 Received: 13 September 2019 Accepted: 20 January 2020 Competing interests The authors declare no competing interests. Author details 1D f 1Department of Electrical and Computer Engineering, UCSD, San Diego, CA, USA. 2Beyster Center for Genomics of Psychiatric Diseases, Department of Psychiatry, UCSD, San Diego, CA, USA. 3Department of Cellular and Molecular Medicine, UCSD, San Diego, CA, USA. 4Department of Pediatrics, UCSD, San Diego, CA, USA. Ethics approval and consent to participate Ethics approval and consent to participate Not applicable. Availability of data and materials Hi-C Contact Matrices by chromosome were deposited into NCBI’s Gene Expression Omnibus (accession GSE128678, https://bit.ly/2NbONMc), in conjunction with our companion study by Gorkin et al. [22]. Details and genotypes for common deletions are provided in the supplementary materials. 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https://openalex.org/W2053049023	https://zenodo.org/records/2192085/files/article.pdf	German	null	Über die Änderung der Dichte und spezifischen Wärme bei Platin und Nickel durch Bearbeitung und über Temperaturabhängigkeit der spezifischen Wärme derselben	Annalen der Physik	1,908	public-domain	2,911	1) F. Richarz, Sitzungsber. d. Physik. Gee. Berlin 24. Febr. 1893; Wied. Ann. 48. p. 708. 1893; 67. p. 704. 1899; Naturw. Rundech. 9. p. 221 u. 237. 1894; 16. p. 221. 1900; LimprichtrFeabchrift Greifew. 1900; Sitzungsber. d. G. z. B. d. g. N. Marb. 6. p. 61. 1904; 7. p. 100. 1905. 2) A. Wigand, hug.-Dies. Marburg 1905; Ann. d. Phye. 22. p. 64. 1907. Abh. der Socihth Batwe, Rotterdam. Qekrirnte Preieschrift. 3) G. W. A. Kahlbaum, K. Roth u. P. Siedler, Zeitechr. Wr anorg. Chemie 29. p. 198. 1902; 0. W. A. Kahlbaum u. E. Stnrm, Zeitechr. fiir anorg. Chemie 46. 1905. 20 1 20 1 9. 8ber die Amdewng der Dichte und spex.lftschen W U m e bei Plnt4n und N4ckeZ d u w h Bearbeitung tmd fiber Tem.perntzcr- abhUng4ykdt der spewlflschem W&me derselben ; vo'lc Wdlh. SchZett. (Auenug aue der Marburger Inaugural- Dieeertation vom 23. Juli 1907.) Annden der Physik. IV. Folge. 26. I. Eigenechaf'ten der Metalle in verechiedenem Bearbeitungeauetand. Ob die Metalle, wenn sie durch mechanische Bearbeitung in verschiedenen Dichten dargestell t werden, sich ahnlich ver- halten wie die allotropen Modifikationen der Metalloide, ins- besondere ob die von F. Richarzl) aus seiner kinetischen Theorie fester Elemente hergeleitete und von A. Wiganda) bestiltigte Regel: ,,Von verschiedenen allotropen Modifikationen eines Elements hat die dichtere Form die geringere spezifische W ilrme" auch auf die Metalle in verschiedenem Bearbeitungs- zustand anwendbar ist, sollte durch die nachfolgenden Unter- suchnngen ermittelt werden. Uber die Veranderlichkeit der physikalischen Eigenschaften bei Metallen liegen zahlreiche Untersuchungen vor. Hervor- znheben sind besonders die schanen Arbeiten von Kahlbanm3), der durch Ziehen, Hammern, Tordieren, Pressen uud Ausgliihen auf seine Metalle einzuwirken suchte, um den Punkt hiichster Dichte festzustellen, den er dann als die jenen Elementen zu- kommende ,,wahre Dichte" setzte. 14 14 02 W S h W. Schlett. 202 Dabei machte er die iiberraschende und auf den ersten Blick schwerverstaindliche Entdeckung, da6 Motalle, nachdem sie sehr hohen Drucken ausgesetzt worden waren, zuerst einen Anstieg der Dichte zeigten, dann aber bei weiterer Steigerung des allseitigen Druckes in einer Olpresse einen Umkehrpunkt und einen darauffolgenden Riickgang deutlich erkennen lieBen. Durch nunmehr vorgenommene Erhitzung erfolgte d a m wieder der Anstieg bis zum Punkt maximaler Dichte, der nach Kahl- b aum der natiirlichsten Anordnung der Molekeln entspricht. Zunlchst werden natiirlich die GuSfebler, Vakuolen , die sich beim GuD bilden konnen , ausgeglichen. Dann aber erfolgt eine Eininwirkung von Atom zu Atom. Es kann also bei Metallen ein Zustand erreicht werden, in dem die Atome einander so nahe rticken, da6 bei dem Versuch weiterer An- nilherung Krafte in Tatigkeit zu treten scheinen, die sie wieder auseinanderschleudern, so dal3 sie in einem graSeren Abstand ihrer Schwerpunkte wieder zur Ruhe kommen. Hand in Hand mit dieser Dichtelndernng geht eine Anderung slimtlicher bisher darauf untersuchten physikalischen Eigenschaften. Elektrische Leitfilhigkeit , Elastizitllts- und Torsionsmodul sind Funktionen der Dichte. Auch die spezifische Warme ist bereits von Regnaultl) und Kahlbaum? in den Bereich dieser Untersuchungen ge- zogen worden, ohne da0 es gelang, ein einhejtliches Verhalten fes tzustellen. Nach dieseh Ergebnissen gelangt man zu der Anschauung, da6 die mechanischen Krlfte, die auf die Metalle einwirken, eine inderung in der inneren Konstitntion bewirken. Wllhrend der gro6ere Teil derseiben sicher in Reibungswilrme umgesetzt wird, wird ein anderer Teil derselben in dem vergnderten molekularen Aufbau als potentielle Energie latent. Um- wandlungswiirmen fur verschiedene Bearbeitungszustande sind bereits bekannt bei Gold, Silber und Eisen. 1) V. Regnault, Ann. chim. 73. p. ?iff. 1840. 2) G. W. A. Kahlbaum, R. Roth u. P. Siedler, 1.c. 3) H. Hort, hug.-Dias. Berlin 1906. 1) G. W. A. Kahlbaum, Xeitecbr. f. anorg. Ch. 46. p. 302. 1905; 2) G. W. A. Kablbsum, zeitechr. f. anorg. Ch. 46. p. 252. 1905. W. Spring, Ber. der &em. D. Gee. 16. p. 2725. 1882; 14* I. Eigenechaf'ten der Metalle in verechiedenem Bearbeitungeauetand. Bei letzterem findet H. Hortq, daB 10 Proz. der aufgewandten mechanischen Energie latent wird. 203 Dichte und spez$sclre 1Farmc bei Platin und Nickel usw. Dichte und spez$sclre 1Farmc bei Platin und Nickel usw. Wird die mechanisch ausgeilbte Kraft bis zu hohen Drucken gesteigert, so verhalten sich die Metalle wie Fliissig- keiten, d. h. sie gehen in den amorphen Zustand tiber, sie flieBen bei gegebener MZSglichkeit ale Drahte aus, die amorph und biegsam sind.') Durch Erhitzen ist die Rtickkehr in den kristallinen Zustand moglich. Man hat es also bei ein nnd demselben Metall in ver- schiedenen Dichten mit iihnlichen Umlagerungen zu tun, wie bei den allotropen Modifikationen der Metalloide. Es erschien daher als eine nicht aussichtslose Aufgabe, die fUr diese geltende Richarzsche Regel anf jene auszudehnen. E'iir die Auswahl der Metalle, die diesen Untersuchungen zugrunde gelegt werden sollten, war einmal das Erfordernie groBer Luftbestiindigkeit mabgebend, sodann die Erfahrungen, die andere Beobachter mit denselben gemacht hsben. Sie fie1 auf Platin und Nickel. Beides sind luftbestllndige Metalle; Platin (Handelsware der Firms Heraeus), ein lluSerst homo- genes Material, das seine Eigenschaften in sehr regelma6iger Weise Lndert. Von Nickel hat Kahlbaurnq eine ziemlich bedeutende Dichteiinderung bei Bearbeitung konstatiert. Aus einem Barren des betreffenden Metalles wnrden un- mittelbar nebeneinander je drei Bolzen ansgeschnitten, mit einem oberen Durchmesser von 8 mm, 40 mm hoch, nach unten zu einer Spitze abgedreht. Von diesen sechs Bolzen wurde spezifisches Gewicht und spezifische WlSrme bestimmt ; darauf wurde von beiden Metallen der Bolzen mit der niedrigsten Dichte zu weiterer Bearbeitnng nach Hanau geschickt. Zu Drilhten von 2mm gehammert und gewdzt, wurden sie in Marburg wieder untertiucht, dann das Platin zu 0,5 mm weiter gezogen, Nickel zu 0,36 mm, wobei der Nickeldraht spr6de and bruchig wnrde und viele Male an den Ziehl6chern ab- ri6: Das Ziehen wurde auf ausdrtickliche Bestellung so aus- gefiihrt, daS der Draht zwischen den einzelnen Stadien nicht ausgegllibt wurde. Es war also ein sogenanntes ,,kaltes Ziehen". p 14* W. Scirlett. 204 Der Ziehprozed besteht aus einem Lilngnngs- und Pressungs- prozeS, Uber dessen Einzelheiten man keine genanen Angaben machen kann. Auderdem wurde durch Qliihen auf die Dichten der iibrigen Bolzen einzuwirken gesucht, was bei einem der Platin- bolzen zu Resultaten fiihrte. 1) 0. Dieterici, Ann. (1. Php. 16. p. 599ff. 1906. ) , y p 2) p. 26 meiner Marburger Dies. Zeile 9 v. u. mufl heiflen 0,031 888 statt 0,s 1888. 1) C. Dieterici, Ann. d. Pbye. 16. p. 599 u. 600. 1905. 2) p. 26 meiner Marburger Dies. Zeile 9 v. u. mufl heiflen 0,031 888 statt 0,s 1888. 11. Die Methoden. Die Messungen der spezifischen Wilrme geschahen rnit dem Bunsenschen Eiskalorimeter. Bei dem Brtu desselben waren alle Erfabrungen im hiesigen Institnt und die in der Literatur sich findenden Angaben berlicksichtigt. Eioe genaue Beschreibung und die Bandhabung desselben findet sich in meiner Marburger Dissertation p. 17 ff: Eine besondere Sorg- falt wurde auf eine miiglichst gesicherte Gangbestimmung ver- wendet und zu diesem Zweck eine von Dietericil) empfohlene Kontaktvorrichtung an der Saugspitze angebracht, die errnag- lichte, den Kontakt der Quecksilberschichten in Rapillare und Wiigeniipfen beim Umwechseln derselben sofort wieder her- zustellen. Als Heizquelle diente ein elektrischer Ofen in einer Form, wie er sich in neuerer Zeit zu derartigen Zwecken gentigsltm bekannt gemacht hat. Die Fallvorrichtung war so erdacht, dad in einem be- stimmten Zeitmoment, der durch Temperaturablesung und Gangbestimmnng gegeben war, durch einen auden am Eeiz- ofen befindlichen Knopf Federn ausgelast werden konnten, die, an Messingarmen anfassend, die Metallbolzen im Innern des Ofene hielten und sie eben in diesem Moment loslieden. Die Temperaturablesuog geschah durch geeichte, in 0, lo geteilte Thermometer mit einer Schiitzungsmaglichkeit von 0,O 1 O. Bei den Versuchen wurden bestimmte Kriterien iiber ihre Zuliissigkeit zu den Resultaten anfgestellt. Nur solche Ver- suche wnrden bei den definitiven Resultaten benutzt , bei denen die mijglichen Versuchsfehler den Bereich der zu er- wartenden Gesamtunterschiede nicht iiberschritten. Dazu mudte man verlangen, da6 der Gang innerhalb der kleinen Qrenzen Die Temperaturablesuog geschah durch geeichte, in 0, lo geteilte Thermometer mit einer Schiitzungsmaglichkeit von 0,O 1 O. Dichte und spez$sche Warme l e i Platin und Nickel usw. 203 vor und nach dem Versuch konstant geblieben war, daE die Temperatur wahrend des Versuches in 15 Minuten nicht mehr a19 um 0,lo geschwankt hatte. Alle spezifischen Wilrmen sind hier bezogen auf die mittlere Bunsensche Kalorie co-loo als Einheit, deren Queck- silberwert von Dietericil) neu bestimmt ist zu 0,015491 g. g Die Resultate sind immer der Mittelwert aus 3 bis 4 Einzel- bestimmungen, mit einem wahrscheinlichen Fehler des Mittel- wertes von weniger als 0,25 Prozea) Die Dichtebestimmungen wurden durch hydrostatische Wbgungen vorgenommen. Die Dilmpfung in Wasser war dabei durch besondere VorsichtsmaEregeln herabgemindert, und der EinfluS der Kapillarkraft des Aufhllngedrahtes elimi- niert (vgl. Marburger Diseertation p. 28 u. 29)) so daS jede aus drei Einzelversuchen bestehende Bestimmung eine groEte Abweichnng vom Mittel von weniger als 0,06 'Proz. hatte. 1) C. Dieterici, Ann. d. Pbye. 16. p. 599 u. 600. 1905. 1) C. Dieterici Ann d Pbye. 16 p 599 u 600 1905 111. Die Reaultate und ihre Diekueeion. Nach einer gro0en Anzahl von Vorversuchen wurden folgende Resultate erhalten. Platinbolzen 111 1, 1 ,, I1 Tabelle I. I I L 0,03'68 I} + 0,2143 - 0,00025 i~~~~~ - 1) 0,1563 - 0,00025 i 21,1296 21,3439 1 21,4802 - i - - Vertinderung der Dichte 1 der sper. W. Substanz 11 Dichte 1 spez. WBrme I 0,1059 - Nickelbolzen I 8,8487 l - fiir die unterstrich. Stellen: Bei den spezifischen WLrmen sind immer die mittleren spezifischen Warmen cao-o miteinander verglichen. 206 W. Schlett. Die Zalilen der Tab. I lassen folgendes erkennen: g Erstens bei Platin, daE bei Proben des Metalles gleicher Provenienz, in unmittelbarer Niihe voneinander genommen, das spezifische aewicht dnrchaus verschiedene Werte haben kann, daE aber auch die spezifische Wiirme eine differierende G r a b ist, und zwar - was bei diesen nicht mit Absicht verschieden bearbeiteten Proben gar nicht erwartet zu werden brauchte - da6 fur diese bereits die Richarzsche Regel ernillt ist: die Dichten sind nach steigenden, die spezifischen Warmen nach fallenden Werten geordnet. g Dies zwingt nun zu der Annahme, da% die anfslngliche Dichteverteilnng in diesem Faile bei Platin nicht, wie Kahl- baum annimmt, auf ,,GuEfehlern", Vakuolen, die sich beim Erstarren aus dem fliissigen Zustand bilden, beruht, sondern zum wesentlichen Teil auf Zustandsdifferenzen zuriickzufnhren ist, die sich durch ungleichmiiEiges Erkalten bilden kbnnen ; denn Vakuolen kijnnten keinen EinfluB auf die spezifische Warme haben. Bekannt ist, daB auch Kupfer frei yon GuB- fehlern ist, und sie in Eisen durch Schmieden beseitigt werden, wahrend bei Messing und GuBeisen solche vorkommen. g Die Nickelstiicke waren ausgegliiht. Das Gllihen hat ja nach Kahlbaum zur Folge, daS die Molekeln eine maglichst natiirliche Anordnung ibrer Gruppen einnehmen, d. h. daS bei Metallindividuen gleicher Reinheit die Dichten sich einem ge- meinsamen Wert nahern, der nach ihm einem Dichtemaximum entspricht. So zeigen denn auch die Dichten keine nennens- werten Unterschiede. Trotz der augenfrilljgen gesetzmafiigen Anderung bei Platin dlirfen wir dieser noch keine direkte Beweiskraft fur die Richarzsche Regel zusprechen. g Wir wollen jetzt sehen, mie sich die Verhaltnisse diidurch anderten, daE die Bolzen gehammert, gewalzt, gezogen und ausgegliiht wurden. Die in der Tab. I1 enthaltenen Resultate sind ausnakms- 10s eine Bestatigung der Richarzschen Theorie und der daraus abgeleiteten Regel, die man auch so aussprechen kann: Die Anderungen des spezifischen Gewichtes und der spezi- fischen Wilrme haben stets entgegengesetztes Vorzeichen bei allotroper Modifizierung eines Elemontes. Also : ___ t, - - cf = 0,030595 + 0,0000282 t . d t ___ t, - - cf = 0,030595 + 0,0000282 t . d t Fur Nickel von Oo bis 300° lauten sie C, = 0,10280 + 0,00004704 t c1 = 0,10280 + 0,0000941 t . Also ist der Temperaturkoeffizient der spezifischen Wiirme (gerade wie der Dichtekoeffizient) etwa 4 ma1 so groS wie fur Platin. Fiir Platin von O0 bis 300° geniigt eine lineare Gleichung nicht mehr, vielmehr stellt die in t quadratische Gleichung angenahert die Abhangigkeit dar. Gegeniiber der zuvor an- gegebenen linearen Gleichung ergibt die Ausgleichung nach der Methode der kleinsten Quadrate auch eine kleine Anderung des konstanten Gliedes. ct = 0,030456 + 0,00002972 t - 0,0000000561 t2 111. Die Reaultate und ihre Diekueeion. 1 Subetanz 1 I '1 11 1 1 platinIII 1 gegossen 21,4802 0,03118 Platin'' {~30Dlin.~eiBgeg~iihl/ 21g32 I 0,03145 I} - 0,1170 I + 0,00027 Veriinderung Bearbeitung Dichte Wiirme der spez. Dichte 1 WPrme gegossen 1 21,1296 0,03168 1 - zu 2mm gehiimmert zu 0,5 mm kalt gezogen - )+ 0,2133 - 0,00034 und gewalzt 21,3429 0,03134 21,3062 0,03150 - 0,0367 + 0,00016 - - - I Ii 11 Nickel 111 ausgegluht zu 2mm gehgmmert und gewalzt zu 0,36 mm kalt gezogen ' I 8,8209 1 0,1068 - - Tab, I1 zeigt ferner, daf3 bei Nickel die spezifische Wiirme starker von der Dichte abhilngig ist als fUr Platin. Rechnen wir die Unterschiede prozentual aus:; epez. Wtirme: Dichte: Platin 111: o,03168] 0,03134 - 1,1 Proz. 21,3429 21J2g6] + 1 Proz. 21,4802 Platin 11: 0,03145 s28442] - 0,27 ,, 8,8209 Mickul 111: 0,1057 o,1068 ] + 1 ,, W ahrend also bei Platin die zusammengeharigen Ande- rungen der Dichte und spezifischen Wilrme fast gleich groS sind, ist bei Nickel der Wert des Differenzenquotienten A c l A D etwa 4 ma1 so grof3 wie bei Platin. Da andererseits nach den bisherigen Beobachtungen such die Temperaturabhiingigkeit der spezitischen Wiirme bei Nickel 208 W. ScMett. weit groSer ist als bei Platin, so regte dieses Resultat zu einem Vergleich beider Abhangigkeiten an. g g g Um fur diese Betrachtungen nicht auf das bei den ein- zelnen Beobachtern stark abweichende Zahlenmaterial an- gewiesen zu sein, wurde eine Nmbestimmung der Abhangig- keit der spezifischen Wiirme bei Platin und Nickel von der Temperatur unternommen. Die Resultate dieser Untersuchungen lieferten folgende Qleichungen, nach der Methode der kleinsten Quadrate be- rechnet: l Von Platin von Oo bis looo fur die mittlere spezifische Warme : C, = 0,030595 + 0,0000141 t . C, = 0,030595 + 0,0000141 t . Zwischen c,,, und der wahren spezifischen Warme cf bei to bestsht: t Also : IV. SchluBfolgerungen und Zueammenfaseung. Wir haben nun zwei Abhangigkeiten der spezifischen Warme gemessen: I. die Abhiingigkeit der spezifischen Warme von der Temperatur, I. die Abhiingigkeit der spezifischen Warme von der Temperatur, Dichte und spexifiche Warme bei Platin und Nickel usw. 209 Dichte und spexifiche Warme bei Platin und Nickel usw. 209 209 XI.. die Abhilngigkeit derselben von der Dichte, soweit diese durch Bearbeitung geibndert wird. Andererseits besteht die Abhhngigkeit der Dichte von der Temperatur oder des spezifischen Volumens von der Tem- peratur: v, = pt, (1 + % D , - t,)) 1 1 = - (1 + 3 4 , - q), oder W8nU Dtl die Dichte bei t: , a, ), 9 ) ,, q! , a, ), 9 ) ,, q! , ) q und 3 tc der kubische Ausdehnungskoeffizient ist. , ) q und 3 tc der kubische Ausdehnungskoeffizient ist. Um dann die beiden Abhilngigkeiten der spezifischen Wilrme miteinander in Beziehung zu bringen, denke ich mir die linderung des spezifischen Volumens, die wir vorher dauernd durch Bearbeitungsdrucke hervorgebracht haben, nun durch die thermische Ausdehnung vor sich gehend. g g Es ist dann die Frage, wird in beiden Fallen die Ver- Lndernng des spezifischen Volumens etwa den gleichen EinfluO auf die spezifische Wilrme haben? , Die Berechnung ergibt, daS die in dieser Weise ver- glichene Abhilngigkeit der spezifischen Warme von der Tern- peratur vie1 gr&r ist als die vom spezifischen Volumen durch Bearbeitung. LieBen sich diese beiden Abhilngigkeiten durch dieselbe Gleichung darstellen, so wilre die spezifische Wilrme ausschlie6lich Funktion des spezifischen Volumens ; dagegen Funktion der Temperatur indirekt nur insofern, ale u eine Funktion deraelben ist. Da dioe, wie die Berechnung ergibt, nicht der Fall ist, ist I: direkt sowohl Funktion des spezifischen Volumens als auch der Temperatur. (Eingegangen 1. April 1908.) c - f(v, 4' Ein weiterer Vergleich zwischen Platin einerseits, Nickel andererseits, lill3t erkennen, da6 bei Platin der EinfluS des spezifischen Volumens oder - wie wir jetzt bei einem Ver- gleich der beiden Elemente sagen wollen - des Atomvolumens allein in der beobachteten Abhangigkeit der spezifischen WZlrme von der Temperatur eine untergeordnete Rolle spielt. Bei ihm, mit dem grbl3eren Atomvolumen, ist eben die Mgglichkeit 210 W; Schleit. Dichte und spez. Warme bci Platin usw. einer Einwirkung der Atome in ihren oszillatorischen Bahnen aufeinander eine geringere. Bei Nickel jedoch, mit dem kleineren Atomvolumen, ist dessen Einflul3 ein merklicher. Die gr6Bere Temperaturabhangigkeit in beiden F U e n riihrt moglicherweise daher, daS bei der thermischen Bus- dehnung eine gewisse WArmemenge zur Arbeitsleistung gegen die molekularen KohLionskrlfte verbraucht wird. Fiir cv wiirde man vielleicht finden, daB beide Abhangigkeiten die- selben sind Die Resultate dieser Untersuchungen sind also : 1. Die Metalle in verschiedenem Bearbeitungszustand ver- halten sich wie die allotropen Modifikationen der Yetalloide. Es gilt insbesondere fir sie die Richarzsche Regel: Die dichtere . Form hat die geringere spezifische Wlirme. 1. Die Metalle in verschiedenem Bearbeitungszustand ver- halten sich wie die allotropen Modifikationen der Yetalloide. Es gilt insbesondere fir sie die Richarzsche Regel: Die dichtere . Form hat die geringere spezifische Wlirme. 2. Bei einem Vergleich der Abhiingigkeit der spezifischen Warme vom spezifischen Volumeu nnd von der Temperatur erkennt man, dal3 bei Temperaturerhohung vie1 eingreifendere Veriinderungen in der Molektilgruppierung vor sich gehen wie bei einfacher Bearbeitung. g g p 2. Bei einem Vergleich der Abhiingigkeit der spezifischen Warme vom spezifischen Volumeu nnd von der Temperatur erkennt man, dal3 bei Temperaturerhohung vie1 eingreifendere Veriinderungen in der Molektilgruppierung vor sich gehen wie bei einfacher Bearbeitung. g g p Die Versuche sind ausgefiihrt im physikalischen Institut zu Marburg nnter Leitung von Hrn. Prof. F. Richarz, dem ich fUr seine liebenswiirdige Faderung, besonders bei den theoretischen Erwkgungen, zu herzlichem Dank verpflichtet bin. (Eingegangen 1. April 1908.)
https://openalex.org/W1974400815	https://bmcvetres.biomedcentral.com/counter/pdf/10.1186/1746-6148-8-173	English	null	The dental cavities of equine cheek teeth: three-dimensional reconstructions based on high resolution micro-computed tomography	BMC veterinary research	2,012	cc-by	10,817	* Correspondence: susan.kopke@tiho-hannover.de 1Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, Hannover D- 30173, Germany Full list of author information is available at the end of the article RESEARCH ARTICLE Open Access Open Access Abstract Background: Recent studies reported on the very complex morphology of the pulp system in equine cheek teeth. The continuous production of secondary dentine leads to distinct age-related changes of the endodontic cavity. Detailed anatomical knowledge of the dental cavities in all ages is required to explain the aetiopathology of typical equine endodontic diseases. Furthermore, data on mandibular and maxillary pulp systems is in high demand to provide a basis for the development of endodontic therapies. However, until now examination of the pulp cavity has been based on either sectioned teeth or clinical computed tomography. More precise results were expected by using micro-computed tomography with a resolution of about 0.1 mm and three-dimensional reconstructions based on previous greyscale analyses and histological verification. The aim of the present study was to describe the physiological configurations of the pulp system within a wide spectrum of tooth ages. Results: Maxillary teeth: All morphological constituents of the endodontic cavity were present in teeth between 4 and 16 years: Triadan 06s displayed six pulp horns and five root canals, Triadan 07-10s five pulp horns and four root canals and Triadan 11s seven pulp horns and four to six root canals. A common pulp chamber was most frequent in teeth ≤5 years, but was found even in a tooth of 9 years. A large variety of pulp configurations was observed within 2.5 and 16 years post eruption, but most commonly a separation into mesial and distal pulp compartments was seen. Maxillary cheek teeth showed up to four separate pulp compartments but the frequency of two, three and four pulp compartments was not related to tooth age (P > 0.05). In Triadan 06s, pulp horn 6 was always connected to pulp horns 1 and 3 and root canal I. In Triadan 11s, pulp horns 7 and 8 were present in variable constitutions. Mandibular teeth: A common pulp chamber was present in teeth up to 15 years, but most commonly seen in teeth ≤5 years. A segmented pulp system was found in 72% of the investigated teeth. Segmentation into separate mesial and distal pulp compartments was most commonly present. Pulp horn 4 coalesced either with the mesial pulp horns 1 and 3 or with the distal pulp horns 2 and 5. Conclusions: Details of the pulpar anatomy of equine cheek teeth are provided, supporting the continuous advancement in endodontic therapy. © 2012 Kopke et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 The dental cavities of equine cheek teeth: three-dimensional reconstructions based on high resolution micro-computed tomography Susan Kopke1, Nina Angrisani2 and Carsten Staszyk3 Scanning parameters E h h Each tooth was scanned individually using a XtremeCTd with following scanning parameters: Cone beam, beam energy 60 kVp, electrical current 1 mA, resolution 82 μm (or 41 μm), integration time 439 ms (or 700 ms), algorithm optimised for bone, field of view 126 mm, maximal image matrix size 3072 x 3072 pixel. Scanning was performed in the coronal plane from the occlusal surface to the apex. Due to the isotropic voxel, slice thickness was equivalent to resolution, and thus 1000 to 2000 two-dimensional images were obtained. Abstract Numerous individual configurations of the pulp system were obtained in maxillary cheek teeth, but much less variability was seen in mandibular cheek teeth. Keywords: Horse, Equine dentistry, Dental anatomy, Dental roots, Pulp system, Root canal Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 2 of 16 Background The mandibular sample pool comprised seven Triadan 06s, eleven Triadan 07s, two Triadan 08s, four Triadan 09s, three Triadan 10s and eight Triadan 11s. Due to their structural similarities [23] the central Triadan posi- tions (Triadan 07–10) within the maxillary and the man- dibular sample pool were grouped. Despite recent studies describing the pulpar anatomy of equine cheek teeth [1,2], knowledge regarding physio- logical configurations and age-related changes of the pulp cavity remains incomplete [3]. Recently erupted mandibular and maxillary cheek teeth feature a single endodontic cavity comprising a very large common pulp chamber which connects all pulp horns [4]. Subsequent to the development of dental roots, root canals are formed and complete the morphological constituents of the pulp system. The continuous deposition of second- ary dentine over all of the pulp cavity walls [5] leads to a segmentation of the pulp cavity into separate pulp com- partments [6]. To our knowledge, no studies reporting on a lifelong progression of the pulpar segmentations in individual cheek teeth have been published. Horses` ages ranged from five to 24 years (median age 15 years). The age of 17 horses was determined by means of the equine ID card; the age of two horses was estimated using the ageing guides by Muylle (2005) [24] and Martin (2007) [25]. Owing to the staggered eruption times of cheek teeth, the dental ages were used for ana- lyses and determined as published by Dixon (2005) [5]. The sample population included teeth between 1.5 and 23 years post eruption with a median age of 12 years in maxillary and 11 years in mandibular teeth. To calculate age-related changes of the pulp system, teeth were clas- sified into one of the following four dental age groups: Cheek teeth affected by pulpar or apical infection are still more commonly extracted than preserved [1,7,8]. Promising attempts were made to restore infected teeth by performing endodontic therapy [9-12]. However, the long-term success rates of apicoectomy followed by endodontic procedures range from 44% [13] to 86% [14]. One reason for poor success rates is insufficient know- ledge of the variable pulpar morphology [3,11]. Indeed, in human dentistry detailed knowledge of the pulp horn and root canal configurations is an essential prerequisite for successful endodontic treatments [15] with impres- sive success rates of up to 95% being achieved [16-18]. Preparation of cheek teeth The maxillas and mandibles were disarticulated and divided along the midline, creating hemimaxillas and hemimandibles using a saber bone sawa. Cheek teeth were extracted intact along with surrounding tissues by sawing through the adjacent cheek teeth using a steel band sawb. To optimise imaging quality the samples were processed, reducing sample size and approaching original tooth size. These further adjustments were per- formed using a diamond-coated water-cooled band sawc. All teeth were fixed with 10% neutrally buffered formalin solution. Studies investigating pulpar anatomy have either been based on sectioned teeth, accepting inevitable loss of dental tissue [19-21] or clinical computed tomography, allowing sections as thin as 1 mm [1,2,4,22]. Even more precise results can be achieved by micro-computed tom- ography. This method provides a resolution of less than 0.1 mm, thus enabling the detection of even delicate interpulpar communications between pulp system com- ponents. The aim of the present study was to identify common physiological configurations of the pulp sys- tems within different Triadan positions, to describe mor- phological features, and to analyse age-related changes. Background Age group “young”: 0 – 5 years Age group “middle-aged”: 5.5 – 12 years Age group “old”: 12.5 – 17 years Age group “senile”: >20 years Age group “young”: 0 – 5 years Age group “old”: 12.5 – 17 years Age group “senile”: >20 years Methods Material A total of 65 cheek teeth (30 maxillary teeth and 35 mandibular teeth) were extracted from the heads of 19 horses of different breeds obtained from several equine clinics in Northern Germany. Horses were subjected to euthanasia on human grounds for non-dental reasons. From each skull, varying numbers of cheek teeth were selected, ranging from one to eleven. Greyscale analyses and three-dimensional reconstruction Two-dimensional images were evaluated using the oper- ating softwaree. For means of thresholding, selected regions of pulp and adjacent dentine were transferred Maxillary and mandibular cheek teeth were analysed independently. The maxillary sample pool included seven Triadan 06s, eight Triadan 07s, one Triadan 08, six Triadan 09s, three Triadan 10s and five Triadan 11s. Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 3 of 16 histological sections, each measuring 6 μm. Measure- ments of pulp horn sizes were performed at three localisations, whereby four μCT models of different grey values (i.e. 1612 HU; 1807 HU; 2001 HU; 2148 HU) were compared with the corresponding pulp in histological sections. These evaluations revealed that a maximal value of 1800 HU for pulpar tissue served well. However, there was still a discrepancy of about 22 – 28%, with the μCT models being thicker than the histologically evaluated pulp horns. This discrepancy is due to using a predetermined value for reconstructions of the pulp system. from a horizontal view to a histogram display. Pulp was delimited physically of dentine by bordering the pulp specific gauss distribution and in a following step the corresponding grey values for pulp were defined. By evaluating pulp in 202 images of six teeth the lower and upper mean values were equivalent with 0 to 1807 Hounsfield units. Reconstructions of three-dimensional models of the pulp system utilised greyscale thresholding based on previous greyscale analyses. Models of the pulp system were viewed using the operating softwaree and evaluated visually for morphological features. Pulpar morphology l d f d A recently modified equine endodontic numbering sys- tem was used for denomination of the pulp horns [22]. To address the root canals, a labelling was proposed using corresponding Roman numerals (for details see Figure 1). The morphology of the pulp systems was eval- uated two- and three-dimensionally. To detect common age-related patterns of pulpar morphology, variations of the pulp systems were classified into categories and Two distinct regions of reference located at two pulp horns of one μCT model were identified to allow for con- secutive measurements in μCT slices and in histological sections. Histological sample blocks were decalcified, sectioned and stained for examination (Masson-Goldner Trichrom). Both μCT slices and corresponding histo- logical sections were evaluated on sagittal planes. The width of one μCT slice (82 μm) was covered by 13 Figure 1 Denomination of root canals and pulp horns for maxillary cheek teeth (top) and mandibular cheek teeth (bottom), adapted from Du Toit et al. (2008) [22]. The occlusal surface is illustrated by dentine (cream), cement (light grey) and enamel (dark grey). Root contours are schematically displayed adjacent to the occlusal surfaces. Positions of pulp horns are indicated with Arabic numerals (1 to 8). Positions of root canals are indicated with Roman numerals (I to IV). Figure 1 Denomination of root canals and pulp horns for maxillary cheek teeth (top) and mandibular cheek teeth (bottom), adapted Figure 1 Denomination of root canals and pulp horns for maxillary cheek teeth (top) and mandibular cheek teeth (bottom), adapted from Du Toit et al. (2008) [22]. The occlusal surface is illustrated by dentine (cream), cement (light grey) and enamel (dark grey). Root contours are schematically displayed adjacent to the occlusal surfaces. Positions of pulp horns are indicated with Arabic numerals (1 to 8). Positions of root canals are indicated with Roman numerals (I to IV). Figure 1 Denomination of root canals and pulp horns for maxillary cheek teeth (top) and mandibular cheek teeth (bottom), adapted from Du Toit et al. (2008) [22]. The occlusal surface is illustrated by dentine (cream), cement (light grey) and enamel (dark grey). Root contours are schematically displayed adjacent to the occlusal surfaces. Positions of pulp horns are indicated with Arabic numerals (1 to 8). Positions of root canals are indicated with Roman numerals (I to IV). Kopke et al. Results The variation in pulp systems fell into one of three groups. The most common configuration within each group is illustrated and described in Figure 2. A com- mon pulp chamber (CPC) which connects all root canals and pulp horns was found in six (23%) of 26 teeth. A partially segmented pulp chamber (PS) was present in 14 (54%) of 26 teeth. Hereby, the pulp cavity was divided into either two or three separate pulp compartments. The resultant pulp compartments did not always com- prise the same communicating root canals and pulp horns. Instead, six variations of a PS were seen. The most common configuration of a PS was observed in five (36%) of 14 teeth: Root canal IV solely contributed to pulp horn 4, root canal II solely contributed to pulp horn 2, and root canals I and III contributed to the coa- lesced pulp horns 1, 3 and 5. In three (21%) of 14 teeth the distal root canals II and IV contributed to the distal pulp horns 2 and 4, and the mesial root canals I and III contributed to the mesial pulp horns 1, 3 and 5 (Figure 3). All structures of the tooth which were located occlusally of the bi- or trifurcation were considered as reserve crown, and all structures located apically to the bi- or trifurcation were considered as roots. Pulp horns were located within the reserve crown of teeth. The central cheek teeth (Triadan 07-10s) contained five pulp horns numbered 1 to 5. The second maxillary and mandibular premolars (Triadan 06s) contained six pulp horns with pulp horn number 6 additionally present at the mesial edge of teeth. The third molars (maxillary and mandibu- lar, Triadan 11s) never displayed an additional pulp horn at the mesial side (referred to as pulp horn number 6). The third maxillary molars contained seven pulp horns with two additional pulp horns present at the distal edge, referred to as pulp horn numbers 7 and 8, whereas the third mandibular molars displayed six pulp horns with one additional pulp horn present at the distal edge, referred to as pulp horn number 7. Characteristically, pulp horns joined each other within the reserve crown showing different patterns of doing so and finally branched into root canals which terminated at their ap- ical foramina. Maxillary cheek teeth Morphology of roots and root canals in maxillary cheek teeth Partial segmentation (PS) – the pulp system is divided, but at least one pulp horn communicates with more than one root canal Three roots were seen in all teeth ≥2 years (25 of 26 teeth), with each of the two buccal roots containing one root canal. The elongated palatal root possessed two root canals in all teeth ≥4 years (20 of 26 teeth). These two palatal root canals (one mesial and one distal) con- tributed to separate pulp compartments in all but one tooth >5 years. Maximal segmentation (MS) – each pulp horn communicates with only one root canal The degree of segmentation was then compared be- tween young, middle-aged, old and senile cheek teeth. Associations between the status of pulpar segmentation (CPC, PS, MS) and age groups were calculated using Fisher Exact Probability Test for small sample sizes. Pulpar morphology l d f d BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 4 of 16 of such root canals would still be located within the re- serve crown. A varying number of root canals contribu- ted to the coalesced pulp horns, with maxillary teeth showing a complex pattern of interpulpar communica- tion compared to the rather simple pattern in mandibu- lar teeth. In seven senile cheek teeth >20 years the pulp horns appeared completely filled with secondary dentine. Thus, rudimentary dental cavities comprising only root canals were observed. Therefore, all following descrip- tions of the pulp system refer to teeth up to 16 years in maxillary teeth and up to 17 years in mandibular teeth, respectively. subsequently associations between categories and tooth age were calculated. To do so, models of the pulp cavity were evaluated regarding the segmentation into separate pulp compartments. A pulp compartment represents one functional unit of the pulp system in a cheek tooth, having no direct communication with other pulp com- partments within the same tooth. One pulp compart- ment comprises at least one pulp horn and one root canal. All specimens were assigned to one of three degrees of pulpar segmentation: Common pulp chamber (CPC) – all root canals and pulp horns communicate within the common pulp chamber Results Some peculiar morphological features were seen, such as distinct double or triple connection canals between two pulp horns or elongated blind end- ings of former connections. In the present study all pul- par tissue located apically of the pulpar coalescences was defined as root canals, knowing that the coronal aspect A maximally segmented pulp cavity (MS) was found in six (23%) of 26 teeth. According to the number of root canals in maxillary cheek teeth, four solitary pulp com- partments were observed. Hereby, no more than two pulp horns were coalesced (Triadan 07-10s) and these being either pulp horns 1 and 3 or pulp horns 3 and 5. The most frequent MS was seen in five (83%) of six teeth, whereby the root canals II, III and IV solely Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 5 of 16 Page 5 of 16 Figure 2 (See legend on next page.) Figure 2 (See legend on next page.) Page 6 of 16 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 (See figure on previous page.) Figure 2 Morphology of pulp horns and root canals in maxillary cheek teeth. Arabic numerals (1 to 8): Pulp horns. Roman numerals (I to IV): Root canals. Colours indicate separate pulp compartments. (a) 3D models of the pulp cavity. Inserted planes p1 to p4 render the position of selected 2D μCT images, coloured planes indicate locations of connections. Sections (p1-p4) demonstrate cross-sectional shape and size of pulp horns and root canals. Dark grey: Pulp tissue; light grey: Dentine and cementum; white: Enamel; rbm = bucco-mesial root; rbd = bucco-distal root; rp = palatal root. No segmentation (Triadan 211, 2 years): A wide common pulp chamber () connects all pulp horns. Root canals are seen at early stage of development. Partial segmentation (Triadan 107, 5 years): Three separate pulp compartments are present. Root canals I and III contribute both to the branching pulp horns 1, 3 and 5. Root canal II solely contributes to pulp horn 2. Root canal IV solely contributes to pulp horn 4. Green ellipse: Pulp horn 2 is split up into a main and a delicate accessory branch. Maximal segmentation (Triadan 107, 8 years): Four solitary pulp compartments are present. Root canal I solely contributes to the coalesced pulp horns 1 and 3. Root canals II, III and IV solely contribute to pulp horns 2, 5 and 4, respectively. Pulp systems in maxillary Triadan 06s and Triadan 11s with respect to pulp horns 6, 7 and 8 Pulp systems in maxillary Triadan 06s and Triadan 11s with respect to pulp horns 6, 7 and 8 Pulp systems in maxillary Triadan 06s and Triadan 11s with respect to pulp horns 6, 7 and 8 Compared to the central maxillary cheek teeth no fur- ther configurations of the pulp horns 1 to 5 were seen in the Triadan 06s and Triadan 11s. Instead, pulp horns 6, 7 and 8 were accessorily connected to the pulp compart- ments observed in the central Triadan positions. In all seven teeth of Triadan position 06 the mesial pulp horn 6 coalesced with its adjacent pulp horns 1 and 3. This coalescence formed just apical of the bottom of the me- sial infundibulum. In the event of maximal pulpar seg- mentation, root canal I solely contributed to pulp horns 1, 3 and 6. Most commonly (in six of the seven teeth), root canal I released pulp horn 6 first, i.e. most apically, and then pulp horns 1 and 3 branched. Triadan 06s showed a furcation of the mesial root canal in three of four middle-aged teeth (Figure 4), with the furcation site Results BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 7 of 16 Page 7 of 16 being on the same level as the root formation and within the enamel layer (in two of the three teeth). pulp compartments was seen in 16 (80%) of 20 teeth having a divided pulp system. Altogether, 12 different configurations of individual pulp compartments were found. Comparing the occurrence of individual pulp compartments it is worth mentioning that the following pulp compartments were most commonly observed: The solitary pulp compartment comprising pulp horn 4 and root canal IV (“4-IV”) was found in 13 (65%) of 20 teeth, “2-II” in 11 teeth (55%) and “5-III” in ten teeth (50%); followed by “I-1-3-5-III” in eight teeth (40%) and “I-1-3- III” in seven teeth (35%). The distally located pulp horns 7 and 8 in teeth of Triadan position 11 appeared smaller and shorter com- pared to pulp horns 1 to 5 (Figure 5). Pulp horn 7 was derived from root canal II and coalesced with pulp horn 2. Pulp horn 8 was derived from root canal IV and coa- lesced with pulp horn 4 (two teeth), was isolated from other pulpal tissue (one tooth, 11.5 years), or was absent (one tooth, 13.5 years). In one of two middle-aged teeth the bucco-distal root canal (II) as well as the palatal root canal (III) was forked, with the furcation being located slightly occlusally to the roots and the enamel layer (Figure 5). Results Green ellipse (a, p1): Pulp horn 1 is split up into a main distal and an accessory mesial branch. frequently with pulp horn 1 (five of six teeth) rather than pulp horn 5 (one of six teeth). contributed to the single pulp horns 2, 5 and 4, respect- ively, and root canal I solely contributed to the coales- cing pulp horns 1 and 3. Pulp horn 3 was always coalesced with the adjacent pulp horn(s), even when solely derived from one root canal, and hereby most Regardless of the Triadan position, the separations of the pulp system were confined to the pulp horns 1 to 5. A major trend of segmentation into mesial and distal Figure 3 Schematic illustrations of the numerous pulpar configurations with respect to commonly observed variations in maxillary cheek teeth of Triadan positions 06 to 11. Degree of segmentation is displayed according to tooth age, starting with the non-segmented pulp cavity on top and the maximally segmented pulp cavity at the bottom. Configurations within each degree of segmentation are displayed in relation to their incidence. The most frequent configurations are highlighted by larger size and multi-colouring. Less common variations are smaller sized and simply coloured. The proposed pattern of pulpar segmentation with age is indicated in green. Connections are demonstrated by a straight line. Red is used for the largest pulp compartment within one tooth. Orange, ocher and purple differentiate between the second, third and fourth pulp compartment in a tooth. Figure 3 Schematic illustrations of the numerous pulpar configurations with respect to commonly observed variations in maxillary cheek teeth of Triadan positions 06 to 11. Degree of segmentation is displayed according to tooth age, starting with the non-segmented pulp cavity on top and the maximally segmented pulp cavity at the bottom. Configurations within each degree of segmentation are displayed in relation to their incidence. The most frequent configurations are highlighted by larger size and multi-colouring. Less common variations are smaller sized and simply coloured. The proposed pattern of pulpar segmentation with age is indicated in green. Connections are demonstrated by a straight line. Red is used for the largest pulp compartment within one tooth. Orange, ocher and purple differentiate between the second, third and fourth pulp compartment in a tooth. merous pulpar configurations with respect to commonly observed variations in maxillary Kopke et al. Degree of pulpar segmentation and tooth age in maxillary cheek teeth Tooth ages and the observed number of pulp compart- ments are documented in Table 1. All teeth ≤2 years showed a common pulp chamber. The youngest tooth having two separate pulp compartments was 2.5 years, followed by a 3-year-old tooth having three separate pulp compartments, and finally four solitary pulp com- partments were found in teeth ≥8 years (Figure 6). The occurrence of the common pulp chamber was age- related, with the risk ratio of a common pulp chamber being present in teeth ≤5 years compared to teeth >5 years being 9.44 (95% CI >1). The degree of segmen- tation was independent of age (P = 0.3913). Thus, the probability of teeth ≤16 years having two, three or four Figure 4 Pulp horn 6 within the solitary mesial pulp compartment maxillary and mandibular second premolars (Triadan 06). Scale bar: 5 mm. Maxillary 106, 9.5 years: Four pulp compartments are developed, with pulp horns 1, 3 and 6 derived from root canal I. Please note the furcation of root canal I into I1 and I2. Mandibular 306, 12.5 years: Two solitary pulp compartments are present. Pulp horn 6 is coalesced with distinct remnants of pulp horns 1, 3 and 4. Figure 4 Pulp horn 6 within the solitary mesial pulp compartment maxillary and mandibular second premolars (Triadan 06). Scale bar: 5 mm. Maxillary 106, 9.5 years: Four pulp compartments are developed, with pulp horns 1, 3 and 6 derived from root canal I. Please note the furcation of root canal I into I1 and I2. Mandibular 306, 12.5 years: Two solitary pulp compartments are present. Pulp horn 6 is coalesced with distinct remnants of pulp horns 1, 3 and 4. Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 8 of 16 Figure 5 Configurations of pulp horns and root canals in maxillary and mandibular third molars (Triadan 11). Scale bar: 5 mm. Maxillary 111, 11.5 years: One mesial and one distal pulp compartment is present. Pulp horn 7 is connected to the adjacent pulp horn 2. Solid arrow (a): Isolated pulp horn 8. Arrowheads (a, p2): Furcation of root canals II and III. Mandibular 311, 13.5 years: Three pulp compartments are only developed in Triadan 11s. Root canal I solely contributes to pulp horns 1, 3 and 4; root canal II solely contributes to pulp horns 2 and 5. Mandibular cheek teeth pulp compartments did not differ significantly between the three age groups. Within age group “young” (tooth age 1.5 – 5 years) the common pulp chamber was the most frequent configuration pattern and was seen in five (56%) of the nine teeth. In teeth >5 years the common pulp chamber was seen in one tooth aged 9 years, but all other teeth among the middle-aged and old cheek teeth (tooth age 8 – 16 years) had a segmented pulp cavity. Degree of pulpar segmentation and tooth age in maxillary cheek teeth The third pulp compartment comprises root canal III which is connected to pulp horn 7. Both distal root canals II and III are placed in the elongated distal root. Figure 5 Configurations of pulp horns and root canals in maxillary and mandibular third molars (Triadan 11). Scale bar: 5 mm. Maxillary 111, 11.5 years: One mesial and one distal pulp compartment is present. Pulp horn 7 is connected to the adjacent pulp horn 2. Solid arrow (a): Isolated pulp horn 8. Arrowheads (a, p2): Furcation of root canals II and III. Mandibular 311, 13.5 years: Three pulp compartments are only developed in Triadan 11s. Root canal I solely contributes to pulp horns 1, 3 and 4; root canal II solely contributes to pulp horns 2 and 5. The third pulp compartment comprises root canal III which is connected to pulp horn 7. Both distal root canals II and III are placed in the elongated distal root. Morphology of roots and root canals in mandibular cheek teeth Formation of two roots was seen in all teeth >2 years (28 of 32 teeth). One root canal within each root was found in all teeth between 2.5 and 4 years. Two branches of the mesial root canal were seen in one tooth aged 4.5 years, but in all teeth ≥7 years (22 of 32 teeth). Table 1 Observed pulp compartments in maxillary cheek teeth of Triadan position 06 to 11 Number of teeth No segmentation (CPC) Partial segmentation (PS) Maximal segmentation (MS) Tooth age (years) Total no. of teeth in group Common pulp chamber Two pulp compartments Three pulp compartments Four pulp compartments (all solitary) 1.5 – 2 2 2 0 0 0 2.5 – 4 5 2 1 2 0 4.5 – 5 2 1 0 1 0 8 1 0 0 0 1 9 – 9.5 3 1 0 1 1 11 – 12 4 0 1 2 1 12.5 – 14 5 0 1 2 2 14.5 – 16 4 0 3 0 1 >20 4 0 0 0 4 Intercommunication between all pulp horns was observed up to 9 years post eruption. Partially segmented pulp systems (PS) were seen in teeth ≥2.5 years post eruption. Configurations of PS varied – solitary, medium sized or large separate pulp compartments were combined. Four pulp compartments (MS) were found in teeth ≥8 years post eruption. In all teeth >20 years rudimentary dental cavities were observed. Table 1 Observed pulp compartments in maxillary cheek teeth of Triadan position 06 to 11 Intercommunication between all pulp horns was observed up to 9 years post eruption. Partially segmented pulp systems (PS) were seen in teeth ≥2.5 years post eruption. Configurations of PS varied – solitary, medium sized or large separate pulp compartments were combined. Four pulp compartments (MS) were found in teeth ≥8 years post eruption. In all teeth >20 years rudimentary dental cavities were observed. Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 9 of 16 Figure 6 Boxplots of the tooth ages of maxillary cheek teeth assigned to three degrees of segmentation. The shaded box represent the interquartile range (25%, 75%), the vertical line the median and the whiskers the range. Figure 6 Boxplots of the tooth ages of maxillary cheek teeth assigned to three degrees of segmentation. The shaded box represent the interquartile range (25%, 75%), the vertical line the median and the whiskers the range. Segmentation into separate pulp compartments in mandibular cheek teeth A common pulp chamber (CPC) connecting all pulp horns and root canals was observed in nine (28%) of 32 teeth. The maximally segmented pulp chamber (MS), i.e. each pulp horn is derived by only one root canal, was found in 22 (69%) of the 32 teeth. The solitary pulp compartments of mandibular teeth comprised up to three coalesced pulp horns being derived from one root canal (Figure 7). The most common configuration of a maximally segmented pulp system was seen in 11 (50%) of 22 teeth, with the mesial root canal I solely contribut- ing to the mesial pulp horns 1 and 3, and the distal root canal II solely contributing to pulp horns 2, 4 and 5. The most common variation was found in eight (36%) of 22 teeth, whereby pulp horn 4 was coalesced with the me- sial pulp horns (Figure 8). The least frequent variation of a maximally segmented pulp system was observed in three (14%) of 22 teeth, with pulp horn 4 being isolated from any pulp compartment or being absent in spite of Pulp systems in mandibular Triadan 06s and Triadan 11s with respect to pulp horns 6 and 7 Compared to the central mandibular cheek teeth no fur- ther configurations of the pulp horns 1 to 5 were seen in the second premolars and the third molars. In all six teeth of Triadan position 06, the mesial pulp horn 6 was accessorily connected to the adjacent pulp horns 1 and 3, with the connection site being located apically of the coalescence of pulp horn 1 and 3. In all Triadan 06s hav- ing a segmented pulp cavity, root canal I solely contribu- ted to the coalesced pulp horns 1, 3, 4 and 6 (Figure 4). In three of seven teeth of Triadan position 11 the distally located pulp horn 7 was connected to the common pulp chamber. However, in all four specimens having roots developed an additional distal root canal III was seen within the distal root. Thus, a third pulp compartment was observed. This typical third pulp compartment was separate in all four teeth and was composed of pulp horn 7 being solely derived from root canal III (Figure 5). Morphology of roots and root canals in mandibular cheek teeth definite encompassing pulp cavity formations (Figure 9). All three variations indicated that pulp horn 4 was the only discontinuity within a segmented pulp cavity. Within the distal root the undivided root canal was found in most teeth <10 years (11 of 12 teeth), whereas in teeth ≥10 years branching of the distal root canal was most commonly present (12 of 16 teeth). Comparing the branching sites of the root canals within mesial and dis- tal roots, division of the distal root canal was located further apically and in most cases was seen even apically of the enamel extension, with this being independently of tooth age. In Triadan 11s an additional root canal within the elongated distal root was seen in teeth >8 years. Within the distal root the undivided root canal was found in most teeth <10 years (11 of 12 teeth), whereas in teeth ≥10 years branching of the distal root canal was most commonly present (12 of 16 teeth). Comparing the branching sites of the root canals within mesial and dis- tal roots, division of the distal root canal was located further apically and in most cases was seen even apically of the enamel extension, with this being independently of tooth age. In Triadan 11s an additional root canal within the elongated distal root was seen in teeth >8 years. Pulp systems in mandibular Triadan 06s and Triadan 11s with respect to pulp horns 6 and 7 Pulp systems in mandibular Triadan 06s and Triadan 11s with respect to pulp horns 6 and 7 Degree of pulpar segmentation and tooth age in mandibular cheek teeth Tooth ages and the observed number of pulp compart- ments are documented in Table 2. All teeth ≤2 years showed a common pulp chamber. The youngest tooth having two separate pulp compartments was 2.5 years (Figure 10). Within age group “young” (tooth age 1.5 – Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 10 of 16 igure 7 (See legend on next page.) Figure 7 (See legend on next page.) Page 11 of 16 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 (See figure on previous page.) Figure 7 Morphology of pulp horns and root canals in mandibular cheek teeth. Arabic numerals (1 to 7): Pulp horns. Roman numerals (I to III): Root canals. Colours indicate separate pulp compartments. (a) 3D images of the pulp cavity. Inserted planes p1 to p6 render the position of selected 2D μCT images, coloured planes indicate locations of connections. Sections (p1 - p6) demonstrate cross-sectional shape and size of pulp horns and root canals. Dark grey: Pulp tissue; light grey: Dentine and cementum; white: Enamel; rm = mesial root, rd = distal root. Scale bar: 5 mm. No segmentation (Triadan 409, 15 years): A narrow common pulp chamber connects all five pulp horns. Two distinct root canals are developed. Maximal segmentation (1) (Triadan 407, 7 years): Two pulp compartments are present, with pulp horn 4 included in the distal pulp compartment. Root canal I solely contributes to pulp horns 1 and 3. Root canal II solely contributes to pulp horns 2, 4 and 5. Maximal segmentation (2) (Triadan 407, 5 years): Two pulp compartments are displayed, with pulp horn 4 connected to the mesial pulp compartment. Root canal I solely contributes to pulp horns 1, 3 and 4. Root canal II solely contributes to pulp horns 2 and 5. 17 years) a maximally segmented pulp cavity was most commonly observed and seen in 18 (82%) of 22 teeth. The probability of cheek teeth having a maximally seg- mented pulp cavity increased with age (P = 0.0463). 5 years) the common pulp chamber was the most fre- quent configuration pattern and was seen in six (60%) of ten teeth. In teeth >5 years (n = 22) the common pulp chamber was seen in two teeth aged 9.5 years and in one tooth aged 15 years. Degree of pulpar segmentation and tooth age in mandibular cheek teeth The occurrence of the common pulp chamber was age-related (P = 0.0125), with the relative risk of a common pulp chamber being present in teeth ≤5 years compared to teeth >5 years being 4.4 (95% CI >1). In middle-aged and old cheek teeth (tooth age 7 – Common pulp chamber The common pulp chamber was found in six (20%) of 30 maxillary teeth and in nine (26%) of 35 mandibular teeth. However, a similar study found a higher preva- lence of 26% in maxillary teeth, but a lower prevalence of 14% in mandibular teeth [2]. Presumably, small inter- missions between pulp compartments in maxillary teeth might not be detected by clinical computed tomography having a resolution of 1 mm, which was used by Wind- ley et al. (2009) [2]. Nonetheless, Windley et al. (2009) [2] stated the common pulp chamber to be the most fre- quent pulpar configuration in maxillary cheek teeth which is in line with data of our study. Figure 9 Isolated pulpar tissue.f Two pulp compartments are developed, with pulp horn 4 being isolated (Triadan 309, 16 years). Scale bar: 5 mm. Figure 9 Isolated pulpar tissue.f Two pulp compartments are developed, with pulp horn 4 being isolated (Triadan 309, 16 years). Scale bar: 5 mm. deposition of secondary dentine around the walls of the dental cavity, and distinct growth processes within the apical third of the tooth, the pulp chamber narrows and subsequently divides into separate pulp compartments. In the present study, variable configurations of the pulp cavity were observed even in teeth of the same age and the same Triadan position. However, major trends of pulpar segmentation were identified for mandibular and maxillary cheek teeth. Generally, the number of separate pulp compartments within one tooth increased signifi- cantly with age. Interestingly, there was a trend of pre- molars having less interpulpar communication in young teeth and more often solitary pulp compartments in aged teeth compared to molars. Isolated and completely filled pulp horns were found only in the third molars (pulp horns 4 and 8 in mandibular and maxillary teeth, respect- ively) and in mandibular Triadan 09s (pulp horn 4). Senile teeth >20 years showed rudimentary endodontic cavities It has been shown that pulp horn coalescences are more common in younger horses [1,4]. The mean dental age of maxillary teeth displaying the common pulp chamber was 4 years in the present study. Dacre et al. (2008) [1] observed pulpar communication in teeth hav- ing a mean dental age of only 2 years. Furthermore, the oldest maxillary tooth in the present study showing the common pulp chamber was 9 years, whereas Windley et al. Discussion The pulp cavity of equine maxillary and mandibular cheek teeth is subject to profound age-related changes throughout its life span [5]. Due to continuous Figure 8 Schematic illustrations of pulpar configurations with respect to commonly observed variations in mandibular cheek teeth of Triadan positions 06 to 11. The most frequent configurations are highlighted in green. Less common variations are smaller sized. Connections are demonstrated by a straight line. Red is used for the largest pulp compartment. Orange represents the second and ocher the third pulp compartment within one tooth. Encircled numerals represent either isolated pulp horns or pulp horns which are completely filled with secondary dentine. In Triadan 06s, pulp horn 6 was always connected to the mesial pulp compartment. In Triadan 11s, pulp horn 7 was always connected to the additional root canal III. Figure 8 Schematic illustrations of pulpar configurations with respect to commonly observed variations in mandibular cheek teeth of Triadan positions 06 to 11. The most frequent configurations are highlighted in green. Less common variations are smaller sized. Connections are demonstrated by a straight line. Red is used for the largest pulp compartment. Orange represents the second and ocher the third pulp compartment within one tooth. Encircled numerals represent either isolated pulp horns or pulp horns which are completely filled with secondary dentine. In Triadan 06s, pulp horn 6 was always connected to the mesial pulp compartment. In Triadan 11s, pulp horn 7 was always connected to the additional root canal III. Page 12 of 16 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Table 2 Observed pulpar segmentation in mandibular cheek teeth of Triadan position 06 to 11 Number of teeth No segmentation (CPC) Maximal segmentation (MS) Tooth age (years) Total no. of teeth in group Common pulp chamber Two and three pulp compartments (all solitary) 1.5 – 2 4 4 0 2.5 – 4 4 2 2 4.5 – 5 2 0 2 7 – 8 2 0 2 8.5 – 10 5 2 2 11 – 12 4 0 4 12.5 – 14 5 0 5 14.5 – 17 6 1 5 >20 3 0 3 In all teeth ≤2 years a common pulp chamber was seen. In all teeth >20 years rudimentary dental cavities were present. Discussion Table 2 Observed pulpar segmentation in mandibular cheek teeth of Triadan position 06 to 11 Figure 9 Isolated pulpar tissue.f Two pulp compartments are developed, with pulp horn 4 being isolated (Triadan 309, 16 years). Scale bar: 5 mm. In all teeth ≤2 years a common pulp chamber was seen. In all teeth >20 years rudimentary dental cavities were present. In all teeth ≤2 years a common pulp chamber was seen. In all teeth >20 years rudimentary dental cavities were present. due to advanced abrasion and attrition, with interpulpar communications being absent. due to advanced abrasion and attrition, with interpulpar communications being absent. Common pulp chamber (2009) [2] observed the common pulp chamber only up to 6 years post eruption. The mean dental age of mandibular teeth displaying the common pulp chamber was 5 years in the present study, this being similar to results documented by Dacre et al. (2008) [1], who found the mean dental age of teeth having pulpar com- munication to be 4.5 years. The common pulp chamber of mandibular teeth was seen up to 15 years post eruption in the present study. Our results differ greatly Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 13 of 16 Figure 10 Box plots of the tooth ages of mandibular cheek teeth assigned to none and maximal segmentation. The shaded box represents the interquartile range (25%, 75%), the vertical line the median and the whiskers the range. Figure 10 Box plots of the tooth ages of mandibular cheek teeth assigned to none and maximal segmentation. The shaded box represents the interquartile range (25%, 75%), the vertical line the median and the whiskers the range. from studies of Kirkland et al. (1996) [4] and Windley et al. (2009) [2], who found the common pulp chamber only in teeth <6 years, and <2 years, respectively. These differing results are probably one effect of the μCT in- herent higher resolution compared to clinical computed tomography (used by Kirkland et al. (1996) [4] and Windley et al. (2009) [2]), which enabled us to detect even delicate and curved endodontic cavities. commonly observed (in ten (50%) of 20 teeth with seg- mented pulp systems): a) the pulp horns 1-3-5, 2 and 4 were separately derived from root canals I-III, II and IV, respectively, and b) pulp horns 1–3, 5, 2 and 4 were solely derived from root canals I, III, II and IV, respectively. The second most common configuration was observed in three teeth (15%), whereby “I-1-3-5-III” and “II-2-4-IV” was seen. In general, all teeth had at least two coalesced pulp horns present, these being most commonly the me- sial pulp horns 1 – 3 (in 19 of 20 divided pulp systems; 95%) and the mesial pulp horns 3 – 5 (in 10 of 20 divided pulp systems; 50%). Similarly, observations of sectioned teeth by Dacre et al. (2008) [1] and studies of donkey teeth by Du Toit et al. (2008) [22] revealed coalescence of pulp horns 3 – 5 to be mostly present. Common pulp chamber It is noteworthy that separation of the pulp cavity into mesially and dis- tally located pulp compartments occurred in 16 (80%) of 20 maxillary teeth. Conversely, coalescence of a mesial and a distal pulp horn was only seen in four (20%) of 20 divided pulp systems, this always being the buccal pulp horns 1 – 2. The maxillary pulp horn 3 was missing a corresponding root canal and therefore was always con- nected to pulp horns 1 and/or 5. Windley et al. (2009) [2] aptly mentioned that pulp horn 3 was split and appeared to be derived from the mesiobuccal and palatal roots. Segmented pulp cavity Once the common pulp chamber was divided into separ- ate pulp compartments, configurations of the dental cav- ities varied and appeared unpredictable [2]. Results indicate that in mandibular teeth >5 years solitary pulp compartments are frequently present, with still a minor risk left of teeth having all pulp horns connected in middle-aged and even in old teeth. Apparently, segmen- tation of the maxillary pulp system into separate solitary pulp compartments increased gradually between 2.5 and 8 years post eruption. As shown, maximal segmentation of the pulp cavity was not observed even in some aged teeth. It probably would be of clinical interest to deter- mine the factors causing delayed pulpar segmentation in cheek teeth. Despite the observed gradual beginning of pulpar segmentation, further separation of the pulp sys- tem, perhaps once the root canals have been developed, may proceed slowly. However, to fully consider this question, further studies are needed providing closely spaced age groups between 5 and 20 years of tooth age, including large numbers of specimens. Within segmented mandibular pulp systems four dif- ferent configurations were observed. As already docu- mented by Kirkland et al. (1996) [4], only two separate pulp compartments were developed in teeth of Triadan positions 06 to 10. In teeth of Triadan position 11, con- figurations of the pulp system within pulp horns 1 to 5 did not display additional patterns of segmentation, but a third solitary pulp compartment was established dis- tally, with pulp horn 7 being derived from root canal III and terminating at an additional apical foramen. Con- versely, Windley et al. (2009) [2] observed pulp horn 7 In maxillary teeth ≤16 years having a segmented pulp cavity eight different configurations were observed in the present study. Hereby, two, three or four separate pulp compartments were displayed, with pulp horns appearing variably coalesced. However, in accordance with findings of Windley et al. (2009) [2], two configurations were most Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 14 of 16 as being always connected to pulp horns 4 and 5 with no additional distal root canal being present. root canals of maxillary teeth appeared shorter compared to mandibular root canals, and were not divided except for the second premolars and the third molars. These teeth are macroscopically of larger size, thus giving space for additional pulpar tissue within roots in order to main- tain the nutritional function. Clinical aspects O l l l Occlusal pulpar exposure is associated with previous pul- par insults followed by reduced or ceased deposition of dentine [26-28]. Multiple pulpar exposure (two and more pulp horns) is considered to indicate that the entire endo- dontic system is affected [29]. However, Joest (1970) [30] stated that due to the pulp configuration a partial pulpitis does not necessarily affect all pulp horns. Casey and Tre- maine (2010) [31] showed multiple defective secondary dentinal areas to be more prevalent in diseased mandibu- lar teeth compared to maxillary teeth. Assumingly, this might be due to developing solitary pulp compartments which are composed of only one pulp horn and one root canal more frequently in maxillary teeth, compared to mandibular teeth which display solitary pulp compart- ments comprising up to three coalesced pulp horns. Fur- thermore, the most commonly defective area identified by Casey and Tremaine (2010) [31] was pulp horn 2 in maxil- lary teeth. This result tallies with findings in the present study whereby the solitary pulp compartment comprising only pulp horn 2 and root canal II was found in 11 (55%) of 20 teeth having a divided pulp cavity, thus being the second most common individual pulp compartment observed in maxillary cheek teeth. As reported in clinical studies the outcome of endo- dontic therapy was more successful in mandibular com- pared to maxillary teeth [11,13,32]. A study by Carmalt and Barber (2004) [33] even reported only one root being affected in 100% of 14 mandibular teeth having a mean age of 5.3 years. Similar to this finding, in 69% of 32 mandibular teeth of the present study, which had a mean age of 9 years, the pulp cavity was maximally divided, presumably preventing the spread of pulpar in- fection to adjacent pulp compartments via mesio-distal connections. However, only in 23% of 26 maxillary teeth of the present study (mean age: 9 years) was maximal segmentation present, thus pulpar infection could spread to unaffected pulps via variable connections in the ma- jority of teeth. Despite such high degree of variable pul- par configurations in maxillary teeth, we observed a division of the pulp cavity into mesially and distally located pulp compartments similar to mandibular teeth, with the solitary pulp compartments “4-IV”, “2-II” and “5-III” being most commonly present. Presumably, the knowledge [11] and selected treatment of solitary pulp compartments could simplify endodontic procedures and therefore improve clinical outcomes. Segmented pulp cavity Generally, in 32 (100%) mandibular teeth ≤17 years, dis- tinct pulp horns were always connected: The mesial pulp horns 1 and 3 were coalesced as well as the distal pulp horns 2 and 5. However, Du Toit et al. (2008) [22] found only 37.5% of teeth having both these communications present, whereas Dacre et al. (2008) [1] only saw this pat- tern in one tooth. In the present study both coalescences were part of solitary pulp compartments. Furthermore, pulp horn 4 (if present and not isolated) was either con- nected to the distal or to the mesial pulp compartment, more commonly being part of the distal pulp compart- ment (11 of 19 teeth; 58%) compared to the mesial pulp compartment (eight of 19 teeth; 42%). Windley et al. (2009) [2] even documented the distal coalescence of pulp horn 4 in 68% of mandibular cheek teeth. Interestingly, only pulp horn 4 was seen to be isolated or absent, as observed by Windley et al. (2009) [2], this being found in three teeth >8 years. In accordance with other studies no further interpulpar communication between the mesial and the distal pulp compartment was seen [1,2,4]. This finding can be explained by the pronounced buccal en- amel infolding (ectoflexid) in mandibular teeth reaching further apically compared to the lingual infolding (lingua- flexid). Consequently, apart from pulp horn 4, the gross anatomy of mandibular teeth does not permit any further connection between the mesial and distal pulp compart- ments. If pulp horn 4 in mandibular teeth is exposed, ei- ther the mesial or the distal pulp cavity might be affected, or this pulp tissue could be necrotic due to isolation of the pulp horn. In turn, if exposure of pulp horns other than number 4 is diagnosed, their corresponding root canal can be clearly determined. Apical morphology Root canals can take various pathways to the apex includ- ing branches, divisions and rejoining, as has been described in great detail in human dentistry. Features de- scribing variations of the root canals comprise accessory, lateral and furcation canals, canal orifices, apical deltas and apical foramina [15]. Many features of the root canals were recently observed in mandibular teeth [21]. In the present study the mesial and distal root canal of man- dibular teeth dispersed into two branches of varying length, width and route with gradually increasing age. Windley et al. (2009) [2] found two branches of the distal root canal even in all teeth >10 years and accordingly observed the root canals to unite occlusally to the roots. Kirkland et al. (1996) [4], who observed teeth up to eight years, found two root canals in the mesial root and one root canal in the distal root in teeth >5 years. Westenber- ger (2002) [20] observed a varying total number of up to three root canals in mandibular cheek teeth. In general, Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Page 15 of 16 Page 15 of 16 Authors' contributions SK designed the study, collected and processed the specimens, conducted the micro-computed tomography scans, assembled and analysed the data, drafted and wrote the manuscript. NA contributed to the study design and the micro-computed tomography examinations. CS contributed to the study design, helped to collect and process the specimens, contributed to data analysis and interpretation, and edited and revised the manuscript. All authors read and approved the manuscript. 15. Vertucci FJ, Haddix JE, Britto LR: Tooth morphology and access cavity preparation. In Pathways of the pulp. 9th edition. Edited by Cohen S, Hargreaves KM. St. Louis: Mosby Elsevier; 2006:200–309. SK designed the study, collected and processed the specimens, conducted the micro-computed tomography scans, assembled and analysed the data, drafted and wrote the manuscript. NA contributed to the study design and the micro-computed tomography examinations. CS contributed to the study 16. Chugal NM, Clive JM, Spangberg LS: A prognostic model for assessment of the outcome of endodontic treatment: effect of biologic and diagnostic variables. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001, 91:342–352. 17. Basmadjian-Charles CL, Farge P, Bourgeois DM, Lebrun T: Factors influencing the long-term results of endodontic treatment: a review of the literature. Int Dent J 2002, 52:81–86. Conclusions macroscopic occlusal or computed tomographic lesions. Equine Vet J 2009, 41:1–8. The present study has highlighted variable pulpar con- figurations of equine cheek teeth and documented pro- found age-related changes to the pulp system. A wide range of tooth ages displaying individual configurations was seen, and major trends were observed. It might be of great value to the clinician to be acquainted with the general configurations of the pulp system, since know- ledge optimises prognostic evaluations of diseased teeth. 3. Dacre IT: Equine dental pathology. In Equine dentistry. 2nd edition. Edited by Baker GJ, Easley J. Edinburgh: Elsevier Saunders; 2005:91–110. 4. Kirkland KD, Baker GJ, Manfra Marretta S, Eurell JA, Losonsky JM: Effects of aging on the endodontic system, reserve crown, and roots of equine mandibular cheek teeth. Am J Vet Res 1996, 57:31–8. aging on the endodontic system, reserve crown, and roots of equine mandibular cheek teeth. Am J Vet Res 1996, 57:31–8. 5. Dixon PM: Dental Anatomy. In Equine dentistry. 2nd edition. Edited by Baker GJ, Easley J. Edinburgh: Elsevier Saunders; 2005:25–48. 6. Baker GJ: Dental decay and endodontic disease. In Equine dentistry. 2nd edition. Edited by Baker GJ, Easley J. Edinburgh: Elsevier Saunders; 2005:121–125. 7. Dixon PM, Tremaine WH, Pickles K, Kuhns L, Hawe C, McCann J, McGorum BC, Railton DI, Brammer S: Equine dental disease Part 4: a long-term study of 400 cases: apical infections of cheek teeth. Equine Vet J 2000, 32:182–194. Endnotes asaber bone saw, type EFA 61, Schmid & Wezel GmbH & Co. KG, Maulbronn, Germany; bsteel band saw, type K 420, Kolbe GmbH, Elchingen, Germany; cdiamond- coated band saw, type MBS 220/E, Proxxon GmbH, Föhren, Germany; dXtremeCT, Scanco Medical AG, Brüttisellen, Switzerland; esoftware μCT Tomography V5.4C, Scanco Medical AG, Brüttisellen, Switzerland; fAMIRA 5.4.2, Visage Imaging GmbH, Berlin, Germany. asaber bone saw, type EFA 61, Schmid & Wezel GmbH & Co. KG, Maulbronn, Germany; bsteel band saw, type K 420, Kolbe GmbH, Elchingen, Germany; cdiamond- coated band saw, type MBS 220/E, Proxxon GmbH, Föhren, Germany; dXtremeCT, Scanco Medical AG, Brüttisellen, Switzerland; esoftware μCT Tomography V5.4C, Scanco Medical AG, Brüttisellen, Switzerland; fAMIRA 5.4.2, Visage Imaging GmbH, Berlin, Germany. 8. Dixon PM, Dacre I: A review of equine dental disorders. Vet J 2005, 169:165–187. 9. Schumacher J, Honnas CM: Dental surgery. Vet Clin North Am Equine Pract 1993, 9:133–152. 10. Baker GJ: Endodontic therapy. In Equine dentistry. 2nd edition. Edited by Baker GJ, Easley J. Edinburgh: Elsevier Saunders; 2005:295–302. 11. Simhofer H, Stoian C, Zetner K: A long-term study of apicoectomy and endodontic treatment of apically infected cheek teeth in 12 horses. Vet J 2008, 178:411–418. 12. Wilke M, Roux P: Amikacin-imprägnierter Knochenzement zur Behandlung apikaler Abszesse der Backenzähne. In Proceedings of the DVG Tagung Fachgruppe Pferdekranheiten: 12–13 March 2010. Edited by Deutsche Veterinärmedizinische Gesellschaft. Hannover: DVG-Verlag; 2010. 30. Abbreviations 2D, Two-dimensional; 3D, Three-dimensional; HU, Hounsfield unit; ID, Identity; kVp, Kilovoltage peak; mA, Miliampere; mm, Milimetres; ms, Miliseconds; n, Number; rbd, Root bucco-distal; rbm, Root bucco-mesial; rd, Root distal; rm, Root mesial; rp, Root palatal; μCT, Micro-computed tomography; μm, Micrometres. ms, Miliseconds; n, Number; rbd, Root bucco-distal; rbm, Root bucco-mesial; rd, Root distal; rm, Root mesial; rp, Root palatal; μCT, Micro-computed tomography; μm, Micrometres. 13. Schramme MC, Boswell JC, Robinson J, Butson RJ, May SA, Smith RK, Platt D, Schumacher J: Endodontic therapy for periapical infection of the cheek teeth: a study of 19 horses. In Proceedings of the Annual Convention of the AAEP: 26–29 November 2000. 46th edition. Edited by AAEP. San Antonio: IVIS; 2000:113–116. Received: 28 February 2012 Accepted: 19 September 2012 Published: 25 September 2012 22. Du Toit N, Kempson SA, Dixon PM: Donkey dental anatomy. Part 1: Gross and computed axial tomography examinations. Vet J 2008, 176:338–344. Competing interests k f Susan Kopke: No financial and non-financial competing interests exist. Nina Angrisani: No financial and non-financial competing interests exist. Carsten Staszyk: No financial and non-financial competing interests exist. 14. Simhofer H, Stoian C, Zetner K: Apicoectomy of maxillary and mandibular cheek teeth in eleven horses - technique and results. In Proceedings of the Equine Dentistry Focus Meeting of the AAEP: September 2006. Edited by AAEP. Indianapolis: IVIS; 2006. Acknowledgements We wish to thank Dr. M. Lüpke for invaluable advice on the present study; G. Wirth and O. Stünkel for expert technical assistance and the Gesellschaft für Pferdemedizin e. V. (GPM) for financially supporting this project. 18. Setzer FC, Kohli MR, Shah SB, Karabucak B, Kim S: Outcome of endodontic surgery: a meta-analysis of the literature–Part 2: Comparison of endodontic microsurgical techniques with and without the use of higher magnification. J Endod 2012, 38:1–10. Author details 1 f Author details 1Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, Hannover D- 30173, Germany. 2Small Animal Hospital, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, Hannover D- 30559, Germany. 3Institute for Veterinary Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Str. 98, Giessen D- 35392, Germany Author details 1Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, Hannover D- 30173, Germany. 2Small Animal Hospital, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, Hannover D- 30559, Germany. 3Institute for Veterinary Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, Justus-Liebig-University Giessen, Frankfurter Str. 98, Giessen D- 35392, Germany. 19. Obiger G: Untersuchungen an den Pulpaästen der mandibulären Backenzähne des Pferdes unter besonderer Berücksichtigung der Ersatzdentinbildung. Thesis: Faculty of Veterinary Medicine of the University Leipzig, Large Animal Clinic for Surgery; 1939. 1Institute of Anatomy, University of Veterinary Medicine Hannover, Foundation, Bischofsholer Damm 15, Hannover D- 30173, Germany. 2Small Animal Hospital, University of Veterinary Medicine Hannover, Foundation, Bünteweg 9, Hannover D- 30559, Germany. 3Institute for Veterinary Anatomy, -Histology and -Embryology, Faculty of Veterinary Medicine, 20. Westenberger E: Cavum dentis und Pulpa dentis mandibulärer und maxillärer Backenzähne bei Pferden verschiedenen Alters. Thesis: University of Veterinary Medicine Hannover, Institute of Anatomy; 2002. Justus-Liebig-University Giessen, Frankfurter Str. 98, Giessen D- 35392, Germany. 21. Gasse H, Westenberger E, Staszyk C: The endodontic system of equine cheek teeth: a re-examination of pulp horns and root canals in view of age-related physiological differences. Pferdeheilkunde 2004, 20:13–18. Received: 28 February 2012 Accepted: 19 September 2012 Published: 25 September 2012 24. Muylle S: Aging. In Equine dentistry. 3rd edition. Edited by Baker GJ, Easley J. London: Saunders Elsevier; 2005:55–69. References 1. Dacre IT, Kempson S, Dixon PM: Pathological studies of cheek teeth apical infections in the horse: 1. Normal endodontic anatomy and dentinal structure of equine cheek teeth. Vet J 2008, 178:311–320. 2. Windley Z, Weller R, Tremain WH, Perkins JD: Two- and three-dimensional computed tomographic anatomy of the enamel, infundibulae and pulp of 126 equine cheek teeth. Part 1: Findings in teeth without 1. Dacre IT, Kempson S, Dixon PM: Pathological studies of cheek teeth apical infections in the horse: 1. Normal endodontic anatomy and dentinal structure of equine cheek teeth. Vet J 2008, 178:311–320. 2. Windley Z, Weller R, Tremain WH, Perkins JD: Two- and three-dimensional computed tomographic anatomy of the enamel, infundibulae and pulp of 126 equine cheek teeth. Part 1: Findings in teeth without 23. Dixon PM: The gross, histological, and ultrastructural anatomy of equine teeth and their relationship to disease. In Proceedings of the 48th Annual Meeting of the AAEP: 4–8 December 2002. Edited by AAEP. Orlando: IVIS; 2002:421–437. 1. Dacre IT, Kempson S, Dixon PM: Pathological studies of cheek teeth apical infections in the horse: 1. Normal endodontic anatomy and dentinal structure of equine cheek teeth. Vet J 2008, 178:311–320. 2. Windley Z, Weller R, Tremain WH, Perkins JD: Two- and three-dimensional computed tomographic anatomy of the enamel, infundibulae and pulp of 126 equine cheek teeth. Part 1: Findings in teeth without 24. Muylle S: Aging. In Equine dentistry. 3rd edition. Edited by Baker GJ, Easley J. London: Saunders Elsevier; 2005:55–69. 24. Muylle S: Aging. In Equine dentistry. 3rd edition. Edited by Baker GJ, Easley J. London: Saunders Elsevier; 2005:55–69. Page 16 of 16 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 Kopke et al. BMC Veterinary Research 2012, 8:173 http://www.biomedcentral.com/1746-6148/8/173 25. Martin MT: Guide for determining the age of the horse. AAEP: Lexington; 2007. 26. Dacre IT, Shaw DJ, Dixon PM: Pathological studies of cheek teeth apical infections in the horse: 3. Quantitative measurements of dentine in apically infected cheek teeth. Vet J 2008, 178:333–340. 27. Dacre IT, Kempson S, Dixon PM: Pathological studies of cheek teeth apical infections in the horse: 4. Aetiopathological findings in 41 apically infected mandibular cheek teeth. Vet J 2008, 178:341–351. 28. Dacre IT, Kempson S, Dixon PM: Pathological studies of cheek teeth apical infections in the horse: 5. References Aetiopathological findings in 57 apically infected maxillary cheek teeth and histological and ultrastructural findings. Vet J 2008, 178:352–363. 29. Dixon PM, Du Toit N, Dacre I: Equine dental pathology. In Equine dentistry. 3rd edition. Edited by Easley J, Dixon PM, Schumacher J. Edinburgh: Saunders Elsevier; 2010:129–148. 29. Dixon PM, Du Toit N, Dacre I: Equine dental pathology. In Equine dentistry. 3rd edition. Edited by Easley J, Dixon PM, Schumacher J. Edinburgh: Saunders Elsevier; 2010:129–148. 30. Joest E: Zähne. In Handbuch der speziellen pathologischen Anatomie der Haustiere. 3rd edition. Edited by Joest E. Berlin: Paul Parey; 1970. 31. Casey MB, Tremaine WH: The prevalence of secondary dentinal lesions in cheek teeth from horses with clinical signs of pulpitis compared to controls. Equine Vet J 2010, 42:30–36. 31. Casey MB, Tremaine WH: The prevalence of secondary dentinal lesions in cheek teeth from horses with clinical signs of pulpitis compared to controls. Equine Vet J 2010, 42:30–36. 32. Baker GJ, Kirkland DK: Endodontic therapy in the horse. In Proceedings of the 38th Annual Meeting of the AAEP: 2 December 1992. Edited by AAEP. Orlando: IVIS; 1992:329–335. 33. Carmalt JL, Barber SM: Periapical curettage: an alternative surgical approach to infected mandibular cheek teeth in horses. Vet Surg 2004, 33:267–271. 33. Carmalt JL, Barber SM: Periapical curettage: an alternative surgical approach to infected mandibular cheek teeth in horses. Vet Surg 2004, 33:267–271. doi:10.1186/1746-6148-8-173 Cite this article as: Kopke et al.: The dental cavities of equine cheek teeth: three-dimensional reconstructions based on high resolution micro-computed tomography. BMC Veterinary Research 2012 8:173. 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https://openalex.org/W2971837699	https://discovery.ucl.ac.uk/10120470/1/ciz474.pdf	English	null	Pediatric Bacterial Meningitis Surveillance in Nigeria From 2010 to 2016, Prior to and During the Phased Introduction of the 10-Valent Pneumococcal Conjugate Vaccine	Clinical infectious diseases/Clinical infectious diseases (Online. University of Chicago. Press)	2,019	cc-by	7,944	Pediatric Bacterial Meningitis Surveillance in Nigeria From 2010 to 2016, Prior to and During the Phased Introduction of the 10-Valent Pneumococcal Conjugate Vaccine Pediatric Bacterial Meningitis Surveillance in Nigeria From 2010 to 2016, Prior to and During the Phased Introduction of the 10-Valent Pneumococcal Conjugate Vaccine Downloaded from https://academic.oup.com/cid/article/69/Supplement_2/S81/5561327 by University College London user on 31 January 2021 Beckie N. Tagbo,1,2,a Rowan E. Bancroft,3,a Iretiola Fajolu,4,5 Mohammed B Abdulkadir,6 Muhammad F. Bashir,7 Olusola P. Okunola,8 Ayodeji H. Isiaka,9 Namadi M. Lawal,10 Benedict O. Edelu,2 Ngozi Onyejiaka,11 Chinonyerem J. Ihuoma,12 Florence Ndu,13 Uchenna C. Ozumba,12 Frances Udeinya,12 Folasade Ogunsola,11 Aishat O. Saka,6 Abayomi Fadeyi,14 Sunday A. Aderibigbe,15 Jimoh Abdulraheem,14 Adamu G. Yusuf,16 Peter Sylvanus Ndow,3 Philomena Ogbogu,17 Chinomnso Kanu,18 Velly Emina,19 Olajumoke J. Makinwa,11 Florian Gehre,2,20 Kabir Yusuf,10 Fiona Braka,21 Jason M. Mwenda,22 Johnson M. Ticha,9 Dorothy Nwodo,9 Archibald Worwui,3 Joseph N. Biey,22 Brenda A. Kwambana-Adams,3 and Martin Antonio3,23; for the African Paediatric Bacterial Meningitis Surveillance Network 1Institute of Child Health, University of Nigeria Teaching Hospital, Ituku-Ozalla, and 2Department of Paediatrics University of Nigeria Teaching Hospital Ituku-Ozalla, Enugu State; 3World Health Organization (WHO) Collaborating Centre for New Vaccines Surveillance, Medical Research Council Unit The Gambia at London School of Hygiene & Tropical Medicine, Banjul; 4Department of Paediatrics, Lagos University Teaching Hospital, 5Department of Paediatrics, College of Medicine, University of Lagos, 6Department of Paediatrics and Child Health, University of Ilorin Teaching Hospital, 7Department of Paediatrics, Abubakar Tafawa Balewa University Teaching Hospital, Bauchi, 8Department of Child Health, University of Benin Teaching Hospital, 9WHO Country office, Abuja, 10Department of Disease Control and Immunization, National Primary Health Care Development Agency, Abuja, 11Department of Medical Microbiology and Parasitology, Lagos University Teaching Hospital, 12Department of Microbiology, University of Nigeria Teaching Hospital, Ituku-Ozalla, Enugu State, 13Mother of Christ Specialist Hospital Enugu, 14Department of Medical Microbiology and Parasitology, University of Ilorin Teaching Hospital, Kwara, 15Department of Epidemiology and Community Health, University of Ilorin Teaching Hospital, Kwara, 16Medical Microbiology Department, Abubakar Tafawa Balewa University Teaching Hospital, Bauchi, 17Department of Medical Microbiology, University of Benin Teaching Hospital, 18Department of Community Health, University of Benin Teaching Hospital, and 19Department of Community Health and Primary Care, Lagos University Teaching Hospital, Nigeria; 20Department of Infectious Disease Epidemiology, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany; 21WHO, Nigeria EPI Cluster Lead; 22WHO Regional Office for Africa WHO/AFRO, Republic of Congo, Brazzaville; and 23Microbiology and Infection Unit, Warwick Medical School, University of Warwick, Coventry, United Kingdom. Background. Historically, Nigeria has experienced large bacterial meningitis outbreaks with high mortality in children. Pediatric Bacterial Meningitis Surveillance in Nigeria From 2010 to 2016, Prior to and During the Phased Introduction of the 10-Valent Pneumococcal Conjugate Vaccine Streptococcus pneumoniae (pneumococcus), Neisseria meningitidis (meningococcus), and Haemophilus influenzae are major causes of this invasive disease. In collaboration with the World Health Organization, we conducted longitudinal surveillance in sentinel hospitals within Nigeria to establish the burden of pediatric bacterial meningitis (PBM). g g Methods. From 2010 to 2016, cerebrospinal fluid was collected from children <5 years of age, admitted to 5 sentinel hospitals in 5 Nigerian states. Microbiological and latex agglutination techniques were performed to detect the presence of pneumococcus, me- ningococcus, and H. influenzae. Species-specific polymerase chain reaction and serotyping/grouping were conducted to determine specific causative agents of PBM. i g Results. A total of 5134 children with suspected meningitis were enrolled at the participating hospitals; of these 153 (2.9%) were confirmed PBM cases. The mortality rate for those infected was 15.0% (23/153). The dominant pathogen was pneumococcus (46.4%: 71/153) followed by meningococcus (34.6%: 53/153) and H. influenzae (19.0%: 29/153). Nearly half the pneumococcal meningitis cases successfully serotyped (46.4%: 13/28) were caused by serotypes that are included in the 10-valent pneumococcal conjugate vac- cine. The most prevalent meningococcal and H. influenzae strains were serogroup W and serotype b, respectively. hl Conclusions. Vaccine-type bacterial meningitis continues to be common among children <5 years in Nigeria. Challenges with vaccine introduction and coverage may explain some of these finding. Continued surveillance is needed to determine the distribu- tion of serotypes/groups of meningeal pathogens across Nigeria and help inform and sustain vaccination policies in the country. Keywords. pediatric; meningitis; Nigeria; pneumococcus; meningococcus. Keywords. pediatric; meningitis; Nigeria; pneumococcus; meningococcus. Keywords. but without adequate treatment, fatalities can be as high as 70% [1, 3]. The African meningitis belt is the most significantly affected area worldwide, with an estimated 400 million people at-risk an- nually. Bacterial meningitis epidemics have occurred frequently across this region for over a century: one of the largest occurred in Nigeria during 1996, with over 109 000 cases and 11 000 deaths [4]. Subsequently, there have been several major outbreaks within Nigeria, over wide geographical areas, most notably in 2009 and 2015 [4, 5]. In December 2016, the Nigeria Centre for Disease Control (NCDC) received reports of a meningitis outbreak. By May 2017, there were 13 420 suspected cases and 1069 deaths (8%), of which 47% were children in the 5–14 year age group [6]. Correspondence: B. N. Tagbo, Institute of Child Health, University of Nigeria Teaching Hospital, Enugu, Nigeria (tagbobeckie@gmail.com). aB. N. T. and R. E. B. contributed equally to this article. Clinical Infectious Diseases® 2019;69(S2):S81–8 © The Author(s) 2019. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciz474 Clinical Infectious Diseases S U P P L E M E N T A R T I C L E Clinical Infectious Diseases Clinical Infectious Diseases Case Enrollment Hospitalized children aged 0–59 months (<5 years) with features of suspected meningitis: rapid onset of fever with axillary or rectal temperatures of >38°C and >38.5°C, combined with any of the following symptoms: impaired consciousness, meningismus (stiff neck), photophobia, bulging fontanelle (infants), and convulsions were enrolled in the surveillance [16]. A lumbar puncture (LP) was performed for routine diagnostic tests, and cerebrospinal fluid (CSF) was collected. Nigeria is the most populous country within Africa, with an estimated 193 million people living in 36 states [10]. The national human immunodeficiency virus (HIV) prevalence among adults aged 15–49 year in Nigeria is 1.4%; however, this varies between states with 5.6% estimated in the southern Akwa Ibom State and 0.3% in the northwestern Katsina State [11, 12]. In 2012, the Nigerian Expanded Program on Immunization (EPI) replaced the diphtheria-tetanus-pertussis and hepatitis B vaccines with a pentavalent vaccine that included H. influenzae type b (Hib) and underwent a phased introduction [13]. Additionally, meningococcal A conjugate vaccine (MenAfriVac) campaigns were carried out from 2011 to 2014 in 19 states in Northern Nigeria. The 2017 outbreak was caused by meningo- coccal serogroup C; thus, a vaccine with longevity against this strain is required [14]. A phased introduction of the 10-valent pneumococcal conjugate vaccine (PCV10) throughout Nigeria, targeting 10 invasive pneumococcal serotypes, commenced in December 2014 and concluded in October 2016. However, the 2017 WHO and United Nations Children’s Fund (UNICEF) na- tional immunization coverage estimates suggest that coverage rates for children who received 3 doses of PCV10 and Hib- containing vaccines remain low, at 36% and 42%, respectively [15]. As part of the IB-VPD network, longitudinal surveillance was established in 2010 within 5 sentinel hospitals in 5 Nigerian states. We have conducted analysis to estimate the prevalence of bacterial meningitis in children <5 years of age and determine the distribution of the causative pathogens from 2010 to 2016. Pediatric Bacterial Meningitis Surveillance in Nigeria From 2010 to 2016, Prior to and During the Phased Introduction of the 10-Valent Pneumococcal Conjugate Vaccine Acute bacterial meningitis is a much-dreaded infectious disease in children and a major cause of morbidity and mortality [1, 2]. The incidence and case-fatality rates for this invasive bacterial disease vary by region, country, causative-agent, and age-group affected, Pediatric Bacterial Meningitis Nigeria • cid 2019:69 (Suppl 2) • S81 The leading causes of bacterial meningitis are Neisseria meningitidis (meningococcus), Streptococcus pneumoniae (pneumococcus), and Haemophilus influenzae; which are usually carried asymptomatically in the nasopharynx and transmitted through respiratory droplets [1]. Normally, the pathogen responsible for epidemics is the Neisseria spe- cies, whereas pneumococcus and H. influenzae are endemic throughout the year [4, 7, 8]. In 2008, the World Health Organization (WHO) established the Global Invasive Bacterial- Vaccine Preventable Diseases (IB-VPD) network building on regional surveillance networks. This global surveillance net- work encompasses 100 sentinel hospital laboratories, aiming to estimate the burden of bacterial meningitis and characterize circulating bacterium within member countries [2, 9]. As part of this, the Medical Research Council Unit The Gambia at the London School of Hygiene and Tropical Medicine (MRCG at LSHTM), a WHO Collaborating Center for New Vaccines Surveillance (WHO CC NVs), provides laboratory support for pediatric bacterial meningitis (PBM) surveillance in 10 coun- tries across West Africa, including Nigeria. surveillance started in Lagos University Teaching Hospital (Lagos State) in 2010, followed by University of Nigeria Teaching Hospital (Enugu State) in 2011. In the following year, 2 more sites were included: Abubakar Tafawa Balewa University Teaching Hospital (Bauchi State) and University of Ilorin Teaching Hospital (Kwara State). Finally, in 2013, University of Benin Teaching Hospital (Edo State) joined sur- veillance. Therefore, surveillance was conducted in 5 states with a combined estimated population of approximately 32 million people. Molecular Analysis CSF samples were transported to the MRCG at LSHTM ac- cording to International Air Transport Association (IATA) regulations [19]. Species-specific quantitative polymerase chain reaction (qPCR) assays for detection of pneumococcus, meningococcus, and H. influenzae were performed, using the autolysin gene (lytA), Cu, Zn superoxide dismutase gene (sodC) and protein D encoding gene (hpd), respectively, as described elsewhere [20]. For amplification, samples were heated at 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. Cycle threshold (CT) values of ≤36 were considered positive results. CSF Analysis CSF samples were processed following the WHO standard op- erating procedure [16]. An aliquot of CSF was centrifuged, and the deposit inoculated onto Columbia blood and chocolate agar plates and incubated overnight. Isolates of the target pathogens were identified using the optochin test (5 μg optochin disk; Oxoid, Basingstoke, UK) for pneumococcus and analytical pro- file index (API NH; Biomerieux, Basingstoke, UK) for meningo- coccus and H. influenzae. The remaining centrifuged pellet was used to prepare smears for Gram staining. Using the supernatant, latex agglutination was performed with the Pastorex meningitis kit (Biorad, Watford, UK), for the detection of pneumococcus, Hib and meningococcus groups A, B, C, Y, and W antigens. When possible, CSF was used to detect the presence of pneumo- coccus using the BINAX® NOW kit (Alere Inc., Waltham, MA, USA). A white blood cell (WBC) count was conducted along with CSF protein and glucose analysis using trichloroacetic acid turbidimetric and glucose oxidase methods [17, 18]. Serotyping and Serogrouping then sent to WHO Afro data managers, and feedback was pro- vided to sites every 3–6 months. WHO Afro also sent regional data to WHO Global data managers. For presentation here, data were analyzed using GraphPad Prism 8.1.1; percentages, pro- portion, means, and standard deviations were calculated as ap- propriate and presented as prose, tables, and figures. Meningococcus and H. influenzae serogrouping/typing was conducted using direct qPCR. Meningococcal gene targets were: sacB, synD, synE, synG, xcbB, synF for serogroups A, B, C, W, X, and Y, respectively. Additionally, for H. influenzae gene targets included acsB, bcsB, ccsD, dscE, ecsH, bexD for serotypes Hia, Hib, Hic, Hid, Hie, and Hif, respectively. CT values of ≤32 were considered positive [21]. For pneumococcus, nucleic acid extraction using Qiagen DNA Mini-kit was performed. Purified DNA underwent sequential triplex qPCR for detection of 21 capsular serotypes as described elsewhere [22]. Nontypeable pneumococci, with CT values ≤32 by qPCR, were further subjected to conventional multiplex serotyping PCR assays. Ethical approval was not a requirement in Nigeria for routine meningitis surveillance including drug susceptibility testing of collected isolates as this is approved within the routine di- agnostic algorithm at the Ministry of Health. However, in- formed consent was sought from caregivers of the surveillance participants. Additionally, the surveillance received overarching ethical approval (SCC1188) by the joint MRC/The Gambia Government ethics board that allowed the analysis of collected West African isolates at MRC Unit, The Gambia. Statistical Analysis Data were collected at the sentinel hospitals using a standardized WHO Regional Office for Africa (Afro) PBM network case report form. Information recorded included, patient demo- graphics, clinical symptoms, vaccination history, laboratory in- formation (CSF microscopy, bacteriological tests, genotyping), and outcome at discharge. Data were subsequently put into a WHO Epi Info-based customized new-vaccine surveillance data module. Data cleaning and analysis were first performed at the sentinel site level before being sent to the national and regional WHO data managers. At the national level, data from sites were merged, cleaned, analyzed, and interpreted. Merged data were Pediatric Bacterial Meningitis Nigeria • cid 2019:69 (Suppl 2) • S83 Demographic and Clinical Characteristics Details of the demographic characteristics of the children enrolled in surveillance are shown in Table 1. A total of 5134 children <5 years of age with suspected bacterial meningitis were enrolled at the sentinel hospitals from 2010 to 2016. Of these, 5008 (97.5%: 5008/5134) children had CSF samples collected, which then underwent diagnostic testing. Overall, 57.8% (2969/5134) of patients were male and the median age Table 1. Summary of Demographic Characteristics of Study Population Total Bauchia Lagosb Edoc Kwarad Enugue Characteristic Category n (%) n (%) n (%) n (%) n (%) n (%) Age 0–11 m 3014 (58.7) 230 (32.4) 1473 (72.3) 588 (67.1) 222 (37.1) 501 (54.9) 12–23 m 798 (15.5) 120 (16.9) 222 (10.9) 120 (13.7) 127 (21.2) 209 (22.9) 24–59 m 1234 (24.0) 358 (50.4) 264 (13.0) 164 (18.7) 247 (41.3) 201 (22.0) Unknown 88 (1.7) 2 (0.3) 79 (3.9) 4 (0.5) 2 (0.3) 1 (0.1) Sex Female 2147 (41.8) 300 (42.3) 843 (41.4) 356 (40.6) 269 (45.0) 379 (41.6) Male 2969 (57.8) 410 (57.7) 1181 (57.9) 516 (58.9) 329 (55.0) 533 (58.4) Unknown 18 (0.4) 0 (0.0) 14 (0.7) 4 (0.5) 0 (0.0) 0 (0.0) Antibiotic before admission Yes 743 (14.5) 62 (8.7) 313 (15.4) 24 (2.7) 85 (14.2) 259 (28.4) No 2972 (57.9) 581 (81.8) 622 (30.5) 834 (95.2) 329 (55.0) 606 (66.4) Unknown 1419 (27.6) 67 (9.4) 1103 (54.1) 18 (2.1) 184 (30.8) 47 (5.2) Outcome diagnosis Meningitis 363 (7.1) 107 (15.1) 87 (4.3) 42 (4.8) 22 (3.7) 105 (11.5) Pneumonia 72 (1.4) 37 (5.2) 11 (0.5) 4 (0.5) 12 (2.0) 8 (0.9) Septicemia 235 (4.6) 26 (3.7) 101 (5.0) 4 (0.5) 34 (5.7) 70 (7.7) Other/multiple 1022 (19.9) 301 (42.4) 120 (5.9) 173 (19.7) 255 (42.6) 173 (19.0) Unknown 3442 (67.0) 239 (33.7) 1719 (84.3) 653 (74.5) 275 (46.0) 556 (61.0) Outcome Discharged Alive 3107 (60.5) 561 (79.0) 935 (45.9) 308 (35.2) 456 (76.3) 847 (92.9) Died 278 (5.4) 93 (13.1) 49 (2.4) 52 (5.9) 39 (6.5) 45 (4.9) Unknown 1749 (34.1) 56 (7.9) 1054 (51.7) 516 (58.9) 103 (17.2) 20 (2.2) Total no. of suspected cases recruitedf 5134 (100.0) 710 (13.8) 2038 (39.7) 876 (17.1) 598 (11.6) 912 (17.8) aAbubakar Tafawa Balewa University Teaching Hospital. bLagos University Teaching Hospital. cUniversity of Benin Teaching Hospital. dUniversity of Ilorin Teaching Hospital. eUniversity of Nigeria Teaching Hospital. fSuspected cases include cases that were defined as probable per World Health Organization case definition guidelines [16]. Surveillance Sites Surveillance of children <5 years admitted to sentinel hospitals with suspected meningitis, was carried out for 7 years. PBM S82 • cid 2019:69 (Suppl 2) • Tagbo et al Serotyping and Serogrouping Serotype and Serogroup Distribution Serotype analysis was attempted on 31 (43.7%: 31/71) pneu- mococcal positive CSF samples, however, 3 samples had a low concentration of DNA with CT values of >32 so could not be serotyped. Of the 28 isolates that were successfully serotyped, a variety of pneumococcal serotypes were responsible for PBM from year to year. For instance, the pneumococcal meningitis cases in 2011 and 2012 were caused by serotype 23F and 14, re- spectively, both of which are targeted by the PCV10 vaccine. We successfully serotyped 2 cases in 2013, which were caused by serotype 23F and 19A, which is a non-PCV10 serotype. In 2014, 10 pneumococcal meningitis cases were serotyped; of these, 1 There were a total of 153 (3.0%: 153/5134) cases of PBM observed at the 5 sentinel hospitals during the surveillance period in Nigeria. Pneumococcus was responsible for 71 cases (46.4%: 71/153) with a mortality rate of 14.1% (10/71). Meningococcal meningitis was confirmed in 53 (34.6%:53/153) pediatric patients with a case fatality rate of 20.7% (11/53). A total of 29 (19.0%: 29/153) children had meningitis caused by H. influenzae, among these 2 patients died; mortality rate of 3.4% (2/29). The overall mortality rate for confirmed bacterial meningitis cases was 15% (23/153). Table 2. Summary of Clinical Characteristics of Patients in Relation to Causative Recruited Tested Pneumococcus Meningococcus Haemophilus influenzae Characteristic n n (%) n (%) n (%) n (%) Cerebrospinal fluid appearance Clear 3338 3281 (98.3) 39 (1.2) 18 (0.5) 18 (0.5) Turbid 418 411 (98.3) 20 (4.9) 18 (4.4) 6 (1.5) Xanthrochromic 635 624 (98.3) 6 (1.0) 9 (1.4) 1 (0.2) Blood stained 614 607 (98.9) 4 (0.7) 8 (1.3) 3 (0.5) Unknown 129 85 (65.9) 2 (2.4) 0 1 (1.2) White blood cell count (cells/mm3) ≤10 3888 3840 (98.8) 33 (0.9) 20 (0.5) 14 (0.4) >10 to 100 352 348 (98.3) 4 (1.1) 6 (1.7) 2 (0.6) >100 196 195 (99.5) 15 (7.7) 11 (5.6) 3 (1.5) Unknown/not done 698 625 (90.0) 19 (3.0) 16 (2.6) 10 (1.6) Protein (mg/dL) ≤100 3010 2952 (98.1) 41 (1.4) 20 (0.7) 20 (0.7) >100 441 432 (98.0) 15 (3.5) 23 (5.3) 4 (0.9) Unknown/not done 1683 1624 (96.5) 15 (0.9) 10 (0.6) 5 (0.3) Glucose (g/dL) ≤40 1279 1261 (98.6) 21 (1.7) 23 (1.8) 13 (1.0) ≥40 2193 2146 (97.9) 35 (1.6) 19 (0.9) 10 (0.5) Unknown/not done 1662 1601 (96.3) 15 (0.9) 11 (0.7) 6 (0.4) Total no. Distribution of Bacterial Pathogens of patients was 22 months (interquartile range: 1–23). The largest proportion of meningitis cases were reported in children from the youngest age group, 0–11 months, with 3014 (58.7%: 3014/5134) suspected cases. Unfortunately, 278 patients with suspected meningitis died in hospital resulting in a case fatality of 5.4% (278/5134). The number of suspected PBM cases observed in each of the 5 sentinel hospitals varied annually with an average of 715.4 cases per year (Figure 1). In 2013, with 5 sentinel hospitals enrolled, the total number of suspected cases peaked at 1204. Lagos State had the highest proportion of suspected meningitis over the study period (39.7%: 2038/5134) and Kwara State the least (11.6%: 598/5134). Additionally, the prevalence of menin- gitis caused by each of the 3 bacterial pathogens varied annually (Figure 2). The clinical characteristics of the children enrolled are de- tailed in Table 2. In summary, 75 (2.2%: 75/3338) children who presented with clear CSF samples had confirmed bacterial meningitis. However, a higher proportion of patients (4.5%: 75/1667) with turbid, xanthrochromic and blood-stained CSF samples had confirmed bacterial meningitis. The percentage of patients with PBM increased with WBC count; 1.7% (67/3888) of patients with a low WBC count (≤10 cells/mm3) had bacte- rial meningitis, whereas 3.4% (12/352) and 14.8% (29/196) of children with >10 to 100 cells/mm3 and >100 cells/mm3, respec- tively, were infected. More patients with PBM had high levels of protein (>100 mg/dL) in their CSF (9.5%: 42/441) compared to those with low protein levels ≤100 mg/dL (2.7%: 81/3010). However, more children with CSF glucose levels of ≤40 g/ dL had PBM (4.5%: 57/1279) compared to 2.9% (64/2193) of patients with lower glucose levels (>40 g/dL). The frequency of confirmed meningitis cases observed fluctuated between months of the year (Figure 3). Presentation of PBM was highest from February to June throughout the sur- veillance period, and the highest number of cases was recorded in April. The prevalence of each causative agent varied from month to month. For instance, from February to April, most cases were caused by meningococcus 34 (51.5%: 34/66) and from May to August the dominant pathogen switched to pneu- mococcus with 28 (59.6%: 28/47) cases. Demographic and Clinical Characteristics Table 1. Summary of Demographic Characteristics of Study Population Distribution of Bacterial Pathogens Distribution of Bacterial Pathogens ned as probable as per World Health Organization case definition guidelines [16]. Serotype and Serogroup Distribution Each hospital commenced surveillance at different time points; however, surveillance continued for all hospitals until 2016. The number of suspected cases of bacterial meningitis and the number of confirmed cases of meningitis (World Health Organization definitions [16]) varied per hospital. The main causative agents for bacterial meningitis were Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. successfully serotyped were caused by serotypes that are not targeted by PCV10 vaccine. case was caused by serotype 4, a PCV10 serotype. However, the remaining 9 (90%: 9/10) were caused by pneumococcal strains that were nontypeable by PCR and, thus, not covered by the current formulations of PCV. The 3 serotyped cases in 2015 were all caused by PCV10 serotypes, serotype’s 1, 5, and 18C. A further 11 cases were serotyped in 2016: 6 cases were caused by 4 PCV10 serotypes (4, 6B, 19F, and 23F), 4 cases were caused by pneumococci that were nontypeable by PCR, and 1 case was caused by serotype 23A, a non-PCV10 pathogen. Overall, half (50.0%: 14/28) of the pneumococcal meningitis cases that were Furthermore, most of the meningococcal meningitis cases were reported from 2014 to 2016, with 41 (77.4%: 41/53) patients across the 3 years and a peak of 17 cases in 2015. In total, 20 (37.7%: 20/53) meningococcal isolates underwent serogrouping analysis. Four serogroups were observed within the surveillance period, the most prevalent was serogroup (50.0%: 14/28) of the pneumococcal meningitis cases that were Figure 2. Proportion of confirmed pediatric bacterial meningitis cases and the pathogens responsible from 2010 to 2016 in 5 Nigerian states. The percentage of confirmed bacterial meningitis cases caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in children <5 years across 5 Nigerian states. A dashed black line indicates the total number of cerebrospinal fluid samples that were tested each year of surveillance. Figure 3. Monthly distribution of suspected pediatric bacterial meningitis cases for the period 2010 to 2016 within 5 sentinel hospitals across Nigeria. The per- centage of pediatric bacterial meningitis (PBM) cases seen across 5 hospitals in 5 Nigerian states, caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae per month. A black dashed line indicates the total number of PBM cases per month throughout the surveillance period. 327 by University College London user on 31 January 2021 Figure 2. Proportion of confirmed pediatric bacterial meningitis cases and the pathogens responsible from 2010 to 2016 in 5 Nigerian states. Serotype and Serogroup Distribution of suspected cases recruiteda 5134 5008 (97.5) 71 (1.4) 53 (1.1) 29 (0.6) aSuspected cases include cases that were defined as probable as per World Health Organization case definition guidelines [16]. Table 2. Summary of Clinical Characteristics of Patients in Relation to Causative S84 • cid 2019:69 (Suppl 2) • Tagbo et al Figure 1. Distribution of suspected pediatric bacterial meningitis cases from 2010 to 2016 within 5 sentinel hospitals in 5 Nigerian states. A total of 5134 suspected pe- diatric bacterial meningitis cases were observed at 5 sentinel hospitals: Lagos University Teaching Hospital (Lagos State), University of Nigeria Teaching Hospital (Enugu State), Abubakar Tafawa Balewa University Teaching Hospital (Bauchi State), University of Ilorin Teaching Hospital (Kwara State), and University of Benin Teaching Hospital (Edo State). Each hospital commenced surveillance at different time points; however, surveillance continued for all hospitals until 2016. The number of suspected cases of bacterial meningitis and the number of confirmed cases of meningitis (World Health Organization definitions [16]) varied per hospital. The main causative agents for bacterial meningitis were Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Figure 1. Distribution of suspected pediatric bacterial meningitis cases from 2010 to 2016 within 5 sentinel hospitals in 5 Nigerian states. A total of 5134 suspected pe- diatric bacterial meningitis cases were observed at 5 sentinel hospitals: Lagos University Teaching Hospital (Lagos State), University of Nigeria Teaching Hospital (Enugu State), Abubakar Tafawa Balewa University Teaching Hospital (Bauchi State), University of Ilorin Teaching Hospital (Kwara State), and University of Benin Teaching Hospital (Edo State). Each hospital commenced surveillance at different time points; however, surveillance continued for all hospitals until 2016. The number of suspected cases of bacterial meningitis and the number of confirmed cases of meningitis (World Health Organization definitions [16]) varied per hospital. The main causative agents for bacterial meningitis were Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. Downloaded from https://academic.oup.com/cid/article/69/Supplement_2/S81/5561327 by University College London user on 31 January 2021 Figure 1. Distribution of suspected pediatric bacterial meningitis cases from 2010 to 2016 within 5 sentinel hospitals in 5 Nigerian states. A total of 5134 suspected pe- diatric bacterial meningitis cases were observed at 5 sentinel hospitals: Lagos University Teaching Hospital (Lagos State), University of Nigeria Teaching Hospital (Enugu State), Abubakar Tafawa Balewa University Teaching Hospital (Bauchi State), University of Ilorin Teaching Hospital (Kwara State), and University of Benin Teaching Hospital (Edo State). DISCUSSION We conducted detailed analysis of longitudinal PBM surveil- lance data from 5 hospitals in 5 states of Nigeria. We found the highest number of suspected bacterial meningitis cases in children from the youngest age group and a lower number of cases reported in children aged 12–23 months but rising in the eldest cohort of children aged 24–59 months (Table 1). This was unexpected, as previous studies in The Gambia, Oman, and Turkey have shown that the incidence of bacterial meningitis decreases with age, due to maturation of the immune response [23–25]. The mortality rate of suspected meningitis was 5.4% (278/5134). However, when excluding data for patients where the outcome at discharge was not recorded (34.1%: 1749/5134), the mortality rate increased to 8.2% (278/3385), suggesting that suspected meningitis patients were severely unwell. Sequelae was only reported for 2% (102/5134) of patients with suspected meningitis; however, this prevalence is likely to be higher given the large number of patients (62.3%: 3245/5134) without fol- low-up after discharge.fi Vaccines targeting the main bacterial pathogens causing meningitis are effective at reducing morbidity and mortality [37, 38]. The Hib vaccine was introduced in phases across Nigeria from May 2012 to May 2014, and 41.4% (12/29) of the H. influenzae associated meningitis cases occurred during this period, with a further 48.3% (14/29) cases postvaccine intro- duction. We identified 11 cases of Hib meningitis; of these, 7 patients had not received the Hib vaccine, and the Hib vacci- nation status for the remaining 4 patients was unknown. The WHO and UNICEF 2017 national immunization coverage estimates for the Hib containing vaccine in Nigeria were <50% [15]. Additionally, in 2017, Nigeria’s National Primary Health Care Development Agency (NPHCDA) found that only Bauchi and Kwara of the 5 states participating in PBM surveillance re- ported >50% coverage of the Hib vaccine, indicating that cov- erage across Nigeria needs to be improved [39]. Diagnosis of acute bacterial meningitis can be difficult in sub-Saharan Africa where resources are limited and clinical features are similar to malarial infections [26]. Analysis of CSF through microbiology culture is the gold standard tech- nique used to detect PBM; however, studies have found low bacterial recovery rates in West Africa [27]. Additionally, 14.5% (743/5134) of patients with suspected meningitis in Nigeria received antibiotics before admission, which may have contributed to low bacteriological recovery. Serotype and Serogroup Distribution W, with 8 cases (40%: 8/20) reported in 2015, followed by a nongroupable strain causing 1 case in 2015 and 5 cases in 2016 (30%: 6/20). Serogroup B meningococcus was responsible for 5 case, 1 each in 2013 and 2015 and a further 3 in 2016. However, serogroup C was identified in 1 case of meningococcal menin- gitis in 2014. when combined with other common symptoms [28]. During surveillance, only 22% (34/153) of meningitis patients had an altered consciousness. Additionally, meningismus (10.5%: 16/153) and bulging fontanelle (8.5%: 13/153) were observed less frequently, highlighting the importance of considering all clinical symptoms when diagnosing PBM as outlined in the WHO guidelines [16]. Finally, of the 29 H. influenzae positive CSF samples, se- rotype analysis was successfully performed on 16 (55.2%: 16/29) of these. The most prevalent serotype was Hib, respon- sible for 1 case in 2012, 2 cases in 2013, and 8 cases in 2016 (69%:11/16). Hia caused 3 infections in 2015, and Hic was re- sponsible for 2 infection in 2015. Interestingly, 2 of the patients with H. influenzae meningitis reported receiving at least 1 dose of the Hib vaccine, 1 case was caused by serotype Hia, and the other was not serotyped. West Africa has a bimodal climate: wet season occurs from mid-April until mid-October, and the remaining months are characterized by dry season. Available data suggest the inten- sity of bacterial meningitis epidemics are associated with the Harmattan winds during dry season; thus, epidemics are rare during rainy season [29, 30]. The seasonal distribution of PBM within Nigeria from 2010 to 2016 is in line with this trend (Figure 3). Previous studies have shown that respiratory syn- cytial virus (RSV) is also seasonal in Nigeria, with cases mainly observed during the dry season and often peaking in November [31, 32]. Conversely, influenza has been shown to have year- round activity within Nigeria [33, 34]. Moreover, the season- ality of malaria transmission within Nigeria differs across the country based on the various ecological regions. Thus, malaria transmission is often year-round in southern Nigeria where there are high levels of mangrove swamps but lasts 3 months or less in northern regions such as the Sahel-savannah [35, 36]. Serotype and Serogroup Distribution The percentage of confirmed bacterial meningitis cases caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in children <5 years across 5 Nigerian states. A dashed black line indicates the total number of cerebrospinal fluid samples that were tested each year of surveillance. Figure 2. Proportion of confirmed pediatric bacterial meningitis cases and the pathogens responsible from 2010 to 2016 in 5 Nigerian states. The percentage of confirmed bacterial meningitis cases caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae in children <5 years across 5 Nigerian states. A dashed black line indicates the total number of cerebrospinal fluid samples that were tested each year of surveillance. Figure 3. Monthly distribution of suspected pediatric bacterial meningitis cases for the period 2010 to 2016 within 5 sentinel hospitals across Nigeria. The per- centage of pediatric bacterial meningitis (PBM) cases seen across 5 hospitals in 5 Nigerian states, caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae per month. A black dashed line indicates the total number of PBM cases per month throughout the surveillance period. Figure 3. Monthly distribution of suspected pediatric bacterial meningitis cases for the period 2010 to 2016 within 5 sentinel hospitals across Nigeria. The per- centage of pediatric bacterial meningitis (PBM) cases seen across 5 hospitals in 5 Nigerian states, caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae per month. A black dashed line indicates the total number of PBM cases per month throughout the surveillance period. Figure 3. Monthly distribution of suspected pediatric bacterial meningitis cases for the period 2010 to 2016 within 5 sentinel hospitals across Nigeria. The per- centage of pediatric bacterial meningitis (PBM) cases seen across 5 hospitals in 5 Nigerian states, caused by Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae per month. A black dashed line indicates the total number of PBM cases per month throughout the surveillance period. Pediatric Bacterial Meningitis Nigeria • cid 2019:69 (Suppl 2) • S85 W, with 8 cases (40%: 8/20) reported in 2015, followed by a nongroupable strain causing 1 case in 2015 and 5 cases in 2016 (30%: 6/20). Serogroup B meningococcus was responsible for 5 case, 1 each in 2013 and 2015 and a further 3 in 2016. However, serogroup C was identified in 1 case of meningococcal menin- gitis in 2014. S86 • cid 2019:69 (Suppl 2) • Tagbo et al Limitations Acknowledgments. The authors thank the WHO Country Office of Nigeria and WHO Inter-country Support Team for coordination, advice and support throughout the project. They thank the surveillance team members as well as patients and their families in Nigeria; the staff and students at the MRCG at London School of Hygiene and Tropical Medicine [LSHTM], as well as the IBD writing group for their advice and input.h During this surveillance, children with suspected meningitis were not followed up after hospital discharge. There were a total of 278 (5.4%: 278/5134) in-hospital deaths; of these, 23 (8.2%: 23/278) were patients with confirmed bacterial meningitis. However, the outcome for 1749 patients (34.1% 1749/5134) was unknown or not recorded, and of these, 26 (1.5%: 26/1749) were patients with confirmed bacterial meningitis. Therefore, the case fatality of 5.4% (278/5134) among children with suspected meningitis may have been higher, but this cannot be confirmed. Additionally, 102 (2%: 102/5134) patients were reported to have long-term sequelae; however, 3245 (63.2%: 3245/5134) were not followed up after dis- charge, and the HIV status was not routinely recorded during the surveillance. Moreover, there were discrepancies between the level of reporting from each sentinel hospital. In future studies, it would be beneficial to record information regarding long-term sequelae, outcome at discharge, and HIV status for all patients with suspected meningitis and to do this in a standardized manner across all sites. This would allow more accurate rates of morbidity and mortality associated with bacterial meningitis within the sur- veillance populations to be estimated. Disclaimer. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions the World Health Organization, the Medical Research Council Unit The Gambia at the London School of Hygiene and Tropical Medicine or the authors’ affiliated institutions. Disclaimer. The opinions expressed by authors contributing to this journal do not necessarily reflect the opinions the World Health Organization, the Medical Research Council Unit The Gambia at the London School of Hygiene and Tropical Medicine or the authors’ affiliated institutions. fi Financial support. Financial support for sentinel site surveillance was provided by the Federal Ministry of Health Nigeria, Gavi - the Vaccine Alliance, through a grant to the WHO for the African Paediatric Bacterial Meningitis Surveillance Network.h Financial support. Limitations Financial support for sentinel site surveillance was provided by the Federal Ministry of Health Nigeria, Gavi - the Vaccine Alliance, through a grant to the WHO for the African Paediatric Bacterial Meningitis Surveillance Network.h Supplement sponsorship. This supplement was supported with funds from Gavi, the Vaccine Alliance through The World Health Organization and the CDC Foundation, and The Medical Research Council Unit The Gambia at the London School of Hygiene and Tropical Medicine.ll Potential conflicts of interest. All authors: No reported conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed. Notes Invasive Bacterial Disease Writing Group members. Brenda Kwambana Adams, Senghore Madikay, Effua Usuf, Archibald Worwui, Uzochukwu Egere, Akram Zaman, Catherine Okoi, Florian Gehre, Leopold Tientcheu, Nuredin Ibrahim Mohammed, Felix Dube, Peter Ndow, Sambou M. Suso, Sheikh Jarju, Dam Khan, Chinelo Ebruke, Rowan Bancroft, Jason M. Mwenda, and Martin Antonio. Author contributions. M. A. and J. M. M. established the World Health Organization Regional Office for Africa–supported Paediatric Bacterial Meningitis Surveillance Network in West Africa. M. A. supervised the overall network including setting up the sentinel surveillance system. B. N. T., I. F., M. B. A., M. F. B., O. P. O., A. H. I., N. M. L., B. O. E., N. O., C. J. I., F. N., U. C. O., F. U., A. O. S., A. F., S. A. A., J. A., A. G. Y., P. O., C. K., V. E., and O. J. M. clinically investigated and recruited the patients at the sentinel sites, collected demographic data, performed microbiological testing, and shipped cerebrospinal fluid and bacterial isolates to the Medical Research Council (MRC) Unit The Gambia (MRCG) for confirmatory testing and molecular analysis supervised by B. K. A. and M. A. B. K. A. and M. A. de- veloped the analysis plan and contributed to analysis and interpretation of data along with the IBD writing group. R. E. B., B. K. A., and M. A. drafted the article along with B. N. T. All authors contributed to the interpretation of the findings and the writing of the final article.hfi CONCLUSIONS nonserogroup A meningococcus, but, as limited serogrouping data were obtained, further work is required to conclude sero- type replacement is occurring [40]. Pneumococcus was responsible for the majority of PBM cases in Nigeria; however, PCV10 has now been introduced within all states included in surveillance, and with improved vaccine coverage it is expected that pneumococcal meningitis rates will start to decline. Hib remains responsible for a significant pro- portion of meningitis despite vaccine introduction. Therefore, our findings emphasize the need for further monitoring to es- tablish the impact of conjugate vaccines on reducing the preva- lence of bacterial meningitis within Nigeria. Further serotype/ group data for PBM cases is required to understand the distri- bution of specific pathogen strains across Nigeria and to en- hance efficacy of target vaccines. yp p g Pneumococcus was the predominant pathogen causing 71 (46.4%: 71/153) PBM cases during surveillance in Nigeria. A high incidence of pneumococcal meningitis is also common in The Gambia [25]. Of the 5 states where surveillance occurred, Edo state commenced PCV10 vaccinations in 2014, and only 2 pneumococcal meningitis cases were reported here afterward. The remaining 4 states received PCV10 in 2016; thus, pre- and postvaccine comparisons are not possible, and we were unable to record the PCV10 vaccination status of patients throughout our surveillance period. However, 46.4% (13/28) of the isolates serotyped were strains that are included in PCV10 and thus could have been prevented by immunization. The 2017 WHO and UNICEF national immunization coverage estimates sug- gest that coverage rates for PCV10 are <50% in Nigeria [15]. For each of the 5 states enrolled in surveillance, coverage of 3 PCV10 doses from January to November 2017 was >50% in Bauchi and Kwara only [39]. We found 1 case of pneumococcal meningitis caused by serotype 19A, targeted by PCV13 but not PCV10, and 13 cases caused by pneumococcal serotypes that were nontypeable and thus not covered by current PCV formulations. Serotype replacement of vaccine serotypes with nonvaccine serotypes is a phenomenon that has been widely reported since the introduction of PCVs [41, 42]. However, due to the limited pneumococcal serotype data reported and the recent introduc- tion of PCV10, surveillance should be continued in Nigeria to monitor the burden of vaccine preventable bacterial meningitis and any potential changes in serotype distribution over time. Downloaded from https://academic.oup.com/cid/article/69/Supplement_2/S81/5561327 by University College London user on 31 January 2021 DISCUSSION Moreover, without the sophisticated laboratory techniques used, diagnosis of bac- terial meningitis would have relied on clinical characteristics. Thus, the 44 (28.8%: 44/153) PBM patients presenting with clear CSF and low WBC count may have been misdiagnosed. A history of fever (84.3%: 129 /153) and presence of seizures (71.2%: 109/153) were the most common symptoms associated with confirmed PBM cases in Nigeria. However, an altered con- sciousness is a more accurate indicator of bacterial meningitis MenAfriVac against serogroup A has not been introduced into routine immunization programs in Nigeria but was given to at risk populations during mass campaigns from 2011 to 2014 in high-risk states. Therefore, the recording of MenAfriVac vac- cination status of patients during surveillance was limited, with 4 patients reporting they had received the vaccines, 650 patients reporting they had not, and 4480 patients where this informa- tion was unknown or not recorded. However, of the 20 isolates we serogrouped, none were serogroup A; instead, serogroup W was the predominant strain, followed by nongroupable strains. 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https://openalex.org/W4390062535	https://www.nature.com/articles/s41597-023-02862-0.pdf	English	null	Author Correction: A Satellite Imagery Dataset for Long-Term Sustainable Development in United States Cities	Scientific data	2,023	cc-by	227	www.nature.com/scientificdata www.nature.com/scientificdata Scientific Data \| (2023) 10:928 \| https://doi.org/10.1038/s41597-023-02862-0 Author Correction: A Satellite Imagery Dataset for Long-Term Sustainable Development in United States Cities OPEN Published: xx xx xxxx Yanxin Xi , Yu Liu , Tong Li , Jingtao Ding , Yunke Zhang , Sasu Tarkoma , Yong Li & Pan Hui Correction to: Scientific Data https://doi.org/10.1038/s41597-023-02576-3, published online 04 December 2023 In this article the author name Jingtao Ding was incorrectly written as Jintao Ding. The original article has been corrected. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2023 © The Author(s) 2023 Scientific Data \| (2023) 10:928 \| https://doi.org/10.1038/s41597-023-02862-0
https://openalex.org/W3040493355	https://www.mdpi.com/2072-6694/12/7/1749/pdf?version=1593658859	English	null	Treatment of Locally Advanced Gastric Cancer (LAGC): Back to Lauren’s Classification in Pan–Cancer Analysis Era?	Cancers	2,020	cc-by	9,226	Received: 30 May 2020; Accepted: 29 June 2020; Published: 1 July 2020 Abstract: Background: Guidelines recommend a perioperative approach in patients with stage II/III gastric cancer, but in real-life many patients receive immediate surgery followed by adjuvant chemotherapy (aCT). Although histologic subtypes may have diﬀerent response to CT, no study has explored the inﬂuence of histotype on the eﬃcacy of perioperative CT (pCT) or aCT. Materials and methods: The objective of the study was to evaluate the impact of clinicopathological features and histology (intestinal or diﬀuse) on survival according to strategy (pCT vs. aCT). The primary endpoint was overall survival (OS) and the secondary endpoint was event-free survival (EFS). Results: Out of 203 patients aﬀected by LAGC, 83 received pCT and 120 aCT. At multivariate, histology and LVI in pCT cohort and positive resection margin in the aCT inﬂuenced both OS and EFS. No diﬀerence in EFS and OS was observed in relation to strategy. However, in the intestinal-type of pCT cohort survival outcomes were signiﬁcantly higher compared to the aCT cohort, whereas in the diﬀuse-type were signiﬁcantly worse in patients receiving pCT compared to those receiving aCT. Conclusions: Although retrospective and small-sized, this study suggests that the beneﬁt of pCT might be limited to the intestinal-type. This hypothesis needs to be conﬁrmed in prospective series. Keywords: gastric cancer; diﬀuse histology; intestinal histology; neo-adjuvant therapy; perioperative therapy; adjuvant therapy cancers cancers cancers www.mdpi.com/journal/cancers Treatment of Locally Advanced Gastric Cancer (LAGC): Back to Lauren’s Classiﬁcation in Pan–Cancer Analysis Era? Ina Valeria Zurlo 1,,† , Michele Basso 2,†, Antonia Strippoli 2, Maria Alessandra Calegari 1,2, Armando Orlandi 2 , Alessandra Cassano 1,2, Mariantonietta Di Salvatore 2, Giovanna Garuﬁ1, Emilio Bria 1,2 , Giampaolo Tortora 1,2, Carlo Barone 1,2 and Carmelo Pozzo 2 1 Comprehensive Cancer Center, Università Cattolica del Sacro Cuore-IRCCS, 00168 Rome, Italy; mariaalessandra.calegari@policlinicogemelli.it (M.A.C.); alessandra.cassano@unicatt.it (A.C.); giovanna.garuﬁ@unicatt.it (G.G.); emilio.bria@unicatt.it (E.B.); giampaolo.tortora@unicatt.it (G.T.); carlo.barone@unicatt.it (C.B.) 1 Comprehensive Cancer Center, Università Cattolica del Sacro Cuore-IRCCS, 00168 Rome, Italy; mariaalessandra.calegari@policlinicogemelli.it (M.A.C.); alessandra.cassano@unicatt.it (A.C.); giovanna.garuﬁ@unicatt.it (G.G.); emilio.bria@unicatt.it (E.B.); giampaolo.tortora@unicatt.it (G.T.); carlo.barone@unicatt.it (C.B.) 2 Comprehensive Cancer Center, Policlinico Universitario “Agostino Gemelli”-IRCSS, 00168 Roma, Italy; michele.basso@policlinicogemelli.it (M.B.); antonia.strippoli@policlinicogemelli.it (A.S.); armando.orlandi@policlinicogemelli.it (A.O.); disalvatore.mariantonietta@gmail.com (M.D.S.); carmelo.pozzo@policlinicogemelli.it (C.P.) Correspondence: valeriazurlo26@gmail.com; Tel.: +39-06-3015-6318 * Correspondence: valeriazurlo26@gmail.com; Tel † These authors equally contributed to this work. 1. Introduction According to Lauren’s classiﬁcation, GC is divided into two histological entities characterized by diﬀerent epidemiology, pathogenesis, biological features and clinical behavior: the intestinal and diﬀuse subtypes [17]. Intestinal-type tumors form gland-like structures; are strongly associated with severe atrophic gastritis, intestinal metaplasia and Helicobacter Pylori infection; and display a better survival [18–20]. On the other hand, diﬀuse histology is associated with cellular discohesion (due to the lack of cadherin E expression) and poor diﬀerentiation and is characterized by chemoresistance, rapid progression and poor prognosis. There are many conﬂicting retrospective data concerning the association between histology and treatment outcome in GC, therefore the Lauren’s classiﬁcation cannot be acknowledged as predictive of response to currently used drugs [20,21]. Therefore, international guidelines recommend an adequate D2 lymphadenectomy eventually preceded by a perioperative strategy for all patients with stage II or III disease. However, despite evidence and recommendations, in real-life, many patients receive immediate surgery followed by aCT. GC is a heterogeneous entity and histology is one of the earliest recognized criteria used for subtyping GC and it is frequently considered as a prognostic factor [16]. According to Lauren’s classiﬁcation, GC is divided into two histological entities characterized by diﬀerent epidemiology, pathogenesis, biological features and clinical behavior: the intestinal and diﬀuse subtypes [17]. Intestinal-type tumors form gland-like structures; are strongly associated with severe atrophic gastritis, intestinal metaplasia and Helicobacter Pylori infection; and display a better survival [18–20]. On the other hand, diﬀuse histology is associated with cellular discohesion (due to the lack of cadherin E expression) and poor diﬀerentiation and is characterized by chemoresistance, rapid progression and poor prognosis. There are many conﬂicting retrospective data concerning the association between histology and treatment outcome in GC, therefore the Lauren’s classiﬁcation cannot be acknowledged as predictive of response to currently used drugs [20,21]. In the modern era of precision medicine, other molecular classiﬁcations have gained prominence, in particular the comprehensive molecular characterization [22]. However, neither randomized nor prospective trials have been conducted selecting or stratifying patients according to molecularly deﬁned categories. Similarly, no analysis has been carried out in order to evaluate whether histologic subtypes of LAGC may be diﬀerently aﬀected by the strategy of treatment. Since it is widely accepted that diﬀerent subtypes of GC represent conditions with diﬀerent biologic and clinical characteristics, it is conceivable that they might respond in a not uniform way to diﬀerent strategies of treatment. 1. Introduction Thus, we have hypothesized that histology might predict a diﬀerent beneﬁt from pCT or aCT, allowing to deﬁne the optimal approach. The primary endpoint of this retrospective analysis was to compare overall survival of patients receiving perioperative chemotherapy according to histology and overall survival of patients receiving immediate surgery and adjuvant chemotherapy according to histology. 1. Introduction Although the incidence of gastric cancer (GC) has been substantially declining for several decades, it remains a major cause of cancer mortality due to poor prognosis. Until 2006, surgery was the only really eﬀective strategy for patients with resectable locally advanced GC (LAGC), with an adequate D2 lymphadenectomy qualifying as the gold standard approach [1,2]. However, despite R0 resection, the rate of postoperative recurrence is high. Consequently, many eﬀorts have been made to improve survival through administration of pCT or aCT [3–8]. The MAGIC and FFCD-FNCLCC 9703 trials showed the superiority of a perioperative strategy compared to surgery alone, due to Cancers 2020, 12, 1749; doi:10.3390/cancers12071749 www.mdpi.com/journal/cancers www.mdpi.com/journal/cancers 2 of 11 Cancers 2020, 12, 1749 tumor downstaging allowing better surgery, about 13% decrease of recurrence and improvement of survival [9,10]. On the other hand, many randomized clinical trials have compared surgery alone with aCT or adjuvant chemoradiotherapy (aCT-RT), but clear evidence of beneﬁt is lacking because survival improvement was only modest and sometimes controversial [3–8,11–13]. The largest beneﬁt from aCT and aCT-RT has been demonstrated in patients who underwent D0 or D1 surgery, while the same beneﬁt is less clear in patients who underwent D2 lymphadenectomy [4,11,13,14]. More recently, combination regimens with docetaxel, oxaliplatin and ﬂuorouracil (FLOT schedule) have demonstrated better outcomes in terms of DFS and OS, when compared to ECF/ECX in the perioperative setting [15] survival [9,10]. On the other hand, many randomized clinical trials have compared surgery alone with aCT or adjuvant chemoradiotherapy (aCT-RT), but clear evidence of beneﬁt is lacking because survival improvement was only modest and sometimes controversial [3–8,11–13]. The largest beneﬁt from aCT and aCT-RT has been demonstrated in patients who underwent D0 or D1 surgery, while the same beneﬁt is less clear in patients who underwent D2 lymphadenectomy [4,11,13,14]. More recently, combination regimens with docetaxel, oxaliplatin and ﬂuorouracil (FLOT schedule) have demonstrated better outcomes in terms of DFS and OS, when compared to ECF/ECX in the perioperative setting [15]. Therefore, international guidelines recommend an adequate D2 lymphadenectomy eventually preceded by a perioperative strategy for all patients with stage II or III disease. However, despite evidence and recommendations, in real-life, many patients receive immediate surgery followed by aCT. GC is a heterogeneous entity and histology is one of the earliest recognized criteria used for subtyping GC and it is frequently considered as a prognostic factor [16]. 2.2. Treatment Procedures Both in pCT and aCT cohort the most frequently used regimens were triplet CT [epirubicin plus oxaliplatin plus capecitabine (EOX), or epirubicin plus cisplatin plus infusional 5-fluorouracil (ECF)] or platinum-containing doublet CT [leucovorin plus oxaliplatin plus bolus and infusional 5-fluorouracil (FOLFOX-6), capecitabine plus oxaliplatin (XELOX) or cisplatin plus 5-fluorouracil (CF)]. Only in few cases of aCT cohort, a fluoropyrimidine monotherapy was employed (DeGramont regimen). In the pCT cohort, restaging was accomplished using CE-CT scan before surgery. All patients underwent gastrectomy (total or subtotal according to tumor extension and location) with an adequate D2 lymphadenectomy. Margin resection was defined as R0 when no tumor was identified on microscopic examination of proximal, distal or circumferential margin and as R1 when microscopic margin involvement was demonstrated. In the pCT cohort, surgery was carried out within 6–8 weeks from the last CT course, whereas in the aCT cohort the first cycle of CT was administered within 8 weeks after surgery. Postoperative morbidity and mortality were recorded. All patients underwent follow-up according international guidelines including clinical examination and CT-scan or abdominal ultrasound every six months. 2.1. Study Population This is a retrospective monocentric study. Clinical records of all patients aﬀected by LAGC treated with aCT or pCT at Medical Oncology Unit of the Fondazione Policlinico Universitario “A. Gemelli”-IRCCS, Rome, Italy, between January 2009 and January 2018 were reviewed. Inclusion criteria were: (1) histologically conﬁrmed adenocarcinoma of the stomach; (2) gastro-esophageal junction, Siewert type 2 or 3 (GEJ) or non-cardia GC (stomach); (3) known histotype according to Lauren’s classiﬁcation (intestinal or diﬀuse); (4) total or sub-total gastrectomy; (5) stage II or III (clinical staging for pCT and pathological staging for aCT); (6) CT administration in perioperative or adjuvant setting; (7) age ≥18 years; (8) no serious concomitant illnesses that could have aﬀected treatment duration, short-time survival or the possibility of surgery; (9) performance status (PS) according to the Eastern Cooperative Oncology Group of 0 or 1; (10) adequate organ function (bone marrow, liver and kidney); (11) left ventricular ejection fraction (LVEF) of ≥50% for anthracycline-containing CT; and (12) informed consent to surgery and chemotherapy according to local practice. Patients whose 3 of 11 Cancers 2020, 12, 1749 histotype according to Lauren’s classiﬁcation was not known or mixed-type, with gastro-esophageal junction, Siewert type 1, with medical history of metastatic disease or other cancers (with the exception of non-melanoma skin cancers and in situ cervical cancer) diagnosed within the previous 5 years, or who received upfront surgery without aCT due to clinical conditions or concomitant illness, were excluded. Patients were divided into two groups according to treatment strategy (perioperative and adjuvant) and outcome was evaluated according to histology (intestinal vs. diﬀuse). All patient data were collected anonymously; the study was conducted in accordance with the Declaration of Helsinki and consent for chemotherapy was obtained by all patients, also including the consent for retrospective analysis of all clinical data, according to the Ethical Committee of the Catholic University School of Medicine. In the pCT group, T staging was determined using endoscopic ultrasonography (EUS) and N and M status were classiﬁed using a contrast-enhanced computer tomography (CE-CT) scan of the abdomen and chest. In the aCT group, TNM staging was assigned based on pathological examination and distant metastases were excluded by CE-CT. 3.1. Patients Characteristics Among 250 consecutive Among 250 consecutive patients aﬀected by stage II or III GC treated at Medical Oncology Unit of the Fondazione Policlinico Universitario “A. Gemelli”–Comprehensive Cancer Center (IRCCS) between January 2009 to January 2018, 203 met all inclusion criteria and were included in this retrospective analysis (Figure 1). Forty-seven were excluded due to incomplete clinical and/or histologic information. Eighty-three patients underwent pCT and 120 received upfront surgery followed by aCT. One hundred thirteen patients were aﬀected by diﬀuse GC, 39 were treated with pCT and 74 with surgery followed by aCT. Ninety patients had intestinal GC, 44 received pCT and 46 underwent upfront surgery followed by aCT. Within the whole population, 52% of patients (105) were male and 48% (98) were female. Median age was 64 years (range 42–78). A diagnostic laparoscopy was performed in 70% of cases to exclude peritoneal carcinomatosis in the pCT cohort. All patients (203) received an adequate D2 lymphadenectomy with a median of 38 lymph nodes evaluated (range 20–60). g p y g gy of the Fondazione Policlinico Universitario “A. Gemelli”–Comprehensive Cancer Center (IRCCS) between January 2009 to January 2018, 203 met all inclusion criteria and were included in this retrospective analysis (Figure 1). Forty-seven were excluded due to incomplete clinical and/or histologic information. Eighty-three patients underwent pCT and 120 received upfront surgery followed by aCT. One hundred thirteen patients were affected by diffuse GC, 39 were treated with pCT and 74 with surgery followed by aCT. Ninety patients had intestinal GC, 44 received pCT and 46 underwent upfront surgery followed by aCT. Within the whole population, 52% of patients (105) were male and 48% (98) were female. Median age was 64 years (range 42–78). A diagnostic laparoscopy was performed in 70% of cases to exclude peritoneal carcinomatosis in the pCT cohort. All patients (203) received an adequate D2 lymphadenectomy with a median of 38 lymph nodes evaluated (range 20–60). Figure 1. Consort diagram. In the pCT group, patients diagnosed with a GEJ or stomach cancer were well-balanced between testinal and diffuse sub-groups. In this group, four patients (4.8%) had a microscopic residual after urgery (R1); all of them belonged to the diffuse sub-group. The percentage of patients with a Figure 1. Consort diagram. In the pCT group, patients diagnosed with a GEJ or stomach cancer were well-balanced between ntestinal and diﬀuse sub-groups. 2.3. Statistical Analysis The objective of the study was to compare survival of patients following pCT or aCT, according to histology in each group (pCT or a CT). The primary endpoint was OS and the secondary endpoints were EFS and tumor regression grade (TRG) for pCT. OS was deﬁned as the time from the onset of treatment for pCT group or from surgery for aCT group to the date of death due to any cause, or censored at the date of last follow-up for alive patients. EFS was deﬁned as the time from the start of treatment for pCT group or from surgery for aCT group to the date of the ﬁrst documented recurrence or progression (local, regional or distant), death due to any cause or discontinuation of treatment for any reason, whichever occurred ﬁrst. TRG was categorized according to the Mandard classiﬁcation system [23]. The Kaplan–Meier method was used to estimate OS and EFS, a Cox regression model was employed to estimate hazard ratios (HRs) and two-sided 95% conﬁdence intervals (CIs) were used for the comparison of survival of diﬀuse vs. intestinal subtype in both perioperative and adjuvant setting. Pearson chi-square test was performed for comparing TRG rate of intestinal and diﬀuse GCs, respectively, within pCT cohort. The statistical signiﬁcance level was set at p < 0.05. Univariate analysis was performed to establish the relationship among survival endpoints and clinic-pathologic variables: age (<65 vs. >65 years), tumor location (proximal vs. distal), histology (diﬀuse vs. intestinal), lymphatic vascular inﬁltration (LVI) (absent vs. present), lymph node involvement (N0 vs. N+), grading (G1–G2 vs. G3), resection margin (R0 vs. R1), CT regimen employed (doublet vs. triplet) and exposure to adjuvant radiotherapy (RT) (absent vs. present). Clinical variables with a p value < 0.5 were included in a multivariate analysis. Data were analyzed using MedCal Statistical software. 4 of 11 Cancers 2020, 12, 1749 software. 3. Results 3 1 Patie t 3.1. Patients Characteristics Among 250 consecutive 3.1. Patients Characteristics Among 250 consecutive In the pCT cohort, 24 patients received RT for R1 surgery (4.8%) or postoperative lymph node N3 status (24%), of whom 4 (4.8%) had an intestinal histology and 20 (24.0%) a diﬀuse cancer. In the pCT cohort, the preferred treatment regimens were in the intestinal subgroup EOX or ECF in 26.5% of patients and CF or FOLFOX in other 26.5%, whereas in the diﬀuse group 34.9% and 12.0%, respectively. Patients received a median of three cycles with EOX, ECF or CF (range 2–4) and a median of six cycles with FOLFOX (range 4–8). No dose reduction was required. Surgery was performed on average 38 days (range 28–54) after the last administration of chemotherapy. In the pCT cohort 36% of patients did not receive any treatment after surgery due to postoperative worsening of PS or poor response to preoperative CT. In the aCT group, patients diagnosed with a GEJ cancer were well-balanced between intestinal and diﬀuse histology. Among those with a stomach cancer, diﬀuse histotype was more frequent (27.5% vs. 49.1%). Even in this group, four patients (3.3%) had a microscopic residual after surgery (R1); all of them belonged to the diﬀuse sub-group once again. LVI was less frequent among patients with an intestinal cancer in comparison with those with a diﬀuse histology (19.1% vs. 30.8%). The cases of pT4 tumors were slightly more frequent in diﬀuse histology (30.0% vs. 17.5%) but there were no diﬀerences concerning patients with N0 status (4.8% vs. 2.4%). The percentage of patients receiving RT after surgery was higher in diﬀuse-type than intestinal histology (15% vs. 28.3%), due to a higher rate of R1 surgery and lymph node N3 status. In the aCT cohort, cytotoxic therapy started on average 52 days (range 38–62) after surgery. In this case, the preferred regimens were: in the intestinal cohort, EOX/ECF (11.6%), FOLFOX or CF (12.5%) and DeGramont regimen (14.1%), while, in the diﬀuse cohort, 29.1%, 14.1% and 18.3%, respectively. Drug reductions were necessary in 25% of patients mainly due to neutropenia or diarrhea. The median follow-up time of whole group of patients was 41 months. Baseline patients and disease characteristics according to histology and treatment strategy are summarized in Table 1. Table 1. Patients’ Characteristics. Characteristic Perioperative Chemotherapy (No. 83) Characteristic Adjuvant Chemotherapy (No. 120) No. Intestinal (No. 44) Diﬀuse (No. 39) No. Intestinal (No. 46) Diﬀuse (No. 3.1. Patients Characteristics Among 250 consecutive In this group, four patients (4.8%) had a microscopic residual fter surgery (R1); all of them belonged to the diﬀuse sub-group. The percentage of patients with a ostoperative lymph node N0 status was higher (32.5% vs. 6.0%), when comparing intestinal and iﬀuse histology while the rate of patients with LVI was inferior (9.6% vs. 20.1%) in patients with an ntestinal histology. Moreover, the cases of yT4 tumors were fewer (6.0% vs. 19.2%) among intestinal Figure 1. Consort diagram. Figure 1. Consort diagram. Figure 1. Consort diagram. Figure 1. Consort diagram. In the pCT group, patients diagnosed with a GEJ or stomach cancer were well-balanced between intestinal and diffuse sub-groups. In this group, four patients (4.8%) had a microscopic residual after surgery (R1); all of them belonged to the diffuse sub-group. The percentage of patients with a In the pCT group, patients diagnosed with a GEJ or stomach cancer were well-balanced between intestinal and diﬀuse sub-groups. In this group, four patients (4.8%) had a microscopic residual after surgery (R1); all of them belonged to the diﬀuse sub-group. The percentage of patients with a postoperative lymph node N0 status was higher (32.5% vs. 6.0%), when comparing intestinal and diﬀuse histology while the rate of patients with LVI was inferior (9.6% vs. 20.1%) in patients with an intestinal histology. Moreover, the cases of yT4 tumors were fewer (6.0% vs. 19.2%) among intestinal In the pCT group, patients diagnosed with a GEJ or stomach cancer were well-balanced between intestinal and diffuse sub-groups. In this group, four patients (4.8%) had a microscopic residual after surgery (R1); all of them belonged to the diffuse sub-group. The percentage of patients with a In the pCT group, patients diagnosed with a GEJ or stomach cancer were well-balanced between intestinal and diﬀuse sub-groups. In this group, four patients (4.8%) had a microscopic residual after surgery (R1); all of them belonged to the diﬀuse sub-group. The percentage of patients with a postoperative lymph node N0 status was higher (32.5% vs. 6.0%), when comparing intestinal and diﬀuse histology while the rate of patients with LVI was inferior (9.6% vs. 20.1%) in patients with an intestinal histology. Moreover, the cases of yT4 tumors were fewer (6.0% vs. 19.2%) among intestinal Cancers 2020, 12, 1749 5 of 11 cancers in comparison to diﬀuse ones. 3.2. Outcome 3.2. Outcome 3.2. Outcome Overall, median OS (mOS) and median EFS (mEFS) were 92 (range 66–110) and 66 (range 45–110) months, respectively. In the pCT cohort, mEFS and mOS were not reached, whereas, in the aCT cohort, mOS and mEFS were 89 (range 56–110 months) and 62 (range 41–110) months, respectively. No statistically signiﬁcant diﬀerence was found both in mOS (p = 0.99) and EFS (p = 0.96) (Figure 2). Overall, median OS (mOS) and median EFS (mEFS) were 92 (range 66–110) and 66 (range 45– 110) months, respectively. In the pCT cohort, mEFS and mOS were not reached, whereas, in the aCT cohort, mOS and mEFS were 89 (range 56–110 months) and 62 (range 41–110) months, respectively. No statistically significant difference was found both in mOS (p = 0.99) and EFS (p = 0.96) (Figure 2). Overall, median OS (mOS) and median EFS (mEFS) were 92 (range 66–110) and 66 (range 45– 110) months, respectively. In the pCT cohort, mEFS and mOS were not reached, whereas, in the aCT cohort, mOS and mEFS were 89 (range 56–110 months) and 62 (range 41–110) months, respectively. No statistically significant difference was found both in mOS (p = 0.99) and EFS (p = 0.96) (Figure 2). Figure 2. Kaplan–Meier curves for OS (A) and EFS (B) according to strategy (pCT vs. aCT). Figure 2. Kaplan–Meier curves for OS (A) and EFS (B) according to strategy (pCT vs. aCT). Figure 2. Kaplan–Meier curves for OS (A) and EFS (B) according to strategy (pCT vs. aCT). ure 2. Kaplan–Meier curves for OS (A) and EFS (B) according to strategy (pCT vs. aCT). Figure 2. Kaplan–Meier curves for OS (A) and EFS (B) according to strategy (pCT vs. aCT). ure 2. Kaplan–Meier curves for OS (A) and EFS (B) according to strategy (pCT vs. aCT). In the pCT cohort, univariate Cox regression analysis, including age, tumor location, histology, LVI, lymph node involvement, grading, resection margin, CT regimen and exposure to adjuvant RT, demonstrated that grading, histology, LVI, resection margin and lymph node status were significantly associated with both EFS and OS (Table 2). Multivariate analysis performed on these variables demonstrated that only histology and LVI remained significantly associated with EFS (p = 0.0023; p = 0.0028) and OS (p = 0.0001; p = 0.0004) (Table 3). 3.2. Outcome 3.2. Outcome 3.2. Outcome Median OS was 31 months (20–47) in the diffuse-type and not reached in the intestinal-type (HR 9.3; 95% CI 4.59–19.13; p < 0.0001), whereas the mEFS was 18 months (13–36) in the diffuse cohort and not reached in the intestinal-type (HR 7.2; 95% CI 3.6–14.27; p < 0.0001) (Figure 3). In the pCT cohort, univariate Cox regression analysis, including age, tumor location, histology, LVI, lymph node involvement, grading, resection margin, CT regimen and exposure to adjuvant RT, demonstrated that grading, histology, LVI, resection margin and lymph node status were signiﬁcantly associated with both EFS and OS (Table 2). Multivariate analysis performed on these variables demonstrated that only histology and LVI remained signiﬁcantly associated with EFS (p = 0.0023; p = 0.0028) and OS (p = 0.0001; p = 0.0004) (Table 3). Median OS was 31 months (20–47) in the diﬀuse-type and not reached in the intestinal-type (HR 9.3; 95% CI 4.59–19.13; p < 0.0001), whereas the mEFS was 18 months (13–36) in the diﬀuse cohort and not reached in the intestinal-type (HR 7.2; 95% CI 3.6–14.27; p < 0.0001) (Figure 3). In the pCT cohort, univariate Cox regression analysis, including age, tumor location, histology, LVI, lymph node involvement, grading, resection margin, CT regimen and exposure to adjuvant RT, demonstrated that grading, histology, LVI, resection margin and lymph node status were significantly associated with both EFS and OS (Table 2). Multivariate analysis performed on these variables demonstrated that only histology and LVI remained significantly associated with EFS (p = 0.0023; p = 0.0028) and OS (p = 0.0001; p = 0.0004) (Table 3). Median OS was 31 months (20–47) in the diffuse-type and not reached in the intestinal-type (HR 9.3; 95% CI 4.59–19.13; p < 0.0001), whereas the mEFS was 18 months (13–36) in the diffuse cohort and not reached in the intestinal-type (HR 7.2; 95% CI 3.6–14.27; p < 0.0001) (Figure 3). Figure 3. Kaplan–Meier curves for OS (A) and EFS (B) in the pCT cohort according histology (intestinal vs. diffuse subtype, HR 9.3; 95% CI 4.59–19.13; p < 0.0001 for OS; HR 7.2; 95% CI 3.6–14.27; p < 0.0001 for EFS). Figure 3. Kaplan–Meier curves for OS (A) and EFS (B) in the pCT cohort according histology (intestinal vs. diffuse subtype, HR 9.3; 95% CI 4.59–19.13; p < 0.0001 for OS; HR 7.2; 95% CI 3.6–14.27; p < 0.0001 for EFS). Figure 3. 3.1. Patients Characteristics Among 250 consecutive 74) Tumor location Tumor location GEJ 41 23 (27.7%) 18 (21.7%) GEJ 28 13 (10.8%) 15 (12.5%) Stomach 42 21 (25.3%) 21 (25.3%) Stomach 92 33 (27.5%) 59 (49.2%) Signet-cell 9 - 9 (10.8%) Signet-cell 18 - 18 (15.0%) LVI 25 8 (9.6%%) 17 (20.4%) LVI 60 23 (19.1%) 37 (30.8%) Total gastrectomy 41 19 (22.8%) 22 (26.5%) Total gastrectomy 48 21 (17.5%) 27 (32.5%) R1 surgery 4 - 4 (4.8%) R1 surgery 4 - 4 (3.3%) T T yT0/T1 8 6 (7.2%) 2 (2.4%) pT0/T1 4 1 (0.8%) 3 (2.5%) yT2 16 12 (14.5%) 4 (4.8%) pT2 13 6 (5.0%) 7 (5.9%) yT3 38 21 (25.3%) 17 (20.5%) pT3 46 18 (15.0%) 28 (23.3%) yT4 21 5 (6.0%) 16 (19.3%) pT4 57 21 (17.5%) 36 (30.0%) N N yN0 32 27 (32.5%) 5 (6.0%) pN0 6 4 (3.3%) 2 (1.7%) yN1 15 8 (9.7%) 7 (8.4%) pN1 24 7 (5.8%) 17 (14.2%) yN2 16 5 (6.0%) 11 (13.3%) pN2 32 17 (14.2%) 15 (12.5%) yN3 20 4 (4.8%) 16 (19.3%) pN3 58 18 (15.0%) 40 (33.3%) TRG TRG1 7 5 (6.0%) 2 (2.4%) TRG2 7 7 (8.4%) - TRG3 28 17 (20.5%) 11 (13.3%) TRG4 34 15 (18.1%) 19 (22.9%) TRG5 7 - 7 (8.4%) Treatment Treatment EOX/ECF 51 22 (26.5%) 29 (34.9%) EOX/ECF 49 14 (11.6%) 35 (29.2%) FOLFOX/CF 32 22 (26.5%) 10 (12.1%) FOLFOX/CF 32 15 (12.5%) 17 (14.2%) DeGramont - - - DeGramont 39 17 (14.2%) 22 (18.3%) Radiotherapy 24 4 (4.8%) 20 (24.0%) Radiotherapy 52 18 (15%) 34 (28.3%) Table 1. Patients’ Characteristics. Table 1. Patients’ Characteristics. Table 1. Patients’ Characteristics. 6 of 11 2 3 Cancers 2020, 12, 1749 CF 32 22 nt - nt - py 24 4 3.2. Outcome 3.2. Outcome 3.2. Outcome Variable OS EFS HR (95% CI) for Progression p Value HR (95% CI) for Mortality p Value pCT Histology 10.95 (3.31–36.24) 0.0001 4.84 (1.76–13.29) 0.0023 LVI 5.57 (2.18–14.26) 0.0004 3.60 (1.56–8.32) 0.0028 aCT Tumor location – – 0.47 (0.26–0.85) 0.013 Resection margin 19.97 (7.04–56.59) <0.001 4.036 (1.44–11.30) 0.0082 In the aCT cohort, univariate analysis including the same variables showed that resection margin was signiﬁcantly associated with both EFS and OS, whereas tumor location was associated only with EFS (Table 2). In multivariate analysis, resection margin conﬁrmed the signiﬁcant association with both EFS (p = 0.0082) and OS (p < 0.001) and also tumor location conﬁrmed the relationship with EFS (p = 0.013) (Table 3). In the aCT cohort no statistically signiﬁcant survival diﬀerence in relation to histology was observed. Median EFS was 89 months (range 41–89) in the intestinal subgroup and 59 months (range 35–110) in diﬀuse subtype (HR 1.12 CI 95% 0.65–1.91; p = 0.67), while mOS was 96 (range 71–96) and 66 (range 38–110) months, respectively (HR 1.2; 95% CI 0.72–2.26; p = 0.40) (Figure 4). Moreover, in the perioperative cohort, a diﬀerent percentage of tumor regression grade (TRG) was also observed according to histology, with a higher response in intestinal GC compared to diﬀuse GC. Over 60% of patients in the intestinal sub-group showed a TRG between 1 and 3 compared to diﬀuse-type, in which a TRG between 4 and 5 was obtained in over 60% of patients (p = 0.01). Table 2. Univariate Analysis of OS and EFS for Clinicopathologic Variables in pCT and aCT Cohort. Variable OS EFS HR (95% CI) for Mortality p Value HR (95% CI) for Progression p Value pCT cohort Age 0.6 (0.3–1.22) 0.1 0.4 (0.21–0.82) 0.01 Grading 2.7 (1.31–5.68) 0.004 3.2 (1.5–6.5) 0.0003 Histology 0.1 (0.05–0.21) <0.0001 0.13 (0.07–0.27) <0.0001 LVI 3.6 (1.55–8.34) 0.0001 3.8 (1.72–8.62) <0.0001 Surgery R1 3.4 (0.71–16.9) 0.006 3.59 (0.71–18.0) 0.004 Tumor location 1.2 (0.61–2.53) 0.53 1.16 (0.59–2.26) 0.65 N status 4.71 (2.30–9.65) 0.0012 3.66 (1.87–7.15) 0.0017 Doublet vs. 3.2. Outcome 3.2. Outcome 3.2. Outcome triplet chemotherapy 1.28 (0.64–2.54) 0.48 1.22 (0.58–2.58) 0.5 Radiotherapy 1.99 (0.91–4.1) 0.06 1.7 (0.86–3.68) 0.08 aCT cohort Age 1.70 (0.95–3.02) 0.06 1.48 (0.87–2.54) 0.12 Grading 1.31 (0.73–2.36) 0.37 1.45 (0.83–2.52) 0.19 Histology 1.28 (0.72–2.26) 0.40 1.12 (0.65–1.91) 0.67 LVI 1.31 (0.70–2.43) 0.66 1.13 (0.63–2.0) 0.66 Surgery R1 4.04 (0.58–27.9) 0.0033 13.2(0.61–28.01) <0.0001 Tumor location 0.59 (0.29–1.21) 0.09 0.55 (0.28–1.07) 0.03 N status 1.5 (0.29–7.67) 0.68 2.03 (0.49–8.34) 0.46 Doublet vs. triplet chemotherapy 1.11 (0.60–2.03) 0.72 1.16 (0.66–2.05) 0.57 Radiotherapy 1.42 (0.82–2.47) 0.19 1.44 (0.85–2.42) 0.16 Table 2. Univariate Analysis of OS and EFS for Clinicopathologic Variables in pCT and aCT Cohort. Variable OS EFS HR (95% CI) for Mortality p Value HR (95% CI) for Progression p Value pCT cohort Age 0.6 (0.3–1.22) 0.1 0.4 (0.21–0.82) 0.01 Grading 2.7 (1.31–5.68) 0.004 3.2 (1.5–6.5) 0.0003 Histology 0.1 (0.05–0.21) <0.0001 0.13 (0.07–0.27) <0.0001 LVI 3.6 (1.55–8.34) 0.0001 3.8 (1.72–8.62) <0.0001 Surgery R1 3.4 (0.71–16.9) 0.006 3.59 (0.71–18.0) 0.004 Tumor location 1.2 (0.61–2.53) 0.53 1.16 (0.59–2.26) 0.65 N status 4.71 (2.30–9.65) 0.0012 3.66 (1.87–7.15) 0.0017 Doublet vs. triplet chemotherapy 1.28 (0.64–2.54) 0.48 1.22 (0.58–2.58) 0.5 Radiotherapy 1.99 (0.91–4.1) 0.06 1.7 (0.86–3.68) 0.08 aCT cohort Age 1.70 (0.95–3.02) 0.06 1.48 (0.87–2.54) 0.12 Grading 1.31 (0.73–2.36) 0.37 1.45 (0.83–2.52) 0.19 Histology 1.28 (0.72–2.26) 0.40 1.12 (0.65–1.91) 0.67 LVI 1.31 (0.70–2.43) 0.66 1.13 (0.63–2.0) 0.66 Surgery R1 4.04 (0.58–27.9) 0.0033 13.2(0.61–28.01) <0.0001 Tumor location 0.59 (0.29–1.21) 0.09 0.55 (0.28–1.07) 0.03 N status 1.5 (0.29–7.67) 0.68 2.03 (0.49–8.34) 0.46 Doublet vs. triplet chemotherapy 1.11 (0.60–2.03) 0.72 1.16 (0.66–2.05) 0.57 Radiotherapy 1.42 (0.82–2.47) 0.19 1.44 (0.85–2.42) 0.16 Table 3. Multivariate Cox Regression Analysis of EFS and OS for Clinicopathologic Variables Resulted Signiﬁcant in Univariate Analysis; pCT and aCT Cohort. Variable OS EFS HR (95% CI) for Progression p Value HR (95% CI) for Mortality p Value pCT Histology 10.95 (3.31–36.24) 0.0001 4.84 (1.76–13.29) 0.0023 LVI 5.57 (2.18–14.26) 0.0004 3.60 (1.56–8.32) 0.0028 aCT Tumor location – – 0.47 (0.26–0.85) 0.013 Table 2. Univariate Analysis of OS and EFS for Clinicopathologic Variables in pCT and aCT Cohort. Table 3. Multivariate Cox Regression Analysis of EFS and OS for Clinicopathologic Variables Resulted Signiﬁcant in Univariate Analysis; pCT and aCT Cohort. 3.2. Outcome 3.2. Outcome 3.2. Outcome Variable OS EFS HR (95% CI) for Progression p Value HR (95% CI) for Mortality p Value pCT Histology 10.95 (3.31–36.24) 0.0001 4.84 (1.76–13.29) 0.0023 LVI 5.57 (2.18–14.26) 0.0004 3.60 (1.56–8.32) 0.0028 aCT Tumor location – – 0.47 (0.26–0.85) 0.013 Resection margin 19.97 (7.04–56.59) <0.001 4.036 (1.44–11.30) 0.0082 In the aCT cohort, univariate analysis including the same variables showed that resection margin was signiﬁcantly associated with both EFS and OS, whereas tumor location was associated only with EFS (Table 2). In multivariate analysis, resection margin conﬁrmed the signiﬁcant association with both EFS (p = 0.0082) and OS (p < 0.001) and also tumor location conﬁrmed the relationship with EFS (p = 0.013) (Table 3). In the aCT cohort no statistically signiﬁcant survival diﬀerence in relation to histology was observed. Median EFS was 89 months (range 41–89) in the intestinal subgroup and 59 months (range 35–110) in diﬀuse subtype (HR 1.12 CI 95% 0.65–1.91; p = 0.67), while mOS was 96 (range 71–96) and 66 (range 38–110) months, respectively (HR 1.2; 95% CI 0.72–2.26; p = 0.40) (Figure 4). M i h i i h diﬀ f i d (TRG) In the aCT cohort, univariate analysis including the same variables showed that resection margin was signiﬁcantly associated with both EFS and OS, whereas tumor location was associated only with EFS (Table 2). In multivariate analysis, resection margin conﬁrmed the signiﬁcant association with both EFS (p = 0.0082) and OS (p < 0.001) and also tumor location conﬁrmed the relationship with EFS (p = 0.013) (Table 3). In the aCT cohort no statistically signiﬁcant survival diﬀerence in relation to histology was observed. Median EFS was 89 months (range 41–89) in the intestinal subgroup and 59 months (range 35–110) in diﬀuse subtype (HR 1.12 CI 95% 0.65–1.91; p = 0.67), while mOS was 96 (range 71–96) and 66 (range 38–110) months, respectively (HR 1.2; 95% CI 0.72–2.26; p = 0.40) (Figure 4). Moreover, in the perioperative cohort, a diﬀerent percentage of tumor regression grade (TRG) was also observed according to histology, with a higher response in intestinal GC compared to diﬀuse GC. Over 60% of patients in the intestinal sub-group showed a TRG between 1 and 3 compared to diﬀuse-type, in which a TRG between 4 and 5 was obtained in over 60% of patients (p = 0.01). No diﬀerences were reported in TRG according diﬀerent pCT regimens. 3.2. Outcome 3.2. Outcome 3.2. Outcome Kaplan–Meier curves for OS (A) and EFS (B) in the pCT cohort according histology (intestinal vs. diﬀuse subtype, HR 9.3; 95% CI 4.59–19.13; p < 0.0001 for OS; HR 7.2; 95% CI 3.6–14.27; p < 0.0001 for EFS). Figure 3. Kaplan–Meier curves for OS (A) and EFS (B) in the pCT cohort according histology (intestinal vs. diffuse subtype, HR 9.3; 95% CI 4.59–19.13; p < 0.0001 for OS; HR 7.2; 95% CI 3.6–14.27; p < 0.0001 for EFS). Figure 3. Kaplan–Meier curves for OS (A) and EFS (B) in the pCT cohort according histology (intestinal vs. diffuse subtype, HR 9.3; 95% CI 4.59–19.13; p < 0.0001 for OS; HR 7.2; 95% CI 3.6–14.27; p < 0.0001 for EFS). Figure 3. Kaplan–Meier curves for OS (A) and EFS (B) in the pCT cohort according histology (intestinal vs. diﬀuse subtype, HR 9.3; 95% CI 4.59–19.13; p < 0.0001 for OS; HR 7.2; 95% CI 3.6–14.27; p < 0.0001 for EFS). 7 of 11 Cancers 2020, 12, 1749 Table 2. Univariate Analysis of OS and EFS for Clinicopathologic Variables in pCT and aCT Cohort. Variable OS EFS HR (95% CI) for Mortality p Value HR (95% CI) for Progression p Value pCT cohort Age 0.6 (0.3–1.22) 0.1 0.4 (0.21–0.82) 0.01 Grading 2.7 (1.31–5.68) 0.004 3.2 (1.5–6.5) 0.0003 Histology 0.1 (0.05–0.21) <0.0001 0.13 (0.07–0.27) <0.0001 LVI 3.6 (1.55–8.34) 0.0001 3.8 (1.72–8.62) <0.0001 Surgery R1 3.4 (0.71–16.9) 0.006 3.59 (0.71–18.0) 0.004 Tumor location 1.2 (0.61–2.53) 0.53 1.16 (0.59–2.26) 0.65 N status 4.71 (2.30–9.65) 0.0012 3.66 (1.87–7.15) 0.0017 Doublet vs. triplet chemotherapy 1.28 (0.64–2.54) 0.48 1.22 (0.58–2.58) 0.5 Radiotherapy 1.99 (0.91–4.1) 0.06 1.7 (0.86–3.68) 0.08 aCT cohort Age 1.70 (0.95–3.02) 0.06 1.48 (0.87–2.54) 0.12 Grading 1.31 (0.73–2.36) 0.37 1.45 (0.83–2.52) 0.19 Histology 1.28 (0.72–2.26) 0.40 1.12 (0.65–1.91) 0.67 LVI 1.31 (0.70–2.43) 0.66 1.13 (0.63–2.0) 0.66 Surgery R1 4.04 (0.58–27.9) 0.0033 13.2(0.61–28.01) <0.0001 Tumor location 0.59 (0.29–1.21) 0.09 0.55 (0.28–1.07) 0.03 N status 1.5 (0.29–7.67) 0.68 2.03 (0.49–8.34) 0.46 Doublet vs. triplet chemotherapy 1.11 (0.60–2.03) 0.72 1.16 (0.66–2.05) 0.57 Radiotherapy 1.42 (0.82–2.47) 0.19 1.44 (0.85–2.42) 0.16 Table 3. Multivariate Cox Regression Analysis of EFS and OS for Clinicopathologic Variables Resulted Signiﬁcant in Univariate Analysis; pCT and aCT Cohort. Moreover 4. Discussion This is in contrast with some previous reports suggesting that five year survival is lower with perioperative in comparison to adjuvant therapy [4 6 9 10] In the whole population of our study, there is no survival diﬀerence among patients in pCT cohort in comparison to those in aCT cohort. This is in contrast with some previous reports suggesting that ﬁve-year survival is lower with perioperative in comparison to adjuvant therapy [4–6,9,10]. However, the high quality of surgery with 100% of patients receiving D2 gastrectomy in the same institution together with accurate staging before surgery (70% laparoscopy rate) might account for this result, strengthening the acknowledged crucial role of surgery in this disease [24]. Interestingly, the pCT diﬀuse histology group showed the worst median OS (31 months) among subgroups, suggesting that stage migration during pCT or underestimation of stage of disease might have played a role for this disappointing outcome. that five-year survival is lower with perioperative in comparison to adjuvant therapy [4–6,9,10]. However, the high quality of surgery with 100% of patients receiving D2 gastrectomy in the same institution together with accurate staging before surgery (70% laparoscopy rate) might account for this result, strengthening the acknowledged crucial role of surgery in this disease [24]. Interestingly, the pCT diffuse histology group showed the worst median OS (31 months) among subgroups, suggesting that stage migration during pCT or underestimation of stage of disease might have played a role for this disappointing outcome. Even if there is no definitive argument explaining our results, we might think that the alternative therapeutic strategies that have been considered might affect subtle molecular and biological differences between histologic subtypes [15]. Updated analysis of INT-0116 shows that intestinal subtype of gastric cancer takes advantage from aCT-RT, but not diffuse histotype, suggesting a different sensitivity to chemotherapy or an intrinsically worse prognosis [25]. More recently, the FLOT4 study has demonstrated the superiority of a triplet-regimen combination with fluorouracil plus leucovorin, oxaliplatin and docetaxel versus fluorouracil or capecitabine plus cisplatin and epirubicin in the perioperative setting of gastric cancer. In this study, which has become practice- changing superseding all previous trials of perioperative chemotherapy patients have been stratified pp g Even if there is no deﬁnitive argument explaining our results, we might think that the alternative therapeutic strategies that have been considered might aﬀect subtle molecular and biological diﬀerences between histologic subtypes [15]. Moreover 4. Discussion p p p g g g ( ) was also observed according to histology, with a higher response in intestinal GC compared to diffuse GC. Over 60% of patients in the intestinal sub-group showed a TRG between 1 and 3 compared to diffuse-type, in which a TRG between 4 and 5 was obtained in over 60% of patients (p = 0.01). No differences were reported in TRG according different pCT regimens. 4. Discussion To our knowledge, this is the first report in real life suggesting the possibility of a histology- driven approach to the treatment of LAGC Although retrospective and small-sized this study To our knowledge, this is the ﬁrst report in real life suggesting the possibility of a histology-driven approach to the treatment of LAGC. Although retrospective and small-sized, this study generates the hypothesis that pCT could be the option of choice for patients with stage II–III intestinal gastric cancer, since they survive much longer when compared to patients with a diﬀuse histology receiving pCT. This ﬁnding does not seem due only to the known prognostic negative eﬀect of diﬀuse histology, since, among patients treated in an adjuvant setting, there was no statistically signiﬁcant diﬀerence concerning OS and EFS between diﬀuse and intestinal histology. Thus, the strategy of therapy (pCT vs. aCT) seems to have aﬀected the outcome in relation to histotype. driven approach to the treatment of LAGC. Although retrospective and small-sized, this study generates the hypothesis that pCT could be the option of choice for patients with stage II–III intestinal gastric cancer, since they survive much longer when compared to patients with a diffuse histology receiving pCT. This finding does not seem due only to the known prognostic negative effect of diffuse histology, since, among patients treated in an adjuvant setting, there was no statistically significant difference concerning OS and EFS between diffuse and intestinal histology. Thus, the strategy of therapy (pCT vs. aCT) seems to have affected the outcome in relation to histotype. In the whole population of our study, there is no survival difference among patients in pCT cohort in comparison to those in aCT cohort. 3.2. Outcome 3.2. Outcome 3.2. Outcome g g p y p g Moreover, in the perioperative cohort, a diﬀerent percentage of tumor regression grade (TRG) was also observed according to histology, with a higher response in intestinal GC compared to diﬀuse GC. Over 60% of patients in the intestinal sub-group showed a TRG between 1 and 3 compared to diﬀuse-type, in which a TRG between 4 and 5 was obtained in over 60% of patients (p = 0.01). No diﬀerences were reported in TRG according diﬀerent pCT regimens. Moreover, in the perioperative cohort, a diﬀerent percentage of tumor regression grade (TRG) was also observed according to histology, with a higher response in intestinal GC compared to diﬀuse GC. Over 60% of patients in the intestinal sub-group showed a TRG between 1 and 3 compared to diﬀuse-type, in which a TRG between 4 and 5 was obtained in over 60% of patients (p = 0.01). No diﬀerences were reported in TRG according diﬀerent pCT regimens. 8 of 11 Cancers 2020, 12, 1749 Figure 4. Kaplan–Meier curves for OS (A) and EFS (B) in aCT cohort according to histology (intestinal diffuse sub–type (HR 1.2; 95% CI 0.72–2.26; p = 0.40 for OS; HR 1.12 CI 95% 0.65–1.91; p = 0.67 for EFS). Figure 4. Kaplan–Meier curves for OS (A) and EFS (B) in aCT cohort according to histology (intestinal diﬀuse sub–type (HR 1.2; 95% CI 0.72–2.26; p = 0.40 for OS; HR 1.12 CI 95% 0.65–1.91; p = 0.67 for EFS). Figure 4. Kaplan–Meier curves for OS (A) and EFS (B) in aCT cohort according to histology (intestinal diffuse sub–type (HR 1.2; 95% CI 0.72–2.26; p = 0.40 for OS; HR 1.12 CI 95% 0.65–1.91; p = 0.67 for EFS). Figure 4. Kaplan–Meier curves for OS (A) and EFS (B) in aCT cohort according to histology (intestinal diﬀuse sub–type (HR 1.2; 95% CI 0.72–2.26; p = 0.40 for OS; HR 1.12 CI 95% 0.65–1.91; p = 0.67 for EFS). Moreover 4. Discussion Updated analysis of INT-0116 shows that intestinal subtype of gastric cancer takes advantage from aCT-RT, but not diﬀuse histotype, suggesting a diﬀerent sensitivity to chemotherapy or an intrinsically worse prognosis [25]. More recently, the FLOT4 study has demonstrated the superiority of a triplet-regimen combination with ﬂuorouracil plus leucovorin, oxaliplatin and docetaxel versus ﬂuorouracil or capecitabine plus cisplatin and epirubicin in the perioperative setting of gastric cancer. In this study, which has become practice-changing superseding all previous trials of perioperative chemotherapy, patients have been stratiﬁed according histology [14]. Although the experimental approach appears equally eﬀective in both histotype, HR for OS is much better in non-diﬀuse histology (0.74 vs. 0.85, p = 0.41). In contrast with our data, the JCOG 0501 phase III study has shown no statistically signiﬁcant diﬀerences between pCT and aCT in poorly diﬀerentiated gastric cancer of an Eastern population, although this study and our study are diﬃcult to be compared since the study has not been published in extenso yet and all patients received adjuvant chemotherapy [26]. changing superseding all previous trials of perioperative chemotherapy, patients have been stratified according histology [14]. Although the experimental approach appears equally effective in both histotype, HR for OS is much better in non-diffuse histology (0.74 vs. 0.85, p = 0.41). In contrast with The highest chemo-sensitivity of intestinal GC might allow a deeper response to neoadjuvant chemotherapy, which, in turn, could lead to smaller tumors at time of surgery, easier resection with a 9 of 11 Cancers 2020, 12, 1749 lower risk of R1 surgery, better control of micrometastatic disease and, ultimately, better outcomes. On the other hand, it might be feasible that in diﬀuse GC delayed surgery in favor of neoadjuvant chemotherapy, through unknown modiﬁcations of tumor microenvironment, could favor tumor cells extravasation and metastasis. Recently, another retrospective analysis suggested an upfront surgery approach highlighting no survival beneﬁt from a pCT strategy in signet-ring cell carcinoma population [27]. Unfortunately, no randomized trial of pCT has evaluated response or survival in histologic subtypes, thus we cannot support our arguments with results of clinical research. However, it should be noticed that in our analysis the rate of patients without postoperative lymph nodes involvement was much higher in patients with intestinal subtype receiving pCT compared not only to patients with diﬀuse GC (32.5% vs. 6.0%) but also to patients with intestinal GC receiving aCT (32.5% vs. 3.3%). Moreover 4. Discussion These observations, together with the higher rate of tumor regression observed in intestinal GCs compared to diﬀuse GCs, suggest that pCT is much more eﬃcacious in patients with intestinal rather than diﬀuse GCs. In multivariate analysis, not only the histologic subtype, but also LVI resulted signiﬁcantly associated with survival in the pCT cohort. Interestingly, the percentage of LVI was about 20–30% in all subgroups of both cohorts with the exception of intestinal subgroup in the pCT cohort (9.6%). Since there is no reason to believe that in such subgroup a population with a diﬀerent biology had been casually selected, we think that it might result from a higher ability of pCT to reduce LVI (as well as lympho-nodes involvement) in the intestinal but not in the diﬀuse subtype. In addition, in all subgroups of our series, about 15–28% of patients received adjuvant RT, with the only exception of patients with intestinal GC treated with pCT. In this subgroup, only 4.8% of patients received adjuvant RT, mainly because the incidence of postoperative node-negative tumors was higher. Taken together, these observations suggest that a histology-driven approach may have an impact not only on the choice of CT strategy, but also modulate the therapeutic program, allowing to reduce the need of adjuvant RT in a subgroup of patients, thus avoiding useless toxicity and saving costs. We are aware that these results have to be conﬁrmed in larger analyses. We also know that in the present genomic era a histology-driven approach to crucial decisions looks simplistic and rather obsolete. However, the increasing cost of new drugs and technology might draw oncologists’ attention to this easy, inexpensive and widely available tool for improving the management of LAGC while waiting for more compelling indications coming from molecular research. Author Contributions: Conceptualization, I.V.Z. and M.B.; Data curation, I.V.Z. and G.G.; Formal analysis, I.V.Z. and A.O.; Methodology, M.B., A.O. and E.B.; Project administration, A.C.; Supervision, E.B., G.T., C.B. and C.P.; Validation, M.B., A.S., C.B. and C.P.; Writing—original draft, I.V.Z., M.B., M.A.C. and C.B.; and Writing—review and editing, M.B., A.S., A.C., M.D.S., E.B., G.T. and C.B. All authors have read and agreed to the published version of the manuscript. Ethical Approval and Consent to Participate: All patient data were collected anonymously. The report does not present identifying images or other personal or clinical details of participants that compromise anonymity. The study was conducted in accordance with the Declaration of Helsinky and consent for chemotherapy was obtained by all patients, also including the consent for retrospective analysis of all clinical data, according to the approval by Ethical Committee of the Catholic University School of Medicine (Policlinico A. Gemelli—Università Cattolica del Sacro Cuore-Roma PROT.0002 approved on 2013). Funding: This research was supported by the non-proﬁt Onlus Association “Stare Accanto-Amici dell’Oncologia Medica del Policlinico A. Gemelli”. References 1. Gee, D.W.; Rattner, D.W. Management of gastroesophageal tumors. Oncologist 2007, 12, 175–185. [CrossRef] [PubMed] 1. Gee, D.W.; Rattner, D.W. Management of gastroesophageal tumors. Oncologist 2007, 12, 175–185. [CrossRef] [PubMed] 2. Siewert, J.R.; Bottcher, K.; Roder, J.D.; Busch, R.; Hermanek, O.; Meyer, H.J. Prognostic relevance of systematic lymph node dissection in gastric carcinoma: German Gastric Carcinoma Study Group. Br. J. Surg. 1993, 80, 1015–1018. [CrossRef] 3. Macdonald, J.S.; Smalley, S.R.; Benedetti, J.; Hundahl, S.A.; Estes, N.C.; Stemmermann, G.N.; Haller, D.G.; Ajani, J.A.; Gunderson, L.L.; Jessup, L.M.; et al. Chemoradiotherapy after surgery compared with surgery alone for adenocarcinoma of the stomach or gastroesophageal junction. N. Engl. J. Med. 2001, 345, 725–730. [CrossRef] 4. Nakajima, T.; Nashimoto, A.; Kitamura, M.; Kito, T.; Iwanaga, T.; Okabayashi, K.; Goto, M. Adjuvant mitomycin and ﬂuorouracil followed by oral uracil plus tegafur in serosa–negative gastric cancer: A randomised trial. The Lancet 1999, 354, 273–277. [CrossRef] 5. Cascinu, S.; Labianca, R.; Barone, C.; Santoro, A.; Carnaghi, C.; Cassano, A.; Beretta, G.; Catalano, V.; Bertetto, O.; Barni, S.; et al. Adjuvant treatment of high-risk, radically resected gastric cancer patients with 5-ﬂuorouracil, leucovorin, cisplatin, and epidoxorubicin in a randomized controlled trial. J. Natl. Cancer Inst. 2007, 99, 601–607. [CrossRef] [PubMed] 6. Lee, J.; Lim, D.H.; Kim, S.; Park, J.O.; Park, J.S.; Lim, H.Y.; Choi, M.G.; Sohn, T.S.; Noh, J.H.; Bae, J.M.; et al. Phase III trial comparing capecitabine plus cisplatin versus capecitabine plus cisplatin with concurrent capecitabine radiotherapy in completely resected gastric cancer with D2 lymph node dissection: The ARTIST trial. J. Clin. Oncol. 2012, 30, 268–273. [CrossRef] [PubMed] 7. Sakuramoto, S.; Sasako, M.; Yamaguchi, T.; Kinoshita, T.; Fujii, M.; Nashimoto, A.; Furukawa, H.; Nakajima, T.; Ohashi, Y.; Imamura, H.; et al. Adjuvant chemotherapy for gastric cancer with S-1, an oral ﬂuoropyrimidine. N. Eng.l J. Med. 2007, 357, 1810–1820. [CrossRef] 8. Bang, Y.J.; Kim, Y.W.; Yang, H.K.; Chung, H.C.; Park, Y.K.; Lee, K.W.; Kim, Y.H.; Noh, S.; Cho, J.Y.; Mok, Y.J.; et al. Adjuvant capecitabine and oxaliplatin for gastric cancer after D2 gastrectomy (CLASSIC): A phase 3 open-label, randomised controlled trial. The Lancet 2012, 379, 315–321. [CrossRef] 9. Cunnigham, D.; Allum, W.H.; Stenning, S.P.; Thompson, J.N.; Van de Velde, C.J.; Nicolson, M.; Howard, S.J.; Lofts, F.J.; Falk, S.J.; Iveson, T.J.; et al. Perioperative chemotherapy versus surgery alone for resectable gastroesophageal cancer. N. Engl. J. Med. 2006, 355, 11–20. [CrossRef] 10. 5. Conclusions Our retrospective analysis seems to suggest that in a Western LAGC population the intestinal histotype might have a better eﬃcacy from a pCT strategy compared to diﬀuse-type to whom an aCT approach might ensure better survival. These results have to be taken with wariness and no conclusive consideration are allowed. Author Contributions: Conceptualization, I.V.Z. and M.B.; Data curation, I.V.Z. and G.G.; Formal analysis, I.V.Z. and A.O.; Methodology, M.B., A.O. and E.B.; Project administration, A.C.; Supervision, E.B., G.T., C.B. and C.P.; Validation, M.B., A.S., C.B. and C.P.; Writing—original draft, I.V.Z., M.B., M.A.C. and C.B.; and Writing—review and editing, M.B., A.S., A.C., M.D.S., E.B., G.T. and C.B. All authors have read and agreed to the published version of the manuscript. Funding: This research was supported by the non-proﬁt Onlus Association “Stare Accanto-Amici dell’Oncologia Medica del Policlinico A. Gemelli”. Conﬂicts of Interest: All authors declare no conﬂict of interest. Conﬂicts of Interest: All authors declare no conﬂict of interest. Ethical Approval and Consent to Participate: All patient data were collected anonymously. The report does not present identifying images or other personal or clinical details of participants that compromise anonymity. The study was conducted in accordance with the Declaration of Helsinky and consent for chemotherapy was obtained by all patients, also including the consent for retrospective analysis of all clinical data, according to the approval by Ethical Committee of the Catholic University School of Medicine (Policlinico A. Gemelli—Università Cattolica del Sacro Cuore-Roma PROT.0002 approved on 2013). 10 of 11 10 of 11 Cancers 2020, 12, 1749 References Ychou, M.; Boige, V.; Pignon, J.P.; Conroy, T.; Bouchè, O.; Lebreton, G.; Ducourtieux, M.; Bedenn, L.; Fabre, L.M.; Saint-Aubert, B.; et al. Perioperative chemotherapy compared with surgery alone for resectable gastroesophageal adenocarcinoma: An FNCLCC and FFCD multicenter phase III trial. J. Clin. Oncol. 2011, 29, 1715–1721. [CrossRef] 11. Macdonald, J.S.; Fleming, T.R.; Peterson, R.F.; Berenberg, J.L.; McClure, S.; Chapman, R.A.; McClure, S.; Chapman, R.A.; Eyre, H.J.; Solanki, D.; et al. 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https://openalex.org/W2767372555	https://link.springer.com/content/pdf/10.1007/s13204-017-0627-2.pdf	English	null	Influence of source parameters on the growth of metal nanoparticles by sputter-gas-aggregation	Applied nanoscience	2,017	cc-by	6,900	Appl Nanosci (2017) 7:875–883 https://doi.org/10.1007/s13204-017-0627-2 Appl Nanosci (2017) 7:875–883 https://doi.org/10.1007/s13204-017-0627-2 ORIGINAL ARTICLE Inﬂuence of source parameters on the growth of metal nanoparticles by sputter-gas-aggregation Malak Khojasteh1,2 • Vitaly V. Kresin1 Received: 24 July 2017 / Accepted: 1 November 2017 / Published online: 7 November 2017 The Author(s) 2017. This article is an open access publication Abstract We describe the production of size-selected manganese nanoclusters using a magnetron sputtering/ag- gregation source. Since nanoparticle production is sensitive to a range of overlapping operating parameters (in partic- ular, the sputtering discharge power, the inert gas ﬂow rates, and the aggregation length), we focus on a detailed map of the inﬂuence of each parameter on the average nanocluster size. In this way, it is possible to identify the main contribution of each parameter to the physical pro- cesses taking place within the source. The discharge power and argon ﬂow supply the metal vapor, and argon also plays a crucial role in the formation of condensation nuclei via three-body collisions. However, the argon ﬂow and the discharge power have a relatively weak effect on the average nanocluster size in the exiting beam. Here the deﬁning role is played by the source residence time, gov- erned by the helium supply (which raises the pressure and density of the gas column inside the source, resulting in more efﬁcient transport of nanoparticles to the exit) and by the aggregation path length. 123 & Vitaly V. Kresin kresin@usc.edu 1 Department of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089-0484, USA 2 Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089-1211, USA 1 Department of Physics and Astronomy, University of Southern California, Los Angeles, CA 90089-0484, USA 2 Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089-1211, USA Introduction To explore precisely how the properties and functionality of nanoscale particles depend on the number of constituent atoms, it is important to have tools which enable full control of particle size, purity, and shape. Consequently, surface deposition of size-selected metal nanoclusters has gained popularity for its ability to tune the particle size and composition over a wide range (Milani and Iannotta 1999; Meiwes-Broer 2000; Binns 2001; Wegner et al. 2006; Vajda and White 2015). A powerful tool for generating beams of neutral and charged nanoclusters covering a range of sizes and mate- rials is the sputtering/aggregation source, also sometimes referred to as the ‘‘terminated gas condensation’’ source (Haberland et al. 1992, 1994; Hutte 2017). It is based on the quenching of atomic vapor produced by magnetron sputtering of the material of interest. The vapor becomes supersaturated due to collisions with the surrounding inert gas atoms which are cooled by the cold walls of the aggregation zone, condenses into nanoclusters, and is car- ried out of the condensation chamber by a continuous ﬂow of gas. This device has been adopted by many research groups and has evolved from a purely home-built instru- ment to a commercial thin-ﬁlm deposition product. Keywords Nanoparticles Nanoclusters Vapor aggregation Mass spectrometry Keywords Nanoparticles Nanoclusters Vapor aggregation Mass spectrometry Understanding the efﬁciency of cluster formation in a source of this type is obviously a nontrivial problem, because it involves the interplay between multiple pro- cesses, including (1) sputtering of atoms and ions, (2) emergence of condensation nuclei, (3) supersaturation and particle growth, (4) transport to the exit aperture and dif- fusion to the aggregation chamber walls, (5) expansion through the aperture into the process vacuum chamber, accompanied by the formation of the nanoparticle beam and termination of growth. Importantly, as nanoparticles & Vitaly V. Kresin kresin@usc.edu 123 Appl Nanosci (2017) 7:875–883 876 move with the gas through the source towards the exit aperture, their local environment continuously changes, adding a degree of non-equilibrium dynamics to the growth process. and visualized with the help of contour plots. Such a map over the permutations and interplay of independent factors is sometimes referred to as a ‘‘factorial design’’ experi- ment. Introduction It allows us to consider and assign the key roles played by the individual parameters listed above, for example the distinct contributions of argon and helium gases to the processes of nanocluster formation and trans- port within the source. These assignments are supple- mented by nanocluster beam velocity measurements. Not surprisingly, therefore, the yield and size distribu- tion of the resulting nanoparticle beam are functions of multiple interrelated operational parameters: source geometry, gas ﬂow rates, discharge power and conﬁgura- tion, aggregation residence time, etc. Thus, to enhance particle production and to steer its size distribution toward the desired range, it is valuable to have both empirical and conceptual insights into the effect of these parameters on the cluster formation process. Many papers have examined the effect of operating conditions on the size, morphology, and kinetic energy of nanoclusters (examples include Hihara and Sumiyama 1998; Morel et al. 2003; Pratontep et al. 2005; Das et al. 2009; Quesnel et al. 2010; Ayesh et al. 2010; Gracia-Pinilla et al. 2010; Nielsen et al. 2010; Ganeva et al. 2012; Luo et al. 2012; Ayesh et al. 2013; Bray et al. 2014; Dutka et al. 2015; Fischer et al. 2015; Kusior et al. 2016; Zhao et al. 2016; Rudd et al. 2017), but each typically looked only at a subset of source parameters. Consequently, a comprehensive multidimensional charac- terization has not yet been presented. 123 Experiment The ions are ﬁltered by a quadrupole mass analyzer equipped with an ion ﬂux measurement grid and enter the deposition chamber 123 877 Appl Nanosci (2017) 7:875–883 Fig. 2 a Tapping mode AFM image of size-selected 4 nm diameter Mn nanoclusters deposited on a Si/SiO2 substrate. b AFM proﬁle of one individual nanoparticle from a, as well as a histogram of the Fig. 2 a Tapping mode AFM image of size-selected 4 nm diameter Mn nanoclusters deposited on a Si/SiO2 substrate. b AFM proﬁle of one individual nanoparticle from a, as well as a histogram of the deposited particles’ heights. Note that the transverse dimension appears artiﬁcially broadened due to tip size convolution diameter proﬁle of m of the deposited particles’ heights. Note that the transverse dimension appears artiﬁcially broadened due to tip size convolution diameter proﬁle of m of the deposited particles’ heights. Note that the transverse dimension appears artiﬁcially broadened due to tip size convolution Fig. 2 a Tapping mode AFM image of size-selected 4 nm diameter Mn nanoclusters deposited on a Si/SiO2 substrate. b AFM proﬁle of one individual nanoparticle from a, as well as a histogram of the deposited particles’ heights. Note that the transverse dimension appears artiﬁcially broadened due to tip size convolution regulated by Alicat MC series mass ﬂow controllers. The ﬂow rate dependence of cluster production will be descri- bed below. Argon is used as the plasma discharge medium, and the roles of argon and helium in the nucleation and clustering process are further discussed below. The outer jacket of the source chamber is maintained full of liquid nitrogen with the help of a funnel ﬁlling system and a liquid level controller. by means of a picoammeter (Keithley 6487). A Faraday cup arrangement (see Appendix) can be positioned down- stream from the quadrupole exit to measure the cluster ions’ kinetic energies. Figure 2 shows, as an example, an atomic-force microscope (AFM) image and proﬁle, and a height histogram, of nanoparticles soft-landed in the deposition chamber when the quadrupole mass spectrom- eter was set to a diameter of 4 nm. The close correspon- dence between the selected and imaged nanoparticle sizes conﬁrms the accuracy of the mass ﬁlter. The gas carries the nanoclusters out of a 5-mm aperture at the source exit, where particle growth is terminated. Experiment Figure 1 shows the scheme of our experimental setup for the production of size-selected nanoclusters. The source is Nanogen-50 from Mantis Deposition Ltd. As mentioned above, nanoparticles are produced by magnetron (dc) sputtering followed by condensation within the environ- ment of a cold inert gas. The magnetron block is equipped with ‘‘magnet set A’’ whose most useful feature, per company speciﬁcations, is that it produces almost exclu- sively ionized clusters (Mantis Deposition 2017), making it possible to ﬁlter and manipulate the entire beam by electric ﬁelds. Clusters are generated from 99.95% Mn targets (ACI Alloys) of 2-in. diameter and 0.125-in. thickness, bonded onto a copper backing plate. The magnetron head is mounted on a linear translator, enabling the aggregation length (the distance between the target and the exit aper- ture) to be varied over a range of 10 cm. Argon and helium gases (both 99.999% purity) are introduced into the source region behind the magnetron head, with ﬂow rates In this paper, we describe a systematic study of the inﬂuence of the parameters of our source on the production of metal nanoclusters, using manganese as an example. Four independently controlled variables (argon and helium ﬂow rates, discharge power, and aggregation length) were varied over a set of discrete levels (corresponding to a total of 720 four-dimensional grid points), and the effect of each combination on the cluster size distribution can be traced Fig. 1 Apparatus schematic. In the magnetron/vapor condensation source, sputtered metal atoms enter the aggregation zone where they undergo collisions with the inert gas and quickly thermalize. Nanocluster ions form and grow, as the mixture moves through the source toward the exit aperture. The ions are ﬁltered by a quadrupole mass analyzer equipped with an ion ﬂux measurement grid and enter the deposition chamber Fig. 1 Apparatus schematic. In the magnetron/vapor condensation source, sputtered metal atoms enter the aggregation zone where they undergo collisions with the inert gas and quickly thermalize. Nanocluster ions form and grow, as the mixture moves through the Fig. 1 Apparatus schematic. In the magnetron/vapor condensation source, sputtered metal atoms enter the aggregation zone where they undergo collisions with the inert gas and quickly thermalize. Nanocluster ions form and grow, as the mixture moves through the source toward the exit aperture. Experiment The resulting directed beam passes through a 6-mm skimmer followed by a high-range/high throughput quadrupole mass ﬁlter (Mantis MesoQ, see also (Baker et al. 1997)) with a manufacturer stated size resolution of * 2%. The standard mass range of the ﬁlter is from 350 amu to * 106 amu, but its performance can be extended somewhat to either side of the standard range. A grid mounted at the quadrupole exit samples the ion ﬂux and an electrometer, included in the mass spectrometer instrumentation package, measures the current corresponding to the selected cluster size. The resolution of the mass ﬁlter is selected by setting the U/ V ratio (i.e., the ratio of the dc and ac amplitudes of the quadrupole’s rod voltages) between 0.001 and 0.168; the rf frequency is then adjusted automatically by the MesoQ power supply and its control software. For the data reported below, the U/V ratio was kept at 0.02. Results and discussion As described above, the magnetron sputtering and cluster formation processes involve the interplay of many source parameters. In our work, the four main factors are the magnetron discharge power P, the aggregation length L, and the Ar and He gas ﬂow rates QAr and QHe. The cluster beam distribution was traced over 5 9 394 9 12 set levels of these variables, respectively, for a total of 720 outcome data points. Such a map enables us to examine both individual effects of the source parameters on the cluster beam distribution as well as, importantly, possible correlations which cannot be detected from separate one-way analyses. Upon passing through the quadrupole, the size-selected nanoclusters ﬁnd themselves in the main deposition chamber (base pressure 10-6 Pa). Here their mass deposition rate, as a function of size, can be measured using a quartz crystal ﬁlm thickness oscillator (McVac Manufacturing) and monitor (Inﬁcon XTC). In addition to the arrangement described in a preliminary report (Kho- jasteh and Kresin 2016), in this work the ion current impinging on the deposition surface can also be measured 123 Argon and helium supply In dc magnetron sputtering, a high negative voltage is applied to the target, accelerating Ar? ions to sputter material off the target (in our case, Mn) surface. Strong magnets positioned behind the target create a specially shaped magnetic ﬁeld designed to lengthen electron paths in front of the target and intensify the plasma. In our 123 Appl Nanosci (2017) 7:875–883 878 Fig. 4 Position of the peak of the Mn nanoparticle size distribution vs. Ar and He ﬂow rates. The ﬂow rates were measured at intervals of 20 sccm, and the values in-between interpolated. The discharge power was 22 W and the aggregation length was 9 cm exploration of the parameter space, we ﬁrst let in only argon gas to determine the dc power needed to produce a stable ﬂux of Mn nanoclusters as detected by the quadru- pole mass ﬁlter. This process was performed gradually to prevent target thermal shock possibly resulting in cracking or debonding from the backing plate. Once the discharge is established, the Ar ﬂow rate can be increased further, and then He admixed gradually. In this way, the variation of cluster sizes as a function of both gas ﬂow rates can be mapped out for a given discharge power and condensation length. We found that the size distribution is quite repro- ducible for each set of operating parameters. Initially, as the supply of pure argon is increased both the ﬂux and the average size of the cluster ions grow, until ﬁnally a stable log-normal-type shape of the distribution becomes established. At this point, the helium supply is turned on, and the response of the nanoparticle beam to increasing helium ﬂow is illustrated in Fig. 3: the overall intensity rises, reaches a maximum, and then starts to decrease, while the average particle size shifts to smaller sizes. At the same time, the width of the beam distribution becomes narrower. Fig. 4 Position of the peak of the Mn nanoparticle size distribution vs. Ar and He ﬂow rates. The ﬂow rates were measured at intervals of 20 sccm, and the values in-between interpolated. The discharge power was 22 W and the aggregation length was 9 cm functions within the cluster source. Argon and helium supply Their roles and inﬂu- ences can be rationalized as follows: As extensively described in the literature, the formation of nanoclusters Mn out of atomic vapor is initiated by nucleation and sustained by supersaturation and growth (see, e.g., Kappes and Leutwyler 1988; Haberland 1994; Pauly 2000; Smirnov 2000; Hutte 2017). The initial step is the formation of a bound dimer M2 which requires a three- body collision for stabilization: M ? M?Ar ? M2 ? Ar. It is well-known that the heavier noble gas atoms are efﬁcient at removing the dimer’s binding energy, and helium is not nearly as effective at enabling nucleation. This is also why heavier carrier gases are better at pro- moting clustering in supersonic expansion sources (Kappes and Leutwyler 1988). The dimers then serve as condensa- tion nuclei for further growth, if the vapor is maintained in a state of supersaturation. In this process, clusters grow by sequential condensation as additional atoms arrive at their surface one by one (with further collisions with noble gas atoms helpful in cooling the cluster seeds by removing the additional condensation energy). At higher nucleation densities, cluster–cluster collisions also can result in the appearance of larger particles. Particles which reach the so- called ‘‘critical size’’ will continue coagulating towards the condensed phase; therefore, if a population of ﬁnite-sized clusters is desired, then the condensation process must be interrupted. In the present source, this comes about as the gas ﬂow carries the atomic vapor through the aperture and out of the condensation zone. Figure 4 puts the inﬂuence of both gases into perspec- tive by simultaneously plotting the effect of Ar and He ﬂows on the peak of the cluster beam distribution. With the helium supply ﬁxed, increasing the argon ﬂow has only a moderate inﬂuence on the average particle size. However (as already illustrated in Fig. 3), an increase in the helium ﬂow shifts the beam distribution toward lower sizes very signiﬁcantly. How can one interpret these different (indeed, opposite) trends? It is evident that helium and argon perform distinct Fig. 3 Effect of He ﬂow rate on the size distribution of Mn nanoclusters. All other source parameters are kept constant: aggre- gation length 9 cm, discharge power 21.8 W, Ar ﬂow rate 150 sccm 1 3 Now we can formulate the separate roles of the two noble gases supplied to the source. Argon and helium supply While the distinction obviously is not sharp, it enables useful qualitative inter- pretation and guidance. Fig. 3 Effect of He ﬂow rate on the size distribution of Mn nanoclusters. All other source parameters are kept constant: aggre- gation length 9 cm, discharge power 21.8 W, Ar ﬂow rate 150 sccm 879 Appl Nanosci (2017) 7:875–883 As just stated, the size of nanoclusters in the beam is to a large degree controlled not by a hypothetical equilibrium distribution, but by the fact that the growth process is interrupted by the transit of the clusters out of the source (hence the aforementioned label ‘‘terminated gas conden- sation source’’). The stage at which particle condensation is interrupted, and therefore the maximum size that is able to be attained, is deﬁned by the residence time in the growth region, i.e., by the speed at which the metal vapor/inert gas mixture is swept from the sputtering area to the source exit aperture. It is this transport which appears to be mostly affected by the amount of helium ﬂow into the source, in such a way that the average cluster size goes down as the helium supply increases. the appearance of condensation nuclei. Hence the argon density strongly affects the overall intensity of the nanoparticle beam, but its roles in supplying metal vapor for coagulation and in promoting cluster drift toward the source exit appear to balance each other out. As a result, the Ar ﬂow rate does not have a sharp inﬂuence on the size distribution of the formed particles. An alternative interpretation of the principal role of helium in a magnetron source was put forward by Pra- tontep et al. (2005). They suggested that the helium gas is itself involved in cluster formation in such a way that, with increased He ﬂow there is a rise in the nucleation of small seeds which results in more but smaller clusters. However, since argon is even more efﬁcient in enabling the formation of small nuclei, under this scenario one might expect a stronger shift towards small sizes not just with He, but also with increasing Ar ﬂow, which is not observed. In what way can the rate of helium gas supply inﬂuence the time a nanoparticle spends inside the source? Magnetron power and aggregation length The dc sputtering discharge power strongly affects nan- ocluster production. In principle, the stronger the dis- charge the greater the supply of raw cluster material into the vapor; however, one also has to be cognizant of heat load limitations on the target as well as of discharge stability and plasma charging dynamics. In Fig. 5a, we examine the inﬂuence of power and helium ﬂow rate on the peak of the beam size distribution. We see that in this representation, the helium supply again plays the most inﬂuential role. Figure 5b plots the variation of the peak of the beam size distribution under the inﬂuence of discharge power and argon ﬂow rate in the absence of helium gas. Note that the size range variation is signiﬁcantly narrower than in the presence of He. A comparison of Fig. 5a, b reafﬁrms that He plays the dominant role in shifting the nanocluster distribution toward smaller sizes. Therefore, the likely reason for the reduction in average cluster size with greater He density inside the source is that the clusters become more effectively entrapped in the gas streamlines. This derives both (1) from the higher number of cluster collisions with the gas atoms in the column drifting to the exit aperture, and (2) from the fact that the rate of cluster diffusion toward the surrounding walls decreases inversely with the diffusion coefﬁcient and therefore inversely with the gas density (Smirnov 2000; Shyjumon et al. 2006). As a result, the growing particles have a greater tendency to persist on their direct trajecto- ries, their residence time decreases, and the growth is ter- minated sooner. Analogous conclusions are drawn from varying the aggregation length L. Figure 6a is a plot of the joint inﬂuence of L and QAr at zero helium ﬂow rate on the peak size of the nanocluster distribution. We see that the aggregation length plays the main role in changing the size, while there is little sensitivity to argon ﬂow. In contrast, Fig. 6b, which follows the joint inﬂuence of L and QHe, demonstrates a strong effect along both axes. Argon and helium supply It might be supposed that a higher mass ﬂow¸ Q, translates into a greater speed of the gas column inside the source, vgas, pulling the particles along and reducing their resi- dence/growth time. However, this is mainly not the case. Indeed, in equilibrium, the gas mass ﬂow through the source is Q = qgasvgasA, where qgas is the gas mass density in the column and A is its effective cross section. At the same time, Q must equal the mass ﬂow through the nozzle aperture into the vacuum chamber, which is proportional to the stagnation pressure in the plane of the nozzle (Miller 1988; Pauly 2000), and thereby to qgas: QPgasqgas. Comparing these two expressions, both of which involve qgas but only one involves vgas, we conclude that raising the inlet gas ﬂow rate should mainly affect the pressure and density of the gas column inside the source but not its velocity. Magnetron power and aggregation length The ﬂow rate was measured at intervals of 20 sccm, and the discharge powers and corresponding discharge currents were P = 7.3, 11.3, 15.8, 21.8, 35 W and I = 35, 55, 75, 100, and 150 mA, respectively; the values in-between are interpolated. The argon ﬂow rate was 190 sccm and the aggregation length was 9 cm. b The peak of the Mn nanocluster size distribution vs. magnetron discharge power and Ar ﬂow rate. The ﬂow rate intervals, and powers, discharge currents and the aggrega- tion length were the same as in a. No helium ﬂow was present for this plot Fig. 6 a The peak of the Mn nanocluster size distribution vs. the aggregation length and Ar ﬂow rate. The ﬂow rate was measured at intervals of 20 sccm, and the investigated lengths were 5, 7, and 9 cm. For this plot, no helium ﬂow was present and the discharge power was 7.3 W. b The peak of the Mn nanocluster size distribution vs. the aggregation length and He ﬂow rate. The ﬂow rate intervals, the investigated aggregation lengths, and the power were the same as in a, and the Ar ﬂow rate was 210 sccm Fig. 6 a The peak of the Mn nanocluster size distribution vs. the aggregation length and Ar ﬂow rate. The ﬂow rate was measured at intervals of 20 sccm, and the investigated lengths were 5, 7, and 9 cm. For this plot, no helium ﬂow was present and the discharge power was 7.3 W. b The peak of the Mn nanocluster size distribution vs. the aggregation length and He ﬂow rate. The ﬂow rate intervals, the investigated aggregation lengths, and the power were the same as in a, and the Ar ﬂow rate was 210 sccm Fig. 5 a Position of the peak of the Mn nanoparticle size distribution vs. magnetron discharge power and He ﬂow rate. The ﬂow rate was measured at intervals of 20 sccm, and the discharge powers and corresponding discharge currents were P = 7.3, 11.3, 15.8, 21.8, 35 W and I = 35, 55, 75, 100, and 150 mA, respectively; the values in-between are interpolated. The argon ﬂow rate was 190 sccm and the aggregation length was 9 cm. b The peak of the Mn nanocluster size distribution vs. magnetron discharge power and Ar ﬂow rate. Magnetron power and aggregation length The marked decrease in average particle size either with increasing He ﬂow rate or with decreasing aggregation length conﬁrms that the source residence time is the most sensitive parameter in determining the extent of particle condensa- tion in the cold, strongly supersaturated, sputtered metal vapor environment, and that the dominant role of helium is in setting the transport time through the aggregation tube, as discussed above. The fact that smaller cluster sizes are congruent with entrapment in the gas is also supported by velocity mea- surements on particles emerging from the source aperture, as described in the Appendix. An increased supply of Ar contributes to cluster trans- port as well; however, it also performs the essential func- tions of (1) enabling the sputtering process, thereby feeding atoms and atomic ions into the vapor, and (2) facilitating 123 123 Appl Nanosci (2017) 7:875–883 880 e Mn nanoparticle size distribution nd He ﬂow rate. The ﬂow rate was m, and the discharge powers and were P = 7.3, 11.3, 15.8, 21.8, d 150 mA, respectively; the values argon ﬂow rate was 190 sccm and b The peak of the Mn nanocluster charge power and Ar ﬂow rate. The ischarge currents and the aggrega- Fig. 6 a The peak of the Mn nanocluster size distribution vs. the aggregation length and Ar ﬂow rate. The ﬂow rate was measured at intervals of 20 sccm, and the investigated lengths were 5, 7, and 9 cm. For this plot, no helium ﬂow was present and the discharge power was 7.3 W. b The peak of the Mn nanocluster size distribution vs. the aggregation length and He ﬂow rate. The ﬂow rate intervals, the investigated aggregation lengths, and the power were the same as in a, and the Ar ﬂow rate was 210 sccm Conclusions W h t d d t il d t d f th i ﬂ f th The sputtering power supplied to the discharge and the argon ﬂow are the crucial parameters for nanocluster pro- duction. The discharge supplies the metal vapor for building the nanoparticles, while argon is not only Fig. 5 a Position of the peak of the Mn nanoparticle size distribution vs. magnetron discharge power and He ﬂow rate. Magnetron power and aggregation length The ﬂow rate intervals, and powers, discharge currents and the aggrega- tion length were the same as in a. No helium ﬂow was present for this plot Fig. 6 a The peak of the Mn nanocluster size distribution vs. the aggregation length and Ar ﬂow rate. The ﬂow rate was measured at intervals of 20 sccm, and the investigated lengths were 5, 7, and 9 cm. For this plot, no helium ﬂow was present and the discharge power was 7.3 W. b The peak of the Mn nanocluster size distribution vs. the aggregation length and He ﬂow rate. The ﬂow rate intervals, the investigated aggregation lengths, and the power were the same as in a, and the Ar ﬂow rate was 210 sccm The sputtering power supplied to the discharge and the argon ﬂow are the crucial parameters for nanocluster pro- duction. The discharge supplies the metal vapor for building the nanoparticles, while argon is not only responsible for the sputtering process but also is the dominant player in three-body collisions that provide the condensation nuclei triggering further growth. 123 Conclusions A measurement of the kinetic energies of nanocluster ions exiting the source supports the preferential entrapment of smaller nanoclusters by the gas ﬂow: 2 nm particles followed the terminal velocity of the gas expansion, while 9 nm ones displayed a signiﬁcant velocity slip. In the context of the present work, it is suggested that increased gas ﬂow promotes the transport of nanoclusters through the interior of the aggregation volume. This decreases the time available for condensation and reduces the average cluster size in the outgoing beam. Therefore, one also would expect that for a given amount of gas ﬂow and a given size distribution, the smaller clusters exit the nozzle with velocities closer to those of the helium and argon atoms, while the larger ones (which are prone to follow the streamlines less efﬁciently and therefore to spend longer within the condensation region) would exhibit a signiﬁcantly larger velocity slip. The two variables, the helium supply rate and the aggregation length (controlled by shifting the magnetron head with respect to the source exit aperture) have the dominant inﬂuence on the average nanocluster size in the outgoing beam. The conclusions guided by systematic studies of source operation are useful for optimizing source performance, and are fruitful in untangling speciﬁc physical processes taking place within the dynamic sputtering/condensation source environment. It would be possible and interesting to gain further insight by exploring the above variables over a still wider range of values, as well as by adding new ones, for example other types of noble gases, variable sources of wall temperature, precise control of internal source pres- sure, etc., and by position- and time-resolved spectroscopy of the contents of the source interior. The velocities of negative cluster ions were measured by the retarding potential technique, using a Faraday cup with two grids, one to repel positively charged clusters and the other to apply a slowing voltage V. The current I of the ion beam was measured by the picoammeter and its kinetic energy distribution was determined by differentiating the I(V) curve and ﬁtting the result with a Gaussian function. A complementary measurement of the ion energies utilizing a quadrupole beam deﬂector resulted in very close values. Details of these experimental arrangements will be described elsewhere. Acknowledgements We would like to thank Dr. Avik Halder for extensive discussions, Akash P. Conclusions Shah for valuable experimental assistance and for constructing the quadrupole ion deﬂector, the USC Machine Shop personnel for expert technical help, and the staff of Mantis Deposition Ltd. for their advice. This research was supported by the Army Research Ofﬁce under Grant Number W911NF-17-1- 0154. The velocity distributions of Mn nanoparticles of 2 and 9 nm diameter are shown in Fig. 7. The gas ﬂows for the two cases corresponded to molar fraction ratios within the source of XAr/XHe = 0.7 and XAr/XHe = 3, respectively. The average velocity of the smaller nanoparticles is 600 m/s, while that of the larger ones is much low- er, * 140 m/s. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The terminal velocity for a gas mixture in the contin- uum expansion regime can be approximated (Cattolica et al. 1979; Miller 1988) by the use of an average mass M ¼ P XiMi, so that for monatomic gases, one has vt ¼ 5kBT0= M ð Þ1=2 [for a purely effusive expansion, the forward beam velocity is & 15% lower (Pauly 2000; Conclusions We have presented a detailed study of the inﬂuence of the main operating parameters of a magnetron/condensation nanocluster source on the particle size. Speciﬁcally, we investigated how the peak size of the nanocluster ensemble responds to changes in the argon and helium gas supply ﬂow rates, in the discharge power, and in the aggregation length. The beneﬁt of such a cross-correlation study is that it allows one to classify the main physical role played by each of the variables. Once the discharge and nucleation processes are stabi- lized, the next dominant factor is the source residence time, i.e., the length of time over which aggregation of the cryogenically cooled highly supersaturated metal vapor is allowed to proceed. If not terminated, it would result in the formation of large ‘‘smoke’’ particles both by addition of 123 881 Appl Nanosci (2017) 7:875–883 During operation, the pressure of the argon and helium mixture inside the source is in the range of 10–100 Pa (Haberland et al. 1992; Hutte 2017). The source walls are cooled by liquid nitrogen, but the stagnation temperature at the exit aperture is expected to be higher. The corre- sponding mean free path l of the gas atoms lies in the range of * 0.05–1.5 mm (Haynes 2016). This corresponds to Knudsen numbers Kn = l/d * 0.01–0.3, where d = 5 mm is the diameter of the exit aperture, placing the expansion in the intermediate to mildly supersonic continuum regime (Hutzler et al. 2012). In this range, atoms and small molecules approach the regime of being fully accelerated by a buffer gas expansion (Hutzler et al. 2012); however, the ‘‘velocity slip’’ phenomenon (Milani and Iannotta 1999) also becomes more and more pronounced as the mass of the diluted species increases. individual atoms and by binary cluster–cluster collisions (Pfau et al. 1982; Zimmermann et al. 1994). Hence for obtaining a population of sufﬁciently small nanoclusters, it is essential to sweep the aggregating medium out of the source at an adequately fast rate. This is the main role of the helium supply. It is much less efﬁcient than argon at promoting nucleation and aggregation, but an increase in the helium ﬂow raises the pressure and density of the gas column inside the source, resulting in stronger entrapment of nanoparticles within the gas streamlines. This reduces their residence time and enhances the population of smaller particles in the beam. 123 Appendix: Cluster velocities A measurement of cluster beam velocities was performed to examine the degree to which nanoparticles are suscep- tible to following the source gas streamlines. 123 Appl Nanosci (2017) 7:875–883 882 Fig. 7 Velocity distributions of Mn nanoparticles emitted by the source, as determined via retarding potential measurements. a Particle diameter 2 nm (aggregation length L = 9 cm, Ar and He ﬂow rates QAr = 150 sccm and QHe = 210 sccm, discharge power P = 15 W). b Particle diameter 9 nm (L = 9 cm, QAr = 150 sccm, QHe = 50 sccm, P = 15 W) Hutzler et al. 2012)]. The aforementioned velocity of 2 nm particles is in sensible agreement with the value vt & 650 m/s obtained for a stagnation temperature T0 = 200 K (as expected, this is somewhat higher than at the source jacket, see above), but the corresponding value for the 9 nm particle source parameters would be & 500 m/s, which is signiﬁcantly greater than the measured velocity. This implies that the larger nanoclusters display a signiﬁcant velocity slip. Other groups (Ayesh et al. 2007; Polonskyi et al. 2012; Ganeva et al. 2013) have reported analogously low velocities and evidence of strong velocity slip for the heavier nanoclusters produced by magnetron aggregation sources. These observations sup- port the picture of a more efﬁcient transport of smaller nanoclusters by the gas ﬂowing through the source. Das SC, Majumdar A, Shripathi T, Hippler R (2009) Development of metal nanocluster ion source based on DC magnetron plasma sputtering at room temperature. Rev Sci Instrum 80:095103 Dutka MV, Turkin AA, Vainchtein DI, De Hosson JThM (2015) On the formation of copper nanoparticles in nanocluster aggregation source. J Vac Sci Technol, A 33:031509 Fischer A, Kruk R, Hahn H (2015) A versatile apparatus for the ﬁne- tuned synthesis of cluster-based materials. Rev Sci Instrum 86:023304 Ganeva M, Peter T, Bornholdt S, Kersten H, Strunskus T, Zaporo- jtchenko V, Faupel F, Hippler R (2012) Mass spectrometric investigations of nano-size cluster ions produced by high pressure magnetron sputtering. Contrib Plasma Phys 52:881–889 Ganeva M, Pipa AV, Smirnov BM, Kashtanov PV, Hippler R (2013) Velocity distribution of mass selected nano-size clusters. Plasma Sources Sci Technol 22:045011 Gracia-Pinilla M, Vidaurri GS, Pe´rez-Tijerina E (2010) Deposition of size-selected Cu nanoparticles by inert gas condensation. Nanoscale Res Lett 5:180–188 Haberland H (1994) Experimental methods. Appendix: Cluster velocities In: Haberland H (ed) Clusters of atoms and molecules: theory, experiment, and clusters of atoms. Springer, Berlin, pp 207–232 Haberland H, Karrais M, Mall M, Thurner Y (1992) Thin ﬁlms from energetic cluster impact: a feasibility study. J Vac Sci Technol, A 10:3266–3271 References Ayesh AI, Lassesson A, Brown SA, Dunbar ADF, Kaufmann M, Partridge JG, Reichel R, Lith JV (2007) Experimental and simulational study of the operation for a high transmission mass ﬁlter. Rev Sci Instrum 78:053906 Haberland H, Mall M, Moseler M, Qiang Y, Reiners T, Thurner Y (1994) Filling of micron-sized contact holes with copper by energetic cluster impact. J Vac Sci Technol, A 12:2925–2930 Haynes W (ed) (2016) CRC handbook of chemistry and physics, 97th edn. CRC Press, Boca Raton Ayesh AI, Qamhieh N, Ghamlouche H, Thaker S, El-Shaer M (2010) Fabrication of size-selected Pd nanoclusters using a magnetron plasma sputtering source. 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Surf Sci Rep 44:1–49 Khojasteh M, Kresin VV (2016) Formation of manganese nanoclus- ters in a sputtering/aggregation source and the roles of individual operating parameters. Proc SPIE 10174:1017407 Bray KR, Jiao CQ, DeCerbo JN (2014) Inﬂuence of carrier gas on the nucleation and growth of Nb nanoclusters formed through plasma gas condensation. J Vac Sci Technol, B 32:031805 Kusior A, Kollbek K, Kowalski K, Borysiewicz M, Wojcie T (2016) Sn and Cu oxide nanoparticles deposited on TiO2 nanoﬂower 3D substrates by inert gas condensation technique. Appl Surf Sci 380:193–202 Cattolica RJ, Gallagher RJ, Anderson JB, Talbot L (1979) Aerody- namic separation of gases by velocity slip in free jet expansions. AIAA J 17:344–355 123 Appl Nanosci (2017) 7:875–883 883 Luo Z, Woodward WH, Smith JC, Castleman AW Jr (2012) Growth kinetics of Al clusters in the gas phase produced by a magnetron- sputtering source. 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https://openalex.org/W4254198015	https://insu.hal.science/insu-01876914/document	English	null	WIRA-C: A compact 142-GHz-radiometer for continuous middle-atmospheric wind measurements	null	2,018	cc-by	16,780	To cite this version: Jonas Hagen, Axel Murk, Rolf Rüfenacht, Sergey Khaykin, Alain Hauchecorne, et al.. WIRA-C: a compact 142-GHz-radiometer for continuous middle-atmospheric wind measurements. Atmospheric Measurement Techniques, 2018, 11 (9), pp.5007 - 5024. 10.5194/amt-11-5007-2018. insu-01876914 WIRA-C: a compact 142-GHz-radiometer for continuous middle-atmospheric wind measurements Jonas Hagen, Axel Murk, Rolf Rüfenacht, Sergey Khaykin, Alain Hauchecorne, Niklaus Kämpfer WIRA-C: a compact 142-GHz-radiometer for continuous middle-atmospheric wind measurements Jonas Hagen, Axel Murk, Rolf Rüfenacht, Sergey Khaykin, Alain Hauchecorne, Niklaus Kämpfer To cite this version: Jonas Hagen, Axel Murk, Rolf Rüfenacht, Sergey Khaykin, Alain Hauchecorne, et al.. WIRA-C: a compact 142-GHz-radiometer for continuous middle-atmospheric wind measurements. Atmospheric Measurement Techniques, 2018, 11 (9), pp.5007 - 5024. 10.5194/amt-11-5007-2018. insu-01876914 Correspondence: Jonas Hagen (jonas.hagen@iap.unibe.ch) Correspondence: Jonas Hagen (jonas.hagen@iap.unibe.ch) Received: 28 February 2018 – Discussion started: 4 April 2018 Revised: 12 July 2018 – Accepted: 31 July 2018 – Published: 4 September 2018 Abstract. Ground-based microwave wind radiometry pro- vides a method to measure horizontal wind speeds at alti- tudes between 35 and 75 km as has been shown by various previous studies. No other method is capable of continuously delivering wind measurements in this altitude region. As op- posed to lidar systems, microwave radiometers operate au- tonomously and independent of daylight and clouds. paign and compare our measurements to model data from the European Centre for Medium-range Weather Forecasts (ECMWF) and coincident measurements of the co-located Rayleigh–Mie Doppler wind lidar. We ﬁnd a good agree- ment between our measurements and the ECMWF opera- tional analysis for the time series, where many features are present in both datasets. The wind proﬁles of the coinci- dent WIRA-C and lidar observations are consistent and agree within their respective uncertainties for the lidar measure- ments with long integration times. In this paper, we present the WIRA-C (Wind Radiometer for Campaigns) instrument that observes the 142.17504 GHz rotational transition line of ozone with a high spectral resolu- tion using a low noise single side band heterodyne receiver. Because the emitting molecules are drifting with the wind, the line is Doppler shifted. Together with the pressure broad- ening effect, this allows the retrieval of altitude resolved wind proﬁles. HAL Id: insu-01876914 https://insu.hal.science/insu-01876914v1 Submitted on 3 Aug 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Atmos. Meas. Tech., 11, 5007–5024, 2018 https://doi.org/10.5194/amt-11-5007-2018 © Author(s) 2018. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Wind is a key parameter of dynamics throughout the atmo- sphere. In the troposphere, wind is directly related to weather phenomena. Dynamics in the stratosphere also have an in- ﬂuence on tropospheric dynamics and thus on weather phe- nomena (Baldwin et al., 2003; Charlton et al., 2004). Hence, many numerical weather prediction models have extended their upper limit to the mesosphere region in the past few years. At the same time, it is a fact that nearly no measure- ments of wind speeds in the upper stratosphere and the lower mesosphere exist. This region roughly corresponds to the so called radar gap, where too few scatterers for radar obser- vations are present. The ﬁrst wind radiometer WIRA proved Doppler microwave radiometry to be a suitable method to achieve wind proﬁle observations between 35 and 75 km al- titude on a campaign basis as well as for long term station- ary measurements (Rüfenacht et al., 2012, 2014). In con- trast, Rayleigh–Mie Doppler wind lidar techniques can also reach the upper stratosphere or even the mesosphere at 80 km The novel WIRA-C instrument represents the newest de- velopment in microwave wind radiometry and implements many improvements over its predecessor, the WIRA instru- ment. The main improvements include the compact structure, lower noise and an advanced retrieval setup. This paper de- scribes the instrument and the data processing with a focus on the retrieval that takes into account a three-dimensional at- mosphere and has never been used in ground-based radiom- etry before. The retrieval yields proﬁles of horizontal wind speeds with a 12 h time resolution and a vertical resolution of 10 km for zonal and 10 to 15 km for meridional wind speeds. We give an error estimate that accounts for the thermal noise on the measured spectra and additionally estimate systematic errors using Monte Carlo methods. WIRA-C has been continuously measuring horizontal wind speeds for 1 year at the Maïdo observatory on Réunion (21.4◦S, 55.9◦E). We present the time series of this cam- 2 Measurement principle WIRA-C measures the spectral intensity of the 142.17504 GHz ozone rotational transition emission line. Wind information is introduced to the emission line by the classical Doppler shift, the linear relation between the line-of-sight speed of an emitter drifting with the wind ﬂow vlos and the observed frequency shift 1ν: Rogers et al. (2016) observed the 11 GHz ozone line using low-cost satellite television electronics and derived seasonal and local solar time aggregated wind speeds at 95 km altitude using 5 years of measurements. Spaceborne instruments like the Microwave Limb Sounder (MLS) measured wind speeds between 70 and 95 km (Wu et al., 2008) using the Doppler shift introduced to the 118 GHz emission line of oxygen and proposed to extend this range towards 40 km by using other emission lines. The Su- perconducting Submillimeter Wave Limb-Emission Sounder (SMILES) observed winds between October 2009 and April 2010 between 30 and 80 km by observing the Doppler shift of the 625 GHz ozone emission line and the HCl emission line at 625 GHz (Baron et al., 2013). 1ν = vlos c ν0. (1) 1ν = vlos c ν0. (1) Further, the emission line is pressure broadened, meaning that information about the altitude of the emitters is encoded in the spectrum. This allows the retrieval of wind proﬁles up to approximately 75 km, where the altitude-independent Doppler broadening effect starts to dominate. Because the Doppler shift is proportional to the emitted frequency ν0, it is advantageous to use a high observation frequency. We chose the 142 GHz emission line of ozone be- cause of its strong magnitude and because the troposphere is more transparent in this frequency range than at higher frequencies. This limits the tropospheric contribution to the observed spectrum and increases the signal-to-noise ratio for middle atmospheric emission signals. Ground-based passive microwave instruments are au- tonomous and independent of daylight or clouds and can thus deliver continuous measurements, even though with lower spacial and temporal resolution compared to lidar. Such measurements are important for the validation of models and other instruments, as demonstrated by Rüfenacht et al. (2018). In addition Le Pichon et al. (2015) showed that mi- crowave wind radiometry is a valuable complement to other techniques like lidar and infrasound at multi instrument sites and contributes to the general understanding of middle atmo- spheric dynamics. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns (Souprayen et al., 1999; Baumgarten, 2010; Yan et al., 2017). Lidar systems can provide wind proﬁles with a high temporal and spacial resolution; however, they always need clear sky conditions and measurements during daytime are difﬁcult to achieve. In addition, they are not operating autonomously and are thus not very well suited for continuous wind mea- surements. is widely used in middle atmospheric research and to coinci- dent lidar measurements. 2 Measurement principle Passive microwave wind radiometers require a stable frequency reference as the ratio between observation fre- quency and the Doppler shift is in the order of 10−8 to 10−7 for typical atmospheric wind speeds of 10 m s−1 or 100 m s−1, respectively. Given our observation frequency of 142.17504 GHz, the Doppler shift introduced by line-of- sight wind speeds is 4.75 kHz per 10 m s−1. Further, we rely on opposing measurement directions, for example eastwards vs. westwards, to derive an absolute wind speed in the pres- ence of possible frequency drifts and shifts not related to wind. This implies that we assume the horizontal wind speed to be constant over the horizontal distance spanned by the two opposing line-of-sights. For an elevation angle of 22◦, this horizontal distance would be 150 km at 30 km altitude and 370 km at 70 km altitude. The WIRA-C instrument (Wind Radiometer for Cam- paigns) presented here, represents the newest development in microwave wind radiometry. It is capable to deliver 12 hourly resolved wind proﬁles in an altitude range of 35 to 75 km. Compared to the WIRA instrument (Rüfenacht et al., 2012), it is more compact, and thus easier to deploy and operate on campaigns. All optical elements, including the calibration target and the corrugated feed horn antenna, are integrated in a single housing with a stable temperature and stay dry and clean at all times, which allows us to resume high-quality observations immediately after rainfall. Furthermore, we ap- ply a three-dimensional retrieval method (Christensen, 2015) that has never been used for ground based radiometry before. Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. 5008 3.1 Receiver optics Figure 2 shows the optical system with its four mirrors. Ra- diation from the sky enters the instrument through the scan drum that contains the ﬂat mirror M3 and is rotatable to se- lect any elevation angle. Together with the azimuthal drive at the bottom of the instrument, all cardinal directions (north, east, south, west) can be observed. This is important for ro- bust wind retrievals, as the observation of opposite directions allows us to compensate for possible shifts in absolute fre- quency scale and also makes the calibration more robust as will be explained in Sect. 4.1. Besides the more compact structure, several technical im- provements have been made over the WIRA prototype pre- sented by Rüfenacht et al. (2012). Firstly, WIRA-C has a bet- ter signal-to-noise ratio than WIRA, thanks to the better low noise ampliﬁer (LNA) in the receiver chain. Secondly, while WIRA observes at a ﬁxed elevation angle of 22◦, WIRA-C can freely select the elevation and azimuth angle to look at the sky thanks to independent elevation and azimuth drives. This makes WIRA-C a true all-sky microwave radiometer, similar to the concept of ASMUWARA (Martin et al., 2006), and we will beneﬁt from this ﬂexibility in the future, e.g. for the characterisation of tropospheric inhomogeneities in the context of tipping curve calibration. At the moment we use the all-sky mode only for the geometrical alignment by scanning the sun. For wind measurements we use the same well-established observation scheme as for WIRA because 22◦elevation provide an optimum in terms of projection of horizontal wind speed to the line-of-sight vs. decreas- ing signal to noise ratio with increasing path length through the troposphere. Further, the ambient temperature calibration target is embedded inside the housing and thus better pro- tected against environmental inﬂuence such as inhomoge- neous heating by solar radiation. In particular, the optics and the calibration target are fully protected against rain. As no highly absorbing water can be deposited on the optical com- ponents, the instrument can resume the measurement imme- diately after rainfall has stopped. In addition many smaller technical improvements have been implemented, for exam- From mirror M3, the radiation is deﬂected by the ﬂat mir- ror M2 and coupled into the feed horn antenna by the ellip- tical mirror M1. 3 The instrument After a short introduction of the measurement principle, we present the instrument, its optics and receiver system in Sect. 3. The data processing and the retrieval process used to obtain wind proﬁles from radiometric measurements is pre- sented in Sect. 4. Also in Sect. 4, we present error estimations for random and systematic errors of our retrieval. Finally, the results from the 1-year campaign of WIRA-C on the Maïdo observatory on Réunion are shown in Sect. 5 and we compare our measurement data to the European Centre for Medium- range Weather Forecasts (ECMWF) operational model that WIRA-C has been designed to be compact and autonomous. As depicted in Fig. 1, it ﬁts into one single housing with the dimensions 0.6×0.75×0.5 m and is set up on a tripod. It only needs an ethernet and a power connection and thus requires no additional laboratory space. Once set up, it measures au- tonomously and we supervise and conﬁgure the measure- ment process via remote connection. This makes WIRA-C an ideal instrument for campaigns at remote locations as well as for long term continuous observations. Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaign [t] Figure 1. The WIRA-C instrument as installed on the Maïdo ob- servatory on Réunion. It measures 0.6 × 0.75 × 0.5 m and contains the optics, the receiver, a spectrometer, a computer and power sup- plies. Radiation from the sky enters the instrument through the scan drum (a), which is at the same time the air outlet. The air ﬁlters (b) are placed below the instrument and the GNSS antenna and a rain sensor (c) are attached on top . J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 5009 M3 M2 M1 M4 Hot load Frontend Y X Z Figure 2. WIRA-C optics with ﬂat mirror M3 (inside the scan- drum), ﬂat mirror M2 (on a linear stage), elliptical mirror M1, ellip- tical mirror M4 (slewable, drawn in inactive state), hot load and the front end with the feed horn. M3 M2 M1 M4 Hot load Frontend Y X Z [t] Figure 2. WIRA-C optics with ﬂat mirror M3 (inside the scan- drum), ﬂat mirror M2 (on a linear stage), elliptical mirror M1, ellip- tical mirror M4 (slewable, drawn in inactive state), hot load and the front end with the feed horn. Figure 1. 3 The instrument The WIRA-C instrument as installed on the Maïdo ob- servatory on Réunion. It measures 0.6 × 0.75 × 0.5 m and contains the optics, the receiver, a spectrometer, a computer and power sup- plies. Radiation from the sky enters the instrument through the scan drum (a), which is at the same time the air outlet. The air ﬁlters (b) are placed below the instrument and the GNSS antenna and a rain sensor (c) are attached on top . ple the path length modulator to mitigate standing waves be- tween calibration target and receiver. The key speciﬁcations of WIRA-C are summarised in Table 1. www.atmos-meas-tech.net/11/5007/2018/ 3.2 Receiver electronics 60 50 40 30 20 10 0 Amplitude [dB, ref max] YZ cut Measurement Simulation 6 4 2 0 2 4 6 Angle [deg] 60 50 40 30 20 10 0 Amplitude [dB, ref max] XZ cut Measurement Simulation (a) (b) The receiver front end (Fig. 4) of WIRA-C contains a temperature-stabilised heterodyne single side-band receiver. The observed radio frequency (RF) of 142 GHz is collected by the feed horn and then ampliﬁed by the low noise am- pliﬁer (LNA) by 20 dB (3.29 dB noise ﬁgure at 142 GHz and 293 K). This LNA has been built by the Fraunhofer IAF based on the 50 nm M-HEMT technology described by Leuther et al. (2012). After subsequent selection of a single side band, the sub-harmonic mixer is fed by a local oscillator (LO) with 72.9 GHz, which gives an intermediate frequency (IF) of 3.65 GHz. The microwave components of the front end are all mounted on a rigid aluminium plate that is tem- perature stabilised by thermo-electrical elements to maintain a stable temperature at 295 K. YZ cut Measurement Simulation We use a Universal Software Radio Peripheral (USRP X310 with CBX-120 daughterboard, see Ettus Research, 2018) as Fast Fourier Transform Spectrometer (FFTS). It has a bandwidth of 200 MHz and a channel width of 12.2 kHz but due to some constraints by ﬁlters in the USRP, only the cen- tral 120 MHz of the full bandwidth can be used for our mea- surements. As shown in Fig. 4, the USRP provides two chan- nels with independent local oscillators and AD converters. In the current setup, the primary channel (channel A) is centred around the resonance frequency of the ozone thermal emis- sion line at 142 GHz while the secondary channel is offset by 120 MHz to extend the spectrum towards the off-resonance frequencies. The Fast Fourier Transformation (FFT) and ac- cumulation algorithms are implemented using LabVIEW and programmed on the FPGA chip aboard the USRP. Figure 3. Measured and simulated far-ﬁeld beam cuts of the whole instrument when pointing to zenith direction. Panel (a) shows the cut along the Y-Z-plane which is also the plane of reﬂection on the last mirror (see Fig. 2 for the coordinate system). Panel (b) shows the perpendicular cut. The gray dashed line marks the −35 dB level. in Murk et al. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns Table 1. Key speciﬁcations of the WIRA-C microwave Wind Ra- diometer for Campaigns. Optics Ultra-Gaussian feed horn + elliptical and ﬂat mirrors Beam width 2.3◦FWHM Receiver type Pre-ampliﬁed single-side band heterodyne Frequency 142.17504 GHz Bandwidth 2 × 120 MHz Backend Ettus Research USRP, FFTS Spectral resolution 12.2 KHz System Temperature 550 K Calibration Hot load + tipping curve Elevation range All sky 60 50 40 30 20 10 0 Amplitude [dB, ref max] YZ cut Measurement Simulation 6 4 2 0 2 4 6 Angle [deg] 60 50 40 30 20 10 0 Amplitude [dB, ref max] XZ cut Measurement Simulation (a) (b) Figure 3. Measured and simulated far-ﬁeld beam cuts of the whole instrument when pointing to zenith direction. Panel (a) shows the cut along the Y-Z-plane which is also the plane of reﬂection on the last mirror (see Fig. 2 for the coordinate system). Panel (b) shows the perpendicular cut. The gray dashed line marks the −35 dB level. Table 1. Key speciﬁcations of the WIRA-C microwave Wind Ra- diometer for Campaigns. We measured the beam pattern of the instrument using a vector network analyzer (VNA) in the near-ﬁeld. The exper- imental setup for this measurement includes an open-ended waveguide probe placed in front of the instrument on a linear scanning stage that allows scanning along the x and y axis (see Fig. 2 for the coordinate system). The VNA source sig- nal at 142 GHz is coupled into the optics by the WIRA-C feed horn. Figure 3 shows the far-ﬁeld transformation of the scanning along the two axes as well as the corresponding physics simulations carried out with GRASP (TICRA, 2015). The measurements and simulations agree on a full width at half maximum of the beam of 2.1◦and conﬁrm the side lobes to be below −35 dB. Optics Ultra-Gaussian feed horn + elliptical and ﬂat mirrors Beam width 2.3◦FWHM Receiver type Pre-ampliﬁed single-side band heterodyne Frequency 142.17504 GHz Bandwidth 2 × 120 MHz Backend Ettus Research USRP, FFTS Spectral resolution 12.2 KHz System Temperature 550 K Calibration Hot load + tipping curve Elevation range All sky 3.1 Receiver optics The mirror M2 is mounted on a linear stage that can be shifted back and forth to make a λ/4 difference in optical path length between two measurements. This path length modulation is especially useful for calibration with the internal hot load as it mitigates standing waves between the receiver and the calibration target by destructive interference. The calibration target is an aluminium wedge with a half angle of 12◦, coated with absorbing material Eccosorb MMI- U. This absorber type from Laird NV is particularly well suited for those frequencies as shown by Fernandez et al. (2015b). Mirror M4 can be moved into the optical path to perform a hot load measurement and because of its ellipti- cal shape focuses the beam to ﬁt the load aperture, which results in a very compact calibration wedge. The calibration wedge is placed with its plane of incidence perpendicular to the electric ﬁeld, which is generally referred to as transversal- magnetic (TM) mode. As measured with the setup described Atmos. Meas. Tech., 11, 5007–5024, 2018 5010 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 1 8 20 dB RF 142 GHz IF 3.65 GHz LO 72.91252 GHz I Q A D A D FFT Σ LO 3.66 GHz USRP backend Ch A Radiometer frontend I Q A D A D FFT Σ LO 3.78 GHz USRP backend Ch B GPS OCXO 10 MHz REF (a) (b) (c) Figure 4. Block diagram of the WIRA-C single side-band receiver with radiometer front end (a) and USRP spectrometer (b) with channels A and B. The oven-controlled and GPS-disciplined crystal oscillator (OCXO) (c) provides the 10 MHz reference frequency for all local oscillators (LO) in the front and back end. 1 8 20 dB RF 142 GHz IF 3.65 GHz LO 72.91252 GHz Radiometer frontend (a) IF 3.65 GHz (b) Figure 4. Block diagram of the WIRA-C single side-band receiver with radiometer front end (a) and USRP spectrometer (b) with channels A and B. The oven-controlled and GPS-disciplined crystal oscillator (OCXO) (c) provides the 10 MHz reference frequency for all local oscillators (LO) in the front and back end. 101 102 103 Integration time [s] 10 4 10 3 10 2 Noise allan 1 B * t Figure 5. Allan variance of the receiver measured for a bandwidth of 14.6 kHz compared to the radiometric noise formula. The mini- mal Allan variance is reached after 4 min of integration. 4 Data processing 101 102 103 Integration time [s] 10 4 10 3 10 2 Noise allan 1 B * t The primary measurement cycle of WIRA-C alternates be- tween the six targets, which are the hot load, zenith (used as cold load), and the four 22◦elevation observations (north, south, east, west). For all six targets the linear stage is placed in two different positions to make a difference in path length of λ/4. The integration time for each position of the linear stage is 10 s and the two measurements are averaged prior to calibration to cancel standing waves. Notably, we use the time during the relatively slow rotation around the azimuthal axis for the zenith and hot-load measurements to save valu- able integration time. The twelve measurements of one cycle are then processed further, as described in the following sec- tions. Figure 5. Allan variance of the receiver measured for a bandwidth of 14.6 kHz compared to the radiometric noise formula. The mini- mal Allan variance is reached after 4 min of integration. 4.1 Calibration Compactness and low maintenance requirements were ma- jor design goals of WIRA-C, ruling out liquid nitrogen or a Peltier calibration target (Fernandez et al., 2015b) as cold reference that is needed in addition to the hot reference for radiometric calibration. This is why we opted for an am- bient temperature hot load complemented with the tipping curve method for the radiometric calibration. Essentially, this method has been explained by Han and Westwater (2000) and uses the sky as cold load by assuming a mean tro- pospheric temperature and ﬁtting the tropospheric opacity to a set of observations at different elevation angles. We use the measurements at 22◦elevation and zenith, and es- timate the mean tropospheric temperature Tm according to The receiver gain typically drifts with time and periodi- cal calibration is important to get consistent measurements. The Allan variance computation scheme (Ossenkopf, 2007) gives a timespan for which a receiver can be considered sta- ble. Figure 5 shows the Allan variance for a 14 h measure- ment with the WIRA-C receiver. The noise of the WIRA-C receiver drops for an integration time up to 4 min for a single channel with a bandwidth of 14.6 kHz, then starts to increase again because of drifts. The duration of one measurement cy- cle was thus chosen to be 2 min. Atmos. Meas. Tech., 11, 5007–5024, 2018 3.2 Receiver electronics (2006), the calibration wedge performs well with a reﬂectivity lower than −60 dB at 142 GHz. A narrow beam with low side lobes is required for a well deﬁned pointing. This is important for ground based radio- metric measurements of the middle atmosphere, as the path length through the troposphere, and thus the tropospheric signal, increases rapidly with decreasing elevation angle, especially at low elevation angles used for wind measure- ments. The antenna of WIRA-C is an ultra low side lobe Gaussian corrugated feed horn with a divergence angle of 2feed = 14.3◦. The elliptical mirror M1 transforms this beam to the near-pencil instrument beam that has a full width at half maximum divergence angle of 2instr = 2.1◦. The system noise temperature of the single-sideband re- ceiver system is 510 K at 142 GHz, as measured in the lab- oratory by a hot–cold calibration using liquid nitrogen and conﬁrmed by the routine tipping curve calibration. This is about 300 K lower than for the WIRA instrument, mainly due to the better quality of the 20 dB low noise ampliﬁer. As wind measurements require a stable frequency refer- ence, we use a GPS disciplined and oven-controlled quartz oscillator to improve the long and short-term stability of the local oscillators of the front end and the back end. www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 5011 4.2 Tropospheric correction The calibrated brightness temperature as seen on the ground, Tb(z0), can be modelled as a sum of the tropospheric contri- bution driven by the same mean temperature Tm used above and a middle-atmospheric contribution Tb(ztrop) that would be observed if the instrument was above the troposphere (In- gold et al., 1998): This gives us an estimate on τ for each observation direc- tion and we can estimate the non-tropospheric contribution through Tb ztrop = Tb(z0) −Tm (1 −exp(−τ/sinη)) exp(−τ/sinη) . (4) (4) Tb(z0) = Tm (1 −exp(−τ/sinη)) + Tb ztrop exp(−τ/sinη), (2) (2) As Eq. (4) is not linear in τ, it does not hold exactly for average values of τ and Tb for long integration times or highly variable tropospheric conditions. We encounter such conditions, for example, on the Maïdo observatory on Réu- nion (21.4◦S, 55.9◦E). There, during nighttime, the condi- tions are optimal for radiometric observations because the observatory is located at 2200 m above sea level and near the free troposphere during the night (Baray et al., 2013). However, during daytime, when microclimatic effects and convection are dominant, the opacity is highly variable as shown in Fig. 6. At the same time the signal-to-noise ratio for wind measurements is quite low and long integration times of several hours are required. The high variability of the opac- ity and the long integration times are the reasons why we apply the tropospheric correction directly to the calibrated spectra before integration and use the 12 h integration of the corrected brightness temperatures Tb(ztrop) for the wind re- trievals. This integration time showed to be suited for the objective of instrument validation, but for other studies one might also consider shorter or longer integration times. where τ is the zenith opacity of the troposphere and η is the elevation angle of the observation. The opacity itself can be estimated in different ways. We are applying the same tech- nique as Fernandez et al. (2015a) and use the brightness tem- perature at the wings of the measured spectra, as far away from the ozone rotational transition resonance frequency as possible. In practice we use an average over 10 MHz at the left wing of the spectrum measured by the second spectrome- ter channel (USRP channel B) depicted in Fig. 4. The Zenith opacity is then given by τ = −sin(η) ln Tm −T off-resonance b Tm −T bg ! www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 5012 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns Ingold et al. (1998) from the ambient temperature Tamb as T 22 m = Tamb −9.8 K and T 90 m = Tamb −10 K, respectively. Ingold et al. (1998) from the ambient temperature Tamb as T 22 m = Tamb −9.8 K and T 90 m = Tamb −10 K, respectively. 0 10 20 30 40 50 60 70 Time, hours since 2017-06-25 UTC 0.0 0.2 0.4 0.6 Opacity 0 10 20 30 40 50 60 70 Time, hours since 2017-09-09 UTC 0.0 0.2 0.4 0.6 Opacity (a) (b) Figure 6. Opacity τ at the off-resonance observation frequency obtained from tipping calibration for three days in June (a) and September (b) at the Maïdo observatory. The gray areas mark night- time, with sunrise and sunset at 02:00 and 14:00 UTC, respectively. 0 10 20 30 40 50 60 70 Time, hours since 2017-06-25 UTC 0.0 0.2 0.4 0.6 Opacity 0 10 20 30 40 50 60 70 Time, hours since 2017-09-09 UTC 0.0 0.2 0.4 0.6 Opacity (a) (b) The temperature of the hot load is measured by two tem- perature sensors mounted on its aluminium backing and fol- lows the internal temperature of the instrument which we sta- bilise at about 10 K above the typical maximum ambient tem- perature by regulating air ﬂow and additional heaters. In order to include as little wind information in the tipping calibration process as possible, we average the northwards and southwards measurement to provide the input for the 22◦ elevation measurement to the tipping curve algorithm. We prefer that in favour of the eastwards and westwards mea- surements, as zonal winds are expected to be stronger and thus the slight broadening of the spectral line when averag- ing the two measurements would be increased. Figure 6. Opacity τ at the off-resonance observation frequency obtained from tipping calibration for three days in June (a) and September (b) at the Maïdo observatory. The gray areas mark night- time, with sunrise and sunset at 02:00 and 14:00 UTC, respectively. www.atmos-meas-tech.net/11/5007/2018/ 4.2 Tropospheric correction , (3) (3) where we set the background temperature T bg to 2.7 K. We apply this estimation for all four cardinal directions indepen- dently and thus account for direction-dependent tropospheric conditions. As described in Sect. 3.2, the second channel of the USRP is offset by 120 MHz, giving us information up to 180 MHz off-resonance. At this offset from the line centre the ozone signal is still relatively strong and we see more than just the microwave background T bg. However, for wind measure- ments we are more interested in a normalisation of the spec- tra of the four cardinal directions against each other to com- pensate for the tropospheric inhomogeneities than in absolute brightness temperature calibration. Figure 7 shows an example of a measured spectrum from one calibration cycle before and after tropospheric correc- tion. Without tropospheric correction, the measurements in eastward and westward direction differ by 20 K because of tropospheric inhomogeneities. If we apply the tropospheric correction as described above using the left wing as refer- ence, the spectra are on the same level and have the same magnitude. While we use the measurement form channel A Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaign 140 160 180 Tb [K] Channel A Channel B East calibrated West calibrated 142.00 142.05 142.10 142.15 142.20 Frequency [GHz] 20 0 20 40 60 Tb [K] Channel A Channel B East corrected West corrected (a) (b) Figure 7. Measured spectrum of the ozone line from a single cal- ibration cycle on 25 June 2017 at 09 h (UTC). Panel (a) shows the eastward and westward measurement after calibration, panel (b) shows the same measurement but with tropospheric correction ap- plied. Channel A of the USRP has 12.2 kHz resolution and is cen- tred around the line centre while channel B has 97.7 kHz resolution and observes the line wing. 5013 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 140 160 180 Tb [K] Channel A Channel B East calibrated West calibrated 142.00 142.05 142.10 142.15 142.20 Frequency [GHz] 20 0 20 40 60 Tb [K] Channel A Channel B East corrected West corrected (a) (b) Fitting one atmosphere to two measurements drastically increases the overdetermination of the retrieval as the number of measurements is increased. 4.3 Retrieval of wind proﬁles x = u xO3, 1 ... xO3, M 1f b⊺, (5) y = T b, east T b, west ⊺, (6) (5) (5) (6) We retrieve wind information from the measured spectra by inverting a radiative transfer model that describes the rela- tion between the atmospheric state vector x and the mea- surement vector y as x = F(y). The inversion thereof is typ- ically ill-posed because many (unphysical) conﬁgurations of the atmosphere lead to the same measured brightness tem- perature. The optimal estimation method uses an a priori value with associated uncertainties for the atmospheric con- ﬁguration to regularise the inverse problem as described by Rodgers (2000). (6) where the elements of x are itself vectors. For ex- ample the zonal wind speed proﬁle is given by u = u(p1) u(p2) ... u(pN) for N pressure levels. Besides the zonal wind proﬁle u, the x vector also contains the pro- ﬁles of volume mixing ratio of ozone xO3 at M different spa- cial grid points as well as the frequency shift parameter 1f and one or more baseline parameters b. Finally, the temper- atures T b, east and T b, west are the calibrated and corrected brightness temperatures from Eq. (4). The WIRA-C retrieval of zonal wind uses the brightness temperature measured in eastern and western direction and combines these measurements to retrieve a single wind pro- ﬁle. The retrieval of the meridional wind is set up analo- gously. This is in contrast to the wind retrieval procedures used for WIRA, where wind proﬁles have been estimated for east and west separately and are then averaged to get a single zonal wind proﬁle (Rüfenacht et al., 2014). By combining both observations in one inversion, we can effectively max- imise the a posteriori likelihood of the wind proﬁle given our two measurements in opposite directions. This is especially important in the presence of frequency shifts or drifts that are not related to wind. Such shifts are of a systematic or ran- dom nature and can originate from instrumental instabilities or offsets or even uncertainties in the molecular resonance frequency. The optimal estimation method then minimizes the cost function χ2 = ˆx −xa ⊺S−1 a ˆx −xa + y −F ˆx ⊺S−1 ϵ y −F ˆx , (7) (7) for ﬁnding the most probable atmospheric state ˆx given the a priori proﬁle xa and the measurement y. 4.2 Tropospheric correction This is explicitly wanted for wind and frequency shift where we need to combine all our measurements, but not ideal for ozone abundance that is also being retrieved to ﬁt the observed line. Fitting one common ozone proﬁle to the eastward and westward direction con- strains the retrieval too much resulting in non-convergence or oscillations of the ozone proﬁles. This might be due to actual spatial variations in ozone abundances, which we consider to be unlikely as they are not expected to be that big in tropical latitudes. More probably, tropospheric inhomogeneities or clouds affecting the eastward and westward observations dif- ferently could have an inﬂuence on the ozone proﬁle. How- ever, this is not expected to have an inﬂuence on the re- trieved wind speed, as the Jacobian of the forward model is completely antisymmetric with regard to wind as elaborated in Rüfenacht et al. (2014). This is why we model a three- dimensional atmosphere and include independent ozone pro- ﬁles, and thus more freedom in our retrieval for the opposing observations. Figure 7. Measured spectrum of the ozone line from a single cal- ibration cycle on 25 June 2017 at 09 h (UTC). Panel (a) shows the eastward and westward measurement after calibration, panel (b) shows the same measurement but with tropospheric correction ap- plied. Channel A of the USRP has 12.2 kHz resolution and is cen- tred around the line centre while channel B has 97.7 kHz resolution and observes the line wing. In case of WIRA-C, the state vector x and the measure- ment vector y have the following form for the zonal wind retrieval (and analogous for the meridional wind retrieval): for the retrieval of wind speeds, channel B is used solely for the tropospheric correction. www.atmos-meas-tech.net/11/5007/2018/ 4.4 A priori data and model parameters For the a priori data for wind, we always use a 0 m s−1 pro- ﬁle. This equalises the probability to retrieve easterly and westerly winds, which is desirable in case of sudden wind reversals like they are observed around equinox and in con- text of sudden atmospheric events. To put it in other words, even though wind speeds in the atmosphere are generally not normally distributed we assume that the wind in the atmo- sphere is (0 ± su) m s−1 and we use climatological statistics from 6 years of ECMWF data at the campaign location to estimate su which then depends on altitude but not on time. The same applies for meridional wind, and sv turns out to be smaller than su because meridional winds are typically slower than zonal winds. We multiply these statistics by a factor of 2 in order not to have a bias towards zero, as elab- orated by Rüfenacht et al. (2014) and additionally we im- pose a vertical correlation length of 0.5 pressure decades to construct the covariance matrix. Like this, our retrieved wind speeds are regularised but in no case biased towards either direction by the a priori wind proﬁle. ˆx = xa + G(y −Kxa), (10) (10) where G is the gain-matrix and describes the sensitivity of the retrieved proﬁle to changes in the spectra: G = ∂ˆx ∂y = KT S−1 ϵ K + S−1 a −1 KT S−1 ϵ . (11) (11) As the frequency shift introduced by wind has a non-linear impact on the brightness temperature, the ﬁnal solution ˆx is found by a Levenberg–Marquardt algorithm and Eq. (10) is applied iteratively while updating the point of linearisation for K but leaving xa ﬁxed. Assuming that Sϵ characterises the radiometric noise on the spectra, the uncertainty of the retrieved proﬁles due to thermal noise, the so called observational error σo, is deﬁned as as σ 2 o = diag GSϵGT . (12) (12) For the ozone a priori data, we rely on a F 2000 WACCM scenario from a simulation by Schanz et al. (2014). This al- lows us to extend the retrieval grid up to 110 km altitude and thus includes the nighttime secondary ozone maximum at 10−3 Pa. 4.3 Retrieval of wind proﬁles It does so using the assigned statistics in form of the covariance matrices Sa and Sϵ for the a priori data and the measurement, respectively. Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ 5014 J. 5014 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns The value σy on the diagonal of Sϵ is directly determined as the Allan-deviation of the measurement vector y by σ 2 y = 1 2⟨ yn+1 −yn 2⟩. F ll i R d y 1 2⟨ yn+1 −yn 2⟩. 1 2⟨ yn+1 −yn 2⟩. F ll i R d (2000) d i li i d f f Following Rodgers (2000) and using a linearised form of the forward model with Jacobian K, the solution that mini- mizes Eq. (7) in a linear case is www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns They are constructed as block diagonal matrices, analo- gous to the x and y vectors in Eqs. (5) and (6): Another measure for quality of our retrieved state ˆx is the averaging kernel matrix given by A = ∂ˆx ∂x = GK. (13) Sa =   Sa, u S1, 1 a, XO3 ... S1, M a,XO3 ... ... ... SM, 1 a, XO3 ... SM, M a,XO3 Sa, 1f Sa, b   , (8) Sϵ = STb, east STb, west = σy I, (9) A = ∂ˆx ∂x = GK. (13) Each row of the matrix A is called an averaging kernel and describes the smoothing of information. We use the averag- ing kernels for quality control as described in Sect. 4.5. (8) g q y The forward model and OEM implementation is provided by ARTS/QPACK2 (version 2.3) (Eriksson et al., 2011). In the current setup for WIRA-C wind retrievals we use 6 ozone proﬁles equally spaced around the instrument location inside the east–west observation plane for zonal wind. We choose M = 6 as this showed to give superior retrieval results in terms of measurement response and altitude resolution than lower values. This is a detail related to the grid interpolations done by ARTS/QPACK2 and the construction of the covari- ance matrix for ozone. The covariance matrices for ozone are set up using separable statistics with a horizontal correlation length of 200 km, which we assume to be height independent. (9) where the off-diagonal elements Si,j a, XO3 (i ̸= j) describe where the off-diagonal elements Si,j a, XO3 (i ̸= j) describe the covariance of the spatially distributed ozone proﬁles. Details about the setup of covariance matrices for multi- dimensional retrievals are described by Christensen (2015). The value σy on the diagonal of Sϵ is directly determined as the Allan-deviation of the measurement vector y by σ 2 y = g a, XO3 ( ̸ j) the covariance of the spatially distributed ozone proﬁles. Details about the setup of covariance matrices for multi- dimensional retrievals are described by Christensen (2015). The value σy on the diagonal of Sϵ is directly determined as the Allan-deviation of the measurement vector y by σ 2 y = 2 dimensional retrievals are described by Christensen (2015). 4.5 Quality control and uncertainty A big advantage of the optimal estimation method over other regularisation methods is the availability of error estimations and quality control information. As expressed by Eq. (13), the averaging kernel matrix (AVKM) describes the sensitivity of our estimated atmo- spheric state ˆx for the true state x. We derive three quantities from the averaging kernel matrix: ﬁrstly, the measurement response that is the sum of the rows of the AVKM and de- scribes the sensitivity of our retrieved state to the true state as can readily be seen in Eq. (13). Ideally it is exactly 1, meaning that a change in the true atmospheric state is exactly represented in the retrieved state. Secondly, the full width at half maximum of the averaging kernels gives information about the spatial smoothing of the data. Ideally these kernels would be delta peaks (which would make the AVKM diag- onal). Finally, we examine the difference between the peak of the averaging kernels to their respective nominal height. In the ideal case (diagonal AVKM), the offset would be zero, meaning that all information is mapped to the correct grid points. We use the information in the AVKM for quality con- trol of the wind retrieval: the measurement response must be between 0.8 and 1.2 and the offset of the peak to the nominal height of the kernel must not exceed 5 km. If these criteria are fulﬁlled for an extended altitude range, the retrieved values are valid. Further, the full width at half maximum (FWHM) of the individual kernels gives information about the altitude resolution. Figure 8. Visualisation of the averaging kernel matrix (AVKM) for the nighttime measurement of 26 June 2017. The individual aver- aging kernels (rows of the AVKM) for each altitude (a) are char- acterized by the measurement response (MR), their full width at half maximum (FWHM) and the difference of their maximum to the nominal height (Offset). The valid ranges for all parameters are marked by the green areas. Valid components that fulﬁll all criteria are shown in colours and others in gray (or dashed lines and hollow markers, respectively). depending on the ozone a priori proﬁle. Even though the measurement response stays within the bounds of validity in these altitudes, offset criteria reject these points reliably. The FWHM in Fig. 8 indicates an altitude resolution be- tween 9 and 11 km for the whole altitude range. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 5015 0.0 0.2 AVK 20 30 40 50 60 70 80 90 100 110 Approx. altitude [km] 0.0 0.5 1.0 MR 0 10 20 FWHM [km] 0 25 Offset [km] (a) (b) (c) (d) Figure 8. Visualisation of the averaging kernel matrix (AVKM) for the nighttime measurement of 26 June 2017. The individual aver- aging kernels (rows of the AVKM) for each altitude (a) are char- acterized by the measurement response (MR), their full width at half maximum (FWHM) and the difference of their maximum to the nominal height (Offset). The valid ranges for all parameters are marked by the green areas. Valid components that fulﬁll all criteria are shown in colours and others in gray (or dashed lines and hollow markers, respectively). We multiply the variance of ozone by a factor of 4 for the same reasons as above and impose a vertical correlation length of 0.3 pressure decades to get the covariance matrix. As explained in Sect. 4.3, the horizontal covariance of ozone is assumed to be height independent with a horizontal corre- lation length of 200 km. The ozone a priori proﬁle and covari- ance matrix thus depend on altitude and time (day or night and time of year). 0.0 0.2 AVK 20 30 40 50 60 70 80 90 100 110 Approx. altitude [km] 0.0 0.5 1.0 MR 0 10 20 FWHM [km] 0 25 Offset [km] (a) (b) (c) (d) The forward model also needs additional information about the atmosphere, namely it includes the tempera- ture proﬁle (from MLS and ECMWF complemented with WACCM) and volume mixing ratio proﬁles for the less crit- ical species N2 and O2 (from standard atmospheres) that are known well enough and thus will not need to be optimised. 4.4 A priori data and model parameters We determine the a priori proﬁle and variance in a window of 11 days around the day-of-year of our measure- ment while only regarding the same hours of the day that we integrated over (either day or nighttime). Extending the re- trieval grid and separating day and nighttime retrievals is im- portant because signals from the secondary ozone maximum can have an inﬂuence on wind retrievals below 75 km if not properly handled as elaborated by Rüfenacht and Kämpfer (2017). We assume that the major contribution to the uncertainty on the retrieved proﬁles is due to radiometric noise and thus use the observational error σo as a measure for the uncer- tainty in this study. It is important to note, that the observa- tional error is inﬂuenced by the a priori statistics via Eq. (11) and the observational error grows with increasing a priori co- variance because then the measurement and its noise have a bigger impact on the retrieved quantity. We accept this as an inherent property of the optimal estimation method: for a given thermal noise on the spectrum, the uncertainty of the retrieved value is smaller if there is less ambiguity in the a priori state. www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 Atmos. Meas. Tech., 11, 5007–5024, 2018 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns www.atmos-meas-tech.net/11/5007/2018/ 4.5 Quality control and uncertainty While the measurement re- sponse is even between 0.9 and 1.1 (as opposed to the qual- ity requirement of 0.8 to 1.2) for nearly the entire altitude domain for zonal wind indicating that our retrieval is highly sensitive to changes in the atmospheric wind speed and l l i d d f h i i ﬁl Th 0 10 20 30 Observation error [m s ] o 35 40 45 50 55 60 65 70 75 Approx. altitude [km] 0 5 10 15 FWHM of AVKs [km] 2017-06-26 day 2017-06-26 night 2017-09-10 day 2017-09-10 night 0.8 1.0 1.2 Measurement response 0 10 20 30 Observation error [m s ] o 35 40 45 50 55 60 65 70 75 Approx. altitude [km] 0 5 10 15 FWHM of AVKs [km] 2017-06-26 day 2017-06-26 night 2017-09-10 day 2017-09-10 night 0.8 1.0 1.2 Measurement response -1 -1 Figure 10. Characterisation of the retrieval quality for zonal and meridional wind for the day and nighttime period of 2 days. The observation error represents the measurement uncertainty. The full width at half maximum (FWHM) of the averaging kernels describes the altitude resolution, which is approximately 10 km, up to 68 km altitude. The measurement response is a measure for the sensitivity of our retrieved wind speeds to changes in actual wind speeds. In the perfect case it would be 1.0 but values between 0.8 and 1.2 are acceptable. 20 40 TB [K] Eastwards observation Westwards observation 75 50 25 0 25 50 f - 142.17504 GHz [MHz] 0.5 0.0 0.5 Residuals [K] 75 50 25 0 25 50 f - 142.17504 GHz [MHz] (a) (b) 20 40 TB [K] Eastwards observation Westwards observation 75 50 25 0 25 50 f - 142.17504 GHz [MHz] 0.5 0.0 0.5 Residuals [K] 75 50 25 0 25 50 f - 142.17504 GHz [MHz] (a) (b) Figure 9. Corrected and integrated (12 h) brightness temperature spectra as used for the retrieval of 26 June 2017 nighttime for eastwards and westwards direction (a) together with the residuals (observed minus computed, b). Smoothed residuals (by binning 50 channels) are shown in black. 0 10 20 30 Observation error [m s ] o 35 40 45 50 55 60 65 70 75 Approx. altitude [km] 0 5 10 15 FWHM of AVKs [km] 2017-06-26 day 2017-06-26 night 2017-09-10 day 2017-09-10 night 0.8 1.0 1.2 Measurement response -1 Figure 9. 4.5 Quality control and uncertainty Corrected and integrated (12 h) brightness temperature spectra as used for the retrieval of 26 June 2017 nighttime for eastwards and westwards direction (a) together with the residuals (observed minus computed, b). Smoothed residuals (by binning 50 channels) are shown in black. 0 10 20 30 Observation error [m s ] o 35 40 45 50 55 60 65 70 75 Approx. altitude [km] 0 5 10 15 FWHM of AVKs [km] 2017-06-26 day 2017-06-26 night 2017-09-10 day 2017-09-10 night 0.8 1.0 1.2 Measurement response -1 ratio of the spectra, the bigger uncertainty for the daytime measurements can be explained by the higher opacity during daytime as is shown in Fig. 6. Also the ozone concentration is lower during daytime as studied for example by Studer et al. (2014), resulting in less emitters and lower signal-to-noise ratio during daytime compared to nighttime. The observa- tion error is smaller for the meridional wind than it is for the zonal wind. As elaborated in Sect. 4.3, this is because the ob- servation error is not independent of the a priori statistics and the covariance for meridional wind is smaller than for zonal wind. The full width at half maximum, that is also shown in Fig. 10, describes the altitude resolution. For zonal wind, the altitude resolution is approximately 10 km up to 68 km. For meridional wind, the resolution is between 10 and 15 km, which is a direct consequence of the more restrictive a pri- ori proﬁle for meridional wind. While the measurement re- sponse is even between 0.9 and 1.1 (as opposed to the qual- ity requirement of 0.8 to 1.2) for nearly the entire altitude domain for zonal wind indicating that our retrieval is highly sensitive to changes in the atmospheric wind speed and largely independent of the a priori proﬁle. The measurement response for the meridional wind is somewhat more variable, which is related to the constriction by the a priori proﬁle, be- cause a smaller a priori covariance also implies less weight on the measurement and thus lower sensitivity. Nevertheless, the quality requirement is fulﬁlled between 38 and 65 km. Figure 10. Characterisation of the retrieval quality for zonal and meridional wind for the day and nighttime period of 2 days. The observation error represents the measurement uncertainty. 4.5 Quality control and uncertainty This is an improvement in comparison to the WIRA retrieval, where the altitude resolution for zonal wind is about 10 to 16 km (Rüfenacht et al., 2014). We attribute this improvement to the lower noise of the instrument and the simultaneous inversion of the two measurements, which gives more independent in- formation than the inversion of one spectrum after the other. Further, Fig. 9 shows the residuals for the same retrieval shown in Fig. 8. The residuals look random, indicating that we properly model our observations. Figure 8 shows the averaging kernels and the derived qual- ity control parameters for one measurement. The retrieved values are considered to be valid between 38 and 75 km al- titude. The measurement response would be acceptable on higher altitudes but the upper points are rejected by the off- set parameter. We see the offset parameter jumping from −7 to 10 km at approximately 80 km altitude. This is because Doppler broadening starts to dominate the pressure broaden- ing above approximately 75 km altitude and signals can not be attributed to the exact height they originate from. This means that they are attributed to lower or higher altitudes Figure 10 shows the observation error σo for four different measurements together with the FWHM and the measure- ment response. We see that the observation error for zonal wind retrievals is approximately 15 m s−1, up to 64 km al- titude for the nighttime measurement with the chosen inte- gration time of 12 h. Below 55 km, the errors of the day and nighttime measurement are nearly identical, but above 60 km the error for the day time measurements increase rapidly. As tropospheric opacity has a big impact on the signal-to-noise www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 0 10 20 30 Observation error [m s ] o 35 40 45 50 55 60 65 70 75 Approx. altitude [km] 0 5 10 15 FWHM of AVKs [km] 2017-06-26 day 2017-06-26 night 2017-09-10 day 2017-09-10 night 0.8 1.0 1.2 Measurement response 0 10 20 30 Observation error [m s ] o 35 40 45 50 55 60 65 70 75 Approx. altitude [km] 0 5 10 15 FWHM of AVKs [km] 2017-06-26 day 2017-06-26 night 2017-09-10 day 2017-09-10 night 0.8 1.0 1.2 Measurement response -1 -1 Figure 10. 4.5 Quality control and uncertainty The full width at half maximum (FWHM) of the averaging kernels describes the altitude resolution, which is approximately 10 km, up to 68 km altitude. The measurement response is a measure for the sensitivity of our retrieved wind speeds to changes in actual wind speeds. In the perfect case it would be 1.0 but values between 0.8 and 1.2 are acceptable. with their expected distribution. The Monte Carlo estimation involves sampling from these distributions and retrieving a wind proﬁle for every sample. The estimated systematic error is then derived from the standard deviation of the retrieved wind speeds. 4.5 Quality control and uncertainty Characterisation of the retrieval quality for zonal and meridional wind for the day and nighttime period of 2 days. The observation error represents the measurement uncertainty. The full width at half maximum (FWHM) of the averaging kernels describes the altitude resolution, which is approximately 10 km, up to 68 km altitude. The measurement response is a measure for the sensitivity of our retrieved wind speeds to changes in actual wind speeds. In the perfect case it would be 1.0 but values between 0.8 and 1.2 are acceptable. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 5016 5016 J. Hagen et al.: WIRA C: Wind Radiometer for Campaigns 20 40 TB [K] Eastwards observation Westwards observation 75 50 25 0 25 50 f - 142.17504 GHz [MHz] 0.5 0.0 0.5 Residuals [K] 75 50 25 0 25 50 f - 142.17504 GHz [MHz] (a) (b) Figure 9. Corrected and integrated (12 h) brightness temperature spectra as used for the retrieval of 26 June 2017 nighttime for eastwards and westwards direction (a) together with the residuals (observed minus computed, b). Smoothed residuals (by binning 50 channels) are shown in black. ratio of the spectra, the bigger uncertainty for the daytime measurements can be explained by the higher opacity during daytime as is shown in Fig. 6. Also the ozone concentration is lower during daytime as studied for example by Studer et al. (2014), resulting in less emitters and lower signal-to-noise ratio during daytime compared to nighttime. The observa- tion error is smaller for the meridional wind than it is for the zonal wind. As elaborated in Sect. 4.3, this is because the ob- servation error is not independent of the a priori statistics and the covariance for meridional wind is smaller than for zonal wind. The full width at half maximum, that is also shown in Fig. 10, describes the altitude resolution. For zonal wind, the altitude resolution is approximately 10 km up to 68 km. For meridional wind, the resolution is between 10 and 15 km, which is a direct consequence of the more restrictive a pri- ori proﬁle for meridional wind. www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns Table 2. Considered uncertainties for the Monte Carlo error analysis together with the estimated error. The resulting error is given as the maximum error in three altitude domains: lower, from 5 to 1 hPa (36 to 48 km), middle, from 1 to 0.2 hPa (48 to 59 km) and upper, from 0.2 to 0.02 hPa (59 to 75 km). Estimated 1σ error, m s−1 Subject Distribution Type Parameters Lower Middle Upper Temperature proﬁle Gaussian absolute 2σ = 10 K 0.86 0.94 0.57 Ozone a priori proﬁle Gaussian absolute 2σ = 0.4 ppm 0.91 1.2 3.2 Ozone covariance Gaussian relative 2σ = 50 % 2.4 4.6 10 Wind covariance Gaussian relative 2σ = 50 % 2.5 3.0 4.3 Elevation uniform absolute ±0.2◦ 3.4 1.5 2.0 Calibration uniform relative [1, 1.3] 2.4 3.0 6.5 Total systematic 5.6 6.6 13 Retrieval noise 15 17 26 2σ = 50 % means σ = 1 4 µ for a Gaussian distribution with mean µ. valuable because they coincide with three lidar measure- ments. consider the systematic part that comes from the fact that our off-resonance frequencies used to determine the tropospheric opacity is still somewhat closer to the line centre than would be desirable (see Sect. 4.2). In our Monte Carlo estimation we simulate this error by introducing a factor in the range [1,1.3] to the y prior to the retrieval, which corresponds to an assumed uncertainty of 10 % of the tropospheric opacity. Further, we neglect all correlations between systematic errors and among systematic and random errors. For all retrievals presented in this section, we used an inte- gration time of 12 h, from 02:00 to 14:00 UT which is 06:00 and 18:00 local time (LT) and roughly corresponds the times of sunrise and sunset in the tropics. We set up the a priori proﬁles and covariances as described in Sect. 4.4 and most notably use an a priori of 0 m s−1 for all pressure levels for zonal and meridional wind. Quality control for the retrieved data is done as described in Sect. 4.5. We performed the Monte Carlo estimation for four differ- ent cases (same as shown in Fig. 10). The results for the setup of one retrieval (26 June 2017, nighttime) is shown in Table 2 for three different altitude domains. 5.2.1 ECMWF model data The ECMWF operational analysis provides atmospheric data on 137 layers up to 80 km altitude. However, the main fo- cus lies on delivering data on the atmospheric layers below 35 km for weather forecasts. Especially above 68 km the data quality is supposed to decrease because the uppermost layers do not assimilate measurements but are artiﬁcially forced to model stability. The ECMWF operational analysis has a higher time and altitude grid resolution than the WIRA-C retrieval. The time resolution is 6 h, whereas WIRA-C has a time resolution of 12 h. Thus, to check the two datasets for consistency we al- ways average the two ECMWF time steps which are within the respective integration period of WIRA-C. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns The biggest systematic error is evident in higher altitude domains and comes from the ozone a priori proﬁle. The inﬂuence of the ozone a pri- ori proﬁle has been thoroughly examined by Rüfenacht and Kämpfer (2017), concluding that a careful choice of ozone a priori and covariance data is important for the retrieval of wind speeds in higher altitudes. The total systematic error is approximately half the retrieval error in the worst case and by just looking at the retrieval noise, we thus underestimate the total error by approximately 10 %. 5.1 The Maïdo campaign From August 2016 until February 2018, the WIRA-C in- strument has been operated at the Maïdo observatory on Réunion(21.4◦S, 55.9◦E) at 2200 m above sea level. After having been operational for a few days in August 2016, a very uncommon failure of the synthesiser–multiplier chain occurred and the campaign could continue only in mid- November. Since then, WIRA-C measured continuously, ex- cept for a period of tropical cyclone alert and some power outages. The few measurements in August 2016 are very To adapt the vertical resolution of the model to our re- trieval, we convolve the model data with the averaging ker- nels of the retrieval. Integrated forecast system cycles Cy41r2, Cy43r1 and Cy43r3 have been used for this study. Atmos. Meas. Tech., 11, 5007–5024, 2018 4.6 Estimation of systematic errors All the proﬁles (temperature, a priori and covariances) are expected to follow a Gaussian distribution as they are derived from statistics as described in Sect. 4.4. We perturb the pro- ﬁles on all altitudes simultaneously using the value sampled from the respective distribution. The elevation is expected to have a systematic error of maximum ±0.2◦, as this is the esti- mated precision we reach when levelling the instrument. The calibration subject in Table 2 accounts for the uncertainty in the calibration and tropospheric correction. This uncer- tainty has a random and a systematic component. We only In the above section, we discussed the random errors caused by thermal noise on the spectrum as determined by the opti- mal estimation method. Additionally we perform a Monte Carlo error estimation to further characterise uncertainties not related to noise. These uncertainties are of systematic na- ture, as they are inherent to the retrieval setup and choice of a priori proﬁles and covariance matrices. Table 2 gives a list of the variables we considered in this analysis together Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ 5017 Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 5018 10 1 100 Pressure [hPa] (a) WIRA-C 2016-12-01 2017-01-01 2017-02-01 2017-03-01 2017-04-01 2017-05-01 2017-06-01 2017-07-01 2017-08-01 2017-09-01 2017-10-01 2017-11-01 2017-12-01 2018-01-01 10 1 100 Pressure [hPa] (b) ECMWF convolved 120 100 80 60 40 20 0 20 40 60 80 100 120 Zonal wind [m s ] 40 50 60 70 Approx. altitude [km] 40 50 60 70 Approx. altitude [km] -1 Figure 11. Time series of zonal wind speeds measured by WIRA- C (a) and ECMWF analysis data (b) between 14 November 2016 and 31 December 2018 for the altitude range of 35 to 75 km with a time resolution of 12 h (day and nighttime). The ECMWF data has been convolved with the averaging kernels of the retrieval in order to get the same spatial and temporal resolution for both datasets. In- valid data points are grayed out, resulting in different altitude ranges for different days. The few data gaps are due to a tropical cyclone and power outages. 10 1 100 Pressure [hPa] (a) WIRA-C 2016-12-01 2017-01-01 2017-02-01 2017-03-01 2017-04-01 2017-05-01 2017-06-01 2017-07-01 2017-08-01 2017-09-01 2017-10-01 2017-11-01 2017-12-01 2018-01-01 10 1 100 Pressure [hPa] (b) ECMWF convolved 120 100 80 60 40 20 0 20 40 60 80 100 120 Zonal wind [m s ] 40 50 60 70 Approx. altitude [km] 40 50 60 70 Approx. altitude [km] -1 100 0 100 35 km / 6.4938 hPa WIRA-C ECMWF conv ECMWF 100 0 100 39 km / 3.6517 hPa 100 0 100 43 km / 2.0535 hPa 100 0 100 47 km / 1.1548 hPa 100 0 100 51 km / 0.6494 hPa 100 0 100 55 km / 0.3652 hPa 100 0 100 59 km / 0.2054 hPa 100 0 100 63 km / 0.1155 hPa 100 0 100 67 km / 0.0649 hPa 2016-12-01 2017-01-01 2017-02-01 2017-03-01 2017-04-01 2017-05-01 2017-06-01 2017-07-01 2017-08-01 2017-09-01 2017-10-01 2017-11-01 2017-12-01 2018-01-01 Time UTC 100 0 100 u [m s ] 71 km / 0.0365 hPa -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 u [m s ] -1 (a) (b) (c) (d) (e) (f) (g) (h) (i) (j) Figure 12. www.atmos-meas-tech.net/11/5007/2018/ Zonal WIRA-C wind measurements on 10 distinct pres- sure levels between 35 km (a) to 71 km (j). The fully convolved ECMWF model data as well as the ECMWF data from the nearest pressure level (but still smoothed in time) is given for comparison. Of h d l d ECMWF i id Ti Figure 11. Time series of zonal wind speeds measured by WIRA- C (a) and ECMWF analysis data (b) between 14 November 2016 and 31 December 2018 for the altitude range of 35 to 75 km with a time resolution of 12 h (day and nighttime). The ECMWF data has been convolved with the averaging kernels of the retrieval in order to get the same spatial and temporal resolution for both datasets. In- valid data points are grayed out, resulting in different altitude ranges for different days. The few data gaps are due to a tropical cyclone and power outages. Figure 11. Time series of zonal wind speeds measured by WIRA- 5.2.2 The Rayleigh–Mie Doppler wind lidar 10 1 100 Pressure [hPa] (a) WIRA-C 2016-12-01 2017-01-01 2017-02-01 2017-03-01 2017-04-01 2017-05-01 2017-06-01 2017-07-01 2017-08-01 2017-09-01 2017-10-01 2017-11-01 2017-12-01 2018-01-01 10 1 100 Pressure [hPa] (b) ECMWF convolved 60 40 20 0 20 40 60 Meridional wind [m s ] 40 50 60 70 Approx. altitude [km] 40 50 60 70 Approx. altitude [km] -1 50 0 50 v [m s ] 43 km / 2.0535 hPa WIRA-C ECMWF conv ECMWF 50 0 50 47 km / 1.1548 hPa 50 0 50 51 km / 0.6494 hPa 50 0 50 55 km / 0.3652 hPa 50 0 50 59 km / 0.2054 hPa 2017-06-07 2017-06-14 2017-06-21 2017-06-28 2017-07-05 2017-07-12 2017-07-19 2017-07-26 2017-08-02 2017-08-09 2017-08-16 2017-08-23 2017-08-30 2017-09-06 2017-09-13 Time UTC 50 0 50 63 km / 0.1155 hPa -1 v [m s ] -1 v [m s ] -1 v [m s ] -1 v [m s ] -1 v [m s ] -1 (a) (b) (c) (d) (e) (f) Figure 13. Same as Fig. 11 but for meridional wind. Time series of meridional wind speeds measured by WIRA-C (a) and ECMWF analysis data (b) between 14 November 2016 and 1 January 2018 for the altitude range of 35 to 75 km with a time resolution of 12 h (day and nighttime). The ECMWF data has been convolved with the averaging kernels of the retrieval in order to get the same spatial and temporal resolution for both datasets. Invalid data points are grayed out, resulting in different altitude ranges for different days. The few data gaps are due to a tropical cyclone and power outages. the two lines-of-sight is not relevant, as WIRA-C retrieves a wind proﬁle that best ﬁts both observations in opposing directions. As the retrieval is not linear, this does not neces- sarily deliver the mean proﬁle but an approximation thereof. For our retrieval and comparisons, we thus assume that the variation of horizontal wind speeds are negligible for 12 h in- tegration time and horizontal distances from 150 km at 30 km altitude up to 370 km at 75 km altitude. 2 2 Time UTC Figure 14. Same as Fig. 12 but for meridional wind. WIRA-C measurements on 10 distinct pressure levels between 35 km (a) to 71 km (f) for 1 June 2017 to 19 September 2017. The data before June 2017 and after September 2017 (shown in Fig. 5.2.2 The Rayleigh–Mie Doppler wind lidar The Rayleigh–Mie Doppler wind lidar is an active sounder, measuring the Doppler shift of backscattered visible light us- ing Fabry–Perot interferometry and can provide wind proﬁles from 5 up to approximately 60 km. Up to 30 km, the vertical resolution is 100 m and the accuracy is better than 1 m s−1 for 1 h integration time. Because of decreasing density of molec- ular backscatters and the inverse-square law of light, the un- certainties of the lidar measurements increase with altitude and ﬁnally limit the altitude domain to approximately 60 km depending on integration time. Between 30 and 60 km, the vertical resolution varies between 0.5 to 3 km and the mea- surement error is 10 m s−1 at 50 km altitude for an integra- tion time of 3 h. The instrument and the retrieval scheme is described in Khaykin et al. (2016) and references therein. The lidar only measures at nighttime and has a variable integration time that depends on meteorological conditions (clear sky) and available man power. As the integration time often is below 4 h, we cannot run a retrieval for the mi- crowave radiometer for exactly the same integration time because of the noise. We currently have no opportunity to adapt our measurement to the short integration times of the lidar and thus we just compare the nighttime measurement of WIRA-C and the lidar while noting the respective integration times. For the vertical resolution we convolve the lidar data with the averaging kernels of the retrieval to have comparable altitude resolution of the proﬁles. 2 2 2 Time UTC Figure 12. Zonal WIRA-C wind measurements on 10 distinct pres- sure levels between 35 km (a) to 71 km (j). The fully convolved ECMWF model data as well as the ECMWF data from the nearest pressure level (but still smoothed in time) is given for comparison. Often the raw and convolved ECMWF curves coincide. Time reso- lution is 12 h (day and nighttime). The light-blue area represents the uncertainty σo of the WIRA-C data. The lidar measures at an elevation angle of 45◦as opposed to the 22◦of WIRA-C. However, the difference between Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 5019 J. 5.2.2 The Rayleigh–Mie Doppler wind lidar 11) is not rep- resented here in order to focus on the period with more variability. The fully convolved ECMWF model data as well as the ECMWF data from the nearest pressure level (but still smoothed in time) is given for comparison. Time resolution is 12 h (day and nighttime). The light-blue area represents the uncertainty σo of the WIRA-C data. 5.2.2 The Rayleigh–Mie Doppler wind lidar Hagen et al.: WIRA-C: Wind Radiometer for Campaign 10 1 100 Pressure [hPa] (a) WIRA-C 2016-12-01 2017-01-01 2017-02-01 2017-03-01 2017-04-01 2017-05-01 2017-06-01 2017-07-01 2017-08-01 2017-09-01 2017-10-01 2017-11-01 2017-12-01 2018-01-01 10 1 100 Pressure [hPa] (b) ECMWF convolved 60 40 20 0 20 40 60 Meridional wind [m s ] 40 50 60 70 Approx. altitude [km] 40 50 60 70 Approx. altitude [km] -1 Figure 13. Same as Fig. 11 but for meridional wind. Time series of meridional wind speeds measured by WIRA-C (a) and ECMWF analysis data (b) between 14 November 2016 and 1 January 2018 for the altitude range of 35 to 75 km with a time resolution of 12 h (day and nighttime). The ECMWF data has been convolved with the averaging kernels of the retrieval in order to get the same spatial and temporal resolution for both datasets. Invalid data points are grayed out, resulting in different altitude ranges for different days. The few data gaps are due to a tropical cyclone and power outages. 50 0 50 v [m s ] 43 km / 2.0535 hPa WIRA-C ECMWF conv ECMWF 50 0 50 47 km / 1.1548 hPa 50 0 50 51 km / 0.6494 hPa 50 0 50 55 km / 0.3652 hPa 50 0 50 59 km / 0.2054 hPa 2017-06-07 2017-06-14 2017-06-21 2017-06-28 2017-07-05 2017-07-12 2017-07-19 2017-07-26 2017-08-02 2017-08-09 2017-08-16 2017-08-23 2017-08-30 2017-09-06 2017-09-13 Time UTC 50 0 50 63 km / 0.1155 hPa -1 v [m s ] -1 v [m s ] -1 v [m s ] -1 v [m s ] -1 v [m s ] -1 (a) (b) (c) (d) (e) (f) Figure 14. Same as Fig. 12 but for meridional wind. WIRA-C measurements on 10 distinct pressure levels between 35 km (a) to 71 km (f) for 1 June 2017 to 19 September 2017. The data before June 2017 and after September 2017 (shown in Fig. 11) is not rep- resented here in order to focus on the period with more variability. The fully convolved ECMWF model data as well as the ECMWF data from the nearest pressure level (but still smoothed in time) is given for comparison. Time resolution is 12 h (day and nighttime). The light-blue area represents the uncertainty σo of the WIRA-C data. www.atmos-meas-tech.net/11/5007/2018/ 5.3 Results Figures 11 and 13 show an overview over the zonal and meridional measurements from the Maïdo campaign, to- gether with the corresponding ECMWF data, convolved in space and time. A more detailed view is given in Figs. 12 and 14 for zonal and meridional wind, respectively. There, besides the convolved ECMWF data, the model data of the nearest level is also given for comparison. At the lowest and highest levels, the difference between the fully convolved and the original ECMWF data is quite obvious. This difference is an indicator for the smoothing error, and is a consequence of the slightly worse altitude resolution and accuracy at the lowest and uppermost levels compared to the central levels where the difference nearly vanishes. riod of approximately 10 days present at the layers between 50 and 60 km in August and September 2017 are also present in both datasets. There are also short periods where we can see a clear dis- crepancy between the model data and the measurement. For example at the layers below 45 km for the end of January and beginning of February 2017, where WIRA-C measured a smaller magnitude of zonal wind than predicted by the model for several days. This might be connected to the tropical cy- clone in the Indian ocean that was the reason for the sub- sequent interruption of the measurement, as the instrument had to be dismounted and protected inside the building. At the uppermost levels, ECMWF has the tendency to predict a higher magnitude in zonal wind speed and to some extent also in meridional wind speed than WIRA-C. Most promi- nently at the end of April 2017, the model predicts a much In general, the zonal and meridional wind for WIRA-C and ECMWF are consistent: ﬁrstly, the zonal wind reversal around equinox is resolved by the model as well as WIRA-C and they agree on the time of this event as well as on the magnitude. Secondly, the well-deﬁned periods of stronger westward winds between 35 and 55 km in June are present in both datasets. Further, the increased variability with a pe- www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 5020 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns ECMWF 12h ECMWF 18h ECMWF 00h ECMWF 06h WIRA-C Lidar Lidar conv Radiosonde 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] 50 0 50 100 150 Zonal wind speed [m s ] 100 101 Pressure [hPa] 2016-08-18 (3.3 h) 50 0 50 100 150 100 101 2016-08-22 (3.0 h) 50 0 50 100 150 100 101 2016-08-23 (2.7 h) 50 0 50 100 150 100 101 2017-06-21 (8.8 h) 50 0 50 100 150 100 101 Pressure [hPa] 2017-06-22 (9.7 h) 50 0 50 100 150 100 101 2017-06-26 (3.5 h) 50 0 50 100 150 100 101 2017-06-27 (3.1 h) -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Figure 15. Seven coincident observations of zonal wind from WIRA-C and Doppler lidar from August 2016 and June 2017 together with radio soundings and ECMWF operational model data at different times, WIRA-C measurements start at 14:00 UT (18:00 LT) and lidar measurements typically between 17:00 and 20:00 UT (21:00 and 24:00 LT). The measurement time for WIRA-C is 12 h for every proﬁle while the measurement time for the lidar observation (given in parenthesis) is typically between 3 and 3.5 h, with the exception of 21 and 22 June 2017, where measurement took 8.8 and 9.7 h, respectively. Source of radiosonde data: Météo-France. 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] 50 0 50 100 150 Zonal wind speed [m s ] 100 101 Pressure [hPa] 2016-08-18 (3.3 h) 50 0 50 100 150 100 101 2016-08-22 (3.0 h) 50 0 50 100 150 100 101 2016-08-23 (2.7 h) 50 0 50 100 150 100 101 2017-06-21 (8.8 h) -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 2016-08-23 (2.7 h) Approx. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns altitude [km] 0 25 0 25 50 Meridional wind speed [m s ] 2016-08-18 (0.9 h) 50 25 0 25 50 100 101 2016-08-22 (1.6 h) 50 25 0 25 50 100 101 2016-08-23 (1.6 h) 50 25 0 25 50 100 101 2017-06-21 (8.8 h) -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 30 35 40 45 50 55 60 30 35 40 45 50 55 60 50 25 0 25 50 100 101 2016-08-22 (1.6 h) 50 25 0 2 100 101 2016-08-23 (1 Meridional wind speed [m s ] -1 Meridional wind spee 30 35 40 45 50 55 60 30 35 40 45 50 55 60 50 m s ] ) 50 25 0 25 50 100 101 2016-08-22 (1.6 h) 50 100 101 -1 Meridional wind speed [m s ] -1 M 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] 50 25 0 25 50 100 101 2016-08-23 (1.6 h) 50 25 0 25 50 100 101 2017-06-21 (8.8 h) Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Approx. altitude [km] 1 Pressure [hPa] Meridional wind speed [m s ] -1 ECMWF 12h UTC ECMWF 18h UTC ECMWF 00h UTC ECMWF 06h UTC WIRA-C Lidar Lidar conv Radiosonde 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] p 50 25 0 25 50 100 101 Pressure [hPa] 2017-06-22 (9.9 h) 50 25 0 25 50 100 101 2017-06-26 (3.7 h) 50 25 0 25 50 100 101 2017-06-27 (3.3 h) Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] Meridional wind speed [m s ] Meridional wind speed [m s ] 1 2017-06-27 (3.3 h) Pressure [hPa] Figure 16. Seven coincident observations of meridional wind from WIRA-C and Doppler lidar from August 2016 and June 2017 together with radio soundings and ECMWF operational model data at different times, WIRA-C measurements start at 14:00 UT (18:00 LT) and lidar measurements typically between 17:00 and 20:00 UT (21:00 and 24:00 LT). J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns altitude [km] Pressure [hPa] Zonal wind speed [m s ] -1 ECMWF 12h ECMWF 18h ECMWF 00h ECMWF 06h WIRA-C Lidar Lidar conv Radiosonde 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] p [ ] 50 0 50 100 150 100 101 Pressure [hPa] 2017-06-22 (9.7 h) 50 0 50 100 150 100 101 2017-06-26 (3.5 h) 50 0 50 100 150 100 101 2017-06-27 (3.1 h) Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] Zonal wind speed [m s ] Zonal wind speed [m s ] -1 Zonal wind speed [m s ] 2017-06-27 (3.1 h) Pressure [hPa] Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Zonal wind speed [m s ] -1 Figure 15. Seven coincident observations of zonal wind from WIRA-C and Doppler lidar from August 2016 and June 2017 together with radio soundings and ECMWF operational model data at different times, WIRA-C measurements start at 14:00 UT (18:00 LT) and lidar measurements typically between 17:00 and 20:00 UT (21:00 and 24:00 LT). The measurement time for WIRA-C is 12 h for every proﬁle while the measurement time for the lidar observation (given in parenthesis) is typically between 3 and 3.5 h, with the exception of 21 and 22 June 2017, where measurement took 8.8 and 9.7 h, respectively. Source of radiosonde data: Météo-France. higher magnitude in zonal wind but a lower variability. This might be an effect of the artiﬁcial forcing in the model at the uppermost layers. At the same time, our observation error in- creases with altitude and we cannot completely rule out, that the variability is caused by retrieval noise. able to date for the zonal and meridional wind component, respectively. The lidar proﬁles have been acquired in Au- gust 2016 during routine measurements and in June 2017 during the LIDEOLE-III campaign. In addition, the corre- sponding ECMWF model data is shown at the four clos- est time steps of the model. In case of zonal wind, these ECMWF proﬁles are nearly identical but for the meridional Figures 15 and 16 show all seven coincident measurements of WIRA-C and the Rayleigh–Mie Doppler wind lidar avail- www.atmos-meas-tech.net/11/5007/2018/ www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns 5021 ECMWF 12h UTC ECMWF 18h UTC ECMWF 00h UTC ECMWF 06h UTC WIRA-C Lidar Lidar conv Radiosonde 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. altitude [km] 50 25 0 25 50 Meridional wind speed [m s ] 100 101 Pressure [hPa] 2016-08-18 (0.9 h) 50 25 0 25 50 100 101 2016-08-22 (1.6 h) 50 25 0 25 50 100 101 2016-08-23 (1.6 h) 50 25 0 25 50 100 101 2017-06-21 (8.8 h) 50 25 0 25 50 100 101 Pressure [hPa] 2017-06-22 (9.9 h) 50 25 0 25 50 100 101 2017-06-26 (3.7 h) 50 25 0 25 50 100 101 2017-06-27 (3.3 h) -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Meridional wind speed [m s ] -1 Figure 16. Seven coincident observations of meridional wind from WIRA-C and Doppler lidar from August 2016 and June 2017 together with radio soundings and ECMWF operational model data at different times, WIRA-C measurements start at 14:00 UT (18:00 LT) and lidar measurements typically between 17:00 and 20:00 UT (21:00 and 24:00 LT). The measurement time for WIRA-C is 12 h for every proﬁle while the measurement time for the lidar observation (given in parenthesis) is typically between 1 and 4 h, with the exception of 21 and 22 June 2017, where measurement took 8.8 and 9.9 h, respectively. Source of radiosonde data: Météo-France. 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 30 35 40 45 50 55 60 Approx. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns The measurement time for WIRA-C is 12 h for every proﬁle while the measurement time for the lidar observation (given in parenthesis) is typically between 1 and 4 h, with the exception of 21 and 22 June 2017, where measurement took 8.8 and 9.9 h, respectively. Source of radiosonde data: Météo-France. wind, they indicate a high temporal variability in the model data. At the lowermost levels, the radiosonde launched at the nearby Gillot airport at noon is given for comparison where available. where the whole night was used for lidar acquisition, the agreement of the two independent measurements is well within their respective uncertainties. We would like to em- phasise that favourable conditions for lidar measurements, namely clear sky and nighttime, also imply lower uncertain- ties for the WIRA-C measurements. Remarkably, the zonal wind measurements from 22 June 2017 of WIRA-C and the For both horizontal wind components, the proﬁles of the three sources (WIRA-C, lidar, ECMWF) are consistent. Es- pecially for the lidar measurements on 21 and 22 June 2017, www.atmos-meas-tech.net/11/5007/2018/ Atmos. Meas. Tech., 11, 5007–5024, 2018 5022 J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns lidar are nearly identical, while the ECMWF model is offset by 20 m s−1 at 55 km altitude. proximately 10 km for zonal and 15 km for meridional wind. Even though we retrieve ozone proﬁles as well, we consider them as a by-product that is only needed to fully ﬁt the spec- trum and discussion of them is not in the scope of this paper. For the meridional wind, the lidar shows some patterns with very large vertical gradients in the wind speeds as on 18 August 2016 and 21, 22 and 26 June 2017 at an altitude around 40, 55, 48 and 47 km, respectively. These patterns are not present in the other datasets and are probably caused by internal gravity waves. For characteristics and details about such structures observed by lidar, see Khaykin et al. (2015). It is conceivable that the vertical structures observed by the lidar are simply not resolved by the ECMWF model and smoothed out by the radiometer. 6 Conclusions WIRA-C is a new passive microwave wind radiometer de- signed for campaigns as well as long-term measurements. With it, the successful prototype WIRA has been replicated and improved. The optical system and the pre-ampliﬁed sin- gle side band heterodyne receiver and the spectrometer are embedded in a single housing with compact dimensions. Cal- ibration is performed with the tipping curve scheme and tropospheric correction accounts for tropospheric inhomo- geneities and normalises the spectra acquired in the four car- dinal directions. The next steps in passive microwave wind radiometry will go towards optimising the retrieval process and explore the lower limits of time resolution. This could include a time series retrieval as performed by Christensen and Eriksson (2013) for a water vapour instrument. Also, the possibility of wind retrievals above the Doppler broadening range could be further explored as a ﬁrst comparison between WIRA and meteor radar measurements (Rüfenacht et al., 2018, Fig. A1) was very promising. We applied an optimal estimation retrieval to combine ob- servations in opposing directions to get a single wind proﬁle that best represents all our measurements. The main beneﬁt of our retrieval scheme is the availability of quality control parameters representing the whole inversion process and the increased altitude resolution of 9 to 11 km (as opposed to 10 to 16 km for WIRA). The observation error gives an estimate on the uncertainty in wind speeds caused by the thermal noise on our measurements. Its 1σ value for zonal wind is typically around 15 m s−1 up to 68 km or 60 km for nighttime and day- time measurements, respectively. The error on the meridional wind is approximately 9 m s−1 due to the smaller covariance of the a priori proﬁle that represents the expected magnitude of the wind speeds. To complement the estimation of the ran- dom error we performed Monte Carlo estimations of possible systematic error sources. These estimations show that the ex- pected systematic errors are lower than the random errors. Data availability. We acknowledge ECMWF for the operational analysis data (www.ecmwf.int, last access: 23 August 2018) as well as NASA for the Aura MLS temperature proﬁles (http://disc. gsfc.nasa.gov/acdisc, last access: 23 August 2018) used in the re- trieval. The WIRA-C data presented in this manuscript can be made available on request and are available via the ARISE data portal (arise-portal.eu, last access: 23 August 2018). Competing interests. J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns For example, for the mea- surement of the meridional wind on 21 and 22 June 2017, we can see that the convolved lidar proﬁle and the WIRA-C measurement agree quite well while the high resolution pro- ﬁle of the lidar shows a layer of wind speeds with higher magnitude at 50 km altitude. This indicates that WIRA-C in- deed smoothes out the feature, but that the two measurements are consistent. The measurements are consistent with the ECMWF oper- ational analysis and also show very good agreement with the available lidar measurements from the co-located Rayleigh– Mie Doppler wind lidar. The main challenge for the compar- isons is to properly account for the different integration times and spatial resolutions, especially for the lidar measurements with short acquisition times. The ﬁner structures in the wind proﬁles as seen by the lidar are not resolved by WIRA-C, but the convolved proﬁles indicate a high consistency of the mea- surements. For the lidar measurements where integration has been performed during the whole night, the two independent measurements agree within their respective errors in the en- tire altitude range of overlap (37 to 50 km). More coincident lidar measurements would certainly be valuable for further validation. 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R.: Analysis and improvement of tipping calibration for ground-based microwave ra- diometers, IEEE T. Geosci. Remote, 38, 1260–1276, https://doi.org/10.1109/36.843018, 2000. Ingold, T., Peter, R., and Kämpfer, N.: Weighted mean tropospheric temperature and transmittance determination at millimeter-wave frequencies for ground-based applications, Radio Sci., 33, 905– 918, https://doi.org/10.1029/98RS01000, 1998. 6 Conclusions The authors declare that they have no conﬂict of interest. Acknowledgements. This study beneﬁted from the excellent sup- port from the dedicated staff at the Maïdo observatory on Réunion. We acknowledge the European Communities, the Région Réunion, CNRS, and Université de la Réunion for their support and con- tributions in the construction phase of the research infrastructure OPAR (Observatoire de Physique de l’Atmosphère de La Réunion). OPAR is presently funded by CNRS (INSU) and Université de The validation campaign on the Maïdo observatory on Réunion proved that WIRA-C can provide continuous mea- surements of horizontal wind speeds in the altitude range of 35 to 70 km. We presented a 1-year dataset of measurements with a time resolution of 12 h and an altitude resolution of ap- Atmos. Meas. Tech., 11, 5007–5024, 2018 www.atmos-meas-tech.net/11/5007/2018/ 5023 www.atmos-meas-tech.net/11/5007/2018/ J. Hagen et al.: WIRA-C: Wind Radiometer for Campaigns posium on Millimeter Waves – TSMMW2006, The 7th MINT Millimeter-Wave International Symposium – MINT- MIS2006, 15–17 February, 2006, Espoo, Finland, 359–364, https://doi.org/10.7892/boris.19296, 2006. posium on Millimeter Waves – TSMMW2006, The 7th MINT Millimeter-Wave International Symposium – MINT- MIS2006, 15–17 February, 2006, Espoo, Finland, 359–364, https://doi.org/10.7892/boris.19296, 2006. 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https://openalex.org/W2527912905	https://academic.oup.com/jamia/article-pdf/24/3/633/13063233/ocw141.pdf	English	null	Human resource information systems in health care: a systematic evidence review	Journal of the American Medical Informatics Association	2,016	cc-by	15,559	Aizhan Tursunbayeva,1 Raluca Bunduchi,2 Massimo Franco,1 and Claudia Pagliari3 1Department of Economics, Management, Society and Institutions, University of Molise, Campobasso, Italy, 2Business School, University of Edinburgh, Edinburgh, UK, and 3eHealth Research Group, Usher Institute of Population Health Sciences and Infor- matics, University of Edinburgh, Edinburgh, UK Corresponding Author: Claudia Pagliari, eHealth Research Group, Usher Institute of Population Health Sciences and Informatics, University of Edinburgh Medical School, Teviot Place, Edinburgh, EH8 9AG, Scotland. E-mail: Claudia. Pagliari@ed.ac.uk; Tel: þ44 131 650 9464 Received 14 April 2016; Revised 11 August 2016; Accepted 23 August 2016 Received 14 April 2016; Revised 11 August 2016; Accepted 23 August 2016 V C The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. V C The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association. Journal of the American Medical Informatics Association, 24(3), 2017, 633–654 doi: 10.1093/jamia/ocw141 Advance Access Publication Date: 5 October 2016 Review Journal of the American Medical Informatics Association, 24(3), 2017, 633–654 doi: 10.1093/jamia/ocw141 Advance Access Publication Date: 5 October 2016 Review Procedure Outputs were stored in EPPI-Reviewer 4 software. After initial screening of titles and abstracts, the full text of potentially relevant articles was examined by 2 reviewers (AT, RB) to assess their fit with the inclusion criteria. Disagreements were resolved through consensus or arbitration by a third reviewer (CP). What HRIS are and why they are so important y y p Staff costs account for 65–80% of health organizations’ total oper- ating budgets.3 Therefore, effective management of human resources (HR) is essential, from both a clinical and financial perspective. HRIS support a variety of HRM practices, including recruitment and performance management, and provide health leaders with cru- cial information guiding effective capacity planning and resource al- location. HRIS can take various forms, ranging from dedicated stand-alone packages (eg, payroll) to components of integrated en- terprise resource planning (ERP) or hospital information systems (HISs). Not perceived as life-critical, HRIS have received very little attention in the health informatics literature, and their development, implementation, use, and impacts in health organizations are poorly understood compared with clinical systems (eg, electronic health re- cords). HRIS research also tends to be distributed across the social (encompassing business and management), information and commu- nications technology (ICT), and health sciences literature. ABSTRACT Objective: This systematic review aimed to: (1) determine the prevalence and scope of existing research on human resource information systems (HRIS) in health organizations; (2) analyze, classify, and synthesize evi- dence on the processes and impacts of HRIS development, implementation, and adoption; and (3) generate recommendations for HRIS research, practice, and policy, with reference to the needs of different stakeholders. Methods: A structured search strategy was used to interrogate 10 electronic databases indexing research from the health, social, management, technology, and interdisciplinary sciences, alongside gray literature sources and reference lists of qualifying studies. There were no restrictions on language or publication year. Two re- viewers screened publications, extracted data, and coded ﬁndings according to the innovation stages covered in the studies. The Critical Appraisal Skills Program checklist was adopted to assess study quality. The process of study selection was charted using a Preferred Items for Systematic Reviews and Meta-Analysis (PRISMA) diagram. Results: Of the 6824 publications identiﬁed by the search strategy, 68, covering 42 studies, were included for ﬁ- nal analysis. Research on HRIS in health was interdisciplinary, often atheoretical, conducted primarily in the hospital sector of high-income economies, and largely focused uncritically on use and realized beneﬁts. Discussion and Conclusions: While studies of HRIS in health exist, the overall lack of evaluative research raises unanswered questions about their capacity to improve quality and efﬁciency and enable learning health systems, as well as how sociotechnical complexity inﬂuences implementation and effectiveness. We offer this analysis to decision makers and managers considering or currently implementing an HRIS, and make recommendations for further research. Trial Registration: International Prospective Register of Systematic Reviews (PROSPERO): CRD42015023581. http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID¼CRD42015023581#.VYu1BPlVjDU. Key words: eHealth, health care management, information systems, systematic review, human resource information systems V C The Author 2016. Published by Oxford University Press on behalf of the American Medical Informatics Association. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommon which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. 633 634 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 Exclusion criteria l d d d We excluded descriptive reports, pure market research, articles fo- cused on software design issues, studies that were not primarily fo- cused on HRIS or that mentioned HRIS without specifying the health sector, and articles examining generic ERP/HIS without refer- ring to HR functionalities. Details of the filters applied at each screening stage are included in the PRISMA flow diagram. Administrative information systems as a topic of research in health Administrative information systems (IS) in health organizations deal with such processes as records management, billing and finance, and aspects of human resource management (HRM), which can also help to support care delivery, quality improvement, and research. Despite their role as enablers of efficient, effective, and, potentially, “learning” health organizations,1 administrative systems have been somewhat neglected as a topic of research in health informatics.2 This systematic review focuses on a key subcategory of administra- tive systems, human resource information systems (HRIS). Inclusion criteria Th l There were 2 inclusion criteria: (1) studies involving a formal or semiformal approach to the investigation or evaluation of HRIS, whether led by academia or industry (eg, consulting sector), or from within the health sector; and (2) studies of broader business/admin- istrative/ERP/HIS systems that explicitly examine their application to HR practices. Although forms of HRIS have been used in the health sector for al- most half a century,4 this is still an evolving area. Increasingly so- phisticated modular HRIS are being procured and implemented in health organizations worldwide,5 often at high expense in terms of technology, support, and change management. While the benefits of these systems have been much vaunted by HRIS vendors6 and policy makers,7 there have also been spectacular failures, where large-scale implementations have encountered huge overspends, weak organiza- tional buy-in, or poor interoperability with existing systems.8 Given the opportunity costs of getting these projects wrong, developers, procurers, and managers require more guidance on the usefulness, effectiveness, and implementation barriers associated with HRIS, as well as how to evaluate them. Thus this systematic review is very timely. Search strategy A comprehensive search strategy was developed and tested itera- tively during a scoping phase (see Supplementary Appendix 1). This was used to interrogate 10 international online databases indexing medical/health (Cochrane Library, MEDLINE, EMBASE); social science (ABI/INFORM, ASSIA, Sociological Abstracts), ICT (IEEE Xplore); and multidisciplinary research (Scopus, Web of Science Core Collection, ScienceDirect). Gray literature sources were also examined, including reports from the World Health Organization (WHO), relevant professional organizations (eg, Chartered Institute of Personnel and Development, Society for Human Resource Man- agement, Healthcare Information and Management Systems Soci- ety), and consulting firms (eg, Deloitte, Ernst & Young, PricewaterhouseCoopers, KPMG). Academic dissertations were searched via Google, and the reference lists of qualifying articles were searched by hand to identify additional relevant studies. No re- strictions were applied to publication year or language. INTRODUCTION development, implementation, and use; and (3) generate recommen- dations for HRIS research, practice, and policy, with reference to the needs of different stakeholders and communities of practice. Administrative information systems as a topic of research in health What is new about this review One author (AT) extracted information from all eligible studies us- ing a structured form containing the following fields: authors, publi- cation year, setting (type of organization, country/region in which the study was conducted), innovation stage, journal discipline, HRIS functionality, research purpose/questions, theoretical basis, HRIS users, study design, and main findings. Extracted information was then verified by all team members (CP, RB, and MF). Our scoping study identified only 2 previous literature reviews spe- cifically examining HRIS in health, both of which were limited in scope.9 We therefore conducted an interdisciplinary systematic re- view utilizing sources of evidence from the ICT, social science, and health research literature, encompassing any ICT used for HR ad- ministration, management, and development practices in health or- ganizations. The specific objectives were to: (1) determine the prevalence and scope of existing research and evaluation pertaining to HRIS in health organizations; (2) analyze, classify, and synthesize existing evidence on the processes and impacts of HRIS To differentiate among HRIS project stages, we borrowed from existing innovation models (eg10,11) and coded the results according to 3 main innovation stages: (1) development (eg, needs assessment, procurement initiation, prototyping, and user acceptance testing), Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 635 (2) implementation (eg, purchasing, systems integration, organiza- tional change management, and training), and (3) use (including adaptation of organizational procedures to accommodate rou- tinization of the innovation as part of day-to-day working practices). were not covered by Scimagojr. 29 articles (71%) were published in a single discipline: 18 in health (44%), 9 in social science (22%), and 2 in ICT (5%). Just under a third (29%) were published in mul- tidisciplinary journals, including 5 covering ICT and health (12%), 3 covering health and social science (7%), and 4 covering social sci- ence and ICT (10%). were not covered by Scimagojr. 29 articles (71%) were published in a single discipline: 18 in health (44%), 9 in social science (22%), and 2 in ICT (5%). Just under a third (29%) were published in mul- tidisciplinary journals, including 5 covering ICT and health (12%), 3 covering health and social science (7%), and 4 covering social sci- ence and ICT (10%). We also coded studies using Parry and Tyson’s12 framework to compare the intended and actual benefits of HRIS adoption. What is new about this review This in- cludes 6 types of goals relating to operational efficiency, service deliv- ery, strategic orientation, manager empowerment, standardization, and organizational image. Additional goals emerging from our analy- sis were added into separate categories. Research designs and study quality Most studies (n ¼ 24) used qualitative methods. Nine employed quantitative designs, while 8 used mixed methods. One study was a systematic literature review (a second review identified by our search did not meet the inclusion criteria; it focused on ICT for en- abling continuing professional development, and e-learning was out of the scope of this review9). Descriptive studies were excluded at the full-text review stage. None of the qualifying studies received a maximum score of 8 on quality assessment. Those scoring highest were quantitative studies and postgraduate research theses; those scoring lower did not ade- quately explain their units of analysis, research methodology, or sources of potential bias. Of the qualitative studies, very few scored higher than 6 (see Table 1 and Supplementary Appendix 2). Critical appraisal techniques Although diverse health organizations were represented, more than half of the studies focused on hospitals in high-income countries, typically taking one hospital as their unit of analysis. Only one study focused on a primary health care organization (see Table 1). Studies in low-income countries mostly reviewed country-wide HRIS and/or systems developed, implemented, and used by government Depart- ments of Health or professional organizations. Following recommendations for systematic reviews of qualitative re- search,15,16 we adapted the qualitative Critical Appraisal Skills Pro- gramme checklist.17 Questions concerning the appropriateness of qualitative methodology and ethical issues were eliminated, since a first reading of the material revealed that most eligible studies were qualitative and lacked ethical considerations (see Supplementary Appendix 2). In addition to the “yes” or “no” answers, we added a “not clear” option (corresponding to scores of 1.0, 0.5, and 0, re- spectively). One reviewer (AT) appraised all eligible studies. A sec- ond reviewer (CP) independently appraised a random 20% sample to assess interrater consistency and facilitate discussion about the process and any ambiguities. Since only a few minor discrepancies were identified, a secondary appraisal focused on studies about which the first reviewer was uncertain. Country The majority of studies were conducted in high-income countries (see Table 1): 17 in Europe (4 each in the Netherlands and the UK, 3 in Finland, 2 in Ireland, and 1 each in Greece, Norway, Spain, and Turkey), 9 in North America (7 in the United States and 2 in Can- ada), and 1 in Australia (although several authors independently studied this case, it was classified as one study). Only 4 studies were conducted in Asia (2 in Pakistan and 1 each in India and Taiwan), 6 in Africa (2 in Kenya, 1 each in Malawi, Uganda, and Tanzania, and 1 covering 9 African countries). One study was conducted in South America (Brazil), and 1 in the Middle East (Saudi Arabia). Three studies either involved several countries across different regions or did not specify the countries covered. Finally, of the various models of HRM practices described in the literature (eg13), including in relation to HRIS (eg5), we chose to adapt Foster’s E-HRM Landscape model14 to classify our studies (see Figure 3), as it covers the majority of the HRM practices men- tioned in the reviewed articles. To the verbs describing core objec- tives of HRIS in the e-HRM Landscape we added “interact,” taking account of HRIS modules described as self-service, HR portals, or HR Intranets. We also added several subcategories reflecting addi- tional functions mentioned in the studies (eg, employee relations and qualifications tracking). RESULTS In all, 6824 results were generated by the search strategy and 6104 titles and abstracts remained after removing 720 duplicates. Of these, 399 qualified for full-text review, 232 due to their potential eligibility and 167 because there was insufficient information in the title or abstract to make a decision. After removing documents that did not meet the inclusion criteria, 68 publications representing 42 separate studies were included in the final analysis (see Table 1). The stages of selection are illustrated in the PRISMA diagram la- beled Figure 1. Theoretical frameworks Over half of the studies (n ¼ 22) did not specify any theoretical per- spective. The other 20 referred to a diversity of frameworks, most specifying only one (see Table 2). Publication characteristics Most qualifying studies (n ¼ 21) examined dedicated HRIS, com- prising one or several modules for supporting particular HRM prac- tices. Sixteen studies focused on generic integrated organizational systems, including modules dedicated to HRM practices. Five did not clarify whether the HRIS were dedicated or components of ge- neric systems (see Table 1). Included articles were published between 1979 and 2014. More than half entered the literature within the last decade, peaking in 2010, when 11 were published (see Figure 2). Out of 68 publications, the vast majority (n ¼ 41) were journal articles. To test our observation that HRIS in health is a multidisci- plinary topic,9 these articles were first classified into subject areas according to the Scimago Journal ranking portal (Scimagojr) and af- terward using broader discipline categories such as health, ICT, and social science. Nine articles were classified manually, as the journals Descriptions of ICT for managing HR in health organizations lacked a common terminology (see Table 1). Organizational sys- tems that included HRM functions were commonly described as Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 636 Table 1. Characteristics of the included studies # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported S1 Altuwaijri and Khorsheed, 201218 (social science) Saudi Arabia (high); Mixedb (gen.: ERP) To propose a new generic model for successful implementation of IT projects Qual. 4 Implementation Barriers: individual, and project Use Realized beneﬁtsc: op- erational, strategic, empowerment, and IT infrastructure S2 Bakar, Sheikh and Sultan, 201219 (ICT/health) Tanzania (low); Ministry of Health (ded.: open-source HRIS) To describe the opportunities and related challenges of integrating an open-source software process in the organization Qual. 5.5 Use Barriers: environ- ment, project, and individual Realized beneﬁtsc: operational, and service Approaches to: technology S3 Bondarouk and Ruel, 200320 (N/A) Netherlands (high); second- ary (hospital) (ded.: personnel and salary ad- ministration system) To explore differences in the adoption of a human manage- ment system be- tween 2 groups of users Qual. Publication characteristics 5.5 Implementation Facilitators: organiza- tion, and project Barriers: organization Dent, 199131 (social science) Approaches to: proj- ect, and technology To examine the devel- opment of comput- i d IT Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported S5 Cockerill and O’Brien-Pallas, 199028 (health) Canada (high); secondary (>1 hospitals) (gen.: nursing work- load measure- ment systems) To develop a proﬁle of use of nursing workload measure- ment systems in Canadian hospitals, assess user satisfac- tion, and identify challenges/per- ceived problems and research issues related to these systems Quant. 6 Implementation Barriers: organization Generic: project, and individual O’Brien-Pallas and Cockerill, 199029 (health) To explore senior nurse executives’ needs and expecta- tions for nursing workload systems Use Realized beneﬁtsc: strategic Satisfaction: familiar- ity with the system, its functions or use of them, and user Use Realized beneﬁtsc: strategic Satisfaction: familiar- ity with the system, its functions or use of them, and user satisfaction varied between roles; sys- tem needs to reﬂect true workload for users to be satisﬁed Approaches to: tech- nology, and individual 5.5 Implementation Facilitators: organiza- tion, and project Barriers: organization Approaches to: proj- ect, and technology individual S6 Dent et al., 199130 (N/A) UK (high); sec- ondary (>1 hospitals) (ded.: manpower IS) To ﬁnd out how dis- trict managements had prepared for and were respond- ing to implementa- tion of 3 corporate computer systems Qual. 5.5 Implementation Facilitators: organiza- tion, and project Barriers: organization Dent, 199131 (social science) Approaches to: proj- ect, and technology To examine the devel- opment of comput- ing and IT strategies within NHS England and Wales S7 Engbersen, 201032 (N/A) Netherlands (high); second- ary (hospital) (gen.: Intranet) To advance under- standing of the spe- cial features of e-HRM implemen- tation and provide insight into the in- ﬂuences e-HRM has on the HRM department and the organization with its HR activities Qual. 6.5 Implementation Recommendations: individual, organi- zation, task, and project Use Barriers: individual, project, task, and organization Outcomes > generic: no change to opera- tional, and strategic S8 Escobar-Perez and Escobar-Rodri- guez, 201033 (social science) Spain (high); secondary (hospital) (gen.: ERP) To analyze the pro- cess of implementa- tion of ERP systems in hospitals as an organization with divided and hetero- geneous functional areas, and to iden- tify the principal technological ob- jectives that were Qual. O’Brien-Pallas and Cockerill, 199029 (health) S7 Engbersen, 201032 (N/A) Publication characteristics 6 Implementation Facilitators: individ- ual, technology, and organization Bondarouk and Sikkel, 200321 (N/A) To apply a theory of a group learning to highlight relevant aspects of imple- mentation of groupware Barriers: organiza- tion, and individual Bondarouk and Sikkel, 200422 (N/A) To look closer at groupware imple- mentation from a learning-oriented approach Bondarouk, 200423(social science/ICT) To describe a project concerning the im- plementation of a personnel manage- ment system Use Facilitators: individ- ual, technology, and organization Bondarouk and Sikkel, 200524 (social science) To validate 5 pro- cesses of adoption of IT through group learning, and to get insights on which of the group processes are most inﬂuential in the system imple- mentation Bondarouk and Ruel, 200825 (N/A) To explore the rela- tionship between the organizational climate for innova- tion and ICT imple- mentation success Bondarouk and Ruel, 200826 (social science) To describe an HRM system that can lead to IT imple- mentation success Barriers: organiza- tion, and individual S4 IntraHealth Int., Inc.,d 200927 (N/A) Nine African countries (low or lower-mid- dle); NHS (ded.: open- source HRIS) To present an over- view of the results achieved by the Capacity project Report (Qual.) 5.5 Use Facilitators: project Realized beneﬁtsc: strategic, and inter- est from other countries (continued) Table 1. Characteristics of the included studies Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 637 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported S5 Cockerill and O’Brien-Pallas, 199028 (health) Canada (high); secondary (>1 hospitals) (gen.: nursing work- load measure- ment systems) To develop a proﬁle of use of nursing workload measure- ment systems in Canadian hospitals, assess user satisfac- tion, and identify challenges/per- ceived problems and research issues related to these systems Quant. 6 Implementation Barriers: organization Generic: project, and individual O’Brien-Pallas and Cockerill, 199029 (health) To explore senior nurse executives’ needs and expecta- tions for nursing workload systems Use Realized beneﬁtsc: strategic Satisfaction: familiar- ity with the system, its functions or use of them, and user satisfaction varied between roles; sys- tem needs to reﬂect true workload for users to be satisﬁed Approaches to: tech- nology, and individual S6 Dent et al., 199130 (N/A) UK (high); sec- ondary (>1 hospitals) (ded.: manpower IS) To ﬁnd out how dis- trict managements had prepared for and were respond- ing to implementa- tion of 3 corporate computer systems Qual. S6 Dent et al., 199130 (N/A) UK (high); sec- ondary (>1 hospitals) (ded.: manpower IS) Dent, 199131 (social science) S8 Escobar-Perez and Escobar-Rodri- guez, 201033 (social science) Spain (high); secondary (hospital) (gen.: ERP) Publication characteristics 5.5 Development Expected beneﬁtsc: strategic Generic: organization, tech- nology, and indi- vidual Implementation Generic: individual Approaches to: indi- vidual, inter-organ- ization, and project Use Barriers: project, and individual (continued) S6 Dent et al., 199130 (N/A) UK (high); sec- ondary (>1 hospitals) (ded.: manpower IS) Dent, 199131 (social science) S6 Dent et al., 199130 (N/A) UK (high); sec- ondary (>1 hospitals) (ded.: manpower IS) To ﬁnd out how dis- trict managements had prepared for and were respond- ing to implementa- tion of 3 corporate computer systems Qual. 5.5 Dent, 199131 (social science) To examine the devel- opment of comput- ing and IT strategies within NHS England and Wales S7 Engbersen, 201032 (N/A) Netherlands (high); second- ary (hospital) (gen.: Intranet) To advance under- standing of the spe- cial features of e-HRM implemen- tation and provide insight into the in- ﬂuences e-HRM has on the HRM department and the organization with its HR activities Qual. 6.5 S8 Escobar-Perez and Escobar-Rodri- guez, 201033 (social science) Spain (high); secondary (hospital) (gen.: ERP) To analyze the pro- cess of implementa- tion of ERP systems in hospitals as an organization with divided and hetero- geneous functional areas, and to iden- tify the principal technological ob- jectives that were Qual. 5.5 5.5 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 638 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported set in the process of implementation, which of those ob- jectives were achieved, and the deﬁciencies that subsequently be- came evident Escobar-Perez et al., 201034 (ICT) Satisfaction: varies between roles Approaches to: technology S9 Evers, 200935 (N/A) Netherlands (high); secondary (hos- pital) (ded.: HR portal) To assess the contri- bution of an HR portal toward HR processes Qual. 6.5 Development Expected beneﬁtsc: strategic, service, and operational Implementation Recommendations: project, task, and individual S16 Kumar et al., 201342 (health) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Recommendations: project, and task S10 Fahey and Bur- bridge, 200836 (health) USA (high); sec- ondary (>1 hospitals) (gen.: daily staff man- agement system) To present a case study of a failed attempt to apply the princi- ples of diffusion of innovation to a soft- ware program Qual. 4.5 Development Generic: technology Implementation Facilitators: organization Barriers: technology, and organization Use Facilitators: organization Barriers: organiza- tion, and task S11 Fehse, 200237 (N/A) Netherlands (high); second- ary (hospital) (ded.: personnel IS) To explore to what extent and how or- ganizational poli- tics explain IS implementation outcomes Qual. 6.5 Development Expected beneﬁtsc: strategic Implementation Facilitators: individual Barriers: organiza- tion, project, and individual Generic: individual, and organization Approaches to: proj- ect, and technology Use Outcomes > generic: no change to opera- tional S12 Gurol et al., 201038 (N/A) Turkey (upper-middle); secondary (hospital) (ded.: e-HRM) To investigate several speciﬁc and critical points that will contribute to a bet- ter understanding of e-HRM and pro- vide a model for implementation of e-HRM Qual. 4.5 Use Realized beneﬁtsc: op- erational, strategic, and empowerment (continued) , gy Use Outcomes > generic: no change to opera- tional S12 Gurol et al., 201038 (N/A) Turkey (upper-middle); secondary (hospital) (ded.: e-HRM) To investigate several speciﬁc and critical points that will contribute to a bet- ter understanding of e-HRM and pro- vide a model for implementation of e-HRM Qual. 4.5 Use Realized beneﬁtsc: op- erational, strategic, and empowerment (continued) Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 639 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported S13 Hawker et al., 199639 (health) Canada (high); secondary (hos- pital) (gen.: workload mea- surement system) To describe the devel- opment and appli- cation of a computerized workload measure- ment tool for use in hospital nursing education departments Qual. 2.5 Use Realized beneﬁtsc: ser- vice, and strategic S14 Helfert, 200940 (social science) Ireland (high); NHS (ded.: per- sonnel payroll attendance and recruitment system) To outline a frame- work for analyzing health care process management projects Qual. 16 Kumar et al., 201342 (health) Pakistan (lower- middle); NHS (NS: HRIS) Pakistan (lower- middle); NHS (NS: HRIS) S20 Pierantoni and Vianna, 200345 (health/social science) Brazil (upper-mid- dle); Depart- ments of Health (NS: HRIMS) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 7 Development Expected beneﬁtsc: operational, ser- vice, strategic, stan- dardization, and empowerment Implementation Facilitators: individ- ual, and project Generic: technology Use Realized beneﬁtsc: op Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported examples of the im- pacts they have on patients, caregivers, and the organiza- tion, and lessons learned Approaches to: technology S19 Parry and Tyson, 201112 (social science) UK (high); sec- ondary (>1 hospitals) (ded.: e-HRM) To examine the goals stated by organiza- tions for introduc- tion of e-HRM, whether they were actually achieved, and the factors af- fecting this Qual. S21 PWC, 201046 (N/A) Queensland, Australia (high); NHS (ded.: payroll system) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 5.5 Implementation Barriers: individual, project, task, inter- organization, organization, and technology Approaches to: inter- organization and project S15 Kazmi and Naara- noja, 201441 (social science) Pakistan (lower- middle); sec- ondary (hospi- tal) (ded.: HRIS) To propose an evalua- tion of how, in a small-business sce- nario, bits and pieces of knowl- edge can be seen scattered at differ- ent work locations and how manage- ment can strategi- cally arrange and manage a viable data resource in the form of existing knowledge base to be retrieved as and when required Quant. 4 Use Satisfaction: majority of users satisﬁed with information system provides S16 Kumar et al., 201342 (health) Pakistan (lower- middle); NHS (NS: HRIS) To document how HR information is currently being col- lected, managed, and reported; to identify the gaps re- lated to HRH in- formation that need to be urgently addressed; and to suggest the tools and processes for managing HR data Quant. 6.5 Development Expected beneﬁtsc: operational, ser- vice, and strategic S17 Lin et al., 201043 (ICT/health) Taiwan (high); secondary (hos- pital) (gen.: nursing assis- tant manage- ment system) To compare the re- sults of manual op- eration and system intervention in as- signing work to nursing assistants, in order to evaluate the system’s perfor- mance Mixed method 4.5 Use Realized beneﬁtsc: operational, and patient care Satisfaction: different categories of users are satisﬁed with the system S18 Memel et al., 200144 (health) USA (high); sec- ondary (>1 hospitals) (gen.: Intranet) To discuss speciﬁc components of the information man- agement and IT infrastructure, Qual. 2 Development Expected beneﬁtsc: operational Use Realized beneﬁtsc: operational, and service Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 639 Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 6.5 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 640 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported examples of the im- pacts they have on patients, caregivers, and the organiza- tion, and lessons learned Approaches to: technology S19 Parry and Tyson, 201112 (social science) UK (high); sec- ondary (>1 hospitals) (ded.: e-HRM) To examine the goals stated by organiza- tions for introduc- tion of e-HRM, whether they were actually achieved, and the factors af- fecting this Qual. KPMG, 201047 (N/A) KPMG, 201048 (N/A) Eden and Sedera, 201454 (N/A) Auditor-General of Queensland, 201051 (N/A) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 7 Development Expected beneﬁtsc: operational, ser- vice, strategic, stan- dardization, and empowerment Implementation Facilitators: individ- ual, and project Generic: technology Use Realized beneﬁtsc: op- erational, service, strategic, and stan- dardization S20 Pierantoni and Vianna, 200345 (health/social science) Brazil (upper-mid- dle); Depart- ments of Health (NS: HRIMS) To evaluate imple- mentation of HRIS in selected health departments and present the imple- mentation evalua- tion methodology; and to identify the limits and possibili- ties for using the system as an HR planning and man- agement tool in lo- cal health systems Mixed method 5.5 Development Expected beneﬁtsc: strategic Implementation Facilitators: environ- ment, and organization Barriers: environ- ment, organization, technology, and individual Use Facilitators: environ- ment and organization Approaches to: task S21 PWC, 201046 (N/A) Queensland, Australia (high); NHS (ded.: payroll system) To review the organi- zation of corporate services under the shared services model and deter- mine the most ap- propriate arrange- ments for the future; to investi- gate and make rec- ommendations on the appropriate governance model for shared services going forward; and to provide recom- mendations for the future rollout of the Corporate Solu- tions Program and the most effective way to deliver it Report (Qual.) 5.5 Development Expected beneﬁtsc: strategic and standardization KPMG, 201047 To summarize the Facilitators: individ- dardization S20 Pierantoni and Vianna, 200345 (health/social science) Brazil (upper-mid- dle); Depart- ments of Health (NS: HRIMS) To evaluate imple- mentation of HRIS in selected health departments and present the imple- mentation evalua- tion methodology; and to identify the limits and possibili- ties for using the system as an HR planning and man- agement tool in lo- cal health systems Mixed method 5.5 Development Expected beneﬁtsc: strategic Implementation Facilitators: environ ment, and organization Barriers: environ- ment, organizatio technology, and individual Use Facilitators: environ ment and organization Approaches to: task S21 PWC, 201046 (N/A) Queensland, Australia (high); NHS (ded.: payroll system) To review the organi- zation of corporate services under the shared services model and deter- mine the most ap- propriate arrange- ments for the future; to investi- gate and make rec- ommendations on the appropriate governance model for shared services going forward; and to provide recom- mendations for the future rollout of the Corporate Solu- tions Program and the most effective way to deliver it Report (Qual.) 5.5 Development Expected beneﬁtsc: strategic and standardization KPMG, 201047 (N/A) To summarize the work undertaken to date on the re- view of the Queens- land Health (QH) payroll implemen- tation project Facilitators: individ ual, and project KPMG, 201048 (N/A) Recommendations: project, technolo environment, task organization, and individual (continue S20 Pierantoni and Vianna, 200345 (health/social science) Brazil (upper-mid- dle); Depart- ments of Health (NS: HRIMS) To evaluate imple- mentation of HRIS in selected health departments and present the imple- mentation evalua- tion methodology; and to identify the limits and possibili- ties for using the system as an HR planning and man- agement tool in lo- cal health systems Mixed method 5.5 S21 PWC, 201046 (N/A) Queensland, Australia (high); NHS (ded.: payroll system) To review the organi- zation of corporate services under the shared services model and deter- mine the most ap- propriate arrange- ments for the future; to investi- gate and make rec- ommendations on the appropriate governance model for shared services going forward; and to provide recom- mendations for the future rollout of the Corporate Solu- tions Program and the most effective way to deliver it Report (Qual.) 5.5 KPMG, 201047 (N/A) To summarize the work undertaken to date on the re- view of the Queens- land Health (QH) payroll implemen- tation project KPMG, 201048 (N/A) KPMG, 201047 (N/A) KPMG, 201048 (N/A) (continued) Journal of the American Medical Informatics Association, 2017, Vol. Chesterman, 201352 (N/A) Silva and Rosem- man, 201253 (N/A) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported payroll implemen- tation project; and to develop a theo- retically and practi- cally derived system develop- ment life cycle model Outcomes > generic: resignation of Min- ister of Health, strikes, improved country ICT strat- egy, and gover- nance procedures Recommendations: inter-organization, # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported payroll implemen- tation project; and to develop a theo- retically and practi- cally derived system develop- ment life cycle model Outcomes > generic: resignation of Min- ister of Health, strikes, improved country ICT strat- egy, and gover- nance procedures Recommendations: inter-organization, organization, proj- ect, task, technol- ogy, and individual S22 Rauhala, 200855 (N/A) Finland (high); secondary mixed (gen.: pa- tient classiﬁca- tion system) To evaluate whether the patient classiﬁ- cation system was valid and feasible enough to be used as a measurement tool for HRM in nursing in the wards of somatic specialized health care Quant. 7.5 Use Approaches to: task Fagerstrom et al., 200056 (health) Fagerstrom et al., 2000 57 (health) Rauhala and Fager- strom, 200458 (health) Rauhala and Fager- strom, 200759 (health) Rauhala et al., 200760 (health) S23 F Fi l d (hi h) T ill h h Q 6 U R li d b ﬁ c Downloaded from https://academic.oup.com/j S22 Rauhala, 200855 (N/A) Finland (high); secondary mixed (gen.: pa- tient classiﬁca- tion system) Fagerstrom et al., 200056 (health) Fagerstrom et al., 2000 57 (health) Rauhala and Fager- strom, 200458 (health) Rauhala and Fager- strom, 200759 (health) Rauhala et al., 200760 (health) S23 Fagerstrom, 200961 (health) Finland (high); secondary (>1 hospitals) (gen.: patient classiﬁ- cation system) S24 Rainio and Ohin- maa, 200562 (health) Finland (high); secondary (hos- pital) (gen.: pa- tient classiﬁca- tion system) 2007 (health) S23 Fagerstrom, 200961 (health) Finland (high); secondary (>1 hospitals) (gen.: patient classiﬁ- cation system) To illustrate how the system can be used to facilitate evi- dence-based HRM Quant. S25 Riley et al., 200763 (health) S24 Rainio and Ohin- maa, 200562 (health) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported payroll implemen- tation project; and to develop a theo- retically and practi- cally derived system develop- ment life cycle model Outcomes > generic: resignation of Min- ister of Health, strikes, improved country ICT strat- egy, and gover- nance procedures Recommendations: inter-organization, organization, proj- ect, task, technol- ogy, and individual S22 Rauhala, 200855 (N/A) Finland (high); secondary mixed (gen.: pa- tient classiﬁca- tion system) To evaluate whether the patient classiﬁ- cation system was valid and feasible enough to be used as a measurement tool for HRM in nursing in the wards of somatic specialized health care Quant. 7.5 Use Approaches to: task Fagerstrom et al., 200056 (health) Fagerstrom et al., 2000 57 (health) Rauhala and Fager- strom, 200458 (health) Rauhala and Fager- strom, 200759 (health) Rauhala et al., 200760 (health) S23 Fagerstrom, 200961 (health) Finland (high); secondary (>1 hospitals) (gen.: patient classiﬁ- cation system) To illustrate how the system can be used to facilitate evi- dence-based HRM Quant. 6 Use Realized beneﬁtsc: strategic Approaches to: task S24 Rainio and Ohin- maa, 200562 (health) Finland (high); secondary (hos- pital) (gen.: pa- tient classiﬁca- tion system) To assess the feasibil- ity of the system in nursing staff man- agement, and whether it can be seen as the transfer- ring of nursing re- sources between wards according to the information re- i d f i Quant. 5.5 Use Approaches to: technology Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported payroll implemen- tation project; and to develop a theo- retically and practi- cally derived system develop- ment life cycle model Outcomes > generic: resignation of Min- ister of Health, strikes, improved country ICT strat- egy, and gover- nance procedures Recommendations: inter-organization, organization, proj- ect, task, technol- ogy, and individual S22 Rauhala, 200855 (N/A) Finland (high); secondary mixed (gen.: pa- tient classiﬁca- tion system) To evaluate whether the patient classiﬁ- cation system was valid and feasible enough to be used as a measurement tool for HRM in nursing in the wards of somatic specialized health care Quant. Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 24, No. 3 641 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported KPMG, 201249 (N/A) To review the current status, proposed so- lutions, strategies, programs of work, and governance frameworks in place for the QH payroll system Approaches to environment E&Y, 201050 (N/A) To conduct a review of QH payroll and rostering systems to establish their on- going suitability for QH, and to ascer- tain what potential options are avail- able to resolve the recently experi- enced payroll problems Implementation Facilitators: project, and individual Auditor-General of Queensland, 201051 (N/A) To evaluate the effec- tiveness of the Department of Pub- lic Works’s pro- gram and project management and QH processes in re- lation to the busi- ness readiness of and transition to new systems Barriers: environ- ment, inter-organi- zation, organiza- tion, project, technology, task, and individual Chesterman, 201352 (N/A) To present a full and careful inquiry into implementation of the QH payroll system Approaches to: proj- ect, inter-organiza- tion, and technology Silva and Rosem- man, 201253 (N/A) To propose an ap- proach to represent the dynamic rela- tions between so- cial and material entities where the latter are divided into technical and organizational entities Qual. Recommendations: inter-organization, project, task, and technology Eden and Sedera, 201454 (N/A) To illustrate the fac- tors that contrib- uted to QH’s disastrous imple- mentation project; and to understand the broader appli- cations of this proj- ect failure on state and national legis- lations as well as industry sectors Use Generic: organization, project, and technology Thi d S dh T i h i A h j Implementation Facilitators: project, and individual Silva and Rosem- man, 201253 (N/A) Thite and Sandhu, 20148 (social science/ICT) (continued) Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 642 Table 1. Finland (high); secondary mixed (gen.: pa- tient classiﬁca- tion system) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 7.5 Use Approaches to: task Fagerstrom et al., 200056 (health) Fagerstrom et al., 2000 57 (health) Rauhala and Fager- strom, 200458 (health) Rauhala and Fager- strom, 200759 (health) Rauhala et al., 200760 (health) S23 Fagerstrom, 200961 (health) Finland (high); secondary (>1 hospitals) (gen.: patient classiﬁ- cation system) To illustrate how the system can be used to facilitate evi- dence-based HRM Quant. 6 Use Realized beneﬁtsc: strategic Approaches to: task S24 Rainio and Ohin- maa, 200562 (health) Finland (high); secondary (hos- pital) (gen.: pa- tient classiﬁca- tion system) To assess the feasibil- ity of the system in nursing staff man- agement, and whether it can be seen as the transfer- ring of nursing re- sources between wards according to the information re- ceived from nursing care intensity classi- ﬁcation Quant. 5.5 Use Approaches to: technology S25 Riley et al., 200763 (health) Kenya (lower- middle); NHS (ded.: nursing workforce database) To describe the devel- opment, initial ﬁnd- ings, and implications of a national nursing workforce database system in Kenya Mixed method 5 Use Facilitators: environ- ment, and organization Realized beneﬁtsc: strategic Approaches to: technology Recommendations: technology S26 Riley et al., 201264 (health/ social science) Int.; NHS (NS: HRIS) To review and assess national practices in HRIS implemen- tation worldwide; identify the main f k Systematic review 6.5 Development Expected beneﬁtsc: strategic Use Approaches to: envi- ronment, organiza- tion, technology, d k Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Table 1. To describe the sys- tem development process Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 6 Use Realized beneﬁtsc: strategic Approaches to: task S24 Rainio and Ohin- maa, 200562 (health) Finland (high); secondary (hos- pital) (gen.: pa- tient classiﬁca- tion system) To assess the feasibil- ity of the system in nursing staff man- agement, and whether it can be seen as the transfer- ring of nursing re- sources between wards according to the information re- ceived from nursing care intensity classi- ﬁcation Quant. 5.5 Use Approaches to: technology S25 Riley et al., 200763 (health) Kenya (lower- middle); NHS (ded.: nursing workforce database) To describe the devel- opment, initial ﬁnd- ings, and implications of a national nursing workforce database system in Kenya Mixed method 5 Use Facilitators: environ- ment, and organization Realized beneﬁtsc: strategic Approaches to: technology Recommendations: technology S26 Riley et al., 201264 (health/ social science) Int.; NHS (NS: HRIS) To review and assess national practices in HRIS implemen- tation worldwide; identify the main areas of weakness in HRIS implemen- tation, with Systematic review 6.5 Development Expected beneﬁtsc: strategic Use Approaches to: envi- ronment, organiza- tion, technology, and task (continued) m/jamia/article/24/3/633/2907914 by guest on 24 October 2024 (continued) Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 643 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported attention to coun- tries facing acute health workforce shortages; and draw upon docu- mented best prac- tices to offer recommendations to decision and pol- icy makers on how to improve the sci- ence and applica- tion of HRIS S27 Rodger et al., 199865 (N/A) USA (high); mixed (ded.: HRIS) To describe the efforts of the HR depart- ment to redesign its HRIS to better meet enterprise- wide goals of cost effectiveness and ef- ﬁciency Mixed method 4.5 Use Satisfaction: users sa isﬁed with distribu tion and collection of HRIS reports and their conﬁden tiality, but not wit complicated proce dures and forms fo HRIS Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 64 Table 1. Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported attention to coun- tries facing acute health workforce shortages; and draw upon docu- mented best prac- tices to offer recommendations to decision and pol- icy makers on how to improve the sci- ence and applica- tion of HRIS S27 Rodger et al., 199865 (N/A) USA (high); mixed (ded.: HRIS) To describe the efforts of the HR depart- ment to redesign its HRIS to better meet enterprise- wide goals of cost effectiveness and ef- ﬁciency Mixed method 4.5 Use Satisfaction: users sat- isﬁed with distribu- tion and collection of HRIS reports and their conﬁden- tiality, but not with complicated proce- dures and forms for HRIS Rodger et al., 199866 (social science/ICT) Approaches to: tech- nology, and task Recommendations: project, task, and individual S28 Ruland, 200167 (ICT/health) Norway (high); secondary (hos- pital) (gen.: de- cision support system) To describe the sys- tem development process Mixed method 5.5 Development Expected beneﬁtsc: strategic, empower- ment, and operational Facilitators: project, and individual Ruland and Ravn, 200168 (ICT/ health) To evaluate the sys- tem’s effect on nursing costs, qual- ity of management information, user satisfaction, and ease of use, and its usefulness as deci- sion support for im- proved ﬁnancial management and decision-making Implementation Facilitators: project, and individual Use Facilitators: organiza- tion, individual, project, and technology Realized beneﬁtsc: operational, and strategic Satisfaction: users satis- ﬁed with system, and information it provides S29 Sammon and Adam, 201069 (social science/ ICT) Ireland (high); NHS (gen.: ERP) To investigate the managers’ level of understanding of ERP project imple- mentation and the preparations that h ld b d t Qual. 6.5 Development Expected beneﬁtsc: strategic Implementation Barriers: project Approaches to: orga- nization, and project draw upon docu mented best prac- tices to offer recommendations to decision and pol- icy makers on how to improve the sci- ence and applica- tion of HRIS S27 Rodger et al., 199865 (N/A) USA (high); mixed (ded.: HRIS) To describe the efforts of the HR depart- ment to redesign its HRIS to better meet enterprise- wide goals of cost effectiveness and ef- ﬁciency Mixed method 4.5 Use S32 Smith et al., 197972 (ICT) S33 Spaulding, 201273 (N/A) USA, Australia, Canada, UK (high); second- ary (>1 hospi- tals) (NS: HRIS) S31 Shukla et al.,d 201471 (N/A) (continued) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Ruland and Ravn, 200168 (ICT/ health) To evaluate the sys- tem’s effect on nursing costs, qual- ity of management information, user satisfaction, and ease of use, and its usefulness as deci- sion support for im- proved ﬁnancial management and decision-making Implementation Facilitators: project, and individual Use Facilitators: organiza- tion, individual, project, and technology Realized beneﬁtsc: operational, and strategic Satisfaction: users satis- ﬁed with system, and information it provides S29 Sammon and Adam, 201069 (social science/ ICT) Ireland (high); NHS (gen.: ERP) To investigate the managers’ level of understanding of ERP project imple- mentation and the preparations that should be made to increase the likeli- hood of success Qual. 6.5 Development Expected beneﬁtsc: strategic Implementation Barriers: project Approaches to: orga- nization, and project S30 Schenck-Yglesias, 200470 (N/A) Malawi (low); NHS (gen.: HRIS) To review the avail- ability of staff de- ployment and training data from routine IS in Ma- lawi and inform the Report (Qual.) 5.5 Development Approaches to: inter- organization, and technology Use Recommendations: task 33/2907914 by guest on 24 October 2024 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 644 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported Ministry of Health and Population of deﬁciencies that would need to be addressed to better inform the develop- ment and ongoing monitoring and de- ployment of train- ing policies and plans S31 Shukla et al.,d 201471 (N/A) India (lower-mid- dle); NHS (ded.: open- source HRIS) To review HRIS across all 28 states and 7 union territo- ries of India to as- sess their purpose, scope, coverage, software technol- ogy, usability, and sustainability Report (Qual.) 5.5 Development Expected beneﬁtsc: operational, and compliance Facilitators: project Use Approaches to: inter- organization, proj- ect, task, and individual S32 Smith et al., 197972 (ICT) USA (high); sec- ondary (hospi- tal) (ded.: computer-based scheduling system) To discuss 3 years’ ex- perience in com- puter-assisted scheduling of nurs- ing personnel Qual. 2.5 Development Expected beneﬁtsc: strategic Implementation Facilitators: individual, and project Approaches to: tech- nology, and indi- vidual U R li d b ﬁ c Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 project S33 Spaulding, 201273 (N/A) USA, Australia, Canada, UK (high); second- ary (>1 hospi- tals) (NS: HRIS) To review existing conceptualizations of HRIS and set forth propositions deﬁning the impact such systems have on individual and organizational per- formance; to test several of those propositions by evaluating hospital HRIS use and hos- pital-acquired con- dition outcomes; and to conduct cost effectiveness analy- sis examining the Quant. 6.5 Use Realized beneﬁtsc: patient care S33 Spaulding, 201273 (N/A) USA, Australia, Canada, UK (high); second- ary (>1 hospi- tals) (NS: HRIS) 6.5 (continued) Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 645 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported compositions of rapid response teams S34 Spero et al., 201174 (health/ social science) Uganda (low); professional or- ganization (ded.: open- source HRIS) To describe Uganda’s transition from a paper ﬁling system to an electronic HRIS; and to de- scribe how HRIS data can be used to address workforce planning questions via an initial analy- sis of the Uganda Nurses and Mid- wives Council training, licensure, and registration records Mixed method 5 Use Realized beneﬁtsc: op- erational, and pa- tient care Approaches to: tech- nology Recommendations: technology S35 Stamouli and Mantas, 200175 (ICT/health) Greece (high); sec- ondary (>1 hospitals) (gen.: IS for the nurs- ing service) To describe the devel- opment and evalua- tion of an IS for the Nursing Service Administration Quant. 4.5 Development Expected beneﬁtsc: strategic, and operational Barriers: individual, and organization Use Facilitators: technol- ogy, and project Realized beneﬁtsc: op- erational, and pa- tient care Approaches to: tech- nology Recommendations: technology 4.5 provides S36 Thouin and Bard- han, 200976 (N/ A) USA (high); sec- ondary (>1 hospitals) (ded.: HRM systems) To study the effect of IT usage on quality improvements in patient outcomes and examine the ef- fect of clinical and administrative IT adoption and usage on ﬁnancial perfor- mance Quant. S36 Thouin and Bard- han, 200976 (N/ A) USA (high); sec- ondary (>1 hospitals) (ded.: HRM systems) Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 6 Use Realized beneﬁtsc: patient care, and operational S37 Valentine et al., 200877 (health) USA (high); sec- ondary (>1 hospitals) (ded.: automated open-shift man- agement program) To discuss how a suc- cessful nursing ini- tiative to apply automation to open-shift schedul- ing and fulﬁllment across a 3-hospital system had a broad enterprise-wide impact Mixed method 2 Implementation Facilitators: individual Approaches to: task Use Realized beneﬁtsc: op- erational, empow- erment, and strategic Approaches to: technology S38 Waring, 200078 (N/A) UK (high); sec- ondary (hospi- tal) (ded.: payroll-person- nel system) To critically investi- gate potential emancipatory prin- ciples for system analysis, design, and development synthesized from the wider literature, then translate these principles into Qual. 7 Development Expected beneﬁtsc: service, compli- ance, and factors beyond organiza- tion’s control Facilitators: project Barriers: organiza- tion, task, and inter-organization ( ti d) S36 Thouin and Bard- han, 200976 (N/ A) USA (high); sec- ondary (>1 hospitals) (ded.: HRM systems) To study the effect of IT usage on quality improvements in patient outcomes and examine the ef- fect of clinical and administrative IT adoption and usage on ﬁnancial perfor- mance Quant. 6 S37 Valentine et al., 200877 (health) USA (high); sec- ondary (>1 hospitals) (ded.: automated open-shift man- agement program) To discuss how a suc- cessful nursing ini- tiative to apply automation to open-shift schedul- ing and fulﬁllment across a 3-hospital system had a broad enterprise-wide impact Mixed method 2 S38 Waring, 200078 (N/A) UK (high); sec- ondary (hospi- tal) (ded.: payroll-person- nel system) To critically investi- gate potential emancipatory prin- ciples for system analysis, design, and development synthesized from the wider literature, then translate these principles into Qual. 7 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 646 Table 1. Continued # Authors, year (discipline) Country (incomea); HO (IS) Research goals Study design Quality score (0–10) Innovation stage Outcomes reported practice within the context of IS imple- mentation Approaches to: inter- organization, and project Waring, 200479 (social science) Implementation Barriers: organiza- tion, and inter- organization Approaches to: proj- ect, and technology S39 Warner et al., 199180 (health) USA (high); sec- ondary (>1 hospitals) (ded.: nurse schedul- ing system) To describe what nursing administra- tion is looking for in an automated scheduling system; and to discuss is- sues of implementa- tion from the viewpoint of nurs- ing administration, including realizable beneﬁts Qual. Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 2 Use Realized beneﬁtsc: strategic, and operational S40 Waters et al., 201381 (health) Kenya (lower- middle); NHS (ded.: open- source HRIS) To document the im- pact of system data on HR policy, planning, and management Mixed method 5.5 Use Realized beneﬁtsc: op- erational, strategic, and compliance S41 West et al., 200482 (health) UK (high); pri- mary (gen.: IS to collect work- load data) To describe the imple- mentation of a computerized IS to collect workload data and discuss feedback from staff evaluation of use and value Qual. 5.5 Use Barriers: organiza- tion, task, and individual S42 WHO, 199083 (N/A) Int.; NHS(NS: HRH IS) To share expertise and experiences in the areas of re- search and health personnel IS and identify strategies for better use of in- formation and re- search in decision- making for HRH development Report (Qual.) 5.5 Development Expected beneﬁtsc: strategic Facilitators: environ- mental Approaches to: envi- ronment and inter- organization Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 Octob aClassiﬁed according to the World Bank’s Country and Lending Groups.84 bPrimary and secondary. cBeneﬁts: operational¼ operational efﬁciency; service¼ service de- livery; strategic¼ strategic orientation; empowerment¼ empowerment of managers and employees; compliance¼ statutory compliance. Abbreviations: HO ¼ health organization; IT ¼ information technology; Qual. ¼ qualitative; Quant. ¼ quantitative; NHS ¼ National Health System; Int. ¼ in- ternational; HRH ¼ Human Resources for Health; HRIMS ¼ human resource information and management system; gen. ¼ generic IS; ded. ¼ dedicated IS; NS ¼ not speciﬁed; N/A ¼ not applicable. aClassiﬁed according to the World Bank’s Country and Lending Groups.84 bPrimary and secondary. cBeneﬁts: operational¼ operational efﬁciency; service¼ service de- livery; strategic¼ strategic orientation; empowerment¼ empowerment of managers and employees; compliance¼ statutory compliance. Abbreviations: HO ¼ health organization; IT ¼ information technology; Qual. ¼ qualitative; Quant. ¼ quantitative; NHS ¼ National Health System; Int. ¼ in- ternational; HRH ¼ Human Resources for Health; HRIMS ¼ human resource information and management system; gen. ¼ generic IS; ded. ¼ dedicated IS; NS ¼ not speciﬁed; N/A ¼ not applicable. HRIS users gGeneric analyses of principles, beneﬁts, requirements, implementation methods of HRIS in health organizations, or pure market research. Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 647 Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Figure 1. PRISMA ﬂow diagram. aDatabase has limitations on the number of keywords, therefore the search had to be run several times to ensure that all search query keywords were included (please see9). bBook reviews, front and back covers, copyright notice, title pages, collection of conference proceedings’ descrip- tions, tables of contents, press releases, announcements, descriptions of issues, advertisements, bulletins, questionnaires, notices of retraction, chair’s mes- sages, keynotes, plenary talks, welcome messages, news published in journals and magazines that have “news” in their title and news published by companies that do not provide any analytical or research materials, presentation description, very brief cases and analytical materials published in newspaper and maga- zines, company proﬁles, advertising/marketing articles. cArticles not related to HRIS in health organizations, research on HR practices in health organizations that do not defer to use of ICT in relation to HR activities. dArticles where no abstract was available or where title and abstract did not give sufﬁcient detail to judge eli- gibility, articles on HRIS that do not specify the industry/sector in which they were implemented, articles on generic ERP/HIS that do not specify the module/func- tionality and/or industry/sector in which they were implemented. ePotentially relevant articles referring to HRIS in health organizations. fArticles focused on computer science models (eg, software speciﬁcation) or management science models (eg, creating algorithms to enable stafﬁng and scheduling in health organi- zations). gGeneric analyses of principles, beneﬁts, requirements, implementation methods of HRIS in health organizations, or pure market research. strategic orientation – being able to use information about HR needs and performance for evidence-based decision-making, to inform HRM policy and planning, or as a means of migrating to a central- ized, enterprise-wide HR shared services approach. This was fol- lowed by operational efficiency – reduction and control of costs, automation or augmentation of manual processes, time saving, and reduced bureaucracy. Improvements in HR service delivery were also expected, such as identifying current levels of provision, resolv- ing issues with external service providers, and/or increasing the qual- ity of information in HRIS. Innovation stages Innovation stage was classified based on our interpretation of a study’s aims and findings rather than any authors’ explicit statements, which often bore little resemblance to the stages described in the study. Half of the studies (n ¼ 21) focused exclusively on a single inno- vation stage, mostly on HRIS use (n ¼ 17), with 2 studies focusing on either development or implementation. The other half encom- passed several innovation stages, 9 covering development, imple- mentation, and use, 5 development and use, 5 implementation and use, and 2 development and implementation. Table 3 indicates the innovation stages covered and shows that the studies focused mainly on (1) approaches to HRIS use, (2) factors of influence during HRIS implementation, (3) HRIS outcomes, such as realized benefits, and (4) drivers for HRIS. The most commonly realized benefits of HRIS implementation related to strategic orientation and operational efficiency improve- ments, followed by empowerment of managers and employees, im- provements in service delivery, standardization, and compliance with regulatory requirements. Another was improvement in patient HRIS users Other expectations driving implementation included standardization of systems, processes, or data; empowerment of managers and/or employees; compliance with statutory requirements for data on the health workforce; and helping to manage macro organizational changes, such as a planned hospital merger. We did not find evidence that health organizations adopted HRIS to improve their organizational image, as suggested in Parry and Tyson’s framework. nurse educators (all with n ¼ 1). Seven studies did not specify any HRIS user categories. strategic orientation – being able to use information about HR needs and performance for evidence-based decision-making, to inform HRM policy and planning, or as a means of migrating to a central- ized, enterprise-wide HR shared services approach. This was fol- lowed by operational efficiency – reduction and control of costs, automation or augmentation of manual processes, time saving, and reduced bureaucracy. Improvements in HR service delivery were also expected, such as identifying current levels of provision, resolv- ing issues with external service providers, and/or increasing the qual- ity of information in HRIS. Other expectations driving implementation included standardization of systems, processes, or data; empowerment of managers and/or employees; compliance with statutory requirements for data on the health workforce; and helping to manage macro organizational changes, such as a planned hospital merger. We did not find evidence that health organizations adopted HRIS to improve their organizational image, as suggested in Parry and Tyson’s framework. nurse educators (all with n ¼ 1). Seven studies did not specify any HRIS user categories. HRIS users ERP (n ¼ 3), patient classification system (n ¼ 3), or Intranet (n ¼ 2). Dedicated systems were described as HRIS (n ¼ 7), payroll/ salary system (n ¼ 4), or electronic-HRM (n ¼ 2). HRIS (n ¼ 3) was used most frequently in studies not specifying whether the system was dedicated or generic. HRIS are designed for a variety of users. The most commonly men- tioned user groups were health sector leaders/decision-makers (n ¼ 6), hospital management, HR department/HR professionals, nurses, nurse managers/administrators, and employees (all with n ¼ 5). Less commonly mentioned were health organizations, gov- ernment//professional authorities, line managers (all with n ¼ 3), staffing clerk/coordinator (n ¼ 2), clinicians, donor agencies, inter- nal temporary employment agencies, rural primary care teams, and HRIS support various HRM practices in health organiza- tions. However, as shown in Figure 3, most qualifying studies focus on operational HRM practices (eg, HR administration or scheduling). Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 647 Figure 1. PRISMA ﬂow diagram. aDatabase has limitations on the number of keywords, therefore the search had to be run several times to ensure that all search query keywords were included (please see9). bBook reviews, front and back covers, copyright notice, title pages, collection of conference proceedings’ descrip- tions, tables of contents, press releases, announcements, descriptions of issues, advertisements, bulletins, questionnaires, notices of retraction, chair’s mes- sages, keynotes, plenary talks, welcome messages, news published in journals and magazines that have “news” in their title and news published by companies that do not provide any analytical or research materials, presentation description, very brief cases and analytical materials published in newspaper and maga- zines, company proﬁles, advertising/marketing articles. cArticles not related to HRIS in health organizations, research on HR practices in health organizations that do not defer to use of ICT in relation to HR activities. dArticles where no abstract was available or where title and abstract did not give sufﬁcient detail to judge eli- gibility, articles on HRIS that do not specify the industry/sector in which they were implemented, articles on generic ERP/HIS that do not specify the module/func- tionality and/or industry/sector in which they were implemented. ePotentially relevant articles referring to HRIS in health organizations. fArticles focused on computer science models (eg, software speciﬁcation) or management science models (eg, creating algorithms to enable stafﬁng and scheduling in health organi- zations). Drivers and realized benefits The majority of studies described HRIS implementation as being driven by expected benefits or goals. The most common related to Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 648 Figure 2. Types of publications on HRIS by year. Figure 2. Types of publications on HRIS by year. Table 2. Theoretical frameworks referred to in qualifying studies Disciplinary perspective Framework Study HR and HR related Concept of experiential learning S3 Central principles of HRM S22 Personnel as resource in HRM theory S23 HRIS impact through drawing from motivation in organizational behavior and theory of work performance S33 Innovation and change Diffusion of innovations S10 Theoretical models of organizational change S11 IS and IS related InnoDiff model based on model for IS success S1 Framework of impacts of technology implementation S8 Technology acceptance model S9 Corporate information factory S18 System development life cycle S21 Concept of mindfulness to develop concept of preparedness in ERP implementation S29 Process-centric role of ICT in terms of its impact on business value S36 Speciﬁc combinations of HR and IS concepts Conceptual framework developed by WHO Study Group linking 3 components: decision-making in the de- velopment of HR for health, research, and IS S42 The role of HRM in ICT implementation S3 Framework for goals for ICT use in HR S19 Framework for ICT effects, enriched with the concept of organizational object and integrating perspective on emergence and enacted practices S21 Other broad management /business Structuration theory S3; S7 Management strategies S6 Game-theoretic model S6 Evaluation framework for business process projects S14 Knowledge-sharing concept S15 Evidence-based health care S23 Emancipatory principles and principles of critical social theory S38 Does not specify S2, S4, S5, S12, S13, S16, S17, S20, S24, S25, S26, S27, S28, S30, S31, S32, S34, S35, S37, S39, S40, S41 Table 2. Theoretical frameworks referred to in qualifying studies Only 5 studies reported whether projects had achieved their ex- pected benefits, and even fewer described failure of the HRIS to in- fluence specific goals, notably operational efficiency (n ¼ 3), strategic orientation (n ¼ 1), and service delivery (n ¼ 1) (see Table 1 for details). care by facilitating minimum standards of nursing care.43 One study reported that hospitals using HRIS had lower rates of vascular cath- eter urinary tract infections.73 Generation of interest from other countries27 and improved ICT infrastructure18 were also reported as beneficial outcomes. care by facilitating minimum standards of nursing care.43 One study reported that hospitals using HRIS had lower rates of vascular cath- eter urinary tract infections.73 Generation of interest from other countries27 and improved ICT infrastructure18 were also reported as beneficial outcomes. Journal of the American Medical Informatics Association, 2017, Vol. 24, No. Summary y The intention of this review was to capture, synthesize, and interpret existing evidence on HRIS in health care organizations. We discov- ered that research in this area ranges across disciplines and varies widely in terms of its objectives, methods, theoretical orientation, quality, and language. As expected, the evidence base is sparse com- pared with clinical information systems research. Most studies fo- cus, somewhat uncritically, on the use and realized benefits of HRIS in practice, rather than sociocontextual or technological factors influencing their development, implementation success, or impacts on strategic decision-making or cost-effectiveness. Most research comes from higher-income countries and examines small-scale sys- tems in individual hospital settings. Nevertheless, several higher- quality studies were found, including one national program evalua- tion, and we were able to adapt and apply existing theoretical frameworks to help organize and interpret the evidence, suggesting Figure 2. Types of publications on HRIS by year. 3 649 Figure 3. HRM practices examined in the included studies. aOut of scope of this review (please see9). bNot mentioned in any of the qualifying studies. Solid line ovals: existing Foster’s e-HRM landscape categories. Dashed line ovals, text in italic: categories added to Foster’s e-HRM landscape. Downloaded from https://academic.oup.com/jamia/article/24/3/633/2907914 by guest on 24 October 2024 Figure 3. HRM practices examined in the included studies. aOut of scope of this review (please see9). bNot mentioned in any of th ovals: existing Foster’s e-HRM landscape categories. Dashed line ovals, text in italic: categories added to Foster’s e-HRM landscape xamined in the included studies. aOut of scope of this review (please see9). bNot mentioned in any of the qualifying studies. Solid HRM landscape categories. Dashed line ovals, text in italic: categories added to Foster’s e-HRM landscape. Figure 3. HRM practices examined in the included studies. aOut of scope of this review (please see9). bNot mentioned in any of the qualifying studies. Solid line ovals: existing Foster’s e-HRM landscape categories. Dashed line ovals, text in italic: categories added to Foster’s e-HRM landscape. Only one study (S9) reported specific adverse effects of HRIS im- plementation within the organization, including negative percep- tions of HR roles and increases in supervisors’ workload associated with changing to new HRIS processes. More general adverse effects were mentioned in another study (S21), which described a region- wide HRIS project as a “catastrophic failure”52 with multiple nega- tive consequences for contractors and government, including staff strikes and the Minister of Health’s resignation. significant barriers. Some studies described technological barriers, including breadth of system functionality, degree of local configura- tion, and interoperability. Barriers associated with existing HR pro- cesses were also mentioned, and several studies recommended simplifying such processes prior to HRIS introduction, although none reported any evidence of this having facilitated a project’s suc- cess. Macro-environmental influences, such as political reforms and inter-organizational relationships, were considered very little. User satisfaction Three studies reported users being satisfied with the system itself, 1 with its functions, and 4 with the information it provides, although 1 noted dissatisfaction with new HRIS procedures and forms. Two described HRIS satisfaction as being dependent upon ease of use, 2 upon types of users, and 1 each on users’ familiarity with the system, time required to judge systems, whether systems reflect true work- load, and time in use, satisfaction increasing with evolving user ca- pabilities and organizational adaptation. Factors shaping HRIS development, implementation, and use Facilitators and barriers were reported across innovation stages (see Table 4). Success was influenced primarily by project-related fac- tors, including governance structure, approaches to project manage- ment, and quality of execution, and by individual factors such as stakeholders’ political behaviors and user involvement. Organiza- tional factors, including organizational size, diversity, culture, de- gree of centralization, and availability of resources, were the most Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 650 Table 3. Innovation stages examined in the included studies Category Development Implementation Use Expected beneﬁts S8, S9, S11, S16, S18, S19, S20, S21, S26, S28, S29, S31, S32, S35, S38, S42 Factors of inﬂuence Facilitators S21, S28, S31, S38, S42 S3, S6, S10, S11, S19, S20, S21, S28, S32, S37 S3, S4, S10, S20, S25, S28, S35 Barriers S35, S38 S1, S3, S5, S6, S10, S11, S14, S20, S21, S29, S38 S2, S3, S7, S8, S10, S41 Generic S8; S10 S5, S8, S11, S19 S21 Approaches to S21; S30; S38; S42 S6, S8, S11, S14, S21, S29, S32, S37, S38 S2, S5, S8, S18, S20, S21, S22, S23, S24, S25, S26, S27, S31, S32, S34, S37 Recommendations S21 S7, S9, S21 S9, S21, S25, S27, S30, S32, S34 Outcomes Realized beneﬁts S1, S2, S4, S5, S9, S12, S13, S17, S18, S19, S23, S25, S28, S32, S33, S34, S36, S37, S39, S40 Satisfaction S5, S8, S9, S15, S17, S27, S28, S32, S35 Generic S7, S9, S11, S21 Downsides S9 Table 3. Innovation stages examined in the included studies that it may be possible to build a more integrated body of research in this area. that it may be possible to build a more integrated body of research in this area. plied projects and do not advance theoretical understanding of HRIS development, implementation, or use. plied projects and do not advance theoretical understanding of HRIS development, implementation, or use. International perspectives The plethora of terms used to describe HRIS, and variation across disciplines, suggests a lack of consensus and makes it difficult to build a coherent evidence base. This may explain why a previous systematic review on HRIS in health64 did not identify any research prior to 2000, whereas our review, using a broader range of search terms, found 7 such studies. Therefore, we recommend that re- searchers go beyond obvious keywords (eg, HRIS) when undertak- ing background research for new projects (for list of relevant keywords, see9). The focus of HRIS research has varied across countries in terms of systems, contexts, and priorities. Most studies from high-income countries have focused on small-scale systems in individual hospital settings, with the key users being internal personnel and managers (clinical/nonclinical), although there are notable exceptions, such as a major program evaluation in Australia.8 Moreover, nearly all user satisfaction studies have come from high-income countries. Research from lower-income countries tends to concentrate on open-source HRIS to collect data at the national and regional levels, focusing on health leaders and decision- and policy-makers as the primary system users. Most studies, especially those from low- income countries, prioritize operational aspects of HRM practices, despite WHO recommending in 2001 that effective HR departments should also undertake managerial or strategic HR activities.89 Types and quality of research Purely descriptive research was excluded at the screening phase, hence the methodological quality of the included studies was higher than in the literature as a whole. Most included studies were published in health journals, but many in social science and ICT journals, with some crossing disci- plines. Over half were qualitative, and of those reporting quantita- tive data, none evaluated cost-effectiveness or return on investment. Given the considerable expenditure on HRIS within the heath sec- tor, this gap is surprising, although it reflects a broader evidence def- icit in the health informatics literature.85,86 We observed a general scarcity of HRIS research in health from East Asia and the Pacific, Eastern Europe, Central Asia, Latin Amer- ica and the Caribbean, the Middle East and North Africa, South Asia, and sub-Saharan Africa. Moreover, we did not identify any study that compared HRIS projects across countries, supporting the call for more international comparisons of ICT research in health.90 Use of theory The majority of existing HRIS studies have concentrated on the use of systems in practice across several innovation stages. Very few fo- cused on the development stage, and even fewer reported measur- able outcomes of HRIS projects. While some studies differentiated between expected and realized benefits, we found no rigorous evalu- ations that compared both systematically. The focus on usage com- pared to development and impact suggests that the importance of user-centered design for the success of health ICT projects and the need for evaluation have not been fully acknowledged. The use of relevant theories was an important consideration for our assessment of HRIS research. Although many studies mentioned one or more theoretical frameworks, half did not, confirming observa- tions from a previous literature review on HRIS.87 Most of the theo- retically informed studies were published in social science journals or as academic dissertations. Of the studies mentioning a theoretical perspective, nearly all referred to different ones. As such, in line with clinical systems studies, which seldom build on prior re- search,88 studies on HRIS research in health mostly represent ap- Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 651 Table 4. Key messages effects on patient outcomes, despite their having been characterized as “the only class of hospital IS that has a dual beneficial impact [on] patient care [and] operating costs.”76 HRIS are underrepresented in the health informatics literature, de- spite their potential to contribute to information-driven learning health systems and the substantial financial investments that are be- ing made in them. Most research is based on softer forms of evi- dence, and there are important gaps in knowledge about the impacts and cost-effectiveness of these systems, which calls for further re- search. Interdisciplinarity is a positive characteristic of this litera- ture, in view of the importance of sociotechnical factors for the success of HRIS projects, but the sheer variety of terminologies and theories represents a barrier to building the coherent evidence base needed to translate evidence into practice. Given the rising cost of health care and the growth in patient traffic, the future sustainability of health systems will depend on making the best use of information to optimize deployment of HR.3 Linking the administrative data from HRIS with data on clinical processes and outcomes offers tremendous opportunities to enable real-time and predictive analytics alongside continuous monitoring and evaluation for smart, efficient, and “learning” health systems.91 Use of theory Summary of inﬂuential factors mentioned in the included studies Technology Organization Project Environment Task Inter- organization Individual Facilitators Development S21, S28, S31, S38 S42 S21, S28 Implementation S3 S3, S6, S10, S20 S6, S19, S21, S28, S32 S20 S3, S11, S19, S21, S28, S32, S37 Use S3, S28, S35 S3, S10, S20, S25, S28 S4, S28, S35 S20, S25 S3, S28 Barriers Development S35, S38 S38 S38 S35 Implementation S10, S14, S20, S21 S3, S5, S6, S10, S11, S14, S20, S21, S38 S1, S11, S14, S21, S29 S20, S21 S14, S21 S14, S21, S38 S1, S3, S11, S14, S20, S21 Use S3, S7, S10, S41 S2, S7, S8 S2 S7, S10, S41 S2, S3, S7, S8, S41 Generic Development S8, S10 S8 S8 Implementation S19 S11 S5 S5, S8, S11 Use S21 S21 S21 Approaches to Development S30 S38 S21, S42 S30, S38, S42 Implementation S6, S11, S21, S32, S38 S29 S6, S8, S11, S14, S21, S29, S38 S37 S8, S14, S21 S8, S32 Use S2, S5, S8, S18, S24, S25, S26, S27, S32, S34, S37 S26 S21, S31 S26 S20, S22, S23, S26, S27, S31 S31 S5, S31, S32 Recommendations Development S21 S21 S21 S21 S21 S21 Implementation S21 S7 S7, S9, S21 S7, S9, S21 S21 S7, S9 Use S21, S25, S34 S21, S32 S9, S21, S27, S32 S32 S9, S21, S27, S30 S21 S21, S27 Table 4. Summary of inﬂuential factors mentioned in the included studies SUPPLEMENTARY MATERIAL 22. Bondarouk T, Sikkel K. The role of group learning in implementation of a personnel management system in a hospital. In: A Sarmento, ed. Issues of Human Computer Interactions. Philadelphia: Idea Group Publishing; 2004:335–62. Supplementary material is available at Journal of the American Medical Informatics Association online. Supplementary material is available at Journal of the American Medical Informatics Association online. FUNDING 13. Patterson M, Rick J, Wood S, Carroll C, Balain S, Booth A. A Systematic review of the links between human resource management practices and 13. Patterson M, Rick J, Wood S, Carroll C, Balain S, Booth A. A Systematic review of the links between human resource management practices and performance. Health Technol Assess. 2010;14(51):1–334. AT’s doctoral research is sponsored by a grant from the University of Molise. CP is a grant holder on the Economic and Social Research Council Adminis- trative Data Research Centre for Scotland, award reference ES/L007487/1. The views expressed in the paper are the authors’ own. performance. Health Technol Assess. 2010;14(51):1–334. 14. Foster S. Making Sense of e-HRM: Technological Frames, Value Creation and Competitive Advantage. University of Hertfordshire; 2009. https:// core.ac.uk/download/pdf/1640360.pdf. Accessed March 23, 2016. 15. Sheikh A, Nurmatov U, Cresswell K, Bates D. Investigating the cost- effectiveness of health information technologies: a systematic review pro- tocol. BMJ Open. 2013;3(12):e003737. Limitations By excluding descriptive HRIS studies, which are published mostly by HR and clinical practitioners, we may have missed applied case Of the many studies in our review, only 4 looked at the potential for HRIS to support wider aspects of health care and their indirect 652 Journal of the American Medical Informatics Association, 2017, Vol. 24, No. 3 ing AT during completion of this research. We also thank medical librarian Marshall Dozier and research fellow Bright Nwaru at the University of Edin- burgh for helpful feedback on our search strategy. studies with valuable insights for the area. The timeline of our re- view means that some recent studies92 are not integrated. While multiple publications have emerged from the United States Agency for International Development’s Capacity and Capacity Plus pro- grams on global health workforce strengthening, we have included 2, the final report for the Capacity project27 and the last available report on the Capacity Plus project,71 which we believe provide a fair representation of the overall findings of this program and its ac- tivities. In common with other systematic reviews, publication bias is a risk, as most of the published studies report only positive results and several were compiled by consulting firms paid by the imple- menting organization. The authors have no competing interests to declare. The authors have no competing interests to declare. 16. Dyba T, Dingsoyr T. Empirical studies of agile software development: a systematic review. Inform Software Technol. 2008;50:833–59. 17. Critical Appraisal Skills Programme (CASP). 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https://openalex.org/W4200287111	https://www.revista.ueg.br/index.php/revelli/article/view/11741/8821	Portuguese	null	GT 16 DA ANPEd	Revelli	2,021	cc-by	8,117	REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 1 GT 16 DA ANPEd: panorama, tendências e desafios no pós-pandemia1 ANPEd GT 16: overview, trends and challenges in the post-pandemic LIMA, Daniela Da Costa Britto Pereira. ALONSO, Katia Morosov. ECHALAR, Jhonny David. ALONSO, Katia Morosov. ECHALAR, Jhonny David. Resumo: Este estudo busca estabelecer um panorama temático do Grupo de Trabalho 16 (GT 16) Educação e Comunicação da Associação Nacional de Pós-Graduação e Pesquisa em Educação (ANPEd), por meio de levantamento bibliográfico dos artigos publicados nos anais dos eventos nacionais e regionais entre os anos de 2011 e 2017, com o objetivo de observar tendências e temáticas daqueles que foram aprovados e, assim, relacionar com os desafios advindos da pandemia da Covid-19. Os resultados apontam que a temática discutida neste GT apresenta um leque de temas com ênfases diversificadas, tanto nas reuniões nacionais quanto nas regionais, o que também se verifica quando se analisam os autores mais citados. Constata- se assim, necessidade de se trabalharem compreensões que possibilitem a apreensão dos processos educacionais, da produção de imagens e uso das linguagens em tais contextos, considerando as tecnologias da informação e comunicação. g ç ç Palavras-chave: ANPEd. GT Educação e Comunicação. Temáticas. Compreensões. Abstract: This study seeks to establish a thematic panorama of the Working Group 16 (GT 16) Education and Communication of the National Association of Graduate Studies and Research in Education (ANPEd), by means of a bibliographic survey of the articles published in the annals of national and regional events between the years 2011 and 2017, in order to observe the understandings and themes/tendencies of those that were approved, and, thus, relate to the challenges arising from the Covid-19 pandemic. The results show that the theme discussed in this GT presents a range of themes with different emphases, both in national and regional meetings, which is also verified when analyzing the most cited authors. Thus, there is a need to work on understandings that possibilitate the seize of the educational processes, of the production of images and of the use of languages in such contexts, considering information and communication technologies. Keywords: ANPEd. GT Education and Communication. Topics. Trends. Thematic. Understanding. LI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 3 2 Disponível em: https://dadosabertos.capes.gov.br. Acesso em: 10 fev. 2019. 1 Para início de conversa 1 Resultado parcial da pesquisa “Políticas de Expansão da Educação a Distância (EaD) no Brasil: Regulação, Qualidade e Inovação em Questão” (2019-2022), com apoio do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). 1 O sistema de pós-graduação no Brasil possui o reconhecimento da comunidade científica tanto nacional quanto internacional, tendo em vista a trajetória das políticas públicas e a organização das instituições que fazem a sua gestão. Tal credibilidade resultou, por exemplo, no financiamento desse nível de formação no país, traduzido em sua expansão com qualidade ao longo dos anos (SANTOS; AZEVEDO, 2009; OLIVEIRA; LIMA, 2018). Desde o Parecer n. 977/1965 do Conselho Federal de Educação (CFE), que define a pós-graduação no país, as ações de planejamento, expansão, fomento, avaliação e consolidação dos cursos de mestrado e doutorado passaram a contar, efetivamente, com a gestão e o financiamento da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes) e do Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), o que redundou em seu desenvolvimento, sobretudo, nas universidades públicas. Nessa trajetória, houve também a concretização das sociedades científicas, que passaram a contar com a participação crescente dos docentes. Houve, portanto, um esforço conjugado do Estado e da comunidade científica no sentido de expandir e consolidar o sistema de pós-graduação no Brasil (OLIVEIRA; LIMA, 2018). Os dados da Tabela 1 a seguir, embora específicos do ano de 2016, demonstram a robustez da pós-graduação no país. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 2 Tabela 1. Número de Cursos de Pós-Graduação Stricto Sensu no Brasil em 2016 ÁREA DO CONHECIMENTO PRIVADA PÚBLICA Total Geral DR ME ME PROF TOTAL DR ME ME PROF TOTAL CIÊNCIAS AGRÁRIAS 13 20 6 39 250 365 32 647 686 CIÊNCIAS BIOLÓGICAS 9 16 3 28 214 274 15 503 531 CIÊNCIAS DA SAÚDE 56 67 49 172 349 441 82 872 1044 CIÊNCIAS EXATAS E DA TERRA 13 19 4 36 191 278 19 488 524 CIÊNCIAS HUMANAS 78 104 18 200 236 402 56 694 894 CIÊNCIAS SOCIAIS APLICADAS 82 148 66 296 145 281 63 489 785 ENGENHARIAS 23 42 21 86 180 301 51 532 618 LINGUÍSTICA, LETRAS E 16 24 2 42 104 172 11 287 329 Tabela 1. 1 Para início de conversa Número de Cursos de Pós-Graduação Stricto Sensu no Brasil em 2016 2 ARTES MULTIDISCIPLINAR 52 91 67 210 190 362 140 692 902 Total Geral 342 531 236 1109 1859 2876 469 5204 6313 Fonte: Elaborada pelos autores, com base nos Dados Abertos/Capes (2019)2. Nota: DR – Doutorado; ME – Mestrado; ME PROF – Mestrado Profissional. ARTES MULTIDISCIPLINAR 52 91 67 210 190 362 140 692 902 Total Geral 342 531 236 1109 1859 2876 469 5204 6313 Fonte: Elaborada pelos autores, com base nos Dados Abertos/Capes (2019)2. Nota: DR – Doutorado; ME – Mestrado; ME PROF – Mestrado Profissional. MULTIDISCIPLINAR No tocante à área da Educação (dentro da grande área de Ciências Humanas), observa- se seu crescimento ao longo do tempo, marcando algumas tendências, como mostra a Tabela 2 a seguir. 3 T b l Tabela 2. Número de Cursos de Pós-Graduação Stricto Sensu na área de Concentração Educação no Brasil de 2013 e 2016 Tabela 2. Número de Cursos de Pós-Graduação Stricto Sensu na área de Concentração Educação no Brasil de 2013 e 2016 Tipo/ Curso IES Privada IES Públicas Diferença de crescimento Federal Estadual Municipal 2013 2016 2013 2016 2013 2016 2013 2016 2013-2016 Mestrado 42 42 50 57 27 28 1 1 8 a mais Mestrado Profissional 3 8 12 19 7 12 -- 3 20 a mais Doutorado 29 30 31 31 13 13 -- -- 1 a mais Total 74 80 93 107 47 53 1 4 Fonte: Elaborada pelos autores, com base nos Dados Abertos/Capes (2019)3. Fonte: Elaborada pelos autores, com base nos Dados Abertos/Capes (2019)3. Fonte: Elaborada pelos autores, com base nos Dados Abertos/Capes (2019)3. Observa-se que o número de cursos de doutorado praticamente se manteve o mesmo, havendo, porém, aumento significativo dos cursos de mestrado (na ordem de 90%), especialmente os profissionais, e de 4,91% no número de cursos de mestrado acadêmico. O evento mais relevante da área da Educação para socializar as produções é promovido pela Associação Nacional de Pós-Graduação e Pesquisa em Educação (ANPEd), lócus de pesquisa deste estudo, que considera, justamente, sua importância no cenário brasileiro. As reuniões nacionais da ANPEd são bianuais4, precedidas e intercaladas por encontros regionais. Por serem realizados em anos alternados, é possível afirmar que, regionais ou nacionais, ambos os encontros promovem a socialização das pesquisas em Educação. 4 A 36ª Reunião Nacional, que ocorreu entre os dias 29 de setembro e 2 de outubro de 2013 na Universidade Federal de Goiás/Goiânia, foi a última realizada em anos consecutivos, uma vez que, como decidido em assembleia extraordinária, ela passaria a ser bianual, em anos intercalados com as reuniões científicas regionais. 5 Os GTs da ANPEd estão vinculados a determinadas temáticas. A abertura deles depende de proposições apreciadas em assembleia geral dos associados, conforme encaminhamentos previstos no Estatuto da Associação em seu CAPÍTULO VII. 3 Disponível em: https://dadosabertos.capes.gov.br. Acesso em: 10 fev. 2019. 4 3 Disponível em: https://dadosabertos.capes.gov.br. Acesso em: 10 fev. 2019. 4 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 4 em seu CAPÍTULO VII. Entre os 23 GTs da ANPEd, o GT 16 – Educação e Comunicação tem sido objeto de estudos de diversos pesquisadores da área6, contribuindo para analisar os avanços, os desafios e as possibilidades sobre a temática que lhe dá base. Nesse sentido, destaca-se o Dossiê “Cibercultura, Educação on-line e Processos Culturais” da Revista Teias de 2012. Nela constam cinco artigos identificados como “encomendados” pelo GT 167 e que tratam da produção de pesquisadores no período de 20 anos de existência do grupo. O Quadro 1 a seguir traz os trabalhos publicados no dossiê ou em outros periódicos, mas que tiveram também como escopo o objeto de estudo do GT 168. QUADRO 1. Artigos sobre as publicações do GT 16 da ANPEd Autores/Ano Título Foco/Objeto Nelson De Luca Pretto 2007 Educação, comunicação e a ANPEd: uma história em movimento Análise do percurso do GT 16, enfoques, desafios. Nelson De Luca Pretto 2009 Educação, Comunicação e informação: uma das tantas histórias Resgata 16 anos de atuação do GT 16 e o conjunto de trabalhos apresentados no período. Maria Helena Silveira Bonilla 2012 A presença da cultura digital no GT Educação e a Comunicação da ANPEd Resgata os trabalhos publicados no GT entre 1994 e 2010 sobre cultura digital com enfoque nas TIC, formação de professores, TIC e escolas e jovens. Marco Silva 2012 Educação a distância (EAD) e Educação on-line (EOL) nas reuniões do GT 16 da ANPEd (2000-2010) Estudo sobre 34 trabalhos publicados pela ANPEd entre 2000 e 2010 no GT 16 sobre EaD e EOL, explorando: formação de professores, mediação docente, material e desenho didático em AVA e avaliação. Nelson De Luca Pretto 2012 Educação e Comunicação: caminhos que cruzam entre si e com as tecnologias Histórico do GT 16 em seus 20 anos de existência, abordando relações entre as temáticas desenvolvidas e as tecnologias. Rosa Maria Bueno Fischer 2012 Rastros de um passado nem tão remoto: mídias audiovisuais em vinte anos de pesquisa Estudo sobre trabalhos do GT 16 com foco nas mídias audiovisuais: televisão, cinema, rádio, vídeo, fotografia e música. Guaracira Gouvêa de Souza 2012 As mídias impressas nas pesquisas em educação e comunicação de 1991 a 2010 Apresenta discussões dos trabalhos do GT 16 entre 1991 e 2010, com análise de livro, fotografia, história em quadrinhos, jornais e revistas. José Anderson Costa Gomes; Maria Francileide de O. Trajano; Verônica Maria de A. Pontes; Francisco das C. S. REVELLI V l 13 2021 D iê lid d i ã d / d ã õ 6 Apesar de no presente trabalho o “foco” ser o GT 16, outros grupos da ANPEd são também objetos de estudos. 7 Nas reuniões da ANPEd, alguns autores são convidados por GTs específicos, ou por um conjunto deles, para apresentar seus trabalhos que tenham aderência à temática que dá base àquele encontro. 8 Em buscas na internet, foram identificados trabalhos com foco no GT 16, porém, relacionados a outros GTs. No Quadro 1 foram priorizados estudos que coadunam com a especificidade do GT 16 da ANPEd, destacando-se a contribuição deles ao estudo em questão. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 5 ç q 1 Para início de conversa As reuniões nacionais possuem 23 Grupos de Trabalho (GT5) com temáticas variadas, como forma de agregar os pesquisadores e permitir o avanço nas pesquisas das respectivas áreas. Cada reunião regional tem organizado os GTs de forma específica, mantendo, porém, os propósitos da associação. 4 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 5 6 Apesar de no presente trabalho o “foco” ser o GT 16, outros grupos da ANPEd são também objetos de estudos. 7 Nas reuniões da ANPEd, alguns autores são convidados por GTs específicos, ou por um conjunto deles, para apresentar seus trabalhos que tenham aderência à temática que dá base àquele encontro. 8 Em buscas na internet, foram identificados trabalhos com foco no GT 16, porém, relacionados a outros GTs. No Quadro 1 foram priorizados estudos que coadunam com a especificidade do GT 16 da ANPEd, destacando-se a contribuição deles ao estudo em questão. A ANPEd e as Tecnologias da Educação e Comunicação: um resgate de sua produção Levantamento dos trabalhos publicados no GT 16 entre 2004 e 2015, observando as tendências das publicações no tocante ao uso das TIC. QUADRO 1. Artigos sobre as publicações do GT 16 da ANPEd 6 Apesar de no presente trabalho o “foco” ser o GT 16, outros grupos da ANPEd são também objetos de estudos. 7 Nas reuniões da ANPEd, alguns autores são convidados por GTs específicos, ou por um conjunto deles, para apresentar seus trabalhos que tenham aderência à temática que dá base àquele encontro. 8 Em buscas na internet, foram identificados trabalhos com foco no GT 16, porém, relacionados a outros GTs. No Quadro 1 foram priorizados estudos que coadunam com a especificidade do GT 16 da ANPEd, destacando-se a contribuição deles ao estudo em questão. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 5 5 Souza 2017 Fonte: Elaborado pelos autores. Souza Fonte: Elaborado pelos autores. Considerando que os trabalhos relevantes que tratam das produções do referido GT datam de 2012 e ao observar que nas produções, na elaboração do Quadro 1, foi dada prioridade aos trabalhos que analisaram as publicações oriundas dos encontros nacionais da ANPEd, ou seja, nenhum deles comparou essas publicações com as das reuniões regionais, o presente estudo tem por objetivo, portanto, trabalhar esse hiato e analisar as produções realizadas entre os anos de 2011 e 2017 no GT 16 – Educação e Comunicação, constituindo um panorama dos temas e tendências das produções apresentadas, tanto nas reuniões nacionais quanto nas regionais, apresentando os debates, reflexões e panoramas que se constituem e os desafios que a pesquisa na área de comunicação, em interface com a educação, apresenta. Assim, este estudo apresenta resultados de um levantamento bibliográfico desenvolvido com foco no que tem sido produzido sobre a relação entre educação e comunicação para “apontar caminhos que vêm sendo tomados e aspectos que são abordados em detrimento de outros” (ROMANOWSKI; ENS, 2006, p. 38), por meio das temáticas e compreensões, considerando os principais autores presentes nos trabalhos publicados pelo GT 16 da ANPEd nos Encontros Nacionais e Regionais entre 2011 e 2017. Importante ressaltar que o presente trabalho é recorte de estudo mais amplo9. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 6 9 Como mencionado, um dos trabalhos das pesquisas do grupo foi a organização de ebook com trabalhos, justamente, de revisões de literatura. O livro está disponível em: https://f3286f62-e14d-4952-ad27- eac5c2feb473.usrfiles.com/ugd/f3286f_948889550e3a475d93c65c738127774c.pdf. Acesso em: 12 mai. 2020. Neste foram levantadas várias outras fontes no sentido de se debater a relação educação e as tecnologias da informação e comunicação (TIC). No entanto, dada a importância da produção científica da ANPEd, bem como as especificidades observadas no que se referiu aos trabalhos do GT16 – Educação e Comunicação, é que se constituiu a pesquisa em tela. O artigo está estruturado considerando a maneira pela qual se deu a coleta e o tratamento dos dados, para evidenciar as temáticas debatidas e compreensões observadas, tendo por base, sobretudo, os autores citados anteriormente. 6 2 Publicações do GT 16 nos eventos nacionais e regionais: levantamento e possibilidades para a pesquisa científica 2 Publicações do GT 16 nos eventos nacionais e regionais: levantamento e possibilidades para a pesquisa científica 2 Publicações do GT 16 nos eventos nacionais e regionais: levantamento e possibilidades para a pesquisa científica A ANPEd foi criada em 1978 e, entre os anos de 1989 e 1992, houve as primeiras mobilizações de pesquisadores para o estabelecimento do GT 16. Segundo Pretto (2007), havia a propagação de trabalhos que traziam a temática Educação e Comunicação por outros GTs, o que causava sua superposição ou fragmentação. Em seu início, a associação sofria com a precariedade de recursos para seu custeio, bem como para a realização das reuniões anuais, apresentando dicotomias entre seu estatuto científico e sua atuação política, considerando o período de redemocratização e o surgimento dos ideais neoliberais no país (PRETTO, 2007). Ainda conforme Pretto (2007, p. 7), o movimento de criação do GT 16 se deu durante a 13ª Reunião Anual, realizada no período de 15 a 19 de outubro de 1990, em Belo Horizonte. O argumento para a criação do GT teve por fundamento a ideia de que “ ... comunicação, tecnologias e temas correlatos não existiam na ANPEd, apesar de já estarem presentes em alguns Programas de P s-Graduação”. Um grupo de 22 pesquisadores se organi ou nesse evento e, por afinidade dos temas – que naquele momento eram os meios de comunicação e a análise de imagens –, propôs a criação de um novo GT. Este foi autorizado a se constituir em assembleia geral da ANPEd ainda em caráter experimental, consolidando-se com aprovação posterior. Fischer (2012) defende a tese de que, desde o início, o GT 16 tem seguido uma trajetória coerente de publicações, e que o artigo de Maria Luíza Belloni, Formação do telespectador: uma experiência de educação para a mídia – um dos textos redigidos sob encomenda durante o primeiro ano de criação da ANPEd – conferiu uma “marca” às produções daquela época. Afirma, também, que a temática Educação e Comunicação, mesmo com distintas justificativas teóricas e pedagógicas, continua discutindo propostas de investigação e de intervenção na escola (FISCHER, 2012). É, portanto, com tal tônica que a história do GT se consolida, e fundamentado em tal premissa é que o levantamento aqui referido se desenvolveu. Para Romanowski e Ens (2006), os estudos com base em „estado da arte‟, „estado do conhecimento‟ e de revisões/levantamentos bibliográficos em geral ganham relevância na REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. 2 Publicações do GT 16 nos eventos nacionais e regionais: levantamento e possibilidades para a pesquisa científica E-202122 7 7 pesquisa em educação por possibilitarem conhecimentos e entendimentos sobre a produção e pesquisas, de modo a indicar possibilidades, lacunas e novas discussões nesse campo. Se, até anos recentes, essas pesquisas eram mais comuns em outras áreas de conhecimento como a saúde, por exemplo, atualmente, e em se tratando da área da educação: Esta intensificação de publicações gera inquietações e questionamentos como: Quais são os temas mais focalizados? Como estes têm sido abordados? Quais as abordagens metodológicas empregadas? Quais contribuições e pertinência destas publicações para a área? O que é de fato específico de uma determinada área da educação, a formação de professores, o currículo, a formação continuada, as políticas educacionais? [...] Com base nos aspectos apontados, pode-se dizer que faltam estudos que realizem um balanço e encaminhem para a necessidade de um mapeamento que desvende e examine o conhecimento já elaborado e apontem os enfoques, os temas mais pesquisados e as lacunas existentes. (ROMANOWSKI; ENS, 2006, p. 38). Diante disso e considerando a emergência de determinadas temáticas, buscou-se, com base em pesquisas já publicadas, temáticas e entendimentos que se consolidam sobre o assunto. De acordo, ainda, com Romanowski e Ens (2006), pesquisas como a aqui organizada, devem considerar as categorias que as identifiquem em cada um dos estudos, ao mesmo tempo em que se observa no conjunto destes as facetas sobre as quais a educação e comunicação (no caso) vem sendo debatida no GT 16 da ANPEd, objetivo do nosso trabalho. Isso poderá contribuir para analisar a continuidade da trajetória político-temática das publicações considerando os objetivos primeiros do GT, conforme Fischer destaca (2012). Para realizar o mapeamento e levantamento das publicações no GT 16 da ANPEd, optamos por realizar a caracterização, análise e sistematização das principais temáticas por meio dos textos apresentados em sua íntegra, visto que as pesquisas ou publicações que possuem como eixo a revisão de literatura e fazem uso de conteúdo de apenas parte dos textos, podem ficar limitadas, em alguns casos com equívocos e informações restritas, insuficientes para a divulgação dos resultados ou análise das contribuições da produção analisada (MEGID, 1999). Diante disso, foi tomada a decisão de tomar a leitura completa dos textos publicados, conforme interstício de tempo antes indicado, de modo a constituir análises de caráter amplo. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. 2 Publicações do GT 16 nos eventos nacionais e regionais: levantamento e possibilidades para a pesquisa científica E-202122 8 Com essa preocupação, foi estabelecida uma metodologia de levantamento e análise dos dados para o corpus do estudo na seguinte sequência: (i) levantamento com buscas diretas nos sites dos eventos nacionais e regionais, no link “Publicações/Anais” dos trabalhos Com essa preocupação, foi estabelecida uma metodologia de levantamento e análise dos dados para o corpus do estudo na seguinte sequência: (i) levantamento com buscas diretas nos sites dos eventos nacionais e regionais, no link “Publicações/Anais” dos trabalhos REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 8 nos sites dos eventos nacionais e regionais, no link Publicações/Anais dos trabalhos 8 8 aprovados no GT; (ii) sistematização dos artigos por meio de arquivamento e organização das informações de todos eles no software NVivo10→ for MAC e em planilha eletrônica; (iii) pautou-se a construção das nuvens de palavras, que considerou as 25 mais incidentes em cada um dos eventos nacionais e regionais; e, finalmente, (iv) observando-se os referenciais teóricos, sobretudo dos autores mais utilizados pelos pesquisadores que tiveram seus trabalhos aprovados, foi possível estabelecer as compreensões sobre as pesquisas divulgadas nos referidos eventos científicos. Para o início da coleta de dados, tivemos que lidar com algumas limitações. A primeira delas está relacionada ao recorte temporal definido para o levantamento: o levantamento dos trabalhos ocorreu entre outubro de 2018 e janeiro de 2019, sendo que, no primeiro momento, objetivou-se realizar a pesquisa nos trabalhos apresentados nos últimos dez anos, ou seja, entre 2007 e 2017. Outra limitação foi a dificuldade de acesso on-line ao evento nacional de 2010, e por isso houve um redirecionamento do levantamento dos dados, que passou a considerar os últimos cinco encontros nacionais, com acesso ininterrupto e no período mencionado. Da mesma maneira, para os eventos regionais, foram considerados os que possibilitaram acesso a todos os trabalhos de todas as regionais11. Mas, entre os trabalhos publicados em eventos das reuniões regionais, só foi possível acessar de todos os eventos pela internet os de 2014. O levantamento dos eventos regionais contemplou, portanto, as reuniões realizadas em 2014 e, em âmbito nacional, as de 2011, 2012, 2013, 2015, 2017. Tabela 3. Encontros regionais e nacionais selecionados e quantidade de trabalhos publicados. ela 3. Encontros regionais e nacionais selecionados e quantidade de trabalhos publicados. 10 Esse software “ ... consiste em um sistema de indexação e categori ação de dados não estruturados, permitindo a descoberta e exploração dos sentidos das informações alfanuméricas, auxiliando o pesquisador desde a definição e organização das categorias e sub-categorias analíticas até o processo de análise” (FREITAS; ARRUDA; FALQUETO, 2017, p. 622). 11 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984 6576 10 Esse software “ ... consiste em um sistema de indexação e categori ação de dados não estruturados, permitindo a descoberta e exploração dos sentidos das informações alfanuméricas, auxiliando o pesquisador desde a definição e organização das categorias e sub-categorias analíticas até o processo de análise” (FREITAS; ARRUDA; FALQUETO, 2017, p. 622). 11 São quatro eventos regionais: Encontro de Pesquisa em Educação/Centro-Oeste; Encontro de Pesquisa em Educação/Sudeste; ANPEd Sul; Encontro de Pesquisa Educacional/ Norte e Nordeste. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 9 Educação/Sudeste; ANPEd Sul; Encontro de Pesquisa Educacional/ Norte e Nordeste. 2 Publicações do GT 16 nos eventos nacionais e regionais: levantamento e possibilidades para a pesquisa científica Ano Regional Quantidade de trabalhos GT 16 Ano Nacional Quantidade de trabalhos GT 16 2014 XII Encontro de Pesquisa em Educação/Centro-Oeste 18 2011 34ª Reunião Anual da ANPEd Natal/RN 22 11° Encontro de Pesquisa em Educação da Região Sudeste 48 2012 35ª Reunião Anual da ANPEd 14 10 Esse software “ ... consiste em um sistema de indexação e categori ação de dados não estruturados, permitindo a descoberta e exploração dos sentidos das informações alfanuméricas, auxiliando o pesquisador desde a definição e organização das categorias e sub-categorias analíticas até o processo de análise” (FREITAS; ARRUDA; FALQUETO, 2017, p. 622). 11 9 Porto de Galinhas/PE X ANPEd Sul 41 2013 36ª Reunião Anual da ANPEd Goiânia/GO 20 XXII Encontro de Pesquisa Educacional Norte e Nordeste 25 2015 37ª Reunião Anual da ANPEd Florianópolis/SC 21 2017 38ª Reunião Anual da ANPEd São Luís/MA 18 Total 132 Total 95 Fonte: Elaborada pelos autores. Fonte: Elaborada pelos autores. Outro elemento limitante importante está no fato de as publicações das reuniões regionais não possuírem padronização quanto a elementos textuais, como indicação de palavras-chave, vínculo institucional, resumo, normas para referências bibliográficas, contrariamente ao que ocorre com as do evento nacional. Muitas vezes, sequer os trabalhos dos diferentes GTs seguem uma padronização, conforme pode ser observado no Quadro 2 a seguir. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 12 12 Em razão do grande número de diferentes autores citados nos trabalhos, priorizaram-se os que apareciam, no mínimo, cinco vezes. 13 A Plataforma Lattes é um sistema de integração de bases de dados de currículos, de grupos de pesquisa e de instituições em um único sistema de informações criado e mantido pelo Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq). Disponível em: http://lattes.cnpq.br/ Fonte: Elaborado pelos autores. Fonte: Elaborado pelos autores. De toda maneira, é importante apontar essas limitações, para que, mais e mais, a produção dos eventos da ANPEd avance em função da qualidade editorial condizente com a divulgação e democratização da ciência. Conforme Ramos, Faria e Faria (2014), as pesquisas do tipo revisão e levantamento bibliográfico têm crescido, ampliando o interesse por questões metodológicas e epistemológicas a elas relacionadas. A ANPEd, com a organização de suas reuniões científicas, é sem dúvida um dos espaços mais importantes para os investigadores da área da Educação no Brasil, merecendo, assim, desenvolvimento de sua padronização das publicações. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 10 QUADRO 2. Limitações nos sites/anais dos eventos regionais pesquisados QUADRO 2. Limitações nos sites/anais dos eventos regionais pesquisados Ano Regional GT Considerado 2014 XII Encontro de Pesquisa em Educação/Centro- Oeste GT 16 – Educação e Comunicação Autores dos trabalhos não apresentam vínculo institucional. Trabalhos possuem resumo e palavras-chave, com exceção de um deles. 11° Encontro de Pesquisa em Educação da Região Sudeste Eixo 9 – Pesquisa, artes, mídias e educação Formatações variadas – alguns trabalhos trazem resumo e palavras- chave, e outros, não; alguns têm vínculo institucional, e outros, não. X ANPEd Sul Eixo 11 – Educação, Comunicação e Tecnologias Padronizado: resumo, palavras-chave, vínculo e e-mail. XXII Encontro de Pesquisa Educacional Norte e Nordeste GT 16 – Educação e Comunicação Os trabalhos apresentam vínculo institucional, mas não possuem resumo e palavras-chave. Fonte: Elaborado pelos autores. Sobre a reunião nacional, também foram detectadas algumas limitações, como pode ser constatado no Quadro 3 a seguir. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 11 QUADRO 3. Limitações dos Anais dos eventos nacionais pesquisados Ano Nacional GT 16 2011 34ª Reunião Anual da ANPEd Natal/RN GT 16 – Educação e Comunicação - Padrão sem resumo e sem palavras-chave; apresenta vínculo dos autores, porém, um trabalho possui resumo/palavras-chave, abstract/key words e outros dois, resumo e palavras-chave. 2012 35ª Reunião Anual da ANPEd Porto de Galinhas/PE GT 16 – Educação e Comunicação - Padrão sem resumo e sem palavras-chave; apresenta vínculo dos autores, porém, um trabalho possui resumo, e outro, resumo e palavras- chave. 2013 36ª Reunião Anual da ANPEd Goiânia/GO GT 16 – Educação e Comunicação - Padrão com resumo, palavras-chave e vínculo dos autores, porém, dois trabalhos estão sem resumo e sem palavras-chave. 2015 37ª Reunião Anual da ANPEd Florianópolis/S C GT 16 – Educação e Comunicação - Padronizado: resumo, palavras-chave e vínculo institucional dos autores. 2017 38ª Reunião Anual da ANPEd - GT 16 – Educação e Comunicação - Padronizado: resumo, palavras-chave e vínculo dos autores. QUADRO 3. Limitações dos Anais dos eventos nacionais pesquisados São Luís/MA 3 Temáticas e compreensões em contexto Para desenvolvimento deste item, foram utilizados, como já mencionado, dois softwares para coleta, sistematização, organização, quantificação e apresentação dos dados: o NVivo→ for MAC e o Microsoft Excel. A finalidade foi levantar as principais temáticas desenvolvidas nos eventos no período de 2010-2017, bem como apresentar perspectivas teóricas dos trabalhos. Neste caso, foram verificados os autores que possuíam cinco ou mais recorrências de referências nos trabalhos publicados12. Para a definição de perspectivas teóricas e áreas de atuação de alguns autores, foram realizadas também buscas em seus currículos na Plataforma Lattes13, de modo a coligir informações pertinentes à produção relativa à temática Educação e Comunicação. Não houve a pretensão de esgotar as discussões com esse levantamento, tampouco observar os trabalhos aprovados, seja no GT16/nacional, seja nos regionais, sob determinado viés teórico. Neste momento, o presente estudo permite registrar indícios sobre a maior ou menor inserção de autores que permeiam as leituras de um coletivo de pesquisadores. Uma posterior análise dos conteúdos dos trabalhos aprovados é “campo aberto”, já que não foram objeto desta pesquisa. 3.1 Sobre as temáticas De posse das frequências das palavras/temas mais recorrentes nos eventos regionais e nacionais pesquisados, foram elaboradas as seguintes nuvens a partir dos textos de introdução e resumo: REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 13 13 Figura1 – Frequência de palavras nos eventos regionais (A) e nacionais (B), considerando a introdução e os resumos dos trabalhos A B Fonte: Elaboradas pelos autores. B Tanto nos eventos regionais quanto nos nacionais, a maior frequência foi a das palavras educação e tecnologias, corroborando a tese levantada por Fischer (2012). A essas palavras, seguiram-se escolas, professores, alunos, processos, uso, digitais, comunicação e práticas. Nos eventos nacionais, as palavras formação, contexto, culturas se sobressaíram em relação aos regionais, enquanto, nestes, as palavras meio, relação, trabalho se sobressaíram em relação àqueles. As palavras mídias e políticas foram recorrentes nos eventos nacionais, enquanto criança, experiência e ambientes apareceram, com maior recorrência, nos eventos regionais. As recorrências são condizentes com as temáticas mais abordadas por trabalho, conforme a Tabela 4 a seguir. Tabela 4. Incidências das temáticas mais abordadas nos trabalhos pesquisados Temas mais abordados por trabalho Nacionais Regionais Mídia 37 Tecnologia, TIC/TDIC 46 Tecnologia, TIC/TDIC 26 Mídia 29 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 14 Tabela 4. Incidências das temáticas mais abordadas nos trabalhos pesquisados Temas mais abordados por trabalho Nacionais Regionais Mídia 37 Tecnologia, TIC/TDIC 46 Tecnologia, TIC/TDIC 26 Mídia 29 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 14 Educação a distância 11 Artes 21 Ciberespaço 11 Educação a distância 19 Redes sociais 9 Ciberespaço 12 Voluntariado 1 Redes sociais 5 Artes 0 Voluntariado 0 Total 95 Total 132 Fonte: Elaborada pelos autores. A Tabela 5 a seguir apresenta, em conjunto, os dados gerais obtidos tanto das reuniões nacionais quanto das regionais da ANPEd e permite observar a inserção dos diferentes autores nas discussões das diferentes esferas desta pesquisa. Nesse sentido, constatou-se uma grande recorrência de documentos oficiais da União em ambas as bases de dados, o que aponta para discussões relacionadas a políticas públicas em âmbito nacional, salientando-se que, não por consequência, estas sejam o objeto de análise dos trabalhos apresentados. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 15 Tabela 5. Recorrência geral de cada autor, com base nas referências bibliográficas dos trabalhos aprovados nos encontros nacionais e regionais da ANPEd. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 13 NACIONAL REGIONAIS AUTOR RECORRÊNCIA AUTOR RECORRÊNCIA LÉVY, Pierre 35 BRASIL 64 FOUCAULT, Michel 30 LÉVY, Pierre 42 BRASIL 28 FREIRE, Paulo 40 LEMOS, André 24 DELEUZE, Giles 35 BAUMAN, Zygmunt 23 FOUCAULT, Michel 23 BAKHTIN, Mikhail 22 MORIN, Edgar 21 SANTAELLA, Lúcia 20 VYGOTSKI, Lev Semyonovich 20 DELEUZE, Gilles 16 ALMEIDA, Maria Elizabeth Bianconcini de 19 COSTA, Marisa Vorraber; PRETTO, Nelson De Luca 14 GUATTARI, Félix 18 CASTELLS, Manuel; GUATTARI, Félix; MARTÍN- 13 MATURANA, Humberto Ramesin 17 abela 5. Recorrência geral de cada autor, com base nas referências bibliográficas dos trabalhos aprovados nos encontros nacionais e regionais da ANPEd. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 16 BARBERO, Jesús FREIRE, Paulo 12 CASTELLS, Manuel 14 BARTHES, Roland 11 BARBOSA, Ana Mae BAUMAN, Zygmunt 13 HABERMAS, Jürgen 12 FISCHER, Rosa Maria Bueno; SOARES, Magda Becker 10 LARROSA, Jorge; SANTAELLA, Lucia 10 ADORNO, Theodor W.; ANDRÉ, Marli; BONILLA, Maria Helena Silveira; NÓVOA, António 9 ADORNO, Theodor W.; HALL, Stuart; VARELA, Francisco J. 9 HALL, Stuart; ROJO, Roxane; SANTOS, Edméa Oliveira dos 8 MACEDO, Roberto Sidnei 8 ALMEIDA, Maria Elizabeth Bianconcini de; ARDOINO, Jacques; CERTEAU, Michel de; CHARTIER, Roger; FREITAS, Maria Teresa; HABERMAS, Jürgen; JENKINS, Henry; KASTRUP, Virgínia; KELLNER, Douglas; LARROSA, Jorge; MACEDO, Roberto Sidnei; RECUERO, Raquel; ROLNIK, Suely; TARDIF, Maurice; VEIGA- NETO, Alfredo; VIGOTSKI, Liev Semionovich 7 COSTA, Marisa Vorraber; KENSKI, Vani Moreira; LEMOS, André; STREET, B.; VALENTE, José Armando 7 BALL, S. J.; BELLONI, Maria Luiza; BUCKINGHAM, David; RANCIÈRE, Jacques; REGO, Sheila, Cristina Ribeiro; SIEMENS, George 6 BAKHTIN, Mikhail; BELLONNI, Maria Luiza; CHARTIER, Roger; DUARTE, Rosália; FISCHER, Rosa Maria Bueno; LACLAU, Ernesto; PRENSKY, Marc 6 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. BARBERO, Jesús FREIRE, Paulo 12 CASTELLS, Manuel 14 BARTHES, Roland 11 BARBOSA, Ana Mae BAUMAN, Zygmunt 13 HABERMAS, Jürgen 12 FISCHER, Rosa Maria Bueno; SOARES, Magda Becker 10 LARROSA, Jorge; SANTAELLA, Lucia 10 ADORNO, Theodor W.; ANDRÉ, Marli; BONILLA, Maria Helena Silveira; NÓVOA, António 9 ADORNO, Theodor W.; HALL, Stuart; VARELA, Francisco J. 9 HALL, Stuart; ROJO, Roxane; SANTOS, Edméa Oliveira dos 8 MACEDO, Roberto Sidnei 8 ALMEIDA, Maria Elizabeth Bianconcini de; ARDOINO, Jacques; CERTEAU, Michel de; CHARTIER, Roger; FREITAS, Maria Teresa; HABERMAS, Jürgen; JENKINS, Henry; KASTRUP, Virgínia; KELLNER, Douglas; LARROSA, Jorge; MACEDO, Roberto Sidnei; RECUERO, Raquel; ROLNIK, Suely; TARDIF, Maurice; VEIGA- NETO, Alfredo; VIGOTSKI, Liev Semionovich 7 COSTA, Marisa Vorraber; KENSKI, Vani Moreira; LEMOS, André; STREET, B.; VALENTE, José Armando 7 BALL, S. J.; BELLONI, Maria Luiza; BUCKINGHAM, David; RANCIÈRE, Jacques; REGO, Sheila, Cristina Ribeiro; SIEMENS, George 6 BAKHTIN, Mikhail; BELLONNI, Maria Luiza; CHARTIER, Roger; DUARTE, Rosália; FISCHER, Rosa Maria Bueno; LACLAU, Ernesto; PRENSKY, Marc 6 COSTA, Marisa Vorraber; KENSKI, Vani Moreira; LEMOS, André; STREET, B.; VALENTE, José Armando 7 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 16 VIGOTSKI, Liev Semionovich BALL, S. J.; BELLONI, Maria Luiza; BUCKINGHAM, David; RANCIÈRE, Jacques; REGO, Sheila, Cristina Ribeiro; SIEMENS, George 6 BAKHTIN, Mikhail; BELLONNI, Maria Luiza; CHARTIER, Roger; DUARTE, Rosália; FISCHER, Rosa Maria Bueno; LACLAU, Ernesto; PRENSKY, Marc 6 FANTIN, M.; GATTI, Bernadete; GIROUX, Henry; GOUVÊA, Guaracira; HORKHEIMER, Max; MARASCHIN, Cleci; MARX, Karl; MILL, D. R. S. ; PEIXOTO, Joana; PESCE, Lucila; PRIMO, Alex; SARAIVA, Karla; WORTMANN, Maria Lúcia Castagna 5 BALL, Stephen J.; BENJAMIN, Walter; CANEVACCI, M.; FERNANDES, Adriana Hoffmann; FREITAS, Maria Teresa de Assunção; LIBÂNEO, José Carlos; LUKÁCS, György; MORAN, José Manoel; NOGUEIRA, Maria Alice; NÓVOA, António; PEIXOTO, Joana; PETERS, Otto; PRETTO, Nelson De Luca; SILVEIRA, Rosa Maria Hessel 5 Fonte: Elaborada pelos autores. BALL, Stephen J.; BENJAMIN, Walter; CANEVACCI, M.; FERNANDES, Adriana Hoffmann; FREITAS, Maria Teresa de Assunção; LIBÂNEO, José Carlos; LUKÁCS, György; MORAN, José Manoel; NOGUEIRA, Maria Alice; NÓVOA, António; PEIXOTO, Joana; PETERS, Otto; PRETTO, Nelson De Luca; SILVEIRA, Rosa Maria Hessel Em ambos os encontros, observou-se que os autores Manuel Castells, Pierre Lévy e Paulo Freire são os que possuem maior recorrência nas referências. Destaca-se a inserção da pesquisadora Vani Moreira Kenski entre os autores com maior recorrência nas referências das reuniões regionais. A pesquisadora/autora atua nas linhas de Educação a Distância, Educação e Comunicação, Tecnologias e Educação on-line, buscando nexos entre Educação e Tecnologia, mas não fundamentada em teorias da comunicação. Outro pesquisador que atua nessa linha e que surge nos dados das reuniões regionais é o professor Nelson De Luca Pretto, docente da Universidade Federal da Bahia (UFBA), que desenvolve seus trabalhos sobre a relação entre Educação e Comunicação, Educação e Cibercultura, Educação a Distância e Informática Educativa, abrindo para temas mais amplos que os condicionados à temática do GT 16. Ainda nessa perspectiva, emerge nos dados a inserção nas referências da professora Maria Luiza Bellonni, que esteve vinculada, até sua aposentadoria, à Universidade Federal de Santa Catarina (UFSC). Por final, entre os autores brasileiros mais referenciados, insere-se o professor Roberto Sidnei Macedo, com vínculo com o GT de Currículo, campo em que desenvolve seus trabalhos. Constata-se, ainda, a tendência de se utilizarem, nos trabalhos das reuniões regionais, pesquisadores com vinculação com essas mesmas regiões, fato que ocorreu com maior ênfase nas regiões Sul e Sudeste. Tal constatação aponta provavelmente para um mesmo eixo teórico REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 17 17 entre os grupos de pesquisa/estudo dessas regiões, assim como para um maior fluxo e intercâmbio de suas produções. entre os grupos de pesquisa/estudo dessas regiões, assim como para um maior fluxo e intercâmbio de suas produções. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 16 VIGOTSKI, Liev Semionovich BALL, S. J.; BELLONI, Maria Luiza; BUCKINGHAM, David; RANCIÈRE, Jacques; REGO, Sheila, Cristina Ribeiro; SIEMENS, George 6 BAKHTIN, Mikhail; BELLONNI, Maria Luiza; CHARTIER, Roger; DUARTE, Rosália; FISCHER, Rosa Maria Bueno; LACLAU, Ernesto; PRENSKY, Marc 6 Entre os dez autores mais referenciados nas duas bases de dados, observa-se que Pierre Lévy, Gilles Deleuze, Michel Foucault e Félix Guattari possuem maior recorrência para além dos documentos oficiais. Nesse sentido, Pierre Lévy é o autor mais utilizado nos trabalhos apresentados tanto nos encontros nacionais quanto nos regionais da ANPEd, apresentando-se, portanto, como referência inequívoca nas discussões desses GTs. Jesús Martín-Barbero aparece, contudo, como um dos autores mais utilizados somente nos encontros nacionais, não ultrapassando cinco recorrências em nenhum dos eventos regionais. Entre os autores de maior recorrência nos encontros nacionais, foi possível observar que Zygmunt Bauman, Mikhail Bakhtin, Manuel Castells, Lucia Santaella, Marisa Vorraber Costa e André Lemos aparecem também com maior recorrência apenas em eventos das regiões Sul e/ou Sudeste. Daí ser possível inferir certa aproximação teórica entre as discussões do GT 16 nacional e as das regiões Sul e Sudeste. De toda maneira, ao observar o quadro geral dos autores mais citados nas reuniões nacionais e regionais, há um dado significativo sobre o contexto dos trabalhos aprovados: a maioria dos autores citados propõe discussões e reflexões em áreas como Filosofia e Sociologia, por exemplo, fato que evidencia que as teorias/teóricos educacionais são pouco utilizadas nas análises. Longe de arbitrar em favor ou não das discussões postas no GT 16, é importante ressaltar o esforço para compreender as aproximações entre os temas Educação e Comunicação e as correntes teóricas implicadas nas referências autorais. Temáticas como mídia, TIC, Educação a Distância (EaD), ciberespaço, redes sociais, voluntariado e artes compõem, efetivamente, o campo da pesquisa em educação/comunicação. Isso, contudo, implica pensar/debater o contexto educativo com elas. Na constituição do presente artigo, fomos apanhados pelo momento da pandemia da Covid-19 e nós, os/as educadores/as, surpreendidos pela necessidade do isolamento social como forma de cuidado de si e dos outros e, com isso, a suspensão das atividades presenciais de ensino. Perplexos ficamos, com nossas solidões, pelo pouco que havíamos nos apropriado do uso mais intenso das TIC no cotidiano das instituições escolares em seus diferentes níveis. Ao trabalhar então com as compreensões que os achados da pesquisa indicavam, sobreveio a constatação das REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 18 18 l i relacionadas àquilo que seria o “lugar” da escola filos fica e sociologicamente no contexto comunicacional presente na relação educativa/pedagógica. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 16 VIGOTSKI, Liev Semionovich BALL, S. J.; BELLONI, Maria Luiza; BUCKINGHAM, David; RANCIÈRE, Jacques; REGO, Sheila, Cristina Ribeiro; SIEMENS, George 6 BAKHTIN, Mikhail; BELLONNI, Maria Luiza; CHARTIER, Roger; DUARTE, Rosália; FISCHER, Rosa Maria Bueno; LACLAU, Ernesto; PRENSKY, Marc 6 Absolutamente não se trata de negar a importância de tal compreensão, no entanto nos perguntávamos sobre quais fundamentos adviriam para se compreender mais o como aprendemos e como nos relacionamos com as mídias, TIC, redes sociais, entre as principais temáticas inscritas no GT. Diante disso, retornamos aos estudos de Peixoto (2015) que alertava para o uso tecnocêntrico e instrumental fosse das TIC na educação, da internet no contexto escolar e da Educação a Distância (EaD) ou, ainda, das perspectivas de neutralidade do uso daquelas tecnologias no processo de aprender e ensinar. Se os estudos com base nos levantamentos bibliográficos, como apontados por Roamnowski e Ens (2006), permitem trazer à tona lacunas a serem pesquisadas, mais do que nunca compreendermos como aprendemos e a maneira pela qual nos relacionamos com os artefatos culturais da cultura digital implicados nos processos educativos é temática relevante a ser pesquisada. Como assinala Peixoto (2015), compreender a abordagem sociotécnica como possibilidade de orientação teórico- metodológica seja nas pesquisas seja no trabalho pedagógico é essencial no momento em que vivemos. Frente a isso, o estudo faz indicar, então, a necessidade de constituirmos, seja no âmbito do GT16, seja no âmbito das pesquisas em Educação, os sentidos e significados que cultura digital, cibercultura, os artefatos culturais que dela advêm nos possibilitam trabalhar perspectivas e, daí entendimentos, que façam prevalecer o caráter democrático e emancipatório da educação. A questão fundamental agora, portanto, é construir e constituir entendimentos que venham a configurar teorias educacionais próprias que façam emergir possibilidades e ampliem sentidos que coadunem com democracia, participação e qualidade da educação no país. 4 Considerações finais Que os estudos com base em levantamento e revisão de bibliografia têm importância no cenário das pesquisas, independentemente das diferentes áreas do conhecimento, é fato indiscutível. Com o crescente volume da produção científica, é imprescindível observar a maneira pela qual debates, reflexões e panoramas se constituem em determinado âmbito. O REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 19 presente trabalho se inclui, assim, em tal perspectiva. Evidenciar, para compreender os objetos que conformam o campo educacional, é tarefa complexa e que exige esforço dos pesquisadores e, neste caso, a ANPEd, como antes mencionado, é espaço significativo na socialização da produção científica das pesquisas em Educação. Isso justifica, portanto, a escolha do lócus. Por outro lado, não há como negar que o campo da Comunicação influencia, mais e mais, os processos educativos quando a escola se percebe atravessada por artefatos tecnológicos, pela mídia e, mais recentemente, pelas mídias digitais. Analisar e compreender tal fenômeno é, ao que indica o levantamento aqui realizado, objeto e objetivo que têm constituído os estudos aprovados no GT 16 da ANPEd. É muito provável que outros GTs da associação também discutam esse mesmo objeto, o que abre a possibilidade de novas empreitadas de estudo; todavia, há de se marcar a identidade do GT 16 na discussão antes caracterizada. É, pois, na identidade do GT 16 que se configura o panorama aqui exposto: temáticas como mídia, TIC, EaD, ciberespaço, redes sociais, voluntariado e artes estão presentes, todo o tempo, nos estudos a ele vinculados, reforçando e consagrando uma “vocação”. Com relação aos autores, inferindo que suas análises consubstanciam apreciações das temáticas/objetos de estudo, fica marcada a tentativa de transcender a especificidade de uma análise que se gostaria mais implicada no educacional/educativo. Pensar a educação em seu caráter histórico, social e político, por exemplo, e a partir daí as suas intencionalidades é, sem dúvida, entender tendências relevantes das pesquisas, no sentido de que a escola possa se apropriar de saberes e fazeres implicados com uma educação pública e de qualidade, consoante o compromisso dos pesquisadores. Considerando a pandemia da Covid-19, o GT 16 torna-se ainda mais relevante para o contexto de ampliação/divulgação de pesquisas que possam se relacionar ainda mais ao que é implicado ao educacional/educativo, demanda de tempo atual. 4 Considerações finais Associado a isso, observar esse foco requer ampliar a inclusão e o escopo dos pesquisadores que atuam no GT. É, então, nesse domínio, que o presente trabalho incita pensar sobre como o tema Educação e Comunicação proporia, ou discutiria, teorias educacionais e pedagógicas que aprofundassem a compreensão do cotidiano escolar em resistência aos movimentos que, REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 20 frequentemente, desvalorizam o espaço da escola pública, apoiando, em consequência, o fortalecimento do trabalho docente com os discentes. Assunto, claro, para ser discutido por nós que frequentamos a ANPEd, mas, sobretudo, por pesquisadores cujo lócus de reflexão é o GT16. frequentemente, desvalorizam o espaço da escola pública, apoiando, em consequência, o fortalecimento do trabalho docente com os discentes. Assunto, claro, para ser discutido por nós que frequentamos a ANPEd, mas, sobretudo, por pesquisadores cujo lócus de reflexão é o GT16. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 21 SANTOS, A. L. F.; AZEVEDO, J. M. L. A pós-graduação no Brasil, a pesquisa em educação e os estudos sobre a política educacional: os contornos da constituição de um campo acadêmico. Revista Brasileira de Educação, Rio de Janeiro, v. 14, n. 42, p. 534-550, 2009. Disponível em: http://www.scielo.br/pdf/rbedu/v14n42/v14n42a10.pdf. Acesso em: 12 mar. 2019. RAMOS, A.; FARIA, P. M.; FARIA, A.. Revisão sistemática de literatura: contributo para a inovação na investigação em ciências da educação. Revista Diálogo Educacional, Curitiba, v. 14, n. 41, p. 17-36, 2014. Disponível em: https://periodicos.pucpr.br/index.php/dialogoeducacional/article/view/2269. Acesso em: 2 abr. 2019. ROMANOWSKI, J. P.; ENS, R. T. As pesquisas denominadas do tipo “Estado da Arte” em Educação. Diálogo Educacional. Curitiba, v. 6, n. 19, 2006. Disponível em: http://www2.pucpr.br/reol/index.php/DIALOGO?dd1=237&dd99=view. Acesso em: 10 jul. 2013. SANTOS, A. L. F.; AZEVEDO, J. M. L. A pós-graduação no Brasil, a pesquisa em educação e os estudos sobre a política educacional: os contornos da constituição de um campo acadêmico. Revista Brasileira de Educação, Rio de Janeiro, v. 14, n. 42, p. 534-550, 2009. Disponível em: http://www.scielo.br/pdf/rbedu/v14n42/v14n42a10.pdf. Acesso em: 12 mar. 2019. Referências BONILLA, M. H. S. A presença da cultura digital no GT Educação e Comunicação da Anped. Revista Teias, Rio de Janeiro, v. 13, n. 30, p. 71-93, 2012. Disponível em: https://www.e-publicacoes.uerj.br/index.php/revistateias/article/view/24272. Acesso em: 10 fev. 2019. FISCHER, R. M. B. Rastros de um passado nem tão remoto: mídias audiovisuais em vinte anos de pesquisa. Revista Teias, Rio de Janeiro, v. 13, n. 30, p. 23-42, 2012. Disponível em: https://www.e-publicacoes.uerj.br/index.php/revistateias/article/view/24270/17249. Acesso em: 10 fev. 2019. FISCHER, R. M. B. Rastros de um passado nem tão remoto: mídias audiovisuais em vinte anos de pesquisa. Revista Teias, Rio de Janeiro, v. 13, n. 30, p. 23-42, 2012. Disponível em: https://www.e-publicacoes.uerj.br/index.php/revistateias/article/view/24270/17249. Acesso em: 10 fev. 2019. FREITAS, L D.; ARRUDA, J. A.; FALQUETO, J. M. Z. Uso do software NVivo→ em investigação qualitativa: ferramenta para pesquisa nas ciências sociais. Atas Investigación Cualitativa en Ciências Sociales, online, v. 3, 2017. Disponível em: https://proceedings.ciaiq.org/index.php/ciaiq2017/article/view/1436. Acesso em: 18 fev. 2019. OLIVEIRA, J. F.; LIMA, D. C. B. P. Pós-Graduação e Educação a Distância: novos fins, natureza e modus operandi em construção. In: CASTRO, A.; CABRAL NETO, A.; CABRITO, B.; CERDEIRA, L.; CHAVES, V. J. (Orgs.). Educação superior em países e regiões de língua portuguesa: desafios em tempo de crise. Lisboa: Educa, 2018. p. 215-242. PEIXOTO, J. Relações entre sujeitos sociais e objetos técnicos uma reflexão necessária para investigar os processos educativos mediados por tecnologias. Revista Brasileira de Educação, v. 20 n. 61 abr.-jun. 2015, p. 317-332. Disponível em: https://www.scielo.br/pdf/rbedu/v20n61/1413-2478-rbedu-20-61-0317.pdf. Acesso em: 28 mai. 2020. PRETTO, N. D. L. Educação, comunicação e a ANPEd: uma história em movimento. 30ª Reunião Nacional da ANPEd. Anais eletrônicos [...]. Caxambú: ANPEd, 2007. Disponível em: PRETTO, N. D. L. Educação, comunicação e a ANPEd: uma história em movimento. 30ª Reunião Nacional da ANPEd. Anais eletrônicos [...]. Caxambú: ANPEd, 2007. Disponível em: http://30reuniao.anped.org.br/trabalhos_encomendados/trabalho%20encomendado%20gt16% 20-%20nelson%20de%20pretto%20-%20int.pdf. Acesso em: 16 fev. 2019. http://30reuniao.anped.org.br/trabalhos_encomendados/trabalho%20encomendado%20gt16% 20-%20nelson%20de%20pretto%20-%20int.pdf. Acesso em: 16 fev. 2019. REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 21 REVELLI, Vol. 13. 2021. Dossiê qualidade e inovação da/na educação: concepções, possibilidades e desafio ISSN 1984-6576. E-202122 1 1
https://openalex.org/W4323315965	https://www.frontiersin.org/articles/10.3389/fviro.2023.1137133/pdf	English	null	Oral intake of heat-killed Lactiplantibacillus pentosus ONRICb0240 partially protects mice against SARS-CoV-2 infection	Frontiers in virology	2,023	cc-by	5,235	TYPE Brief Research Report PUBLISHED 06 March 2023 DOI 10.3389/fviro.2023.1137133 TYPE Brief Research Report PUBLISHED 06 March 2023 DOI 10.3389/fviro.2023.1137133 TYPE Brief Research Report PUBLISHED 06 March 2023 DOI 10.3389/fviro.2023.1137133 Front. Virol. 3:1137133. doi: 10.3389/fviro.2023.1137133 COPYRIGHT © 2023 Kiso, Uraki, Ito, Yamayoshi, Kotani, Imai, Kohda and Kawaoka. This is an open- access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. SARS-CoV-2, probiotics, Lactiplantibacillus pentosus ONRICb0240, mouse model, host responses SARS-CoV-2, probiotics, Lactiplantibacillus pentosus ONRICb0240, mouse model host responses Oral intake of heat-killed Lactiplantibacillus pentosus ONRICb0240 partially protects mice against SARS-CoV-2 infection OPEN ACCESS EDITED BY Keita Matsuno, Hokkaido University, Japan REVIEWED BY Kei Miyakawa, National Institute of Infectious Diseases (NIID), Japan Elahe Abdolalipour, Pasteur Institute of Iran, Iran CORRESPONDENCE Yoshihiro Kawaoka yoshihiro.kawaoka@wisc.edu SPECIALTY SECTION This article was submitted to Emerging and Reemerging Viruses, a section of the journal Frontiers in Virology RECEIVED 04 January 2023 ACCEPTED 20 February 2023 PUBLISHED 06 March 2023 CITATION Kiso M, Uraki R, Ito M, Yamayoshi S, Kotani Y, Imai M, Kohda N and Kawaoka Y (2023) Oral intake of heat-killed Lactiplantibacillus pentosus ONRICb0240 partially protects mice against SARS-CoV-2 infection. Front. Virol. 3:1137133. doi: 10 3389/fviro 2023 1137133 Maki Kiso 1, Ryuta Uraki 1,2, Mutsumi Ito 1, Seiya Yamayoshi 1,2, Yoshifumi Kotani 3, Masaki Imai 1,2, Noriyuki Kohda 3 and Yoshihiro Kawaoka 1,3,4,5 1Division of Virology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, 2The Research Center for Global Viral Diseases, National Center for Global Health and Medicine Research Institute, Tokyo, Japan, 3Otsu Nutraceuticals Research Institute, Nutraceuticals Division, Otsuka Pharmaceutical Co., Ltd., Shiga, Japan, 4Inﬂuenza Research Institute, Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, WI, United States, 5The University of Tokyo, Pandemic Preparedness, Infection and Advanced Research Center, Tokyo, Japan CITATION Kiso M, Uraki R, Ito M, Yamayoshi S, Kotani Y, Imai M, Kohda N and Kawaoka Y (2023) Oral intake of heat-killed Lactiplantibacillus pentosus ONRICb0240 partially protects mice against SARS-CoV-2 infection. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the ongoing coronavirus pandemic. Besides vaccines and antiviral drugs, probiotics have attracted attention for prevention of SARS-CoV-2 infection. Here, we examined the efﬁcacy of heat-killed Lactiplantibacillus pentosus ONRICb0240 (b240) against SARS-CoV-2 infection in mice. We observed that oral intake of heat-killed b240 did not affect virus titers in the respiratory organs of SARS-CoV-2-infected mice, but did provide partial protection against SARS- CoV-2 infection. In addition, heat-killed b240 treatment suppressed the expression of IL-6, a key proinﬂammatory cytokine, on Day 2 post-infection. Our results highlight the promising protective role of heat-killed b240 and suggest a possible mechanism by which heat-killed b240 partially protects against SARS-CoV-2 infection by modulating host responses. Front. Virol. 3:1137133. doi: 10.3389/fviro.2023.1137133 frontiersin.org Viruses Excised animal tissues were ﬁxed in 4% paraformaldehyde in PBS and processed for parafﬁn embedding. The parafﬁn blocks were cut into 3-µm-thick sections and mounted on silane-coated glass slides for histopathological examination. The sections were stained with hematoxylin and eosin. Mouse-adapted SARS-CoV-2 was generated by serial passages of SARS-CoV-2 (gamma: hCoV-19/Japan/TY7-501/2021) (18) in BALB/c mice. The detailed methods of mouse adaptation are currently unpublished (manuscript in preparation). Mouse- adapted SARS-CoV-2 was propagated in VeroE6/TMPRSS2 cells in VP-SFM (Thermo Fisher Scientiﬁc). Animal experiments and approvals Animal studies were carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocols were approved by the Animal Experiment Committee of the Institute of Medical Science, the University of Tokyo (approval number PA19-72). All animals were housed under speciﬁc pathogen-free conditions in a temperature control environment with a 12 h: 12h light: dark cycle, with 50% humidity and ad libitum access to water and standard laboratory chow. Virus inoculations were performed under anesthesia, and all efforts were made to minimize animal suffering. Probiotics are deﬁned as live microorganisms that provide health beneﬁts to the host when administered in adequate amounts, (3); they include several genera of bacteria and yeast such as Lactobacillus, Biﬁdobacterium, Leuconostoc, Pediococcus, and Enterococcus (4, 5). Probiotics play an important role in balancing the intestinal microﬂora, which leads to modulation of the immune system. Previous studies have shown that probiotics have antiviral activity against respiratory viruses such as rhinovirus, inﬂuenza virus, respiratory syncytial virus, and SARS-CoV-2 (6–8). Although probiotics provide physiological beneﬁts to the host, their safety proﬁles remain controversial, because they are live strains (9). Therefore, there is increasing interest in non-viable microorganisms or microbial cell extracts to avoid the risks of using live microorganisms. Lactiplantibacillus pentosus ONRICb0240 (b240) is an anaerobic, non-sporulating, Gram-positive bacterium originally isolated from fermented tea leaves. Clinical trials have demonstrated that heat-killed b240 enhances salivary IgA secretion, reduces the incidence of the common cold, and alleviates allergic symptoms (10–12). In addition, we and other groups have previously reported that oral intake of heat-killed b240 modulates mucosal immunity, which provides protection against inﬂuenza virus, Streptococcus pneumoniae, and Salmonella infection (13–16). Here, we evaluated the protective efﬁcacy of heat-killed b240 against SARS- CoV-2 infection in mice. Cells VeroE6/TMPRSS2 (JCRB 1819) cells (17) were propagated in the presence of 1 mg/ml geneticin (G418; In vivogen) and 5 mg/ml plasmocin prophylactic (In vivogen) in Dulbecco’s modiﬁed Eagle’s medium (DMEM) containing 10% Fetal Calf Serum (FCS). VeroE6/ TMPRSS2 cells were maintained at 37 °C with 5% CO2 and regularly tested for mycoplasma contamination by using PCR, and conﬁrmed to be mycoplasma-free. Introduction Coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), emerged in China at the end of 2019 and has continued to spread throughout the world. The World Health Organization (WHO) reported that as of September 2022, about 600 million cases of COVID-19 and 6.4 million associated deaths have occurred. Vaccination against COVID-19 is currently the most- Frontiers in Virology 01 frontiersin.org Kiso et al. 10.3389/fviro.2023.1137133 Diseases, Japan, which are approved for such use by the Ministry of Agriculture, Forestry, and Fisheries, Japan. effective ﬁrst line of defense against severe disease and death; however, the antigenicity of circulating SARS-CoV-2 variants affects the efﬁcacy of the COVID-19 vaccines. Therapeutic monoclonal antibodies and antiviral drugs are available for the treatment of COVID-19 (1, 2); however, the risk of emerging escape or resistant viruses drives the need for alternative approaches. Frontiers in Virology Experimental infection of mice Six-week-old female BALB/c mice (Japan SLC Inc., Shizuoka, Japan) were used in the study. Oral administration of heat-killed b240 was initiated in mice at six weeks of age. Mice were orally administered heat-killed b240 every day at a dose of 10 mg/mouse, which corresponds to 1010 cell counts of heat-killed microbe, in 200 ml of buffered saline for 5 weeks. The control group received saline. The b240 dose was determined on the basis of previous studies (14, 15). On Day 21 of heat-killed b240 administration, mice were intranasally infected with PBS, or with 0.3 or 0.6 MLD50 of mouse-adapted SARS-CoV-2 [MLD50 = 103.3 plaque forming units (PFU).] under isoﬂurane anesthesia. To determine the effects of oral administration of heat-killed b240 on mouse mortality, mice were infected with 0.3 or 0.6 MLD50 of SARS- CoV-2 and their body weight and survival were monitored daily for 10 days post-infection (n = 20 for 0.3 MLD50; n = 10 for 0.6 MLD50). To investigate the effects of oral administration of heat- killed b240 on viral replication and host immune responses, the animals infected with 0.6 MLD50 of mouse-adapted SARS-CoV-2 were euthanized on Days 2 and 5 post-infection, and the virus titers in the nasal turbinates and lungs were determined by using plaque assays on VeroE6/TMPRSS2 cells. frontiersin.org Results D (Roche, Basel, Switzerland)) for 30 min at 37°C. The single-cell suspension was ﬁltered through a 70-µm cell strainer and washed twice with 5 ml of RPMI 1640. Leukocytes were enriched by centrifugation (14 min, 700 ×g) on a 33% Percoll gradient (Cytiva, Marlborough, MA, USA) in HBSS, and red blood cells (RBCs) were lysed by RBC lysis buffer (pluriSelect Life Science UG & Co.KG, Leipzig, Germany). Cells were then incubated with anti- CD16/32 Ab (93) to block Fc receptors and stained with antibodies speciﬁc to CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD11b (M1/70), CD8a (53–6.7), CD11c (N418), I-A/I-E (M5/114.15.2), Ly6G (1A8), and Ly6C (HK1.4) from Biolegend or eBioscience (San Diego, CA, USA) and Live/Dead ﬁxable aqua (Thermo Fisher Scientiﬁc, Waltham, MA, USA). D (Roche, Basel, Switzerland)) for 30 min at 37°C. The single-cell suspension was ﬁltered through a 70-µm cell strainer and washed twice with 5 ml of RPMI 1640. Leukocytes were enriched by centrifugation (14 min, 700 ×g) on a 33% Percoll gradient (Cytiva, Marlborough, MA, USA) in HBSS, and red blood cells (RBCs) were lysed by RBC lysis buffer (pluriSelect Life Science UG & Co.KG, Leipzig, Germany). Cells were then incubated with anti- CD16/32 Ab (93) to block Fc receptors and stained with antibodies speciﬁc to CD3 (17A2), CD45 (30-F11), CD4 (RM4-5), CD11b (M1/70), CD8a (53–6.7), CD11c (N418), I-A/I-E (M5/114.15.2), Ly6G (1A8), and Ly6C (HK1.4) from Biolegend or eBioscience (San Diego, CA, USA) and Live/Dead ﬁxable aqua (Thermo Fisher Scientiﬁc, Waltham, MA, USA). To evaluate the prophylactic effects of heat-killed b240 against SARS-CoV-2, we orally administered heat-killed b240 (10 mg/ mouse) to Balb/c mice once daily for 21 days before intranasal infection with 0.3 or 0.6 MLD50 (50% mouse lethal dose) of mouse- adapted SARS-CoV-2. Thereafter, heat-killed b240 was administered once daily for 10 days. When mice were infected with 0.3 MLD50, the heat-killed b240 showed statistically signiﬁcant improvement in body weight changes and survival (Figure 1A). In mice infected with 0.6 MLD50, although no signiﬁcant differences in body weight or survival were observed between the animals that were treated with heat-killed b240 and those treated with saline (control), we observed smaller body weight reductions and better survival compared with 0.6 MLD50 infection of saline-treated mice (Figure 1B). Overall, these results suggest that heat-killed b240 may partially protect against SARS-CoV-2 infection. Cytokine and chemokine measurement Under isoﬂurane anesthesia, twelve mice per group were infected with 0.6 MLD50 of mouse-adapted SARS-CoV-2 on Day 21 of heat-killed b240 administration. On Day 0 (pre) prior to the infection, and Days 2 and 5 post-infection, animals were euthanized and their lungs were collected. For cytokine and chemokine measurements, homogenates of mouse lungs were processed with the Bio-Plex Mouse Cytokine 23-Plex (Bio-Rad Laboratories). We next assessed the effect of heat-killed b240 on virus replication in the respiratory tract of mice infected with the higher dose (0.6 MLD50) of mouse-adapted SARS-CoV-2. No obvious differences in virus titers in the nasal turbinates and lungs were found between the animals that received heat-killed b240 and those that received saline on Days 2 and 5 post-infection (Figure 2A). Furthermore, there were no differences in lung histology between heat-killed b240-treated mice and control mice on Day 5 post-infection (Figure 2B). Cell preparation and ﬂow cytometry All experiments with SARS-CoV-2 were performed in enhanced biosafety level 3 (BSL3) containment laboratories at the University of Tokyo and the National Institute of Infectious To isolate single cells from lungs, lung tissue was minced, and fragments were digested in 5 ml of DMEM containing collagenase 02 frontiersin.org Kiso et al. Kiso et al. 10.3389/fviro.2023.1137133 10.3389/fviro.2023.1137133 10.3389/fviro.2023.1137133 Results For detailed analyses, we chose the higher dose (i.e., 0.6 MLD50) to compare virus replication and host responses under more severe conditions. Data were acquired with CytoFLEX S (Beckman Coulter Inc., Brea, CA, USA) and data analysis was performed using FlowJo software (FlowJo, Ashland, OR, USA). Reagent availability Probiotics have regulatory effects on host innate and adaptive immune responses (19, 20). Therefore, to assess whether heat-killed b240 can alter immune cell recruitment to the lungs following infection with SARS-CoV-2 in mice, heat-killed b240-treated mice were intranasally inoculated with 0.6 MLD50 of mouse-adapted SARS-CoV-2, and whole lungs were harvested pre-infection (Day 0) and on Days 2 and 5 post-infection. Flow cytometry analysis revealed no differences in CD4+T and CD8+T cell numbers in the lungs between heat-killed b240- and control vehicle-treated mice at any timepoints (Figures 3A, B). A rapid increase in neutrophil recruitment was observed in the lungs of infected mice treated with All materials are available from the authors or from commercially available sources. Statistical analysis GraphPad Prism software was used to analyze the data. Statistical analysis included unpaired Student’s t-tests, Mann- Whitney tests, the Log-rank (Mantel-Cox) test, and ANOVA with post-hoc tests. Differences among groups were considered signiﬁcant for P values < 0.05. A B FIGURE 1 Efﬁcacy of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were administered heat-killed b240 at a dose of 10 mg/mouse daily for 21 days prior to infection and for 14 days after infection. Mice in the control group were administered saline. Mice were then intranasally infected with 0.3 (A) or 0.6 MLD50 (B) of mouse-adapted SARS-CoV-2 on Day 21 of heat-killed b240 administration. Body weight (left panels) and survival (right panels) were monitored daily for 10 days. The data are presented as the mean percentages of the starting weight ± s.e.m. Weight data were analyzed by using a two-way ANOVA followed by Dunnett’s test. Survival data were analyzed by using the Log-rank (Mantel-Cox) test. n = 20 for 0.3 MLD50, n = 10 for 0.6 MLD50. B A B B B FIGURE 1 Efﬁcacy of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were administered heat-killed b240 at a dose of 10 mg/mouse daily for 21 days prior to infection and for 14 days after infection. Mice in the control group were administered saline. Mice were then intranasally infected with 0.3 (A) or 0.6 MLD50 (B) of mouse-adapted SARS-CoV-2 on Day 21 of heat-killed b240 administration. Body weight (left panels) and survival (right panels) were monitored daily for 10 days. The data are presented as the mean percentages of the starting weight ± s.e.m. Weight data were analyzed by using a two-way ANOVA followed by Dunnett’s test. Survival data were analyzed by using the Log-rank (Mantel-Cox) test. n = 20 for 0.3 MLD50, n = 10 for 0.6 MLD50. FIGURE 1 Efﬁcacy of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were administered heat-killed b240 at a dose of 10 mg/mouse daily for 21 days prior to infection and for 14 days after infection. Mice in the control group were administered saline. Mice were then intranasally infected with 0.3 (A) or 0.6 MLD50 (B) of mouse-adapted SARS-CoV-2 on Day 21 of heat-killed b240 administration. Body weight (left panels) and survival (right panels) were monitored daily for 10 days. The data are presented as the mean percentages of the starting weight ± s.e.m. Statistical analysis Weight data were analyzed by using a two-way ANOVA followed by Dunnett’s test. Survival data were analyzed by using the Log-rank (Mantel-Cox) test. n = 20 for 0.3 MLD50, n = 10 for 0.6 MLD50. 03 Frontiers in Virology frontiersin.org Kiso et al. Kiso et al. 10.3389/fviro.2023.1137133 A B FIGURE 2 Virologic effect of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were infected with 0.6 MLD50 of mouse-adapted SARS-CoV-2 on Day 21 of heat-killed b240 administration and euthanized on Days 2 and 5 post-infection. (A) Virus burdens in the lungs and nasal turbinates were determined by performing plaque assays. The values are means ± s.e.m. (n = 4). Points indicate data from individual mice. The lower limit of detection is indicated by the horizontal dashed line. Statistical signiﬁcance was determined with a two-tailed Student’s t-test (lung) or the Mann- Whitney test (nasal turbinate). (B) Histopathologic examination of the lungs of infected mice (n = 3/group) on Day 5 post-infection. Representative images of infected mice are shown. A B B B FIGURE 2 Virologic effect of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were infected with 0.6 MLD50 of mouse-adapted SARS-CoV-2 on Day 21 of heat-killed b240 administration and euthanized on Days 2 and 5 post-infection. (A) Virus burdens in the lungs and nasal turbinates were determined by performing plaque assays. The values are means ± s.e.m. (n = 4). Points indicate data from individual mice. The lower limit of detection is indicated by the horizontal dashed line. Statistical signiﬁcance was determined with a two-tailed Student’s t-test (lung) or the Mann- Whitney test (nasal turbinate). (B) Histopathologic examination of the lungs of infected mice (n = 3/group) on Day 5 post-infection. Representative images of infected mice are shown. heat-killed b240 and the control mice on Day 2 post-infection, although no statistically signiﬁcant difference in the percentage of neutrophils was observed between the two groups (Figures 3C, D). We also saw no difference in the percentage of Ly6chiCD11b+ or Ly6c+CD11b+ monocytes between the two groups (Figure 3D). Interestingly, however, the percentage of dendritic cells (DCs) was signiﬁcantly higher for infected mice treated with heat-killed b240 compared with the infected control mice on Day 2 post- infection (Figure 3D). to sites of infection. These mediators are also associated with pulmonary inﬂammation and lung damage. Statistical analysis Elevated levels of proinﬂammatory cytokines such as IL-6 and TNFa have been reported in patients with severe COVID-19 (21, 23, 24). We therefore examined the effects of oral administration of heat- killed b240 on the expression levels of pro-inﬂammatory cytokines (i.e., IL-1a, IL-1b, IL-2, IL-6, IL-12p40, IL-12p70, IL- 17A, TNFa, and IFNg) in mice infected with SARS-CoV-2 (Figure 3E). Consistent with our previous report (14), no signiﬁcant differences in cytokine levels in the lungs were found between non-infected mice that were treated with heat-killed b240 Proinﬂammatory cytokines (21, 22), which are the central host mediators of innate immunity, are essential to recruit immune cells Frontiers in Virology 04 frontiersin.org Kiso et al. Kiso et al. 10.3389/fviro.2023.1137133 A B D E C URE 3 munologic effect of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were infected with 0.6 MLD50 of mouse-adapted SARS- V-2 on Day 21 of heat-killed b240 administration. On Day 0 (pre) prior to the infection and Days 2 and 5 post-infection, mice were euthanized d their lungs were harvested. (A–D) Frequency of immune cells in lungs examined by use of ﬂow cytometry. Representative gating strategies used dentify CD4+T and CD8+T cells (A), and neutrophil, monocytes, and dendritic cells (C) are shown. Cell frequency data are shown (n = 4/group) D). The values are means ± s.e.m. (n = 4). Points indicate data from individual mice. Statistical signiﬁcance was determined with a two-way OVA followed by Tukey’s multiple comparisons test. (E) The expression of proinﬂammatory cytokines in mouse lungs is shown. Vertical bars show mean ± s.e.m (n = 4). Points indicate data from individual mice. Data were analyzed by using a two-way ANOVA with Tukey’s multiple mparisons test. All values were normalized to the mean value of the saline-treated mice on Day 0 (pre) prior to the infection. A B C B A B C C C D D E E FIGURE 3 Immunologic effect of oral intake of heat-killed b240 in SARS-CoV-2-infected mice. Mice were infected with 0.6 MLD50 of mouse-adapted SARS- CoV-2 on Day 21 of heat-killed b240 administration. On Day 0 (pre) prior to the infection and Days 2 and 5 post-infection, mice were euthanized and their lungs were harvested. (A–D) Frequency of immune cells in lungs examined by use of ﬂow cytometry. Representative gating strategies used to identify CD4+T and CD8+T cells (A), and neutrophil, monocytes, and dendritic cells (C) are shown. Statistical analysis Cell frequency data are shown (n = 4/group) (B, D). The values are means ± s.e.m. (n = 4). Points indicate data from individual mice. Statistical signiﬁcance was determined with a two-way ANOVA followed by Tukey’s multiple comparisons test. (E) The expression of proinﬂammatory cytokines in mouse lungs is shown. Vertical bars show the mean ± s.e.m (n = 4). Points indicate data from individual mice. Data were analyzed by using a two-way ANOVA with Tukey’s multiple comparisons test. All values were normalized to the mean value of the saline-treated mice on Day 0 (pre) prior to the infection. Publisher’s note All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Conﬂict of interest YKa is a co-founder of FluGen and has received related funding support from Otsuka Pharmaceutical Co., Ltd. as well as unrelated funding support from Daiichi Sankyo Pharmaceutical, Toya-ma Chemical, Tauns Laboratories, Inc., Shionogi & Co. LTD, KM Biolog-ics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio. YKo and NK are employees of Otsuka Pharmaceutical Co., Ltd. In conclusion, our data suggest that oral intake of heat-killed Lactiplantibacillus pentosus ONRICb0240 promotes the survival of SARS-CoV-2-infected mice. Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Funding This work was supported by the Japan Program for Infectious Diseases Research and Infrastructure (JP22wm0125002) and the Japan Initiative for World-leading Vaccine Research and Development Centers (JP223fa627001) from the Japan Agency for Medical Research and Development. Acknowledgments We thank Susan Watson for scientiﬁc editing. We also thank Rie Onoue, Madoka Yoshikawa, Naoko Mizutani, and Kengo Kajiyama for technical assistance, and Sept.Sapie Co., Ltd. for pathological analyses of the lungs of the mice. We found that DCs were slightly but signiﬁcantly increased in heat-killed b240-treated mice on Day 2 post-infection. Previous studies have demonstrated that DCs induce cytotoxic T lymphocytes (CTL)-mediated antiviral immunity (32, 33), which suggest that increased levels of DCs may contribute to protection upon SARS-CoV-2 infection. How DCs are recruited or induced in the lungs of SARS-CoV-2-infected mice upon oral administration of heat-killed b240 should be examined in a future study. Author contributions inﬂammatory cytokines such as IL-6 and TNF-a after Streptococcus pneumoniae infection in mice (10) and that probiotics such as Biﬁdobacterium longum MM-2 and Lactobacillus plantarum 06CC2 inhibit the production of pro-inﬂammatory cytokines such as IL-6 and TNF-a after inﬂuenza virus infection in mice (25, 26). In addition, we previously showed that heat-killed b240 modulates the expression levels of genes involved in metabolism and antiviral responses in mice, which may result in the partial protection of pdmH1N1 inﬂuenza virus-infected mice by heat-killed b240 (14). Recent studies indicated that tightly regulated microbiota-host interplay inﬂuences the establishment of the immune system, which affect the outcome after pathogen-infection (27–30). Therefore, it is possible that heat-killed b240 could have altered the microbiota-host interaction, leading to the inhibition of IL-6 production in SARS-CoV-2-infected animals. Further investigation is needed to assess how heat-killed b240 treatment leads to the suppression of IL-6 production in virus- infected mice. Overall, these ﬁndings suggest that oral administration of heat-killed b240 may modulate the host immune responses in lungs infected with respiratory viruses such as inﬂuenza virus and SARS-CoV-2. Recently, several studies have reported improved outcomes in COVID-19 patients who received probiotics in clinical trials, suggesting promising beneﬁcial effects of probiotics as part of COVID-19 management (31). It would be interesting to extend our study and examine the effect of b240 in COVID-19 patients. MK, RU, YKo, MIm, NK, and Y.Ka conceived and designed the research. Y.Ko and NK contributed reagents. MK, RU, MIt, and SY performed the experiments and analyzed the data. RU, MIm, and YKa wrote the initial draft, with all other authors providing editorial comments. All authors contributed to the article and approved the submitted version. Discussion and those given the control vehicle, indicating that oral administration of heat-killed b240 does not induce inﬂammatory responses (Figure 3E). The expression levels of proinﬂammatory cytokines in the lungs of infected mice treated with heat-killed b240 were similar to those in infected mice given the control vehicle, except for IL-6 on Day 2 post-infection; the IL-6 expression level was signiﬁcantly lower for the former than for the latter (Figure 3E). These results suggest that heat-killed b240 may reduce the early host inﬂammatory responses including IL-6-mediated proinﬂammatory signaling caused by SARS-CoV-2 infection, leading to partial protection against SARS-CoV-2 infection. Our previous study showed that oral administration of heat-killed b240 enhanced protection against a lethal inﬂuenza A(H1N1) pdm virus in a mouse model (14). In the present study, we found that the oral intake of heat-killed b240 partially protects mice from SARS- CoV-2 infection. The heat-killed b240 treatment did not affect the virus titers in the respiratory organs of the mice infected with SARS- CoV-2; however, this treatment suppressed the expression of proinﬂammatory cytokines in the lungs. Previous studies have reported that heat-killed b240 inhibits the production of pro- Frontiers in Virology 05 frontiersin.org Kiso et al. Kiso et al. 10.3389/fviro.2023.1137133 References 1. Ho C, Lee PC. COVID-19 treatment-current status, advances, and gap. 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https://openalex.org/W3210271961	https://journals.sfu.ca/cob/index.php/files/article/download/395/pdf_76	English	null	Mental Health and Negative Sexual Experiences of Bahamian University Students	The International journal of Bahamian studies	2,021	cc-by	9,543	ERROR: type should be string, got "https://doi.org/10.15362/ijbs.v27i0.395  W. Fielding & V. Ballance, 2021.\nJournal compilation International Journal of Bahamian Studies, 2021. University of The Bahamas University of The Bahamas Abstract This study investigates the mental health of victims of sexual abuse, particularly rape, in college \nstudent populations. The study identifies the connection between sexual abuse/rape and elevated \nscores of mental health. It indicates that even with other stressful events occurring in the lives of \nrespondents, sexual abuse/rape has a detrimental effect on the mental health of both males and \nfemales. A death in the family was the most commonly reported stressful event for males and \nfemales, and females were more likely than males to have suffered from a sexual attack. Early \nnegative experiences of sexual intercourse can apparently have long lasting negative effects on the \nvictim’s mental health. These findings require society to look beyond the physical consequences \nof sexual abuse and rape to ensure that the long-term mental health of victims, both male and \nfemale children and adults, is not overlooked. ORIGINAL ARTICLES \nMental Health and Negative Sexual Experiences of \nBahamian University Students Elizabeth J. Williams \nWilliam J. Fielding \n 0000-0001-5433-9673 \nVirginia C. Ballance \n 0000-0003-1067-8205 Introduction Bahamas highlighted that many victims of \nrape are unaware that they have been raped \nand that females can engage in unwanted \nsexual intercourse due to being afraid of their \nintimate partner. Bethel and Fielding (2020) \nalso found that men can be forceful in getting \ntheir intimate partners to participate in sexual \nintercourse, even when it is unwanted. They \nalso found that college students still \nsubscribe to myths with regard to sexual \nintercourse, such as strangers are most likely \nto rape a woman. Although there have been many studies do\nin The Bahamas on risky sexual behavio\n(Deveaux & Rolle, 2016), other aspects\nsexual abuse and rape appear to have be\nless studied. Gender-based violence in T\nBahamas is a national concern. The relativ\nhigh incidence of rape has been recogniz\nthrough the Bahamas National Task Force \nGender-based \nViolence \nreport \n(201\nAlthough women in particular are worr\nabout being victims of rape, their fear be\nno relationship to the reported crime figu\non rape. This suggests that even a singu\nevent causes great worry, and/or th\npresume that rape is more common than \nofficial figures suggest, a position that\nconfirmed by both Aranha (2016) and Bet\nand Fielding (2020). The study by Bethel a\nFielding (2020) on college students in T\nTable 1 \nNumber of Rapes, by Sex, Reported t\nYear \nMale \nn = \nFe\n2017 \n0 \n2018 \n4 \n2019 \n3 \nNote. Figures subject to change, Royal Baham\nBethel and Fielding (2020) found in th\nstudy that none of the men who were rap\nreported their victimization, so they would\nunlikely to benefit from any formal supp\nto cope with their trauma. Even though m\nrape is less common than female rape\nshould not be overlooked because it may\neven more traumatic for men than wom\nMusevenzi and Musevenzi (2018) illustr\nthis in their study of men in Zimbabwe w\nwere raped and fear being seen as femin\nand no longer being masculine. Afric\nchildren’s experiences of physical violen Although there have been many studies done \nin The Bahamas on risky sexual behaviours \n(Deveaux & Rolle, 2016), other aspects of \nsexual abuse and rape appear to have been \nless studied. Gender-based violence in The \nBahamas is a national concern. The relatively \nhigh incidence of rape has been recognized \nthrough the Bahamas National Task Force for \nGender-based \nViolence \nreport \n(2015). Dedication Dr. Elizabeth Williams joined The College of The Bahamas on September 1, 2004 as a lecturer in \nthe Critical Care Nursing programme. Her doctoral dissertation was entitled The Influence of \nKnowledge and Self-efficacy on Bahamian Women’s Adherence to a Hypertensive Medication \nRegime (2011) which recognized the importance of hypertension in our community. Her gentle \nmanner made her a favourite with students, who voted her as the best lecturer in the School of \nNursing and Allied Health Professions. Her love of research was evident in the way she taught the \nhigher-level research classes and her enthusiasm and dedication made her a popular teacher of \nresearch. She encouraged her students to study important topics, and this last research project of \nhers is a testament to her willingness to address “hard” issues which impact women’s health. This paper, dedicated to the life and memory of Dr. Elizabeth Williams, is the project that she was \nworking on before her untimely death. The completion of this project would not have been possible \nwithout the exceptional support of the co-authors. I hope that the findings of this study will fill an \nimportant gap in our knowledge of mental health in The Bahamas and add to the current body of \nknowledge. Ingrid Mobley, Ph.D., ARNP https://doi.org/10.15362/ijbs.v27i0.395  W. Fielding & V. Ballance, 2021. Journal compilation International Journal of Bahamian Studies, 2021. 2 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. Bahamas highlighted that many victims of \nrape are unaware that they have been raped \nand that females can engage in unwanted \nsexual intercourse due to being afraid of their \nintimate partner. Bethel and Fielding (2020) \nalso found that men can be forceful in getting \ntheir intimate partners to participate in sexual \nintercourse, even when it is unwanted. They \nalso found that college students still \nsubscribe to myths with regard to sexual \nintercourse, such as strangers are most likely \nto rape a woman. Method poorer mental health among women (Potter \net al., 2020) and may result in depression and \nanxiety (Nickerson et al., 2013). The long-\nterm effects of child sexual abuse on the \nmental health of adult females include anger, \ndepression, and sexual problems (Hailes et \nal., 2019). An Internet-based survey was devised that \nincorporated some of the questions asked by \nBethel and Fielding (2020). These included \nstandard \nitems \non \nrespondents’ \ndemographics as well as questions about their \nlifetime experiences of sexual intercourse \nand their attitudes towards rape. The \nquestionnaire also included 19 aspects \nrelating to the mental health of the \nrespondent. These \naspects \nincluded \nstatements such as, “I wish I were somebody \nelse,” “I feel like I am inferior to others,” and \n“I blame myself for being sexually abused.” \nThe writing authors believe that these \nquestions may have been drawn from a \nnumber of stress/trauma related scales (such \nas \nAmirkhan, \n1990; \nSharma, \n2018; \nSwahnberg & Wijma, 2003) and possibly \nreworded to be culturally appropriate for a \nBahamian student population. The scale was \na Likert response scale with a frequency \nresponse scale from 1 (never) to 5 (always), \nso the minimum score in this scale was 19 \nand the maximum score 95. In this study, the \nCronbach’s α for the scale was .94. The \nsurvey included additional questions about \nevents in the life of participants that may have \nbeen stressful, such as a death in the family \nor being a victim of crime. An Internet-based survey was devised that \nincorporated some of the questions asked by \nBethel and Fielding (2020). These included \nstandard \nitems \non \nrespondents’ \ndemographics as well as questions about their \nlifetime experiences of sexual intercourse \nand their attitudes towards rape. The \nquestionnaire also included 19 aspects \nrelating to the mental health of the \nrespondent. These \naspects \nincluded \nstatements such as, “I wish I were somebody \nelse,” “I feel like I am inferior to others,” and \n“I blame myself for being sexually abused.” In the Caribbean, Pilgrim and Blum (2012) \nidentified a number of environmental factors, \nincluding peers who engaged in violence, as \nhaving negative influences on mental health; \nthe importance of mental health issues has \nresulted in the Caribbean medical literature \ndevoting special issues to the topic (Barton, \n2012). Introduction Although women in particular are worried \nabout being victims of rape, their fear bears \nno relationship to the reported crime figures \non rape. This suggests that even a singular \nevent causes great worry, and/or they \npresume that rape is more common than the \nofficial figures suggest, a position that is \nconfirmed by both Aranha (2016) and Bethel \nand Fielding (2020). The study by Bethel and \nFielding (2020) on college students in The Another rape related myth that persists in The \nBahamas is that only females can be victims \nof rape (Rolle, 2020). Unpublished statistics \nprovided by the Royal Bahamas Police Force \n(see Table 1) confirm that male rape occurs, \nalthough it is accepted that male and female \nrape is under-reported (Bethel & Fielding, \n2020). Table 1 \nNumber of Rapes, by Sex, Reported to the Royal Bahamas Police Force \nYear \nMale \nn = \nFemale \n n = \nMales raped \n% \n2017 \n0 \n56 \n0% \n2018 \n4 \n56 \n6.7% \n2019 \n3 \n44 \n6.4% \nNote. Figures subject to change, Royal Bahamas Police Force (unpublished). Number of Rapes, by Sex, Reported to the Royal Bahamas Police Force and sexual violence have been related to risky \nsexual behaviour and mental health concerns \n(Smith et al., 2020). This demonstrates that \nadverse sexual experiences in both boys and \ngirls can be detrimental to their mental \nhealth. It has also been demonstrated in the \nUnited States that forced sexual intercourse \nhas detrimental effects beyond physical and \nmental trauma, such as negative academic \noutcomes—effects that can be life-long and \nhave implications for the development of a \ncountry (Rees & Sabia, 2013). In the United \nStates, the experience of rape was linked to Bethel and Fielding (2020) found in their \nstudy that none of the men who were raped \nreported their victimization, so they would be \nunlikely to benefit from any formal support \nto cope with their trauma. Even though male \nrape is less common than female rape, it \nshould not be overlooked because it may be \neven more traumatic for men than women; \nMusevenzi and Musevenzi (2018) illustrate \nthis in their study of men in Zimbabwe who \nwere raped and fear being seen as feminine \nand no longer being masculine. African \nchildren’s experiences of physical violence International Journal of Bahamian Studies Vol. 27 (2021) E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences 3 Findings There were 1,240 starts in the survey. One \nperson who entered the survey declined to \nparticipate. After cleaning the data so that \nonly respondents who were members of the \ntarget \npopulation \nwere \nincluded, \n865 \nrespondents were retained. Not all surveys \nwere completed, so this is the maximum \nnumber of respondents. Two respondents \ngave their sex as “other” and this group was \nomitted due the small number of responses. Of the remainder, most respondents were \nfemale (82.1% of N = 860). Although most of \nthe results are presented disaggregated by \nsex, in the case of males, in some cases the \nnumber of responses was small, so we then \njust present the results from the female \nparticipants. The modal age group was 21-24 \nyears (42.3%). Overall, 84.4% of participants \nhad participated in sexual intercourse; this Method After cleaning the data so that \nonly respondents who were members of the \ntarget \npopulation \nwere \nincluded, \n865 \nrespondents were retained. Not all surveys \nwere completed, so this is the maximum \nnumber of respondents. Two respondents \ngave their sex as “other” and this group was \nomitted due the small number of responses. Of the remainder, most respondents were \nfemale (82.1% of N = 860). Although most of \nthe results are presented disaggregated by \nsex, in the case of males, in some cases the \nnumber of responses was small, so we then \njust present the results from the female \nparticipants. The modal age group was 21-24 \nyears (42.3%). Overall, 84.4% of participants \nhad participated in sexual intercourse; this \nTable 2 \nMean Ranked Fear of Being a Victim of Se\nCrime, mean rank \nBeing raped \nBeing shot \nBeing sexually abused \nBeing held up and robbed \nBeing physically attacked by another \nHaving your home broken into \nHaving your car stolen \nHaving something stolen from your car \nNote. 1= most fearful, 8 least fearful; p values from \nAttitudes Toward Rape \nFemales were more aware than men of the \nimportance of consent with regard to sexual Context of the Fear of Rape Fear of rape is one of many anxieties, so \nrespondents were asked to rank their \nconcerns; they were more concerned about \ncrimes against the person than property (see \nTable 2). The fear of being a victim of crime \nwas dissimilar between the sexes. Table 2 \nshows that rape is the crime most feared by \nwomen, whereas men are most fearful of \nbeing shot. As demonstrated by Bethel and \nFielding (2020), the fear of crime does not \nreflect the statistical occurrence of crimes \nreported by the Royal Bahamas Police Force \n(2020). Table 2 \nMean Ranked Fear of Being a Victim of Selected Crimes Table 2 \nMean Ranked Fear of Being a Victim of Selected Crimes \nCrime, mean rank \nMale \nN ≈ 147 \nFemale \nN ≈ 665 \np = \nM \nM \nBeing raped \n4.2 \n2.6 \n< .001 \nBeing shot \n3.0 \n3.6 \n< .001 \nBeing sexually abused \n5.3 \n3.8 \n< .001 \nBeing held up and robbed \n3.5 \n4.2 \n< .001 \nBeing physically attacked by another \n4.2 \n4.3 \n.47 \nHaving your home broken into \n4.0 \n4.7 \n< .001 \nHaving your car stolen \n5.2 \n5.9 \n< .001 \nHaving something stolen from your car \n6.6 \n6.8 \n.081 \nNote. 1= most fearful, 8 least fearful; p values from t-test. International Journal of Bahamian Studies Vol. 27 (2021) \nTable 2 \nMean Ranked Fear of Being a Victim of Selected Crimes \nCrime, mean rank \nMale \nN ≈ 147 \nFemale \nN ≈ 665 \np = \nM \nM \nBeing raped \n4.2 \n2.6 \n< .001 \nBeing shot \n3.0 \n3.6 \n< .001 \nBeing sexually abused \n5.3 \n3.8 \n< .001 \nBeing held up and robbed \n3.5 \n4.2 \n< .001 \nBeing physically attacked by another \n4.2 \n4.3 \n.47 \nHaving your home broken into \n4.0 \n4.7 \n< .001 \nHaving your car stolen \n5.2 \n5.9 \n< .001 \nHaving something stolen from your car \n6.6 \n6.8 \n.081 \nNote. 1= most fearful, 8 least fearful; p values from t-test. Attitudes Toward Rape \nFemales were more aware than men of the \nimportance of consent with regard to sexual \nintercourse, and men were more likely to \nview rape as requiring physical resistance by Table 2 \nMean Ranked Fear of Being a Victim of Selected Crimes Method The 2017 World Health Organization \nreport on mental health indicates that, in The \nBahamas, many of its indicators have not \nbeen reported on, and there appears to be a \nlack of mental health programmes (World \nHealth Organization, 2017). The literature on violence and rape often \nfocusses on university students living on \nresidential campuses. However, the study \npopulation in this research project, although \nuniversity-aged students, typically live in \ndomestic \nsettings, \nnot \nstudent \naccommodation, so their experiences can be \nexpected to reflect those of non-resident \ncampus populations, such as that of students \nattending community colleges (Voth Schrag \n& Edmond, 2018; Potter et al., 2020). Therefore, the responses of participants \nshould not be assumed to reflect sexual \nexperiences in a specific place. The target population was enrolled college-\nlevel students in The Bahamas. Students from \na nursing research class used their social \nmedia contacts to recruit students, with credit \nbeing given to students for getting students to \nparticipate in the survey via a link to the \nSurveyMonkey™ questionnaire. Although \nsuch a student population does not reflect the \nwider population, its members are drawn \nfrom across the country, particularly New \nProvidence, so it predominately represents an \nurban population. The students reflect a range \nof economic backgrounds, and many receive \nfinancial aid from the government to attend \nuniversity. The study was approved by the The purpose of this cross-sectional study was \nto focus on the mental health of victims of \nrape or sexual abuse in college students. It \naimed to identify links, if any, between being \na victim of rape and the victim’s mental \nhealth. It also aimed to assess the impact of \nother traumatic events to put the event of rape \nin a wider context. International Journal of Bahamian Studies Vol. 27 (2021) 4 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences Institutional Review Board of University of \nThe Bahamas. percentage was similar for both sexes (χ2 = \n.54, df = 1, N = 858, p = .816). However, \nfemales were more likely than males to be \nparticipating in stable relationships (sexually \ninvolved with the same partner for 12 months \nor more): 59.1% compared with 47.3% of \nmales (χ2 = 7.03, df = 1, N = 853, p = .008). International Journal of Bah\nInstitutional Review Board of University of \nThe Bahamas. Findings \nThere were 1,240 starts in the survey. One \nperson who entered the survey declined to \nparticipate. Table 3 Table 3 \nPercentage of Respondents, Within Sex, Agreeing to Various Aspects of Rape \nAspects of rape \nMale Female \nχ2 \n \n% \n% \np = \nRape occurs when one of those engaged in the sexual intercourse \ndid not consent \n92.1 \n96.3 \n.019 \nRape occurs when one of those engaged in sexual intercourse \nconsents due to threats or fear of bodily harm from the other person \n86.8 \n85.3 \n.483 \nA married couple cannot rape each other \n39.6 \n43.8 \n.221 \nRape only occurs when the victim tries to fight off their attacker \n15.9 \n9.7 \n.011 \nOnly females can be raped \n4.0 \n1.3 \n.085 p = .82). Males tended to engage in sexual \nintercourse at an earlier age than females (see \nFigure 1). We also note that 30.7% of males \nand 15.7% of females who had participated \nin sexual intercourse did so before age 16. p = .82). Males tended to engage in sexual \nintercourse at an earlier age than females (see \nFigure 1). We also note that 30.7% of males \nand 15.7% of females who had participated \nin sexual intercourse did so before age 16. Attitudes Toward Rape intercourse, and men were more likely to \nview rape as requiring physical resistance by Females were more aware than men of the \nimportance of consent with regard to sexual International Journal of Bahamian Studies Vol. 27 (2021) E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 5 the victim to make the unwanted sex rape (see \nTable 3). Close to 40% of both males and \nfemales agreed that rape cannot occur within \nmarriage; this indicates the considerable \ndisagreement on this topic in a society where \nthe law recognizes rape only outside of \nmarriage, as demonstrated in debates on \nmartial rape (Benjamin & LeGrand, 2012). Some respondents in this study indicated that they may have been sexually abused by their \nhusbands. Although this study did not ask \nabout marital status directly, when we \ngrouped respondents who were aged over 30 \nand in long-term relationships (one year or \nmore) together with those who were married, \n7.2% of these 69 females reported having sex \nagainst their will with their partner in their \nmost recent sexual encounter. Participation in Sexual Intercourse Similar percentages of males (16.2%) and \nfemales (15.5%) had never participated in \nsexual intercourse (χ2 = .054, df = 1, N = 858, International Journal of Bahamian Studies Vol. 27 (2021) \nFigure 1 \nPercentage of Males and Females by Age at First Experience of Sexual Intercourse \n \n0%\n5%\n10%\n15%\n20%\n25%\nUnder 14\n14\n15\n16\n17\n18\n19-24\nOver 24\nMale\nFemale International Journal of Bahamian Studies Vol. 27 (2021) 6 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. Males and females reported having different \nexperiences of their first participation in \nsexual intercourse. Females were more likely \nthan males to agree that their first experience \nof sexual intercourse was not agreeable and, \non reflection, was abuse (see Table 4). However, it should be noted that both males \nand females felt that they had been abused \nwhen they first had sexual intercourse and \nboth males and females had various degrees \nof negative experiences. Table 4 \nPercentage of Respondents Agreeing to Various Aspects of their First Experience of \nSexual Intercourse \nFirst experience of sexual intercourse \nMale \nFemale \nΧ2 \n \n% \n% \np = \nI had sexual intercourse against my will \n4.8 \n12.6 \n.048 \nI found the experience enjoyable \n73.6 \n41 \n< .001 \nI did not enjoy it but my partner did \n13.6 \n38.1 \n< .001 \nI clearly agreed to have sexual intercourse \n93.5 \n78.3 \n.001 \nAfterwards, I wished I had not agreed to the sexual \nintercourse \n9.6 \n37.0 \n< .001 \nI knew the person well with whom I had sexual intercourse \n76.6 \n88.0 \n.017 \nAfterwards, I wanted to repeat the experience \n80.6 \n45.8 \n< .001 \nOn reflection, I feel that I was sexually abused when I first \nhad sexual intercourse \n5.6 \n14.1 \n.009 Respondents Agreeing to Various Aspects of their First Experience of Although clear consent prior to having sex \nwas given by most males and females, \nrespondents indicated that they sometimes \nwere unable to give consent or gave consent \nout of fear of their partner (see Table 5). Several differences in the experiences \nsurrounding sexual intercourse were reported \nby males and females as seen in Table 5. Females reported being hurt by their partners, \n23.6% of females had been hit by their \nintimate partners. Participating in sexual \nintercourse under circumstances of fear or \nincapacitation invalidates the consent and \nmakes it rape. Participation in Sexual Intercourse Table 6 \nPercentage of Respondents Agreeing to Various Aspects of their Most Recent \nExperience of Sexual Intercourse \nAgreeing to \nMale Female \nΧ2 \n \n% \n% \np = \nI had sexual intercourse against my will \n4.1 \n5.7 \n.273 \nI found the experience enjoyable \n87.9 \n82.4 \n.163 \nI did not enjoy it but my partner did \n11.3 \n12.5 \n.197 \nI clearly agreed to have sexual intercourse \n96.8 \n91.0 \n.072 \nAfterwards, I wished I had not agreed to the sexual intercourse \n7.4 \n11.4 \n.055 \nI knew the person well with whom I had sexual intercourse \n91.0 \n92.0 \n.63 \nAfterwards, I wanted to repeat the experience \n81.8 \n80.3 \n.202 \nOn reflection, I feel that I was sexually abused when I had sexual \nintercourse \n2.5 \n3.7 \n.299 \nTable 7 \nChoice Sex of Intimate Partner by Sex of Respondent. Sex of those with whom respondents had sexual intercourse \nMale % Female % \nOnly male \n4.2 \n87.0 \nOnly female \n89.2 \n0.7 Table 5 \nPercentage of Respondents Agreeing to Various Aspects of their Experiences of Sexual \nIntercourse \nAspect \nMale \nFemale \nΧ2 \n \n% \n% \np = \nDo you always give clear consent (verbal or non-verbal permission) \nbefore having sexual intercourse? 79.8 \n81 \n.61 \nHave you ever had sexual intercourse with an individual because \nyou were afraid of them? 1.7 \n15 \n< .001 \nHave you ever had sexual intercourse when you were physically or \nmentally unable to give consent? (e.g. drunk or high) \n24.4 \n25.6 \n.032 \nHave you ever had sexual intercourse when your partner was \nphysically or mentally unable to give consent? (e.g. drunk or high) \n18.5 \n15.5 \n.44 \nHave you ever had sexual intercourse with a person under the age \nof 16? 6.7 \n1.4 \n< .001 \nHave you been hit or physically hurt by your intimate partner? 10.1 \n23.6 \n< .001 \nNote. Options include Not sure except when marked §. Respondents Agreeing to Various Aspects of their Experiences of Sexual International Journal of Bahamian Studies Vol. Participation in Sexual Intercourse Table 5 also indicates that \n6.7% of males raped females due to having \nsex with underage partners. In their most recent experience of sexual \nintercourse, there were no statistically \nsignificant differences between the sexes in \ntheir attitudes towards their experience (see \nTable 6). This is a marked contrast to their \nrecollections of their first experience of \nsexual intercourse as seen in Table 4. In their most recent experience of sexual \nintercourse, there were no statistically \nsignificant differences between the sexes in \ntheir attitudes towards their experience (see \nTable 6). This is a marked contrast to their \nrecollections of their first experience of \nsexual intercourse as seen in Table 4. Significantly \nmore \nfemale \nthan \nmale \nrespondents reported having sexual partners \nof both sexes (χ2 = 564.9, df = 2, N = 674, p \n< .001; see Table 7). This finding is \nconsistent with Bethel and Fielding (2020) \nand broadly in line with female choice of \npartner in the United States (Tansill et al., \n2012). International Journal of Bahamian Studies Vol. 27 (2021) E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 7 Table 5 \nPercentage of Respondents Agreeing to Various Aspects of their Experiences of Sexual \nIntercourse \nAspect \nMale \nFemale \nΧ2 \n \n% \n% \np = \nDo you always give clear consent (verbal or non-verbal permission) \nbefore having sexual intercourse? 79.8 \n81 \n.61 \nHave you ever had sexual intercourse with an individual because \nyou were afraid of them? 1.7 \n15 \n< .001 \nHave you ever had sexual intercourse when you were physically or \nmentally unable to give consent? (e.g. drunk or high) \n24.4 \n25.6 \n.032 \nHave you ever had sexual intercourse when your partner was \nphysically or mentally unable to give consent? (e.g. drunk or high) \n18.5 \n15.5 \n.44 \nHave you ever had sexual intercourse with a person under the age \nof 16? 6.7 \n1.4 \n< .001 \nHave you been hit or physically hurt by your intimate partner? 10.1 \n23.6 \n< .001 \nNote. Options include Not sure except when marked §. Participation in Sexual Intercourse 27 (2021) \nTable 6 \nPercentage of Respondents Agreeing to Various Aspects of their Most Recent \nExperience of Sexual Intercourse \nAgreeing to \nMale Female \nΧ2 \n \n% \n% \np = \nI had sexual intercourse against my will \n4.1 \n5.7 \n.273 \nI found the experience enjoyable \n87.9 \n82.4 \n.163 \nI did not enjoy it but my partner did \n11.3 \n12.5 \n.197 \nI clearly agreed to have sexual intercourse \n96.8 \n91.0 \n.072 \nAfterwards, I wished I had not agreed to the sexual intercourse \n7.4 \n11.4 \n.055 \nI knew the person well with whom I had sexual intercourse \n91.0 \n92.0 \n.63 \nAfterwards, I wanted to repeat the experience \n81.8 \n80.3 \n.202 \nOn reflection, I feel that I was sexually abused when I had sexual \nintercourse \n2.5 \n3.7 \n.299 \nTable 7 \nChoice Sex of Intimate Partner by Sex of Respondent. Sex of those with whom respondents had sexual intercourse \nMale % Female % \nOnly male \n4.2 \n87.0 \nOnly female \n89.2 \n0.7 \nBoth male and female \n6.7 \n12.3 Respondents Agreeing to Various Aspects of their Most Recent \nS\nl I t International Journal of Bahamian Studies Vol. 27 (2021) \nTable 7 \nChoice Sex of Intimate Partner by Sex of Respondent. Sex of those with whom respondents had sexual intercourse \nMale % Female % \nOnly male \n4.2 \n87.0 \nOnly female \n89.2 \n0.7 \nBoth male and female \n6.7 \n12.3 Table 7 International Journal of Bahamian Studies Vol. 27 (2021) \nTable 7 \nChoice Sex of Intimate Partner by Sex of Respondent. Sex of those with whom respondents had sexual intercourse \nMale % Female % \nOnly male \n4.2 \n87.0 \nOnly female \n89.2 \n0.7 \nBoth male and female \n6.7 \n12.3 8 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 8 International Journal of Bahamian Studies Vol. 27 (2021) Events Impacting Mental Health Females were more likely than males to \nreport being a victim of physical violence \nfrom their intimate partner: 23.6% of females \nand 10.1% of males (χ2 = 10.8, df = 1, N = \n673, p = .001). A larger percentage of female \nthan male participants (47.9% of 368 \nresponses, compared to 33.1% of 103 \nresponses; χ2 = 7.15, df = 1, N = 471, p = \n.007) had participated in sexual intercourse \nagainst their will; in the case of females who \nhad engaged in unwanted sexual intercourse, \n27.8% had done so in the last month. Similarly, 23.7% of 536 females and 7% of \n113 males admitted to having been raped at \nleast once (χ2 = 16.2, df = 4, N = 649, p = \n.003). Only one of 11 male respondents \nobtained medical help for physical reasons \nfollowing the sexual abuse. In the case of \nfemales, 5.6% of 88 respondents sought help \nfor mental health reasons after being sexually \nabused, and 4.3% sought help for physical \nreasons following the abuse. Of the 24 males \nwho had suffered sexual abuse/rape, 58.3% \nagreed that it had affected their mental health; \nin the case of females, 81.7% of 241 \nrespondents agreed that it has affected their \nmental health. Various events in our lives can have negative \nimpacts upon us and some of these events \nwere included in the study. Here we consider \nevents that respondents thought had impacted \ntheir mental health. Females were more likely \nthan males to have suffered at least one event \nin Table 8 that they thought negatively \naffected their mental health in the previous 10 \nyears: 54.8% of females and 40.3% of males \n(χ2 = 10.77, df = 1, N = 856, p = .001). The \nmost commonly reported stressful event \nrelated to a death of a friend or family \nmember. However, the negative impact \nassociated with being a victim of a sexual \nattack was clearly different for males and \nfemales. Overall, it was apparent that females \nwere more likely than males to report events \nin their lives that affected their mental health \n(χ2 = 46.6, df = 1, N = 860, p < .001, see Table \n8). The other category included natural \ndisasters, such as Hurricane Dorian and the \nCovid-19 pandemic, and life events, such as \nending a relationship (break-up) and taking \nuniversity examinations. Table 8 Table 9 \nPercentage of Female Respondents Reporting Being a Victim of Rape and Also \nExperiencing Events that Had Negatively Affected Their Mental Health, Percentages \nWithin Victim of Rape \nVictim of rape \nA death of a \nfriend/ family \nmember \nVictim of \nproperty \ncrime \nVictim of a \nphysical \nattack \nVictim of a \nsexual attack \nVictim of \nbullying \n \n \n% \n% \n% \n% \n% \nN \nNo, never \n27.9 \n4.9 \n4.9 \n4.2 \n8.1 \n409 \nOnly once \n43.9 \n10.6 \n10.6 \n33.3 \n12.1 \n66 \nSometimes \n26.7 \n4.4 \n15.6 \n40.0 \n15.6 \n45 \nOften \n33.3 \n8.3 \n41.7 \n75.0 \n33.3 \n12 \nFrequently \n50.0 \n0% \n25.0 \n25.0 \n0 \n4 Table 9 \nPercentage of Female Respondents Reporting Being a Victim of R\nExperiencing Events that Had Negatively Affected Their Mental Healt\nWithin Victim of Rape Female Respondents Reporting Being a Victim of Rape and Also \nvents that Had Negatively Affected Their Mental Health, Percentages Table 8 Table 8 \nPercentage of Respondents Reporting Events that Had Negatively Affected Their Mental \nHealth \nNegative event \nMale \nFemale \n \n \n% \n% \np = \nA death of a friend/family member \n27.9 \n29.6 \n.68 \nVictim of bullying \n7.1 \n10.9 \n.16 \nVictim of a sexual attack \n2.6 \n10.8 \n.002 \nVictim of a physical attack \n3.2 \n6.9 \n.087 \nVictim of property crime \n5.2 \n5.1 \n.96 \nOther \n9.7 \n18.1 \n.006 \nN \n154 \n706 \n \nNote. Multiple answers allowed. Table 8 \nPercentage of Respondents Reporting Events that Had Negatively Affected Their Mental \nHealth \nN\nti\nt\nM l\nF\nl Respondents Reporting Events that Had Negatively Affected Their Mental E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 9 9 reverse may be the case. In Table 9, we only focused on females being \nvictims of rape due to the relatively small \nnumber of males who were victims of rape. Being a victim of rape is associated with \nelevated chances of experiencing other \nevents \nthat \nrespondents \nconsidered \ndetrimental to their mental health (χ2 = 55.5, \ndf = 16, N = 350, p < .001, see Table 9). Using \nthe responses of participants who had never \nbeen victims of rape as a benchmark, it can \nbe appreciated that as the occurrence of rape \nincreases, so do other stressful events, or the Being a victim of a sexual attack resulted in \nthe largest differences between the mental \nhealth scores of victims and non-victims of \nany of the negative events in Table 9 \n(victims, M = 48.5, non-victims, M = 35.9, \nt(630) = 7.48, p < .001). Analysis of \ncovariance, \ntaking \ninto \naccount \nthe \noccurrence of the events in Table 9, resulted \nin adjusted means (victims, M = 45.9, non-\nvictims, M = 36.3, p < .001). Mental Health Scores It would appear that there may be long lasting \neffects on the mental health of females who \nfelt abused when they first participated in \nsexual intercourse, as those who felt sexually \nabused when first initiated into sex had a \nhigher mean mental health score than those \nwho did not feel abused (see Table 10). This \nwould suggest that even though other events \nhad a negative impact on the mental health of \nfemale participants, it was still possible to \ndetect the residual negative effect of their \nfirst sexual encounter. The 19 questions on mental health were used \nto calculate a mental health score, with lower \nscores indicative of better mental health than \nhigher scores. The minimum score in this \nscale was 19 and the maximum score 95. The \noverall mean mental health score was 37.3 \n(SE = .54). However, females had a higher \nmental health score than males (females, M = \n37.9, SE = .6, males, M = 34.3, SE = 1.26, t = \n-2.57, df = 627, p = .01). This result is \nconsistent with other literature from the \nCaribbean (Pilgrim & Blum, 2012). International Journal of Bahamian Studies Vol. 27 (2021) 10 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 10 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 10 Table 10 \nFemale Experience of First Sexual \nIntercourse and Mental Health Score \nSexually abused at \nfirst sexual \nintercourse \nM \nSE \np = \n1 Strongly agree \n44.8 2.05 \n<.001 \n2 \n46.2 6.3 \n3 Cannot say \n39.9 1.86 \n4 \n40.2 2.54 \n5 Strongly disagree \n36.2 .65 \nNote. p value from analysis of variance. Table 10 \nFemale Experience of First Sexual \nIntercourse and Mental Health Score partner was associated with a higher mean \nmental health score (t = 3.81, df = 629, p < \n.001). partner was associated with a higher mean \nmental health score (t = 3.81, df = 629, p < \n.001). In the case of rape, both males and females \nhad higher mean mental health scores, even \nwhen the scores were adjusted for other \nnegative events in their lives, other than \nsexual abuse (see Table 12). Mental Health Scores Although the \nmean mental health score for males who had \nbeen raped sometimes has a high standard \nerror due to the small sample size, the fact \nthat it is the largest mean in the table may still \nindicate the detrimental effect rape has on \nmales. This is more evident when we \nappreciate that, overall, males had a lower \nmental health score than females (males, \n34.3, SE = 1.26, females, 37.9, SE = .60), a \nmean difference of 3.6. Females who had experienced unwanted \nsexual intercourse reported having higher \nmental health concerns the more frequently \nthey had had unwanted sex (see Table 11); \ntherefore, within the group of females \nparticipating in unwanted sex, the repetition \nof the event is linked with a higher mean \nmental health score. In the case of females, \nthe more frequently they had had sex against \ntheir will, the higher their mental health \nscore: r = .16 (p < .001). Although the mental health scores between \nrespondents who had had sexual relations \nwith others of the same sex were not \nstatistically different (ANOVA, F(2, 622) = \n1.21, p = .3), the interaction between sex of \nrespondent and sexual partner (F(2, 622) = \n2.2, p = .112) was suggestive of greater \ntrauma associated with respondents who \nparticipated exclusively in non-heterosexual \nsex (see Table 13). The relatively small \nsample size of the number of respondents \nwho reported sexual intercourse with both \nsexes or the same sex as themselves may \naccount for the lack of statistical significance. Table 11 \nMean Mental Health Score and Lifetime \nExperience of Unwanted Sex of Female \nRespondents \nHad sex against \nyour will: \nM \nSE \np = \nOnly once \n37.4 \n.89 \n \n.001 \nA few times \n41.6 \n1.44 \nOften \n46.4 \n5.14 \nFrequently \n49.8 \n6.09 \nNote. p value from analysis of variance. N = 324 Table 11 \nMean Mental Health Score and Lifetime \nExperience of Unwanted Sex of Female \nRespondents Female study participants who had been \nsexually abused indicated that the most likely \nvictimizers were people known to them, in \nparticular current or ex-boyfriends or friends. However, it should be noted that relatives and \nthose with trusted access to the victim were \nreported as victimizers by about a quarter of \nthe abused respondents. Relatively few \nfemales had been victimized by strangers \n(see Table 14). This finding is consistent with \nBethel and Fielding (2020) and in line with a \nstudy from the United States (Brooks, 2001). Mental Health Scores In the case of their most recent sexual \nencounter, those females who felt that they \nhad been abused had higher mental health \nscores than those who did not feel abused \n(Kruskal-Wallis H = 12.6, df = 4, p = .013). Also, having been hit by their intimate International Journal of Bahamian Studies Vol. 27 (2021) E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 11 Table 12 \nMean Mental Health Score and Respondent Lifetime Experience of Rape, by Sex of \nRespondent \nSex \nRaped \nM \nSE \nN \np = \nMale \nNever \n33.6 \n1.18 \n101 \n.003 \nOnly once \n35.5 \n5.98 \n4 \nSometimes \n58.4 \n6.96 \n3 \nFemale \nNever \n36.2 \n.66 \n389 \n.007 \nOnly once \n40.3 \n1.67 \n62 \nSometimes \n43.1 \n1.99 \n44 \nOften \n46.6 \n4.06 \n11 \nFrequently \n40.6 \n6.35 \n4 \nNote: Means adjusted for occurrence of negative events in the lives of the respondents in Table 7, excluding \nsexual abuse. p values from analysis of variance. Health Score and Respondent Lifetime Experience of Rape, by Sex of Note: Means adjusted for occurrence of negative events in the lives of the respondents in Table 7, excluding \nsexual abuse. p values from analysis of variance. International Journal of Bahamian Studies Vol. 27 (2021) \nTable 13 \nMean Mental Scores of Respondents, by Their Sex and the Sex of Their Intimate Partners \nSex of those with whom respondents had sexual intercourse \nMale \nFemale \n \nM \nM \nOnly male \n49.6 \n37.3 \nOnly female \n33.0 \n38.7 \nBoth male and female \n40.6 \n42.3 \nTable 14 \nAssociation Between the Victimizer and Female Sex Abuse Victim \nFemale participants having had sex against their will with: \n% reports \nGroup % \nBoyfriend \n35.9 \n \nBest friend \n0.5 \n \nBoyfriend (first time) \n0.5 \n \nBoyfriend and uncle \n0.5 \n \nBoyfriend, brother \n0.5 \n \nBoyfriend/Guy I had frequent sex with \n0.5 \n \nBoyfriends, cousins, uncles \n0.5 \n \nIntimate partner/ friends with benefits \n0.5 \n \nEx-boyfriend \n4.6 \n \nFirst ex-boyfriend \n0.5 \n \nNow ex-boyfriend \n0.5 \n \nDate \n1.5 \n46.7 \nGroup n = \n91.0 \n \nFriend \n1.8 \n \nFamily friend \n3.1 \n \nFriend, brother \n1.0 Table 13 \nMean Mental Scores of Respondents, by Their Sex and the Sex of Their Intimate Partners \nSex of those with whom respondents had sexual intercourse \nMale \nFemale \n \nM \nM \nOnly male \n49.6 \n37.3 \nOnly female \n33.0 \n38.7 \nBoth male and female \n40.6 \n42.3 International Journal of Bahamian Studies Vol. Mental Health Scores 27 (2021) \nTable 14 \nAssociation Between the Victimizer and Female Sex Abuse Victim \nFemale participants having had sex against their will with: \n% reports \nGroup % \nBoyfriend \n35.9 \n \nBest friend \n0.5 \n \nBoyfriend (first time) \n0.5 \n \nBoyfriend and uncle \n0.5 \n \nBoyfriend, brother \n0.5 \n \nBoyfriend/Guy I had frequent sex with \n0.5 \n \nBoyfriends, cousins, uncles \n0.5 \n \nIntimate partner/ friends with benefits \n0.5 \n \nEx-boyfriend \n4.6 \n \nFirst ex-boyfriend \n0.5 \n \nNow ex-boyfriend \n0.5 \n \nDate \n1.5 \n46.7 \nGroup n = \n91.0 \n \nFriend \n1.8 \n \nFamily friend \n3.1 \n \nFriend, brother \n1.0 12 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 12 Female participants having had sex against their will with: \n% reports \nGroup % \n“Friend” and boyfriend \n1.0 \n \n“Friend” \n0.5 \n \n“Trusted” friend \n0.5 \n \nFriend, we were never actually together, we never make it official \n0.5 \n \nFriend/Neighbor \n0.5 \n \nA guy who was my friend but isn’t my friend anymore \n0.5 \n \nEx Best friend \n0.5 \n \nFiancé and Old Friend \n0.5 \n \nNot intercourse, but assaulted or touched against my will by someone I \nwas friends with \n0.5 \n20 \nGroup n = \n39 \n \nHusband \n4.6 \n \nUncle \n1.5 \n \nUncle, close friend at former high school \n0.5 \n \nMy children [sic] daddy \n0.5 \n \nFather \n0.5 \n \nStepfather \n2.6 \n \nGodfather \n0.5 \n \nBrother \n2.6 \n \nCousin \n6.7 \n \nCousin (male) \n1.0 \n \nCousin and girl in primary school \n0.5 \n \nRape and Sexual Abuse- Male cousin \n0.5 \n \nRelatives \n0.5 \n \nGod brothers \n0.5 \n23.1 \nGroup n = \n45 \n \nNeighbour \n1.5 \n \nA situationship I was in [verbatim, not clear to authors] \n0.5 \n \nBabysitters [sic] son, uncle, brother \n0.5 \n \nCan't say \n0.5 \n \nCo worker \n0.5 \n \nDa dog outside \n0.5 \n \nEx friend’s uncle \n0.5 \n \nJust sexually assaulted/abused \n0.5 \n \nSexually abused never raped. First time it was my cousin. Second a \npolice officer. Third a priest. 0.5 \n \nOlder neighbour \n0.5 \n \nThe entire world, police and family \n0.5 \n6.7 \nGroup n = \n13 \n \nStranger \n3.1 \n \nStranger I met once prior \n0.5 \n3.6 \nGroup n= \n7 \n \nN = \n195 International Journal of Bahamian Studies Vol. 27 (2021) E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 13 Bethel and Fielding (2020), who reported on \nthe life-time sexual experiences of college \nstudents in The Bahamas. The fear of rape \nwas again found to be the crime of greatest \nconcern to females, even though, according \nto police statistics, it is not a commonly \nreported crime (Bethel & Fielding, 2020). Mental Health Scores The current study confirms that sexual \nintercourse is an activity that is not without \nrisk of violence, particularly for females, and \nsome females, even if not admitting to being \nraped, clearly agreed to participate in sexual \nintercourse out of fear. This may help to \nexplain the fear that females have of rape, \neven if they do not feel that they have been \nraped. The violence to which they are \nsubjected by their intimate partner may \nultimately be manifested by rape. Therefore, \nviolence appears to be a normative aspect of \nfemale life. The fact that about 25% of the \nfemale participants had unwanted sex in the \nprevious month indicates that unwanted \nencounters are not uncommon. As Bethel and \nFielding (2020) found, circumstances such as \nthis increase the percentage of individuals \nwho are legally raped beyond the percentage \nof people who admit to being raped. Discussion Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 14 As might have been expected, respondents \nhad experienced a number of events that they \nfelt had negatively affected their mental \nhealth; the most common of these was a death \nof someone whom they knew. Respondents \nwho had reported being raped were also more \nlikely to have suffered other events in their \nlives that had negative impacts on their \nmental health. As noted in Norway, \nchildhood experiences of violence increase \nthe risk of these children being victimized as \nadults and increase their risk of suffering \nfrom mental health concerns (Thoresen et al., \n2015). This study cannot explain why this \nmight be, but it may be associated with \npersons who have less coping capability than \nothers with respect to negative events. This \nresult warrants further study. Although the first sexual experience of \nfemales was less satisfactory than for males, \nit is apparent that the most recent sexual \nencounters of males and females were \nexperienced with equal approval, even \nthough some respondents may have felt \nabused. The study confirmed that persons \nknown to the victim were the most likely \ngroup to inflict sexual abuse/rape. Family \nmembers accounted for about 25% of \nvictimizers identified by respondents (Table \n14), indicating that family members used \ntheir privileged position in the family to \nvictimize others. This helps to explain why \nsexual abuse/rape is under reported, as it is \nconcealed behind the walls of the home, \nwhich hinders reporting and investigation of \ncases. Dating violence in adolescent women \nhas been demonstrated to be associated with \ngreater mental health concerns (Hébert et al., \n2008). What is of interest, and merits further study \nin the Bahamian context, is that it appears \nthat the negative aspects of the respondents’ \nsexual initiation appear to be detectable today \nthrough their mental health score, a finding \nconsistent with Dovran et al. (2016) and \nBurgić Radmanović (2020). This suggests \nthat a person’s early sexual experience can \nhave life-long consequences. This would \nsuggest that every effort should be made to \nensure that a person is appropriately prepared \nfor engaging in sexual intercourse, even if the \nact is consensual, or how a participant might \nprotect themselves from abuse, if it is not \nconsensual. Discussion In considering the results, we should be \naware that the target population was college-\nlevel students, not the wider population, so, \nalthough the study provides useful insights \nwith regard to sexual experiences and mental \nhealth, it may not reflect what occurs in the \nwider and more diverse population of The \nBahamas, particularly with respect to older \npeople. Notwithstanding these limitations, \nthis study goes some way to contributing to \nthe need identified in the Strategic Plan to \nAddress Gender-Based Violence (Bahamas \nNational Task Force for Gender-based \nViolence, 2015) to undertake further research \non gender-based violence in The Bahamas. Although interpersonal violence has many \nnegative \nphysical \nimpacts \non \nvictims \n(Campbell, 2002) that cannot be separated \nfrom mental trauma, this study only aimed to \nidentify possible links between the mental \nhealth of Bahamian college students and their \nnegative experiences of sexual intercourse; \nalso, it allows additional linkages to be made, \nas it considers other traumas that may \ninfluence mental health. Although this is a \nlimitation of the study, it does help to \ncomplement the study on physical trauma in \nThe Bahamas by Burnett-Garraway (2001). Although it is easy to focus on female rape \nand abuse, this study confirms the results of \nBethel and Fielding (2020) and unpublished \ndata provided by the Royal Bahamas Police \nForce (see Table 1) that males are also \nsubjected to rape. As indicated by others, for \nexample, Musevenzi and Musevenzi (2018), \nrape can be even more traumatic for male \nvictims than for female victims due to its \nassociated stigma. Additionally, as has been \nreported elsewhere, for example, Pakistan \n(Ali et al., 2013), it is not unusual for victims \nto be unwilling to seek help. Consequently, \nthis suggests that there should be increased \ngreater concern for appreciating that only \nfemales are victims of rape and need help. The methodology of this study is not \nnecessarily the most appropriate to identify \nlinkages \nbetween \nnegative \nsexual \nexperiences and mental health, but in the \nabsence of a longitudinal study, which could \nhave identified the changes in mental health \nover time, this study may still be useful in \nproviding preliminary information on how \nnegative sexual experiences affect mental \nhealth. The study findings are similar to those \nreported by researchers in the United States \n(Voth Schrag & Edmond, 2018) and by International Journal of Bahamian Studies Vol. 27 (2021) 14 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 14 E. Discussion This may mean that additional \nefforts need to be made with respect to sex \neducation for school children due to the fact \nthat some 25% of female respondents’ first \nexperience was during their school years, \nand, for females in particular, this experience \nwould most certainly be classified legally as \nrape (Table 4). Dating violence may be what is being \ndescribed in Table 14, where boyfriends, etc. were \nreported \nas \nbeing \ncommonly \nresponsible for the violence inflicted upon \nour study respondents, a figure consistent \nwith the range reported by Bergen and \nBarnhill (2006). Beyond dating violence, we \nshould note that in the United States, 14% of \nmarried women have reported being raped by \ntheir husbands (Brooks, 2001). Therefore, \ngiven the expected small number of married \nfemales in a college population, the fact that \n7.2% of older female respondents reported \nbeing victimized by their husband/long-term \npartner is noteworthy, and the occurrence of \nmartial rape requires further study in The \nBahamas. It is apparent that intimate partners \ncan be violent towards those with whom they \nhave relationships, and this violence is also \nassociated with increased mental health \nscores. Consequently, \nvarious \nnegative \naspects of imitate relationships, violence, \nsexual abuse, and rape can contribute to \nhigher mental health scores. As others have indicated, victims who fail to \nseek help from such a traumatic event may \nfind that their mental health problems \nbecoming worse (Tansill et al., 2012). These \nnegative impacts can also affect the children International Journal of Bahamian Studies Vol. 27 (2021) E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 15 of sexually abused mothers, as they are less \nlikely to praise their children, i.e., use \npositive parenting techniques (Fujiwara et al., \n2012). elevated mental health concerns (Pereira & \nCosta, 2016). These observations reflect in \npart the stigma of being what is loosely \ntermed “gay” or not engaging solely in \nheterosexual sex (“The Truth About Being \nGay,” 2016), a stigma that has resulted in \npersons being attacked in the streets \n(Turnquest, \n2016). Consequently, \nthis \nmarginalized group may be in even greater \nneed of mental health care than the \nheterosexual group. Although most participants were more \npositive about their most recent sexual \nexperience, those who felt abused or raped \nhad higher mental health scores than those \nwho had not experienced this trauma. Discussion Although the study methodology does not \nallow for national estimates of the occurrence \nof sexual abuse or rape to be made, the fact \nthat study participants reported having sexual \nintercourse against their will in the previous \nfour weeks starts to give an idea of the \npossible frequency of sexual abuse or rape. There was also an indication that persons \nwho were not “straight” in their sexual \nbehaviour had higher mental scores, a result \nconsistent with Szalacha et al. (2017), who \nalso found differences in the mental health of \nfemales who reported having sex with other \nfemales. In Portugal, the stigma of being non-\nheterosexual has been associated with Overall, \nthe \nresults \nfrom \nthis \nstudy \ndemonstrate the link between negative sexual \nencounters and mental health and its long-\nterm nature in the Bahamian context. Consequently, the recommendations of \nNaylor et al. (2012), which focus on \nintegrating mental health care with primary \nhealth care, may be beneficial. The authors \nhope that these results will assist with \nincreasing the awareness of the hidden harm \nof sexual trauma and encourage society to \nsensitively engage victims of negative sexual \nexperiences. Dr. Williams died on November 26, 2020, aged 55. International Journal of Bahamian Studies Vol. 27 (2021) Acknowledgments We would like to acknowledge the students in Nursing Research 409 Spring 2020 class, which \nwas taught by Dr. Elizabeth Williams, who participated in the data collection and shared their final \npapers with us. Dr. Williams died before she could write up this investigation, so we have had the \nhonour of writing this paper on her behalf and have attempted to present the data in a way \nconsistent with her IRB submission. We are grateful for the feedback of Dr. Michelle Bettin on an \nearlier draft and for the comments of the referees. International Journal of Bahamian Studies Vol. 27 (2021) 16 E. Williams, W. Fielding & V. Ballance. Mental Health and Negative Sexual Experiences. 16 References National Resource Center on Domestic \nViolence, Pennsylvania Coalition Against \nDomestic Violence. http://www.ncdsv.org/images/VAWnet_\nMaritalRapeNewResearchDirections_2-\n2006.pdf National Resource Center on Domestic \nViolence, Pennsylvania Coalition Against \nDomestic Violence. http://www.ncdsv.org/images/VAWnet_\nMaritalRapeNewResearchDirections_2-\n2006.pdf Ali, T. S., Mogren, I., & Krantz, G. (2013). 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https://openalex.org/W4361809620	https://aacr.figshare.com/ndownloader/files/39841680	English	null	Supplementary Figures 9 - 10 from MicroRNAs Regulate Tumor Angiogenesis Modulated by Endothelial Progenitor Cells	null	2,023	cc-by	161	FigS9 API CD31/DAPI Mixed/DAPI Plummer_FigS9 C Anti-DIG-FITC/DAPI miR-10b miR-196b 63x CD31/DAPI Positive Scrambled Mixed/DAPI CD31/DAPI Mixed/DAPI Plummer_Fig DAPI iR-196b ositive Plummer_Fig DAPI iR-196b ositive Plum Anti-DIG-FITC/DAPI R-10b miR-196b Positive rambled Anti-DIG-FITC/ R-10b m P ambled I CD31/DAPI Mixed/DAPI C 63x D31/DAPI Mixed/DAPI D31/DAPI Mixed/DAPI B d/DAPI miR-10b-FITC/ DAPI CD31/DAPI Mixed/DAPI DCIS E miR-196b miR-10b Anti-DIG-FITC/DAPI DCIS ed/DAPI miR-196b-FITC/ DAPI CD31/DAPI Mixed/DAPI miR-132 miR-152 D Positive Scrambled CIS .0001 7.5 IDC Grade III 10 DCIS P<0.0001 P=0.2389 miR field field 5 0 2.5 5 0 2.5 7.5 miR- 7 Number of vessels/f Number of vessels/f miR-10b- miR-196b+miR-196b- 0 miR-196b+ miR-196b- 0 Plummer_FigS10 I Mixed/DAPI E miR-196b miR-10b Anti-DIG-FITC/DAPI Mixed/DAPI iR 132 iR 152 igS10 Positive Scrambled DCIS P<0.0001 iR-196b+ miR-196b- e B API miR-10b-FITC/ DAPI CD31/DA DCIS DCIS API miR-196b-FITC/ DAPI CD31/DAP D 01 7.5 IDC Grade III 10 P=0.2389 miR field field 5 0 2.5 5 0 2.5 7.5 miR- 7 Number of vessels/f Number of vessels/f iR-10b- miR-196b+miR-196b- 0 0
https://openalex.org/W2888311184	https://www.bio-conferences.org/10.1051/bioconf/20181100045/pdf	English	null	Thematic interpretation of the Northern Dvina River floodplain meadow ecosystem using remote sensing	Bio web of conferences/BIO web of conferences	2,018	cc-by	1,433	Thematic interpretation of the Northern Dvina River floodplain meadow ecosystem using remote sensing Northern (Arctic) Federal University named after M.V. Lomonosov. Higher School of Natural Sciences and Technologies. Department of biology, ecology and biotechnology. Arkhangelsk, Russian Federation Abstract. In this work we represent the possibility of using satellite images of medium spatial resolution for thematic interpretation of meadow ecosystems located in the flood plain of the Northern Dvina river. We have used multidimensional scaling to study the differences in spectral characteristics. Classes such as arable land, water, mineral soils, forests and swamps are most well-classified. However, the meadow ecosystems overlap with reed beds according to spectral characteristics. Therefore, for more accurate identification of meadows, it is recommended to use different vegetative indices, for example NDVI and Cl Green. As a result of the research, a map of the distribution of meadow ecosystems was created using GIS. © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). * Corresponding author: a.g.volkov@narfu.ru BIO Web of Conferences 11, 00045 (2018) Prospects of Development and Challenges of Modern Botany BIO Web of Conferences 11, 00045 (2018) Prospects of Development and Challenges of Modern Botany https://doi.org/10.1051/bioconf/20181100045 1 Introduction One of the traditional tasks for geobotany is the mapping of vegetation, including meadow, which is the object of our many years of research. Large-scale data on areas of meadow ecosystems in the Russian Federation refer mainly to the 1960-1980th years and at the present time do not adequately reflect the current situation [1]. Meadows in the floodplain are of high natural and economic value. Among other ecosystems, they are particularly dynamic and undergo significant changes over time. Their areas, the degree of moistening, the composition of the herbage are constantly changing. Therefore, it is important to have a reliable and affordable way of remote meadow registration and evaluation of their areas. The aim of this study is to identify criteria that allow deciphering the meadow communities and create a map of the floodplain of the Northern Dvina River. According to this, the following tasks are set: to determine the spectral characteristics of meadows from satellite images, to compare them with other classes of objects, to identify differences, to establish the relationship of spectral characteristics to the values of vegetation indices, and to conduct thematic interpretation with the subsequent compilation of a map of the flood plain of the Northern Dvina River. https://doi.org/10.1051/bioconf/20181100045 BIO Web of Conferences 11, 00045 (2018) Prospects of Development and Challenges of Modern Botany Where NIR – near infrared channel; RED – red channel; GREEN – green channel. Where NIR – near infrared channel; RED – red channel; GREEN – green channel. Using ArcGIS, we created a vector polygonal layer with known natural objects: meadows, arable land, forests, reed beds, marshes, water and anthropogenic. For each class we created 1000 random points and extract values from all data sets. Then we exported the data to csv file and analysed in R. Multidimensional scaling with Bray-Curtis distance used for searching the differences between classes. Multidimensional scaling makes it possible to visualize multidimensional feature data in a two-dimensional space of coordinates based on the distance matrix. In addition, it is possible to find a correlation with the factors, determine their influence on the differentiation of objects and display them in the form of vectors on the graph. g p The interpretation was also carried out in R using the "random forest" method [5] which is an ensemble learning method for classification that operates by constructing a multitude of decision trees at training time and outputting the class that is the mode. 2 Materials and Methods The meadows in the floodplain of the Severnaya Dvina River representing grassy communities with more or less contiguous herbage and formed mainly by long-term mesophilous plants [2] were chosen as the research subject. We have used several data sources for thematic interpretation of meadow ecosystems. Firstly during field expeditions in 2011 2017 in the floodplain of the Severnaya Dvina We have used several data sources for thematic interpretation of meadow ecosystems. Firstly, during field expeditions in 2011-2017, in the floodplain of the Severnaya Dvina River we have detected meadows and located their coordinates Later they were added on We have used several data sources for thematic interpretation of meadow ecosystems. Firstly, during field expeditions in 2011-2017, in the floodplain of the Severnaya Dvina River, we have detected meadows and located their coordinates. Later they were added on the satellite image and used as reference sites. Secondly, we used satellite images from the Sentinel-2 (07/24/2017) with a spatial resolution of 10, 20 and 60 m / pixel, depending on the channel. All spectral channels from MSI were included in the analysis. Thirdly, to improve the accuracy of the classification, we used satellite-derived data, such as the vegetation index. First of all, this is NDVI (1), which reflects the relative amount of photosynthetically active biomass (2). And also Cl Green (2) − the index of photosynthetic activity of the vegetation cover, used in assessing the chlorophyll a and b content in plant leaves [3, 4]. NDVI = (NIR – RED) / (NIR + RED) (1) Cl Green = NIR / Green – 1 (2) (1) (2) 3 Results This project is supported by grant from the Russian Foundation for Basic Research (RFBR) and the government of Arkhangelsk region № 17-44-290111 (project manager: Prof. Elena Nakvasina). 3 Results The above classification of the satellite image revealed significant differences in the spectral characteristics of various deciphered objects. As expected, classes of arable land, water, mineral soils (such as sandbanks in the delta of the river), forests and marshes are best separated. At the same time, it is quite difficult to distinguish between meadow ecosystems and the so-called reed (Figure 1A), which are heavily waterlogged areas with periodic seasonal underflooding, where reed Phragmites australis (Cav.) Trin. ex Steud. L. predominates. Vegetative indices can be used for more accurate interpretation of meadow ecosystems and reed thickets. In our work we used NDVI and Cl Green. In R we calculated the correlation coefficients between these indices and multidimensional scaling axes, containing compressed information on the spectral characteristics of the objects. Meadow communities as a whole have higher values of the applied indices (Figure 1B), which 2 BIO Web of Conferences 11, 00045 (2018) https://doi.org/10.1051/bioconf/20181100045 Prospects of Development and Challenges of Modern Botany corresponds to more phytomass and chlorophyll content in meadow grass plants than in coarse phytomass of reeds. Fig. 1. The results of multidimensional scaling: A − for all classes of objects, B − for meadows and reed thickets with superposition of vectors of vegetative indices. Fig. 1. The results of multidimensional scaling: A − for all classes of objects, B − for meadows and reed thickets with superposition of vectors of vegetative indices. The obtained information on spectral characteristics and vegetation indices was used to decode the entire satellite image. Based on the results of the thematic interpretation, we have created a vector map of the meadow ecosystems of the floodplain of the Northern Dvina River. The purpose of compiling any map is to present, at a certain scale, the objects of observation for their recording, as well as to study the patterns of their spread in connection with various natural factors. As a result of the study, spectral characteristics of meadow ecosystems located in the floodplain of the Northern Dvina River were determined. It is revealed that the meadows as a whole are well interpreted with respect to other classes of objects, such as forest, bog, arable and water. However, for the separation of meadows from classes with similar vegetation, it is necessary to use vegetative indices. 5. T. Hastie, R. Tibshirani, J. Friedman, Random Forests. The Elements of Statistical Learning: Data Mining, Inference, and Prediction (Springer-Verlag, 2009) References 1. T.K. Yurkovskaya, Actual problems of geobotany, 43-71 (Petrozavodsk, 2007) 2. B.M. Mirkin, L.M. Gareyeva, Bulletin of the Moscow Society of Naturalists 83, 38-50 (1978) 3. J. Rouse, R. Haas, J. Schell, D. Deering, 3rd ERTS Symposium, NASA SP-351, 1, 309-317 (1973) 1. T.K. Yurkovskaya, Actual problems of geobotany, 43-71 (Petrozavodsk, 2007) 1. T.K. Yurkovskaya, Actual problems of geobotany, 43-71 (Petrozavodsk, 2007) 2. B.M. Mirkin, L.M. Gareyeva, Bulletin of the Moscow Society of Naturalists 83, 38-50 (1978) 3. J. Rouse, R. Haas, J. Schell, D. Deering, 3rd ERTS Symposium, NASA SP-351, 1, 309-317 (1973) 3. J. Rouse, R. Haas, J. Schell, D. Deering, 3rd ERTS Symposium, NASA SP-351, 1, 309-317 (1973) 4. G. Dall’Olmo, A.A. Gitelson, Applied Optics 44, 412-422 (2005) 3 3
https://openalex.org/W2472958708	https://europepmc.org/articles/pmc4936706?pdf=render	English	null	Retinal Image Enhancement Using Robust Inverse Diffusion Equation and Self-Similarity Filtering	PloS one	2,016	cc-by	5,924	Lu Wang1☯, Guohua Liu2☯, Shujun Fu3, Lingzhong Xu1, Kun Zhao4, Caiming Zhang5,6 1 School of Public Health, Shandong University, Jinan 250012, China, 2 Department of Ophthalmology, Qilu Children’s Hospital of Shandong University, Jinan 250022, China, 3 School of Mathematics, Shandong University, Jinan 250100, China, 4 Department of Medical Imaging, The Second Hospital of Shandong University, Jinan 250033, China, 5 School of Computer Science and Technology, Shandong University of Finance and Economics, Jinan 250061, China, 6 School of Computer Science and Technology, Shandong University, Jinan 250101, China a1111 ☯These authors contributed equally to this work. ☯These authors contributed equally to this work. shujunfu@163.com * shujunfu@163.com OPEN ACCESS As a common ocular complication for diabetic patients, diabetic retinopathy has become an important public health problem in the world. Early diagnosis and early treatment with the help of fundus imaging technology is an effective control method. In this paper, a robust inverse diffusion equation combining a self-similarity filtering is presented to detect and evaluate diabetic retinopathy using retinal image enhancement. A flux corrected transport technique is used to control diffusion flux adaptively, which eliminates overshoots inherent in the Laplacian operation. Feature preserving denoising by the self-similarity filtering ensures a robust enhancement of noisy and blurry retinal images. Experimental results demonstrate that this algorithm can enhance important details of retinal image data effec- tively, affording an opportunity for better medical interpretation and subsequent processing. Citation: Wang L, Liu G, Fu S, Xu L, Zhao K, Zhang C (2016) Retinal Image Enhancement Using Robust Inverse Diffusion Equation and Self-Similarity Filtering. PLoS ONE 11(7): e0158480. doi:10.1371/ journal.pone.0158480 Citation: Wang L, Liu G, Fu S, Xu L, Zhao K, Zhang C (2016) Retinal Image Enhancement Using Robust Inverse Diffusion Equation and Self-Similarity Filtering. PLoS ONE 11(7): e0158480. doi:10.1371/ journal.pone.0158480 Editor: Yuanquan Wang, Beijing University of Technology, CHINA Received: February 23, 2016 Accepted: June 17, 2016 Published: July 7, 2016 Editor: Yuanquan Wang, Beijing University of Technology, CHINA Editor: Yuanquan Wang, Beijing University of Technology, CHINA Received: February 23, 2016 Accepted: June 17, 2016 Published: July 7, 2016 Copyright: © 2016 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Introduction Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Retinal Image Enhancement Using Robust Inverse Diffusion Equation and Self-Similarity Filtering Lu Wang1☯, Guohua Liu2☯, Shujun Fu3, Lingzhong Xu1, Kun Zhao4, Caiming Zhang5,6 ☯These authors contributed equally to this work. shujunfu@163.com Data Availability Statement: All relevant data are within the paper and its Supporting Information files. With the development of economy and the aging population, people’s visual impairments has become a major public health problem all over the world. All kinds of ophthalmic diseases causing visual defects not only increase the burden of public health care system, more impor- tantly, they also pose serious threats to social and economic activities [1, 2]. Among them, as one of main blinding eye diseases, diabetic retinopathy is the most common ocular complica- tion in diabetic patients, which includes a series of typical lesions with retinal microvascular and neuron damages caused by sugar metabolic abnormalities. It is a chronic and progressive blinding fundus disease which can be characterized by such clinical features as decreased vision, fundus bleeding and exudation, macular edema and hyperplastic lesions [3]. The overall prevalence of diabetic retinopathy was 34.6% according to a meta-analysis of 35 international renowned epidemiological studies (22896 cases of diabetes) of the world [4]. The fact of high Funding: The research has been supported in part by the National Natural Science Foundation of China (61272239, 61070094, 61020106001); the NSFC Joint Fund with Guangdong (U1201258); the Science and Technology Development Project of Shandong Province of China (2014GGX101024); and the Fundamental Research Funds of Shandong University (2014JC012). Competing Interests: The authors have declared that no competing interests exist. 1 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation prevalence, high blindness rate, high fashion trend, high social and economic burden, and low cognition rate makes things worse [2]. Early diagnosis and early treatment is an effective method for the control of diabetic retinop- athy [5, 6]. Fundus imaging by digital fundus camera is a standard diagnostic mode in ophthal- mology, which captures the intensity of light reflected from the retinal surface in three different wavelength ranges [7, 8]. By reason of imaging mechanism and system of fundus retina imaging itself, and the disturbance of various noise in image formation process, one often obtain noisy and blurry retinal image with nonuniform and distorted illumination, which is difficult to inter- pret medically and to process subsequently [7, 9–11]. Thus, it is indispensable to remove noise and disturbances, to improve signal-to-noise rate of image, to adjust image contrast and to enhance vessels and fine details of retinal image data [9, 12, 13]. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. By above image preprocessing, useful information in retinal image is highlighted, while useless one is weakened or removed, to make the result more suitable to clinical diagnosis and treatment [3, 7, 9, 14–17]. Many different methods have been put forth for retinal image denoising and enhancement [7, 9, 18, 19], such as the Gamma transformation [18], histogram equalization [20, 21], sharp- ening by the Laplacian operation [22], filtering methods in transformation fields [19], varia- tional methods and partial differential equations (PDEs) [13, 23, 24]. However, one of major challenges faced by these methods is, how to avoid enhancing noise, producing overshoot arti- facts around edges, and erasing fine details in enhanced images [23, 25, 26]. Important medical information of a retinal image lies in its retinal vessel network and local fine details. In order to accurately detect and evaluate diabetic retinopathy as soon as possible, it is very crucial to properly enhance possible retinal pathological features such as microaneur- ysm, bleeding and exudating spots. In this paper, a robust inverse diffusion equation is pre- sented, which combines a powerful self-similarity filtering [27, 28] for detail preserving image denoising. A flux corrected transport (FCT) technique [25, 29] is used to control diffusion flux adaptively, which effectively eliminates overshoots inherent in the Laplacian operation. The proposed method extends our previous work [25] to enhance noisy images while avoiding annoying overshoots and noise magnification. We organize this paper as follows. In Section II, related image enhancement methods by the Gamma transformation, the shock computing and the self-similarity filtering are introduced. In Section III, the robust inverse diffusion equation is built to enhance noisy and blurry retinal image data, where the flux corrected transport technique is elaborated in a subsequential pro- cess including three main steps. In Section IV, experiments on retinal images with typical dia- betic retinopathy are carried out to verify the effectiveness of the proposed algorithm. Finally, conclusions and future work are included to end this paper in section V. Related image enhancement methods (II) Intensity transformation is the simplest technique in image enhancement through mapping a pixel value r into a pixel value z, among which the Gamma (power-law) transformation is one of basic transformation functions [18]. It is defined as z ¼ brg; ð1Þ ð1Þ z ¼ brg; where, b and γ are positive constants. The Gamma transformation with fractional values of γ can map a narrow range of input values into a wider range of output values. In a variety of devices the Gamma transformation is used to appropriately enhance image contrast and details for image capture, printing and display. where, b and γ are positive constants. The Gamma transformation with fractional values of γ can map a narrow range of input values into a wider range of output values. In a variety of devices the Gamma transformation is used to appropriately enhance image contrast and details for image capture, printing and display. In the past decades there has been an increasing research on partial differential equations (PDEs) in image enhancement [23, 30–32]. A great deal of successful applications of nonlinear 2 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation evolving PDEs in image enhancement can mainly be attributed to their two basic characteris- tics: local operation and iterative processing. Osher and Rudin introduced a novel image sharp- ening technique, called the shock filter (SF) [30], which simulates the shock wave calculation in computational fluid mechanics: @u @t ¼ signðuNNÞjruj; uNN ¼ 1 jruj 2 ðu2 xuxx þ 2uxuyuxy þ u2 yuyyÞ; ð2Þ ð2Þ where sign is a sign function, r is a gradient operator, and uNN is the second directional deriva- tive of image along local normal direction to isophote line. It detects an image edge using the zero-crossing of uNN, where a shock is formed at a speed of the gradient magnitude \|ru\|. Related image enhancement methods (II) Considering image noise in the estimation of edges, Alvarez and Mazorra added a smooth- ing kernel and coupled the anisotropic diffusion with the shock filter (ADSF) [31, 32] for noise elimination and edge sharpening: @u @t ¼ signðGs uNNÞjruj þ cuTT; uTT ¼ Du uNN; ð3Þ ð3Þ where Δ is a Laplacian operator, Gσ is a Gaussian kernel with standard deviation σ, uTT is the second directional derivative of image along local tangent direction, and c is a constant to bal- ance the anisotropic diffusion and the shock ﬁltering. On the other hand, in order to effectively denoise images while preserving image details, a powerful non-local means algorithm was proposed by Buades et al. [27], which fully utilizes the big redundancy and the self-similarity of natural images in the photometric range. The dis- crete expression of the self-similarity filtering (SSF) algorithm is as follows. Let u be a noisy image defined in a discrete grid O R2. The denoised intensity at the pixel (i, j) is expressed by SSFðuijÞ ¼ P ðm;nÞ2Owijðm; nÞumn P ðm;nÞ2Owijðm; nÞ . ; ð4Þ ð4Þ where wij(m, n) is an average weight which is determined by the similarity between the pixels (i, j) and (m, n), and is adopted as wijðm; nÞ ¼ exp k uðNijÞ uðNmnÞ k2 2;a =h2 n o ; ð5Þ ð5Þ where Nij and Nmn are similarity windows of size (2s + 1) × (2s + 1) centered at pixels (i, j) and (m, n), respectively. The term u(Nij) denotes an image patch restricted in the similarity window Nij. The notation k k2,a denotes a Gaussian weighted Euclidean distance between two image patches, where a is the standard deviation of the Gaussian function. The parameter h denotes a smoothing factor that controls the decay of the exponential function in the Eq (5). To reduce the computational burden and to improve the efﬁciency of the SSF ﬁltering, the search window is always restricted to a proper local neighborhood (2f + 1) × (2f + 1) in O. The denominator in the Eq (4) is a normalizing factor. Robust inverse diffusion equation (III) As special inverse diffusion processing [23], although the shock computing [30–33] can effec- tively sharpen image edges and remove image noise, there are some inherent weaknesses for it to enhance retinal image. Firstly, for noisy and blurry retinal image data, it is difficult to estimate its local tangential and normal directions; and for finer details these directions are difficult to define and estimate. Secondly, in order to enhance tiny lesions important for the detection of diabetic retinopathy, where the value of image gradient is very small, it is 3 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation improper for the shock computing at a speed of the gradient magnitude. Thirdly, the Gauss- ian and tangential smoothings in the shock computing easily erase important details when removing noise and smoothing the second directional derivative. Finally, unnatural artifacts may be produced around image edges where shocks are formed by the shock computing [23]. These defects will be compared and shown in following experiments of enhancing retinal images. In order to overcome above difficulties, we present the following robust inverse diffusion (RID) equation: @u @t ¼ jrujDu; ð6Þ ð6Þ When solving numerically a nonlinear inverse diffusion equation like Eq (6) using a differ- ence scheme, it must be discretized carefully because it is an instable process. Otherwise, numerical blowing up will appear inevitably. A strategy is to try to stop from numerical fluc- tuations before they appear, which is based on the Total Variation Diminishing (TVD) and nonlinear limiters [32, 34]. The main idea of above flux corrected transport technique is to use a limiter function to control the change of the numerical solution by a nonlinear way, and the corresponding schemes satisfy the TVD condition and consequently eliminate above disadvantage effects. An explicit Euler method with central difference scheme is used to approximate the Eq (6) except the gradient term. Below we detail a approach to it numerically. On the image grid O, the approximate solution is to satisfy: uk ij uðil; jl; kDtÞ; i; j; k 2 Zþ; ð7Þ uk ij uðil; jl; kDtÞ; i; j; k 2 Zþ; ð7Þ where l and Δt are spatial and temporal steps. Let l = 1, and dþuk ij and duk ij are forward and backward difference schemes of uk ij, respectively. Robust inverse diffusion equation (III) For example, along the x direction, dþ x uk ij ¼ uk ðiþ1Þj uk ij, d x uk ij ¼ uk ij uk ði1Þj; the case is similar along the y direction. A limiter func- tion M(p, q) is used to approximate the gradient term (see Fig 1): p p p ij ij backward difference schemes of uk ij, respectively. For example, along the x direction, dþ x uk ij ¼ uk ðiþ1Þj uk ij, d x uk ij ¼ uk ij uk ði1Þj; the case is similar along the y direction. A limiter func- tion M(p, q) is used to approximate the gradient term (see Fig 1): j dþ x uk ij ¼ uk ðiþ1Þj uk ij, d x uk ij ¼ uk ij uk ði1Þj; the case is similar along the y direction. A limiter func- tion M(p, q) is used to approximate the gradient term (see Fig 1): jruj k ij ¼ min Mðdþ x uk ij; d x uk ijÞ; Mðdþ y uk ij; d y uk ijÞ n o ; ð8Þ Fig 1. Illustration of limiter function M(p, q): two situations for forward and backward difference schemes. M function stops Eq (6) from numerical fluctuations (overshoots) along data points (Xi−1, Xi, Xi+1). jruj k ij ¼ min Mðdþ x uk ij; d x uk ijÞ; Mðdþ y uk ij; d y uk ijÞ n o ; ð8Þ ð8Þ Fig 1. Illustration of limiter function M(p, q): two situations for forward and backward difference schemes. M function stops Eq (6) from numerical fluctuations (overshoots) along data points (Xi−1, Xi, Xi+1). doi:10.1371/journal.pone.0158480.g001 4 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 2. Flow chart of our proposed algorithm. A degraded image is enhanced through three steps in sequence: Gamma manipulation (Gamma), self-similarity filtering (SSF) and robust inverse diffusion (RID), respectively. Fig 2. Flow chart of our proposed algorithm. A degraded image is enhanced through three steps in sequence: Gamma manipulation (Gamma), self-similarity filtering (SSF) and robust inverse diffusion (RID), respectively. where Mðp; qÞ ¼ l; pq > 0 0; pq 0: ( ð9Þ ð9Þ Here, λ is a constant to guarantee that tiny important details can also be enhanced effectively regardless of its small gradient magnitude. Robust inverse diffusion equation (III) After the numerical discretization of Eq (6), it can be considered as an enhancement process by an iterative constrained Laplacian operation [25]. In order to improve the contrast of retinal image for the detection of tiny lesions, a Gamma transformation [18] is first used to enhance the image within proper gray levels. Then, the pow- erful self-similarity filtering [27] is employed to remove image noise, especially in the regions of interest (ROI). Finally, the proposed robust inverse diffusion is carried out to further sharpen important details of retinal image while avoiding overshoot artifacts. A whole flow chart of our proposed algorithm is shown in Fig 2. PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Experimental results and analyses (IV) Although retinal images can be represented in many color spaces (RGB, HSI, HSV, etc.), the selection of them highly depends on the application. In this paper, a retinal image enhancement algorithm is designed to help physicians in their task of early diagnose of retinopathy, and there- fore the selected space must be as close as possible to human perception [35]. A well-established agreement is that the green channel in the RGB color space provides more blood vessel struc- tural information and is less subject to non-uniform illumination, while the HSV color space does not preserve the fidelity of retinal images [35–37]. Because green light is absorbed by the blood and reflected by the retinal pigment epithelium, providing a good contrast for visualizing retinal vascular network, bleeding and exudation, we routinely extract and enhance the green channel (in the gray range of [0, 1]) from a RGB color fundus photograph [7]. In Fig 3, a retinal image with tiny microaneurysms of size 465 × 600 is enhanced for the early detection of diabetic retinopathy. Through sequential processings of the Gamma manipu- lation (γ = 0.6), the self-similarity filtering (h = 0.01, f = 5, s = 3) and the robust inverse diffu- sion (λ = 0.6, Δt = 0.15, k = 7), tiny microaneurysms and microvasculature are shown clearly due to image contrast improvement and noise removal. Moreover, our method produces fewer overshoot artifacts while avoiding noise magnification. A further comparison is carried out with the histogram equalization [18], the ADSF filtering (c = 0.2, Δt = 0.5, n = 10) after the Gamma manipulation, and the Laplacian operation after the Gamma manipulation and the self-similarity filtering in Figs 4 and 5, where enhancement results and their zoomed local parts are shown when enhancing the macular area by these methods. The tiny microaneurysms are not too clear in the original image shown in Fig 3. The Gamma transformation improves image contrasts on the whole, but fine spots and details remain blurry without being highlighted compared with their surrounding regions. Although the histogram equalization can enhance image contrasts to some extent, it produces 5 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 3. Retinal image enhancement for detection of microaneurysms in diabetic retinopathy (from top- left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, successively. Experimental results and analyses (IV) doi:10 1371/journal pone 0158480 g003 Fig 3. Retinal image enhancement for detection of microaneurysms in diabetic retinopathy (from top- left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, successively. doi:10.1371/journal.pone.0158480.g003 Fig 4. Retinal image enhancement for detection of microaneurysms in diabetic retinopathy (from top- left to bottom-right): results by histogram equalization, ADSF filtering after Gamma manipulation, Laplace operation after Gamma manipulation and self-similarity filtering, and robust inverse diffusion, respectively. Fig 4. Retinal image enhancement for detection of microaneurysms in diabetic retinopathy (from top- left to bottom-right): results by histogram equalization, ADSF filtering after Gamma manipulation, Laplace operation after Gamma manipulation and self-similarity filtering, and robust inverse diffusion, respectively. Fig 4. Retinal image enhancement for detection of microaneurysms in diabetic retinopathy (from top- left to bottom-right): results by histogram equalization, ADSF filtering after Gamma manipulation, Laplace operation after Gamma manipulation and self-similarity filtering, and robust inverse diffusion, respectively. doi:10.1371/journal.pone.0158480.g004 6 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 5. Zoomed parts of enhanced retinal images for microaneurysm detection (from top-left to bottom-right): original image, results by Gamma manipulation, histogram equalization, ADSF filtering after Gamma manipulation, Laplace operation after Gamma manipulation and self-similarity filtering, and robust inverse diffusion, respectively. doi:10.1371/journal.pone.0158480.g005 neurysm detection (from top-left to bottom-right): original Fig 5. Zoomed parts of enhanced retinal images for microaneurysm detection (from top-left to bottom-right): original image, results by Gamma manipulation, histogram equalization, ADSF filtering after Gamma manipulation, Laplace operation after Gamma manipulation and self-similarity filtering, and robust inverse diffusion, respectively. doi:10.1371/journal.pone.0158480.g005 doi:10.1371/journal.pone.0158480.g005 7 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 6. Retinal image enhancement: local comparison of profiles (350th row, 250-300 columns) of enhanced images by robust inverse diffusion, ADSF filtering and Laplacian operation, respectively. doi:10.1371/journal.pone.0158480.g006 Fig 6. Retinal image enhancement: local comparison of profiles (350th row, 250-300 columns) of enhanced images by robust inverse diffusion, ADSF filtering and Laplacian operation, respectively. doi:10.1371/journal.pone.0158480.g006 doi:10.1371/journal.pone.0158480.g006 doi:10.1371/journal.pone.0158480.g006 doi:10.1371/journal.pone.0158480.g006 nonuniform illumination distribution and noise magnification concealing some fine details. The noise magnification and artifacts (overshoots and halos) from the over-enhancing by the Laplacian operation and the ADSF filtering can be obviously observed. Moreover, the numeri- cal blowing up will quickly come out for the multiple Laplacian operations [25], especially at bigger gradients around image edges. PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Experimental results and analyses (IV) Retinal image enhancement for detection of soft exudations in diabetic retinopathy (from top- left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. doi:10.1371/journal.pone.0158480.g007 doi:10.1371/journal.pone.0158480.g007 produce overshoots and halos around two vessels. The proposed method does not produce annoying artifacts, ensuring a retinal image enhancement as faithful as possible. Finally, both retinal images are enhanced by the SF filtering (Δt = 0.5, n = 10) after the Gamma manipulation in Fig 10. As discussed above, although the SF filtering enhances images by sharpening their edges, noise magnification and over-enhanced artifacts by the shock com- puting can be obviously observed. False over-enhanced details will make it difficult for a physi- cian to identify abnormal pathologic changes correctly. It is important to point out that the parameters in the proposed method will greatly affect the results of retinal image enhancement. For a specific system of fundus retina imaging, the parameters in the proposed method can be fixed by a certain amount of data simulations and tests. Experimental results and analyses (IV) Only by the proposed method are tiny microaneurysms clearly shown while avoiding the artifacts and noise magnification. In order to observe enhancement effects by these methods more clearly, local profiles (350th row, 250-300 columns) of different results are shown in Fig 6. One can see that, the Laplace operation produces overshoots and halos around two vessels. Because smaller uNN will also be enhanced indiscriminately for shock computing, the ADSF filtering over enhances image differences and leads to annoying artifacts and false edges in flat areas of image [23]. The proposed method does not produce annoying artifacts own to its proper constrained enhance- ment, providing a chance to early detect the diabetic retinopathy faithfully by image enhancement. Next, in Fig 7, a retinal image with soft exudations of size 768 × 768 is enhanced to verify the proposed robust inverse diffusion for highlighting important medical features such as reti- nal vascular networks and local fine details. Through sequential processings of the Gamma manipulation (γ = 0.6), the self-similarity filtering (h = 0.03, f = 5, s = 3) and the robust inverse diffusion (λ = 0.5, Δt = 0.15, k = 7), one can see that, the proposed method removes noise effec- tively and preserves important image details. At the same time, vascular networks and exuda- tive spots are shown more clearly while producing no artifacts. In Fig 8, zoomed local parts of results by the proposed method are shown. Obviously, through sequential processings in three steps the degraded retinal image is greatly enhanced: image contrasts are improved, image noise is removed, and overshoots and halos are not pro- duced, which further verify the advantages of the proposed method. Local profiles (500th row, 510-560 columns) of different enhancement results are also shown in Fig 9. Similarly, one can see that, both the Laplace operation and the ADSF filtering 8 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 7. Retinal image enhancement for detection of soft exudations in diabetic retinopathy (from top- left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. doi:10 1371/journal pone 0158480 g007 Fig 7. Retinal image enhancement for detection of soft exudations in diabetic retinopathy (from top- left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. Fig 7. Supporting Information S1 Fig. Test data in diabetic retinopathy: left, image with microaneurysms; right, image with soft exudations. (DOC) S1 Fig. Test data in diabetic retinopathy: left, image with microaneurysms; right, image with soft exudations. Author Contributions Conceived and designed the experiments: LW GL SF KZ. Performed the experiments: LW GL SF KZ. Analyzed the data: LW GL SF LX KZ CZ. Contributed reagents/materials/analysis tools: GL KZ. Wrote the paper: LW GL SF. Designed the software used in analysis: LW SF. Conclusions (V) doi:10.1371/journal.pone.0158480.g010 Experimental results demonstrate that this algorithm can enhance important details of image data effectively without overshoots and noise magnification, affording an opportunity for bet- ter medical interpretation and subsequent processing. Experimental results demonstrate that this algorithm can enhance important details of image data effectively without overshoots and noise magnification, affording an opportunity for bet- ter medical interpretation and subsequent processing. For future research, we will further try to optimize the algorithm in the process of adaptive image enhancement according to the gray-level distribution of retinal lesions. Conclusions (V) In retinal image data enhancement for early detection of diabetic retinopathy, it is crucial to highlight important pathological features such as microaneurysm, bleeding and exudating spots. In this paper, a robust inverse diffusion equation is presented by combining a powerful self-similarity filtering, where the flux corrected transport technique is used to eliminate over- shoots inherent in the Laplacian operation. At the same time, the self-similarity filtering not only effectively removes image noise, but also avoids noise magnification common in image enhancement methods, resulting in a robust processing of noisy and blurry retinal image data. 9 / 13 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 8. Zoomed parts of enhanced retinal images for detection of soft exudations (from top-left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. Fig 8. Zoomed parts of enhanced retinal images for detection of soft exudations (from top-left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. Fig 8. Zoomed parts of enhanced retinal images for detection of soft exudations (from top-left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. Fig 8. Zoomed parts of enhanced retinal images for detection of soft exudations (from top-left to bottom-right): original image, results by Gamma manipulation, self-similarity filtering and robust inverse diffusion, respectively. doi:10.1371/journal.pone.0158480.g008 Fig 9. Retinal image enhancement: local comparison of profiles (350th row, 250-300 columns) of enhanced images by robust inverse diffusion, ADSF filtering and Laplacian operation, respectively. doi:10 1371/journal pone 0158480 g009 Fig 9. Retinal image enhancement: local comparison of profiles (350th row, 250-300 columns) of enhanced images by robust inverse diffusion, ADSF filtering and Laplacian operation, respectively. Fig 9. Retinal image enhancement: local comparison of profiles (350th row, 250-300 columns) of enhanced images by robust inverse diffusion, ADSF filtering and Laplacian operation, respectively. doi:10.1371/journal.pone.0158480.g009 doi:10.1371/journal.pone.0158480.g009 PLOS ONE \| DOI:10.1371/journal.pone.0158480 July 7, 2016 10 / 13 Retinal Image Enhancement Using Robust Inverse Diffusion Equation Fig 10. Retinal image enhancement for detection of diabetic retinopathy: top, original images; bottom, results by SF filtering after Gamma manipulation, respectively. doi:10.1371/journal.pone.0158480.g010 Fig 10. Retinal image enhancement for detection of diabetic retinopathy: top, original images; bottom, results by SF filtering after Gamma manipulation, respectively. 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https://openalex.org/W4232338640	https://figshare.com/articles/figure/Sovereign_Saint_and_City_Honor_and_Reuse_of_Textiles_in_the_Treasury_of_San_Isidoro_Leon_/8088644/1/files/15086150.pdf	English	null	Sovereign, Saint, and City: Honor and Reuse of Textiles in the Treasury of San Isidoro (León)	BRILL eBooks	2,020	cc-by	743	brill.com/me Medieval Jewish, Christian and Muslim Culture Encounters in Confluence and Dialogue brill.com/me Medieval Jewish, Christian and Muslim Culture Encounters in Confluence and Dialogue Medieval Encounters 25 (2019) 96–123 Sovereign, Saint, and City: Honor and Reuse of Textiles in the Treasury of San Isidoro (Leon) María Judith Feliciano Independent Scholar María Judith Feliciano Independent Scholar Keywords saints’ relics – textile reuse – Arabic epigraphy – ṭirāz – reliquary linings Supplementary Material Abstract The textiles currently housed in the treasury of the collegiate church of San Isidoro de León are part of a centuries-long process of collecting relics at the heart of an emblem- atic royal mausoleum. Instead of studying them through the prism of the “charter of 1063,” this investigation proposes a wider look at the place of luxurious textiles in Cas- tile and León before and after the arrival of Saint Isidore’s relics. What emerges is an intricate relationship between the mythification of the city of León as a civitas regia, the Leonese monarchy’s concept of antiquity in legitimizing their rule, and the refine- ment associated with the royal court and its sacred performance of the cults of saints. Supplementary Material Figure 1 Textile lining the reliquary of San Isidoro Photo: Ana Cabrera Figure 1 Textile lining the reliquary of San Isidoro Photo: Ana Cabrera Figure 2 Embroidery lining the lid of the reliquary of San Isidoro Photo: Therese Martin Figure 2 Embroidery lining the lid of the reliquary of San Isidoro Photo: Therese Martin Figure 3 Textile fragment from Colls. 11th century. Silk and gold thread. 51.8 × 58.5 cm. Museo de Huesca, no. 1542. ure 3 Textile fragment from Colls. 11th century. Silk and gold thread. 51.8 × 58 cm. Figure 3 Textile fragment from Colls. 11th century. Silk and gold thread. 51.8 × 58.5 cm. Museo de Huesca, no. 1542. Museo de Huesca, no. 1542. Figure 4 An assortment of liturgical treasures from Serradaui, Huesca Photo: Adolf Mas, c Arxiu Mas/Institut Amatller d’Art Hispanic Figure 5 Textiles with enamel casket ca. 1931, San Isidoro de Leon Photo: Adolf Mas, c Arxiu Mas/Instituto Amatller d’Art Hispanic Figure 4 An assortment of liturgical treasures from Serradaui, Huesca Photo: Adolf Mas, c Arxiu Mas/Institut Amatller d’Art Hispanic Figure 5 Textiles with enamel casket ca. 1931, San Isidoro de Leon Photo: Adolf Mas, c Arxiu Mas/Instituto Amatller d’Art Hispanic Figure 4 An assortment of liturgical treasures from Serradaui, Huesca Photo: Adolf Mas, c Arxiu Mas/Institut Amatller d’Art Hispanic Figure 5 Textiles with enamel casket ca. 1931, San Isidoro de Leon Photo: Adolf Mas, c Arxiu Mas/Instituto Amatller d’Art Hispanic Figure 5 Textiles with enamel casket ca. 1931, San Isidoro de Leon Figure 6 Lining of the reliquary of San Juan Bautista and San Pelayo, San Isidoro de Leon Photo: Ana Cabrera Figure 6 Lining of the reliquary of San Juan Bautista and San Pelayo, San Isidoro de Leon Photo: Ana Cabrera Figure 7 Elephant fragment or so-called Baghdad Silk, San Isidoro de Leon Photo: Ana Cabrera Figure 7 Elephant fragment or so-called Baghdad Silk, San Isidoro de Leon Photo: Ana Cabrera Photo: Ana Cabrera Photo: Ana Cabrera Figure 8 Textile fragment with wrestling lions and harpies from the tomb of Pedro de Osma. Spain, probably Almeria, early 12th century. Silk lampas with supplementary discontinuous metal-wrapped patterning wefts, 50 × 43 cm. Boston, Museum of Fine Arts, 33.371. Ellen Page Hall Fund. Photo: c Museum of Fine Arts, Boston 8 Textile fragment with wrestling lions and harpies from the tomb of Pedro de Osma. Supplementary Material Photo: c Museum of Fine Arts, Boston Figure 9 Detail, textile from the reliquary of San Juan Baustista y San Pelayo Photo: Ana Cabrera Figure 9 Detail, textile from the reliquary of San Juan Baustista y San Pelayo Photo: Ana Cabrera Figure 10 Reliquary of San Marcelo/San Claudio, early twentieth century, with textiles still in situ Photo: Archivo Gomez-Moreno, CSIC Figure 10 Reliquary of San Marcelo/San Claudio, early twentieth century, with textiles still in situ Photo: Archivo Gomez-Moreno, CSIC Photo: Archivo Gomez-Moreno, CSIC Photo: Archivo Gomez-Moreno, CSIC Figure 11 Detail of San Marcelo/San Claudio, showing a standing male figure with a knife Photo: Therese Martin Figure 11 Detail of San Marcelo/San Claudio, showing a standing male figure with a knife Photo: Therese Martin Figure 12 Detail of San Marcelo/San Claudio, showing a female figure on a beast Photo: Therese Martin Figure 12 Detail of San Marcelo/San Claudio, showing a female figure on a beast Photo: Therese Martin
https://openalex.org/W3210084948	https://eprints.gla.ac.uk/257792/1/257792.pdf	English	null	Identifying novel candidates and configurations for human helminth vaccines	Expert review of vaccines	2,021	cc-by	5,025	Expert Review of Vaccines Expert Review of Vaccines ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ierv20 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ierv20 Rick M. Maizels Rick M. Maizels Wellcome Centre for Integrative Parasitology; Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK ARTICLE HISTORY Received 18 August 2021 Accepted 26 October 2021 KEYWORDS Cestode; nematode; trematode; extracellular vesicle; Th2 immunity ARTICLE HISTORY Received 18 August 2021 Accepted 26 October 2021 ABSTRACT Introduction: : Human infections with helminth worm parasites are extraordinarily prevalent across tropical and subtropical parts of the world, and control relies primarily on drugs that offer short-term suppression of infection. There is an urgent need for new vaccines that would confer long-lived immunity, protecting children in particular and minimizing community transmission. KEYWORDS Cestode; nematode; trematode; extracellular vesicle; Th2 immunity y p g p g y Areas covered: : This article discusses the development of helminth vaccines, from the first successful veterinary vaccines that demonstrated the feasibility of inducing protective immunity to helminths, to more recent initiatives to test human helminth antigens. The field has focussed primarily on evaluating individual antigens that could constitute targets amenable to antibody attack to inhibit parasite establishment. In a new direction, vaccines employing extracellular vesicles released by helminths have also given exciting results. g g Expert opinion: : Taking into account the complex life cycles and sophisticated immune evasion strategies of many helminths, a combination of antigens and approaches designed to target essential functional pathways of the parasite will be required to achieve a high level of protection in future anti- helminth vaccines. 1. Introduction Despite these successes, we still lack vaccines for human helminth infections. In part, irradiated larval strategies could not be applied to humans on any scale, and there are far greater regulatory hurdles than in the veterinary setting. Nevertheless, hundreds of candidate antigens have been tested in model systems for filariasis [15,16], schistosomiasis [17–19], soil-transmitted nematodes [20] and other human helminth parasites [21], with trials in endemic populations under way for hookworm and schistosomiasis [22]. But there remain some fundamental obstacles to achieve fully effective vaccines against human helminth parasites. Vaccines against human helminth parasites remain one of the greatest challenges in global medicine [1,2]. Helminth round worm (nematodes) and flatworm (cestodes and trematodes) infect over a billion people across many low/middle-income countries [3], with high prevalence in children and a myriad of pathological effects [4]. Drug treatments are of limited effi cacy, with populations rapidly reinfected and showing little sign of acquired immunity from natural exposure to infections [5,6]. Hence, there is a burning need for vaccines that would interrupt transmission and confer lasting benefits on many of the poorest communities in the world [7,8]. Efforts to generate vaccines against helminths date back 50 years to the development of a dog hookworm vaccine using irradiated infective larvae of Ancylostoma caninum [9,10]. While not a commercial success (as the vaccine did not entirely prevent transmission), this demonstrated that vaccine-induced immunity was possible, and paved the way for a similar irradiated larval vaccine (Huskvac) for lungworm (Dictyocaulus viviparus) in cattle [11]. A separate initiative, also in the veterinary arena, led to the development of a vaccine based on purified intestinal antigens of Haemonchus contortus (the ‘Barber’s Pole’ worm), now mar keted as Barbervax [12,13]. Finally, successful recombinant vaccines have been pioneered against cestode tapeworms of livestock [14], now marketed in a number of tropical countries as CysVax (for Taenia solium in pigs) and Hidatil (for Echinococcus granulosus in ruminants). CONTACT Rick M. Maizels rick.maizels@glasgow.ac.uk Wellcome Centre for Integrative Parasitology; Institute of Infection, Immunity and Inflammation, University of Glasgow, 120 University Place, Glasgow G12 8TA, UK © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Identifying novel candidates and configurations for human helminth vaccines Rick M. Maizels To cite this article: Rick M. Maizels (2021) Identifying novel candidates and configurations for human helminth vaccines, Expert Review of Vaccines, 20:11, 1389-1393, DOI: 10.1080/14760584.2021.1999810 To link to this article: https://doi.org/10.1080/14760584.2021.1999810 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 09 Nov 2021. Submit your article to this journal Article views: 453 View related articles View Crossmark data © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. Published online: 09 Nov 2021. Submit your article to this journal Article views: 453 View related articles View Crossmark data EXPERT REVIEW OF VACCINES 2021, VOL. 20, NO. 11, 1389–1393 https://doi.org/10.1080/14760584.2021.1999810 3. Mechanism-led approaches g p ●Extracellular vesicles may represent conserved targets suitable for vaccine development. Historically, helminth antigen identification strategies have been empiricalfor example, screening for antigens recog nized by serum antibodies from exposed but uninfected (‘putatively immune’) individuals, with less consideration given to biological context (e.g. expression through the life cycle, essential function and accessibility to the immune system. With the explosion in helminth genomics, transcriptomics and proteomics, candidate antigens are plentiful and require thoughtful criteria to select the most effective. would have to be sufficiently expressed over time, and be essential for survival if a vaccine were not simply to select for parasites that lose expression of the antigen. It is also to be expected that many helminth antigens will show polymorph isms across natural populations; while this dimension is rarely evaluated, a combination approach will minimize the danger of vaccine failure due to antigenic variation of the key target epitope. p p A further problem with single subunit strategies is that all induce partial rather than complete immunity. Even in animal models, with little variation in host or parasite, immunization with individual antigens is often considered successful if 50% worm load reductions are achieved, and trials in endemic human populations with single antigen vaccines have so far been disappointing [23]. Vaccines that reduce worm loads by 50% may not compromise the ability of helminths to down-reg ulate the immune system, and will do little to break transmission; those that block egg production while leaving adult worms unscathed may do little to benefit the individual receiving the vaccine. Hence, a synergistic approach will often be needed. The cestode vaccines that succeed with a single target (To45 or Eg95), are exceptional as the target oncosphere stage is small enough (~20 µm) to be lysed by complement, and expresses a minimal number of products essential for its invasion of the intestinal mucosa [24]. Among other helminth organisms, the schistosome protein Sm-p80 (calpain) individually induces high levels of pro tection in baboons and is currently awaiting clinical trials [25,26], but no other single protein has proved so effective. Most anti- helminth vaccines may therefore need to be multi-component ‘cocktails,’ as successfully applied to a sheep nematode [27] and more recently explored with the human filarial parasite Brugia malayi [28]. 3. Mechanism-led approaches Although posing considerable obstacles, for example, if each individual component must be produced and validated under GMP conditions, new technologies such as mRNA vaccines [29] offer more rapid development and could facilitate the combi nation of multiple antigens into a single immunogen. This brings to the fore the question of mechanism; a clear understanding of how the immune system targets and elim inates helminth parasites is required for a rational choice of vaccine antigens. Again, adopting paradigms of antibody- mediated cytotoxicity or lytic T cells from the microbial world can be misleading – rather than sudden death, hel minths are degraded more slowly and by the mass effect of innate immune cells, either by swarming and surrounding in the tissues, or bathing in mucus and inhibitory products in the lumen of the gut. Over time, these progressively compromise parasite fitness, first reducing egg production (the ‘anti- fecundity’ effect) and eventually causing the death of the whole organism [34]. Choosing antigens that represent key vulnerabilities of the parasite, as considered below, is of cen tral importance; so too is the recognition of responder T cell modes (primarily but not always Th2). However, few discus sions of helminth vaccines have as yet included pathways to promote mobilization of the innate effector populations (macrophages, eosinophils, and even neutrophils) that would be required to block worm establishment [35]. Across the helminth field, two lines of approach have been able to integrate mechanism with rational antigen selection. A long-standing example is that of targetting intestinal enzymes of hematophagous parasites such as H. contortus in sheep, and N. americanus in humans, or of flatworms established in the vasculature or tissues [36,37]. Here, the logic is that internal parasite constituents exposed on their intestinal membrane represent ‘hidden antigens’ which have not been subject to immune pressure, and for which even infected individuals have no preexisting antibody. Hence, vaccination will generate antibodies that, when ingested with blood by the parasites, will interfere with worm nutrition and eventually cause their demise. This principle underlies the Barbervax vaccine, using industrial-scale extraction from native parasites, and the sec ond generation human hookworm vaccine currently under evaluation [38]. The latter is a bi-valent entity with recombi nant APR-1 (aspartyl protease which digests hemoglobin) and GST-1 (gluathione S-transferase which detoxifies heme). Article highlights ●Helminth vaccines have been shown to be effective in the veterinary arena ●Subunit vaccines with single antigens are less likely to succeed for human helminth parasites p ●Vaccine design should be informed by mechanistic understanding of immunity y ●Combinatorial antigens are likely to be required for effective vaccina tion, and would offer advantages of vaccines targeting multiple stages and species 2. Challenges for helminth vaccines A primary consideration is the nature of helminth infections, in which parasites migrate through different tissues, maturing from infective stages to adult worms, presenting a ‘moving target’ in more senses than one. Secondly, helminths are generally large, resilient organisms that may require a sus tained and multi-pronged immune assault, rather than the one-off ‘lethal hit’ that the immune system can deliver to a virus-infected cell. Thirdly, they express multiple immune eva sion strategies, at many levels and most likely in a redundant fashion, that subvert and defuse vaccine-induced immunity. These factors militate against the conventional single sub unit vaccine development approach that has proved success ful for microbial pathogens. In contrast, for a single helminth antigen to induce effective protective immunity, that target 1390 R. M. MAIZELS Article highlights ●Helminth vaccines have been shown to be effective in the veterinary arena ●Subunit vaccines with single antigens are less likely to succeed for human helminth parasites ●Vaccine design should be informed by mechanistic understanding of immunity ●Combinatorial antigens are likely to be required for effective vaccina tion, and would offer advantages of vaccines targeting multiple stages and species ●Extracellular vesicles may represent conserved targets suitable for vaccine development. that may persist in dampening immunity, particularly as infec tions are known to inhibit responses to microbial vaccines [32,33]. Thus, prior infection could confound vaccination either by hyper-reactivity to vaccine antigens, or conversely by ‘immunoregulatory memory’ that would represent a form of antigen-specific immune tolerance that could prevent responses to a helminth vaccine. 3. Mechanism-led approaches polygyrus EVs revealed a prominent set of components associated with the apical intestinal epithelium of nematodes [43], including homologs of antigens represented in Barbervax, the prepara tion of H. contortus gut material, such as H11, as well tetra spanins discussed above which are already candidate vaccines for S. mansoni. Immunogenic tetraspanins also were abundant in a proteomic analysis of S. haematobium EVs, alongside other vaccine candidates such as GST [48], giving credence to the possibility that immunization with these antigens targets EVs as well as parasite tissues. A second mechanistic approach with a strong immuno logical foundation has been to target the suite of factors that helminths release to modify their environment and down-modulate the host immune system. Indeed, many of the first experimental vaccines in different helminth models utilized released molecules (termed excretory/secretory or ES products) and achieved high levels of protection [39]. However, these are generally complex mixtures and in very few cases have individual ES proteins been shown to induce protective immunity. Most probably, the redundancy of parasite products is responsible for lack of efficacy, return ing the discussion to the need for multicomponent vac cines. In a study on a murine model system, Heligmosomoides polygyrus, immunization with ES products elicited sterile immunity to challenge infection; from the mixture, a combination of 3 proteins (VAL-1/2/3) were suffi cient to drive immunity [40]. Interestingly these are mem bers of the same gene family as N. americanus ASP-2, which was an earlier human vaccine candidate, and a number of homologs tested as vaccines for veterinary nematodes [41]. It remains to be determined if there is functional homology between members of this gene family in the different host- parasite combinations, but their prominence as effective immunogens does indicate a key role in parasite modula tion of the host [41]. Furthermore, vaccination with individual EV antigens, rather than intact EVs, may suffice to generate an antibody response that will neutralize the total immunomodulatory cargo – including small RNAs [44]. In this setting, parasites release combinations of immune modulators that are pack aged into the EVs, to target host cells such as the macrophage and block protective immunity. Due to the discrete nature of the vesicle, antibody binding to any of its surface epitopes should result in uptake of EVs by phagocytes, and thereby destruction of the whole entity. 5. Expert opinion Historically, the field of human helminth vaccine development has been dominated by the search for individual antigens that would generate protective immunity against a single species of parasite. However, helminths are complex organisms with sophisticated immune evasion mechanisms, and immunity is likely to require a co-ordinated attack on many fronts. Hence, we may already have in hand key antigenic targets but need to consider how to employ them to best effect, in combina tions and with optimal adjuvants that induce the most effec tive mode of immune response. Hence, it may be that the era of discovering new candidate antigens for the major human helminths will soon draw to a close, replaced by a strategy of selecting, combining, and configuring the known candidates, 3. Mechanism-led approaches Hence, targetting EVs by vaccinating against a single surface antigen could achieve exactly the combinatorial effect that is required for immunity. In practice, an anti-EV vaccine would be most potent target ting multiple-surface constituents, especially if the vesicles are heterogeneous with respect to membrane protein expression, or (as it likely) differ in composition between immature and mature parasite stages. 3. Mechanism-led approaches In A widely discussed challenge for helminth vaccines is the immunization of individuals who have been previously exposed, and/or are currently infected with parasites that profoundly manipulate the host immune system [30]. Preexisting IgE responses in adult recipients of the N. amer icanus ASP-2 antigen led its discontinuation in the first itera tion of the human hookworm vaccine [31], although it may still be suitable for vaccination of infants prior to their first exposure. Developing successful vaccines against helminths also requires appreciation of the immunomodulatory effects EXPERT REVIEW OF VACCINES 1391 regulating the activation of macrophages involved in innate immunity to helminths [45]. combination, antibodies are thought to prevent parasites from digesting blood normally. While this is an attractive strategy, with proven efficacy in the case of H. contortus, it is limited to those species that feed on blood, and targets the mature stages of those species, rather than immature, tissue-migra tory larvae. In addition, if the target antigens are never pre sented to the immune system, there will be no natural boosting by subsequent parasite infection or exposure, neces sitating re-vaccination to maintain antibody levels. In the model H. polygyrus system, secreted EVs were found to evoke a strong serum antibody response, and a high degree (>80%) of protective immunity when administered with alum adjuvant [45]. In vitro, anti-EV antibodies promoted uptake of vesicles by macrophages into the lysosomal pathway, dis abling them from exerting modulatory effects. Thus, in the presence of antibodies macrophages were protected from immunosuppression and retained their type 2 profile. Subsequently, further studies reported that immunization with EVs from the trematode Opisthorchis viverrini, and the nematode Trichuris muris, induce a protective immune response [46,47]. Further examples of this physiology-led approach are found in the current generation of Schistosome vaccines, including Sm-p80, which is highly expressed on the adult worm tegument being implicated in surface turnover and immune evasion, and as mentioned above generated a high level of protection in baboons [25,26]. Other candidates being actively pursued are tetraspanin (TSP)-2, again linked to the tegumental membrane, FABP/Sm-14, a widely expressed fatty acid binding and uptake protein, as well as the glutathione S- transferase Sh28GST, which in Schistosomes is also involved with muscle function [30]. Interestingly, there are suggestions that the existing S. mansoni vaccine antigens should be employed in combination [22]. Analysis of the proteins associated with H. •• Insightful and informative overview of the helminth vaccine field. •• Insightful and informative overview of the helminth vaccine field. 3. James S, Disease GBD, Injury I, et al. Global, regional, and national incidence, prevalence, and years lived with disability for 354 dis eases and injuries for 195 countries and territories, 1990–2017: a systematic analysis for the Global burden of disease study 2017. Lancet. 2018 Nov 10;392(10159):1789–1858. Combinatorial vaccines, however, imply a substantial growth in permutations that will require testing, shining a greater spotlight on screening and trial capacity. The pro blem is accentuated because animal models are distant from the human setting for most helminths, although Schistosoma mansoni infects rodents and the human and mouse Trichuris species are closely related. On a positive note, veterinary helminth vaccines are being actively pur sued [50] and can act as excellent pathfinders across a range of issues, from antigen validation, selection of combi nations, and optimal mode of immune response induction, each against a backdrop of a pan-helminth vaccine agenda. Moreover, exciting progress has been made on controlled human infection with hookworm [51,52,53] and schisto somes [54] that will more quickly and rigorously evaluate front-running vaccine formulations. 4. Hotez PJ, Brindley PJ, Bethony JM, et al. Helminth infections: the great neglected tropical diseases. 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McKeand JB. Vaccine development and diagnostics of Dictyocaulus viviparus. Parasitology. 2000;120:S17–23. Undoubtedly, the pipeline to realizing a final vaccine for human use is long and difficult, with multiple hurdles to progress each candidate through the ‘critical path’ to approval [2]. References Papers of special note have been highlighted as either of interest (•) or of considerable interest (••) to readers. In the case of functional ‘hidden antigens’ and those from EVs, there is more likelihood of conserved epitopes shared across species so that a pan-helminth vaccine might be pos sible, and there is a further possibility of a compound vaccine presenting homologous antigens from multiple species to create the pan-helminth vaccine discussed previously [49]. Strategically, a vaccine that targetted multiple helminth spe cies would be not only of logistical value in delivering protec tion to low-income communities, but would also avoid the possibility that clearance of one helminth species could pro vide greater opportunities for others. 1. Hewitson JP, Maizels RM. Vaccination against helminth parasite infections. Expert Rev Vaccines. 2014;13:473–487. 1. Hewitson JP, Maizels RM. Vaccination against helminth parasite infections. Expert Rev Vaccines. 2014;13:473–487. 2. Diemert DJ, Bottazzi ME, Plieskatt J, et al. Lessons along the critical path: developing vaccines against human helminths. Trends Parasitol. 2018 Sep;34(9):747–758. Reviewer disclosures Peer reviewers on this manuscript have no relevant financial or other relationships to disclose. Declaration of interest The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. Whether based on conventional protein antigens, or EV- based components, vaccines can be developed that target both larvae and adults, preempting both pathology and transmission; immunity to helminths can act to degrade their fitness, as observed in ‘anti-fecundity immunity’ in which surviving adult worms produce fewer eggs. Accordingly, it may be expected that even if parasites survive immune attack as larvae, they will be less able to resist expulsion once they mature to adults. In this setting, multi-stage vaccines should show a synergistic effect, and would be particularly desirable where tissue-migrating lar vae are as pathogenic as intestinal adults of the same species. 4. Extracellular vesicles – a new target Most recently, a new dimension in helminth vaccines has emerged with the discovery that many parasites release within their ES material not only soluble macromolecules, but lipid- bound vesicles containing a cargo of proteins and small RNAs [42–44]. Importantly, these extracellular vesicles (EVs) exert significant immunomodulatory effectsfor example, inhibiting the expression of the receptor for IL-33 that is required to initiate the type 2 immune response [43], and down- 1392 R. M. MAIZELS R. M. MAIZELS R. M. MAIZELS 1392 potentially through modifications to maximize immunogeni city and minimize any safety concerns. •• Insightful and informative overview of the helminth vaccine field. 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https://openalex.org/W1780914609	https://europepmc.org/articles/pmc1379650?pdf=render	English	null	Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines	BMC cancer	2,006	cc-by	11,085	Open Acc Research article Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines Sharon E Campbell1, William L Stone2, Steven Lee2, Sarah Whaley1, Hongsong Yang2, Min Qui2, Paige Goforth1, Devin Sherman1, Derek McHaffie1 and Koyamangalath Krishnan3 Address: 1Department of Internal Medicine, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA, 2Department of Pediatrics, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA and 3Department of Clinical Cancer Prevention and Gastrointestinal Medical Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX 770303, USA Email: Sharon E Campbell - campbese@etsu.edu; William L Stone - stone@etsu.edu; Steven Lee - microbes99@yahoo.com; Sarah Whaley - whaleys@etsu.edu; Hongsong Yang - YANGH@mail.etsu.edu; Min Qui - QUI@mail.etsu.edu; Paige Goforth - pmgoforth@charter.net; Devin Sherman - devinsherman@hotmail.com; Derek McHaffie - zdrm16@imail.etsu.edu; Koyamangalath Krishnan - KKrishna@mdanderson.org mail: Sharon E Campbell* - campbese@etsu.edu; William L Stone - stone@etsu.edu; Steven Lee - microbes99@yah arah Whaley - whaleys@etsu.edu; Hongsong Yang - YANGH@mail.etsu.edu; Min Qui - QUI@mail.etsu.edu; Sarah Whaley whaleys@etsu.edu; Hongsong Yang YANGH@mail.etsu.edu; Min Qui QUI@mail.etsu.edu; Paige Goforth - pmgoforth@charter.net; Devin Sherman - devinsherman@hotmail.com; Derek McHaffie - zdrm16@imail.etsu.edu; Koyamangalath Krishnan - KKrishna@mdanderson.org aige Goforth - pmgoforth@charter.net; Devin Sherman - devinsherman@hotmail.com; Derek McHaffie - zdrm16@ oyamangalath Krishnan - KKrishna@mdanderson.org * Corresponding author Received: 15 August 2005 Accepted: 17 January 2006 Received: 15 August 2005 Accepted: 17 January 2006 Published: 17 January 2006 BMC Cancer 2006, 6:13 doi:10.1186/1471-2407-6-13 BMC Cancer 2006, 6:13 doi:10.1186/1471-2407-6-13 This article is available from: http://www.biomedcentral.com/1471-2407/6/13 © 2006 Campbell et al; licensee BioMed Central Ltd. p This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. BioMed Central BioMed Central BioMed Central BMC Cancer Open Access Research article Open Acc Research article Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines Sharon E Campbell1, William L Stone2, Steven Lee2, Sarah Whaley1, Hongsong Yang2, Min Qui2, Paige Goforth1, Devin Sherman1, Derek McHaffie1 and Koyamangalath Krishnan3 Background To date, no study has compared the anti-prolifera- tive and apoptotic effects of α- and γ-tocopherol on colon cancer cell lines with differences in molecular features. Gysin et al. demonstrated the inhibition of cell prolifera- tion by RRR-γ-tocopherol treatment at 25 µM on CaCo-2 (colon carcinoma), DU-145 and LNCap (prostate carci- noma) and SaOs-2 (osteosarcoma) cells, but demon- strated no apoptosis in any of the cell lines tested. While most clinical studies with vitamin E have used all- rac-α-tocopherol, recent studies show that RRR-γ-toco- pherol also may play a unique role in preventing colon cancer [5]. Epidemiological evidence shows that RRR-γ- tocopherol levels in plasma correlate with a reduced risk for both colon and prostate cancer [6-8]. Animal studies measuring direct end-points of cancer (i.e., survival stud- ies, tumor reduction, tumor prevention) with RRR-γ-toco- pherol are lacking. Vitamin E was demonstrated a chemopreventive in animal models of chemically induced colon cancer [9], but these studies tested only α-tocophe- rol or α-tocopherol acetate (RRR- or all-rac- not specified). Animal studies measuring the potential chemopreventive mechanisms of RRR-γ-tocopherol have used surrogate biomarkers of carcinogenesis. For example, RRR-γ-toco- pherol can suppress the expression of ras p-21 in rat colonocytes in vivo [10]. Using Wistar rats, Jiang has dem- onstrated that RRR-γ-tocopherol, but not α-tocopherol (RRR- or all-rac- not specified) decreases the proinflam- matory eicosanoid PGE2 (which is known to play a role in the progression of colorectal cancer through inflamma- tion). Further, hemodialysis patients administered RRR-γ- enriched tocopherols showed a consistently lower level of C-reactive protein (a biomarker of inflammation) while the administration of α-enriched tocopherols (RRR- or all-rac- not specified) did not [11]. In this study, we compared the anti-proliferative and apoptotic effects of RRR-α-tocopherol and RRR-γ-toco- pherol in four colon cancer cell lines with varying molec- ular characteristics, SW480 (APC, type I truncation and COX-2 deficient), HCT-15 (COX-2 deficient), HCT-116 (APC, wild-type and COX-2 inducible), and HT-29 (APC, type II truncation and COX-2 constitutive expression) and normal untransformed colon cells (CCD-12CoN). Prolif- eration studies demonstrated that RRR-γ-tocopherol is able to inhibit proliferation and cause cell death in all four cancer cell lines, but not in the normal cells. In the colon cancer cell lines tested for apoptosis, we have shown that RRR-γ-tocopherol activates cleavage of PARP and caspase 3, 7 and 8, but not caspase 9. Dietary RRR-γ- Cooney et al. Background g Differences in the incidence of colorectal cancer through- out the world suggest that diet has a role in colorectal car- cinogenesis [1]. Mediterranean diets, rich in vitamin E isoforms, are associated with a lower incidence of colon cancer [2,3]. Vitamin E refers to any of four tocopherols or tocotrienols (α, β, δ, and γ) isoforms. All of the tocopherol isoforms found in nature are the RRR-forms since all three chiral carbons on the side chain attached to the chroman head group have the "R-" configuration rather then the "S- " configuration. Synthetic vitamin E (typically found in dietary supplements) almost always refers to all-rac- alpha-tocopherol which is a racemic mixture containing eight stereo isomers, one eighth of which is the biologi- cally active RRR-isoform. The isoform found in highest concentration in the serum is the RRR-α-tocopherol. The primary form of vitamin E found in the North American diet is RRR-γ-tocopherol, which is present at levels 2–4 times higher than that of RRR-α-tocopherol [4]. Vitamin E isoforms (and metabolites) have had varying effectiveness at inhibiting cell growth and inducing apop- tosis. RRR-γ-Tocopherol is superior to all-rac-α-tocophe- rol at cell growth inhibition in vitro in human prostate cancer cells [16]. Vitamin E succinate (VES) and tocot- rienols demonstrate potent apoptotic inducing properties [17]. Zu et al. [18] have shown differential synergistic effects of selenium with vitamin E isoforms on cell growth and apoptosis in PC-3 human prostate cancer cells. VES was the most effective form tested and synergizes with selenium, while RRR-α-tocopherol and RRR-α-tocopheryl acetate were weaker in their effects on suppressing growth and inducing apoptosis in PC-3 prostate cancer cells. α- Tocopherol (RRR or all-rac not specified) is a poor inducer of apoptosis in the colon cancer xenograft nude mouse model, while the synthetic form, α-tocopheryl succinate (a redox inactive analogue of vitamin E) is a strong inducer of apoptosis [19]. In addition, carboxyethyl hydroxychromans (CEHC) metabolites of γ-tocopherol are powerful inducers of apoptosis and inhibit cell growth and down regulate cyclin expression in PC-3 prostate can- cer cell lines [20]. The performance of vitamin E deriva- tives and analogues with regard to apoptosis also vary among tissue types. VES and a vitamin E analogue, 2,5,7,8-tetramethyl-2R-(4R,8R,12-trimethyltridecyl)chro- man-6-yloxy acetic acid (α-TEA) can induce human breast, prostate, colon, lung, cervical, and endometrial tumor cells, but the apoptotic-inducing effects of α-TEA are greater in human ovarian and cervical cancer cells [21]. http://www.biomedcentral.com/1471-2407/6/13 http://www.biomedcentral.com/1471-2407/6/13 RRR-γ-tocopherol is taken up by RAW 264.7 macrophages to a much greater extent than RRR-α-tocopherol [15]. RRR-γ-tocopherol is taken up by RAW 264.7 macrophages to a much greater extent than RRR-α-tocopherol [15]. Abstract Background: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR- γ-tocopherol isoform is found primarily in the US diet, while RRR-α-tocopherol is highest in the plasma. Methods: The effectiveness of RRR-α- and RRR-γ-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD- 112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-α- or RRR-γ- tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. Results: Treatment with RRR-γ-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-α- tocopherol did not. Further, RRR-γ-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-γ-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-γ-tocopherol to induce cell death. Conclusion: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-γ-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-γ-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-γ-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-γ-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents. Page 1 of 14 (page number not for citation purposes) BMC Cancer 2006, 6:13 Cell viability and cell death analysis y y SW480, HT-29, HCT-116 and HCT-15 cells were seeded at 1.5 × 105 cells/well in 12 well plates 24 hours prior to tocopherol treatment for time intervals to 72 hours. Toco- pherol-enriched media was added to a concentration up to 100 µM tocopherol. This concentration was selected after performing Live-Dead Assays (Molecular Probes) and determining that it was the lowest concentration where cell death was mediated by α-tocopherol. We selected this concentration to determine if α-tocopherol could induce apoptosis. Cells were removed from flasks by trypsinization at indicated times and counted with a hemocytometer using trypan blue staining and the Beck- man Coulter Z2 cell counter. The cells were assayed by trypan blue exclusion stain as described above. At lower tocopherol concentrations and incubation times the cells were seeded at 1 × 104 cells/well. Tocopherol enriched media was added to the appropriate concentration at the zero time interval and was replenished every 72 hours. Cell viability was assayed hemocytometer cell counting with trypan blue dye. Troglitazone, 15-deoxy ∆12,14- PGJ2, and camptothecin are capable of inducing cell death and apoptosis in colon cancer cells and were used as pos- itive controls for cell death and apoptosis. Cell viability was measured by live cell counts as a function of time and compared to the vehicle, while cell death was measured as the percentage of dead cells at each time point measured. Intracellular analysis of vitamin E by HPLC analysis y f y y The tocopherol content of the treated cell lysates was measured using HPLC analysis with highly sensitive elec- trochemical detection as previously described [22]. The response factors of tocopherols relative to tocol (an inter- nal standard) were determined in triplicate. The concen- trations of RRR-α-tocopherol and RRR-γ-tocopherol were measured using a Spectronic Genesys 5 spectrophotome- ter and published extinction coefficients [23]. http://www.biomedcentral.com/1471-2407/6/13 http://www.biomedcentral.com/1471-2407/6/13 BMC Cancer 2006, 6:13 http://www.biomedcentral.com/1471-2407/6/13 tocopherol may be an effective colon cancer preventive agent while pharmacological concentrations of RRR-γ- tocopherol may be useful as an adjuvant chemotherapy agent resulting lower dose of agents that are cytotoxic to normal cells. exclusion assay. EthD-1 dye and cell concentrations were optimized according to manufacturers' instructions. SW480 cells were seeded at a density of 5 × 104 cells/well while HCT-116 cells were plated at a density of 2 × 104 cells/well in 96-well plates for 24 hours before treatment. Vitamin E-enriched PBS (previously described) was added at 25, 50, 100, 150 and 200 µM concentrations. Cells were treated for five hours. Percent dead assay Percent cell death was measured by using the Live-Dead Assay according to the manufacturer's instructions (Molecular Probes, Eugene, OR) with a Gemini XS fluor- imeter (Molecular Devices, Sunnyvale, CA). The percent- age of cell death was calculated by measuring the fluorescence intensity of the ethidium bromide homodimer (EthD-1) that is excluded from live cells, but enters the cells with damaged membranes and undergoes a fluorescence enhancement upon binding to nucleic acids in dead cells (λex = 530 nm and λem = 645 nm). Con- trol samples for percent dead calculations were verified for viability before and after plating using the trypan blue Methods Chemicals α-Tocopherol (Eastman Chemical, Kingsport, TN, 99% pure RRR-α-tocopherol), γ-tocopherol (Tama Biochemi- cal, Tokyo, Japan, 97% pure RRR-γ-tocopherol), troglita- zone (BioMol Research Lab, Plymouth Meeting, PA), 15- deoxy∆12,14-PGJ2 (BioMol Research Lab, Plymouth Meeting, PA), bovine serum albumin (Gibco BRL, Gaith- ersburg, MD), proteinase K (Sigma Chemical, St. Louis, MO), RNAse A (Sigma Chemical, St. Louis, MO), and camptothecin (Sigma Chemical, St. Louis, MO) were obtained from the indicated sources. Enrichment of tocopherol into tissue culture medium Enrichment of tocopherol into tissue culture medium Prior to treatment, the cell culture medium was enriched with tocopherol by adding the appropriate amount of tocopherol in ethanol, followed by five volumes of bovine serum albumin (BSA). The BSA/tocopherol mixture was vortexed and added to appropriate culture medium. In the vehicle-treated cells, the tocopherol was omitted from the BSA/ethanol mixture which was added to the appropriate culture medium supplemented with 10% FBS. Cell culture h l The colon cancer cell lines SW480 (RPMI 1640), HCT-116 (McCoy's), HT-29 (Dulbecco's Modified Eagle Medium), and HCT-15 (RPMI 1640) were purchased from American Type Culture Collection, ATCC (Manassas, VA) and grown in the indicated media supplemented with 10% FBS and 50 IU penicillin/streptomycin in a humidified atmosphere of 5% CO2 at 37°C. The CCD-112CoN (nor- mal primary cells, nontransformed phenotype) were grown in a humidified atmosphere of 5% CO2 at 37°C with Dulbecco's Modified Eagle's and supplemented with 10% FBS. Background found that RRR-γ-tocopherol was a much more potent inhibitor of neoplastic transformation in 3- methycholanthrene-treated C3H/H10T/1/2 murine fibroblasts than α-tocopherol (RRR- or all-rac- not speci- fied) [12]. In addition, RRR-γ-tocopherol is a potent inhibitor of COX-2 activity and inhibits human cancer cell cycle progression and cell proliferation by down-reg- ulation of cyclins [13,14]. Stone et al. have found that Page 2 of 14 (page number not for citation purposes) Page 2 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) Apoptosis assays Annexin V and propidium iodide (PI) double staining assay HCT-116 and SW480 cells were plated in 100 × 20 mm plates at cell densities between 3 × 106 and 5 × 106 cells/ plate. HCT-116 cells and SW480 cells were treated as described in the trypan blue staining assays. After treat- ment, cells were removed from the plate with trypsin and analyzed for phosphatidylserine externalization by an Page 3 of 14 (page number not for citation purposes) Page 3 of 14 (page number not for citation purposes) BMC Cancer 2006, 6:13 http://www.biomedcentral.com/1471-2407/6/13 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead vehicle alpha tocopherol gamma tocopherol troglitazone C. 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead vehicle alpha tocopherol gamma tocopherol troglitazone 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead vehicle alpha tocopherol gamma tocopherol troglitazone A. * * * * * * * * * * * * B. * * * * * * * * * * * * * * * * 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead vehicle alpha tocopherol gamma tocopherol troglitazone * A. * * * * * * * * * * * * B. * * * * * * * * * * 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead * A. * * * * * * * * * * * * A. vehicle alpha tocopherol gamma tocopherol troglitazone 0 5 10 15 20 25 30 25 50 100 150 200 Concentration (µµµµM) % Dead vehicle alpha tocopherol gamma tocopherol troglitazone C. * * * * * * SW480, HCT-116, and HT-29 cells were treated for 5 hours with varying concentrations of tocopherol or the PPAR γ ligand, troglitazone (positive control) Figure 1 SW480, HCT-116, and HT-29 cells were treated for 5 hours with varying concentrations of tocopherol or the PPAR γ ligand, troglitazone (positive control). The percent of dead cells for SW480 cells (A), HCT-116 cells (B) or HT-29(C) was measured using the Live-Dead assay (Molecular Probes, CA). (Data are representative of three independent trials performed in triplicate. PARP and caspase cleavage by western blot analysis PARP and caspase cleavage by western blot analysis p g y y The protein concentration of the cells lysates was deter- mined by the BCA protein assay (Pierce Biotechnology, Rockford, IL). Total protein was separated by electro- phoresis on a 12% SDS polyacrylamide gel and electro transferred onto Hybond-ECL nitrocellulose membrane using an X cell II Mini Cell Blot module (San Diego, CA). Blotted membranes were incubated with the primary anti- bodies indicated as follows: caspase 3, 7, 8 and 9 (Cell Sig- naling Technology, Beverly, MA) and probed with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, Beverly, MA). The signal was revealed on hyperfilm using the ECL Western blotting detection reagents 1 and 2 (1 mL each) detection system (Amersham Biosciences, Arlington Heights, IL). To con- trol for consistent loading, membranes were probed with the β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) after stripping the blot with Restore solution (Pierce Biotechnology, Rockford, IL) for 30 minutes at 37°C. Since our goal was to determine if both vitamin E iso- forms (RRR-α- and RRR-γ-tocopherol) could induce apop- tosis, a concentration was selected whereby cell death could be obtained with both isoforms. When treated with RRR-α-tocopherol, cells did not exhibit cell death at con- centrations lower than 100 µM (Figure 1). Effects of RRR-α- and RRR-γ-tocopherol treatment on cell proliferation in human colon cancer cells Effects of RRR-α- and RRR-γ-tocopherol treatment on cell proliferation in human colon cancer cells Since cell death and cell proliferation are different proc- esses, live cell counts were monitored over time in the four colon cancer cell lines, SW480, HCT-116, HCT-15, and HT-29 with 100 µM RRR-α- and RRR-γ-tocopherol treat- ment (Figure 2A–D). This was designed to assess the effects of the tocopherols on cell proliferation. In the HCT-116 and HT-29 (COX-2 positive) cell lines, RRR-α- tocopherol treatment resulted in a statistically significant http://www.biomedcentral.com/1471-2407/6/13 annexin V and propidium iodide apoptosis kit (Oncogene Research Products, San Diego, CA) according to the man- ufacturers' instructions using a Becton Dickson FACS Cal- ibur Flow Cytometer (Becton Dickson, San Diego, CA). Apoptotic cells were counted by flow cytometry and the resulting data analyzed using WinMDI freeware. annexin V and propidium iodide apoptosis kit (Oncogene Research Products, San Diego, CA) according to the man- ufacturers' instructions using a Becton Dickson FACS Cal- ibur Flow Cytometer (Becton Dickson, San Diego, CA). Apoptotic cells were counted by flow cytometry and the resulting data analyzed using WinMDI freeware. from annexin V staining and cell cycle data are measured as a percentage of the 10,000 events in the analysis. The apoptosis in the control cells are subtracted from the treated samples. The percentage values represent an aver- age percentage apoptotic cells over the control (set at 0%) of two independent assays performed in duplicate. Apoptosis assays p < 0.05 vs. vehicle at corresponding concentration.) SW480, HCT-116, and HT-29 cells were treated for 5 hours with varying concentrations of tocopherol or the PPAR γ ligand, troglitazone (positive control) Figure 1 SW480, HCT-116, and HT-29 cells were treated for 5 hours with varying concentrations of tocopherol or the PPAR γ ligand, troglitazone (positive control). The percent of dead cells for SW480 cells (A), HCT-116 cells (B) or HT-29(C) was measured using the Live-Dead assay (Molecular Probes, CA). (Data are representative of three independent trials performed in triplicate. p < 0.05 vs. vehicle at corresponding concentration.) Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) Page 4 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/6/13 BMC Cancer 2006, 6:13 Effects of RRR-α- and RRR-γ-tocopherol treatment on cell death in human colon cancer cells Effects of RRR-α- and RRR-γ-tocopherol treatment on cell death in human colon cancer cells DNA fragmentation was measured by gel electrophoresis by the standard published procedures. In brief, the cells were plated at a density of 5 × 106 cells/plate and treated with 100 µM tocopherol-enriched, vehicle-enriched media or 10 µM camptothecin (positive control) for 48 hours. A concentration of 1 × 106 cells/mL were pelleted and resuspended in lysis buffer (50 mM Tris HCl, 10 mM EDTA, 0.5% SDS). The cell lysates were treated with 20 mg/mL proteinase K and incubated at 55°C for 1 hour fol- lowed by addition of 0.5 mg/mL RNAse A and heated to 70°C for 5 minutes. DNA was precipitated with isopropa- nol, mixed with loading dye (10 mM EDTA, pH 8.0, 40 % sucrose, 0.25 % bromophenol blue) and analyzed on a 2% agarose gel containing 0.5 µg/mL ethidium bromide. Gel images were captured using an Alpha Innotech digital camera equipped with a transilluminator and Alpha Ease 5.5 software (Alpha Innotech Corporation, San Leandro, CA). Using the Live-Dead Assay (Molecular Probes) cell death was monitored with physiological and pharmacological concentrations of Vitamin E after 5 hours of treatment (Figure 1). The results varied among the cell lines, how- ever RRR-γ-tocopherol demonstrated a concentration- dependent increase in cell death over the vehicle in all cell lines tested. RRR-α-tocopherol did not demonstrate sig- nificant cell death over the vehicle at any concentration tested in the HCT-116 and was only significant at 200 µM in the HT-29 cells. The RRR-γ-tocopherol treatment resulted in significant cell death above the vehicle-treated cells in the SW480 and HCT-116 at all concentrations tested (25 µM through 200 µM). For the HT-29 cell line, RRR-γ-tocopherol at levels of 50 µM were effective at inducing cell death. The RRR-α-tocopherol treatment resulted in significant cell death at 100 µM in the SW480 and HCT-116 cell lines, while the HT-29 cells required 200 µM α-tocopherol to increase cell death above the con- trol. One-way ANOVA and tukey's statistical analysis was used to compare the dead cell means for each treatment to that of the vehicle. Asterisks above the bars demonstrate p-values less than 0.05 for cell mean comparison with the vehicle treatment. Post-hoc analysis (Tukey's test) revealed that RRR-γ-tocopherol was significantly better at inducing cell death than RRR-α-tocopherol in SW480 (at all concentrations tested) and HCT-116 cells (at concen- trations higher than 50 µM). Statistics Data is displayed as means with error bars representing standard deviation (SD). One-way analysis of variance (ANOVA) followed by Tukey's test was used to compare the means of live cells in the cell proliferation assays and percentage cell death. Probability levels (p-values) of < 0.05 indicate statistical significance. The values obtained Page 5 of 14 (page number not for citation purposes) BMC Cancer 2006, 6:13 http://www.biomedcentral.com/1471-2407/6/13 owth curves for SW480 cells (A) HCT-116 cells (B), HCT-15 cells (C), HT-29 (D) were plotted as average cell counts o e after treatment with 100 µM tocopherol at 1, 2, and 3 days gure 2 owth curves for SW480 cells (A) HCT-116 cells (B), HCT-15 cells (C), HT-29 (D) were plotted as average cell counts o e after treatment with 100 µM tocopherol at 1, 2, and 3 days. and Live Cell Counts in CCD-112CoN cells following 100 opherol treatment at 72 hours (E). Values plotted are averages of three independent trials. Error bars represent stand viation of the means. Positive controls used included (15 deoxy ∆ 12,14 PGJ2, troglitzone and camptothecin). (Data are entative of three independent trials performed in triplicate. p < 0.05 vs. vehicle at corresponding concentration.) HCT-116 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 10 20 30 40 vehicle troglitazone alpha tocopherol gamma tocopherol HT-29 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 10 20 30 40 vehicle troglitazone alpha tocopherol gamma tocopherol HCT-15 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 1 2 3 4 5 6 7 vehicle camptothecin alpha tocopherol gamma tocopherol SW480 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 1 2 3 4 5 6 7 vehicle 15 deoxy 12,14 PGJ2 alpha tocopherol gamma tocopherol A. B. D. C. CCD-112No 72 hours Live Cell Counts (millions) 0 10 20 30 40 vehicle alpha tocopherol camptothecin gamma tocopherol E. * * * * * * * * * * * * * HCT-116 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 10 20 30 40 vehicle troglitazone alpha tocopherol gamma tocopherol HT-29 cells B. * * * HCT-15 cells SW480 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 1 2 3 4 5 6 7 vehicle 15 deoxy 12,14 PGJ2 alpha tocopherol gamma tocopherol A. C * * * A. B. Effects of RRR-α- and RRR-γ-tocopherol treatment on cell viability in human colon cancer cell lines compared with human normal colon cell line CCD-112CoN Effects of RRR-α- and RRR-γ-tocopherol treatment on cell viability in human colon cancer cell lines compared with human normal colon cell line CCD-112CoN p p The effects of RRR-α-tocopherol and RRR-γ-tocopherol on apoptosis were measured by three independent assays on two selected colon cancer cell lines SW480 (COX-2 defi- cient) and HCT-116 (COX-2 inducible). In addition to the variable COX-2 expression, the SW480 and HCT-116 cell lines were selected for the apoptosis studies because these cell lines resulted in significant RRR-α-tocopherol-medi- ated cell death above the vehicle at 72 hours. It was rea- soned that if RRR-α-tocopherol could mediate apoptosis; it would be seen in these two cell lines. First, annexin V/ propidium iodide (PI) double staining was performed (Figure 4). This method identifies the percentage of cells that are in early (annexin V positive cells in lower right quadrant) and late apoptosis (annexin V and PI positive cells, double stained in the upper right quadrant). Both cell lines demonstrate significant apoptosis occurring fol- lowing treatment with RRR-γ-tocopherol, but much less with the RRR-α-tocopherol treatment. In fact, the SW480 The effects of 100 µM RRR-α- and RRR-γ-tocopherol treat- ments on cell viability were monitored with hemocytom- eter cell counts and the use of trypan blue dye following 72 hours in the normal nontransformed colon cell line CCD-112CoN (Figure 2E). RRR-γ-tocopherol treatment resulted in no cytotoxicity to normal colon cells, but dem- onstrated selectivity against cancer cell growth in all four colon cancer cell lines treated (Figure 2). RRR-α-tocophe- rol resulted in a statistically significant increase in CCD- 112CoN cells. Statistics paring the trypan blue live-cell means of the tococpherol-treated sampleswith vehicle-treated sample at µM tocopherol treatment Table 1: Statistical results comparing the trypan blue live-cell means of the tococpherol-treated sampleswith vehicle-treated sample at 48 and 72 hours following 100 µM tocopherol treatment tistical results comparing the trypan blue live-cell means of the tococpherol-treated sampleswith vehicle-tre ours following 100 µM tocopherol treatment Comparison of means was performed using one-way anova and a Tukey's post hoc analysis. Data are representative of three independent trials performed in triplicate. concentrations over longer time intervals. SW480, HCT- 116, and HT-29 cells were treated with 25 µM of either RRR-γ-tocopherol or RRR-α-tocopherol for 9–10 days. Figure 3 shows the live cell counts as a function of time for the SW480 and HT-29 cell lines. RRR-γ-tocopherol treat- ment in the SW480 cells resulted in significantly less live cells compared to the vehicle treatment after 96 hours and time periods beyond 144 hours (p < 0.05). In the SW480 cells, RRR-γ-tocopherol-treated cell numbers were signifi- cantly less than the RRR-α-tocopherol treated up to 216 hours. In the HT-29 cell line, the RRR-γ-tocopherol treat- ment is statistically less at time periods longer then 216 hours. The effects of RRR-α-tocopherol, in the HT-29 cells, are not statistically different from that of vehicle-treated cells. The HCT-116 cells showed no statistical difference from the control for either RRR-α-tocopherol or RRR-γ- tocopherol treatment at any time point tested (data not shown). decrease in cell proliferation, but the RRR-γ-tocopherol treatment resulted in a more pronounced decrease in cell proliferation compared with RRR-α-tocopherol. In fact, RRR-γ-tocopherol was statistically different from RRR-α- tocopherol in every cell line tested at 72 hours and in SW480, HCT-116, and HT-29 at 48 hours as demon- strated by post hoc analyses (Table 1). In the HCT-15 (COX-2 deficient) and SW480 (COX-2 deficient) cell lines, RRR-α-tocopherol treatment resulted in a statisti- cally significant increase in cell growth compared with the vehicle, which was most evident at the 72-hour time inter- val. RRR-γ-tocopherol treatment resulted in a statistically significant reduction in proliferation with every cancer cell line tested. The HT-29 (COX-2 positive) cells demon- strated a slight resistance to RRR-γ-tocopherol treatment, initially (24-hour treatment) however, by 48 hours RRR- γ-tocopherol was as effective in HT-29 cells as in the other cells treated. Statistics HCT-15 cells Ti (d ) 1.0 2.0 3.0 Live Cell Counts (millions) 0 1 2 3 4 5 6 7 vehicle camptothecin alpha tocopherol gamma tocopherol C. * HT-29 cells Time (days) 1.0 2.0 3.0 Live Cell Counts (millions) 0 10 20 30 40 vehicle troglitazone alpha tocopherol gamma tocopherol D. * * * * 40 D. C. E. Live Cell Counts (millions) 0 10 20 30 40 vehicle alpha tocopherol camptothecin gamma tocopherol * * Growth curves for SW480 cells (A) HCT 116 cells (B), HCT 15 cells (C), HT 29 (D) were plotted as average cell counts over time after treatment with 100 µM tocopherol at 1, 2, and 3 days Figure 2 Growth curves for SW480 cells (A) HCT-116 cells (B), HCT-15 cells (C), HT-29 (D) were plotted as average cell counts over time after treatment with 100 µM tocopherol at 1, 2, and 3 days. and Live Cell Counts in CCD-112CoN cells following 100 µM tocopherol treatment at 72 hours (E). Values plotted are averages of three independent trials. Error bars represent standard deviation of the means. Positive controls used included (15 deoxy ∆ 12,14 PGJ2, troglitzone and camptothecin). (Data are rep- resentative of three independent trials performed in triplicate. p < 0.05 vs. vehicle at corresponding concentration.) Page 6 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/6/13 BMC Cancer 2006, 6:13 BMC Cancer 2006, 6:13 Table 1: Statistical results comparing the trypan blue live-cell means of the tococpherol-treated sampleswith vehicle-treated sample at 48 and 72 hours following 100 µM tocopherol treatment SW480 HCT-15 HCT-116 HT-29 CCD- 112CoN Treatment Agent p-value 48 hour p-value 72 hour p-value 48 hour p-value 72 hour p-value 48 hour p-value 72 hour p-value 48 hour p-value 72 hour p-value 72 hour alpha tocopherol 0.034 <0.001 0.775 <0.0001 0.996 0.084 0.000 <0.0001 0.020 gamma tocopherol 0.0001 <0.001 0.112 <0.0001 0.0005 0.005 <0.0001 <0.0001 0.927 15-deoxy ∆12, 14 PGJ2 0.0004 <0.001 N/A N/A N/A N/A N/A N/A N/A troglitazone N/A N/A N/A N/A 0.0003 0.0005 0.0000 <0.0001 <0.0001 camptothecin N/A N/A 0.853 <0.001 N/A N/A N/A N/A N/A alpha vs. gamma <0.018 <0.001 0.416 <0.001 0.0005 0.001 0.0000 <0.0001 0.0117 Comparison of means was performed using one-way anova and a Tukey's post hoc analysis. Data are representative of three independent trials performed in triplicate. Effects of RRR-α- and RRR-γ-tocopherol treatment on apoptosis in human colon cancer cell lines Effects of RRR-α- and RRR-γ-tocopherol treatment on cell viability in human colon cancer cell lines compared with human normal colon cell line CCD-112CoN Effects of RRR-α- and RRR-γ-tocopherol treatment on apoptosis in human colon cancer cell lines Effects of RRR-α- and RRR-γ-tocopherol treatment on apoptosis in human colon cancer cell lines http://www.biomedcentral.com/1471-2407/6/13 http://www.biomedcentral.com/1471-2407/6/13 BMC Cancer 2006, 6:13 Live Cell Counts following a 25 µM α- and γ-tocopherol treatment at varying times up to ten days in SW480 cells (A) and HT-29 cells (B) Figure 3 Live Cell Counts following a 25 µM α- and γ-tocopherol treatment at varying times up to ten days in SW480 cells (A) and HT-29 cells (B). (Data are representative of two inde- pendent trials performed in triplicate. p < 0.05 vs. vehicle at corresponding concentration). SW480 cell counts 25 µM Time (days) 2 3 4 5 6 7 8 9 10 Live Cell Counts (millions) -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 vehicle alpha tocopherol gamma tocopherol camptothecin HT-29 cell counts 25 µM Time (days) 2 3 4 5 6 7 8 9 10 Live Cell Counts (millions) -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 vehicle alpha tocopherol gamma tocopherol camptothecin A. B. * * * * * * SW480 cell counts 25 µM Time (days) 2 3 4 5 6 7 8 9 10 Live Cell Counts (millions) -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 vehicle alpha tocopherol gamma tocopherol camptothecin A. * * * * * Further, we sought to determine which apoptotic path- way(s) are involved in the RRR-γ-tocopherol-mediated apoptosis, by monitoring caspase activation. Western blot analyses demonstrated that PARP, caspase 3, 7 and 8 are activated by cleavage in both cell lines with RRR-γ-toco- pherol treatment, but not RRR-α-tocopherol treatment (Figure 6). Caspase 9 was not activated by cleavage in either cell line (data not shown). SW480 cell counts 25 µM Further, we sought to determine which apoptotic path- way(s) are involved in the RRR-γ-tocopherol-mediated apoptosis, by monitoring caspase activation. Western blot analyses demonstrated that PARP, caspase 3, 7 and 8 are activated by cleavage in both cell lines with RRR-γ-toco- pherol treatment, but not RRR-α-tocopherol treatment (Figure 6). Caspase 9 was not activated by cleavage in either cell line (data not shown). A. Intracellular accumulation of RRR-α- and RRR-γ- tocopherol treatment is both time and concentration dependent The intracellular accumulation of RRR-α- and RRR-γ-toco- pherol in SW480 cells was determined as a function of the tocopherol concentration in the media (Figure 7A) and as a function of time (Figure 7B). Figure 7A demonstrates that the uptake of vitamin E is concentration dependent. We wanted to determine if the cellular up take during our treatment conditions (100 µM RRR-α- or RRR-γ-tocophe- rol at 24, 48 and 72 hours) followed a linear time depend- ent relationship. As shown in Figure 7B, the cellular uptake of RRR-α- and RRR-γ-tocopherol with respect to time is linear. The intracellular concentration of the vita- min E isoforms were normalized to the number of live cells in the sample at each time point. This was required since the tocopherols resulted in cell death at 100 µM. The intracellular concentrations of RRR-α- and RRR-γ-toco- pherol were very low (femtomoles/cell) when compared to the amount of tocopherol added to the media (100 µM) but the RRR-γ-tocopherol intracellular uptake was significantly higher than that of RRR-α-tocopherol. Time (days) 2 3 4 5 6 7 8 9 10 Live Cell Counts (millions) -0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 vehicle alpha tocopherol gamma tocopherol camptothecin B. * * B. Time (days) Effects of lower RRR-α- and RRR-γ-tocopherol treatment concentrations for extended time intervals on human colon cancer cell proliferation Since 100 µM is not a physiologically achievable concen- tration for dietary RRR-γ-tocopherol, we wanted to deter- mine if similar results could be obtained with lower Page 7 of 14 (page number not for citation purposes) Page 7 of 14 (page number not for citation purposes) Discussion Recently published work has demonstrated disparity among the vitamin E isoforms, analogs, and metabolites to induce apoptosis. For example, VES and CEHC metab- olites of vitamin E are strong apoptotic inducers, while α- tocopherol acetate (RRR- or all-rac- not specified), α-toco- pheryl acetate (RRR- or all-rac- not specified) and α-toco- pherol (RRR- or all-rac- not specified) are weak apoptotic inducers [17,18,20]. Gysin et al. used 25 µM RRR-γ-, RRR- α- and RRR-β-tocopherol, compared the proliferation effects, and determined that RRR-γ-tocopherol was best at inhibiting cell proliferation in human prostate carcinoma cells (DU-145 and LNCaP), human colorectal adenocarci- noma cells (CaCo2), and human osteosarcoma cells (SaOs-2). In this study, Gysin did not test the colon cancer cell line for apoptosis. They found no apparent cell death in the CaCo2 cells when using the trypan blue assay to test with 25 µM tocopherols. They further tested the DU-145 and LNCaP cell lines for apoptosis at the 25 µM RRR-γ- tocopherol concentration and found less than 3% of the cells were apoptotic. When employing 100 µM RRR-γ- tocopherol treatment to HCT-116 and SW480 cell lines we found significant apoptosis (Figures 4, 5 and 6). We The annex for 24 hou Figure 4 The annexin V/propidium iodide double staining assay following 100 µM tocopherol treatment in HCT-116 cells (Panels A-D) for 24 hours and SW480 (Panels E-H) cells for 70 hours Figure 4 The annexin V/propidium iodide double staining assay following 100 µM tocopherol treatment in HCT-116 cells (Panels A-D) for 24 hours and SW480 (Panels E-H) cells for 70 hours. These data show RRR-γ-tocopherol is superior to RRR-α-tocopherol at inducing apoptosis. The percentages in right quadrants represent percentage apoptosis over the blank and are an average of at least two independent trials. The annexin V/propidium iodide double staining assay following 100 µM tocopherol treatment in HCT 116 cells (Panels A D) for 24 hours and SW480 (Panels E H) cells for 70 hours Figure 4 The annexin V/propidium iodide double staining assay following 100 µM tocopherol treatment in HCT-116 cells (Panels A-D) for 24 hours and SW480 (Panels E-H) cells for 70 hours. These data show RRR-γ-tocopherol is superior to RRR-α-tocopherol at inducing apoptosis. The percentages in right quadrants represent percentage apoptosis over the blank and are an average of at least two independent trials. dant liposomes could by-pass the bioselective processes of the liver and rapidly increase plasma and tissue levels of RRR-γ-tocopherol beyond what can be achieved by oral administration. In vivo experiments in a rat model suggest that colonocytes receive RRR-γ-tocopherol from both suspect the reason for this discrepancy involves the cellu- lar uptake of RRR-γ-tocopherol. We demonstrated that uptake of the tocopherols is concentration dependent. We suspect that the variation in apoptotic induction is due to the fact that we used a tocopherol concentration that was four times higher thereby significantly increasing cellular tocopherol uptake. Page 9 of 14 (page number not for citation purposes) γ-Tocopherol treatment results in DNA laddering in HCT- 116 cells (Panel A) and SW480 cells (Panel B) following a 100 µM tocopherol treatment for 48 hours (V = vehicle, C = camptothecin (+) control, A = alpha tocopherol, G = gamma tocopherol) Figure 5 γ-Tocopherol treatment results in DNA laddering in HCT- 116 cells (Panel A) and SW480 cells (Panel B) following a 100 µM tocopherol treatment for 48 hours (V = vehicle, C = camptothecin (+) control, A = alpha tocopherol, G = gamma tocopherol). A. B. V C A G V C A G A. V C A G B. V C A G B. The annex for 24 hou Figure 4 A number of studies have demonstrated that RRR-γ-toco- pherol has chemopreventive properties not shared with RRR-α-tocopherol. For example, can lower levels of C- reactive protein [11], inhibit neoplastic transformation [12], suppress ras p-21 [10], inhibit COX-2 activity [13], down regulate cyclins [14], and up regulate PPAR γ [22]. Our study is the first to demonstrate that RRR-γ-tocophe- rol can result in cell death and apoptosis to colon cancer cell lines, with no significant cytotoxic effects to normal colon cells. Live Cell C treatment and HT-29 Figure 3 g µ γ p y g p y ( ) ( ) g Live Cell Counts following a 25 µM α- and γ-tocopherol treatment at varying times up to ten days in SW480 cells (A) and HT-29 cells (B). (Data are representative of two inde- pendent trials performed in triplicate. *p < 0.05 vs. vehicle at corresponding concentration). cells treated with RRR-α-tocopherol have no more than 11% of the cells undergoing early and late stage apoptosis combined. In both cell lines tested the ratio of RRR-γ- tocopherol-mediated late stage apoptosis is five times that of RRR-α-tocopherol. To confirm that apoptosis resulted from tocopherol treat- ment, a DNA laddering assay was performed (Figure 5). DNA laddering is only evident in the RRR-γ-tocopherol treated sample, but not the RRR-α-tocopherol treated sample in both SW480 and HCT-116 cell lines. Page 8 of 14 (page number not for citation purposes) Page 8 of 14 (page number not for citation purposes) BMC Cancer 2006, 6:13 http://www.biomedcentral.com/1471-2407/6/13 The annexin V/propidium iodide double staining assay following 100 µM tocopherol treatment in HCT-116 cells (Panels A-D) for 24 hours and SW480 (Panels E-H) cells for 70 hours Figure 4 The annexin V/propidium iodide double staining assay following 100 µM tocopherol treatment in HCT-116 cells (Panels A-D) for 24 hours and SW480 (Panels E-H) cells for 70 hours. These data show RRR-γ-tocopherol is superior to RRR-α-tocopherol at inducing apoptosis. The percentages in right quadrants represent percentage apoptosis over the blank and are an average of at least two independent trials. 23% 32% camptothecin 15.66% 32.3% camptothecin 25% 10% D-tocopherol 5.1% 5.1% D-tocopherol 16.6% 24.9% J-tocopherol 42% 10% J-tocopherol vehicle 1% 0% vehicle 1.1% 1.2% A. B. C. D. H. G. F. E. Annexin V Propidium Iodide 23% 32% camptothecin 25% 10% D-tocopherol 42% 10% J-tocopherol vehicle 1.1% 1.2% A. B. C. D. Iodide D. B. Annexin V Page 9 of 14 (page number not for citation purposes) γ-Tocophe 116 cells ( µM tocop camptothe tocophero Figure 5 ProCaspase 7 ( 37 kd) β-actin 35 kd ProCaspase 8 (55kd) V A G C Caspase 3 17/19 kd β-actin 35 kd Cleaved Caspase 7 20kd Pro Caspase 7 37 kd b-actin 35 kd Pro Caspase 8 55 kd Cleaved Caspase 8 43/41 kd β-actin 35 kd Cleaved Caspase 7 20kd Cleaved Caspase 8 43/41 kd V A G C 88 kD Cleaved PARP CleavedCaspase 3 (17/19 kd) 88 kD β-actin 35 kd Cleaved PARP ProCaspase 7 ( 37 kd) β-actin 35 kd ProCaspase 8 (55kd) V A G C Cleaved Caspase 7 20kd Cleaved Caspase 8 43/41 kd CleavedCaspase (17/19 kd) Western Blot analysis of SW480 (left) and HCT-116 (right) cell lysates following treatment with 100 µM tocopherols for 24 hours blotted with the antibodies to caspase 3, caspase 7, caspase 8, PARP, and β-actin as a loading control Figure 6 Western Blot analysis of SW480 (left) and HCT-116 (right) cell lysates following treatment with 100 µM tocopherols for 24 hours blotted with the antibodies to caspase 3, caspase 7, caspase 8, PARP, and β-actin as a loading control. β-actin 35 kd β-actin 35 kd Caspase 8 55 kd Cleaved Caspase 8 43/41 kd β-actin 35 kd Caspase 8 43/41 kd 88 kD Cleaved PARP 88 kD β-actin 35 kd Cleaved PARP Cleaved PARP 88 kD β-actin 35 kd Western Blot analysis of SW480 (left) and HCT-116 (right) cell lysates following treatment with 100 µM tocopherols for 24 hours blotted with the antibodies to caspase 3, caspase 7, caspase 8, PARP, and β-actin as a loading control Figure 6 Western Blot analysis of SW480 (left) and HCT-116 (right) cell lysates following treatment with 100 µM tocopherols for 24 hours blotted with the antibodies to caspase 3, caspase 7, caspase 8, PARP, and β-actin as a loading control. plasma and the contents of the digestive tract [10]. In humans, consuming a Western diet the dietary levels of RRR-γ-tocopherol are much higher than that of RRR-α- tocopherol. It would be expected, therefore, that RRR-γ- tocopherol would make a significant contribution to the total tocopherol content of colonocytes. Levels of RRR-γ- tocopherol in human colonocytes are indeed higher than that of RRR-α-tocopherol [25]. These data suggest that lev- els of RRR-γ-tocopherol in colonocytes could be much higher than reflected by plasma levels where ratio of RRR- γ-tocopherol to RRR-α-tocopherol is about 1 to 10 [10,25]. γ-Tocophe 116 cells ( µM tocop camptothe tocophero Figure 5 γ p g ( ) ( ) g µ p ( , p ( ) , p p , g p ) g γ-Tocopherol treatment results in DNA laddering in HCT- 116 cells (Panel A) and SW480 cells (Panel B) following a 100 µM tocopherol treatment for 48 hours (V = vehicle, C = camptothecin (+) control, A = alpha tocopherol, G = gamma tocopherol). γ p g ( ) ( ) g µ p ( p ( ) p p g p ) g γ-Tocopherol treatment results in DNA laddering in HCT- 116 cells (Panel A) and SW480 cells (Panel B) following a 100 µM tocopherol treatment for 48 hours (V = vehicle, C = camptothecin (+) control, A = alpha tocopherol, G = gamma tocopherol). We acknowledge that 100 µM concentrations of tocophe- rols used in cell culture experiments is not an achievable plasma level through oral administration in humans but could be achieved by other methods such as intravenous vitamin E-liposomes [24]. The intravenous use of antioxi- Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) Page 9 of 14 (page number not for citation purposes) BMC Cancer 2006, 6:13 http://www.biomedcentral.com/1471-2407/6/13 Western Blot analysis of SW480 (left) and HCT-116 (right) cell lysates following treatment with 100 µM tocopherols for 24 hours blotted with the antibodies to caspase 3, caspase 7, caspase 8, PARP, and β-actin as a loading control Figure 6 Western Blot analysis of SW480 (left) and HCT-116 (right) cell lysates following treatment with 100 µM tocopherols for 24 hours blotted with the antibodies to caspase 3, caspase 7, caspase 8, PARP, and β-actin as a loading control. http://www.biomedcentral.com/1471-2407/6/13 BMC Cancer 2006, 6:13 Cellular up take of RRR-α- and RRR-γ-tocopherol is time and concentration dependent, with RRR-γ-tocopherol accumulat ing to a higher intracellular concentration Figure 7 Cellular up take of RRR-α- and RRR-γ-tocopherol is time and concentration dependent, with RRR-γ-tocopherol accumulat ing to a higher intracellular concentration. A) SW480 cells were treated with varying concentrations of RRR-α- and RRR-γ-tocopherol for 24 hours. B) SW480 cells were treated with 100 µM RRR-α- or RRR-γ-tocopherol, col- lected, counted and subjected to HPLC analysis after 24, 48 and 72 hour treatments. Values are average of three inde- pendent experiments and error bars represent standard deviation. Time-dependent HPLC values were normalized to cell counts due to cell death at longer time intervals. forms, the higher the intracellular accumulation of vitamin E. With this in mind, we demonstrated that colon cancer cell lines exposed to physiological concentrations of RRR-γ-tocopherol for extended periods of time undergo reduced cell proliferation. These data suggest that dietary RRR-γ-tocopherol could have a protective effect over a long time span through a gradual tissue accumulation reaching the femtomole/cell range and thereby be chem- opreventive. We can only speculate at this time on the molecular basis for the differences in the growth inhibitory effects on the different colon cancer cell lines. There are, however, some interesting data correlations that can be made between the molecular characteristics of the cell lines and the effects of the tocopherols in this study. For example, HCT-15 and SW480 cells are COX-2 negative, HCT-116 cells are COX- 2 inducible and HT-29 cells over express COX-2. The growth inhibitory effect of RRR-α-tocopherol is more pro- nounced in cells that over express COX-2 (HT-29 cells). Preliminary work in our laboratories indicate that toco- pherols suppress COX-2 in HT-29 and CaCo2 cells (unpublished) and it is possible that some of the apop- totic effects of tocopherols are mediated by COX-2 sup- pression and hence are more apparent in cells that express COX-2. The HT-29 cells are slightly more resistant to cell death than the other cell lines as demonstrated by the increase in cell number over the vehicle at 24 hours. This may be due to the COX-2 expression in HT-29 cells. It is known that the expression of COX-2 can mediate apop- totic resistance in cancer cells [26]. γ-Tocophe 116 cells ( µM tocop camptothe tocophero Figure 5 Page 10 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/6/13 Page 11 of 14 (page number not for citation purposes) http://www.biomedcentral.com/1471-2407/6/13 Further investigation is underway in our laboratories to explore if the effects of RRR-α- and RRR-γ-tocopherol on COX-2 expression and apoptosis in these cell lines. Jiang et al. have demonstrated that RRR-γ-tocopherol, but not RRR-α-tocopherol decreases the activity of COX-2 in IL-1 and LPS-stimulated macrophages. Jiang et al. also demonstrated that, in mac- rophages, neither COX-2 protein or mRNA expression was affected by tocopherol treatment, leading to the spec- ulation that RRR-γ-tocopherol may compete for the ara- chidonic acid binding site which leads to protein inhibition and reduced activity [13]. Additionally, SW480 and HT-29 cells have a truncated APC gene, while the HCT-116 cells have a wild type APC gene. The APC pro- tein is involved in the regulation of β-catenin. In cancer, mutations in the APC gene allow for accumulation of β- catenin. β-Catenin is a nuclear transcription factor that modulates genes associated with cell proliferation. Accu- mulation of β-catenin protein leads to enhanced cell pro- liferation by dysregulation of the wnt pathway [27]. Our data show that HCT-116 cells are not affected by 25 µM tocopherol treatment at any time point tested (maximum 10 days), while the SW480 and HT-29 cell lines show a reduction in cell proliferation, indicating that RRR-γ-toco- pherol may have an effect on cell proliferation by regula- Cellular up concentrat ing to a hig Figure 7 Cellular up concentrat ing to a hig Figure 7 Cellular up take of RRR α and RRR γ tocopherol is time and concentration dependent, with RRR γ tocopherol accumulat ing to a higher intracellular concentration Figure 7 Cellular up take of RRR-α- and RRR-γ-tocopherol is time and concentration dependent, with RRR-γ-tocopherol accumulat- ing to a higher intracellular concentration. A) SW480 cells were treated with varying concentrations of RRR-α- and RRR-γ-tocopherol for 24 hours. B) SW480 cells were treated with 100 µM RRR-α- or RRR-γ-tocopherol, col- lected, counted and subjected to HPLC analysis after 24, 48 and 72 hour treatments. Values are average of three inde- pendent experiments and error bars represent standard deviation. Time-dependent HPLC values were normalized to cell counts due to cell death at longer time intervals. Ce u a up ta e o α a γ tocop e o s t e a co ce t at o epe e t, w t γ tocop e o accu u at g to a g e t ace u a co ce t at o gu e 7 Cellular up take of RRR-α- and RRR-γ-tocopherol is time and concentration dependent, with RRR-γ-tocopherol accumulat- ing to a higher intracellular concentration. A) SW480 cells were treated with varying concentrations of RRR-α- and RRR-γ-tocopherol for 24 hours. B) SW480 cells were treated with 100 µM RRR-α- or RRR-γ-tocopherol, col- lected, counted and subjected to HPLC analysis after 24, 48 and 72 hour treatments. Values are average of three inde- pendent experiments and error bars represent standard deviation. Time-dependent HPLC values were normalized to cell counts due to cell death at longer time intervals. Our HPLC results also indicate that at the 100 µM vitamin E concentrations used to treat these colon cancer cells only femtomoles of vitamin E are available inside the cell, yet, at these intracellular concentrations (femtomoles/ cell), cell death and apoptosis were induced. It is a signif- icant finding that intracellular concentrations in the fem- tomole range can induce cell death in colon cancer cells. To date, it is not known whether these intracellular con- centrations of femtomoles can be achieved in vivo through dietary means. Further research needs to be per- formed to determine what physiological intracellular and tissue concentrations of vitamin E isoforms are achievable within various tissues. http://www.biomedcentral.com/1471-2407/6/13 http://www.biomedcentral.com/1471-2407/6/13 http://www.biomedcentral.com/1471-2407/6/13 tion of the wnt pathway. It is well documented that disruption of the wnt pathway occurs early in the carcino- genesis process. The potential modulation of the wnt pathway by RRR-γ-tocopherol needs to be investigated further. This mutation makes them resistant to ligand activation. Our data (Figure 2) shows that RRR-γ-tocopherol induces cell death in the HCT-15 cells, therefore we suspect that the RRR-γ-tocopherol mediated cell death does not occur through a PPAR γ dependent mechanism only but that other pathways are involved. It is possible that vitamin E may activate multiple pathways in concert resulting in apoptosis and cell death of colon cancer cells of different prosurvival factors result in the variations among cell lines to induce apoptosis (as in the 24-hour data obtained with the HT-29 cell line). The potential pathways of tocopherol activity under investigation in our laboratories include mechanisms related to lipid metabolism (COX-2, lipoxy- genase, and sphingolipid pathways) and to pathways not related to lipid metabolism (wnt pathway). Jiang et al. has performed mechanistic studies suggesting that RRR-γ-tocopherol induces prostate cancer cell death by disrupting the de novo sphingolipid pathway which is important for the biosynthesis of ceramide [28]. Cera- mide has received considerable attention as a potential second messenger important for inducing apoptosis [29- 31]. In the sphingolipid study, vitamin E did not show an apoptotic effect on LNCaP prostate cells until 72 hours following treatment. The apoptotic event correlated with the increase and accumulation of dihydroceramide and dihydrosphingosine. Our data demonstrates a similar time differential before apoptosis occurs in the colon can- cer cell lines we tested. It is possible that RRR-γ-tocopherol may interfere with the de novo sphingolipid pathway in the colon cells as well as prostate cells to induce apoptosis by production of ceramide. There are different compart- mentalized pathways of ceramide signaling, each having unique molecular signatures for apoptosis/proapoptosis [32]. For example, lysosomal ceramide generation results in Cathespin D and BID-mediated activation of caspase 9 and 3. The generation of ceramide in the ER or mitochon- dria will cause protein phosphatase 1-mediated activation of caspase 9. Ceramide generation in the lipid membrane can affect signaling pathways generated by receptors such as Fas resulting in the activation of caspase 8. Our data dif- fers from that of Jiang et al. with respect to caspase 9 cleav- age. Competing interests p g Our laboratories have no financial interest, arrangement or affiliation with any product or organization that could be perceived as a real or apparent conflict of interest in the context of this manuscript. Our research group previously found that RRR-γ-tocophe- rol up regulates the expression of peroxisome proliferator activator receptor-gamma (PPAR-γ) more effectively than RRR-α-tocopherol [22]. PPAR γ is a key molecular target for cancer chemoprevention. Many in vitro and xenograft studies have demonstrated that PPAR γ ligands are anti- tumorigenic due to anti-proliferative, pro-differentiation and anti-angiogenic effects [33-38]. RRR-γ-tocopherol mediated cell death and apoptosis may follow a PPAR γ dependent mechanism in HCT-116, SW480 and HT-29 cell lines, but not in HCT-15 cells which have a point mutation (K422Q) and express a mutant form of PPAR γ. http://www.biomedcentral.com/1471-2407/6/13 We did not observe caspase 9 cleavage in the colon cells whereas Jiang et al. observed caspase 9 cleavage in the prostate cells. This means that if vitamin E is following a pathway involving ceramide biosynthesis, the cellular sublocalization of vitamin E mediation of ceramide is dif- ferent in colon from that of the prostate gland. It is likely that if RRR-γ-tocopherol interacts to interfere with sphin- golipid synthesis in the colon it happens in the plasma membrane as we have detected cleavage of caspase 8. This pathway requires further investigation in the colon. Conclusion h ff The effectiveness of γ-tocopherol to inhibit cell prolifera- tion in these human colon cell lines vary according the individual molecular characteristics of each line. This study indicates that RRR-γ-tocopherol mediated apoptosis in colon cells largely follows a death receptor pathway since caspase 8, but not caspase 9 cleavage was detected. This study demonstrates that both physiological (25 µM) and pharmacological concentrations (100 µM) of RRR-γ- tocopherol effectively reduced cell proliferation in malig- nant colon cancer cell lines that possess different molecu- lar signatures without damage to normal colon cells. Based on our studies, it is possible that both physiological and pharmacological concentrations of RRR-γ-tocopherol may have a role in the prevention and therapy of colorec- tal cancer. Physiological concentrations of RRR-γ-toco- pherol as taken in the diet may be chemopreventive, while pharmacological concentrations of RRR-γ-tocopherol may be used in conjunction with chemotherapeutic agents as adjunctive therapy enabling the reduction in the concen- trations of toxic chemotherapeutic agents, maintaining the levels of cell cancer cell death while reducing the death of normal colon cells. Cellular up concentrat ing to a hig Figure 7 In addition, our HPLC results indi- cate that the longer the cells are exposed to vitamin E iso- Page 11 of 14 (page number not for citation purposes) Page 11 of 14 (page number not for citation purposes) BMC Cancer 2006, 6:13 References 1. Potter JD: Nutrition and colorectal cancer. Cancer Causes Control 1996, 1:127-146. 22. Campbell SE, Stone WL, Whaley SG, Qui M, Krishnan K: Gamma tocopherol upregulates peroxisome proliferator activated receptor (PPAR) gamma expression in SW 480 human colon cancer cell lines. BMC Cancer 2003, 3:. 2. Berrino F, Muti P: Mediterranean diet and cancer. Eur J Clin Nutr 1989, 43:49-55. 3. Khlat M: Cancer in Mediterranean migrants – based on stud- ies in France and Australia. Cancer Causes Control 1995, 6:525-531. 23. Podda M, Weber C, Traber MG, Milbradt R, Packer L: Sensitive high-performance liquid chromatography techniques for simultaneous determination of tocopherols, tocotrienols, ubiquinols, and ubiquinones in biological samples. Methods Enzymol 1999, 299:330-341. 4. Stone WL, Papas AM: Tocopherols and the etiology of colon cancer. J Natl Cancer Inst 1997, 89:1006-1014. 5. Campbell S, Stone W, Whaley S, Krishnan K: Development of gamma-tocopherol as a colorectal cancer chemopreventive agent. Critical Reviews in Oncology/Hematology 2003, 47:249-259. y 24. Stone WL, Smith M: Therapeutic uses of antioxidant lipo- somes. Mol Biotechnol 2004, 27:217-230. 25. Nair PP, Lohani A, Norkus EP, Feagins H, Bhagavan HN: Uptake and distribution of carotenoids, retinol, and tocopherols in human colonic epithelial cells in vivo. Cancer Epidemiol Biomark- ers Prev 1996, 5:913-916. g gy gy 6. Bostick RM, Potter JD, McKenzie DR, Sellers TA, Kushi LH, Steinmetz KA, Folsom AR: Reduced risk of colon cancer with high intake of vitamin E: the Iowa Women's Health Study. Cancer Res 1993, 53:4230-4237. 26. Krysan K, Dalwadi H, Sharma S, Pold M, Dubinett S: Cyclooxygen- ase 2-dependent expression of survivin is critical for apopto- sis resistance in non-small cell lung cancer. Cancer Research 2004, 64:6359-6362. 7. Helzlsouer KJ, Huang HY, Alberg AJ, Hoffman S, Burke A, Norkus EP, Morris JS, Comstock GW: Association between alpha-tocophe- rol, gamma-tocopherol, selenium, and subsequent prostate cancer. J Natl Cancer Inst 2000, 92:2018-2022. 27. Li H, Pamukcu R, Thompson WJ: Beta-catenin signaling: thera- peutic strategies in oncology. Cancer Biol Ther 2002, 1:621-625. J 8. Ingles SA, Bird CL, Shikany JM, Frankl HD, Lee ER, Haile RW: Plasma tocopherol and prevalence of colorectal adenomas in a mul- tiethnic population. Cancer Res 1998, 58:661-666. p g gy 28. Jiang Q, Wong J, Fyrst H, Saba JD, Ames BN: Gamma-tocopherol or combinations of vitamin E forms induce cell death in human prostate cancer cells by interrupting sphingolipid synthesis. Proceedings of the National Academy of Sciences 2004, 101:17825-17830. 9. References Steele VE, Moon RC, Lubet RA, Grubbs CJ, Reddy BS, Wargovich M, McCormick DL, Pereira MA, Crowell JA, Bagheri D.: Preclinical efficacy evaluation of potential chemopreventive agents in animal carcinogenesis models: methods and results from the NCI Chemoprevention Drug Development Program. J Cell Biochem Suppl 1994, 20:32-54. 29. Andrieu-Abadie N, Gouaze V, Salvayre R, Levade T: Ceramide in apoptosis signaling: relationship with oxidative stress. Free Radical Biology and Medicine 2001, 31:717-728. pp 10. Stone WL, Papas AM, LeClair IO, Qui M, Ponder T: The influence of dietary iron and tocopherols on oxidative stress and ras- p21 levels in the colon. Cancer Detect Prev 2002, 26:78-84. gy 30. Macchia M, Bertini S, Fogli S, Giovannetti E, Minutolo F, Rapposelli S, Romano D: Ceramide analogues in apoptosis: a new strategy for anticancer drug development. Il Farmaco 2003, 58:205-211. 11. Himmelfarb J, Kane J, McMonagle E, Zaltas E, Bobzin S, Boddupalli S, Phinney S, Miller G: Alpha and gamma tocopherol metabolism in healthy subjects and patients with end-stage renal disease. Kidney Int 2005, 64:978-991. 31. Levade T, Malagarie-Cazenave S, Gouaze V, Segui B, Tardy C, Betito S, Andrieu-Abadie N, Cuvillier O: Ceramide in apoptosis: a revis- ited role. Neurochem Res 2002, 27:601-607. 32. Ogretmen B, Hannun YA: Biologically active sphingolipids in cancer pathogenesis and treatment. Nat Rev Cancer 2004, 4:604-616. y 12. Cooney RV, Harwood PJ, Franke AA, Narala K, Sundstrom AK, Berg- gren PO, Mordan LJ: Products of gamma-tocopherol reaction with NO2 and their formation in rat insulinoma (RINm5F) cells. Free Radic Biol Med 1995, 19:259-269. 33. Rumi MA, Ishihara S, Kazumori H, Kadowaki Y, Kinoshita Y: Can PPAR gamma ligands be used in cancer therapy? Curr Med Chem Anti-Canc Agents 2004, 4:465-477. 13. Jiang Q, Elson-Schwab I, Courtemanche C, Ames BN: Gamma- tocopherol and its major metabolite, in contrast to alpha- tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc Natl Acad Sci U S A 2000, 97:11494-11499. 14. Gysin R, Azzi A, Visarius T: Gamma tocopherol inhibits human cancer cell cycle progression and cell proliferation by down- regulation of cyclins. FASEB J 2002, 16:1952-1954. 13. Jiang Q, Elson-Schwab I, Courtemanche C, Ames BN: Gamma- tocopherol and its major metabolite, in contrast to alpha- tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc Natl Acad Sci U S A 2000, 97:11494-11499. g 34. Acknowledgements The work was supported by a post-doctoral fellowship grant from Cancer Research and Prevention Foundation of America (SC and KK) and a Pro- gram Exploration Hypothesis Development Award #PC030061 (WLS) through the Department of Defense. 21. Anderson K, Simmons-Menchaca M, Lawson KA, Atkinson J, Sanders BG, Kline K: Differential response of human ovarian cancer cells to induction of apoptosis by Vitamin E Succinate and Vitamin E Analogue, {alpha}-TEA. Cancer Research 2004, 64:4263-4269. http://www.biomedcentral.com/1471-2407/6/13 study along side SC, participated in the study design and guided trouble shooting assays, and manuscript prepara- tion. All authors read and approved the final manuscript. 19. Neuzil J, Weber T, Schroder A, Lu M, Ostermann G, Gellert N, Mayne G, Olenjnicka B, Negre-salvayre A, Sticha M, Coffey R, Weber C: Induction of cancer cell apoptosis by alpha tocopheryl suc- cinate: molecular pathways and structural requirements. FASEB 2002, 15:403-412. 20. 20. Galli F, Stabile AM, Betti M, Conte C, Pistilli A, Rende M, Floridi A, Azzi A: The effect of [alpha]- and [gamma]-tocopherol and their carboxyethyl hydroxychroman metabolites on pros- tate cancer cell proliferation. Archives of Biochemistry and Biophys- ics 2004, 423:97-102. Authors' contributions SC conceived the study, and participated in the study design, performed data analysis, aided in troubleshooting assays and drafted the manuscript. WS participated in the design and coordination of the delivery of the vitamin E isoforms to the cells and the HPLC analysis and manu- script preparation. SW, DS, DM and PG carried out the cell proliferation and apoptosis assays, while QM, SL and HY performed the western blot analyses. 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Moyad MA, Brumfield SK, Pienta KJ: Vitamin E, alpha- and gamma-tocopherol, and prostate cancer. Semin Urol Oncol 1999, 17:85-90. 36. Hase T, Yoshimura R, Mitsuhashi M, Segawa Y, Kawahito Y, Wada S, Nakatani T, Sano H: Expression of peroxisome proliferator- activated receptors in human testicular cancer and growth inhibition by its agonists. Urology 2002, 60:542-547. 17. Weber T, Lu M, Andera L, Lahm H, Gellert N, Fariss MW, Korinek V, Sattler W, Ucker DS, Terman A, Schroder A, Erl W, Brunk UT, Coffey RJ, Weber C, Neuzil J: Vitamin E succinate is a potent novel antineoplastic agent with high selectivity and coopera- tivity with tumor necrosis factor-related apoptosis-inducing ligand (Apo2 ligand) in vivo. Clin Cancer Res 2002, 8:863-869. g gy 37. Bull AW: The role of peroxisome proliferator-activated receptor gamma in colon cancer and inflammatory bowel disease. Arch Pathol Lab Med 2003, 127:1121-1123. 38. Li MY, Deng H, Zhao JM, Dai D, Tan XY: Peroxisome prolifera- tor-activated receptor gamma ligands inhibit cell growth and induce apoptosis in human liver cancer BEL-7402 cells. World J Gastroenterol 2003, 9:1683-1688. 18. Zu K, Ip C: Synergy between Selenium and Vitamin E in apop- tosis induction is associated with activation of distinctive ini- tiator caspases in human prostate cancer cells. Cancer Research 2003, 63:6988-6995. 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https://openalex.org/W4288357441	https://zenodo.org/records/2756209/files/2019LACANIZING.pdf	English	null	Lacanizing Marxism: the Effects of Lacan in Readings of Marx and Marxist Thinkers	Zenodo (CERN European Organization for Nuclear Research)	2,019	cc-by	14,014	Abstract In this essay I discuss the ways that Marxism is read through the lens of Lacanian theory by Lacan’s followers and not by Lacan himself. I distinguish between different Lacanian approaches to Marxism and between Lacan’s diverse effects on the subjects that are approached. I scrutinize five affirmative effects, namely those of problematising, historicising, generalising, confirming and completing what is read. I first explicate these effects briefly in discussing classic works of the 1980s and then at length in presenting my own Lacanian approach to Marxism. I show how the realisation of such effects implies a Lacanization of Marxism and the resulting constitution of a Lacanian Marxism that I openly assume. Keywords: Marxism, psychoanalysis, politics, Marx, Lacan. 2 E. g. Laclau & Mouffe 1985, Žižek 1989, Močnik 1991, Dolar 1993, Silveira 2002, Özselçuk & Madra 2010, Lippi & Landman 2013, Žižek, Ruda & Hamza, 2018. 1 E. g. Regnault 2005, Bruno 2010, Pavón Cuéllar 2013, Tomšič 2015, Vighi 2016. 2 E. g. Laclau & Mouffe 1985, Žižek 1989, Močnik 1991, Dolar 1993, Silveira 2002, Özselçuk & Madra 2010, Lippi & Landman 2013, Žižek, Ruda & Hamza, 2018. Lacanizing Marxism: the Effects of Lacan in Readings of Marx and Marxist Thinkers David Pavón Cuéllar Abstract 1 E. g. Regnault 2005, Bruno 2010, Pavón Cuéllar 2013, Tomšič 2015, Vighi 2016. Other effects We see that Lacan can inhibit reading Marxism, deviate from it and make us believe that it is unnecessary. It can also happen, however, that Lacan pushes us to read Marx and the Marxists to try to resolve his enigmatic assertions about them. It is possible, in addition, that he transforms our reading, making us read them or reread them in another way: in a Lacanian way. This last possibility is the one that interests us here. How is it that ordinary Lacanians know the errors of Marxism so well? Obviously, they have not detected them by themselves, but have learned them from Lacan or perhaps from other ordinary Lacanians who repeat again and again what they have learned from Lacan. Among what they have learned are the errors of Marx and the Marxists, almost always the misses and almost never the hits, according to a selective criterion that might be revealing the single thought, pensée unique, in which Marxism is necessarily related to error. When read through a Lacanian lens, Marx and the Marxists are transfigured and shown in a totally different light. It’s almost as if they become others or mad. Suddenly, their speeches are full of nonsense, paradoxes, tensions, uncertainties and enigmas. Their theories are reconfigured. Certain parts are revalued and others are devalued and reduced to absurdity. Some of their lateral ideas become decisive, while their central ideas lose importance. Their seemingly simple and obvious notions cease to seem that way; they become complicated, they move away from common sense, they are carried to their ultimate consequences and reappear with new nuances and an unfathomable depth. It happens that their perspective becomes more solid, radicalises and reinvigorates, acquires greater scope and reveals unexpected aspects. We are surprised again by what no longer surprised us. We stop understanding what we understood perfectly. In dissolving Lacan into something as anti-Lacanian as mainstream ideological conformism, an average Lacanian may well confine Marx and the Marxists to error. It does not matter, of course, that he or she has not read Marx and the Marxists. Why read them when you can read Lacan to know that they were wrong? Normal effects disfigured to be later corrected, revised, rectified and refuted with his own ideas: with the surplus enjoyment, with the revolutionary circularity, with the symptom, with the lack of metalanguage. Those who do so, usually in schools and associations of Lacanian psychoanalysis, betray not only that they have not read Marx, but also that they have misread Lacan, who always ends up giving credit to Marx for his discoveries.6 The average Lacanian is familiar with at least some of the ‘mistakes’ of Marxism. He or she is even able to recite them from memory. Marx and the Marxists were wrong to postpone desire and turn it into a post- revolutionary issue, to imagine that the problem was capitalism and not language and its discontent, to calculate the incalculable surplus enjoyment and thus transform it into surplus value, to interpret the symptom as a sign and not as a signifier, to fail to see that revolutions return to the point of departure, to believe that they aspired to freedom when they wanted a master, and to remain trapped in the master discourse. 3 Lacan 1946, p. 192; 1959-1960, p. 245. Introduction Jacques Lacan’s reading of Karl Marx has already been thoroughly analysed in the literature.1 The analyses are usually accompanied by extensive reflections inspired by Lacan. Of course, these reflections can also be found without analyses of Lacan’s reading of Marx, which is replaced by another reading conducted in Lacanian style. This manner of proceeding has been extraordinarily fruitful in the last thirty years, giving rise to a myriad of Lacanian readings of Marx and Marxist thinkers.2 This essay precisely addresses the way that Marxism is read not by Lacan but by authors inspired by him. The subject is not embraced in all its breadth and depth, but approached in terms of one of its most insignificant expressions, the one I know best: my own Lacanian reading of Marx and Marxist thinkers. However, before grappling with this specific reading, I will briefly examine different Lacanian approaches to Marxism and Lacan’s diverse effects on the subjects that are approached. Most of the essay will focus on five rather affirmative effects of Lacan in the reading of Marx and Marxist thinkers, namely those of problematising, historicising, generalising, confirming and completing what is read. These effects will first be explicated briefly in a discussion of classic works of the 1980s and then at length in a presentation of my own Lacanian approach to Marxism. I will try to show how the realisation of such effects implies a Lacanization of Marxism and the resulting constitution of something as problematic and scandalous as the Lacanian Marxism that I have openly assumed. For now, before reaching the Lacanian left, let’s review a little of what happened with Marx and the Marxists in conventional Lacanism, which is usually right-wing or supposedly apolitical. Lacanizing Marxism 263 Normal effects 4 Marx 1852. 6 Lacan 1968-1969, 1969-1970, 1971. Other effects Some of the best examples of this kind of generalisation can be found in the Lacanian reading of Marx provided by Slavoj Žižek. For example, when Žižek Lacanianly read Marx’s famous reflections on fetishism and the relations between things that replace relations between people, he moved beyond the specific framework of the functioning of commodities in capitalism and scrutinised the general fact of the objectivity and radical exteriority of ‘beliefs, superstitions and metaphysical mystifications’, as well as the ‘most intimate emotions’.9 The exterior of the interior, the objective of the subjective and the impersonal of the personal appeared as a universal truth that Marx discovered through the fetishism of commodities in capitalism. The fetishistic configuration, by which things believe and feel instead of us, was no longer a specific situation of the industrial capitalist system studied by Marx, but a general condition of humanity ranging from Greek theatre to television and the social networks. This general condition is what we can read in Marx when reading him through Lacan. Lacanian theory confirms the Marxist concept on a general level. discuss the Marxist conception of the subject, of society or of the working class, as a given empirical being. The Lacanian perspective makes this subject problematic, showing its ‘precariousness and absence of suture’ and its ‘ambiguous, incomplete, polysemical’ character as ‘discursive identity’, which is what makes a ‘hegemonic articulation’ possible and necessary to ‘construct nodal points’ that ‘fix the meaning of the subject’.7 Thanks to Lacan, we can see a theoretical and practical problem, a matter of controversy and a challenge for the socialist strategy, whereas before, for some though not all Marxists, there was evidence of the revolutionary nature of the subject. The indisputable became questionable. This did not inhibit reading Marxism, but quite the opposite. Laclau and Mouffe approached the Marxists and read them carefully to discuss the subject with them, disagreeing with some, like Kautsky or Plekhanov, but also coinciding with others, such as Sorel and particularly Gramsci. • Instead of merely dismissing Marxism by considering it to have been surpassed by the Lacanian perspective, we can historicise it by resituating it in a certain historical context with the help of Lacan. Other effects Lacan is used here not to read Marx and the Marxists, but to avoid them, to discard and ignore them, to put them aside or, more precisely, to leave them behind, because average Lacanians are convinced that they have surpassed Marx thanks to Lacan, having forgotten what Lacan himself taught them: that Marx is ‘always new’, that he ‘cannot be overcome’.3 p y Lacan had many more possible effects on readings of Marx and Marxist thinkers, among them the typically postmodern solvent or deconstructive effects, such as disorganising, disarticulating, fragmenting and volatilising. And, in the antipodes of these negative effects, there are five rather affirmative effects, namely to problematise, historicise, generalise, confirm and complete, upon which I would like to concentrate, and which are perhaps not very consonant with the typical vision of the Lacanian spirit as being essentially characterised by negativity. Let us review an example of each of these effects in the already classic works of well-known authors whose readings of Marx and Marxism had a marked Lacanian tonality: In order to overcome Marx, average Lacanians imagine that they have exhausted him, and in imagining this outcome, they do not see that he is inexhaustible. They simplify and trivialise him. He is represented as a naive thinker. They replace him with a caricature of who he was. They make him contemptible to hold him in contempt. They end up being certain that he did not know what he knew very well, such as the circularity of revolutions4 or the difference between surplus value and surplus enjoyment.5 Many Lacanians mutilate Marx. They steal his ideas and attribute them to Lacan. Then they use these same ideas to argue against a defenceless, weakened, impoverished, unrecognizable Marx. Marx is • Instead of simply avoiding Marxism by considering it Lacanianly irrelevant, it is possible to problematise its ideas as Ernesto Laclau and Chantal Mouffe did by using Lacan to Instead of simply avoiding Marxism by considering it Lacanianly irrelevant, it is possible to problematise its ideas as Ernesto Laclau and Chantal Mouffe did by using Lacan to 6 Lacan 1968-1969, 1969-1970, 1971. Lacanizing Marxism Lacanizing Marxism 265 264 • If Lacan prevents us from relegating Marxist discoveries to the past, it is because he makes us generalise them by allowing us to recognise the universal scope they possess. 9 Žižek 1989, 31-33. 10 Jameson 1981, 111-112. 7 Laclau & Mouffe 1985, 112-122. 8 Askofaré 1989, 121-138. 9 Žižek 1989, 31-33. 7 Laclau & Mouffe 1985, 112-122. Other effects This is what happened, for instance, when Alain Badiou, rejecting both Freudo-Marxism and Marxist psychology, found in Lacan’s work the ‘theory of the subject’ required by Marxism: a theory developed successively as an algebra of the lack and as a topology of excess with which we can elucidate Marxist categories as those of the masses or the party.11 Marxism demanded a theory of the subject like the one that it receives from Lacan. The Lacanian reading of Marx and Marxism responds to what is read to complete it, complement it, ground it, justify it, answer its questions, satisfy its needs and continue it in the sense that it represents. Since the 1980s the Lacanian reading of Marx, as we have just shown in the previous examples, has allowed for the effective development of Marxist ideas by addressing them in a positive manner. When problematised, historicised, generalised, confirmed or completed, each idea was explained or justified, deepened or extended, nuanced or detailed, prolonged or evolved—that is, it was developed. Each Marxist idea was developed through the consideration, respectively, of the problems it posed, the historical conditions in which it arose, its general scope, the situations that confirmed it or the unexplored dimensions that might have completed it. However powerful some of the aforementioned arguments may be, I have stubbornly sustained myself as a Lacanian Marxist for a decade. I have done so based first on certain personal positions and convictions: because I am a communist, I am in the Marxist tradition and I would never renounce Marxism to adopt a Lacanian theory, which interests me mainly for the service it can render to communism. Other effects After the death of Lacan in 1981, from the 1980s until now, the Lacanization of Marxism has led to one of the most fertile political-intellectual currents of our time, which is sometimes designated with the vague expression the ‘Lacanian left’.13 Many of its adherents could be called ‘Lacanian Marxists’, but they avoid calling themselves that for several reasons: because they do not seem to want to name themselves in any way, because their adhesion to Marx or their interest in his ideas does not necessarily imply an inclination for Marxism, because they are all too aware of the errors of Marx and Marxism that they learned from Lacan, because there is something that prevents them from recognising the greater successes of Marx and Marxism that Lacan also taught them, because they remain faithful to Lacan, who was not a Marxist and criticised Marxism, because their very fidelity to Lacan makes them reject any alliance between Marxism and psychoanalysis that reminds them of the Freudo-Marxism rejected by Lacan because Lacan demonstrates that Marxism and psychoanalysis cannot connect to one another without being embroiled, because the homology between Marx and Lacan makes a Lacanian Marxism as redundant as a Marxist Marxism or a Lacanian Lacanism because Marxism implies a positivity that contradicts the negativity accentuated in the dominant reading of Lacan, because we no longer live in a time when being a Marxist intellectual is fashionable or means something like what it meant before, and because of the postmodern discrediting of Marxism and any -ism in general. • Sometimes Lacanian concepts do not express what Marxist concepts refer to, but their correlates, their counterparts, their complements, the solutions to some of their problems or something else that has a precise place and that attracts our attention for being undesignated. We then have an opportunity to complete the theories of Marx or his followers with Lacan’s theory. The Lacanian theoretical contribution comes to correct a lack or to deal with a slope in Marxism. Other effects This is what Sidi Askofaré did by showing how the ‘emergence’ of the proletariat as a ‘historical figure’ of truth and dispossession of knowledge, at the moment of the transition from feudalism to capitalism, allowed Marx to discover a ‘social symptom’ that ‘connotes the universality of the function of the symptom’ in which the subject suffers from a particular truth irreducible to what can be universally known about it.8 This truth is obviously different for each subject and that is why it resists universal knowledge. It is for the same reason that the symptom is invariably particular, that is, universally particular. But this universality of particularity is precisely the insurmountable sense of the social symptom discovered by Marx thanks to a unique conjuncture of history. To historicise is here to recognise the role of history in Marxist discoveries, which were, in fact, discoveries by history and not only of history. The historical world, in short, discovered itself through Marx’s findings. However, as we have just seen, this does not compromise the universality of what was discovered and should not make us relegate it to the past. History is never behind us. We are simply in another moment of the same history. • Generalisation is not the only possible Lacanian way of confirming what we read in Marx. There are other possibilities, among them the opposite of generalisation, specification, by which Lacanian ideas or postulates constitute specific cases with which general Marxist ideas or postulates are confirmed. Fredric Jameson offered us a good example of this process when he realised that Lacan’s ‘critique of the subject’, with his idea of subjective ‘decentring’ and with his conception of consciousness as an ‘effect of structure’, theoretically confirmed the Marxist non-individualist notion of the subject in the specific historical context of the ‘dissolution of an essentially bourgeois ideology of the subject and of psychic unity or identity’.10 The crisis of individualism that manifests itself in Lacan allows us to confirm the critique of individualism that we can read in Marx and Marxist thinkers. Marxism demonstrates its truth in the very categories through which it is Lacanianly read. The Lacanian concepts are a specific expression of what the Marxist concepts refer to. Lacanizing Marxism Lacanizing Marxism 267 266 readers and disciples. Other effects Now, in addition to my ‘subjective’ political reasons, there are also ‘objective’ theoretical reasons and dogmatic rationalisations that appear to me to provide unquestionable evidence to embrace Lacanian Marxism: because there seems to be nothing insuperably incompatible between the Lacanian and Marxist discourses, because Marxism does not cease to be consistent by rectifying the errors that Lacan imputes to it, because this rectification can only purify and reaffirm the Marxist theory, because this theory needs to develop in a Lacanian direction in order to deal with much of what it encounters in the current world, because the new forms of domination and subjectification pose difficulties that Marxism cannot even conceive without being Lacanized, because Lacanian psychoanalysis requires radical positions such as Marxism so as not to degrade itself by dissolving into psychology or dominant ideology, because many of the ideas of Marx and his followers underlay Lacanian theory, because to go deeper into 13 E. g. Stavrakakis 2006, Alemán 2013. 268 11 Badiou 1982, 132-133, 144-151, 195-197, 245. 12 E. g. Pavón-Cuéllar 2009, 2014a, Lacanizing Marxism 12 E. g. Pavón-Cuéllar 2009, 2014a, 11 Badiou 1982, 132-133, 144-151, 195-197, 245. Effect of effects: the Lacanization of Marxism and Lacanian Marxism Certain effects of Lacan in the reading of Marx and his followers are positive as they positively develop Marxist ideas. But the important result is that they develop them in a specifically Lacanian direction. This development implies a Lacanization of Marxism, which, in turn, logically produces what I have obstinately called ‘Lacanian Marxism’.12 The emergence of the Lacanian Marxist orientation, one of the strangest episodes in the history of Marxism, has its origins in Lacan’s own approach to Marx, as well as in the work of some of Lacan’s first Lacanizing Marxism 8 Lacanizing Marxism 269 268 Lacan inevitably leads us to Marx, because the entanglement produced by adding Marxism to psychoanalysis only mirrors the opacity and complexity of the material reality for those who try to conceive it, because Lacanian Marxism can only be redundant for those who accept its truth, and because most of the reasons to avoid Lacanian Marxism seem more suspicious and tempting than persuasive or dissuasive. to being what is reflected, unconsciously modifies the reflective surface that ideologically distorts the reflection, which is its own reflection.15 Mental distortions, therefore, are of ideological external origin and not only internal, mental or cerebral. In fact, by adopting a Lacanian symbolic materialism in which I radicalise Engelsian dialectical materialism and agree with Plekhanov in his ephemeral hieroglyphic materialism, I consider that the mental can only exist in an ideologically distorted form because it is formed by its own distortion, because it must distort what it interprets, because it must translate and thus betray what it reflects, because its images are narrated, because it is discourse, because it is determined symbolically, because its structure is language and not a supposed reality independent from language, and because its elements are signifiers and not just reflections.16 In addition to so many compelling reasons to speak of Lacanian Marxism, there is the decisive factor that I have already referred to, namely that Lacanian Marxism already exists; it is already there since it has been created by the rather affirmative effects of Lacan’s theory in readings of Marx and Marxist thinkers. These effects, as we have seen, can be synthesised in a single theoretical effect: that of Lacanizing Marxist ideas. Problematising Although existent and justified, Lacanian Marxism is extremely problematic, doubtful and controversial, both for internal and external causes, that is, both for its constitution and its position in the field of knowledge. One of the main internal causes of this problematicity is that Lacan’s ideas cannot come into contact with the Marxist ones without problematising them. We have already referred to the problematisation of the Marxist notion of the subject in the Lacanian sensibility of Laclau and Mouffe. Under a totally different Lacan influence, I have also problematised the subject of Marxism, as well as various theories related to it, among them one on which I would like to dwell a moment: the Marxist theory of reflection in which it is postulated that consciousness reflects the external world.l y Interiority is nothing more than a kind of crease or fold of exteriority. It is the same language because there is no metalanguage.19 There is no reflection that would be different from what is reflected, but rather, as Korsch pointed out in criticising the theory of reflection, there is a ‘very special part of the whole’.20 Or, better still, there is a moment of the not-all. We cannot even say that this moment is differentiated from the rest by being composed of qualitatively different elements, mental elements such as ideas, since these elements also make up the exterior, as Pannekoek noted in the same Western Marxist tradition of Korsch.21 In its simplest version, the one elaborated by Lenin, the reflection theory epistemologically generalises and legitimises a particular interpretation of a deceptive subjective experience of the specular imaginary in Lacan: the internal world is conceived as a conscious surface on which the external world is reflected as in a photograph, and if there are errors or mental distortions in the reflection, it is surely because of imperfections in the cerebral surface that reflects it.14 In problematising this theory of reflection, my Lacanian reading of Lenin coincides with the arguments of other Marxists. Let us consider some of these overlaps. The recognition of the ‘mental’ aspect of exteriority, which betrays more of a materialist conception of the mind than an idealistic conception 15 See Engels 1888. 16 Pavón-Cuéllar 2009, 2012. 17 See Vygotsky 1934 and Pavón-Cuéllar 2010. 18 See Pavón-Cuéllar 2017a. 19 Lacan 1960-1961. 20 Korsch 1923. 21 Pannekoek 1938. Effect of effects: the Lacanization of Marxism and Lacanian Marxism The resulting Lacanian Marxism is Lacanized Marxism, that is, Lacanianly problematised, historicised, generalised, confirmed and completed Marxism These five effects, previously illustrated in the works of Badiou, Jameson, Žižek, Askofaré and Laclau and Mouffe, will now be exemplified through my own theoretical work, in which, navigating against the air of the times, the effect of the effects, Lacanian Marxism, is assumed and elaborated upon explicitly, deliberately and systematically. There is at least one point, that of the discrepancy with the Leninist theory of reflection, at which my Lacanian vision agrees with the Vygotskian vision: the psyche cannot reflect the exteriority without interpreting it, signifying it or, better yet, signifierising it, symbolising it according to codes and structures that derive from the same cultural exteriority and, in particular, language.17 It is, then, the symbolic external world itself that manifests itself symbolically in what it makes us conceive as an internal world. We can suppose, therefore, just as Vygotsky supposed, that thought is internalised speech, but perhaps it does not make much sense to pose the concept that way since interiority itself is an internalisation of exteriority.18 14 Lenin 1908. 15 See Engels 1888. 16 Pavón-Cuéllar 2009, 2012. 17 See Vygotsky 1934 and Pavón-Cuéllar 2010. 18 See Pavón-Cuéllar 2017a. 19 Lacan 1960-1961. 20 Korsch 1923. 21 Pannekoek 1938. Volume 6 / Issue 1 17 See Vygotsky 1934 and Pavón-Cuéllar 2010. 15 See Engels 1888. 21 Pannekoek 1938. 16 Pavón-Cuéllar 2009, 2012. 20 Korsch 1923. Problematising In other words, when I go deeper into my Leninist inner reflection, I cross it and come to the same place where I arrive by delving into the outside and crossing it: to the field of research of Marxism, which is also that of psychoanalysis, that is, the most radical exteriority that is also the deepest intimacy, the extimacy, which is behind the interior and exterior mirrors of the imaginary.l It is not deplorable that the psyche is also madly reflected in the world instead of only reflecting it. This madness allows us to attend to our desire in reality and not only in dreams. Or, rather, it helps to revolutionise reality by realising the dream in which desire is fulfilled. Hence such madness was the goal of revolutions in the political program of surrealist Freudian Marxism to which my Lacanian Marxist perspective also adheres, but only in its imaginary front, which is not the only or the most important front, as we shall see in the next section. In the imaginary my orientation aspires to a communist idea that should be insanely reflected in the world besides reflecting it with a strategic dose of sanity. My orientation thus diverges from strategies based unilaterally on the Leninist theory of reflection. The revolutionary conscience, if it wants to be truly revolutionary, cannot limit itself to reflecting reality by adapting or adjusting to it. This is something that Lenin understood very well, but that many Marxist-Leninists forgot. What they forgot is that reflecting reality is nothing more than a way of reproducing it. Of course, such reproduction is necessary for success, but it may end up compromising that success. This is how real socialism, through state capitalism, ended up successfully repeating in its own way, in one country, the capitalist reality of the world with which it maintained its aggressive, imaginary rivalry. In my Lacanian problematisation of the Leninist theory of reflection, not only the exterior is reflected in the interior, but, as the Marxist-Freudian surrealist Karel Teige wonderfully expressed it, the psyche ‘makes the material world its reflection and image, the illustration and manifestation of its desire’.24 It cannot be otherwise when we admit what Lacan thought regarding the imaginary and the specular reflection. The reflection exists in both senses. Problematising For me, as for Engels, there is no internal world clearly separated and differentiated from the external world since the external, in addition Lacanizing Marxism Lacanizing Marxism 271 270 of the external world, is a fundamental point of my Lacanian Marxist vision. This point, as we have seen, is not new in the Marxist tradition: it has already been developed in Western Marxism, but also in the Soviet field and particularly in the theory of activity constructed by Sergei Rubinstein, who deeply examined the ‘psychological contents’ of ‘material external activity’.22 Taking Rubinstein’s theory to its ultimate consequences, to the Lacanization of his ideas, we even realise that the most external can be the most intimate component of the subject, the ‘extimate’, as Lacan would say.23i Our more or less shared delusions internally organise our world by deploying the transindividual exteriority that constitutes our unconscious. This is what makes us all crazy at least in some way and to some extent. The reason for this madness is well explained by Attila József in his original Freudo-Marxist perspective: we are all crazy because it is not our conscience that always responds to our existence, as some Marxists would like, because it is not our psyche that always reflects our world, as Lenin explained, but constantly, as Freud showed us, it is our world that reflects our psyche, which, in a pathological way, ‘forms’ and ‘deforms’ our world.27l The concept of extimacy overcomes the apparent, superficial distinction between the interior and the exterior since it designates a single and unique space that is beyond the exterior and beyond the interior. The interior and the exterior are here only specular reflections that reflect each other in an imaginary game while covering the extimate behind them, namely the capitalist system studied by Marx, but also the symbolic system of culture and the unconscious studied by Freud and Lacan. 272 22 Rubinstein 1945, p. 169; 1959, p. 340. 23 Lacan 1968-1969. 24 Teige 1945, p. 296. 25 Breton 1932, 123-129. 26 Lacan 1954-1955. Lacanizing Marxism 22 Rubinstein 1945, p. 169; 1959, p. 340. 27 József 1934. 28 Lacan 1977. 27 József 1934. 24 Teige 1945, p. 296. 23 Lacan 1968-1969. 25 Breton 1932, 123-129. Volume 6 / Issue 1 26 Lacan 1954-1955. 28 Lacan 1977. Historising In fact, as we have seen, it is the world itself that resists history, that reacts to conserve itself and that reproduces itself by reflecting itself through its consciousness. This consciousness is part of the world that logically seeks to persevere in its being, but the world is also historical and historically transforms itself through consciousness by not only reflecting on it. On the one hand, as we saw in the previous section, in addition to the reflection of the world in consciousness, there is the reflection of consciousness in the world. However, on the other hand, something more interesting may also happen: what we can describe as breaking the mirror that allows us to symptomatically discover the logical space of extimacy that lies behind the inner surface of consciousness, beyond the game of the reflections of the imaginary. This symptomatic discovery in turn produces what Lacan has described as a subversion with which the revolution is triggered and, more importantly, the revolutionary circle is opened, something changes and a historical spiral movement is assured. However, in addition to what is cognitively reflected, there is what is symptomatically discovered: the covered-discovered by the reflection, the extimate processes that underlie external or internal states, the production of the product and the enunciation of the enunciated, but also the negativity of positivity, the misery of wealth and the abstract character of the most concrete. The discovery is made in the same reflection, in the open and hollow structure, in the imperceptible matter that must be calculated through the microscope of ‘abstraction’.32 It is here, in the abstract, mathematical, empty and unfounded material structure, where we discover that the most apparent is the least apparent, that the evident is contradictory, that the whole is not-all, that the Other is barred and that the king is naked, that he is a proletarian, a subject without attributes, except to be alive. We have, then, in addition to the reflection, the possibility of a symptomatic discovery of what is behind the mirror: a discovery that provokes a transformation. This is what we find eloquently illustrated in Marx’s work when we read it in a Lacanian manner. This reading allows us to historicise Marx when we see how the world and its history are manifested, debated, realised and revolutionised in his thoughts and through what his thoughts do not only reflect, but simultaneously reflect and discover. Problematising The psyche reflects the external world based on the reality principle, while reality reflects the psyche based on what is desired according to the pleasure principle. The problem is, of course, that it is practically impossible to distinguish one from the other. The external world, like the internal world, responds to our desire. As André Breton pointed out, the same processes of ‘condensation, displacement, substitution, retouching’ by which desire forms the dream also allows it to create the reality that surrounds us when we are awake.25 This surrealist conception coincides with the notion of Lacan, a worthy heir of surrealism, that reality is imaginary and that we somehow dream or delude our world.26 No matter how successful they are in the world, the scrupulously realistic, neurotically- obsessively realistic revolutionaries fail to transform it in a ‘historical’, ‘hysterical’ way, according to the revealing Lacanian pun.28 Realists cannot enact more than a small revolution that only describes a circular movement in order to finish at the starting point. It is the circularity inherent in any specular game. As in Lampedusa’s Leopard, everything has to change so that everything remains the same. Another revolutionary process, one that is fully historical, open and spiralling, cannot be based exclusively on the Leninist theory of reflection. Consciousness that only reflects tends to be conservative, reactionary, and surely ahistorical, even anti-historical. 272 Lacanizing Marxism Lacanizing Marxism Lacanizing Marxism 273 272 an Other30 and a closed and unidimensional system comprised only of one qualitative dimension and its quantitative variations and proportions, devoid of otherness and negativity, as Marcuse already showed.31 In short, Marx’s structuralism and materialism belonged to nineteenth-century capitalism, to capitalist modernity. This historical world was the one that faithfully reflected itself in the work of Marx. Historising 30 Lacan 1968-1969, 1969-1970. 29 See Habermas 1968. 31 Marcuse 1964. 30 Lacan 1968-1969, 1969-1970. 31 Marcuse 1964. 32 Marx 1867, p. xiii. Generalising What happens is that our life experience cannot be transferred to the Other who takes our life because the Other, behind his fetishised appearance, is pure insensitive language that cannot experience anything. Instead of experiencing our life, the Other simply enjoys, possess a labour force in which our life and possible experience are dissolved. Correlatively, instead of the experience of our life, we experience our alienation in the fetishism of the signifier. We suffer the dispossession of our life in its possession by language. We feel our inertia in the Other’s jouissance, in its enjoyment of our life, in the satisfaction of the death drive. The universal proletarian is at the centre of my proposal of Lacanian Marxism. Among the effects that Lacan has on my reading of Marx, one of the most important is the generalisation of the category of the proletariat.33 This category allows me to describe a general experience of the subject and not only the particular situation of the industrial worker who does not have his own means of production, who has only his own life, and is forced to sell it as a labour force in exchange for a salary. Like the proletarian studied by Marx, the subject conceptualised by Lacan ($) must detach himself from his life that will be exploited as a labour force to execute the work of the unconscious, to pronounce the discourse of the Other, to express what is articulated by language (S1-S2). The subject, the universal proletarian, is thus exploited by the symbolic system of culture as the worker studied by Marx is exploited by the capitalist system, which is a historical particularisation of the symbolic system of culture. In both cases, while life belongs to the subject who loses it by selling it as a labour force, the work done by such force is owned and used by the Other, by language, by the system. j y If jouissance is the satisfaction of the death drive, the plus-de-jouir is the surplus of jouissance that is produced by losing life, by transmuting the living into the dead, the real into the symbolic, the vital existence of the worker into the death essence of capital and capitalism, the life spent by the subject on the surplus value gained by the Other, the experience of life in the possession of labour power, the generous life experience converted into deadly possessive jouissance. Generalising This is how having supplants being, private property replaces the community and the sexual relationship and the social bond are replaced by the signifying chain between things. But this is not something that is only experienced by the workers exploited in capitalism. The proletarian condition is widespread. Language uses the work performed by the subject to produce a surplus value, a surplus of symbolic value, by which discourse is signifying or significant (S1-S2) and not insignificant or tautological (S1-S1). We confirm here the Lacanian idea that it is language that ‘employs’ the subject to express it instead of being the subject who utilises language to communicate.34 In other words, it is not language that has a use value as a communication tool for the subject, but rather it is the subject who has a use value as an enunciating labour force exploited by language. We know from Lacan that proletarianisation is the only ‘social symptom’.35 Everyone in society is, in a way, a proletarian. Even the capitalists lose the community, the social bond, the being and the experience of their life that is converted into the possession and enjoyment of capital. This was something that Marx understood very well when he showed how the will and consciousness of the capitalists were possessed by capital or, rather, how the vampire of capital derived its existence from the capitalists who obtained their enjoyment, their possessive essence, from capital, but at the price of the experience of their own lives.36 The use value of the labour force is the expression of all the signifiers articulated by language (S2). As for the exchange value, as with Marx’s proletarian, it is the price of the subject’s existence (S1). It is the signifier that allows the subject to exist in the symbolic system, the signifier with which he is identified, the only signifier he receives in exchange for the arduous work of the expression of all signifiers. While the capitalists gain their enjoyment from capital, capital obtains its very life from the capitalists. This exchange is found in the different relationships that we establish in the symbolic system. In all relationships, subjects embody what represents them. 35 Lacan 1975, p. 187 33 Pavón-Cuéllar 2009, 2010. Historising The symptomatic discovery of Marx is what makes him not simply materialistic and structuralist, but what has been called, roughly, ‘dialectical’ and ‘historical’. What is important here is that the structure and its economic materiality appear in Marx as what they are: precarious, transitory, crossed by history, by conflicts and contradictions, by tensions and struggles, by movement and by life, by disrupting desires and corrosive drives and also, on a genetic level, as products of negation, destruction and alienation, expropriation and privatisation, exploitation and pauperisation, fetishisation and reification. We can reject some of these conceptualisations, but we cannot deny that they designate in a more or less accurate and adequate manner what is revealingly embodied by the proletarian and understood as the historical truth of capitalism, as a symptom of how bourgeois society strips and reveals to Marx everything that he discovers. What is reflected and what is discovered of the historical world through Marx? What is reflected with frightening fidelity is what had already begun to be reflected in the English liberal political economy: the structure of the capitalist system in the nineteenth century. This material structure must have reached the development it had in Marx’s time to be able to externalise itself as it did in the structuralism and materialism of Marx. If Marx was materialistic, it was not only because of everything we already know, but also, as Habermas and others have shown, because he lived in a materialistic world in which materiality reigned impudently; brazen material interest guided all actions, money bought everything, and the economic determined the ideological and dominated the social and the political.29 Similarly, as Lacan showed, if Marx was the first structuralist, that was because globalised capitalism offered the best example of the structure of structuralists: a set of relations between exchange values determined by their differences and mutual relations, a symbolic universe without an exterior, a language without a metalanguage, an Other without The discovery of Marx is also a discovery of history. It is as historic, then, as the reflection. However, no matter how historical it is, it is not limited to the moment in which it occurs. Its moment is also ours. History does not stop being our history. We discover ourselves in the proletariat that is discovered through Marx. The particular discovery acquires a universal character. Lacanizing Marxism Lacanizing Marxism 275 275 274 Generalising 35 Lacan 1975, p. 187 36 Marx 1867. Volume 6 / Issue 1 34 Lacan 1969-1970, pp. 74-75. Generalising Marx himself generalised it when he unravelled the operation of the successive systems of production at the very centre of subjectivity, when he saw an ‘open book of psychology’ in the field of industry37 or when he referred to the machines that absorb knowledge, skills and the other capacities of the ‘social brain’.38 We have a general theory, which, by being Lacanized, can only be confirmed. This confirmation is the most that Lacanian Marxism can offer here. First, in quantitative terms, subjects give the Other more existence than the being they receive from it. While we give our whole life to express all the discourse of the Other and all the signifiers articulated by language (S2), the Other pays us only the signifiers we need to identify with them and be who we are in the symbolic sense (S1). This general surplus of the predicates over the subject, of the signifying chain over a single link in the chain, is the general situation that is revealed in the particular case of the surplus of use value over the exchange value of the labour force of the proletarian. From the perspective of Lacanian Marxism, as in that of Marx, we think on the outside through language, with the symbolic system of culture. It is out there, not inside our head, where our thinking organ resides. It is an external, cultural-symbolic device, not an internal, organic-cerebral organ. We do not think with the cells of the brain, but with enunciations, with social interactions, with historical events or with economic operations. These are the constitutive elements and the basic processes of our psyche. Our inner life is external. We come, once again, to the Lacanian concept of extimacy: the most intimate is external. I leave myself when I go deeper into myself, the ego is two-dimensional and I cannot enter myself without crossing through my imaginary appearance in the mirror.39 Secondly, in qualitative terms, there is another injustice in the relation of the subject with the signifier. While the signifier obtains its existence effectively from the subject, the subject receives its being from the signifier only in an apparent manner. The subject, in fact, will never be the signifier that represents it. The signifier will never coincide with the subject. The subject will never be completely absorbed by discourse. Generalising The signifier receives from the subject its literalness, its conscience and its will, its body and its life, while the subject acquires the deadly enjoyment of its identity, the very being of the signifier that will represent it for another signifier. The identification of the subject with the signifier, his gain of an identity to exist symbolically, causes him to lose his life, which is alienated in the discourse of the Other. This life is used as a labour force for the production of a symbolic surplus value, a surplus of significance, which will certainly be earned by the Other, but at the price of a surplus enjoyment (a). What we have here, in the plus-de-jouir, is the surplus of jouissance that is gained when we lose the experience of our life, which is reduced to a role in the labour force of the Other, that is, the workforce of the unconscious. i The exchange is apparently fair: while the signifier gives a being to the subject, the subject gives an existence to the signifier. And yet, Marx shows us here that there is a trap, a scam and an injustice. Where is this 36 Marx 1867. Lacanizing Marxism Lacanizing Marxism 276 277 the most important component, the capital of capital, variable capital. To be generalised, this conception of Marx does not require a Lacanian reading. Marx himself generalised it when he unravelled the operation of the successive systems of production at the very centre of subjectivity, when he saw an ‘open book of psychology’ in the field of industry37 or when he referred to the machines that absorb knowledge, skills and the other capacities of the ‘social brain’.38 We have a general theory, which, by being Lacanized, can only be confirmed. This confirmation is the most that Lacanian Marxism can offer here. injustice that justifies the frustration, indignation and insurrection of the subject? the most important component, the capital of capital, variable capital. To be generalised, this conception of Marx does not require a Lacanian reading. Generalising Hence our alienation (Entfremdung) in the Other (S2) implies our division (Entäusserung) as subjects ($). We are never what we are. We never get confused with what we have. This is also why there can be exploitation: the subject can be exploited because he is excluded from what exploits him. W h f d l l f h h l d Extimacy is only one of multiple concepts, among which there is also that of the unconscious understood as exteriority or as politics or as the discourse of the Other, through which a Lacanian reading can confirm the traditional monistic orientation of Marxism and its correlative opposition to any dualistic perspective that remains trapped in the inner/ outer or mind/body dualities. In the same sense, Lacan can also serve to confirm the convincing historical explanation of dualism we read in Marx and especially in Engels, with its three acts: first, at the origin of civilisation, the division of classes; then, on the basis of class division, the division between manual and intellectual work, with the dominant class monopolising the intellectual work and condemning the dominated class to do the manual labour, in such a way that people belonging to the former class think with their minds what people belonging to the latter class perform with their bodies; and finally, because of the division of labour, the mind/body duality appears since the mental and the corporeal, when situated and developed separately into two classes, begin to be separated and differentiated one from the other.40 We come here to a fundamental rule of the system: the exploited could not be exploited if he were not excluded with respect to the fruits of his exploitation. The surplus value cannot be accessible to those who produce it. Exploitation requires exclusion. This is why inequality necessitates a separation between the unequal, discrimination requires segregation, workers must remain in their poor suburbs, and high border walls and harsh migratory laws must protect the wealth of Europeans and Americans against Asians, Africans and Latin Americans. In general, there is no place for the producers in the world that they themselves have contributed to produce. The discourse must eradicate the being that has enunciated it. Linguistics abstracts from its enunciators. The experience of our life does not belong to us, but is forbidden to us; it is the enjoyment of the Other. 37 Marx 1844, p. 151. 38 Marx 1857-1858, p. 220. 39 Lacan 1954-1955. 40 See Engels 1876. Confirming The symbolic system, both in general and in its capitalist particularisation, excludes the same subjects who are exploited by it. And, nevertheless, these subjects are possessed by the system as if by a demon. The Other manifests in their actions, in their words, in their thoughts and even in their deepest feelings. Nothing seems to escape the Other. In Marx, for example, the capitalist system, capital itself, is the one that acts, speaks, thinks and feels through the capitalist, but it is also the one that works with the labour force of the worker, which, for that reason, is a component of capital, Lacanizing Marxism Lacanizing Marxism 278 279 A Lacanian reading allows us to confirm the process described by Marx and Engels by rediscovering it at another level through the Hegelian relationship between the master and the slave. As in Marxism, the position of the master, that of power, will have the privilege of consciousness, while the position of the slave is that of the body, that of the unconscious. The soul/body duality has its origin, here also, in a dominant/dominated duality. Everything begins with a dialectic of domination that unfolds in the discourse of the master with its difference between the master-signifier (S1) of the consciousness that dominates and all the other signifiers (S2), namely those of the discourse of the Other, those of the unconscious that works with the labour force of the subject, with his life and with his body ($).41 correspond to our desires or our interests. What dominates us through our soul is rather something that possesses us, represents us and usurps our identity in such a way that we can act against our interests and against our wishes. The soul is necessary, therefore, for the subjects to turn against themselves and help their master to master them. Our domination requires, then, the support of our soul. This can be well seen, as I have tried to show, in Spanish colonialism that uses evangelisation to generate a soul, to dig an internal world, to build a mental prison in those Amerindian, though not all of them, who had managed to resist the stupid temptation of the soul. In the indigenous communities in which the soul did not exist, the non-existence of a soul correlated with the absence of private property, of social classes and of the division of labour. Completing The development of psychology is inseparable from the advance of capitalism. The advancing capital is personified by the capitalist, who, as a bourgeois, is also the prototype of the homo psychologicus, the man identified with his soul or psyche, that is, with the fact of being intelligent, thoughtful, calculating, self-absorbed, introspective, depressed, stressed, frustrated, sentimental, in love, jealous, possessive, interested, capricious and so on. Marx and Engels demonstrated that the ego, with its personality, ideas and emotions, constitutes the most intimate private property of the bourgeoisie, the possession of its own existence, the enjoyment of itself, the confusion of being with having in the psychological objectification of the subject.45 However, by completing the Marxist demonstration with a Lacanian observation, we should add that the bourgeois cannot limit themselves to enjoy this self, but must verify it again and again through their own reflection on the surface of the mirror, which makes them impose it on the whole society through disciplinary devices, ideological apparatuses of the State, various sectors of the cultural industry and many other specular means. The soul, whether it is conceived as such or as consciousness or spirit or reason or the psyche or otherwise, is the fundamental seat of power. This usually goes unnoticed because the mental domination usually takes the opposite form of freedom for a subject identified with his soul, be it homo religiosus, spiritualis, rationalis or psychologicus. In all cases, something dominates us when we believe that we are dominating ourselves and thus freeing ourselves, and even when we believe that we are ‘freeing our own body’, as Marcuse showed.43 The homo psychologicus becomes as universal as its internal mental world. However, as we have seen, the imposed and universalised mind is not neutral. It is inseparably linked with the dominant class, reflects it and can serve as a means for the dominated to help dominate them by dominating themselves. This may be the case for many reasons, including the origin of the mental sphere as a class privilege, its imaginary specular constitution and its monopolisation and production-reproduction by the What dominates us through the soul? It does not matter whether we respond by referring to the Lacanian concept of the master-signifier or to the Marxist-Engelsian notion of the dominant ideology understood as the ideology of the ruling class. Confirming The processes of colonisation, appropriation and primitive accumulation demanded, and continue demanding at every moment and in each one of us, a process of psychologisation.44 The interesting thing about the Lacanian reading is that it allows us to appreciate the way in which the historical explanation given by Marx and Engels not only refers to the origin of human civilisation, but also to each discursive gesture, to each enunciation, by which subjects are situated in a position of power, move away from their body and appear as pure souls or psyches, as agents of cognition or thought, by pretending to overcome their unconscious, control their discourse and dominate their body, as if it belonged to them and not to the Other. Thus, a power relationship, a class division with the correlative separation between mental and manual labour, constantly divides each subject between an authoritarian mind and an obedient body. The perspectives of Marx and Engels are confirmed through a Lacanian reading that also allows us to confirm the Foucaultian inversion of the Platonic description of the body as a jail of the soul. The truth is the reverse: ‘the soul is the prison of the body’.42 41 Lacan 1969-1970. 42 Foucault 1975, p. 34. 43 Marcuse 1964, pp. 89-11. 44 Pavón-Cuéllar 2016. 41 Lacan 1969-1970. Volume 6 / Issue 1 45 Marx and Engels 1846, chapter III. Completing How is it that private property and the resulting oppressive appropriation of the other originate at the same time as the possession and oppression of women in the monogamous family? We know the Engelsian explanation of the father who exercises his power over the woman to be sure that the heirs of his private property will also be his children.50 This explanation is crucial, but incomplete, because it already presupposes the existence of private property that should still be clarified and it thus frames a situation in which there is no longer either matriarchy or communism, which were practically the same thing and which ceased to exist at the same time and not one after the other. We must still explain why the matriarchal community disappears and gives way to patriarchy and private property. A present task of my Lacanian Marxist proposal is to examine how one of the consequences of the Marxist findings, the emergence of the inconceivable proletarian soul under the form of class consciousness, is perfectly correlated with the Freudian discovery, the revelation of the unconscious where the body of the bourgeois has been confined. The problem is that such discoveries seem to have led not to the reconstitution and liberation of total humanity through the overcoming of the mental-bourgeois and corporal-proletarian human halves, but to the proletarianisation of the bourgeois and the embourgeoisement of the proletariat that Lacan perceived so well in showing how the working class regained a master consciousness, a ‘master knowledge’, while the bourgeois recovered a ‘slave body’.47 After all, in the kind of society in which we live, there are only models of dominant souls and dominated bodies. No other models are available! This is also something that can be deduced from the point raised by Lacan. What if there was a strictly logical relationship between patriarchy and private property, between patriarchal masculinity and possession- possessiveness, between having the phallus and having in general understood as phallic enjoyment, as well as a strictly logical relationship between being the phallus and the being that is at stake in desire, between being a woman and an inevitably common and singular being, between femininity and community, between matriarchy and communism? Completing The important thing is to understand that it is something that is not us, does not concern us and does not even Lacanizing Marxism Lacanizing Marxism 281 280 ruling class, whose members devote their lives to cultivating their mind and spreading it in society. subjectivity, society, history and culture. This was already observed by the Frankfurtians, particularly by Adorno, and made them opt for the theoretical critique of the dualist-class division, of the tearing of the individual and society, instead of a monist-communist solution that could only come from practice and that in any case still did not seem possible.48 In modern society, just as psychology is predominantly bourgeois, so the bourgeoisie is preponderantly psychological. Let us reiterate that the bourgeois class is the homo psychologicus class. It is, so to speak, a mental class that sometimes seems to have neither body nor external world and to obey exclusively the ‘psychological factor’, the ideas, emotions and other impulses coming from the internal world, as Plekhanov observed in certain literature of the nineteenth century.46 There is nothing here but intrigues in which souls without bodies participate. The corporal, particularly in its sexual expression, is repressed and reappears in a symptomatic way in Freud’s hysterics. As if by chance, this symptomatic return of the body and its drives repressed in the mental class, in the dominant class composed of the intellectual workers, occurs at approximately the same time as the symptomatic return of the repressed mental potentialities of the dominated class, the corporal class of the manual workers of Marx and Marxism. In both cases, among the bourgeoisie and among the workers, the symptom is the irruption of the truth of monism in the dualistic ideological constructions. Why would it seem that it is still impossible today, and perhaps always impossible, to overcome dualism and classism? We know the Lacanian response that refers to the real as impossible, to castration, sexuation and the non-existence of the sexual relationship.49 This response can complete the forgotten intuition of Marx and Engels about the deep link between patriarchy and class society. Marx and Engels highlight the conjectural simultaneous emergence of the exploitation of man by man and the exploitation of woman by man. According to this hypothesis, the transition from matriarchy to patriarchy coincides with the dissolution of the original community and primitive communism. 283 48 E. g. Adorno 1955. 49 Lacan 1968-1969, 1969-1970, 1971, 1971-1972. 50 Engels 1884. 51 Pavón-Cuéllar 2017b. Lacanizing Marxism 282 46 Plekhanov 1907, pp. 98-99. 47 Lacan 1968-1969, pp. 172-173, 1969-1970, pp. 20-35. Lacanizing Marxism 46 Plekhanov 1907, pp. 98-99. 49 Lacan 1968-1969, 1969-1970, 1971, 1971-1972. 47 Lacan 1968-1969, pp. 172-173, 1969-1970, pp. 20-35. Completing While the not- all respects the singularity on a case-by-case basis, the for-all reduces the singular to the exceptional, to the exception to the rule, or tends to dissolve it into a generality in which there are no singular cases that are qualitatively different from each other, but simply individual expressions of the general category or units that can be counted and calculated in quantitative terms. The totalised individuality, closed on itself in its general definition, is the antithesis of what remains incomplete, open, undefined, pending, in suspense. The subjects of psychoanalysis and communism respond to the not-all by which they doubt, ignore, desire, struggle, organise and knot with others, make and unmake groups, discuss in endless assemblies and try in vain to complete themselves. On the contrary, the individual of capitalism and psychology obeys the for-all rule that guides most of the psychological tests, prêt-à-porter diagnoses of the DSM, opinion polls, mass production for undifferentiated consumers, emoticons and likes of social networks, the bourgeois democracy of the summable votes and the anatomo-political and biopolitical devices elucidated by Foucault. Marx and several of his followers have studied how for several centuries, since capitalism has progressed unstoppably in the world, the quantitative dimension of money and exchange value has tended to gain ground over the qualitative dimension of things themselves and their use value. This evolution implies the most diverse transformations, such as those that make us go from the unquantifiable truth to a supposedly quantifiable reality or from knowledge to data and information. My Lacanian reading of such evolution, which aims to complete the Marxist vision, has not only raised the insufficient and arguable hypothesis of a progressive symbolisation and derealisation of the world, but also the conjecture of the advancement of the generalising and homogenising masculine logic of the for-all to the detriment of the irreducibly singular feminine element of the not-all. This conjecture is politically relevant because it could serve to explain the development of the masses at the expense of the communities, that is, the progression of aggregated and massified interchangeable individuals at the expense of community integrations between different subjects. The same conjecture could also explain the development of quantitative inequalities between income or capabilities or anything else over the qualitative differences between subjects who are so different that they cannot be judged unequal. Completing These relationships, which must be nuanced and complicated through the Lacanian logic of sexuation, have already served me to Lacanianly complete what was just outlined by Marx and Engels.51 The Lacanian Marxist result already has several old precedents in the field of Freudian A Lacanian reading allows us to complete Marx and Engels, not only by considering the bourgeois unconscious correlate of the class consciousness of the proletarians, but also by strategically foreseeing the consequences of both correlative expressions of the return of the repressed. We may fear, for instance, that such symptomatic irruptions of the truth of monism do not have the expected subversive effects because of an irremediably dualistic and classist functioning of Lacanizing Marxism Lacanizing Marxism 283 282 In the absence of conclusion Marxism, among which those of Erich Fromm52 and Oswald de Andrade53 stand out. They and others elucidated what would later be well sensed in feminism: that the anti-capitalist struggle is futile as long as it is not also anti-patriarchal. Both the development of inequality at the expense of difference and the advancement of the masses at the expense of communities are victories of capitalism over the subject of communism, but also over the subject of psychoanalysis. The irreducibly singular and absolutely different Freudian subject is the only one that can effectively organise with others to fight for communism or to coherently join and knot with others to form the community for which the communists fight. And this subject has nothing to do with the generalised, interchangeable and summable individuals of the masses, of capitalism and psychology. p If we must face patriarchy to fight efficaciously against capitalism, it is not only because capital rests on the possessive logical element of the patriarchal function, but because this same function involves another element that is also at the base of the capitalist system, an element that was pointed out by Lacan54 and emphasised by Jorge Alemán,55 and that has also recently allowed me to add something to help completing the Marxist intuition of the link between capitalism and patriarchy.56 I refer to the masculine for-all and its contradiction to the feminine not-all. Volume 6 / Issue 1 57 Marx 1845, Marx and Engels 1846. 284 52 Fromm 1934. 53 Andrade 1950. 54 Lacan 1972-1973. 55 Alemán 2013. 56 Pavón-Cuéllar and Boggio Éwanjé-Épée 2018. Lacanizing Marxism 58 Freud 1921. 56 Pavón-Cuéllar and Boggio Éwanjé-Épée 2018. Completing Thus Lacan may also help to prevent capitalism from reabsorbing and recovering what Marx and his followers discovered. Lacanian Marxism should be for now, at least for now, an entity that is still too irrational to be assimilated to capitalist rationality. Perhaps it can never be rationalised, just as it was never possible to carry out the rationalisation of the encounter between Marxism and psychoanalysis in surrealism, in which, as if by chance, we found the first Lacanian Marxist, the brilliant René Crevel63, who perhaps should have been our starting point. allow themselves to be caught in any signifier, be it the race, the nation, the father or man of patriarchy or any symbol of power, success, health or normality, especially the most important and irresistible of all in capitalism, namely money, something whose only use value is its exchange value, pure possession, principle of possessiveness or quintessence of enjoyment. Money is the most powerful of the signifiers because it is the most perfect, the purest, the most arbitrary, that is, as Lacan pointed out, the ‘most destructive of any significance’, the least dependent on a precise meaning, since it can mean everything by being able to buy everything.60 However, as Marx remarked brilliantly, money is never enough to buy everything, as its ‘quantitative limitation’ always prevents the realisation of its ‘qualitatively unlimited nature’.61 This inherent characteristic of the signifier produces the insatiable avidity, the typical enjoyment of capitalism, for which we try to possess more and more, to have more and more money to fulfil all that the signifier is and offers us, to really possess it, a result that is impossible to realise in any way. By resisting and not just giving in to the enjoyment of money, the subject of Marxism and psychoanalysis, the subject of history and desire, is radically subversive to capitalism. Of course, capital always finds a way to recover what subverts it. There is no need to remember what the communist parties and ego psychologies have been. However, in addition to what is recoverable, there is always something irrecoverable, incurable, in the truth that is revealed symptomatically through Marx and Freud. This makes everything in the capitalist reality conjure itself against the revelation. Everything is like an immense reactive formation to refute Freud and especially Marx. Completing The dominant ideology in capitalism, the same that gives rise to psychology, makes us imagine that it is the collective that is composed of individuals, while Marxism and psychoanalysis have taught us that it is individuality that is made up of group components that are knotted in it, namely social relations for Marx57 or mass identifications for Freud.58 It is the Other who becomes One, who makes the One exist, and not the One who already exists and relates to the Other. There is, then, no socialisation of the individual, as Piaget thought, but an individualisation of the social, as Vygotsky recognised.59 Or better yet, there is a generation of individuals in a discourse of the Other that only retroactively, après coup, appears as transindividual. In this discourse that does not close in a totality and that is not the same for all, that is not-all and different for everyone, the subject of Marx and Freud is not an individual subject among others, but the result that is always still postponed, always indefinable and evasive of a convergence and unique combination, which is irreducibly singular and absolutely different, of innumerable signifiers corresponding to individual identifications. i The subjects of Marx and Freud are intrinsically subversive because they resist in one way or another that which defines them. They do not 284 52 Fromm 1934. 53 Andrade 1950. 54 Lacan 1972-1973. 55 Alemán 2013. 56 Pavón-Cuéllar and Boggio Éwanjé-Épée 2018. Lacanizing Marxism Lacanizing Marxism Lacanizing Marxism 285 285 284 C R I S I S & C R I T I Q U E / Volume 6 / Issue 1 serve to preserve the truth of what is Lacanized, preserving it as it is: incomprehensible, counterintuitive, strange. Thus Lacan may also help to prevent capitalism from reabsorbing and recovering what Marx and his followers discovered. Lacanian Marxism should be for now, at least for now, an entity that is still too irrational to be assimilated to capitalist rationality. Perhaps it can never be rationalised, just as it was never possible to carry out the rationalisation of the encounter between Marxism and psychoanalysis in surrealism, in which, as if by chance, we found the first Lacanian Marxist, the brilliant René Crevel63, who perhaps should have been our starting point. serve to preserve the truth of what is Lacanized, preserving it as it is: incomprehensible, counterintuitive, strange. 60 Lacan 1956, p. 37. 63 Crevel 1933, see Pavón-Cuéllar 2014b. 60 Lacan 1956, p. 37. 61 Marx 1867, p. 91. 62 Pavón-Cuéllar 2017c. Completing Marxismo y psicoanálisis ante el fundamento sexual-familiar de la opresión política- Pavón-Cuéllar, David 2017b, ¿Por qué la violencia de género no puede explicarse por la de clase? Marxismo y psicoanálisis ante el fundamento sexual-familiar de la opresión política- económica. Teoría y Crítica de la Psicología 9, 244-253. de clase? Marxismo y psicoanálisis ante el fundamento sexual-familiar de la opresión política- económica. Teoría y Crítica de la Psicología 9, 244-253. Pavón-Cuéllar, David 2017c, Tokio y la eterna permanencia de la Revolución de Octubre. Crisis C íti 1(1) 43 53 económica. Teoría y Crítica de la Psicología 9, 244-253. Pavón-Cuéllar David 2017c Tokio y la eterna pe Crisis of Psychoanalysis: Essays on Freud, Marx, and Social Psychology (pp. 109-135), New York: Henry Holt, 1970. Crisis of Psychoanalysis: Essays on Freud, Marx, and Social Psychology (pp. 109-135), New York: Henry Holt, 1970. Habermas, Jürgen, 1968, Ciencia y técnica como ideología, Madrid: Tecnos, 2013. Jameson, Fredric, 1983, The Political Unconscious, Narrative as a Socially Symbolic Act, London, Crisis of Psychoanalysis: Essays on Freud, Marx, and Social Psychology (pp. 109-135), New Y Holt, 1970. Habermas, Jürgen, 1968, Ciencia y técnica como ideología, Madrid: Tecnos, 2013. J F d i 1983 Th P liti l U i N ti S i ll S b li Pavón-Cuéllar, David 2017c, Tokio y la eterna permanencia de la Revolución de Octubre. Crisis e Crítica 1(1), 43-53. Pavón-Cuéllar, David, and Félix Boggio Éwanjé-Épée 2018, Que peut la psychanalyse Pavón Cuéllar, David 2017c, Tokio y la eterna permanencia de la Revolución de Octubre. Crisis e Crítica 1(1), 43-53. Pavón-Cuéllar David and Félix Boggio Éwanjé-Épée 2018 Que peut la psychanalyse Habermas, Jürgen, 1968, Ciencia y técnica como ideología, Madrid: Tecnos, 2013. e Crítica 1(1), 43-53. Pavón-Cuéllar, David, and Félix Boggio Éwanjé-Épée 2018, Que peut la psychanalyse aujourd’hui? Entretien avec David Pavón-Cuéllar Période retrieved from http://revueperiode net/que- Pavón-Cuéllar, David, and Félix Boggio Éwanjé-Épée 2018, Que peut la psychanalyse aujourd’hui? Entretien avec David Pavón-Cuéllar, Période, retrieved from http://revueperiode.net/que- Jameson, Fredric, 1983, The Political Unconscious, Narrative as a Socially Symbolic Act, London, Routledge, 2002. Jameson, Fredric, 1983, The Political Unconscious, Narrative as a Socially Symbolic Ac Routledge, 2002. József, Attila 1934, Hegel, Marx, Freud. Action Poétique 49, 1972, 68–75. Korsch, Karl 1923, Marxismo y filosofía, Mexico City, Era, 1977. É peut-la-psychanalyse-aujourdhui-entretien-avec-david-pavon-cuellar/ Regnault, François, 2005, Le Marx de Lacan. La Lettre Mensuelle de l’ECF 242, 4-6. Completing Everything is as it is to show that there is no truth in the truth of our uniqueness and our community. As I tried to explain it once when describing an experience in Tokyo, communism is a truth, the one posed as such by Sen Katayama, that internally moulds, in a negative way, everything that works so impeccably in the Japanese manifestation of capitalism, everything that is possible and visible, everything that is done so that the truth is invisible and impossible.62 Here we must understand the principle of negativity whereby truth is not confused with a reality that Lacan correctly describes as imaginary. Reality is always so wrong and misleading, especially in capitalism, that it cannot but differ from the truth and contradict it. In fact, especially in capitalist society, it is precisely to contradict the truth that reality is what it is when it is constituted ideologically. That is why the truth always has a strange, counterintuitive, incomprehensible aspect, as in the work of Lacan. g , , p p , The Lacanization of Marxism, like that of psychoanalysis, can also Lacanizing Marxism Lacanizing Marxism 287 286 C REFERENCES Marx, Karl 1867, El Capital I, Mexico City: FCE, 2008. Marx, Karl, and Friedrich Engels 1846, La ideología alemana, Madrid: Akal, 2014. Adorno, Theodor 1955, Acerca de la relación entre sociología y psicología, in H. Jensen (Ed), Teoría crítica del sujeto (pp. 36-76), Mexico City: Siglo XXI, 1986. Alemán, Jorge 2013, Conjeturas sobre una izquierda lacaniana, Buenos Aires, Grama. 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https://openalex.org/W2993123771	http://eprints.whiterose.ac.uk/154245/1/28-Article%20Text-903-1-10-20191203.pdf	English	null	Degassing at Sabancaya volcano measured by UV cameras and the NOVAC network	Volcanica	2,019	cc-by	10,731	Article: Article: Ilanko, T. orcid.org/0000-0001-6535-1117, Pering, T. orcid.org/0000-0001-6028-308X, Wilkes, T. orcid.org/0000-0002-3448-6067 et al. (9 more authors) (2019) Degassing at Sabancaya volcano measured by UV cameras and the NOVAC network. Volcanica, 2 (2). pp. 239-252. ISSN 2610-3540 10 30909/vol 02 02 239252 Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ This is a repository copy of Degassing at Sabancaya volcano measured by UV cameras and the NOVAC network. White Rose Research Online URL for this paper: http://eprints.whiterose.ac.uk/154245/ Version: Published Version Version: Published Version ∗Corresponding author: tehnuka@volcanoﬁles.com Resumen Utilizamos cámaras ultravioletas (UV) Raspberry Pi para medir los ﬂujos de dióxido de azufre (SO2) en el volcán Sabancaya, Perú, durante la actividad del 27 abril 2018. La corrección por dilución de luz se realizó midiendo simul- táneamente en dos sitios a diferentes distancias. Los ﬂujos promedio (27.1 kgs−1) son superiores a los reportados previamente, probablemente debido al actual episodio explosivo. Cada evento tuvo frecuentes emanaciones ricas en ceniza y gas, emitiendo 3.0–8.2 toneladas de SO2. La desgasiﬁcación sostenida, sin sobrepresión, indica una chi- menea abierta. Estos ﬂujos son similares a los medidos en una estación permanente de NOVAC (25.9 kgs−1) debajo de la pluma. La diferencia restante es por velocidad del viento estimada y la frecuencia de la muestreo. Nuestro trabajo muestra la importancia de modelar con precisión la dilución de luz y velocidad del viento, y que co-instalar cámaras UV y espectrómetros permanentes podrían dar velocidades del viento más exactos. Keywords: Sulphur dioxide; PiCam; UV camera; NOVAC; DOAS; Sabancaya Abstract We used low-cost Raspberry Pi ultraviolet (UV) cameras to measure sulphur dioxide (SO2) ﬂuxes from Saban- caya volcano, Peru, during eruptive activity on 27 April 2018. Light dilution corrections were made by operating instruments at two distances simultaneously. Estimated SO2 ﬂuxes of 27.1 kgs−1 are higher than previously re- ported, likely due to the current eruptive episode (ongoing since November 2016). Each eruptive event included frequent (2–3 per minute), ash-rich emissions, forming gas pulses with masses of 3.0–8.2 tonnes SO2. Sustained degassing and lack of overpressure suggest open-vent activity. Mean ﬂuxes are consistent with those measured by a permanent NOVAC station (25.9 kgs−1) located under the plume, with remaining diﬀerences likely due to wind- speed estimates and sampling rate. Our work highlights the importance of accurate light dilution and windspeed modelling in SO2 retrievals and suggests that co-location of UV cameras with permanent scanning spectrometers may be valuable in providing accurate windspeeds. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ eprints@whiterose.ac.uk https://eprints.whiterose.ac.uk/ O V L O V NI O V O V L O V NI O V O V L O V NI O V RESEARCH ARTICLE RESEARCH ARTICLE RESEARCH ARTICLE Degassing at Sabancaya volcano measured by UV cameras and the NOVAC network Tehnuka Ilanko∗α, Thomas D. Peringα, Thomas C. Wilkesα, Fredy E. Apaza Choquehuaytaβ, Christoph Kernγ, Alejandro Díaz Morenoδ, Silvio De Angelisδ, Susana Layanaε, ζ, Felipe Rojasη, Felipe Aguileraǫ, η, Freddy Vasconezϑ, Andrew J. S. McGonigleα, ι, κ αDepartment of Geography, University of Sheﬃeld, UK ⋆Full list of aﬃliations given in Aﬃliations section. 1 Introduction age sequences [e.g. Gliß et al. 2018; Peters and Op- penheimer 2018], whereas windspeeds for DOAS ﬂux calculations often require the use of multiple scanners and cross-correlation [McGonigle et al. 2009; Galle et al. 2010], independent windspeeds from in situ mea- surements [e.g. Prata 2013], or forecast models [Lübcke et al. 2013]. Long-term ground-based monitoring of volcanic SO2 emissions, however, with rare exceptions (Burton et al. [2015] at Stromboli; Kern et al. [2018] at K¯ılauea Volcano; D’Aleo et al. [2016] at Mt Etna), uses scanning DOAS. The development of low-cost UV cam- era alternatives is recent, and despite the advantages of higher temporal and spatial resolutions, challenges re- main with image-based measurements. For example, regular calibration is needed with changing light con- Small, low cost, ultraviolet (UV) cameras with low power requirements [Wilkes et al. 2016; Wilkes et al. 2017] have facilitated quantiﬁcation of sulphur dioxide (SO2) in volcanic plumes, while also providing time- series images that can be used for tracking plume fea- tures and explosions [e.g. Peters et al. 2015]. Higher temporal resolutions can be achieved with a UV camera than with diﬀerential optical absorption spectroscopy (DOAS) based methods that require scanning through the plume [e.g. Galle et al. 2003; McGonigle et al. 2009; McGonigle et al. 2017]. Windspeeds for calculating SO2 ﬂuxes can also be obtained directly from UV im- Degassing at Sabancaya volcano Ilanko et al., 2019 decade. Using UV cameras and scanning DOAS, Mous- sallam et al. [2017] found higher SO2 ﬂuxes from 1.5 hours of data in November 2015, with a notable diﬀer- ence between the lower DOAS and higher UV camera ﬂuxes (Table 1), which they attribute to diﬀerences in temporal resolution and retrieval procedures. Finally, Kern et al. [2017] calculated an average of 890 td−1 from two hours of DOAS data in May 2016, six months before the start of the current eruptive episode. The range of these reported ﬂuxes likely reﬂect diﬀerences in measurement and retrieval techniques as well as changes in activity. Leading up to the start of the erup- tion in November 2016, new fumarolic activity was ob- served in the summit area, and an increase in daily SO2 ﬂuxes was identiﬁed by the Observatorio Volcanológico del INGEMMET using the scanning DOAS stations in the NOVAC network [Ramos Palomino et al. 2016]. 1 Introduction Fur- ther details on the onset of this eruptive episode are available in reports by the Observatorio Volcanológico del INGEMMET [Ramos Palomino et al. 2016] and the Global Volcanism Program. Data reported here were collected by PiCams deployed on 27 April 2018 during a NOVAC workshop [Kern et al. 2018], and from NO- VAC scans over the same period. ditions, using SO2-ﬁlled gas cells or a co-aligned DOAS instrument, and clouds in front of the plume can pre- vent accurate SO2 retrievals [Lübcke et al. 2013]. [ ] The Network for Observation of Volcanic and Atmo- spheric Change, NOVAC [Galle et al. 2010], operates a network of ground-based permanent scanning DOAS spectrometers for automated SO2 ﬂux measurements that currently monitors 42 volcanoes (https://novac- community.org/volcanoes, accessed on 10 June 2019). The system includes software providing automated cal- culations of windspeed and ﬂuxes. Ideally, spectrom- eters scan perpendicular to the wind direction with diﬀerent viewing angles to measure SO2 slant column amounts [Galle et al. 2010]. The primary source of error under favourable meteorological conditions is in windspeed estimates, which use either the lag be- tween column amounts for two scanners or an external windspeed model. However, plume height estimates, light dilution and scattering may also contribute sig- niﬁcantly to errors [Galle et al. 2010], with the magni- tude of errors being dependent on time- and location- speciﬁc parameters. In addition, a lack of plume-free sky will result in reference spectra contaminated by SO2, causing ﬂuxes to be underestimated [Lübcke et al. 2016]. While data from the network are valuable for permanent monitoring of SO2 emissions and compar- ison of degassing at diﬀerent sites, it is also useful to compare these with data collected by other methods. Here, therefore, we demonstrate the use of low-cost UV cameras [PiCams: Wilkes et al. 2016; Wilkes et al. 2017] in a comparison with SO2 ﬂuxes collected by NOVAC permanent DOAS scanning stations at Sabancaya vol- cano, Peru, in April 2018. We provide here the ﬁrst comparison of contempo- raneous UV camera- and NOVAC-derived volcanic SO2 ﬂuxes, and use this example to explore sources of un- certainty that pose ongoing challenges to SO2 ﬂux es- timates, due to the diﬃculty in quantifying the associ- ated errors. We also consider eruption mechanisms that could explain the qualitative observations of activity at Sabancaya during these measurements. 1.1 Activity at Sabancaya volcano The following sections provide details of data collec- tion from UV cameras (Subsection 2.1) and the NO- VAC scanning network (Subsection 2.2). Infrasound data (Subsection 2.3) were also available for this time period from a permanent station ~2.7 km SW of the ac- tive vent. Sabancaya is an active, persistently degassing strato- volcano in the south of the Peruvian volcanic arc (Fig- ure 1). Episodes of heightened activity over the past three decades have been explosive [e.g. Samaniego et al. 2016] and the latest such episode, which started in 2016, includes regular and sustained ash- rich explo- sions producing plumes averaging 2–4 km in height [OVI-INGEMMET & IGP 2018]. These frequent explo- sions (~20–30 explosions/day during the week of 23 April 2018), are associated with increases in SO2 emis- sions from the summit area. producing plumes averag- ing 2–4 km in height [OVI-INGEMMET & IGP 2018]. However, of the 26 explosions identiﬁed by long pe- riod earthquakes and tremors on 27 April 2018, none occurred during the measurement period, with several hours’ interval between one explosion at 13:41 UTC and the next at 16:32 UTC. 2.1 PiCams, ﬁeld methods and analyses To view the plume, which was moving to the northeast, three Raspberry Pi (PiCam)-based UV camera units [Wilkes et al. 2016; Wilkes et al. 2017] were set up about 10.29 km from the summit crater of Sabancaya (Figure 1, ‘Far camera site’). Data from two of these cameras, Cameras A and B, are reported here. A fourth unit was operated approximately 4.25 km from the summit (Ampato station/near camera). We give a brief outline here of the instrument setup and retrievals, but thorough discussion of this and alternative methods are given by Kantzas et al. [2010] and McGonigle et al. [2017]. Mean SO2 ﬂuxes from Sabancaya for the period 2005–2015 were reported from the Ozone Monitor- ing Instrument (OMI) satellite retrievals by Carn et al. [2017] to be 87 td−1 with 1 s.d. of 158 td−1. Their an- nual data show an increase in SO2 ﬂuxes over the past Each UV camera instrument contains two PiCams Presses universitaires de rasbourg Page 240 2(2): 239 – 252. doi: 10.30909/vol.02.02.239252 Volcanica Table 1 – Recent published SO2 ﬂux data for Sabancaya bulk plume. Study Duration & dates Method Flux (td−1) 1 s.d. td−1 Carn et al. [2017] 2015 mean OMI satellite 518 1600 Moussallam et al. [2017] 1.5 hrs, Nov 2015 DOAS 947 332 UV camera 1 1394 352 UV camera 2 1663 318 Kern et al. [2017] 2 hrs, Apr 2016 DOAS 890 240 Ramos Palomino et al. [2016] Daily means, July–Oct 2016 NOVAC 600–7100 Not reported Ramos Palomino et al. [2016] Daily means, Nov 2016 NOVAC 1200–7000 Not reported Figure 1: Satellite view of Ampato–Sabancaya complex showing NOVAC installations [Masías Alvarez and Apaz Choquehuayta 2018], infrasound station, UV camera sites from this study, with dashed lines showing camer Table 1 – Recent published SO2 ﬂux data for Sabancaya bulk plume. Study Duration & dates Method Flux (td−1) 1 s.d. td−1 Carn et al. [2017] 2015 mean OMI satellite 518 1600 Moussallam et al. [2017] 1.5 hrs, Nov 2015 DOAS 947 332 UV camera 1 1394 352 UV camera 2 1663 318 Kern et al. [2017] 2 hrs, Apr 2016 DOAS 890 240 Ramos Palomino et al. [2016] Daily means, July–Oct 2016 NOVAC 600–7100 Not reported Ramos Palomino et al. [2016] Daily means, Nov 2016 NOVAC 1200–7000 Not reported Table 1 – Recent published SO2 ﬂux data for Sabancaya bulk plume. 2.1 PiCams, ﬁeld methods and analyses sorbance (near site: r2 of 0.98; far site: r2 of 1.00 in cal- ibrations for all data presented here). Acquisition rates were set separately for each camera pair at 0.25 to 0.2 Hz. (pressure, humidity, and aerosol concentration), dis- tance of the instrument from the plume, and conditions within the plume (SO2 concentration, aerosol concen- tration and composition). Light is scattered diﬀerently by air molecules and aerosols, depending on their size relative to the wavelength of the light. Light dilution becomes an issue when a signiﬁcant portion of the mea- sured radiation is scattered into the ﬁeld of view in be- tween the plume and the instrument, and thus has not passed through the plume [Mori et al. 2006; Kern et al. 2010]. In UV camera images, this causes systematic underestimation of ICAs [e.g. Bluth et al. 2007; Cam- pion et al. 2018]. The two most recently published methods for correcting light dilution in UV camera im- ages are calibration by co-located DOAS [Kern et al. 2013], which were not available for the data presented here, or correction using scattering coeﬃcients, derived from background intensities of the ground in the same UV camera images [Campion et al. 2015]. The latter method has the advantage of not requiring simultane- ous DOAS measurements. However, it requires that the UV images show terrain of constant albedo at varying distances from the camera. With irregular snow cover in our images, it was not possible to ﬁnd a uniform slope on the ground from which to calculate scattering coeﬃcients. Instead, to estimate the eﬀect of light di- lution on our measurements, we compared ICAs along cross sections close to the vent from two time series col- Retrievals for integrated column amounts (ICAs) fol- low Wilkes et al. [2017] using a MATLAB script that permits a plume-free region of the images to be selected for background correction. Column densities (e.g. Fig- ure 2) are integrated along a user-deﬁned plume cross- section to obtain ICAs (in units of kgm−1). Integration lines for the cameras at distance (far camera site) were taken about 1 km from the crater, where there was less light occlusion by ash from explosions. However, the resulting ICAs and ﬂuxes should be considered lower bounds, as we could not correct directly for light dilu- tion or eliminate the eﬀect of ash. 2.1 PiCams, ﬁeld methods and analyses Figure 1: Satellite view of Ampato–Sabancaya complex showing NOVAC installations [Masías Alvarez and Apaz Choquehuayta 2018], infrasound station, UV camera sites from this study, with dashed lines showing camer ﬁelds of view at the two camera sites, arrow showing plume direction with additional solid lines indicating th extent reached by the plume from the Sabancaya summit crater during the measurement period, and dotted lines with circles showing NOVAC scan intercept with plume; inset shows the Sabancaya plume viewed from fa camera site on 27 April 2018. Figure 1: Satellite view of Ampato–Sabancaya complex showing NOVAC installations [Masías Alvarez and Apaza Choquehuayta 2018], infrasound station, UV camera sites from this study, with dashed lines showing camera ﬁelds of view at the two camera sites, arrow showing plume direction with additional solid lines indicating the extent reached by the plume from the Sabancaya summit crater during the measurement period, and dotted lines with circles showing NOVAC scan intercept with plume; inset shows the Sabancaya plume viewed from far camera site on 27 April 2018. with bandpass ﬁlters at 330 nm and 310 nm (10 nm full width at half maximum), the latter of which is an SO2 absorption band commonly used for UV retrievals. Cal- ibration, clear sky, and dark images were collected, and shutter speeds were manually selected for each cam- era. The calibration images used at least three gas-ﬁlled quartz cells with known SO2 concentrations (near site: 110, 1000, 2500, 3500, and 4600 ppm; far site: 100, 467, and 1989 ppm) and showed good linear relation- ships between known concentrations and apparent ab- Presses universitaires de rasbourg Presses universitaires de rasbourg Page 241 Ilanko et al., 2019 Degassing at Sabancaya volcano Figure 2: Example of scaled absorbance image from a UV camera; integration line marked in white. Colour bar on right shows calculated column density of SO2 (ppm·m). At this distance, with a f.o.v of 23.1°, the width of the frame is about 4.2 km. Note the ash-rich central region of the rising plume (rectangle with dashed red line). Figure 2: Example of scaled absorbance image from a UV camera; integration line marked in white. Colour bar on right shows calculated column density of SO2 (ppm·m). At this distance, with a f.o.v of 23.1°, the width of the frame is about 4.2 km. Note the ash-rich central region of the rising plume (rectangle with dashed red line). 2.1 PiCams, ﬁeld methods and analyses Multiplying the ICA by plume velocity perpendicular to the integration line yields SO2 ﬂux. Velocities used here are based on man- ual tracking of plume features by following, where pos- sible, high SO2 concentration regions or plume features as they crossed the integration line. Figure 2 shows an example of a calibrated absorbance image with the in- tegration line used for calculating ICAs. Presses universitaires de rasbourg 2.1.1 Light dilution correction The 1-degree database (GDAS1) has a spatial resolution of 1 decimal degree, with data gathered three times per hour at pres- sure increments of 50 hPa and includes corresponding height, wind direction, and windspeeds. Windspeeds during the scanning periods are derived from linear in- terpolation between two windspeeds directly above the vent at the plume height, three hours apart. UV camera images show the base of the plume at about 1 km above the ground at the furthest distance from the vent visi- ble in images (close to, but not at, the SAD3 plume in- tersect), so we consider this a reasonable estimate. Fur- ther details of the retrieval procedure can be found in the Supplementary Materials. We also note that the integration line used for this calculation was not the same as that used for ﬂux deter- mination. We needed to select a line visible from both near and far cameras and were thus restricted by the ﬁeld of view of the near camera to a distance about 600 m from the vent. However, the plume ash content and condensation of emitted water vapour are expected to give a higher optical thickness here than farther down- wind, potentially also aﬀecting scattering. Therefore, we used an integration line 1 km downwind of the vent for our ﬁnal ﬂux estimates. One sequence from cam- era B (see results), had a ﬁeld of view slightly further downwind, and thus further from the plume. The ex- act distance is unknown as there are no geographical features in these images to which the distances can be referenced. We therefore used the same light dilution factor for all sequences taken from the far camera site. We consider this an acceptable estimate since, for an integration line 3 km downwind, the distance from the far camera site to the plume is about 10.5 km. The light dilution factor does not change signiﬁcantly from that at 10.3 km and mean ﬂuxes for this sequence would in- crease by less than 0.5 kgs−1. For comparison, light di- lution for Sabancaya was modelled by Kern et al. [2017] to aﬀect their DOAS measurements by about 10 %, and Moussallam et al. [2017] estimated up to 38 % for their UV camera measurements at a distance of 4.1 km from the plume, under diﬀerent measurement conditions. 2.3 Infrasound data Over the last two decades, infrasound (atmospheric acoustic waves with frequencies <20 Hz) has emerged as a powerful and robust tool for volcano monitoring [Fee and Matoza 2013]. Among the numerous natu- ral processes that can generate acoustic waves, volca- noes are proliﬁc radiators of infrasound, in particular when large volumes of gas and ash are violently ejected into the atmosphere. The use of infrasound for volcano monitoring in the near-ﬁeld (<10 km) has become in- creasingly popular due to its unmatched temporal res- olution [Johnson 2004]. Infrasound applications in- clude detecting, locating and tracking explosive vol- canic eruptions, and recent eﬀorts towards providing estimates of eruption source parameters hold promis- ing results [De Angelis et al. 2019]. At Sabancaya, data from one permanent infrasound station (Figure 1) were available during the UV camera measurement period. The station recorded at 100Hz and 24-bit resolution using an iTem prs100 microphone [Delle Donne and Ripepe 2012]. The acoustic ﬁnger- print of major explosions (i.e. those reported by the ob- servatory) at Sabancaya, from infrasound data recorded outside of the measurement period, is characterized by relatively low excess pressures (<10 Pa) lasting an av- erage of 1–3 minutes. Typical waveforms show an ini- tial compression followed by a sustained complex coda, probably shaped by the turbulence of the plume. How- ever, no major explosions with these typical waveforms were registered during the UV camera measurements. 2.2 NOVAC data The NOVAC installation at Sabancaya [Masías Alvarez and Apaza Choquehuayta 2018] includes three DOAS instruments located around the volcano (Figure 1). Data were available for two of these and are presented here for comparison. The plume was roughly over Hornillos during these measurements, with the scan plane intersecting the plume about 3 km downwind, while the near UV camera site was at Ampato at a much greater distance of about 11 km from the plume (8 km downwind). Flux calculations used the NOVAC 2.1.1 Light dilution correction The quantitative eﬀect of radiation scattering through the atmosphere on optical plume measurements de- pends on atmospheric conditions in front of the plume Presses universitaires de rasbourg Page 242 2(2): 239 – 252. doi: 10.30909/vol.02.02.239252 Volcanica lected at near and far measurement sites. Over the 7- minute overlap between the datasets, ICAs along these cross sections are about 1.7 times higher at the near site (Figure 3). The relationship between ICAs at diﬀerent distances can be used to calculate an extinction coeﬃ- cient and estimate the eﬀect of light dilution [Bluth et al. 2007; Lübcke et al. 2013; Smekens et al. 2015a]. Fol- lowing Lübcke et al. [2013, Appendix D], this ratio and the diﬀerence in distance to the plume is used to calcu- late an extinction coeﬃcient of 0.0878 km−1. This coef- ﬁcient represents the loss in measured column amounts due to light dilution with distance. We use the same process to determine that column amounts and ﬂuxes measured at 10.3 km distance from the plume could be underestimated by a factor of 2.5. At a distance of 4.25 km from the plume, this factor is lower at 1.5, but still indicates a signiﬁcant eﬀect from light dilution. software (available at https://novac-community.org/), assuming plume heights of 1 km above the scanner and a windspeed model from the National Oceanic and Atmospheric Administration (NOAA) through the Global Data Assimilation System (GDAS). The 1-degree database (GDAS1) has a spatial resolution of 1 decimal degree, with data gathered three times per hour at pres- sure increments of 50 hPa and includes corresponding height, wind direction, and windspeeds. Windspeeds during the scanning periods are derived from linear in- terpolation between two windspeeds directly above the vent at the plume height, three hours apart. UV camera images show the base of the plume at about 1 km above the ground at the furthest distance from the vent visi- ble in images (close to, but not at, the SAD3 plume in- tersect), so we consider this a reasonable estimate. Fur- ther details of the retrieval procedure can be found in the Supplementary Materials. software (available at https://novac-community.org/), assuming plume heights of 1 km above the scanner and a windspeed model from the National Oceanic and Atmospheric Administration (NOAA) through the Global Data Assimilation System (GDAS). Presses universitaires de rasbourg these emission events. ing peak in SO2 ﬂuxes. Coincident scan ﬂuxes from Hornillos NOVAC station are very similar to those from the UV cameras, averaging 2266 td−1 with a standard deviation of ± 472 td−1 over the measurement period of about 1.5 hrs. NOVAC ﬂuxes from the scanner at Ampato for our measurement period are lower than the UV camera ﬂuxes, averaging 1394 td−1. The compari- son also shows the advantage of the UV cameras’ higher temporal resolution, which permits identiﬁcation of in- dividual gas pulses in time series data. This is largely due to the frequency of imaging compared to DOAS scan times (yielding a ﬂux measurement about every 15 minutes). The ability to select camera integration lines close to the vent also helps in distinguishing individual events before the plume mixes downwind. The time series ﬂux data from three UV camera se- quences and NOVAC scanners at Sabancaya are shown in Figure 4. Two camera sequences from cameras A and B were taken with the vent at the centre of the image, while another sequence from camera B was taken fully in the plume. As this ﬁeld of view corresponds to a lag of several minutes in the time stamps, a fourth se- quence from Camera A was used to match it to the pre- vious time series. This shows the plume, and retrieved data capture the same trends in degassing; however, this sequence was excluded from quantitative analy- ses due to features appearing in the background, likely caused by reﬂections from an object in the near-ﬁeld. The UV camera-derived ﬂuxes after corrections for light dilution (mean of 27.1 kg−1) extrapolate to a daily mean of about 2340 td−1, nearly double that of re- cent UV camera and DOAS measurements (Table 1) made before the onset of the current eruptive episode in November 2016. The decline in apparent ﬂuxes to- wards the end of the measurement period is due to clouds in the ﬁeld of view (Figure 4, grey shading), and data from 15:28:30 UTC onwards are excluded from these calculations for this reason. Ash is visible cross- ing the integration line around 14:29:00 UTC (Figure 4, grey shading), following an ash-rich pulse. This is likely to cause underestimation during the correspond- 3 Over nearly two hours of measurements, several dis- crete pulses of ash and gas were observed (see Sup- plementary Materials for an example UV camera ab- sorbance video). Passive degassing continued between Presses universitaires de rasbourg Presses universitaires de rasbourg Page 243 Ilanko et al., 2019 Degassing at Sabancaya volcano Figure 3: Light dilution is estimated using [A] the relationship between ICAs at [B] far and [C] near sites as explained in the text. Masses were calculated along an integration line that is visible in both sets of absorbance images, selected to also minimize interference from the central column of ash; [B] images at 15:07:00 UTC from Camera B at the far site and [C] from the near site camera; the start of an eruption pulse is visible in both images, where column density appears lower due to ash; note that images have diﬀerent colour scales and diﬀerent viewing angles. Figure 3: Light dilution is estimated using [A] the relationship between ICAs at [B] far and [C] near sites as explained in the text. Masses were calculated along an integration line that is visible in both sets of absorbance images, selected to also minimize interference from the central column of ash; [B] images at 15:07:00 UTC from Camera B at the far site and [C] from the near site camera; the start of an eruption pulse is visible in both images, where column density appears lower due to ash; note that images have diﬀerent colour scales and diﬀerent viewing angles. 3.1 Sources of uncertainty in UV camera and NOVAC methods Grey shaded boxes indicate times when retrievals may be aﬀected by ash and cloud, as labelled on the plot, causing underestimation of ﬂux. Vertical arrows indicate times when the ﬁrst pulse of gas from an explosion crosses the integration line for the UV cameras, corresponding to times indicated in Table 2, for the three explosions where this ﬁrst gas pulse can be isolated. Note that this occurs several minutes after the explosion gas pulse ﬁrst appears above the vent. instrument’s conical scanning plane intersected the plume at an oblique angle 11 km from the vent (Fig- ure 1). At such great distances, light will enter the ﬁeld of view between the plume and the instrument and di- lute the measured absorption signal, despite the rela- tively clear conditions in the high-elevation Peruvian desert. Kern et al. [2010] showed that dilution by pure Rayleigh scattering in the absence of any atmospheric aerosols could lead to about a 35 % reduction in mea- sured SO2 column densities at a distance of 5 km (see their Figure 4). However, this number was calculated for sea-level conditions. Given that our Sabancaya mea- surements were made at approximately half the pres- sure of sea-level, we expect a similar dilution would occur at about twice the plume distance. Therefore, the diﬀerences between our results from Ampato and those obtained by the Hornillos scanner directly beneath the plume are in qualitative agreement with this rough es- timate of light dilution. of error and uncertainty—namely, light dilution and wind velocity—that are important considerations in ob- taining reliable measurements of SO2 ﬂux by either method. The explanation for the lower ﬂuxes at Ampato sta- tion compared to that at Hornillos is twofold. Firstly, the location of Ampato station, to the south-southeast of the active vent, was unfavourable for capturing the gas plume at the time of measurement. The instru- ment’s orientation is such that the scanning plane in- tersects the plume at fairly low scan elevation angles (often >75 degrees from zenith). It is therefore likely that part of the plume was behind the visible horizon and was missed by the instrument. Based on an auto- matic plume characterization scheme incorporated in the NOVAC software, the plume completeness was de- termined to be less than 85 % for all Ampato measure- ments (Figure 4). 3.1 Sources of uncertainty in UV camera and NOVAC methods Fluxes obtained by the NOVAC scanner at Hornillos are consistent with those from the UV camera mea- surements, with the exception of short-term varia- tions that the scanner was not able to capture due to its limited data acquisition rate. Next, we out- line reasons for the mismatch between these data and the lower ﬂuxes recorded at the Ampato NOVAC sta- tion, followed by a discussion of potential sources Presses universitaires de rasbourg Page 244 2(2): 239 – 252. doi: 10.30909/vol.02.02.239252 Volcanica Figure 4: Retrieved raw and light-dilution-corrected SO2 ﬂuxes from two UV cameras at far camera site, and two NOVAC scan sites. Note that the ﬁrst two UV camera sequences are from integration lines about 1 km downwind (camera A from 14:03 to 14:41 and camera B from 14:41 to 15:08), while the last sequence (camera B from 15:11 UTC onwards) is calculated further downwind in the plume. An additional sequence, which is not shown as a variable background aﬀected retrieved ICAs, was used to match the time stamps across these sequences. Points marked with grey circles indicate scans for which the NOVAC software retrieved a plume completeness >85 %, indicating the entire plume was likely captured. Grey shaded boxes indicate times when retrievals may be aﬀected by ash and cloud, as labelled on the plot, causing underestimation of ﬂux. Vertical arrows indicate times when the ﬁrst pulse of gas from an explosion crosses the integration line for the UV cameras, corresponding to times indicated in Table 2, for the three explosions where this ﬁrst gas pulse can be isolated. Note that this occurs several minutes after the explosion gas pulse ﬁrst appears above the vent. Figure 4: Retrieved raw and light-dilution-corrected SO2 ﬂuxes from two UV cameras at far camera site, and two NOVAC scan sites. Note that the ﬁrst two UV camera sequences are from integration lines about 1 km downwind (camera A from 14:03 to 14:41 and camera B from 14:41 to 15:08), while the last sequence (camera B from 15:11 UTC onwards) is calculated further downwind in the plume. An additional sequence, which is not shown as a variable background aﬀected retrieved ICAs, was used to match the time stamps across these sequences. Points marked with grey circles indicate scans for which the NOVAC software retrieved a plume completeness >85 %, indicating the entire plume was likely captured. Presses universitaires de rasbourg 3.1 Sources of uncertainty in UV camera and NOVAC methods Although deriving ﬂuxes from the UV camera would require calibration to changing con- ditions, no calibration is required for tracking plume movement. Further experiments with co-locating UV cameras and scanning DOAS would be required to un- derstand how this would be implemented. Speciﬁcally, to obtain windspeed at the same transect as the SO2 ICAs, the camera ﬁeld of view would need to include the possible DOAS scan planes. We note that in the ex- ample presented, a direct multiplication of UV camera- derived windspeed with NOVAC scan data would result in lower ﬂuxes than those reported here—whether from the UV camera, or from NOVAC scans with modelled NOAA windspeeds—likely because of the diﬀerence in the scan transect compared with the camera transect used for column amounts and windspeed. Correcting for light dilution is still key, however, particularly at several kilometres’ distance from the plume, as demonstrated here. For UV camera images, unless the ﬁeld of view includes ground surfaces suit- able for corrections following Campion et al. [2015] or co-located DOAS data are available, corrections require images taken at diﬀerent distances. Using multiple UV cameras for simultaneous data capture has enabled us to estimate light dilution by this method as well as to track features through the plume. We note that our light dilution estimate is based on a brief overlap of 7 minutes (81 data points) but for longer time series with changing atmospheric conditions, the methods men- tioned above [Kern et al. 2013; Campion et al. 2015] would provide continuous correction factors and may be simpler than operating two cameras continuously. Inconsistent windspeed information is another po- tential source for discrepancies between our data streams. Despite the good match between UV camera and Hornillos NOVAC data, the absolute windspeeds used with NOVAC are about 1.6 times higher than the perpendicular speed component derived from manual plume tracking in the UV camera images, with a me- dian of 4.8 ms−1 compared to 2.9 ms−1, respectively (means of 4.8 and 3.1 ms−1, respectively). Such a dis- crepancy would be expected if the plume were not per- pendicular to the camera’s viewing plane. However, the scanning data suggest that the plume was gen- erally over the Hornillos station during the measure- ment period, a conﬁguration that would lead to a near- perpendicular view by the cameras. 3.1 Sources of uncertainty in UV camera and NOVAC methods Plume completeness is one of the main criteria used by observatories to assess the qual- ity of NOVAC data, and measurements with low plume completeness would normally be ﬁltered out and not reported. However, we elect to show the data here in order to discuss the limitations of the measurements. Without more detailed information on atmospheric and plume conditions at the time of the measurements, we cannot isolate these eﬀects to identify whether in- complete plume scans or light dilution was dominant in causing the discrepancy between the Ampato and Hornillos measurements, but both likely contribute to Secondly, light dilution is also expected to signif- icantly aﬀect the measurements from Ampato. The Presses universitaires de rasbourg Page 245 Degassing at Sabancaya volcano Ilanko et al., 2019 across the image sensor [Klein et al. 2017] However, this eﬀect should be very minor given our nearly perpendic- ular viewing geometry and the fact that we performed our manual tracking of the plume near the centre of the images. some degree. We also know that the measurements at Hornillos are expected to be more representative of the true emission rates due to the favourable location of the scanner directly beneath the plume, thus minimizing the impact of either of these error sources. It is there- fore reassuring that the UV camera measurements are consistent with these data after the images have been corrected for light dilution. g It is unclear whether uncertainties in windspeed af- fected our comparison in a signiﬁcant way, but this discussion highlights the importance of accurate wind- speed and wind direction information when analyzing scanning DOAS data. In situations where NOVAC scan- ners are outﬁtted with dual spectrometers, the plume is directly over the instruments and travelling in a con- sistent direction, and the telemetry link between the observatory and the NOVAC station is functional, the NOVAC instruments can determine the plume speed using a dual-beam correlation approach [Galle et al. 2010]. However, these conditions are often not met and, as is the case here, alternate approaches for obtain- ing accurate wind information are often required. Our experiment shows that simple, inexpensive UV cam- eras co-located with NOVAC scanners could in many situations provide more accurate windspeed data for analysis of SO2 emission rates than is often available from other sources. 3.1 Sources of uncertainty in UV camera and NOVAC methods Other possible explanations for this discrepancy in windspeed are (1) that the windspeed given by the GDAS model is too high and/or (2) that the plume changes speed and/or direction on its path between the summit vent and the NOVAC station. It is im- portant to note that the 1-degree grid of the GDAS is too coarse to account for atmospheric dynamics caused by topographic features such as the ediﬁce of the Am- pato/Sabancaya volcanic complex. Such features might cause signiﬁcant disruptions to the large-scale wind ﬁeld, potentially aﬀecting both speed and direction on local to regional scales. Given the agreement be- tween the ﬂuxes calculated for both instruments, it is possible in this example that the scale of the GDAS-1 model data was appropriate for the measurement pro- ﬁle of the NOVAC scan and to capture changes in wind- speed or plume spreading at the scan location. Finally, second-order errors can be induced by variations in the camera’s viewing angle relative to the plume direction 3.2 Eruptive activity Such signals have been often linked to degassing pulses in open vent systems [e.g. Lees and Ruiz 2008]. It appears that the initial increase in SO2 ﬂux at 14:13 UTC is at least partially associated with an event that occurred before the start of the measurement sequence, as it takes 10–20 minutes for gas emissions to cross the UV camera integration line. Ash and a vertical SO2 column are visible at the start of our image sequence that support this increase in SO2 being caused by a discrete pulse. However, background ﬂux remains elevated and appears to increase throughout the sequence following this, due either to the frequency of ash and gas pulses or to sustained passive degassing. Both this background increase and the high ﬂuxes in the subsequent gas pulses (the ﬁrst of which is detected at 14:26) may be linked to the chugging signal. y g g g The activity at Sabancaya during the measurement period is characterised by sustained gas emissions with additional discrete pulses of gas and ash. The in- frasound data show a lack of overpressure. Juvenile ash sampled earlier in the eruption, during 2017, was andesitic [Manrique et al. 2018], and previous erup- tions have been andesitic-to-rhyolitic [Samaniego et al. 2016]. Ballistics were reported in 2017; activity at the time was described as vulcanian, with a transition in ash composition after vent-clearing wherein the pro- portion of lithics decreased while that of juvenile mate- rial increased [Manrique et al. 2018]. INGEMMET re- ports that seismic activity during the week of 23 April was dominated by long period earthquakes and tremor, with few hybrid earthquakes [OVI-INGEMMET & IGP 2018]. As parts of the plume move at diﬀerent speeds, gas from a single event cannot be cleanly isolated, but tracking of SO2-rich pulses as they move horizontally in UV camera absorbance videos, together with peaks in the calculated SO2 ﬂux, allows us to estimate gas mass from some discrete events (see Supplementary Materials for calculations). Increases in ﬂux are ob- served following each emission event and, while it was necessary to exclude the later data due to clouds cross- ing the integration lines from around 15:18:05 UTC, some of our data capture the contrast between back- ground degassing and the SO2 ﬂux associated with an event (Table 2). 3.2 Eruptive activity Visual observations suggest that ash and gas emission events during the measurement period are explosions; however, infrasound data during the observation pe- riod do not show clear discrete explosions above the level of noise, and we note that noise levels are partic- ularly high from 15:18:00 UTC, coinciding with chang- ing weather conditions. In the images, pulses of ash appear frequently (up to 2–3 times per minute) above the top of the summit, and in calculating gas masses for these pulses, we assume that the combined ash and gas emissions that produce discrete pulses in the vertical column are single events. One instance of acoustic chugging occurs at 14:14:55–14:15:10 UTC, preceding a large increase in SO2 degassing (Figure 5). Chugging is a sequence of simple acoustic (and sometimes seismic) pulses Presses universitaires de rasbourg Page 246 2(2): 239 – 252. doi: 10.30909/vol.02.02.239252 Volcanica Table 2 – SO2 masses for gas pulses. Time of appearance above vent, time that the leading edge and back of the gas pulse cross the integration line, and gas masses adjusted for estimated light dilution are also listed. Note that the SO2 masses for the three pulses from 14:14:04 UTC are a mean, as degassing is sustained over this period and pulses could not be separated. Masses are rounded to 3 s.f; see Supplementary Materials for details of calculations. Table 2 – SO2 masses for gas pulses. Time of appearance above vent, time that the leading edge and back of the gas pulse cross the integration line, and gas masses adjusted for estimated light dilution are also listed. Note that the SO2 masses for the three pulses from 14:14:04 UTC are a mean, as degassing is sustained over this period and pulses could not be separated. Masses are rounded to 3 s.f; see Supplementary Materials for details of calculations. Time of emitted pulse (UTC) Instrument Pulse leading edge (UTC) Pulse back (UTC) SO2 mass (kg) Adjusted SO2 mass (kg) ? Camera A 14:13:36 14:26:20 3340 8240 14:14:04 Camera A 14:26:24 14:42:36 1390 3430 14:19:56 (ﬁrst pulse) (last pulse) 1390 3430 14:25:44 1390 3430 14:33:56 Camera B 14:55:40 15:08:08 1200 2950 about 14:26 to 14:52 UTC, aﬀecting the three pulses ob- served over this period and that the reported masses are not fully corrected for increased background degassing. recorded at regular and short intervals. 3.2 Eruptive activity Emitted masses were calculated by summing the ﬂuxes (in kilograms per second) over the time taken for each gas pulse to cross the integration line. For each measurement, the lowest ﬂux before or after the pulse was subtracted, as it was assumed to represent background degassing. For ﬁve events, the masses corrected for light dilution range from about 2950 kg to 8240 kg. Given the potential for under- estimation of emissions when imaging optically thick plumes [Kern et al. 2013] and the fact that there is ash clearly visible crossing the integration line, the actual masses may be higher than these estimates. It is also possible that chugging occurring early in the sequence may have caused an elevated background SO2 ﬂux from Sustained SO2 degassing during the measurement period suggests an open vent, with gas sourced from within the conduit. The lack of ballistics, which could indicate fragmentation of a sealed conduit, also sup- ports an open conduit. Erupted magma appears to be solely in the form of ash, which could be juvenile or remobilised from earlier explosions. The pulses of in- creased gas and ash emission indicates a ﬂuctuating gas supply. The SO2 ﬂuxes reported during similar erup- tive episodes at other volcanoes typically focus on ex- plosions, which we did not capture in our measure- ments at Sabancaya. Nonetheless, the observed activity merits comparison to other volcanoes due to the high SO2 ﬂuxes and to the fact that it occurs between, and may be related to the processes that cause, frequent ex- plosions. The SO2 masses we measure during the pulses at Sabancaya are signiﬁcantly higher (2.95–8.24 t) than those reported from explosions with some similar char- acteristics at Karymsky volcano [1.05–1.53 t; Fischer et al. 2002] Santa María volcano (Santiaguito dome com- plex) [0.37–1.27 t; Holland et al. 2011] or Gunung Se- meru [0.2–1.46 t; Smekens et al. 2015b]. At these volca- noes, explosive events (which are also characterised by Presses universitaires de rasbourg Page 247 Ilanko et al., 2019 Degassing at Sabancaya volcano Degassing at Sabancaya volcano Ilanko et al., 2019 Figure 5: Acoustic infrasound data recorded on 27 April 2018 during UV camera observations (SO2 ﬂux in top panel, infrasound in second panel from top); no explosion signals are observed above the level of noise. Author affiliations α Department of Geography, University of Sheﬃeld, UK. α Department of Geography, University of Sheﬃeld, UK. α Department of Geography, University of Sheﬃeld, UK. Windspeeds generated by the NOAA GDAS1 model and used with the NOVAC data were, on average, 1.6 times higher than those manually estimated from tracking plume motion on camera images. Since the camera direction is approximately perpendicular to the plume, this discrepancy suggests that the model may have overestimated windspeeds above the NOVAC scanners. This is concerning, given that errors in the windspeed linearly aﬀect retrieved emission rates. The 1-degree grid and the low temporal resolution may not have captured local and short-term variations in wind- speed. Even if our measurements at Sabancaya were not severely aﬀected, obtaining accurate wind speeds is challenging with DOAS scanners alone, and requires several speciﬁc conditions. Instead, co-location of UV cameras with permanent DOAS scanners–ideally, view- ing perpendicular to the scan plane–could prove to be the best approach to obtaining independent local plume speed estimates. β Instituto Geológico Minero y Metalúrgico, Lima, Perú. β Instituto Geológico Minero y Metalúrgico, Lima, Perú. γ Cascades Volcano Observatory, U.S. Geological Survey, Vancouver, WA, USA. γ Cascades Volcano Observatory, U.S. Geological Survey, Vancouver, WA, USA. δ Department of Earth, Ocean & Ecological Sciences, Uni- versity of Liverpool, UK. ε Ckelar Volcanes – Núcleo de Investigación en Riesgo Vol- cánico, Universidad Católica del Norte, Antofagasta, Chile. f g ζ Programa de Doctorado en Ciencias mención Geología, Universidad Católica del Norte, Antofagasta, Chile. ζ Programa de Doctorado en Ciencias mención Geología, Universidad Católica del Norte, Antofagasta, Chile. f g η Departamento de Ciencias Geológicas, Universidad Católica del Norte, Antofagasta, Chile. η Departamento de Ciencias Geológicas, Universidad Católica del Norte, Antofagasta, Chile. ϑ Instituto Geofísico de la Escuela Politécnica Nacional, Quito, Ecuador. ϑ Instituto Geofísico de la Escuela Politécnica Nacional, Quito, Ecuador. ι School of Geosciences, The University of Sydney, Sydney, NSW 2006, Australia. ι School of Geosciences, The University of Sydney, Sydney, NSW 2006, Australia. κ Faculty of Health, Engineering and Sciences, University of Southern Queensland, Toowoomba, QLD 4350, Aus- tralia. κ Faculty of Health, Engineering and Sciences, University of Southern Queensland, Toowoomba, QLD 4350, Aus- tralia. Light dilution remains a challenge that both tech- niques must overcome to obtain accurate results. Acknowledgements TI is a Commonwealth Rutherford Fellow, supported by the UK government. AJSMcG acknowledges the support of Rolex, and TDP a Royal Society Research Grant (RG170226). ADM is funded by NERC Grant NE/P00105X/1. FA acknowledges the support of the PCI-CONICYT project REDES170174. FA and FR acknowledge the support of Proyectos Especiales 2017 Vicerrectoría de Investigación y Desarrollo Tec- nológico de la Universidad Católica del Norte “Proce- sos que controlan el balance de masa en volcanes ac- tivos del norte de Chile”. SL is funded by a CONICYT- PCHA/Doctorado Nacional/2016- 21160276 scholar- ship. 4 Conclusions Our comparison of UV camera and NOVAC DOAS data highlight the advantages and limitations of the two techniques. While the NOVAC scanners operate con- tinuously without need for user interaction, typically measure the plume further downwind where ash is less of a concern, and provide full spectral information, the UV cameras provide higher time resolution and im- ages from which plume speed can be calculated di- rectly. This allowed us to identify SO2 emissions as- sociated with individual gas pulses at Sabancaya that could not be detected in the DOAS data. The USAID Oﬃce of Foreign Disaster Assistance is gratefully acknowledged for its support of the 2018 NOVAC workshop. We thank Patricia Nadeau and Si- mon Carn for their insightful reviews. Author affiliations For the UV camera data collected in this study, light dilu- tion could be estimated due to the availability of simul- taneous imagery at diﬀerent distances, with scope for error due to the angle of the plume and variations over 3.2 Eruptive activity However, we are limited in interpreting the eruption mechanisms at Sa- bancaya by a lack of information about the magma rhe- ology and our relatively short dataset, which does not characterise the full range of activity at Sabancaya dur- ing the current eruptive episode. Speciﬁcally, our gas data do not capture any of the explosions detected in the seismic data, and this transition could be impor- tant to understanding the eruption mechanisms. The high SO2 masses associated with the observed pulses may relate to conduit processes following previous ex- plosions. For example, chugging at Karymsky has been proposed to result from depressurisation in the conduit and continued degassing after an explosion [Johnson et al. 1998]. We also cannot rule out the possibility that multiple conduits and vents are present [e.g. Nadeau et al. 2011], such that active and passive degassing occur simultaneously through separate pathways. tures, leading to a larger explosion. However, we are limited in interpreting the eruption mechanisms at Sa- bancaya by a lack of information about the magma rhe- ology and our relatively short dataset, which does not characterise the full range of activity at Sabancaya dur- ing the current eruptive episode. Speciﬁcally, our gas data do not capture any of the explosions detected in the seismic data, and this transition could be impor- tant to understanding the eruption mechanisms. The high SO2 masses associated with the observed pulses may relate to conduit processes following previous ex- plosions. For example, chugging at Karymsky has been proposed to result from depressurisation in the conduit and continued degassing after an explosion [Johnson et al. 1998]. We also cannot rule out the possibility that multiple conduits and vents are present [e.g. Nadeau et al. 2011], such that active and passive degassing occur simultaneously through separate pathways. time. However, locating cameras at diﬀerent distances may not always be eﬃcient, and further approaches to dealing with dilution eﬀects, or at least ﬁltering poor data, should be explored. Finally, our data show a SO2 emission rate of about 27 kgs−1 at Sabancaya during the measurement period, with sustained SO2 degassing as well as discrete pulses with higher SO2 masses and ash. 3.2 Eruptive activity Expan- sion shows infrasound waveforms (third panel from top) and spectrogram (bottom panel) of a chugging sequence potentially associated with gas puﬃng or the occurrence of small bubble explosions (colour scale indicates rela- tive power/frequency). Chugging coincides with sustained increases in SO2 ﬂux after the ﬁrst gas pulse detected in UV camera data at 14:14 UTC. Figure 5: Acoustic infrasound data recorded on 27 April 2018 during UV camera observations (SO2 ﬂux in top panel, infrasound in second panel from top); no explosion signals are observed above the level of noise. Expan- sion shows infrasound waveforms (third panel from top) and spectrogram (bottom panel) of a chugging sequence potentially associated with gas puﬃng or the occurrence of small bubble explosions (colour scale indicates rela- tive power/frequency). Chugging coincides with sustained increases in SO2 ﬂux after the ﬁrst gas pulse detected in UV camera data at 14:14 UTC. multiple pulses of ash visible in the absorbance images, has some similarity to this description. The mecha- nism for explosions with multiple pulses proposed by Smekens et al. [2015b] is higher magma supply rate compared to single explosions. Campion et al. [2018] measured SO2 masses of 1.3–6.9 t from explosions at Popocatéptl. They propose that ‘gas slug’ rise and co- alescence within a permeable fracture network, kept open by high passive degassing ﬂux, could account for this type of behaviour. This implies that a decrease in degassing could cause sealing of the conduit frac- discrete pulses of gas) are attributed to viscous plug for- mation followed by overpressure from buildup of gas and magma causing rupture [Fischer et al. 2002; Hol- land et al. 2011; Smekens et al. 2015b]. Smekens et al. [2015b] identify two clusters of higher and lower SO2 masses in explosions at Semeru, and link the higher masses to longer duration explosive events lasting about 15 minutes with multiple pulses and sustained increases in degassing. While pulses are not clearly vis- ible in SO2 ﬂuxes measured 1 km down-vent at Saban- caya, the duration and magnitude of the events, with Presses universitaires de rasbourg Page 248 2(2): 239 – 252. doi: 10.30909/vol.02.02.239252 Volcanica tures, leading to a larger explosion. Presses universitaires de rasbourg References Bluth, G., J. Shannon, I. Watson, A. Prata, and V. Real- muto (2007). “Development of an ultra-violet digi- tal camera for volcanic SO2 imaging”. Journal of Vol- canology and Geothermal Research 161.1-2, pp. 47–56. doi: 10.1016/j.jvolgeores.2006.11.004. Galle, B., C. Oppenheimer, A. Geyer, A. J. McGonigle, M. Edmonds, and L. Horrocks (2003). “A minia- turised ultraviolet spectrometer for remote sensing of SO2 ﬂuxes: a new tool for volcano surveillance”. Journal of Volcanology and Geothermal Research 119.1- 4, pp. 241–254. doi: 10 . 1016 / s0377 - 0273(02 ) 00356-6. Burton, M., G. Salerno, L. D’Auria, T. Caltabiano, F. Murè, and R. Maugeri (2015). “SO 2 ﬂux monitor- ing at Stromboli with the new permanent INGV SO 2 camera system: A comparison with the FLAME net- work and seismological data”. Journal of Volcanology and Geothermal Research 300, pp. 95–102. doi: 10. 1016/j.jvolgeores.2015.02.006. Gliß, J., K. Stebel, A. Kylling, and A. Sudbø (2018). “Improved optical ﬂow velocity analysis in SO2 cam- era images of volcanic plumes – implications for emission-rate retrievals investigated at Mt Etna, Italy and Guallatiri, Chile”. Atmospheric Measurement Techniques 11.2, pp. 781–801. doi: 10.5194/amt- 11-781-2018. Campion, R., H. Delgado-Granados, D. Legrand, N. Taquet, T. Boulesteix, S. Pedraza-Espitıa, and T. Lecocq (2018). “Breathing and Coughing: The Ex- traordinarily High Degassing of Popocatépetl Vol- cano Investigated With an SO2 Camera”. Frontiers in Earth Science 6. doi: 10.3389/feart.2018.00163. Campion, R., H. Delgado-Granados, and T. Mori (2015). “Image-based correction of the light dilution eﬀect for SO 2 camera measurements”. Journal of Volcanol- ogy and Geothermal Research 300, pp. 48–57. doi: 10. 1016/j.jvolgeores.2015.01.004. Holland, A. P., I. M. Watson, J. C. Phillips, L. Caric- chi, and M. P. Dalton (2011). “Degassing processes during lava dome growth: Insights from Santiaguito lava dome, Guatemala”. Journal of Volcanology and Geothermal Research 202.1-2, pp. 153–166. doi: 10. 1016/j.jvolgeores.2011.02.004. Carn, S. A., V. E. Fioletov, C. A. McLinden, C. Li, and N. A. Krotkov (2017). “A decade of global volcanic SO2 emissions measured from space”. Scientiﬁc Re- ports 7.1. doi: 10.1038/srep44095. Johnson, J. B. (2004). “Volcanic eruptions observed with infrasound”. Geophysical Research Letters 31.14. doi: 10.1029/2004gl020020. D’Aleo, R., M. Bitetto, D. D. Donne, G. Tamburello, A. Battaglia, M. Coltelli, D. Patanè, M. Prestiﬁlippo, M. Sciotto, and A. Aiuppa (2016). “Spatially resolved SO2 ﬂux emissions from Mt Etna”. Geophysical Re- search Letters 43.14, pp. 7511–7519. doi: 10.1002/ 2016gl069938. Johnson, J. B., J. M. Lees, and E. I. Gordeev (1998). Author contributions UV camera images were collected by TP, TW, FAC, SL, FR, FA, and FV, and processed by TI using code writ- ten by TW. Infrasound data were provided by ADM Presses universitaires de rasbourg Page 249 Ilanko et al., 2019 Degassing at Sabancaya volcano De Angelis, S., A. Diaz-Moreno, and L. Zuccarello (2019). “Recent Developments and Applications of Acoustic Infrasound to Monitor Volcanic Emissions”. Remote Sensing 11.11, p. 1302. doi: 10 . 3390 / rs11111302. and SDA. NOVAC data were provided and processed by FAC and CK. The manuscript was written by TI, CK, TP, and AJSMcG. and SDA. NOVAC data were provided and processed by FAC and CK. The manuscript was written by TI, CK, TP, and AJSMcG. Copyright notice © The Author(s) 2019. This article is distributed un- der the terms of the Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, pro- vided you give appropriate credit to the original au- thor(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Fischer, T. P., K. Roggensack, and P. R. Kyle (2002). “Open and almost shut case for explosive eruptions: Vent processes determined by SO2 emission rates at Karymsky volcano, Kamchatka”. Geology 30.12, p. 1059. doi: 10.1130/0091-7613(2002)030<1059: oaascf>2.0.co;2. Galle, B., M. Johansson, C. Rivera, Y. Zhang, M. Kihlman, C. Kern, T. Lehmann, U. Platt, S. Arel- lano, and S. Hidalgo (2010). “Network for Obser- vation of Volcanic and Atmospheric Change (NO- VAC)—A global network for volcanic gas monitoring: Network layout and instrument description”. Jour- nal of Geophysical Research 115.D5. doi: 10.1029/ 2009jd011823. Data availability Delle Donne, D. and M. Ripepe (2012). “High-frame rate thermal imagery of Strombolian explosions: Im- plications for explosive and infrasonic source dy- namics”. Journal of Geophysical Research: Solid Earth 117.B9. doi: 10.1029/2011jb008987. 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https://openalex.org/W218282353	https://escholarship.org/content/qt8zv541j1/qt8zv541j1.pdf?t=mrvja5	English	null	Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp.	Transmodernity	2,013	cc-by	2,820	Peer reviewed Peer reviewed https://escholarship.org/uc/item/8zv541j1 Copyright Information Copyright 2013 by the author(s). All rights reserved unless otherwise indicated. Contact the author(s) for any necessary permissions. Learn more at https://escholarship.org/terms Peer reviewed Title Title Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. UC Merced TRANSMODERNITY: Journal of Peripheral Cultural Production of the Luso-Hispanic World Title Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. Permalink https://escholarship.org/uc/item/8zv541j1 Journal TRANSMODERNITY: Journal of Peripheral Cultural Production of the Luso-Hispanic World, 2(2) ISSN 2154-1353 Author Lifshey, Adam Publication Date 2013 DOI 10.5070/T422018505 Copyright Information Copyright 2013 by the author(s). All rights reserved unless otherwise indicated. Contact the author(s) for any necessary permissions. Learn more at https://escholarship.org/terms Peer reviewed UC Merced TRANSMODERNITY: Journal of Peripheral Cultural Production of the Luso-Hispanic World Title Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. Permalink https://escholarship.org/uc/item/8zv541j1 Journal TRANSMODERNITY: Journal of Peripheral Cultural Production of the Luso-Hispanic World, 2(2) ISSN 2154-1353 Author Lifshey, Adam Publication Date 2013 DOI 10.5070/T422018505 Copyright Information Copyright 2013 by the author(s). All rights reserved unless otherwise indicated. Contact the author(s) for any necessary permissions. Learn more at https://escholarship.org/terms Peer reviewed UC Merced TRANSMODERNITY: Journal of Peripheral Cultural Production of the Luso-Hispanic World Title Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. Permalink https://escholarship.org/uc/item/8zv541j1 Journal TRANSMODERNITY: Journal of Peripheral Cultural Production of the Luso-Hispanic World, 2(2) ISSN 2154-1353 Author Lifshey, Adam Publication Date 2013 DOI 10.5070/T422018505 Copyright Information Copyright 2013 by the author(s). All rights reserved unless otherwise indicated. Contact the author(s) for any necessary permissions. Learn more at https://escholarship.org/terms Peer reviewed Powered by the California Digital Library University of California eScholarship.org eScholarship.org Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. Rizo, Elisa, ed. Caminos y veredas: narrativas de Guinea Ecuatorial. Mexico City: UNAM, 2011. Print. 164 pp. ADAM LIFSHEY GEORGETOWN UNIVERSITY Among the more notable aspects of the minimal attention given to African literature in Spanish is the dearth of approaches marked by Latin American referents. The publication in Mexico of Caminos y veredas: narrativas de Guinea Ecuatorial, the first anthology of hispanophone West African fiction to see print in the Americas, offers as a result an opportunity to alter the basic parameters of academic and popular reception of such texts. That reception, in global terms, is scarce to begin with: few are the literary scholars or general readers who know that Equatoguinean fiction exists. Those who do, however, including the novelists and short story writers themselves, historically have tended to place it in an interior monologue concerned with national or ethnic identity, or in dialogue, implicitly or explicitly, with the cultural and sociopolitical contexts of Spain. That is where the authors often live and work; that is the nation whose literary legacy is repeatedly brought up as relevant. The gaze tends to be northerly or southerly, or some combination thereof, depending on whether a writer, academic or creative, decides to cast it from an ideated Equatorial Guinea or an ideated Spain. The time to transgress that template is now. The transatlantic and Latin American juxtapositions that may be prompted by the appearance of Caminos y veredas could start in any direction. Among the most obvious points of departure might be the contexts of dictatorship, a phenomenon all too resonant with the dead of Latin America and the literatures produced in their wakes. Since independence in 1968, Equatorial Guinea has been run by a hyperbolically tyrannical man and, since a coup in 1979, by his standard-issue tyrannical nephew. For people familiar with major Latin American literary output on such realities, it is impossible to read a text like the one that initiates Caminos y veredas, the opening fragment of a novel by Donato Ndongo, without thinking of La fiesta del chivo by Mario Vargas Llosa or El otoño del patriarca by Gabriel García Márquez. The excerpt by Ndongo begins with the sudden death, during attempted sex with a teenage wife, of a long-ruling, panoptic and murderous despot from whose cadaver 130 A. Lifshey. ADAM LIFSHEY GEORGETOWN UNIVERSITY Transmodernity (Spring 2013) “sobresalía altanero el órgano erecto que taladraba el aire como un desafío a la nada, compendio de su poder, fundamento de su autoridad, símbolo inequívoco de su portentosa virilidad, vestigio de su enérgica dominación secular sobre cualquier ser viviente” (34). An omniscient narrator then takes the reader around the minds of the conniving relatives of the dictator, who are standing in the room, staring at the man who had utterly dominated their lives, each of them weighing their odds at taking his place or surviving the fallout of his fatality. The fact of dictatorship, however, should not condition automatically the reception of Equatoguinean texts any more than should the ethnological interest that likely will cause some readers to pick up Caminos y veredas in the first place. It would be unfortunate if the anthology were read primarily by armchair anthropologists interested in assimilating, at a safe distance, the seemingly exotic: an unfamiliar country with unknown peoples who have produced, seemingly unexpectedly, literature in a language unassociated with Africa. Of course, there is nothing exotic about tyranny and certainly nothing exotic about writers of different ethnic backgrounds with diverse oral traditions emerging from artificially amalgamated African countries to produce fiction in erstwhile European languages. Yet it seems probable that the reason why this book will land in some libraries and reach some readership is because of the novelty, even at this late date of colonial history, of literature in Spanish from Africa. That is the trap that scholars all too frequently set for themselves in this microfield: academics produce prose with the aim of identifying and contextualizing obscure authors in the most traditional of ways for an audience that, though barely in existence, is assumed to expect pretty much the same. A bit of biography here, a bit of semiprofessional sociology there, a consensus valuation of costumbrismo all around, and everyone can go home ensconced in more or less the same place as before. This is too little to ask. The great value of a text such as Caminos y veredas is how it initiates the publication of collected Equatoguinean fiction in the Americas with just enough introduction so as to situate a reader desiring of such situation and just enough hints at the larger issues at stake, and then lets the texts speak for themselves and with each other. ADAM LIFSHEY GEORGETOWN UNIVERSITY Elisa Rizo, the editor of the anthology, though a fine scholar in her own right and one of the few to think beyond what usually passes for analysis of hispanophone African literature, limits her own voice here to a brief prologue divided into five sections. The first section suggests the idea, radical even now in Equatoguinean literary criticism, that the aim of reading these 131 A. Lifshey. Transmodernity (Spring 2013) texts is not to reach “cualquier noción esencialista sobre lo auténtico” (13-14). The remaining four sections hone in on the contents of the volume at hand. Rizo provides an overview of the history of the country, followed by outlines of its tradition in the novel and the short story, and then a sketch of the particular pieces in Caminos y veredas. At this point, Rizo withdraws from the stage, save for inserting very concise summaries of the backgrounds and titles of the authors as their fictions come up in turn. She has chosen, judiciously, to not turn the anthology into an academic text complete with footnotes and further glosses. This allows the book to appeal to a general readership that might be willing to enjoy a collection of fiction for its own sake. Expanding the audience for African literature in Spanish is a laudatory goal, since the first order of business for Equatoguinean writers, unlike, for example, their Mexican counterparts, is often to prove that they and their country exist. The lack of additional academic framing devices in the anthology also effects the salutary aim of eradicating the hierarchy implicitly established between footnoter and footnoted. Such discrepancies in power are a persistent issue in academic treatments of Equatoguinean literature, which are usually produced by scholars in comfortable institutions of the West about cultural artifacts created by individuals in conditions that range among exile and dictatorship and poverty. From an aesthetic standpoint of readability alone, the absence of persistent fourth wall penetration by the editor is most welcome. Caminos y veredas is bookended with pieces by two prolific Equatoguinean writers who, with the possible exception of María Nsue Angüe and her novel Ekomo, have attracted in general the most attention in academic circles. These authors are Ndongo, with his novel fragment, and Juan Tomás Ávila Laurel, with two short stories. ADAM LIFSHEY GEORGETOWN UNIVERSITY The texts of three other writers appear in between, including short stories by Justo Bolekia Boleka, José Fernando Siale Djangany and Recaredo Silebo Boturu. The fluid and entertaining texts by Siale, a relatively overlooked author in terms of scholarship produced around his work, are the most pleasant surprise of the anthology. They suggest an author with an eclectic range of narrative skills whose fictions bear several re-readings well. The opening text by Ndongo is the commencement of a novel, Los hijos de la tribu, that when published will be the final installment of a trilogy that began with Las tinieblas de tu memoria negra in 1987. The fragment, entitled “Cero,” is a polished piece by an author apparently well-versed in literary predecessors who have written on similar themes. The urge 132 A. Lifshey. Transmodernity (Spring 2013) among many readers familiar with Equatorial Guinea and with Ndongo in particular–he has been the most prominent intellectual in hispanophone African literature for decades now– will be to interpret the text as an undisguised indictment of national politics, perhaps even a roman à clef. A far more interesting move, however, would be to read it against the Latin American representations of dictatorship with which it so obviously shares stylistic and topical commonalities. An ethical question that might be considered as well is whether the fragment gains its rhythm and readability from the dramatic tension of which of the profiled characters will seize the power of the dead autocrat rather than from the development of a multilayered assessment of why such tyranny emerges and endures. “Cero,” in other words, reads as a mystery thriller at least as much as a depiction of despotism. The second author in the anthology, Justo Bolekia Boleka, presents a contrast in his two stories to the smooth craftings of Ndongo. Bolekia has many noteworthy academic publications to his credit but his fiction here appears forced. Both his texts open with a first person narrator intent on offering some truism or expounding to the reader, often via stilted parenthetical commentary, on something he just voiced. ADAM LIFSHEY GEORGETOWN UNIVERSITY The flavor of the prose is exemplified by the metatextual moment near the start of “Mi sobrino consorte Anfiloquio,” the second text, when the narrator explains, “Me encontraba sentado en mi despacho de profesor universitario, todo un privilegio para un inmigrante ya integrado, asimilado, vinculado, doblemente deculturado, y también condenado a vivir para siempre pensando, comiendo, viviendo y soñando como cualquiera de las personas de su tierra de acogida” (82). Though efficiency is hardly a required or even desirable characteristic of good fiction (whatever that is), subsequent passages such as the following do not seem to gain anything by their verbosity: “mi sobrina Pitusa-Claudia, hija, como ya dije antes, de mi primo- hermano Vicky, regidor que fue – como ya tengo dicho – de la antigua Clarence City, hoy Malabo, y una vez Santa Isabel” (83). The better story, “Los mensajeros de Moka,” does have a strong suit: its powerful depiction of the gruesome body count compiled by a venomous regime and its henchmen. The three narratives by Siale really anchor the anthology in its middle. The first and most substantive story, “La visitante de la bahía,” is according to Rizo inspired by a local narrative about a sea goddess (26). Rizo describes Siale as possessing “an ojo crítico y lúdico” and this seems accurate at various levels (26). When “La visitante de la bahía” opens, the story appears to revolve around a young couple. The sea goddess only slips into the 133 A. Lifshey. Transmodernity (Spring 2013) narrative in mid-paragraph a few pages later, an entry so subtle that a first-time reader might easily take her as just a colorful background character in a micro-parable about development. She then disappears in the text, seemingly never to return, as the focus swings back to the young couple. The surprise ending allows for an entirely new read of the story, almost compelling thereby a return to the opening page to understand the tale in a new light. This narrative playfulness is generally absent in the equally skillful but more straightforward text by the much more famous Ndongo that begins the anthology. The reception of “Cero” seems unlikely to change between a first reading and a second because the possibilities of relatively discrete interpretive frameworks appears less ample. ADAM LIFSHEY GEORGETOWN UNIVERSITY “La visitante de la bahía,” in contrast, can be read as an attractive legend in its own right, as an adept reimagination of an oral tradition, and/or as a critique of changing economic and sociopolitical orders in Equatorial Guinea. The only weak moments are its somewhat cumbersome attempts at erotica. “La visitante de la bahía” is followed by two other, much shorter texts by Siale that feature the entirely different tones of satire and faux academicism. These brief narratives, “El mandato” and “El Negruzco,” read as perhaps partially inspired by Borges, though without the abstractions: their subjects stay rooted in African contexts. The fourth writer featured in the anthology, Boturu, offers in “La danza de la abuela” a folk tale complete with a concluding moral. The story is nested by a grandmother who narrates it to her grandchildren. Of all the texts in Caminos y veredas, this one has the most circumscribed ambitions as a literary endeavor. Perhaps it is meant to be read as a plea for fraternity across a larger fractured society, but otherwise it does not seem particularly compelling. The narratorial tone is uneven and the storytelling is somewhat wooden. The final author, Ávila Laurel, offers two pieces that demonstrate his dexterity in assuming unusual perspectives. “Mi boda mozambicana” is a quasi-journalistic sketch voiced by a dryly amused first-person narrator who wanders away from an official summit of international cultural cooperation and into a church where a wedding is being held. The narrator decides to pass as a wedding guest and, in a mix of observational playfulness and intellect, comments the goings-on to the reader. In a rather deep emotional contrast, “De cuando Cecilia era nadadora” is a viscerally compelling short story narrated by a recently conceived fetus. As the narrator grows in the womb, her perspective on matters such as intercourse, masturbation and abortion are powerfully conveyed in tones of wit as well as pathos. Together, the texts by Ávila Laurel have the virtue of disassembling the expectations 134 A. Lifshey. Transmodernity (Spring 2013) of readers who would approach the anthology with anticipations of stories featuring Equatoguinean dictatorship and ethnography. Caminos y veredas ends well with these texts because their diversity in form and function suggests an open-endedness to the types of narratives that might emerge from Equatorial Guinea. ADAM LIFSHEY GEORGETOWN UNIVERSITY If the polish of Ndongo in “Cero” indicates that African literature in Spanish can match in achievement that of canonical Latin American literature on the same theme, then the adroitness of Ávila Laurel reveals that Equatoguinean literature can surface on canvases as varied as human life itself. The lack of a valedictory comment in the anthology by the editor or someone else is a wise move, for that absence thereby proposes that the African voices in consideration do not need a final word administered by academics from beyond. Introducing unfamiliar voices in the Americas, which is what the publication in Mexico of Caminos y veredas accomplishes, is a move toward opening space; so too is leaving to readers whatever thoughts they may carry away. Perhaps some people might take “De cuando Cecilia era nadadora” as an opportunity to align Ávila Laurel aside Carlos Fuentes and the fetal narrator of Cristóbal Nonato. Perhaps others would juxtapose “Mi boda mozambicana” to assorted crónicas of Latin America. Whatever the readings that come out of Caminos y veredas, the result, hopefully, will be to allow African fiction in Spanish to circulate with the same possibilities of reach and influence as short stories and novels from, say, Mexico itself.
https://openalex.org/W2782441151	https://www.nepjol.info/index.php/OJN/article/download/18894/15428	English	null	Distribution of Malocclusion Traits among Orthodontic Patients in a Tertiary Care Center of Western Nepal	Orthodontic journal of Nepal/Orthodontic Journal of Nepal	2,017	cc-by	2,279	Research Article Research Article ABSTRACT Objectives: To determine the pattern of distribution of dental malocclusion in a sample of Nepalese orthodontic patients in Western Nepal. Materials & Method: A sample consisted of 200 study casts of patient who visited Department of Orthodontics, UCMS, Bhairahawa who had never undergone orthodontic treatment previously. The total sample size was divided into male and female groups, age was divided into 3 groups: 8-11 years, 12-17 years, and 18-36 years. All data was recorded and analyzed with SPSS software version 22. Result: Among the total 200 casts examined; the distribution of malocclusion according to Angle’s classification was: Class I malocclusion in 101(50.5%), Class II malocclusion in 90(45%) sample and Class III in 9(4.5%) samples. Conclusion: Angle’s Class I malocclusion was the most prevalent malocclusion among orthodontic patients visiting UCMS College of Dental Surgery. Conclusion: Angle’s Class I malocclusion was the most prevalent malocclusion among orthodontic patients visiting UCMS College of Dental Surgery. Keywords: Angle’s Class I, Angle’s Class II, Angle’s Class III, malocclusion, prevalence INTRODUCTION when evaluating and advising potential patient regarding the desirability of the treatment. Also epidemiological data is essential in assessing the resource required for orthodontic services and can provide valuable information regarding the etiology of malocclusion. Crowded, irregular and protruding teeth have been a problem for some individual since antiquity and attempts to correct this disorder go back at least to 1000 BC. Angle’s classification of malocclusion in 1890s was an important step in the development of orthodontics because it not only subdivided major types of malocclusion but also included the first clear and simple definition of normal occlusion in the natural dentition.1 According to Angle if the teeth were arranged on a smoothly curving lines of occlusion and class I molar relation existed, then normal occlusion would result. Angle’s classification has four Classes i.e. Normal occlusion, Class I malocclusion, Class II malocclusion and Class III malocclusion.2 Various epidemiological studies have been conducted worldwide to assess the prevalence of malocclusion and to describe the occlusal trait.3,4 Data from the third National Health and Nutrition Examination Survey (NHANES-III) provide a clear picture of malocclusion in the US population in the 1990s.4 In this context very few studies have been conducted to assess the pattern of distribution of dental malocclusion among the population of western region of Nepal.5 The aim of this study was to determine the pattern of distribution of dental malocclusion in a sample of Nepalese orthodontic patient visiting a tertiary care center of Western Nepal. Difference in malocclusion characteristics or variation in dentofacial traits among various populations would be expected because of the difference in racial and ethnic composition. Information regarding most typical variation in dentofacial trait can be derived from studying how widely the trait is distributed i.e. prevalence and the frequency with which it is found i.e. incidence. It provides an important perspective for the practicing orthodontist Distribution of Malocclusion Traits among Orthodontic Patients in a Tertiary Care Center of Western Nepal Dr Hemant Kumar Halwai,1 Dr Vanita Gautam2 1Asso Prof, Dept of Orthodontics, 2Asso Prof, Dept. of Conservative Dentistry & Endodontics UCMS College of Dental Surgery, Bhairahawa, Nepal Correspondence: Dr. Hemant Kumar Halwai; Email: drhemanthalway@gmail.com Correspondence: Dr. Hemant Kumar Halwai; Email: drhemanthalway@gmail.com MATERIALS AND METHOD The sample consisted of 200 study models of patient with the age group of 8-36 years who have visited Department of Orthodontics, Universal College of Medical Sciences- 11 Orthodontic Journal of Nepal, Vol. 7 No. 1, June 2017 Halwai HK, Gautam V: Distribution of Malocclusion Traits among Orthodontic Patients in a Tertiary Care Center of Western Nepal College of Dental Surgery, Bhairahawa for orthodontic treatment. The study was conducted by evaluating the study model of the patients. was 8-36 years with the mean of 18.74 years. Age groups were divided into 3 groups: 8-11 years, 12-17 years and 18- 36 years. Among them the age group of 18-36 (61.5%) was the most to seek the orthodontic treatment while the age group 8-11years (3%) was the least (Table 2). Samples having full complement of permanent teeth with the exception of third molars were included in the study. Dentitions with missing molars, any history of jaw trauma, presence of masticatory disharmony, and temporomandibular joint disorders were exclude from the study. The distribution of malocclusion according to Angle’s classification showed that; Class I malocclusion was most prevalent with 101(50.5%) sample followed by Class II malocclusion in 90(45%) and Class III in 9(4.5%) (Table3). According to gender distribution, all forms of malocclusions were more prevalent in female subjects (Table 4). Among the age groups; in 8-11 and 12-17 years Class II malocclusion was the most prevalent, however in age group 18-36 the most prevalent malocclusion was Class I (Table 5). The study model was examined to classify into Class I, Class II and Class III malocclusion according to Angle’s classification system. The classification was done using Angle’s molar relationship on study models of each patient to describe the malocclusion in anterior-posterior plane and data were recorded in the data collection sheet. All the data were analyzed with SPSS software version 22. To test the association between distribution of malocclusion and gender; Pearson chi square value was calculated. At ∝ value set at 0.05 and df of 2, the p-value was 0.62(p<0.05); thus the association was non- significant (Table 4). However, the association between the distribution of malocclusion and age was statistically significant (p=0.023) (Table 5). RESULT Out of 200 study casts examined 74 study casts were of male and 126 were of female. The ratio between male to female was 0.58:1 (Table 1). The age range in the study Table 1: Gender distribution of the subjects Gender Frequency Percentage (%) Male 74 37 Female 126 63 Total 200 100 Table 2: Age distribution of the subjects Age range Frequency Percentage (%) 8-11 6 3 12-17 71 35.5 18-36 123 61.5 Total 200 100 Table 3: Distribution of malocclusion according to Angle’s classification Angle’s Classification Frequency Percentage (%) Class I 101 50.5 Class II 90 45 Class III 9 4.5 Total 200 100 Table 4: Association of malocclusion with gender Angle’s Classification Male Female X2 Value p-Value Class I 33 (32.67%) 68(67.32%) 5.53 0.62 (NS) Class II 40 (44.44%) 50(55.55%) Class III 1(11.11%) 8(88.88 %) Total 74 126 NS: non-significant Table 5: Association of malocclusion with age group Age group Class I Class II Class III X2 Value p-Value 8-11 yrs 1 (16.66%) 4 (66.66%) 1 (16.66%) 11.27 0.023* 12-17 yrs 30(42.25%) 35(49.29%) 6(8.45%) 18-36 yrs 70(56.91%) 51(41.46%) 2(1.62%) Total 101 90 9 Statistically significant at p<0.05 Table 1: Gender distribution of the subjects Gender Frequency Percentage (%) Male 74 37 Female 126 63 Total 200 100 Table 1: Gender distribution of the subjects DISCUSSION The prevalence of malocclusion is found to vary with different population, racial and ethnic origin. This type of study particularly is helpful in determining and planning the type of orthodontic service that might be provided on the basis of distribution of malocclusion in a population. Another similar study was conducted by Shrestha S and Shrestha RM,9 in which the study models of 464 orthodontic patients (165 male and 299 female) of the age ranging from 11 to 30 years were studied to evaluate the prevalence of malocclusion using Angle’s classification. The malocclusion status among the Nepalese seeking orthodontic treatment was 54.7% Class I, 36.9% Class II, and 8.4% Class III. Compared to this study, the present study showed similar distribution with Angle’s Class I malocclusion. Nainan10 found Angle’s Class I malocclusion in 49.9%; which was most prevalent as compared to the other type of malocclusions. The finding is similar to the pattern and distribution of malocclusion as compared to the present study. Present study showed 50.5% having Angle’s Class I malocclusion, 45% Class II malocclusion and 4.5% Class III malocclusion among the sample of western Nepali population. The pattern of distribution of malocclusion in the present study is similar to the study conducted by Sharma6 who found 62.28 % Class I malocclusion, 29.4% Class II malocclusion and 8.2% Class III malocclusion; however the frequency of Class II malocclusion is higher in our study. Similar pattern of distribution of malocclusion had been reported by National Health and Nutrition Examination Survey (NHANES). NHANES data suggests that greatest number are Class I malocclusion (50%-55%), next highest group are of class II (15%) and the least number are of Class III (less than 1 %) and the remainder of population had normal occlusion (30%).4 Frequency of Class II malocclusion was higher in our study as compared to the NHANES report. Since our study used the study cast of only the patient seeking orthodontic treatment none of them had normal occlusion. Table 1: Gender distribution of the subjects Table 3: Distribution of malocclusion according to Angle’s classification Table 2: Age distribution of the subjects Table 2: Age distribution of the subjects Age range Frequency Percentage (%) 8-11 6 3 12-17 71 35.5 18-36 123 61.5 Total 200 100 Angle’s Classification Frequency Percentage (%) Class I 101 50.5 Class II 90 45 Class III 9 4.5 Total 200 100 Table 4: Association of malocclusion with gender Table 4: Association of malocclusion with gender Angle’s Classification Male Female X2 Value p-Value Class I 33 (32.67%) 68(67.32%) 5.53 0.62 (NS) Class II 40 (44.44%) 50(55.55%) Class III 1(11.11%) 8(88.88 %) Total 74 126 NS: non-significant Table 5: Association of malocclusion with age group Age group Class I Class II Class III X2 Value p-Value 8-11 yrs 1 (16.66%) 4 (66.66%) 1 (16.66%) 11.27 0.023 12-17 yrs 30(42.25%) 35(49.29%) 6(8.45%) 18-36 yrs 70(56.91%) 51(41.46%) 2(1.62%) Total 101 90 9 *Statistically significant at p<0.05 Table 5: Association of malocclusion with age group 12 Halwai HK, Gautam V: Distribution of Malocclusion Traits among Orthodontic Patients in a Tertiary Care Center of Western Nepal conducted by Piya et al with the exception of Class III malocclusion which was higher in the later. This may be the result of ethnic variation because of higher population of mongoloids present in Kathmandu valley in comparison to Terai region of Nepal. CONCLUSION Based on the pretreatment records of the patients seeking orthodontic treatment in a tertiary care teaching hospital; the status of malocclusion in a sample of Western region of Nepal is: 1. The frequency of Angle’s Class I, Class II and Class III malocclusion was found to be 50.5%, 45% and 4.5% respectively, with Class I malocclusion being the most prevalent. According to the study conducted by Shrestha BK7 among 937 children in different schools of Kathmandu valley; normal occlusion was present in 27%, Class I malocclusion in 59% followed by Class II in 25 % and Class III in 16 %. Another study conducted by Piya et al8 among patient seeking orthodontic treatment in Nepal Medical College, Class I malocclusion was seen in 59.5% followed by Class II 26.7% and Class III in 13.7%. The pattern of distribution of malocclusion in the present study is similar to the study 2. Most of the numbers of patients seeking orthodontic treatment were female (63%). 3. Most of the numbers of patients seeking orthodontic treatment were of the age group 18-35(61.5%). 1. Proffit WR, Fields HW, Sarver DM. Contemporary Orthodontics. 4th ed. India: Mosby, Health Science imprints of Elsevier; 2 2. Angle EH. Treatment of malocclusion of the teeth and fractures of the maxillae. In: Angle’s System, Ed. 6; Philadelphia: SS White Dental Mfg Co; 1990 REFERENCES 2. Angle EH. Treatment of malocclusion of the teeth and fractures of the maxillae. In: Angle’s System, Ed. 6; Philadelphia: SS White Dental Mfg Co; 1990 3. Tak M, Nagarajappa R, Sharda AJ, Asawa K, Tak A, Jalihal S. Prevalence of malocclusion and orthodontic treatment needs among 12-15 years old school children of Udaipur, India. Eur J Dent; 2013; 7:45-53. 3. Tak M, Nagarajappa R, Sharda AJ, Asawa K, Tak A, Jalihal S. Prevalence of malocclusion and orthodontic treatment needs among 12-15 years old school children of Udaipur, India. Eur J Dent; 2013; 7:45-53. 4. Profit WR, Fields HW, Moray LJ. Prevalence of malocclusion and orthodontic treatment need in the United States: Estimates from NHANS III. Int J Adult Orthod Orthogn Surg, 1998; 13:97-106 4. Profit WR, Fields HW, Moray LJ. Prevalence of malocclusion and orthodontic treatment need in the United States: Estimates from NHANS III. Int J Adult Orthod Orthogn Surg, 1998; 13:97-106 . Baral P. Prevalence of malocclusion in Western Nepal. Orth 6. Sharma J N. Pattern of distribution of malocclusions in patients seeking orthodontic treatment at BPKIHS from Sunsari District of Nepal. Health Renaissance, 2010; 8:2:93-96. 6. Sharma J N. Pattern of distribution of malocclusions in patients seeking orthodontic treatment at BPKIHS from Sunsari District of Nepal. Health Renaissance, 2010; 8:2:93-96. a B.K, Yadav R, Basel P. Prevalence of malocclusion among high school students in Kathmandu Valley. Orthod J Nep, 2012; 2:1:1 7. Shrestha B.K, Yadav R, Basel P. Prevalence of malocclusion among high school students in Kathmandu Valley. Orthod estha VB, Acharya J, Khanal S, Bhattarai P. Pattern of distribution of malocclusion among patients seeking orthodontic at Dental College- Nepal Medical College. J Nep Dent Asso, 2013; 13:2:36-41. 8. Piya A, Shrestha VB, Acharya J, Khanal S, Bhattarai P. Pattern of distribution of malocclusion among patients s treatment at Dental College- Nepal Medical College. J Nep Dent Asso, 2013; 13:2:36-41. 9. Shrestha S, Shrestha RM. An analysis of malocclusion and occlusal characteristics in Nepalese orthodontic patients. Orthod J Nep, 2013; 3:1:19-25. 9. Shrestha S, Shrestha RM. An analysis of malocclusion and occlusal characteristics in Nepalese orthodontic patients. Orthod J 10. Nainan O, Singh S, Mitra R, Basanar DR. Evaluation of malocclusion pattern and dentofacial characteristics in orthodontically referred urban Indians. J Ind Orthod Soc, 2013; 47:4:328-34. 13
https://openalex.org/W1964620049	https://zenodo.org/records/2146971/files/article.pdf	English	null	Suggestions from the<i>quarterly</i>	The Quarterly journal of speech/Quarterly journal of speech	1,916	public-domain	728	Quarterly Journal of Speech Quarterly Journal of Speech ISSN: 0033-5630 (Print) 1479-5779 (Online) Journal homepage: http://www.tandfonline.com/loi/rqjs20 Suggestions from the quarterly Mrs. Perle Shale Kingsley Date: 20 June 2016, At: 21:13 Mrs. Perle Shale Kingsley To cite this article: Mrs. Perle Shale Kingsley (1916) Suggestions from the quarterly , Quarterly Journal of Speech, 2:2, 203-204, DOI: 10.1080/00335631609360540 To link to this article: http://dx doi org/10 1080/00335631609360540 To cite this article: Mrs. Perle Shale Kingsley (1916) Suggestions from the quarterly , Quarterly Journal of Speech, 2:2, 203-204, DOI: 10.1080/00335631609360540 To link to this article: http://dx.doi.org/10.1080/00335631609360540 Published online: 05 Jun 2009. Submit your article to this journal Article views: 5 View related articles Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rqjs20 Download by: [University of California Santa Barbara] Date: 20 June 2016, At: 21:13 Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=rqjs20 Download by: [University of California Santa Barbara] THE FORUM 203 In conclusion I wish to state most positively that if anyone who reads my writings is in doubt as to my meaning that doubt may be at once cleared up by the use of any standard English dictionary* FLOYD S. MUCKEY SUGGESTIONS FROM THE QUARTERLY 10 THE isolated teacher of public speaking the Quarterly Jour- nal is invaluable. By the isolated teacher I mean the one who singly constitutes the staff of his department» This teacher must depend upon himself for ideas for the advancement of his work, and because he must cover so broad a field as is covered by the department of public speaking, he may often find himself so exhausted that he is absolutely without ideas. At the University of Denver we have recently used,, for judging our Junior-Senior oratorical contest, the plan suggested by Pro- fessor Shaw in the first number of the Quarterly Journal. We found the plan the most satisfactory we have ever used, and, because of its popularity with the students, we expect to use it in all the intramural contests this year. It has even been suggested by some students that we use this method in the intercollegiate debates, where, of course, students from the home college would always constitute a majority of the audience. The other suggestion from the Quarterly Journal which we have put into practice is also to be found in the first number» It is Professor Pearson's plan for individual instruction by students given in the report of the Eastern Public-Speaking Conference. I might- say that I had thought of a similar plan, but had never had the courage to propose it to my Dean. And just here is where the Quarterly Journal is of practical help. To find that other teachers are succeeding with certain plans gives us courage to propose them. I have seven students, men and women, in this teachers' train- ing course. They were invited last spring to begin this work in the fall. We meet one afternoon each week, spending from one and one-half to two and one-half hours together. These students are given definite reading assignments for their own preparation THE QUARTERLY JOUMMAL OF PUBLIC SFEAKING 2O4 to supplement the practice and discussion of the class hour—this practice and discussion arising from consideration of the problems they meet when instructing the younger students. Each student who teaches six hours per week is given three hours5 credit for the course^ each one who teaches four hours is given two hours5 credit^ and each one who teachers two hours is given one hour?s credit. SUGGESTIONS FROM THE QUARTERLY The support which the isolated teacher receives from the inter- est, enthusiasm^ and ideas of this student-staff is exceedingly helpful. MRS. PERLE SHALE KINGSLEY UNIVERSITY OF DENVER UNIVERSITY OF DENVER UNIVERSITY OF DENVER The University of Iowa -and Northwestern University have entered into a dual debate league in which the teams will be com- posed of two men each. The teams will be made up of men who have had no experience in inter-collegiate debate^ and they will be coached by no one who has ever taken part in an inter-collegiate debate^ taught public speakings or done teaching of any kind for which pay was received. Really, the purpose is to have • non- coached teams. CORRECTIONS On page 104, line 19, for best read bent. OB page 12, lines 25-26, for expansion read contraction.
https://openalex.org/W4224219180	https://www.nature.com/articles/s41419-022-04705-z.pdf	English	null	Correction: The anthelmintic drug niclosamide induces GSK-β-mediated β-catenin degradation to potentiate gemcitabine activity, reduce immune evasion ability and suppress pancreatic cancer progression	Cell death and disease	2,022	cc-by	516	www.nature.com/cddis CORRECTION OPEN Correction: The anthelmintic drug niclosamide induces GSK- β-mediated β-catenin degradation to potentiate gemcitabine activity, reduce immune evasion ability and suppress pancreatic cancer progression © The Author(s) 2022 Cell Death and Disease (2022) 13:366 ; https://doi.org/10.1038/s41419-022-04705-z Correction to: Cell Death and Disease https://doi.org/10.1038/ s41419-022-04573-7, published online 03 February 2022. The original version of this article contained a mistake in ﬁgure 8. The correct ﬁgure can be found below. The original article has been corrected. Ofﬁcial journal of CDDpress Y. Guo et al. 2 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, daptation, distribution and reproduction in any medium or format, as long as you give ppropriate credit to the original author(s) and the source, provide a link to the Creative ommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless ndicated otherwise in a credit line to the material. If material is not included in the rticle’s Creative Commons license and your intended use is not permitted by statutory egulation or exceeds the permitted use, you will need to obtain permission directly om the copyright holder. To view a copy of this license, visit http://creativecommons. rg/licenses/by/4.0/. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless ndicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly f th i ht h ld T i f thi li i it htt // ti Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. © The Author(s) 2022 CORRECTION OPEN Correction: The anthelmintic drug niclosamide induces GSK- β-mediated β-catenin degradation to potentiate gemcitabine activity, reduce immune evasion ability and suppress pancreatic cancer progression The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. © The Author(s) 2022 Cell Death and Disease (2022) 13:366
https://openalex.org/W1989586229	https://www.ias-iss.org/ojs/IAS/article/download/911/827	English	null	LOCAL STEREOLOGY OF EXTREMES	Image analysis & stereology	2,012	cc-by	7,026	INTRODUCTION of extremes, only isotropic uniform random probes were considered. In Wicksell’s corpuscle problem (Wicksell, 1925), the prediction of maximum size of spherical particles was studied by Drees and Reiss (1992) and Takahashi and Sibuya (1996; 1998; 2001) while the behaviour of minimum size was investigated in K¨otzer and Molchanov (2006). An extension to spheroidal particles leads to the prediction of not only extremal size but also extremal shape. This was considered in Hlubinka (2003a;b; 2006a); Hlubinka and Kotz (2010) for oblate spheroidal particles and in Hlubinka (2006b;c) for prolate spheroidal particles. The novel contribution of this paper is the study of stereology of extremes for spheroidal particles under different stereological sampling design. Our aim is to investigate the stereological estimation of the tail of particle shape and size distribution based on the local probes. We derive the relation between the particle parameters and the parameters of particle central section (also called proﬁle). Subsequently, we show how the limiting distribution of the extremal particle parameters is related to the limiting distribution of the extremal proﬁle parameters. It turns out that the distribution of proﬁle parameters belongs to the same domain of attraction as the distribution of particle parameters. Local stereological methods form quite modern branch of stereology, see Vedel Jensen (1998) for a comprehensive exposition. They require that we associate a reference point to each particle and accomplish sectioning through this reference point. We speak about central sections or local probes. Information about the particle population is then extracted from these local probes. In contrast to traditional sampling, where the system of particles is sectioned by an isotropic uniform probe, the local sample is representative for the particle population. This fact was exploited in Pawlas et al. (2009) for the development of a statistical procedure for obtaining information about particle size distribution from central sections without speciﬁc assumptions about particle shape. The motivation for local stereology comes from the study of biological tissues. The particles are cells and the centres of cell nuclei or nucleoli serve as reference points. Local techniques are most easily implemented if optical sectioning is available. One of the possible applications in stereology is to estimate extremes of particle parameters from the observation of test probes of lower dimension. This ﬁeld is referred to as stereology of extremes (Beneˇs and Rataj, 2004). In practical applications it is often important to analyze extremes of the particle parameters. doi:10.5566/ias.v31.p99-108 doi:10.5566/ias.v31.p99-108 doi:10.5566/ias.v31.p99-108 Image Anal Stereol 2012;31:99-108 Original Research Paper ABSTRACT Local stereology uses information obtained from central sections passing through a reference point of the particle. The aim of this paper is to investigate the prediction of extremes of shape and size parameters based on the central sections. We consider the particle population formed by spheroids (either prolate or oblate) and assume that the reference point is the centre of the spheroid. A relation between shape and size parameters of the particles and their planar sections is derived and consequently stability properties of the domain of attractions are proved. eywords: extremes, local stereology, maximum domain of attraction, shape and size parameters, spheroids ZBYNˇEK PAWLASB Department of Probability and Mathematical Statistics, Faculty of Mathematics and Physics, C Sokolovsk´a 83, 18675 Prague, Czech Republic Department of Probability and Mathematical Statistics, Faculty of Mathematics and Physics, Charles University, Sokolovsk´a 83, 18675 Prague, Czech Republic e-mail: pawlas@karlin.mff.cuni.cz (Received November 28, 2011; revised April 2, 2012; accepted April 4, 2012) (Received November 28, 2011; revised April 2, 2012; accepted April 4, 2012) LOCAL STEREOLOGY OF EXTREMES ZBYNˇEK PAWLASB PARTICLES Local stereological estimates are based on information collected on a section plane through a ﬁxed reference point of the particle. In the present paper, we will concentrate on three-dimensional particles and two-dimensional section planes. By a particle K we understand a non-empty compact subset of R3. We associate a reference point x to the particle K. A section plane through the reference point takes the form x + L, where L is a ﬁxed two- dimensional linear subspace of R3. The information about the particle is deduced from the planar central section K ∩(x+L). In this paper we pursue the model- based approach. However, all results remain valid in the same way for the design-based approach. In the latter, the particle K is deterministic while the section plane is random. If Ξ0 is a triaxial ellipsoid with semiaxes of lengths A, B and C (A ≥B ≥C) and the corresponding reference point X0 is the centre of the ellipsoid, then the observed planar proﬁle Ξ0 ∩(X0 +L) is an ellipse. In particular, we will consider the case of two equal semiaxes, i.e., Ξ0 is a spheroid. It can be either prolate (B = C, two equal minor semiaxes) or oblate (A = B, two equal major semiaxes). A possible choice for p describing shape of the typical particle is given by the shape factor The class of all particles equipped with the Hausdorff metric forms a separable metric space. Thus, it is possible to deﬁne random particles, their distribution and independence. For more details on theory of random sets we refer to Molchanov (2005). S = A2 C2 −1 . This parameter is commonly used for spheroids, see, e.g., Beneˇs and Rataj (2004); Hlubinka (2003a; 2006a;b). For spherical particles (A = C) we get S = 0, more elongated spheroids lead to larger value of S. In similar way, we can deﬁne the function q to quantify the shape of the planar proﬁle. We will denote the proﬁle shape factor by T. We consider a population Ξ1,...,Ξn of random particles with reference points X1,...,Xn. The particles are not observed directly, only their planar proﬁles Ξi ∩(Xi + L) are available. An illustration for two- dimensional particles and one-dimensional central sections is shown in Fig. 1. INTRODUCTION For example, the damage of materials is related rather to extremal than mean characteristics of microstructure. The application of stereology of extremes to metallurgy is discussed e.g., in Takahashi and Sibuya (2002) or Bortot et al. (2007). So far in the literature concerning stereology Particular attention is devoted to the population of spheroids (either prolate or oblate). If we consider a spheroid with two semiaxes of length a and one semiaxis of length c, then its planar section through the centre is an ellipse with semiaxes of lengths a and d, where min(a,c) ≤d ≤max(a,c). In other words, the length of one semiaxis is directly observed at the central section while the second semiaxis appears shorter (for prolate spheroid) or longer (for oblate 99 PAWLAS Z: Local stereology of extremes Ξi Xi + L Fig. 1. A two-dimensional illustration of local sectioning applied to the particle population under study. spheroid). Therefore, proﬁles have more circular shape than original particles projected to the plane perpendicular to one of semiaxes of length a. This paper is organized as follows. At ﬁrst, we derive the relation between particle and proﬁle shape and size parameters in the population of spheroidal particles. Then, we summarize basic facts from extreme value theory which will be needed to obtain main results stated in the subsequent section. We make the inference about the extremal domains of attraction under local sampling design. We conclude with an example showing the behaviour of maximal shape parameter in the population of simulated oblate spheroids. Fig. 1. A two-dimensional illustration of local sectioning applied to the particle population under study. tail behaviour of the typical particle parameter p(Ξ0). Since the parameters p(Ξ1),..., p(Ξn) describing random shape or size of particles cannot be directly observed, we have to use the particle proﬁle parameters q(Ξ1 ∩(X1 + L)),...,q(Ξn ∩(Xn + L)) derived from local probes. In what follows, we will consider uniform randomly oriented (isotropic) particles. If the particles cannot be regarded as isotropic, we may instead randomize the orientation of the section plane (use isotropic subspace L) and work under design-based setting. PARTICLES Based on the extreme value theory we are going to study the tail behaviour of shape factor. and A for oblate spheroid. Shape factor of the ellipse obtained from central section is and A for oblate spheroid. Shape factor of the ellipse obtained from central section is T = D2 C2 −1 = Ssin2 Θ 1+Scos2 Θ It is natural to use the semiaxes lengths as the size parameters. Since one semiaxis can be recovered from central section, we condition on the knowledge of its length and we are interested in the other semiaxis length, that is, A for prolate spheroids, C for oblate spheroids and D for proﬁles. Similarly as for the shape factors, we can derive the relation between the conditional distributions of particle size parameter and proﬁle size parameter D. For prolate spheroids, we have from Eq. 1, (2) for prolate spheroid and T = A2 D2 −1 = Ssin2 Θ for oblate spheroid. Note that T ≤S in both cases. It means that the proﬁle shape factor is always smaller or equal to the particle shape factor. Equality occurs if and only if the section plane is perpendicular to the plane spanned by two semiaxes of the same length. 1−FD\|C(d \| c) = P(D > d \| C = c) = P µ sin2 Θ > A2(d2 −c2) d2(A2 −c2) ¶ = Z ∞ d Z π/2 arcsin r a2(d2−c2) d2(a2−c2) sinθ dθ FA\|C(da \| c) = c d Z ∞ d s a2 −d2 a2 −c2 FA\|C(da \| c) , d ≥c . Since the spheroid Ξ0 is assumed to have isotropic orientation, the density of Θ is fΘ(θ) = sinθ, θ ∈ [0,π/2]. Exploiting the independence of Θ and S, we obtain the complementary distribution functions of T: 1−FT(t) = P µ Ssin2 Θ 1+Scos2 Θ > t ¶ = Z ∞ t Z π/2 arcsin√ t(1+s)/s(1+t) sinθ dθ FS(ds) = 1 √1+t Z ∞ t r s−t s dFS(s) , t ≥0 , We apply the integration by parts and obtain We apply the integration by parts and obtain 1−FD\|C(d \| c) = c(d2 −c2) d Z ∞ d a(1−FA\|C(a \| c)) (a2 −c2)3/2√ a2 −d2 da , for prolate case and d > c . PARTICLES Each spheroid is uniquely determined by the centre X (reference point), the length A of major semiaxis, the length C of minor semiaxis, and the angles Θ and Φ which describe its orientation. Let Θ be chosen as the angle between the norm vector of L and the major (for prolate case) or minor (for oblate case) axis, i.e., Θ is the latitude and Φ is the longitude. In what follows we assume that the random vectors (A,C) and (Θ,Φ) are independent. The proﬁle observed on planar central We assume that the particles Ξ1,...,Ξn are independent and identically distributed. Denote by Ξ0 a random particle with the same distribution as the Ξi. We will refer to Ξ0 as a typical particle. Let p be a real measurable function deﬁned on the space of all particles. It will be used to describe shape or size parameters of particles. Our aim is to investigate the 100 Image Anal Stereol 2012;31:99-108 section is an ellipse and it can be easily shown that it has semiaxes of lengths which for t > 0 becomes t Z ∞1 F (s) lipse and it can be easily shown that it f l th which for t > 0 becomes ction is an ellipse and it can be easily shown that it as semiaxes of lengths which for t > 0 becomes hat it which for t > 0 becomes which for t > 0 becomes section is an ellipse and it can be easily shown that it has semiaxes of lengths 1−FT(t) = t √1+t Z ∞ t 1−FS(s) 2s3/2√s−t ds, t > 0 . (3) g D = s A2C2 C2 sin2 Θ+A2 cos2 Θ (1) 1−FT(t) = t √1+t Z t 1−FS(s) 2s3/2√s−t ds, t > 0 . (3) For oblate spheroid we end up with the following formula D = s A2C2 C2 sin2 Θ+A2 cos2 Θ (1) For oblate spheroid we end up with the following formula and C in the case of prolate spheroid and and C in the case of prolate spheroid and 1−FT(t) = t Z ∞ t 1−FS(s) 2s3/2√s−t ds, t > 0 . (4) (4) D = s A2C2 A2 sin2 Θ+C2 cos2 Θ We deal with the stereological unfolding problem. The estimation of unknown particle shape distribution function FS based on the estimator of FT is an ill-posed problem. EXTREME VALUE THEORY where ηF = inf{z ∈R : F(z) > 0} is the left endpoint of F. PARTICLES The distribution function F belongs to the maximum domain of attraction of G3 if and only if there exists some positive function b such that PARTICLES (5) 1−FT(t) = P(Ssin2 Θ > t) = Z ∞ t Z arcsin√ t/s 0 sinθ dθ FS(ds) = Z ∞ t r s−t s dFS(s) , t ≥0 , Clearly, FD\|C(d \| c) = 0 if d < c. For oblate spheroids we have FD\|A(d \| a) = P(D ≤d \| A = a) = P µ sin2 Θ ≥C2(a2 −d2) d2(a2 −C2) ¶ = Z d 0 Z π/2 arcsin r (a2−d2)c2 d2(a2−c2) sinθ dθ FC\|A(dc \| a) = Z d 0 a d s d2 −c2 a2 −c2 FC\|A(dc \| a) , d ≤a . for oblate case, where FS is the distribution function of S. It will turn out to be useful to rewrite these formulas using integration by parts. For prolate spheroid it yields 1−FT(t) = 1 √1+t − t √1+t Z ∞ t FS(s) 2s3/2√s−t ds , 101 PAWLAS Z: Local stereology of extremes If FC\|A(0 \| a) = 0, then the integration by parts implies If FC\|A(0 \| a) = 0, then the integration by parts implies If FC\|A(0 \| a) = 0, then the integration by parts implies The normalizing constants can be chosen such that cn = F←(1 −1/n) and dn = 0. The distribution function F belongs to the maximum domain of attraction of G2,γ if and only if ωF < ∞and FD\|A(d \| a) = a(a2 −d2) d Z d 0 cFC\|A(c \| a) √ d2 −c2(a2 −c2)3/2 dc , d < a . (6) Clearly, FD\|A(d \| a) = 1 if d > a. FD\|A(d \| a) = a(a2 −d2) d Z d 0 cFC\|A(c \| a) √ d2 −c2(a2 −c2)3/2 dc , d (6 FD\|A(d \| a) lim u→0+ 1−F(ωF −uz) 1−F(ωF −u) = zγ, for all z > 0 . d < a . (6) d < a . (6) d < a . (6) (6) Clearly, FD\|A(d \| a) = 1 if d > a. Clearly, FD\|A(d \| a) = 1 if d > a. The normalizing constants can be chosen such that cn = ωF −F←(1−1/n) and dn = ωF. The distribution function F belongs to the maximum domain of attraction of G3 if and only if there exists some positive function b such that The normalizing constants can be chosen such that cn = ωF −F←(1−1/n) and dn = ωF. EXTREME VALUE THEORY Let Z1,...,Zn be independent identically distributed random variables with common distribution function F. We are interested in the behaviour of the sample maximum Mn = max(Z1,...,Zn). We say that F belongs to the maximum domain of attraction of a distribution function G if there exist normalizing constants cn > 0 and dn ∈R such that for all z ∈R: lim u→ωF− 1−F(u+zb(u)) 1−F(u) = e−z, for all z ∈R . (8) The auxiliary function b may be chosen to be differentiable on (−∞,ωF) such that limu→ωF−b′(u) = 0, limu→∞b(u)/u = 0 if ωF = ∞ and limu→ωF−b(u)/(ωF −u) = 0 if ωF < ∞. The normalizing constants can be chosen such that dn = F←(1−1/n) and cn = b(dn). The auxiliary function b may be chosen to be differentiable on (−∞,ωF) such that limu→ωF−b′(u) = 0, limu→∞b(u)/u = 0 if ωF = ∞ and limu→ωF−b(u)/(ωF −u) = 0 if ωF < ∞. The normalizing constants can be chosen such that dn = F←(1−1/n) and cn = b(dn). lim n→∞F(cnz+dn)n = G(z) . (7) lim n→∞F(cnz+dn)n = G(z) . (7) Since P(Mn−dn cn ≤z) = P(Mn ≤cnz + dn) = F(cnz + dn)n, the limit relation Eq. 7 says that the distribution function of Mn−dn cn converges to the distribution function G. We shall write F ∈MDA(G). The normalizing constants cn and dn are not given uniquely. For example, if we consider c′ n > 0 and d′ n such that Analogous considerations can be carried out for sample minima. A given distribution function belongs to the minimum domain of attraction of one of three distributions (Fr´echet, Weibull or Gumbel). We give the characterization of the minimum domain of attraction of Weibull distribution. A distribution function F belongs to the minimum domain of attraction of G2,γ if and only if ηF > −∞and lim n→∞ c′ n cn = 1 , lim n→∞ d′ n −dn cn = 0 , lim u→0+ 1−F(ηF +uz) 1−F(ηF +u) = zγ, for all z > 0 , then Eq. 7 holds with cn replaced by c′ n and dn replaced by d′ n. It is well-known that there are three possible non-degenerate limit distributions (Fisher- Tippett theorem, cf. Embrechts et al. (1997), Theorem 3.2.3), G belongs to the type of one of the following distribution functions (γ > 0): where ηF = inf{z ∈R : F(z) > 0} is the left endpoint of F. TAIL BEHAVIOUR OF SHAPE AND SIZE PARAMETERS lim u→ω− 1−FT(u+zb(u)) 1−FT(u) = lim u→ω− u+zb(u) √ 1+u+zb(u) R ω u+zb(u) 1−FS(s) 2s3/2√ s−u−zb(u) ds u √1+u R ω u 1−FS(s) 2s3/2√s−u ds = lim u→ω− (u+zb(u))√1+u u p 1+u+zb(u) · R g−1(ω) u 1−FS(v+zb(v)) (v+zb(v))3/2√ v+zb(v)−u−zb(u)(1+zb′(v))dv R ω u 1−FS(v) v3/2√v−u dv . lim u→ω− 1−FT(u+zb(u)) 1−FT(u) = lim u→ω− u+zb(u) √ 1+u+zb(u) R ω u+zb(u) 1−FS(s) 2s3/2√ s−u−zb(u) ds u √1+u R ω u 1−FS(s) 2s3/2√s−u ds = lim u→ω− (u+zb(u))√1+u u p 1+u+zb(u) · R g−1(ω) u 1−FS(v+zb(v)) (v+zb(v))3/2√ v+zb(v)−u−zb(u)(1+zb′(v))dv R ω u 1−FS(v) v3/2√v−u dv . Theorem 1. Let Ξ1,...,Ξn be independent and identically distributed prolate spheroids with isotropic orientation. Assume that the orientation is independent of semiaxes lengths and that the Ξi are not spheres with positive probability. Then the following assertions hold. • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ+1/2). • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ+1/2). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G3), then FT ∈MDA(G3). • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ+1/2). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ+1/2). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G3), then FT ∈MDA(G3). If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ+1/2). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G3), then FT ∈MDA(G3). The properties of b and Eq. 8 imply limu→ω− (u+zb(u))√1+u u√ 1+u+zb(u) = 1, limu→ω−(1 + zb′(u)) = 1, limu→ω− u3/2 (u+zb(u))3/2 = 1 and limu→ω− 1−FS(u+zb(u)) 1−FS(u) = e−z. Applying the mean value theorem and Lemma 1, we conclude with limu→ω− 1−FT (u+zb(u)) 1−FT (u) = e−z. Proof. From Eq. 2 we observe that the right endpoints of FT and FS coincide, we denote them by ω. Since we excluded the case S = 0 a.s., we have ω > 0. Proof. From Eq. 2 we observe that the right endpoints of FT and FS coincide, we denote them by ω. Since we excluded the case S = 0 a.s., we have ω > 0. ( ( )) e−z. Applying the mean value theorem and Lemma 1, we conclude with limu→ω− 1−FT (u+zb(u)) 1−FT (u) = e−z. First we consider the maximum domain of attraction of Fr´echet distribution. Applying Eq. TAIL BEHAVIOUR OF SHAPE AND SIZE PARAMETERS – Fr´echet: G1,γ(z) = exp{−z−γ}, z > 0, – Weibull: G2,γ(z) = exp{−(−z)γ}, z ≤0, – Gumbel: G3(z) = exp{−e−z}, z ∈R. – Fr´echet: G1,γ(z) = exp{−z−γ}, z > 0, – Weibull: G2,γ(z) = exp{−(−z)γ}, z ≤0, – Gumbel: G3(z) = exp{−e−z}, z ∈R. In this section we show the relation between the maximum domains of attraction of the shape and size parameters of proﬁles and particles. In the proofs we will often use the following lemma. It is a generalization of Lemma 1.2.1 in de Haan (1975) and it can also be found in K¨otzer and Molchanov (2006) as Lemma 2.4. We summarize several results concerning the characterization of MDA(G), for more details we refer to de Haan (1975) or Embrechts et al. (1997), Section 3.3. Let ωF = sup{z ∈R : F(z) < 1} be the right endpoint of F and let F←(u) = inf{z ∈ R : F(z) ≥u}, u ∈(0,1), be the quantile function. The distribution function F belongs to the maximum domain of attraction of G1,γ if and only if ωF = ∞and Lemma 1. Let f(·,·) and g(·,·) be positive functions such that both lim u→∞ 1−F(uz) 1−F(u) = z−γ, for all z > 0 . Z ω 0 f(s,t)dt and Z ω 0 g(s,t)dt 102 Image Anal Stereol 2012;31:99-108 are ﬁnite for some ω ∈(0,∞] and for s ≤t < ω, lim s→ω f(s,t) g(s,t) = c with c ∈[0,∞] . are ﬁnite for some ω ∈(0,∞] and for s ≤t < ω, are ﬁnite for some ω ∈(0,∞] and for s ≤t < ω, lim s→ω f(s,t) g(s,t) = c with c ∈[0,∞] . Finally, we consider a Gumbel limiting distribution. Let b be the auxiliary function from Eq. 8. Using the same arguments as in Drees and Reiss (1992), p. 211, we deduce that for some u0 ∈(0,ω), the function g : v 7→v + zb(v) is strictly increasing on [u0,ω). Substituting s = v+zb(v) we get Then Then lim s→ω R ω s f(s,t)dt R ω s g(s,t)dt = c . Now we are ready to prove the stability of MDA for shape factors of spheroids. We start with prolate case. Proof. The proof is analogous to that of Theorem 1. We only have to use Eq. 4 instead of Eq. 3. TAIL BEHAVIOUR OF SHAPE AND SIZE PARAMETERS 3 and Lemma 1 we ﬁnd that lim u→∞ 1−FT(uz) 1−FT(u) = lim u→∞ uz √1+uz R ∞ uz 1−FS(s) 2s3/2√s−uz ds u √1+u R ∞ u 1−FS(s) 2s3/2√s−u ds = lim u→∞ z√1+u √1+uz · R ∞ u 1−FS(vz) z2v3/2√v−uzdv R ∞ u 1−FS(v) v3/2√v−u dv = lim u→∞ √1+u √1+uz · 1−FS(uz) 1−FS(u) = z−1/2 ·z−γ = z−(γ+1/2). It means that the distribution function of proﬁle shapes belongs to the same maximum domain of attraction as the distribution function of particle shapes, only the parameter γ may differ. Similar result holds for the population of oblate spheroids. Theorem 2. Let Ξ1,...,Ξn be independent and identically distributed oblate spheroids with isotropic orientation. Assume that the orientation is independent of semiaxes lengths and that the Ξi are not spheres with positive probability. Then the following assertions hold. The case of MDA of Weibull distribution can be treated in a similar way: lim u→0+ 1−FT(ω −uz) 1−FT(ω −u) lim u→0+ 1−FT(ω −uz) 1−FT(ω −u) = lim u→0+ ω−uz √1+ω−uz R ω ω−uz 1−FS(s) 2s3/2√s−ω+uz ds ω−u √1+ω−u R ω ω−u 1−FS(s) 2s3/2√s−ω+u ds = lim u→0+ (ω −uz)√1+ω −u (ω −u)√1+ω −uz · R u 0 1−FS(ω−vz) (ω−vz)3/2√ (u−v)zzdv R u 0 1−FS(ω−v) (ω−v)3/2√u−v dv = √z lim u→0+ (ω −uz)√1+ω −u (ω −u)√1+ω −uz · 1−FS(ω −uz) 1−FS(ω −u) = zγ+1/2. ( ) = lim u→0+ ω−uz √1+ω−uz R ω ω−uz 1−FS(s) 2s3/2√s−ω+uz ds ω−u √1+ω−u R ω ω−u 1−FS(s) 2s3/2√s−ω+u ds = lim u→0+ (ω −uz)√1+ω −u (ω −u)√1+ω −uz · R u 0 1−FS(ω−vz) (ω−vz)3/2√ (u−v)zzdv R u 0 1−FS(ω−v) (ω−v)3/2√u−v dv = √z lim u→0+ (ω −uz)√1+ω −u (ω −u)√1+ω −uz · 1−FS(ω −uz) 1−FS(ω −u) • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G3), then FT ∈MDA(G3). • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G3), then FT ∈MDA(G3). • If FS ∈MDA(G1,γ), then FT ∈MDA(G1,γ). • If FS ∈MDA(G2,γ), then FT ∈MDA(G2,γ+1/2). • If FS ∈MDA(G3), then FT ∈MDA(G3). Proof. The proof is analogous to that of Theorem 1. We only have to use Eq. 4 instead of Eq. 3. 103 PAWLAS Z: Local stereology of extremes In the remainder of this section we are interested in the relation of tail distributions of size parameters of proﬁles and spheroids. TAIL BEHAVIOUR OF SHAPE AND SIZE PARAMETERS We use the lengths of semiaxes as the size parameters. Recall that one semiaxis is always recovered from central section. Therefore, we condition on the knowledge of its length in our considerations. For the population of prolate spheroids we observe minor semiaxis of length C. Since D ≤A, large major semiaxis of the section proﬁle can only be observed if the corresponding spheroid major semiaxis is large. Similarly as for the shape parameters, it turns out that the stability of the maximum domain of attraction follows. be observed if the corresponding spheroid minor semiaxis is small. Hence, we may investigate the lower tail behaviour of size parameters. Since the sizes are positive, we don’t have to care about the minimum domain of attraction of Fr´echet distribution. The following result states the stability property of the minimum domain of attraction of Weibull distribution. Theorem 4. Let Ξ1,...,Ξn be independent and identically distributed oblate spheroids with isotropic orientation. Assume that the orientation is independent of semiaxes lengths A and C. Let the conditional distribution function FC\|A of C given A have the left endpoint ηF = 0 and FC\|A(0 \| a) = 0. If FC\|A belongs to the minimum domain of attraction of G2,γ, then FD\|A belongs to the minimum domain of attraction of G2,γ as well. Theorem 3. Let Ξ1,...,Ξn be independent and identically distributed prolate spheroids with isotropic orientation. Assume that the orientation is independent of semiaxes lengths A and C and that the Ξi are not spheres with positive probability. Then the following assertions hold. Proof. Using a characterization of the minimum domain of attraction we get • If FA\|C ∈MDA(G1,γ), then FD\|C ∈MDA(G1,γ+1). • If FA\|C ∈MDA(G2,γ), then FD\|C ∈MDA(G2,γ+1/2). • If FA\|C ∈MDA(G3), then FD\|C ∈MDA(G3). lim u→0+ FC\|A(uz \| a) FC\|A(u \| a) = zγ lim u→0+ FC\|A(uz \| a) FC\|A(u \| a) = zγ for any z > 0. Applying Eq. 6 we can write for any z > 0. Applying Eq. 6 we can write lim u→0+ FD\|A(uz \| a) FD\|A(u \| a) = lim u→0+ R uz 0 ac(a2−u2z2) uz√ u2z2−c2(a2−c2)3/2 FC\|A(c \| a)dc R u 0 ac(a2−u2) u√ u2−c2(a2−c2)3/2 FC\|A(c \| a)dc = lim u→0+ R u 0 vz(a2−u2z2) z2√ u2−v2(a2−v2z2)3/2 FC\|A(vz \| a)zdv R u 0 v(a2−u2) √ u2−v2(a2−v2)3/2 FC\|A(v \| a)dv = zγ, which completes the proof. lim u→0+ FD\|A(uz \| a) FD\|A(u \| a) Proof. TAIL BEHAVIOUR OF SHAPE AND SIZE PARAMETERS We have to show that TAIL BEHAVIOUR OF SHAPE AND SIZE PARAMETERS The proof proceeds along the same lines as that of Theorem 1, except that we use Eq. 5. We just show how this works for the maximum domain of attraction of Fr´echet distribution: = lim u→0+ R uz 0 ac(a2−u2z2) uz√ u2z2−c2(a2−c2)3/2 FC\|A(c \| a)dc R u 0 ac(a2−u2) u√ u2−c2(a2−c2)3/2 FC\|A(c \| a)dc = lim u→0+ R u 0 vz(a2−u2z2) z2√ u2−v2(a2−v2z2)3/2 FC\|A(vz \| a)zdv R u 0 v(a2−u2) √ u2−v2(a2−v2)3/2 FC\|A(v \| a)dv = zγ, lim u→∞ 1−FD\|C(uz \| c) 1−FD\|C(u \| c) = lim u→∞ c(u2z2−c2) uz R ∞ uz a(1−FA\|C(a\|c)) (a2−c2)3/2√ a2−u2z2 da c(u2−c2) u R ∞ u a(1−FA\|C(a\|c)) (a2−c2)3/2√ a2−u2 da = lim u→∞ u2z2 −c2 z(u2 −c2) · R ∞ u vz(1−FA\|C(vz\|ca)) (v2z2−c2)3/2z√ v2−u2 zdv R ∞ u v(1−FA\|C(v\|c)) (v2−c2)3/2√ v2−u2 dv = z−1 lim u→∞ 1−FA\|C(uz \| c) 1−FA\|C(u \| c) = z−(γ+1). lim u→∞ 1−FD\|C(uz \| c) 1−FD\|C(u \| c) = lim u→∞ c(u2z2−c2) uz R ∞ uz a(1−FA\|C(a\|c)) (a2−c2)3/2√ a2−u2z2 da c(u2−c2) u R ∞ u a(1−FA\|C(a\|c)) (a2−c2)3/2√ a2−u2 da = lim u→∞ u2z2 −c2 z(u2 −c2) · R ∞ u vz(1−FA\|C(vz\|ca)) (v2z2−c2)3/2z√ v2−u2 zdv R ∞ u v(1−FA\|C(v\|c)) (v2−c2)3/2√ v2−u2 dv = z−1 lim u→∞ 1−FA\|C(uz \| c) 1−FA\|C(u \| c) = z−(γ+1). D\|C( \| ) = lim u→∞ c(u2z2−c2) uz R ∞ uz a(1−FA\|C(a\|c)) (a2−c2)3/2√ a2−u2z2 da c(u2−c2) u R ∞ u a(1−FA\|C(a\|c)) (a2−c2)3/2√ a2−u2 da = lim u→∞ u2z2 −c2 z(u2 −c2) · R ∞ u vz(1−FA\|C(vz\|ca)) (v2z2−c2)3/2z√ v2−u2 zdv R ∞ u v(1−FA\|C(v\|c)) (v2−c2)3/2√ v2−u2 dv = z−1 lim u→∞ 1−FA\|C(uz \| c) 1−FA\|C(u \| c) which completes the proof. which completes the proof. which completes the proof. We also consider the maximum domain of attraction of Weibull distribution. Theorem 5. Let Ξ1,...,Ξn be independent and identically distributed oblate spheroids with isotropic orientation. Assume that the orientation is independent of semiaxes lengths A and C. Let FC\|A(0 \| a) = 0. If FC\|A belongs to the maximum domain of attraction of G2,γ with γ > 1, then FD\|A belongs to the maximum domain of attraction of G2,1. When considering oblate spheroids, the situation is reversed compared to the prolate case. We observe major semiaxis directly and minor semiaxis of the section proﬁle is larger than minor semiaxis of the spheroid. It means that large minor semiaxis can be observed even if spheroid minor semiaxis is small. On the other hand, small minor semiaxis can only Proof. Proof. We have to show that lim u→0+ 1−FD\|A(a−ud \| a) 1−FD\|A(a−u \| a) = d. 104 Image Anal Stereol 2012;31:99-108 From Eq. 6, we get Proof. First we rewrite the complementary distribution function of FT using substitution y = (ω −s)/(ω −t), 1−FD\|A(a−u \| a) = ua(2a−u) a−u Z a−u 0 c(1−FC\|A(c \| a)) p (a−u)2 −c2(a2 −c2)3/2 dc . 1−FT(t) (ω −t)γ+1/2 = Z ω t tK(ω −s)γ 2s3/2√s−t(ω −t)γ+1/2 ds = Z 1 0 tKyγ(1−y)−1/2 2(ω −(ω −t)y)3/2 dy . The assumption FC\|A ∈MDA(G2,γ) ensures that 1 − FC\|A(c \| a) = (a−c)γL(a−c) for some slowly varying function L, i.e., limu→0+ L(uz)/L(u) = 1 for any z > 0. Hence, we may apply the dominated convergence theorem and deduce that The statement of the lemma now follows by noting that The statement of the lemma now follows by noting that lim t→ω− t (ω −(ω −t)y)3/2 = 1 √ω lim u→0+ 1−FD\|A(a−u \| a) u = 2a Z a 0 c(1−FC\|A(c \| a)) (a2 −c2)2 dc < ∞. lim t→ω− t (ω −(ω −t)y)3/2 and Z 1 yγ(1−y)−1/2 dy = B(γ lim u→0+ 1−FD\|A(a−u \| a) u = 2a Z a 0 c(1−FC\|A(c \| a)) (a2 −c2)2 dc < ∞. and and Z 1 0 yγ(1−y)−1/2 dy = B(γ +1,1/2) . Therefore, we can conclude that Therefore, we can conclude that lim u→0+ 1−FD\|A(a−ud \| a) 1−FD\|A(a−u \| a) = d lim u→0+ 1−FD\|A(a−ud \| a) ud u 1−FD\|A(a−u \| a) lim u→0+ 1−FD\|A(a−ud \| a) 1−FD\|A(a−u \| a) = d lim u→0+ 1−FD\|A(a−ud \| a) ud u 1−FD\|A(a−u \| a) = d . Recall that the normalizing constants for FS can be chosen such that cn = ω −F← S (1 −1/n) and dn = ω. By Eq. 9, we get cn = (nK)−1/γ, see also Embrechts et al. (1997), Example 3.3.16. Lemma 2 yields that the possible choice of normalizing constants for FT is ˜cn = (n ˜K)−1/ ˜γ and ˜dn = ω, where ˜γ = γ + 1/2 and ˜K = K 2√ω B( ˜γ + 1/2,1/2). Since we want to estimate cn from estimate of ˜cn, the following relation turns out to be useful, = d lim u→0+ 1−FD\|A(a−ud \| a) ud u 1−FD\|A(a−u \| a) WORKED EXAMPLE (13) We generate a sample of n = 250 independent isotropic oblate spheroids with semiaxes lengths distributed according to Eq. 11. For each spheroid a local section through its centre is performed, resulting in the collection of ellipses (proﬁles). Let S1,...,Sn Profile shapes T Density 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 Profile shapes T Density 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 evaluating integral in Eq. 4, FT(t) = 1− √3−t √ 3 − √ 3 18 t √ 3−t + t(12−t) 18 log √3−t + √ 3 √t , 0 < t ≤3 . (13) Profile shapes Density 0.5 1.0 1.5 Profile shapes Profile shapes FT(t) = 1− √3−t √ 3 − √ 3 18 t √ 3−t + t(12−t) 18 log √3−t + √ 3 √t , 0 < t ≤3 . (13) p Density 0.5 1.0 1.5 Density We generate a sample of n = 250 independent isotropic oblate spheroids with semiaxes lengths distributed according to Eq. 11. For each spheroid a local section through its centre is performed, resulting in the collection of ellipses (proﬁles). Let S1,...,Sn be the shape factors of simulated particles and let T1,...,Tn be the shape factors of their proﬁles. Fig. 2 shows the histogram of particle shape factors together with theoretical density function. Of course, both are not available when observing only sectional data. On the other hand, Fig. 3 shows the histogram of shape factors obtained from central sections, this is the information that we have in practice when dealing with real data. For comparison, the theoretical density function (in practice unknown) of proﬁle shapes is also depicted. T Fig. 3. Histogram of proﬁle shape factors in simulated population of oblate spheroids together with theoretical density function derived from the distribution function given by Eq. 13. Denote by Mn = max(S1,...,Sn) the maximal shape parameter. Our aim is to predict the distribution of Mn based on observations of local section shapes T1,...,Tn. We order them from the largest to the smallest, i.e., T(1) ≥T(2) ≥··· ≥T(n) is the order statistics. We suppose that FS ∈MDA(G2,γ) and that the right endpoint ω = 3 is known. By Theorem 2, FT ∈MDA(G2, ˜γ), where ˜γ = γ +1/2. WORKED EXAMPLE The index ˜γ can be estimated from the k largest proﬁle shape factors as Particle shapes S Density 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Fig. 2. Histogram of particle shape factors in simulated population of oblate spheroids together with theoretical density function corresponding to the distribution function given by Eq. 12. Particle shapes S Density 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Particle shapes WORKED EXAMPLE cn = Ã ˜cγ+1/2 n B(γ +1, 1 2) 2√ω ! 1 γ . (10) In this section we demonstrate the utility of our theoretical results on the following example. We consider a population of oblate spheroids and assume that the shape factor S has distribution function FS with ﬁnite right endpoint 0 < ω < ∞and power law behaviour at ω. Speciﬁcally, (10) In what follows, we consider a particular example that leads to the shape factor with power law behaviour at the right endpoint. A typical particle Ξ0 is assumed to be oblate spheroid with semiaxes lengths A and C having joint density function 1−FS(s) = K(ω −s)α, 0 ≤ω −K−1/γ ≤s ≤ω , (9) , (9) fA,C(a,c) =        8 9 ³a c ´3 , for 1 ≤c ≤a ≤2 , 0, otherwise . (11 ( ) for some K,γ > 0. This ensures that FS belongs to the Weibull maximum domain of attraction MDA(G2,γ). By Theorem 2, distribution function FT of proﬁle shape factor T belongs to MDA(G2,γ+1/2). Using Eq. 4 we show that FT is tail-equivalent to a distribution function with power law behaviour at ω. (11) After straightforward calculation one obtains distribution function FS of the shape factor S = A2/C2 −1, Lemma 2. Let FS satisfy Eq. 9 and FT be given by Eq. 4, then FS(s) = 1−1 9(3−s)2, 0 ≤s ≤3 . (12) lim t→ω− 1−FT(t) (ω −t)γ+1/2 = K 2√ω B µ γ +1, 1 2 ¶ , (12) We see that it is of the form Eq. 9 with γ = 2, K = 1/9 and ω = 3. It is also possible to express FT by where B(·,·) is the beta function. where B(·,·) is the beta function. 105 PAWLAS Z: Local stereology of extremes Profile shapes T Density 0.0 0.5 1.0 1.5 2.0 2.5 0.0 0.5 1.0 1.5 Fig. 3. Histogram of proﬁle shape factors in simulated population of oblate spheroids together with theoretical density function derived from the distribution function given by Eq. 13. evaluating integral in Eq. 4, evaluating integral in Eq. 4, FT(t) = 1− √3−t √ 3 − √ 3 18 t √ 3−t + t(12−t) 18 log √3−t + √ 3 √t , 0 < t ≤3 . Distribution function of maximal particle shape Distribution function of maximal particle shape 2.75 2.80 2.85 2.90 2.95 3.00 0.2 0.4 0.6 0.8 1.0 Particle shapes b˜γ = Ã log(ω −T(k))−1 k k ∑ i=1 log(ω −T(i)) !−1 (Weissman, 1978). Then the estimate of γ is ˆγ = b˜γ −1 2. Furthermore, we estimate ˜cn by Density b˜cn = n −1 b˜γ ω . Finally, the estimator of cn is obtained by plugging the estimates ˆγ and b˜cn into Eq. 10: ˆcn = Ã b˜c ˆγ+1/2 n B( ˆγ +1, 1 2) 2√ω ! 1 ˆγ . S The estimators depend on the choice of k. For our purposes, we take k = 10. Fig. 2. Histogram of particle shape factors in simulated population of oblate spheroids together with theoretical density function corresponding to the distribution function given by Eq. 12. The distribution function of Mn is FMn(s) = P(Mn ≤s) = FS(s)n and for large n it can be approximated by G2,γ(s−dn cn ), cf. Eq. 7. By replacing 106 Image Anal Stereol 2012;31:99-108 γ and cn with their estimates, we obtain the asymptotic approximation of the distribution function of Mn in the form then estimated by ˆcn and ˆdn using the relations between (cn,dn) and (˜cn, ˜dn) and estimators b˜cn and b˜dn. Finally, the distribution of extremal spheroid parameter is approximated by the limiting distribution (Fr´echet, Weibull or Gumbel) with normalizing constants ˆcn and ˆdn. ˆFMn(s) = G2, ˆγ µs−ω ˆcn ¶ , s ≤ω . The graph of this function is shown in Fig. 4. It is compared with true (unknown) distribution function of Mn. REFERENCES Beneˇs V, Rataj J (2004). Stochastic geometry: Selected topics. Boston: Kluwer Academic Publishers. Bortot P, Coles SG, Sisson SA (2007). Inference for stereological extremes. J Am Stat Assoc 102:84–92. de Haan L (1975). On regular variation and its application to the weak convergence of sample extremes, 3rd printing. Mathematical Centre Tracts 32, Mathematisch Centrum, Amsterdam. Drees H, Reiss RD (1992). Tail behaviour in Wicksell’s corpuscle problem. In: Galambos J, K´atai I, eds. Probability Theory and Applications – Essays to the Memory of J´ozsef Megyor´odi. Dordrecht: Kluwer Academic Publishers, 205–20. Fig. 4. The resulting asymptotic approximation (dashed line) of FMn compared with true distribution function (solid line). Embrechts P, Kl¨uppelberg C, Mikosch T (1997). Modelling extremal events. Berlin: Springer. ACKNOWLEDGEMENTS The work is supported by the Grant Agency of the Czech Republic, project P201/10/0472. An earlier (non-reviewed) version of this paper was presented at the 13th International Congress for Stereology and appeared in the Congress proceedings (Pawlas, 2011). 2.75 2.80 2.85 2.90 2.95 3.00 0.2 0.4 0.6 0.8 1.0 Distribution function of maximal particle shape s Fig. 4. The resulting asymptotic approximation (dashed line) of FMn compared with true distribution function (solid line). CONCLUSION Hlubinka D (2003a). Stereology of extremes; shape factor of spheroids. Extremes 6:5–24. We proved stability properties of the domain of attraction for shape and size parameters of spheroidal particles under local stereological sampling design. They can be helpful for estimating stereologically the tail of a particle parameter distribution. Practical applications are illustrated by an example applied to the shape factor of oblate spheroids generated by computer simulations. Analogous applications for the case of isotropic uniform random probes are discussed in Hlubinka (2003b) or Hlubinka (2006b). First we choose a parametric model for spheroid parameter of interest. It belongs to the domain of attraction of some distribution function. Then our theoretical results say that proﬁle parameter belongs to the same domain of attraction with possibly different parameter γ. From sectional data we ﬁnd the estimates b˜cn and b˜dn of normalizing constants ˜cn and ˜dn for the observed proﬁle parameter. The normalizing constants cn and dn for distribution function of spheroid parameter are Hlubinka D (2003b). Stereology of extremes; size of spheroids. Math Bohem 128:419–38. Hlubinka D (2006a). Extremes of spheroid shape factor based on two dimensional proﬁles. Kybernetika 62:77– 94. Hlubinka D (2006b). Shape factor extremes for prolate spheroids. Kybernetika 62:557–68. Hlubinka D (2006c). Size and shape factor extremes of spheroids. Image Anal Stereol 25:145–54. Hlubinka D, Kotz S (2010). The generalized FGM distribution and its application to stereology of extremes. Appl Math 55:495–512. K¨otzer S, Molchanov I (2006). On the domain of attraction for the lower tail in Wicksell’s corpuscle problem. In: Lechnerov´a R, Saxl I, Beneˇs V, eds. Proc S4G. Prague: Union of Czech Mathematicians and Physicists. 91–6. Molchanov I (2005). Theory of random sets. London: Springer. 107 PAWLAS Z: Local stereology of extremes Pawlas Z, Nyengaard JR, Vedel Jensen EB (2009). Particle sizes from sectional data. Biometrics 65:216–24. Math 53:647–60. Math 53:647–60. Takahashi R, Sibuya M (2002). Metal fatigue, Wicksell transform and extreme values. Appl Stochast Models Bus Ind 18:301–12. Pawlas Z (2011). Local stereology of extremes. In: Guoquan Liu et al., eds. Proc 13th Int Congr Stereol. Beijing: Chinese Society for Stereology. Vedel Jensen EB (1998). Local stereology. Singapore: World Scientiﬁc. Takahashi R, Sibuya M (1996). The maximum size of the planar sections of random spheres and its application to metallurgy. Ann Inst Stat Math 48:127–44. Weissman I (1978). Estimation of parameters and larger quantiles based on the k largest observations. J Am Stat Assoc 73:812–5. CONCLUSION Takahashi R, Sibuya M (1998). Prediction of the maximum size in Wicksell’s corpuscle problem. Ann Inst Stat Math 50:361–77. Wicksell SD (1925). The corpuscle problem. A mathematical study of a biometric problem. Biometrika 17:84–99. Takahashi R, Sibuya M (2001). Prediction of the maximum size in Wicksell’s corpuscle problem, II. Ann Inst Stat 108
https://openalex.org/W4362626991	https://figshare.com/articles/journal_contribution/Supplementary_Table_S1_from_Targeting_YAP-Dependent_MDSC_Infiltration_Impairs_Tumor_Progression/22530987/1/files/39994164.pdf	unk	null	Supplementary Table S1 from Targeting YAP-Dependent MDSC Infiltration Impairs Tumor Progression	null	2,023	cc-by	163	Table S1. Antibody list for CyTOF. Table S1. Antibody list for CyTOF. Table S1. Antibody list for CyTOF. Marker Clone Label Vendor Cat# Used for Fig. 1c-d Used for Fig. 1e-f Gr-1 RB6-8C5 141Pr DVS sciences 3141005B YES CD11c N418 142Nd DVS sciences 3142003B YES YES CD69 H1.2F3 143Nd DVS sciences 3143004B YES CD4 RM4-5 145Nd DVS sciences 3145002B YES YES CD8a 53-6.7 146Nd DVS sciences 3146003B YES YES CD45 30-F11 147Sm DVS sciences 3147003B YES YES CD11b M1/70 148Nd DVS sciences 3148003B YES YES CD19 6D5 149Sm DVS sciences 3149002B YES YES CD25 3C7 151Eu DVS sciences 3151007B YES CD3e 145-2C11 152Sm DVS sciences 3152004B YES CD62L MEL-14 160Gd DVS sciences 3160008B YES Ter119 TER119 162Dy DVS sciences 3162003B YES TCRβ H57-597 169Tm DVS sciences 3169002B YES YES NK1.1 PK136 170Er DVS sciences 3170002B YES YES CD44 1M7 171Yb DVS sciences 3171003B YES Ep- CAM G8.8 174Yb BioLegend 118201 (metal- labeled in- house) YES B220 RA3-6B2 176Yb DVS sciences 3176002B YES YES
https://openalex.org/W4304460108	https://zenodo.org/records/7186199/files/WJARR-2022-0366.pdf	English	null	Clostridioides (Clostridium) difficile infection: Review of literature	Zenodo (CERN European Organization for Nuclear Research)	2,022	cc-by	6,487	Abstract Clostridioides (Clostridium) difficile (C. difficile) is a gram-positive bacterium that infects the large intestine. The number of clostridium difficile infections has increased in the recent years due to multiple risk factors including frequent use of antibiotics and proton pump inhibitors. The virulence of C. difficile comes from its production of toxins. Treatment for C. difficile infection includes the use of antibiotics, monoclonal antibodies, or a fecal transplant. Keywords: Clostridioides Difficile; Clostridium difficile; Antibiotics; Toxin; Diagnosis; Treatment; Monoclonal Antibodies Clostridioides (Clostridium) difficile infection: Review of literature 1 PGY2, Department of Internal Medicine Sunrise Health GME Consortium 2880 N Tenaya Way 2nd Floor Las Vegas, NV, USA. 2 PGY3, Department of Internal Medicine Sunrise Health GME Consortium Las Vegas, NV, USA 3PGY1, Department of Internal Medicine Sunrise Health GME Consortium Las Vegas, NV, USA. 3 PGY1, Department of Internal Medicine Sunrise Health GME Consortium Las Vegas, NV, USA. 4 Faculty Attending, Department of Internal Medicine Sunrise Health GME Consortium Las Vegas, NV, USA. 5 Mountain View Medical Center. 6 HCA Healthcare Nashville TN World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 Publication history: Received on 22 March 2022; revised on 26 April 2022; accepted on 28 April 2022 ublication history: Received on 22 March 2022; revised on 26 April 2022; accepted on 28 April 2022 Article DOI: https://doi.org/10.30574/wjarr.2022.14.2.0366  Corresponding author: George Trad Department of Internal Medicine Sunrise Health GME Consortium 2880 N Tenaya Way 2nd Floor Las Vegas, NV, USA. Copyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. opyright © 2022 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attr or(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. Corresponding author: George Trad epartment of Internal Medicine Sunrise Health GME Consortium 2880 N Tenaya Way 2nd Floor Las Vegas, NV,  Corresponding author: George Trad 3.1 Antibiotic use As mentioned above, the main risk factor for a C. difficile infection is the use of pharmacological agents, mainly antibiotics (figure 1). The idea is that antibiotics alter the composition of normal colonic microbial populations, providing a niche for C. difficile to flourish and produce toxins. Many antibiotics can cause C. difficile infection, but there are few that tend to be frequently associated with the infection such as clindamycin, fluoroquinolones, and cephalosporins. Figure 1 Antibiotics that are associated with C. difficile infection Figure 1 Antibiotics that are associated with C. difficile infection 1. Introduction Clostridioides (Clostridium) difficile (C. difficile), also known as the difficult clostridium, was initially identified in 1935 as Bacillus difficilis where it was found in the fecal flora of healthy infants [1]. In 1977, C. difficile was identified as the cause of human infections, but it was only found in sporadic cases at that time. In 2000, C. difficile became an urgent public health threat due to patients presenting with severe symptoms ultimately causing death [2]. In 2005, the first outbreak from C. difficile was officially reported [3]. C. difficile survives in a resistant spore form once outside the colon. These spores are heat, acid, and antibiotic resistant. Once in the intestine, C. difficile spores convert to their fully functional vegetative form. These toxins, known as enterotoxin and cytotoxin, are released by C. difficile leading to severe inflammation and necrosis of the mucosa [4]. When it comes to the risk of developing an active C. difficile infection (CDI), pharmacological agents are known to be the main risk factor. Other factors include a history of diabetes mellitus, inflammatory bowel disease, chronic kidney disease, or cancer. To formally diagnose CDI, the bacteria need to be identified, as well as the genes associated with toxin production, and/or detect the toxin produced by bacteria in watery stool. Treatment of C. difficile is very complex and requires an alternating antibiotic regimen depending on the patient's comorbidities, allergies, presentation, and bacterial resistance to antibiotics. Treatment usually involves an infectious disease specialist to closely monitor the patient's clinical course. World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 2. Epidemiology Until 2000, C. difficile infection was not a well-known infection, but due to the emergence of a previously rare virulent strain B1/NAP1/027, the incidence of C. difficile infection has increased dramatically, making it a global health challenge [5]. It has been estimated that C. difficile is responsible for 15-25% of cases of antibiotic associated diarrhea, and all cases of antibiotic-associated pseudomembranous colitis [6]. In terms of individuals who acquire CDI, it is estimated that C. difficile causes approximately 500,000 infections annually in the United States, of which 290,000 are hospital associated infections, and 160,000 are community acquired [7]. Up to 4.8 billion dollars are spent each year on treating C. difficile infections in U.S. hospitals [8]. Patients over 65 years of age were noted to have a dramatic increase in CDI with double the number of cases for 85- year-olds and above. In 2017, community acquired CDI was also reported in 49% of all CDI cases which is a significantly higher percentage compared to previous years. One of the main factors that lead to the increased CDI in community settings is that C. difficile has a highly fluid genome that can modify its content and make it more adaptable to the environment. Community acquired CDI represents a growing public health threat and burden on the health care systems [7]. In addition, there was a decline in the incidence of healthcare associated CDI from 99.6 to 73.3 per 100,000 population primarily driven by a decrease in health care associated infections [9]. An estimated 83,000 patients with CDI have at least one recurrence, and 29,300 patients will die. Up to 25% of patients experience recurrent CDI within 30 days of treatment. Less commonly, recurrent CDI can occur as late as two months after discontinuation of treatment [7]. 3.2 Advanced age Advanced age is not only a risk for developing C. difficile infection, but these patients are also at an increased risk of morbidity from CDI [10]. An observation study was done in 2002 following the outbreak in Quebec, it was postulated 147 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 that persons over 65 years of age were 10-fold higher in frequency at acquiring CDI than younger adults. The reasons behind this association are unclear but may be multifactorial. Host factors of older individuals such as a reduced immune response to CDI, hospitalization, frequent antibiotic use, or inherent dysbiosis may be among the factors that play a role [11]. that persons over 65 years of age were 10-fold higher in frequency at acquiring CDI than younger adults. The reasons behind this association are unclear but may be multifactorial. Host factors of older individuals such as a reduced immune response to CDI, hospitalization, frequent antibiotic use, or inherent dysbiosis may be among the factors that play a role [11]. 4. Pathogenesis C. difficile is a spore-forming anaerobe that is found in the intestinal flora. Primary, CDI can be triggered using antibiotics to treat another condition. Additionally, CDI can be a secondary infection by the ingestion of spores from the environment, hence cross-contamination via health-care staff [10]. When in its spore state, C. difficile remains dormant, but also resistant to a variety of environmental factors such as heat, acid, and antibiotics. When C. difficile is activated by bile acid, it converts to its vegetative state that produces two principal toxins which can cause severe diarrhea and eventually cause life threatening pseudomembranous colitis [20, 21]. The virulence of C. difficile comes from its production of toxins. The toxins, Toxin A and B, act on intestinal epithelial and inflammatory cells causing tissue destruction and inflammation. The mechanism behind colonocyte death is through inactivation of the Rho family of guanosine tri-phosphatases which subsequently leads to loss of intestinal barrier function and colitis. Toxin A binds to the brush border of the colonocyte and disrupts the cytoskeleton integrity. This then leads to intestinal fluid secretion and mucosal injury, which causes the patient to have copious amounts of watery diarrhea. Mediators of this pathway include arachidonic acid metabolites, substance P, tumor necrosis factor, IL-8, IL-6, and IL-1. Neutrophils are also directly activated by Toxin A and promote chemotaxis to localize in the pseudo membrane and mucosal layer. Toxin B, found to be cytotoxic, directly destroys enterocyte cytoskeleton structure and causes a pseudomembrane to develop [22]. 3.3 Gastric acid suppression Proton pump inhibitors (PPIs) are widely used in the hospital setting as prophylaxis for gastric ulcer disease. PPIs have been associated with C. difficile infection. In fact, C. difficile infection has become one of the scariest side effects when it comes to prescribing PPIs. The risk of CDI increases to 1.4 to 2.75 times higher in patients with PPI exposure than with those without PPI exposure, associating gastric acid suppression to an increased risk of CDI [12, 13, 14]. Currently, there are no randomized clinical trials that can state that PPIs cause C. difficile infection, but multiple observational studies have shown the association. 3.4 Liver cirrhosis Patients with liver cirrhosis are at a higher risk of CDI due to recurrent hospitalization visits, frequently administered prophylaxis antimicrobial therapies, chronic PPI use, and immunodeficiency [15]. The use of antibiotics as well as PPIs significantly increase the risk of CDI in healthy populations; therefore, it is expected to increase the risk in cirrhosis patients as well, especially since they are immunodeficient and require frequent hospitalizations. Not only that, but cirrhosis patients are at higher risk of comorbidities and mortality rates from CDI as well. Other risk factors for acquiring C. difficile infection include enteral feeding, gastrointestinal surgery, obesity, cancer chemotherapy, hematopoietic stem cell transplantation, or inflammatory bowel disease [9, 16, 17, 18, 19]. 5. Diagnosis No single test can accurately diagnose CDI; therefore the European Society of Clinical Microbiology and Infectious Diseases (ESCMID) recommends a two or three step algorithm consisting of a screening test with high sensitivity followed by a more specific test to detect free toxins (figure 2) [10]. Not every patient presenting with diarrhea should be tested for C. difficile. Patients should be tested if they are presenting with acute watery diarrhea (> 3 loose stools) and have associated risk factors such as recent use of antibiotics, recent hospitalization, or are over 65 years old. Liquid stool, noted to be within 24 hours, is the only appropriate specimen for testing [23]. The first test performed to diagnose C. difficile is an enzyme immunoassay (EIA) of glutamate dehydrogenase (GDH) antigen test which is a rapid and highly sensitive test. If the test is positive, the following test should be the EIA for Toxin A and B. If one of the toxins is positive, then it is confirmatory for CDI. If the EIA for toxins is negative, but there is still a high suspicion of CDI, it is recommended to obtain a cell culture cytotoxicity assay or nucleic-acid amplification test (NAAT). Both cytotoxicity assay and NAAT are highly sensitive, but cytotoxicity assay takes a long time to run while NAAT can over diagnose CDI by detecting a colonization state. Cell culture is not obtained as a standalone test due to 148 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 the inability to confirm whether toxins are present or not. Fecal leukocyte testing is considered to have poor sensitivity when compared to toxin assay and is no longer recommended in the screening for C. difficile [24]. Figure 2 C. difficile testing protocol Figure 2 C. difficile testing protocol Figure 2 C. difficile testing protocol 8.2 Presence of ileus In patients with fulminant colitis without ileus consideration of addition of vancomycin rectally or fecal microbiota transplant is warranted. However, the risk of colonic perforation is also present with these treatments and therefore should be restricted to the patients who are unresponsive to the standard therapy. If rectal vancomycin is given, it is in addition to the oral vancomycin [32, 33, 34 44-46]. The dosing for rectal vancomycin is not yet established and is often given as a retention enema (500 mg in 100 cc of normal saline every six hours). If recovery is delayed, treatment can be extended up to 14 days from 10 days. In one case series which included nine patients with refractory symptoms, toxic megacolon, or fulminant colitis; eight patients had complete resolution of symptoms and one died from multi-organ failure [32]. 6. Treatment Metronidazole and oral vancomycin have been the mainstay of treatment for C. difficile infection since the 1970s. However, there have been marked increases in the failure associated with metronidazole, especially in patients with the BI/NAP1/027 strain [23]. Due to the failure of metronidazole, the guidelines for managing C. difficile was updated in 2018 with oral metronidazole as no longer the first-line therapy for adults. Currently, fidaxomicin and oral vancomycin are first-line treatments [24]. Choosing which antibiotic is the right treatment for CDI, physicians need to assess the severity of the disease which can be classified into non-fulminant vs fulminant colitis. Non-fulminant can be broken into a non-severe disease which presents with white blood cell count ≤15,000 cells/mL and serum creatinine <1.5 mg/dL, while severe disease presents with a white blood cell count >15,000 cells/mL and/or serum creatinine ≥1.5 mg/dL. Fulminant colitis on the other hand presents with shock symptoms such as hypotension as well possible ileus and toxic megacolon. The main indication for treatment is when a patient presents with acute diarrhea ≥3 loose stools in 24 hours with no other obvious explanation and a positive diagnostic assay. The use of laxatives should be excluded while diagnosing CDI. In addition, if the clinical suspicion is high, treatment can be started empirically while waiting laboratory confirmation. 149 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 10. Recurrent CDI Recurrent C. difficile is defined as re-appearance of symptoms within two months after complete resolution of infection after appropriate therapy. The approach to the recurrent C. difficile remains the same but varies depending on the number of recurrences. 8.1 Absence of ileus Patients with fulminant colitis without ileus can be treated with oral vancomycin 500 mg every six hours in addition to intravenous (IV) metronidazole 500 mg every eight hours. Data regarding the IV metronidazole is limited. A lower mortality rate was observed among the patients who received dual therapy than those that received monotherapy (36% versus 16%) [31, 32]. 7. Non-Fulminant Colitis Following a meta-analysis study that included 22 randomized trails, researchers found that patients presenting with non-severe non-fulminant CDI will benefit from one of the following treatments: oral vancomycin (bacteriostatic): 125 mg every six hours for 10 days or fidaxomicin (bactericidal): 200 mg every 12 hours for 10 days [25]. The recurrence rate of C. diff infection with fidaxomicin has been lower as compared to oral vancomycin with non-NAP1 strain. However, the recurrence rate has been similar with vancomycin and fidaxomicin in NAP1 strain. [26, 27, 28]. For severe non-fulminant C. difficile infection, the treatment remains the same as non-severe along with the close monitoring and supportive care of the patients. In addition, surgery should also be consulted. 8. Fulminant Colitis Surgery consultation along with antibiotics and symptomatic treatment are the mainstay of treatment. Early surgical consultation helps in diagnosing severe disease, who will benefit from surgery, and timely operative management if the condition worsens. The approach to the antibiotics therapy depends on if ileus is present. 9. Indications for Surgical Consultation  Hypotension with or without use of vasopressors, fever > 38.5 C, ileus, peritonitis, encephalopathy, WBC > 20,000 cells/ml, lactic acid > 2.2, ICU admission, end organ dysfunction, or failure to improve after three to five days of maximum medical therapy.  Hypotension with or without use of vasopressors, fever > 38.5 C, ileus, peritonitis, encephalopathy, WBC > 20,000 cells/ml, lactic acid > 2.2, ICU admission, end organ dysfunction, or failure to improve after three to five days of maximum medical therapy.  Toxic megacolon should be suspected if the patient develops abdominal distension with minimum diarrhea which is due to paralytic ileus resulting from loss of colonic tone.  Toxic megacolon should be suspected if the patient develops abdominal distension with minimum diarrhea which is due to paralytic ileus resulting from loss of colonic tone. 10.1.1 First recurrence  If oral Vancomycin was used, treatment needs to be tapered off over six weeks: o 125 mg every six hours for 10 to 14 days followed by o 125 mg twice daily for seven days followed by o 125 mg once daily for seven days followed by o 125 mg every two to three days for two weeks  Alternative: fidaxomicin 200 mg twice daily for 10 days  If oral Vancomycin was used, treatment needs to be tapered off over six weeks: o 125 mg every six hours for 10 to 14 days followed by o 125 mg twice daily for seven days followed by o 125 mg once daily for seven days followed by o 125 mg every two to three days for two weeks  Alternative: fidaxomicin 200 mg twice daily for 10 days 150 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 10.1.3 Third recurrence Consider fecal microbiota transplant  Fulminant C. difficile: treatment remains the same as that of primary or first episode of CDI.  Fulminant C. difficile: treatment remains the same as that of primary or first episode of CDI.  Pulse-tapered dosing of vancomycin is believed to facilitate a gradual return of the normal colonic flora. Again, the data on the pulse-tapered regimen is very limited. Vancomycin followed by rifaximin was evaluated in two small studies. In one series, 7 out of 8 patients who were treated had no further recurrence of infection [35]. Pulse tapered dosing of vancomycin is believed to facilitate a gradual return of the normal colonic flora. Again, the data on the pulse-tapered regimen is very limited. Vancomycin followed by rifaximin was evaluated in two small studies. In one series, 7 out of 8 patients who were treated had no further recurrence of infection [35]. 11. Microbial replacement therapy It is well understood that antibiotics disrupt the normal flora of the gut which facilitates the proliferation of C. difficile and in doing so, the production toxins increase the risk of C. difficile infection and recurrence. Due to the alteration of the microbiome, the pathogenesis of C. difficile infection has led to the development of the microbial replacement therapies (MRT).  Nasogastric, nasoduodenal tube, colonoscopy and enema based microbial replacement  Capsule-based therapies In a systemic review, which included seven randomized controlled trials and 30 case series, capsule-based therapies proved to be beneficial in 92% of the patient treated with MRT [36]. MRT is administered in multiple ways as mentioned above. A study done in Netherlands showed clinically significant results in 81% of patients after duodenal fecal infusion MRT versus 31% in vancomycin group ( However, there is not sufficient data to suggest the use of MRT for treatment of refractory severe CDI. A case series of nine patients showed a resolution of CDI in all the patients with recurrence in one patient who was receiving antibiotics [37]. Currently, there are clinical trials to assess the use of MRT as a primary treatment of moderate to severe CDI or its use before antibiotics. 10.1.2 Second recurrence Vancomycin pulsed tapered as above or Fidaxomicin 200 mg twice daily for 10 days or Vancomycin 125 mg every six hours for 10 days followed by rifaximin 200 mg every eight hours for 20 days or fecal microbiota transplant. 11.2 Capsule-based therapy SER-109 is an oral capsule that contains spores derived from stool of healthy donors. A study was done in 30 patients with rCDI who were treated with oral antibiotics which showed a resolution of symptoms in 96.7% [40]. The diversity of the gut microbiome increased after SER-109 administration. For example, Bacteroides, a dominant bacterium in healthy populations that is not present in SER-109, increased in 38% of the patients. The prevalence of some pathogens like Klebsiella decreased by 92% by fourth week. Adverse effects of capsule-based therapy include nausea, mild diarrhea, and abdominal pain. However, phase two trials did not show any superiority to the placebo. Phase three trials are currently ongoing. Similar products are CP101, RBX7455, SER-262 and all have ongoing trials. 11.1 Enema based microbial replacement RBX2660, a microbiota-based suspension derived from donor stool, is being studied in clinical trials for treatment of recurrent CDI (rCDI). Data from phase two trial showed that RBX2660 was superior to placebo with no adverse events. It showed that 51.6 % of patients recovered after the first treatment, and 78.6% who received the second treatment. The overall response was 87.1% [38]. In another phase two study of RBX2660, it met the primary end point of preventing rCDI at eight weeks with success rate of 78.8% compared with 51.8% (p<0.001) in historical control treated with antibiotics alone [39]. Phase three is currently ongoing. 12. Role of probiotics Probiotics are the live micro-organisms that are given to restore the gut flora. Many physicians’ debate on prescribing probiotics due to lack of objective data stating their benefit. A pilot randomized controlled trial was done which included 33 participants from February 2013 to February 2015. This trial found that probiotic adjunctive therapy was associated with significant improvement in diarrhea outcomes; however, there was no significant difference in the rate of CDI or functional improvement over time between treatment groups [44]. In a meta-analysis which included all the published reports from 1966 to 2007, four studies met the inclusion criteria. One out of these four studies showed a benefit of adding probiotics as adjunctive therapy while the other three showed no significance data [45]. 14. Prevention Antibiotic stewardship plays one of the most important roles in preventing CDI. Minimizing the use of antibiotics has been shown to decrease CDI [46]. One way to prevent CDI is minimizing use of gastric acid suppression medication. In a meta-analysis which included 7703 patients with CDI, 1525 patients (19.8%) developed rCDI. The rate of rCDI in patients with gastric acid suppression was 22.1% (892 of 4038 patients) compared with 17.3% (633 of 3665) in patients without gastric acid suppression, which indicated an increased risk (odds ratio [OR], 1.52; 95% CI, 1.20-1.94; P < .001) [46]. World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 Bezlotoxumab is the first FDA approved human monoclonal antibody which has shown to reduce rCDI in patients 18 years or older receiving antibacterial drug treatment. The mechanism of action is targeted towards Toxin B and neutralizes its effect [41]. Bezlotoxumab is used in conjunction with an antibiotic regimen. In phase three trials, the use of anti-toxin A was not shown to reduce the likelihood of rCDI [42]. During these trials, cases of heart failure were reported. So, it should be used with caution in patients with history of heart failure if the benefits outweigh the risks. In patients with a history of heart failure, there were more deaths in Bezlotoxumab treated patients 19.5% (n=23/118) than in placebo-treated patients 12.5% (n=13/104). The causes of death varied and included cardiac failure, infections, and respiratory failure [43]. Pooled intravenous immunoglobulin likely has C. difficile antitoxin and has been used as an adjunctive therapy in severe and refractory CDI [44]. There is a very limited data available that shows the benefit of IVIG. A retrospective study which included 18 patients with severe CDI who received IVIG in addition to IV metronidazole and/or oral/rectal vancomycin and 61 patients did not receive IVIG therapy. This study showed no significant difference in the study groups regarding mortality or length of stay in the hospital [44]. 13. Other antimicrobials There are several other antibiotics that have been used clinically in patients but due to limited data, cost, and questionable outcomes their use is not recommended. These antibiotics are Tigecycline, Teicoplanin, Ramoplanin, Rifaximin, Nitazoxanide, Teicoplanin, Bacitracin, Cadazolid, and Surotomycin. 11.3 Antibody-based therapy The immune response against the C. difficile toxin is an important factor that determines outcomes. The higher the concentration of the antibodies in the serum, the lower the risk of rCDI. Current treatment options include:  Bezlotoxumab  Intravenous immunoglobulin 151 References [1] Curry S. Clostridium difficile. Clin Lab Med. 2010 Mar; 30(1):329-42. doi: 10.1016/j.cll.2010.04.001. PMID: 20513554; PMCID: PMC5501328. [1] Curry S. Clostridium difficile. Clin Lab Med. 2010 Mar; 30(1):329-42. doi: 10.1016/j.cll.2010.04.001. PMID: 20513554; PMCID: PMC5501328. 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Treatment of C. difficile is dependent on patient’s presentation. Complication can arise following C. difficile infection as well due to medications that are used to treat C. difficile, therefore patients with C. difficile infection must be monitored closely. 152 World Journal of Advanced Research and Reviews, 2022, 14(02), 146–155 Acknowledgments This research was supported (in whole or in part) by HCA Healthcare and/or an HCA Healthcare affiliated entity. The views expressed in this publication represent those of the author(s) and do not necessarily represent the official views of HCA Healthcare or any of its affiliated entities. Disclosure of conflict of interest This research was supported (in whole or in part) by HCA Healthcare and/or an HCA Healthcare affiliated entity. The views expressed in this publication represent those of the author(s) and do not necessarily represent the official views of HCA Healthcare or any of its affiliated entities. 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https://openalex.org/W4387402531	https://www.nature.com/articles/s41598-023-43964-4.pdf	English	null	Impact of humid climate on rheumatoid arthritis faecal microbiome and metabolites	Scientific reports	2,023	cc-by	9,257	Impact of humid climate on rheumatoid arthritis faecal microbiome and metabolites Dingnan Wang 1,3, Zhili Zheng 1,3, Han Yu 2,3, Dou Dou 1, Yining Gao 1, Shuang Xu 1, Zhiming L Lili Sun 1, Xudong Qiu 1 & Xianggen Zhong 1* OPEN Studies have shown that high humidity is a condition that aggravates the pain of rheumatoid arthritis (RA), but the relevant mechanism is controversial. Currently, there is a lack of experimental animal studies on high humidity as an adverse factor related to the pathogenesis of RA. We used healthy SD rats and collagen-induced arthritis (CIA) rats to investigate the effects of high humidity on arthritis. Integrated metabolomics analyses of faeces and 16S rRNA sequencing of the faecal microbiota were performed to comprehensively assess the diversity of the faecal microbiota and metabolites in healthy and CIA rats. In this study, high humidity aggravated arthritis in CIA rats, which manifested as articular cartilage lesions, increased arthritis scores, and an increase in proinflammatory cytokines. High humidity had a certain effect on the articular cartilage extent, arthritis score and proinflammatory cytokines of healthy rats as well. Furthermore, high humidity caused significant changes in faecal microbes and metabolites in both healthy and CIA rats. 16S rRNA sequencing of faecal samples showed that high humidity increased the amount of inflammation-related bacteria in healthy and CIA rats. Faecal metabolomics results showed that high humidity significantly altered the level of faecal metabolites in healthy rats and CIA rats, and the changes in biological functions were mainly related to the inflammatory response and oxidative stress. Combined analysis showed that there was a strong correlation between the faecal microbiota and faecal metabolites. High humidity is an adverse factor for the onset and development of RA, and its mechanism is related to the inflammatory response and oxidative stress. However, the question of how high humidity impacts RA pathogenesis needs to be further investigated. Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory changes in synovial tissues, cartilage, and bone1; chronic destructive polyarthritis is the main clinical manifestation of RA. The aetiology of RA remains unclear, and previous studies have shown that genetic and environmental factors can promote the development of RA2,3. Periodontal disease, smoking and diet can induce RA onset in genetically susceptible individuals4–6. It is also commonly reported that climate and the environment are associated RA pathogenic factors7. Cold and humidity are climate and environmental factors associated with an increased risk for RA8–11. Studies have shown that weather can affect the pain of RA in middle-aged patients12, especially in female patients aged 41–65 years13. Low temperature, high air pressure and high humidity are significantly correlated with pain in RA patients14. www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2023) 13:16846 www.nature.com/scientificreports/ that is increased based on relative humidity. In a clinical study from 1998 to 2001, humid conditions at a health centre directly induced rheumatic symptoms, including RA19.h The microbiome is critical to the balance of the human immune system and regulates a variety of functions as part of the human immune system20,21. The structure of healthy faecal microbiota can maintain immune balance and inhibit inflammatory responses. The microbiota significantly influences the development of joint lesions in inflammatory diseases, including RA and osteoarthritis22,23. Furthermore, dysregulated faecal microbes are associated with a variety of autoimmune diseases, including psoriasis, RA, and other immune system diseases24,25. The faecal microbiota and RA are closely related. On the one hand, dysbiosis of the faecal microbiota is common in RA patients26,27; on the other hand, regulating a faecal microbiota imbalance can effectively alleviate the occur- rence and development of RA28. Clinical studies have shown that actinomycetes are bacteria that may directly induce RA29. Moreover, early biomarkers of RA found in the body’s blood or other body and tissue fluids can be used for the early diagnosis of RA patients and the prediction of treatment effects30. The metabolism of the host is regulated by its own genes and the gene composition of the symbiotic microbiota in the body; further- more, metabolomics can efficiently screen biomarkers and comprehensively analyse the molecular mechanism underlying the health of the body31. The combination of microbiome and metabolome analyses can provide a comprehensive picture of host-microbiome interactions32. p p In this study, to investigate the relationship between humidity and the onset and development of RA, healthy SD rats and collagen-induced arthritis (CIA) rats were exposed to high humidity (80 ± 5%). Foot and ankle histopathology and joint analysis were used to evaluate the effect of high humidity on the onset and devel- opment of arthritis in healthy and CIA rats. The abundance of faecal microorganisms was analysed by 16S rRNA high-throughput sequencing technology. Differentially activated metabolic pathways and differentially expressed metabolites were identified by untargeted metabolomics via gas chromatography‒mass spectrometry (GC–MS). Effects of high humidity on colonic symptoms and faecal moisture levels in healthy rats and CIA model rats We analyzed colonic pathology and fecal moisture levels on Day 28 and Day 60 to assess potential early changes in fecal characteristics. The impact of elevated humidity on the colon is depicted in Fig. 1. Notably, the HH group (Fig. 2b,f.) displayed enhanced accumulation of inflammatory cells compared to the CON group (Fig. 2a,e). Fur- thermore, compared to the CIA group (Fig. 2c,g), the H + CIA group (Fig. 2d,h) exhibited pronounced mucosal surface damage along with exacerbated accumulation of inflammatory cells. Notably, on Day 60, the severity of inflammation and tissue damage was considerably more prominent in the HH group, CIA group, and H + CIA (Fig. 2f,g,h) group compared to Day 28. The histological scores are presented in Fig. 2i.i g g g p p yh g p g Regarding faecal moisture content, a significant increase was observed in the HH group relative to the CON group. Similarly, the H + CIA group showed a significantly higher faecal moisture content than the CIA group (Fig. 2j). Of particular interest was a significant elevation in faecal moisture levels on Day 60 compared to Day 28 in the HH group, CIA group, and H + CIA group. Results Effects of high humidity on arthritis symptoms and inflammation in healthy rats and CIA model ratsf To evaluate the effect of high humidity on arthritis, we observed changes in arthritis symptoms in healthy rats and CIA rats under normal-humidity (CON, 50 ± 5%) and high-humidity (HH, 80 ± 5%) conditions. Compared to those in the CON group (1a), ankle redness and swelling were observed in the HH group (1b). Compared to the CIA group (1c), the HH + CIA group exhibited more severe ankle swelling and joint deformation (1d). The effects of high humidity on articular cartilage in healthy rats and CIA rats are shown in Fig. 1. Compared to that in the CON group (1e), the staining of the cartilage in the HH group (1f.) was reduced. Compared to the CIA group (1 g), many of the cartilage samples were not stained, and chondrocytes were significantly reduced in the HH + CIA group (1 h). This result suggests that high humidity can cause or aggravate cartilage damage. Compared to those in the CIA group, arthritis scores were significantly increased in the HH + CIA group at Days 42, 49 and 56. Compared to those in the CON group, arthritis scores were significantly increased in the HH group at Days 49 and 56 (1i).ll g p y We investigated inflammation based on three proinflammatory cytokines in the rats of each group. Compared to the CON group, high humidity caused the upregulation of TNF-α in the HH group, and the difference was statistically significant (Fig. 1j). High humidity led to the upregulation of IL-6 and IL-17 in the HH group, but the difference was not statistically significant (Fig. 1k; 1 l). Compared with the CIA group, high humidity led to the upregulation of IL-6, IL-17 and TNF-α in the HH + CIA group (Fig. 1k; 1 l; 1 m), and the difference was statistically significant. Impact of humid climate on rheumatoid arthritis faecal microbiome and metabolites Dingnan Wang 1,3, Zhili Zheng 1,3, Han Yu 2,3, Dou Dou 1, Yining Gao 1, Shuang Xu 1, Zhiming L Lili Sun 1, Xudong Qiu 1 & Xianggen Zhong 1* OPEN Humidity is a frequently studied climate and environmental factor. An animal experiment showed that high humidity aggravated the severity of arthritis in CIA mice by upregulating xylitol and L-pyroglutamate expression15. The results confirmed clinical observations that high humidity could aggravate pain and stiffness in RA patients16. Research has shown that the combination of temperature and humidity creates a microclimate near the skin, and the humidity of the microclimate is affected by skin sweat glands on the water vapour surface. The microclimate could increase pain in RA patients by producing local vapour pressure. There is a positive correlation between humidity in the microclimate and RA-related pain17. Furthermore, researchers found that women living in damp houses had a higher risk of knee aches18, which may be linked to the autoimmune response 1Institute of Synopsis of Golden Chamber Department, School of Chinese Medicine College, Beijing University of Chinese Medicine, Beijing 100029, People’s Republic of China. 2Formulas of Chinese Medicine, Basic Medical College of Chengdu University of Traditional Chinese Medicine, Chengdu 611137, Sichuan, People’s Republic of China. 3These authors contributed equally: Dingnan Wang, Zhili Zheng and Han Yu. email: zhongxg@ bucm.edu.cn \| https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 www.nature.com/scientificreports/ www.nature.com/scientificreports/ This study aimed to (1) explore the effects of high humidity on the joints of healthy rats and CIA rats, (2) determine the composition and abundance within the faecal microbiota in healthy rats and CIA rats under high-humidity conditions, (3) measure the composition of faecal metabolites in healthy rats and CIA rats under high-humidity conditions, and 4) correlate faecal microbial diversity with GC–MS untargeted metabolomics results to explore the relationship between high humidity and the onset and development of RA. Furthermore, this study aimed to determine the potential biological impact of high humidity to provide novel insights into the impact of high humidity on RA. Faecal Microbiota Analysis Alpha diversity analysis usually reflects faecal microbiome abundance. Good’s diversity index (3a), Shannon’s diversity index (3b) and the Specaccum species accumulation curve (3c) are shown in Fig. 3. The results showed https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 \| www.nature.com/scientificreports/ ww.nature.com/scientificreports/ Figure 1. Effects of high humidity on arthritis symptoms and inflammation in healthy rats and CIA model rats. Representative images of ankle joints. (a) CON; (b) HH; (c) CIA; (d) H + CIA. Histopathological examination of articular cartilage by Saffron O staining. (e) CON; (f) HH; (g) CIA; (h) H + CIA. (i) arthritis scores. (j) Serum proinflammatory cytokines levels of rats in the each group. (k) IL-6; (l) IL-17; (m) TNF-α. Compared to that in the CON group, p < 0.05, *p < 0.01; compared to that in the CIA group, #p,0.05, ##p < 0.01; N.S, represents no significance. Figure 1. Effects of high humidity on arthritis symptoms and inflammation in healthy rats and CIA model rats. Representative images of ankle joints. (a) CON; (b) HH; (c) CIA; (d) H + CIA. Histopathological examination of articular cartilage by Saffron O staining. (e) CON; (f) HH; (g) CIA; (h) H + CIA. (i) arthritis scores. (j) Serum proinflammatory cytokines levels of rats in the each group. (k) IL-6; (l) IL-17; (m) TNF-α. Compared to that in the CON group, p < 0.05, *p < 0.01; compared to that in the CIA group, #p,0.05, ##p < 0.01; N.S, represents no significance. that the sequencing data fully reflected the information about bacterial communities in the samples. The results of beta diversity analysis reflected sample differences between groups. Principal coordinate analysis (PCoA, unweighted UniFrac, Fig. 3d) and nonmetric multidimensional scaling (NMDS) (Fig. 3e) were used to analyse the effects of humidity on the microbial communities of each group. The results showed that there were signifi- cant differences among the groups, and high humidity had significant effects on the microbial communities of healthy rats and CIA rats.hf Then, linear discriminant analysis coupled with effect size measurement analysis (LEfSe) was used to identify biomarkers with significantly different abundances between each group (Fig. 4c). Compared to those in the CIA group, the most common bacteria in the HH + CIA group were Prevotella, Clostridial spore-forming bacteria and Ruminococcaceae_UCG_010 (Fig. 4b); compared to those in the CON group, the most common bacteria in the HH group were Deltaproteobacteria, Bacteroidaceae and Actinobacteria (Fig. 4a). Faecal Microbiota Analysis Notably, compared with that in the CON group, the abundance of Prevotella species in the HH group was reduced. This information is collectively shown in Fig. 4d. Multivariate analysis of faecal metabolite profileshif The score chart of PLS-DA showed significant differences (spectrum separation) between each group, and the ratios of R2 X (CUM), R2 Y (CUM) and Q2 (CUM) are shown in Fig. 5a. PLS-DA was used to screen out dif- ferential variables (Fig. 5b-d).fh Differentially expressed metabolites were determined based on a VIP > 1 and an adjusted p value < 0.05. results showed that a total of 108 metabolites were significantly expressed between the HH group and CON gro Scientific Reports \| (2023) 13:16846 \| https://doi.org/10.1038/s41598-023-43964-4 www.nature.com/scientificreports/ w.nature.com/scientificreports/ Figure 2. Effects of high humidity on colonic symptoms and faecal moisture levels were evaluated in healthy rats and CIA model rats. (a) CON 28 days; (b) HH 28 days; (c) CIA 28 days; (d) H + CIA 28 days; (e) CON 60 days; (f) HH 60 days; (g) CIA 60 days; (h) H + CIA 60 days. Histopathological examination of the colon by Hematein Eosin staining. (i) Histological scores. (j) Faecal moisture levels. Compared to that in the CON group, p < 0.05, *p < 0.01; compared to that in the CIA group, #p,0.05, ##p < 0.01; compared to that in the 60 days, $p < 0.05, $$p < 0.01, N.S, represents no significance. Figure 2. Effects of high humidity on colonic symptoms and faecal moisture levels were evaluated in healthy rats and CIA model rats. (a) CON 28 days; (b) HH 28 days; (c) CIA 28 days; (d) H + CIA 28 days; (e) CON 60 days; (f) HH 60 days; (g) CIA 60 days; (h) H + CIA 60 days. Histopathological examination of the colon by Hematein Eosin staining. (i) Histological scores. (j) Faecal moisture levels. Compared to that in the CON group, p < 0.05, *p < 0.01; compared to that in the CIA group, #p,0.05, ##p < 0.01; compared to that in the 60 days, $p < 0.05, $$p < 0.01, N.S, represents no significance. Figure 2. Effects of high humidity on colonic symptoms and faecal moisture levels were evaluated in healthy rats and CIA model rats. (a) CON 28 days; (b) HH 28 days; (c) CIA 28 days; (d) H + CIA 28 days; (e) CON 60 days; (f) HH 60 days; (g) CIA 60 days; (h) H + CIA 60 days. Histopathological examination of the colon by Hematein Eosin staining. (i) Histological scores. (j) Faecal moisture levels. Multivariate analysis of faecal metabolite profileshif Compared to that in the CON group, p < 0.05, *p < 0.01; compared to that in the CIA group, #p,0.05, ##p < 0.01; compared to that in the 60 days, $p < 0.05, $$p < 0.01, N.S, represents no significance. and 119 different metabolites were significantly differentially expressed in the HH + CIA group and CIA group. In the HH group vs. CON group comparison, the subclasses of differentially expressed metabolites were associated with amino acids, peptides and analogues (18 differentially expressed metabolites), carbohydrates and conjugates (8 differentially expressed metabolites), and fatty acids and conjugates (6 differentially expressed metabolites). In the HH + CIA group vs. CIA group comparison, the subclasses of differentially expressed metabolites were associated with organic acids and derivatives (24 differential metabolites), lipids and function-like molecules (18 differential metabolites), and organic oxygen compounds (16 differential metabolites). The top 50 differentially expressed metabolites between each group are presented as a heatmap in Fig. 6. Pathway analysis of differentially expressed metabolites y yf y p Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was used to further analyse the function of differ- ent metabolites, and the results of significant pathway enrichment analysis are presented on a scatterplot graph (Fig. 7). The following 8 pathways of the top 20 pathways were activated when comparing to the HH vs. CON groups and the HH + CIA vs. CIA groups: ABC transporter pathways, purine metabolism, carbohydrate diges- tion and absorption, glutathione metabolism, steroid biosynthesis, basal cell carcinoma pathways, central carbon metabolism pathways in cancer, and primary bile acid biosynthesis. Discussion Weather and environmental factors can directly impact human health. Relative humidity has been shown to play an important role in the study of climate sensitivity in patients with chronic pain. Studies have shown that high humidity is a direct or indirect factor affecting many diseases34,35. To date, there has been a lack of experimental studies regarding high humidity as an adverse factor associated with the pathogenesis of RA. In this study, healthy and CIA rats were exposed to high-humidity, and it was confirmed that high humidity can aggravate the extent of arthritis symptoms, including pathological changes, arthritis score and inflammatory factor expression. At the same time, the mechanism of the effect of high humidity on the joints of healthy rats and CIA rats was explored from the perspective of faecal microbial and metabolite homeostasis. To account for these changes, faecal microbiome analysis was performed on the faeces of rats from each group. The faecal microbiota may indicate environmental risk factors for RA36. In this study, Prevotella, Clostridia, and Ruminococcaceae_UCG_010 were the most common bacteria after high humidity intervention in CIA rats, while Deltaproteobacteria, Bacteroidaceae, and Actinobacteria were the most common bacteria in high humidity intervention healthy rats. The faecal microbiome influences innate and adaptive immunity, and its imbalance can trigger inflammatory responses and increase the risk of autoimmune disease, leading to joint damage37,38. Animal experiments have shown that Prevotella, Ruminococcaceae and Clostridia were associated with RA because they induce the inflammatory response mediated by IL-6, IL-17 and IFN-γ cytokines39. In addition, studies have shown that Prevotella bacteria exhibit properties that increase inflammatory responses, possibly related to its ability to drive immune responses to Th17 cytokines40. In this study, high humidity aggravated joint injury in CIA rats, which may be related to the high abundance of bacterial species such as Prevotella, Clostridia, and Ruminococcaceae_UCG_010 induced by high humidity, which could promote the immune response of Th17 cells. It is possible that Th17 cell differentiation is activated and inflammatory cytokines increase, which would then induce an inflammatory response and aggravate joint injury. Notably, Deltaproteobacteria, Bacteroidaceae and Actinobacteria were significantly abundant in the HH group in this study. Previous studies have shown that Bacteroidaceae and Deltaproteobacteria are associated with RA4,41,42, while Actinobacteria, as a pathogen closely related to RA, may be an important factor that directly induces RA in damp environments19,29. Correlation of the faecal microbiota and metabolites To comprehensively analyse the relationship between faecal metabolism and the faecal microbiota, Spearman correlation analysis was performed for the HH vs. CON groups (Fig. 8a) and the HH + CIA vs. CIA groups (Fig. 8b). A correlation matrix network was constructed. The results showed that the paired correlations between the HH vs. CON groups and the HH + CIA vs. CIA groups indicated a strong correlation between the faecal microbiota and faecal metabolites. Prevotellaceae was identified as a significantly regulated microbial taxon at the genus level in the comparison between the HH and CON groups. It showed a significant negative correla- tion with Ciliatine. In the comparison between the CIA and H + CIA groups, Ruminococcaceae was found to be a significantly regulated microbial taxon. Specifically, within the Ruminococcaceae family, the Ruminococ- caceae_NK4A214_group exhibited a positive correlation with D-fucose while displaying a negative correlation https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 \| www.nature.com/scientificreports/ Figure 3. Species abundance and diversity. (a) Good’s diversity analysis; (b) Shannon’s diversity analysis; (c) Specaccum species accumulation curve; (d) NMDS analysis (Strees:0.08); (e) PCoA analysis (P = 0.0019). Figure 3. Species abundance and diversity. (a) Good’s diversity analysis; (b) Shannon’s diversity analysis; (c) Specaccum species accumulation curve; (d) NMDS analysis (Strees:0.08); (e) PCoA analysis (P = 0.0019). with 13 amino acids, including L-glutamic acid. Furthermore, the lactobacillus species, closely associated with RA, negatively correlated with L-lysine, oxamic acid, and methylamine. with 13 amino acids, including L-glutamic acid. Furthermore, the lactobacillus species, closely associated with RA, negatively correlated with L-lysine, oxamic acid, and methylamine. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Statistical analysis of microbial multivariate. (a) Differential species score map of HH vs. CON groups; (b) differential species score map of H + CIA vs. CIA groups; (c) example diagram of annotated branches of different species; (d) histogram of relative abundance. The solid line is the mean value of the relative abundance, and the dashed line is the median value of relative abundance. CIA VS. H + CIA group: d1, Prevotella; d2, Clostridial; d3, Ruminococcaceae_UCG_010. CON VS. HH group: d4, Deltaproteobacteria; d5, Bacteroidaceae; d6, Actinobacteria; d7, Prevotella. al multivariate. (a) Differential species score map of HH vs. CON p of H + CIA vs. CIA groups; (c) example diagram of annotated am of relative abundance. The solid line is the mean value of the s the median value of relative abundance. CIA VS. H + CIA group: d1, ccaceae_UCG_010. CON VS. HH group: d4, Deltaproteobacteria; d5, Prevotella. Figure 4. Statistical analysis of microbial multivariate. (a) Differential species score map of HH vs. CON groups; (b) differential species score map of H + CIA vs. CIA groups; (c) example diagram of annotated branches of different species; (d) histogram of relative abundance. The solid line is the mean value of the relative abundance, and the dashed line is the median value of relative abundance. CIA VS. H + CIA group: d1, Prevotella; d2, Clostridial; d3, Ruminococcaceae_UCG_010. CON VS. HH group: d4, Deltaproteobacteria; d5, Bacteroidaceae; d6, Actinobacteria; d7, Prevotella. Previous animal studies have shown that the abundance of Prevotellaceae decreased during the immune activa- tion stage of CIA43, which is consistent with our results. We speculate that high humidity may cause a certain degree of immune response, which may be related to RA. However, the objectivity of this conclusion still needs to be further explored. p Combined studies on the microbiome and metabolome are considered to be one of the best ways to assess host-microbiome interactions44. The pathogenesis of RA is multifactorial, and oxidative stress and inflamma- tory responses are associated with the onset and development of RA. In this study, ABC transporter pathways, primary bile acid biosynthesis, and glutathione metabolism were found to be the metabolic pathways that were significantly enriched, and all of these are associated with oxidative stress45. Carbohydrate digestion and absorp- tion and steroid biosynthesis were also metabolic pathways with significant enrichment, and these pathways are both related to digestion and absorption of the diet. Discussion At the same time, compared with the CON group, the abundance of Prevotellaceae in the HH group was significantly reduced, suggesting that high humidity intervention reduced the abundance of Prevotellaceae in healthy rats. https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 7. Scatterplot diagrams of KEGG pathways33. (a) HH vs. CON groups; (b) H + CIA vs. CIA groups. The x-axis shows the Rich factor, the colour of each circle indicates the p value, and the size of each circle reflects the number of metabolites of each pathway. Figure 7. Scatterplot diagrams of KEGG pathways33. (a) HH vs. CON groups; (b) H + CIA vs. CIA groups. The x-axis shows the Rich factor, the colour of each circle indicates the p value, and the size of each circle reflects the number of metabolites of each pathway. Figure 8. Correlation of the faecal microbiota and faecal metabolites. (a) HH vs. CON groups; (b) H + CIA vs. CIA groups. p < 0.05, *p < 0.01. The correlation matrices were obtained from the cloud platform of Shanghai OE Biotech, Inc. (https://cloud.oebiotech.cn/task/detail/correlation-multiomics-oehw/, version 1.8). Figure 8. Correlation of the faecal microbiota and faecal metabolites. (a) HH vs. CON groups; (b) H + CIA vs. CIA groups. p < 0.05, *p < 0.01. The correlation matrices were obtained from the cloud platform of Shanghai OE Biotech, Inc. (https://cloud.oebiotech.cn/task/detail/correlation-multiomics-oehw/, version 1.8). arthritis in CIA rats. In conclusion, high humidity can lead to the occurrence and development of RA by affecting oxidative stress and promoting the inflammatory response. Interestingly, these pathways also appeared in the comparison between the HH and CON groups. We speculated that high humidity may impact RA by inducing oxidative stress and promoting the inflammatory response; however, this result still needs further verification. In addition, Spearman correlation analysis was used to establish a correlation matrix to comprehensively analyse the relationship between faecal metabolites and the faecal microbiota. Our results suggest a potential interrela- tionship between faecal metabolites and faecal microbes. arthritis in CIA rats. In conclusion, high humidity can lead to the occurrence and development of RA by affecting oxidative stress and promoting the inflammatory response. Interestingly, these pathways also appeared in the comparison between the HH and CON groups. We speculated that high humidity may impact RA by inducing oxidative stress and promoting the inflammatory response; however, this result still needs further verification. In addition, Spearman correlation analysis was used to establish a correlation matrix to comprehensively analyse the relationship between faecal metabolites and the faecal microbiota. Our results suggest a potential interrela- tionship between faecal metabolites and faecal microbes. www.nature.com/scientificreports/ Thus, we provide the following two explanations for these phenomena: 1. It has been confirmed in relevant reports46 and our previous reports47,48 that cholestasis may cause oxidative stress. ABC transporters play an important role in the pathogenesis of cholestasis, as they participate in the regulation of tryptophan metabolism and promote the synthesis of glutathione. Inhibition of Scientific Reports \| (2023) 13:16846 \| https://doi.org/10.1038/s41598-023-43964-4 www.nature.com/scientificreports/ Figure 5. Multivariate statistical analysis of metabolic profiles in each group. (a) PCA parameters; (b-d) PLS-DA analysis results of each group. Figure 5. Multivariate statistical analysis of metabolic profiles in each group. (a) PCA parameters; (b-d) PLS-DA analysis results of each group. Figure 6. Heatmap of the differential metabolites. (a, b) Heatmap of HH vs. CON groups, H + CIA vs. CIA groups, respectively. Heatmaps were obtained from the cloud platform of Shanghai OE Biotech, Inc. ( https:// cloud.oebiotech.cn/task/detail/heatmap/, version 1.26). Figure 6. Heatmap of the differential metabolites. (a, b) Heatmap of HH vs. CON groups, H + CIA vs. CIA groups, respectively. Heatmaps were obtained from the cloud platform of Shanghai OE Biotech, Inc. ( https:// cloud.oebiotech.cn/task/detail/heatmap/, version 1.26). ABC transporters could cause an inflammatory response, which would induce the production of reactive oxygen species (ROS). The content of mitochondrial ROS in monocytes of RA patients increases fivefold. Oxidative stress caused by ROS may be associated with the pathogenesis of RA. The onset and development of RA in the HH and HH + CIA groups may be closely related to oxidative stress. The mechanism by which high humidity interferes with ABC transporter pathways and primary bile acid biosynthesis could be related to ROS production and glutathione synthesis disorder, which are induced by the abnormal metabolism of bile acid. 2. Diet metabolism is closely related to the inflammatory process. Studies have shown that the abnormal metabolism of carbohy- drates and fatty acids can promote inflammatory responses49. Disturbed carbohydrate absorption50 stimulates the inflammatory response by interfering with TNF-α and IL-6 production. Dysregulation of lipid metabolism homeostasis causes an immune reaction by increasing inflammatory factors such as TNF-α51. Therefore, high humidity may promote the inflammatory response by regulating metabolic pathways such as digestion and absorption, steroid biosynthesis and the biosynthesis of other metabolites, thus promoting the development of https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 \| Animals and husbandry details Six-week-old healthy male SD rats were used in accordance with the Guidelines for the Care and Use of Labora- tory Animals of the Institute of Laboratory Animal Resources, Institutional Animal Care and Use Committee of Beijing University of Chinese Medicine. The study is reported in accordance with ARRIVE guidelines. The animal weights were between 135 and 165 g at the beginning of the first study. All rats (n = 40) were housed with 5 animals per cage on a 12 h/12 h light/dark cycle in the animal facility of the Experimental Center of Beijing University of Chinese Medicine (room temperature: 25 ± 1 °C, humidity: 50 ± 5%). All animals were allowed ad libitum access to food and water. Treatment and sample collection p Bovine type II collagen (CII; 2 mg/ml) (Chondrex Inc., WA, United States) emulsified with complete Freund’s adjuvant (CFA; 2 mg/ml) (Sigma–Aldrich Co., St. Louis, United States) was obtained. All animals were divided into the following 4 groups (N = 20) with the random number method after acclimation for 1 week: ① control (CON) group, kept at 25 ± 1 °C, 50 ± 5% humidity and injected with 0.9% NaCl solution (0.2 ml/rat) on Days 7 and 14; ② high humidity (HH) group, kept at 25 ± 1 °C, 80 ± 5% humidity and injected with 0.9% NaCl solution (0.2 ml/rat) on Days 7 and 14; ③ CIA model (CIA) group, kept at 25 ± 1 °C, 50 ± 5% humidity and injected with CII-CFA emulsion (1 mg/ml, 0.2 ml/rat) on Days 7 and 14; and ④ CIA model with high humidity (HH + CIA) group, kept at 25 ± 1 °C, 80 ± 5% humidity and injected with CII-CFA emulsion (1 mg/ml, 0.2 ml/rat) on Days 7 and 14. Arthritis scores were measured every 7 days after Day 2115,33. Two fresh stool pellets from each rat were collected after Day 59. Faecal moisture levels were measured on Day 28 and Day 56. Samples were placed in sterile conical tubes and immediately frozen at −80 °C. Rats were anaesthetized by intraperitoneal injection of 2% sodium pentobarbital (0.2 ml/100 g). Ten rats from each group were randomly selected and euthanized after 28 days to isolate colonic tissues to observe histopathological changes. The remaining rats were euthanized and the hind limbs of the rats were collected on Day 60. Blood samples were collected from the inferior vena cava of rats and centrifuged at 3500×g and 4 °C for 15 min to collect the serum, which was immediately frozen at −80 °C. The group information and experimental process are shown in Fig. 8 are shown in Fig. 9. www.nature.com/scientificreports/ p In conclusion, the results of our study indicate that high humidity can impact the onset and development of RA, and the mechanism is likely related to the inflammatory response and oxidative stress. Nevertheless, it is crucial to acknowledge that our study represents a preliminary exploration, warranting further investigation into the impact of high humidity on the risk of RA in healthy rats. Next, we need to make experiments on how high humidity causes faecal microbiota changes and how these changes affect joint inflammation. In particular, Scientific Reports \| (2023) 13:16846 \| https://doi.org/10.1038/s41598-023-43964-4 www.nature.com/scientificreports/ the relationship between environmental humidity and the onset and development of RA can be further explored by extending the experimental observation period, adjusting the environmental humidity and verifying these results in clinical trials, cell and microorganism, and vitro and vivo mechanism validation experiments. www.nature.com/scientificreports/ embedded in paraffin, and sliced into 4 μm sections by pathological sectioning. Then, the slides were stained with safranin O to assess articular cartilage damage. embedded in paraffin, and sliced into 4 μm sections by pathological sectioning. Then, the slides were stained with safranin O to assess articular cartilage damage. Measurements of serum proinflammatory cytokinesh pl y y The levels of TNF-α, IL-6 and IL-17 in serum were measured by enzyme-linked immunosorbent assay (ELISA). All reagents were purchased from CUSABIO (CUSABIO Technology LLC, Wuhan, China), and analyses were conducted according to the manufacturer’s instructions. The absorbance was measured at 450 nm. Operational taxonomic unit (OTU) clustering and species annotation g Raw sequencing data were in FASTQ format. Paired-end reads were then pre-processed using Trimmomatic software (version 0.35) to detect and cut off ambiguous bases (N). The sliding window method was used to evalu- ate the average base quality. If the average mass value in the window was lower than 20, the back-end base was cut from the window. Paired-end reads were assembled using FLASH software (version 1.2.11). The stitching parameters were as follows: the smallest overlap was 10 bp, the largest overlap was 200 bp, and the maximum error matching rate was 20%. Sequences were further filtered as follows: sequences containing ambiguity were removed and reads with 75% of bases above Q20 were retained. Moreover, Quantitative Insights into Microbial Ecology (QIIME, version 1.8) was used to detect and remove chimeric sequences.tt After the sequencing data were pre-processed to generate high-quality sequences, Vsearch software (Ver- sion 2.4.2) was used to classify the sequences into multiple operational taxonomic units (OTUs) according to their similarity. A parameter sequence similarity greater than or equal to 97% was classified as an OTU unit. The QIIME software package was used to select representative sequences of each OTU, and all representative sequences were annotated and blasted against the Unite database (ITS rDNA) using pynast (v0.1). DNA extraction and 16S rRNA sequencing q g According to the manufacturer’s instructions, a DNeasy PowerSoil kit (Cat. No. 12888; QIAGEN, Dusseldorf, Germany) was utilized to extract DNA from different samples. The purity and concentration of DNA were detected by agarose gel electrophoresis. Extracted DNA was diluted to a concentration of 1 ng/μl and stored at −20 °C for further processing. The DNA genome was utilized as a template for PCR amplification to ensure the efficiency and accuracy of amplification with barcode-specific primers and Takara Ex Taq hi-fi enzyme (Cat. No. RR001Q; Takara, Dalian, China). The corresponding areas of bacterial diversity identification were as follows: The V3-V4 region of the 16S rRNA genes was amplified with universally primed 343F (5’ -TACgGRaggCAG CAGG-3’) and 798R (5’ -AgggTATCtaatCCT-3’) using a commercial PCR kit (Cat. No. 51531; Qiagen, Dus- seldorf, Germany).ith y PCR products were detected by gel electrophoresis and purified by AMPure XP beads after detection. The purified products were used as second round PCR templates and amplified by a second round of PCR. After a second purification step with AMPure XP beads, PCR products were quantitatively analysed by a Qubit dsDNA detection kit (Cat. No. Q32854; Thermo Fisher Scientific, MA, United States). The samples were mixed in equal quantities according to the concentration of PCR products and then sequenced. An equal amount of purified amplicon was pooled for sequencing with a NovaSeq PE250 instrument. Assessment of arthritis variables Arthritis scores were measured every 7 days after Day 21. The assessment criteria of arthritis scores were as follows: 0, no redness and swelling in the foot joints and claws; 1, mild swelling or redness of the foot joint and claws; 2, moderate swelling or mild redness of the foot joint and claws; 3, the claws are red and swollen below the ankle joint; and 4, severe redness, swelling and deformation of the ankle foot joint.hii j g j The articular cartilage was fixed in 4.0% paraformaldehyde for 3 days and then decalcified in 10% ethylen- ediamine tetraacetic acid (EDTA) decalcification solution. The tissues were dehydrated by an ethanol gradient, Figure 9. Group information, time course and experimental procedure. D, day. Isolators for maintaining a rat’s humid climate provide a complete barrier around rat cages. The isolators allow for the adjustment of different humidity and temperature levels. Figure 9. Group information, time course and experimental procedure. D, day. Isolators for maintaining a rat’s humid climate provide a complete barrier around rat cages. The isolators allow for the adjustment of different humidity and temperature levels. Scientific Reports \| (2023) 13:16846 \| https://doi.org/10.1038/s41598-023-43964-4 www.nature.com/scientificreports/ Sample preparation and GC–MS analysish p p p y The samples stored at −80 °C were thawed on ice, 60 mg of stool sample was accurately weighed and placed into a 1.5-ml centrifugation tube, 40 μl of internal standard (l-2-chloro-phenylalanine, 0.3 mg/ml, methanol configuration) was added to each sample, and 2 small steel balls and 360 μl of cold methanol were successively added. Samples were stored at −20 °C for 2 min and ground in a grinding machine (60 Hz, 2 min). Samples were sonicated in an ice water bath for 30 min, 200 μl of chloroform was added, and the mixture was vortexed (60 Hz, 2 min). Then, 400 μl of water was added, and the mixture was vortexed (60 Hz, 2 min). Ultrasonic extraction was performed in an ice water bath for 30 min, and the samples were allowed to stand at −20 °C for 30 min. Then, the extract was centrifuged for 10 min (13,000×g, at 4 °C), and 300 μl of the supernatant was put into a glass-derived bottle and dried in a centrifugal freeze dryer. Next, 80 μl of methoxamine hydrochloride pyridine solution (15 mg/ml) was added to each sample, followed by vortexing for 2 min and ice water ultrasonic treatment for 3 min. The oxime reaction was carried out for 90 min in an incubating shaker at 37 °C. A total of 80 μl of trifluoroacetamide (containing 1% chlorotrimethylsilane) derivatizing reagent and 20 μl n-hexane were added, and 11 internal standards (C8/C9/C10/C12/C14/C16, 0.8 mg/mL; C18/C20/C22/C24/C26, 0.4 mg/ml, all prepared in chloroform) were added at a volume of 10 μl, followed by vortexing for 2 min and reaction at 70 °C for 60 min. After the samples were removed, they were placed at room temperature for 30 min for GC–MS metabolomics analyses. All extraction reagents were precooled at −20 °C before use. Meteorological chromatography‑mass spectrometric conditions b l l d b h h Meteorological chromatography‑mass spectrometric conditions Metabolite analysis was carried out by a gas chromatograph-mass spectrometer (7890B-5977A; Agilent J&W Sci- entific, Folsom, CA, United States). Separation was performed by loading a 30-m × 0.25-mm × 0.25-μm DB-5MS fused silica capillary column (Agilent J&W Scientific, Folsom, CA, United States). The flow rate of the carrier gas, high-purity helium (purity not less than 99.999%), was 1.0 ml/min, and the inlet temperature was 260 °C. The injection volume was 1 μl, and the solvent delay was 5 min. The temperature programme was achieved using the following gradient: The initial temperature of the GC oven was 60 °C, and then the temperature was ramped to 125 °C at 8 °C/min, to 210 °C at 5 °C/min, to 270 °C at 10 °C/min, and to 305 °C at 20 °C/min and held steady for 5 min. Mass spectrometric conditions were as follows: ionization source, electron impact ionization; ion source https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 \| www.nature.com/scientificreports/ temperature, 230 °C; quadrupole temperature, 150 °C; collision energy, 70 eV; solvent delay, 3 min; scan mode, full scan (scan mode); and mass scan range, m/z 50–500. temperature, 230 °C; quadrupole temperature, 150 °C; collision energy, 70 eV; solvent delay, 3 min; scan mode, full scan (scan mode); and mass scan range, m/z 50–500. Ethics approval and consent to participateh pp p p The animal study was reviewed and approved by Ethics Committee of Beijing University of Traditional Chinese Medicine (approved animal experimental protocol number, BUCM-4-2020092905-3119). Data pre‑processing and statistical analysis p p g y Raw GC–MS mass spectra were converted to ABF format files by Analysis Base File Converter software (version 4.0). Then, the data were imported into MS-DIAL software (version 3.9) for pre-processing. Finally, the raw data matrix including the sample information, the name of each peak, retention time, mass-to-charge ratio, and mass spectral response intensity (peak area) were derived. The NIST database (https://webbook.nist.gov/chemi stry/) was used for material qualitative analysis. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were performed to visualize the changes in metabolites between the experi- mental groups after mean centering (Ctr) and Pareto variance (Par) scaling, respectively. Variable importance in the projection (VIP) values were calculated according to the PLS-DA model. A VIP > 1 was used to identify potential biomarkers. The PLS-DA model was tested for 200 response sequencing tests; the x-matrix was fixed, the variables of the previously defined classification Y matrix (such as 0 or 1) were randomly arranged n times (n = 200), and the corresponding PLS-DA model was established to obtain R2 and Q2 values of the random model. Linear regression was performed with R2Y and Q2Y of the original model, and the intercept values of the regression line and Y-axis were R2 and Q2, respectively, which were used to measure whether the model was overfitting. The VIP values were calculated based on the PLS-DA model, and p values were derived from a two-tailed Student’s t test using the normalized peak areas. The criteria for screening differentially expressed metabolites were VIP > 1 and p < 0.05. Statistical analysis y Student’s t test for unpaired data (95% confidence interval) was used for comparisons between each group using GraphPad Prism (Version 9.3; GraphPad Software, San Diego, CA, USA). The data are expressed as the mean ± standard deviation of the mean (S.D.). One-way analysis of variance (ANOVA) was used for measurement data of multiple groups, and least-significant difference (LSD) was used for pial comparison between groups. If homogeneity of variance was not satisfied, the rank sum test was used for comparison of multiple independ- ent samples. Error bars represent the standard deviation. The degree of significance is indicated as p < 0.05, **p < 0.01, #p < 0.05 and ##p < 0.01. Data availabilityh y The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: The 16 s rRNA dataset presented in this study has been deposited at https://www.ncbi.nlm.nih.gov/bioproject/PRJNA823862; Metabolights [accession: MTBLS4649]. Received: 1 July 2022; Accepted: 30 September 2023 Received: 1 July 2022; Accepted: 30 September 2023 References References 1. Nygaard, G. & Firestein, G. 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Periodontal disease and rheumatoid arthritis: The evidence accumulates for complex pathobiologic interactions. Curr. Opin. Rheumatol. 25, 345–353 (2013).i interactions. Curr. Opin. Rheumatol. 25, 345–353 (2013).i p 43. Li, Z. X. et al. Traditional Tibetan medicine twenty-five Wei p 43. Li, Z. X. et al. Acknowledgements We thank the Shanghai Luming biological technology co., LTD (Shanghai, China) for providing metabolomics services and the Shanghai Luming biological technology co., LTD (Shanghai, China) for their enthusiastic sup- port of this metabolomics analysis. We also would like to thank the staff of Shanghai OE Biotech, Inc. (Shanghai, China), MS. Ruirui Li and Jing Wang for their technical support. References & Ichchou, L. Seasonal and weather effects on rheumatoid arthritis: Myth or reality?. Pain Res. Manag. 2020, 576 (2020). 10. Yimer, B. B. et al. Heterogeneity in the association between weather and pain severity among patients with chronic pain: A Bayesian multilevel regression analysis. Pain Rep. 7, e963 (2022).f g y p 1. Zhao, D. S. et al. Effect of ambient temperature on outpatient admission for osteoarthritis and rheumatoid arthritis in a subtropica Chinese city. BMC Public Health. 22, 172 (2022).hf 12. Abasolo, L. et al. Weather conditions may worsen symptoms in rheumatoid arthritis patients: The possible effect of temperature. Reumatol. Clin. 9, 226–228 (2013). 3. de Brito, R. S., Baldo, D. C. & Andrade, L. E. C. Clinical and pathophysiologic relevance of autoantibodies in rheumatoid arthritis Adv. Rheumatol. 59, 2 (2019).l Adv. Rheumatol. 59, 2 (2019). 14. Strusberg, I., Mendelberg, R. C., Serra, H. A. & Strusberg, A. M. Influence of weather conditions on rheumatic pain. J. Rheumatol. 29, 335–338 (2002). 4. Strusberg, I., Mendelberg, R. C., Serra, H. A. & Strusberg, A. M. Influence of weather conditions on rheumatic pain. J. Rheumatol 29, 335–338 (2002). ( ) 5. Wang, M. et al. High humidity aggravates the severity of arthritis in collagen-induced arthritis mice by upregulating xylitol and L-pyroglutamic acid. Arthritis Res. Ther. 23, 292 (2021).h py gh ( ) 6. Aikman, H. The association between arthritis and the weather. Int. J. Biometeorol. 40, 192–199 (1997). https://doi.org/10.1038/s41598-023-43964-4 Scientific Reports \| (2023) 13:16846 \| Fundingh g This study was supported by the following fund: Beijing University of Chinese Medicine New Olympics Award Fund Project (No. 2019-XAJLJJ-004) and National Natural Science Foundation (No. 82374312). Scientific Reports \| (2023) 13:16846 \| https://doi.org/10.1038/s41598-023-43964-4 www.nature.com/scientificreports/ Additional information Correspondence and requests for materials should be addressed to X.Z. Reprints and permissions information is available at www.nature.com/reprints. 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https://openalex.org/W4285180421	https://www.shs-conferences.org/articles/shsconf/pdf/2022/11/shsconf_mtde2022_01011.pdf	English	null	Digital capital as an indicator of the effectiveness of the use of digital technologies in the management of socio-economic systems	SHS web of conferences	2,022	cc-by	4,336	Digital capital as an indicator of the effectiveness of the use of digital technologies in the management of socio-economic systems Galina Merzlikina, and Natalya Mogharbel Volgograd State Technical University, Volgograd, Russia Volgograd State Technical University, Volgograd, Russia Abstract. Digitalization (the use of digital technologies in the management of socio-economic systems) is currently recognized as a prerequisite for effective economic development and competitiveness. A comparative analysis of digitalization assessment methods has shown that in most cases the level of dissemination and application of digital technologies, provision of equipment, software products, Internet access, the possibility of forming and using databases is assessed. The methods do not involve evaluating the effectiveness of digitalization – comparing the result and the costs of digitalization. In this article, it is proposed to use digital capital as an indicator of the effectiveness of the use of digital technologies in the management of socio-economic systems. A comparative analysis of the definitions of "digital capital" proposed by various scientists is carried out and a refined definition is proposed: a set of tangible and intangible identifiable and unidentifiable (the concept of digital goodwill is proposed) digital assets and digital competencies of employees that allow them to successfully implement digital technologies and increase labor productivity. Possible methods of digital capital assessment and features of digital capital assessment of various socio-economic systems are considered. 1. Introduction The purpose of the study. The study of the concept of "digital capital", the definition of the content and structure, the study of the possibility of using the indicator of digital capital to assess the effectiveness of digitalization, the use of digital technologies in the management of socio-economic systems. y To achieve the purpose of the study, the following tasks are set: To conduct a comparative analysis of methods for evaluating the effectiveness and/or performance of digitalization, to clarify the content of the concept of "digital capital" and determine its structure, to consider possible methods for evaluating digital capital. The object and subject of the study. As an object of the study, the authors consider socio- economic systems (countries, regions, enterprises) using digital technologies in their activities. https://doi.org/10.1051/shsconf/202214101011 https://doi.org/10.1051/shsconf/202214101011 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). SHS Web of Conferences 141, 01011 (2022) MTDE 2022 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 The subject of this study is the process of clarifying the content of the concept of "digital capital", assessing the possibility of using the digital capital indicator as an indicator of the effectiveness of digitalization. The subject of this study is the process of clarifying the content of the concept of "digital capital", assessing the possibility of using the digital capital indicator as an indicator of the effectiveness of digitalization. Relevance. Currently, the development of the management of socio-economic systems (country, region, enterprise) is carried out primarily in one direction – the wider use of digital technologies (digitalization). Digitalization, widespread use of Internet resources, provision with digital equipment and software products are becoming a prerequisite for development and competitiveness. However, the effectiveness of the use is not as high as expected, perhaps this is the influence of the digital lag and so far the result of digitalization has not yet simply matured. In addition, it is necessary to take into account possible unforeseen circumstances ("dark corners" [1]) that require improved management in the digital economy in favor of structural changes, including the capital structure of the enterprise. The process of digitalization itself is not always carried out "as planned"; it was assumed that digitalization would develop exponentially [2], become more active as the scale increases, but this did not happen; digital technologies are used quite actively by organizations and households (judging by statistical reporting and analytics), but the effectiveness of digitalization is still low. Process measurement tools are always important in management, perhaps in this case it is necessary to change the tools for measuring the effectiveness of digitalization? Many studies have been devoted to the study and evaluation of the effectiveness of the use of digital processes [3-10]; however, a detailed and comparative analysis of digitalization indicators has shown that they are all about the effectiveness of the digitalization process, i.e. about the implementation of the process, not about its performance and efficiency. There are some scientific papers on evaluating the effectiveness of digitalization [11-13], but either classical indicators of investment analysis or indicators based on expert assessments are used. In this article, it is proposed to use the digital capital indicator as an indicator of the effectiveness of the use of digital technologies. SHS Web of Conferences 141, 01011 (2022) MTDE 2022 The concept of "digital capital" is considered in some scientific papers, its structure is determined [9-10,14-20]; summarizing various points of view, the authors propose a refined definition of the concept of "digital capital" as tangible and intangible identifiable assets and unidentifiable digital assets (digital goodwill and digital competencies of employees), allowing the implementation of digital technologies and contributing to the improvement of labor productivity; the concept of digital goodwill is proposed for the first time. Possible methods of assessing digital capital are considered: from classical methods of assessing the value of a business, assessing excess profits to expert assessments of digital competencies. The features of digital capital assessment for different types of socio-economic systems (country, region, enterprise) are differentiated. Thus, it is necessary to substantiate the use of digital capital as an indicator of the effectiveness of the use of digital technologies, clarify the content of the concept of "digital capital" and its structure, and further create methodological support for the assessment and management of digital capital. 2 Research methodology This research is based on the use of the main provisions of the theory of economics and management of socio-economic systems at various levels (country, region, enterprise), the theory of digital economy, balanced development, scientific foundations of crisis management, the theory of valuation and management of business value and intangible assets, methods of statistical observation, analysis and measurement, the methodology of capital formation of the enterprise, presented in scientific publications of foreign and Russian scientists. Corresponding author: merzlikina@vstu.ru © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). https://doi.org/10.1051/shsconf/202214101011 3 Research results The article presents new scientific results: p - it has been revealed that the abundance of existing methods for assessing digitalization, the use of digital technologies does not solve the problem of evaluating the effectiveness of digitalization, since they are devoted to assessing the coverage of any socio-economic system (country, region, industry, enterprise) with digital technologies, and in Russia and abroad they assume an assessment of the provision of digital assets, Internet resources, software, however, purely statistical accounting of digital assets dominates; - it is revealed that in some scientific papers the problem of evaluating the effectiveness of digitalization is considered, but classical methods of evaluating investment projects are proposed for use; - it is revealed that in some scientific papers the problem of evaluating the effectiveness of digitalization is considered, but classical methods of evaluating investment projects are proposed for use; - it is proposed to use the concept/indicator of "digital capital" as an indicator of the effectiveness of the use of digital technologies in the management of socio-economic systems, as tangible and intangible identifiable assets and unidentifiable digital assets (digital goodwill and digital competencies of employees), allowing the implementation of digital technologies and contributing to increased productivity; the concept of digital goodwill is proposed for the first time; - possible methods of digital capital assessment are considered; - the features of digital capital assessment for various socio-economic systems (country, region, enterprise) are determined. Further development of the theory of digital capital management involves the formation of methodological support (assessment tools and methods). 2 2 https://doi.org/10.1051/shsconf/202214101011 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 SHS Web of Conferences 141, 01011 (2022) 4.1 Evaluation of the effectiveness of the use of digital technologies in the management of socio-economic systems Digitalization, the use of digital technologies in the management of any socio-economic system (country, region, enterprise) gives positive results, manifested in the growth of labor productivity, cost reduction, expansion of opportunities to enter new markets, successful formation and use of large information arrays, which indicates a "multiplicative effect of economic development" [3]. The authors conducted a comparative analysis of performance assessment methods (this is what the use of digital technologies is about). Our comparative analysis of methods for evaluating the effectiveness of digitalization [4- 10]; all methods (and almost every work is a detailed and comparative analysis of Russian and foreign methods) involve an assessment of the level of digitalization, digital technology coverage of the activities of economic entities, region, country (Internet availability, cloud services, electronic planning systems, participation in electronic commerce). The disadvantages of the international digital economy development indices (namely, they are used as the main indicators) include the inability to take into account the peculiarities of individual countries, the methods evaluate the "technical side" [12] by determining the development of the digital economy by the development of digital infrastructure and the readiness of workers and the population to work in a "digital" format. But the use of digital technologies implies (and one should agree with this) deeper transformations and very diverse effects. Scientific developments are also underway to improve the digitalization assessment indices, the most popular is the business digitalization index. Based on the principles of qualimetry, criteria for assessing the level of digitalization of socio-economic systems 3 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214101011 (country, region, enterprise) are proposed (a set of indicators) [4]; resource (tangible and intangible resources) indicators of digitalization performance have been developed and an integral indicator of digitalization based on completeness, availability of data in open sources, absence of derivatives of GDP and GNP among the indicators [5] has been proposed. (country, region, enterprise) are proposed (a set of indicators) [4]; resource (tangible and intangible resources) indicators of digitalization performance have been developed and an integral indicator of digitalization based on completeness, availability of data in open sources, absence of derivatives of GDP and GNP among the indicators [5] has been proposed. 4.1 Evaluation of the effectiveness of the use of digital technologies in the management of socio-economic systems There is a well-known business digitalization index based on five indicators of business use of information technologies: the share of organizations using broadband Internet, using cloud technologies, using RFID technologies, resource planning systems (Enterprise Resource planning or ERP), carrying out electronic sales[6]. Another business digitalization index [7] is based on the analysis of five auxiliary indices characterizing the channels of transmission and storage of information, human capital, information security, the use of digital tools for the development and promotion of the company, the level of implementation of digital technologies; note that the "attention zone" has been expanded to the concepts of human capital and information security. The World Bank offers 22 indicators for all socio- economic systems (country, region, enterprise) [8]. There have also been works on the assessment of a new concept – "digital maturity", a very indicative concept, since it essentially defines a "certain necessary requirement" for the development of digital technologies, a kind of "threshold" value, but, nevertheless, the level of development of digital technologies, not the effectiveness of their use. [ 9-10]. In all the methods considered, the level of development, digital technologies, coverage of territories with digital technologies is analyzed, which probably cannot be called the effectiveness of digitalization, it is rather the creation of conditions for digitalization. 4.3 Digital capital: concept and structure Currently, there is a process of formation of new factors of production. Classical factors of production (fixed, working capital, human capital, entrepreneurship and information) retain their importance, but new ones are emerging to solve modern economic problems: innovative, intellectual, patent, social, environmental, capital and digital capital. Digital capital should be attributed to the most popular of the new factors of production; there is a lot of talk about it in the context of the need for total digitalization. However, the theoretical foundations of the formation and use of digital capital are only being developed and are at the stage of comprehension, refinement of the conceptual apparatus, formation of the evaluation methodology. The definition of "digital capital" was first proposed by experts analysts of the McKinsey company, which was supposed to mean resources for the creation of new products and services in the digital economy. The definition of digital capital is vague, general, which does not allow not only to estimate, but also to assume the types of resources needed in the digital economy. Digital capital, in our opinion, can be used to assess the effectiveness of digitalization, since it is "capital" that will "combine" both the results achieved and the costs incurred for this (when forming a revenue stream, which will be capitalized to form the desired capital). In a certain sense, the use of digital capital is consonant with the cost approach of evaluating management efficiency based on the balanced scorecard of D. Norton and R. Kaplan. Scientists are actively working on the formation of the theory of digital capital, its content and structure are being clarified, evaluation methods are being considered. In [14], digital capital is defined as working with Big Data, involving their processing and analysis, work on the formation of information systems; digital capital is considered as an opportunity to access the digital information environment and digital technologies [15]. Digital capital is proposed [16] to be represented as a system of accumulation of digital, information, digital communication competencies, digital security competencies and digital technologies. In [17], digital capital is already supposed to accumulate (creating capital!) due to the growth of digital competencies and the use of digital technologies, which can be isolated, so it can be identified, evaluated, and managed. 4.2 Evaluation of the effectiveness of the use of digital technologies There were also works on evaluating the effectiveness of the use of digital technologies in enterprise management; [11] presents a methodology based on the analysis of six enlarged business processes, each of which is refined by a number of subprocesses (personnel management, production, performance of works, provision of services, marketing, logistics, finance and accounting, general economic activity), the assessment is carried out based on the results of the survey; based on the results of the survey, an assessment is carried out according to the levels of digitalization: local, partial complex, smart, digital ecosystem, but economic financial evaluation of the results of digitalization is not supposed to be carried out. In another work [12], a methodology is proposed for evaluating the effectiveness of the use of digital assets (we are talking about digital tangible assets) to assess the use of digital technologies in industry; moreover, it is noted that for different socio-economic systems, the tools for evaluating the effectiveness of digitalization should be different: the assessment of digitalization at the country, region level will be based on the gross domestic product (absolute value and share of the digital economy), and for industry, it is necessary to assess digitalization according to the readiness of industries and their current contribution (investment, development) to digitalization, in addition, it is necessary to take into account the so-called indirect effects of investments in digital assets, since they are significant, but do not fall into the "field vision" of the digitalization result. In [13], methodological approaches to assessing the effectiveness of digital transformation of one of the varieties of socio-economic systems - enterprises of high-tech industries are formulated; based on the analysis of eight domestic methods for evaluating the effectiveness of the use of digital technologies (investment analysis, dynamic models, value added calculation, expert), it is proposed to evaluate the effectiveness of digitalization taking into account the stages of the digital transformation strategy or taking into account the delayed time lag of the manifestation of the digitalization effect. It should be agreed with the author that the 4 4 https://doi.org/10.1051/shsconf/202214101011 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 efficiency assessment should take into account all the effects and all the costs of implementing digitalization projects. 4.2 Evaluation of the effectiveness of the use of digital technologies Thus, a comparative analysis of methods and indicators for evaluating the efficiency and effectiveness of the use of digital technologies in the management of socio-economic systems (there are indeed many methods, more references to the literature used, since all scientific works include a detailed and comparative analysis of many Russian and foreign methods) showed that (generally) indicators are used as the main indicators providing digital technologies, if this is the purpose of digitalization, in increasing digital equipment, software availability, Internet usage activity – then the performance indicators of digitalization can be used. However, it is necessary to search for indicators of the efficiency of using digital technologies, comparing results and costs. In this paper, it is proposed to use the indicator of "digital capital" for this purpose. SHS Web of Conferences 141, 01011 (2022) MTDE 2022 https://doi.org/10.1051/shsconf/202214101011 A brief overview of the definitions of the concept of "digital capital", its content and components, allowed the authors to propose their own definition of this concept: digital capital is tangible and intangible digital assets that allow the implementation of digital technologies and contribute to increasing labor productivity. The proposal on the content of digital capital – tangible and intangible digital assets implies the need to allocate tangible identified digital assets – digital equipment, networks, communications, data processing, intangible identified assets (for example, software products), intangible unidentifiable digital assets (we propose to introduce the concept of digital image, digital reputation, digital goodwill) and digital competencies of employees (knowledge, experience, maintaining knowledge in force, return), as, probably, a new unidentifiable intangible digital asset. The authors' proposal to introduce a new concept of "digital goodwill" is still only at the proposal stage, adapting the well-known definition of goodwill, we propose that "digital goodwill" is the good name of the company, consisting of the reputation of the company, prestige, customer relations, location, product range and others, based on the active use of digital technologies in management and providing a significant impact on its income. In [17], the digital capital index is proposed to be determined on the basis of two indices – the index of the level of access to digital technologies and the index of digital competencies of digital technology users. That is, there are two main components of digital capital: tangible and intangible digital assets and digital competencies. Digital competencies of employees are still out of sight of the digitalization assessment procedure, and it is they who "revive" tangible digital assets and "make" them work. Having considered the components of digital capital, we will clarify its definition, digital capital is tangible and intangible identifiable assets and unidentifiable digital assets (digital goodwill and digital competencies of employees), allowing the implementation of digital technologies and contributing to increased productivity. 4.3 Digital capital: concept and structure In [18], it is noted that the organization of information technologies requires very significant capital resources and current costs, which can be represented, among other things, in the form of intangible, digital capital. Digital capital is considered [19] as a set of conditions that determine the ability of people to access, use and interact with digital services. They include in digital capital [20] all resources that are important for the development of new goods (products and services) (recall McKinsey's definition of digital capital), attention is drawn to the significant complexity of evaluating both tangible digital assets and even more intangible digital assets. 5 5 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 4.4 Evaluation of digital capital The authors found it necessary to at least briefly outline the main methods of assessing digital capital. Tangible (identifiable) digital assets can be evaluated by any methods of all three classical approaches to business valuation (costly, market, profitable), here there can be problems only in the formation of an information base and the collection and generalization of evaluation experience. To evaluate digital goodwill, you can try using the well-known Excess earnings method, but there may be problems in assessing the "normalization" (that's right!) of profits and the discount rate of excess profits due to simply lack of experience and information. The assessment of digital competencies of employees is not particularly difficult (there are various, as a rule, expert assessment methods), but the problem will be in the possibility of valuing this component of digital capital, since capital, although digital, requires a valuation. But this is further work on the formation of a methodology for assessing digital capital. Features of digital capital assessment of various socio-economic systems. In this article, the authors intended to explore the possibility of using the concept/indicator of "digital capital" for any socio-economic system: country, region, enterprise. Although, it should be recognized that different types of socio-economic systems will require the use of different indicators; the digital capital of an enterprise can be evaluated first of all, other indicators will be needed to assess the digital capital of a region and a country. But the content of the concept of "digital capital" for any socio-economic system should be unchanged: tangible and intangible identifiable assets and unidentifiable digital assets (digital goodwill and digital competencies of employees), allowing the implementation of digital technologies and contributing to increased productivity. 6 6 6 https://doi.org/10.1051/shsconf/202214101011 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 5 Conclusion The authors in their article came to the following conclusions: - the methods currently used to assess digitalization, and there are quite a lot of them, do not imply an assessment of the effectiveness of the use of digital technologies, but only allow us to assess the coverage of digital assets, the availability of digital equipment, Internet resources, software, i.e. these are methods for evaluating the process, not the effectiveness of the process; - the methods currently used to assess digitalization, and there are quite a lot of them, do imply an assessment of the effectiveness of the use of digital technologies, but only allow p - several scientific papers have been identified that offer methods for evaluating the effectiveness of digitalization, but only methods and indicators for evaluating investment projects are offered; - several scientific papers have been identified that offer methods for evaluating the effectiveness of digitalization, but only methods and indicators for evaluating investment projects are offered; - it is proposed to use the concept/indicator of "digital capital" as an indicator of the effectiveness of the use of digital technologies in the management of socio-economic systems, as tangible and intangible identifiable assets and unidentifiable digital assets (digital goodwill and digital competencies of employees), allowing the implementation of digital technologies and contributing to increased productivity; the concept of digital goodwill is proposed for the first time; - possible methods of assessing digital capital are considered (business valuation methods and the method of excess profits for evaluating the digital goodwill indicator); - possible methods of assessing digital capital are considered (business valuation methods and the method of excess profits for evaluating the digital goodwill indicator); - the features of digital capital assessment for various socio-economic systems (country, - possible methods of assessing digital capital are considered (business valuation methods and the method of excess profits for evaluating the digital goodwill indicator); - the features of digital capital assessment for various socio-economic systems (country, region, enterprise) are determined. and the method of excess profits for evaluating the digital goodwill indicator); - the features of digital capital assessment for various socio-economic systems (country, region, enterprise) are determined. Further development of the theory of digital capital management involves the formation of methodological support (assessment tools and methods). Further development of the theory of digital capital management involves the formation of methodological support (assessment tools and methods). 20. Jacques Bughin, James Manyika, Measuring the full impact of digital capital. https://www.mckinsey.com/industries/technology-media-and-telecommunications/our- insights/measuring-the-full-impact-of-digital-capital References 1. O. Kovacs, Technology in Society 55, 140 (2018). DOI: 10.1016/j.techsoc.2018.07.0 1. O. Kovacs, Technology in Society 55, 140 (2018). DOI: 10.1016/j.techsoc.2018.07.009 2. K. Mainzer, Foresight and STI Governance 14(4), 10 (2020). DOI: 10.17323/2500- 2597.2020.4.10.19 2. K. Mainzer, Foresight and STI Governance 14(4), 10 (2020). DOI: 10.17323/2500- 2597.2020.4.10.19 3. S.A. Starykh, S.V. Solovyov, A.A. Baroyan, Region: Systems, Economics, Management 2(49),130 (2020) 4. A.V. Tebekin, Qualimetric assessment of the level of digitalization of the economy in the Russian Federation. https://vestnik.astu.org/temp/f6e4be5ee1fd3c982f83cd141ec79281.pdf 5. Ya. E. Pile, Economics, Entrepreneurship and Law 10(2), 253 (2020). doi: 10.18334/epp.10.2.100473 6. Business Digitalization Index. Institute of Statistical Research and Economics of Knowledge of the Higher School of Economics. https://issek.hse.ru/news/244878024.html 7. NAFI and Otkritie Bank. The index of readiness of Russian business for the digital economy. Nafi.ru. https://nafi.ru/en/projects/predprinimatelstvo/indeks-peremen- gotovnost-rossiyskikh-kompaniy-k-tsifrovoy-ekonomike/ 8. World Bank 2016, Digital Adoption Index. https://www.worldbank.org/en/publication/wdr2016/Digital-Adoption-Index 9. G. Remane, A. Hanelt, G. Wiesboeck Remane, A. Hanelt, F. Wiesboeck, Lutz Kolbe, Twenty-Fifth European Conference on Information Systems (ECIS), (Guimarães, Portugal, 2017). https://www.researchgate.net/publication/316687803_DIGITAL_MATURITY_IN_TR ADITIONAL_INDUSTRIES_-_AN_EXPLORATORY_ANALYSIS 7 7 https://doi.org/10.1051/shsconf/202214101011 SHS Web of Conferences 141, 01011 (2022) MTDE 2022 10. T. А. Gileva, USNTU Bulletin. Science, education, economics. Economics series 1(27), 38 (2019). DOI: 10.17122/2541-8904-2019-1-27-38-52 11. I.Yu. Merzlov, E.V. Shilova, E.A. Sannikova, M.A. Sedinin, Economics, Entrepreneurship and Law 10(9), 2379 (2020). doi: 10.18334/epp.10.9.110856 12. E.A. Istomina, Bulletin of Chelyabinsk State University 12 (422), Economic sciences. Issue 63, 108 (2018). DOI 10.24411/1994-2796-2018-11212 13. T.V. Kokuitseva, O.P. Ovchinnikova, Creative Economy 15(6), 2413 (2021). doi: 10.18334/ce.15.6.112192 14. V.K. Krutikov, E.V. Geraeva, Economics and Entrepreneurship 3, 703 (2018) 15. E.L. Vartanova, A.A. Gladkova, Mediascope 1, (2020). DOI: 10.30547/mediascope.1.2020.8 16. M. Massimo Ragnedda, Telematics and Informatics 35(8), 2366 (2018). DOI:10.1016/j.tele.2018.10.006 17. M. Ragnedda, M.L. Ruiu, F. Addeo, New Media and Society 1, 1 (2019) doi.org/10.1177/1461444819869604 18. Prasanna Tambe, Lorin Hitt, Daniel Rock,Erik Brynjolfsson,Prasanna Tambe, Lorin Hitt, Daniel Rock, Erik Brynjolfsson, Working Paper, 28285 (2020). DOI 10.3386/w28285 19. Sora Park Digital Capital. London: Palgrave Macmillan, (2017). DOI:10.1057/978-1- 137-59332-0 20. Jacques Bughin, James Manyika, Measuring the full impact of digital capital. https://www.mckinsey.com/industries/technology-media-and-telecommunications/our- insights/measuring-the-full-impact-of-digital-capital 8 8
https://openalex.org/W2792699808	https://www.frontiersin.org/articles/10.3389/fped.2018.00005/pdf	English	null	Feasibility of Physical Activity Assessment with Wearable Devices in Children Aged 4–10 Years—A Pilot Study	Frontiers in pediatrics	2,018	cc-by	3,504	Correspondence: Jan Müller Specialty section: This article was submitted to Child Health and Human Development, a section of the journal Frontiers in Pediatrics Keywords: daily activity, wearable, Garmin vivo jr, applicability, feasibility Feasibility of Physical Activity Assessment with Wearable Devices in Children Aged 4–10 Years—A Pilot Study n Müller, Anna-Maria Hoch, Vanessa Zoller and Renate Oberhoffer Jan Müller, Anna-Maria Hoch, Vanessa Zoller and Renate Oberhoffer Faculty of Sport and Health Sciences, Institute of Preventive Pediatrics, Technical University of Munich, Munich, Germany Objective: Physical activity (PA) is associated with multiple beneficial health outcomes. Unfortunately, current studies report an alarming decrease of PA throughout all age groups. This study aims to assess general feasibility and PA levels of kindergarten and primary school children with wearable technology specifically manufactured for young children. Original Research published: 26 January 2018 doi: 10.3389/fped.2018.00005 INTRODUCTION Received: 14 November 2017 Accepted: 09 January 2018 Published: 26 January 2018 Being physically active is one of the most important cornerstones for people of all ages to maintain physical and mental health (1). Lack of physical activity (PA) is associated with multiple of non- communicable diseases (NCD) and was therefore named the fourth leading risk factor of NCD by the WHO in 2009 (2). Since the first PA recommendations for adults were published in 1995 from the Center for Disease Control (CDC) (3), several modifications have been issued and specific recommendations addressed, in particular for children and elderly people (4, 5). In addi- tion, many countries have established national recommendations and guidelines to increase PA throughout the population. This was mainly caused by a lot of sectional and epidemiological stud- ies reporting continuously decreasing PA in adults, adolescents, and children (6). Unfortunately, Edited by: y Frederick Robert Carrick, Bedfordshire Centre for Mental Health Research in Association with University of Cambridge, United Kingdom Patients and methods: From April 2017 to August 2017, a total of 59 children (7.1 ± 1.7 years, 34 girls) recorded their PA for seven consecutive day wearing a wearable bracelet (Garmin vivofit jr). Afterward, they filled out a short, child-oriented questionnaire to rate the feasibility. Reviewed by: Carlos Teixeira Brandt, Federal Rural University of Pernambuco, Brazil Heather Macdonald, University of British Columbia, Canada Results: The general feasibility of the devices was rated as rather well regarding size, materials, and wearing comfort. Moreover, children achieved a mean of 83 ± 18 min of moderate-to-vigorous physical activity (MVPA) and 12.202 ± 2.675 steps per day on a weekly average. Therefore, 52 (88.1%) children, and almost all boys (96%), fulfilled the WHO criteria of 60 min of MVPA per day on a weekly average. Hamilton Roschel, University of São Paulo, Brazil Conclusion: Wearables bracelets seem to be feasible devices for PA assessment even in young children. Nevertheless, their potential to increase PA for primary and secondary prevention of cardiovascular disease, as well as the long-term compliance needs to be clarified in further studies. Correspondence: Jan Müller j.mueller@tum.de Keywords: daily activity, wearable, Garmin vivo jr, applicability, feasibility Data Analyses The aim of this pilot study is to assess general feasibility and PA levels in children aged 4–10 years with a wearable bracelet specifically manufactured for this age group. Data are described by mean ± SD for all variables. All analyses were performed using SPSS 23.0 software (IBM Inc., Armonk, NY, USA). Citation: Müller J, Hoch A-M, Zoller V and Oberhoffer R (2018) Feasibility of Physical Activity Assessment with Wearable Devices in Children Aged 4–10 Years—A Pilot Study. Front. Pediatr. 6:5. doi: 10.3389/fped.2018.00005 January 2018 \| Volume 6 \| Article 5 Frontiers in Pediatrics \| www.frontiersin.org 1 Müller et al. Wearables in Children the parents. The device has shown to be accurate in assessing PA and steps (12–14) but not energy expenditure (13, 15). measuring PA is challenging and methodologies range from labor intensive direct observation, over secondary measures of heart rate monitoring or accelerometry, to subjective measures of self-report (7).f Children and their guardians were instructed with a standard- ized information sheet on how to pair the bracelet with an app on their mobile phone. They were also instructed to wear the bracelet on seven consecutive days even during leisure time and school sport. The only time the watch could be removed was overnight. In light of these different approaches, studies showed that recalled PA in children is difficult and flawed (8, 9). More than that, accelerometer-based devices that are worn around the hip appear to be too technical and are described as awkward to wear. However, over the past few years more and more consumer appealing commercial wearable activity trackers (e.g., Fitbit, Jawbone, TomTom, Garmin) for adults have entered the marked, also expanding opportunities to integrate such new technology into research. In 2016, Garmin has pioneered with the vívofit® jr as the first wearable bracelet just for children below the age of 10. With controversial reactions in media forums, some praised the technical innovation with its potential benefits of encouraging the very young to more PA. However, others argued it is simply a commercial overkill and a new panoptical tool for helicopter parents. As some of these arguments are understandable, the potential for early childhood prevention and opportunity to fundamentally shape children’s relationship with PA cannot be overlooked (10).h For statistical purposes, MVPA minutes and steps for every day were analyzed and also computed to a weekly average. Assessment of Feasibilityt Feasibility was assessed after the 7-day trial with a short question- naire in which children had to answer five questions regarding the convenience of the bracelet. In detail, the questions were as follows: “Did you like to wear the watch?” “Was the watch com- fortable for you?” “How have you tolerated the material of the watch?” “How do you like the size of the watch?” and “Has the watch motivated you to be more active?” Children responded on a 5-point pictorial (smiley) Likert scale that was later transformed in a scale from “−2,” “−1,” “0,” “+1,” “+2” with “0” as neutral anchor. Study Subjects The feasibility of the wearable was rated rather well from the chil- dren in regard to size, materials, and wearing comfort (Table 1). From April 2017 to August 2017, a convenience sample of 59 healthy children (7.1 ± 1.7 years, 34 girls) participated in a wearable-based PA assessment to estimate the feasibility of the devices. Participants were therefore recruited in several Kindergartens and after-school care centers in Bavaria and Baden- Wüerttemberg to voluntary participate in a 1-week trial. Weight and height were assessed in the morning and body mass index (BMI) calculated by weight in kilograms divided by the square of the height in meters. BMI values were transformed into z-scores according to German reference values from Kromeyer-Hauschild et al. (11).h g g Children achieved a mean of 83 ± 18 min of MVPA and a total 12.202 ± 2.675 steps on a weekly average of 7 days. In addition, 52 Table 1 \| Study subjects. Anthropometrics Sex (girls) 34 (57.6%) Age (years) 7.1 ± 1.7 Body height (cm) 125.8 ± 10.8 Body weight (kg) 26.5 ± 7.8 Body mass index (z-score) 0.09 ± 0.91 School type Kindergarten 35 (59.3%) Primary school 18 (30.5%) All-day school 6 (10.2%) Feasibility Did you like to wear the watch? 1.8 ± 0.4 Was the watch comfortable for you? 1.0 ± 1.2 How have you tolerated the material of the watch? 1.8 ± 0.4 How do you find the size of the watch? 1.3 ± 0.8 Has the watch motivated you to be more active? 0.6 ± 1.2 The study was designed in accordance with the declaration of Helsinki (revision 2008) and approved by the local ethical board of the Technical University of Munich (project number: 314/14). All children were verbally informed about the meaning and purpose of the study and agreed to participate. Written informed consent was obtained from all guardians. Frontiers in Pediatrics \| www.frontiersin.org DISCUSSION This study outlined that the feasibility of the wearable was rather well in regard to size, materials, and wearing comfort. Moreover, the majority of children and almost all boys fulfilled the WHO (5) criteria of 60 min of MVPA per day on a weekly average and 31 children (53.4%) reached at least 60 min of MVPA on every day of the week. In young children, however, this is certainly different. Seven- year-old children are not able to understand the rational of PA for health outcomes, nor to interpret the PA measures on the watch at all. Therefore, it is unlikely that they are more active just because of the bracelet. This assumption is even reflected in the neutral response to the question in our questionnaire: “Has the watch motivated you to be more active?” On the other hand, fashion concerns were already present in this young age group which might be a reason why the feasibility of this children-friendly designed wearable turned out to be good to very good in regard to materials, comfort, and size and why the children reported they liked to wear the bracelet very much. Assessing PA levels in children is challenging. Previous studies point out that subjective measured PA levels recalled in question- naires or activity logs showed only a weak correlation to measured PA by accelerometry (8). However, in big cohort studies self- report is still the only applicable method, whereas accelerometry is the method of choice in research projects with a smaller sample size (16). The latter is currently also the gold standard of objective measurement when taking feasibility, applicability, and salary in different settings into account (8, 16, 17). f Nowadays, wearable technology is also more and more used to assess PA since an appealing design and consumer friendly usage have led the line between consumer health wearables and medical devices begin to blur (10, 18). Our pilot study outlines now that the feasibility and acceptance of those devices is very good during a 1-week trial. Especially, the fact that children reported that they liked to wear the watch and that they had no problems with the materials is promising for the long-term use. Physical Activity Assessmenthi The Garmin vivofit® jr is a wearable bracelet designed specifically for children from 4 to 9 years of age to track PA in terms of steps and moderate-to-vigorous physical activity (MVPA) in minutes per day. According to the manufacturer, the children-friendly design is comfortable, durable, and waterproof. The wearable had to be paired with a mobile phone for the parents and children to interact with the device and control the settings. In addition, the app offers an interactive gamification concept in which children can earn coins to redeem for agreed-upon rewards managed by January 2018 \| Volume 6 \| Article 5 Frontiers in Pediatrics \| www.frontiersin.org 2 Müller et al. Wearables in Children with a belt around the hip (19). The appealing design and accessory character are also crucial factors for long-term compliance, which in fact is an aspect that needs to be proven in further studies. That inconspicuous design is also beneficial when it comes to medical application of those wearables, because wearing such a device does not evoke inconvenient questions for the patients. Even in older adults the commercially available wearable appeared to be useful and acceptable (20). Regarding PA assessment, there is just one major concern in general. Wearing such a device for the first time leads to a higher activity in the first days or week of wearing which then declines to normal PA levels over time (21).f (88.1%) children, and almost all boys (96%), fulfilled the WHO criteria of 60 min of MVPA per day on a weekly average. There was a slight incline in MVPA from Monday to Friday that was more present in boys (Figure 1). REFERENCES 13. Price K, Bird SR, Lythgo N, Raj IS, Wong JY, Lynch C. Validation of the Fitbit One, Garmin Vivofit and Jawbone UP activity tracker in estimation of energy expenditure during treadmill walking and running. J Med Eng Technol (2017) 41:208–15. doi:10.1080/03091902.2016.1253795 1. Hallal PC, Andersen LB, Bull FC, Guthold R, Haskell W, Ekelund U, et al. Global physical activity levels: surveillance progress, pitfalls, and prospects. Lancet (2012) 380:247–57. doi:10.1016/S0140-6736(12)60646-1 1. Hallal PC, Andersen LB, Bull FC, Guthold R, Haskell W, Ekelund U, et al. Global physical activity levels: surveillance progress, pitfalls, and prospects. Lancet (2012) 380:247–57. doi:10.1016/S0140-6736(12)60646-1 14. El-Amrawy F, Nounou MI. Are currently available wearable devices for activity tracking and heart rate monitoring accurate, precise, and medically beneficial? Healthc Inform Res (2015) 21:315–20. doi:10.4258/hir.2015.21.4.315 14. El-Amrawy F, Nounou MI. Are currently available wearable devices for activity tracking and heart rate monitoring accurate, precise, and medically beneficial? Healthc Inform Res (2015) 21:315–20. doi:10.4258/hir.2015.21.4.315 2. WHO. Global Health Risks: Mortality and Burden of Disease Attributable to Selected Major Risks. Geneva: World Health Organization (2009). 15. Evenson KR, Goto MM, Furberg RD. Systematic review of the validity and reliability of consumer-wearable activity trackers. Int J Behav Nutr Phys Act (2015) 12:159. doi:10.1186/s12966-015-0314-1 3. Pate RR, Pratt M, Blair SN, Haskell WL, Macera CA, Bouchard C, et al. Physical activity and public health. A recommendation from the Centers for Disease Control and Prevention and the American College of Sports Medicine. JAMA (1995) 273:402–7. doi:10.1001/jama.1995.03520290054029 16. Strath SJ, Kaminsky LA, Ainsworth BE, Ekelund U, Freedson PS, Gary RA, et al. Guide to the assessment of physical activity: clinical and research applica- tions: a scientific statement from the American Heart Association. Circulation (2013) 128:2259–79. doi:10.1161/01.cir.0000435708.67487.da 4. Haskell WL, Lee IM, Pate RR, Powell KE, Blair SN, Franklin BA, et al. Physical activity and public health: updated recommendation for adults from the American College of Sports Medicine and the American Heart Association. Circulation (2007) 116:1081–93. doi:10.1161/CIRCULATIONAHA.107.185649 17. Strath SJ, Pfeiffer KA, Whitt-Glover MC. Accelerometer use with children, older adults, and adults with functional limitations. Med Sci Sports Exerc (2012) 44:S77–85. doi:10.1249/MSS.0b013e3182399eb1 5. WHO. Global Recommendations on Physical Activity for Health. Geneva: World Health Organization (2010). 18. Cortez NG, Cohen IG, Kesselheim AS. FDA regulation of mobile health technologies. N Engl J Med (2014) 371:372–9. doi:10.1056/ NEJMhle1403384 6. Cameron C, Craig CL, Bauman A, Tudor-Locke C. FUNDING Accelerometer-assessed PA is biased due to different devices and utilization of different cutoff points to determine MVPA when using accelerometry (25–28). These drawbacks make a comparison of our data possible only within our own cohort. This study was supported by the “Wilhelmine Holzapfel Stiftung” from Munich. This work was supported by the German Research Foundation (DFG) and the Technical University of Munich (TUM) in the framework of the Open Access Publishing Program. CONCLUSION Overall, wearable bracelets seem to be feasible devices for PA assessment even in young children. The appealing design and the relatively low price in comparison to triaxial or heart-rate-based accelerometers make them applicable in broader cohort studies. Nevertheless, their potential to increase PA for primary and sec- ondary prevention of cardiovascular disease needs to be clarified in further studies. DISCUSSION Initial interest at recruitment and compliance with wear time protocols is much improved for wrist-based commercial activity trackers, as com- pared with the less-appealing research-grade accelerometer worn Regarding the technical applicability, studies have shown that wearable bracelets or watches track steps and activity minutes at the wrist as accurate as accelerometers (12–14), although tech- nological details and algorithms of the manufactures are kept confidential. However, it should be mentioned that agreement between devices from different manufacturers is generally poor. This is due to different interpretation of guidelines and the use of different cut points (thresholds) for MVPA. This ambiguity resulting in a span from almost 0 to 95% in preschool children complying with PA recommendations (22) makes generalization Figure 1 \| Daily moderate-to-vigorous physical activity minutes according to weekdays. Figure 1 \| Daily moderate-to-vigorous physical activity minutes according to weekdays. Frontiers in Pediatrics \| www.frontiersin.org January 2018 \| Volume 6 \| Article 5 3 Wearables in Children Müller et al. ETHICS STATEMENT impossible which is why it is recommended that estimates are usable only within their own cohort of measurement (4, 16). The study was prospectively designed in accordance with the declaration of Helsinki (revision 2008) and approved by the local ethical board of the Technical University of Munich (project number 314/14). All children were orally informed on the mean- ing and purpose of the study and agreed to participate. Written informed consent was obtained from all guardians. AUTHOR CONTRIBUTIONS JM was responsible for conception, design of the study, sampled parts of the data, analyzed the data, and drafted the manuscript. A-MH and VZ sampled the data and gave important input for drafting and revising the manuscript. RO was responsible for conception and design of the study and gave important input for revising the manuscript. Study Limitations Several limitations are reported with the use of pictorial Likert scales in children (23, 24) and a possible scoring bias in feasibility could not entirely be ruled out. Moreover, the questionnaire lacks standardization and the voluntary recruitment of participants may have led to an overestimation of PA in this cohort. 27. Ainsworth BE, Bassett DR Jr, Strath SJ, Swartz AM, O’Brien WL, Thompson RW, et al. Comparison of three methods for measuring the time spent in physical activity. Med Sci Sports Exerc (2000) 32:S457–64. doi:10.1097/00005768- 200009001-00004 REFERENCES CANPLAY study: secular trends in steps/day amongst 5-19year-old Canadians between 2005 and 2014. Prev Med (2016) 86:28–33. doi:10.1016/j.ypmed.2015.12.020 19. Voss C, Gardner RF, Dean PH, Harris KC. Validity of commercial activity trackers in children with congenital heart disease. Can J Cardiol (2016) 33(6):799–805. doi:10.1016/j.cjca.2016.11.024 7. Sirard JR, Pate RR. Physical activity assessment in children and adolescents. Sports Med (2001) 31:439–54. doi:10.2165/00007256-200131060-00004 8. Sylvia LG, Bernstein EE, Hubbard JL, Keating L, Anderson EJ. Practical guide to measuring physical activity. J Acad Nutr Diet (2014) 114:199–208. doi:10.1016/j.jand.2013.09.018 f 20. Mercer K, Giangregorio L, Schneider E, Chilana P, Li M, Grindrod K. Acceptance of commercially available wearable activity trackers among adults aged over 50 and with chronic illness: a mixed-methods evaluation. JMIR Mhealth Uhealth (2016) 4:e7. doi:10.2196/mhealth.4225 9. Hardy LL, Hills AP, Timperio A, Cliff D, Lubans D, Morgan PJ, et al. A hitchhiker’s guide to assessing sedentary behaviour among young people: deci ding what method to use. J Sci Med Sport (2013) 16:28–35. doi:10.1016/j.jsams. 2012.05.010 21. Schaefer S, Ching C, Breen H, German J. Wearing, thinking, and moving: testing the feasibility of fitness tracking with urban youth. Am J Health Educ (2016) 47:8–16. doi:10.1080/19325037.2015.1111174 10. Piwek L, Ellis DA, Andrews S, Joinson A. The rise of consumer health wear- ables: promises and barriers. PLoS Med (2016) 13:e1001953. doi:10.1371/ journal.pmed.1001953 22. Beets MW, Bornstein D, Dowda M, Pate RR. Compliance with national guidelines for physical activity in U.S. preschoolers: measurement and inter- pretation. Pediatrics (2011) 127:658–64. doi:10.1542/peds.2010-2021 h 11. Kromeyer-Hauschild K, Wabitsch M, Kunzem D, Geller F, Geiß HC, Hesse V, et al. Perzentile für den Body-mass-Index für das Kindes- und Jugendalter unter Heranziehung verschiedener deutscher Stichproben. Monatsschr Kinderheilkd (2001) 149:807–18. doi:10.1007/s001120170107 23. Mellor D, Moore KA. The use of Likert scales with children. J Pediatr Psychol (2014) 39:369–79. doi:10.1093/jpepsy/jst079 24. van Laerhoven H, van der Zaag-Loonen HJ, Derkx BH. A comparison of Likert scale and visual analogue scales as response options in children’s questionnaires. Acta Paediatr (2004) 93:830–5. doi:10.1111/j.1651-2227.2004. tb03026.x 12. Alsubheen SA, George AM, Baker A, Rohr LE, Basset FA. Accuracy of the vivofit activity tracker. J Med Eng Technol (2016) 40:298–306. doi:10.1080/ 03091902.2016.1193238 January 2018 \| Volume 6 \| Article 5 Frontiers in Pediatrics \| www.frontiersin.org 4 Müller et al. Wearables in Children 28. Ekelund U, Tomkinson G, Armstrong N. What proportion of youth are phys- ically active? Measurement issues, levels and recent time trends. REFERENCES Br J Sports Med (2011) 45:859–65. doi:10.1136/bjsports-2011-090190 28. Ekelund U, Tomkinson G, Armstrong N. What proportion of youth are phys- ically active? Measurement issues, levels and recent time trends. Br J Sports Med (2011) 45:859–65. doi:10.1136/bjsports-2011-090190 25. Van Hecke L, Loyen A, Verloigne M, van der Ploeg HP, Lakerveld J, Brug J, et al. Variation in population levels of physical activity in European children and adolescents according to cross-European studies: a systematic literature review within DEDIPAC. Int J Behav Nutr Phys Act (2016) 13:70. doi:10.1186/ s12966-016-0396-4 Conflict of Interest Statement: The authors declare that the research was con- ducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 26. Vanhelst J, Beghin L, Salleron J, Ruiz JR, Ortega FB, Ottevaere C, et al. Impact of the choice of threshold on physical activity patterns in free living conditions among adolescents measured using a uniaxial accelerometer: the HELENA study. J Sports Sci (2014) 32:110–5. doi:10.1080/02640414.2013. 809473 Copyright © 2018 Müller, Hoch, Zoller and Oberhoffer. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. January 2018 \| Volume 6 \| Article 5 Frontiers in Pediatrics \| www.frontiersin.org 5
https://openalex.org/W3044884323	https://formative.jmir.org/2020/9/e20165/PDF	English	null	Feasibility of Self-Monitoring Rheumatoid Arthritis With a Smartphone App: Results of Two Mixed-Methods Pilot Studies	JMIR formative research	2,020	cc-by	9,063	Abstract Background: Several mobile apps that monitor symptoms of rheumatoid arthritis (RA) exist, but a recent systematic review indicated that high-quality apps are lacking. When patients self-monitor their own disease with patient-reported outcomes (PROs) and self-initiate care at the right moment, it may be possible to reduce the frequency of their clinic visits, which would reduce health care burden and costs. We developed an app, that is, the MijnReuma Reade app, for this purpose and performed 2 pilot tests with weekly self-monitoring. Objective: The primary objective of this study was to design, develop, and evaluate the usability, satisfaction, and usage of the MijnReuma Reade app—an app that allows patients with RA to monitor their own disease. The secondary objective was to review the patients’ perspectives on app usage and its intended purpose. Methods: This app was designed in collaboration with patients with RA, rheumatologists, and information technology experts. Two 1-month pilot studies were performed, after which satisfaction (0-10 scale), usability (system usability scale, 0-100), and usage (proportion of completed questionnaires) of this app were assessed. After the second pilot study, semistructured interviews were performed to determine patients’ perspectives and the promoters and barriers of app usage. Results: In the first and second pilot study, 42 and 27 patients were included, respectively. Overall, the patients were satisfied (medians, 8 and 7) and found the app usable (mean system usability scores, 76 and 71) in pilot studies 1 and 2, respectively. App usage declined over time in both the pilot studies; 61% (17/28) and 37% (10/27) of the patients who disclosed their usage statistics completed the final weekly questionnaire in pilot study 1 and pilot study 2, respectively. Approximately 81% (25/31) of the patients indicated they would like to skip hospital visits if the self-monitored disease activity is low. In the semistructured interviews, technical problems, internal resistance (respondent fatigue, the app reminded them of their disease), and a lack of symptoms were identified as barriers for usage. Patients reported that “experiencing more grip on their disease” and “improved communication with their physician” were promoters for usage. Patients reported that pain positively mediated usage, that is, more pain promoted and less pain discouraged app usage. Conclusions: This study illustrates the feasibility of the MijnReuma Reade app that enables self-monitoring of the disease activity in patients with RA with the overarching aim to allocate clinical consultations according to need. Corresponding Author: Corresponding Author: Bart F Seppen, MD Amsterdam Rheumatology and Immunology Center Reade Dr Jan van Breemenstraat 2 Amsterdam Netherlands Phone: 31 616796208 Email: b.seppen@reade.nl http://formative.jmir.org/2020/9/e20165/ Original Paper Original Paper JMIR FORMATIVE RESEARCH JMIR FORMATIVE RESEARCH Seppen et al JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 1 (page number not for citation purposes) Feasibility of Self-Monitoring Rheumatoid Arthritis With a Smartphone App: Results of Two Mixed-Methods Pilot Studies Bart F Seppen1,2, MD; Jimmy Wiegel1,2, MD; Merel J L'ami1, MSc, PhD; Sharon Duarte dos Santos Rico1, MD; Fabio S Catarinella3, MD; Franktien Turkstra1, MD, PhD; Maarten Boers1,4, MD, Prof Dr; Wouter H Bos1, MD, PhD 1Amsterdam Rheumatology and Immunology Center, Reade, Amsterdam, Netherlands 2Department of Rheumatology, VU Medical Center, Amsterdam UMC, Amsterdam, Netherlands 3Brightfish Ltd, Amsterdam, Netherlands 4Department of Epidemiology & Biostatistics, Amsterdam Public Health, Vrije Universiteit Amsterdam, Amsterdam UMC, Amsterdam, Netherlands Corresponding Author: Bart F Seppen, MD Amsterdam Rheumatology and Immunology Center Reade Dr Jan van Breemenstraat 2 Amsterdam Netherlands Phone: 31 616796208 Email: b.seppen@reade.nl Outcome Measures and Data Collection The primary outcomes of the pilot studies were satisfaction, usability, and app usage. Overall satisfaction was measured on a 10-point Likert scale (eg, How would you rate the app?). Alternatively, patient satisfaction was measured with the Net Promoter Score (NPS); this tool allows patients to rate the extent to which they would recommend the use of the app to a friend or colleague [20]. This tool, often used in customer loyalty research, predicts how likely a customer would recommend a product on an 11-point Likert scale. Patients who scored the app 9 or 10 were considered as promoters of the app, 7 or 8 were considered as neutrals or passive enthusiasts, and 0-6 were considered as detractors. Grouping patients into these 3 categories, that is, promoters, passive enthusiasts, and detractors, provides a simple intuitive scheme that accurately predicts the users’ behavior (ie, in business: the repurchase rate). The NPS is calculated by subtracting the proportion of critics from the proportion of promoters. Usability was evaluated with the system usability scale. The system usability scale has proved to be a valuable evaluation tool since it is highly robust and reliable [21,22]. The average system usability scale score is 68; a mean score of 52 indicates OK usability and 72 indicates good usability [23]. The final questionnaire included 2 additional questions regarding usability rated on a 10-point Likert scale ranging from “definitely agree, 10” to “definitely disagree, 1” (eg, “I use every function in the app” and “I think the explanation on how the app works is clear”). As proxy for app 1. Primary research question: Is it feasible to let patients with RA self-monitor their disease with the use of the MijnReuma Reade app, in terms of satisfaction, usability, and app usage? 2. Secondary research question: What are the perspectives of the patients regarding the app and self-monitoring with the purpose of reducing unnecessary consultations? KEYWORDS mHealth; eHealth; patient-reported outcome, smartphone app; rheumatoid arthritis; self-monitoring; telemonitoring; mobile phone mHealth; eHealth; patient-reported outcome, smartphone app; rheumatoid arthritis; self-monitoring; telemonitoring; mobile phone mHealth; eHealth; patient-reported outcome, smartphone app; rheumatoid arthritis; self-monitoring; telemo invited to participate in the pilot studies during regular outpatient clinic consultations by their treating rheumatologists. When patients indicated an interest to participate in these pilot studies to their rheumatologist, they were called by a researcher (SR). Interested patients were included if they met the following criteria: diagnosed with RA, 18 years or older, able to read Dutch, and own a smartphone or tablet with an Android or iPhone operating system. No exclusion criteria were set. All patients signed informed consent. Study Design Patients were asked to download the app from the app store and complete a questionnaire in the app every week for 4 weeks. In pilot study 1, the weekly questionnaire comprised the full multidimensional Health Assessment Questionnaire (HAQ, including an RA disease activity index and symptom list). In pilot study 2, we downsized the weekly questionnaire to Routine Assessment of Patient Index Data 3 (RAPID3) with additional questions regarding fatigue, sleep, morning stiffness, anxiety, stress, and social participation as found in the HAQ-II. After 4 weeks, a questionnaire was sent to the patients through email to evaluate the usability, satisfaction, and qualitative outcomes. Patients who stopped the study or never installed the app were not sent the final questionnaire. Technical problems reported by patients were recorded in an Excel logbook. The local Reade/Slotervaart hospital medical ethical committee issued a waiver for this study. Abstract Satisfaction with the app and usability of the app were found to be high; however, app usage declined over time. Patients acknowledged the potential of the app to self-monitor their own disease and would like to be able to skip clinic visits if the monitored disease activity is low. To evaluate this strategy, a randomized controlled trial is underway. (JMIR Form Res 2020;4(9):e20165) doi: 10.2196/20165 (JMIR Form Res 2020;4(9):e20165) doi: 10.2196/20165 JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 1 (page number not for citation purposes) JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 1 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Seppen et al Introduction eHealth—the health care practice supported by electronic processes and communication—is an upcoming theme in medicine [1]. One of the quickly developing fields within eHealth is mobile health (mHealth) care. mHealth promises to provide medical support for patients through mobile devices such as smartphones or tablets [2]. In rheumatology, we can use mHealth to enable patients to self-monitor their own conditions with patient-reported outcomes (PROs) [3-5], which in turn could support self-initiated care [6]. Most patients visit their rheumatologist every 3-6 months to evaluate disease activity [7]. The value of many of these consultations might be low, as many patients, at least in the affluent societies, have minimal disease activity [8]. Furthermore, due to the capricious nature of rheumatoid arthritis (RA), clinically relevant flares that occur between visits may be missed when patients visit the outpatient clinic according to predetermined schedules [9]. With mHealth, it is possible to monitor disease activity frequently [4,10], and thus, it may improve the clinical management of patients by better allocating clinical consultations according to need [11,12]. Several mobile apps that self-monitor disease activity already exist [13]. However, multiple studies have indicated that there is still a lack of high-quality apps for self-monitoring RA disease activity [13-15]. The quality of the apps can only be confirmed after a thorough and repeated clinical evaluation. This paper reports the development of an app to self-monitor RA disease activity and the results of 2 mixed-methods pilot studies. The research questions in the pilot studies were as follows: JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 2 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ Setting and Subjects The pilot studies were performed at Reade, a center for rheumatology and rehabilitation in Amsterdam. The city of Amsterdam along with its surroundings is an ideal setting for mHealth studies, as network coverage is excellent, download speeds rank 6th worldwide, and 87% of the adult population in Amsterdam own a smartphone [16-18]. In 2015, Reade started improving its information technology infrastructure [19]. The first goal was to digitize PROs. This facilitates electronic questionnaire assessment and integration of clinical data such as laboratory results, radiology reports, and severity scores with the PROs. Reade has now set the aim to extend the electronic PRO infrastructure to outside the walls of the hospital. In order to do this, we built an app that allows patients to access and complete PROs. Patients were informed about the app and http://formative.jmir.org/2020/9/e20165/ XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Seppen et al usage, we used the weekly response rate for RAPID3. All outcomes were presented in descriptive statistics. usage, we used the weekly response rate for RAPID3. All outcomes were presented in descriptive statistics. guidance for developing and evaluating complex interventions [26]. The 3 phases were as follows: (1) setting design requirements, building the prototype, and the first evaluation, (2) improvement of the prototype and re-evaluation, (3) further improvement of the app and a randomized controlled trial. Phase 3 has been described previously [6]. Design of the Prototype The prototype was developed in 2016. As recommended [14], this was done by a collaboration of patients (enthusiastic volunteers), nurses, rheumatologists, and information technology experts (Brightfish Ltd). The following design requirements were set. 1. Integration of a validated PRO. 1. Integration of a validated PRO. 2. Short weekly 5-minute questionnaires. Semistructured Interviews in Pilot Study 2 No areas of confusion or problems that required immediate repair were noted; therefore, we concluded that the app could be used in the first pilot study. After the first pilot study, a new phase of development took place. First, we shortened the questionnaire after pilot study 1 to meet the 5-minute requirement. Second, the app was integrated with the electronic medical record. Patients were now able to see their laboratory results and appointments. Furthermore, the filled out questionnaires were now visible in the patients’ medical files at Reade. This version of the app was used in the second pilot study. Patients’ Perspectives in Pilot Study 1 To assess the patients’ perspectives, the final survey included statements regarding the app, its purpose and possible features, and a free text field. The statements were adapted from Vorrink et al [24] for use in rheumatology and are presented in Multimedia Appendix 1. Patients were presented 17 “overall feedback” statements and 9 “privacy statements,” which they could score on a 10-point Likert scale (ranging from 1=definitely do not agree to 10=definitely agree). In the next section of the questionnaire, patients could (optionally) provide their opinion on what aspects of the app were unnecessary, unclear, or could be improved and what sections were useful and clear in a free text field. Semistructured Interviews in Pilot Study 2 3. High usability and user satisfaction. Patients in the second pilot study were asked to take part in a semistructured interview to explore their perspectives on the app, its intended purpose, and app usage. Patients were purposefully selected to form a varied group that included patients who frequently used the app, patients who discontinued use during the study, and patients who did not use the app more than once. The recruitment of patients continued until data saturation. One team member (BS) conducted telephone interviews (15 minutes) in November and December 2018. BS was not previously known to the patients and was not involved in the feasibility studies. Patients gave verbal consent for audio recording. Patients’experiences of using the app were explored following a 7-question interview guide (Multimedia Appendix 2). The questions were in part derived from themes in the mobile app rating scale and in part through discussion between authors BS and WB [25]. Questions were intended to guide the conversation, rather than to be prescriptive. The interviewer responded to patients’ comments and encouraged them to talk freely to maximize informative comments. All interviews were audiotaped and transcribed (BS). Patients’ perspectives on the app, app usage, and its intended purpose were thematically coded. The coding and thematic analysis were performed by BS; subsequently, 2 investigators (BS/WB) discussed the data. Illustrative comments were selected to illustrate the patients’ perspectives and the identified barriers and promoters of usage. 4. Multiplatform (native iPhone, native Android, and web-based operating systems). 5. Provision of helpful information for patients about RA. 5. Provision of helpful information for patients about RA. 6. Integration with the electronic medical record. A prototype MijnReuma Reade app was built by an information technology company (Figure 1). This prototype met design requirements 1-5. As a validated PRO, the multidimensional RAPID3-HAQ-II was chosen by the study team [27]. In the app, all the domains of disease activity are displayed in illustrative graphs over time [28,29]. BrightFish developed the interface to be easy to use and intuitive. Before the initiation of the pilot study, we performed a small pretest. The goal was to explore areas of confusion and areas to improve user experience. Fifteen volunteers completed the questionnaire in the app, while being observed by a rheumatologist (WB) and an information technology expert. Patient Characteristics For pilot study 1 and 2, 42 and 27 patients signed informed consent, respectively; 24 of the 27 patients in the second pilot study had also participated in the first pilot study. Patient characteristics are summarized in Table 1. During the first pilot study (n=42), 5 patients dropped out (12%), leaving 37 patients. The reasons for dropout were as follows: never downloaded or used the app (n=2) and technical problems with (n=1) or without Table 1. Patient characteristics. Pilot study 2, n=27 Pilot study 1, n=42 Patient characteristics 52 (11) 54 (13) Age (years), mean (SD) 21 (78) 36 (86) Females, n (%) 2.6 (1.48) 2.88 (1.28) Baseline disease activity score in 28 joints, mean (SD) 7 (3,8) 9 (4,13) Disease duration (years), median (25th percentile, 75th percentile) 16 (59) 27 (64) Biological use (yes), n (%) Primary Outcomes The primary outcomes are summarized in Table 2. Overall, the app was rated with satisfaction scores of 8.0 (IQR 7.0-9.0) and 7.0 (IQR 6.0-8.0) in the first and second pilot study, respectively. In the first pilot study, the NPS of the app was –9 (9/31 [29%] promoters, 10/31 [32%] passives, and 39% [12/31] detractors); in the second pilot study, the NPS was neutral (37% [7/19] promoters, 26% [5/19] passives, and 37% [7/19] detractors). The completion rates of the weekly in-app questionnaires declined over time in both pilot studies. In the first and second pilot study, the completion rates declined from 100% (28/28) and 78% (21/27) in week 1 to 61% (17/28) and 37% (10/27) in week 4, respectively. Table 1. Patient characteristics. Pilot study 2, n=27 Pilot study 1, n=42 Patient characteristics 52 (11) 54 (13) Age (years), mean (SD) 21 (78) 36 (86) Females, n (%) 2.6 (1.48) 2.88 (1.28) Baseline disease activity score in 28 joints, mean (SD) 7 (3,8) 9 (4,13) Disease duration (years), median (25th percentile, 75th percentile) 16 (59) 27 (64) Biological use (yes), n (%) Biological use (yes), n (%) App Development During the first pilot study (n=42), 5 patients dropped out (12%), leaving 37 patients. The reasons for dropout were as follows: never downloaded or used the app (n=2) and technical problems with (n=1) or without (n=2) direct relationship to the app (n=2). Of the 37 patients who completed the first pilot study, 31 patients filled the questionnaire to evaluate the app, while 6 patients did not respond after several reminders. Three patients did not provide their app ID in the questionnaire; the app ID was necessary to request the usage statistics from the software company. Thus, we analyzed the app usage of 28 patients. In the second pilot study, 2 patients never installed the app, 1 decided not to participate after consenting, and 5 did not complete the final questionnaire, leaving 19 patients for analysis. Results (n=2) direct relationship to the app (n=2). Of the 37 patients who completed the first pilot study, 31 patients filled the questionnaire to evaluate the app, while 6 patients did not respond after several reminders. Three patients did not provide their app ID in the questionnaire; the app ID was necessary to request the usage statistics from the software company. Thus, we analyzed the app usage of 28 patients. In the second pilot study, 2 patients never installed the app, 1 decided not to participate after consenting, and 5 did not complete the final questionnaire, leaving 19 patients for analysis. App Development The development and evaluation of the app were carried out in 3 distinct phases according to the Medical Research Council JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 3 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ http://formative.jmir.org/2020/9/e20165/ JMIR FORMATIVE RESEARCH Seppen et al Figure 1. The MijnReuma Reade App prototype. A. The activity module where due questionnaires are found. B. The dashboard module that displays several disease outcomes over time. C. The dossier module that displays the numerical answers given to the different questionnaires. D. The information module that provides information on the app and rheumatoid arthritis. English translation ("Dutch translation"); walking test ("Looptest’’); disease activity ("ziekte-activiteit’’); pain score ("pijn score’’). d 27 patients signed informed 27 patients in the second pilot the first pilot study. Patient n Table 1. During the first pilot out (12%), leaving 37 patients. follows: never downloaded or problems with (n=1) or without (n=2) direct relationship to the app (n=2). Of the 37 patients who completed the first pilot study, 31 patients filled the questionnaire to evaluate the app, while 6 patients did not respond after several reminders. Three patients did not provide their app ID in the questionnaire; the app ID was necessary to request the usage statistics from the software company. Thus, we analyzed the app usage of 28 patients. In the second pilot study, 2 patients never installed the app, 1 decided not to participate after consenting, and 5 did not complete the final questionnaire, leaving 19 patients for analysis. (n=2) direct relationship to the app (n=2). Of the 37 patients who completed the first pilot study, 31 patients filled the questionnaire to evaluate the app, while 6 patients did not respond after several reminders. Three patients did not provide their app ID in the questionnaire; the app ID was necessary to request the usage statistics from the software company. Thus, we analyzed the app usage of 28 patients. In the second pilot study, 2 patients never installed the app, 1 decided not to participate after consenting, and 5 did not complete the final questionnaire, leaving 19 patients for analysis. Results Patient Characteristics For pilot study 1 and 2, 42 and 27 patients signed informed consent, respectively; 24 of the 27 patients in the second pilot study had also participated in the first pilot study. Patient characteristics are summarized in Table 1. JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 4 (page number not for citation purposes) Qualitative Outcomes of Pilot Study 1 Patient opinions on the feedback statements have been shown in a heat map (Multimedia Appendix 3). In the open feedback fields, several patients reported that the HAQ-II was too long (over 5 minutes), which meant that it did not meet the set design requirements. No other issues with the app were reported. Patients indicated that the graphs (displaying outcomes over time) were “useful” and that “the interface was clear.” To optimize the app, several patients indicated that they desired an open field to disclose some notes with their submitted questionnaires, as they sometimes felt that the questionnaire did not fully capture their symptoms or that symptoms might be caused by something else. Other desires were a medication alarm/reminder, touch ID to log in, more graphs to display outcomes over time, a two-way chat function, or a change in questionnaires. Conflicting opinions were given regarding the addition of game-like elements to the app. Some opinions were positive such as “good, if it helps to me to fill out more questionnaires,” and “fun, if I can win something,” while some opinions indicated that the patients did not see any point in the addition of game-like elements, such as “I do not see additional value” or “not interesting.” Usability aScale of 1-10. The higher the score, the higher the satisfaction. bPilot study 1, n=31; Pilot study 2, n=19. cScale of 0-100. dPercentage of weekly questionnaires that were completed in the app. Pilot study 1, n=28; Pilot study 2, n=27. also identified 3 promoters for app usage: experiencing more grip on the disease, better communication with the physician, and an increase in disease activity. It can be noted that symptoms anecdotally play a modulating role in usage, as more symptoms induce usage whereas a lack of symptoms functions as a barrier for usage. A total of 5 illustrative quotes were chosen, which are presented in Table 3. Primary Outcomes The primary outcomes are summarized in Table 2. Overall, the app was rated with satisfaction scores of 8.0 (IQR 7.0-9.0) and 7.0 (IQR 6.0-8.0) in the first and second pilot study, respectively. In the first pilot study, the NPS of the app was –9 (9/31 [29%] promoters, 10/31 [32%] passives, and 39% [12/31] detractors); p p The completion rates of the weekly in-app questionnaires declined over time in both pilot studies. In the first and second pilot study, the completion rates declined from 100% (28/28) and 78% (21/27) in week 1 to 61% (17/28) and 37% (10/27) in week 4, respectively. JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 4 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ http://formative.jmir.org/2020/9/e20165/ XSL•FO RenderX Table 2. Primary outcomes of the pilot studies. Pilot study 2 Pilot study 1 Outcomes 7 (6,8) 8 (7,9) Overall satisfaction score, median (25th percentile, 75th percentile)a Net promoter scoreb 0 –9 Total score 7 (37) 9 (29) Promoters, n (%) 7 (37) 12 (39) Detractors, n (%) Usability 71 (20) 76 (15) System usability score, mean (SD)c Usaged 21 (78) 28 (100) Week 1, n (%) 11 (41) 26 (93) Week 2, n (%) 11 (41) 21 (75) Week 3, n (%) 10 (37) 17 (61) Week 4, n (%) aScale of 1-10. The higher the score, the higher the satisfaction. bPilot study 1, n=31; Pilot study 2, n=19. cScale of 0-100. dPercentage of weekly questionnaires that were completed in the app. Pilot study 1, n=28; Pilot study 2, n=27. Secondary Outcomes Qualitative Outcomes of Pilot Study 1 Patient opinions on the feedback statements have been shown in a heat map (Multimedia Appendix 3). In the open feedback fields, several patients reported that the HAQ-II was too long (over 5 minutes), which meant that it did not meet the set design requirements. No other issues with the app were reported. Patients indicated that the graphs (displaying outcomes over time) were “useful” and that “the interface was clear.” Qualitative Outcomes of Pilot Study 2: Semistructured Interviews In general, the app was described as “clear,” “easy to use,” and “user friendly.” Patients acknowledged that the app had the potential to improve insight in disease activity over time and that it could help to reduce the burden of unnecessary outpatient clinic visits in time. Usage of the app varied between the patients. Primary Outcomes When asked to state reasons for not using the app, the following barriers for app usage were identified: technical problems, internal resistance (respondent fatigue, the app reminded them of their disease), and a lack of symptoms. We also identified 3 promoters for app usage: experiencing more grip on the disease, better communication with the physician, and an increase in disease activity. It can be noted that symptoms anecdotally play a modulating role in usage, as more symptoms induce usage whereas a lack of symptoms functions as a barrier for usage. A total of 5 illustrative quotes were chosen, which are presented in Table 3. To optimize the app, several patients indicated that they desired an open field to disclose some notes with their submitted questionnaires, as they sometimes felt that the questionnaire did not fully capture their symptoms or that symptoms might be caused by something else. Other desires were a medication alarm/reminder, touch ID to log in, more graphs to display outcomes over time, a two-way chat function, or a change in questionnaires. Conflicting opinions were given regarding the addition of game-like elements to the app. Some opinions were positive such as “good, if it helps to me to fill out more questionnaires,” and “fun, if I can win something,” while some opinions indicated that the patients did not see any point in the addition of game-like elements, such as “I do not see additional value” or “not interesting.” Seppen et al JMIR FORMATIVE RESEARCH JMIR FORMATIVE RESEARCH Table 2. Primary outcomes of the pilot studies. Pilot study 2 Pilot study 1 Outcomes 7 (6,8) 8 (7,9) Overall satisfaction score, median (25th percentile, 75th percentile)a Net promoter scoreb 0 –9 Total score 7 (37) 9 (29) Promoters, n (%) 7 (37) 12 (39) Detractors, n (%) Usability 71 (20) 76 (15) System usability score, mean (SD)c Usaged 21 (78) 28 (100) Week 1, n (%) 11 (41) 26 (93) Week 2, n (%) 11 (41) 21 (75) Week 3, n (%) 10 (37) 17 (61) Week 4, n (%) aScale of 1-10. The higher the score, the higher the satisfaction. bPilot study 1, n=31; Pilot study 2, n=19. cScale of 0-100. dPercentage of weekly questionnaires that were completed in the app. Pilot study 1, n=28; Pilot study 2, n=27. Table 2. Primary outcomes of the pilot studies. Table 2. Primary outcomes of the pilot studies. Summary This study shows the design, development, and evaluation of a smartphone app that allows patients with RA to monitor their disease activity off-site. This app was developed in line with the recommendations by the European League Against Rheumatism taskforce for development of mHealth apps, which were published after the current pilot studies were performed [30]. The pilot studies showed promising satisfaction (overall) and usability ratings; however, the app usage rates remain a challenge. Furthermore, patients indicated that they agreed with self-monitoring to be able to better allocate clinical consultations according to need. The overarching aim of the app is to reduce the frequency of clinic visits if the self-monitored disease activity is low, thereby reducing the health care burden for patients, and healthcare costs.We believe that the app is ready to evaluate these anticipated benefits in a randomized controlled trial, as the overall satisfaction and usability ratings were very promising. The NPS showed less positive results, with a negative and a neutral score, which may indicate that patients would not likely recommend the app to others. The discrepancy between the NPS and the overall satisfaction rating may be caused by the cultural differences in scoring. The Dutch or the Europeans tend to give less extreme scores compared to the Americans, and the NPS originated in the United States [31]. If 8 was also considered a promoter score instead of a neutral score and 6 as a neutral score instead of a negative score, both pilot studies would have had a positive NPS rating. The positive NPS rating would better match the overall satisfaction rating. We believe that the proposed implementation strategy is also supported by patients because patients in this study and in previous research studies acknowledge that apps could assist allocation of clinic visits according to need [32]. Furthermore, they approved of self-monitoring (27/31, 87%) and would like to skip hospital visits if the self-monitored disease activity is low (25/31, 81%). There were no concerns with regard to data privacy and security with this app, and a majority of the patients intended to keep using this app in the future. Qualitative Outcomes of Pilot Study 2: Semistructured Interviews If we get at least one questionnaire per patient per month for a year, we will still have 4 times more updates on their disease activity than when patients visit the outpatient clinic every 3 months. In the qualitative part of our study, we reported several factors that could play a role in the declining usage, including a lack of symptoms, technical barriers, and respondent fatigue, which are endorsed by previous research [32]. Possible ways to increase usage would include providing shorter questionnaires or adaptive questionnaires, improving persuasive and gamified app designs, adding reminder notifications, and limiting technical problems [33,38,39]. Furthermore, as patients reported that more disease activity stimulated usage, it is possible that patients mainly use the app in case of impending flares. This could mean that although usage is low, no flares are missed. This hypothesis should be further examined in larger observational studies. http://formative.jmir.org/2020/9/e20165/ Qualitative Outcomes of Pilot Study 2: Semistructured Interviews Qualitative Outcomes of Pilot Study 2: Semistructured Interviews In general, the app was described as “clear,” “easy to use,” and “user friendly.” Patients acknowledged that the app had the potential to improve insight in disease activity over time and that it could help to reduce the burden of unnecessary outpatient clinic visits in time. Usage of the app varied between the patients. When asked to state reasons for not using the app, the following barriers for app usage were identified: technical problems, internal resistance (respondent fatigue, the app reminded them of their disease), and a lack of symptoms. We JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 5 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ http://formative.jmir.org/2020/9/e20165/ JMIR FORMATIVE RESEARCH Table 3. Illustrative quotes of the patients. Patients with a similar quote (n) Indicative quote Identified barriers and promoters 5 …Improves interaction with my doctor, as the complaints I have had in the past month are now clearer. Grip on disease and better communication with physician 5 …When my pain relapses, I would be more inclined to fill out the questionnaire. Disease activity 4 …Technical problems prevented me from further usage. Technical problems 4 …It is the same (questionnaire) every time. Respondent fatigue 3 …When I fill out the questionnaire, it makes me feel like a patient, I prefer not to feel like a patient this often. App reminds patients of their disease Seppen et al JMIR FORMATIVE RESEARCH Patients with a similar quote (n) of the users drop out before completion or stop using the app [33-35]. The frequency of usage was previously evaluated in 2 apps for patients with RA; the median completion rates were 91% of the daily questionnaires over 3 months and 79% of the daily questionnaires over 6 months [36,37]. Our completion rates were lower; this could be (partly) due to the difference in the intended usage frequency (daily versus weekly). One review shows that more frequent intended usage predicts better adherence [33]. For now, it is unclear how often a patient has to be monitored to better target consultation according to need. Hypothetically, if one questionnaire per month would be needed, it might be recommendable to set the intended usage to once a week to make sure that sufficient questionnaires are collected. Even considering the limited usage, we did collect at least one questionnaire per patient. JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 6 (page number not for citation purposes) Acknowledgments The authors would like to thank all patient partners who helped with the development of the app. This study is supported by AbbVie. AbbVie had no role in the design of the study and did not have any role during its execution, analyses, interpretation of the data, or decision to submit results. Limitations As these were pilot studies, several limitations are present. First, the app is only available for patients of Reade, which limits generalizability. To improve this, we have, as a starting point, made our prototype available for other designers and health care centers. This will help others create a similar app. Second, it is possible that patients with enthusiasm for eHealth were more likely to participate. Therefore, it may be possible that the volunteers had above average technical skills and motivation to use the app. This warrants larger observational studies and controlled experiments in the future. Third, we cannot preclude that patients provided favorable feedback to the investigators. We did try to minimize this limitation by performing semistructured interviews and pilot studies with different researchers so that both had no previous relationships with the Conclusion Two pilot studies demonstrated that self-monitoring RA disease activity with the MijnReuma Reade app is feasible in terms of overall (patient) satisfaction and usability; however, the app usage rates remain a challenge. Patients acknowledged that the app had the potential to help them self-monitor their own disease so that they could reduce their frequency of clinic visits in case of low disease activity. Conflicts of Interest FSC is CMIO at Brightfish Ltd, the company that developed the MijnReuma Reade app. Authors' Contributions SR and WB performed the pilot studies. BS and JW analyzed the data and wrote and edited the manuscript. All other authors were involved in the design of the study, the development of the app, and editing of the manuscript. Multimedia Appendix 2 Interview guide. Multimedia Appendix 3 Heatmap of the patients' perspectives. [DOCX File , 25 KB-Multimedia Appendix 3] Multimedia Appendix 1 Statements of patients' perspectives. Statements of patients' perspectives. [DOCX File , 14 KB-Multimedia Appendix 1] JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 7 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ Strengths We performed 2 pilot studies with different qualitative and quantitative approaches to evaluate the app. This optimized our understanding of the patients’ perspectives toward the app and its purpose and gave insights into the overall functioning of the app. We think these data provide meaningful insights to aspiring medical app designers and rheumatologists who are considering to prescribe apps to specific patient populations. Furthermore, the overall strengths of our project are patient involvement in all stages of the app development and integration of the app with the existing Reade electronic medical record. Ultimately, we have developed a mobile app that facilitates easy data entry for patients, and visualization of that data for both patients and Declining adherence is a challenge with our app and for medical apps in general. In any eHealth trial, a substantial proportion XSL•FO RenderX XSL•FO RenderX JMIR FORMATIVE RESEARCH Seppen et al patients. Fourth, the semistructured interviews were rather short; however, after 9 interviews, no new opinions and data arose. We feel we have covered the most important opinions and experiences with patients. However, it could be possible that with longer interviews, more data would have been gathered. The last limitation is that patients who did not install the app were not included in the final questionnaire of the first pilot study—this may have led to an overestimation of the positive effects. To collect valuable information on the nonusers, we did purposefully include that specific group in the semistructured interviews to examine their barriers for adherence. physicians. The repetitive collection of PROs with the app combined with statistics in the patients’ existing electronic medical record has enormous research potential. This has been recognized before, but this integration is often not accomplished [14,40,41]. JMIR FORMATIVE RESEARCH Seppen et al 4. Crouthamel M, Quattrocchi E, Watts S, Wang S, Berry P, Garcia-Gancedo L, et al. Using a ResearchKit Smartphone App to Collect Rheumatoid Arthritis Symptoms From Real-World Participants: Feasibility Study. JMIR Mhealth Uhealth 2018 Sep 13;6(9):e177 [FREE Full text] [doi: 10.2196/mhealth.9656] [Medline: 30213779] 4. Crouthamel M, Quattrocchi E, Watts S, Wang S, Berry P, Garcia-Gancedo L, et al. Using a ResearchKit Smartphone App to Collect Rheumatoid Arthritis Symptoms From Real-World Participants: Feasibility Study. 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JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 10 (page number not for citation purposes) Abbreviations Abbreviations HAQ: Health Assessment Questionnaire mHealth: mobile health NPS: Net Promoter Score PRO: patient-reported outcome RAPID3: Routine Assessment of Patient Index Data 3 JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 9 (page number not for citation purposes) JMIR Form Res 2020 \| vol. 4 \| iss. 9 \| e20165 \| p. 9 (page number not for citation purposes) http://formative.jmir.org/2020/9/e20165/ http://formative.jmir.org/2020/9/e20165/ JMIR FORMATIVE RESEARCH Seppen et al Edited by G Eysenbach; submitted 15.05.20; peer-reviewed by J Knitza, R Grainger; comments to author 22.06.20; r received 21.07.20; accepted 22.07.20; published 21.09.20 ysenbach; submitted 15.05.20; peer-reviewed by J Knitza, R Grainger; comments to author 22.06.20; revised version 20; accepted 22.07.20; published 21.09.20 Please cite as: Seppen BF, Wiegel J, L'ami MJ, Duarte dos Santos Rico S, Catarinella FS, Turkstra F, Boers M, Bos WH Feasibility of Self-Monitoring Rheumatoid Arthritis With a Smartphone App: Results of Two Mixed-Methods Pilot Studies JMIR Form Res 2020;4(9):e20165 URL: http://formative.jmir.org/2020/9/e20165/ doi: 10.2196/20165 PMID: 32955447 ©Bart F Seppen, Jimmy Wiegel, Merel J L'ami, Sharon Duarte dos Santos Rico, Fabio S Catarinella, Franktien Turkstra, Maarten Boers, Wouter H Bos. Originally published in JMIR Formative Research (http://formative.jmir.org), 21.09.2020. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Formative Research, is properly cited. The complete bibliographic information, a link to the original publication on http://formative.jmir.org, as well as this copyright and license information must be included. ©Bart F Seppen, Jimmy Wiegel, Merel J L'ami, Sharon Duarte dos Santos Rico, Fabio S Catarinella, Franktien Turkstra, Maarten Boers, Wouter H Bos. Originally published in JMIR Formative Research (http://formative.jmir.org), 21.09.2020. 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https://openalex.org/W3162318924	https://hal.science/hal-03227563/document	English	null	Impact of early low-calorie low-protein versus standard-calorie standard-protein feeding on outcomes of ventilated adults with shock: design and conduct of a randomised, controlled, multicentre, open-label, parallel-group trial (NUTRIREA-3)	BMJ open	2,021	cc-by	212	Impact of early low-calorie low-protein versus standard-calorie standard-protein feeding on outcomes of ventilated adults with shock: design and conduct of a randomised, controlled, multicentre, open-label, parallel-group trial (NUTRIREA-3) Jean Reignier, Amélie Le Gouge, Jean-Baptiste Lascarrou, Djillali Annane, Laurent Argaud, Yannick Hourmant, Pierre Asfar, Julio Badie, Mai-Anh Nay, Nicolae-Vlad Botoc, et al. To cite this version: Jean Reignier, Amélie Le Gouge, Jean-Baptiste Lascarrou, Djillali Annane, Laurent Argaud, et al.. Impact of early low-calorie low-protein versus standard-calorie standard-protein feeding on outcomes of ventilated adults with shock: design and conduct of a randomised, controlled, multicentre, open-label, parallel-group trial (NUTRIREA-3). BMJ Open, 2021, 11 (5), pp.e045041. 10.1136/bmjopen-2020- 045041. hal-03227563 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-03227563 https://hal.science/hal-03227563v1 Submitted on 17 May 2021 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License
https://openalex.org/W2770600750	https://europepmc.org/articles/pmc5778210?pdf=render	English	null	Nutritional, physicochemical, and functional properties of protein concentrate and isolate of newly‐developed Bambara groundnut (<i>Vigna subterrenea</i> L.) cultivars	Food science & nutrition	2,017	cc-by	10,830	Received: 16 August 2017 \| Revised: 20 October 2017 \| Accepted: 31 October 2017 Received: 16 August 2017 \| Revised: 20 October 2017 \| Accepted: 31 October 2017 Received: 16 August 2017 \| Revised: 20 October 2017 \| Accepted: 31 October 2017 DOI: 10.1002/fsn3.552 DOI: 10.1002/fsn3.552 Olaposi R. Adeleke1 \| Oladipupo Q. Adiamo1 \| Olumide S. Fawale2 Higher protein content was observed in BGW (20.73%) and BGB (20.14%) as compared to BGC (18.50%). Also, the fat and ash contents of BGB and BGW were higher than that of BGC. Also, the new varieties were found to contain higher levels of some essential fatty acids such as linoleic and linolenic acids. The concentration of thiamine, riboflavin, niacin, pantothenic, ascorbic acids, pyrodoxine, alpha tocopherol, and vitamin K were also significantly higher in the two new varieties. The new varieties were good sources of magnesium, calcium, iron, manganese, sodium, and potassium. The oil and water absorption and swelling capaci- ties of whole, defatted, and protein concentrate flour of the new varieties increase with increase in temperature. The defatted flour and protein concentrate of brown Bambara groundnut was found to exhibit high emulsifying activity and stability at dif- ferent pH’s and salt concentrations. The new varieties possess significantly higher foaming capacity and stability than the commercial variety. The results obtained from this study have shown the potential for the industrial and household use of the new Bambara groundnut cultivars into shelf stable protein products and could be a useful ingredient in food formulations. Olaposi R. Adeleke1 \| Oladipupo Q. Adiamo1 \| Olumide S. Fawale2 Olaposi R. Adeleke1 \| Oladipupo Q. Adiamo1 \| Olumide S. Fawale2 1Department of Food Science and Technology, Faculty of Technology, Obafemi Awolowo University, Ile-Ife, Osun State, Nigeria Abstract Bambara groundnut is an indigenous African vegetable grown mainly for human food and animal feed due to its high protein content. Different factors like varieties and ori- gin can influence the chemical composition of Bambara groundnut cultivars. Therefore, the aims of this study are to produce defatted flour and protein concentrate from newly developed Bambara groundnut cultivars [Accessions No: TVSU 5 – Bambara Groundnut White (BGW) and TVSU 146 – Bambara Groundnut Brown (BGB)] and compare their nutritional, physicochemical, and functional properties with market sample [Bambara groundnut commercial (BGC)]. Higher protein content was observed in BGW (20.73%) and BGB (20.14%) as compared to BGC (18.50%). Also, the fat and ash contents of BGB and BGW were higher than that of BGC. Also, the new varieties were found to contain higher levels of some essential fatty acids such as linoleic and linolenic acids. The concentration of thiamine, riboflavin, niacin, pantothenic, ascorbic acids, pyrodoxine, alpha tocopherol, and vitamin K were also significantly higher in the two new varieties. The new varieties were good sources of magnesium, calcium, iron, manganese, sodium, and potassium. The oil and water absorption and swelling capaci- ties of whole, defatted, and protein concentrate flour of the new varieties increase with increase in temperature. The defatted flour and protein concentrate of brown Bambara groundnut was found to exhibit high emulsifying activity and stability at dif- ferent pH’s and salt concentrations. The new varieties possess significantly higher foaming capacity and stability than the commercial variety. The results obtained from this study have shown the potential for the industrial and household use of the new Bambara groundnut cultivars into shelf stable protein products and could be a useful ingredient in food formulations. Abstract Bambara groundnut is an indigenous African vegetable grown mainly for human food and animal feed due to its high protein content. Different factors like varieties and ori- gin can influence the chemical composition of Bambara groundnut cultivars. Therefore, the aims of this study are to produce defatted flour and protein concentrate from newly developed Bambara groundnut cultivars [Accessions No: TVSU 5 – Bambara Groundnut White (BGW) and TVSU 146 – Bambara Groundnut Brown (BGB)] and compare their nutritional, physicochemical, and functional properties with market sample [Bambara groundnut commercial (BGC)]. Food Sci Nutr. 2018;6:229–242. \| 229 www.foodscience-nutrition.com This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2017 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. www.foodscience-nutrition.com 230 \| 1 \| INTRODUCTION 230 ADELEKE et al. – Bambara Groundnut Brown (BGB) used for this study were col- lected from the International Institute of Tropical Agriculture (IITA), Ibadan, Oyo State, Nigeria. The seeds were planted on a farmland at Kelebe, Osogbo, Osun State, Nigeria and the ripe, matured dried pods were harvested after 160 days. The seeds were dehulled from the sundried pods while the Bambara groundnut commercial (BGC) seeds were purchased from a local market in Oyo, Nigeria. Adhering dirt were removed from the seeds by washing with clean tap water, sun dried and stored in airtight containers for analysis. All chemicals used were of analytical grade and obtained from Sigma chemicals, (St. Louis, MO, U.S.A.). In human diet, legumes are being ranked next to cereals as sources of calorie and protein. With an exponential increase in population growth, decline per head capital availability and bad weather conditions, the production of common legumes like cowpeas might be inadequate (Atiku & Mohammed, 2004). The need therefore arises for the develop- ment and utilization of underutilized legume seeds which are abundant in Nigeria and are under-exploited. Bambara groundnut (Vigna subterre- nea, L.) falls into this group of underutilized species of plants. Bambara groundnut (Vigna subterrenea, L.) is an indigenous African vegetable cultivated principally by farmers as a “famine food.” The high tolerance for drought and poor soil enables Bambara to grow under con- ditions unsuitable for groundnut (Agbenorhevi, Oduro, Ellis, Aodakpi, & Eleblu, 2007). In Nigeria, Bambara groundnut has found various food uses: it can be fried or boiled and eaten as snack or pounded into flour and used in the preparation of soup, porridge, and various fried or steamed food products such as “akara, moin-moin” and “okpa.” It has also been used in the preparation of local food drink such as “kunnu” and “tuwo.” Reports has it that Bambara groundnut flour has been used in making bread in Zambia (Adebowale, Adeyemi, & Oshodi, 2005) and it was also observed that milk prepared from Bambara groundnut gave a preferred flavor when compared with milk from soybean, cow- pea, and pigeon pea (Piyarat, 2008). The animal feed and haulm po- tentials of Bambara groundnut has long been discovered (Adebowale & Lawal, 2004) and its suitability for animal grazing which is basically due to the abundance of nitrogen and phosphorus in its leaves has also been reported (Bamishaiye, Adegbola, & Bamishaiye, 2011). 2.4 \| Physicochemical and functional properties Bulk density (BD) was determined by the method described by Okezie and Bello (1988). A method described by Adepeju, Gbadamosi, Adeniran, and Omobuwajo (2011) was used in determining the water absorption capacity (WAC) at room temperature (25°C) and vary- ing temperature (60–90°C). Two gram (2 g) sample was mixed with 10 ml distilled water, centrifuged at 4,000g for 30 min and the water absorption was expressed as percentage increase in the sample 2.2.1 \| Preparation of defatted flour Bambara groundnut defatted flour was prepared using a modified method of Sathe (1994). The cleaned Bambara groundnut were ground in a Warring blender (BLG-450, Binatone, Shenzhen, China) and the flour was defatted with cold (4°C) acetone (flour to solvent ratio 1:5 w/v) with constant magnetic stirring provided for 4 hr. The trace of residual acetone was removed by placing the defatted flour inside a fume cupboard for 6 hr to dry. A fine powder (moisture con- tent: 10.92%) was obtained by grounding the defatted flakes, sieved through a size mesh of 150 μm, packed in plastic tubes and stored at −10°C. 2.2.2 \| Preparation of protein concentrate Bambara groundnut concentrate samples were prepared by a modi- fication of the method described by Cheftel, Lug, and Lorient (1985). A ratio of 1:10 flour to water was stirred on a magnetic stirrer for 10 min, and the resultant slurry was adjusted to pH 4.0 and centri- fuged (MSE, Harrier 15/80, Sydenham, London, U.K.) at 3,500g for 30 min. The precipitate pH was adjusted to 7.0, was washed twice with distilled water, centrifuged at 3,500g for 10 min and dried in an oven at 45°C for 8 hr to obtain the protein concentrate. However, available data on the chemical composition and nutritional properties are limited to the commonly used variety (commercial sample) and these parameters may vary among same sample of different vari- eties. The changes in the properties may be influenced by the variety/ genetic origin of the varieties as well as climatic conditions, soil, pesti- cides, and fertilizers employed in the production of the crop. Therefore, the objective of the present research was to study the chemical com- positions and nutritional properties of two newly developed Bambara groundnut cultivars and evaluate the functional properties of their defatted flour and protein concentrate. This study could provide some basic information, which would help determine an application for the newly developed cultivars as source of plant protein in food products. 2.3 \| Proximate composition Crude protein, moisture, fat, and ash contents of whole and defatted flour of BGB, BGW, and BGC were determined according to AOAC (2000). 230 \| 1 \| INTRODUCTION Atiku and Mohammed (2004) noted that in the North Eastern Nigeria, Bambara groundnut is not only consumed as food but also used for medicinal purposes. Research efforts in Nigeria have only focused on the agron- omy and little or no attention has been paid to the chemical composition and functional properties of protein products of some newly developed varieties of Bambara groundnut in spite of its growing importance. K E Y W O R D S bambara groundnut cultivars, Defatted flour, Functional properties, Nutritional properties, Physicochemical properties, Protein concentrate under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, properly cited. \| 229 www.foodscience-nutrition.com \| www.foodscience-nutrition.com Food Sci Nutr. 2018;6:229–242. Sample preparation The water-soluble vitamins were extracted by weighing exactly 0.100 g sample into 100 ml volumetric flasks; then 80 ml of water was added. After 15 min of extraction, water was added to the mark and the solution was filtered through a 0.2 μm filter prior to injection into the HPLC. About 0.125 g of Bambara groundnut flours were weighed separately into 10 ml volumetric flasks followed by addition of 8 ml of CH3OH:CH2Cl2 (1:1, v/v) to each flask to extract the fat soluble vitamins. After 15 min of extraction, CH3OH:CH2Cl2 (1:1, v/v) was added to the mark. The so- lution was filtered through 0.2 μm filter and the sample solutions were stored in a dark cupboard prior to injection into the HPLC. Standard preparation A stock solution of 1.0 mg/ml of each water soluble vitamin stand- ards (thiamine (Vitamin B1), riboflavin (Vitamin B2), nicotinamide (Vitamin B3), pantothenic acid (Vitamin B5), pyridoxine (Vitamin B6), cyanocobalamin (Vitamin B12), and ascorbic acid (vitamin C)) was prepared by weighing 10 mg of each vitamin powder and 10 ml of deionized water was added. The concentration of stock solution of vitamin B2 was decreased to 0.25 mg/ml in deionized water while vitamin B9 was prepared using 0.2 mol/L of KHCO3 instead of deionized water to make a solution of 0.5 mg/ml due to their limited solubility in water. A stock solution of 1.0 mg/ml for each fat-soluble vitamin standards of vitamins A, D, and E was prepared by weighing 10 mg of each standard vitamin and 10 ml of CH3OH was added. Acetone and CH2Cl2 was used in preparing the standard for vitamin K1 instead of CH3OH. The stock standards of fat soluble vitamins were stored in the dark. A mixture of CH3OH:CH2Cl2 (1:1, v/v) was used for preparation of working standard for fat-soluble vitamins while the water-soluble vitamin working standards were prepared from the stock standards on the day of use by diluting with deion- ized water. weight. Oil absorption capacity (OAC) was determined by the cen- trifugal method described by Beuchat (1977). The method of Sathe and Salunkhe (1981) was employed for the determination of gelling concentration. The swelling capacity (SC) was determined using the method described by Takashi and Sieb (1988) with slight modification. Exactly, 1 g of sample was mixed with 10 ml distilled water and the slurry was heated at a constant temperature (60, 70, 80, and 90°C) in a water bath for 15 min, centrifuged at 3,000g for 10 min and the SC was expressed as percentage increase in sample weight. Vol: 20 μl; UV Detection: Absorbance at 210, 245, 265, and 280 nm. Also the conditions for determination of fat-soluble vitamins include; Column: Acclaim C18, 5 μm, 120 Å, 4.6 × 150 mm (P/N 059133); Temperature: 25°C; Mobile phase: A: CH3OH:CH3CN (8:2, v/v); B: MTBE; Flow Rate: 1.0 ml/min; Injection Vol: 20 μl; UV Detection: Wavelength-switching absorbance at 265, 325, and 450 nm. 2.5.2 \| Minerals content analysis The main and trace elements of sample was determined using the method described in the AOAC (2000). Exactly 0.5 g of sample was weighed into a digestion tube and 10 ml of nitric/perchloric acid was added prior to mineral determination. The samples were digested at 150°C until a clear fume was obtained and washed into a standard 50 ml volumetric flask and distilled water was added up to the mark. Atomic absorption spectrophotometer (210VGP, Buck Scientific incorporation, Norwalk, Connecticut, USA) was used in the deter- mination of calcium (Ca), magnesium (Mg), copper (Cu), zinc (Zn), manganese (Mn), cadmium (Cd), chromium (Cr), and iron (Fe) was determined using a spectrophotometer (Spectro 20D plus RS-232C, Labomed incorporation, Culver city, CA, USA) and sodium (Na) and potassium (K) were determined using a flame photometer (2655-00, Coring Inc., USA.). 2.5.3 \| Fatty acid determination The fatty acid composition of the samples was determined using Gas Chromatography (GC) of the methyl ester method (Sun, Han, Yan, Yang, & Tetsuo, 2008). Exactly, 0.5 g of each sample was weighed, mixed with 1.5 ml hexane overnight and the mixture was centrifuged at 7,000 rpm for 5 min. About 350 lL of sodium methoxide solution was added to the supernatant, vortexed thoroughly and mixed for 1 hr. The supernatant obtained after centrifuging at 7,000 rpm for 5 min was filtered into the special sample bottle for GC detectors. HP INNOWax Column (30 mm × 0.25 mm × 0.25 mm) was used for the GC analysis with nitrogen, hydrogen, and air as the carrier gases for 20 min and the injection volume was 1 lL. The percentage of fatty acid components was calculated using the area normalization method. 2.1 \| Collection of raw materials The two new varieties of Bambara groundnut seeds (Accessions No: TVSU 5 – Bambara Groundnut White (BGW) and TVSU 146 ADELEKE et al. 231 2.6 \| Effect of pH and NaCl concentration on emulsifying activity index and emulsion stability index of defatted and protein concentrate of Bambara groundnut cultivars The effect of pH and salt concentration on emulsifying activity index (EAI) was determined by the method described by Gbadamosi, Abiose, and Aluko (2012) with some modifications. At different NaCl concen- trations (0.0, 0.5, and 1.0 mol/L), approximately 500 mg of the sam- ples was dispersed in 100 ml of distilled water. The pH of the protein solution was then adjusted with either 1 N HCl or 1 N NaOH to pH 2, 4, 6, 8, and 10 separately. The protein solution was homogenized using a blender (SN2200 Qlink, Beijing, China) set at high speed for Emulsion stability index (%)= AA×Δt A−AA where A is the absorbance at 0 min after homogenization; AA is the absorbance at 10 min after homogenization; Δt = 10 min; and ΔA = A–AA. where A is the absorbance at 0 min after homogenization; AA is the absorbance at 10 min after homogenization; Δt = 10 min; and ΔA = A–AA. 2.7 \| Effect of pH and NaCl concentration on foam capacity and stability of defatted and protein concentrate of Bambara groundnut cultivars The effect of pH and salt concentration on foam capacity (FC) and foam stability (FS) was determined by a modification of the method described by Chavan, McKenzie, and Shahidi (2001). At different NaCl concentrations (0.0, 0.5, and 1.0 mol/L), approximately 500 mg of the samples was dispersed in 100 ml of distilled water. The pH of the protein solution was then adjusted with either 1 N HCl or 1 N NaOH to pH 2, 4, 6, 8, and 10 separately. The protein solution was homogenized using a blender (SN2200 Qlink, Beijing, China) set at high speed for 2 min and then poured into 250 ml measuring cylinder. The percentage ratio of the volume increase to that of the original volume of protein solution in the measuring cylinder was calculated and expressed as FC while the FS was expressed as percentage of the volume of foam remaining in the measuring cylinder to that of the original volume after 30 min of quiescent period. \| RESULTS AND DISCUSSION 3 60 s after been mixed with 50 ml of pure Gino oil. After homogeniza- tion, 50 µl of the aliquot of the emulsion was transferred from the bottom of the blender and mixed with 5 ml of 0.1% sodium dodecyl sulfate (SDS) solution. The absorbance of the resulting solution was measured at 500 nm using spectrophotometer (722-2000 Spectronic 20D, Jiangsu, China). The EAI was calculated from the absorbance ob- tained as shown in the formula below: 3.2.1 \| Vitamins composition The vitamin contents of samples BGW, BGB, and BGC are shown in Table 1. The result of the B-vitamins showed that BGW and BGB had significantly (p < .05) higher B-vitamin contents as compared to the commercial sample, BGC. Vitamin B3 was highest among all the vitamins in the three samples studied with BGW exhibiting highest value of 13.05 as compared to that BGB (12.74) and BGC (11.40). The results of this investigation were significantly (p < .05) higher than the value of 0.88 mg/100 g reported for vitamin B3 in Bambara groundnut seed by Odeghe, Adumanya, Obi-Adumanya, and Chukwu (2012). Varietal differences as well as determination procedure could be the reason for the difference. According to FAO/WHO (2004), the recommended daily allowance for vitamin B3 (water soluble vitamin) that occurs in the form of niacin, nicotinic acid, and nicotinamide in adult males and female is 14–16 mg nia- cin equivalent. Therefore, the high vitamin B3 content found in the Foaming capacity (%) = (volume after whipping−volume before whipping )ml (volume before whipping )ml ×100 Foaming stability (%) = (volume after whipping−volume after standing )ml (volume after whipping−volume before whipping)ml ×100 Foaming capacity (%) ( l f hi (volume before whipping )ml HPLC conditions For determination of water-soluble vitamins, the conditions were; Column: Acclaim PA2, 5 μm, 120 Å, 4.6 × 150 mm (P/N 063197); Temperature: 25°C Mobile phase: A: CH3CN B: 0.25 mol/L phosphate buffer (about 3.4 g KH2PO4 was dissolved in 1 L of water, and pH was adjusted to 3.2 with H3PO4); Flow Rate: 1.0 ml/min; Injection 232 ADELEKE et al. 3.1 \| Chemical composition The chemical composition of the Bambara groundnut cultivars is shown in Table 1. The protein content of the samples ranged be- tween 18.50% and 20.73%, with the commercial sample (BGC) ex- hibiting the lowest (18.50%) and BGW the highest (20.73%). The protein contents of BGW (20.73%) and BGB (20.14%) were signifi- cantly (p < .05) higher than that of the commercial sample (BGC: 18.50%). The values obtained in this study agree with what was reported for commercial Bambara groundnut seeds by Abdulsalami and Sherriff (2010). According to the FAO/WHO (2007), adequate dietary protein is essential during growth when new tissue proteins are being synthesized. The two new varieties owing to their high contents of protein could be utilized in the formulation of protein rich diet or for the supplementation of diets low in protein contents. The fat contents of the samples ranged between 6.01% and 7.93% with BGB exhibiting significantly (p < .05) highest value. These values obtained were comparable with that obtained for Bambara groundnut seeds (Eltayeb, Ali, Abou-Arab, & Abu-Salem, 2011). Essential fatty acids and energy which serves as a vehicle for fat- soluble vitamins and facilitates their absorption are been produced by dietary fat. The ash composition of sample BGW (5.15%) was sig- nificantly (p < .05) higher than that of BGB (4.33%) and BGC (4.18%) and these values were within the range (3.0%–4.5%) reported by Fadahunsi, Jonathan, and Garuba (2010) for Bambara groundnut. Ash is an indication of the mineral contents of the samples and the high values obtained in these new varieties indicates that it may serve as sources of micro and macro elements. Commercial sam- ple (BGC) had the highest carbohydrate content and energy values as compared to that of BGB and BGW (p < .05). The values ob- tained for BGW and BGB were still higher than 46.5% reported by Okonkwo and Opara (2011) for Bambara groundnut. Emulsifying activity index ( m2∕g ) = 2×2.303×A 0.25×sample weight (g) To determine the emulsion stability, the emulsions was allowed to stand for 10 min at room temperature and the ESI was determined as described above and it was expressed based on the absorbance at 0, 10 min and the time difference as shown in the formula below: Emulsion stability index (%)= AA×Δt A−AA 2.8 2.8 Statistical analysis of data collected in triplicate was carried out using Data collected analysis of variance (ANOVA) and the differences be- tween the treatment means were separated using Duncan’s multiple range tests at a level considered to be significant at p < .05. 233 ADELEKE et al. 2.8 TABLE 1 Nutritional and chemical composition* of commercial and newly developed Bambara groundnut cultivars Sample Moisture (%) Protein (%) Fat (%) Ash (%) Crude fiber (%) Carbohydrate (%) Energy value (kcal) Dry matter (g/100 g) BGC 7.19 ± 0.50a 18.50 ± 0.26a 6.01 ± 0.34a 4.18 ± 0.10a 3.64 ± 0.27c 60.48 ± 0.50c 6.08 ± 0.11c 92.81 ± 0.43c BGW 10.10 ± 0.17b 20.73 ± 0.57c 7.93 ± 0.64b 5.15 ± 0.64c 2.11 ± 0.11a 53.98 ± 1.13b 5.43 ± 0.09b 89.90 ± 0.17b BGB 11.87 ± 0.85c 20.14 ± 0.88b 8.73 ± 1.17c 4.33 ± 0.76b 2.20 ± 0.10b 52.73 ± 1.58a 4.12 ± 0.01a 88.13 ± 0.32a Sample Vitamin B1 Vitamin B2 Vitamin B3 Vitamin B5 Vitamin B6 Vitamin B9 Vitamin C Vitamin E Vitamin K BGC 0.56 ± 0.09a 0.10 ± 0.00a 11.40 ± 0.34a 1.25 ± 0.05a 0.35 ± 0.01a 0.21 ± 0.08a 0.22 ± 0.04a 8.33 ± 0.33c 0.00 ± 0.00a BGW 0.64 ± 0.08bc 0.12 ± 0.00b 13.05 ± 0.45c 1.84 ± 0.08bc 0.43 ± 0.05c 0.25 ± 0.01b 0.27 ± 0.08b 3.31 ± 0.12a 0.001 ± 0.00a BGB 0.61 ± 0.01b 0.13 ± 0.09c 12.74 ± 0.21b 1.80 ± 0.04b 0.44 ± 0.08bc 0.25 ± 0.01b 0.27 ± 0.01b 3.42 ± 0.67b 0.001 ± 0.00a Sample Magnesium Calcium Sodium Copper Iron Zinc Manganese Potassium Cadmium Chromium BGC 7.02 ± 0.10a 0.23 ± 0.50a 2.18 ± 0.14a 0.44 ± 0.05c 2.51 ± 0.02a 0.81 ± 0.50c 0.06 ± 0.01a 92.43 ± 1.14a 2.02 ± 0.50a 1.98 ± 0.00a BGW 8.56 ± 4.50c 1.40 ± 0.10b 2.30 ± 0.00b 0.13 ± 0.00b 6.67 ± 0.29c 0.23 ± 0.00b 0.18 ± 0.00b 147.00 ± 2.50b 2.00 ± 0.00a 2.00 ± 0.01ab BGB 7.58 ± 0.10b 1.60 ± 0.00c 3.60 ± 0.00c 0.09 ± 0.00a 5.52 ± 0.01b 0.27 ± 0.00a 0.26 ± 0.00c 183.00 ± 1.50c 2.50 ± 0.00b 2.00 ± 0.00ab Sample Myristic Palmitic Behenic Stearic SFA (%) Oleic Linoleic Linolenic Palmitoleic UFA (%) BGC 0.27 ± 0.04c 9.97 ± 0.69b 5.41 ± 0.47c 4.14 ± 0.22c 19.55 54.17 ± 0.77c 21.14 ± 0.99a 0.82 ± 0.01b 3.41 ± 047c 79.86 BGW 0.21 ± 0.04b 10.37 ± 0.14c 3.00 ± 0.49a 4.10 ± 0.42b 17.63 52.02 ± 0.42b 25.95 ± 0.70c 0.30 ± 0.01a 0.29 ± 0.18a 82.33 BGB 0.14 ± 0.02a 9.72 ± 0.22a 4.41 ± 0.23b 3.72 ± 0.33a 10.99 51.98 ± 0.20a 25.10 ± 0.40b 0.87 ± 0.20c 0.65 ± 0.12b 83.01 Values are means ± standard deviation of three determinations. 3.2.2 \| Minerals composition The results of the mineral composition showed that the newly devel- oped Bambara groundnut cultivars have significantly higher (p < .05) macro mineral (magnesium, calcium, sodium, and potassium) contents compared to the commercial sample (Table 1). The amount of potas- sium was the highest among the minerals in all the three samples with BGB having significantly (p < .05) highest value of 183 ppm. The results obtained were higher than 92.43 ppm reported for Bambara groundnut seeds by Abdulsalami and Sherriff (2010) and 160.00 ppm reported by Bamishaiye et al. (2011). Potassium is an essential min- eral, widespread in nature and the recommended daily allowance (RDA) in adult is 3.5 g/day (Bender, 2006). Therefore, the potassium contents obtained in the three samples were lower than the RDA in adult. However, the lowest among the macro minerals is calcium with BGC, BGW, and BGB having values of 0.23, 1.40, and 1.60, respec- tively, (p < .05). The results obtained for the two new varieties were still higher than 0.23 ppm and 0.79 ppm reported by Abdulsalami and Sherriff (2010) and Odeghe et al. (2012), respectively, for raw Bambara groundnut cultivars. Among the micro minerals, iron content was the in the three samples and this ranged between 2.51 and 6.67 ppm, with BGW having significantly (p < .05) highest value. The body requires about 1.5–2.2 mg/day of iron (Belitz, Grosch, & Schierberle, 2010). According to Abdulsalami and Sherriff (2010), raw and processed Bambara groundnut cultivars contained 5.5 and 2.96 ppm of iron, respectively. Hemoglobin and myoglobin pigments as well as some enzymes which are the major constituents of iron must be taken at about 15 mg/day by an average human being (Rao, 2007). The lowest micro mineral was copper with commercial sample, BGC (0.44 ppm) exhibiting significantly (p < .05) higher value compared to BGW (0.13 ppm) and BGB (0.09 ppm). The results obtained for samples BGW and BGB were lower than that of Bambara ground- nut seed (0.41 ppm) reported by Abdulsalami and Sherriff (2010). In the formation of oxido-reductase enzyme, copper has been seen to be a vital element as it helps in the catalysis of iron II to iron III. This reaction has been found to be very important because it is only in this form that protein can be transported to the liver (Bender, 2006). 3.2.3 \| Fatty acids profile The fatty acids profile of samples BGC, BGB, and BGW is shown in Table 1. Palmitic, oleic, and linoleic acids were found to be the most abundant in the samples. The monounsaturated fatty acid (oleic) was the most abundant fatty acid in Bambara groundnut constitut- ing more than half of the total fatty acids. The commercial sample showed the highest level of oleic acid when compared to the two new varieties and the differences among the samples with respect to oleic acid were significant (p < .05). The levels of linolenic, palmi- toleic and myristic in samples BGC, BGW, and BGB were 0.82, 3.41 and 0.27%, 0.30, 0.29 and 0.21% and 0.87, 0.65 and 0.14%, respec- tively. The results obtained were in conformity with the report by Okonkwo and Opara (2011) and Minka and Bruneteau (2000) that linoleic, palmitic, and linolenic were predominant types of fatty acids present in Bambara groundnut seeds. It was also observed that the saturated fatty acids constitute about 20% of the total fatty acids while unsaturated fatty acids make up about 80%. For generation of cellular energy and biosynthesis of membrane lipids and lipid me- diators in the body, fatty acids are used (Ratnayake & Galli, 2009). Two of the essential fatty acids (Linoleic and oleic acids) were found to be present in abundant quantities in the new Bambara ground- nut seeds while the stability of the Bambara groundnut oil might be adversely affected by the higher proportion of unsaturated fatty acids present. Oxidation of the unsaturated fatty acids at the dou- ble bonds makes this oil to be prone to the development of rancid flavor. The suitability of such oil for light cooking might be due to the awareness of the consumption of diet containing higher ratio of polyunsaturated fatty acids to saturated fatty acids which are now being advocated due to the high incidence of coronary diseases such as atherosclerosis. 3.2.2 \| Minerals composition According to Chiplonkar, Agte, and Mengale (2003), the rec- ommended dietary allowance (RDA) of copper is about 2 mg/day, but in all the samples copper contents was found to be lower than the RDA level. 2.8 Means followed by the same letter within the same rows are not significantly (p < .05) different according to LSD test. White bambara groundnut sample (BGW); Brown bambara groundnut sample (BGB); Control bambara groundnut sample (BGC). ADELEKE et al. 234 two new varieties implies they could be a better source of niacin compared to the commercial sample. Also, the vitamin C of BGC (0.22) were significantly (p < .05) increased to 0.27 in BGW and BGB. However, a significantly (p < .05) lower value was observed in the vitamin E content of BGW (3.31) and BGB (3.42) as compared to BGC (8.33). Dietary deficiency of vitamin E is not normally encoun- tered (FAO/WHO, 2004). The requirement of vitamin E suggested was 8–10 mg tocopherol per day depending on the edible oil used. Therefore, the values obtained in these new varieties were lower than the recommended daily allowance of vitamin E. 3.3.2 \| Oil absorption capacity (OAC) The OAC of samples BGW and BGB were found to be 79.54 and 63.44%, respectively, while that of sample BGC was 69.07% (Table 2). The OAC of Bambara protein concentrate of BGB was the highest (103.66%). The OAC of defatted and protein concentrate of the three samples were higher than those of their raw samples. The OAC of Bambara groundnut flour products was higher than that of sandbox seed flour (65.50%–107.63%) as reported by Osungbade, Gbadamosi, and Adiamo (2016) but lower than that of jackfruit flour (230 and 350%) as reported for jackfruit flour by Odoemelam (2005). The low OAC of Bambara groundnut products flour might be due to low levels of hydrophobic proteins which show superior binding of lipids. The bridge caused by protein in fat and water emulsion may not make Bambara groundnut flour products a suitable ingredient in the cold meat industry particularly for sausages. , Oil Absorption Capacity; WAC, Water Absorption Capacity; SC, Swelling Capacity; LGC, Least Gelling Concentration; EAI, Emulsion Activity Index; ESI, Emulsifying Stability Index; FC, m Stability. ndard deviation of three determinations. Means followed by the same letter within the same rows are not significantly (p < .05) different according to LSD test. White bambara groundnut BGB) were acidic as shown in Table 2. The pH values range between 6.30 and 6.67. The pH values of the aqueous solutions of the protein concentrate (BGC, BGW, and BGB) ranged between 7.12 and 7.25. The adjustment of pH to 7 during the preparation of concentrate may influence its pH values in aqueous solution. Adepeju et al. (2011) re- ported that some functional properties such as solubility, emulsifying activity and foaming properties are affected by pH. 3.3.3 \| Water absorption capacity (WAC) The WAC ranged from 108.67 to 311.43% (Table 2). The concen- trate of the three samples had the highest WAC which implies that increase in protein level increases the WAC of the products with sample BGW exhibiting the highest value of 311.43%. The higher WAC of Bambara groundnut concentrate flour may be due to the higher polar amino acid residues of protein having an affinity for water molecules (Yusuf, Ayedun, & Sanni, 2007). Protein con- centrate was also observed by Gbadamosi (2008) to exhibit bet- ter water binding capacity to that of the raw conophor nut flour. Osundahunsi, Fagbemi, Kesselman, and Shimoni (2003) attributed this to the fact that protein concentrate had greater ability to swell, dissociate and unfold exposing additional binding sites, whereas the carbohydrate and other compounds of the protein flour may impair it. The incorporation of the new Bambara groundnut seed flours and its protein products into aqueous food formulations, especially those involving soup and dough handling might be due to its good WAC which is the ability of flour to absorb water and swell for im- proved consistency in food. 3.3.1 \| Bulk density (BD) and pH The BD ranged between 0.58 and 0.73 g/ml; 0.77–0.82 g/ml and 0.62–0.70 g/ml for whole, defatted and concentrates flour, respec- tively, (Table 2). The defatted sample (BGB) exhibited the highest BD (0.82 g/ml) while the whole flour (BGC) had the lowest bulk density (0.58 g/ml). The values obtained were comparable to 0.56 and 0.62 g/ ml reported by Eltayeb et al. (2011) for Bambara groundnut flour but higher than that of conophor flour (0.41 g/ml) and kariya protein con- centrates (0.57 g/ml) as reported by Gbadamosi (2008) and Adiamo, Gbadamosi, and Abiose (2016a), respectively. The presence of higher proportion of carbohydrate in defatted flour may be responsible for the high BD demonstrated by defatted Bambara flour samples. Starch polymer structure has been seen to influence BD and loose starch polymer could result in low bulk density (Plaanmi, 1997). The increase observed in the rate of dispersion has a result of high bulk density is important in the reconstitution of flour in hot water to produce dough (Akinjayeju & Enude, 2002). The aqueous solutions of whole and defatted flour of Bambara groundnut seeds (BGC, BGW, and ADELEKE et al. 235 BD, Bulk Density; OAC, Oil Absorption Capacity; WAC, Water Absorption Capacity; SC, Swelling Capacity; LGC, Least Gelling Concentration; EAI, Emulsion Activity Index; ESI, Emulsifying Stability Index; FC, Foam Capacity; FS, Foam Stability. Values are means ± standard deviation of three determinations. Means followed by the same letter within the same rows are not significantly (p < .05) different according to LSD test. White bambara groundnut sample (BGW); Brown bambara groundnut sample (BGB); Control bambara groundnut sample (BGC) TABLE 2 Functional properties* of whole, defatted, and protein concentrate of commercial and newly developed Bambara groundnut flour Functional Properties Whole Defatted Concentrate BGC BGW BGB BGC BGW BGB BGC BGW BGB BD (g/ml) 0.58 ± 0.02a 0.64 ± 0.04b 0.73 ± 0.01c 0.77 ± 0.00a 0.79 ± 0.00b 0.82 ± 0.32c 0.62 ± 0.01a 0.63 ± 0.01ab 0.70 ± 0.04c OAC (%) 69.07 ± 0.09b 73.32 ± 0.43c 63.44 ± 0.06a 79.71 ± 0.07b 79.54 ± 0.56b 70.94 ± 0.17a 89.18 ± 0.21a 89.72 ± 0.03ab 103.66 ± 0.03c pH 6.67 ± 0.05c 6.65 ± 0.15b 6.40 ± 0.00a 6.30 ± 0.00a 6.40 ± 0.00b 6.50 ± 0.00c 7.12 ± 0.05a 7.25 ± 0.0bc 7.20 ± 0.00b WAC (%) 137.67 ± 0.15b 139.33 ± 0.08c 108.67 ± 0.05a 168.67 ± 0.00c 149.33 ± 0.05a 161.33 ± 0.05b 286.67 ± 0.05b 311.43 ± 0.25c 174.65 ± 0.05a SC 2.52 ± 0.06b 2.71 ± 0.04c 2.39 ± 0.01a 9.72 ± 0.01a 10.14 ± 0.03b 10.94 ± 0.01c 2.64 ± 0.01b 3.19 ± 0.03c 1.96 ± 0.01a LGC (%) 20.00 ± 0.00b 9.00 ± 0.00a 20.00 ± 0.00b 20.00 ± 0.00a 20.00 ± 0.00a 20.00 ± 0.00a 20.00 ± 0.00c 15.00 ± 0.00a 13.00 ± 0.00b EAI (m2/g) 14.37 ± 0.23b 13.21 ± 0.44a 17.56 ± 0.28c 19.29 ± 0.11a 19.27 ± 0.34a 21.02 ± 0.21b 22.04 ± 0.28c 17.91 ± 0.10a 21.24 ± 0.24b ESI (%) 127.14 ± 0.56b 82.16 ± 0.68a 164.800.77c 30.12 ± 0.02b 26.83 ± 0.68a 54.83 ± 0.50c 60.90 ± 0.38a 81.47 ± 0.30c 79.56 ± 0.55b FC (%) 8.41 ± 0.11a 12.75 ± 0.05c 9.90 ± 0.57b 12.00 ± 0.034a 12.71 ± 0.06a 14.21 ± 0.06b 13.57 ± 0.03a 14.01 ± 0.01b 23.56 ± 0.09c FS (%) 42.43 ± 0.10a 54.13 ± 0.47c 47.31 ± 0.50b 25.47 ± 0.42a 40.70 ± 0.55b 52.63 ± 0.06c 89.76 ± 0.03a 92.50 ± 0.01b 97.82 ± 0.05c BD, Bulk Density; OAC, Oil Absorption Capacity; WAC, Water Absorption Capacity; SC, Swelling Capacity; LGC, Least Gelling Concentration; EAI, Emulsion Activity Index; ESI, Emulsifying Stability Index; FC, Foam Capacity; FS, Foam Stability. Values are means ± standard deviation of three determinations. Means followed by the same letter within the same rows are not significantly (p < .05) different according to LSD test. White bambara groundnut sample (BGW); Brown bambara groundnut sample (BGB); Control bambara groundnut sample (BGC). TABLE 2 Functional properties of whole, defatted, and protein concentrate of commercial and newly developed Bambara groundnut flour Functional Properties Whole Defatted Concentrate BGC BGW BGB BGC BGW BGB BGC BGW BGB BD (g/ml) 0.58 ± 0.02a 0.64 ± 0.04b 0.73 ± 0.01c 0.77 ± 0.00a 0.79 ± 0.00b 0.82 ± 0.32c 0.62 ± 0.01a 0.63 ± 0.01ab 0.70 ± 0.04c OAC (%) 69.07 ± 0.09b 73.32 ± 0.43c 63.44 ± 0.06a 79.71 ± 0.07b 79.54 ± 0.56b 70.94 ± 0.17a 89.18 ± 0.21a 89.72 ± 0.03ab 103.66 ± 0.03c pH 6.67 ± 0.05c 6.65 ± 0.15b 6.40 ± 0.00a 6.30 ± 0.00a 6.40 ± 0.00b 6.50 ± 0.00c 7.12 ± 0.05a 7.25 ± 0.0bc 7.20 ± 0.00b WAC (%) 137.67 ± 0.15b 139.33 ± 0.08c 108.67 ± 0.05a 168.67 ± 0.00c 149.33 ± 0.05a 161.33 ± 0.05b 286.67 ± 0.05b 311.43 ± 0.25c 174.65 ± 0.05a SC 2.52 ± 0.06b 2.71 ± 0.04c 2.39 ± 0.01a 9.72 ± 0.01a 10.14 ± 0.03b 10.94 ± 0.01c 2.64 ± 0.01b 3.19 ± 0.03c 1.96 ± 0.01a LGC (%) 20.00 ± 0.00b 9.00 ± 0.00a 20.00 ± 0.00b 20.00 ± 0.00a 20.00 ± 0.00a 20.00 ± 0.00a 20.00 ± 0.00c 15.00 ± 0.00a 13.00 ± 0.00b EAI (m2/g) 14.37 ± 0.23b 13.21 ± 0.44a 17.56 ± 0.28c 19.29 ± 0.11a 19.27 ± 0.34a 21.02 ± 0.21b 22.04 ± 0.28c 17.91 ± 0.10a 21.24 ± 0.24b ESI (%) 127.14 ± 0.56b 82.16 ± 0.68a 164.800.77c 30.12 ± 0.02b 26.83 ± 0.68a 54.83 ± 0.50c 60.90 ± 0.38a 81.47 ± 0.30c 79.56 ± 0.55b FC (%) 8.41 ± 0.11a 12.75 ± 0.05c 9.90 ± 0.57b 12.00 ± 0.034a 12.71 ± 0.06a 14.21 ± 0.06b 13.57 ± 0.03a 14.01 ± 0.01b 23.56 ± 0.09c FS (%) 42.43 ± 0.10a 54.13 ± 0.47c 47.31 ± 0.50b 25.47 ± 0.42a 40.70 ± 0.55b 52.63 ± 0.06c 89.76 ± 0.03a 92.50 ± 0.01b 97.82 ± 0.05c BD, Bulk Density; OAC, Oil Absorption Capacity; WAC, Water Absorption Capacity; SC, Swelling Capacity; LGC, Least Gelling Concentration; EAI, Emulsion Activity Index; ESI, Emulsifying Stability Index; FC, Foam Capacity; FS, Foam Stability. *Values are means ± standard deviation of three determinations. Means followed by the same letter within the same rows are not significantly (p < .05) different according to LSD test. White bambara groundnut 3.3.4 \| The LGC in this study ranged from 9.0 to 20.0% with raw BGW hav- ing the lowest value (Table 2). Processing methods, the relative ratio of different constituents- protein, carbohydrate and lipids and the interac- tion between such components might be due to the variations observed in the LGC values and these may affect functional properties (Aremu, 236 \| ADELEKE et al. FIGURE 1 Influence of temperature on water absorption capacity (WAC) and swelling capacity (SC) of (a, d) whole, (b, e) defatted and (c, f) concentrate of Bambara groundnut cultivars, respectively 236 \| ADELEKE et al. ADELEKE et al. FIGURE 1 Influence of temperature on water absorption capacity (WAC) and swelling capacity (SC) of (a, d) whole, (b, e) defatted and (c, f) concentrate of Bambara groundnut cultivars, respectively Olaofe, & Akintayo, 2006). (Aremu et al., 2006) reported that the LGC of some legume ranged between 13.0 and 16.0%. The LGC is the lowest protein concentration at which gel remained in inverted tube. Lower LGC results in better gelation ability of protein ingredient (Akintayo, Oshodi, & Esuruoso, 1999). The use of BGW flour for the formation of curd or as an additive to other gel forming materials in food products may be an asset owing to its low gelation concentration (Akintayo et al., 2002). is shown in Figure 1. The water absorption capacities of whole, de- fatted, and concentrate flours increased with increase in temperature from 60 to 90°C. The samples of protein concentrate demonstrated the highest capacity to absorb water with sample BGW exhibiting the highest value (391.43%) at 90°C. The high WAC of both the defat- ted and the concentrate of Bambara groundnut flour suggest that it can be used as thickener in liquid and semi liquid foods because of their ability to absorb water for improved consistency in food (Fasasi, Adeyemi, & Fagbenro,2007; Osundahunsi et al., 2003). 3.3.5 \| Effect of temperature on water absorption and swelling capacity Mune-Mune, Minka, Mbome, and Etoa (2011) reported 0.95%–3.76% for Bambara groundnut flour while 91.17%–103.7% was reported for cowpea and yam-bean (Agunbiade & Longe, 1999). The high swelling 3.3.5 \| Effect of temperature on water absorption and swelling capacity The results showing the effect of different temperatures on the swelling capacity (SW) of Bambara groundnut samples are shown in Figure 1. As temperature increases, there is significant increase in the SW of whole, defatted, and protein concentrate in samples BGC, The effect of temperature on water absorption capacity of whole, de- fatted and protein concentrate of Bambara groundnut flour samples \| 237 ADELEKE et al. 237 \| 237 ADELEKE et al. FIGURE 2 Emulsifying activity index (m2/g) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH FIGURE 2 Emulsifying activity index (m2/g) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH power achieved at 90 ºC suggests that water penetration into the starch granules and proteins can be achieved at elevated temperature. This could be useful in the manufacture of confectionery foods (that requires high swelling power). BGW, and BGB increased with increase in temperature from 60 to 90°C with defatted samples having the greatest values. The highest swelling capacity occurs in BGC concentrate (25.47) as compared to other samples at 90°C. The increase in temperature indicating differ- ences in molecular organization within the granules results in an in- crease un swelling capacities of the flour (Agunbiade & Longe, 1999). Process conditions, nature of materials and type of treatment are the main conditions on which swelling capacities are dependent. The swelling capacities of samples increased with increase in temperature. Mune-Mune, Minka, Mbome, and Etoa (2011) reported 0.95%–3.76% for Bambara groundnut flour while 91.17%–103.7% was reported for cowpea and yam-bean (Agunbiade & Longe, 1999). The high swelling BGW, and BGB increased with increase in temperature from 60 to 90°C with defatted samples having the greatest values. The highest swelling capacity occurs in BGC concentrate (25.47) as compared to other samples at 90°C. The increase in temperature indicating differ- ences in molecular organization within the granules results in an in- crease un swelling capacities of the flour (Agunbiade & Longe, 1999). Process conditions, nature of materials and type of treatment are the main conditions on which swelling capacities are dependent. The swelling capacities of samples increased with increase in temperature. 3.4 \| Effect of pH and NaCl concentration on emulsifying activity and emulsifying stability index Emulsifying activity index (EAI) and emulsifying stability index (ESI) of defatted Bambara groundnut and protein concentrate flour was measured as a function of pH and NaCl concentration. Various con- centrations of sodium chloride salt affected EAI and ESI of Bambara 238 \| ADELEKE et al. 238 \| ADELEKE et al. FIGURE 3 Emulsifying stability index (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH FIGURE 3 Emulsifying stability index (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH their isoelectric pH of 4.0 with concomitant increase in emulsion activ- ity below and above this pH. The highest EAI of protein concentrate BGC, BGW and BGB at 0.0, 0.5 and 1.0 mol/L were 40.40, 45.47 and 34.08 m2/g, respectively, at pH 10. However, the highest emulsion stability index (ESI) of defatted BGC, BGW, and BGB at 0.0, 0.5, and 1.0 mol/L were 60.52, 69.16, and 40.08%, respectively, at pH 10.0. The lowest ESI of defatted BGC, BGW, and BGB were 18.91, 16.47 and 6.04%, respectively, at their isoelectric region of pH 4.0. There was an increase in the ESI values above and below this region. Meanwhile, the lowest ESI of Bambara groundnut concentrate of samples BGC, BGW, and BGB were 17.88, 6.73, and 5.52%, respectively while the highest ESI of samples BGC, BGW, and BGB were 103.04, 86.78, and 48.36%, their isoelectric pH of 4.0 with concomitant increase in emulsion activ- ity below and above this pH. The highest EAI of protein concentrate BGC, BGW and BGB at 0.0, 0.5 and 1.0 mol/L were 40.40, 45.47 and 34.08 m2/g, respectively, at pH 10. However, the highest emulsion stability index (ESI) of defatted BGC, BGW, and BGB at 0.0, 0.5, and 1.0 mol/L were 60.52, 69.16, and 40.08%, respectively, at pH 10.0. The lowest ESI of defatted BGC, BGW, and BGB were 18.91, 16.47 and 6.04%, respectively, at their isoelectric region of pH 4.0. There was an increase in the ESI values above and below this region. 3.4 \| Effect of pH and NaCl concentration on emulsifying activity and emulsifying stability index Meanwhile, the lowest ESI of Bambara groundnut concentrate of samples BGC, BGW, and BGB were 17.88, 6.73, and 5.52%, respectively while the highest ESI of samples BGC, BGW, and BGB were 103.04, 86.78, and 48.36%, groundnut flour samples as shown in Figure 2 and 3, respectively. The lowest EAI of defatted BGC, BGW, and BGB at NaCl concentrations of 0.0, 0.5, and 1.0 mol/L were 2.80, 3.32, and 4.70 m2/g and it oc- curred around their isoelectric region (about pH 4.0). EAI of defatted BGC, BGW, and BGB increased at pH values above this region. The highest EAI of defatted BGC, BGW, and BGB at 0, 0.5, and 1.0 mol/L NaCl concentrations were 34.43, 38.06, and 32.76 m2/g and occurred at the alkaline pH of 10.0. Aremu, Olonisakin, Bako, and Madu (2008) reported that emulsion activity depends mostly on salt concentration and the type of the salt under consideration. The lowest EAI of pro- tein concentrate BGC, BGW, and BGB at different concentrations of 0.0, 0.5, and 1.0 mol/L were 3.39, 3.59, and 3.26 m2/g, respectively, at \| 239 ADELEKE et al. FIGURE 4 Foaming capacity (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH ADELEKE et al. 239 \| FIGURE 4 Foaming capacity (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH FIGURE 4 Foaming capacity (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH between water and oil in the emulsion is based on the surfactancy of proteins (Oshodi & Ojokan, 1997). The ease with which protein can migrate to, adsorb at, unfold and rearrange at an interface is a function of the surface activity and presumably salts reduce the surface activity of the flour and thereby increase the interfacial tension which leads to decrease in emulsion capacity. Charge repulsion between the proteins may also be reduced by salt which will in-turn enhance hydrophilic association at the interface (Kinsella, Damodaran, & German, 1985). 3.4 \| Effect of pH and NaCl concentration on emulsifying activity and emulsifying stability index The differences in foaming properties of defatted BGC, BGB, and BGW and their concentrate samples may be due to the level of proteins present in their flour. The increase in solubility and surface activity of the soluble protein after removing lipids may be due to the improvement in foaming properties at higher pH (Jitngarmkusol, BGB at pH 10 and 0.0 mol/L NaCl concentration were 40.00, 84.00, and 112.50%, respectively, while it was 9.08, 25.00, and 57.89%, respec- tively, at pH 4.0 (Figure 4d). As the NaCl concentration increased from 0.0 to 1.0 mol/L, the FC of the samples also increases at all pH values. Generally, the defatted Bambara groundnut samples of BGC, BGB, and BGW exhibited a similar pattern of foaming capacity at the different salt concentrations and pH values to those of Bambara protein concentrates samples (BGC, BGW, and BGB). The differences in foaming properties of defatted BGC, BGB, and BGW and their concentrate samples may be due to the level of proteins present in their flour. The increase in solubility and surface activity of the soluble protein after removing lipids may be due to the improvement in foaming properties at higher pH (Jitngarmkusol, 3.4 \| Effect of pH and NaCl concentration on emulsifying activity and emulsifying stability index At isoelectric region, the decrease in emulsion stability may be due to increase contact leading to coalescence which thereby reduces stabil- ity (Parker, 1987). When moved away from their isoelectric pH, these proteins may, however, be effective emulsifiers. respectively. Good emulsifying activity of a protein is related to high solubility (El Nasri & El Tinay, 2007) while the pH-emulsifying proper- ties profile resembles the pH-solubility profile (Ogunwolu, Henshaw, Mock, Santros, & Awonorin, 2009). This is because at isoelectric pH, most food proteins are sparingly soluble; poorly hydrated, lack elec- trostatic repulsive forces and are generally poor emulsifier. At this pH, the net charge of peptide will be minimized and peptide movement to the interface would not be rapid since the lowest solubility occurred at the isoelectric point. In the formation of stable emulsion, (Aremu et al., 2008) reported three mechanisms that may appear to be involved: (1) reduction of interfacial tension; (2) formation of a rigid interfacial film; and (3) electrical charge. The ability to lower the interfacial tension 240 \| ADELEKE et al. FIGURE 5 Foaming stability (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH ADELEKE et al. 240 \| ADELEKE et al. FIGURE 5 Foaming stability (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH FIGURE 5 Foaming stability (%) of defatted and protein concentrate of Bambara groundnut cultivars at (a, d) 0.0 mol/L, (b, e) 0.5 mol/L, and (c, f) 1.0 mol/L NaCl concentration as a function of pH BGB at pH 10 and 0.0 mol/L NaCl concentration were 40.00, 84.00, and 112.50%, respectively, while it was 9.08, 25.00, and 57.89%, respec- tively, at pH 4.0 (Figure 4d). As the NaCl concentration increased from 0.0 to 1.0 mol/L, the FC of the samples also increases at all pH values. Generally, the defatted Bambara groundnut samples of BGC, BGB, and BGW exhibited a similar pattern of foaming capacity at the different salt concentrations and pH values to those of Bambara protein concentrates samples (BGC, BGW, and BGB). REFERENCES Abdulsalami, M. S., & Sherriff, H. B. (2010). Effect of processing on the proximate composition and mineral content of Bambara groundnut (Voandezeia subterranea). Bayero Journal of Pure and Applied Sciences, 3(1), 188–190. The FS of BGC (45.00%), BGW (66.66%), and BGB (140.00%) at pH 10 and 0.05 mol/L salt concentration were lower than the values of 80.25, 80.27, and 153.33%, respectively obtained at 1.0 mol/L salt con- centration (Figure 5). The FS of defatted samples BGC (28.89%), BGW (25.00%), and BGB (50.03%) increased to 40.00, 55.56, and 55.58%, re- spectively at pH 10 and 0.5 mol/L NaCl concentration while it was also increased to 50.00, 68.73, and 68.75%, respectively, at 1.0 mol/L NaCl concentration. The FS of protein concentrate BGC, BGW, and BGB were 68.89, 81.58, and 126.67%, respectively, at pH 10 and 1.0 mol/L salt concentration while it was lower at pH 10 and 0.0 mol/L salt (30.00, 40.22, and 88.24%). Generally, the FS followed a similar pattern to foaming capacity where the maximum and minimum FS occurred at pH 10. Also sample BGB of both the defatted and concentrates exhibited the highest stability while the commercial sample (BGC) of the defatted and concentrates also exhibited the minimum FS. Increase in salt con- centration from 0.5 mol/L to 1.0 mol/L also increased the FS including the FS around pH 4. The results obtained showed that the highest FS and FC was observed at alkaline pH 10 for sample BGB protein con- centrate. A direct relationship was observed between foam capacity and foam stability (Mao & Hua, 2012). Large air bubbles surrounded by thinner and less flexible protein films could be formed by flour with high foaming ability while these air bubbles might be easier to collapse and consequently lowered the foaming stability (Jitngarmkusol et al., 2008). The results obtained in this study suggest that Bambara ground- nut defatted flour and concentrate may be useful in food system to im- prove textural and leavening characteristics of foods such as ice cream, whipped topping, cakes and confectionery products where foaming properties are important as reported by Eltayeb et al. (2011). Adebowale, Y. A., Adeyemi, I. A., & Oshodi, A. A. (2005). Functional and physicochemical properties of flours of six mucuna species. African Journal of Biotechnology, 4, 416–468. Adebowale, K. O., & Lawal, O. S. (2004). REFERENCES Comparative study of the func- tional characteristics of some underutilized African legume flours: Bambara groundnut (Voandzeia subterranea) Jack bean (Canavalia eni- formis) and Mucuna bean (Mucuna prulens). Food Research International, 37, 335–365. Adepeju, A. B., Gbadamosi, S. O., Adeniran, A. H., & Omobuwajo, T. O. (2011). Functional and pasting characteristics of breadfruit (Artocarpus altilis) flours. African Journal of Food Science, 5(9), 529–535. Adiamo, O. Q., Gbadamosi, O. S., & Abiose, S. H. (2016a). Functional properties and protein digestibility of protein concentrates and iso- lates produced from kariya (Hildergadia bateri) seed. Journal of Food Processing and Preservation, 40(5), 979–989.https://doi.org/10.1111/ jfpp.12678 Adiamo, O. Q., Gbadamosi, O. S., & Abiose, S. H. (2016b). Antioxidative and functional properties of kariya (Hildergadia bateri) protein hydrolysates obtained with two different proteolytic enzymes. J. Food Pro. Pres., 40(2), 202–211.https://doi.org/10.1111/jfpp.12597 Agbenorhevi, J. K., Oduro, I., Ellis, W. O., Aodakpi, V. D., & Eleblu, S. E. (2007). Effect of soaking, autoclaving and repeated boiling on oligosac- charides in cowpea. Nigerian Food Journal, 25(2), 88–94. Agunbiade, S. O., & Longe, O. G. (1999). The physico-functional characteris- tics of starches from cowpea, pigeon pea and yam bean. Food Chemistry, 65, 469–474. https://doi.org/10.1016/S0308-8146(98)00200-3 Akinjayeju, O., & Enude, O. T. (2002). Effects of de-hulling on some proper- ties of cowpea (Vigna unguiculata walp L.) flours. Italian. Journal of Food Science, 14(1), 53–58. Akintayo, E. T., Adebayo, E. A., & Arogundade, L. A. (2002). Chemical com- position, physico-chemical and functional properties of akaa pulp and seed flours. Food Chemistry, 77, 333–336. Akintayo, E. T., Oshodi, A. A., & Esuruoso, K. O. (1999). Effect of NaCl, ionic strength and pH on the foaming and gelation of pigeon pea (Cajanus cajan) protein concentrates. Food Chemistry, 66, 51–56. https://doi. org/10.1016/S0308-8146(98)00155-1 AOAC. (2000). Official Methods of Analysis’ Association of Official Analytical Chemists. Washington, DC: AOAC. 4 \| CONCLUSION Aremu, M. O., Olaofe, O., & Akintayo, E. T. (2006). Mineral and amino acid composition of Bambara groundnut (Vigna subterranean) and Kerstings groundnut (Kerstingiella geocarpa) flour. International Journal of Chemistry, 16, 57–64. In conclusion, the protein and fat contents of the new varieties of Bambara groundnut samples were found to be higher than the commer- cial sample. Also, the new varieties were found to contain higher levels of essential fatty acids, minerals and vitamins. Better functional proper- ties were observed in the newly developed Bambara groundnut culti- vars as compared to the commercial sample. The results obtained from this study have shown the potential for the industrial and household use of the newly developed Bambara groundnut cultivars into shelf stable protein products and could be a useful ingredient in food formulations. Aremu, M. O., Olonisakin, A., Bako, D. A., & Madu, P. C. (2008). Compositional studies and physicochemical characteristics of cashew nut (Anarcardium occidentale) flour. Pakistan Journal of Nutrition, 5, 34–38. Aremu, M. O., Olonisakin, A., Bako, D. A., & Madu, P. C. (2008). Compositional studies and physicochemical characteristics of cashew nut (Anarcardium ( ) p studies and physicochemical characteristics of cashew nut (Anarcardiu occidentale) flour. Pakistan Journal of Nutrition, 5, 34–38. occidentale) flour. Pakistan Journal of Nutrition, 5, 34–38. occidentale) flour. Pakistan Journal of Nutrition, 5, 34–38. Atiku, S., & Mohammed, K. (2004). Cultivation of Bambara groundnut in the Nigeria. ABU, Zaria: Report of a field study. Bamishaiye, O. M., Adegbola, J. A., & Bamishaiye, E. I. (2011). Bambara groundnut: An under-utilized nut in Africa. Advances Agric. Biotech., 1, 42–51. Belitz, H. D., Grosch, W., & Schierberle, P. (2010). Food Chem, 4th ed.. Verlag Berlin Heidelberg: Springer. Olaposi R. Adeleke http://orcid.org/0000-0003-0948-8965 Olaposi R. Adeleke http://orcid.org/0000-0003-0948-8965 Hongsuwankul, & Tananuwong, 2008) while at aqueous phase, greater concentration of soluble protein could enhance foam formulation (Eltayeb et al., 2011). The increase in foaming properties observed at alkaline pH of walnut protein isolate and concentrate (Mao & Hua, 2012) and kariya protein hydrolysates Adiamo, Gbadamosi, and Abiose (2016b) in the re- port of, agrees to the assertion that foaming properties are pH depend- ent. The two new varieties of Bambara groundnut samples (BGB and BGW) showed superior foaming capacities over the commercial sample. ORCID ORCID The authors declared no conflict of interest. 3.5 \| Effect of pH and NaCl concentration on foam capacity and stability The results showing the effect of pH and salt concentration on the foam capacity (FC) and foam stability (FS) of defatted and protein concentrate flour of samples BGC, BGW, and BGB are depicted in Figure 4 and 5, respectively. Figure 4a shows that the maximum FC at 0.0 mol/L sodium chloride concentration occurred at pH 10 for defatted Bambara ground- nut flour samples BGC, BGW, and BGB and the values were 38.89, 62.5 and 57.89%, respectively. The lowest FC for samples BGC, BGW, and BGB were 16.32, 23.91, and 33.33%, respectively, and it occurred at pH 4.0. The FC of Bambara protein concentrate samples of BGC, BGW, and \| 241 ADELEKE et al. 241 (2004). Vitamin and mineral requirements in human nutrition. 2nd Edition. Geneva: FAO/WHO. Parker, N. S. (1987). Properties and functions of stabilizing agents in food emulsions. 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Isolation, partial characteri- zation and modification of Great Northern Bean (Phaseolus vul- garis L.) starch. Journal of Food Science, 46, 617–621. https://doi. org/10.1111/j.1365-2621.1981.tb04924.x Jitngarmkusol, S., Hongsuwankul, J., & Tananuwong, K. (2008). Chemical compositions, functional properties, and microstructure of defat- ted macadamia flours. Food Chemistry, 110, 23–30. https://doi. org/10.1016/j.foodchem.2008.01.050 Kinsella, J. E., Damodaran, S., & German, B. (1985). Physicochemical and functional properties of oil seed proteins with emphasis on soy pro- teins. In A.M. Altschul & H.L. Wilcke (Eds.), New protein foods (pp. 107– 179). New York: Academic Press. Sun, J., Han, F., Yan, S., Yang, H., & Tetsuo, S. (2008). Ogunwolu, S. O., Henshaw, F. O., Mock, H., Santros, A., & Awonorin, S. O. (2009). Functional properties of proteins concentrate and isolates produced from cashew (Anacardium occidentale L.) nut. 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The analysis of Bambara nut (Voandzeia subterranea (L) Thouars) for sustainability in Africa. Research Journal of Applied Sciences, 5(6), 394–396. Chiplonkar, S. A., Agte, V. V., & Mengale, S. S. (2003). Relative importance of micronutrient deficiency in deficiency anaemia. Nutr. Res., 23, 1355– 1367. https://doi.org/10.1016/S0271-5317(03)00151-9 Oshodi, A. A., & Ojokan, E. (1997). Effect of salts on some of the functional properties of bovine plasma protein concentrate. Food Chemistry, 59, 333–338. https://doi.org/10.1016/S0308-8146(96)00102-1 El Nasri, N. A., & El Tinay, A. H. (2007). Functional properties of fenugreek (Trigonella foenumgraecum) protein concentrate. Food Chemistry, 103, 582–589. https://doi.org/10.1016/j.foodchem.2006.09.003 Osundahunsi, O. F., Fagbemi, T. N., Kesselman, E., & Shimoni, E. (2003). Comparison of the physico-chemical properties and pasting character- istics of flour and starch from red and white sweet potato cultivars. 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https://openalex.org/W3111315855	https://europepmc.org/articles/pmc7803102?pdf=render	English	null	Detection of CD39 and a Highly Glycosylated Isoform of Soluble CD73 in the Plasma of Patients with Cervical Cancer: Correlation with Disease Progression	Mediators of inflammation	2,020	cc-by	12,179	Hindawi Mediators of Inﬂammation Volume 2020, Article ID 1678780, 14 pages https://doi.org/10.1155/2020/1678780 Hindawi Mediators of Inﬂammation Volume 2020, Article ID 1678780, 14 pages https://doi.org/10.1155/2020/1678780 Research Article Detection of CD39 and a Highly Glycosylated Isoform of Soluble CD73 in the Plasma of Patients with Cervical Cancer: Correlation with Disease Progression Ricardo Muñóz-Godínez ,1,2 María de Lourdes Mora-García ,3 Benny Weiss-Steider ,3 Juan José Montesinos-Montesinos ,4 Adriana del Carmen Aguilar-Lemarroy ,5 Rosario García-Rocha ,3 Jorge Hernández-Montes ,3 Christian Azucena Don-López ,3 Luis Roberto Ávila-Ibarra ,1,3 Daniela Berenice Torres-Pineda ,1 Gabriela Molina-Castillo ,3 Rommel Chacón-Salinas ,6,7 Luis Vallejo-Castillo ,6,8 Sonia Mayra Pérez-Tapia ,6,7,9 and Alberto Monroy-García 1,2,3 1Laboratorio de Inmunología y Cáncer, Unidad de Investigación Médica en Enfermedades Oncológicas, CMN SXXI, Instituto Mexicano del Seguro Social, Ciudad de México, Mexico 1Laboratorio de Inmunología y Cáncer, Unidad de Investigación Médica en Enfermedades Oncológicas, CMN SXXI, Instituto Mexicano del Seguro Social, Ciudad de México, Mexico g Programa de Posgrado en Ciencias Biológicas, UNAM, Ciudad de México, Mexico 2Programa de Posgrado en Ciencias Biológicas, UNAM, Ciudad de México, Mexico Laboratorio de Inmunobiología, UIDCC-UMIEZ, FES-Zaragoza, UNAM, Ciudad de México, Mexico 4Laboratorio de Células Troncales Mesenquimales, Unidad de Investigación Médica en Enfermedades Oncológi Instituto Mexicano del Seguro Social, Ciudad de México, Mexico 5Centro de Investigación Biomédica de Occidente División de Inmunología Sierra Mojada, No. 800, Col. Independencia, C.P. 44340 Guadalajara, Jalisco, Mexico 6Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI), Instituto Politécnico Nacional, Ciudad de México, Mexico 7Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, ENCB-IPN, 6Unidad de Desarrollo e Investigación en Bioprocesos (UDIBI), Instituto Politécnico Nacional, Ciudad de México, Mexico 7Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, ENCB-IPN, Ciudad de México, Mexico Departamento de Farmacología, Centro de Investigación y de Estudios Avanzados del IPN (Cinvestav-IPN), Ciudad de México, Mexico 8Departamento de Farmacología, Centro de Investigación y de Estudios Avanzados del IPN (Cinvestav-IPN), Ciudad de México, Mexico 9Laboratorio Nacional para Servicios Especializados de Investigacioón, Desarrollo e Innovación (I + D + i) para Farmoquímicos y Biotecnológicos (LANSEIDI-FarBiotec-CONACyT), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, 9Laboratorio Nacional para Servicios Especializados de Investigacioón, Desarrollo e Innovación (I + D + i) para Farmoquímicos y Biotecnológicos (LANSEIDI-FarBiotec-CONACyT), Escuela Nacional de Ciencias Biológicas, Instituto Politécnico Nacional, Mexico City, Mexico Correspondence should be addressed to María de Lourdes Mora-García; lulumora@yahoo.com and Alberto Monroy-García; albertomon@yahoo.com 1. Introduction Likewise, the presence of high levels of CD73 in the plasma of cancer patients has been correlated with advanced stages of the disease [21–24], suggesting that extracellular adenosi- nergic activity may play an important role in the pathophys- iology of cancer. Likewise, the presence of high levels of CD73 in the plasma of cancer patients has been correlated with advanced stages of the disease [21–24], suggesting that extracellular adenosi- nergic activity may play an important role in the pathophys- iology of cancer. Cervical cancer (CC) is the fourth most common type of cancer in women and represents a major public health prob- lem worldwide because more than 500,000 new cases and approximately 250,000 deaths are reported each year, more than 80% of which occur in developing countries [1]. Persis- tent infection by high-risk human papillomavirus (HR-HPV) is the main factor in the development of low-grade squamous intraepithelial lesions (LSILs), which can progress to high- grade lesions (HSILs) and eventually to CC [2]. To date, more than 200 HPV genotypes have been identiﬁed, and HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-45, HPV-51, HPV-52, HPV-56, and HPV-58, which are considered HR-HPVs, are associated with anogenital cancer [3, 4]. Although the immune response against HPV antigens eliminates most infections and precursor lesions, some women exposed to HR-HPV will develop cancer, sug- gesting that other risk factors may be involved [5]. A growing number of studies have suggested that immunoregulation may play an important role in the development of CC. Recently, the adenosinergic pathway has been proposed to play an important role in essential signaling related to tumor growth, immunosuppression, and metastasis in cancer [6, 7]. In this pathway, the nucleotides adenosine triphosphate (ATP) and adenosine diphosphate (ADP) increase to high concentrations greater than 50 μM in response to stress sig- nals, such as hypoxia, damage, and inﬂammation in the tumor microenvironment (TME), and are hydrolyzed by the ectoenzyme CD39 (ectonucleoside triphosphate dipho- sphohydrolase-1, ENTPD1; EC 3.6.1.5) to AMP and subsequently to adenosine (Ado) by the activity of 5′-ecto- nucleotidase (CD73, EC 3.1.3.5) [8, 9]. Most Ado extracellu- lar signaling activities are mediated by four receptor subtypes (adenosine receptors (ARs): A1R, A2AR, A2BR, and A3R) coupled to G proteins in the target cell membrane [10, 11]. Correspondence should be addressed to María de Lourdes Mora-García; lulumora@yahoo.com and Alberto Monroy-García; albertomon@yahoo.com inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-β (TGF-β) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 (r = 0:4432, p < 0:001) and CD73 (r = 0:5786, p < 0:001). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-β promote the expression of CD39 and CD73 to favor CC progression through Ado generation. Correspondence should be addressed to María de Lourdes Mora-García; lulumora@yahoo.com and Alberto Monroy-García; albertomon@yahoo.com Correspondence should be addressed to María de Lourdes Mora-García; lulumora@yahoo.com and Alberto Monroy-García; albertomon@yahoo.com Received 18 June 2020; Revised 2 November 2020; Accepted 9 November 2020; Published 7 December 2020 Academic Editor: Giuseppe Valacchi Academic Editor: Giuseppe Valacchi Copyright © 2020 Ricardo Muñóz-Godínez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Persistent infection with high-risk human papillomavirus (HR-HPV) is the main factor in the development of cervical cancer (CC). The presence of immunosuppressive factors plays an important role in the development of this type of cancer. To determine whether CD39 and CD73, which participate in the production of immunosuppressive adenosine (Ado), are involved in the progression of CC, we compared the concentrations and hydrolytic activity of these ectonucleotidases in platelet-free plasma (PFP) samples between patients with low-grade squamous intraepithelial lesions (LSILs) (n = 18), high-grade squamous intraepithelial lesions (HSILs) (n = 12), and CC (n = 19) and normal donors (NDs) (n = 15). The concentrations of CD39 and CD73 in PFP increased with disease progression (r = 0:5929, p < 0:001). The PFP of patients with HSILs or CC showed the highest concentrations of CD39 (2.3 and 2.2 times that of the NDs, respectively) and CD73 (1.7 and 2.68 times that of the NDs, respectively), which were associated with a high capacity to generate Ado from the hydrolysis of adenosine diphosphate (ADP) and adenosine monophosphate (AMP). The addition of POM-1 and APCP, speciﬁc inhibitors of CD39 and CD73, respectively, 2 Mediators of Inﬂammation Mediators of Inﬂammation Mediators of Inﬂammation inhibited the ADPase and AMPase activity of PFP by more than 90%. A high level of the 90 kD isoform of CD73 was detected in the PFP of patients with HSILs or CC. Digestion with endoglycosidase H and N-glycanase generated CD73 with weights of approximately 90 kD, 85 kD, 80 kD, and 70 kD. In addition, the levels of transforming grow factor-β (TGF-β) in the PFPs of patients with LSIL, HSIL and CC positively correlated with those of CD39 (r = 0:4432, p < 0:001) and CD73 (r = 0:5786, p < 0:001). These results suggest that persistent infection by HR-HPV and the concomitant production of TGF-β promote the expression of CD39 and CD73 to favor CC progression through Ado generation. 1. Introduction In the TME, high concentrations of Ado (10–100 μM) are generated through adenosinergic activity and exert impor- tant eﬀects on the host, such as an immunosuppressive eﬀect on CD8+ cytotoxic T lymphocytes (CTLs), NK cells, B cells, and dendritic cells by interacting with the high-aﬃnity A2AR [12]. In addition, Ado can promote tumor growth by inducing the proliferation, invasion, and metastasis of tumor cells, mainly through its interaction with A1R, A2AR, and A2BR [13]. Upregulation of CD39 and CD73 in several types of tumors has been associated with a poor clinical prognosis [14, 15]. In addition, extracellular vesicles derived from squa- mous cell carcinoma of the head and neck [16], prostate can- cer [17], neuroblastoma [18], and other types of neoplastic cells [19, 20] can generate an immunosuppressive environ- ment through the generation of Ado from ATP hydrolysis. gy Recently, we provided evidence that cells obtained from cervical samples of patients with low-grade intraepithelial neoplasms (CINI) positive for HPV-16 showed higher con- centrations of CD39 and CD73 than cells from samples of patients with CINI negative for HPV-16 and from normal donors (NDs). The solubilized cervical mucus of these patients also showed higher concentrations of soluble CD39 and CD73, which was associated with a greater capacity to produce Ado through the hydrolysis of ATP and AMP [25]. To determine whether CD39 and CD73 are involved in the development of CC, we analyzed the concentrations and hydrolytic activity of these ectonucleotidases in plasma samples from patients with LSILs, HSILs, or CC. For compar- ison, ND plasma samples were also analyzed. Recently, we provided evidence that cells obtained from cervical samples of patients with low-grade intraepithelial neoplasms (CINI) positive for HPV-16 showed higher con- centrations of CD39 and CD73 than cells from samples of patients with CINI negative for HPV-16 and from normal donors (NDs). The solubilized cervical mucus of these patients also showed higher concentrations of soluble CD39 and CD73, which was associated with a greater capacity to produce Ado through the hydrolysis of ATP and AMP [25]. To determine whether CD39 and CD73 are involved in the development of CC, we analyzed the concentrations and hydrolytic activity of these ectonucleotidases in plasma samples from patients with LSILs, HSILs, or CC. For compar- ison, ND plasma samples were also analyzed. Mediators of Inﬂammation The blood plasma was centrifuged at 2422 × g for 10 min at 4°C to separate platelets as previously reported [29]. Soluble CD39 and CD73 were quantiﬁed in platelet-free plasma (PFP). 2.3. Hydrolytic Activity of Soluble CD39 and CD73. To deter- mine the hydrolytic activity of CD39 and CD73 ectonucleoti- dases contained in the PFP, 5 μl of each PFP sample was incubated in the presence of ADP or AMP at a ﬁnal concen- tration of 5 mM. After 72 h of incubation, Ado production was evaluated. To inhibit the enzymatic activity of CD39 and CD73, the speciﬁc inhibitors polyoxotungstate sodium (POM-1, Sigma-Aldrich, St. Louis, MO, USA) and adenosine 5′-(α,β-methylene) diphosphate (APCP, Sigma-Aldrich), respectively, were used at a ﬁnal concentration of 5 mM, as previously described [30]. The total volume of each reaction was 100 μl. The amount of Ado produced by each sample incubated with ATP or AMP was evaluated through ultra- high-performance liquid chromatography (UPLC) after applying 25μl of each reaction to a chromatograph (UPLC Acquity, Waters Corporation, Milford, MA, USA) using a mobile phase composed of 0.5% acetonitrile, 5% methanol, and 94.5% sodium acetate (0.25 M and pH 6.3). Before read- ing, the samples were ﬁltered through 3000 D Amicon ﬁlters (Millipore Corporation, USA). A standard Ado curve was prepared in Empower 3 (Waters Corporation, Milford, MA, USA) to evaluate the Ado concentrations in the diﬀerent samples. 2.2. Detection and Quantiﬁcation of Soluble CD39 and CD73 in PFP. CD39 and CD73 were detected in PFP by the enzyme-linked immunosorbent assay (ELISA). The data were interpolated in type curves using diﬀerent concentra- tions (1-35 ng/ml) of human recombinant enzymes (rhCD39 and rhCD73, R&D Systems, Minneapolis, MN, USA) diluted in phosphate-buﬀered saline (PBS). PFP was diluted with PBS, and CD39 was detected in PFP diluted to 1 : 40,000 and CD73 to 1 : 25,000. Samples of 100 μl of the diﬀerent dilutions were placed in triplicate in 96-well ﬂat-bottomed ELISA/radioimmunoassay plates (Corning Inc., USA). The plates were incubated for 1 h at 37°C and then overnight at 4°C. The plates were washed with a washing solution (PBS 0.1% Tween-20) and then incubated with a blocking solution (2% BSA w/v in PBS 0.1% Tween-20) for 2 h at 37°C. After washing, an anti-CD39 or anti-CD73 antibody (Novus Bio- logicals, USA) was added at a 1 : 1000 dilution in blocking solution and incubated for 2 h at 37°C. Mediators of Inﬂammation 3 Table 1: Clinical data of normal donors. DNA extraction. The samples were analyzed by conventional single-round polymerase chain reaction (PCR) to rule out the presence of HPV using the MY09/MY11 primers [27]; women who were consistently negative in clinical and molec- ular tests were considered NDs. As a positive control, DNA from the HeLa cell line (HPV-18+) was used as previously reported [28]. 10% (pH 9 8) Finally the reading was performed at a Cervical sample number HPV genotypes Age (years) Number of sexual partners Number of pregnancies 1 — 32 2 2 2 — 31 4 0 3 — 27 2 1 4 — 28 1 1 5 — 37 2 2 6 — 28 3 0 7 — 32 2 1 8 — 26 1 0 9 — 41 3 3 10 — 25 4 0 11 — 31 2 0 12 — 22 1 0 13 — 29 2 0 14 — 41 3 2 15 — 39 2 3 Averages — 31.2 2.26 1 The cervical samples of women diagnosed with SILs or CC were subjected to molecular analysis by PCR using the LINEAR ARRAY® HPV kit (Roche Diagnostics, CA, USA) for genotyping of the 37 main types of HPV that infect the anogenital region (HPV-6, HPV-11, HPV-16, HPV-18, HPV-26, HPV-31, HPV-33, HPV-35, HPV-39, HPV-40, HPV-42, HPV-45, HPV-51, HPV-52, HPV-53, HPV-54, HPV-55 (HPV-44 subtype), HPV-56, HPV-58, HPV-59, HPV-61, HPV-62, HPV-64 (HPV-34 subtype), HPV-66, HPV-67, HPV-68, HPV-69, HPV-70, HPV-71, HPV-72, HPV-73 (MM9), HPV-81, HPV-82 (MM4), HPV-83 (MM7), HPV-84 (MM8), IS39 (HPV-84 variant), and HPV-89 (CP6108)). In each sample, the human beta-globin gene was ampliﬁed as an internal control. After the hybridi- zation reaction, the strips were visually read against a refer- ence guide. All procedures followed the manufacturer’s instructions. 10% (pH 9.8). Finally, the reading was performed at a wavelength of 405 nm in an ELISA plate reader. 10% (pH 9.8). Finally, the reading was performed at a wavelength of 405 nm in an ELISA plate reader. The peripheral blood samples used in this study were obtained from 15 NDs, 18 patients with LSILs, 12 patients with HSILs, and 19 patients with CC (Tables 1 and 2). The samples were collected in vacutainer tubes with an ACD anti- coagulant (Becton Dickinson, USA) and centrifuged at 385 × g for 10 min to isolate the plasma. 2. Methods 2.1. Biological Material. Biological samples were obtained from women who attended early detection programs at the Gynecology and Obstetrics Hospital No. 4 of the Mexican Social Security Institute (Instituto Mexicano del Seguro Social (IMSS)), the gynecology service of the Oncology Hospital of CMN SXXI of the IMSS, Mexico City, Mexico, and the West- ern National Medical Center (CMNO-IMSS) in Guadalajara, Jalisco, Mexico, between April 2016 and May 2018 after signing the informed consent form endorsed by the local bioethics committee. Women without cervical lesions were diagnosed by conventional cytology (Papanicolaou stain) and colposcopy. In cases of precancerous lesions or CC, the diagnosis was conﬁrmed by histopathology. The cytologies and biopsies were analyzed by the pathologists of each clinic and were classiﬁed according to the Bethesda System 2001 [26] as follows: negative for intraepithelial lesion or malig- nancy (NILM); cervical intraepithelial neoplasia grades I, II, and III (CINI, CINII, and CINIII, respectively); and invasive CC. To analyze the data, the lesions were grouped according to the classiﬁcation of the squamous intraepithelial lesion (SIL), either low-grade SIL (LSIL, corresponding to the histological classiﬁcation of CINI) or high-grade SIL (HSIL, corresponding to the histological classiﬁcation of CINII and CINIII). Samples diagnosed as CC constituted the last group. p g g p Cervical samples from women diagnosed with NILM were collected during gynecological examinations with a cytobrush (Cytobrush®, STERYLMEDICAL Co., Yangon, Myanmar) and then placed in transport medium (PreservCyt Solution; Hologic, Bedford, MA) to be stored at 4°C until Mediators of Inﬂammation Cervical sample number HPV genotypes Age (years) Number of sexual partners Number of pregnancies LSIL 1 16, 58, 61 36 2 1 2 53 29 2 2 3 16, 33, 35, 52, 58 43 3 2 4 33, 71 32 3 2 5 16, 54 25 3 3 6 67 39 3 3 7 16 28 4 2 8 16, 33 43 2 4 9 66 27 3 2 10 73, 83 28 2 2 11 84 29 3 2 12 89 31 1 2 13 59, 66, 73 21 2 1 14 59 34 2 1 15 40, 52, 53, 66, 70 24 2 1 16 53 40 3 2 17 83 41 2 1 18 16 40 2 1 Averages — 32.7 2.44 1.88 HSIL 1 16 36 5 3 2 51, 52 29 3 4 3 45, 33 37 4 3 4 16 42 2 4 5 56, 66 44 3 2 6 16, 39 41 3 5 7 18 39 4 3 8 16 46 2 5 9 16, 45 33 3 2 10 18, 33, 45, 52 27 3 4 11 18, 53 30 4 4 12 16 34 4 3 Averages — 36.5 3.3 3.5 SCC 1 45, 84 42 3 4 2 33 53 2 3 3 16 39 4 4 4 16 49 2 5 5 16, 52 57 2 6 6 31 48 4 3 7 69, 71, 81, 84 62 3 4 buﬀer and analyzed by 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane (Amersham Protran, 0.2μm), which was incubated with blocking solution (TBS 0.1% Tween-20 and 5% BSA) for 60 min at room temperature. The membrane was washed twice with washing solution (TBS 0.1% Tween-20) and then incubated with anti-CD73 (Novus Biologicals, USA) at a 1 : 1000 dilution in blocking solution overnight at 5°C. After ﬁve washes, the membrane was incubated with the secondary antibody (HRP goat anti- rabbit) at a 1 : 1500 dilution with blocking solution for 1 h. After ﬁve washes, the presence of CD73 in the membrane was revealed in ChemiDoc (Life Science Research Bio-Rad, USA) using the Chemiluminescent Peroxidase Substrate (Sigma-Aldrich, USA). 2.6. Enzymatic Deglycosylation. To deglycosylate CD73 contained in PFP samples, these samples were incubated for 5 min at 37°C in the presence of 1.5 μl of a denaturing solution (1M β-mercaptoethanol and 2% SDS). Mediators of Inﬂammation Next, the endoglycanase H (Genzyme, Co., USA) and N-glycanase (Genzyme, Co., USA) enzymes were added to a ﬁnal concen- tration of 0.05 U/ml and incubated for 18 h at 37°C. Last, the products of enzymatic digestion were analyzed by Western blot using the anti-CD73 antibody as described above. 2.7. Statistical Analysis. All numerical data are presented as the mean value ± standard error of the mean (SEM) of three independent experiments. The comparisons and correlations were evaluated by multivariate statistical analysis with GraphPad Prism version 7 (La Jolla, CA, USA). Mediators of Inﬂammation The plates were washed six times and incubated with the secondary goat anti-mouse or anti-rabbit IgG bound to alkaline phosphatase (Thermo Fisher Scientiﬁc, Waltham, MA, USA) in a 1 : 500 dilution in blocking solution and incubated for 2 h at 37°C. After eight washes, the substrate for alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA) diluted in diethanola- mine (Sigma-Aldrich, St. Louis, MO, USA) was added to 2.4. Quantiﬁcation of TGF-β1. To quantify the TGF-β1 in PFP samples, the Quantikine human TGF-β1 ELISA kit (R&D Systems) was used according to the manufacturer’s protocol. 2.5. Western Blot. To analyze the presence of CD73, samples of 3 μl of each PFP or 20ng of CD73 contained in the PFP (based on the rhCD73 type curve) were treated with Laemmli 4 Mediators of Inﬂammation Mediators of Inﬂammation buﬀer and analyzed by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were tr to a nitrocellulose membrane (Amersham Protran, which was incubated with blocking solution (T Tween-20 and 5% BSA) for 60 min at room tem The membrane was washed twice with washing (TBS 0.1% Tween-20) and then incubated with an (Novus Biologicals, USA) at a 1 : 1000 dilution in solution overnight at 5°C. After ﬁve washes, the m was incubated with the secondary antibody (HRP g rabbit) at a 1 : 1500 dilution with blocking solution After ﬁve washes, the presence of CD73 in the m was revealed in ChemiDoc (Life Science Research USA) using the Chemiluminescent Peroxidase (Sigma-Aldrich, USA). 2.6. Enzymatic Deglycosylation. To deglycosylat contained in PFP samples, these samples were incu 5 min at 37°C in the presence of 1.5 μl of a de solution (1M β-mercaptoethanol and 2% SDS). N endoglycanase H (Genzyme, Co., USA) and N-g (Genzyme, Co., USA) enzymes were added to a ﬁna tration of 0.05 U/ml and incubated for 18 h at 37°C. products of enzymatic digestion were analyzed by blot using the anti-CD73 antibody as described abo 2.7. Statistical Analysis. All numerical data are pre the mean value ± standard error of the mean (SEM) independent experiments. The comparisons and cor were evaluated by multivariate statistical analy GraphPad Prism version 7 (La Jolla, CA, USA). 3. Results 3.1. Characteristics of the Participants. The present s carried out with 64 cervical and peripheral blood from women who attended early cancer detection p at the Gynecology and Obstetrics Hospital No. Mediators of Inﬂammation 4 of Table 2: Clinical data of patients with diﬀerent cervical squamous cell abnormalities. Cervical sample number HPV genotypes Age (years) Number of sexual partners Number of pregnancies LSIL 1 16, 58, 61 36 2 1 2 53 29 2 2 3 16, 33, 35, 52, 58 43 3 2 4 33, 71 32 3 2 5 16, 54 25 3 3 6 67 39 3 3 7 16 28 4 2 8 16, 33 43 2 4 9 66 27 3 2 10 73, 83 28 2 2 11 84 29 3 2 12 89 31 1 2 13 59, 66, 73 21 2 1 14 59 34 2 1 15 40, 52, 53, 66, 70 24 2 1 16 53 40 3 2 17 83 41 2 1 18 16 40 2 1 Averages — 32.7 2.44 1.88 HSIL 1 16 36 5 3 2 51, 52 29 3 4 3 45, 33 37 4 3 4 16 42 2 4 5 56, 66 44 3 2 6 16, 39 41 3 5 7 18 39 4 3 8 16 46 2 5 9 16, 45 33 3 2 10 18, 33, 45, 52 27 3 4 11 18, 53 30 4 4 12 16 34 4 3 Averages — 36.5 3.3 3.5 SCC 1 45, 84 42 3 4 2 33 53 2 3 3 16 39 4 4 4 16 49 2 5 5 16, 52 57 2 6 6 31 48 4 3 7 69, 71, 81, 84 62 3 4 Table 2: Continued. Cervical sample number HPV genotypes Age (years) Number of sexual partners Nu pre 8 39, 68 53 2 9 16 45 4 10 16, 18 38 3 11 16 46 3 12 45 40 2 13 45, 72 42 2 14 16, 62 52 2 15 16, 18, 52 51 3 16 16 35 2 17 18, 45 49 3 18 16 55 2 19 16, 52 50 3 Averages — 47.68 2.68 buﬀer and analyzed by 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis. Proteins were transferred to a nitrocellulose membrane (Amersham Protran, 0.2μm), which was incubated with blocking solution (TBS 0.1% Tween-20 and 5% BSA) for 60 min at room temperature. Mediators of Inﬂammation The membrane was washed twice with washing solution (TBS 0.1% Tween-20) and then incubated with anti-CD73 (Novus Biologicals, USA) at a 1 : 1000 dilution in blocking solution overnight at 5°C. After ﬁve washes, the membrane was incubated with the secondary antibody (HRP goat anti- rabbit) at a 1 : 1500 dilution with blocking solution for 1 h. After ﬁve washes, the presence of CD73 in the membrane was revealed in ChemiDoc (Life Science Research Bio-Rad, USA) using the Chemiluminescent Peroxidase Substrate (Sigma-Aldrich, USA). Table 2: Continued. Cervical sample number HPV genotypes Age (years) Number of sexual partners Number of pregnancies 8 39, 68 53 2 2 9 16 45 4 4 10 16, 18 38 3 5 11 16 46 3 6 12 45 40 2 5 13 45, 72 42 2 6 14 16, 62 52 2 4 15 16, 18, 52 51 3 3 16 16 35 2 4 17 18, 45 49 3 3 18 16 55 2 5 19 16, 52 50 3 4 Averages — 47.68 2.68 4.2 Table 2: Clinical data of patients with diﬀerent cervical squamous cell abnormalities. Table 2: Continued. Table 2: Clinical data of patients with diﬀerent cervical squamous cell abnormalities. 3. Results To detect CD39 and CD73, the PFP of the NDs and of the women with LSILs, HSILs, or CC were diluted with PBS at ratios of 1 : 40,000 and 1 : 25,000, respectively. The amounts of soluble CD39 and CD73 in the PFP increased according to the degree of disease progression. Patients with LSILs, HSILs, and CC showed sig- niﬁcantly more CD39 (averages, 6459 ± 1232 ng/ml, 8333 ± 1440 ng/ml, and 7728 ± 1198 ng/ml, respectively) than the NDs, whose average was 3839 ± 824 ng/ml (Figure 1(b)). The CD39 concentration in the PFP of patients with HSILs or CC, who showed the highest CD39, was 2.3 and 2.2 times that of the ND group, respectively (Figure 1(b)). Patients with LSILs, HSILs, or CC also showed signiﬁcantly higher amounts of CD73 in the PFP (averages, 2638 ± 630 ng/ml, 3181 ± 863 ng/ml, and 5053 ± 396 ng/ml, respectively) than NDs who showed an average of 1880 ± 228 ng/ml (Figure 1(d)). The CD73 concentration in the PFP of patients with HSILs or CC, who showed the highest CD73, was 1.7 and 2.68 times that of the ND group, respectively (Figure 1(d)). Unlike NDs, who showed a low correlation between the levels of CD39 and CD73 in PFP (r = 0:1104, p < 0:001) (Figure 2(a)), the LSIL, HSIL, and CC groups had a positive correlation between the concentrations of these ectonucleotidases in PFP (r = 0:5929, p < 0:001) (Figure 2(b)). p g To rule out the presence of HPV infection in the samples of NILM women, DNA was obtained from the cervical sam- ples for molecular analysis by PCR using the consensus oligo- nucleotides MY09 and MY11, which amplify a conserved fragment of 450 bp of the gene coding the L1 protein of the diﬀerent HPV genotypes. As a positive control, DNA from the HeLa cell line (HPV-18+) was used (Supplementary Figure 1). Nineteen cervical samples from NILM women were analyzed, 15 of which were negative for HPV infection and were included in the study as ND samples. The average age of the NDs was 31.2 (range 22-41) years (Table 1), that of women with LSILs was 32.7 (range 21-43) years, that of women with HSILs was 36.5 (range 27-46) years, and that of women with CC was 47.68 (range 35-62) years (Table 2). 3. Results 3.1. Characteristics of the Participants. The present study was carried out with 64 cervical and peripheral blood samples from women who attended early cancer detection programs at the Gynecology and Obstetrics Hospital No. 4 of the IMSS Mediators of Inﬂammation 5 Table 3: Correlations between clinical data of NDs and patients with diﬀerent cervical squamous cell abnormalities. The clinical data ND Patients LSIL HSIL SCC p values Age (years) 31:26 ± 5:86 32:77 ± 6:94 36:5 ± 6:11a 47:68 ± 7:08b,c,d a < 0:03 vs. ND b < 0:0001 vs. ND c < 0:0001 vs. LSIL d < 0:0001 vs. HSIL Number of sexual partners 2:26 ± 0:96 2:44 ± 0:7 3:3 ± 0:88e,f 2:6 ± 0:75g e < 0:006 vs. ND f < 0:005 vs. LSIL g < 0:03 vs. HSIL Number of pregnancies 1:13 ± 0:2 1:88 ± 0:83h 3:5 ± 1i,j 4:2 ± 1:1k,l h < 0:01 vs. ND i < 0:0001 vs. ND j <0.0001 vs. LSIL k < 0:0001 vs. ND l < 0:001 vs. LSIL ND: normal donor; LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; SCC: squamous cell carcinoma. p values were calculated using the Wilcoxon signed-rank test and Student’s t-test. : Correlations between clinical data of NDs and patients with diﬀerent cervical squamous cell abnormalities. (52.6%) were in patients with CC. Patients with HSILs or CC also reported signiﬁcantly greater numbers of sexual partners (averages of 3.3 and 2.6, respectively) and pregnancies (3.5 and 4.2, respectively) than NDs, whose averages were 2.26 and 1.13, respectively (Table 3). and the gynecology service of the Oncology Hospital of CMN SXXI of the IMSS, Mexico City, Mexico, and the Western National Medical Center (CMNO-IMSS) in Guadalajara, Jalisco, Mexico. According to the cytological and histopatho- logical analysis, 15 samples came from ND women (Table 1), 18 LSIL patients, 12 HSIL patients, and 19 CC patients (Table 2). All samples and clinical data of the participants were taken after obtaining informed consent according to ethical requirements and conﬁdentiality related to the sampling of humans in the institutions involved. 3.2. PFP Samples from Patients with HSILs or CC Are High in Soluble CD39 and CD73. To measure soluble CD39 and CD73 in the PFP, rhCD39 curves (Figure 1(a)) and rhCD73 curves (Figure 1(c)) were established using concentrations of 5-30 ng/ml of each recombinant protein. 3. Results The average numbers of sexual partners and pregnancies in the ND group were 2.26 (range 1-4) and 1 (range 0-3), respectively (Table 1). In patients with LSILs, the average numbers were 2.44 (range 1-4) and 1.88 (range 1-4), respectively; in patients with HSILs, the average numbers were 3.3 (range 2-5) and 3.5 (range 2-5), respectively; and in patients with CC, the average numbers were 2.68 (range 2-4) and 4.2 (range 3-6), respectively (Table 2). All cervical samples from patients with SILs were positive for HR-HPV. The most frequent genotypes were HPV-16 (22/49, 44%), HPV-45 and HPV-52 (7/49, 14%), HPV-18 and HPV-33 (6/49, 12%), and HPV-53 (3/49, 6%) (Table 2). Likewise, 25/49 patients had coinfection with two or more HPV genotypes, 8/18 (44.4%) of which were in patients with LSILs, 7/12 (58%) were in patients with HSILs, and 10/19 3.3. PFP Samples from Patients with HSILs and CC Show a High Capacity to Generate Ado through Hydrolysis of ADP and AMP. To analyze the hydrolytic activity of the CD39 6 Mediators of Inﬂammation r = 0.9925 p < 0.001 OD 0 0 1 2 3 4 5 10 rhCD39 (ng/ml) 20 30 (a) ⁎⁎⁎⁎ ⁎⁎⁎⁎ ⁎⁎⁎⁎ CD39 (ng/ml) ND 0 5000 10000 15000 LSIL PFP samples HSIL CC LSIL ns CC HSIL ⁎⁎⁎ ⁎⁎ (b) r = 0.9952 p < 0.001 OD 0 10 rhCD39 (ng/ml) 20 30 0 1 2 3 4 5 (c) ⁎⁎⁎⁎ ⁎⁎⁎⁎ ⁎⁎⁎⁎ CD73 (ng/ml) ND 0 2000 4000 6000 LSIL PFP samples LSIL CC ns HSIL HSIL CC ⁎⁎⁎⁎ ⁎⁎⁎⁎ (d) Figure 1: CD39 and CD73 concentrations in PFP samples from NDs and patients with LSILs, HSILs, or CC. The type curves for soluble CD39 and CD73 were established by ELISA using diﬀerent concentrations (5-30 ng/ml) of human recombinant proteins CD39 (rhCD39) (a) and CD73 (rhCD73) (c), whose correlations with absorbance (optical density) were r = 0:9925 (p < 0:001) and r = 0:9952 (p < 0:001), respectively. For the detection of CD39 and CD73, the PFP samples of the NDs and women with LSILs, HSILs, or CC were diluted with PBS at ratios of 1 : 40,000 and 1 : 25,000, respectively. The data are representative of three independent experiments. The means ± SEMs of the concentrations of soluble CD39 (b) and CD73 (d) detected in the PFP of the NDs and patients with LSILs, HSILs, or CC are shown. 3. Results Signiﬁcant diﬀerences are indicated by ∗(p < 0:05), ∗∗(p < 0:001), and ∗∗∗(p < 0:0001). ns: not signiﬁcant. r = 0.9925 p < 0.001 OD 0 0 1 2 3 4 5 10 rhCD39 (ng/ml) 20 30 ⁎⁎⁎⁎ ⁎⁎⁎⁎ ⁎⁎⁎⁎ CD39 (ng/ml) ND 0 5000 10000 15000 LSIL PFP samples HSIL CC CD39 (ng/ml) (a) r = 0.9952 p < 0.001 OD 0 10 rhCD39 (ng/ml) 20 30 0 1 2 3 4 5 ( ) ⁎⁎⁎⁎ ⁎⁎⁎⁎ ⁎⁎⁎⁎ CD73 (ng/ml) ND 0 2000 4000 6000 LSIL PFP samples LSIL CC ns HSIL HSIL CC ⁎⁎⁎⁎ ⁎⁎⁎⁎ (d) ⁎⁎⁎⁎ ⁎⁎⁎⁎ ⁎⁎⁎⁎ CD73 (ng/ml) ND 0 2000 4000 6000 LSIL PFP samples HSIL CC (c) (d) Figure 1: CD39 and CD73 concentrations in PFP samples from NDs and patients with LSILs, HSILs, or CC. The type curves for soluble CD39 and CD73 were established by ELISA using diﬀerent concentrations (5-30 ng/ml) of human recombinant proteins CD39 (rhCD39) (a) and CD73 (rhCD73) (c), whose correlations with absorbance (optical density) were r = 0:9925 (p < 0:001) and r = 0:9952 (p < 0:001), respectively. For the detection of CD39 and CD73, the PFP samples of the NDs and women with LSILs, HSILs, or CC were diluted with PBS at ratios of 1 : 40,000 and 1 : 25,000, respectively. The data are representative of three independent experiments. The means ± SEMs of the concentrations of soluble CD39 (b) and CD73 (d) detected in the PFP of the NDs and patients with LSILs, HSILs, or CC are shown. Signiﬁcant diﬀerences are indicated by ∗(p < 0:05), ∗∗(p < 0:001), and ∗∗∗(p < 0:0001). ns: not signiﬁcant. tively (Figure 3(b)). Interestingly, the addition of 5 mM POM-I or APCP decreased the ability of PFP to hydrolyze ADP and AMP by more than 90% in all cases (Figure 3(b)). and CD73 ectonucleotidases contained in the PFP samples from NDs and patients with LSILs, HSILs, or CC, samples of 5 μl of each PFP were incubated in the presence of 5 mM ADP or AMP (total volume 100μl) and in the presence or absence of POM-1 or APCP, speciﬁc inhibitors of CD39 and CD73, respectively. Aliquots of each reaction were taken at the beginning and after 72h of incubation to evaluate Ado production through UPLC. 3. Results Using diﬀerent concentrations (0.1-10 μM) of synthetic Ado as reference standards (Figure 3(a)), we found that the PFP derived from patients with HSIL or CC generated signiﬁcantly higher amounts of Ado than the PFP of NDs or LSIL patients. The average Ado concentration produced by the ND PFP when incubated in the presence of ADP or AMP was 0 or 12:63 ± 2:3 μM, respectively (Figure 3(b)), and that of the patient groups was 0 or 53:9 ± 7:88 (LSIL), 52:1 ± 1:3 or 202:9 ± 13:10 (HSIL), and 143:2 ± 7:01 or 401:3 ± 22:7 μM (CC), respec- 3.4. The PFP of Patients with HSILs or CC Contains Highly Glycosylated CD73. CD73 has four N-glycosylation motifs, and changes in glycosylation at one or more of these sites can alter its AMPase activity [31]. To determine whether the strong hydrolytic activity of CD73 detected in the PFP of patients with HSILs or CC was related to the degree of gly- cosylation, samples of 3μl of PFP (Figure 4(a)) or 20 ng of CD73 contained in the PFP (Figure 4(b)) were analyzed by Western blot using anti-CD73 antibodies. Two bands with weights of 70kD and 90kD approximately were revealed. Interestingly, the density of the 90kD band detected in the PFP increased with disease progression (Figures 4(a) and 4(b)). Likewise, the 70 kD band showed higher density in 7 Mediators of Inﬂammation (a) ND 0 1000 1800 2600 3400 4200 5000 400 800 1200 1600 r = 0.1104 p < 0.001 CD73 (ng/ml) Patients (b) CD39 (ng/ml) 0 0 2000 4000 6000 8000 10000 r = 0.5929 p < 0.001 2000 4000 6000 Figure 2: Correlations between the concentrations of CD39 and CD73 in the PFP of NDs and in the PFP of patients (LSILs, HSILs, and CC together). The correlations (r) between the concentrations of CD39 and CD73 in the ND PFP samples (open circles) are shown (r = 0:1104, p < 0:001) (a), as well as the correlations for patients with LSILs (white diamonds), HSILs (black and white diamonds), and CC (black diamonds) (b) (r = 0:5929, p < 0:001). 3. Results (a) ND 0 1000 1800 2600 3400 4200 5000 400 800 1200 1600 r = 0.1104 p < 0.001 CD73 (ng/ml) Patients (b) 0 0 2000 4000 6000 8000 10000 r = 0.5929 p < 0.001 2000 4000 6000 Figure 2: Correlations between the concentrations of CD39 and CD73 in the PFP of NDs and in the PFP of patients (LSILs, HSILs, and CC together). The correlations (r) between the concentrations of CD39 and CD73 in the ND PFP samples (open circles) are shown (r = 0:1104, p < 0:001) (a), as well as the correlations for patients with LSILs (white diamonds), HSILs (black and white diamonds), and CC (black diamonds) (b) (r = 0:5929, p < 0:001). the PFP of patients with HSILs or CC than in that of the LSIL or ND groups when equal volumes of PFP were analyzed (Figure 4(a)). On the other hand, when using the same amount of CD73 (20 ng), the density of the 70 kD band was similar between the PFP samples. However, a greater density of the 90 kD band was noted in the samples of the patients with LSILs, HSILs, or CC than in the ND samples (Figure 4(b)). To determine whether the 90 kD band corre- sponded to a highly glycosylated CD73 isoform, 20ng samples of CD73 contained in the PFP were subjected to deglycosylation using the endoglycosidase H and N- glycanase enzymes. Enzymatic digestion of PFP samples with these enzymes resulted in products of approximately 90, 85, 80, and 70kD according to Western blot analysis with the anti-CD73 antibody (Figure 4(c)). Samples from patients with CC showed bands corresponding to these four products. Notably, the samples of patients with HSILs and CC pre- sented an 85 kD product, which was not detected in the sam- ples of patients with LSILs or NDs, while an 80 kD product was detected in the samples of LSIL patients and NDs (Figure 4(c)). These results suggest that the greater hydrolytic capacity of CD73 detected in the PFP of patients with HSILs or CC may be related to a higher concentration of a highly glycosylated CD73 isoform. important role in promoting HPV infection and local sup- pression in HPV-associated neoplasms [32]. Likewise, the levels of TGF-β increase with the severity of cervical lesions, and the strong expression of this cytokine has been associated with poor survival in patients with CC [33, 34]. 3. Results Aliquo ars), patients with HSILs (diagonal lines), and patients with CC (horizo otal volume 100 μl) and in the presence or absence of POM-1 or APCP o produced by hydrolysis of the nucleotides was quantiﬁed after upper). A representative linear relationship between the Ado concent Ado produced during the incubation of PFP with ADP (upper) or AM wn. Diﬀerences in Ado concentrations were analyzed by two-way ANO experiments OD Retention time (min) 0.00 10.000 𝜇M 7.000 𝜇M 5.000 𝜇M 3.000 𝜇M 2.000 𝜇M 1.000 𝜇M Adenosina - 2.313 0.500 𝜇M 0.100 𝜇M 0.00 0.04 0.08 0.10 0.14 0.18 0.20 0.24 0.50 1.50 1.00 2.00 2.50 3.00 3.50 OD Retention time (min) 0.00 0.00 0.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Ado (𝜇M) r2= 0.999988 𝜇 Aden 0.500 𝜇M 0.100 𝜇M 0.00 0.04 0.50 1.50 1.00 2.00 2.50 3.00 3.50 (a) Adenosine (𝜇M) ND 0 50 100 150 LSIL ADP ADP+POM-1 HSIL CC (A) (B) ND LSIL HSIL CC ⁎ ⁎⁎ ND 0 100 300 500 LSIL AMP PFP samples ADP+APCP HSIL CC ND LSIL HSIL CC 200 400 ⁎ ⁎⁎ ⁎⁎ (b) Figure 3: Catalytic activity of CD39 and CD73 in PFP of NDs and patients with LSILs, HSILs, or CC. Aliquots of 5 μl of PFP from NDs (black bars), patients with LSILs (white bars), patients with HSILs (diagonal lines), and patients with CC (horizontal lines) were incubated in the presence of 5 mM ADP or AMP (total volume 100 μl) and in the presence or absence of POM-1 or APCP, speciﬁc inhibitors of CD39 and CD73, respectively. (a) The Ado produced by hydrolysis of the nucleotides was quantiﬁed after 72 h by UPLC using standard concentrations of synthetic Ado (upper). A representative linear relationship between the Ado concentration and the optical density is shown (lower). (b) The amount of Ado produced during the incubation of PFP with ADP (upper) or AMP (lower) and in the presence or absence of POM-1 or APCP is shown. Diﬀerences in Ado concentrations were analyzed by two-way ANOVA. ∗p < 0:01, ∗∗p < 0:0001. The data represent three independent experiments. 3. Results On the other hand, we recently reported that in patients with low-grade cervical neoplasms and persistent infection by HR-HPV, the highest level of expression of CD73 in cervical cells was associated with higher plasma TGF-β in relation to that found in NDs [25]. Likewise, we reported that TGF-β is important in inducing and maintaining the expression of CD73 in CC tumor cells [35]. Therefore, we proceeded to analyze the levels of this cytokine in the PFP of patients with LSILs, HSILs, or CC and compare these values with the ND value. We observed that patients with HSILs and CC, who showed the highest levels of CD39 and CD73, also showed the highest levels of TGF-β. The average concentration of TGF-β contained in the ND PFP was 350 ± 61 pg/ml, while that of LSIL, HSIL, and CC was 634 ± 122, 749 ± 155, and 954 ± 152 pg/ml, respectively (Figure 5(a)). In addition, we found a positive correlation between the concentrations of TGF-β with CD39 or CD73, in the PFP of patients with LSIL, HSIL, or CC (r = 0:4432, p < 0:001 (Figure 5(c)) and r = 0:5786, p < 0:001 (Figure 5(e)), respectively). In the PFP of the ND groups, the correlations were r = 0:2647 (p < 0:001) (Figure 5(b)) and r = 0:2502 (p < 0:001) (Figure 5(d)), respectively. 3.5. The PFP of Patients with HSILs or CC Has Higher TGF-β than the PFP of Patients with LSILs and NDs. TGF-β plays an Mediators of Inﬂammation 8 OD Retention time (min) 0.00 0.00 0.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 10.000 𝜇M 7.000 𝜇M 5.000 𝜇M 3.000 𝜇M Ado (𝜇M) r2= 0.999988 2.000 𝜇M 1.000 𝜇M Adenosina - 2.313 0.500 𝜇M 0.100 𝜇M 0.00 0.04 0.08 0.10 0.14 0.18 0.20 0.24 0.50 1.50 1.00 2.00 2.50 3.00 3.50 (a) Adenosine (𝜇M) ND 0 50 100 150 LSIL ADP ADP+POM-1 HSIL CC (A) (B) ND LSIL HSIL CC ⁎ ⁎⁎ ND 0 100 300 500 LSIL AMP PFP samples ADP+APCP HSIL CC ND LSIL HSIL CC 200 400 ⁎ ⁎⁎ ⁎⁎ (b) 9 and CD73 in PFP of NDs and patients with LSILs, HSILs, or CC. 3. Results 0.00 0.0 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 Ado (𝜇M) r2= 0.999988 Ado (𝜇M) (a) Adenosine (𝜇M) ND 0 50 100 150 LSIL ADP ADP+POM-1 HSIL CC (A) (B) ND LSIL HSIL CC ⁎ ⁎⁎ ND 0 100 300 500 LSIL AMP PFP samples ADP+APCP HSIL CC ND LSIL HSIL CC 200 400 ⁎ ⁎⁎ ⁎⁎ (b) (b) Figure 3: Catalytic activity of CD39 and CD73 in PFP of NDs and patients with LSILs, HSILs, or CC. Aliquots of 5 μl of PFP from NDs (black bars), patients with LSILs (white bars), patients with HSILs (diagonal lines), and patients with CC (horizontal lines) were incubated in the presence of 5 mM ADP or AMP (total volume 100 μl) and in the presence or absence of POM-1 or APCP, speciﬁc inhibitors of CD39 and CD73, respectively. (a) The Ado produced by hydrolysis of the nucleotides was quantiﬁed after 72 h by UPLC using standard concentrations of synthetic Ado (upper). A representative linear relationship between the Ado concentration and the optical density is shown (lower). (b) The amount of Ado produced during the incubation of PFP with ADP (upper) or AMP (lower) and in the presence or absence of POM-1 or APCP is shown. Diﬀerences in Ado concentrations were analyzed by two-way ANOVA. ∗p < 0:01, ∗∗p < 0:0001. The data represent three independent experiments. 9 9 Mediators of Inﬂammation 100 kD 75 kD 50 kD ND 0 LSIL PFP samples Relative density HSIL CC MW 20 40 60 70 kD 90 kD (a) MW ND LSIL PFP samples Relative density HSIL CC 0 1 2 3 4 5 70 kD 90 kD 100 kD 75 kD 50 kD (b) Relative density MW ND 0 LSIL PFP samples HSIL CC 1 2 3 4 5 70 kD 80 kD 85 kD 90 kD 100 kD 75 kD 50 kD (c) Figure 4: Detection of CD73 in the PFP of NDs and patients with LSILs, HSILs, or CC. Samples of 3 μl of PFP (a) or 20 ng of CD73 contained in the PFP (b) of the ND, LSIL, HSIL, and CC groups were analyzed by Western blot using the anti-CD73 antibody. The densities of the 70 kD (black bars) and 90 kD (white bars) bands detected in the samples of patients with LSILs, HSILs, or CC relative to those of NDs (set to 1) are shown. 3. Results (c) Twenty-nanogram samples of CD73 contained in PFP were subjected to deglycosylation using the enzymes endoglycosidase H and N-glycanase. The densities of the 70 kD (black bars), 80 kD (bars with horizontal lines), 85 kD (bars with diagonals), and 90 kD (white bars) bands detected in the LSIL, HSIL, and CC samples relative to the ND samples (set to 1) are shown. MW: molecular weight. A representative test of three independent tests is shown. 9 Mediators of Inﬂammation (b) (c) Figure 4: Detection of CD73 in the PFP of NDs and patients with LSILs, HSILs, or CC. Samples of 3 μl of PFP (a) or 20 ng of CD73 contained in the PFP (b) of the ND, LSIL, HSIL, and CC groups were analyzed by Western blot using the anti-CD73 antibody. The densities of the 70 kD (black bars) and 90 kD (white bars) bands detected in the samples of patients with LSILs, HSILs, or CC relative to those of NDs (set to 1) are shown. (c) Twenty-nanogram samples of CD73 contained in PFP were subjected to deglycosylation using the enzymes endoglycosidase H and N-glycanase. The densities of the 70 kD (black bars), 80 kD (bars with horizontal lines), 85 kD (bars with diagonals), and 90 kD (white bars) bands detected in the LSIL, HSIL, and CC samples relative to the ND samples (set to 1) are shown. MW: molecular weight. A representative test of three independent tests is shown. 4. Discussion For patients with LSILs, HSILs, or CC, the correlations were r = 0:4432 (p < 0:001) (c) and r = 0:5786 (p < 0:001) (e), respectively. The coordinates of the TGF-β1 concentration with the CD39 or CD73 concentration are indicated by white circles in the ND group, white diamonds in the LSIL group, white and black diamonds in the HSIL group, and black diamonds in the CC group. 10 Mediators of Inﬂammation 0 200 400 600 800 1000 1200 ND LSIL (a) HSIL CC ⁎ ⁎⁎ ⁎⁎ (b) 0 100 200 300 400 500 0 500 1000 1500 2000 r = 0.2647 𝜌 < 0.001 -𝛽 (pg/ml) (d) 0 100 200 300 400 500 0 500 1000 1500 2000 r = 0.2502 p < 0.001 (c) 0 200 400 600 800 1000 1200 1400 0 4000 8000 12000 CD39 TGF-𝛽 r = 0.4432 p < 0.001 (e) 0 200 400 600 800 1000 1200 1400 0 2000 4000 6000 CD73 r = 0.5786 p < 0.001 Figure 5: TGF-β concentrations in PFP samples from NDs and patients with LSILs, HSILs, or CC and their correlations with the concentrations of CD39 and CD73. (a) The TGF-β1 content is shown in PFP samples taken from NDs and patients with LSILs, HSILs, or CC. The data are representative of three independent experiments, and the means ± SEMs are shown. ∗p < 0:05, ∗∗p < 0:001 for the concentration of TGF-β1 in a patient group relative to the ND group. The correlations (r) between the levels of TGF-β1 and CD39 or CD73 in the PFP of NDs were r = 0:2647 (p < 0:001) (b) and r = 0:2502 (p < 0:001) (d), respectively. For patients with LSILs, HSILs, or CC, the correlations were r = 0:4432 (p < 0:001) (c) and r = 0:5786 (p < 0:001) (e), respectively. The coordinates of the TGF-β1 concentration with the CD39 or CD73 concentration are indicated by white circles in the ND group, white diamonds in the LSIL group, white and black diamonds in the HSIL group, and black diamonds in the CC group. respectively, suggesting that during the development of CC, alteration of nucleotide metabolism is promoted to generate increased levels of circulating Ado through the activity of these ectonucleotidases. 4. Discussion levels. Thus, the concentrations and activity of circulating ectoenzymes in the bloodstream of cancer patients may be useful as biomarkers of disease progression and to devise therapeutic strategies [38]. In the last 10 years, cancer research has especially focused on the role played by Ado-, CD39-, and CD73-producing ectoenzymes in immunomodulation and evasion of the antitumor immune response. In fact, inhibition of the adeno- sinergic pathway in the tumor microenvironment has been proposed as an indispensable alternative in oncological ther- apy [36, 37]. However, large gaps in knowledge prevent the development of eﬀective Ado-based therapies, such as the roles of redundant pathways that control ATP and Ado Our working group recently reported higher expression levels of CD39 and CD73 in cells obtained from cervical sam- ples of patients with CINI positive for HPV-16 than in cells from HPV-16-negative samples and ND samples, which cor- related with the presence of higher levels of CD39 and soluble CD73 in cervical mucus with the capacity to produce Ado through hydrolysis of ATP and AMP [25]. However, to 10 Mediators of Inﬂammation 0 200 400 600 800 1000 1200 ND LSIL (a) (b) (d) (c) (e) HSIL CC ⁎ ⁎⁎ ⁎⁎ 0 100 200 300 400 500 0 500 1000 1500 2000 r = 0.2647 𝜌 < 0.001 0 200 400 600 800 1000 1200 1400 0 4000 8000 12000 CD39 TGF-𝛽 (pg/ml) r = 0.4432 p < 0.001 0 100 200 300 400 500 0 500 1000 1500 2000 r = 0.2502 p < 0.001 0 200 400 600 800 1000 1200 1400 0 2000 4000 6000 CD73 r = 0.5786 p < 0.001 Figure 5: TGF-β concentrations in PFP samples from NDs and patients with LSILs, HSILs, or CC and their correlations with the concentrations of CD39 and CD73. (a) The TGF-β1 content is shown in PFP samples taken from NDs and patients with LSILs, HSILs, or CC. The data are representative of three independent experiments, and the means ± SEMs are shown. ∗p < 0:05, ∗∗p < 0:001 for the concentration of TGF-β1 in a patient group relative to the ND group. The correlations (r) between the levels of TGF-β1 and CD39 or CD73 in the PFP of NDs were r = 0:2647 (p < 0:001) (b) and r = 0:2502 (p < 0:001) (d), respectively. 4. Discussion Nucleotidase Normal donors SIL patients p values Age (years) Number of sexual partners Number of pregnancies Age (years) Number of sexual partners Number of pregnancies CD39 -0.06017 -0.2257 0.0721 0.1308 0.3312a 0.3512b a < 0:0201 b < 0:0134 c < 0:0001 d < 0:0001 CD73 0.3028 0.2277 0.2284 0.5868c 0.1542 0.6387d Values of Pearson’s coeﬃcient (r) are shown. Values of Pearson’s coeﬃcient (r) are shown. of TGF-β in cells infected with HPV [35]. In this study, we found a signiﬁcantly higher level of TGF-β in the PFP of patients with LSILs, HSILs, or CC than in that of NDs. In fact, a positive correlation was observed between the TGF-β con- centration of these patients and the CD39 and CD73 concen- trations detected in PFP. In addition, we observed that patients with HSILs or CC, who presented the highest plasma concentrations of TGF-β, CD39, and CD73, showed the highest numbers of sexual partners (averages of 3.3 and 2.6, respectively) and pregnancies (averages of 3.5 and 4.2, respectively) in contrast to numbers of 2.2 and 1.1, respec- tively, in the ND group. A strong correlation was also observed between the number of sexual partners and CD39, as well as between the number of pregnancies and the expres- sion of CD39 and CD73, in patients with LSILs, HSILs, or CC (Table 4), which is consistent with previous reports stating that more sexual partners and pregnancies are the main risk factors associated with persistent infection by HR-HPV and the development of cervical dysplasia and CC [47, 48]. The increase in soluble CD73 found in PFP samples from patients with HSILs or CC in our study is consistent with reports of increased hydrolytic activity of AMP in the plasma of patients with advanced cancer [22, 42]. Interestingly, the increased AMPase activity found in the PFP of patients with HSILs or CC was also associated with a high concentration of a highly glycosylated 90kD protein as revealed by Western blot assays using the anti-CD73 antibody. CD73 has four consensus N-glycosylation motifs, 53NAS, 311NSS, 333NYS, and 403NGT, and changes in glycosylation in one or more of these sites can alter the hydrolytic activity of CD73 because the three of them (N311, N333, and N403) are found in the C-terminal catalytic domain of the molecule [31]. In pathological states, the CD73 protein can undergo posttrans- lational changes, generating diﬀerent isoforms with catalyti- cally diﬀerent properties. 4. Discussion For example, the increase in AMPase activity in muscular dystrophy has been associated with a higher concentration of an active form of CD73 (72 kD) than an inactive form (62 kD) [43]. On the other hand, in hepatocellular carcinoma, altered CD73 glycosyla- tion is associated with attenuated AMPase activity due to greater production of an isoform that is 50 amino acids shorter than the complete protein [44, 45]. We found that enzymatic digestion of the PFP samples using endoglycosi- dase H and N-glycanase resulted in products of 90, 85, 80, and 70 kD, which were observed mainly in the PFP samples of patients with HSILs or CC, suggesting that the highest degree of CD73 glycosylation in the plasma of these patients was associated with a higher AMPase capacity. In addition, the high level of CD39 and high glycosylation of CD73 in the PFP of these patients suggest that both ectonucleotidases act in a coordinated manner to generate an immunosuppres- sive environment through the generation of Ado, as has been proposed in other cancers [22, 42]. TGF-β increases the levels of CD39 and CD73 in activated T cells and myeloid suppressor cells [13]. The expression of TGF-β1 in CC has been directly correlated with the degree of disease progres- sion [34] and with the expression of the HR-HPV E6 and E7 oncogenes, which induce activation of the human TGF- β1 promoter by recognizing the Sp1 sequence [46]. We previously reported that CC tumor cells infected with HR- HPV constitutively produce TGF-β, which is important for inducing and maintaining CD73 expression. We also demon- strated that Ado generated by the enzymatic activity of CD73 induced the production of TGF-β in tumor cells by interact- ing with A2AR and A2BR, suggesting an important connec- tion between the adenosinergic pathway and the production Therefore, the results obtained in this study suggest that the production of TGF-β associated with persistent infection by HR-HPV may be an important factor inducing and main- taining the expression of the CD39 and CD73 ectonucleoti- dases during the development of CC. 4. Discussion Considering the important role of the adenosinergic pathway in the suppres- sion of the antitumor immune response through Ado gener- ation [13] and that the presence of high concentrations of extracellular nucleotides in the TME of CC seems to interfere with the regulation, proliferation, diﬀerentiation, and apo- ptosis of cancer cells of the cervix [49], the concentrations and activity of CD39 and CD73 in the plasma of patients with CC may be valuable biomarkers of disease progression and may direct the choice of clinical treatment for these patients. 4. Discussion ATPase/ADPase and AMPase activ- ities mediated by CD39 and CD73, respectively, in the body ﬂuids of patients with inﬂammatory diseases and cancer have been recently reported [39, 40]. In the context of cancer, increased expression and activity of CD39 and CD73 in tissues and/or biological ﬂuids can lead to high levels of Ado that potently suppress the T cell-mediated antitumor immune response and promote tumor progression through stimulation of ARs [7, 41]. In fact, higher serum CD73 in patients with metastatic melanoma has been associated with lower eﬀective- ness of nivolumab-based immunotherapy [24]. determine whether adenosinergic activity is associated with the degree of disease progression, here, we analyzed the con- centrations and activity of CD39 and CD73 in the PFP of patients with LSILs, HSILs, or CC and compared them with those of NDs. Interestingly, we found that the concentrations of CD39 and CD73 in PFP increased with the degree of dis- ease progression. The PFP of patients with HSILs or CC showed a CD39 content 2.3 and 2.2 times that of the NDs, respectively, and a CD73 content 1.7 and 2.68 times that of the NDs. The concentrations of CD39 and CD73 in the PFP of these patients were associated with a high capacity to generate Ado from the hydrolysis of ADP and AMP. ADPase and AMPase activities were reduced by the addition of POM-1 and APCP, speciﬁc inhibitors of CD39 and CD73, Mediators of Inﬂammation 11 Table 4: Correlation analysis of the clinical data of normal donors and patients with SIL and the plasmatic contents of the nucleotidases CD39 and CD73. Nucleotidase Normal donors SIL patients p values Age (years) Number of sexual partners Number of pregnancies Age (years) Number of sexual partners Number of pregnancies CD39 -0.06017 -0.2257 0.0721 0.1308 0.3312a 0.3512b a < 0:0201 b < 0:0134 c < 0:0001 d < 0:0001 CD73 0.3028 0.2277 0.2284 0.5868c 0.1542 0.6387d Values of Pearson’s coeﬃcient (r) are shown. Table 4: Correlation analysis of the clinical data of normal donors and patients with SIL and the plasmatic contents of the nucleotidases CD39 and CD73. Mediators of Inﬂammation Mediators of Inﬂammation Conflicts of Interest The authors have no conﬂicts of interest to declare. [4] N. Muñoz, F. X. Bosch, S. de Sanjosé et al., “Epidemiologic classiﬁcation of human papillomavirus types associated with cervical cancer,” New England Journal of Medicine, vol. 348, no. 6, pp. 518–527, 2003. Supplementary Materials Supplementary Figure 1: detection of the HPV L1 gene in cervical samples from NILM women. Expression of the L1 gene in cervical samples was detected by PCR as described in Section 2. The absence (left) or presence (right) of L1 is shown in samples from NILM women. As a positive control, DNA from the HeLa cell line (HPV-18+) was used. The expression of the β-actin gene in all samples was used as an internal control. The size of the fragment (bp) of each gene is indicated. (Supplementary Materials) Data Availability [2] C. Woodman, S. Collins, and L. Young, “The natural history of cervical HPV infection: unresolved issues,” Nature Reviews Cancer, vol. 7, no. 1, pp. 11–22, 2007. The data used to support the ﬁndings of this study are available from the corresponding author upon request. [3] IARC Working Group, “Biological agents. Volume 100 B. A review of human carcinogens,” IARC monographs on the eval- uation of carcinogenic risks to humans/World Health Organi- zation, International Agency for Research on Cancer, vol. 100, pp. 1–441, 2012. References [1] F. Bray, J. Ferlay, I. Soerjomataram, R. L. Siegel, L. A. Torre, and A. Jemal, “Global cancer statistics 2018: GLOBOCAN esti- mates of incidence and mortality worldwide for 36 cancers in 185 countries,” CA: A Cancer Journal of Clinicians, vol. 68, no. 6, pp. 394–424, 2018. Abbreviations Ado: Adenosine ADP: Adenosine diphosphate AMP: Adenosine monophosphate APCP: 5′-(α,β-Methylene) diphosphate ARs (A1R, A2AR, A2BR, and A3R): Adenosine receptors ATP: Adenosine triphosphate CC: Cervical cancer CD39: Ectonucleoside triphosphate diphosphohydrolase-1 CD73: 5′-Nucleotidase CINI, CINII, and CINIII: Cervical intraepithelial neoplasia grades I, II, and III, respectively CTL: Cytotoxic T lymphocyte HR-HPV: High-risk human papillomavirus HSIL: High-grade squamous intrae- pithelial lesion LSIL: Low-grade squamous intrae- pithelial lesion NDs: Normal donors NILM: Negative for intraepithelial lesion or malignancy PCR: Polymerase chain reaction PFP: Platelet-free plasma POM-1: Sodium polyoxotungstate TGF-β: Transforming growth factor-β UPLC: Ultra-high-performance liquid chromatography. Ado: Adenosine ADP: Adenosine diphosphate AMP: Adenosine monophosphate APCP: 5′-(α,β-Methylene) diphosphate ARs (A1R, A2AR, A2BR, and A3R): Adenosine receptors ATP: Adenosine triphosphate CC: Cervical cancer CD39: Ectonucleoside triphosphate diphosphohydrolase-1 CD73: 5′-Nucleotidase CINI, CINII, and CINIII: Cervical intraepithelial neoplasia grades I, II, and III, respectively CTL: Cytotoxic T lymphocyte HR-HPV: High-risk human papillomavirus HSIL: High-grade squamous intrae- pithelial lesion LSIL: Low-grade squamous intrae- pithelial lesion NDs: Normal donors NILM: Negative for intraepithelial lesion or malignancy PCR: Polymerase chain reaction PFP: Platelet-free plasma POM-1: Sodium polyoxotungstate TGF-β: Transforming growth factor-β UPLC: Ultra-high-performance liquid chromatography. Acknowledgments the production of TGF-β associated with persistent infection by HR-HPV, which is present in more than 99% of CC cases [1, 50], is a factor that promotes the expression of CD39 and CD73 to favor CC progression through Ado generation. the production of TGF-β associated with persistent infection by HR-HPV, which is present in more than 99% of CC cases [1, 50], is a factor that promotes the expression of CD39 and CD73 to favor CC progression through Ado generation. The present study was carried out with the following funding: Mexican Social Security Institute (Instituto Mexicano del Seguro Social (IMSS)) Grants FIS-G14/1314 and FIS/IMSS/PROT/PRIO/19/114, National Council of Science and Technology (Consejo Nacional de Ciencia y Tecnología (CONACyT)) Grant No. 240635 to AMG and Grant No. SALUD-2016-1-272793 to JJMM, and DGAPA- PAPIIT Grant No. IN225519 to MLMG. We also appreciate the support given to M. en C. Ricardo Muñóz Godínez for Doctorate Scholarship No. 288691 from CONACyT. Finally, the authors thank the “Laboratorio Nacional para Servicios Especializados de Investigación, Desarrollo e Innovación (I+ D+ i) para Farmoquímicos y Biotecnológicos (LAN- SEIDIFarBiotec-CONACyT)” for facilitating the use of the UPLC system. 5. Conclusions This study provides the ﬁrst evidence that the concentrations of the PFP-soluble CD39 and CD73 ectonucleotidases in patients in diﬀerent stages of CC development positively cor- related with disease progression and the capacity to generate Ado from the hydrolysis of ADP and AMP. The greater AMPase activity found in the PFP of patients with HSILs or CC was associated with a high concentration of a highly gly- cosylated 90kD CD73 isoform. The level of TGF-β in the PFP of patients with LSILs, HSILs, or CC was signiﬁcantly higher than that of NDs and showed positive correlations with the levels of CD39 and CD73. These results suggest that 12 Mediators of Inﬂammation 13 [24] S. Morello, M. Capone, C. Sorrentino et al., “Soluble CD73 as biomarker in patients with metastatic melanoma patients treated with nivolumab,” Journal of Translational Medicine, vol. 15, no. 244, pp. 1–9, 2017. [8] S. Robson, J. Sévigny, and H. Zimmermann, “The E-NTPDase family of ectonucleotidases: structure function relationships and pathophysiological signiﬁcance,” Purinergic Signal, vol. 2, no. 2, pp. 409–430, 2006. [25] M. de Lourdes Mora-García, S. López-Cisneros, V. Gutiérrez- Serrano et al., “HPV-16 infection is associated with a high con- tent of CD39 and CD73 ectonucleotidases in cervical samples from patients with CIN-1,” Mediators of Inﬂammation, vol. 2019, 13 pages, 2019. [9] H. Zimmermann, M. Zebisch, and N. Strater, “Cellular func- tion and molecular structure of ecto-nucleotidases,” Purinergic Signal, vol. 8, no. 3, pp. 437–502, 2012. [10] F. Di Virgilio and F. 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https://openalex.org/W3212908986	https://economyandsociety.in.ua/index.php/journal/article/download/715/687	Ukrainian	null	КРИПТОВАЛЮТНІ ВІДНОСИНИ В УКРАЇНІ: ЕКОНОМІКО-ПРАВОВИЙ АНАЛІЗ	Ekonomìka ta suspìlʹstvo	2,021	cc-by	5,294	Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Овсієнко Ольга Вікторівна кандидатка економічних наук, доцентка, Національний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-6467-9396 Kovtun Volodymyr, Ovsiienko Olha Yaroslav the Wise National Law University У статті розглядаються передумови формування та розвитку криптовалютних відносин, стан їх правового регулювання в Україні. Доведено, що правове регулювання криптовалютних відносин в Україні недостатнє, що має негативні економічні наслідки у вигляді шахрайства, недоотримання державним бюджетом коштів. Обґрун- товано шляхи удосконалення нормативно-правового регулювання криптовалютних відносин, а саме: упорядку- вання термінологічної бази; визначення напрямів роботи з криптовлютними активами; встановлення дозволу юридичним особам на роботу з криптовалютою; регламентація оподаткування криптовалютних операцій. Ви- значено позитивні соціально-економічні наслідки унормування криптовалютного обігу – введення криптовалют у господарський обіг, запобігання економічному шахрайству, збільшення надходжень до державного бюджету. Ключові слова: криптовалюта, криптовалютні відносини, цифрова валюта, блокчейн, правове регулю- вання криптовалютних відносин в Україні. у д р у р у д д д д р д у Ключові слова: криптовалюта, криптовалютні відносини, цифрова валюта, блокчейн, правове регулю- вання криптовалютних відносин в Україні. В статье рассматриваются предпосылки формирования и развития криптовалютных отношений, их правового регулирования в Украине. Доказано, что правовое регулирование криптовалютных отношений в Украине недостаточно, следствием чего являются негативные экономические последствия в виде мошен- ничества, недополучение государственным бюджетом средств. Обоснованы пути усовершенствования нор- мативно-правового регулирования криптовалютных отношений, а именно: составление терминологической базы; определение направлений работы с криптовлютными активами; унормирование выдачи разрешений юридическим лицам работать с криптовалютой; регламентация налогообложения криптовалютных опера- ций. Определены положительные социально-экономические последствия унормирования криптовалютного оборота – введение криптовалют в хозяйственный оборот, предотвращение экономического мошенничества, увеличение поступлений в государственный бюджет. у у уд р д Ключевые слова: криптовалюта, криптовалютные отношения, цифровая валюта, блокчейн, правовое регулирование криптовалютных отношений в Украине. The article is devoted to the consideration of the key prerequisites for the formation and development of cryp- tocurrency, the principles and methods of its creation, its economic nature, the state of legal regulation of crypto- currency relations in Ukraine. The relevance of considering this issue arises, since cryptocurrency is becoming a widespread phenomenon in our country and the world as a whole, which is caused by scientific and technical progress, which has become a dotic phenomenon in all spheres of human life. ttps://doi.org/10.32782/2524-0072/2021-31-50 36.743:351.82 КРИПТОВАЛЮТНІ ВІДНОСИНИ В УКРАЇНІ: ЕКОНОМІКО-ПРАВОВИЙ АНАЛІЗ CRYPTOCURRENCY RELATIONS IN UKRAINE: ECONOMIC AND LEGAL ANALYSIS Ковтун Володимир Володимирович бакалаврант, Національний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Овсієнко Ольга Вікторівна кандидатка економічних наук, доцентка, Національний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-6467-9396 DOI: https://doi.org/10.32782/2524-0072/2021-31-50 УДК 336.743:351.82 © Ковтун В.В., Овсієнко О.В. КРИПТОВАЛЮТНІ ВІДНОСИНИ В УКРАЇНІ: ЕКОНОМІКО-ПРАВОВИЙ АНАЛІЗ CRYPTOCURRENCY RELATIONS IN UKRAINE: ECONOMIC AND LEGAL ANALYSIS Ковтун Володимир Володимирович бакалаврант, Національний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Овсієнко Ольга Вікторівна кандидатка економічних наук, доцентка, Національний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-6467-9396 Випуск # 31 / 2021 Випуск # 31 / 2021 ЕКОНОМІКА ТА СУСПІЛЬСТВО Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Ways to improve the legislative regulation of cryptocurrency relations are proposed, since in our country it is imperfect, in particular in civil law (the introduction of cryptocurrency as an object of civil circulation), commercial law (the ability to provide legal entities with the necessary licenses to work with cryptocurrency in the process of commercial and other activities) , criminal law (creation of prerequisites for the introduction of cryptocurrency as an object of special confiscation, and so on), tax law (the problem of taxation of cryptocurrency transactions and the establishment of the required amount of tax duty on them), improvement of the terminological base, a pattern is indicated in determining the directions of work ЕКОНОМІКА © Ковтун В.В., Овсієнко О.В. ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 31 / 2021 with cryptocurrency assets, defined their potential economic consequences for the economy of our state. Conclu- sions are made about the positive features of cryptocurrency, the risks of this economic phenomenon are identified, the possibility of leveling the harmful consequences of risks by regulating these processes at the legislative level is considered. Analyzed the work of famous scientists working with finance, financial law in the field of the essence of cryptocurrency and related processes, their economic and legal nature. The approaches of various scientists re- garding the settlement of cryptocurrency relations in our country were investigated. Conclusions are made about the state of cryptocurrency relations in the country based on statistical data. Through the prism of history, the evolution of the development of legislative thought in Ukraine regarding the normalization of cryptocurrency relations was considered, errors were indicated during the development of these bills. The international experience of different countries in relation to cryptocurrency and its introduction into financial circulation, establishing control over these operations has been investigated. p g Keywords: cryptocurrency, cryptocurrency relations, digital currency, blockchain, legal regulation of cryptocur- rency relations in Ukraine Постановка проблеми. ХХІ століття – період кардинальних змін в усіх сферах еконо- міки. Так, у грошово-кредитній сфері поступо- вого розповсюдження набуває криптовалюта. За даними Р. Родіна, щоденний обіг криптова- люти у світі становить близька чверті трильйону доларів США [1]. Україна також бере активну участь в операціях з криптовалютою. У 2021 р. наша країна увійшла в ТОП-5 Рейтингу впро- вадження криптовалюти (The 2021 Global Crypto Adoption Index) та посіла четверте місце за обсягом операцій з нею [2]. Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Проте правове регулювання цих відносин поки що недостатнє, зокрема не створено чіткого понятійного апа- рату в цій сфері, не унормовано процедури використання криптовалюти як платіжного засобу, не встановлені правила оподатку- вання операцій з криптовалютою тощо. Через це суб’єкти криптовалютних відносин стика- ються з численними ризиками – шахрайством, неможливістю здійснювати трансакції у право- вому полі. Законодавча база криптовалютного обігу потребує удосконалення, що зумовлює необхідність системного економіко-правового аналізу крипто валютних відносин. наукового пошуку у сфері регулювання крип- товалютних відносин, дискусійним питанням є економіко-правовий зміст понять «крип- товалюта» «блокчейн», «майнинг», а також деяких криптовалютних процесів. Недостат- ньо дослідженими залишаються соціально- економічні наслідки різних варіантів унорму- вання криптовалютних відносин. наукового пошуку у сфері регулювання крип- товалютних відносин, дискусійним питанням є економіко-правовий зміст понять «крип- товалюта» «блокчейн», «майнинг», а також деяких криптовалютних процесів. Недостат- ньо дослідженими залишаються соціально- економічні наслідки різних варіантів унорму- вання криптовалютних відносин. Формулювання цілей статті. Метою дослідження є уточнення змісту криптовалют- них відносин, обґрунтування напрямів удоско- налення їх правового регулювання, а також визначення соціально-економічних наслідків конкретних способів унормування криптова- лютних відносин. Виклад основного матеріалу дослід- ження. Криптовалюту часто вважають різно- видом цифрових валют [1; 2; 4, с. 86; 6, с. 54; 12, с. 175–176]. Її можна визначити як різновид цифрової валюти, емісія та облік якої викону- ється децентралізованою платіжною систе- мою повністю в автоматичному режимі (без можливості внутрішнього або зовнішнього адміністрування) [5, с. 25–26; 6, с. 54–55]. Емісія та облік криптовалюти здійснюються на основі криптографічних методів, напри- клад, асиметричному шифруванні та методі захисту Proof-of-Work. Для того, щоб створити таку «монету», необхідно встановити спеці- альне програмне забезпечення. Це програмне забезпечення певний час генерує («майнить») унікальний ключ, складність якого залежить від величини навантаження на мережу та загальної потужності майнерів. Саме склад- ність добування криптовалюти забезпечує її цінність. Результат генерації – криптогра- фічна послідовність, вона і є кінцевою валю- тою. Феноменальність криптовалюти полягає в її протоколі, який автоматизує стимули до вигідної системі поведінки. Звернемо увагу на відмінності між фіатною валютою та криптова- лютою. Фіатна валюта – платіжний засіб, цін- ність якого штучно встановлюється державою. НОМІКА Аналіз останніх досліджень і публіка- цій. Криптовалюта є новим поняттям для еко- номічної науки та права. Дослідження цього феномену розпочалися не так давно. У робо- тах А. Генкіна та А. Михеєва [3], І. Дороніна [4], Л. Островерх та В. Нарогана [5], В. Усо- ського [6] та інших визначається роль крип- товалюти у платіжній системі країни. Г. Гіл- мен (G. Hileman) та М. Рочс (M. Rauchs) [7], А. Кудь, М. Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Кучерявенко, Є. Смичок [8], Р. Фарел (R. Farell) [9], К. Ярова [10] аналізу- ють механізми криптовалютного обігу. Зна- чна увага вітчизняних науковців приділяється правовому регулюванню криптовалют в Укра- їні (Д. Кобильник, А. Бурчак [11], В. Радінська [12], Р. Родін [1], М. Шаренко [13] та інші). ЕКОНОМІКА Виділення невирішених раніше частин загальної проблеми. Попри активізацію ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 31 / 2021 Що важливо, цінність цієї валюти не забез- печується прив'язкою до конкретних активів. Якщо раніше, наприклад, цінність валюти певної країни визначалася її золотим запа- сом, то тепер єдиний гарант вартості – авто- ритет уряду [4, с. 86; 5, с. 27–28; 6, с. 54–55]. Порівняно з фіатною валютою, криптовалюта не є платіжною вимогою до емітента. Фіатна валюта є зобов’язанням центрального банку або комерційних банків [9, с. 9–10]. потрібна згода всіх трьох або довільних двох сторін. Учасники угоди можуть добровільно тимчасово взаємно блокувати свої кошти як заставу. Точніше, криптовалюта буде перера- хована на спеціальний рахунок, для розпоря- дження яким потрібна згода всіх або кількох сторін. При цьому будь-яка сторона самостійно неспроможна виконати будь-яку операцію; – наявність верхньої межі загального обсягу емісії. Проте існують певні види криптовалюти, у яких ця верхня межа відсутня, і можлива як емісія за рахунок накопичень, так і демісія шляхом обов'язкового знищення невеликої фіксованої суми в кожній транзакції. – наявність верхньої межі загального обсягу емісії. Проте існують певні види криптовалюти, у яких ця верхня межа відсутня, і можлива як емісія за рахунок накопичень, так і демісія шляхом обов'язкового знищення невеликої фіксованої суми в кожній транзакції. Некоректним, на нашу думку, є ототож- нення криптовалюти з електронними гро- шима, адже ці поняття діаметрально проти- лежні в дискурсі валютного ринку. Електронні гроші – це різновид грошей чи фінансових зобов'язань, обмін та взаєморозрахунки за якими проводяться за допомогою інфор- маційних технологій (наприклад, гроші, які перебувають на банківських рахунках). Роз- повсюдження електронних грошей навіть уможливило зникнення та нівелювало необ- хідність «живих» банківських відділень. Так, в додатку Universal bank для онлайн-банкінгу «monobank», всі операції здійснюються за допомогою додатку в смартфоні, що є надзви- чайно зручним. До речі, нещодавно керівни- цтво Universal bank заявило, що в «monobank» буде можливо створювати електронні гаманці для зберігання криптовалюти та купувати її через цей додаток. Слід зауважити, що сума грошей, яка перебуває в електронній формі, еквівалентна кількості паперових грошей, які знаходяться в банку, чого не можна сказати про криптовалюту. Захищеність криптовалют зумовлена техно- логією блокчейн, яка забезпечує стабільність криптовалюти. У монографії А. Кудя, М. Кучеря- венка та Є. Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Смичка «Цифрові активи та їх пра- вове регулювання» охарактеризовано головні переваги блокчейну, серед яких [8, с. 11]: Захищеність криптовалют зумовлена техно- логією блокчейн, яка забезпечує стабільність криптовалюти. У монографії А. Кудя, М. Кучеря- венка та Є. Смичка «Цифрові активи та їх пра- вове регулювання» охарактеризовано головні переваги блокчейну, серед яких [8, с. 11]: 1. Блокчейн є розподіленим реєстром і буде функціонувати до останнього активного мережевого вузла. 2. Доступ до історії транзакцій блокчейну мають всі учасники мережі, а повного контр- олю над ним немає ніхто. 3. У мережі блокчейн відсутня ієрархія, тобто серед безлічі мережевих вузлів нема головного. 4. За своєю природою блокчейн здатен поєднати відкритість та захищенність даних користувачів. Високий ступінь надійності забезпечується за допомогою прогресивних методів шифрування. 5. Дані в мережі блокчейн неможливо вида- лити або замінити, бо вони підтверджуються безліччю мережевих вузлів. До основних властивостей криптовалюти можна віднести такі: – відсутність центрального органу управ- ління. Криптовалюта, на відміну від фіатної валюти, не регулюється центральною гро- шово-кредитною установою. Контроль за процесами використання криптовалюти базу- ється на тому, що учасники тримають іден- тичні повні бази даних балансів усіх гаманців, і змінити базу можливо лише за умови повного консенсусу системи. Саме такий механізм збільшує довіру до цього виду віртуальної валюти, оскільки майже унеможливлює про- цес підробки криптовалюти або скасування відповідного коду; 6. Технологія блокчейн забезпечує абсо- лютну прозорість, оскільки доступ до інфор- мації про всі операції відкритий і кожен може перевірити її вірогідність. 7. Мережа блокчейн є «довірчою» систе- мою, бо транзакції здійснюються прямо між її учасниками, автоматично перевіряються та підтверджуються безліччю вузлів мережі й не потребують посередників, що повністю виключає недовіру до однієї організації-посе- редника. Як наслідок, це приводить до зна- чного зменшення вартості транзакції за раху- нок скорочення комісійних витрат, а також до збільшення швидкості транзакцій внаслідок скорочення витрат часу. – у криптовалютах немає примусового повернення платежів, оскільки немає адміні- стрування, кошти не можуть бути примусово заморожені або вилучені без доступу до при- ватного ключа власника. Однак є можли- вості укладання угод за участю посередника, коли для завершення або скасування угоди Водночас особливості криптовалютного обігу зумовлюють численні ризики, а саме: – можливість шахрайських дій. Один з най- успішніших та найбагатших інвесторів у світі ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 31 / 2021 Випуск # 31 / 2021 По-перше, право є фундаментальним каркасом цивілізованого розвитку криптовалютних відно- син. По-друге, правове регулювання активно сприяє створенню та забезпеченню гарантій безпечного використання криптовалюти (дер- жава не може захистити права власників без належної законодавчої бази). Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 По-третє, оподат- кування деяких криптовалютних операцій може значно поповнити державний бюджет. Воррен Баффет висловив таку думку про крип- товалюти: «Дивіться, ось я відриваю ґудзик, і ми використовуємо його, як невеликий токен. Я пропоную вам його за $1000 і сподіваюся, що під кінець дня він виросте до $2000. Люди, звичайно, будуть створювати їх (такі токени) у величезних кількостях. Це інструмент для азартних ігор, з ним пов'язано багато шахрай- ства, і люди втрачали великі гроші» [14]; В Україні були здійснені спроби правового регулювання криптовалютних відносин. Пер- шою ініціативою був проєкт Закону України (далі ЗУ) «Про обіг криптовалюти в Україні». В ньому було визначено зміст основних понять у сфері криптовалютних відносин, запропоно- вані механізми впровадження криптовалюти в обіг, однак законопроєкт було відхилено та від- правлено на доопрацювання. Далі на розгляд Верховній Раді України було подано проєкт ЗУ «Про стимулювання ринку криптовалют та їх похідних в Україні». Він мав на меті регла- ментацію загальних засад функціонування та правового регулювання ринку криптовалюти та похідних, однак також не був ухвалений та відправлений на доопрацювання. Ще однією законодавчою ініціативою був проєкт ЗУ «Про внесення змін до Податкового кодексу України щодо оподаткування операцій з віртуальними активами в Україні». Термінологічна база про- єкту заслуговує на увагу, оскільки було здій- снено спробу хоча б частково заповнити про- галину в законодавстві. Разом з тим, мають місце окремі термінологічні неточності, а саме [1; 8, с. 140–142; 13, с. 50–54]: – складність конвертації у паперову валюту, оскільки цей механізм є витратним та недо- статньо законодавчо врегульованим; – нестабільність курсу криптовалюти по відношенню до звичайної валюти. Це можна спостерігати на прикладі Bitcoin. Курс цього виду криптовалюти є нестабільним, мінли- вим. Він навіть змінюється від постів в соці- альних мережах (від постів Ілона Маска курс як стрімко зростав, так і починав падати), що не можна сказати про фіатну валюту (долари, євро), курс яких все ж можна спрогнозувати з певною похибкою. В Україні криптовалютні відносини активно розвиваються. Наша країна посідає 4-те місце за криптовалютною активністю – інтеграль- ним показником, розрахованим Chainalysis для 154 країн світу (табл. 1). Цей показник визначається на основі таких даних: загаль- ної кількості криптовалют, отриманих країною; активності непрофесійних індивідуальних користувачів криптовалюти (обсяг криптова- лютних операцій у порівнянні з багатством середньої людини в країні); обсягу біржової торгівлі криптовалютою. Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Оскільки суд не зможе без чіткого визначення криптовалюти надати ква- ліфікацію діянням, то є висока вірогідність виправдання обвинуваченого. Щоб розв’язати цю проблему, необхідно прийняти певний «загальний» закон, який буде чітко визначати понятійний апарат криптовалюти та похідних для неї термінів (блок-чейн, майнинг тощо), встановить основні риси криптовалютних від- носин та вирішить інші загально-теоретичні проблеми. Мають бути законодавчо встанов- лені механізми майнингу криптовалюти, меха- нізми отримання права власності на ці активи. лення від оподаткування та відповідальності. Наприклад, у разі відсутності термінологіч- ного визначення, буде набагато простіше в процесі судового розгляду уникнути відпові- дальності, доводячи, що діяння не пов’язані з криптовалютою. Оскільки суд не зможе без чіткого визначення криптовалюти надати ква- ліфікацію діянням, то є висока вірогідність виправдання обвинуваченого. Щоб розв’язати цю проблему, необхідно прийняти певний «загальний» закон, який буде чітко визначати понятійний апарат криптовалюти та похідних для неї термінів (блок-чейн, майнинг тощо), встановить основні риси криптовалютних від- носин та вирішить інші загально-теоретичні проблеми. Мають бути законодавчо встанов- лені механізми майнингу криптовалюти, меха- нізми отримання права власності на ці активи. Проривом у сфері унормування криптова- лютних відносин є прийнятий в другому читанні та в цілому проєкт ЗУ «Про віртуальні активи». Проривом у сфері унормування криптова- лютних відносин є прийнятий в другому читанні та в цілому проєкт ЗУ «Про віртуальні активи». Він врегульовує: права та обов’язки учасників ринку вірту ц у р Він врегульовує: – права та обов’язки учасників ринку вірту- альних активів; – послуги зі зберігання або адміністру- вання віртуальних активів або ключів вірту- альних активів; – послуги з обміну віртуальних активів; – послуги з переказу віртуальних активів; – фінансові послуги, пов’язані з публічною про- позицією та/або продажем віртуальних активів. Центральними органами влади, який фор- мує і реалізує державну політику в сфері обігу віртуальних активів, є Національний банк України та Національна комісія з цінних паперів та фондового ринку. На них покладені завдання, зокрема, здійснення державного регулювання та контролю за обігом вірту- альних активів на території України, забезпе- чення та здійснення контролю за виконанням, в межах компетенції, учасниками ринку вірту- альних активів законодавства у сфері запо- бігання та протидії легалізації (відмиванню) доходів, одержаних злочинним шляхом, фінансуванню тероризму та фінансуванню розповсюдження зброї масового знищення. 2. Внесення змін у Цивільний кодекс України (далі – ЦКУ). У ЦКУ потрібно вказати, що саме можна робити з криптовалютними активами. Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 – при визначенні поняття «віртуальний актив» майже ототожнюються несумісні поняття «криптовалюти» та токен-активів; – при визначенні поняття «віртуальний актив» майже ототожнюються несумісні поняття «криптовалюти» та токен-активів; – визначення поняття «майнинг» не міс- тить вказівки на мету та форму цієї діяльності; – визначення поняття «реєстр» є недостат- ньо конкретним та однозначним, що може при- звести до хибного зіставлення з цим понят- тям великої кількості об’єктів, які не мають до нього жодного відношення. ЕКОНОМІКА Таблиця 1 Криптовалюта у світі Країна Індексний бал Загальний рейтинговий індекс В’єтнам 1 1 Індія 0,37 2 Пакистан 0,36 3 Україна 0,29 4 Кенія 0,28 5 Нігерія 0,26 6 Венесуела 0,25 7 США 0,22 8 Того 0,19 9 Аргентина 0, 19 10 Джерело: складено авторами на основі [2] Необхідність правового регулювання крипто валютних відносин визнається у бага- тьох дослідженнях [8; 10, с. 1119; 11, с. 24; 13]. ЕКОНОМІКА Таблиця 1 Криптовалюта у світі Країна Індексний бал Загальний рейтинговий індекс В’єтнам 1 1 Індія 0,37 2 Пакистан 0,36 3 Україна 0,29 4 Кенія 0,28 5 Нігерія 0,26 6 Венесуела 0,25 7 США 0,22 8 Того 0,19 9 Аргентина 0, 19 10 Джерело: складено авторами на основі [2] Необхідність правового регулювання крипто валютних відносин визнається у бага- тьох дослідженнях [8; 10, с. 1119; 11, с. 24; 13]. – визначення поняття «майнинг» не міс- тить вказівки на мету та форму цієї діяльності; – визначення поняття «майнинг» не міс- тить вказівки на мету та форму цієї діяльності; – визначення поняття «реєстр» є недостат- ньо конкретним та однозначним, що може при- звести до хибного зіставлення з цим понят- тям великої кількості об’єктів, які не мають до нього жодного відношення. Серед інших законодавчих ініціатив, що без- посередньо пов’язані з регулюванням крипто- валютних відносин, можна виділити ухвалений в другому читанні та в цілому проєкт ЗУ «Про платіжні послуги». Відповідно до ст. 3 законо- проєкту грошові кошти існують в Україні у готів- ковій (формі грошових знаків) та безготівковій (формі записів на рахунках) формах. Грошові кошти включають також електронні гроші та цифрові гроші, які існують лише в безготівко- вій формі. Також цей законопроєкт впрова- джує механізм отримання ліцензії від НБУ для ЕКОНОМІКА ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 31 / 2021 випуску цифрових грошей. Однак законопро- єкт не регулює в повному обсязі сам процес використання криптовалюти. лення від оподаткування та відповідальності. Наприклад, у разі відсутності термінологіч- ного визначення, буде набагато простіше в процесі судового розгляду уникнути відпові- дальності, доводячи, що діяння не пов’язані з криптовалютою. Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 У Податковому кодексі Випуск # 31 / 2021 ЕКОНОМІКА ТА СУСПІЛЬСТВО Випуск # 31 / 2021 люти лежить механізм генерації коду, цінність криптовалюти визначається кількістю витра- ченої електроенергії та попиту на цю валюту. В Україні попит на криптовалюту невисокий, тому в нашій країни існує надлишок криптова- люти. Правове регулювання криптовалютних відносин в Україні недостатнє, що має нега- тивні економічні наслідки у вигляді шахрай- ства, недоотримання державним бюджетом коштів. Основними напрямами удосконалення нормативно-правового регулювання криптова- лютних відносин мають стати: упорядкування термінологічної бази; визначення напрямів роботи з криптовлютними активами; встанов- лення дозволу юридичним особам на роботу з криптовалютою; регламентація оподаткування криптовалютних операцій. люти лежить механізм генерації коду, цінність криптовалюти визначається кількістю витра- ченої електроенергії та попиту на цю валюту. В Україні попит на криптовалюту невисокий, тому в нашій країни існує надлишок криптова- люти. Правове регулювання криптовалютних відносин в Україні недостатнє, що має нега- тивні економічні наслідки у вигляді шахрай- ства, недоотримання державним бюджетом коштів. Основними напрямами удосконалення нормативно-правового регулювання криптова- лютних відносин мають стати: упорядкування термінологічної бази; визначення напрямів роботи з криптовлютними активами; встанов- лення дозволу юридичним особам на роботу з криптовалютою; регламентація оподаткування криптовалютних операцій. України необхідно встановити розмір податку, методи оподаткування криптовалютних дохо- дів та операцій. Необхідно запровадити про- зорий механізм ліцензування діяльності з криптовалютою для юридичних осіб. 5. Встановлення кримінальної відпові- дальності за злочинні дії з криптовалютою (незаконний майнинг, шахрайські махінації тощо). Також потрібно визначити статус крип- товалюти в кримінальному праві: визнати її предметом спеціальної конфіскації, описати сферу застосування криптовалюти в коруп- ційних злочинах та інші заходи. Чи можна частково нівелювати ризики крип- товалюти за допомогою права? Однозначно. Правове регулювання зменшує шахрайські ризики, у користувачів з’явиться можливість відстоювати свої права в сфері криптова- лютних відносин. Якщо наша країна визнає криптовалюту на законодавчому рівні, це сприятиме стабілізації курсу по відношенню до фіатних валют, у подальшому спричинить розвиток конвертаційних механізмів. Враховуючи те, що завдяки унормуванню криптовалютних відносин їх спектр може сут- тєво розширитися, подальші дослідження у цій сфері доцільно зосередити на пошуку оптимальних форм та способів інтеграції від- повідних правових норм у чинну систему зако- нодавства, а також на визначені соціально- економічних наслідків нормативно-правового регулювання криптовалюти. Висновки. Можна стверджувати, що крипто- валюта стає поширеним явищем в нашій країні та у світі загалом. В основі створення криптова- Ковтун Володимир Володимирович бакалаврант, ціональний юридичний університет імені Ярослава Мудрого ORCID: https://orcid.org/0000-0002-0660-1320 Суб’єкт правових відносин має розуміти, які у нього будуть можливості в разі декларування криптовалюти: чи можна буде передавати її у спадок, дарувати, розраховуватися, відстою- вати право на володіння в суді тощо. Тому, для початку, потрібно внести зміни до статті 177 ЦКУ, а саме, на нашу думку, викласти її в такій редак- ції: «Об'єктами цивільних прав є речі, у тому числі гроші (в електронному та паперовому вигляді), віртуальні активи, цифрова валюта (криптовалюта та інші) та цінні папери, інше майно, майнові права, результати робіт, послуги, результати інтелектуальної, творчої діяльності, інформація, а також інші матеріальні і нематері- альні блага». Така зміна допоможе ввести крип- товалюту в повноцінний обіг, зробити її повноцін- ним об’єктом цивільних правовідносин. Законопроєкт визначає правовий статус та вимоги до постачальників послуг, пов'язаних з віртуальними активами, через проходження процедур державної реєстрації одного або декількох видів діяльності: – зберігання або адміністрування віртуаль- них активів та ключів віртуальних активів; – зберігання або адміністрування віртуаль- них активів та ключів віртуальних активів; 3. Внесення змін до нормативно-правових актів у сфері господарського та банківського законодавства для встановлення дозволу юридичним особам на роботу з криптовалю- тою. На даний момент жодна фірма, банк або інші організації не мають права працювати з криптовалютою. Зміни в цих галузях необхідні для уможливлення роботи з криптовалютою та визначення дозволених принципів, методів та способів регламентації криптовалютних опе- рацій. Все це сприятиме зменшенню нелегаль- них доходів, які не проходять оподаткування, підвищить темпи розвитку ринку криптова- люти. Також фізичні особи зможуть викорис- товувати криптовалюту як платіжний засіб, а також зберігати її на банківських рахунках. – обмін віртуальних активів; – обмін віртуальних активів; – переказ віртуальних активів; – переказ віртуальних активів; – участь і надання фінансових послуг, пов’язаних із пропозицією емітента та/або продажем віртуальних активів. Однак не про- писана повноцінна процедура, за допомогою якої можна добувати криптовалюту. На нашу думку, основними напрямами удо- сконалення нормативно-правового регулювання криптовалютних відносин мають стати такі: На нашу думку, основними напрямами удо- сконалення нормативно-правового регулювання криптовалютних відносин мають стати такі: 1. Упорядкування термінологічної бази. Потрібно чітко встановити, що таке крипто- валюта, які в неї ознаки тощо. Це необхідно зробити для уникнення колізій в регулюванні, адже без розуміння суті предмету регулю- вання, неможливо розбудувати повноцінну законодавчу базу. Також це унеможливить спекулятивні махінації, спрямовані на ухи- ЕКОНОМІКА 4. Регламентація оподаткування крипто- валютних операцій. СПИСОК ВИКОРИСТАНИХ ДЖЕРЕЛ: 1. Родін Р. Чому Україна легалізує криптовалюту не з того кінця? URL: https://www.epravda.com.ua/rus/ columns/2021/01/26/670382/ 2. The 2021 Global Crypto Adoption Index: Worldwide Adoption Jumps Over 880 % With P2P Platforms Driving Cryptocurrency Usage in Emerging Markets. URL: https://blog.chainalysis.com/reports/2021-global-crypto-adoption-index 2. The 2021 Global Crypto Adoption Index: Worldwide Adoption Jumps Over 880 % With P2P Platforms Driving Cryptocurrency Usage in Emerging Markets. URL: https://blog.chainalysis.com/reports/2021-global-crypto-adoption-index 3. Генкин А.С., Михеев А.А. Блокчейн. Как работает и что ждет нас завтра : монография. Москва : ООО «Альпина Паблишер», 2018. 281 с. І.М. Криптовалюти: соціально-економічні фактори, право та функції держави. Інформація і № 3. С. 85–93. 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The ratio of material and procedural in the regula- tion of financial relations (Chernivtsi, October 4-5th, 2017) (pр. 247–248). Chernivtsi. (in Ukrainian) 14. Kuzyuk, D. (2019) Uorren Baffet: Bitkoin nichem ne otlichaetsya ot morskoj rakushki [Warren Buffett: Bitcoin is no different from a seashell]. Portal 2Bitcoins.ru. Retrieved from: https://2bitcoins.ru/bitkoin-morskaja-rakushka/ (in Russian) КОНОМІКА 14. Kuzyuk, D. (2019) Uorren Baffet: Bitkoin nichem ne otlichaetsya ot morskoj rakushki [Warren Buffett: Bitcoin is no different from a seashell]. Portal 2Bitcoins.ru. Retrieved from: https://2bitcoins.ru/bitkoin-morskaja-rakushka/ (in Russian) 15. Kriptovalyuta v Ukrayini: tehnologiya, rinok, regulyuvannya (2018) Proyekt USAID «Transformaciya finansovogo sektoru» [Cryptocurrency in Ukraine: technology, market, regulation. USAID Financial Sector Transformation Project]. Retrieved from: https://zakon.help/files/article/9634/Cryptocurrency_Paper_Sept2018_ua.pdf (in Ukrainian) 15. Kriptovalyuta v Ukrayini: tehnologiya, rinok, regulyuvannya (2018) Proyekt USAID «Transformaciya finansovogo sektoru» [Cryptocurrency in Ukraine: technology, market, regulation. USAID Financial Sector Transformation Project]. Retrieved from: https://zakon.help/files/article/9634/Cryptocurrency_Paper_Sept2018_ua.pdf (in Ukrainian)
W4386610952.txt	https://www.cambridge.org/core/services/aop-cambridge-core/content/view/65157599555491B7F2EB65311B899F54/S2054425123000444a.pdf/div-class-title-adolescent-pursuit-of-health-information-online-during-the-covid-19-pandemic-the-roles-played-by-ehealth-literacy-and-psychological-distress-div.pdf	en		Review: Adolescent pursuit of health information online during the COVID-19 pandemic: The roles played by eHealth literacy and psychological distress — R0/PR2	null	2,023	cc-by	7,782	Cambridge Prisms: Global Mental Health www.cambridge.org/gmh Adolescent pursuit of health information online during the COVID-19 pandemic: The roles played by eHealth literacy and psychological distress Fong-Ching Chang1 Research Article Cite this article: Chang F-C, Chang C and Tao C-C (2023). Adolescent pursuit of health information online during the COVID-19 pandemic: The roles played by eHealth literacy and psychological distress. Cambridge Prisms: Global Mental Health, 10, e51, 1–10 https://doi.org/10.1017/gmh.2023.44 Received: 28 January 2023 Revised: 31 May 2023 Accepted: 07 August 2023 Keywords: eHealth literacy; online health information seeking; mental health; adolescence; COVID-19 Corresponding author: Fong-Ching Chang; Email: fongchingchang@ntnu.edu.tw , Chingching Chang2 and Chen-Chao Tao3 1 Department of Health Promotion and Health Education, National Taiwan Normal University, Taipei, Taiwan; 2Research Center for Humanities and Social Sciences, Academia Sinica, Taipei, Taiwan and 3Department of Communication and Technology, National Yang Ming Chiao Tung University, Hsinchu, Taiwan Abstract COVID-19 has led to an increase in mental health problems for adolescents. In this study, we examined the factors related to the eHealth literacy of adolescents and how that impacted their pursuit of health information and mental health information online during the COVID-19 pandemic. We analyzed data from the 2020 Taiwan Communication Survey, which involved a total of 1,250 national representative adolescents who completed an online questionnaire. The results showed that two-thirds of adolescents reported searching for health information online, and about half of them reported searching for mental health information online during the COVID-19 pandemic. Multivariate analysis results indicated that adolescents who spent more time learning online, had higher levels of bonding social capital and self-determination, and had higher levels of parental active internet mediation were more likely to have higher levels of eHealth literacy. In addition, multivariate analysis results showed that adolescents who had higher levels of eHealth literacy and had higher depression and anxiety were more likely to seek health information and mental health information online. In conclusion, the levels of eHealth literacy and psychological distress of adolescents played a crucial role in their pursuit of health information and mental health information online during the COVID-19 pandemic. Impact statement Adolescents are showing an increase in vulnerability to mental health illness, and studies have documented an increase in psychological distress among adolescents during the COVID-19 pandemic. Adolescents spent more time online, and their pursuit of mental health information online has facilitated their autonomy to control their help-seeking journey and allow them to connect with others with more privacy and less stigma. The abundance of misinformation online, however, particularly during the COVID-19 outbreak, has enhanced the need for eHealth literacy in seeking health information online, appraising health information, making healthy decisions and implementing protective behaviors. This study examined the role of factors such as self-determination, eHealth literacy and psychological distress in determining adolescents’ pursuit of health / mental health information online during the COVID-19 pandemic. Introduction © The Author(s), 2023. Published by Cambridge University Press. This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http:// creativecommons.org/licenses/by/4.0), which permits unrestricted re-use, distribution and reproduction, provided the original article is properly cited. Adolescents are showing an increase in vulnerability to mental health illness (Gunnell et al., 2018), and studies have documented an increase in psychological distress among adolescents during the COVID-19 pandemic (Nearchou et al., 2020). School closures, social distancing measures and isolation had negatively impacted the mental health of children and adolescents during the COVID-19 pandemic (Meherali et al., 2021). Adolescents tend to be reluctant to seek mental health services in person, but may seek mental health information online due to less stigma and more privacy (Pretorius et al., 2019). Studies have shown that the recent pursuit of mental health information online has been high among adolescents (Park and Kwon, 2018; Rideout et al., 2018), and that individuals with psychological distress and mental health problems were more likely to pursue health information online (Gallagher and Doherty, 2009; Rowlands et al., 2015; Pretorius et al., 2019). A review study found that for young people with a preference for self-reliance, the pursuit of mental health information online is either therapeutic or acts as a gateway to further help (Pretorius et al., 2019). Children and adolescents spend more time online than adults, which is a global phenomenon. The 2020 EU Kids Online survey showed that in many countries the time that children report spending online almost doubled compared with the 2010 findings (Smahel et al., 2020). In particular, during the COVID-19 pandemic adolescents spent more time than ever on the https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press 2 internet, which they used for socializing, entertainment and learning (Ofcom, 2020). The internet is the main source of information for adolescents, while a review study found that the primary purpose for adolescents’ health-related use of the internet is the pursuit of health information (Park and Kwon, 2018). Studies showed that about half of adolescents have reported using the internet to search for health information (Jiménez-Pernett et al., 2010; Gazibara et al., 2020), but many of those adolescents also reported a lack of search skills to find reliable web pages and had difficulties in filtering overabundant content and determining the quality of information (Esmaeilzadeh et al., 2018; Patterson et al., 2019). The COVID-19 pandemic has provoked a greater number of healthcare organizations to provide eHealth resources and to stress the importance of individual eHealth literacy for the use of eHealth services. The abundance of misinformation online, particularly during the COVID-19 outbreak, has enhanced the role of eHealth literacy in appraising online information, making health decisions and implementing protective behaviors (Brørs et al., 2020). eHealth literacy refers to an individual’s ability to seek out, understand, appraise and apply electronic health information to solve health problems (Norman and Skinner, 2006). A study has shown that individuals with higher eHealth literacy were more likely to search for COVID-19 information online and adopt preventive behaviors (Guo et al., 2021). Prior studies also found a positive association between higher levels of eHealth literacy and the pursuit of health information online (Gazibara et al., 2020), healthy lifestyle behaviors in adolescents (Gürkan and Ayar, 2020; Eyimaya et al., 2021) and positive mental health effects (Chen et al., 2020). Most studies assessed eHealth literacy using selfreporting of perceived skills, while few studies have measured performance-based eHealth literacy through the testing of actual performance to determine eHealth literacy levels, such as the completion of computerized simulation tasks. Prior studies showed that perceived and performed eHealth literacy were either moderately (Neter and Brainin, 2017) or weakly correlated (van der Vaart et al., 2011). Some studies have shown a large discrepancy between perceived and performance-based eHealth literacy, which has highlighted the levels of poor critical self-awareness among adolescents (Maitz et al., 2020; McKinnon et al., 2020. During the COVID-19 pandemic, parents and adolescents were living with increased stress (Cluver et al., 2020). Parents have reported increases in the difficulties associated with controlling their children’s screen time and increased levels of concern related to the online risks to children (Ofcom, 2021). When adolescents spend more time using the internet, online risks and psychological distress increase (Deslandes and Coutinho, 2020; Guessoum et al., 2020). Parents play a crucial role in the use of the internet by their children. A review study found that the parent–child relationship, parental mediation practices and parents’ own use of media influences children’s media use, attitudes and effects (Coyne et al., 2017). A study found that parental eHealth literacy, active parental internet mediation and adolescent internet health information literacy all were related to the pursuit of health information online by adolescents (Chang et al., 2015). Self-determination theory emphasizes that the satisfaction of basic psychological needs including competence, autonomy and relatedness were associated with development, health-behavior change and better mental health (Deci and Ryan, 2000). A prior study associated a higher level of self-determination with seeking online health information (Lee and Lin, 2016). Despite studies that have examined the relationships between eHealth literacy and the pursuit of health information online and related factors, most https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press Fong-Ching Chang et al. research has focused on adults. By comparison, only a scant amount of research has explored adolescent pursuit of health information online and examined the influence of self-determination, eHealth literacy, psychological distress and parental mediation. For the present study, we analyzed data from the 2020 Taiwan Communication Survey conducted during the COVID-19 pandemic. Our aims included (1) an assessment of adolescents’ pursuit of health information and health topics online during the COVID-19 pandemic; (2) an examination of the relationships between internet use, selfdetermination and parental internet mediation with adolescents’ eHeatlh literacy; and (3) a further examination into how selfdetermination, eHealth literacy, psychological distress and parental internet mediation are associated with adolescents’ pursuit of health information online (i.e., the pursuit of health/ mental health information online and the number of health topics searched online). Methods Participants This study analyzed data from the 2020 Taiwan Communication Survey (second phase, fourth wave) (Chang, 2022). The cluster sampling method was used, while the six socioeconomic strata from Taiwan townships (Hou et al., 2008) were adopted as a sampling scheme to draw the sample schools. A total of 19 elementary schools, 20 middle schools and 23 high schools were randomly selected from the strata and invited to join the survey. The classes were randomly selected from the sample schools. Teachers gave students consent forms to take home to parents requesting consent for their children to participate in the survey. Students were assured that their information would be protected and anonymous. This study analyzed 1,250 national representative adolescents from 43 middle schools (n = 582) and high schools (n = 668). Adolescents completed an online self-administered questionnaire between November 2020 and January 2021, during the COVID-19 pandemic. Approval was obtained from the Institutional Review Board at Academia Sinica, Taiwan. A self-administered questionnaire was developed based on previous studies (Norman and Skinner, 2006; Williams, 2006; Löwe et al., 2010; Nishimura and Suzuki, 2016; Rideout et al., 2018). A group of experts was invited to assess the content validity of the questionnaire. In addition, a pilot survey was conducted at one middle school and two high schools in order to assess the appropriateness of the survey questions and to evaluate the reliability of the data that the questionnaire would yield. Pursuit of health information online Questions concerning the pursuit of health information online were adapted from a study found in the literature (Rideout et al., 2018). Participants were asked, “Have you used the internet to search the following health topics?” If participants answered that they did not use the internet to search for any health topics, then they were categorized as not seeking health information online. If participants answered that they use the internet to search for health topics (i.e., exercise, physical fitness, nutrition, sexual health, stress management, depression, anxiety, mental health and others), then they were categorized as having sought health information online. In addition, if participants answered that they used the internet to search for any mental health topics (i.e., sexual health, stress management, depression, anxiety and mental health), then they were categorized as having sought mental health information online. Cambridge Prisms: Global Mental Health Internet use time Internet use time was measured using six items. Participants were asked how much time (amount of hours and minutes) they spend on computers, tablets and/or smartphones for learning and recreation, respectively. Sample questions follow: “How long do you surf the internet using a computer (excluding the usage of smartphones and tablets; only including your internet-surfing screen time) for learning and working purposes every day?”; “Excluding your learning and working time online, how long do you surf the internet using a computer for entertainment and leisure purposes every day on average?” The sum of internet use time on a computer, a tablet, or a smartphone for learning and working purposes was considered internet learning time. On the other hand, the sum of time using the internet on a computer, a tablet, or a smartphone for entertainment and leisure purposes was considered internet entertainment and leisure time. eHealth literacy eHealth Literacy (5 items) was measured by adapting a previously developed eHealth literacy scale (Norman and Skinner, 2006) to measure participants’ perceived skills at finding, evaluating and applying electronic health information to health problems. Participants were asked about their opinions and experiences using the internet for health information. Sample statements follow: “I know how to find helpful health resources on the internet”; “I know how to use the internet to answer my health questions”; “I know how to use the health information I find on the internet to help me”; “I can tell high-quality from low-quality health resources on the internet”; “I feel confident in using information from the internet to make health decisions.” The response options were graded on a 5-point Likert-type scale that ranged from strongly disagree (scoring 1) to strongly agree (scoring 5), with higher scores indicating a higher level of eHealth literacy. The Cronbach’s alpha of eHealth literacy was 0.87. Parental internet mediation Parental internet mediation (six items) was measured by adapting scales from prior studies (Nathanson, 1999; Valkenburg et al., 1999). Parental internet mediation included parental restrictive internet mediation (three items) and parental active internet mediation (three items). Participants were asked about their experience regarding their parents’ internet mediation. Parental restrictive internet mediation statements follow: “My parents don’t allow me to visit certain websites”; “My parents set rules regarding when I can use the internet and when I cannot”; “My parents set a limit on how long I use the internet.” Active parental internet mediation statements follow: “My parents encourage me to use the internet”; “My parents discuss internet use experiences with me”; “My parents discuss online stories and events with me.” The response options included never (scoring 1), seldom (scoring 2), sometimes (scoring 3), often (scoring 4), with higher scores indicating a higher level of parental restrictive/active mediation. The Cronbach’s alpha scores for parental restrictive internet mediation and for active parental internet mediation were 0.76 and 0.68, respectively. Bonding social capital Bonding social capital (one item) was measured by adapting an approach from a social capital study (Williams, 2006) to measure participants’ perceived emotional support and access to resources from strong-tie networks. Participants were asked to complete the following statement: “There are several people I trust to help solve my problems.” The response options were graded on a 5-point https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press 3 Likert-type scale that ranged from strongly disagree (scoring 1) to strongly agree (scoring 5), with higher scores indicating a higher level of bonding social capital. Self-determination Self-determination (three items) was measured by factors that included competence, autonomy and relatedness, and was adapted from a study found in the literature (Nishimura and Suzuki, 2016). Participants were asked their opinions and experiences regarding the following statements: “I feel confident that I can do things well”; “I feel I have been doing what really interests me”; “I feel close and connected with other people who are important to me (e.g. family, friends).” The response options were graded on a 5-point Likerttype scale that ranged from strongly disagree (scoring 1) to strongly agree (scoring 5), with higher scores indicating higher levels of selfdetermination (competence, autonomy and relatedness). Depression and anxiety Depression and anxiety (four items) were measured using a scale developed in a previous study (Löwe et al., 2010). Participants were asked “Over the last week, how often have you been bothered by the following problems? (1) Little interest or pleasure in doing things; (2) Feeling down, depressed or hopeless; (3) Feeling nervous, anxious or on edge; (4) Not being able to stop or control worrying.” The response options ranged from 0 (scoring 0) to 7 days (scoring 7), with higher scores indicating a higher level of depression and anxiety. The Cronbach’s alpha of depression and anxiety was 0.90. Physical and emotional exhaustion To gauge physical and emotional exhaustion, two items were adopted from the Copenhagen Burnout Inventory (Kristensen et al., 2005). The questions were “How often are you physically exhausted?” and “How often are you emotionally exhausted (such as feeling helpless or frustrated)?” The response options included never (scoring 1), seldom (scoring 2), sometimes (scoring 3) and often (scoring 4), with higher scores indicating a higher level of physical and emotional exhaustion. Characteristics of the adolescents The characteristics of the adolescents who participated in this study included gender (male or female), age, school type (middle school or high school) and academic performance (very good, good, average, poor, very poor). Statistical analysis SAS was used to perform the statistical analysis. A series of t-tests were conducted to compare adolescents’ eHealth literacy, internet use time and related factors by gender and by school type. Chisquare tests were conducted to compare adolescents’ pursuit of health information and mental health online. In addition, multiple regression was conducted to examine the factors related to adolescents’ eHealth literacy and the number of health topics searched online. Multiple logistic regression was conducted to examine factors related to adolescents’ pursuit of health information online and the factors associated with the pursuit of mental health information online. A 95% confidence interval (95% C.I.) and p value was presented. The outcome variable was the pursuit of health information online including the pursuit of health information online, pursuit of mental health information online and number of health topics searched online. The independent variables included internet use time, eHealth literacy, parental internet 4 Fong-Ching Chang et al. mediation, bonding social capital, self-determination, depression and anxiety, physical and emotional exhaustion. The covariate variables were adolescents’ characteristics including gender, age and academic performance. Results for recreation and 12.5 h per week using the internet for learning. In addition, adolescents mainly accessed the internet via smartphones (22.2 h/week for recreation and 8.3 h/week for learning). By gender, boys spent more time using computers to go online than girls. By school type, high school students spent more time using computers and smartphones to go online than middle school students (Table 1). Internet use by adolescents Of the 1,250 participant students, 655 were boys (52.4%) and 595 were girls (47.6%). The age range of participant students was 12–18 years, while the mean age of middle school students and high school students was 12.9 years, and 16.1 years, respectively. Overall, adolescents spent 29.8 h per week using the internet eHealth literacy of adolescents and related factors The eHealth literacy of the adolescent participants was above average (Mean = 3.69) (Table 1). The participants had higher scores for the searching of health information than for appraising and Table 1. Adolescent’s internet use, eHealth literacy and health status Age Internet learning time (hr/wk) Computer(hr/wk) Tablet(hr/wk) Smartphone(hr/wk) Internet recreation time (hr/wk) Computer(hr/wk) Tablet(hr/wk) Smartphone(hr/wk) Health topic searches EHealth literacy Bonding social capital Competence Autonomy Relatedness Depression and anxiety Total Mean (SD) Girl Mean (SD) Boy Mean (SD) t-test p value Middle school Mean (SD) High school Mean (SD) t-test p value 14.60 14.51 14.68 0.0987 12.91 16.08 <0.0001 (1.80) (1.81) (1.79) (0.84) (0.97) 12.47 12.37 12.57 10.14 14.50 (15.98) (15.93) (16.03) (14.87) (16.63) 3.36 2.67 3.99 (6.97) (5.97) (7.72) 0.79 0.63 0.93 (3.27) (2.83) (3.61) 8.32 9.07 7.64 (12.15) (12.93) (11.36) 29.84 27.65 31.84 (27.83) (25.77) (29.46) 5.98 3.41 8.31 (12.94) (8.55) (15.56) 1.72 1.50 1.92 (6.98) (6.78) (7.16) 22.15 22.74 21.61 (20.79) (20.91) (20.69) 2.21 2.24 2.19 (2.06) (2.08) (2.04) 3.69 3.64 3.73 (0.65) (0.60) (0.69) 3.88 3.90 3.87 (0.90) (0.90) (0.89) 3.47 3.35 3.58 (0.88) (0.84) (0.90) 3.53 3.42 3.62 (0.96) (0.94) (0.97) 4.00 4.04 3.96 (0.87) (0.84) (0.90) 1.56 1.79 1.36 (1.66) (1.73) (1.58) 0.8277 0.0007 0.0936 0.0393 0.0075 <0.000 1 0.2913 0.3355 0.6534 0.0118 0.4889 <0.0001 0.0003 0.0992 <0.0001 2.13 4.43 (5.57) (7.84) 1.04 0.57 (3.90) (2.57) 6.97 9.50 (11.31) (12.72) 24.33 34.65 (27.15) (27.54) 3.80 7.88 (10.38) (14.57) 2.18 1.32 (8.21) (5.67) 18.35 25.46 (20.78) (20.25) 1.76 2.60 (1.84) (2.15) 3.68 3.70 (0.71) (0.59) 3.84 3.91 (0.98) (0.82) 3.47 3.48 (0.86) (0.89) 3.59 3.47 (0.94) (0.97) 3.94 4.04 (0.90) (0.84) 1.30 1.80 (1.56) (1.72) <0.0001 <0.0001 0.0139 0.0002 <0.0001 <0.0001 0.0335 <0.0001 <0.0001 0.6957 0.1787 0.7647 0.0189 0.0393 <0.0001 (Continued) https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press Cambridge Prisms: Global Mental Health 5 Table 1. (Continued) Physical exhaustion Emotional exhaustion Parental restrictive internet mediation Parental active internet mediation Total Mean (SD) Girl Mean (SD) Boy Mean (SD) t-test p value Middle school Mean (SD) High school Mean (SD) t-test p value 2.88 2.96 2.81 0.0010 2.67 3.06 <0.0001 (0.77) (0.75) (0.78) (0.77) (0.72) 2.89 3.02 2.78 (0.82) (0.79) (0.82) 2.13 2.10 2.16 (0.85) (0.82) (0.87) 2.28 2.37 2.18 (0.76) (0.76) (0.76) <0.000 1 0.2171 <0.000 1 2.69 3.07 (0.83) (0.76) 2.50 1.83 (0.86) (0.71) 2.28 2.27 (0.76) (0.77) <0.0001 <0.0001 0.8638 Note: Boy n = 655 girl n = 595, middle school n = 582 high school n = 668. applying the health information they found. By gender, boys had slightly higher levels of eHealth literacy (Mean = 3.73) than girls (Mean = 3.64) (Table 1). Multiple regression results indicated that adolescents who were boys, who spent more time using the internet for learning, who had higher levels of bonding social capital, who higher selfdetermination (competence, relatedness), and who had higher levels of active parental internet mediation were more likely to have higher levels of eHealth literacy (Table 3). Pursuit of health information online by adolescents and related factors The rates of adolescents’ pursuit of health information online are listed in Table 2. Overall, 75.6% of adolescents searched health information online, while 47.7% of adolescents searched mental health information online during the COVID-19 pandemic. The percentage of girls who searched mental health topics online (50.8%) was higher than that for boys (44.9%). The percentage of high school students who searched health topics online (79.5%) was higher than that for middle school students (71.5%). Similarly, the percentage of high school students who searched mental health topics online (60.1%) was higher than that for middle school students (33.5%) (Table 2). Multiple logistic regression results showed that adolescents who were boys, who had higher levels of eHealth literacy, who had higher levels of depression and anxiety, or who had higher levels of parental active internet mediation were more likely to seek health information online. In addition, adolescents who were high school students, who had higher levels of eHealth literacy, who had higher Table 2. Adolescents’ pursuit of health information online and health topics searched Total n (%) Girl n (%) Boy n (%) 305 159 146 (24.4) (26.7) (22.3) Pursuit of health information online No Yes Yes Middle school n (%) High school n (%) 0.0684 945 436 509 (75.6) (73.3) (77.7) Pursuit of mental health information online No Chi-square p value 0.0015 166 (28.5) 416 (71.5) 139 (20.8) 529 (79.2) 0.0379 654 293 361 (52.3) (49.2) (55.11) 596 302 294 (47.7) (50.8) (44.9) 663 272 391 (53.0) (45.7) (59.7) Chi-square p value <0.0001 387 (66.5) 195 (33.5) 267 (40.0) 401 (60.0) Health topics searched Exercise Physical fitness Nutrition 272 96 176 (21.8) (16.1) (26.9) 352 163 189 (28.2) (27.4) (28.9) 315 (54.1) 138 (23.7) 145 (24.9) 348 (52.5) 134 (20.1) 207 (31.0) (Continued) https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press 6 Fong-Ching Chang et al. Table 2. (Continued) Total n (%) Girl n (%) Boy n (%) 163 36 127 (13.0) (6.1) (19.4) (6.4) 110 Sexual health Stress management 388 210 178 (31.0) (35.3) (27.2) 288 182 106 (23.0) (30.6) (16.2) Depression Anxiety 240 147 93 (19.2) (24.7) (14.2) Mental health 398 172 226 (31.8) (26.3) (38.0) Chi-square p value Middle school n (%) 37 (18.9) 83 (14.3) 68 (11.7) 126 (21.7) High school n (%) Chi-square p value 126 (18.9) 278 (41.6) 205 (30.7) 172 (25.8) 272 (40.7) Note: Boy n = 655 girl n = 595, middle school n = 582 high school n = 668. Discussion Table 3. Factors related to adolescents’ eHealth literacy β 95% C.I. p value Intercept 2.06 1.65–2.48 <0.0001 Gender (boy = 1, girl = 0) 0.10 0.03–0.17 0.0040 Age 0.00 0.02–0.02 0.9873 Academic performance 0.01 0.03–0.05 0.7134 Internet learning time 0.01 0.01–0.01 0.0178 Internet recreation time 0.01 0.01–0.01 0.3104 Bonding social capital 0.17 0.13–0.22 <0.0001 Competence 0.10 0.06–0.15 <0.0001 Autonomy 0.01 0.04–0.05 0.7887 Relatedness Parental restrictive internet mediation Parental active internet mediation 0.08 0.01 0.09 0.03–0.12 0.0010 0.05–0.03 0.6695 0.05–0.14 <0.0001 Note: (1) N = 1,236. (2) Multiple regression was conducted. levels of depression and anxiety, and who had higher levels of emotional exhaustion were more likely to seek mental health information online (Table 4). Health topics searched by adolescents and related factors The health topics that adolescents searched included exercise (53.0%), mental health (31.8%), stress (31.0%), nutrition (28.2%), depression (23.0%), physical fitness (21.8%), anxiety (19.2%), and sexual health (13.0%) (Table 2). The average number of health topics adolescents searched was 2.2. High school students searched more health topics (mean = 2.60) than middle school students (1.76) (Table 1). Multiple regression results showed that adolescents who were high school students, who had higher levels of eHealth literacy, who had higher competence, who had higher levels of depression and anxiety, who had higher levels of physical and emotional exhaustion, and who had higher levels of active parental internet mediation were more likely to search more health topics online (Table 5). https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press This study found that two-thirds of adolescents searched health topics online during the COVID-19 pandemic. Prior studies conducted in the United States (Rideout et al., 2018), Serbia (Gazibara et al., 2020), Saudi Arabia (Neumark et al., 2013) and Spain (Jiménez-Pernett et al., 2010) also found that more than half of adolescents reported seeking health information online. In addition, the results found in this study are consistent with prior studies that found adolescents searched a variety of health topics including exercise, fitness, nutrition, and mental and sexual health, with exercise and fitness being the most common health topics searched (Jiménez-Pernett et al., 2010; Neumark et al., 2013; Park and Kwon, 2018; Rideout et al., 2018). These results indicated that online information was an important source for adolescents to obtain health information. However, a study reviewed websites and found that very few webpages were written specifically for adolescents and suggested that governments invest in co-designing excellentquality and more interactive health information online that better targets an adolescent audience (Ruan et al., 2021). In addition, the present study found that half of adolescents searched mental health topics online, and that adolescents who had higher levels of depression and anxiety were more likely to search health information and mental health information online. Prior studies also found that individuals with psychological distress were more likely to engage in seeking help online (Pretorius et al., 2019) and in searching for health information (Gallagher and Doherty, 2009; Rowlands et al., 2015). An Australian study found that young women experiencing “stigmatized” conditions were more likely to search health information online (Rowlands et al., 2015). A review study indicated that the benefits for young people who use online help-seeking searches of mental health included anonymity, immediacy, ease of access, inclusivity, shared experiences and a sense of control over the help-seeking journey (Pretorius et al., 2019). These results were consistent with helpseeking models (Rickwood et al., 2005) that showed that when young people had psychological needs and could easily access online mental health information, they were more willing to seek mental health resources online. Studies have established that young people were open to accessing mental health information online, as well as pursuing mental health support online (Oh et al., 2009; Horgan and Sweeney, 2010). Cambridge Prisms: Global Mental Health 7 Table 4. Factors related to adolescent pursuit of health information online Pursuit of health information online OR 95% C.I. p value Pursuit of mental health information online OR 95% C.I. p value Gender (boy = 1, girl = 0) 1.35 1.01–1.79 0.0395 0.90 0.69–1.18 0.4459 Age 1.11 1.02–1.22 0.0207 1.28 1.18–1.39 <.0001 Academic performance 0.93 0.79–1.10 0.4213 0.97 0.84–1.14 0.7383 Internet learning time 1.00 0.99–1.01 0.4833 1.00 0.99–1.01 0.8523 Internet recreation time 1.00 0.99–1.00 0.4968 1.00 1.00–1.01 0.5161 Bonding social capital 0.84 0.70–1.01 0.0647 0.95 0.80–1.13 0.5600 Competence 1.03 0.84–1.25 0.8098 1.11 0.93–1.33 0.2458 Autonomy 1.09 0.92–1.30 0.3195 1.06 0.90–1.24 0.5057 Relatedness 0.93 0.77–1.13 0.4898 1.07 0.89–1.28 0.4701 eHealth literacy 2.19 1.71–2.79 <.0001 1.38 1.11–1.71 0.0032 Depression and anxiety 1.17 1.04–1.32 0.0111 1.34 1.20–1.49 <.0001 Physical exhaustion 1.13 0.91–1.41 0.2553 1.19 0.98–1.45 0.0866 Emotional exhaustion 1.15 0.92–1.44 0.2089 1.67 1.35–2.06 <.0001 Parental restrictive Internet mediation 1.02 0.84–1.22 0.8774 1.03 0.87–1.22 0.7663 Parental active internet mediation 1.22 1.01–1.48 0.0443 1.12 0.94–1.33 0.2017 Note: (1) N = 1,236. (2) Multiple logistic regression was conducted. (3) Pursuit of health information online: yes n = 931 no n = 305, pursuit of mental health information online: yes n = 596 no n = 640. Table 5. Factors related to the number of health topics searched online β 95% C.I. p value 1.94 5.35–2.55 <0.0001 Gender (boy = 1, girl = 0) 0.09 0.14–0.30 0.4795 Age 0.48 0.08–0.22 <0.0001 0.11 0.23–0.02 0.0991 Internet learning time 0.01 0.01–0.01 0.7758 Internet recreation time 0.01 0.01–0.01 0.9089 0.10 0.25–0.03 0.1283 Intercept Academic performance Bonding social capital Competence Autonomy Relatedness 0.18 0.03–0.32 0.0214 0.01 0.13–0.14 0.9576 0.00 0.14–0.15 0.9383 eHealth literacy 0.52 0.34–0.69 <0.0001 Depression and anxiety 0.28 0.20–0.36 <0.0001 Physical exhaustion 0.17 0.01–0.33 0.0539 Emotional exhaustion 0.26 0.07–0.42 0.0052 Parental restrictive internet mediation 0.00 0.11–0.17 0.6572 Parental active internet mediation 0.18 0.03–0.32 0.0155 Note: (1) N = 1,236. (2) Multiple regression was conducted. Moreover, the results of this study were consistent with those of prior studies (James and Harville II, 2016; Wong and Cheung, 2019; Gazibara et al., 2020) that found individuals with better eHealth literacy were more likely to pursue health information online. Another study also found that the internet skill level of adolescents was associated with their pursuit of health information online https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press (Neumark et al., 2013). Results of the present study have shown, however, that adolescents were not as adept at appraising and applying health information as they are at searching for it. Other studies also found that adolescents seldom evaluated search results, had difficulty in selecting appropriate search strings and also had difficulty determining the quality of the information they acquired, which suggests an overall lack of appraisal strategies (Walraven et al., 2009; Jiménez-Pernett et al., 2010; Esmaeilzadeh et al., 2018; Freeman et al., 2018). In addition, in previous studies, adolescents judged their own eHealth literacy much higher than its actual value, and those studies suggested implementing education to strengthen adolescents’ eHealth and critical literacy (Maitz et al., 2020; McKinnon et al., 2020). At least one study associated exposure to credible sources of health information online with higher eHealth literacy and suggested that credible health information resources online be incorporated into school health education curricula (Ghaddar et al., 2012). Schools could implement eHealth literacy combined with critical media literacy programs to strengthen adolescents’ eHealth literacy competence and enhance their pursuit of health information online. This study positively associated active parental internet mediation and bonding social capita with adolescent eHealth literacy. These results indicated the crucial roles of parents and significant others in supporting and providing resources to help adolescents solve online problems and enhance their eHealth literacy. Prior studies have also related active parental internet mediation to adolescent eHealth literacy (Chang et al., 2015). In addition, previous studies have positively associated individual social capital with health information self-efficacy, the scope of health information sources and intentions to pursue health information (Kim et al., 2015). Social capital has also been shown to have a positive effect on technological literacy (Yang et al., 2012). These results underscore the importance of strengthening individual social capital to enhance eHealth literacy. Governments could implement parental internet mediation and eHealth literacy training to 8 improve eHealth and the online pursuit of health information, which would decrease the digital divide and health inequality among children and adolescents. The results of this study positively associated internet learning time and self-determination factors such as competence and relatedness with higher levels of eHealth literacy among adolescents. Prior studies also positively related self-efficacy to eHealth literacy (Holch and Marwood, 2020; Maitz et al., 2020). These results were consistent with self-determination theory (Deci and Ryan, 2000), which addresses adolescent competence, autonomy and relatedness, and shows these factors to be crucial for developing eHealth literacy and enhancing the pursuit of health information online. Our results showed that boys reported higher levels of eHealth literacy than girls; however, this difference could have been related to their self-determinism, such as when boys perceived higher levels of competence in appraising the information on the internet. In addition, at least one study has applied selfdetermination theory and found that the pursuit of health information online offers individuals greater autonomy, competence and relatedness compared with face-to-face office visits with a physician (Lee and Lin, 2016). Similarly, another study found that individual competence increases with the use of technology, and learning with peers was more engaging when using digital tools to pursue health information to meet health needs (Scott Duncan et al., 2019). These results suggest that eHealth services design could incorporate a self-determination perspective to promote adolescent eHealth use. Limitations This study had some limitations. First, this was a cross-sectional study, which limits the information that can be used to infer causality. Second, this study analyzed the dataset from the 2020 Taiwan Communication Survey, and some variables such as the psychological distress scale had a limited number of items, which could reduce the reliability. Third, eHealth literacy was measured based on the adolescents’ perceptions, and gaps could exist between perceived eHealth literacy and actual capabilities of searching and evaluating health information. Future studies could assess adolescents in an experimental setting to test their capabilities in searching and appraising health information combined with self-report eHealth literacy. Finally, parental internet mediation was measured based on adolescents’ reporting, and future studies could conduct a parent–child dyad study to examine parental influences on adolescents’ eHealth literacy, the pursuit of health information online, and physical and mental health outcomes. Despite these limitations, the present study adds to the limited amount of literature that addresses adolescents’ pursuit of health information online and the roles of self-determination, eHealth literacy, psychological distress and parental internet mediation. Conclusions During the COVID-19 pandemic, adolescents were vulnerable to problems with mental health. The rates of adolescent pursuit of health information / mental health information and health-related topics online during the COVID-19 pandemic were unknown, as were the associations of self-determination, eHealth literacy, psychological distress and parental internet mediation with adolescents’ online health and mental health information seeking. Our results showed that two-thirds of adolescents reported searching for https://doi.org/10.1017/gmh.2023.44 Published online by Cambridge University Press Fong-Ching Chang et al. health information online, and about half of adolescents searched mental health information online during the COVID-19 pandemic. The results of this study revealed the significance of the roles of bonding social capital, self-determination and parental active internet mediation in enhancing adolescents’ eHealth literacy. Adolescents’ levels of competence, eHealth literacy, psychological distress and active parental internet mediation played crucial roles in increasing their pursuit of health and mental health information online. These results implied the need to implement eHealth literacy combined with critical media literacy programs to strengthen adolescents’ eHealth literacy competence. Future research could promote a self-determination perspective to develop eHealth literacy intervention, promote the seeking of online health information and underscore the value of appraisal to adolescents. Open peer review. To view the open peer review materials for this article, please visit http://doi.org/10.1017/gmh.2023.44. Data availability statement. The data that support the findings of this study are openly available in the 2020 Taiwan Communication Survey (Phase Two, Year Four): New Communication Technologies & Life Boundary Expansion (D00216) (Data file). Further information is available from the Survey Research Data Archive, Academia Sinica. https://doi.org/10.6141/TW-SRDA-D00216-2 (Chang, 2022). Acknowledgements. Many thanks go to the participant schools and students. Author contribution. Professor Fong-Ching Chang was responsible for the conception and analysis of the study and for the writing of the manuscript. Professor Chingching Chang was the chair to design the Taiwan Communication Survey and the conception of this work. Professor Chen-Chao Tao was the co-chair to conduct the Taiwan Communication Survey and the conception of this work. All authors contributed to manuscript preparation and approved the final manuscript. Financial support. This research received no specific grant from any funding agency, commercial or not-for-profit sectors. Competing interest. The authors declare no conflicts of interest. Ethics statement. Approval of this research was obtained from the Institutional Review Board at Academia Sinica, Taiwan. References Brørs G, Norman CD and Norekvål TM (2020) Accelerated importance of eHealth literacy in the COVID-19 outbreak and beyond. 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https://openalex.org/W1964873032	https://zenodo.org/records/1690810/files/article.pdf	Javanese	null	Die Pflanzen-Alkaloide	Nature	1,901	public-domain	10	© 1901 Nature Publishing Group © 1901 Nature Publishing Group
https://openalex.org/W4246614120	https://europepmc.org/articles/pmc6657203?pdf=render	English	null	Correction to: Transmission risk beyond the village: entomological and human factors contributing to residual malaria transmission in an area approaching malaria elimination on the Thailand–Myanmar border	Malaria journal	2,019	cc-by	647	Reference 1. Edwards HM, Sriwichai P, Kirabittir K, Prachumsri J, Chavez IF, Hii J. Trans- mission risk beyond the village: entomological and human factors con- tributing to residual malaria transmission in an area approaching malaria elimination on the Thailand–Myanmar border. Malar J. 2019;18:221. https ://doi.org/10.1186/s12936-019-2852-5. “As part of a concurrent longitudinal study in the area from Mahidol University (ICEMR and D43 funded Projects U19AI0819672 and D43TW006571), preva- Correction to: Malar J (2019) 18:221 https://doi.org/10.1186/s12936-019-2852-5 Following publication of the original article [1], the authors advised of two errors present in the article: one concerning two author names and the other missing funding details. The errors are: However, the sentence should read as: However, the sentence should read as: Correction to: Malar J (2019) 18:221 lence by microscopy in the three villages was found to be 0.71%, 0.89% and 0.27% in January, May and November 2016, respectively (Fig. 1); all infections were caused by Plasmodium vivax.” Edwards et al. Malar J (2019) 18:248 https://doi.org/10.1186/s12936-019-2881-0 Edwards et al. Malar J (2019) 18:248 https://doi.org/10.1186/s12936-019-2881-0 Malaria Journal Open Access The authors apologize for these errors. 1. The named authors of ‘Jetsumon Prachumsri’ and ‘Kirakorn Kirabittir’ are incorrect and should read respectively as: Author details 1 Department of Disease Epidemiology, Imperial College London, London, UK. 2 Malaria Consortium Asia, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. 3 Department of Entomol- ogy, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. 4 Mahidol Vivax Research Unit, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. 5 Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University, 420/6 Rajvithi Road, Bangkok 10400, Thailand. ‘Jetsumon Sattabongkot’ and ‘Kirakorn Kiattibutr’ 2. Under the ‘Study area’ sub-section of the ‘Methods’ section, this sentence is missing funding details: The original article can be found online at https://doi.org/10.1186/s1293 6-019-2852-5. The original article can be found online at https://doi.org/10.1186/s1293 6-019-2852-5. “As part of a concurrent longitudinal study in the area, prevalence by microscopy in the three villages was found to be 0.71%, 0.89% and 0.27% in January, May and November 2016, respectively (Fig. 1); all infections were caused by Plasmodium vivax.” Correction to: Transmission risk beyond the village: entomological and human factors contributing to residual malaria transmission in an area approaching malaria elimination on the Thailand–Myanmar border Hannah M. Edwards1,2* , Patchara Sriwichai3, Kirakorn Kirabittir4, Jetsumon Sattabongkot4, Irwin F. Chavez5 and Jeffrey Hii1 Publisher’s Note S i N i © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Correspondence: hannah.edwards11@imperial.ac.uk 1 Department of Disease Epidemiology, Imperial College London, London, UK Full list of author information is available at the end of the article Correspondence: hannah.edwards11@imperial.ac.uk *Correspondence: hannah.edwards11@imperial.ac.uk 1 Department of Disease Epidemiology, Imperial College London, London UK Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. , Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
https://openalex.org/W4389297482	https://jsd.sdevelopment4.com/index.php/jsd/article/download/155/157	Arabic	null	The Problem of Terminology and its Significance in Arabic Criticism	Majalat altatwir aleilmii	2,023	cc-by	9,983	:اتريخ النشر01 / 12 / 2023 :اتريخ االستالم04 / 07 / 2023 ملخص: كثر اجلدل يف الساحة العربية فهزّ حياة النقد العريب ،وهذا ما جعل القارئ العريب يعيش يف اضطراب مستمر بسبب اجلدل القائم بني الرتاث واملعاصرة وال سيما بعد مناداة أنصار املعاصرة بتحديث العقل العريب وقطع الصلة مع الرتاث وذلك النبهارهم ابحلدا ثة الغربية . فيما متسك آخرون ابلرتاث العر يب مع حماوله إلحيائه وتوظيفه يف أعماهلم الفكرية واألدبية . ويف خضم هذا الصراع الفكري الذي ساد مضمار النقد العريب املعاصر أصبح الواقع يعيش أزمة حقيقية متثلت أبرز مظاهرها يف فقدان النقد خصوصيته وحىت هويته الفكرية ، وكان ذلك نتيجة للمصطلحات الوافدة من الثقافة الغربية إىل الثقافة العربية جرّاء املثاقفة ،إال أن هذا االنتقال كان له أثر سليب يف املمارسة النقدية ،حيث انتقلت هذه املصطلحات حمملة ومشحونة حبمولة مفاهيمية كبرية على مجيع املستوايت .كما أنّ هذه املصطلحات الوافدة بقيت تدور يف فلك مل يستقم بعد . وظل النقد ا لعريب يتخبط بني غياب صيغة مصطلحية موحدة من جهة ،وبني ترمجة هذه املصطلحات أو نقلها من الدراسات الغربية من جهة أخرى .مما ينعكس سلبا على كفاية املصطلح اإلجرائية ، ودوره الفعال . يف توحيد املعلومات وتيسري تداوهلا فما داللة املصطلح يف الساحة النقدية العربية ؟ و ما السبيل إىل ضبطه وتوحيده ؟ وسنحاول يف ورقتنا البحثية هذه– إبذن هللا- الوقوف على واقع املصطلح النقدي العريب وإشكاالته من خالل العناصر :اآلتية ماهية املصطلح النقدي وإشكاالته ،واآلليات املعتمدة يف صياغة املصطلح النقدي ،وجتربة بعض النقاد العرب يف جمال املصطلح النقدي ، و احللول املقرتحة الحتواء إشكالية املصطلح .النقدي : الكلمات املفتاحية املصطلح– األدب– النقد- املناهج– . احلداثة : الكلمات املفتاحية املصطلح– األدب– النقد- املناهج– . احلداثة اجمللد 4 / العـــدد : 16 ( 2023 ) ، Volume 4, Issue 16 ص64 - 80 جملـة التطوير العلمي للدراسات والبحوث Journal of Scientific Development for Studies and Research (JSD) P- ISSN 2709-1635 E-ISSN 2958-7328 إشكالية املصطلح وداللته يف النقد العريب .ط. د أمهين نيشد جامعة أمحد بن بلة1 – وهران (اجلزائر) The Problem of Terminology and its Significance in Arabic Criticism NICHED M'HENNI 710X - 2199 - 0004 - https://orcid.org/0009 Ahmed Ben Bella University 1 - Oran (Algeria), mhennini9688@gmail.com ااااا اجمللد 4 / العـــدد : 16 ( 2023 ) ، Volume 4, Issue 16 ص64 - 80 اجمللد 4 / العـــدد : 16 ( 2023 ) ، Volume 4, Issue 16 ص64 - 80 إشكالية املصطلح وداللته يف النقد العريب .ط. د أمهين نيشد جامعة أمحد بن بلة1 – وهران (اجلزائر) مج The Problem of Terminology and its Significance in Arabic Criticism NICHED M'HENNI 710X - 2199 - 0004 - https://orcid.org/0009 Ahmed Ben Bella University 1 - Oran (Algeria), mhennini9688@gmail.com :اتريخ االستالم04 / 07 / 2023 :اتريخ القبول22 / 08 / 2023 :اتريخ النشر01 / 12 / 2023 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 intellectual conflict that dominated the field of contemporary Arab criticism, reality is experiencing a genuine crisis, manifested in the loss of the distinctiveness and intellectual identity of criticism. This was a result of the influx of Western cultural terms into Arab culture through the process of acculturation. However, this transition had a negative impact on the practice of criticism, as these imported terms carried significant conceptual baggage at all levels. Moreover, these imported terms remained in a state of flux and the Arab criticism continued to struggle between the absence of a unified terminological formula and the translation or adaptation of these terms from Western studies. This has had a negative effect on the effectiveness of procedural terminology and its active role in unifying and facilitating the circulation of information. What is the significance of terminology in the Arab critical arena? And how can it be regulated and unified? In this research paper, we will attempt, God willing, to examine the reality of Arab critical terminology and its challenges through the following elements: 1. The nature of critical terminology and its challenges. 2. The mechanisms used in formulating critical terminology. 3. The experiences of some Arab critics in the field of critical terminology. 4. Proposed solutions to address the problematic nature of critical terminology. intellectual conflict that dominated the field of contemporary Arab criticism, reality is experiencing a genuine crisis, manifested in the loss of the distinctiveness and intellectual identity of criticism. This was a result of the influx of Western cultural terms into Arab culture through the process of acculturation. However, this transition had a negative impact on the practice of criticism, as these imported terms carried significant conceptual baggage at all levels. Moreover, these imported terms remained in a state of flux and the Arab criticism continued to struggle between the absence of a unified terminological formula and the translation or adaptation of these terms from Western studies. This has had a negative effect on the effectiveness of procedural terminology and its active role in unifying and facilitating the circulation of information. What is the significance of terminology in the Arab critical arena? And how can it be regulated and unified? In this research paper, we will attempt, God willing, to examine the reality of Arab critical terminology and its challenges through the following elements: 1. The nature of critical terminology and its challenges. 2. إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 The mechanisms used in formulating critical terminology. 3. The experiences of some Arab critics in the field of critical terminology. 4. Proposed solutions to address the problematic nature of critical terminology. eywords: terminology, literature, criticism, methodologies, modernity. Abstract: The controversy in the Arab arena has stirred up the world of Arab criticism causing Arab readers to live in constant turmoil due to the ongoing debate between heritage and modernity, particularly after advocates of modernity called for updating the Arab mind and severing ties with tradition, captivated by Western modernity. On the other hand, others have clung to Arab heritage, attempting to revive it and employ it in their intellectual and literary works. Amidst this 64 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 1 : .املصطلح لغة واصطالحا 1 : .املصطلح لغة واصطالحا ا مص ح وص يقول اجلاجظ :"لكل صناعة ألفاظ ،ومن البديهي أال تفهم آاثر أولئك القوم أو تلك الصناعة إال مبعرفة تلك " األلفاظ ،(اجلاحظ1998 ، صفحة136 ) من هذا املنطلق فإن اجلاحظ يريد ابأللفاظ يف كالمه هذا األسامي واملصطلحات واملفاهيم "فمفاهيم العلوم مصطلحاهتا ،ومصطلحات العلوم مثارها القصوى فهي جممع حقائقها املعرفية وعنوان ما يتميز به كل واحد منها عما سواه ،وليس يف مسلك يتوسّل به اإلنسان على منطق العلم غ ري " ألفاظه االصطالحية ،(عزام2010 ، صفحة06 ) كما أحسّ علماء العرب منذ القدمي أبمهية املصطلح وضرورة االتفاق على مفاهيم واصطالحات معينة بدقة مدركني بذلك العالقة القوية اليت تربط املصطلحات ابلعلوم املختلفة وانطالقا من إدراكهم هذا راحوا يؤلّفون الكتب ويضعون القواميس واملعاجم اليت تعىن ابملصطلحات العلمية املختلفة ،فتوفّر لنا من جهودهم يف ذلك كتب نفيسة أبرزها :كتاب مفاتيح العلوم للخوارزمي (ت387ه) ،وكتاب التعريفات للشريف اجلرجاين(ت816 ه ) وال ننسى جهود آخرين يف هذا الباب كالرماين(ت386ه ) .وقد اتّفق العلماء على أنه ال داللة للمصطلح إال داخل عرفه اخلاص وال ق يمة له خارجه ،ألنه يصبح حينئذ وح دة من وحدات العرف العام واملفهوم الشائع ،(وغليسي2008 ، صفحة18 ) . وبذلك جند أن داللة امل صطلح يف الطرح القدمي هو يف جوهره مواضعة منبثقة عن طائفة متخصّصة يف صناعة من الصناعات ،وفن من فنون املعرفة عن طريق وضع اللفظ إبزاء املعىن واملواضعة ال تكون عندهم إال إبمجاع . واعتبارا من أنّ أهل الصناعات والعلوم إذا توصّلوا إىل بعض التصورات كان لزاما عليهم وضع اصطالحات هلا على تعبري ابن جين ،(مطلوب1989 ، صفحة140 ) .والذي ميثل لذلك ما جنده من اخرتا عات النحاة ملفاهيم من األمساء مثل :الفاعل ،املفعول به التميي ز احلال ،الصفة ،النعت ...فهذه األمساء يف تصور القدماء إّنّا بياانت لفظية حتمل . :مقدمة يكتسي املصطلح أمهية كبرية يف كل حقل من احلقول املعرفية ،وذلك ابعتباره الركيزة األساسية اليت يبىن عليها العلم .كما يعمل املصطلح على تكوين مفاهيم موح د ة ومرتابطة لدى الدارسني يف أي اختصاص إذ يعد أجبدية للتواصل بني أهل االختصاص الواحد ،إال أنه ويف اآلونة األخرية ونتيجة ملا أفرزته ظاهرة املثاقفة والرتمجة واالطالع على الدراسات الغربية قد خلق فوضى مصطلحية على مستوى الساحة العربية يف جممل الدراسات وخنصّ ابلذك ر الساحة النقدية العربية .ومن بني النماذج املصطلحية اليت أاثرت اجلدل ، بني الباحثني املصطلح النقدي حب يث شغل هذا األخري جزءا غري يسري من الدراسة ،وذلك ألنه شكل معظلة لدى القارئ العريب ،فتناوله أهل االختصاص ابلبحث يف إشكاالته ،ولعل أكرب إشكاالته تتمثل يف غياب التنسيق والتوفيق بني الباحثني والنقاد إضافة إىل اعتمادهم مفهومات أوروبية ال تتخذ ترمجتها صيغة هنائي ة ،وكذا صعوبة ترمجة املصطلح النقدي من لغة املصدر إىل لغة اهلدف ،ومن أهم اإلشكاالت أيضا جلوء الباحثني إىل االشتقاق والتعريب والنحت وهنا يدخل احلس اللغوي ّوالذوق الفردي واملعرفة ابللغات إضافة إىل ثقافة املرتجم ،وتعد د املقابل يف العربية للمصطلح الواحد من اللغات األجنبية ،ولعل أهم ما ضاعف املشكلة وخلق الفوضى على مستوى الساحة النقدية العربية هو تعدّد املدارس النقدية 65 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 واختالف املناهج .ومن هنا تراءت لنا اإلشكاالت التالية :ما املصطلح ؟وما إشكاالته ؟ وما الوسائل املعتمدة يف صياغة املصطلح النقدي ؟وما السبيل إىل ضبط إشكالية املصطلح يف النقد العريب ؟ ولدراسة موضوع البحث مت اعتماد املنهج الوصفي التحليلي ؛إذ متثل الوصف يف رصد املفاهيم اللغوية واالصطالحية ،أما التحليل فتمثل يف البحث يف إشكالية املصطلح النقدي وطرق صياغته الداللية ،ألن املصطلح مرتبط بتحقيق الداللة . . والداللة قد تكون مباشرة أو رمزية وللسياق دور يف حتقيق هذه الداللة ما : أ.ماهية املصطلح وإشكاالته : أ.ماهية املصطلح وإشكاالته إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 من الفخر الرازي ،وابن حزم عندما يقول هذا األخري :"ال بد ألهل كل علم وأهل كل صناعة من ألفاظ خيتصون " هبا للتعبري عن مرادهم وليختصروا هبا معاين كثرية ،(مطلوب1989 ، صفحة9) وعليه فإنه ال ميكن فهم أي حقل من حقول العلم واملعرفة ما مل يتم التعرّف على مصطلحاته واستيعاب مدلوالهتا ،األمر الذ ي جعلهم ميكنون لظهور املصطلحات ، ويعملون على ضبط وبلورة املفاهيم خدمة للعلوم والفكر واحلياة .يقول أمحد مطلوب :"إن املصطلح "مهم يف حتصيل العلوم ،ألنه حيدد قصد الباحث أو املتحدّث ،وكان السلف يعنون به كثريا وإن هذا التنبه من املفكرين العرب القدامى ألمهية املصطلح ساهم بشكل كبري يف ضبطه ورسم حدوده كما نتلمّس ذلك مع اجلاحظ وهو يتحدّث عن مصطلحات املتكلمني ،أو أمحد بن فارس يف تفريقاته بني املفهوم اللغوي واملفهوم االصطالحي . لأللفاظ 1 : .املصطلح لغة واصطالحا تصوّرات ومفاهيم خاصة بصناعة من الصناعات وأييت ضمن هذا السياق ما أبدعه اخلليل بن أمحد يف لغة العروض من أمساء ومفاهيم كالطويل والبسيط واملديد ...كما ذكر األواتد واألسباب و.اخلرم والعلل ،(اجلاحظ1998 ، صفحة140 ) ويذهب يف هذا املنحى كال يقول اجلاجظ :"لكل صناعة ألفاظ ،ومن البديهي أال تفهم آاثر أولئك القوم أو تلك الصناعة إال مبعرفة تلك " األلفاظ ،(اجلاحظ1998 ، صفحة136 ) من هذا املنطلق فإن اجلاحظ يريد ابأللفاظ يف كالمه هذا األسامي واملصطلحات واملفاهيم "فمفاهيم العلوم مصطلحاهتا ،ومصطلحات العلوم مثارها القصوى فهي جممع حقائقها املعرفية وعنوان ما يتميز به كل واحد منها عما سواه ،وليس يف مسلك يتوسّل به اإلنسان على منطق العلم غ ري " ألفاظه االصطالحية ،(عزام2010 ، صفحة06 ) كما أحسّ علماء العرب منذ القدمي أبمهية املصطلح وضرورة االتفاق على مفاهيم واصطالحات معينة بدقة مدركني بذلك العالقة القوية اليت تربط املصطلحات ابلعلوم املختلفة وانطالقا من إدراكهم هذا راحوا يؤلّفون الكتب ويضعون القواميس واملعاجم اليت تعىن ابملصطلحات العلمية املختلفة ،فتوفّر لنا من جهودهم يف ذلك كتب نفيسة أبرزها :كتاب مفاتيح العلوم للخوارزمي (ت387ه) ،وكتاب التعريفات للشريف اجلرجاين(ت816 ه ) وال ننسى جهود آخرين يف هذا الباب كالرماين(ت386ه ) .وقد اتّفق العلماء على أنه ال داللة للمصطلح إال داخل عرفه اخلاص وال ق يمة له خارجه ،ألنه يصبح حينئذ وح دة من وحدات العرف العام واملفهوم الشائع ،(وغليسي2008 ، صفحة18 ) . وبذلك جند أن داللة امل صطلح يف الطرح القدمي هو يف جوهره مواضعة منبثقة عن طائفة متخصّصة يف صناعة من الصناعات ،وفن من فنون املعرفة عن طريق وضع اللفظ إبزاء املعىن واملواضعة ال تكون عندهم إال إبمجاع . واعتبارا من أنّ أهل الصناعات والعلوم إذا توصّلوا إىل بعض التصورات كان لزاما عليهم وضع اصطالحات هلا على تعبري ابن جين ،(مطلوب1989 ، صفحة140 ) .والذي ميثل لذلك ما جنده من اخرتا عات النحاة ملفاهيم من األمساء مثل :الفاعل ،املفعول به التميي ز احلال ،الصفة ،النعت ...فهذه األمساء يف تصور القدماء إّنّا بياانت لفظية حتمل . تصوّرات ومفاهيم خاصة بصناعة من الصناعات وأييت ضمن هذا السياق ما أبدعه اخلليل بن أمحد يف لغة العروض من أمساء ومفاهيم كالطويل والبسيط واملديد ...كما ذكر األواتد واألسباب و.اخلرم والعلل ،(اجلاحظ1998 ، صفحة140 ) ويذهب يف هذا املنحى كال 66 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 :املصطلح لغة يفهم من قويل اجلرجاين أن املصطلح هو انتقال داللة اللفظة من معىن لغوي سابق (قدمي) إىل معىن جديد مع وجود صلة بني املعنيني القدمي .واحلديث منمنح ويرى عبد السالم املسدي :ّأبن "املصطلحات هي جمموعة األلفاظ اليت يصطلح هبا أهل علم من العلوم على متصوراهتم الذهنية اخلاصة ابحلقل املعريف الذي يشتغلون فيه، وينهظون أبعبائه، وأيمتنهم الناس عليه، وال حيق ألحد أن يتداوهلا مبجرد إضمار النية أبهنا مصطلحات يف ذلك الفن إال إذا طابق بني ما ينشده من داللة هلا وما حدّده "أهل ذلك االختصاص هلا من مقاصد تطابقا اتما ،(املسدي2004 ، صفحة146 ) . ويرى عبد السالم املسدي :ّأبن "املصطلحات هي جمموعة األلفاظ اليت يصطلح هبا أهل علم من العلوم على متصوراهتم الذهنية اخلاصة ابحلقل املعريف الذي يشتغلون فيه، وينهظون أبعبائه، وأيمتنهم الناس عليه، وال حيق ألحد أن يتداوهلا مبجرد إضمار النية أبهنا مصطلحات يف ذلك الفن إال إذا طابق بني ما ينشده من داللة هلا وما حدّده "أهل ذلك االختصاص هلا من مقاصد تطابقا اتما ،(املسدي2004 ، صفحة146 ) . وقد أورد أمحد بوحسون تعريفا للمصطلح، يقول: "املصطل ح لفظ خاص يستعمل يف حقل من حقول املعرفة أو جمموعة من االكلمات تتجاوز داللتها اللفظية واملعجمية إىل أتطري تصورا ت فكرية، وتسميتها يف إطار معني وتقوى على " تشخيص وضبط املفاهيم اليت تنتجها ممارسة ما يف حلظات معينة الغريب ،(بوحسون1989 ) يتضح مما سبق أن املصطلح لفظ خاص يستعمل يف حقل أو اختصاص معني ويتم إمجاع أهل العلم . واالختصاص ليدل على معىن مقصود يتبادر إىل الذهن عند إطالق ذلك اللفظ 2 : .إشكالية املصطلح النقدي ا نه صا ل يم () وقد أورد أمحد بوحسون تعريفا للمصطلح، يقول: "املصطل ح لفظ خاص يستعمل يف حقل من حقول املعرفة أو جمموعة من االكلمات تتجاوز داللتها اللفظية واملعجمية إىل أتطري تصورا ت فكرية، وتسميتها يف إطار معني وتقوى على " تشخيص وضبط املفاهيم اليت تنتجها ممارسة ما يف حلظات معينة الغريب ،(بوحسون1989 ) يتضح مما سبق أن املصطلح لفظ خاص يستعمل يف حقل أو اختصاص معني ويتم إمجاع أهل العلم . واالختصاص ليدل على معىن مقصود يتبادر إىل الذهن عند إطالق ذلك اللفظ م يعيش الواقع النقدي العريب اضطرااب مستمر ا بسبب اجلدل القائم بني الرت اث واملعاصرة واملتجسد أساسا يف ذلك االستقطاب بني أنصار املعاصرة الداعني إىل حتديث العقل العريب وقطع الصلة مع الرتاث من جهة .وبني طرف آخر يدعو إىل ال تمسك ابلرتاث العريب مع حماوله إلحيائه وتوظيفه يف أعماهلم الفكرية واألدبية . :املصطلح لغة م من خالل االطالع على املعاجم اللغوية القدمية واحلديثة تبني أنّ كلمة "مصطلح"مأخوذة من املادة اللغوية "صلح" الدالة على صالح الشيء وصلوحه ،أي أنه انفع ،إذ جند يف املعجم الوسيط :صلح ،صالحا ،وصلوحا : : زال عنه الفساد .واصطالح القوم زال ... مابينهم من خالف واتفقوا م من خالل االطالع على املعاجم اللغوية القدمية واحلديثة تبني أنّ كلمة "مصطلح"مأخوذة من املادة اللغوية "صلح" الدالة على صالح الشيء وصلوحه ،أي أنه انفع ،إذ جند يف املعجم الوسيط :صلح ،صالحا ،وصلوحا : : زال عنه الفساد .واصطالح القوم زال ... مابينهم من خالف واتفقوا واالصطالح مصدر اصطلح وهو اتفاق طائفة على شيء خمصوص ،ولكل علم اصطالحاته ،(العربية 2004 ، صفحة520 ) ،(مطلوب1989 ، صفحة9 ) .أما يف القاموس امل نجد فاالصطالح هو : العرف اخلاص أي اتّفاق طائف ة خمصوصة من القوم على وضع الشيء أو الكلمة ، (املنجد يف اللغة العربية1989 ، صفحة432 ) وأورد ابن منظور أن الصالح ضدّ الفساد .أي:اصطلحوا وصاحلوا واصّلحوا ،وتصّاحلوا مع تشديد الصاد مث قلبوا التاء صادا مع إدغامها يف الصاد مبعىن واحد ،واملصلحة ضد املفسدة (منظور، صفحة2480 ). ويف أساس البالغة مادة صلح يقال صلح فالن بعد الفساد وصاحل العدو إذا وقع بينهما الصلح واملصاحلة على كذا وتصاحلا عليه واصطلحا فهم لنا صلح أي متصاحلون ،(الزخمشري1998 ، صفحة554 ) وهذا املفهوم هو الوارد يف أكثر املعاجم . العربية وأن االستقصاء املعجمي ملعاين اجلذر اللغوي "ص.ل.ح"مل خيرج عن معىن الصلح واالتفاق والتواضع : املصطلح اصطالحا لقد أمجعت بعض اآلراء على أن بوادر لفظة "مصطلح" ظهرت مع اجلاحظ يف كتابه البيان والتبيني ،حني أشاد خبطب املتكلمني العظماء ،وأبهنم خت ريّوا تلك األلفاظ لتلك املعاين ،(اجلاحظ1998 ، صفحة139 ) بلفظ آخر مناسبة الدال للمدلول .أما الشريف اجلرجاين فيعرف املصطلح يف كتابه "التعريفات" أبنه "عبارة عن اتفاق قوم على "تسمية الشيء ابسم ما ينقل عن موضعه األول. (اجلرجاين، صفحة22 ) كما يعرفه أيضا فيقول: "أبن ه إخراج اللفظ من معىن لغوي إىل آخر ملناسبة بينهما وقيل االصطالح اتفاق طائفة على وضع اللفظ إبزاء املعىن، وقيل االصطالح 67 67 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إخراج الشيء عن معىن لغوي إىل معىن آخر لبيان املراد، وقيل "االصطالح لفظ معني بني قوم معينني ،(اجلرجاين صفحة22 ) . :املصطلح لغة ويف خضم هذا الصراع الفكري أصبح الواقع النقدي يعيش أزمة حقيقية متثلت أبرز مظاهرها يف فقدان النقد العريب خصوصيته وحىت هويته الفكرية نتيجة ما وفد إىل الثقافة العربية من مصطلحات من الثقافة الغربية جرّاء املثاقفة ،وقد كان هلذا االنتقال أثر سليب يف املمارسة النقدية ،حيث انتقل ت هذه املصطلحات حمملة ومشحونة حب مولة مفاهيمية كبرية على مجيع املستوايت .ومن أمثلة املصطلحات الوافدة من الدراسات الغربية إىل الساحة النقدية العربية : اللسان يات التفكيكية ،التأويل ،التناص اإلنتاجية ،فضاء ...إال أن هذه املصطلحات الوافدة بقيت تدور يف فلك مل يستقم بعد وظل الصراع قائما بني النقاد العرب حول حتديد الصيغة املصطلحية املناسبة للمصطلح العريب مع احملافظة على داللتها .ومبا أنّ النقد العريب شهد تداخال يف كيفية صياغة املصطلحات "منذ قدوم النظرية اللسا نية العربية وابلضبط بعد أن استلهمت احلداثة العربية أدواهتا اإلجرائية من املنجز الغريب ،(بوخامت2005 ، صفحة22 ) .فلقد أصبح نشاط 68 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 الباحثني العرب يف هذا النوع من الدراسات "ضراب من الفوضى الثقافية ،فقد حاول بعضهم التأصيل ملا يرنو به " ابلعودة إىل الرتاث العريب إلبراز جوانبه احلداثية ،(محودة1998 ، صفحة64 ) . كما أنّ النقد العريب أصبح يتخبط بني غياب صيغة مصطلحية موحدة من جهة ،وبني ترمجة هذه املصطلحات أو نقلها من الدراسات الغربية من جهة أخرى .حبيث أصبح النقد العريب املعاصر خيضع لصراع املفاهيم ،وكذلك ، االستهالك االصطالحي القادم من الغرب ح يث ينقل الباحثون العرب تلك املصطلحات يف عزلة عن خلفياهتا الفكرية والفلسفية ،حيث تفرغ من داللتها وتفقد القدرة على حتديد املعىن املناسب ،أما إذا نقلت هذه املصطلحات خبلفياهتا الفلسفية أدّت إىل الفوضى واالضطراب "إذ أن القيم املعرفية القادمة من املصطلح ختتلف ب ل تتعارض "أحياان مع القيم املعرفية اليت طورها الفكر العريب املختلف ،(بوساحة2005 ، صفحة63 ) . كما جند الكثري من الباحثني يقومون بوضع مصطلحات فردية تتسم ابلفوضوية، وهبذا يفقد املصطلح محولته الداللية املوضوعية املرتبطة مبرجعية حمددة ليستبدهلا أبخرايت متعددة بتعدّد واضعيها واختالف مستوايهتم مما ينعكس سلبا على كفاية املصطلح اإلجرائ ي ، ودوره .الفعال يف توحيد املعلومات وتيسري تداوهلا 3 : .داللة املصطلح يف حقل النقد مأاامم الباحثني العرب يف هذا النوع من الدراسات "ضراب من الفوضى الثقافية ،فقد حاول بعضهم التأصيل ملا يرنو به " ابلعودة إىل الرتاث العريب إلبراز جوانبه احلداثية ،(محودة1998 ، صفحة64 ) . كما أنّ النقد العريب أصبح يتخبط بني غياب صيغة مصطلحية موحدة من جهة ،وبني ترمجة هذه املصطلحات أو نقلها من الدراسات الغربية من جهة أخرى .حبيث أصبح النقد العريب املعاصر خيضع لصراع املفاهيم ،وكذلك ، االستهالك االصطالحي القادم من الغرب ح يث ينقل الباحثون العرب تلك املصطلحات يف عزلة عن خلفياهتا الفكرية والفلسفية ،حيث تفرغ من داللتها وتفقد القدرة على حتديد املعىن املناسب ،أما إذا نقلت هذه املصطلحات خبلفياهتا الفلسفية أدّت إىل الفوضى واالضطراب "إذ أن القيم املعرفية القادمة من املصطلح ختتلف ب ل تتعارض "أحياان مع القيم املعرفية اليت طورها الفكر العريب املختلف ،(بوساحة2005 ، صفحة63 ) . يشكل املصطلح النقدي إح دى العتبات واملداخل األساسية للنص النقدي بدءا من العنوان وابمتالكه ميتلك املتلقي املفتاح احلقيقي للدخول إىل عامله ،وفهم مكامنه ،ولعل طبيعة هذا اإلدراك أبمهية املصطلح ومكانته هو الذي كان وراء احتفاء النقاد به لدرجة أن يبسط سلطته على النص ،ويفرض نفسه كعتبة ال ميكن جتاوزها وختطيها ،وهلذا السبب أبدع النقاد يف وضعه وابتكاره (جعنيد، صفحة11 ) . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 "الصناعتني" لـ"أيب هالل العسكري" (ت395 )ه (جعنيد، صفحة65 ) وقد كان لظهور هذه الكتب األثر الكبري . والدافع النفتاح ابب النقد واشتداد اخلصومات األدبية اليت مهدت للنقد العلمي املوضوعي إن هذا التطور يف الوضع االصطالحي الذي أحدثه ابن املعتز جعل انقدا كـ"حممد مندور "يثين عليه :قائال ن "وم الواجب االعرتاف البن املعتز جبهده اهلام ومشاركته اإلجيابية يف هذا اجملال ليس ألنه نظّم البديع فحسب، بل كذلك ألنه سعى أكثر من غريه لتحديد االصطالحات وتدقيقها، ولعل بظهور هذا الرجل الناقد والشاعر يف اآلن نفسه تشكلت الثوابت املعرفية املتمثلة ابملنظومة االصطالحية للنقد العريب املستندة إىل األسس املوضوعية ،(مندور 1996 ، صفحة156 ) . ّوحبسب إدريس الناقوري فإن ّالنقاد العرب إّن ا شرعوا منذ القرن الثالث اهلجري فقط يف وضع اصطالحات نقدية ّوبالغية متأسني يف ذلك ابلفالسفة واملتكلمني ،وبتأثري الثقافة اليواننية .وهذا مل يدم األمر كثريا حيث أنّه ما إن أطل ّعلينا القرن الرابع اهلجري حىت تشك لت مالمح املصطلح النقدي ،واستمرّت املنظومة االصطالحية يف ّنوه ا واتّساعها يف املراحل الالحقة وهكذا جند أنفسنا أمام رجال كان هلم حضورهم القوي يف النقد العريب القدمي من حيث اخللق . واإلبداع واإلجياد للمصطلح النقدي بطريقة علمية موضوعية بعيدة عن الذوقية واالنطباعية 4 : .اآلليات املعتمدة يف صياغة املصطلح اغة امل طل النقدي فق آل ة االش قاق أ يعد االشتقاق من أهم اآلليات اللغوية املستعملة يف صياغة املصطلح ،فقد استعان به اللغويون والنقاد يف إثراء املعجم العريب ،خاصة يف النقد السيميائي الذي ينتابه الغموض ،واتّسم إبشكالية االصطالح منذ ولوجه إىل ساحة النقد العريب املعاصر. إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 ي إن ظهور النقد كعلم له مصطلحاته اخلاصة أتخر نوعا ما مقارنة ابلعلوم اللغوية والعلوم الدينية اليت كانت هلا األسبقية يف االهتمام والرعاية من طرف العلماء ،ولع ل أهم كتاب ينسب إىل علم النقد ،وحيمل بني طياته طابعا نقداي كتاب "فحول الشعراء" لألصمعي .ونعتقد أنه أخذ تلك التسمية من الكلمة املعروفة إذ ذاك الشاعر الفحل وأخذت بعد ذلك ترتى املصطلحات النقدي ة ،وأييت بعد األصمعي الناقد األمل عي "ابن سالم اجلمحي (ت237ه) " "وكتابه "طبقات فحول الشعراء وهو كاتب استفاد كثريا من الرتاكمات اليت سبقته على أ يدي اللغويني والنحويني والرواة ،لكن رغم ذلك ظل املصطلح النقدي ميشي على استحياء ومل ترتسّخ مفاهيمه ومبادئه بعد .ألن مرحلة ّالتشكل والبناء الفعلي للمصطلحات النقدية إّن ا بدأت بذورها يف احتكاك احلضارة العربية اإلسالمية ابحلضارات األخرى عرب جدلية التأثري والتأثر بني الداخل واخلارج ،(النبهان30 أفريل1995 ، صفحة17 ). خنررأ وتوالت املؤلفات بعد عبد هللا بن املعتز (ت296) ومن أمهها كتاب "نقد الشعر" لـ"قدامى (ت337 ")ه وكتاب "املوازنة بني الطائيني" لـ"اآلمدي"(ت371ه) ،وكتاب "الوساطة" لـ"القاضي اجلرجاين"(ت392 ه) وكتاب 69 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 مقابلpoétique ،يتولد مفهوم دال على اإلطار العام الذي ينزل فيه األدب ،فقد ظلت الالحقة االشتقاقية قائمة مقام لفظ العلم ،كما لو كان هذا اللفظ يتجه صوب ختصيص السمة اإلبداعية بصاحبها ،(بوخامت2005 ، صفحة 110 ). : ب.صياغة املصطلح النقدي وفق آلية النحت يعترب النحت إجراء عمليا لتوليد املصطلح يف اللغة العربية ،فقد استخدم قدميا وحديثا يف نقل ما استجدّ من مفاهيم معرفية لدى األمم األخرى ،وابتباع األسلوب اجلديد آللية النحت (األقرب إىل مفهوم الرتكيب يف اللغات األخرى خاصة االجنليزية والفرنسية) استطاع النقاد الع رب املعاصرون إجياد بعض املصطلحات السيميائية مثل :لغة اللغة ميطاليسانية ،نقد النقد ،قراءة القراءة الزمكان ...وغريها من . املصطلحات : ج.تعريب املصطلح النقدي لقد تعامل النقاد العرب املعاصرون مع هذه اآللية بشكل أوسع من اآلليات األخرى فاستخدموها يف تعريب املصطلحات الغربية بداية ابملفاهيم اللسانية ؛مثل :فونيم ،ومونيم ومورفيم ...وغريها .وهناك بعض املصطلحات املعربة جتاوزت مرحلة اللفظ الدخيل ،واندجمت يف سياق اللفظ ا لعريب حىت أنّ السامع يظنها ( كذلك ،وهي يف أصلها غري عربية مثل مصطلح أيقونةicone .) : ج.تعريب املصطلح النقدي لقد تعامل النقاد العرب املعاصرون مع هذه اآللية بشكل أوسع من اآلليات األخرى فاستخدموها يف تعريب املصطلحات الغربية بداية ابملفاهيم اللسانية ؛مثل :فونيم ،ومونيم ومورفيم ...وغريها .وهناك بعض املصطلحات املعربة جتاوزت مرحلة اللفظ الدخيل ،واندجمت يف سياق اللفظ ا لعريب حىت أنّ السامع يظنها ( كذلك ،وهي يف أصلها غري عربية مثل مصطلح أيقونةicone .) د. ترمج ة املصطلح:النقدي تعد الرت مجة رافدا مهما، وعامال أساسيا لنهضة األمة؛ ألهنا تساعد يف رصد كل ما استحدث لدى األمم األخرى ومسايرة التطور احلضاري ،(دقاجبي2008 2009 ، صفحة33 ). فمنذ نشأة النقد احلديث عرفت اللغة العربية مصطلحات جديدة عن ( طريق تفعيل آلية الرتمجة ؛ومنها : التشاكلisotopie ) ،حيث شهد هذا املفهوم تداخال يف االصطالح ،(دقاجبي2008 2009 ، صفحة34 ) . ، فظهر ت ألفاظ أخرى بصيغة خمتلفة، وهلا الداللة ذاهتا كلفظ املشاكلة، كما ."عربه البعض بلفظ "ازوتوبيا وترجم مصطلحpoétique بعدة كلمات عربية ؛منها : الشعرية ،اإلنشائية ،الشا عرية ،علم األدب ،الفن اإلبداعي ّفن الن ّظم ،فن الشعر ،نظرية الشعر ،كما عر( ب ببعض األلفاظ (البيوطيقا ،البويتيك) .أما مصطلحsème )فقد ترجم بعد ألفاظ يف اللغة العربية ؛منها:مسة ،نواة داللية ...وغريها من املصطلحات ،(دقاجبي2008 2009 ، صفحة 34 ) . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 فقد بذل نقادان املعا صرون قصارى جهدهم (سواء على مستوى األفراد أو اجلماعات )أو اهليئات إلجياد املصطلح النقدي املناسب بتوظيف آلية االشتقاق ابلرغم من اختالفهم يف حتديد صيغة موحدة :للمصطلح ،وضبط داللته ؛ومن تلك اجلهود نذكر مصطلح شعريةpoétique : تتداخل داللة هذا املفهوم كغريه من املصطلحات النقدية ،فمن النقاد من يرى أنه مستنبط من اللغة العربية عن طريق االشتقاق ؛أي مادة (ش ع ر) ،كما قال عبد السالم املسدي :"فهذا اجلذر الثالثي يف داللته حول كل ما له ارتباط ابحلس من حيث هو خامس مخسة مل جند ما نعرب به عنها إال لفظة احلواس ذاهتا ،وال ينفك عن هذا املعىن التأثيلي ما تدل عليه املادة اللغوية يف جدوليها الكبريين أبي تقليب من تقليبات "الوزن الصريف يف أخذها (املسدي ع.، االزدواج و املماثلة يف املصطلح النقدي، صفحة36 ) .ومل يعرف العرب هذا اللفظ )(شعرية ّ،وإّن ّا تداول عندهم مصطلحات أخرى تد ل على شاعرية الشاعر ؛ومنها :الشاعرية ،شعر الشاعر ،القول الشعري (اجلمحي، صفحة97 ) ولفظ شعرية يف النقد املعاصر رمبا حيمل داللة معرفية ؛ألنّ اختيار هذا اللفظ كبديل يعد االشتقاق من أهم اآلليات اللغوية املستعملة يف صياغة املصطلح ،فقد استعان به اللغويون والنقاد يف إثراء املعجم العريب ،خاصة يف النقد السيميائي الذي ينتابه الغموض ،واتّسم إبشكالية االصطالح منذ ولوجه إىل ساحة النقد العريب املعاصر. فقد بذل نقادان املعا صرون قصارى جهدهم (سواء على مستوى األفراد أو اجلماعات )أو اهليئات اجإلجياد املصطلح النقدي املناسب بتوظيف آلية االشتقاق ابلرغم من اختالفهم يف حتديد صيغة موحدة جا ح مصطلح شعريةpoétique : تتداخل داللة هذا املفهوم كغريه من املصطلحات النقدية ،فمن النقاد من يرى أنه مستنبط من اللغة العربية عن طريق االشتقاق ؛أي مادة (ش ع ر) ،كما قال عبد السالم املسدي :"فهذا اجلذر الثالثي يف داللته حول كل ما له ارتباط ابحلس من حيث هو خامس مخسة مل جند ما نعرب به عنها إال لفظة احلواس ذاهتا ،وال ينفك عن هذا املعىن التأثيلي ما تدل عليه املادة اللغوية يف جدوليها الكبريين أبي تقليب من تقليبات "الوزن الصريف يف أخذها (املسدي ع.، االزدواج و املماثلة يف املصطلح النقدي، صفحة36 ) .ومل يعرف العرب هذا اللفظ )(شعرية ّ،وإّن ّا تداول عندهم مصطلحات أخرى تد ل على شاعرية الشاعر ؛ومنها :الشاعرية ،شعر الشاعر ،القول الشعري (اجلمحي، صفحة97 ) ولفظ شعرية يف النقد املعاصر رمبا حيمل داللة معرفية ؛ألنّ اختيار هذا اللفظ كبديل 70 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 املشاكلة : هي من مصطلحات البالغة العربية القدمية "وهي أن يذكر الشيء بلفظ غريه لوقوعه يف صحبته كقوله / تعاىل :"وال تكونوا كالذين نسوا هللا فأنساهم أنفسهم أولئك هم الفاسقون " احلشر19 .أي أمهلهم فذكر اإلمهال هنا بلفظ النسيان لوقوعه يف صحبته ،(اهلامشي2003 ، صفحة309 ) : .وقد ذكر شعر أليب الرقمع قال فيه أصحابنا قصدوا الصبوح بسحرة * وأتى رسوهلم إيل خصيصا قالوا اقرتح شيئا جند لك طبخة * قلت اطبخوا يل جبة وقميصا أي :خيطوا يل جبة وقميصا ،فذكر اخلياطة بلفظ الطبخ لوقوعه يف صحبة طبخ الطعام ،ألنه كان فقريا ليس له كسوة تقيه من الربد ،(اهلامشي2003 ، صفحة309 ) . : و.توليد املصطلح النقدي عن طريق اجملاز إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 ي ن رق إل حم و رغم اجلهود املبذولة من أجل إحياء الرتاث الفكري العريب ،إال أنه مازال هنالك الكثري من الكنوز املعرفية ّاملد ّخرة منها واملهملة اليت مل يكشف عنها بعد حتتاج إىل حبث وتنقيب ،ودراسة معم قة كي تبعث من جديد ومن : بني املصطلحات النقدية اليت أوجدها النقاد العرب املعاصرون بتوظيف هذه اآللية إحياء للرتاث جند 71 71 : و.توليد املصطلح النقدي عن طريق اجملاز يعترب اجملاز وسيلة هامة تستخدم من أجل توسيع املعىن اللغوي للكلمة ،وحتميلها معىن جديدا فقد جلأ النقاد : إىل هذه اآللية إلثراء اللغة ؛حيث شهدت املصطلحات النقدية السيميائية ضرواب من األلفاظ اجملازية ؛نذكر منها ( "االنزايح" كلفظ بديل عن املصطلح األجنيبEcart ) والذي "مسي الفارق ،واالجنراف ،والبعد ،والفجوة وهي كلمات يف أصوهلا اللغوية خمتلفة ،ألن الفارق=بون ،واالحنراف=زيغ ،والبعد=جفاء والبعيد=انء والفجوة=شرخ .أما "االنزايح فهو املصطلح األقرب إىل العدول ،ولكن املصطلحات السالفة الذكر تعترب مقبولة جمازاي ،(مراتض2006 ، صفحة251 ) . ويف خضم اجلدل القائم بني النقاد يف تناوهلم للمصطلح السيميائي جمازا ،فأشاروا إىل الوتد األلسين ،والسلم الصويت واملاء الشعري ،والتقويضية ،واملفتاح السردي ... وغدت هذه املصطلحات موظفة توظيفا مكرّرا يف كتاابهتم بديال عن عن املصطلحات التالية :اجلملة املل فوظة (الوحدة الكالمية) ،التفاوت ال لغوي ،العناصر األدبية اجلمالية ّالتفكيكية ،مث حل " ت العقدة يف النص القصصي ،(مراتض، ألف ليلة و ليلة حتليل سيميائي تكتيكي حلكاية محال بغداد صفحة96 ) . 5 .جتربة بعض النقاد يف جمال املصطلح : النقدي بني القدامى واحملدثني : )أ.جهود القدامى (قدامة بن جعفر أمنوذجا : إسهامات قدامة بن جعفر النقدية يعترب قدامة بن جعفر من خالل كتابه "نقد الشعر" قيمة مضافة يف عصره (ق4 ه)اعتبارا ملا أضفاه من صبغة موضوعية على العملية النقدية ،وما أضافه من جهد حمرتم يف اتريخ النقد العريب . فكتابه "نقد الشعر" الذي اعرتف به الكثري من العلماء يف جمال النقد ملا له من أ مهية ومكانة ،ذلك أنه شكل أول حبث من نوعه يف اتريخ الدراسات األدبية العربية مما دفع مبعاصريه ومؤرخيه إىل اإلشادة به ،ونعته ابلبالغة واالنفراد ابلنقد ،والقدرة على 5 .جتربة بعض النقاد يف جمال املصطلح : النقدي بني القدامى واحملدثني : )أ.جهود القدامى (قدامة بن جعفر أمنوذجا يعترب قدامة بن جعفر من خالل كتابه "نقد الشعر" قيمة مضافة يف عصره (ق4 ه)اعتبارا ملا أضفاه من صبغة موضوعية على العملية النقدية ،وما أضافه من جهد حمرتم يف اتريخ النقد العريب . فكتابه "نقد الشعر" الذي اعرتف به الكثري من العلماء يف جمال النقد ملا له من أ مهية ومكانة ،ذلك أنه شكل أول حبث من نوعه يف اتريخ الدراسات األدبية العربية مما دفع مبعاصريه ومؤرخيه إىل اإلشادة به ،ونعته ابلبالغة واالنفراد ابلنقد ،والقدرة على 72 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. : و.توليد املصطلح النقدي عن طريق اجملاز أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 دراسة الشعر ،بعد أن وصفوه ابإلمام املقتدى به يف هذا الشأن ،إذ أن كتابه كان نقطة حتول يف الدراسات النقدية عند العرب ،ابعتباره أول حبث علمي منظم يف النقد ،ملا وضع فيه من معامل واضحة ،وأصول اثبتة للنقد ،(طبانة 2011 ، صفحة426 ) . ميكن أن جنمل إسهامات قدامة النقدية من خالل كتابه "نقد الشعر" يف العوامل التالية (جعنيد، صفحة127 ) : _إن قدامة استطاع أن حيول النقد والبالغة إىل علمني مستقلني قائمني على أساس نظري متني ،خاصة وأن كل . اجلهود السابقة له .إّنا كانت جتعل من الذوق واحلس متكأ هلا _إن قدامة استطاع أن حيول النقد والبالغة إىل علمني مستقلني قائمني على أساس نظري متني ،خاصة وأن كل . اجلهود السابقة له .إّنا كانت جتعل من الذوق واحلس متكأ هلا _أن منهجه النقدي تتجلى فيه بوضوح أتثري الثقافة اليواننية املرتسمة يف تدقيقه يف احلدود والتعريفات واملفاهيم . واملصطلحات هحجّج _أن منهجه النقدي تتجلى فيه بوضوح أتثري الثقافة اليواننية املرتسمة يف تدقيقه يف احلدود والتعريفات واملفاهيم . واملصطلحات _كما يظهر يف عمله النقدي ذلك التمازج الكبري بني النقد والبالغة ،والتفاعل والتجاور العجيب بني املصطلحات . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 وال ننسى أن عبد املالك مراتض متأثر إىل حد ما ابلثقافة الرتاثية العربية وهذا ما نلمسه يف كثري من كتاابته سواء اإلبداعية أو النقدية ؛ فلقد وصف مصطلح التناص ب"االقتباس"،وهذا اللفظ له جذور وامتداد يف املوروث النقدي والبالغي العريب ،كما يقول الباحث ،"...وهو إن شئت (أي التناص) اقتباس ،وهذا املصطلح بالغي حمض ّبل إهن ا أحلقت أيضا األدب املقارن نفسه بنظرية التن" اص ،وبكل جرأة ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة188 ). و بح اوز ن إ ر جي و نى ي و يع و و 12 .االستحالة والتناقض : وهو من عيوب املعاين كما قال قدامة ،وذلك أبن يذكر الشاعر الشيء فيجمع بينه وبني . املقابل له من جهة واحدة ... وغريها من املصطلحات اليت مانت من مستخرجات قدامة ب.جهود احملدثني وأعماهلم يف جمال املصطلح النقدي )(عبد املالك مراتض أمنوذجا : لقد حظي املصطلح النقدي يف العصر احلديث ابهتمام ابلغ من قبل النقاد العرب أمثا ل "جمدي وهبة"يف معجم مصطلحات األ دب ،وإدريس الناقوري يف "املصطلح النقدي يف نقد الشعر"،وحممد رشاد احلمزاوي يف"املصطلحات اللغوية احلديثة يف اللغة العربية" ،ومسري حجازي يف "قاموس مصطلحات النقد األديب"،ومحود فهمي حجازي يف"األسس اللغوية لعلم املصطلح" ،وعبد السالم املسدي يف "املصطلح النقدي" ،وحم مد عناين يف "املصطلحات األدبية احلديثة" ،ورشيد بن مالك يف "قاموس مصطلحات التحليل السيميائي" ،وعزت حممود جاد يف "نظرية املصطلح النقدي" ، ويوس ف وغليسي يف "إلشكالية املصطلح يف اخلطاب النقدي العريب اجلديد ،وكما جند عبد املالك مراتض قد ترجم العديد من املفاهيم الغربية مبعانيها األصلية منخالل حتويلها من اللغات الغربية . : و.توليد املصطلح النقدي عن طريق اجملاز النقدية والبالغية ضبط املصطلحات النقدية عند قدامة بن :جعفر ضبط املصطلحات النقدية عند قدامة بن :جعفر بعد الرجوع لكتاب نقد الشعر ،ومن خالل القراءة املستفيضة لبعض الكتب واملؤلفات اليت تناولت "نقد الشعر" ابلتحقيق والتعليق خاصة ما كتبه بدوي طبانة ،وعبد املنعم خفاجي ،وبعد املقارنة بني ما ورد يف هذه الكتب من مصطلحات وما تضمنته كتب أخرى تناولت أيضا املصطل ح النقدي بشكل عام مثل كتاب"املصطلح النقدي يف الرتاث األديب العريب" حملمد عزام ،وكتاب "معجم النقد العريب القدمي" ألمحد مطلوب ،وكتاابت أخرى جديثة تبني لنا أبن قدامة بن جعفر كانت له الكثري من املفاهيم االصطالحية يف امليدان النقدي نورد منها 1 ..صحة التقسيم : ويسمى أيضا التقسيم وهو من املصطلحات اليت انفرد هبا قدامة ضبط املصطلحات النقدية عند قدامة بن :جعفر بعد الرجوع لكتاب نقد الشعر ،ومن خالل القراءة املستفيضة لبعض الكتب واملؤلفات اليت تناولت "نقد الشعر" ابلتحقيق والتعليق خاصة ما كتبه بدوي طبانة ،وعبد املنعم خفاجي ،وبعد املقارنة بني ما ورد يف هذه الكتب من مصطلحات وما تضمنته كتب أخرى تناولت أيضا املصطل ح النقدي بشكل عام مثل كتاب"املصطلح النقدي يف الرتاث األديب العريب" حملمد عزام ،وكتاب "معجم النقد العريب القدمي" ألمحد مطلوب ،وكتاابت أخرى جديثة تبني لنا أبن قدامة بن جعفر كانت له الكثري من املفاهيم االصطالحية يف امليدان النقدي نورد منها 1 ..صحة التقسيم : ويسمى أيضا التقسيم وهو من املصطلحات اليت انفرد هبا قدامة 2 . ".صحة املقابالت : ومساها غريه من البالغيني"املقابلة 3 . .صحة التفسري : وهو التبيني والشرح زالتوضيح 4 .التمام : ومساه احلامتي ب"التتمي م"،ومساه أبو هالل العسكري حتت اسم "التكميل" ،وابن رشيق القريواين حتت مسمى" االحرتاس" و" االحتياط" . 5 . .املبالغة : ومساها ابن املعتز "اإلفراط يف الصفة" .واملبالغة هي أن يبلغ الشاعر ابملعىن أقصى غايته ومنتهاه 6 . .التكافؤ : وهو ما يسميه البالغيون ابلطباق أو املقابلة 7 . .االلتفات : ويسمى االستدراك وهو أن أيخذ الشاعر يف معىن فيأخذه الشك فيعود على ما قدّمه 8 . .االستغراب والطرافة : ومسي من بعده ابلتطريف 9 .اإلشارة : يعترب قدامة أول من تكلم عنها ،وعرفها أبهنا :اشتمال اللفظ القليل على املعىن الكثري ابللمحة الدالة وتسمى اإلجياز أو اإلمياء عند غريه. ماا 7.االلتفات : ويسمى االستدراك وهو أن أيخذ الشاعر يف معىن فيأخذه الشك فيعود على ما قدّم 8 . .االستغراب والطرافة : ومسي من بعده ابلتطريف اما 9 .اإلشارة : يعترب قدامة أول من تكلم عنها ،وعرفها أبهنا :اشتمال اللفظ القليل على املعىن الكثري ابللمحة الدالة وتسمى اإلجياز أو اإلمياء عند غريه. 73 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 ييامي اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 10 . .اإلخالل : وهو أن يرتك من اللفظ ما به يتم املعىن 11 . .التخليع : وهو أن يكون معىن الشعر جيدا ولفظه حسن ،إال أن وزنه قد شانه وقبح حسنه 12 .االستحالة والتناقض : وهو من عيوب املعاين كما قال قدامة ،وذلك أبن يذكر الشاعر الشيء فيجمع بينه وبني . املقابل له من جهة واحدة ... وغريها من املصطلحات اليت مانت من مستخرجات قدامة ب.جهود احملدثني وأعماهلم يف جمال املصطلح النقدي )(عبد املالك مراتض أمنوذجا : لقد حظي املصطلح النقدي يف العصر احلديث ابهتمام ابلغ من قبل النقاد العرب أمثا ل "جمدي وهبة"يف معجم مصطلحات األ دب ،وإدريس الناقوري يف "املصطلح النقدي يف نقد الشعر"،وحممد رشاد احلمزاوي يف"املصطلحات اللغوية احلديثة يف اللغة العربية" ،ومسري حجازي يف "قاموس مصطلحات النقد األديب"،ومحود فهمي حجازي يف"األسس اللغوية لعلم املصطلح" ،وعبد السالم املسدي يف "املصطلح النقدي" ،وحم مد عناين يف "املصطلحات األدبية احلديثة" ،ورشيد بن مالك يف "قاموس مصطلحات التحليل السيميائي" ،وعزت حممود جاد يف "نظرية املصطلح النقدي" ، ويوس ف وغليسي يف "إلشكالية املصطلح يف اخلطاب النقدي العريب اجلديد ،وكما جند عبد املالك مراتض قد ترجم العديد من املفاهيم الغربية مبعانيها األصلية منخالل حتويلها من اللغات الغربية . _وخصوصا الفرنسية _إىل ما يقابلها يف اللغة العربية ؛ومنها مصطلح التناص ( لقد اكتسب هذا املصطلح مكانة حساسة يف جمال الشعرية احلديثةpoétique ) والتحليل البنيوي ،وهو من املفاهيم اليت اعتمد عليها الكثري من النقاد يف الدرس النقدي على شاكلة عبد املالك مراتض اليت تعامل مع هذا املصطلح النقدي كآلية إجرائية لتحليل اخلطاب األديب ،حيث اعترب التناص مبثابة األ كسجني من الصعب مشّه أو رؤيته على الرغم من انتشاره يف أماكن متعدّدة ،وال وجود لعاقل ينكر هذه احلقيقة .فهو يرى أن هذا املصطلح مثرة ( من مثرات الرتمجة الفرنسية ،حيث أشار إىل لفظ التناصintertextualité) كمفه ّوم فيه نص ان أو أكثر يتعارض ان أو يتضارابن أو يتنافسان ّ،فمعرفة أن ه تبادر للتأثري ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة196 ) ّوقد عر ّفه عبد املالك مراتض "أبن ّه تفاعل وتبادل للعالقة بني نص وآخر ،إم ا على سبيل االقتباس أو املعارضة ّأو التضاد " ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة195 ). إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 أطلق"عبد السالم املسدي " "على هذا املفهوم مصطلح "العدول كما هو سائد يف البالغة العربية ،.(املسدي ع ، األسلوب و األسلوبية1977 ، صفحة158 ) حيث وجد عبد املالك مراتض أن هذه ، املصطلحات "ماهي إال مرتادفات ملفهوم واحد ، وهي الترقى يف داللتها األدبية واجلمالية وحىت املعرفية إىل مستوى االستعمال املتداول بني النقاد العرب ...؛ "فمصطلح "االنزايح ّرمبا يظل ، األيسر بني الناس وهو األسلم لغواي ومعرفيا ، (مراتض، فكرة السرقات األدبية ونظرية التناص1991 ، صفحة05 ) .ويف كل هذه املصطلحات ما يدل على احنراف املتكلم أو الكاتب عن كل ما هو مألوف ومتداول يف االستعمال األسلويب املعتاد أو اللغة املباشرة إىل شيء آخ غري ذلك وبناء على ما سبق– ومن خالل نبشه للرتاث العريب– "طرح عبد املالك مراتض مسألة "السرقات األدبية كمرادف تطرق إليه بوضوح البالغيون والنقاد العرب القدامى ،على غرار ابن سالم اجلمحي (ت232 ه) وقدامة بن جعفر(ت337ه) ،وابن رشيق القريواين(ت456ه) ،وعبد القاهر اجلرج اين(ت471 ه) ،وغريهم .كما نبّه الكاتب إىل عملية (حفظ النصوص) ونسياهنا يف تصور ابن خلدون (ت808 ه) ،على أساس أهنا "فكرة تناصية "وليس النص نفسه ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة188 ) ،وقد أبدى بعض املالحظات حني حاول الربط بني فكرة (ا )لسرقات األدبية ، (مراتض، فكرة السرقات األدبية ونظرية التناص1991 ، 70ل ل - إن فكرة السرقات األدبية ال تستبعد التأثري وهي مل تكن تتناول املعىن فقط ّ،وإّن ا تناولت اللفظ أكثر من املعىن ، وإذا كان مصطلح ا ّلتناص حيىي على التناصي ّة ،فإن كثريا من امل صطلحات النقدية حتتاج إىل مراجع مصطلحاتية .وكذلك التناص يقوع يف بعض أطواره على التضاد ،كما هو احلال مع فكرة (السرقات األدبية)اليت . تقوم أيضا على التضاد واالختالف وقد توافق آراء ابن خلدون–يف مسألة نسيان احملفوظ- مع فكرة (روالن )ابرت و ." يف تصرحيه :"أان أكتب ألين نسيت ايأاحي : مفهوم االنزايح ( االنزايح أو االحنراف هو مصطلح سيميائي بديل عن لفظl’écart ) يف اللغة الفرنسية وقد ترمجه بعض النقاد املعاصرين إىل مصطلحات منها "الفجوة" و"االبتعاد" (مراتض، مفاهيم سيميائية مبصطلحات ،بالغية2006 ، صفحة04 ) . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 _وخصوصا الفرنسية _إىل ما يقابلها يف اللغة العربية ؛ومنها مصطلح التناص ( لقد اكتسب هذا املصطلح مكانة حساسة يف جمال الشعرية احلديثةpoétique ) والتحليل البنيوي ،وهو من املفاهيم اليت اعتمد عليها الكثري من النقاد يف الدرس النقدي على شاكلة عبد املالك مراتض اليت تعامل مع هذا املصطلح النقدي كآلية إجرائية لتحليل اخلطاب األديب ،حيث اعترب التناص مبثابة األ كسجني من الصعب مشّه أو رؤيته على الرغم من انتشاره يف أماكن متعدّدة ،وال وجود لعاقل ينكر هذه احلقيقة .فهو يرى أن هذا املصطلح مثرة ( من مثرات الرتمجة الفرنسية ،حيث أشار إىل لفظ التناصintertextualité) كمفه ّوم فيه نص ان أو أكثر يتعارض ان أو يتضارابن أو يتنافسان ّ،فمعرفة أن ه تبادر للتأثري ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة196 ) ّوقد عر ّفه عبد املالك مراتض "أبن ّه تفاعل وتبادل للعالقة بني نص وآخر ،إم ا على سبيل االقتباس أو املعارضة ّأو التضاد " ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة195 ). وال ننسى أن عبد املالك مراتض متأثر إىل حد ما ابلثقافة الرتاثية العربية وهذا ما نلمسه يف كثري من كتاابته سواء اإلبداعية أو النقدية ؛ فلقد وصف مصطلح التناص ب"االقتباس"،وهذا اللفظ له جذور وامتداد يف املوروث النقدي والبالغي العريب ،كما يقول الباحث ،"...وهو إن شئت (أي التناص) اقتباس ،وهذا املصطلح بالغي حمض ّبل إهن ا أحلقت أيضا األدب املقارن نفسه بنظرية التن" اص ،وبكل جرأة ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة188 ). 74 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 وبناء على ما سبق– ومن خالل نبشه للرتاث العريب– "طرح عبد املالك مراتض مسألة "السرقات األدبية كمرادف تطرق إليه بوضوح البالغيون والنقاد العرب القدامى ،على غرار ابن سالم اجلمحي (ت232 ه) وقدامة بن جعفر(ت337ه) ،وابن رشيق القريواين(ت456ه) ،وعبد القاهر اجلرج اين(ت471 ه) ،وغريهم .كما نبّه الكاتب إىل عملية (حفظ النصوص) ونسياهنا يف تصور ابن خلدون (ت808 ه) ،على أساس أهنا "فكرة تناصية "وليس النص نفسه ،(مراتض، بني التناص و التكاتب املاهية و التطور1996 ، صفحة188 ) ،وقد أبدى بعض املالحظات حني حاول الربط بني فكرة (ا )لسرقات األدبية ، (مراتض، فكرة السرقات األدبية ونظرية التناص1991 ، صفحة70 ) ،ومفهوم التناص :ومنها ما يلي - إن فكرة السرقات األدبية ال تستبعد التأثري وهي مل تكن تتناول املعىن فقط ّ،وإّن ا تناولت اللفظ أكثر من املعىن ، وإذا كان مصطلح ا ّلتناص حيىي على التناصي ّة ،فإن كثريا من امل صطلحات النقدية حتتاج إىل مراجع مصطلحاتية .وكذلك التناص يقوع يف بعض أطواره على التضاد ،كما هو احلال مع فكرة (السرقات األدبية)اليت . تقوم أيضا على التضاد واالختالف وقد توافق آراء ابن خلدون–يف مسألة نسيان احملفوظ- مع فكرة (روالن )ابرت و ." يف تصرحيه :"أان أكتب ألين نسيت : مفهوم االنزايح ( االنزايح أو االحنراف هو مصطلح سيميائي بديل عن لفظl’écart ) يف اللغة الفرنسية وقد ترمجه بعض النقاد املعاصرين إىل مصطلحات منها "الفجوة" و"االبتعاد" (مراتض، مفاهيم سيميائية مبصطلحات ،بالغية2006 ، صفحة04 ) . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 ، ويبقى هذا املفهوم يف غاية الغموض "وقد اعرتف "غرمياس" و"كورتيس الزاعمان أبن هذا املفهوم قد جاء من ،"إنتاج مستعملي اللغة انطالقا من أتمالت "ديسو سري غري أن عبد املالك مراتض استند إىل آراء ومقرتحات ،""جان ماري سشيفر ّفالحظ أن ه عاجل هذا املفهوم بتفصيل ووضوح ؛ألنه اتبع دراسته ملا هو شائع يف آخر النظرات السيميائية ،حيث وجد أن لالنزايح أنواعا متعدّدة هي االنزايح البالغي ،واال نزايح النحوي ،والنزايح الوصفي . والالنزايح األسلويب البالغي الذي هو يف أصله السيميائي الراهن : "مصطلح "لغة اللغة ( يعترب لفظmétalangage ) من املصطلحات اللسانية احلديثة ،وهو مركب من ( مادتني لغويتني ؛مهاMéta ) اليت تعين (ما بعد) ،أو (ما وراء) ،وهي عنصر حنوي حيدّد (ما فوق اللغة) أما كلمة ( langage ) ،فتعين اللغة اللغة أو اللسان . وقد ترجم هذا املفهوم إىل ألفاظ عربية عدة من قبل النقاد االعرب وقد احتج ع ، ليها الباحث عبد املالك مراتض وآثر مصطلح "لغة اللغة" على ما ورد من مص طلحات مثل : (ما وراء " اللغة) ،و لغة واصفة" و"مابعد اللغة"،حيث رأى الباحث أن هذا خمالف ألدىن مواصفات هذا املصطلح يف أصله ، الغريب أل ّن ( السابقةMéta ، ) يف حقل العلوم اإلنسانية تعين : االنتماء واالحتواء : وهي كلمة إغريقية تعين )ما يشمل اللغة كمفهوم اصطنعه الفالسفة األملان يف مدرسة "فيينا" واألنسب حينئذ القول للمصطلح (لغة اللغة "أو"اللغة الواصفة" أو "اللغة اجلامعة ،(يوسف2008 ، صفحة315 ) . : مصطلح العالمة من املصطلحات اليت مت تفعيلها عن طريق اإلحياء كإجراء لسد بعض الثغرات املوجودة يف جمال االصطالح ،وقد استحسن الباحث عبد املالك مراتض هذه التقنية ،وأوجد العديد من املصطلحات ومنها )مصطلح (مسة :( signe ) ، وهو من صور االضطراب الناتج عن اختالف الرؤى حول املقابل العريب ملصطلح ( signe ( ) يف الثقافة الغربية ،وأصل هذا اللفظ التيينsignun ، ) وهو مرادف للعالمة واألمارة . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 أطلق"عبد السالم املسدي " "على هذا املفهوم مصطلح "العدول كما هو سائد يف البالغة العربية ،.(املسدي ع ، األسلوب و األسلوبية1977 ، صفحة158 ) حيث وجد عبد املالك مراتض أن هذه ، املصطلحات "ماهي إال مرتادفات ملفهوم واحد ، وهي الترقى يف داللتها األدبية واجلمالية وحىت املعرفية إىل مستوى االستعمال املتداول بني النقاد العرب ...؛ "فمصطلح "االنزايح ّرمبا يظل ، األيسر بني الناس وهو األسلم لغواي ومعرفيا ، (مراتض، فكرة السرقات األدبية ونظرية التناص1991 ، صفحة05 ) .ويف كل هذه املصطلحات ما يدل على راحنراف املتكلم أو الكاتب عن كل ما هو مألوف ومتداول يف االستعمال األسلويب املعتاد أو اللغة املباشرة إىل شيء و يف خضم هذا التداخل االصطالحي حول مفهوم االنزايح أضاف الباحث عبد املالك مراتض لفظا مماثال "وهو "االلتفات ، واستشهد بكالم الزخمشري (ت528 ّه) الذي حتد ث عن االلتفات وبني وظيفته البالغية ،بكوهنا تنق ال من أسلوب إىل أسلوب واحد ،(حممود2009 ، صفحة441 ) .،والكالم إ ذا كان كذلك يعترب أحسن تطرية ، لنشاط السامع وإيقاظا لإلصغ.اء إليه من إجرائه على أسلوب واحد وبناء على ماهو يف كتب الرتاث العريب توصل" عبد املالك مراتض إىل أن االنزايح جتاوز االقتصار على املراوحة بني استعمال الضمائر املختلفة داخل كالم واحد إىل . التوسع غري احملدود يف التعامل مع اللغة األدبية يف أسلوب الكالم 75 يأ إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 وقد اعتىن مراتض هبذا املصطلح النقدي حيث حاول التأصيل له مبا استوحاه من الرتاث العريب مرتكزا على فكرة احلداثة السائد فهو يرى أن العلماء العرب القدامى قد وظفوا ه دا املفهوم أبسلو ب إشاري ،أو اب لأل وان للداللة على أمر ما سواء يف ّالسر ّاء أم الضر اء مبا يف ذلك التعبري عن إقامة األفراح وإقامة املمارسات الطقوسية والشعائر الدينية (مراتض، مفاهيم ،سيميائية مبصطلحات بالغية2006 ، صفحة09 ) ،وخالل دراسته هلذا املصطلح أجرى مقاربة ملصطلحات أخرى ( استعملت لدى البعض للداللة على مصطلحsigne ") فمنهم من استعمل لفظ "العالمة" ؛ولكنه فضل كلمة "مسة ( ألنه رأى أهنا األقرب للداللة على هذا املصطلح بدال من لفظة "عالمة" اليت تدل على مصطلحmarque ) ، ،وذلك ألنه يعترب "السمة ظاهرة طبيعية تدرك بصفة مباشرة ّفالل ون الداكن الذي يسم وجه السماء هو "مسة" أو ""قرينة ، لعاصفة وشيكة احلدوث ... كما أضاف يف مواضع أخرى مصطلحني آخرين للداللة ذاهتا . "ومها :"مؤشر"و"علمية يقول الدكتور موالي علي بوخامت :"إن الدكتور عبد املالك مراتض أراد أن يساو ي بني 76 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 جمموعة من املفاهيم من حيث داللتها ، وأمهها (مسة- قرينة-مؤشر-علمية) ،واعت ربها مصطلحات متباينة لسانياتيا ومتشاهبة مفهوميا ،(بوخامت، الدرس السيميائي املغاريب2005 ، صفحة125 ) . إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 ، فإشكالية املصطلح النقدي أسالت الكثري من احلرب وفق التباين والتماثل املنهجي ّوإن كثفت اجلهود ووح دت ّاملفاهيم سيتم ّاالت فاق على كيفية اختيار املصطلحات ّالنقدية املناسبة للداللة على معىن حمد د حبسب املعىن الذي ّيصبو إليه القارئ حىت ال يلتبس عليه الفهم ،وهبذا تتم . احملافظة على استمرارية النقد العريب وتطوره : التوصيات إن عملية وضع املصطلح الواحد للمفهوم الواحد اب ت، فاق عام أمر مثايل ّومن الصعب أن يتحق، ق ولتجاوز ّهذه اإلشكاليات يتوج، ب على املنشغلني ابلبحث يف املصطلح النقدي تكثيف اجلهود ّواستعمال كل ما هو ميس ر ومفيد ؛ ، إلجياد مصطلح نقدي عريب موحد ، ليكون بديال عن املفاهيم الغربية : وذلك وفق 1 _إعادة فحص املصطلح النقدي واللساين والبالغي ّاملوروث والعمل على إعادة تشغيل بعض املفردات جتن با للقطيعة احلاصلة . يف الوقت احلاضر بني املصطلح املوروث واملصطلح احلديث 2 ، _السعي إىل تداول مصطلح موحد ّوجتنب التعد . د الداليل للمصطلح الواحد 3_إعادة فتح الرصيد االصطالحي عند خمتلف النقاد ومال ح ظة سريورة تداولية للمصطلحات املختلفة ،كما جرى . مؤخرا عند دراسة اخلطاب النقدي لدى :طه حسني ، العقاد ،ومندور 4 _السعي لنشر الثقافة املصطلحية واملعجمية ،والوقوف ضد حماولة جتاهل العقد االصطالحي أو التصرف . االعتباطي العشوائي ابملصطلح النقدي 5 _مواصلة اجلهود الرامية إىل وضع مصطلحات تتسم ابلبساطة والوضوح ،مع احملافظة على سالمة اللفظ لغواي سواء أكان مشتقا أم مولدا ،أم مرتمجا بطرق أخرى . 6 _درء االختالف املوجود يف جمال صياغة املصطلحات ،ألن تعدد األلفاظ العربية اليت تعرب عن املصطلح األعجمي . الواحد تسببت يف اتساع الفجوة املوجودة أصال بني النقاد العرب املعاصرين 7 - الدعوة إىل ضرورة النهوض واالهتمام مبوضوع املصطلح من خمتلف جوانبه ؛وذلك من خالل تنظيم امللتقيات ، والندوات الدراسية . والبحوث األكادميية ورسائل التخرج وبرامج التكوين يف اجلامعات ومعاهد اللغة العربية وآداهبا ويف ّخضم هذا االختالف الذي متخ، ضت عنه إشكالية املصطلح النقدي ّفض ل بعض الباحثني العرب املزج ، بني املوروث البالغي والنقدي العريب وبني املفاهيم الواردة من الدراسات احلداثية الغربية كما أن البحث املصطلحي ّشهد إقباال واسعا وأجنزت العديد من الدراسات العربية يف هذا اجملال ،ولكن ّها تعرب يف غالبيتها عن آراء ذاتية ،يف حني لو تضافرت اجلهود لتمكننا من جتاوز هذه اإلشكالية ألن اللغة العربية هلا كل املؤهالت ا ليت تسمح هلا مبسايرة ّ كل مستجد من معارف و . :اخلـاتـمة خ شهدت الساحة النقدية العربية حتوال كبريا بعد دخول املناهج الغربية احلداثية ،وهذا يدل على تالقح فكري ضروري وليس تبعية مطلقة ،فقد أعاد النقاد سبكها وتكييفها ؛حيث ألقوا عليها من مسات العربية وخصائصها كما مت حتوير وت طوير مناهج النقد العريب احلديث، فقد عدلت املناهج ال: نقدية الغربية التارخيي النفسي ... واالجتماعي من قبل رواد احلركة النقدية العربية احلديثة ؛ أمثال طه حسني والعقاد ومندور ...حيث مت تطويعها مبا يناسب فكران . العريب ورغم اجلهود املبذولة يف هذا اجملال إال أن املصطلح النقدي العريب مازال يعاين مجلة من العوائق اليت حتول دون بلوغه املستوى املنشود ،وهي إشكاالت متعددة األوجه ،حنصر منها :التوليد ،والتحديد والتوحيد ... وهذا ما ساهم بشكل مباشر يف وجود حالة الفوضى املصطلح . ية فاألصل يف تسمية املفاهيم وصياغة املصطلحات يكمن ، يف جعل مصطلح أو رمز لغوي واحد أمام كل مفهوم وهذا بتوافق أهل االختصاص ّوإن كان للمفهوم الواحد عد ة ، أمساء ّأو كان اللفظ الواحد داال ، على معان كثرية . فإن التواصل الفكري سيضطرب : نتائج الدراسة من خالل البحث والدراسة استخلصنا إىل مجلة من النتائج املتمثلة أساسا يف احليثيات واألسباب اليت ترتجم . واقع املصطلح يف الساحة األدبية العريبة وإشكاالته : وأمهها من خالل البحث والدراسة استخلصنا إىل مجلة من النتائج املتمثلة أساسا يف احليثيات واألسباب اليت ترتجم . واقع املصطلح يف الساحة األدبية العريبة وإشكاالته : وأمهها 1 _تعدد املنابع اليت تصدر املصطلحات يف الوطن العريب سواء من قبل اهليئات العلم ية كاجملامع اللغوية واجلامعات أو اجلهود الفردية ك . :النقاد واملعجميني ،واملرتمجني 2 ّ_اختالف املناهج والطرق املستعملة يف توليد املصطلح ،فبعض الباحثني يفض لون اللجوء إىل املصطلح الرتاثي ، وبعضهم يلجأ إىل آلية االشتقاق والنحت لتوليد املصطلحات ، وحاول آخرون توليد املصطلحات الدخيلة أو . وضع األلفاظ اجملازية للداللة على املفاهيم الغربية 3 ّ_تعد د املصادر الغربية اليت ينقل منها املصطلح ،كالفرنسية واالجنليزية ولكل منهما خصائصها اللسانية وضوابطها . الداللية 4 . _غياب التنسيق بني املشتغلني على املصطلح النقدي يف الوطن العريب 5 _بطء، االستجابة للمصطلحات اجلديدة ّمما قد يضي . ع علينا فرصة االستفادة من تلك املفاهيم حني ظهورها 77 77 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 مفاهيم وألفاظ مهما كان مصدرها إن عملية وضع املصطلح الواحد للمفهوم الواحد اب ت، فاق عام أمر مثايل ّومن الصعب أن يتحق، ق ولتجاوز ّهذه اإلشكاليات يتوج، ب على املنشغلني ابلبحث يف املصطلح النقدي تكثيف اجلهود ّواستعمال كل ما هو ميس ر ومفيد ؛ ، إلجياد مصطلح نقدي عريب موحد ، ليكون بديال عن املفاهيم الغربية : وذلك وفق بي ي حجإ مم نا 1 _إعادة فحص املصطلح النقدي واللساين والبالغي ّاملوروث والعمل على إعادة تشغيل بعض املفردات جتن با للقطيعة احلاصلة . يف الوقت احلاضر بني املصطلح املوروث واملصطلح احلديث منام عي 7 - الدعوة إىل ضرورة النهوض واالهتمام مبوضوع املصطلح من خمتلف جوانبه ؛وذلك من خالل تنظيم امللتقيات ، والندوات الدراسية . والبحوث األكادميية ورسائل التخرج وبرامج التكوين يف اجلامعات ومعاهد اللغة العربية وآداهبا ويف ّخضم هذا االختالف الذي متخ، ضت عنه إشكالية املصطلح النقدي ّفض ل بعض الباحثني العرب املزج ، بني املوروث البالغي والنقدي العريب وبني املفاهيم الواردة من الدراسات احلداثية الغربية كما أن البحث املصطلحي ّشهد إقباال واسعا وأجنزت العديد من الدراسات العربية يف هذا اجملال ،ولكن ّها تعرب يف غالبيتها عن آراء ذاتية ،يف حني لو تضافرت اجلهود لتمكننا من جتاوز هذه اإلشكالية ألن اللغة العربية هلا كل املؤهالت ا ليت تسمح هلا مبسايرة ّ كل مستجد من معارف و . مفاهيم وألفاظ مهما كان مصدرها 78 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 1 -اجلاحظ. (1998). البيان والتبيني . تح: عبد السالم حممد هارون .ج1 .ط7 القاهرة.مصر: مكتبة اخلاجني. 2 - اجلرجاين( 1357 )ه. .التعريفات .مصر مطبعة مصطفى الباي احلليب و أوالده. 3 - اجلمحي) 1998 ( .طبقات فحول الشعراء .تح:حممود حممد شاكر .ج1. دار.املدين .جدة .اململكة السعودية 4 -الزخمشري, أ. ا. (1998). أساس البالغة. تح: حممد ابسل عيون السود. ج1 .ط1 .. . منشورات دار الكتب العاملية . بريوت لبنان 5 -الزخمشري. (2009). الكشاف عن حقائق غوامض التنزيل و عيون األقاويل يف وجوه التنزيل .ط3 . . دار املعرفة 6 -منظور, ا ( 1119 )ه. لسان العرب . تح: جمموعة من األساتذة منهم : عبد هللا حممد الكبري ،و جممد الشاذيل. ج28 . دار املعارف . القاهرة . مصر. ب- :املراجع ب- :املراجع 1 - العربية, م. ا. (2004). املعجم الوسيط . ط1. مصر . مكتبة الشروق الدولية. 2 -املسدي, ع. ا. (1977). إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 األسلوب و األسلوبية . الدارالعربية للكتاب .تونس . ط3. 3 -املسدي, ع. ا. (2004). األدب و خطاب النقد .ط1 . بريوت لبنان: دار الكتاب اجلديد املتحدة. م( )مم 2 -املسدي, ع. ا. (1977). األسلوب و األسلوبية . الدارالعربية للكتاب .تونس . ط3. 3 -املسدي, ع. ا. (2004). األدب و خطاب النقد .ط1 . بريوت لبنان: دار الكتاب اجلديد املتحدة. 4 - املنجد يف اللغة العربية. (1989). بريوت لبنان: دار املشرق. 5 -اهلامشي, ا. أ. (2003). جواهر البالغة. بيورت لبنان: املكتبة العصرية. 6 - بوخامت, م. ع. (2005). الدرس السييميائي املغاريب. اجلزائر: . ديوان املطبوعات اجلامعية 7 -جعنيد, ع. ا. (n.d.). . املصطلح النقدي قضااي و إشكاالت . عامل الكتب احلديث . اململكة املعربية 8 -محودة, ع. ا. (1998). . املرااي احملدبة. الكويت سلسة عامل املعرفة مطابع الوطن. 9 -دقاجبي, ص. (2008 2009 , جامعة اجلياليل اليابس . إشكالية املصطلح السيميائي. ماجستري, p. 33. 10 -طبانة, ب. (2011). . قدامة بن جعفر و النقد األديب .إربد . األردن دار علم الكتب احلديث. 11 -عزام, م. (2010). املصطلح النقدي يف الرتاث األديب العريب. بريوت. .لبنان دار الشرق العريب. 12 -مراتض, ع. ا. (n.d.). ألف ليلة و ليلة حتليل سيميائي تكتيكي حلكاية محال بغداد. اجلزائر . ديوان املطبوعات اجلامعية. 79 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 إشكالية املصطلح وداللته يف النقد العريب/ ط. د. أمهين نيشد اجمللد4 / العـــدد : 16 ( 2023 ،) Volume 4, Issue 16 13 -مطلوب, أ. (1989). يف معجم النقد العريب القدمي .ج1 .ط1. .بغداد دار الشؤون الثقافية العامة. 14مندور, م. (1996). النقد املنهجي عند العرب. . القاهرة دار هنضة مصر للطباعة و النشر و التوزيع. 15 -وغليسي, ي. (2008). إشكالية املصطلح يف اخلطاب النقدي العريب اجلديد. .اجلزائر الدار العربية للعلوم انشرون. ط1 . 16 -وغليسي ، ي. (2008). . إشكالية املنهج و املصطلح يف جتربة عبد املالك مراتض النقدية . منشورات االختالف . اجلزائر الدار العربية للعلوم. ط1 . ج- : الرسائل 1 -املذخوري, م. ف. (2004). أشكالية املصطلح البالغي دراسة تطبيقية يف املصطلح. . ماجستري السعودية. 2 -دقاجبي, ص. (2008 2009 , جامعة اجلياليل اليابس . إشكالية املصطلح السيميائي. ماجستري p 33 . د- : اجملالت ج- : الرسائل 1 -املذخوري, م. ف. (2004). أشكالية املصطلح البالغي دراسة تطبيقية يف املصطلح. . ماجستري السعودية. 2 -دقاجبي, ص. (2008 2009 , جامعة اجلياليل اليابس . إشكالية املصطلح السيميائي. ماجستري p 33 . د- : اجملالت 1 -املسدي, ع. ا. (n.d.). . االزدواج و املماثلة يف املصطلح النقدي . املنظمة العربية للرتبية و الثقافة و العلوم . تونس اجمللد13 . العدد24 2 - النبهان, ع. ( . ا1995 ) . األسس املوضوعية لنشأة املصطلح يف النقد العريب القدمي .احتاد الكتاب العرب . سوراي اجمللد15 . العدد59 . 30 افر يل1995 . 2 - النبهان, ع. ( . ا1995 ) . األسس املوضوعية لنشأة املصطلح يف النقد العريب القدمي .احتاد الكتاب العرب . سوراي اجمللد15 . العدد59 . 30 افر يل1995 . 3 - بوحسون, أ. (1989). . علم املصطلح جملة الفكر العريب املعاصر .العدد61 . مركز اإلّناء القومي . بريوت. لبنان. 4 -بوساحة, ع. (2005). . حتت أنقاض حداثة اليباب جملة املوقف األديب . العدد413 . . دمشق . سوراي 5 -مراتض, ع. ا. (1991). . فكرة السرقات األدبية ونظرية التناص جملة عالمات . العدد1 . . جدة . السعودية 6 -مراتض, ع. ا. (1996). بني التناص و التكاتب املاهية و التطور. جملة عالمات . العدد7 . .النادي األديب . جدة . السعودية 7 -مراتض, ع. ا. (2006). مفاهيم سيميائية مبصطلحات بالغية. جملة سيميائيات . العدد2 . . جامعة وهران . اجلزائر 80
https://openalex.org/W4295693130	https://periodicos.uff.br/antropolitica/article/download/53113/32618	Portuguese	null	Duas vezes Jorge: o silenciamento como permanência do autoritarismo na República Dominicana	Antropolítica	2,022	cc-by	12,597	Twice Jorge: silencing as the permanence of the authoritarianism in the Dominican Republic Twice Jorge: silencing as the permanence of the authoritarianism in the Dominican Republic Victor Miguel Castillo de Macedo Pós-Graduação em Antropologia Social, Universidade de São Paulo, São Paulo, SP, Brasil or Miguel Castillo de Macedo Graduação em Antropologia Social, Universidade de São Paulo, São Paulo, SP, Brasil Victor Miguel Castillo de Macedo RESUMO Neste artigo analiso a ausência de transição política na República Dominicana da década de 1960 através de fragmentos da vida de um revolucionário e seu filho. Este é um desdobramento de minha tese sobre ex-combatentes da Revolução de Abril de 1965 em Santo Domingo, capital do país. Durante a pesquisa de campo, conheci pessoalmente o senhor que era um pré-adolescente quando foi preso em 1967 pelo serviço secreto do governo de Joaquín Balaguer (1966-1978). A história, comentada nos jornais da época, levanta um problema quanto ao entendimento atual do que foi a revolução: se para muitos dos ex-combatentes foi bem-sucedida e hoje é reconhecida pelo governo dominicano como uma efeméride pátria, parte de seus efeitos foram silenciados. A transição democrática esperada pelos revolucionários se transformou em 12 anos de tirania, mesmo tendo sido chancelada pelo voto popular. O caso traz a chave temporal dos efeitos estendidos do pós- revolução de 1965, o retorno do autoritarismo e sua diluição num regime supostamente democrático. Meu objetivo é demonstrar que nas trajetórias desses revolucionários se encontram fragmentos e enredos que permitem um entendimento das permanências do autoritarismo na República Dominicana. Palavras-chave: Silêncio, Reparação, Autoritarismo, Transição política, Repúblic Dominicana. Palavras-chave: Silêncio, Reparação, Autoritarismo, Transição política, República Dominicana. Dominicana. 1 Este texto é uma versão com algumas modificações da reflexão apresentada primeiramente no GT “80 Transições Demo cráticas e Controle Social: repensando marcações temporais”, da 32ª Reunião Brasileira de Antropologia. Parte desta reflexão também compôs o quarto capítulo de minha tese de doutorado (CASTILLO DE MACEDO, 2021a). Vale frisar, no entanto, que apesar de semelhante pelos materiais que se utiliza, o texto tem direcionamentos distintos em seu objetivo e suas conclusões. A pesquisa que permitiu esse texto recebeu apoio do Projeto de Pesquisa de Bancada coordenado por Olivia Gomes da Cunha, com apoio da FAPERJ (CNE2018-E-26/202.758/2018) e do CNPq (EU-439103/2018-5). Durante a escrita recebi apoio da bol sa de pós-doutorado FAPESP (processo 2021/05444-0) com a supervisão de João Felipe Gonçalves. Aproveito para agradecer os excelentes pareceres da Revista Antropolítica, que permitiram o rearranjo do texto. Recebido em 11 de fevereiro de 2022. Avaliador A: 28 de abril de 2022. Avaliador B: 25 de maio de 2022. Aceito em 30 de junho de 2022. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 ABSTRACT In this article I analyze the absence of political transition in the Dominican Republic from the 1960’s from the fragments of the life of a revolutionary and his son. This is a development of my PhD dissertation about former combatants of the 1965 April Revolution, in Santo Domingo, the Dominican capital. During the fieldwork I met the man who was a teenager when he was arrested by Joaquín Balaguer government’s (1966- 1978) secret service in 1967. The story, addressed by the newspapers of that time, brings about a problem to the contemporary understanding of the revolution: while for many of the former combatants it was a successful endeavor, also recognized by the current government as a national event, part of this history remains silenced. The democratic transition expected by the revolutionaries turned into 12 years of tyranny, although it was endorsed by popular vote. The case presents the temporal key of the extended effects of the aftermath of the 1965 revolution, the authoritarian comeback, and its dissolution towards a supposedly democratic regime. I aim to demonstrate that in the stories of these elders one can find many common fragments and plots that allow an understanding of the permanencies of the authoritarianism in the Dominican Republic. 211 Victor Miguel Castillo de Macedo In this article I analyze the absence of political transition in the Dominican Republic from the 1960’s from the fragments of the life of a revolutionary and his son. This is a development of my PhD dissertation about former combatants of the 1965 April Revolution, in Santo Domingo, the Dominican capital. During the fieldwork I met the man who was a teenager when he was arrested by Joaquín Balaguer government’s (1966- 1978) secret service in 1967. The story, addressed by the newspapers of that time, brings about a problem to the contemporary understanding of the revolution: while for many of the former combatants it was a successful endeavor, also recognized by the current government as a national event, part of this history remains silenced. The democratic transition expected by the revolutionaries turned into 12 years of tyranny, although it was endorsed by popular vote. The case presents the temporal key of the extended effects of the aftermath of the 1965 revolution, the authoritarian comeback, and its dissolution towards a supposedly democratic regime. I aim to demonstrate that in the stories of these elders one can find many common fragments and plots that allow an understanding of the permanencies of the authoritarianism in the Dominican Republic. Keywords: Silence, Redress, Authoritarianism, Political transition, Dominican Republic. ABSTRACT INTRODUÇÃO “Tristeza não tem fim, felicidade sim...”. Eu acabara de conhecer Jorge Puello Soriano. Ao ouvi-lo cantar esse trecho da canção A felicidade, de Vinícius de Moraes e Tom Jobim, uma das músicas brasileiras que conheceu durante o exílio na Itália nos anos 1970, senti saudades de casa. Estávamos separados pelas grades de ferro da Fundación de Solidaridad con los Héroes de Abril (Fusha), em Santo Domingo, na República Dominicana. Aguardávamos a volta do presidente da fundação, Tirso Medrano, que havia saído para fazer um pagamento no banco com a chave da grade que nos separava. A voz marcante e bem ritmada do poeta, músico e dançarino foi uma surpresa agradável. Naquele momento, sem saber, eu ouvia a voz de uma das pessoas que sofreram diretamente com as consequências da revolução dominicana de abril de 1965. * * Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 212 Victor Miguel Castillo de Macedo O presente trabalho analisa fragmentos da vida de Jorge Antonio Puello Soriano (Don Jorge ou Jorgito), a pessoa mais jovem a ser torturada pelo governo dominicano nos anos 1960, e seu pai, Jorge Puello Soriano, El Men, revolucionário de 1965, como índices da ausência de transição política dominicana. Conheci Don Jorge durante a pesquisa de campo com ex-combatentes da revolução de abril de 1965 em Santo Domingo (capital da República Dominicana). Na pesquisa acompanhei o dia a dia da Fusha e sua luta pelo reconhecimento e pela reparação das condições de vida dos ex-combatentes idosos. Don Jorge era uma criança de 14 anos na primeira vez que foi preso pelas forças paramilitares do governo de Joaquín Balaguer (1966-1978) e tinha 15 ao ser torturado pela primeira vez. Sua história, comentada nos jornais da época, levanta um problema para o entendimento atual do que foi a revolução: há um enorme silêncio a respeito de diversas violências estatais e paraestatais que decorreram dos conflitos da revolução. Para explicar como a Revolução de Abril de 1965 se articula a uma linha temporal marcada por lutas contra o autoritarismo e a opressão na República Dominicana, retomo parte da trajetória do pai de Don Jorge. INTRODUÇÃO Apesar do nome homônimo, Jorge Puello, seu pai, é conhecido pelos ex-combatentes como El Men, devido à sua bravura na luta revolucionária (alcunha que poderia ser traduzida como “o cara”; no entanto manterei o uso do apelido em espanhol, para evitar a confusão com o filho). Através dos percursos de El Men e seu filho, quero explicitar a articulação entre o fim da ditadura de Rafael Leónidas Trujillo (1930-1961), a revolução de 1965 e os 12 anos da ditadura de Balaguer (antigo braço direito de Trujillo) iniciada em 1966. Ao passar da história do pai para o envolvimento do filho, acompanho também as articulações e movimentações das organizações da esquerda dominicana no período autoritário. As várias prisões que culminaram no exílio de El Men para a Europa, primeiramente na França e em seguida na Itália, são descritas de acordo com o nível de detalhe que pude captar em nossa conversa, via ligação de WhatsApp, uma vez que não pude voltar a campo, devido à pandemia de Covid-19. Apesar da condição adversa em que ocorreu o diálogo, sigo a indicação de Sidney Mintz, quando comenta que “o empreendimento etnográfico deve voltar-se para algum objetivo relacional” (MINTZ, 1984, p. 49). Ele segue explicando que fatos “dificilmente” existem sem relações. Com isso, o antropólogo quer dizer, ao defender o método de “história de vida”, que não só o acesso a determinados acontecimentos e sua narrativa ocorrem em campo; seu entendimento e possível enquadramento se dão por causa das relações construídas na pesquisa. Ainda que aqui não pretenda replicar uma análise de “história de vida”, esse apontamento metodológico ajuda a conectar diferentes períodos dominicanos. O que é exposto neste texto é um olhar sobre “aquilo que ficou do que foi vivido”, como propõe Cynthia Sarti (2019, p. 507). Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 213 Victor Miguel Castillo de Macedo O caso de Don Jorge é exemplar, porque procurei conviver com ele antes de apressar a conversa sobre seus itinerários. Só assim pude entender que, por suas relações, sua trajetória e a abominável violência que sofreu, sua história contém elementos comuns à experiência de muitos ex-combatentes e/ou pessoas ligadas à resistência democrática na República Dominicana. É por isso que a narração de sua trajetória expõe certa conformidade com a herança dos 12 anos de autoritarismo que se instalaram à medida que o tempo passou. Aqui dialogo também com as circunstâncias propostas por Christian Krohn-Hansen (2009, p. 5) para evitar a reprodução de análises clássicas sobre o autoritarismo: primeiro, não é um homem que detém todo o poder; segundo, a análise do regime deve se basear em dinâmicas do cotidiano; terceiro, Estados autoritários devem ser vistos como conjuntos de práticas culturais. O autoritarismo aparece, portanto, também como efeito de um conjunto de relações. O silenciamento, tanto em termos práticos (em nome da ordem) quanto no sentido analítico de dispositivos de saber-poder, como descrito no primeiro capítulo da obra de Michel-Rolph Trouillot (1995, p. 26-29), pode ser um meio para compreender as maquinações autoritárias. Meu objetivo é demonstrar a forma como o silenciamento dessas histórias fabrica uma realidade comum a muitos dominicanos e dominicanas. Um lugar onde não houve tribunais nem comissões para a busca de verdade, justiça e memória pede que escutemos seus sobreviventes a partir dos quadros que eles nos oferecem. Antes de passar às histórias do grande combatente El Men, quero fazer um comentário sobre a República Dominicana e a ausência de transições políticas de facto. 2 A presença de dominicanos em Nova York foi analisada pelo sociólogo Jesse Hoffnug-Garskoff (2013). O aumento de pedidos de visto no pós-revolução é um ponto que não abordarei neste texto. O papel do Brasil na Revolução de Abril de 1965, apoiando o exército estadunidense, foi observado por Thomas Skidmore (1982, p. 397) como uma prova das relações entre militares dos dois países. Carlos Fico também tece alguns comentários (2008, p. 34 e 155-166), mas é no pequeno livro de Raimundo Caruso que se encontra uma análise mais detida de EBULIÇÕES/EFERVESCÊNCIAS DOMINICANAS A Revolução de Abril de 1965 é um evento crítico no sentido explorado por Veena Das (1995, p. 6). Seus efeitos são multiplicidades espalhadas por passados e futuros, alcançando não só a paisagem de ruínas da cidade de Santo Domingo, mas também os bairros de Nova York (caso do predominantemente dominicano Washington Heights) e os silêncios da assombrosa história recente da ditadura brasileira2. A título de aproximação, vale a pena perpassar por alguns Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 214 Victor Miguel Castillo de Macedo acontecimentos e dados, para informar olhares brasileiros sobre as questões a serem tratadas. A República Dominicana divide uma ilha com o Haiti. Foi na porção de terra que tem Cuba a noroeste, Porto Rico a sudeste e Jamaica à leste que se consolidaram as primeiras instâncias/instituições coloniais/colonizadoras do hemisfério, tais como a cidade no modelo de tabuleiro, a catedral e a universidade, entre outras. O nome da ilha – para alguns, Hispaniola ou Quisqueya; para outros, Ayti – é objeto de controvérsias o suficiente para compreendermos que se trata de um território marcado por convulsões sociais3. Na região de Santo Domingo, hoje a capital da República Dominicana, em 1521, houve a primeira revolta de escravizados africanos na ilha (ACEVEDO, 2019). Praticamente três séculos depois, em 1804, a República do Haiti foi criada, com a libertação de todos os escravizados da ilha. Em 1822, o domínio haitiano expande-se ao lado do território que pertencia à Espanha. Essa ocupação permanece até 27 de fevereiro de 1844, data que marca a independência comemorada pelos dominicanos. Em 1861, parte da elite que restou do país articula o retorno do jugo espanhol, que durou até 1865. É o ano do fim da chamada Guerra de Restauração, quando os dominicanos conseguiram se livrar do domínio espanhol. Para recobrar as demandas econômicas de tantos anos em estado de guerra, produtores de tabaco e cana-de- açúcar do país recorrem ao pujante vizinho do norte – os Estados Unidos, nas últimas décadas do século XIX. Desde o início dessas relações, o interesse maior dos estadunidenses era anexar a ilha toda a seu império. Diversas justificativas eram mobilizadas, mas a tratativa não chegou a se concretizar. Não foi necessário. 3 Para um trabalho que explora as fundações do antagonismo e as solidariedades entre dominicanos e haitianos, ver a obra de Ann Eller (2016, p. 238) We dream together. Florestan Fernandes (1988), entre outras entrevistas e textos. EBULIÇÕES/EFERVESCÊNCIAS DOMINICANAS Devido aos créditos oferecidos aos produtores dominicanos, que não puderam dar conta das dívidas acumuladas, o governo dos Estados Unidos negociou o controle alfandegário dominicano com os governos da virada do século. Essa situação se arrasta até 1916. Naquele ano, os Estados Unidos, em nome dessas mesmas dívidas acumuladas, ocupam o território dominicano, obtendo o controle total do país. O mesmo ocorre com o vizinho Haiti, um ano antes. A ocupação se estende até 1924, quando muitas empresas estadunidenses se consolidam na produção de açúcar e a fronteira com o Haiti é delimitada e controlada pelos militares imperialistas. Nessa época, muitos dominicanos se formavam nas academias militares dos norte-americanos. Um deles foi Rafael Leónidas Trujillo – o ditador subiu ao poder em 1930 e, com o apoio dos Estados Unidos e da Igreja Católica, se tornou um dos ditadores mais violentos e poderosos de todo o hemisfério. Por meio de um Estado autoritário, empreendeu Florestan Fernandes (1988), entre outras entrevistas e textos. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 215 Victor Miguel Castillo de Macedo uma série de reformas econômicas e de infraestrutura para modernizar a economia dominicana. O crescimento econômico que gerou também foi lucrativo para sua família, que passou a ser a principal acionista de todas as empresas estatais e a maior detentora de terras do país. Christian Krohn-Hansen (1997), inspirado por Maurice Bloch, faz uma leitura a respeito do simbolismo de violência que contém esse emaranhado de agenciamentos sob a alcunha de Estado no caso dominicano: sua origem, para o autor, estaria no massacre de mais de 30 mil haitianos na fronteira do país com o Haiti em 1937. Como um sacrifício inaugural do Estado enquanto detentor do monopólio da violência, o episódio legitima um tipo de poder que, segundo a leitura de Krohn-Hansen, se reproduz de diversas outras formas na vida dominicana até hoje. Talvez o rito do massacre tenha performado o caráter racializado do Estado dominicano. No entanto, essa relação não se resume aos termos Haiti-República Dominicana; contém também os Estados Unidos como outro ponto de apoio. Somente em 1961 o ditador foi assassinado ou “ajusticiado”, como dizem os dominicanos progressistas. Boa parte do aparato estatal autoritário que ele criou se manteve, bem como o cultivo de um nacionalismo anti-haitianista/racista e uma aversão anticomunista. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 EBULIÇÕES/EFERVESCÊNCIAS DOMINICANAS O país conheceu suas primeiras eleições democráticas em 1962, quando foi eleito Juan Bosch, intelectual opositor do antigo regime de Trujillo. A Constituição aprovada pelo governo Bosch tinha como um de seus diferenciais a separação entre Estado e Igreja. Seu governo propôs também uma série de reformas para dirimir os abismos sociais do país. Iniciado em janeiro de 1963, não passou do mês de setembro. Bosch foi deposto por uma articulação entre militares e grupos da oligarquia dominicana, inclusive alguns que concorreram contra ele nas eleições de 1962. Em seu lugar, foi posto um triunvirato formado por notáveis da sociedade que não durou até o ano de 1964 e foi dissolvido num Conselho de Estado. O conselho era encabeçado por Donald Reid Cabral, jovem empresário e membro da oligarquia. Seu breve comando foi marcado por muitas greves e pela crise econômica, devido à seca nos campos de cana-de-açúcar. Muitas articulações foram feitas para derrubar o Conselho de Estado. Foi necessária uma revolta entre soldados, tenentes, sargentos e outros membros do baixo escalão do exército dominicano, em 24 de abril de 1965, para que as tensões escalassem ao combate franco. Naquele dia, no programa de rádio de destaque nacional de José Francisco Peña Gómez, ouviu-se a convocação do povo às ruas em apoio ao grupo de militares. A consignação do grupo era o retorno à Constituição de 1963 e a volta de Juan Bosch à Presidência. Começando no sábado na hora do almoço, a rebelião reuniu populares e grupos de esquerda favoráveis à causa. As primeiras batalhas serviram para impedir que as tropas leais ao governo oligarca Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 216 Victor Miguel Castillo de Macedo entrassem na capital. O palácio da Presidência foi ocupado e todo o centro da cidade de Santo Domingo já pertencia aos rebeldes. Em meio a esses combates, o nome de Francisco Alberto Caamaño, um jovem militar de uma família próxima ao ex-ditador, se destacou como uma liderança importante. Foi acordado que, enquanto Bosch não voltasse, Caamaño seria o novo presidente do país e líder da revolução. Na quarta-feira da semana seguinte (28 de abril), os primeiros soldados estadunidenses desembarcavam no país em nome da segurança de seus cidadãos. Caamaño e seus companheiros do “comitê de transição” – primeiro nome do governo revolucionário, uma vez que no início imaginavam que a revolta não duraria mais que alguns dias – haviam pedido apoio ao embaixador dos Estados Unidos. Entretanto o retorno de Bosch poderia significar “uma nova Cuba” no Caribe. A luta perdurou até setembro de 1965. Em torno de 30 mil soldados estadunidenses foram mobilizados. Além dos EUA, governos alinhados ao imperialismo como Brasil, Honduras, Paraguai, Nicarágua e Costa Rica enviaram contingentes menores para formar a Força Interamericana de Paz (FIP). Do Brasil partiram 1.300 soldados – o segundo maior contingente enviado ao país4. Ambos os lados tiveram muitas baixas, mas os revolucionários se viram numa situação mais fragilizada pela falta de recursos, treinamento e poderio militar. Em setembro foi assinado um acordo que determinou novas eleições em 1966, além da anistia aos revolucionários. Minha pesquisa investigou a fabricação da noção de ex-combatentes enquanto parte dos efeitos da revolução, no contexto da Fundación de Solidaridad con los Héroes de Abril (Fusha). A fundação trabalha com a produção de relatos a respeito dos civis ex-combatentes dos bairros operários do período da revolução. Apesar de seus membros serem respeitados por outras organizações e pesquisadores da revolução, suas condições são precárias e a Fusha só foi reconhecida pelo governo dominicano no ano de 2021. O acontecimento fundante desses agenciamentos (a revolução) é tomado aqui como uma tragédia, no sentido que o antropólogo jamaicano David Scott atribui à Revolução de Granada, em que “a ação trágica costura preocupações com o tempo da reparação justa” (SCOTT, 2014, p. 28). 4 Honduras enviou 250 soldados, Paraguai, 178, Nicarágua, 159, e Costa Rica, 21 policiais. O menor contingente foi enviado por El Salvador, com apenas três militares. 5 A reflexão do livro de David Scott se aproxima dos temas tratados aqui; é difícil definir somente um ponto de 5 A reflexão do livro de David Scott se aproxima dos temas tratados aqui; é difícil definir somente Scott inspira-se nos debates desenvolvidos pela filosofia ocidental e pela teoria política contemporânea a respeito da irredutibilidade da história ao tempo (vice-versa) para expressar a necessidade de compreender questões que envolvem traumas, memória e justiça como partes de uma crise de tempo e experiência temporal (SCOTT, 2014, p. 22)5. No caso da ilha caribenha de 4 Honduras enviou 250 soldados, Paraguai, 178, Nicarágua, 159, e Costa Rica, 21 policiais. O menor contingente foi enviado por El Salvador, com apenas três militares. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 217 Victor Miguel Castillo de Macedo Granada, Scott descreve a tragédia dos desentendimentos internos de uma revolução socialista que termina com uma invasão comandada pelas tropas de Ronald Reagan. Sua leitura é também informada pela análise que fez em sua obra anterior, dedicada ao clássico Os jacobinos negros, de C. L. R. James, a respeito da Revolução Haitiana (SCOTT, 2004) – inspira-se, ainda, nas tragédias gregas, sobretudo nas mudanças da segunda edição dessa obra clássica. Quanto à ideia de transição, Scott questiona o “autoentendimento ideológico da justiça transicional e sua relação com as circunstâncias numa ordem global liberalizante” (SCOTT, 2014, p. 135, tradução nossa). Ao observar o fim da revolução socialista iniciada em 1979 na ilha de Granada, Scott explora as sequências de fatos que levaram à morte de Maurice Bishop, líder do New Jewel Movement (NJM), em 1983. Em seguida, o exército norte-americano invadiu a ilha para controlar os conflitos entre civis e membros das diferentes linhas do movimento libertador. Além de dissolver e controlar o governo, os estadunidenses atribuíram o mando dos crimes políticos a um grupo de 17 ex-dirigentes do NJM. Condenados à época, hoje estão soltos, após as revisões das condenações. David Scott procurou problematizar as dimensões morais do que foi tomado como um caso de justiça transicional ou de transição bem-sucedido. Classificado como uma “transição política liberalizante”, o caso de Granada é um dos primeiros experimentos dessa engenharia política de transformação de regimes “iliberais”. Aqui ele é tomado como referencial por sua proximidade temporal e geográfica com a República Dominicana. Do mesmo modo, o incômodo com a ideia de transição na crítica de Scott a autoras como Ruti Teitel, uma das criadoras da noção de “justiça de transição”, por conta da forma espetacularizada do julgamento dos “17 de Granada”, é algo que pretendo conservar como pano de fundo. conexão. Para evitar uma exposição pormenorizada das questões locais ou teorias que o autor trata, portanto, indico que minha inspiração reside na constatação nada ingênua de que tragédias são o resultado de uma pluralidade de ações concatenadas. Da colisão de tais ações, surgem suas justificações e seus efeitos (SCOTT, 2014, p. 22, tradução nossa). Aquilo que Scott chama de “presságios de adversidade” em sua análise da derrocada da Revolução de Granada comportaria um trabalho da significação dos fatos e de modulação semântica que se aproxima das formas de silenciamentos descritas por Trouillot (1995). Scott critica a defesa irrefletida do liberalismo como universal civilizacional. E argumenta que a aparente incapacidade de aplicar os critérios da justiça de transição a regimes supostamente democráticos como o dos Estados Unidos (marcado por políticas do mal como torturas e genocídios) expõe a arbitrariedade dessas leituras. No contexto discreto dessa reflexão, as experiências de Don Jorge Puello Soriano são trazidas de modo a oferecer uma perspectiva crítica a respeito do caso dominicano, em que os seus algozes não experimentaram nenhum tipo de responsabilização por seus atos. Nem a Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 6 Aqui me inspiro na discussão empreendida pelas organizadoras da coletânea “Trouillot Remixed”, intitulada “Overture” (2021). Elas relacionam diretamente a ideia de “provincializar a Europa”, do historiador Dipesh Chakrabarty (2000), com o que Michel-Rolph Trouillot fez ao longo de sua obra. O uso do termo é próximo do que propõe o historiador: tomar determinada experiência histórica (do Atlântico Norte) pela sua especificidade e estranhar a sua centralidade para a interpretação e entendimento de acontecimentos em outros lugares. 218 Victor Miguel Castillo de Macedo violência da revolução de 1965, nem o período de 12 anos de autoritarismo de Joaquín Balaguer foram submetidos à comissões de verdade, memória e justiça. A transição, para David Scott, bem como sua ausência na República Dominicana, ressoa à perspectiva de Michel-Rolph Trouillot diante de palavras como “democracia ou progresso”, que ele chama de “ficções universais do Atlântico Norte” (TROUILLOT, 2003, p. 35). Tais palavras dizem muito mais sobre experiências locais ou particulares. A forma como esses termos e expressões nos afetam está ligada aos processos que Trouillot chama de “geografia da administração” e “geografia da imaginação” (TROUILLOT, 2003, p. 38). Ambas, entrelaçadas em seu funcionamento, fabricam a distribuição espacial de uma administração da imaginação que nos impede de provincializar experiências e palavras (vide “transição”, “democracia”, “liberalismo”)6. Isso sem mencionar as situações em que pedidos coletivos de desculpa histórica performados por Estados terminam por produzir uma espécie de “ritual abortivo” (TROUILLOT, 2021), tão comum nas últimas décadas do século XX e no início do XXI (AHMED, 2005). No caso dominicano, experimentos políticos que não tem o liberalismo nem os Estados Unidos como medida para existir tendem a ser silenciados. Meu esforço é apontar para essa prática de silenciamento como parte da longa história autoritária dominicana. Para entender como Don Jorge se envolve nos movimentos políticos da época, mesmo tão jovem, portanto, vale a pena retornar às redes de relações de seu pai, El Men. EL MEN, UM HERÓI DA REVOLUÇÃO Na conversa que tivemos via WhatsApp, Don Jorge não trouxe muitos elementos para um relato substancial sobre seu pai. Por isso utilizarei algumas das histórias que estão nos relatos que Tirso Medrano, presidente da Fusha, fez a partir de sua entrevista com El Men. Como figura que é parte do panteão dos heróis revolucionários de 1965, ele foi entrevistado muitas vezes. Por isso, já há um corpo de informações conhecidas sobre ele. Jorge Puello Soriano (pai) nasceu em 1925. Durante a ditadura de Trujillo, trabalhava na principal fábrica de sapatos da capital, uma das mais antigas no setor industrial da República Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 219 Victor Miguel Castillo de Macedo Dominicana. Jorgito complementava a renda com trabalhos como sapateiro que realizava em casa e desde a infância começou a se vincular a movimentos políticos contrários à ditadura. Como disse meu interlocutor: “era um homem alto, forte e musculoso – de personalidade forte”. Através de alguns conhecidos como Andrés Ramos Guerrero, que lutou com Fidel Castro em Cuba, se conectou aos grupos socialistas do Caribe. Após a morte do ditador Trujillo, El Men decide se filiar ao Movimento Popular Dominicano (MPD), um partido de denominação marxista-leninista criado em Cuba em 1956. Em 1962, ele vai a Cuba para receber treinamento militar. No ano seguinte, apesar de fazer parte de um grupo crítico ao governo liberal de Juan Bosch, El Men, junto com o MPD, se opõe ao golpe de Estado sofrido pelo primeiro presidente eleito no pós-ditadura. Nessa época, Don Jorge foi se envolvendo com diversas ações do meio de atuação de seu pai: acompanhava reuniões e levava os coturnos feitos por seu pai para costurá-los, antes do envio aos combatentes. El Men foi preso em 1963 por apoiar as guerrilhas que se formaram em oposição ao governo golpista. A principal delas foi o Movimiento Revolucionario 14 de Junio, que teve parte de seus guerrilheiros mortos nas montanhas. Esse movimento era composto em sua maioria por jovens de classe média e média alta que fizeram parte da oposição ao ditador Trujillo. O líder, Manolo Tavárez Justo, foi um dos mortos na ação ocorrida em 1963. Ficou conhecido primeiramente por perder sua esposa, Minerva Mirabal, assassinada pelo ditador junto com suas irmãs7. Depois da queda de Trujillo, Tavárez Justo se projetou como uma das grandes lideranças do país. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 EL MEN, UM HERÓI DA REVOLUÇÃO Após a morte do líder do 14 de Junio, a perseguição aos simpatizantes se tornou mais intensa. Don Jorge contou que as primeiras manifestações de que participou foram nesse período. Foi seguindo o movimento pela anistia aos presos políticos que seu envolvimento começou a ganhar contornos mais significativos: passou a prestar mais atenção aos conceitos que permeavam os debates da esquerda dominicana. El Men foi solto em 1964 e voltou às atividades de mobilização contra o governo do Conselho de Estado. Como afirmei anteriormente, eram muitos os planos para derrubar o governo encabeçado por Donald Reid Cabral. Ainda assim, o estouro da revolução pegou muitos grupos de surpresa. Tirso Medrano relatou a maneira como El Men soube da revolução de 1965: Estando no pátio da minha casa, por volta das três da tarde, chegaram Marcos Santana Estando no pátio da minha casa, por volta das três da tarde, chegaram Marcos Santana Estando no pátio da minha casa, por volta das três da tarde, chegaram Marcos Santana 7 A história das irmãs Mirabal repercutiu de tal forma que foi um dos principais fatores que levaram à derrocada do ditador Trujillo. O 25 de novembro de 1960 em que elas foram mortas serviu de referência para a definição do Dia Internacional do Combate à Violência Contra a Mulher. Para uma versão ficcional da história delas, ver Júlia Alvarez (2001). 7 A história das irmãs Mirabal repercutiu de tal forma que foi um dos principais fatores que levaram à derrocada do ditador Trujillo. O 25 de novembro de 1960 em que elas foram mortas serviu de referência para a definição do Dia Internacional do Combate à Violência Contra a Mulher. Para uma versão ficcional da história delas, ver Júlia Alvarez (2001). Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 202 220 Victor Miguel Castillo de Macedo e Teresa Rojas para me dizer que Peña Gómez estava exortando o povo a se lançar às ruas. Eu era membro do comitê central do MPD, e fui imediatamente à escola Arzobispo Valera, onde começamos a nos reunir Maximiliano Gómez (El Moreno), Otto Morales, Prim Montás, Baldemiro Santana, Pablo Robles, Pachiro e outros membros mais cujo nome não me lembro agora [...], mas eu ainda não tinha armas. No dia 26 de abril, estando na rua Benigno Del Castillo, esquina com Salcedo, vejo que vem um guarda que se via cansado e disse a mim mesmo “esse é o meu fuzil”. Me aproximei dele e disse “irmão, vem cá, você está cansado!” e fiz ele se sentar num colmado [boteco/mercearia]. Ofereci um refresco e aí lhe tirei a metralhadora San Cristóbal. Essa foi minha primeira arma. (MEDRANO, 2016). Os revolucionários se organizaram em grupos chamados comandos. Eram agrupamentos quase segmentares, seguindo a lógica das relações interpessoais dos combatentes (grupos familiares, vizinhos, amigos de bairro) e/ou formatos de agregados institucionalizados (partidos, sindicatos, agrupamentos militares). O que importa para a discussão é o diferencial das posições internas nos comandos. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Estando no pátio da minha casa, por volta das três da tarde, chegaram Marcos Santana Ser o comandante não só oferecia reputação, também colocando à prova a capacidade de liderar combatentes mal treinados, com armas e munições limitadas, diante de boa parte do exército dominicano, das tropas estadunidenses e de alguns destacamentos de outros países latinos (como o Brasil). El Men foi o comandante do “comando do MPD”. Ao longo das batalhas, se firmou como um líder e combatente de respeito. Uma das histórias que contou a Tirso Medrano e Don Jorge também mencionou em nossa conversa ocorreu no dia 6 de maio de 1965, quando um grupo de soldados ianques, com um comboio blindado e um jipe, avançaram sobre a zona constitucionalista (título do território dos rebeldes, uma vez que defendiam a Constituição de 1963). A ordem do líder da revolução, Francisco Caamaño, era abater qualquer estadunidense que entrasse nos perímetros dos revolucionários. El Men habilmente enviou dois motoqueiros para averiguar a situação, descobrir o número de homens e o tipo de armas que levavam. Segundo conta, eram mais ou menos sete soldados, bem armados com metralhadoras calibre 50, um canhão 105 mm no Jipe e fuzis AR-15. O comando do MPD incluía 16 homens com metralhadoras Thompson, San Cristóbal, revólveres Enriquillo e fuzis Mauser. Apesar da diferença numérica, aspectos relacionados às armas, aos equipamentos e à preparação exigiam cuidado no ataque (como disse o comandante, foi uma briga nas sombras). A guerra de guerrilha se caracteriza por essas táticas de provocação e emboscada para o aniquilamento. O ataque foi rápido; durou menos de 20 minutos, mas foi o suficiente para matar três soldados invasores. Como não houve baixas da parte do comando do MPD, o então presidente Caamaño condecorou El Men por sua astúcia e sua tática. Esse ataque também foi descrito no livro do jornalista Tad Szulc, correspondente do The New York Times na época (SZULC, 2015, p. 194). Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo 221 Ainda na revolução, ele foi convocado a levar a luta para o interior do país. Além de chegar com alguma dificuldade à segunda maior cidade dominicana, Santiago de los Caballeros, foi preso junto com seus companheiros e levado por avião até a base onde estavam os estadunidenses. Lá testemunhou o enorme contingente mobilizado para derrubar o esforço revolucionário: de lá partiam de 20 a 30 helicópteros por dia. Após a revolução, a luta continuou para El Men. Talvez seja possível dizer que a situação piorou nos anos seguintes. O melhor resumo vem do próprio El Men, quando conta a Tirso Medrano que, depois de 1965, “passava mais tempo preso do que solto”. De tantos golpes que recebeu, terminou a vida cego. Na entrevista ele afirma perdoar a todos os seus detratores em nome do objetivo final de suas lutas, a “torre universal do socialismo”. No livro De la calle a los estrados por justicia y libertad (2008), o advogado que defendeu El Men ao longo dos anos Balaguer, Ramón Antonio (Negro) Veras, relata que o ex-combatente foi submetido a todo tipo de humilhação. Chegou a ficar três meses sem ver o sol e recebia alimentos uma vez ao dia. Além das torturas, teve o tempo na sua prisão prorrogado, com a postergação dos julgamentos. No dia 3 de setembro de 1965, o coronel Caamaño resumiu bem o resultado imediato da revolução em seu último discurso como presidente: “Porque o povo me deu o poder, ao povo venho devolver o que lhe pertence. Não pudemos vencer, tampouco pudemos ser vencidos”. Não pôde permanecer no país, no entanto. Foi “enviado” como embaixador para Londres. As eleições em 1966 foram marcadas pela violência dos grupos articulados com Joaquín Balaguer. Juan Bosch se candidatou novamente, mas, devido às ameaças constantes, decidiu fazer a campanha pelo rádio de sua casa. O resultado foi a eleição de Balaguer e a readequação das estruturas estatais a uma lógica a que já estavam acostumadas. Como fora o braço direito do ditador Trujillo, Balaguer pôde dar continuidade ao projeto fundado por seu mentor8. O uso da força foi realocado para um espaço narrativo tanto mais tecnocrático quanto mais romântico. Balaguer não se preocupava em atuar uma performance de dominância, no sentido de um poder masculino. 8 Em minha pesquisa de doutorado, concordo com a análise de Christian Krohn-Hansen sobre esse período conhecido na República Dominicana como “trujillismo sem Trujillo” (KROHN-HANSEN, 2009, p. 2). Apesar de o livro em que escreve sobre essas ligações ter argumentos próximos aos meus, objeto que sua abordagem de uma localidade mais próxima à fronteira sudoeste com o Haiti demandaria questões que fogem ao escopo deste texto. As realidades descritas nesta reflexão pertencem ao contexto da capital, um contexto, portanto, mais urbano. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Ele era a vítima de suas vítimas. Era uma relação esquizoide que fabricou traumas, tragédias e mortos sob um véu de docilidade (retornarei a esse ponto). Trujillo, por sua vez, não demonstrava passividade ou reatividade nesses casos. Era o varão da nação, segundo Lauren Derby (2009). 8 Em minha pesquisa de doutorado, concordo com a análise de Christian Krohn-Hansen sobre esse período conhecido na República Dominicana como “trujillismo sem Trujillo” (KROHN-HANSEN, 2009, p. 2). Apesar de o livro em que escreve sobre essas ligações ter argumentos próximos aos meus, objeto que sua abordagem de uma localidade mais próxima à fronteira sudoeste com o Haiti demandaria questões que fogem ao escopo deste texto. As realidades descritas nesta reflexão pertencem ao contexto da capital, um contexto, portanto, mais urbano. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo 222 Na entrevista concedida à Tirso Medrano, El Men, já com 91 anos, pouco tempo antes de seu falecimento, procurou evitar o rancor. Ressaltou o respeito, o reconhecimento de seus inimigos e até de alguns carcereiros entre as muitas histórias contadas. Apesar das entrevistas, dos livros e das comemorações em seu nome, parece que sua trajetória de lutas terminou por ocultar as violências sofridas por seu filho Jorgito. Em uma matéria publicada no periódico Acento de 14 de abril de 2016, que acompanha a foto exposta abaixo, a história de Don Jorge é apresentada a partir do mesmo título dos jornais da época de sua primeira prisão: “O anjinho negro: o menino que foi preso político nos doze anos de Balaguer” (RAMOS, 2016, on-line, tradução nossa). O texto escrito pelo historiador Alejandro Paulino Ramos oferece um exemplo dos efeitos de ocultamento que as ações de Jorgito sofreram. Apesar de falar sobre ele, o texto traz El Men como o grande sujeito de toda a trama de acontecimentos. Para complementar as informações, o historiador se baseou numa entrevista feita pelo Departamento de História Oral do Archivo General de la Nación (AGN) em 2015. Figura 1. El Men numa entrevista, alguns anos antes de seu falecimento Figura 1. El Men numa entrevista, alguns anos antes de seu falecimento Fonte: site Acento (2016). Fonte: site Acento (2016). A ausência de Jorge Puello filho, o sujeito do tema da matéria, seja na ausência de imagens, seja na falta de indicações a respeito de sua vida contemporânea, opera como a consolidação de seu lugar na página de um arquivo ausente. Trouillot comenta que “presenças e ausências corporificadas nas fontes (artefatos e corpos que transformam o evento em um fato) ou arquivos (fatos coletados, tematizados e processados como documentos e monumentos) Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo 223 não são neutros nem naturais” (TROUILLOT, 1995, p. 48, tradução nossa). O silêncio como processo “ativo e transitivo”9, nesse caso, cala sobre a trajetória posterior à primeira prisão de Don Jorge. A única foto contemporânea da matéria é a de El Men, já com idade avançada. Dirijo minha atenção à voz de Don Jorge a seguir, para compreender as formações do silenciamento em sua experiência. 9 Trouillot estabelece quatro momentos em que os silêncios operam na produção histórica: na criação do fato (elaboração de fontes); na composição do fato (elaboração de arquivos); na recuperação dos fatos (elaboração de narrativas); e na significância retroativa, a elaboração da história em última instância (TROUILLOT, 1995, p. 61). Ele chega a essas sínteses depois de propor uma teoria da narrativa histórica que compreenda processo e narrativa. Nos materiais produzidos com Jorgito, o filho, há tanto os momentos de elaboração das fontes como sua significância retroativa. Ou seja, não pude acompanhar diretamente a elaboração dos arquivos nem a recuperação dos fatos em narrativas. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 EL ANGELITO NEGRO A conversa que tivemos ocorreu depois que voltei ao Brasil. O contato foi facilitado pelo vizinho de Don Jorge (o venezuelano Jesús, que emprestou seu celular para a nossa conversa). Através de uma ligação, ele respondeu durante mais ou menos duas horas a algumas provocações e perguntas. Pedi, de início, que me contasse sobre seu pai e a relação com a militância que herdou dele. Não demorou muito para que contasse alguns de seus sonhos, as lutas que enfrentou e as mudanças de perspectiva que vivenciou a partir do exílio. O enredo de sua narrativa sobre si parece ter sido organizado intencionalmente em três momentos: no primeiro, se envolve com as atividades revolucionárias; no segundo, expande suas visões de mundo, movido pela experiência do exílio; no último, vive a melancolia dos últimos anos de volta à terra natal. Na introdução, mencionei que, nascido em 1951, um Don Jorge pré-adolescente já participava com seu pai das movimentações políticas que seguiram à morte do ditador Trujillo. Quando estourou a revolução, quis continuar ativo, mas seu pai não permitiu, mesmo que fosse para buscar armas ou ajudar a roubá-las de membros do Estado dominicano. Por isso, quando o conflito aberto de 1965 cessou, Jorgito se envolveu de maneira mais direta com os movimentos estudantis e a organização de células do MPD nas escolas públicas. Ele descobriu, porém, que suas atividades vinham sendo monitoradas. Em 1967, seu Ele descobriu, porém, que suas atividades vinham sendo monitoradas. Em 1967, seu 10 Na conversa, ele mencionou outras situações em que sua habilidade com as palavras mobilizou justificativas para o uso da força para manter a ordem: a morte de Francisco Caamaño em 1973, também nas montanhas, reunindo guerrilheiros para derrubar o regime, quando o ditador disse que “não havia prisão grande o suficiente para recebê-lo”; e o assassinato do líder estudantil universitário Orlando Martinez, cujos artigos jornalísticos “não o deixavam trabalhar”. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo 224 pai decide tirá-lo do país, para que continuasse seus estudos em Cuba. Eles organizaram em segredo uma viagem rumo à França, de onde tomariam o voo para Cuba. No entanto, quando arrumavam as malas para partir, um espião da inteligência do governo os seguiu e alertou as autoridades. Don Jorge foi retirado de dentro do avião com outros dois revolucionários adultos, companheiros de seu pai. Sua mãe, que desconhecia os planos, descobriu pela rádio que o filho fora preso. Em sua bagagem havia documentos sobre a esquerda dominicana, a ser entregues a membros de organizações socialistas e comunistas europeias. Foram utilizados por Balaguer para denunciar o MPD e outras organizações de ferir a soberania nacional. Em fevereiro de 1967, o então presidente fez uma declaração em que acusava os membros do partido de aliciarem jovens como o “Angelito Negro”. Ao me contar da repercussão nacional de sua prisão, Don Jorge explica que foi um acontecimento apoteótico em sua vida. Para ele, Balaguer, com sua habilidade de comunicador e intelectual, se utilizou do eufemismo “angelito” para expressar o que na verdade queria dizer: “diablito” (diabinho)10. A pretensão de suavizar as acusações voltadas ao menino foi bem-sucedida – o que preocupava eram os “comunistas maus”, aliciadores de crianças; nesse meio, o presidente autoritário seria a vítima. Essa história é recuperada no texto de Alejandro Paulino Ramos. Aliás, consolida-se nessa recuperação um certo congelamento da primeira prisão de Don Jorge no conjunto de eventos dessa época, um confortável lugar no passado que parece não incomodar o presente. Diferentemente da imagem trazida acima, do velho revolucionário El Men ativo, mesmo cego, Jorgito parece ter se tornado um tema do passado. Algo do uso dessas imagens parece reiterar aquilo que os próprios apelidos indicam: El Men, “o cara” ou “o homem” ainda passível de representação; por outro lado, Jorgito, um pequeno anjo negro quando foi preso, surge como um quadro do passado, irrelevante para o presente. Do mesmo modo, os opositores da atuação de cada um deles indicam tipos distintos de disposição corporal: El Men contra Trujillo (uma disputa masculinizada, de varões); e Jorgito contra Balaguer (ambos menos vigorosos que seus antecessores, supostas “vítimas do comunismo” na época do embate). 10 Na conversa, ele mencionou outras situações em que sua habilidade com as palavras mobilizou justificativas para o uso da força para manter a ordem: a morte de Francisco Caamaño em 1973, também nas montanhas, reunindo guerrilheiros para derrubar o regime, quando o ditador disse que “não havia prisão grande o suficiente para recebê-lo”; e o assassinato do líder estudantil universitário Orlando Martinez, cujos artigos jornalísticos “não o deixavam trabalhar”. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 225 Victor Miguel Castillo de Macedo Figuras 2 e 3 – Capa do jornal El Nacional após a primeira libertação de Jorgito (1967); ficha da detenção de Jorge Puello (2016) Figuras 2 e 3 – Capa do jornal El Nacional após a primeira libertação de Jorgito (1967); ficha da detenção de Jorge Puello (2016) Fonte: site Acento (2016). Fonte: site Acento (2016). Fonte: site Acento (2016). Fonte: site Acento (2016). A imprensa da época se surpreendeu com a capacidade do pequeno Jorgito de mobilizar conceitos marxistas como a dialética para justificar seu envolvimento nas atividades do MPD. No mesmo ano, Jorge Puello voltou a ser preso e passou a ser torturado “por até 4 horas, com golpes na cabeça”, como me disse. Dos 15 aos 18 anos, foi preso diversas vezes, e na maioria das vezes quem o torturava era o chefe do serviço secreto. Esses detalhes, ausentes da matéria do site Acento, reiteram a continuidade da violência dos anos de Trujillo no período Balaguer – uma violência especialmente direcionada a corpos negros. À medida que as detenções ocorriam, como Don Jorge também me descreveu, maior era sua ligação com as discussões da esquerda dominicana naquele período. Para ele, o principal intelectual operário que se produziu dentro das estruturas do MPD foi Maximiliano Gómez El Moreno, “obrero, negro y muy humilde”, conforme me descreveu. As ideias de El Moreno, que defendiam a nacionalização das problemáticas marxistas para o contexto dominicano, foram a maior inspiração de Jorge em seus anos de formação. Após a morte de Che Guevara em 1967, a tática dos focos revolucionários já não bastava, segundo o pensador. Era necessário forjar alianças estratégicas com outros setores para derrubar o ditador, ultrapassando as querelas entre Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo 226 maoístas e leninistas. Sua tese era de que o país vivia em um “subdesenvolvimento econômico híbrido”, formado pelo capitalismo desenvolvido, o protocapitalismo e o semifeudalismo. As críticas ao que El Moreno chamou de colonialismo ideológico da esquerda dominicana chegaram à “alma e ao coração”, me disse Jorge Puello. Inspirado por essas ideias, ele continuou trabalhando para o MPD, ao longo da segunda metade da década de 1960, organizando a Unión de Estudiantes Revolucionários (UER). Em 1969, esteve encarcerado na famosa La Victoria, prisão criada por Trujillo onde também estava El Moreno. Nessa ocasião, os membros do partido sabiam que a prisão do líder era uma estratégia do regime balaguerista para matá-lo. Em resposta, sequestraram o embaixador estadunidense Joseph Crowley, demandando que os membros do partido presos fossem enviados a Cuba. Antes que as negociações terminassem, Jorge conseguiu um habeas corpus. Uma vez fora da cadeia, ele tentou articular um grupo paramilitar. Seu pai, que estava no interior, mobilizando e organizando grupos sindicais no norte do país, onde se produz tabaco, mandou buscar Jorge, para que se escondesse por um tempo fora da capital. O início dos anos 1970 foi marcado por uma escalada da violência do regime de Balaguer, e a resposta de seus opositores também mudou de tom. Em 1973, com a morte de Caamaño, houve muitas divisões no MPD. Em sua última prisão, nesse mesmo ano, Don Jorge foi deportado para a França. Um novo horizonte de expectativas começara a se desenhar a partir dessa mudança em sua trajetória. Meu interlocutor foi para a França com três ou quatro companheiros. Como o partido estava mais organizado na Itália, eles foram para lá depois de três meses. Como delegado, ele participava da estrutura internacional do partido e enviava informes para o MPD na República Dominicana. Ele não detalhou sua saída dos quadros do partido. Somente explicou que já não via mais sentido na militância longe do país e que havia passado muitos anos discutindo as mesmas ideias. Nessa época, se vinculou à contracultura italiana e acompanhou a chegada das ideias do guru Osho no país, sem se engajar na organização destes movimentos. Envolveu-se com expressões artísticas como o teatro e a música. Andava sempre com um bongô, o que o levou a ser convidado para participar de um grupo de salsa, a convite do equatoriano Juan Lopez, que conheceu numa festa. No grupo, cantava e dançava. Juntos fizeram apresentações em diversos lugares da Itália e da Europa. Com a popularidade, também conseguiu trabalhos como professor de salsa. Disse-me que, por ser negro, chamavam-no para muitos trabalhos – para além desses, se sustentava como cozinheiro, lavador de carros, professor de espanhol e atendente de livraria. Entre essas diversas atuações, chegou a aparecer na televisão na época em que vivia na região Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 de Milão. No início dos anos 1980, foi da Itália para o México e se apaixonou pelo país e sua cultura indígena. Foi quando, conforme me disse, o “bichinho da viagem” o picou. Foi para Guatemala, onde conheceu argentinos com os quais montou um grupo musical. Com eles foi até Honduras; de lá, partiu sozinho para a Nicarágua, descendo até o Panamá, passando pela Costa Rica. Do Panamá, voltou brevemente para a República Dominicana, em 1981, nessa época governada por António Guzmán Fernandez, do Partido Revolucionário Dominicano (PRD), primeiro partido de Juan Bosch. Foi o primeiro governo eleito depois do domínio de Balaguer, que saiu como se os 12 anos no poder tivessem sido legítimos. Naquele momento, a economia dominicana já estava completamente comprometida com o capital de empresas multinacionais. A tentativa balaguerista de fortalecer a burguesia industrial nacional – que o sociólogo Wilfredo Lozano (2018) chamou de “reformismo dependente” – terminou por vincular forças econômicas estrangeiras à burguesia financeira. Como muitos dominicanos nesse período, Don Jorge foi a Nova Iorque, após conseguir o visto que lhe havia sido negado enquanto estava no México. Ao passar o final de 1981 nos Estados Unidos, desistiu de morar lá por causa do frio. Em 1982, voltou ao México, onde morou por três anos, quando lhe saiu a “veia de compositor e pintor”. Em 1985, voltou a seu país de origem e lá permaneceu até março de 1987. Joaquín Balaguer havia retornado ao poder após o desastroso governo de Salvador Jorge Blanco (que sucedeu a Guzmán e era do PRD). Nesse período culminaram os efeitos das políticas urbanas iniciadas no final dos anos 1960, com crise econômica e de abastecimento nas grandes cidades dominicanas. De 23 a 26 de abril de 1984, uma revolta tomou conta da população da periferia da cidade de Santo Domingo, no que talvez tenha sido a maior manifestação de insatisfação política das últimas décadas do século XX no país. Um amigo porto-riquenho de Jorge Puello o convidou para tocar em Toulouse, na França. Como a passagem estava paga, decidiu ir. Não foi uma boa viagem, no entanto. De lá ele foi para a Espanha, voltou a Itália e se estabeleceu por um tempo na ilha de Sardenha. Trabalhou como músico e professor de dança. Tentou voltar ao México em 1991, mas só pôde visitar o país por alguns meses, por não conseguir o visto de permanência. 227 Victor Miguel Castillo de Macedo de Milão. Retornou à República Dominicana no mesmo ano, passando antes pela Europa. Ficou lá até novembro de 1992, quando um amigo conseguiu o contato para a residência no México. Porém, conforme me disse, “já não era a mesma coisa”. Com a crise da produção da tequila e o levante zapatista em 1994, o clima do país estava muito tensionado. Dessa vez, voltou para ficar 23 anos na terra de seu pai. Quando lhe perguntei em que situação havia conhecido Tirso (o presidente da Fusha Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo 228 me respondeu que o conheceu provavelmente em 1985. Foi durante algum evento promovido por ONGs, como o Octubre Mulato (outubro mulato). O presidente da fundação era então somente um poeta e participava dos eventos como declamador. Conheceu-o, pois, “coincidiam em lugares”. Demoraram alguns anos até que se aproximassem e compartilhassem o interesse comum nos eventos da revolução de 1965. Em nossa conversa, a volta à República Dominicana soou como um momento menor. Dentre as tantas coisas extraordinárias que haviam acontecido à Jorge Puello, aquela parecia a menos interessante. Voltou ao país ainda governado pelo homem que causou seu exílio, Joaquín Balaguer, cego, já com 90 anos (ironicamente, situação similar à de seu pai nessa idade, anos depois). A passagem dos anos 1990 para a década de 2000 só é mencionada por Don Jorge como o momento em que buscou o Estado dominicano para conseguir apoio para seus projetos artísticos (enviou currículo e propostas). Foi estranho voltar como um desconhecido a um país que começara a sentir os efeitos das reformas neoliberais e seus ajustes estruturais, impostos pelo Banco Mundial e pelo Fundo Monetário Internacional. Parecia que já não havia mais lugar para os sonhos de uma sociedade mais justa. A relação com Tirso se fortaleceu de 2015 em diante, conforme me explicou. Isso coincidiu com a criação da fundação, com as buscas por ex-combatentes da revolução e a entrevista com El Men. Mas, diferentemente dos órgãos estatais de produção de arquivos como o AGN, Tirso projetou a possibilidade de reparações e financiamentos do governo para o projeto da fundação. Don Jorge, apesar de não ter lutado nas linhas de frente da revolução, recebeu um certificado de Herói, como tantos outros combatentes. Desse modo, o presidente da Fusha operou uma importante extensão dos significados da Revolução de Abril de 1965. Se a vida de Jorge Puello, tomada como um ato sacrificial em nome da criação de condições de vida mais justas no país, podia ser compreendida como a trajetória de um herói, então o próprio sentido da revolução se estendera. Mas outras permanências também ficam mais aparentes. O que se passa de um ano ao outro (de 1965 a 1966), não é a instauração de um novo regime. O Estado dominicano teve sua lógica autoritária suspensa por alguns meses em 1963. Com Balaguer, o argumento foi mantido para o uso da violência: a preocupação com a manutenção da ordem e do progresso. A articulação com a oligarquia, tática para evitar golpes de Estado, foi restabelecida, e os setores da burguesia e do exército foram fundamentais para a manutenção de Balaguer no poder (LOZANO, 2018). i O que foi alterado no estado de espírito daqueles que sofreram com o fim da revolução foi a necessidade de continuar a luta através de articulações e movimentos organizados. Assim, parece que a extensão operada por Tirso com o trabalho da Fusha ata laços que pareciam Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 229 Victor Miguel Castillo de Macedo desfeitos, os elos entre a revolução e a luta para derrubar o regime autoritário de Balaguer. E ele faz isso quando dá espaço a Jorge Puello para contar sua história e ser visto como herói (já conhecido, porém esquecido). A socióloga Laura Faxas (2007) desenvolveu uma análise conjuntural desse contexto, considerando as linhas de permanências dos anos 1960 aos anos 1990. Para ela (FAXAS, 2007, p. 159), a derrocada da revolta popular de 1984 significou a morte do que chamou de “mito populista” dominicano. O retorno de Balaguer em 1986 o manteve no poder até 1996. Para tanto, foi necessário fraudar as eleições de 1990, quando José Francisco Peña Gómez, candidato do PRD (o mesmo que fez o chamamento via rádio para a revolução), era considerado o favorito. Nessas eleições, também era candidato Juan Bosch, pelo partido que criou em 1973, o Partido de la Liberación Dominicana (PLD), que fez eco às acusações da fraude de Balaguer. Era um momento em que as organizações da sociedade civil estavam mais bem organizadas e menos suscetíveis aos arroubos autoritários, situação também causada pela perda de força organizativa dos setores militares e da fraca articulação com a classe política. O PLD, que teve quadros de centro-esquerda durante sua formação, herdou, num primeiro momento, a desconfiança de Bosch do imperialismo norte-americano. No pleito de 1994, Peña Gomez quase foi eleito. Balaguer venceu pela terceira vez seguida, o que gerou uma enorme crise política, cuja resposta foi um acordo que estabeleceu a necessidade de uma reforma eleitoral e um mandato de dois anos (para o candidato supostamente eleito). As eleições de 1996 foram as primeiras que tiveram segundo turno e não incluíram o direito à reeleição. Peña Gomez concorreu no segundo turno contra o candidato do PLD, o jovem intelectual Leonel Fernandez. Ele foi eleito com a articulação e o apoio de Balaguer, que iniciou o novo ciclo da economia política dominicana mantendo seu protagonismo: encerrando o período que ele próprio criou, deixou como legado o cinismo clientelista da classe política e o neoliberalismo como modelo econômico11. Fernandez e o PLD dominaram a vida política institucional nas décadas seguintes, até as eleições de 2021. 11 Há que se dizer também que esse projeto inclui a manutenção de brancos na cadeira presidencial. Fernandez não é um homem branco, mas os acenos que fez a Washington antes da eleição e o fato de ter um diploma universitário o colocavam num lugar objetivamente branco na sociedade dominicana. Ademais, ganhar de José Francisco Peña Gómez – o único candidato negro dos últimos 50 anos a ter reais chances de vitória – é um ponto relevante. O parentesco haitiano de Peña Gomez foi sempre tomado como sua maior debilidade eleitoral. O sociólogo Franklin Franco observa comparativamente, em entrevista de 1979, que tanto Bosch quanto Balaguer vinham de famílias estrangeiras (FRANCO PICHARDO, 2003, p. 131), portanto ser negro de origem haitiana era um impeditivo para Peña Gómez. Seu sucessor nesse período, Danilo Medina, do mesmo partido, foi o responsável pelas celebrações vultosas à memória da revolução no cinquentenário de 2015. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Em 2022, Tirso Medrano tem organizado tertúlias para divulgar as histórias de ex-combatentes, contadas por eles próprios. 12 Da forma como a compreendo, a relação da Fusha com o Estado carrega diversos tipos de ambiguidade. É um agrupamento que passou a ser reconhecido pelo Ministério da Cultura como fundação no final de 2021, entrando no orçamento geral da pasta para o ano seguinte. Em 2022, Tirso Medrano tem organizado tertúlias para divulgar as histórias de ex-combatentes, contadas por eles próprios. 230 Victor Miguel Castillo de Macedo Não é preciso repetir que muitos dos ex-combatentes da periferia não foram lembrados Na perspectiva de Don Jorge, sua relação com a fundação quando conversamos em 2020 estava “um pouco fria”. Não é surpreendente que as condições que se desenharam nos anos da pandemia tenham afetado de maneira aguda a estrutura da fundação, que já era precária. As atividades dessa organização funcionam através de doações. As reparações mínimas que operam advêm de pequenos gestos como a entrega de cestas de Natal no fim do ano, o pagamento de remédios, a impressão dos certificados de heróis e a divulgação das histórias nas redes sociais. Entre os futuros sonhados do passado, a inconstância que impedia a adaptação a novos lugares e as melancolias da volta ao seu país, esse projeto de reparação12 também pode frustrar as expectativas do antigo militante do MPD. E, ainda assim, é nas páginas da rede social da fundação que sua imagem circula (ver abaixo) – entre os “relatos de heróis anônimos” da Revolução de Abril de 1965. Essa forma de herói que habita os oxímoros do poeta Tirso Medrano carrega outras versões possíveis do que foi o tempo de Balaguer e de suas silenciosas permanências. Figura 4. Don Jorge, como membro da fundação, circulando em relato e imagem pelas redes (tanto na página “Fundación”, como na página “Relatos”) Figura 4. Don Jorge, como membro da fundação, circulando em relato e imagem pelas redes (tanto na página “Fundación”, como na página “Relatos”) Figura 4. Don Jorge, como membro da fundação, circulando em relato e imagem pelas redes (tanto na página “Fundación”, como na página “Relatos”) Fonte: Fusha (2020). 12 Da forma como a compreendo, a relação da Fusha com o Estado carrega diversos tipos de ambiguidade. É um agrupamento que passou a ser reconhecido pelo Ministério da Cultura como fundação no final de 2021, entrando no orçamento geral da pasta para o ano seguinte. Em 2022, Tirso Medrano tem organizado tertúlias para divulgar as histórias de ex-combatentes, contadas por eles próprios. Fonte: Fusha (2020). 12 Da forma como a compreendo, a relação da Fusha com o Estado carrega diversos tipos de ambiguidade. É um agrupamento que passou a ser reconhecido pelo Ministério da Cultura como fundação no final de 2021, entrando no orçamento geral da pasta para o ano seguinte. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 231 Victor Miguel Castillo de Macedo 13 Nesse aspecto, a reflexão de Dixa Ramírez (2018) oferece uma análise da lógica dos “grandes homens” na República Dominicana no terceiro capítulo do livro Colonial phantoms, especialmente a dos homens brancos, como Bosch e Caamaño. Não me parece uma coincidência ela ter focado, entre outras coisas, no monumento Faro a Colón, em homenagem a Cristóvão Colombo, que fez parte das grandes obras do projeto urbano-cultural de Balaguer. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 SILENCIAMENTOS E A TRANSIÇÃO INACABADA: À GUISA DE CONCLUSÃO La libertad como un antiguo espejo roto en la luz, se multiplica más, y cada vez que un trozo da un reflejo el tiempo nuevo le repite al viejo: Ni un paso atrás. Pedro Mir, Ni un paso atrás (1995) Na recuperação da história de Don Jorge por Alejandro Paulino Ramos feita em 2016, o historiador se utiliza do material gravado pelo AGN que faz parte de um projeto de história oral. Durante minha pesquisa em 2019, contatei os pesquisadores que estiveram à frente desse projeto, chamado Vozes de Abril. Apesar de as entrevistas serem bem-feitas e tratarem de questões pertinentes, muitos de seus entrevistados que conheci em campo sentiam que aquele esforço havia sido em vão. Não são arquivos de fácil acesso e não levaram a uma mobilização pelo reconhecimento nem pela reparação das violências sofridas. Terminam, portanto, por contribuir para a manutenção de silêncios. As histórias de El Men também suscitam questões, quando confrontadas com a trajetória de seu filho no artigo escrito por Ramos. Ele certamente teve seu reconhecimento em vida, ao contrário do que ocorreu com o filho, até ser interpelado pelos membros da Fusha. Parece-me que essas histórias evidenciam a consolidação de um herói, no sentido reservado aos grandes homens da República Dominicana13. Ademais, o fato de ser um homem negro fica praticamente em segundo plano nos relatos sobre ele – talvez não chegue a ser um caso tão acintoso de branqueamento como o descrito por Ramírez (2018) a respeito da poetisa dominicana do século XIX, Salomé Ureña. Mas certamente há algo que o diminui da mesma maneira que diminui Maximiliano Gómez – descrito por Jorgito como “negro, operário e humilde” – diante de outros nomes como Caamaño e Bosch (ambos brancos). É como se fossem personagens coadjuvantes da luta contra o autoritarismo de Balaguer nos anos 1970. Diante desse quadro mais amplo de silenciamento, vale a pena retornar à questão das Diante desse quadro mais amplo de silenciamento, vale a pena retornar à questão das Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 232 Victor Miguel Castillo de Macedo torturas sofridas por Don Jorge em sua adolescência. Sua capacidade de enunciá-las, ainda que sem detalhes, é enquadrada por motivações maiores que seu próprio corpo negro (SARTI, 2019) como parte da luta pelo socialismo. Se sua prisão tivesse ocorrido secretamente ou se não tivesse deixado nenhum registro, a posição de desamparo em que ele se encontra hoje talvez fosse concebível. No entanto, suas primeiras detenções foram noticiadas em cadeia nacional. Com o passaporte dele em mãos, Balaguer justificou-se num pronunciamento para todo o país, culpando-o como “futuro arquiteto do comunismo”, de novo se construindo como vítima ou possível alvo. Apesar disso, era Balaguer que atuava como arquiteto do futuro neoliberalismo dominicano, levando consigo o projeto de branqueamento do ditador Rafael Trujillo. Trabalhei com mais detalhe a passagem para o período do PLD no poder em outra discussão (CASTILLO DE MACEDO, 2021b), em que expus como Balaguer encerra o governo de 12 anos procurando estimular a substituição de importações. Esse movimento leva ao fortalecimento das grandes multinacionais no território dominicano. Quando volta em 1986, eleito por um conchavo de setores do catolicismo internacional, convida para ocuparem pastas de seu governo diversos nomes da contrarrevolução de 1965 (Donald Reid Cabral e os militares António Imbert e Elías Wessin y Wessin). Independentemente da permanência dos mesmos homens brancos no poder – Joaquín Balaguer fica no cargo até 1996 –, a década de 2000 consolida a privatização da previdência social e o estabelecimento da capitalização individual como regra para a aposentadoria. A derrota revolucionária de 1965 parece ter sido a primeira de uma série distribuída na segunda metade do século XX. Essas derrotas culminam na ausência de reparação e assistência social para ex-combatentes idosos. Ao longo do artigo, procurei apresentar fragmentos das vidas de El Men e Jorgito como fontes de reflexão sobre processos de silenciamento como ausência de transição política no caso dominicano. Partindo de um sobrevoo por acontecimentos daquilo que se compreende como história dominicana, procurei localizar os leitores. Um aspecto comum a diversas experiências de outros países caribenhos e latino-americanos é a interferência de forças estrangeiras, coloniais ou imperialistas em assuntos nacionais. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 O golpe brasileiro de 1964, por exemplo, também está relacionado ao medo americano de uma “nova Cuba” – depois, é o Exército brasileiro que auxilia a invasão estadunidense na República Dominicana. As especificidades se desenham nas personagens e nos efeitos que têm na vida dos diferentes estratos da população. Assumi os perigos do reducionismo em minha descrição para ressaltar o caráter violento que permanece no cotidiano dominicano entre um ditador e outro, de Trujillo a Balaguer. Busquei destacar especialmente como duas gerações de uma família, pai e filho, são submetidas ao mesmo tipo de violência, embora em diferentes graus, ao longo das Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 233 Victor Miguel Castillo de Macedo transformações institucionais que afetaram o país num curto espaço de tempo. Em contrapartida, ambos são construídos nas narrativas da fundação como heróis, apesar dos itinerários distintos. A relação entre eles, trazida pela leitura de Don Jorge, não deixa de ter seus conflitos, apesar do engajamento político em comum – El Men evitou que Jorgito se radicalizasse e pegasse em armas e desde sua juventude articulou planos para tirá-lo do país. A própria condição de nosso contato, no âmbito da fundação, talvez não lhe permitisse falar mais sobre suas discordâncias com o pai. Afinal, ele era um herói da revolução. Exceto pelas evidentes características específicas aos heroísmos revolucionários, este texto poderia ter descrito histórias de um pai e um filho colombianos, brasileiros ou haitianos. A sensação de que a democracia idealizada não se concretizará é constante para as populações negras dos países latino-americanos e caribenhos. Talvez palavras como “silenciamento”, a que o haitiano Trouillot (1995) deu tanta ênfase em sua obra mais conhecida – possam compor novas gramáticas e práticas de pesquisa para pensar “transições políticas” periféricas. * Meu convívio com Jorge Puello segue através de nossas redes sociais. Vi-o passar por momentos difíceis durante o primeiro ano da pandemia de Covid-19 em 2020. No entanto, parece que no segundo ano, após as primeiras doses das vacinas distribuídas na República Dominicana, o filho do grande El Men se reinventou. Numa postagem do dia 4 de abril de 2021, em uma de suas redes, uma foto o mostrava conduzindo, segundo a descrição, uma aula de dança e consciência rítmica para um grupo de crianças do bairro periférico de Villa Mella. Ao longo do ano, outras fotos dessas novas danças foram aparecendo. Por enquanto, parece que seus passos encontraram novos ritmos e outras formas para seguir resistindo. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 REFERÊNCIAS 1. ACEVEDO, Anthony Stevens. The Santo Domingo slave revolt of 1521 and the Slave Laws of 1522: black slavery and black resistance in the early colonial Americas. New York: CUNY Dominican Studies Institute – Research Monograph, 2019. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 234 Victor Miguel Castillo de Macedo 2. AHMED, Sara. The politics of bad feeling. Australian Critical Race and Whiteness Studies Association Journal, Melbourne, v. 1, p. 72-85, 2005. 3. ALVAREZ, Julia. No tempo das borboletas. São Paulo: Editora Rocco, 2001. 3. ALVAREZ, Julia. No tempo das borboletas. São Paulo: Editora Rocco, 2001. 4. BONILLA, Yarimar; BECKETT, Greg; FERNANDO, Mayanthi. Overture. Trouillot Remixed. In: Trouillot remixed. The Michel-Rolph Trouillot reader. Durham: Duke University Press, 2021. p. 14-49. 5. CARUSO, Raimundo. 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Construction of Dominican State power and symbolisms Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 235 Victor Miguel Castillo de Macedo of violence. Ethnos: Journal of Anthropology, v. 62, n. 3/4, p. 49-78, 1997. 17. KROHN-HANSEN, Christian. Political authoritarianism in the Dominican Republic. New York: Palgrave Macmillan, 2009. Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 Victor Miguel Castillo de Macedo Doutor em Antropologia Social pelo Museu Nacional da Universidade Federal do Rio de Janeiro. Pesquisador de Pós-doutorado no Programa de Pós-Graduação em Antropologia Social da Faculdade de Filosofia, Letras e Ciências Humanas da Universidade de São Paulo e Bolsista da Fundação de Amparo à Pesquisa do Estado de São Paulo. ID ORCID: https://orcid.org/0000-0001-6923-0734. E-mail: victormcmacedo2@gmail.com Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 17. KROHN-HANSEN, Christian. Political authoritarianism in the Dominican Republic. New York: Palgrave Macmillan, 2009. 18. LOZANO, Wilfredo. El Gobierno de los Doce Años: 1966-1978. Entre el autoritarismo y la reforma conservadora. In: Historia General del Pueblo Dominicano. Santo Domingo: Academia de la Historia Dominicana, 2018. t. 6. p. 187-276. 19. MEDRANO, Tirso. Relato de Jorge Puello Soriano (El Men). Santo Domingo, 18 de maio de 2016. Fundacion de solidaridad com los heroes de abril 1965. (Página do Facebook). Disponível em: https://bit.ly/3PtXEqD. Acesso em: 28 de mai. 2019. 20. MINTZ, Sidney. Encontrando Taso, me descobrindo. Dados, Revista de Ciências Sociais, Rio de Janeiro, v. 27, n. 1, p. 45-59, 1984. 21. RAMÍREZ, Dixa. Colonial phantoms. Belonging and refusal in the Dominican Americas, from the 19th Century to the Present. New York: New York University Press, 2018. 22. RAMOS, Alejandro Paulino. El Angelito Negro: el niño que fue preso político en los doce años de Balaguer. Acento, Santo Domingo, 12 de abril de 2016. Disponível em: https://acento.com.do/politica/angelito-negro-nino-fue-preso-politico-los-doce-anos- balaguer-8339402.html. Acesso em: 28 mai. 2019. 23. SARTI, Cynthia. Enunciações da tortura: memórias da ditadura brasileira. Revista de Antropologia, São Paulo, 2019. p. 505-529. 24. SCOTT, David. Conscripts of modernity. The tragedy of colonial enlightenment. Durham: Duke University Press, 2004. 25. SCOTT, David. Omens of adversity. Tragedy, time, memory, justice. Durham: Duke University Press, 2014. 26. SKIDMORE, Thomas. Brasil: de Getúlio Vargas a Castelo Branco, 1930-1964. Rio de Janeiro: Paz e Terra, 1982. 27. SZULC, Tad. Diario de la Guerra de Abril. Santo Domingo: Academia de la Historia, 2015. 28. TROUILLOT, Michel-Rolph. Silencing the past: power and the production of history. Boston: Beacon Press, 1995. 29. TROUILLOT, Michel-Rolph. North Atlantic fictions: global transformations, 1492- 1945. In: Global transformations: Anthropology and the modern world. New York: Palgrave Macmillan, 2003. p.29-47. 30. TROUILLOT, Michel-Rolph. Abortive rituals: Historical apologies in the global era. In: BONILLA, Yarimar; BECKETT, Greg; FERNANDO, Mayanthi (eds.). Trouillot Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022 236 Victor Miguel Castillo de Macedo remixed. The Michel-Rolph Trouillot reader. Durham: Duke University Press, 2021. p.386-406. remixed. The Michel-Rolph Trouillot reader. Durham: Duke University Press, 202 p.386-406. 31. VERAS, Ramon Antonio. De la calle a los estrados por justicia y libertad. Santo Domingo: Archivo General de la Nación, 2008. Victor Miguel Castillo de Macedo Doutor em Antropologia Social pelo Museu Nacional da Universidade Federal do Rio de Janeiro. Pesquisador de Pós-doutorado no Programa de Pós-Graduação em Antropologia Social da Faculdade de Filosofia, Letras e Ciências Humanas da Universidade de São Paulo e Bolsista da Fundação de Amparo à Pesquisa do Estado de São Paulo. ID ORCID: https://orcid.org/0000-0001-6923-0734. E-mail: victormcmacedo2@gmail.com Revista Antropolítica, v. 54, n. 2, Niterói, p. 210-236, 2. quadri., mai./ago., 2022
https://openalex.org/W3089450715	https://observatorio.fm.usp.br/bitstream/OPI/38365/1/art_GOMES_Access_to_drinking_water_and_sewage_treatment_in_2020.PDF.pdf	English	null	Access to drinking water and sewage treatment in Brazil: a challenge for the control of waterborne infectious diseases	Revista do Instituto de Medicina Tropical de São Paulo	2,020	cc-by	2,390	Dear Editor Considering the worldwide advance of COVID-19 and the urgent need to alert the population to the main preventive measures, which include social distancing, the use of masks and hand washing, it is even more urgent for governments to take responsibility to provide conditions for the population to comply these recommendations. It is noteworthy that the current pandemic caused by the new coronavirus (SARS‑CoV-2) also reflects the reality of other Water, Sanitation, and Hygiene (WaSH)-related infections and diseases, which need access to drinking water, hygiene and proper waste disposal for their prevention and control1. 1Universidade de São Paulo, Faculdade de Medicina, Departamento de Pediatria, São Paulo, São Paulo, Brazil One in three people worldwide still do not have access to drinking water, two in five do not have adequate basic facilities to wash their hands with soap and water, and more than 673 million people do not have toilets or latrines2. 2Universidade de São Paulo, Instituto de Medicina Tropical de São Paulo, Laboratório de Pesquisa Médica de Imunopatologia da Esquistossomose e Outras Parasitoses (LIM06), São Paulo, São Paulo, Brazil In Brazil, the recent disclosure of results from the Continuous National Household Sample Survey (PNAD), referring to basic sanitation conditions in 2019, demonstrates current unresolved needs3. Some indicators support this statement. The general water distribution network, which served 85.8% of households in 2016, has remained practically unchanged at 85.5% in 2019. In addition, regional distribution of the general water network is uneven, varying from 58.8% in the North region of the country to 92.3% in the Southeast3. 3Universidade de São Paulo, Faculdade de Medicina, Departamento de Moléstias Infecciosas e Parasitárias, Núcleo de Medicina Tropical, São Paulo, São Paulo, Brazil Analysis of the water sources and infrastructure used to supply Brazilian municipalities, shows that 31% of the population live in places of low water security, that is, they face rationing, collapse or warning in periods of drought; and 41% live in regions where production systems require expansion. Only 27% of the population live in municipalities where the supply was considered satisfactory. Water distribution across income brackets is very unequal, as 40% of the unserved population are in the 1 minimum wage or less income bracket4. LETTER TO THE EDITOR http://doi.org/10.1590/S1678-9946202062071 Dear Editor 4Universidade de São Paulo, Escola de Enfermagem, Departamento de Enfermagem em Saúde Coletiva, São Paulo, São Paulo, Brazil 5Universidade de São Paulo, Faculdade de Medicina, Departamento de Moléstias Infecciosas e Parasitárias, Laboratório de Pesquisa Médica em Imunologia (LIM48), São Paulo, São Paulo, Brazil These regional inequalities are more intensely noticed when looking at the proportion of households with access to the general sewage system: in 2019, the North and Northeast regions of the country had the lowest coverage, with 27.4% and 47.2%, respectively, whilst coverage in the Southeast region reached an estimated 88.9%; and the South and Midwest regions had the same coverage of 68.7%3. This is an open-access article distributed under the terms of the Creative Commons Attribution License. Access to drinking water and sewage treatment in Brazil: a challenge for the control of waterborne infectious diseases Sao Paulo, September 7th, 2020 LETTER TO THE EDITOR Gomes et al. Brazil, totaling 0.7% of total Unified Health System (SUS) spending on hospitalizations in that period5. ecological crisis, disasters and associated risks, such as the global warming in addition to the presence of polluting industries and technologies and their effects on water, soil, air and food, reducing the biodiversity and destroying ecosystems15. Overall, in 2015, diarrhea was estimated to be one of the main causes of death in all age groups (1.31 million), and one of the main causes of Disability-adjusted life years - DALYs (71.59 million DALYs) due to its disproportionate impact on children under 5 years old. In general, rotavirus is the leading cause of death from this disease, followed by Shigella spp and Salmonella spp6. The goal 6, one of the Sustainable Development Goals, proposes: “Ensuring the availability and sustainable management of water and sanitation for all”4. However, without an universal access to quality water and sewage treatment, which could allow hand hygiene minimally, we will not reduce morbity and mortality caused by waterborne diseases and in the future, we will be exposed again to other emerging pandemics, which will impact the quality of life of populations in the planet, and which could be avoided through basic actions such as an universal access to drinking water and sewage treatment. The World Health Organization reported that inadequate water, sanitation and hygiene conditions were responsible for 829,000 deaths from diarrhea in the world in 20167. Some waterborne diseases, such as gastroenteritis, are on the Brazilian list of primary care-sensitive conditions5. Hospitalizations for primary care-sensitive conditions represent potentially preventable conditions, which can reduce the risk of unnecessary hospitalizations and are a powerful indicator of primary care access and quality8. Thus, according to Heller16, the recent approval by the federal government of the new Legal Framework for Basic Sanitation, Bill Nº 4162/2019, causes anxiety by breaking the fundamental principles of the current legal framework: universality, completeness, social control and the use of appropriate technologies. Diseases that are still common in many regions of the world are associated with deficiencies in hygiene, sanitation and water supply. The group of diseases potentially transmitted by water contains both, viral (hepatitis A, E and F, polio and viral diarrhea) and bacterial diseases (campylobacteriosis, cholera, legionellosis, leptospirosis, pathogenic E. coli, and salmonellosis such as typhoid and paratyphoid fever). Gomes et al. In the latter group, it is important to highlight that, since 1817, cholera has caused seven pandemics, the most recent being the one that started in 1961, responsible for epidemics in Haiti and the current devastating epidemic in Yemen9,10. Basic sanitation covers water supply, sanitation, drainage and rainwater management, urban cleaning and solid waste management. For Moraes17, an advisor to the National Observatory on the Rights to Water and Sanitation, the concession of public services to private companies and public-private partnerships (PPP) are already permitted by Laws 8,987/1995 and 11,079/2004, but this new Law 4162/2019 substantially favors the private sector, whose only interest in the water supply and sewage sectors is where it can make the most profit. It also highlights that one of the most impactful changes in this law is to remove the autonomy of States and municipalities in hiring companies to distribute water and take care of the solid waste. In addition, this new law does not serve rural regions and, therefore, does not contribute to universalization17. Protozoa also cause diseases through water transmission (amoebiasis, cryptosporidiosis, and giardiasis). Among helminthiasis, schistosomiasis stands out, an endemic disease directly related to the lack of basic sanitation in several regions of the country and still presenting with many natural foci for transmission of the disease11. Water and sanitation are a world problem, resulting from rapid global urbanization, and finding solutions in large cities is particularly urgent. It is noteworthy that the majority of urban growth occurs in slums, in developing countries. The complexity of this issue is the reality for approximately one billion people (13% of the world population) living in urban slums7,12,13. The challenge imposed by the COVID-19 pandemic highlights the urgency of establishing public policies aimed at reducing social inequalities and providing access to basic conditions that guarantee quality of life and health, as the 1988 Brazilian Constitution establishes that health is a right for all and a duty of the State. When referring to the COVID-19 pandemic, it is essential to recognize that social distancing and frequent hand washing may not be possible for millions of people living in densely populated communities, with precarious housing conditions, and difficulties in the access to water and sewage treatment14. Correspondence to: Francisco Oscar de Siqueira França Countrywide, 19.1% of households are connected to septic tanks but they are not connected to the general network; substantial regional variations are seen, with 42.9% of households in the North, 30.7% in the Northeast, and 5.5% in the Southeast using this modality of connection, thus corroborating the evident heterogeneity in the access to this essential service3. Universidade de São Paulo, Faculdade de Medicina, Departamento de Moléstias Infecciosas e Parasitárias, Laboratório de Pesquisa Médica em Imunologia (LIM48), Av. Dr. Enéas de Carvalho Aguiar, 470, Térreo, CEP 05403000, São Paulo, SP, Brazil Approximately 9 million households (12.6%) had a ditch, rudimentary cesspit, river, lake or sea, in addition to other forms of waste disposal. In the North region, 29.6% of households (1.6 million) were in this condition, exceeding the estimated 27.4% of households connected to the general network3. The relevance of sanitation infrastructure in the health-disease process was highlighted in a study on hospitalizations due waterborne diseases. It was estimated that, in 2015, these diseases corresponded to 2.35% of all hospitalizations in Rev Inst Med Trop São Paulo. 2020;62:e71 Page 1 of 3 Page 1 of 3 Gomes et al. Page 2 of 3 REFERENCES 13. Ross AG, Rahman M, Alam M, Zaman K, Qadri F. Can we ‘WaSH’ infectious diseases out of slums? Int J Infect Dis. 2020;92:130- 2. 1. World Health Organization. Safer water, better health. Geneva: WHO; 2019. [cited 2020 Sep 9]. Available from: https://apps. who.int/iris/bitstream/handle/10665/329905/9789241516891- eng.pdf?ua=1 1. World Health Organization. Safer water, better health. Geneva: WHO; 2019. [cited 2020 Sep 9]. Available from: https://apps. who.int/iris/bitstream/handle/10665/329905/9789241516891- eng.pdf?ua=1 14. The Lancet. Redefining vulnerability in the era of COVID-19. Lancet. 2020;395:1089. 15. Porto MF. Crise das utopias e as quatro justiças: ecologias, epistemologias e emancipação social para reinventar a saúde coletiva. Cienc Saude Coletiva. 2019;24:4449-58. 2. United Nations. Sustainable Development Goals. Goal 6: ensure access to water and sanitation for all. [cited 2020 Sep 9]. Available from: https://www.un.org/sustainabledevelopment/ water-and-sanitation/ 2. United Nations. Sustainable Development Goals. Goal 6: ensure access to water and sanitation for all. [cited 2020 Sep 9]. Available from: https://www.un.org/sustainabledevelopment/ water-and-sanitation/ 16. Associação Brasileira de Saúde Coletiva. “Enfoque foi inserir a iniciativa privada”, aponta Léo Heller sobre PL 4162/19. Rio de Janeiro: ABRASCO; 2020 [cited 2020 Sep 9]. Available from: https://www.abrasco.org.br/site/noticias/enfoque-foi-inserir-a- iniciativa-privada-aponta-leo-heller-sobre-pl-4162-19/49633/ 3. Instituto Brasileiro de Geografia e Estatística. Pesquisa Nacional por Amostra de Domicílios Contínua. Características gerais dos domicílios e dos moradores 2019. Brasil: IBGE; 2020. [cited 2020 Sep 9]. Available from: https://biblioteca.ibge.gov.br/ visualizacao/livros/liv101707_informativo.pdf 17. Associação Brasileira de Saúde Coletiva. Água limpa e saneamento básico são direitos de todos e não mercadorias! Não ao PL 4162/19!. Rio de Janeiro: ABRASCO; 2020 [cited 2020 Sep 9]. Available from: https://www.abrasco.org. br/site/noticias/ecologia-e-meio-ambiente/agua-limpa-e-o- saneamento-basico-sao-direitos-de-todos-e-nao-mercadorias- nao-ao-pl-4162-19/49593/ 4. Brasil. Agência Nacional de Águas. ODS 6 no Brasil: visão da ANA sobre os indicadores. Brasília: ANA; 2019. [cited 2020 Sep 9]. Available from: https://www.ana.gov.br/acesso-a- informacao/institucional/publicacoes/ods6/ods6.pdf 5. Paiva RF, Souza MF. Associação entre condições socioeconômicas, sanitárias e de atenção básica e a morbidade hospitalar por doenças de veiculação hídrica no Brasil. Cad Saude Publica. 2018;34:e00017316. 18. Machado CV, Silva GA. Political struggles for a universal health system in Brazil: successes and limits in the reduction of inequalities. Global Health. 2019;15 Suppl 1:77. 6. Troeger C, Forouzanfar M, Rao PC, Khalil I, Brown A, Reiner Jr RC, et al. Estimates of global, regional, and national morbidity, mortality, and aetiologies of diarrhoeal diseases: a systematic analysis for the Global Burden of Disease Study 2015. Lancet Infect Dis. 2017;17:909-48. 19. United Nations. UN-Water Decade Programme on Advocacy and Communication. The human right to water and sanitation: milestones. [cited 2020 Sep 9]. Gomes et al. The already precarious access for the most vulnerable populations to health services has been aggravated by the recent neoliberal measures that have worsened some health indicators, such as the increase in infant mortality in all but the Southern region of the country; and the increase in infant mortality from preventable causes, such as diarrhea18. Additionally there is the worsening water crisis in several regions of the world, as well as the evident In July 2010, the UN General Assembly recognized, for the first time, in Resolution 64/292, the right to water and Page 2 of 3 Page 2 of 3 Page 2 of 3 Rev Inst Med Trop São Paulo. 2020;62:e71 Access to drinking water and sewage treatment in Brazil: a challenge for the control of waterborne infectious diseases sanitation, admitting that drinking water and sanitation are essential for the accomplishment of all human rights19,20. sanitation, admitting that drinking water and sanitation are essential for the accomplishment of all human rights19,20. Access to drinking water and sewage treatment is a response to the pressing need to consider health-disease as a process that goes beyond merely a biological concept, but which is, above all, socially determined. Overcoming social inequalities requires public policies fundamentally oriented to reach the largest portion of social groups that, historically, and particularly in recent years, have been deprived of the social rights that ensure citizenship. 8. Alfradique AM, Bonolo PF, Dourado I, Lima-Costa MF, Macinko J, Mendonça CS, et al. Internações por condições sensíveis à atenção primária: a construção da lista brasileira como ferramenta para medir o desempenho do sistema de saúde (Projeto ICSAP – Brasil). Cad Saude Publica. 2009;25:1337- 49. Access to drinking water and sewage treatment is a response to the pressing need to consider health-disease as a process that goes beyond merely a biological concept, but which is, above all, socially determined. Overcoming social inequalities requires public policies fundamentally oriented to reach the largest portion of social groups that, historically, and particularly in recent years, have been deprived of the social rights that ensure citizenship. 9. Harris JB, LaRocque RC, Qadri F, Ryan ET, Calderwood SB. Cholera. Lancet. 2012;379:2466-76. 10. Deen J, Mengel MA, Clemens JD. Epidemiology of cholera. Vaccine. 2020;38 Suppl 1:A31-40. 11. Bartram J, Cairncross S. Hygiene, sanitation, and water: forgotten foundations of health. PLoS Med. 2010;7:e1000367. Gomes et al. Filumena Maria da Silva Gomes 1 Maria Cristina Carvalho do Espírito Santo 2 Ronaldo César Borges Gryschek 2,3 Maria Rita Bertolozzi 4 Francisco Oscar de Siqueira França 3,5 12. World Health Organization. Global report on urban health: equitable, healthier cities for sustainable development. Geneva: WHO; 2016. [cited 2020 Sep 9]. Available from: https://www. who.int/gender-equity-rights/knowledge/global-report-on- urban-health/en/ REFERENCES Available from: https://www. un.org/waterforlifedecade/pdf/human_right_to_water_and_ sanitation_milestones.pdf 20. United Nations. Resolution adopted by the General Assembly on 28 July 2010: 64/292. The human right to water and sanitation. [cited 2020 Sep 9]. Available from: https://www.un.org/ga/ search/view_doc.asp?symbol=A/RES/64/292 7. World Health Organization. Integrating health in urban and territorial planning: a sourcebook. Geneva: WHO; 2020. [cited 2020 Sep 9]. Available from: https://unhabitat.org/integrating- health-in-urban-and-territorial-planning-a-sourcebook-for- urban-leaders-health-and Page 3 of 3 Rev Inst Med Trop São Paulo. 2020;62:e71
https://openalex.org/W3012296058	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0230454&type=printable	English	null	Built environment correlates of physical activity in low- and middle-income countries: A systematic review	PloS one	2,020	cc-by	11,961	PLOS ONE RESEARCH ARTICLE RESEARCH ARTICLE Built environment correlates of physical activity in low- and middle-income countries: A systematic review Sarah ElshahatID1, Michael O’Rorke2, Deepti Adlakha3 1 School of Medicine, Dentistry and Biomedical Sciences, Centre for Public Health, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom, 2 Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, Iowa, United States of America, 3 School of Natural and Built Environment, Queen’s University Belfast, Belfast, Northern Ireland, United Kingdom a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 selshahat01@qub.ac.uk * selshahat01@qub.ac.uk * selshahat01@qub.ac.uk Abstract Insufficient physical activity (PA) is the fourth major risk factor for many non-communicable diseases and premature mortality worldwide. Features of the built environment (BE) play a considerable role in determining population PA behaviors. The majority of evidence for PA- BE relationships comes from high-income countries and may not be generalizable to low- and middle-income countries (LMICs). We aim to systematically review the literature and assess the associations between perceived and/or objective BE characteristics and PA domains in LMICs. This review adopted a systematic search strategy for English language articles published between January 2000 and June 2019 from four electronic databases— Medline, Embase, Web of Science and PubMed—adhering to the PRISMA guidelines. Studies addressing the associations between self-reported and/or objective BE and PA were only included if they were conducted in LMICs, according to the World Bank classifica- tion list. Articles investigating PA-BE relationships across any age groups were included, and all study designs were eligible, except for qualitative studies and reviews. Thirty-three studies were included for evidence synthesis. Cross-sectional studies were the most pre- vailing study design (97%), revealing a notable gap in longitudinal PA-BE research in LMICs. A majority of the BE factors were not associated with different PA domains while others (e.g., density, proximity to services, aesthetics) exhibited an inconsistent association. Land-use mix diversity was positively associated with transport PA and the presence of recreation facilities resulted in an increase in PA during leisure-time. Increased safety from crime at night consistently increased total PA and walking levels. Research exploring the associations between BE attributes and PA behaviors in LMICs appears to be limited and is primarily cross-sectional. Longitudinal research studies with objective measures are needed for inferring well-grounded PA-BE causal relationships and informing the design of evidence-based environmental interventions for increasing PA levels in LMICs. Editor: Chaisiri Angkurawaranon, Chiang Mai University Faculty of Medicine, THAILAND Editor: Chaisiri Angkurawaranon, Chiang Mai University Faculty of Medicine, THAILAND Received: October 29, 2019 Accepted: March 1, 2020 Published: March 17, 2020 Copyright: © 2020 Elshahat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Editor: Chaisiri Angkurawaranon, Chiang Mai University Faculty of Medicine, THAILAND Received: October 29, 2019 Accepted: March 1, 2020 Published: March 17, 2020 Copyright: © 2020 Elshahat et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Elshahat S, O’Rorke M, Adlakha D (2020) Built environment correlates of physical activity in low- and middle-income countries: A systematic review. PLoS ONE 15(3): e0230454. https://doi. org/10.1371/journal.pone.0230454 PLOS ONE PLOS ONE 1. Introduction and analysis, decision to publish, or preparation of the manuscript. and analysis, decision to publish, or preparation of the manuscript. Physical inactivity is a global public health issue and the fourth leading risk factor for many non-communicable diseases (NCDs), including obesity, diabetes, cardiovascular disease, stroke and some cancers [1]. The global health care costs of physical inactivity have been esti- mated to exceed international $53 billion annually [2]. Worldwide, about 3.2 million deaths are attributed to insufficient physical activity (PA) annually, with low- and middle-income countries (LMICs) carrying a disproportionate share of the disease burden [3]. Approximately 82% of the global NCDs deaths take place in LMICs, where the risk of premature NCD mortal- ity is 1.5 times higher than high-income country populations [4]. Regular physical activity directly contributes to achieving the UN Sustainable Development Goal 3 (good health and well-being) as it helps prevent and treat NCDs [5]. Recognizing the importance of PA for NCD prevention, the WHO member states made an agreement to jointly work towards a rela- tive decline in the global prevalence of inadequate PA by 10% by 2025 [6]. However, a recent study of 168 countries by Guthold et al. [7] did not reveal a significant decrease in the world- wide prevalence of insufficient PA between 2001 and 2016, (28.5 vs 27.5%, respectively), indi- cating slow progress towards meeting the global target and highlighting an urgent need for accelerated actions and policy changes for reversing these trends. Competing interests: The authors have declared that no competing interests exist. Characteristics of the built environment (BE) (human-made physical parts of the environ- ment where people live and work, e.g., homes, buildings, streets, parks, open spaces, and trans- port infrastructure) play a vital role in determining individuals’ PA behaviors across four separate life-domains: domestic or home-based PA, occupational or work-related PA, travel PA, and leisure PA. Examples of the BE attributes include residential density, diversity and access to a mix of land uses and public services (e.g. parks, greenways, transit stops), pleasant neighborhood surroundings (e.g. trees, shrubs, rivers), as well as crime and traffic safety [8]. For example, accessible public parks and recreational amenities provide settings for leisure- time PA for families, individuals and institutions [9]. Availability of high-quality infrastructure for walking and cycling (e.g. sidewalks, bike paths, crosswalks) promote travel PA amongst populations [9]. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: SE and DA received support from a Global Challenges Research Award from the UK Department for Economy titled, “Age-friendly cities in the Global South” (project code R3145NBE). DA was responsible for funding acquisition. URL of funder: https://www.economy-ni.gov.uk/ publications/global-challenges-research-fund- northern-ireland-universities-strategies. The funders had no role in study design, data collection 1 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 1. Introduction Research investigating the associations between PA and BE attributes has increased rapidly over the last two decades and has become an international priority [10,11]. The vast majority of evidence in this area has primarily been limited to developed countries in Europe, North America, and Australia, with recent evidence from some LMICs in Asia, Africa and South America [8,12–14]. For instance, a systematic review of 70 studies from Europe revealed a con- sistent evidence for positive associations between accessible public services and PA, and reported a gap in knowledge of the BE correlates across different PA domains [15]. Similarly, Smith et al. systematically reviewed 28 studies from the US and Australia, and reported signifi- cant positive relationships between destination accessibility, travel and total PA [1]. A recent systematic review of 25 studies by Zhang et al. [16] investigated the impact of BE characteris- tics in parks in North America, Australia, and Europe, and reported significant positive associ- ations between park-based leisure PA, park lighting and walking and cycling trails. However, these findings may not generalize to LMIC contexts, where BE attributes are distinct from developed countries with respect to patterns of land-use, density, design and urbanization [17]. Population growth and economic development have spurred rapid absolute increases in the number of motor vehicles in LMICs, with marked shifts in travel patterns that are moving away from public transport, walking and cycling, to private motorized vehicles [12]. Rapid, unplanned urban expansion has contributed to environmental and health hazards, including road traffic casualties, air pollution, and diminished safe spaces for walking and cycling [18]. In the last decade, Latin America has witnessed major urban changes such as an increase in PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 2 / 19 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries utilities infrastructure and expansion in transport networks, influencing lifestyle changes and PA behaviors in populations [19,20]. For example, studies showed that residential density and street connectivity were associated with reduced likelihood of PA in urban Brazil and Colom- bia, respectively. [21,22]. These socio-ecological issues are associated with reduced PA levels and sedentary lifestyles, making populations of LMICs highly predisposed to NCDs and pre- mature mortality [23]. 1. Introduction Despite the well-documented benefits of PA, there is a gap in the current knowledge of the pathways through which the BE can promote or constrain PA across different life domains (e.g., home, work, travel and leisure) in LMICs populations. Previous reviews on this topic have been constrained by several limitations including challenges in summarising studies given their heterogeneity in terms of study design and quality and approach to evidence syn- thesis [24,25]. For instance, Day [24] conducted a review to investigate the impact of different BE features (e.g. density, availability of parks) and non-BE factors (e.g. weather, stray animals) on PA levels in developing countries. However, the review was narrative in nature, was not restricted to LMICs, and did not follow PRISMA guidelines or assess the quality of included studies. The present systematic review aims to more robustly investigate the associations between BE characteristics and PA domains amongst different populations exclusively from LMICs, in order to inform and direct future research and necessary policy change towards sustainable design of the BE and to foster and promote more physically active populations in these devel- oping regions. 2. Methodology This systematic review was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (Registration no. CRD42019141945) [26]. 2.1. Search strategy This review was conducted following the PRISMA guidelines for reporting of systematic reviews [27] (S1 Appendix). Various search terms and phrases were selected to reflect the key concepts of the review (PA, BE, LMICs), and combined using Boolean operators AND/OR (S2 Appendix). Four electronic databases were searched: Medline, Embase, Web of Science and PubMed. The search was limited to human studies and restricted to English Language papers published between January 2000 and June 2019 to summarize contemporaneous literature in the past two decades. A manual search of the reference lists of potentially relevant articles was also conducted. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 2.3. Data extraction A pre-defined data collection form was used for data extraction. This comprised authorship, study origin (country), characteristics of study subjects, study design, sampling methods, covariates, PA and BE variables, as well as key findings (S3 Appendix). Data was classified according to the PA domains, namely, leisure-time PA, travel PA, leisure-time walking, trans- port-related walking, transport-related cycling, and total PA (S4 Appendix). For each PA domain, BE characteristics were categorized into six categories according to the Neighborhood Environment Walkability Scale [29]: land-use mix diversity, density, land-use mix access, aes- thetics, infrastructure for walking and cycling, as well as crime and traffic safety. Additional categories for addressing associations with income and urbanicity (degree to which a given geographical area is urban) were added. 2.2. Study selection Studies were considered for inclusion only if they were conducted in LMICs, according to the World Bank classification list [28]. The included studies investigated the association between objectively and/or subjectively measured BE attributes and PA. All PA domains across all age groups were included. All study designs were eligible, with the exception of qualitative studies and any types of reviews. Studies investigating the impact of PA on health outcomes without any BE attributes were excluded. A two-step procedure was adopted for selection of eligible articles from among the retrieved results. Firstly, all authors (SE, MOR, DA) screened titles and abstracts of the retrieved articles after duplicate removal. Secondly, all potentially relevant studies were re-assessed in full, applying the previously established inclusion/exclusion criteria (Fig 1). Disagreements were resolved via discussion. 3 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries Fig 1. PRISMA flow chart of the research results. https://doi.org/10.1371/journal.pone.0230454.g001 Fig 1. PRISMA flow chart of the research results. https://doi.org/10.1371/journal.pone.0230454.g001 https://doi.org/10.1371/journal.pone.0230454.g001 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 3.1. Study characteristics and quality assessment The literature search yielded a total of 6,324 articles. In addition, six papers were identified from the bibliographies of relevant studies. Of these, only 33 articles met the eligibility criteria and were included in the final analysis (Fig 1). Most of these studies were carried out in Brazil (43%), followed by both Colombia (9%) and India (9%) (S1 Table). Approximately 94% of the included studies adopted a cross-sectional design. Most studies were considered to be of high quality (55%), and about 30% were of moderate quality (S5 Appendix). Potential socio-demographic confounders (e.g., age, gender, socioeco- nomic status) were controlled for in 88% of the included studies. Only 27% of the eligible arti- cles reported a reliable response rate (80%). Only 9% of the studies measured PA objectively (i.e., using an accelerometer), whereas 42% considered assessing BE attributes through objec- tive methods (i.e., geographical information systems (GIS)). Leisure PA was the most com- monly investigated PA domain across studies (48%), followed by leisure walking (30%) (S1 Table). Transport cycling was only investigated in four studies [21,33–35]. The most predomi- nant BE attributes were safety from traffic and crime safety (45% each). We classified the results into four main subsections according to PA domains: leisure PA, travel PA, walking and cycling, as well as total PA. 2.4. Quality assessment and evidence synthesis An eight-item checklist was constructed for assessing the quality of the included studies (S5 Appendix). Six items were adapted from both the Center for Evidence-Based Management quality appraisal guidelines, and a previously developed checklist by Barnett et al. [30,31]. These included: [1] study design [weight: cross-sectional or case study = 1, longitudinal or quasi design = 2], [2] reliable response rate (80%) [yes = 1, no = 0], [3] stratification of recruitment areas by suitable environmental characteristics to maintain generalizability [yes = 1, no = 0], [4] controlling for socio-demographic confounders [yes = 1, no = 0], [5] calculation of confidence interval for main results [yes = 1, no = 0], and [6] assessment of sta- tistical significance (p value) [yes = 1, no = 0]. In order to improve the appropriateness of appraising the studies included in the current review, two additional items were considered; [7] method of PA measurement (weight: subjective = 0.5, objective = 1), and [8] method of BE measurement (weight: subjective = 0.5, objective = 1). Higher scorings indicate better quality: 4 (low quality), 4.1–5.9 (intermediate quality), 6–6.9 (high quality), and 7 (very high PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 4 / 19 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries quality). The review adapted the criteria developed by Sallis et al. [32] for synthesizing evidence in relation to PA-BE associations. PLOS ONE Table 1. Relationships between leisure PA and BE attributes across included studies. BE attributes PA-BE relationships % studies supporting the predicted association ab Summary codes c Significant (+) Significant (-) Non-significant Land-use mix diversity N/A N/A [8,36] 0/2 = 0% 0 Residential density N/A [8] [37] 1/2 = 50% ? Park density [19] N/A [38] 1/2 = 50% ? Leisure amenities availability [39–42 d,43] [44] d N/A 4/4 = 100% ++ Land-use mix access N/A N/A [8] 0/1 = 0% 0 Leisure facilities proximity [39,45] [46] e [36,37, 42 d,43] 2/5 = 40% ? Transit stops proximity N/A N/A [19,36,38] 0/3 = 0% 00 Services proximity N/A N/A [33,41] 0/2 = 0% 0 Aesthetics [36,37,39] N/A [8,41,46] 3/6 = 50% ? Infrastructure to walk and cycle N/A N/A [8,36,41, 42 d] 0/3 = 0% 00 Walkability [47] N/A N/A 1/1 = 100% + Street connectivity N/A N/A [8,44 d] 0/1 = 0% 0 Sidewalks [33] [46] e [41,44 d] 1/2 = 50% ? Terrain slope N/A [19] 1/1 = 100% - Bike path availability N/A [19,41] 0/2 = 0% 0 Crime safety [37,44 df] [38] f [8,33,36,41,46 f,48 f] 1/5 = 20% 000 Traffic safety N/A N/A [8,33,36,41,44 d,46 f] 0/4 = 0% 00 District income [43,47] N/A N/A 2/2 = 100% + etween leisure PA and BE attributes across included studies. a Number of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. e reduced/lack of access. f feeling unsafe. https://doi.org/10.1371/journal.pone.0230454.t001 https://doi.org/10.1371/journal.pone.0230454.t001 whereas it did not show any significant relationships in urban India (Chennai), Uganda (Kam- pala) and Brazil (Recife) [8,41,46]. 3.2.5. Infrastructure for walking and cycling. Studies revealed no significant relation- ships between leisure PA and either street connectivity or presence of infrastructure for walk- ing and cycling (Table 1). When investigating perceived sidewalks availability, Parra et al. [33] detected a positive relationship with self-reported leisure PA, whereas, Vancampfort et al. [41] did not note any significant association. Finally, a study by Gomez et al. [19] in Colombia (Bogota´) found a significant negative association between objectively-measured land slope of 4% and self-reported leisure PA. 3.2.6. Crime and traffic safety. Traffic safety was not significantly associated with leisure PA across LMICs (Table 1). Crime safety was also not related to self-reported leisure PA across studies, with the exception of one study by Rech et al. umber of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. d f c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d association with physical inactivity. 3.2. Leisure PA 3.2.1. Land-use mix diversity. Two studies showed that land-use mix diversity was not significantly related to self-reported leisure PA (Table 1). A cross-sectional study by Adlakha et al. [8] did not find any significant associations between perceived land-use mix diversity and self-reported leisure PA among urban Indian adults. Likewise, Ja´uregui et al. [36] did not detect any significant relationships between both in urban Mexico (Cuernavaca). 3.2.2. Density. Perceived residential density showed an inconsistent association with self- reported leisure PA (Table 1). A significant negative association between both was found in urban India by Adlakha et al. [8], whereas, a study by Rech et al. [37] in urban Brazil did not detect any significant relationships. Availability of recreational amenities (e.g. parks, fitness centers) was a consistently positive feature (100%) of leisure PA across LMICs (Table 1). Amorim et al [44], also showed that reduced perception of green spaces was significantly asso- ciated with lower leisure PA in urban Brazil (Pelotas). 3.2.3. Land-use mix access and proximity to services. A cross-sectional study by Adlakha et al. did not reveal any significant association between land-use mix access and leisure PA among urban Indian adults [8]. Studies revealed inconsistent associations between proximity of recreational amenities and leisure PA (Table 1). While studies by both Akpinar et al. [39] and Chen et al. [45] detected significant positive associations for urban green spaces proximity, Ja´uregui et al. [36], Hino et al. [43] and Rech et al. [37] did find any significant relationships. Studies did not demonstrate any significant relationships between proximity of services (i.e., transit stops, stores) and leisure PA (Table 1). 3.2.4. Aesthetics. Studies revealed inconsistent relationships between perceived aesthetics and self-reported leisure PA in different LMICs (Table 1). Aesthetics was a significant positive predictor in urban Turkey (Aydın), Mexico (Cuernavaca) and Brazil (Curitiba) [36,37,39], 3.2.4. Aesthetics. Studies revealed inconsistent relationships between perceived aesthetics and self-reported leisure PA in different LMICs (Table 1). Aesthetics was a significant positive predictor in urban Turkey (Aydın), Mexico (Cuernavaca) and Brazil (Curitiba) [36,37,39], 5 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 Built environment correlates of physical activity in low- and middle-income countries PLOS ONE ariables signed d, e, or f was not considered in the coding unless all studies solely investigated the same variable. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 a Number of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. PLOS ONE Table 2. Relationships between travel PA and BE attributes across included studies. BE attributes PA-BE relationships % studies supporting the predicted association ab Summary codes c Significant (+) Significant (-) Non-significant Land-use mix diversity [8,49] N/A [36] 2/3 = 67% ++ Residential density [8] N/A [49] 1/2 = 50% ? Leisure amenities availability N/A N/A [44]d 0/1 = 0% 0d Land-use mix access N/A N/A [8,49] 0/2 = 0% 0 Recreational facilities proximity N/A [36] [46] 1/2 = 50% ? Transit stops proximity [49] N/A [36] 1/2 = 50% ? Aesthetics [46]f [8,49] [36] 2/3 = 67% -- Infrastructure to walk/cycle N/A N/A [8,36,49] 0/3 = 0% 00 Street connectivity N/A [8,49] [44] d 2/2 = 100% - Sidewalks N/A N/A [44 d,46] 0/1 = 0% 0 Crime safety N/A [8,49] [36,44 d,46 e] 2/3 = 67% -- Traffic safety [44] d N/A [8,36,46 e,49] 0/3 = 0% 00 Table 2. Relationships between travel PA and BE attributes across included studies. etween travel PA and BE attributes across included studies. a Number of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d association with physical inactivity. https://doi.org/10.1371/journal.pone.0230454.t002 Stud es vest gat g va ab es s g ed , , o was ot co s de ed t e cod g u ess a stud es so e y vest gated t e sa e va ab e. c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d association with physical inactivity. e f l f PLOS ONE [37] who reported a significant positive association in urban Brazil. 3.2.7. District income. Only two studies investigated the relationship between district income (assessed through GIS) and leisure-time PA. These showed a significant positive asso- ciation between high area income and self-reported leisure PA in southern Brazil (Curitiba) [43,47]. 3.2.7. District income. Only two studies investigated the relationship between district income (assessed through GIS) and leisure-time PA. These showed a significant positive asso- ciation between high area income and self-reported leisure PA in southern Brazil (Curitiba) [43,47]. 6 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 Built environment correlates of physical activity in low- and middle-income countries PLOS ONE PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 er of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. a Number of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. b d f 3.4. Walking and cycling 3.4.1. Density. Objectively measured residential density (GIS) predicted increased and decreased odds of transport walking and transport cycling in urban Brazil, respectively (Table 3). Hino et al. [21] reported that commercial density in Brazil (Curitiba) was significantly posi- tively associated with self-reported transport walking, but it was not significantly related to transport cycling. Street density was a significant positive predictor for leisure walking in urban Brazil [50], yet it showed inconsistent associations with both transport walking and cycling across LMICs (Table 1). When combining both transport and leisure walking, Gomez et al. [22] found a positive relationship with objectively assessed park density (GIS) in urban Columbia. 3.4.2. Land-use mix access and proximity to services. Studies did not reveal any signifi- cant relationships between proximity to transit stops and leisure walking, transport walking or transport cycling (Table 1). Perceived proximity to public services (e.g. stores, parks and trans- port stations) in suburban China (Shanghai) was associated with significantly increased odds for both transport and leisure walking [51]. Parra et al. [33], however, did not show significant relationships between perceived presence of public services with a 10-min walk in urban Brazil and either transport or leisure walking, yet a significant positive association was found for transport cycling. 3.4.3. Aesthetics. Perceived aesthetics was not significantly related to either leisure or transport walking across LMICs (Table 1). Jia et al. [51] did not detect any significant relation- ships between aesthetics and either self-reported transport or leisure walking in urban Chinese adults. A survey by Hallal et al. [46] also did not reveal any significant associations between perceived presence of pleasant surroundings and leisure walking in urban Brazil. 3.4.4. Infrastructure for walking and cycling. Studies did not detect any significant rela- tionships for presence of infrastructure for walking and cycling and leisure walking, transport walking or transport cycling (Table 1). Giehl et al. [50], however, reported significant positive associations between transport walking and street connectivity, sidewalks or paved streets (assessed through GIS) among Brazilian older adults. When examining the impact of lack of sidewalks on self-reported leisure walking among urban Brazilian adults, Hallal et al. [46] reported a negative association, whereas Gomes et al. [20] noted a positive relationship. Bike path existence within 500-m buffers in Brazil (Sao Paulo) was associated with significantly increased odds for leisure walking in a study by Florindo et al. [14]. 3.3. Travel PA 3.3.1. Land-use mix diversity. Two separate studies by Adlakha et al. [8,49] showed sig- nificant positive relationships between perceived land-use mix diversity and self-reported travel PA in urban Indian adults. Ja´uregui et al [36], on the other hand, did not note any signif- icant associations between both in urban Mexico. 3.3.2. Density. While residential density predicted significantly increased odds for travel PA in India (Chennai), it was not significantly related to multi-modal or active commuting in the same Indian City [8,49]. 3.3.3. Land-use mix access and proximity to services. Adlakha et al. [8] reported no sig- nificant relationships between land-use mix access and travel PA in urban India (Chennai). While Adlakha et al. [49] reported a strong positive association between transit stop proximity and travel PA in urban India, a study by Ja´uregui et al. [36] did not demonstrate a significant association between both in urban Mexico. 3.3.4. Aesthetics. Three studies showed significant negative relationships between neigh- borhood aesthetics and travel PA (Table 2). Aesthetics predicted significantly reduced odds of transport PA among urban Indian adults in studies by Adlakha et al. [8,49]. Hallal et al. [46] also reported an inverse relationship between aesthetics and travel PA in urban Brazil. Aesthetics predicted significantly reduced odds of transport PA among urban Indian adults in studies by Adlakha et al. [8,49]. Hallal et al. [46] also reported an inverse relationship between aesthetics and travel PA in urban Brazil. 3.3.5. Infrastructure for walking and cycling. Studies did not detect any significant rela- tionships between presence of infrastructure for walking and bicycling and self-reported travel PA across LMICs (Table 1). Adlakha et al. [8,49] reported a significant negative association between perceived street connectivity and self-reported travel PA in India (Chennai). 3.3.5. Infrastructure for walking and cycling. Studies did not detect any significant rela- tionships between presence of infrastructure for walking and bicycling and self-reported travel PA across LMICs (Table 1). Adlakha et al. [8,49] reported a significant negative association between perceived street connectivity and self-reported travel PA in India (Chennai). 7 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries 3.3.6. Crime and traffic safety. Perceived traffic safety was unrelated to travel PA across LMICs (Table 1). On the other hand, surveys by Adlakha et al. 3.3. Travel PA [8,49] showed that higher per- ceived crime safety in urban India was significantly negatively associated with travel PA. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 PLOS ONE PLOS ONE Table 3. Relationships between walking/ cycling and BE attributes across included studies. PA type BE attributes PA-BE relationships % studies supporting the predicted association ab Summary codes c Significant (+) Significant (-) Non-significant Transport walking Residential density [21,50] N/A N/A 2/2 = 100% + Commercial density [21] N/A N/A 1/1 = 100% + Street density [35] N/A [21,50] 1/3 = 33% ? Leisure amenities availability N/A N/A [50] 0/1 = 0% 0 Transit stops proximity N/A N/A [21] 0/1 = 0% 0 Services proximity [51] N/A [33] 1/2 = 50% ? Aesthetics N/A N/A [51] 0/1 = 0% 0 Infrastructure for walking N/A N/A [34] 0/1 = 0% 0 Walkability [47] N/A N/A 1/1 = 100% + Street connectivity [50] N/A [21] 1/2 = 50% ? Sidewalks [50] N/A [33,34] 1/3 = 33% ? Terrain slope N/A N/A [21,33] 0/2 = 0% 0 Paved streets [50] N/A 1/1 = 100% + Bike path availability N/A N/A [21,33] 0/2 = 0% 0 Bike path proximity N/A N/A [21] 0/1 = 0% 0 Traffic safety N/A N/A [21,33,34,51] 0/4 = 0% 00 Total crime safety [33] N/A [51] 1/2 = 50% ? Crime safety during day N/A N/A [48] e 0/1 = 0% 0 e Crime safety at night N/A [48] e N/A 1/1 = 100% - e District income N/A N/A [21,47,50] 0/3 = 0% 00 Leisure walking Residential density N/A N/A [50] 0/1 = 0% 0 Street density [50] N/A N/A 1/1 = 100% + Leisure amenities availability [43] N/A [50] 1/2 = 50% ? Recreational facilities proximity [43] N/A [14,36,46] 1/4 = 25% 00 Transit stops proximity N/A N/A [36] 0/1 = 0% 0 Services proximity [51] N/A [33] 1/2 = 50% ? PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 3.4. Walking and cycling On the other hand, terrain slope of 5% and high street connectivity (measured using GIS) predicted reduced odds of total walking among urban Columbian adults [22]. 3.4.5. Crime and traffic safety. Studies did not show significant associations between traffic safety and leisure walking, transport walking or transport cycling across LMICs (Table 1). While perceived crime-related safety did not exhibit any association with either lei- sure walking or transport cycling, it was a significant positive predictor for self-reported trans- port walking in Brazil (Curitiba) [33]. When combining both transport and leisure walking, Oyeyemi et al. [52] noted significant positive associations with both perceived crime safety during day and at night. 3.4.6. District income. Studies did not show any significant associations between district income and transport walking (Table 1). On the other hand, objectively assessed medium and PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 8 / 19 Built environment correlates of physical activity in low- and middle-income countries PLOS ONE https://doi.org/10.1371/journal.pone.0230454.t003 high area income in Brazil predicted significantly increased odds of self-reported leisure walk- ing in Curitiba and Floriano´polis, respectively [43,50]. In contrast, high district income was inversely associated with transport cycling among urban Brazilian adults [21]. high area income in Brazil predicted significantly increased odds of self-reported leisure walk- ing in Curitiba and Floriano´polis, respectively [43,50]. In contrast, high district income was inversely associated with transport cycling among urban Brazilian adults [21]. PLOS ONE Squares proximity N/A N/A [14] 0/1 = 0% 0 Aesthetics [36] N/A [46,51] 1/3 = 33% 00 Infrastructure for safe walking N/A N/A [20,36] 0/2 = 0% 0 Walkability N/A N/A [47] 0/1 = 0% 0 Street connectivity N/A N/A [50] 0/1 = 0% 0 Sidewalks [20] d [46] d [33,50] 0/2 = 0% 0 Paved streets N/A N/A [50] 0/1 = 0% 0 Bike path proximity [14] N/A N/A 1/1 = 100% + Crime safety N/A N/A [33,36,46,48 e,51] 0/4 = 0% 00 Traffic safety [36] N/A [20,33,46,51] 1/5 = 20% 000 District income [43,50] N/A [47] 2/3 = 67% ++ Total walking Residential density N/A N/A [12] 0/1 = 0% 0 Park density [22] N/A 1/1 = 100% + Leisure amenities availability N/A N/A [12,41] 0/2 = 0% 0 Transit stops proximity N/A N/A [12,22] 0/2 = 0% 0 Services proximity [41] N/A [12] 1/2 = 50% ? Aesthetics [12] N/A [41] 1/2 = 50% ? Infrastructure for safe walking N/A N/A [41] 0/1 = 0% 0 Street connectivity N/A [22] [12] 1/2 = 50% ? Sidewalks [41] N/A [12,22] 1/3 = 33% 00 Terrain slope N/A [22] N/A 1/1 = 100% - Bike path availability N/A N/A [12] 0/1 = 0% 0 Bike path proximity N/A N/A [41] 0/1 = 0% 0 Crime safety during day [52] N/A [12,41] 1/3 = 33% 00 Crime safety at night [12,52] N/A [41] 2/3 = 67% ++ Traffic safety [22] N/A [12,41,52] 1/4 = 25% 00 (Continued) Table 3. Relationships between walking/ cycling and BE attributes across included studies. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 9 / 19 Built environment correlates of physical activity in low- and middle-income countries a Number of articles supporting the predicted association divided by the entire number of studies investigated each BE varia PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d reduced/lack of access. gating variables signed d, or e was not considered in the coding unless all studies solely investigated the same variable. b Studies investigating variables signed d, or e was not considered in the coding unless all studies solely investigated the same variable. PLOS ONE Table 3. (Continued) PA type BE attributes PA-BE relationships % studies supporting the predicted association ab Summary codes c Significant (+) Significant (-) Non-significant Transport cycling Residential density N/A [21] N/A 1/1 = 100% - Commercial density N/A N/A [21] 0/1 = 0% 0 Street density [35] N/A [21] 1/2 = 50% ? Transit stops proximity N/A N/A [21] 0/1 = 0% 0 Services proximity [33] N/A N/A 1/1 = 100% + Infrastructure for cycling N/A N/A [34] 0/1 = 0% 0 Street connectivity N/A N/A [21] 0/1 = 0% 0 Sidewalks N/A N/A [33,34] 0/2 = 0% 0 Terrain slope N/A N/A [21,33] 0/2 = 0% 0 Bike path availability N/A N/A [21,33] 0/2 = 0% 0 Bike path proximity N/A N/A [21] 0/1 = 0% 0 Crime safety N/A N/A [33] 0/1 = 0% 0 Traffic safety N/A [21] [33,34] 1/3 = 33% 00 District income N/A [21] N/A 1/1 = 100% - b Studies investigating variables signed d, or e was not considered in the coding unless all studies solely investigated the same variable. c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d reduced/lack of access. c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d reduced/lack of access. e feeling unsafe. PLOS ONE Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d association with physical inactivity. https://doi.org/10.1371/journal.pone.0230454.t004 3.5.5. Crime and traffic safety. While perceived crime safety during the day showed inconsistent relationships, crime safety at night was consistently positively associated with total PA across LMICs (Table 1). Two studies did not show any significant relationship between perceived traffic safety and total PA among urban Mexican and Brazilian adults (Table 1). Oyeyemi et al. [12] however, reported a significant unexpected negative association between perceived traffic safety and self-reported total PA (excluding walking) among young Nigerian adults. 3.5.6. Urbanicity. Three studies demonstrated a consistent significant positive association (100%) between urbanicity and self-reported physical inactivity (Table 1). A cross sectional analysis of data from 46 LMICs showed a significant positive association between urbanicity and not meeting the WHO PA guidelines [56]. Similarly, urbanization was a significant posi- tive predictor for self-reported physical inactivity in separate studies by Katulanda et al. (in Sri Lanka) [57] and Allender et al. (in India) [58]. 3.5. Total PA 3.5.1. Density. A study in Nigerian students (University of Ibadan) by Oyeyemi et al. [12] did not reveal any significant association between perceived residential density or existence of leisure facilities and total PA (Table 4). 3.5.2. Land-use mix access and proximity to services. Proximity of public services and transit stops did not exhibit any relationships with total PA (Table 1). Proximity to recreational amenities, however, significantly increased odds for total PA among urban Mexican adults [53]. 3.5.3. Aesthetics. Studies did not report any significant relationships between perceived aesthetics and total PA (Table 1). Ja´uregui et al. [53] did not detect any significant relationship between aesthetics and objectively measured total PA (accelerometer) in urban Mexican adults. Perceived pleasant surroundings was also not related to self-reported PA in young Nigerian adults [12]. 3.5.4. Infrastructure for walking and cycling. Parra et al. [33] noted significant positive associations between total PA and presence of sidewalks, bike paths or steep terrain among urban Brazilian adults. Perceived lack of four-way intersections predicted significantly reduced odds of participation in total PA in Nigerian university students [12]. 10 / 19 PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 Built environment correlates of physical activity in low- and middle-income countries umber of articles supporting the predicted association divided by the entire number of studies investigated each BE variable. bles signed d or e was not considered in the coding unless all studies solely investigated the same variable. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 PLOS ONE PLOS ONE Table 4. Relationships between total PA and BE attributes across included studies. BE attributes PA-BE relationships % studies supporting the predicted association ab Summary codes c Significant (+) Significant (-) Non-significant Residential density N/A N/A [12] 0/1 = 0% 0 Leisure amenities availability N/A N/A [12] 0/1 = 0% 0 Recreational facilities proximity [53] N/A N/A 1/1 = 100% + Transit stops proximity [12] [53] [54] 1/3 = 33% 00 Services proximity N/A [54] [12,33] 1/3 = 33% 00 Aesthetics N/A N/A [12,53] 0/2 = 0% 0 Infrastructure for cycling/walking [55] d N/A N/A 1/1 = 100% + d Street connectivity N/A [12] e N/A 1/1 = 100% −e Sidewalks [33] N/A [12,53] 1/3 = 33% 00 Terrain slope [33] N/A 1/1 = 100% + Bike path availability [33] N/A [12] 1/2 = 50% ? Crime safety during day [33,53] [52] [12] 2/4 = 50% ? Crime safety at night [33,52,53] N/A [12] 3/4 = 75% ++ Traffic safety N/A [12] [33,53] 1/3 = 33% 00 Urbanicity [56–58]d N/A N/A 3/3 = 100% ++ d Table 4. Relationships between total PA and BE attributes across included studies. vestigating variables signed d or e was not considered in the coding unless all studies solely investigated the same variable. b Studies investigating variables signed d or e was not considered in the coding unless all studies solely investigated the same variable. c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d association with physical inactivity. c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). c The principles of evidence summary coding were adapted from Sallis et al [32]; +/- = positive or negative association (60–100% of articles supporting the predicted association); 0 = No relationship (0–33% of articles supporting the predicted association); ? = inconsistent relationship (34–59% of articles supporting the predicted association). Single signed codes (+, - or 0) were given for BE variables that were investigated only by 1–2 studies with respect to certain PA domains; When relationships were investigated in (3–4) or > 4 studies, double (++, - or 00) and triple (+++, - or 000) signed summary coding was applied, respectively. d association with physical inactivity. https://doi.org/10.1371/journal.pone.0230454.t004 s investigating variables signed d or e was not considered in the coding unless all studies solely investigated the same variable 4. Discussion This systematic review aimed to provide a better understanding of the impact of BE attributes on PA behaviors in LMICs populations. We identified a supportive role of the BE environment on PA domains, however, there were considerable variations in BE correlates across different PA domains in LMICs. All eligible studies included in this review were descriptive in nature (cross-sectional and case-series design), revealing the gap in longitudinal and analytical research in LMICs. More than half of included studies (58%) were conducted in Latin America PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 11 / 19 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries (Mexico, Brazil, and Columbia), highlighting the paucity of research on this topic in other developing countries. Perceived land-use mix diversity was not significantly related to leisure PA, however, there was a consistent positive association with travel PA across LMICs. Our findings produced lim- ited data concerning land-use mix access and did not show any significant relationships with either leisure PA or travel PA. Similarly, in a systematic review of 64 studies from both high- income and LMICs, Cleland et al [59] revealed no significant relationship between land-use mix access and either leisure or travel PA in older adults. Residential density was significantly positively related to transport walking, but negatively associated with transport cycling. A possible reason for this inverse relationship with cycling may be the lack of bicycling infrastructure such as segregated bike paths, specialized traffic signs, and signals and secured bike racks in highly dense residential areas [60]. In many LMICs, perceptions that roads are dangerous and uncomfortable, largely due to high volumes and high speeds of motorised traffic are well-established barriers to cycling [61]. Transport- related walking and bicycling are still common means of transportation especially in lower income groups, accounting for 50% to 70% of commuter trips in some urban areas, yet social prejudice against bicycle use prevails, and car ownership is still seen as a sign of social status [62]. Similar to our study, a survey by Forsyth et al. [63] revealed that residential density was significantly positively correlated with travel walking in the US (Minnesota). Commercial density was investigated only in one study in urban Brazil, where it predicted significantly increased odds of transport walking, yet it was not significantly related to transport cycling [21]. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 4. Discussion For example, PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 12 / 19 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries cycling is perceived as more appropriate for younger men, but an improper and unacceptable activity for women to engage in as per societal traditions in some LMICs [72]. Reviewed literature showed that perceived crime safety was not significantly related to lei- sure PA, yet it was significantly inversely associated with travel PA. These inconclusive results were also reported in previous studies, highlighting the need for further investigation of the influence of crime-related safety on different PA domains [73]. Perceived traffic safety was not significantly related to any PA domain across LMICs. On the other hand, perceived traffic- related safety predicted increased odds of participation in PA among Dutch adults in a study by Jongeneel-Grimen et al. [74]. These variations may be attributed to differences in perceived meaning of traffic safety among populations of different countries, where LMICs populations are more accustomed to high traffic volumes. Similar to Jongeneel-Grimen et al.’s study, a narrative review by Day [24] reported an association between traffic safety and travel PA in developing countries. However, we believe that findings from this review may have been inconclusive due to inconsistencies in evidence synthesis. Along with BE features, this review also examined non-BE factors (e.g. stray animals, weather), which may have impacted the overall study findings and conclusions. Some studies showed that high district income was significantly positively associated with both leisure PA and leisure walking. Kari et al. [75] also reported that higher district income was a significant positive predictor for leisure PA among Finnish adults. High-income popula- tions often have the resources to pay for memberships in private, well-maintained leisure cen- tres, offering a wide variety of recreational activities (e.g. swimming and fitness classes, gymnasium, golf courts) [76]. Studies revealed that urbanicity significantly increased the likelihood of physical inactivity across LMICs. In both developed and developing countries, there is strong evidence that urba- nicity is associated with increased sedentary behavior and diminishing levels of PA [58]. Urba- nicity was also a significant positive predictor of physical inactivity among older populations in Europe [77]. 4. Discussion Across LMICs, availability of recreational amenities showed a consistent positive influ- ence (100%) on leisure PA only. Existence of leisure amenities was also a significant positive predictor for leisure PA in Australia (Adelaide) and the US (North Carolina) [64,65]. This review showed that subjectively or objectively measured proximity to transit stops was not significantly associated with PA domains across LMICs. A meta-analysis of nine studies from North America and the UK, however, concluded that access to public transit was signifi- cantly positively correlated with participation in PA [66]. In contrast to developed countries, the lack of association between proximity to transit stops and PA in LMICs may be due to the widespread availability of alternative privately-operated ride-hailing services (e.g., minibuses, taxis, autorickshaws or tuk-tuks) providing fast connections to transit stops and minimizing PA levels [67]. Perceived aesthetics showed a significant association (negative direction) only with travel PA. In contrast, the presence of pleasant surroundings increased the likelihood of participation in PA among Japanese adults [68]. These conflicting results may be explained by discrepancies in different populations’ perceptions about aesthetics and its importance in pro- moting PA behavior. Our results reveal that availability of infrastructure for safe walking and cycling was not sig- nificantly associated with any PA domains across LMICs. A study in older American adults also did not find significant relationships between perceived presence of walking and cycling facilities and self-reported total PA in seven rural areas in North Carolina [69]. Self-reported and/or objective street connectivity did not show significant associations with leisure PA, lei- sure walking or transport cycling, however, it was significantly inversely related to travel PA. The study findings demonstrated that presence of bike paths was not significantly related to any PA domains. The 2012 Active People Survey in England, however, showed that bike path networks were significantly positively related to self-reported cycling [70]. These variations may be explained by the juxtaposition of different means of transportation (pedestrians, bicy- clists, and drivers), poor road infrastructure, lack of walking and cycling paths, weak road reg- ulations and law enforcement in LMICs [71]. These inconsistent findings may also be related to socio-cultural differences and gendered norms in developing countries. 4. Discussion Urban environment factors such as high population density, motorization, lack of safety from traffic and crime, diminishing space for walking, cycling and recreational activities, and excessive involvement in screen-based leisure time activities (e.g., television watching, video games, computer use, etc.) are associated with reduced PA and higher seden- tary behaviors [23]. Measures of urbanicity have limitations as they only consider basic aspects of urbanization [78]. In LMIC contexts, these measures may not be capturing the context accu- rately as neighborhoods may have differing types of BE infrastructure (e.g., number of paved roads, proximity to markets, etc.), highlighting the need to create a hierarchy of BE features corresponding to levels of urbanicity for LMICs. PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 5. Strengths & limitations This systematic review focuses on addressing the relationship between BE and PA in LMICs, which have thus far garnered limited research attention. Given that there are no gold standards for the selection of databases in the conduct of systematic reviews, we adopted a rigorous search strategy across four electronic databases including MEDLINE, PubMED, EMBASE and Web of Science. In conjunction with a robust search strategy, we feel confident that no perti- nent articles were missed. This review was registered in PROSPERO to increase its transpar- ency and minimize reporting bias. The findings were categorized by PA domains in order to allow effective reporting on the relationship between each separate domain and with BE fea- tures. This study, however, has some limitations for consideration. Given the large number of PA and BE variables examined, different statistical methods adopted by studies, and the incon- sistency in the measurement of different PA domains and BE characteristics across the PLOS ONE \| https://doi.org/10.1371/journal.pone.0230454 March 17, 2020 13 / 19 PLOS ONE Built environment correlates of physical activity in low- and middle-income countries included studies, it was not feasible to do a meta-analysis. The majority of evidence in the cur- rent study was based on cross-sectional designs, which cannot infer causation [79]. Further- more, more than half of the included studies relied on self-reported questionnaires for measuring BE and/or PA, which made our results subject to different types of bias (e.g. social desirability, recall bias) [80]. This review only included English publications, which posed the likelihood of excluding other relevant non-English articles from LMICs. 6. Conclusions and recommendations Research investigating the relationship between BE and PA behaviors amongst LMICs popula- tions appears to be limited. The majority of evidence in this field comes from cross-sectional studies, highlighting the gap in longitudinal, analytical and intervention research in LMICs. The majority of the investigated BE attributes were either unrelated or showed inconsistent relationships with all PA domains. Based on the findings of this systematic review, we propose that future BE-PA research and policy making in LMICs must focus on longitudinal assessments in different geographical areas (both rural and urban), with sub-group variations (e.g., age, gender, socioeconomic sta- tus and ethnicity) in order to infer reliable, temporal BE-PA relationships that will inform pub- lic health interventions and practice. Utilizing a combination of both validated/standardized objective and self-reported measures for assessing BE attributes and different PA domains can minimize any potential information bias. Recommendations include the development of tai- lored environmental interventions for promoting PA amongst LMICs populations, with feasi- bility studies for testing, and intervention evaluation before scaling up. S1 Appendix. PRISMA checklist. (DOCX) S2 Appendix. The search terms used in the searched databases. (DOCX) S2 Appendix. The search terms used in the searched databases. (DOCX) S3 Appendix. The standardized form with data extracted from the 33 eligible studies. (DOCX) S3 Appendix. The standardized form with data extracted from the 33 eligible studies. (DOCX) S5 Appendix. Quality assessment of the included studies. (DOCX) S1 Table. Numerical distribution and characteristics of the included studies. (DOCX) References 1. Smith M, Hosking J, Woodward A, Witten K, Macmillan A, Field A, et al. Systematic literature review of built environment effects on physical activity and active transport–an update and new findings on health equity. Int J Behav Nutr Phys Act. 2017; 14: 158. https://doi.org/10.1186/s12966-017-0613-9 PMID: 29145884 2. Ding D, Lawson K, Kolbe-Alexander T, Finkelstein E, Katzmarzyk P, Mechelen W, et al. The economic burden of physical inactivity: A global analysis of major non-communicable diseases. Lancet. 2016; 388. https://doi.org/10.1016/S0140-6736(16)30383-X 3. WHO. 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https://openalex.org/W2098136715	https://aab.copernicus.org/articles/46/187/2003/aab-46-187-2003.pdf	English	null	Follicular dynamic and ovulation in cattle – a review	Archives animal breeding/Archiv für Tierzucht	2,003	cc-by	7,613	Titel der Arbeit: Follikeldynamik und Ovulation beim Rind – eine Übersicht Titel der Arbeit: Follikeldynamik und Ovulation beim Rind – eine Übersicht Der Artikel gibt einen Überblick über Follikelpopulationen und Aspekte der Follikeldynamik beim Rind. In den Eierstöcken von Rindern existieren zwei Follikelpopulationen, die entweder dem ruhenden oder dem wachsenden Pool angehören. Während der reproduktiven Phase gehen ruhende Follikel in den wachsenden Pool über. Nachdem ein Follikel mit dem Wachstum begonnen hat, wird er entweder atretisch oder gelangt zur Ovulation. Das Wachstum der obligatorisch gonadotropinabhängigen Follikel erfolgt dabei in sogenannten Follikelreifungswellen. Diese sind durch die Prozesse der Rekrutierung, Selektion und Dominanz gekennzeichnet. Die Mechanismen und Faktoren, welche für die drei genannten Prozesse verantwortlich sind werden diskutiert. Schlüsselwörter: Rind, Follikelpopulationen, Follikeldynamik Summary A review is given about follicular populations and aspects of follicular development in cattle. Ovaries of cattle contain two different pools of follicles, the non-growing pool and the growing pool. Entry of primordial follicles into the growth phase occurs throughout the reproductive life. Once follicles are recruited to grow, they are destined to undergo atresia or ovulation. Growth of obligatory gonadotropin-dependent follicles occurs in a wave like pattern. The growth waves are characterised by the processes of recruitment, selection and dominance. The known mechanisms responsible for these three processes are discussed. Key Words: cattle, follicular populations, follicular dynamic Follicular dynamic and ovulation in cattle – a review Dedicated to Prof. Dr. agr. habil. Dr. h. c. mult. Georg Schönmuth on the occasion of his 75th birthday Arch. Tierz., Dummerstorf 46 (2003) 2, 187-198 Arch. Tierz., Dummerstorf 46 (2003) 2, 187-198 Research Institute for the Biology of Farm Animals, Department of Reproductive Biology, Dummerstorf, Germany Follicular populations Ovaries of cattle contain two different pools of follicles, the non-growing pool and the growing pool (Fig. 1). The non-growing pool contains the primordial follicles, whereas the growing pool contains the primary, secondary and tertiary follicles (KANITZ et al., 2001). Entry of primordial follicles into the growth phase occurs throughout the reproductive life. The primordial follicles continuously leave the arrested pool and undergo the primordial to primary follicle transition. The oocytes increase in size and the surrounding squamous pre-granulosa cells become cuboidal and proliferate to form a layer of cuboidal cells around the growing oocyte (FORTUNE et al., 2000). The follicle is called now a primary follicle. The mechanisms responsible for the initiation of follicular growth are poorly understood although some candidate molecules (gonadotropins, growth factors, c-kit) have been discussed (WEBB et al., 1999). In vitro studies with rat ovarian tissue showed that 188 KANITZ: Follicular dynamic and ovulation in cattle leukaemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF) are able to promote the primordial to primary follicle transition (NILSSON et al., 2001, 2002). But the finding that during culture of isolated bovine or primate ovarian cortex almost all primordial follicles activate and develop into primary follicles suggests that non- cortical portions of the ovary may regulate the flow of follicles from the resting reservoir (FORTUNE et al., 1999). The number of follicles commencing growth in a given time is predictable because it is a function of the size of the primordial store, which declines exponentially with time (WEBB et al., 1999). Recruitment Growth factors? Gonadotropins ? non - growing pool ( primordial follicles, 35 –100 µm ) Size of the pool depends on: oogonia multiplication, time of meiosis initiation, loss of germ cells by apoptosis gonadotropin- dependent growing pool ( primary, secondary and tertiary follicles ) antral follicles ( antrum formation: ∅~ 0.3 mm ) gonadotropin- obligatory dependent (follicular waves) ∅~2 mm ovulatory follicles Recruitment Growth factors? Gonadotropins ? non - growing pool ( primordial follicles, 35 –100 µm ) Size of the pool depends on: oogonia multiplication, time of meiosis initiation, loss of germ cells by apoptosis gonadotropin- dependent growing pool ( primary, secondary and tertiary follicles ) antral follicles ( antrum formation: ∅~ 0.3 mm ) gonadotropin- obligatory dependent (follicular waves) gonadotropin- obligatory dependent (follicular waves) obligatory dependent (follicular waves) ∅~2 mm ovulatory follicles Fig. Follicular populations 1: Schematic diagram of follicular populations in cattle (Schematische Darstellung der Follikelpopulationen beim Rind) Fig. 1: Schematic diagram of follicular populations in cattle (Schematische Darstellung der Follikelpopulationen beim Rind) During recruitment of follicles into the growing pool theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. In this process granulosa cell-derived kit-ligand appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles (PARROTT and SKINNER, 2000). In the process of theca cell differentiation cells become epitheloid and acquire organelles characteristic for steroid secreting cells. Once follicles are recruited to grow, they are destined to undergo apoptosis unless rescued by survival factors (HSUEH et al., 1996). The Fas antigen and the Fas ligand system seem to play an important role in mediating apoptosis (PORTER et al., 2001). During recruitment of follicles into the growing pool theca cells organize into distinct layers around early developing follicles and establish essential cell-cell interactions with granulosa cells. In this process granulosa cell-derived kit-ligand appears to promote the formation of theca cell layers around small (i.e., primary) ovarian follicles (PARROTT and SKINNER, 2000). In the process of theca cell differentiation cells become epitheloid and acquire organelles characteristic for steroid secreting cells. p q g g Once follicles are recruited to grow, they are destined to undergo apoptosis unless rescued by survival factors (HSUEH et al., 1996). The Fas antigen and the Fas ligand system seem to play an important role in mediating apoptosis (PORTER et al., 2001). Follicular waves second wave 1st follicular wave S e l e c t i o n S e l e c t i o n Dominanc Dominanc A t r e s i a 2nd follicular wave FSH FSH Inhibin LH Estradiol progesterone Ovulation Progesteone concentration (ng/ml) PGF 2α Recruitment Recruitment 0 1 0 2 4 6 8 10 12 14 16 Follicular diameter (mm) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 Days of oestrous cycle dom. foll. first wave dom. foll. second wave 1st follicular wave S e l e c t i o n S e l e c t i o n Dominanc Dominanc A t r e s i a 2nd follicular wave FSH FSH Inhibin LH Estradiol progesterone Ovulation Progesteone concentration (ng/ml) PGF 2α Recruitment Recruitment 0 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 Days of oestrous cycle 0 1 0 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 Days of oestrous cycle Fig. 2: Schematic diagram of events of follicular growth in waves during an interovulatory interva (Schematische Darstellung von Ereignissen in Follikelreifungswellen während eines Interöstrusintervalls) dom. foll. first wave dom. foll. second wave progesterone dom. foll. first wave dom. foll. second wave progesterone Fig. 2: Schematic diagram of events of follicular growth in waves during an interovulatory interval (Schematische Darstellung von Ereignissen in Follikelreifungswellen während eines Interöstrusintervalls) p g p g Changes in mRNA expression for the gonadotropin receptors, key steroidogenic enzymes and growth factors (IGF-I and -II) and their binding proteins (IGFBP) have been associated with different stages of follicular growth and atresia. In general, expression of mRNA for the gonadotropin receptors, steroidogenic enzymes, and steroidogenic acute regulatory protein (StAR) increase with progressive follicular development and is highest when dominant follicles approach maximum size. Expression of mRNA declines rapidly and becomes low or undetectable in atretic follicles. The IGF-I (granulosa cells) and IGF-II (theca cells) are increased, whereas IGFBP-2 (granulosa cells) is reduced, in dominant follicles. Recruitment of a cohort of follicles is associated with initiation of expression of mRNA for P450scc and P450arom in granulosa cells. Follicular waves In cattle the growth of obligatory gonadotropin-dependent follicles occurs in a wave like pattern (DRIANCOURT, 2001). Waves of growth can be observed during the prepubertal period (ADAMS et al., 1994; EVANS et al., 1994; MELVIN et al., 1999), in pregnant cattle (TAYLOR and RAJAMAHENDRAN, 1991), in postpartum periods (MURPHY et al., 1990) and during oestrous cycles (ROCHE et al., 1999). During one interovulatory interval two (GINTHER et al., 1989; KNOPF et al., 1989; RAJAMAHENDRAN and TAYLOR, 1991; AHMAD et al, 1997, BURKE et al., 2000; BELLMANN 2001), three (SAVIO et al., 1988; SIROIS and FORTUNE, 1988; AHMAD et al., 1997, BURKE et al., 2000; BELLMANN, 2001) or four waves (RHODES et al., 1995) have been observed. Cycles with three waves were on average 189 Arch. Tierz. 46 (2003) 2 1.1 day longer and Corpora lutea regressed later than in animals with two waves. Moreover, interval from detection of dominant follicle to ovulation and duration of dominance were shorter in animals with three waves (AHMAD et al., 1997). ADAMS (1999) concluded from the available data that greater than 95 % of oestrous cycles are composed of either two or three follicular waves. Normally three to six follicles with a diameter of 4 to 5 mm occur after recruitment of follicles into in a follicular wave (SAVIO et al., 1988; SIROIS and FORTUNE, 1988; SUNDERLAND et al., 1994). However, the number of follicles, which are recruited in a wave seems to be higher (ADAMS, 1999; BELLMANN, 2001). From the cohort, one follicle is selected for continued growth and becomes dominant. If luteolysis occurs during the growth phase of dominant follicles, final maturation and ovulation occurs. If luteolysis does not occur during the growing and maintenance phase of follicles, the fate is atresia (Fig. 2). Fig. 2: Schematic diagram of events of follicular growth in waves during an interovulatory interval (Schematische Darstellung von Ereignissen in Follikelreifungswellen während eines Interöstrusintervalls) 0 1 0 2 4 6 8 10 12 14 16 Follicular diameter (mm) 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 0 1 Days of oestrous cycle dom. foll. first wave dom. foll. Follicular waves Selection of dominant follicles is associated with expression of mRNA for LH receptors and 3β-HSD in granulosa cells. Thus, changes in gene expression likely are important to recruitment, selection, dominance, and atresia in ovarian follicles (BAO and GARVERICK, 1998; BEG et al., 2001, 2002). Recruitment Recruitment 190 KANITZ: Follicular dynamic and ovulation in cattle The concept recruitment is used for the entrance of follicles in the growing pool, but also for the processes associated with the entrance of follicles in a wave like growth pattern. FSH is the key hormone for the endocrine initiation of follicular wave occurrence (ADAMS et al., 1992; SUNDERLAND et al., 1994; FRICKE et al., 1997). ADAMS et al. (1992) found that two-wave heifers had two FSH surges and three- wave heifers had three apparent FSH surges during the interovulatory interval. Results of the cautery and follicular fluid experiments indicated that a surge in FSH necessarily preceded the emergence of a wave. The FSH surges in treated and control heifers began 2 - 4 days before the detectable (ultrasound) emergence of a follicular wave (follicles of 4 and 5 mm), peaked 1 or 2 days before emergence and began to decrease approximately when the follicles of a wave begin to diverge into a dominant follicle and subordinate follicles (follicles 6 - 7 mm). GINTHER et al. (2000) found also that the surge reaches a peak by the time the follicles attain 4 mm in diameter. In accordance with this CROWE et al., (1998) observed that the emergence of each follicle wave postpartum was preceded by a 2- to 4-day rise in FSH concentrations. The pattern of FSH isoform distribution did not differ between the pre- and postpartum periods. As follicular recruitment proceeds, mRNA for P450 aromatase increases (FORTUNE et al., 2001). Selection 46 (2003) 2 means of 8.5 and 7.7 mm in diameter at the end of the selection period. Thereafter they begin to undergo deviation in diameters, which is characterized by continued growth of the largest follicle to become the dominant follicle and reduced or terminated growth of the remaining follicles to become subordinate follicles. At the beginning of follicular deviation the largest follicle releases increased estradiol into the blood, and the released estradiol is involved in the continuing depression of FSH concentrations to below the requirement of the smaller follicles but not the largest follicle (GINTHER et al., 1999; 2000). In addition aspiration of the dominant follicle at any stage of the cycle affected circulating FSH but did not appear to influence the mean LH concentration (AMIRIDIS et al., 1999). GINTHER et al. (2001a) reported that apparently both estradiol and inhibin contribute to the continuing FSH decline from this time. Moreover it was found that elevated concentrations of LH and reduced concentrations of FSH were present 32 to 16 h before to at least 24 h after the beginning of follicle deviation GINTHER et al. (1998). The increased secretion of estradiol into the circulation and the increase in estradiol and IGF-I and decrease in IGFBP-2 concentrations in the follicular fluid at the start of deviation are functions of the transient increase in LH concentrations that encompasses follicle deviation GINTHER et al. (2001b). In heifers, receptors for LH appeared in the granulosa cells of the future dominant follicle about 8 h before the beginning of deviation. In addition, LH-receptor mRNA expression in the granulosa cell layer was limited to follicles that also expressed mRNAs for P450scc and P450arom in the granulosa cells (BAO et al., 1997). The LH stimulates the production of estradiol and insulin-like growth factor-1. These intrafollicular factors and perhaps others account for the responsiveness of the largest follicle to the low concentrations of FSH. The smaller follicles have not reached a similar developmental stage and because of their continued and close dependency on FSH become susceptible to the low concentrations (GINTHER et al., 2001a). In the past decade, numerous intrafollicular growth factors, such as inhibins, activins, and insulin-like growth factors and their binding proteins, have been identified in follicular fluid of individual bovine follicles (CHAMBERLAIN and SPICER, 2001; MIHM et al., 2002; SCHAMS et al., 2002). Selection Selection means that the number of growing follicles is brought into line with the species-specific ovulation number (FORTUNE, 1994). After recruitment fewer and fewer recruited follicles continue in growth until one follicle is selected to become dominant while the remaining members of the recruited follicles become static and undergo atresia via apoptosis. The processes of selection occur under declining FSH concentrations and take 2 to 3 days (EVANS et al., 1997; AUSTIN et al., 2001). GIBBONS et al. (1999) found that Follicles ≤ 3 mm had no detectable capacity to suppress FSH. As follicles grew from 3 to 5 mm, they gained the capacity to suppress FSH; however, as follicles grew beyond 5 mm, FSH-suppressing capacity did not increase. At the beginning of selection all of the growing follicles ≥ 5 mm contribute to the decline in FSH concentrations (GIBBONS et al., 1997; GINTHER, 2000). These results of KANEKO et al. (1997) demonstrated that inhibin neutralization during the early luteal phase produces hypersecretion of FSH with a coincident stimulation of follicular development, indicating that inhibin is an important factor for the negative regulation of FSH secretion during the early luteal phase when secretion of estradiol and progesterone are normally high. Also GINTHER et al. (2001a) stated that inhibin is the primary FSH suppressant at this time. IRELAND and IRELAND (1994), IRELAND et al. (1994) and GOOD et al. (1995) observed multiple forms of inhibin/activin subunit mRNA and peptide in follicular tissue and fluid. AUSTIN et al. (2001) found that the dominant follicle maintains high amounts of higher molecular weight inhibins while the subordinate follicles have increased amounts of the smallest (34 kDa) inhibin. KULIK et al. (1999) reported that deviation occurred when the 2 largest follicles were 8.3 ± 0.2 and 7.8 ± 0.2 mm in diameter. This was observed at 61.0 ± 3.7 h after wave emergence. Very similar data about follicular diameter at the time of deviation were reported by GINTHER et al. (2000). The authors found that the largest follicles reach KULIK et al. (1999) reported that deviation occurred when the 2 largest follicles were 8.3 ± 0.2 and 7.8 ± 0.2 mm in diameter. This was observed at 61.0 ± 3.7 h after wave emergence. Very similar data about follicular diameter at the time of deviation were reported by GINTHER et al. (2000). The authors found that the largest follicles reach 191 Arch. Tierz. Selection The IGF stimulate ovarian function by acting synergistically with gonadotropins to promote growth and steroidogenesis of ovarian cells. Actions of IGF-I and -II are restrained by a series of IGF binding proteins (IGFBP) that either originate from the blood or are synthesized locally within the follicle. Degradation and differential synthesis of IGFBP are important mechanisms regulating IGFBP amounts. The relative amounts of IGFBP may ultimately determine ovarian IGF action (LUCY, 2000). MIHM et al. (2000) found that the future dominant follicle in most cohorts had the highest estradiol and lowest IGFBP-4 concentrations compared with future subordinate follicles. The authors concluded that IGFBP-4 and estradiol may have key roles in determining the physiological fate of follicles during selection of the first wave dominant follicle and that both are reliable markers to predict which follicle in a growing cohort of 5 to 8.5 mm follicles becomes dominant. In addition, maintenance of low amounts of intrafollicular IGFBP4 may constitute an important mechanism in the future DF to attain FSH independence (MIHM and AUSTIN, 2002; MIHM et al., 2002). In contrast to that atresia of subordinate follicles appears to be associated with increased expression of the IGFBP2 gene (YUAN et al., 1998). Results of BERISHA et al. 192 KANITZ: Follicular dynamic and ovulation in cattle (2000) suggest that VEGF and FGF families are involved in the proliferation of capillaries that accompanies the selection of the preovulatory follicle resulting in an increased supply of nutrients and precursors, and therefore supporting the growth of the dominant follicle. In vitro studies complete the results obtained in vivo. In these studies it was demonstrated that growth factors could have endocrine, autocrine or paracrine actions that modify gonadotropin-stimulated follicular growth and differentiation. FORTUNE et al. (2001) found that granulosa cells from the dominant follicle produce more estradiol in vitro than cells from subordinate follicles. Shortly after selection, dominant follicles have higher levels of mRNAs for gonadotropin receptors and steroidogenic enzymes. In conclusion, the decline in FSH beginning after Day 2 of the heifer oestrous cycle causes differential alterations in FSH dependent growth factors and hormones within the cohort of preselection follicles, simultaneously inducing growth and enhanced estradiol producing capacity of the DF and atresia of subordinate follicles (MIHM et al., 1997). Dominance Follicles are functionally dominant (capable of ovulating after luteal regression) while they are still growing and early during their plateau in growth (FORTUNE et al., 1991). Follicles acquired ovulatory capacity at about 10 mm, corresponding to about 1 day after the start of follicular deviation, but they required a greater LH dose to induce ovulation compared with larger follicles. It was speculated that acquisition of ovulatory capacity may involve an increased expression of LH receptors on granulosa cells of the dominant follicle and that this change may also be important for further growth of the dominant follicle (SARTORI et al., 2001). Observations of GINTHER et al. (1997) indicate that the future dominant follicle cannot be identified reliably by either its diameter or estradiol production before the deviation in growth rates between the two largest follicles. Dominance appears to be maintained by negative feedback effects of products of the dominant follicle on circulating FSH. Selection and dominance are accompanied by progressive increases in the ability of theca cells to produce androgen and granulosa cells to aromatise androgen to estradiol. Dominant follicles grow to a much larger size than all the other follicles (from 8.5 mm at the end of selection to 12 – 20 mm). This takes 3 to 4 days. LH pulses are indispensable for follicle development beyond 9 mm in diameter (GONG et al., 1996; KANITZ et al., 2001). Endocrine pattern of gonadotropins is followed by characteristic changes in follicles. Concentrations of estradiol-17 beta in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles compared to subordinate follicles. Aromatase activity in first-wave dominant follicles was higher at Days 5 and 8 compared to Day 12 (BADINGA et al., 1992). Observations of GINTHER et al. (1997) indicate that the future dominant follicle cannot be identified reliably by either its diameter or estradiol production before the deviation in growth rates between the two largest follicles. Dominance appears to be maintained by negative feedback effects of products of the dominant follicle on circulating FSH. Selection and dominance are accompanied by progressive increases in the ability of theca cells to produce androgen and granulosa cells to aromatise androgen to estradiol. Dominant follicles grow to a much larger size than all the other follicles (from 8.5 mm at the end of selection to 12 – 20 mm). This takes 3 to 4 days. Dominance LH pulses are indispensable for follicle development beyond 9 mm in diameter (GONG et al., 1996; KANITZ et al., 2001). Endocrine pattern of gonadotropins is followed by characteristic changes in follicles. Concentrations of estradiol-17 beta in follicular fluid and aromatase activity of follicular walls were higher in dominant follicles compared to subordinate follicles. Aromatase activity in first-wave dominant follicles was higher at Days 5 and 8 compared to Day 12 (BADINGA et al., 1992). ASSEY et al. (1994) investigated the structure of oocytes aspirated from the dominant and its subordinate follicles. Therefore oocytes were aspirated from the dominant (largest) and two largest subordinate follicles and processed for transmission electron microscopy. Follicular fluids were analysed for concentrations of estradiol-17 beta (E2) and progesterone (P4). Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2-dominated. ASSEY et al. (1994) investigated the structure of oocytes aspirated from the dominant and its subordinate follicles. Therefore oocytes were aspirated from the dominant (largest) and two largest subordinate follicles and processed for transmission electron microscopy. Follicular fluids were analysed for concentrations of estradiol-17 beta (E2) and progesterone (P4). Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2-dominated. 193 Arch. Tierz. 46 (2003) 2 Subordinate follicles ceased growing at about days 3 - 4 and their follicular fluid had low E2:P4 ratio or was P4-dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. Subordinate follicles ceased growing at about days 3 - 4 and their follicular fluid had low E2:P4 ratio or was P4-dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. The fate of the dominant follicle depends on function of the Corpus luteum. In the cases of elevated progesterone concentrations the dominant follicle becomes atretic due to the negative influence of the progesterone on pulsatility of LH secretion (IRERLAND et al., 2000). Under these circumstances functional dominance is lost some time between the early and late plateau phases (between days 7 and 9 of the oestrous cycle), while the follicle is still morphologically dominant (i.e. the largest follicle). Dominance Loss of dominance occurs after a decline in estradiol secretion (around day 6) of the dominant follicle of the first follicular wave (SUNDERLAND et al., 1994). A decrease in follicular estradiol and inhibin-A secretion is going in front of the new wave of follicular growth (GINTHER et al., 1996; MIHM et al., 2002). From the available data following conclusions were drawn (ADAMS, 1999): From the available data following conclusions were drawn (ADAMS, 1999): (1) follicles grow in a wave-like fashion; (2) periodic surges in circulating FSH are associated with follicular wave emergence; (3) selection of a dominant follicle involves a decline in FSH and acquisition of LH responsiveness; (4) periodic anovulatory follicular waves continue to emerge until occurrence of an LH surge; (5) within species, there is a positive relationship between the duration of the oestrous cycle and the number of follicular waves; (6) progesterone suppresses LH secretion and growth of the dominant follicle; (7) the duration of the interwave interval is a function of follicular dominance, and is negatively correlated with circulating FSH; (8) follicular dominance in all species is more pronounced during the first and last follicular waves of the oestrous cycle and (9) pregnancy, the prepubertal period and seasonal anoestrus are characterized by regular, periodic surges in FSH and emergence of anovulatory follicular waves. Final follicular maturation and ovulation Ovulation is induced by an increase in LH secretion (KANITZ et al., 2001). The LH surge triggers a biochemical cascade. The results of DIELEMAN and BLANKENSTEIN (1984, 1985) indicate that in preovulatory bovine follicles inhibition of aromatisation takes place at about 14 h after the preovulatory LH peak and progesterone concentrations increase before ovulation. Also progesterone receptor mRNA expression is upregulated specifically in the granulosa layer of bovine preovulatory follicles following the LH surge (CASSAR et al., 2002). Moreover results of DOW et al. (2002) show that mRNA expression and enzyme activity for both tPA and uPA are increased in a temporally and spatially specific manner in bovine preovulatory follicles after exposure to a gonadotropin surge. Increased plasminogen activator and plasmin activity may be a contributing factor in the mechanisms of follicular rupture in cattle. In sum the processes lead to the rupture of the preovulatory follicle, the expulsion of the oocyte and the formation of the Corpora luteum. Local regulation of ovulation involves the interaction of LH and intrafollicular factors including steroids, prostaglandins, and peptides derived from endothelial cells, leukocytes, fibroblasts, and steroidogenic cells. An increase of prostaglandins 194 KANITZ: Follicular dynamic and ovulation in cattle (PGE2 and PGF2α) in follicular fluid of preovulatory follicles in the cow has been demonstrated by ALGIRE et al. (1992). Results from ACOSTA et al. (1998, 2000) suggest that interactions among ET-1, PGE2, and cytokines may have key roles in a local intermediatory/amplifying system of the LH- triggered ovulatory cascade in the bovine preovulatory follicle. FORTUNE al. (1993) found that oxytocin is also part of the LH induced biochemical cascade for ovulation. In cattle PETERS and BENBOULAID (1998) investigated the occurrence of ovulation after PGF2α/GnRH application in some animals by means of ultrasound. Ovulation occurred between 24 to 48 h after GnRH injection. More recently, we examined the time of ovulation after PGF2α/GnRH application in heifers. Ultrasonographic examinations of ovaries were done every 6 hours during the periovulatory period. The mean interval from GnRH to ovulation was 25 to 33 hours. Our data and results from PETERS and BENBOULAID (1998) indicate, that ovulatory follicles have a diameter between 15 and 20 mm. The results of KOT and GINTHER (1999) show, that the mean time from beginning to completion of evacuation of ovulatory follicles was 4.3 ± 3.3 min (min. 6 s; max. 14.5 min.). Some authors investigated results of A.I. Final follicular maturation and ovulation in dependence on number of follicular waves or duration of dominance. AHMAD et al. 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https://openalex.org/W1965116262	https://figshare.com/articles/journal_contribution/A_FAK_scaffold_inhibitor_disrupts_FAK_and_VEGFR_3_signaling_and_blocks_melanoma_growth_by_targeting_both_tumor_and_endothelial_cells/1246746/2/files/3246266.pdf	English	null	A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells	Cell cycle/Cell cycle (Georgetown, Tex. Online)	2,014	cc-by	2,730	A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells A FAK scaffold inhibitor disrupts FAK and VEGFR-3 signaling and blocks melanoma growth by targeting both tumor and endothelial cells Elena Kurenova1,6, Deniz Ucar5, Jianqun Liao1, Michael Yemma1, Gogate Priyanka1, Wiam Bshara2 , Ulas Sunar3, Mukund Seshadri4, Steven N. Hochwald1 and William G. Cance1,6 . Antibodies and reagents VEGFR-3 and p-VEGFR-3 rabbit polyclonal antibody from Cell Aplications, Inc. and Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Cell Signaling Technology (Danvers, MA, USA): Pro-caspase- 8, -3; PARP. Motility wound healing assay For the measurement of cell migration during wound healing, human C8161 cells were seeded on 24-well plates. Confluent cell cultures were incubated in serum-free medium for 24 h and were wounded by scraping with a 100-μL pipette tip, washed to remove debris and incubated in medium containing 10% FBS with or without C4 (10 and 50 μM), as indicated. Cells were photographed after wounding by phase contrast microscopy (Axiovert200, Zeiss) at 12, 24 and 48 h. Experiments were done in duplicate and two fields of each well were recorded. Image analysis was done with WimScratch module of Wimasis software. Apoptosis assay After treatment of cells for 24, 48, and 72 hours cells were collected, counted and prepared for terminal uridine deoxynucleotidyl transferase (TUNEL) assay by utilizing an APO-BRDU kit (BD Pharmingen, San Diego, CA) according to the manufacturer's instructions. Stained cells were analyzed with a FACSCalibur cytometer (Becton Dickinson, San Jose, CA). Calculation of the percentage of apoptotic cells in the sample was completed with CellQuest software (BD Biosciences). Development of fluorescence polarization (FP)-based binding assays for FAK protein Sensitive and quantitative FP-based binding assay was developed to determine the binding affinity of FAK scaffold inhibitors to the recombinant FAK protein using the Perkin Elmer EnVision 2103 Multilabel Reader and software Wallac Envision Manager 1.12. Determine Kd value of TAMRA probe to protein The 12-mer VEGFR-3 derived peptide AV3 (1) was 5-carboxytetramethylrhodamine (TAMRA)- conjugated (Biomatik, Wilmington, DE) and used as the fluorescent probe in the FP-based binding assays (TAMRA- Acp-WHWRPWTPCKMF-NH2). The corresponding unlabeled peptide AV3 (Biomatik, Wilmington, DE) was used in the competition assay. The Kd value was determined to FAK protein with a fixed concentration of the tracer (175 nM of AV3-TAMRA) and increasing concentrations (0.97 µM - 200 µM) of FAT domain protein, in a final volume of 125 μl in the assay buffer (5 mM dithiothreitol (DTT), 0.05% BSA, and 0.05% Triton X-100 in PBS at pH 6.5). Plates (384 well non-binding surface flat bottom black well plates, Corning) were mixed and incubated at room temperature for 1 hour and the polarization values in millipolarization units (mP) were measured at an excitation wavelength of 531 nm and an emission wavelength of 579 nm. Equilibrium dissociation constants (Kd) were calculated by using the non-linear curve fitting algorithms of Graphpad Prism 6.0 software (Graphpad Software). Equilibrium dissociation constants (Kd) were calculated by using the non-linear curve fitting algorithms of Graphpad Prism 6.0 software (Graphpad Software). Determine IC50 values of FAK scaffold inhibitors 1 Based on the Kd values, the concentrations of the FAT protein used in the competitive binding experiments was 40 µM. The fluorescent probe AV3-TAMRA were fixed at 175 nM for all assays. A range of concentrations of unlabeled AV3 (0.39 µM – 200 µM), C4 (0.97 µM – 250 µM), and C1 (0.97 µM – 250 µM) were used for competition assays. 100 μL of protein/probe complex in the assay buffer (5 mM dithiothreitol (DTT), 0.05% BSA, and 0.05% Triton X-100 in PBS at pH 6.5) were added to assay plates (384 well non-binding surface flat bottom black well plates, Corning), incubated at room temperature for 1 h and the polarization values (mP) were measured at an excitation wavelength of 531 nm and an emission wavelength of 579 nm. Non-linear curve fitting algorithms were used to calculate the inhibition constants (one site log IC50) using GraphPad Prism 6. Magnetic Resonance Imaging MRI examinations were performed in mice bearing C8161 melanoma xenografts with matched tumor volumes (n = 3 per group). Experimental MRI examinations were carried out in a 4.7 T/33-cm horizontal bore magnet (GE NMR Instruments, Fremont, CA) incorporating AVANCE digital electronics 2 (Bruker Biospec with ParaVision 3.0.2; Bruker Medical Inc., Billerica, MA) and a removable gradient coil insert (G060, Bruker Medical Inc., Billerica, MA) generating maximum field strength of 950 mT/m and a custom-designed 35-mm RF transmit-receive coil. Mice were placed on a form-fitted MR-compatible sled (Dazai Research Instruments, Toronto, Canada) and supplied with 2% isoflurane during image acquisition. Respiration rates and core-body temperature were monitored continuously while mice were in the scanner. Preliminary scout images were acquired on the sagittal and axial planes to assist in slice prescription for subsequent scans. Multislice non contrast-enhanced T2-weighted images were acquired on the axial planes with the following parameters: TEeff = 41 ms, TR = 2500 ms, FOV = 3.2 x 3.2 cm, matrix size = 256 x 192, 25 slices, slice thickness 1 mm). T1-weighted (T1W) contrast-enhanced MRI was performed using albumin-gadopentetate dimeglumine (albumin-GdDTPA) as described previously (2-4). A series of eight T1-weighted images (3 precontrast and 5 post contrast) were acquired over ~45 minutes. Albumin-Gd- DTPA was administered at a dose of 0.05 mmol/kg as a bolus via tail-vein injection after completion of 3 baseline precontrast images. The vascular relaxation enhancement after administration of albumin- GdDTPA was measured using a balanced steady state precession, inversion recovery technique (TrueFISP) as described previously (3). Raw image datasets were transferred to a workstation for post processing using AnalyzeTM (AnalyzeDirect, Overland Park, KS) and MATLAB (Version 7.0, Mathworks Inc., Natick, MA). Regions of interest (ROI) were manually defined for the entire tumor, blood vessel, kidneys and murine muscle tissues at each time point. At least 2–3 slices were evaluated for each tumor. The change in R1 (ΔR1) following contrast agent injection was assumed to be proportional to the tissue concentration of the contrast agent. Blood R1 curves were fitted to monoexponential decay. Kinetic analysis of ΔR1tumor (normalized to the fit values of ΔR1 in blood) was performed to estimate the fractional blood volume (BV; y-intercept) (2, 3). Relative blood volume maps were calculated on a pixel-by-pixel basis using MATLAB (Version 7.0, Mathworks Inc., Natick, MA) and pseudo-colorized in Analyze™. Diffuse Correlation Spectroscopy for Blood Flow Quantification 3 Tissue blood flow was measured using a previously described and validated diffuse correlation spectroscopy (DCS) instrument (5, 6), which measures rapid light intensity temporal fluctuations in tissue and then uses the autocorrelation functions associated with these fluctuations to extract information about the speed of moving tissue scatterers, in this case red blood cells (7). The decay rate of the autocorrelation 3 function is related to blood flow (8). Optical blood flow measurements were performed with a system comprised of three main components: a long coherence length laser operating at 785 nm, a photon- counting detector, and a custom built autocorrelator board. The optical probe contained one multimode laser source fiber and four single-mode detector fibers so that four source-detector separations ranging from 2mm to 4mm were obtained. For each measurement, the four detector signals were averaged to obtain average blood flow. Skeletal muscle away from the tumor was measured for comparison. Five independent measurements were performed by placing the optical probe at slightly different spatial positions. Photodetector outputs were fed into a correlator board, and intensity autocorrelation functions and photon arrival times were recorded by a computer for further processing to extract blood-flow-related parameters. We generally report relative blood flow, , to describe blood flow changes during therapy: is a blood flow parameter measured relative to its pre-treatment value. Blood flow was measured before treatment to obtain baseline levels and subsequent measurements were performed 7 days and 14 days after treatment. Measurements were normalized to baseline values to quantify changes in relative blood flow due to C4 treatment. ; 2. Seshadri M, Mazurchuk R, Spernyak JA, Bhattacharya A, Rustum YM, Bellnier DA. Activity of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid against human head and neck carcinoma xenografts. Neoplasia. 2006;8:534-42. ure S4. FAK scaffold inhibitor C4 sensitized melanoma cells to cytotoxic therapy Supplementary figure S5. FAK scaffold inhibitor C4 inhibited tumor growth in vivo. A. Mice weight, IP treatment with PBS or C4 50 mg/kg for 62 days. B. Mice weight in PBS and C4 50 mg/kg treatment groups at the end of the experiment at day 62. C. Heart, lung, liver, spleen, kidney weight in PBS and C4 50 mg/kg treatment groups at the end of the experiment at day 62. D. Panel represents mean C8161 tumor weight +SEM at the end of the experiment at day 23. Female nude mice were subcutaneously inoculated with C8161 cancer cells (5 mice/ group). IP with compound C4 (25mg/kg, daily), DTIC (8 mg/kg, Q4D) and combination were started when tumor reach 100 mm3. * P<0.05 and ** P<0.01 relative to untreated control, @ relative to C4 alone, # relative to chemotherapy alone. E. Panel represents mean A375 tumor weight +SEM at the end of the experiment at day 31. Female nude mice were subcutaneously inoculated with melanoma A375 cells (5 mice/group). IP with compound C4 (25mg/kg, daily), DTIC (8 mg/kg, Q4D) and combination were started when tumor reached 100 mm3 and continued for 31 days. 1. Garces CA, Kurenova EV, Golubovskaya VM, Cance WG. Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells. Cancer Res. 2006;66:1446-54. 1. Garces CA, Kurenova EV, Golubovskaya VM, Cance WG. Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells. Cancer Res. 2006;66:1446-54. 2. Seshadri M, Mazurchuk R, Spernyak JA, Bhattacharya A, Rustum YM, Bellnier DA. Activity of the vascular-disrupting agent 5,6-dimethylxanthenone-4-acetic acid against human head and neck carcinoma xenografts Neoplasia 2006;8:534 42 Supplementary Figures. Supplementary Figure S1. FAK scaffold inhibitor C4 reduced phosphorylation of VEGFR-3 and decreased FAK-VEGFR3 complex formation. C4 dose-dependent decrease of FAK-VEGFR-3 association in melanoma C8161 cells. Cells were treated with increased concentrations C4 for 24 h. A. VEGFR-3 protein complexes were immunoprecipitated (IP) with VEGFR-3 antibody (Millipore, clone 9D9) and precipitates were analyzed in consequent western blotting (WB) for VEGFR-3 phosphorylation with pan-phospho-tyrosine antibody 4G10 (P-Tyr) and for the presence of FAK protein with FAK specific antibody (Millipore, clone 4.47). B. Densitometry was performed for each IP experiment and data are presented at the bottom of the western blot image. Left panel - decrease of P-Tyr phosphorylation of VEGFR-3; right panel - decrease of FAK protein in VEGFR-3 co-precipitates. 4 Supplementary figure S2. FAK scaffold inhibitor C4 inhibited motility of melanoma cells. A. The wound healing assay of C4 treated C8161 cells. Image analysis was done with WimScratch module of Wimasis software. Data shown are representative of three independent experiments. B. Graphical representation of the quantitative values of the wound size at different doses and at different time points post-C4 treatment. The error bar represents +SEM. Supplementary figure S3. FAK scaffold inhibitor C4 caused apoptosis in melanoma cell lines. C8161 and A375 cells were treated with increased concentration of C4 for 48 h and 72 h and percentage of TUNEL positive cells were determined by FACS. STS – staurosporin was used as a positive control. Supplementary figure S3. FAK scaffold inhibitor C4 caused apoptosis in melanoma cell lines. C8161 and A375 cells were treated with increased concentration of C4 for 48 h and 72 h and percentage of TUNEL positive cells were determined by FACS. STS – staurosporin was used as a positive control. Supplementary figure S4. FAK scaffold inhibitor C4 sensitized melanoma cells to cytotoxic therapy and caused apoptosis in melanoma cell lines. Western blot analysis of biochemical markers of the apoptotic pathway. A. C8161 cells were treated for 48 h with 60 μM of C4 and 10 μM DTIC alone or in combination and activation of Caspase 8, Caspase 3 and PARP cleavage are analyzed. B. A375 cells were treated for 48 h with 60 μM of C4 and 20 μM DTIC alone or in combination. All shown figures are representative of experiments performed in triplicate. REFERENSES 1. Garces CA, Kurenova EV, Golubovskaya VM, Cance WG. Vascular endothelial growth factor receptor-3 and focal adhesion kinase bind and suppress apoptosis in breast cancer cells. Cancer Res. 2006;66:1446-54. 5 3. 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Scattering and Imaging with Diffusing Temporal Field Correlations. Phys Rev Lett. 1995;75:1855-8. 8. Mesquita RC, Durduran T, Yu G, Buckley EM, Kim MN, Zhou C, et al. Direct measurement of tissue blood flow and metabolism with diffuse optics. Philosophical transactions Series A, Mathematical, physical, and engineering sciences. 2011;369:4390-406. 6 6 Supplementary Figure S1 IP: VEGFR-3 VEGFR-3 FAK WB: P-Tyr Ab PBS 0 10 50 100 mM C4 150 kDa 150 kDa 125 kDa A B A A IP: VEGFR-3 VEGFR-3 FAK WB: P-Tyr Ab PBS 0 10 50 100 mM C4 150 kDa 150 kDa 125 kDa B B B Supplementary Figure S2 Supplementary Figure S2 Supplementary Figure S2 Supplementary F Cell covered area 0 20 40 60 80 100 0 24 48 h 0 24 48 h 0 24 48 Control C4 10 uM C4 50 uM A B A A B B Cell covered area 0 20 40 60 80 100 0 24 48 h 0 24 48 h 0 24 48 Control C4 10 uM C4 50 uM Cell covered area 0 20 40 60 80 100 0 24 48 h 0 24 48 h 0 24 48 Control C4 10 uM C4 50 uM Supplementary Figure S3 Supplementary Figure S3 A 0 10 20 30 40 50 0 10 30 60 100 STS Apoptosis (%TUNEL positive) C4 concentration µM C8161 48h C8161 72h 0 10 20 30 40 50 0 10 30 60 100 STS Apoptosis (%TUNEL positive) C4 concentration µM A375 48h A375 72h 0 10 20 30 40 50 0 10 30 60 100 STS Apoptosis (%TUNEL positive) C4 concentration µM C8161 48h C8161 72h 0 10 20 30 40 50 0 10 30 60 100 STS Apoptosis (%TUNEL positive) C4 concentration µM A375 48h A375 72h B 0 10 20 30 40 50 0 10 30 60 100 STS Apoptosis (%TUNEL positive) C8161 48h C8161 72h B Supplementary Figure S4 Supplementary Figure Caspase 8 Caspase 3 PARP GAPDH C8161 C4 - + - + DTIC - - + + kDa 57 35 115 37 Caspase 8 Caspase 3 PARP GAPDH A375 C4 - + - + DTIC - - + + kDa 57 35 115 37 Caspase 8 Caspase 3 PARP GAPDH C8161 C4 - + - + DTIC - - + + kDa 57 35 115 37 A B Caspase 8 Caspase 3 PARP GAPDH A375 C4 - + - + DTIC - - + + kDa 57 35 115 37 115 37 37 Supplementary Figure S5 A B 0 0.2 0.4 0.6 0.8 1 1.2 PBS C4 50 mg/kg organ weight (g) heart,lung liver kidney spleen C D E Supplementary Figure S5 Supplementary Figure S5 Supplementary Figure A A B 0 0.2 0.4 0.6 0.8 1 1.2 PBS C4 50 mg/kg organ weight (g) heart,lung liver kidney spleen C D E B B 0 0.2 0.4 0.6 0.8 1 1.2 PBS C4 50 mg/kg organ weight (g) heart,lung liver kidney spleen C C E D E
https://openalex.org/W4361833292	https://figshare.com/articles/journal_contribution/Supplementary_Figs_S7_8_from_ERBB3_and_IGF1R_Signaling_Are_Required_for_Nrf2-Dependent_Growth_in_KEAP1-Mutant_Lung_Cancer/22422464/1/files/39868706.pdf	English	null	Supplementary Figs S1-4 from ERBB3 and IGF1R Signaling Are Required for Nrf2-Dependent Growth in KEAP1-Mutant Lung Cancer	null	2,023	cc-by	725	KEAP1 WT KEAP1 mut 0.1 1 10 IC50 (μM) erastin IC50 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 Cell ATP levels (AUC) erastin viability p=0.03 p=0.03 Figure S7 A KEAP1 WT KEAP1 mut 0.01 0.1 1 10 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 p=0.12 p=0.07 B BPTES IC50 BPTES viability IC50 (μM) 1.0 p=0.23 C BSO viability 10 15 of cell lines BSO sensitivity WT KEAP1 p=0.14 Cell ATP levels (AUC) vels (AUC) KEAP1 WT KEAP1 mut 0.1 1 10 IC50 (μM) erastin IC50 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 Cell ATP levels (AUC) erastin viability p=0.03 p=0.03 Figure S7 A KEAP1 WT KEAP1 mut 0.01 0.1 1 10 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 p=0.12 p=0.07 B BPTES IC50 BPTES viability IC50 (μM) 1.0 p=0.23 C BSO viability 10 15 of cell lines BSO sensitivity WT KEAP1 p=0.14 Cell ATP levels (AUC) vels (AUC) KEAP1 WT KEAP1 mut 0.1 1 10 IC50 (μM) erastin IC50 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 Cell ATP levels (AUC) erastin viability p=0.03 p=0.03 Figure S7 A A KEAP1 WT KEAP1 mut 0.1 1 KEAP1 WT KEAP1 mut 0.0 0.5 Cell ATP KEAP1 WT KEAP1 mut 0.01 0.1 1 10 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 p=0.12 p=0.07 B BPTES IC50 BPTES viability IC50 (μM) KEAP1 WT KEAP1 mut 0.0 0.5 1.0 p=0.23 C BSO viability Sensitive Insensitive 0 5 10 15 Number of cell lines BSO sensitivity WT KEAP1 p=0.14 Cell ATP levels (AUC) Cell ATP levels (AUC) KEAP1 WT KEAP1 mut 0.1 KEAP1 WT KEAP1 mut 0.01 0.1 1 10 KEAP1 WT KEAP1 mut 0.0 0.5 1.0 p=0.12 p=0.07 B BPTES IC50 BPTES viability IC50 (μM) Cell ATP levels (AUC) B 0 07 BPTES viability KEAP1 WT KEAP1 mut 0.0 0.5 1.0 p=0.23 C BSO viability Sensitive Insensitive 0 5 10 15 Number of cell lines BSO sensitivity WT KEAP1 p=0.14 Cell ATP levels (AUC) C Figure S8 p=0.002 A H C E 100 n (RPKM) 10 100 1000 TXN expresssion (RPKM) 10 100 1000 TXNRD1 expresssion (RPKM) 100 1000 PRDX1 expresssion (RPKM) p<0.001 B KEAP1 WT KEAP1 mut p<0.001 KEAP1 WT KEAP1 mut KEAP1 WT KEAP1 mut D F p<0.001 G KEAP1 WT KEAP1 mut J p=0.049 I ERBB3 expresssion (RPKM) 0.1 1 10 100 0.01 100 to NTC) IGF1R NTC_24 sh1 24 A549 H441 0 50 100 150 RNA levels (relative to NTC) TXN NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 A549 H441 0 50 100 RNA levels (relative to NTC) PRDX1 NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 A549 H441 0 50 100 RNA levels (relative to NTC) TXNRD1 NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 A549 H441 0 50 100 RNA levels (relative to NTC) ERBB3 NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 * * * * * ** * * * * * * * * * * *** * * * * Figure S8 A 10 100 1000 TXN expresssion (RPKM) p<0.001 B KEAP1 WT KEAP1 mut A549 H441 0 50 100 150 RNA levels (relative to NTC) TXN NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 * * * * 10 100 1000 TXN expresssion (RPKM) p<0.001 B KEAP1 WT KEAP1 mut B C 100 1000 PRDX1 expresssion (RPKM) WT mut p<0.001 KEAP1 WT KEAP1 mut D A549 H441 A549 H441 0 50 100 RNA levels (relative to NTC) PRDX1 NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 * ** * * C E 10 100 1000 TXNRD1 expresssion (RPKM) KEAP1 WT KEAP1 mut F p<0.001 A549 H441 0 50 100 RNA levels (relative to NTC) TXNRD1 NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 * * * * * * * * E H WT mut KEAP1 WT KEAP1 mut p=0.049 ERBB3 expresssion (RPKM) 0.1 1 10 100 0.01 H G KEAP1 WT KEAP1 mut p=0.049 ERBB3 expresssion (RPKM) 0.1 1 10 100 0.01 A549 H441 0 50 100 RNA levels (relative to NTC) ERBB3 NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 *** * * * G p=0.002 0.1 1 10 100 IGF1R expresssion (RPKM) KEAP1 WT KEAP1 mut J p=0.002 0.1 1 10 100 IGF1R expresssion (RPKM) KEAP1 WT KEAP1 mut J I A549 H441 0 50 100 RNA levels (relative to NTC) IGF1R NTC_24 sh1_24 sh10_24 NTC_48 sh1_48 sh10_48 * * J
https://openalex.org/W2116586479	https://europepmc.org/articles/pmc2903497?pdf=render	English	null	Development and evaluation of an open source Delphi-based software for morphometric quantification of liver fibrosis	Fibrogenesis & tissue repair	2,010	cc-by	6,000	RESEARCH Open Access © 2010 Huss et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Computer-based morphometry can minimize subjectivity in the assessment of liver fibrosis. An image processing program was developed with Delphi for the quantification of fibrosis in liver tissue samples stained with Sirius Red. Bile duct ligated and sham operated wild type C57BL/6 mice served as a model of time-dependent induction of liver fibrosis. Formation of fibrosis was determined with the developed software at day 0, 3, 7, 10, 14, 20, 30 and 60. The results were compared to a semi-quantitative scoring system. Results: Quantitative accumulation of collagen fibres was observed from day 3 to day 14, with a slight further increase thereafter. During ongoing fibrogenesis, there was a significant elevation of alanine aminotransferase (ALT), aspartate transaminase (AST) and bilirubin. The results from our computer-based morphometric analysis were highly correlated with the results that were obtained in a standardized pathology semi-quantitative scoring system (R 2 = 0.89, n = 38). Conclusions: Using our Delphi-based image analysing software, the morphometric assessment of fibrosis is as precise as semi-quantitative scoring by an experienced pathologist. This program can be a valuable tool in any kind of experimental or clinical setting for standardized quantitative assessment of fibrosis. Results: Quantitative accumulation of collagen fibres was observed from day 3 to day 14, with a slight further increase thereafter. During ongoing fibrogenesis, there was a significant elevation of alanine aminotransferase (ALT), aspartate transaminase (AST) and bilirubin. The results from our computer-based morphometric analysis were highly correlated with the results that were obtained in a standardized pathology semi-quantitative scoring system (R 2 = 0.89, n = 38). Conclusions: Using our Delphi-based image analysing software, the morphometric assessment of fibrosis is as precise as semi-quantitative scoring by an experienced pathologist. This program can be a valuable tool in any kind of experimental or clinical setting for standardized quantitative assessment of fibrosis. Background not in use for standard clinical diagnostics but is some- times used scientifically in experimental models [4-8]. Liver fibrosis is characterized by an increase of collage- nous matrix (the quantitative aspect of fibrosis). There is also a reduction of the vascular bed, pathologic perfusion, atrophy and regeneration of parenchyma leading to a fun- damental rebuilding of tissue architecture (qualitative aspect of fibrosis). Complete organ fibrosis represents the final course of chronic progressive liver diseases. The primary goal of the present study was the develop- ment of highly user-friendly, charge-free and open source computer software to assess the quantitative aspect of liver fibrosis in a standardized and reproducible manner. Therefore, we used the model of bile duct ligation (BDL) to induct liver fibrosis in C57/BL6 mice. The experimen- tal model has been well described and evaluated in rats and mice [9,10] and has been widely used to study chole- static liver injury [11,12] and fibrogenesis [9,13,14]. We analysed time-related quantitative and semi-quantitative aspects of murine liver fibrosis and evaluated the differ- ent measurement techniques. Pathologists describe changes in these two dimensions subjectively or semi-quantitatively by a variety of scoring systems depending on the underlying disease causing dif- ferent histological patterns. For example, in chronic hep- atitis the Ishak score [1] is used. In alcoholic or non alcoholic steatohepatitis, fibrotic progression has been quantified by Brunt and colleagues [2]. Cholangiode- structive and cholangitic diseases are scored according to Portmann and Nakanuma [3]. A computer-based mor- phometry for the assessment of liver fibrosis is currently Research Development and evaluation of an open source Delphi-based software for morphometric quantification of liver fibrosis Sebastian Huss1, Jörg Schmitz1, Diane Goltz1, Hans-Peter Fischer1, Reinhard Büttner1 and Ralf Weiskirchen2 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Discussion Liver fibrosis is a major parameter guiding the diagnosis and prognosis of chronic liver diseases and liver biopsy and its histological evaluation remains the gold standard for diagnosis and prognosis. Therefore, accurate qualita- tive and quantitative assessment of fibrosis is essential. Many scoring systems were designed to classify and stage different chronic liver diseases [1,2,15]. One major flaw of these scoring systems is that they are dependent on the visual interpretation of the observer. In addition, the observer must be an experienced pathologist. In order to avoid these pitfalls, over the last decade quantification of fibrosis by multiple computer assisted methods has been introduced [5,7,16,17]. The principle behind these meth- ods is to stain a section with a specific tissue stain that highlights collagen fibres. Then, with the aid of the soft- ware, the fibrotic area is calculated. Ideally, the correct assessment of total fibrosis should be possible fully com- puterized that rules out intra- and inter-observer varia- tions. Dahab et al. used the commercial program Adobe Photoshop to calculate a fibrosis index [6]. In addition, there are different companies (Soft Imaging Systems, Münster, Germany or Aperio, CA, USA) who sell pro- grams which have been designed specially for image anal- ysis purpose. These programs are usually highly expensive and are not easily adjustable. Figure 1 Study design and experimental setting. Bile duct ligation was performed on day 0. Analysis was performed on day 3 and 7 after the procedure to measure short time effects. Time-points after 10, 14 and 20 days were chosen to display intermediate and 30 and 60 days to analyse long-term effects. moribund and were killed before the planned end point. The activity of sham-operated animals was nearly normal 24 h after the lapratomy while the animals subjected to BDL showed reduced activity during the first 72 h but regained normal activity thereafter. Jaundiced skin was already apparent in all animals 24 h after BDL. Impact of bile duct ligation on survival, activity and jaundice We have performed BDL in 40 animals and analysed ongoing hepatic fibrogenesis in respective animals at fixed time points (Figure 1). All sham-operated animals survived but two of 40 mice in the BDL group became Correspondence: sebastian.huss@ukb.uni-bonn.de 1 Institute of Pathology, University of Bonn, Bonn, Germany Full list of author information is available at the end of the article Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Page 2 of 8 Figure 1 Study design and experimental setting. Bile duct ligation was performed on day 0. Analysis was performed on day 3 and 7 after the procedure to measure short time effects. Time-points after 10, 14 and 20 days were chosen to display intermediate and 30 and 60 days to analyse long-term effects. sion curve (R2 = 0.89 versus R2 = 0.86; n = 38). However, semi-quantitative scoring of perisinusoidal fibrosis showed a lower correlation (R2 = 0.64, n = 38) (Figure 4). sion curve (R2 = 0.89 versus R2 = 0.86; n = 38). However, semi-quantitative scoring of perisinusoidal fibrosis showed a lower correlation (R2 = 0.64, n = 38) (Figure 4). Hepatocellular injury and cholestasis after BDL Alanine aminotransferase (ALT) and aspartate transami- nase (AST) increased rapidly after BDL, peaking at 7 respectively twenty days after the surgery. After the peak- ing, ALT and AST decreased steadily until day 30; serum levels remained almost unchanged after 60 days. Bilirubin levels steadily elevated and reached a plateau after 7 days. Total protein serum levels had a greater variability with a slight decrease after 7 days (Figure 2). Therefore, we have written a new simple, charge free, computer-based morphometric assessment tool to evalu- ate liver fibrosis. This software (Fibromat) is written in Delphi - one of the best known and widely used program- ming tools - to create software and applications for Microsoft Windows computers. With Delphi it is possible to create small powerful applications which do not need to be installed, do not depend on Active X controls or special DLLS. Therefore, there are no problems with installation, as usually observed with software from other programming utilities (for example, Java, .NET, Visual Basic or Visual C++). The program allows the determina- tion of different colour intensities that are necessary when morphometric scoring is necessary, for example, Sirus Red stained specimen. In order to perform a quanti- tative analysis, respective images of such a stain are first transformed into a grey scale and then further modified into a colour image in which the calculated background is depicted in black, liver parenchyma in light green and fibrous deposits in red. After this conversion, a precise and rapid mathematical quantification (in %) of individ- ual colours is possible. Development of liver fibrosis The stage of fibrosis was assessed semi-quantitatively. Periportal fibrosis was staged 0-4 and perisinusoidal fibrosis was scored 0-2 by an experienced pathologist (HPF), giving a maximum possible of 6 (Table 1). The morphometric computer-based assessment of fibrosis showed 0.13 ± 0.037% fibrosis-index in the sham oper- ated group. A strong accumulation of collagen fibres was observed between day 3 (0.10 ± 0.03%) and 14 (4.75 ± 0.35%), with a slight further increase thereafter (Table 2, Figure 3). The mean fibrosis score in sham operated ani- mals was 0.00 ± 0. It increased steadily until day 60 to 4.83 ± 0.17. The maximum of periportal fibrosis (stage 3; complete lamellae) was reached at day 20 (3.0 ± 0.0). Peripsinusoidal fibrosis was absent during the first 10 days and was established after 14 days (1.0 ± 0.0). It increased steadily until day 60 to 1.8 ± 0.17 (Table 2, Fig- ure 3). Both the total and periportal semi-quantitative scoring values showed a good correlation with the computer- based assessment and could be fitted to a linear regres- Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Page 3 of 8 Figure 2 Serum markers of cholestasis and hepatocellular injury during bile duct ligation. Biochemical markers are shown for (A) bilirubin, (B) total protein, (C) alanine aminotransferase and (D) aspartate transaminase. Figure 2 Serum markers of cholestasis and hepatocellular injury during bile duct ligation. Biochemical markers are shown for (A) bilirubin, (B) total protein, (C) alanine aminotransferase and (D) aspartate transaminase. One major advantage of our program is that it is open source. Therefore, the complete source code of the pro- gram is accessibly by downloading it (T Additional file 1) and it is easily adapted it to specific needs. For example one can choose different colors for the morphometrical analysis and make the program suitable for Massons Tric- rome Staining or any immunohistochemical staining. With little effort an analysis of different subtypes of colla- gen is possible. along with the proliferation of neoductules and peri- ductular fibrosis, leads to porto-portal septae [18]. A semi-quantitative scoring system for PBC was first described in 1965 by Rubin et al. [19]. They described four successive stages, which were modified by others [20-22] and summarized by Portmann and Nakanuma [3]. Other work groups scored their BDL-experiments according to one of these systems [23,24]. Development of liver fibrosis However, the scoring systems discussed were originally designed to score PBC, an autoimmune liver disease with an unknown aetiology where selectively small intrahe- patic bile ducts are destroyed. The pathogenic mecha- nism has to be distinguished from that occurring in BDL, where the common bile duct is ligated. Secondary to this procedure, small bile ducts react and form neoductuli [9]. As previously mentioned, the pattern of fibrosis depends on the aetiology, severity and duration of the underlying disease. We investigated the qualitative and quantitative aspects of murine liver fibrosis caused by bile duct ligation using a semi-quantitative score. The pattern of fibrosis caused by this procedure can be compared, to a certain degree, to the pattern occurring in primary biliary cirrhosis (PBC). There is a type of periportal fibrosis that, We propose an alternative approach to assess fibrotic changes after murine bile duct ligation with a two-tired Page 4 of 8 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Table 1: Semi-quantitative scoring of fibrosis: a two-tired scoring system for histopathological liver changes after murine bile duct ligation Periportal fibrosis Score No fibrosis 0 Scattered periportal and perineoductular fibrosis (incomplete lamellae) 1 Periportal, perineoductular fibrosis (complete lamellae) +/- beginning septa 2 Periportal, perineoductular fibrosis with portal-portal septa * 3 Complete cirrhosis 4 Perisinusoidal fibrosis No fibrosis 0 Mild fibrosis (fibres in < 50% of the perisinusoidal spaces) 1 Severe fibrosis (fibres in > 50% of the perisinusoidal spaces) 2 *Three or more portal-portal septa have to be found per 10 portal fields. analyses [26,27]. Also adding the numbers representing different grading components (periportal and perisinu- soidal) together to create a total score can lead to inaccu- racies. At best, a total grading score can give only an approximate idea of the severity of disease [28]. We, therefore, correlated every dimension as well as the total score of our two-tired scoring system with the calculated fibrotic-index. We could show that the periportal fibrosis, as well as total score, is highly correlated but perisinusoi- dal fibrosis is lower. These findings suggest that a qualita- tive assessment of the computerized fibrosis pattern might still be necessary for an accurate interpretation of computerized fibrosis ratio, because a merely quantita- tive fibrotic-index of a specimen does not display all the information supplied by a visual interpretation of the slide. Conclusions d d We designed an easy-to-use, charge free and open source Delphi-based computer program to assess the quantita- tive aspect of liver fibrosis in a standardized and repro- ducible way. The program was evaluated in an experimental setting of murine liver fibrosis following bile duct ligation. It can also be used for the analysis of fibrosis due to other aetiopathologies. scoring system. Modified after Portmann and Nakanuma [3] we staged periportal fibrosis as follows: 0, no fibrosis; stage 1, focal periportal and perineoductular fibrosis that build incomplete lamellae around affected portal fields; stage 2, fully established periportal and perineoductular fibrosis building complete lamellae, with or without spo- radic portal-portal bridging; stage 3, an extension of the portal-portal bridging (three or more bridges per 10 por- tal fields); and stage 4, fully developed cirrhosis (Addi- tional File 2). However, stage 4 was not observed in our experimental setting. This finding is different to descrip- tions of the rat model. Here fibrosis is progressive and cirrhosis can develop within 15 days after BDL [25]. Fur- thermore, we staged perisinusoidal fibrosis as follows: 0, no fibrosis; stage 1, a mild fibrosis with less than 50% of the lobule affected; and stage 2, a severe fibrosis with more than 50% of the lobule affected. scoring system. Modified after Portmann and Nakanuma [3] we staged periportal fibrosis as follows: 0, no fibrosis; stage 1, focal periportal and perineoductular fibrosis that build incomplete lamellae around affected portal fields; stage 2, fully established periportal and perineoductular fibrosis building complete lamellae, with or without spo- radic portal-portal bridging; stage 3, an extension of the portal-portal bridging (three or more bridges per 10 por- tal fields); and stage 4, fully developed cirrhosis (Addi- tional File 2). However, stage 4 was not observed in our experimental setting. This finding is different to descrip- tions of the rat model. Here fibrosis is progressive and cirrhosis can develop within 15 days after BDL [25]. Fur- thermore, we staged perisinusoidal fibrosis as follows: 0, no fibrosis; stage 1, a mild fibrosis with less than 50% of the lobule affected; and stage 2, a severe fibrosis with more than 50% of the lobule affected. Methods Animals Male C57BL/6 mice (Harlan Laboratories, Eystrup, Ger- many) aged 10-12 weeks were kept under controlled envi- ronmental conditions with a 12-h light-dark cycle for a minimum of 7 days before surgery. Mice were fed on a standard laboratory diet with food and water ad libitum. All experiments were approved by the Landesamt für Natur, Umwelt und Verbraucherschutz NRW, Reckling- hausen, Germany (AZ 50.203.2 AC20, 13/06 and AZ 8.87-50.10.37.09.248). In our model, periportal fibrosis was fully developed after 20 days; persinusoidal fibrosis become evident at day 10 and increased until the end of the experiment (day 60). When semi-quantitative scoring data are evaluated, it is important to bear in mind that the numbers repre- sent categories rather than measurements. They, there- fore, cannot be used as real numbers in statistical Development of liver fibrosis Nevertheless both, the semi-quantitatively scoring and the computer-based assessment (fibrotic-index) showed good correlation in depicting increased collagen deposition as a consequence of ongoing fibrogenesis. Therefore, we propose this highly user-friendly image analysis tool for the accurate quantification of collagen deposits in Sirius Red stained liver sections. The image processing is computerized and, for this reason, more insensitive to intra- and inter-observer variations than a semi-quantitative scoring system. Procedures BDL After midline skin lapratomy the liver was gently removed and the common bile duct was mobilized. In the region above the pancreas two 4-0 nylon sutures were Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Page 5 of 8 Table 2: Fibrosis index and semi-quantitative score Table 2: Fibrosis index and semi-quantitative score Time after bile duct ligation (days) Fibrosis index (%) Semi-quantitative score Portal fibrosis Perisinusoidal fibrosis Total score 0 (n = 3) 0.13 ± 0.04 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 3 (n = 5) 0.10 ± 0.03 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 7 (n = 5) 0.76 ± 0.11 0.60 ± 0.25 0.0 ± 0.0 0.60 ± 0.25 10 (n = 5) 1.65 ± 0.22 1.40 ± 0.25 0.25 ± 0.25 1.67 ± 0.25 14 (n = 5) 4.75 ± 0.35 2.4 ± 0.25 1.0 ± 0.0 3.40 ± 0.24 20 (n = 4) 5.02 ± 0.47 3.0 ± 0.0 1.0 ± 0.0 4.0 ± 0.0 30 (n = 5) 5.45 ± 0.35 2.8 ± 0.2 1.4 ± 0.25 4.20 ± 0.20 60 (n = 6) 5.63 ± 0.25 3.0 ± 0.0 1.8 ± 0.17 4.83 ± 0.17 Fibrosis index (% Sirius Red stain) and semi-quantitative score consisting of portal fibrosis score, perisinusoidal score and total score (mean +/- standard error of mean; see Table 1) Periportal fibrosis was staged 0-4 and perisinusoidal fibrosis was scored 0-2, giving a maximum possible of 6 (Table 1). placed around the bile duct and carefully tightened to avoid rupture. Sham lapratomy (control) Lapratomy and mobilization of the common bile duct were performed as in the former group but without liga- tion. Development of the image analysing program The principle behind computer-based morphometry is the different staining pattern of cells, nuclei and fibres following Sirius Red staining. Collagen fibres, as well as cell nuclei, appear red, while the hepatocellular cyto- plasm becomes pale and yellowish. In order to measure short time effects of the BDL, mice were sacrificed at day 3 and 7 after the surgery. In order to indicate intermediate time effects mice were sacrificed after 14 and 20 days, to assess long term effects after 30 and 60 days. Control animals were killed 20 days after the sham lapratomy. At indicated time points, blood was drawn from the right ventricle, centrifuged and the serum was stored at -80°C until further analysis. The liver was removed and fixed in 4% buffered formalin for 24 h for histological analysis. y For the analysis, 10 photographs of random high-power fields (100 × magnifications) were taken of each liver sample. Large bile ducts and vessels were excluded. Pho- tographs were stored as 1280 × 1024 pixel RGB-bitmaps (bmp) with a colour-resolution of 24 bits per pixel. These pictures were analysed with our Delphi-based program as follows. After a shading correction, a grey transformation - derived from the green color channel only - is calculated from the original image. Then a blurring filter based on an arithmetic mean filter is applied. Thresholding of the positively stained collagen fibres is performed by a histo- gram analysis of the grey value distribution resulting in a binary image. As cell nuclei and fibres appear to have the same staining intensity, binary object detection is per- formed and all objects with an area lower than a specific threshold are eliminated, distinguishing between fibres and nuclei. The total area of the combined fibres is expressed as a percentage of the total parenchyma area. Measurement of serum parameters Blood biochemical parameters (bilirubin, ALT, AST and total protein) were measured using the Modular Pre- Analytics (MPA) system (Roche Diagnostics, Mannheim, Germany) Histology After being fixed in 4% buffered formalin for 24 h, the liver tissue was embedded into paraffin wax. A histologi- cal semi-quantitative examination of the liver was per- formed on sections after standard Sirius Red staining. Page 6 of 8 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 These steps are performed automatically and the dis- played resulting image shows background in black, liver parenchyma in a light green and fibrosis intense red (Fig- ures 5 and 6). Nevertheless, the user has several options to interact with the program, eliminate different common errors and to simplify his work. (i) Sirius Red staining can have different intensities due to staining time and/or thickness of the histological slide. The user can modify the threshold of the grey transformation to minimize these effects. (ii) If random sections of a sample were taken there may be flapped areas or other artifacts giving a wrong signal. The user can exclude obviously mistaken objects by a simple mouse click. All steps of the complete procedure are visualized in one display and are therefore easy to follow. (iii) Lumina of small vessels, as well as the background, are automatically excluded from the total area of the parenchyma. (iv) Unlimited photographs can be analysed one after another. The user can save and reopen the current working file or send it to a co-worker who can proceed or reevaluate the work. (v) The values (0.00-1.00, that is 0% to 100% fibrosis) can be exported to a text file and re-imported into Excel, GraphPad, SPSS or another program for statistical analysis. The software, which is compatible with Microsoft Windows 7, Micro- soft Vista SP2, Microsoft Windows XP Home or Profes- sional (SP2 or SP3), can be downloaded from the journal home page (Additional File 1). Details of programming are available on request Figure 3 Semi-quantitative analysis versus computer-based mor- phometrical analysis. (A) A semi-quantitative score that considers both periportal and perisinusoidal fibrosis was determined and com- pared to (B) the morphometric analysis determined in Sirius Red stained specimen. Figure 4 Correlation between semi-quantitative and computer- based analysis. Linear regression fit between semi-quantitative total (A; R2 = 0.89), periportal (B; R2 = 0.86) and perisinuidal (C; R2 = 0.64) score and morphometric assessment (Percentage of positively stained surface, Sirius Red Staining; n = 38.) Figure 3 Semi-quantitative analysis versus computer-based mor- phometrical analysis. Histology (A) A semi-quantitative score that considers both periportal and perisinusoidal fibrosis was determined and com- pared to (B) the morphometric analysis determined in Sirius Red stained specimen. Figure 4 Correlation between semi-quantitative and computer- based analysis. Linear regression fit between semi-quantitative total (A; R2 = 0.89), periportal (B; R2 = 0.86) and perisinuidal (C; R2 = 0.64) score and morphometric assessment (Percentage of positively stained surface, Sirius Red Staining; n = 38.) Figure 3 Semi-quantitative analysis versus computer-based mor- phometrical analysis. (A) A semi-quantitative score that considers both periportal and perisinusoidal fibrosis was determined and com- pared to (B) the morphometric analysis determined in Sirius Red stained specimen. These steps are performed automatically and the dis- played resulting image shows background in black, liver parenchyma in a light green and fibrosis intense red (Fig- ures 5 and 6). Nevertheless, the user has several options to interact with the program, eliminate different common errors and to simplify his work. (i) Sirius Red staining can have different intensities due to staining time and/or thickness of the histological slide. The user can modify the threshold of the grey transformation to minimize these effects. (ii) If random sections of a sample were taken there may be flapped areas or other artifacts giving a wrong signal. The user can exclude obviously mistaken objects by a simple mouse click. All steps of the complete procedure are visualized in one display and are therefore easy to follow. (iii) Lumina of small vessels, as well as the background, are automatically excluded from the total area of the parenchyma. (iv) Unlimited photographs can be analysed one after another. The user can save and reopen the current working file or send it to a co-worker who can proceed or reevaluate the work. (v) The values Figure 4 Correlation between semi-quantitative and computer- based analysis. Linear regression fit between semi-quantitative total (A; R2 = 0.89), periportal (B; R2 = 0.86) and perisinuidal (C; R2 = 0.64) score and morphometric assessment (Percentage of positively stained surface, Sirius Red Staining; n = 38.) 4 Correlation between semi-quantitative and computer- (0.00-1.00, that is 0% to 100% fibrosis) can be exported to a text file and re-imported into Excel, GraphPad, SPSS or another program for statistical analysis. Histology The software, which is compatible with Microsoft Windows 7, Micro- soft Vista SP2, Microsoft Windows XP Home or Profes- sional (SP2 or SP3), can be downloaded from the journal home page (Additional File 1). Details of programming are available on request. Page 7 of 8 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 Figure 5 Computer-based morphometry. (A) Photograph of a Sirius Red stain of a liver section (100 × magnifications). (B) Grey transforma- tion, blurring and shading of respective photograph. (C) Resulting im- age of respective photograph. Background is black, liver parenchyma light green and fibrosis intuitively red. Figure 6 Screenshot of program interface. T Figure 6 Screenshot of program interface. The original photo- graphs are shown in the upper left corner. In the upper right corner the same picture is shown after adding the grey transformation, blurring and shading. The histogram analysis is shown in the lower right corner. The image in the lower middle shows the calculated background in black, the liver parenchyma in a light green and fibrosis intuitively red. The percentage of fibrous tissue is calculated on the lower left side (black arrow). Figure 6 Screenshot of program interface. The original photo- graphs are shown in the upper left corner. In the upper right corner the same picture is shown after adding the grey transformation, blurring and shading. The histogram analysis is shown in the lower right corner. The image in the lower middle shows the calculated background in black, the liver parenchyma in a light green and fibrosis intuitively red. The percentage of fibrous tissue is calculated on the lower left side (black arrow). Statistical analysis All results are expressed as the mean values ± standard error of mean, except for serum parameters of the ani- mals, which are expressed as mean values ± standard deviations. Semi-quantitative scores were correlated with the values from the computer assisted morphometrical analysis using Graph Pad Prism software. p g The authors declare that they have no competing interests. Figure 5 Computer-based morphometry. (A) Photograph of a Sirius Red stain of a liver section (100 × magnifications). (B) Grey transforma- tion, blurring and shading of respective photograph. (C) Resulting im- age of respective photograph. Background is black, liver parenchyma light green and fibrosis intuitively red. Authors' contributions SH did the animal experiments, helped with the development of the program and drafted the manuscript. JS was the principle programmer and performed the quantitative analysis. DG helped with the animal experiments. RB super- vised the animal experiments and participated in the study design. HPF per- formed the histopathological semi-quantitative scoring. RW was responsible for the serum analysis and was involved in the study design as well as in revis- ing the manuscript for important intellectual content. All authors carefully read and approved the final manuscript. Abbreviations ALT: alanine aminotransferase; AST: aspartate transaminase; BDL: bile duct liga- tion; bmp: bitmaps; PBC: primary biliary cirrhosis. Additional material Additional file 1 The Fibromat, an open source Delphi-based soft- ware for computer-based morphometry. Download the file and unzip it Additional file 1 The Fibromat, an open source Delphi-based soft- ware for computer-based morphometry. Download the file and unzip it to your hard disc. Open Delphi (installation is required; program can be obtained from Borland, Texas, USA). Open the file 'ihc.exe' and start the pro- gram. If you have any questions on how to use or modify the program feel free to contact us via the corresponding author. Additional file 2 Scoring periportal fibrosis. (A) A normal portal field without fibrosis (stage 0). (B) Focal periportal and perineoductular fibrosis (incomplete lamellae, stage 1). (C) Fully established periportal and perineo- ductular fibrosis building complete lamellae, with or without sporadic por- tal-portal bridging (stage 2). (D) Extension of the portal-portal bridging (three or more bridges per 10 portal fields, stage 3); complete cirrhosis (stage 4) is not shown. Competing interests Competing interests The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. p g The authors declare that they have no competing interests. References Dahab GM, Kheriza MM, El-Beltagi HM, Fouda AM, El-Din OA: Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software. J Gastroenterol Hepatol 2004, 19:78-85. doi: 10.1186/1755-1536-3-10 Cite this article as: Huss et al., Development and evaluation of an open source Delphi-based software for morphometric quantification of liver fibro- sis Fibrogenesis & Tissue Repair 2010, 3:10 7. Masseroli M, Caballero T, O'Valle F, Del Moral RM, Perez-Milena A, Del Moral RG: Automatic quantification of liver fibrosis: design and validation of a new image analysis method: comparison with semi- quantitative indexes of fibrosis. J Hepatol 2000, 32:453-464. 8. Abdalla AF, Zalata KR, Ismail AF, Shiha G, Attiya M, Abo-Alyazeed A: Regression of fibrosis in paediatric autoimmune hepatitis: morphometric assessment of fibrosis versus semiquantiatative methods. Fibrogenesis Tissue Repair 2009, 2:2. g p 9. Georgiev P, Jochum W, Heinrich S, Jang JH, Nocito A, Dahm F, Clavien PA: Characterization of time-related changes after experimental bile duct ligation. Br J Surg 2008, 95:646-656. 9. Georgiev P, Jochum W, Heinrich S, Jang JH, Nocito A, Dahm F, Clavien PA: Characterization of time-related changes after experimental bile duct ligation. Br J Surg 2008, 95:646-656. g g 10. Johnstone JM, Lee EG: A quantitative assessment of the structural changes the rat's liver following obstruction of the common bile duct. Br J Exp Pathol 1976, 57:85-94. 10. Johnstone JM, Lee EG: A quantitative assessment of the structural changes the rat's liver following obstruction of the common bile duct. Br J Exp Pathol 1976, 57:85-94. 11. Wang H, Vohra BP, Zhang Y, Heuckeroth RO: Transcriptional profiling after bile duct ligation identifies PAI-1 as a contributor to cholestatic injury in mice. Hepatology 2005, 42:1099-1108. 12. Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Weiglein AH, Lammert F, Marschall HU, Tsybrovskyy O, Zatloukal K, Denk H, Trauner M: Ursodeoxycholic acid aggravates bile infarcts in bile duct-ligated and Mdr2 knockout mice via disruption of cholangioles. Gastroenterology 2002, 123:1238-1251. 13. Neumeier M, Hellerbrand C, Gabele E, Buettner R, Bollheimer C, Weigert J, Schaffler A, Weiss TS, Lichtenauer M, Scholmerich J, Buechler C: Adiponectin and its receptors in rodent models of fatty liver disease and liver cirrhosis. World J Gastroenterol 2006, 12:5490-5494. 14. Arias M, Sauer-Lehnen S, Treptau J, Janoschek N, Theuerkauf I, Buettner R, Gressner AM, Weiskirchen R: Adenoviral expression of a transforming growth factor-beta1 antisense mRNA is effective in preventing liver fibrosis in bile-duct ligated rats. References 1. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, Desmet V, Korb G, MacSween RN, et al.: Histological grading and staging of chronic hepatitis. J Hepatol 1995, 22:696-699. 1. Ishak K, Baptista A, Bianchi L, Callea F, De Groote J, Gudat F, Denk H, Desmet V, Korb G, MacSween RN, et al.: Histological grading and staging of chronic hepatitis. J Hepatol 1995, 22:696-699. Desmet V, Korb G, MacSween RN, et al.: Histological grading and staging of chronic hepatitis. J Hepatol 1995, 22:696-699. 24. Park DH, Baik SK, Choi YH, Kim MY, Rhim DW, Kim JW, Kwon SO, Cho MY, Kim CH, Ahn SC: Inhibitory effect of angiotensin blockade on hepatic fibrosis in common bile duct-ligated rats. Korean J Hepatol 2007, 13:61-69. 2. Brunt EM: Nonalcoholic steatohepatitis: definition and pathology. Semin Liver Dis 2001, 21:3-16. 2. Brunt EM: Nonalcoholic steatohepatitis: definition and pathology. Semin Liver Dis 2001, 21:3-16. 3. Diseases of the Bile Ducts. 5th edition. Edited by: Portmann BC, Nakanuma Y. New York: Churchill Livingstone; 2007. 25. Kountouras J, Billing BH, Scheuer PJ: Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 1984, 65:305-311. 4. Zaitoun AM, Mardini H Al, Awad S, Ukabam S, Makadisi S, Record CO: Quantitative assessment of fibrosis and steatosis in liver biopsies from patients with chronic hepatitis C. J Clin Pathol 2001, 54:461-465. 26. Cross SS: Grading and scoring in histopathology. Histopathology 1998, 33:99-106. 5. 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Dahab GM, Kheriza MM, El-Beltagi HM, Fouda AM, El-Din OA: Digital quantification of fibrosis in liver biopsy sections: description of a new method by Photoshop software. J Gastroenterol Hepatol 2004, 19:78-85. 6. Acknowledgements The study was supported by SFB/TRR57 (projects P05 and Q1) from the Deutsche Forschungsgemeinschaft (DFG). The authors thank Christiane Esch and Claudine Neumann for their excellent technical support. Page 8 of 8 Huss et al. Fibrogenesis & Tissue Repair 2010, 3:10 http://www.fibrogenesis.com/content/3/1/10 19. Rubin E, Schaffner F, Popper H: Primary Biliary Cirrhosis. Chronic Non- Suppurative Destructive Cholangitis. Am J Pathol 1965, 46:387-407. Received: 22 April 2010 Accepted: 17 June 2010 Published: 17 June 2010 22. Scheuer P: Primary biliary cirrhosis. Proc R Soc Med 1967, 60:1257-1260. 23. 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https://openalex.org/W2419108215	https://journals.iium.edu.my/ejournal/index.php/iiumej/article/download/571/430	English	null	A REVIEW ON LOWER APPENDICULAR MUSCULOSKELETAL SYSTEM OF HUMAN BODY	IIUM engineering journal	2,016	cc-by	8,434	A REVIEW ON LOWER APPENDICULAR MUSCULOSKELETAL SYSTEM OF HUMAN BODY * ABSTRACT: Rehabilitation engineering plays an important role in designing various autonomous robots to provide better therapeutic exercise to disabled patients. Hence it is necessary to study human musculoskeletal system and also needs to be presented in scientific manner in order to describe and analyze the biomechanics of human body motion. This review focuses on lower appendicular musculoskeletal structure of human body to represent joints and links architectures; to identify muscle attachments and functions; and to illustrate muscle groups which are responsible for a particular joint movement. Firstly, human lower skeletal structure, linking systems, joint mechanisms, and their functions are described with a conceptual representation of joint architecture of human skeleton. This section also represents joints and limbs by comparing with mechanical systems. Characteristics of ligaments and their functions to construct skeletal joints are also discussed briefly in this part. Secondly, the study focuses on muscular system of human lower limbs where muscle structure, functions, roles in moving endoskeleton structure, and supporting mechanisms are presented ellaborately. Thirdly, muscle groups are tabulated based on functions that provide mobility to different joints of lower limbs. Finally, for a particular movement action of lower extremity, muscles are also grouped and tabulated to have a better understanding on functions of individual muscle. Basically the study presents an overview of the structure of human lower limbs by characterizing and classifying skeletal and muscular systems. ABSTRAK: Kejuruteraan pemulihan memainkan peranan yang penting dalam mereka bentuk pelbagai robot autonomi untuk menyediakan latihan terapeutik yang lebih baik untuk melumpuhkan pesakit. Jadi, ia adalah perlu untuk mengkaji sistem otot manusia dan perlu disampaikan secara saintifik untuk menerangkan, dan menganalisis biomekanik gerakan tubuh manusia. Kajian ini memberi tumpuan kepada struktur otot appendicular bawah badan manusia untuk mewakili sendi dan menghubungkan seni bina; mengenal pasti lampiran otot dan fungsi; dan menggambarkan kumpulan otot yang bertanggungjawab untuk pergerakan sendi tertentu. Pertama, struktur rangka yang lebih rendah manusia, sistem yang menghubungkan, mekanisme sendi dan fungsi mereka adalah seperti yang dinyatakan dengan perwakilan konsep seni bina bersama rangka. Seksyen ini juga mewakili sendi dan anggota badan dengan membandingkan dengan sistem mekanikal. Ciri-ciri daripada ligamen dan fungsi mereka untuk membina sendi tulang juga dibincangkan secara ringkas di bahagian ini. Kedua, kajian memberi tumpuan kepada sistem otot anggota badan manusia yang lebih rendah. Struktur otot, fungsi, peranan dalam menggerakkan struktur rangka dalam, dan menyokong mekanisme yang dibentangkan di dalam bahagian ini. IIUM Engineering Journal, Vol. 17, No. 1, 2016 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. A REVIEW ON LOWER APPENDICULAR MUSCULOSKELETAL SYSTEM OF HUMAN BODY M. AKHTARUZZAMAN, A. A. SHAFIE, AND M. R. KHAN Department of Mechatronics Engineering, Faculty of Engineering, International Islamic University Malaysia, 53100 Kuala Lumpur, Malaysia. akhter900@gmail.com, aashafie@iium.edu.my, and raisuddin@iium.edu.my (Received: 20 Jul. 2015; Accepted: 24 Dec. 2015; Published on-line: 30 Apr. 2016) A REVIEW ON LOWER APPENDICULAR MUSCULOSKELETAL SYSTEM OF HUMAN BODY * Ketiga, kumpulan otot adalah jadual berdasarkan fungsi yang menyediakan mobiliti kepada sendi yang berbeza anggota badan yang lebih rendah. Akhir sekali, untuk tindakan gerakan tertentu hujung yang lebih rendah, otot juga dikumpulkan dan dijadualkan untuk mempunyai pemahaman yang lebih baik mengenai fungsi otot 83 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. individu. Pada asasnya kajian mewakili gambaran keseluruhan struktur anggota bawah manusia dengan mencirikan dan mengelaskan sistem rangka dan otot. individu. Pada asasnya kajian mewakili gambaran keseluruhan struktur anggota bawah manusia dengan mencirikan dan mengelaskan sistem rangka dan otot. KEYWORDS: Musculoskeletal system; Human lower limbs; Muscle groups; Joint motion; Biomechatronics; Rehabilitation. 1. INTRODUCTION becomes very important in biomechatronics engineering research. In this review, only musculoskeletal structure of human lower extremity is studied to present endoskeletal joint structures, muscle attachments, muscle groups, and muscle functionalities of a particular movement of lower limbs. Studies on biomechanics and biomechatronics of musculoskeletal system provides better understanding of joint movements, trajectories, joint functions, and dynamic properties of biological systems [16]. Appropriate design of various mechanical and robotic systems such as artificial joint, orthotic devices, medical therapeutic devices, robot assisted system, exoskeleton etc. are also required for a proper analysis and understanding of musculoskeletal systems [17] and functions of biological systems so that any secondary injury can be avoided. 1. INTRODUCTION Stroke is one of the major causes for morbidity and disability, both in the developed and developing countries [1]. It is a common neurological disorder that causes post-stroke depression among survivors. Many survivors suffer from physical and psychological disabilities. One study presents that 66% of 80 stroke patients were in post-stroke depression where 51% were in mild, 11% were in moderate and 4% were in severe condition [2]. About 15 million people globally suffer from stroke where 5 million people die and 5 million become permanently disabled [3, 4]. Neurological impairment of post-stroke patient often conducts to hemiparesis (partial paralysis of one side of body). Hemiparesis can lead to profoundly impaired functional performance of daily activities such as running, walking, standing, speaking, and eating. It is observed that, at 6 months of post-stroke, about 50% of survivors get hemiplegia, 30% are unable to walk and 26% become dependent for their daily activities [5]. Nowadays robot assisted therapy is increasingly attractive in stroke rehabilitation both for upper and lower limbs of human body [6, 7]. Robotic systems unfold a wide range of opportunities to study on functional adaptation of various limbs of post-stroke patients [8- 10]. Robotic tools also provide opportunity to observe and measure improvements of functionality of a particular muscle as well as limbs [11-14]. Robot assisted therapy for lower limbs shows successful improvements in functional limitations of stroke patients with reduction of motor impairments [5, 15]. Basically, robotic skeletal arm provides external force to perform limb motions of lower extremity so that muscle and joint system mobility remains active. This therapeutic exercise also improves the nervous system of human lower limbs. The activity can be represented in a block diagram as presented in Fig. 1. Fig. 1: Block diagram of musculoskeletal system activity process versus rehabilitation. Fig. 1: Block diagram of musculoskeletal system activity process versus rehabilitation. It is very important to keep in mind that simply movement (passive exercise) of impaired limbs do not provide maximum recovery level for a stroke patient. Recognition of cortical functionalities, feedback control, and learning of motors are also involved in intense use of impaired limbs. Studies on the response of complex biological systems, in terms of its mechanics and electronics, is the focus of biomechatronics engineering. Therefore, understanding musculoskeletal structure of biological systems especially the human body 84 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. IIUM Engineering Journal, Vol. 17, No. 1, 2016 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Human body musculoskeletal structure is formed based on endoskeleton of human body that consists of rigid bones. Skeletal system not only provides structural support but also ensures protection, functions as levers, works as storage (of minerals and fats), and helps in blood cell formation [18]. Lower extremity of human skeletal system consists of pelvic girdle segment, thigh (proximal segment), shank (distal segment/lower leg), and foot. Connection portions of segments are called joints. Most of the joints of lower limbs are synovial joints (joint so articulated as to move freely) with cavities that contain synovial fluid [18,19]. All of these joints are almost frictionless (Fr ≈ 0) comparing to any other systems in the World and are also highly efficient shock absorbers [19]. Hip, knee, and ankle are the largest articulated joints of lower extremity of human endoskeleton. These joints are holding segments (bones) in place through a system of cartilage, tendons, and ligaments [18-20]. Figure 3 shows a conceptual architecture of the joints of human musculo-skeletal system. Fig. 3: Basic and conceptual architecture of joint in human musculoskeletal system. Fig. 3: Basic and conceptual architecture of joint in human musculoskeletal system. 2. SKELETAL SYSTEM OF HUMAN LOWER LIMBS The human musculoskeletal system has a primary responsibility to interact with physical environment to produce normal movement. Balancing body and normal locomotion, especially walking, are ensured by proper functioning musculoskeletal structure of the lower limbs of human body. The lower limbs also support superior parts of a body such as neck, head, and torso. Basically, human body is the accumulation of a number of independent systems. Holding structure and balancing body movements are controlled by nervous system, vascular system, skeletal system, muscular system, ligaments, and tendons [18]. For the purpose of lower limbs rehabilitation, it is important to understand the functions of lower torso of human body as well as functions of independent systems of musculoskeletal structure. Study on functions of lower extremity not only focuses on a single joint movement, but also refers to the structure and behavior of limbs, pelvis, and trunk [18]. Figure 2 presents structure of human endoskeletal system. Fig. 2: Anterior and posterior view of human endoskeletal structure focusing on the lower extremity of the system [22]. Fig. 2: Anterior and posterior view of human endoskeletal structure focusing on the lower extremity of the system [22]. Fig. 2: Anterior and posterior view of human endoskeletal structure focusing on the lower extremity of the system [22]. 85 2.2. Hip Joint (Coxal Joint) Joint that is formed at the meeting point of femur head and hip’s acetabulum socket is a perfect example of ball and socket joint as presented in Fig. 4(b). This is a type of synovial joint (diarthrotic joint) that allows triaxial movement of lower limbs. Synovial joints are the most mobile comparing to all other joints in human skeleton [22]. Triaxial movement of lower limbs can be characterized as flexion, extension, abduction, adduction, medial rotation, lateral rotation, and circumduction. Figure 5 shows the ligaments of pelvis and hip structure of human endoskeleton. (a) (b) Fig. 5: Ligaments of pelvis and hip of human skeletal system [23], (a) Anterior view of pelvis and hip, (b) Posterior view of pelvis and hip. (b) (a) Fig. 5: Ligaments of pelvis and hip of human skeletal system [23], (a) Anterior view of pelvis and hip, (b) Posterior view of pelvis and hip. 2.1. Pelvis Girdle Between torso and lower limbs, pelvic girdle plays an important role as a crucial linking system that acts as a fulcrum in between actions of upper and lower extremities [18,19]. Movement of one leg is counter balanced by other leg through the anatomical structure of pelvic. Hip joint mechanism of musculoskeletal system is structurally supported by the pelvic girdle [20,21]. Pelvis is formed by three bones; Ilium, Ischium, and pubic symphysis [21,22] as shown in Fig. 4(a). Pubic symphysis is a kind of cartilaginous joint. This type of joint has slight movement capability which separates articulating surface of contingent bones [22]. This type of joint exists between bodies of vertebrae and also in structure of ribcage. Slight movement capability of joint allows pelvic girdle work as a suspension system while walking, running, and jumping especially on uneven surface. (a) (a) (b) Fig. 4: Pelvic girdle and hip (coxal) joint structure [22], (a) Skeleton of pelvis, and (b) Hip joint structure (synovial joint). (b) (a) (b) Fig. 4: Pelvic girdle and hip (coxal) joint structure [22], (a) Skeleton of pelvis, and (b) Hip joint structure (synovial joint). 86 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. 2.3. Knee Joint (Tibiofemoral Joint) Lower portion of femur and upper side of tibia (shin bone) are connected together with multiple ligaments at knee joint. There is a small bone at the anterior portion of knee joint which is called as patella. These three bones form the synovial joint (diarthrotic joint) for knee which is an example of a hinge joint with ellipsoidal shape allowing roll and glide movement capability [18,19,22]. Patella has sliding movement capability on femoral groove as it is connected with tibia through Patella tendon at the bottom side and with quadriceps muscles through quadriceps tendon on the upper side [23]. Articular cartilage at the surface of opposing bones provides principal interface of articulation [25]. Multiple ligaments at knee ensure stability of the joint [24]. Furthermore, lateral and medial meniscus (have loose peripheral attachment to the joint capsule) of knee joint provide extra structural support. Studies show that meniscus has about 50% of intrinsic elastic modulus and 10% to 16% of permeability of articular cartilage [19]. So, compressive viscoelastic creep behavior of meniscus is regulated, to a great extent, by interstitial fluid that flows through tissue during compression. Two characteristics, low compressive stiffness and low permeability, of menisci prove that the system is able to function as a robust and efficient shock absorber. Most of the mechanical shocks are generated at knee joint and absorbed by meniscus. Total mass of menisci is much greater than articular cartilage-bearing load across knee joint (femoromeniscotibial articulation) [19]. Deformable characteristic of menisci with low compressive, shear stiffness, and permeability ensures well distribution of load at knee joint. Tibiofemoral joint (knee joint) allows one degree of freedom (DoF) of movement which can be characterized as flexion and extension [22]. Knee joint also allows a slight medial and lateral rotation which characterize the joint as biaxial (2 DoF) [18]. Figure 6 presents knee joint motions and example of hinge joint resembling tibiofemoral joint. Ligaments involved to stable tibiofemoral joint are presented in Fig. 7. 87 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. (a) (b) Fig. 6: Tibiofemoral joint (knee joint) structure and movements [22], (a) Lateral view of tibiofemoral joint representing roll and glide of tibiofemoral joint while flexion and extension, (b) Convex-concave surface of a hinge joint that follows the convex-concave rule to determine direction of glide and roll movement. (b) d [22] ( ) L (a) (a) (b) Fig. 2.3. Knee Joint (Tibiofemoral Joint) 6: Tibiofemoral joint (knee joint) structure and movements [22], (a) Lateral view of tibiofemoral joint representing roll and glide of tibiofemoral joint while flexion and extension, (b) Convex-concave surface of a hinge joint that follows the convex-concave rule to determine direction of glide and roll movement. (b) (a) (a) (b) Fig. 7: Ligaments of tibiofemoral joint [23], (a) Anterior and (b) Posterior view of right knee joint. (a) (b) Fig. 7: Ligaments of tibiofemoral joint [23], (a) Anterior and (b) Posterior view of right knee joint. 2.4. Leg (Syndesmosis) Leg of human skeleton is formed with two bones, tibia and fibula. These two bones are also firmly attached by interosseus membrane and ligaments. This is fibrous type joint called syndesmosis [22]. This type of joint is more stable as bones are connected tightly together and there is a little (or no) movement at this joint. Figure 8 shows skeletal architecture of lower leg and Fig. 9 presents syndesmosis (fibrous joint) between tibia and fibula. (a) (b) Fig. 8: Skeletal structure of human leg, ankle, and foot [26], (a) Anterior, and (b) Posterior view. (a) (b) Fig. 8: Skeletal structure of human leg, ankle, and foot [26], (a) Anterior, and (b) Posterior view. 88 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Fig. 9: Syndesmosis (fibrous joint) of leg formed by ligaments and interosseous membrane [22]. Fig. 9: Syndesmosis (fibrous joint) of leg formed by ligaments and interosseous membrane [22]. 2.5. Ankle Joint (Talocrural Articulation) Connection point between lower leg and foot is called ankle joint which also works like a hinge joint. Medial malleolus and lateral malleolus of ankle joint are formed by inferior portion of two bones, tibia and fibula, of leg. Both malleolus of ankle is connected and attached to main ankle-foot tarsal bone called talus. Ankle joint is uniaxial and has only one DoF. Mobility of ankle joint is characterized as planter flexion and dorsiflexion [18,26]. These movement capabilities of ankle hinge joint drive body forward or backward on sagittal plane. Foot is one of the most mobile structures having complex joint mechanisms that supports body to absorb shock of ground contact force and adapt with various surfaces [26]. Complex multi joint structure has in total of seven tarsal bones which ensure triaxial movement (3 DoF) of a foot. As a result, foot movements are also characterized as inversion and eversion. These tarsal bones are attached to each other such as, Talas with Calcaneus (heel bone), Talas with Navicular, Calcaneus with Cuboid, Calcaneus with Navicular, Navicular with three Cuneiforms (medial, internal and lateral), Navicular with Cuboid, and cuneiform bones with each other. Calcaneus bone of tarsal group supports almost 50% of total body weight [18,26]. Skeletal structure of a foot is presented in Fig. 10. Tarso-metatarsal joint comprises four tarsal bones (three cuneiforms and cuboid) connected to five metatarsal bones of toes as shown in Fig. 10. First three metatarsal bones are connected with three cuneiform bones accordingly. Fourth metatarsal is connected with both lateral cuneiform and cuboid bones. Finally, lateral metatarsal is solely connected to cuboid tarsal [18,26]. First and second metatarsals support about 25% of total body weight. Rest of the body weight (about 25%) is absorbed by 3rd, 4th, and 5th metatarsals [18]. Figure 11 to 14 show ligament structure of a foot skeleton. Phalanges of five toes are contacted with five metatarsal bones where each of the phalanges, from 2nd to 5th has three bones (proximal phalanx, middle phalanx, and distal phalanx) and 1st one has two bones, proximal phalanx and distal phalanx, as presented in Fig. 10. Phalange joints are also considered as hinge joints having 1 DoF and movements are characterized as flexion and extension. 89 IIUM Engineering Journal, Vol. 17, No. 1, 2016 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. UM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. 90 (a) (b) Fig. 10: Skeletal structure of right foot [26]. (a) Lateral view, and (b) Medial view. Fig. 11: Ligaments of right foot (lateral view) [26]. Fig. 12: Ligaments of right foot (medial view) [26]. (a) (b) Fig. 10: Skeletal structure of right foot [26]. (a) Lateral view, and (b) Medial view. (a) (a) (b) Fi 10 Sk l t l t t f i ht f t [26] ( ) L t l i d (b) M di l i (b) Fig. 10: Skeletal structure of right foot [26]. (a) Lateral view, and (b) Medial view. Fig. 11: Ligaments of right foot (lateral view) [26]. Fig. 12: Ligaments of right foot (medial view) [26]. (b) Fig. 10: Skeletal structure of right foot [26]. (a) Lateral view, and (b) Medial view. Fig. 10: Skeletal structure of right foot [26]. (a) Lateral view, and (b) Medial view. Fig. 11: Ligaments of right foot (lateral view) [26]. Fig. 11: Ligaments of right foot (lateral view) [26]. Fig. 12: Ligaments of right foot (medial view) [26]. Fig. 12: Ligaments of right foot (medial view) [26]. Fig. 12: Ligaments of right foot (medial view) [26]. 90 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Fig. 13: Ligaments of right foot (posterior view) [26]. Fig. 14: Ligaments of right foot (inferior view) [26]. Fig. 13: Ligaments of right foot (posterior view) [26]. Fig. 13: Ligaments of right foot (posterior view) [26]. Fig. 14: Ligaments of right foot (inferior view) [26]. 3. MUSCULAR SYSTEM OF HUMAN LOWER LIMBS Skeletal muscle plays an important role in protecting endoskeleton against injury, producing joint movements and locomotion. Skeletal muscle forms about 40% to 45% of total body weight [21]. Rudimentary structural element of skeletal muscle is muscle fiber, a single cell, which is apparently very long but usually shorter than individual muscle [21]. Each fiber is surrounded by endomysium layer and fibers are bundled together called fascicles. Fascicles are surrounded by perimysium and finally multiple perimysium are layered by epimysium which is tough enough, friction less, and anchors muscle fibers to tendons at both ends of a muscle. Other end of each tendon is attached with endoskeleton. A muscle could be attached with bones through one or more joints [18,21,27]. Figure 15 and 16 present conceptual architecture and anatomy of skeletal muscle respectively. (a) (b) Fig. 15: Conceptual architecture and connectivity of skeletal muscle, (a) tendon and bone connectivity concept, and (b) cross sectional architecture concept. (b) (a) (b) (a) ( ) ( ) Fig. 15: Conceptual architecture and connectivity of skeletal muscle, (a) tendon and bone connectivity concept, and (b) cross sectional architecture concept. 91 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Fig. 16: Basic anatomy of skeletal muscle [27]. Fig. 16: Basic anatomy of skeletal muscle [27]. Primary uses of skeletal muscles of lower extremity are leverage and locomotion by exerting a pull on bones. During both dynamic activities and static posture, skeletal muscle plays a significant role to stabilize skeletal segments and joint structures [18]. Skeletal muscles, such as abdominal muscle, protect underlying structure and organs of human body. Other two important activities of skeletal muscles are, producing body heat (thermogenesis), and vascular pumping. About 75% of total energy generated by muscle tissue is heat which keeps the body warm [27]. On the other hand, contraction of skeletal muscle helps to propel fluids of lymphatic vessels and veins having comparatively low pressure than arteries. Basically, skeletal muscle congregates properties of extensibility (stretching without sustaining damage), elasticity (ability to return to its original shape), excitability (ability to response to a stimulus by generating electrical signals), conductivity (ability to propagate electrical signals), and contractility (ability to produce force by shorten or thicken), which ensures unique ability of skeletal muscles. Endoskeleton is a multi-joint system of rigid bones acting as levers and with the muscle system it produces internal forces to move it. 3. MUSCULAR SYSTEM OF HUMAN LOWER LIMBS (Asst), LR.(Asst.) Inferior Gemellus O: Proximal part of ischial tuberosity. I: Medial surface of greater trochanter of femur. Obturator Internus Sacral plexus; L5, S1, S2. O: Inferior surface of obturator membrane of ischium. I: Medial surface of greater trochanter of femur. Ab., LR. Obturator Externus Obturator nerve; L5, S1. O: Pubis and ischium, superior and inferior rami. I: Trochanteric fossa of femur. LR. Quadratus Femoris Sacral plexus; L4 - S2. O: Lateral part of ischial tuberosity. I: Between greater and lesser trochanters of femur. Biceps Femoris Posterior Femoral (Hamstring) Sciatic nerve; L5, S1 - S3. O: Long head: ischial tuberosity, Short head: lateral lip of linea aspera. I: Head of fibula. Ext., LR.(Asst.) Semimembranosus Tibial division of sciatic nerve; L5, S1, S2. O: Ischial tuberosity. I: Posteromedial portion of medial tibial condyle. Ext., MR.(Asst.) Semitendinosus O: Ischial tuberosity. I: Medial tibial shaft via pes anserine tendon. O: Origin. I: Insertion. Flx.: Flexion. Ext.: Extension. Ab.: Abduction. Add.: Adduction. MR: Medial Rotation. LR: Lateral Rotation. Asst.: Assisting Muscle. Table 1: Muscles acting on lower extremity Hip Joint [23,30-32] Muscles (Hip joint) Group Innervation Attachments Functions Psoas (Major and Minor) Iliopsoas Femoral nerve; L2, L3, L4. O: Transverse processes, lateral bodies, and corresponding intevertberal disks of Tl2, L1 - L5. I: Lesser trochanter of femur. Flx., LR.(Asst.) Iliacus O: Iliac fossa and ala of sacrum. I: Lesser trochanter of femur. Rectus Femoris Quadriceps O: Anterior inferior iliac spine (AIIS). I: Tibial tuberosity via patellar tendon. Flx., Ab.(Asst.) Sartorius Anterior Femoral Femoral nerve; L2, L3. O: Anterior superior iliac spine (ASIS). I: Medial shaft of tibia via pes anserine tendon. Flx., Ab.(Asst.), LR Pectineus Adductor Femoral and obturator nerves; L2, L3, L4. O: Superior ramus of pubis. I: Pectineal line of femur. Flx., Add. Adductor Brevis Obturator nerve; L2, L3, L4. O: Outer surface of inferior ramus of pubis. I: Pectineal line and proximal part of medial lip of linea aspera of femur. Add., Flx., LR.(Asst) Adductor Longus O: Between pubic crest and symphysis. I: Middle one-third of medial lip of linea aspera of femur. Add., Flx., MR.(Asst.) Adductor Magnus Tibial division of sciatic nerve; L2, L3, L4. O: Inferior ramus of pubis, ramus of ischium, and ischial tuberosity. I: Medial lip of linea aspera of femur, medial supracondylar line, and adductor tubercle. Ext.(Asst.), Add., Flx.(Asst), MR Gracilis Obturator nerve; L2, L3, L4. O: Inferior ramus of pubis. 3. MUSCULAR SYSTEM OF HUMAN LOWER LIMBS This mechanism is comparable with a composite system of levers [27] where joints act as axes of levers. Common resistances (external opposite force) of muscle force are gravity and friction. There are three types of levers in human musculoskeletal system, (a) First-class lever, (b) Second-class lever, and (c) Third-class lever [27,28]. In a first-class lever axis is in between the force and the resistance, can be represented as Force-Axis-Resistance (FAR). This type of lever mechanism is used in skeletal structure for balancing. A second-class lever is Force- Resistance-Axis (FRA) type where resistance is in the middle of force and axis. This type of lever mechanism is found at ankle joint when a human being try to stand on the tips of their feet. Second-class lever is very powerful in terms of motion range and speed. Finally, third-class lever mechanism has force in between resistance and axis, reflecting Resistance- Force-Axis (RFA) type. This is the most common type of lever found in human musculoskeletal structure. Knee joint movements are managed by this type of lever mechanism. Muscles of lower extremity can be grouped depending on various criteria such as, (a) corresponding segments [18], (b) actions or movements [23], (c) innervation [23], (d) corresponding joints [29] etc. Figure 17 and 18 show muscles of the lower musculoskeletal system of a human body. 92 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Fig. 17: Muscles of human body lower extremity (Anterior view) [27]. Fig. 17: Muscles of human body lower extremity (Anterior view) [27]. Fig. 17: Muscles of human body lower extremity (Anterior view) [27]. 93 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Fig. 18: Muscles of human body lower extremity (Posterior view) [27]. Fig. 18: Muscles of human body lower extremity (Posterior view) [27]. Fig. 18: Muscles of human body lower extremity (Posterior view) [27]. Muscles acting on various joints of the lower extremity of a human body are grouped and characterized based on their functions of particular joint movements, as presented in Table 1, Table 2, and Table 3. Muscles are also characterized based on their attachments, origins (O) and insertions (I), with corresponding bones, and their innervation system. In terms of robotic therapy systems, these significant characteristics are very important to understand the complex system of humanoid lower extremity. 94 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. 3. MUSCULAR SYSTEM OF HUMAN LOWER LIMBS Table 1: Muscles acting on lower extremity Hip Joint [23,30-32] Muscles (Hip joint) Group Innervation Attachments Functions Psoas (Major and Minor) Iliopsoas Femoral nerve; L2, L3, L4. O: Transverse processes, lateral bodies, and corresponding intevertberal disks of Tl2, L1 - L5. I: Lesser trochanter of femur. Flx., LR.(Asst.) Iliacus O: Iliac fossa and ala of sacrum. I: Lesser trochanter of femur. Rectus Femoris Quadriceps O: Anterior inferior iliac spine (AIIS). I: Tibial tuberosity via patellar tendon. Flx., Ab.(Asst.) Sartorius Anterior Femoral Femoral nerve; L2, L3. O: Anterior superior iliac spine (ASIS). I: Medial shaft of tibia via pes anserine tendon. Flx., Ab.(Asst.), LR Pectineus Adductor Femoral and obturator nerves; L2, L3, L4. O: Superior ramus of pubis. I: Pectineal line of femur. Flx., Add. Adductor Brevis Obturator nerve; L2, L3, L4. O: Outer surface of inferior ramus of pubis. I: Pectineal line and proximal part of medial lip of linea aspera of femur. Add., Flx., LR.(Asst) Adductor Longus O: Between pubic crest and symphysis. I: Middle one-third of medial lip of linea aspera of femur. Add., Flx., MR.(Asst.) Adductor Magnus Tibial division of sciatic nerve; L2, L3, L4. O: Inferior ramus of pubis, ramus of ischium, and ischial tuberosity. I: Medial lip of linea aspera of femur, medial supracondylar line, and adductor tubercle. Ext.(Asst.), Add., Flx.(Asst), MR Gracilis Obturator nerve; L2, L3, L4. O: Inferior ramus of pubis. I: Medial shaft of tibia via pes anserine tendon. Add., MR.(Asst.) Gluteus Maximus Gluteal Inferior gluteal nerve; L5, S1, S2. O: Posterior iliac crest, sacrum, and sacrotuberous ligament. I: Greater trochanter of femur, gluteal tuberosity, and lateral tibial condyle via iliotibial band. Ext., LR., Ab. (upper fibers), Add. (lower fibers) Gluteus Medius Superior gluteal nerve; L4, L5, S1. O: External surface of ilium between anterior and posterior gluteal lines. I: Lateral surface of greater trochanter of femur. Ab., Asst: (MR., Flx., Ext., LR.) Gluteus Minimus O: External surface of ilium between anterior and inferior gluteal lines. I: Anterior border of greater trochanter of femur. Ab., MR., Flx.(Asst) Tensor Fasciae Latae O: Anterolateral lip of iliac crest. I: Lateral condyle of tibia via iliotibial band. Flx.(Asst.), Ab.(Asst.), MR Piriformis Lateral Rotator Sacral plexus; S1, S2. O: Anterior surface of sacrum. I: Superior border of greater trochanter of femur. Ab.(Asst.), LR. Superior Gemellus Sacral plexus; L5, S1. O: External surface of ischium. I: Medial surface of greater trochanter of femur. Ab. 3. MUSCULAR SYSTEM OF HUMAN LOWER LIMBS I: Medial shaft of tibia via pes anserine tendon. Add., MR.(Asst.) Gluteus Maximus Gluteal Inferior gluteal nerve; L5, S1, S2. O: Posterior iliac crest, sacrum, and sacrotuberous ligament. I: Greater trochanter of femur, gluteal tuberosity, and lateral tibial condyle via iliotibial band. Ext., LR., Ab. (upper fibers), Add. (lower fibers) Gluteus Medius Superior gluteal nerve; L4, L5, S1. O: External surface of ilium between anterior and posterior gluteal lines. I: Lateral surface of greater trochanter of femur. Ab., Asst: (MR., Flx., Ext., LR.) Gluteus Minimus O: External surface of ilium between anterior and inferior gluteal lines. I: Anterior border of greater trochanter of femur. Ab., MR., Flx.(Asst) Tensor Fasciae Latae O: Anterolateral lip of iliac crest. I: Lateral condyle of tibia via iliotibial band. Flx.(Asst.), Ab.(Asst.), MR Piriformis Lateral Rotator Sacral plexus; S1, S2. O: Anterior surface of sacrum. I: Superior border of greater trochanter of femur. Ab.(Asst.), LR. Superior Gemellus Sacral plexus; L5, S1. O: External surface of ischium. I: Medial surface of greater trochanter of femur. Ab. (Asst), LR.(Asst.) Inferior Gemellus O: Proximal part of ischial tuberosity. I: Medial surface of greater trochanter of femur. Obturator Internus Sacral plexus; L5, S1, S2. O: Inferior surface of obturator membrane of ischium. I: Medial surface of greater trochanter of femur. Ab., LR. Obturator Externus Obturator nerve; L5, S1. O: Pubis and ischium, superior and inferior rami. I: Trochanteric fossa of femur. LR. Quadratus Femoris Sacral plexus; L4 - S2. O: Lateral part of ischial tuberosity. I: Between greater and lesser trochanters of femur. Biceps Femoris Posterior Femoral (Hamstring) Sciatic nerve; L5, S1 - S3. O: Long head: ischial tuberosity, Short head: lateral lip of linea aspera. I: Head of fibula. Ext., LR.(Asst.) Semimembranosus Tibial division of sciatic nerve; L5, S1, S2. O: Ischial tuberosity. I: Posteromedial portion of medial tibial condyle. Ext., MR.(Asst.) Semitendinosus O: Ischial tuberosity. I: Medial tibial shaft via pes anserine tendon. O: Origin. I: Insertion. Flx.: Flexion. Ext.: Extension. Ab.: Abduction. Add.: Adduction. MR: Medial Rotation. LR: Lateral Rotation. Asst.: Assisting Muscle. Table 1: Muscles acting on lower extremity Hip Joint [23,30-32] 95 IIUM Engineering Journal, Vol. 17, No. 1, 2016 O: Origin. I: Insertion. Flx.: Flexion. Ext.: Extension. Ab.: Abduction. Add.: Adduction. MR: Medial Rotation. LR: Lateral Rotation. Asst.: Assisting Muscle. IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. IIUM Engineering Journal, Vol. 17, No. 1, 2016 Table 3: Muscles acting on lower extremity Ankle Joint [26,30-32] Table 3: Muscles acting on lower extremity Ankle Joint [26,30-32] Muscles (Ankle joint) Group Innervation Attachments Functions Tibialis Anterior Extrinsic Deep peroneal nerve; L4 – L5. O: Lateral condyle and proximal half of tibia and interosseous membrane. I: Plantar surface of medial cuneiform and base of first metatarsal. Ank. Df., Ft. Inv. Extensor Digitorum Longus Deep peroneal nerve; L4 – L5. O: Lateral tibial condyle, proximal anterior fibula, and interosseous membrane. I: Middle and distal phalanges of digits 2-5, by four tendons to dorsal surfaces. Ank. Df., Toe 2-5 Plf., Ft. Ev. Extensor Hallucis Longus Deep peroneal nerve; L4 – L5. O: Middle of anterior surface of fibula and membrane. I: Base of first distal phalanx, dorsal side. Ank. Df., Big Toe Plf., Ft. Add. (Inv.) Peroneus Longus Superficial peroneal nerve; IA – 52. O: Head and lateral two-thirds of fibula. I: Lateral sides of first metatarsal and medial cuneiform. Ank. Plf., Ft. Ab., Ft. Ev. Peroneus Bravis Superficial peroneal nerve; L5 – S2. O: Distal two-thirds of lateral surface of fibula. I: Tuberosity on base of fifth metatarsal. Ank. Plf., Ft. Ev. Peroneus Tertius Deep peroneal nerve; L4 – L5. O: Distal one-third of anterior surface of fibula and interosseous membrane. I: Dorsal surface of base of fifth metatarsal. Ank. Df., Ft. Ev. Gastrocnemius Extrinsic & Triceps surae Tibial nerve; S1 – S2. O: Medial head: posterior surface of medial femoral condyle; Lateral head: posterior surface of lateral femoral condyle. I: Posterior surface of calcaneus via Achilles tendon. Ank. Plf. Soleus O: Soleal line and posterior surface of tibia and posterior head and proximal surface of fibula. I: Posterior surface of calcaneus via Achilles tendon. Plantaris Tibial nerve; S1 – S2 O: Distal part of lateral supracondylar line of femur . I: Posterior surface of calcaneus via Achilles tendon. Ank. Plf. (Asst.) Tibialis Posterior Intrinsic Tibial nerve; L4 – L5 O: Lateral, posterior tibia, proximal two- thirds of medial fibula, and interosseous membrane. I: Navicular tuberosity, cuneiforms l-3, cuboid, and bases of metatarsals 2-4. Ank. Plf. (Asst), Ft. Inv. Flexor Digitorum Longus Tibial nerve; S2 – S3 O: Middle of posterior surface of tibia. I: Base of distal phalanges of digits 2-5,by four tendons to plantar surfaces. Toe 2-5 Df., Ank. Plf. (Asst), Ft. Inv. IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Table 2: Muscles acting on lower extremity Knee Joint [23,30-32] Muscles (Knee joint) Group Innervation Attachments Functions Rectus Femoris Quadriceps Femoral nerve; L2 – L4. O: Anterior inferior iliac spine (AIIS). I: Tibial tuberosity via patellar tendon. Ext. Vastus Lateralis O: Greater trochanter of femur, gluteal tuberosity, and proximal, lateral lip of linea aspera. I: Tibial tuberosity via patellar tendon. Vastus Medialis O: Intertrochanteric line and medial lip of linea aspera of femur. I: Tibial tuberosity via patellar tendon. Vastus Intermedius O: Proximal two-thirds of anterior shaft of femur. I: Tibial tuberosity via patellar tendon. Sartorius Anterior Femoral Femoral nerve; L2 – L3. O: Anterior superior iliac spine (ASIS). I: Medial shaft of tibia via pes anserine tendon. Flx. (Asst.), MR. (Pronation) Biceps Femoris Posterior Femoral (Hamstring) Tibial division (long head) and common peroneal division (short head) of sciatic nerve; L5, S1 – S3. O: Long head: ischial tuberosity; Short head: lateral lip of linea aspera. I: Head of fibula. Flx., LR. (Supinaiton) Semimembranosus Tibial division of sciatic nerve; L5 – S2. O: Ischial tuberosity. I: Posteromedial portion of medial tibial condyle. Flx., MR. (Pronation) Semitendinosus O: Ischial tuberosity. I: Medial tibial shaft via pes anserine tendon. Popliteus Lateral Rotator Tibial nerve; L4 – S3. O: Lateral femoral condyle. I: Proximal posterior surface of tibia. Flx. (Asst), MR. (Pronation) Gracilis Adductor Obturator nerve; L2 – L4. O: Inferior ramus of pubis. I: Medial shaft of tibia via pes anserine tendon. Gastrocnemius Triceps surae Tibial nerve; S1 – S2. O: Medial head: posterior surface of medial femoral condyle; Lateral head: posterior surface of lateral femoral condyle. I: Posterior surface of calcaneus via Achilles tendon. Flx. (Asst.) Plantaris O: Distal part of lateral supracondylar line of femur. I: Posterior surface of calcaneus via Achilles tendon. Flx. (Asst) O: Origin. I: Insertion. Flx.: Flexion. Ext.: Extension. Ab.: Abduction. Add.: Adduction. MR: Medial Rotation. LR: Lateral Rotation. Asst.: Assisting Muscle. able 2: Muscles acting on lower extremity Knee Joint [23,30-32] 96 IIUM Engineering Journal, Vol. 17, No. 1, 2016 (Asst.) Flexor Hallucis Longus Tibial nerve; S2 – S3 O: Distal posterior surface of fibula and interosseous membrane. I: Base of first distal phalanx, plantar surface. Big Toe Df., Ank. Plf. (Asst), Ft. Ad., Ft. Inv. (Asst.) O: Origin. I: Insertion. Ank..: Ankle. Df.: Dirsiflexion. Plf..: Plantarflexion. Add.: Adduction. Ab.: Abduction. Inv.: Inversion. Ev.: Eversion. Ft.: Foot. Asst.: Assisting Muscle. Movements of joints occur along any of three section planes of human body, Sagittal plane (Lateral plane), Frontal plane (Coronal plane), and Transverse plane (Axial plane) [28,33]. Each of these three planes has corresponding axis around which a particular O: Origin. I: Insertion. Ank..: Ankle. Df.: Dirsiflexion. Plf..: Plantarflexion. Add.: Adduction. Ab.: Abduction. Inv.: Inversion. Ev.: Eversion. Ft.: Foot. Asst.: Assisting Muscle. O: Origin. I: Insertion. Ank..: Ankle. Df.: Dirsiflexion. Plf..: Plantarflexion. Add.: Adduction. Ab.: Abduction. Inv.: Inversion. Ev.: Eversion. Ft.: Foot. *Asst.: Assisting Muscle. Movements of joints occur along any of three section planes of human body, Sagittal plane (Lateral plane), Frontal plane (Coronal plane), and Transverse plane (Axial plane) [28,33]. Each of these three planes has corresponding axis around which a particular 97 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. movement occurs. Movements, Flexion and Extension (front-back) occur on Sagittal plane along Frontal axis, Abduction and Adduction (side-side) occur on Frontal plane along Sagittal axis, and rotational movements, Lateral rotation and Medial rotation, occur on Transverse plane along Longitudinal axis. Muscles that are involved in particular movements of limbs can be grouped together to identify and monitor performances of affected muscles during rehabilitation or therapy. Muscle groups are presented in Table 4, Table 5, and Table 6, according to the movements of the lower extremity of a human body. ab e 5, a d ab e 6, acco d g to t e ove e ts o t e owe e t e ty o a u a body. Table 4: Hip joint movements and muscle groups [23,32] Hip Movements Muscle groups Abductor 1. Sartorius 2. Tensor fascia latae 3. Piriformis 4. Giuteus maximus (upper fibers) 5. Gluteus medius 6. Gluteus minimus 7. Gemellus superior 8. Gemellus inferior 9. Obturator internus Adductor 1. Pectineus 2. Adductor brevis 3. Adductor longus 4. Gracilis 5. Adductor magnus 6. Gluteus maximus (lower fibers) Extensor 1. Adductor magnus (posterior fibers) 2. Gluteus maximus 3. Gluteus medius (posterior fibers) 4. Biceps femoris (long head) 5. Semimembranosus 6. IIUM Engineering Journal, Vol. 17, No. 1, 2016 Semitendinosus Flexor 1. Psoas 2. Iliacus 3. Sartorius 4. Tensor fascia latae 5. Rectus femoris 6. Pectineus 7. Adductor brevis 8. Adductor longus 9. Adductor magnus (anterior fibers) 10. Gluteus medius (anterior fibers) 11. Gluteus minimus Lateral Rotator 1. Psoas 2. Iliacus 3. Sartorius 4. Adductor brevis 5. Gluteus maximus 6. Gluteus medius (posterior fibers) 7. Piriformis 8. Gemellus superior 9. Gemellus inferior 10. Obturator internus 11. Obturator extemus 12. Quadratus femoris 13. Biceps femoris (long head) Medial Rotator 1. Tensor fascia latae 2. Gluteus medius (anterior fibers) 3. Gluteus minimus 4. Semimembranosus 5. Semitendinosus Table 4: Hip joint movements and muscle groups [23,32] Table 4: Hip joint movements and muscle groups [23,32] Hip Movements Muscle groups Abductor 1. Sartorius 2. Tensor fascia latae 3. Piriformis 4. Giuteus maximus (upper fibers) 5. Gluteus medius 6. Gluteus minimus 7. Gemellus superior 8. Gemellus inferior 9. Obturator internus Adductor 1. Pectineus 2. Adductor brevis 3. Adductor longus 4. Gracilis 5. Adductor magnus 6. Gluteus maximus (lower fibers) Extensor 1. Adductor magnus (posterior fibers) 2. Gluteus maximus 3. Gluteus medius (posterior fibers) 4. Biceps femoris (long head) 5. Semimembranosus 6. Semitendinosus Flexor 1. Psoas 2. Iliacus 3. Sartorius 4. Tensor fascia latae 5. Rectus femoris 6. Pectineus 7. Adductor brevis 8. Adductor longus 9. Adductor magnus (anterior fibers) 10. Gluteus medius (anterior fibers) 11. Gluteus minimus Lateral Rotator 1. Psoas 2. Iliacus 3. Sartorius 4. Adductor brevis 5. Gluteus maximus 6. Gluteus medius (posterior fibers) 7. Piriformis 8. Gemellus superior 9. Gemellus inferior 10. Obturator internus 11. Obturator extemus 12. Quadratus femoris 13. Biceps femoris (long head) Medial Rotator 1. Tensor fascia latae 2. Gluteus medius (anterior fibers) 3. Gluteus minimus 4. Semimembranosus 5. Semitendinosus 98 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. 4. CONCLUSION The main focus of lower limbs rehabilitation is to maintain normal mobility of lower extremity joints by providing external force in harmony to ensure repetitive and persistent movement actions within the range of musculoskeletal motion. Therefore, the lower extremity maintains its normal functions instead of muscles being shrunk and will improve muscle performance as well as the nervous system. As an automated rehabilitation system should provide better facility to do exercise without any risk of accident or secondary injury, it is very important to study human musculoskeletal structure. Considering this point, the review presents a basic overview on musculoskeletal structure of human lower limbs by characterizing and classifying skeletal and muscular system based on various types of functionalities, movements, and joint architecture. ACKNOWLEDGEMENT The authors would like to express their gratitude to the Ministry of Education (MOE), Malaysia for funding the project through the Fundamental Research Grant Scheme (FRGS). The authors would like to express their gratitude to the Ministry of Education (MOE), Malaysia for funding the project through the Fundamental Research Grant Scheme (FRGS). IIUM Engineering Journal, Vol. 17, No. 1, 2016 IIUM Engineering Journal, Vol. 17, No. 1, 2016 Akhtaruzzaman et al. Intrafusal fiber is a special muscle fiber of muscle spindle which is surrounded by a coil of sensory nerve endings, ‘Gamma motor neuron’ and ‘Afferent fibers’. Muscle spindles are special nerves distributed in muscles and tendons that monitor stimuli regarding changes of position, movement, and magnitude of stretch of muscle tissues [27]. If any external force is applied, ‘Extrafusal fibers’ of muscle spindle acknowledge ‘Alpha motor neuron’ to adjust length of muscles in order to protect muscle fiber from damage. This reaction is known as Myotatic Reflex. On the other hand, tension of muscle spindle is adapted by ‘Gamma motor neurons’ to manage length monitoring function [27,34]. In terms of therapy, it is important to consider that a fast or strong force or stretch that cross a certain limit may cause potential damage of tissues [27,34]. Force-velocity relationship of muscle shows inverse characteristics as increment of external force will decrease motion velocity [35,36]. So, it is important to consider and analyze these biological functions and reactions in designing robot assisted rehabilitation systems. Table 5: Knee joint movements and muscle groups [23,32] Table 5: Knee joint movements and muscle groups [23,32] Table 5: Knee joint movements and muscle groups [23,32] Knee Movements Muscle groups Extensor 1. Rectus femoris 2. Vastus lateralis 3. Vastus intermedius 4. Vastus medialis Flexor 1. Sartorius 2. Gracilis 3. Biceps femoris 4. Semimembranosus 5. Semitendinosus 6. Plantaris 7. Popliteus 8. Gastrocnemius Lateral Rotator 1. Biceps femoris Medial Rotator 1. Gracilis 2. Sartorius 3. Semimembranosus 4. Semitendinosus 5. Popliteus Table 5: Knee joint movements and muscle groups [23,32] Knee Movements Muscle groups Extensor 1. Rectus femoris 2. Vastus lateralis 3. Vastus intermedius 4. Vastus medialis Flexor 1. Sartorius 2. Gracilis 3. Biceps femoris 4. Semimembranosus 5. Semitendinosus 6. Plantaris 7. Popliteus 8. Gastrocnemius Lateral Rotator 1. Biceps femoris Medial Rotator 1. Gracilis 2. Sartorius 3. Semimembranosus 4. Semitendinosus 5. Popliteus 1 99 Table 6: Ankle and foot movements and muscle groups [26,32] Foot Movements Muscle groups Dorsiflexor (Flexor) 1. Tibialis anterior 2. Extensor digitorum longus 3. Extensor hallucis longus 4. Peroneus tertius Plantar flexor (Extensor) 1. Gastrocnemius 2. Soleus 3. Plantaris 4. Peroneus longus 5. Peroneus brevis 6. Tibialis posterior 7. Flexor digitorum longus 8. Flexor hallucis longus Foot invertor 1. Tibialis anterior 2. Extensor hallucis longus 3. Tibialis posterior 4. Flexor digitorum longus 5. Flexor hallucis longus Foot evertor 1. Extensor digitorum longus 2. Peroneus longus 3. Peroneus brevis 4. Peroneus tertius Toe extensor (Plantar flx.) 1. Extensor digitorum longus (2-5) 2. Extensor hallucis longus (1) Toe flexor (Dorsiflx.) 1. Flexor digitorum longus 2. (2-5) 3. Flexor hallucis longus (1) Table 6: Ankle and foot movements and muscle groups [26,32] Table 6: Ankle and foot movements and muscle groups [26,32] 99 REFERENCES [1] Venketasubramanian N. (1998) The epidemiology of stroke in ASEAN countries – A Review. Neurol J. Southeast Asia, 3:9-14. [1] Venketasubramanian N. (1998) The epidemiology of stroke in ASEAN countries – A Review. Neurol J. Southeast Asia, 3:9-14. [2] Glamcevski MT, Tan CT. (2000) Prevalence of post-stroke depression, a Malaysian study. Neurol J. Southeast Asia, 5:51-53. [3] Tan KS, Tan CT, Churilov L, Mackay M, Donnan GA. 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W4391692366.txt	https://www.researchsquare.com/article/rs-3927383/latest.pdf	en		Efficient Encryption using Quondam Signature Algorithm and Modified Lean Six Sigma for Sustainability with Supply Chain Management	Research Square (Research Square)	2,024	cc-by	6,689	Efficient Encryption using Quondam Signature Algorithm and Modified Lean Six Sigma for Sustainability with Supply Chain Management Fateh Bahadur Kunwar Hitendra Singh Rakesh Kumar Yadav Research Article Keywords: Blockchain, IIIoT Security, Federated Learning, Efficient Encryption, Quondam Signature Algorithm, Modified Lean, Six Sigma, Sustainability, Supply Chain Management Posted Date: February 9th, 2024 DOI: https://doi.org/10.21203/rs.3.rs-3927383/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Page 1/19 Abstract This paper presents a novel blockchain-based Industrial Internet of Things (IIIoT) security approach. The proposed architecture uses an eco-friendly federated learning AI-driven paradigm with cutting-edge Quondam Signature Algorithm technology and efficient encryption. The research also uses Modified Lean Six Sigma for sustainable supply chain management. The framework combines these aspects to synergistically increase IIIoT systems cyber resilience and environmental sustainability. The proposed work explores advanced encryption and process optimization methods to develop robust security architecture and advance safe and environmentally friendly IIIoT applications in supply chain management. We analyze cost-in-communication systems, focusing on federated learning and encryption. The suggested method uses an online/offline system, an elliptic curve digital signature technique, and online phase signature generation to combine offline phase calculations and optimize temporal complexity. Since it uses federated learning and encryption, the proposed method looks to improve efficiency and lower communication costs. This proposed work also shows that the recommended technique reduces communication costs, improves analytical capabilities, and protects IIoT systems from various attacks. 1. Introduction New levels of connection, efficiency, and creativity have been introduced into the ever-changing world of Industry 4.0 with the advent of blockchain technology and the Industrial Internet of Things (IIIoT). Unfortunately, the integration of these revolutionary technologies is not without its security risks, and it is vital that we find solutions to these problems.[4] Addressing this critical requirement, this article presents a novel strategy: "Strengthening Blockchain IIIoT Security with an Eco-Friendly Federated Learning AIDriven Framework." The goal of this groundbreaking approach is to create a more reliable and robust industrial ecosystem by improving the security of blockchain-enabled IIIoT devices. Essentially, the system trains models jointly across decentralised nodes via federated learning, a new paradigm in AI that ensures data privacy. Because of this, the system becomes more resilient, and it also conforms to ecofriendly principles by reducing the amount of electricity that is often connected with AI's central processing unit.[5] The use of effective encryption methods is a critical component of this strategy, since it guarantees the privacy and authenticity of data transmitted inside the IIIoT network. By providing a fresh and robust approach to cryptographic signatures, the Quondam Signature Algorithm considerably enhances the security architecture. Combining this state-of-the-art technology does double duty: it strengthens the blockchain's IIIoT security and establishes a standard for environmentally friendly digital transformation practises. In addition, this framework incorporates aspects of the Modified Lean Six Sigma technique, acknowledging the vital importance of sustainability in modern business practises. This key addition strengthens the IIIoT ecosystem's supply chain management by improving efficiency and cutting down on waste. [6] Page 2/19 1.1 Background and Motivation The widespread use of IIIoT devices has revolutionised several industries by improving efficiency and encouraging new forms of corporate organisation. The safety of vital infrastructure and private information is becoming more of a worry as a result of these technological developments. Production delays, compromised safety, and massive financial losses are all possible outcomes of cyberattacks against IIIoT systems.[7] To protect the privacy and authenticity of information in IIIoT settings, cuttingedge security protocols are essential. 1.2 Brief Overview of IIIoT Security Challenges IIIoT security covers a wide range of issues, including as ensuring the authenticity, confidentiality, and integrity of data, as well as protecting it from harmful assaults. Due to their dispersed and networked design, IIIoT devices are open to a host of security risks, including data manipulation and illegal access. A comprehensive strategy that extends beyond traditional security measures is necessary to tackle these difficulties.[8] Device Proliferation and Heterogeneity: It is difficult to provide uniform security requirements across all devices in an IIoT ecosystem due to the vast quantity and diversity of devices. This problem may be lessened by putting in place-standardised security frameworks and standards, doing routine device audits, and making sure secure onboarding procedures are followed. Data Integrity and Confidentiality: IIoT devices produce large volumes of sensitive data. It is essential to protect the confidentiality and integrity of this data in order to stop illegal access, alteration, or disclosure. Data integrity and confidentiality may be protected by encrypting data while it's in transit and at rest, putting access restrictions in place, and routinely upgrading security measures. Network Security: Securing the complete communication channel is difficult since IIoT devices frequently communicate via a variety of networks, including public, private, and hybrid networks. To guard against illegal access and data interception, use strong network security measures like firewalls, intrusion detection systems, and secure communication protocols. Lack of Standardization: Inconsistencies and vulnerabilities may arise from the lack of standardised security procedures among various IIoT devices and platforms. Establishing and implementing common security standards through industry collaboration may improve interoperability and guarantee a more uniform and safe IIoT ecosystem. Endpoint Security: Since IIoT devices frequently have low processing and memory capacities, it might be challenging to immediately install robust security mechanisms. To improve endpoint security in IIoT systems, secure boot procedures, frequent firmware updates, and network-level security implementation are necessary. Supply Chain Risks: Vulnerabilities may arise from the intricate supply chain that is involved in producing IIoT devices as hacked components may find their way into the finished products. Supply Page 3/19 chain risks may be reduced by establishing a secure supply chain with thorough vendor evaluations, keeping an eye out for any threats, and putting safe device manufacturing procedures in place. Human Factors: Human error may have a major influence on IIoT security, including using weak passwords, making setup mistakes, and being unaware of things. Human elements in IIoT security may be addressed by performing frequent security audits, implementing robust authentication procedures, and offering cybersecurity training to staff members. Regulatory Compliance: IIoT technology is constantly changing, making it difficult for laws to follow up, which might result in compliance gaps. Regulatory problems in IIoT security may be addressed by keeping up with and abiding by pertinent industry rules, as well as by proactively modifying security measures to conform with changing requirements. In order to effectively address the security concerns associated with the Internet of Things, industry collaboration, technical solutions, and continuous attempts to stay ahead of emerging threats are all necessary. 1.3 The Role of Blockchain in Enhancing IIIoT Security A new approach to protecting IIIoT ecosystems is available with blockchain technology, which is known for its decentralized and unchangeable character. Blockchain technology reduces vulnerabilities related to centralised points of failure by offering an immutable and publicly accessible ledger. This study delves into the idea of using blockchain technology as a first line of defence to strengthen the security of IIIoT systems, guaranteeing the authenticity and integrity of data all throughout the network. [9] 1.4 Importance of Sustainability in Supply Chain Management This research acknowledges the critical role of sustainability in modern supply chain management, going beyond the security of the Internet of Things. New frameworks are required to meet the growing demand for environmentally conscious practices and the pressing need to lessen our impact on the planet. In order to promote a balance between efficiency and environmental responsibility, this article primarily focuses on how to improve supply chain operations with a modified Lean Six Sigma approach. [10] 2. Review of literature Khurshid, A. Alsaaidi, R. (2022) several electronic components, including software, actuators, and sensors, make up the Internet of Things (IoT). A distributed system of embedded, heterogeneous "things" that can exchange data digitally and interact with each other is shown by it. Industrial Internet of Things (IIoT) devices, which include sensors and need processing power, could be useful for agricultural monitoring in a variety of settings, including woods, farms, and industries. There is a plethora of IIoT applications; some examples include smart grids, smart parking, and smart healthcare. In 2013, the International Data Corporation released a report predicting that by 2020, the IIoT market will have grown by 8.9 trillion dollars, with 41 billion devices connected to the network. Page 4/19 Sood, K. Karmakar, K.K. (2020)Due to the fact that IIoT devices bring in a new era of disruption, they pose serious privacy and security risks to consumers to varying degrees. This situation occurs because IIoT devices have a lot of potential. These devices not only track users' daily activities and whereabouts (e.g., when they went on vacation, where they stayed, what they ate, etc.), but they also attack and collect personal information (e.g., names, addresses, born date, phone numbers, etc.). Users should exercise caution while storing large quantities of personally identifiable information in databases linked to open or public cloud platforms, considering the spate of high-profile data breaches that have occurred recently [11]. Kornaros, G. (2022) investigates the world of industrial internet of things (IIoT) networks and investigates how using blockchain technology might improve security and privacy [12]. The paper presents a solution to protect sensitive data and secure communications in IIoT contexts by utilising the decentralised and tamper-resistant properties of blockchain. This work's original contribution is the approach it takes to addressing issues brought about by privacy breaches and security holes in IIoT systems addresses the need for an improved security measure for IIoT networks with the implementation of a secure and lightweight protocol. Rathee, G. Ahmad, F. (2023) Internet of Things (IoT) is Rotocol's primary focus. The study lays forth a strategy to safeguard data integrity and avoid unauthorised access by combining encryption technology with efficient communication methods. Finding a happy medium between security and resource efficiency is the primary focus of this essay as it addresses the limitations of IIoT devices [13]. Since these limitations are discussed in this work, it holds great significance. Vangala, A.; Das, (2023) suggests a secure method that integrates machine learning with blockchain technology [14]. The paper proposes a way to detect and counteract security risks in real-time by utilising the immutability of blockchain technology and the adaptive capabilities of machine learning. This study makes a significant contribution by enhancing the resilience of IIoT systems against growing cyber threats through its novel combination of blockchain and machine learning. Zhang, X.; Zhong, H (2022) In its most basic form, the Internet of Things (IoT) aims to connect everyday objects with people and other networks of interconnected smart devices [15]. Recent advances in novel Internet techniques, wireless communication technology, and affordable tiny sensors have piqued a great deal of interest in this area. Medical and healthcare, environmental monitoring, logistics, smart buildings and residences, and smart transportation systems are just a few of the many fields that have used Internet of Things approaches. An important feature of IIoT systems is their ability to take context into account. As with other areas of sensory data collection, geographical information is crucial. Iqbal, W.; Abbas, H (2021) A number of methods for encrypting and decrypting data are employed to ensure data security and privacy [16]. On the other hand, traditional technologies often make do with inadequate resources, which limit their processing speeds and power. Devices in the Internet of Things scenario, which often have low resources, are not suitable for using these technologies. Cryptographic algorithms that are both small and powerful enough to meet the needs of these devices have been Page 5/19 designed to address this issue. Block cyphers have shown to be the most effective of the various cypher blocks. Breitenbacher, D. Homoliak, I. (2022) secure two-way communication is made possible by the authentication of user credentials. As supplementary data, authentication systems are required for oneway hash functions to perform pre-shared, random key pre-distribution, and existing asymmetric functions. Among the many possible authentication methods, symmetric and asymmetric cyphers include biological features and identity-based authentication. Stability, naturalness, non-repudiation, tamper-resistance, and other desirable biological traits can be attained by the use of these exceptional binding qualities. Biometric authentication methods, such as those based on a person's face, iris, retina, DNA, or fingerprints, are all the rage in the security certification industry. Authentication functions based on hashes or message authentication codes are part of function-based encryption [17]. Cui, J. Wang, F. (2021) The PUF technique is the foundation of several fuzzy extraction algorithm-based schemes. Furthermore, the designs for future IIoT systems' architectures are explained via PUF-based mutual authentication techniques. New security and other issues have emerged as a result of the IIoT's fast expansion in recent years, which has resulted in a large increase in the number of linked devices. To ensure that only authorized devices are used, there exist systems that use radio-frequency identification (RFID). These technologies are essential for the readers and tags to function, and they also work well for short-range wireless radio communication. Tags allow readers to get access to their identity by retrieving their credentials from memory [18]. An impediment to the usual, complex ways is the scheme's operation, which is operated by secure cryptographic activities done by the user's device. Xenofontos, C.; Zografopoulos, I (2022) In PUF, biological features are also used in authentication techniques. These can achieve the benefits of microstructure-based electronic components, which are resistant to damage and have the characteristics of randomization and uniqueness. The only time its stimulus-response behaviour changes—and every PUF goes into incentive response mode—is when an attacker tries to break into a PUF device. The PUF's challenge response features allow for mutual authentication between entities, which solves these difficulties [19]. An additional perk is that the PUF system may be made more unpredictable and random by using the same challenge answer. Data encryption and decryption keys can also be generated using such approaches [20] [21]. A physical oneway function was the initial form of PUF. 3. Statement of the problem Consistent data transfer across various networks and smart devices is critical in the Industrial Internet of Things (IIoT). The administration of keys, encryption and decryption of data, aggregation of data, and authentication are just a few of the many obstacles that crop up while attempting to secure digital and physical assets. Power consumption, computing speed, and storage space are all examples of intrinsic resource constraints. Data encryption and decryption are given new approaches by this study. Data authentication using one-time accessible keys is the novel premise around which these techniques are Page 6/19 built. The strategy described in this discourse outperforms current approaches and guarantees data integrity while bestowing communication cost-effectiveness. The results show how great and novel the proposed method is, which has a new way of encrypting and decrypting data called the "Quondam Signature Algorithm (QSA)." With this function, IIoT apps and real devices may conduct secure transactions. The main objective of this technique is to decrease vulnerability to man-in-the-middle attacks. 4. Significance of the study Internet of Things (IoT) security measures are top priority throughout rollout. One of the most important things is keeping users and systems safe from cybercrime and assaults. Threat actors are getting more creative in their attempts to have user data compromised as IIoT technology develops. There must be an immediate solution to the problem of weak authentication procedures in network security that can be used by both little local networks and massive cloud servers. Defending against sophisticated cyberattacks is becoming more important as the IIoT continues its meteoric rise. There must be a strong emphasis on originality, sophistication, and ease of use in the final authentication tool. Since IIoT devices are dispersed across vast distances and users often leave them unattended, there are times when attackers manage to physically access these devices. The transmission of data also becomes an easy target for hackers, especially in wireless connection situations when there are open areas where data assaults may be launched. To develop novel authentication protocols with low communication overhead, PUF methods serve as a springboard. Given this, the current research proposes a novel approach to within IIoT device data access and user authentication. Due to the high frequency of cyberattacks, safeguarding systems linked to the Internet of Things is crucial. A new authentication procedure that works for both small and big networks is offered as an answer to these problems. The goal of this study is to find innovative and affordable ways to authenticate users and grant them access to data in IIoT systems by utilising strategies like the Physical Unclonable Function (PUF). 5. Proposed Methodology A new encryption/decryption technique is introduced in this study to help IIoT systems withstand assaults. 5.1 Proposed Method To prevent man-in-the-middle (MiM) attacks, our proposed technique incorporates a signature formation mechanism with a unique, one-time usability component. The purpose of developing this component was to protect against assaults like these. The term used to describe this method is "Quondam Signature Algorithm," or "QSA" for short. To begin authenticating devices, a connection request must first be made. Our system generates the one-time usable signature using the current displayed date and time. A timestamp is generated by combining the system date with the corresponding time. The next step in the encryption process is to multiply the timestamp vector by a substitution window (S). The new container Page 7/19 follows the basic structure of a 12x12 diagonal matrix. You may get this 12-by-12-dimensional Quondam Matrix (QM) by multiplying vectors using the substitution method. Corners of the Quondam Matrix that are diagonally opposite one another form the Quondam Signature (QS). The Quondam Signature (QS) may be enhanced to include a Media Access Control (MAC) address or other physical identification, allowing clients to verify their devices before sending them to the server. Making device authentication easier is the main objective here. Only authorised users' personal computers will have an algorithm and a "Substitution Box" to choose from. Integral to the experimental setup are real-time clocks (RTCs), desktop computers (PCs), and nodeMCUs. This configuration has the NodeMCU acting as the server and the PC as the client. A precise record of the current time and date may be obtained via the system's real-time clock. Visual representation of the suggested system in motion is shown in Fig. 1. 5.2 Proposed Algorithm There are two main components to the operation's methodology: first, selecting the format, and second, creating the signature. Format setup using Algorithm 1 is as follows. Page 8/19 Algorithm 1: Quondam Signature Algorithm (QSA) for Federated Systems Step 1: Start Step 2: Gather data from the client on the Time Stamp (TS), Client Identity (CI), and Client Connection Request (CR). Step 3: Divide Client Identity (CI) into two parts: - MAC ADD: To get the MAC address, use the first eight digits of the CI. - Eight-Digit Hexadecimal: MAC addresses should be converted to eight-digit hexadecimal values (each digit equals four bits). Step 4:Set up the settings for federated learning: - Explain the global model. - Indicate the number of devices (N) that are involved. - Establish communication rounds (R). Step 5: For every cycle of communication r in [1, R]: For every gadget i that takes part in [1, N]: - To device I, send the global model. - Device I uses the global model it got and its own data to update the local model. - Device I uses federated learning to train its local model. - Device I uses the modified local model to calculate a local Quondam Signature (LQS). Step 6:Combine Local Quondam Signatures in One Batch: - Gather every device that is a participant's local Quondam Signature (LQS). - Once the LQS have been aggregated, calculate the global Quondam Signature (GQS). Step 7:Execute the Device Authentication process: - GQS should be appended to the client's connection request (CR). - Send the server a CR along with GQS to authenticate the device. Step 8: End Federal learning, PUFs, and the Quondam Signature Algorithm (QSA) are the three components that make up the proposed method to deal with IoT issues that arise in real time. Security and efficiency are ensured for time-sensitive IIoT applications using this strategy. An essential part of the method is federated learning, which is designed to handle data in IIoT situations in real-time. When devices work together to train ML models, federated learning takes place without a central repository for raw data. On the contrary, Page 9/19 devices may access and exchange model updates, enabling real-time local computations and adjustments. The Internet of Industrialised Things (IIoT) allows for constant learning and adaptation without overwhelming a central server with data by improving real-time responsiveness and lowering latency. 5.3 Algorithm for Signature Generation Step 1: For retrieving the system's date and time in the formats (D [] and (T []), respectively. Step 2: Formulate the time stamp vector TS [] as TS [] D [] + T [], where D is the current date and T is the current system time. Step 3: Multiply the substitution matrix S [] by the time stamp vector TS [] to complete the process. Step 4: The replacement matrix S must be pre-installed for authorised users.. Step 5: A Quondam matrix, denoted as QM, is produced. Step 6: Compute the value of QM12 × 12 [ ]. Step 7: Extract the diagonal elements from QM12 × 12 [ ] to construct a 12-character-long QS [ ]. Step 8: Combine QS [ ] with CI [ ] and send the resulting data to the server as M20 × 1, where M20 × 1 = [M1 M2 M3…M20]. Step 9: The client transmits the message M20 × 1 to the server. Step 10: Decryption is complete. Step 11: The server segregates QS [ ] and CI [ ] from M20 × 1 as follows: M20 × 1 = QS [ ] 12 × 1 + CI [ ] 8 × 1. Step 12: Formulate the Quondam signature as DM [ ] 12 × 12. Step 13: Derive TS [ ] using the equation: TS [ ] = QM [ ] × S − 1. Step 14: The process of decryption is complete. 5.4 Federated Learning with PUF and QSA Modern information systems may benefit from this study's innovative and safe architecture, which incorporates federated learning, the Quondam Signature Algorithm, and Physical Unclonable Functions in a novel way. Utilising the physical variances in electrical components to provide device-specific solutions, this integration is based on the basic principle gained from PUF. These keys are the foundation of improved security measures since they are almost hard to copy. Numerous benefits are encapsulated by the intentional incorporation of PUFs into the proposed system. First and foremost, they play a crucial Page 10/19 part in improving the device authentication procedure. An extra safeguard is built into the communication channel by using keys obtained from PUFs during the connection formation phase. The inherent uniqueness of PUF answers guarantees that every device has its own identity, which improves system integrity and reduces the danger of unauthorised access. Within the complex dance that is the Quondam Signature Algorithm (QSA), PUFs play a crucial role. The QSA's authentication, decryption, and data encryption processes rely on these PUF-derived keys as their foundation. These keys are one-time accessible. By including PUFs, the QSA's resilience is greatly enhanced, leading to a dynamic synergy that strengthens the system's security. 6. Results The real-time IIoT network was important in the successful validation of the Quondam Signature technique. In order to prevent unauthorised users from gaining access, this system efficiently distinguishes between legitimate and malicious devices. The implementation procedure also involved calculating potential communication costs. Our proposed approach requires between 192 and 220 bits for successful transmission. In order to determine the effectiveness of the Quondam Signature Algorithm, a carefully planned experimental setup was used within a real-time IIoT network. A wide variety of IIoT devices, each with its own specific function within the network, were included into the design. By properly linking IIoT sensors, actuators, gateways, and intermediate nodes, a preset network design facilitated interactions akin to real-world IIoT situations. Modifying the data rates and frequencies would cause the MQTT protocol to act as event-triggered communication and real-time data delivery. To guarantee it could faithfully represent a diverse and realistic network, the experimental setup's IIoT architecture was thoroughly examined. A total of fifty IIoT devices, such as sensors, actuators, and gateways, were thoughtfully distributed over the network. This heterogeneous ensemble has the potential to mimic complex interactions inside the IIoT ecosystem. Key creation, signature, and verification are only a few examples of the computationally and energy intensive processes that may be measured in Table 1. Potentially used in the offline signature approach are the Inversion Operation and the Pairing Operation. By using inverted values, the IO hides the Oracle outputs. Po, also known as bilinear mapping, is an effective method for creating sophisticated cryptographic protocols. Scalar multiplication applied to the elliptical curve further reveals the brief fingerprints mostly generated by the PO. In the offline method, scalars (SM) are multiplied by the hash function (H) that G1 generates. Taken together, SM is exactly an elliptical curve at point Q multiplied by a finite field. Multiplying it by its scalar value is the best way to characterize a point. The value of the intermediate result may be increased by adding any rational point Q to a certain elliptical curve. An SMbased binary validation technique is Elliptic Curve Cryptography (ECC). The execution of a point-doubling operation is indicated by a value of '0' for outcomes reported as bits. When the message's secret is compromised, the attacker is able to distinguish between the point-doubling and point-adding operations; a bit value of '1' indicates that point addition with doubling is executed. Page 11/19 Table 1 Comparison between the key size and the evaluation cost Methods [22] [23] [24] [25] [26] [27] Proposed Scheme Online evaluation 3A + M 2A + M 2A + M 2A + M A + 3M 3M 2M Offline evaluation 3MP + 1M M 3MP + 1 MM 3MP + 1 MM 3MP + 1 MM 3MP + 1 MM 3MP + 1 MM 3MP + 1 MM Ciphertext length 2144 6464 2144 4320 3424 2144 1280 Offline storage 2624 5056 2624 5056 2624 3632 1312 No. of pairing for decryption 8 7 5 5 4 4 2 Table 2 provides a summary of the energy-efficient performance metrics achieved by the proposed technique during execution, which is below one bit joule. The signature methods add some temporal complexity to the process of verifying the validity and authenticity of messages exchanged between nodes. Table 2 Computing expenses for different plans Scheme Parameter Generation Signing Verification (SM) (H) (PO) (SM) (IO) (H) (PO) (SM) (IO) (H) HMAC [1] 1 0 0 1 1 1 1 0 1 1 AES [28] 1 0 0 1 0 0 0 2 0 1 DES [29] 1 0 2 1 1 1 0 4 1 1 IDEA [30] 1 0 0 1 1 1 1 1 0 1 MPKG [3] 1 1 0 1 1 1 1 1 0 1 Diffe Hellman [2] 1 0 2 1 1 1 0 2 0 1 Proposed Scheme 1 1 0 0 1 1 1 1 1 1 The proposed strategy, however, substantially simplifies the temporal aspects. Furthermore, as the key size increases, ECC's performance benefits over RSA become more apparent, especially with regard to execution time and energy consumption. Table 3 displays the outcomes of comparing the suggested system to previous designs of sensor networks. Page 12/19 Table 3 Duration of various schemes' execution Scheme Time Taken (Seconds) HMAC 0.0987741 AES 0.1091002 DES 0.0970710 IDEA 0.1121050 MPKG 0.0456012 Diffe Hellman 0.0570011 Proposed Scheme 0.0342860 Table 3 shows the results of evaluating and comparing the temporal complexity of different systems. The time complexity of the suggested technique is significantly lower than that of the other schemes since it generates the signature during the online phase and does complicated computations during the offline phase. The suggested method differs from others that use an online-based elliptic curve digital signature technique by using an offline signature methodology to reduce the time required for key creation, signing, and verification. So far, the SBM method has shown the slowest performance of the systems we've covered. At almost 25 rounds, 1500 signatures generated, and 1500 verifications, the aforementioned numbers show how lengthy the procedure is. Reducing computational cost and signature size during signature production is a major advantage of identity-based online/offline techniques. To ensure that the user's public key and signature are of the same size, the proposed approach checks them. An ID-based online/offline method produces a 160-bit key that is 20 bytes long. 7. Discussion The experiment included a wide variety of real-world scenarios that pertain to IIoT settings. The configuration of devices was done with great care, and the hardware requirements included things like memory, processing power, and other parts. A Signature Algorithm for Quondam, with its complex linkages between signature production and verification, was meticulously distributed to these devices using federated learning techniques and Physical Unclonable Functions (PUFs) as necessary. Skillfully implementing access control techniques was a programming priority since authentication procedures are crucial for differentiating between legitimate and malicious devices. We were able to properly establish a vital quantity called communication cost by collecting and evaluating communication data. The suggested method was already successful in real-time IIoT settings, and its communication cost range of 192–220 bits only proved it. Page 13/19 In order to measure how well the Quondam Signature Algorithm worked, it was evaluated using a number of criteria. This collection of metrics includes things like authentication accuracy, false positives/negatives, and transmission overhead. Results showed that the approach improved IIoT network security via real-time device authentication. The algorithm was able to handle real-time demands while strengthening IIoT security, according to rigorous assessment, careful implementation, and a wellplanned testing environment. As a crucial part of their communication features, the devices' data rates and frequencies were fine-tuned to imitate the dynamics of the real IIoT. The data transmission intervals for the sensor devices were configured at 5 s intervals to allow for frequent updates. Actuators began event-triggered communication in reaction to thresholds that were predefined, with real-time responsiveness being the primary emphasis. Because IIoT devices often have minimal resources, the communication was carried out utilising the famedly lightweight and publish-subscribe MQTT protocol. This sophisticated IIoT architecture accommodates a broad range of devices and has communication characteristics tuned for real-time interactions. It has also developed a robust experimental framework. We made sure the suggested Quondam Signature Algorithm could handle the rigorous and constantly changing conditions of real IIoT installations by simulating their sophisticated configuration. One of the main applications of the PO, sometimes called bilinear mapping, is the building of complicated cryptographic protocols. The brief signatures mostly generated by the PO are further revealed by applying scalar multiplication to the elliptical curve. The offline method involves multiplying the hash function (H) that is created by G1 with scalars (SM). An first step in solving for SM is to multiply a point Q on an elliptical curve across a limited field. When depicting a point, it is more realistic to multiply its scalar value by itself. We choose a point Q from a collection of rational points on an elliptical curve and add the intermediate result to it such that it becomes more valuable. To verify binary data using the SM, one may use Elliptic Curve Cryptography (ECC). Zero will be the value of the bits-expressed outcomes after a point-doubling operation. One bit value of '1' indicates that point addition with doubling is done when the message's secret is compromised, while another bit value of '0' indicates that point addition is used. The results show that the algorithms work well to lower communication costs, as measured in bits transferred, and that there is potential for further progress in this area. The study highlights the significance of the results by contrasting and comparing different approaches to communication cost. Other common attacks against IIoT systems might be included into the suggested method. To satisfy the demands of IIoT devices and mobile phones, it offers an encrypted command protocol with configurable parameters that guarantee a high level of security. 8. Conclusion Combining a Modified Lean Six Sigma methodology with an Eco-Friendly Federated Learning AI-Driven Framework that includes the Quondam Signature Algorithm and Efficient Encryption offers a strong Page 14/19 defence for Blockchain IIIoT security. With its environmentally friendly characteristics, this comprehensive approach not only improves data protection but also advances sustainability. By combining cutting-edge technology with an emphasis on environmental responsibility and using Lean Six Sigma concepts, supply chain management is made more efficient and sustainable while maintaining a strong security paradigm. 8.1 Scope for further research This study offers cutting-edge data encryption and decryption techniques to solve the problem of random assaults on IIoT systems. By generating device signatures with the help of the once-recoverable Quondam Signature Algorithm (QSA), the proposed techniques reduce the likelihood of man-in-the-middle attacks. The results demonstrate that the algorithms substantially reduced communication costs, as assessed in bits transmitted, and they also reveal that there is space for more optimization. By looking at several ways to figure out gearbox cost, the study highlights how practical the results are. We may address additional typical risks to IIoT systems by extending the given strategy. Its safe command protocol has customizable settings and guarantees a high level of security, which can be enough for IIoT devices and smartphones. The use of shorter device signatures for authentication is one area that might be investigated in future studies. Compressed physical identities, including signatures from central processing units (CPUs), coprocessors, memory units (MUs), and others, may be analysed using this technology. It is also possible to do more research into the compression of generated timestamps in order to achieve a far more substantial reduction in transmission costs. Declarations Conflicts of Interest The author declares no conflict of interest Funding Statement This research received no external funding. Author Contribution Author A: Developed the research idea, designed the methodology, Conducted data collection, Contributed to the conceptualization, performed data analysis, and contributed to the writing of the manuscript.Author B: Reviewed and edited the manuscript, supervised the project and created visualizations.Author C: supervised the project administration, and provided critical feedback during the Page 15/19 writing and editing process.Each author's specific contributions can vary based on their expertise, role, and involvement in different aspects of the research project. References 1. Khurshid, A., Alsaaidi, R., Aslam, M., & Raza, S. (2022). EU Cybersecurity Act and IIoT Certification: Landscape, Perspective and a Proposed Template Scheme. IEEE Access, 10, 129932–129948. 2. Sood, K., Karmakar, K. K., Yu, S., Varadharajan, V., Pokhrel, S. R., & Xiang, Y. (2020). Alleviating Heterogeneity in SDN-IIoT Networks to Maintain QoS and Enhance Security. 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Figures Figure 1 Application flow for socket programming Page 18/19 Figure 2 Execution time of different schemes Page 19/19
https://openalex.org/W2923107797	https://www.frontiersin.org/articles/10.3389/fphar.2019.00352/pdf	English	null	Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury	Frontiers in pharmacology	2,019	cc-by	8,459	Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury Carmela Belardo1†, Monica Iannotta1†, Serena Boccella1†, Rosamaria Cristina Rubino2, Flavia Ricciardi1, Rosmara Infantino1, Gorizio Pieretti3, Luigi Stella4, Salvatore Paino1, Ida Marabese1, Rosa Maisto1, Livio Luongo1, Sabatino Maione1* and Francesca Guida1* Carmela Belardo1†, Monica Iannotta1†, Serena Boccella1†, Rosamaria Cristina Rubino2, Flavia Ricciardi1, Rosmara Infantino1, Gorizio Pieretti3, Luigi Stella4, Salvatore Paino1, Ida Marabese1, Rosa Maisto1, Livio Luongo1, Sabatino Maione1* and Francesca Guida1* 1 Department of Experimental Medicine, University of Campania Luigi Vanvitelli, Naples, Italy, 2 Enecta s.r.l., Bologna, Italy, 3 Department of Plastic Surgery, University of Campania Luigi Vanvitelli, Naples, Italy, 4 Drug Addiction Unit (SerT), Naples, Italy Edited by: Marialessandra Contino, University of Bari Aldo Moro, Italy Edited by: Marialessandra Contino, University of Bari Aldo Moro, Italy Neurological dysfunctions are the most impactful and persistent consequences of traumatic brain injury (TBI). Indeed, previous reports suggest that an association between TBI and chronic pain syndromes, as well anxio-depressive behaviors, tends to be more common in patients with mild forms of TBI. At present, no effective treatment options are available for these symptoms. In the present study, we used a weight drop mild TBI mouse model to investigate the effect of a commercially available 10% Cannabidiol (CBD) oil on both the sensorial and neuropsychiatric dysfunctions associated with mild TBI through behavioral and biomolecular approaches. TBI mice developed chronic pain associated with anxious and aggressive behavior, followed by a late depressive-like behavior and impaired social interaction. Such behaviors were related with speciﬁc changes in neurotransmitters release at cortical levels. CBD oral treatment restored the behavioral alterations and partially normalized the cortical biochemical changes. In conclusion, our data show some of the brain modiﬁcations probably responsible for the behavioral phenotype associated with TBI and suggest the CBD as a pharmacological tool to improve neurological dysfunctions caused by the trauma. Reviewed by: Faramarz Dehghani, Martin Luther University of Halle-Wittenberg, Germany Joseph T. McCabe, Uniformed Services University of the Health Sciences, United States Correspondence: Sabatino Maione sabatino.maione@unicampania.it Francesca Guida franc.guida@gmail.com †These authors have contributed equally to this work Reviewed by: Faramarz Dehghani, Martin Luther University of Halle-Wittenberg, Germany Joseph T. McCabe, Uniformed Services University of the Health Sciences, United States Correspondence: Sabatino Maione sabatino.maione@unicampania.it Francesca Guida franc.guida@gmail.com †These authors have contributed equally to this work Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 19 December 2018 Accepted: 21 March 2019 Published: 16 April 2019 Keywords: cannabidiol, traumatic brain injury, pain, behavior, microdialysis ORIGINAL RESEARCH published: 16 April 2019 doi: 10.3389/fphar.2019.00352 Citation: Belardo C, Iannotta M, Boccella S, Rubino RC, Ricciardi F, Infantino R, Pieretti G, Stella L, Paino S, Marabese I, Maisto R, Luongo L, Maione S and Guida F (2019) Oral Cannabidiol Prevents Allodynia and Neurological Dysfunctions in a Mouse Model of Mild Traumatic Brain Injury. Front. Pharmacol. 10:352. doi: 10.3389/fphar.2019.00352 The phytocannabinoid cannabidiol (CBD), the major non-psychoactive constituent of Cannabis sativa, exhibits a broad spectrum of potential therapeutic properties, including neuroprotective eﬀects in Central Nervous System (CNS) disorders (Fernández-Ruiz et al., 2013; De Gregorio et al., 2018; Schonhofen et al., 2018). Through a multitarget mechanism, CBD shows potent anti-inﬂammatory and anti-oxidant properties which have been previously demonstrated in diﬀerent models of neurodegenerative diseases and in acute episodes of brain damage (i.e., hypoxia-ischemia) (Hayakawa et al., 2007, 2010; Castillo et al., 2010). CBD has very low aﬃnity for cannabinoid receptors type 1 (CB1) and type 2 (CB2), whereas diﬀerent mechanisms, such as inhibition of anandamide uptake and enzymatic hydrolysis (Lastres-Becker et al., 2005), and April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 1 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. decrease of adenosine reuptake (Carrier et al., 2006), are believed to be responsible for its neuroprotective eﬀects. not ﬁxed. After a midline longitudinal incision, the skull was exposed to locate the area of impact and placed under a metal tube device where the opening was positioned directly over the animal’s head. The injury was induced by dropping a cylindrical metal weight (50 g), through a vertical metal guide tube from a height of 20 cm. The point of impact was between the anterior coronal suture (Bregma) and posterior coronal suture (Lambda). Immediately following injury, the skin was closed with surgical wound clips and mice were placed back in their cages to allow for recovery from the anesthesia and mTBI. Sham mice were submitted to the same procedure as described for mTBI, but without release of the weight. Sham and mTBI animals did not receive analgesic drugs after surgery. No animals have been excluded from the study. p p Traumatic brain injury (TBI) is a complex injury with a number of symptoms accompanied by inﬂammatory process and cell death (Arciniegas, 2011; Liu et al., 2019). Experimental Design Time points of evaluations were based on our previous study (Guida et al., 2017a). A total number of 80 mice were divided in four experimental groups: Sham/vehicle, mTBI/vehicle, Sham/CBD and mTBI/CBD. Behavioral tasks were performed at diﬀerent time points and scheduled in order to avoid carry-over eﬀects from prior testing experience. The number of animals for each experiment is represented in Table 1. Tactile Allodynia y Tactile allodynia was evaluated at a series of calibrated nylon von Frey monoﬁlaments (Semmes-Weinstein monoﬁlaments, 2 Biological Instruments, Italy). Mice were allowed 7, 14 and 21 days after mTBI or sham surgery by to move freely in the compartment of the enclosure positioned on the metal mesh surface. Mice were adapted to the testing environment for 30 min before any measurement was taken. The monoﬁlaments, starting from the 0.008 g monoﬁlament, was applied perpendicularly to the plantar surface of each hind- paw in a series of ascending forces (0.008, 0.02, 0.04, 0.07, 0.16, 0.40, 0.60, 1.0, 1.4, 2.0, and 4.0 g). Each stimulus was applied for approximately 1 s with an interstimulus interval of 5 s. Withdrawal responses evoked by each monoﬁlament was obtained from ﬁve consecutive trials. Data (gr) were expressed as a mean of right and left response hind paws. Drugs Cannabidiol and vehicle were kindly provided by Enecta Group, Bologna (BO), Italy https://www.enecta.eu/?lang CBD was dissolved in hemp seed oil and natural tocopherols, used as vehicle. CBD (30 µl, oil 10%) was administered via gavage from day 1 to day 14 and from day 50 to day 60. Those time points represent the pathological windows in which we previously observed the main features of the mTBI, such as aggressiveness, recklessness and/or sensorial changes in the ﬁrst phase, and the depressive-like behavior in a late phase (Guida et al., 2017a). Animals Male C57BL/6 mice (Charles River, Italy) weighing 18–20 g were housed three per cage under controlled illumination (12 h light/dark cycle; light on 6:00 A.M.) and standard environmental conditions (ambient temperature 20– 22◦C, humidity 55–60%) for at least 1 week before the commencement of experiments. Mice chow and tap water were available ad libitum. The experimental procedures were approved by the Animal Ethics Committee of University of Campania “L. Vanvitelli,” Naples. Animal care was in compliance with Italian (D.L. 116/92) and European Commission (O.J. of E.C. L358/1 18/12/86) regulations on the protection of laboratory animals. All eﬀorts were made to reduce both animal numbers and suﬀering during the experiments. Following behavioral testing, mice were scariﬁed for microdialysis experiments at 14 or 60 day after mTBI induction. The timeline of mTBI induction, treatments and behavioral and biochemical characterization is given in the Figure 1. Observers were blind to the treatments in each experiment. Citation: It is characterized by an initial neuroinﬂammation, mediated by a rapid glia cells activation, peripheral immune cells recruitment and secretion of inﬂammatory cytokines, followed by the late appearance of psychologically debilitating symptoms and cognitive impairments (Woodcock and Morganti-Kossmann, 2013). Despite recent advances in the knowledge of TBI pathophysiology, no adequate pharmacotherapies are currently available (Loane and Faden, 2010). It is assumed that the secondary neuropsychiatric changes that occur as a consequence of trauma are associated with plastic rearrangements at hippocampal and cortical circuitry (Schwarzbold et al., 2008). In these brain regions endocannabinoid (EC) molecules induce pro-homeostatic and neuroprotective eﬀects, by aﬀecting neuroplasticity in cognitive and aﬀective processes (Vigano et al., 2009; Boccella et al., 2019). A growing body of evidence suggests that the pharmacological manipulation of EC attenuates neuroinﬂammation and improve the recovery of neurobehavioral function during the early weeks after TBI (Shohami et al., 2011; Mayeux et al., 2017; Schurman and Lichtman, 2017). To our knowledge, no study has evaluated the eﬀects of CBD on the neurological dysfunctions associated with the TBI. In particular, we coupled behavioral tasks and biochemical evaluations to assess the CBD eﬀects on long-term cognitive and emotional responses induced by trauma. Frontiers in Pharmacology \| www.frontiersin.org Mild TBI Induction Groups Days 0 7 13–15 21 34 59–61 Sham/vehicle N = 20 N = 12 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) Sham/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBl/vehicle N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBI/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 11 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive.) N = 13 (Depression) N = 5 (Pain) N = 6 (Rotarod) FIGURE 1 \| Timeline of mTBI induction, treatments and behavioral and biochemical characterization. FIGURE 1 \| Timeline of mTBI induction, treatments and behavioral and biochemical characterization. of permanence of the mouse on the cylinder was expressed as latency time (s). Voluntary movement, associated with the locomotion, was not counted as a withdrawal response. Tactile allodynia was deﬁned as a signiﬁcant decrease in the withdrawal threshold to the von Frey hair application. Each mouse served as its own control, the responses being measured both before and after surgical procedures. Open Field Test Motor activity was also evaluated by open ﬁeld test in sham and mTBI mice. Brieﬂy, each mouse was individually monitored for 5 min in an open arena (l × w × h: 25 cm × 25 cm divided into 16 square grids). Parameters evaluated included: (1) number of transitions; and (2) number of rearings; and (3) time spent in the periphery or center (s). Rotarod Test Possible motor coordination impairment was evaluated at 7, 21, and 60 days after mTBI or sham surgery by Rotarod test (Ugo Basile). Mice was measured for the time (s) of equilibrium before falling on a rotary cylinder by a magnet that, activated from the fall of the mouse on the plate, allows to record the time of permanence on the cylinder. After a period of adaptation of 30 s, the spin speed gradually increases from 5 to 40 rpm for a maximum time of 5 min. The animals were analyzed by two separate tests at 1-hour interval in the same day. The experiment was performed for every group of animals the day before the surgical procedure and the days before the behavioral tests in order to avoid stress. The time Mild TBI Induction Experimental mTBI was performed using a weight-drop device developed in our laboratory. Mice were anesthetized with intraperitoneal injection of Tribromoethanol (250 mg/kg) and placed in a prone position on a spongy support. The head was April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 2 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. TABLE 1 \| Numbers of animals used in each experiment. Groups Days 0 7 13–15 21 34 59–61 Sham/vehicle N = 20 N = 12 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) Sham/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 5 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBl/vehicle N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 10 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive) N = 12 (Depression) mTBI/CBD N = 20 N = 13 (Pain) N = 10 (Rotarod) N = 11 (Pain) N = 10 (Rotarod) N = 6 (Anxiety) N = 10 (Aggressive) N = 9 (Depression) N = 5 (Pain) N = 6 (Rotarod) N = 5 (Pain) N = 5 (Rotarod) N = 3 (Sociability) N = 5 (Aggressive.) N = 13 (Depression) FIGURE 1 \| Timeline of mTBI induction, treatments and behavioral and biochemical characterization. Voluntary movement, associated with the locomotion, was not counted as a withdrawal response. Tactile allodynia was deﬁned of permanence of the mouse on the cylinder was expressed as latency time (s). TABLE 1 \| Numbers of animals used in each experiment. TABLE 1 \| Numbers of animals used in each experiment. Frontiers in Pharmacology \| www.frontiersin.org Three Chambers Sociability Microdialysis experiments were performed in awake and freely moving mice. In brief, mice were anesthetized with pentobarbital (50 mg/kg, i.p.) and stereotaxically implanted with concentric microdialysis probes into the mPFC using the coordinates: AP: 1.4–1.8 mm, L: 0.3–05 mm from bregma and V: 3.0 mm below the dura (Paxinos and Franklin, 2004). Dialysis probes, were constructed with 25G (0.3 mm inner diameter, 0.5 mm outer diameter) stainless steel tubing (A-M Systems). Inlet and outlet cannulae (0.04 mm inner diameter, 0.14 mm outer diameter) consisted of fused silica tubing (Scientiﬁc Glass Engineering). The probe had a tubular dialysis membrane (Enka AG, Wuppertal, Germany) 1.3 mm in length. Following a recovery period of 24 h, dialysis was commenced with aCSF (NaCl 147 mM, CaCl2 2.2, KCl 4 mM; pH 7.2) perfused at a rate of 1 µL/min by a Harvard Apparatus infusion pump. The neurotransmitters release was evaluated after chronic treatment performed with CBD or Vehicle in m-TBI or sham animals. Data were expressed as the average of six repeated measurements (each 30 min) to give a more accurate value. No appreciable diﬀerences were observed between the diﬀerent six dyslisate samples collected during the single experiment. At the end of experiments, mice were anesthetized and their brains perfused ﬁxed via the left cardiac ventricle with heparinized paraformaldehyde saline (4%). Brains were dissected out and ﬁxed in a 10% formaldehyde solution for 2 days and included in OCT compound. The brain was cut in 40-µm thick slices and observed under a light microscope to identify Test at 60 days after mTBI or sham surgery, mice were tested for social interaction using a three-chambered social interaction apparatus. A plexi-glass three-chambered box was custom-built as follows: doorways in the two dividing walls had sliding covers to control access to the outer-side chambers. The test consisted of two consecutive stages of 5 and 10 min each. During the 5-minute ﬁrst stage of habituation the mouse was allowed to freely explore the three chambers of the apparatus, detecting at this stage any innate side preference. After that the mouse was gently encouraged into the central chamber and conﬁned there brieﬂy by closing the side chamber doors. During the following 10-minute stage sessions, a custom made stainless-steel barred cup (6.5 cm × 15 cm) was placed upside down in one of the side chambers. Three Chambers Sociability A never before-met intruder, previously habituated, was placed into an upside-down identical cup in the other chamber. The time spent sniﬃng each upside-down cup, the time spent in each chamber and the number of entries into each chamber were recorded. Resident-Intruder At 14 and 60 days after mTBI or sham surgery, mice were tested for aggressive behavior using a resident intruder test. Mice were individually housed for 1 week in Plexiglas cages to establish a home territory and to increase the aggression of the resident experimental mice. To begin, food containers were removed and an intruder mouse of the same gender was placed in a resident home cage and resident–intruder interactions were analyzed for 10 min. The April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 3 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. of the 6-minute test by a time recorder. Immobility time was deﬁned as the absence of escape-oriented behavior. Mice were considered to be immobile when they did not show any body movement, hung passively and completely motionless. aggressive behavior of resident socially-isolated mice was characterized by an initial pattern of exploratory activity around the intruder, which was followed by rearing and tail rattle, accompanied in a few seconds by wrestling and/or a violent biting attack. The number of these attacks and latency to the ﬁrst attack during the 10 min observation period was recorded. CBD Effects on Motor Coordination and Anxiety in mTBI Mice the probe locations (Figure 2). The concentrations of D- Aspartate, L-glutamate and GABA contained in the dialysate were analyzed using by HPLC coupled with ﬂuorimetric detection method. The system comprised two Gilson pumps (model no. 303), a C-18 reverse-phase column, and a Gilson ﬂuorimetric detector (model no. 121). Dialysates were pre-column derivatized with o-phthaldialdehyde- N-acetylcysteine (OPA-NAC) (10 µl dialysate + 5 µl OPA-NAC + 10 µl borate buﬀer 10%). The mobile phase consisted of two components: (A) 0.2 M Na2HPO4, 0.2 M citric acid and 20% methanol and (B) 90% acetonitrile. Gradient composition was determined using an Apple microcomputer installed with Gilson gradient management software. Mobile phase ﬂow rate was maintained at 1.2 ml/min. Data were collected using a Dell Corporation PC system 310 interfaced to the detector via a Drew data-collection unit. Data were expressed as mean ± SEM of six samples for each mouse. the probe locations (Figure 2). The concentrations of D- Aspartate, L-glutamate and GABA contained in the dialysate were analyzed using by HPLC coupled with ﬂuorimetric detection method. The system comprised two Gilson pumps (model no. 303), a C-18 reverse-phase column, and a Gilson ﬂuorimetric detector (model no. 121). Dialysates were pre-column derivatized with o-phthaldialdehyde- N-acetylcysteine (OPA-NAC) (10 µl dialysate + 5 µl OPA-NAC + 10 µl borate buﬀer 10%). The mobile phase consisted of two components: (A) 0.2 M Na2HPO4, 0.2 M citric acid and 20% methanol and (B) 90% acetonitrile. Gradient composition was determined using an Apple microcomputer installed with Gilson gradient management software. Mobile phase ﬂow rate was maintained at 1.2 ml/min. Data were collected using a Dell Corporation PC system 310 interfaced to the detector via a Drew data-collection unit. Data were expressed as mean ± SEM of six samples for each mouse. y In the rotarod test, no diﬀerence in riding time was observed between any of the treatment groups (Figure 3B), indicating no impairments in motor coordination. Statistical Analysis Data were represented as mean ± SEM. Behavioral data were analyzed by using one-way ANOVA, followed by Bonferroni’s multiple comparison. Newman–Keuls post hoc test was used as post hoc test in microdialysis analysis. P values < 0.05 were considered statistically signiﬁcant. Statistical analysis was carried out using Prism/Graphpad (GraphPad Software, Inc.,) software. Numbers of animals used in each experiment is given in Table 1. Tail Suspension Test (TST) The Depression like behavior was evaluated at 14 days and 60 days after mTBI or sham surgery, mice were individually suspended by the tail on a horizontal bar (55 cm from ﬂoor) using adhesive tape placed approximately 4 cm from the tip of the tail. The duration of immobility, recorded in seconds, was monitored during the last 4 min FIGURE 2 \| Microdialysis probe location. (A) Shows a panoramic picture of the pre-frontal cortex, the star indicates the prelimbic area. (B) Shows a high magniﬁcation of the microdialysis probe location for amino acid collection within the pre/infra-limbic cortex. FIGURE 2 \| Microdialysis probe location. (A) Shows a panoramic picture of the pre-frontal cortex, the star indicates the prelimbic area. (B) Shows a high magniﬁcation of the microdialysis probe location for amino acid collection within the pre/infra-limbic cortex. April 2019 \| Volume 10 \| Article 352 4 Frontiers in Pharmacology \| www.frontiersin.org Cannabidiol Effects in Traumatic Brain Injury Belardo et al. RESULTS CBD Effects on Allodynia in mTBI Mice A signiﬁcant decrease of TWT was observed in vehicle- treated mTBI mice at 7, 14, and 21 days after trauma induction [0.14 g ± 0.025, F(3,47) = 11.06, P < 0.0001; 0.11 g ± 0.04, F(3,37) = 12.40, P < 0.0001; 0.11 g ± 0.04, F(3,16) = 8.833, P = 0.0011, at 7, 14, and 21 days, respectively] compared to the sham group (0.63 g ± 0.12, 0.61 g ± 0.09, 0.62 g ± 0.1, at 7, 14, and 21 days, respectively) (Figure 3A). Moreover, a physiological re-establishment of normal pain response was observed 34 days after trauma induction [Sham/vehicle 0.62 g ± 0.06; Sham/CBD 0.65 g ± 0.1; mTBI/vehicle 0.64 g ± 0.22; mTBI/CBD 0.62 g ± 0.07 F(3,16) = 0.01345, P = 0.9978] (Figure 3A). No diﬀerence in pain threshold was observed between right and left paw (see Supplementary Table S1). Oral CBD treatment signiﬁcantly reduced the tactile allodynia in mTBI mice at 14 and 21 days (0.28 g ± 0.04; 0.41 g ± 0.04; 0.46 ± 0.02, at 7, 14, and 21 days, respectively) as compared with vehicle (0.14 g ± 0.025; 0.11 g ± 0.04;0.11 g ± 0.04, at 7, 14, and 21 days, respectively) (Figure 3A). The CBD administration in sham mice did not change the pain response (0.71 g ± 0.17; 0.51g ± 0.07; 0.41g ± 0.09; 0.65 ± 0.1 at 7, 14 and 21, and 34 days, respectively) compared to sham/vehicle mice (0.63 g ± 0.12; 0.61 g ± 0.09; 0.62 g ± 0.1; 062 ± 0.06, at 7, 14, 21, and 34 days, respectively). CBD Effects on Motor Coordination and Anxiety in mTBI Mice In the open ﬁeld test, used to assay general locomotor activity levels, but also anxiety, one-way ANOVA, followed by Bonferroni post hoc test, revealed no signiﬁcant changes in the time spent in the center [sham/vehicle 73.33 s ± 15.63; sham/CBD 56.83 s ± 5.31; mTBI/ vehicle 53.80 s ± 19.51; mTBI/CBD 46.67 s ± 10.05, F(3,19) = 0.7663, P = 0.5269) or in the periphery [sham/vehicle 241.7 s ± 9.86; sham/CBD 243.2 s ± 5.31; mTBI/ vehicle 268.8 s ± 12.89; mTBI/CBD 263.3 s ± 11.12, F(3,19) = 1.896, P = 0.1646] or for the number of transitions [sham/vehicle 204.5 ± 17.50; sham/CBD 239.8 ± 10.41; mTBI/ vehicle 226.8 ± 13.62; mTBI/CBD 238.8 ± 14.06, F(3,20) = 1.354, P = 0.2854] after trauma or any treatment (Figures 3C,E,F). However, mTBI/vehicle [52.5 ± 2.38; F(3,20) = 24.00, P < 0.0001] mice showed an increase in the number of rearing as compared to sham/vehicle (12.50 ± 3.17) and this eﬀect was signiﬁcantly reduced by CBD treatment (33.33 ± 4.10). CBD did not change the number of rearing in sham animals (25.33 ± 3.77) (Figure 3D). CBD Effects on Aggressive Behavior in mTBI Mice No diﬀerence in the latency to the ﬁrst attack in all groups of mice at 14 and 60 days after brain injury was observed [sham/vehicle 470.3 s ± 38.56; sham/CBD 424.6 s ± 71.80; mTBI/ vehicle 463.4 s ± 37.26; mTBI/CBD 424.5 s ± 34.10, F(3,36) = 0.2630, P = 0.8516; sham/vehicle 227.4 s ± 48.53; sham/CBD 200.4 s ± 53.56; mTBI/ vehicle 186.4 s ± 53.51; mTBI/CBD 226.0 s ± 45.33, F(3,16) = 0.1588, P = 0.9225] (Figures 4A,C). However, 14 days after the trauma, mTBI mice showed an increased number of attacks [20.20 ± 2.99, F(3,36) = 5.353, P = 0.0037], as compared to the controls (10.60 ± 1.36) (Figure 4B). CBD treatment signiﬁcantly reduced this eﬀect (9.9 ± 1.84) as compared with vehicle (20.20 ± 2.99). At 60 days after trauma, no signiﬁcant change was observed in the number of attacks [sham/vehicle 0.6 ± 0.4; sham/CBD 0.8 ± 0.37; mTBI/ vehicle 0.8 ± 0.37; mTBI/CBD 0.4 ± 0.24, F(3,16) = 0.2933, P = 0.8296]. Sham mice treated with CBD did not show any change in the latency to the ﬁrst attack or number of attacks compared to sham/vehicle mice (Figures 4C,D). CBD Effects on Depressive-Like Behavior in mTBI Mice mTBI mice showed an increased immobility time, measured as the lack of escape-oriented activity (169.4 s ± 5.93) compared to the sham mice (132.4 s ± 4.15) 60 days post trauma (Figure 5A). CBD treatment signiﬁcantly reduced the immobility in mTBI condition (125.1 s ± 8.95) compared to the vehicle (169.4 s ± 5.93) (Figure 5A). Sham mice treated with CBD did not show any change in the duration of immobility compared to vehicle-treated April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 5 Belardo et al. Cannabidiol Effects in Traumatic Brain Injury FIGURE 3 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows Tactile withdrawal thresholds (TWT) measured through Von Frey monoﬁlaments, (B) shows the latency to fall in the rotarod test, (C–F) show the number of transitions, number of rearing, the time spent in the periphery or in the center, in the open ﬁeld test, respectively. Data are represented as mean ± SEM of 10–11 mice per group. ∗, # and ◦indicate signiﬁcant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc tests. Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 3 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows Tactile withdrawal thresholds (TWT) measured through Von Frey monoﬁlaments, (B) shows the latency to fall in the rotarod test, (C–F) show the number of transitions, number of rearing, the time spent in the periphery or in the t i th ﬁld t t ti l D t t d ± SEM f 10 11 i ∗ # d ◦i di t i iﬁ t diff d t FIGURE 3 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows Tactile withdrawal thresholds (TWT) measured through Von Frey monoﬁlaments, (B) shows the latency to fall in the rotarod test, (C–F) show the number of transitions, number of rearing, the time spent in the periphery or in the center, in the open ﬁeld test, respectively. Data are represented as mean ± SEM of 10–11 mice per group. ∗, # and ◦indicate signiﬁcant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc tests. CBD Effects on Depressive-Like Behavior in mTBI Mice mice [110.4 s ± 10.53, F(3,45) = 13.64, P < 0.0001] (Figure 5A). At 14 days mTBI mice did not shown any change in the immobility time as compared with sham animals (Supplementary Figure S1). In the three chambers sociability test, no diﬀerence in the time spent in each chamber or in the number of transitions was observed in mTBI and Sham mice treated with vehicle or CBD (Supplementary Table S2). However, mTBI mice had reduced sociability level, spending a higher time in interacting with the object during the recorded time [27.33 s ± 4.1; F(3,8) = 11.40, P < 0.0029], compared to sham animals (4.67 s ± 0.88) (Figure 5B, session I). This eﬀect was signiﬁcantly improved in mTBI CBD-treated animals (5.0 s ± 1.0). Moreover, mTBI mice [interaction CBD Effects on Social Behavior in mTBI Mice At 60 days post mTBI, while GABA and D-Asp dialysate were not changed [GABA: sham/vehicle 2.21 pmol/µl ± 0.01; sham/CBD 3.65 pmol/µl ± 1.08; mTBI/vehicle 1.51 pmol/µl ± 0.36; mTBI/CBD 2.11 pmol/µl ± 0.36, F(3,7) = 2.025 P = 0.1990; D-Asp sham/vehicle 1.08 pmol/µl ± 0.53; sham/CBD 0.61 pmol/µl ± 0.18; mTBI/vehicle 0.47 pmol/µl ± 0.10; mTBI/CBD 0.29 pmol/µl ± 0.06, F(3,7) = 2.176 P = 0.1789] (Figures 6D,F), Glu levels were still high, but CBD did not revert this eﬀect [Glu: sham/vehicle 10.81 pmol/µl ± 5.33; mTBI/vehicle 47.29 pmol/µl ± 11.14; mTBI/CBD 73.62 pmol/µl ± 4.80, F(3,5) = 15.26 P = 0.0060] (Figure 6E). Finally, we found that CBD increased per sè Glu levels in the mPFC of sham mice at both 14- and 60-days post trauma (sham/CBD: 17.12 pmol/µl ± 1.40; 47.02 pmol/µl ± 6.09, 14 and 60 days, respectively), as compared with vehicle (sham/Vehicle: 7.12 pmol/µl ± 0.26; 10.81 pmol/µl ± 5.33, 14 and 60 days, respectively) (Figures 6B,E). (Figures 6A,B). On the contrary, GABA levels were decreased by TBI, and CBD signiﬁcantly reverted this eﬀect (sham/vehicle 3.25 pmol/µl ± 0.6; sham/CBD 2.62 pmol/µl ± 0.31; mTBI/vehicle 0.191 pmol/µl ± 0.01; mTBI/CBD 1.38 pmol/µl ± 0.13, F(3,12) = 15.74 P = 0.0002) (Figure 6C). At 60 days post mTBI, while GABA and D-Asp dialysate were not changed [GABA: sham/vehicle 2.21 pmol/µl ± 0.01; sham/CBD 3.65 pmol/µl ± 1.08; mTBI/vehicle 1.51 pmol/µl ± 0.36; mTBI/CBD 2.11 pmol/µl ± 0.36, F(3,7) = 2.025 P = 0.1990; D-Asp sham/vehicle 1.08 pmol/µl ± 0.53; sham/CBD 0.61 pmol/µl ± 0.18; mTBI/vehicle 0.47 pmol/µl ± 0.10; mTBI/CBD 0.29 pmol/µl ± 0.06, F(3,7) = 2.176 P = 0.1789] (Figures 6D,F), Glu levels were still high, but CBD did not revert this eﬀect [Glu: sham/vehicle 10.81 pmol/µl ± 5.33; mTBI/vehicle 47.29 pmol/µl ± 11.14; mTBI/CBD 73.62 pmol/µl ± 4.80, F(3,5) = 15.26 P = 0.0060] (Figure 6E). Finally, we found that CBD increased per sè Glu levels in the mPFC of sham mice at both 14- and 60-days post trauma (sham/CBD: 17.12 pmol/µl ± 1.40; 47.02 pmol/µl ± 6.09, 14 and 60 days, respectively), as compared with vehicle (sham/Vehicle: 7.12 pmol/µl ± 0.26; 10.81 pmol/µl ± 5.33, 14 and 60 days, respectively) (Figures 6B,E). CBD Effects on Social Behavior in mTBI Mice with mouse II: 24.0 s ± 11.55 F(3,8) = 1.308, P = 0.3373] did not show signiﬁcantly altered preference for social novelty compared with control mice (interaction with mouse II: 40.67 s ± 11.05) (Figure 5C, session II). The CBD treatment did not induce any change in sociability, in the time spent in the two chambers or in the number of transitions between the chambers compared to vehicle in sham or mTBI animals (Sham/CBD and mTBI/CBD: interaction with mouse II: 39.67 s ± 7.86 and 48.67 ± 2.91) (Figures 5A,B). CBD Effects on Social Behavior in mTBI Mice Analysis of the social preference revealed an impairment of social interaction which occurred 60 days post trauma. April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 6 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 4 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A-D) show the latency to the ﬁrst attack and the number of attacks in the resident intruder test, respectively, at 14- and 60-days post mTBI. Data are represented as mean ± SEM of 10 mice per group. ∗and ◦indicate signiﬁcant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Tukey post hoc test. FIGURE 4 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A-D) show the latency to the ﬁrst attack and the number of attacks in the resident intruder test, respectively, at 14- and 60-days post mTBI. Data are represented as mean ± SEM of 10 mice per group. ∗and ◦indicate signiﬁcant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Tukey post hoc test. FIGURE 4 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A-D) show the latency to the ﬁrst attack and the number of attacks in the resident intruder test, respectively, at 14- and 60-days post mTBI. Data are represented as mean ± SEM of 10 mice per group. ∗and ◦indicate signiﬁcant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Tukey post hoc test. (Figures 6A,B). On the contrary, GABA levels were decreased by TBI, and CBD signiﬁcantly reverted this eﬀect (sham/vehicle 3.25 pmol/µl ± 0.6; sham/CBD 2.62 pmol/µl ± 0.31; mTBI/vehicle 0.191 pmol/µl ± 0.01; mTBI/CBD 1.38 pmol/µl ± 0.13, F(3,12) = 15.74 P = 0.0002) (Figure 6C). CBD Effects on Neurotransmitters Release mTBI Mice In vivo microdialysis was used to assess the amino acids contents in the m-PFC of mTBI mice. HPLC analysis revealed a notable increase of extracellular glutamate (Glu) and D-Aspartate (D-Asp) levels in the mPFC of 14 days mTBI animals [Glu: 32.05 pmol/µl ± 1.33 F(3,12) = 123.1, P < 0.0001; D-Asp: 2.29 pmol/µl ± 0.38; F(3,8) = 7.922, P = 0.0088], as compared with controls (Glu: 7.12 pmol/µl ± 0.26; D-Asp: 0.93 pmol/µl ± 0.42). Remarkably, CBD treatment normalized both Glu and D-Asp levels (Glu: 9.43 pmol/µl ± 0.55; D-Asp: 0.28 pmol/µl ± 0.06) April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 7 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 5 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows the duration of immobility in the tail suspension test. (B,C) Show the duration of the time spent with an object or mouse in the three chambers sociability apparatus. Data are expressed in seconds and represented as mean ± SEM of 10–12 mice per group. ∗and ◦indicate signiﬁcant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc test. FIGURE 5 \| Effect of CBD on behavioral evaluations in sham and mTBI mice. (A) Shows the duration of immobility in the tail suspension test. (B,C) Show the duration of the time spent with an object or mouse in the three chambers sociability apparatus. Data are expressed in seconds and represented as mean ± SEM of 10–12 mice per group. ∗and ◦indicate signiﬁcant differences compared to sham/vehicle or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One-way ANOVA, followed by Bonferroni’s Multiple Comparison post hoc test. Frontiers in Pharmacology \| www.frontiersin.org DISCUSSION Though brain trauma did not aﬀect the motor coordination or the April 2019 \| Volume 10 \| Article 352 8 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. FIGURE 6 \| Effect of CBD on the release of glutamate (B,E), GABA (C,F) and D-Aspartate (A,D) in sham or mTBI mice at 14 and 60 days after trauma. The values of extracellular amino acids in the mPFC were expressed as pmol in 10 µl of perfusate. Each point represents the mean ± SEM of 3–4 animals per group. ∗, # and ◦ indicate signiﬁcant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One- Way ANOVA, post hoc test Newman-Keuls Multiple Comparison. FIGURE 6 \| Effect of CBD on the release of glutamate (B,E), GABA (C,F) and D-Aspartate (A,D) in sham or mTBI mice at 14 and 60 days after trauma. The values of extracellular amino acids in the mPFC were expressed as pmol in 10 µl of perfusate. Each point represents the mean ± SEM of 3–4 animals per group. ∗, # and ◦ indicate signiﬁcant differences compared to sham/vehicle, sham/CBD 10 or TBI/vehicle, respectively. P < 0.05 was considered statistically signiﬁcant. One- Way ANOVA, post hoc test Newman-Keuls Multiple Comparison. that mTBI may be responsible for hyper-functional glutamate/D- aspartate signaling at the supraspinal level and, possibly, of the trauma-associated negative state (aggressive phenotype), at least at this time point. Indeed, while CBD reduced the depression and the impaired sociability, it was not able to change glutamate levels that were still high 60 days after trauma. This suggests the involvement of other brain areas, including the hippocampus, and/or other neurotransmissions (i.e., serotoninergic) in the altered neuropsychiatric behavioral proﬁle of mTBI mice. Moreover, we demonstrated that CBD treatment also increased glutamate in sham mice, at both 14 and 60 days. Indeed, although it does not alter behavior in selected tasks, we cannot exclude that CBD may play a physiological role in other neuropsychological functions regulated by cortical processing, such as cognition, memory and reward. after trauma, indicating an impaired recognition memory. A positive trend to the increase was given by CBD treatment. TBI strongly aﬀects the cortical neuronal plasticity. DISCUSSION Indeed, we have previously shown that the altered behaviors following mTBI correlated with the biphasic ﬁring activity of the pyramidal neurons in the mPFC, considered a key area regulating chronic pain (Giordano et al., 2011; Luongo et al., 2013) and negative aﬀective states, such as anxiety and depression (Vialou et al., 2014; Apps and Strata, 2015; Guida et al., 2015). Remarkably, microdialysis/HPLC analysis revealed that mTBI (14 days) induced an increase of extracellular glutamate levels in the mPFC, which strengthens, the concept that plastic changes and novel neural remodeling may occur after trauma. Conversely, GABA levels were decreased, possibly as a counterbalance of the glutamate-mediated excitation. Remarkable, CBD normalized both GABA and glutamate levels. These latter data are in line with previous reports showing the protective eﬀects of cannabinoids on the excitoxicity and inﬂammation correlated with glutamatergic system dysregulation in diverse neurodegenerative diseases (Guida et al., 2015, 2017b; Palazzo et al., 2015). In particular, the neuroprotective and antioxidant properties of CBD have been shown in high glutamate induced- toxicity in rat cortical neurons (Hampson et al., 1998). Our data also indicated that the extracellular levels of D-aspartic acid (D-Asp), endogenous NMDA receptor agonist, involved in pain and synaptic plasticity (Guida et al., 2015; D’Aniello et al., 2017) enhanced in mTBI mice. This eﬀect was decreased in CBD- treated mTBI mice. Therefore, collectively these ﬁndings indicate DISCUSSION exploratory activity, we found an increased rearing activity in mTBI mice, which was counteracted by CBD treatment. The signiﬁcance of rearing movements seems to be strongly related to the speciﬁc surrounding environment. Rearing may reﬂect attentive processes underlying the assembling of information in novel situations (Aspide et al., 1998), however, in some circumstances, it may simply represent an escape motivation (Lever et al., 2006). It is possible that the rearing activity in our model may reﬂect a kind of recklessness-like behavior, as previously reported in TBI mice (Guida et al., 2017a) and humans (DSM V). mTBI mice presented a typical phenotype, characterized by an aggressive behavior followed by a depressive-like behavior. Indeed, the aggressiveness of mTBI animals, revealed by an increased number of attacks on intruder mouse, was followed by a depressive-like behavior, manifested as enhanced immobility in the tail suspension test (14 and 60 days after trauma, respectively). The impaired social activity was also observed in the three-chamber sociability task, suggesting a general illness, often reported in patients with TBI. CBD signiﬁcantly prevented all these eﬀects. Interestingly, mTBI mice showed a reduced interest for social novelty, compared with controls. In fact, even if was not signiﬁcant, we found that the time spent with the novel mouse (stranger) was reduced Cognitive and emotional dysfunctions are the most impactful and persistent consequences of TBI. Indeed, motor and sensory deﬁcits and psychiatric disorders may endure for weeks, as a consequence of the traumatic damage to the underlying brain structures. We previously showed that mTBI induced late (up to 60 days) neurological dysfunctions in mice and identiﬁed electrophysiological changes at the cortical level possibly associated with symptomatology (Guida et al., 2017a). In the present study, we demonstrated that the repeated treatment with commercially available 10% CBD oil exerts beneﬁcial eﬀects on the behavioral dysfunctions associated with TBI. Moreover, at the dose tested, CBD does not change the normal attitude, in term of locomotion, nociception or emotional behavior, in not injured animals. As previously shown (Guida et al., 2017a), 2 weeks-mTBI mice displayed abnormal pain response after innocuous stimuli to the paw (mechanical allodynia), probably due to the overall inﬂammatory condition (Feliciano et al., 2014). The daily treatment with CBD signiﬁcantly reduced pain behavior, which, in fact, spontaneously disappeared in 30 days. REFERENCES doi: 10.1016/j.nbd.2009.10.023 Lever, C., Burton, S., and O’Keefe, J. (2006). Rearing on hind legs, environmental novelty, and the hippocampal formation. Rev. Neurosci. 17, 111–134. doi: 10.1515/revneuro.2006.17.1-2.111 D’Aniello, A., Luongo, L., Romano, R., Iannotta, M., Marabese, I., Boccella, S., et al. (2017). d-Aspartic acid ameliorates painful and neuropsychiatric changes and reduces beta-amyloid abeta1-42 peptide in a long lasting model of neuropathic pain. Neurosci. Lett. 651, 151–158. doi: 10.1016/j.neulet.2017.04.041 Liu, J., Xiong, X., and Sui, Y. (2019). 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MMPIP, an mGluR7-selective negative allosteric modulator, alleviates pain and normalizes aﬀective and cognitive behavior in neuropathic mice. Pain 156, 1060–1073. doi: 10.1097/j.pain.0000000000000150 Giordano, C., Cristino, L., Luongo, L., Siniscalco, D., Petrosino, S., Piscitelli, F., et al. (2011). TRPV1-Dependent and -independent alterations in the limbic cortex of neuropathic mice: impact on glial caspases and pain perception. Cereb. Cortex 22, 2495–2518. doi: 10.1093/cercor/bhr328 Paxinos, G., and Franklin, K. B. (2004). The Mouse Brain in Stereotaxic Coordinates. Houston, TX: Gulf professional publishing. REFERENCES Guida, F., Palazzo, E., Luongo, L., Marabese, I., de Novellis, V., Maione, S., et al. (2017b). Supraspinal metabotropic glutamate receptors: an endogenous substrate for alleviating chronic pain and related aﬀective disorders. mGLU Receptors 31, 15–31. Apps, R., and Strata, P. (2015). Neuronal circuits for fear and anxiety-the missing link. Nat. Rev. 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Inhibition of an equilibrative nucleoside transporter by cannabidiol: a mechanism of cannabinoid immunosuppression. Proc. Natl. Acad. Sci. U.S.A. 103, 7895–7900. doi: 10.1073/pnas.0511232103 ochem. 102, 1488–1496. doi: 10.1111/j.1471-4159.2007.04565.x Lastres-Becker, I., Molina-Holgado, F., Ramos, J. A., Mechoulam, R., and Fernández-Ruiz, J. (2005). Cannabinoids provide neuroprotection against 6- hydroxydopamine toxicity in vivo and in vitro: relevance to Parkinson’s disease. Neurobiol. Dis. 19, 96–107. doi: 10.1016/j.nbd.2004.11.009 Castillo, A., Tolón, M., Fernández-Ruiz, J., Romero, J., and Martinez- Orgado, J. (2010). The neuroprotective eﬀect of cannabidiol in an in vitro model of newborn hypoxic–ischemic brain damage in mice is mediated by CB2 and adenosine receptors. Neurobiol. Dis. 37, 434–440. CONCLUSION In conclusion, our data demonstrate that mTBI causes late sensorial aﬀective/cognitive deﬁciencies linked to altered neurotransmitter release at cortical level. Moreover, we showed that chronic CBD treatment reduces behavioral dysfunctions by restoring at least in part cortical biochemical processes. Taken together, our results suggest that CBD could represent a novel approach for the management of neuropsychiatric disorders associated with TBI. In conclusion, our data demonstrate that mTBI causes late sensorial aﬀective/cognitive deﬁciencies linked to altered neurotransmitter release at cortical level. Moreover, we showed that chronic CBD treatment reduces behavioral dysfunctions by restoring at least in part cortical biochemical processes. Taken together, our results suggest that CBD could represent a novel approach for the management of neuropsychiatric disorders associated with TBI. April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 9 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. SUPPLEMENTARY MATERIAL CB, LL, and RR conceived and designed the experiments. CB, MI, SB, FR, RI, and RM performed the experiments. GP, LS, SP, RR, LL, and IM analyzed the data and contributed to materials and analysis tools. CB, FG, and SM wrote the manuscript. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphar. 2019.00352/full#supplementary-material FIGURE S1 \| Effect of CBD on depressive-like behavior in sham and mTBI mice. The duration of immobility is measured in the tail suspension test at 14 days after mTBI induction. TABLE S1 \| Effect of CBD on pain behavior in sham and mTBI mice. Left and right paw tactile withdrawal thresholds (TWT) are measured through Von Frey monoﬁlaments at 7, 14, 21, and 34 days after mTBI induction. ACKNOWLEDGMENTS We thank Enecta Group for providing Cannabidiol. We also thank the Department of Experimental Medicine of Universisty of Campania for publication fees. We thank Enecta Group for providing Cannabidiol. We also thank the Department of Experimental Medicine of Universisty of Campania for publication fees. TABLE S2 \| Effect of CBD on social behavior in sham and mTBI mice. The time spent in each chamber (A) or in the number of transitions (B) in the three chambers sociability apparatus. REFERENCES Guida, F., Boccella, S., Iannotta, M., De Gregorio, D., Giordano, C., Belardo, C., et al. (2017a). Palmitoylethanolamide reduces neuropsychiatric behaviors by restoring cortical electrophysiological activity in a mouse model of mild traumatic brain injury. Front. Pharmacol. 8:95. doi: 10.3389/fphar.2017.00095 Schonhofen, P., Bristot, I. J., Crippa, J. A., Hallak, J. E. C., Zuardi, A. W., Parsons, R. B., et al. (2018). Cannabinoid-based therapies and brain development: potential harmful eﬀect of early modulation of the endocannabinoid system. CNS Drugs 32, 697–712. doi: 10.1007/s40263-018-0550-4 April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 10 Cannabidiol Effects in Traumatic Brain Injury Belardo et al. Schurman, L. D., and Lichtman, A. H. (2017). Endocannabinoids: a promising impact for traumatic brain injury. Front. Pharmacol. 8:69. doi: 10.3389/fphar. 2017.00069 Woodcock, T., and Morganti-Kossmann, C. (2013). The role of markers of inﬂammation in traumatic braininjury. Front. Neurol. 4:18. doi: 10.3389/fneur. 2013.00018 Schwarzbold, M., Diaz, A., Martins, E. T., Ruﬁno, A., Amante, L. N., Thais, M. E., et al. (2008). Psychiatric disorders and traumatic brain injury. Neuropsychiatr. Dis. Treat. 4, 797–816. Conﬂict of Interest Statement: RR is Enecta Group staﬀ. CB is supported by a grant provided by Enecta Group. The experiments were not supported by Enecta. Conﬂict of Interest Statement: RR is Enecta Group staﬀ. CB is supported by a grant provided by Enecta Group. The experiments were not supported by Enecta. Shohami, E., Cohen-Yeshurun, A., Magid, L., Algali, M., and Mechoulam, R. (2011). Endocannabinoids and traumatic brain injury. Br. J. Pharmacol. 163, 1402–1410. doi: 10.1111/j.1476-5381.2011. 01343.x The remaining authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. Vialou, V., Bagot, R. C., Cahill, M. E., Ferguson, D., Robison, A. J., Dietz, D. M., et al. (2014). Prefrontal cortical circuit for depression- and anxiety- related behaviors mediated by cholecystokinin: role of deltafosb. J. Neurosci. 34, 3878–3887. doi: 10.1523/JNEUROSCI.1787-13.2014 Copyright © 2019 Belardo, Iannotta, Boccella, Rubino, Ricciardi, Infantino, Pieretti, Stella, Paino, Marabese, Maisto, Luongo, Maione and Guida. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. Frontiers in Pharmacology \| www.frontiersin.org REFERENCES No use, distribution or reproduction is permitted which does not comply with these terms. Vigano, D., Guidali, C., Petrosino, S., Realini, N., Rubino, T., Di Marzo, V., et al. (2009). Involvement of the endocannabinoid system in phencyclidine-induced cognitive deﬁcits modelling schizophrenia. Int. J. Neuropsychopharmacol. 12, 599–614. doi: 10.1017/S1461145708009371 April 2019 \| Volume 10 \| Article 352 Frontiers in Pharmacology \| www.frontiersin.org 11
https://openalex.org/W3105033282	https://hal.science/hal-01226485/file/espirito_12636.pdf	English	null	A Coinductive Approach to Proof Search	arXiv (Cornell University)	2,013	cc-by	10,227	To cite this version: José Espírito Santo, Ralph Matthes, Luís Pinto. A Coinductive Approach to Proof Search. Fixed Points in Computer Science (FICS 2013), Sep 2013, Turin, Italy. pp. 28-43. hal-01226485 HAL Id: hal-01226485 https://hal.science/hal-01226485v1 Submitted on 9 Nov 2015 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. A Coinductive Approach to Proof Search Ralph Matthes Institut de Recherche en Informatique de Toulouse (IRIT) C.N.R.S. and University of Toulouse France Lu´ıs Pinto Centro de Matem´atica Universidade do Minho Portugal Lu´ıs Pinto Centro de Matem´atica Universidade do Minho Portugal Jos´e Esp´ırito Santo Centro de Matem´atica Universidade do Minho Portugal Jos´e Esp´ırito Santo Centro de Matem´atica Universidade do Minho Portugal We propose to study proof search from a coinductive point of view. In this paper, we consider intuitionistic logic and a focused system based on Herbelin’s LJT for the implicational fragment. We introduce a variant of lambda calculus with potentially inﬁnitely deep terms and a means of expressing alternatives for the description of the “solution spaces” (called B¨ohm forests), which are a representation of all (not necessarily well-founded but still locally well-formed) proofs of a given formula (more generally: of a given sequent). As main result we obtain, for each given formula, the reduction of a coinductive deﬁnition of the solution space to a effective coinductive description in a ﬁnitary term calculus with a formal greatest ﬁxed-point operator. This reduction works in a quite direct manner for the case of Horn formulas. For the general case, the naive extension would not even be true. We need to study “co-contraction” of contexts (contraction bottom-up) for dealing with the varying contexts needed beyond the Horn fragment, and we point out the appropriate ﬁnitary calculus, where ﬁxed-point variables are typed with sequents. Co-contraction enters the interpretation of the formal greatest ﬁxed points - curiously in the semantic interpretation of ﬁxed-point variables and not of the ﬁxed-point operator. Open Archive TOULOUSE Archive Ouverte (OATAO) OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. This is an author-deposited version published in : http://oatao.univ-toulouse.fr/ Eprints ID : 12636 Official URL: http://dx.doi.org/10.4204/EPTCS.126.3 To cite this version : Espírito Santo, José and Matthes, Ralph and Pinto, Luís A Coinductive Approach to Proof Search. (2013) In: Fixed Points in Computer Science (FICS 2013), 1 September 2013 (Turin, Italy). OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible. To cite this version : Espírito Santo, José and Matthes, Ralph and Pinto, Luís A Coinductive Approach to Proof Search. (2013) In: Fixed Points in Computer Science (FICS 2013), 1 September 2013 (Turin, Italy). Any correspondance concerning this service should be sent to the repository administrator: staff-oatao@listes-diff.inp-toulouse.fr c⃝J. Esp´ırito Santo and R. Matthes and L. Pinto This work is licensed under the Creative Commons Attribution License. 1 Introduction Proof theory starts with the observation that a proof is more than just the truth value of a theorem. A valid theorem can have many proofs, and several of them can be interesting. In this paper, we somehow extend this to the limit and study all proofs of a given proposition. Of course, who studies proofs can also study any of them (or count them, if there are only ﬁnitely many possible proofs, or try to enumerate them in the countable case). But we do this study somehow simultaneously: we introduce a language to express the full “solution space” of proof search. And since we focus on the generative aspects of proof search, it would seem awkward to ﬁlter out failed proof attempts from the outset. This does not mean that we pursue impossible paths in the proof search (which would hardly make sense) but that we allow to follow inﬁnite paths. An inﬁnite path does not correspond to a successful proof, but it is a structure of locally correct proof steps. In other words, we use coinductive syntax to model all locally correct proof ﬁgures. This gives rise to a not necessarily wellfounded search tree. However, to keep the technical effort simpler, we have chosen a logic where this tree is ﬁnitely branching, namely the implicational fragment of intuitionistic propositional logic (with proof system given by the cut-free fragment of the system λ by Herbelin [3]). Lambda terms or variants of them (expressions that may have bound variables) are a natural means to express proofs (an observation that is called the Curry-Howard isomorphism) in implicational logic. Proof alternatives (locally, there are only ﬁnitely many of them since our logic has no quantiﬁer that ranges over inﬁnitely many individuals) can be formally represented by a ﬁnite sum of such solution space expressions, and it is natural to consider those sums up to equivalence of the set of the alternatives. Since inﬁnite lambda-terms are involved and since whole solution spaces are being modeled, we call these coinductive terms B¨ohm forests. By their coinductive nature, B¨ohm forests are no proper syntactic objects: they can be deﬁned by all mathematical (meta-theoretic) means and are thus not “concrete”, as would be expected from syntactic elements. 1 Introduction This freedom of deﬁnition will be demonstrated and exploited in the canonical deﬁnition (Deﬁnition 6) of B¨ohm forests as solutions to the task of proving a sequent (a formula A in a given context Γ). In a certain sense, nothing is gained by this representation: although one can calculate on a case-by-case basis the B¨ohm forest for a formula of interest and see that it is described as ﬁxed point of a system of equations (involving auxiliary B¨ohm forests as solutions for the other meta-variables that appear in those equations), an arbitrary B¨ohm forest can only be observed to any ﬁnite depth, without ever knowing whether it is the expansion of a regular cyclic graph structure (the latter being a ﬁnite structure). ) Our main result is that the B¨ohm forests that appear as solution spaces of sequents have such a ﬁnitary nature: more precisely, they can be interpreted as semantics of a ﬁnite term in a variant of lambda calculus with alternatives and formal greatest ﬁxed-points. For the Horn fragment (where nesting of implications to the left is disallowed), this works very smoothly without surprises (Theorem 15). The full implicational case, however, needs some subtleties concerning the ﬁxed-point variables over which the greatest ﬁxed points are formed and about capturing redundancy that comes from the introduction of several hypotheses that suppose the same formula. The interpretation of the ﬁnite expressions in terms of B¨ohm forests needs a special operation that we call co-contraction (contraction bottom-up). However, this operation is already deﬁnable in terms of B¨ohm forests. Without this operation, certain repetitive patterns in the solution spaces due to the presence of negative occurrences of implications could not be identiﬁed. With it, we obtain the ﬁnitary representation (Theorem 24). In the next section, we quickly recapitulate syntax and typing rules of the cut-free fragment of system λ and also carefully describe its restriction to Horn formulas. Section 3 has the deﬁnition of the not necessarily well-founded proofs, corresponding to a coinduc- tive reading of λ (including its typing system). This is system λ co. Elimination alternatives are then added to this system (yielding the B¨ohm forests), which directly allow the deﬁnition of the solution spaces for the proof search for sequents. We give several examples and then show that the deﬁned solution spaces adequately represent all the λ co proofs of a sequent. 1 Introduction In Section 4, we present ﬁrst the ﬁnitary system to capture the Horn fragment and then modify it to get the main result for full implicational logic. The paper closes with discussions on related and future work in Section 5. 2 Background We recall below the cut-free fragment of system λ (a.k.a. LJT), a sequent calculus for intuitionistic implication by Herbelin [3]. Letters p,q,r are used to range over a base set of propositional variables (which we also call atoms). Letters A,B,C are used to range over the set of formulas (= types) built from propositional variables using the implication connective (that we write A ⊃B) that is parenthesized to the right. Often we will use the fact that any implicational formula can be uniquely decomposed as A1 ⊃A2 ⊃... ⊃An ⊃p with n ≥0, also written in vectorial notation as ⃗A ⊃p. For example, if the vector ⃗A is empty the notation means simply p, and if ⃗A = A1,A2, the notation means A1 ⊃(A2 ⊃p). The cut-free expressions of λ are separated into terms and lists, and are given by: (terms) t,u ::= xl \|λxA.t (lists) l ::= ⟨⟩\|u :: l Figure 1: Typing rules of λ Γ\|⟨⟩: p ⊢p LAx Γ ⊢u : A Γ\|l : B ⊢p Γ\|u :: l : A ⊃B ⊢p LIntro Γ,x : A ⊢t : B Γ ⊢λxA.t : A ⊃B RIntro Γ\|l : A ⊢p (y : A) ∈Γ Γ ⊢yl : p App where a countably inﬁnite set of variables ranged over by letters x, y, w, z is assumed. Note that in lambda- abstractions we adopt a domain-full presentation, annotating the bound variable with a formula. The term constructor xl is usually called application. Usually in the meta-level we prefer to write x⟨t1,...,tn⟩ (with n ∈N0) to range over application constructions, and avoid speaking about lists explicitly (where obviously, the notation ⟨t1,...,tn⟩means ⟨⟩if n = 0 and t1 :: l, if ⟨t2,...,tn⟩means l). In the meta-level, when we know n = 0, instead of x⟨t1,...,tn⟩, we simply write the variable x. where a countably inﬁnite set of variables ranged over by letters x, y, w, z is assumed. Note that in lambda- abstractions we adopt a domain-full presentation, annotating the bound variable with a formula. The term constructor xl is usually called application. Usually in the meta-level we prefer to write x⟨t1,...,tn⟩ (with n ∈N0) to range over application constructions, and avoid speaking about lists explicitly (where obviously, the notation ⟨t1,...,tn⟩means ⟨⟩if n = 0 and t1 :: l, if ⟨t2,...,tn⟩means l). In the meta-level, when we know n = 0, instead of x⟨t1,...,tn⟩, we simply write the variable x. 2 Background We will view contexts Γ as ﬁnite lists of declarations x : A, where no variable x occurs twice. The context Γ,x : A is obtained from Γ by adding the declaration x : A, and will only be written if this yields again a valid context, i. e., if x is not declared in Γ. The system has a form of sequent for each class of expressions: Γ ⊢t : A Γ\|l : A ⊢p. Note the restriction to atomic sequents (the RHS formula is an atom) in the case of list sequents. The rules of λ for deriving sequents are in Figure 1. Note that, as list sequents are atomic, the conclusion of the application rule is also atomic. This is not the case in Herbelin’s original system [3], where list sequents can have a non-atomic formula on the RHS. In the variant of cut-free λ we adopted, the only rule available for deriving a term sequent whose RHS is an implication is RIntro. Still, our atomic restriction will not cause loss of completeness of the system for intuitionistic implication. This restriction is typically adopted in systems tailored for proof search, as for example systems of focused proofs. In fact, λ corresponds to a focused backward chaining system where all atoms are asynchronous (see e. g. Liang and Miller [7]). We will need the following properties of λ. We will need the following properties of λ. Lemma 1 (Type uniqueness) 1. Given Γ and t, there is at most one A such that Γ ⊢t : A. 2. Given Γ, l and A, there is at most one p such that Γ\|l : A ⊢p. 2. Given Γ, l and A, there is at most one p such that Γ\|l : A ⊢p. Proof Simultaneous induction on derivability. □ Proof Simultaneous induction on derivability. □ Since the empty list ⟨⟩has no type index, we need to know A in the second statement of the previous lemma. mma 2 (Inversion of typing) In λ: (Horn formulas) H ::= p\| p ⊃H where p ranges over the set of propositional variables. Note that for Horn formulas, in the vectorial notation ⃗H ⊃p, the vector components Hi are necessarily propositional variables, i. e., any Horn formula is of the form ⃗q ⊃p. where p ranges over the set of propositional variables. Note that for Horn formulas, in the vectorial notation ⃗H ⊃p, the vector components Hi are necessarily propositional variables, i. e., any Horn formula is of the form ⃗q ⊃p. The Horn fragment is obtained by restricting sequents as follows: The Horn fragment is obtained by restricting sequents as follows: 1. contexts are restricted to Horn contexts, i. e., contexts where all formulas are Horn formulas; 1. contexts are restricted to Horn contexts, i. e., contexts where all formulas are Horn formulas; 2. term sequents are restricted to atomic sequents, i. e., term sequents are of the form Γ ⊢t : p. As a consequence, the λ-abstraction construction and the rule RIntro, that types it, are no longer needed. The restricted typing rules are presented in Figure 2. Proof By induction on t, with the help of Lemma 2. Lemma 2 (Inversion of typing) In λ: 1. Γ ⊢λxA.t : B iff there exists C s.t. B = A ⊃C and Γ,x : A ⊢t : C; 1. Γ ⊢λxA.t : B iff there exists C s.t. B = A ⊃C and Γ,x : A ⊢t : C; 2. Γ ⊢x⟨t1,...,tk⟩: A iff A = p and there exists ⃗B s.t. x : ⃗B ⊃p ∈Γ and Γ ⊢ti : Bi, for any i. Proof 1. is immediate and 2. follows with the help of the fact that: Γ\|⟨t1,...,tk⟩: B ⊢p iff there exist B1,...,Bk s.t. B = B1 ⊃... ⊃Bk ⊃p and, for any i, Γ ⊢ti : Bi (proved by induction on k). □ Proof 1. is immediate and 2. follows with the help of the fact that: Γ\|⟨t1,...,tk⟩: B ⊢p iff there exist B1,...,Bk s.t. B = B1 ⊃... ⊃Bk ⊃p and, for any i, Γ ⊢ti : Bi (proved by induction on k). □ Figure 2: Typing rules of λ Horn Γ\|⟨⟩: p ⊢p LAx Γ ⊢u : p Γ\|l : H ⊢q Γ\|u :: l : p ⊃H ⊢q LIntro Γ\|l : H ⊢p (y : H) ∈Γ Γ ⊢yl : p App Now we identify the Horn fragment of cut-free λ, that we denote by λ Horn. The class of Horn formulas (also called Horn clauses) is given by the grammar: (Horn formulas) H ::= p\| p ⊃H 3 Coinductive representation of proof search in lambda-bar We want to represent the whole search space for cut-free proofs in λ. This is proﬁtably done with coinductive structures. Of course, we only consider locally correct proofs. Since proof search may fail when inﬁnite branches occur (depth-ﬁrst search could be trapped there), we will consider such inﬁnite proofs as proofs in an extended sense and represent them as well, thus we will introduce expressions that comprise all the possible well-founded and non-wellfounded proofs in cut-free λ. p p p The raw syntax of these possibly non-wellfounded proofs is presented as follows N ::=co λxA.N \|x⟨N1,...,Nk⟩, yielding the (co)terms of system λ co (read coinductively, as indicated by the index co). Note that instead of a formal class of lists l as in the λ-system, we adopt here the more intuitive notation ⟨N1,...,Nk⟩to represent ﬁnite lists. yielding the (co)terms of system λ co (read coinductively, as indicated by the index co). Note that instead of a formal class of lists l as in the λ-system, we adopt here the more intuitive notation ⟨N1,...,Nk⟩to represent ﬁnite lists. Since the raw syntax is interpreted coinductively, also the typing rules have to be interpreted coin- ductively, which is symbolized by the double horizontal line in Figure 3, a notation that we learnt from Nakata, Uustalu and Bezem [9]. (Of course, the formulas/types stay inductive.) As expected, the restric- tion of the typing relation to the ﬁnite λ-terms coincides with the typing relation of the λ system: Lemma 3 For any t ∈λ, Γ ⊢t : A in λ iff Γ ⊢t : A in λ co. Proof By induction on t, with the help of Lemma 2. □ Proof By induction on t, with the help of Lemma 2. □ Γ,x : A ⊢t : B Γ ⊢λxA.t : A ⊃B RIntro RIntro Figure 4: Extra typing rule of λ co Σ w. r. t. λ co Γ ⊢Ei : p,i = 1,...,n Γ ⊢E1 +···+En : p Alts Example 4 Consider ω := λ f p⊃p.λxp.N with N = f⟨N⟩of type p. This inﬁnite term N is also denoted f ∞. It is quite common to describe elements of coinductive syntax by (systems of) ﬁxed point equations. As a notation on the meta-level for unique solutions of ﬁxed-point equations, we will use the binder ν for the solution, writing ν N.M, where N typically occurs in the term M. 1The division into two syntactic categories also forbids the generation of an inﬁnite sum (for which n = 2 would sufﬁce had the categories for N and E been amalgamated). 3 Coinductive representation of proof search in lambda-bar Intuitively, ν N.M is the N s. t. N = M. (The letter ν indicates interpretation in coinductive syntax.) Example 5 ω of Example 4 can be written as λ f p⊃p.λxp.ν N. f⟨N⟩. Γ, f : p ⊃p,x : p ⊢ν N. f⟨N⟩: p is seen coinductively, so we get Γ ⊢ω : (p ⊃p) ⊃p ⊃p. We now come to the representation of whole search spaces. The set of coinductive cut-free λ-terms with ﬁnite numbers of elimination alternatives is denoted by λ co Σ and is given by the following grammar: (co-terms) N ::=co λxA.N \|E1 +···+En (elim. alternatives) E ::=co x⟨N1,...,Nk⟩ (co-terms) N ::=co λxA.N \|E1 +···+En (elim. alternatives) E ::=co x⟨N1,...,Nk⟩ where both n,k ≥0 are arbitrary. Note that summands cannot be lambda-abstractions.1 We will often use ∑ i Ei instead of E1 +··· +En if the dependency of Ei on i is clear, as well as the number of elements. Likewise, we write ⟨Ni⟩i instead of ⟨N1,...,Nk⟩. If n = 0, we write O for E1 +···+En. If n = 1, we write E1 for E1 +···+En (in particular this injects the category of elimination alternatives into the category of co-terms) and do as if + was a binary operation on (co)terms. However, this will always have a unique reading in terms of our raw syntax of λ co Σ . In particular, this reading makes + associative and O its neutral element. co co Co-terms of λ co Σ will also be called B¨ohm forests. Their coinductive typing rules are the ones of λ co, together with the rule given in Figure 4, where the sequents for (co)terms and elimination alternatives are not distinguished notationally. Notice that Γ ⊢O : p for all Γ and p. Notice that Γ ⊢O : p for all Γ and p. Below we consider sequents Γ ⇒A with Γ a context and A an implicational formula (corresponding to term sequents of λ without proof terms – in fact, Γ ⇒A is nothing but the pair consisting of Γ and A, but which is viewed as a problem description: to prove formula A in context Γ). since RIntro is the only way to prove the implication. since RIntro is the only way to prove the implication. In the case of an atom p, for the deﬁnition of S (Γ ⇒p), let yi : Ai be the i-th variable in Γ with Ai of the form ⃗Bi ⊃p. Let ⃗Bi = Bi,1,...,Bi,ki. Deﬁne Ni,j := S (Γ ⇒Bi,j). Then, Ei := yi⟨Ni,j⟩j, and ﬁnally, S (Γ ⇒p) := ∑ i Ei . This is more sloppily written as S (Γ ⇒p) := ∑ y:⃗B⊃p∈Γ y⟨S (Γ ⇒B j)⟩j . In this manner, we can even write the whole deﬁnition in one line: S (Γ ⇒⃗A ⊃p) := λ⃗x : ⃗A. ∑ y:⃗B⊃p∈∆ y⟨S (∆⇒B j)⟩j with ∆:= Γ,⃗x : ⃗A This is a well-formed deﬁnition: for every Γ and A, S (Γ ⇒A) is a B¨ohm forest and as such rather a semantic object. Lemma 7 Given Γ and A, the typing Γ ⊢S (Γ ⇒A) : A holds in λ co Σ . Lemma 7 Given Γ and A, the typing Γ ⊢S (Γ ⇒A) : A holds in λ co Σ . Let us illustrate the function S at work with some examples. Example 8 We consider ﬁrst the formula A = (p ⊃p) ⊃p ⊃p and the empty context. We have: Let us illustrate the function S at work with some examples. p Example 8 We consider ﬁrst the formula A = (p ⊃p) ⊃p ⊃p and the empty context. We have: Example 8 We consider ﬁrst the formula A = (p ⊃p) ⊃p ⊃p and the empty context. We have: S (⇒(p ⊃p) ⊃p ⊃p) = λ f p⊃p.λxp.S ( f : p ⊃p,x : p ⇒p) Now, observe that S ( f : p ⊃p,x : p ⇒p) = f⟨S ( f : p ⊃p,x : p ⇒p)⟩+x. We identify S ( f : p ⊃p,x : p ⇒p) as the solution for N of the equation N = f⟨N⟩+x. Using ν as means to communicate solutions of ﬁxed-point equations on the meta-level as for λ co, we have S (⇒(p ⊃p) ⊃p ⊃p) = λ f p⊃p.λxp.ν N. f⟨N⟩+x By unfolding of the ﬁxpoint and by making a choice at each of the elimination alternatives, we can collect from this coterm as the ﬁnitary solutions of the sequent all the Church numerals (λ f p⊃p.λxp. f n⟨x⟩ with n ∈N0), together with the inﬁnitary solution λ f p⊃p.λxp. 3 Coinductive representation of proof search in lambda-bar Deﬁnition 6 The function S , which takes a sequent Γ ⇒A and produces a B¨ohm forest which is a coinductive representation of the sequent’s solution space, is given corecursively as follows: In the case of an implication, S (Γ ⇒A ⊃B) := λxA.S (Γ,x : A ⇒B) since RIntro is the only way to prove the implication. f ∞, studied before as example for λ co (corresponding to always making the f-choice at the elimination alternatives). Example 9 We consider now an example in the Horn fragment. Let Γ = x : p ⊃q ⊃p,y : q ⊃p ⊃q,z : p (again with p ̸= q). Note that the solution spaces of p and q relative to this sequent are mutually dependent and they give rise to the following system of equations: Np = x⟨Np,Nq⟩+z Nq = y⟨Nq,Np⟩ Figure 5: Membership relations Figure 5: Membership relations mem(M,N) mem(λxA.M,λxA.N) memE(M,Ei) mem(M,E1 +···+En) (for some i) mem(M1,N1) ... mem(Mk,Nk) memE(x⟨M1,...,Mk⟩,x⟨N1,...,Nk⟩) mem(M,N) mem(λxA.M,λxA.N) memE(M,Ei) mem(M,E1 +···+En) (for some i) and so we have and so we have S (Γ ⇒p) = ν Np.x⟨Np,ν Nq.y⟨Nq,Np⟩⟩+z S (Γ ⇒q) = ν Nq.y⟨Nq,ν Np.x⟨Np,Nq⟩+z⟩ Whereas for p we can collect one ﬁnite solution (z), for q we can only collect inﬁnite solutions. Because in the Horn case the recursive calls of the S function are all relative to the same (initial) context, in this fragment the solution space of a sequent can always be expressed as a ﬁnite system of equations (one for each atom occurring in the sequent), see Theorem 15. Example 10 Let us consider one further example where A = ((((p ⊃q) ⊃p) ⊃p) ⊃q) ⊃q (a formula that can be viewed as double negation of Pierce’s law, when q is viewed as absurdity). We have the following (where in sequents we omit formulas on the LHS) N0 = S (⇒A) = λx(((p⊃q)⊃p)⊃p)⊃q.N1 N1 = S (x ⇒q) = x⟨N2⟩ N2 = S ! x ⇒((p ⊃q) ⊃p) ⊃p = λy(p⊃q)⊃p.N3 N3 = S (x,y ⇒p) = y⟨N4⟩ N4 = S (x,y ⇒p ⊃q) = λzp.N5 N5 = S (x,y,z ⇒q) = x⟨N6⟩ N6 = S ! x,y,z ⇒((p ⊃q) ⊃p) ⊃p = λy(p⊃q)⊃p 1 .N7 N7 = S (x,y,z,y1 ⇒p) = y⟨N8⟩+z+y1⟨N8⟩ N8 = S (x,y,z,y1 ⇒p ⊃q) = λzp 1.N9 N9 = S (x,y,z,y1,z1 ⇒q) Now, in N9 observe that y,y1 both have type (p ⊃q) ⊃p and z,z1 both have type p, and we are back at N5 but with the duplicates y1 of y and z1 of z. Later, we will call this duplication phenomenon co-contraction, and we will give a ﬁnitary description of N0 and, more generally, of all S (Γ ⇒A), see Theorem 24. Corollary 13 (Adequacy of the co-inductive representation of proof search in λ) For any t ∈λ, we have mem(t,S (Γ ⇒A)) iff Γ ⊢t : A (where the latter is the inductive typing relation of λ). Proof By the proposition above and Lemma 3. □ since RIntro is the only way to prove the implication. Consider (N,S (Γ ⇒A)) ∈R, with S (Γ ⇒A) = E1 + ··· + En. We must show that, for some i, (N,Ei) ∈RE. From S (Γ ⇒A) = E1 +··· +En, we must have A = p. Now, from Γ ⊢N : p, there must exist (x : B1,...,Bk ⊃p) ∈Γ and N1,...,Nk s. t. N = x⟨N1,...,Nk⟩. By deﬁnition of S (Γ ⇒A), there is i s. t. Ei = x⟨S (Γ ⇒B1),...,S (Γ ⇒Bk)⟩. co “Only if”. By coinduction on the typing relation of λ co. This is conceptually easier than the other direction since ⊢is a single coinductively deﬁned notion. We deﬁne a relation R for which it is sufﬁcient to prove R ⊆⊢: R := {(Γ,N,A) \| mem(N,S (Γ ⇒A))} Proving R ⊆⊢by coinduction amounts to showing that R is backwards closed – with respect to the typing relation of λ co, i. e., we have to show: Proving R ⊆⊢by coinduction amounts to showing that R is backwards closed – with respect to the typing relation of λ co, i. e., we have to show: 1. (Γ,λxA.t,A ⊃B) ∈R implies ((Γ,x : A),t,B) ∈R; 1. (Γ,λxA.t,A ⊃B) ∈R implies ((Γ,x : A),t,B) ∈R; 2. (Γ,x⟨N1,...,Nk⟩, p) ∈R implies the existence of B1,...,Bk s. t. (x : B1,...,Bk ⊃p) ∈Γ and, for all i = 1,...,k, (Γ,Ni,Bi) ∈R. 2. (Γ,x⟨N1,...,Nk⟩, p) ∈R implies the existence of B1,...,Bk s. t. (x : B1,...,Bk ⊃p) ∈Γ and, for all i = 1,...,k, (Γ,Ni,Bi) ∈R. 2. (Γ,x⟨N1,...,Nk⟩, p) ∈R implies the existence of B1,...,Bk s. t. (x : B1,...,Bk ⊃p) ∈Γ and, for all i = 1,...,k, (Γ,Ni,Bi) ∈R. We show the second case (relative to rule LVecIntro). So, we have mem(N,S (Γ ⇒A)) with N = x⟨N1,...,Nk⟩and A = p, and we need to show that, for some (x : B1,...,Bk ⊃p) ∈Γ, we have, for all i, mem(Ni,S (Γ ⇒Bi)). Since A = p, S (Γ ⇒A) = E1 + ··· + En. Hence, the second rule for mem was used to infer mem(N,S (Γ ⇒A)), i. e., there is a j s. t. memE(N,E j). Therefore, E j = x⟨M1,...,Mk⟩ with terms M1, . . . , Mk, and, for all i, mem(Ni,Mi). By the deﬁnition of S (Γ ⇒A), this means that there are formulas B1, . . . , Bk s. t. since RIntro is the only way to prove the implication. (x : B1,...,Bk ⊃p) ∈Γ and, for all i, Mi = S (Γ ⇒Bi). □ Example 12 Let us consider the case of Pierce’s law that is not valid intuitionistically. We have (for p ̸= q): p p ̸= q): S (⇒((p ⊃q) ⊃p) ⊃p) = λx(p⊃q)⊃p.x⟨λyp.O⟩ The fact that we arrived at O and found no elimination alternatives on the way annihilates the co-term and implies there are no terms in the solution space of ⇒((p ⊃q) ⊃p) ⊃p (hence no proofs, not even inﬁnite ones). Corollary 13 (Adequacy of the co-inductive representation of proof search in λ) For any t ∈λ, we have mem(t,S (Γ ⇒A)) iff Γ ⊢t : A (where the latter is the inductive typing relation of λ). since RIntro is the only way to prove the implication. Of course, by taking the middle alternative in N7, we obtain a ﬁnite proof, showing that A is provable in λ. We now deﬁne a membership semantics for co-terms and elimination alternatives of λ co Σ in terms of sets of (co)terms in λ co. co co co co The membership relations mem(M,N) and memE(M,E) are contained in λ co ×λ co Σ and λ co ×Eλ co Σ respectively (where Eλ co Σ stands for the set of elimination alternatives of λ co Σ ) and are given coinductively by the rules in Fig. 5. Proposition 11 For any N ∈λ co, mem(N,S (Γ ⇒A)) iff Γ ⊢N : A in λ co. Proposition 11 For any N ∈λ co, mem(N,S (Γ ⇒A)) iff Γ ⊢N : A in λ co. Proof “If”. Consider the relations R := {(N,S (Γ ⇒A)) \| Γ ⊢N : A} RE := {(x⟨Ni⟩i,x⟨S (Γ ⇒Bi)⟩i) \| (x : B1,...,Bk ⊃p) ∈Γ∧Γ ⊢x⟨N1,...,Nk⟩: p} It sufﬁces to show that R ⊆mem, but this cannot be proven alone since mem and memE are deﬁned si- multaneously. We also prove RE ⊆memE, and to prove both by coinduction on the membership relations, it sufﬁces to show that the relations R, RE are backwards closed, i. e.: It sufﬁces to show that R ⊆mem, but this cannot be proven alone since mem and memE are deﬁned si- multaneously. We also prove RE ⊆memE, and to prove both by coinduction on the membership relations, it sufﬁces to show that the relations R, RE are backwards closed, i. e.: 1. (λxA.M,λxA.N) ∈R implies (M,N) ∈R; A.M,λxA.N) ∈R implies (M,N) ∈R; 2. (M,E1 +···+En) ∈R implies for some i, (M,Ei) ∈RE; 2. (M,E1 +···+En) ∈R implies for some i, (M,Ei) ∈RE; 3. (x⟨M1,...,Mk⟩,x⟨N1,...,Nk⟩) ∈RE implies for all i, (Mi,Ni) ∈R 3. (x⟨M1,...,Mk⟩,x⟨N1,...,Nk⟩) ∈RE implies for all i, (Mi,Ni) ∈R We illustrate one case. Consider (N,S (Γ ⇒A)) ∈R, with S (Γ ⇒A) = E1 + ··· + En. We must show that, for some i, (N,Ei) ∈RE. From S (Γ ⇒A) = E1 +··· +En, we must have A = p. Now, from Γ ⊢N : p, there must exist (x : B1,...,Bk ⊃p) ∈Γ and N1,...,Nk s. t. N = x⟨N1,...,Nk⟩. By deﬁnition of S (Γ ⇒A), there is i s. t. Ei = x⟨S (Γ ⇒B1),...,S (Γ ⇒Bk)⟩. co We illustrate one case. 4 Finitary representation of proof search in lambda-bar In the ﬁrst section we deﬁne a calculus of ﬁnitary representations. In the third section we obtain our main result (Theorem 24): given Γ ⇒C, there is a ﬁnitary representation of S (Γ ⇒C) in the ﬁnitary calculus. To make the proof easier to understand, we ﬁrst develop in the second section the particular case of the Horn fragment. where the notation ξ ∪[X 7→N] stands for the environment obtained from ξ by setting X to N where the notation ξ ∪[X 7→N] stands for the environment obtained from ξ by setting X to N. Remark that the recursive deﬁnition above has an embedded corecursive case (pertaining to the gfp- operator). Its deﬁnition is well-formed since every elimination alternative starts with a head/application variable and the occurrences of N are thus guarded. When a ﬁnitary term N has no free occurrences of ﬁxpoint variables, all environments determine the same coterm, and in this case we simply write [[N]] to denote that coterm. 4.2 Equivalence of the representations: Horn case Theorem 15 (Equivalence for the Horn fragment) Let Γ be a Horn context. Then, for any atom r, there exists Nr ∈λ gfp Σ with no free occurrences of ﬁxpoint variables such that [[Nr]] = S (Γ ⇒r). 4.1 The ﬁnitary calculus The set of inductive cut-free λ-terms with ﬁnite numbers of elimination alternatives, and a ﬁxpoint operator is denoted by λ gfp Σ and is given by the following grammar (read inductively): (terms) N ::= λxA.N \|gfpX.E1 +···+En\|X (elim. alternatives) E ::= x⟨N1,...,Nk⟩ (terms) N ::= λxA.N \|gfpX.E1 +···+En\|X (elim. alternatives) E ::= x⟨N1,...,Nk⟩ where X is assumed to range over a countably inﬁnite set of ﬁxpoint variables (letters Y, Z will also be used to range over ﬁxpoint variables that may also be thought of as meta-variables), and where both f where X is assumed to range over a countably inﬁnite set of ﬁxpoint variables (letters Y, Z will also be used to range over ﬁxpoint variables that may also be thought of as meta-variables), and where both f n,k ≥0 are arbitrary. Below, when we refer to ﬁnitary terms we have in mind the terms of λ gfp Σ . The ﬁxed-point operator is called gfp (“greatest ﬁxed point”) to indicate that its semantics is (now) deﬁned in terms of inﬁnitary syntax, but there, ﬁxed points are unique. Hence, the reader may just read this as “the ﬁxed point”. f We now give a straightforward interpretation of the formal ﬁxed points (built with gfp) of λ gfp Σ in terms of the coinductive syntax of λ co Σ (using the ν operation on the meta-level). Deﬁnition 14 We call environment a function from the set of ﬁxpoint variables into the set of (co)terms of λ co Σ . The interpretation of a ﬁnitary term (relative to an environment) is a (co)term of λ co Σ given via a family of functions [[−]]ξ : λ gfp Σ →λ co Σ indexed by environments, which is recursively deﬁned as follows: [[X]]ξ = ξ(X) [[λxA.N]]ξ = λxA.[[N]]ξ [[gfpX.∑ i Ei]]ξ = ν N.∑ i [[Ei]]ξ∪[X7→N] [[x⟨N1,...,Nk⟩]]ξ = x⟨[[N1]]ξ,...,[[Nk]]ξ⟩ ξ ξ [[gfpX.∑ i Ei]]ξ = ν N.∑ i [[Ei]]ξ∪[X7→N] where the notation ξ ∪[X 7→N] stands for the environment obtained from ξ by setting X to N. Proof S (Γ ⇒p) = ∑ (y:−→r ⊃p)∈Γ y⟨[[Nrj(−−→ X : q,Xp : p)]]ξ∪[Xp7→S (Γ⇒p)]⟩j (3) S (Γ ⇒p) = ∑ (y:−→r ⊃p)∈Γ y⟨S (Γ ⇒rj)⟩j (4) ∑ (y:−→r ⊃p)∈Γ y⟨[[Nrj(−−→ X : q,Xp : p)]]ξ∪[Xp7→S (Γ⇒p)]⟩j (3) ∑ (y:−→r ⊃p)∈Γ y⟨[[Nrj(−−→ X : q,Xp : p)]]ξ∪[Xp7→S (Γ⇒p)]⟩j (3) ∑ (y:−→r ⊃p)∈Γ y⟨S (Γ ⇒rj)⟩j (4) ∑ (y:−→r ⊃p)∈Γ y⟨[[Nrj(−−→ X : q,Xp : p)]]ξ∪[Xp7→S (Γ⇒p)]⟩j (3) ∑ y⟨S (Γ ⇒rj)⟩j (4) S (Γ ⇒p) = (3) Nrj( → X : q,Xp : p)]]ξ∪[Xp7→S (Γ⇒p)]⟩j (3) ∑ (y:−→r ⊃p)∈Γ y⟨S (Γ ⇒rj)⟩j (4) S (Γ ⇒p) = ∑ (y:−→r ⊃p)∈Γ y⟨S (Γ ⇒rj)⟩j (4) ∑ (y:−→r ⊃p)∈Γ y⟨S (Γ ⇒rj)⟩j (4) S (Γ ⇒p) = (4) By deﬁnition of S (Γ ⇒p), (4) holds; hence – because of (3) – S (Γ ⇒p) is the solution N∞of (2), concluding the proof that LHS = RHS. Finally, the theorem follows as the particular case of (1) where p = r and the vector of ﬁxpoint variable declarations is empty. □ Proof Let us assume there are k atoms occurring in Γ ⇒r. We deﬁne simultaneously k functions Np( → X : q) (one for each atom p occurring in Γ ⇒r), parameterized by a vector of declarations of the form X : q. The vector is written −−→ X : q and is such that no ﬁxpoint variable and no atom occurs twice. The simultaneous deﬁnition is by recursion on the number of atoms of Γ ⇒r not occurring in −−→ X : q, and is as follows: Np(−−→ X : q) =    Xi if p = qi gfpXp. ∑ (y:−→r ⊃p)∈Γ y⟨Nrj(−−→ X : q,Xp : p)⟩j otherwise if p = qi where vector −−→ X : q,Xp : p is obtained by adding the component Xp : p to the vector −−→ X : q. Observe that only ﬁxpoint variables among the ﬁxpoint variables declared in the vector have free occurrences in Np(−−→ X : q). where vector −−→ X : q,Xp : p is obtained by adding the component Xp : p to the vector −−→ X : q. Observe that only ﬁxpoint variables among the ﬁxpoint variables declared in the vector have free occurrences in Np(−−→ X : q). → ) induction on the number of atoms of (the ﬁxed sequent) Γ ⇒r not in (the variable) −−→ X : q, we h t By induction on the number of atoms of (the ﬁxed sequent) Γ ⇒r not in (the variable) −−→ X : q, we prove that: → [[Np(−−→ X : q)]]ξ = S (Γ ⇒p) if ξ(Xi) = S (Γ ⇒qi), for any i. (1) (1) Case p = qi, for some i. Then, LHS = [[Xi]]ξ = ξ(Xi) = S (Γ ⇒qi) = RHS. LHS = [[Xi]]ξ = ξ(Xi) = S (Γ ⇒qi) = RHS. Otherwise, LHS = [[gfpXp. ∑ (y:−→r ⊃p)∈Γ y⟨Nrj(−−→ X : q,Xp : p)⟩j]]ξ = N∞ where N∞is given as the unique solution of the following equation: where N∞is given as the unique solution of the following equation: N∞= ∑ (y:−→r ⊃p)∈Γ y⟨[[Nrj(−−→ X : q,Xp : p)]]ξ∪[Xp7→N∞]⟩j (2) ∑ (y:−→r ⊃p)∈Γ y⟨[[Nrj(−−→ X : q,Xp : p)]]ξ∪[Xp7→N∞]⟩j (2) (2) Now observe that, by I.H., the following equations (3) and (4) are equivalent. Now observe that, by I.H., the following equations (3) and (4) are equivalent. Lemma 17 (Co-contraction: invertibility of contraction) If x1,x2,y /∈Γ, then Lemma 17 (Co-contraction: invertibility of contraction) If x1,x2,y /∈Γ, then S (Γ,x1 : A,x2 : A ⇒C) = [x1 +x2/y]S (Γ,y : A ⇒C) . Proof The proof is omitted since Lemma 20 below is essentially a generalization of this result. □ We now capture when a context Γ′ is an inessential extension of context Γ: We now capture when a context Γ′ is an inessential extension of context Γ: We now capture when a context Γ′ is an inessential extension of context Γ: 1. \|Γ\| = {A : ∃x s.t.(x : A) ∈Γ}. 1. \|Γ\| = {A : ∃x s.t.(x : A) ∈Γ}. 2. Γ ≤Γ′ if Γ ⊆Γ′ and \|Γ\| = \|Γ′\|. 2. Γ ≤Γ′ if Γ ⊆Γ′ and \|Γ\| = \|Γ′\|. 3 (Γ ⇒p) ≤(Γ′ ⇒p′) if Γ ≤Γ′ and p = p′ 3. (Γ ⇒p) ≤(Γ′ ⇒p′) if Γ ≤Γ′ and p = p′. 3. (Γ ⇒p) ≤(Γ′ ⇒p′) if Γ ≤Γ′ and p = p′. Let σ range over sequents of the form Γ ⇒p. Thus, the last deﬁnition clause deﬁnes in general when σ ≤σ ′. Deﬁnition 19 1. Let Γ ≤Γ′. For N and E in λ co Σ , we deﬁne [Γ′/Γ]N and [Γ′/Γ]E by simultaneous corecursion as follows: Deﬁnition 19 1. Let Γ ≤Γ′. For N and E in λ co Σ , we deﬁne [Γ′/Γ]N and [Γ′/Γ]E by simultaneous corecursion as follows: [Γ′/Γ](λxA.N) = λxA.[Γ′,(x : A)/Γ,(x : A)]N [Γ′/Γ]∑ i Ei = ∑ i [Γ′/Γ]Ei [Γ′/Γ] ! z⟨Ni⟩i = z⟨[Γ′/Γ]Ni⟩i if z /∈dom(Γ) [Γ′/Γ] ! z⟨Ni⟩i = ∑ (w:Γ(z))∈Γ′ w⟨[Γ′/Γ]Ni⟩i if z ∈dom(Γ) [Γ′/Γ](λxA.N) = λxA.[Γ′,(x : A)/Γ,(x : A)]N [Γ′/Γ]∑ i Ei = ∑ i [Γ′/Γ]Ei [Γ′/Γ] ! z⟨Ni⟩i = z⟨[Γ′/Γ]Ni⟩i if z /∈dom(Γ) [Γ′/Γ] ! z⟨Ni⟩i = ∑ (w:Γ(z))∈Γ′ w⟨[Γ′/Γ]Ni⟩i if z ∈dom(Γ) 2. Let σ ≤σ ′. [σ ′/σ]N = [Γ′/Γ]N where σ = (Γ ⇒p) and σ ′ = (Γ′ ⇒p). Similarly for [σ ′/σ]E. Lemma 20 (Co-contraction) If Γ ≤Γ′ then S (Γ′ ⇒C) = [Γ′/Γ](S (Γ ⇒C)). Proof Let R := {(S (Γ′ ⇒C),[Γ′/Γ](S (Γ ⇒C))) \| Γ ≤Γ′,C arbitrary}. We prove that R is backward closed relative to the canonical equivalence = generated by the coinductive deﬁnition of terms of λ co Σ (but see the comments following the proof), whence R ⊆=. S (Γ′ ⇒C) = λzA1 1 ···zAn n . 4.3 Equivalence of the representations: full implicational case The main difference with exhaustive proof search in the case of Horn formulas is that the backwards application of RIntro brings new variables into the context that may have the same type as an already existing declaration, and so, for the purpose of proof search, they should be treated the same way. We illustrate this phenomenon with the following deﬁnition and lemma and then generalize it to the form that will be needed for the main theorem (Theorem 24). Deﬁnition 16 For N and E in λ co Σ , we deﬁne [x1 + ···+ xn/y]N and [x1 + ··· + xn/y]E by simultaneous corecursion as follows: [x1 +···+xn/y](λxA.N) = λxA.[x1 +···+xn/y]N [x1 +···+xn/y]∑ i Ei = ∑ i [x1 +···+xn/y]Ei [x1 +···+xn/y] ! z⟨Ni⟩i = z⟨[x1 +···+xn/y]Ni⟩i if z ̸= y [x1 +···+xn/y] ! y⟨Ni⟩i = ∑ 1≤j≤n xj⟨[x1 +···+xn/y]Ni⟩i Lemma 17 (Co-contraction: invertibility of contraction) If x1,x2,y /∈Γ, then ∑ (z:⃗B⊃p)∈∆′ z⟨S (∆′ ⇒B j)⟩j (5) (5) and and [Γ′/Γ](S (Γ ⇒C)) = λzA1 1 ···zAn n . ∑ (y:⃗B⊃p)∈∆ ∑ (w:∆(y))∈∆′ w⟨[∆′/∆]S (∆⇒B j)⟩j (6) [Γ′/Γ](S (Γ ⇒C)) = λzA1 1 ···zAn n . ∑ (y:⃗B⊃p)∈∆ ∑ (w:∆(y))∈∆′ w⟨[∆′/∆]S (∆⇒B j)⟩j (6) (6) where ∆:= Γ∪{z1 : A1,··· ,zn : An} and ∆′ := Γ′ ∪{z1 : A1,··· ,zn : An}. From Γ ≤Γ′ we get ∆≤∆′, hence (S (∆′ ⇒B j),[∆′/∆]S (∆⇒B j)) ∈R . To conclude the proof, it sufﬁces to show that (i) each head-variable z that is a “capability” of the sum- mation in (5) is matched by a head-variable w that is a “capability” of the summation in (6); and (ii) vice-versa. To conclude the proof, it sufﬁces to show that (i) each head-variable z that is a “capability” of the sum- mation in (5) is matched by a head-variable w that is a “capability” of the summation in (6); and (ii) vice-versa. (i) Let z ∈dom(∆′). We have to exhibit y ∈dom(∆) such that (z : ∆(y)) ∈∆′. First case: z ∈dom(∆). By ∆≤∆′, (z : ∆(z)) ∈∆′. So we may take y = z. Second and last case: z ∈Γ′\Γ. By Γ ≤Γ′, there is y ∈Γ such that (z : Γ(y)) ∈Γ′. But then (z : ∆(y)) ∈∆′. ( ( )) ( ( )) (ii) We have to show that, for all y ∈dom(∆), and all (w : ∆(y)) ∈∆′, w ∈dom(∆′). But this is immediate. □ □ Notice that we cannot expect that the summands appear in the same order in (5) and (6). Therefore, we have to be more careful with the notion of equality of B¨ohm forests. It is not just bisimilarity, but we assume that the sums of elimination alternatives are treated as if they were sets of alternatives, i. e., we further assume that + is symmetric and idempotent. It has been shown by Picard and the second author [10] that bisimulation up to permutations in unbounded lists of children can be managed in a coinductive type even with the interactive proof assistant Coq. In analogy, this coarser notion of equality (even abstracting away from the number of occurrences of an alternative) should not present a major obstacle for a fully formal presentation. Lemma 17 (Co-contraction: invertibility of contraction) If x1,x2,y /∈Γ, then In the rest of the paper – in particular in Theorem 24 – we assume that sums of alternatives are treated as if they were sets. Example 21 (Example 10 continued) Thanks to the preceding lemma, N9 is obtained by co-contraction from N5: N9 = [x : ·,y : (p ⊃q) ⊃p,z : p,y1 : (p ⊃q) ⊃p,z1 : p/x : ·,y : (p ⊃q) ⊃p,z : p]N5 , where the type of x has been omitted. Hence, N6, N7, N8 and N9 can be eliminated, and N5 can b expressed as the (meta-level) ﬁxed point: where the type of x has been omitted. Hence, N6, N7, N8 and N9 can be eliminated, and N5 can be expressed as the (meta-level) ﬁxed point: N5 = ν N.x⟨λy(p⊃q)⊃p 1 .y⟨λzp 1.[x,y,z,y1,z1/x,y,z]N⟩+z+y1⟨λzp 1.[x,y,z,y1,z1/x,y,z]N⟩⟩, S (⇒A) = λx(((p⊃q)⊃p)⊃p)⊃q.x⟨λy(p⊃q)⊃p.y⟨λzp.N5⟩⟩ The question is now how to give a ﬁnitary meaning to terms like N5 in the example above, which are deﬁned by ﬁxed points over variables subject to context substitution. We might expect to use the equation deﬁning N5 to obtain a ﬁnitary representation in λ gfp Σ , provided context substitution is deﬁned on this system. But how to do that? Applying say [x,y,z,y1,z1/x,y,z] to a plain ﬁxed-point variable cannot make much sense. The desired ﬁnitary representation in the full implicational case is obtained by adjusting the terms of λ gfp Σ used in the Horn case as follows: (terms) N ::= (···)\|gfpXσ.E1 +···+En \|Xσ Hence ﬁxpoint variables are “typed” with sequents σ. Hence ﬁxpoint variables are “typed” with sequents σ. Different free occurrences of the same X may be ”typed” with different σ’s, as long as a lower bound of these σ’s can be found w.r.t. ≤(Deﬁnition 18). co Relatively to Deﬁnition 14, an environment ξ now assigns (co)terms N of λ co Σ to “typed” ﬁxpoint variables Xσ, provided X does not occur with two different “types” in the domain of ξ, for all X; we also change the following clauses: [[Xσ′]]ξ = [σ ′/σ]ξ(Xσ) if σ ≤σ ′ [[gfpXσ.∑ i Ei]]ξ = ν N.∑ i [[Ei]]ξ∪[Xσ 7→N] We will have to assign some default value to Xσ′ in case there is no such σ, but this will not play a role in the main result below. → We will have to assign some default value to Xσ′ in case there is no such σ, but this will not play a role in the main result below. → Map Np(−−→ X : q) used in the proof of Theorem 15 is replaced by the following: Deﬁnition 22 Let Ξ := −−−−−−→ X : Θ ⇒q be a vector of m ≥0 declarations (Xi : Θi ⇒qi) where no ﬁxpoint variable and no sequent occurs twice. NΓ⇒⃗A⊃p(Ξ) is deﬁned as follows: If, for some 1 ≤i ≤m, p = qi and Θi ⊆Γ and \|Θi\| = \|∆\|, then Deﬁnition 22 Let Ξ := −−−−−−→ X : Θ ⇒q be a vector of m ≥0 declarations (Xi : Θi ⇒qi) where no ﬁxpoint variable and no sequent occurs twice. NΓ⇒⃗A⊃p(Ξ) is deﬁned as follows: If for some 1 ≤i ≤m p = q and Θ ⊆Γ and \|Θ \| = \|∆\| then NΓ⇒⃗A⊃p(Ξ) = λzA1 1 ···zAn n .Xσ i otherwise, otherwise, NΓ⇒⃗A⊃p(Ξ) = λzA1 1 ···zAn n .gfpY σ. ∑ (y:⃗B⊃p)∈∆ y⟨N∆⇒Bj(Ξ,Y : σ)⟩j where, in both cases, ∆:= Γ∪{z1 : A1,··· ,zn : An} and σ := ∆⇒p. where, in both cases, ∆:= Γ∪{z1 : A1,··· ,zn : An} and σ := ∆⇒p. The deﬁnition of Np(−−→ X : q) in the proof of Theorem 15 was by recursion on a certain number of atoms. The following lemma spells out the measure that is recursively decreasing in the deﬁnition of NΓ⇒C(Ξ). Lemma 23 For all Γ ⇒C, NΓ⇒C(·) is well-deﬁned, where · denotes the empty vector. Hence ﬁxpoint variables are “typed” with sequents σ. Proof Let us call recursive call a “reduction” NΓ⇒⃗A⊃p(−−−−−−→ X : Θ ⇒q) ⇝N∆⇒Bj(−−−−−−→ X : Θ ⇒q,Y : σ) (7) (7) where the if-guard in Def. 22 fails; ∆and σ are deﬁned as in the same deﬁnition; and, for some y, (y : ⃗B ⊃p) ∈∆. We want to prove that every sequence of recursive calls from NΓ⇒C(·) is ﬁnite. First we introduce some deﬁnitions. A sub := {B \| there is A ∈A such that B is subformula of A}, for A a ﬁnite set of formulas. We say A is subformula-closed if A sub = A . A stripped sequent is a pair (B, p), where B is a ﬁnite set of formulas. If σ = Γ ⇒p, then \|σ\| denotes the stripped sequent (\|Γ\|, p). We say (B, p) is over A if B ⊆A and p ∈A . There are size(A ) := a·2k stripped sequents over A , if a (resp. k) is the number of atoms (resp. formulas) in A . → Let A be subformula-closed. We say Γ ⇒C and Ξ := −−−−−−→ X : Θ ⇒q satisfy the A -invariant if: Let A be subformula-closed. We say Γ ⇒C and Ξ := −−−−−−→ X : Θ ⇒q satisfy the A -invariant if: (i) \|Γ\|∪{C} ⊆A ; (i) \|Γ\|∪{C} ⊆A ; (ii) Θ1 ⊆Θ2 ⊆··· ⊆Θm = Γ (if m = 0 then this is meant to be vacuously true); (iii) For 1 ≤j ≤m, qj ∈\|Γ\|sub, where m ≥0 is the length of vector Ξ (if m = 0, also item (iii) is vacuously true). In particular, \|σ\| is over A , for all σ ∈Ξ. We prove that, if Γ ⇒C and Ξ satisfy the A -invariant for some A , then every sequence of recursive calls from NΓ⇒C(Ξ) is ﬁnite. The proof is by induction on size(A )−size(Ξ), where size(Ξ) is the number of elements of \|Ξ\| and \|Ξ\| := {\|σ\| : σ ∈Ξ}. Let C = ⃗A ⊃p. We analyze an arbitrary recursive call (7) and prove that every sequence of recursive calls from N∆⇒Bj(Ξ,Y : σ) is ﬁnite. This is achieved by proving: (I) ∆⇒B j and Ξ,Y : σ satisfy the A -invariant; (II) size(Ξ,Y : σ) > size(Ξ). (I) ∆⇒B j and Ξ,Y : σ satisfy the A -invariant; (II) size(Ξ,Y : σ) > size(Ξ). (II) size(Ξ,Y : σ) > size(Ξ). Proof of (I). Hence ﬁxpoint variables are “typed” with sequents σ. □ Theorem 24 (Equivalence) For any Γ and C, there exists NΓ⇒C ∈λ gfp Σ with no free occurrences of ﬁxpoint variables such that [[NΓ⇒C]] = S (Γ ⇒C). Theorem 24 (Equivalence) For any Γ and C, there exists NΓ⇒C ∈λ gfp Σ with no free occurrences of ﬁxpoint variables such that [[NΓ⇒C]] = S (Γ ⇒C). [[NΓ⇒⃗A⊃p(Ξ)]]ξ = S (Γ ⇒⃗A ⊃p) , (8) (8) where Ξ := −−−−−−→ X : Θ ⇒q. In this proof we re-use the concepts introduced in the proof of Lemma 23. Let A := (\|Γ\|∪{⃗A ⊃p})sub. The proof is by induction on size(A )−size(Ξ). qi and Θ′ i ⊆Γ and \|Θ′ i\| = \|∆\|, for some 1 ≤i ≤m, with m the length of Ξ. Then, = λzA1 1 ···zAn n .[[X∆⇒qi i ]]ξ (by deﬁnition) = λzA1 1 ···zAn n .[∆⇒qi/Θi ⇒qi]ξ(XΘi⇒qi i ) (by deﬁnition and () below) = λzA1 1 ···zAn n .[∆⇒qi/Θi ⇒qi]S (Θi ⇒qi) (by assumption) = λzA1 1 ···zAn n .S (∆⇒qi) (by Lemma 20 and ()) = RHS (by deﬁnition) where ∆:= Γ∪{z1 : A1,··· ,zn : An}, which implies (Θi ⇒qi) ≤(∆⇒qi). The latter fact is the justiﬁca- tion () used above. where ∆:= Γ∪{z1 : A1,··· ,zn : An}, which implies (Θi ⇒qi) ≤(∆⇒qi). The latter fact is the justiﬁca- tion () used above. The inductive case is an easy extension of the inductive case in Theorem 15. Suppose the case above holds for no 1 ≤i ≤m. Then LHS = λzA1 1 ···zAn n .N∞, where N∞is the unique solution of the following equation N∞ = ∑ (y:−→B ⊃p)∈∆ y⟨[[N∆⇒Bj(Ξ,Y : σ)]]ξ∪[Y σ7→N∞]⟩j (9) (9) and, again, ∆:= Γ ∪{z1 : A1,··· ,zn : An}. Now observe that, by I.H., the following equations (10) and (11) are equivalent. and, again, ∆:= Γ ∪{z1 : A1,··· ,zn : An}. Now observe that, by I.H., the following equations (10) and (11) are equivalent. S (∆⇒p) = ∑ (y:−→ B ⊃p)∈∆ y⟨[[N∆⇒Bj(Ξ,Y : σ)]]ξ∪[Y σ7→S (∆⇒p)]⟩j (10) S (∆⇒p) = ∑ (y:−→ B ⊃p)∈∆ y⟨S (∆⇒B j)⟩j (11) (10) (11) By deﬁnition of S (∆⇒p), (11) holds; hence - because of (10) - S (∆⇒p) is the solution N∞of (9). Therefore LHS = λzA1 1 ···zAn n .S (∆⇒p), and the latter is RHS by deﬁnition of S (Γ ⇒⃗A ⊃p). Hence ﬁxpoint variables are “typed” with sequents σ. By assumption, (i), (ii), and (iii) above hold. We want to prove: Proof of (I). By assumption, (i), (ii), and (iii) above hold. We want to prove: (i’) \|∆\|∪{B j} ⊆A ; (ii’) Θ1 ⊆Θ2 ⊆··· ⊆Θm ⊆∆= ∆; (iii’) For 1 ≤j ≤m+1, qj ∈\|∆\|sub. (ii’) Θ1 ⊆Θ2 ⊆··· ⊆Θm ⊆∆= ∆; (iii’) For 1 ≤j ≤m+1, qj ∈\|∆\|sub. Proof of (i’). \|∆\| = \|Γ\| ∪{A1,··· ,An} ⊆A by (i) and A subformula-closed. B j is a subformul Proof of (i’). \|∆\| = \|Γ\| ∪{A1,··· ,An} ⊆A by (i) and A subformula-closed. B j is a subformula of ⃗B ⊃p and ⃗B ⊃p ∈\|∆\| because (y : ⃗B ⊃p) ∈∆, for some y. \| \| \| \| { } j ⃗B ⊃p and ⃗B ⊃p ∈\|∆\| because (y : ⃗B ⊃p) ∈∆, for some y. ⃗B ⊃p and ⃗B ⊃p ∈\|∆\| because (y : ⃗B ⊃p) ∈∆, Proof of (ii’). Immediate by (ii) and Γ ⊆∆. Proof of (ii’). Immediate by (ii) and Γ ⊆∆. b Proof of (iii’). For 1 ≤j ≤m, qj ∈\|Γ\|sub ⊆\|∆\|sub, by (iii) and Γ ⊆∆. On the other hand, qj+1 = p ∈ \|∆\|sub because (y : ⃗B ⊃p) ∈∆, for some y. \| \| (y p) y Proof of (II). Given that the if-guard of Def. 22 fails, and that Θi ⊆Γ due to (ii), we conclude: for all 1 ≤i ≤m, p ̸= qi or \|Θi\| ̸= \|∆\|. But this means that \|∆⇒p\| /∈\|Ξ\|, hence size(Ξ,Y : σ) > size(Ξ). \| \| ( ) Proof of (II). Given that the if-guard of Def. 22 fails, and that Θi ⊆Γ due to (ii), we conclude: for all 1 ≤i ≤m, p ̸= qi or \|Θi\| ̸= \|∆\|. But this means that \|∆⇒p\| /∈\|Ξ\|, hence size(Ξ,Y : σ) > size(Ξ). \| \| \| \| \| \| / \| \| ( ) ( ) Now, by I.H., every sequence of recursive calls from N∆⇒Bj(Ξ,Y : σ) is ﬁnite. This concludes the proof by induction. Now, by I.H., every sequence of recursive calls from N∆⇒Bj(Ξ,Y : σ) is ﬁnite. This concludes the proof by induction. p y Finally let A = (\|Γ\|∪{C})sub and observe that Γ ⇒C and Ξ = · satisfy the A -invariant. Finally let A = (\|Γ\|∪{C})sub and observe that Γ ⇒C and Ξ = · satisfy the A -invariant. ally, the theorem follows as the particular case of (8) where C = ⃗A ⊃p and the vector of ﬁxpoint e declarations is empty. □ 5 Conclusion We proposed a coinductive approach to proof search, which we illustrated in the case of the cut-free system LJT for intuitionistic implication (and its proof-annotated version λ). As the fundamental tool, we introduced the coinductive calculus λ co Σ , which besides the coinductive reading of λ, introduces a construction for ﬁnite alternatives. The (co)terms of this calculus (also called B¨ohm forests) are used to represent the solution space of proof search for LJT-sequents, and this is achieved by means of a corecursive function, whose deﬁnition arises naturally by taking a reductive view of the inference rules and by using the ﬁnite alternatives construction to account for multiple alternatives in deriving a given sequent. We offered also a ﬁnitary representation of proof search in LJT, based on the inductive calculus λ gfp Σ with ﬁnite alternatives and a ﬁxed point construction, and showed equivalence of the representations. The equivalence results turned out to be an easy task in the case of the Horn fragment, but demanded for co-contraction of contexts (contraction bottom-up) in the case of full implication. With Pym and Ritter [11] we share the general goal of setting a framework for studying proof search, and the reductive view of inference rules, by which each inference rule is seen as a reduction opera- tor (from a putative conclusion to a collection of sufﬁcient premises), and reduction (the process of repeatedly applying reduction operators) may fail to yield a (ﬁnite) proof. However, the methods are very different. Instead of using a coinductive approach, Pym and Ritter introduce the λµνε-calculus for classical sequent calculus as the means for representing derivations and for studying intuitionistic proof search (a task that is carried out both in the context of the sequent calculus LJ and of intuitionistic resolution). In the context of logic programming with classical ﬁrst-order Horn clauses, and building on their previous work [6, 4], Komendantskaya and Power [5] establish a coalgebraic semantics uniform for both ﬁnite and inﬁnite SLD-resolutions. In particular, a notion of coinductive (and-or) derivation tree of an atomic goal w. r. t. a (ﬁxed) program is introduced. Soundness and completeness results of SLD- resolution relative to coinductive derivation trees and to the coalgebraic semantics are also proved. Logic programming is viewed as search for uniform proofs in sequent calculus by Miller et al. [8]. Hence ﬁxpoint variables are “typed” with sequents σ. ⃗ Finally, the theorem follows as the particular case of (8) where C = ⃗A ⊃p and the vector of ﬁxpoint variable declarations is empty. □ □ 5 Conclusion For intuition- istic implication, uniform proofs correspond to the class of (η-)expanded normal natural deductions (see Dyckoff and Pinto [2]), hence to the typed λ-terms we considered in this paper (recall the restriction to atoms in rule Der of Fig. 1 for typing application). Under this view, our work relates to Komendantskaya and Power [5], as both works adopt a coinductive approach in the context of proof search. However, the two approaches are different in methods and in goals. As the basis of the coinductive representation of the search space, instead of and-or inﬁnite trees, we follow the Curry-Howard view of proofs as terms, and propose the use of a typed calculus of coinductive lambda-terms. Whereas Komendantskaya and Power [5] are already capable of addressing ﬁrst-order quantiﬁcation, we only consider intuitionistic im- plication. Still, as we consider full intuitionistic implication, our study is not contained in classical Horn logic. The fact that we need to treat negative occurrences of implication, raises on the logic programming side the need for dealing with programs to which clauses can be added dynamically. As a priority for future work, we plan to develop notions of normalisation for the calculi λ co Σ and λ gfp Σ in connection with aspects of proof search like pruning search spaces and reading off (ﬁnite) proofs. In order to test for the generality of our approach, we intend to extend it to treat the ﬁrst-order case. Staying within intuitionistic implication, but changing the proofs searched for, another case study we intend to investigate is Dyckhoff’s contraction-free system [1]. Acknowledgments We thank our anonymous referees for their helpful comments. Jos´e Esp´ırito Santo and Lu´ıs Pinto have been ﬁnanced by FEDER funds through “Programa Operacional Factores de Com- petitividade – COMPETE” and by Portuguese funds through FCT – “Fundac¸˜ao para a Ciˆencia e a Tec- nologia”, within the project PEst-C/MAT/UI0013/2011. Ralph Matthes thanks the Centro de Matem´atica of Universidade do Minho for funding research visits to Jos´e Esp´ırito Santo and Lu´ıs Pinto to start this research (2011/2012). Subsequently, he has been funded by the Climt project (ANR-11-BS02-016 of the French Agence Nationale de la Recherche). References [1] Roy Dyckhoff (1992): Contraction-Free Sequent Calculi for Intuitionistic Logic. J. Symb. Log. 57(3) 795–807, doi:10.2307/2275431. [2] Roy Dyckhoff & Lu´ıs Pinto (1994): Uniform Proofs and Natural Deductions. In Didier Galmiche & Lincoln Wallen, editors: Proceedings of CADE–12 Workshop on Proof Search in Type-Theoretic Languages, IN- RIA Lorraine – CRIN, pp. 717–23. Available at http://citeseerx.ist.psu.edu/viewdoc/summary? doi=10.1.1.43.9659. [3] H. Herbelin (1995): A λ-calculus structure isomorphic to a Gentzen-style sequent calculus structure. In L. Pacholski & J. Tiuryn, editors: Proceedings of CSL’94, Lecture Notes in Computer Science 933, Springer- Verlag, pp. 61–75, doi:10.1007/BFb0022247. [4] Ekaterina Komendantskaya, Guy McCusker & John Power (2010): Coalgebraic Semantics for Parallel Derivation Strategies in Logic Programming. In Michael Johnson & Dusko Pavlovic, editors: AMAST, Lecture Notes in Computer Science 6486, Springer, pp. 111–127, doi:10.1007/978-3-642-17796-5_7. [5] Ekaterina Komendantskaya & John Power (2011): Coalgebraic Derivations in Logic Programming. In Marc Bezem, editor: CSL, LIPIcs 12, Schloss Dagstuhl - Leibniz-Zentrum f¨ur Informatik, pp. 352–366, doi:10. 4230/LIPIcs.CSL.2011.352. [6] Ekaterina Komendantskaya & John Power (2011): Coalgebraic Semantics for Derivations in Logic Program- ming. In Andrea Corradini, Bartek Klin & Corina Cˆırstea, editors: CALCO, Lecture Notes in Computer Science 6859, Springer, pp. 268–282, doi:10.1007/978-3-642-22944-2_19. [7] Chuck Liang & Dale Miller (2009): Focusing and Polarization in Linear, Intuitionistic, and Classical Logic. Theoretical Computer Science 410, pp. 4747–4768, doi:10.1016/j.tcs.2009.07.041. [8] Dale Miller, Gopalan Nadathur, Frank Pfenning & Andre Scedrov (1991): Uniform Proofs as a Founda- tion for Logic Programming. Annals of Pure and Applied Logic 51(1-2), pp. 125–157, doi:10.1016/ 0168-0072(91)90068-W. [9] Keiko Nakata, Tarmo Uustalu & Marc Bezem (2011): A Proof Pearl with the Fan Theorem and Bar Induction - Walking through Inﬁnite Trees with Mixed Induction and Coinduction. In Hongseok Yang, editor: APLAS, LNCS 7078, Springer, pp. 353–368, doi:10.1007/978-3-642-25318-8_26. [10] Celia Picard & Ralph Matthes (2012): Permutations in Coinductive Graph Representation. In Dirk Pattin- son & Lutz Schr¨oder, editors: Coalgebraic Methods in Computer Science (CMCS 2012), Lecture Notes in Computer Science, IFIP subseries 7399, Springer, pp. 218–237, doi:10.1007/978-3-642-32784-1_12. [11] D.J. Pym & E. Ritter (2004): Reductive Logic and Proof-search: Proof Theory, Semantics, and Control. Oxford Logic Guides, Oxford University Press, Incorporated, doi:10.1093/acprof:oso/9780198526339. 001.0001.
https://openalex.org/W3084816013	https://link.springer.com/content/pdf/10.1007/s10533-020-00699-y.pdf	English	null	Nitrogen dynamics after two years of elevated CO2 in phosphorus limited Eucalyptus woodland	Biogeochemistry	2,020	cc-by	11,523	Nitrogen dynamics after two years of elevated CO2 in phosphorus limited Eucalyptus woodland Louise C. Andresen . Yolima Carrillo . Catriona A. Macdonald . Louise C. Andresen . Yolima Carrillo . Catriona A. Macdonald . Laura Castan˜eda-Go´mez . Samuel Bode´ . Tobias Ru¨tting Received: 20 December 2019 / Accepted: 28 August 2020 / Published online: 10 September 2020 The Author(s) 2020 free amino acid (FAA) pool had a fast turnover time (4 h) compared to that of ammonium (NH4 ?) which was 11 h. Both NH4-N and FAA-N were important N pools; however, protein depolymerization rate was three times faster than gross N mineralization rates, indicating that organic N is directly important in the internal ecosystem N cycle. Hence, the depolymer- ization was the major provider of plant available N, while the gross N mineralization rate was the constraining factor for inorganic N. After two years of elevated CO2, no major effects on the pools and rates of the soil N cycle were found in spring (November) or at the end of summer (March). The limited response of N pools or N transformation rates to elevated CO2 suggest that N availability was not the limiting factor behind the lack of plant growth response to elevated CO2, previously observed at the site. Abstract It is uncertain how the predicted further rise of atmospheric carbon dioxide (CO2) concentra- tion will affect plant nutrient availability in the future through indirect effects on the gross rates of nitrogen (N) mineralization (production of ammonium) and depolymerization (production of free amino acids) in soil. The response of soil nutrient availability to increasing atmospheric CO2 is particularly important for nutrient poor ecosystems. Within a FACE (Free- Air Carbon dioxide Enrichment) experiment in a native, nutrient poor Eucalyptus woodland (Euc- FACE) with low soil organic matter (B 3%), our results suggested there was no shortage of N. Despite this, microbial N use efﬁciency was high (c. 90%). The Responsible Editor: Stephen D. Sebestyen. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10533-020-00699-y) con- tains supplementary material, which is available to authorized users. Keywords Gross N mineralization rate Depolymerization Free amino acids Phosphorus limitation L. C. Andresen (&) T. Ru¨tting Department of Earth Sciences, University of Gothenburg, Gothenburg, Sweden e-mail: louise.andresen@gu.se Biogeochemistry (2020) 150:297–312 https://doi.org/10.1007/s10533-020-00699-y (0123456789().,-volV)( 01234567 89().,-volV) Introduction Recently reviewed evidence suggest that the CO2 response in plant biomass is controlled by stocks of plant available nitrogen (N) and phosphorus (P) (Terrer 2019). Hence, C, N and P responses to CO2 are likely to be intimately linked. However, if plant growth is limited by one or more nutrient (Chapin et al. 1987; Cˇ apek 2018), an increased plant growth under elevated CO2 is only possible if nutrient mobilization also increases. Even- tually, a down-regulation of growth responses (pro- gressive nutrient limitation, PNL) might occur through long-term changes in nutrient cycles. However, PNL under eCO2 has only been conﬁrmed in few cases (Norby 2011; Za¨hle 2014). Beginning with photosyn- thesis, CO2 can stimulate a cascade of potential effects in an ecosystem, leading to an increase in plant growth and in belowground allocation (root growth) (An- dresen et al. 2016b; Ko¨rner 2018). Subsequently, increased rhizodeposition could stimulate organic matter decomposition (rhizosphere priming) and nutrient mineralization, leading to increased nutrient availability, to meet the extra nutrient demand (Dijk- stra et al. 2013; Kuzyakov 2015; Jilling et al. 2018; Moreau et al. 2019; Schleppi 2019). A meta-analysis found that gross N mineralization rate was stimulated by CO2 across N limited ecosystems, but not in ecosystems limited by P (Ru¨tting & Andresen 2015), potentially because the cascade response to labile C deposition switches from N mining to P mining (Dijkstra et al. 2013). Investigation of FAA pools and production rates under elevated CO2 are rare (Chen et al. 2014; Wild et al. 2018). Hypothetically, the abundance of FAAs in soil can increase as rhizodeposition increases. Such an increase in FAAs can occur directly from an increase in the release of FAAs in root exudates (Xionga 2019), or indirectly from an increase in the depolymerization rate. To our knowledge, no theory is available to explain why some individual FAAs would be more abundant than others. Further, individual FAA abun- dance and production rates are unknown for most FACE sites. In fact, most conclusions about the responses of the N cycle in FACE studies are based on Organic nitrogen (Norg) plays a key role in soil N cycling and amino acids constitute an important direct resource of N for microbes and plants across ecosys- tems (Chapin III 1995; Kuzyakov 2013). It is known that many vascular species, including Eucalyptus species (Warren 2006, 2009), can take up intact amino acids (Andresen et al. Introduction Y. Carrillo C. A. Macdonald L. Castan˜eda-Go´mez Hawkesbury Institute for the Environment (HIE), Western Sydney University, Richmond, Australia Ecosystem effects of elevated atmospheric carbon dioxide (CO2) concentration are a global concern, with the atmospheric CO2 concentrations recently having S. Bode´ Department of Green Chemistry and Technology, Ghent University, Gent, Belgium 12 3 3 Biogeochemistry (2020) 150:297–312 298 surpassed 415 ppm (McGee 2019). Worldwide, sev- eral experiments manipulate atmospheric CO2 con- centration at the ﬁeld scale in open air by the FACE (Free Air Carbon dioxide Enrichment) technique (Miglietta 2001; Ellsworth et al. 2017) to investigate direct physiological responses of vegetation and effects on whole ecosystems. The question of whether plant growth is limited by carbon (C) or nutrient supply is relevant as both nutrient loading and elevated atmospheric CO2 currently alter natural terrestrial environments (Steffen 2015; Stevens 2019). Recently reviewed evidence suggest that the CO2 response in plant biomass is controlled by stocks of plant available nitrogen (N) and phosphorus (P) (Terrer 2019). Hence, C, N and P responses to CO2 are likely to be intimately linked. However, if plant growth is limited by one or more nutrient (Chapin et al. 1987; Cˇ apek 2018), an increased plant growth under elevated CO2 is only possible if nutrient mobilization also increases. Even- tually, a down-regulation of growth responses (pro- gressive nutrient limitation, PNL) might occur through long-term changes in nutrient cycles. However, PNL under eCO2 has only been conﬁrmed in few cases (Norby 2011; Za¨hle 2014). Beginning with photosyn- thesis, CO2 can stimulate a cascade of potential effects in an ecosystem, leading to an increase in plant growth and in belowground allocation (root growth) (An- dresen et al. 2016b; Ko¨rner 2018). Subsequently, increased rhizodeposition could stimulate organic matter decomposition (rhizosphere priming) and nutrient mineralization, leading to increased nutrient availability, to meet the extra nutrient demand (Dijk- stra et al. 2013; Kuzyakov 2015; Jilling et al. 2018; Moreau et al. 2019; Schleppi 2019). A meta-analysis found that gross N mineralization rate was stimulated by CO2 across N limited ecosystems, but not in ecosystems limited by P (Ru¨tting & Andresen 2015), potentially because the cascade response to labile C deposition switches from N mining to P mining (Dijkstra et al. 2013). Introduction Organic nitrogen (Norg) plays a key role in soil N acids are as abundant as Nin in a Eucalyptus woodland with Brown Sodosol soils in New South Wales (Australia) (Prendergast-Miller et al. 2015). Similarly, amino acids were reported as abundant as Nin in Eucalyptus, Melaleuca and Banksia dry bush with Aridisol soils in Western Australia (Farrell 2013). Hence amino acids might be as relevant an N source as Nin across Australia (Lee et al. 2018). The transfor- mation of Norg via N-rich polymers to free amino acids (FAA) directly from readily available detritus, is driven by soil microbes. Additionally, Norg in mineral associated organic matter (MAOM) can be destabi- lized for further microbial transformation by organic acids in plant exudates (Schimel & Bennett 2004; Kuzyakov 2015; Jilling et al. 2018). Traditionally, for ecosystems with N limited vegetation having typical high soil C to N ratio, the liberation of FAAs from proteins during depolymerization is seen as the rate- limiting step in the transformation pathway of N from Norg to Nin. Contrastingly, in more fertile ecosystems, both gross N mineralization and depolymerization rates can constrain Nin availability (Schimel & Ben- nett 2004; Jones et al. 2009). Recently it has been noted that even liberation of FAA from MAOM can be a limiting factor for gross N mineralization rate (Jilling et al. 2018). Hence, FAA and Nin pools can have several sources and sinks and it is unclear if these pool sizes are good indicators of the gross rates (Ga¨rdena¨s et al. 2011; Hobbie & Hobbie 2012). Gross rates determined by the 15N pool dilution technique allow the estimation of instantaneous rates, accounting for all sources (aided by all types of functional enzymes) and sinks together. For Australian ecosystems, only limited knowledge about gross rates driving the soil N cycle exist, and their responses to future climate change such as elevated CO2 are unknown. surpassed 415 ppm (McGee 2019). Worldwide, sev- eral experiments manipulate atmospheric CO2 con- centration at the ﬁeld scale in open air by the FACE (Free Air Carbon dioxide Enrichment) technique (Miglietta 2001; Ellsworth et al. 2017) to investigate direct physiological responses of vegetation and effects on whole ecosystems. The question of whether plant growth is limited by carbon (C) or nutrient supply is relevant as both nutrient loading and elevated atmospheric CO2 currently alter natural terrestrial environments (Steffen 2015; Stevens 2019). Introduction 2011; Na¨sholm et al. 2009) as well as inorganic N (Nin) sources (ammonium and nitrate). It has recently been suggested that amino 123 299 Biogeochemistry (2020) 150:297–312 total of 86 species present (Hasegawa et al. 2018). The climate is a humid temperate-subtropical transitional climate. In the southern hemisphere spring months are September to November, summer months are Decem- ber to February, autumn months are March to May and winter months are June to August (Drake et al. 2016). Growing season can be deﬁned as August to March but this has much variation and relies on rainfall patterns and soil moisture (Collins et al. 2018). Soil volumetric water content is highly variable at the site, ranging from (rare) ﬂooded conditions of 35% to exceptionally dry soils of 5% (Drake et al. 2018; Gimeno et al. 2018). This variation in soil volumetric water content arises mostly from variable weather patterns at the site, but typically, the summer receives most rainfall. The soil is formed from weakly organised alluvial deposits and primarily an Aeric Podosol with areas of Densic Podosol (Australian soil classiﬁcation) (Ross 2020). The soil is a slightly acidic loamy sand with low soil organic matter content and there is a hard impermeable clay layer that varies in depth across the site from 35 to 75 cm depth. Nin, leaving a large knowledge gap on Norg responses to CO2. Studies on rates of gross N mineralization and depolymerization are lacking in Australia, and as many soils in Australia are considered as nutrient poor, because of a long history of weathering (Turner & Condron 2013), further understanding the importance of Norg in N cycling in such nutrient poor ecosystems is important. At the Cumberland Woodland site central to this study, measurements have shown seasonal variations of the nutrient pools, but with minimal effects from FACE (ambient ? 150 ppm CO2 increase; Hasegawa et al. 2016; Ochoa-Hueso et al. 2017). The study site soil has a low P status (* 60 mg kg -1; Crous et al. 2015) and is considered to have P-limited vegetation, as Eucalyptus stem diameter (Crous et al. 2015) and root biomass (Nielsen et al. 2015) increased signiﬁ- cantly following 2 years of P fertilization (50 kg Pha-1 yr-1). Introduction We hypothesised that (i) Norg would be an important factor in N cycling in these nutrient poor soils, and based on the outcome of a meta analysis of gross N mineralization rates (Ru¨tting & Andresen 2015) that (ii) gross N mineralization and depolymer- ization rates would show minimal response to elevated CO2. We used 15N pool dilution to assess gross N mineralization and depolymerization rates, in combi- nation with quantiﬁcation of the Nin and FAA pools, in order to determine the role of FAAs in the N cycle and its response to elevated CO2 in a P limited Cumberland Plain woodland (EucFACE). Field site The EucFACE experiment is located within a Cum- berland Plain Woodland in Western Sydney, New South Wales (NSW), Australia. The site is character- ized by a relatively open canopy of evergreen Eucalyptus tereticornis (Forest Redgum) trees of 17 to 23 m height, at a low density of 600 to 1000 trees ha-1 with a basal area of 27 m2 ha-1 (Crous et al. 2015; Duursma et al. 2016; Ellsworth et al. 2017). Cumberland Woodland is listed as an endangered ecological community in Commonwealth Legislation (under Sect. 181 of the Environment Protection and Biodiversity Conservation Act 1999; EPBC Act). The understory is a diverse mix of grasses and forbs with a FACE treatment The main EucFACE experiment consists of six instrumented circular rings of 25 m diameter, con- nected by a set of vertical pipes that release CO2 into the ecosystem in a controlled manner using the FACE technique with CO2 pre-dilution (Hendrey 2006). Carbon dioxide is injected in three randomly selected rings. Three other rings are instrumented controls where CO2 is not added. The rings are separated by 80 to 100 m. The target of the FACE treatment is 150 ppm above ambient atmospheric CO2 concentra- tion. Injection of CO2 began in September 2012 and gradually reached ? 150 ppm in February 2013. Soil sampling Soil was sampled 26 and 30 months after CO2 treatment began, on November 17, 2014 (late spring) and on March 9, 2015 (early autumn). Within each of four 2 m 9 2 m sub-plots dedicated for soil sampling, located within each ring, four soil cores were sampled to a depth of 10 cm using a 5 cm diameter metal auger. In parallel, separate samples were collected from the top 2 cm. For each depth, replicates from within each sub-plot were mixed resulting in four replicate 12 3 3 300 Biogeochemistry (2020) 150:297–312 samples per ring. Leaf and bark litter as well as roots and stones were removed by hand. Soil was then sieved through a 2 mm sieve and kept at 4 C for up to 2 weeks until labelling or extraction took place. The average ﬁeld soil temperature from November 1, 2014 until March 31, 2015 was 22.5 C ± 0.1 with a minimum of 18.8 C and a maximum of 25.8 C. The incubating soils were kept at room temperature, varying between 22 and 25 C, for acclimation prior to the labelling. During calculations (see later), an average of the four sub-plots was used to cover the spatial variation in one plot. Bulk density (g dry soil cm-3) of the top 10 cm was determined for each ring in August 2017. Air dried and ground soil (approximately 60 lg) was accurately weighed in triplicate into tin capsules and analysed by the IRMS (GSL elemental analyzer coupled to a 20–22 isotope ratio mass spectrometer Sercon Ltd., Crewe, UK) for total N and C and for 15N and 13C abundance. Uncertainties of d15N and d13C were lower than 0.3 %. Free amino acids were extracted from fresh soil (10 g) from each sup-plot using 20 mL of a 10 mMolL-1 CaSO4 (120 rpm, 1 h) and ﬁltered (What- man qualitative ﬁlter papers no. 1) after sedimentation for 30 min. An internal standard (200 lL) consisting of norvaline (0.072 mg mL-1), norleucine (0.071 mg mL-1) and 4-chlorphenylalanine (0.096 mg mL-1) was added to the CaSO4 extracts and the soil water, which were then transferred to solid phase extraction (SPE) columns (Dionex OnGuard II H, 1 mL cartridge, ThermoScientiﬁc) by vacuum suction. The SPE columns were then frozen and shipped to Ghent, Belgium (ISOFYS laboratories; Andresen et al. 2016a) for analysis. Nitrogen turnover rates by pool dilution technique Two isotope labellings were performed in the labora- tory to study gross soil N turnover rates: 1. gross depolymerization rate (amino acid production rate) by 15N amino acid pool dilution, and 2. gross N miner- alization rate by 15N–NH4 pool dilution. Gross N mineralization rates were assessed in November and March but depolymerization only in March. For each sub-plot (four sub-plots per treatment ring), sub- samples of fresh soil (10 g) were weighed into 6 cm diameter dark ﬂasks, producing a layer of soil c. 5 mm thick. Samples were then labelled by adding the isotope enriched label solution to the soil. In Novem- ber (but not March), the soil moisture was so low that applying the isotope label was difﬁcult as the soil was hydrophobic. Therefore, 1 mL deionized water was slowly added dropwise to each soil one day before labelling. The two 15N labels were: 1. a 20 amino acids-mixture (AA; ‘cell free powder’ from Cam- bridge isotope laboratories; see (Andresen et al. 2015)) with 99% 15N, dissolved in 0.1 MolL-1 HCl, containing 0.36 g AA L-1 corresponding to 3 lg AA-N g-1 soil.; and 2. ammonium sulphate with 99.8% 15N, amended at a rate of 2 lg N g-1 soil. Label addition was done by dripping 1.5 mL of the 15N enriched label onto the soil surface in an even Soil water sampling To measure soil water amino acid content, we obtained soil water from each of the four sub-plots within each ring, using permanently installed lysime- ters with ceramic suction cups at two different depths (1900 soil water sampler from Soil Moisture Equip- ment Corp., Santa Barbara, California). The ﬁrst was installed to a depth of 10 to 15 cm (upper cup) and the second immediately above the clay layer (lower cup). The day before sampling (December 10, 2014) a vacuum was applied to the cup using a hand pump to pull soil water into the cup. The following day, the pumped soil water was collected using PVC piping and a hand pump. Samples were sterilized by ﬁltering (0.22 lm SLGP033RB, Millipore, OH, USA) and stored at 4 C until analysis. All water samples were analysed for individual amino acid concentration. 123 Soil properties Gravimetric soil water was determined by drying 5 g fresh soil at 105 C until weight constancy. Soil organic matter (SOM) content was determined by loss on ignition (550 C, 5 h). Extractable inorganic N was determined from fresh soil (2 g) for each sub-plot following extraction with 2 MolL-1 KCl. Nitrate (NO3 -) and ammonium (NH4 ?) concentrations were determined by colorimetry (AQ2 Discrete Analyser, SEAL Analytical, Mequon, WI, USA). Nutrient content is presented as mg (NO3 - or NH4 ?) kg-1 dry soil, and by use of bulk density, on a m2 basis in the top 10 cm. 12 123 Biogeochemistry (2020) 150:297–312 301 spatial pattern (in November only 0.5 mL of label was added to account for the 1 mL of deionized water already added). After label addition, the ﬂasks were incubated in a dark chamber at 25 C. At ten minutes and at 7 h after label addition soils from sub-plots were extracted as follows. The incubations with the 15N-NH4 label with 20 mL of 1 MolL-1 KCl (120 rpm, 60 min), and incubations with the 15N- amino acid label with 20 mL of 3.7% formaldehyde in 10 mMolL-1 CaSO4 solution (120 rpm, 60 min). After 30 min, the slurries were decanted and ﬁltered (Whatmann 42 ashless, diam. 125 mm). The KCl extracts were frozen and transported to ISOGOT labs in Sweden for analysis. The CaSO4 extracts were transferred to SPE columns as above, then frozen and transported to ISOFYS for analysis. block the polar groups of amino acid in order to make them volatile for GC seperation. Finally, the individ- ual FAAs were measured by gas chromatography– mass-spectrometry (GC–MS, Trace GC–DSQ, Thermo Fisher). Detection limit for individual amino acids is lower than 0.02 mgkg-1 soil. block the polar groups of amino acid in order to make them volatile for GC seperation. Finally, the individ- ual FAAs were measured by gas chromatography– mass-spectrometry (GC–MS, Trace GC–DSQ, Thermo Fisher). Detection limit for individual amino acids is lower than 0.02 mgkg-1 soil. Abundance and isotopic composition of individual amino acid analysis by GC–MS Abundance and isotopic composition of individual amino acid analysis by GC–MS Calculations Depolymerization rate (DSON, amino acid production rate), gross N mineralization rate (M, NH4 ? produc- tion rate) and the consumption rates of amino acids (CFAA) and ammonium (CNH4) were calculated using the analytical equations (Kirkham 1954) for gross mineralization or depolymerization (Wanek et al. 2011). In most cases the formula for decreasing concentration between time steps was used as the difference of concentration was greater than the detection limit. The rates are expressed as mg Nkg-1 dry soilh-1; or on a soil carbon (C) basis as mg NkgC-1h-1; and on m2 basis by using the bulk density. Measurement of NH4 ? and 15N-NH4 by SPINMASS The KCl extracts were analysed for 15N-NH4 using the SpinMass (Sample Preparation of Inorganic Nitrogen MASSpectrometer) at ISOGOT labs by automated determination of NH4-N and its 15N abundance in liquid samples. The procedure is a reaction of the KCl extracted sample (c. 9 mL) containing NH4 ? with NaOBr solution (c. 1 mL) by the Rittenberg reaction (Stange 2007) and then transfer of the produced N2 gas into the quadrupole mass spectrometer (QMS, GAM 400, InProcess Instruments GmbH, Bremen). The QMS determines 14N14N (mass 28), 14N15N (mass 29) and 15N15N (mass 30) in N2 gas from which the 15N enrichment and concentration of NH4 ? was calcu- lated. The minimum amount required (detection limit) at SpinMass for d15N in ammonium is 1 lg N. The turnover time was calculated by dividing the pool size (NH4-N or FAA-N) by the production rate (depolymerization rate or gross N mineralization rate). Half-life (t1/2) of a pool is the time when half of a pool size would be produced (t1/2 = turnover/2). Half-life is a measure that compares to results from studies of amino acids with 14C methods. The microbial N use efﬁciency (NUE) is the efﬁciency of N immobilization and is calculated as NUE = (CFAA ? CNH4–M)/(CFAA ? CNH4); con- sumption of FAA and NH4 ? express the microbial N uptake and gross N mineralization (M) express the microbial N loss (Wild et al. 2018). Free amino acids The total FAAs content from the soil extracts (on dry soil base) was 2 to 3 times higher in March compared to November (not signiﬁcant; Table 1). Individual FAAs in soil extracts were more variable in March than in November (Table S1) and were dominated by the presence of aspartic acid, glutamic acid, serine and threonine on both dates (Fig. 2a). Ten out of 14 FAAs were either signiﬁcantly, or by tendency, different at the two dates, but with no signiﬁcant effect of CO2 treatment (Table S2). Soil water total FAA concen- tration was c. 30% higher in the lower than in the upper lysimeter (Table S1). Soil water was dominated by the amino acids serine, glycine and lysine (Fig. 2b and Table S1), with signiﬁcant effect of time only for glycine and lysine (Table S2). Statistical testing The effect of the factors (CO2 treatment, time and depth when available) on the variables was assessed using ‘‘R’’ software, by ﬁtting linear mixed effect models with the function ‘‘lmer’’ from package ‘‘lm4’’ (Bates 2017). When only one time point was available, ring was included as random effect. For variables with repeated measures, the ﬁt of a model with sub-plot within ring as a random effect was tested against a model with only ring as random effect. AIC (Akaike Information Criterion) of the latter was always lower and thus, only ring was retained as a random effect in The method to assess the individual free amino acid (FAA) content and isotopic enrichment is described in detail elsewhere (Wanek et al. 2010; Andresen et al. 2015, 2016a). In short, at arrival at ISOFYS lab, the SPE columns were washed with 10 mL of ultrapure water after which the FAAs were eluted with 30 mL 3 MolL-1 NH4OH. The NH4OH extraction solvent was removed by evaporation at reduced pressure (c. 30 mbar by rotovap), and amino acids were deriva- tized by ethyl chloroformate in ethanol/pyridine to 12 3 3 Biogeochemistry (2020) 150:297–312 302 affected by elevated CO2 treatment or time (month), either on the dry soil weight or the soil carbon base (Table 2). Only the ammonium consumption rate (CNH4) signiﬁcantly differed between the two dates (0.007) while the interaction of sampling date and treatment had marginal difference (p = 0.058) (Table 2). In the aCO2 treatment, CNH4 was higher in November compared to March (time p = 0.007 and time 9 treatment; Table 2). this case. The reason is the low variability across sub- plots within rings. The normality of the residuals of each model was tested and log transformations performed when necessary. The predicted values from the model output were extracted using the ‘‘predict’’ function from base R. Finally, the signiﬁcance of the effect of the factors was tested performing an ANOVA (analyses of variance) with the ‘‘Anova’’ function [‘‘car’’ package (Fox 2017)] and Kenward-Roger degrees of freedom. A signiﬁcant effect is reported if p \ 0.05; and 0.1 [ p [ 0.05 as a tendency. Soil properties The soil C to N ratio was unaffected by CO2 treatments (aCO2: 13.4 ± 3.0 mean and standard error; n = 12 and eCO2: 14.1 ± 3.6; n = 12). Total soil N (0.02%) and C (0.2%) was higher in March than in November (p = 0.0015 and p = 0.0007 respectively; Table 1). Soil NO3 - and NH4 ? content and gravimetric soil moisture, at either depth or date, did not signiﬁcantly respond to CO2 treatment (Fig. 1; Table 1). The soil nitrogen cycle Based on our observations we can characterize the main pools and ﬂuxes of N in this ecosystem (Fig. 4 and Tables 3 and 4). The pool size (g N m-2) of Nin was two to ﬁve times larger than the FAA pool (F(1,41) = 415.7, p \ 0.001; linear mixed model), especially in November, but was not signiﬁcantly different between the two dates or CO2 treatments. Depolymerization rate DSON was a factor three to four greater than M (F(1,41) = 79.3, p \ 0.001; linear mixed model), and the CFAA was a factor 8 to 12 greater than CNH4 (F(1,40) = 340, p \ 0.001; linear mixed model). Consequently, the turnover of the FAA amino acid pool was faster (4 h) than the turnover of the NH4 ? pool (11 h) (Fig. 4 and Table 4). The half-life of the NH4 ? pool was on average 5 h and was around 1.5 h for the FAA pool (98 min; Table 4). In March, NUE was 84% in elevated CO2 and 90% in control treatments. Discussion This study is one of the ﬁrst relating gross N mineralization and depolymerization rates and N pools in Australian soils using 15N techniques. Using a 14C approach, Farrell et al. (2013) determined amino acid and peptide half-life (alanine, dialanine and trialanine) across grassland, bush and forest in Western Australia. With 15N techniques, Ru¨tting & Hovenden (2020) determined gross N mineralization rate in a Tasmanian grassland (TasFACE). Our study is however, the ﬁrst conducted in Australian woodland within an eCO2 ﬁeld facility (EucFACE). Results Soil properties Gross rates Soil moisture and soil organic matter measures were made only at one time point and thus tim and interactive effects were not determined n.d. Data was log transformed when necessary g y Table 1 Soil properties in 0 to 10 cm depth Gravimetric soil moisture (%, by weight loss at 100 C), soil organic matter content (%, by loss on ignition at 550 C), total soil nitrogen and carbon (%, by IR mass spectrometry), total soil d15N (%) and d13C (%, by isotope ratio mass spectrometry), soil NO3, NH4, PO4 and total free amino acids (FAA, mg kg-1 dry soil). The average (n = 3) with standard error in parenthesis for ambient (aCO2) and elevated (eCO2) CO2 treatments (EucFACE) sampled on November 17, 2014 and March 10, 2015 Statistical signiﬁcance was tested by linear mixed effect model and ANOVA with treatment, time and the interaction of time and treatment as factors. F values are presented with P value in parenthesis, when a signiﬁcant effect (\ 0.05) was observed. Non- signiﬁcant effects are indicated as ns. Soil moisture and soil organic matter measures were made only at one time point and thus time and interactive effects were not determined n.d. Data was log transformed when necessary Statistical signiﬁcance was tested by linear mixed effect model and ANOVA with treatment, time and the interaction of time and treatment as factors. F values are presented with P value in parenthesis, when a signiﬁcant effect (\ 0.05) was observed. Non- signiﬁcant effects are indicated as ns. Soil moisture and soil organic matter measures were made only at one time point and thus time and interactive effects were not determined n.d. Data was log transformed when necessary was very high (84% in eCO2 to 90% in aCO2), thus leaving a 10–16% margin of nutrients potentially available for plants. High NUE is usually related to N limitation of microbes, but a wide range in NUE has been observed for soils with C to N ratios below 20 (Mooshammer et al. 2014). The EucFACE soil C to N ratio of 13 to 15 suggest this is not an ecosystem with N limited plant growth. Thus according to the meta- study by Terrer et al. (2019), a positive biomass organic sources of N are available in soil for plant uptake. Gross rates Our results serve to enhance the understanding of soil N cycling and ecosystem response to elevated CO2. By determining available soil N stocks and their turnover rate, we showed that both inorganic and Both gross N mineralization, depolymerization and the amino acid consumption rates (M, DSON and CFAA; Fig. 3) were highly variable but were not signiﬁcantly 12 123 Biogeochemistry (2020) 150:297–312 303 organic sources of N are available in soil for plant uptake Even though our 15N labelling experiment did was very high (84% in eCO2 to 90% in aCO2), thu leaving a 10 16% margin of nutrients potential Table 1 Soil properties in 0 to 10 cm depth aCO2 eCO2 time treatment time*treatme Soil moisture (%) November 2.0 (0.3) 1.9 (0.2) n.d n.d n.d March 3.6 (0.5) 4.1 (0.6) Soil organic matter (%) November n.d n.d n.d n.d n.d March 2.7 (0.2) 2.9 (0.3) Soil nitrogen (%) November 0.10 (0.01) 0.10 (0.01) 11.591 (0.0015) ns ns March 0.12 (0.01) 0.11 (0.01) Soil carbon (%) November 1.3 (0.1) 1.4 (0.1) 13.67 (0.0007) ns ns March 1.5 (0.1) 1.6 (0.2) Soil d15N (%) November 2.4 (0.2) 2.7 (0.2) ns ns ns March 2.3 (0.2) 2.7(0.3) Soil d13C (%) November -25.6 (0.2) -26.1 (0.2) ns ns ns March -25.8 (0.2) -26.4 (0.2) Soil NO3 (mg•kg-1) November 1.71 (0.15) 1.92 (0.19) ns ns ns March 2.04 (0.33) 1.68 (0.35) Soil NH4 (mgkg-1) November 1.29 (0.45) 1.74 (0.61) ns ns ns March 1.56 (0.55) 1.40 (0.17) FAA (mgkg-1) November 0.6 (0.2) 0.3 (0.1) ns ns ns March 1.2 (0.5) 1.0 (0.6) Gravimetric soil moisture (%, by weight loss at 100 C), soil organic matter content (%, by loss on ignition at 550 C), total so nitrogen and carbon (%, by IR mass spectrometry), total soil d15N (%) and d13C (%, by isotope ratio mass spectrometry), soil NO NH4, PO4 and total free amino acids (FAA, mg kg-1 dry soil). The average (n = 3) with standard error in parenthesis for ambie (aCO2) and elevated (eCO2) CO2 treatments (EucFACE) sampled on November 17, 2014 and March 10, 2015 Statistical signiﬁcance was tested by linear mixed effect model and ANOVA with treatment, time and the interaction of time a treatment as factors. F values are presented with P value in parenthesis, when a signiﬁcant effect (\ 0.05) was observed. No signiﬁcant effects are indicated as ns. Gross rates Even though our 15N labelling experiment did not directly involve plants, results from other exper- iments have demonstrated Eucalyptus plant uptake of intact FAAs (Warren 2006, 2009). Other experiments using 15N tracing suggest that the competition between plants and microbes for N sources is dominated by microbial uptake (Warren 2009; Kuzyakov 2013). We found that the microbial nutrient use efﬁciency NUE 12 3 3 Biogeochemistry (2020) 150:297–312 304 Fig. 1 Nitrate and ammonium content (mgkg-1 dry soil) in November (Nov) and in March, in the 0 to 2 cm and the 0 to 10 cm soil depths, in ambient CO2 (aCO2) and elevated CO2 (eCO2) treatments. The black horizontal lines inside boxes represent the median and the upper and lower quartiles are represented by bottom and top borders. Whiskers represent the range and dots represent outliers that are 1.5 times the interquartile range. F and P values are presented for nitrate indicating a tendency of effect of depth and of an interaction between depth and treatment (linear mixed effect models and ANOVAs) range and dots represent outliers that are 1.5 times the interquartile range. F and P values are presented for nitrate indicating a tendency of effect of depth and of an interaction between depth and treatment (linear mixed effect models and ANOVAs) Fig. 1 Nitrate and ammonium content (mgkg-1 dry soil) in November (Nov) and in March, in the 0 to 2 cm and the 0 to 10 cm soil depths, in ambient CO2 (aCO2) and elevated CO2 (eCO2) treatments. The black horizontal lines inside boxes represent the median and the upper and lower quartiles are represented by bottom and top borders. Whiskers represent the response to eCO2 is likely, based on this soil’s C to N ratio. plantations in Portugal on Cambisols (Mediterranean climate) (Go´mez-Rey et al. 2010). Finally, the half- life of FAA in EucFACE soils were within the same range as those observed in 14C-labelled amino acids experiments in Australian woodland soils (Farrell 2013; Prendergast-Miller et al. 2015). Hence, we consider the N dynamics of this Cumberland Plain woodland to be typical of woodlands in similar climatic regions or similar soils. In the lab incubations, the amount of plant accessible N made available through depolymerization (e.g. FAA-N release) was three to four times the amount made available by gross N mineralization (e.g. NH4-N release). Gross rates Hence, the amino acid pathway is more efﬁcient than the gross N mineralization in making N available for plant uptake (Warren 2006, 2009). This plant driven short circuit- ing of the N-cycle (Chapin III 1995), is thus an important part of the N cycle as we hypothesized. With the gross N mineralization rate being slower than depolymerisation in our soils, gross mineralization, rather than depolymerisation, is the limiting step for Nin availability. Hence, according to the paradigm suggested by Schimel and Bennett (2004), and mod- iﬁed by Jilling et al. (2018), this Cumberland Wood- land has signiﬁcant NH4 ? and monomer N sources, either from readily available detritus or from MAOM. Further, both gross N mineralization and depolymer- ization rates are potential controlling factors for plant N availability (Schimel & Bennett 2004; Jilling et al. 2018) The EucFACE soil pool of FAA-N was smaller than the Nin pool. Only a few studies of natural (unfertilized) Mediterranean or Australian soils pro- vide parallel N content on FAA and Nin pools. Soil content of FAAs and Nin across three woodlands on Brown Sodosoil (with soil C to N ratios of 19 to 30) in New South Wales had smaller or similar content of FAA as Nin (Prendergast-Miller et al. 2015). Smaller or similar FAA content as Nin was found across natural soils in Western Australia (Farrell 2013), and a trend with less FAA-N than Nin was found in a grassy woodland ecosystem dominated by Eucalyptus mel- liodora, and Blakely’s Red Gum (Eucalyptus blakelyi) near Canberra (with soil C to N ratio of 12 to 17) (Macdonald et al. 2014). Gross N mineralization rates in the EucFACE soils were within the same order of magnitude as rates measured in an Eucalyptus forest on Durustalf soil with a clayey texture in Western Australia (Mediter- ranean climate) (Banning et al. 2008). In addition, gross N mineralization and ammonium consumption rates in our study were within one order of magnitude as measured in a native forest in Queensland (Aus- tralia), that had a subtropical climate and higher soil moisture (Burton et al. 2007) compared to our study. Furthermore, the EucFACE gross N mineralization rate fell within the high-end range of gross N mineralization rate measured in ﬁve Eucalyptus 123 123 305 Biogeochemistry (2020) 150:297–312 Fig. Gross rates The black horizontal lines inside boxes represent the median and the upper and lower quartiles are represented by bottom and top borders. Whiskers represent the range. Dots represent outliers that are 1.5 times the interquartile range. F and P values are presented for CFAA indicating signiﬁcant effect of time and the tendency of an interaction between time and treatment (linear mixed effect models and ANOVAs) Fig. 3 Gross N mineralization rate (M) and NH4 ? consumption rate (CNH4) in November and March (depth 0 to 10 cm), and depolymerization rate (DSON) and amino acid consumption rate (CFAA) in March (depth 0 to 10 cm) in ambient CO2 and elevated CO2 treatments. All rates are in mg N kg-1 dry soil hour-1. The black horizontal lines inside boxes represent the median and the upper and lower quartiles are represented by bottom and top borders. Whiskers represent the range. Dots represent outliers that are 1.5 times the interquartile range. F and P values are presented for CFAA indicating signiﬁcant effect of time and the tendency of an interaction between time and treatment (linear mixed effect models and ANOVAs) Individual amino acids from the two pools (CaSO4 extracts and soil lysimeter water) represent the pool of available FAAs to plants and are made available by depolymerization and rhizodeposition. In addition, cell destruction may potentially bias the content as an artefact (Hobbie & Hobbie 2012). Soil FAA extracts were dominated at both sampling times by aspartic acid, glutamic acid, serine and threonine. These are acidic (aspartic and glutamic acid) and polar (serine and threonine) amino acids which, due to their electronegativity (soil pH 5.5), are mobilized by extraction (Warren & Taranto 2010). The soil water did have a different set of dominant FAAs compared to the soil CaSO4 extracts. The basic amino acid lysine, which is a positively charged compound, dominated in the soil water along with serine and glycine. These are both low molecular weight amino acids and will likely emerge from degradation and destabilization of larger amino acid molecules prior to leaching (glutamine, asparagine and threonine have serine as basic struc- ture; likewise, glycine is the smallest fundamental structure of all amino acids). The larger FAA concen- tration in the lower than upper lysimeter water suggest either a leaking of FAAs down in the soil or a less intense FAA consumption and gross N mineralization in the lower soil layer. Gross rates 2 Free a amino acid nitrogen (lg amino acid Ng-1 dry oil) in soil extracts (10 mMolL-1 CaSO4) in November and March (0 to10 cm depth). Data is presented as average of ambient (aCO2) and elevated (eCO2) treatments within each ime point; b amino acid nitrogen (lg N amino acid L-1) in soil water in November from upper (15 to 20 cm) and lo 60 cm) lysimeters. Data is presented as average (aCO2) and elevated (eCO2) treatments. Error bars a error. Data is presented in Table S1 and signiﬁcan Table S2 Biogeochemistry (2020) 150:297–312 Fig. 2 Free a amino acid nitrogen (lg amino acid Ng-1 dry soil) in soil extracts (10 mMolL-1 CaSO4) in November and March (0 to10 cm depth). Data is presented as average of ambient (aCO2) and elevated (eCO2) treatments within each time point; b amino acid nitrogen (lg N amino acid L-1) in soil water in November from upper (15 to 20 cm) and lower (30 to 60 cm) lysimeters. Data is presented as average of ambient (aCO2) and elevated (eCO2) treatments. Error bars are standard error. Data is presented in Table S1 and signiﬁcant effects in Table S2 1 Fig. 2 Free a amino acid nitrogen (lg amino acid Ng-1 dry soil) in soil extracts (10 mMolL-1 CaSO4) in November and March (0 to10 cm depth). Data is presented as average of ambient (aCO2) and elevated (eCO2) treatments within each time point; b amino acid nitrogen (lg N amino acid L-1) in soil water in November from upper (15 to 20 cm) and lower (30 to 60 cm) lysimeters. Data is presented as average of ambient (aCO2) and elevated (eCO2) treatments. Error bars are standard error. Data is presented in Table S1 and signiﬁcant effects in Table S2 water in November from upper (15 to 20 cm) and lower (30 to 60 cm) lysimeters. Data is presented as average of ambient (aCO2) and elevated (eCO2) treatments. Error bars are standard error. Data is presented in Table S1 and signiﬁcant effects in Table S2 12 3 306 Biogeochemistry (2020) 150:297–312 Fig. 3 Gross N mineralization rate (M) and NH4 ? consumption rate (CNH4) in November and March (depth 0 to 10 cm), and depolymerization rate (DSON) and amino acid consumption rate (CFAA) in March (depth 0 to 10 cm) in ambient CO2 and elevated CO2 treatments. All rates are in mg N kg-1 dry soil hour-1. Gross rates The time steps 10 min to 7 h were used for calculating all rates by the analytical equations developed by Kirkham and Bartholomew (1954) Statistical signiﬁcance was tested by linear mixed effect model and ANOVA with treatment (for DSON and CFAA with only one time point) and time, and the interaction of time and treatment, were factors. F values are presented with P-value in parenthesis, when a signiﬁcant effect (\ 0.05) or a tendency (\ 0.1). Non-signiﬁcant effects are indicated as ns and n.d. means not determined. Data was log transformed when necessary litter chemistry (Crous et al. 2019). Water availability, which was low during both samplings, controls degradation and biological activity, along with root exudation (Bengtson et al. 2012). These factors are both theoretically enhanced under elevated CO2 (Gimeno et al. 2018). Even though observations at the EucFACE indicated no CO2 stimulation of plant growth, there were ﬂuctuating stimulations of photo- synthesis and belowground C allocation. Therefore, potential stimulation of priming of SOM and MAOM degradation followed by mineralization would theo- retically have been possible (Duursma et al. 2016; Ellsworth et al. 2017; Collins et al. 2018; Drake et al. 2018; Jilling et al. 2018). As a parallel, a lack of response in depolymerization and gross N litter chemistry (Crous et al. 2019). Water availability, which was low during both samplings, controls degradation and biological activity, along with root exudation (Bengtson et al. 2012). These factors are both theoretically enhanced under elevated CO2 (Gimeno et al. 2018). Even though observations at the EucFACE indicated no CO2 stimulation of plant growth, there were ﬂuctuating stimulations of photo- synthesis and belowground C allocation. Therefore, potential stimulation of priming of SOM and MAOM degradation followed by mineralization would theo- retically have been possible (Duursma et al. 2016; Ellsworth et al. 2017; Collins et al. 2018; Drake et al. 2018; Jilling et al. 2018). As a parallel, a lack of response in depolymerization and gross N mineralization rates to CO2 was also found in a nutrient poor heathland (Larsen et al. 2011; Holmstrup et al. 2017; Wild et al. 2018). Our study spanned surface soils (0–10 cm), where most of the root inﬂuence occurs in this system (Ochoa-Hueso et al. 2017). Gross rates From the spring (November) to the late summer (March) sampling time, soil properties did change slightly, whereby higher total C, total N and FAA content were observed in late summer, which corre- sponded to a smaller ammonium consumption in late summer. However, the lack of strong responses in N transformation rates to eCO2 at EucFACE were in accordance with our hypothesis and with the observed lack of increase in plant growth, litter inputs (Ells- worth et al. 2017; Jiang 2020) and changes in senesced 12 123 Biogeochemistry (2020) 150:297–312 307 Table 2 Nitrogen turnover rates in the EucFACE soils Month CO2 treatment M gross N mineralization rate CNH4 gross N consumption rate DSON depolymerization rate CFAA amino acid consumption rate mg Nkg-1 dry soilh-1 mg Nkg-1 dry soilh-1 mg Nkg-1 dry soilh-1 mg Nkg-1 dry soilh-1 mg NkgC-1h-1 mg NkgC-1h-1 mg NkgC-1h-1 mg NkgC-1h-1 November aCO2 0.098 (0.01) 0.234 (0.06) 7.721 (0.79) 16.531(3.27) n.d n.d November eCO2 0.081 (0.02) 0.108 (0.02) 6.407 (1.74) 7.789 (1.09) n.d n.d March aCO2 0.111 (0.02) 0.115 (0.02) 0.460 (0.04) 0.954 (0.05) 7.429 (1.16) 7.683 (0.92) 30.174 (1.81) 64.047 (3.19) March eCO2 0.159 (0.07) 0.083 (0.01) 0.419 (0.04) 0.894 (0.08) 10.563 (4.08) 5.908 (1.13) 30.554 (4.62) 67.247 (9.12) Linear mixed model time: ns time: 8.38 (0.007) treatment: ns treatment: ns treatment: ns treatment: ns time 9 treatment: ns time 9 treatment: 3.83 (0.058) time: ns time: 10.17 (0.003) treatment: ns treatment: ns treatment: ns treatment: ns time 9 treatment: ns time 9 treatment: ns Table 2 Nitrogen turnover rates in the EucFACE soils Data is presented as mean values with standard errors in parenthesis. Gross nitrogen mineralization rate (M) and consumption rate (CNH4) are expressed as mg NH4-Nkg-1 dry soilh-1 and in italics mg NH4-NkgC-1h-1. Gross depolymerization rate DSON (amino acid production) and amino acid consumption rate CFAA are expressed as mg AA-Nkg-1dry soilh-1 and in italics as mg AA- Nkg-1h-1. M and CNH4 were obtained by the pool dilution assay using 15N-labelled NH4 in November and March. DSON and CFAA were obtained by the pool dilution assay using 15N-labelled amino acids only in March. Gross rates However, as N supplies and root density (and thus activity) changes with depth, we cannot rule out that a response of soil N cycling to CO2 occurs in deeper soil layers (Za¨hle 2014). Although root C and N dynamics (Castan˜eda- Go´mez et al. 2020) were not part of our study, the behaviour of the soil microbes in the short-term incubation is driven by impacts from long-term exposure to experimental treatments which includes 12 123 123 308 Biogeochemistry (2020) 150:297–312 4 Conceptual diagram for an area of one (m2) in the top m soil layer of average soil nitrogen (N) pools and ﬂuxes, he aCO2 (a) and eCO2 (b) treatments in March 2014. Pools l soil N, amino acid N (AA-N), ammonium-N (NH4 ?) and te–N (NO3 -)) are presented in gNm-2. Fluxes (gross N ralization rate (M), NH4 ? consumption rate (CNH4), depolymerization rate (DSON) and amino acid consumption (CFAA)) are presented in gNm-2day-1. Turnover ti indicated by circular arrows for AA-N and NH4 ? pools, is time needed to produce the entire pool. Data and standard er are presented in Table 3 Biogeochemistry (2020) 150:297– Fig. 4 Conceptual diagram for an area of one (m2) in the top 10 cm soil layer of average soil nitrogen (N) pools and ﬂuxes, for the aCO2 (a) and eCO2 (b) treatments in March 2014. Pools (total soil N, amino acid N (AA-N), ammonium-N (NH4 ?) and nitrate–N (NO3 -)) are presented in gNm-2. Fluxes (gross N mineralization rate (M), NH4 ? consumption rate (CNH4), depolymerization rate (DSON) and amino acid consump (CFAA)) are presented in gNm-2day-1. Turnove indicated by circular arrows for AA-N and NH4 ? pool time needed to produce the entire pool. Data and standa are presented in Table 3 Fig. 4 Conceptual diagram for an area of one (m2) in the top 10 cm soil layer of average soil nitrogen (N) pools and ﬂuxes, for the aCO2 (a) and eCO2 (b) treatments in March 2014. Pools (total soil N, amino acid N (AA-N), ammonium-N (NH4 ?) and nitrate–N (NO3 -)) are presented in gNm-2. Fluxes (gross N mineralization rate (M), NH4 ? consumption rate (CNH4), depolymerization rate (DSON) and amino acid consumption rate (CFAA)) are presented in gNm-2day-1. Turnover time, indicated by circular arrows for AA-N and NH4 ? pools, is the time needed to produce the entire pool. Gross rates Data and standard errors are presented in Table 3 depolymerization rate (DSON) and amino acid consumption rate (CFAA)) are presented in gNm-2day-1. Turnover time, indicated by circular arrows for AA-N and NH4 ? pools, is the time needed to produce the entire pool. Data and standard errors are presented in Table 3 Fig. 4 Conceptual diagram for an area of one (m2) in the top 10 cm soil layer of average soil nitrogen (N) pools and ﬂuxes, for the aCO2 (a) and eCO2 (b) treatments in March 2014. Pools (total soil N, amino acid N (AA-N), ammonium-N (NH4 ?) and nitrate–N (NO3 -)) are presented in gNm-2. Fluxes (gross N mineralization rate (M), NH4 ? consumption rate (CNH4), 12 23 Biogeochemistry (2020) 150:297–312 309 Table 3 Mean soil nitrogen (N) pools and rates on area (m2) basis in the 0 to 10 cm soil depth of the ambient (aCO2) and elevated CO2 (eCO2) treatments Nutrient pool or ﬂux Treatment November 2014 March 2015 Total soil N g N m-2 aCO2 133.8 (20.7) 160.3 (29.4) Total soil N g N m-2 eCO2 130.8 (14.7) 146.2 (26.1) Inorganic N (NO3 ? NH4) g N m-2 aCO2 0.20 (0.04) 0.24 (0.05) Inorganic N (NO3 ? NH4) g N m-2 eCO2 0.25 (0.08) 0.20 (0.01) Amino acid N g N m-2 aCO2 0.115 (0.04) 0.219 (0.10) Amino acid N g N m-2 eCO2 0.050 (0.02) 0.175 (0.10) NH4 consumption rate g AA-N m-2 day -1 aCO2 0.671 (0.382) 0.388 (0.109) NH4 consumption rate g AA-N m-2 day -1 eCO2 0.342 (0.009) 0.269 (0.071) Gross N-mineralization rate g AA-N m-2 day -1 aCO2 0.314 (0. Gross rates 061) 0.376 (0.067) Gross N-mineralization rate g AA-N m-2 day -1 eCO2 0.281 (0.045) 0.535 (0.201) Depolymerization rate g AA-N m-2 day -1 aCO2 n.d 1.477 (0.145) Depolymerization rate g AA-N m-2 day -1 eCO2 n.d 1.428 (0.146) AA-N consumption rate g AA-N m-2 day -1 aCO2 n.d 3.197 (0.281) AA-N consumption rate g AA-N m-2 day -1 eCO2 n.d 3.168 (0.351) Numbers in parenthesis are standard error (n = 3) Table 4 Nitrogen (N) turnover time (hours) for the ammonium (NH4-N) and the free amino acid (FAA-N) pools based on gross N mineralization and depolymerization rate respectively, for the ambient (aCO2) and elevated CO2 (eCO2) treatments Turnover Treatment November 2014 March 2015 t1/2 NH4-N aCO2 11 11 5 eCO2 16 7 3 FAA-N aCO2 n.d 4 107 (min) eCO2 n.d 3 88 (min) Data is presented in Table 3. Half-life (t1/2) of the pool in March in hours for NH4 and in minutes for AAs rnover time (hours) for the ammonium (NH4-N) and the free amino acid (FAA-N) pools based on gross N ymerization rate respectively, for the ambient (aCO2) and elevated CO2 (eCO2) treatments Table 4 Nitrogen (N) turnover time (hours) for the ammonium (NH4-N) and the free amino acid (FAA-N) mineralization and depolymerization rate respectively, for the ambient (aCO2) and elevated CO2 (eCO2) tr a is presented in Table 3. Half-life (t1/2) of the pool in March in hours for NH4 and in minutes for AAs the direct and indirect root inﬂuence (Bengtson et al. 2012; Ochoa-Hueso et al. 2017). increase in both above ground and below ground plant growth following P additions (Crous et al. 2015; Nielsen et al. 2015). When P (and not N) is limiting, increased rhizodeposition may not result in enhanced soil organic matter decomposition. Thus a stimulation of N mineralization is prevented because microbial P limitation forces microorganisms to target P rather than N (Dijkstra et al. 2013). Therefore future studies should target the P dynamics in the EucFACE experiment to better understand ecosystem response to eCO2 in nutrient poor systems. The weak or missing N cycle response under FACE treatment in some ecosystems – grassland, heathland or forest observed here and elsewhere (Wild et al. 2018; Schleppi 2019; Ru¨tting 2020), could be due to limitation of another nutrient, such as P (Dijkstra et al. 2013; Ru¨tting & Andresen 2015). Gross rates At EucFACE, evidence derived from Eucalyptus leaf C:N:P stoi- chiometry revealed strong P re-translocation com- pared to N in both ambient and CO2 treated trees (Crous et al. 2019), emphasising that P is highly limiting for the trees in this ecosystem. Phosphorus limitation within this ecosystem was further supported by evidence from a P-addition experiment near the experimental rings that demonstrated a signiﬁcant Acknowledgements The Hawkesbury Institute for the Environment’s Research Exchange grant to Tobias Ru¨tting and Louise C. Andresen in 2014. EucFACE is supported by the Australian Commonwealth government in collaboration with the Western Sydney University. This is part of a TERN Super- 12 3 3 Biogeochemistry (2020) 150:297–312 310 MC (2016b) Shifting impacts of climate change. In: Dumbrell AJ, Kordas RL & Woodward G (eds) Large- scale ecology: model systems to global perspectives. Advances in Ecological Research, p 437–473 site facility. EucFACE was built as an initiative of the Australian government as part of the Nation-building Economic Stimulus Package. This research attributes to the Swedish strategic research area ‘Biodiversity and Ecosystem services in a Changing Climate’ BECC. We thank John Drake, Vinod Kumar and Craig McNamara for help and advice at the EucFACE ﬁeld site in 2014 and Phillipp Schleussner, Anna- Karin Bjo¨rsne and Joseﬁna Carlberg for help with the SpinMass analysis and Stijn Vandervoorde for help with GC–MS analysis. site facility. EucFACE was built as an initiative of the Australian government as part of the Nation-building Economic Stimulus Package. This research attributes to the Swedish strategic research area ‘Biodiversity and Ecosystem services in a Changing Climate’ BECC. We thank John Drake, Vinod Kumar and Craig McNamara for help and advice at the EucFACE ﬁeld site in 2014 and Phillipp Schleussner, Anna- Karin Bjo¨rsne and Joseﬁna Carlberg for help with the SpinMass analysis and Stijn Vandervoorde for help with GC–MS analysis. Banning NC, Grant CD, Jones DL, Murphy DV (2008) Recovery of soil organic matter, organic matter turnover and nitrogen cycling in a post-mining forest rehabilitation chronosequence. Soil Biol Biochem 40(8):2021–2031 Bates D, Maechler M, Bolker B, Walker S, Christensen RHB, Singmann H, Green, P (2017) lme4: linear mixed-effects models using ‘‘eigen’’ and S4 (Version 1.1–15) Author contribution All authors planned or carried out the 15N-labelling experiments at the HIE with soils from the EucFACE experiment in Richmond, NSW Australia. Analysis of NH4, NO3 and PO4 was carried out at HIE by CM. Funding Open access funding provided by University of Gothenburg. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Castan˜eda-Go´mez L, Walker JKM, Powell JR, Ellsworth DS, Pendall E, Carrillo Y (2020) Impacts of elevated carbon dioxide on carbon gains and losses from soil and associated microbes in a Eucalyptus woodland. Soil Biol Biochem 143:107734 Chapin FS, Bloom AJ, Field CB, Waring RH (1987) Plant responses to multiple environmental factors. Bioscience 37(1):49–57 Chapin FS III (1995) New cog in the nitrogen cycle. Nature 377:199–200 Chen J, Zelikova TJ, Pendall E, Morgan JA, Williams DG (2014) Daily and seasonal changes in soil amino acid composition in a semiarid grassland exposed to elevated CO2 and warming. Biogeochemistry 123(1–2):135–146 Collins L, Bradstock RA, Resco de Dios V, Duursma RA, Velasco S, Boer MM (2018) Understorey productivity in temperate grassy woodland responds to soil water avail- ability but not to elevated [CO2]. Glob Chang Biol 24(6):2366–2376 Gross rates Analysis of amino acids was carried out at ISOFYS labs in Ghent, Belgium by LCA and SB. Analysis of 15N-NH4 extracts was carried out at ISOGOT in Gothenburg, Sweden by LCA and TR. Calculations and statistical analysis done by LCA, TR and LCG. LCA wrote the ﬁrst draft and all authors contributed to the development of the current paper. Bengtson P, Barker J, Grayston SJ (2012) Evidence of a strong coupling between root exudation, C and N availability, and stimulated SOM decomposition caused by rhizosphere priming effects. Ecol Evol 2(8):1843–1852 Burton J, Chen C, Xu Z, Ghadiri H (2007) Gross nitrogen transformations in adjacent native and plantation forests of subtropical Australia. Soil Biol Biochem 39(2):426–433 ˇ Cˇ apek P, Manzoni S, Kasˇtovska´ E, Wild B, Dia´kova´ K, Ba´rta J, Schnecker J, Biasi C, Martikainen PJ, Alves RJE, Guggenberger G, Gentsch N, Hugelius G, Palmtag J, Mikutta R, Shibistova O, Urich T, Schleper C, Richter A, Sˇantru˚cˇkova´ H (2018) A plant–microbe interaction framework explaining nutrient effects on primary produc- tion. Nat Ecol Evol 2:1588–1596 Funding Open access funding provided by University of Gothenburg. Funding Open access funding provided by University of Gothenburg. 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https://openalex.org/W1995141374	https://bmcbiophys.biomedcentral.com/counter/pdf/10.1186/2046-1682-4-12	English	null	Mechanism of PhosphoThreonine/Serine Recognition and Specificity for Modular Domains from All-atom Molecular Dynamics	BMC biophysics	2,011	cc-by	10,010	RESEARCH ARTICLE Open Access © 2011 Huang and Chang; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. * Correspondence: chiaenc@ucr.edu Department of Chemistry, University of California, Riverside, CA92521, USA Abstract Background: Phosphopeptide-binding domains mediate many vital cellular processes such as signal transduction and protein recognition. We studied three well-known domains important for signal transduction: BRCT repeats, WW domain and forkhead-associated (FHA) domain. The first two recognize both phosphothreonine (pThr) and phosphoserine (pSer) residues, but FHA has high specificity for pThr residues. Here we used molecular dynamics (MD) simulations to reveal how FHA exclusively chooses pThr and how BRCT and WW recognize both pThr/pSer. The work also investigated the energies and thermodynamic information of intermolecular interactions. Results: Simulations carried out included wide-type and mutated systems. Through analysis of MD simulations, we found that the conserved His residue defines dual loops feature of the FHA domain, which creates a small cavity reserved for only the methyl group of pThr. These well-organized loop interactions directly response to the pThr binding selectivity, while single loop (the 2nd phosphobinding site of FHA) or in combination with a-helix (BRCT repeats) or b-sheet (WW domain) fail to differentiate pThr/pSer. Conclusions: Understanding the domain pre-organizations constructed by conserved residues and the driving force of domain-phosphopeptide recognition provides structural insight into pThr specific binding, which also helps in engineering proteins and designing peptide inhibitors. (FHA) domain [9]. Among them, the FHA domain differ- entiates pThr-containing peptides from pSer-containing peptides, although the difference is only one methyl group [10-15]. Because Ser/Thr kinase phosphorylates both residues, the FHA domain can efficiently reduce potential interaction sites by specifically binding to pThr- containing regions. Although experimental structures and recent studies have shown important interactions involve in the binding of the methyl group of pThr [14,16], the detailed mechanisms of the phosphoresidue recognition of different domains and how the FHA domain can reserve non-polar interactions for a small non-polar methyl group are not fully understood. Mechanism of PhosphoThreonine/Serine Recognition and Specificity for Modular Domains from All-atom Molecular Dynamics Yu-ming M Huang and Chia-en A Chang* Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Background Protein phosphorylation is widely exploited in DNA damage repair, signal transduction, cell growth and cell cycle regulation; the cascades of downstream signals can be triggered by grabbing a certain phosphoprotein [1-6]. Elucidating the characteristics of phosphopeptide recogni- tion is fundamental to study cellular functions [7]. The phosphoproteins are usually classified into two families, phosphotyrosine (pTyr)-containing and phosphoserine (pSer)/phosphothreonine (pThr)-containing sequences which are phosphorylated and dephosphorylated by dif- ferent categories of kinases (e.g., pThr/pSer kinase and pThr kinase) and phosphatases [8]. Recent studies discov- ered a few modular domains that particularly recognize pThr/pSer- or pThr-containing sequences, such as the breast-cancer-associated protein BRCA1 C-terminal (BRCT) repeats, WW domain and forkhead-associated The FHA domain is associated with proteins of diverse functions in different organisms. For example, the Rad53-FHA1 domain interacts with phosphorylated Rad9 in response to DNA damage, and the Dun1-FHA domain interacts with SCD1 of Rad53, which leads to activation of Dun1in response to DNA damage response [17]. Instead of binding to a single pThr, the formation Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 2 of 13 BRCT repeats in BRCA1 are considered to be related to breast cancer [24-27]. The structure of the tandem BRCA1-BRCT repeats bind to phosphorylated protein that contains pSer or pThr, although binding to pSer is preferred [28]. Several structural studies have revealed a conserved structure for the repeats, mainly composed of a helixes, b sheets and loops that link secondary struc- tures. The phosphoresidue-recognized site is located between b1 and a2 (Figure 1 (b)) [29-31]. of the Dun1-FHA:SCD1 complex requires two phos- phoresidues, which suggests a potential mechanism whereby sequential signaling events could be triggered through the recognition of multiple phosphoresidue- binding sites. Similar to Dun1-FHA, Ki67-FHA domain also recognizes dual phosphorylated residues at the same time [18], and the sequence identities of Dun1- FHA and the Ki67-FHA to Rad53-FHA1 are both 34%. The structure of the FHA domain is mostly organized into a twisted b sandwich of 11 well-defined b sheets, five in the front and six at the back (Figure 1 (a)) [12,14,16,19-23]. The domain contains ~120 to 140 resi- dues, but only five to ten residues are conserved. Six loops connected to the secondary b strands constructing the pThr binding site are the main difference between distinct FHA domains. Background Experimental structures show that the synthetic peptides bind to the loops between b3-b4, b4-b5, b6-b7 and b10-b11, and the conserved pThr binding site locates between loops b4-b5 and b6- b7 (Figure 1 (a)) [12,14]. The WW domain in Pin1 is essential for mitotic pro- gression [32]. The domain has only about 40 residues and is one of the smallest pThr/pSer binding domains [3]. It specifically binds to pThr-Pro- or pSer-Pro-containing motifs with slightly higher affinity for pThr-Pro-containing peptides [33]. For example, in the Pin1-WW domain, the aromatic rings of Tyr23 and Trp34 define a steric clamp to confer a Pro adjacent to pSer/pThr [34]. The WW domain folds into three anti-parallel b stands and Arg21 and Ser22 residues in the loops between b1 and b2 are the phosphate group recognition sites (Figure 1 (c)) [3,4,34]. Figure 1 Overall architecture of signaling domains. (a) Snapshot of Rad53-FHA1 molecular dynamics (MD) simulations. Pink and blue represent front and back loop/b-strand respectively. (b) Snapshot of an MD simulation for BRCT repeats in BRCA1. (c) WW domain in Pin1 protein. Figure 1 Overall architecture of signaling domains. (a) Snapsh Figure 1 Overall architecture of signaling domains. (a) Snapshot of Rad53-FHA1 molecular dynamics (MD) simulations. Pink and blue represent front and back loop/b-strand respectively. (b) Snapshot of an MD simulation for BRCT repeats in BRCA1. (c) WW domain in Pin1 t i Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 3 of 13 phosphoresidues are not defined in the ff99sb force field, we used the pThr and pSer force field developed by Homeyer et al. [39]. All simulations of wild-type sequences were initialed from six experimental com- plexes. In this work, we studied six un-mutated com- plexes and eight mutated structures. The protonation states were checked by the MCCE program [40]. All complexes were solvated in a rectangular box of 12 Å TIP3P water with the tleap program in Amber10 [41]. The placement of counter-ions of Na+ was based on the Columbic potential to keep the whole system neutral. Particle Mesh Ewald was used to consider the long- range electrostatic interactions [42]. Following 10,000 and 20,000 steps of minimization of the water and sys- tem, respectively, the systems were gradually heated for each complex from 250 K for 20-ps, 275 K for 20-ps and 300 K for 200-ps. Molecular systems We selected three FHA domains in different families, BRCT repeats and the WW domain. One of the FHA domains is the human Chk2 homolog in yeast, Rad53- FHA1, involved in checkpoint signaling in Saccharo- myces. Serevisiae. The target protein of Rad53, Rad9, is phosphorylated in response to DNA damage and inter- acts with the C-terminal FHA1 domain of Rad53. Two initial structures are from crystallographic coordinates (Protein Data Bank (PDB) code 1G6G) [12] and NMR structure (PDB code 1K3Q) [16]. Both Rad53-FHA1 domains share the same protein sequence. Another FHA domain is from the Dun1 checkpoint kinase. The Dun1-FHA domain interacts with phosphorylated SCD1 of Rad53, which leads to activation of Dun1. The Dun1- FHA and SCD1complex of the domain-peptide structure is acquired from the PDB code 2JQL[35]. The other sys- tem of the FHA domain near the N-terminus of human Ki67 antigen protein that interacts with human nucleo- lar protein hNIFK was studied a few years ago. The structural complex of Ki67-FHA and a 44-residues frag- ment in phosphorylated hNIFK is explored by the coor- dinates of the PDB code 2AFF[18]. We chose to study the pThr/pSer binding modular domain (PDB code 1T2V) of the complex of the BRCT domain in the BRCA1 C-terminus and its target binding partner, BRCH1 [29]. Another phosphodomain, the WW domain from the Pin1 N-terminus, interacts with the heptapho- phorylated peptide in the CTD domain (PDB code 1F8A) [34]. Although the peptide contains two pSer residues, only one binds to the domain. All peptide sequences are in Table 1. To study the recognition for phosphoresidue and how FHA domains differentiate the pThr/pSer residue, we manually mutated pThr to pSer or pSer to pThr in phosphopeptides. The mutated sites are shown in Table 1. Binding energy calculation by MM-PBSA methods and entropy calculation To quantify the stability of the phosphopeptide binding to the domain, we performed end-point energy calcula- tions, also known as MM-PBSA/MM-GBSA calculations [44-50]. A simple thermodynamic cycle and single-tra- jectory post-processing allows for efficiently computing the various contributions to the domain-peptide binding. We used the structural ensemble obtained from the final 1-ns of each random number seed to demonstrate the post-energy calculations. The binding interaction energy, ΔEbind, associated with the binding of a domain to its cognate peptide to form a protein-peptide complex is calculated as follows: Background To initial the mutated structures, after equilibrium from 300 K, we manually added or deleted the methyl group and changed the residue name accordingly, then used the Amber program to build the prmtop files for mutants. A quick 100-steps minimiza- tion was applied to the mutants, then we preformed 20- ps equilibrium at 300 K. All MD simulations for each wild-type and mutated complex was performed in 1 ns by five different random number seeds to generate dif- ferent initial velocity. The resulting trajectories were col- lected every 1 ps and the time step was 2-fs. The NPT ensemble was applied, and periodic boundary conditions were used throughout the MD simulations. A tempera- ture of 300 K was maintained with use of a Langevin thermostat, with a damping constant of 2 ps-1, and the hybrid Nose-Hoover Langevin piston method was used to control the pressure at 1 atm. The SHAKE procedure was used to constrain hydrogen atoms during MD simu- lations [43]. Because FHA domains are pThr specific modular domains, this study focused on how FHA domains dis- play selective for pThr/pSer residues and comparison with BRCT and WW domains. We study the dynamic and conformational changes of the free domain and the complexes of Rad53-FHA1:Rad9, Dun1-FHA:SCD1, Ki67-FHA:hNIFK, BRCA1-BRCT:BRCH1 and Pin1-WW: CTD systems. We also computed the interaction energy between pThr/pSer and the domains to disclose the driving force of pThr/pSer binding. We propose a model for pThr specificity and potential applications. Results and discussion Ser and Thr have very similar sidechains, so the Ser/Thr protein kinases phosphorylate the OH group of either residue without discriminating between them. Similarly, most pSer/pThr binding modules, such as BRCT and WW domains, can specifically bind to short pSer/pThr- containing motifs equally well. Not unsurprisingly, one more methyl group of Thr has few effects on the overall binding and molecular recognition. However, uniquely, most FHA domains recognize only pThr residues in tar- get proteins, and substitution of pSer for pThr in model peptides severely weakens binding. The pThr specific recognition may function as a filter to further select the protein partner. representing the changes in valance (v) energy (bond, angle, dihedral, and improper dihedral energies), van der Waal (vdw) interactions, Coulombic (Coul) interactions, and solvation free energy ΔWPB/ΔWGB and ΔWnp. We note that the binding energy computed here includes the solvation free energy which considers water entropy, and the valance energies cancel out in Eq. 2 due to the single trajectory approach. The solvation free energy can be further decomposed into the polar term, ΔWPB/ ΔWGB, and non-polar cavity term, ΔWnp term [44,51]. Here, we demonstrate two methods, Poisson-Boltzmann (PB) and Generalized-Born (GB), to estimate the polar solvation term [45,46]. PB was calculated by solving the PB equation in the PBSA model of Amber11. The dielectric constants of the interior and exterior protein were set to 1 and 80 respectively. GB (igb = 1) was used in the sander program of Amber11 package. The non- polar solvation term was calculated by the solvent-acces- sible surface area (SASA) model. All energy involved a 40 Å cutoff value for non-bonded interactions. One of our aims was to investigate how FHA domains can distinguish the tiny difference between Ser and Thr in their binding partner. The work helps gain deeper understanding in molecular recognition and provides valuable insight into strategies of protein engineering. Although we have simulated the entire FHA, WW and BRCT systems, we do not attempt to draw conclusions on properties of the whole system. Instead, we focus on the phosphoresidue binding region, in particular pThr or pSer. Multiple 1 ns MD simulations were performed for each system with different initial velocity so that the simulations evolve independently of each other to reduce potential bias caused by the initial conformation. where X denotes each dihedral angle, such as phi, psi, omega and sidechain. Ebind = Ev + Evdw + ECoul + WPB/WGB + Wnp, (2) average energy on molecular interactions can be decom- posed as follows: where X denotes each dihedral angle, such as phi, psi, omega and sidechain. TSX = TSX, mutated −TSX, wild-type, (4) Molecular dynamics simulation (1) Ebind =< Ecomplex > −< Edomain > −< Ephosphopeptide > (1) We performed molecular dynamics (MD) simulations using the Amber10 and NAMD2.6 simulation packages with the ff99sb amber force field [36-38]. Because The bracket <E > denotes the average energy com- puted from a given MD trajectory. The changes in Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 4 of 13 Table 1 Peptide sequences of domain-phosphopeptide complexes domain Protein PDB ID Method Phosphopeptide Kd(μM) Ref. FHA1 Rad53 1G6G X-ray LEV(pT)EADATFAK 0.53 (12) FHA1 Rad53 1K3Q NMR SLEV(pT)EADATFVQ 0.3 (16) FHA Dun1 2JQL NMR NI(pT)QP(pT)QQST 0.3-1.2 (35) FHA Ki67 2AFF NMR KTVD(pS)QGP(pT)PVC(pT)PTFLERRKSQVAELNDDDKDDEIVFKQPISC 0.077 (18) BRCT BRCA1 1T2V X-ray AAYDI(pS)QVFPFA 0.4 (29) WW Pin1 1F8A X-ray Y(pS)PT(pS)PS 34 (34) Residues in the bracket are either pT or pS. The first mutation site is represented by both underline and bold, and the second mutation site is underlined. Secondary structures, a helix and b sheet, are labeled as italic with underline and italic, respectively. Table 1 Peptide sequences of domain-phosphopeptide complexes Residues in the bracket are either pT or pS. The first mutation site is represented by both underline and bold, and the second Secondary structures, a helix and b sheet, are labeled as italic with underline and italic, respectively. average energy on molecular interactions can be decom- posed as follows: Revealing the specificity of the main pThr-binding site in FHA domains In the FHA domain family, the loops that link two b- sheets appear to play a pivotal role in constructing the binding pocket for pThr-containing peptides. Despite the variability of primitive sequences in the loop area in different FHA-containing proteins such as Rad53-FHA1, Dun1-FHA and Ki67-FHA, analysis of FHA domain conformations suggests a conserved structure in the main pThr-binding site formed by two loops between b3-b4 and b6-b7 (see Figure 2 (A1)). We substituted pThr with pSer in silico, and the glo- bal binding energy calculations show that pSer-contain- ing peptides have 3-6 kcal/mol higher binding energies than the pThr-containing peptide. Notably, the binding energy calculations are for potential (MM) and solvation (PB/SA) energy only, and the entropic changes upon binding are not included here. Of interest is knowing which energy term contributes more to weaken the FHA domain-peptide interaction. Because the only dif- ference between pThr and pSer is one non-polar methyl group, the pSer-containing peptide may reasonably result in less favorable van der Waals attraction. How- ever, the trend is not clear, which suggests that repla- cing pThr by pSer may affect interactions between pThr/pSer and the domain, and the stability of the entire peptide binding to the protein. For example, pSer-containing peptides have weaker van der Waals interactions between Rad53-FHA1 and Ki67-FHA (Table 2), but the interaction is in the opposite direction for Dun1-FHA. Moreover, the Ki67-FHA:pSer-peptide com- plex shows slightly more favorable electrostatic attrac- tion (-0.98 kcal/mol) as compared with the Ki67-FHA1: pThr-peptide complex. However, the Rad53-FHA1:pSer- peptide and Dun1-FHA1:pSer-peptide complexes have weaker electrostatic interactions. Of note, the polar Of note, although experimental structures demon- strate a pocket to accept the pThr methyl group, the static conformation cannot ascertain that pSer fails to form equally good interactions with the nearby residues, because the protein is dynamic and may fill the space by slightly changing the protein conformations. Neverthe- less, our MD simulations show that the cavity is highly suited to pThr, and small changes in this particular resi- due can diminish the domain-peptide interactions. Because pSer shows less perfect geometry complemen- tary to the binding cavity of the FHA domain, we also studied whether the local flexibility is changed because of the lack of the methyl group. The rotameric states of each sidechain of phosphopeptides, as well as their con- figuration entropy, were calculated. Results and discussion Because the short phosphopeptides are highly flexible, carrying out short simulations allows the peptide to stay in similar local energy well during different runs [54,55]. The root mean square deviation (RMSD) of selected sys- tems is shown in Figure S1 of Additional file 1. The configuration entropy S consisted of phi, psi, omega and sidechain dihedrals, which include both con- formational (number of energy wells) and vibrational entropy (width of an energy well) [47,48,52]. We com- puted each dihedral angle entropy using the Gibbs entropy formula: S = −R p(x) ln p(x)dx (3) (3) where p(x) is the probability distribution of dihedral x, and R is the gas constant. T-analyst was used to com- pute the Gibbs entropy [53]. We considered only the internal dihedral degree of freedom of each dihedral, and the coupling between dihedrals was ignored. The change in configuration entropy during the mutation can be presented as follows: To obtain an estimate of the differences in interaction energies of the domain with its pSer- or pThr-contain- ing peptides, we post-processed our MD trajectories and computed the domain-peptide interaction energies, including the potential energy and solvation energy. The method is usually called the MM-PBSA method. We computed intermolecular interactions between the (4) TSX = TSX, mutated −TSX, wild-type, (4) Page 5 of 13 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 whole domain and the entire peptide, termed the “global binding energy calculation”. In our global energy calcu- lations, the solvation energy term includes a PB term, WPB, for electrostatic solvation free energy, and a cavity/ surface area term, Wnp, for nonpolar solvation free energy. Because we are particularly interested in study- ing the local region that contributes significantly to pThr or pSer recognition, we also selected residues within 5 to 7 Å of the phosphate group (Table S3 in Additional file 1) to calculate interaction energies between the residues chosen. We called the calculations between these selected residues “local interaction energy calculation”. Because the calculations involved only resi- dues near the binding sites, the solvation energies com- puted by the PB or GB model are similar. As a result, we used the GB model in our local energy calculation to speed up the calculation. contributions from the solvation model (PB term) are mostly compensated with the Coulombic term. Results and discussion There- fore, although all FHA:pSer-peptide complexes can form more stable Coulombic interactions, they also result in less stable solvation energy (PB term). In summary, in considering all energy terms, pThr-containing peptides are still highly favored, and our results are in good agreement with other experiments [12,16]. g p We also performed local interaction energy calcula- tions and focused on the interactions between pSer/ pThr and residues around the phosphoresidue to reveal how FHA can discriminate between them. Although the only difference between the Thr and Ser residue is one methyl group, which is usually considered not signifi- cant, our study indicates that the methyl group directly interacts with residues of loops b4-b5 and b6-b7 of the FHA domain (see Figure 2 (A2) and (A3)). Again, the trend agrees with the global binding energy calculations, and the local interaction energy is less favorable when pThr is replaced by pSer. The local interaction energy calculations show that van der Waals interactions are weakened considerably by the lack of a single methyl group of pSer; the loss of the van der Waals attraction can be weakened by ~3 kcal/mol (Table 3). The interac- tion between the methyl group of pThr and the nearby residues are unlikely to be 3 kcal/mol, but instead, the computed energy reveals the crucial role of the methyl group to stabilize the complex conformation locally. Interestingly, although the phosphate group of pSer still retains hydrogen bonding between the nearby residues of FHA, the electrostatic attractions are still weakened. This again supports that solely forming H-bonds between the phosphate group of the phosphoresidue is not enough for phosphopeptide and FHA domain bind- ing, and lacking the methyl group destabilizes the com- plex. As illustrated in Figure 1 (A2) (A3) and Figure S2 in Additional file 1, fewer contacts can be formed when pSer is present in the peptide. Revealing the specificity of the main pThr-binding site in FHA domains Most sidechain dihe- drals stay in the same rotametic states for both pSer/ Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 6 of 13 Figure 2 Detailed illustration of pThr binding in Rad53-FHA1 main binding site (A), Dun1-FHA second binding site (B), BRCT repeats (C) and WW domain (D). The binding areas are circled in red (see (1) on the left side). Residues surrounding pThr and pSer residues are in (2) and (3), respectively. Atom pairs that have charge interactions with phosphoresidues are shown with a blue dashed line. Figures are a snapshot of our MD simulations. Figure 2 Detailed illustration of pThr binding in Rad53-FHA1 main binding site (A), Dun1-FHA second binding site (B), BRCT repeats (C) and WW domain (D). The binding areas are circled in red (see (1) on the left side). Residues surrounding pThr and pSer residues are in (2) and (3), respectively. Atom pairs that have charge interactions with phosphoresidues are shown with a blue dashed line. Figures are a snapshot of our MD simulations. Figure 2 Detailed illustration of pThr binding in Rad53-FHA1 main binding site (A), Dun1-FHA second binding site (B), BRCT repeats (C) and WW domain (D). The binding areas are circled in red (see (1) on the left side). Residues surrounding pThr and pSer residues are in (2) and (3), respectively. Atom pairs that have charge interactions with phosphoresidues are shown with a blue dashed line. Figures are a snapshot of our MD simulations. Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 7 of 13 Table 2 Global MM-PBSA energy calculations domain mutated site mutation ΔΔUVDW ΔΔUCoul ΔΔWPB ΔΔEele ΔΔEtot-np ΔΔWnp ΔΔEtot Rad53-FHA1 1 pT®pS 2.50 -34.34 37.83 3.49 5.99 0.75 6.74 Rad53-FHA1 1 pT®pS 0.44 -13.74 16.08 2.33 2.77 0.72 3.49 Dun1-FHA 1 pT®pS -2.63 -13.70 18.84 5.15 2.52 2.34 4.86 Ki67-FHA 1 pT®pS 4.30 -8.77 7.79 -0.98 3.32 -0.84 2.48 Dun1-FHA 2 pT®pS -2.54 -59.30 63.39 4.09 1.55 1.95 3.50 Ki67-FHA 2 pS®pT 0.94 19.77 -20.15 -0.38 0.55 0.36 0.91 BRCT 1 pS®pT -3.33 28.77 -23.67 5.10 1.77 0.84 2.61 WW 1 pS®pT -1.03 -39.75 36.78 -2.96 -3.99 -0.01 -4.00 ΔΔUCoul and ΔΔUvdw are the electrostatic and van der Waals interactions, respectively, between the wild-type and mutants; ΔΔWPB and ΔΔWnp are the polar and non-polar contributions from the solvation energy. ΔΔEele represents the sum of ΔΔUCoul and ΔΔWPB. Revealing the specificity of the main pThr-binding site in FHA domains Figure 3 (B1) and (B2) shows the distribution of the sidechain dihedral angle of pThr and pSer in Rad53-FHA1 peptide. The dihedral of pSer deviates from pThr with an angle shift from 115 to 162 degrees, and also has wider distribution, so the dihedral is more flexible. Clearly, the methyl group of pThr allows the phosphoresidue to fill the entire pocket of the binding site, and no room is available for spacious vibration of the pThr sidechain. In contrast, the space released by the absence of pSer cannot be adequately filled by protein sidechains, which creates room for the dihedral of pSer to be more flexible. Although side- chains of the dual loops are mobile, the conserved His, located at the N-terminus of b5, uses the imidazole ring and polar interactions to form stable interactions with residues of loops b4-b5 and b6-b7. For example, Figure 4 (C) shows that His can interact with the conserved Ser85 of loop b4-b5 and Ile104 and Gly108 of loop b6- b7 in Rad53-FHA1. During our MD simulations, the sidechains moved between the two loops, which strengthens the interactions between residues around His (e.g., Ser85 and Thr106) and generates a proper space exclusively for the methyl group. The same move- ment and enhanced interactions for forming a cavity are observed in multiple 50 ns simulations (data not To quantify the flexibility of dihedral rotation and vibration of pSer/pThr, we performed Gibbs entropy calculations. As illustrated in Table 4, the sidechain dihedral entropy increased ~0.7-1.5 kcal/mol after muta- tion and the configuration entropy of the entire pSer residue increased nearly 2 kcal/mol as compared with pThr. Among all the dihedral angles, the entropy increase is mostly contributed by sidechain dihedrals. Notably, the local entropy increase when pThr is substi- tuted by pSer is only a local effect, and the entropy loss of the whole system with phosphopeptide binding was not computed and compared in this work. The local interaction energy and local entropy calculations offer quantitative comparison for pSer- and pThr-containing peptide binding, and we do not suggest that the binding energy loss may be fully compensated by the local entropy gain in this study. Revealing the specificity of the main pThr-binding site in FHA domains ΔΔ indicates the changes between two calculations of the mutated and non-mutated state. For example, ΔΔUVDW = ΔUVDW,pSer-ΔUVDW,pThr, where ΔUVDW is the interaction energy between the phosphopeptide and the domain. Table 2 Global MM-PBSA energy calculations domain mutated site mutation Δ ΔΔUCoul and ΔΔUvdw are the electrostatic and van der Waals interactions, respectively, between the wild-type and mutants; ΔΔWPB and ΔΔWnp are the polar and non-polar contributions from the solvation energy. ΔΔEele represents the sum of ΔΔUCoul and ΔΔWPB. ΔΔ indicates the changes between two calculations of the mutated and non-mutated state. For example, ΔΔUVDW = ΔUVDW,pSer-ΔUVDW,pThr, where ΔUVDW is the interaction energy between the phosphopeptide and the domain. shown). Therefore, our simulations explain how FHA makes use of the conserved His to stabilize the dual loop and form a structural room to dock the methyl group and discriminate pThr/pSer. pThr residues, but the second pThr sidechain dihedral angle (see Figure 3 (A)) differs. Figure 3 (B1) and (B2) shows the distribution of the sidechain dihedral angle of pThr and pSer in Rad53-FHA1 peptide. The dihedral of pSer deviates from pThr with an angle shift from 115 to 162 degrees, and also has wider distribution, so the dihedral is more flexible. Clearly, the methyl group of pThr allows the phosphoresidue to fill the entire pocket of the binding site, and no room is available for spacious vibration of the pThr sidechain. In contrast, the space released by the absence of pSer cannot be adequately filled by protein sidechains, which creates room for the dihedral of pSer to be more flexible. Although side- chains of the dual loops are mobile, the conserved His, located at the N-terminus of b5, uses the imidazole ring and polar interactions to form stable interactions with residues of loops b4-b5 and b6-b7. For example, Figure 4 (C) shows that His can interact with the conserved Ser85 of loop b4-b5 and Ile104 and Gly108 of loop b6- b7 in Rad53-FHA1. During our MD simulations, the sidechains moved between the two loops, which strengthens the interactions between residues around His (e.g., Ser85 and Thr106) and generates a proper space exclusively for the methyl group. The same move- ment and enhanced interactions for forming a cavity are observed in multiple 50 ns simulations (data not pThr residues, but the second pThr sidechain dihedral angle (see Figure 3 (A)) differs. We selected residues within 5 to 7 Å around pThr/pSer residues. The residues selected are in the Additional file 1. Table S3. The not 2 We selected residues within 5 to 7 Å around pThr/pSer residues. The residues selected are in the Additional file 1. Table S3 2. We selected residues within 5 to 7 Å around pThr/pSer residues. The residues selected are in the Additional file 1. Table S3. The notations are the same in Table 2. hin 5 to 7 Å around pThr/pSer residues. The residues selected are in the Additional file 1. Table S3. The notations are the same in Tab The second phosphoresidue-binding site of Dun1-FHA and Ki67-FHA Some FHA domains also show the second phosphore- sidue-binding site, and knowing whether the second Some FHA domains also show the second phosphore- sidue-binding site, and knowing whether the second Table 3 Local MM-PBSA energy calculations domain mutated site mutation ΔΔUVDW ΔΔUCoul ΔΔWGB ΔΔEele ΔΔEtot-np ΔΔWnp ΔΔEtot Rad53-FHA1 1 pT®pS 3.12 -8.36 5.71 -2.65 0.46 0.03 0.49 Rad53-FHA1 1 pT®pS 0.23 -9.64 10.93 1.29 1.52 -0.12 1.40 Dun1-FHA 1 pT®pS 2.30 3.00 -0.71 2.29 4.59 0.06 4.66 Ki67-FHA 1 pT®pS 3.22 2.25 -1.67 0.58 3.80 0.14 3.94 Dun1-FHA 2 pT®pS 0.49 -1.30 -0.26 -1.57 -1.07 0.00 -1.07 Ki67-FHA 2 pS®pT 0.70 3.33 -2.02 1.30 2.01 0.09 2.10 BRCT 1 pS®pT 0.06 7.17 -6.75 0.41 0.47 0.08 0.56 WW 1 pS®pT -1.85 1.67 0.10 1.78 -0.07 0.05 -0.02 We selected residues within 5 to 7 Å around pThr/pSer residues The residues selected are in the Additional file 1 Table S3 The notations are the same in Table Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 8 of 13 Figure 3 Distribution of a dihedral angle of the phosphoresidue. (i) Plots of the dihedral angle with five seeds are shown in frame index 1- 100, 101-200, 201-300, 301-400 and 401-500, respectively and (ii) corresponding population distributions. Column (A) shows the dihedral angle of pThr (1) or pSer (2) used for plotting. (B), (C), (D) and (E) indicate the binding area in the main binding site of FHA, the second binding site of FHA the pThr/pSer binding site of BRCT repeats and the WW domain respectively Figure 3 Distribution of a dihedral angle of the phosphoresidue. (i) Plots of the dihedral angle with five seeds are shown in frame index 1- 100, 101-200, 201-300, 301-400 and 401-500, respectively and (ii) corresponding population distributions. Column (A) shows the dihedral angle of pThr (1) or pSer (2) used for plotting. (B), (C), (D) and (E) indicate the binding area in the main binding site of FHA, the second binding site of FHA, the pThr/pSer binding site of BRCT repeats and the WW domain, respectively. Figure 3 Distribution of a dihedral angle of the phosphoresidue. (i) Plots of the dihedral angle with five seeds are shown in frame index 1- 100, 101-200, 201-300, 301-400 and 401-500, respectively and (ii) corresponding population distributions. Column (A) shows the dihedral angle of pThr (1) or pSer (2) used for plotting. The second phosphoresidue-binding site of Dun1-FHA and Ki67-FHA (B), (C), (D) and (E) indicate the binding area in the main binding site of FHA, the second binding site of FHA, the pThr/pSer binding site of BRCT repeats and the WW domain, respectively. Overall, the global binding energy calculations show that for the second phosphoresidue-binding site, the mutations worsen binding affinities (see Table 2), but the changes are smaller than the values for the main pThr-binding site. However, the local interaction energy calculations do not show the same trend, and the muta- tion of Dun1-FHA is preferable. Therefore, the calcula- tions do not directly support that the domain strongly prefers either pThr or pSer in the second phosphoresi- due-binding site. The local interaction energy calcula- tions suggest that pSer can have good interactions with the domain, which are contributed mainly from the electrostatic attractions, and by losing the methyl group, the van der Waal interactions are weakened, but not sig- nificantly. As illustrated in Figure 2 (B2) and (B3), the site can discriminate pThr and pSer is of interest. We therefore studied two diphosphoresidue-recognized FHA domains, Dun1-FHA and Ki67-FHA. Both domains have one pThr binding to the main pThr- binding site, but they also have one more phosphoresi- due, pSer or pThr, in the peptide sequences. One main difference between the main pThr and the second phosphoresidue-binding site is that the main pThr- binding site consists of two loops that form a well- defined pocket, whereas the second binding site is located in areas with a single loop. To understand whether the difference contributes to residue specifi- city, we mutated the phosphoresidue available in the experiment, pThr of Dun1-FHA and pSer of Ki67-FHA to pSer and pThr, respectively. Page 9 of 13 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Figure 4 Detailed illustration of conserved His interactions in Rad53-FHA1. (A) Overall FHA structure. Conserved His is in red; other residues around His that contribute to form a pThr binding pocket are in orange. The phosphopeptide and pThr are in cyan. (B) Cartoon representation of two loops held by His and the nearby residues. Red dashed lines indicate interactions between residues. (C) MD simulation snapshots with time. Atom pairs with electrostatic attractions are labeled with blue dashed lines. Figure 4 Detailed illustration of conserved His interactions in Rad53-FHA1. (A) Overall FHA structure. The second phosphoresidue-binding site of Dun1-FHA and Ki67-FHA Conserved His is in red; other residues around His that contribute to form a pThr binding pocket are in orange. The phosphopeptide and pThr are in cyan. (B) Cartoon representation of two loops held by His and the nearby residues. Red dashed lines indicate interactions between residues. (C) MD simulation snapshots with time. Atom pairs with electrostatic attractions are labeled with blue dashed lines. Figure 4 Detailed illustration of conserved His interactions in Rad53-FHA1. (A) Overall FHA structure. Conserved His is in red; other residues around His that contribute to form a pThr binding pocket are in orange. The phosphopeptide and pThr are in cyan. (B) Cartoon representation of two loops held by His and the nearby residues. Red dashed lines indicate interactions between residues. (C) MD simulation snapshots with time. Atom pairs with electrostatic attractions are labeled with blue dashed lines. second phosphoresidue-binding site in Dun1-FHA uses two Arg residues, Arg62 and Arg64, to recognize pThr or pSer and form multiple H-bonds with the phosphate group. Therefore, the electrostatic attractions are the major driving forces in pThr/pSer binding is not sur- prising [56]. In addition, both Arg residues are located in one single loop, which is a flexible region of FHA domains, so the protein is freely adjustable to adopt both pThr and pSer. Although the methyl group of pThr forms non-polar attractions with the alkane branch of Arg62 shown in Figure 2 (B2), the binding site does not hold a small pocket when pThr is substi- tuted by pSer (Figure 2 (B3)), because the space is filled by the nearby FHA domain sidechains. The second phosphoresidue-binding site is located in a single loop, b3-b4, and without spatial constraint, the second site allows the FHA domain to rearrange side- chains to optimize both pThr and pSer binding. The second phosphoresidue-binding site of Dun1-FHA and Ki67-FHA We Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 Page 10 of 13 Table 4 Configuration entropy changes domain mutated site mutation TΔSphi TΔSpsi TΔSomega TΔSsidechain TΔStotal Rad53-FHA1 1 pT®pS 0.18 0.29 0.03 1.39 1.91 Rad53-FHA1 1 pT®pS 0.37 0.37 0.14 1.26 2.16 Dun1-FHA 1 pT®pS -0.25 -0.08 0.00 0.72 0.45 Ki67-FHA 1 pT®pS 0.25 0.32 0.14 1.50 2.23 Dun1-FHA 2 pT®pS 0.10 -0.01 0.00 1.03 1.12 Ki67-FHA 2 pS®pT -0.04 -0.06 0.01 -0.47 -0.57 BRCT 1 pS®pT -0.08 0.01 0.00 0.35 0.28 WW 1 pS®pT -0.15 -0.01 -0.12 -0.71 -1.01 Configuration entropy changes (kcal/mol) for phi, psi, omega and sidechain dihedrals of phosphoresidue in the wild-type and mutants. Table 4 Configuration entropy changes function as phosphopeptide binding modules, but both have a subset that binds to phosphopeptides. Both domains can bind to particular sequences that contain pSer or pThr, but in general, tandem BRCT domains bind stronger to pSer than pThr and WW domains have a preference for pThr preceding a Pro [28,33]. Note that pSer or pThr must be followed by Pro, for pSer/pThr-Pro sequences for binding to WW domains. In contrast to FHA domains, which bind exclusively to pThr-containing peptides, BRCT and WW domains do not recognize solely pThr- or pSer-containing peptides. Although proteins that treat Ser and Thr as similar resi- dues may be common, knowing how both domains have a specific or non-specific pThr/pSer recognition is of interest. therefore examined changes in local flexibility when a different phosphoresidue stays in this binding site, and we focused on dihedral angles of the phosphate group of pThr and pSer. Both residues do not expressly reveal dynamic motions in either complex, but the most popu- lated angles modeled from our MD simulations shift more than 20° (Figure 3 (C1) (C2)). Although the dihe- dral angle has only one rotameric states in both cases, pThr has smaller vibration range and the configuration entropy is 0.5-1.0 kcal/mol smaller than pSer, presum- ably due to a bulkier methyl group. The entropy changes between pThr and pSer is less pronounced in the second phosphoresidue-binding site than in the main one. Moreover, the motions of backbone dihedrals remain the same, which indicates the negligible influ- ence of the mutation. Both global binding energy and local interaction energy calculations suggest that the tandem BRCT domains pre- fer the pSer- than pThr-containing sequence, although the preference is not strong. The second phosphoresidue-binding site of Dun1-FHA and Ki67-FHA The local interaction ener- gies shown in Table 3 suggest that pSer can form a more favorable electrostatic attraction, ~0.5 kcal/mol more negative than that contributed by pThr binding, but the difference is relatively small. In addition, our simulations show that most of the time, the methyl group of pThr does not directly interact with the domain, as demon- strated by a representative complex conformation in Fig- ure 2 (C2). As a result, we see a negligible difference in van der Waal interactions in the local interaction energy calculations (see Table 3), and the electrostatic attractions are the main driving forces to recruit phosphopeptides binding to tandem BRCT domains (Table S2 in Addi- tional file 1). The phospho recognition is through form- ing interactions with Lys1702 in an a helix and Ser1655 and Gly1656 near the loop, where no small cavity is reserved for the methyl group of pThr. Although the methyl group is not directly involved in binding, the local arrangement of the phosphate group is changed, but the overall flexibility of the phosphate group remains similar (Figure 2 (C)). In conclusion, the main pThr-binding site has a unique feature to recognize pThr, and a special pocket built by linking two loops with the conserved His is reserved for the methyl group of pThr, which plays a crucial role in distinguishing between pThr and pSer. However, the second phosphoresidue binding site is positioned in a single loop near the N-terminus, which uses two Arg residues to recognize a phosphate group but lacks a well structured binding cavity to identify only pThr or pSer. Our simulations show that the pro- tein sidechain of the binding site changes when pThr or pSer binds to the domain. Therefore, a single loop used to provide a phosphoresidue-binding site cannot discri- minate pThr/pSer but can bind to both residues. Other domains, such as the WW domain, also use a similar strategy, as discussed in the next section. Of note, although not within the scope of this paper, the promis- cuous domain has preferences for selected sequences, and the peptide sequences also play an important role in the phosphopeptide-binding site. BRCT repeats and WW domain recognize both pThr and pSer BRCT repeats and WW domain recognize both pThr and pSer BRCT repeats and WW domain recognize both pThr and pSer The BRCT and WW domains are distinct pSer/pThr binding domains. Not all BRCT and WW domains Although the WW domains are able to recognize both pSer- and pThr-containing peptides, global binding Page 11 of 13 Huang and Chang BMC Biophysics 2011, 4:12 http://www.biomedcentral.com/2046-1682/4/12 energy calculations suggest that the domain favors pThr because of the more preferable van der Waals attrac- tions. The trend is in agreement with experimental results [33]. However, the local interaction energy calcu- lations show that the favorable van der Waals attrac- tions are mostly compensated by weaker electrostatic interactions. As shown in Figure 2 (D2) and (D3), two conserved aromatic residues of the domain, Tyr and Trp, create a cavity, but no sidechains of phosphopep- tides could nicely fit into the cavity during our simula- tions. Interestingly, the conserved Pro residue adjacent to the phosphoresidue is clamped by Tyr and Trp, which stays in the cavity and further restricts nearby phosphopeptide conformations. The confined region formed by rings of Tyr, Trp and Pro is conserved regardless of the presence of pSer or pThr (see Figure 2 (D2) and (D3)), which also explains the crucial roles of Pro. Because of the bulky ring conformations, an empty space is observed during the course of the simulations. The empty space can be partially filled by the methyl group of pThr, thus resulting in more favorable van der Waals interactions and a less flexible sidechain while pThr is binding. However, the Pro residue but not the methyl group of pThr primarily occupies the cavity in phosphopeptide recognition. Therefore, the domains do not show significant discrimination between pSer and pThr. phosphate group and geometry complementary in the binding surface. Of note, the binding affinities of phosphopeptide binding to these domains are in general weak, in the micromolar range (see Table 1); therefore, weakened attractions by a few kcal/mol can completely diminish the phosphopeptides binding. Therefore, although sub- stitution of pThr by pSer mainly reduces van der Waals attractions in the main pThr binding site of FHA domains, the pSer-containing peptide cannot form the domain-peptide complex. Biological implication d l d Modular domains are common regulators in important biological processes. This work studied three important domains for DNA damage responses, FHA, BRCT and WW domains, all with a phosphopeptide binding site to relay the damage signal and trigger further repair. The specific peptide can be recognized by three different proteins: a protein kinase to phosphorylate Ser/Thr, a modular domain that binds to the phosphopeptide for a downstream process, and a phosphatase to dephosphor- ylate the phosphoresidue. Kinases involved in the DNA damage response, ATM and ATR, can phosphorylate both Ser and Thr of a substrate. Similar to kinases, phosphatases work for both pThr and pSer [57], and most phosphodomains can also bind to both pThr- and pSer-containing peptides. FHA domains have evolved a simple but remarkable mechanism to specifically recog- nize pThr to further select particular partners after kinase phosphorylation. For example, a binding partner of FHA domains, the SCD protein family, contains rich Thr-glutamine (TQ) and Ser-glutamine (SQ) repeat motifs [58]. Although the kinase phosphorylates both Thr and Ser, Rad53-FHA1 can bind only to regions that have pThr. How binding to the particular pThr region triggers further responses is unclear, but the pThr-only recognition may play a role in regulation. The mechan- ism contributing to pThr binding brings insights into how modular domains differentiate pThr and pSer or BRCT repeats and WW domain recognize both pThr and pSer Different from other phosphopeptide binding sites, sidechain rearran- gements cannot bring other attraction forces to com- pensate for the loss of the van der Waals interactions because of the rigid structure formed by His and the dual loops. In addition to energy calculations, our local entropy calculations suggest that binding pThr to the main pThr-binding site of FHA can reduce the mobi- lity significantly, which indicates stronger attraction and more geometry confinement. However, the entropy changes between pThr and pSer binding to other domains show smaller differences, which sug- gests that the system retains a similar dynamic beha- vior that may help balance energy loss by gaining other attractions. Conclusions 5. Seet BT, Dikic I, Zhou MM, Pawson T: Reading protein modifications with interaction domains. 2006, 7. In this study, we performed dynamic-guided process for FHA, BRCT and WW domain-peptide structures. The components of detailed interaction energies were calcu- lated by MM-PBSA/MM-GBSA method. The main pThr-binding cavity is identical in four different FHA complexes. Our results reveal FHA domain uses the conserved His residue to define a dual loops structure which shows strong favor for pThr because of the geo- metry of methyl group embedded in deep binding pocket nicely. The dynamics simulations, energy and entropy calculations indicate that the phosphoresidue binding site of FHA is highly suited to pThr, and small changes of pThr to pSer can diminish the domain-pep- tide interactions due to the pre-organized binding cavity. On the other hand, BRCT repeats and WW domain uti- lize the combination a single loop with a-helix or b- sheet which allows effectively sidechain rearrangement to accept both pSer and pThr. The results highlight broader implications in recognition pathway of kinase/ phosphotase and also help to engineer proteins and design peptide inhibitors. 6. Jorgensen C, Linding R: Directional and quantitative phosphorylation networks. Briefings in Functional Genomics & Proteomics 2008, 7(1):17-26. 7. Narayanan A, Jacobson MP: Computational studies of protein regulation by post-translational phosphorylation. Current Opinion in Structural Biology 2009, 19(2):156-163. 8. Virshup DM, Shenolikar S: From Promiscuity to Precision: Protein Phosphatases Get a Makeover. Molecular Cell 2009, 33(5):537-545. 9. Diella F, Haslam N, Chica C, Budd A, Michael S, Brown NP, Trave G, Gibson TJ: Understanding eukaryotic linear motifs and their role in cell signaling and regulation. Frontiers in Bioscience 2008, 13:6580-6603. signaling and regulation. Frontiers in Bioscience 2008, 13:6580-66 10. Liao H, Byeon IJL, Tsai MD: Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53. 1999, 294. 11. Liao H, Yuan CH, Su MI, Yongkiettrakul S, Qin DY, Li HY, Byeon IJL, Pei DH, Tsai MD: Structure of the FHA1 domain of yeast Rad53 and identification of binding sites for both FHA1 and its target protein Rad9. Journal of Molecular Biology 2000, 304(5):941-951. 12. Durocher D, Taylor IA, Sarbassova D, Haire LF, Westcott SL, Jackson SP, Smerdon SJ, Yaffe MB: The molecular basis of FHA Domain: Phosphopeptide binding specificity and implications for phospho- dependent signaling mechanisms. 2000, 6. 13. Liang XY, Van Doren SR: Mechanistic insights into phosphoprotein- binding FHA domains. 2008, 41. 14. Acknowledgements g We thank Dr. Ming-Daw Tsai for valuable suggestions and discussion. This research was supported in part by start-up funds from the University of California, Riverside and the National Science Foundation (MCB-0919586) through TeraGrid resources provided by the National Center for Supercomputing Application under grant number (TG-MCB080039N) and ShaRCS, the University of California Shared Research Computing Services Cluster, which is technically supported by multiple UC information technology divisions and managed by the University of California, Office of the President. 19. 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Pennell S, Westcott S, Ortiz-Lombardia M, Patel D, Li JJ, Nott TJ, Mohammed D, Buxton RS, Yaffe MB, Verma C, Smerdon SJ: Structural and Functional Analysis of Phosphothreonine-Dependent FHA Domain Interactions. Structure 2010, 18(12):1587-1595. Additional material 16. Yuan CH, Yongkiettrakul S, Byeon IJL, Zhou SZ, Tsai MD: Solution structures of two FHA1-phosphothreonine peptide complexes provide insight into the structural basis of the ligand specificity of FHA1 from yeast Rad53. 2001, 314. Additional file 1: Table S1: MM-PBSA energy calculations for each seed Table S2: Local interaction energy calculations (MM-GBSA) for wild-type and mutated MD trajectory Table S3: List of residues selected around phosphoresidue Figure S1: RMSD plot of Rad53-FHA1 Figure S2: Detailed illustration of pThr/pSer peptide binding to FHA Additional file 1: Table S1: MM-PBSA energy calculations for each seed Table S2: Local interaction energy calculations (MM-GBSA) for wild-type and mutated MD trajectory Table S3: List of residues selected around phosphoresidue Figure S1: RMSD plot of Rad53-FHA1 Figure S2: Detailed illustration of pThr/pSer peptide binding to FHA 17. Lee SJ, Schwartz MF, Duong JK, Stern DF: Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling. Molecular and Cellular Biology 2003, 23(17):6300-6314. 17. Lee SJ, Schwartz MF, Duong JK, Stern DF: Rad53 phosphorylation site clusters are important for Rad53 regulation and signaling. 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Without the methyl group, neither pSer binding nor rearranging sidechains of FHA near the methyl binding site can effectively fill the pocket, which results in unsuccessful binding. The sec- ond binding site of FHA domains makes use of a single loop to recognize pThr/pSer, and WW and BRCT domains combine a single loop and a nearby a helix (BRCT domains) or b sheet (WW domains) to bind to pThr/pSer. The structures of these phosphoresidue binding sites allow the protein sidechains to be adjusta- ble to both pThr and pSer residues. Notably, although loops are usually considered flexible regions of a protein, the dual loops in the main pThr-binding site of FHA domains show an interaction network between the loops to form a pre-organized binding cavity for pThr (data not shown). 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https://openalex.org/W4372338093	https://www.researchsquare.com/article/rs-2663338/latest.pdf	English	null	The whole-cell proteome shows the characteristics of macrolides-resistant Bordetella pertussis in China linked to the biofilm formation	Archives of microbiology	2,023	cc-by	5,918	The whole-cell proteome shows the characteristics of macrolides-resistant Bordetella pertussis in China linked to the biofilm formation The whole-cell proteome shows the charac macrolides-resistant Bordetella pertussis i linked to the biofilm formation Zhe Lv Department of Epidemiology and Biostatistics, School of Public Health, Xi'an Jiaoton Science Center Sha Yin National Regional Children's Medical Center (Northwest), Key Laboratory of Precision Diseases of Shaanxi Province, Xi'an Key Laboratory of Children's Health and Diseases Kaichong Jiang National Regional Children's Medical Center (Northwest), Key Laboratory of Precision Diseases of Shaanxi Province, Xi'an Key Laboratory of Children's Health and Diseases Wei Wang Department of Clinical Laboratory, Xi'an Children's Hospital Yang Luan Xi’an Center for Disease Control and Prevention Shuang Wu Department of Clinical Laboratory, Xi'an Children's Hospital Jianfei Shi Department of Clinical Laboratory, Xi'an Children's Hospital Zhe Li Department of Diphtheria, Tetanus and Pertussis Vaccine and Toxins, National Instit Control Xiao Ma Department of Diphtheria, Tetanus and Pertussis Vaccine and Toxins, National Instit Control Zengguo Wang (  william_wzg@126.com ) Department of Clinical Laboratory, Xi'an Children's Hospital Hong Yan Department of Epidemiology and Biostatistics, School of Public Health, Xi'an Jiaoton Science Center Zhe Lv Department of Epidemiology and Biostatistics, School of Public Health, Xi'an Jiaotong University Health Science Center Sha Yin Diseases of Shaanxi Province, Xi an Key Laboratory of Childrens Health and Diseases, Shaanxi Kaichong Jiang National Regional Children's Medical Center (Northwest), Key Laboratory of Precision Medicine to Pediatric Diseases of Shaanxi Province, Xi'an Key Laboratory of Children's Health and Diseases, Shaanxi Wei Wang Department of Clinical Laboratory, Xi'an Children's Hospital Yang Luan Xi’an Center for Disease Control and Prevention Shuang Wu Department of Clinical Laboratory, Xi'an Children's Hospital Jianfei Shi Department of Clinical Laboratory, Xi'an Children's Hospital Zhe Li Department of Diphtheria, Tetanus and Pertussis Vaccine and Toxins, National Institute for Food and Drug Control Xiao Ma Department of Diphtheria, Tetanus and Pertussis Vaccine and Toxins, National Institute for Food and Drug Control Zengguo Wang (  william_wzg@126.com ) Department of Clinical Laboratory, Xi'an Children's Hospital Hong Yan Department of Epidemiology and Biostatistics, School of Public Health, Xi'an Jiaotong University Health Science Center Research Article Version of Record: A version of this preprint was published at Archives of Microbiology on May 6th, 2023. See the published version at https://doi.org/10.1007/s00203-023-03566-0. Abstract The macrolides-resistant Bordetella pertussis (MR-Bp) isolates in China evolved from the ptxP1/fhaB3 allele and rapidly became predominant, suggestive of an adaptive transmission ability. This was different from the global prevalent ptxP3 strains, in which MR-Bp was rarely reported. The study aimed to determine the underlying mechanism responsible for fitness and resistance in these two strains. We identify proteomic differences between ptxP1/fhaB3 and ptxP3/fhaB1 strains using TMT-based proteomics. We then performed in-depth bioinformatic analysis to determine differentially expressed genes (DEGs), followed by gene ontology (GO), and protein-protein interaction (PPI) network analysis. Further PRM analysis confirmed the expression of 4 target proteins. Finally, the crystal violet method was used to determine biofilm-forming ability. The results showed that the main significant different proteins between the two represent isolates were related to biofilm formation. Furthermore, we have confirmed that ptxP1/fhaB3 showed hyperbiofilm formation in comparison with ptxP3/fhaB1. It is suggested that the resistance and adaptability of ptxP1/fhaB3 strains may be related to the formation of biofilm through proteomics. In a word, we determined the significantly different proteins between the ptxP1/fhaB3 and ptxP3/fhaB1 strains through whole-cell proteome, which were related to biofilm formation. Research Article Page 1/16 Page 1/16 Page 1/16 Posted Date: March 16th, 2023 DOI: https://doi.org/10.21203/rs.3.rs-2663338/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Additional Declarations: No competing interests reported. Additional Declarations: No competing interests reported. Version of Record: A version of this preprint was published at Archives of Microbiology on May 6th, 2023. See the published version at https://doi.org/10.1007/s00203-023-03566-0. Page 2/16 Abstract • Important Notes This study identified proteomic differences between ptxP1/fhaB3 (Macrolides-resistant Bordetella pertussis prevalent in China) and ptxP3/fhaB1 (Macrolides-sensitivity Bordetella pertussis prevalent in global) strains using TMT-based proteomics and PRM-based protein quantification. The results showed that the main significant different proteins between the two represent isolates were related to biofilm formation. Furthermore, we have confirmed that ptxP1/fhaB3 showed hyperbiofilm formation in comparison with ptxP3/fhaB1. It is suggested that the resistance and adaptability of ptxP1/fhaB3 strains may be related to the formation of biofilm. 1 Background Bordetella pertussis (Bp) is the pathogen of pertussis, a severe respiratory infectious disease. Although pertussis has been well controlled globally through pertussis vaccination, the incidence of pertussis has increased significantly in recent years, showing the characteristics of a "resurgence"(Clark 2014). PtxP3/fhaB1 Bp lineage has almost replaced resident ptxP1-Bp lineage resulting in an almost worldwide selective sweep(Consortium 2019; Xu, Octavia, et al. 2019). Moreover, the macrolides-resistant (MR) ptxP3/fhaB1 Bp strains were occasionally reported without further dissemination(Guillot et al. 2012), which may be related to the fitness cost (greatly reduced pathogenicity) of Bp after MR. However, there has become an explosive increase after the emergence of macrolides-resistant B. pertussis (MR-Bp) in China in 2011(Wang et al. 2013; Wang et al. 2014; Liu et al. 2018). From our previous genomic results, we found that all MR-Bp strains in China have evolved from ptxP1/fhaB3 lineage(Xu, Wang, et al. 2019). Recently, the MR-Bp strain in Japan and Vietnam was reported and related to the ptxP1/fhaB3 lineage, which raises the possibility that MR-Bp could spread all over the world(Kamachi et al. 2020; Page 3/16 Page 3/16 Yamaguchi et al. 2020; Feng et al. 2021). Thus, ptxP1/fhaB3 MR-Bp became the dominant strain in China, which is contrary to the phenomenon of the fitness cost of loss of virulence after macrolides resistance of Bp(Weiss and Falkow 1984). However, the mechanism of this phenomenon was under dispute. Microbial biofilm is an organized aggregate of microorganisms, which is resistant to an extreme environment, such as increasing microbial resistance against various antibiotics(Yin et al. 2019). Bp is more resistant to macrolide antibiotics in the biofilm and biofilm spatial structure confers a consistent and robust fitness advantage(Dorji et al. 2016; Deschaine et al. 2018), suggesting that the adaptive transmission ability of ptxP1/fhaB3 MR-Bp may be related to the ability of biofilm formation. Therefore, in order to reveal the possible mechanism of the fitness of prevalent MR-Bp, mass spectrometry- based proteomics of ptxP1/fhaB3 MR-Bp and the globally prevalent macrolides-sensitive (MS) ptxP3/fhaB1 Bp were performed in this study to screen the characteristic proteins of prevalent MR-Bp and detect the biofilm-forming ability of the two representative isolates. 2.1 Bacterial culture Bordetella pertussis strains 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1) from glycerol stocks were grown on Bordet-Gendou agar plates in parallel and incubated at 37°C for 3 days. Pure cultures were obtained by sub-culturing a single clone onto second Bordet-Gendou agar plates and incubated again at 37°C for 2–3 days. A loopful of pure colonies was inoculated into 40 ml of SS with 1% Heptakis ((2,6-O- dimethyl) β-cyclodextrin) and incubated for 24 hours with shaking at 37°C with the starting OD600 adjusted to 0.1. 2 Methods 2.1 Bacterial culture 2.2 Sample preparation After incubation, the whole cell and supernatant were separated by centrifugation at 3000 × g at 4°C for 15 min. The whole-cell pellet was washed 3 times with phosphate-buffered saline (PBS) and then frozen quickly with liquid nitrogen. The whole-cell pellet is stored at -80°C for subsequent TMT-based proteomics analysis. 2.3 TMT-based proteomics analysis 2.3 TMT-based proteomics analysis 2.3 TMT-based proteomics analysis Page 4/16 The pellet was lysed with SDT (4% (w/v) SDS, 100mM Tris/HCl pH7.6, 0.1M DTT) and the number of proteins was quantified with the BCA Protein Assay Kit (Bio-Rad, USA). Subsequently, the proteins were digested with trypsin to obtain peptides(Chou and Schwartz 2011), which were further desalted by C18 Cartridgeand. After lyophilization, the peptides were reconstituted with 40µL 0.1% formic acid solution and were quantified (OD280). TMT reagent (Thermo Fisher Scientific) was used to label the resulting 100 µg peptides, according to the manufacturer’s instructions. Labeled peptides were fractionated by the High pH Reversed-Phase Peptide Fractionation Kit (Thermo Scientific). Then, the samples were separated using an EASY-nLC liquid chromatography instrument (Thermo Fisher Scientific) and LC-MS/MS analysis was performed on a Q Exactive mass spectrometer (Thermo Scientific). The protein identification and quantification were accomplished using Mascot2.2 and Proteome Discoverer1.4. We find the protein Page 4/16 Page 4/16 sequence according to "uniprot_Bordetella_pertusis_34672_20190712. fasta", and the protein ratios are calculated as the median of only unique peptides of the protein. Three biological replicates were performed for each strain. 2.5 PRM-based protein quantification Parallel reaction monitoring (PRM) is a liquid chromatography-mass spectrometry-based targeted peptide/protein quantification method. Proteins that were identified to be significantly different in TMT experiments were confirmed using PRM measurements. The proteins of whole-cell were trypsin digested, an HPLC system was used for chromatographic separation, and Q-Exactive HF mass spectrometer (Thermo Scientific) was used for mass spectrometry analysis. Here, we selected 11 node proteins in the PPI network (Cpn10, RpoZ, RpsC, HscA, RpoD, RplB, RpsD, prn, fim2, fim3, and fimD) for PRM verification, of which four target proteins can monitor credible peptides, namely Prn, Fim2, Fim3, and RpoD. The list of target peptides and sub ions for PRM quantification is shown in Supplementary Table 1. 2.4 Bioinformatics analysis CELLO (http://cello.life.nctu.edu.tw/), which is a multi-class SVM classification system, was used to predict protein subcellular localization. The protein sequences of the selected differentially expressed proteins were locally searched using the NCBI BLAST + client software (NCBI-blast-2.2.28+-win32.exe) and InterProScan to find homolog sequences, then gene ontology (GO) terms were mapped and sequences were annotated using the software program Blast2GO. The Fisher’s Exact Test is used to compare the distribution of each GO classification in the protein set and perform the enrichment analysis of GO annotation annotations on the target protein set. Upregulated and downregulated proteins were defined as having fold changes (FC) > 1.2 and < 0.82, respectively. A two-tail Student t-test was then performed with p < 0.05 assigned as significant. Three biological replicates were used for each strain. In order to explore the role of the functional link between different proteins of the two strains, we performed protein-protein interaction (PPI) network on the significantly different proteins. The PPI information of the studied proteins was based on the STRING database (http://string-db.org/) and Cytoscape software (version 3.8.2). The GO annotation results and volcano results were plotted by R scripts. 2.6 Biofilm formation assay To determine the biofilm formation of 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1), the strains were grown for 4 days at 37℃ and stained with crystal violet every 24h. The specific steps were as follows. Bp from overnight SS liquid cultures was diluted to OD600 = 0.1. 100ul Bp culture were added to 96 well plates and incubated for 96h at 37℃ at rest. The culture medium was discarded every 24h and the cells were washed three times with PBS, supplemented with fresh culture medium. After incubation, 96 well plates were washed three times with PBS and left to dry. Crystal violet (1% w/v, 150 µL) was added to each well and the plate was incubated for 10 min at room temperature. The supernatant was then discarded and 200 µL of ethanol containing 10% acetic acid was added. After 10 minutes of incubation at room temperature, biofilm formation was then visualized by measuring the OD600. Five biological replicates were used at each time point. All statistical analyses were completed using GraphPad Prism 8.0. Page 5/16 Page 5/16 3.2 Function analysis of significantly different proteins in whole-cell The outer membrane acts as the first line of defense against the penetration of multiple toxic compounds, including several antimicrobial agents(Munita and Arias 2016). The differential characteristics of outer membrane proteins have been identified in antibiotic-resistant bacteria, which form a specific pattern of antibiotics(Peng, Li, and Peng 2019). In addition, the loss of some outer membrane proteins reduced the virulence and fitness of bacteria(Smani, Dominguez-Herrera, and Pachón 2013). Therefore, we further analyzed the subcellular localization of significantly different proteins in the whole cell (Fig. 2a). There are five kinds of proteins located in the outer membrane, namely FimD (A0A381A3A6), BN118_1423 (A0A0T7CMT4), HscA (Q7VXG7), AZ26_1060 (A0A171JW15), Prn1 (S5TUY6), and Prn1 (D2WF61). In order to further analyze the protein characteristics of MR-Bp, we performed a GO cluster analysis on the significantly different proteins in the whole-cell proteome. The top 20 enriched GO terms are presented in Fig. 2b. The most noteworthy is the iron-sulfur cluster and metallo-sulfur cluster assembly. The significant differences in metallo-sulfur cluster between the two strains are HscA (Q7VXG7), CyaY (Q7VT96), and IscA (A0A0U0VXP3). 3 Results 3.1 Overview of the significantly different proteins in whole-cell 3.1 Overview of the significantly different proteins in whole-cell TMT was performed to characterize and compare the whole-cell proteome of 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1). There are a total of 68 significantly different proteins between the two strains (Supplementary Table 2). Compared with 19068 (ptxP3/fhaB1), 40 proteins in 19147 (ptxP1/fhaB3) are up- regulated and 28 proteins are down-regulated. Differentially expressed proteins were analyzed by volcano plot (Fig. 1a). The heat map shows the clustering of significantly differentially expressed proteins between the two strains (Fig. 1b). Macrolide resistance is mainly caused by target modifications such as the methylation in a 23S ribosomal RNA (rRNA) adenine residue and the mutation in ribosomal protein L4 or L22(Fyfe et al. 2016).To explore the key proteins that may be involved, we focused on macrolides-resistant related molecules. Although the Erm, an enzyme that catalyzes A2058 methylation to preclude antibiotic binding, was not detected in the two pertussis strain, the expression of RumA (A0A0N2IMR5) protein was found to be significantly different in the two strains (Fig. 1b). In addition, we found that the expression of ribosomal protein L2, also known as RplB (A0A171JW48), was significantly different in expression between the two strains. 3.2 Function analysis of significantly different proteins in whole-cell 3.3 Protein interaction analysis of the significantly different proteins in whole-cell 2016; Deschaine et al. 2018). Crystal violet staining was used to evaluate whether biofilm formation changed in 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1), of which ATCC strain 9797 was used as the standard strain. Interestingly, crystal violet staining revealed that 19147 (ptxP1/fhaB3) had a significant increase the biofilm formation compared to 19068 (ptxP3/fhaB1) (Fig. 4). 3.3 Protein interaction analysis of the significantly different proteins in whole-cell P 6/16 The functional connection of the significantly different proteins between 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1) may be a breakthrough to answer the reason for the enhanced resistance and adaptability in MR-Bp. The PPI network was constructed using the String website and the figures were generated by Cytoscape (MCODE plug-in). The most significant module (MCODE score = 5.333) contained 7 nodes and 16 edges (Fig. 3a). In the whole-cell proteome in pertussis, Cpn10 (P0A339) and RpoZ (Q7VXZ4) were significantly higher in 19147 (ptxP1/fhaB3) resistant strain compared to 19068 (ptxP3/fhaB1) sensitive Page 6/16 Page 6/16 strains, and the other 5 proteins RpsC (Q7VTC7), HscA (Q7VXG7), RpoD (A0A0T7CNP5), RplB (A0A171JW48) and RpsD (P0A4C5) were significantly lower than 19068 (ptxP3/fhaB1) indicating the role of key proteins in the macrolides resistance of Bp (Fig. 1b). strains, and the other 5 proteins RpsC (Q7VTC7), HscA (Q7VXG7), RpoD (A0A0T7CNP5), RplB (A0A171JW48) and RpsD (P0A4C5) were significantly lower than 19068 (ptxP3/fhaB1) indicating the role of key proteins in the macrolides resistance of Bp (Fig. 1b). The next significant module (MCODE score = 4) contained four genes, which are prn, fim2, fim3, and fimD (Fig. 3b). Prn, fim2, and fim3 have internationally recognized pertussis virulence genes, and fim2/fim3 represents distinct serotypes of B. pertussis. In this experiment, compared with 19068 (ptxP3/fhaB1), the expression of Fim3 and FimD increased in 19147 (ptxP1/fhaB3), and the expression of Fim2 and Prn decreased. 3.4 PRM verifies significantly different protein expression levels in whole-cell To confirm the reliability of the quantitative proteomics analyses, the eleven candidate proteins selected in PPI were evaluated by PRM analyses. The mass spectrometry identification results show that the four target proteins can be accurately identified namely Prn, Fim2, Fim3, and RpoD. The fold changes of four proteins by PRM were listed in Table 1. Fim2, Prn, and RpoD were downregulated in 19147 (ptxP1/fhaB3), while Fim3 was upregulated. The results of the relative quantification demonstrated that the target proteins displayed similar trends between the TMT and PRM analyses, thus supporting the reliability of the proteomics data. 3.5 The different proteins in whole-cell are related to the biofilm formation In this study, many differential proteins related to macrolide-resistance are closely related to the formation of biofilm, such as metallo-sulfur cluster. Moreover, it has been reported that biofilm-grown Bp confers increased tolerance to antimicrobial agents compared with planktonic cultures and biofilm formation provides a fitness advantage to bacteria(Dorji et al. 4 Discussion In this study, we determined the significantly different proteins between the dominant lineage of Bp strains between China and other countries nearly all over the world through TMT-based proteomics and PRM-based protein quantification. The specific key proteins of MR-Bp strain include ribosome-associated proteins (RumA, RplB), outer membrane proteins (FimD, BN118_1423, HscA, AZ26_1060, Prn), Fe/S clusters proteins (HscA, CyaY, IscA), RNA polymerase proteins (RpoD, RpoZ) and virulence proteins (Prn, Fim2, Fim3, FimD). Interestingly, the deletion and mutation of multiple resistance genes encoding these proteins are closely related to the formation of biofilms in other bacterial species. Moreover, the biofilm formation of 19147 (ptxP1/fhaB3) was increased compared with 19068 (ptxP3/fhaB1) (Fig. 4). Based on our limited current Page 7/16 Page 7/16 Page 7/16 knowledge, this is the first report screening the potential mechanisms of macrolides resistance and the fitness of prevalent MR-Bp through proteomics. More and more evidence shows that defects in Fe/S proteins assembly and maturation are closely related to biofilm formation and antibiotic resistance(Mashruwala et al. 2016; Deshpande et al. 2020; Ellepola et al. 2021). Among the three significantly different proteins in the Fe/S cluster, HscA and IscA are involved in the iron-sulfur cluster assembly(Zeng et al. 2007; Mayer 2021), and either hscA mutation or loss of iscA will change the formation of bacterial biofilm(Vasil'eva and Strel'tsova 2013; Rondeau et al. 2019). In the PPI network, RNA polymerase related to the Fe/S cluster contributes to concern, and the RpoZ and RpoD in Bp strains may dynamically change macrolides resistance. RpoZ and RpoD, called ω and σ70 factors respectively, are the subunits of the RNA polymerase core enzyme. We found that the RpoZ expression of the 19147 (ptxP1/fhaB3) resistant isolates was increased and the RpoD was decreased correspondingly. Although ω-encoding rpoZ evolves in the ecosystem during colder and drier periods, resulting in an increased rate of biofilm production of rpoZ variants in vitro(Cui et al. 2020), whether it is in Gram-positive or Gram-negative bacteria, defects of rpoZ have been confirmed to impair the ability to completely form biofilms and affect antibiotic resistance(Mukherjee and Chatterji 2008; Weiss et al. 2017; Bhardwaj, Syal, and Chatterji 2018). Another important function of the ω factor is involved in σ factor recruitment. RpoD mutation is associated with antibiotic resistance(Palace et al. 2020), and we have verified the low expression of RpoD in 19147 (ptxP1/fhaB3) by PRM-based protein quantification. 7.1 Funding This work was supported by the Natural Science Foundation of China [Grant number 82172312]; the Key Research and Development Program of Shaanxi Province [Grant number 2021SF-003]; and the Xi'an Science and technology project [Grant number 20YXYJ0006(1)]. 4 Discussion Besides the proteins from the cells, the outer membrane vesicles (OMVs) from the culture supernatant were also closely attributed to the biofilm formations. Therefore, it is impossible to rule out that some proteins related to the biofilm formations have not been discovered. 7.2 Competing interests The authors have no relevant financial or non-financial interests to disclose. 4 Discussion In the PPI network, except for RNA polymerase (RpoZ and RpoD), both RpsC and RpsD are part of the 30S ribosomal subunit, and their changes will affect antibiotic resistance(Björkman et al. 1999; Gupta et al. 2016). Horizontal gene transfer (HGT) of rplB, also known as ribosomal protein L2, has been shown to play a key role in the genesis of antimicrobial resistance(Gentry and Holmes 2008; Manoharan-Basil et al. 2021). Cpn10, also known as GroES, and over-expression of Cpn10 promote streptomycin resistance(Goltermann, Sarusie, and Bentin 2015). As we all know, biofilms are communities of microorganisms attached to a surface that is significantly less susceptible to antimicrobial agents than non-adherent planktonic cells(Hall and Mah 2017). It has recently been discovered that biofilm formation was associated with Bp resistance. Compared with planktonic cultures, biofilm growing Bp conferred increased tolerance to antimicrobial agents(Dorji et al. 2016), which may be a possible reason for the emergence of macrolides resistance of the ptxP1/fhaB3 strain in China. After antibiotic resistance, the adaptability of some bacteria will increase rather than decrease, which is usually related to a compensatory mutation in evolution(Durão, Balbontín, and Gordo 2018). The mutation rate of ptxP1/fhaB3 strain in China is much higher than the rest of the global ptxP1-fhaB1 isolates(Xu, Wang, et al. 2019). Therefore, the adaptability of the Chinese MR-Bp strains may also be produced by compensation mutation, but it needs to be further verified. Another aspect that deserves attention: the fitness advantage of bacteria is related to biofilm formation. When replication and virulence of drug-resistant bacteria were significantly attenuated, their biofilm formation decreased(Biot et al. 2020). Other studies have reported that hyperbiofilm formation is associated with enhanced virulence traits in Bp(Cattelan et al. 2017). The higher biofilm-forming strains will exhibit Page 8/16 increased cellular adherence to epithelial cells and contribute to enhanced respiratory tract colonization. FimD (fimbrial subunits), highly expressed in 19147 (ptxP1/fhaB3) strain, is critical in adherence to airway cells and can also serve as specific residues of the chaperone-usher pathway to mediate biofilm formation(Volkan et al. 2013; Guevara et al. 2016). Therefore, the link between the hyperbiofilm-forming ability of Bp and enhanced pathogenic phenotypes indirectly suggested that the significant difference in proteins related to biofilm formation may be a crucial factor for the adaptive transmission ability of ptxP1/fhaB3/MR-Bp. However, the limitation of this study is that the proteomics from the culture supernatant was not performed. 5 Conclusions In summary, in the proteomics of 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1) strains, the significantly different proteins are probably related to biofilm formation. The bacterium growing in biofilm is more resistant to macrolides and contributed to the persistence and transmission, which may be the reason for macrolides resistance and the fitness of ptxP1/fhaB3 MR-Bp strains in China. Further studies focused on biofilms need to be explored much more. 7.3 Author Contributions All authors contributed to the study conception and design. The study was designed by Hong Yan and Zengguo Wang and mainly performed by Zhe Lv and Sha Yin. Material preparation were performed by Kaichong Jiang and Yang Luan. Data collection were performed by Wei Wang, Shuang Wu and Jianfei Shi. Data analysis were performed by Zhe Li and Xiao Ma. The first draft of the manuscript was written by Zhe Lv and Sha Yin. And all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Page 9/16 Page 9/16 7.4 Data Availability The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request References 1. Bhardwaj N, Syal K, and Chatterji D (2018) The role of ω-subunit of Escherichia coli RNA polymerase in stress response. Genes Cells 23: 357–69. 2. Biot FV, Bachert BA, Mlynek KD et al. (2020) Evolution of Antibiotic Resistance in Surrogates of Francisella tularensis (LVS and Francisella novicida): Effects on Biofilm Formation and Fitness. Front Microbiol 11: 593542. 3. Björkman J, Samuelsson P, Andersson DI et al. (1999) Novel ribosomal mutations affecting translational accuracy, antibiotic resistance and virulence of Salmonella typhimurium. Mol Microbiol 31: 53–8. 3. 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(2019) Biofilms: The Microbial "Protective Clothing" in Extreme Environments. Int J Mol Sci 20. 43. Zeng J, Geng M, Jiang H et al. (2007) The IscA from Acidithiobacillus ferrooxidans is an iron-sulfur protein which assemble the [Fe4S4] cluster with intracellular iron and sulfur. Arch Biochem Biophys 463: 237–44. 43. Zeng J, Geng M, Jiang H et al. (2007) The IscA from Acidithiobacillus ferrooxidans is an iron-sulfur protein which assemble the [Fe4S4] cluster with intracellular iron and sulfur. Arch Biochem Biophys 463: 237–44. Tables Page 12/16 Table 1 Comparison of the quantification results between TMT and PRM for the four candidate proteins. Protein name TMT result PRM result 19147 _average 19068 _average Ratio_ 19147/19068 19147 _average 19068 _average Ratio_ 19147/19068 A0A0T7CNP5(RpoD) 0.9198 1.2183 0.7550 0.0908 0.1262 0.7191 B6DYY0(Fim2) 0.4898 1.3462 0.3639 0.0030 0.5669 0.0053 B6DYY5(Fim3) 1.4421 0.6918 2.0846 2.0524 0.0132 154.9690 D2WF61(Prn) 0.4172 1.0482 0.3980 0.0491 1.1896 0.0412 uantification results between TMT and PRM for the four candidate proteins. Figures Page 13/16 Figure 1 Th h t i ti f i ifi tl diff t t i i h l Figure 1 The characteristics of significantly different proteins in whole-cell (a). Differentially expressed proteins were analyzed by volcano plot between 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1). (b). The heat map shows two groups of significantly different proteins. Page 14/16 Figure 2 Functional analysis of 19147 (ptxP1/fhaB3) and 19068 (ptxP3/fhaB1) proteins detected in the whole-cell (a). The subcellular structure of significantly different proteins between the two groups. (b). The top 20 enriched Gene Ontology (GO) terms of significantly different proteins. BP: biological process; MF: molecular function; CC: cellular component. Figure 3 The protein-protein interaction (PPI) network on the significantly different proteins in the whole cell (a). The PPI in the most significant module. (b). The PPI in a second significant module. Figure 3 Figure 4 The fhaB3 allele MR-Bp increases the formation of biofilm The crystal violet staining revealed the biofilm formation among 19147 (ptxP1/fhaB3), 19068 (ptxP3/fhaB1), and 9797. The fhaB3 allele MR-Bp increases the formation of biofilm The crystal violet staining revealed the biofilm formation among 19147 (ptxP1/fhaB3), 19068 (ptxP3/fhaB1), and 9797. Figure 3 The protein-protein interaction (PPI) network on the significantly different proteins in the whole cell (a). The PPI in the most significant module. (b). The PPI in a second significant module. Page 15/16 Page 15/16 Figure 4 The fhaB3 allele MR-Bp increases the formation of biofilm The crystal violet staining revealed the biofilm formation among 19147 (ptxP1/fhaB3), 19068 (ptxP3/fhaB1), and 9797. Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementaryTable1.xls SupplementaryTable2.xls Page 16/16
https://openalex.org/W2126090181	https://hal.archives-ouvertes.fr/hal-01185655/file/2015_Guo_Biotechnol%20Biofuels_%7BCBA8D6E6-37B1-47DC-A148-3A5C09D2C7F9%7D.pdf	English	null	Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes	Biotechnology for biofuels	2,015	cc-by	12,428	Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes Zhongpeng Guo, Sophie Duquesne, Sophie Bozonnet, Gianluca Cioci, Jean-Marc Nicaud, Alain Marty, Michael O’Donohue Development of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes Zhongpeng Guo, Sophie Duquesne, Sophie Bozonnet, Gianluca Cioci, Jean-Marc Nicaud, Alain Marty, Michael O’Donohue To cite this version: Zhongpeng Guo, Sophie Duquesne, Sophie Bozonnet, Gianluca Cioci, Jean-Marc Nicaud, et al.. De- velopment of cellobiose-degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes. Biotechnology for Biofuels, 2015, 8, pp.1-16. 10.1186/s13068-015-0289-9. hal-01185655 © 2015 Guo et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. HAL Id: hal-01185655 https://hal.science/hal-01185655v1 Submitted on 20 Aug 2015 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Guo et al. Biotechnol Biofuels (2015) 8:109 DOI 10.1186/s13068-015-0289-9 Open Access Abstract Background: Yarrowia lipolytica, one of the most widely studied “nonconventional” oleaginous yeast species, is unable to grow on cellobiose. Engineering cellobiose-degrading ability into this yeast is a vital step towards the devel‑ opment of cellulolytic biocatalysts suitable for consolidated bioprocessing. Results: In the present work, we identified six genes encoding putative β-glucosidases in the Y. lipolytica genome. To study these, homologous expression was attempted in Y. lipolytica JMY1212 Zeta. Two strains overexpressing BGL1 (YALI0F16027g) and BGL2 (YALI0B14289g) produced β-glucosidase activity and were able to degrade cellobiose, while the other four did not display any detectable activity. The two active β-glucosidases, one of which was mainly cell-associated while the other was present in the extracellular medium, were purified and characterized. The two Bgls were most active at 40–45°C and pH 4.0–4.5, and exhibited hydrolytic activity on various β-glycoside substrates. Specifically, Bgl1 displayed 12.5-fold higher catalytic efficiency on cellobiose than Bgl2. Significantly, in experiments where cellobiose or cellulose (performed in the presence of a β-glucosidase-deficient commercial cellulase cocktail produced by Trichoderma reseei) was used as carbon source for aerobic cultivation, Y. lipolytica ∆pox co-expressing BGL1 and BGL2 grew better than the Y. lipolytica strains expressing single BGLs. The specific growth rate and biomass yield of Y. lipolytica JMY1212 co-expressing BGL1 and BGL2 were 0.15 h−1 and 0.50 g-DCW/g-cellobiose, respectively, similar to that of the control grown on glucose. Conclusions: We conclude that the bi-functional Y. lipolytica developed in the current study represents a vital step towards the creation of a cellulolytic yeast strain that can be used for lipid production from lignocellulosic biomass. When used in combination with commercial cellulolytic cocktails, this strain will no doubt reduce enzyme require‑ ments and thus costs. Keywords: Lignocellulosic biomass, Oleaginous yeast, Lipids, Enzymatic hydrolysis, Cellulases costly pretreatment and enzymatic hydrolysis steps, the latter requiring the action of several types of enzymes [1, 2]. Indeed, the hydrolysis of cellulose alone requires the synergistic action of endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21) [3]. Endoglucanases are active on the internal bonds in cellulose and release free reducing and non- reducing extremities, which are used by cellobiohydro- lases as starting points for exo-processive hydrolysis that yields cellodextrins as products. Finally, β-glucosidases convert cellodextrins into glucose [4]. Development of cellobiose‑degrading ability in Yarrowia lipolytica strain by overexpression of endogenous genes Zhongpeng Guo1,2,3, Sophie Duquesne1,2,3, Sophie Bozonnet1,2,3, Gianluca Cioci1,2,3, Jean‑Marc Nicaud4,5, Alain Marty1,2,3 and Michael Joseph O’Donohue1,2,3 © 2015 Guo et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background It is widely recognized that lignocellulosic biomass (or LC biomass) will form an important part of the future bio- economy. However, the use of this renewable resource as feedstock for industrial activities poses a major challenge, because its deconstruction to sugars and lignin is com- plex, requiring a series of unit operations. These include Correspondence: zguo@insa‑toulouse.fr; marty@insa‑toulouse.fr 1 LISBP‑Biocatalysis Group, INSA/INRA UMR 792, Université de Toulouse, 135 Avenue de Rangueil, 31077 Toulouse, France Full list of author information is available at the end of the article Correspondence: zguo@insa‑toulouse.fr; marty@insa‑toulouse.fr 1 LISBP‑Biocatalysis Group, INSA/INRA UMR 792, Université de Toulouse, 135 Avenue de Rangueil, 31077 Toulouse, France Full list of author information is available at the end of the article Page 2 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 One strategy to reduce investment and operational costs in LC biomass processing is to internalize enzyme production and combine enzymatic hydrolysis with fer- mentation. This is known as consolidated bioprocessing (CBP) and can be achieved using a microorganism that possesses the dual ability to produce biomass-hydrolyz- ing enzymes and ferment sugars to products of commer- cial interest, thus allowing a one-pot type bioconversion process in which process integration is maximized [5]. While CBP is considered to be an ultimate aim for biorefining, the ways to achieve this goal are not simple. Although the number of naturally occurring, biomass- degrading microorganisms is no doubt large, those that possess the ability to hydrolyze LC biomass and ferment free sugars into target products, such as ethanol, butanol, hydrogen, fatty acid ethyl esters (FAEE) or isopropanol, at industrially compatible rates and titers, are probably very rare and so far undiscovered [6]. Additionally, many of the best known biomass-degrading microorganisms display low β-glucosidase (cellobiase) activity, meaning that the hydrolysis of cellobiose constitutes a rate-lim- iting step during the enzymatic processing of cellulose [7–9]. Therefore, engineering cellobiose-degrading ability into microorganisms is a vital step towards the develop- ment of cellulolytic biocatalysts suitable for CBP. In this respect, examples of recent work performed on Saccha- romyces cerevisiae, the current workhorse of biotech- nological processes, are noteworthy [10–12]. In these studies, even though the engineered S. cerevisiae strains exhibited poor cellulose-degrading ability, the fact that they both produce significant cellobiase activity means that their incorporation into a simultaneous saccharifi- cation and fermentation (SSF) process is likely to reduce the loading of external cellulases and thus overall process cost [10]. Resultsi Although ethanol is the target molecule in many biore- finery concepts, Fatty Acid Esters (FAEs) such as those used in biodiesel, are also attractive targets. This is because FAEs display high energy density and are well- tolerated by production strains [13]. Currently, FAEs are mainly produced by transesterification of plant oils using an alcohol (methanol or ethanol) and base, acid or enzyme catalysts [14]. However, the high cost of this process and various issues surrounding the production of plant oils for non-food purposes make the search for alternative routes both attractive and strategically per- tinent. In this respect, microbial production of biofuels (so-called microdiesel and microkerosene) represents a sustainable and quite economical way to produce FAEs. For this purpose, both Escherichia coli and S. cerevisiae have been engineered to produce structurally tailored fatty esters [15–17]. However, neither of these microor- ganisms is naturally able to accumulate high amounts of Background lipids, nor able to degrade cellulose. Moreover, in these microorganisms the biosynthesis of fatty acid is highly regulated [18], thus limiting the possibility to improve lipid production [16, 17, 19]. So-called oleaginous microorganisms, which natu- rally accumulate lipids to more than 20% of their dry cell weight (DCW) [20, 21], have already been exploited for the production of commercially useful lipids, such as substitutes for cocoa butter and polyunsaturated fatty acids [22]. Therefore, it is unsurprising that microbial lipid or single cell oil is also being considered for bio- diesel production, especially because this route implies shorter production times, reduced labor costs and sim- pler scale-up [23]. Prominent among the oleaginous microorganisms, Yarrowia lipolytica has been extensively studied and is known to accumulate lipids up to 50% of its dry weight depending on culture conditions [20, 21, 24]. Advantageously, since Y. lipolytica is already widely used in the detergent, food, pharmaceutical and environ- mental industries it has been classified by the FDA (Food and Drug Administration) as “Generally Recognized as Safe” (GRAS) for numerous processes [25]. Nevertheless, despite these advantages, Y. lipolytica displays limited ability for sugar use and is unable to use cellulose as car- bon source [26]. In a recent paper, the use of cellobiose by Y. lipolyt- ica was tackled for the first time, thus opening the way towards the development of an efficient yeast-based CBP microorganism capable of consuming cellulose- derived glucose and converting it into lipids and deriva- tives thereof [27]. Herein, we present work that shares this aim, but which has employed a different strategy that relies upon the activation of endogenous β-glucosidase activity (Fig. 1). Identification of genes encoding active β‑glucosidases in Y. lipolytica y Analysis of the Y. lipolytica genome using BLAST revealed the presence of six sequences that were identi- fied as putative family GH3 β-glucosidases (see Addi- tional file 1: Table S1; Additional file 2: Fig. S1) on the basis of high amino acid sequence identity with other yeast β-glucosidases (Fig. 2, Additional file 2: Fig. S1). However, in the absence of biochemical data it was impossible to assert at this stage that these sequences actually encode β-glucosidases, since family GH3 con- tains glycoside hydrolases that display other specifici- ties. Moreover, Y. lipolytica does not grow on cellobiose and has not been found to express a detectable level of β-glucosidase activity (Additional file 2: Fig. S2), despite the fact that preliminary transcriptional analysis revealed that the six genes are weakly transcribed when Y. Guo et al. Biotechnol Biofuels (2015) 8:109 Page 3 of 16 lipolytica is grown on glucose, although no further induc- i b d i h f ll bi (Addi gratifying to observe that overexpression of BGL1 (YAL- I0F16027 ) BGL2 (YALI0B14289 ) i Y li l i Fig. 1 The strategies used in the current study to develop the cellobiose-degrading ability in Y. lipolytica. B. subtilus Bgl YALI0D18381 YALI0B14333 S. venezuelae Bgl YALI0B14289 (Bgl2) K. marxianus Bgl A. aculeatus Bgl YALI0E20185 T. neapolitana Bgl T. reesei Bgl YALI0F01672 YALI0F16027 (Bgl1) 10 Fig. 2 Phylogenetic tree of the structurally characterized family three glycosyl hydrolases. Multiple-sequence alignment was performed using CLUSTALW (http://www.genome.jp/tools/clustalw/), and a phylogenetic tree was constructed using MEGA5 (http://www.megasoftware.net/). Sequences are: Bacillus subtilus Bgl (PDB accession number 4GYJ_A); Streptomyces venezuelae Bgl (PDB accession number 4I3G_A); Kluyveromyces marxianus Bgl (PDB accession number 3AC0_A); Aspergillus aculeatus Bgl (PDB accession number 4IIB_A); Thermotoga neapolitana Bgl (PDB acces‑ sion number 2X42_A); Trichoderma reesei Bgl (PDB accession number 4I8D_A); and include the 6 putative GH3 sequences from Yarrowia lipolytica genome. Fig. 1 The strategies used in the current study to develop the cellobiose-degrading ability in Y. lipolytica. Fig. 1 The strategies used in the current study to develop the cellobiose-degrading ability in Y. lipolytica. Fig. 1 The strategies used in the current study to develop the cellobiose-degrading ability in Y. lipolytica. B. subtilus Bgl YALI0D18381 YALI0B14333 S. venezuelae Bgl YALI0B14289 (Bgl2) K. marxianus Bgl A. aculeatus Bgl YALI0E20185 T. neapolitana Bgl T. reesei Bgl YALI0F01672 YALI0F16027 (Bgl1) 10 Fig. 2 Phylogenetic tree of the structurally characterized family three glycosyl hydrolases. Identification of genes encoding active β‑glucosidases in Y. lipolytica Multiple-sequence alignment was performed using CLUSTALW (http://www.genome.jp/tools/clustalw/), and a phylogenetic tree was constructed using MEGA5 (http://www.megasoftware.net/). Sequences are: Bacillus subtilus Bgl (PDB accession number 4GYJ_A); Streptomyces venezuelae Bgl (PDB accession number 4I3G_A); Kluyveromyces marxianus Bgl (PDB accession number 3AC0_A); Aspergillus aculeatus Bgl (PDB accession number 4IIB_A); Thermotoga neapolitana Bgl (PDB acces‑ sion number 2X42_A); Trichoderma reesei Bgl (PDB accession number 4I8D_A); and include the 6 putative GH3 sequences from Yarrowia lipolytica genome. B. subtilus Bgl YALI0D18381 YALI0B14333 S. venezuelae Bgl YALI0B14289 (Bgl2) K. marxianus Bgl A. aculeatus Bgl YALI0E20185 T. neapolitana Bgl T. reesei Bgl YALI0F01672 YALI0F16027 (Bgl1) 10 S. venezuelae Bgl YALI0B14289 (Bgl2) YALI0F01672 Fig. 2 Phylogenetic tree of the structurally characterized family three glycosyl hydrolases. Multiple-sequence alignment was performed using CLUSTALW (http://www.genome.jp/tools/clustalw/), and a phylogenetic tree was constructed using MEGA5 (http://www.megasoftware.net/). Sequences are: Bacillus subtilus Bgl (PDB accession number 4GYJ_A); Streptomyces venezuelae Bgl (PDB accession number 4I3G_A); Kluyveromyces marxianus Bgl (PDB accession number 3AC0_A); Aspergillus aculeatus Bgl (PDB accession number 4IIB_A); Thermotoga neapolitana Bgl (PDB acces‑ sion number 2X42_A); Trichoderma reesei Bgl (PDB accession number 4I8D_A); and include the 6 putative GH3 sequences from Yarrowia lipolytica genome. Fig. 2 Phylogenetic tree of the structurally characterized family three glycosyl hydrolases. Multiple-sequence alignment was performed using CLUSTALW (http://www.genome.jp/tools/clustalw/), and a phylogenetic tree was constructed using MEGA5 (http://www.megasoftware.net/). Sequences are: Bacillus subtilus Bgl (PDB accession number 4GYJ_A); Streptomyces venezuelae Bgl (PDB accession number 4I3G_A); Kluyveromyces marxianus Bgl (PDB accession number 3AC0_A); Aspergillus aculeatus Bgl (PDB accession number 4IIB_A); Thermotoga neapolitana Bgl (PDB acces‑ sion number 2X42_A); Trichoderma reesei Bgl (PDB accession number 4I8D_A); and include the 6 putative GH3 sequences from Yarrowia lipolytica genome. gratifying to observe that overexpression of BGL1 (YAL- I0F16027g) or BGL2 (YALI0B14289g) in Y. lipolytica (strains ZetaB 1 and ZetaB 2, respectively) enhanced lipolytica is grown on glucose, although no further induc- tion was observed in the presence of cellobiose (Addi- tional file 2: Fig. S3). In this respect, it was particularly Page 4 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 the transcription of the genes and conferred the ability to grow on solid medium containing cellobiose as the sole carbon source (Additional file 2: Figs. S2, 3). Addi- tionally, when these recombinant strains were grown on YNB-pNP-βGlc (p-nitrophenyl-β-d-glucoside) plates, yellow halos surrounding the colonies were clearly visu- alized, indicating β-glucosidase activity (Additional file 2: Fig. S3). Identification of genes encoding active β‑glucosidases in Y. lipolytica Finally, after growth in liquid YTD medium, β-glucosidase activity could be measured in the cell extract of ZetaB 1 (3.2 ± 0.2 IU/mg) and in the culture supernatant of ZetaB 2 (2.6 ± 0.1 U/mL), while much lower activities were measured in the culture supernatant of ZetaB 1 (0.33 ± 0.02 U/mL) and in the cell extract of ZetaB 2 (0.42 ± 0.01 IU/mg). Regarding the remaining four putative β-glucosidases, expression of their encod- ing sequences (YALI0F01672g, YALI0D18381g, YAL- I0B14333g and YALI0E20185g) in Y. lipolytica failed to produce any detectable β-glucosidase activity or sustain yeast growth on solid medium containing cellobiose as the sole carbon source. (Fig. 3d), consistent with the fact that no β-glucosidase activity was detected in this fraction. Significantly, the expression of the native BGL2 sequence (i.e. without the His6-tag) provided much more satisfactory expres- sion, implying that the presence of the His6-tag on Bgl2 somehow impairs the expression and/or secretion of this protein. Localization of β‑glucosidases in ZetaB 1 and ZetaB 2 Localization of β‑glucosidases in ZetaB 1 and ZetaB 2 To determine the localization of Bgl1 and Bgl2, yeast cells producing these enzymes (without His6 tag) were fractionated, generating on the one hand extracellular samples (culture supernatant), and on the other cell- associated periplasmic, cytoplasmic and membrane frac- tions. Measurement of the β-glucosidase activities in each of these fractions revealed that Bgl1 was primarily localized in the periplasm (61%), but was also present in the cytoplasm (30%), while Bgl2 was mainly in the supernatant (80%), and to a lesser extent (26%) in the periplasm (Table 1). The apparent ambiguity of these results is undoubtedly due to the separation method that was employed to isolate the different cellular fractions, which inevitably led to a low level of cross-contamina- tion between the samples. Nevertheless, taking this into account, it is reasonable to deduce that Bgl1 is primarily localized in the periplasmic space, while Bgl2 is secreted into the culture medium. To further investigate the production of the six β-glucosidases, western blot analysis was carried out using anti-His6 antibodies. This revealed the presence of Bgl1-His6 in both the culture supernatant and cell extract (Fig. 3a, b). Bgl2-His6 could only be detected in concentrated (tenfold) cell extract (Fig. 3c). However, this method failed to detect Bgl2-His6 in the culture media Fig. 3 Western blot detection of the expressed β-glucosidases a M, molecular weight standards; lane 1, intracellular Bgl1, b lane 1, extracellular Bgl1-His6, c lane 1, intracellular Bgl2-His6, d lane 1, extracellular Bgl2-His6, and SDS-PAGE analysis of the purified β-glucosidases from Y. lipolytica JMY1212 transformants e lane 1, purified Bgl1-His6, and f lane 1, purified Bgl2; lane 2, endo-H treated Bgl2 (The lower band in lane 2 represents the expected size of Endo-H). Fig. 3 Western blot detection of the expressed β-glucosidases a M, molecular weight standards; lane 1, intracellular Bgl1, b lane 1, extracellular Bgl1-His6, c lane 1, intracellular Bgl2-His6, d lane 1, extracellular Bgl2-His6, and SDS-PAGE analysis of the purified β-glucosidases from Y. lipolytica JMY1212 transformants e lane 1, purified Bgl1-His6, and f lane 1, purified Bgl2; lane 2, endo-H treated Bgl2 (The lower band in lane 2 represents the expected size of Endo-H). Page 5 of 16 Guo et al. Localization of β‑glucosidases in ZetaB 1 and ZetaB 2 Biotechnol Biofuels (2015) 8:109 Table 1 Distribution of β-glucosidase activity in recombi- nant strains ZetaB 1 and ZetaB 2 Table 1 Distribution of β-glucosidase activity in recombi- nant strains ZetaB 1 and ZetaB 2 ±, the standard deviation. a Triplicate experiments. Activity was assayed with pNP-βGlc. Fraction Relative enzyme activitya Bgl1 (%) Bgl2 (%) Total 100 100 Growth medium 2.3 ± 0.4 79.6 ± 1.2 Periplasm 60.7 ± 1.0 25.8 ± 1.0 Cytoplasm 30.0 ± 0.5 4.8 ± 0.8 Membrane 8.1 ± 0.7 3.7 ± 0.3 purified Bgl2 protein with endoglycosidase H (EndoH) and migrating it on a SDS-PAGE. This analysis revealed that the Mr of the recombinant EndoH-treated Bgl2 was approximately 95 kDa, quite consistent with the theoreti- cal Mr of 92.9 kDa (Fig. 3f). Finally, N-terminal amino acid sequence analysis of Bgl1 and Bgl2 confirmed that the signal sequences of both proteins had been cleaved and allowed the accurate localization of the cleavage sites (Additional file 2: Fig. S4). i Preliminary characterization of Bgl1 and Bgl2 using pNP-βGlc as substrate revealed that Bgl1 was 5-fold more active (102.8 U/mg) on this substrate than Bgl2 (25.8 U/ mg). The activity of Bgl1 was highest at approximately pH 4.5 and 45°C, and was stable in the pH range of 4.0–5.0 and below 40°C. Regarding Bgl2, it was found to display highest activity at pH 4.0 and 50°C and was stable in the pH range of 3.5–7.0 and below 50°C (Additional file 2: Figs. S5, S6). It is noteworthy that deglycosylation of Bgl2 led to a 60% decrease in specific activity, which was prob- ably due to its instability at 40°C (Additional file 2: Fig. S7). a Triplicate experiments. Activity was assayed with pNP-βGlc. Production, purification and characterization of Bgl1 and Bgl2 Production of Bgl1-His6 and native Bgl2 was achieved by growing the appropriate Y. lipolytica strains on YTD, with expression of both enzymes increasing until com- plete depletion of glucose was reached (36 h).i Regarding purification of Bgl1-His6, yeast cells from a 200-mL culture volume yielded approximately 550 U of enzyme in the crude cell extract. However, after one step of affinity-purification, only 17% of Bgl1 was recov- ered (Table 2). In the case of Bgl2, a two-step protocol using anion exchange chromatography and gel filtration allowed its purification to near homogeneity, but led to significant loss of protein (8.8% recovery). SDS-PAGE analysis of the two purified protein samples revealed that while the Mr of Bgl1 was consistent with the expected value (i.e. Mr of 90.4 kDa for the protein lacking the putative signal peptide), that of Bgl2 was significantly higher (>250 kDa) (Fig. 3e, f). To investigate whether this discrepancy was due to glycosylation, the amino acid sequence of Bgl2 was analyzed using the glyco- sylation predictor GlycoEP (http://www.imtech.res.in/ raghava/glycoep/) [28]. This revealed that Bgl2 harbors 18 potential N-glycosylation sites (Additional file 1: Table S2). Glycosylation was finally confirmed by treating the a Specific activity was tested on pNP-βGlc. Substrate specificity and kinetic parameters of Bgl1 and Bgl2hii The substrate specificity of the purified β-glucosidases was examined using different substrates displaying α and β configurations. The results showed that both β-glucosidases were maximally active against pNP- βGlc (Fig. 4). However, using activity on pNP-βGlc as benchmark, it is noteworthy that both enzymes were active on pNP-β-d-cellobioside (Bgl1, 24% and Bgl2, 27%), but only Bgl1 displayed significant activ- ity (10%) on pNP-β-d-xylopyranoside. Neither enzyme displayed activity on pNP-β-d-galactopyranoside and pNP-α-d-glucopyranoside. When the activity of Bgl1 and Bgl2 on cellobiose was compared with that on other oligosaccharides, it was found that both enzymes displayed highest activity on laminaribiose (β-1, 3-linkage), followed by gentiobiose n of intracellular Bgl1-His6 and extracellular Bgl2 produced by Y. lipolytica overexpressing strains ble 2 Purification of intracellular Bgl1-His6 and extracellular Bgl2 produced by Y. lipolytica overexpre i g g p y p y p g a Specific activity was tested on pNP-βGlc. Enzyme and purification method Total protein (mg) Total activity (U) Specific activity (U/mg) Fold purification Yield (%) recovery Bgl1-His6 Filtrate 169.7 543.0 – – 100 TALON His-taga 0.9 92.5 102.8 32.1 17.0 Bgl2 Culture supernatant 2302.5 530.2 – – 100 Ultra filtration 1986.4 510.5 – 1.1 96.3 Ion exchange 235.3 478.5 – 7.7 90.2 Gel filtration 1.8 46.4 25.8 112.2 8.8 on method Total protein (mg) Total activity (U) Specific activity (U/mg) Fold purification Yield (%) recove zyme and purification method Total protein (mg) Total activity (U) Specific activity (U/mg) Fold purification Page 6 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 Fig. 4 Comparison of the hydrolytic activity of β-glucosidases from Y. lipolytica JMY1212. Bgl1-His and Bgl2 on a pNP-derived substrates, and b natural glycosyl substrates with different β-configurations. (β-1, 6-linkage), octylglucoside, sophorose (β-1, 2-link- age), cello-oligosaccharides (β-1, 4-linkage, trisaccha- ride and higher)) and cellobiose (β-1, 4-linkage). It is noteworthy that the hydrolytic activity of Bgl1 was less dependent on the chain length of cello-oligosaccharides, while hydrolytic activity of Bgl2 increased as the length of cello-oligosaccharides increased. Both enzymes rec- ognized methylglucoside as substrate, but the hydrolytic activities were low compared with the other substrates (Fig. 4), indicating that correct occupation of subsite +1 is important for catalysis. substrates for Bgl1 and Bgl2, respectively. Additionally, the performance constant of Bgl1 measured on cellobiose was 12.5-fold higher than that describing Bgl2. Regard- ing other glucosyl substrates (i.e. Substrate specificity and kinetic parameters of Bgl1 and Bgl2hii those containing link- ages other than β-1, 4), both Bgls displayed the highest performance constants on laminaribiose. Nevertheless, comparison of the performance constants on each of the substrates revealed that Bgl2 is less regioselective, since the kcat/KM values were always lower in reactions cata- lyzed by Bgl1 (86% for sophorose, 47% for laminaribiose, 37% for gentiobiose, 18% for methylglucoside and 45% for octylglucoside). Finally, the lowest performance con- stants for both Bgls were measured for reactions contain- ing methylglucoside. The determination of the apparent kinetic parameters of reactions catalyzed by Bgl1 and Bgl2 and containing various glucosyl disaccharides and cello-oligosaccharides revealed that the values of KM(app) and kcat/KM for Bgl2- catalyzed reactions increased as a function of degree of polymerization (DP) of the cello-oligosaccharides (Table 3). In the case of Bgl1, increased DP was associ- ated with increased KM(app) values, but not kcat/KM values. Overall, considering the performance constant (kcat/KM), cellobiose and cellohexaose were the best Cellobiose and cello‑oligosaccharide fermentation with Y. lipolytica recombinant strains Yeast strains ZetaB 1 and ZetaB 2, expressing BGL1 and BGL2, respectively, were grown in micro cultivation plates under aerobic conditions in the presence of cellobiose or cellodextrins (until Glc × 6) as sole carbon sources, using Page 7 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 Table 3 Kinetic parameters of Y. lipolytica Bgls for various glycoside-substrates The mean values of three independent experiments are shown and the standard deviation is below 10%. Hydrolytic activities for the substrate were determined from the amount of released glucose and the kinetic parameters were calculated as described in “Methods”. Substrate Linkage Bgl1 Bgl2 KM (mM) kcat (s−1) kcat/KM (mM−1 s−1) KM (mM) kcat (s−1) kcat/KM (mM−1 s−1) Cellobiose Glc × 2, β-l, 4 0.26 21.1 81.1 0.79 5.1 6.5 Cellotriose Glc × 3, β-1, 4 0.43 20.5 47.7 0.99 9.5 9.6 Cellotetraose Glc × 4, β-1, 4 1.89 30.9 16.3 1.86 20.6 11 Cellopentaose Glc × 5, β-1, 4 2.18 29.5 13.5 2.24 27.5 12.3 Cellohexaose Glc × 6, β-1, 4 3.01 31.5 10.5 2.37 30.5 12.9 Sophorose Glc × 2, β-1, 2 2.25 28.4 14.8 2.4 41.2 17.2 Laminaribiose Glc × 2, β-1, 3 0.68 75.6 110.7 0.89 211.1 237.2 Gentiobiose Glc × 2, β-1, 6 1.16 43.6 37.6 1.84 186.5 101.4 Methylglucoside C = l 15 15 1 6.23 34.1 5.5 Octylglucoside C = 8 0.86 32.8 38.1 1.3 111.1 85.2 Table 3 Kinetic parameters of Y. lipolytica Bgls for various glycoside-substrates The mean values of three independent experiments are shown and the standard deviation is below 10%. Hydrolytic activities for the substrate were determined from the amount of released glucose and the kinetic parameters were calculated as described in “Methods”. The mean values of three independent experiments are shown and the standard deviation is below 10% the amount of released glucose and the kinetic parameters were calculated as described in “Methods”. previously described strategy to increase lipid accumu- lation, involving the deletion of the 6 POX genes (POX1 to POX6) that encode the peroxisomal acyl-coenzyme oxidases involved in lipid β-oxydation, was adopted [29]. Accordingly, Y. lipolytica ∆poxB1, ∆poxB2 and ∆poxB12 were constructed and grown on cellulose in the presence of Celluclast 1.5L. Even though this cocktail is reputedly β-glucosidase-deficient, to avoid any problems (i.e. Cellobiose and cello‑oligosaccharide fermentation with Y. lipolytica recombinant strains In contrast, ZetaB 2 consumed all of the cellobi- ose over 60 h. Furthermore, ZetaB 1 sustained a specific growth rate (μmax) of 0.10 h−1, whereas ZetaB 2 exhibited a long lag phase on cellobiose after which two subsequent growth phases (μmax values of 0.08 h−1 and then 0.16 h−1) were observed (Fig. 6; Table 4). In order to combine the advantages procured by the overexpression of BGL1 and BGL2 (i.e. shorter lag phase and higher cellobiose utiliza- tion, respectively), the two BGL sequences were cloned into JMY1212, thus yielding ZetaB 12. During cultiva- tion on cellobiose, the performance of ZetaB 12 was the best among all the recombinant strains. It showed similar growth rate to that of the control grown on glucose and consumed 10 g/L of cellobiose within 40 h. Cellobiose and cello‑oligosaccharide fermentation with Y. lipolytica recombinant strains spuri- ous results linked to the presence of β-glucosidase in Cel- luclast) the Celluclast loading was kept low (7.5 FPU/g cellulose), and control experiments containing the proto- trophic Y. lipolytica ∆poxW strain grown in the presence of Celluclast 1.5L with or without β-glucosidase supple- mentation were performed. During the initial 6 h of culti- vation an accumulation of reducing sugars was observed in all of the cultures, which was attributed to Celluclast 1.5L-mediated cellulose hydrolysis. However, further monitoring revealed that after 12-h growth, less reduc- ing sugars were present in the Y. lipolytica ∆poxB12 cul- ture (2.7 g/L) compared to the other cultures (Fig. 7a). Moreover, this observation was correlated with contin- ued yeast growth, whereas the growth of the other cul- tures stagnated over the same period (Fig. 7b). After 60 h of cultivation, the growth of ∆poxB12 reached a station- ary phase. At this point the amount of FAMEs obtained after the methylation of Total fatty acids produced by this yeast had reached 0.8 g/L (Fig. 7c), but further growth did not result in an increase in cellular lipid content, reflect- ing a limitation of the available energy source. Besides the longer lag phase, the growth of ∆poxB1 and ∆poxB2 was similar to that of the control culture supplemented with β-glucosidase. Regarding the control culture, in the absence of β-glucosidase supplementation, growth ceased after 60 h and the cell density of the culture was approximately half that of the other cultures. Moreover, wild-type Y. lipolytica ZetaW as the control. The maxi- mum specific growth rates (μmax) of the transformants on cellobiose were essentially the same as that of the con- trol grown on glucose (Fig. 5a, b). ZetaB 1 grew faster than ZetaB 2 on cellobiose and cellodextrins (Fig. 5b–f), while the control was unable to grow on either of these substrates. Surprisingly, despite indications that the wild- type strain cannot grow on cellobiose or cellodextrins, the control culture (ZetaW) reached an OD value of 2.0. However, further investigation using a defined medium revealed that this unexpected growth could be attrib- uted to the presence in the medium of 0.2% w/v casamino acids, which acted as a suitable carbon source. In defined medium, ZetaB 1 consumed 80% cellobiose over 48 h. However, upon further incubation, the remaining 20% cel- lobiose (at the concentration of 2 g/L) was not consumed (Fig. 6). Characterization of cellulose‑based lipid production by recombinant Y. lipolytica strains To investigate whether the BGLs described in this study could be used to construct a lipid-producing strain, a Guo et al. Biotechnol Biofuels (2015) 8:109 Page 8 of 16 Fig. 5 Comparison of Y. lipolytica ZetaW (control), ZetaB 1 (PTEF-BGL1) and ZetaB 2 (PTEF-BGL2) during aerobic growth on 5 g/L a glucose, b cel‑ lobiose, c cellotriose, d cellotetraose, e cellopentaose and f cellohexaose as carbon and energy source. Shown is OD600nm, optical density at 600 nm, versus time. Each data point represents the mean of at least three independent experiments and the standard deviation is less than 5%. Fig. 5 Comparison of Y. lipolytica ZetaW (control), ZetaB 1 (PTEF-BGL1) and ZetaB 2 (PTEF-BGL2) during aerobic growth on 5 g/L a glucose, b cel‑ lobiose, c cellotriose, d cellotetraose, e cellopentaose and f cellohexaose as carbon and energy source. Shown is OD600nm, optical density at 600 nm, versus time. Each data point represents the mean of at least three independent experiments and the standard deviation is less than 5%. sources for the production of lipids [34]. Nevertheless, Y. lipolytica is also known for its inability to grow on cel- lulose or even cellobiose. Therefore, it also constitutes an attractive target for strain engineering work. continuous addition of cellulases to the control culture did not procure any obvious increase in growth. When the control was supplemented with β-glucosidase, the amount of cellulose that remained unconsumed (25 g/L) was similar to that of cultures of the ∆pox strain express- ing β-glucosidases after 5 days of growth. Interestingly, comparative genomics has revealed that Y. lipolytica is only distantly related to the majority of yeast species and instead shares a number of common properties with filamentous fungi [35]. Therefore, based on this observation, we investigated whether Y. lipol- ytica harbors genes that allow cellobiose degradation and thus the possibility to confer cellobiose-degrading ability to Y. lipolytica through the use of endogenous β-glucosidases. Gratifyingly, our data clearly revealed that at least two genes, designated BGL1 and BGL2, encode β-glycosidases, active under the control of TEF promoter, that hydrolyze cellobiose, although our results also indicate that these enzymes do not exclusively cleave β-1, 4 linkages. In this respect, it is noteworthy that many cellobiolytic yeasts, such as Debaryomyces vanrijiae [36], Candida peltata [37], Monascus purpureus [38], Kluyveromyces fragilis [39] and Metschnikowia pulcher- rima [40], only produce one active Bgl and even in the case of S. Characterization of cellulose‑based lipid production by recombinant Y. lipolytica strains fibuligera, which produces two active Bgls, only Discussion Even though fungal β-glucosidases can be produced at relatively low cost via solid-state fermentation [30], cel- lulases still account for almost 50% of the cost of cellulose hydrolysis processes [31]. Moreover, taking into account the fact that the T. reesei secretome is rather deficient in β-glucosidases, it is particularly relevant to engineer microorganisms that are self-sufficient with regard to this type of enzyme activity. To date, S. cerevisiae has been the main target for engi- neering aimed at the creation of a cellulolytic yeast strain for consolidated bioprocessing purposes [32, 33]. How- ever, Y. lipolytica is one of the most widely studied “non conventional” oleaginous yeast species, which is well characterized for its ability to use hydrophobic substrates (e.g. alkanes, FAs, oils), glucose and glycerol as carbon Guo et al. Biotechnol Biofuels (2015) 8:109 Page 9 of 16 Fig. 6 Comparison of Y. lipolytica a ZetaW (control), b ZetaB 1 (PTEF-BGL1), c ZetaB 2 (PTEF-BGL2) and d Zeta-B12 (PTEF-BGL1, PTEF-BGL2) during aerobic growth on 10 g/L cellobiose. Shown are OD600nm, optical density at 600 nm, and cellobiose concentration versus time. Each data point represents the mean of five independent experiments and the error bar indicates the standard deviation. Fig. 6 Comparison of Y. lipolytica a ZetaW (control), b ZetaB 1 (PTEF-BGL1), c ZetaB 2 (PTEF-BGL2) and d Zeta-B12 (PTEF-BGL1, PTEF-BGL2) during aerobic growth on 10 g/L cellobiose. Shown are OD600nm, optical density at 600 nm, and cellobiose concentration versus time. Each data point represents the mean of five independent experiments and the error bar indicates the standard deviation. Table 4 Comparison of growth and biomass yield of Y. lipolytica JMY1212 control and recombinant strains in aer- obic cellobiose cultivation on defined medium ±, the standard deviation. NA not available. Parameter Control ZetaB 1 ZetaB 2 ZetaB 12 μmax (h−1) on glucose 0.15 ± 0.01 0.16 ± 0.01 0.16 ± 0.01 0.16 ± 0.01 μmax (h−1) on cellobiose NA 0.09 ± 0.01 0.14 ± 0.01 0.15 ± 0.01 YX/S (DCW-g/g cello) NA 0.52 ± 0.01 0.53 ± 0.01 0.50 ± 0.01 Residue cellobiose 60 h (%) NA 17.2 ± 1.0 7.2 ± 0.1 1.0 ± 0.3 Table 4 Comparison of growth and biomass yield of Y. lipolytica JMY1212 control and recombinant strains in aer- obic cellobiose cultivation on defined medium ±, the standard deviation. NA not available. Discussion Parameter Control ZetaB 1 ZetaB 2 ZetaB 12 μmax (h−1) on glucose 0.15 ± 0.01 0.16 ± 0.01 0.16 ± 0.01 0.16 ± 0.01 μmax (h−1) on cellobiose NA 0.09 ± 0.01 0.14 ± 0.01 0.15 ± 0.01 YX/S (DCW-g/g cello) NA 0.52 ± 0.01 0.53 ± 0.01 0.50 ± 0.01 Residue cellobiose 60 h (%) NA 17.2 ± 1.0 7.2 ± 0.1 1.0 ± 0.3 Table 4 Comparison of growth and biomass yield of Y. lipolytica JMY1212 control and recombinant strains in aer- obic cellobiose cultivation on defined medium revealed by western blot analysis. To pursue this further it would no doubt be useful to take a closer look at tran- scription, but this was not performed since the successful expression of two BGLs was more than enough to con- tinue the present study.hi The multiplicity of β-glucosidases in filamentous fungi is common and is often due to the presence of multiple genes, or differential post transcriptional modifications [42, 43]. However, our study provides the first evidence of possible β-glucosidase multiplicity in yeast. In filamentous fungi, the apparent redundancy of multiple cellulolytic enzymes can be explained by the ability of these microorganisms to adapt to differ- ent biomass resources and culture conditions [43] and is probably essential in fungal metabolism and survival. Similarly, in the case of Y. lipolytica, the presence of multiple putative β-glycosidase genes (two of which have been shown to be β-glucosidases) could be the result of adaptation of the yeast to changing environ- ments and may help to explain the different evolution- ary history of this yeast. one of these (SfBgl1) actually hydrolyzes β-1,4 glycosidic bonds [41]. In this respect it is also intriguing to note that while Bgl1 (described herein) displays good activity on both cellobiose and cellodextrin, its homolog from S. fibuligera (SfBgl2, 49.5% identity) is inactive on cellobiose. Regarding the remaining four putative β-glucosidases, at this stage it is unclear why no recombinant products were Guo et al. Biotechnol Biofuels (2015) 8:109 Page 10 of 16 Fig. 7 Growth and lipid production on cellulose medium of Y. lipolytica strains. Growth during SSF on 50 g/L cellulose supplemented with Cel‑ luclast 1.5L. a the concentration of reduced sugar versus time; b growth expressed as cell number versus time; c lipid content at 60 h. Strains are Y. Discussion lipolytica ∆poxB1 (PTEF-BGL1), ∆poxB2 (PTEF-BGL2), ∆poxB12 (PTEF-BGL1, PTEF-BGL2) and ∆poxW (wild type) under the same condition without (control) or with (control + Bgl) extra β-glucosidase (Novozyme 188). Each data point represents the mean of at least three independent experiments and the error bars indicate the standard deviation. Fig. 7 Growth and lipid production on cellulose medium of Y. lipolytica strains. Growth during SSF on 50 g/L cellulose supplemented with Cel‑ luclast 1.5L. a the concentration of reduced sugar versus time; b growth expressed as cell number versus time; c lipid content at 60 h. Strains are Y. lipolytica ∆poxB1 (PTEF-BGL1), ∆poxB2 (PTEF-BGL2), ∆poxB12 (PTEF-BGL1, PTEF-BGL2) and ∆poxW (wild type) under the same condition without (control) or with (control + Bgl) extra β-glucosidase (Novozyme 188). Each data point represents the mean of at least three independent experiments and the error bars indicate the standard deviation. β-glucosidase from Aspergillus niger (5.2 units/mg pro- tein), the enzyme that is generally used to complement the cellulolytic cocktail of T. reesei [44], is rather flat- tering for the former. Moreover, the KM values describ- ing the cellobiolytic reactions catalyzed by Bgl1 and 2 are approximately 10 and 3.4-fold lower than those of the β-glucosidases from S. fibuligera (2.8 mM Bgl1) and A. niger (2.7 mM) [44], meaning that the minimum con- centration of cellobiose required for effective catalysis to occur is much lower. Likewise, comparing the appar- ent performance constants, kcat/KM, of Y. lipolytica Bgls with those of other reported β-glucosidases [36–41, 44] suggests that the enzymes described in this study hydro- lyze cellodextrins more efficiently. In this respect, it is also interesting to consider the fact that Bgl1 appar- ently exhibits higher catalytic performance on low DP Considering the physicochemical characteristics of the two Y. lipolytica β-glucosidases described in this study, it is possible to tentatively correlate these with the cellular localization of the enzymes. Bgl2 was more stable in the assay conditions employed (50°C, pH 4.0), which might arguably be logical for an extracellular enzyme that needs to show a certain resilience to environmental challenges. On the other hand, the relative fragility of Bgl1 can be explained by the fact that its natural intracellular loca- tion probably protects it from major temperature and pH changes. Conclusionsh This study has provided a clear demonstration that Y. lipolytica does not naturally use cellobiose, despite the fact that this strain contains the genetic potential to do so. This is intriguing because the protein products of these genes are active on glucose-based oligosaccha- rides, including cello-oligosaccharides. Moreover, our data clearly show that upon expression of BGL1 and BGL2 under the control of the constitutive well charac- terized TEF promoter in Y. lipolytica, growth on cellobi- ose becomes possible. These encouraging findings render plausible the creation of an engineered Y. lipolytica strain that could be useful in advanced generation biorefinery schemes involving the use of lignocellulosic hydrolysates as feedstock for the production of bioenergy and valuable chemicals. Strains and mediah The genotypes of the microbial strains used in the pre- sent study are summarized in Table 5. E. coli DH5 were purchased from Invitrogen (Paisley, UK) and used for plasmid construction. The Y. lipolytica strains were rou- tinely grown in YPD (1% yeast extract, 2% bacto pep- tone, and 2% glucose). Solid YPD medium contains 1.5% agar. Transformants were selected on solid YNB medium (0.17% w/v YNB, 1% glucose or cellobiose w/v, 0.5% w/v ammonium chloride, with (for Ura+) or with- out (for Leu+) 0.2% w/v casamino acids and 50 mM sodium–potassium phosphate buffer, pH 6.8), supple- mented with uracil (440 mg/L) or leucine (440 mg/L) depending on the auxotrophic requirements. The detec- tion of β-glucosidase activity in solid YNBcasa medium was achieved by incorporating 1.0 mM p-nitrophenyl- β-d-glucoside (pNP-βGlc) [45]. For β-glucosidase char- acterization, enzymes were produced in YTD medium (1% w/v yeast extract, 2% w/v tryptone, 5% w/v glucose and 100 mM phosphate buffer, pH 6.8). To compare the efficiency of recombinant β-glucosidase to degrade cello- biose and cellodextrin with respect to cell growth, yeasts were aerobically cultivated in YNBcasa medium, contain- ing 5 g/L cellobiose or cello-oligosaccharides (C3–C6), and defined medium containing vitamins, trace elements [46] and salts, including 3.5 g/L (NH4)2SO4, 3.0 g/L K2HPO4, 3.0 g/L NaH2PO4 and 1.0 g/L MgSO4∙7H2O with 10 g/L cellobiose. For lipid production using cel- lulose as the carbon source, Y. lipolytica strains were grown in defined media supplemented with 50 g/L Avicel PH-101. Apparently, in the lipid production experiments performed in this study, the activity of the Yarrowia β-glucosidases adequately satisfied the requirements for exo-cellulase activity, since no accumulation of reducing sugars was observed. However, lipid production was not sustained, since growth ceased before the cellulose was consumed. This failure suggests that cellulose degrada- tion was the limiting factor, with a part of the cellulose being recalcitrant to further hydrolysis by the Celluclast cocktail, even when Bgl1 and 2 were correctly expressed and thus available. Discussion In this respect the different cellular locations of the enzymes is no doubt beneficial to the yeast, since it provides optimal activity.hi The comparison of the specific activities of Bgl1 and Bgl2 on cellobiose (108 units/mg and 25 units/mg pro- tein, respectively) with that of the commercially available Page 11 of 16 Page 11 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 cellodextrins, while Bgl2 is more active on higher DP ones. This difference in substrate preference is consist- ent with the cellular location of the two enzymes (i.e. the extracellular enzymes deals with the longer oligosaccha- rides that cannot be transported into the cell) and possi- bly forms the basis of the superior performance of ZetaB 12 on Avicel compared to control cultures containing the β-glucosidase from Novozymes 188. b His-tag introduced into the corresponding genes. Plasmid constructionsh The plasmids constructed in the present study are sum- marized in Table 6, and all primers are listed in Table 7. Table 5 Microbial strains used in the present study Strains Relevant genotype Source of reference E. coli DH5 Φ80dlacZΔm15, recA1, endA1, gyrA96, thi-1, hsdR17 (rk−, mk+), supE44, relA1, deoR, Δ(lacZYA-argF) U169 Invitrogen Y. lipolytica JMY1212 (Zeta) MATA, ura3-302, leu2-270-LEU2-zeta, xpr2-322 ∆lip2, ∆lip7, ∆lip8 [50] Y. lipolytica ∆pox JMY1233 MATA, leu2-270, ura3-302, xpr2-322, pox1-6∆ [29] ZetaW MATA, xpr2-322, ∆lip2, ∆lip7, ∆lip8 This investigation ZetaB 1 PTEF-BGL1 This investigation ZetaB 2 PTEF-BGL2 This investigation ZetaB 12 PTEF-BGL1, PTEF-BGL2 This investigation ∆poxW MATA, xpr2-322, pox1-6∆ This investigation ∆poxB1 PTEF-BGL1 This investigation ∆poxB2 PTEF-BGL2 This investigation ∆poxB12 PTEF-BGL1, PTEF-BGL2 This investigation Table 5 Microbial strains used in the present study Page 12 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 The plasmids used for expression of the putative β-glucosidases were constructed using the expression vectors JMP62UraTEF and JMP62LeuTEF, which are derivatives of a previously described vector [47]. Briefly, these vectors contain the Y. lipolytica TEF promoter and either the URA3ex or LEU2ex excisable selection markers, which are flanked by loxP sites and a Zeta fragment that serves as the homologous integration site [48]. Regarding β-glucosidases, six putative gene candidates (Sequences YALI0F16027g, YALI0F01672g, YALI0D18381g, YAL- I0B14289g, YALI0B14333g, YALI0E20185g available at Genome Resources from Yeast Chromosomes: http:// gryc.inra.fr/) were identified (see Additional file 1: Table S1). For the expression of wild-type and His6-tagged pro- teins, the genes were amplified by PCR using FA (1–6) as forward primers and RB (1–6) or RB-His (1–6) as reverse primers, respectively. The PCR fragments were digested Table 6 Plasmids used or created in the present study Plasmids Description Source of reference JMP62UraTEF URA3, TEFP-XPRT [56] JMP62LeuTEF LEU2, TEFP-XPRT [57] JMP62UraTB1 URA3, TEFP-BGL1-XPRT This investigation JMP62UraTB2 URA3, TEFP-BGL2-XPRT This investigation JMP62LeuTB2 LEU2, TEFP-BGL2-XPRT This investigation JMP62UraTB12 URA3, TEFP-BGL1-XPRT, TEFP-BGL2- XPRT This investigation Table 7 The sequences of the oligonucleotide primers used in a Restriction site with corresponding restriction enzyme. b His-tag introduced into the corresponding genes. a Restriction site with corresponding restriction enzyme. Plasmid constructionsh Primer names Sequence (5′–3′) restriction sites are italic/underlin FA1 CGaGGATCCCGCGATGATCTTCTCTCTGCAACTACTAC RB1 CGCCTAGGCTACAAAGTGAAAGTCTCACATAGC FA2 CCCAAGCTTGGGTTTGGAGGGGGTGAAAAA RB2 CCCAAGCTTGGGCTAAAGACCTAACCAATTCTTAGTCT FA3 CGGGATCCCGCGATGATTGCAAAAATACCCC RB3 CGCCTAGGCTACTGGAGAGTAAAGGACTCG FA4 CGGGATCCCGCGATGCTCGCATTCGTCCTAC RB4 CGGGATCCCGCTACTTGAGAGTGAAGCTGGTG FA5 CGGGATCCCGCGATGGCTCCACCCCCGCCTCCT RB5 CGCCTAGGTTAAGCAATCGTGATGCGACCAAGG FA6 CGCCTAGGCGCGATGGAGGAATTATCGGAGGC RB6 CGCCTAGGCTACCGGCTGAACTTCTCTTC RB-His1 bCGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCG RB-His2 CCCAAGCTTGGGTTAATGATGGTGATGATGGTGGCTGCCG RB-His3 CGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCG RB-His4 CGGGATCCCGTTAATGATGGTGATGATGGTGGCTGCCGCG RB-His5 CGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCG RB-His6 CGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCG Table 6 Plasmids used or created in the present study Plasmids Description Source of reference JMP62UraTEF URA3, TEFP-XPRT [56] JMP62LeuTEF LEU2, TEFP-XPRT [57] JMP62UraTB1 URA3, TEFP-BGL1-XPRT This investigation JMP62UraTB2 URA3, TEFP-BGL2-XPRT This investigation JMP62LeuTB2 LEU2, TEFP-BGL2-XPRT This investigation JMP62UraTB12 URA3, TEFP-BGL1-XPRT, TEFP-BGL2- XPRT This investigation Table 6 Plasmids used or created in the present study using either BamHI/AvrII or HindIII/AvrII and inserted into the plasmid JMP62 UraTEF at the corresponding sites. using either BamHI/AvrII or HindIII/AvrII and inserted into the plasmid JMP62 UraTEF at the corresponding sites. using either BamHI/AvrII or HindIII/AvrII and inserted into the plasmid JMP62 UraTEF at the corresponding sites. After construction, all expression vectors were veri- fied by DNA sequencing (GATC Biotech, Konstanz, Germany). For Y. lipolytica transformation, vectors were digested using NotI, thus generating a linear DNA with Zeta sequences at both extremities, and purified. Then the linear DNA fragments were introduced into the Zeta docking platform of Y. lipolytica JMY1212 Zeta or ran- domly into the genome of ∆pox strain using the lithium acetate method [49]. Transformants were tested for β-glucosidase activity on YNB glucose plate containing pNP-βGlc and for growth on cellobiose using solid YNB cellobiose plates. Clones displaying both activities were retained for further analysis. The plasmids used for expression of the putative β-glucosidases were constructed using the expression vectors JMP62UraTEF and JMP62LeuTEF, which are derivatives of a previously described vector [47]. Briefly, these vectors contain the Y. lipolytica TEF promoter and either the URA3ex or LEU2ex excisable selection markers, which are flanked by loxP sites and a Zeta fragment that serves as the homologous integration site [48]. Regarding β-glucosidases, six putative gene candidates (Sequences YALI0F16027g, YALI0F01672g, YALI0D18381g, YAL- I0B14289g, YALI0B14333g, YALI0E20185g available at Genome Resources from Yeast Chromosomes: http:// gryc.inra.fr/) were identified (see Additional file 1: Table S1). For the expression of wild-type and His6-tagged pro- teins, the genes were amplified by PCR using FA (1–6) as forward primers and RB (1–6) or RB-His (1–6) as reverse primers, respectively. The PCR fragments were digested Western blot analysis Western blotting of proteins was performed as described previously [51]. Crude supernatant and cell-free extracts of Y. lipolytica JMY1212 expressing putative β-glucosidases fused with the His6 tag were concentrated 10-fold using an ultra-centrifugation filter unit (Amicon® Ultra-4 10 kDa cut-off, Merk Millipore, Bedford, MA, USA). Blots were sequentially treated with mouse non position-specific His- Tag antibody 1:2,500 (THE™ from Genscript, Piscataway, NJ, USA) and the alkaline phosphatase-conjugated goat anti-mouse IgG. f For purification of Bgl2, the culture supernatant was concentrated fivefold using an Amicon® Ultra-4 Cen- trifugal Filter Unit with 30 kDa cut-off (Merk Millipore, Bedford, MA, USA). The concentrated sample was then loaded onto a Q Sepharose™ High Performance col- umn (Hiload, 1.6 × 10 cm, Pharmacia Biotech), equili- brated with Tris-buffer (20 mM, pH8.0). The column was washed first with equilibration buffer (2 bed vol- umes) before applying a linear gradient of 0–1.0 M NaCl in Tris-buffer (20 mM, pH7.4) at a flow rate of 1.0 mL/ min (Pharmacia Biotech ÄKTA). Eluted fractions were collected and assayed for β-glucosidase activity. All frac- tions displaying activity were pooled, desalted and con- centrated using an Amicon ultra-filtration unit equipped with a PM-10 membrane (Millipore), before being applied to a Superdex 200 column (1.0 × 30 cm, Pharma- cia Biotech) equilibrated in Tris-sodium buffer (20 mM Tris–HCl, 150 mM NaCl, pH 7.4). Protein species were separated at a flow rate of 0.5 mL/min. Fractions were collected and analyzed by SDS-PAGE to ascertain purity and estimate the approximate molecular weights of Bgl1- His6 and Bgl2. All fractions satisfying the purity criterion (>95% purity) were pooled and retained for further work. Transcriptional analysis Y. lipolytica JMY1212 wide type and recombinant strains overexpressing BGL1 and BGL2 were grown to mid- exponential phase in defined media and then transferred into fresh medium containing either glucose or cellobi- ose as the sole carbon source. Cells were recovered from the medium at 20 min and 1 h, respectively, and rapidly frozen in liquid nitrogen and stored at −80°C until use. Total mRNA was isolated using RNeasy Plus Mini Kit (QIAGEN) and reverse transcription was performed with iScript™ cDNA Synthesis Kit (BIO-RAD) according Table 7 The sequences of the oligonucleotide primers used in this study Primer names Sequence (5′–3′) restriction sites are italic/underlined Restriction sites FA1 CGaGGATCCCGCGATGATCTTCTCTCTGCAACTACTAC BamHI RB1 CGCCTAGGCTACAAAGTGAAAGTCTCACATAGC AvrII FA2 CCCAAGCTTGGGTTTGGAGGGGGTGAAAAA HindIII RB2 CCCAAGCTTGGGCTAAAGACCTAACCAATTCTTAGTCT HindIII FA3 CGGGATCCCGCGATGATTGCAAAAATACCCC BamHI RB3 CGCCTAGGCTACTGGAGAGTAAAGGACTCG AvrII FA4 CGGGATCCCGCGATGCTCGCATTCGTCCTAC BamHI RB4 CGGGATCCCGCTACTTGAGAGTGAAGCTGGTG BamHI FA5 CGGGATCCCGCGATGGCTCCACCCCCGCCTCCT BamHI RB5 CGCCTAGGTTAAGCAATCGTGATGCGACCAAGG AvrII FA6 CGCCTAGGCGCGATGGAGGAATTATCGGAGGC AvrII RB6 CGCCTAGGCTACCGGCTGAACTTCTCTTC AvrII RB-His1 bCGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCGGCACCAGCCTAGGCAAAGTGAAAGTCTCA RB-His2 CCCAAGCTTGGGTTAATGATGGTGATGATGGTGGCTGCCGCGCGGCACCAGCCTAGGAAGACCTAACCAATTCTTA RB-His3 CGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCGGCACCAGCCTAGGCTGGAGAGTAAAGGA RB-His4 CGGGATCCCGTTAATGATGGTGATGATGGTGGCTGCCGCGCGGCACCAGCCTAGGCTTGAGAGTGAAGCT RB-His5 CGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCGGCACCAGCCTAGGAGCAATCGTGATGC RB-His6 CGCCTAGGTTAATGATGGTGATGATGGTGGCTGCCGCGCGGCACCAGCCTAGGCTGAACTTCTCTTCC Table 7 The sequences of the oligonucleotide primers used in this study Table 7 The sequences of the oligonucleotide primers used in this study Page 13 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 to the manufacturer’s instructions. Transcription of the BGLs was analyzed by PCR, using gene-specific primers and sequencing of the PCR products (Additional file 1: Table S3). vortex in the presence of glass beads (0.4–0.45 mm). The homogenate was centrifuged (20,000×g for 2 h at 4°C) and the supernatant and solid fractions were designated as the cytoplasmic and membrane fraction, respectively. Prior to enzyme assays, the membrane fraction was sus- pended in citrate buffer. Purification of β‑glucosidases Y. lipolytica JMY1212 overproducing Bgl1-His6 and Bgl2 was grown in 200 mL YTD medium at 130 rpm, 28°C for 36 h before centrifugation at 8,000×g for 5 min. For purification of His6-Bgl1, the cell pellet was washed, suspended in 50 mL phosphate buffer (50 mM, pH 7.4) and homogenized over a 3-min period using a MP Fast- Prep-24 Instrument. After centrifugation (8,000×g for 5 min at 4°C), the supernatant was applied to 2 mL of TALON Metal Affinity Resin (Clontech, Takara-Bio, Kyoto, Japan) and protein was eluted using imidazole buffer according to the manufacturer’s instructions.i Measurement of enzyme activity β-Glucosidase activity was measured by quantifying the release of pNP (p-nitrophenol) from pNP-βGlc as described previously [50]. One unit of pNP-βGlcase activity was defined as the amount of enzyme required to release 1 μmol pNP per min. All protein concentrations were measured using the Bradford method and bovine serum albumin as a standard [47]. Subcellular fractionation and enzyme localization Fractionation of yeast cells was carried out as described by Cummings and Fowler [52], with slight modifica- tions. Briefly, yeasts were cultivated until a cellular den- sity of 6 × 107 cells/mL was reached. Then, to quantify total β-glucosidase activity, a 50-mL sample was taken and subjected to centrifugation at 8,000×g for 5 min at 4°C thus isolating a cell pellet and supernatant. The cell pellet was disrupted in Tris–HCl buffer (50 mM, pH 7.4, 3 mM EDTA and 0.5 mM PMSF) using a MP FastPrep-24 Instrument (MP Biomedicals Inc.). β-Glucosidase activ- ity in both the cell lysate and the supernatant was deter- mined as described earlier to estimate total β-glucosidase activity. Using a second 50 mL yeast culture, a cell pellet containing approximately 2 × 108 cells/mL was obtained by centrifugation and then treated with zymolyase 100T at 10 mg/mL (Seikagaku corp coger) in 15 mL of sorbi- tol buffer (1 M sorbitol, 50 mM Tris–HCl, pH 7.4, 2 mM dithiothreitol, 10 mM MgCl2, 20 mM-sodium azide, 0.5 mM PMSF) at 30°C with gentle shaking. Protoplast formation was monitored using a microscope until ≥99% of the cells was lysed when SDS was added (1% SDS w/v). The solid protoplast fraction was then separated from the supernatant by centrifugation (1,000 rpm for 5 min at 4°C) and the latter was designated as the periplasmic fraction. The protoplasts were re-suspended in Tris– HCl buffer (50 mM Tris–HCl, pH 7.4) and disrupted by Substrate specificity and enzyme kineticshi The substrate specificity of Bgl1-His6 and Bgl2 was inves- tigated by assaying for activity on the aryl-glycosides pNP-β-d-glucopyranoside, pNP-α-d-glucopyranoside, pNP-β-d-galactopyranoside, pNP-β-d-xylopyranoside and pNP-β-d-cellobioside, and on the oligosaccha- rides cellobiose, cellotriose, cellotetraose, cellopenta- ose, cellohexaose, sophorose, laminaribiose, gentiobiose, methylglucoside and octylglucoside. When using aryl- substrates, the standard assay method was employed, simply replacing pNP-βGlc by another substrate as appropriate. For oligosaccharides, the release of glu- cose was quantified using an enzyme kit (d-Fructose/d- Glucose Assay Kit, liquid stable, Megazyme). To study the Michaelis–Menten parameters KM, Vmax and kcat, Bgl1 (0.120 nM) or Bgl2 (0.13 nM) were added to reaction mixtures containing different substrate concentrations: 0.25–5 mM cellobiose, 0.25–5 mM cel- lotriose, 0.25–5 mM cellotetraose, 0.25–5 mM cellopen- taose, 0.25–5 mM cellohexaose, 0.2–4 mM sophorose, 0.1–2 mM laminaribiose, 0.1–2 mM gentiobiose, 0.5– 20 mM methylglucoside and 0.2–4 mM octylglucoside. Initial rates were fitted to the Michaelis–Menten kinetic equation using a nonlinear regression (SigmaPlot 10) to extract the apparent KM and kcat [53]. Physicochemical characteristics of β‑glucosidases reader (Spectrostar Omega, BMG Labtech, Germany) at 30°C with continuous shaking (150 rpm) and automatic OD600 recording. Similarly, yeast growth on cellobiose was also performed on 30 ml defined medium containing 10 g/L cellobiose in 250 mL Erlenmeyer flasks. Physicochemical characteristics of β‑glucosidases Optimal temperatures and pH for the activity of Bgl1 and Bgl2 were determined using pNPGlc as the sub- strate. Assays were either performed at pH 5.0 and vari- ous temperatures (30–70°C) or at 30°C in variable pH conditions (2.0–8.0) using either 50 mM glycine–HCl (pH 2.0), 50 mM citrate/acetate (pH 3.0–7.2) or potas- sium phosphate (pH 7.0–8.2) buffer. When the tempera- ture was varied, the pH of the citrate buffer was adjusted accordingly. Stability of Bgl1 and Bgl2 depending on pH and temperature was analyzed as follows: enzymes were incubated at 30°C for up to 2 h at various pH values (2.0– 8.0) or at various temperatures (30–70°C) for up to 2 h in 50 mM citrate buffer, pH 5.0. Residual glucosidase activ- ity was then assayed at 30°C in 50 mM citrate buffer, pH 5.0. g yl For lipid production a fresh yeast culture in exponen- tial phase was used to inoculate 50 mL defined medium containing 50 g/L Avicel in Erlenmeyer flasks, achieving an initial OD600 of 1.0. Celluclast 1.5L (60 FPU/mL, gift from Novozymes, Denmark) was added (7.5 U/g cellu- lose) and growth was pursued for 5 days (30°C, 150 rpm). Samples were taken at regular intervals to determine concentrations of biomass, glucose, cellobiose and citric acid. In parallel, two control experiments were conducted under the same conditions, with or without the addition of extra β-glucosidase (810 IU/mL Novozyme 188, gift from Novozyme, Denmark) at 12.0 IU/g cellulose as rec- ommended [54]. Analysis of product formation and determination of dry cell weight To determine the concentration of substrates and extra- cellular metabolites, three aliquots (1.5 mL each) of cul- tures were rapidly frozen in liquid nitrogen and then thawed on ice before centrifugation (8,000×g for 5 min at 4°C) to recover supernatants for analysis. Glucose, cel- lobiose and citric acid were measured using an Aminex HPX87-H column (Bio-Rad Laboratories, Germany), operating at 50°C using a mobile phase (5 mM H2SO4) flowing at a rate of 0.5 mL/min. Glucose and cellobiose were detected using a Shodex RI-101 refractive index detector (Showa Denko, New York, NY, USA), while cit- ric acid was detected using an UV detector at 210 nm (Dionex, Sunnyvale, CA, USA). To determine the dry cell weight, three aliquots (5 mL each) of cultures were filtered through pre-weighed PES filters (0.45 μm; Sartorius Biolab, Germany). The biomass retained by the filters was washed, dried in a microwave oven at 150 W for 15 min and then placed in a desiccator before weighing. The biomass yield was calculated as the ratio of the amount of biomass obtained divided by the amount of carbon source consumed. Lipids were extracted from freeze-dried cells (~10 mg) and methylated as described previously [55]. During the lipid extraction, C17:0 (Sigma) (50 μg) was added as the internal standard and fatty acid methyl esters (FAMEs) were analyzed by gas chromatography (6890 N Network GC System, Agilent, USA). The measurements were per- formed in a split mode (1 μL at 250°C), with helium as the carrier gas (2 mL/min). FAMEs were separated on a HP-5 GC column (30 m × 0.32 mm I.D., 0.5-μm film thickness, Agilent, USA). The temperature program was 120°C, ramped to 180°C (10°C/min) for 6 min, 183°C Deglycosylation and N‑terminal amino acid sequencingi Deglycosylation and N‑terminal amino acid sequencing Purified Bgl1-His6 and Bgl2 were treated with endogly- cosidase H (New England Biolabs, Beverly, MA, USA) according to the manufacturer’s instructions. After deglycosylation, the protein species displaying Mr (rela- tive molecular mass) closest to those of the theoretical Mr (predicted using Protparam, http://web.expasy.org/ protparam/) of Bgl1-His6 and Bgl2 were excised and sub- mitted to N-terminal amino acid sequencing (PISSARO platform, Rouen, France). Page 14 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 Compliance with ethical guidelines Additional file 1: Table S1. Six putative β-glucosidase coding genes identified harboring conserved glycosyl hydrolase family 3 N and/or 3C terminal domain. Table S2. The predicted N-glycosylation sites in sequence of Bgl2 by GlycoEP. Table S3. The sequences of the oligonu‑ cleotide primers used in verification of transcriptions. Yeast growth and lipid production Yeast growth on cellobiose and cellodextrins was per- formed in a 40-well microplate. A single colony from a fresh YPD plate was transferred into 5 mL of defined medium containing 10 g/L of glucose and pre-cultured until the mid-exponential phase. The cells were then har- vested, washed, suspended in sterile water and used to inoculate 200 μL YNBcasa media containing 5 g/L cello- biose or cellodextrins in the microplate, achieving an ini- tial OD600 of 0.1. This culture was grown in a microplate Page 15 of 16 Guo et al. Biotechnol Biofuels (2015) 8:109 (0.33°C/min) for 9 min and 250°C (15°C/min) for 5 min. Detection was performed using a flame ionization detec- tor (FID) at 270°C (2.0 pA). FAMEs were quantified by comparing their profiles with that of standards of known concentration. Acknowledgements The authors would like to express their gratitude to Nelly Monties for her help with chromatographic analyses. They thank the ICEO facility dedicated to enzyme screening and discovery and part of the Integrated Screening Plat‑ form of Toulouse (PICT, IBiSA) for providing access to its protein purification and analytical facilities. This work was funded by the Agence Nationale de la Recherche (Investissements d’Avenir program; reference ANR-11-BTBR-0003). Dr. Guo is a recipient of a postdoctoral fellowship from the Institut National de la Recherche Agronomique. Abbreviations LC biomass: lignocellulosic biomass; CBP: consolidated bioprocessing; FAEE: fatty acid ethyl esters; SSF: simultaneous saccharification and fermentation; FAEs: Fatty Acid Esters; DCW: dry cell weight; SCO: single cell oil; FDA: Food and Drug Administration; GRAS: generally recognized as safe; pNP-βGlc: p-nitrophenyl-β-d-glucoside; Mr: relative molecular mass; Bgl: β-glucosidase. 12. Tang H, Hou J, Shen Y, Xu L, Yang H, Fang X et al (2013) High β-glucosidase secretion in Saccharomyces cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simul‑ taneous saccharification and fermentation. J Microbiol Biotechnol 23(11):1577–1585 13. Zhang F, Carothers JM, Keasling JD (2012) Design of a dynamic sensor- regulator system for production of chemicals and fuels derived from fatty acids. Nat Biotechnol 30(4):354–359 References 1. Pedersen M, Meyer AS (2010) Lignocellulose pretreatment severity-relat‑ ing pH to biomatrix opening. N Biotechnol 27(6):739–750 1. Pedersen M, Meyer AS (2010) Lignocellulose pretreatment severity-relat‑ ing pH to biomatrix opening. N Biotechnol 27(6):739–750 2. Wilson DB (2009) Cellulases and biofuels. Curr Opin Biotechnol 20(3):295–299 2. Wilson DB (2009) Cellulases and biofuels. Curr Opin Biotechnol 20(3):295–299 3. Tomme P, Warren RA, Gilkes NR (1995) Cellulose hydrolysis by bacteria and fungi. 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Duff SJB, Cooper DG, Fuller OM (1985) Cellulase and beta-glucosidase production by mixed culture of Trichoderma reesei Rut C30 and Aspergillus phoenicis. Biotechnol Lett 7:185–190 7. Duff SJB, Cooper DG, Fuller OM (1985) Cellulase and beta-glucosidase production by mixed culture of Trichoderma reesei Rut C30 and Aspergillus phoenicis. Biotechnol Lett 7:185–190 8. Holtzapple M, Cognata M, Shu Y, Hendrickson C (1990) Inhibition of Trichoderma reesei cellulase by sugars and solvents. Biotechnol Bioeng 36(3):275–287 9. Stockton DC, Mitchell DJ, Grohmann K (1991) Optimum β-glucosidase supplementation of cellulose for efficient conversion of cellulose to glucose. Biotechnol Lett 13:57–62 10. Lee WH, Nan H, Kim HJ, Jin YS (2013) Simultaneous saccharification and fermentation by engineered Saccharomyces cerevisiae without supple‑ menting extracellular β-glucosidase. J Biotechnol 167(3):316–322 11. Lian J, Li Y, HamediRad M, Zhao H (2014) Directed evolution of a cellodex‑ trin transporter for improved biofuel production under anaerobic condi‑ tions in Saccharomyces cerevisiae. Biotechnol Bioeng 111(8):1521–1531 Competing interests lipolytica expressing the 6 putative β-glucosidases on (a) indication plate containing YNBcasa medium supplemented with 1 mM p-nitrophenyl-β-D-glucoside (pNP-βGlc), and (b, c) YNBC plate with cel‑ lobiose as sole carbon source. Figure S3. Transcriptional analysis of the expression of the six putative BGLs in wild type strain on glucose (A) and cellobiose (B), and recombinant strain overexpression of BGL1 and BGL2 (C). Figure S4. N-terminal amino acid sequences of Y. lipolytica Bgl1 (A) and Bgl2 (B). The first 50 N-terminal amino acid sequences are indicated with the predicted signal sequence determined with signal P (underlined), the cleavage site predicted with a, and the N-terminal AA sequence of the purified protein determined by direct sequencing (in bold). Figure S5. Optimal pH (a) and temperature (b) of Bgl1 (square) and Bgl2 (diamond) from Y. lipolytica JMY1212. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Figure S6. Stability of Bgl1 (a) and Bgl2 (b) from Y. lipolytica JMY1212 at pH from 2.0–8.0 as a function of time at 30ºC, and stability of Bgl1 (c) and Bgl2 (d) at temperature from 30ºC to 60ºC as a function of time at pH 5. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Please note that only one curve is given to represent the stability of Bgl2 at pH 4.0, 5.0 and 6.0 (b) and at 30ºC and 40ºC (d) as 100% of enzyme activity remained for these conditions. Figure S7. The hydrolytic activity of Bgl2 on pNP-βGlc (a) and the stability of Bgl2 at 40ºC as a function of time at pH5.0 before and after deglycosylation. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Authors’ contributions ZPG, SB, SD, JMN, AM, MJD conceived of the study and participated in its design. ZPG designed the constructs, carried out all the experiments and drafted the manuscript. SD participated in the western blot analysis of the expressed proteins. GC participated in the protein purification. SB, SD, JMN, AM, MJD revised the manuscript. All authors read and approved the final manuscript. 14. Demirbaş A (2003) Biodiesel fuels from vegetable oils via catalytic and non-catalytic supercritical alcohol transesterifications and other methods: a survey. Energy Convers Manage 44:2093–2109 15. Steen EJ, Kang Y, Bokinsky G, Hu Z, Schirmer A, McClure A et al (2010) Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature 463:559–562 Competing interests The authors declare that they have no competing interests. The authors declare that they have no competing interests. Received: 24 March 2015 Accepted: 22 July 2015 Received: 24 March 2015 Accepted: 22 July 2015 Additional file 2: Figure S1. Multiple alignments of putative conserved domains of the family 3 glycosyl hydrolases of S. fibuligera (Bgl1, GenBank Accession numbers: AAA34314.1) against Yarrowia genome. Figure S2. Screening of Y. lipolytica expressing the 6 putative β-glucosidases on (a) indication plate containing YNBcasa medium supplemented with 1 mM p-nitrophenyl-β-D-glucoside (pNP-βGlc), and (b, c) YNBC plate with cel‑ lobiose as sole carbon source. Figure S3. Transcriptional analysis of the expression of the six putative BGLs in wild type strain on glucose (A) and cellobiose (B), and recombinant strain overexpression of BGL1 and BGL2 (C). Figure S4. N-terminal amino acid sequences of Y. lipolytica Bgl1 (A) and Bgl2 (B). The first 50 N-terminal amino acid sequences are indicated with the predicted signal sequence determined with signal P (underlined), the cleavage site predicted with a, and the N-terminal AA sequence of the purified protein determined by direct sequencing (in bold). Figure S5. Optimal pH (a) and temperature (b) of Bgl1 (square) and Bgl2 (diamond) from Y. lipolytica JMY1212. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Figure S6. Stability of Bgl1 (a) and Bgl2 (b) from Y. lipolytica JMY1212 at pH from 2.0–8.0 as a function of time at 30ºC, and stability of Bgl1 (c) and Bgl2 (d) at temperature from 30ºC to 60ºC as a function of time at pH 5. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Please note that only one curve is given to represent the stability of Bgl2 at pH 4.0, 5.0 and 6.0 (b) and at 30ºC and 40ºC (d) as 100% of enzyme activity remained for these conditions. Figure S7. The hydrolytic activity of Bgl2 on pNP-βGlc (a) and the stability of Bgl2 at 40ºC as a function of time at pH5.0 before and after deglycosylation. Each data point represents the mean of three independent experiments and the error bar indicates the standard deviation. Additional file 2: Figure S1. 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https://openalex.org/W3113012996	https://boris.unibe.ch/169239/1/ciaa1647.pdf	English	null	Brain Magnetic Resonance Imaging Reveals Different Courses of Disease in Pediatric and Adult Cerebral Malaria	Clinical infectious diseases/Clinical infectious diseases (Online. University of Chicago. Press)	2,020	cc-by	8,463	(See the Editorial Commentary by John on pages e2397–8.) Background. Cerebral malaria is a common presentation of severe Plasmodium falciparum infection and remains an important cause of death in the tropics. Key aspects of its pathogenesis are still incompletely understood, but severe brain swelling identified by magnetic resonance imaging (MRI) was associated with a fatal outcome in African children. In contrast, neuroimaging investiga- tions failed to identify cerebral features associated with fatality in Asian adults. ffi Methods. Quantitative MRI with brain volume assessment and apparent diffusion coefficient (ADC) histogram analyses were performed for the first time in 65 patients with cerebral malaria to compare disease signatures between children and adults from the same cohort, as well as between fatal and nonfatal cases. Results. We found an age-dependent decrease in brain swelling during acute cerebral malaria, and brain volumes did not differ between fatal and nonfatal cases across both age groups. In nonfatal disease, reversible, hypoxia-induced cytotoxic edema occurred predominantly in the white matter in children, and in the basal ganglia in adults. In fatal cases, quantitative ADC histogram analyses also demonstrated different end-stage patterns between adults and children: Severe hypoxia, evidenced by global ADC decrease and elevated plasma levels of lipocalin-2 and microRNA-150, was associated with a fatal outcome in adults. In fatal pediatric disease, our results corroborate an increase in brain volume, leading to augmented cerebral pressure, brainstem herniation, and death. i g g pi dings suggest distinct pathogenic patterns in pediatric and adult cerebral malaria with a stronger cytotox porting the development of age-specific adjunct therapies.ffi i Keywords. cerebral malaria; Plasmodium falciparum; magnetic resonance imaging; apparent diffusion coefficient maps; hypoxia. including seizures and impaired consciousness and has a fa- tality rate up to 30% in treated patients [2]. The range and type of CM-associated complications vary between the 2 age groups: While children more frequently develop cerebral involvement as mono-organ failure, adults often present with additional organ dysfunctions such as acute kidney injury, jaundice, and acute respiratory distress syndrome. Such differences in clinical presentation were reported between cohorts from different geo- graphic areas [3], as well as within the same cohort [4].h Falciparum malaria remains the most important parasitic di- sease globally. In high-transmission settings in sub-Saharan Africa, falciparum malaria is a pediatric disease; in lower- transmission settings such as Southeast Asia, all age groups are affected, owing to differences in antimalaria immunity building [1]. Brain Magnetic Resonance Imaging Reveals Different Courses of Disease in Pediatric and Adult Cerebral Malaria Praveen K. Sahu,1,a Angelika Hoffmann,2,3,a Megharay Majhi,4 Rajyabardhan Pattnaik,5 Catriona Patterson,6 Kishore C. Mahanta,4 Akshaya K. Mohanty,7 Rashmi R. Mohanty,8 Sonia Joshi,8 Anita Mohanty,5 Jabamani Bage,1 Sameer Maharana,1 Angelika Seitz,2 Martin Bendszus,2 Steven A. Sullivan,9 Ian W. Turnbull,10 Arjen M. Dondorp,11,12 Himanshu Gupta,6 Lukas Pirpamer,6,13 Sanjib Mohanty,1,a and Samuel C. Wassmer6,a, 1Center for the Study of Complex Malaria in India, Ispat General Hospital, Rourkela, Odisha, India, 2Department of Neuroradiology, University Hospital Heidelberg, Heidelberg, Germany, 3University Institute of Diagnostic and Interventional Neuroradiology, University Hospital Bern, Inselspital, University of Bern, Switzerland, 4Department of Radiology, Ispat General Hospital, Rourkela, Odisha, India, 5Department of Intensive Care, Ispat General Hospital, Rourkela, Odisha, India, 6Department of Infection Biology, London School of Hygiene and Tropical Medicine, London, United Kingdom, 7Infectious Diseases Biology Unit, Institute of Life Sciences, Bhubaneswar, Odisha, India, 8Department of Ophthalmology, Ispat General Hospital, Rourkela, Odisha, India, 9Department of Biology, New York University, New York, New York, USA, 10North Manchester General Hospital, Manchester, United Kingdom, 11Mahidol Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand, 12Centre for Tropical Medicine and Global Health, Nuffield Department of Clinical Medicine, Oxford, United Kingdom, and 13Department of Neurology, Division of Neurogeriatrics, Medical University of Graz, Graz, Austria Received 23 June 2020; editorial decision 30 September 2020; published online 16 December 2020. Correspondence: S. C. Wassmer, London School of Hygiene and Tropical Medicine, Department of Infection Biology, Keppel St, Rm 236a, London WC1E 7HT, UK (sam.wassmer@ lshtm.ac.uk). aP. K. S., A. H., S. Mo., and S. C. W. contributed equally to this work. Clinical Infectious Diseases® 2021;73(7):e2387–96 © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciaa1647 Clinical Infectious Diseases Clinical Infectious Diseases M A J O R A R T I C L E Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2387 Clinical Infectious Diseases Clinical Infectious Diseases MRI and Analysis Imaging of the brain was performed using a 1.5T Siemens Symphony MRI scanner (Siemens AG, Erlangen, Germany). ADC maps were generated and used for differentiation between cytotoxic and vasogenic edema [15]. Each MRI was interpreted by 1 radiologist on site (M. M.) and 2 neuroradiologists off- site (I. W. T. and A. H.). T1-weighted images were used for automated volume analysis using the freely available program SIENAX to assess brain volume, and SIENA to compare admis- sion and follow-up scans (Supplementary Figures 2 and 3) [16]. Assessments were performed blindly by an experienced MRI postprocessing image analysis expert (L. P.). Normalized ADC histograms were created for whole brain, and the peak location of whole-brain histograms, corresponding to the most common ADC value in the brain tissue [17], was used for the analyses. Additional details are available in the Supplementary Materials. We provide the first comprehensive comparative analysis of CM-associated structural and functional brain changes in a co- hort of both pediatric and adult Indian patients, and investigate patterns associated with survival and mortality by combining MRI with quantitative brain volume and ADC histogram ana- lyses, complemented with an assessment of parasite biomass and hypoxia biomarkers. (See the Editorial Commentary by John on pages e2397–8.) Conversely, vasogenic edema is characterized by an increase in ADC and expansion of the extracellular space after leakage of fluid from the blood-brain barrier to the paren- chymal tissue. To date, no systematic quantitative ADC studies have been performed in CM patients to distinguish between these different etiologies of brain swelling. antimalarial treatment. In this cohort, the case fatality rate in patients with CM was 7 of 65 (10.8%). frequently in both adult [8, 9] and pediatric CM [10]. In African children, the increase in cerebral volume can be severe and result in brain stem herniation, leading to death by respiratory arrest [11]. This differs markedly from Southeast Asian adults, who usually present milder cerebral swelling not leading to coma or death [12]. • UM (n = 26): fully conscious UM patients (Glasgow Coma Score = 15/15 for adults) infected with P. falciparum with fever (axillary temperature, ≥37.5°C) or history of fever in the preceding 24 hours, and no signs of complicated malaria, were eligible for inclusion. Because of the difficulty to obtain quality MRI scans in young children with febrile illness, pe- diatric UM control patients were not included in this study. Specific magnetic resonance imaging (MRI) techniques may help to get further insights into disease pathology. Apparent diffusion coefficient (ADC) is a measure of the magnitude of diffusion of water molecules within a tissue. ADC maps are commonly calculated clinically using MRI with diffusion- weighted imaging and allow the discrimination between cyto- toxic and vasogenic edema. Cytotoxic edema is characterized by ADC decrease due to restricted diffusion of water molecules [13]: ATP pumps cease to operate following a hypoxic/hypo- glycemic injury, leading to a shift of fluid from the extracellular to the intracellular compartment and shrinkage of the extracel- lular space [14]. Conversely, vasogenic edema is characterized by an increase in ADC and expansion of the extracellular space after leakage of fluid from the blood-brain barrier to the paren- chymal tissue. To date, no systematic quantitative ADC studies have been performed in CM patients to distinguish between these different etiologies of brain swelling.i Plasma Level Evaluation of Lipocalin-2, MicroRNA-150, and P. falciparum Histidine-Rich Protein 2 Plasma levels of lipocalin-2, a recently described marker of brain hypoxia [18], microRNA-150 (miRNA-150), a regulator of hypoxia-induced factor 1α [19], and P. falciparum histidine- rich protein 2 (PfHRP2), an indicator of the total parasite bio- mass [20], were assessed in all patients from the cohort using commercially available kits (Supplementary Materials). All as- says were performed according to the manufacturer protocols, in duplicate with results averaged for analyses, and by individ- uals blinded to study endpoints. • CM (n = 65): All CM patients fulfilled the strict clin- ical definition, according to the World Health Organization criteria [2], including a Glasgow Coma Score ≤9/15 for adults and a Blantyre Coma Score ≤2 for young preverbal chil- dren. Inclusion and exclusion criteria are detailed in the Supplementary Materials. Two patients were first diagnosed with uncomplicated malaria (UM), but developed CM after Study Site and Patients The study was carried out at Ispat General Hospital (IGH) in Rourkela, India, from October 2013 to November 2019 (Supplementary Table 1). Written consent was obtained from all enrolled subjects or their families prior to inclusion in the study. Ethical approval was obtained from IGH, the Indian Council of Medical Research (TDR589/2010/ECDII), New York University School of Medicine (S12-03016), the London School of Hygiene and Tropical Medicine, and Heidelberg University Hospital. Eighty-five patients were enrolled, and MRI was car- ried out within 10 hours of admission. Sixty-five patients (76%) underwent MRI a second time (Table 1; Supplementary Figure 1). Patients were classified as follows: Plasma Level Evaluation of Lipocalin-2, MicroRNA-150, and P. falciparum Histidine-Rich Protein 2 Study Procedures and Clinical Care On admission, a full medical history and physical examination including funduscopy were conducted and recorded on a stand- ardized clinical record form. Blood samples were collected for complete blood count, parasite count, hemoglobin, hematocrit, glucose, and biochemistry. Antimalarial treatments were in ac- cordance with the national drug policy of the government of India. Additional details on funduscopy and drug regimens are available in the Supplementary Materials. (See the Editorial Commentary by John on pages e2397–8.) Cerebral malaria (CM) is the presenting syn- drome in around half of the patients with severe malaria, both in children and adults. CM leads to neurological dysfunctions The fundamental pathogenesis of fatal CM is still incom- pletely understood. The mechanical obstruction of cerebral microvessels by sequestered Plasmodium falciparum–parasit- ized red blood cells (pRBCs) is central to its pathogenesis [5], and hyperactivation of host immune cells leading to the exces- sive release of proinflammatory cytokines, as well as critical he- matologic dysfunctions, has also been proposed [6].h Received 23 June 2020; editorial decision 30 September 2020; published online 16 December 2020. Correspondence: S. C. Wassmer, London School of Hygiene and Tropical Medicine, Department of Infection Biology, Keppel St, Rm 236a, London WC1E 7HT, UK (sam.wassmer@ lshtm.ac.uk). aP. K. S., A. H., S. Mo., and S. C. W. contributed equally to this work. Clinical Infectious Diseases® 2021;73(7):e2387–96 © The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. DOI: 10.1093/cid/ciaa1647 The causes and the contribution of brain swelling to neurological symptoms have been a source of debate [7]. Brain swelling occurs Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2387 frequently in both adult [8, 9] and pediatric CM [10]. In African children, the increase in cerebral volume can be severe and result in brain stem herniation, leading to death by respiratory arrest [11]. This differs markedly from Southeast Asian adults, who usually present milder cerebral swelling not leading to coma or death [12]. Specific magnetic resonance imaging (MRI) techniques may help to get further insights into disease pathology. Apparent diffusion coefficient (ADC) is a measure of the magnitude of diffusion of water molecules within a tissue. ADC maps are commonly calculated clinically using MRI with diffusion- weighted imaging and allow the discrimination between cyto- toxic and vasogenic edema. Cytotoxic edema is characterized by ADC decrease due to restricted diffusion of water molecules [13]: ATP pumps cease to operate following a hypoxic/hypo- glycemic injury, leading to a shift of fluid from the extracellular to the intracellular compartment and shrinkage of the extracel- lular space [14]. Statistical Analyses 2 The χ 2 test was used to compare categorical variables. Depending on the normality distribution, an unpaired Student t test or Mann-Whitney test was used to compare 2 groups. e2388 • cid 2021:73 (1 October) • Sahu et al C b l M l i i Ad lt d Child cid 2021 73 (1 O t b ) Table 1. Summary of Clinical and Imaging Findings in 91 Indian Patients with CM or UM Children Adults Demographics Fatal CM Non-fatal CM Fatal CM Non-fatal CM UM No. 3 24 4 34 20 Age (mean, (SD)) 5 (0) 7.8 (3.9) 38 (9.3) 31 (9.1) 36 (13) Sex (female, male) 0, 3 6, 18 0, 4 10, 22 6, 14 Parasite burden Parasitemia: No. 3 22 4 32 19 Parasitemia (×1000/uL): median (range) 0.43 (0.17 71.36) 4.33 (0.41 20.78) 97.50 (1.48 294.02) 4.08 (0.39 22.59) 28.48 (0.60 192.01) PfHRP2: No. 3 19 3 20 9 PfHRP2 (ng/mL): median (range) 13.71 (9.05 1019.72) 933.50 (140.13 1186.64) 10.34 (9.10 606.95) 933.77 (133.73 1293.01) 275.57 (9.06 640.75) Clinical parameters No. 3 24 4 34 20 Platelet count (×1000/uL): median (range) 47.00 (31.25 48.50) 47.50 (27.00 81.50) 8.00 (0.00 40.00) 36.25 (15.00 60.00) 58.50 (38.00 85.50) Hb (g/dL): median (range) 5.60 (4.03 7.25) 6.65 (6.00 7.85) 9.00 (7.50 10.25) 7.40 (6.20 9.80) 10.75 (9.40 12.20) Bilirubin (mg/dL): median (range) 1.40 (0.65 2.75) 1.95 (0.65 2.90) 26.90 (23.65 29.60) 3.60 (2.10 7.50) 1.30 (0.95 1.75) Creatinine (mg/dL): median (range) 1.70 (0.95 4.48) 0.60 (0.50 0.90) 6.75 (5.05 8.10) 2.35 (1.20 5.00) 1.00 (0.80 1.10) Retinopathies: hemmorhages (1/0): No. (%) 3 (33%) 23 (57%) 4 (50%) 30 (50%) 18 (11%) Retinopathies: whitening (1/0): No. (%) 3 (0%) 23 (9%) 4 (0%) 30 (17%) 18 (0%) Retinopathies: papilledema: (1/0): No. (%) 3 (0%) 23 (4%) 4 (0%) 30 (13%) 18 (0%) Quantitative MRI parameters Brain volume First scan No. 2 15 4 23 18 Normalized brain volume (cm3): mean (SD) 2070.73 (5.04) 1684.94 (695.98) 1594.94 (148.09) 1639.53 (118.35) 1572.64 (109.13) Second scan No. NA 48.11 (44.22 74.98) NA 47.92 (46.89 63.25) 50.37 (48.23 71.00) Time between first and second scan (hours): median (IQR) NA 1627.95 (701.05) NA 1665.14 (148.47) 1554.95 (80.97) Normalised brain volume (cm3): mean (SD) ADC 3 16 3 28 17 First scan No. Statistical Analyses 2 781.50 (637.50 797.62) 782.00 (759.75 798.75) 598.50 (579.00 630.00) 677.50 (627.50 709.75) 742.50 (732.88 760.00) Whole brain adcpeak (10˗6 mm2/s): median (range) 0 14 0 19 14 Second scan No. NA 50.28 (47.18 96.03) NA 48.02 (47.42 66.75) 50.33 (48.10 70.88) Time between first & second scan (hours): median (range) NA 778.50 (757.50 800.00) NA 718.50 (672.75 734.50) 716.25 (700.50 739.50) Whole brain ADCpeak (10˗6 mm2/s): median (range) NA 48.11 (44.22 74.98) NA 47.92 (46.89 63.25) 50.37 (48.23 71.00) Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2389 of brain stem herniation (Figure 1C). The other 2 patients developed end-stage disease 47 and 49 hours after the scan, respectively. Fatal adult CM patients showed moderate (2/4) or no brain swelling (2/4), and no signs of brain stem herni- ation were observed (Figure 1C). In nonfatal CM, reversible brain swelling after treatment was evidenced by a rapid de- crease in brain volume at the follow-up MRI compared to the volume measured on admission (P = .014; Supplementary Figure 4A and 4C), which is consistent with the previously reported semi-quantitative assessment of 11 cases from the same cohort [8]. UM patients showed no significant change in brain volume between the 2 scans (Supplementary Figure 4B and 4D). Pearson correlation coefficients were calculated for correlation analyses. A 2-sided P < .05 was considered statistically signif- icant. All statistical analyses were performed using GraphPad Prism 8.3 (GraphPad Software). Data Availability Anonymized evaluation data are available upon request. Brain Volume Increase in CM Is Age-Dependent and Not Associated With Mortality Downloaded from https://academic.oup.com/cid/article/73/7/e2387/6035083 by Universitätsbibliothek Bern user on 14 April 2022 Normalized brain volume on admission was higher in pa- tients with nonfatal CM (1753.7 ± 192.5 cm3, P = .0003) and fatal CM (1753.5 ± 271.2 cm3, P = .019) compared with UM patients (1570 ± 103 cm3), irrespective of age. There was no significant difference in volume between fatal and nonfatal CM, and large intragroup variations were observed, ranging from 1396.6 to 2084.4 cm3 in nonfatal CM, and from 1458.9 to 2074.3 cm3 in fatal CM (Figure 1A). The normalized brain volume on admission was negatively correlated with age, ir- respective of the outcome (Figure 1B; R2 = 0.66, P < .0001 for nonfatal CM; R2 = 0.73, P = .029 for fatal CM). At the time of MRI, 1 of 3 fatal pediatric CM patients showed signs Nonfatal Pediatric and Adult CM Patients Show Reversible Cytotoxic Edema in Different Brain Regions Nonfatal Pediatric and Adult CM Patients Show Reversible Cytotoxic Edema in Different Brain Regions In nonfatal pediatric CM, the most prominent finding was diffusion restriction in the deep and subcortical white matter characterized by a decrease in ADC, indicating hypoxia- related cytotoxic edema (Figure 2A). In contrast, an ADC de- crease in the basal ganglia was the hallmark of nonfatal adult CM, indicating focal hypoxia-related cytotoxic edema in deep gray matter structures (Figure 2B). UM patients did not article/73/7/e2387/6035083 by Universitätsbibliothek Bern user on 14 April 2022 Figure 1. Comparison of brain volumes on admission between age and disease groups. A, Normalized brain volumes in uncomplicated malaria (UM) and nonfatal and fatal cerebral malaria (CM). B, Correlation between age and normalized brain volume in nonfatal and fatal CM. C, Representative sagittal T2-weighted magnetic resonance imaging of patients with UM (first row) and CM (second and third rows). In pediatric CM patients, the outer cerebrospinal fluid spaces are more reduced due to brain swelling compared with adults. One fatal pediatric CM case showed brain stem herniation with no remaining cerebrospinal fluid space at the craniocervical junction (arrows). P < .05; *P < .0005.. Figure 1. Comparison of brain volumes on admission between age and disease groups. A, Normalized brain volumes in uncomplicated malaria (UM) and nonfatal and fatal cerebral malaria (CM). B, Correlation between age and normalized brain volume in nonfatal and fatal CM. C, Representative sagittal T2-weighted magnetic resonance imaging of patients with UM (first row) and CM (second and third rows). In pediatric CM patients, the outer cerebrospinal fluid spaces are more reduced due to brain swelling compared with adults. One fatal pediatric CM case showed brain stem herniation with no remaining cerebrospinal fluid space at the craniocervical junction (arrows). P < .05; **P < .0005.. e2390 • cid 2021:73 (1 October) • Sahu et al Figure 2. Evolution of apparent diffusion coefficient (ADC) between admission and follow-up magnetic resonance imaging (MRI). A, C, and E, Representative sequential ADC maps at the same window level (130) and width (290) from 3 cases of pediatric cerebral malaria (CM), adult CM, and uncomplicated malaria (UM). A, Pediatric CM pa- tient showing decreased ADC values in the white matter on admission (left, arrows) that resolved at follow-up (right). C, Adult CM patient showing decreased ADC values in the basal ganglia on admission (left) that reversed at follow-up (right). E, UM patient without pathological ADC changes on admission (left) or at follow-up (right). Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2391 Cytotoxic Edema Is Associated With Adult CM and Is More Severe in Fatal Disease present any pathological features noticeable by visual inspec- tion (Figure 2E). In both age groups, serial ADC measures revealed rapid ADC reversal in follow-up scans, with only subtle remaining changes in a few patients (median of 47.9 hours [interquartile range {IQR}, 7.8 hours] after the first scan), indicative of reversible cytotoxic edema (Figure 2D and 2E; Supplementary Figure 5; Table 1). In pediatric CM, ADCpeak values increased at follow-up in 50% of pediatric CM cases (5/10). The low ADCpeak values in these children were mainly located in the cerebral white matter. Thirty per- cent of pediatric CM patients (3/10) with increased ADCpeak values on admission showed decreased values at follow-up, suggesting a predominantly vasogenic component during the acute disease. In 20% of cases (2/10), ADCpeak values re- mained constant (Figure 2A and 2D). In adult CM patients, ADCpeak values increased significantly at follow-up compared to admission (677.50 × 10–6mm2/second [IQR, 82.25] vs 718.50 × 10–6mm2/second [IQR, 61.75]; P = .0004; Figure 2E), reaching values similar to ADCpeak values observed in most UM patients (724.25 × 10–6mm2/second [IQR, 39.00]). ADCpeak values in UM patients slightly decreased at follow-up (P = .012), indicating a resolution of mild vasogenic edema posttreatment (Figure 2C and 2F; Table 1). present any pathological features noticeable by visual inspec- tion (Figure 2E). In both age groups, serial ADC measures revealed rapid ADC reversal in follow-up scans, with only subtle remaining changes in a few patients (median of 47.9 hours [interquartile range {IQR}, 7.8 hours] after the first scan), indicative of reversible cytotoxic edema (Figure 2D and 2E; Supplementary Figure 5; Table 1). In pediatric CM, ADCpeak values increased at follow-up in 50% of pediatric CM cases (5/10). The low ADCpeak values in these children were mainly located in the cerebral white matter. Thirty per- cent of pediatric CM patients (3/10) with increased ADCpeak values on admission showed decreased values at follow-up, suggesting a predominantly vasogenic component during the acute disease. In 20% of cases (2/10), ADCpeak values re- mained constant (Figure 2A and 2D). In adult CM patients, ADCpeak values increased significantly at follow-up compared to admission (677.50 × 10–6mm2/second [IQR, 82.25] vs 718.50 × 10–6mm2/second [IQR, 61.75]; P = .0004; Figure 2E), reaching values similar to ADCpeak values observed in most UM patients (724.25 × 10–6mm2/second [IQR, 39.00]). Nonfatal Pediatric and Adult CM Patients Show Reversible Cytotoxic Edema in Different Brain Regions Whole-brain ADCpeak values on admission and at follow-up are displayed and grouped according to age and disease categories: nonfatal pediatric CM (B), nonfatal adult CM (D), and UM (F). P < .05; **P < .0005; ns, not significant. Downloaded from https://academic.oup.com/cid/article/73/7/e2387/6035083 by Universitätsbibliothek Bern user on 14 April 2022 Figure 2. Evolution of apparent diffusion coefficient (ADC) between admission and follow-up magnetic resonance imaging (MRI). A, C, and E, Representative sequential ADC maps at the same window level (130) and width (290) from 3 cases of pediatric cerebral malaria (CM), adult CM, and uncomplicated malaria (UM). A, Pediatric CM pa- tient showing decreased ADC values in the white matter on admission (left, arrows) that resolved at follow-up (right). C, Adult CM patient showing decreased ADC values in the basal ganglia on admission (left) that reversed at follow-up (right). E, UM patient without pathological ADC changes on admission (left) or at follow-up (right). Whole-brain ADCpeak values on admission and at follow-up are displayed and grouped according to age and disease categories: nonfatal pediatric CM (B), nonfatal adult CM (D), and UM (F). P < .05; **P < .0005; ns, not significant. Cytotoxic Edema Is Associated With Adult CM and Is More Severe in Fatal Disease Cytotoxic Edema Is Associated With Adult CM and Is More Severe in Fatal Disease ADCpeak values in UM patients slightly decreased at follow-up (P = .012), indicating a resolution of mild vasogenic edema posttreatment (Figure 2C and 2F; Table 1). Adult patients with fatal CM had significantly lower ADCpeak values compared to patients from the same age group who sur- vived (P = .026; Figure 3A; Table 1). UM had no pathological ADC alteration by visual inspection, whereas in adults with nonfatal CM, local areas of ADC decrease were observed in the subcortical white matter and the basal ganglia (Figure 3B). In contrast, patients with fatal disease showed a homogeneous, global ADC decrease affecting all brain structures (Figure 3B). Such homogeneous, symmetric diffusion alterations can only be detected by measurement of ADC values, and this was evi- denced by a shift of ADC histograms to lower values (Figure 3C; Supplementary Figure 6). Pediatric CM patients showed slightly decreased, normal and high ADCpeak values (Figure 3D and 3E; Supplementary Figure 6; Table 1). Compared to adults, they presented a less pronounced cytotoxic component. ADC de- crease affecting a large proportion of the white matter was seen in patients with slightly decreased ADCpeak values (Figure 3D). Two of 3 fatal CM cases had no or only little subcortical ADC decrease and showed normal to high ADCpeak values and thus no cytotoxic edema, but rather an increased water content, con- sistent with vasogenic or interstitial edema (Figure 3D and 3E; gure 3. Apparent diffusion coefficient (ADC) alterations in adult and pediatric cerebral malaria (CM). A, Whole-brain ADCpeak values (mean and standard deviation) of dult patients with uncomplicated malaria (UM), nonfatal CM, and fatal CM. B, Representative ADC maps at the same window level (130) and width (290) from 1 represen- ative UM patient (framed in green), 1 case of nonfatal adult CM (framed in blue), and 1 case of fatal adult CM (framed in red) are shown with the corresponding ADCpeak alue listed below the image. The ADC map of the representative UM patient does not show pathological alterations, whereas the nonfatal case exhibits an ADC decrease the basal ganglia (white arrowheads) and the subcortical white matter (white arrows). In the fatal CM case, a global ADC decrease, which is hardly detectable by visual spection, results in low whole-brain ADCpeak values. C, Mean histograms with mean and standard deviation of UM, nonfatal adult CM, and fatal adult CM. Cytotoxic Edema Is Associated With Adult CM and Is More Severe in Fatal Disease ADCpeak values were the lowest in fatal CM, followed by nonfatal CM and UM. D, Whole-brain ADCpeak values (mean and standard deviation) of pediatric patients with nonfatal CM and fatal M. E, Representative ADC images at the same window level (130) and width (290) from 2 nonfatal pediatric cases (framed in blue) and 2 fatal pediatric CM cases (framed red) with the corresponding ADCpeak value listed below the image. The displayed ADC map of a nonfatal case illustrates a strong ADC decrease in the white matter (white rrows). The ADC map of 1 fatal pediatric case shows globally elevated ADC values with subtle ADC decrease in the subcortical white matter (white arrows). F, The mean stogram with mean and standard deviation of nonfatal pediatric CM and the mean histogram of fatal pediatric CM cases as well as the individual histograms of fatal pedi- tric CM cases. P < .05; ***P < .0001; ns, not significant. Downloaded from https://academic.oup.com/cid/article/73/7/e2387/6035083 by Universitätsbibliothek Bern user on 14 April 2022 Figure 3. Apparent diffusion coefficient (ADC) alterations in adult and pediatric cerebral malaria (CM). A, Whole-brain ADCpeak values (mean and standard deviation) of adult patients with uncomplicated malaria (UM), nonfatal CM, and fatal CM. B, Representative ADC maps at the same window level (130) and width (290) from 1 represen- tative UM patient (framed in green), 1 case of nonfatal adult CM (framed in blue), and 1 case of fatal adult CM (framed in red) are shown with the corresponding ADCpeak value listed below the image. The ADC map of the representative UM patient does not show pathological alterations, whereas the nonfatal case exhibits an ADC decrease in the basal ganglia (white arrowheads) and the subcortical white matter (white arrows). In the fatal CM case, a global ADC decrease, which is hardly detectable by visual inspection, results in low whole-brain ADCpeak values. C, Mean histograms with mean and standard deviation of UM, nonfatal adult CM, and fatal adult CM. ADCpeak values were the lowest in fatal CM, followed by nonfatal CM and UM. D, Whole-brain ADCpeak values (mean and standard deviation) of pediatric patients with nonfatal CM and fatal CM. E, Representative ADC images at the same window level (130) and width (290) from 2 nonfatal pediatric cases (framed in blue) and 2 fatal pediatric CM cases (framed in red) with the corresponding ADCpeak value listed below the image. Cytotoxic Edema Is Associated With Adult CM and Is More Severe in Fatal Disease The displayed ADC map of a nonfatal case illustrates a strong ADC decrease in the white matter (white arrows). The ADC map of 1 fatal pediatric case shows globally elevated ADC values with subtle ADC decrease in the subcortical white matter (white arrows). F, The mean histogram with mean and standard deviation of nonfatal pediatric CM and the mean histogram of fatal pediatric CM cases as well as the individual histograms of fatal pedi- atric CM cases. P < .05; ***P < .0001; ns, not significant. Supplementary Figure 6; Table 1). In the fatal case with brain stem herniation, ADCpeak values were significantly decreased, suggesting severe cytotoxic brain swelling caused by brain stem herniation as an end stage of fatal pediatric CM (Supplementary Figure 7). age group. The concentration of miRNA-150 on admission discriminated between fatal and nonfatal disease in adults (Figure 4A; median of 25.4 vs 8.5 RELs, P = .003), but not in children. Plasma concentrations of lipocalin-2 were not significantly increased in children with CM compared to UM patients, irrespective of the outcome. However, the con- centrations of plasma lipocalin-2 were significantly higher in adult patients with CM compared to both pediatric CM and UM cases (median, 266 776 vs 108 797 pg/mL, P = .001 and vs 146 062 pg/mL, P = .003, respectively). They also dis- criminated between nonfatal and fatal disease in adult CM Plasma Biomarkers of Hypoxia Reflect Changes in ADC During CM Plasma concentrations of miRNA-150 assessed on admission were significantly lower in UM compared to CM patients (me- dian, 4.7 vs 10.6 relative expression levels [RELs], P = .002), both with a fatal and nonfatal outcome, and irrespective of e2392 • cid 2021:73 (1 October) • Sahu et al Figure 4. Plasma biomarkers of hypoxia in cerebral malaria (CM). Levels of microRNA 150 (miRNA-150; A) and lipocalin-2 (B) were measured in the plasma of all patients and plotted according to age and disease category. C, Both miRNA-150 and lipocalin-2 were plotted against plasma levels of Plasmodium falciparum histidine-rich protein 2 and grouped according to age. Statistical significance was obtained from Mann-Whitney U test (A and B) and Spearman correlation analysis (C). P < .05; P < .005; P < .0005. Abbreviations: CM, cerebral malaria; PfHRP2, Plasmodium falciparum histidine-rich protein 2; RELs, relative expression levels; UM, uncomplicated malaria. Figure 4. Plasma biomarkers of hypoxia in cerebral malaria (CM). Levels of microRNA 150 (miRNA-150; A) and lipocalin-2 (B) were measured in the plasma of all patients and plotted according to age and disease category. C, Both miRNA-150 and lipocalin-2 were plotted against plasma levels of Plasmodium falciparum histidine-rich protein 2 and grouped according to age. Statistical significance was obtained from Mann-Whitney U test (A and B) and Spearman correlation analysis (C). P < .05; P < .005; *P < .0005. Abbreviations: CM, cerebral malaria; PfHRP2, Plasmodium falciparum histidine-rich protein 2; RELs, relative expression levels; UM, uncomplicated malaria. (Figure 4B; median, 227 181 vs 636 871 pg/mL, P = .0227). Plasma levels of miRNA-150 correlated negatively with PfHRP2 in children (r = –0.68, P = .002), and lipocalin-2 cor- related positively with PfHRP2 in adults (r = 0.44, P = .001) (Figure 4C). (Figure 4B; median, 227 181 vs 636 871 pg/mL, P = .0227). Plasma levels of miRNA-150 correlated negatively with PfHRP2 in children (r = –0.68, P = .002), and lipocalin-2 cor- related positively with PfHRP2 in adults (r = 0.44, P = .001) (Figure 4C). In survivors, isolated ADC decrease in hypoxia-sensitive re- gions was commonly observed in CM patients. Sequestration of pRBCs in the cerebral microvasculature is a hallmark of CM and has long been postulated to alter blood flow, likely re- sulting in the hypoxic injury we describe [5]. Plasma Biomarkers of Hypoxia Reflect Changes in ADC During CM However, these regions differed with age: children predominantly showed re- stricted diffusion in the white matter, whereas the basal ganglia were mainly affected in adults (Figure 5). The age-related sus- ceptibility of white matter to hypoxia during the acute phase of the pediatric disease may result from active myelination in children, an energy-intensive process sensitive to metabolic disturbances that extends into the third decade of life [24, 25]. Furthermore, the stage of the disease and the degree of hypoxia may contribute to the distinct ADC distribution we identi- fied. Experiments on perinatal primates showed that a milder, more gradual insult resulted in white matter injury sparing the basal ganglia [26, 27], and similar findings were also reported in newborns with neonatal hypoxic-ischemic encephalopathy [28]. In pediatric CM, white matter diffusion restriction may thus reflect prolonged mild/moderate hypoxia. In contrast, a combination of white matter involvement and ADC decrease Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2393 DISCUSSION Differences in apparent diffusion coefficient (ADC) values and pathogenic patterns between adults and children with fatal and nonfatal cerebral malaria (CM). An increase in ADC values is associated with extracellular water accumulation in the cerebral tissue. In CM, this is likely to result from vasogenic edema: following blood- brain barrier breakdown, there is a transfer of fluid from the circulation to the brain parenchyma (a). Decreased ADC values are the signature of cytotoxic edema, which is triggered by the obstruction of circulation by sequestered Plasmodium falciparum–parasitized red blood cells in CM, resulting in hypoxic and hypoglycemic conditions in the surrounding cerebral tissue (in red). Due to the decreased energy supply, cellular ATP pumps cease to work, causing an osmotic transfer of water inside the cells and their subsequent swelling (b). In nonfatal CM, specific hypoxia-sensitive regions of the brain are affected, and these differ with age: cytotoxic edema evidenced by ADC decrease develops in the white matter in children (c) and in the basal ganglia in adults (d). In both age groups, it reverses rapidly upon antimalaria treatment. In fatal pediatric CM, 2 different patterns were observed: Brain swelling is associated with globally increased ADC signal, indicating diffuse vasogenic edema (e). In contrast, when brain stem herniation occurs, it leads to severe cytotoxic brain swelling with ADC decrease (f). Fatal CM in adult is associated with global, severe hypoxia evidenced by the decreased ADC signal and mild or no brain swelling (g). in the basal ganglia may indicate a more advanced stage of di- sease [29]. Basal ganglia are areas of high metabolic activity and are highly susceptible to hypoxic changes as they are sup- plied by end arteries with low collateral blood supply [30]. This may explain the ADC decrease in the basal ganglia of nonfatal adult CM, as similar observations were reported in adults after global hypoxic-ischemic injury [31]. Overall, ADC values in adults were lower upon admission compared to children and increased during recovery, suggesting a stronger cytotoxic component. Plasma levels of miRNA-150, a marker of hypoxia, were significantly higher in CM patients irrespective of the age group, confirming that hypoxia is a frequent occurrence in this neurological syndrome. proven unsuccessful as an adjuvant therapy [9]. In addition, a decrease in blood flow leads to elevated concentrations of the excitotoxic neurotransmitter glutamate [34], causing cell death if glutamate reuptake fails. DISCUSSION In this study of both children and adult patients with CM in India, we used quantitative MRI analyses to compare the course of disease between age groups and clinical outcomes. Brain vol- umes on admission were higher in pediatric compared to adult CM patients and the swelling reversed rapidly in survivors, cor- roborating previous reports in Malawian children [11]. This age-related difference in brain swelling during acute CM may relate to the more loosely organized extracellular spaces in chil- dren, which occupy about 20% of total brain volume and enable a more rapid development of brain swelling compared to adults [21], as described in other diseases [22, 23]. In adult patients with CM, our findings confirm the lack of association between mortality and brain swelling [9, 12]. Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2393 Figure 5. Differences in apparent diffusion coefficient (ADC) values and pathogenic patterns between adults and children with fatal and nonfatal cerebral malaria (CM). An increase in ADC values is associated with extracellular water accumulation in the cerebral tissue. In CM, this is likely to result from vasogenic edema: following blood- brain barrier breakdown, there is a transfer of fluid from the circulation to the brain parenchyma (a). Decreased ADC values are the signature of cytotoxic edema, which is triggered by the obstruction of circulation by sequestered Plasmodium falciparum–parasitized red blood cells in CM, resulting in hypoxic and hypoglycemic conditions in the surrounding cerebral tissue (in red). Due to the decreased energy supply, cellular ATP pumps cease to work, causing an osmotic transfer of water inside the cells and their subsequent swelling (b). In nonfatal CM, specific hypoxia-sensitive regions of the brain are affected, and these differ with age: cytotoxic edema evidenced by ADC decrease develops in the white matter in children (c) and in the basal ganglia in adults (d). In both age groups, it reverses rapidly upon antimalaria treatment. In fatal pediatric CM, 2 different patterns were observed: Brain swelling is associated with globally increased ADC signal, indicating diffuse vasogenic edema (e). In contrast, when brain stem herniation occurs, it leads to severe cytotoxic brain swelling with ADC decrease (f). Fatal CM in adult is associated with global, severe hypoxia evidenced by the decreased ADC signal and mild or no brain swelling (g). Downloaded from https://academic.oup.com/cid/article/73/7/e2387/6035083 by Universitätsbibliothek Bern user on 14 April 2022 Figure 5. e2394 • cid 2021:73 (1 October) • Sahu et al Notes Acknowledgments. The authors thank the patients and their guard- ians/families for their participation in this study, as well as Nakul Chandra Khatua and Tapas Kar, the magnetic resonance technicians at Ispat General Hospital, Rourkela, Odisha, India, for their enthusiasm and logistical sup- port. The authors acknowledge Dr Saroj Mishra (deceased) and Professor Frederik Barkhof for their invaluable help during the set-up of the project; the Director in Charge and the clinical staff of Ispat General Hospital in Rourkela for their support and dedication; and the Director of the Institute of Life Sciences in Bhubaneswar for allowing us to use its Infectious Disease Biology Unit to conduct laboratory work in Rourkela, Odisha, India. 21. Syková E, Nicholson C. Diffusion in brain extracellular space. Physiol Rev 2008; 88:1277–340. 22. Aldrich EF, Eisenberg HM, Saydjari C, et al. Diffuse brain swelling in severely head-injured children. A report from the NIH Traumatic Coma Data Bank. J Neurosurg 1992; 76:450–4. 23. Wolfsdorf J, Glaser N, Sperling MA; American Diabetes Association. Diabetic ketoacidosis in infants, children, and adolescents: a consensus statement from the American Diabetes Association. Diabetes Care 2006; 29:1150–9. American Diabetes Association. Diabetes Care 2006; 29:1150–9. 24. Paus T, Zijdenbos A, Worsley K, et al. Structural maturation of neural pathw children and adolescents: in vivo study. Science 1999; 283:1908–11. 24. Paus T, Zijdenbos A, Worsley K, et al. Structural maturation of neu children and adolescents: in vivo study. Science 1999; 283:1908–1 25. Watanabe M, Liao JH, Jara H, Sakai O. Multispectral quantitative MR imaging of the human brain: lifetime age-related effects. Radiographics 2013; 33:1305–19. Financial support. Research reported in this publication was sup- ported by the National Institute Of Allergy And Infectious Diseases of the National Institutes of Health under Award Numbers U19AI089676 and R21AI142472. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. S. C. W. is supported by the Medical Research Council, United Kingdom (award number MR/S009450/1) and A. H. is supported by a clin- ical leave stipend from the German Centre for Infection Research. 26. Myers RE. Two patterns of perinatal brain damage and their conditions of occur- rence. Am J Obstet Gynecol 1972; 112:246–76. 27. Myers RE. Four patterns of perinatal brain damage and their conditions of occur- rence in primates. Adv Neurol 1975; 10:223–34. 28. Li AM, Chau V, Poskitt KJ, et al. DISCUSSION In the adult CM group, plasma levels of lipocalin-2 also correl- ated positively with PfHRP2, further suggesting that in adults cerebral hypoxia is linked to high parasite burdens and results in more prominent decrease in blood flow compared to chil- dren. Although additional studies are warranted in children, this conclusion is in line with the hypothesis that hypoxia may be triggered by a mechanical obstruction of cerebral microvessels by sequestered pRBCs [5], platelets, clumps, and rosettes [39], and immune cells [40].ii Potential conflicts of interest. The authors: No potential conflicts of interest. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. References Mohanty S, Mishra SK, Patnaik R, et al. Brain swelling and mannitol therapy in adult cerebral malaria: a randomized trial. Clin Infect Dis 2011; 53:349–55. 10. Potchen MJ, Kampondeni SD, Seydel KB, et al. Acute brain MRI findings in 120 Malawian children with cerebral malaria: new insights into an ancient disease. AJNR Am J Neuroradiol 2012; 33:1740–6. 11. Seydel KB, Kampondeni SD, Valim C, et al. Brain swelling and death in children with cerebral malaria. N Engl J Med 2015; 372:1126–37. Our findings suggest for the first time that these distinct disease courses may be differentially targeted by specific ad- junctive therapy according to age group. Approaches focusing on a reduction of brain swelling would be more relevant in children and could be achieved by reducing perivascular in- flammation. In adults, adjunctive approaches aimed at either ameliorating cytotoxic edema, like glutamate agonists [36], or improving neuroprotection and survival of brain cells may re- duce mortality. 12. Maude RJ, Barkhof F, Hassan MU, et al. Magnetic resonance imaging of the brain in adults with severe falciparum malaria. Malar J 2014; 13:177. 13. Ebisu T, Naruse S, Horikawa Y, et al. Discrimination between different types of white matter edema with diffusion-weighted MR imaging. J Magn Reson Imaging 1993; 3:863–8. 14. Sevick RJ, Kanda F, Mintorovitch J, et al. Cytotoxic brain edema: assessment with diffusion-weighted MR imaging. Radiology 1992; 185:687–90. 15. Le Bihan D, Breton E, Lallemand D, Grenier P, Cabanis E, Laval-Jeantet M. MR imaging of intravoxel incoherent motions: application to diffusion and perfusion in neurologic disorders. Radiology 1986; 161:401–7. 16. Smith SM, De Stefano N, Jenkinson M, Matthews PM. Normalized accurate meas- urement of longitudinal brain change. J Comput Assist Tomogr 2001; 25:466–75. 17. Tofts PS, Davies GR, Dehmeshki J. Histograms: measuring subtle diffuse disease. In: Tofts P, ed. Quantitative MRI of the brain: measuring changes caused by di- sease. New York City, USA: John Wiley & Sons, Ltd, 2003. sease. New York City, USA: John Wiley & Sons, Ltd, 2003. Supplementary Data 18. Ranjbar Taklimie F, Gasterich N, Scheld M, et al. Hypoxia induces astrocyte- derived lipocalin-2 in ischemic stroke. Int J Mol Sci 2019; 20:1271. Supplementary materials are available at Clinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author. 19. Chen M, Shen C, Zhang Y, Shu H. MicroRNA-150 attenuates hypoxia-induced excessive proliferation and migration of pulmonary arterial smooth muscle cells through reducing HIF-1α expression. Biomed Pharmacother 2017; 93:861–8. 20. Dondorp AM, Desakorn V, Pongtavornpinyo W, et al. Estimation of the total par- asite biomass in acute falciparum malaria from plasma PfHRP2. PLoS Med 2005; 2:e204. DISCUSSION Glutamine is catalyzed by glutamine synthetase to form glutamate [35]. A recent study showed that treatment with a new glutamine antagonist led to a net de- crease of glutamate build-up and prevented mice infected with Plasmodium berghei ANKA from developing experimental CM [36], further supporting the role of reversible cytotoxic edema in CM. Remarkably, the UM group in our cohort also showed a subtle whole-brain ADC increase upon admission that re- versed after treatment. The slightly elevated ADC values on the first scan suggest mild endothelial dysfunction and vasogenic edema, 2 features that have not previously been reported in fully conscious, nonsevere malaria patients. In nonfatal disease, ADC values rapidly normalized following treatment with artesunate, indicating reversal of cytotoxic edema, presumably through removal of sequestered pRBCs and restoration of the cerebral microcirculatory blood flow. Some patients showed subtle increased ADC, and this vasogenic com- ponent could result from vascular leakage through damaged endothelium after reperfusion [32]. The rapid ADC normaliza- tion after treatment and clinical improvement of patients with nonfatal CM within 24–48 hours strongly suggest the involve- ment of reversible cytotoxic edema [33], and explains why treat- ment with mannitol aimed at ameliorating vasogenic edema has We show that fatal CM is associated with global ADC al- terations in both age groups, with a more prominent signal decrease in adults. In the absence of brain swelling, this is consistent with a profound global hypoxic injury, likely in- duced by blood sludging due to sequestered pRBCs [37]. These results contrasted with 2 of 3 pediatric patients who had brain swelling and high ADC values, suggestive of a global accumulation of extracellular fluid. One fatal pedi- atric case was admitted and scanned at end-stage disease e2394 • cid 2021:73 (1 October) • Sahu et al consistent with previous reports [11], with brain stem herni- ation, consecutive low ADC values due to ceasing blood flow, and resultant cytotoxic edema. Plasma lipocalin-2 levels were significantly higher in adult CM and discriminated between fatal and nonfatal outcomes, confirming severe and global brain hypoxia in fatal adult disease. Lipocalin-2 is released during excitotoxic neuronal injury by neurons and astrocytes [38] and is associated with cerebral hypoxic injury [18]. These previously unreported results demonstrate that profound brain hypoxia measurable by low ADC values is associated with fatality in adult CM, and high plasma levels of miRNA- 150 and lipocalin-2 are predictive of negative outcomes. Cerebral Malaria in Adults and Children • cid 2021:73 (1 October) • e2395 References 1. Reyburn H, Mbatia R, Drakeley C, et al. Association of transmission intensity and age with clinical manifestations and case fatality of severe Plasmodium falciparum malaria. JAMA 2005; 293:1461–70. 1. Reyburn H, Mbatia R, Drakeley C, et al. 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https://openalex.org/W2057500830	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0033907&type=printable	English	null	Clinically Isolated Syndromes Suggestive of Multiple Sclerosis: An Optical Coherence Tomography Study	PloS one	2,012	cc-by	6,088	Abstract This is an open-access article distributed under the unrestricted use, distribution, and reproduction in any medium, provided the original auth Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: orejacbn@gmail.com Competing Interests: The authors have declared that no competing interests exist. * E-mail: orejacbn@gmail.com Celia Oreja-Guevara1, Susana Noval2, Juan Alvarez-Linera3, Laura Gabaldo´ n4, Beatriz Manzano2, Beatriz Chamorro1, Exuperio Diez-Tejedor1 Celia Oreja-Guevara1, Susana Noval2, Juan Alvarez-Linera3, Laura Gabaldo´ n4, Beatriz Manzano2, Beatriz Chamorro1, Exuperio Diez-Tejedor1 1 Neuroimmunology and Multiple Sclerosis Unit, Department of Neurology, Idipaz Health Research Institute, University Hospital La Paz, Madrid, Spain, 2 Neuro Ophthalmology Unit, Department of Ophthalmology, Idipaz Health Research Institute, University Hospital La Paz, Madrid, Spain, 3 Department of Radiology, Hospit Ruber Internacional, Madrid, Spain, 4 Department of Neurology, Hospital de Denia, Alicante, Spain PLoS ONE \| www.plosone.org Citation: Oreja-Guevara C, Noval S, Alvarez-Linera J, Gabaldo´n L, Manzano B, et al. (2012) Clinically Isolated Syndromes Suggestive of Multiple Sclerosis: An Optical Coherence Tomography Study. PLoS ONE 7(3): e33907. doi:10.1371/journal.pone.0033907 Editor: Pablo Villoslada, Institute Biomedical Research August Pi Sunyer (IDIBAPS) - Hospital Clinic of Barcelona, Spain Received November 4, 2011; Accepted February 20, 2012; Published March 20, 2012 Copyright: 2012 Oreja-Guevara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: The authors have no support or funding to report. Competing Interests: The authors have declared that no competing interests exist. * E-mail: orejacbn@gmail.com Abstract Background: Optical coherence tomography (OCT) is a simple, high-resolution technique to quantify the thickness of retinal nerve fiber layer (RNFL), which provides an indirect measurement of axonal damage in multiple sclerosis (MS). This study aimed to evaluate RNFL thickness in patients at presentation with clinically isolated syndromes (CIS) suggestive of MS. Methodology: This was a cross-sectional study. Twenty-four patients with CIS suggestive of MS (8 optic neuritis [ON], 6 spinal cord syndromes, 5 brainstem symptoms and 5 with sensory and other syndromes) were prospectively studied. The main outcome evaluated was RNFL thickness at CIS onset. Secondary objectives were to study the relationship between RNFL thickness and MRI criteria for disease dissemination in space (DIS) as well as the presence of oligoclonal bands in the cerebrospinal fluid. Principal Findings: Thirteen patients had decreased RNFL thickness in at least one quadrant. Mean RNFL thickness was 101.67610.72 mm in retrobulbar ON eyes and 96.93610.54 in unaffected eyes. Three of the 6 patients with myelitis had at least one abnormal quadrant in one of the two eyes. Eight CIS patients fulfilled DIS MRI criteria. The presence of at least one quadrant of an optic nerve with a RNFL thickness at a P,5% cut-off value had a sensitivity of 75% and a specificity of 56% for predicting DIS MRI. Conclusions: The findings from this study show that axonal damage measured by OCT is present in any type of CIS; even in myelitis forms, not only in ON as seen up to now. OCT can detect axonal damage in very early stages of disease and seems to have high sensitivity and moderate specificity for predicting DIS MRI. Studies with prospective long-term follow-up would be needed to establish the prognostic value of baseline OCT findings. Received November 4, 2011; Accepted February 20, 2012; Published March 20, 2012 a-Guevara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits tion, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Oreja-Guevara et al. This is an open-access article distributed under the terms of the Creative Commons At unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: 2012 Oreja-Guevara et al. MRI CIS patients were divided into two groups according to their clinical onset: CIS onset with ON (CIS-ON) and CIS onset without ON (CIS-nON). In CIS-ON patients, the eye which had suffered ON was defined as affected eye and was studied independently from the fellow eye. Left eyes of CIS-nON patients and fellow eyes of CIS-ON patients were grouped as unaffected eyes to include only one eye of each patient and therefore, avoiding design bias. The following MRI sequences of the brain were acquired using a 3 Tesla scanner (GE, USA): a) dual-echo axial variable echo fast spin echo (FSE): repetition time (TR) = 2940 ms; first echo time (TE) = 10 ms; second TE = 110 ms; echo train length (ETL) = 16, FOV = 24, 44 slices, slice thickness = 3.0 mm, interleaved slices, matrix = 2566256), b) axial 3D T1 Inversion recovery (IR) Prep: first TE = 10 ms; T1 = 750 ms; TR = 10 ms, flip angle = 12, FOV = 24, slice thickness = 1 mm, 24 slices, matrix = 2406240), c) axial fast fluid attenuated inversion recovery (FLAIR): TR = 9000, TE = 95, T1 = 2200; 3-mm-thick slices, FOV = 24, d) pre- and post-contrast T1-weighted conventional spin-echo (TR = 680 ms; TE = 14 ms, FOV = 24, slice thickness = 3.0, interleaved), 5 minutes after the intravenous administration of 0.1 mmol/kg gadopentetate dimeglumine. Statistical analysis was performed using the SPSS 12.0 program for Windows (SPSS Inc., Chicago, IL, USA). Visual acuity (VA) was measured in Snellen and expressed as a decimal value, but it was transformed to LogMAR system to be analyzed. VA was considered normal when it was of 1 expressed by decimal scale or 0 by LogMAR system. The Shapiro-Wilk test was used to verify if the distribution of variables values fit the normal distribution. Nonparametric tests were applied since most variables were not normally distributed. The Wilcoxon test was used to analyze differences between the eyes of each patient. A Mann-Whitney test was used for independent samples. The total level of significance was set at 0.05. Patients All consecutive patients with a single episode of CIS attending the Neurology Department of our center during 2008 were invited to participate in this study. OCT scanning was performed within the first month from the onset of the clinical episode with the Stratus OCT (Carl Zeiss Meditec, Dublin, CA) after induction of pharmacological mydriasis. Image acquisition was performed with the Fast Retinal Nerve Fiber Layer (RNFL) Thickness (3.46) and RNFL thickness values (measured in micrometers [mm]) were obtained using the RNFL Thickness Average Analysis protocol. The Stratus OCT built-in software compares a color-coded graph that displays the RNFL measurements with the age-matched data of a normalized database. Assuming a normal distribution of the RNFL, the thickest 5% of measurements are colored white (white .95%) and the thinnest 1% of measurements fall in the red area. Measurements in red are considered outside normal limits (red ,1%, outside normal limits). Five percent of measurements fall in the yellow area or below (1%# yellow ,5%, suspect) and 90% of measurements fall in the green area (5%# green #95%). Thickness values falling into the green area are considered normal. The yellow area marks thickness values that are 5% or less than all thickness values measured in the normative database. Thickness measurements in the red area are considered pathologic. The diagnosis of ON was based on clinical criteria, including visual loss in the affected eye, relative afferent pupillary defect, a visual field defect and pain that increases with eye movement [29]. Exclusion criteria included the presence of an ocular pathology other than ON, intraocular pressure higher than 21 mmHg and a refractive error greater than 5.0 diopters (D) of spherical equivalent or 3.0 D of astigmatism in either eye. CIS with a clinical onset other than an ON were diagnosed on the basis of clinical criteria and, when needed, confirmed by the presence of lesions on brain or spinal cord MRI. Appropriate investigations were carried out as necessary to exclude alternative diagnoses, and all patients were carefully interviewed for a previous demyelinating event. Patients treated with corticosteroids for relapses before OCT and lumbar puncture examination were not included. All patients underwent a complete neurological examination (within 7 days from symptoms onset) and neurological disability was measured using the Expanded Disability Status Scale (EDSS) [30]. Introduction the development of clinical disability over time, biomarkers of early axonal degeneration in patients at presentation with CIS would therefore be of great interest. A clinically isolated syndrome (CIS) involving the optic nerve, spinal cord, brainstem or other portions of the brain, is the most frequent initial presentation of multiple sclerosis (MS). Several studies have tried to identify the risk factors associated with the development of clinically definite MS (CDMS) after a first acute demyelinating attack [1–11]. The presence and extent of lesions on baseline magnetic resonance imaging (MRI) of the brain is strongly related to the probability of developing MS [1,2,5,9,10]. In addition, due to its high sensitivity in detecting disease-related abnormalities, MRI has been formally included in the diagnostic work-up of MS patients, to exclude alternative diagnoses and aid in the demonstration of disease dissemination in space (DIS) and time (DIT), which are the main criteria for a diagnosis of CDMS [12,13]. P [14 15] d MRI [16 18] di h d Monitoring axonal loss has become a priority in MS. It has recently demonstrated that analysis of the retinal nerve fiber layer (RNFL) thickness may be useful to detect degenerative process in the central nervous system [19,20]. Optical coherence tomography (OCT) has emerged as a simple accurate noninvasive technique that can be used to measure RNFL thickness. Several studies have consistently demonstrated that OCT is a useful tool to detect retinal axonal loss following an acute episode of optic neuritis (ON), a very common, often initial symptom of MS [21–25]. In addition, this technique can help to detect subclinical axonal loss, as suggested by the demonstration of a decreased RFNL thickness in fellow ON eyes or reduced RFNL thickness in MS patients without a history of ON [26–28]. ( ) g [ ] Post-mortem [14,15] and MRI [16–18] studies have demon- strated early axonal loss in MS patients. Since axonal damage is a key contributor to the clinical manifestations of the disease and to The aim of this study was to measure RNFL thickness in patients at presentation with CIS suggestive of MS and to PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33907 1 March 2012 \| Volume 7 \| Issue 3 \| e33907 Optical Coherence Tomography in CIS the fulfillment of IP criteria for DIS [31]. Introduction According to the original IP criteria, the presence of two brain MRI lesions consistent with MS together with positive oligoclonal bands (OCB) was also considered as DIS. investigate the relationship between RNFL thickness abnormalities and clinical manifestations at disease onset, MRI features according to the International Panel (IP) criteria for DIS [14] and cerebrospinal fluid (CSF) profile. Ophtalmologic evaluation All study patients underwent a complete ophthalmologic evaluation, including best corrected Snellen visual acuity (VA), biomicroscopy of the anterior and posterior segments, funduscopy, intraocular pressure measurement and Humphrey visual field testing, using the Swedish Interactive Threshold Algorithm standard 24-2 strategy (Carl Zeiss Meditec, Dublin, CA). Affection of the visual field was defined according to the Optic Neuritis Trial criteria [32] and quantified using mean deviation (MD) (expressed in decibels [dB]). Ethics statement The study was approved by the local Ethics Committee (CEIC Hospital La Paz) and written informed consent was obtained from all subjects. The study complies with the guidelines of the Declaration of Helsinki. MRI The following sequences were also acquired for cervical cord: a) sagittal T2 FSE (TE = 99, TR = 3000 ms; ETL = 24; 4-mm-thick slices with an interslice gap of 0.4 mm), b) sagittal T1 FSE (TE = 22, TR = 575; ETL = 3; 4-mm-thick slices with an interslice gap of 0.4 mm), c) sagittal PD-weighted (TE = 38, TR = 2000; TE = 38; ETL = 8; 4-mm-thick slices with an interslice gap of 0.4 mm) and an oblique two-dimensional GE (TR = 300; FOV = 20, 4-mm-thick slices with an interslice gap of 1 mm) was a post-contrast T1w scan acquired for the cord. Patients Within the first two months from the attack, all the subjects underwent lumbar puncture (whenever possible) and brain and spinal cord MRI. Sensitivity and specificity were calculated for RNFL thickness for the quadrant sectors at p,5% and p,1% cut-off values. PLoS ONE \| www.plosone.org Discussion In patients diagnosed with MS it has been demonstrated that axonal loss occurs in the early stages of the disease. Therefore, a large effort has been focused on early detection of patients with high risk of developing MS at the first CIS in order to start early immunomodulatory treatment to reduce the accumulation of irreversible axonal loss. RNFL thickness analysis provided the following results: 54.2% of all patients and 56.3% of the CIS-nON patients presented at least one quadrant of an optic nerve with a decreased RNFL thickness. Mean RNFL thickness was 142.13 mm (SD 13.56) in anterior ON eyes and it was 101.67 mm (SD 10.72) in retrobulbar ON eyes (p = 0.05). Mean RNFL thickness was 96.93 (SD 10.54) in unaffected eyes of CIS-nON patients and ON patients. OCT has demonstrated to be a useful tool to detect axonal loss as a thinning of the RNFL following an initial episode of ON. To our knowledge only one study have addressed the use of OCT on patients with other types of CIS different from ON, like myelitis or brainstem syndromes [21]. RNFL thickness increase in acute anterior ON and progressive axonal loss in anterior and retrobulbar ON has already been demonstrated [21,33]. Mean Table 1 shows mean RNFL thickness and the color assigned by the age-normalized Stratus-OCT database for each group of eyes. Table 1 shows mean RNFL thickness and the color assigned by the age-normalized Stratus-OCT database for each group of eyes. Three of the 6 patients with myelitis had at least one abnormal quadrant in one of the two eyes. Three of the 8 (37.5%) ON Three of the 6 patients with myelitis had at least one abnormal quadrant in one of the two eyes. Three of the 8 (37.5%) ON Table 1. Mean RNFL thickness and its color assigned by the normalized Stratus-OCT database according to age and proportion of eyes with at least one quadrant P,5% and P,1% cut-off values. Optical Coherence Tomography in CIS Optical Coherence Tomography in CIS affected eyes and 6 of 24 (25%) unaffected eyes presented at least one quadrant with a thickness at a p,5% cut-off value. Three of 24 (12.5%) unaffected eyes presented at least one quadrant with a thickness at a p,1% cut-off value. When we analyzed the results by patients, 13 (54.2%) and 7 (29.2%) patients had at least one quadrant with a thickness at a p,5% and a p,1% cut-off values, respectively. (21%) with sensory and other syndromes. There were thirteen (54%) women and 11 (46%) men, with a median age of 38 years (range 19–57 years). Their EDSS score ranged from 0.0 to 4.0, with a median of 1.0. Lumbar puncture was performed in 21 patients because three of them refused. Oligoclonal IgG bands were found in 12 (57.1%) patients and the IgG index was increased in 10 (47.6%) patients. p y Crosstabulation between OCT measures and MRI criteria and alternative criteria (at least two MRI lesions and OCB presence) for DIS are shown in Table 2. Eight CIS patients fulfilled DIS MRI criteria and 9 patients met alternative criteria for DIS based on MRI and OCB presence, Six out of 8 patients who fulfilled MRI criteria for DIS presented at least one quadrant with a RNFL thickness of less than 5% than all thickness values measured in the normative database. The presence of at least one quadrant of an optic nerve with a RNFL thickness at a p,5% cut-off value had a sensitivity of 75% and a specificity of 56% for predicting DIS according to the MRI Barkhof criteria. Specificity increased to 81% whereas sensitivity decreased to 50% when the cut-off value was set at p,1%. Sensitivity decreased to 67% at the p,5% cut- off value and to 33% at the p,1% cut-off value according to MIR criteria and OCB presence. Specificity was 58% at the p,5% cut- off value and increased to 67% at the p,1% cut-off value. Table 3 shows the sensibility and specificity for OCT findings according to 3/4 Barkhof criteria and alternative criteria based on MIR evidence and OCB presence. Eight patients (33%) fulfilled IP criteria for DIS, whereas 8 patients had normal brain MRI scans. Cervical MRI was pathological in all 6 patients with spinal cord CIS and in two patients with DIS. Optical Coherence Tomography in CIS Nine of the patients who underwent CSF analysis (37.5%) showed two or more MRI lesions consistent with MS and positive oligoclonal bands. Two CIS-ON patients had an anterior and 6 a retrobulbar form of ON, defined by the presence or absence of edema of the optic nerve head respectively. In the 8 patients with ON-affected eyes, mean VA was 0.36 (SD 0.31) and all presented affected visual fields, with a MD of 214.62 dB (SD 7.82). All the fellow eyes of CIS-ON patients had normal VA (p = 0.03) and visual fields were affected in 5 out of 8 (62.5%), with a MD of 22.22 dB (SD 2.31) (p = 0.01). Visual acuity was normal in 21 out of 24 (87.5%) of unaffected eyes; it was 0.8 in two and 0.5 in one eye. Eight (33.3%) unaffected eyes of CIS-nON patients and ON patients had abnormal visual fields, with MD of 22.64 dB (SD 4.82). Visual acuity was normal in 21 out of 24 (87.5%) of unaffected eyes; it was 0.8 in two and 0.5 in one eye. Five right eyes and 3 left eyes had abnormal visual fields, with a MD of 22.22 dB (SD 2.31) and 22.35 dB (SD 4.81), respectively. March 2012 \| Volume 7 \| Issue 3 \| e33907 Results Twenty-four patients with CIS were recruited, including 8 (33%) patients with unilateral ON, 6 (25%) with spinal cord syndrome (myelitis), 5 (21%) with brainstem symptoms and 5 Brain and cord MRI scans were assessed by two radiologists blinded to neurological and ophthalmological examinations and March 2012 \| Volume 7 \| Issue 3 \| e33907 March 2012 \| Volume 7 \| Issue 3 \| e33907 2 Discussion To our knowledge, this is the first study to demonstrate early retinal axonal layer thinning detected by OCT in patients who have suffered from any type of CIS, including patients with isolated myelitis. As expected, this thinning was mild but identifiable by the software of the Stratus-OCT. At the moment, the presence and extent of demyelinating lesions in the MRI of patients with CIS is the main predictor of conversion to CDMS. Retinal axonal thinning present at the time of CIS presentation might be considered a signal of wider subclinical axonal damage and early neurodegeneration. Since the current study was conducted within the first two months of CIS presentation, we could only consider OCT and MRI pathological findings as measures of dissemination in space. Our results are in contrast to the only available study using OCT on CIS patients [41] that did not revealed retinal axonal loss at the earliest clinical stage of MS and did not predict conversion to MS at 6 months. Nevertheless, our study cannot be directly compared with the abovementioned study due to a substantial difference in disease duration. In this previous study, patients had a disease duration up to one year whereas our study population consisted of CIS patients who underwent OCT in the first month and MRI and lumbar puncture in the first two months. Additionally, the study population was very different to our CIS group since 23% of the patients had clinically definite MS and 71% presented dissemination in space according to the revised McDonald criteria. Nevertheless, it was shown that 25% of the patients had atrophy in at least one quadrant and 4 patients in two quadrants, which reflects a slight axonal damage. In our study, mean RNFL thickness was 101.67 mm and 97.44 mm in affected and fellow non affected eyes of the CIS-ON patients respectively. The increased thickness obtained in affected eyes could be explained by the presence of two anterior forms since RNFL thickness increases in optic nerve edema [37,38]. Mean RNFL thickness was 92.58 and 97.44 mm in right CIS-nON eyes and fellow ON-eyes respectively, suggesting subclinical retinal axonal damage in CIS patients that occurs in the absence of optic ON. The findings are consistent with those of previous studies that described retinal axonal loss in patients with MS without previous ON history when compared with a healthy control population [27,28,39]. Discussion CIS-ON patients CIS-nON patients Affected eyes Fellow eyes Right eyes Left eyes (N = 8) (N = 8) (N = 16) (N = 16) Mean RFNL thickness ± SD (mm) 101.67610.72 97.44613.53 92.5869.49 96.6769.21 RFNL thickness color, n (%) White 0 (0) 0 (0) 0 (0) 0 (0) Green 6 (75) 7 (87.5) 13 (81.3) 15 (93.8) Yellow 0 (0) 1 (12.5) 2 (12.5) 1 (6.3) Red 0 (0) 0 (0) 1 (6.3) 0 (0) At least one quadrant at p,5% and p,1% cut-off values, n (%) $1 quadrant p,5% 3 (37.5) 3 (37.5) 9 (56,3) 3 (18.8) $1 quadrant P,5% 0 2 (25) 5 (31.3) 1 (6.3) CIS: Clinically isolated syndrome; N: Number of eyes; ON: optic neuritis; RNFL: retinal nerve fiber layer. doi:10.1371/journal.pone.0033907.t001 d its color assigned by the normalized Stratus-OCT database according to age and proportion of ,5% and P,1% cut-off values. March 2012 \| Volume 7 \| Issue 3 \| e33907 PLoS ONE \| www.plosone.org 3 Optical Coherence Tomography in CIS Table 2. Crosstabulation between the two different criteria of spatial dissemination applied and OCT findings. Table 2. Crosstabulation between the two different criteria of spatial dissemination applied and OCT findings. Quadrants ,5% Quadrants ,1% DIS MRI criteria None $1 None $1 Total Fulfilled 2 6 4 4 8 Not fulfilled 9 7 13 3 16 Total 11 13 17 7 24 Alternative criteria Quadrants ,5% Quadrants ,1% (OCB+ at least two lesions in MRI) None $1 None $1 Total Fulfilled 3 6 6 3 9 Not fulfilled 5 7 8 4 12 Total 8 13 14 7 21 DIS: dissemination in space; OCB: oligoclonal bands. doi:10.1371/journal.pone.0033907.t002 DIS: dissemination in space; OCB: oligoclonal bands. doi:10.1371/journal.pone.0033907.t002 transfer, axonal damage has been demonstrated in patients presenting with CIS at baseline [17,18,40]. RNFL thickness in the fellow non-affected eyes has ranged between 92.9 mm (SD 11.4) to 99.8 mm (SD 32.50) in studies focused on patients with isolated ON [23,24,34]. Statistical significant differences have not been found when compared non- affected eyes with control eyes, despite that their RNFL thickness mean values were above 100 mm. [25,34–36]. Most studies have been unable to demonstrate a significant thinning of the RNFL of the fellow eyes when compared to healthy controls. March 2012 \| Volume 7 \| Issue 3 \| e33907 Discussion When we analyzed the color-code ordinal scale of RNFL thickness, it was found that only 4 out of 48 eyes had a mean RNFL thickness below the 5% cut-off value. This finding is consistent with previous absolute values in control groups [25,34– 36]. However, in our study, 18 out of 48 eyes had at least one quadrant in which RNFL thickness was below the 5% cut-off values and RNFL thickness was below the 1% cut-off values in at least one quadrant in 8 out of 48 eyes.These results suggest a mild axonal loss present in CIS even within the first week after its presentation. In brain studies, performed with proton magnetic resonance spectroscopy, diffusion tensor MRI and magnetization g g Six out of 8 patients who fulfilled DIS MRI criteria presented at least one quadrant with less than 5% probability of normal RNFL thickness. This provides a sensitivity of 75%, which decreased to 50% when a higher probability of real RNFL atrophy is considered. However, when the cut-off value is set at p,1%, specificity increased to 81%, since 13 out 16 patients who did not fulfill DIS MRI criteria did not present any red-colored quadrant either. These findings reflect that OCT seems to have high sensitivity and moderate specificity for predicting DIS MRI. The sensitivity of DIS MRI criteria was higher than the criteria based on the finding of OCB in CSF plus two MRI lesions for DIS. However, this alternative criterion based on OCB detection yielded a slightly higher specificity than DIS-MRI at p,5% cut off value. These data therefore reinforce the role of CSF study in MS diagnosis. Table 3. Sensibility and specificity for OCT findings according to MIR Barkhof criteria and MIR and OCB criteria for DIS. Table 3. Sensibility and specificity for OCT findings according to MIR Barkhof criteria and MIR and OCB criteria for DIS. Table 3. Sensibility and specificity for OCT findings according to MIR Barkhof criteria and MIR and OCB criteria for DIS. L Barkhof MIR criteria MIR and OCB criteria ,5% ,1% ,5% ,1% Sensitivity (%) 75 50% 66.67% 33.33% Specificity (%) 56.25% 81.25% 58.33% 66.67% doi:10.1371/journal.pone.0033907.t003 March 2012 \| Volume 7 \| Issue 3 \| e33907 4 Optical Coherence Tomography in CIS In our series, 57% of CIS patients presented positive OCB. This result is in line with the proportions of CIS patients with positive OCB reported in previous studies. References 1. Optic neuritis study group (2008) Multiple sclerosis risk after optic neuritis: final optic neuritis treatment trial follow-up. Arch Neurol 65: 727–732. 16. Filippi M, Bozzali M, Rovaris M, Gonen O, Kesavadas C, et al. (2003) Evidence for widespread axonal damage at the earliest clinical stage of multiple sclerosis. Brain 126: 433–437. 2. Brex PA, Miszkiel KA, O’Riordan JI, Plant GT, Moseley IF, et al. (2001) Assessing the risk of early multiple sclerosis in patients with clinically isolated syndromes: the role of a follow up MRI. 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Acknowledgments We thank Mara Rocca and Massimo Filippi for their critical review of the manuscript. We are grateful to Javier Galvez for his technical support. Discussion Tintore et al. [11] found that 61% of patients had positive OCB, considering that 41% of those with normal MRI did not undergo a lumbar puncture. Masjuan et al. [7] reported that 63% of patients were positive for OCB (7 out of 52 presented a normal scan and 7 had negative OCB) and Rojas et al. [42] detected positive OCB in 53% of CIS patients. would be needed to establish the prognostic value of baseline OCT findings in patients with CIS, when dissemination in time and CDMS diagnosis could be determined. In conclusion, axonal damage measured by OCT is present in any type of CIS, even in myelitis forms, not only in ON as seen up now. Although OCT has shown to detect axonal damage in very early stages of disease, its capacity to predict conversion in CDMS has not yet been demonstrated. Additional longitudinal studies would establish its role as prognostic marker of neurological disability. Thus, OCT could represent a potential tool that could be used to detect and monitor axonal protective effects of new neuroprotective therapies. The present study is limited to a relatively small sample size. A larger sample was not achieved probably due to the fact that patients were included in the study within the first 4 weeks after the first event, and additionally, CSF and OCT had to be available before the initiation of treatment with corticosteroids in order to avoid false negative results owing to the effect of treatment. However, patients usually receive treatment before OCT and CSF analyses are performed when they arrive at the emergency room and the time elapsed until both examinations are performed is generally 6 weeks in routine clinical practice. In our study, patients underwent OCT, MRI and CSF examination within the two first months after the first event. Author Contributions Conceived and designed the experiments: COG SN JAL. Performed the experiments: COG SN JAL LG BM BC. Analyzed the data: COG SN JAL EDT. Contributed reagents/materials/analysis tools: COG SN JAL. Wrote the paper: COG SN JAL EDT. In the view of evidence, the relationship between RNFL thinning in CIS and progression to MS is still unclear, since only long-term follow-up will determine if these changes are clinically relevant. Therefore, prospective studies with long-term follow-up Optical Coherence Tomography in CIS Optical Coherence Tomography in CIS 31. Barkhof F, Filippi M, Miller DH, Scheltens P, Campi A, et al. (1997) Comparison of MRI criteria at first presentation to predict conversion to clinically definite multiple sclerosis. Brain 120: 2059–2069. 37. Savini G, Bellusci C, Carbonelli M, Zanini M, Carelli V, et al. (2006) Detection and quantification of retinal nerve fiber layer thickness in optic disc edema using stratus OCT. 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Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, et al. (2005) Diagnostic criteria for multiple sclerosis: 2005 revisions to the ‘‘McDonald Criteria’’. Ann Neurol 58: 840–846. 10.1002/ana.20703 [doi]. 29. Smith CH (2005) Optic neuritis. In: Miller NRNN, ed. Walhs & Hoyt Clinical Neuro-ophthalmology. Philadelphia, USA: Lipincott Williams & Willians. pp 293–347. 15. Trapp BD, Peterson J, Ransohoff RM, Rudick R, Mork S, et al. (1998) Axonal transection in the lesions of multiple sclerosis. N Engl J Med 338: 278–285. 10.1056/NEJM199801293380502 [doi]. 30. Kurtzke JF (1983) Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology 33: 1444–1452. PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33907 5 March 2012 \| Volume 7 \| Issue 3 \| e33907 Optical Coherence Tomography in CIS Optical Coherence Tomography in CIS PLoS ONE \| www.plosone.org March 2012 \| Volume 7 \| Issue 3 \| e33907 6
https://openalex.org/W314319712	https://inria.hal.science/hal-01342151/document	English	null	Data Mining Challenges in the Management of Aviation Safety	IFIP advances in information and communication technology	2,014	cc-by	5,410	To cite this version: Olli Sjöblom. Data Mining Challenges in the Management of Aviation Safety. 13th Conference on e-Business, e-Services and e-Society (I3E), Nov 2014, Sanya, China. pp.213-223, 10.1007/978-3-662- 45526-5_21. hal-01342151 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-01342151 https://inria.hal.science/hal-01342151v1 Submitted on 5 Jul 2016 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License 1 Introduction Organisational decision making, especially in safety-critical systems, such as nuclear power and air traffic, is a complicated task. For successful operations, an acceptable air safety record has been required from the airline [1]. Air traffic has generally been forecasted to grow 5 – 6 % annually over the next two decades [2], or even over the next 10 – 15 years, the global air travel will probably double [3]. Consequently, the number of accidents will respectively increase if nothing were done to improve it, which development would, clearly, be unacceptable. This is why new and efficient ways for improving air safety need to be explored [4]. The conventional safety tools and methods based on data collection have reached their peak performance because of their inability to create new knowledge. Usually, data accumulates faster than it can be processed [5]. For further improvements new methods and tools are urgently need- ed [6]. Turku University School of Economics, Turku, Finland oljusj@utu.fi Turku University School of Economics, Turku, Finland oljusj@utu.fi Abstract. This paper introduces aviation safety data analysis as an important application area for data mining. Safety is a key strategic management concern for safety-critical industries and management needs new, more efficient tools and methods for more effective management routines. The aviation field is con- fronted with increasing challenges to provide safe and fluent services. Air travel has grown steadily during the last decades with a direct impact on the air traffic control. At the same time, the competition has become tougher because of in- creasing fuel prices and growing demand for air travel. Keywords: Management, Flight Safety, Strategic Management, Data Mining, Text Mining, Analysis Method Keywords: Management, Flight Safety, Strategic Management, Data Mining, Text Mining, Analysis Method Data Mining Challenges in the Management of Aviation Safety Olli Sjöblom Turku University School of Economics, Turku, Finland oljusj@utu.fi 2 Management in safety-related context Any system can be recognised to consist of elements, or factors, or parts that make up the whole [7]. Managing the organisation is exercised largely through management processes, in which the means of managerial communication inter-links with the envi- ronment. Johnsen (2002) defines the management process as “the interaction between people who want to attain mutual ends through mutual means.” [8]. The strategy of the corporation is according to Johnson et al. [9] to concern the organisation’s mis- sion, vision and objectives, developing plans and policies to use resources for enhanc- ing the performance of the organisation. Kettunen et al. [10] emphasise the managerial challenges in the safety-critical in- dustries, which are typically related to finding a balance between diverging demands and expectations, like economy- and safety-related objects without forgetting the priorities-setting and maintaining focus on these components. The key action is a continuous balancing between taking risks and allocating resources for risk manage- ment. A scale with theoretical ends can be displayed, where at one end there is a situation where risks do not exist because the resources allocated are infinite; at the other end no resources are allocated because the risks are ignored and thus they are (practically) infinite. The reality is found somewhere in between, but no fixed loca- tion can be defined because all environments are somewhat unique and are also changing all the time. In daily operations perhaps existing hidden threats produce the need to maintain extra safety level naturally causing additional costs. In studying risk management, the concept of tension cannot be ignored. It refers to the challenges of balancing conflicting objectives or expectations, like safety and other goals. These might exist for various reasons, even in the situation in which the executives of the organisation have set a high safety level as the priority official goal [11].In case warning signals appear, responding to those should happen without delay allocating safety resources to the critical area. The safety decisions in an air traffic company follow the same pattern as other stra- tegic decisions. Risk management should be carried out in parallel with safety man- agement, referring to measures seeking to identify, assess and control risks on the organisational level having the goal to ensure the organisational and environmental safety. The executive management is responsible for recognising the safety signifi- cance of the ways the organisation is operated and maintained [12]. 2 Management in safety-related context Managing risk and safety has been problematic in air transport: very high levels of safety are too costly – high levels of risk are unacceptable. Therefore, safety reports have been col- lected through decades to investigate and assess risks and to define risk standards, which are consistent with the value systems of the society [13, 14]. The value of safety cannot be estimated in any traditional way, because it has no determined price. Theoretically, limitless resources should be allocated to it, because one single failure may lead to significant losses in the form of missed business possi- bilities and claims for covering the damage caused to a third party. Kaplanski and Haim [15] have presented some estimates for the accident costs. A very large disaster with hundreds of casualties will cause a loss of about $1 billion for an airline com- pany. However, the observed market effect has been found to be about 60 times lar- ger; Kaplanski and Haim (2010) have found the evidence of a significant negative effect with an average market loss of more than $60 billion per aviation disaster. However, budget constraints set limits in practise and therefore a certain risk has to be accepted by achieving a sufficient safety level. There is never a 0-level risk. In case sufficient resources could not be allocated to achieve the required level of safety, the whole air traffic business would be critical. When confronting such a situation, the operations are to be adjusted by diminishing or changing them to correspond with the allocable safety resources so that a sufficient safety level is maintained. Estimating the significance and importance of different alternatives in managing risks also needs tools, the exact definition of which is important for making strategic decisions. After the executive management has set goals as the thresholds of achievement, there must be methods and models to measure to what degree the achievements have been realised. In the decision process, there is always question about evaluating different alternatives. Any matter having significance enough to be taken into account in the evaluation process should be considered for evaluation [16]. Rumsfeld [17] has defined (simply expressed) three categories for knowledge: first, we know what we do know; second, we know what we do not know; and, finally, we do not know what we do not know. 2 Management in safety-related context The hidden dangers belong to the last group, so in case we know what we are searching for, we obviously have means to reach it, but otherwise we need tools for finding something we do not know we are looking for. Thus, a deeper understanding is required for developing better methods and refining rules and practices that will contribute to higher levels of safety. The unknown lethal factors brought into daylight could be eliminated; at least a significant part of them and a sufficient safety level could be reached with reduced investment allocation. For air traffic, there is theoretically no upper limit to allocate resources to safety in different forms. The relation between safety and cost efficiency could be illustrated explicitly comparing the costs between comprehensive mainte- nance programs and maintenance-induced accidents, the benefits that outweigh the accident costs [18]. The process for allocating extra resources to special projects might become even more troublesome in case there are interdependencies among the projects [16]. 3 Flight Safety According to the ICAO Safety Management manual [19], safety is defined as “a state in which the risk of harm to persons or property damage is reduced to, and main- tained at or below, an acceptable level through a continuing process of hazard identi- fication and risk management“. Safety is not a matter-of-course, but the result of a rather complicated, carefully structured and comprehensive management process approaching to all airline safety aspects, particularly those of flight operations. Air traffic is full of incidents and deviations that do not contain any hazard as such, but need to be reported and investigated to find out potential lethal trends. These un- desirable, but very minor events are valuable investigation subjects for risk and safety specialists to build an understanding about their causes and to detect unsafe trends. Investigation also reveals whether countermeasures are warranted and how to reduce or eliminate potential accidents [20]. The appearance of similar recurring cases (a cluster, cf. Chapter 6) may indicate a hazardous trend that should be analysed very carefully to find out whether a real danger exists or not. The possibly existing lethal trends are trying to penetrate through the layers of defences, barriers and safeguards (cf. Figure 1) that, fortunately, usually stop them from proceeding. Because serious incidents and even accidents do happen, it can be presumed that after a certain amount of time they pass all the layers but the last one; then they will pass the last layer as well, which leads to accidents. Finding trends from flight safety data, especially from narrative data has required significant human involvement. Thus, the analysis process and its possible results rely on the skill, memory and experience of the safety officers [21]. Watson [22] found that with conventional techniques it might take years to find meaningful relationships. Before text mining systems (one sub-class of data mining) were developed, there were no tools for analysing textual data with computers. Data mining provides a worthy analysis method in order to illustrate the safety indicators and to reveal undesired trends. 4 Safety Tools and Systems Accident analysis as well as flight and operations modelling and simulation enhance the understanding of risk, but this is usually reactive and produces knowledge about causal factors potentially at the human and/or financial cost. Risk modelling typically collects knowledge resulting from flight safety analysis, human experience and theo- retical and empirical studies. The goal of aviation risk assessment is to be comprehen- sive, timely and proactive, and this is why the analysis methods should be enhanced [23]. In aviation, the quantitative assessment of risk is particularly challenging, because the deviation events are extremely rare and the causal factors are non-linearly related to the events which makes them difficult to quantify [23]. The eventuality for the incident or accident occurring may be markedly reduced in case the risks can be effi- ciently diagnosed [24]. Then the question is: how to find and identify deviations lead- ing to incidents and those leading to accidents? Reason [25] has modelled the process for the occurrence of accidents in his Swiss Cheese model, which is presented in Fig- ure 1. The hazards appear from the right-hand side. Normally, their progress is stopped by successive layers of defences, barriers and lifeguards. If the process goes through all of these ‘holes in the cheese slices’, formally called the limited windows of accident opportunities, an accident will happen. Kettunen et al. [10] regard redundancy as a method in improving safety by the du- plication and overlap of critical factors like systems, functions and/or personnel. In general, redundancy can augment safety as such, but may also have counter- productive or unexpected effects, especially in case it is not managed properly. These unwanted effects can increase the complexity of the systems, which may hide indi- vidual failures and make them latent, so that they remain unnoticed and uncorrected and may even accumulate over time. Under these circumstances, a rather rare event might act as a trigger for an avalanche of unexpected events, which may be difficult to handle [10]. For situations of this kind, the Reason’s Swiss Cheese model would work out excellently. Losses Hazards Holes due to active failures Other holes due Other holes due Figure. 1. The Swiss Cheese model (adapted from Reason 1997, 2000 [25, 26]) Figure. 1. The Swiss Cheese model (adapted from Reason 1997, 2000 [25, 26]) 6 Testing Three Tools - Data Mining in Finnish 6 The basic idea of cluster analysis is that all the texts within each cluster have a high similarity in content [31]. This method was chosen for this study because it is an es- sential mining function in searching for similar documents, able to reveal a recurring hazard that might lead to an accident. It explores the data set and determines the struc- ture of natural groupings without any preliminary assumptions. Another reason for its choice was the direct applicability to Reason’s Swiss Cheese model presented in Fig- ure 1. A third reason was that English literature gives several examples about using clustering in mining flight safety reports. These results have proved its better per- formance compared with more traditional statistical methods [32]. The beginning was finding text mining tools for processing Finnish. Three differ- ent systems seemed to be appropriate for benchmarking. The author was aware of one prototype (GILTA), one commercial product (TEMIS) with a Finnish module proto- type, and one commercial system (PolyVista) with encouraging results mining Span- ish, which seemed worth testing in Finnish. The Finnish Civil Aviation Authority granted the test data of 1240 cases (Target data on Figure 2), which created “a critical mass” for study. The pre-processing produced filtered data containing 10572 word tokens, numbers and special characters, call signs, headings, the temperature, etc. The amount could be reduced to 8294 when parentheses and other similar characters without relevance were removed. The next procedure was preparing the lists of stop words (those to be ignored because of having no information) and synonyms. No transformation was needed because the data was extracted from one database. The first round produced already promising results. Due to the Finnish module of TEMIS, no pre-processing was necessary. It created 26 clusters, their size varying between 108 and 21 reports. As the biggest cluster contained more than 100 clusters, the operator allowed the tool divide it into two sub-clusters with 58 and 50 docu- ments. After the division, the biggest cluster included 78 reports. The similarity (range 5-1) of the five closest clusters varied from 3.41 to 2.07 %, which supports the assumption that the clusters are different from each other and thus this method in this data selection is reliable. 5 Data Mining in Flight safety Several different methods are recognised as data mining methods and a mining sys- tem can use the combinations of several of these methods. Parsaye [27] describes data mining as searching in the data for the patterns of information to guide a decision support process. These, often called “the nuggets of knowledge”, are hidden in vast amounts of data and are practically undiscoverable with conventional techniques [22]. Using mining software, knowledge of data is combined by an analyst with advanced machine learning technologies to discover the relationships. In the discovery process to find hidden patterns, there are neither hypotheses nor any other predetermined model of the characteristics of the patterns. Obviously large databases, like those of aviation incidents and other deviations, contain a large number of patterns, so that the user of the discovery system can practically never ask the right question. The mining process acts as a decision support system that will not give straight answers to the questions; that is why skilled analytical and technical specialists are still required to interpret the created output [28]. The process contains several steps or phases (cf. Figure 2) that must be gone through to form knowledge from raw data. To be under- standable the information must be presented with reports, graphs or in other suitable forms once found. Figure. 2. The Knowledge Discovery in a Database Process (adapted from Fayyad et al. 1996 [29]) Figure. 2. The Knowledge Discovery in a Database Process (adapted from Fayyad et al. 1996 [29]) With structured data, the explanation of a case usually tells the truth to a certain ex- tent, but completed with narrative data it can be close to 100 %, at least theoretically. Mining combined with other methods will give significant contributions to the deci- sion processes. The idea to use text mining in the analysis of flight safety reports oc- curred along the need to analyse large amounts of narrative reports and when reports about successful text mining projects in the flight safety data analysis of English nar- ratives were published [21, 30]. 6 Testing Three Tools - Data Mining in Finnish Because the maximum degrees of explanation of the clus- ters, about 18 %, are relatively high, they prove that the clusters are composed of relevant reports and the most explaining reports alone might well reveal a trend that should be examined more thoroughly. As Kloptchenko [33] says, interpreting the mining results is more art and common sense than science. The one single mining round of TEMIS made the direct compari- son of the results challenging. Despite it, due to the high efficiency of the system with its in-built module for Finnish and because the mining results did not seem to require major changes, missing the second mining round was not considered a cause for los- ing significant information. The smallest clusters began to produce some directly applicable information indi- cating that the sizes of the clusters play a significant role in the applicability of the results. This must, however, be scaled with the amount of production data. Addition- ally, a couple of similar cases found do not automatically create a dangerous trend; the way they occur and the reasons causing them can only be estimated by a thorough examination and investigation by human analysts. The results of TEMIS ought to be examined differently from the two other systems due to its interface and way of pro- ducing results which differ remarkably from the others. This, however, does not mean that these mining results would not be coherent with those of the other ones. GILTA (manaGIng Large Text mAsses) divided the data on both rounds into 100 clusters (named classes) on the basis of the nine most significant words. Hence, on the first round 63 clusters contained less than 10 reports. These were easily analysable by a human analyst and could already be considered good mining results, proving clus- tering to be a useful method for this type of data. Some of the bigger classes could be interpreted as being real clusters, but according to experience the sizes should be re- duced to less than 20. The results that were produced in Excel-form made it possible to carry out a comprehensible analysis and comparison of them with the results found with other tools. The system left out four reports beyond defined clusters. PolyVista was originally built for using in English, but due to encouraging results with Spanish, its applicability for Finnish was tested, too. 6 Testing Three Tools - Data Mining in Finnish The system set score 100 for the most content describing word of the cluster and correspondent values to the others. The scores of the ten most important words of each cluster were only avail- able, not the reports. The reports of the clusters could be ‘guessed’ by comparing the scores with the most important words in GILTA changing their relative weights for comparison. The data was processed determining the number of clusters first to be 6 and then raising it up to 20 in a second step. When there were 20 clusters, the smallest of them contained 10 reports and the biggest 232. In the case of 20 clusters, in eleven of them the scores of the three most important words were more than 50. In the last cluster containing 10 reports, the scores of the 10 most important words were 50 or more, which can be considered a good mining result. As one result of the first mining round, the need for tuning, especially the defini- tion of stop words and synonyms was discovered. Some pure mistakes, like some common stop words and synonyms forgotten from the list, were noticed. A more sig- nificant problem was the appearance of some frequently used “common” words (like ‘plane’ with its synonyms ‘airplane’ and ‘aircraft’) skewing the results. Their role in the data was carefully analysed [34], using an application called NVivo to get a deep- er analysis. NVivo itself has no mining characteristics, but is used in analysing quali- tative information, especially meeting the requirements of deep levels of analyses on different quantities of data, varying between a couple of sentences and thousands of text rows. In this context, the most important feature was cross-examining the mining results applying its search engine and query functions. Almost one hundred checking procedures were made with synonyms and stop words to prepare the data for the sec- ond round. After the careful estimation of the impact of possible changes, no major ones were made to keep the process unchanged but making the results more accurate. After the second mining round with GILTA and PolyVista was performed, the re- sults were studied carefully using the professional skills of a flight safety inspector. The coherent clusters were taken into more detailed inspection. 6 Testing Three Tools - Data Mining in Finnish The progress as the change of distribution can be recognised through the increased percentage of ‘sense making’1 clusters, as for GILTA, illustrated in Table 1 displaying the minor, but per- ceptible change. First, the number of the relevant clusters increased from 9 to 11, and their average size diminished from 11.9 to 10.5 reports per cluster, shown in columns two and three. Further, the average weight of the nine most important words increased from 5.88 to 6.44 and the correspondent standard deviation diminished from 5.588 to 5.065, as shown in the two next columns. All these changes indicate the movement towards the aimed more homogenous clusters. Table 1. Results illustration in GILTA rounds I and II Table 1. Results illustration in GILTA rounds I and II Round Clusters Average size Average weight Correspondent Standard Deviation I 9 11.9 5.88 5.588 II 11 10.5 6.44 5.065 As already mentioned, the mining results of PolyVista must be analysed differ- ently. Although comparing the weights of the most significant words is a cursory method, it was noticed to be relevant in this context. The results are illustrated in Ta- ble 2 showing an obvious progress between the two rounds. On the first round, 40.0 % of the clusters seemed to belong to the ‘sense making’ clusters, on the second 52.3 %. The size of the clusters did not seem to have any linear impact, but on both rounds those were found among the smallest ones. The average sizes changed from the first round being 37.9 compared with 62.5 of all clusters to the second, being then 20.6 compared with 28.2. These numbers illustrate that more information is achieved from the results of round II. Table 2. Cluster distribution change between rounds 1 and 2 in PolyVista Table 2. Cluster distribution change between rounds 1 and 2 in PolyVista Criteria / Round # 1 2 ‘Sense making’ clusters of all content 40.0 % 52.3 % Average size of all clusters (reports) Average size of ‘sense making’ clusters 62.5 37.2 28.2 20.6 Average size of ‘sense making’ clusters Proceeding with the same test and putting the results in a graphic presentation in Excel, the increased homogeneity was seen also from the ‘centre of gravity’ moving from the beginning of the rows rightwards as well as from the top downwards, when the clusters were sorted by the weight of the most significant words. 1Clusters, from which information can be seen clearly as such 7 Results and Discussion As already expressed before, the mining process does not give straight answers to the questions, but it acts as a support system for producing information for decision mak- ing. That is why experienced analytical and technical specialists are needed to inter- pret the created output. The testing process proved that data mining is neither an easy nor a fast method, but might be the only one for uncovering hidden information. All the results support the premise that it could reveal important safety information from fast accumulating, vast amounts of data, not accessible with other methods, to be used as an essential factor for strategic safety management. It is worth noticing that the test data was that contained no lethal trends, but in other case they could have been dis- covered and revealed using the method and tools as done in this study. An additional detail is worth noting - all the used tools left out almost the same reports as outliers. g p The research process confirmed that text mining is a challenging task, especially in small language groups, where tools for text mining are scarcer than for big languages such as English which is an “easy” language for search technologies. Narrative text mining is generally demanding due to the multiplicity of languages spoken in the world. Especially languages with small user groups, such as Finnish, have to wait for efficient tools being developed much longer than the major languages. The search technologies are challenged by inflected forms and compounds. In Finnish, for exam- ple, the words may have thousands of inflected forms and in addition to that, they can be parts of compounds in almost countless combinations [35]. On average, every seventh word can be found in its basic form in fluent Finnish texts [36]. From the point of view of language processing, two significant results were achieved: first, Finnish texts were successfully mined with a tool originally to be used in an English environment. Secondly, the Finnish module for TEMIS was successfully production tested with real Finnish production data. The number of clusters proved to be significant in the process: the more clusters, the better results. Mining is an iterative process although it makes no sense to increase the amount of rounds too much. 6 Testing Three Tools - Data Mining in Finnish It means that the number of clusters having more significant words increased. This occurred with both systems, indicating a slight improvement using this method, too. Based on the professional skills and experience of the author, in case the safety personnel know what they are looking for, business intelligence (BI) methods could be applicable, allowing database queries using numerous keywords to search for known cases of a certain type or their combinations. BI could also be applied as a complementary method when mining is used to find something worth examining. References 1. Liou, J.J.H., L. Yen, and G.-H. Tzeng, Building an effective safety management system for airlines. Journal of Air Transport Management, 2008. 14(1): p. 20–26. 2. Netjasov, F. and M. Janic, A review of research on risk and safety modelling in civil avia- tion. Journal of Air Transport Management, 2008. 14(4): p. 213– 220. 3. Global Airline Industry Program. Analysis: The Airline Industry. Global Airline Industry Program [WWW-page] 2008 [cited 2011 9.5.]; Available from: http://web.mit.edu/airlines/analysis/analysis_airline_industry.html. 4. European Commission, Proposal for a DIRECTIVE OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL on occurrence reporting in civil aviation, Commission of the European Communities, Editor 2000: Brussels. 5. Wang, X., et al., LSSVM with Fuzzy Pre-processing Model Based Aero Engine Data Min- ing Technology, in Advanced Data Mining and Applications2007, Springer Berlin / Hei- delberg: Heidelberg. p. 100-109. 6. Evans, B., A.I. Glendon, and P.A. Creed, Development and initial validation of an Avia- tion Safety Climate Scale. Journal of Safety Research, 2007. 38(6): p. 675–682. 7. Barnard, C.I., The Functions of the Executive. Thirtieth Anniversary ed1938, Cambridge, MA and London, UK: Harvard University Press. 334. 8. Johnsen, E., Managing the Managerial Process. A Participative Process2002, Copenhagen: DJØF Publishing. 606. 9. Johnson, G., K. Scholes, and R. Whittington, Exploring Corporate Strategy2005: Pearson Education Limited. 10. Kettunen, J., T. Reiman, and B. Wahlström, Safety management challenges and tensions in the European nuclear power industry. Scandinavian Journal of Management, 2007. 23(4): p. 424-444. 11. Sagan, S.D., The limits of safety. Organizations, accidents, and nuclear weapons.1993, Princeton, NJ: Princeton University Press. 12. OECD/NEA, State-of-the-art report on systematic approaches to safety management, O.N.E. Agency, Editor 2006, OECD Nuclear Energy Agency: Issy-les-Moulineaux. 13. Janic, M., An assessment of risk and safety in civil aviation. Journal of Air Transport Management, 2000. 6(1): p. 43-50 14. . Sage, A.P. and E.B. White, Methodologies for Risk and Hazard Assessment: A Survey and Status Report. IEEE Transaction on Systems, Man, and Cybernetics, 1980. SMC- 10(8): p. 425-446 15. Kaplanski, G. and L. Haim, Sentiment and stock prices: The case of aviation disasters. Journal of Financial Economics, 2010. 95(2): p. 174-201. 16. Kirkwood, C.W., Strategic Decision Making1997, Belmont, CA: Wadsworth Publishing Company. 17. Rumsfeld, D.H., News Transcript, 2002, U.S. Department of Defense Office of the Assis- tant Secretary of Defense (Public Affairs). 18. Castro, R., A Holistic Approach to Aviation Safety, in Flight Safety Digest1988. p. 1-12. 19. 7 Results and Discussion Although this study has offered data mining as one solution to growing challenges, it is to be noticed that it is only one among several methods. Its special characteristic simply expressed is the ability to find something that is not known but expected to exist. Data mining has been used successfully for several years by a couple of airlines and other actors in the aviation industry. The process chain, beginning from the collection of safety data and ending in revised regulations for improving flight safety, going through several mining rounds and analyses to produce issued aviation rules and instructions, is rather long and demand- ing. Despite its complexity, it is worth going through, even for avoiding one single accident. References ICAO, Safety Management Manual, 2009, International Civil Aviation Organization: Montreal, Canada. p. 264. 20. Kirwan, B., Incident reduction and risk migration. Safety Science, 2011. 49(1): p. 11–20 21. Nazeri, Z., Application of Aviation Safety Data Mining Workbench at American Airlines. Proof-of-Concept Demonstration of Data and Text Mining., 2003, Center for Advanced Aviation Systems Development, MITRE Corporation Inc.: McLean, Virginia, US. 22. Watson, R.T., Data Management: Databases and Organizations. 2nd Edition ed, ed. J.W.S. 2nd Edition, 1999.1999: John Wiley & Sons. 23. Hadjimichael, M., A fuzzy expert system for aviation risk assessment. Expert Systems with Applications, 2009. 36(3): p. 6512–6519. 24. Lee, W.-K., Risk assessment modeling in aviation safety management. Journal of Air Transport Management, 2006. 12(5): p. 267–273. 25. Reason, J.T., Managing the Risks of Organizational Accidents1997, Aldershot: Ashgate Publishing Limited. 252. 26. Reason, J.T., Human error: models and management. British Medical Journal, 2000. 320(7237): p. 768-770. 27. Parsaye, K., A Characterization of Data Mining Technologies and Processes. Journal of Data Warehousing, 1997. 2(3): p. 2-15. g p 28. Kutais, B.G., ed. Focus on the Internet. 2006, Nova Science Publishers, Inc. 225. 29. Fayyad, U., G. Piatetsky-Shapiro, and P. Smyth, From Data Mining to Knowledge Discov- ery in Databases. AI Magazine, 1996. 17(3): p. 18. 30. Megaputer Intelligence. Flight safety data analysis for Southwest Airlines. 2004 [cited 2004 17 December]; Available from: http://www.megaputer.com/company/cases/ south- west.php3 31. Rosell, M., Text Clustering Exploration. Swedish Text Representation and Clustering Re- sults Unraveled, in School of Computer Science and Communication2009, Kungliga Tek- niska Högskolan: Stockholm. p. 71. 32. Saracoglu, R., K. Tütünkü, and N. Allahverdi, A new approach on search for similar doc- uments with multiple categories using fuzzy clustering. Expert Systems with Applications: An International Journal, 2008. 34(4): p. 2545-2554. 33. Kloptchenko, A., Text Mining Based on the Prototype Matching Method, in Turku Centre for Computer Science2003, Åbo Akademi University: Turku. p. 117 plus additional pages including original papers. 34. Lindén, K., Word Sense Discovery and Disambiguation, in General Linguistics2005, Uni- versity of Helsinki: Helsinki. p. 191. 35. Karlsson, F., Yleinen kielitiede1994, Helsinki: Yliopistopaino. 36. Karlsson, F., Finnish grammar1987, Porvoo: WSOY.
https://openalex.org/W3095405164	https://revistes.uab.cat/da/article/download/v11-n3-meriggi/505-pdf-en	English	null	The harmonization of animal protection during transport in the European Union - Analysis of the sanctioning systems in Italy, Romania and Spain	Derecho animal	2,020	cc-by	9,151	Abstract In the last sixty years, countries in Europe1 developed a common legal framework for the protection of so-called “farmed” Animals2: on farms, during transport and at the time of their killing. This document describes the most relevant aspects of the sanctioning systems implementing the legislation on the protection of Animals during transport3 in three countries: Italy, Spain and Romania. These nations were chosen in connection with the author’s collaboration with the German non-governmental organization, Animals´ Angels. The association has been investigating animal transports at the international level since 1998, with particular attention to these three countries. The article draws on findings collected from the organization’s field experience as well as perspectives that have emerged over time during the analysis of the various countries. This document aims to lay out the positive and the negative points of each penalty system, as a basis for a wider analysis, and to formulate proposals for a better and uniform application of a dissuasive sanctioning system in Europe. The fact that penalties are left to the competence of the member states, which are culturally different and have different legal systems, has led to an alarmingly irregular implementation. From another perspective, with protection being put in second place, this leads to competitive distortion in the territory of the European Union. The article examines efforts made by European institutions and member states to improve the harmonisation of the application of Regulation (EC) No. 1/2005. It also offers practical inputs that can be used in future negotiations of the existing laws on animal transport. p Keywords: animal transport; sanctioning system; European Union; animal protection; farmed animals; harmonization. Keywords: animal transport; sanctioning system; European Union; animal protection; farmed animals; harmonization. Resumen - La armonización de la protección animal durante el transporte en la Unión Europea – Análisis del sistema de sanciones en Italia, Rumanía y España En los últimos sesenta años, los países europeos desarrollaron un marco legal común para la protección de los animales “de granja”: en las granjas, durante el transporte y en el momento de su matanza. Este documento pretende describir los aspectos más relevantes de los sistemas de sanción que aplican la legislación sobre la protección de los animales durante el transporte en tres países: Italia, España y Rumanía. La razón por la que elegí estas naciones reside en mi colaboración con la organización no gubernamental alemana Animals' Angels. dA.Derecho Animal (Forum of Animal Law Studies) 2020, vol. 11/3 125-133 dA.Derecho Animal (Forum of Animal Law Studies) 2020, vol. 11/3 125-133 The harmonization of animal protection during transport in the European Union - Analysis of the sanctioning systems in Italy, Romania and Spain Silvia Meriggi MA Animal Law and society (UAB) Investigator, Animals´ Angels Received: May 2020 Accepted: September 2020 Received: May 2020 Accepted: September 2020 Received: May 2020 Accepted: September 2020 Recommended citation. MERIGGI, S., The harmonization of animal protection during transport in the European Union - Analysis of the sanctioning systems in Italy, Romania and Spain, dA. Derecho Animal (Forum of Animal Law Studies) 11/3 (2020). - DOI https://doi.org/10.5565/rev/da.505 Recommended citation. MERIGGI, S., The harmonization of animal protection during transport in the European Union - Analysis of the sanctioning systems in Italy, Romania and Spain, dA. Derecho Animal (Forum of Animal Law Studies) 11/3 (2020). - DOI https://doi.org/10.5565/rev/da.505 https://doi.org/10.5565/rev/da.505 ISSN 2462-7518 1 First as the European Economic Community, then as the European Community and finally as the European Union 2 Juridical and common language indicate with the term “farm” animals those destined for production of food and commodities in the European Union, such as cattle, sheep, pigs, horses, chickens and rabbits. The use of a capital letter for Animals is intentional, taking it as a license to give them the importance they deserve. 3 Council Regulation No. 1/2005 on the protection of animals during transport and related operations, hereinafter only Reg. 1/2005 A. Protection of farmed animals during transport in Europe In the 1960s, when the European Communities were created, live animals kept for farming purposes were already traded over long distances. Civil society was concerned about their transport conditions. The WSPA4 lobbied for the approval of the first European law to protect farmed animals during transport, to balance economic and ethical concerns. The result was a Convention opened for signatures in 19685. The voluntary format of the agreement soon revealed its limits. Meanwhile, the Communities developed another text, with the same aim of the Convention, which became law in 1977: Directive No. 77/489/EEC. It was the first directive for the protection of animals during transport. Subsequently this law was replaced by Directive No. 91/628/EEC, amended in 19956.These attempts aimed at solving the same old problem that was well expressed among the whereas of Directive No. 77/489 which state7: “there exist between the national Laws at present in force in the field of animal transport disparities affecting the functioning of the common market”, “in order to eliminate the resultant technical barriers to trade in live animals, the laws of the member states should be harmonized…”. “in order to eliminate the resultant technical barriers to trade in live animals, the laws of the member states should be harmonized…”. Nevertheless, in the early 2000s, transportation of animals increased inside, even outside the EU. Business interests seemed to prevail over ethical values expressed by the public. The EU institutions realized that member states did not enforce the directives8, and in addition, diseases were spreading9 with the traffic of animals. Consequently, the European Council invited the European Commission to make a proposal with more stringent and uniform requirements. In 2007, Council Regulation No. 1/2005 on the protection for animals during transport entered into force. As a Regulation, it has the advantage of being directly applicable in all EU Member States. This is in contrast to directives, which have to be implemented by national laws, which leads to variation among the different countries. Unfortunately, its strong binding effects did not include provisions concerning penalties, leaving autonomy to every country10 to decide which consequences derived from the violation of the Regulation11. Forty years passed (1968-2007) from the drafting of the first agreement, to limit the suffering of farmed animals during their transport in the EU, to the emergence of Regulation No. 1/2005. Abstract Esta asociación lleva investigando los transportes de animales desde 1998, a nivel internacional, con especial atención a estos tres países. Me gustaría utilizar algunos de los hallazgos recogidos de nuestra experiencia directa en el campo y las opiniones consolidadas durante el análisis de los distintos países. El presente documento tiene por objeto extrapolar lo positivo y lo negativo de cada sistema de sanciones, como punto de partida para un análisis más amplio, a fin de formular propuestas para una aplicación mejor y más uniforme de un sistema de sanciones disuasivas en Europa. El hecho de que las sanciones se dejen a la competencia de los Estados miembros, que son culturalmente diferentes y tienen sistemas jurídicos distintos, da lugar a una preocupante ISSN 2462-7518 ISSN 2462-7518 Silvia Meriggi The harmonization of animal protection during transport in the European Union falta de homogeneidad en la aplicación en la actualidad. Desde otra perspectiva, en relación a la necesidad de protección, esto lleva a una distorsión de la competencia en el territorio de la Unión Europea. Examinaré los esfuerzos realizados por las instituciones europeas y los Estados Miembros, para mejorar la armonización de la aplicación del Reglamento (CE) Nº 1/2005. Además, tengo la intención de ofrecer algunas aportaciones prácticas que espero sean tomadas en serio en caso de futuras negociaciones de las actuales leyes sobre transporte de animales. Palabras clave: transporte de animales; sistema de sanciones; Unión Europea; protección de los animales; animales de granja; armonización. 126 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 4 World Society for the Protection of Animals, charity based in United Kingdom and now known as World Animal Protection 5 European convention for the protection of Animals during international transport 6 Directive No. 91/628/EEC was amended by Directive 95/29/EC 7 Last two whereas, before article 1 of Directive No. 77/489/EEC of 18 July 1977 8 COM/2000/0809 final and Memo 01/124, Brussels, 6 April 2001 (European Commission. Webpage: europa.eu/rapid/press- release_MEMO-01-124_en.pdf) [last access: 09.09.2020] 9 Council Resolution of 19 June 2001 on the protection of animals during transport Official Journal C 273, 28/09/2001 10 Article 25 of Reg. 1/2005 11 “Law without enforcement is only good advice” (Abraham Lincoln) 12 The only exception is the paper drafted by two NGOs, WSPA and Eyes on Animals: “Weaknesses in the animal-transport monetary sanctions - A comparative study of the effectiveness, proportion and dissuasiveness of the monetary penalties applicable to infringements of Regulation EC 1/2005 among major players of the EU”. Webpage: https://www.eyesonanimals.com/wp-content/uploads/2011/12/Downloads_WEAKNESS_IN_MONETARY_ SANCTIONS_OF_ANIMAL_TRANSPORT(1).pdf) [last access: 09.09.2020] 13 “…the humane treatment of animals is one of the hall-marks of Western civilization…”: Recommendation No. 287/1961 issued by the Consultative Assembly of the Council of Europe. 14 This includes the authorities in charge of to enforcing the protection of animals during transport: ministries of health or agriculture and veterinary authorities 15 Webpage: www.animals-angels.de [last access: 09.09.2020] 16 Various proposals suggested to modify article 9 of the Italian Constitution, to insert, expressly, the protection of environment and of animals. The most recent proposal dates back to 2019, webpage: https://www.animal-law.it/rivista/diritto/la-tutela-degli-animali- potrebbe-entrare-nella-costituzione/ [last access: 09.09.2020] 17 “Country Profile of Italy – Organisation of official controls”, DG Health and Food Safety, European Commission 18 “Protocollo d’intesa per il potenziamento dci controlli di legalità nel settore del trasporto internazionale degli animali”, 3 October 2011 A. Protection of farmed animals during transport in Europe The concerns expressed in the introduction of the Convention, in the subsequent directives and in the related reports of the Council, have remained the same and continue to be discussed in recent documents: • increase of animal transports; • difficulty in implementing the rules; • uneven application of the law; • spread of diseases due to increased transports; • need to harmonize rules to avoid distortion of competition. • increase of animal transports; • uneven application of the law; • spread of diseases due to increased transports; • need to harmonize rules to avoid distortion of competition. Silvia Meriggi The harmonization of animal protection during transport in the European Union Despite many years of experience of different legal tools and of animal transports, no official document could be found recommending practical solutions, for common rules, for a uniform sanctioning system in EU12. The author therefore decided to undertake research in order to suggest concrete actions towards real harmonization. Despite many years of experience of different legal tools and of animal transports, no official document could be found recommending practical solutions, for common rules, for a uniform sanctioning system in EU12. The author therefore decided to undertake research in order to suggest concrete actions towards real harmonization. B. Rationale and method of research As of today, after 50 years of demands for a uniform protection of animals in Europe, and despite the proclamation that society cares about Animals13, they are not yet benefitting consistently from the basic transport conditions they deserve. The author therefore conducted research on the official web pages of the competent national authorities14 and among the findings collected by Animals´ Angels15, the non- governmental organization for which the author has worked since 2011. Animals’ Angels has been investigating animal transports for 20 years, observing animals in trucks, reporting findings to, and meeting with, authorities. The sanctioning systems of Italy, Spain and Romania were analyzed for two reasons: because Animals´ Angels has worked in these countries or for reasons of language comprehension. The analysis led to a compilation of a list of relevant points that characterize and influence the sanctioning systems of Regulation 1/2005: for example, the subjects of controls (inspectors) and their empowerment, strategies, legal characterization of the violations and type of penalties, training efforts. These points were used to suggest strengths of the different national sanctioning systems. It is hoped that this list of points can be useful in the near future, to design a new unique and uniform sanctioning system, directly applicable in all EU Member States. 12 The only exception is the paper drafted by two NGOs, WSPA and Eyes on Animals: “Weaknesses in the animal-transport monetary sanctions - A comparative study of the effectiveness, proportion and dissuasiveness of the monetary penalties applicable to infringements of Regulation EC 1/2005 among major players of the EU”. Webpage: C. Findings C.01 Italy Italy was one of the founders of the European Communities and is a parliamentary republic, divided in 20 administrative regions. The Italian Constitution does not expressly protect animals16. Nevertheless, the protection of animals also has sanitary implications, of which the competence is shared between the State and the Regions. The Italian Law No. 151/2007 implements article 25 of Reg. 1/2005, providing penalties. The main authorities in charge of enforcing these laws are the Ministry of Health and the Regional Veterinary Offices, at different levels17. The Ministry coordinates the activities of the regions, defining the national policy. The regions enforce the law through their veterinary inspectors in the field. Beside the Ministry of Health, and thanks to an agreement with it18, the Ministry of the Interior supplements the veterinary inspectors with police inspectors. In practice, both, veterinary and police field officials, have the competence to check animal transports, verify if the law is complied with and in case of violations, to levy fines. p , y p , y Sanctions for the violations of Reg. 1/2005 are administrative and mainly monetary fines, in the first instance, applied to all stakeholders involved in the organization of the transport (farmers, transporters, organisers). Fines vary between 1000 and 6000 euros: the most expensive concern the fitness of animals to be transported or their mistreatment but also vehicle requirements for long distances or the lack of the necessary authorizations. Sanctions are criminal when violations break penal provisions. Accessory sanctions entail the suspension or withdrawal of authorizations or the seizure of the vehicles and animals. The Italian sanctioning system has a good legal characterization of sanctions, quite faithful to the provisions of the Regulation. The best categorization of sanctions would refer to each paragraph of the original text of Reg. 1/2005, indicating Derecho Animal. Forum of Animal Law Studies, vol. 11/3 127 The harmonization of animal protection during transport in the European Union Silvia Meriggi Silvia Meriggi the correspondent penalty. The amounts of Italian monetary fines are relatively high, compared to the profit of the offenders, thus sufficiently dissuasive. When animal transporters are fined in Italy for violating Reg. 1/2005, and trucks are registered overseas, immediate payment must be performed19. Otherwise, the vehicle and the animals are seized. The vehicle is taken to a garage, the animals are unloaded in a place where their can be taken care of, at the expenses of the lawbreaker. C. Findings C.01 Italy the correspondent penalty. The amounts of Italian monetary fines are relatively high, compared to the profit of the offenders, thus sufficiently dissuasive. When animal transporters are fined in Italy for violating Reg. 1/2005, and trucks are registered overseas, immediate payment must be performed19. Otherwise, the vehicle and the animals are seized. The vehicle is taken to a garage, the animals are unloaded in a place where their can be taken care of, at the expenses of the lawbreaker. p Enforcement is uneven across the regions, due to their autonomy in sanitary and veterinary matters. Enforcement is uneven also from another point of view: the number of checks carried out by police is much higher than checks carried out by veterinary officers. This was also confirmed by auditors of the EU Commission20. The application of accessory sanctions is complicated and thus very rare21. A peculiarity of Italy is the availability 24/7 of highway police to inspect animal trucks and, in some regions, of a veterinarian on shift, who can be called on by the police for joint inspections. Animals´ Angels experienced this firsthand, having contacted the police in many cases, at any time of day, when serious breaches of Reg. 1/2005 were observed. The Ministries of Health and Interior have shown themselves to be open-minded, extending their collaboration to external experts22 and distributing to inspectors a practical pocket-size manual on checks on animal transports, with useful tables. This manual has helped inspectors to quickly identify violations and correspondent sanctions. Animals´ Angels also organized the first series of courses to train police agents to become expert at Reg. 1/2005, some of which are now training new police officers. In general, the level of transparency concerning enforcement activities and their outcome is satisfactory in Italy. Various reports (the mandatory annual report23 and an internal one24), of the veterinary authorities, are published on the official website of the Ministry of Health. A national plan25 stipulates the minimum percentages of trucks to inspect yearly and sets a list of risks that should be taken into account, by each region, to schedule checks. Unfortunately, not all of them drew up a regional plan, based on the specific risks, linked to the characteristics of their territory (e.g. intense presence of certain types of farms and slaughterhouses; presence of control posts; if they are bordering regions or transit regions affected by specific movements). 19 The Italian national law, D. Lgs. No. 151/2007, article 12, refers to the Italian Road Traffic Code, article 207 20 EU Commission audit report DG(SANCO) 2011-6048 21 This can happen if the offender pays the fine, or in case of appeal, when the courts release the final decision (res judicata). 22 Annual report on the protection of animals during transport of Italy, 2012, page 38: “Regular training courses continue to be organized with the active participation of experts from the Ministry veterinary services, the Regions and the local health authorities, and experts from the animal protection organizations of Lav and Animals´ Angels” 23 Mandatory on the base of Regulation (EC) No 1/2005, art. 27 24 http://www.salute.gov.it/imgs/C_17_pubblicazioni_2865_allegato.pdf [last access: 09.09.2020] 25 Published in the official website of the Ministry of Health, webpage: http://www.salute.gov.it/pianoNazionaleIntegrato2015/homePianoNazionaleIntegrato2015.jsp [last access: 09.09.2020] See also: http://www.salute.gov.it/relazioneAnnuale2018/homeRA2018.jsp [last access: 09.09.2020] 26 Autoritaea Nationala Sanitara Veterinara si pentru Siguranta Alimentelor (the national sanitary, veterinary and food safety authority of Romania) 27 Decision No 984/2005 concerning the violations of the sanitary veterinary and food safety laws in Romania had been amended in g 0 EU Commission audit report DG(SANCO) 2011-6048 27 Decision No. 984/2005, concerning the violations of the sanitary-veterinary and food safety laws in Romania, had 2008 by Government Decision No. 30, to introduce the sanctions connected to the violation of Reg. 1/2005 y , 28 See Ordinance No. 2/2001, article 15 and Ordinance No. 42/2004, Chapter III 128 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 19 The Italian national law, D. Lgs. No. 151/2007, article 12, refers to the Italian Road Traffic Code, article 207 20 EU Commission audit report DG(SANCO) 2011-6048 21 This can happen if the offender pays the fine, or in case of appeal, when the courts release the final decision (res judicata). 22 Annual report on the protection of animals during transport of Italy, 2012, page 38: “Regular training courses continue to be organized with the active participation of experts from the Ministry veterinary services, the Regions and the local health authorities, and experts from the animal protection organizations of Lav and Animals´ Angels” 23 Mandatory on the base of Regulation (EC) No 1/2005, art. 27 24 http://www.salute.gov.it/imgs/C_17_pubblicazioni_2865_allegato.pdf [last access: 09.09.2020] 25 Published in the official website of the Ministry of Health, webpage: http://www.salute.gov.it/pianoNazionaleIntegrato2015/homePianoNazionaleIntegrato2015.jsp [last access: 09.09.2020] See also: http://www.salute.gov.it/relazioneAnnuale2018/homeRA2018.jsp [last access: 09.09.2020] 26 Autoritaea Nationala Sanitara Veterinara si pentru Siguranta Alimentelor (the national sanitary, veterinary and food safety authority of Romania) 27 Decision No. 984/2005, concerning the violations of the sanitary-veterinary and food safety laws in Romania, had been amended in 2008 by Government Decision No. 30, to introduce the sanctions connected to the violation of Reg. 1/2005 28 See Ordinance No. 2/2001, article 15 and Ordinance No. 42/2004, Chapter III 29 Example: letter of DSVSA Suceava, protocol No 8254/19.05.2016 to ANSVSA. See webpages: https://www.digi24.ro/stiri/actualitate/social/animalele-chinuite-neprotejate-de-lege-606900, https://www.ziaruldeiasi.ro/stiri/stiti- ca-exista-politia-animalelor-in-ia-i-are-un-singur-angajat--140341.html [last access: 09.09.2020], as more examples. 30 Decision No. 984/2005, article 11(4): “Contravenţiile prevăzute la art. 3 lit. a) pct. 2, lit. b) pct. 1, 2 şi 9 şi la lit. c) pct. 3 se pot constata şi sancţiona şi de către poliţişti” 128 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 , , p 29 Example: letter of DSVSA Suceava, protocol No 8254/19.05.2016 to ANSVSA. See webpages: https://www.digi24.ro/stiri/actualitate/social/animalele-chinuite-neprotejate-de-lege-606900, https://www.ziaruldeiasi.ro/stiri/stiti- ca-exista-politia-animalelor-in-ia-i-are-un-singur-angajat--140341.html [last access: 09.09.2020], as more examples. 30 Decision No. 984/2005, article 11(4): “Contravenţiile prevăzute la art. 3 lit. a) pct. 2, lit. b) pct. 1, 2 şi 9 şi la lit. c) pct. 3 se pot constata şi sancţiona şi de către poliţişti” 19 The Italian national law, D. Lgs. No. 151/2007, article 12, refers to the Italian Road Traffic Code, article 207 20 EU Commission audit report DG(SANCO) 2011-6048 http://www.salute.gov.it/relazioneAnnuale2018/homeRA2018.jsp [last access: 09.09.2020] 26 Autoritaea Nationala Sanitara Veterinara si pentru Siguranta Alimentelor (the national sanitary, veterinary and food safety authority of Romania) C.02 Romania Romania is a semi-presidential republic, which joined the European Union in 2007. It is divided into 41 administrative counties. The central authority of ANSVSA26 is subordinated to the government and coordinated by the Prime Minister, having competences in veterinary and food safety, including animal welfare. Despite the legislative power belonging to the parliament, the government has the power of initiative and also the power to adopt decisions for the execution of laws. In fact, ANSVSA adopted Decision No. 984/200527 to implement article 25 of Reg. 1/2005. The only authorities in charge of enforcing the Regulation on the spot are official veterinarians entitled28 to carry out inspections and levy fines. They are employed by the county veterinary offices. Unfortunately, human resources are very limited and burdened with other duties29. Agreements with police inspectorates have been concluded so that in theory police officers will help veterinarians, to stop trucks during road-checks and to keep order and security. Nevertheless, Romanian police officers have a limited competence for some documental checks30. In one instance, Animals´ Angels informed the border police of south Romania of trucks appearing to be in breach of the Regulation. The organization See Ordinance No. 2/2001, article 15 and Ordinance No. 42/2004, Chapter III 29 Example: letter of DSVSA Suceava, protocol No 8254/19.05.2016 to ANSVSA. See webpages: https://www.digi24.ro/stiri/actualitate/social/animalele-chinuite-neprotejate-de-lege-606900, https://www.ziaruldeiasi.ro/stiri/stiti- ca-exista-politia-animalelor-in-ia-i-are-un-singur-angajat--140341.html [last access: 09.09.2020], as more examples. 30 Decision No. 984/2005, article 11(4): “Contravenţiile prevăzute la art. 3 lit. a) pct. 2, lit. b) pct. 1, 2 şi 9 şi la lit. c) pct. 3 se pot constata şi sancţiona şi de către poliţişti” 28 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 8 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 Silvia Meriggi The harmonization of animal protection during transport in the European Union asked the police to stop the trucks and call the veterinarians for a joint check, but they denied their competence to do so, apparently not aware of any agreement. Police officers are neither trained neither empowered to enforce Reg. 1/2005 in Romania. Sanctions are basically pecuniary contraventions. Offenders can be individuals or legal persons. Fines are much heavier when they are committed by companies, varying approximately between 6000 and 30000 lei31. The most expensive are levied in case of violations concerning unfit animals (injured/ill animals), emergency plans, navigation systems, journey time and mandatory intervals, transport practices, requirements of vehicles and accompanying documents. 31 Romanian currency equals to 0.21 euro, according to google extracted on 18.01.2019 at 13:34 UTC 32 Annual reports on the protection of animals during transport of Romania, according to art. 27 of Reg. 1/2005 33 Example: Letters of veterinary directorate (DSVSA) of county Giurgiu, protocols No. 1360 of 20.01.2016 and No. 4200/11.03.2016 34 Animals´ Angels reports: “Formal complaint by Animals´ Angels to the European Commission, concerning systematic failure by Romanian authorities to secure compliance with Council Regulation EC No. 1/2005 for the protection of animals during transport and related operations in animal markets in Romania”, November 2014 and Letter of Animals´ Angels to the EU Commission concerning animal transport to Romanian traditional markets – update of Complaint CHAP(2014)03700, 15.02.2016 35 Animals´ Angels reports: “Easter slaughter in Romania - An investigation of Animals´ Angels 7-14 April 2017” and Animals´ Angels report: “Transport of sheep for slaughter, from Romania to Greece, 19.-20.12.2017” and “Transport of sheep for slaughter, from Romania to Greece, 17. – 18.12.2017” and “Transport of sheep for slaughter, from Romania to Bulgaria 22.12.2017 “ and ” Transport of lambs for slaughter from Romania to Greece, 01./03.04.2018” 36 Letter of the EU Commission, Ref. Ares (2016)7135068 - 22/12/2016, Audit Report of the EU Commission, Ref. Ares(2013)1846550 - 07/06/2013 and Fact Finding Mission of EU Commission in Romania from 26 to 30 October 2015 and Letter Ref. Ares(2016)7135068 - 22/12/2016 and Audit Report of the EU Commission, Ref. Ares(2018)2908268 - 04/06/2018 37 Webpage of Ansvsa: www.ansvsa.org [last access: 09.09.2020] 38 See webpage: http://www.ansvsa.ro/download/pncui/pncui-plan_anual/2017-2019-Planul-National-Multianual-Unic-De-Control- Integrat-Pentru-Romania.pdf [last access: 09.09.2020] 39 According to articles 110(b) and 111(2) of Regulation 2017/625/EU on official controls on animal welfare C.02 Romania The amount of the monetary fines is heavy in general, especial for companies, thus dissuasive. On the other hand, according to the official documents32, the number of checks and sanctions applied is scarce, compared to Italy, for example. This manifests itself in an imbalance that leads to distortion of competition among transporters in EU Member States. Furthermore, according to the research carried out for this article, there is no provision in the Romanian road code, contrarily to the Italian system, in which foreign transporters must pay fines immediately on the spot, when infringements of Reg. 1/2005 are detected. Because of the lack of this provision, foreign transporters may decide not to pay the fines, counting of the fact that execution procedures outside Romania are complicated. This causes an unfair treatment between Romanian and foreign transporters. Despite the quite good legal tool to enforce Reg. 1/2005 in Romania, there seems to be a lack of specific attention to the welfare of the animals, in favor to documental formalities and food safety issues33. This aspect stands out in the annual reports on the protection of animals during transport. Animals’ Angels complained about it, reporting unlawful transport conditions of animals at local markets34 but also during the intense export of sheep35 to other EU countries. This limitation was also noted by the European Commission36 and recognized by the Romanian authorities themselves. y p g y It proved extremely difficult to find documents reporting the outcome of the inspections on official websites37. The official website of ANSVSA published the new MANCP of Romania38. Like the previous versions of it, it contains little information about checks on animal welfare during transport and it still lacks essential aspects such as risk analysis and strategies39 based on previous outcomes and on territorial peculiarities of Romania. For example, Romania is a transit country, has intense movements to export lambs in certain seasons and temperatures that reach extreme values in summer and winter. Neither the most relevant types of transports to check or the period of the year are mentioned in any paragraph of the planning. The mandatory annual reports contain very poor analysis and plans of initiatives to improve enforcement. 31 Romanian currency equals to 0.21 euro, according to google extracted on 18.01.2019 at 13:34 UTC 32 32 Annual reports on the protection of animals during transport of Romania, according to art. 27 of Reg. 1/2005 33 Example: Letters of veterinary directorate (DSVSA) of county Giurgiu, protocols No. 1360 of 20.01.2016 and No g p [ ] 39 According to articles 110(b) and 111(2) of Regulation 2017/625/EU on official controls on animal welfare Example: Letters of veterinary directorate (DSVSA) of county Giurgiu, protocols No. 1360 of 20.01.2016 and No. 4200/11.03.2016 34 Animals´ Angels reports: “Formal complaint by Animals´ Angels to the European Commission, concerning systematic failure by Romanian authorities to secure compliance with Council Regulation EC No. 1/2005 for the protection of animals during transport and related operations in animal markets in Romania”, November 2014 and Letter of Animals´ Angels to the EU Commission concerning animal transport to Romanian traditional markets – update of Complaint CHAP(2014)03700, 15.02.2016 C.03 Spain Spain is a constitutional monarchy organised in 17 autonomous communities with their own government. The central Ministry of Agriculture coordinates the autonomous ministries on animal welfare policies. Each community is responsible for implementing animal welfare legislation through its veterinary units. Spanish veterinary officers checking animal transports are not empowered to levy fines on the spot, if they ascertain violations of Reg. 1/2005. In some Spanish autonomous communities, police forces can also carry out checks on animal transports, but they are not empowered to levy sanctions either. Police must call a veterinary officer who, in any case, does not have the power to levy sanctions on the spot. The central veterinary offices oversee, analyzing the findings of the officers involved in the checks and, if necessary, raising the corresponding penalties. The communities may also issue their own legislation. Consequently, many laws overlap, and their hierarchy is not very clear at first glance. p p p ( ) 35 Animals´ Angels reports: “Easter slaughter in Romania - An investigation of Animals´ Angels 7-14 April 2017” and Animals´ Angels report: “Transport of sheep for slaughter, from Romania to Greece, 19.-20.12.2017” and “Transport of sheep for slaughter, from Romania to Greece, 17. – 18.12.2017” and “Transport of sheep for slaughter, from Romania to Bulgaria 22.12.2017 “ and ” Transport of lambs for slaughter from Romania to Greece, 01./03.04.2018” p p p ( ) 35 Animals´ Angels reports: “Easter slaughter in Romania - An investigation of Animals´ Angels 7-14 April 2017” and Animals´ Angels report: “Transport of sheep for slaughter, from Romania to Greece, 19.-20.12.2017” and “Transport of sheep for slaughter, from Romania to Greece, 17. – 18.12.2017” and “Transport of sheep for slaughter, from Romania to Bulgaria 22.12.2017 “ and ” Transport of lambs for slaughter from Romania to Greece, 01./03.04.2018” p g , 36 Letter of the EU Commission, Ref. Ares (2016)7135068 - 22/12/2016, Audit Report of the EU Commission, Ref. Ares(2013)1846550 - 07/06/2013 and Fact Finding Mission of EU Commission in Romania from 26 to 30 October 2015 and Letter Ref. Ares(2016)7135068 - 22/12/2016 and Audit Report of the EU Commission, Ref. Ares(2018)2908268 - 04/06/2018 37 Webpage of Ansvsa: www.ansvsa.org [last access: 09.09.2020] See webpage: http://www.ansvsa.ro/download/pncui/pncui-plan_anual/2017-2019-Planul-National-Multianual-Unic tegrat-Pentru-Romania.pdf [last access: 09.09.2020] 39 According to articles 110(b) and 111(2) of Regulation 2017/625/EU on official controls on animal welfare Derecho Animal. Forum of Animal Law Studies, vol. 11/3 Derecho Animal. 31 Romanian currency equals to 0.21 euro, according to google extracted on 18.01.2019 at 13:34 UTC 32 Annual reports on the protection of animals during transport of Romania, according to art. 27 of Reg. 1/2005 33 Example: Letters of veterinary directorate (DSVSA) of county Giurgiu, protocols No. 1360 of 20.01.2016 and No. 4200/11.03.2016 34 Animals´ Angels reports: “Formal complaint by Animals´ Angels to the European Commission, concerning systematic failure by Romanian authorities to secure compliance with Council Regulation EC No. 1/2005 for the protection of animals during transport and related operations in animal markets in Romania”, November 2014 and Letter of Animals´ Angels to the EU Commission concerning animal transport to Romanian traditional markets – update of Complaint CHAP(2014)03700, 15.02.2016 35 Animals´ Angels reports: “Easter sla ghter in Romania An in estigation of Animals´ Angels 7 14 April 2017” and Animals´ Transport of lambs for slaughter from Romania to Greece, 01./03.04.2018 36 Letter of the EU Commission, Ref. Ares (2016)7135068 - 22/12/2016, Audit Report of the EU Commission, Ref. Ares(2013)1846550 - 07/06/2013 and Fact Finding Mission of EU Commission in Romania from 26 to 30 October 2015 and Letter Ref. Ares(2016)7135068 - 22/12/2016 and Audit Report of the EU Commission, Ref. Ares(2018)2908268 - 04/06/2018 37 Webpage of Ansvsa: www.ansvsa.org [last access: 09.09.2020] 38 S b htt // /d l d/ i/ i l l/2017 2019 Pl l N ti l M lti l U i D C t l C.03 Spain Forum of Animal Law Studies, vol. 11/3 129 The harmonization of animal protection during transport in the European Union Silvia Meriggi Before Reg. 1/2005 came into force, and at the time of the previous European directives on the protection of animals during transport, nearly all Autonomous Communities set their rules on the matter, between 1990 and 2003. These texts have not been apparently updated, after the Regulation. Instead, at the national level, Law n. 32/2007 “para el cuidado de los animales, en su explotación, transporte, experimentación y sacrificio40” provided sanctions for the violation of the Regulation and of other laws concerning animal protection (on farms and during slaughter, for example). A very general provision includes violations concerning animal welfare during transport, referring very generally to “failure to comply with the obligations required by animal protection standards for the care and handling of animals”. It calls for minor, serious or most serious sanctions, depending on, respectively, if there is no permanent damage caused to the Animals, if there are or if there is the intention to torture or to cause death to them. Violations concerning animal transport are generally minor, therefore accessory sanctions, which apply only to serious offences, apparently never apply for most animal transport breaches. In the case of minor infringements, like those violating Reg. 1/2005, a fine not exceeding 600 euros or a warning shall be imposed. In the case of the laws of the autonomous communities, most of the violations concerning animal transports fell under minor offences and only in case of mistreatment, injuries and death they are serious. Generally, the amounts of minor offences are a maximum of 150.25 or 300.51 euros. Sanctions in Spain are not at all dissuasive41, something that was also confirmed by the auditors of the European Commission42. y p As one reads the Multi Annual National Control Plan of Spain, called “Official control of the food chain” of 2016-2020, the impression is that the Spanish sanctioning system is very fragmentary and uneven. The MANCP puts little effort into trying to keep track of all the challenging differences. According to this document, Spain does not even have a general manual, valid for all field inspectors, providing written procedures, concerning checks on animal transports for veterinarians. Instead, a guide for the police forces was published in 2017. As in Romania, annual reports lack a real analysis and action plan. 40 i.e., in English: for the protection of animals on the farm, during transport, vivisection and slaughter 41 “legal departments of the ACs can impose a maximum fine of €600 as this offence is graded in the annex to the Law on Sanctions as a light offence. In practice sanctions for farmers or transporters were usually €150-200 after appeal which are not proving to be effective at addressing this issue”, pages 13-14 of the audit report DG(SANCO) 2014-7079. 42 Recommendation 9 of audit report DG(SANCO) 2008-8047 43 See footnote 39 C.03 Spain Those accompanying Spanish reports are meagre and superficial. 40 i.e., in English: for the protection of animals on the farm, during transport, vivisection and slaughter 130 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 D. Outcome of the comparison The sanctioning systems of Spain, Italy and Romania are very different from each other, beginning with the legal definition of the offences, through to the different figures in charge of enforcement and empowered to levy fines. y All three countries provide mainly monetary sanctions, in case of violations of Reg. 1/2005, of very different amounts. To give some examples: VIOLATION PENALTY IN ITALY PENALTY IN ROMANIA PENALTY IN SPAIN Too many animals loaded 1000 euro 1292.52 euro (6000 lei) Up to 600 euro or a warning Water system not functioning 2000 euro 1292.52 euro Up to 600 euro or a warning Unfit animals 2000 38777.57 euro (18000 lei) Up to 600 euro or a warning The national control plans of all the three countries do not fulfill the legal provisions regarding risk analysis and risk-based checks. The annual reports reflect such a lack, in particular because they are accompanied by very superficial analysis and action plans. The Italian MANCP and accompanying analysis get closer to the legal requirements43, than the Romanian and Spanish. The multi annual planning mentions, at least, some risks for the Regions to keep into consideration, when drafting their official controls on animal transport. The analysis of the annual reports in Italy are definitely longer and apparently more structured than the Romanian and Spanish ones. Nevertheless, at a deeper look, they also do not contain a real statistical study and interpretation of the collected data. 130 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 Silvia Meriggi The harmonization of animal protection during transport in the European Union The following table was compiled after analysing strengths and weaknesses of the three compared countries. It summarizes key factors influencing, in general, an effective sanctioning system for the violation of Reg. 1/2005: STRATEGY Thorough44 national and regional plans Annual reports with professional analysis and action plan TRAINING Pocket-size manual for inspectors Standardized training Training provided by an expert group of inspectors EMPOWERMENT Veterinary and police field inspectors empowered to levy fines COORDINATION Agreements between police and veterinarians/respective ministries Contact persons for information exchange EFFECTIVENESS High monetary sanctions Three levels of intensity: minor, serious, most serious infringements/correspondent sanctions Clear and thorough legal categorization of offences, based on Reg. D. Outcome of the comparison 1/2005 definitions Easy procedure to impose accessory sanctions European database to track repeat offences On-the-spot payment and seizure of trucks and animals for foreign offenders TRANSPARENCY National and regional plans, official reports and contact for animal transport checks published on government websites E. Theory for the harmonization of the sanctioning system of Reg. 1/2005 How can the sanctions for the violation of Regulation (EC) No. 1/2005 be harmonized in all Member States of the EU? How can practices and enforcement be improved? The production of the Transport Guidelines in 2017 took three years, from 2015 to 2018, and required an investment of 990.000 euros of EU funds, but it is soft law. Drivers, who were among the final recipients, are not familiar with them, after 3 years, and veterinary inspectors claim that they do not have legal force. Should every national sanctioning system be modified? This would be too complicated and time-consuming and would again be left to the discretion of each member state, as was the case with article 25 of the Regulation. g It took 40 years to conclude that a common legal framework was the only solution to harmonize the protection of animals during transport. It should now be clear that a regulation directly applicable to all EU Member States is the only solution to harmonise the correspondent sanctions. To the question of whether such harmonisation is possible, the answer is yes. European laws must be applied to members and citizens of the European Union equally, as per the Treaty on the Functioning of the European Union, article 8. This does not happen for Reg. 1/2005, as animal transporters meet with different legal consequences, depending on the country in which they are checked by authorities. In the same way, farmed animals, which are transported for commercial reasons, are treated differently, depending on the country in which they are inspected. Articles 114, 115, 116, 117 of the TFEU point to the need to harmonise regulations and administrative actions in the EU Member States, when they concern the functioning of the internal market and when competition is distorted. Furthermore, the Council and European Parliament shall set out appropriate measures to implement the common transport policy (articles 90, 91 of TFEU). Derecho Animal. Forum of Animal Law Studies, vol. 11/3 131 Velarde, C. Fuentes, A. Truar, J.L. Otero, E. Di Fede, P. Dalla Villa 45 The White Paper of 2010, of the European Commission, mentions the same principles to harmonise penalties in the context of 44 Risk assessment in animal welfare – Especially referring to animal transport M. Marahrens,∗, N. Kleinschmidt Velarde, C. Fuentes, A. Truar, J.L. Otero, E. Di Fede, P. Dalla Villa F. Proposals The European Union must issue a regulation containing the sanctions for the violation of Reg. 1/2005, on the basis of the above mentioned articles of the TFEU because: • at present, different sanctions apply to the offenders of the same provision, when they are fined in different member states; different member states; • for this reason (and others), the internal market of live transport is distorted45. Derecho Animal. Forum of Animal Law Studies, vol. 11/3 131 44 Risk assessment in animal welfare – Especially referring to animal transport M. Marahrens,∗, N. Kleinschmidt, A. Di Nardo, A. Velarde, C. Fuentes, A. Truar, J.L. Otero, E. Di Fede, P. Dalla Villa 45 The White Paper of 2010, of the European Commission, mentions the same principles to harmonise penalties in the context of Silvia Meriggi The harmonization of animal protection during transport in the European Union The European Union has already issued directives and regulations containing sanctions, or indications for harmonized sanctions, for various important matters, which needed to be harmonized in EU (e.g. data protection, market abuse, professional transport), to align the differing competitive conditions. Particularly interesting is the case of Commission Regulation (EU) No. 2016/40346. This law set up a list of categories, types and degrees of seriousness of serious infringements of Union rules, which may lead to the loss of good repute of the road transporter undertaking or the transport manager. According to the Commission, this was a step forward ensuring fair competition and harmonised enforcement. In 2013 a law firm published a comparative study, for the European Commission47, that highlighted relevant disparities, among the different national laws concerning road transport. The regulation of 2016 has been an attempt to harmonise transport sanctions in the EU. This paper proposes: 1) to include animal transport in the EU transport policy on commercial road transport and its harmonization process; 1) to include animal transport in the EU transport policy on commercial road transport and its harmonization process; 2) that the European Commission, DG Sante, commissions a comparative study from a law firm, to analyse and compare each national sanctioning system for animal transport, in the 27 EU Member States48, including legal categorizations of violations, sanctions, empowerment and concluding with a proposal for their harmonization; 3) to directly amend Regulation 1/2005 or to issue a new regulation, providing sanctions of different levels of intensity, for minor, serious and most serious infringements of Reg. F. Proposals 1/2005. The Regulation must provide minimum and maximum limits of the amount of the monetary penalties. It shall also indicate supervising authorities, including police forces and not only veterinary officers. It shall grant investigative and corrective powers to all field inspectors. It shall supply tables listing offences and sanctions, in order to simplify the application of the law. It shall also provide templates for the compilation of professional control plans and of analysis and action plans accompanying annual reports. 132 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 commercial road transport (pages 13, 22, 25). 46 Commission Regulation (EU) 2016/403 of 18 March 2016 supplementing Regulation (EC) No 1071/2009 of the European Parliament and of the Council with regard to the classification of serious infringements of the Union rules, which may lead to the loss of good repute by the road transport operator 47 “Study on sanctions in the field of commercial road transport” – Contract no 2011/MOVE/D3/ETU/514-2011/SI2.612723 – February 2013, submitted by Studio Legale Grimaldi to the European Commission 48 Report from the Commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport: “such a comparison has not been carried out by the Commission. However, estimation based on the information available shows significant variations when it comes to the level of penalties for infringements of the Regulation across the European Union”, page 12 49 See „Interinstitutional agreement between the European Parliament, the council of the European Union and the European Commission on better Law-making interinstitutional agreement of 13 April 2016 on Better Law-Making and REFIT – making EU law simpler and less costly. “The European Commission's regulatory fitness and performance (REFIT) programme aims to ensure that EU legislation delivers results for citizens and businesses effectively, efficiently and at minimum cost. REFIT aims to keep EU law simple, remove unnecessary burdens and adapt existing legislation without compromising on policy objectives”. Webpage https://ec.europa.eu/info/law/law-making-process/evaluating-and-improving-existing-laws/refit-making-eu-law-simpler-and-less- costly_en [last access: 09.09.2020] 50 See webpage: https://www.animals- angels.de/fileadmin/user_upload/03_Publikationen/Dokumentationen/Animals_Angels_Myth_of_Enforcement.pdf [last access: 09.09.2020] 51 As an example, see “Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” of 2011 of the European Commission, page 15 “…ambiguities and inefficiencies in the current animal welfare legislation…” commercial road transport (pages 13, 22, 25). 46 g p , p g 49 See „Interinstitutional agreement between the European Parliament, the council of the European Union and the European Commission on better Law-making interinstitutional agreement of 13 April 2016 on Better Law-Making and REFIT – making EU law simpler and less costly. “The European Commission's regulatory fitness and performance (REFIT) programme aims to ensure that EU legislation delivers results for citizens and businesses effectively, efficiently and at minimum cost. REFIT aims to keep EU law simple, remove unnecessary burdens and adapt existing legislation without compromising on policy objectives”. Webpage https://ec.europa.eu/info/law/law-making-process/evaluating-and-improving-existing-laws/refit-making-eu-law-simpler-and-less- costly_en [last access: 09.09.2020] 50 See webpage: commercial road transport (pages 13, 22, 25). 46 Commission Regulation (EU) 2016/403 of 18 March 2016 supplementing Regulation (EC) No 1071/2009 of the European Parliament and of the Council with regard to the classification of serious infringements of the Union rules, which may lead to the loss of good repute by the road transport operator 47 “Study on sanctions in the field of commercial road transport” – Contract no 2011/MOVE/D3/ETU/514-2011/SI2.612723 – February 2013, submitted by Studio Legale Grimaldi to the European Commission 48 Report from the Commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport: “such a comparison has not been carried out by the Commission. However, estimation based on the information available shows significant variations when it comes to the level of penalties for infringements of the Regulation across the European Union”, page 12 49 See „Interinstitutional agreement between the European Parliament, the council of the European Union and the European Commission on better Law-making interinstitutional agreement of 13 April 2016 on Better Law-Making and REFIT – making EU law simpler and less costly. “The European Commission's regulatory fitness and performance (REFIT) programme aims to ensure that EU legislation delivers results for citizens and businesses effectively, efficiently and at minimum cost. REFIT aims to keep EU law simple, remove unnecessary burdens and adapt existing legislation without compromising on policy objectives”. Webpage https://ec.europa.eu/info/law/law-making-process/evaluating-and-improving-existing-laws/refit-making-eu-law-simpler-and-less- costly_en [last access: 09.09.2020] 50 See webpage: https://www.animals- G. Conclusion Regulation No. 1/2005 on the protection of Animals during transport and related operations is not easy to interpret, thus not easy to implement, despite the EU policy to keep the law simple 49. This, in addition to the differences among the Member States of the European Union, impedes the effective enforcement of the protection of farmed Animals. That the Regulation needs to be amended to improve its applicability was argued extensively by the organisation Animals´ Angels, in its many complaints as well as its document, “The Myth of Enforcement”50. Despite the awareness of the member states, and of the European Commission too51, g p , p g 49 See „Interinstitutional agreement between the European Parliament, the council of the European Union and the European Commission on better Law-making interinstitutional agreement of 13 April 2016 on Better Law-Making and REFIT – making EU law simpler and less costly. “The European Commission's regulatory fitness and performance (REFIT) programme aims to ensure that EU legislation delivers results for citizens and businesses effectively, efficiently and at minimum cost. REFIT aims to keep EU law simple, remove unnecessary burdens and adapt existing legislation without compromising on policy objectives”. Webpage https://ec.europa.eu/info/law/law-making-process/evaluating-and-improving-existing-laws/refit-making-eu-law-simpler-and-less- costly_en [last access: 09.09.2020] p angels.de/fileadmin/user_upload/03_Publikationen/Dokumentationen/Animals_Angels_Myth_of_Enforcement.pdf [last access: 09.09.2020] p angels.de/fileadmin/user_upload/03_Publikationen/Dokumentationen/Animals_Angels_Myth_of_Enforcement.pdf [last access: 09.09.2020] 51 As an example, see “Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” of 2011 of the European Commission, page 15 “…ambiguities and inefficiencies in the current animal welfare legislation…” 51 As an example, see “Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” of 2011 of the European Commission, page 15 “…ambiguities and inefficiencies in the current animal welfare legislation…” 132 Derecho Animal. Forum of Animal Law Studies, vol. 11/3 The harmonization of animal protection during transport in the European Union Silvia Meriggi of the limits of this Regulation, the policy of the latter has been, so far, since Reg. 1/2005, to produce soft law and improve enforcement. It is clear, however, that non-binding rules can´t change binding rules. udy visits to improve Member State controls on animal welfare during transport” of the European Commission, 2015 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” 2011 of the European Commission, page 14 54 One of the identified best practices was the possibility to impose sanctions on the spot to foreign transporters, in case of violations of Reg. 1/2005 56 In 2011, Animals´ Angels launched the 8hours petition, asking that animals are not transported for more than eight hours in total. 1,103,248 signatures were handed in to the European Commissioner for Health in June 2012. Over 140 MEPs expressed support for the campaign. The European Parliament adopted a Written Declaration No 49/2011 in March 2012, demanding the 8-hour limit. 55 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” 2011 of the European Commission, page 12 the protection of animals during transport 2011 of the European Commission, page 14 54 One of the identified best practices was the possibility to impose sanctions on the spot to foreign transporters, in case of violations of Reg. 1/2005 53 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” 2011 of the European Commission, page 14 54 One of the identified best practices was the possibility to impose sanctions on the spot to foreign transporters, in case of violations G. Conclusion The European Commission carried out a series of study visits to exchange best practices among the Member States, concerning the enforcement of the protection of animal transport in 2013-201452, as scheduled in the impact report53. Nevertheless, we are still far from a harmonised application of the regulation and of the “better practices” exchanged in those visits54. The European Commission recognized the need for a comparative study55, concerning the penalty system of Reg. 1/2005 of all Member States, but it has not yet been commissioned. Thirteen years have passed, since the Regulation has come into force, authorities have become aware of its limits and efforts to improve enforcement have been made. Over the last sixty years, attempts have been made to protect farmed Animals in Europe during transport and to formulate a harmonized approach. Now it is time to act coherently. Regulation No. 1/2005 must be amended and must carry uniform penalties or criteria for penalties that are implemented in all Member States. Even when the basic protection provided by the Regulation is finally enforced, European farmed Animals will not be protected according to the real meaning of the word “protection”. Farmed Animals will be truly protected when people will stop farming them and they will not be traded like goods, transported all over the world to be fattened or killed, deprived of their own personality and dignity and right to exist. Nevertheless, society has demanded that these Animals be protected somehow, meaning that, at the very least, they be spared unnecessary suffering. Legal protection is the minimum that European and national institutions should provide, using all their powers to fulfill the request of their citizens56 and to fulfill the content of their own laws. While Animals continue to travel crammed in trucks and without space to lie down or reach drinking devices, and while authorities consider this to be in compliance with the law, there will be protection only of the economic interests of a very small part of society, which makes profits out of the loopholes in the law. This is simply unfair. 54 One of the identified best practices was the possibility to impose sanctions on the spot to foreign transporters, in of Reg. 1/2005 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) e protection of animals during transport” 2011 of the European Commission, page 14 Study visits to improve Member State controls on animal welfare during transport of the European Commission, 2015 53 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” 2011 of the European Commission, page 14 54 One of the identified best practices was the possibility to impose sanctions on the spot to foreign transporters, in case of violations of Reg. 1/2005 55 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” 2011 of the European Commission, page 12 56 In 2011, Animals´ Angels launched the 8hours petition, asking that animals are not transported for more than eight hours in total. 1,103,248 signatures were handed in to the European Commissioner for Health in June 2012. Over 140 MEPs expressed support for Derecho Animal. Forum of Animal Law Studies, vol. 11/3 1 52 “Study visits to improve Member State controls on animal welfare during transport” of the Eu G. Conclusion 55 Report from the commission to the European Parliament and the Council on the impact of Council Regulation (EC) No 1/2005 on the protection of animals during transport” 2011 of the European Commission, page 12 56 In 2011, Animals´ Angels launched the 8hours petition, asking that animals are not transported for more than eight hours in total. 1,103,248 signatures were handed in to the European Commissioner for Health in June 2012. Over 140 MEPs expressed support for the campaign. The European Parliament adopted a Written Declaration No 49/2011 in March 2012, demanding the 8-hour limit. Derecho Animal. Forum of Animal Law Studies, vol. 11/3 133 133
https://openalex.org/W4389990497	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0295378&type=printable	English	null	Initial programme theory development: The first step in a realist evaluation of a cross-sectoral intervention for expectant Danish parents living with psychosocial risks	PloS one	2,023	cc-by	13,122	PLOS ONE PLOS ONE RESEARCH ARTICLE Initial programme theory development: The first step in a realist evaluation of a cross- sectoral intervention for expectant Danish parents living with psychosocial risks Sara Mandahl EllehaveID1,2, Louise Lund Holm Thomsen1, Marianne Stistrup Frederiksen1, Charlotte Overgaard3 1 Public Health and Epidemiology Group, Department of Health Science and Technology, Aalborg University, Aalborg East, Denmark, 2 Clinical Nursing Research Unit, Aalborg University Hospital, Aalborg, Denmark, 3 The Unit of Health Promotion, Department of Public Health, University of Southern Denmark, Esbjerg, Denmark a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 s.ellehave@rn.dk * s.ellehave@rn.dk OPEN ACCESS A distinct inequality in maternity care exists, and women with psychosocial risks are at a greater risk of adverse birth outcomes. In several high-income countries, a psychosocial risk assessment early in pregnancy is recommended so that expectant parents are offered an appropriate level of care which facilitates relevant, tailored interventions for those in need. In 2017, a cross-sectoral and interdisciplinary intervention for expectant parents with psycho- social risks was developed and implemented in the North Denmark Region. The develop- ment process of the intervention has not been reported and theory-based knowledge about how supportive interventions bring about change for expectant parents with psychosocial risks is scarce. Through the initial phase of a realist evaluation, we aimed to elicit key con- texts and mechanisms of change regarding the intervention for expectant parents with psy- chosocial risks. Through an initial programme theory, this article illustrates how, for whom and in which contexts the intervention is intended to work. Data is comprised of intervention documents, 14 pilot observations and 29 realist interviews with key stakeholders. A thematic analytical approach inspired by retroductive thinking was applied to identify and analyse pat- terns related to the incentive of the intervention, its structure, intended outcomes, generative mechanisms and contextual matters. Generative mechanisms responsible for bringing about change in the intervention were identified as healthcare professionals’ approach, con- tinuity, trust, early intervention and social network. Cross-sectoral collaboration and health- care professionals’ competencies were assumed to be central stimulating contextual factors. The initial programme theory developed in this study will serve as the basis for fur- ther refinement via empirical testing in a later phase of the realist evaluation. Citation: Ellehave SM, Thomsen LLH, Frederiksen MS, Overgaard C (2023) Initial programme theory development: The first step in a realist evaluation of a cross-sectoral intervention for expectant Danish parents living with psychosocial risks. PLoS ONE 18(12): e0295378. https://doi.org/10.1371/journal. pone.0295378 Editor: Doris Vero´nica Ortega-Altamirano, National Institute of Public Health: Instituto Nacional de Salud Publica, MEXICO Editor: Doris Vero´nica Ortega-Altamirano, National Institute of Public Health: Instituto Nacional de Salud Publica, MEXICO Introduction Maternal and child health is characterised by significant and consistent social inequality [1, 2]. Women with psychosocial risks due to, e.g., low educational level, poor living conditions, pov- erty, psychological distress, social isolation, substance abuse or exposure to violence or abuse are at an increased risk of maternal morbidity and adverse birth outcomes such as preterm birth, stillbirth, low birth weight and perinatal mortality [3–5]. Their children’s long-term health and life chances are also negatively affected [6, 7]. These women and their partners’ needs are often complex and antenatal services should therefore be individualised and tailored [8, 9]. Funding: This work was supported by the Danish Regions Funds for Research in Health Promotion and Illness Prevention [grant number A3293]. The senior author of the paper (CO) was financially supported by the University of Aalborg through the entire research period. The funders had no role in study design, data collection, and analysis, decision to publish, or preparation of the manuscript. There was no additional external funding received for this study. In several high-income countries, national guidelines recommend a psychosocial risk assessment early in pregnancy in order to assign expectant parents an appropriate level of care and offer relevant, tailored interventions [10–12]. The methods used for psychosocial risk assessment have however been questioned [13, 14]. Specifically, the use of checklists or close- ended questions may not allow expectant parents to share sensitive and potentially retrauma- tising issues thus preventing disclosure of important information or engagement with services [14]. Psychosocial risk assessment may furthermore trigger negative feelings of, e.g., fear, stigma or loss of self-esteem among expectant parents [15–18]. Awareness of such potential negative consequences of public health interventions is important to ensure that expectant parents receive quality care that meets their support needs. Interventions or programmes for expectant parents with psychosocial risks are rarely the focus of in-depth studies and often lack formal evaluation [9, 19, 20]. Competing interests: The authors have declared that no competing interests exist. In Denmark, maternity care is provided at four levels according to the nature and severity of parents’ psychosocial risk factors [11]. Following Danish national guidelines [11], the Regional Health Authority and the municipalities of the North Denmark Region developed and implemented a cross-sectoral and interdisciplinary intervention for expectant parents with psychosocial risks. Editor: Doris Vero´nica Ortega-Altamirano, National Institute of Public Health: Instituto Nacional de Salud Publica, MEXICO Editor: Doris Vero´nica Ortega-Altamirano, National Institute of Public Health: Instituto Nacional de Salud Publica, MEXICO Received: September 21, 2022 Accepted: November 21, 2023 Published: December 20, 2023 Copyright: © 2023 Ellehave et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: September 21, 2022 Accepted: November 21, 2023 Published: December 20, 2023 Copyright: © 2023 Ellehave et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Due to EU and Danish legislation, data cannot be shared publicly because the participants have not provided written, informed consent for the transcripts of their interview to be shared publicly. The transcripts contain medical information and confidential personal information, and it is not possible to fully anonymise the data due to the highly individual position and/or context of the participants. Researchers who meet the criteria for access to 1 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks confidential data can obtain it from the Contract Department of Aalborg University (contact: persondata@adm.aau.dk). PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 Competing interests: The authors have declared that no competing interests exist. Introduction This intervention, as assessed in this study, was delivered by midwives (regional sector) and health visitors (municipal sector) in 2017 [21] and is still currently used in the region. The target group for the intervention was expectant parents with complex men- tal health problems or social disadvantages of either an economic or social character [11, 21]. The intervention was organised into three sequential activities. First, all expectant women and their partners (if attending) would undergo a systematic psychosocial risk assessment by use of a dialogue-based, semi structured interview guide at the first antenatal midwife consultation in order to identify psychosocial risk factors as well as protective factors and resources in the fam- ily. If risk factors were identified, the expectant parents were invited for a cross-sectoral team meeting involving the parents, their health visitor and their midwife with the aim of planning a targeted and tailored support plan. Supportive services were then offered, ranging from high-frequency consultations with the health visitor or midwife to cross-sectoral and interpro- fessional services such as courses specifically tailored for parents with psychosocial risks and offered in small groups and with continuity of course leaders. Overall, the intervention was designed to improve the parenting and coping skills of the expectant parents and reduce adverse birth outcomes and perinatal mental health problems by, e.g., reducing parental stress levels [21]. The intervention was based on the notions of a local intervention reported to show promis- ing results [22]. It was adapted to the wider context of the North Denmark Region and upscaled to include all three maternity units and 11 municipalities in the region. Essential components were changed during the upscaling process. The adaption from a local to a regional setting was not preceded by scientific evaluation, nor were pilot or feasibility studies undertaken [21]. The intervention components have been described in a brief report targeting 2 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks health professionals [21] and in lay information material, but the development process of the intervention has not been reported in writing. Potentially unintended and negative conse- quences of the psychosocial risk assessment and service tailoring for expectant parents with psychosocial risks have been highlighted in recent studies [15, 17, 23]. These uncertainties challenge the adaptive and scalable potential [24] of the intervention and therefore warrant sci- entific evaluation. General study design y g We conducted the initial phase of a realist evaluation using a qualitative approach. The realist methodology draws on critical realism and scientific realism, respectively, [25, 26] and pro- vides a generative understanding of the causality of complex interventions through which explaining how and why an intervention works is possible [27, 28]. Critical realism projects the understanding of a stratified mind-independent reality, divided into three related domains —the real, actual and empirical–and focuses on exploring, via retroduction and abduction [29], underlying, mind independent structures and mechanisms (the real domain) that when activated by certain stimulating contexts will generate outcomes (the actual domain) that potentially become observed (the empirical domain) [28, 30]. Critical realism hence contrib- utes a vertical, ontologically deep and generative understanding of causality rather than the more linear, simple explanation of causal relations [31]. Scientific realism shares the under- standing of a mind independent reality being accessed via retroduction [32] and further con- tributes to realistic evaluation through the notions of the nature of reality being captured by scientific theories and of the best, most credible theories approximating the truth [26, 32]. The purpose of realist revaluation is thus to generate ontologically deep scientific explanations about how a programme works by unpacking the generative mechanisms and their conducive contexts responsible for outcomes of interest as well as unintended outcomes of a programme [32]. More specifically, a realist evaluation goes a step further than standard causal explana- tions by allowing us to understand how the effects of a programme are produced by opening the “black box” and via constructs of context-mechanism-outcome configurations (CMOc) illustrating what (and how) underlying mechanisms and structures affect the process between input and outcome [33]. A context is defined as the external individual, organisational or envi- ronmental circumstance or condition in which the programme is implemented [28]. Mecha- nisms are defined as the resources provided by a programmes’ modalities and the way people respond to those resources [28, 32, 34], while outcomes are defined as the intended and unin- tended effects generated by certain combinations of contexts and mechanisms [28]. CMO con- figurations make the substance of the programme theory in realist evaluations [28], which is an essential prerequisite of realist evaluation [28]. Introduction The aim of our study was to elicit an initial programme theory as the first phase of a realist evaluation of the intervention for expectant parents with psychosocial risks in the North Denmark Region. PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 Data collection Following the realist evaluation cycle, the first step represents the theory gleaning phase, also referred to as theory elicitation [28, 38] (Fig 1). During this phase, we developed an initial pro- gramme theory which captured the assumptions of key stakeholders and programme develop- ers regarding how the intervention for expectant parents with psychosocial risks is intended to work. To obtain and articulate a preliminary understanding of how, why and under what cir- cumstances the intervention works, we adopted an elucidating research approach which allowed for a row of data collection methods and sources of information [39]. First, formal doc- uments describing the intervention were collected from key stakeholders. These documents, included the written reciprocal health agreement framing the intervention, an implementation guideline for the intervention and the interview guide used for the systematic dialogue based psychosocial risk assessment [21]. Additionally, the first author conducted supplementary searches of relevant databases (Google Scholar, PubMed, Scopus, Sociological Abstracts) and web pages (Australian Government–Department of Health and Aged Care, National Institute for Health and Care Excellence, and Danish Health Authorities [40–42]) using the search terms “intervention”, “programme”, “guideline”, “antenatal”, “perinatal”, “pregnant” and “psychoso- cial risks”. These searches were made to identify similar interventions worldwide and interna- tional evidence or guidelines regarding support for expectant parents with psychosocial risks and helped us identify key policy reports and relevant research articles on the topic. To further enhance and articulate our preliminary understanding of relevant contexts and mechanisms triggering the outcomes of the intervention, pilot observations [43] of the delivery of key modalities of the intervention were conducted. Two authors (SME & LLHT) observed midwifery consultations with psychosocial risk assessment of expectant parents and team meetings. These observations were made with the overall purpose of the researchers learning about the field and allowed us to gain insight into the delivery of the intervention and the dynamics between expectant parents and midwives and/or health visitors. Twenty-nine realist interviews were then conducted [38, 44, 45]. The interviews were, like the observations, con- ducted by two authors (LLHT & SME) both of whom were experienced in qualitative research methodology. The interview guide was developed with the purpose of strengthening and elab- orating the initial CMOcs elicited from the intervention documents and research articles. The interview guide started off with exploratory questions with attention being given to particular context as the intervention was implemented across multiple settings. General study design The overarching theory or model of how an intervention is expected to work is described as a programme’s rationale and assumptions about contexts and mechanisms that connect the programme’s inputs to outcomes–both intended and unintended [35]. The ‘theory’ in a programme theory “may be an articulation of practice wisdom or of tacit assumptions–that is, not only a formal, research-based theory” [33, p. 33]. The programme theory we developed drew on Funnel & Rogers’ elaboration of how to present programme theories [36]. Intended and unintended effects of the intervention may also become apparent through in-depth theorisation of a programme theory [28, 37]. Knowl- edge about both the intentional and unintentional workings of an intervention is a consider- able advantage of adopting the realist approach. This is partly because the contribution will 3 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks Fig 1. Process of the gleaning phase: development of the initial programme theory. https://doi.org/10.1371/journal.pone.0295378.g001 Fig 1. Process of the gleaning phase: development of the initial programme theory. https://doi.org/10.1371/journal.pone.0295378.g001 ensure that the benefits of the intervention outweigh its harms and partly because a detailed understanding of generative mechanisms may facilitate refinement and improvement of the intervention and thus enable upscaling and replication processes [28]. Following the under- standing of realist evaluation, i.e., that people are not passive recipients of interventions and that a programmes’ ability to bring about change is highly dependent on its participants’ com- mitment, [28] adopting a realist perspective on evaluation of the intervention for expectant parents with psychosocial risks also enhanced our attention regarding social interaction between the expectant parents and healthcare professionals (HPs). PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 Data collection This was followed by more targeted questions which explored the impact of contextual factors and mechanisms through questions such as “Please try to explain to me what you think it is in the aforementioned PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 4 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks activity that makes the pregnant woman benefit from it?” and “In your experience, what circum- stance are in place for this activity to be successful/when this activity is not successful?” Finally, questions about potentially unintended consequences of the intervention were asked, e.g., “Are there any situations you can think of where this (activity) does not lead to the intended effect?”. The interviews lasted between 32–57 minutes. Parallel to the realist interviews, SME and LLHT observed 14 antenatal midwife consulta- tions and team meetings that aimed at strengthening the refinement of elicited contexts and generative mechanisms important to the intervention and eliciting potential new ones. The authors were focused on both verbal and physical behaviours. Questions like “How is the mid- wife’s phrasing concerning the assessment for psychosocial risk factors?”,”How does the pregnant woman respond (verbally and non-verbally)?” and “What is the role division between health visi- tor and the midwife during the team meetings?” served to focus our observations which were documented as field notes [43, 46]. Realist interviews and observations were conducted over an eight-month period (from November 2020 to June 2021) with the aim of gaining a deep insight into the stakeholders’ assumptions regarding the workings of the intervention. Due to COVID-19 restrictions, not all midwife consultations allowed for observations due to small consultation rooms. Most informants were interviewed via Microsoft Teams or by telephone. Nonetheless, some face-to- face interviews were completed. For the final part of the theory gleaning phase of the evaluation and to decide on in- and exclusion of content in the initial programme theory, frontline workers and other key stake- holders were invited for workshops where researchers and programme providers met face-to- face to comment, discuss, adjust and refine the initial programme theory. PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks Table 1. Characteristics of data collecting activities and informants contributing to the initial programme theory. Data collection activity Informant ID Professional characteristics of informants Total number of informants Regional level individual interviews I_1 Administrative/regional level manager of the intervention 1 I_15 Representative of general practitioners 1 I_16 Obstetrician 1 I_2, I_3, I, 11 Chief midwives 3 I_5, I_9, I_17, I_18, I_19, I_20, I_23, I_24, I_25, I_26, I_27, I_28 Frontline midwives 12 Municipal level individual interviews I_6, I_7, I_8, I_12, I_13, I_14 Health visitor managers 6 I_4, I_10, I_20, I_21 Front-line health visitors 4 I_29 Health visitor specialist 1 Pilot observation ID Type of activity Total number of activities Regional level pilot observations PO_1, PO_3 Midwife consultations–psychosocial risk assessments 2 PO_2, PO_4, PO_5, PO_6, PO_7, PO_8, PO_9, PO_10, PO_11, PO_12 Team meetings 12 Workshop ID Professional characteristics of participants Total number of participants Regional and municipal level workshop W_2 Frontline midwives and health visitors, a health visitor manager, a chief midwife, and a vice chief midwife 19 Regional level workshops W_1 Front-line midwives and a chief midwife 10 W_3 Front-line midwives and a chief midwife 21 Municipal workshops W_4 Front-line health visitors and a health visitor manager 14 W_5 Front-line health visitors and a health visitor manager 6 W_6 Front-line health visitors and a health visitor manager 6 W_7 Front-line health visitors and a health visitor manager 13 W_8 Front-line health visitors 5 W_9 Front-line health visitors and a health visitor manager 9 https://doi.org/10.1371/journal.pone.0295378.t001 Sampling and recruitment Programmes are often developed and implemented within organisations, and members of an organisation are thus essential for explaining the anticipated impact of a programme [36, 47]. The intervention was implemented in the entire North Denmark Region and comprised of three maternity units, midwifery out-patient clinics in all larger towns in the region and 11 municipal health visiting units. Eight of the 11 regional municipalities agreed to participate in the initial phase of the evaluation. We strove to achieve maximum variation in the data to accommodate expected implementation differences. Using a purposive sampling strategy [48], informants were recruited for interviews because of how their position within healthcare related to the intervention. Considering that this was the initial phase, informants represented expert level of experience with the intervention and were key representatives from the regional reference group. These experts included the regional project manager, chief midwives from each of the three maternity units, managers of the involved municipal health visiting units, a representative of general practitioners and a regional outpatient clinic for families with psy- chosocial risks. Informants primarily represented the decision level to secure insight into the incentives for and the development and implementation of the intervention [28, 36]. Key stakeholders from the decision level helped identify other relevant stakeholders who were actively engaged in the intervention through their daily practice; hence, more organisational levels were represented. The informants were considered to hold similar professional charac- teristics, emphasising a sample with high specificity [49]. Informants holding characteristics that were highly specific for the study aim underpinned the information power [49]. Twenty- nine informants were recruited for participation in the realist interviews (Table 1). Expectant parents will be included in a subsequent sub study with the aim of testing some of the initial programme theories derived in this current study. 5 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 https://doi.org/10.1371/journal.pone.0295378.t001 Data management and analysis The different sources of data (Fig 1) played different roles in the process of uncovering genera- tive causation. The formal documents describing the design and implementation strategy of the intervention was analysed mainly to identify themes attributed to the deliverers of the intervention, the intervention modalities and expected outcomes. The studies identified through the initial literature search were reviewed regarding the identification of CMOcs for development of the initial programme theory. As these studies mainly covered parents’ per- spectives, the studies provided invaluable insights into the contexts and mechanisms poten- tially responsible for the workings of the intervention from the perspectives of expectant parents in particular. The pseudonymised field notes obtained during the observations of both midwife consulta- tions and team meetings were all read thoroughly several times by the first and second author (SME & LLHT) before being analysed using the thematic analysis approach [50]. These data provided important insights into the design and implementation of the intervention as well as the unspoken (not formally written) characteristics of contextual circumstances and generative mechanisms being responsible for the workings of the intervention. All interviews were pseu- donymised in pursuance of EU legislation on data protection [51], transcribed verbatim and 6 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks imported into the Qualitative Data Analysis Software NVivo 11 by one author (SME). We used a thematic analysis approach [50] to identify and analyse data patterns in the interview transcripts. The analysis was performed using researcher triangulation. Two researchers (SME & LLHT) individually coded the transcripts following a coding manual. Initial codes were gen- erated abductively based on the realist understanding of elements in a programme theory [28, 52, 53]. Data excerpts related to the theoretical background of the intervention, activities embedded in it, possible mechanisms, intended outcomes and influential contexts were coded. Any disagreements or ambiguities related to how to code data were discussed by the team. The thematic analysis was an iterative process during which we checked if the codes worked in relation to the entire data set and captured new emerging mechanisms. Coding occurred when an observable CMOc or parts of it was found in the data and memos were added in NVivo for review/refinement. Data management and analysis Thus, the analysis evolved inductively when new patterns added to con- texts, mechanisms or outcomes as they appeared from the data. The identified themes formed the foundation for formulating hypothesised CMOcs using retroduction. Within the realist tradition, retroduction includes investigating the underlying generative mechanisms leading to potential outcomes even though these mechanisms may not be directly observable empiri- cally [27]. Retroduction is an empirical process of developing a theory which requires moving transcendentally from information collected by a researcher regarding a concrete phenome- non to the reconstruction of the basics for a deeper causal understanding [29, 54]. We there- fore used our spontaneous interpretations [29] of information gathered through the formal documents of the intervention, other research on the topic, our field observations and inter- views to generate our hypotheses of the generative causation of the intervention for expectant parents with psychosocial risks. We did this by attentively exploring how certain outcomes were expected to arise by both stakeholders and developers. We also examined what it was about the midwives’ and health visitors’ actions and practices they assumed would matter for reaching intended outcomes [32]. To assist this retroductive inquiry, we aimed to identify the mechanisms responsible for the effects of the intervention in certain contextual settings as inspired by Dalkin et al.’s (2015) elaboration detailing how resources and reasonings are mutu- ally constitutive of a mechanism. We therefore disaggregated the mechanism into resources and reasonings whereby mechanisms could be identified by looking for statements indicating how a resource of the intervention introduced among the expectant parents with psychosocial risks was expected to trigger a change in their reasoning eventually forming the outcome. We looked for patterns in the data and were thereby able to connect resources and reasonings to outcomes that were related to certain contextual factors and were thus able to develop the CMOcs of the intervention. Any CMOcs resembling already identified CMOCcs were com- bined and, if appropriate, further refinement occurred. Th ti f th t d f ll d th COREQ lid t d it i f ti lit The reporting of the study followed the COREQ consolidated criteria for reporting qualita- tive research [55] and reporting standards for realist evaluations [56]. Results Five dominant CMOcs suggesting different pathways through which expectant parents with psychosocial risks and their partners were expected to achieve the intended outcomes as well as potential unintended consequences were derived. They are visualised in the programme theory presented in Fig 2. Ethical considerations Under Danish legislation, qualitative studies are based solely on informed participant consent and are not subject to approval by a national health research ethics committee [57]. This study was complied with the General Data Protection Regulations of the European Parliament and the Council of The European Union [51]. In accordance with the principles outlined in the Helsinki Declaration [58], all informants were informed about the purpose of the study, how their data would be protected and that they had the opportunity to withdraw from the study at any time for any reason. Subsequently, all informants gave their written informed consent to participate [51]. 7 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks Fig 2. Initial programme theory of the cross-sectoral intervention for expectant parents living with psychosocial risks in the North Denmark Region. Elements relating to the organisational part of the programme theory are marked with dotted lines. Potential unintended consequences are marked with the lighter coloured lines. https://doi.org/10.1371/journal.pone.0295378.g002 Fig 2. Initial programme theory of the cross-sectoral intervention for expectant parents living with psychosocial risks in the North Denmark Region. Elements relating to the organisational part of the programme theory are marked with dotted lines. Potential unintended consequences are marked with the lighter coloured lines. https://doi.org/10.1371/journal.pone.0295378.g002 https://doi.org/10.1371/journal.pone.0295378.g002 CMOc 1 –Acknowledgement and respect Previous experiences with the social services (C) were identified as a generic contextual factor with the potential of affecting the woman and her partner’s inclination to engage with HPs during the psychosocial risk assessment and team meetings (O) as explained by a health visitor manager: They live on the experiences they carry: If dad has been in a marriage previously where the child was removed, then of course there is resistance (I_8). HPs were aware that engaging with the intervention was not necessarily easy for parents due to their past experiences and that there was an inherent power imbalance in the relation- ship with the HPs: PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 8 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks The parents are well aware that this is about them becoming parents and that is where the fear comes in[. . .] We can work with their fear, but I don’t think we can eliminate it. I don’t think we are able to remove the premise and the imbalance or structure of power that lies within this assessment (the psychosocial risks assessment), but we can work with their fear and communicate openly about the purpose of the assessment. Namely, we give the parents the best conditions for pregnancy and parenthood (I_2). Due to parents’ past experiences and the potential fear they may have felt, HPs’ knew that their approach towards the parents was important to make them feel safe and secure. HPs’ showing acknowledgement and belief in the couple as capable parents (M resource) and sup- porting their confidence was assumed to sustain self-belief and subsequently sense of parental skills (O). A midwife explained how she found a respectful approach crucial when she was asked about the generative mechanism for a trustful relationship between herself and the expectant parents: When the screening and dialogue work well, she (the pregnant woman) will leave the con- sultation with a sense of trust of the system, trust in the midwife, and a feeling of empower- ment in relation to what she has talked to the midwife about. She will be confident in the midwife being there for her regarding what she brought up at the consultation [. . CMOc 1 –Acknowledgement and respect .] (And she may) experience that the midwife has helped her by pointing out precisely what her sit- uation is, which is important to recognize when having a child and to her position in gen- eral. The mother may feel that she is not on her own while dealing with her challenges. She may feel that the midwife wishes her well and that she [the midwife] can help her receive support from the system, e.g., gain a social network, speak to the right healthcare workers, and receive the support she needs (I_3). This excerpt illustrates the assumption that a respectful, non-judgmental and empowering attitude on the part of the HPs (M resource) may allow the expectant parents to feel under- stood and safe enough in their relationship with the HPs (M reasoning) to open up, share sen- sitive information and discuss support needs (O). Another excerpt showed how sensitive building the initial trust can be: When I get close to what is sensitive, I am rejected. I point out the vulnerabilities and then it is not me they want to visit the next time [. . .] When I get close to the heart, I have experi- enced [being rejected] a few times since we started screening the families. It may be because I am too crude, too old . . .or because I dare to ask these questions. That is a little difficult, actually (I_27) Overall, acknowledgement (M resource) and sensitive articulation of the expectant parents‘ potential fear of being labelled as inadequate or being reported to the social services and even- tually having their child removed was described as essential for the parents to feel safe in the relation to the HP (M reasoning) and was assumed a necessity for them to engage with the HPs and for the effect of the intervention. PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 And: It is our experience that we will not be able to build a trustful relationship with the couples until we are able to help the pregnant woman or her partner comprehend what we are doing, why we are doing it and what they may get out of it (I_3). These citations underpin the assumption that HPs who speak openly and are informative (M resource) may sustain a feeling of respect and eventually trust in their relationship with the expectant parents (M reasoning). A midwife explained how clear communication and the tim- ing of the risk assessment during the first consultation was important for her to accommodate expectant parents: Telling them the purpose is one of the most important things I do to make them feel safe enough to share with me [. . .] I also put an effort into establishing a relation before con- ducting the psychosocial risk assessment. When I was a student and introduced to the assessment, I sat with a midwife who did it for the first time, and I had to learn to do it [myself]. Back then, we did it as the first thing when they arrived. I do not do that any lon- ger because I think building the relationship is crucial for me to be allowed to ask these questions and allow them to build trust in me. Therefore, I always place it last (I_26). The midwives expressed how they frequently felt uncomfortable and inadequate when assessing the expectant parents for psychosocial risk factors. These experiences were generally shared by the midwives, and many referred to the interview guide as overwhelming. A midwife at the workshop expanded this perspective in the following manner: That is how I experience it, too; it gets better the more you practice. In the beginning, I asked my colleagues and sought professional feedback quite a bit because I found that it was uncomfortable. I thought it was a strenuous task. I did not think it was my responsibil- ity at all (Midwife during workshop W_1). The data showed how specially trained HPs with qualified communicative skills (C) repre- sent important moderating contextual factors to ensure well-timed and informative communi- cation (M resource) assumed to be essential in making expectant parents feel safe (M reasoning) and share their stories (O). CMOc 2 –Clear and careful communication Having a history with social services (C) and experiencing uncertainty or even fear in the pres- ence of authorities was assumed to sometimes lead expectant parents to feel a loss of control or feelings of unworthiness as parents. Clear and careful communication during psychosocial assessment and team meetings (M resource) was seen to contribute to the expectant parents’ PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 9 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks feelings of being heard, valued and respected as well as their ability to feel as if they were in control of their lives (M reasoning) in general. Feeling in control, valued and respected was assumed vital for expectant parents having a voice which pointed to their perpetual right to choose and framed engagement with the intervention as safe and harmless (O). Accordingly, it was considered essential to build trust for the expectant parents by HPs being transparent about the purpose of the meeting: When we talk about what it is. . . why are we here. [. . .] It is about articulating why we are doing the things we are, so that there is no hidden agenda. [. . .] It is all about being open about the things we are doing so that the expectant parents have no doubt about our position. . . They will have no reason to think that I am doing anything behind their back. . . [. . .] This gives a trusting collabora- tion (I_5). CMOc 3 –Continuity of caregiver In the target group, many had multiple interactions with professionals and various authorities in relation to childbearing and other aspects of their lives. In some cases, these contacts led to distrust. Some women or their partners suffered from anxiety, social phobia, a history of loss or neglect which led to them being challenged regarding their ability to build relationships and trust in general. Negative experiences with professionals (C) together with challenged social networking abilities (C) were considered contextual factors that may affect expectant parents’ inclination to engage with HPs (O). Care by known HPs (M resource) was described as an essential generative mechanism that strengthened expectant parents’ inclination to reach out and share their experiences (O), as familiarity and predictability were assumed essential for expectant parents to feel comfortable and safe (M reasoning). This made these characteristics a prerequisite for building trust with the HPs (O). How a sense of security and familiarity with HPs was perceived by HPs as important for the woman and her partner is illustrated by the fol- lowing excerpt: A sense of security is linked to the fact that they know us. You will have the courage to join the cafe´ though you are challenged and do not attend frequently (I_5). Knowing the HP may affect expectant parents with psychosocial risks and encourage them to feel safe and attend services which may otherwise trigger a sense of insecurity in these parents. One health visitor manager explained how they try to avoid change in caregiver to accommodate for continuity: We believe in the value of the professional relation and the security that lies within this rela- tion. Therefore, we strive to work in a way that allows us to avoid shifts in the professional relations if possible. That is a sustaining principle to us (I_7). Continuity of HP was described as better facilitating tailored and more individualised ser- vices, as continuity ensured a well-prepared HP held in-depth insights into the family’s chal- lenges and support needs. Continuity of HP (M resource) was thus considered a key mechanism for expectant parents to experience the intervention as meaningful (M reasoning). Not having to repeat one’s story to shifting HPs was additionally described as triggering feel- ings of meaningfulness, comfort and trust on the part of the expectant parents (M reasoning). And: For the midwives to be able to ensure well-timed and informative communication, and covering all the themes in the interview guide, having 10 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks enough time for the psychosocial risk assessment was pointed out as extremely important. Time available (C) was thereby identified as another contextual factor affecting the intervention. CMOc 3 –Continuity of caregiver This assumption was supported by an observation of a team meeting (PO_4) with a woman with prior postnatal depression at which the woman shared her prior experiences of disconti- nuity and of receiving care from three different health visitors. She explained how discontinu- ity did not allow her to develop a trusting relationship causing her to refrain from maternity care. CMOc 4 –Early initiation Let’s pretend that you have a relationship and share your innermost thoughts with a completely new caregiver right after you have given birth and have arrived home. I think having met before, can make it a little easier for both the mother and the health visitor (I_2). I think it makes sense to a lot of pregnant women that we are initiating support in their pregnancies. They may have issues that they worry about in their pregnancy that need to be talked about. That is why it is good to intervene early. It is important for our relationship. Let’s pretend that you have a relationship and share your innermost thoughts with a completely new caregiver right after you have given birth and have arrived home. I think having met before, can make it a little easier for both the mother and the health visitor (I_2). Meeting the health visitor or other relevant actors before birth was assumed to emphasise feelings of being offered coherent and safe support (M reasoning). Again, these reasonings were described to facilitate trustful relationships between the expectant parents and the HPs and to increase the parents’ chances of benefitting from the intervention (O). Health visitors and midwives alike described that initiating pregnancy visits by health visi- tors and performing a coherent intervention relied on well-functioning cross-sectoral collabo- rations as expressed in the following excerpt: I think it adds to our job satisfaction knowing each other well. My colleague (a health visi- tor) takes over for me in the situations that I find difficult. In other situations, I may be the one to provide expert knowledge or suggest a different approach. We support each other through thick and thin (I_19) Settings where cross-sectoral collaboration was close and integrated through light commu- nication pathways and frequent meetings between HPs (C) were thus identified as positive contexts promoting the implementation of a coherent intervention. CMOc 4 –Early initiation Another generic contextual factor identified in the data was the so-called “Window of opportu- nity” (C), as women were assumed to have a higher degree of mental surplus early in preg- nancy than late in pregnancy when focus is on birth-related issues and practical planning. Therefore, the time of initiation was assumed to affect the ability of the intervention to bring PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 11 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks about change. Including the women or couples in the intervention early (M resource), i.e., in gestational week 15–18, was hence assumed to evoke the expectant parents’ wish to give their child the best possible start in life, trigger reflections about their coming role as parents and motivate them to make potential changes in life (M reasoning) as outlined by a health visitor: When you get pregnant and are expecting your first child. . ., well, then the mental struc- tures are open and that is the most wonderful room for collaboration, because there is also a strong desire to do better than. . . or to do the best you have learned. There is an opening for collaboration, which is unique in this exact part of life (I_4). In this excerpt, early intervention (M resource) is described as enhancing the chances for engaging the expectant parents (O). More informants pointed out how addressing the families’ needs early in pregnancy (M resource) was assumed to increase the likelihood of a good initia- tion of the collaboration and prevent psychosocial risks from developing into circumstances that would be harmful for the children. Positive, motivating experiences at this stage of life (M reasoning) were considered strong determinants of future positive maternal health and wellbe- ing (O) and thereby of positive parenting and expedient child development (O). Early initiation of the intervention (M resource) was also assumed to facilitate gentle bridg- ing between sectors as described by this midwife: I think it makes sense to a lot of pregnant women that we are initiating support in their pregnancies. They may have issues that they worry about in their pregnancy that need to be talked about. That is why it is good to intervene early. It is important for our relationship. CMOc 5 –Social networks Both the existence and quality of networks (C) were experienced by the professionals to affect the expectant parents’ possible intervention outcomes. Some expectant parents in the target group may have been challenged in their ability to form and sustain social relationships, 12 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks leaving them with a compromised social network. Referring the expectant parents to group courses that typically included attendance of a known HP in informal settings facilitated a social network in safe and non-judgmental surroundings (M resource) as described by a mid- wife facilitating a baby cafe´ for women with psychosocial risks: The cafe´ can be used to facilitate a maternal group because it is a neutral place with no demands. They can come as they are (I_6). Ultimately, with the accessibility of such a social network, the expectant parents were more likely to experience a sense of commonness and a feeling that they were important to another person (M reasoning). This was expressed by a health visitor in an interview: Something great about a group course is the development of networks. Suddenly, they make friends [. . .] Many of them are lonely [. . .] They make friends, someone they can talk to, and I know that the health visitor has seen them walk together [. . .] The part about get- ting to know a few, whom you have something in common with, that is great (I_13). Experiencing commonness and a sense of being important to another person was consid- ered mechanisms for the expectant parents to benefit from the intervention, as this feeling of commonness was described as generating mirrors, allowing for the exchange of knowledge among parents (O). An informant stated: The group dynamics are essential as they learn from each other. We see how they learn from each other [. . .] What is going on in these groups add so much more than us visiting the family at home. They are meeting somebody else who more or less face similar chal- lenges being a mum or dad, or who also carries a complicated personal story. Group dynamics are highly essential in this matter (I_14). CMOc 5 –Social networks Group dynamics were formed by participants with different personalities who had issues fundamentally rooted in the same challenges, which was essential for expectant parents to feel safe and engage in the group sessions. Social networks (M resource) were thus considered as contributing to sustaining the expectant parents’ benefit from the intervention (O). PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 Discussion We applied a realist methodology to identify configurations of contextual factors, mechanisms and outcomes articulated by key stakeholders. Through the development of an initial pro- gramme theory, the realist methodology took our findings deep and suggested CMOcs that unearth explanations of how this complex intervention may work, for whom and under which circumstances. The knowledge of how interventions work often remains silent. By eliciting configurations of context, mechanisms and outcomes, this study contributes with the initial step of a realist evaluation that supports the development of an explicit middle range theory base for early interventions for expectant parents with psychosocial risks. The initial pro- gramme theory is left to be tested and refined in different contexts with attention being given to both intended and unintended outcomes as well as harmful consequences thus contributing to the safety of such interventions. CMOc 1 suggests that acknowledgement, use of non-judgmental communication and a respectful, sensitive approach by HPs are generative mechanisms for the intervention to pro- duce effect. This finding is in line with other recent studies that show such a care approach is key to ensuring that expectant parents with psychosocial risks engage with antenatal services PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 13 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks [18, 23, 59] as experiences of being judged or labelled by HPs are frequent among this group [15, 17]. Being offered participation in a supportive intervention may be a stigmatising experi- ence for expectant parents with psychosocial risk factors since the professional risk-assessment may not be in line with their own assessment and may clash with their self-image [17, 60]. The intervention may also negatively affect their self-belief and perception of parental skills [15, 23]. As reported by Mule et al. [18] and Frederiksen et al. [17], fear of being judged or per- ceived as bad parents may lead expectant parents to not share important information with pro- fessionals or to avoid disclosing problems they are experiencing. These studies underpin why HPs emphasise the value of keeping a respectful and non-judgmental approach when caring for expectant parents. They also help identify a potentially unintended consequence of the intervention, i.e., that expectant parents identified with psychosocial risk factors may feel stig- matised or labelled into a category they do not recognise. Discussion CMOc 2 shows that clear and well-timed communication may contribute to a feeling of being in control among expectant parents with psychosocial risks. Accordingly, Rayment- Jones et al. found that women with complex social risk factors may perceive maternity services as a system of surveillance rather than as support and that building relationships with HPs may be intimidating and appear as a loss of control in their lives [59]. Freedom of choice in antenatal interventions is questioned by some authors [61]. Specifically, these authors found that offers of support are not always experienced as optional [61] due to the power relation that evolves within all social relations [62]. This reflection emphasises that expectant parents with psychosocial risks may attend supportive services, not simply from freewill but due to a fear of consequences if they decline to participate. Another potentially unintended conse- quence of the intervention may be that expectant parents may feel forced to attend the inter- vention regardless of their personal interests. Their concern and distress may furthermore be exacerbated by worrying about possible implications related to sharing of sensitive informa- tion or declining participation in supportive services. g p p pp Through CMOc 3, continuity of caregiver was described as an important generative mecha- nism for expectant parents feeling safe and developing trustful relationships with their care providers. With continuity of HP as a central ingredient, the assumption underlying this CMOc gains support from studies which show how antenatal care programmes based on con- tinuity of care provider appear to be successful in addressing complex care needs of the women with psychosocial risk factors thereby improving birth outcomes such as gestation and birth weight [19, 63, 64]. An ongoing relationship between a patient and a care provider, defined as relational continuity [65], does not constitute continuity or ensure care coherence alone. Other important aspects relate to transfer of information (information continuity) and the overall organisation of care (management continuity) [65]. This is especially important when care is received from various providers [65], as seen in the intervention discussed here, and management continuity therefore becomes extraordinarily important. This finding was confirmed in a recent study of continuity of care for expectant parents receiving the present intervention [66]. It is thus likely that a systematic approach to cross-sectoral collaboration combined with well-established collaboration between the involved HPs may bring about a coherent and meaningful antenatal care pathway. Discussion This perspective emphasises a key intention of the intervention: Bridging the gaps between the sectors and fostering a gentle transition from midwife (regional service) to health visitors (municipal service) and other relevant municipal or regional actors to whom the pregnant woman may be referred. Through CMOc 3, continuity of caregiver was described as an important generative mecha- nism for expectant parents feeling safe and developing trustful relationships with their care providers. With continuity of HP as a central ingredient, the assumption underlying this CMOc gains support from studies which show how antenatal care programmes based on con- tinuity of care provider appear to be successful in addressing complex care needs of the women with psychosocial risk factors thereby improving birth outcomes such as gestation and birth weight [19, 63, 64]. An ongoing relationship between a patient and a care provider, CMOc 4 is closely linked with CMOc 3 as early initiation leaves time for early introduction to relevant care providers, the identification of needs and initiation of support, collaboration and continuity across sectors, and services when parents are open to change. In accordance with research literature [65, 67], early initiation also supports the building of trust as an PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 14 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks outcome of continuity because it leaves time for familiarity between parents and caregivers. Continuity of care models have been identified as important in the contexts of detecting peri- natal mental health problems, leaving room for establishing a trustful relationship and giving HPs the possibility to draw on previous encounters with the family [19, 67]. Continuity of care models are, however, time demanding. Considering that midwives and nurses in perinatal care settings indicate limited time as the most dominant barrier to addressing mental health issues with pregnant women [68], attention should be given to potential challenges such as time restrictions when aiming to provide continuity in the care for the expectant parents in the intervention undergoing this evaluation. CMOc 5 suggests that available social networks may lead to better learning opportunities for parents because of both the chances for mirroring and exchanging of knowledge and the positive and supportive aspect that a feeling of belonging provides. This hypothesis is further emphasised by Berkman et al. Discussion [69] who elaborate on social networks as an opportunity for social support which has been shown to impact health behaviour. A key element in Berkman et al.’s theory is the distinction between supportive and non-supportive social relations [69] as exemplified through research literature showing that perceived social support is a significant protective factor for maternal mental health including post partum depression [70]. Thus, research literature supports our findings by emphasising the positive impact of social support among new parents. Our findings suggest that relational difficulties exist particularly among expectant parents with psychosocial risks. Components of the intervention acting to increase accessibility of social networks are therefore assumed to provide opportunity for expectant parents to reach out to other likeminded parents to fulfil their need for support and belonging. For whom, how, why and under what circumstances these opportunities may work will be tested in the evaluation, which is ongoing. HPs possessing specialised knowledge and communicative skills are suggested as an impor- tant moderating contextual factor in the initial programme theory. This assumption is sup- ported by Turienzo et al. [64] who found that organisational infrastructure, such as training and support of the staff and access to guidelines, was essential to ensuring high quality of care and for addressing the complex care needs of pregnant women with either low socioeconomic status or social risk factors. In building the programme theory, front-line midwives repeatedly highlighted the importance of training and experience affecting their delivery of the interven- tion with confidence. The importance of this contextual factor is underpinned by international studies reporting that midwives do not feel equipped to provide mental healthcare or perform psychosocial risk assessments [67, 68, 71]. The same studies also provide support for the assumption that having enough time to build trust and conduct quality psychosocial risk assessment is important for ensuring that expectant parents experience the intended interven- tion outcomes. Additionally, studies indicate that limited ability to perform quality maternity care may be a source of stress, reduce job satisfaction and ultimately cause job resignation [72, 73] among HPs. Such potentially unintended and harmful consequences warrant further attention in the theory-testing phase of this evaluation. From a socioecological perspective [74], the intervention mainly works at an individual level and targets expectant parents with psychosocial risks. PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 Discussion The initial programme theory, however, indicated pathways reaching beyond the expectant parents’ engagement with the intervention, including organisational level components such as improved professional com- petencies and cross-sectoral collaboration and the accessibility and quality of a supportive social network. Early indications suggest that there may be differences in parents’ experiences depending on their psychosocial risks and life experiences influenced by structural, organisa- tional and individual contextual factors. 15 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks The intervention positions itself as a high-risk prevention strategy [75] as it aims to improve the maternity care for expectant parents who are at a high risk of experiencing adverse birth outcomes and negative maternal and child health outcomes. The intervention strives to reduce this risk by improving the woman and her partner’s ability to attach to their child. This is achieved by supporting and improving their coping ability and parental skills via extended and/or shared consultation, health visits and individual- and group based tailored services. “The priority of concern should always be the discovery and control of the causes of disease” [71: p. 432]. By taking into account that health is widely influenced by distal, societal structures, the effect and—more importantly—the sustainability of an intervention may be supported by focusing on eliminating fundamental causes of disease rather than proximal risk factors [76, 77]. According to the initial programme theory, the intervention is identified with only limited focus on more fundamental causes of disease. Following both a complex system [78] and a socioecological perspective [74], a risk exists that the intervention was designed to focus on casual pathways that do not hold the power to provide sustainable, long-term outcomes. Another limitation of high-risk prevention strategies is their dependency on the individual’s contribution thus leaving the intervention user with the responsibility for benefitting from the intervention [75]. In this respect, the intervention may cause an effect opposite to what was intended and increase social inequality in maternity, as expectant parents with psychosocial risk factors may not have the resources to meet the demands embedded in the intervention. PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 Conclusion This study constitutes the starting point of an evaluation of an intervention for expectant parents with psychosocial risk factors by theorising how, for whom and under which circum- stances it is expected to work. The intervention is expected to reduce adverse birth outcomes and support positive child development. Through the initial programme theory, we identified the key assumption that trust building is essential for expectant parents living with psychoso- cial risks. Trust building allows expectant parents to feel sufficiently safe to engage with the ser- vices thereby improving their parental skills and ensuring that they feel less stressed. This study constitutes the starting point of an evaluation of an intervention for expectant parents with psychosocial risk factors by theorising how, for whom and under which circum- stances it is expected to work. The intervention is expected to reduce adverse birth outcomes and support positive child development. Through the initial programme theory, we identified the key assumption that trust building is essential for expectant parents living with psychoso- cial risks. Trust building allows expectant parents to feel sufficiently safe to engage with the ser- vices thereby improving their parental skills and ensuring that they feel less stressed. Qualifications of the HPs, such as their communicative skills, were identified as contextual fac- tors expected to stimulate an acknowledging, respectful and non-judgemental approach which provided room for the HPs to establish a trustful relationship with the expectant parents. A generative contextual condition seemed to be HPs’ ability to establish interprofessional and cross-sectoral collaboration as a well-functioning collaboration across professions which was essential in regard to early initiation, gentle bridging and continuity of care. Finally, the expec- tant parents’ past experiences with the healthcare system were considered as an influential con- text for how they respond to the intervention. This relates to the acknowledging, respectful and non-judgemental approach of the HPs involving timely communication. Potential unin- tended consequences that warrant further attendance in the theory-testing phase of the evalua- tion include the parents’ experiences of stigma, marginalisation or loss of control. Such experiences may potentially threaten the intended intervention outcomes and ultimately increase health inequality for both parents and children. Acknowledgments We would like to take this opportunity to express our gratitude to the participating stakehold- ers for their support for the evaluation and for their collaboration. Conclusion y p g p g y Qualifications of the HPs, such as their communicative skills, were identified as contextual fac- tors expected to stimulate an acknowledging, respectful and non-judgemental approach which provided room for the HPs to establish a trustful relationship with the expectant parents. A generative contextual condition seemed to be HPs’ ability to establish interprofessional and cross-sectoral collaboration as a well-functioning collaboration across professions which was essential in regard to early initiation, gentle bridging and continuity of care. Finally, the expec- tant parents’ past experiences with the healthcare system were considered as an influential con- text for how they respond to the intervention. This relates to the acknowledging, respectful and non-judgemental approach of the HPs involving timely communication. Potential unin- tended consequences that warrant further attendance in the theory-testing phase of the evalua- tion include the parents’ experiences of stigma, marginalisation or loss of control. Such experiences may potentially threaten the intended intervention outcomes and ultimately increase health inequality for both parents and children. Discussion PLOS ONE \| https://doi.org/10.1371/journal.pone.0295378 December 20, 2023 16 / 21 PLOS ONE Realist evaluation of an intervention for expectant parents with psychosocial risks Discussion Adding a focus on underlying and fundamental risk factors via collaboration with relevant ser- vices across sectors and supporting parents’ access to e.g., financial support [79], education or labour market may increase the likelihood that the intervention reaches its long-term overall objective of reducing social inequity of health. Attention to the reach of the intervention in relation to the parents’ risk factors will be a focus of the ongoing realist evaluation. We identi- fied the five most dominant generative mechanisms assumed to be essential for the interven- tion to produce the intended outcomes. While considering potentially unintended consequences identified through this study, the CMOcs constitute the framework for the ongoing evaluation [37]. A remarkable strength of this study is that the theories generated in this first phase of the realist evaluation were based on multiple data collection methods and sources of evidence. In qualitative studies, validity may be addressed through the extent of triangulation. Triangula- tion of the data collection methods was thus expected to promote rich data with an appropriate scope while capturing the complexity that resembled the intervention and contributed to a strong theoretical base [80]. Another strength of this study is the use of multiple stakeholders’ involvement in prioritisa- tion of the elements of the programme theory. It may be difficult to determine how changes are brought about because multiple stakeholders are involved, and their responses are often not aligned [81]. To reduce the risk of identifying and prioritising incorrectly, we included key stakeholders in workshops thus facilitating the processes of choosing the aspects that were con- sidered pivotal in the initial programme theory. The workshops helped us gain a better under- standing of the interdependencies of the mechanisms and allowed us to grasp how they were often embedded within one another. Stakeholder involvement leads to mutual understandings and influence [82]. The workshops eventually allowed the stakeholders to reflect on and gain insight into the entire intervention beyond their own setting. A potential limitation of the study relates to the fact that although we obtained assumptions from programme designers and stakeholders representing the decisive level and front line workers, we did not include that of expectant parents. Parents from all participating munici- palities will be included as stakeholders in the theory-testing phase of the evaluation. [29, 54]. References 1. 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Canberra; 2020. Available: https:// www.health.gov.au/sites/default/files/documents/2022/06/pregnancy-care-guidelines-pregnancy-care- guidelines_0.pdf 11. Danish Health Authority. Anbefalinger for Svangreomsorgen [Recommendations for Maternity Care]. København; 2021. Available: https://www.sst.dk/-/media/Udgivelser/2021/Anbefalinger- svangreomsorgen/Anbefalinger-for-svangreomsorgen.ashx?sc_lang=da&hash= E4195822AAB3A79AAC1F10969B8695AD 12. National Institute for Health and Care Excellence. Author Contributions Conceptualization: Sara Mandahl Ellehave, Charlotte Overgaard. Conceptualization: Sara Mandahl Ellehave, Charlotte Overgaard. Data curation: Sara Mandahl Ellehave, Louise Lund Holm Thomsen. Data curation: Sara Mandahl Ellehave, Louise Lund Holm Thomsen. Formal analysis: Sara Mandahl Ellehave, Louise Lund Holm Thomsen. Formal analysis: Sara Mandahl Ellehave, Louise Lund Holm Thomsen. Funding acquisition: Charlotte Overgaard. Investigation: Sara Mandahl Ellehave, Louise Lund Holm Thomsen, Marianne Stistrup Frederiksen. Investigation: Sara Mandahl Ellehave, Louise Lund Holm Thomsen, Marianne Stistrup Frederiksen. Methodology: Sara Mandahl Ellehave, Louise Lund Holm Thomsen, Charlotte Overgaard. Methodology: Sara Mandahl Ellehave, Louise Lund Holm Thomsen, Charlotte Overgaard. Project administration: Sara Mandahl Ellehave. Project administration: Sara Mandahl Ellehave. Resources: Sara Mandahl Ellehave. Resources: Sara Mandahl Ellehave. Software: Sara Mandahl Ellehave. Supervision: Louise Lund Holm Thomsen, Marianne Stistrup Frederiksen, Charlotte Overgaard. Visualization: Sara Mandahl Ellehave. Writing – original draft: Sara Mandahl Ellehave. 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https://openalex.org/W2558848267	https://pcmp.springeropen.com/track/pdf/10.1186/s41601-016-0026-9	English	null	Adaptive concentric power swing blocker	Protection and control of modern power systems	2,016	cc-by	6,348	© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 DOI 10.1186/s41601-016-0026-9 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 DOI 10.1186/s41601-016-0026-9 Protection and Control of Modern Power Systems Open Access Open Access Abstract The main purpose of power swing blocking is to distinguish faults from power swings. However, the faults occur during a power swing should be detected and cleared promptly. This paper proposes an adaptive concentric power swing blocker (PSB) to overcome incapability of traditional concentric PSB in detecting symmetrical fault during power swing. Based on proposed method, two pairs of concentric characteristics are anticipated which the first one is placed in a stationary position (outer of zone3) but the position of the second pair is adjustable. In order to find the position of the second pair of characteristic, Static Phasor Estimation Error (SPEE) of current signal is utilized in this paper. The proposed method detects the abrupt change in SPEE and puts the second pair of characteristic in location of impedance trajectory correspondingly. Second concentric characteristic records travelling time of impedance trajectory between outer and inner zones and compares to threshold value to detect symmetrical fault during power swing. If recorded time is lower than threshold, three-phase fault is detected during power swing. Intensive studies have been performed and the merit of the method is demonstrated by some test signals simulations. Keywords: Concentric PSB, Phasor estimation error, Power swing, Symmetrical fault during power swing Keywords: Concentric PSB, Phasor estimation error, Power swing, Symmetrical fault during power swing Adaptive concentric power swing blocker Jalal Khodaparast and Mojtaba Khederzadeh* * Correspondence: m_khederzadeh@sbu.ac.ir Electrical Engineering Department, Shahid Beheshti University, Tehran 165895371, Iran Static phasor estimation error Static phasor estimation error Static Phasor Estimation Error (SPEE) is calculated by static phasor estimation process in every sample and can be used as a quality measure of phasor es- timation. In phasor calculation process, windowed signal is utlized for every sample of time. According to Fig. 1, when a transient occurs in the power sys- tem, there will be a seri of windows, contain pre and post transient data which are illustrated in shaded box in Fig. 1. It is obvious that the calcu- lated phasors resulted from just pre or just post data of transient periods are accurate, which are il- lustrated in unshaded box in Fig. 1. The calculated phasors based on the shaded windows (boxes) are not accurate which can be used as a detecter of transient in any signal. Therefore SPEE can be formulated as: SPEEn ¼ X n¼r n¼r−N1 Sn j −S ⌢ n ð1Þ ð1Þ where r is the first sample of time window, Sn is the real sample, which is measured by relay and S^n is recum- puted sample of Sn obtained based on static phasor estimation. Introduction Mathematical morphology is presented in [11] for detecting symmetrical fault during power swing and it is based on monitoring shape of signal. Al- though this method uses time domain transform- ation, selection of processing function and its length is difficult. Moving average is a low-pass fil- ter that is presented in [12] to discriminate power swing from fault. The moving average varies period- ically during power swing, while it becomes either positive or negative consistently during fault. How- ever, utilization of all three phase currents even in symmetrical fault increases computational burden in this method. In [13], a method based on maximum rate of change of three-phase active and reactive powers is proposed. However, the mathematical demonstration of the proposed index is based on a somewhat impractical hypothesis that considers im- pedance without resistive component. Combination of Park,s transformation and moving data window is presented in [14] to extract power coefficents dur- ing fault and power swing. These coefficient are ap- proximately zero during power swing and significant during fault. Computational burden of calculation of power coefficients limits the application of this method. Reference [15] proposes a method based on fundamental frequency component that is cre- ated in instantaneous three-phase active power after inception of a symmetrical fault. However, it as- sumed that the fault resistance is negligible. Refer- ence [16] proposes a technique based on negative sequence component of current and cumulative sum (CUSUM) for detecting three-phase fault during power swing in series compensated line. A new method based on extracting created transient of current signal by least square dynamic phasor esti- mation is proposed in [17]. The challenge of this method is high computational burden of dynamic phasor estimation. The purpose of this paper is to modify the traditional concentric PSB to enable it for detecting three-phase used in this method for detecting fault during power swing, which these indices complete each other. The first index (IX1) is transient monitor that shows occur- rence of transient in signal and determines the location of second pair of characteristic and the second index (IX2) that is the output of second concentric characteris- tics as final index for detecting three-phase fault during power swing. regression technique. Application of time frequency transforms is another solution. Introduction fault during power swing and unblock distance relay. However, due to the symmetric nature of power swing, detection of symmetrical faults during power swing is more difficult than unsymmetrical faults. Therefore, this issue attracts attentions of many researchers at the moment. Distance relay malfunction has raised concerns about blackouts in power systems. Distance relays make decisions based on entering of impedance trajectory in protected zones. When a fault occurs in a protected line, the impedance trajectory enters in distance relay zones and the relay operates. However, this impedance penetration may also occur during power swing condition. During a power swing the voltage and current fluctuate simultaneously, causing fluctuation in the measured apparent impedance at the distance relay, which may enter the relay tripping zones. This condition causes relay malfunction and may lead to consecu- tive events (cascading outages) and even a blackout eventually [1–3]. There are various suggestions in the literature as to how to deal with this issue. The most traditional method is utilizing rate of change of impedance for power swing detection [4]. However, this method cannot detect fault during power swing when im- pedance trajectory crosses concentric characteristics during power swing (it is exemplified in Fig. 4). New methods based on voltage phase angle are presented in [5] and [6] but high resistance and symmetrical faults are not considered in these refer- ences. Fault detection based on differential power is another proposed method that makes use of auto- regression technique to predict samples in the fu- ture [7]. However this method needs lots of simula- tions to select appropriate parameters for the auto- To avoid this malfunction, Power Swing Blocker (PSB) is installed in modern distance relay [4]. The main task of PSB is discriminating power swing from fault and block distance relay from operating during power swing. Moreover, it should detect any Page 2 of 12 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 2 of 12 Page 2 of 12 regression technique. Application of time frequency transforms is another solution. Wavelet transform and S-transform are presented in [8] and [9] re- spectively, to detect power swing but they require high sampling rate, which is a requirement of most Time-Frequency transforms. Another method based on adaptive neuro-fuzzy system is proposed in [10]. This method requires many simulations in different conditions for training and even retraining in new case. Introduction Wavelet transform and S-transform are presented in [8] and [9] re- spectively, to detect power swing but they require high sampling rate, which is a requirement of most Time-Frequency transforms. Another method based on adaptive neuro-fuzzy system is proposed in [10]. This method requires many simulations in different conditions for training and even retraining in new case. Mathematical morphology is presented in [11] for detecting symmetrical fault during power swing and it is based on monitoring shape of signal. Al- though this method uses time domain transform- ation, selection of processing function and its length is difficult. Moving average is a low-pass fil- ter that is presented in [12] to discriminate power swing from fault. The moving average varies period- ically during power swing, while it becomes either positive or negative consistently during fault. How- ever, utilization of all three phase currents even in symmetrical fault increases computational burden in this method. In [13], a method based on maximum rate of change of three-phase active and reactive powers is proposed. However, the mathematical demonstration of the proposed index is based on a somewhat impractical hypothesis that considers im- pedance without resistive component. Combination of Park,s transformation and moving data window is presented in [14] to extract power coefficents dur- ing fault and power swing. These coefficient are ap- proximately zero during power swing and significant during fault. Computational burden of calculation of power coefficients limits the application of this method. Reference [15] proposes a method based on fundamental frequency component that is cre- ated in instantaneous three-phase active power after inception of a symmetrical fault. However, it as- sumed that the fault resistance is negligible. Refer- ence [16] proposes a technique based on negative sequence component of current and cumulative sum (CUSUM) for detecting three-phase fault during power swing in series compensated line. A new method based on extracting created transient of current signal by least square dynamic phasor esti- mation is proposed in [17]. The challenge of this method is high computational burden of dynamic phasor estimation. Limitation of traditional concentric PSB In normal situation, the measured impedance is far away from the distance relay protection zones. However, when a fault initiates, the measured im- pedance moves in the complex plane (R, X) rapidly from load point to characteristic of line impedance. As a result of the electrical property of a fault, the rate of change of impedance is very high but it is very slow during power swing a result of the mech- anical property of power swing. Traditional concen- tric PSB utilizes this difference to discriminate power swing from fault. To achieve this goal, two concentric impedance characteristics (outer and inner zones) along with a timer are used in trad- itional concentric PSB. The required time for impedance movement between outer and inner zones during quickest power swing is considered as threshold value. If the recorded time is lower than The purpose of this paper is to modify the traditional concentric PSB to enable it for detecting three-phase fault during power swing. In this paper, Phasor Estima- tion Error (PEE) is employed as a quantity with high abrupt at fault initiation that helps the proposed method in determining the location of second pair of concentric PSB. According to proposed method, two indices are Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 3 of 12 Fig. 1 Concept of PEE (N1 is sample number in one cycle and r is the last sample inserted in window) ncept of PEE (N1 is sample number in one cycle and r is the last sample inserted in window) frequency. Impedance trajectory of this case is shown in Fig. 3. According to this figure, power swing starts at t = 0.4 s with impedance value 1.88– 0.24i, which is outside of the relay outermost zone. After power swing initiation, impedance starts to move and come near the relay zones. Before enter- ing the relay’s outermost zone, two circular concen- tric characteristics (outer and inner zones) are located to record the travelling time of impedance trajectory between outer and inner zones. This re- corded time is compared with threshold value for detecting power swing. Therefore, if threshold value is selected accurately (threshold value is selected based on traveling time in fastest power swing), traditional CPSB can detect power swing in this condition. Limitation of traditional concentric PSB the threshold value, it is detected as a fault and in contrast, if the recorded time is higher than thresh- old, it is detected as power swing. In order to analyze the performance of traditional concentric PSB in discriminating power swing from fault, a series of tests are carried out on a two- machine equivalent system, shown in Fig. 2. The data of the power system, are: EB = 1∟0, EA = 1∠δ(t), ZA = 0.25∠750, ZB = 0.25∠750, ZLine = 0.5∠750. The power system frequency is 50 Hz and simula- tion time step is 500 μs. Case1: first test is programmed to examine capability of traditional CPSB in detecting power swing. In order to simulate the power swing, displacement angle of source A is considered as: δ tð Þ ¼ δ0 þ k⋅e−t=τ⋅sin 2π⋅f slip⋅t ð2Þ ð2Þ Case2: A second test is programmed to show capability of traditional CPSB in detecting symmetrical fault during power swing in special condition. Similar to the previous case, power swing is simulated by displacement angle of where k = 5 is constant scaling coefficient, τ = 0.3 is the damping time constant and fslip = 1Hz is the slip Fig. 2 Simple two-machine-system Fig. 2 Simple two-machine-system Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 4 of 12 Page 4 of 12 Fig. 4 Capability of concentric PSB in power swing detection and fault during power swing Fig. 3 Capability of concentric PSB in pure power swing detection Fig. 3 Capability of concentric PSB in pure power swing detection Fig. 3 Capability of concentric PSB in pure power swing detection Fig. 3 Capability of concentric PSB in pure power swing detection Fig. 4 Capability of concentric PSB in power swing detection and fault during power swing Fig. 4 Capability of concentric PSB in power swing detection and fault during power swing Fig. 4 Capability of concentric PSB in power swing detection and fault during power swing Fig. 5 Incapability of concentric PSB for detecting fault during power swing Fig. 5 Incapability of concentric PSB for detecting fault during power swing Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 5 of 12 Page 5 of 12 Fig. Limitation of traditional concentric PSB 6 Capability of proposed new concentric PSB for detecting symmetrical fault during power swing Fig. 6 Capability of proposed new concentric PSB for detecting symmetrical fault during power swing source A as Eq. (2). A three-phase fault is simulated in right end of the protected line at t = 0.85 s during power swing. Next a three-phase fault occurs at t = 0.85 s during power swing. This causes the impedance trajectory crosses CPSB again during fault and so a new trav- elling time is recorded by timer, which can be used for detecting fault individually. Hence, traditional CPSB can detect both power swing and fault dur- ing power swing in this case. The impedance trajectory of this case is shown in Fig. 4. According to the figure, after power swing initiation, impedance moves toward distance zones so that it crosses the CPSB for the first time and then the timer records the traveling time between outer and inner zones. Therefore, power swing can be detected by comparing the recorded time with threshold value and then distance relay is blocked. As a consequence of power swing, impedance tra- jectory moves back and gets away from the dis- tance zones and so leaves the outer zone of CPSB. Case3: A third test is simulated to show the con- dition in which traditional CPSB cannot detect a three-phase fault during power swing. Impedance trajectory of this case is shown in Fig. 5. Power swing is programmed similar to the two previous cases. According to Fig. 5, as a result of power swing, impedance trajectory crosses CPSB for the first time and the travelling time is recorded by timer, which can be used for detecting power swing. However, a fault occurs at t = 0.55 s, when the impedance trajectory is inside the inner zone of CPSB. According to Fig. 5, impedance trajectory does not cross the traditional CPSB again during fault. This condition shows inability of traditional CPSB in three-phase fault detection during power swing. Fault during power swing If time1 > h1 If PEE > h2 Compute the difference between actual and estimated of current (Phasor Estimation Error) (PEEn) Record travelling time by traditional concentric characteristics Fault Power swing Record travelling time by new concentric characteristics If time2 < h1 Fig. 7 Flowchart of proposed method CPSB in three-phase fault detection during power swing. Limitation of traditional concentric PSB Although, distance relay can easily detect unsym- metrical faults with various faulted loops by Fig. 8 SMIB power system with two parallel transmission lines Fault during power swing If time1 > h1 If PEE > h2 Compute the difference between actual and estimated of current (Phasor Estimation Error) (PEEn) Fault Power swing Record travelling time by new concentric characteristics If time2 < h1 Fig. 7 Flowchart of proposed method g Although, distance relay can easily detect unsym- metrical faults with various faulted loops by Fig. 8 SMIB power system with two parallel transmission lines Record travelling time by new concentric characteristics Fig. 8 SMIB power system with two parallel transmission lines Fig. 7 Flowchart of proposed method Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 6 of 12 Page 6 of 12 Fig. 9 Impedance trajectory for power swing at =1 sec and three-phase fault at t = 1.9 sec at 25% line during stable power swing in SMIB Fig. 9 Impedance trajectory for power swing at =1 sec and three-phase fault at t = 1.9 sec at 25% line during stable pow ry for power swing at =1 sec and three-phase fault at t = 1.9 sec at 25% line during stable power swing in SMIB assessing the negative sequence of current signal, it is faced by challenge in symmetrical faults during power swing because of inconsiderable amount of negative sequence during three-phase fault. its location is adapted by PEE, for detecting sym- metrical fault during power swing. This idea is shown in Fig. 6. According to this figure, proposed method provides two independent pairs of CPSB for power swing and symmetrical fault during power swing. Therefore, recorded time by second PSB is used for detecting fault during power swing. Proposed adaptive concentric psb Another key point of this proposed method is de- tection of the location of impedances trajectory (the place in complex plane) for placing the second CPSB. In order to achieve this goal, phasor estimation error (PEE) is employed in this paper. By monitoring PEE during power swing, abrupt change of PEE can be used as primary indicator of symmetrical fault initi- ation and then the second CPSB is set at correspond- ing impedance in complex plane. According to motioned simulations and explanations, traditional concentric PSB has limitation for detect- ing symmetrical fault during power swing and cannot detect it in special condition. When a sym- metrical fault occurs, while impedance trajectory of power swing is inside of inner zone of CPSB, this kind of CPSB cannot detect fault because there is no second cross through zones of the CPSB during fault period. Hence, the proposed method in this paper includes two steps. In the first step, first CPSB is placed farther zone3 to discriminate power swing from fault. Recorded time by this CPSB is compared to predefined threshold In order to solve this problem, adaptive CPSB is proposed in this paper. According to proposed method, second pair of CPSB is programmed, which Fig. 10 Recorded time by first concentric characteristic Page 7 of 12 Page 7 of 12 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Fig. 11 Analyzing PEE during power swing in SMIB power system three-phase fault during power swing is examined in sin- gle machine to infinite bus (SMIB) and in the second part; the performance of the proposed method in three- machine power system is verified and in the last section, the performance of the proposed method is examined in IEE 39-Bus power system. Simulation results of the proposed method in single machine to infinite bus (SMIB) In order to validate performance of the proposed method (shown in flowchart (Fig. 7)) in discriminat- ing three-phase fault from power swing, power sys- tem shown in Fig. 8 is considered, which its data are presented in [8]. A distance relay is considered at bus 1 in the upper line (line with impedance 76.8 + 469.98i). A three phase fault (F1) is simulated at the middle of lower line which occurs at t = 1 s and is cleared after 0.03 s by opening the breakers at both ends (CB1, CB2). This event causes a stable power swing in the line between buses 1 and 2 and is observed by the relay R. Therefore, distance relay should be blocked by power swing blocker during power swing. Moreover, A three-phase fault (F2) initiates at t = 1.9 s (at 25% protected line) during power swing which should be detected by power swing blocker and then distance relay should be unblocked. (h1) so that it is detected as power swing if it is higher than threshold otherwise it is detected as fault. The sec- ond step of proposed method is employed when power swing is detected by first step. In the second step, PEE is calculated during power swing continuously and ana- lyzed (compare to predefined threshold (h2)) to antici- pate three-phase fault during power swing. In order to verify this anticipation, the second CPSB is placed where impedance trajectory presents at this time. Recorded time by the second CPSB is compared to the predefined threshold (h1) so that it is detected as symmetrical fault during power swing if it is lower than threshold value. Therefore combination of these two pairs of CPSB pro- vides a complete method which can detect power swing and three-phase fault during power swing in different situ- ations. Flowchart of the proposed method is shown in Fig. 7. Impedance trajectory of this condition is shown in Fig. 9. According to this figure, the stable power swing causes the impedance trajectory enters into protected zone 3, which could lead to malfunction of the distance relay. In order to prevent this malfunction, first pair of CPSB is designed farther zone3 to detect power swing. Travelling time be- tween first-outer and first-inner zone is shown in Fig. 10. According to this figure, impedance Results Simulation part of this paper is divided into three parts. In the first part, the proposed method for detecting Fig. 12 Recorded time by second concentric characteristic in SMIB Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 8 of 1 Page 8 of 12 Fig. 13 Three-machine power system Fig. 14 Impedance trajectory for power swing at =1 sec and three-phase fault at t = 1.9 sec at 25% line during stable power swing in Three- machine power system Fig. 13 Three-machine power system Fig. 14 Impedance trajectory for power swing at =1 sec and three-phase fault at t = 1.9 sec at 25% line during stable power swing in Three- Fig. 14 Impedance trajectory for power swing at =1 sec and three-phase fault at t = 1.9 sec at 25% line during stable power swing in Three- machine power system Fig. 15 Recorded time by first concentric characteristic Fig. 15 Recorded time by first concentric characteristic Page 9 of 12 Page 9 of 12 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Fig. 16 Analyzing PEE during power swing in Three-machine power system mulation results of three-machine power system Simulation results of three-machine power system Simulation results of three machine power system In order to examine the proposed method in larger power system, the three-machine power system shown in Fig. 13 is considered [18]. A three-phase fault is simulated at 90% of the line connecting buses 5 and 1. The fault (F1) occurs at t = 1 s and is cleared after 0.25 s. This event causes an unstable power swing that is observed by the distance relay. Moreover, another three-phase fault (F2) initiates at t = 2.1 s (during unstable power swing) in 57% pro- tected line. Impedance trajectory of this condition is shown in Fig. 14. According to this figure, the unstable power swing causes the impedance trajectory enters into protected zone 3. First CPSB is designed farther zone3 at the first step of proposed method and the process of recording time by CPSB (between first- outer and first-inner zones) is shown in Fig. 15. Ac- cording to this figure, impedance trajectory needs 0.026 s for travelling between first-outer and first- inner zones. Results By comparing the recorded time with threshold value (0.005 sec), it can be understand that this is power swing. Fig. 16 Analyzing PEE during power swing in Three-machine power system trajectory enters first-outer zone at t = 1.465 s and enters first-inner zone at t = 1.54 s that results in 0.085 s recorded time by first CPSB. By comparing recorded time with threshold value (0.01 sec), power swing can be detected. Based on the proposed method, PEE of current signal is monitored continuously during power swing. PEE of the current signal is shown in Fig. 11. According to this figure, a new transient happens at t = 1.9 s which is anticipated to be three-phase fault. In order to verify this anticipation, the second CPSB is designed (as shown in Fig. 9) and recorded time by this CPSB is shown in Fig. 12. According to this figure, impedance trajectory enters outer zone at t = 1.906 s and enters second-inner zone at t = 1.907 s which result in recorded time close to 0.0013se. By comparing recorded time with threshold value (0.01 sec), it can be detected that this is symmetrical fault during power swing. p g PEE of current signal is monitored during unstable power swing detection. PEE of current signal is shown in Fig. 16. According to this figure, a new transient hap- pens at t = 2.1 s which is anticipated to be a three-phase fault. In order to verify this anticipation, the second CPSB is designed (as shown in Fig. 14) and recorded time by this CPSB is shown in Fig. 17. According to this figure, impedance trajectory needs 0.0008 sec for travel- ling between two zones of second CPSB. By comparing recorded time with threshold value (0.005 sec), it can be understudied that this is symmetrical fault during power swing. Fig. 17 Recorded time by second concentric characteristic in Three-machine power system Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 10 of 12 Page 10 of 12 Page 10 of 12 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 protective relay’s characteristics of distance relay due to the power swing. In this condition, the distance relay is blocked by the first CPSB; meanwhile, another three- phase fault (F2) occurs at t = 1.9 s. Results So the impedance leaves the power swing locus immediately and reaches to the fault impedance point. As is shown in Fig. 19, since the three-phase fault occurs after the impedance trajec- tory leaves the first CPSB characteristics (special condi- tion); the first CPSB is not capable of detecting the fault. Simulation results of IEEE 39-Bus power system IEEE 39-Bus power system is examined as a large test system (Fig. 18) in this paper. A three-phase fault (F1) is simulated at 50% of the line connecting buses 10 and 13. The fault occurs at t = 1 s and is cleared after 0.2 s. This event causes an unstable power swing and is observed by the distance relay (R). In order to examine the per- formance of the proposed CPSB, another three-phase fault (F2) is simulated at 100% of the protected line (line connecting buses 4 and 14) during unstable power swing. The impedance locus for this condition is shown in Fig. 19. According to this figure, at the first the im- pedance starts to move at t = 1.2 s and enters into the p g According to Fig. 19 and proposed strategy, first CPSB characteristics are designed farther zone3 for a distance relay to discriminate fault from power swing. Travelling time between first-outer and first- Fig. 18 IEEE 39-Bus power system Fig. 18 IEEE 39-Bus power system Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 11 of 12 Page 11 of 12 inner zone is shown in Fig. 20. According to this figure, impedance trajectory enters outer zone at t = 1.78 s and enters inner zone at t = 1.8 s which result in 0.02 s recorded time by first CPSB. By comparing recorded time with threshold value (0.005 sec), it can be understand that this is power swing. Based on proposed strategy, PEE of current signal is monitored during time after power swing detection. PEE of current signal is shown in Fig. 21. According to this figure a new transient starts at Fig. 20 Recorded time by first CPSB in IEEE 39-Bus Fig. 19 Impedance locus of three-phase fault during power swing in IEEE 39-Bus Fig. 19 Impedance locus of three-phase fault during power swing in IEEE 39-Bus Fig. 17. Khodapaast, J., & Khederzadeh, M. (2015). “Three-Phase Fault Detection During Power Swing by Transient Monitor”. IEEE Transactions on power system, 30(5), 2558–2565. Results 19 Impedance locus of three-phase fault during power swing in IEEE 39-Bus Fig. 20 Recorded time by first CPSB in IEEE 39-Bus Fig. 20 Recorded time by first CPSB in IEEE 39-Bus Fig. 20 Recorded time by first CPSB in IEEE 39-Bus inner zone is shown in Fig. 20. According to this figure, impedance trajectory enters outer zone at t = 1.78 s and enters inner zone at t = 1.8 s which result in 0.02 s recorded time by first CPSB. By comparing recorded time with threshold value (0.005 sec), it can be understand that this is power swing. Based on proposed strategy, PEE of current signal is monitored during time after power swing detection. PEE of current signal is shown in Fig. 21. According to this figure, a new transient starts at t = 1.9 s which is anticipated to be a three-phase fault. In order to verify this anticipation, second CPSB is designed (as shown in Fig. 19) and re- corded time by this CPSB is shown in Fig. 22. Ac- cording to this figure, impedance trajectory enters second-outer zone at t = 1.907 s and enters second- inner zone at t = 1.908 s which result in 0.001 s Fig. 21 PEE during power swing in IEEE 39-Bus rol of Modern Power Systems (2016) 1:16 Page 12 of 12 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 12 of 1 Khodaparast and Khederzadeh Protection and Control of Modern Power Systems (2016) 1:16 Page 12 of 12 Fig. 22 Recorded time by second CPSB in IEEE 39-Bus recorded time by second CPSB. By comparing recorded time with threshold value (0.005 sec), it can be understudied that this is symmetrical fault during power swing. 1. Kundu, P., & Pradhan, K. (2014). “Synchrophasor-assisted zone 3 operation”. IEEE Trans Power Del, 29(2), 660–667. 1. Kundu, P., & Pradhan, K. (2014). “Synchrophasor-assisted zone 3 operation”. IEEE Trans Power Del, 29(2), 660–667. 2. Nayak, P., Pradhan, K., & Bajpai, P. (2015). “Secured zone 3 protection during stressed condition”. IEEE Trans Power Del, 30(1), 89–96. 3. Horowitz, S., & Phadke, A. (2006). “Third zone revisited”. IEEE Trans Power Del, 21(1), 23–29. Conclusion “A novel power swing blocking scheme using adaptive neuro-fuzzy inference system”. Electr Power Syst Res, 78(7), 1138–1146. 10. Zade, H., & Li, Z. (2008). “A novel power swing blocking scheme using adaptive neuro-fuzzy inference system”. Electr Power Syst Res, 78(7), 1138–1146. 11. Gautam, S., & Brahma, S. (2012). “Out-of-step blocking function in distance relay using mathematical morphology”. IET Generation, Transmission & Distribution, 6(4), 313–319. 12. Rao, J., & Pradhan, K. (2015). “Power swing detection using moving averaging of current signals”. IEEE Trans Power Del, 30(1), 368–376. 13. Lin, X., Gao, Y., & Liu, P. (2008). “A novel scheme to identify symmetrical fault occurring during power swings”. IEEE Trans Power Del, 21(1), 73–78. 13. Lin, X., Gao, Y., & Liu, P. (2008). “A novel scheme to identify symmetrical fault occurring during power swings”. IEEE Trans Power Del, 21(1), 73–78. 14. Andanapalli, K., & Varma, B.R.K. (2014). “Parks transformation based symmetrical fault detection during power swing”,(pp. 1-5). Guwahati: Power Systems Conference (NPSC) Eighteenth National. Authors’ contributions JK, Ph.D. student, brings up the idea of adaptive procedure, performed the primary simulations and drafted the manuscript. MK, JK Ph.D. supervisor, participated in enriching the manuscript (in theoretical idea and simulation section (IEEE 39-Bus power system)) and carried out the revising the manuscript (response to the reviewers and editing grammatical and lexical mistakes). Both authors read and approved the final manuscript. 15. Mahamedi, B., & Zhu, J. (2012). “A novel approach to detect symmetrical faults occurring during power swings by using frequency components of instantaneous three phase active power”. IEEE Trans Power Del, 27(3), 1368–1376. 16. Nayak, P., & Bajpai, P. (2013). “A fault detection technique for the series- compensated line during power swing”. IEEE Trans Power Del, 28(2), 714–722. 17. Khodapaast, J., & Khederzadeh, M. (2015). “Three-Phase Fault Detection During Power Swing by Transient Monitor”. IEEE Transactions on power system, 30(5), 2558–2565. 16. Nayak, P., & Bajpai, P. (2013). “A fault detection technique for the series- compensated line during power swing”. IEEE Trans Power Del, 28(2), 714–722. 15. Mahamedi, B., & Zhu, J. (2012). “A novel approach to detect symmetrical faults occurring during power swings by using frequency components of instantaneous three phase active power”. IEEE Trans Power Del, 27(3), 1368–1376. Conclusion 4. IEEE PSRC WG D6. (2005). “Power swing and out of step considerations on transmission lines”, A report to power system relaying committee of the IEEE power engineering society. IEEE PES (Power and Energy Society). Measured apparent impedance by a distance relay moves into relay operating zones during power swing as a consequence of disturbance in power system that causes malfunction of distance relay. Traditional CPSB is designed inside of distance relay to prevent this malfunction by blocking distance relay during power swing. However, if a fault occurs during power swing, it should be detected and distance relay is blocked. Traditional CPSB is a common method for detecting power swing. However, it has limitation in detecting symmetrical fault during power swing. Therefore, adjustable concept of this method is pro- posed in this paper to overcome this difficulty. Ac- cording to the proposed method, two pairs of CPSB are employed; the first CPSB is used for discriminat- ing fault from power swing and the second CPSB is used for detecting symmetrical fault during power swing. According to results, the proposed method demonstrates its ability to unblock distance relay in three-phase fault during power swing. y y y 5. Mechraoui, A., & Thomas, D. W. P. (1995). “A New Blocking Principle with Phase and Earth Fault Detection during Fast Power Swings for Distance Protection”, IEEE Transactions on Power Delivery, 10(3). 6. Mechraoui, A., & Thomas, D. W. P. (1997). “A New Principle for High Resistance Earth Fault Detection during Fast Power Swings for Distance Protection”, IEEE Transactions on Power Delivery, 12(4). 7. Ganeswara, J., & Pradhan, A. (2012). “Differential power based symmetrical fault detection during power swing”. IEEE Trans Power Del, 27(3), 1557–11564. 8. Brahma, S. (2007). “Distance relay with out of step blocking function using wavelet transform”. IEEE Trans Power Del, 22(3), 1360–1366. 8. Brahma, S. (2007). “Distance relay with out of step blocking function using wavelet transform”. IEEE Trans Power Del, 22(3), 1360–1366. 9. Mohamad, N., Abidin, A., & Musirin, I. (2014). “Intelligent power swing detection scheme to prevent false relay tripping using S_Transform”. International Journal of Emerging Electrical Power systems, 15(3), 195–311. 9. Mohamad, N., Abidin, A., & Musirin, I. (2014). “Intelligent power swing detection scheme to prevent false relay tripping using S_Transform”. International Journal of Emerging Electrical Power systems, 15(3), 195–311. 10. Zade, H., & Li, Z. (2008). 10. Zade, H., & Li, Z. (2008). “A novel power swing blocking scheme using adaptive neuro-fuzzy inference system”. Electr Power Syst Res, 78(7), 1138–1146. 14. Andanapalli, K., & Varma, B.R.K. (2014). “Parks transformation based symmetrical fault detection during power swing”,(pp. 1-5). Guwahati: Power Systems Conference (NPSC) Eighteenth National. 12. Rao, J., & Pradhan, K. (2015). “Power swing detection using moving averaging of current signals”. IEEE Trans Power Del, 30(1), 368–376. 8. Brahma, S. (2007). “Distance relay with out of step blocking function using wavelet transform”. IEEE Trans Power Del, 22(3), 1360–1366. 13. Lin, X., Gao, Y., & Liu, P. (2008). “A novel scheme to identify symmetrical fault occurring during power swings”. IEEE Trans Power Del, 21(1), 73–78. Received: 26 May 2016 Accepted: 15 November 2016 11. Gautam, S., & Brahma, S. (2012). “Out-of-step blocking function in distance relay using mathematical morphology”. IET Generation, Transmission & Distribution, 6(4), 313–319. Competing interests p g The authors declare that they have no competing interests. 18. Moravej, Z., Pazoki, M., & Khederzadeh, M. (2014). "Impact of UPFC on Power Swing Characteristic and Distance Relay Behavior". Power Delivery IEEE Transactions on, 29, 261–268. Received: 26 May 2016 Accepted: 15 November 2016 Received: 26 May 2016 Accepted: 15 November 2016
https://openalex.org/W3004276465	https://link.springer.com/content/pdf/10.1007/s42113-020-00082-y.pdf	English	null	Bayesian Inference for Multidimensional Scaling Representations with Psychologically Interpretable Metrics	Computational brain & behavior/Computational Brain & Behavior	2,020	cc-by	13,346	ERROR: type should be string, got "https://doi.org/10.1007/s42113-020-00082-y\nComputational Brain & Behavior (2020) 3:322–340 https://doi.org/10.1007/s42113-020-00082-y\nComputational Brain & Behavior (2020) 3:322–340 ORIGINAL PAPER Abstract Multidimensional scaling (MDS) models represent stimuli as points in a space consisting of a number of psychological\ndimensions, such that the distance between pairs of points corresponds to the dissimilarity between the stimuli. Two\nfundamental challenges in inferring MDS representations from data involve inferring the appropriate number of dimensions\nand the metric structure of the space used to measure distance. We approach both challenges as Bayesian model-\nselection problems. Treating MDS as a generative model, we define priors needed for model identifiability under metrics\ncorresponding to psychologically separable and psychologically integral stimulus domains. We then apply a differential\nevolution Markov-chain Monte Carlo (DE-MCMC) method for parameter inference, and a Warp-III method for model\nselection. We apply these methods to five previous data sets, which collectively test the ability of the methods to infer an\nappropriate dimensionality and to infer whether stimuli are psychologically separable or integral. We demonstrate that our\nmethods produce sensible results, but note a number of remaining technical challenges that need to be solved before the\nmethod can easily and generally be applied. We also note the theoretical promise of the generative modeling perspective,\ndiscussing new and extended models of MDS representation that could be developed. Keywords Multidimensional scaling · Bayesian model selection · MDS dimensionality · Separable and integral stimuli ·\nW\nIII b id\nli Keywords Multidimensional scaling · Bayesian model selection · MDS dimensionality · Separable and integral stimuli ·\nWarp-III bridge sampling Bayesian Inference for Multidimensional Scaling Representations\nwith Psychologically Interpretable Metrics Quentin F. Gronau1 · Michael D. Lee2 © The Author(s) 2020\nPublished online: 8 July 2020 © The Author(s) 2020\nPublished online: 8 July 2020 \u0002 Quentin F. Gronau\nQuentin.F.Gronau@gmail.com 2\nDepartment of Cognitive Sciences, University of California\nIrvine, Irvine, CA 92697, USA 1\nDepartment of Psychological Methods, University of\nAmsterdam, 1012 WX Amsterdam, Netherlands Introduction knowledge, and the capability of the mind to make adaptive\npredictions about properties and consequences (Shepard\n1987). For these reasons, mental representations found\nvia MDS methods have been and remain widely used in\ncognitive process models of identification, categorization,\nand decision making (e.g., Nosofsky 1992). Multidimensional scaling (MDS) was developed in the\n1950s in cognitive psychology as a statistical method for\nmaking inferences about human mental representations\n(Shepard 1957, 1962; Kruskal 1964) MDS models the\nsimilarities or psychological proximities between pairs\nof stimuli, representing each stimulus as a point in a\nmultidimensional space, such that more similar stimuli are\nnearer each other. The core psychological motivation is\nthat the similarities reflect the basic cognitive process of\ngeneralization. Generalization can be thought of as the\nability to treat two stimuli as being the same, and has been\nargued to serve as a basis for the mental organization of Soon after its development in cognitive psychology,\nhowever, MDS algorithms found application as a statistical\nmethod that produces a low-dimensional representation of\na set of objects, based on a measure of the similarities\nbetween them. As a data-reduction or visualization method,\nMDS has been applied in the natural, biological, and human\nsciences, with application areas as diverse as representing\nthe similarities of skulls in archaeology, the tastes of\ncolas in marketing, and the voting patterns of senators in\npolitics (e.g., Borg and Groenen 1997; Cox and Cox 1994;\nSchiffman et al. 1981). \u0002 Quentin F. Gronau\nQuentin.F.Gronau@gmail.com Whether viewed as a model of psychological represen-\ntation or a data-reduction method, a foundational challenge\nin MDS modeling is determining the dimensionality M of\nthe representational space. In his 1974 Presidential Address\nto the Psychometric Society, Roger Shepard identified six 323 Comput Brain Behav (2020) 3:322–340 Integral Fig. 1 MDS representations\nwith integral and separable\nmetric structures\nIntegral\nSeparable Integral\nSeparable Fig. 1 MDS representations\nwith integral and separable\nmetric structures Separable Integral Separable 1991) emphasizes the role that the metric structure of\nthe space plays in capturing key psychological properties\nof the stimuli. In particular, the idea is that different\nmetrics capture the theoretical and empirical distinction\nbetween separable and integral stimuli (Attneave 1950;\nGarner 1974). Separable stimuli are those for which the\ncomponent dimensions can be attended to separately. An\nexample is different shapes of different sizes, since it is\npossible for people to attend selectively to either shape or\nthe size. Introduction Integral stimuli, by contrast, are those for which the\ncomponent dimensions cannot be attended to independently. The standard example is color, since it is typically not\npossible for people to attend selectively to the underlying\nhue, saturation, and brightness components. basic challenges for MDS, the third of which was “The\nproblem of determining the proper number of dimensions\nfor the coordinate embedding space” (Shepard 1974, p. 377). A number of methods for solving the problem of\nMDS dimensionality have been developed in both statistics\nand psychology. The most common approach is a scree test\nthat aims to identify an “elbow” in the goodness-of-fit as\ndimensionality increases (Cox and Cox 1994; Kruskal 1964;\nSchiffman et al. 1981). Steyvers (2006) suggests the use of\ncross-validation methods, although this approach does not\nseem to be widely used. Since choosing the correct dimensionality of an MDS\nis naturally regarded as a model-selection problem—\nthat is, choosing between a one-dimensional versus two-\ndimensional versus three-dimensional representation, and\nso on—the statistically principled approach offered by\nBayes factors should provide a solution (Kass and Raftery\n1995). Along these lines, Lee (2001) implements an\napproach based on the Bayesian Information Criterion\n(BIC). The difference between BIC values for representa-\ntions with different dimensionality provides a crude approx-\nimation to the Bayes factor. Oh and Raftery (2001) provide\na different approach to approximation by computing the\nmarginal likelihoods of different representations using plug-\nin point estimates for the stimulus locations. This is an\napproximation because the exact Bayes factor requires an\nintegration across the stimulus location parameters. Oh\n(2012) developed a method based on spike-and-slab priors,\nin which the dimensionality is determined by the marginal\nposterior probabilities for each dimension that the coordi-\nnate locations are not zero for all stimuli. Figure 1 shows how different metric structures are used\nto represent integral and separable stimuli. In the left\npanel, there are four stimuli, represented by the points\np1, . . . , p4. The pairwise distances between these points,\nsuch as d12 between the first point and the second point,\nare modeled using the Euclidean metric, and so correspond\nto standard straight lines. In the right panel, there are\nthree stimuli, and the pairwise distances between them are\nmodeled according to the city-block metric. Intuitively, this\ncorresponds to comparing the stimuli on each underlying\ndimension independently, then adding those dissimarilities\nto get an overall measure of dissimilarity. Introduction Admittedly, this account of integrality and separability\nis a theoretical and empirical caricature, and much more\nnuanced and detailed accounts are possible (Shepard 1991;\nTversky and Gati 1982). The point is that psychological\nrepresentations based on MDS need to make assumptions\nabout the metric structure of the space, and use metrics\nother than the Euclidean metric. As J¨akel et al. (2008, p. 2)\npoint out, from the origins of MDS as a psychological\nmodel, “There was no a priori reason to believe that mental From the perspective of MDS as psychological models\nhowever, none of these approaches qualifies as being\nprincipled and complete. The key issue is that the theory of\nmental representation developed by Shepard (1957, 1987, 324 Comput Brain Behav (2020) 3:322–340 the development of joint prior distributions on the stimulus\nlocation parameters for the Euclidean metric, and non-\nEuclidean metrics other than the city-block metric. With\nthese priors established, we apply an approach to Bayesian\ninference using differential evolution Markov-chain Monte\nCarlo (DE-MCMC) computational sampling methods. The\nDE-MCMC method helps address the difficulties inher-\nent in inferring MDS representations, which are especially\nevident in non-Euclidean cases. We then use the Warp-\nIII bridge sampling method to approximate the marginal\ndensities needed to determine Bayes factors. We apply the\nmethod to five previously studied data sets, differing in\nthe type of stimuli and expected dimensionality of their\nMDS representation. For all five applications, the method\nmakes sensible inferences about dimensionality, and pro-\nduces interpretable stimulus representations. We conclude\nwith a discussion of remaining statistical and computa-\ntional challenges, and potential directions for refining and\nextending the approach. representations should be Euclidean.” Previous methods\nfor determining the dimensionality of MDS representations\nusing Bayesian model selection, however, have either been\ninsensitive to the metric structure of the representation (Lee\n2001) or have focused on the Euclidean metric (Oh 2012;\nOh and Raftery 2001). The use of non-Euclidean metrics raises another chal-\nlenge, related to inferring MDS representations themselves. There is evidence that it can be computationally difficult\nto find multidimensional city-block MDS representations\n(Groenen et al. 1998; Hubert et al. 1992), as well as find-\ning unidimensional MDS representations (Mair and Leeuw\n2014). Given that these difficulties stem from basic geomet-\nric properties of the MDS representations, it seems likely\nthey will continue to present an issue for Bayesian methods\nof inference. Introduction Finally, there is the challenge of inferring the appropriate\nmetric structure for an MDS representation. Shepard (1991)\nreviews the original statistical approach to this problem,\nwhich involved applying non-metric MDS algorithms for\na large number of different metrics, and choosing the one\nwith the best goodness-of-fit. As Lee (2008) pointed out,\nthis approach neglects to account for the component of\nmodel complexity that arises from the functional form\nof parameter interaction (Pitt et al. 2006), which is\noften the only difference between MDS models using\ndifferent psychologically interpretable metrics. Lee (2008)\ndeveloped a Bayesian approach in which the possible\nmetrics correspond to a parameter that is inferred jointly\nwith the coordinate location parameters that represent the\nstimuli. Okada and Shigemasu (2010) developed and tested\nthis approach further, and showed it is capable of recovering\nthe correct metric in simulation studies. Both the Lee (2008)\nand Okada and Shigemasu (2010) methods, however, failed\nto resolve basic challenges in model identifiability that arise\nfrom treating the choice of metric structure as a parameter\ninference problem. It is possible these identifiability issues\ncould be addressed by considering the choice as a model-\nselection problem, and restricting the set of possibilities to\na few interpretable metrics. The Identiﬁability Problem σ ∼TruncatedGaussian\n\u0006\n0.15,\n1\n0.22\n\u0007\nT (0, ) ,\n(3) (3) where the T (0, ) indicates the sampled value is truncated to\nbe a positive real number. This is an informative prior (Lee\nand Vanpaemel 2018), consistent with previous data and\nmodeling. Intuitively, σ corresponds to the average standard\ndeviation of different individual ratings of the same pair\nof stimuli. Empirical estimates of this standard deviation\nin previous data tend to range from about 0.1 to about 0.2\n(Lee 2001; Lee and Pope 2003).2 Accordingly, the prior is\ncentered on 0.15, but allows a wide range of possibilities. Most other methods, in contrast, assume the MDS\nspace is Euclidean. The post-processing of the coordinate\nlocation parameters used by both Oh and Raftery (2001)\nand Oh (2012) assumes a Euclidean space and controls for\ntranslation, reflection, and rotation. Okada and Mayekawa\n(2018) extend the approach developed by Okada (2012),\nwhich relies on Procrustes analysis. Their post-processing\nuses a loss function to align posterior samples of the\ncoordinate location, but again assumes a Euclidean space. We note that this MDS model does not incorporate\nindividual differences. It is assumed that the same point\npi represents the ith stimulus for all participants. We also\nemphasize, however, that individual-level proximity data\ndijk are modeled, rather than averaged or aggregated data\nacross participants. The problems inherent in averaging\ndata have long been understood (Estes 1956), and have\nbeen studied in the specific cognitive modeling context\nprovided by MDS representations (Lee and Pope 2003). Our approach is to require the same underlying MDS\nrepresentation to provide an account of each individual\nproximity matrix. Besides the lack of flexibility in the nature of the\ndistance metric, post-processing methods have the effect\nof implementing modeling assumptions without explicitly\nspecifying those assumptions as part of the model. While\nthis is often practical, it is theoretically inelegant, and\ncontrary to the goals of generative modeling. Ideally, the\nconstraints required for model identifiability should be part\nof the model itself. In the case of MDS models, these\nconstraints are naturally imposed through the specification\nof a joint prior over the coordinate location parameters\nthat addresses the transformational invariances, removes\nthe need for post-processing, and makes bridge sampling\nfeasible. To complete the generative model, a straightforward\napproach would be to give all of the coordinate locations\nfor the representational points uniform priors pim\n∼\nUniform\n\b\n−1, 1\n\t\n. The Identiﬁability Problem Formally, suppose there are N stimuli to be represented,\nbased on observed proximity data from P participants,\nwith dijk measuring the proximity between the ith and jth\nstimulus provided by the kth participant. We assume these\nobserved proximities are normalized to lie between 0 and 1. The point representing the ith stimulus in a M-dimensional\nspace is pi = (pi1, . . . , piM) and the distance between\npoints pi and pj is measured by the Minkowski metric with\nmetric parameter r, so that ˆdijk =\n\u0002 M\n\u0003\nm=1\n\u0004\u0004pim −pjm\n\u0004\u0004r\n\u00051/r\n. (1) (1) The Minkowski metric has special cases of the city-block\nmetric when r = 1 and the Euclidean metric when r = 2. Values of r between 1 and 2 can potentially be interpreted\nas intermediate assumptions about the independence of\nstimulus dimensions between the end point of complete\nseparability and complete integrality. ˆ Accordingly, the goals of this article are to examine the\nimplementation of MDS models that use psychologically\ninterpretable metrics, including both the Euclidean and a\nnon-Euclidean metric, and explore the possibility of infer-\nring the appropriate dimensionality and metric structure\nof these representations using Bayesian model-selection\nmethods. The structure of the remainder of the article is\nas follows. In the next section, we define MDS models,\nand address the issue of model identifiability under differ-\nent metrics. Consistent with previous literature, we argue\nthat the city-block metric presents fundamental problems\nin making MDS representations identifiable. This leads to The goal of MDS is for the modeled distances ˆdijk to\ncorrespond to the observed proximities dijk. We use the\nprobabilistic model dijk ∼Gaussian\n\u0006\nˆdijk, 1\nσ 2\n\u0007\n,\n(2) (2) 325 Comput Brain Behav (2020) 3:322–340 where σ is the standard deviation with which the observed\nproximities are measured.1 It is assumed to be the same for\nall of the proximities, and is given a prior location parameters to control for translation, reflection,\nand permutation. For example, to control for translation,\nthe method zero centers every posterior sample of the\nsets of coordinate location. The Lee (2008) method does\nnot control for rotation, which is problematic, because the\nmethod also attempts to infer the r metric parameter, and\nso the inferred representational space can have a Euclidean\nmetric, which requires rotational invariance. The Identiﬁability Problem These priors, however, made the model\nnon-identifiable, because the distances between points\nare invariant under transformations (Borg and Groenen\n1997, Ch. 2). The distances between points are preserved\nunder translation, reflection, axes permutation (for non-\nEuclidean metrics), and rotation (for the Euclidean metric). A principled Bayesian approach for controlling these\ninvariances to ensure model identifiability constrains the\ncoordinate location parameters through a joint prior\ndistribution that depends on the assumed metric. This generative approach is used by the “parameter\nfixing” method considered by Okada and Mayekawa\n(2018), who evaluate it as a contrast with the Procrustes\nmethods that are their focus. Parameter fixing corresponds\nto setting a structured joint prior over the coordinate\nlocation parameters. Okada and Mayekawa (2018) define\nthe appropriate prior for a Euclidean space using results\nprovided by Bakker and Poole (2013), which were derived\nusing an analytic method based on matrix properties. Our goal is to extend this approach to include non-\nEuclidean representations. We start by considering one-\ndimensional MDS representations, before considering mul-\ntidimensional representations in both Euclidean and non-\nEuclidean metric spaces. We take a geometric approach\nto identifying the required joint priors for invariance con-\nstraints, complementing the non-geometric approach of\nBakker and Poole (2013) for the Euclidean metric. Previous Approaches Existing MDS modeling methods that use Bayesian\ninference almost always rely on post-processing to address\nthe issue of identifiability. The method developed by Lee\n(2008) post-processes posterior samples of the coordinate 1We parameterize the Gaussian distribution in terms of mean and\nprecision parameters, for consistency with the JAGS graphical\nmodeling language. 2See also the data repository at https://osf.io/ey9vp/ One-dimensional Representation 1We parameterize the Gaussian distribution in terms of mean and\nprecision parameters, for consistency with the JAGS graphical\nmodeling language. For a one-dimensional representation, all of the psycho-\nlogically interpretable metrics we consider give the same 326 Comput Brain Behav (2020) 3:322–340 Fig. 2 Identification constraints\nfor a one-dimensional\nrepresentation coordinate location to zero, “+” denotes constraining it to be\npositive, and “R” denotes imposing no constraint. coordinate location to zero, “+” denotes constraining it to be\npositive, and “R” denotes imposing no constraint. distances. The required constraints on the points are shown\nin Fig. 2, with one point fixed at the origin to control\ntranslation, and second point restricted to be positive to\ncontrol reflection. These constraints can be formalized by a joint prior with p1 = 0\np2 ∼Uniform\n\b\n0, 1\n\t\np3, . . . , pN ∼Uniform\n\b\n−1, 1\n\t\n. (4) (4) p3, . . . , pN ∼Uniform\n\b\n−1, 1\n\t\n. (4) Non-Euclidean Multidimensional Representations emphasized in the seminal text by Borg and Groenen (1997,\npp. 369–372). Finding constraints for invariance in non-Euclidean metrics\nis more complicated, and is especially difficult for the\ncity-block metric. The basic geometric problem was noted\nas early as Arnold (1971), and discussed in Shepard’s\n(1974) presidential address. A simple demonstration of the\nfundamental problem is provided by Fig. 4. The three panels\ncorrespond to Euclidean (r = 2), city-block (r = 1), and\na general non-Euclidean (r = 1.5) metric, and show unit\niso-distance contours around the same two points in each\nmetric, shown as black dots. These iso-distance contours are\nthe “unit circles” of each metric, showing all the points in\nthe space that are the same distance from the two points. For the Euclidean metric, these contours are familiar circles,\nand coincide at only one point, shown by the white dot. This\nmeans that there is a unique point in the space that is equally\ndistant from the two points shown by black dots. In the\ncontext of an MDS representation, a stimulus that is equally\ndifferent to both of the points can be uniquely identified. Figure 5 provides a concrete example, based on the\nmore general configuration examined by Borg and Groe-\nnen (1997, Figure 17.6). Each panel shows a representa-\ntion of six fictitious people in terms of two underlying\ndimensions. The city-block distance between each pair of\npeople is identical in both configurations. This means,\nof course, that this proximity matrix is equally consis-\ntent with both representations, and either could be inferred\nfrom the data. But, the two representations are substan-\ntively different, in non-trivial ways. The representations\ndo not differ simply by changing the axes, and have\nbasic structural differences. For example: Cedric, Ding-\nbats, and Ethelred are co-linear in the first representation,\nbut not in the second, where Dingbats, Ethelred and Fiona\nbecome co-linear; the ordering of Albert and Beowulf\nchanges on both dimensions between the configurations;\nand so on. In fact, once the lack of invariance revealed\nby the Borg and Groenen (1997, Figure 17.6) analysis is\nunderstood, it is clear that many additional representations\nfor the proximity between the six people could be con-\nstructed, supporting a wide range of different meaningful\ninterpretations. For the city-block case, however, the iso-distance\ncontours are diamonds, and there are infinitely many points\nthat are equally different. Euclidean Multidimensional Representations Figure 3 shows the constraints needed to identify Euclidean\nMDS representations in two and three dimensions. In\nthe two-dimensional case, the first point p1 is fixed at\nthe origin, to control translation, the second point p2 is\nconstrained to the positive x-axis, to control reflection in\nthe y-axis and rotation, and the third point p3 is constrained\nto have a positive y-value to control reflection in the x-\naxis. The same logic is applied in the three-dimensional\ncase, with p1 controlling translation, p2 and p3 controlling\nreflection and rotation in successive axes, and p4 controlling\nthe final reflection. Formally, these constraints in D dimensions correspond\nto the joint prior p11, . . . , p1D = 0\np21 ∼Uniform\n\b\n0, 1\n\t\np22, . . . , p2D = 0\np31 ∼Uniform\n\b\n−1, 1\n\t\np32 ∼Uniform\n\b\n0, 1\n\t\np33, . . . , p3D = 0\np41, p42 ∼Uniform\n\b\n−1, 1\n\t\np43 ∼Uniform\n\b\n0, 1\n\t\np44, . . . , p4D = 0\n. . . p11, . . . , p1D = 0\np21 ∼Uniform\n\b\n0, 1\n\t\np22, . . . , p2D = 0\np31 ∼Uniform\n\b\n−1, 1\n\t\np32 ∼Uniform\n\b\n0, 1\n\t\np33, . . . , p3D = 0\np41, p42 ∼Uniform\n\b\n−1, 1\n\t\np43 ∼Uniform\n\b\n0, 1\n\t\np44, . . . , p4D = 0\n. . . (5) These are the first two cases of a general pattern, clear by\ninduction, that applies to a M-dimensional representation,\nand corresponds to the matrix result provided by Bakker\nand Poole (2013). An intuitive presentation of the inductive\npattern is shown below, where “0” denotes fixing a (5) Fig. 3 Identification constraints for Euclidean representations in two dimensions (left) and three dimensions (right) Fig. 3 Identification constraints for Euclidean representations in two dimensions (left) and three dimensions (right) 327 Comput Brain Behav (2020) 3:322–340 Fig. 4 The nature of iso-distance curves and the identifiability of mid-points for the three Minkowski metrics corresponding to r = 2 (Euclidean),\nr = 1 (city-block), and r = 1.5 tance curves and the identifiability of mid-points for the three Minkowski metrics corresponding to r = 2 (Euclidean)\n1 5 Fig. 4 The nature of iso-distance curves and the identifiability of mid-points for the three Minkowski metrics corresponding to r = 2 (Euclidean),\nr = 1 (city-block), and r = 1.5 Non-Euclidean Multidimensional Representations Three specific possibilities are\nshown by white dots, but clearly any point along the line\nwhere the iso-distance contours coincide is possible. In the\ncontext of an MDS representation, this means that there is\na fundamental difficulty in identifying a stimulus that is\nequally different to both of the points. This basic problem\nis not, in general, solved by the introduction of additional\nstimuli that provide additional constraints. Indeed, the\nproblem compounds for potential city-block representations\nwith many stimuli. Bortz (1974, see, especially, Figures 2\nand 3) provides compelling examples, and the same point is A practical approach for identifying city-block repre-\nsentations, used by Nosofsky (1985), relies on determining\nthe values of some stimuli on some dimensions, by means\nexternal to the MDS modeling. Ultimately, this strategy\ncan solve the problem, if it is possible to find the val-\nues of every stimulus on every dimension. But, Fig. 5\nsuggests the strategy may not be effective in situations\nwhere the identification of just a few stimuli is possible. In both representations, Dingbats is at the same location, 328 Comput Brain Behav (2020) 3:322–340 Dimension 1\nDimension 2\n Albert\n Beowulf\n Cedric\n Dingbats Ethelred\n Fiona\nDimension 1\nDimension 2\n Albert\n Beowulf\n Cedric\n Dingbats\n Ethelred\n Fiona\nFig. 5 Two city-block representations of six fictitious people in terms of two dimensions. Both representations have identical proximity matrices Albert Fig. 5 Two city-block representations of six fictitious people in terms of two dimensions. Both representations have identical proximity matrices Fig. 5 Two city-block representations of six fictitious people in terms of two dimensions. Both representa coincide at only one point. The asymmetry of these contours\nmakes clear they do not have the rotational invariance of the\nEuclidean r = 2 metric. In this way, general non-Euclidean\nmetrics, such as r\n=\n1.5, capture the psychological\nidea that the dimensions in an MDS representation have\nmeaning and allow selective attention, while avoiding the\ndegenerate lack of identifiability inherent in the city-block\nmetric. consistent with values on dimensions having been exter-\nnally determined, yet the locations of the remaining stimuli\nare under-determined. In addition, if, for example, Albert\nwas additionally identified as being located in the position\nshown in the first representation, that would constrain the\ninference about Beowulf and Cedric, but would not con-\nstrain Ethelred and Fiona, who could still be inferred to be at\neither of the possibilities shown in the two representations. 3These order constraints can be imposed either in decreasing manner,\nas shown in Fig. 6 for easier visualization, or in an increasing manner,\nas they are in our code. Non-Euclidean Multidimensional Representations Thus, while the addition of stimuli, or the identification of\ndimension values for some stimuli, may work in some spe-\ncific circumstances, we do not believe either represents a\ngeneral approach to making city-block MDS representations\nidentifiable. Figure 6 shows the constraints needed to identify these\nsort of non-Euclidean MDS representations in two and three\ndimensions. In the two-dimensional case, the first point p1\nis once again fixed at the origin, to control translation, the\nsecond point p2 is constrained to the positive quadrant to\ncontrol reflection. In addition, the constraint that p22 ≤p21\nis imposed, requiring the value of the second stimulus on\nthe y-axis not to be larger than its value on the x-axis. This constraint controls for axis permutation, preventing the\ntwo dimensions from being swapped, and so allocates a We do not know how to solve the problem of MDS\nmodel invariance for the city-block metric. As the right-\nmost panel of Fig. 4 makes clear, however, the problem\ndoes not occur for Minkowski-metric parameters r > 1. For the r = 1.5 metric, the iso-distance contours again Fig. 6 Identification constraints for non-Euclidean representations in two dimensions (left) and three dimensions (right) Fig. 6 Identification constraints for non-Euclidean representations in two dimensions (left) and three dimensions (right) 329 Comput Brain Behav (2020) 3:322–340 Marginal Likelihood Comparing MDS models with different dimensions and\nmetrics via Bayes factors and posterior model probabilities\nrequires the computation of the marginal likelihood for all\nof the models, Mm,r, being considered where m denotes\nthe dimensionality and r the metric. Let D denote the\nobserved data (i.e., the pairwise dissimilarity ratings dijk)\nand P denote the N × m matrix with the latent stimulus\ncoordinates for each stimulus. The marginal likelihood for\nmodel Mm,r corresponds to the normalizing constant of the\njoint posterior distribution for θ = (P , σ): These first two cases once again make clear a general\npattern, in which the coordinate values of the second\npoint are positive and order constrained.3\nFormally,\nthe constraints for non-city-block but non-Euclidean D\ndimensions are p11, . . . , p1D = 0\np21, . . . , p2D ∼Uniform\n\b\n0, 1\n\t\n:\np21 ≥. . . ≥p2D\np31, . . . , p3D ∼Uniform\n\b\n−1, 1\n\t\n. . . (6 (6) p(D \| Mm,r) =\n\nq(θ \| D, Mm,r) dθ\n=\n\n \np(D\|P, σ, Mm,r)\n\u000b\n\f\r\n\u000e p(P \|Mm,r)\n\u000b\n\f\r\n\u000e p(σ \|Mm,r)\n\u000b\n\f\r\n\u000e dPdσ, p(D \| Mm,r) =\n\nq(θ \| D, Mm,r) dθ\n=\n\n \np(D\|P, σ, Mm,r)\n\u000b\n\f\r\n\u000e\nLikelihood\np(P \|Mm,r)\n\u000b\n\f\r\n\u000e\nJoint Prior on\nStimulus Locations\np(σ \|Mm,r)\n\u000b\n\f\r\n\u000e\nPrior on\nImprecision\ndPdσ Bayesian MDS Inference via DE-MCMC (7) When posterior samples for MDS models are obtained\nusing conventional Markov-chain Monte Carlo algorithms\n(MCMC; e.g., Gamerman & Lopes, 2006), it can occur\nthat chains get stuck in local maxima. In our experience,\nthe reason is typically that the stimuli that are constrained\nare similar to each other. To prevent local maxima, we\nimplemented a heuristic to order the stimuli in a way that\nthose defining the constraints are dissimilar. We motivate\nand describe this heuristic in detail in Appendix 1. In\naddition, to improve sampling, we used the differential\nevolution Markov-chain Monte Carlo algorithm (DE-\nMCMC; e.g., Heathcote et al. in press; Turner et al. 2013)\nthat helps to guide the chains to regions of high posterior\ndensity. where q(θ\n\| D, Mm,r) denotes the unnormalized joint\nposterior density. where q(θ\n\| D, Mm,r) denotes the unnormalized joint\nposterior density. Bayesian Model Comparison via Bridge\nSampling specific underlying stimulus dimension to each axis. The\nthree-dimensional case extends this logic by requiring that\nthe z-axis value of the second point be positive, to prevent\nreflection, and be less than the value of the second point on\nthe y-axis, to prevent permutation. Bridge Sampling Since the marginal likelihood in Eq. 7 is not available\nanalytically, we use Warp-III bridge sampling (Meng\nand Schilling 2002) to estimate this potentially high-\ndimensional integral. Bridge sampling (Meng and Wong\n1996; for a recent tutorial, see Gronau et al. 2017) is based\non the following identity: p(D \| Mm,r) = Eg(θ)\n\u000f\nh(θ) q(θ \| D, Mm,r)\n\u0010\nEp(θ\|D,Mm,r) [h(θ) g(θ)]\n,\n(8) (8) DE-MCMC is a population-based MCMC algorithm that\ngenerates efficient proposals via a population of interacting\nchains (Turner et al. 2013). One strength of the algorithm\nis that it works well for highly correlated target distri-\nbutions. However, we used DE-MCMC primarily for the\nreason that the interacting chains can guide each other to\nregions of high posterior density which helps to avoid the\nissue of chains getting stuck in local maxima. Specifically,\nduring burn-in, we used a migration step that remedies\nthe problem of outlier chains in an effective manner (for\ndetails, see Turner et al. 2013, Appendix 2). We found that\nthe combination of the ordering heuristic and DE-MCMC\nprovides effective sampling consistently for the Euclidean\nmetric,\nand\nis\npartially\neffective\nfor\nnon-Euclidean\nmetrics. where the numerator is an expected value with respect\nto a proposal distribution g(θ), the denominator is an\nexpected value with respect to the parameter posterior\ndistribution p(θ \| D, Mm,r), and h(θ) is a function such\nthat 0 <\n\u0004\u0004\nh(θ) p(θ \| D, Mm,r) g(θ) dθ\n\u0004\u0004 < ∞. The\nbridge sampling estimate is obtained by sampling from\nthe proposal distribution g(θ) and the posterior distribution\np(θ \| D, Mm,r) to approximate the two expected values. Meng and Wong (1996) showed that the optimal choice for\nh(θ) is given by ho(θ) ∝\n\u000f\ns1 q(θ \| D, Mm,r) + s2 p(D \| Mm,r) g(θ)\n\u0010−1 ,\n(9) (9) where si\n= ni/(n1 + n2), i ∈{1, 2}, n1 denotes the\nnumber of samples from the posterior p(θ \| D, Mm,r),\nand n2 denotes the number of samples from the proposal\ng(θ). The optimal choice for h(θ) depends on the marginal 330 Comput Brain Behav (2020) 3:322–340 likelihood of interest. Therefore, in practice, the bridge\nsampling estimate is obtained via an iterative scheme,\npresented below, that updates an initial guess of the marginal\nlikelihood until convergence. first moment of the proposal and the posterior distribution,\nas shown in the upper-right panel. Bridge Sampling To compute the\nWarp-III estimate, one obtains 2n1 posterior samples: the\nfirst half of these samples is used to approximate μ and\nC with their sample versions ˆμ and ˆC, the second half\nof the posterior samples is used in the iterative scheme\n(i.e., Eq. 11). We use the bridgesampling R package\n(Gronau et al. in press) to compute the bridge sampling\nestimate in Eq. 11. η = b C−1 (ζ −μ) ,\n(10) (10) where b\n∼\nBernoulli (0.5) on {−1, 1}, μ denotes the\nexpected value vector of the posterior samples, and \u0005 =\nCC⊤denotes the posterior covariance matrix (i.e., C is\nobtained via a Cholesky decomposition). Figure 7 illustrates the warping approach for the\nunivariate case. In the upper-left panel, the solid line\ncorresponds to the standard Gaussian proposal distribution\nand the gray histogram depicts synthetic posterior samples. Subtracting the posterior mean from all samples matches the 5The function f needs to be one-to-one and its inverse f −1 needs to\nhave a well-defined Jacobian. 4Note that other proposal distributions are conceivable. The only\nconstraints are that the proposal has a zero mean vector, an identity\ncovariance matrix, and exhibits no skewness. 4Note that other proposal distributions are conceivable. The only\nconstraints are that the proposal has a zero mean vector, an identity\ncovariance matrix, and exhibits no skewness.\n5The function f needs to be one-to-one and its inverse f −1 needs to\nhave a well-defined Jacobian.\n6We use a function f that applies a log transformation to σ and\n(scaled) probit transformations to the non-zero elements of P . The\ntransformation for the ordered coordinates of the second stimulus for\nthe non-Euclidean case is described in Appendix 2. Note that it is\nirrelevant whether the coordinates are ordered as decreasing, as shown\nin Fig. 6 for easier visualization, or increasing, as implemented in\nour code. The transformation described in the appendix assumes the\nlatter. These transformations can be applied after having obtained\nposterior samples for θ. Furthermore, where necessary, the expressions\nare adjusted by the relevant Jacobian term \|det Jf −1(ζ)\|. Bridge Sampling Dividing all samples by\nthe posterior standard deviation matches the second moment\nof the two distributions, as shown in the lower-right panel. Finally, attaching a minus sign with probability 0.5 to the\nposterior samples achieves symmetry and thus matches the\nthird moment of the proposal and the posterior distribution,\nas shown in the lower-left panel. The variability of the bridge sampling estimate is\ngoverned not only by the number of samples but also,\nby the overlap between the proposal and the posterior\ndistribution. To obtain estimates with low variability, it\nis therefore prudent to maximize the overlap between\nthese two distributions. The Warp-III approach attempts to\ncreate a large overlap by fixing the proposal to a standard\nmultivariate Gaussian distribution and then manipulating\n(i.e., “warping”) the posterior in a way that matches the\nfirst three moments of the two distributions.4 Crucially, the\nwarping procedure retains the normalizing constant of the\nposterior (i.e., the marginal likelihood of interest). The Warp-III bridge sampling estimate based on ho(θ)\nis computed via an iterative scheme where the value of the\nestimate at iteration t is given by (for more details see,\nGronau et al. 2019): ˆp(D \| Mm,r)(t+1) =\n1\nn2\nn2\n\u0011\ni=1\nl2,i\ns1 l2,i+s2 ˆp(D\|Mm,r)(t)\n1\nn1\nn1\n\u0011\nj=1\n1\ns1 l1,j +s2 ˆp(D\|Mm,r)(t)\n,\n(11) (11) A prerequisite for the warping procedure is that all\nelements of the parameter vector are allowed to range\nacross the entire real line. This can be achieved via a\nchange-of-variables of the form ζ\n=\nf (θ), where f\nis a suitable5 vector-valued function that transforms the\nconstrained elements of θ so that all elements of ζ are\nunconstrained.6 The Warp-III procedure is based on the\nfollowing stochastic transformation of the unconstrained\nparameter vector ζ: l1,j =\n\| ˆC\|\n2\n\u0012\nq(2 ˆμ−ζ ∗\nj \|D,Mm,r)+q(ζ ∗\nj \|D,Mm,r)\n\u0013\ng\n\u0014\nˆC\n−1\u0014\nζ ∗\nj −ˆμ\n\u0015\u0015\n,\n(12)\nand\nl2,i =\n\| ˆC\|\n2\n\u0012\nq( ˆμ−ˆC ˜ηi\|D,Mm,r)+q( ˆμ+ ˆC ˜ηi\|D,Mm,r)\n\u0013\ng(˜ηi)\n. (13) (12) (13) In Eqs. 12–13, q(· \| D, Mm,r) denotes the unnormalized\nposterior density with respect to the unconstrained parame-\nter vector ζ, {ζ ∗\n1, ζ ∗\n2, . . . , ζ ∗\nn1} denote n1 posterior samples,\nand {˜η1, ˜η2, . . . , ˜ηn2} denote n2 samples from the standard\nmultivariate Gaussian proposal distribution. Applications 4Note that other proposal distributions are conceivable. The only\nconstraints are that the proposal has a zero mean vector, an identity\ncovariance matrix, and exhibits no skewness. In this section, we present applications of our method to\nfive existing data sets. For each application, we describe the\nstimuli and the nature of the data, as well as make clear\nour expectations about the MDS representation that will be\ninferred. In particular, we state our expectations about both\nthe dimensionality and metric structure of the representation\nwhenever possible. The results we present are based on\nconsidering MDS models up to and beyond this expected\ndimensionality, so that the inference our method makes\nis clear. Where possible, we apply our method under the\nassumption that the metric space is both Euclidean (r = 2)\nand non-Euclidean (r = 1.5) so that an inference can also 5The function f needs to be one-to-one and its inverse f −1 needs to\nhave a well-defined Jacobian. 6We use a function f that applies a log transformation to σ and\n(scaled) probit transformations to the non-zero elements of P . The\ntransformation for the ordered coordinates of the second stimulus for\nthe non-Euclidean case is described in Appendix 2. Note that it is\nirrelevant whether the coordinates are ordered as decreasing, as shown\nin Fig. 6 for easier visualization, or increasing, as implemented in\nour code. The transformation described in the appendix assumes the\nlatter. These transformations can be applied after having obtained\nposterior samples for θ. Furthermore, where necessary, the expressions\nare adjusted by the relevant Jacobian term \|det Jf −1(ζ)\|. 331 Comput Brain Behav (2020) 3:322–340 Fig. 7 Illustration of the Warp-III procedure. The black solid line shows the standard Gaussian proposal distribution and the gray histogram shows\nsynthetic posterior samples. Available at https://tinyurl.com/y7owvsz3 under CC license https://creativecommons.org/licenses/by/2.0/ Fig. 7 Illustration of the Warp-III procedure. The black solid line shows the standard Gaussian proposal distribution and the gray histogram shows Fig. 7 Illustration of the Warp-III procedure. The black solid line shows the standard Gaussian proposal distribution and the gray histogram shows\nsynthetic posterior samples. Available at https://tinyurl.com/y7owvsz3 under CC license https://creativecommons.org/licenses/by/2.0/ assuming a Euclidean metric.7 As for all of our applications,\nwe used 15 chains and 500 burn-in samples. During burn-\nin, the probability of a migration step was set to 0.05. 7We note, however, for completeness that we had difficulty with\nconvergence using the r = 1.5 metric for these data. Applications After\nburn-in, migration was switched off, and the algorithm was\nrun for 9000 iterations. We only retained every third sample\nso that we ended up with 3000 samples per chain for further\nuse (i.e., a total of 45,000 samples collapsed across chains). be made about the integrality or separability of the stimulus\ndomain. For some applications, we were unable to generate\nsamples with acceptable convergence for the r = 1.5 metric. In those cases, we only report results assuming the r = 2\nmetric. Line Length Because\nof the assumptions of equal prior probabilities, the ratio of\nany pair of posterior probabilities is naturally interpreted\nas a Bayes factor. The key result is that the expected\none-dimensional representation is inferred, with a posterior\nprobability near one. We expect the MDS representation to use the Euclidean\nmetric, consistent with the integral nature of the color\nstimulus domain. We also expect a two-dimensional\nrepresentation, following the color circle found by previous\nMDS analyses of these and other color similarity data,\nsuch as the Shepard (1962) original MDS analysis of data\nreported by Ekman (1954). The right panel of Fig. 8 shows the inferred one-\ndimensional MDS representation. The black lines show\nthe stimuli in terms of their physical line lengths,\nlocated at the posterior mean of their location in the\npsychological\nspace. The\nblue\nhistograms\nshow\nthe\nmarginal posterior distributions for each line stimulus. The\nMDS representation arranges the line stimuli in order of\ntheir length, but they are not evenly spaced, despite the\nlines increasing in constant physical increments. Instead,\nthe psychological representation shows compression for the\nlonger lines, consistent with basic psychophysics (Fechner\n1966). This compression is large enough that the posterior\ndistributions begin to overlap for the longest line stimuli. Figure 9 shows the results of applying our method,\nassuming a Euclidean metric. This was a case in which\nwe were unable to generate samples with acceptable\nconvergence for the r = 1.5 metric. For the Euclidean\nmetric, there is uncertainty regarding the dimensionality,\nwith a three-dimensional representation having probability\na little over 0.6 and a two-dimensional representation having\nalmost all of the remaining probability. The inferred three-\ndimensional representation is shown by pairing the first two\ndimensions as a two-dimensional plot in the center of Fig. 9,\nand showing the remaining third dimension separately to\nthe right along an axis. Because of our ordering heuristic,\nthe yellow and purple-blue stimuli were fixed at the origin\nand on the first axis. These assignments mean that the\nfirst two dimensions effectively represent the expected color\ncircle that “bends” the visible physical spectrum from red\nto purple colors into a circle that reflects the psychological\nsimilarity between the end points. Line Length The left panel of Fig. 8 shows posterior model\nprobabilities, assuming equal prior probabilities, for one-,\ntwo-, and three-dimensional MDS representations. To\nassess the stability of the posterior model probability\nestimates, we ran the Warp-III procedure five times based on\nnew samples from the proposal distribution (we always used Our first application involves the similarity judgments\nbetween nine lines of equally increasing length provided\nby 27 participants, as reported in Cohen et al. (2001). We expect these stimuli to have a one-dimensional MDS\nrepresentation, corresponding to line length. Because the\nMinkowski metrics are all equivalent in a one-dimensional\nspace, we do not have any expectations about the metric\nstructure. Thus, we applied our method to these data by 332 Comput Brain Behav (2020) 3:322–340 1\n2\n3\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. 8 Results for line-length similarity data from Cohen et al. (2001). The left panel shows the posterior model probabilities for one- through\nthree-dimensional MDS representations. The right panel shows the 1\n2\n3\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. 8 Results for line-length similarity data from Cohen et al. (2001). The left panel shows the posterior model probabilities for one- through\nthree-dimensional MDS representations. The right panel shows the\ninferred one-dimensional representation with black lines showing the\nline stimuli at their inferred locations and blue histograms showing the\nmarginal posterior distributions for these locations 0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability inferred one-dimensional representation with black lines showing the\nline stimuli at their inferred locations and blue histograms showing the\nmarginal posterior distributions for these locations inferred one-dimensional representation with black lines showing the\nline stimuli at their inferred locations and blue histograms showing the\nmarginal posterior distributions for these locations Fig. 8 Results for line-length similarity data from Cohen et al. (2001). The left panel shows the posterior model probabilities for one- through\nthree-dimensional MDS representations. The right panel shows the been considered in the MDS literature (e.g., Borg and\nGroenen 1997; Carrol and Wish 1974). We consider only\nthe data from the ten participants with normal color vision. the same set of posterior samples). These five repetitions\nare drawn as separate lines but, in this case, the results are\nso similar that they are visually indistinguishable. Line Length The third dimension,\nwhich we did not expect, could correspond to something\nlike luminance, since low luminance purple-like colors are\ngenerally located at one end of the dimension and high\nluminance yellow-like colors are generally located at the\nother end. Colors Our second application considers classic data reported\nby Helm (1964), involving the similarities between ten\ncolors. The experimental procedure involved trials in which\nparticipants were presented with physical tiles of three\ndifferent colors, and moved one of the tiles to reflect their\nperceived overall similarity of the color of this tile to the\ncolors of the other two tiles. Based on these responses, Helm\n(1964) calculated measures of pairwise similarities between\nthe colors, and the resulting proximity data have previously 333 Comput Brain Behav (2020) 3:322–340 yell\n pur\n green-yellow-2\n blue\n green\n green-yellow\n1\n2\n3\n4\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. 9 Results for color similarity data from color-normal subjects\nreported by Helm (1964). The left panel shows the posterior proba-\nbilities for one- through four-dimensional MDS representations. The\nright panel shows the inferred three-dimensional representation, with\ntwo dimensions shown as a two-dimensional plot in the center, and the\nthird dim\nlabels s\n95% cre\ndimensi yellow\n purple-blue\n red-purple\n green-yellow-2\n red-orange\n blue\n purple-2\n green\n green-yellow-1\n purple-1\n purple-1\n red-orange\n purple-2\n yellow\n purple-blue\n red-purple\n blue\n green\n green-yellow-1\n green-yellow-2\n1\n2\n3\n4\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability yellow\n purple-blue\n red-purple\n green-yellow-2\n red-orange\n blue\n purple-2\n green\n green-yellow-1\n purple-1\n purple-1\n red-orange\n purple-2\n yellow\n purple-blue\n red-purple\n blue\n green\n green-yellow-1\n green-yellow-2\n1\n2\n3\n4\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. 9 Results for color similarity data from color-normal subjects\nreported by Helm (1964). The left panel shows the posterior proba-\nbilities for one- through four-dimensional MDS representations. The\nright panel shows the inferred three-dimensional representation, with\nthird dimension shown along an axis to the right. Circular markers and\nlabels show the inferred locations of each stimulus and error bars show\n95% credible intervals for the marginal posterior distribution for each\ndimension Dimensions third dimension shown along an axis to the right. Circular markers and\nlabels show the inferred locations of each stimulus and error bars show\n95% credible intervals for the marginal posterior distribution for each\ndimension Fig. 9 Results for color similarity data from color-normal subjects\nreported by Helm (1964). The left panel shows the posterior proba-\nbilities for one- through four-dimensional MDS representations. The\nright panel shows the inferred three-dimensional representation, with\ntwo dimensions shown as a two-dimensional plot in the center, and the 8For these stimuli, we did not have access to information about the\nprecise physical values of the radius and angles, and so the depictions\nin Fig. 11 are approximate. Rectangles with Line Segments analyses of these data, we expect a two-dimensional MDS\nrepresentation. We also expect the two stimulus dimensions\nto be psychologically separable. Our third application involves data reported by Kruschke\n(1993) involving the similarity between eight geometric\nstimuli. These stimuli consisted of rectangles with interior\nline segments, and varied in terms of the height of the\nrectangle and the horizontal location of the line segment. A\ntotal of 50 participants provided similarity ratings on a nine-\npoint scale for all 28 stimulus pairs. Based on the original\n(Kruschke 1993) and subsequent (e.g., Lee 2001, 2008) Figure 10 shows the results of applying our method\nassuming both the r = 1.5 and r = 2 metrics. It is clear\nthat a two-dimensional representation with the separable\nr = 1.5 metric is inferred. It has essentially all of the\nposterior probability, with one- and three-dimensional r =\n1.5 representations, and all of the r = 2 representations\nhaving essentially no posterior probability. The inferred 1\n2\n3\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. 10 Results for rectangles with interior line segments data reported\nby Kruschke (1993). The left panel shows the posterior probabilities\nfor one- through three-dimensional MDS representations, for both the\nMinkowski metrics with r = 1.5 and r = 2. The right panel shows\nthe inferred two-dimensional representation. The stimuli are shown at\ntheir inferred locations and error bars show 95% credible intervals for\nthe marginal posterior distribution for each dimension the inferred two-dimensional representation. The stimuli are shown at\ntheir inferred locations and error bars show 95% credible intervals for\nthe marginal posterior distribution for each dimension 1\n2\n3\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability Fig. 10 Results for rectangles with interior line segments data reported\nby Kruschke (1993). The left panel shows the posterior probabilities\nfor one- through three-dimensional MDS representations, for both the\nMinkowski metrics with r = 1.5 and r = 2. The right panel shows the inferred two-dimensional representation. The stimuli are shown at\ntheir inferred locations and error bars show 95% credible intervals for\nthe marginal posterior distribution for each dimension 334 Comput Brain Behav (2020) 3:322–340 the inferred two-dimensional representation. The stimuli are shown at\ntheir inferred locations and error bars show 95% credible intervals for\nthe marginal posterior distribution for each dimension 1\n2\n3\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. Rectangles with Line Segments 11 Results for the Shepard circles data collected by Treat\net al. (2001). The left panel shows the posterior probabilities for\none- through three-dimensional MDS representations, for both the\nMinkowski metrics with r = 1.5 and r = 2. The right panel shows\nthe inferred two-dimensional representation. The stimuli are shown at\ntheir inferred locations and error bars show 95% credible intervals for\nthe marginal posterior distribution for each dimension 1\n2\n3\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability Dimensions the inferred two-dimensional representation. The stimuli are shown at\ntheir inferred locations and error bars show 95% credible intervals for\nthe marginal posterior distribution for each dimension Fig. 11 Results for the Shepard circles data collected by Treat\net al. (2001). The left panel shows the posterior probabilities for\none- through three-dimensional MDS representations, for both the\nMinkowski metrics with r = 1.5 and r = 2. The right panel shows Colored Shapes representation closely matches the ways in which the stimuli\nphysically vary, with each psychological axis corresponding\nto an interpretable stimulus dimension. The horizontal axis\ncorresponds to the position of the line segment and the\nvertical axis corresponds to the height of the rectangle. Our final application considers similarity data for nine\ncolored shape stimuli collected by Lee and Navarro (2002). The stimuli were circles, squares, and triangles that were\ncolored red, green, and blue. The data were collected from\n20 participants, each of whom rated the similarity of each\npair of stimuli on a five-point scale. Shepard Circles The middle and right panels show the inferred four-dimensional\nrepresentation, with two dimensions shown in each panel. The col-\nored shapes show the inferred locations of each stimulus and error bars\nshow 95% credible intervals for the marginal posterior distribution for\neach dimension 1\n2\n3\n4\n5\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability Dimensions representation, with two dimensions shown in each panel. The col-\nored shapes show the inferred locations of each stimulus and error bars\nshow 95% credible intervals for the marginal posterior distribution for\neach dimension Fig. 12 Results for colored shapes data reported by Lee and Navarro\n(2002). The left panel shows the posterior probabilities for one-\nthrough five-dimensional MDS representations for the Euclidean met-\nric. The middle and right panels show the inferred four-dimensional stimulus set. The corresponding approximately equilateral\ntriangles could be equally well accommodated by any of\nthe Minkowski metrics we are considering. Thus, from\na statistical perspective—without regard to the theory of\nseparable and integral stimuli—we expect the simplest\nmetric to be inferred. Since all metrics should be able\nto fit the data, the one with the smallest functional form\ncomplexity should be preferred. expectations, with the exception of the color application. In addition, where inferences about whether a Euclidean or\nnon-Euclidean metric structure were made, they matched\ntheoretical expectations. It is interesting to note that all\nof the applications for which non-Euclidean metrics made\ninference difficult involved stimulus domains for which the\nexpectation was that the Euclidean metric was appropriate. We also think that the five applications serve to\ndemonstrate the usefulness of our approach to determining\ndimensionality and metric structure. Our approach is to\ntreat these determinations as Bayesian model-selection\nproblems and use Bayesian posterior probabilities to make\ninferences. Complete Bayes posterior probabilities have\nnot been used in this way previously to determine either\ndimensionality or metric structure, and our introduction of\nthe Warp-III method to solve the difficult computational\napproximation problems involved represents progress on\nthese long-standing challenges in MDS modeling. We found that this was a third case in which we were\nunable to generate samples with acceptable convergence for\nthe r = 1.5 metric. Accordingly, Fig. 12 shows the results\nof applying our method assuming the Euclidean metric. A four-dimensional representation is clearly favored. This\nrepresentation is shown in terms of two two-dimensional\nsubspaces, and has the expected structure. The middle panel\nof Fig. Shepard Circles Our fourth application involves data collected by Treat et al. (2001), involving the similarity between nine geometric\nstimuli known as “Shepard circles.” These stimuli consist\nof a closed semi-circle with an interior ray from the\ncenter to the perimeter. The nine stimuli are constructed\nby exhaustively varying three different radius lengths and\nthree different angles for the internal ray. As for the\nrectangles with line segments, we expect a separable two-\ndimensional MDS representation. For these stimuli, we\nexpect the dimensions to correspond to the radius and angle\ndimensions. Following the previous analysis in Lee and Navarro\n(2002), we expect a four-dimensional representation. This\nrepresentation is best understood as being the product\nof a pair of two-dimensional representations, with one\nrepresenting the similarities between the shapes, and the\nother representing the similarities between the colors. There\nare only three shapes and three colors, and neither set of\nthree has a natural ordering. Instead, the circle, square, and\ntriangle are all approximately equally different from one\nanother, and the same is true of the red, green, and blue\ncolors. These equal similarities are naturally represented by\ntwo-dimensional approximately equilateral triangles. The\nfour-dimensional representation we expect is simply the\nindependent combination of these two two-dimensional\nsubspaces. Figure 11 shows the results of applying our method\nassuming both the r = 1.5 and r = 2 metrics.8 It is\nclear, once again, that a two-dimensional representation\nwith the separable r\n=\n1.5 metric is inferred. The\ninferred representation also again closely matches the ways\nin which the stimuli physically vary, with the horizontal\naxis corresponding to the radius of the semi-circle and the\nvertical axis corresponding to the angle of the ray. Our expectations for the metric structure of the MDS\nrepresentations are less straightforward. Theoretically, the\ninteraction between the shape and color dimensions is a\nclassic example of a separable relationship. The metric\nstructure within the color subspace, however, is theoretically\nintegral, as for the previous application. Countering these\ntheoretical expectations is the fact that there are only three\nvalues for the color and shape dimensions present in the Comput Brain Behav (2020) 3:322–340 335 1\n2\n3\n4\n5\nDimensions\n0\n0.2\n0.4\n0.6\n0.8\n1\nModel Probability\nFig. 12 Results for colored shapes data reported by Lee and Navarro\n(2002). The left panel shows the posterior probabilities for one-\nthrough five-dimensional MDS representations for the Euclidean met-\nric. Shepard Circles 12 shows a subspace that captures the similarity\nrelationships between the red, green, and blue colors. The\nright panel shows a subspace that captures the similarity\nrelationships between the circle, square, and triangle shapes. These subspaces were found using an orthogonal Procrustes\nmethod (Borg and Groenen 1997, p. 162). In particular,\nwe solved for the orthogonal transformation matrix that\nmost closely mapped the inferred coordinate locations\nto the expected representational structure, defined as the\nproduct of two subspaces each with an equilateral triangle\nconfiguration. Despite this progress, we think the greatest contribution\nof the current work is to highlight fundamental challenges\nin MDS models of mental representation, and suggest new\navenues for theoretical development. The challenges largely\nstem from our insistence on fully Bayesian inference,\nwhich has enormous advantages in terms of reaching\ncomplete, coherent, and principled conclusions, but also\nraises technical hurdles. The opportunities largely stem\nfrom our adoption of a generative modeling approach (Lee\n2018). In particular, we think there are many remaining\npossibilities relating to the use of different metrics in MDS\nrepresentations, and that there is an opportunity to extend\nthe generative approach to develop more complete cognitive\nprocess models for inferring MDS representations. We\nconclude by discussing some of these challenges and\nopportunities. Other Representations We did not consider Minkowski metrics with r < 1. This\npossibility has been proposed as a way of representing\nstimulus domains in which the component dimensions\ncompete for attention (Shepard 1987, 1991; Tversky and\nGati 1982). The identifiability constraints for this metric\npresent an open research challenge, and it is not clear\nhow well DE-MCMC sampling methods will perform in\ninferring representations. Our current approach to determining the appropriate\nmetric treats this inference as a model-selection problem,\nand only considers the possibilities r\n= 1.5 and r\n=\n2. Allowing for other metrics is theoretically interesting,\nbut computationally difficult. One obvious cost is the\nneed to generate posterior probabilities across a larger set\nof candidate models. But it also seems likely that some\nmodels will be difficult to make inferences about. We\ntried our DE-MCMC approach for r = 1.1 on a number\nof data sets, and were not able to achieve satisfactory\nconvergence. Furthermore, as explained above, for a few\nof the applications, we were also not able to achieve\nsatisfactory convergence for r = 1.5. These challenging\ncases involved stimulus domains for which the expectation\nwas that the Euclidean metric was appropriate, which\nleads to a speculative suggestion that failure is related\nto model mis-specification. This is a potential example\nof a general aspect of Bayesian model comparison that\ncan be computationally challenging: in order to rule out\nmodels that are likely mis-specified, one needs to be able\nto infer them well enough that they can be part of the\nmodel comparison. Although we believe that DE-MCMC\nis a powerful sampling algorithm which substantially helps\nalleviate the issue of non-converging chains, future research\nshould explore different sampling algorithms that may\nperform better, particularly for non-Euclidean metrics. There is also the possibility of moving beyond the\nMinkowski family of metrics. In his presidential address,\nShepard (1974, Figure 11) presented a taxonomy of\nmetric spaces, each of which makes different fundamental\nrepresentational assumptions that could be appropriate for at\nleast some stimulus domains. There has been relatively little\nwork in exploring these possibilities. Lindman and Caelli\n(1978) investigated MDS representations using Riemannian\nspaces with constant curvature, and Cox and Cox (1991)\npresented compelling applications for a special case of this\napproach involving MDS representations on a sphere. Discussion Collectively, the five applications demonstrate that our\nmethod is able to make reasonable inferences about MDS\nrepresentations. The inferred number of dimensions, and the\ninferred stimulus locations, generally matched theoretical 336 Comput Brain Behav (2020) 3:322–340 Technical Challenges insight into how people represent the real-world stimuli. The successful applications we presented—in which there\nwere clear expectations about dimensionality, metric, and\nrepresentational structure—provide a basis for believing the\nBayesian framework can provide this insight to situations\nwhere, because there are no clear theoretical expectations,\nanswers must be inferred from data, if and when the\ncomputational technical hurdles are overcome. Developing a generative MDS model in a Bayesian setting\nrequired the key issue of identifiability and invariance to\nbe solved in terms of prior information, rather than more\nheuristically through post-processing. We used an exist-\ning solution to this challenge for the Euclidean metric,\nand proposed a solution for psychologically interpretable\nnon-Euclidean metrics with 1 < r < 2. We also high-\nlighted, however, the fundamental intractability of MDS\nrepresentations using the city-block metric. This intractabil-\nity has been documented before (Bortz 1974; Frank 2006,\nFigure 5.4; Shepard 1974, Figure 11), but has not pre-\nvented the use of MDS representations inferred based on the\ncity-block metric in the cognitive modeling literature (e.g.,\nKruschke 1993; Lee and Wetzels 2010). Conclusion One example, involving the line-length application, was\npresented in a preliminary form by Lee (2014). A simple\nplot of the raw behavioral data suggests that one of the\n27 participants appears to have reversed the scale that was\nused to judge similarity. This means that their judgments\ncontaminate the inference of the MDS representation. Lee\n(2014) used a simple latent-mixture model extension of the\nbasic MDS generative model, in which either the scale was\nused correctly or reversed. One participant was inferred\nto have reversed the scale, as expected. Perhaps more\nimportantly, however, the resulting inference about the one-\ndimensional MDS representation was shown to have less\nuncertainty than the one shown in Fig. 8. In this way,\nthe introduction of individual differences in the cognitive\nprocess of similarity judgment helped decontaminate the\ninference about the representation of stimuli. We adopted a Bayesian model-selection approach to the\nproblem of determining the dimensionality and metric struc-\nture of MDS representations, while considering psycholog-\nically interpretable Euclidean and non-Euclidean metrics. Our methods for inferring the representations and choosing\ntheir dimensionality and metric structure show the promise\nof the approach, but computational challenges remain a\nbarrier in terms of an easy-to-use general capability. Our\nmethods and applications also show the promise of plac-\ning MDS representations in a generative cognitive modeling\nframework, offering the possibility of new models of how\npeople represent stimuli, and how those representations help\nguide behavior. Acknowledgments All code is available on the Open Science\nFramework: https://osf.io/82g3r/. We thank Rob Nosofsky and Mike\nD’Zmura for helpful discussions. QFG acknowledges the support\nby a Netherlands Organization for Scientific Research (NWO)\ngrant (406.16.528). Correspondence should be sent to Quentin F. Gronau, University of Amsterdam, Nieuwe Achtergracht 129 B,\n1018 WT Amsterdam, The Netherlands. E-mail may be sent to\nQuentin.F.Gronau@gmail.com. Acknowledgments All code is available on the Open Science\nFramework: https://osf.io/82g3r/. We thank Rob Nosofsky and Mike\nD’Zmura for helpful discussions. QFG acknowledges the support\nby a Netherlands Organization for Scientific Research (NWO)\ngrant (406.16.528). Correspondence should be sent to Quentin F. Gronau, University of Amsterdam, Nieuwe Achtergracht 129 B,\n1018 WT Amsterdam, The Netherlands. E-mail may be sent to\nQuentin.F.Gronau@gmail.com. The same basic generative approach could support much\nmore general cognitive process modeling using MDS rep-\nresentations. Other Representations A new idea raised by our application to the colored shape\nstimuli involves the possibility of different metric structures\nwithin the same representation. These stimuli involved\ntwo sorts of stimulus dimensions: those representing color,\nwhich are usually considered to be integral, and those\nrepresenting qualitatively different shapes, which seems\nmore separable. Certainly the interaction between the color\ndimensions and the shape dimensions would be expected\nto be separable, since it seems likely people can selectively\nattend to either the color or the shape of a stimulus,\ndepending upon the cognitive context. This suggests a\ngeneralization of the MDS models in which each pair of\ndimensions is associated with a metric. Collectively, these technical challenges mean that our\napproach cannot currently be applied to large naturalistic\nstimulus domains. For example, Nosofsky et al. (2018)\nconsider MDS representations based on sparse matrices\nof pairwise similarity judgments for a set of 360 images\nof rocks, and Hebart et al. (2020) report extensive\ncrowd-sourced triadic comparison similarity data for 1854\nimages of real-world objects. Being able to determine\nthe dimensionality, metric structure, and psychological\nrepresentations of MDS representations of these domains\nusing the Bayesian framework would potentially offer deep Finally, there are alternative representational models,\nwhich do not assume stimuli are represented by values on\ndimensions, that can compete with or complement MDS\nmodels. These alternatives include feature-based represen-\ntations (Tversky 1977), such as those found by additive\nclustering and related methods (Shepard and Arabie 1979)\nand special cases such as tree-based models (Corter 1996;\nShepard 1980). One attraction of the Warp-III approach\nwe used is that it could estimate Bayes factors between\nfundamentally different sorts of representations—such as Comput Brain Behav (2020) 3:322–340 337 individual differences, such as INDSCAL (Carroll and\nChang 1970; Carroll 1972). These would be easy to imple-\nment within our generative modeling framework. A model\nlike INDSCAL, which assumes individuals weight the latent\nstimulus dimensions differently, relies on the appropriate\nnumber of dimensions being inferred, and evidence that the\nstimulus domain is separable. In this way, the potential of\nour method to make these inferences is especially important. As a final example, the rectangle and line segment stim-\nuli are used by Kruschke (1993) to study category learning,\nbut the similarity data and category learning data are ana-\nlyzed independently. Other Representations In effect, the similarity data are used to\ngenerate the MDS representation, and that representation is\nthen assumed to provide the fixed basis for category learn-\ning. An alternative approach would be to infer the MDS\nrepresentation jointly from both the similarity judgments\nand the category learning choices. This sort of flexibility\nraises the possibility of tackling more complicated cogni-\ntive phenomena, such as the ability to adapt representations\nin response to changes in the external environment, or the\ncurrent context or goals. comparing dimensional and featural representations—since\nit operates directly on posterior samples for each model\napplied independently to the data. Even further, Navarro and\nLee (2003) proposed a hybrid model of stimulus represen-\ntation that combined both dimensions and features, and it\nwould be conceptually elegant to choose between all of the\ncandidate models, with various combinations of dimensions\nand features, using our methods. Navarro and Lee (2003)\nused an approximate analytic approach for this purpose,\nwhich would be significantly improved by an approach\nbased on Bayes factors. MDS Cognitive Process Models Our modeling approach is generative, but is based\non an extremely simple cognitive model. In essence,\nwe assume that all participants have the same MDS\nrepresentation, and produce dissimilarity judgments for\npairs of stimuli that directly reflect the distances between\nthose stimuli in the representation. It is likely that much\nbetter generative models can be developed by considering\nmore realistic processing assumptions, and especially by\nincluding individual differences. Appendix 1. The ordering heuristic Figure 13 provides a concrete example to motivate the\nneed for the ordering heuristic. It is clear this is an\ninferior representation to the one presented in Fig. 8. In\nFig. 13, the first- and second-line stimuli, which are the two\nshortest, are located at almost the same point, rather than\nbeing appropriately spaced to reflect their psychological\ndissimilarity. Consistent with this intuition, the posterior\ndensity is worse for the representation in Fig. 13 than the\nrepresentation in Fig. 8. We used this ordering heuristic for the colors and colored\nshapes applications. For the line-length application, we used\nthe heuristic as described but then, in an additional step,\nswitched the first stimulus with the second stimulus. This\nswitch helped prevent the posterior for the ninth stimulus,\ncorresponding to the longest line, push against the upper\nbound of 1. For the rectangles with interior line segments\nand Shepard circles applications, we used the heuristic as\na starting point, but we then reordered some of the stimuli\nmanually since it seemed to help with convergence. This suboptimality is caused by the naive application of\nthe constraints identified in Fig. 2 for a one-dimensional\nrepresentation. The first stimulus is fixed at the origin, and\nthe second stimulus is constrained to be positive. It is clear\nfrom Fig. 13 that the second stimulus is indeed inferred to be\npositive, but is extremely close to zero, with the remaining\nlonger line stimuli “flipping” to negative values in the MDS\nspace. This configuration still satisfies the proximity data\nreasonably well, because the required distance between the\nfirst two stimuli is small, and the distances from the first\nand second stimuli to all of the others is approximately\nconserved. Thus, it is the choice of the two similar stimuli\nas those that are constrained that leads to this potential for a\nlocal maximum and suboptimal representation. Conclusion The hierarchical, latent mixture, and common\ncause model structures advocated by Lee (2018) could allow\nfor rich accounts of individual differences in judgment\nprocesses or stimulus representations, and allow for mod-\nels that extend beyond the judgment of similarity to other\ncognitive capabilities like categorization and inference. As\none example, Ennis (1992) considers extended assump-\ntions about MDS representations that allow for the noisy\nrepresentation of perceptual stimuli, which could be incor-\nporated by adding hierarchical structure to the coordinate\nlocations. As another example, there are extensions of the\nbasic MDS model we considered that allow for structured Open Access This article is licensed under a Creative Commons\nAttribution 4.0 International License, which permits use, sharing,\nadaptation, distribution and reproduction in any medium or format, as\nlong as you give appropriate credit to the original author(s) and the\nsource, provide a link to the Creative Commons licence, and indicate\nif changes were made. The images or other third party material in this\narticle are included in the article’s Creative Commons licence, unless\nindicated otherwise in a credit line to the material. If material is not\nincluded in the article’s Creative Commons licence and your intended\nuse is not permitted by statutory regulation or exceeds the permitted Comput Brain Behav (2020) 3:322–340 338 stimuli. Our heuristic for doing this is based on the across\nparticipants averaged pairwise dissimilarity ratings. The\nfirst two stimuli are chosen to be the ones with the largest\naveraged pairwise dissimilarity. The remaining stimuli are\nchosen, one at a time, by considering the minimum averaged\npairwise dissimilarity to the already selected stimuli. Specifically, the next stimulus is always chosen to be the\none with the maximum value for the minimum averaged\npairwise dissimilarity to the already selected stimuli. use, you will need to obtain permission directly from the copyright\nholder. To view a copy of this licence, visit http://creativecommons. org/licenses/by/4.0/. Appendix 2. Transformation ordered vector\n(0–1 bounded) The constrained vector x, 0 ≤x1 ≤x2 ≤. . . ≤xK ≤1,\ncan be transformed to an unconstrained vector y ∈ℜK as\nfollows: yk =\n\u0016\n\u0003−1 (xk)\nif k = 1,\n\u0003−1 \u0014\nxk−xk−1\n1−xk−1\n\u0015\nif 1 < k ≤K, Accordingly, we developed an ordering heuristic to try\nand assign the constraints for the various dimensionalities\nand metrics to stimuli that are sufficiently dissimilar. Because higher dimensionalities place constraints on more\nthan two stimuli, the general approach is to order all of the where \u0003−1(·) denotes the inverse of the normal CDF. The\ninverse transformation is given by: where \u0003−1(·) denotes the inverse of the normal CDF. The\ninverse transformation is given by: xk =\n\u0017 \u0003 (yk)\nif k = 1,\nxk−1 + (1 −xk−1) \u0003 (yk) if 1 < k ≤K, if k = 1, 0\nFig. 13 A suboptimal one-dimensional representation of the line-\nlength similarity data from Cohen et al. (2001), motivating the\nneed for the ordering heuristic. The black lines show the stimuli\nat their inferred locations in the representation, and the blu\nhistograms show the marginal posterior distributions for thes\nlocations at their inferred locations in the representation, and the blue\nhistograms show the marginal posterior distributions for these\nlocations Fig. 13 A suboptimal one-dimensional representation of the line-\nlength similarity data from Cohen et al. (2001), motivating the\nneed for the ordering heuristic. The black lines show the stimuli 339 Comput Brain Behav (2020) 3:322–340 where \u0003(·) denotes the normal CDF. Note that xk is a func-\ntion of y1, y2, . . . , yk (the dependence on y1, y2, . . . , yk−1\nis “hidden” in xk−1). Crucially, xk does not depend\non yk+1, yk+2, . . . , yK. Consequently, the Jacobian matrix\nJ of the transformation is lower triangular so that its\ndeterminant \|J \| is obtained by multiplying its diagonal\nentries. 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https://openalex.org/W4381854395	https://hal.science/hal-04268202/document	English	null	Family psychoeducation to improve outcome in caregivers and patients with schizophrenia: a randomized clinical trial	Frontiers in psychiatry	2,023	cc-by	8,046	Family psychoeducation to improve outcome in caregivers and patients with schizophrenia: a randomized clinical trial Arnaud Tessier, Karine Roger, Alexandra Gregoire, Pauline Desnavailles, David Misdrahi To cite this version: Arnaud Tessier, Karine Roger, Alexandra Gregoire, Pauline Desnavailles, David Misdrahi. Family psychoeducation to improve outcome in caregivers and patients with schizophrenia: a randomized clinical trial. Frontiers in Psychiatry, 2023, 14, pp.1171661. 10.3389/fpsyt.2023.1171661. hal- 04268202 Distributed under a Creative Commons Attribution 4.0 International License OPEN ACCESS OPEN ACCESS EDITED BY Yann Hode, Association Psychoeducation Profamille, France REVIEWED BY Mario Luciano, University of Campania Luigi Vanvitelli, Italy Massimo Casacchia, University of L’Aquila, Italy CORRESPONDENCE Arnaud Tessier atessier@ch-perrens.fr RECEIVED 22 February 2023 ACCEPTED 06 June 2023 PUBLISHED 23 June 2023 CITATION Tessier A, Roger K, Gregoire A, Desnavailles P and Misdrahi D (2023) Family psychoeducation to improve outcome in caregivers and patients with schizophrenia: a randomized clinical trial. Front. Psychiatry 14:1171661. doi: 10.3389/fpsyt.2023.1171661 OPEN ACCESS EDITED BY Yann Hode, Association Psychoeducation Profamille, France REVIEWED BY Mario Luciano, University of Campania Luigi Vanvitelli, Italy Massimo Casacchia, University of L’Aquila, Italy CORRESPONDENCE Arnaud Tessier atessier@ch-perrens.fr RECEIVED 22 February 2023 ACCEPTED 06 June 2023 PUBLISHED 23 June 2023 Arnaud Tessier 1,2,3, Karine Roger 1, Alexandra Gregoire 1, Pauline Desnavailles 1 and David Misdrahi 1,2,3 1 Department of Adult Psychiatry, Charles Perrens Hospital, Bordeaux, France, 2 Aquitaine Institute for Cognitive and Integrative Neuroscience, Bordeaux, France, 3 Fondation Fondamental, Créteil, France Introduction: Schizophrenia is recognized for its severe impact on both patients and caregivers. In a 12-month follow-up randomized clinical trial, we aimed to measure the efficacy of a brief family psychoeducation program in terms of reducing relapse risk and improving medication adherence in patients, as well as reducing caregiver burden, depression and increasing knowledge of the illness. Methods: A total of 25 days of patients with schizophrenia (DSM-IV-TR) and family primary caregivers were recruited in a single regional psychiatric outpatient facility located in Bordeaux. In the active group, caregivers received a psychoeducational intervention consisting of six sessions spread over 1.5 months, while the control group was placed on a waiting list. Sociodemographic, symptom severity (PANSS) and medication adherence (MARS) from patients were assessed at baseline and relapse rates was recorded during the 12 months follow-up period. Caregivers’ burden (ZBI), depression (CES-D), quality of life (S-CGQoL), knowledge of the disease (KAST) and therapeutic alliance (4PAS-C) were assessed at baseline, three and 6 months. COPYRIGHT © 2023 Tessier, Roger, Gregoire, Desnavailles and Misdrahi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. HAL Id: hal-04268202 https://hal.science/hal-04268202v1 Submitted on 2 Nov 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License TYPE Clinical Trial PUBLISHED 23 June 2023 DOI 10.3389/fpsyt.2023.1171661 OPEN ACCESS Results: On the 25 patients included, the mean age was 33.3 years (SD = 9.7) with a mean duration of disease of 7.48 years (SD = 7.1). On the 25 caregivers included, the mean age was 50.6 years (SD = 14.0). Twenty-one were female (84.0%), 12 were married (48.0%) and 11 lived alone (44.0%). For patients, the family psychoeducation intervention significantly reduced the risk of relapse with a significant effect found at 12 months follow-up (p = 0.014). No change was observed on medication adherence. For caregivers, the intervention reduced the burden (p = 0.031), decreased the depression (p = 0.019), and increased the knowledge on schizophrenia (p = 0.024). Analyzes for repeated measures showed a statistically significant difference in therapeutic alliance (p = 0.035). Conclusion: As confirmed by previous studies, the brief multifamily program (consisting of six sessions over a period of 1.5 months) was found to be effective in improving outcomes for caregivers (e.g., burden, depression, knowledge) and patients (e.g., preventing relapse) in the context of routine care. Given its short duration, this program is expected to be easily implementable within the community. Clinical trial registration: https://clinicaltrials.gov/, NCT03000985. Clinical trial registration: https://clinicaltrials.gov/, NCT03000985. psychoeducation, caregivers, schizophrenia, relapse, burden, depression, therapeutic alliance, quality of life Study site and participants Schizophrenia is a chronic and severe mental disorder which has serious consequences for both the patient and caregivers. The burden of schizophrenia on caregivers had been demonstrated (1–4) and justify that family have to be included in the care plan with adequate information and support (5). Therefore, family intervention should be developed to reduce the burden of caregivers and enhance patients’ prognosis. For patients, family psychoeducation has been effective in improving outcomes in schizophrenia with a better level of global functioning, medication adherence, and a reduction in the use of healthcare resources and the frequency of relapse (6–8). A recent systematic review including 11 studies demonstrates consistent improvement in many outcome measures of patients, such as relapse rates and medication adherence, but heterogeneity in symptoms reliefs (9). Its effectiveness has also been demonstrated for individuals at clinical high risk for psychosis although rigorous further studies are required (10). Through psychoeducation, a better understanding of the illness was associated with a better insight and medication adherence (11). For caregivers, increased knowledge of the disease reduces aspects related to stigma, stress and burden which contributes to a supportive social environment to increase the patient’s awareness of the disease and adapted care (12, 13). This randomized single-blind controlled trial adopted a two arms parallel groups design. The controlled trial was conducted at a single regional psychiatric outpatient facility located in Bordeaux, France, registered in ClinicalTrials.gov (Ref.: NCT03000985). A total of 25 dyads of patients and family primary caregivers were recruited between December 2014 and December 2019. The inclusion criteria for patients were (i) a diagnosis of schizophrenia or schizoaffective disorder according to DSM-IV-TR criteria (20), (ii) age of at least 18 years, and (iii) being in a stable phase and receiving outpatients’ routine care. Exclusion criteria consisted of a history of traumatic head injury, any current or past major medical or neurological illness, and mental retardation. The inclusion criteria for caregivers were males or females aged 18 years or older who were currently caring for a relative diagnosed with schizophrenia or schizoaffective disorder and receiving appropriate outpatient clinical care. Caregivers who had previously received a standardized psychoeducational intervention or had intellectual disability, dementia or any other psychiatric condition were excluded from the study. Participants were assigned to one of the two study groups through a computer-generated random process. Study site and participants On the 25 caregivers 11 were the mother, 7 the father, 2 the sister, 1 the brother, 1 spouse/husband, 1 the aunt and finally 2 were the child of patient. In the active group, caregivers received a psychoeducational intervention consisting of six sessions spread over 1.5 months, while the control group, which received treatment as usual (TAU), was placed on a waiting list without any additional strategies. All participants including patients and caregivers signed an informed consent form prior to randomization and trial inclusion. The study protocol involving human participant was reviewed and approved by the local human subject research ethics committee. It has been demonstrated that family psychoeducation is effective and is considered part of the guideline recommendations in the treatment of schizophrenia (5, 14, 15). A Cochrane review confirmed a 20% reduction in relapse rates compared with usual care (7). Caregivers’ outcomes from family psychoeducation are less commonly studied. The only meta-analysis of family outcomes found considerable positive effects on relatives’ burden and psychological distress, the relationship between relatives and the patient, and family functioning (16). A review of family psychoeducation programs suggested that it was more likely to be effective in families if knowledge of the disease and other outcomes such as burden, family functioning, emotional response etc. were systematically assessed to reflect the specific goals of the intervention (8). Despite recommendations and significant results, family psychoeducation is not a widely accessible option in mental health services, often due to limitations including a lack of interest from families, limited availability of care staff, or a shortage of trained professionals in these programs (5, 17, 18). A review of more than 30 randomized clinical trials (RCTs) pointed out that application of family psychoeducation in routine settings remains limited, reflecting attitudinal, knowledge, practice and systemic barriers to implementation (19). Frontiers in Psychiatry frontiersin.org Clinical trial registration: h psychoeducation, caregivers, schizophrenia, relapse, burden, depression, therapeutic alliance, quality of life 01 Frontiers in Psychiatry frontiersin.org Tessier et al. 10.3389/fpsyt.2023.1171661 Patient’s assessment Analyzes were conducted based on the “intention-to-treat” principle; patients and caregivers were analyzed according to the randomization group they were allocated to and regardless of the intervention they followed. Baseline characteristics of patients and caregivers were compared between intervention and control groups using appropriate tests. They were no dropout at follow-up visits. When normality assumption was not rejected, an independent t-test was used for continuous variables. Otherwise, the Mann– Whitney U test was performed. Categorical data were compared using a chi-squared test or a Fisher’s exact test if necessary. Longitudinal repeated measures (baseline, 3 and 6 months) of caregivers’ scales were analyzed in each randomized group using whether repeated measures ANOVA (for normal distributed data) or Friedman test (for non-normal distributed data), followed by a post-hoc analysis using the Wilcoxon signed-rank test with Benjamini-Hochberg correction. Patients were assessed once at inclusion. Sociodemographic information (gender, age, marital status, level of education, living situation) and illness history (duration of illness, lifetime suicide attempt, history of treatments, BMI) were collected. Symptom severity was assessed using the Positive And Negative Syndrome Scale (PANSS) (21) and medication adherence was evaluated with the Medication Adherence Rating Scale (MARS) (Kuder–Richardson-20: 0.60) (22). With the agreement of the authors (23), our team translated the scale into French, then validated it by reverse translation into English with the author. The scale was then used and validated in a large national cohort of 319 patients suffering from schizophrenia (24, 25). Relapses, defined as new psychiatric hospitalization, were recorded by reviewing the computerized medical records at 3, 6, and 12 months from baseline. Intervention The questionnaire yields two subscores (Empathy and Psychoeducation) and a Visual Analog Scale score (0 to 100) (30); the Compliance Rating Scale (CRS), a seven-point rating scale, score ranging from 1 (complete refusal) to 7 (active participation) (31). Alliance Scale – Caregiver (4PAS-C) is an 11-item questionnaire (e.g., “I believe my doctor is helping us”; “I have a better understanding of the symptoms of my relative’s illness.”) scored using a Likert-type format. Responses range from 1 (“strongly disagree”) to 4 (“strongly agree”) and scores range from 11 to 44, with higher scores indicating of a more positive alliance (Cronbach’s alpha: 0.91). The questionnaire yields two subscores (Empathy and Psychoeducation) and a Visual Analog Scale score (0 to 100) (30); the Compliance Rating Scale (CRS), a seven-point rating scale, score ranging from 1 (complete refusal) to 7 (active participation) (31). patient’s daily life, i.e., the psychosocial and cognitive consequences, as well as negative symptoms hindering a good rehabilitation. Solutions for care, networks and associations are presented. The sixth session focuses on the caregivers’ experiences: the weight of the disease, verbalization of feelings. Afterward, a review of the program is made and answers to the last questions are given. A booster session is conducted six months after the initial phase. This session allows to improve the program’s efficacy and to monitor the implementation of daily effective strategies. The Schiz’Aides program has a complete manual with a session guide, which can be provided upon request. Caregivers’ assessment All p values were two-sided, and the level of statistical significance was set to 5%. Statistical analyzes were performed with IBM SPSS statistics, version 26.0. Complete test statistics are displayed in the respective tables and Supplementary data. Caregivers were assessed at baseline, and at 3 and 6 months after the completion of the psychoeducational program. Self-administered questionnaires were used to assess the different dimensions: the knowledge of the disease using the Knowledge About Schizophrenia Test (KAST). The KAST is an 18-item multiple-choice questionnaire (e.g., “Medicines that are used for hearing voices are called …”; “A person strongly believes that the FBI has put a computer chip in his/ her body. This symptom is called a …”) with five response options and the score ranges from 0 to a maximum possible score of 21 (indicating good knowledge) (Kuder–Richardson-20: 0.82) (26); the Quality of Life with Schizophrenia-Caregiver Quality of Life questionnaire (S-CGQoL) contains 25 items scored with a six-point Likert scale and describing seven dimensions, with 100 indicating the best possible level of QoL and 0 the worst (27); the burden of disease was assessed with the Zarit Burden Interview (ZBI). The ZBI includes 22 statements (e.g., “Do you feel that because of the time you spend with your relative that you do not have enough time for yourself?”; “Do you feel your health has suffered because of your involvement with your relative?”) recorded in a 0–4 Likert scale (total score range 0 to 88), that rates the subjective component of burden (Cronbach’s alpha: 0.92) (28); the Center for Epidemiologic Studies – Depression Scale (CES-D) is a 20-item questionnaire that measures depressive symptoms and related behaviors experienced over the past week, with each item rated on a 0–3 Likert scale. Possible scores range from 0 to 60, with higher scores indicating more severe depressive symptoms. A cutoff score of 16 or greater is indicative of individuals at risk for clinical depression (Cronbach’s alpha: 0.90) (29); the 4-Point ordinal Frontiers in Psychiatry Intervention The Schiz’Aides program is a multifamily psychoeducational program, which was built for caregivers of patients with schizophrenia, consisting of six sessions over a 1.5 months period. Each session lasted an average of 1 h and 30 min and was delivered in a group format led by a nurse and a psychologist who were trained to provide psychoeducation. One specific session of the program included the participation of a psychiatrist and a social worker. The program follows international guidelines concerning these interventions (5). The first session focuses on the presentation of each family and the experience of their relative’s illness. It also provides an opportunity to gather families’ expectations and to present the program’s themes. The second session is dedicated in understanding the disease. The objective is to give caregivers the criteria to identify symptoms, as well as etiological factors (multifactorial hypothesis). The third session focuses on drug treatments (role, forms, efficacy, side effects), non-drug treatments (mainly psychotherapies such as cognitive remediation) and finally forms/modes of hospitalization. The fourth session allows the caregiver to better manage their patients’ crisis states. The objective is to identify the warning signs and the adaptive reaction to adopt. The sharing of experiences between the different families is strongly encouraged. The fifth session focuses on the The objective of this study was to compare the efficacy of a new brief family psychoeducation program (consisting of 6 sessions over 1.5 months) to treatment as usual (TAU) in a single center randomized clinical trial. Our aim was to measure the impact of the intervention on both caregivers and patients over a 12-month follow-up period, in comparison to usual care. We hypothesized that the psychoeducation program would reduce the risk of relapse and improve medication adherence in patients with schizophrenia, while also enhancing the quality of life, reducing caregiver burden, improving the therapeutic alliance, increasing knowledge of the illness, and reducing depression in caregivers. 02 frontiersin.org Tessier et al. 10.3389/fpsyt.2023.1171661 10.3389/fpsyt.2023.1171661 Alliance Scale – Caregiver (4PAS-C) is an 11-item questionnaire (e.g., “I believe my doctor is helping us”; “I have a better understanding of the symptoms of my relative’s illness.”) scored using a Likert-type format. Responses range from 1 (“strongly disagree”) to 4 (“strongly agree”) and scores range from 11 to 44, with higher scores indicating of a more positive alliance (Cronbach’s alpha: 0.91). frontiersin.org Sample characteristics at baseline On the 25 patients included, the mean age was 33.3 years (SD = 9.7). They were 6 females (24.0%) with a mean duration of disease of 7.48 years (SD = 7.1). The majority were single (n = 24, 96%), lived alone (n = 15, 60%) and were unemployed (n = 22, 88%). Regarding the clinical variables, patients had a mean duration of illness of 7.5 years (SD = 7.1), a mean number of antipsychotics of 1.20 (SD = 0.5) and a mean antipsychotic dose of 4.55 mg (SD = 4.2) chlorpromazine equivalent. All patients were treated with a second- generation antipsychotic. The mean total PANSS score was 70.76 (SD = 11.8) and the mean score on the MARS scale was 6.52 (SD = 1.9) (moderate compliance). At baseline the two study groups were significant different on PANSS scores, antipsychotic dose, and BMI. The sociodemographic and clinical characteristics of the sample are presented in Table 1. On the 25 caregivers included, the mean age was 50.6 years (SD = 14.0). Twenty-one were female (84.0%), 12 were married (48.0%) and 11 lived alone (44.0%). Twenty-two had a level of education higher than the high school (88%). The mean quality of life 03 frontiersin.org Tessier et al. 10.3389/fpsyt.2023.1171661 TABLE 1 Demographic and clinical characteristics of patients and caregivers included in the study. TABLE 1 Demographic and clinical characteristics of patients and caregivers included in the study. Patients Caregivers Fisher’s exact test Fisher’s exact test Active (PsyEduc) (N = 12) Control (TAU) (N = 13) Active (PsyEduc) (N = 12) Control (TAU) (N = 13) VARIABLES n (%) n (%) p n (%) n (%) p Gender, male 8 (66.7) 11 (84.6) 0.561 3 (25.0) 1 (7.7) 0.603 Marital status, married 0 (0.0) 1 (7.7) 1.000 8 (66.7) 4 (30.8) 0.567 Housing, alone (vs. accompanied) 8 (66.7) 7 (53.8) 0.806 8 (66.7) 3 (23.1) 0.284 Level of education, high school or lower 9 (75.0) 7 (53.8) 0.494 1 (8.3) 2 (15.4) 0.838 Employment status, work (vs. unemployed) 1 (8.3) 2 (15.4) 1.000 Second generation of antipsychotics (vs. Sample characteristics at baseline first) 11 (91.7) 10 (76.9) 0.593 Mann–Whitney U-test for independent samples Mann–Whitney U-test for independent samples Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p Age (years) 36.17 (11.3) 30.62 (7.7) 0.161 45.92 (15.7) 56.89 (8.5) 0.055 BMI (kg/m2) 27.06 (5.0) 24.06 (3.5) 0.034 Number of children 0.17 (0.4) 0.00 (0.0) 0.148 Duration of illness (years) 7.83 (6.2) 7.15 (8.0) 0.298 Number of suicide attempts (lifetime) 0.08 (0.3) 0.15 (0.4) 0.629 Average number of antipsychotics 1.25 (0.6) 1.15 (0.4) 0.898 Antipsychotic dose (1 mg chlorpromazine equivalent) 3.54 (4.0) 5.48 (4.3) 0.043* PANSS 64.58 (10.2) 76.46 (10.5) 0.009* Positive symptoms 13.50 (3.6) 18.77 (4.3) 0.003* Negative symptoms 17.50 (4.9) 19.31 (6.7) 0.453 General psychopathology 33.58 (4.5) 38.38 (4.0) 0.010* MARS 6.78 (1.7) 6.33 (2.0) 0.601 Medication adherence behavior 2.67 (0.9) 2.67 (1.1) 0.969 Attitude to taking medication 3.11 (0.6) 2.83 (1.3) 0.970 Negative side-effects 1.00 (1.0) 0.83 (0.9) 0.726 Mean (SD): mean +/− standard deviation. BMI, Body Mass Index; PANSS, Positive and Negative Syndrome Scale; MARS, Medication Adherence Rating Scale. Significant difference with p < 0.05 are in bold text. (ZBI) and the level of depression (CES-D) [β = 0.78, CI = (0.06, 1.50), p = 0.036]. was 70.80 (SD = 16.5). Burden was scored as medium with a mean of 37.75 (SD = 7.9). Knowledge of the disease was rated as “moderate” with a mean score of 13.1 (SD = 2.3) and therapeutic alliance was rated as “good” with a mean sore of 30.94 (SD = 7.4). Depression was rated as “moderate” with a mean score on CES-D of 20.37 (SD = 11.4). No significant difference was found between groups for sociodemographic and psychometrics scores of caregivers at baseline. A positive significant association was found, between the burden Frontiers in Psychiatry frontiersin.org Caregiver’s outcome The results indicated a significant difference in the total score of the KAST at 1.5 months (p = 0.024). Caregivers who received psychoeducation had higher scores (mean: 15.45; SD: 1.4) than caregivers in the control group (mean: 12.38; SD: 3.4). This difference was not observed at 6 months (p = 0.098). Two significant results were found at 6 months: first, the ZBI score (burden) (p = 0.031), showed a significant reduction for caregivers in the active group (mean: 20.17; SD: 8.0) compared to the control group (mean: 35.60; SD: 12.1); secondly, the CES-D score (depression) showed a significant reduction (p = 0.019), for caregivers in the active group (mean: 6.20; SD: 4.9) compared to the control group (mean: 14.20; SD: 3.6). No significant differences were found for quality of life and therapeutic alliance, regardless of the visit. Additional details are provided in Table 3.i Despite expectations, we did not show any impact of the family psychoeducation on medication adherence. This result is consistent with a previous study where carers’ knowledge about schizophrenia appeared to be not related to compliance (35). Medication adherence which is recognized as complex and multi-determined phenomenon cannot be resolved by a single, non-specific intervention. Moreover, the caregiver’s judgment of their relative’s medication intake may be influenced by factors such as the amount of time spent together and the patient’s regimen (oral or injectable antipsychotic). This presents a limitation for the interpretation of scores on the CRS and highlights the need for a combination of objective measures (e.g., pill counts, serum levels) and validated self-report scales to accurately assess medication adherence (25, 36). Nevertheless, the level of caregiver burden may also have an impact on medication adherence, as demonstrated by recent studies, which underscore the importance of supportive programs for caregivers (9, 37). Analyzes for repeated measures showed a statistically significant difference in therapeutic alliance (4PAS-C total score) (p = 0.035) and two sub-scores: Visual Analogic Scale (p = 0.015), and Psychoeducation (p = 0.036). There was also a significant improvement in the burden, as evaluated by ZBI (p = 0.040; see Table 4 for details). Post hoc analysis did not yield any significant results, despite an overall perceived improvement of therapeutic alliance between caregiver and the healthcare team and a reduction in the burden (Supplementary data). Patient’s outcome A lower rate of relapse was observed at 3, 6 and 12 months for patients whose caregivers participated in the intervention group. The 04 frontiersin.org 10.3389/fpsyt.2023.1171661 Tessier et al. parison of patients’ outcomes (cumulative relapse (re-hospitalization) rates and perceived medication adherence) following time M3 M6 M12 Active (PsyEduc) (N = 12) Control (TAU) (N = 13) Active (PsyEduc) (N = 12) Control (TAU) (N = 13) Active (PsyEduc) (N = 12) Control (TAU) (N = 13) n (%) n (%) p n (%) n (%) p n (%) n (%) p Cumulative relapses 0 (0.0) 3 (37.5) 0.058 0 (0.0) 3 (37,5) 0.082 0 (0.0) 4 (50.0) 0.014 Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p CRS 6.22 (1.4) 5.88 (1.6) 0.778 5.82 (1.7) 6.13 (1.5) 0.747 5.80 (2.2) 6.75 (0.5) 0.661 SD, Standard Deviation; CRS, Compliance Rating Scale. Percentages are adjusted for lost and missing data. Significant difference with p < 0.05 are in bold text. M3 M6 M12 Active (PsyEduc) (N = 12) Control (TAU) (N = 13) Active (PsyEduc) (N = 12) Control (TAU) (N = 13) Active (PsyEduc) (N = 12) Control (TAU) (N = 13) n (%) n (%) p n (%) n (%) p n (%) n (%) p Cumulative relapses 0 (0.0) 3 (37.5) 0.058 0 (0.0) 3 (37,5) 0.082 0 (0.0) 4 (50.0) 0.014 Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p CRS 6.22 (1.4) 5.88 (1.6) 0.778 5.82 (1.7) 6.13 (1.5) 0.747 5.80 (2.2) 6.75 (0.5) 0.661 SD, Standard Deviation; CRS, Compliance Rating Scale. Percentages are adjusted for lost and missing data. Significant difference with p < 0.05 are in bold text. The efficacy of family psychoeducation in patient with schizophrenia has been well established in previous studies (9, 19). A review of the literature showed that family intervention can improve relapse and hospital admission rates in early psychosis (32). Additionally, studies conducted in chronic schizophrenia found that family psychoeducation can reduce the risk of relapse (25, 26, 33, 34). These interventions have also been shown to be cost saving and are included in international treatment guidelines (14, 15). These findings highlight the importance of incorporating family psychoeducation as a part of the comprehensive treatment plan for patients with schizophrenia. difference was significant at 12 months (p = 0.014). Patient’s outcome Medication adherence assessed by CRS estimated by caregivers was not modified by the intervention (see details in Table 2). Caregiver’s outcome In caregivers we found at baseline a significant association between the level of depression and the burden estimated with the Zarit Burden Interview. This association is confirmed by the study from Mittendorfer-Rutz (4) in a nationwide comparative study of parents of offspring with schizophrenia, rheumatoid arthritis, multiple sclerosis, epilepsy, and healthy controls. The results of this study showed that the parents of a patient with schizophrenia were at a higher risk for burden and had a 2.7 times higher risk of needing specialized psychiatric health care. We found a significant effect in caregiver’s burden at 6 months follow-up, suggesting a temporal learning effect with a gradual reduction in burden over time attributed to the family psychoeducation program. The impact of schizophrenia on caregivers can result in a significant burden including emotional, psychological, physical and financial strain associated with feelings of shame, embarrassment, guilt and blame (16, 37). Reducing caregiver burden is crucial for patient management, and for the caregiver Frontiers in Psychiatry frontiersin.org Discussion The efficacy of a family psychoeducational program was assessed through a randomized clinical trial (RCT) on a community dwelling patient with schizophrenia and their caregivers. Principal findings should be summarized as follows: (i) For patients, the family psychoeducation intervention reduced the risk of relapse with a significant effect found at 12 months follow-up. However, no change was observed on medication adherence; (ii) For caregivers, the intervention reduced the burden, decreased the depression, increased the knowledge on schizophrenia, and strengthened the therapeutic alliance. 05 frontiersin.org 10.3389/fpsyt.2023.1171661 Tessier et al. TABLE 3 Comparisons of caregiver’s outcomes (scores on psychometric scales) at each visit. TABLE 3 Comparisons of caregiver’s outcomes (scores on psychometric scales) at each visit. Discussion Mann–Whitney U-test for independent samples M0 M3 M6 Active (PsyEduc) Control (TAU) Active (PsyEduc) Control (TAU) Active (PsyEduc) Control (TAU) (N = 12) (N = 13) (N = 12) (N = 13) (N = 12) (N = 13) VARIABLES Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p Mean (SD) Mean (SD) p S-CGQoL 73.17 (20.5) 67.25 (7.4) 0.376 79.64 (20.3) 74.62 (6.6) 0.458 87.83 (14.8) 70.60 (14.0) 0.080 Psychological and physical well-being 15.00 (4.6) 12.00 (3.3) 0.128 15.55 (4.3) 14.00 (4.4) 0.452 19.17 (2.6) 13.60 (6.1) 0.073 Psychological burden and daily life 21.25 (7.5) 20.38 (6.8) 0.794 23.45 (6.2) 23.25 (6.6) 0.946 24.83 (3.5) 20.00 (8.2) 0.221 Relationships with spouse 8.25 (5.6) 5.75 (5.8) 0.415 8.18 (5.3) 6.00 (5.6) 0.405 10.33 (4.1) 6.00 (5.0) 0.149 Relationships with psychiatric team 8.00 (4.9) 9.88 (3.6) 0.367 10.45 (3.6) 9.00 (3.9) 0.415 11.00 (4.3) 10.80 (4.0) 0.939 Relationships with family 6.50 (2.6) 4.63 (3.8) 0.204 6.36 (2.8) 6.50 (2.8) 1.000 7.83 (2.0) 5.60 (3.2) 0.194 Relationships with friends 5.67 (3.3) 5.25 (3.2) 0.783 7.44 (2.4) 6.25 (2.9) 0.375 6.67 (2.1) 5.40 (3.4) 0.468 Material burden 8.50 (6.3) 9.38 (2.3) 0.666 8.18 (6.1) 9.63 (2.9) 0.867 8.00 (6.8) 9.20 (1.8) 0.692 ZBI 37.67 (17.3) 37.88 (19.9) 0.980 30.45 (16.9) 35.38 (21.4) 0.582 20.17 (8.0) 35.60 (12.1) 0.031 KAST 13.67 (1.6) 12.25 (3.1) 0.192 15.45 (1.4) 12.38 (3.4) 0.024* 15.33 (1.9) 12.80 (2.7) 0.098 4PAS-C 30.30 (7.8) 31.75 (7.1) 0.691 36.67 (7.8) 35.50 (6.8) 0.749 36.40 (10.7) 32.50 (9.9) 0.593 Visual analogic scale 50.50 (34.3) 49.38 (30.5) 0.943 71.11 (25.0) 67.50 (26.3) 0.776 71.00 (34.0) 49.50 (46.2) 0.321 Empathy 14.20 (3.4) 15.50 (3.4) 0.435 17.11 (3.3) 16.63 (3.1) 0.626 17.00 (5.2) 15.00 (4.2) 0.521 Psychoeducation 16.10 (4.6) 16.25 (3.9) 0.942 19.56 (4.5) 18.88 (3.9) 0.745 19.40 (5.7) 17.50 (5.9) 0.641 CES-D 20.27 (13.3) 20.50 (8.9) 0.967 14.91 (10.0) 23.54 (11.3) 0.118 6.20 (4.9) 14.20 (3.6) 0.019* SD, Standard Deviation; S-CGQoL, Schizophrenia Caregiver Quality of Life questionnaire; ZBI, Zarit Burden Interview; KAST, Knowledge About Schizophrenia Test; 4PAS-C, 4-Point ordinal Alliance Scale–Caregiver; CES-D, Center for Epidemiologic Studies–Depression scale.Significant difference with p < 0.05 are in bold text. himself, as it can help to reduce depression, burden-related care and associated costs (38). caregivers in the intervention group. This demonstrates the relevance of the information delivered during psychoeducation and its directly measurable effect (33). Frontiers in Psychiatry Discussion S-CGSQoL ZARIT KAST 4PAS-C CES-D CRS Friedman’s ANOVA Total PsPhW PsBDL RS RPT RFa RFr MB Total Total Total VAS Em PE Total Total Active (PsyEduc) Khi-square 0.785 1.634 0.290 0.231 1.433 0.556 0.900 0.545 4.507 3.756 5.218 7.354 2.000 5.167 1.852 0.545 p 0.482 0.243 0.754 0.798 0.284 0.590 0.638 0.761 0.040 0.061 0.035* 0.015* 0.368 0.036* 0.236 0.761 Control (TAU) Khi-square 1.071 0.161 0.498 0.054 0.615 3.797 2.308 1.000 0.460 0.762 0.743 0.594 5.167 1.588 1.476 2.000 p 0.387 0.854 0.626 0.948 0.564 0.069 0.200 0.410 0.647 0.498 0.515 0.497 0.755 0.297 0.291 0.368 S-CGQoL–PsPhW, Psychological and Physical Well-being; PsBDL, Psychological Burden and Daily Life; RS, Relationships with Spouse; RPT, Relationships with Psychiatric Team; RFa: Relationships with Family; RFr: Relationships with Friends; MB: Material Burden; I d l b l S CGQ L S CGQ L i Therapeutic alliance has been found to be enhanced by the family psychoeducation intervention. To our best knowledge, the therapeutic alliance between caregivers and healthcare staff has never been investigated in cohorts of patients with schizophrenia. Interactions with the health care staff during the family psychoeducation group strengthened the relationship and understanding with the caregiver. This was explored in other pathologies such as cancer (42).i In contrast to the study by Savanaud et al., we did not find any impact of the intervention on quality of life (38, 43). Quality of life may be influenced by the heterogeneous relational degree of the caregiver (parents, partner, child…). Indeed, it has been shown that parents of a patient have a lower quality of life than the patient’s siblings as demonstrated in a recent Croatian study (44). Although 72% of the included caregivers in our sample were the ill loved one, no significant results emerged regarding their quality of life. Most studies with similar design have focused on the patient’s quality of life rather than the caregiver’s (45, 46). We found one recent Indian RCT, that showed a significant improvement in overall quality of life scores in the experimental group caregivers compared to control group at the end of the program, after 6 months (47).hfi Thus, our study provides evidence of the efficacy of a short multifamily program (six sessions over 1.5 months) for caregivers (depression, knowledge) and patients (preventing relapse) in the context of routine care. This program can be repeated multiple times during the year. Discussion Previous RCTs and Evidence-Based Medicine (EBM) have considered the same primary evidence (9); however, the implementation of this evidence in routine care is limited (19). Moreover, To the best of our knowledge, and within the context of French mental health, psychoeducational programs for patients have been more extensively developed than those for caregivers. The brevity of this program should facilitate its implementation in the community. Discussion However, at 6 months, the difference between the two groups was no longer significant. There was a spontaneous improvement in the score in the control group, which may be related to the caregivers’ self-training by different resources (books, internet, meeting with the treating psychiatrist...). Knowledge of the disease have been found to be associated with a better medication adherence in patients (11). The Cochrane review highlights the benefits of patient psychoeducation in reducing relapse and readmission rates, promoting medication adherence, and shortening hospital stays. These findings suggest that psychoeducation not only has a positive impact on patients, but also on their family caregivers, making it a clinically effective and cost-beneficial intervention (39). The central role of family support in care was recently coroborated in an Italian multicenter study of 136 caregivers, where caregivers’ personal growth was associated with good family functioning and adequate professional support (40). Another study focused on the functioning pointed out that interpersonal relationships and work skills are the impaired functional areas in both patients and caregivers (41). At baseline, the level of depression is evaluated as “moderate” in both groups, with a score above the threshold fixed at 16 on CES-D scale. Previous studies had shown that caregivers of patients with schizophrenia are at higher risk for developing depression (4, 38). In a large survey, Gupta et al. found a 10% increased of depression in caregivers of patients with schizophrenia compared to non-caregivers and caregivers of adults with other conditions (38). At 6 months, in comparison with TAU, the intervention was found to significantly reduce depression. Depressive symptoms can have a negative impact on family interactions and lead to maladaptive behaviors toward the patient. Our findings is consistent with a previous RCT showed the usefulness of a family intervention in reducing caregiver’s depressive symptoms as measured by the CES-D, and a moderate effect in reducing the subjective burden as assessed by the ZBI over an 8 months follow-up period (2). The purpose of family psychoeducation is to increase caregiver’s knowledge and understanding of illness and treatment. A significant improvement in the knowledge of the disease (KAST) was found in 06 frontiersin.org Tessier et al. 10.3389/fpsyt.2023.1171661 10.3389/fpsyt.2023.1171661 TABLE 4 Evolutions in psychometric scales’ scores by group: Friedman’s ANOVA for repeated measures. Data availability statement Health (PHRIP “2013–0017–PsyEduc”). The funding source had no role in the conduct or publication of the study. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Publisher’s note KR and DM were involved in generating hypotheses, the management of the study and the development of the psychoeducational program. KR, AG, and DM were involved in the conduction of the psychoeducational program (Schiz’Aides). AT and PD conducted statistical analyzes. AT, PD, and DM wrote the first complete manuscript. All authors were involved in the patients’ recruitment, the clinical evaluation, acquisition of the clinical data, modified the manuscript and approved the final version. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Ethics statement The authors thank the team of the CMP de Talence (Pôle PGU of the CH Charles Perrens) in the elaboration and the conduction of the program, particularly: the psychiatrist M. Lescarret, J. Libert, and M-C Levassor; the nurses I. Tanguy, M-L Bamas, and F. Campedel; the psychologists A. Rebollo and H. Tastet; the social worker A. Bonnat. The studies involving human participants were reviewed and approved by Committee for the Protection of Persons–South West and Overseas 3 (CPP-SOOM3) Study folder: 2014/58. Department of Clinical Pharmacology, Bordeaux Hospital, Place Amélie Raba Léon 33076 BORDEAUX Cedex FRANCE Tel: +33 (0)5 57 81 76 07 Email: cpp.soom3@u-bordeaux.fr Web: http://www.cpp-soom3.u-bordeaux2. fr/. The patients/participants provided their written informed consent to participate in this study. Limitations Although our study has several strengths, there are some limitations that must be acknowledged. Our limited sample size could limit the reliability of our results. Additionally, the single-center design of our study may introduce a selection bias, which may affect the representativeness of our sample. To fully understand the effects of the intervention and to confirm our encouraging results, a larger, multi-center study should be done. However, in the context of the French mental health psychiatric service, the regional psychiatric outpatient facility should be considering as a good representation of the population of a community dwelling patient suffering of schizophrenia. Although patients were considered to be in a stable phase with only minor modifications to their prescribed medication during follow-up, this issue was not controlled and should be considered as a limitation in assessing the effectiveness of the psychoeducational program in preventing relapse. In conclusion, as confirmed by previous studies, the brief multifamily program (consisting of six sessions over a period of 1.5 months) was found to be effective in improving outcomes for caregivers (e.g., burden, depression, knowledge) and patients (e.g., preventing relapse) in the context of routine care. Given its short duration, this program is expected to be easily implementable within the community. frontiersin.org 07 Frontiers in Psychiatry Tessier et al. 10.3389/fpsyt.2023.1171661 Conflict of interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Funding The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fpsyt.2023.1171661/ full#supplementary-material This work was supported by a grant from the Nurses and Paramedics Research Hospital Program from the French Ministry of References 9. 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https://openalex.org/W2421571241	https://ris.utwente.nl/ws/files/6944837/Kline_et_al-2016-GCB_Bioenergy.pdf	English	null	Reconciling food security and bioenergy: priorities for action	Global change biology. Bioenergy/GCB bioenergy	2,016	cc-by	18,464	Abstract Understanding the complex interactions among food security, bioenergy sustainability, and resource manage- ment requires a focus on speciﬁc contextual problems and opportunities. The United Nations’ 2030 Sustainable Development Goals place a high priority on food and energy security; bioenergy plays an important role in achieving both goals. Effective food security programs begin by clearly deﬁning the problem and asking, ‘What can be done to assist people at high risk?’ Simplistic global analyses, headlines, and cartoons that blame biofuels for food insecurity may reﬂect good intentions but mislead the public and policymakers because they obscure the main drivers of local food insecurity and ignore opportunities for bioenergy to contribute to solutions. Applying sustainability guidelines to bioenergy will help achieve near- and long-term goals to eradicate hunger. Priorities for achieving successful synergies between bioenergy and food security include the following: (1) clari- fying communications with clear and consistent terms, (2) recognizing that food and bioenergy need not com- pete for land and, instead, should be integrated to improve resource management, (3) investing in technology, rural extension, and innovations to build capacity and infrastructure, (4) promoting stable prices that incentivize local production, (5) adopting ﬂex crops that can provide food along with other products and services to society, and (6) engaging stakeholders to identify and assess speciﬁc opportunities for biofuels to improve food security. Systematic monitoring and analysis to support adaptive management and continual improvement are essential elements to build synergies and help society equitably meet growing demands for both food and energy. Keywords: bioenergy, biofuels, energy, ﬂex crops, food insecurity, food security and nutrition, natural resource management, poverty reduction, sustainable development goals Received 23 December 2015; accepted 8 March 2016 The most serious mistakes are not being made as a result of wrong answers. The truly dangerous thing is asking the wrong questions. —Peter Drucker (1971) †This manuscript was coauthored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. Department of Energy. The Uni- ted States Government retains and the publisher, by accepting the article for publication, acknowledges that the United States Government retains a non-exclusive, paid-up, irrevocable, world-wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy. gov/downloads/doe-public-access-plan). Reconciling food security and bioenergy: priorities for action K E I T H L . K L I N E 1 , † , S I W A M S A N G I 2 , V I R G I N I A H . D A L E 3 , J E R E M Y W O O D S 4 , G L A U C I A M . S O U Z A 5 , P A T R I C I A O S S E W E I J E R 6 , J O Y S . C L A N C Y 7 , J O R G E A . H I L B E R T 8 , F R A N C I S X . J O H N S O N 9 , P A T R I C K C . M C D O N N E L L 1 0 and HARRIET K. MUGERA 1 1 1Environmental Science Division, Climate Change Science Institute, Oak Ridge National Laboratory, TN 37831, USA, 2International Food Policy Research Institute, 2033 K St NW, Washington, DC 20006, USA, 3Center for Bioenergy Sustainability, Environmental Science Division, ORNL, Oak Ridge, TN 37831, USA, 4Centre for Environmental Policy, Imperial College London, Exhibition Road, London SW7 1NA, UK, 5Instituto de Quımica, Universidade de S~ao Paulo, Av. Prof. Lineu Prestes 748, S~ao Paulo, Brazil, 6Department of Biotechnology, Delft University of Technology, 2628 BC Delft, The Netherlands, 7CSTM, University of Twente, 7500AE Enschede, The Netherlands, 8Instituto de Ingenierıa Rural INTA, cc 25 1712 Castelar, Buenos Aires, Argentina, 9Stockholm Environment Institute Africa Centre, World Agroforestry Centre (ICRAF), United Nations Avenue, Gigiri PO Box 30677, Nairobi, Kenya, 10BEE Energy, 2000 Nicasio Valley Rd., Nicasio, CA 94946, USA, 11World Bank, 1818 H Street NW, Washington, DC 20433, USA © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. GCB Bioenergy (2016), doi: 10.1111/gcbb.12366 R E P O R T Deﬁnitions and measures of food security The deﬁnitions used for food and food security are impor- tant determinants of the scope and outcomes of analyses. The oft-cited deﬁnition from the Food and Agriculture Organization of the United Nations (FAO) reﬂects broad aspirational goals (FAO 1996, Table 1). Four dimensions of food security emerge from this deﬁnition, namely, avail- ability, accessibility, stability, and utilization (Table 2). Thus, one approach to assessing impacts of biofuels on food security examines interactions across these four dimensions. However, many other factors including distri- butional and contextual issues affect vulnerability and hunger (von Grebmer et al., 2014). A science-based examination of evidence linking food security and bioenergy illuminates practical solutions when problems are well deﬁned. Good science is essen- tial to inform decisions in a world of strong beliefs (Hecht et al., 2009). An initial step must be to under- stand relationships between biomass production, food production, and hunger. Food security is recognized as a fundamental human right (UN General Assembly, 2015) with modern energy services being an essential component of food production, supply, and preparation (Woods et al., 2010). Measuring food insecurity. While the concept of food security is intuitive, underlying data are fraught with uncertainties due to large variations in diets and bio- physical conditions, making food security difﬁcult to measure and monitor. Therefore, manifestations of food insecurity that can be observed and veriﬁed are often used as proxy indicators of hunger and are monitored, rather than monitoring food security itself. For example, three international organizations collaborate to produce annual reports on the ‘State of Food Insecurity in the World’ (SOFI) (e.g., FAO, IFAD and WFP 2015a, 2014, 2013, FAO, WFP, IFAD, 2012, and previous years). This study describes the complexities in assessing sustainability as related to energy and food security in four parts: (1) food security, (2) interactions among food security, biofuels, and resource management, (3) priori- ties and conditions for achieving positive synergies, and (4) conclusions and recommendations. We begin by rec- ognizing that food insecurity is typically the indicator, so linkages among resource management, biofuels, and strategies to reduce food insecurity are relevant. We highlight where conventional wisdom could be mislead- ing and identify areas where further research should be a priority. The paper concludes with recommendations for enhancing food and energy security as complemen- tary goals for sustainable development. Introduction Understanding the nexus of food security, bioenergy sustainability, and resource management facilitates achievement of the 2030 Sustainable Development Goals (SDGs) to end hunger and ensure access to modern energy for all (United Nations (UN) 2015), as well as the Paris Agreement under the UN Convention on Correspondence: Keith L. Kline, tel. +1 865 574 4230, fax +1 865 241 4078, e-mail: klinekl@ornl.gov © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd. © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properl 1 p , which permits use, distribution and reproduction in any medium, provided the original work is properly cited 2 K. L. KLINE et al. and assumptions about relationships among biofuels, prices, food, and land security. It is important to ana- lyze the reasons for divergence and to ﬁnd common ground (Rosillo-Calle & Johnson, 2010). Climate Change. Contextual conditions determine costs, beneﬁts, and strategic opportunities that foster food and energy security for all (DeRose et al., 1998; FAO, 2015b; FAO, IFAD and WFP 2014). However, it is important to acknowledge that public perception about the interac- tion of bioenergy, in particular biofuels, and food secu- rity is mostly negative. Popular media reinforce beliefs reﬂected in the assumption used in economic models that biofuels produced from crops or on cropland com- pete with food production and increase food prices. Cartoons of hungry children juxtaposed to corn being ‘fed’ to cars have generated an emotional response to biofuel policies that is difﬁcult to overcome (Osseweijer et al., 2015; The Economist, 2015). Sensational news gar- ners attention while subsequent corrections are over- looked (Flipse & Osseweijer, 2013). In this report, we review the underlying evidential and theoretical basis concerning the impacts of bioenergy, in general, and biofuels, in particular, on food security and offer steps that can help society achieve SDGs for food and energy security. © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 FOOD SECURITY AND BIOENERGY SYNERGIES 3 Price indices alone are not indicators of food security. Given market equilibrium models, they have been widely used Table 1 Deﬁnitions relating to food security (based on IPC Global Partners 2012 and other sources as noted) Term Deﬁnition/Examples Anthropometry Study of the measurements and proportions of the human body; used as an indicator of malnutrition. Examples include child underweight (weight for age), stunting (height for age), and wasting (weight for height), compared with reference standards (United Nations World Food Program (WFP) Hunger Glossary, 2015) Commodity Traded item, especially unprocessed materials. Relevant examples include crude palm oil, raw sugar, #2 yellow corn, wheat, soybeans Commodity price index Mathematical value used to measure commodity price movements over a deﬁned time period; typically based on prices registered between suppliers or nations Consumer food price index Mathematical measure of price movements over a deﬁned time period for a ﬁxed basket of food items in a given nation, state, region, or group Famine Food insecurity causing or potentially causing death in the near term Food Source of nutrients required for energy and growth FAO food price index Monthly change in international prices of a basket of ﬁve food commodity groups (cereals, oils, dairy, meats, sugar), weighted per average export share values of each group for a given period, for example, 2002–2004 (FAO, 2013a) Flex crop Cultivated plant grown for both food and nonfood markets. Food security Condition that exists when all people, at all times, have physical and economic access to sufﬁcient safe and nutritious food that meets their dietary needs and food preferences for an active and healthy life (FAO, 1996) Food insecurity (chronic or transitory) Absence of food security; condition exists when people suffer or are at risk of suffering from inadequate consumption to meet nutritional requirements; may be classiﬁed as chronic (long term), acute (transitory), cyclical, or critical (see famine); typically measured via multiple indicators of malnutrition Hunger (or ‘food deprivation’) Degree of discomfort or unpleasant physical sensation associated with insufﬁcient food consumption. World Food Program deﬁnes hunger as ‘Not having enough to eat to meet energy requirements.’ The World Hunger Education Service (2015) refers to hunger as ‘aggregated food scarcity exempliﬁed by malnutrition.’ Malnutrition Condition arising from deﬁciencies, excesses, or imbalances in the consumption of important macro- and micronutrients. Deﬁnitions and measures of food security p y The terms food security and food insecurity are often used loosely or interchangeably; however, the deﬁni- tions and approaches for their measurement vary con- siderably (DeRose et al., 1998). Anthropometric measures of food insecurity are complemented by quali- tative surveys of behavior from census data on house- hold income and expenditures. Undernourishment, a common measure of food insecurity, is the probability that an individual in the population is undernourished (FAO, 2015a), while other measures focus on household food purchases (USDA, 2015; Coleman-Jensen et al., 2015). A global hunger index combines three equally weighted indicators: (1) undernourishment, deﬁned as people with insufﬁcient caloric intake (percentage of population); (2) children under the age of ﬁve with low weight for their age; and (3) mortality rate for children under age ﬁve (von Grebmer et al., 2014, Gautam, 2014). The effects of biofuels or a given policy on ‘food insecu- rity’ thus depend on the measures used to deﬁne who is ‘food insecure.’ An international workshop (IFPRI, 2015) helped frame the key issues evaluated here and underscored the importance of clear deﬁnitions and consistent use of terminology. The workshop focused on liquid biofuels, but the discussion and conclusions in this paper aim to be broadly applicable to food security interactions with an expanding bio-based economy. Polarization in the food-vs.-fuel debate begins with differing deﬁnitions 16 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 FOOD SECURITY AND BIOENERGY SYNERGIES 3 Malnutrition can arise directly from food insecurity or be a result of (1) inadequate childcare practices, (2) inadequate health services, (3) a harmful environment, or (4) excessive intake of unhealthy food Poverty State of being that encompasses multiple dimensions of deprivation relating to human capacity and capability, including consumption and food security, health, education, rights, security, dignity, and decent work Social safety nets Public programs that provide assistance, often as income transfers, to families or individuals who are unable to work or are temporarily affected by natural disasters, political crises, or other adverse conditions. Programs may involve (1) direct and targeted feeding (school meals, soup kitchens, or food distribution centers), (2) food-for-work programs, (3) cash or in-kind transfers (e.g., food vouchers), (4) subsidized rations, or (5) other support to targeted households Staple food Principal or recurring food ingredient in a regional diet Monthly change in international prices of a basket of ﬁve food commodity groups (cereals, oils, dairy, meats, sugar), weighted per average export share values of each group for a given period, for example, 2002–2004 (FAO, 2013a) Cultivated plant grown for both food and nonfood markets. Cultivated plant grown for both food and nonfood markets. Condition that exists when all people, at all times, have physical and economic access to sufﬁcient safe and nutritious food that meets their dietary needs and food preferences for an active and healthy life (FAO, 1996) Absence of food security; condition exists when people suffer or are at risk of suffering from inadequate consumption to meet nutritional requirements; may be classiﬁed as chronic (long term), acute (transitory), cyclical, or critical (see famine); typically measured via multiple indicators of malnutrition Absence of food security; condition exists when people suffer or are at risk of suffering from inadequate consumption to meet nutritional requirements; may be classiﬁed as chronic (long term), acute (transitory), cyclical, or critical (see famine); typically measured via multiple indicators of malnutrition Degree of discomfort or unpleasant physical sensation associated with insufﬁcient food consumption. World Food Program deﬁnes hunger as ‘Not having enough to eat to meet energy requirements.’ Degree of discomfort or unpleasant physical sensation associated with insufﬁcient food consumption. FOOD SECURITY AND BIOENERGY SYNERGIES 3 Table 2 Questions and trade-offs to consider when assessing effects of bioenergy across four dimensions of food security (food security dimensions based on FAO, 1996, 2008) Dimensions of food security Key questions: Does the proposed project increase or decrease. . . Assessment considerations Trade-offs Availability: quantity available for consumption in markets or within households The quantity of food, especially staples, available for household consumption? coping mechanisms and institutional capacity to respond in times of crisis? quantity of food required for traditional cultural practices and identity? Which dimensions of food security are the primary causes for food insecurity or risk of insecurity in this area? Which households/subgroups of the local population are most food insecure at present and why? Which households and subgroups are at highest risk of becoming food insecure, given current local trends and the context of the proposed project? How does local energy use interact with food production, transport, preparation, and processing? Can improvement in one dimension offset reductions in another? Will critical aspects for local food security or insecurity be affected? Will the project make clean energy services more affordable or widely available? Who gains and who loses in each dimension? How are project impacts distributed among groups, particularly food-insecure and at- risk groups? Accessibility: affordability or other aspects of securing available food Affordability of food, particularly for low-income households or other at-risk groups? investment in roads, bridges, public transport, or other features that facilitate access to markets and services (particularly in times of crisis)? factors that have caused disruptions in access to food in the past for this area? Stability: volatility in prices, availability, or affordability Market ‘ﬂoors’ or ‘ceilings’ that reduce price ﬂuctuation in staple foods? diversity of markets for producers (e.g., higher or lower dependence on single buyer or use)? diversity of food sources? diversity of sources of income? the base area of production of staple foods (e.g., changing susceptibility to localized extreme weather events)? other price and supply volatility impacts? Utilization: retention and use of the nutrients in consumed food to sustain health and well-being Nutritional value of diet for at-risk population? health and sanitation services? education for at-risk populations? micronutrient deﬁciencies? food safety, general health, and other factors inﬂuential in utilization? FOOD SECURITY AND BIOENERGY SYNERGIES 3 World Food Program deﬁnes hunger as ‘Not having enough to eat to meet energy requirements.’ The World Hunger Education Service (2015) refers to hunger as ‘aggregated food scarcity exempliﬁed by malnutrition.’ o oo og a e es ge as No a g e o g o ea o ee e e gy eq e e s The World Hunger Education Service (2015) refers to hunger as ‘aggregated food scarcity exempliﬁed by malnutrition.’ Condition arising from deﬁciencies, excesses, or imbalances in the consumption of important macro- and micronutrients. Malnutrition can arise directly from food insecurity or be a result of (1) inadequate childcare practices, (2) inadequate health services, (3) a harmful environment, or (4) excessive intake of unhealthy food State of being that encompasses multiple dimensions of deprivation relating to human capacity and capability, including consumption and food security, health, education, rights, security, dignity, and decent work Public programs that provide assistance, often as income transfers, to families or individuals who are unable to work or are temporarily affected by natural disasters, political crises, or other adverse conditions. Programs may involve (1) direct and targeted feeding (school meals, soup kitchens, or food distribution centers), (2) food-for-work programs, (3) cash or in-kind transfers (e.g., food vouchers), (4) subsidized rations, or (5) other support to targeted households Principal or recurring food ingredient in a regional diet Price indices alone are not indicators of food security. Given the high cost and complexity of ﬁeld measurements, broad indicators related to prices and regional balances of commodity supplies and utilization are often used for food market assessments. Price, supply, and trade data are readily available from existing sources and do not require primary ﬁeldwork to gather. Further, because these data can be easily plugged into existing market equilibrium models, they have been widely used to estimate the effects of biofuels on food security. Yet, as discussed below, there is little evidence that price indices can tell us much about who actually suffers from malnutrition due to food insecurity or its primary causes. Despite correlations, changes in global commod- ity prices are distinct from changes in consumer food price indices (Fig. 1). The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 4 K. L. KLINE et al. FOOD SECURITY AND BIOENERGY SYNERGIES 3 1 The FAO global Food Price Index (FPI) based on commodities vs. the FAO global food Consumer Price Index (CPI), 2000– 2015 (FAOStat, 2015). See Table 1 for deﬁnitions. Percentage change is relative to the 2002–2004 average for FPI and year 2000 for CPI (FAO, 2015c). The food CPI increased each year at an average annual rate of 6% (2000–2015), while the annual average global FPI var- ied sharply and was negative in 7 of the 15 years. Fig. 1 The FAO global Food Price Index (FPI) based on commodities vs. the FAO global food Consumer Price Index (CPI), 2000– 2015 (FAOStat, 2015). See Table 1 for deﬁnitions. Percentage change is relative to the 2002–2004 average for FPI and year 2000 for CPI (FAO, 2015c). The food CPI increased each year at an average annual rate of 6% (2000–2015), while the annual average global FPI var- ied sharply and was negative in 7 of the 15 years. FAO notes that its food price index (FPI) is not an indi- cator of food insecurity. Rather, the FPI is based on weighted indices of trade data (Table 1) which may not reﬂect: (1) foods needed by food-insecure countries; (2) price changes relevant to food security; and (3) the actual prices for households which ‘may be quite different from the border prices’ (FAO, 2013a). Furthermore, in nations where high numbers of people are food insecure, staples such as rice are managed or regulated explicitly to pro- tect local consumers from external price ﬂuctuations (FAO, 2014, 2015c). Finally, FPI weighting creates bias favoring expensive commodities that are less important for populations at risk; for example, meat has the highest weight, 0.35, while sugar has a weight of 0.07. hungry reside (FAO, 2015a), but their local prices have minimal inﬂuence on CFPI values. When these staples are grown and consumed locally, they are omitted by both the aggregate trade models and CFPIs, despite being crucial sources of nutrition for vulnerable house- holds. The annual SOFI reports highlight dozens of context- speciﬁc factors, other than CFPI changes, that determine who goes hungry in times of crisis (e.g., FAO, WFP, 2010). Malnutrition is associated with many factors other than food intake (e.g., Smith & Haddad, 2000; Gautam, 2014; Lombard, 2014). FOOD SECURITY AND BIOENERGY SYNERGIES 3 Table 2 Questions and trade-offs to consider when assessing effects of bioenergy across four dimensions of food security (food security dimensions based on FAO, 1996, 2008) Dimensions of food security Key questions: Does the proposed project increase or decrease. . . Assessment considerations Trade-offs Availability: quantity available for consumption in markets or within households The quantity of food, especially staples, available for household consumption? coping mechanisms and institutional capacity to respond in times of crisis? quantity of food required for traditional cultural practices and identity? Which dimensions of food security are the primary causes for food insecurity or risk of insecurity in this area? Which households/subgroups of the local population are most food insecure at present and why? Which households and subgroups are at highest risk of becoming food insecure, given current local trends and the context of the proposed project? How does local energy use interact with food production, transport, preparation, and processing? Can improvement in one dimension offset reductions in another? Will critical aspects for local food security or insecurity be affected? Will the project make clean energy services more affordable or widely available? Who gains and who loses in each dimension? How are project impacts distributed among groups, particularly food-insecure and at- risk groups? Accessibility: affordability or other aspects of securing available food Affordability of food, particularly for low-income households or other at-risk groups? investment in roads, bridges, public transport, or other features that facilitate access to markets and services (particularly in times of crisis)? factors that have caused disruptions in access to food in the past for this area? Stability: volatility in prices, availability, or affordability Market ‘ﬂoors’ or ‘ceilings’ that reduce price ﬂuctuation in staple foods? diversity of markets for producers (e.g., higher or lower dependence on single buyer or use)? diversity of food sources? diversity of sources of income? the base area of production of staple foods (e.g., changing susceptibility to localized extreme weather events)? other price and supply volatility impacts? Utilization: retention and use of the nutrients in consumed food to sustain health and well-being Nutritional value of diet for at-risk population? health and sanitation services? education for at-risk populations? micronutrient deﬁciencies? food safety, general health, and other factors inﬂuential in utilization? © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: FOOD SECURITY AND BIOENERGY SYNERGIES 5 Fig. FOOD SECURITY AND BIOENERGY SYNERGIES 3 Thus, biofuel effects on food security could be determined by a project’s inﬂu- ence on physical infrastructure, asset accrual, institu- tional capacity, training, technologies that enhance food safety or resilience, ecosystem stability, cultural well- being, or other drivers and coping mechanisms omitted from food price indices (Rose, 1999; RTI, 2014; Cole- man-Jensen et al., 2015; Gustafson et al., 2016). National and regional ‘consumer food price indices’ (CFPIs) provide a higher resolution than the FPI but are still insufﬁcient indicators of food insecurity due to similar dollar-value weighting bias and reliance on formal market prices. The people most susceptible to severe food insecurity typically live in isolated areas and rely on informal markets or subsistence produc- tion (Rose, 1999; FAO, 2015a; FAO, IFAD and WFP, 2015b). Rice, wheat, millet, white maize, and yams are staples in Asia and Africa, where 94% of the world’s Finally, analyses that rely on FPIs tend to focus on price spikes while ignoring long periods of depressed prices. This can mislead policymakers and the public because depressed prices discourage agricultural invest- ment and can be more detrimental to long-term food 016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.1236 6 K. L. KLINE et al. Fig. 2 Global biofuel consumption (billion liters) 2000–2014 grew steadily, although fuel ethanol production dipped slightly in 2010–2012 due to global recession and poor weather in Brazil (in 2011) and the USA (in 2012). Still, average annual growth in global production over 2009–2014 remained robust, at 5.2% and 11% for fuel ethanol and biodiesel, respectively (REN21, 2015). Chart based on U.S. Energy Information Administration (2015) and REN21, 2015. road). Thus, the degree to which biofuel production and processing may inﬂuence food security depends on the interaction of many variables within a local context including, among others: what feedstocks are grown and where and how feedstocks are distributed, what investments are made, management practices, who ben- eﬁts, and who loses (Table 2). Biofuels and food security: short-term correlations vs. long- term trends. The high-proﬁle expansion of ethanol pro- duction in the United States and Brazil, in tandem with a global price spike in food and commodities in 2007– 2008, led many to contend that a causal relationship exists between biofuels expansion and food insecurity (e.g., Mitchell, 2008; Tenenbaum, 2008; Wenzlau, 2013). FOOD SECURITY AND BIOENERGY SYNERGIES 3 The apparent short-term correlations are often cited as evidence of negative impacts of biofuels on food secu- rity (e.g., EPI, 2014; Searchinger & Heimlich, 2015). There are several problems with such assertions (Zilber- man et al., 2013). First, many studies attribute the food price spikes in 2008 primarily to other factors such as oil prices, economic growth, currency exchange rates, and trade policies (e.g., Baffes & Dennis, 2013; Konan- dreas, 2012; HLPE, 2011; Foresight, 2011; Trostle et al., 2011; DEFRA, 2010). Speculation in food commodities also contributed to price spikes in 2008 and 2011 (Lagi et al., 2011; Hajkowicz et al., 2012). Second, the correla- tions did not persist as global biofuel consumption con- tinued to grow (Fig. 2) and cereal prices fell or showed distinct patterns over the last 6 years driven by oil price, national agricultural policies, and exchange rates (FAO, 2015a,c, The Economist Intelligence Unit, 2015). Causation cannot be assumed based on correlation, but the divergence in recent trends is notable, and models using the same data can reach opposing conclusions (Table 3). Fig. 2 Global biofuel consumption (billion liters) 2000–2014 grew steadily, although fuel ethanol production dipped slightly in 2010–2012 due to global recession and poor weather in Brazil (in 2011) and the USA (in 2012). Still, average annual growth in global production over 2009–2014 remained robust, at 5.2% and 11% for fuel ethanol and biodiesel, respectively (REN21, 2015). Chart based on U.S. Energy Information Administration (2015) and REN21, 2015. security than price spikes (see, e.g., the SOFI reports and Roser, 2015). Projects that contribute to price stabil- ity at a level high enough to motivate local investment in food production and its associated infrastructure will improve resilience and food security over the long term (FAO, IFAD, WFP, 2002). Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interactions Thus, the impacts would be detrimental to health if biofuels drove sugar and yellow maize prices down so as to marginally increase overconsumption of red meat and sweeteners Hypothesis 1: The effect of biofuel production on the price of food is most pronounced for commodities that compete directly with bioenergy feedstock. Sugarcane and yellow maize are the two most important biofuel feedstocks. The primary foods derived from sugarcane and yellow maize are sugar and other sweeteners (such as high-fructose corn syrup used globally), and red meat (most yellow maize is fed to cattle). These foods are among the primary sources of malnutrition from overeating (WHO, 2015). If biofuels cause higher prices and higher prices marginally reduce overconsumption, then the expected impacts on health would be beneﬁcial Conclusion: None of the hypotheses above can be endorsed because they are not supported by evidence of price transmissions to the speciﬁc populations at risk. Despite a rapid increase in biofuel production, there is no evidence of biofuel impacts on food-related health, either beneﬁcial or detrimental. Models that simulate demand shocks from biofuels necessarily show price transmission and reduced consumption, but evidence is lacking to support either the assumed ‘shock’ or the assumed impacts on people at risk. To test a hypothesis, the problem must be clearly deﬁned and the linkages between biofuels and impacts on behavior veriﬁed constructive efforts to improve data (Gibson, 2013) and to identify effective mechanisms to address the food security issues that matter most, namely those having an impact on human health and morbidity. improvement in food security when assessing the sus- tainability of biofuels projects (Dale et al., 2013). Bioenergy projects that improve resilience can reduce vulnerabilities that lead to food insecurity (Gustafson et al., 2016). Resilience refers to the ability of the system to recover following disturbance, and vulnerability refers to inability to withstand a hostile situation. Reducing risk exposure might take the form of facilitat- ing the transition of households from livelihoods that are subject to high levels of variability – such as low- level subsistence farming dependent on a single crop – toward more stable sources of revenue and income. Priority actions to reduce risks of food insecurity. Biofuel projects can address food security concerns by apply- ing best practices that reduce exposure to risks of food insecurity (Table 4). Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interactions Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interactions Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interacti Observations: Despite population growth, 167 million fewer people suffered from hunger and undernourishment in 2015 than a decade earlier (FAO, 2015a). Over the same decade, biofuel production expanded rapidly along with the number of people suffering early mortality and disease from consuming too much of the wrong foods. Today, more people are malnourished from overconsumption than are undernourished due to insufﬁcient food. Over the coming decade, the global population suffering from hunger is projected to decline, while the number suffering from diseases caused by overconsumption is projected to steadily rise (WHO, 2015) Hypothesis 3 (conventional wisdom): The effect of biofuel production on the price of food is most pronounced for commodities such as maize that compete directly with bioenergy feedstock. Biofuels also compete for land, reducing production of other crops. This reduces food supply or increases food prices, thereby contributing to increased hunger. Evidence cited in this paper refutes most assumptions underlying this hypothesis. Whether the issue is hunger or overconsumption, who is impacted depends on who is at risk of malnutrition and other contextual conditions that determine causal relationships. Speciﬁc nutrition problems must be clearly deﬁned to identify effective solutions Hypothesis 3 (conventional wisdom): The effect of biofuel production on the price of food is most pronounced for commodities such as maize that compete directly with bioenergy feedstock. Biofuels also compete for land, reducing production of other crops. This reduces food supply or increases food prices, thereby contributing to increased hunger. Evidence cited in this paper refutes most assumptions underlying this hypothesis. Whether the issue is hunger or overconsumption, who is impacted depends on who is at risk of malnutrition and other contextual conditions that determine causal relationships. Speciﬁc nutrition problems must be clearly deﬁned to identify effective solutions Hypothesis 2: The effect of biofuel production on the price of food is most pronounced for commodities that compete directly with bioenergy feedstock. Sugarcane and yellow maize are the two most important biofuel feedstocks. Bioenergy markets bolster investment and innovation, reducing long-term costs and increasing global supplies of said commodities. The primary foods derived from sugarcane and yellow maize (sugar, sweeteners, red meat) are more widely available at lower prices than would occur without biofuels. Effective food security strategies address relevant risk factors To assess how a policy or project affects food security, an understanding of risk factors that lead to food inse- curity is needed. As described above, analysis of aggre- gate commodity data may generate conﬂicting conclusions, because correlations with biofuels are often extraneous to the causes of local food insecurity. Under- standing why and how people become food insecure is prerequisite to developing effective responses. Food insecurity may involve distinct risk factors depending on whether effects are long term (chronic) or short term (acute or transitory). A majority of papers and reports that assert that bio- fuels harm food security rely on assumed relationships between biofuels, rising global ‘food’ commodity prices, and food insecurity over relatively short time spans (e.g., on the order of months) (Boddiger, 2007; Rajagopal et al., 2007; Tenenbaum, 2008; Wenzlau, 2013). Interest- ingly, organizations wishing to show that biofuels do not raise food prices often cite the same FAO ‘food com- modity’ data over similar time spans (e.g., see Zhang et al., 2010; Mueller et al., 2011; and GRFA, 2015). The assumptions underlying both sides of this food-vs.-fuel debate are questionable and subjective (Table 3). Long- term trends (over years and decades) for food insecurity and food commodity prices illustrate that the world’s most severe famines (Roser, 2015) occur during extended periods of depressed global food prices (Sum- ner, 2009). The emphasis on biofuels and food commod- ity price spikes has diverted attention from more The type and cause of food insecurity in a particular context determine appropriate responses (IPC Global Partners, 2012) and how the effects of a bioenergy pro- ject on food security should be assessed (Table 2). Addressing chronic food insecurity requires coordi- nated commitments to long-term strategies that reduce household vulnerabilities. Transitory food insecurity requires investments that mitigate or prevent sudden events that can limit access to adequate food for short periods. Transitory food insecurity may be caused by events that impede distribution from areas of food sur- plus to areas of need (e.g., loss of critical bridge or 16 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 8 K. L. KLINE et al. Bio-based fuels and improved stoves for healthy food preparation Clean, affordable, and reliable energy for value-added processing Equitable and open energy markets Recognition that problems and solutions are context speciﬁc Focus on women, the poor, and small producers Transparency Access to ﬁnancial, technical, ‘safety nets’ and other social services Environmental sustainability Employment productive resources and to markets that are remunera- tive and beneﬁcial to smallholders’ (FAO, 2015d). Assessments should compare the relative merits of alternative trajectories in meeting goals. The trade-offs depend on historical developments and prevailing local economic, social, environmental, political, and cultural conditions (Efroymson et al., 2013). Because sustainabil- ity is context speciﬁc, local stakeholders should help set priorities, deﬁne the purposes of the assessment, and establish the temporal and spatial boundaries for con- sideration (Tarka-Sanchez et al., 2012; Dale et al., 2015). For example, dimensions of sustainability for bioenergy include soil quality, water quality and quantity, green- house gases, biodiversity, air quality, productivity, social well-being, energy security, trade, proﬁtability, Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interactions Many recommendations for investments in biofuels tailored for developing nations have been published (UNCTAD, 2014; FAO 2010, 2011a, 2015b; FAO, IFAD, WFP, 2002, FAO, IFAD, WFP 2013). Exposure to risk can also be reduced by programs that help build rural assets and diversify income sources. If the exposure of households to environmental or socioeconomic shocks cannot be reduced, then a bioenergy project might aim to increase the capacity of vulnerable households to cope with shocks when they arise. Resilience is achieved by ‘strengthening sustain- able local food systems, and fostering access to Lifting people out of poverty is essential to reduce hunger (von Braun et al., 2009, FAO, IFAD, WFP, 2014, 2015b; Coleman-Jensen et al., 2015). The creation of stable, gainful, rural employment is a high-priority, poverty-reduction strategy (Conway and Wilson, 2012; FAO, IFAD, WFP, 2015b). Improvement in rural house- hold incomes is proposed as a proxy indicator for rs. 8 K. L. KLINE et al. 8 K. L. KLINE et al. Table 4 Examples of convergence among recommended practices to enhance food security and to produce sustainable biomass for bioenergy (based on FAO, IFAD, WFP 2002; FAO, 2010, 2011a, 2013b, 2014b, 2015b, 2015e; FAO, IFAD, WFP 2013, 2015b; IMF, 2013; UNCTAD, 2013, 2014; World Bank, 2015) Dimension Recommended practices Access to land, water, and markets Consultation with stakeholders including smallholders Mapping of customary rights and communal environmental services 8 K. L. KLINE et al. Table 4 Examples of convergence among recommended practices to enhance food security and to produce sustainable biomass for bioenergy (based on FAO, IFAD, WFP 2002; FAO, 2010, 2011a, 2013b, 2014b, 2015b, 2015e; FAO, IFAD, WFP 2013, 2015b; IMF, 2013; UNCTAD, 2013, 2014; World Bank, 2015) Dimension Recommended practices Table 4 Examples of convergence among recommended practices to enhance food security and to produce sustainable biomass for bioenergy (based on FAO, IFAD, WFP 2002; FAO, 2010, 2011a, 2013b, 2014b, 2015b, 2015e; FAO, IFAD, WFP 2013, 2015b; IMF, 2013; UNCTAD, 2013, 2014; World Bank, 2015) Recommended practices Consultation with stakeholders including smallholders Mapping of customary rights and communal environmental services Fair compensation to owners and traditional users Rule of law and fair mechanisms for conﬂict resolution Infrastructure to access inputs and markets Adherence to international conventions (e.g., International Labour Organization guidelines) Reliable local jobs and healthy working conditions Access to education, vocational skills, and safety Incentives to expand local production Removal of barriers to trade and market information Contracts with local goods and service providers (e.g., proﬁt-sharing options) Freedom of association and collective bargaining Access to credit and business management training Fair and transparent pricing Stable regulatory environment Integrated food and energy systems Improved output and nutritional value from urban gardens and small farms Provision of agricultural inputs, technologies, and equipment Training that is relevant for developing coping strategies (asset building, etc.) Distribution and storage systems Improved local infrastructure (transportation, water, schools, etc.) Women in leadership positions Health and safety services and emergency assistance Microlending and ﬁnancial support mechanisms Social welfare organizations Improved energy infrastructure and maintenance Energy for agricultural technology: cultivation, marketing, irrigation, etc. Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interactions Table 4 Examples of convergence among recommended practices to enhance food security and bioenergy (based on FAO, IFAD, WFP 2002; FAO, 2010, 2011a, 2013b, 2014b, 2015b, 2015e; FAO, UNCTAD, 2013, 2014; World Bank, 2015) Dimension Recommended practices Access to land, water, and markets Consultation with stakeholders including smallholders Mapping of customary rights and communal environm Fair compensation to owners and traditional users Rule of law and fair mechanisms for conﬂict resolution Infrastructure to access inputs and markets Employment Adherence to international conventions (e.g., Internatio Reliable local jobs and healthy working conditions Access to education, vocational skills, and safety Incentives to expand local production Removal of barriers to trade and market information Income generation Contracts with local goods and service providers (e.g., p Freedom of association and collective bargaining Access to credit and business management training Fair and transparent pricing Stable regulatory environment Local food security Integrated food and energy systems Improved output and nutritional value from urban gard Provision of agricultural inputs, technologies, and equip Training that is relevant for developing coping strategie Distribution and storage systems Community development Improved local infrastructure (transportation, water, sch Women in leadership positions Health and safety services and emergency assistance Microlending and ﬁnancial support mechanisms Social welfare organizations Energy security Improved energy infrastructure and maintenance Energy for agricultural technology: cultivation, marketi Bio-based fuels and improved stoves for healthy food p Clean, affordable, and reliable energy for value-added p Equitable and open energy markets Cross-cutting aspects Recognition that problems and solutions are context spe Focus on women, the poor, and small producers Transparency Access to ﬁnancial, technical, ‘safety nets’ and other soc Environmental sustainability 8 K. L. KLINE et al. Table 3 Identical data can support contradicting hypotheses about nutritional effects of biofuel-food interactions Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 Table 4 Examples of convergence among recommended practices to enhance food security and to produce sustainable biomass for bioenergy (based on FAO, IFAD, WFP 2002; FAO, 2010, 2011a, 2013b, 2014b, 2015b, 2015e; FAO, IFAD, WFP 2013, 2015b; IMF, 2013; UNCTAD, 2013, 2014; World Bank, 2015) Dimension Recommended practices Access to land, water, and markets Consultation with stakeholders including smallholders Mapping of customary rights and communal environmental services Fair compensation to owners and traditional users Rule of law and fair mechanisms for conﬂict resolution Infrastructure to access inputs and markets Employment Adherence to international conventions (e.g., International Labour Organization guidelines) Reliable local jobs and healthy working conditions Access to education, vocational skills, and safety Incentives to expand local production Removal of barriers to trade and market information Income generation Contracts with local goods and service providers (e.g., proﬁt-sharing options) Freedom of association and collective bargaining Access to credit and business management training Fair and transparent pricing Stable regulatory environment Local food security Integrated food and energy systems Improved output and nutritional value from urban gardens and small farms Provision of agricultural inputs, technologies, and equipment Training that is relevant for developing coping strategies (asset building, etc.) Distribution and storage systems Community development Improved local infrastructure (transportation, water, schools, etc.) Women in leadership positions Health and safety services and emergency assistance Microlending and ﬁnancial support mechanisms Social welfare organizations Energy security Improved energy infrastructure and maintenance Energy for agricultural technology: cultivation, marketing, irrigation, etc. Bio-based fuels and improved stoves for healthy food preparation Clean, affordable, and reliable energy for value-added processing Equitable and open energy markets Cross-cutting aspects Recognition that problems and solutions are context speciﬁc Focus on women, the poor, and small producers Transparency Access to ﬁnancial, technical, ‘safety nets’ and other social services Environmental sustainability 8 K. L. KLINE et al. Interactions among bioenergy, food security, and resource management, focusing on more sustainable systems Making progress toward sustainable development goals requires attention to provision of social and ecosystem services as well as economics across integrated produc- tion systems. Sustainability involves assessing trade-offs among multiple dynamic goals and striving for contin- ual improvement, rather than achieving a speciﬁc state. The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 FOOD SECURITY AND BIOENERGY SYNERGIES 9 FOOD SECURITY AND BIOENERGY SYNERGIES 9 resource conservation, and social acceptability (McBride et al., 2011; Dale et al., 2013). Two-way linkages Past and future resource management goals help deﬁne both opportunities and constraints for cultivating more sus- tainable feedstock crops. legislation passed in part due to recognition of latent productive capacity for maize. In the decade leading up to 2012, U.S. maize production increased steadily and exceeded targets for fuel blending under national legis- lation. In 2012, the U.S. experienced the most extensive drought recorded since the 1950s (IMF, 2012; USDA, 2013). As impacts of the drought became evident, mar- kets responded; some ethanol plants reduced output; others shut down temporarily. Thanks, in part, to the ethanol ‘supply cushion’ and market ﬂexibility, there was not a notable jump in commodity prices as the 2012–2013 crop was harvested, despite a drought affect- ing 80% of U.S. agricultural land. Resource management effects on food security. Good resource management underpins food security. Increased efﬁciency and productivity of crops enhance resilience and are essential for secure food availability. Similar to biofuel sustainability, good resource manage- ment allows identiﬁcation of place-based opportunities and constraints and enhances the efﬁciency of resource use. While several studies discuss potential negative effects of biofuels, few examine the ways that biofuels can positively inﬂuence food security. First, adequately planned biofuel production can add value, stabilize, and diversify rural production systems (Kline et al., 2009). Additionally, energy is required throughout the food supply chain; therefore, to the degree that biofu- els enhance sustainability and accessibility of energy supplies, particularly energy for households most at risk from poverty, they enhance food security. Further- more, as long as farmers and agro-industry are free to respond, diversiﬁed markets for products can spread risk and reduce price volatility compared with more narrow markets. Adding bioenergy markets to existing uses of local produce can thereby increase price stabil- ity. Finally, efforts to enhance sustainability of biofuels have generated spin-off effects in other sectors and placed greater scrutiny on resource management asso- ciated with conventional production (Woods & Kalas, 2014). The result is improved sustainability for many nonbiofuel products that constitute the majority of ﬁnal uses for palm oil, sugar cane, soybean, and maize. Food security effects on bioenergy. Food security can affect biomass resource management in many ways. A secure, healthy diet provides the biophysical and socioeco- nomic basis for managing soil, water, nutrients, and related resources. Two-way linkages Two-way linkages Bioenergy effects on food security. Bioenergy can foster social development, which is a precondition for food security and sustainability. Bioenergy provides energy security not only for transport (and hence broader access to food, selling markets, employment, and ser- vices) but also for food processing, business develop- ment, and drying and storage of surplus production (Durham et al., 2012; Lynd et al., 2015). The latter, pro- viding an outlet for surplus, diversiﬁes sources of income and improves supply resilience in the event of market shocks or shortages. Innovation is stimulated as new institutions and actors are empowered to engage in expanding biomass production. The early investments made by developed, developing, and emerging econo- mies alike in biofuels illustrate the universal nature of the linkages between energy security and development (Johnson & Silveira, 2014). Choices inevitably involve trade-offs. Improving one aspect of sustainability may compromise another, and beneﬁts for one group may involve costs for another (Table 2). Complete transformation chains rather than single bioenergy products should be analyzed to under- stand the interactions across sectors and industries that may inﬂuence system efﬁciencies for bioenergy and food security (Hilbert, 2014). A key goal is to identify opportunities where collective progress can be achieved – sometimes referred to as the triple bottom line of social, economic, and environmental beneﬁts. Resource management practices are more important in determining many environmental impacts than crop type (Davis et al., 2013). Wise management of available resources supports both bioenergy sustainability and food security (Manning et al., 2014). Hence, interactions among resource management, bioenergy sustainability, and food security are discussed with paired interactions considered ﬁrst, followed by the three-way nexus (Fig. 3). The capacity for biofuels to help balance another com- modity market has been demonstrated by the Brazilian sugar–ethanol industries. Similarly, U.S. ethanol Fig. 3 The nexus of resource management, bioenergy sustainability, and food security. Key aspects of the six two-way interactions frame the nexus at the center. 016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.1236 10 K. L. KLINE et al. 10 sustainability by increasing the efﬁciency and produc- tivity of supply chains. Improved management of soils and water permits higher output of bioenergy, food, and other products coupled to enhanced nutrient and water use efﬁciencies (FAO-UNEP, 2011). Two-way linkages Excess production, desirable to enhance food security as a precautionary measure, can be absorbed by bioenergy markets and expand income opportunities for farmers when that supply cushion is not needed for sustenance. Food security effects on resource management. Improving food security can reduce pressures on forests and mar- ginal lands, thereby avoiding erosion and other negative consequences for soils, water, and ecosystem functions. Food-secure families are less inclined to risk health and livelihood to set off to distant frontiers and clear new land, whereas migration is often a last resort for food- insecure families. Food-secure families are also less likely to feel a need to cultivate on steep slopes and other fragile areas that involve physical and legal risks (parks and reserves). Desperate actions required to address food crises or famine can lead to displaced pop- ulations and emergency actions that have environmen- tal consequences. Finally, food security provides the foundation required for effective outreach and learning about systematic approaches to improving natural resource management. Bioenergy effects on resource management. Bioenergy has spurred well-known efforts to develop best practices that reduce greenhouse gas emissions and negative impacts on soil and water. However, bioenergy sustain- ability has also called attention to land-use planning and biodiversity protection and provided increased incentives for land restoration (Souza et al., 2015). Speciﬁcally, bioenergy sustainability calls for considera- tion of a diverse set of potential effects on water, soils, air, and biodiversity, with emphasis on understanding baseline conditions and setting targets for continual improvement. These are key steps toward implementa- tion of resource management systems that are resilient and adaptable to climate change. The three-way nexus between resource management, bioenergy sustainability, and food security The three-way nexus between resource management, bioenergy sustainability, and food security The interactions between these three factors form the central region of the Venn diagram in Fig. 3. Good gov- ernance incorporates both political commitment and the institutional capacity to provide effective services and security under the rule of law. Good governance is Resource management effects on bioenergy. In turn, improving resource management inﬂuences bioenergy © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 FOOD SECURITY AND BIOENERGY SYNERGIES 11 11 essential for effective resource management, food secu- rity, and bioenergy sustainability. Government institu- tions provide ‘social safety nets,’ or create conditions that allow nongovernment organizations to ﬁll this role, to help vulnerable populations cope in times of food cri- sis. These coping mechanism become unavailable or inoperable when governance fails or is undermined by corruption. Several initiatives promoting sustainable bioenergy (e.g., GBEP, 2011; RSB, 2011; FAO, 2011a) acknowledge this nexus by considering governance, participation of civil society, and development of insti- tutional capacity. agricultural systems over the last decade (Fuglie & Rada, 2013). Integrated system design can also help to identify opportunities to utilize what might otherwise be considered waste from one part of the system, as input for other parts (Berndes et al., 2015). Reduction in and reallocation of waste offer signiﬁcant beneﬁts, par- ticularly if the waste would otherwise be burned or require costly removal. Diverse ecosystem services are inﬂuenced by the interactions among resource management, food, and biofuel feedstock production (Gasparatos et al., 2011). For example, enhanced water and air quality, improved soil conditions, stable jobs, and economic beneﬁts can all accrue if the agricultural system is designed and deployed in a way that efﬁciently meets the demand for food, ﬁber, and feedstocks (Berndes et al., 2015; Souza et al., 2015). Respect for peoples’ rights to land and resources is interwoven with good governance and prerequisite for any project promoting more sustainable production (FAO, 2011a; Dale et al., 2013). The ‘Global Commercial Pressures on Land Project’ found that failures of gover- nance were causal factors leading to ‘land grabs’ (Anseeuw et al., 2011). Traditional uses of land and other natural resources by the poor are of special concern when designing policies and projects to enhance food security. Guidelines are available to ensure that biofuels develop- ment takes traditional land rights into consideration (e.g., FAO, 2011a, 2013b). The three-way nexus between resource management, bioenergy sustainability, and food security Properly applying these guidelines would avoid problems such as the displacement of small- holder farmers by agro-industrial developments as tran- spired in Colombia (Clancy et al., 2013). The occurrence of extreme weather events is unpre- dictable, but their intensity and frequency are expected to increase because of climate change (IPCC, 2014). Resi- lience to extreme events is enhanced through diversiﬁed production systems and multiple suppliers with ﬂexibil- ity to adjust based on the linkages between resource management, food security, and sustainable bioenergy production systems. This buoyancy can occur whether the disturbance is due to natural events (e.g., hurri- canes, droughts, ﬁres), market forces (e.g., sudden sharp decline or rise in prices), or human-induced disasters (social or political conﬂicts). More diversiﬁed produc- tion systems have also been shown to be more adapt- able to change than traditional monoculture production systems (Woods et al., 2015). Investments in infrastructure and advances in tech- nology are necessary for all parts of the system. Food security requires the means to produce, package, and distribute high-quality food. Biofuel sustainability relies on efﬁcient systems for production, transport, and processing. As documented in Brazil, investments in bioethanol industries can support spin-off beneﬁts for neighboring productive sectors and local econo- mies. In rural areas where biomass and labor are abundant, but infrastructure is limited by lack of funding, bioenergy investments help ﬁll gaps and facilitate economic development (Batidzirai & Johnson, 2012; Moraes & Zilberman, 2014). In Malawi and Tan- zania, contracting with smallholders was found to effectively improve household incomes and commu- nity welfare (Sulle & Nelson, 2009; Hermann & Grote, 2015). By understanding the nexus and intentionally design- ing systems to promote beneﬁcial linkages among resource management, bioenergy sustainability, and food security, we can enhance the resilience and adapt- ability of biofuel and food production systems and the coping mechanisms required in times of crisis. Such integrated systems should be designed to apply best practices and support critical local priorities including food security (Tables 4 and 5). 016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.1236 Priorities and conditions to achieve positive synergies Integrated crop management and production systems are necessary for efﬁcient provision of food, feed, ﬁber, and energy feedstocks. Integration helps minimize use of inputs such as fertilizer or pesticides and helps opti- mize use of assets such as natural, social, physical, and ﬁnancial capital (e.g., Pretty, 2008; and Mueller et al., 2012). Combining the goals of food security and biofuel sustainability with other local priorities contributes to increases in total factor productivity that are responsible for the majority of growth in output from global Many challenges in reconciling bioenergy and food security also present opportunities. Achieving positive synergies between bioenergy and food production requires science-based clariﬁcations about context-speci- ﬁc problems. This also demands science-based valida- tion of assumptions and clear deﬁnitions. Therefore, in addition to techno-economic challenges of multiproduct agricultural systems, we also should resolve barriers to social acceptance, clarify terminology, and verify that © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 12 K. L. KLINE et al. FOOD SECURITY AND BIOENERGY SYNERGIES 13 FOOD SECURITY AND BIOENERGY SYNERGIES 13 Sorting out complex causal relationships for defor- estation is difﬁcult (Pacheco et al., 2012). Quantitative models are facilitated by the convenience of remote sensing data and the simplicity of the conventional assumption that causation can be determined by the apparent land cover following deforestation; however, oversimpliﬁcations in such models often lead to faulty conclusions (Dale & Kline, 2013). Correlations between deforestation and increasing ‘agricultural area’ are assumed to reﬂect agricultural land scarcity. Several studies that use models to support the hypothesis that biofuels compete globally for land with food (Boddiger, 2007; Tenenbaum, 2008; Searchinger & Heimlich, 2015) rely on assumptions that contradict empirical evidence (Kline et al., 2011; Souza et al., 2015). scientiﬁcally sound approaches are applied to address real problems. Focusing on positive synergies urges us to ask the right questions and to identify mechanisms for energy investments that improve food security. Use accurate and consistent terms for analysis and communications Robust scientiﬁc analysis should be grounded in a clear deﬁnition of the problem to be assessed and a systemic approach to resolving it. The results of many studies rely on faulty assumptions such as: Global land area is the limiting factor for food production; producing more commodities in the United States will alleviate global hunger; or any increase in commodity prices will cause food insecurity. Furthermore, policymakers and the public are misled by terms used in reporting research about food security. For example, #2 yellow corn, the subject of many reports about U.S. biofuel impacts on ‘food security,’ is a feed grain unﬁt for direct use as food. U.S. maize grown for human consumption (sweet corn, white corn, popcorn) represents about 3% of total U.S. corn production (Hansen & Brester, 2012), and from 2010 to 2014, represented only 2% of total U.S. maize exports (USDA-GATS, 2015). Simpliﬁed models confuse #2 yellow industrial feed with food. Resulting communications promulgate misconceptions, for exam- ple, that food insecurity increases with increasing com- modity prices of corn or sugar (Table 3). Authentic communication requires that appropriate terminology is deﬁned clearly and used consistently. Indeed, policymakers in major food-producing nations have been challenged by waste, overproduction, and depressed farm-commodity prices for decades. As a result of excess production, policies were developed in the 1980s and 1990s to reduce spoilage, waste, and ﬁnancial losses associated with excessive stocks of major food commodities. Priorities and conditions to achieve positive synergies Table 5 Beneﬁts arising from the systemic integration of food production and bioenergy (based on Dale et al., 2014, and case studies cited in this paper) Main principle Land-efﬁcient food production and consumption Integrated bioenergy production and use Comments Food security Increased food supply, decreased pressure on land Direct provision of energy services and income from existing land Enhanced coping capacity also essential; requires planning for optimized use of limited resources (capital, water, inputs, time) Climate security Reduced land clearing and land-use change Supply of low-carbon energy to agriculture and rural communities Enhanced soil and above-ground carbon stocks; increased resilience Energy security and supply Increased land for bioenergy and ecological habitats Increased provision of local energy services Bioenergy providing low-cost drying, processing energy, and transport energy Preservation of habitat, wild places Reduced expansion of managed lands Enhanced vegetation cover, species diversity, and wildlife corridors Introduction of perennial cropping in riparian zones, on steeper slopes and in vulnerable zones in water catchment Enhanced soil quality Increased resiliency and crop yields Beneﬁts of perennial bioenergy crops in landscape and cropping strategies with greater diversity of options Novel crop rotations, increased use of perennials to enhance soil organic matter and reduce soil disturbance Enhanced environmental quality Increased intensity of production with reduced environmental impacts Novel landscape planning and cropping strategies to reduce erosion, enhanced nutrient and water availability, and decreased leaching Beneﬁts not a default outcome but will require careful planning and implementation combined with improved extension, knowledge transfer, and IT-based decision tools Poverty alleviation Greater and more resilient yields, reduced storage losses, and improved tillage and transport logistics raise income and reduce economic losses Enhanced reliability and resilience of local energy supply; hedging strategies provided in case of damaged, condemned, or contaminated crops; improved use of residues to raise income Direct beneﬁts to rural farmers, processors, and traders. Care required with emerging economies of scale and marginalization of the most vulnerable/poor Rural economic development Increased competitiveness, enhanced knowledge and innovation capacity Increased local economic activity and critical mass Beneﬁts to urban poor and rural poor © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Lt FOOD SECURITY AND BIOENERGY SYNERGIES 13 Those policies emphasized land set- asides and environmental protection rather than increased production. Furthermore, food security in some less developed nations was impaired by food ‘aid’ and subsidized export of surplus production (Thurow & Kilman, 2009; FAO, WFP, 2010). Since the 1990s, inno- vations in technology, system integration, and logistics have allowed producers to meet the growing global demands for food without requiring additional land (Alexandratos & Bruinsma, 2012; Conway & Wilson, 2012). Yet the belief that biofuel production directly competes with food production and increases food prices remains widely held (e.g., Hajkowicz et al., 2012). © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 Invest in technological innovation to build capacity and infrastructure One of the most persistent recommendations for improving food security is to invest in rural agricultural technology, as discussed in the SOFI reports and reﬂected in multiple recent initiatives to ‘feed the future’ (Godfray et al., 2010; IMF, 2013; USG, 2015; World Bank, 2015). However, during periods of historically low real prices for food producers, there is limited motivation for investments in technology or yield improvement. Declining support for agricultural research around the globe since the 1970s is a concern, and the ‘signiﬁcant decline in annual investment in high-income countries between 1991 and 2000 is especially troubling’ (Beachy, 2014). Case studies in Brazil have illustrated the poten- tial for investments in bioenergy technology and infras- tructure to simultaneously reduce hunger, expand food commodity exports, and promote socioeconomic devel- opment (Souza et al., 2015). 14 K. L. KLINE et al. 14 K. L. KLINE et al. (Zumkehr & Campbell, 2015). Rooftops and other small urban gardens illustrate that far higher yields per hec- tare are possible, potentially reducing land requirements to as little as 0.01 ha per capita (Orsini et al., 2014; Rockwell, 2015). Still less land would be required for intensive, closed-loop agricultural systems that recycle water and nutrients. Given current trends, some researchers expect that the agricultural area required to support global food needs will decline over coming decades (Roser, 2015). Promote stable prices high enough to incentivize local food production Price volatility in a food security context is deﬁned as large, sudden changes in the prices of staples on which at-risk populations depend. Sudden price increases make staples less accessible to urban at-risk groups, while sudden decreases undermine smallholder produc- ers’ livelihoods and household incomes in rural areas. More predictable staple prices that create incentives for local investment in food production are important to improving food security (IFPRI, 2015). Declines in prices are more detrimental to food security than temporary price spikes because (1) capacity and investment in local food production supply chains are undermined, (2) over 70% of the global population living with hunger is in rural areas (FAO, 2014, 2015b), and (3) price crashes cat- alyze rural-to-urban migration, which can further undermine existing productive capacity. Rural areas and uncharted neighborhoods created by recent migrants are more difﬁcult and costly to reach with food assistance than well-established, urban popula- tions. Farmers and agro-industries have demonstrated capacity to respond to local market signals for products that can be grown proﬁtably. When considering land, context is critical. Local com- petition for land reﬂects historic inertia and can be polit- ically and socially sensitive. Even though no further deforestation is required to feed humanity well into the future, deforestation continues due in part to poor understanding of the local causes. Effective policies to conserve natural areas do not require reducing food or biomass production but may involve incentives for efﬁ- cient resource management and recycling of water and nutrients. Recognize that food and bioenergy need not compete for land The idea that bioenergy competes with food for land is predicated on several correlations and assumptions, beginning with land being a limiting factor for global food production. The land scarcity concept is based, in part, on conventional wisdom (‘Buy land, they aren’t making more of it!’) and on an oversimpliﬁed interpre- tation of historical land clearing. Many analyses assume incorrectly that a land-cover class indicates the cause of clearing. In such analyses, forest cover typically change to agricultural cover classiﬁed as crops or pastures, and deforestation is attributed to agricultural demand. Yet, when viewed from social and historical perspectives, the actual causes of deforestation can be attributed to many other drivers such as colonization and tenure policies, market-distorting subsidies, speculation based on intrinsic value, new infrastructure, customary prac- tices for claiming frontier land, migration, and extrac- tive enterprises (Scouvart et al., 2007; Kline & Dale, 2008). It becomes clear that global land area is not the limit- ing factor for food and bioenergy production when con- sistent data on land cover, land use, and productive potential are applied to the analysis (Babcock, 2011; Woods et al., 2015). Despite ongoing population growth and deforestation, the total land area used to feed the world has remained steady since 1990 (Ausubel et al., 2013; FAOStat, 2015). The average area of cropland used to feed one person has fallen from 0.45 ha in 1961 to 0.22 ha in 2006 (FAO, 2011b) and is projected to be close to 0.19 ha at present, based on FAOStat 2015. At 0.19 ha per capita, 1.7 billion hectares, or about a third of all arable land available today, could feed the population of 9 billion projected for 2050. Output from most agricultural land is far below potential yields (Mueller et al., 2012). Thus, the land required to feed humanity is a fraction of that currently classiﬁed as agricultural (Woods et al., 2015). Most U.S. cities could be fed from a 50-mile-radius ‘foodshed’ 016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.1236 Adopt ﬂex crops that can provide food and other products Extreme weather events such as drought and ﬂood are inevitable and cause unpredictable supply shocks in affected areas. Trade combined with surplus production from diverse regions can help alleviate such vulnerabili- ties to extreme events. Remote sensing tools and com- munication platforms that share crop progress and projected harvest data are increasingly allowing far- ﬂung regions to respond quickly to supply shocks. Producers with competitive technologies and access to markets can boost yields or plant a second crop on existing ﬁelds. The supply shock caused by the 2012 drought in North America was offset in part by planting second crops on existing ﬁelds in the Southern Hemi- sphere (USDA, 2013). The increasingly interconnected world is better informed and responsive to arising crises, helping to reduce casualties from famine over the last two decades (Roser, 2015). Investments in innovation and local infrastructure are promoted at the nexus of sustainable bioenergy, food security, and resource management. Innovations in technology and integrated production systems charac- terized recent biofuels expansion in the United States and Brazil (Gee & McMeekin, 2011). Bio-based indus- tries that can entice new investments are a prominent part of many rural development strategies (UNCTAD, 2014). Investment is required to complement the land and labor that tend to be plentiful in rural areas at risk to food insecurity (FAO, 2015a). Key constraints, capital and technology, can be alleviated by investments in strong, growing markets. Biofuel markets have been proposed as one mecha- nism that can absorb the surplus production in normal years and provide a cushion in years of unexpected supply disruptions. The opportunities offered and prob- lems created by ‘ﬂex crops’ that can serve food and other markets merit further study. International organi- zations concerned with food security (e.g., FAO, IFAD, IMF, OECD, UNCTAD, WFP, the World Bank, WTO, IFPRI) support policies or market mechanisms that The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 FOOD SECURITY AND BIOENERGY SYNERGIES 15 allow feedstocks to be diverted from biofuel production to uses that could dampen volatility of food commodity prices (see for example the Committee on Food Security report, HLPE 2013; Locke et al., 2013). This ability to shift end use of available supply as a ‘safety valve’ to reduce price volatility (Wright, 2011) has been a corner- stone of Brazilian strategies for maintaining strong bio- fuels and sugar industries (Osseweijer et al., 2015). Ask the right questions g j Similarly, U.S. maize production capacity expanded from 2002 to 2011 in part as a response to federal bio- fuel mandates. Investments made during this period in technologies such as precision agriculture, irrigation, and grain storage would have been impossible without favorable proﬁt margins. Federal support to expand bio- fuel markets increased conﬁdence in the ability to sell crops at a proﬁt. The investments increased efﬁciency and reduced long-term production costs. Investments in irrigation and storage between 2002 and 2012 also helped to moderate price volatility in the face of the worst drought to hit U.S. farms in more than 50 years (USDA, 2013). A drought of this magnitude represents a ‘supply shock’ that could have triggered a global food price crisis, but market responses helped avoid a major price spike. Moreover, the drought and its effects were monitored and communicated widely, which allowed Southern Hemisphere nations to respond with second crops of maize. There is growing recognition of the value of ﬂex crops combined with good market intelli- gence to support predictable and relatively stable com- modity prices, as this information inﬂuences decisions of buyers and sellers in futures markets (FAO-UNEP, 2011; UNCTAD, 2014). Analysis must consider local contextual conditions to understand the drivers of food insecurity. Multiple causal factors should be addressed using a holistic approach. Developing a bioenergy policy or project designed to improve food security requires that answers to the follow- ing questions be applied to a well-deﬁned, local context. g pp Developing a bioenergy policy or project designed to improve food security requires that answers to the follow- ing questions be applied to a well-deﬁned, local context. 1. Who is most at risk from food insecurity? 1. Who is most at risk from food insecurity? 2. What factors are causing or increasing the risk of speciﬁc food security problems? How do these fac- tors relate to energy and fuels? 3. What actions are feasible and likely to effectively address the causal risk factors? 4. What can be done to mitigate hunger problems in the near term while also building resilience to reduce future risk of food insecurity? And how do these actions and those identiﬁed in response to question 3 relate to potential (bio)energy/fuels? 5. How can a bioenergy policy or project be designed to address the local causal risk factors and contribute to reduced food insecurity? 6. Ask the right questions Is a regional development plan that integrates sustain- able bioenergy more effective and efﬁcient in achiev- ing food security goals than one without bioenergy? Adopt ﬂex crops that can provide food and other products development goals including the eradication of hunger. The following recommendations aim to facilitate syn- ergies between food security and energy security through careful planning and development of bioenergy projects and policies. Identify conditions under which bioenergy improves food security Identify conditions under which bioenergy improves food security Consensus-based principles of sustainable global food security underscore the importance of developing pro- jects with local ownership that consider the needs of the most vulnerable populations (FAO, 2015a) (Table 4). Stakeholders can help identify ways in which bioenergy investments can reinforce efﬁcient local food production and other services. Stakeholder engagement also sup- ports adaptive decision-making to enhance goal achievement (Dale et al., 2015). Integration of land- and resource-efﬁcient food and bioenergy production will increase the sustainability of the system and extend beneﬁts across multiple value- added product chains (Table 5). Conclusions and recommendations Relationships among food security and biofuel policies are complex and context speciﬁc. Such nuanced local relationships cannot be captured in global-scale analy- ses, and the validity of simple models for useful policy guidance is questionable. Assessing impacts requires an understanding of the interactions among factors rele- vant to food security within a speciﬁc place and time. The debate needs to transition from irreconcilable gen- eralizations about whether biofuels are ‘good or bad’ for food security, to constructive understandings of where and how biofuels can help achieve sustainable Apply adaptive management and promote continual improvement Apply adaptive management and promote continual improvement Adaptive management involves learning from ongoing monitoring so that decisions can be adjusted to changing conditions and needs. Timely information about environ- mental, social, and economic conditions, local crops, and market intelligence can support more sustainable food and energy production. It is important to collect data and monitor indicators of food and energy security that are most relevant to local context and stakeholders. Local monitoring helps to verify progress, ﬂag problems, and signal requirements for corrective actions. The informa- tion gained needs to inform adjustments in management practices and plans that support adaptation to changing conditions. Accurate and timely data on prices, stocks, futures markets, and weather are essential to support monitoring and adaptive management. Crop monitoring and timely information sharing can also help address unplanned supply shortfalls and reduce price volatility, as observed when Southern Hemisphere nations such as Brazil and Argentina planted second crops in response to early reports of the 2012 U.S. drought. Diversifying sources of production and end uses of agricultural products enhances local food security. More efﬁcient production of nutritious staples can be pro- moted through integrated production systems that offer a diversity of coproducts for bioenergy and other mar- kets. Crops that can serve multiple markets reduce risks for producers and possibly enhance food safety by pro- viding noon-food outlets for contaminated or damaged food. It may be beneﬁcial to promote strategic supply chains in order to facilitate access to multiple markets for such ‘ﬂex crops.’ Investments in better technology and more efﬁcient production (e.g., precision agriculture and efﬁcient irrigation) can help producers respond to market signals for different crops as well as adapt to disturbances such as those caused by weather. Diversity in the geospatial distribution of production and types of production can reduce price sensitivities caused by dis- ruptive events (e.g., political upheaval, ﬂood, or drought). Encourage coproduct complementarity, diversity, and stable markets Relatively stable and predictable prices for food and energy are essential for food security. Access to afford- able energy supports food security goals, while energy price volatility can exacerbate food crises. Building con- ﬁdence with long-term policies allows markets to work hange Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12366 016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.1236 16 K. L. KLINE et al. 16 supports 4.5 million jobs, improves livelihoods, and promotes rural infrastructure and development (Moraes & Zilberman, 2014). effectively. For example, to the degree that biofuel poli- cies support a more stable and proﬁtable market-driven price ﬂoor, local production can be incentivized by mar- kets that can absorb increasing output. If price caps are used to protect consumers, mechanisms to support local producers may be needed lest food security be under- mined. As price crashes are often more detrimental to long-term food security than price spikes, sudden shifts in policies that reduce investment in agricultural pro- duction should be avoided. effectively. For example, to the degree that biofuel poli- cies support a more stable and proﬁtable market-driven price ﬂoor, local production can be incentivized by mar- kets that can absorb increasing output. If price caps are used to protect consumers, mechanisms to support local producers may be needed lest food security be under- mined. As price crashes are often more detrimental to long-term food security than price spikes, sudden shifts in policies that reduce investment in agricultural pro- duction should be avoided. Communicate clearly about barriers and opportunities to address local needs Support planning and implementation of landscapes designed for multiple uses and waste minimization How food and food security are discussed shapes pub- lic opinion. Clear deﬁnitions, consistent use of terminol- ogy, science-based problem identiﬁcation, and validation of assumptions help reduce confusing and conﬂicting messages. Data need to be relevant; commu- nications focusing on global commodity prices may have little bearing on the factors that determine when and where local food insecurity becomes a problem. Reliance on readily available aggregate data distracts attention from aspects of food insecurity that matter most for peoples’ health and well-being. Timely infor- mation on the status of indicators for environmental, social, and economic effects of development projects needs to be publicly accessible. Long-term commitments to food security, energy security, and environmental quality need to be broadly communicated, and deﬁned goals should be shared widely. Apply landscape design to help stakeholders assess trade-offs when making choices about locations, types, and management of crops, as well as transport, reﬁning, and distribution of products and services. Landscape design refers to a spatially explicit, collaborative plan for management of landscapes and supply chains for food, energy, and other services (Dale et al., 2016), which respects traditional landholdings and farming practices. Proactive resource-use planning can support improvements in management and provision of services based on a set of deﬁned goals (Dale et al., 2014). Such planning should consider shared infrastructure to meet the needs for food, energy, and other markets in a way that reduces costs and waste. Reduction in agricultural wastes provides a means for more efﬁcient crop produc- tion. Agro-ecological zoning developed in response to biofuel sustainability concerns in Brazil has inﬂuenced other agricultural sectors and helped protect biodiver- sity and forests, which are important sources for sus- tained food production in rural areas (Sunderland et al., 2013). The sugarcane–ethanol industry in Brazil Collaborate with local development programs on common goals Bioenergy policies can support progress toward the 2030 Sustainable Development Goals of doubling of © 2016 The Authors. Global Change Biology Bioenergy Published by John Wiley & Sons Ltd., doi: 10.1111/gcbb.12 FOOD SECURITY AND BIOENERGY SYNERGIES 1 17 agricultural productivity, improving incomes of small- scale food producers, and providing clean energy for all (UN, 2015). Research should provide relevant lessons drawn from bioenergyfood interactions over the last decade to guide efforts to provide food and energy while reducing greenhouse gas emissions (Dale et al., 2011). Communicate clearly about barriers and opportunities to address local needs The 2015 assessment of progress toward Millen- nium Development Goals (MDGs) found that several countries with domestic biofuel production policies, such as Brazil, China, Indonesia, Malawi, Malaysia, and Peru, also achieved or exceeded challenging hunger- reduction goals (FAO, IFAD, WFP, 2015a). Other coun- tries with notable bioenergy potential, but where biofuel policies were not effectively implemented, such as Zam- bia, Senegal, and Guatemala, fell short on MDG hunger- reduction targets (Tay, 2013; Mukanga, 2014; UNCTAD, 2014; World Bank, 2015). Biofuel projects responsive to site-speciﬁc needs in developing nations offer opportu- nities to support food and energy security goals (Kline et al., 2009; Gasparatos et al., 2011; Mitchell, 2011). incentives. Case studies that document actual conditions before and after project implementation can support more integrated project designs and adaptive manage- ment (FAO, 2011a; Elbehri et al., 2013). Transparent doc- umentation of the problem, hypotheses, research methods, input data sources, and assumptions is essen- tial to avoid potential misrepresentation of analytical results (Dale & Kline, 2013). Build on and improve existing systems Bioenergy is already an integral part of global food pro- duction, processing, and consumption systems. Experience indicates that investments in bioenergy can help expand local food supplies, infrastructure, and productive capac- ity and thereby reduce risks of hunger for speciﬁc groups and situations (FAO, 2011a; Durham et al., 2012; Moraes & Zilberman, 2014). The nexus of bioenergy, food security, and resource management is especially signiﬁcant for the rural poor. Dependence on subsistence agriculture and inefﬁcient traditional biomass use leaves rural populations vulnerable and deepens impoverishment through resource degradation. Current practices can transition and trans- form through continual improvements to meet the needs of society in a changing world. Institutional capacity for learning and sharing experiences should be developed across the supply chain. Applying science to support con- tinual improvement will help feed more people and pro- vide them with more sustainable energy resources for the future. Acknowledgements Work by Keith L. Kline and Virginia H. Dale was supported by the U.S. Department of Energy (DOE) under the Bioenergy Technologies Ofﬁce; ORNL is managed by UT-Battelle, LLC, for DOE under contract DE-AC05-00OR22725. Harriet K. Mugera was supported by the World Bank. Jeremy Woods was supported by Climate-KIC and Imperial College London. Glau- cia M. Souza was supported by a grant from the Sao Paulo State Research Foundation (FAPESP 2012/23765-0) and a Pro- ductivity Fellowship from the Brazilian National Council for Scientiﬁc and Technological Development (CNPq). Erica Atkin and Gina Busby are thanked for editorial assistance. References Alexandratos N, Bruinsma J (2012) World agriculture towards 2030/2050: the 2012 revision. ESA Working paper No. 12-03. Rome, FAO. Conclusions Effectively addressing food security and bioenergy sus- tainability requires a renewed focus on populations at risk. 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https://openalex.org/W2113020449	https://europepmc.org/articles/pmc3432005?pdf=render	English	null	Radiotherapy alone for stage I-III low grade follicular lymphoma: long-term outcome and comparison of extended field and total nodal irradiation	Radiation oncology	2,012	cc-by	5,449	RESEARCH Open Access Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 © 2012 Guckenberger et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Radiotherapy alone for stage I-III low grade follicular lymphoma: long-term outcome and comparison of extended field and total nodal irradiation Matthias Guckenberger, Nikolaus Alexandrow and Michael Flentje Matthias Guckenberger, Nikolaus Alexandrow and Michael Flentje * Correspondence: Guckenberger_M@klinik.uni-wuerzburg.de Department of Radiation Oncology, University of Würzburg, Josef-Schneider-Str. 11, 9708 Würzburg, Germany * Correspondence: Guckenberger_M@klinik.uni-wuerzburg.de Department of Radiation Oncology, University of Würzburg, Josef-Schneider-Str. 11, 9708 Würzburg, Germany © 2012 Guckenberger et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: To analyze long-term results of radiotherapy alone for stage I-III low grade follicular lymphoma and to compare outcome after extended field irradiation (EFI) and total nodal irradiation (TNI). Methods and materials: Between 1982 and 2007, 107 patients were treated with radiotherapy alone for low grade follicular lymphoma at Ann Arbor stage I (n = 50), II (n = 36) and III (n = 21); 48 and 59 patients were treated with EFI and TNI, respectively. The median total dose in the first treatment series of the diaphragmatic side with larger lymphoma burden was 38 Gy (25 Gy – 50 Gy) and after an interval of median 30 days, a total dose of 28 Gy (12.6 Gy – 45 Gy) was given in the second treatment series completing TNI. Results: After a median follow-up of 14 years for living patients, 10-years and 15-years overall survival (OS) were 64% and 50%, respectively. Survival was not significantly different between stages I, II and III. TNI and EFI resulted in 15-years OS of 65% and 34% but patients treated with TNI were younger, had better performance status and higher stage of disease compared to patients treated with EFI. In multivariate analysis, only age at diagnosis (p < 0.001, relative risk [RR] 1.06) and Karnofsky performance status (p = 0.04, RR = 0.96) were significantly correlated with OS. Freedom from progression (FFP) was 58% and 56% after 10-years and 15-years, respectively. Recurrences outside the irradiated volume were significantly reduced after TNI compared to EFI; however, increased rates of in-field recurrences and extra-nodal out-of-field recurrence counterbalanced this effect resulting in no significant difference in FFP between TNI and EFI. In univariate analysis, FFP was significantly improved in stage I compared to stage II but no differences were observed between stages I/II and stage III. In multivariate analysis no patient or treatment parameter was correlated with FFP. Acute toxicity was significantly increased after TNI compared to EFI with a trend to increased late toxicity as well. Conclusions: Radiotherapy alone for stage I and II follicular lymphoma resulted in long-term OS with high rates of disease control; no benefit of TNI over EFI was observed. For stage III follicular lymphoma, TNI achieved promising OS and FFP and should be considered as a potentially curative treatment option. Keywords: Follicular lymphoma, Total nodal irradiation, Extended field irradiation Page 2 of 8 Guckenberger et al. Background Follicular lymphoma (FL) is after diffuse large B-cell lymphoma the second most common subtype of non- Hodgkin’s lymphomas in adults (~ 20–25%). Radiother- apy alone is an established curative treatment option for patients with stage I-II FL [1-3]. However, the volume, which needs to be irradiated, is still an issue of contro- versy. As the site of treatment failure is most frequently nodal outside irradiated involved volumes, extended- field irradiation (EFI) or even total nodal irradiation (TNI) have been proposed to improve outcome com- pared to involved-field irradiation (IFI). While studies reported improved progression free survival (PFS) after treatment of larger volumes, this did not transfer into improved overall survival (OS) [4,5]. Despite the major- ity of relapses are observed within 5 years after radio- therapy, late recurrences 15 – 20 years after treatment [4-6] indicate the need for studies with long-term follow-up, which are scarce in the literature. Statistical analysis Patient and treatment characteristics were compared be- tween the two groups treated with EFI and TNI and be- tween stage I/II and stage III using Chi-squared test for categorical variables and Mann-Withney U-test for con- tinuous variables using Statistica X software (Statsoft, Tulsa, OK, USA). Survival and recurrence data were cal- culated by the Kaplan–Meier method: overall survival (OS) and freedom from progression (FFP) were calcu- lated. Results between subgroups were compared using the log-rank test and the influence of patient and treat- ment factors on OS and FFP was evaluated using the multivariate Cox proportional-hazards regression model with backward exclusion of non-significant variables. Results with p < 0.05 were considered as statistically significant. Stage III disease is considered as non-curative and chemotherapy or wait and see strategies are most fre- quently perused. Nevertheless, studies reported promis- ing disease control and overall survival rates after radiotherapy alone for stage III FL [7,8]. Recent data reported that lower radiation doses are sufficient for dis- ease control [9], which might further reduce acute and especially long term toxicity. In this context of poten- tially reduced toxicity, radiotherapy in form of TNI might become a more attractive treatment strategy for advanced stage of disease. It was consequently the aim of this retrospective single institution study to describe long-term outcome after radiotherapy alone for stage I- III FL and to compare patterns of failure and OS after EFI and TNI. Abstract Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 burden or elective irradiation (second series). Total lymphatic irradiation with routine treatment of the Wal- deyer’s tonsillar ring and mesenteric lymph nodes was not practiced. The spleen and Waldeyer's tonsillar ring were irradiated in 12% and 36% of the patients, respect- ively. Single fraction doses ranged between 1.5 Gy −2.0 Gy with the majority of patients treated with 1.8 Gy or 2.0 Gy. Lymph node regions without macroscopic in- volvement were treated with a total dose of 30 Gy and a boost of 6 - 8 Gy was given to macroscopic involved lymph node regions: the median total dose in the first treatment series was 38 Gy (25 Gy – 50 Gy) and after a median interval of 30 days, a total dose of 28 Gy (12.6 Gy – 45 Gy) was given in the second treatment series. Material and methods Patient characteristics and involved lymph node regions are summarized in Tables 1 and 2. Differences between EFI and TNI reflect the decision making process for the two approaches. Younger patients with better perform- ance status were treated predominantly with TNI. Pres- ence of B-symptoms, elevated serum LDH and bulky disease were not different between ENI and TNI. Add- itionally, no differences in total irradiation doses were observed. Between 1982 and 2007, 107 patients were treated with radiotherapy alone for low grade follicular lymphoma. Of these 107 patients, Ann Arbor stage was I (n = 50), II (n = 36) and III (n = 21). Ann Arbor stage was based on contrast enhanced whole body CT imaging and bone marrow biopsy in all patients. All patients were treated with radiotherapy alone as primary treatment; patients who received chemotherapy as part of the primary treatment were excluded from this analysis. Patients were treated with either EFI of all central lymph node regions on the involved side of the diaphragm (supra-diaphragmatic: cervical, supra- and infraclavicular, mediastinal and hilar; infra- diaphragmatic: paraaortal, iliacal, inguinal) or with TNI (sequential supra-diaphragmatic and infra-diaphragmatic irradiation). Treatment was always started at the dia- phragmatic side with higher lymphoma burden (first series) followed by the side with lower lymphoma The median number of involved lymph node regions was 2 (range 2–5) and 4 (range 2–11) in stage II and III disease, respectively. Patients at stage III were younger compared to stage I/II and elevated LDH was observed more frequently in stage III patients; there were no dif- ference in the rate of bulky disease and irradiation doses. Of patients with stage III disease, 18/21 were treated with TNI; one patient denied infra-diaphragmatic after supra-diaphragmatic irradiation and the reason for two patients is unknown. Guckenberger et al. Material and methods Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Page 3 of 8 Page 3 of 8 Table 1 Patient and treatment characteristics with comparison between EFI and TNI and between stages I/II and stage III disease All EFI TNI p-value Stage I/II Stage III p-value # patients 107 48 59 86 21 Age <0.001 <0.001 median/range [years] 57 26 - 83 66 37 - 83 51 26 - 74 61 27 – 83 51 26 - 68 ≥60 years 48 45% 35 73% 13 22% 45 52% 3 14% Sex [male] 59 57% 26 55% 33 56% NS 49 57% 10 48% Karnofsky Performance status <0.001 NS median/range 90 50 - 100 90 50 - 100 100 80 - 100 90 50 – 100 90 70 – 100 B-Symptoms 1 1% 0 0% 1 2% NS 0 0% 1 5% NS LDH NS 0.03 Elevated 16 15% 9 19% 7 12% 8 9% 8 38% Normal 63 59% 29 60% 34 58% 55 64% 8 24% unknown 28 26% 10 21% 18 30% 23 27% 5 38% Ann Arbor stage 0.04 I 50 47% 27 56% 23 39.0% II 36 34% 18 38% 18 30.5% III 21 20% 3 6% 18 30.5% Bulk ≥7.5 cm 5 5% 2 4% 3 5% NS 3 3% 2 9% NS Radiotherapy dose median/range [Gy] 38 25 - 50 38 27 - 50 37 25 - 46 NS 37 25 - 50 37 29 - 46 NS Table 1 Patient and treatment characteristics with comparison between EFI and TNI and III disease 1 Patient and treatment characteristics with comparison between EFI and TNI and between stages eatment characteristics with comparison between EFI and TNI and between stages I/II and stage FFP was 76%, 58% and 56% after 5-years, 10-years and 15-years (Figure 1), respectively. Only one recurrence was observed after 10 year (12.8 years). FFP at 10 years was not different between TNI and EFI with 62% and 54% (p = 0.50) (Figure 3a), respectively. FFP at 10 years was 74%, 40% and 62% for stage I, stage II and stage III (Figure 3b), respectively, and results of stage II were significantly worse compared to stage I (p = 0.001). In Cox proportional-hazards analysis (with Ann Arbor stage, age and TNI vs ENI included into the modelling), no patient or treatment characteristic was correlated with FFP. Material and methods Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Page 4 of 8 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Overall survival Freedom from progression Figure 1 Kaplan Meier Curves showing overall survival (OS) and freedom from disease progression (FFP) for all patients. Overall survival Freedom from progression Figure 1 Kaplan Meier Curves showing overall survival (OS) and freedom from disease progression (FFP) for was significantly increased with 1 patient (2%) develop- ing grade IV toxicity (thrombocytopenia) and 8 patients (14%) suffering from grade III toxicity (leukopenia n = 3, thrombocytopenia n = 2, gastrointestinal mucositis n = 2, nausea and vomiting n = 1). irradiation of the involved side or both sides of the dia- phragm [4]. After a median follow-up of 7.7 years, FFP at 10 year were 36% and 67% for treatment of the involved side only and both sides of the diaphragm, re- spectively, which was highly significant. However, this improvement in disease control did not transfer into increased OS. Late toxicity was maximum grade II in all patients and grade II toxicity was observed in 27 patients (25%). The most frequent grade II toxicity was pulmonary (n = 12; pulmonary fibrosis, dyspnoea, cough) and gastro- intestinal (n = 9; dry mouth, dysphagia, dental, hepatitis, colitis). Two patients developed chronic haematological toxicity and one patient generalized dry skin and fatigue. Any grade II toxicity was increased after TNI (n = 18; 31%) compared to EFI (n = 8; 17%) but this difference did not reach statistical significance. In our own study, evaluating the impact of TNI and EFI on FFP and OS was difficult because of a correlation between the target volume concept and established prognostic parameters: age of the patients, performance status and stage of disease. No difference of FFP was observed between TNI and EFI in univariate and multi- variate analysis. In agreement to Stuschke et al. [10], irradiation of uninvolved lymph node regions with a median dose of 30 Gy was effective in reducing out-of- field recurrences. However, an increased rate of in-field recurrences and extra-nodal out-of-field recurrence counterbalanced this beneficial effect of TNI. Material and methods Median follow-up was 10 years for all patients (0.3 – 26 years) and 14 years (2–26 years) for living patients. Follow-up was not different between EFI and TNI and between stages I/II and stage III. For the total patient population, 10-years and 15-years OS were 64% and 50% (Figure 1), respectively. In uni- variate analysis, age > median of 60 years (p = 0.0001), EFI versus TNI (p = 0.001) and Karnofsky performance status <100 (p = 0.0003) were correlated with decreased OS. Median age of 60 years differentiated between 15- years OS of 67% and 29%; TNI and EFI resulted in 15- years OS of 65% and 34% (Figure 2a), respectively. Overall survival was not significantly different between stages I-III: 15-years OS were 51%, 45% and 54% for patients with stage I, II and III (Figure 2b), respectively. In Cox proportional-hazards analysis (with age, TNI vs ENI, Karnofsky performance status and Ann Arbor stage included into the modelling), age at diagnosis (p < 0.001 and relative risk of 1.06) and Karnofsky performance sta- tus (p = 0.04 and relative risk of 0.96) were significantly correlated with OS (Table 3). A total of 35 recurrences were observed with crude rates of 33% and 32% after EFI and TNI, respectively. 14/16 (87.5%) and 8/19 (42%) of the recurrences after EFI and TNI were located outside the irradiated volume, which was highly significant (p = 0.01). During the first irradiation series, 5 patients (5%) developed acute toxicity grade III (gastrointestinal mucositis n = 4; nausea and vomiting n = 1). During the second irradiation series in TNI patients, acute toxicity Table 2 Distribution of involved lymph node regions separately for patients with stage I, stage II and stage III follicular lymphoma Neck Axilla Mediastinum Paraaortal Iliakal Inguinal Unknown Stage I (n = 50) 46% 8% 0% 0% 2% 32% 12% Stage II (n = 36) 81% 39% 31% 36% 36% 56% 3% Stag III (n = 21) 76% 62% 24% 67% 29% 48% 0% Table 2 Distribution of involved lymph node regions separately for patients with stage I, stage II lymphoma Guckenberger et al. Material and methods Regarding OS, patients treated with TNI were younger and had a better performance status and as a consequence, 15-years OS was 65% and 34% after TNI and EFI, respectively. However, only age and Karnofsky performance status and not the target volume concept remained statistically significant in multivariate analysis. Discussion This study confirms previous data, that treatment of lar- ger irradiation fields does not improve overall survival in stage I and II follicular lymphoma. Wilder et al. reported the M.D. Anderson experience of 80 patients treated with IFI (9%), regional RT (54%; treatment of 1–3 adja- cent, grossly uninvolved nodal regions) or EFI (37%); TNI irradiation was not practiced [5]. After a median follow-up of 19 years for living patients, no difference in OS was observed between EFI and IFI/regional RT: 49% versus 40% at 15 years. Recurrences were more fre- quently observed in patients treated with EFI; however, there were imbalances of stage distribution and total ir- radiation dose and no multivariate analysis was per- formed. Mac Manus et al. described the Stanford experience of 177 patients and differentiated between The 10-years OS of 64% for the patient population with stages I/II disease is considered as representative for early stage follicular lymphoma because our depart- ment is the only radiotherapy centre within a radius of minimum 50 – 100 km and rather strict referral patters in the geographical region; a potential patient selection bias is consequently limited. The observed OS is in good Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Page 5 of 8 agreement with published data in the literature about irradiation was well tolerated with only 5% of the 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients TNI EFI a b 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Stage I Stage 2 Stage 3 Figure 2 Kaplan Meier Curves showing overall survival in relationship to TNI versus EFI (no significant difference) (Figure 2a) and stage of disease (no significant differences) (Figure 2b). Discussion Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Page 6 of 8 Table 3 Univariate and multivariate analysis of patient and treatment factors influencing overall survival and freedom from disease progression Overall Survival Freedom from disease progression Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis Age p = 0.0001 p = 0.0001 RR = 1.06 NS Karnofsky performance status p = 0.0003 P = 0.04 RR = 0.96 NS Stage of disease NS P = 0.001 NS NS TNI vs EFI p = 0.001 NS NS 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients TNI ENI 0.6 0.7 0.8 0.9 1.0 of patients Stage I Stage II Stage III a b Table 3 Univariate and multivariate analysis of patient and treatment factors influencing overall survival and freedom from disease progression Overall Survival Freedom from disease progression Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis Age p = 0.0001 p = 0.0001 RR = 1.06 NS Karnofsky performance status p = 0.0003 P = 0.04 RR = 0.96 NS Stage of disease NS P = 0.001 NS NS TNI vs EFI p = 0.001 NS NS Table 3 Univariate and multivariate analysis of patient and treatment factors influencing overall survival and freedom from disease progression Overall Survival Freedom from disease progression Univariate analysis Multivariate analysis Univariate analysis Multivariate analysis Age p = 0.0001 p = 0.0001 RR = 1.06 NS Karnofsky performance status p = 0.0003 P = 0.04 RR = 0.96 NS Stage of disease NS P = 0.001 NS NS TNI vs EFI p = 0.001 NS NS d multivariate analysis of patient and treatment factors influencing overall survival and freedom i 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients TNI ENI 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Stage I Stage II Stage III a b Figure 3 Kaplan Meier Curves showing freedom from disease progression in relationship to TNI versus EFI (no significant difference) (Figure 2a) and stage of disease (significant difference between stage I and stage II) (Figure 2b). Discussion 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients TNI EFI a a Follow-up [months] b 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Stage I Stage 2 Stage 3 b 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Stage I Stage 2 Stage 3 b p Figure 2 Kaplan Meier Curves showing overall survival in relationship to TNI versus EFI (no significant difference) (Figure 2a) and stage of disease (no significant differences) (Figure 2b). irradiation was well tolerated with only 5% of the patients developing grade III toxicity, 16% of the patients suffered from acute toxicity grade ≥III during the second series, which completed the TNI. Additionally, chronic toxicity grade II was observed more frequently after TNI (31%) compared to EFI (17%). agreement with published data in the literature about primary radiotherapy for stages I/II follicular lymphoma: two recent literature reviews reported 10-years OS rates ranging between 46% and 79% (weighted average of 63% in Heinzelmann et al.) after primary radiotherapy using mostly IFI or EFI [11,12]. No obvious correlation of OS and irradiated volume is seen in this literature review. This further supports the hypothesis of no OS benefit after TNI. Randomized data about the question of the appropri- ate irradiation volume are missing. A German multicen- tre phase III trial, which randomized between TNI and EFI, closed in 2007 and final results are awaited. Until results of this trial are available, increased acute and The larger treatment volumes of TNI came at the price of increased toxicity. While the first series of EFI Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Guckenberger et al. Authors’ contributions MG d i d h d MG designed the study, participated in data collection and performed the data analysis. NA collected the data and participated in the data analysis. MF participated in the design of the study. All authors performed critical review of the manuscript and finally approved the manuscript. Acknowledgments h bl Outcome was favourable with 10-years OS and FFP of 61% and 62%, respectively. OS and FFP were not signifi- cantly different to stages I/II. This favourable outcome is in good agreement with data in the literature [7,8,13,14], where 10-years OS ranged between 47% and 65%. Only a small number of patients with stage III disease was at risk after 10 years in our study; however, the Stanford experience of 66 stage III patients treated with radio- therapy alone described no disease recurrence after 10 years and only 5/29 lymphoma deaths after 10 years [8]. These data are promising especially in the light of the recently published British trial, which showed that radiotherapy doses for indolent lymphomas can be reduced without negatively affecting clinical outcome. Consequently, TNI with reduced irradiation may have the potential of long term disease control and survival with simultaneously decreased acute and chronic toxicity [9]. Of course, such concepts need to be validated within clinical trials. This publication was funded by the German Research Foundation (DFG) and the University of Wuerzburg in the funding programme Open Access Publishing. Received: 16 April 2012 Accepted: 17 June 2012 Published: 24 June 2012 Received: 16 April 2012 Accepted: 17 June 2012 Published: 24 June 2012 References Guadagnolo BA, Li S, Neuberg D, Ng A, Hua L, Silver B, Stevenson MA, Mauch P: Long-term outcome and mortality trends in early-stage, Grade 1–2 follicular lymphoma treated with radiation therapy. Int J Radiat Oncol Biol Phys 2006, 64(3):928–934. y 7. Jacobs JP, Murray KJ, Schultz CJ, Wilson JF, Goswitz MS, Stevens CW, Cox JD: Central lymphatic irradiation for stage III nodular malignant lymphoma: long-term results. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 1993, 11(2):233–238. 7. Jacobs JP, Murray KJ, Schultz CJ, Wilson JF, Goswitz MS, Stevens CW, Cox JD: Central lymphatic irradiation for stage III nodular malignant lymphoma: long-term results. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 1993, 11(2):233–238. 8. Murtha AD, Rupnow BA, Hansosn J, Knox SJ, Hoppe R: Long-term follow- up of patients with Stage III follicular lymphoma treated with primary radiotherapy at Stanford University. Int J Radiat Oncol Biol Phys 2001, 49 (1):3–15. 8. Murtha AD, Rupnow BA, Hansosn J, Knox SJ, Hoppe R: Long-term follow- up of patients with Stage III follicular lymphoma treated with primary radiotherapy at Stanford University. Int J Radiat Oncol Biol Phys 2001, 49 (1):3–15. 9. Lowry L, Smith P, Qian W, Falk S, Benstead K, Illidge T, Linch D, Robinson M, Jack A, Hoskin P: Reduced dose radiotherapy for local control in non- Hodgkin lymphoma: a randomised phase III trial. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology 2011, 100(1):86–92. References 1. Kelsey SM, Newland AC, Hudson GV, Jelliffe AM: A British National Lymphoma Investigation randomised trial of single agent chlorambucil plus radiotherapy versus radiotherapy alone in low grade, localised non- Hodgkins lymphoma. Med Oncol 1994, 11(1):19–25. 2. Pugh TJ, Ballonoff A, Newman F, Rabinovitch R: Improved survival in patients with early stage low-grade follicular lymphoma treated with radiation: a Surveillance, Epidemiology, and End Results database analysis. Cancer 2010, 116(16):3843–3851. 2. Pugh TJ, Ballonoff A, Newman F, Rabinovitch R: Improved survival in patients with early stage low-grade follicular lymphoma treated with radiation: a Surveillance, Epidemiology, and End Results database analysis. Cancer 2010, 116(16):3843–3851. y 3. Nissen NI, Ersboll J, Hansen HS, Walbom-Jorgensen S, Pedersen-Bjergaard J, Hansen MM, Rygard J: A randomized study of radiotherapy versus radiotherapy plus chemotherapy in stage I-II non-Hodgkin’s lymphomas. Cancer 1983, 52(1):1–7. 4. Mac Manus MP, Hoppe RT: Is radiotherapy curative for stage I and II low- grade follicular lymphoma? Results of a long-term follow-up study of patients treated at Stanford University. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 1996, 14(4):1282–1290. j y gy 5. Wilder RB, Jones D, Tucker SL, Fuller LM, Ha CS, McLaughlin P, Hess MA, Cabanillas F, Cox JD: Long-term results with radiotherapy for Stage I-II follicular lymphomas. Int J Radiat Oncol Biol Phys 2001, 51(5):1219–1227. Some limitations of our study need to be discussed. The retrospective nature of this study is associated with well-known limitations. The decision making process be- tween EFI and TNI considered well established prognos- tic factors like age, performance status and stage of FL, which made the statistical comparison of the two target volume concepts difficult. Additionally, the reconstruc- tion of the decision making process in favour of radio- therapy and against chemotherapy in stage III FL could not be fully reconstructed: the ratio of stage I + II : stage III patients in the International Lymphoma classification project was 2:1 [15] compared to 4:1 in our study, which suggests some selection process for radiotherapy. 6. Guadagnolo BA, Li S, Neuberg D, Ng A, Hua L, Silver B, Stevenson MA, Mauch P: Long-term outcome and mortality trends in early-stage, Grade 1–2 follicular lymphoma treated with radiation therapy. Int J Radiat Oncol Biol Phys 2006, 64(3):928–934. 6. Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Page 7 of 8 associated with increased rates of acute and chronic tox- icity. Based on this study and data from the literature, EFI or IFI are the preferred treatments for stage I and II follicular lymphoma. In patients with stage III FL, TNI resulted in promising tumor control and overall survival and should be considered as a potentially curative treat- ment option. chronic toxicity with no apparent benefit in terms of survival and disease control as observed in this study do not support the concept of TNI compared to EFI for stage I and II FL. Twenty one patients with stage III FL were treated with radiotherapy alone and were included into this study. Unfortunately, prognostic factors were not fully balanced between stages I/II and stage III. Patients at stage III FL were younger indicating that age was a se- lection criterion for radiotherapy alone in stage III patients. As expected based on the increased lymphoma burden, LDH was more frequently elevated in stage III patients. TNI was the planned treatment in all stage III patients and was successfully completed in almost 90% of the patients. Competing interests The authors declare that they have no competing interest. Discussion 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients TNI ENI a a Follow-up [months] p [ ] 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Stage I Stage II Stage III b K l M i C h i f d f di i i l i hi TNI EFI ( i ifi diff ) 0 12 24 36 48 60 72 84 96 108 120 132 144 156 168 180 192 Follow-up [months] 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Proportion of patients Stage I Stage II Stage III b b Follow-up [months] Figure 3 Kaplan Meier Curves showing freedom from disease progression in relationship to TNI versus EFI (no significant difference) (Figure 2a) and stage of disease (significant difference between stage I and stage II) (Figure 2b). Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Conclusions 10. Stuschke M, Hoederath A, Sack H, Potter R, Muller RP, Schulz U, Karstens J, Makoski HB: Extended field and total central lymphatic radiotherapy in the treatment of early stage lymph node centroblastic-centrocytic lymphomas: results of a prospective multicenter study, Study Group NHL-fruhe Stadien. Cancer 1997, 80(12):2273–2284. 10. Stuschke M, Hoederath A, Sack H, Potter R, Muller RP, Schulz U, Karstens J, Makoski HB: Extended field and total central lymphatic radiotherapy in the treatment of early stage lymph node centroblastic-centrocytic lymphomas: results of a prospective multicenter study, Study Group NHL-fruhe Stadien. Cancer 1997, 80(12):2273–2284. Radiotherapy alone for stage I and II follicular lymph- oma was an effective and curative treatment with high rates of disease control. Larger irradiation fields covering both sides of the diaphragm did not improve tumor control and overall survival. Additionally, TNI was 11. Eich HT, Heimann M, Stutzer H, Kriz J, Reiser M, Muller RP: Long-term outcome and prognostic factors in early-stage nodal low-grade 11. Eich HT, Heimann M, Stutzer H, Kriz J, Reiser M, Muller RP: Long-term outcome and prognostic factors in early-stage nodal low-grade Page 8 of 8 Page 8 of 8 Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 Guckenberger et al. Radiation Oncology 2012, 7:103 http://www.ro-journal.com/content/7/1/103 non-hodgkin's lymphomas treated with radiation therapy. Strahlentherapie und Onkologie: Organ der Deutschen Rontgengesellschaft [et al] 2009, 185(5):288–295. 12. Heinzelmann F, Engelhard M, Ottinger H, Bamberg M, Weinmann M: Nodal follicular lymphoma: the role of radiotherapy for stages I and II. Strahlentherapie und Onkologie: Organ der Deutschen Rontgengesellschaft [et al] 2010, 186(4):191–196. 13. Paryani SB, Hoppe RT, Cox RS, Colby TV, Kaplan HS: The role of radiation therapy in the management of stage III follicular lymphomas. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 1984, 2(7):841–848. 14. Ha CS, Kong JS, McLaughlin P, Tucker SL, Fayad LE, Hess MA, Wilder RB, Cabanillas F, Cox JD: Stage III follicular lymphoma: long-term follow-up and patterns of failure. Int J Radiat Oncol Biol Phys 2003, 57(3):748–754. 15. Armitage JO, Weisenburger DD: New approach to classifying non- Hodgkin’s lymphomas: clinical features of the major histologic subtypes, Non-Hodgkin’s Lymphoma Classification Project. Journal of clinical oncology: official journal of the American Society of Clinical Oncology 1998, 16(8):2780–2795. doi:10.1186/1748-717X-7-103 Cite this article as: Guckenberger et al.: Radiotherapy alone for stage I-III low grade follicular lymphoma: long-term outcome and comparison of extended field and total nodal irradiation. Radiation Oncology 2012 7:103. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W3021960211	https://refubium.fu-berlin.de/bitstream/fub188/28305/1/s41598-020-64045-w.pdf	English	null	Impact of groundwater depth and soil salinity on riparian plant diversity and distribution in an arid area of China	Scientific reports	2,020	cc-by	9,615	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Impact of groundwater depth and soil salinity on riparian plant diversity and distribution in an arid area of China Yong Zeng1,2,3, Chengyi Zhao4,1 ✉, Fengzhi Shi 1, Michael Schneider5, Guanghui Lv2 Riparian plant diversity in arid regions is sensitive to changes in groundwater. Although it is well known that groundwater has a significant influence on plant diversity, there have been few studies on how groundwater and soil salinity impact plant community in desert riparian ecosystems. Therefore, we surveyed 77 quadrats (100 m × 100 m) to examine the relationship between groundwater depth, groundwater salinity, soil salinity and plant community in the upper reaches of the Tarim River. Data were analyzed with two-way indicator species analysis (TWINSPAN), detrended canonical correspondence analysis (DCCA) and principal component analysis (PCA). The results indicated that Populus euphratica, Tamarix ramosissima, and Phragmites australis were the dominant plants among trees, shrubs and herbs, respectively. Five plant community types were classified. There were significant differences in species diversity, soil moisture, soil salinity, groundwater depth and groundwater salinity across the community types. The composition and distribution of plant community are significantly influenced by groundwater depth, groundwater salinity, soil moisture, distances from the river to the quadrats, soil pH, electrical conductivity, total salt, CO3 2−, Cl−, SO4 2−, Ca2+, Mg2+, Na+ and K+. Shallow groundwater depth, low groundwater salinity, and high soil moisture and soil salinity were associated with higher plant diversity. Riparian areas are in the transition zone between aquatic and terrestrial ecosystems and play a significant role in the energy and nutrient fluxes between the two types of ecosystems1. Riparian habitats comprise a diverse collec- tion of valuable species and are regarded as biodiversity corridors2. Riparian vegetation plays an important role in protecting biodiversity, providing animal food and habitats for animals, and maintaining ecological balance3. However, riparian vegetation has become less stable as groundwater tables have dropped, leading to declines in arid desert river systems4. Therefore, the analysis of the changes in species composition and community distribu- tion is crucial for protecting the biodiversity of riparian ecosystems5.f p g y p y Riparian vegetation in arid regions is mainly controlled by precipitation, surface runoff, and groundwater6. High rates of evapotranspiration and low annual precipitation are characteristic of arid desert river basins6. The low precipitation and limited surface runoff, both spatially and temporally, in extremely arid regions do not pro- vide any significant source of water for plant growth7. Thus, groundwater constitutes the main water source for vegetation in arid river ecosystems8. Impact of groundwater depth and soil salinity on riparian plant diversity and distribution in an arid area of China g y Riparian plant species, as groundwater-dependent vegetation, are referred to as phreatophytes9. Riparian vegetation productivity, biomass, competitiveness, composition, structure, and abundance are controlled by the groundwater10. Increases in water table depth has imposed drought stress on vegetation and reduced plant cover, diversity and richness11,12. Salt accumulation associated with high rate of evaporation of shallow groundwater through the unsaturated zone has been shown to influence plant composition in many arid riparian systems13. The area proximity to river had high salt accumulation12. The soil moisture content, electrical conductivity and pH in the areas nearer to the riverbank were generally higher than other areas14. Riparian plant species richness 1State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi, 830011, Xinjiang, China. 2College of Resources and Environmental Sciences, Xinjiang University, Urumqi, 830046, Xinjiang, China. 3University of Chinese Academy of Sciences, Beijing, 100049, China. 4Nanjing University of Information Science and Technology, Nanjing, 210044, China. 5Earth Sciences, Freie Universität Berlin (FUB), Malteserstr. 74-100, 12249, Berlin, Germany. ✉e-mail: zhaocy@nuist.edu.cn Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ No. Species RD% RF% RDM/RBC% IVI% Rank Tree layer 100 100 100 100 1 Populus euphratica 76.71 80.00 83.29 80.31 1 2 P. pruinosa 23.22 19.18 16.66 19.69 2 Shrub layer 100 100 100 100 1 Tamarix ramosissima 56.09 59.40 31.72 65.71 1 2 T. hispida 3.03 9.25 4.68 5.64 5 3 T. arceuthoides 10.64 1.79 8.81 7.07 4 4 Haloxylon ammodendron 0.08 2.09 0.14 0.76 7 5 Lycium ruthenicum 14.41 12.54 0.80 9.24 3 6 Halimodendron halodendron 0.15 2.99 0.06 1.05 6 7 Halostachys caspica 15.618 11.94 3.79 10.53 2 Herblayer 100 100 100 100 1 Glycyrrhiza inflata 15.73 5.39 0.62 7.27 5 2 Poacynum hendersonii 1.27 2.70 0.87 1.50 9 3 Hexinia polydichotoma 0.08 1.08 0.02 0.40 11 4 Karelinia caspia 19.83 15.10 21.28 18.74 3 5 Cynanchum sibiricum 0.04 1.08 0.01 0.38 13 6 Calamagrostis pseudophragmites 1.80 1.62 1.15 1.53 10 7 Inula salsoloides 0.08 1.08 0.01 0.40 12 8 Phragmites australis 33.14 12.40 34.44 26.67 1 9 Apocynum venetum 5.63 6.47 20.16 10.76 4 10 Alhagi sparsifolia 17.42 31.27 20.95 23.22 2 11 Halocnemum strobilaceum 0.36 4.85 0.11 1.78 8 12 Salsola ruthenica 2.57 10.78 0.31 4.56 6 13 Halogeton arachnoideus 2.06 6.20 0.08 2.79 7 Table 1. Impact of groundwater depth and soil salinity on riparian plant diversity and distribution in an arid area of China The important value index (IVI%), relative density (RD%), relative frequency (RF dominance/relative basal coverage (RDM%/RBC%) was calculated for each species at each l d ( ) ( ) Table 1. The important value index (IVI%), relative density (RD%), relative frequency (RF%), relative dominance/relative basal coverage (RDM%/RBC%) was calculated for each species at each tree, shrub and herb layer in 77 quadrats. IVITree =(RD + RF + RDM)/3, IVIShrub or herb =(RD + RF + RBC)/3. h ominance/relative basal coverage (RDM%/RBC%) was calculated for each species at each tree, shrub and herb ayer in 77 quadrats. IVITree =(RD + RF + RDM)/3, IVIShrub or herb =(RD + RF + RBC)/3. was positively associated with high soil pH in a riparian forest14,15. Therefore, it is necessary to understand the elationship between groundwater, soil salinity and the plant community in arid riparian ecosystems.h g y y y The Tarim River, located in the Tarim Basin, which is the most arid basin in China, is 1321 km long and is the second largest sandy desert on earth6,12. For the period from 1957 to 2000, the average annual inflow along the upper and lower reaches was 4.74 km3/a and 1.42 km3/a, respectively, while the environmental flow was 1.65 km3/a and 0.18 km3/a16, respectively. However, due to the severe misuse of water resources, the annual run-off in the upper reaches of the Tarim River has declined17, causing a reduction in the groundwater level in the upper and lower reaches18. The forests along the lower reaches have already been strongly degraded or even completely destroyed19. To restore and reconstruct the natural degraded arid riparian ecosystems, a 1.8 billion US dollar water diversion project has been invested in by the Chinese government since 2000. The restoration effort has been successful within 800 m from the river channel20. The groundwater depth declined from 12.6 m to 5.5–6.2 m between 2000 and 2015 in the lower reaches21. The riparian ecosystem plays a significant role instabilizing the water balance of the desert oasis and limiting desertification22,23. Many studies have examined the relationship between community and groundwater depth in the lower reaches of the Tarim River6,11,18. For example, Hao et al.6 found that richness and diversity declined with increasing groundwater depth. Li et al.24 found that the commu- nity structure changed from trees/shrubs/herbs to trees/shrubs when the groundwater depth increased from shal- low to deep. Impact of groundwater depth and soil salinity on riparian plant diversity and distribution in an arid area of China Although it is well known that groundwater has a significant influence on plant diversity, there have been few studies on how groundwater and soil salinity impact the plant community in desert riparian ecosystems. Furthermore, the upper reaches, constituting the core area of the Tarim River riparian zone, are less well studied25. The objectives of the present study are (1) to characterize the plant composition and community along the upper reaches of the Tarim River and (2) to determine the influences of groundwater depth and soil salinity on the plant communities. Our study provides a scientific foundation for informing government decisions related to pp g p The objectives of the present study are (1) to characterize the plant composition and community along the upper reaches of the Tarim River and (2) to determine the influences of groundwater depth and soil salinity on the plant communities. Our study provides a scientific foundation for informing government decisions related to ecological protection in arid riparian regions. Results Pl t Plant community composition. The plant composition categories in the upper reaches of the Tarim River included trees, shrubs, and herbs (Table 1). Twenty-two species were found in the 77 investigated quadrats: 2 tree species, 7 shrub species, and 13 herbaceous species. In the tree layer, the relative density, relative frequency, and relative dominance of P. euphratica were larger than those of P. pruinosa, and the importance value of P. euphratica was 80.31%. In the shrub and herbaceous layers, T. ramosissima and P. australis possessed the largest importance values (65.71% and 26.67%, respectively). Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ Figure 1. (a) location of the study area, and (b) spatial distribution of quadrats for five pant community classes as determined using the TWINSPAN clustering classifier in upper reaches of Tarim River. The source of map was from the resource and environment data cloud platform. The URL for the source of the map is http://www. resdc.cn/data.aspx?DATAID=184. Figure 1. (a) location of the study area, and (b) spatial distribution of quadrats for five pant community classes as determined using the TWINSPAN clustering classifier in upper reaches of Tarim River. The source of map was from the resource and environment data cloud platform. The URL for the source of the map is http://www. resdc.cn/data.aspx?DATAID=184. Figure 1. (a) location of the study area, and (b) spatial distribution of quadrats for five pant community classes as determined using the TWINSPAN clustering classifier in upper reaches of Tarim River. The source of map was from the resource and environment data cloud platform. The URL for the source of the map is http://www. resdc.cn/data.aspx?DATAID=184. Figure 2. Dendrogram of TWINSPAN analysis classification of 77 quadrats in upper reaches of Tarim River. Arabic numbers in solid rectangle represent individual quadrats and arabic numbers in the dashed rectangle represent plant community classifications. Figure 2. Dendrogram of TWINSPAN analysis classification of 77 quadrats in upper reaches of Tarim River. Arabic numbers in solid rectangle represent individual quadrats and arabic numbers in the dashed rectangle represent plant community classifications. Classification of the plant communities. Five plant community classes were identified using TWINSPAN (Figs. 1b and 2; Fig. A1). Class 1: P. euphratica + Tamarix spp., L. ruthenicum, H. caspica, H. haloden- dron + A. sparsifolia, K. caspia, H. strobilaceum, C. sibiricum, P. hendersonii, I. salsoloides, and H. polydichotoma. P. euphratica (tree layer), Tamarix spp., L. ruthenicum, H. caspica, H. Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w Results Pl t halodendron (shrub layer) and the herbaceous layer constitute the plant community (Table 2). Class 1 includes eight subclasses, with 17 plant quadrats that were mainly distributed in the direction of the oasis, close to the river channel (Fig. 1b). y ( g ) Class 2: Populus spp.+ Tamarix spp.+ A. sparsifolia, H. polydichotoma, K. caspia, C. pseudophragmites, P. hen- dersonii, and P. australis. P. euphratica and P. pruinosa (tree layer), Tamarix spp. (shrub layer) and the herbaceous layer constitute the plant community (Table 2). Class 2 includes seven subclasses, with 29 plant quadrats that were mainly distributed in the direction of the desert, close to the river channel (Fig. 1b).l y g Class 3: Populus spp. + T. ramosissima + A. sparsifolia, G. inflata, and A. venetum. Populusspp. (tree layer), T. ramosissima (shrub layer), and few herbaceous plants constitute the plant community (Table 2). Class 3 includes 10 subclasses, with 17 plant quadrats that were mainly distributed an average distance of approximately 5 km away from the river channel (Fig. 1b). g Class 4: Tamarix spp., H. ammodendron + H. arachnoideus, and S. ruthenica. This plant community comprises Tamarix spp., H. ammodendron (shrub layer) and few herbaceous plants (Table 2). Class 4 includes six subclasses, with 10 plant quadrats that were mainly distributed an average distance of approximately 23 km away from the river channel (Fig. 1b). Results Pl t Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ Community classification Sub- classes Name Number of quadrats Class 1 1 Pop eup + Tam his, Tam ram, Lyc rut, Hal cas + Alh spa, Karcas, Hal str 1 2 Pop eup + Tam ram, Lyc rut, Hal cas + Alh spa, Karcas, Cyn sib 2 3 Pop eup + Tam ram, Lyc rut + Alh spa, Karcas, Hex pol 2 4 Pop eup + Tam ram, Lyc rut, Hal hal + Alh spa, Karcas, Hex pol, Poa hen, Apo ven, Inusal 2 5 Pop eup + Tam ram, Lyc rut + Alh spa, Karcas, Hex pol 1 6 Pop eup + Tam ram, Hal cas + Alh spa, Karcas 2 7 Pop eup + Tam ram, Hal cas + Hal str, Alh spa 3 8 Pop eup + Tam ram, Hal cas + Hal str 4 Class 2 1 Pop eup + Tam his, Tam ram + Alh spa, Hex pol, 4 2 Pop eup + Tamarc + Alh spa, Karcas, 2 3 Pop eup, Pop pru + Tam ram + Alh spa, Karcas,Poa hen, Cal pse, Inusal, Apo ven 2 4 Pop eup, Pop pru + Tam ram + Alh spa, Karcas, Phraus, Poa hen, Cal pse, Apo ven 2 5 Pop eup, Pop pru + Tam ram + Karcas, Phraus, Cal pse, Alh spa 11 6 Pop eup + Tam his + Alh spa, Karcas 4 7 Pop eup, Pop pru + Tam ram + Phraus 4 Class 3 1 Pop eup, Pop pru + Tam ram + Alh spa 1 2 Pop eup, Pop pru + Tam ram + Glyinf 4 3 Pop eup, Pop pru + Tam ram + Glyinf, Apo ven 1 4 Pop eup,Poppru + Tam ram + Apo ven 1 5 Pop eup + Tam ram + Glyinf 4 6 Pop eup + Tam ram + Alh spa 1 7 Pop eup + Tam ram + Alh spa, Apo ven 1 8 Pop eup, Pop pru + Alh spa, Glyinf 1 9 Pop eup, Pop pru + Apo ven 1 10 Pop eup + Glyinf 2 Class 4 1 Tam ram, Hal amm + Hal ara,Sal rut 3 2 Tam ram + Hal ara, Sal rut 4 3 Tam ram + Sal rut 1 4 Tam his + Hal ara, Sal rut 1 5 Tam his 1 Class 5 1 Pop eup + Hal ara, Sal rut 1 2 Pop eup + Sal rut 1 3 Pop eup + Hal ara 1 4 Sal rut 1 Table 2. Results Pl t Plant diversity index of different community category (a Shannon-Wiener index, b Simpson index, c Species evenness, d Species richness). igure 3. Plant diversity index of different community category (a Shannon-Wiener index, b Simpson index, c pecies evenness, d Species richness). Environmental parameters Class 1 Class 2 Class 3 Class 4 Class 5 DistR. km 2.01 ± 1.77b 2.06 ± 1.69b 4.96 ± 1.76b 23.56 ± 8.65a 22.69 ± 5.70a GWD. m 3.57 ± 0.68b 4.31 ± 1.46b 4.69 ± 1.49b 14.64 ± 5.63a 13.75 ± 6.24a GS (g/L) 1.34 ± 0.19c 1.51 ± 0.26c 1.57 ± 0.91c 3.55 ± 1.24b 5.20 ± 0.75a GEC (g/L) 1.42 ± 0.21c 1.51 ± 0.19c 1.74 ± 0.92c 3.63 ± 1.29b 5.52 ± 0.76a SM (%) 19.67 ± 2.17a 11.10 ± 2.73ab 10.59 ± 2.10ab 3.61 ± 1.51b 2.79 ± 1.14b TS (g/kg) 36.08 ± 8.34a 31.85 ± 9.30a 18.72 ± 2.93b 13.20 ± 2.56b 10.62 ± 1.28b PH 8.76 ± 0.31a 8.69 ± 0.26a 8.26 ± 0.29b 8.14 ± 0.29c 7.80 ± 0.04c EC (ms/cm) 10.26 ± 3.24a 8.91 ± 1.66a 3.88 ± 1.07b 3.31 ± 1.49b 2.61 ± 0.49b CO3 2− (g/kg) 0.02 ± 0.004a 0.02 ± 0.013a 0.00 ± 0.001b 0.00 ± 0.001b 0.00 ± 0.000b HCO3 − (g/kg) 0.20 ± 0.056a 0.21 ± 0.074a 0.22 ± 0.049a 0.20 ± 0.050a 0.18 ± 0.013a Cl− (g/kg) 15.37 ± 4.14a 12.64 ± 4.51a 8.43 ± 1.79b 3.57 ± 1.16c 2.31 ± 0.54c SO4 2− (g/kg) 13.44 ± 3.03a 12.09 ± 3.50a 7.56 ± 1.33b 5.02 ± 2.38b 4.81 ± 2.29b Ca2+ (g/kg) 2.19 ± 1.04a 2.04 ± 1.05a 0.63 ± 0.35b 1.62 ± 0.86ab 1.72 ± 0.44ab Mg2+ (g/kg) 0.37 ± 0.10a 0.47 ± 0.09a 0.20 ± 0.04a 0.19 ± 0.13a 0.16 ± 0.09a Na+ (g/kg) 4.06 ± 2.24a 4.04 ± 2.24a 1.53 ± 1.00b 2.44 ± 0.90b 1.29 ± 1.01b K+ (g/kg) 0.41 ± 0.34a 0.33 ± 0.27a 0.14 ± 0.12a 0.15 ± 0.08a 0.13 ± 0.10a Table 3. Groundwater and surface soil salinity of different community category. Letters above means represent the results of pairwise contrasts betweenthefive community classes. All data are mean±SD. Results Pl t Five plant community class identified using TWINSPAN, community compositio quadrats. Species names are abbreviated using the first three letters of genus and species na Table 2. Five plant community class identified using TWINSPAN, community composition and number of quadrats. Species names are abbreviated using the first three letters of genus and species names; full species names are listed in Table 1. Class 5: P. euphratica + H. arachnoideus, and S. ruthenica. This plant community comprises P. euphratica (tree layer) and few herbaceous plants (Table 2). Class 5 includes four subclasses, with four plant quadrats that were mainly distributed an average distance of approximately 22 km away from the river channel (Fig. 1b). Class 5: P. euphratica + H. arachnoideus, and S. ruthenica. This plant community comprises P. euphratica (tree layer) and few herbaceous plants (Table 2). Class 5 includes four subclasses, with four plant quadrats that were mainly distributed an average distance of approximately 22 km away from the river channel (Fig. 1b). Plant diversity and environmental factors under different plant community types. Significant differences in the Shannon-Weiner index, Simpson index, evenness index, richness index, groundwater depth, distance from the river channel, soil pH, electrical conductivity, total salt, CO3 2−, Cl−, and SO4 2− were found among the five plant community types (Fig. 3; Table 3). The values of the plant diversity indices, groundwater and soil salinity for each community were ranked from the highest to the lowest values. The plant diversity indices, soil moisture, pH, EC, TS, CO3 2−, Cl−, and SO4 2− were ranked as follows: class 1, class 2, class 3, class 4, and class 5; distance from the river channel and groundwater depth: class 4, class 5, class 3, class 2, and class 1; groundwater salinity: class 5, class 4, class 3, class 2, and class 1. DCCA analysis of the plant community and environmental factors. The results of the DCCA are displayed in ordination diagrams, with 77 quadrats or 22 species (Fig. 4). The triangles represent the spe- cies, and the vectors represent the 15 environmental parameters. The eigenvalues of the first two ordinations were 0.935 and 0.832. The first DCCA represents a gradient with increasing groundwater depth, distance from the river channel to the quadrat, and groundwater salinity, while soil moisture declines from left to right. The Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ Figure 3. Results Pl t DistR is distance from the river channel to the quadrat; GWD is the groundwater depth; GS is the groundwater salinity; SM is the soil moisture; TS is the soil total salt; EC is the soil electrical conductivity; GEC is the groundwater electrical conductivity, the conversion factor is 0.515. Table 3. Groundwater and surface soil salinity of different community category. Letters above means represent the results of pairwise contrasts betweenthefive community classes. All data are mean±SD. DistR is distance from the river channel to the quadrat; GWD is the groundwater depth; GS is the groundwater salinity; SM is the soil moisture; TS is the soil total salt; EC is the soil electrical conductivity; GEC is the groundwater electrical conductivity, the conversion factor is 0.515. corresponding plant communities shift from classes 1 and 2 to classes 5 and 4. This suggests that plant commu- nity changes from high water consumers to drought-tolerant species. The community structure shifts from a tree-shrub-herb structure to a tree-herb or shrub-herb structure. The dominant plant species changed from P. euphratica, T. ramosissima and L. ruthenicum to P. euphratica or T. hispida as the distance from the river channel increased (Fig. 4a; A1). The composition of the herbaceous species changed from P. australis, K. caspia, H. strobi- laceum and C. pseudophragmites to S. ruthenica and H. arachnoideus.h corresponding plant communities shift from classes 1 and 2 to classes 5 and 4. This suggests that plant commu- nity changes from high water consumers to drought-tolerant species. The community structure shifts from a tree-shrub-herb structure to a tree-herb or shrub-herb structure. The dominant plant species changed from P. euphratica, T. ramosissima and L. ruthenicum to P. euphratica or T. hispida as the distance from the river channel increased (Fig. 4a; A1). The composition of the herbaceous species changed from P. australis, K. caspia, H. strobi- laceum and C. pseudophragmites to S. ruthenica and H. arachnoideus. p p g The second DCCA represents a gradient within declining soil salt (pH, EC, TS, CO3 2−, Cl−, SO4 2−, Ca2+, Mg2+, Na+, and K+), while groundwater salinity increases from top to bottom. The plant communities changed from classes 2 and 1 to class 3. The community structure changed from a tree-shrub-herb structure to a tree-shrub struc- ture. The dominant species of the plant communities showed almost no change, but the soil salinity and groundwater salinity affected the herbaceous layer. Results Pl t DistR is distance from the river channel to the quadrat; GWD is the groundwater depth; GS is the groundwater salinity; SM is the soil moisture; TS is the soil total salt; EC is the soil electrical conductivity; GEC is the groundwater electrical conductivity, the conversion factor is 0.515. Environmental parameters Class 1 Class 2 Class 3 Class 4 Class 5 DistR. km 2.01 ± 1.77b 2.06 ± 1.69b 4.96 ± 1.76b 23.56 ± 8.65a 22.69 ± 5.70a GWD. m 3.57 ± 0.68b 4.31 ± 1.46b 4.69 ± 1.49b 14.64 ± 5.63a 13.75 ± 6.24a GS (g/L) 1.34 ± 0.19c 1.51 ± 0.26c 1.57 ± 0.91c 3.55 ± 1.24b 5.20 ± 0.75a GEC (g/L) 1.42 ± 0.21c 1.51 ± 0.19c 1.74 ± 0.92c 3.63 ± 1.29b 5.52 ± 0.76a SM (%) 19.67 ± 2.17a 11.10 ± 2.73ab 10.59 ± 2.10ab 3.61 ± 1.51b 2.79 ± 1.14b TS (g/kg) 36.08 ± 8.34a 31.85 ± 9.30a 18.72 ± 2.93b 13.20 ± 2.56b 10.62 ± 1.28b PH 8.76 ± 0.31a 8.69 ± 0.26a 8.26 ± 0.29b 8.14 ± 0.29c 7.80 ± 0.04c EC (ms/cm) 10.26 ± 3.24a 8.91 ± 1.66a 3.88 ± 1.07b 3.31 ± 1.49b 2.61 ± 0.49b CO3 2− (g/kg) 0.02 ± 0.004a 0.02 ± 0.013a 0.00 ± 0.001b 0.00 ± 0.001b 0.00 ± 0.000b HCO3 − (g/kg) 0.20 ± 0.056a 0.21 ± 0.074a 0.22 ± 0.049a 0.20 ± 0.050a 0.18 ± 0.013a Cl− (g/kg) 15.37 ± 4.14a 12.64 ± 4.51a 8.43 ± 1.79b 3.57 ± 1.16c 2.31 ± 0.54c SO4 2− (g/kg) 13.44 ± 3.03a 12.09 ± 3.50a 7.56 ± 1.33b 5.02 ± 2.38b 4.81 ± 2.29b Ca2+ (g/kg) 2.19 ± 1.04a 2.04 ± 1.05a 0.63 ± 0.35b 1.62 ± 0.86ab 1.72 ± 0.44ab Mg2+ (g/kg) 0.37 ± 0.10a 0.47 ± 0.09a 0.20 ± 0.04a 0.19 ± 0.13a 0.16 ± 0.09a Na+ (g/kg) 4.06 ± 2.24a 4.04 ± 2.24a 1.53 ± 1.00b 2.44 ± 0.90b 1.29 ± 1.01b K+ (g/kg) 0.41 ± 0.34a 0.33 ± 0.27a 0.14 ± 0.12a 0.15 ± 0.08a 0.13 ± 0.10a Table 3. Groundwater and surface soil salinity of different community category. Letters above means represent the results of pairwise contrasts betweenthefive community classes. All data are mean±SD. Discussion Th T The Tarim River is China’s largest river and is the world’s fifth largest endorheic river20. In the present study, 22 plant species were found in the upper reaches of the Tarim River, which is higher than the number of species recorded in the lower reaches33. The plant species richness in the Tarim River is similar to that in the Syr Darya and Amu Darya Rivers34,35 but is low compared to that in the Gurbantünggüt Desert of the Junggar Basin in China36. In this study, the plant importance value analysis showed that P. euphratica and T. ramosissima were the most significant species in the tree and shrub layers, respectively (Table 1). This suggests that P. euphratica and T. ramosissima are dominant species in the upper reaches of the Tarim River, which corroborates the study of Hao et al.33 in the lower reaches of the Tarim River. It is possible that P. euphratica and T. ramosissima utilize a “sit-and-wait” strategy to avoid the disturbances from river runoff, resulting in them being the dominant species in the riparian plant communities of the upper and lower reaches37. In the riparian forests of the upper reaches of the Tarim River, 2 trees, 7 shrubs and 13 herbaceous species were found during the survey. TWINSPAN successfully distinguished the riparian forests into five classes, which is greater than that recorded in the lower reaches of the Tarim River38. A partial overlap in species composition among the five classes was identified, indicating that some species exhibit broad environmental tolerance. For example, the keystone species P. euphratica and Tamarix spp. can exist from the riverside to the oasis and desert margins9,12. It is possible that P. euphratica and Tamarix spp. are flood-tolerant species37,39,40 and that they have evolved a unique allocation strategy that allows them to withstand flooding. For example, they often lose part of their aboveground biomass during flooding and increase the allocation of biomass to their roots during favorable times41. This supports the storage effect theory that carbohydrates stored in belowground tissue during favorable times allow the plants to survive flooding. Additionally, P. euphratica and Tamarix spp. are drought-tolerant species, and P. euphratica was found to growing in locations with a groundwater depth of up to 13 m (Table 3), which was in agreement with the finding of Gries et al.42 and Thomas et al.43. Tamarix spp. Results Pl t There were few herbaceous plants, such as A. venetum and G. inflata, in class 3. PCA of the environmental factors in the different plant communities. Groundwater depth, groundwater salinity, soil moisture and soil salinity in the five plant communities were assessed using PCA (Tables 4 and 5). Five principal components (g1, g2, g3, g4, and g5) were extracted with eigenvalues > 1.0, and their cumulative contribution rate reached 95.95%. The orders of the comprehensive appraisal value scores of the envi- ronmental factors were as follows: class 1 > class 2 > class 3 > class 4 > class 5 (Table 5), which is consistent with the plant diversity index result (Fig. 3). Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ Figure 4. DCCA analysis of data from 22 plant species in upper reaches of Tarim River. Species names are listed in Table 1. (a) species are shown as triangles and labeled with their first three letters of the generic name and first three letters of the specific name, and environment characteristics are shown as arrow (where the DistR is the distance from the river channel to the quadrat, GWD is the groundwater depth, GS is the groundwater salinity, SM is the soil moisture, TS is the soil total salt; EC is the soil electrical conductivity); (b) DCCA analysis of plant quadrats of different classes and environment characteristics. The quadrats are divided into five classes (same as Table 2). Figure 4. DCCA analysis of data from 22 plant species in upper reaches of Tarim River. Species names are listed in Table 1. (a) species are shown as triangles and labeled with their first three letters of the generic name and first three letters of the specific name, and environment characteristics are shown as arrow (where the DistR is the distance from the river channel to the quadrat, GWD is the groundwater depth, GS is the groundwater salinity, SM is the soil moisture, TS is the soil total salt; EC is the soil electrical conductivity); (b) DCCA analysis of plant quadrats of different classes and environment characteristics. The quadrats are divided into five classes (same as Table 2). Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w Discussion Th T were found growing in locations with a groundwater depth of more than 14 m and had a greater ability than that of the other species to extract water from a relatively dry soil5, which was in agreement with the results presented by Gries et al.42. y y g y Water availability plays an important role in the composition and distribution of plant communities, particu- larly in arid and semi-arid regions43. The DCCA indicated that the plant communities changed from classes 1and 2 to classes 5 and 4, transitioning from a tree-shrub dominated communities to a tree or shrub dominated com- munities as the water conditions changed from good to poor. The herbaceous species changed from P. australis, K. caspia, H. strobilaceum and C. pseudophragmites to S. ruthenica and H. arachnoideus as the distance from the river channel increased. This may be because herbaceous plants with shallow root systems are eliminated when the groundwater depth is too deep45,46. However, the herbaceous species S. ruthenica and H. arachnoideus can grow in desert habitats. These two herbaceous species exist under the dominant species P. euphratica and Tamarix spp., which have a significant “fertility island” effect44. For example, the plants trap nutrient rich sediments transported during floods, provide a sheltered microhabitat and reduce the surface temperature of the soil in the summer47. Therefore, P. euphratica and Tamarix spp.were the “nurse plants” for these two herbs. h p pp p In this study, the dominant species showed almost no change when the plant communities changed from classes 1 and 2 to class 3 as the soil salinity changed from high to low. This may be because the dominant species, P. euphratica and T. ramosissima, have deep roots and are able to access the less saline, shallow groundwater. However, soil salinity affected the herbaceous layer. This may be because the herbaceous plants may be more affected by changes in surface soil salinity because their roots are unable to access the less saline groundwater. There were few herbaceous plants, such as A. venetum and G. inflata, in class 3. A. venetum not only grows in class Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ Component Initial eigenvalues Extraction sums of squared loadings Eigenvalues (λ) Variance (%) Cumulative variance (%) Eigenvalues (λ) Variance (%) Cumulative variance (%) 1 DistR. km 7.28 46.86 46.86 7.28 46.86 46.86 2GWD. Discussion Th T m 4.19 15.21 62.07 4.19 15.21 62.07 3GS (g/L) 2.28 13.44 75.51 2.28 13.44 75.51 4 SM (%) 1.52 10.22 85.73 1.52 10.22 85.73 5 TS (g/kg) 1.19 8.60 95.95 1.19 8.60 95.95 6 PH 0.34 2.66 98.61 7 EC (ms/cm) 0.18 1.39 100.00 8 CO3 2− (g/kg) 4.75E-16 3.65E-15 100.00 9 HCO3 − (g/kg) 1.51E-16 1.16E-15 100.00 10 Cl− (g/kg) 1.34E-16 1.03E-15 100.00 11 SO4 2− (g/kg) − 5.20E-17 − 4.00E-16 100.00 12 Ca2+ (g/kg) − 1.45E-16 − 1.12E-15 100.00 13 Mg2+ (g/kg) − 1.92E-16 − 1.48E-15 100.00 14 Na+ (g/kg) − 2.99E-16 − 2.30E-15 100.00 15 K+ (g/kg) − 4.00E-16 - 3.08E-15 100.00 Table 4. Total variance explained and component matrixes (five principal component selected) for 15 parameters from the five plant communities using PCA. Table 4. Total variance explained and component matrixes (five principal component selected) for 15 parameters from the five plant communities using PCA. Class Principal component scores Comprehensive appraisal value (g) Rank g1 g2 g3 g4 g5 Class 1 17.04 0.82 3.20 7.61 1.89 9.03 1 Class 2 14.01 −1.04 3.38 −3.30 2.01 6.24 2 Class 3 9.27 −2.86 −1.80 −4.37 1.32 2.81 3 Class 4 −1.52 7.34 8.36 1.05 0.96 2.52 4 Class 5 −1.27 3.74 5.85 0.97 0.88 1.35 5 Table 5. Principal component score matrix of 15 parameters from five plant communities and their comprehensive appraisal value (g) of groundwater and soil salinity. Table 5. Principal component score matrix of 15 parameters from five plant communities and their comprehensive appraisal value (g) of groundwater and soil salinity. Table 5. Principal component score matrix of 15 parameters from five plant communities and their comprehensive appraisal value (g) of groundwater and soil salinity. 3 but is also found in classes 1 and 2. This result indicated that A. venetum is distributed widely across the study area. Therefore, the different soil salinity requirements (i.e., niche differences) of the herbaceous plant reflect the soil salinity can determine the distributions of the herbaceous plants.lf 3 but is also found in classes 1 and 2. This result indicated that A. venetum is distributed widely across the study area. Therefore, the different soil salinity requirements (i.e., niche differences) of the herbaceous plant reflect the soil salinity can determine the distributions of the herbaceous plants.lf Environmental variability is considered to have an important influence on species diversity due to its effects on plant growth, development, and regeneration37,45,48. Discussion Th T In this study, we analyzed the environmental characteristics of different plant communities using principal component analysis (PCA). The comprehensive appraisal value scores of the environmental factors of the five communities were ranked as follows: class 1 > class 2 > class 3 > class 4 > class 5. Plant diversity may change in response to environmental gradients49. The quadrats in classes 1 and 2 were mainly distributed close to the river; this area is associated with shallow groundwater depth, low groundwater salinity, and high soil moisture and soil salinity. These environmental factors have positive effects on species diversity14,20,21,49. It is also possible that the quadrats close to the river experienced flooding disturbances, and as plant diversity is highest at moderate flooding stress, this supports the intermediate disturbance hypothe- sis2,50. The quadrats in classes 4 and 5 were mainly distributed at the edge of the desert; this area is characterized by a deep groundwater depth, high groundwater salinity, and low soil moisture. These environmental factors have negative effects on species diversity10,38,43. The seedlings of the dominant species, P. euphratica and Tamarix spp., were mainly established in a moist environment near the river channel5, while almost no seedlings had estab- lished at the edge of the desert12. The spatial variation in key environmental variables resulted in different plant assemblages at the patch scale which contributes to plant diversity at larger spatial scales. Therefore, the environ- mental factors that are creating the habitat heterogeneity which in turn affects plant diversity.hih g g yf p y The extent of riparian vegetation has declined significantly in response to changes in the environment. The area of the tugai forest declined by 3.0 × 105 ha from 1958 to 1978 in the Tarim Basin and by 4.3× 105 ha from 1950 to 1998 in the Aral Sea Basin51. Furthermore, the P. euphratica forest has decreased from 5.4 × 104 hm2 to 0.67 × 104 hm224, and this species has been listed as an endangered national level three protected plant in China48. The tugai forests thus constitute a highly threatened ecosystem52. Plant species diversity and richness are consid- ered to be the primary objectives of successful restoration53. Our study demonstrates that the plant diversity indi- ces in classes 1 and 2 were higher than those in classes 4 and 5 (Fig. 3). Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w Material and methods Study area. In this study, the upper reaches of the Tarim River were selected as the study area (Fig. 1). The elevation ranges from 900 m to 1050 m above sea level; the annual precipitation ranges from 50 mm to 70 mm; and the annual pan evaporation is more than 2100 mm12. The average annual temperature is 10.6–11.5 °C, with a minimum and maximum temperature of −27.5 °C and 43.6 °C25, respectively. The vegetation mainly includes Populus euphratica, Tamarix spp., and Alhagi sparsifolia5,12. Plant quadrats and measurements. In this study, the survey work was performed in July 2016 in the upper reaches of the Tarim River. There are obvious differences in plant diversity from the river channel to the edge of the desert in this area12. The distance from the river channel to the edge of the desert is approximately 30 km12. Therefore, to fully understand the correlations between the plant assemblage and the environmental variables, 77 quadrats were investigated. Nuclear magnetic resonance (GMR, Vista Clara Inc., WA, USA) and ground penetrating radar (RIS-2K, IDS Ingegneria dei Sistemi S.p.A., Italy) were used to ascertain the ground- water depth. Groundwater salinity (GS) was determined based on the method reported in Zhou26. The size of the plant quadrats was 100 m × 100 m. Sixteen sub-quadrats of 25 m × 25 m were used for recording the characteris- tics of the tree and shrub plants in each plant quadrat. For example, the diameter of trees at breast height (DBH) (breast height = 1.3 m) was recorded for each tree (≥5 cm DBH)12. The height, width, and number of species were recorded for the tree layer and shrub layer. Four sampling quadrats of 5 m×5 m were used for recording the number, height, and width of herbs in each sub-quadrat (25 m ×25 m). A GPS was used to record the quadrat locations. Soil sampling and measurement. In each quadrat, the soil samples were randomly collected from five location in the upper 20 cm soil layer. The samples were air-dried and then passed through 2 mm sieves before the soil analyses. The soil pH, electrical conductivity (EC), and total salt (TS) were determined using a suspension of the soil sample and deionized water (ratio of 1: 5)27,28. Material and methods A glass electrode pH meter was used to determine the soil pH27, the dry residue method was used to determine the TS, anda conductivity meter was used to determine the EC28. The neutral double indicator method was used to test for bicarbonate (HCO3 −) and carbonate (CO3 2−). AgNO3 titration and EDTA indirect titration were used to determine sulfate (SO4 2−) and chloride (Cl−), respec- tively. Complexometry was used to determine the calcium (Ca2+) and magnesium (Mg2+), the flame photometer method was used to determine the sodium (Na+) and potassium (K+), and the soil moisture was determined by oven-drying the samples. Calculation of diversity. The plant species diversity was determined using the Simpson diversity index (DS)29, Shannon-Weiner diversity index (H)30, and Pielou evenness index (JSW)31. The following formulae were used: ∑ = − D P 1 (1) S i 2 ∑ = − H P P ln (2) i i = J H lnS / (3) SW (1) (2) (3) where S is the number of species, and N is the number of individuals of all the species in a community. In = P n N / i i , ni is the importance value of species i in a community, and N is the sum of the importance values of all the species. where S is the number of species, and N is the number of individuals of all the species in a community. In = P n N / i i , ni is the importance value of species i in a community, and N is the sum of the importance values of all the species. Calculation of the relationship between the environment and plant community. Two-Way indi- cator species analysis (TWINSPAN) method was used to identify the riparian plant communities based on the importance value of the species in all the quadrats7. The plant importance value was calculated according to the following equation7,12: = + + Importance value (relative density relativefrequency relative dominance)/3 (4 = + + Importance value (relative density relativefrequency relative dominance)/3 (4) (4) The diameter at breast height was used for the determination of the relative dominance of the trees, while basal coverage was used for the shrubs and herbs. TWINSPAN was performed using PC-ORD5.0. Detrended canonical correspondence analysis (DCCA) wasused to analyze the relationship between the environmental factors and the plant community composi- tion7. Two data matrices are required for DCCA. Discussion Th T This might indicate that the environmental factors of classes 1 and 2, such as groundwater depth, groundwater salinity, and soil moisture, were more suitable for plant growth than those of classes 4 and 5. Classes 1 and 2 were characterized as tree-shrub-herb structures, which are highly stable and have a stronger sand stabilization ability than that of classes 4 and 538. However, classes 4 and 5 were characterized as shrub-herb and tree-herb structures, respectively. These structures are also Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ effective at sand stabilization. Therefore, we suggest that to protect the riparian plant community, all habitats, rather than some, should be considered for conservation. Conservation managers need to ensure that a sufficient amount of habitat is maintained for the structural and functional sustainability of the riparian forest. This finding has great significance for the restoration and protection of damaged desert riparian ecosystems. effective at sand stabilization. Therefore, we suggest that to protect the riparian plant community, all habitats, rather than some, should be considered for conservation. Conservation managers need to ensure that a sufficient amount of habitat is maintained for the structural and functional sustainability of the riparian forest. This finding has great significance for the restoration and protection of damaged desert riparian ecosystems. Material and methods Oneisa species-by-quadrats matrix, and the other one is an environment-by-quadrats matrix. The ordination program CANOCO 4.5 was used to perform the DCCA7. The differences in the species diversity indices, groundwater and soil salinity between the five plant community classes analyzed here were compared individually using multiple comparisons [Tukey’shonest significant difference (HSD) tests at P < 0.05]. Scientific Reports \| (2020) 10:7272 \| https://doi.org/10.1038/s41598-020-64045-w www.nature.com/scientificreports/ www.nature.com/scientificreports/ Principal component analysis (PCA)32 method was used to assess the comprehensive appraisal value (g) of groundwater and soil salinity in different plant communities. The following formulae was used: ∑ ∑ =    = = g x x g / ) (5) i n i i n i i 1 1 ∑ ∑ =    = = g x x g / ) i n i i n i i 1 1 (5) where g is the value of the comprehensive appraisal of the environmental characteristic, n is the number of prin- cipal components, xi is the eigenvalue of the ith principal component, ∑= x x /i i n i 1 is the weighing factor of the ith principal component, and gi is the ith principal component score. All the principal components extracted from the variables with eigenvalues > 1.0 and a cumulative contribution rate of extraction ≥ 85% were retained32. 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Relationship between ecological stoichiometry and community diversity of plant ecosystems in the u reaches of the Tarim River, China. BioRxiv 2018, 432278 (2018). Acknowledgementsh g This study was funded by the Key National Natural Science Foundation project (41671030, U1403281), the This study was funded by the Key National Natural Science Foundation project (41671030, U1403281), the Chinese Academy of Sciences (CAS) Project (Y52410) and the project of Thousand Young Talents Program (Chinese Academy of Sciences) (Y772121). Author contributions Y.Z., C.Z. designed the study; Y.Z., C.Z., and F.S. performed the experiments; Y.Z. and C.Z. analyzed the data; and Y.Z., C.Z., F.S., M.S., G.L. and Y.L. wrote the manuscript. All authors read and approved the final manuscript. Y.Z., C.Z. designed the study; Y.Z., C.Z., and F.S. performed the experiments; Y.Z. and C.Z. analyzed the data; and Y.Z., C.Z., F.S., M.S., G.L. and Y.L. wrote the manuscript. All authors read and approved the final manuscript. Additional information Supplementary information is available for this paper at https://doi.org/10.1038/s41598-020-64045-w. Correspondence and requests for materials should be addressed to C.Z. Correspondence and requests for materials should be addressed to C.Z. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. The authors have no competing interests as defined by Nature Research, or other interests that might be perceived to influence the results and/or discussion reported in this paper. 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https://openalex.org/W2079826041	https://europepmc.org/articles/pmc4256574?pdf=render	English	null	Lactic acid bacteria and yeasts involved in the fermentation of <i>amabere amaruranu</i>, a Kenyan fermented milk	Food science & nutrition	2,014	cc-by	5,739	ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Lactic acid bacteria and yeasts involved in the fermentation of amabere amaruranu, a Kenyan fermented milk Bitutu Nyambane1,2, William M. Thari1, John Wangoh2 & Patrick M. K. Njage2 1Food Technology Division, Kenya Industrial Research and Development Institute, PO Box 30650-00100, Nairobi 2Department of Food Science, Nutrition and Technology, University of Nairobi, PO Box 29053-00100, Nairobi Lactic acid bacteria and yeasts involved in the fermentation of amabere amaruranu, a Kenyan fermented milk Bitutu Nyambane1,2, William M. Thari1, John Wangoh2 & Patrick M. K. Njage2 1Food Technology Division, Kenya Industrial Research and Development Institute, PO Box 30650-00100, Nairobi 2Department of Food Science, Nutrition and Technology, University of Nairobi, PO Box 29053-00100, Nairobi doi: 10.1002/fsn3.162 and Streptococcus thermophilus (El-Baradei et al. 2008). The most commonly encountered yeast genera were Saccharo- myces, Candida, and Trichosporon (Beukes et al. 2001; Lore et al. 2005; Njage et al. 2011). Correspondence Bitutu Nyambane, Food Technology Division, Kenya Industrial Research and Development Institute, PO Box 30650-00100, Nairobi. Tel: +254 720 777 154; Fax: +254 20 600 3493; E-mail: Bitutuo@yahoo.com Bitutu Nyambane, Food Technology Division, Kenya Industrial Research and Development Institute, PO Box 30650-00100, Nairobi. Tel: +254 720 777 154; Fax: +254 20 600 3493; E-mail: Bitutuo@yahoo.com E-mail: Bitutuo@yahoo.com Keywords Amabere amaruranu, container type, Enterobacteriaceae, identiﬁcation, lactic acid bacteria, traditional fermentation, yeasts and molds Indigenous fermented milk products contain microbiota composed of techno- logically important species and strains which are gradually getting lost with new technologies. We investigated the microbial diversity in amabere amaruranu, a traditionally fermented milk product from Kenya. Sixteen samples of the prod- uct from different containers were obtained. One hundred and twenty isolates of lactic acid bacteria (LAB) and 67 strains of yeasts were identiﬁed using API 50 CH and API 20 C AUX identiﬁcation kits, respectively. The average pH of all the traditional fermented samples was 4.00 0.93. Lactobacilli, yeasts, and molds as well as Enterobacteriaceae counts from the plastic containers were sig- niﬁcantly higher (P < 0.05) than those from gourd. Enterobacteriaceae were below 1.00 1.11 log10 cfu/mL in products from the gourds and 2.17 1.92 log10 cfu/mL from the plastic containers. The LAB species were identiﬁed as Streptococcus thermophilus (25%), Lactobacillus plantarum (20%), and Leuconostoc mesenteroides (20%). The predominant yeasts were Saccharomy- ces cerevisiae (25%), Trichosporum mucoides (15%), Candida famata (10%), and Candida albicans (10%). The type of vessel used for fermentation had no signif- icant inﬂuence on the type of isolated and identiﬁed species. The diverse mix- ture of LAB and yeasts microﬂora forms a potential consortium for further product innovation in amabere amaruranu and other fermented milk products. Food Science & Nutrition 2014; 2(6): 692– 699 Food Science & Nutrition 2014; 2(6): 692– 699 Food Science & Nutrition 2014; 2(6): 692– 699 Food Science & Nutrition 2014; 2(6): 692– 699 doi: 10.1002/fsn3.162 Funding Information Kenya Industrial Research and Development Institute (KIRDI) is hereby acknowledged for funding this study. Received: 17 February 2014; Revised: 4 June 2014; Accepted: 12 June 2014 Received: 17 February 2014; Revised: 4 June 2014; Accepted: 12 June 2014 Introduction Modern socio-economic changes mean that some tradi- tional technologies for the production of fermented foods might eventually be lost together with the associated microorganisms (Akabanda et al. 2013). This underscores the importance of studying indigenous fermented products for their microbiota which might yield technologically important species and strains. Microorganisms present in traditionally fermented milk products have been docu- mented in various studies (Gonfa et al. 1999; Beukes et al. 2001; Lore et al. 2005; El-Baradei et al. 2008; Mathara et al. 2008; Njage et al. 2011; Akabanda et al. 2013). The most predominant lactic acid bacterial (LAB) genera that were isolated from these products included Lactobacillus fermen- tum (Beukes et al. 2001), Lactobacillus plantarum (Mathara et al. 2008), Leuconostoc mesenteroides (Lore et al. 2005), Amabere amaruranu is a fermented milk product that is prepared by spontaneous fermentation of milk using a gourd made from the hollowed out fruit of Lagenaria spp. Amabere amaruranu is popular among members of Abagusii, who inhabit the Kisii highlands on the south- western part of Kenya. It is made from cow’s milk that is heated and held at boiling point for 10 min. The milk is then added to a small portion of fermented milk from a previous batch after cooling for 10–20 min and left to ferment at ambient temperature ranging from 10 to 32°C. Two types of containers are used for fermentation, gourd and plastic containers. Milk fermented using the gourd is more popular of the two. The product is white 692 Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. Mumbai, India), pH 7.0 using a stomacher (Stomacher- Bagmixer, Buch and Holm, Interscience, France) for 30 sec, at a preset speed. Ten-fold serial dilutions (101 to 108) were made with the same diluent and 1 mL was pour plated in duplicates on various media for enumera- tion of isolates as described by Harrigan and McCance (1998).Total viable counts were enumerated on pour plates of plate count agar (PCA) (Oxoid Ltd, Basingstoke, UK) and incubated at 30°C for 48 h. Mesophilic lactoba- cilli and Leuconostocs were enumerated on pour plates of de Man, Rogosa and Sharpe (MRS) agar (Oxoid) and incubated anaerobically at 35°C for 48 h, using the gas- generating kit anaerobic system (Oxoid). Lactococci were enumerated on M17 agar (Oxoid) followed by the anaero- bic method and incubated at 35°C for 48 h. Laboratory-based production of amabere amaruranu Discrete colonies from pour plates of the highest dilution of each media were selected and isolated based on their shape, size, colour, and gloss. The isolated colonies were puriﬁed by repetitive streaking thrice on isolation media and stored in 0.25 mol/L sucrose solution at 18°C until required for identiﬁcation. In addition, pure yeast cul- tures were also sub-cultured onto PDA slants, incubated at 25°C and stored at 4–6°C for further identiﬁcation up to the genus and species level. Amabere amaruranu was prepared using the traditional method. Brieﬂy, the milk was heated and held at boiling point for 10 min. The milk was then added to a small portion of fermented milk from a previous batch at 50°C and left to ferment at ambient temperature ranging from 18 to 32°C. A total of four laboratory produced samples were prepared, two using the gourds and the other two using the plastic containers, that had been collected from fermented milk processors and had previously been used for fermentation. The gourd that was used for fermenta- tion was prepared by the traditional milk processors and cleaned using pebbles and hot water before they were handed over. The plastic containers were cleaned using hot water . In the laboratory, all the vessels were cleaned using boiling hot water and left to dry at ambient tem- perature. The pH and microbial counts (total viable counts, LAB, Enterobacteriaceae, and yeasts and molds) were determined after 0, 4, 8, 24, 48, 72, and 96 h. All the laboratory experiments were replicated twice. Sample collection A total of 16 samples of traditionally prepared amabere a- maruranu were collected from several randomly identiﬁed milk processors in the Kisii Region. Eight samples were obtained from the traditional gourd and eight of the sam- ples were from plastic containers. The milk was collected in sterile bottles and transported to the Kenya Industrial Research and Development Institute (KIRDI) microbiology laboratories in a cool box and stored at 4–6°C before analy- sis. The samples were analyzed within 24 h of collection. Identiﬁcation of lactic acid bacteria and yeasts LAB strains were characterized according to methods rec- ommended by Harrigan and McCance (1998). All strains were subjected to Gram staining, microscopic cell mor- phology, the catalase test, growth at 15°C and 45°C, and gas production from glucose and arginine hydrolysis. The API 50 CH (BioMerieux, Marcy l’Etoile, France) strips were used to identify the LAB isolates to the species level. Primary classiﬁcation of yeast colonies from PDA plates was based on colony characteristics (color and shape), mode of vegetative propagation, formation of hyphae or pseudohyphae, and ascospore production. Identiﬁcation of the yeast isolates to the species level was done using API 20 C AUX (BioMerieux). APILAB PLUS V3.2.2 soft- ware database was used for interpretation of the results. Introduction Yeasts were enumerated on potato dextrose agar (PDA) (Oxoid), acid- iﬁed to pH 3.7 using 10% tartaric acid (Lobachemie), and incubated at 25°C for 5 days. Enterobacteriaceae were enu- merated on pour plates of violet red bile glucose agar (VRBGA) (Oxoid), after incubation at 37°C for 24 h. Typical Enterobacteriaceae consisted of red to dark purple pin-point colonies surrounded by a dark purple halo on VRBGA. in color, has a grain-like appearance, low viscosity, is lumpy in nature, and acidic in taste (O. Arasa, personal communication). There is no substantial information available concerning the microbiological analysis of amabere amaruranu. The objective of this study was to isolate, enumerate, and identify the dominant microorganisms in the fer- mented amabere amaruranu. Laboratory fermentation replicating the traditional fermentation was also carried out and monitored. Acidiﬁcation of amabere amaruranu There was no signiﬁcant difference (P > 0.05) in the mean pH between samples from the two fermentation vessels. The mean pH of the samples obtained from gourd was 4.54 0.88 while that of samples from plastic containers was 4.53 1.05 after 96 h of fermentation. The pH of the milk decreased from 5.84 0.10 to 3.64 0.45 in the gourd while it decreased from 6.10 0.01 to 3.44 0.55 in the plastic container after 96 h. The pH of the milk fermented in the plastic con- tainer decreased faster than that fermented using the gourd while milk fermented in the gourd emerged with a lower ﬁnal coliform count than the plastic container. However, there was no signiﬁcant difference between the two ﬁnal counts (P > 0.05) after 96 h. The milk fer- mented in the gourd was expected to have a higher load of Enterobacteriaceae, yeasts, and LAB considering the dif- ﬁculties experienced while cleaning it. Enumeration of microorganisms Twenty ﬁve milliliters of each sample was homogenized in 225 mL sterile diluent (0.1% bacteriological peptone (Hi- media, Mumbai, India) and 0.85% NaCl (Loba Chemie, 693 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. Statistical analysis The data on microbial counts were ﬁrst transformed by a logarithmic (log10) transformation before computing the mean log10 count and standard deviations. The indepen- dent t-test was used to determine whether or not a signif- icant difference existed between the milk fermented using the gourd and that fermented using a plastic container, with respect to the pH and microbial counts. The level of signiﬁcance was 5%. Statistical analysis was done using Microsoft Excel 2010. The microbiological study of amabere amaruranu revealed the dominance of LAB in laboratory samples during the entire duration of fermentation, although yeasts were also present in considerably high numbers. Figure 1 shows the changes on average in the microbial numbers and pH during laboratory fermentation using gourds and plastic vessels. LAB counts increased from an initial level of 6.42 0.01 in the gourd to 8.32 0.45 log10 cfu/mL, while they increased from 5.41 0.53 to 7.86 0.50 log10 cfu/mL in the plastic container after 96 h of fermen- tation. The yeast population in both containers also increased with time from 5.50 at 0 h to 6.65 log10 cfu/mL at 96 h in the gourds and from 4.98 0.19 at 0 h to 7.62 0.50 log10 cfu/mL in the plastic vessels at 96 h. There was no signiﬁcant difference (P > 0.05) in the LAB and yeast counts between the two fermentation vessel types after 96 h of fermentation. ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Enumeration of microorganisms associated with amabere amaruranu 694 Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. (A) (B) Figure 1. Changes on average in microbial numbers and pH during 96 h laboratory fermentation using (A) gourd and (B) the plastic containers. (A) (B) Figure 1. Changes on average in microbial numbers and pH during 96 h laboratory fermentation using (A) gourd and (B) the plastic containers. Figure 1. Changes on average in microbial numbers and pH during 96 h laboratory fermentation using (A) gourd and (B) the plastic containers Figure 1. Changes on average in microbial numbers and pH during 96 h laboratory fermentation using (A) gourd and (B 8.93 log10 cfu/mL in suusac while Mathara et al. (2008) found average values of 8.0 log10 cfu/mL in the study of kule naoto. Beukes et al. (2001) found mean counts rang- ing from 7.05 to 7.7 log10 cfu/mL in traditionally fer- mented milk in South Africa. respectively, to almost undetectable levels after 24 h, with a decrease in pH of both products to below 4.5. A decrease in pH occasioned by the production of organic acids in fermented milk products leads to inhibition of Escherichia coli and other coliforms (Gran et al. 2003). The yeast population increased steadily from 5.50 to 6.65 log10 cfu/mL in the gourd, while they increased from 6.65 to 7.62 log10 cfu/mL in the plastic container. The yeast counts recorded in this report were in similar range to those reported by Mathara et al. (2008) who found yeasts counts of <1.0–7.4 log10 cfu/mL. The presence of yeast in traditionally fermented milk products, in varying numbers, has been reported elsewhere (Beukes et al. 2001; Lore et al. 2005; Kebede et al. 2007; Njage et al. 2011). The frequent concurrence of LAB and yeasts has led to the suggestion that there could be interactions that may inﬂuence the product characteristics and quality (Narvhus and Gadaga 2003). However, after 48 h, there was an emergence of Entero- bacteriaceae colonies, as identiﬁed by colony morphology, in both vessels and their population increased remarkably for the rest of the fermentation process. High prevalence of Enterobacteriaceae was also reported by Mathara et al. (2008) in the study of kule naoto. When the backslope method was used, acid-resistant strains of E. coli and coli- forms may be present in the inoculum, which may explain the high numbers of E. Enumeration of microorganisms associated with amabere amaruranu Table 1 shows total viable counts, LAB, yeasts and molds, and Enterobacteriaceae in the ﬁeld samples. High total viable counts were observed in all the samples. There was no signiﬁcant difference (P > 0.05) in the numbers of total viable microorganisms and the LAB between the samples from gourds and those from plastic containers. However, the total viable counts, yeasts and molds as well as Enterobacteriaceae counts from the plastic contain- ers were signiﬁcantly higher (P < 0.05) than those from gourds. While Enterobacteriaceae were less than 1.00 1.11 log10 cfu/mL in the samples from the gourds, they were present in those from plastic containers at 2.17 1.92 log10 cfu/mL. During the laboratory fermentation, the ﬁnal LAB counts for the milk fermented using the gourd at 96 h were 8.32 0.45 log10 cfu/mL, which reﬂected the growth of the LAB present in the backslope (8.3 0.51 log10 cfu/ mL). This was also observed in the plastic container where the LAB counts in the backslope (fermented milk from a previous batch) were 7.25 0.50 log10 cfu/mL, and this increased to 7.86 0.50 log10 cfu/mL in the ﬁnal labora- tory fermented milk. Table 1. Microbiological counts of samples from traditionally fermented amabere amaruranu. Microbial group Log count (log10 cfu/mL) Gourds (n = 8) Plastic containers (n = 8) Total viable counts 8.06 0.59 8.24 0.40 Lactobacilli 8.08 0.531 7.26 0.591 Lactoccoci 7.85 0.67 7.12 0.95 Yeasts and molds 4.65 1.041 6.07 0.521 Coliforms 0.59 1.111 2.17 1.921 1Signiﬁcant difference between samples from gourd and those from plastic containers. Table 1. Microbiological counts of samples from traditionally fermented amabere amaruranu. The high numbers of LAB, accompanied by the low pH observed in samples obtained from the ﬁeld and dur- ing the laboratory replication process (pH 3.64 for the gourd and pH 3.44 for the plastic) may be responsible for the sour taste, ﬂavor and unique aroma of the product. The production of lactic acid gives the fermented product a sour taste and results in the formation of a coagulum (Narvhus and Gadaga 2003). The LAB counts (105 to 107) are similar in range to other studies on traditionally fermented milk products. Lore et al. (2005) found counts ranging from 6.77 to 1Signiﬁcant difference between samples from gourd and those from plastic containers. ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Enumeration of microorganisms associated with amabere amaruranu coli and coliforms in the fermented product (Gran et al. 2003). Enterobacteriaceae are normally associated with poor hygiene and their presence may be a pointer toward a potential health risk. Dirar (1993) observes that lack of pasteurization in traditionally fermented milk products is a major risk-enhancing factor. In our study, even though Enterobacteriaceae decreased from 4.73 and 4.32 log10 cfu/mL in the gourd and plastic container, 695 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. Table 2. Phenotypic properties of lactic acid bacteria from amabere amaruranu. Table 2. Phenotypic properties of lactic acid bacteria from amabere amaruranu. Property Group 1 2 3 4 5 6 7 Cell morphology Cocci Cocci Rods Rods Rods Rods Cocci/Rods CO2 from glucose + + V NH3 from arginine Growth at 15°C + + V Growth at 45°C + + + + V Substrate fermentation D-arabinose + a Ribose + + + a D-xylose + + a Galactose + + + + a Glucose + + + + + + a Fructose + + + + + + a Mannose + + + + W a Rhamnose a Mannitol + a Esculin + + + + a Salicin + a Cellobiose + + + a Maltose + + + + + a Lactose + + + + + + a Melibiose + + + a Sucrose + + + + a Trehalose + + + + a Melezitose + a D-rafﬁnose + + + + a 2-keto-glucose a Identity Streptococcus thermophilus (30 isolates) Leuconostoc mesenteroides subsp. Mesenteroides (24 isolates) Lactobacillus plantarum (24 isolates) Lactobacillus bulgaricus (18 isolates) Lactobacillus helviticus (6 isolates) Lactobacillus fermentum (6 isolates) Not identiﬁed (12 isolates) +, positive reaction; , negative reaction; w, weak reaction; v, variable reaction. +, positive reaction; , negative reaction; w, weak reaction; v, variable reaction. Enumeration of microorganisms associated with amabere amaruranu belong to three genera namely Lactobacillus (45%), Strep- tococcus (25%), and Leuconostoc (20%). the milk is boiled for prolonged periods of time, this is insufﬁcient to minimize the risk of contamination, coli- forms were still detected, an indication of postheat treat- ment contamination. The most frequently isolated species comprising of 25% of all LAB was S. thermophilus, which was evenly distrib- uted in samples from both containers. These organisms were characterized by the lens-shaped colonies growing on M17 agar and could not grow at 15°C but were able to grow at 45°C. Tests using API 50 CH galleries identi- ﬁed these strains at 90% level of certainty as S. thermophi- lus. It was observed that the plastic container began to accumulate high levels of gases and swell, and this coin- cided with the rapid increase in the numbers of the atypi- cal coliforms. Atypical coliform colonies were not detected in the ﬁrst 24 h. This could also be due to the presence of coliforms, such as Enterobacter aerogenes which produces a frothy product (Nout 1994). Streptococcus thermophilus has been reported to play a prominent role in the fermentation of dairy products. It was also reported as the most dominant species in the fermentation of zabady a fermented milk product from Egypt (El-Baradei et al. 2008) and ergo, a traditional fer- mented milk product from Ethiopia (Gonfa et al. 1999). ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Identiﬁcation of lactic acid bacteria Table 2 provides a summary of the phenotypic properties of LAB that were isolated from amabere amaruranu sam- ples. All the isolates that were Gram positive, catalase negative, were rod or coccus shaped, and were considered to be LAB. They were further identiﬁed and shown to Lactobacillus bulgaricus subsp. bulgaricus made up 15% of the total number of lactic acid bacterial isolates. Strep- tococcus thermophilus in combination with Lactobacillus 696 Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. B. Nyambane et al. Table 3. Phenotypic properties of yeasts isolated from amabere amaruranu. Property Yeast isolates Group 1 Group 2 Group 3 Group 4 Group 5 Colony color White White White White White Ascospores Present Absent Absent Absent Absent Budding cells Present Present Present Absent V Hyphae/pseudohyphae Present Present Absent Present V Substrate fermentation Glucose + + + + + Glycerol + + + 2-keto-D-gluconate + + + L-arabinose + + D-Xylose + + + Adonitol + + + Xylitol + + + Galactose + + + + + Inositol + + Sorbitol + + + a-methyl-D-glucoside + + + N-acetyl-glucosamine + + + + Cellobiose + + + Lactose + + + Maltose + + + + + Sucrose + + + Trehalose + + + Melezitose + + + Rafﬁnose + + Identity Saccharomyces cerevisiae (17 isolates) Trichospora mucoides (10 isolates) Candida famata (7 isolates) Candida albicans (7 isolates) Low discrimination (26 isolates) +, positive reaction; , negative reaction; v, variable reaction. Table 3. Phenotypic properties of yeasts isolated from amabere amaruranu. +, positive reaction; , negative reaction; v, variable reaction. was the most dominant lactobacilli in kule naoto. It was also isolated by Beukes et al. (2001) from South African traditional fermented milk products and Lore et al. (2005) from suusac. Lactobacillus plantarum is commonly associated with plant-based fermentations (Holzapfel 1997) such as production of lactic acid in pickles and sauerkraut (Stiles and Holzapfel 1997). Their presence in fermented milk products could be due to the use of plant materials such as the gourd for fermentation and the adaptation of strains to milk (Mathara et al. 2008). ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Identiﬁcation of yeasts Table 3 provides a summary of the phenotypic properties of the yeasts isolated from amabere amaruranu samples. All the yeast species isolated in this study were present in samples were obtained from both the gourds and the plastic containers. This shows uniformity in the microbial diversity in the milk fermented using both containers. No molds were detected. The source of yeast in traditionally fermented milk products could be contamination from the environment and also from the equipment associated with milking and processing equipment, especially the fermentation vessel (Narvhus and Gadaga 2003). In a study conducted by Kebede et al. (2007), the type of container used for fer- mentation did not necessarily inﬂuence the yeast counts, but had an effect on the diversity of yeasts isolated from the different containers used. They established that a clay pot gave a product of diverse ﬂavors due to the many dif- ferent microorganisms isolated. The yeast species that were identiﬁed belonged to the genera Saccharomyces (25%), Candida (20%), and Tricho- sporon (15%). Similar genera of yeasts isolated from amabere amaruranu were reported by Njage et al. (2011) who characterized the yeasts associated with fermented camel milk. The most frequently isolated species included Saccharomyces cerevisiae (25%:55% from the gourd and 45% from the plastic container), Trichosporon mucoides (15%:55% for the gourd and 45% from the plastic con- tainer), Candida famata (10%:50% from the gourd and 50% from the plastic vessel) and Candida albicans (10%:50% from the gourd and 50% from the plastic ves- sel). These isolates were unambiguously identiﬁed using the API software database. Saccharomyces cerevisiae has been associated with the production of alcohols and other aroma compounds, stimulation of LAB, improvement of nutritional value, and inhibition of undesirable microorganisms (Jespersen 2003). However, the yeasts present in this product need to be investigated further to establish their exact role in the fermentation process, including their interaction with LAB and their metabolic properties. The presence of C. albicans is of particular concern since it is rarely isolated from fermented milk products. It is mostly known as an opportunistic pathogen that can cause superﬁcial, localized, and/or systemic infections in humans (Ryan 1990). Its presence in this product requires further investigations because its signiﬁcant growth pre- sents a safety concern. In the present study, S. cerevisiae isolates were able to ferment galactose, sucrose, rafﬁnose, and glucose but failed to ferment lactose, while they showed the presence of pseudohyphae. Identiﬁcation of yeasts Nonlactose fermenting yeasts, such as S. cerevisiae utilize galactose that is mainly secreted by most of the lactobacilli (Hickey et al. 1986). The API 20C AUX was able to identify 60% of the iso- lates accurately while the remaining isolates were assigned two to three yeast species at different identity levels. Simi- lar ﬁndings were reported by Njage et al. (2011). The fact that species that are found in milk might not be present in databases of commercial identiﬁcation methods such as API kits could explain the limited capacity of the com- mercial kits to identify the yeast isolates accurately (Njage et al. 2011). The API 20C AUX was unable to conclusively identify 40% of the yeast isolates and these were assigned pre- sumptively to a number of yeast species at different iden- tity levels. Four isolates showed similarities to C. albicans (78% identiﬁcation percentage) and also Candida krusei/ inconspicua (5.4% identiﬁcation percentage) from the API software database. Three other isolates were identiﬁed as Candida pelliculosa (61.6% identiﬁcation percentage) and Candida sphaerica (30.6% identiﬁcation percentage). The remaining isolates could not be accurately identiﬁed using API kits and were found to be either Cryptococcus lauren- tii (46.1% identiﬁcation percentage) Candida humicolus (29.5% identiﬁcation percentage) or T. mucoides (23.4% identiﬁcation percentage). Due to the limited capacity of API system to satisfactorily identify these strains, the use of molecular tools for more accurate typing of these organisms is recommended. Identiﬁcation of lactic acid bacteria Lacto- bacillus plantarum is also homofermentative, fermenting lactose to produce lactic acid as the main metabolic prod- uct. This could suggest that it also plays a signiﬁcant role in lactic fermentation of amabere amaruranu. delbrueckii subsp. bulgaricus play a key role in the fermentation of yoghurt and need to be viable and abun- dant in the ﬁnal product (CAC 1994). Leuconostoc mesenteroides subsp. Mesenteroides consisted of 20% of all the isolates. It was also evenly distributed in samples from the gourd and plastic containers. Leuconos- toc mesenteroides subsp. mesenteroides fermented most of the sugars present except D-Arabinose, rhamnose, manni- tol, salicin, melezitose, and 2-keto-gluconate. It was simi- larly one of the most frequently isolated species in suusac, a traditional fermented camel milk product (Lore et al. 2005). It has also been isolated from nunu (Akabanda et al. 2013), a Ghanaian fermented milk product. Leuco- nostoc spp. are able to convert citrate into aroma com- pounds such as acetoin and diacetyl (Lore et al. 2005). This may indicate an important functional characteristic of this organism in amabere amaruranu. A further 5% of the isolates were identiﬁed as Lactoba- cillus helveticus and were isolated from milk samples obtained from the plastic container. Lactobacillus helveti- cus has been associated with the production of bacterioc- ins that inhibit the growth of Staphylococcus aureus, Salmonella species, and E. coli (Stiles and Holzapfel 1997). It has also been isolated from nunu (Akabanda et al. 2013). Another highly prevalent Lactobacillus in amabere a- maruranu was L. plantarum, which comprised 20% of the isolates. A total of 60% of the L. plantarum isolates were from the gourd while the rest were from plastic containers. Mathara et al. (2008) found that L. plantarum 697 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. T. mucoides, C. incospicua, and C. famata were found in suusac (Njage et al. 2011). In the present study, the most dominant species was S. cerevisiae. T. mucoides, C. incospicua, and C. famata were found in suusac (Njage et al. 2011). 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FEMS Microbiol. Rev. 87:175–188. Nout, M. J. R. 1994. Fermented foods and food safety. Food Res. Int. 27:291–298. J. Food Microbiol. 121:295–301. Gilliland, S. E. 1990. Health and nutritional beneﬁts from lactic acid bacteria. FEMS Microbiol. Rev. 87:175–188. Ryan, K. J. 1990. Candida and other opportunistic Fungi. Pp. 651–657 in J. C. Sherris ed., Medical microbiology: an introduction to infectious diseases, Elsevier Science Publishing Co. Inc., New York, NY. Gonfa, A., A. Fite, K. Urga, and B. A. Gashe. 1999. The microbiological aspects of ergo (ititu) fermentation. SINET. Ethiop. J. Sci. 22:2. Stiles, M. E., and W. Holzapfel. 1997. Lactic acid bacteria of food and their current taxonomy. Review. Int. J. Food Microbiol. 36:1–27. Gran, H. M., H. T. Gadaga, and J. A. Narvuhs. 2003. Conclusions Microorganisms involved in amabere amaruranu fermen- tation were found to consist of LAB from the three gen- era Lactobacillus, Leuconostoc, and Streptococcus. The most prevalent genus was Lactobacillus while the most domi- nant species were S. thermophilus and L. mesenteroides subsp. Mesenteroides. Yeasts included Candida, Saccharo- myces and Trichosporon. The most predominant yeast spe- cies was S. cerevisiae. These microorganisms should be tested for their technological properties, microbial interac- tions and possible inhibitory effects against spoilage and pathogenic microorganisms. This will enable product development and innovation for a more predictable fer- The presence of yeasts in traditionally fermented milk products has been reported by many investigators (Lore et al. 2005; Njage et al. 2011; Akabanda et al. 2013). They consist of lactose fermenting and nonlactose fermenting species (Gilliland 1990). The predominant yeast species encountered in nunu were S. cerevisiae and Pichia kudriavzevii (Akabanda et al. 2013), whereas S. cerevisiae, 698 Lactic Acid Bacteria and Yeasts in amabere amaruranu B. Nyambane et al. mentation and quality using starter cultures of mixed LAB and yeast cultures. amasi, a Zimbabwean naturally fermented raw milk fermented product. Int. J. Food Microbiol. 88:19–28. amasi, a Zimbabwean naturally fermented raw milk fermented product. Int. J. Food Microbiol. 88:19–28. Harrigan, W. F., and M. E. McCance. 1998. Laboratory methods in food and dairy microbiology. Academic Press, London. Acknowledgments Hickey, M. W., A. J. Hillier, and G. R. Jago. 1986. Transport and metabolism off lactose, glucose and galactose by homofermentative lactobacilli. Appl. Environ. Microbiol. 51:825–831. Kenya Industrial Research and Development Institute (KIRDI) is hereby acknowledged for funding this study. The authors also thank all the families and individuals from Kisii who took part in the survey exercise for their enthusiasm and all the invaluable information they pro- vided. The authors also extend their gratitude to Obedy Arasa (Ministry of Agriculture) and Samuel Warui (KIRDI) for their inputs. Holzapfel, W. 1997. Use of starter cultures in fermentation on a household scale. Food Control 8:241–258. Jespersen, L. 2003. Occurrence and taxonomic characteristics of strains of Saccharomyces cerevisiae predominant in African indigenous fermented foods and beverages. FEMS Yeast Res. 3:191–200. Conﬂict of Interest Kebede, A., B. C. Viljoen, T. H. Gadaga, J. A. Narvhus, and A. Lourens-Hattingh. 2007. The effect of container type on the growth of yeast and lactic acid bacteria during production of sethemi, South African spontaneously fermented milk. Food Res. Int. 40:33–38. None declared. ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc. References Utilization of various starter cultures in the production of 699 ª 2014 The Authors. Food Science & Nutrition published by Wiley Periodicals, Inc.
https://openalex.org/W4393040093	https://www.nature.com/articles/s41598-024-57180-1.pdf	English	null	Publisher Correction: Proteomics of appetite-regulating system influenced by menstrual cycle and intensive exercise in female athletes: a pilot study	Scientific reports	2,024	cc-by	602	www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Publisher Correction: Proteomics of appetite‑regulating system influenced by menstrual cycle and intensive exercise in female athletes: a pilot study Kazuhiro Tanabe , Kayoko Kamemoto , Yoshimasa Kawaguchi , Kai Fushimi , Sing Ying Wong , Nodoka Ikegami , Mikako Sakamaki‑Sunaga & Nobuhiro Hayashi Correction to: Scientific Reports https://doi.org/10.1038/s41598-024-54572-1, published online 20 February 2024 In the original version of this Article a previous rendition of Figure 4 was published. The original Fig. 4 and accompanying legend appear below. The original Article has been corrected. EN Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Publisher Correction: Proteomics of appetite‑regulating system influenced by menstrual cycle and intensive exercise in female athletes: a pilot study OPEN In the original version of this Article a previous rendition of Figure 4 was published. The original Fig. 4 and accompanying legend appear below. \| https://doi.org/10.1038/s41598-024-57180-1 Scientific Reports \| (2024) 14:6798 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Heatmap analysis and transition pattern classification. (A) Heatmap analysis depicting the expression levels of 511 proteins across 50 samples: red: up-regulated, green: down-regulated. Proteins and samples were categorized by cluster analysis. (B) Three orthogonal transition bases obtained by PCA analysis. (C) Contributions of each principal components to the original data. (D) Transitional pattern heatmap with cluster analysis; Each protein transition pattern during exercise dissolved into three basic patterns, and the cosines to the three bases were used for categorization. Cosines were further converted to RGB colors to visualize the categorization. Figure 4. Heatmap analysis and transition pattern classification. (A) Heatmap analysis depicting the expression levels of 511 proteins across 50 samples: red: up-regulated, green: down-regulated. Proteins and samples were categorized by cluster analysis. (B) Three orthogonal transition bases obtained by PCA analysis. (C) Contributions of each principal components to the original data. (D) Transitional pattern heatmap with cluster analysis; Each protein transition pattern during exercise dissolved into three basic patterns, and the cosines to the three bases were used for categorization. Cosines were further converted to RGB colors to visualize the categorization. https://doi.org/10.1038/s41598-024-57180-1 Scientific Reports \| (2024) 14:6798 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2024 https://doi.org/10.1038/s41598-024-57180-1 Scientific Reports \| (2024) 14:6798 \|
https://openalex.org/W4391279553	https://comserva.publikasiindonesia.id/index.php/comserva/article/download/1159/1483	Indonesian	null	Penolakan Fraksi PKS dalam Pengesahan Revisi RUU IKN : Studi Kasus Optimalisasi Wewenang Fraksi dalam Legislasi	COMSERVA	2,024	cc-by-sa	4,362	ABSTRAK Fraksi partai politik dalam Dewan Perwakilan Rakyat merupakan sebuah institusi partai politik yang juga menjadi alat bagi partai politik untuk memperjuangkan nilai-nilai dasar masing-masing partai. Fraksi parati politik haruslah menjadi wakil dari kepentingan rakyat. Fungsi dan kewenangan fraksi telah diatur untuk mengoptimalisasikan kinerja mereka bukan hanya untuk partai masing-masing tapi juga untuk kepentingan rakyat. Penolakan fraksi PKS terhadap pengesahan RUU IKN merupakan wujud kineraja Fraksi PKS sebagai institusi dari partai politik, di mana partai politik menjadi wadah bagi aspirasi rakyat. Dalam penelitian ini, peneliti memfokuskan pada peran dan fungsi fraksi dalam berbagai keputusan legislasi. Tujuan penelitian ini untuk meneliti dan menganalisis secara mendalam peran Fraksi Partai dalam proses pengesahan RUU, khususnya terkait Revisi UU IKN. Fokus pada pemahaman bagaimana fraksi-fraksi di DPR RI berkontribusi dalam penetapan keputusan pengesahan atau penolakan RUU dan Meneliti dengan cermat alasan yang diutarakan oleh Fraksi PKS dalam menolak Revisi UU IKN. Penelitian ini menggunkan metode kualitatif dengan menggunakan berbagai literatur untuk mendeskripsikan fungsi dan wewenang fraksi partai politik dalam menggunakan haknya sebagai wakil rakyat yang berorientasi pada kepentingan rakyat. Dalam hal penolakan Fraksi Partai PKS terhadap revisi UU IKN, merupakan sebuah wujud di mana fraksi parti di DPR-RI memiliki wewnang penuh dalam menentukan arah mereka, hal tersbeut dilakukan sebagai wujud bahwa fraksi merupakan kepanjangan tangan dari partai politik yang mewakili berbagai kepentingan rakyat. Kata Kunci: Fraksi Partai, Fraksi PKS, Revisi UU IKN, Optimalisasi, Kewenangan. Kata Kunci: Fraksi Partai, Fraksi PKS, Revisi UU IKN, Optimalisasi, Kewenangan. Penolakan Fraksi PKS dalam Sidang Paripurna Pengesahan Revisi RUU IKN: Sebuah Studi Kasus terhadap Optimalisasi Kewenangan Fraksi dalam Bidang Legislasi 3658 Email:Muhammadaqshabs25@gmail.com Correspondence: Muhammad Aqsha DOI: 10.59141/comserva.v3i09.1159 Kata Kunci: Fraksi Partai, Fraksi PKS, Revisi UU IKN, Optimalisasi, Kewenangan. e-ISSN: 2798-5210 p-ISSN: 2798-5652 ejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation Muhammad Aqsha Universitas Indonesia, Indonesia Email:Muhammadaqshabs25@gmail.com Correspondence: Muhammad Aqsha PENDAHULUAN Pengesahan perubahan RUU atas Undang-Undang Nomor 3 Tahun 2022 tentang Ibu Kota Nusantara menjadi Undang-Undang melalui Rapat Paripurna DPR RI Ke-7 Masa Persidangan I Tahun Sidang 2023-2024. Pengesahan UU tersebut disetujui oleh tujuh Fraksi Partai yang berada di DPR RI, satu Fraksi menyetujui dengan catatan sedangkan satu Fraksi lagi menolok. Tujuh Fraksi yang menyetujui pengesahan tersebut adalah Fraksi Partai Golkar, PDIP, PKB, Partai Gerindra, Partai NasDem, PPP, PAN dan Satu Partai yang menyetujui dengan catatan adalah Partai Demokrat. Sedangkan PKS merupakan satu-satunya partai yang menolak keputusan tersebut. Penolakan PKS terhadap pengesahan UU tersebut, merupakan sebuah wewenang Fraksi yang dimiliki oleh fraksi manapun (Tweedie, 2008). Fraksi memiliki kedudukan yang amat penting dalam segala pengembilan keputusan penetapan Undang-Undang (Ramadani, 2022). Namun, masih belum ada tata tertib dari DPR yang mengatur tentang tugas-tugas fraksi secara teknis, tidak adanya petunjuk operasional yang rinci tentang bagaimana fraksi melaksanakan tugasnya sesuai dengan tugas dan wewenangnya dalam membantuk pengoptimalan kewenangan DPR. Penetapan Undang-Undang ditetapkan disahkan, ketika mayoritas anggota parlemen telah menyetujui rancangan undang-undang tersebut. Para anggota yang tidak memberi suara persetujuannya,maka secara langsung tidak menghendaki isi undang-undang tersebut (Hans Kalsen 2011). Keberadaan fraksi memiliki peran dalam setiap agenda Dewan Perwakilan Rakyat, terutama dalam fungsi legislasi (Isra, 2010). Akan tetapi, fraksi bukan hanya sebagai pelengkap DPR, lebih dari itu fraksi memiliki peran yang sangat dominan dalam penentuan proses dan substansi Rancangan Undang-Undang (Asmawi & Faizin, 2017). Tata tertib DPR tahun 2009 menyatakan, fraksi dapat menolak RUU dalam sidang paripurna penyempurnaan RUU, bahkan dalam penolakannya tidak disertakan alasan penolakan fraksi. Sebelum rapat Paripurna penentapan RUU menjadi UU. Terlebih dahulu, ada pembahasan Rancangan Undang- Undang, pengambilan keputusan dilakukan oleh fraksi pada pembahasan tingkat pertama, yang biasanya dilakukan secara tertutup. Kecuali, jika ada hal-hal yang tidak dapat disepakati, pembahasan tingkat kedua hanya formalitas berupa pembacaan pandangan umum fraksi. Rancangan Undnag-Undang yang berisikan revisi terhadap UU telah ditolak oleh Fraksi PKS sejak awal mula proses pembahasannya yaitu pada proses rapat tertutup yang hanya dihadiri oleh anggota fraksi dan menolak untuk dilanjutkan pada pembahasan kedua (Zulkarnain, 2019). Sesuai dengan Pasal 135 ayat 6 Tata Tertib DPR tahun 2009 Fraksi ditempatkan dalam tempat yang strategis pada pembahasan proses legislasi yaitu di tahapan pembahasan RUU. Fraksi mempunyai kewenangan yang luas mulai dari pembahasan RUU, daftar inventarisasi masalah sampai pada tingkat persetujuan (Melati, 2023). ABSTRACT The political party faction in the House of Representatives is a political party institution which is also a tool for political parties to fight for the basic values of each party. Also, political party factions must represent the interests of the people. The functions and authorities of factions have been regulated to optimize their performance not only for their respective parties but also for the interests of the people. The PKS faction's rejection of the revision of the IKN law is a manifestation of the PKS faction's performance as a political party institution, where political parties are a forum for the aspirations of the people. In this research, researchers focus on the role and function of factions in various legislative decisions. As well as, highlighting the points that are rejected by PKS as a party faction within the DPR RI, carrying out its function as a representative of the party that has been elected by the people to voice their rights. This research Penolakan Fraksi PKS dalam Sidang Paripurna Pengesahan Revisi RUU IKN: Sebuah Studi Kasus terhadap Optimalisasi Kewenangan Fraksi dalam Bidang Legislasi 3658 3658 q Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation uses qualitative methods using various literature to describe the function and the authority of political party factions to exercise their rights as representatives of the people oriented towards the interests of the people. Keywords: Party Faction, PKS Faction, Revision of the IKN Law, Optimalitation, Authority. Keywords: Party Faction, PKS Faction, Revision of the IKN Law, Optimalitation, Authority. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) PENDAHULUAN Maka, dalam hal ini keputusan penolakan Fraksi PKS terhadap Revisi UU IKN, sudah melalui proses semestinya dan telah menggunakan wewenangnya sebagai Fraksi dari partai yang mendapatkan kursi di DPR RI. OMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) 3659 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation Penolakan yang dilakukan oleh Fraksi PKS terhadap Pengesahan revisi UU IKN, dikarenakan Fraksi tersebut menganggap bahwa dalam salah satu pasal yang berada di dalam revisi UU IKN bertentangan dengan konstitusi dan mengganggap Revisi UU IKN berpihak kepada pemodal karena tidak adanya sanksi terhadap HGU di tanah IKN (Purnama & Fadli, 2007). Fraksi merupakan pengelompokan anggota dewan perwakilan rakyat baik ditingkatan pusat maupun daerah yang mencerminkan konfigurasi partai politik (Saleh et al., 2021) (Andriyansyah, 2020). Dalam sistem perwakilan di Indonesia, setiap anggota dewan harus menjadi anggota salah satu fraksi (Cahyawati et al., 2021). Pembentukan fraksi memudahkan anggota dewan dalam membuat model sebuah pengambilan keputusan di tingkat parlemen (Maarotong, 2020). Banyaknya anggota dewan di sebuah lembaga legislatif baik tingkat pusat maupun daerah, fraksi digunakan sebagai pengontrol vote di dalam pengambilan keputusan sehingga pengambilan keputusan akan lebih efektif dan efisien (Mubiina, 2020). Hal tersebut juga semakin mempermudah partai politik pemenang pemilu untuk mencapai tujuannya dalam melaksanakan tugas dan fungsinya Sebagai dasar hukum pembentukan fraksi terdapat dalam Pasal 80 Undang-Undang Nomor 27 tahun 2009 dalam pembentukan fraksi diperuntukkan dalam memaksimalkan kinerja DPR mengoptimalkan pelaksanaan fungsi, tugas dan wewenang DPR, serta hak dan kewajiban anggota DPR, dibentuk fraksi sebagai wadah dengan baik fraksi sebagai alat untuk mempersatukan para anggota partai yang sama dalam memperjuangkan kepentingan rakyat. Keberadaan fraksi diperkuat lagi dalam tata tertib DPR tahun 2009 dalam pasal 18 yang menyatakan fraksi dibentuk dalam optimalisasi dan keefektifan pelaksanaan tugas dan wewenang DPR serta hak dan kewajiaban anggota (Kelsen, 2011). Penelitian ini mencoba untuk melihat sejauh mana peran dan fungsi Fraksi Partai dalam tugas dan wewenangnya sebagai anggota DPR dan wakil partai. Mengingat dalam studi kasus pengesahan Revisi UU IKN hanya partai PKS yang menolak dengan tegas. Tujuan penelitian ini meneliti dan menganalisis secara mendalam peran Fraksi Partai dalam proses pengesahan RUU, khususnya terkait Revisi UU IKN. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) METODE Penelitian ini menggunakan metode penelitian kualitatif dengan menggunakan pendekatan studi literatur. Pendekatan studi literature akan memungkinkan penelitian ini untuk menganalisis isu yang ada dan mendalaminya melalui tinjauan pustaka yang komprehensif. Untuk mengumpulkan data dalam penulisan jurnal ini dilakukan dengan tahapan sebagai berikut: Identifikasi Sumber: Langkah awal dalam penulisan jurnal ini adalah mengidentifikasi sumber-sumberl iteratur yang relevan. Ini mencakup buku, makalah ilmiah, jurnal, tesis, dan sumber-sumber lain yang berkaitan dengan Fraksi Partai, keterwakilan rakyat, dan sistem politik Indonesia. Pengumpulan Data: Data diperoleh dari literatur yang Peneliti akan mengumpulkan informasi, kutipan dan temuan yang relevan dari literature untuk mendukung argumen dan analisis yang akan disajikan dalam jurnal. Analisis Data: Analisis data dalam ametode ini melibatkan evaluasi kritis terhadap temuan yang ada dalam literatur, mengidentifikasi pola-pola, persamaan, perbedaan, dan relevansi terhadap topic penelitian. Analisis ini akan membantu dalam mengembangkan kerangka teoritis dan argument jurnal. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) PENDAHULUAN Fokus pada pemahaman bagaimana fraksi- fraksi di DPR RI berkontribusi dalam penetapan keputusan pengesahan atau penolakan RUU, mengevaluasi proses pembentukan dan kewenangan Fraksi di DPR RI. memahami apakah tata tertib yang ada sudah memberikan panduan yang cukup jelas terkait tugas dan wewenang fraksi dalam pembentukan dan penetapan RUU, dan meneliti dengan cermat alasan yang diutarakan oleh Fraksi PKS dalam menolak Revisi UU IKN. Menyelidiki argumen-argumen yang mendasari keputusan penolakan tersebut, khususnya terkait dengan keberpihakan terhadap pemodal dan konstitusionalitas. Penelitian ini diharapkan dapat memberikan pemahaman yang lebih mendalam tentang peran fraksi-fraksi di DPR RI dalam proses pengesahan RUU. Ini dapat menjadi landasan untuk meningkatkan efektivitas fraksi dalam menjalankan tugas dan wewenangnya, Penelitian ini diharapkan dapat memberikan wawasan mendalam terkait alasan penolakan Fraksi PKS terhadap Revisi UU IKN. Informasi ini dapat menjadi dasar untuk perdebatan dan dialog lebih lanjut terkait substansi RUU, Hasil penelitian dapat menjadi dasar untuk mendorong evaluasi dan perbaikan tata tertib DPR RI, terutama yang berkaitan dengan tugas dan wewenang fraksi. Hal ini dapat membantu menciptakan pedoman yang lebih jelas bagi fraksi dalam menjalankan peran mereka dan penelitian ini dapat memberikan kontribusi pada pengembangan sistem perwakilan di Indonesia, khususnya terkait peran fraksi dalam pembahasan dan pengambilan keputusan terkait RUU. Diharapkan hasil penelitian dapat menjadi masukan untuk perbaikan kebijakan terkait fungsi fraksi di DPR. 3660 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation HASIL DAN PEMBAHASAN Peran dan Fungsi Fraksi Partai Politik dalam Pengambilan Keputusan Legislatif Peran dan Fungsi Fraksi Partai Politik dalam Pengambilan Keputusan Legislatif Hubungan antara Dewan Perwakilan Rakyat dan fraksi berlandaskan Undang-undang mengenai lembaga perwakilan rakyat yaitu Undang-Undang Nomor 2 Tahun 2018 Tentang Revisi Kedua terhadap Undang-Undang Nomor 17 Tahun 2014 Tentang Majelis Permusyawaratan Rakyat, Dewan Perwakilan Rakyat, Dewan Perwakilan Daerah dan Dewan Perwakilan Rakyat Daerah. Pasal 82 menyatakan bahwa: (1) Fraksi merupakan pengelompokan anggota berdasarkan konfigurasi anggota partai politik peserta pemilu; (2) Setiap anggota Dewan Perwakilan Rakyat harus menjadi anggota fraksi;Fraksi dibentuk oleh partai politik yang memenuhi ambang batas; (3) perolehan suara dalam penentuan perolehan kursi Dewan Perwakilan Rakyat; (4) Fraksi dibentuk untuk mengoptimalkan pelaksanaan fungsi, wewenang, tugas Dewan Perwakilan Rakyat serta hak dan kewajiban anggota Dewan Perwakilan Rakyat; (5) Fraksi didukung oleh sekertariat dan tenaga ahli; (6) Sekertariat jenderal Dewan Perwakilan Rakyat menyediakan sarana, anggaran dan tenaga ahli guna kelancaran pelaksanaan tugas fraksi; (7) Ketentuan lebih lanjut mengenai sarana dan tenaga ahli fraksi sebagaimana diatur dalam ayat (6) diatur dalam peraturan Dewan Perwakilan Rakyat. Selain Undang-Undang Nomor 2 Tahun 2018 Tentang Peubahan Kedua Atas Undang-Undang Nomor 17 Tahun 2014 Tentang Majelis Permusyawaratan Rakyat, Dewan Perwakilan Rakyat, Dewan Perwakilan Daerah dan Dewan Perwakilan Rakyat Daerah, ketentuan mengenai fraksi partai politik juga diatur dalam Peraturan Dewan Perwakilan Rakyat Republik Indonesia Nomor 2 Tahun 2018 Tentang Perubahan Ketiga Atas Peraturan Dewan Perwakilan Rakyat Republik Indonesia Nomor 1 Tahun 2014 Tentang Tata Tertib Dewan Perwakilan Rakyat, yang merumuskan bahwa ; Pasal 20 (1) Fraksi dibentuk untuk mengoptimalkan pelaksanaan fungsi, wewenang dan tugas Dewan Perwakilan Rakyat serta hak dan kewajiban anggota. (2) Fraksi dibentuk oleh partai politik yang memenuhi ambang batas perolehan suara dalam penentuan perolehan kursi Dewan Perwakilan Rakyat. (3) Fraksi juga dapat dibentuk oleh gabungan dari dua atau lebih partai politik sebagaimana dimaksud ayat (2). (4) Setiap anggota harus menjadi salah satu fraksi. (5) Fraksi bertugas mengkoordinasikan kegiatan anggotanya dalam melaksanakan wewenang dan tugas Dewan Perwakilan Rakyat serta meningkatkan kemampuan, disiplin, keefektifan dan efisiensi kerja anggotanya dalam melaksanakan tugas yang tercermin dalam setiap kegiatan Dewan Perwakilan Rakyat. (6) Fraksi melakukan evaluasi terhadap kinerja anggotanya dan melaporkan kepada public 3661 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation paling sedikit satu kali dalam satu tahun sidang. (7) Pimpinan fraksi di tetapkan oleh fraksinya masing- masing. HASIL DAN PEMBAHASAN (8) Fraksi membentuk aturan tata kerja internal sesuai dengan ketentuan peraturan perundang- undangan. Pasal 21 (1) Fraksi didukung oleh sekertariat dan tenaga ahli. (2) Sekertariat fraksi ditetapkan oleh sekertaris jenderal Dewan Perwakilan Rakyat dengan persetujuan pimpinan fraksi. (3) Tenaga ahli pada setiap fraksi paling sedikit sejumlah alat kelengkapan Dewan Perwakilan Rakyat dan mendapat tambahan secara proposional berdasarkan jumlah anggota setiap fraksi. (4) Rekrutmen tenaga ahli fraksi dilakukan oleh pimpinan fraksi dan hasil rekrutmen disampaikan kepada sekertaris jenderal Dewan Perwakilan Rakyat untuk ditetapkan dengan keputusan sekertaris jenderal Dewan Perwakilan Rakyat. (5) Rekrutmen tenaga ahli fraksi sebagaimana dimaksud pada ayat (4) didasarkan pada kompetisi keahlian yang ditentukan oleh pimpinan fraksi. (6) Fraksi mengajukan anggaran serta kebutuhan sekertariat dan tenaga ahli fraksi kepada Badan Urusan Rumah Tangga. (7) Badan Urusan Rumah Tangga meneruskan usulan fraksi sebagaimana dimaksud pada ayat (6) kepada sekertaris jenderal Dewan Perwakilan Rakyat untuk di tindak lanjuti. Berdasarkan Undang- Undang yang telah tercantum di atas, dapat diketahui bahwa fraksi partai politik merupakan pengelompokan anggota-anggota fraksi partai politik yang ada di dalam kelembagaan Dewan Perwakilan Rakyat. Mengartikan, bahwa, fraksi partai politik merupakan bentuk partai politik secara institusional dalam kelembagaan DPR atau dengan kata lain merupakan wakil partai politik untuk menyuarakan aspirasi masing-masing partai di dalam tubuh DPR. Maka, fungsi fraksi partai politik dalam pengambilan keputusan legislatif, memiliki wewenang penuh dalam setiap keputusannya. Setiap keputusannya menyarakan juga, suara partai yang diwakili oleh setiap anggota fraksi. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation Poin Penolakan Fraksi PKS dalam Pengesahan Revisi UU IKN Beberapa poin yang ditolak oleh Fraksi PKS, merupakan sebuah poin penolakan yang memiliki landasan cukup kuat. Hal tersebut menunjukkan bagaimana Fraksi PKS di DPR RI menjalankan fungsinya sebagai representasi partai. Di mana dalam keputusan Fraksi PKS mereka menggunkan fungsi mereka berupa wakil rakyat yang memperdulikan nasib kesejahteraan rakyat (Basir, 2022). Beberapa poin yang ditolak oleh Fraksi PKS adalah: Pertama, meneurut Fraksi PKS posisi IKN yang dicantumkan pada Pasal 6 UU 3/2022 berisikan, bahwa, posisi Ibukota Nusantara secara geografis berada pada lintang dan bujur yang telah ditentukan. Secara konseptual, memiliki pemahaman antara letak geografis dan astronomis. Lalu, jika menurut geografis, letak atau posisi suatu tempat terkait dengan kedudukannya di permukaan bumi. Lebih lanjut, dari segi astronomis posisi sebuah tempat dipandang berdasarkan garis lintang dan bujur. Maka, berdasarkan istilah yang telah digunakan, fraksi PKS memiliki kesimpulan masih ada kekeliruan yang perlu diselesaikan Kedua, wewenang khusus yang diberikan kepada Otorita IKN, tercantum dalam Pasal 12 ayat (1) UU 3/2022, terdapat kewenangan bagi urusan pemerintah pusat dan pemerintah daerah dalam hal pelaksanaan persiapan, pembangunan, dan pemindahan IKN. Serta penyelenggaraan pemerintah daerah khusus ibukota Nusantara kecuali oleh peraturan perundang-undangan ditentukan sebagai urusan pemerintahan secara absolut. Ketentuantersebut, oleh Fraksi PKS dianggap bertentangan dengan prinsip negara kesatuan sebagaimana diatur Pasal 1 ayat (1) dan prinsip penyelenggaraan pemerintah daerah sebagaimana daitur Pasal 18 UUD 1945. “Adanya klausul yang memberikan kewenangan kepada otorita IKN berupa pemberian fasilitas khusus kepada pihak yang mendukung pembiayaan 3662 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation dalam rangka kegiatan persiapan, pembangunan, dan pemindahan Ibu Kota Negara, serta pengembangan Ibu Kota Nusantara dan daerah mitra Ibu Kota Nusantara, sebagaimana tercantum dalam Pasal 12 ayat (2). Menurut Fraksi PKS, hal tersebut juga berpotensi terjadinya abuse of power dalam pasal kewenangan tersebut (Nugrohosudin, 2022). Ketiga, keberadaan Otorita IKN dalam pengelolaan aset IKN. adanya aset dalam penguasaan (ADP) Otorita IKN yakni tanah wilayah IKN yang tidak berhubungan langsung dengan lokasi penyelenggara pemerintahan IKN. Terhadap ADP tersebut, Pasal 14 Perpres 65/2022 mengatur lahan ADP yang ditetapkan pemerintah dikelola oleh Otorita IKN sesuai peraturan. Tanah ADP tersebut diberikan HPL (Hak Pengelolaan Tanah) kepada Otorita IKN. Pengelolaan tanah di IKN sangat erat kaitannya dengan HPL. Dalam hal ini, mengartikan, bahwa Otorita IKN sudah diberikan sepetak atau bagian dari hak menguasai negara yang selanjutnya disebut juga HMN (Harsono, 2018; Hutagalung & Sitorus, 2011; Sumardjono, 2008). COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) Poin Penolakan Fraksi PKS dalam Pengesahan Revisi UU IKN Hal tersebut, tentu saja memberikan beban kepada APBN 3663 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation di tengah pembangunan negara yang masih belum merata, namun sudah mengalokasikan anggaran untuk pembangunan yang masih belum tentu dirasakan manfaatnya oleh rakyat. di tengah pembangunan negara yang masih belum merata, namun sudah mengalokasikan anggaran untuk pembangunan yang masih belum tentu dirasakan manfaatnya oleh rakyat. COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) Poin Penolakan Fraksi PKS dalam Pengesahan Revisi UU IKN Keempat, perihal tata kelola pemberian hak atas tanah Otorita dalam kawasan IKN diantaranya berupa bentuk HAT di kawasan IKN, dan perpanjangan HAT di kawasan IKN. Otorita IKN berwenang dalam penyusunan perencanaan pemanfaatan, peruntukan, untuk penggunaan tanah sesuai dengan rencana tata ruang dan rencana induk IKN serta perincian rencana induk IKN. Maka, pada penyusunan rencana induk, Otorita IKN wajib memperhatikan dan mengacu pada Rencana Tata Ruang Kawasan Strategis Nasional (RTR KSN) IKN dan Rencana Detail Tata Ruang (RDTR) IKN. Perencanaan tata ruang dan tata peruntukan tanah yang baik di IKN oleh Otorita IKN, memiliki pengaruh untuk memberikan kepastian arah pembangunan kota dan berpeluang menarik penanam modal, untuk penanam modal lokal maupun penanam modal luar negeri ke IKN. Kelima, peraturan jangka waktu hak atas tanah dan memiliki penambahan untuk jangka waktunya. Hal tersebut, berupa HGU bertambah dari 90 menjadi 95 tahun HGB. Hal tersebut, tentu saja menampakkan keberpihakan pemerintah pada pemilik modal dengan memanjakan investor dan tentu saja, mengenyampingkan kepentingan rakyat yang lebih luas. Juga, tidak sesuai dengan semangat yang tercantum dalam UU No.5 Tahun 1960 tentang Peraturan Dasar-Dasar Pokok Agraria yang menyatakan dengan jelas, bahwa pemberian hak dilakukan secara berangsur dan bersyarat. Fraksi partai PKS melihat pemberian konsesi langsung dalam satu kali tahapan 95 tahun lalu, pada siklus berikutnya ditambah 95 tahun, sehingga total mendapat konsesi waktu 190 tahun. Begitu juga pemberian HAT dalam bentuk hak pakai yang berkonsesi total mencapai 160 tahun. Pemberian konsesi itu juga tidak memiliki mekanisme kontrol berupa pemberian sanksi dan pencabutan hak serta evaluasi yang jelas kepada pemegang HGU dan hak pakai. Keenam, terkait dengan pendanaan untuk persiapan, pembangunan, dan pemimdahan Ibu Kota Negara. Serta penyelenggaraan pemerintahan daerah khusus Ibu Kota Nusantara. Ketujuh, terkait pendanaan, khususnya soal pembiayaan utang Ibu Kota Nusantara. Kedelapan, terkait persiapan, pembangunan, pemindahan, dan penyelenggaraan pemerintahan Ibukota Nusantara (IKN) menjadi program prioritas nasional selama 10 tahun, fraksi PKS menolak ketentuan itu. Sebab pembangunan Ibukota Nusantara berpotensi menambah beban beat APBN, utang negara, sehingga menjadi masalah bagi pemerintahan berikutnya. Pada poin keenam, ketujuh dan kedelapan, penolakan dari Fraksi PKS cukup jelas, dikarenakan dalam ketiga poin tersbeut menyebutkan perihal pendanaan, 53,5 persen dan 46,5 persen sisanya menggunakan dana lain-lain yang bersumber dari kerja sama pemerintah dan badan usaha (KPBU), BUMN, serta swasta (Website IKN, 2022). Optimalisasi Kewenangan Fraksi Parti dalam Bidang Legislasi Konteks menghubungkan aspirasi dan kehendak rakyat dengan penyelenggaraan negara, partai politik merupakan wadah dalam proses pemilihan umum, yang salah satunya untuk memilih wakil rakyat yang duduk di DPR. Juga, dipahami bahwa partai politik adalah kendaraan bagi masyarakat untuk berpartisipasi dalam proses yang demokratis (pemilihan umum), yang jika terpilih maka akan menduduki jabatan atau keanggotaan tertentu, seperti DPR, DPRD, Presiden dan/atau Wakil Presiden, Gubernur, dan Bupati serta Walikota. Hal yang demikian sejalan dengan apa yang dikemukakan oleh Ramlan Surbakti (Surbakti, 1992), bahwa Fungsi utama partai politik ialah mencari dan mempertahankan kekuasaan guna mewujudkan program- program yang disusun berdasarkan ideologi tertentu. Penjelasan tersebut merupakan sebuah cara yang digunakan oleh partai politik di dalam sistem politik demokrasi dalam proses mendapatkan dan mempertahankan kekuasaan dengan keikutsertaan dalam pemilihan umum (Marijan, 2019). Saat partai politik melaksanakan fungsinya dalam sistem politik demokrasi beberapa hal yang harus dilakukan, yaitu melakukan rekruitmen, kampanye, dan melaksanakan fungsi pemerintahan. Hal tersebut dilakukan dalam rangka, menunjang pelaksanaan kedaulatan rakyat yang berorientasi pada kepentingan rakyat. Fraksi yang ada di dalam kelembagaan Dewan Perwakilan Rakyat merupakan bagian dari struktural partai politik, bukan hanya perpanjangan tangan partai politik, namun juga alat perjuangan partai politik yang memiliki kursi di Dewan Perwakilan Rakyat yang mana komposisi pengurus fraksi diangkat, disahkan dan diberhentikan oleh ketua umum partai politik. Bahkan pengurus partai politik yang bukan anggota Dewan Perwakilan Rakyat dapat memimpin rapat, memberi arahan, memberi pendapat dan kebijakan fraksi dari partai politik. Oleh karena itu, dalam memberikan arahan maupun kebijakan untuk mengoptimalkan pelaksanaan fungsi Dewan Perwakilan Rakyat, fraksi partai politik dituntut untuk memepentingkan kepentingan rakyat. Keberadaan fraksi dalam kelembagaan DPR, terkait juga dengan fungsi partai politik itu sendiri. Sejalan dengan hal tersebut, Miriam Budiardjo mengemukakan (Budiardjo, 1994), bahwa pada umumnya partai politik juga diharapkan akan melaksanakan fungsi seperti dinegara yang sudah mapan kehidupan politiknya. Diharapkan menjadi alat penting untuk mengorganisir kekuasaan politik, mempengaruhi keputusan-keputusan pemerintah serta turut melaksanakannya, menghubungkan secara efektif masyarakat umum dengan proses politik, merumuskan aspirasi dan tuntutan rakyat serta memasukannya kedalam proses pembuatan keputusan. 3664 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation SIMPULAN Fraksi merupakan sebuah wadah berhimpunnya anggota dewan yang mempunyai tanggung jawab besar dalam menampung segala aspirasi rakyat atau konstitunenya. Anggota dewan dituntut untuk mengambil keputusan atas nama rakyat karena mereka telah secara langsung dipilih oleh rakyat sebagai konstituen mereka. Fraksi mempunyai peran yang sangat strategis dalm mendukung pelaksanaan fungsi-fungsi dewan di DPR. Dukungan peran dan kinerja fraksi yang dilakukan secara efektif akan dapat membantu memaksimalkan pelaksanaan fungsi-fungsi anggota dewan dalam bidang legislasi. Mulai dari dari tahap awal penjaringan aspirasi dan turun ke daerah-daerah pada masa reses yang menghasilkan DIM (Daftar Inventarisasi Masalah) hingga pembahasan sampai penentuan keputusan legislasi melibatkan peran fraksi. 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Sekretariat Jenderal, Dewan Perwakilan Rakyat COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) 3666 Muhammad Aqsha Rejection of PKS Faction in Ratification of the Revision of the IKN Bill: Case Study of Optimizing Faction Authority in Legislation COMSERVA: (Jurnal Penelitian dan Pengabdian Masyarakat) - Vol. 3 (9) Januari 2024 - (3658-3667) Republik Indonesia Bekerjasama Zulkarnain, N. (2019). Analisis Siyasah Dusturiyah Terhadap Kewenangan Dewan Perwakilan Daerah (Dpd) Dalam Proses Legislasi Menurut Uu Nomor 17 Tahun 2014. Iain Padangsidimpuan. © 2024 by the authors. Submitted for possible open access publication under the terms and conditions of the Creative Commons Attribution (CC BY SA) license (https://creativecommons.org/licenses/by-sa/4.0/). 3667
https://openalex.org/W2141976382	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_7_from_Osteoblast-Derived_Factors_Induce_an_Expression_Signature_that_Identifies_Prostate_Cancer_Metastasis_and_Hormonal_Progression/22379859/1/files/39825258.pdf	Lithuanian	null	Osteoblast-Derived Factors Induce an Expression Signature that Identifies Prostate Cancer Metastasis and Hormonal Progression	Cancer research	2,009	cc-by	633	VEGF STK6 SMC4L1 PLK1 MCM5 MALT1 HECA ACPP Benign LocCaP MetCaP ASNS Benign LocCaP MetCaP AURKB Benign LocCaP MetCaP BCAP29 Benign LocCaP MetCaP BIRC5 Benign LocCaP MetCaP BUB1 Benign LocCaP MetCaP CCNA2 Benign LocCaP MetCaP CCNB1 Benign LocCaP MetCaP CCNB2 Benign LocCaP MetCaP CCNE2 Benign LocCaP MetCaP CDC2 Benign LocCaP MetCaP CDC6 Benign LocCaP MetCaP CDKN3 Benign LocCaP MetCaP CHEK1 Benign LocCaP MetCaP CRADD Benign LocCaP MetCaP DEPDC1 Benign LocCaP MetCaP ESPL1 Benign LocCaP MetCaP F2R Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP BUB1B Benign LocCaP MetCaP C6orf210 Benign LocCaP MetCaP CDC20 Benign LocCaP MetCaP CDC25C Benign LocCaP MetCaP GTSE1 Benign LocCaP MetCaP HCAP-G Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP ACPP CCNB1 CDC20 CHEK1 STK3 SMC4L1 HCAP-G ESPL1 AD AI AD AI AD AI AD AI AD AI AD AI AD AI AD AI VEGF STK6 SMC4L1 PLK1 MCM5 MALT1 HECA ACPP Benign LocCaP MetCaP ASNS Benign LocCaP MetCaP AURKB Benign LocCaP MetCaP BCAP29 Benign LocCaP MetCaP BIRC5 Benign LocCaP MetCaP BUB1 Benign LocCaP MetCaP CCNA2 Benign LocCaP MetCaP CCNB1 Benign LocCaP MetCaP CCNB2 Benign LocCaP MetCaP CCNE2 Benign LocCaP MetCaP CDC2 Benign LocCaP MetCaP CDC6 Benign LocCaP MetCaP CDKN3 Benign LocCaP MetCaP CHEK1 Benign LocCaP MetCaP CRADD Benign LocCaP MetCaP DEPDC1 Benign LocCaP MetCaP ESPL1 Benign LocCaP MetCaP F2R Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP BUB1B Benign LocCaP MetCaP C6orf210 Benign LocCaP MetCaP CDC20 Benign LocCaP MetCaP CDC25C Benign LocCaP MetCaP GTSE1 Benign LocCaP MetCaP HCAP-G Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP ACPP CCNB1 CDC20 CHEK1 STK3 SMC4L1 HCAP-G ESPL1 AD AI AD AI AD AI AD AI AD AI AD AI AD AI AD AI VEGF STK6 SMC4L1 PLK1 MCM5 MALT1 HECA ACPP Benign LocCaP MetCaP ASNS Benign LocCaP MetCaP AURKB Benign LocCaP MetCaP BCAP29 Benign LocCaP MetCaP BIRC5 Benign LocCaP MetCaP BUB1 Benign LocCaP MetCaP CCNA2 Benign LocCaP MetCaP CCNB1 Benign LocCaP MetCaP CCNB2 Benign LocCaP MetCaP CCNE2 Benign LocCaP MetCaP CDC2 Benign LocCaP MetCaP CDC6 Benign LocCaP MetCaP CDKN3 Benign LocCaP MetCaP CHEK1 Benign LocCaP MetCaP CRADD Benign LocCaP MetCaP DEPDC1 Benign LocCaP MetCaP ESPL1 Benign LocCaP MetCaP F2R Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP BUB1B Benign LocCaP MetCaP C6orf210 Benign LocCaP MetCaP CDC20 Benign LocCaP MetCaP CDC25C Benign LocCaP MetCaP GTSE1 Benign LocCaP MetCaP HCAP-G Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP P P MetCaP ASNS Benign LocCaP MetCaP AURKB Benign LocCaP MetCaP BCAP29 Benign LocCaP MetCaP BIRC5 Benign LocCaP MetCaP BUB1 Benign LocCaP MetCaP 2 MetCaP CCNB1 Benign LocCaP MetCaP CCNB2 Benign LocCaP MetCaP CCNE2 Benign LocCaP MetCaP CDC2 Benign LocCaP MetCaP CDC6 Benign LocCaP MetCaP BUB1B Benign LocCaP MetCaP C6orf210 Benign LocCaP MetCaP CDC20 Benign LocCaP MetCaP CDC25C Benign LocCaP MetCaP VEGF STK6 SMC4L1 PLK1 MCM5 MALT1 HECA ACPP n LocCaP MetCaP ASNS Benign LocCaP MetCaP AURKB Benign LocCaP MetCaP BCAP29 Benign LocCaP MetCaP BIRC5 Benign LocCaP MetCaP BUB1 Benign LocCaP MetCaP CCNA2 n LocCaP MetCaP CCNB1 Benign LocCaP MetCaP CCNB2 Benign LocCaP MetCaP CCNE2 Benign LocCaP MetCaP CDC2 Benign LocCaP MetCaP CDC6 Benign LocCaP MetCaP CDKN3 n LocCaP MetCaP CHEK1 Benign LocCaP MetCaP CRADD Benign LocCaP MetCaP DEPDC1 Benign LocCaP MetCaP ESPL1 Benign LocCaP MetCaP F2R Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP BUB1B Benign LocCaP MetCaP C6orf210 Benign LocCaP MetCaP CDC20 Benign LocCaP MetCaP CDC25C Benign LocCaP MetCaP GTSE1 Benign LocCaP MetCaP HCAP-G Benign LocCaP MetCaP Benign LocCaP MetCaP Benign LocCaP MetCaP A C6orf210 HCAP-G HECA B P CCNB1 CDC20 CHEK1 STK3 SMC4L1 HCAP-G ESPL1 AI AD AI AD AI AD AI AD AI AD AI AD AI AD AI
https://openalex.org/W2161859419	https://dash.harvard.edu/bitstream/1/8156564/1/2628696.pdf	English	null	Egg Consumption and Risk of Type 2 Diabetes in Men and Women	Diabetes care	2,009	cc-by	6,799	Citation Djoussé, Luc, J. Michael Gaziano, Julie E. Buring, and I-Min Lee. 2009. Egg consumption and risk of type 2 diabetes in men and women. Diabetes Care 32(2): 295-300. Published Version doi://10.2337/dc08-1271 http://nrs.harvard.edu/urn-3:HUL.InstRepos:8156564 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Egg Consumption and Risk of Type 2 Diabetes in Men and Women JULIE E. BURING, SCD1,2,4,5 I-MIN LEE, MBBS, SCD2,5 JULIE E. BURING, SCD1,2,4,5 I-MIN LEE, MBBS, SCD2,5 LUC DJOUSS´E, MD, DSC1 1 2 3 Study and a 49% increased risk of CHD among women in the Nurses’ Health Study, compared with intake of less than one per week. Furthermore, we have re- ported similar ﬁndings in U.S. male phy- sicians with type 2 diabetes but not in those without type 2 diabetes (7), sug- gesting that frequent egg consumption may have negative health effects among individuals with type 2 diabetes. How- ever, it is not known whether egg con- sumption increases the risk of type 2 diabetes itself. In animal experiments, a diet rich in fat has been shown to induce hyperglycemia and hyperinsulinemia (8). In addition, a diet enriched with egg yolk was associated with elevated plasma glu- cose compared with a control diet in rats (9). Data from the Zutphen Study (10) have indicated a positive association be- tween egg consumption or dietary choles- terol and fasting glucose. However, in a randomized trial of 28 overweight or obese patients on a carbohydrate-restricted diet, consumption of three eggs per day had no effectsonfastingglucosecomparedwithab- stentionfromeggs(11).Currentdataonthe effects of dietary cholesterol on serum cho- lesterol have been inconsistent, ranging from positive associations (2,12) to lack of effect (12–14) and may be partly due to a largevariabilityinindividualresponsetodi- etary cholesterol (14,15). , J. MICHAEL GAZIANO, MD1,2,3 J. MICHAEL GAZIANO, MD1,2,3 OBJECTIVE — Whereas limited and inconsistent ﬁndings have been reported on the relation between dietary cholesterol or egg consumption and fasting glucose, no previous study has examined the association between egg consumption and type 2 diabetes. This project sought to examine the relation between egg intake and the risk of type 2 diabetes in two large prospective cohorts. RESEARCH DESIGN AND METHODS — In this prospective study, we used data from two completed randomized trials: 20,703 men from the Physicians’ Health Study I (1982–2007) and 36,295 women from the Women’s Health Study (1992–2007). Egg consumption was ascertained using questionnaires, and we used the Cox proportional hazard model to estimate relative risks of type 2 diabetes. RESULTS — During mean follow-up of 20.0 years in men and 11.7 years in women, 1,921 men and 2,112 women developed type 2 diabetes. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . Accessibility E p i d e m i o l o g y / H e a l t h S e r v i c e s R e s e a r c h O R I G I N A L A R T I C L E ●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●●● From the 1Division of Aging, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; the 2Division of Preventive Medicine, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts; the 3Massachusetts Veterans Epidemiology and Research Information Center (MAVERIC), Boston Veterans Affairs Healthcare System, Jamaica Plain, Massachusetts; the 4Department of Ambulatory Care and Prevention, Harvard Medical School, Boston, Massachusetts; and the 5Department of Epidemiology, Harvard School of Public Health, Boston, Massachusetts. Corresponding author: Luc Djousse´, ldjousse@rics.bwh.harvard.edu. Received 9 July 2008 and accepted 8 November 2008. DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 Diabetes Care 32:295–300, 2009 Diabetes Care 32:295–300, 2009 T h several of these nutrients have been asso- ciated with an increased risk of type 2 diabetes (i.e., saturated fat and cholesterol [4,5]), other nutrients may confer a lower risk of type 2 diabetes (i.e., polyunsatu- rated fat [4]). T ype 2 diabetes is highly prevalent and is associated with high health care costs and societal burden (1). Therefore, it is important to identify mod- iﬁable risk factors that may help reduce the risk of type 2 diabetes. Eggs are not only major sources of dietary cholesterol (200 mg/egg) but also contain other im- portant nutrients such as minerals, vita- mins, proteins, carotenoids, and saturated (1.5 g/egg), polyunsaturated (0.7 g/egg), and monounsaturated (1.9 g/egg) fatty acids (2,3). Whereas To our knowledge, no previous study has examined the association between egg consumption and the incidence of type 2 diabetes in a large prospective cohort of men and women. Because eggs can serve as a good source for vitamins, proteins, and other nutrients in the U.S., it is im- portant to determine the net degree of beneﬁt and harm of egg consumption on the risk of type 2 diabetes. The current study examines the association between egg consumption and incident type 2 diabetes among men and women who participated in two large completed randomized control trials. Whereas egg consumption was not associated with coronary heart disease (CHD) or stroke overall, Hu et al. (6) re- ported a twofold increased risk of CHD for egg consumption of more than one per week among men with type 2 diabetes in the Health Professionals’ Follow-up Egg Consumption and Risk of Type 2 Diabetes in Men and Women Compared with no egg consumption, mul- tivariable adjusted hazard ratios for type 2 diabetes were 1.09 (95% CI 0.87–1.37), 1.09 (0.88– 1.34), 1.18 (0.95–1.45), 1.46 (1.14–1.86), and 1.58 (1.25–2.01) for consumption of 1, 1, 2–4, 5–6, and 7 eggs/week, respectively, in men (P for trend 0.0001). Corresponding multivariable hazard ratios for women were 1.06 (0.92–1.22), 0.97 (0.83–1.12), 1.19 (1.03– 1.38), 1.18 (0.88–1.58), and 1.77 (1.28–2.43), respectively (P for trend 0.0001). CONCLUSIONS — CONCLUSIONS — These data suggest that high levels of egg consumption (daily) are as- sociated with an increased risk of type 2 diabetes in men and women. Conﬁrmation of these ﬁndings in other populations is warranted. Diabetes Care 32:295–300, 2009 RESEARCH DESIGN AND METHODS W d d f Corresponding author: Luc Djousse´, ldjousse@rics.bwh.harvard.edu. R i d 9 J l 2008 d d 8 N b 2008 Corresponding author: Luc Djousse´, ldjousse@rics.bwh Received 9 July 2008 and accepted 8 November 2008. RESEARCH DESIGN AND METHODS — We used data from the Physicians’ Health Study (PHS) I and the Women’s Health Study (WHS), two com- pleted randomized, double-blind, place- bo-controlled trials designed to study the effects of aspirin and -carotene (PHS) or Published ahead of print at http://care.diabetesjournals.org on 18 November 2008. DOI: 10.2337/dc08- 1271. Published ahead of print at http://care.diabetesjournals.org on 18 November 2008. DOI: 10.2337/dc08- 1271. Published ahead of print at http://care.diabetesjournals.org on 18 November 2008. DOI: 10.2337/dc08- 1271. © 2009 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for proﬁt, and the work is not altered. See http://creativecommons. org/licenses/by-nc-nd/3.0/ for details. g y The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. g y The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 295 Egg intake and type 2 diabetes trolled for age (continuous), BMI (25, 25–29, 30 kg/m2), smoking (never, former, and current smokers), alcohol consumption (0, 1–3 drinks/month, 1–6 drinks/week, 1 drinks/day), physical activity (vigorous exercise 0, 1, 1–3, 4 times per week in men and quintiles of kilocalories per week expended in lei- sure-time physical activity in women), and history of hypercholesterolemia and hypertension. Because detailed informa- tion on diet and family history was avail- able for women, the multivariable model in women also adjusted for family history of diabetes, energy intake (quintiles), in- take of fruits and vegetables (quintiles), red meat consumption (0.5, 0.5–0.9, and 1 serving/day), and intake of poly- unsaturated fats (quintiles), saturated fats (quintiles), and trans fats (quintiles). To examine whether the relation between egg and diabetes was mediated by dietary cho- lesterol, we evaluated the risk of diabetes associated with dietary cholesterol and also included dietary cholesterol in the multiva- riable model in women. RESEARCH DESIGN AND METHODS W d d f A similar approach was used for saturated fat. A P value for lin- ear trend was obtained by ﬁtting a continu- ous variable that assigned the median egg consumption in each egg category in a Cox regression model. low-dose aspirin and vitamin E (WHS) in the prevention of cardiovascular disease and cancer. Detailed description of the PHS I and WHS has been published pre- viously (16–18). Brieﬂy, a total of 22,071 U.S. male physicians aged 40 years at entry (1982) were randomized using a 22 factorial design to aspirin (325 mg every other day), -carotene (50 mg every other day), or their corresponding place- bos. Similarly, 39,876 female health pro- fessionals aged 45 years at entry (1992– 1995) were randomized to low-dose aspirin (100 mg on alternate days), vita- min E (600 IU on alternate days), or their corresponding placebos. Each participant gave written informed consent, and the institutional review board at Brigham and Women’s Hospital approved both study protocols. For the present analyses, we excluded 1,368 men because of prevalent type 2 diabetes (n 641), missing data on egg consumption (n 365), or missing data on potential confounders: smoking, alcohol intake, BMI, exercise, hyperten- sion, and fruits and vegetables (n 362). Among women, we excluded 3,581 be- cause of prevalent type 2 diabetes (n 1,171), missing data on egg consumption (n852),ormissingdataonpotentialcon- founders: BMI, exercise, smoking, energy intake, fruits and vegetables, nutrients, al- cohol consumption, and hypertension (n 1,558). Thus, a ﬁnal sample of 20,703 men and 36,295 women was used in the current analyses. women), we combined categories of one per day and beyond for stable estimates. The validity of food-frequency question- naires in similar populations has been published elsewhere (19,20). The corre- lation of egg consumption with dietary cholesterol was 0.61 (P 0.0001) and with saturated fat among women was 0.26 (P 0.0001). Ascertainment of incident type 2 diabetes Type 2 diabetes was ascertained by self- report on annual follow-up question- naires in both men and women. Follow-up and ascertainment of type 2 diabetes cases were completed in March 2007. Because all men were physicians, self-report was deemed sufﬁcient. Among the female health professionals, self- reports of type 2 diabetes were validated using American Diabetes Association cri- teria, for which additional information was obtained using telephone interviews, supplemental questionnaires, or review of medical records from treating physicians (21,22). Overall, the positive predictive value for type 2 diabetes validation was 91% (21). Egg consumption f Among men, information on egg con- sumption was self-reported at baseline using a simple abbreviated semiquantita- tive food-frequency questionnaire. Partic- ipants were asked to report how often, on average, they had eaten one egg during the past year. Possible response categories included “rarely/never,” “1–3/month,” “1/ week,” “2–4/week,” “5–6/week,” “daily,” and “2/day.” This information was ob- tained at baseline and at 24, 48, 72, 96, and 120 months after randomization. Among women, information on egg consumption was self-reported using a 131-item validated food-frequency questionnaire (19) at baseline. Women were asked to report their average con- sumption of eggs over the past year. Pos- sible response categories were “Never or 1/month,” “1–3/month,” “1/week,” “2– 4/week,” “5–6/week,” “1/day,” “2–3/day,” “4–5/day,” and “6/day.” Because very few subjects consumed one or more eggs per day (7.8% for men and 1.0% for Other variables h d Demographic data were collected at base- line. In addition, information on prevalence of hypertension, hypercholesterolemia, family history of diabetes (WHS only), smoking, exercise, and alcohol consump- tion was obtained at baseline. Whereas lim- ited data on foods were available in men, detailed dietary information was collected in the WHS, allowing estimation of energy intake and nutrients. In secondary analyses, we examined possible effect modiﬁcation by prevalent hypercholesterolemia (yes/no) and amount of energy from carbohydrate (low vs. high), using median energy from car- bohydrate as cut point in women only, where data were available. We tested for statistical interaction by including the main effects and the product terms be- tween egg consumption and hypercholes- terolemia in a hierarchical Cox regression model (PROC TPHREG in SAS). We also conducted sensitivity analyses by exclud- ing subjects with less than 2 years of fol- low-up. We repeated the main analysis using updated egg consumption at 24, 48, 72, 96, and 120 months in a time- dependent Cox model in men only, where repeated measures on egg consumption were available. Lastly, we used general- ized linear models and polytomous logis- tic regression to impute missing values for continuous and categorical variables, re- spectively. All analyses were completed using SAS (version 9; SAS Institute, Cary, NC). Signiﬁcance level was set at 0.05. Statistical analyses l ﬁd h We classiﬁed each subject according to the following categories of egg consump- tion per week: 0, 1, 1, 2–4, 5–6, and 7. We computed person-time of fol- low-up from baseline until the ﬁrst occur- rence of 1) type 2 diabetes, 2) death, or 3) censoring date, the date of receipt of the last follow-up questionnaire (March 2007). Within each egg-consumption group, we calculated the incidence rate of type 2 diabetes by dividing the number of cases by the corresponding person-time. We used Cox proportional hazard models to compute multivariable adjusted hazard ratios (HRs) with corresponding 95% CIs using subjects in the lowest category of egg consumption as the reference group. Statistical analyses l ﬁd h The initial model adjusted for age, whereas the multivariable model con- RESULTS — The mean SD age at randomization was 53.5 9.4 years (range 39.7– 85.9) in the PHS I and 54.5 7.0 years (38.7–89.9) in the RESULTS — The mean SD age at randomization was 53.5 9.4 years (range 39.7– 85.9) in the PHS I and 54.5 7.0 years (38.7–89.9) in the DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 296 Table 1—Baseline characteristics of 20,703 men and 36,295 women according to egg consumption Table 1 Baseline characteristics of 20,703 men and 36,295 women according to egg consumption Eggs per week 0 1 1 2–4 5–6 7 Men n 1,430 3,025 6,466 6,792 1,378 1,612 Age (years) 53.1 9.2 52.8 9.3 53.2 9.3 53.4 9.4 53.8 9.3 56.4 10.0 BMI (kg/m2) 24.1 2.7 24.6 2.6 24.7 2.7 24.9 2.7 25.1 2.9 24.9 3.1 Fruits and vegetables per week 15.2 8.4 14.1 7.4 14.8 7.1 15.3 6.9 15.8 7.4 17.0 8.5 Whole milk 16.7 31.1 37.1 42.8 50.9 54.2 Skim milk 60.8 61.9 67.7 65.9 61.4 53.5 Nut intake* 72.2 77.9 80.4 81.8 82.7 77.8 Breakfast cereal 60.7 62.1 74.2 77.7 74.2 58.9 Smoking 6.4 8.8 9.8 12.0 14.6 16.8 Never smokers 56.9 51.0 51.1 48.6 46.9 41.6 Exercise 84.4 86.2 87.4 87.6 87.4 84.9 Current drinkers of 1 per day 21.5 23.3 23.9 25.8 26.4 30.7 Hypertension 22.8 20.3 22.8 22.9 24.7 26.2 High cholesterol 14.6 12.9 12.0 11.1 10.4 10.4 Women n 6,381 10,758 9,222 8,921 647 366 Age (years) 55.2 7.2 54.3 7.0 54.4 6.9 54.5 7.0 54.6 7.0 55.1 7.2 BMI (kg/m2) 25.1 4.6 25.7 4.8 25.8 4.8 26.6 5.2 27.4 6.0 26.9 6.0 Fruits and vegetables per week 6.2 3.6 5.7 3.2 6.0 3.1 6.3 3.1 6.4 3.5 6.4 3.8 Red meat (servings/day) 0.45 0.45 0.62 0.46 0.75 0.49 0.93 0.59 1.15 0.76 1.26 0.83 Energy intake (kcal/day) 1,547 506 1,614 498 1,758 503 1,925 530 2,043 585 2,072 605 Exercise (kcal/week) 1,078 1,282 931 1,172 911 1,145 882 1,104 833 1,073 800 1,040 Dietary cholesterol (g/day)† 0.17 0.06 0.20 0.05 0.22 0.05 0.28 0.05 0.35 0.07 0.44 0.15 Trans fat (g/day)† 1.95 1.11 2.28 1.08 2.35 1.01 2.40 1.00 2.55 1.11 2.41 0.98 Polyunsaturated fat (g/day)† 10.6 3.2 11.0 2.9 11.2 2.7 11.4 2.7 11.8 2.8 11.9 3.1 Saturated fat (g/day)† 17.3 5.2 19.4 4.7 20.0 4.40 20.9 4.4 22.4 4.8 23.1 5.5 Smoking 10.8 12.1 11.8 15.3 19.8 23.5 Current drinkers of 1 per day 10.2 9.8 10.8 11.5 10.5 10.4 Hypertension 24.7 23.6 23.4 26.6 29.5 27.1 High cholesterol 38.0 28.9 26.4 25.3 26.1 19.1 Family history of diabetes 24.6 24.2 24.1 25.9 27.5 23.5 Data are mean SD or %. Statistical analyses l ﬁd h Nut consumption assessed 1 year after randomization. †Energy adjusted. women, 2,112 new cases of type 2 diabe- tes occurred during a mean follow-up of 11.7 years. From the lowest to the highest category of egg consumption, crude inci- dence rates of diabetes were 35.8, 41.3, 42.7, 46.8, 62.4, and 67.0 cases per 10,000 person-years in the PHS I. A sim- ilar increase in rates of type 2 diabetes with egg consumption was observed in women, with corresponding crude inci- dence rates of 39.6, 45.8, 43.3, 64.8, 76.8, and 112.7 cases per 10,000 person- years, respectively. Whereas consump- tion of up to one egg per week was generally not associated with an increased risk of type 2 diabetes in either sex in mul- tivariate analyses, more frequent con- sumption of eggs was associated with an increased risk of type 2 diabetes (Table 2). Compared with subjects who did not re- port egg consumption, intake of seven or more eggs per week was associated with a 58% increased risk of type 2 diabetes in men and a 77% increased risk of type 2 diabetes in women after adjustment for po- tential confounders (Table 2). Updating egg consumption using time-dependent Cox regression (PHS I) yielded a stronger rela- tion between egg consumption and inci- dent type 2 diabetes in men with HRs of 1.0 (reference), 1.10 (95% CI 0.99– 1.23), 1.31 (1.16–1.47), 1.40 (1.10– 1.77), 1.77 (1.39–2.26), and 1.99 (1.23– 3.23), from the lowest to the highest category of egg consumption, respec- tively, using a multivariable model as women, 2,112 new cases of type 2 diabe- tes occurred during a mean follow-up of 11.7 years. From the lowest to the highest category of egg consumption, crude inci- dence rates of diabetes were 35.8, 41.3, 42.7, 46.8, 62.4, and 67.0 cases per 10,000 person-years in the PHS I. A sim- ilar increase in rates of type 2 diabetes with egg consumption was observed in women, with corresponding crude inci- dence rates of 39.6, 45.8, 43.3, 64.8, 76.8, and 112.7 cases per 10,000 person- years, respectively. Whereas consump- tion of up to one egg per week was generally not associated with an increased risk of type 2 diabetes in either sex in mul- tivariate analyses, more frequent con- sumption of eggs was associated with an WHS. Among egg consumers, the median egg consumption was approximately one egg per week in men and women. DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 Statistical analyses l ﬁd h Table 1 presents baseline characteristics of the study participants. Frequent consump- tion of eggs was associated with higher BMI, higher proportion of current smok- ing, higher prevalence of hypertension, and lower prevalence of hypercholester- olemia. In addition, frequent consump- tion of eggs was associated with older age and more alcohol consumption in men and higher energy intake, as well as in- takes of saturated and trans fatty acids, and dietary cholesterol in women. A total of 1,921 new cases of type 2 diabetes were documented in men during a mean follow-up of 20.0 years. Among DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 297 Table 2—HR (95% CI) of type 2 diabetes according to egg consumption in men and women Table 2—HR (95% CI) of type 2 diabetes according to egg consumption in men and women Men Women n Age adjusted Model 1 n Age adjusted Model 1† Egg intake per week 0 104 1.0 1.0 295 1.0 1.0 1 254 1.16 (0.92–1.45) 1.09 (0.87–1.37) 576 1.16 (1.01–1.34) 1.06 (0.92–1.22) 1 560 1.19 (0.96–1.46) 1.09 (0.88–1.34) 470 1.10 (0.95–1.27) 0.97 (0.83–1.12) 2–4 637 1.30 (1.06–1.61) 1.18 (0.95–1.45) 669 1.65 (1.44–1.89) 1.19 (1.03–1.38) 5–6 169 1.73 (1.36–2.21) 1.46 (1.14–1.86) 56 1.97 (1.48–2.63) 1.18 (0.88–1.58) 7 197 1.82 (1.44–2.31) 1.58 (1.25–2.01) 46 2.88 (2.11–3.94) 1.77 (1.28–2.43) P for trend 0.0001 0.0001 0.0001 0.0001 n cases of type 2 diabetes. Adjusted for age (continuous), BMI (25, 25–29.9, and 30 kg/m2), smoking (never, former, and current smokers), alcohol consumption (0, 1–3 drinks/month, 1–6 drinks/week, and 1 drink/day), vigorous exercise (0, 1, 1–3, and 4 times per week), and history of hypercholester- olemia and hypertension. †Adjusted for age (continuous), BMI (25, 25–29.9, and 30 kg/m2), smoking (never, former, and current smokers), alcohol consump- tion (0, 1–3 drinks/month, 1–6 drinks/week, and 1 drink/day), exercise (quintiles of kilocalories per week), red meat intake (0.5, 0.5–0.9, and 1 servings/day), quintiles of energy intake, fruits and vegetables, saturated fatty acids, trans fatty acids, polyunsaturated fatty acids, family history of diabetes, and history of hypercholesterolemia and hypertension. sociated with type 2 diabetes (multivari- able adjusted HR 1.0, 1.03 [0.87–1.21], 1.00 [0.84–1.19], 1.00 [0.84–1.20], and 1.10 [0.92–1.33], from the lowest to highest quintile of energy-adjusted satu- rated fat, respectively). CONCLUSIONS — CONCLUSIONS — In this large pro- spective study, we have demonstrated that daily consumption of at least one egg is associated with an increased risk of type 2 diabetes in both men and women, inde- pendently of traditional risk factors for type 2 diabetes. Furthermore, the ob- served association between egg consump- tion and incident type 2 diabetes was not modiﬁed by prevalent hypercholesterol- emia in either sex. In a secondary analysis stratiﬁed by prevalent hypercholesterolemia at base- line (Table 3), similar patterns were ob- served in subjects of either sex with and without hypercholesterolemia (P for in- teraction 0.37 for men and 0.13 for women). Similar relations were observed between egg consumption and type 2 di- To the best of our knowledge, this is the ﬁrst study to examine prospectively le 3—Hazard ratios of diabetes according to prevalent hypercholesterolemia and egg consumption Table 3—Hazard ratios of diabetes according to prevalent hypercholesterolemia and egg consumption Men Women Normal cholesterol High or treated cholesterol Normal cholesterol High or treated cholesterol Egg consumption per week 0 1.0 1.0 1.0 1.0 1 1.09 (0.84–1.42) 1.11 (0.70–1.74) 1.11 (0.91–1.37) 1.02 (0.83–1.25) 1 1.03 (0.80–1.31) 1.28 (0.84–1.94) 1.00 (0.80–1.24) 0.98 (0.79–1.22) 2–4 1.16 (0.92–1.48) 1.19 (0.79–1.81) 1.26 (1.02–1.55) 1.14 (0.92–1.42) 5–6 1.34 (1.01–1.79) 1.78 (1.11–2.87) 0.88 (0.57–1.36) 1.68 (1.13–2.51) 7 1.47 (1.11–1.94) 1.96 (1.23–3.12) 1.84 (1.24–2.75) 1.72 (0.98–3.02) P for trend 0.0001 0.0001 0.0045 0.0028 Adjusted for age (continuous), BMI (25, 25–29.9, and 30 kg/m2), smoking (never, former, and current smokers), alcohol consumption (none, 1–3 drinks/ month, 1–6 drinks/week, and 1 drink/day), vigorous exercise (0, 1, 1–3, and 4 times per week), and history of hypertension. †Adjusted for age (continuous), BMI (25, 25–29.9, and 30 kg/m2), smoking (never, former, and current smokers), alcohol consumption (none, 1–3 drinks/month, 1–6 drinks/week, and 1 drink/day), exercise (quintiles of kilocalories per week), red meat intake (0.5, 0.5–0.9, and 1 serving/day), quintiles of energy intake, fruits and vegetables, saturated fatty acids, trans fatty acids, polyunsaturated fatty acids, family history of diabetes, and history of hypertension. *Adjusted for age (continuous), BMI (25, 25–29.9, and 30 kg/m2), smoking (never, former, and current smokers), alcohol consumption (none, 1–3 drinks/ month, 1–6 drinks/week, and 1 drink/day), vigorous exercise (0, 1, 1–3, and 4 times per week), and history of hypertension. Statistical analyses l ﬁd h Additional control for saturated fat did not alter the results (e.g., HR of 1.78 [1.30–2.45] without and 1.77 [1.28–2.43] with additional control for saturated fat, comparing the highest with the lowest egg consumption categories). Imputing missing data did not change the ﬁndings (online appendix Table A1, available at http://dx.doi.org/ 10.2337/dc08-1271). abetes when data were stratiﬁed by low energy from carbohydrate (P for linear trend 0.0004 for low energy from car- bohydrate and 0.12 for high energy from carbohydrate) in women only (data were not available to estimate carbohydrate in- take in men), and these ﬁndings were not altered when restricted to overweight or obese subjects (online appendix Table A2). above (this was not done for women due to lack of updated information on egg consumption). Lastly, exclusion of sub- jects with follow-up time 2 years in ei- ther cohort did not alter the results (P for trend 0.0001 in men and 0.0001 in women). Dietary cholesterol was positively as- sociated with the risk of diabetes (multi- variable adjusted HR 1.00 [reference], 0.94 [95% CI 0.80–1.11], 1.03 [0.88– 1.21], 1.07 [0.91–1.25], and 1.28 [1.10– 1.50], from the lowest to the highest quintile of dietary cholesterol, respec- tively (P for trend 0.0001). Additional adjustment for dietary cholesterol in women attenuated the point estimates in the multivariable model with corre- sponding HRs of 1.00 (reference), 1.05 (0.91–1.21), 0.94 (0.80–1.10), 1.07 (0.90–1.27), 1.00 (0.73–1.37), and 1.49 (1.06–2.09), respectively (P for trend 0.10). However, saturated fat was not as- CONCLUSIONS — In an animal ex- periment, a diet rich in fat was shown to induce hyperglycemia and hyperinsu- linemia (8). Furthermore, Adamopou- los et al. (9) demonstrated that a diet enriched with egg yolk resulted in ele- vated plasma glucose compared with a control diet in male Wistar albino rats. Data from the Zutphen Study (10) showed a positive association between egg consumption or dietary cholesterol and fasting glucose. These animal studies and data from the Zutphen Study are con- sistent with our ﬁndings. In contrast, in a randomized trial of 28 overweight or obese subjects on a carbohydrate- restricted diet, consumption of three eggs per day had no effects on fasting glucose compared with no egg consumption (11). Because the positive associations de- scribed above were observed in studies without restricted consumption of carbo- hydrates, it is possible that the hypergly- cemic effect of frequent egg consumption might only occur with a diet rich in car- bohydrates. However, our secondary data analysis provided no evidence for such a hypothesis in that we observed similar in- creased risk of type 2 diabetes with con- sumption of one or more eggs per day in women with low or high energy intake from carbohydrate. Further restriction to women with BMI 25 kg/m2, to mimic the above trial of 28 overweight or obese subjects on restricted carbohydrate diet (11), did not alter these ﬁndings. Under the premise that our observed ﬁndings were driven by dietary cholesterol con- tained in eggs, one possible explanation for the inconsistency in reported data on the association between egg consumption and glucose metabolism could be the large variability of individual response to dietary cholesterol (14,15,23). Whereas dietary cholesterol has been shown to in- crease plasma cholesterol in hyperre- sponders (2,12,24), no effect was documented among hyporesponders (12–14). CONCLUSIONS — Second, the lack of an effect of egg consumption on fasting glucose among obese or overweight subjects in the only human randomized trial (11) may imply differential physiological ef- fects of eggs in lean versus overweight or Overall, the observed increased risk of type 2 diabetes with daily consumption of eggs in the current study raises the pos- sibility of undesirable health effects with high rates of egg consumption and may help explain previously reported in- creased risk of CHD that was restricted to individuals with type 2 diabetes in the Health Professional Follow-up Study (6), the Nurses’ Health Study (6), and in our earlier publication from the PHS I show- ing an increased risk of mortality (and suggesting increased risk of CHD and stroke) with frequent egg consumption by subjects with prevalent type 2 diabe- tes (7). It is possible that frequent egg consumption may potentiate the risk of cardiovascular disease by inducing im- paired glucose metabolism and insulin resistance. Future investigations into underlying physiological mechanisms are warranted. Besides dietary cholesterol, eggs con- tain other important nutrients that have been shown to increase (i.e., saturated fat and cholesterol [4,5,25]) or decrease (i.e., polyunsaturated fat [4]) the risk of type 2 diabetes. It is possible that the individual contribution from each of these compo- nents as derived not just from eggs but also from other foods may play a role in determining the net effect of egg con- sumption. Unfortunately, as noted above, we did not have repeated data on fasting glucose, fasting insulin, and other bi- omarkers of glucose metabolism in either cohort to comprehensively examine pos- sible physiological mechanisms by which egg consumption might inﬂuence the risk of type 2 diabetes in our cohort. However, in women, where we had data on dietary cholesterol, there was attenuation of the association after additional adjustment for dietary cholesterol. This suggests that the observed relation between egg intake and diabetes may be partially explained by the cholesterol content of eggs. In con- trast, saturated fat was not associated with type 2 diabetes, and adjustment for this did not attenuate the results. In conclusion, our data are consistent with possible detrimental effects of daily consumption of eggs on the risk of type 2 diabetes in both men and women. CONCLUSIONS — †Adjusted for age (continuous), BMI (25, 25–29.9, and 30 kg/m2), smoking (never, former, and current smokers), alcohol consumption (none, 1–3 drinks/month, 1–6 drinks/week, and 1 drink/day), exercise (quintiles of kilocalories per week), red meat intake (0.5, 0.5–0.9, and 1 serving/day), quintiles of energy intake, fruits and vegetables, saturated fatty acids, trans fatty acids, polyunsaturated fatty acids, family history of diabetes, and history of hypertension. DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 298 Djousse´ and Associates reporting bias in the present study. How- ever, because information on egg con- sumption was collected before the occurrence of type 2 diabetes, such re- porting bias is more likely to be nondif- ferential and thus bias the results toward the null. We did not collect information on whether participants consumed egg yolk (rich in cholesterol) to further exam- ine the contribution of dietary cholesterol from eggs on type 2 diabetes risk in this study. In addition, we had limited dietary data for men to further assess the inter- play of eggs and other foods, energy, and nutrients with the risk of type 2 diabetes. The generalizability of our ﬁnding is lim- ited as both PHS I and WHS consist of homogeneous groups (male physicians and female health professionals, respec- tively) with the possibility that their be- haviors may differ from those of the general population. Furthermore, over 90% of the study participants were Cau- casian. Given the self-report nature of type 2 diabetes, we cannot exclude mis- classiﬁcation of the outcome in these data, especially in the WHS where not all participants were physicians, as was the case in the PHS. However, in the WHS, we had a 91% positive predictive value in a validation study of self-reported type 2 diabetes using American Diabetes Association criteria, for which data were attained by telephone interview, supple- mental questionnaire, or review of medi- cal records from treating physicians (21). Moreover, egg consumption was col- lected before the diagnosis of diabetes; thus, it is likely that any misclassiﬁcation of diabetes would be nondifferential and bias the results toward the null. Neverthe- less, the large sample size, the long dura- tion of follow-up, the repeated and standardized methods for data collection in both cohorts, and the robustness of the ﬁndings in sensitivity analyses are major strengths of this study. CONCLUSIONS — In conclusion, our data are consistent with possible detrimental effects of daily peated data on fasting glucose in men and women in the present study prevented us from further exploring the relation be- tween adiposity, egg consumption, and fasting glucose. reporting bias in the present study. How- ever, because information on egg con- sumption was collected before the occurrence of type 2 diabetes, such re- porting bias is more likely to be nondif- ferential and thus bias the results toward the null. We did not collect information on whether participants consumed egg yolk (rich in cholesterol) to further exam- ine the contribution of dietary cholesterol from eggs on type 2 diabetes risk in this study. In addition, we had limited dietary data for men to further assess the inter- play of eggs and other foods, energy, and nutrients with the risk of type 2 diabetes. The generalizability of our ﬁnding is lim- ited as both PHS I and WHS consist of homogeneous groups (male physicians and female health professionals, respec- tively) with the possibility that their be- haviors may differ from those of the general population. Furthermore, over 90% of the study participants were Cau- casian. Given the self-report nature of type 2 diabetes, we cannot exclude mis- classiﬁcation of the outcome in these data, especially in the WHS where not all participants were physicians, as was the case in the PHS. However, in the WHS, we had a 91% positive predictive value in a validation study of self-reported type 2 diabetes using American Diabetes Association criteria, for which data were attained by telephone interview, supple- mental questionnaire, or review of medi- cal records from treating physicians (21). Moreover, egg consumption was col- lected before the diagnosis of diabetes; thus, it is likely that any misclassiﬁcation of diabetes would be nondifferential and bias the results toward the null. Neverthe- less, the large sample size, the long dura- tion of follow-up, the repeated and standardized methods for data collection in both cohorts, and the robustness of the ﬁndings in sensitivity analyses are major strengths of this study. the association between egg consumption and incident type 2 diabetes in a large population of men and women. Before the current study, limited and inconsistent data (mainly from animal models) have been reported in the literature on the effects of eggs or dietary cholesterol on glucose metabolism. References 1. American Diabetes Association: Eco- nomic costs of diabetes in the U.S. in 2007. Diabetes Care 31:596–615, 2008 19. Willett W: Nutritional Epidemiology. New York, Oxford University Press, 1998 11. Mutungi G, Ratliff J, Puglisi M, Torres- Gonzalez M, Vaishnav U, Leite JO, Quann E, Volek JS, Fernandez ML: Dietary cho- lesterol from eggs increases plasma HDL cholesterol in overweight men consuming a carbohydrate-restricted diet. J Nutr 138: 272–276, 2008 20. Salvini S, Hunter DJ, Sampson L, Stampfer MJ, Colditz GA, Rosner B, Wil- lett WC: Food-based validation of a dietary questionnaire: the effects of week- to-week variation in food consumption. Int J Epidemiol 18:858–867, 1989 2. Herron KL, McGrane MM, Waters D, Lofgren IE, Clark RM, Ordovas JM, Fer- nandez ML: The ABCG5 polymorphism contributes to individual responses to di- etary cholesterol and carotenoids in eggs. J Nutr 136:1161–1165, 2006 12. Chakrabarty G, Manjunatha S, Bijlani RL, Ray RB, Mahapatra SC, Mehta N, Lak- shmy R, Vashisht S, Manchanda SC: The effect of ingestion of egg on the serum lipid proﬁle of healthy young Indians. In- dian J Physiol Pharmacol 48:286–292, 2004 21. Ding EL, Song Y, Manson JE, Pradhan AD, Buring JE, Liu S: Accuracy of administra- tive coding for type 2 diabetes in children, adolescents, and young adults: response to Rhodes et al. (Letter). Diabetes Care 30: e98, 2007 3. Song WO, Kerver JM: Nutritional contri- bution of eggs to American diets. J Am Coll Nutr 19:556S–562S, 2000 4. Salmeron J, Hu FB, Manson JE, Stampfer MJ, Colditz GA, Rimm EB, Willett WC: Dietary fat intake and risk of type 2 dia- betes in women. Am J Clin Nutr 73:1019– 1026, 2001 22. Liu S, Lee IM, Song Y, Van Denburgh M, Cook NR, Manson JE, Buring JE: Vitamin E and risk of type 2 diabetes in the Wom- en’s Health Study randomized controlled trial. Diabetes 55:2856–2862, 2006 13. Fernandez ML: Dietary cholesterol pro- vided by eggs and plasma lipoproteins in healthy populations. Curr Opin Clin Nutr Metab Care 9:8–12, 2006 5. Song Y, Manson JE, Buring JE, Liu S: A prospective study of red meat consump- tion and type 2 diabetes in middle-aged and elderly women: the women’s health study. Diabetes Care 27:2108–2115, 2004 14. Chakrabarty G, Bijlani RL, Mahapatra SC, Mehta N, Lakshmy R, Vashisht S, Man- chanda SC: The effect of ingestion of egg on serum lipid proﬁle in healthy young free-living subjects. Egg intake and type 2 diabetes Am J Clin Nutr 87:964–969, 2008 port on the aspirin component of the on- going Physicians’ Health Study. N Engl J Med 321:129–135, 1989 sible underlying biological mechanisms are warranted. sible underlying biological mechanisms are warranted. 8. Wu L, Vikramadithyan R, Yu S, Pau C, Hu Y, Goldberg IJ, Dansky HM: Addition of dietary fat to cholesterol in the diets of LDL receptor knockout mice: effects on plasma insulin, lipoproteins, and athero- sclerosis. J Lipid Res 47:2215–2222, 2006 17. 17. Lee IM, Cook NR, Gaziano JM, Gordon D, Ridker PM, Manson JE, Hennekens CH, Buring JE: Vitamin E in the primary pre- vention of cardiovascular disease and can- cer: the Women’s Health Study: a randomized controlled trial. JAMA 294: 56–65, 2005 Acknowledgments— This study was sup- ported by grants CA-34944, CA-40360, CA- 047988, and CA-097193 from the National Cancer Institute and grants HL-26490, HL- 43851, HL-080467, and HL-34595 from the National Heart, Lung, and Blood Institute, Be- thesda, Maryland. Acknowledgments— This study was sup- ported by grants CA-34944, CA-40360, CA- 047988, and CA-097193 from the National Cancer Institute and grants HL-26490, HL- 43851, HL-080467, and HL-34595 from the National Heart, Lung, and Blood Institute, Be- thesda, Maryland. 9. Adamopoulos PN, Papamichael CM, Zampelas A, Moulopoulos SD: Choles- terol and unsaturated fat diets inﬂuence lipid and glucose concentrations in rats. Comp Biochem Physiol B Biochem Mol Biol 113:659–663, 1996 18. Ridker PM, Cook NR, Lee IM, Gordon D, Gaziano JM, Manson JE, Hennekens CH, Buring JE: A randomized trial of low-dose aspirin in the primary prevention of car- diovascular disease in women. N Engl J Med 352:1293–1304, 2005 No potential conﬂicts of interest relevant to this article were reported. No potential conﬂicts of interest relevant to this article were reported. 10. Feskens EJ, Kromhout D: Habitual dietary intake and glucose tolerance in euglycae- mic men: the Zutphen Study. Int J Epide- miol 19:953–959, 1990 CONCLUSIONS — Be- cause the median egg consumption in this population (one egg per week for men and women) fell within a range not asso- ciated with an increased risk of type 2 diabetes, dietary advice to reduce egg consumption may target individuals who consume one or more eggs per day if these ﬁndings are conﬁrmed in other studies. Given the societal burden of type 2 diabe- tes, conﬁrmation of these ﬁndings in other populations and exploration of pos- Additional limitations of the present study include the observational nature of the study design in which residual con- founding or unmeasured confounding could partly or completely explain our re- sults. In addition, because egg consump- tion was self-reported, we cannot exclude DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 299 DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009 References Indian J Physiol Phar- macol 46:492–498, 2002 23. Pyorala K: Dietary cholesterol in relation to plasma cholesterol and coronary heart disease. Am J Clin Nutr 45:1176–1184, 1987 6. Hu FB, Stampfer MJ, Rimm EB, Manson JE, Ascherio A, Colditz GA, Rosner BA, Spiegelman D, Speizer FE, Sacks FM, Hennekens CH, Willett WC: A prospec- tive study of egg consumption and risk of cardiovascular disease in men and women. JAMA 281:1387–1394, 1999 24. Sacks FM, Salazar J, Miller L, Foster JM, Sutherland M, Samonds KW, Albers JJ, Kass EH: Ingestion of egg raises plasma low density lipoproteins in free-living subjects. Lancet 1:647–649, 1984 15. Ballesteros MN, Cabrera RM, Saucedo MS, Fernandez ML: Dietary cholesterol does not increase biomarkers for chronic disease in a pediatric population from northern Mexico. Am J Clin Nutr 80:855– 861, 2004 25. Meyer KA, Kushi LH, Jacobs DR Jr, Fol- som AR: Dietary fat and incidence of type 2 diabetes in older Iowa women. Diabetes Care 24:1528–1535, 2001 7. Djousse´ L, Gaziano JM: Egg consumption in relation to cardiovascular disease and mortality: the Physicians’ Health Study. 16. Steering Committee of the Physicians’ Health Study Research Group: Final re- 300 DIABETES CARE, VOLUME 32, NUMBER 2, FEBRUARY 2009
https://openalex.org/W2782267807	https://hal.inrae.fr/hal-02626379/document	English	null	Prion Strains and Transmission Barrier Phenomena	Pathogens	2,018	cc-by	22,582	To cite this version: Angelique Egalon, Vincent Béringue, Human Rezaei, Pierre Sibille. Prion Strains and Transmission Barrier Phenomena. Pathogens, 2018, 7 (1), 10.3390/pathogens7010005. hal-02626379 Distributed under a Creative Commons Attribution 4.0 International License Prion Strains and Transmission Barrier Phenomen Received: 23 October 2017; Accepted: 26 December 2017; Published: 1 January 2018 Abstract: Several experimental evidences show that prions are non-conventional pathogens, which physical support consists only in proteins. This ﬁnding raised questions regarding the observed prion strain-to-strain variations and the species barrier that happened to be crossed with dramatic consequences on human health and veterinary policies during the last 3 decades. This review presents a focus on a few advances in the ﬁeld of prion structure and prion strains characterization: from the historical approaches that allowed the concept of prion strains to emerge, to the last results demonstrating that a prion strain may in fact be a combination of a few quasi species with subtle biophysical speciﬁcities. Then, we will focus on the current knowledge on the factors that impact species barrier strength and species barrier crossing. Finally, we present probable scenarios on how the interaction of strain properties with host characteristics may account for differential selection of new conformer variants and eventually species barrier crossing. Keywords: prion strain; species barrier; strain adaptation; zoonosis; Darwinian evolution; deformed templating; structural elementary brick Pathogens 2018, 7, 5; doi:10.3390/pathogens7010005 www.mdpi.com/journal/pathogens HAL Id: hal-02626379 https://hal.inrae.fr/hal-02626379v1 Submitted on 26 May 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License pathogens Review 1. Introduction Scientists have been intensively working on prion diseases, nevertheless, several aspects of this transmissible neurodegenerative affection remain obscure. Among these black boxes, the biophysical support for prion strain variation and the species barrier have stand as one of the last accessible achievements. Although being known for a long time, scrapie and related diseases really went in the light in the 90’s: society urged scientists for answers to the questions raised by the sudden outbreak of mad cow disease (caused by Bovine Spongiform Encephalopathy (BSE) prions), which could result in a Creutzfeldt-Jakob Disease (CJD)—like disease in humans. At that time, the potential transmission of BSE from cattle to humans had been assessed persistently, leading to perplexing and even opposite results (sometimes coming from the very same lab) [1,2]. Rather than the consequence of an urgent need for results, these inconsistent data underlined the complexity of the addressed question that we are still trying to answer for the last 30 years. This review aims at presenting the main knowledge and the latest milestones in the ﬁeld of prion strains and species barrier phenomena, both topics being intimately linked. Prion diseases are fatal neurodegenerative disorders. At the beginning of the 20th century, Drs Creutzfeldt and Jakob ﬁrst described the pathology in humans as a sporadic disease. Then the disease was identiﬁed as the main responsible for an epidemic of neurodegenerative cases among the Fore population of Papua New Guinea, leading the scientists (Gajdusek, Gibbs and Alpers) to propose an infectious spread of the disease (due to endocannibalistic rituals) [3]. Soon after, Grifﬁth proposed a self-replicating model for the related scrapie disease [4] but the formal conceptualization of prion as a protein only infectious agent, responsible for the misfolding of the host cellular prion protein into a pathologic conformer had to wait for Prusiner’s seminal work (1982) [5]. The prevalence of the disease in human population is rather low (~1 case per million people per year, mostly among aged www.mdpi.com/journal/pathogens 2 of 29 Pathogens 2018, 7, 5 population). Some cases (~15%) are genetically linked, due to point mutation in the prion protein gene (PRNP). This disease is also long known to affect ruminants, including sheep and goats with scrapie, cattle with bovine spongiform encephalopathy and cervids with chronic wasting diseases (CWD). 2.1. Early Cases of Prion Interspecies Transmission The ﬁrst report on experimental prion transmission in animals focused on reproducing disease-speciﬁc clinical signs by inoculating infected brain extract [6]. The studied parameters were the incubation time until disease end stage, the nature of the clinical signs and the anatomic distribution of the lesions that were reported on a score proﬁle. The ﬁrst experiments have been reported in sheep [7]. Then, Pattison and colleagues reported several successful experimental inoculations of sheep scrapie to goats [8,9]. By that time, it was found that some prions could pass from one species to the other (e.g., mink to small ruminants [10]). As susceptible to infection with most prion strains, the bank vole turned out to act like a “universal acceptor” [11–13] (see also Sections 4.1 and 4.2). Conversely, several studies reported on the difﬁculty to pass prions from one species to others (e.g., certain scrapie isolates to cattle [14]). With the development of transgenic mouse engineering, expression of foreign PrP (in the presence or in the absence of endogenous mouse PrP) considerably enlarged the possibilities for studying zoonotic transmission of prions. These approaches proved to be versatile, since it was demonstrated that development of prion pathology relied solely on the presence of convertible PrP [1,15–17]. In many cases, these experimental setups made emerge the idea that almost every prion could adapt to almost every PrP substrate, provided that some critical parameters (presented below) have been set up in order to adapt the strain to its new host PrP. 1. Introduction No crossing of the species barrier between human and ruminant prions have been reported until the implementation of new biophysical parameters in the process for recycling the livestock carcasses into the ruminant alimentary chain: these modiﬁcations resulted in incomplete inactivation of the BSE prions and paved the way for this unconventional agent to cross barrier species and to spread in humans in an outbreak known as the mad cow disease and the variant CJD (vCJD), respectively. And prompted scientists to further study the propensity of this agent to adapt from one host to the other. 2.2. Emergence of the Prion Strain Concept Interestingly they also noticed some reproducibility of the observed clinical signs, depending on the inoculated isolate. The conclusion of their papers proposed that “certain “strains” of the scrapie agent will produce the nervous syndrome, while others will produce the scratching syndrome”. Soon in the prion scientiﬁc history appeared the fact that prion agents could share common strain features with conventional DNA-encoded pathogens. These prion strain features could be distinguished from each other based on a number of parameters such as incubation time and titration, which were remarkably reproducible among strains in a given host. Addition of anatomical data such as the localization and intensity of vacuolation allowed to isolate and further characterize prion strains, that were secondarily used to study their adaptation when passing from a host to the other [18]. With the reﬁnements in biochemical and biophysical analysis methods, several parameters are now available for the extensive study on prions that will be detailed in the following sections. 3 of 29 Pathogens 2018, 7, 5 Pathogens 2018, 7, 5 3.1.1. Incubation Time, Clinical Signs, Vacuolation Tissue Tropism 3.1.1. Incubation Time, Clinical Signs, Vacuolation Tissue Tropism Prion diseases are ﬁrst characterized by clinical signs observed on the affected individuals. In humans, after a long pre-clinical period is ended, affected individuals usually complain with vague sensory feelings, such as depression. Progressive motor paralysis and dementia then rapidly follow. Cerebellar ataxia is often found in the course of the disease [19]. In animals, clinical signs include progressive ataxia as well but some features like itching and scratching seem to be animal-speciﬁc. Behaviour modiﬁcations, including aggressiveness or enhanced tameness are also recorded [9]. But these general features are markedly inﬂuenced by the strain of prion infecting individuals [8,20]. Distribution and abundance of the lesions in speciﬁc brain areas, which appeared to be remarkably stable, were used to score and attribute a “lesion” proﬁle to prion strains [16]. Incubation period for a given strain in a given host was highly reproducible and rapidly served as the ﬁrst criterion for the characterization of prion strains [21]: for instance, 263 K strain kills golden hamster in 65 days, whereas 139H strain kills the same species in 130 days (after intracranial inoculation). It is worth mentioning that inoculum dilution inﬂuences the incubation time [22,23]. Thus, a combination of criterions is necessary for efﬁcient discrimination. Immunoreactivity of antibodies reacting with the diseased form of the PrP (PrPSc) allowed for signiﬁcant reﬁnement in the prion strain characterization [20]. Although prions ultimately accumulate in the Central Nervous System (CNS) of their host, peripheral accumulation may also be associated with some prion strains, contrasting with the ones that are only detected in the brain: in this respect, the most relevant tissues turned out to be the secondary lymphoid organs such as lymph nodes and spleen [24]. This lymphotropism may be used for discrimination between two strains that otherwise would look identical in the brain tissue [25]. 3. From Prion Strain Characterization 3.1. First Approaches 3.1.2. Biophysical Parameters (Circular Dichroism, Infra Red, . . . ) This distribution of prion types is however generally not exclusive: several CJD cases are actually a mix of T1 and T2 [31]. Whether this co-occurrence aroused from biochemical reasons or just by chance remains to be addressed. The size of the resistant-core depends on the prion strain and its evaluation still remains the gold standard of prion analysis. Antigenic epitope mapping of PrPSc raised against different prion strains showed speciﬁc immunoreactivity [32], indicating conformational differences within PrPSc assemblies. Even if several monoclonal antibodies (mAbs) have been raised against PrP antigens from various species, in most cases however, mAbs were poor discriminants and cross reactivities were often recorded between mouse, hamster, human and most common ruminants, as it is the case for the conformational antibody 15B3 [33]. What’s more, these mAbs would hardly discriminate between normal and pathological PrPs, which would have been of enormous interest for diagnostic or laboratory purposes. In addition to providing antibodies more or less selective for a given PrP protein, alternative approaches were designed for the study and conformational screening of prion strains [34]. Known as the conformation-dependent immunoassay (CDI), this technique depends on differential recognition of unmodiﬁed PrPSc or altered PrPSc epitopes to determine a ratio that can be used for direct quantitation of prion in a sample. This technique shows that more than 90% of PrPSc present in sCJD patients are PK-sensitive [35]; moreover, this ratio may reveal variations from strain to strain, as observed for eight characterized hamster-prion strains [34]. In particular, this approach allowed for a better discrimination between prion strains that otherwise would have been indistinguishable using incubation time and PK-resistance as analysis criterion. However, this approach still failed to discriminate between closely related prions, although showing that PrPSc assemblies present different degree of stability in presence of chaotropic treatments that reveal epitopes [36]. Post translational protein modiﬁcations have been proposed to account for the PrP strain-to-strain variations. For instance, prion protein contains two glycosylation sites located in the structured C-terminal part of the protein. Both N-glycosylation sites are conserved in the PrnP gene among species, suggesting that N-glycans play an important role in the protein function. These sites, however, are not systematically glycosylated, as shown by the 3 detected bands in western blots performed with anti-PrP antibodies. 3.1.2. Biophysical Parameters (Circular Dichroism, Infra Red, . . . ) Several biophysical approaches have been employed for elucidating the parameters underneath the biological strain phenomenon. Due to its major insolubility and to the highly heterogeneous aspects of the prion material, all conventional approaches (X-ray crystallography, nuclear magnetic resonance (NMR)) mainly revealed unsuccessful in providing good quality crystals or homogenous solutions for the determination of a pathological PrPSc structure. Though, infra-red approaches ﬁrst identiﬁed strain differences [26]: absorption spectra from Hyper, Drowsy and 263 K hamster strains revealed a difference between the Drowsy strain and the two others. These results were interpreted as differences in β-sheet secondary structures. Using a combination of several investigation techniques, the structural properties of two sorts of ﬁbrils formed under different experimental conditions was further shown to correspond to differential folding patterns of β strands [27]. NMR imaging proved limited in exploring the PrPSc structure because of insolubility matters. Conversely, Hydrogen/Deuterium exchange proved helpful with solid NMR approaches [28]. Mass spectrometry using acetylation of accessible lysines in PrPSc assemblies recently added some arguments in favour with a β-solenoid form of the pathologic prion structure [29]. In addition, the approach was able to reveal structural differences between several common prion strains. Notably, Sc237 hamster prion strain is supposed to have an N-terminal fragment reacting less with the core prion protein than the others hamster strains tested. This view is further supported by the fact that Sc237 infectivity is less sensitive to PK-digestion (partial resistance of the PrPSc protease digestion is used as the gold standard for the detection of infected samples) than the others. Pathogens 2018, 7, 5 4 of 29 3.1.3. Biochemical Methods (Western Blot, Resistance to Chaotropic Agents, Conformation-Dependent Immunoassays . . . ) 3.1.3. Biochemical Methods (Western Blot, Resistance to Chaotropic Agents, Conformation-Dependent Immunoassays . . . ) At ﬁrst, proteinase-K resistance of the pathological PrPSc form served as a diagnostic tool, since most of the antibodies raised against the PrP could not discriminate between normal PrPC and pathological PrPSc forms of the prion protein in western blot. The PrPSc isoform partially resists to the cleavage by the most common proteases, among which proteinase K (PK): three major conformation have been reported according to the protease-resistant core size observed by western blotting: 21 kDa (type-1), 19 kDa (type-2) or 8 kDa with some speciﬁc antibodies specially recognizing some of these types such as 12B2, a type 1-speciﬁc antibody [30]. 3.2.1. Templating Activity In 1996, Prusiner’s team published the observation that FFI (Familial Fatal Insomnia) or sporadic CJD (sCJD) inoculation to a mouse expressing a chimeric human-mouse PrP gene reproduced both PK-resistant 19 kD deglycosylated band pattern for FFI and the 21 kD band pattern for the sCJD, respectively [42]. This basic observation paved the way to the concept of templating activity of prions. This concept has been explored in the ﬁeld of yeast and fungi prions (the yeast prions will voluntarily not be documented here) but had been curiously neglected until the very recent years, when Baskakov’s team reported conformational switches within individual amyloids [43]. In this work, two strains of ﬁbrils made from the identical recombinant hamster PrP showed various individual characteristics, derived only from the different conditions of formation: R strain was obtained under rotation of the monomers and displayed straight shape and polymorphous (twisted or not twisted morphology), while S strain was obtained under shaking and displayed a curvy simple line. When incubating these different ﬁbril strains with heterologous mouse recombinant PrP monomers, they observed that whatever the original strain, the ﬁbrils adopted the straight complex forms of the R phenotype. In addition, FTIR (Fourrier–transform Infra-Red) spectroscopy properties of the daughter ﬁbrils were similar to the ones of the R phenotype. By contrast, R and S ﬁbrils incubated with the homologous hamster monomers yielded the expected parental forms (microscopy and FTIR). Thus, monomer origin is able to imprint a new conformation and new properties to a given inoculum, resulting in the change or adaptation of the strain to its new host. However, these data also suggest that the host PrP could restrict rather than enlarge the conformational panel available for PrPSc. Owing to their very long incubation periods, prion diseases remain difﬁcult to study in vivo. Although cell-based systems have been developed in several laboratories, they proved to be difﬁcult to set up, particularly because this approach was not possible to implement to every kind of prion strain. Despite these difﬁculties, Weissman’s groups produced seminal data on prion strain adaptation and selection in vitro [44–48]. They ﬁrst showed that neuron cell lines chronically infected by two prion strains (RML and 22 L) could be derived into several different cell lines with their own response to various other prion strains [44]. 3.2. New Insights 3.2.1. Templating Activity 3.1.2. Biophysical Parameters (Circular Dichroism, Infra Red, . . . ) Glycosylation pattern changes from one strain to the other, with variations in the relative abundance of the Di-, Mono- and Un-glycosylated forms and even the normal PrPC glycosylation is differentially affected by depending on the prion strain [37]. However, glycosylation deﬁciency at either one or other glycosylation sites does not alter the susceptibility of the host to scrapie prions [38]. Moreover, strain characters were not modiﬁed when these glycosylation mutant mice were used for bio assays. This result was later supported by our work in vitro reporting that several PrP glycosylation mutants are faithful templates for PMCA (protein misfolding cyclic ampliﬁcation, see Section 3.2.1) [39]. Other studies however reported that sialic acids that are deposited on glycan chains may signiﬁcantly account in the prion replication: desialylated PrPSc was mainly found and eliminated in the liver while normal prions were targeted toward secondary lymphoid organs [40]. In the meantime, the desialylation of prions is reported to reduce the species barrier [41]. These data are particularly relevant with respect to cross-species transmission fate. 5 of 29 Pathogens 2018, 7, 5 Pathogens 2018, 7, 5 Pathogens 2018, 7, 5 3.2. New Insights 3.2. New Insights 3.2.1. Templating Activity Thus this in vitro ampliﬁcation tool proved to be a valuable tool for the assessment of cross barrier crossing, a main advantage over bioassays being the extreme shortening of the time required for experimentation. Species barrier crossing could be demonstrated between mouse prion and “cervid” model mice, which would have required much more time with the bio assay [56]. PMCA has been described using brain lysate of transgenic mice as a source of healthy PrPC. But versatility of the system may be further increased by the use of cultured cell lysates. This was used in our lab to further dissect the requirements for PrPSc conversion [57,58]. Additional experiments determined that the conversion and ampliﬁcation of recombinant PrP protein could occur in the presence of only RNA or phospholipids as adjuvant molecules [55,56,59–62]. p y p p p j Simultaneously, RT-QuIC was developed as another technique for the in vitro ampliﬁcation of prion conversion [63]. In brief, recombinant PrP molecules are driven to ﬁbrillation through alternative shaking and incubating with thioﬂavin-T (ThT) as a ﬂuorescent marker: an increase in ﬂuorescence emission, that could be observed in real time, is the sign of amyloid formation and PrP conversion. This approach proved to be sensitive enough to detect prion particles within blood cells [64] and diverse body ﬂuids [65]. An important difference between PMCA and RT-QuIC is that the recombinant PrP that is converted in QuIC experiments is poorly infectious, whereas PMCA ampliﬁed products are usually as highly infectious as the inoculum. Noteworthy, this technique does not faithfully replicate the species barrier phenomenon that is recorded in target animals. Nonetheless, RT-QuIC has proved useful in discriminating among prion strains: Bank Vole PrP could be converted by almost every strain tested in RT-QuIC experiment. Lag phase and ﬁnal ﬂuorescence signals could be used for discrimination between different prion strains, though [13]. These observations parallel those made with transgenic mice expressing bank vole PrP [66]. On the contrary, the use of several different substrates could be used as a screen to differentiate between closely similar strains: atypical L-type BSE and classical BSE for instance [67]. A ﬂuorescent approach to discriminate between prion strains was provided by a chemist group from Sweden using oligo-/poly-thiophene derivates [68]. 3.2.1. Templating Activity Although they could somehow stabilize cell-adapted prion features different from that of brain–adapted prions, they observed the occurrence of a “Darwinian selection” that allowed for the transition from one prion to the other [45]. Further selection could even be achieved using selective prion drugs inhibitors such as swainsonin, demonstrating that the strain features observed ﬁnely depend on the prion production conditions [45,48]. In such a context, Weissmann and colleagues further enforced the concept of quasi-species proposed by Collinge [49], consisting of a major component and many variants, which are constantly being generated and selected against in a particular environment: changing of conditions may result in the selection of a new variant with different features. Several aspects of PMCA and RT-QuIC (real time quaking-induced conversion) ampliﬁcations have been studied and used for prion detection in body ﬂuids or for assessing and validating decontamination procedures that will not be emphasized here. A focus on some of these topic has however been recently published [50]. PMCA approach was ﬁrst described by C. Soto in 2001 [51] and involves the cyclic ampliﬁcation of minute amounts of infectious material diluted in a brain lysate containing solubilized forms of PrPC. The repetition of a few seconds ultrasonication bursts followed by an incubation period at 37 ◦C produces the exponential transconformation of the PrPC present in the normal brain lysate into a PK-resistant form. PrPSc is then detected on Dot or Western blot after PK-digestion. Practically, ampliﬁcation factors and titration capabilities have been reached that extend far beyond that obtained with bioassays: 1012 ampliﬁcations were routinely obtained with laboratory scrapie strains [52] and the system allows for the ampliﬁcation of a large number of strains, sometimes to a lesser extent, though. This reduced ampliﬁcation level could however be largely compensated after several rounds of ampliﬁcation. The difﬁculty to amplify sporadic MM1 Pathogens 2018, 7, 5 6 of 29 CJD, appeared as a notable exception until Safar and colleagues reported that a modiﬁed PMCA using unglycosylated PrP was able to selectively amplify type 1 sCJD [53]. CJD, appeared as a notable exception until Safar and colleagues reported that a modiﬁed PMCA using unglycosylated PrP was able to selectively amplify type 1 sCJD [53]. Castilla and colleagues proved the relatively high strain ﬁdelity of the PMCA-driven prion ampliﬁcation regarding currently available tools (incubation time, brain lesion scores, western blot proﬁle, PK-resistance) [54,55]. 3.2.1. Templating Activity Murine scrapie and CWD have been compared for excitation/emission spectra as well as ﬂuorescence life-time of a few compounds: this parameter is modiﬁed in response to conformational restriction of the thiophene backbone following interaction with the different aggregates. All these methods could provide reﬁned tools to differentiate strains that are difﬁcult to by strain typing in animals. 3.2.2. Size Distribution of Aggregates (Quaternary Structure) Biophysical approaches focused on size-distribution analysis of the prion particles. If not entirely carried on primary or secondary sequence, strain information should be somehow related to the tertiary or quaternary structure of the PrPSc assemblies. Several studies have already pointed the necessary role played by the PrP structure in the pathological process of prion transconformation [2,26,55,69,70]. Sedimentation velocity centrifugation in density gradients proved to be a valuable tool to separate and analyse prion fragments according to their size and/or shape, while preserving as much as possible the “natural” multimerization state of the prion particles and minimizing artefacts due to improper membrane solubilisation [71,72]: this later point is crucial for the reliability of the technique, since the presence of residual membrane lipids would modify the assemblies’ apparent density and lead to improper interpretation of the data. Sedimentation velocity-based fractionation will discriminate dense heavy aggregates that will sediment to the bottom of the gradient from the lighter fractions containing small aggregates or particles of low density. This technique allowed the precise discrimination between several ovine and hamster strains (Figure 1). The disconnection between infectivity level of the fractions (monitored by bioassay) and the PrPSc abundance (estimated by western blot) speciﬁcally for the ‘fast’ 7 of 29 Pathogens 2018, 7, 5 ovine and hamster strains constituted a striking ﬁnding of this approach [71,72]. These experiments led to the view that prions are formed of a strain-speciﬁed collection of non-uniform PrPSc assemblies with speciﬁc activities. Pathogens 2017, 6, 5 7 of 30 h speciﬁc activities. Pathogens 2017, 6, 5 7 of 30 Figure 1. Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. Fractions collected from the gradient were analysed for PK-resistant PrPSc content (black line) and for infectivity (red line). For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis). The sedimentation peaks of standard molecular mass markers (MM markers) are indicated on the top of the graph. From [71]. Figure 1. Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. 3.2.2. Size Distribution of Aggregates (Quaternary Structure) Fractions collected from the gradient were analysed for PK-resistant PrPSc content (black line) and for infectivity (red line). For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis). The sedimentation peaks of standard molecular mass markers (MM markers) are indicated on the top of the graph. From [71]. p Pathogens 2017, 6, 5 7 of 30 Figure 1 Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 Figure 1. Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. Fractions collected from the gradient were analysed for PK-resistant PrPSc content (black line) and for infectivity (red line). For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis). The sedimentation peaks of standard molecular mass markers (MM markers) are indicated on the top of the graph. From [71]. Figure 1. Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. Fractions collected from the gradient were analysed for PK-resistant PrPSc content (black line) and for infectivity (red line). For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis). The sedimentation peaks of standard molecular mass markers (MM markers) are indicated on the top of the graph. From [71]. Figure 1. Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. Fractions collected from the gradient were analysed for PK-resistant PrPSc content (black line) and for infectivity (red line). For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). Of prion isolates, strains and types • Isolate: we refer here as to biological material that has been obtained through sampling of infected individuals; • Isolate: we refer here as to biological material that has been obtained through sampling of infected individuals; • Strain: the term corresponds to a deﬁned prion population isolated from one speciﬁed animal, with regards to the precision of the investigation technique: from basic observations (clinical signs incubation time and so on) to ﬁne biochemical and biophysical parameters that are now becoming precise enough to allow for the discrimination of quasi-species within one strain; for the sake of simplicity, one regularly and erroneously omit the name of the host from which the strain has been originally isolated, even though a totally different prion population may have been selected when passed to the new host. y p p p y p • Type: refers more particularly to a combination of biochemical parameters (mainly to the size of the unglycosylated PrPSc fragment after proteinase K partial digestion) that are independent from the host. y p p p y p • Type: refers more particularly to a combination of biochemical parameters (mainly to the size of the unglycosylated PrPSc fragment after proteinase K partial digestion) that are independent from the host. The prion phenotype characterization is as precise as the accuracy of observation tools used. In some cases, and despite the combination of several investigation methods, two strains may not be discriminated (Box 1). For instance, it has been observed that some scrapie strains could display the same pattern than that of BSE . This raised the question whether BSE could in fact originate from a scrapie strain [73]. Later on, the identiﬁcation of an atypical L-type BSE in bovine raised again doubts on the potential transmission of BSE to small ruminants [74]. First described within a ﬂock in Italy, its behaviour in cattle is very different from that of the classical BSE (presence of amyloid plaques, low non-glycosylated PrPSc fragment). Upon passage on ovinized animals (VRQ allele), however, this strain turned out to be fully similar to C-BSE, which prompted the investigators to speculate that the mad cow outbreak of the late 80’s could have arisen from a passage by the small ruminants. 3.2.2. Size Distribution of Aggregates (Quaternary Structure) For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis). The sedimentation peaks of standard molecular mass markers (MM markers) are indicated on the top of the graph. From [71]. Figure 1. Brain homogenates from tg338 mice infected with LA21K (A); 127S (B); LA19K (C); Nor98 (D) and sheep BSE (E) were solubilized and fractionated by sedimentation velocity. Fractions collected from the gradient were analysed for PK-resistant PrPSc content (black line) and for infectivity (red line). For each fraction, the percentage of total PK-resistant PrPSc detected on the immunoblot is presented (left axis). For each fraction of each strain, infectivity was determined by measuring mean survival times in reporter tg338 mice (mean ± SEM; right, red axis). The sedimentation peaks of standard molecular mass markers (MM markers) are indicated on the top of the graph. From [71]. 8 of 29 Pathogens 2018, 7, 5 3.3. When Two Strains Look the Same Box 1. Of prion isolates, strains and types. Box 1. Of prion isolates, strains and types. Of prion isolates, strains and types • Isolate: we refer here as to biological material that has been obtained through sampling of infected individuals; • Strain: the term corresponds to a deﬁned prion population isolated from one speciﬁed animal, with regards to the precision of the investigation technique: from basic observations (clinical signs incubation time and so on) to ﬁne biochemical and biophysical parameters that are now becoming precise enough to allow for the discrimination of quasi-species within one strain; for the sake of simplicity, one regularly and erroneously omit the name of the host from which the strain has been originally isolated, even though a totally different prion population may have been selected when passed to the new host. • Type: refers more particularly to a combination of biochemical parameters (mainly to the size of the unglycosylated PrPSc fragment after proteinase K partial digestion) that are independent from the host. Of prion isolates, strains and types In fact, these two strains, although they look virtually the same in the ovinized mice, keep their species characteristics, since they still can be differentiated when further back-passaged on bovinized mice (our unpublished observations). In addition, L-BSE could be differentiated from the classical one through its high propensity to colonize lymphoid compartments (our personal observations). Thus, study of prion replication in the lymphoid tissue and back-passage experiments (or to another intermediate species) can be useful to discriminate between truly identical strains and closely resembling strains. 3.3. When Two Strains Look the Same 4.1. Some Great Examples Illustrating the complexity of the species barrier in frame with the prion strain characterization is a difﬁcult task: a few examples are provided below, that have been chosen for the historical role they played in the prion ﬁeld or because of their highly possible or demonstrated impact on human health (in terms of zoonotic risk). Historically, one of the ﬁrst prion species barrier to be studied with laboratory rodents has been the hamster to mouse transmission: early in the 70’s Kimberlin noticed that a hamster scrapie strain named 263 K hardly passed on mice [22,77]. However, years later, asymptomatic replication of hamster prions was demonstrated in mice [78,79]. Despite the absence of clinical signs, half of the inoculated mice had detectable levels of PrPSc in the brain and also showed the presence of speciﬁc prion disease lesions in the brain. This work was the ﬁrst to further include the search for prion speciﬁc signature in the target organ with or without clinical signs for the evaluation of the barrier species. In seminal experiments, the use of transgenic mice expressing hamster PrP allowed hamster prion transmission to mice, indicating that PrP amino acid differences contributed to the species barrier [80]. Then, PMCA approaches conﬁrmed that mouse (RML strain) prions could progressively be adapted to hamster species [81]. The in vitro adaptation could be achieved within 4–6 rounds (~2 weeks, while in vivo adaptation would have required more than 3 years). Similarly, hamster prions (263 K strain) could be adapted to mouse. Interestingly, both directions of adaptation yielded prions showing ﬁne variations in incubation time/PK-resistance/brain deposition pattern/glycosylation proﬁle, etc. These results suggest that although very similar to what is observed in vivo, PMCA-driven adaptation process is different from what is observed in living conditions. Mad cow disease in the 90s triggered very intense research on the mechanisms involved in the crossing of the species barrier for some prion strains. Investigating the capacity of BSE prions to propagate in new host species led to the initial conclusion that the BSE agent was not able to infect mice expressing only human PrP (HuPrP+/+ PrnP0/0, valine allele at position 129) [1]. Looking closer, however, revealed that the BSE prions ﬁnally needed more time than CJD prions to install and replicate in mice expressing human PrP. 4. To the Study of Species Barrier in Prion Transmission What we currently know from the species barrier crossing is that the phenomenon can take a long time to be observed: adapted prions usually kill all the inoculated animals within a few weeks or months, out of a total incubation time that could range between 60 days for the fastest and up to the entire life of the animals (over 700 days for some mice); by contrast, non-adapted prions usually have incomplete attack rates and incubation times greatly increased and much more variable as compared to the original strain. During the adaptation process, however, these hallmarks faint and newly adapted prions recover full attack rate and reduced as well as highly reproducible incubation time, although several other factors may have been changed in the new host (ﬁnal incubation time, tissue distribution, PK resistance . . . ). Strain-to-strain variation is often associated to the passing of one species to the other. In some instances, this can even result in the adaptation of several different strains: inoculation of transmissible mink encephalopathy (TME) to hamsters resulted in the selection of two different strains, depending on the dilution of the inoculum [75] (see Section 4.3 Coinfections). Recent work report on the inﬂuence of PrPC expression level for the selection of different prion populations (see Section 4.4.1 PrP expression 9 of 29 Pathogens 2018, 7, 5 level) [76]. This is highly questioning in regards with the zoonotic and epidemic risks of such diseases (BSE, variant CJD, for instance). level) [76]. This is highly questioning in regards with the zoonotic and epidemic risks of such diseases (BSE, variant CJD, for instance). 4.1. Some Great Examples After one passage, strain seems to be stabilized, with mice repeatedly succumbing 220 days after inoculation, all other features of the disease were looking similar to the genuine CWD, including the spleen tropism. Recent data further evidenced a threat of possible transmission to humans: Herbst and colleagues described the different behaviours of two different CWD strains—CDW1 strain could pass to hamsters but not to mice. By contrast H95+ strain could infect efﬁciently the mice, while the hamster was less susceptible [90]. Humans’ natural resistance to CWD infection has been hypothesized to rely on a speciﬁc amino acid stretch in the β2 loop of the PrP; by swapping this domain with that of elk PrP, Kurt and colleagues rendered the mice expressing this mutant PrP fully susceptible to CWD [91]. Notably, susceptibility to CJD was inversely reduced. A very recent work presented to the Neuroprion meeting reports that CWD can be passed orally to cynomologus macaques [92]. Scandinavia [88]. Considering the data obtained with scrapie and particularly with the fact that some prion strains apparently can circulate without species barrier, it was of importance to determine to what extent this CWD agent is conﬁned to wild cervids. It is already known that CWD can adapt to certain strains of mice, expressing high level of murine PrP [89]. After one passage, strain seems to be stabilized, with mice repeatedly succumbing 220 days after inoculation, all other features of the disease were looking similar to the genuine CWD, including the spleen tropism. Recent data further evidenced a threat of possible transmission to humans: Herbst and colleagues described the different behaviours of two different CWD strains—CDW1 strain could pass to hamsters but not to mice. By contrast H95+ strain could infect efﬁciently the mice, while the hamster was less susceptible [90]. Humans’ natural resistance to CWD infection has been hypothesized to rely on a speciﬁc amino acid stretch in the β2 loop of the PrP; by swapping this domain with that of elk PrP, Kurt and colleagues rendered the mice expressing this mutant PrP fully susceptible to CWD [91]. Notably, susceptibility to CJD was inversely reduced. A very recent work presented to the Neuroprion meeting reports that CWD can be passed orally to cynomologus macaques [92]. 4.1. Some Great Examples Conversely, there are host species that are susceptible to almost every strain: for example, bank vole rodent turned out to stand as a mammal particularly tolerant to many prions [93], including notably sporadic CJD [11]. Transmission of these prions to bank voles results in a disease at full attack rate, with little or no species barrier and in a tempo similar to transgenic mice overexpressing human PrP. Bank vole and human PrP amino acid sequence are differing by 12%. This indicates that the bank vole PrP conformation is per se prone to conversion by human CJD prions. Recent transmission of a library of prion strains to transgenic mice expressing bank vole PrP (M109) further support the view that bank vole PrP can be converted by many abnormal PrPSc conformations. To strengthen the demonstration, so-called ‘resistant’ animals could be rendered compliant with prion infection after introgression of a genuine or modiﬁed prion protein—for instance, the Drowsy strain affecting Syrian hamsters but not Wild-Type (WT) mice could readily infect mice expressing a chimeric hamster-mouse PrP [94]. The species barriers depend in part on host’s PrP primary structure and in part on the strain itself. Up to date, every one of these species barriers has shown they could be crossed, provided that enough time, correct animal or organ target and passage numbers have been taken into account: hamster prions can pass onto mice [95]; TME can pass to cattle [14]; BSE and vCJD both can adapt to Guinea Pig [96]; CWD transmission from cervids to human is every day more probable, at least under certain speciﬁed conditions [97]: CWD can pass in vitro with PMCA to human brain homogenate but efﬁciency is greatly enhanced if ampliﬁcation/adaptation of the CWD prion has been previously performed through PMCA using CWD template [98]; experimental infections to non-primate monkeys were also efﬁcient [99,100]. In conclusion, the strength of the species barrier is mainly dependent on the parameters that are addressed: a closer look to subclinical disease either centrally or peripherally may lead to the assumption that there is ﬁnally no absolute species barrier, at least to the experimental level. What are the factors that render prions adaptable to their new host? What features allow BSE to stand today as the sole prion that adapts easily to other species? Despite signiﬁcant efforts to improve our knowledge, the answer is still largely unknown. 4.1. Some Great Examples However, several leads have been followed that may help building models for the structure of what we call PrPSc. Some of these models will be evoked at the end of the paper but the few following sections will ﬁrst focus on the experimental parameters that demonstrated their inﬂuence on the strength of the barrier species. 4.1. Some Great Examples The same lab detected transmission of BSE to macaque or cats, while their PrnP0/0 HuPrP+/+ still were alive (after more than 500 days post-inoculation) [2]. Thus, BSE prions were more and more amenable to infect humanized animals. The year after, Bruce and colleagues reported that both BSE and vCJD agents replicated similarly in certain lines of conventional mice and induced the same strain phenotype, thus strengthening the link between the epizootic burst of BSE and the outbreak of British and French vCJD cases [82]. A few years later, Prusiner’s team provided another evidence that BSE and vCJD were etiologically linked [83]: they observed that bovinized mice developed similar pathologies when inoculated with either BSE or vCJD agents (incubation time, lesion distribution and score, PK-resistance proﬁle), these proﬁles being absolutely different from those observed after scrapie infection. These observations conﬁrm that two agents isolated from different species could eventually be the same strain. Other lines of transgenic mice overexpressing the Met allele of human PrP were developed and further provide evidence of the intricate link between BSE and vCJD. In two lines, the disease occurred at incomplete penetrance and was mostly subclinical [84,85]. However, one of the lines showed a higher attack rate extraneurally in the spleen tissue (see below). The cross-species capacities and zoonotic potential of CWD prions are another emerging public health concern. This disease affecting cervids is known for long but got only recently media coverage when concerns about the passage from wild ruminants to human started to get conceivable. While CWD was reported in the early 90’s to mainly circulate between captive wild cervids, [86] a warning was raised against the possible epidemic extension of the disease, which truly occurred in the USA during the last 3 decades (reviewed by Watts [87]) and even recently popped up in 10 of 29 Pathogens 2018, 7, 5 Scandinavia [88]. Considering the data obtained with scrapie and particularly with the fact that some prion strains apparently can circulate without species barrier, it was of importance to determine to what extent this CWD agent is conﬁned to wild cervids. It is already known that CWD can adapt to certain strains of mice, expressing high level of murine PrP [89]. 4.2. Importance of Primary Sequence, Aminoacid Polymorphism The primary sequence of the PrP stands naturally in front line, since it is accepted by almost all the scientiﬁc community that this endogenous protein is the only responsible for the disease. The primary sequence (allelic variations, point mutations) would obviously be the main vector ruling the host’s susceptibility to a given strain. With the introduction of transgenic animals, it became easy to test whether one given PrP sequence could account for the susceptibility to prion diseases: 11 of 29 Pathogens 2018, 7, 5 several studies report the acquired susceptibility of mice following expression of recombinant or chimeric hamster protein [101,102]. Homology between the inoculated prion and the host’s PrP looks like a prerequisite [80,103,104]. In the latter case, transgenic rabbits expressing ovine PrP were fully susceptible to scrapie, showing that the rabbit environment is not, per se, incompatible with prion transconformation, although the animal is known to be naturally resistant to prion infection. However, with the notable example of the bank vole being a universal acceptor, the common view is that structural compatibility between host PrP and the infecting prion strain governs the cross-species transmission of prions. Polymorphism within one species have a dramatic impact on the susceptibility of the host (see for instance how ovine polymorphism is governing scrapie transmission [105,106]). The extreme cases are illustrated by the spontaneous prion conversion attributable to familial point mutation of the human PrP gene responsible for genetic CJD or FFI (for a review, see [107]), or by the I109 M point mutation affecting the bank vole PrP: mice expressing I109 Bank vole PrP spontaneously develop a prion disease within 4 months of age [108]. This phenomenon does not strictly apply to the species barrier paradigm. However, it had been shown that the mutations reported in humans seem to cluster in several groups depending on their ability to cross species barrier and infect mice [109]. In that particular case, species barrier was regarded as a tool for the characterization of different prions strains. From an epidemiological and clinical point of view, it is relevant to consider these point mutations, since they condition full sensitivity or resistance to prion disease. The homozygous methionine or valine in position 129 of the human PrP is determinant for the sensitivity to CJD [110]. To date, all clinical cases of vCJD have only occurred in patients homozygous for methionine at codon 129 [111]. 4.2. Importance of Primary Sequence, Aminoacid Polymorphism However the V129 genotype does not protect against vCJD, despite full protection against BSE [112] Polymorphism in position 219 (E/K) is also associated with resistance to prion infection in Asian population [113]. More recently a G/V polymorphism has also been reported to be responsible for full resistance to Kuru infection and was proposed to result from naturally-driven selection process that increased resistance against Kuru in the exposed population [114]. In addition to governing intra species susceptibility to prion diseases PrP polymorphism greatly inﬂuences the susceptibility of the host to exogenous prions. Noteworthy however, this polymorphism has an impact on the population genetics, with breeds being historically more susceptible to scrapie than others because of genotype variations in positions 136/154/171. The M/V polymorphism at position 129 for human PrP similarly controls the susceptibility of individuals to vCJD [110]. Green et al. report that the elk prion codon 132 polymorphism controls cervid and scrapie prion propagation [115]. In brief, transgenic mice expressing the elk mutant L132 are resistant to CWD prion inoculation (no clinical sign at up to 600 days post infection). However, the authors report the detection of small amounts of PK-resistant PrPSc in the brains of the animals inoculated with M/M132 inoculum but not with M/L132. This suggests that L132 genotype is not resistant to infection but rather requires adaptation passages in order to select a fully adapted prion. It is worth noting that in contrast to elk prions, hamster prions adapted easily to the 132 L elk PrP. Using the Bank vole as a model, Agrimi’s team observed that I/M109 heterozygote animals incorporated equal quantities of both allelic forms of PrP in the prion particles [116]. Thus, at least in some cases, it seems that polymorphism does not entirely rule the susceptibility to prions. Beside natural polymorphism, experiments have been performed to test for the mutual inﬂuence of different PrP molecules to diverse strains of prions. Several experiments of cross species inoculation have been performed by Kimberlin and colleagues to assess the infectious potential of scrapie prions to infect mice, hamsters, etc. [117–119]. Later, when the ﬁrst transgenic mice started to be available, the transgenic PrP was co expressed along with the endogenous one. It rapidly appeared that the expression of both the endogenous and the transgene could result in odd responses to prion infection. 4.2. Importance of Primary Sequence, Aminoacid Polymorphism Chesebro’s team published several reports mentioning the interference of heterologous PrP on the accumulation of prions in a cell culture assay [120,121]. A single amino acid substitution could be responsible for drastic inhibition of prion production in cell culture. But other studies also report on 12 of 29 Pathogens 2018, 7, 5 the inﬂuence of additional factors for the efﬁcient replication of prions [122]. These observations paved the way for a long series of publications mentioning a hypothetical protein X as cofactor in the prion mechanism [123]. But recent in vitro conversion experiments invalidated this hypothesis [124]. 4.4. Inﬂuence of Expression Level and Post Translational Modiﬁcations 4.4. Inﬂuence of Expression Level and Post Translational Modiﬁcations Considering there is a competition for substrate between two different strains, then the amount of available PrP certainly inﬂuences the susceptibility, or to a lesser extent the kinetic of the disease. In addition, the several post translational modiﬁcations that undergoes the PrPC during its journey toward the membrane may also inﬂuence the susceptibility/convertibility of PrP to prions. 4.3. Co-Infections The inﬂuence of co-infection, although not strictly relevant in the species barrier topic, is questioning: since several prion strains can infect a given host, what could a multiple infection look like? One would logically suppose for example that the fastest strain would take over the slowest strain. Surprisingly it happened to be quite the opposite: fast strain 22A inoculated to mice that had ﬁrst received a slow 22C strain showed delayed incubation periods [125]; when a Hyper/Drowsy combination of TME prions was peripherally inoculated to hamster, the fastest Hyper strain progression was delayed when co-administered with the slowest strain Drowsy [126]. The authors proposed a competition between both strains for a limiting PrP resource [127].This competition did not occur when both prions were simultaneously inoculated at different locations [128]. 4.4.1. PrP Expression Level The inﬂuence of PrPC expression level has been assessed on incubation time, infectivity titre and lesion distribution in 3 strains of mice, whose expression level varied from 1⁄2 to 8x [129]. The incubation period was reduced when PrPC level was increased: during an exponential phase, rapid accumulation of PK-resistant PrPSc occurs, followed by a plateau phase, whose length is proportional to the PrPC expression level. The authors suggest the accumulation of toxic forms of the PrP that ultimately induce the clinical signs once a certain threshold has been reached. Recently, our group reported the characterization of three independent prions selected from a single scrapie isolate inoculated to transgenic mice expressing various levels of strictly homologous VRQ allelic forms of ovine PrP. The strains were mainly phenotyped according to their PK-digestion proﬁle and their incubation duration [76]. Upon transmission of a 21 K isolate to overexpressing mice (>3.5×), a new 19 K phenotype is progressively selected, with frequency and PrP levels raising accordingly. A third phenotype (21 K fast) is reported to emerge in a stochastic fashion that outcompetes 19 K prions in high expressor hosts. Although these studies were done in homotypic transmission context, they highlight the key role of PrPC levels in the disease tempo and in the prion selection and emergence. It is likely that PrPC would similarly be at play in heterotypic transmission events. 4.5. Prion Route May Inﬂuence Prion Transmission Fate Lymphoid tropism of some prion strains has been described for a long time [24]. Prions have been shown to replicate and accumulate in follicular dendritic cells (FDC) from the germinal centres of lymph nodes and the spleen. Several other immune cells (like B-cells, macrophages) have been shown to carry prion infectivity, however, it has been demonstrated that FDCs are necessary and sufﬁcient for prion replication in the spleen [136]. Of note, the FDCs are not of lymphoid nor myeloid origin, despite their pivotal role in the initiation and maintenance of immune response: rather, they derive from sub-endothelial cells and differentiate upon trophic interactions with B-cells [137]. Macrophages and B-cells also stain positive for PrPSc but they are mainly involved in PrPSc scavenging and transport, respectively [138,139]. Therefore, lymphoid compartment will have a balanced inﬂuence on peripherally acquired prion fate: in one hand, macrophage and in particular splenic scavenging functions will actively degrade or neutralize prion infectivity, while, on the other hand, FDCs will actively replicate prions that are then shuttled to the terminal nerves ending near the germinal centres [140]. Thus, the resulting lymphoid tropism could be the net result from this balance, as claimed recently by Bartz and colleagues [141], who were able to detect Drowsy infectivity within hours after inoculation but also reported that these prions disappeared thereafter as a consequence of an increased susceptibility to proteases (in vitro PK assay). Prion replication in periphery could otherwise be dictated by strain features. It is for instance well known that vCJD, as opposed to other strains of CJD remarkably replicate within the human lymphoid system [142]. Thus, oral contamination by the vCJD strain, which targets the Peyer’s patches beneath the jejunal epithelium, is much more prone to occur than with other sporadic forms of CJD. However, recent analysis of the last documented Kuru case (in 2003) reveals that despite a certain food borne contamination, the Kuru patient did not exhibit the marked vCJD-typical colonization of the digestive tract. This suggests that peripheral pathogenesis of Kuru is similar to that seen in classical CJD rather than vCJD [143], although a more recent study ﬁnally considered that sCJD and vCJD accumulated similarly in the lymphoid system [144]. Thus, strain lymphotropism does not necessarily reﬂect the preferred inoculation route. 4.5. Prion Route May Inﬂuence Prion Transmission Fate This question had been addressed with comparison of oral and intracerebral routes of BSE inoculation in macaque as a model of species barrier transmission [145,146] and the general conclusions that may be derived from these studies is that lymphoid tropism does not facilitate, per se, the crossing of barrier species. 4.4.2. Secondary Modiﬁcations: Glycosylation, Sialylation, Protease Digestion . . . 4.4.2. Secondary Modiﬁcations: Glycosylation, Sialylation, Protease Digestion . . . Glycosylations and other post translational processings have been shown to play a signiﬁcant role in the transmission and adaptation of prions to a new host. Recent work with PrP mutated to the ﬁrst or the second glycosylation site dramatically increased or suppressed the species barrier upon infection with 2 human prions (MM2CJD and vCJD) or 263 K hamster scrapie [130]. It is worth noting, however, that the amino acid mutations designed for glycosylation alterations may also account for the observed phenotype [131]. In vitro conversion experiments using hamster prions in presence of various mouse PrP constructs suggest that heterologous conversion favours unglycosylated PrP incorporation, while autologous conversion results in the usual 3-banded proﬁle [132]. Glycosylations may be further modiﬁed through sialylation of the sugars. It has recently been shown that sialylation of prion protein could modify the species barrier [41]: while normal mouse brain homogenate needed more than 13 of 29 13 of 29 Pathogens 2018, 7, 5 4 rounds of PMCA to reach a steady state level of ampliﬁcation using a 263 K hamster strain, the same desialylated brain homogenate reaches the plateau in one single round. Reciprocally, hamster brain homogenate failed to amplify 22L or ME7 mouse strains even after 10 rounds but upon desialylation, the PMCA reaches the plateau in 3–4 rounds. It is furthermore shown that sialylation participates in a host/tissue and cell-speciﬁc manner to the regulation of PrP [40,133]. Sialylation process is shown to occur even after the PrP has been converted [40,134]. GlycoPhosphatidyl Inositol (GPI) anchor, which attaches PrP to the membrane through its C-terminus participates also to the conformational landscape of the prions upon infection: upon passage on GPI-/- mice, most of the prions retained their specific characteristics when passaged back to their original host, except CWD which gain in PK resistance and chaotropic [GdnHCl]1/2.stability [135]. 4.7. Cell/organ Selectivity As evoked in the previous section, prions can replicate and their expression could be drastically modulated in several organs outside the central nervous system. In addition to that, peripheral organs may, per se, have a different behaviour with respect to the invading prion and the inoculation route. These observations have been made in our laboratory when we monitored the fate of a scrapie prion strain following inoculation by intracranial or intraperitoneal routes [154]: the disease greatly differed in clinical signs, abnormal prion protein levels and neuropathology. In another study, we monitored brain and spleen of ovinized and humanized mice for the presence of infectivity or PK resistant PrP after inoculation with hamster sc237, CWD or BSE [85]. Overall, the three strains, which are not transmissible to either ovine or human PrP mice, did not indeed replicate efﬁciently in the brains of the inoculated animals: for instance, only 2 out of 29 ovine mice infected with CWD were positive by western blot at the end of their lifespan. By contrast, spleens of these animals were almost consistently positive (Figure 2) and from the early time of infection onward. Overall, the spleen appeared 9–10 fold more permissive than the brain to foreign prions. The reason for such tissue-dependent strength of the species barrier remains to be determined. Spleen PrPC might be more prone to heterotypic conversion than brain PrPC, due to conformational variations. The spleen environment might constitute a better niche, due to prolonged possibility of interactions between brain and spleen (through axonal terminations located close to the germinal centres) or presence of co-factors, such as complement. Ultimately, absence of cell prion toxicity outside the brain could also account for an efﬁcient replication of PrPSc. Whatever the reasons, these features allow prion extending its host range. It also provides and experimental explanation to the high number of asymptomatic individuals exposed to BSE agent in the UK population showing pronounced accumulation of PrPSc in their lymphoid tissue [155]. Pathogens 2017, 6, 5 14 of 30 4.7. Cell/organ Selectivity As evoked in the previous section, prions can replicate and their expression could be drastically modulated in several organs outside the central nervous system. In addition to that, peripheral organs may, per se, have a different behaviour with respect to the invading prion and the inoculation route. 4.7. Cell/organ Selectivity These observations have been made in our laboratory when we monitored the fate of a scrapie prion strain following inoculation by intracranial or intraperitoneal routes [154]: the disease greatly differed in clinical signs, abnormal prion protein levels and neuropathology. In another study, we monitored brain and spleen of ovinized and humanized mice for the presence of infectivity or PK resistant PrP after inoculation with hamster sc237, CWD or BSE [85]. Overall, the three strains, which are not transmissible to either ovine or human PrP mice, did not indeed replicate efficiently in the brains of the inoculated animals: for instance, only 2 out of 29 ovine mice infected with CWD were positive by western blot at the end of their lifespan. By contrast, spleens of these animals were almost consistently positive (Figure 2) and from the early time of infection onward. Overall, the spleen appeared 9–10 fold more permissive than the brain to foreign prions. The reason for such tissue- dependent strength of the species barrier remains to be determined. Spleen PrPC might be more prone to heterotypic conversion than brain PrPC, due to conformational variations. The spleen environment might constitute a better niche, due to prolonged possibility of interactions between brain and spleen (through axonal terminations located close to the germinal centres) or presence of co-factors, such as complement. Ultimately, absence of cell prion toxicity outside the brain could also account for an efficient replication of PrPSc. Whatever the reasons, these features allow prion extending its host range. It also provides and experimental explanation to the high number of asymptomatic individuals exposed to BSE agent in the UK population showing pronounced accumulation of PrPSc in their lymphoid tissue [155] Figure 2. The spleen is much more permissive than the brain to the passage of heterologous prions: percentage of diseased animals and western Blot-positive tissue of BSE (A) or CWD (B) prion agents inoculated intraperitoneally to bovinized, humanized or ovinized mice. Spleen and brain were collected at the death of the animals. Tissue infection was diagnosed upon the detection of PrPSc by western blot. From [85]. Other hypotheses involve a tissue specific clearance metabolism as responsible for the different st ai t opis [141] As e tio ed p e iously i a fo e sectio the autho s desc ibe the successful Figure 2. 4.6. Immune Status, Age of the Host It is for long known that immune status and host age or developmental stage inﬂuence the PrP expression in target organs such as the brain but also and particularly the lymphoid organs [147–149]. As a consequence, susceptibility of young sheep to oral contamination with BSE is drastically decreased after weaning [150]. On the other side of the lifespan, aged mice infected intraperitoneally with RML prions show signiﬁcant longer incubation time than their younger littermates [151]. This is to be related to the decrease of follicular germinal centres with age [152]. Thus, depending on the age at the time of infection, prion replication may be signiﬁcantly affected, with obvious effects on the crossing (or not) of the species barrier. For instance, the transmission of BSE was much more efﬁcient on young mice, 14 of 29 Pathogens 2018, 7, 5 while older animals remained free of clinical sign all along their lifespan [153]. Noteworthy, some aged mice could still replicate at visible levels the prion in their spleen. Thus, in that particular case, the modiﬁcation of the species barrier should be regarded as a consequence of a receptor abundance modulation. Still, these observations are of interest when considering the exposed groups within the whole population. 5. Consequences on Prion Adaptation to New Host Following a substantial species barrier, different scenarios may be possible: (i) Silent passing—The prion may be silently passing as it does when Sc237 is inoculated to ovine PrP mice (see previous section). Several studies have reported that some strains require very long adaptation periods and iterative passaging in order to successfully replicate in one given host [159–161]. Three or more reinoculation steps are often needed to get successful isolation of poor prion transmitters. In such cases, the receptor animals also play a pivotal role, in particular with respect to the amount of target PrPC that could be expressed. In some instances, the resulting prions retain the features of the parental strain [159]. In other instances, the isolated prions differ significantly from the source inoculum [161]. (ii) Progressive evolution—Mutational events. In that case, evolution could concern more than two species: for instance, when questioning the origin of the BSE and vCJD epidemics that hit Western Europe a few decades ago. An atypical form of BSE (BASE) has been shown to evolve into a form that is indistinguishable from BSE in wild-type mice [162]. Others described the progressive evolution from a H-BSE to a classical BSE when the strain was serially inoculated to mice [163]. It has also been reported that passage through an intermediate small ruminant allows BSE to better adapt to humanized mice [164,165]. Overall, BSE prions, whether atypical or classical, seem to display a unique behaviour with regards to species barrier, since it is the only prion strain capable of adaptation to a great variety of hosts [166]. In other situations, evolution may be more abrupt (incubation duration deceased rapidly between 1st and 2nd passage: see for instance, the emergence of T1-Ov and T2-Ov strains following experimental passage of MM2 CJD to ovine PrP mice) [167]. The progressive evolution of prions was further supported by Weissmann’s group, who argued for a Darwinian evolution of prions, through silent (or not) “mutations” [45]. These data were supported by in vitro experiments that establish prion evolution induced by chemical prionostatic drugs and selection of prion ‘quasi species’ from an initially homogenous prion strain [168]. As mentioned earlier, other groups support the concept of a ‘portfolio of conformations’ for a given strain, that could match (or not) with a portfolio of possible conformations for the receptor PrP [49,169]. 5. Consequences on Prion Adaptation to New Host The observation that H-BSE phenotype is lost upon passage to hamster mice and restored when passaged back to bovine fits this model [170]. An evolution of the concept emerged from the observation that two strains could too closely look similar to be distinguished, albeit showing different incubation times. In addition, these strains could constantly evolve form one strain to the other, rendering them impossible to clone [171]. 4.7. Cell/organ Selectivity The spleen is much more permissive than the brain to the passage of heterologous prions: percentage of diseased animals and western Blot-positive tissue of BSE (A) or CWD (B) prion agents inoculated intraperitoneally to bovinized, humanized or ovinized mice. Spleen and brain were collected at the death of the animals. Tissue infection was diagnosed upon the detection of PrPSc by western blot. From [85]. Figure 2. The spleen is much more permissive than the brain to the passage of heterologous prions: percentage of diseased animals and western Blot-positive tissue of BSE (A) or CWD (B) prion agents inoculated intraperitoneally to bovinized, humanized or ovinized mice. Spleen and brain were collected at the death of the animals. Tissue infection was diagnosed upon the detection of PrPSc by western blot. From [85]. Other hypotheses involve a tissue specific clearance metabolism as responsible for the different Figure 2. The spleen is much more permissive than the brain to the passage of heterologous prions: percentage of diseased animals and western Blot-positive tissue of BSE (A) or CWD (B) prion agents inoculated intraperitoneally to bovinized, humanized or ovinized mice. Spleen and brain were collected at the death of the animals. Tissue infection was diagnosed upon the detection of PrPSc by western blot. From [85]. Pathogens 2018, 7, 5 Pathogens 2018, 7, 5 15 of 29 Other hypotheses involve a tissue speciﬁc clearance metabolism as responsible for the different strain tropism [141]. As mentioned previously in a former section, the authors describe the successful PMCA ampliﬁcation of Drowsy TME in the spleen of peripherally-inoculated hamsters, although this strain was not supposed to replicate in that tissue. They conclude that the strain selectivity against Drowsy prions in the lymphoid organs is a consequence of the strain-speciﬁc efﬁcient removal of the infectious material. This hypothesis however does not seem to be valid when brain prion distribution was concerned [156,157]: both studies argue at 20 years interval and with different tools against a brain tissue selectivity. Cell culture experiments have also been set up in order to evaluate strain selectivity but the overall resistance of cell lines or even primary cultures to prion infection remains to be overridden [158]. 5.1. De Novo Synthesis and In Vitro Assessment of Species Barrier The experiments reported in the previous sections required the massive use of laboratory animals for the evaluation of species barrier. However, even with the use of transgenic mice that considerably shorten the incubation time in comparison to what is recorded with the target animals, the time needed to obtain such data with 4 and even 5 successive passages is extremely long, not to mention the high number of animals to be included . . . For that reason, alternative in vitro methods were eagerly needed. Most of them were conducted using PMCA or QuIC methods. As a recent and comprehensive review Pathogens 2018, 7, 5 16 of 29 has recently been published on the topic [172], we will mainly focus on the latest highlights of in vitro transmission barrier studies. The ability of PMCA to create de novo prions was certainly one of the most interesting contribution of this technique [62]. Besides the deﬁnitive proof of the proteinaceous origin of the prion disease, it allowed to determine a few RNA and phospholipid cofactors that are crucially needed for the conversion to take place. But several other cofactors have been evaluated, whose inﬂuence in strain selection appeared to be determinant [173,174]. In that later case, removal of phosphatidylserine provoked the phenotypic convergence of the 3 strains tested, as well as a 5 log10 reduction in infectivity. This phenotypic convergence has been reported elsewhere [175], suggesting that the in vitro environment needs to be better controlled in order to properly mimic what is observed in vivo. In addition to these ﬁdelity problems, several studies mentioned the reduction or absence of infectivity resulting from the QuIC ampliﬁcation of prions [176,177]. 5.2. In Vitro Assessment of High Species Barrier Barria et al. reported the generation of cervid prions that can replicate on human matrix, provided that they have been previously submitted to 2 rounds of PMCA using cervid brain lysate [98]. Conversely, PMCA ampliﬁcation of 263K scrapie using partially deglycosylated PrP matrix produced a mixture of classical and atypical PrPSc proﬁles that suggested the development of a new strain. If true, however, this new strain was fully restored to the classical 263 K proﬁle after a series of 10 rounds in classical conditions [178]. PMCA was also used to generate in vitro prions using PrP from mammals known to be naturally resistant to the disease [179–181]. In addition to prove that allegedly resistant animals produced transconformation-prone PrP proteins, these studies established that BSE could induce in vitro the conversion of these so-called resistant PrP and that this PrPSc was fully infectious when administered to the target animal. Similarly, PMCA was used to render mice susceptible to hamster prions and vice versa [95]. While attempting to reduce the number of passages needed for the adaptation of scrapie strains to ovine mice, it was noted that some strains were compliant with a PMCA shortcut, while others produced prions with divergent features [182]. PMCA should therefore not be regarded as a fully effective method to faithfully reproduce the prions obtains with bioassays. 6. Proposed Mechanisms for PrP Conversion, Strain Determination and Species Barrier Crossing roposed Mechanisms for PrP Conversion, Strain Determination and Species Barrier Crossing Despite this signiﬁcant amount of experimental data, we still are waiting for some unifying model for prion conversion. Several hypotheses have been proposed for the transconformation and the elongation of infectious prions following the introduction of a seed. A ﬁrst generation of models, based on a Prusiner’s hypothesis [183] invokes the faithful reproduction of a template. This model however does not account for the emergence of variant prions strains from a given cloned parent. Therefore, a mechanism should be at work to allow this diversity to occur. 6.1. Prion Diversity from a Structural Portfolio and Selection of Mutants upon Species Barrier Crossing 6.2. Deformed Templating 6.2. Deformed Templating Based on observations published in 2012, Makarava and colleagues proposed a model in which recombinant hamster ﬁbrils induce PrPC to form what they call an atypical PrPres [184]. In that model, two major successive steps are at work for the induction of PrPSc and the generation of a new strain of prion: in their experiment, the ﬁrst step results from the formation of atypical PrPres triggered by 0.5 M GdnHCl. This atypical PrPres was detected in the brain of mice at their end of life but was not associated to any clinical sign. Then, the PrPres could trigger the slow conversion of PrPC into PrPSc. The kinetics for the production of both PrPres and PrPSc are quite different: generation of atypical PrPres is RNA independent and its FTIR structure closely resembles that of parental ﬁbrils; the second step, which was named “deformed templating” is more stochastic and less described. It is postulated that the strain structural diversity is acquired depending on the environmental constraints. The authors added that once formed, PrPSc does not require atypical PrPres anymore and outcompetes its rival thanks to favourable kinetic constants (Figure 3). Since these two steps are independent, they could occur in separate animals. This hypothesis would thus explain why the crossing of the species barrier may be achieved even after several successive passages in recipient animals in which no classical PrPSc could be detected. According to Makarava, though, one should be able to detect the atypical PrPres. At the moment, this was conﬁrmed in the case of 263 K scrapie prions [185]. In addition, the authors propose that, in contrast to the ﬁrst step, the rate of deformed templating is not inﬂuenced by PrPC concentration. This model does not address however the mechanisms that concur to the production of this atypical PrPres. Based on observations published in 2012, Makarava and colleagues proposed a model in which recombinant hamster fibrils induce PrPC to form what they call an atypical PrPres [184]. In that model, two major successive steps are at work for the induction of PrPSc and the generation of a new strain of prion: in their experiment, the first step results from the formation of atypical PrPres triggered by 0.5 M GdnHCl. This atypical PrPres was detected in the brain of mice at their end of life but was not associated to any clinical sign. 6.2. Deformed Templating 6.2. Deformed Templating Then, the PrPres could trigger the slow conversion of PrPC into PrPSc. The kinetics for the production of both PrPres and PrPSc are quite different: generation of atypical PrPres is RNA independent and its FTIR structure closely resembles that of parental fibrils; the second step, which was named “deformed templating” is more stochastic and less described. It is postulated that the strain structural diversity is acquired depending on the environmental constraints. The authors added that once formed, PrPSc does not require atypical PrPres anymore and outcompetes its rival thanks to favourable kinetic constants (Figure 3). Since these two steps are independent, they could occur in separate animals. This hypothesis would thus explain why the crossing of the species barrier may be achieved even after several successive passages in recipient animals in which no classical PrPSc could be detected. According to Makarava, though, one should be able to detect the atypical PrPres. At the moment, this was confirmed in the case of 263 K scrapie prions [185]. In addition, the authors propose that, in contrast to the first step, the rate of deformed templating is not influenced by PrPC concentration. This model does not address however the mechanisms that concur to the production of this atypical PrPres. Figure 3. Schematic presentation of the mechanism, illustrating genesis of PrPSc triggered by rPrP fibrils. In a first step, rPrP fibrils seeded atypical PrPres, a transmissible form of PrP that replicates silently without causing clinical disease. Replication of atypical PrPres occasionally produces PrPSc in seeding events that appears to be rare and stochastic as described for a deformed templating mechanism. PrPSc replicates faster than atypical PrPres and eventually replaces it during serial passages. The two forms atypical PrPres and PrPSc can be distinguished after PK treatment via staining Western blot analyses with discriminating antibodies. Atypical PrPres, alternative self-replicating state of prion protein; PrPSc, prion protein scrapie isoform; rPrP, recombinant prion protein. From [185]. Figure 3. Schematic presentation of the mechanism, illustrating genesis of PrPSc triggered by rPrP ﬁbrils. In a ﬁrst step, rPrP ﬁbrils seeded atypical PrPres, a transmissible form of PrP that replicates silently without causing clinical disease. Replication of atypical PrPres occasionally produces PrPSc in seeding events that appears to be rare and stochastic as described for a deformed templating mechanism. PrPSc replicates faster than atypical PrPres and eventually replaces it during serial passages. 6.1. Prion Diversity from a Structural Portfolio and Selection of Mutants upon Species Barrier Crossing 6.1. Prion Diversity from a Structural Portfolio and Selection of Mutants upon Species Barrier Crossin Collinge and Clarke proposed in 2007 a model where prions are described as a panel of thermodynamically favourable conformations, referred to as a portfolio, of which some structures may or may not be selected when passing from one host to another [49]. According to this hypothesis, the strength of the transmission barrier reﬂects the overlap between the available portfolios of a given primary PrP sequence in two different hosts: the larger they overlap, the lower the species barrier. Some questions still remain to be addressed: in particular, what were the mechanisms at work in the generation of this primary diversity and why the resulting phenotype always displays clonal properties. In addition, observations with promiscuous strains like BSE or universal acceptors like the Bank vole question this view: for instance, one should expect from the bank vole that accepts almost every prion to display a conformational portfolio that includes most of the prion strains portfolios; if true, then no change should be seen when the strains will be passed from an host to the bank vole, which eventually was not the case [12]. When passed on bank vole, most of the strains showed an 17 of 29 Pathogens 2018, 7, 5 evolution in their characters but each time the character that emerged was alone, as if every other conformational state was drastically silenced (although it could express in the context of another inoculated prion). Therefore, we still needed a model that ﬁts the puzzling data. The following sections will delineate the factors that have been taken into account in the available studies. Pathogens 2017, 6, 5 17 of 30 6.2. Deformed Templating 6.2. Deformed Templating The two forms atypical PrPres and PrPSc can be distinguished after PK treatment via staining Western blot analyses with discriminating antibodies. Atypical PrPres, alternative self-replicating state of prion protein; PrPSc, prion protein scrapie isoform; rPrP, recombinant prion protein. From [185]. Figure 3. Schematic presentation of the mechanism, illustrating genesis of PrPSc triggered by rPrP fibrils. In a first step, rPrP fibrils seeded atypical PrPres, a transmissible form of PrP that replicates silently without causing clinical disease. Replication of atypical PrPres occasionally produces PrPSc in seeding events that appears to be rare and stochastic as described for a deformed templating mechanism. PrPSc replicates faster than atypical PrPres and eventually replaces it during serial passages. The two forms atypical PrPres and PrPSc can be distinguished after PK treatment via staining Western blot analyses with discriminating antibodies. Atypical PrPres, alternative self-replicating state of prion protein; PrPSc, prion protein scrapie isoform; rPrP, recombinant prion protein. From [185]. Figure 3. Schematic presentation of the mechanism, illustrating genesis of PrPSc triggered by rPrP ﬁbrils. In a ﬁrst step, rPrP ﬁbrils seeded atypical PrPres, a transmissible form of PrP that replicates silently without causing clinical disease. Replication of atypical PrPres occasionally produces PrPSc in seeding events that appears to be rare and stochastic as described for a deformed templating mechanism. PrPSc replicates faster than atypical PrPres and eventually replaces it during serial passages. The two forms atypical PrPres and PrPSc can be distinguished after PK treatment via staining Western blot analyses with discriminating antibodies. Atypical PrPres, alternative self-replicating state of prion protein; PrPSc, prion protein scrapie isoform; rPrP, recombinant prion protein. From [185]. Figure 3. Schematic presentation of the mechanism, illustrating genesis of PrPSc triggered by rPrP fibrils. In a first step, rPrP fibrils seeded atypical PrPres, a transmissible form of PrP that replicates silently without causing clinical disease. Replication of atypical PrPres occasionally produces PrPSc in seeding events that appears to be rare and stochastic as described for a deformed templating mechanism. PrPSc replicates faster than atypical PrPres and eventually replaces it during serial passages. The two forms atypical PrPres and PrPSc can be distinguished after PK treatment via staining Western blot analyses with discriminating antibodies. Atypical PrPres, alternative self-replicating state of prion protein; PrPSc, prion protein scrapie isoform; rPrP, recombinant prion protein. From [185]. Figure 3. Schematic presentation of the mechanism, illustrating genesis of PrPSc triggered by rPrP ﬁbrils. 6.2. Deformed Templating 6.2. Deformed Templating In a ﬁrst step, rPrP ﬁbrils seeded atypical PrPres, a transmissible form of PrP that replicates silently without causing clinical disease. Replication of atypical PrPres occasionally produces PrPSc in seeding events that appears to be rare and stochastic as described for a deformed templating mechanism. PrPSc replicates faster than atypical PrPres and eventually replaces it during serial passages. The two forms atypical PrPres and PrPSc can be distinguished after PK treatment via staining Western blot analyses with discriminating antibodies. Atypical PrPres, alternative self-replicating state of prion protein; PrPSc, prion protein scrapie isoform; rPrP, recombinant prion protein. From [185]. In an attempt to obtain insight into the quaternary structure of the PrPSc assemblies, our team recently published data obtained using velocity sedimentation gradients on urea-denatured and refolded purified PrPSc associated to the assessment of their specific infectivity [186]: we demonstrated the existence of stable packs of oligomeric subunits (suPrP) that encode the main strain structural determinants: when PrPSc aggregates were denatured under increasing concentrations of urea, the velocity sedimentation gradients evolved from large polydisperse aggregates toward the generation of small elements, presumably trimers that were named suPrP. Upon dialysis refolding, In an attempt to obtain insight into the quaternary structure of the PrPSc assemblies, our team recently published data obtained using velocity sedimentation gradients on urea-denatured and refolded puriﬁed PrPSc associated to the assessment of their speciﬁc infectivity [186]: we demonstrated the existence of stable packs of oligomeric subunits (suPrP) that encode the main strain structural determinants: when PrPSc aggregates were denatured under increasing concentrations of urea, the velocity sedimentation gradients evolved from large polydisperse aggregates toward the generation 18 of 29 Pathogens 2018, 7, 5 of small elements, presumably trimers that were named suPrP. Upon dialysis refolding, the velocity sedimentation gradients identiﬁed condensation of refolded aggregates (rfPrP) but with a different distribution from that before denaturation. SuPrP bricks turned out to be fully PK-sensitive and unable to template infectivity either in vitro or in bio assays. However, upon condensation the suPrP bricks regained full infectivity and PK-resistance properties of the parental strain. One of the most important ﬁndings was the fact that suPrP, rfPrP and PrPSc shared a dynamic equilibrium: upon dilution of 263 K PrPSc in physiological buffer, a rapid decrease of the light scattered by the oligomer solutions showed a signiﬁcant reduction in the size of the particles, resulting from the dissociation of the PrPSc into suPrP. 6.2. Deformed Templating 6.2. Deformed Templating When local suPrP concentration was restored and urea removed, condensation of the suPrP into rfPrP could be observed by western blot and infectivity restored as assessed with PMCA. Thus, the results presented in this work suggest the existence of two organization levels within prion assemblies (Figure 4), one suPrP oligomeric subunit (that could contain 3–5 monomers) and a meta assembly that gathers the suPrP subunits and supports the strain infectivity level and structural conformation features. Whether the suPrP pre-exist to the PrPSc before being included in the elongating polymer or the suPrP results from the incorporation of PrPC into the polymer remains to be addressed. The former hypothesis however implies that PrPC and suPrP shall be separated in normal conditions. This mechanistic proposition for the generation of elementary infectious prion bricks that co-exist as an equilibrium with larger assemblies is compatible with the portfolio model of Collinge. We propose the initial coexistence of several structurally different prions within a single brain homogenate [76]: the emergence of a new strain after prion inoculation to a strictly homologous recipient animal results from a difference in PrPC expression level between inoculum donor and the recipient transgenic mice; the original 21 K strain may be favoured in low PrP expressor animals because the elementary brick could be more efﬁcient at recruiting PrPC at low concentration, while the bricks that lead to 19 K phenotype would beneﬁt the advantage of high PrP expression. Alternatively, stochastic events mays also produce a third strain that could outcompete the two others. It is probable that in a heterologous transmission, prion inoculum will ﬁrst depolymerize just after injection and produce the main suPrP that is observed in the gradient experiments. Then, the elementary bricks would have to recruit PrPC that may or may not accommodate the suPrP. This phenomenon could be highly stochastic, the probability that host PrP adopts a conformational state compatible with the foreign suPrP should be related to the proximity of prion strain and host. The generated assemblies could be rapidly stabilized and ampliﬁed, thus producing an infection with no apparent species barrier; conversely, when host PrP could not ﬁt the topological constraints imposed by inoculum suPrP, the process would need longer time to produce and test pseudo stabilized oligomers or new suPrP that would ultimately emerge as a new prion strain. 19 of 29 Pathogens 2018, 7, 5 Figure 4. 6.2. Deformed Templating 6.2. Deformed Templating (A) Evolution of PK resistance and templating propensities of different types of PrP assemblies obtained after sequential unfolding and refolding of the parental prion. PrPSc is the native prion; suPrP is the elementary oligomeric PrP subunit; and rfPrP is the refolded conformer formed after the polymerization of suPrP. The process of conversion of suPrP into rfPrP requires a conformational change in the PrP protomer of suPrP (represented here as a sphere) to form infectious and PK-resistant assemblies (represented as stack of torus); (B) The conserved differential proteolytic pattern of rfPrPT1-Ov-21K and rfPrPT2-Ov-19K suggests that their respective suPrPs (represented respectively as yellow and red spheres) exhibit distinct conformations. During the refolding step (C), two modes of organization contribute to the cohesion within PrPSc assemblies. Weak interactions (in blue) are involved in maintaining the overall quaternary structure by stacking suPrPs, when strong interactions are involved in the cohesion of PrP protomers in suPrP oligomers. The weakness of the interactions interlinking suPrP means that PrPSc assembly and disassembly are highly dynamic events, even in the absence of a chaotropic agent and free suPrP could exist in equilibrium with infectious assemblies. From [186]. Figure 4. The role of suPrP in the dynamics of PrPSc assemblies. (A) Evolution of PK resistance and templating propensities of different types of PrP assemblies obtained after sequential unfolding and refolding of the parental prion. PrPSc is the native prion; suPrP is the elementary oligomeric PrP subunit; and rfPrP is the refolded conformer formed after the polymerization of suPrP. The process of conversion of suPrP into rfPrP requires a conformational change in the PrP protomer of suPrP (represented here as a sphere) to form infectious and PK-resistant assemblies (represented as stack of torus); (B) The conserved differential proteolytic pattern of rfPrPT1-Ov-21K and rfPrPT2-Ov-19K suggests that their respective suPrPs (represented respectively as yellow and red spheres) exhibit distinct conformations. During the refolding step (C), two modes of organization contribute to the cohesion within PrPSc assemblies. Weak interactions (in blue) are involved in maintaining the overall quaternary structure by stacking suPrPs, when strong interactions are involved in the cohesion of PrP protomers in suPrP oligomers. The weakness of the interactions interlinking suPrP means that PrPSc assembly and disassembly are highly dynamic events, even in the absence of a chaotropic agent and free suPrP could exist in equilibrium with infectious assemblies. From [186]. Figure 4. 6.2. Deformed Templating 6.2. Deformed Templating The role of suPrP in the dynamics of PrPSc assemblies. (A) Evolution of PK resistance and templating propensities of different types of PrP assemblies obtained after sequential unfolding and refolding of the parental prion. PrPSc is the native prion; suPrP is the elementary oligomeric PrP subunit; and rfPrP is the refolded conformer formed after the polymerization of suPrP. The process of conversion of suPrP into rfPrP requires a conformational change in the PrP protomer of suPrP (represented here as a sphere) to form infectious and PK-resistant assemblies (represented as stack of torus); (B) The conserved differential proteolytic pattern of rfPrPT1-Ov-21K and rfPrPT2-Ov-19K suggests that their respective suPrPs (represented respectively as yellow and red spheres) exhibit distinct conformations. During the refolding step (C), two modes of organization contribute to the cohesion within PrPSc assemblies. Weak interactions (in blue) are involved in maintaining the overall quaternary structure by stacking suPrPs, when strong interactions are involved in the cohesion of PrP protomers in suPrP oligomers. The weakness of the interactions interlinking suPrP means that PrPSc Figure 4. The role of suPrP in the dynamics of PrPSc assemblies. (A) Evolution of PK resistance and templating propensities of different types of PrP assemblies obtained after sequential unfolding and refolding of the parental prion. PrPSc is the native prion; suPrP is the elementary oligomeric PrP subunit; and rfPrP is the refolded conformer formed after the polymerization of suPrP. The process of conversion of suPrP into rfPrP requires a conformational change in the PrP protomer of suPrP (represented here as a sphere) to form infectious and PK-resistant assemblies (represented as stack of torus); (B) The conserved differential proteolytic pattern of rfPrPT1-Ov-21K and rfPrPT2-Ov-19K suggests that their respective suPrPs (represented respectively as yellow and red spheres) exhibit distinct conformations. During the refolding step (C), two modes of organization contribute to the cohesion within PrPSc assemblies. Weak interactions (in blue) are involved in maintaining the overall quaternary structure by stacking suPrPs, when strong interactions are involved in the cohesion of PrP protomers in suPrP oligomers. The weakness of the interactions interlinking suPrP means that PrPSc Figure 4 The role of suPrP in the dynamics of PrPSc assemblies (A) Evolution of PK resistance and Figure 4. The role of suPrP in the dynamics of PrPSc assemblies. (A) Evolution of PK resistance and Figure 4. The role of suPrP in the dynamics of PrPSc assemblies. 6.2. Deformed Templating 6.2. Deformed Templating Pathogens 2018, 7, 5 20 of 29 20 of 29 7. Conclusions Prion strains and species barrier phenomena still remain difﬁcult questions to address. Much knowledge has been gained regarding the characterization of the strains (thanks in particular to the PMCA in vitro methods and to the gradient fractionation techniques). The model proposed by our lab identiﬁed some features for the faithful reproduction of prions and proposed a kind of generic polymerization principle where prion ﬁbres exist in equilibrium with one or possibly several sub-units that contain the structural strain determinants. Crossing of species barrier could result from the emergence of one of these sub-assemblies: this hypothesis is in good agreement with the quasi species theory proposed by Weissmann and colleagues. Another non-exclusive hypothesis relies on the ability of the prion inoculum to undergo progressive templating deformation by iterative adjustment of the host PrP structure upon oligomerization. This hypothesis also ﬁts the model proposed by Makarava et al., which focuses on the strain adaptation. It would explain why and how a given strain, adapted to its host, is able to infect a new host, sometimes rapidly and sometimes very slowly. 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The process of conversion of suPrP into rfPrP requires a conformational change in the PrP protomer of suPrP (represented here as a sphere) to form infectious and PK-resistant assemblies (represented as stack of torus); (B) The conserved differential proteolytic pattern of rfPrPT1-Ov-21K and rfPrPT2-Ov-19K suggests that their respective suPrPs (represented respectively as yellow and red spheres) exhibit distinct conformations. During the refolding step (C), two modes of organization contribute to the cohesion within PrPSc assemblies. Weak interactions (in blue) are involved in maintaining the overall quaternary structure by stacking suPrPs, when strong interactions are involved in the cohesion of PrP protomers in suPrP oligomers. The weakness of the interactions interlinking suPrP means that PrPSc assembly and disassembly are highly dynamic events, even in the absence of a chaotropic agent and free suPrP could exist in equilibrium with infectious assemblies From [186] Figure 4. The role of suPrP in the dynamics of PrPSc assemblies. (A) Evolution of PK resistance and templating propensities of different types of PrP assemblies obtained after sequential unfolding and refolding of the parental prion. PrPSc is the native prion; suPrP is the elementary oligomeric PrP subunit; and rfPrP is the refolded conformer formed after the polymerization of suPrP. The process of conversion of suPrP into rfPrP requires a conformational change in the PrP protomer of suPrP (represented here as a sphere) to form infectious and PK-resistant assemblies (represented as stack of torus); (B) The conserved differential proteolytic pattern of rfPrPT1-Ov-21K and rfPrPT2-Ov-19K suggests that their respective suPrPs (represented respectively as yellow and red spheres) exhibit distinct conformations. During the refolding step (C), two modes of organization contribute to the cohesion within PrPSc assemblies. 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https://openalex.org/W2058946094	https://annals-general-psychiatry.biomedcentral.com/track/pdf/10.1186/1744-859X-9-S1-S154	English	null	Regional grey matter volumetric changes in forensic schizophrenia patients: a magnetic resonance imaging study comparing the brain structure of patients who have seriously and violently offended with those of patients who have not	Annals of general psychiatry	2,010	cc-by	706	Results The two groups were matched with respect to age, gender and illness duration, but the group with a history of serious violence was on average receiving a higher dose of antipsychotic medication than the other group. There were local regions of reduced grey matter volume in the group with a history of serious and violent offending, compared with the other group without such a history. Significant voxels (p < 0.05, corrected for multiple comparisons) were noted bilaterally in the cerebellum and in BA 39 and 40. The aim of this study was to carry out the first voxel- based morphometry study of grey matter changes in the whole brain in schizophrenia associated with a history of seriously and violently offending. Puri et al. Annals of General Psychiatry 2010, 9(Suppl 1):S154 http://www.annals-general-psychiatry.com/content/9/S1/S154 4Head of Forensic Neurosciences, Lipid Neuroscience Group, Imperial College London and Three Bridges Medium Secure Unit, West London Mental Health NHS Trust, Middlesex, UK Regional grey matter volumetric changes in forensic schizophrenia patients: a magnetic resonance imaging study comparing the brain structure of patients who have seriously and violently offended with those of patients who have not Basant K Puri1, Serena J Counsell1, Nadeem Saeed1, Marcelo G Bustos2, Graeme M Bydder3, Ian H Treasaden4* From 1st International Congress on Neurobiology and Clinical Psychopharmacology and European Psychiatric Association Conference on Treatment Guidance Thessaloniki, Greece. 19-22 November 2009 Materials and methods Structural cerebral MRI scans of 26 patients with schi- zophrenia were analyzed using voxel-based morphome- try: 13 of the patients had seriously and violently offended directly as a result of schizophrenia prior to admission, the offences consisting of homicide, attempted murder or wounding with intent to cause grievous bodily harm; the other 13 patients did not have a history of violence. There was no history of comorbid psychoactive substance misuse disorder in any of the patients. Voxelwise generalized linear mod- elling was applied to the processed magnetic resonance data using permutation-based non-parametric testing, forming clusters at t > 2.3 and testing clusters for significance at p < 0.05, corrected for multiple compar- isons across space. © 2009 Puri et al.; licensee BioMed Central Ltd. Acknowledgements h k h h Acknowledgements We thank the Three Bridges Medium Secure Unit and the MRC. abnormality in neural circuits involved in verbal working memory in this group of patients. abnormality in neural circuits involved in verbal working memory in this group of patients. Conclusions These regions are important in verbal working memory. The cerebellum may integrate inputs from ventrolateral prefrontal cortex and parietal regions, providing a cor- rective signal that refines the process of rehearing the contents of the phonological store. A strong connection has been hypothesized between the supramarginal region corresponding to BA 39/40 and Broca’s area, which may correspond largely to the arcuate fasciculus, with the connectional pattern of the language regions of this model fitting the network of parietotemporal-pre- frontal connections that participate in working memory. Therefore our results point to the possibility of an © 2009 Puri et al.; licensee BioMed Central Ltd. © 2009 Puri et al.; licensee BioMed Central Ltd. Page 2 of 2 Page 2 of 2 Puri et al. Annals of General Psychiatry 2010, 9(Suppl 1):S154 http://www.annals-general-psychiatry.com/content/9/S1/S154 Author details 1 Author details 1Imaging Sciences Department, Hammersmith Hospital, Imperial College London, UK. 2Three Bridges Medium Secure Unit, West London Mental Health NHS Trust, Middlesex, UK. 3Department of Radiology, University of California, San Diego, School of Medicine, San Diego, CA, USA. 4Head of Forensic Neurosciences, Lipid Neuroscience Group, Imperial College London and Three Bridges Medium Secure Unit, West London Mental Health NHS Trust, Middlesex, UK. Published: 22 April 2010 doi:10.1186/1744-859X-9-S1-S154 doi:10.1186/1744-859X-9-S1-S154 Cite this article as: Puri et al.: Regional grey matter volumetric changes in forensic schizophrenia patients: a magnetic resonance imaging study comparing the brain structure of patients who have seriously and violently offended with those of patients who have not. Annals of General Psychiatry 2010 9(Suppl 1):S154. 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https://openalex.org/W2066131038	https://europepmc.org/articles/pmc3814761?pdf=render	English	null	Bovine Lactoferricin-induced Anti-inflammation Is, in Part, via Up-regulation of Interleukin-11 by Secondary Activation of STAT3 in Human Articular Cartilage	Journal of biological chemistry/The Journal of biological chemistry	2,013	cc-by	10,881	Bovine Lactoferricin-induced Anti-inflammation Is, in Part, via Up-regulation of Interleukin-11 by Secondary Activation of STAT3 in Human Articular Cartilage* g publication,July 29, 2013, and in revised form, September 4, 2013 Published, JBC Papers in Press,September 13, 2013, DOI 10.107 Dongyao Yan‡, Ranjan Kc‡, Di Chen‡, Guozhi Xiao‡, and Hee-Jeong Im‡§¶1 gy j g From the Departments of ‡Biochemistry, §Orthopedic Surgery, and ¶Internal Medicine, Section of Rheumatology, Rush University Medical Center, Chicago, Illinois 60612 and the Department of Bioengineering, University of Illinois at Chicago, Chicago, Illinois 60612 Background: Bovine lactoferricin (LfcinB) promotes anti-catabolism and anti-inflammation in articular cartilage. Results: LfcinB induces IL-11 via AP-1, which in turn induces TIMP-1 via STAT3. Conclusion: LfcinB sequentially regulates IL-11 and TIMP-1 expression through distinct mechanisms in artic chondrocytes. Significance: These findings further suggest the potential of LfcinB as a novel therapeutic agent in ost Bovine lactoferricin (LfcinB), a multifunctional peptide, was recently demonstrated to be anti-catabolic and anti-inflamma- tory in human articular cartilage. LfcinB blocks IL-1-mediated proteoglycan depletion, matrix-degrading enzyme expression, and pro-inflammatory mediator induction. LfcinB selectively activates ERK1/2, p38 (but not JNK), and Akt signaling. How- ever, the relationship between these pathways and LfcinB target genes has never been explored. In this study, we uncovered the remarkable ability of LfcinB in the induction of an anti-inflam- matory cytokine, IL-11. LfcinB binds to cell surface heparan sul- fate to initiate ERK1/2 signaling and activate AP-1 complexes composed of c-Fos and JunD, which transactivate the IL-11 gene. The induced IL-11 functions as an anti-inflammatory and chondroprotective cytokine in articular chondrocytes. Our data show that IL-11 directly attenuates IL-1-mediated catabolic and inflammatory processes ex vivo and in vitro. Moreover, IL-11 activates STAT3 signaling pathway to critically up-regulate TIMP-1 expression, as a consecutive secondary cellular re- sponse after IL-11 induction by LfcinB-ERK-AP-1 axis in human adult articular chondrocytes. The pathological relevance of IL-11 signaling to osteoarthritis is evidenced by significant down-regulation of its cognate receptor expression in osteoar- thritic chondrocytes. Together, our results suggest a two-step mechanism, whereby LfcinB induces TIMP-1 through an IL-11- dependent pathway involving transcription factor AP-1 and STAT3. to disruption by various noxious stimuli, such as pro-inflamma- tory cytokines. In particular, IL-1 is considered as a prominent cytokine that perturbs cartilage homeostasis and results in ECM degradation. IL-1 has been shown to inhibit ECM synthe- sis (1), induce cartilage-degrading proteases and inflammatory mediators (2), and enhance chondrocyte apoptosis (3). * Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth Grants AR053220 (to H.-J. I.), AR062136 (to H.-J. I.), and AR055915 (to D. C.). 1 To whom correspondence should be addressed: Cohn Research BD 516, 1735 W. Harrison, Rush University Medical Center, Chicago, IL 60612. Tel.: 312-942-3091; Fax: 312-942-3053; E-mail: Hee-Jeong_Sampen@rush.edu. 2 The abbreviations used are: ECM, extracellular matrix; TLR, Toll-like receptor; OA, osteoarthritis; TIMP, tissue inhibitor of metalloproteinase; MMP, matrix THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 44, pp. 31655–31669, November 1, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 44, pp. 31655–31669, November 1, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 44, pp. 31655–31669, November 1, 2013 © 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A. metalloproteinase; qPCR, quantitative PCR; PG, proteoglycan; HS, heparan sulfate; AP-1, activator protein 1; HSPG, heparan sulfate proteoglycan. EXPERIMENTAL PROCEDURES Materials—Anti-MMP-1 and anti-MMP-13 antibodies were sent from Dr. Gillian Murphy’s laboratory. Phospho-STAT3 (Tyr705) and STAT3 antibodies were purchased from Cell Sig- naling Technology (Danvers, MA); TIMP-1 and GAPDH anti- bodies were purchased from Abcam (Cambridge, MA). Anti- IL-11, anti-c-Fos, anti-c-Jun, and anti-JunD antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). IL-11R (interleukin 11 receptor subunit) antibody was pur- chased from R&D Systems (Minneapolis, MN). PD98059, SB203580, LY294002, and Akt inhibitor IV were purchased from EMD Chemicals (Gibbstown, NJ). Recombinant human IL-1 and IL-11 were purchased from PeproTech (Rocky Hill, NJ). LfcinB was purchased from BioSynthesis (Lewisville, TX). Heparin, heparan sulfate, and heparinase III were purchased from Sigma. siRNA targeting IL11, FOS, and STAT3 were acquired from Invitrogen. y IL-8 ELISA—The IL-8 ELISA kit used in this study has a sen- sitivity of 2 pg/ml and an assay range of 25.6–1000 pg/ml, according to the manufacturer’s descriptions (Thermo Fisher Scientific). This assay does not cross-react with other interleu- kins. After adding standards and samples in duplicates, multi- well plates were covered and incubated at room temperature for 1 h. Then wells were washed three times, followed by the addition of 50 l of biotinylated antibody reagent into each well. The plates were incubated for another hour at room tem- perature. After washing the wells three times, 100 l of strepta- vidin-HRP working solution was added into each well, followed by 30-min incubation at room temperature. Plates were then washed three times, and 100 l of TMB substrate was added to each well. The reactions were allowed to proceed in the dark for 30 min at room temperature before stop solution was added. Absorbance of each well was measured at 450 and 550 nm. q g Tissue Acquisition and Chondrocyte Isolation—Post-mortem human femoral cartilage (age ranging from 40 to 70) was obtained through the Gift of Hope Organ and Tissue Donor Network (Elmhurst, IL) within 72 h. Prior approval by the local ethics committee and consent from donor families were acquired. Before processing, each specimen was graded follow- ing a 5-point scale modified from that of Collins (15). Cartilage for this study was graded 0 or 1, unless otherwise specified. Osteoarthritic femoral and tibial cartilage was obtained from patients (age ranging from 40 to 70) through the Orthopedic Tissue and Implant Repository Study (Chicago, IL) with prior consent from the patients. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation MMP-3 production, up-regulates TIMP-1, and inhibits TNF- production in the presence of soluble IL-11 receptor (9). Another cytokine IL-13 also blocks collagenolysis in the pres- ence of IL-1 and increases TIMP activity (10). Despite a lack of in vivo assessments, these findings suggest that these anti-in- flammatory cytokines could be candidates for effective OA therapy. tilage was digested in DMEM/Ham’s F-12 (1:1) medium with 0.2% Pronase for 1 h, followed by overnight digestion with 0.025% Collagenase P supplemented with 5% FBS in a humidi- fied atmosphere with 5% CO2 and continuous agitation. Chon- drocytes released from enzymatic digestion were resuspended to a density of 3 106 cells/ml in DMEM/Ham’s F-12 medium (1:1) supplemented with 10% FBS (complete medium). For short term monolayer culture, the cells were then plated onto 6-well (2 ml/well), 12-well (1 ml/well), or 24-well (0.5 ml/well) plates. After 3-day culture, the media were replaced with serum-free DMEM/Ham F-12 medium (1:1). After another 24 h, culture media were replaced with fresh serum-free medium again, 2 h prior to treatments. LfcinB concentrations used in monolayer culture were 50 and 100 g/ml. FGF-2 (100 ng/ml) or IL-1 (5 ng/ml) was also applied wherever appropri- ate. The cells were harvested after 24-h stimulation and sub- jected to downstream analyses as detailed below. y Previously we characterized bovine lactoferricin (LfcinB), a 25-amino acid peptide derived from the glycoprotein bovine lactoferrin, in human cartilage and synovium. LfcinB blocks IL-1-mediated catabolic and inflammatory processes in vitro and ex vivo, possibly through a heparan sulfate-dependent mechanism (11). In nucleus pulposus cells, LfcinB inhibits the detrimental activities of IL-1 and LPS and synergizes with bone morphogenetic protein-7 in promoting anabolic processes (12, 13). Gene expression analyses indicated that LfcinB down-reg- ulates several MMPs, aggrecanases, and pro-inflammatory mediators (11). LfcinB also up-regulates two anti-inflammatory cytokines, IL-4 and IL-10, as well as TIMP-3 (11, 14). LfcinB specifically triggers ERK1/2, p38, and Akt signaling, with the ERK1/2 response being the most robust (11, 14). However, it still remained undefined how these pathways regulate LfcinB target genes. In this current study, we aimed to (i) further define the anti-inflammatory property of LfcinB and (ii) provide a link between LfcinB-specific signaling pathways and its chondro- protective activity. Immunoblotting—Cell lysates were prepared using a modi- fied radioimmune precipitation buffer (16). Total protein con- centrations were determined by a BCA protein assay (Pierce). LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation Equal amounts of protein were resolved by 10% SDS-PAGE and transferred to nitrocellulose membrane for immunoblotting. Immunoreactivity was visualized using the ECL system (Amer- sham Biosciences) and the Signal Visual Enhancer system (Pierce) to magnify the signal. g y g Reverse Transcription and Real Time Polymerase Chain Reaction—Total RNA from normal and osteoarthritic articular chondrocytes was isolated using TRIzol reagent (Invitrogen) according to the instructions provided by the manufacturer. RT was carried out with 1 g of total RNA using ThermoScriptTM RT-PCR system (Invitrogen) for first strand cDNA synthesis. For real time PCR, cDNA was amplified using a MyiQ real time PCR detection system (Bio-Rad). Relative gene expression was determined using the CT method, as detailed by manufac- turer guidelines (Bio-Rad). 18 S rRNA and GAPDH were used as internal controls in the reactions for normalization. The standard deviations in samples represent at least five different donors from independent experiments. The primer sequences used in this study are listed in Table 1. Bovine Lactoferricin-induced Anti-inflammation Is, in Part, via Up-regulation of Interleukin-11 by Secondary Activation of STAT3 in Human Articular Cartilage* Patho- logical ramifications from aberrant IL-1 signaling include amplification of inflammatory responses in chondrocytes by supernormally induced pro-inflammatory mediators, such as IL-6, IL-8, and Toll-like receptor 2 (TLR2) (2, 4). ECM frag- ments as the result of protease-mediated degeneration also pro- mote catabolic effects through TLR2/TLR4 (5), thus helping perpetuate cartilage degradation. Pro-inflammatory mediators including IL-1 have been implicated in several degenerative joint diseases, such as osteoarthritis (OA). Pharmaceutical tar- geting of these inflammatory mediators is being actively explored in OA therapy, and the anti-IL-1 strategy using IL-1 receptor antagonist serves as a typical example. Anti-inflammatory cytokines have been demonstrated to attenuate inflammatory responses and thus joint damage in OA and rheumatoid arthritis conditions. IL-1 receptor antagonist represents the best understood cytokine in this category in car- tilage biology. IL-1 receptor antagonist directly dampens IL-1 signaling, thus protecting chondrocytes from excessive cata- bolic and inflammatory activities. In experimental models, IL-1 receptor antagonist effectively inhibits IL-1-mediated cartilage destruction (6). The other proposed anti-inflammatory cyto- kines, by contrast, are inadequately characterized in the context of OA pathogenesis. IL-4 and IL-10 appear to be chondropro- tective in vivo, and a negative correlation between their expres- sion and that of TNF has been reported in OA cartilage (7). IL-11 generated by chondrocytes stimulates the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1), an endoge- nous inhibitor of matrix metalloproteinase (MMP) (8). In rheu- matoid arthritis synovium, IL-11 directly inhibits MMP-1 and Homeostatic chondrocytes maintain a delicate balance between catabolic and anabolic processes, which is character- ized by dynamic and steady turnover of molecules in its extra- cellular matrix (ECM).2 Such a balance, however, is susceptible metalloproteinase; qPCR, quantitative PCR; PG, proteoglycan; HS, heparan sulfate; AP-1, activator protein 1; HSPG, heparan sulfate proteoglycan. 31655 JOURNAL OF BIOLOGICAL CHEMISTRY 31655 NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 RESULTS LfcinB Potently Induces IL-11, an Anti-inflammatory Cyto- kine, in Human Articular Chondrocytes—Our previous studies revealed that LfcinB represses the expression of cartilage- degrading proteases (e.g., MMP-13 and ADAMTS-5) and pro-inflammatory mediators (e.g., IL-1, IL-8, and TLR2). Simultaneously, LfcinB significantly induces multiple anti- inflammatory cytokines (IL-4 and IL-10), providing a molec- ular basis on which LfcinB counteracts the catabolic and inflammatory activities promoted by IL-1 and FGF-2 in articular cartilage (11). Our previous promising results moti- vated us to further investigate LfcinB-controlled signaling pathways and its downstream target genes that may mediate its chondroprotective effects in human primary chondrocytes. When surveying the mRNA expression of other potential anti- inflammatory targets, we observed a striking 40- and 120-fold induction of IL-11 upon stimulation with two different concen- trations of LfcinB (50 and 100 g/ml, respectively) (Fig. 1A; p 0.01 and p 0.001, respectively). This finding was confirmed by concurrent increases in IL-11 protein production in both intra- cellular and extracellular compartments (Fig. 1B). In both com- partments, the molecular weight of IL-11 is 19 kDa, corre- sponding to the mature form of IL-11 with its signal peptide removed. To determine whether such an induction is tempo- were removed, and washed cells were lysed in 1 ml of buffer A. After incubation on ice for 10 min, lysates were centrifuged at 14,000 g at 4 °C for 3 min. Supernatants were discarded, and cell pellets were resuspended in 40 l of buffer B. Then samples were incubated on ice for 1 h before centrifugation at 14,000 g, 4 °C for 5 min. The supernatants were aliquoted and stored at 80 °C. Total protein concentration in each sample was deter- mined by BCA assay (Pierce) right before EMSA. EMSA—The nuclear extracts were prepared after stimula- tion as described above. The EMSA kit (Affymetrix) was used according to the manufacturer’s instructions. EMSA was per- formed by incubating labeled biotin-conjugated probes with 5 g of nuclear extract. Samples were resolved in 6% nondena- turing polyacrylamide gels. Then proteins were transferred to Pall Biodyne B membrane. The membrane was blocked with 1 blocking buffer and then incubated with streptavidin-HRP conjugate. After proper washing, the signals were visualized in a chemiluminescence imaging system. In each experiment, a sep- arate reaction using unlabeled double-stranded DNA (cold probe) was set up to demonstrate binding specificity. EXPERIMENTAL PROCEDURES Human tissues were handled based on the guidelines of the Human Investigation Committee of Rush University Medical Center. After aseptic dissection, car- Preparation of Nuclear Extract—A nuclear extraction kit (Affymetrix, Santa Clara, CA) was used based on the manufac- turer’s instructions. Upon the completion of stimulation, media VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 31656 JOURNAL OF BIOLOGICAL CHEMISTRY 31656 VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 TABLE 1 Primers used in real time PCR analyses Cartilage Explant Culture—Full-thickness explants of 4-mm diameters were prepared from freshly isolated, healthy femoral cartilage strips. Explants then recovered in DMEM/F-12 (1:1, supplemented with 10% FBS) for 48 h. Culture media were changed to DMEM/F-12 (1:1) supplemented with 1% mini- ITS premix (BD Biosciences, San Jose, CA) 48 h before treat- ments started. The explants were treated with FGF-2 (100 ng/ml) or IL-1 (5 ng/ml), in the presence or absence of LfcinB (50 and 100 g/ml). ( g ) Histology—After dissection, human articular cartilage explants were fixed in 4% paraformaldehyde. After embedding the explants in paraffin, serial sections of 8-m thickness were prepared and placed onto slides. Then sections were deparaffinized in xylene, followed by stepwise rehydration in ethanol and distilled water. For Safranin-O Fast Green staining, sections were immersed in 0.1% Fast Green for 3 min, followed by 1% Safranin-O for 15 min. Statistical Analyses—All experiments were performed with 3–5 biological replicates, without pooling samples from differ- ent donors. Statistical significance was determined by Student’s t test or one-way repeated measures analysis of variance fol- lowed by Sidak post hoc test, using the SPSS 17 software (IBM Corporation, Somers, NY). p values lower than 0.05 were con- sidered to be statistically significant in each test. Each value in the figures is presented as the mean standard deviation. Histology—After dissection, human articular cartilage explants were fixed in 4% paraformaldehyde. After embedding the explants in paraffin, serial sections of 8-m thickness were prepared and placed onto slides. Then sections were deparaffinized in xylene, followed by stepwise rehydration in ethanol and distilled water. For Safranin-O Fast Green staining, sections were immersed in 0.1% Fast Green for 3 min, followed by 1% Safranin-O for 15 min. Statistical Analyses—All experiments were performed with 3–5 biological replicates, without pooling samples from differ- ent donors. Statistical significance was determined by Student’s t test or one-way repeated measures analysis of variance fol- lowed by Sidak post hoc test, using the SPSS 17 software (IBM Corporation, Somers, NY). p values lower than 0.05 were con- sidered to be statistically significant in each test. Each value in the figures is presented as the mean standard deviation. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation TABLE 1 Primers used in real time PCR analyses Gene Primer sequence (5 3 3) NCBI reference no. GAPDH NM_002046.4 Forward: TCGACAGTCAGCCGCATCTTCTTT Reverse: GCCCAATACGACCAAATCCGTTGA 18 S rRNA NR_003286.2 Forward: CGGCTACCACATCCAAGGAA Reverse: GCTGGAATTACCGCGGCT ACAN NM_001135.3 Forward: TCTTGGAGAAGGGAGTCCAACTCT Reverse: ACAGCTGCAGTGATGACCCTCAGA MMP1 NM_002421.3 Forward: AGTGACTGGGAAACCAGATGCTGA Reverse: GCTCTTGGCAAATCTGGCGTGTAA MMP13 NM_002427.3 Forward: ACCCTGGAGCACTCATGTTTCCTA Reverse: TGGCATCAAGGGATAAGGAAGGGT IL1B NM_000576.2 Forward: ATGACCTGAGCACCTTCTTTCCCT Reverse: GCATCGTGCACATAAGCCTCGTTA IL6 NM_000600.3 Forward: AAGCCAGAGCTGTGCAGATGAGTA Reverse: TTCGTCAGCAGGCTGGCATTTGT IL8 NM_000584.3 Forward: TCTTGGCAGCCTTCCTGATTTCTG Reverse: GGGTGGAAAGGTTTGGAGTATGTC IL11 NM_000641.3 Forward: TACCCGTATGGGACAAAGCTGCAA Reverse: TGCACCATGTTGCTTAACCCTCAC TIMP1 NM_003254.2 Forward: CATCCTGTTGTTGCTGTGGCTGAT Reverse: AAGGTGGTCTGGTTGACTTCTGGT IL11RA NM_001142784.2 Forward: CTCAAGTTCCGTTTGCAGTACCGT Reverse: TCCAGGTGCCAGCATCTAGAAAGT FOS NM_005252.3 Forward: AGATTGCCAACCTGCTGAAGGAGA Reverse: AAGCCACAGACATCTCTTCTGGGA STAT3 NM_139276.2 Forward: ATGGAAGAATCCAACAACGGCAGC Reverse: TCCTCAGTCACAATCAGGGAAGCA NucleofectorTM kit (Lonza, Walkersville, MD) as described previously (16, 17). Chondrocytes were cultivated for 3 days before transfection. For knockdown experiments, siRNA at a concentration of 200 nM (20 pmol/sample) was used for trans- fection. After 48 h, cell lysates were subjected to quantitative PCR (qPCR) and immunoblotting for validation of successful knockdown. In parallel, stimulations were performed 48 h after transfection. Cell lysates, total RNA, and conditioned media were collected for downstream analyses. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation In addition, IL-11 did not elicit cytotoxicity in chondro- cytes (data not shown). g p p IL-11 Antagonizes IL-1-induced Proteoglycan Depletion in Human Articular Cartilage ex Vivo Organ Culture—IL-11 was previously shown to be an anti-inflammatory cytokine and chondroprotective (18). Nevertheless, its biological roles in joint homeostasis had never been examined in detail. We first determined the potency of IL-11 using an ex vivo culture model, in which full thickness human articular cartilage explants were incubated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (100 and 200 ng/ml) for 11 days. Our histological analyses revealed that IL-1-elicited proteoglycan (PG) depletion was dose-dependently counteracted by IL-11, as indicated by the PG retention in the presence of IL-1 challenge (Fig. 2A, panel b versus panels c and d). This finding suggests that LfcinB-me- diated anti-inflammatory action is, at least in part, achieved through the induction of IL-11 in human articular cartilage. y IL-11 Acts as a Chondroprotective Cytokine Antagonizing Autocrine and Paracrine Catabolic Action of IL-1 in Human Articular Chondrocytes—IL-1 acts in autocrine and paracrine manners by inducing IL-1 itself as well as multiple pro-inflam- matory cytokines and chemokines (e.g., IL-6 and IL-8) in many cell types, including human articular chondrocytes (19). We next determined whether IL-11 also attenuates IL-1-stimu- lated pro-inflammatory cytokine expression in human primary articular chondrocytes. Our results revealed that IL-1-medi- ated up-regulation of IL-1, IL-6, and IL-8 was significantly compromised by IL-11 co-treatment (100 ng/ml) on mRNA levels (Fig. 3, A and B; p 0.05). Consequently, the highly induced IL-8 level in conditioned medium was significantly down-regulated by co-treatment with IL-11, as assessed by ELISA (Fig. 3C; p 0.001). IL-1 significantly induces cartilage-degrading enzymes such as MMP-1 and MMP-13 in human adult articular chon- drocytes. Thus, we tested whether IL-11 also mitigates IL-1- induced ECM protease expression. The presence of IL-11 at 100 ng/ml effectively attenuated IL-1-stimulated collagenase expression at mRNA level (Fig. 3D; p 0.05). Correspondingly at the protein levels, the enhanced expression of MMP-1 and Spurred by this finding, we next characterized whether such PG preservation by IL-11 is due to its biological effects on aggrecan gene expression. Consistent with our previous obser- vation, IL-1 caused marked suppression of aggrecan gene expression (Fig. 2B; p 0.01). This effect, however, was signif- icantly attenuated by IL-11 co-treatment at the concentration of 100 ng/ml (Fig. 2B; p 0.05). LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation FIGURE 1. LfcinB induces IL-11 expression in human articular chondrocytes. A, chondrocytes were incubated with LfcinB (50 and 100 g/ml) for 24 h. Transcripts of IL-11 were quantified by qPCR. B, the abundance of intracellular and extracellular IL-11 was analyzed by immunoblotting using cell lysates and conditioned media, respectively. C, chondrocytes were stimulated with LfcinB (100 g/ml) for 24, 48, and 72 h. IL-11 mRNA expression was measured by qPCR. , p 0.01; , p 0.001. FIGURE 1. LfcinB induces IL-11 expression in human articular chondrocytes. A, chondrocytes were incubated with LfcinB (50 and 100 g/ml) for 24 h. Transcripts of IL-11 were quantified by qPCR. B, the abundance of intracellular and extracellular IL-11 was analyzed by immunoblotting using cell lysates and conditioned media, respectively. C, chondrocytes were stimulated with LfcinB (100 g/ml) for 24, 48, and 72 h. IL-11 mRNA expression was measured by qPCR. , p 0.01; *, p 0.001. rally robust, we stimulated chondrocytes with LfcinB (100 g/ml) for different durations (24, 48, and 72 h). Our data show that IL-11 induction by LfcinB peaked at the 24-h time point and was sustained over 72 h (Fig. 1C; p 0.001 and p 0.01). IL-11 Antagonizes IL-1-induced Proteoglycan Depletion in Human Articular Cartilage ex Vivo Organ Culture—IL-11 was previously shown to be an anti-inflammatory cytokine and chondroprotective (18). Nevertheless, its biological roles in joint homeostasis had never been examined in detail. We first determined the potency of IL-11 using an ex vivo culture model, in which full thickness human articular cartilage explants were incubated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (100 and 200 ng/ml) for 11 days. Our histological analyses revealed that IL-1-elicited proteoglycan (PG) depletion was dose-dependently counteracted by IL-11, as indicated by the PG retention in the presence of IL-1 challenge (Fig. 2A, panel b versus panels c and d). This finding suggests that LfcinB-me- diated anti-inflammatory action is, at least in part, achieved through the induction of IL-11 in human articular cartilage. rally robust, we stimulated chondrocytes with LfcinB (100 g/ml) for different durations (24, 48, and 72 h). Our data show that IL-11 induction by LfcinB peaked at the 24-h time point and was sustained over 72 h (Fig. 1C; p 0.001 and p 0.01). aggrecan expression, suggesting chondroprotection exerted by IL-11 is mainly via anti-inflammatory, but not pro-anabolic action. RESULTS Transfection of siRNA—Nucleofection was optimized for human articular chondrocytes based on the manual of the NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 31657 JOURNAL OF BIOLOGICAL CHEMISTRY 31657 LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation FIGURE2.IL-11counteractsIL-1-inducedPGdepletion.A,fullthicknesscartilageexplantsweremaintainedinDMEM/F-12mediumsupplementedwith1% mini-ITS premix and stimulated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (100 and 200 ng/ml). After 11 days, sections from each group were stained with Safranin-O Fast Green dyes to reveal gross PG content. B, chondrocytes in monolayer were stimulated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (50 and 100 ng/ml) or IL-11 alone (50 and 100 ng/ml). Aggrecan (ACAN) transcripts were quantified by qPCR. , p 0.05; *, p 0.01. Ctrl, control. FIGURE2.IL-11counteractsIL-1-inducedPGdepletion.A,fullthicknesscartilageexplantsweremaintainedinDMEM/F-12mediumsupplementedwith1% mini-ITS premix and stimulated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (100 and 200 ng/ml). After 11 days, sections from each group were stained with Safranin-O Fast Green dyes to reveal gross PG content. B, chondrocytes in monolayer were stimulated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (50 and 100 ng/ml) or IL-11 alone (50 and 100 ng/ml). Aggrecan (ACAN) transcripts were quantified by qPCR. , p 0.05; *, p 0.01. Ctrl, control. MMP-13 by IL-1 was notably reversed by co-incubation with IL-11 in a concentration-dependent manner (Fig. 3E). induction of MMP-1, MMP-13, and IL-1 was essentially abro- gated in LfcinB-primed chondrocytes (Fig. 4, D and E; p 0.01). In IL-1-primed cells, LfcinB was still able to significantly down-regulate MMP-13 (Fig. 4F; p 0.01) and IL-1 (Fig. 4G; p 0.05). These data further confirm that LfcinB promotes anti-inflammatory processes through primary and secondary mechanisms in chondrocytes. p g LfcinB-induced Anti-inflammatory Effects Are Mediated, in Part, via Up-regulation of IL-11 in Human Articular Chondrocytes—Because LfcinB markedly up-regulates IL-11 and IL-11 antagonizes IL-1-induced catabolism, we then hypothesized that the induction of IL-11 by LfcinB may at least partially account for LfcinB-induced anti-inflammation. To directly evaluate the link between the biological impact of LfcinB and IL-11, we knocked down IL-11 gene by siRNA fol- lowed by stimulation with IL-1 (5 ng/ml) in the presence or absence of LfcinB (50 g/ml) for 24 h. We were able to achieve efficient knockdown of IL-11, as reflected by the levels of IL-11 in untreated and LfcinB-stimulated cells (Fig. 4A; p 0.01). Importantly, the antagonistic effects of LfcinB on IL-1-in- duced targets (i.e., MMP-1 and MMP-13) were diminished by IL-11 knockdown on both mRNA (Fig. 4B; p 0.05) and pro- tein levels (Fig. 4C). LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation IL-11 alone did not enhance 31658 31658 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation These data provide direct evidence that LfcinB-induced anti-inflammatory effects are mediated, in part, via up-regulation of IL-11 in human primary articular chondrocytes. LfcinB-mediated IL-11 Induction Is Heparan Sulfate-dependent— LfcinB contains a heparan sulfate (HS)-binding motif, and it has been demonstrated that this motif mediates some of its biolog- ical effects (20). Recently, we showed that LfcinB is capable of antagonizing two HS-dependent factors, FGF-2 and IL-1 (11), suggesting that LfcinB-mediated antagonism against catabolic cytokines is possibly through competitive bindings to HS on cell surface. Hence, we hypothesized that LfcinB requires HS on the plasma membrane of chondrocytes to elicit downstream responses. To test this hypothesis, chondrocytes were pre- treated with heparinase, which removed cell surface HS, before they were stimulated with LfcinB (50 g/ml) for 24 h. Hepari- nase pretreatment resulted in significant attenuation of LfcinB- mediated effects, such as IL-11 induction (Fig. 5, A and B; p 0.01) and MMP repression (Fig. 5, C and D; p 0.05), on mRNA and protein levels. To determine whether LfcinB requires cell- bound HS to exert its functions, LfcinB was preincubated with exogenous heparin or HS before it was applied onto chondro- cytes. Exogenous heparin and HS not only dramatically atten- uated LfcinB-mediated ERK1/2, p38 and Akt signaling (Fig. 5E), but also abolished LfcinB-mediated IL-11 induction and MMP Based on our previous studies, LfcinB can directly antagonize IL-1 within 24 h (11), possibly independent of secondary IL-11 actions. To further examine how LfcinB exerts anti-inflamma- tory effects in chondrocytes, we primed cells with LfcinB (100 g/ml, 24 h) or IL-1 (5 ng/ml, 48 h). We then stimulated LfcinB-primed cells with IL-1 (5 ng/ml), or IL-1-primed cells with LfcinB (100 g/ml), for 24 h. Strikingly, IL-1-mediated NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 31659 JOURNAL OF BIOLOGICAL CHEMISTRY NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 FIGURE 3. IL-11 moderately antagonizes IL-1-mediated inflammatory and catabolic effects. A, chondrocytes in monolayer were stimulated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (50 and 100 ng/ml) or IL-11 alone (50 and 100 ng/ml). IL-1 and IL-6 transcripts were quantified by qPCR. B, chondrocytes were stimulated with IL-1 (1 ng/ml) in the presence or absence of IL-11 (50 and 100 ng/ml) or IL-11 alone (50 and 100 ng/ml). IL-8 transcripts were measured by qPCR. C, in parallel, IL-8 concentrations in the conditioned media were determined by ELISA. Samples were properly diluted to fit the assay range. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation Dilution factors were used to calculate the original concentrations. D, chondrocytes treated as described above were subjected to qPCR quantification of MMP-1 and MMP-13 transcripts. E, conditioned media were collected from chondrocytes treated as described above. Secreted MMP-1 and MMP-13 were assessed by immunoblotting. , p 0.05; , p 0.01; , p 0.001. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation FIGURE 3. IL-11 moderately antagonizes IL-1-mediated inflammatory and catabolic effects. A, chondrocytes in monolayer were stimulated with IL-1 (5 ng/ml) in the presence or absence of IL-11 (50 and 100 ng/ml) or IL-11 alone (50 and 100 ng/ml). IL-1 and IL-6 transcripts were quantified by qPCR. B, chondrocytes were stimulated with IL-1 (1 ng/ml) in the presence or absence of IL-11 (50 and 100 ng/ml) or IL-11 alone (50 and 100 ng/ml). IL-8 transcripts were measured by qPCR. C, in parallel, IL-8 concentrations in the conditioned media were determined by ELISA. Samples were properly diluted to fit the assay range. Dilution factors were used to calculate the original concentrations. D, chondrocytes treated as described above were subjected to qPCR quantification of MMP-1 and MMP-13 transcripts. E, conditioned media were collected from chondrocytes treated as described above. Secreted MMP-1 and MMP-13 were assessed by immunoblotting. , p 0.05; , p 0.01; , p 0.001. cubated with individual pathway inhibitors of ERK1/2, p38, and Akt, followed by LfcinB stimulation for 24 h. Our qPCR results demonstrate that only ERK1/2 inhibition led to a prominent reversal of the IL-11 expression (Fig. 6A; p 0.01), suggesting ERK1/2 is the key regulatory pathway. ERK1/2 is an upstream regulatory kinase of AP-1. Two functional proximal activator protein 1 (AP-1) elements residing side by side within the 82 to 92 region of IL-11 promoter had been reported in human IL-11 transcription (Fig. 6B) (21). To directly examine AP-1 activation status, we analyzed AP-1 protein-DNA interaction by EMSA using nuclear extracts from chondrocytes stimulated with LfcinB (1 h) in the presence or absence of individual path- way inhibitors. We observed enhanced binding of AP-1 to its consensus DNA sequence after stimulation, indicating that LfcinB effectively activates AP-1 (Fig. 6C, lane 2). The enhanced AP-1 binding was specifically abolished upon ERK1/2 inhibi- tion (Fig. 6C, lane 3) but not by inhibitors of p38 (lane 4) or Akt (lane 5). LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation Our data suggest that the ERK1/2 signaling pathway plays a primary role in regulating AP-1 activity after LfcinB stimulation. repression on mRNA and protein levels (Fig. 5, F–I), suggesting that LfcinB forms a complex with membrane-bound HS to trig- ger cellular responses. Together, these findings strongly suggest that LfcinB critically depends on cell surface HS to exert its bioactivities. LfcinB Induces IL-11 via the ERK1/2-c-Fos/JunD Axis—Pre- viously, we reported robust activation of ERK1/2 and Akt by LfcinB, which sustains for 60 min in human articular chon- drocytes (11). Unlike ERK1/2 and Akt, LfcinB induced p38 acti- vation only in a transient manner (less than 60 min; data not shown). In addition, we did not observe activation of JNK, NFB, SMAD1/5/8, SMAD2/3, or STAT3 pathway within 2 h after LfcinB stimulation (data not shown). Our results suggest that the major signaling pathways induced by LfcinB are ERK1/2, Akt, and potentially p38 in human articular chondrocytes. Because the IL-11 induction is the most dramatic and robust cellular response elicited by LfcinB in articular chondrocytes (maximal 120-fold), we wished to determine the responsible signaling pathway. Human articular chondrocytes were prein- 31660 31660 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 FIGURE 4. IL-11 acts as a secondary molecule in LfcinB-mediated responses. A, chondrocytes transfected with siRNA targeting IL-11 were cultured in th presence or absence of LfcinB (50 g/ml). IL-11 transcripts were quantified by qPCR to reveal knockdown efficiency. B, chondrocytes transfected with IL-1 siRNA were incubated with IL-1 (5 ng/ml) and LfcinB (50 g/ml) for 24 h. MMP-1 and MMP-13 transcripts were measured by qPCR. C, in parallel, secrete MMP-1 and MMP-13 were analyzed using conditioned media by immunoblotting. D, chondrocytes were first incubated with LfcinB (100 g/ml) for 24 h an then stimulated with IL-1 (5 ng/ml) for another 24 h. MMP-1 and MMP-13 transcripts were quantified by qPCR. E, IL-1 mRNA level was quantified by qPCR F, chondrocytes were primed with IL-1 (5 ng/ml) for 48 h before LfcinB (100 g/ml) stimulation for 24 h. MMP-1 and MMP-13 mRNA levels were quantified b qPCR. G, likewise, IL-1 transcript was quantified by qPCR. , p 0.05; , p 0.01; , p 0.001. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation FIGURE 4. IL-11 acts as a secondary molecule in LfcinB-mediated responses. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation A, chondrocytes transfected with siRNA targeting IL-11 were cultured in the presence or absence of LfcinB (50 g/ml). IL-11 transcripts were quantified by qPCR to reveal knockdown efficiency. B, chondrocytes transfected with IL-11 siRNA were incubated with IL-1 (5 ng/ml) and LfcinB (50 g/ml) for 24 h. MMP-1 and MMP-13 transcripts were measured by qPCR. C, in parallel, secreted MMP-1 and MMP-13 were analyzed using conditioned media by immunoblotting. D, chondrocytes were first incubated with LfcinB (100 g/ml) for 24 h and then stimulated with IL-1 (5 ng/ml) for another 24 h. MMP-1 and MMP-13 transcripts were quantified by qPCR. E, IL-1 mRNA level was quantified by qPCR. F, chondrocytes were primed with IL-1 (5 ng/ml) for 48 h before LfcinB (100 g/ml) stimulation for 24 h. MMP-1 and MMP-13 mRNA levels were quantified by qPCR. G, likewise, IL-1 transcript was quantified by qPCR. , p 0.05; , p 0.01; , p 0.001. JOURNAL OF BIOLOGICAL CHEMISTRY 31661 JOURNAL OF BIOLOGICAL CHEMISTRY 31661 JOURNAL OF BIOLOGICAL CHEMISTRY 31661 NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation 31662 JOURNAL OF BIOLOGICAL CHEMISTRY LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-medi- ated induction of AP-1 components appeared to be limited to c-Fos, because we did not observe similar stimulation of JunD, the other component of AP-1 heterodimers, by LfcinB (data not shown). Activation of STAT3 has been demonstrated to transactivate TIMP-1 (24, 25). In our initial experiments, treatment of artic- ular chondrocytes with LfcinB showed no activation of STAT3 within 2 h. Cells stimulated with IL-11, however, rapidly acti- vate STAT3 signaling, as represented by the phosphorylation of Tyr705 within 30 min, and the activation sustained for 24 h (Fig. 7G, upper panel). Although we did not observe immediate STAT3 activation upon LfcinB stimulation, it remained possi- ble that STAT3 can be activated after a sufficient amount of IL-11 is produced. To test this notion, chondrocytes were incu- bated with LfcinB for different time periods (24, 48, and 72 h), and STAT3 phosphorylation was analyzed by immunoblotting. To our surprise, STAT3 was markedly phosphorylated after 24 h, and the activation sustained till 72 h after LfcinB stim- ulation (Fig. 7G, lower panel). Such kinetics clearly demon- strates sequential activation profile of STAT3 in which LfcinB activates STAT3 via IL-11 stimulation as a secondary signaling event. Because c-fos expression was specifically up-regulated by LfcinB, we further determined the role of c-fos in LfcinB-medi- ated IL-11 expression. Nucleofection of siRNA successfully knocked down c-fos at both mRNA (data not shown) and pro- tein levels, in contrast with scrambled siRNA (Fig. 6G). Our data show that reduced c-fos by siRNA significantly impaired the extent of IL-11 induction by LfcinB on mRNA level (Fig. 6H; p 0.01). Correspondingly, intra- and extracellular levels of IL-11 protein were also reversed upon c-fos knockdown (Fig. 6I). Collectively, our results evidence that LfcinB-induced c-Fos plays a critical role in the transcriptional regulation of IL-11 expression. LfcinB Up-regulates TIMP-1 via IL-11/STAT3-dependent Manner as a Secondary Stimulatory Event—TIMP family members (TIMP-1–4) are the key natural chondroprotective inhibitors of cartilage-degrading enzyme activities (i.e., MMPs and ADAMTS). Although stimulation with IL-11 significantly up-regulates TIMP-1 at both mRNA and protein levels within 24 h (Fig. 7, A and B; p 0.01), in our initial experiments we were unable to detect significant TIMP-1 induction by LfcinB during the same time frame in human articular chondrocytes. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation The AP-1 complex can be either a homo- or a heterodimer, depending on stimuli and cellular context (22). Thus, next we further defined the components of LfcinB-induced AP-1 com- plex. Human primary chondrocytes were incubated with LfcinB for 1 h, and supershift assays were performed using nuclear extracts that were sequentially incubated with AP-1 probes and individual antibodies against potential AP-1 com- ponents (i.e., c-Fos, c-Jun, and JunD). Our results demonstrated that only antibodies against c-Fos and JunD specifically caused supershift of the protein-DNA complexes, suggesting that LfcinB-induced AP-1 heterodimer primarily consists of c-Fos and JunD (Fig. 6D, lanes 3 and 4). critical level capable of triggering notable TIMP-1 expression at a later time point. To test this notion, articular chondrocytes were treated with LfcinB for different durations (24, 48, and 72 h), followed by qPCR analyses of TIMP-1 expression. Indeed, we observed significant TIMP-1 induction at the 48-h point, and the induced TIMP-1 mRNA and protein levels were sustained until 72 h after LfcinB stimulation (Fig. 7, C and D; p 0.05). Our finding suggests that LfcinB-mediated TIMP-1 expression is a secondary stimulatory event and is highly likely to be mediated by the induced IL-11. We next sought to determine the contribution of induced IL-11 in such delayed TIMP-1 up-regulation by LfcinB. Chon- drocytes were incubated with LfcinB in the presence or absence of IL-11 neutralization antibody for 48 h, followed by analyses of expression levels of TIMP-1. Our qPCR and immunoblotting results indicate that blockage of IL-11 action essentially abol- ished TIMP-1 induction at the mRNA and protein levels after LfcinB stimulation (Fig. 7, E and F; p 0.05). This finding high- lights the importance of induced IL-11 in mediating secondary cellular responses to LfcinB in chondrocytes. g It has been reported that elevated c-fos expression can lead to augmented AP-1 transcriptional activity (23). Therefore, we also determined whether LfcinB regulates the expression of AP-1 component c-fos, which may influence gross AP-1 activ- ity. Cells were cultured with LfcinB (50 and 100 g/ml) for 24 h followed by quantification of c-fos transcripts. Our qPCR data revealed that c-fos mRNA level was increased by 3-fold in the presence of LfcinB (Fig. 6E; p 0.01). Correspondingly, c-Fos protein level was notably augmented upon LfcinB stimulation in a concentration-dependent manner (Fig. 6F). FIGURE 5. Heparan sulfate is required for LfcinB signaling and downstream effects. A, chondrocytes were incubated with heparinase III (1 Sigma unit/ml) in serum-free DMEM overnight. Then chondrocytes were stimulated with LfcinB (50 g/ml) in the presence of heparinase III (1 Sigma unit/ml) in serum-free DMEMfor24h.IL-11transcriptswerequantifiedbyqPCR.B,IL-11inconditionedmediaandwholecelllysateswereexaminedbyimmunoblotting.C,transcripts of MMP-1, MMP-3, and MMP-13 were quantified by qPCR. D, MMP-1, MMP-3, and MMP-13 in the conditioned media were analyzed by immunoblotting. E, LfcinB (50 g/ml) was preincubated with heparin (50 g/ml) or HS (50 g/ml) for 40 min and then used to stimulate chondrocytes for 30 min and 60 min. The levels of activated and total ERK1/2, p38, and Akt were analyzed by immunoblotting. F, LfcinB (50 g/ml) was preincubated with heparin (50 g/ml) or HS (50 g/ml) for 40 min and then used to stimulate chondrocytes for 24 h. IL-11 transcripts were quantified by qPCR. G, IL-11 in conditioned media, and whole cell lysates were examined by immunoblotting. H, transcripts of MMP-1, MMP-3, and MMP-13 were quantified by qPCR. I, MMP-1, MMP-3, and MMP-13 in the conditioned media were analyzed by immunoblotting. Hep, heparin. , p 0.05; , p 0.01. 31662 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation This apparent discrepancy led us to conjecture that LfcinB stimulates TIMP-1 through IL-11 production as a secondary event only when extracellular IL-11 concentration reaches a To directly ascertain the participation of STAT3 in TIMP-1 regulation, STAT3 was knocked down in human primary chon- drocytes by nucleofection using siRNA targeting STAT3, and then the cells were stimulated with LfcinB for 24 and 48 h. Analyses were conducted with qPCR for mRNA level and immunoblotting for protein level of TIMP-1. Knockdown of STAT3 significantly impaired TIMP-1 induction by LfcinB at the 48-h time point (Fig. 7, H and I; p 0.01). By contrast, LfcinB- mediated IL-11 induction was not affected by STAT3 knockdown at the 24-h time point (Fig. 7, J and K), suggesting that STAT3 specifically mediates TIMP-1 expression in response to LfcinB via induced IL-11. Thus, our data together demonstrate that LfcinB 31663 JOURNAL OF BIOLOGICAL CHEMISTRY 31663 NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 DISCUSSION As previously shown, LfcinB bears potential as a therapeutic peptide for OA intervention. In human articular cartilage and synovium, LfcinB effectively antagonized IL-1 and FGF-2, thus conferring chondroprotection (11). Here, we uncovered another important mechanism indicating that LfcinB-medi- ated anti-catabolic and chondroprotective actions are in part through IL-11 induction. LfcinB robustly activates ERK1/2, which in turn dramatically elevates IL-11 expression (100- fold induction by 100 g/ml LfcinB; p 0.001) in a c-Fos/JunD- dependent manner. Induced IL-11 serves as a secondary anti- inflammatory mediator in chondrocytes that are stimulated with LfcinB. IL-11 also promotes anti-catabolism via induction of TIMP-1, hence contributing to protection of cartilage from degradation. Although STAT3 signaling is not required for LfcinB-mediated IL-11 expression, it is essential to IL-11-me- diated TIMP-1 expression. Moreover, we show that IL-11 sig- naling may be suppressed as a result of IL-11R down-regula- tion in OA chondrocytes. Evidence from a handful of studies suggests the utility of IL-11 as a means of reducing arthritis inflammation or diseases with an arthritic component (9, 26). The beneficial bioactivities of IL-11 observed in these studies may be linked to its modulatory function in macrophage (27, 28), which represent one of the predominant cell populations in synovium in the pathological joint condition. Our findings not only corroborate the protective role of IL-11 in synovial joint but also unveiled cartilage as a target tissue of this anti-inflam- matory cytokine. IL-11 exhibits moderate yet significant antag- onism against IL-1 in PG metabolism, collagenase expression, and inflammatory mediator expression. It is worth noting that our findings from in vitro experiments are not in perfect accordance with results acquired from ex vivo experiments using cartilage explants, where striking chondroprotection was achieved with IL-11. Such an apparent discrepancy may arise from at least two sources. First, prolonged treatment with IL-11 y The HS binding affinity of LfcinB allows it to interact with chondrocytes via cell surface heparan sulfate proteoglycans (HSPGs). Here, we directly demonstrate that cell surface HS is critical to LfcinB-mediated biological effects in human primary chondrocytes. The binding of LfcinB to HS appears to be the furthest upstream event, because disruption of this interaction profoundly inhibits LfcinB-mediated intracellular signaling and target gene regulation. Together with our previous findings (11, 14), we propose that the interactions between LfcinB and cell-bound HSPGs are essential to its antagonism against FGF-2 and IL-1, as well as its self-contained activities. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation We conjecture that there may be components inside the native ECM that can potentiate IL-11-mediated responses. It is also worth mentioning that data from two studies do not support the anti-inflammatory role of IL-11 in synovial joint (29, 30). However, caveats exist in these studies. The randomized trial of recombinant human IL-11 in rheumatoid arthritis treatment suffered from considerable pla- cebo effects, which confounded the possible therapeutic benefit of IL-11 (29). The other study, which utilized an acute arthritic model, adopted an experimental scheme with frequent intra- articular injections of supranormal amounts of IL-11 (30). Such an ultra high dose of IL-11 may not generate a translatable outcome. Therefore, further in vivo studies using arthritic ani- mal models are warranted to elucidate the beneficial effects mediated by IL-11. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation in ex vivo culture may allow for delayed cellular responses, which could not be assessed in the monolayer short term cul- ture model. The delayed responses possibly cooperate with the immediate transcriptional events to promote stronger protec- tive activities. Second, chondrocytes reside in very different microenvironments between monolayer culture and ex vivo culture. In monolayer, chondrocytes synthesize their ECM de novo, whereas in cartilage explants, chondrocytes are sur- rounded by preformed intact ECM. We conjecture that there may be components inside the native ECM that can potentiate IL-11-mediated responses. It is also worth mentioning that data from two studies do not support the anti-inflammatory role of IL-11 in synovial joint (29, 30). However, caveats exist in these studies. The randomized trial of recombinant human IL-11 in rheumatoid arthritis treatment suffered from considerable pla- cebo effects, which confounded the possible therapeutic benefit of IL-11 (29). The other study, which utilized an acute arthritic model, adopted an experimental scheme with frequent intra- articular injections of supranormal amounts of IL-11 (30). Such an ultra high dose of IL-11 may not generate a translatable outcome. Therefore, further in vivo studies using arthritic ani- mal models are warranted to elucidate the beneficial effects mediated by IL-11. Using total RNA and cell lysates prepared from age- and grade- matched healthy (grades 0 and 1) and OA chondrocytes, we observed statistically significant up-regulation of IL-11 tran- scripts in OA cells (Fig. 8A; p 0.05). This change may repre- sent a reparative effort in OA chondrocytes to curb inflamma- tory processes. Nonetheless, when we analyzed the level of IL-11R (a specific cognate receptor of IL-11), we found mark- edly reduced IL-11R in OA chondrocytes compared with healthy donor samples at both mRNA (Fig. 8B; p 0.01) and protein levels (Fig. 8C), suggesting that IL-11 and IL-11R dys- regulation in chondrocytes are associated with OA. in ex vivo culture may allow for delayed cellular responses, which could not be assessed in the monolayer short term cul- ture model. The delayed responses possibly cooperate with the immediate transcriptional events to promote stronger protec- tive activities. Second, chondrocytes reside in very different microenvironments between monolayer culture and ex vivo culture. In monolayer, chondrocytes synthesize their ECM de novo, whereas in cartilage explants, chondrocytes are sur- rounded by preformed intact ECM. FIGURE 6. LfcinB potently induces IL-11 expression via the ERK1/2-c-Fos/JunD axis. A, chondrocytes in monolayer were preincubated with individual pharmacological inhibitors for 1 h, before stimulation with LfcinB (50 g/ml) for 24 h. IL-11 transcripts were quantified by qPCR. B, structure of IL-11 promoter. TwoadjacentAP-1elementsarepresentbetween80and100.C,chondrocyteswerepreincubatedwithindividualpharmacologicalinhibitorsfor1hbefore LfcinB (50 g/ml) stimulation for another hour. A binding reaction using unlabeled (cold) AP-1 probe was set up for each EMSA assay to demonstrate binding specificity. The AP-1-DNA complex was visualized using a chemiluminescence imaging system. D, nuclear extracts from articular chondrocytes were sequen- tially incubated with AP-1 probe and individual antibodies against c-Fos, JunD, and c-Jun. A control reaction using IgG was set up to demonstrate antibody specificity. Another binding reaction using unlabeled (cold) AP-1 probe was also performed to demonstrate probe binding specificity. E, chondrocytes were incubated with LfcinB (50 and 100 g/ml) for 24 h. Fos transcripts and protein expression were assessed by qPCR. F, in parallel, conditioned media were analyzed by immunoblotting for IL-11 expression. G, chondrocytes were transfected with siRNA targeting fos. Fos protein expression was then examined by immunoblotting to validate knockdown efficiency. Chondrocytes were also transfected with siRNA with scrambled sequences as a control. H, chondrocytes transfected with fos siRNA were stimulated with LfcinB (50 g/ml) for 24 h. IL-11 transcripts were measured by qPCR. I, after treating chondrocytes as described above, IL-11 protein expression was examined using cell lysates. GAPDH was used as loading control. Scr, scrambled. , p 0.01. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation primarily utilizes the ERK1/2-AP-1 axis to potently induce IL-11 expression, which in turn subsequently up-regulates TIMP-1 expression through the STAT3 signaling pathway in articular chondrocytes (summarized in Fig. 9). IL-11 and Its Cognate Receptor IL-11R Are Dysregulated in Human OA Chondrocytes—Our observations that LfcinB strik- ingly induces IL-11 inspired the question of whether such mod- ulated genetic responses in fact bring benefits to OA therapy. 31664 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 DISCUSSION Although challenging, it is of great interest to identify which HSPGs specifically bind to LfcinB in chondrocytes. Among known HSPGs, perhaps syndecans are the most probable bind- ing partners of LfcinB, because their expression has been reported in chondrocytes (31) and involved in OA pathogenesis (32). LfcinB binding to syndecans may generate a chondropro- tective effect (e.g., induction of TIMPs and IL-11) while mini- mizing FGF-2 and IL-1 binding opportunity leading to reduc- tion of catabolism. Future studies are warranted to elucidate this aspect of LfcinB biology. Our results indicate that IL-11 induction takes place during the first phase after LfcinB stimulation, and then an elevation of NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 31665 NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 JOURNAL OF BIOLOGICAL CHEMISTRY 31665 LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation FIGURE 8. IL-11 signaling is disrupted in OA chondrocytes. A, IL-11 transcripts were quantified using age-matched (40–70 years old) normal femoral and OA femoral/tibial chondrocytes (n 7). B, likewise, IL-11R transcripts were quantified by qPCR. C, IL-11R protein expression in these individuals was analyzed by immunoblotting. GAPDH was used as a loading control. * p 0.05; ** p 0.01. an interesting model of LfcinB action. The immediate IL-11 induction by LfcinB is achieved via the ERK1/2-AP-1 axis. Our EMSA data unequivocally indicate that ERK1/2, not p38 or Akt, determines AP-1 activity. A given AP-1 heterodimer consists of components from Fos, Jun, activating transcription factor, or Jun dimerization protein families (33). Our supershift data revealed that c-Fos and JunD, but not c-Jun, comprise the AP-1 complex upon LfcinB stimulation. This finding recapitulates the specificity of MAP kinases in AP-1 activation. From a struc- tural standpoint, c-Jun only possesses a D domain necessary for JNK targeting, whereas JunD contains a D domain and a DEF motif, rendering itself a favored substrate for both JNK and ERK. Fos is also a phosphorylation substrate of ERK (34). The fact that LfcinB activates ERK1/2 but not JNK corresponds to the finding that JunD rather than c-Jun is incorporated into the AP-1 dimer. The c-Fos/JunD model in LfcinB-mediated IL-11 induction is also supported by two more documented phenom- ena: (i) JunD occupies IL-11 promoter, although not exclusively (21), and (ii) c-Fos/JunD dimer is able to result in strong trans- activation (35). The recruitment of JunD instead of c-Jun may impart transcriptional specificity to LfcinB-induced AP-1, hence not producing the same detrimental effects as c-Fos/c- Jun complex does in chondrocytes (36–39). STAT3 does not act as a transcription co-factor in this context. Thus, our data provide a mechanistic explanation for IL-11- mediated TIMP-1 expression in chondrocytes. Recently, we reported that LfcinB stimulates other anti-in- flammatory cytokines such as IL-4 and IL-10 (11). In addition, we also found that LfcinB markedly up-regulates TIMP-3 expression during the first phase of cellular response in chon- drocytes via activation of Sp1 (14). FIGURE 7. LfcinB up-regulates IL-11, which in turn induces TIMP-1 expression via STAT3 signaling pathway. A, chondrocytes in monolayer were incu- bated with IL-11 (50 ng/ml) for 24 h. TIMP-1 transcripts were quantified by qPCR. B, extracellular and intracellular TIMP-1 protein was analyzed by immuno- blotting. C, chondrocytes were incubated with LfcinB (100 g/ml) for different durations (24, 48, and 72 h). TIMP-1 transcripts were quantified by qPCR. D, chondrocytes were treated as described above. TIMP-1 protein expression in both intracellular and extracellular fractions was examined by immunoblotting. E, chondrocytes in monolayer were treated with LfcinB (100 g/ml) in the presence or absence of IL-11 neutralization antibody (10 g/ml). TIMP-1 mRNA expression was examined by qPCR. F, TIMP-1 protein expression was examined by immunoblotting. G, chondrocytes were treated with either IL-11 (50 ng/ml) or LfcinB (50 g/ml) for various durations (0.5, 1, 24, 48, and 72 h). STAT3 activation was examined by immunoblotting using an anti-phospho-STAT3 antibody. Total STAT3 levels were used as loading controls. H, naive chondrocytes and chondrocytes transfected with STAT3 siRNA were incubated with LfcinB (100 g/ml) for 24 h. TIMP-1 mRNA levels were measured by qPCR. I, extracellular and intracellular TIMP-1 protein levels were analyzed by immunoblotting. J and K, IL-11 mRNA (J) and protein levels (K) were assessed by qPCR and immunoblotting using the same set of samples as described above. Scr, scrambled. , p 0.05; , p 0.01; *, p 0.001. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation ERK1/2 activation by LfcinB can explain why IL-11 is up-regu- lated to this magnitude. The transcriptional cascade governing IL-11 induction and subsequent TIMP-1 expression represents extracellular IL-11 level will trigger secondary responses to sus- tain chondroprotection. IL-11 induction represents the most dramatic response triggered by LfcinB. The robustness of AL OF BIOLOGICAL CHEMISTRY VOLUME 288•NUMBER 44• VOLUME 288•NUMBER 44•NOVEMBER 1, 2013 31666 JOURNAL OF BIOLOGICAL CHEMISTRY FIGURE 8. IL-11 signaling is disrupted in OA chondrocytes. A, IL-11 transcripts were quantified using age-matched (40–70 years old) normal femoral and OA femoral/tibial chondrocytes (n 7). B, likewise, IL-11R transcripts were quantified by qPCR. C, IL-11R protein expression in these individuals was analyzed by immunoblotting. GAPDH was used as a loading control. p 0.05; ** p 0.01. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation Combined with our previous findings (11), we propose that LfcinB promotes anti-catabolic and anti-inflammatory activities via three routes: (i) competi- tive binding to heparan sulfate PGs on chondrocyte plasma membrane, thus blocking IL-1 and FGF-2 actions; (ii) immedi- ate induction of multiple anti-inflammatory cytokines, includ- ing IL-11, which directly antagonizes IL-1-mediated inflamma- tion and up-regulates TIMP-1; and (iii) early induction of TIMP-3 to further limit endogenous protease activities. A pro- posed model is illustrated in Fig. 9 to summarize the mode of action of LfcinB in human articular chondrocytes. In keeping with this hypothesis, our experiments using LfcinB-pretreated chondrocytes show that when LfcinB-triggered primary and secondary mechanisms are fully operating, IL-1 fails to launch characteristic catabolic and inflammatory programs in these cells. By contrast, 24-h administration of LfcinB partially inhib- its IL-1 effects in IL-1-primed chondrocytes, suggesting that secondary actions of LfcinB-induced targets are indeed impor- tant. Interestingly, LfcinB-mediated genetic responses appear to compensate for some deficiencies in OA chondrocytes. The dedicated receptor of IL-11, IL-11R, is significantly down-reg- ulated in OA chondrocytes, which probably leads to partial loss of IL-11 anti-inflammatory signaling. The down-regulation of IL-11R, however, does not alter the response of OA chondro- The induced IL-11 initiates STAT3 pathway to up-regulate TIMP-1. Prominent STAT3 activation was noted 24 h after LfcinB stimulation, yet the increase in TIMP-1 expression could not be detected until 24 h later. Such a delay suggests that persistent STAT3 signaling is required for TIMP-1 induction by IL-11. Active STAT3 may not solely account for TIMP-1 induction, but it appears to be essential. Knockdown of STAT3 results in complete reversal of TIMP-1 levels, suggesting NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 31667 JOURNAL OF BIOLOGICAL CHEMISTRY 31667 NOVEMBER 1, 2013•VOLUME 288•NUMBER 44 LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation FIGURE 9. Hypothetical model of LfcinB-mediated chondroprotection. Evidence suggests that LfcinB exerts its chondroprotective activities through multiple mechanisms, including (i) competitive binding to the co-receptors of IL-1 and FGF-2; (ii) induction of anti-inflammatory cytokines, such as IL-11; and (iii) induction of anti-catabolic TIMP-3. apoptosis. Arthritis Res. Ther. 11, R165 apoptosis. Arthritis Res. Ther. 11, R165 apoptosis. Arthritis Res. Ther. 11, R165 4. Su, S. L., Tsai, C. D., Lee, C. H., Salter, D. M., and Lee, H. S. (2005) Expres- sion and regulation of Toll-like receptor 2 by IL-1 and fibronectin frag- ments in human articular chondrocytes. Osteoarthritis Cartilage 13, 879–886 5. Liu-Bryan, R., and Terkeltaub, R. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation We also observed repression of TIMP-3 expression in OA, suggesting a lack of control of proteolytic activities in this disease state (14). There- fore, LfcinB conduces to the restoration of chondrocyte home- ostasis through replenishing IL-11R signaling and TIMP-3. Taken altogether, our findings again demonstrate the potential of LfcinB as a chondroprotective molecule, and our current understanding of its mode of action provides a mechanistic basis for future in vivo investigations. 12. Kim, J. S., Ellman, M. B., Yan, D., An, H. S., Kc, R., Li, X., Chen, D., Xiao, G., Cs-Szabo, G., Hoskin, D. 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(2007) Basic fibroblast growth factor activates the MAPK and NFB path- ways that converge on Elk-1 to control production of matrix metallopro- teinase-13 by human adult articular chondrocytes. J. Biol. Chem. 282, 31409–31421 Acknowledgments—We thank the Gift of Hope Organ Tissue Donor Network, as well as Drs. Chubinskaya and Margulis, for making human tissues available, and we also extend our appreciation to the tissue donor families who made it possible. We thank Dr. Gabriella Cs-Szabo and David Gerard for time and efforts in OA tissue acquisition. 17. Loeser, R. F., Yammani, R. R., Carlson, C. S., Chen, H., Cole, A., Im, H. J., Bursch, L. S., and Yan, S. D. (2005) Articular chondrocytes express the receptor for advanced glycation end products. Potential role in osteoar- thritis. Arthritis Rheum. 52, 2376–2385 18. Walmsley, M., Butler, D. M., Marinova-Mutafchieva, L., and Feldmann, M. LfcinB-induced IL-11-STAT3 Axis Exerts Anti-inflammation (2010) Chondrocyte innate immune myeloid differentiation factor 88-dependent signaling drives procatabolic effects of the endogenous Toll-like receptor 2/Toll-like receptor 4 ligands low molecular weight hyaluronan and high mobility group box chromo- somal protein 1 in mice. Arthritis Rheum. 62, 2004–2012 6. Calich, A. L., Domiciano, D. S., and Fuller, R. (2010) Osteoarthritis. Can anti-cytokine therapy play a role in treatment? Clin. Rheumatol. 29, 451–455 7. Moos, V., Fickert, S., Müller, B., Weber, U., and Sieper, J. (1999) Immuno- histological analysis of cytokine expression in human osteoarthritic and healthy cartilage. J. Rheumatol. 26, 870–879 7. Moos, V., Fickert, S., Müller, B., Weber, U., and Sieper, J. (1999) Immuno- histological analysis of cytokine expression in human osteoarthritic and healthy cartilage. J. Rheumatol. 26, 870–879 8. Maier, R., Ganu, V., and Lotz, M. (1993) Interleukin-11, an inducible cy- tokine in human articular chondrocytes and synoviocytes, stimulates the production of the tissue inhibitor of metalloproteinases. J. Biol. Chem. 268, 21527–21532 9. Hermann, J. A., Hall, M. A., Maini, R. N., Feldmann, M., and Brennan, F. M. (1998) Important immunoregulatory role of interleukin-11 in the inflammatory process in rheumatoid arthritis. Arthritis Rheum. 41, 1388–1397 FIGURE 9. Hypothetical model of LfcinB-mediated chondroprotection. Evidence suggests that LfcinB exerts its chondroprotective activities through multiple mechanisms, including (i) competitive binding to the co-receptors of IL-1 and FGF-2; (ii) induction of anti-inflammatory cytokines, such as IL-11; and (iii) induction of anti-catabolic TIMP-3. FIGURE 9. Hypothetical model of LfcinB-mediated chondroprotection. Evidence suggests that LfcinB exerts its chondroprotective activities through multiple mechanisms, including (i) competitive binding to the co-receptors of IL-1 and FGF-2; (ii) induction of anti-inflammatory cytokines, such as IL-11; and (iii) induction of anti-catabolic TIMP-3. 10. Cleaver, C. S., Rowan, A. D., and Cawston, T. E. (2001) Interleukin 13 blocks the release of collagen from bovine nasal cartilage treated with proinflammatory cytokines. Ann. Rheum. Dis. 60, 150–157 11. Yan, D., Chen, D., Shen, J., Xiao, G., van Wijnen, A. J., and Im, H. J. (2013) Bovine lactoferricin is anti-inflammatory and anti-catabolic in human ar- ticular cartilage and synovium. J. Cell. Physiol. 228, 447–456 cytes to exogenous IL-11, because we observed that OA chon- drocytes highly up-regulate TIMP-1 expression upon IL-11 stimulation (data not shown). 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https://openalex.org/W2581952271	https://seer.ufrgs.br/index.php/cadernosdoil/article/download/67782/pdf	English	null	Alice Meets the Antipathies: relations between "The Magic Pudding" and the "Alice" books	Cadernos do IL	2,017	cc-by	11,130	Submetido em 09 de setembro de 2016. Submetido em 09 de setembro de 2016. Submetido em 09 de setembro de 2016. Aceito para publicação em 10 de novembro de 2016. Cadernos do IL, Porto Alegre, n.º 53, janeiro de 2017. p. 281-297 Cadernos do IL, Porto Alegre, n.º 53, janeiro de 2017. p. 281-297 POLÍTICA DE DIREITO AUTORAL Autores que publicam nesta revista concordam com os seguintes termos: Autores que publicam nesta revista concordam c (a) Os autores mantêm os direitos autorais e concedem à revista o direito de primeira publicação, com o trabalho simultaneamente licenciado sob a Creative Commons Attribution License, permitindo o compartilhamento do trabalho com reconhecimento da autoria do trabalho e publicação inicial nesta revista. (b) Os autores têm autorização para assumir contratos adicionais separadamente, para distribuição não exclusiva da versão do trabalho publicada nesta revista (ex.: publicar em repositório institucional ou como capítulo de livro), com reconhecimento de autoria e publicação inicial nesta revista. (c) Os autores têm permissão e são estimulados a publicar e distribuir seu trabalho online (ex.: em repositórios institucionais ou na sua página pessoal) a qualquer ponto antes ou durante o processo editorial, já que isso pode gerar alterações produtivas, bem como aumentar o impacto e a citação do trabalho publicado. p (d) Os autores estão conscientes de que a revista não se responsabiliza pela solicitação ou pelo pagamento de direitos autorais referentes às imagens incorporadas ao artigo. A obtenção de autorização para a publicação de imagens, de autoria do próprio autor do artigo ou de terceiros, é de responsabilidade do autor. Por esta razão, para todos os artigos que contenham imagens, o autor deve ter uma autorização do uso da imagem, sem qualquer ônus financeiro para os Cadernos do IL. Alice meets the antipathies: relations between The Magic Pudding and the Alice books Liziane Kugland de Souza 1 Liziane Kugland de Souza holds a Teaching Degree in English Language and Literature from UFRGS (Federal University of Rio Grande do Sul), Porto Alegre, Brazil, and is currently a Master’s student in Literature in Modern Foreign Languages at UFRGS. ALICE ENCONTRA OS ANTIPATHIES: RELAÇÕES ENTRE THE MAGIC PUDDING E OS LIVROS DE ALICE Liziane Kugland de Souza1 RESUMO: As conexões com Alice’s Adventures in Wonderland e Through the Looking-Glass estabelecidas pelo clássico infantil australiano The Magic Pudding incluem temas consagrados em histórias infantis, como “comida” e “animais falantes”, e episódios como um julgamento em que figuras de autoridade são ridicularizadas. Antes de servirem como instrumentos para ensinamentos morais ou comportamentais, as três histórias parecem focar na diversão infantil e na criança como tomadora de decisões. O conceito de “intertextualidade” conforme abordado por Julia Kristeva proporciona a base teórica para a análise das relações entre esses livros infantis, dois deles canônicos e um periférico, demonstrando que ambos os lados são aprofundados e ampliados quando lidos conjuntamente. PALAVRAS-CHAVE: Alice’s Adventures in Wonderland; intertextualidade; The Magic Pudding; Through the Looking-Glass. ABSTRACT: The connections to Alice’s Adventures in Wonderland and Through the Looking-Glass established by the Australian children’s classic The Magic Pudding include themes often seen in children’s stories like “food” and “talking animals,” as well as episodes such as a trial in which figures of authority are ridiculed. Rather than instruments for moral and behavioral lessons, the three stories seem to focus on children’s amusement and on the child as a decision maker. The concept of “intertextuality” as approached by Julia Kristeva provides a framework for the analysis of relations between these canonical and peripheral children’s books, demonstrating that both sides are deepened and amplified when read together. KEYWORDS: Alice’s Adventures in Wonderland; intertextuality; The Magic Pudding; Through the Looking-Glass. POLÍTICA DE ACESSO LIVRE POLÍTICA DE ACESSO LIVRE Esta revista oferece acesso livre imediato ao seu conteúdo, seguindo o princípio de que disponibilizar gratuitamente o conhecimento científico ao público proporciona sua democratização. http://seer.ufrgs.br/cadernosdoil/index Quinta-feira, 19 de janeiro de 2017 15:59:59 http://seer.ufrgs.br/cadernosdoil/index Quinta-feira, 19 de janeiro de 2017 15:59:59 281 1. Reading Alice through the Pudding: intertextual relations This article analyses three children’s books (two canonical and one non- canonical) in order to demonstrate that the dialogue established between them by the newest of them changes the way the world classics are read. Julia Kristeva’s concept of intertextuality provides a framework for the reflections on how texts can be modified by other texts, even subsequent ones. One of Julia Kristeva’s greatest contributions to the field of literary studies is her view on the relationships established between works, authors or cultural systems. The 282 term “intertextuality” was first proposed in her 1966 essay “Word, Dialogues and Novel” (MOI, 1986), in which she defends that any and every text exists in relation to others. By amplifying Bakhtin’s thoughts on “dialogism,” she has deepened the way texts were studied: “any text is constructed as a mosaic of quotations; any text is the absorption and the transformation of another. The notion of intertextuality replaces that of intersubjectivity, and poetic language is read as at least double.” (MOI, 1986, p. 37) Poet T. S. Eliot had already pointed out that the most individual works, or the most authorial parts of an author’s work, could be those in which the touch of her/his ancestors is more visible; and that all literary works are somehow simultaneous, subject to a constant process of adjusting and re-adjusting relations among them (1971, 784 – 785). The text-text relationships do not happen only in one direction, and can operate a posteriori, with new texts influencing the reading of other texts already in existence. p , g g y The works herein analysed are Alice in Wonderland and Through the Looking- Glass, by Lewis Carroll – canonized –, and The Magic Pudding, by Norman Lindsay – non-canonical in the universal literature, but part of the Australian canon. Although the Alice books were written decades before The Magic Pudding, a number of elements present in Lindsay’s tale work as a response to Carroll’s Alices. This response established by the very existence of The Magic Pudding – whether intentional or coincidental – has transformed both the canon and the periphery of the literary world (here, specifically but not exclusively, children’s literature) in a way that can only be perceived when we look closely into an apparently unimportant detail in Carroll’s first novel: the brief mention to the inhabitants of the other side of Carroll’s / Alice’s world. 1. Reading Alice through the Pudding: intertextual relations On the other side, there were the Australians and the New-Zealanders, the “Antipodes,” the “others,” with their own culture, people, literature and voice. If, according to Kristeva, every text holds traces of other texts, while it may become the inspiration or the starting point for a number of others, both “centre” and “periphery” are mutually aggrandized and deepened when read together. The voice of the Antipodes is discussed herein through the abundant examples of points of intersection between the English and the Australian stories, which is the reason why this article is mostly descriptive. More specifically, among the intertextual relations between the three, one element stands out, the form that both authors saw the child reader: not only as a passive receptor of educational and social principals decided by adults, but as an individual with their own thoughts and wishes, which not always coincide with what is expected from them. 2. The English and the Australians In Alice’s Adventures in Wonderland (1865), as Alice falls down the rabbit-hole, she wonders about the other side: “I wonder if I shall fall right through the earth! How funny it’ll seem to come out among the people that walk with their heads downwards! The Antipathies, I think” – (she was rather glad there was no one listening, this time, as it didn't sound at all the right word) “– but I shall have to ask them what the name of 283 the country is, you know. Please, Ma’am, is this New Zealand or Australia?” (CARROLL, 2000, p. 13 – 14) Alice is a Victorian English girl and, like many people in the Northern Hemisphere, especially in Europe, she thinks that the antipodal point to home is Australia and New Zealand. As a recurring feature throughout the tale, Alice has difficulty remembering words and calls their inhabitants “the Antipathies” meaning “the Antipodes” or “Antipodeans,” as they were referred to in England. According to her logic, if they live on the other side of the planet, they must “walk with their heads downwards” (CARROLL, 2000, p. 13). What concerns her is the proper way to behave in their society, which indicates that she expects to find people, regardless of their appearance. When she arrives in Wonderland, she meets living playing cards, talking animals and foods, and all kind of real or legendary creatures, including human beings, but no Australians or New Zealanders, who are never be mentioned again. g The first Alice novel is established in the literary canon and is still popular all over the world, in English and in countless translations and adaptations into over 170 languages, and into abridged and unabridged versions, picture books, live-action and animated films, ballets, operas, musicals and all imaginable kinds of media, for children or adults. The second Alice, Through the Looking Glass (1871) – henceforth referred to as TTLG –, is also popular and often confused with the first one, especially after the 1951 Disney movie, which combines characters and events from both stories. In her second adventure, Alice has a dream again and travels to another fantastic land, now on the other side of the mirror, where she also meets all kinds of strange creatures, including talking foods. 2. The English and the Australians Everything seems to be backwards or upside-down in this world, not in the way the girl imagines while falling down the rabbit-hole in the first book, but in various other manners. There, she realizes that she is about to cross a giant chessboard on which she is initially a pawn, but, according to the rules of the game, upon getting to the opposite edge of the board, she can be promoted to Queen. A pudding talking to a diner (and disagreeing with the latter), in a cartoon published in the Punch in January 1861, might have influenced Carroll to create a dialogue between Alice and a plum pudding at a dinner party in TTLG. This dinner- party episode is said (KELLEN, 2007, p. 334) to have been the inspiration for Australian author and artist Norman Lindsay to create the Australian children’s classic The Magic Pudding: Being The Adventures of Bunyip Bluegum and his friends Bill Barnacle and Sam Sawnoff, or simply The Magic Pudding – henceforth referred to as TMP –, in 1918. “Antipodean” children can still enjoy the Alices as the rest of the world, but they also have a beloved classic of their own, a story set in the Australian bush, with Australian characters, illustrations, songs and peculiar humor. g p Bunyip Bluegum is a young koala that leaves the tree house where he lives with his Uncle Wattleberry in order to see the world. On the road, he meets Albert (the Magic Pudding) and his owners and joins the “Noble Society of Puddin’-owners” (LINDSAY, 2006, p. 35). When the Pudding is stolen, they must recover him and, on their way, they meet the other characters – a bandicoot, a kookaburra, a rooster, a parrot, and a dog, among other Australian animals and human beings – and are eventually arrested and taken to a Court House to be judged for riot and disorder. Similarly to the endings of the two Carroll’s novels, in which Alice wakes up from her dreams in scenes of conflict – a legal trial and a dinner party respectively –, in TMP, after a huge fight involving all the characters, including the Judge, the city Mayor and a Constable, they manage to escape with their Pudding. 2. The English and the Australians Like the two Alice books, 284 the story develops as a journey; however, while Alice is only dreaming, and returns home at the end, Bunyip Bluegum moves to another tree house to live with his new friends and the Pudding. g In spite of the fact that TMP is a celebrated classic in its own country, and still in print, it has not become part of the world literary canon nor has it been translated into so many languages as the Alices – except for Spanish, Japanese, French and German – remaining unknown to children all over the world. The plot is apparently not meant to educate child readers in terms of social rules or appropriate behavior, such as obeying their parents or not fighting, for instance. It has rather a picaresque tone, and the characters cannot be clearly identified as heroes – the illustrations depict animals smoking; the plot is full of verbal and physical fights; and there is even the suspicion of a murder committed by the “heroes” – which might account for its lack of popularity outside of Australia. Nevertheless, it is a classic worth reading by today’s children, the same who enjoy the Alice books, considering the remarkable number of similar or identical elements shared by the three stories, which must be given due attention. That is why this article discusses the intertextual relationships between these works as well as between their authors. 3. Lindsay and Carroll, authors and artists It is a well-known fact that Lewis Carroll – Charles Lutwidge Dodgson’s pen name – wrote and illustrated the original manuscript of Alice, Alice’s Adventures Under Ground, for a ten-year-old girl named Alice Liddell. Dodgson was also a very talented and prolific amateur photographer, but his drawing talent was not comparable to his photographic or poetic skills, so the book was rewritten, amplified and then illustrated by John Tenniel – a professional artist, illustrator and cartoonist for the Punch. In 1865, signed by Lewis Carroll, the manuscript became Alice’s Adventures in Wonderland (henceforth referred to as AAW), followed by TTLG in 1871, also illustrated by Tenniel (JONES; GLADSTONE, 1998). Whereas Dodgson was a mathematician, teacher and photographer who became a writer, Norman Lindsay was a prolific poet and novelist, and a multi-talented artist, who worked for over fifty years as an illustrator for the Bulletin, as well as for novels and collections of poems by other authors, mostly for adults. He produced drawings, paintings, sculptures, etchings and lithographs, as the ones in black and white for TMP, his second literary work – after the novel A Curate in Bohemia (1913) – and first of two children’s books – followed by Flyaway Highway (1936). His fine and avant-garde style is one of the greatest features in the book, as much as Tenniel’s work in the Alice books. In the three books, the illustrations are not mere accessories, but they play an important role in the narrative: as Alice says at the opening paragraph of AAW, “what is the use of a book without pictures or conversations?” (CARROLL, 2000, p. 11) A detail worth noticing is that while Carroll wrote his stories to the Liddell girls, Lindsay wrote TMP to amuse his nephew Peter, and to prove to a co-worker at the Bulletin that children preferred food to fairies (STEWART, 2012, p. 14). I would not go so far as to affirm that the Alice books are (or originally were) meant for girls and TMP for boys; however, this thought should not be ruled out, since we talk about times when these gender divisions were broadly accepted and thus much more common than today. The protagonists and most of the characters in TMP are male humans or animals 285 involved in proudly manly activities. 3. Lindsay and Carroll, authors and artists The protagonist of Carroll’s books is a girl, and her main antagonists are female – the Queen of Hearts and the Duchess in AAW, the Red and the White Queens in TTLG –, whereas there are only three female characters in TMP, two women and one hen. Although they are all depicted in the illustrations, they do not have an active role in the narrative: the hen is only a rooster’s wife, referred to as “the wife” (LINDSAY, 2006, p. 57 – 58); the others are respectively a penguin’s human bride – “the penguin’s bride” (LINDSAY, 2006, p. 66 – 68) – and a human sailor’s bride – “the bo’sun’s bride” (LINDSAY, 2006, p.104) –, both of them only mentioned in songs sung by male characters. g g y Norman Lindsay wrote TMP in 1918, during the First World War, while he was drawing war cartoons for the Bulletin and posters for Australia’s recruitment drives. His brother had been killed in the Somme a year earlier and he was devastated (BLOOMFIELD, 1979, p. 28): arguably, a children’s book could be a sort of escape from the war horrors to which he was submitted in his daily life. If food is an issue of great interest for children in general, lack of it during the war poses a threat to everyone; thus, the idea of never-ending food sounds comforting and sensible for a children’s tale. In 1865, when AAW was published, the latest famine known in Europe had been the Great Famine in Ireland in from 1846 to 1851, a time too far in the past for young children, but perhaps close enough to haunt their parents’ memories. Although the two authors lived in diverse contexts, what is undeniable is that they took food into serious consideration and treated it in a humorous as well as potentially frightening way – since living, aggressive, dubious, out-of-control food may amuse children as well as it may make them feel insecure. 4. Food Admittedly, food is a popular theme in children’s stories, films, cartoons and advertisements, but it is so ubiquitous in these three books, especially in TMP and AAW, that it deserves close attention. There is not one chapter in either of these stories in which food is not mentioned, and in TTLG, albeit not so often, it is also present as a key element, in addition to possibly being a source of inspiration for the title character. The main character in TMP is Albert, a grumpy and rude anthropomorphic pudding whose greatest pleasure is to be constantly eaten. Even though Albert in his living form does not “walk with [his] head downwards” (CARROLL, 2000, p. 13), as Alice imagined the “Antipathies,” his basin is not positioned under him, but on top of his head, as a hat, and his legs and arms come right out of his head, which is in fact his entire body. Therefore, it is reasonable to think that if the basin were regularly positioned, the Pudding’s legs would be upwards and consequently, his head would be downwards. Albert is magic because, by means of two whistles and a turn of his basin, his flavor changes and he can be made into any other kind of pudding – from a steak- and-kidney pudding to jam rolls or apple dumplings; at the end of the meal, he is reformed to his original round shape without a mark left on his body: “It’s a cut-an’- come-again Puddin’” (LINDSAY, 2006, p. 17). Such a wonder is naturally coveted by pudding thieves – a Possum and a Wombat –, who will take every opportunity to steal him from his “Noble Pudding- owners,” Bill Barnacle (a man) and Sam Sawnoff (a penguin), both former sailors, who also stole him from his original owner. According to the theme, the book is divided into 286 four Slices, which end with the characters eating and singing rowing songs, mostly about food and fights. These are actually the two main themes in the narrative: not only do the characters need to fight to recover their Pudding whenever he is stolen, they also seem to enjoy engaging in a brawl as much as they enjoy sitting round a camp fire eating and singing songs about food and rows. The word “pudding” encompasses a range of dishes, savory or sweet. 4. Food In England, it usually denotes a dessert course, such as a bread-and-butter pudding, but the steak and kidney pudding is also a British classic. Lindsay used the variety of dishes called “puddings” in Australia as inspiration for his Magic Pudding. Nevertheless, puddings are not the only foods mentioned in the story. On their way after the Pudding, through rural towns in the Australian bush, they meet several animals, among them Henderson Hedgehog, a nearly deaf hedgehog who is “horticulturing a cabbage” (LINDSAY, 2006, p. 49) in his garden; and an elderly dog named Benjimen Brandysnap carrying a basket of eggs (LINDSAY, 2006, p. 84). The dog owns a market garden (where Albert and his owners move in together at the end of the story), and grows vegetables, which he describes in a peculiar manner: “the radishes swarmed on the angry air” (LINDSAY, 2006, p. 124). Incidentally, he is one of the characters named after food – “brandy snaps” are sweet tubular casings filled with cream –; and another is Curry and Rice, a human cook from whom the pudding-owners have supposedly stolen Albert. Towards the ending, after one of several fights, both owners and thieves are arrested by a Constable, who is accompanied by the city Mayor, and the former constantly eats bananas and also thrusts them into the latter’s mouth to calm him down (LINDSAY, 2006, p. 110 – 112). When they enter the Court House, they meet the Judge and the Usher, who are enjoying a game of cards over a bottle of port (LINDSAY, 2006, p. 115) – another relation to the Alices, since playing cards are personages in AAW. The food theme pervades AAW as well, from the beginning, as Alice falls into the rabbit hole, to the end, when she wakes up from her dream and is called for tea by her sister. She tries a great variety of foods and drinks, which, while making her grow large and small, establish a rhythm to the narrative: whenever she drinks or eats anything, she knows that “something interesting is sure to happen” (CARROLL, 2000, p. 38). Apparently, she feels hungry all the time during her travel to Wonderland, since she only has a chance to eat or drink anything at the beginning of the adventure. 4. Food 110); soon, a witness enters drinking tea and eating a piece of bread-and-butter (CARROLL, 2000, p. 113). The trial finishes when Alice confronts the Queen of Hearts and wakes up from her dream to have tea like a good English girl. In TTLG, food is not as frequently mentioned as in AAW, but it is also present in several passages. Alice learns about peculiar insects, whose bodies are made of plum pudding, raisins, frumenty, mince pie, and bread-and-butter (“a bread-and-butter-fly”) (CARROLL, 2000, p. 174 – 175), and hears a song about oysters (CARROLL, 2000, p. 183 – 187). After some brief mentions to eggs and jam, food is again the main conversational topic, with mentions to a ham sandwich, white and brown bread, bread- and-butter, and a plum cake (CARROLL, 2000, p. 223 – 230). This cake is not magic as the Magic Pudding, but it also has a sort of magic quality, because, being a Looking- glass cake, it should be handed “round first, and cut […] afterwards” (CARROLL, 2000, p. 231). Sandwiches and carrots are among the things carried by the White Knight, who has invented “a new pudding during the meat-course” (CARROLL, 2000, p. 242), made of blotting-paper, gunpowder and sealing-wax (CARROLL, 2000, p. 242 – 243), and who sings a song which mentions batter, dough, buttered rolls, wine, and mutton-pies made of butterflies (CARROLL, 2000, p. 244 – 246). At the end, after reaching the opposite edge of the chessboard, Alice becomes a Queen, and attends a dinner party, where she is introduced to a living leg of mutton and a talking plum pudding (CARROLL, 2000, p. 261 – 262). p g p Even though the Pudding in TTLG has a brief appearance and Albert is the Magic Pudding in the title, they both have a voice and personality, and they both sound rude as well as amusing. Unlike Albert, who often urges his companions to keep eating him, the pudding in TTLG feels outraged by the suggestion: “What impertinence!” said the Pudding. “I wonder how you’d like it, if I were to cut a slice out of you, you creature!” (CARROLL, 2000, p. 263). Naturally, Alice feels uncomfortable to be introduced to the mutton or the pudding, since she would no longer be able to eat them after making their acquaintance (CARROLL, 2000, p. 262 – 263). 4. Food Indeed, the only foods she eats are the bits of mushroom that change her size; a liquid from a bottle labeled “DRINK ME,” which tastes like “a sort of mixed flavor of cherry-tart, custard, pine-apple, roast turkey, toffee, and hot buttered toast” (CARROLL, 2000, p. 16 – 17) – a sort of “magical drink” like the multi-flavored Pudding in TMP; “a very small cake, on which the words ‘EAT ME’ were beautifully marked in currants,” (CARROLL, 2000, p. 18 – 19); and an unspecified liquid from another bottle that also makes her grow large and get stuck in a room (p. 38). If she is not actually drinking or eating, she sees, thinks or talks about it. While she is falling down the rabbit-hole, she takes a jar labeled “ORANGE MARMALADE” (CARROLL, 2000, p. 13) from a shelf, which, to her disappointment, is empty; next, she worries about her cat Dinah’s “saucer of milk at tea-time,” and wonders if “cats eat bats” (CARROLL, 2000, p. 14). She talks about food, for example, when she meets the Hatter, the March Hare and the Dormouse at a tea party (CARROLL, 2000, p. 69) – an occasion in which only the hosts have tea, and not the guest –, they discuss various topics, including the lack of food on the table or the effect of time on meals 287 (CARROLL, 2000, p. 75). Later on, she talks about fish – “they have their tails in their mouths, and they’re all over crumbs” (CARROLL, 2000, p. 103 – 104) – with the Gryphon and the Mock Turtle – “the thing Mock Turtle Soup is made from” (CARROLL, 2000, p. 94) –, who sing a song about a pie – “pie-crust, and gravy, and meat” (CARROLL, 2000, p. 107) – and another about soup: “Beautiful Soup, so rich and green” (CARROLL, 2000, p. 108). Towards the end of the novel, Alice attends a trial, which, according to the title of chapter XI (“Who Stole the Tarts?”), is held to punish whoever has stolen some tarts. Again, food is a fundamental issue, and, again, it is food that she has in mind when she enters the courtroom. As she looks around trying to understand what is happening, she sees the tarts on a platter, which make her hungry: “I wish they’d get the trial done (…) and hand round the refreshments!” (CARROLL, 2000, p. 5. Fighting As demonstrated above, like in many children’s stories, food is an important topic throughout Carroll’s and Lindsay’s books, and so is fighting, the other of the two major themes in the TMP. Even though the fights in Alice are mostly verbal arguments, whereas in TMP the characters often resort to kicking and punching, the three stories are similarly full of belligerence. In TMP, Bunyip Bluegum leaves home because his Uncle’s whiskers annoy him – they blow about in the wind and get into the soup – and, when he meets Albert, the Pudding, the latter immediately shows his bad manners and “his rough and ready way” (LINDSAY, 2006, p. 17). Then, they sing a song to tell Bunyip about the circumstances in which they became pudding-owners: by pushing the original owner off an iceberg and stealing the Pudding – Sam and Bill disagree on some points, “[f]or the night was dark and the flare went out” (LINDSAY, 2006, p. 21), but it is clearly suggested to the reader that the characters are not ideal role models. Also, the first time the thieves, the Wombat and the Possum, appear in the story, they have not stolen anything yet, but the owners decide to fight them because they suspect that they are “professional puddin’-thieves,” (LINDSAY, 2006, p. 24). The narrative of TMP is told in prose and poetry, mostly in song form, along with illustrations. Since two of the characters are former sailors, they often sing rowing songs about their rough life in the seas, which included physical and verbal abuses. The traumatic experience has probably influenced their rather inconsiderate attitudes, even toward people they seem to like and with whom they have fun and share meals. Their idea of having fun is often brutal, like in a demonstration of “breakfast humor,” when Sam pushes Bill’s face “into the Puddin’ with great violence” (LINDSAY, 2006, p. 38 –39). After Albert has been stolen by the thieves, they must find him, and not everyone they accost to ask for information is kind; some of them, such as a Parrot and a Kookaburra, are very intimidating, and when the pudding-owners meet Bunyip’s uncle on the road, they mistake him for a thieve in disguise and beat him. 4. Food The characters in TMP, on the other hand, are not required such scrupulous manners and eat their Pudding heartily. Nevertheless, there is a moment when Albert reveals he might not be so enthusiastic about his condition, echoing his counterpart’s words in TTLG: “‘It's all very fine,’ said the Puddin’ gloomily, ‘singing about the joys of being penguins and pirates, but how’d you like to be a Puddin’ and be eaten all day long?’” (LINDSAY, 2006, p. 34) The dinner party is the climax of TTLG and, after another song about fish, all the foods, dishes, bottles, forks and candles come to life and Alice’s dream ends in a 288 turmoil, with her defying the Red and the White Queens’ power, since they are mere chess pieces, after all. Whether this episode served as inspiration for Lindsay or not, for those who read both stories now, a relationship is established between them, or, at least, between the pudding characters. From a “walk-on part” in TTLG to the main character in TMP, in which Albert is the centre of all the other character’s attention and the reason over which they fight. Out sprang Bill and Sam and set about the puddin'-thieves like a pair of windmills, giving them such a clip-clap clouting and a flip-flap flouting, that what with being punched and pounded, and clipped and clapped, they had only enough breath left to give two shrieks of despair while scrambling back into Watkin Wombat's Summer Residence, and banging the door behind them. (LINDSAY, 2006, p. 62 – 63) 5. Fighting The fights between Pudding owners and thieves are described in detail: In the Fourth (and last) Slice, Albert, his owners and thieves are all arrested in the town of Tooraloo for fighting in the streets and, after realizing the Judge is not 289 trustworthy and intends to put everyone in jail and eat the Pudding, the owners decide to dismiss him, assume control of the trial and judge the thieves. The trial, like the one in AAW, naturally ends up with everybody fighting; the owners manage to escape with the Pudding and decide to live together with Albert and Bunyip in Brandysnap’s market garden, where Albert enjoys throwing “bits of bark at the cabbages” and pulling “faces at the little pickle onions, in order to make them squeak with terror.” (LINDSAY, 2006, p. 137) In AAW, Alice argues with herself and with most “people” she meets; often because they try to make her behave in a way with which she disagrees, or because she cannot understand their customs and ends up offending her interlocutors. She causes a misunderstanding by mentioning cats to a Mouse (CARROLL, 2000, p. 26 – 27) and has arguments with a Lory, who also argues with the Mouse, who disagrees with a Duck, while an Eaglet and a Dodo have a quarrel, and so do an old Crab and her daughter (CARROLL, 2000, p. 29 – 36). Alice is patronized by the Duchess, by the Caterpillar (CARROLL, 2000, p. 48) and by the Mock Turtle and the Gryphon, who provoke her by doubting her knowledge and memory; she is confronted by the Hatter, the March Hare and the Dormouse at the “Mad Tea-Party”, and leaves after hearing she “shouldn’t talk,” a “piece of rudeness [that] was more than [she] could bear” (CARROLL, 2000, p. 77). She is also mistreated by living playing cards who, as apparently everyone else, are afraid of the Queen of Hearts (CARROLL, 2000, p. 80); and by the Queen of Hearts herself, who, “crimson with fury” at Alice, screams, “Off with her head!” (CARROLL, 2000, p. 81 – 82) The croquet game in which Alice takes part involves fights between the “balls” (actually, hedgehogs, an animal also featured in TMP), this time physically. When she first meets the Duchess, a violent scene ends with the Duchess’s cook throwing objects and Alice running away (CARROLL, 2000, p. 65 – 66). 5. Fighting The final two chapters take place in the uproarious courthouse – like the one in TMP –, where everybody fights with everybody (CARROLL, 2000, p. 110 – 127) until Alice puts an end to the situation by confronting the pack of cards, and wakes up by the side of her sister on the riverbank. In TTLG, quarrels are very common as well. Alice is conscious of proper manners and does not miss an opportunity to teach a lesson on how to behave to her cats or to the inhabitants of the other side of the mirror. They often try to teach her lessons, which usually lead to a disagreement. Live flowers criticize her (CARROLL, 2000, p. 157 – 161); she contradicts the Red Queen for talking nonsense (CARROLL, 2000, p. 162); the Guard on a train she catches is rude (CARROLL, 2000, p. 169 – 170); and, when she meets the twin brothers Tweedledum and Tweedledee, the very topic of the chapter is fighting. This chapter and its characters, as well as chapters VI (“Humpty Dumpty”) and VIII (“The Lion and the Unicorn”) are based on nursery rhymes popular in England at the time the book was published, being promptly recognizable by the Victorian child reader. The twin brothers’ nursery rhyme talks about a battle over Tweedledum’s rattle, supposedly spoiled by his brother; in the end, they are frightened away by a “monstrous crow” (CARROLL, 2000, p.181) and the battle never takes place. Humpty Dumpty is another character recognized because of his nursery rhyme – Humpty Dumpty sat on a wall: / Humpty Dumpty had a great fall – (CARROLL, 2000, p. 207). He criticizes Alice’s name and ordinary face (CARROLL, 2000, p. 219), scolds her for being semantically inaccurate, and finishes the conversation in an unfriendly way (CARROLL, 2000, p. 220). The next chapter is also about a fight between characters of a nursery rhyme and, again, several characters row with Alice 290 and with each other. The Lion, symbolizing England, and the Unicorn, Scotland, “were fighting for the crown; / The Lion beat the Unicorn all round the town” (CARROLL, 2000, p. 226), and Alice is among the spectators of the fight. This is the first time that there is actually a physical fight, although it is not described in detail as those in TMP. They soon stop “for refreshments” (CARROLL, 2000, p. 5. Fighting 227) and immediately start intimidating Alice by criticizing her appearance – “monster” – and inability to cut the looking-glass cake (CARROLL, 2000, p. 228 – 230). The next chapter also contains a physical fight, a horse battle between the Red and the White Knights (CARROLL, 2000, p. 234 – 235). This time, there is more violence suggested: “they began banging away at such fury that Alice got behind a tree to be out of the way of the blows” (CARROLL, 2000, p. 234), but the battle finishes with the opponents shaking hands. Chapter IX (“Queen Alice”) is the long end chapter, when the dream ends, after a sequence of disagreements with the Queens. When Alice decides she will not stand that behavior any longer, she “jump[s] up and seize[s] the tablecloth with both hands: one good pull, and plates, dishes, guests, and candles c[o]me crashing down together in a heap on the floor” (CARROLL, 2000, p. 266). Even though the fights are mostly arguments, they are as an important element in the narrative of both Alice books as they are in TMP. Apart from serving as further evidence of Lindsay’s inspiration for TMP, it also supports his thoughts concerning what children enjoy about stories: food and fighting – not only in Victorian England or early-twentieth-century Australia, but still in our days, considering the three books’ lasting popularity, as well as the ubiquity of these themes in children’s stories, comic books, films and cartoons. 6. Poetry, Songs and Parody ‘Let your conduct be noble, and never sing the National Anthem to people wearing bell-toppers.’ (LINDSAY, 2006, p. 107) ‘No singing “God save the King”, neither,’ said the other bell-topperer. ‘Let your conduct be noble, and never sing the National Anthem to people wearing bell-toppers.’ (LINDSAY, 2006, p. 107) The national anthem is “God Save the Queen”, then sung equally by English and Australian subjects, and only replaced with “Advance Australia Fair” in 1984. In 1918, when TMP was published, the monarch was a King, George V, and thus the anthem is called “God Save the King”. Top hats are mentioned for a reason here, since, after a period in 1910 working for the Punch in London, where his talent did not receive the respect and reverence he thought he deserved, Lindsay had a serious argument that ended with him “jumping on his hated, unworn top-hat until it was shapeless – a top-hat was a symbol of what Linsday hated most in Britain and in the British” (JENSEN, 1989, p. 4). The author’s feelings might have been another reason for him to “respond” to the English classics and to his antipodean fellow poet. p g p p The two Alices open with a poem, and TTLG also closes with one, like TMP. Musical and poetic parodies are used by the two authors: in fact, most poems in both Alice books are parodies, such as How doth the little crocodile / Improve his shining tail (AAW), a parody of “Against Idleness and Mischief” (1715) by Isaac Watts (1674 – 1748): How doth the little busy bee / Improve each shining hour. Watts was a theologian, a writer of religious hymns and a Christian man, like Carroll, who was an Anglican deacon, a position that did not prevent him from satirizing his society, and its religious and moral lessons. “You are old, Father William” is a parody of “The Old Man’s Comforts and How He Gained Them” by Robert Southey (1774 – 1843), which ends with the verses In the days of my youth I remember’d my God! / And He hath not forgotten my age (GARDNER, 2000, p. 49), whereas Carroll’s parody ends with the verses Do you think I can listen all day to such a stuff? / Be off, or I’ll kick you downstairs! (CARROLL, 2000, p. 50). 6. Poetry, Songs and Parody Food and fighting are not the only elements shared by the three stories. All of them are narrated in prose and poetry, with a great number of songs, as well as poetic elements, such as assonance, alliteration and rhyming, within the prose. Carroll and Lindsay were poets, and the latter wrote all the poems and songs in TMP; however, only one of the songs is possibly, and partly, a parody: “The Penguin Bold” – To see the penguin out at sea, / And watch how he behaves, / Would prove that penguins cannot be / And never shall be slaves (LINDSAY, 2006, p. 32) –, which sounds similar to “Rule, Britannia”, the patriotic British naval song by James Thomson and Thomas Arne (1740) – Rule, Britannia! Rule the waves:/ Britons never will be slaves. As stated above, TMP was written and published during World War I, a period when Australia, as a dominion of the British Empire, and thus part of the Britons, sent troops to fight Germany. The relationship between the English and Australians, both ruled by the same crown – the former representing the center of an empire and the latter, the periphery, the “other” or “the Antipodean” –, is diversely approached in Carroll and Lindsay. pp y Whereas the Antipodes are briefly mentioned in AAW, and never appear in the story, in the Australian narrative, “Englishness” is treated as part of their culture. The pudding thieves are not described as actually being Englishmen, but they allege to be so in order to avoid taking off their hats and having their disguise unveiled: “An Englishman's hat is his castle, and Top-hats are sacred things” (LINDSAY, 2006, p. 107). Earlier on, having hidden the Pudding under his hat, the Wombat was forced to 291 take it off as the National Anthem was sung (LINDSAY, 2006, p. 94 – 95), and now they want to prevent the pudding owners from using the same strategy: take it off as the National Anthem was sung (LINDSAY, 2006, p. 94 – 95), and now they want to prevent the pudding owners from using the same strategy: ‘No singing “God save the King”, neither,’ said the other bell-topperer. ‘Let your conduct be noble, and never sing the National Anthem to people wearing bell-toppers.’ (LINDSAY, 2006, p. 107) ‘No singing “God save the King”, neither,’ said the other bell-topperer. 7. Satire of Authority The Alice books have been object of innumerable fields of study and scholarly perspectives, ranging from philosophy and psychology to linguistics, terminology and history, and have been read by both adults and children for various reasons, humor certainly among them. Humor, whether in form of song parody, puns, nonsense, or even satire of religion and politics, has proved to be appealing to both child and adult readers. All children are somehow submitted to adult authority and, at home or at school, they are taught about conventions, hierarchy, proper social behavior, etiquette and obedience. Frequently, perhaps out of a sense of responsibility, authors and publishers treat children’s books as educational tools, a point of view which is not a problem in itself, but which may hamper children’s amusement for the sake of a faint idea of duty. This line of thought prioritizes correctness, or adequacy, based on adult criteria, undervaluing the child’s point of view. Carroll was a pioneer in children’s literature when he challenged this norm, by putting himself in the child’s position and writing to rather than for the child, which apparently was Lindsay’s idea as well. The three stories satirize social conventions and even legal norms, with several figures of authority depicted as objects of ridicule. The moral and behavioral lessons to which Alice is constantly submitted are not passively received by her, on the contrary, she tends to question orders or thoughts that do not seem logic or reasonable, which means that she is able to perceive patterns of behavior and hierarchy in the society where she lives, and that she will not necessarily agree with all of them. In a well-known passage of AAW, while the Duchess tries to find a moral in everything, Alice thinks to herself: “How fond she is of finding morals in things!” (CARROLL, 2000, p. 22). This hint of irony reflects the author’s views concerning children’s stories, which were usually meant to control or manipulate the readers into acting according to what society, their families and teachers expect from them. As Finnish author and illustrator of children’s and picture books Riitta Oittinen (2000, p. 125) points out, “the story is a parody that intentionally throws mud on all our ‘sacred cows’ like school, religion, babyhood and family life.” In TMP the satire of institutions and authorities is even more striking and also a source of amusement. 6. Poetry, Songs and Parody Other examples in AAW are Speak roughly to your little boy, the weird lullaby which the Duchess sings to her baby (CARROLL, 2000, p. 62), a parody of “Speak Gently,” attributed to G. W. Langford or David Bates (circa 1849); and Twinkle, twinkle, little bat, sung by the Hatter, a parody of “The Star” by Jane Taylor – Twinkle, twinkle, little star (GARDNER, 2000, p. 74). y y p TTLG presents the aforementioned nursery rhymes in their original version, but it contains poems inspired by previous poems and songs. One example is the poem repeated by Humpty Dumpty, which contains the verses In summer, when the days are long, / Perhaps you understand the song, probably inspired by “Summer Days” by Wathen Mark Wilks Call (1717 – 1870): In summer, when the days were long, / We walked two friends, in field and wood (GARDNER, 2000, p. 216 – 217). Other examples are: “Sitting on a Gate” (GARDNER, 2000, p. 244 – 247), a parody of William Wordsworth’s “Resolution and Independence” (GARDNER, 2000, p. 246); and To the Looking-Glass world it was Alice that said /‘I’ve a scepter in hand I’ve a crown on my head (CARROLL, 2000, p. 260), a parody of “Bonny Dundee”, a song from the play “The Doom of Devergoil” by Walter Scott (1771 – 1832): To the Lords of Convention ‘twas Claver’se who spoke (GARDNER, 2000, p. 260). f p p Another important point in Kristeva’s account of the Bakhtinian concept of “dialogism” is what she called its dynamic and revolutionary essence, including criticism of dogma and subversion of authority, elements also present in the children’s books discussed in this article – which makes her views subjectively adequate to guide this reflection. What the parodies in the three stories have in common is a sort 292 of contempt for rules and authority, especially when the time they were written and their authors’ backgrounds are taken into account. Carroll was a Victorian teacher and a religious man, and Lindsay could not be considered a progressive thinker either. Nonetheless, both authors revolutionized children’s literature by taking the child’s side against the figures of authority in their cultures. By burlesquing these figures as well as institutions and their rules, they also challenged the authority of their readers’ parents and teachers, who were, and still are, responsible for the decisions about what children should read. 7. Satire of Authority Whereas Alice is aware of conventions such as only addressing someone to whom one has been introduced or the proper way to curtsey, the bush characters in TMP jeer the koalas’ manners and praise a way of life more natural and free of strict conventions. A rude Parrot they meet on the road asks them for some tobacco, but judges by the looks of Bunyip Bluegum, the aristocratic koala, that he might not have any: “‘You ain't got any tobacco,’ he said scornfully to Bunyip Bluegum. ‘I can see that at a glance. You’re one of the non-smoking sort, all fur and feathers’” (LINDSAY, 2006, p.52). The koalas, Bunyip and his Uncle Wattleberry, 293 are fonder of social rules than the Pudding, his owners and most personages they meet on the road, not to mention the professional thieves. When Bunyip leaves home, outraged by his uncle’s whiskers, he worries about the proper way to be called, “a Traveller or a swagman?” (LINDSAY, 2006, p. 12), since he does not carry a swag or a bag. After joining the Pudding Society, his habits change as he learns to enjoy pleasures he did not know in his own environment. Although his knowledge and oratory are valued by his companions and help them to deceive the thieves and recover the Pudding, what they most heartily praise is freedom, which they associate with simple life and habits such as smoking, eating and singing. Comfort is not fundamental or even welcomed: “Why, as I always say,” said Bill, “if there's one thing more entrancin’ than sittin’ round a camp fire in the evenin’ it’s sitting round a camp fire in the mornin’. No bed and blankets and breakfast tables for Bill Barnacle.” (LINDSAY, 2006, p. 37) “Why, as I always say,” said Bill, “if there's one thing more entrancin’ than sittin’ round a camp fire in the evenin’ it’s sitting round a camp fire in the mornin’. No bed and blankets and breakfast tables for Bill Barnacle.” (LINDSAY, 2006, p. 37) Alice, on the other hand, is keen to look after her kittens’ hygiene and attitude, and in TTLG, she feels empathetic towards the White Queen and helps the latter when her shawl is blown away and a brush is entangled in her hair. 7. Satire of Authority Alice’s attitude shows that she is a polite and considerate girl, but it does not prevent her from questioning the Queens or any other figure of authority, whenever she finds it necessary. She wakes up from her two dreams after defying the authority of Kings and Queens – in AAW, a pack of playing cards, and in TTLG, a set of chess pieces –, which is indeed her usual reaction throughout the two adventures: she conquers her fear and bravely confronts everyone who tries to intimidate her. Showing a keen sense of logic and justice, she does not take for granted the authority of those who act or speak nonsensically or unfairly – such as the verdict coming before hearing the witnesses in the trial in AAW, or the Queen comparing a hill to a valley in TTLG. Chapter III – “A Caucus-Race and a Long Tale”– in AAW is full of these doubtful figures of authority, and the events satirize power and politics: a group of animals, including a Mouse, a Lory (incidentally, an Australian parrot), a Crab and a Dodo, all wet with giant Alice’s tears, must find a way to get dry, and do not know what to do. The girl takes part in the discussion and “after a few minutes it seemed quite natural to Alice to find herself talking familiarly with them, as if she had known them all her life” (CARROLL, 2000, p. 29). More than feeling comfortable among strangers, she will question their decisions and the very foundations of their power: Indeed, she had a long argument with the Lory, who at last turned sulky, and would only say “I’m older than you, and must know better”. And this Alice would not allow, without knowing how old it was, and, as the Lory positively refused to tell its age, there was no more to be said. (CARROLL, 2000, p. 29) Arguably, since the book was dedicated to a real Alice Liddell and her sisters, and it is full of characters and references to the girls’ life, the fictional Alice’s personality and some of the events and conversations in the story are based on actual ones. According to Martin Gardner (2000, p. 7. Satire of Authority 27), editor of The Annotated Alice, these animals are caricatures of people close to the Liddells, including the author – as the Dodo – and the girls – Lorina, Alice’s older sister, as the Lory, and Edith, her little sister, as the Eaglet. The argument between Alice and the Lory reflects a dispute over authority that is based in the feeble argument of age, thus questionable: she would not take orders from her older sister only because of her age. In fact, there is a phase in 294 any children’s lives when they detect and challenge their parents’, other adults’ or older children’s authority, which makes it an interesting issue to be raised in a children’s book. In Carroll’s novels, this challenge is a key feature in the character’s personality and pervades the whole narrative. Immediately after the argument with the Lory, the Mouse, described by the narrator as someone “who seemed to be a person of some authority among them” (CARROLL, 2000, p. 29), suggests he will manage to dry everyone by telling them a dry story. They are all wet after falling in a pool of giant Alice’s tears and the girl agrees to listen to him, who starts to narrate an episode of the English History involving William the Conqueror and other historical figures. Since the names are difficult to pronounce, the events impossible to be followed by the audience, and the entire strategy inefficient to dry them, the Mouse’s alleged authority is soon questioned and dismissed. Authority per se is thus not acknowledged, unless it can be proved in actuality rather than in theory or appearance. p y y pp In TMP, although the characters are not children like Alice, their views and attitude toward figures of authority are not distinct from hers, and include familial, legal and political authorities. If on the one hand Bunyip Bluegum leaves home claiming that he opposes his uncle’s habit of growing whiskers, on the other hand, he declares he wants to see the world, indicating that the whiskers might be only an excuse for him to live his own life and get rid of familial rule. Later on, when the pudding-owners meet Uncle Wattleberry on the road, he is mistaken for a thief, and accordingly, spanked and humiliated. 7. Satire of Authority He demands respect for his status, claiming he is “an Uncle” (LINDSAY, 2006, p.78), and feels the more offended because his own nephew, although not taking part in the assault, does not condemn his companions emphatically enough. However, the arguments of family or age as hierarchical rights do not suffice, because they are deemed illogic, unreasonable and thus dismissible. Eventually, as he feels so outraged that he will not forgive them or admit his own responsibility, he is simply left behind: Seeing that there was no possibility of inducing Uncle Wattleberry to look at the affair in a reasonable light, they walked off and left him to continue his bounding and plunging for the amusement of the people of Bungledoo, who brought their chairs out on to the footpath in order to enjoy the sight at their ease. (LINDSAY, 2006, p. 77) If the members of the “Noble Society of Puddin’-owners” have acquired their pudding by surreptitious, illegal procedures, they probably would not respect the law and its representatives, not even when they are supposed to be on their side. Accordingly, the representatives of Law, especially the Judges, are depicted as stupid, contemptible men, who seem to be more interested in food than in their duty. The episode of the trial in AAW shows the King of Hearts as an inept Judge, as well as the jurors, who do not know what they are supposed to do and are considered “stupid things” by Alice (CARROLL, 2000, p. 111). Besides, the Queen of Hearts, who keeps threatening and frightening her subjects, is not to be feared either, as the Gryphon tells Alice: “It’s all her fancy, that: they never executes nobody.” (p. 95). Not only the Judge, but also all the authorities in the town of Tooraloo in TMP – the Mayor, the Constable, and the Usher – are all equally incompetent and cowardly, so much so that the Pudding owners decide to judge the thieves without the interference of the authorities, following Bunyip’s advice: 295 “My advice,” he said, “is this: try the case without the Judge; or, in other words, assume the legal functions of this defaulting personage in the bag- wig who is at present engaged in distending himself illegally with our Puddin'.” (LINDSAY, 2006, p. 7. Satire of Authority 120) The judge’s wig is mentioned in AAW as well: when Alice enters the room and starts to identify the people and elements in the court of justice, she knows that the King of Hearts is the judge because he is wearing his crown over the wig, which was “certainly not becoming” (CARROLL, 2000, p. 111). His appearance matches his ineptitude and doubtful authority, allowing Alice to disobey his rules again. As demonstrated, in the Alices as well as in TMP, authority must have a plausible reason to be acknowledged and respected, or it will be challenged, if not dismissed altogether. Nevertheless, Alice, as a child, is often divided between abiding by the rules (and thus behaving appropriately and not offending anybody) and departing from them. The bush characters, on the other hand, seem to have the matter of social conventions sorted out, as put succinctly by Albert: Politeness be sugared, politeness be hanged, Politeness be jumbled and tumbled and banged. It's simply a matter of putting on pace, Politeness has nothing to do with the case. (LINDSAY, 20006, p. 16) 8. Alice after the Pudding After reading these works together and attentively, what becomes clear is that while children’s stories are often meant for educational purposes, according to what adults – parents, educators, publishers – deem to be convenient for a child, both Carroll and Lindsay are more concerned about what and how children think and enjoy. More importantly, the three stories allow the child reader to question the adults’ orders and behavior, and ultimately, their power over children. Lindsay goes even further in the satire of adults, society and authority by mocking the English and “Englishness” itself as a sign of affectation and phoniness. Besides, he does it from a peripheral point of view, which sounds like a blatant disregard for whatever authority might mean – be it political, legal or parental –, and precisely for the same reason, it also sounds irreverent and highly amusing. If his characters are not respectable English subjects, but only “the others”, “the Antipodes”, they are proudly conscious of their position and will not abase themselves before anyone or feel ashamed of their “rude and ready way” (LINDSAY, 2006, p. 17). Another sign of this kind of complicity established between author/text and child reader is that the three stories offer plenty of mentions to food and fighting, without any sort of advice on nutrition or manners, for example. All Alice knows is that she should not have something labeled “poison” or that some foods may cause changes in her size. The foods and drinks are presented in the three narratives as elements of amusement or of magic – sometimes dangerous, but not for the same reasons they would be dangerous in the real world –, and proper manners are often questioned and ridiculed. The characters in TMP are not classical heroes, who would risk their lives to help other people or who could be seen as role models. They are the Antipodes, after all, or rather the “Antipathies,” with their deprecatory remarks, their contempt for comforts or good manners, their pride to be rude and often violent. Although they are sometimes in the position of victims (e.g., when they are robbed by the thieves), they 296 are not easily told apart from the so-called villains of the story. They cannot even be seen as brave, although they do manage to conquer their fear in order to recover their food. 8. Alice after the Pudding In a word, they cannot be called noble, although they affirm to be so, which might lead young readers to identify with them, since they look like some actual people. They might lack nobility, but show abundant charisma, albeit not the usual, “desirable”, sort of charisma, if one considers parents or teachers who are concerned about providing their children with positive, educational examples by means of literature. Alice is certainly the heroine of her two adventures, and not a “criminal”, as the gang of the Pudding, but she is not the ideal role model either, if one expects a well-behaved, obedient Victorian girl. Her courage to defy all kinds of authority might be the greatest lesson this character can teach a child reader, which is evidence of how revolutionary Carroll was at his time. He made a choice for the child reader, by thinking of the child’s amusement first. The same choice was made by Lindsay, decades later: beyond the similarities concerning themes, motifs, events and narrative choices, the most important feature uniting these three classical books is the way the authors almost dismiss the adults, who are usually in charge, to focus on the very reader to whom they wrote. Moreover, both authors seem to respect the readers’ intelligence and write directly to them rather than for them, regardless of the interference of adults in the decision-making process. g p It cannot be truly ascertained whether Lindsay consciously wrote TMP as an antipodean response to the English Alices, whether the allusions to Carroll’s works and the British were intentional, whether the mention to the “Antipathies” in AAW provoked the Australian author into showing their side, or even whether the dinner- party episode indeed triggered the creation of Albert (since talking foods are a recurring motif in children’s stories, games or cartoons). Be that as it may, his book established inter-textual relationships between the two universes to such an extent that it has cast new light on the Alices. After TMP, Alice’s thoughts about “the Antipathies” in AAW, for example, have been re-signified, now illustrated with the bush characters and her peculiar manners, which work as foils to Victorian English Alice and her environment. 8. Alice after the Pudding In addition, her encounters and arguments with the inhabitants of Wonderland and of the Looking-Glass world have been enriched by the encounters and arguments in TMP, not to mention the new perspective on the relations between periphery and centre, and between children and their figures of authority. Following Kristeva’s thoughts, the possibility of re-reading classical novels as the Alices through the points of intersection with a new one such as TMP transforms both sides of this conversation. In addition to raising questions about originality and influence, this way to look at these three children’s books, no longer only as individual works, invites readers and scholars to investigate why the Australian classic has not gained a similar status or popularity among the readers of the Alices all over the world. Furthermore, it justifies the translation of the former into more languages than the four into which it has been translated so far. BLOOMFIELD, Lin. The World of Norman Lindsay. Sidney: Macmillan, 1979. CARROLL, Lewis. Alice’s Adventures in Wonderland. In: GARDNER, Martin (Intr. and notes). “The Annotated Alice”. New York: W.W.Norton & Company, 2000. REFERENCES BLOOMFIELD, Lin. The World of Norman Lindsay. Sidney: Macmillan, 1979. CARROLL, Lewis. Alice’s Adventures in Wonderland. In: GARDNER, Martin (Intr. and notes). “The Annotated Alice”. New York: W.W.Norton & Company, 2000. 297 ______. Through the Looking-Glass (and what Alice found there). In: GARDNER, Martin (Intr. and notes). “The Annotated Alice”. New York: W.W. Norton & Company, 2000. ______. Through the Looking-Glass (and what Alice found there). In: GARDNER, Martin (Intr. and notes). “The Annotated Alice”. New York: W.W. Norton & Company, 2000. ELIOT, T. S. Tradition and the Individual Talent. In: ADAMS, Hazard (ed.). “Critical Theory Since Plato”. Translation S. H. Butcher. San Diego: Harcourt, 1971, p. 784 – 787. GARDNER, Martin (ed., introd. and notes). The Annotated Alice. New York: W.W.Norton & Company, 2000. JENSEN, John. Australasian Cartoonist in Britain – Working paper nº 43. In: “Working Papers in Australian Studies”. London: University of London, 1989. JONES, Jo E.; GLADSTONE, J. Francis. The Alice Companion: a Guide to Lewis Carroll’s Alice Books. London: Macmillan, 1998. KELLEN, Christopher. The Magic Pudding: A Mirror of Our Fondest Wishes. JASAL – Journal of the Association for the Study of Australian Literature, Sidney, v. 6, p. 65 – 78, 2007. KRISTEVA, Julia. Word, Dialogue and Novel. In: MOI, Toril (ed.). “The Kristeva Reader”. Translation: Sean Hand. New York: Columbia University Press, 1986. MOI, Toril (ed.). The Kristeva Reader. New York: Columbia University Press, 1986. LINDSAY, Norman. The Magic Pudding: Being The Adventures of Bunyip Bluegum and his friends Bill Barnacle and Sam Sawnoff. New York: Dover Publications, 2006. OITTINEN, Riitta. Translating for Children. New York: Garland Publishing, 2000. STEWART, Douglas. Norman Lindsay: A Personal Memoir. Melbourne: Allen & Unwin House of Books, 2012. ELIOT, T. S. Tradition and the Individual Talent. In: ADAMS, Hazard (ed.). “Critical Theory Since Plato”. Translation S. H. Butcher. San Diego: Harcourt, 1971, p. 784 – 787. GARDNER, Martin (ed., introd. and notes). The Annotated Alice. New York W.W.Norton & Company, 2000. JENSEN, John. Australasian Cartoonist in Britain – Working paper nº 43. In “Working Papers in Australian Studies”. London: University of London, 1989. JENSEN, John. Australasian Cartoonist in Britain – Working paper n 43. In: “Working Papers in Australian Studies”. London: University of London, 1989. JONES, Jo E.; GLADSTONE, J. Francis. The Alice Companion: a Guide to Lewis ONES, Jo E.; GLADSTONE, J. Francis. The Alice Companion: a Guide to Lewis Carroll’s Alice Books. London: Macmillan, 1998. REFERENCES KELLEN, Christopher. The Magic Pudding: A Mirror of Our Fondest Wishes. JASAL – Journal of the Association for the Study of Australian Literature, Sidney, v. 6, p. 65 – 78, 2007. KRISTEVA, Julia. Word, Dialogue and Novel. In: MOI, Toril (ed.). “The Kristeva Reader”. Translation: Sean Hand. New York: Columbia University Press, 1986. KRISTEVA, Julia. Word, Dialogue and Novel. In: MOI, Toril (ed.). The Kristeva Reader”. Translation: Sean Hand. New York: Columbia University Press, 1986. MOI, Toril (ed.). The Kristeva Reader. New York: Columbia University Press, 1986. LINDSAY, Norman. The Magic Pudding: Being The Adventures of Bunyip Bluegum and his friends Bill Barnacle and Sam Sawnoff. New York: Dover Publications, 2006. OITTINEN, Riitta. Translating for Children. New York: Garland Publishing, 2000. STEWART, Douglas. Norman Lindsay: A Personal Memoir. Melbourne: Allen & Reader . Translation: Sean Hand. New York: Columbia University Press, 1986. MOI, Toril (ed.). The Kristeva Reader. New York: Columbia University Press, 1986. LINDSAY, Norman. The Magic Pudding: Being The Adventures of Bunyip Bluegum and his friends Bill Barnacle and Sam Sawnoff. New York: Dover Publications, 2006. OITTINEN, Riitta. Translating for Children. New York: Garland Publishing, 2000. MOI, Toril (ed.). The Kristeva Reader. New York: Columbia University Press, 1986. LINDSAY, Norman. The Magic Pudding: Being The Adventures of Bunyip Bluegum and his friends Bill Barnacle and Sam Sawnoff. New York: Dover Publications, 2006. OITTINEN, Riitta. Translating for Children. New York: Garland Publishing, 2000. and his friends Bill Barnacle and Sam Sawnoff. New York: Dover Publications, 2006. OITTINEN, Riitta. Translating for Children. New York: Garland Publishing, 2000. and his friends Bill Barnacle and Sam Sawnoff. New York: Dover Publications, 2006. OITTINEN, Riitta. Translating for Children. New York: Garland Publishing, 2000. STEWART, Douglas. Norman Lindsay: A Personal Memoir. Melbourne: Allen & Unwin House of Books, 2012.
https://openalex.org/W3143096508	https://iris.uniroma1.it/bitstream/11573/1627650/1/The%20CMS%20Collaboration_Measurement%20of%20prompt%20D0%20and%20D0_2021.pdf	English	null	Measurement of prompt D0 and <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si1.svg"><mml:msup><mml:mrow><mml:mover accent="true"><mml:mrow><mml:mi mathvariant="normal">D</mml:mi></mml:mrow><mml:mo>‾</mml:mo></mml:mover></mml:mrow><mml:mrow><mml:mn>0</mml:mn></mml:mrow></mml:msup></mml:math> meson azimuthal anisotropy and search for strong electric fields in PbPb collisions at <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML" altimg="si2.svg"><mml:msqrt><mml:mrow><mml:…	Physics letters. B	2,021	cc-by	18,570	1. Introduction pected to receive important contributions from medium energy loss and coalescence effects [17,18]. In ultrarelativistic heavy ion collisions, very strong and tran- sient (∼10−1 fm/c) magnetic and electric ﬁelds are expected to be induced by the collision spectators and participants [19]. Such electromagnetic (EM) ﬁelds are predicted to produce a difference in the vn harmonics for positively and negatively charged parti- cles [19]. In such a picture, the magnetic ﬁeld is mainly responsible for splitting the rapidity (y)-odd directed ﬂow (v1) [19,20]. The electric ﬁeld is predicted to induce a charge-dependent splitting in the v2 coeﬃcient and in the average transverse momentum (⟨pT⟩) values of the emitted particles [19]. As charm quarks are expected to be created very early in the collision, they have a higher proba- bility of interacting with this strong EM ﬁeld than the light ﬂavor quarks [20,21]. The observation of a strongly-coupled quark-gluon plasma (QGP), a state of matter composed of deconﬁned quarks and glu- ons, was established by experiments investigating ultrarelativistic heavy ion collisions at the BNL RHIC [1–4] and CERN LHC [5,6]. The azimuthal particle correlations constitute an effective tool to probe the properties of the QGP [1–9]. These correlations are pa- rameterized by a Fourier expansion [10–12], with the magnitude of the Fourier coeﬃcients, vn, providing information about the initial collision geometry and its ﬂuctuations [12]. The second- (v2) and third- (v3) order Fourier coeﬃcients are referred to as “elliptic” and “triangular” ﬂow harmonics, respectively. Measuring these coeﬃcients for particle species with different quark compo- sition provides additional information about this hot and dense medium [13]. Because of their large mass, charm and bottom quarks are produced earlier in the collisions than the light quarks (up and down) [14,15]. In addition, the charm and bottom quarks have masses many times larger than the typical temperatures in the QGP [16]. These heavy quarks experience the full evolution of the medium until the hadronization phase. As a consequence, the vn of charmed D0 (uc) and D 0 (uc) mesons (henceforth referred to as D0 mesons, except where explicitly stated otherwise) are ex- In this letter, measurements of the v2 and v3 coeﬃcients as functions of D0 meson rapidity, pT, and lead-lead (PbPb) collision centrality are presented. a r t i c l e i n f o Article history: Received 29 September 2020 Received in revised form 21 February 2021 Accepted 25 March 2021 Available online 29 March 2021 Editor: M. Doser Keywords: CMS Heavy-ﬂavor Charm Electromagnetic ﬁelds The strong Coulomb ﬁeld created in ultrarelativistic heavy ion collisions is expected to produce a rapidity- dependent difference (v2) in the second Fourier coeﬃcient of the azimuthal distribution (elliptic ﬂow, Article history: Received 29 September 2020 Received in revised form 21 February 2021 Accepted 25 March 2021 Available online 29 March 2021 Editor: M. Doser The strong Coulomb ﬁeld created in ultrarelativistic heavy ion collisions is expected to produce a rapidity- dependent difference (v2) in the second Fourier coeﬃcient of the azimuthal distribution (elliptic ﬂow, v2) between D0 (uc) and D 0 (uc) mesons. Motivated by the search for evidence of this ﬁeld, the CMS detector at the LHC is used to perform the ﬁrst measurement of v2. The rapidity-averaged value is found to be ⟨v2⟩ = 0.001 ±0.001 (stat)±0.003 (syst) in PbPb collisions at √ sNN = 5.02 TeV. In addition, 0 v2) between D0 (uc) and D 0 (uc) mesons. Motivated by the search for evidence of this ﬁeld, the CMS detector at the LHC is used to perform the ﬁrst measurement of v2. The rapidity-averaged value is found to be ⟨v2⟩ = 0.001 ±0.001 (stat)±0.003 (syst) in PbPb collisions at √ sNN = 5.02 TeV. In addition, 0 the inﬂuence of the collision geometry is explored by measuring the D0 and D 0mesons v2 and triangular ﬂow coeﬃcient (v3) as functions of rapidity, transverse momentum (pT), and event centrality (a measure of the overlap of the two Pb nuclei). A clear centrality dependence of prompt D0 meson v2 values is observed, while the v3 is largely independent of centrality. These trends are consistent with expectations of ﬂow driven by the initial-state geometry. Keywords: CMS Heavy-ﬂavor Charm Electromagnetic ﬁelds © 2021 The Author. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. Measurement of prompt D0 and D 0 meson azimuthal anisotropy and search for strong electric ﬁelds in PbPb collisions at √sNN = 5.02 TeV Measurement of prompt D0 and D 0 meson azimuthal anisotropy and search for strong electric ﬁelds in PbPb collisions at √sNN = 5.02 TeV .The CMS Collaboration ⋆ CERN, Switzerland CERN, Switzerland Contents lists available at ScienceDirect Contents lists available at ScienceDirect Physics Letters B 816 (2021) 136253 Physics Letters B 816 (2021) 136253 ⋆E-mail address: cms -publication -committee -chair @cern .ch. considered as a systematic uncertainty. Using the data recorded in PbPb collisions during the 2018 LHC run period, corresponding to https://doi.org/10.1016/j.physletb.2021.136253 0370-2693/© 2021 The Author. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by SCOAP3. ⋆E-mail address: cms -publication -committee -chair @cern .ch. ed by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Funded by 1. Introduction The collision centrality bins are given in percentage ranges of the total inelastic hadronic cross section, with the 0–10% centrality bin corresponding to the 10% of collisions having the largest overlap of the two nuclei. The ﬂow harmonics are measured using the scalar product method [22,23]. In this anal- ysis, the selection of D0 mesons uses multivariate methods [24] for selecting D0 candidates and their antiparticles. The contamina- tion from nonprompt D0 candidates, arising from B meson decay, is considered as a systematic uncertainty. Using the data recorded in PbPb collisions during the 2018 LHC run period, corresponding to The CMS Collaboration Physics Letters B 816 (2021) 136253 of tracks from oppositely charged particles and requiring an in- variant mass (minv) within a ±200 MeV/c2 window of the world- average D0 meson mass of (1864.83 ± 0.05) MeV/c2 [33]. For each pair of selected tracks, two possible candidates for D0 and D 0 mesons are considered by assuming one of the tracks has the pion mass, while the other track has the kaon mass, and vice versa. Kinematic vertex ﬁts are performed to reconstruct the secondary vertices of D0 candidate decays. 0.58 nb−1 of integrated luminosity, the ﬂow coeﬃcients are mea- sured within the rapidity range \|y\| < 2, which is twice as large as achieved in previous CMS measurements [25]. The extension of the measurements to this larger rapidity range, together with smaller statistical uncertainties provided by a larger data set, furnish im- portant inputs for a better understanding of the three-dimensional evolution of the QGP formed in heavy ion collisions. Measurements of the v2 difference between D0 and D 0 mesons, v2, as a func- tion of rapidity are presented as a method to probe possible effects originating from the Coulomb ﬁelds. of tracks from oppositely charged particles and requiring an in- variant mass (minv) within a ±200 MeV/c2 window of the world- average D0 meson mass of (1864.83 ± 0.05) MeV/c2 [33]. For each 0 pair of selected tracks, two possible candidates for D0 and D 0 mesons are considered by assuming one of the tracks has the pion mass, while the other track has the kaon mass, and vice versa. Kinematic vertex ﬁts are performed to reconstruct the secondary vertices of D0 candidate decays. 4. Analysis technique The elliptic and triangular ﬂow coeﬃcients of D0 mesons are extracted using the scalar product (SP) method, similarly to what was done in a previous CMS publication [25]. In this method, the vn coeﬃcients of D0 candidates (including backgrounds) are mea- sured using Events from Monte Carlo (MC) simulations are used to study both prompt and nonprompt D0 meson processes. The events are generated using an embedding procedure, in which D0 mesons generated by pythia 8.212 [29] (tune CP5 [30]) are embedded into MB events from hydjet 1.9 [31]. A full simulation of the CMS de- tector is performed using Geant4 [32]. The prompt D0 meson MC simulation is employed to deﬁne signal selections and measure ef- ﬁciency corrections, while the nonprompt D0 meson MC sample is used to estimate systematic uncertainties coming from nonprompt D0 contamination. vn{SP} ≡ ⟨Q D0 n Q ∗ nA⟩ ⟨Q nA Q ∗ nB⟩⟨Q nA Q ∗ nC⟩ ⟨Q nB Q ∗ nC⟩ , (1) (1) with the Q -vectors expressed as Q n ≡M j=1 wjeinφj, where the sum is over the total number (M) of HF towers above a certain energy threshold (with the weights wj taken as the energy de- posited in the HF tower at azimuthal angle φj), of tracks with pT above a certain threshold (with wj taken as track pT in φj angle), or of selected D0 meson candidates (with wj taken equal to 1). 2. Experimental apparatus and data sample The central feature of the CMS apparatus is a superconduct- ing solenoid of 6 m internal diameter, providing a magnetic ﬁeld of 3.8 T. Within the solenoid volume, there are four primary subdetectors including a silicon pixel and strip tracker detector, a lead tungstate crystal electromagnetic calorimeter, and a brass and scintillator hadron calorimeter, each composed of a barrel and two endcap sections. Iron and quartz-ﬁber Cherenkov hadron forward (HF) calorimeters cover the pseudorapidity range 2.9 < \|η\| < 5.2. The HF calorimeters are segmented to form 0.175×0.175 (η×φ) towers. Muons are measured in gas-ionization detec- tors embedded in the steel ﬂux-return yoke outside the solenoid. The silicon tracker measures charged particles within the range \|η\| < 2.5. A detailed description of the CMS detector, together with a deﬁnition of the coordinate system used and the relevant kine- matic variables, can be found in Ref. [26]. The analysis presented in this letter uses approximately 4.27 × 109 minimum bias (MB) PbPb collision events collected by the CMS experiment during the 2018 LHC run. The MB events are triggered by requiring signals in both forward and backward sides of the HF calorimeters [27]. Further selections are applied oﬄine to reject events from background processes (beam-gas interactions and non- hadronic collisions), see Ref. [28] for details. Events are required to have at least one interaction vertex, reconstructed based on two tracks or more, and with a distance of less than 15 cm from the center of the nominal interaction point along the beam axis. The primary interaction vertex is deﬁned as the one with the high- est track multiplicity in the event. The shapes of the clusters in the pixel detector have to be compatible with those expected from particles produced at the primary vertex location. The PbPb colli- sion events are also required to have at least two calorimeter tow- ers in each HF detector with energy deposits of more than 4 GeV per tower. These criteria select (99 ±2)% of inelastic hadronic PbPb collisions. The possibility to have values higher than 100% reﬂects the possible presence of ultra-peripheral (nonhadronic) collisions in the selected event sample. 1. Introduction y After the D0 candidate reconstruction, a selection using a boosted decision tree (BDT) algorithm from the tmva pack- age [24] is employed. For the BDT training, misidentiﬁed D0 candi- dates in data events, where pion and kaon have the same charge, are used to mimic the combinatorial background. The signal candi- dates are taken from MC simulations of prompt D0 mesons and are required to match D0 particles at the generator level. The variables related to D0 mesons used to discriminate the signal from the background are: χ 2 probability for the D0 vertex ﬁt, 3D distance between the secondary and primary vertices and its signiﬁcance, the decay length signiﬁcance projected in the xy-plane, and the angle in two and three dimensions between the momentum of the D0 meson candidate and the line connecting the primary and the secondary vertices (pointing angle). Related to the decay products of the D0 meson candidate, the variables used are: the uncertainty in pT returned by the track ﬁtting procedure, the signiﬁcance of the z and the xy distances of closest approach to the primary ver- tex, and the number of hits in the tracker detector. These variables are chosen by analyzing their BDT ranking (variables more fre- quently used in the decision tree) and correlation matrix among all variables. Different BDT boost algorithms are tested, choosing the adaptive boost algorithm [24] as default. Overtraining checks are done for all analysis bins by comparing the BDT distributions from training and testing D0 meson samples. In addition, a BDT cut optimization is performed in bins of centrality, pT, and rapidity, doing a scan in different BDT scores and ﬁnding the one resulting in maximal D0 mesons signal signiﬁcance for each analysis bin. Compared to a cutoff-based procedure, this BDT selection almost doubles the signal signiﬁcance for D0 mesons in 1 < \|y\| < 2, and increases the signal signiﬁcance by 30% for D0 mesons in \|y\| < 1, for events with collision centrality in the range 0–30%. For the remaining analysis bins a similar performance of BDT and cutoff- based methods is observed. 5. Systematic uncertainties vsig+bkg n (minv) = α(minv)vsig n + [1 −α(minv)]vbkg n (minv). (3) (3) The sources of systematic uncertainties include the D0 identi- ﬁcation requirements (BDT selection); the probability distribution function (PDF) for modeling the background in the invariant mass ﬁt; the impact of acceptance and eﬃciency of the D0 meson yield; the variation of the PDF for modeling the background vn; and the remaining nonprompt D0 contamination. With the exception of the last component, the uncertainties are quoted as absolute values of vn and vn after comparing the default analysis conﬁguration with the variations. To diminish the inﬂuence of statistical ﬂuctua- tions, after observing no special trends in the deviations from the default measurements, the systematic uncertainties are evaluated by averaging the deviations with a constant ﬁt as a function of the analysis bins. The α(minv) parameter, which characterizes the signal fraction as a function of mass, is deﬁned as follows: The α(minv) parameter, which characterizes the signal fraction as a function of mass, is deﬁned as follows: α(minv) = [S(minv) + SW (minv)]/[S(minv) + SW (minv) + B(minv)] = αsignal(minv) + αswap(minv). (4 (4) For extracting the difference vsig n , the following expression is em- ployed: For extracting the difference vsig n , the following expression is em- ployed: vsig+bkg n (minv) = vsig n (αsignal(minv) −αswap(minv)) + const. vsig+bkg n (minv) = vsig n (αsignal(minv) −αswap(minv)) + const. In order to take into account the systematic uncertainty associ- ated with the BDT selection, the BDT cut is varied up and down by the maximal deviation between the BDT optimized selection based on MC simulations and data. The BDT cuts (and variations for sys- tematic uncertainties) are deﬁned in bins of collision centrality, pT, and rapidity, ranging from 0.28 to 0.47 (±0.02–0.03). Regarding the effect of the background mass modeling, either an exponential function together with a second order polynomial, or just a second order polynomial, are considered instead of the default ﬁt function using a third-order polynomial. To ﬁt vn as a function of mass, the default conﬁguration using a linear function is replaced by either a constant or a second order polynomial. Although the D0 meson selection eﬃciency essentially cancels in vn measurements, a sys- tematic uncertainty is assigned by comparing the results with and without applying corrections based on MC simulations in bins of pT and rapidity. 3. Reconstruction and selection of D0 mesons The Q -vectors related to HF and the tracker are measured and corrected for detector irregularities by applying a ﬂattening and a recentering procedure [12,34]. The Q nA and Q nB are deﬁned using the event-plane measurements from the negative (−5 < η < −3, HF−) and the positive (3 < η < 5, HF+) sides of HF, and Q nC is measured using the tracker information in the region of \|η\| < 0.75, Prompt D0 mesons are reconstructed from the decay D0 → π+ + K−and D 0 →π−+ K+ with a branching fraction of (3.94 ± 0.04)%, using selected tracks with pT > 1.0 GeV/c and within the acceptance of \|η\| < 2.4. Candidates are formed by combining pairs 2 The CMS Collaboration Physics Letters B 816 (2021) 136253 allowing to minimize the correlations among the three regions, with a gap of more than two units of rapidity. The Q D0 n vector is deﬁned for each D0 meson candidate. The averages ⟨Q nA Q ∗ nB⟩, ⟨Q nA Q ∗ nC⟩, and ⟨Q nB Q ∗ nC⟩are made considering all selected events, while the average ⟨Q D0 n Q ∗ nA⟩is made considering all D0 meson candidates in all selected events. To avoid autocorrelations, the terms ⟨Q D0 n Q ∗ nA⟩and ⟨Q nA Q ∗ nB⟩use A = HF−(HF+) when the D0 meson candidate is at positive (negative) pseudorapidity. The nonprompt D0 meson contamination is taken into account as a systematic uncertainty, by checking that the nonprompt D0 meson fraction is always smaller than 12% (i.e., comparable to the un- certainties in the reconstructed D0 meson yield). This implies that the central values of vn will not be considerably affected by this component, being compatible within statistical uncertainties. Such a low fraction arises from the use of prompt D0 meson signals in the BDT training, together with variables that are highly correlated with the distance of closest approach (DCA) to the primary ver- tex, which is deﬁned as the ﬂight distance of the D0 particle times the sine of the pointing angle in three dimensions. Additional DCA selection and dedicated training, involving prompt and nonprompt D0 meson signals, do not bring considerable improvements in per- formance. The prompt and nonprompt D0 meson fractions are ob- tained using the DCA variable. 3. Reconstruction and selection of D0 mesons For prompt D0 mesons, the nonzero DCA corresponds to the detector resolution, and is expected to be concentrated around zero. For nonprompt D0 mesons, larger val- ues of DCA result from the B meson decay. To extract the prompt and nonprompt D0 meson fractions, a ﬁt to the DCA distributions is performed in data considering DCA shapes from MC simulations for prompt and nonprompt D0 meson components. The nonprompt D0 meson vn is estimated by considering two regions in the DCA: one with very low fraction (2.7–8.0%) of nonprompt D0 particles (DCA < 0.012 cm), and one with a high fraction (62.0–88.0%) of nonprompt D0 particles (DCA > 0.012 cm). Using this information together with Eq. (6), it is possible to estimate vnonprompt n by solv- ing a system of two equations from the two DCA regions. In the current analysis this procedure can only be done in wide pT, cen- trality, and rapidity bins, because of the limited amount of data available in the region with DCA > 0.012 cm. One goal of this analysis is to measure the difference (vn) between D0 and D 0meson ﬂow coeﬃcients, vn, as a function of rapidity, to probe effects from EM ﬁelds. The difference vn is measured as: vn{SP} ≡⟨Q D0 n Q ∗ nA⟩−⟨Q D 0 n Q ∗ nA⟩ ⟨Q nA Q ∗ nB⟩⟨Q nA Q ∗ nC⟩ ⟨Q nB Q ∗ nC⟩ . (2) (2) The vn and vn of D0 meson candidates are ﬁrst measured as a function of their minv. The extraction of the D0 mesons signal vn (vn), vsig n (vsig n ), is performed via a simultaneous binned χ 2 ﬁt of the minv distribution and of vn (vn). The minv distri- bution is ﬁt with three components: a third-order polynomial to model the combinatorial background, B(minv); two Gaussians with the same mean but different widths to describe the minv in dif- ferent kinematic regions for the D0 mesons signal, S(minv); and one additional Gaussian distribution for the swap component cor- responding to the incorrect mass assignment for the assumed pion and kaon particles, SW (minv). The width of SW (minv) and the ra- tio between the yields of SW (minv) and S(minv) are ﬁxed by the values extracted from MC simulations. In this case, the following expression can be used for extracting vsig n : vsig n = fpromptvprompt n + (1 −fprompt)vnonprompt n . Table 1 Table 1 Summary of systematic uncertainties in absolute values for v2, v3, and v2. Ranges of the variation of uncertainties for all the bins are presented. The cells ﬁlled with “—” refer to the cases where the uncertainty cancels out. Systematic sources pT bins y bins Centrality bins v2 BDT selection 0.002–0.014 0.0065 0.005 Bkg. mass PDF 0.0002–0.0017 0.0007–0.0015 0.0007–0.0011 Bkg. vn PDF 0.01–0.05 0.004–0.007 0.003–0.005 D0 eﬃciency correction — 0.004–0.007 0.0040–0.0045 Nonprompt D0 meson contamination 0.0002–0.0077 0.004 0.002–0.005 v3 BDT selection 0.002–0.023 0.001–0.009 0.002–0.006 Bkg. mass PDF 0.0001–0.0040 0.0005–0.0008 0.0012–0.0040 Bkg. vn PDF 0.01–0.05 0.003–0.004 0.0011 D0 eﬃciency correction — 0.002–0.004 0.003–0.005 Nonprompt D0 meson contamination 0.0001–0.0090 0.0010–0.0015 0.0001–0.0008 v2 BDT selection 0.001–0.009 Bkg. mass PDF 0.00015–0.00030 D0 eﬃciency correction 0.001–0.004 Nonprompt D0 meson contamination 0.00002–0.00010 Table 1 Summary of systematic uncertainties in absolute values for v2, v3, and v2. Ranges of the variation of uncertainties for all the bins are presented. The cells ﬁlled with “—” refer to the cases where the uncertainty cancels out. lute values for v2, v3, and v2. Ranges of the variation of uncertainties for all the bins are presented. The cells ﬁlled with “—” cels out certainties in absolute values for v2, v3, and v2. Ranges of the variation of uncertainties for all the bins are presented. The cells ﬁ e uncertainty cancels out matic uncertainties in absolute values for v2, v3, and v2. Ranges of the variation of uncertainties for all the bins are presented. Th where the uncertainty cancels out. BDT selection Bkg. mass PDF D0 eﬃciency correction Nonprompt D0 meson contamination Calculations from theoretical models at midrapidity (\|y\| < 1) are also presented. These models use different assumptions of the QGP properties, for example in the thermal evolution of the colli- sion system and in the initial-state conditions before the formation of the QGP. In addition, different mechanisms are considered re- garding the interaction of heavy quarks with the medium and for the hadronization process. Results from the models LBT [36], CUJET 3.0 [37], and SUBATECH [38] include collisional and ra- diative energy losses, while those from the models TAMU [39], PHSD [15], and TAMU SMCs [40] include only collisional energy loss. Initial-state ﬂuctuations are included in the calculations by LBT, SUBATECH, and PHSD, and calculations for the v3 coeﬃcient are only available from these three models. Table 1 Coalescence mecha- nisms are also included in LBT, SUBATECH, TAMU, PHSD, and TAMU SMCs. While most models seem to capture the qualitative trend of the data (except for the v2 description provided by TAMU in the 10–50% centrality range), most of the models do not provide a quantitative description over the full range, except for TAMU SMCs. The TAMU SMCs version improves the TAMU model by implement- ing event-by-event space-momentum correlations (SMCs) between charm quarks and the high-ﬂow partons in the QGP medium [40]. Since it does not include initial-state ﬂuctuations, TAMU SMCs does not provide v2 calculations for centrality values between 0–10%. This puts more stringent constraints on the development of the collective ﬂow for charm quarks in the QGP medium, giving meson vn in wide bins of pT, rapidity, and centrality. A relative systematic uncertainty is obtained by comparing vn from mixed prompt and nonprompt D0 mesons to the vn derived from non- prompt D0 mesons. Table 1 summarizes the estimates of systematic uncertainties in absolute values for v2, v3, and v2. The ranges of variation of the uncertainties are presented for each binning. 5. Systematic uncertainties The D0 meson selection eﬃciency times accep- tance varies from 0.5 to 12.5% in the pT range of 1.0–8.0 GeV/c, reaching a plateau of approximately 17.0% for pT > 15.0 GeV/c. (5) The term vbkg n (minv) from Eq. (3) is modeled with a linear function, while the constant parameter const in Eq. (5) is added to account for possible ﬂuctuations in the background vn component. The rel- evance of this const parameter was investigated by redoing vn measurements in MC simulation (without azimuthal correlations or effects from EM ﬁelds), indicating that this parameter improves the ﬁt quality and does not introduce artiﬁcial signals. A cross- check is performed by redoing the measurements using a linear function instead of a constant. No signiﬁcant changes in the cen- tral values of v2 and on their uncertainties are observed. Fig. 1 shows an example of a simultaneous ﬁt for v2 and v2. After performing the ﬁts for extracting the signal vn, there is still a sizable fraction of nonprompt D0 mesons embedded in vsig n . The extracted vn can be written as The systematic uncertainties regarding contamination from nonprompt D0 mesons are estimated by measuring nonprompt D0 (6) 3 Physics Letters B 816 (2021) 136253 The CMS Collaboration Fig. 1. Simultaneous ﬁt of the πK invariant mass (left) and v2 (v2) as function of invariant mass (right) for 3.0 < pT < 3.5 GeV/c, centrality 20–70%, and −0.6 < y < 0.0. Fig. 1. Simultaneous ﬁt of the πK invariant mass (left) and v2 (v2) as function of invariant mass (right) for 3.0 < pT < 3.5 GeV/c, Fig. 1. Simultaneous ﬁt of the πK invariant mass (left) and v2 (v2) as function of invariant mass (right) for 3.0 < pT < 3.5 GeV/c, centrality 20–70%, and −0.6 < y < 0.0. 6. Results Results for prompt D0 meson v2 and v3 anisotropic ﬂow co- eﬃcients, obtained with 2018 PbPb data, as functions of pT and for \|y\| < 1, are shown in Fig. 2 for three centrality ranges: 0–10%, 10–30%, and 30–50%. The results extend previously published data from CMS [25], by extending the high-pT coverage to ∼60.0 GeV/c and by providing ﬁner pT bins. These high-precision data are com- patible with previous measurements from Ref. [25], and a clear trend of rise and fall from low to high pT is observed for both v2 and v3 across the full centrality range. This behavior is simi- lar to that observed for inclusive charged particles [35] for \|η\| < 1.0, also shown in Fig. 2. For noncentral collisions (i.e., central- ity 10–50%), values of prompt D0 meson v2 are positive up to pT ∼30.0–40.0 GeV/c, whereas the v3 values become consistent with zero at pT ∼10.0 GeV/c. 4 Physics Letters B 816 (2021) 136253 The CMS Collaboration Fig. 2. Prompt D0 meson and charged particle ﬂow coeﬃcients v2 (upper) and v3 (lower) at midrapidity (\|y\| < 1.0 for prompt D0 mesons and \|η\| < 1.0 for charged particles) for the centrality classes 0–10% (left), 10–30% (middle), and 30–50% (right). The vertical bars and open boxes represent the statistical and systematic uncertainties, respectively. The horizontal bars represent the width of each pT bin. Theoretical calculations for vn coeﬃcients of prompt D0 mesons are also plotted for comparison: LBT [36], CUJET 3.0 [37], SUBATECH [38], TAMU [39], PHSD [15]. The TAMU SMCs model [40] is available only in the 10–50% centrality bins. Fig. 2. Prompt D0 meson and charged particle ﬂow coeﬃcients v2 (upper) and v3 (lower) at midrapidity (\|y\| < 1.0 for prompt D0 mesons and \|η\| < 1.0 for charged particles) for the centrality classes 0–10% (left), 10–30% (middle), and 30–50% (right). The vertical bars and open boxes represent the statistical and systematic uncertainties, respectively. The horizontal bars represent the width of each pT bin. Theoretical calculations for vn coeﬃcients of prompt D0 mesons are also plotted for comparison: LBT [36], CUJET 3.0 [37], SUBATECH [38], TAMU [39], PHSD [15]. The TAMU SMCs model [40] is available only in the 10–50% centrality bins. Fig. 3. 6. Results 4), and can be understood in terms of colli- sion geometry and viscosity effects. In particular, a faster increase of v2 is observed from central to peripheral collisions for charged particles compared to prompt D0 mesons. This feature was also ob- served when comparing v2 of low-pT J/ψ with charged pions [42], where it is claimed that this could be understood in terms of two phenomena: one, associated with transport models predicting an increasing fraction of regenerated J/ψ at low-pT, when going from peripheral to central collisions; the other, not related to regener- ation, is associated with a possible partial or later thermalization of charm quarks compared to light quarks [42]. The v3 shows no centrality dependence, which is also consistent with expectations from collision geometry ﬂuctuations [43]. Fig. 4 (right) presents results for the rapidity dependence of prompt D0 meson v2 and v3, for centrality 20–70%, averaged over 2.0 < pT < 8.0 GeV/c. A weak rapidity dependence of v2 and v3 is observed in the data. Fig. 5. Prompt D0 meson v2 as a function of rapidity, for 2.0 < pT < 8.0 GeV/c and centrality 20–70%. The vertical bars represent statistical uncertainties and open boxes represent systematic uncertainties. The horizontal bars represent the width of each bin. Finally, to search for effects of strong EM ﬁelds, the difference v2 between the v2 values of D0 and D 0mesons is measured. These results are presented in Fig. 5, as a function of rapidity, av- eraged over 2.0 < pT < 8.0 GeV/c and for centrality 20–70%. For all rapidity bins, the v2 values are compatible with zero. The aver- age over the full rapidity region is ⟨v2⟩= 0.001 ± 0.001 (stat) ± 0.003 (syst). In Ref. [19], the predicted v2 splitting for inclusive charged particles due to electric ﬁelds is ∼0.001 at the LHC ener- gies. While quantitative predictions for v2 splitting of D0 mesons are not yet available, they are expected to be much larger than those for inclusive charged particles. In the case of v1, the ALICE collaboration reported results about three orders of magnitude larger than measurements for charged hadrons [44], although the uncertainties prevent a clear conclusion. 6. Results The main reason is that heavy-ﬂavor quarks are usually produced much earlier than light- ﬂavor quarks, the former being predominantly produced soon after the collision takes place, when the EM ﬁeld strength is several or- ders of magnitude stronger [20]. The results presented here pose constraints on possible EM effects on charm quarks. 6. Results Prompt D0 meson ﬂow coeﬃcients v2 (upper) and v3 (lower) at midrapidity (\|y\| < 1, red open circles) and forward rapidity (1 < \|y\| < 2, blue open diamonds) for the centrality classes 0–10% (left), 10–30% (middle), and 30–50% (right). The vertical bars and open boxes represent the statistical and systematic uncertainties, respectively. The horizontal bars represent the width of each pT bin. Fig. 3. Prompt D0 meson ﬂow coeﬃcients v2 (upper) and v3 (lower) at midrapidity (\|y\| < 1, red open circles) and forward rapidity (1 < \|y\| < 2, blue open diamonds) for the centrality classes 0–10% (left), 10–30% (middle), and 30–50% (right). The vertical bars and open boxes represent the statistical and systematic uncertainties, respectively. The horizontal bars represent the width of each pT bin. further inputs for understanding heavy-quark interactions with the medium (for example, energy loss and coalescence mechanisms). observation is similar to that for inclusive charged-hadron mea- surements [41]. In Fig. 4 (left), results for prompt D0 mesons v2 and v3, av- eraged over 2.0 < pT < 8.0 GeV/c, for \|y\| < 1 and 1 < \|y\| < 2, are presented as a function of collision centrality. This pT range is cho- sen in order to cover the widest possible pT range, while maximiz- ing the D0 meson signal yield signiﬁcance. These pT- and rapidity- Results for the rapidity dependence of heavy-ﬂavor collective ﬂow are presented for the ﬁrst time for prompt D0 meson v2 and v3 as functions of pT, both at midrapidity (\|y\| < 1) and in the forward (1 < \|y\| < 2) region, as shown in Fig. 3. No clear rapidity dependence is observed for both v2 and v3 as functions of pT. This 5 Physics Letters B 816 (2021) 136253 The CMS Collaboration The CMS Collaboration Physics Letters B 816 (2021) 136253 Fig. 4. Prompt D0 meson v2 and v3 as functions of centrality, for 2.0 < pT < 8.0 GeV/c and for rapidity ranges \|y\| < 1 and 1 < \|y\| < 2. The results are compared with charged particle v2 and v3 in the same pT range and with \|η\| < 1 (left). Prompt D0 v2 and v3 as functions of rapidity, for 2.0 < pT < 8.0 GeV/c and for centrality 20–70% (right). The vertical bars represent statistical uncertainties and open boxes represent systematic uncertainties. The horizontal bars represent the width of each bin. Fig. 6. Results 4. Prompt D0 meson v2 and v3 as functions of centrality, for 2.0 < pT < 8.0 GeV/c and for rapidity ranges \|y\| < 1 and 1 < \|y\| < 2. The results are compared with charged particle v2 and v3 in the same pT range and with \|η\| < 1 (left). Prompt D0 v2 and v3 as functions of rapidity, for 2.0 < pT < 8.0 GeV/c and for centrality 20–70% (right). The vertical bars represent statistical uncertainties and open boxes represent systematic uncertainties. The horizontal bars represent the width of each bin. Fig. 5. Prompt D0 meson v2 as a function of rapidity, for 2.0 < pT < 8.0 GeV/c and centrality 20–70%. The vertical bars represent statistical uncertainties and open boxes represent systematic uncertainties. The horizontal bars represent the width of each bin. integrated results include an additional centrality bin (50–70%), which has an insuﬃcient number of events for the full differential analysis. For both mid- and forward-rapidity regions, the v2 results show a clear increase from the most central to mid-central events, and then a declining trend toward the most peripheral events. This trend is similar to that observed for inclusive charged particles (also shown in Fig. 4), and can be understood in terms of colli- sion geometry and viscosity effects. In particular, a faster increase of v2 is observed from central to peripheral collisions for charged particles compared to prompt D0 mesons. This feature was also ob- served when comparing v2 of low-pT J/ψ with charged pions [42], where it is claimed that this could be understood in terms of two phenomena: one, associated with transport models predicting an increasing fraction of regenerated J/ψ at low-pT, when going from peripheral to central collisions; the other, not related to regener- ation, is associated with a possible partial or later thermalization of charm quarks compared to light quarks [42]. The v3 shows no centrality dependence, which is also consistent with expectations from collision geometry ﬂuctuations [43]. integrated results include an additional centrality bin (50–70%), which has an insuﬃcient number of events for the full differential analysis. For both mid- and forward-rapidity regions, the v2 results show a clear increase from the most central to mid-central events, and then a declining trend toward the most peripheral events. This trend is similar to that observed for inclusive charged particles (also shown in Fig. References [1] I. Arsene, et al., BRAHMS, Quark-gluon plasma and color glass condensate at RHIC? The perspective from the BRAHMS experiment, Nucl. Phys. A 757 (2005) 1, https://doi .org /10 .1016 /j .nuclphysa .2005 .02 .130, arXiv:nucl -ex /0410020. [2] B.B. Back, et al., PHOBOS, The PHOBOS perspective on discoveries at RHIC, Nucl. Phys. A 757 (2005) 28, https://doi .org /10 .1016 /j .nuclphysa .2005 .03 .084, arXiv: nucl -ex /0410022. [3] J. Adams, et al., STAR, Experimental and theoretical challenges in the search for the quark gluon plasma: the STAR Collaboration’s critical assessment of the evidence from RHIC collisions, Nucl. Phys. A 757 (2005) 102, https://doi .org /10 . 1016 /j .nuclphysa .2005 .03 .085, arXiv:nucl -ex /0501009. 1016 /j .nuclphysa .2005 .03 .085, arXiv:nucl -ex /0501009. [4] K. Adcox, et al., PHENIX, Formation of dense partonic matter in relativistic nucleus-nucleus collisions at RHIC: experimental evaluation by the PHENIX col- laboration, Nucl. Phys. A 757 (2005) 184, https://doi .org /10 .1016 /j .nuclphysa . 2005 .03 .086, arXiv:nucl -ex /0410003. [5] B. Muller, J. Schukraft, B. Wyslouch, First results from PbPb collisions at the LHC, Annu. Rev. Nucl. Part. Sci. 62 (2012) 361, https://doi .org /10 .1146 /annurev- nucl -102711 -094910, arXiv:1202 .3233. [6] N. Armesto, E. Scomparin, Heavy-ion collisions at the Large Hadron Collider: a review of the results from Run 1, Eur. Phys. J. Plus 131 (2016) 52, https:// doi .org /10 .1140 /epjp /i2016 -16052 -4, arXiv:1511.02151. doi .org /10 .1140 /epjp /i2016 -16052 -4, arXiv:1511.02151. [7] ALICE Collaboration, Elliptic ﬂow of charged particles in PbPb collisions at 2.76 TeV, Phys. Rev. Lett. 105 (2010) 252302, https://doi .org /10 .1103 / PhysRevLett .105 .252302, arXiv:1011.3914. [8] ATLAS Collaboration, Measurement of the pseudorapidity and transverse mo- mentum dependence of the elliptic ﬂow of charged particles in lead-lead col- lisions at √ sNN = 2.76 TeV with the ATLAS detector, Phys. Lett. B 707 (2012) 330, https://doi .org /10 .1016 /j .physletb .2011.12 .056, arXiv:1108 .6018. [9] CMS Collaboration, Measurement of the elliptic anisotropy of charged particles produced in PbPb collisions at √ sNN = 2.76 TeV, Phys. Rev. C 87 (2013) 014902, https://doi .org /10 .1103 /PhysRevC .87.014902, arXiv:1204 .1409. Individuals have received support from the Marie-Curie pro- gram and the European Research Council and Horizon 2020 Grant, contract Nos. 675440, 752730, and 765710 (European Union); the Leventis Foundation; the A.P. 7. Summary Measurements of the elliptic (v2) and triangular (v3) ﬂow coef- ﬁcients of prompt D0 mesons are presented as functions of trans- verse momentum (pT), rapidity, and collision centrality, in PbPb collisions at √ sNN = 5.02 TeV. The results improve previously pub- lished CMS data by extending the pT and rapidity coverage and by providing more differential information in pT, rapidity, and cen- trality. A clear centrality dependence of prompt D0 meson v2 is observed, while v3 is largely centrality independent. These trends are consistent with the expectation that v2 and v3 are driven by initial-state geometry. A weak rapidity dependence of prompt D0 6 The CMS Collaboration Physics Letters B 816 (2021) 136253 FIA research grants 123842, 123959, 124845, 124850, 125105, 128713, 128786, and 129058 (Hungary); the Council of Science and Industrial Research, India; the HOMING PLUS program of the Foundation for Polish Science, coﬁnanced from European Union, Regional Development Fund, the Mobility Plus program of the Ministry of Science and Higher Education, the National Science Center (Poland), contracts Harmonia 2014/14/M/ST2/00428, Opus 2014/13/B/ST2/02543, 2014/15/B/ST2/03998, and 2015/19/B/ST2/ 02861, Sonata-bis 2012/07/E/ST2/01406; the National Priorities Re- search Program by Qatar National Research Fund; the Ministry of Science and Higher Education, project no. 02.a03.21.0005 (Russia); the Tomsk Polytechnic University Competitiveness Enhancement Program; the Programa Estatal de Fomento de la Investigación Cientíﬁca y Técnica de Excelencia María de Maeztu, grant MDM- 2015-0509 and the Programa Severo Ochoa del Principado de Asturias; the Thalis and Aristeia programs coﬁnanced by EU-ESF and the Greek NSRF; the Rachadapisek Sompot Fund for Postdoc- toral Fellowship, Chulalongkorn University and the Chulalongkorn Academic into Its 2nd Century Project Advancement Project (Thai- land); the Kavli Foundation; the Nvidia Corporation; the SuperMi- cro Corporation; the Welch Foundation, contract C-1845; and the Weston Havens Foundation (USA). meson v2 and v3 is observed. When comparing various theoret- ical calculations to the data at midrapidity, no model is able to describe the data over the full centrality and pT ranges. Motivated by the search for evidence of the strong electric ﬁeld expected in PbPb collisions, a ﬁrst measurement of the v2 ﬂow coeﬃcient difference (v2) between D0 and D 0mesons as a func- tion of rapidity is presented. The rapidity-averaged v2 difference is measured to be ⟨v2⟩ = 0.001 ±0.001 (stat)±0.003 (syst). Declaration of competing interest The authors declare that they have no known competing ﬁnan- cial interests or personal relationships that could have appeared to inﬂuence the work reported in this paper. 7. Summary This in- dicates that there is no evidence that charm hadron collective ﬂow is affected by the strong Coulomb ﬁeld created in ultrarelativistic heavy ion collisions. Future comparisons of theoretical models with these results may provide constraints on the electric conductivity of the quark-gluon plasma. Acknowledgements We congratulate our colleagues in the CERN accelerator depart- ments for the excellent performance of the LHC and thank the technical and administrative staffs at CERN and at other CMS in- stitutes for their contributions to the success of the CMS effort. In addition, we gratefully acknowledge the computing centers and personnel of the Worldwide LHC Computing Grid for delivering so effectively the computing infrastructure essential to our analyses. Finally, we acknowledge the enduring support for the construc- tion and operation of the LHC and the CMS detector provided by the following funding agencies: BMBWF and FWF (Austria); FNRS and FWO (Belgium); CNPq, CAPES, FAPERJ, FAPERGS, and FAPESP (Brazil); MES (Bulgaria); CERN; CAS, MOST, and NSFC (China); COLCIENCIAS (Colombia); MSES and CSF (Croatia); RIF (Cyprus); SENESCYT (Ecuador); MoER, ERC IUT, PUT and ERDF (Estonia); Academy of Finland, MEC, and HIP (Finland); CEA and CNRS/IN2P3 (France); BMBF, DFG, and HGF (Germany); GSRT (Greece); NK- FIA (Hungary); DAE and DST (India); IPM (Iran); SFI (Ireland); INFN (Italy); MSIP and NRF (Republic of Korea); MES (Latvia); LAS (Lithuania); MOE and UM (Malaysia); BUAP, CINVESTAV, CONACYT, LNS, SEP, and UASLP-FAI (Mexico); MOS (Montenegro); MBIE (New Zealand); PAEC (Pakistan); MSHE and NSC (Poland); FCT (Portu- gal); JINR (Dubna); MON, ROSATOM, RAS, RFBR, and NRC KI (Rus- sia); MESTD (Serbia); SEIDI, CPAN, PCTI, and FEDER (Spain); MoSTR (Sri Lanka); Swiss Funding Agencies (Switzerland); MST (Taipei); ThEPCenter, IPST, STAR, and NSTDA (Thailand); TÜBITAK and TAEK (Turkey); NASU (Ukraine); STFC (United Kingdom); DOE and NSF (USA). 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Zacharopoulou National Technical University of Athens, Athens, Greece I. Evangelou, C. Foudas, P. Gianneios, P. Katsoulis, P. Kokkas, S. Mallios, K. Manitara, N. Manthos, I. Papadopoulos, J. Strologas, F.A. Triantis, D. Tsitsonis University of Ioánnina, Ioánnina, Greece M. Bartók 23, R. Chudasama, M. Csanad, M.M.A. Gadallah 24, P. Major, K. Mandal, A. Mehta, G. Pasztor, O. Surányi, G.I. Veres MTA-ELTE Lendület CMS Particle and Nuclear Physics Group, Eötvös Loránd University, Budapest, Hungary G. Bencze, C. Hajdu, D. Horvath 25, F. Sikler, V. Veszpremi, G. Vesztergombi † Wigner Research Centre for Physics, Budapest, Hungary N. Beni, S. Czellar, J. Karancsi 23, J. Molnar, Z. Szillasi, D. Teyssier Institute of Nuclear Research ATOMKI, Debrecen, Hungary P. Raics, Z.L. Trocsanyi, B. Ujvari Institute of Physics, University of Debrecen, Debrecen, Hungary T. Csorgo, S. Lökös 26, F. Nemes, T. Novak Eszterhazy Karoly University, Karoly Robert Campus, Gyongyos, Hungary S. Choudhury, J.R. Komaragiri, D. Kumar, L. Panwar, P.C. Tiwari Indian Institute of Science (IISc), Bangalore, India S. Bahinipati 27, C. Kar, P. Mal, T. Mishra, V.K. Muraleedharan Nair Bindhu, A. Nayak 28, D.K. Sahoo 27, N. Sur, S.K. H. Bakhshiansohi 34 Department of Physics, Isfahan University of Technology, Isfahan, Iran S. Chenarani 35, S.M. Etesami, M. Khakzad, M. Mohammadi Najafabadi, M. Naseri Institute for Research in Fundamental Sciences (IPM), Tehran, Iran M. Abbrescia a,b, R. Aly a,b,36, C. Calabria a,b, A. Colaleo a, D. Creanza a,c, N. De Filippis a,c, M. De Palma a,b, A. Di Florio a,b, A. Di Pilato a,b, W. Elmetenawee a,b, L. Fiore a, A. Gelmi a,b, G. Iaselli a,c, M. Ince a,b, S. Lezki a,b, G. Maggi a,c, M. Maggi a, I. Margjeka a,b, J.A. Merlin a, G. Miniello a,b, S. My a,b, S. Nuzzo a,b, A. Pompili a,b, G. Pugliese a,c, A. Ranieri a, G. Selvaggi a,b, L. Silvestris a, F.M. Simone a,b, R. Venditti a, P. Verwilligen a a INFN Sezione di Bari, Bari, Italy b Università di Bari, Bari, Italy c Politecnico di Bari, Bari, Italy P. Verwilligen a a INFN Sezione di Bari, Bari, Italy b Università di Bari, Bari, Italy c Politecnico di Bari, Bari, Italy G. Abbiendi a, C. Battilana a,b, D. Bonacorsi a,b, L. Borgonovi a,b, R. Campanini a,b, P. Capiluppi a,b, A. Castro a,b, F.R. Cavallo a, C. Ciocca a, M. Cuﬃani a,b, G.M. Dallavalle a, T. Diotalevi a,b, F. Fabbri a, A. Fanfani a,b, E. Fontanesi a,b, P. Giacomelli a, L. Giommi a,b, C. Grandi a, L. Guiducci a,b, F. Iemmi a,b S. Lo Meo a,37, S. Marcellini a, G. Masetti a, F.L. Navarria a,b, A. Perrotta a, F. Primavera a,b, T. Rovelli a G.P. Siroli a,b, N. Tosi a a INFN Sezione di Bologna, Bologna, Italy b Università di Bologna, Bologna, Italy S. Albergo a,b,38, S. Costa a,b, A. Di Mattia a, R. Potenza a,b, A. Tricomi a,b,38, C. Tuve a,b a INFN Sezione di Catania, Catania, Italy b Università di Catania, Catania, Italy G. Barbagli a, A. Cassese a, R. 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Sharma University of Delhi, Delhi, India 12 Physics Letters B 816 (2021) 136253 The CMS Collaboration M. Bharti 30, R. Bhattacharya, S. Bhattacharya, D. Bhowmik, S. Dutta, S. Ghosh, B. Gomber 31, M. Maity 32, K. Mondal, S. Nandan, P. Palit, A. Purohit, P.K. Rout, G. Saha, S. Sarkar, M. Sharan, B. Singh 30, S. Thakur 30 Saha Institute of Nuclear Physics, HBNI, Kolkata, India P.K. Behera, S.C. Behera, P. Kalbhor, A. Muhammad, R. Pradhan, P.R. Pujahari, A. Sharma, A.K. Sikdar Indian Institute of Technology Madras, Madras, India D. Dutta, V. Jha, D.K. Mishra, K. Naskar 33, P.K. Netrakanti, L.M. Pant, P. Shukla Bhabha Atomic Research Centre, Mumbai, India T. Aziz, M.A. Bhat, S. Dugad, R. Kumar Verma, U. Sarkar Tata Institute of Fundamental Research-A, Mumbai, India S. Banerjee, S. Bhattacharya, S. Chatterjee, P. Das, M. Guchait, S. Karmakar, S. Kumar, G. Majumder, K. Mazumdar, S. Mukherjee, D. Roy, N. Sahoo Tata Institute of Fundamental Research-B, Mumbai, India S. Dube, B. Kansal, A. Kapoor, K. Kothekar, S. Pandey, A. Rane, A. Rastogi, S. Sharma Indian Institute of Science Education and Research (IISER), Pune, India L. Benussi, S. Bianco, D. Piccolo INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, D. Piccolo INFN Laboratori Nazionali di Frascati, Frascati, Italy L. Benussi, S. Bianco, D. Piccolo INFN Laboratori Nazionali di Frascati, Frascati, Italy M. Bozzo a,b, F. Ferro a, R. Mulargia a,b, E. Robutti a, S. Tosi a,b M. Bozzo a,b, F. Ferro a, R. Mulargia a,b, E. Robutti a, S. Tosi a,b M. Bozzo a,b, F. Ferro a, R. Mulargia a,b, E. Robutti a, S. Tosi a,b a INFN Sezione di Genova, Genova, Italy b Università di Genova, Genova, Italy a INFN Sezione di Genova, Genova, Italy b Università di Genova, Genova, Italy A. Benaglia a, A. Beschi a,b, F. Brivio a,b, F. Cetorelli a,b, V. Ciriolo a,b,17, F. De Guio a,b, M.E. Dinardo a,b, P. Dini a, S. Gennai a, A. Ghezzi a,b, P. Govoni a,b, L. Guzzi a,b, M. Malberti a, S. Malvezzi a, D. Menasce a, F. Monti a,b, L. Moroni a, M. Paganoni a,b, D. Pedrini a, S. Ragazzi a,b, T. Tabarelli de Fatis a,b, D. Valsecchi a,b,17, D. Zuolo a,b a INFN Sezione di Milano-Bicocca, Milano, Italy b Università di Milano-Bicocca, Milano, Italy S. Buontempo a, N. Cavallo a,c, A. De Iorio a,b, F. Fabozzi a,c, F. Fienga a, G. Galati a, A.O.M. Iorio a,b, L. Layer a,b, L. Lista a,b, S. Meola a,d,17, P. Paolucci a,17, B. Rossi a, C. Sciacca a,b, E. Voevodina a,b a INFN Sezione di Napoli, Napoli, Italy b Università di Napoli ‘Federico II’, Napoli, Italy c Università della Basilicata, Potenza, Italy d Università G. Marconi, Roma, Italy c Università della Basilicata, Potenza, Italy d Università G. Marconi, Roma, Italy P. Azzi a, N. Bacchetta a, D. Bisello a,b, A. Boletti a,b, A. Bragagnolo a,b, R. Carlin a,b, P. Checchia a, P. De Castro Manzano a, T. Dorigo a, U. Dosselli a, F. Gasparini a,b, U. Gasparini a,b, S.Y. Hoh a,b, M. Margoni a,b, A.T. Meneguzzo a,b, M. Presilla b, P. Ronchese a,b, R. Rossin a,b, F. Simonetto a,b, G. Strong, A. Tiko a, M. Tosi a,b, M. Zanetti a,b, P. Zotto a,b, A. Zucchetta a,b, G. Zumerle a,b a INFN Sezione di Padova, Padova, Italy b Università di Padova, Padova, Italy c Università di Trento, Trento, Italy A. Braghieri a, S. Calzaferri a,b, D. Fiorina a,b, P. Montagna a,b, S.P. Ratti a,b, V. Re a, M. Ressegotti a,b, C. Riccardi a,b, P. Salvini a, I. Vai a, P. H. Bakhshiansohi 34 Giacomelli a, L. Giommi a,b, C. Grandi a, L. Guiducci a,b, F. Iemmi a,b, S. Lo Meo a,37, S. Marcellini a, G. Masetti a, F.L. Navarria a,b, A. Perrotta a, F. Primavera a,b, T. Rovelli a,b, G.P. Siroli a,b, N. Tosi a a INFN Sezione di Bologna, Bologna, Italy b U i ità di B l B l It l S. Albergo a,b,38, S. Costa a,b, A. Di Mattia a, R. Potenza a,b, A. Tricomi a,b,38, C. Tuve a,b a INFN Sezione di Catania, Catania, Italy b Università di Catania, Catania, Italy G. Barbagli a, A. Cassese a, R. Ceccarelli a,b, V. Ciulli a,b, C. Civinini a, R. D’Alessandro a,b, F. Fiori a, E. Focardi a,b, G. Latino a,b, P. Lenzi a,b, M. Lizzo a,b, M. Meschini a, S. Paoletti a, R. Seidita a,b, G. Sguazzoni a, L. Viliani a a INFN Sezione di Firenze, Firenze, Italy 13 Physics Letters B 816 (2021) 136253 The CMS Collaboration b Università di Firenze, Firenze, Italy b Università di Firenze, Firenze, Italy b Università di Firenze, Firenze, Italy L. Benussi, S. Bianco, D. Piccolo INFN Laboratori Nazionali di Frascati, Frascati, Italy Vitulo a,b a INFN Sezione di Pavia, Pavia, Italy b Università di Pavia, Pavia, Italy A. Braghieri a, S. Calzaferri a,b, D. Fiorina a,b, P. Montagna a,b, S.P. Ratti a,b, V. Re a, M. Ressegotti a,b, C. Riccardi a,b, P. Salvini a, I. Vai a, P. Vitulo a,b a INFN S i di P i P i It l M. Biasini a,b, G.M. Bilei a, D. Ciangottini a,b, L. Fanò a,b, P. Lariccia a,b, G. Mantovani a,b, V. Mariani a M. Menichelli a, A. Rossi a,b, A. Santocchia a,b, D. Spiga a, T. Tedeschi a,b a INFN Sezione di Perugia, Perugia, Italy b Università di Perugia, Perugia, Italy K. Androsov a, P. Azzurri a, G. Bagliesi a, V. Bertacchi a,c, L. Bianchini a, T. Boccali a, R. Castaldi a, M.A. Ciocci a,b, R. Dell’Orso a, M.R. Di Domenico a,b, S. Donato a, L. Giannini a,c, A. Giassi a, M.T. Gri F. Ligabue a,c, E. Manca a,c, G. Mandorli a,c, A. Messineo a,b, F. Palla a, A. Rizzi a,b, G. Rolandi a,c, S. Roy Chowdhury a,c, A. Scribano a, N. Shaﬁei a,b, P. Spagnolo a, R. Tenchini a, G. Tonelli a,b, N. Turi A. Venturi a, P.G. Verdini a a INFN Sezione di Pisa, Pisa, Italy b Università di Pisa, Pisa, Italy c Scuola Normale Superiore di Pisa, Pisa, Italy F. Cavallari a, M. Cipriani a,b, D. Del Re a,b, E. Di Marco a, M. Diemoz a, E. Longo a,b, P. Meridiani a, G. Organtini a,b, F. Pandolﬁa, R. Paramatti a,b, C. Quaranta a,b, S. Rahatlou a,b, C. Rovelli a, F. Santanastasio a,b, L. Soﬃa,b, R. Tramontano a,b a INFN Sezione di Roma, Rome, Italy b Sapienza Università di Roma, Rome, Italy M. Biasini a,b, G.M. Bilei a, D. Ciangottini a,b, L. Fanò a,b, P. Lariccia a,b, G. Mantovani a,b, V. Mariani a,b, M. Menichelli a, A. Rossi a,b, A. Santocchia a,b, D. Spiga a, T. Tedeschi a,b a INFN Sezione di Perugia, Perugia, Italy b Università di Perugia, Perugia, Italy K. Androsov a, P. Azzurri a, G. Bagliesi a, V. Bertacchi a,c, L. Bianchini a, T. Boccali a, R. Castaldi a, M.A. Ciocci a,b, R. Dell’Orso a, M.R. Di Domenico a,b, S. Donato a, L. Giannini a,c, A. Giassi a, M.T. Grippo a, F. Ligabue a,c, E. Manca a,c, G. Mandorli a,c, A. Messineo a,b, F. Palla a, A. Rizzi a,b, G. Rolandi a,c, S. Roy Chowdhury a,c, A. L. Benussi, S. Bianco, D. Piccolo INFN Laboratori Nazionali di Frascati, Frascati, Italy Scribano a, N. Shaﬁei a,b, P. Spagnolo a, R. Tenchini a, G. Tonelli a,b, N. Turini a, A. Venturi a, P.G. Verdini a a INFN Sezione di Pisa, Pisa, Italy b Università di Pisa, Pisa, Italy c Scuola Normale Superiore di Pisa, Pisa, Italy F. Cavallari a, M. Cipriani a,b, D. Del Re a,b, E. Di Marco a, M. Diemoz a, E. Longo a,b, P. Meridiani a, G. Organtini a,b, F. Pandolﬁa, R. Paramatti a,b, C. Quaranta a,b, S. Rahatlou a,b, C. Rovelli a, F. Santanastasio a,b, L. Soﬃa,b, R. Tramontano a,b a INFN Sezione di Roma, Rome, Italy b Sapienza Università di Roma, Rome, Italy K. Androsov a, P. Azzurri a, G. Bagliesi a, V. Bertacchi a,c, L. Bianchini a, T. Boccali a, R. Castaldi a, M.A. Ciocci a,b, R. Dell’Orso a, M.R. Di Domenico a,b, S. Donato a, L. Giannini a,c, A. Giassi a, M.T. Grippo a, F. Ligabue a,c, E. Manca a,c, G. Mandorli a,c, A. Messineo a,b, F. Palla a, A. Rizzi a,b, G. Rolandi a,c, S. Roy Chowdhury a,c, A. Scribano a, N. Shaﬁei a,b, P. Spagnolo a, R. Tenchini a, G. Tonelli a,b, N. Turini a, A. Venturi a, P.G. Verdini a a INFN Sezione di Pisa, Pisa, Italy b U i ità di Pi Pi It l F. Cavallari a, M. Cipriani a,b, D. Del Re a,b, E. Di Marco a, M. Diemoz a, E. Longo a,b, P. Meridiani a, G. Organtini a,b, F. Pandolﬁa, R. Paramatti a,b, C. Quaranta a,b, S. Rahatlou a,b, C. Rovelli a, F. Santanastasio a,b, L. Soﬃa,b, R. Tramontano a,b a INFN Sezione di Roma, Rome, Italy 14 Physics Letters B 816 (2021) 136253 The CMS Collaboration N. Amapane a,b, R. Arcidiacono a,c, S. Argiro a,b, M. Arneodo a,c, N. Bartosik a, R. Bellan a,b, A. Bellora a,b, C. Biino a, A. Cappati a,b, N. Cartiglia a, S. Cometti a, M. Costa a,b, R. Covarelli a,b, N. Demaria a, B. Kiani a,b, F. Legger a, C. Mariotti a, S. Maselli a, E. Migliore a,b, V. Monaco a,b, E. Monteil a,b, M. Monteno a, M.M. Obertino a,b, G. Ortona a, L. Pacher a,b, N. Pastrone a, M. Pelliccioni a, G.L. Pinna Angioni a,b, M. Ruspa a,c, R. Salvatico a,b, F. Siviero a,b, V. Sola a, A. Solano a,b, D. Soldi a,b, A. Staiano a, D. J. Goh, A. Gurtu H.S. Kim, Y. Kim Sejong University, Seoul, Republic of Korea J. Almond, J.H. Bhyun, J. Choi, S. Jeon, J. Kim, J.S. Kim, S. Ko, H. Kwon, H B.H. Oh, M. Oh, S.B. Oh, B.C. Radburn-Smith, H. Seo, U.K. Yang, I. Yoon Seoul National University, Seoul, Republic of Korea H. Kim, D.H. Moon B. Francois, T.J. Kim, J. Park Hanyang University, Seoul, Republic of Korea S. Cho, S. Choi, Y. Go, S. Ha, B. Hong, K. Lee, K.S. Lee, J. Lim, J. Park, S.K. Park, Y. Roh, J. Yoo Korea University, Seoul, Republic of Korea L. Benussi, S. Bianco, D. Piccolo INFN Laboratori Nazionali di Frascati, Frascati, Italy Trocino a,b a INFN Sezione di Torino, Torino, Italy b Università di Torino, Torino, Italy c Università del Piemonte Orientale, Novara, Italy y b Università di Torino, Torino, Italy c Università del Piemonte Orientale, Novara, Italy S. Belforte a, V. Candelise a,b, M. Casarsa a, F. Cossutti a, A. Da Rold a,b, G. Della Ricca a,b, F. Vazzoler a,b a INFN Sezione di Trieste, Trieste, Italy b Università di Trieste, Trieste, Italy a INFN Sezione di Trieste, Trieste, Italy b Università di Trieste, Trieste, Italy S. Dogra, C. Huh, B. Kim, D.H. Kim, G.N. Kim, J. Lee, S.W. Lee, C.S. Moon, Y.D. Oh, S.I. Pak, S. Sekmen, D.C. Son, Y.C. Yang Kyungpook National University, Daegu, Republic of Korea Kyungpook National University, Daegu, Republic of Korea Y. Maghrbi 15 The CMS Collaboration Physics Letters B 816 (202 W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia J.F. Benitez, A. Castaneda Hernandez, J.A. Murillo Quijada, L. Valencia Palomo Universidad de Sonora (UNISON), Hermosillo, Mexico H. Castilla-Valdez, E. De La Cruz-Burelo, I. Heredia-De La Cruz 40, R. Lopez-Fernandez, A. Sanchez-Hernandez Centro de Investigacion y de Estudios Avanzados del IPN, Mexico City, Mexico S. Carrillo Moreno, C. Oropeza Barrera, M. Ramirez-Garcia, F. Vazquez Valencia Universidad Iberoamericana, Mexico City, Mexico J. Eysermans, I. Pedraza, H.A. Salazar Ibarguen, C. Uribe Estrada Benemerita Universidad Autonoma de Puebla, Puebla, Mexico A. Morelos Pineda Universidad Autónoma de San Luis Potosí, San Luis Potosí, Mexico J. Mijuskovic 3, N. Raicevic University of Montenegro, Podgorica, Montenegro D. Krofcheck University of Auckland, Auckland, New Zealand S. Bheesette, P.H. Butler University of Canterbury, Christchurch, New Zealand A. Ahmad, M. Ahmad, M.I. Asghar, M.I.M. Awan, Q. Hassan, H.R. Hoorani, W.A. Khan, M.A. Shah, M. Shoaib, M. Waqas National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan V. Avati, L. Grzanka, M. Malawski AGH University of Science and Technology Faculty of Computer Science, Electronics and Telecommunications, Krakow, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. Górski, M. Kazana, M. Szleper, P. Traczyk, P. Zale National Centre for Nuclear Research, Swierk, Poland K. Bunkowski, A. Byszuk 41, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Olszewski, M. Walczak Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland M. Araujo, P. Bargassa, D. Bastos, A. Di Francesco, P. Faccioli, B. Galinhas, M. Gallinaro, J. Hollar, N. Leonardo, T. Niknejad, J. Seixas, K. Shchelina, O. Toldaiev, J. Varela Laboratório de Instrumentação e Física Experimental de Partículas, Lisboa, Portugal A. Baginyan, A. Golunov, I. Golutvin, I. Gorbunov, V. Karjavine, I. Kashunin, A. Lanev, A. Malakhov, V. Matveev 42,43, V.V. Mitsyn, P. Moisenz, V. Palichik, V. Perelygin, S. Shmatov, O. Teryaev, V. Troﬁm N. Voytishin, B.S. Yuldashev 44, A. Zarubin, V. Zhiltsov The CMS Collaboration Physics Letters B 816 (2021) 136253 W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia W.A.T. Wan Abdullah, M.N. Yusli, Z. Zolkapli National Centre for Particle Physics, Universiti Malaya, Kuala Lumpur, Malaysia J.F. Benitez, A. Castaneda Hernandez, J.A. Murillo Quijada, L. Valencia Palomo Universidad de Sonora (UNISON), Hermosillo, Mexico A. Ahmad, M. Ahmad, M.I. Asghar, M.I.M. Awan, Q. Hassan, H.R. Hoorani, W.A. Khan, M.A. Shah, M. Shoaib, M. Waqas National Centre for Physics, Quaid-I-Azam University, Islamabad, Pakistan V. Avati, L. Grzanka, M. Malawski AGH University of Science and Technology Faculty of Computer Science, Electronics and Telecommunications, Krakow, Poland H. Bialkowska, M. Bluj, B. Boimska, T. Frueboes, M. Górski, M. Kazana, M. Szleper, P. Traczyk, P. Zalewski National Centre for Nuclear Research, Swierk, Poland K. Bunkowski, A. Byszuk 41, K. Doroba, A. Kalinowski, M. Konecki, J. Krolikowski, M. Olszewski, M. Walczak Institute of Experimental Physics, Faculty of Physics, University of Warsaw, Warsaw, Poland M. Araujo, P. Bargassa, D. Bastos, A. Di Francesco, P. Faccioli, B. Galinhas, M. Gallinaro, J. Hollar, N. Leonardo, T. Niknejad, J. Seixas, K. Shchelina, O. Toldaiev, J. Varela Laboratório de Instrumentação e Física Experimental de Partículas, Lisboa, Portugal A. Baginyan, A. Golunov, I. Golutvin, I. Gorbunov, V. Karjavine, I. Kashunin, A. Lanev, A. Malakhov, V. Matveev 42,43, V.V. Mitsyn, P. Moisenz, V. Palichik, V. Perelygin, S. Shmatov, O. Teryaev, V. Troﬁmov, N. Voytishin, B.S. Yuldashev 44, A. Zarubin, V. Zhiltsov Joint Institute for Nuclear Research, Dubna, Russia 16 Physics Letters B 816 (2021) 136253 The CMS Collaboration G. Gavrilov, V. Golovtcov, Y. Ivanov, V. Kim 45, E. Kuznetsova 46, V. Murzin, V. Oreshkin, I. Smirnov, D. Sosnov, V. Sulimov, L. Uvarov, S. Volkov, A. Vorobyev Petersburg Nuclear Physics Institute, Gatchina (St. Petersburg), Russia Yu. Andreev, A. Dermenev, S. Gninenko, N. Golubev, A. Karneyeu, M. Kirsanov, N. Krasnikov, A. Pashenkov, G. Pivovarov, D. Tlisov, A. Toropin Institute for Nuclear Research, Moscow, Russia V. Epshteyn, V. Gavrilov, N. Lychkovskaya, A. Nikitenko 47, V. Popov, I. Pozdnyakov, G. Safronov, A. Spiridonov, A. Stepennov, M. Toms, E. Vlasov, A. Zhokin Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of NRC ‘Kurchatov Institute’, Moscow, Russia T. Aushev Moscow Institute of Physics and Technology, Moscow, Russia O. Bychkova, R. Chistov 48, M. Danilov 48, A. Oskin, P. Parygin, S. Polikarpov 48 National Research Nuclear University ‘Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia V. Andreev, M. Azarkin, I. Dremin, M. Kirakosyan, A. Terkulov P.N. Lebedev Physical Institute, Moscow, Russia A. Belyaev, E. Boos, A. Demiyanov, A. Ershov, A. Gribushin, O. Kodolova, V. Korotkikh, I. Lokhtin, S. Obraztsov, S. Petrushanko, V. Savrin, A. Snigirev, I. Vardanyan Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia I. Azhgirey, I. Bayshev, S. Bitioukov, V. Kachanov, A. Kalinin, D. Konstantinov, V. Petrov, R. Ryutin, A. Sobol, S. Troshin, N. Tyurin, A. Uzunian, A. Volkov Institute for High Energy Physics of National Research Centre ‘Kurchatov Institute’, Protvino, Russia A. Babaev, A. Iuzhakov, V. Okhotnikov National Research Tomsk Polytechnic University, Tomsk, Russia W.G.D. Dharmaratna, K. Liyanage, N. Perera, N. Wickramage University of Ruhuna, Department of Physics, Matara, Sri Lanka University of Ruhuna, Department of Physics, Matara, Sri Lanka T.K. Aarrestad, D. Abbaneo, B. Akgun, E. Auffray, G. Auzinger, J. Baechler, P. Baillon, A.H. Ball, D. Barney, J. Bendavid, M. Bianco, A. Bocci, P. Bortignon, E. Bossini, E. Brondolin, T. Camporesi, G. Cerminara, L. Cristella, D. d’Enterria, A. Dabrowski, N. Daci, V. Daponte, A. David, A. De Roeck, M. Deile, R. Di Maria M. Dobson, M. Dünser, N. Dupont, A. Elliott-Peisert, N. Emriskova, F. Fallavollita 53, D. Fasanella, S. Fiorendi, G. Franzoni, J. Fulcher, W. Funk, S. Giani, D. Gigi, K. Gill, F. Glege, L. Gouskos, M. Gruchala, M. Guilbaud, D. Gulhan, J. Hegeman, C. Heidegger, Y. Iiyama, V. Innocente, T. James, P. Janot, J. Kaspar, J. Kieseler, M. Komm, N. Kratochwil, C. Lange, P. Lecoq, K. Long, C. Lourenço, L. Malgeri, M. Mannelli, A. Massironi, F. Meijers, S. Mersi, E. Meschi, F. Moortgat, M. Mulders, J. Ngadiuba, J. Niedziela, S. Orfanelli, L. Orsini, F. Pantaleo 17, L. Pape, E. Perez, M. Peruzzi, A. Petrilli, G. Petrucciani, A. Pfeiffer, M. Pierini, F.M. Pitters, D. Rabady, A. Racz, M. Rovere, H. Sakulin, J. Salfeld-Nebgen, S. Scarﬁ, C. Schäfer, C. Schwick, M. Selvaggi, A. Sharma, P. Silva, W. Snoeys, P. Sphicas 54, J. Steggemann, S. Summers, V.R. Tavolaro, D. Treille, A. Tsirou, G.P. Van Onsem, A. Vartak, M. Verzetti, K.A. Wozniak, W.D. Zeuner CERN, European Organization for Nuclear Research, Geneva, Switzerland L. Caminada 55, K. Deiters, W. Erdmann, R. Horisberger, Q. Ingram, H.C. Kaestli, D. Kotlinski, U. Langenegger, T. Rohe Paul Scherrer Institut, Villigen, Switzerland M. Backhaus, P. Berger, A. Calandri, N. Chernyavskaya, G. Dissertori, M. Dittmar, M. Donegà, C. Dorfer, T. Gadek, T.A. Gómez Espinosa, C. Grab, D. Hits, W. Lustermann, A.-M. Lyon, R.A. Manzoni, M.T. Meinhard, F. Micheli, P. Musella, F. Nessi-Tedaldi, F. Pauss, V. Perovic, G. Perrin, L. Perrozzi, S. Pigazzini, M.G. Ratti, M. Reichmann, C. Reissel, T. Reitenspiess, B. Ristic, D. Ruini, D.A. Sanz Becerra, M. Schönenberger, L. Shchutska, V. Stampf, M.L. Vesterbacka Olsson, R. Wallny, D.H. Zhu ETH Zurich – Institute for Particle Physics and Astrophysics (IPA), Zurich, Switzerland C. Amsler 56, C. Botta, D. Brzhechko, M.F. Canelli, A. De Cosa, R. Del Burgo, J.K. Heikkilä, M. Huwiler, B. Kilminster, S. Leontsinis, A. Macchiolo, V.M. Mikuni, I. Neutelings, G. Rauco, P. Robmann, K. Schweiger, Y. Takahashi, S. Wertz Universität Zürich, Zurich, Switzerland C. Adloff 57, C.M. Kuo, W. Lin, A. Roy, T. Sarkar 32, S.S. V. Borchsh, V. Ivanchenko, E. Tcherniaev Tomsk State University, Tomsk, Russia 17 Physics Letters B 816 (2021) 136253 The CMS Collaboration B. Alvarez Gonzalez, J. Cuevas, C. Erice, J. Fernandez Menendez, S. Folgueras, I. Gonzalez Caballero, E. Palencia Cortezon, C. Ramón Álvarez, V. Rodríguez Bouza, S. Sanchez Cruz Universidad de Oviedo, Instituto Universitario de Ciencias y Tecnologías Espaciales de Asturias (ICTEA), Oviedo, Spain I.J. Cabrillo, A. Calderon, B. Chazin Quero, J. Duarte Campderros, M. Fernandez, P.J. Fernández Manteca, A. García Alonso, G. Gomez, C. Martinez Rivero, P. Martinez Ruiz del Arbol, F. Matorras, J. Piedra Gomez, C. Prieels, F. Ricci-Tam, T. Rodrigo, A. Ruiz-Jimeno, L. Russo 51, L. Scodellaro, I. Vila, J.M. Vizan Garcia Instituto de Física de Cantabria (IFCA), CSIC-Universidad de Cantabria, Santander, Spain M.K. Jayananda, B. Kailasapathy 52, D.U.J. Sonnadara, D.D.C. Wickramarathna University of Colombo, Colombo, Sri Lanka W.G.D. Dharmaratna, K. Liyanage, N. Perera, N. Wickramage University of Ruhuna, Department of Physics, Matara, Sri Lanka W.G.D. Dharmaratna, K. Liyanage, N. Perera, N. Wickramage University of Ruhuna, Department of Physics, Matara, Sri Lanka Yu National Central University, Chung-Li, Taiwan L. Ceard, P. Chang, Y. Chao, K.F. Chen, P.H. Chen, W.-S. Hou, Y.y. Li, R.-S. Lu, E. Paganis, A. Psallidas, A. Steen National Taiwan University (NTU), Taipei, Taiwan 18 18 Physics Letters B 816 (2021) 136253 The CMS Collaboration B. Asavapibhop, C. Asawatangtrakuldee, N. Srimanobhas Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand B. Asavapibhop, C. Asawatangtrakuldee, N. Srimanobhas Chulalongkorn University, Faculty of Science, Department of Physics, Bangkok, Thailand A. Bat, F. Boran, S. Damarseckin 58, Z.S. Demiroglu, F. Dolek, C. Dozen 59, I. Dumanoglu 60, E. Eskut, G. Gokbulut, Y. Guler, E. Gurpinar Guler 61, I. Hos 62, C. Isik, E.E. Kangal 63, O. Kara, A. Kayis Topaksu, U. Kiminsu, G. Onengut, K. Ozdemir 64, A. Polatoz, A.E. Simsek, B. Tali 65, U.G. Tok, S. Turkcapar, I.S. Zorbakir, C. Zorbilmez Çukurova University, Physics Department, Science and Art Faculty, Adana, Turkey B. Isildak 66, G. Karapinar 67, K. Ocalan 68, M. Yalvac 69 Middle East Technical University, Physics Department, Ankara, Turkey I.O. Atakisi, E. Gülmez, M. Kaya 70, O. Kaya 71, Ö. Özçelik, S. Tekten 72, E.A. Yetkin 73 Bogazici University, Istanbul, Turkey A. Cakir, K. Cankocak 60, Y. Komurcu, S. Sen 74 Istanbul Technical University, Istanbul, Turkey F. Aydogmus Sen, S. Cerci 65, B. Kaynak, S. Ozkorucuklu, D. Sunar Cerci 65 Istanbul University, Istanbul, Turkey L. Levchuk E. Bhal, S. Bologna, J.J. Brooke, D. Burns 75, E. Clement, D. Cussans, H. Flacher, J. Goldstein, G.P. Heath, H.F. Heath, L. Kreczko, B. Krikler, S. Paramesvaran, T. Sakuma, S. Seif El Nasr-Storey, V.J. Smith, J. Taylor, A. Titterton University of Bristol, Bristol, United Kingdom K.W. Bell, A. Belyaev 76, C. Brew, R.M. Brown, D.J.A. Cockerill, K.V. Ellis, K. Harder, S. Harper, J. Linacre, K. Manolopoulos, D.M. Newbold, E. Olaiya, D. Petyt, T. Reis, T. Schuh, C.H. Shepherd-Themistocleous, A. Thea, I.R. Tomalin, T. Williams Rutherford Appleton Laboratory, Didcot, United Kingdom R. Bainbridge, P. Bloch, S. Bonomally, J. Borg, S. Breeze, O. Buchmuller, A. Bundock, V. Cepaitis, G.S. Chahal 77, D. Colling, P. Dauncey, G. Davies, M. Della Negra, P. Everaerts, G. Fedi, G. Hall, G. Iles, J. Langford, L. Lyons, A.-M. Magnan, S. Malik, A. Martelli, V. Milosevic, A. Morton, J. Nash 78, V. Palladino, M. Pesaresi, D.M. Raymond, A. Richards, A. Rose, E. Scott, C. Seez, A. Shtipliyski, M. Stoye, A. Tapper, K. Uchida, T. Virdee 17, N. Wardle, S.N. Webb, D. Winterbottom, A.G. Zecchinelli, S.C. Zenz Imperial College, London, United Kingdom J.E. Cole, P.R. Hobson, A. Khan, P. Kyberd, C.K. Mackay, I.D. Reid, L. Teodorescu, S. Zahid Brunel University, Uxbridge, United Kingdom A. Brinkerhoff, K. Call, B. Caraway, J. Dittmann, K. Hatakeyama, C. Madrid, B. McMaster, N. Pastika, C. Smith Baylor University, Waco, USA R. Bartek, A. Dominguez, R. Uniyal, A.M. Vargas Hernandez Catholic University of America, Washington, DC, USA 19 Physics Letters B 816 (2021) 136253 The CMS Collaboration A. Buccilli, O. Charaf, S.I. Cooper, S.V. Gleyzer, C. Henderson, P. Rumerio, C. West A. Buccilli, O. Charaf, S.I. Cooper, S.V. Gleyzer, C. Henderson, P. Rumerio, C. West The University of Alabama, Tuscaloosa, USA A. Albert, D. Arcaro, Z. Demiragli, D. Gastler, C. Richardson, J. Rohlf, D. Sperka, D. Spitzbart, I. Suarez, D. Zou Boston University, Boston, USA G. Benelli, B. Burkle, X. Coubez 18, D. Cutts, Y.t. Duh, M. Hadley, U. Heintz, J.M. Hogan 79, K.H.M. Kwok, E. Laird, G. Landsberg, K.T. Lau, J. Lee, M. Narain, S. Sagir 80, R. Syarif, E. Usai, W.Y. Wong, D. Yu, W. Zhang Brown University, Providence, USA R. Band, C. Brainerd, R. Breedon, M. Calderon De La Barca Sanchez, M. Chertok, J. Conway, R. Conway, P.T. Cox, R. Erbacher, C. Flores, G. Funk, F. Jensen, W. Ko †, O. Kukral, R. Lander, M. Mulhearn, D. Pellett, J. Pilot, M. L. Levchuk Shi, D. Taylor, K. Tos, M. Tripathi, Z. Wang, Y. Yao, F. Zhang University of California, Davis, Davis, USA M. Bachtis, C. Bravo, R. Cousins, A. Dasgupta, A. Florent, D. Hamilton, J. Hauser, M. Ignatenko, T. Lam, N. Mccoll, W.A. Nash, S. Regnard, D. Saltzberg, C. Schnaible, B. Stone, V. Valuev University of California, Los Angeles, USA K. Burt, Y. Chen, R. Clare, J.W. Gary, S.M.A. Ghiasi Shirazi, G. Hanson, G. Karapostoli, O.R. Long, N. Manganelli, M. Olmedo Negrete, M.I. Paneva, W. Si, S. Wimpenny, Y. Zhang University of California, Riverside, Riverside, USA J.G. Branson, P. Chang, S. Cittolin, S. Cooperstein, N. Deelen, M. Derdzinski, J. Duarte, R. Gerosa, D. Gilbert, B. Hashemi, D. Klein, V. Krutelyov, J. Letts, M. Masciovecchio, S. May, S. Padhi, M. Pieri, V. Sharma, M. Tadel, F. Würthwein, A. Yagil University of California, San Diego, La Jolla, USA N. Amin, R. Bhandari, C. Campagnari, M. Citron, A. Dorsett, V. Dutta, J. Incandela, B. Marsh, H. Mei, A. Ovcharova, H. Qu, J. Richman, U. Sarica, D. Stuart, S. Wang University of California, Santa Barbara – Department of Physics, Santa Barbara, USA D. Anderson, A. Bornheim, O. Cerri, I. Dutta, J.M. Lawhorn, N. Lu, J. Mao, H.B. Newman, T.Q. Nguyen, J. Pata, M. Spiropulu, J.R. Vlimant, S. Xie, Z. Zhang, R.Y. Zhu California Institute of Technology, Pasadena, USA J. Alison, M.B. Andrews, T. Ferguson, T. Mudholkar, M. Paulini, M. Sun, I. Vorobiev, M. Weinberg Carnegie Mellon University, Pittsburgh, USA J.P. Cumalat, W.T. Ford, E. MacDonald, T. Mulholland, R. Patel, A. Perloff, K. Stenson, K.A. Ulmer, S.R. Wagner University of Colorado Boulder, Boulder, USA J. Alexander, Y. Cheng, J. Chu, A. Datta, A. Frankenthal, K. Mcdermott, J. Monroy, J.R. Patterson, D. Quach, A. Ryd, W. Sun, S.M. Tan, Z. Tao, J. Thom, P. Wittich, M. Zientek Cornell University, Ithaca, USA S. Abdullin, M. Albrow, M. Alyari, G. Apollinari, A. Apresyan, A. Apyan, S. Banerjee, L.A.T. Bauerdick, A. Beretvas, D. Berry, J. Berryhill, P.C. Bhat, K. Burkett, J.N. Butler, A. Canepa, G.B. Cerati, H.W.K. Cheung, S. Abdullin, M. Albrow, M. Alyari, G. Apollinari, A. Apresyan, A. Apyan, S. Banerjee, L.A.T. Bauerdick, A. Beretvas, D. Berry, J. Berryhill, P.C. Bhat, K. Burkett, J.N. Butler, A. Canepa, G.B. Cerati, H.W.K. Cheung, F. Chlebana, M. Cremonesi, V.D. Elvira, J. Freeman, Z. Gecse, E. Gottschalk, L. Gray, D. L. Levchuk Green, S G ü d hl O G h R M H i S H R H ll T C H i J Hi h B J il k 20 Physics Letters B 816 (2021) 136253 The CMS Collaboration S. Jindariani, M. Johnson, U. Joshi, T. Klijnsma, B. Klima, M.J. Kortelainen, S. Lammel, J. Lewis, D. Lincoln, R. Lipton, M. Liu, T. Liu, J. Lykken, K. Maeshima, J.M. Marraﬃno, D. Mason, P. McBride, P. Merkel, S. Mrenna, S. Nahn, V. O’Dell, V. Papadimitriou, K. Pedro, C. Pena 81, O. Prokofyev, F. Ravera, A. Reinsvold Hall, L. Ristori, B. Schneider, E. Sexton-Kennedy, N. Smith, A. Soha, W.J. Spalding, L. Spiegel, S. Stoynev, J. Strait, L. Taylor, S. Tkaczyk, N.V. Tran, L. Uplegger, E.W. Vaandering, M. Wang, H.A. Weber, A. Woodard Fermi National Accelerator Laboratory, Batavia, USA D. Acosta, P. Avery, D. Bourilkov, L. Cadamuro, V. Cherepanov, F. Errico, R.D. Field, D. Guerrero, B.M. Joshi, M. Kim, J. Konigsberg, A. Korytov, K.H. Lo, K. Matchev, N. Menendez, G. Mitselmakher, D. Rosenzweig, K. Shi, J. Wang, S. Wang, X. Zuo i i f l id G i ill SA E. Adams, A. Baden, O. Baron, A. Belloni, S.C. Eno, Y. Feng, N.J. Hadley, S. Jabeen, G.Y. Jeng, R.G. Kellogg, T. Koeth, A.C. Mignerey, S. Nabili, M. Seidel, A. Skuja, S.C. Tonwar, L. Wang, K. Wong University of Maryland, College Park, USA Y.R. Joshi Florida International University, Miami, USA Florida International University, Miami, USA Florida International University, Miami, USA T. Adams, A. Askew, D. Diaz, R. Habibullah, S. Hagopian, V. Hagopian, K.F. Johnson, R. Khurana, T. Kolberg, G. Martinez, H. Prosper, C. Schiber, R. Yohay, J. Zhang Florida State University, Tallahassee, USA M.M. Baarmand, S. Butalla, T. Elkafrawy 13, M. Hohlmann, D. Noonan, M. Rahmani, M. Saunders, F. Yumiceva Florida Institute of Technology, Melbourne, USA M.R. Adams, L. Apanasevich, H. Becerril Gonzalez, R.R. Betts, R. Cavanaugh, X. Chen, S. Dittmer, O. Evdokimov, C.E. Gerber, D.A. Hangal, D.J. Hofman, V. Kumar, C. Mills, G. Oh, T. Roy, M.B. Tonjes, N. Varelas, J. Viinikainen, H. Wang, X. Wang, Z. Wu University of Illinois at Chicago (UIC), Chicago, USA M. Alhusseini, B. Bilki 61, K. Dilsiz 82, S. Durgut, R.P. Gandrajula, M. Haytmyradov, V. Khristenko, O.K. Köseyan, J.-P. Merlo, A. Mestvirishvili 83, A. Moeller, J. Nachtman, H. Ogul 84, Y. Onel, F. Ozok 85, A. Penzo, C. Snyder, E. Tiras, J. Wetzel, K. Yi 86 The University of Iowa, Iowa City, USA O. Amram, B. Blumenfeld, L. Corcodilos, M. Eminizer, A.V. Gritsan, S. Kyriacou, P. Maksimovic, C. Mantilla, J. Roskes, M. Swartz, T.Á. Vámi Johns Hopkins University, Baltimore, USA C. Baldenegro Barrera, P. Baringer, A. Bean, S. Boren, A. Bylinkin, T. Isidori, S. Khalil, J. King, G. Krintiras, A. Kropivnitskaya, C. Lindsey, W. Mcbrayer, N. Minafra, M. Murray, C. Rogan, C. Royon, S. Sanders, E. Schmitz, J.D. Tapia Takaki, Q. Wang, J. Williams, G. Wilson The University of Kansas, Lawrence, USA S. Duric, A. Ivanov, K. Kaadze, D. Kim, Y. Maravin, D.R. Mendis, T. Mitchell, A. Modak, A. Mohammadi Kansas State University, Manhattan, USA F. Rebassoo, D. Wright S. Duric, A. Ivanov, K. Kaadze, D. Kim, Y. Maravin, D.R. Mendis, T. Mitchell, A. Modak, A. Mohammadi Kansas State University, Manhattan, USA F. Rebassoo, D. Wright Lawrence Livermore National Laboratory, Livermore, USA E. Adams, A. Baden, O. Baron, A. Belloni, S.C. Eno, Y. Feng, N.J. Hadley, S. Jabeen, G.Y. Jeng, R.G. Kellogg, T. Koeth, A.C. Mignerey, S. Nabili, M. Seidel, A. Skuja, S.C. Tonwar, L. Wang, K. Wong University of Maryland, College Park, USA 21 Physics Letters B 816 (2021) 136253 The CMS Collaboration D. Abercrombie, B. Allen, R. Bi, S. Brandt, W. Busza, I.A. Cali, Y. Chen, M. D’Alfonso, G. Gomez Ceballos, M. Goncharov, P. Harris, D. Hsu, M. Hu, M. Klute, D. Kovalskyi, J. Krupa, Y.-J. Lee, P.D. Luckey, B. Maier, A.C. Marini, C. Mcginn, C. Florida International University, Miami, USA Mironov, S. Narayanan, X. Niu, C. Paus, D. Rankin, C. Roland, G. Roland, Z. Shi, G.S.F. Stephans, K. Sumorok, K. Tatar, D. Velicanu, J. Wang, T.W. Wang, B. Wyslouch Massachusetts Institute of Technology, Cambridge, USA R.M. Chatterjee, A. Evans, S. Guts †, P. Hansen, J. Hiltbrand, Sh. Jain, M. Krohn, Y. Kubota, Z. Lesko, J. Mans, M. Revering, R. Rusack, R. Saradhy, N. Schroeder, N. Strobbe, M.A. Wadud University of Minnesota, Minneapolis, USA J.G. Acosta, S. Oliveros University of Mississippi, Oxford, USA K. Bloom, S. Chauhan, D.R. Claes, C. Fangmeier, L. Finco, F. Golf, J.R. González Fernández, I. Kravchenko, J.E. Siado, G.R. Snow †, B. Stieger, W. Tabb University of Nebraska-Lincoln, Lincoln, USA G. Agarwal, C. Harrington, I. Iashvili, A. Kharchilava, C. McLean, D. Nguyen, A. Parker, J. Pekkanen, S. Rappoccio, B. Roozbahani State University of New York at Buffalo, Buffalo, USA G. Alverson, E. Barberis, C. Freer, Y. Haddad, A. Hortiangtham, G. Madigan, B. Marzocchi, D.M. Morse, V. Nguyen, T. Orimoto, L. Skinnari, A. Tishelman-Charny, T. Wamorkar, B. Wang, A. Wisecarver, D. Wood Northeastern University, Boston, USA S. Bhattacharya, J. Bueghly, Z. Chen, A. Gilbert, T. Gunter, K.A. Hahn, N. Odell, M.H. Schmitt, K. Sung, M. Velasco Northwestern University, Evanston, USA R. Bucci, N. Dev, R. Goldouzian, M. Hildreth, K. Hurtado Anampa, C. Jessop, D.J. Karmgard, K. Lannon, W. Li, N. Loukas, N. Marinelli, I. Mcalister, F. Meng, K. Mohrman, Y. Musienko 42, R. Ruchti, P. Siddireddy, S. Taroni, M. Wayne, A. Wightman, M. Wolf, L. Zygala University of Notre Dame, Notre Dame, USA J. Alimena, B. Bylsma, B. Cardwell, L.S. Durkin, B. Francis, C. Hill, W. Ji, A. Lefeld, B.L. Winer, B.R. Yates The Ohio State University, Columbus, USA G. Dezoort, P. Elmer, N. Haubrich, S. Higginbotham, A. Kalogeropoulos, G. Kopp, S. Kwan, D. Lange, M.T. Lucchini, J. Luo, D. Marlow, K. Mei, I. Ojalvo, J. Olsen, C. Palmer, P. Piroué, D. Stickland, C. Tully Princeton University, Princeton, USA S. Malik, S. Norberg University of Puerto Rico, Mayaguez, USA V.E. Barnes, R. Chawla, S. Das, L. Gutay, M. Jones, A.W. Jung, B. Mahakud, G. Negro, N. Neumeister, C.C. Peng, S. Piperov, H. Qiu, J.F. Schulte, N. Trevisani, F. Wang, R. Xiao, W. Xie Purdue University, West Lafayette, USA T. Cheng, J. Dolen, N. Parashar Purdue University Northwest, Hammond, USA 22 Physics Letters B 816 (2021) 136253 The CMS Collaboration A. Baty, S. Dildick, K.M. Ecklund, S. Freed, F.J.M. Florida International University, Miami, USA Geurts, M. Kilpatrick, A. Kumar, W. Li, B.P. Padley, R. Redjimi, J. Roberts †, J. Rorie, W. Shi, A.G. Stahl Leiton, Z. Tu, A. Zhang Rice University, Houston, USA A. Bodek, P. de Barbaro, R. Demina, J.L. Dulemba, C. Fallon, T. Ferbel, M. Galanti, A. Garcia-Bellido, O. Hindrichs, A. Khukhunaishvili, E. Ranken, R. Taus University of Rochester, Rochester, USA B. Chiarito, J.P. Chou, A. Gandrakota, Y. Gershtein, E. Halkiadakis, A. Hart, M. Heindl, E. Hughes, S. Kaplan, O. Karacheban 21, I. Laﬂotte, A. Lath, R. Montalvo, K. Nash, M. Osherson, S. Salur, S. Schnetzer, S. Somalwar, R. Stone, S. Thomas Rutgers, The State University of New Jersey, Piscataway, USA University of Wisconsin – Madison, Madison, WI, USA University of Wisconsin – Madison, Madison, WI, USA † Deceased. 1 Also at Vienna University of Technology, Vienna, Austria. 2 Also at Université Libre de Bruxelles, Bruxelles, Belgium. 3 Also at IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France. 4 Also at Universidade Estadual de Campinas, Campinas, Brazil. 5 Also at Federal University of Rio Grande do Sul, Porto Alegre, Brazil. 6 Also at UFMS, Nova Andradina, Brazil. 7 Also at Universidade Federal de Pelotas, Pelotas, Brazil. 8 Also at University of Chinese Academy of Sciences, Beijing, China. 9 Also at Institute for Theoretical and Experimental Physics named by A.I. Alikhan 10 Also at Joint Institute for Nuclear Research, Dubna, Russia. 11 Also at Helwan University, Cairo, Egypt. 12 Now at Zewail City of Science and Technology, Zewail, Egypt. 13 Also at Ain Shams University, Cairo, Egypt. 14 Also at Purdue University, West Lafayette, USA. † Deceased. 1 Also at Vienna University of Technology, Vienna, Austria. 2 Also at Université Libre de Bruxelles, Bruxelles, Belgium. 3 Also at IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France. 4 Also at Universidade Estadual de Campinas, Campinas, Brazil. 5 Also at Federal University of Rio Grande do Sul, Porto Alegre, Brazil. 6 Also at UFMS, Nova Andradina, Brazil. 7 Also at Universidade Federal de Pelotas, Pelotas, Brazil. 8 Also at University of Chinese Academy of Sciences, Beijing, China. 9 Also at Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of NRC ‘Kurchatov Institute’, Moscow, 10 Also at Joint Institute for Nuclear Research, Dubna, Russia. 11 Also at Helwan University, Cairo, Egypt. 12 Now at Zewail City of Science and Technology, Zewail, Egypt. 13 Also at Ain Shams University, Cairo, Egypt. 14 Also at Purdue University, West Lafayette, USA. † Deceased. 1 Also at Vienna University of Technology, Vienna, Austria. 1 Also at Vienna University of Technology, Vienna, Austria. 2 Also at Université Libre de Bruxelles, Bruxelles, Belgium. 2 Also at Université Libre de Bruxelles, Bruxelles, Belgium. 3 Also at IRFU, CEA, Université Paris-Saclay, Gif-sur-Yvette, France. 4 Also at Universidade Estadual de Campinas, Campinas, Brazil. 5 Also at Federal University of Rio Grande do Sul, Porto Alegre, Brazil. 5 Also at Federal University of Rio Grande do Sul, Porto Alegre, Brazil. 6 Also at UFMS, Nova Andradina, Brazil. 7 Also at Universidade Federal de Pelotas, Pelotas, Brazil. 8 Also at University of Chinese Academy of Sciences, Beijing, China. P.E. Karchin, N. Poudyal, J. Sturdy, P. Thapa Wayne State University, Detroit, USA K. Black, T. Bose, J. Buchanan, C. Caillol, S. Dasu, I. De Bruyn, L. Dodd, C. Galloni, H. He, M. Herndon, A. Hervé, U. Hussain, A. Lanaro, A. Loeliger, R. Loveless, J. Madhusudanan Sreekala, A. Mallampalli, D. Pinna, T. Ruggles, A. Savin, V. Shang, V. Sharma, W.H. Smith, D. Teague, S. Trembath-reichert, W. Vetens K. Black, T. Bose, J. Buchanan, C. Caillol, S. Dasu, I. De Bruyn, L. Dodd, C. Galloni, H. He, M. Herndon, A. Hervé, U. Hussain, A. Lanaro, A. Loeliger, R. Loveless, J. Madhusudanan Sreekala, A. Mallampalli, D. Pinna, T. Ruggles, A. Savin, V. Shang, V. Sharma, W.H. Smith, D. Teague, S. Trembath-reichert, W. Vetens K. Black, T. Bose, J. Buchanan, C. Caillol, S. Dasu, I. De Bruyn, L. Dodd, C. Galloni, H. He, M. Herndon, A. Hervé, U. Hussain, A. Lanaro, A. Loeliger, R. Loveless, J. Madhusudanan Sreekala, A. Mallampalli, D. Pinna, T. Ruggles, A. Savin, V. Shang, V. Sharma, W.H. Smith, D. Teague, S. Trembath-reichert, W. Vetens University of Wisconsin – Madison, Madison, WI, USA H. Acharya, A.G. Delannoy, S. Spanier University of Tennessee, Knoxville, USA H. Acharya, A.G. Delannoy, S. Spanier University of Tennessee, Knoxville, USA O. Bouhali 87, M. Dalchenko, A. Delgado, R. Eusebi, J. Gilmore, T. Huang, T. Kamon 88, H. Kim, S. Luo, S. Malhotra, D. Marley, R. Mueller, D. Overton, L. Perniè, D. Rathjens, A. Safonov Texas A&M University, College Station, USA N. Akchurin, J. Damgov, V. Hegde, S. Kunori, K. Lamichhane, S.W. Lee, T. Mengke, S. Muthumuni, T. Peltola, S. Undleeb, I. Volobouev, Z. Wang, A. Whitbeck Texas Tech University, Lubbock, USA E. Appelt, S. Greene, A. Gurrola, R. Janjam, W. Johns, C. Maguire, A. Melo, H. Ni, K. Padeken, F. Romeo, P. Sheldon, S. Tuo, J. Velkovska, M. Verweij Vanderbilt University, Nashville, USA L. Ang, M.W. Arenton, B. Cox, G. Cummings, J. Hakala, R. Hirosky, M. Joyce, A. Ledovskoy, C. Neu, B. Tannenwald, Y. Wang, E. Wolfe, F. Xia University of Virginia, Charlottesville, USA P.E. Karchin, N. Poudyal, J. Sturdy, P. Thapa Wayne State University, Detroit, USA Also at Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of NRC ‘Kurchatov Institute’, Moscow, Russia. Al J i I i f N l R h D b R i University of Wisconsin – Madison, Madison, WI, USA Petersburg, Russia. 45 Also at St. Petersburg State Polytechnical University, St. Petersburg, Russia. 46 Also at University of Florida, Gainesville, USA. 47 Also at Imperial College, London, United Kingdom. 48 Also at P.N. Lebedev Physical Institute, Moscow, Russia. 49 Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia. 49 Also at Budker Institute of Nuclear Physics, Novosibirsk, Russia. 50 Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia. 50 Also at Faculty of Physics, University of Belgrade, Belgrade, Serbia. 51 Also at Università degli Studi di Siena, Siena, Italy. 52 Also at Trincomalee Campus, Eastern University, Sri Lanka, Nilaveli, Sri Lanka. 52 Also at Trincomalee Campus, Eastern University, Sri Lanka, Nilaveli, Sri Lanka. 52 Also at Trincomalee Campus, Eastern University, Sri Lank 53 Also at INFN Sezione di Pavia a, Università di Pavia b, Pavia, Italy, Pavia, Italy. 53 Also at INFN Sezione di Pavia a, Università di Pavia b, Pavia, Italy, Pavia, Italy. 54 Also at National and Kapodistrian University of Athens, Athens, Greece. 54 Also at National and Kapodistrian University of Athens, Athens, Greece. 55 Also at Universität Zürich, Zurich, Switzerland. 56 Also at Stefan Meyer Institute for Subatomic Physics, Vienna, Austria, Vienna, Austria 56 Also at Stefan Meyer Institute for Subatomic Physics, Vienna, Austria, Vienna, Austria oire d’Annecy-le-Vieux de Physique des Particules, IN2P3-CNRS, An 57 Also at Laboratoire d’Annecy-le-Vieux de Physique des Particules, IN2P3-CNRS, Annecy-le-Vieux, France. 57 Also at Laboratoire d’Annecy-le-Vieux de Physique des Particules, IN2P3-CNRS, Annecy-l 58 Also at ¸Sırnak University, Sirnak, Turkey. 59 Also at Department of Physics, Tsinghua University, Beijing, China, Beijing, Ch 59 Also at Department of Physics, Tsinghua University, Beijing, China, Beijing, China. 60 Also at Near East University, Research Center of Experimental Health Science, Nicosia, Turk 60 Also at Near East University, Research Center of Experimental Health Science, Nicosia, Turkey. 60 Also at Near East University, Research Center of Experimental 61 Also at Beykent University, Istanbul, Turkey, Istanbul, Turkey. 61 Also at Beykent University, Istanbul, Turkey, Istanbul, Turkey. 62 Also at Istanbul Aydin University, Application and Research Center for Advanced Studies (App. & Res. Cent. for Advanced Studies), Istanbul, Turkey. 63 Also at Mersin University, Mersin, Turkey. 62 Also at Istanbul Aydin University, Application and Research Cen 62 Also at Istanbul Aydin University, Application and Research Center for Advanced Studies (App. & Res. Cent. for Advanced Studies), Istanbul, Turkey. 63 63 Also at Mersin University, Mersin, Turkey. University of Wisconsin – Madison, Madison, WI, USA 27 Al IIT Bh b Bh b I di Bh b I di 26 Also at MTA-ELTE Lendület CMS Particle and Nuclear Physics Group, Eötvös Loránd University, Budapest, Hunga 26 Also at MTA-ELTE Lendület CMS Particle and Nuclear Physics Group, Eötvös Loránd University, Budapest, Hungary, Budapest, Hun 26 Also at MTA-ELTE Lendület CMS Particle and Nuclear Physics Group, E 27 Also at IIT Bhubaneswar, Bhubaneswar, India, Bhubaneswar, India. 27 Also at IIT Bhubaneswar, Bhubaneswar, India, Bhubaneswar, India. 28 Also at Institute of Physics, Bhubaneswar, India. 29 Also at G.H.G. Khalsa College, Punjab, India. 30 Also at Shoolini University, Solan, India. 31 Also at University of Hyderabad, Hyderabad, India. 32 Also at University of Visva-Bharati, Santiniketan, India. 33 Also at Indian Institute of Technology (IIT), Mumbai, India. 34 Also at Deutsches Elektronen-Synchrotron, Hamburg, Germany. 35 Also at Department of Physics, University of Science and Technology of Mazandaran, Behshahr, Iran. 35 Also at Department of Physics, University of Science and Technology of Mazandaran, Behshahr, Iran. 6 Now at INFN Sezione di Bari a, Università di Bari b, Politecnico d 37 Also at Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Bologna, Italy. 37 Also at Italian National Agency for New Technologies, Energy and Sustainable Economic Development, Bologna, Italy. 37 Also at Italian National Agency for New Technologies, Energy and Sustainable Econom 38 Also at Centro Siciliano di Fisica Nucleare e di Struttura Della Materia, Catania, Italy. 38 Also at Centro Siciliano di Fisica Nucleare e di Struttura Della Materia, Catania, Italy. 38 Also at Centro Siciliano di Fisica Nucleare e di Struttura Della 39 Also at Riga Technical University, Riga, Latvia, Riga, Latvia. 40 Also at Consejo Nacional de Ciencia y Tecnología, Mexico City 40 Also at Consejo Nacional de Ciencia y Tecnología, Mexico City, Mexico. 41 Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Poland. 41 Also at Warsaw University of Technology, Institute of Electronic Systems, Warsaw, Poland. 41 Also at Warsaw University of Technology, Institute of Ele 42 Also at Institute for Nuclear Research, Moscow, Russia. 43 Now at National Research Nuclear University ‘Moscow Engineering Physics Institute’ (MEPhI), Moscow, Russia. 43 Now at National Research Nuclear University ‘Moscow Engineering Physics Institute’ (MEPhI), Moscow 43 Now at National Research Nuclear University ‘Moscow Engin 4 Also at Institute of Nuclear Physics of the Uzbekistan Academy o 45 Also at St. Petersburg State Polytechnical University, St. University of Wisconsin – Madison, Madison, WI, USA 9 Also at Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of NRC ‘Kurchatov Institute’, Moscow, Rus 10 9 Also at Institute for Theoretical and Experimental Physics named by A.I. Alikhanov of NRC ‘Kurch 9 Also at Institute for Theoretical and Experimental Physics name 10 Also at Joint Institute for Nuclear Research, Dubna, Russia. 11 Also at Helwan University, Cairo, Egypt. 12 Now at Zewail City of Science and Technology, Zewail, Egypt. 13 Also at Ain Shams University, Cairo, Egypt. 14 Also at Purdue University, West Lafayette, USA. 23 Physics Letters B 816 (2021) 136253 The CMS Collaboration 15 Also at Université de Haute Alsace, Mulhouse, France. 15 Also at Université de Haute Alsace, Mulhouse, France. 16 Also at Erzincan Binali Yildirim University, Erzincan, Turkey. 16 Also at Erzincan Binali Yildirim University, Erzincan, Turkey. 17 Also at CERN, European Organization for Nuclear Research, Ge 18 Also at RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany. 18 Also at RWTH Aachen University, III. Physikalisches Institut A, Aachen, Germany. 18 Also at RWTH Aachen University, III. Physikalisches In 19 Also at University of Hamburg, Hamburg, Germany. y g g y 20 Also at Department of Physics, Isfahan University of Technology, Isfahan, Iran, Isfahan, Iran. 20 Also at Department of Physics, Isfahan University of Technology, Isfahan, Iran, Isfahan, Iran. 20 Also at Department of Physics, Isfahan University of Te 1 Also at Brandenburg University of Technology, Cottbus, Germany g y gy y 22 Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia. 22 Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Moscow State University, Moscow, Russia 2 Also at Skobeltsyn Institute of Nuclear Physics, Lomonosov Mosc 23 Also at Institute of Physics, University of Debrecen, Debrecen, Hungary, Debrecen, Hungary. 23 Also at Institute of Physics, University of Debrecen, Debrecen, Hungary, Debrecen, Hungary. 3 Also at Institute of Physics, University of Debrecen, Debrecen, H 24 Also at Physics Department, Faculty of Science, Assiut University, Assiut, Egypt. 4 Also at Physics Department, Faculty of Science, Assiut University 5 Also at Institute of Nuclear Research ATOMKI, Debrecen, Hungar 26 Also at MTA-ELTE Lendület CMS Particle and Nuclear Physics Group, Eötvös Loránd University, Budapest, Hungary, Budapest, Hungary. 86 Also at Nanjing Normal University Department of Physics, Nanjing, China. 87 Also at Texas A&M University at Qatar, Doha, Qatar. 88 Also at Kyungpook National University, Daegu, Korea, Daegu, Republic of Korea. The CMS Collaboration 86 Also at Nanjing Normal University Department of Physics, Nanjing, China. 87 Also at Texas A&M University at Qatar, Doha, Qatar. 88 Also at Kyungpook National University, Daegu, Korea, Daegu, Republic of Korea. University of Wisconsin – Madison, Madison, WI, USA 64 Also at Piri Reis University, Istanbul, Turkey. 65 Also at Adiyaman University, Adiyaman, Turkey. 65 Also at Adiyaman University, Adiyaman, Turkey. 66 Also at Ozyegin University, Istanbul, Turkey. 67 Also at Izmir Institute of Technology, Izmir, Turke 67 Also at Izmir Institute of Technology, Izmir, Turkey. 68 Also at Necmettin Erbakan University, Konya, Turkey. 68 Also at Necmettin Erbakan University, Konya, Turkey. 69 Also at Bozok Universitetesi Rektörlügü, Yozgat, Turkey. 69 Also at Bozok Universitetesi Rektörlügü, Yozgat, Turkey. 70 Also at Marmara University, Istanbul, Turkey. 71 Also at Milli Savunma University, Istanbul, Turk 72 Also at Kafkas University, Kars, Turkey. 73 Also at Istanbul Bilgi University, Istanbul, Turkey. 73 Also at Istanbul Bilgi University, Istanbul, Turkey. 74 Also at Hacettepe University, Ankara, Turkey. 75 Also at Vrije Universiteit Brussel, Brussel, Belgium. 75 Also at Vrije Universiteit Brussel, Brussel, Belgium. 76 Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdom. 76 Also at School of Physics and Astronomy, University o 76 Also at School of Physics and Astronomy, University of Southampton, Southampton, United Kingdo 77 Also at IPPP Durham University, Durham, United Kingdom. 77 Also at IPPP Durham University, Durham, United Kingdom. 78 Also at Monash University, Faculty of Science, Clayton, Austral 78 Also at Monash University, Faculty of Science, Clayton, Austr 79 Also at Bethel University, St. Paul, Minneapolis, USA, St. Paul, USA. 79 Also at Bethel University, St. Paul, Minneapolis, USA, St. Paul, USA. 80 Also at Karamano˘glu Mehmetbey University, Karaman, Turkey. 80 Also at Karamano˘glu Mehmetbey University, Karaman, Turkey. 81 Also at California Institute of Technology, Pasadena, USA. 81 Also at California Institute of Technology, Pasadena, USA. 82 Also at Bingol University, Bingol, Turkey. 83 Also at Georgian Technical University, Tbilisi, Georgia. 83 Also at Georgian Technical University, Tbilisi, Georgia. 84 Also at Sinop University, Sinop, Turkey. 85 Also at Mimar Sinan University, Istanbul, Istanbul, Turkey. 85 Also at Mimar Sinan University, Istanbul, Istanbul, Turkey. 24 Physics Letters B 816 (2021) 136253 25
https://openalex.org/W4255444486	https://journals.vgtu.lt/index.php/MMA/article/download/6726/7372	English	null	INVERSE PROBLEMS FOR A GENERALIZED SUBDIFFUSION EQUATION WITH FINAL OVERDETERMINATION	Mathematical modelling and analysis/Mathematical modeling and analysis	2,019	cc-by	15,690	AMS Subject Classiﬁcation: 35R30; 35R11. AMS Subject Classiﬁcation: 35R30; 35R11. Mathematical Modelling and Analysis Volume 24, Issue 2, 236–262, 2019 https://doi.org/10.3846/mma.2019.016 Mathematical Modelling and Analysis Volume 24, Issue 2, 236–262, 2019 https://doi.org/10.3846/mma.2019.016 Mathematical Modelling and Analysis Volume 24, Issue 2, 236–262, 2019 https://doi.org/10.3846/mma.2019.016 http://mma.vgtu.lt ISSN: 1392-6292 eISSN: 1648-3510 http://mma.vgtu.lt ISSN: 1392-6292 eISSN: 1648-3510 Nataliia Kinash and Jaan Janno Nataliia Kinash and Jaan Janno Abstract. We consider two inverse problems for a generalized subdiﬀusion equa- tion that use the ﬁnal overdetermination condition. Firstly, we study a problem of reconstruction of a speciﬁc space-dependent component in a source term. We prove existence, uniqueness and stability of the solution to this problem. Based on these results, we consider an inverse problem of identiﬁcation of a space-dependent coef- ﬁcient of a linear reaction term. We prove the uniqueness and local existence and stability of the solution to this problem. Keywords: inverse problem, subdiﬀusion, ﬁnal overdetermination, fractional diﬀusion. AMS Subject Classiﬁcation: 35R30; 35R11. 1 Introduction Anomalous diﬀusion processes are described by diﬀerent models [6]. Among them stands out the time (or space-time) fractional diﬀusion equation that is the most common way to represent a subdiﬀusion. For some situations such approach does not work [19]. Therefore, more general models that unify wider range of subdiﬀusion processes are introduced [19,25]. [ ] In this paper we use an operator that is more general than the fractional time derivative: D{k} t v = d dtk ∗v, (1.1) (1.1) where ∗denotes the time convolution, i.e. (v1 ∗v2)(t) = R t 0 v1(t −τ)v2(τ)dτ. Taken k = t−β Γ (1−β), (1.1) transforms into a well-known Riemann-Liouville frac- where ∗denotes the time convolution, i.e. (v1 ∗v2)(t) = R t 0 v1(t −τ)v2(τ)dτ. Taken k = t−β Γ (1−β), (1.1) transforms into a well-known Riemann-Liouville frac- ■ Copyright c⃝2019 The Author(s). Published by VGTU Press ■ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribu- tion, and reproduction in any medium, provided the original author and source are credited. Inverse Problems for a Generalized Subdiﬀusion Equation 237 tional derivative. The operator corresponding to the Caputo fractional deriva- tive denoted as DC (k)v was introduced by Luchko and Yamamoto in [23] and also in [15]. tional derivative. The operator corresponding to the Caputo fractional deriva- tive denoted as DC (k)v was introduced by Luchko and Yamamoto in [23] and also in [15]. The toolkit for treating such a type of derivative have been developed by Pr¨uss et al. [5]. They have created a setting to introduce the operator inverse to D{k} t through the concept of Completely Positive kernels [5]: a kernel M ∈ L1,loc(R+) is called completely positive if there are k0 ≥0 and nonnegative and nonincreasing k1 ∈L1,loc(R+) such that M ∗(k0δ + k1) = 1 holds. The applications of this concept can be found in [1, 33, 34]. Another approach to this issue has been developed by Kochubei [19]. Often parameters of models are unknown. Then additional observations are performed and inverse problems solved to reconstruct unknown quantities [12, 13, 16, 17, 20, 21]. In the present paper we consider two inverse problems (IPs) that use ﬁnal observation data: IP1 is to identify a space-dependent factor f of a source term g(t, x)f(x); IP2 is to reconstruct a coeﬃcient r(x) of a linear reaction term. 1 Introduction IP1 for fractional and perturbed fractional diﬀusion equations is studied in several papers. Theoretical and numerical results are obtained in the particular case g = g(t) [7,17,18,26] and in the case g = g(t, x) [30,32]. In latter papers the existence and uniqueness of solutions are proved for almost all scalar diﬀusion coeﬃcients. IP1 for a semilinear fractional diﬀusion equation is considered in [15]. Uniqueness of the solution is proved. In this paper we consider IP1 for a more general diﬀusion equation that includes the operator (1.1) instead of the fractional derivative. We prove the uniqueness of the solution to IP1 by applying a modiﬁed version of the positivity principle from [15]. That falls into category of maximum principle results [13, 20, 22]. Similar approaches to the inverse problems are well-known in the domain of parabolic equations [2,12]. Next we prove the existence and stability of the solution of IP1 by means of the Fredholm alternative. The uniqueness of solution of IP2 follows from the IP1-results. Finally, we prove local existence and stability of the solution to IP2 by means of the contraction argument. 2 Formulation of direct and inverse problems Let us consider the generalized subdiﬀusion equation Ut(t, x) = (M ∗LU)t(t, x) + Q(t, x), (2.1) (2.1) where U physical state, t is the time, x ∈Rn is a space variable, Q is a source term, the operator L = L(x) is such that where U physical state, t is the time, x ∈Rn is a space variable, Q is a source term, the operator L = L(x) is such that L(x) = L1(x) + r(x)I, where L1(x) = n X i,j=1 aij(x) ∂2 ∂xi∂xj + n X j=1 aj(x) ∂ ∂xj and I is the unity operator. The kernel M is a memory function related to a non-locality of the diﬀusion process. Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. 238 N. Kinash and J. Janno There are two ways to derive the equation (2.1) from physical laws. One method consists in modelling continuous time random walk processes in micro- level and taking a continuous limit in a macro-level [4] and another one uses conservative laws and speciﬁc constitutive relations with memory [27]. There are two ways to derive the equation (2.1) from physical laws. One method consists in modelling continuous time random walk processes in micro- level and taking a continuous limit in a macro-level [4] and another one uses conservative laws and speciﬁc constitutive relations with memory [27]. Real world applications of the equation (2.1) include diﬀusion in fractal and porous media, e.g. propagation of pollution, heat ﬂow in media with memory, dynamics of protein in cells, transport in dielectrics and semiconductors, usage of optical tweezers, Hamiltonian chaos etc. [3,4,6,27,31]. Let us assume that there is a function k such that k ∗M = M ∗k = 1. Then if we apply k∗to (2.1), we obtain an equation that contains the explicit diﬀerential operator L and is called the normal form of (2.1): k ∗Ut(t, x) = LU(t, x)+H(t, x), where H(x, t) := k∗Q(t, x). The term k∗Ut can be rewritten in the form D{k} t (U −U(0, ·)) that does not contain the 1st order derivative of U. Therefore, we get the equation D{k} t (U −U(0, ·)) = LU(t, x) + H(t, x). (2.2) (2.2) Conversely, in case of suﬃciently regular U, the equation (2.1) follows from (2.2) by means of the application of the operator ∂ ∂tM∗. The equation (2.1) and its analogue (2.2) incorporate the following possi- bilities: 1. 2 Formulation of direct and inverse problems The kernel M(t) = tβ−1 Γ (β), 0 < β < 1, represents a power-type mem- ory. Then (2.1) becomes the celebrated time fractional diﬀusion equation Ut = κD1−βLU + Q, where D1−βv = tβ−1 Γ (β) ∗v t is the Riemann- Liouville fractional derivative of the order 1 −β [4, 17, 20, 26]. For such M, it holds k = t−β Γ (1−β) and [k ∗(v −v(0)]t = k ∗vt = ∂β t v is the Caputo fractional derivative. 1. The kernel M(t) = tβ−1 Γ (β), 0 < β < 1, represents a power-type mem- ory. Then (2.1) becomes the celebrated time fractional diﬀusion equation Ut = κD1−βLU + Q, where D1−βv = tβ−1 Γ (β) ∗v t is the Riemann- Liouville fractional derivative of the order 1 −β [4, 17, 20, 26]. For such M, it holds k = t−β Γ (1−β) and [k ∗(v −v(0)]t = k ∗vt = ∂β t v is the Caputo fractional derivative. 2. The kernel M or its associate k is a linear combination of power functions [25,31]: 2. The kernel M or its associate k is a linear combination of power functions [25,31]: M(t) = tβ−1 Γ(β) + l X j=1 pj tβj−1 Γ(βj), 0 < β < βj < 1, pj ≥0, k(t) = t−β Γ(1 −β) + l X j=1 qj t−βj Γ(1 −βj), 0 < βj < β < 1, qj ≥0. 3. The kernel M has the form M(t) = R 1 0 p(s) ts−1 Γ (s)ds where p ≥0 is a nonvanishing integrable function (cf. [3, 25, 31]). Such a kernel stands for the distributed order fractional derivative that is used for modeling diﬀusion with a logarithmic growth of the mean square displacement [19]. 4. Tempered fractional calculus [29], that is another way to generalize a fractional calculus, falls into the case M(t) = 1 Γ(β)e−λttβ−1 + λ Γ(β) Z t 0 e−λττ β−1dτ, λ > 0. Inverse Problems for a Generalized Subdiﬀusion Equation 239 This type of kernel is used for modelling the transition from anomalous to normal diﬀusion. Every presented example of M (or k) has a completely monotonic associate k (or M) that solves k ∗M = 1 (see Section 3). Let Ω∈Rn be an open bounded domain with the boundary ∂Ω. 2 Formulation of direct and inverse problems In direct problem we have to ﬁnd a function u that solves the initial-boundary value problem D{k} t (U −Φ)(t, x) = LU(t, x) + H(t, x), x ∈Ω, t ∈(0, T), U(0, x) = Φ(x), x ∈Ω, (2.3) B(U −b)(t, x) = 0, x ∈∂Ω, t ∈(0, T). (2.3) Here Φ and b are given functions and Here Φ and b are given functions and Bv(x) = v(x) or Bv(x) = ω(x) · ∇v(x), with ω · ν > 0 and ν(x) denoting the outer normal of ∂Ωat x ∈Ω. An important particular case is ω = Pn j=1 aijνj\|i=1,...,n . Then the condition B(U −b)\|(t,x)∈(0,T )×∂Ω= 0 corresponds to the ﬂux speciﬁed at ∂Ω. ( , ) ( , ) Let us proceed to inverse problems. To this end we introduce the conditio U(T, x) = Ψ(x), x ∈Ω, (2.4) (2.4) with a given observation function Ψ. Firstly, we formulate of an inverse source problem. Let H(t, x) = g(t, x)f(x) + h0(t, x), (2.5) (2.5) where the components gf and h0 may correspond to diﬀerent sources or sinks. The factor f is unknown and to be reconstructed by means of the data (2.4). Since the whole function U is also unknown, the ﬁrst inverse problem consists in determination a pair of functions (f, U) that satisﬁes (2.3), (2.4) and (2.5). I th d i bl i i t id tif th ﬃi t f where the components gf and h0 may correspond to diﬀerent sources or sinks. The factor f is unknown and to be reconstructed by means of the data (2.4). Since the whole function U is also unknown, the ﬁrst inverse problem consists in determination a pair of functions (f, U) that satisﬁes (2.3), (2.4) and (2.5). In the second inverse problem, our aim is to identify the coeﬃcient r of the linear reaction term rU. In the mathematical formulation, the problem consists in ﬁnding a pair (r, U) that satisﬁes (2.3) and (2.4). We can handle the case of zero initial condition Φ = 0 (for details, see the end of Section 6). Methods to be used in this paper require homogeneous boundary conditions. Therefore, we perform the change of the second unknown u = U −b in our problems. It brings along shifts of data by addends containing b. Math. Model. Anal., 24(2):236–262, 2019. 2 Formulation of direct and inverse problems p g g y g Firstly, from (2.3) we obtain the following problem for u = U −b: D{k} t (u −ϕ)(t, x) = Lu(t, x) + F(t, x), x ∈Ω, t ∈(0, T), u(0, x) = ϕ(x), x ∈Ω, (2 Bu(t, x) = 0, x ∈∂Ω, t ∈(0, T), D{k} t (u −ϕ)(t, x) = Lu(t, x) + F(t, x), x ∈Ω, t ∈(0, T), u(0, x) = ϕ(x), x ∈Ω, (2.6) Bu(t, x) = 0, x ∈∂Ω, t ∈(0, T), where ϕ(x) = Φ(x) −b(0, x), (2.7) F(t, x) = H(t, x) + Lb(t, x) −D{k} t (b −b(0, ·))(t, x). (2.8) (2.7) (2.8) (2.8) 240 N. Kinash and J. Janno The overdetermination condition (2.4) in terms of u has the form u(T, x) = ψ(x), x ∈Ω, (2.9) u(T, x) = ψ(x), x ∈Ω, (2.9) where ψ(x) = Ψ(x) −b(T, x). Plugging (2.5) into (2.8) we obtain where ψ(x) = Ψ(x) −b(T, x). Plugging (2.5) into (2.8) we obtain F(t, x) = g(t, x)f(x) + h(t, x), (2.10) F(t, x) = g(t, x)f(x) + h(t, x), (2.10) where h(t, x) = h0(t, x) + Lb(t, x) −D{k} t (b −b(0, ·))(t, x). where h(t, x) = h0(t, x) + Lb(t, x) −D{k} t (b −b(0, ·))(t, x). In the reformulated ﬁrst inverse problem (IP1), we seek for the pair of functions (f, u) that satisﬁes (2.6), (2.9) and (2.10). Let us reformulate the second inverse problem, too. From the relations (2.3), (2.4) with Φ = 0 by means of the change of variable u = U −b, we obtain the following problem for the pair (r, u): D{k} t u(t, x) = L1u(t, x) + r(x)(u + b)(t, x) + F1(t, x) x ∈Ω, t ∈(0, T), u(0, x) = 0, x ∈Ω, Bu(t, x) = 0, x ∈∂Ω, t ∈(0, T), (2.11) u(T, x) = ψ(x), x ∈Ω, (2.11) where b(0, x) = 0, x ∈Ω, the function ψ is expressed by ψ(x) = Ψ(x) −b(T, x) and F1(t, x) = H(t, x) + L1b(t, x) −D{k} t b(t, x). where b(0, x) = 0, x ∈Ω, the function ψ is expressed by ψ(x) = Ψ(x) −b(T, x) and F1(t, x) = H(t, x) + L1b(t, x) −D{k} t b(t, x). Thus, the reformulated second inverse problem (IP2) is to ﬁnd the pair of functions (r, u) that satisﬁes (2.11). 3 Basic assumptions In this section we collect basic conditions on the domain, operator L and kernels k and M that will be assumed throughout the paper. We assume that ∂Ωis uniformly of the class C2 and ω ∈(C1(∂Ω))n. Moreover, we assume that aij, aj, r ∈C(Ω) and the principal part of L is uniformly elliptic, i.e. nP i,j=1 aij(x)ξiξj ≥c\|ξ\|2 ∀ξ ∈Rn, x ∈Ωfor some c > 0. Concerning the function k, we assume that Concerning the function k, we assume that 1. k belongs to L1,loc(0, ∞) and is a solution of the equation M ∗k = 1 with a kernel M ∈L1,loc(0, ∞) that satisﬁes the conditions M ∈C1(0, ∞), lim t→0+M(t) = ∞, M > 0, M ′ ≤0, (3.1) (3.1) −M ′ is nonincreasing and convex; (3.1) −M ′ is nonincreasing and convex; (3.1) −M ′ is nonincreasing and convex; −M ′ is nonincreasing and convex; 2. k has the following properties: 2. k has the following properties: 2. k has the following properties: k ∈C(0, ∞), lim t→0+k(t) = ∞, k > 0, k is nonincreasing, (3.2) k ∈C(0, ∞), lim t→0+k(t) = ∞, k > 0, k is nonincreasing, (3.2) ∃tk > 0 : k(t) is strictly decreasing in (0, tk). (3.3) ∃tk > 0 : k(t) is strictly decreasing in (0, tk). (3.3) (3.3) 241 Inverse Problems for a Generalized Subdiﬀusion Equation The assumptions (3.1) ensure the existence of a suﬃciently regular solution of the direct problem (see Lemma 3) and the assumptions (3.2), (3.3) are needed for the application of a positivity principle to this solution. The assumptions (3.1) ensure the existence of a suﬃciently regular solution of the direct problem (see Lemma 3) and the assumptions (3.2), (3.3) are needed for the application of a positivity principle to this solution. We mention that restricting generality a bit it is possible to reduce all conditions 1 and 2 to the single kernel M. Firstly, M ∈L1,loc(0, ∞) and (3.1) imply the existence of a unique solution k ∈L1,loc(0, ∞) of the equation k ∗M = 1 ( [10], Ch. 5, Corollary 5.6). Secondly, all properties (3.2), (3.3) follow from conditions that are a bit stronger than (3.1). It is shown in the following lemma. Proof is in Appendix. Lemma 1. Let M ∈L1,loc(0, ∞) satisfy (3.1) and M ′ < 0, log M - convex, log(−M ′) - convex. Then the solution of M ∗k = 1 satisﬁes (3.2), (3.3). The imposed assumptions on M and k hold for weakly singular completely monotonic kernels from CM={z ∈L1,loc(0, ∞) \ C∞(0, ∞) : lim t→0+z(t)=∞, (−1)iz(i) >0, i=0, 1, . . .}. CM={z ∈L1,loc(0, ∞) \ C∞(0, ∞) : lim t→0+z(t)=∞, (−1)iz(i) >0, i=0, 1, . . .} For M and k satisfying M ∗k = 1, it holds M ∈CM if and only if k ∈CM ( [9], Theorem 3). All l f M d k i i S i 2 b l CM For M and k satisfying M ∗k = 1, it holds M ∈CM if and only if k ∈CM ( [9], Theorem 3). All examples of M and k given in Section 2 belong to CM. 1 The symbol L stands for the space of linear and bounded operators. 4.1 Functional spaces Let X be a Banach space. Since k ∗M = 1, we have D{k} t (M ∗v) = d dtk ∗M ∗v = d dt1 ∗v = v, ∀v ∈L1((0, T); X), (4.1) (4.1) where L1((0, T); X) is the space of functions u : (0, T) →X that are integrable in the Bochner sense on (0, T). This means that the operator M∗is a one-to- one mapping from L1((0, T); X) to {M ∗v : v ∈L1((0, T); X)} and D{k} t is the inverse of M∗. where L1((0, T); X) is the space of functions u : (0, T) →X that are integrable in the Bochner sense on (0, T). This means that the operator M∗is a one-to- one mapping from L1((0, T); X) to {M ∗v : v ∈L1((0, T); X)} and D{k} t is the inverse of M∗. As usual, let C([0, T]; X) stand for the Banach space of functions u : [0, T] →X that are continuous on [0, T] with the norm ∥u∥C([0,T ];X) = max t∈[0,T ] ∥u(t)∥X and C0([0, T]; X) = {u ∈C([0, T]; X) : u(0) = 0}. Based on the relation (4.1), we introduce the functional space C{k} 0 ([0, T]; X) := M ∗C([0, T]; X) = {M ∗v : v ∈C([0, T]; X)}. It is a Banach space with the norm It is a Banach space with the norm ∥u∥C{k} 0 ([0,T ];X) = ∥D{k} t u∥C([0,T ];X). Since M∗∈L(C([0, T]; X), C0([0, T]; X)), it holds C{k} 0 ([0, T]; X) ,→C0([0, T]; X). Math. Model. Anal., 24(2):236–262, 2019. 242 N. Kinash and J. Janno We also deﬁne the space We also deﬁne the space C{k}([0, T]; X) := C{k} 0 ([0, T]; X) + X = {u : u(t) = u1(t) + u2, u1 ∈C{k} 0 ([0, T]; X), u2 ∈X} (4.2) (4.2) that is a Banach space with the norm that is a Banach space with the norm ∥u∥C{k}([0,T ];X) = ∥u −u(0)∥C{k} 0 ([0,T ];X) + ∥u(0)∥X. 4.1 Functional spaces Next we introduce the abstract H¨older spaces with corresponding norms Cα 0 ([0, T]; X) = n u ∈C0([0, T]; X) : n ∥u∥Cα 0 ([0,T ];X) := sup 0<t1<t2<T ∥u(t2) −u(t1)∥X (t2 −t1)α < ∞ o , Cα([0, T]; X) = Cα 0 ([0, T]; X) + X, n ∥u∥Cα 0 ([0,T ];X) := sup 0<t1<t2<T ∥u(t2) −u(t1)∥X (t2 −t1)α < ∞ o , Cα([0, T]; X) = Cα 0 ([0, T]; X) + X, ∥u∥Cα([0,T ];X) = ∥u −u(0)∥Cα 0 ([0,T ];X) + ∥u(0)∥X, ∥u∥Cα([0,T ];X) = ∥u −u(0)∥Cα 0 ([0,T ];X) + ∥u(0)∥X, where 0 < α < 1, and deﬁne the Banach spaces with norms where 0 < α < 1, and deﬁne the Banach spaces with norms C{k},α 0 ([0, T]; X) = M ∗Cα 0 ([0, T]; X), (4.3) ∥u∥C{k},α 0 ([0,T ];X) = ∥D{k} t u∥Cα 0 ([0,T ];X), C{k},α([0, T]; X) = M ∗Cα([0, T]; X) + X, ∥u∥C{k},α([0,T ];X) = ∥D{k} t (u −u(0))∥Cα([0,T ];X) + ∥u(0)∥X. (4.3) Let us establish some connections between the spaces (4.2), (4.3) and the usual C, C1- and H¨older spaces. For C{k}([0, T]; X) the embeddings C1([0, T]; X) ,→C{k}([0, T]; X) ,→C([0, T]; X) (4.4) (4.4) are valid. The right embedding follows from M∗∈L(C([0, T]; X))1 . To prove the left embedding, we choose some u ∈C1([0, T]; X). Then are valid. The right embedding follows from M∗∈L(C([0, T]; X))1 . To prove the left embedding, we choose some u ∈C1([0, T]; X). Then ∥u∥C{k}([0,T ];X) =∥u−u(0)∥C{k} 0 ([0,T ];X)+∥u(0)∥X =∥k∗u′∥C0([0,T ];X)+∥u(0)∥X are valid. The right embedding follows from M∗∈L(C([0, T]; X))1 . To prove the left embedding, we choose some u ∈C1([0, T]; X). Then ∥u∥C{k}([0,T ];X) =∥u−u(0)∥C{k} 0 ([0,T ];X)+∥u(0)∥X =∥k∗u′∥C0([0,T ];X)+∥u(0)∥X and since k∗∈L(C([0, T]; X), C0([0, T]; X)), the left relation in (4.4) follows. Analogous relations for the space C{k},α([0 T]; X) are ∥u∥C{k}([0,T ];X) =∥u−u(0)∥C{k} 0 ([0,T ];X)+∥u(0)∥X =∥k∗u′∥C0([0,T ];X)+∥u(0)∥X and since k∗∈L(C([0, T]; X), C0([0, T]; X)), the left relation in (4.4) follows. A l l ti f th C{k},α([0 T] X) ∥u∥C{k}([0,T ];X) =∥u−u(0)∥C{k} 0 ([0,T ];X)+∥u(0)∥X =∥k∗u′∥C0([0,T ];X)+∥u(0)∥X and since k∗∈L(C([0, T]; X), C0([0, T]; X)), the left relation in (4.4) follows. Analogous relations for the space C{k},α 0 ([0, T]; X) are nd since k∗∈L(C([0, T]; X), C0([0, T]; X)), the left relation in (4.4) follows. 4.1 Functional spaces {k} α nd since k∗∈L(C([0, T]; X), C0([0, T]; X)), the left relation in (4.4) follows. Analogous relations for the space C{k},α 0 ([0, T]; X) are ( ([ ] ) ([ ] )) ( ) Analogous relations for the space C{k},α 0 ([0, T]; X) are Analogous relations for the space C{k},α 0 ([0, T]; X) are C1+α 0 ([0, T]; X) ,→C{k},α 0 ([0, T]; X) ,→Cα 0 ([0, T]; X) (4.5) (4.5) where C1+α 0 ([0, T]; X) = {u : u, u′ ∈Cα 0 ([0, T]; X)}. The right embedding in (4.5) is a consequence of the fact that M∗∈ L(Cα 0 ([0, T]; X)) (see Lemma 4.2 in [14]) and the left embedding in (4.5) can be proved similarly to the left embedding in (4.4). The right embedding in (4.5) is a consequence of the fact that M∗∈ L(Cα 0 ([0, T]; X)) (see Lemma 4.2 in [14]) and the left embedding in (4.5) can be proved similarly to the left embedding in (4.4). Under additional assumptions on M it is possible to show that the operator M∗increases the order of H¨older continuity of a function. Namely, the following lemma is valid. Its proof is deferred to Appendix. 243 Inverse Problems for a Generalized Subdiﬀusion Equation Lemma 2. If M(t) ≤c1tβ−1, \|M ′(t)\| ≤c2tβ−2, t ∈(0, T) for some c1, c2 ∈ R+, 0 < β ≤α < 1 then M∗∈L(Cα−β 0 ([0, T]; X), Cα 0 ([0, T]; X)). Under conditions of Lemma 2, C{k},α−β 0 ([0, T]; X) ,→Cα 0 ([0, T]; X). In the particular case M(t) = tβ−1 Γ (β) (then M∗is the fractional integral of the order β), it holds the equality C{k},α−β 0 ([0, T]; X) = Cα 0 ([0, T]; X) [15]. By exchanging M and k in above relations, we obtain deﬁnitions and em- beddings of spaces that contain {M} instead of {k} in the superscript. 4.2 Abstract Cauchy problem Let A : D(A) →X be a linear densely deﬁned operator in a Banach space X. We say that A belongs to the class S(η, θ) for η ∈R, θ ∈(0, π) if Let A : D(A) →X be a linear densely deﬁned operator in a Banach space X. We say that A belongs to the class S(η, θ) for η ∈R, θ ∈(0, π) if ρ(A) ⊃Σ(η, θ) = {λ ∈C : λ ̸= η, arg\|λ −η\| < θ} and ∥(µ −A)−1∥L(X) ≤ C \|µ −η\| ∀µ ∈Σ(η, θ) for some constant C > 0. An operator A ∈S(η, θ) is closed. This implies that XA := D(A) is a Banach space with the graph norm ∥w∥XA = ∥w∥X + ∥Aw∥X. An operator A ∈S(η, θ) is closed. This implies that XA := D(A) is a Banach space with the graph norm ∥w∥XA = ∥w∥X + ∥Aw∥X. Obviously, S(η, θ1) ⊂S(η, θ2) for θ1 > θ2. Operators of the class S(η, θ), θ ∈ π 2 , π , are the sectorial operators that generate analytic semigroups. Now let us consider the Cauchy problem Now let us consider the Cauchy problem D{k} t (u −ϕ)(t) = Au(t) + F(t), t ∈[0, T], u(0) = ϕ, (4.6) with given F : [0, T] →X and ϕ ∈X. (4.6) Lemma 3. Let A ∈S(η, π 2 ) for some η ∈R. Then the following statements are valid. (i) (uniqueness) Let u ∈C{k}([0, T]; X) T C([0, T]; XA) solve (4.6) and ϕ = 0, F = 0. Then u = 0. (ii) Let F ∈Cα([0 T]; X) and ϕ = 0 Then (4 6) has a solution u in the space F 0. Then u 0. (ii) Let F ∈Cα 0 ([0, T]; X) and ϕ = 0. Then (4.6) has a solution u in the space C{k},α 0 ([0, T]; X) T Cα 0 ([0, T]; XA). This solution satisﬁes the estimate (ii) Let F ∈Cα 0 ([0, T]; X) and ϕ = 0. Then (4.6) has a solution u in the space C{k},α 0 ([0, T]; X) T Cα 0 ([0, T]; XA). This solution satisﬁes the estimate ii) Let F ∈Cα 0 ([0, T]; X) and ϕ = 0. Then (4.6) has a solution u in the space C{k},α 0 ([0, T]; X) T Cα 0 ([0, T]; XA). 4.2 Abstract Cauchy problem This proves the existence of the solution u ∈ C{k},α 0 ([0, T]; X) T Cα 0 ([0, T]; XA) of (4.6). The estimate (4.7) follows from the bounded inverse theorem. (iii) It is suﬃcient to prove this assertion in case F(t) ≡ξ ∈X, because the problem with given pair of data (F, ϕ) can be splitted into two problems with the data (F −F(0), 0) and (F(0), ϕ), respectively. For the ﬁrst problem, the assertion (ii) applies. Having proved (iii) for the second one, u is expressed as (ii) Theorem 3.3 (i) [28] implies that for F ∈Cα 0 ([0, T]; X) there exists a solution v ∈V α of (4.9). This proves the existence of the solution u ∈ C{k},α 0 ([0, T]; X) T Cα 0 ([0, T]; XA) of (4.6). The estimate (4.7) follows from the bounded inverse theorem. (iii) It is suﬃcient to prove this assertion in case F(t) ≡ξ ∈X, because the problem with given pair of data (F, ϕ) can be splitted into two problems with the data (F −F(0), 0) and (F(0), ϕ), respectively. For the ﬁrst problem, the assertion (ii) applies. Having proved (iii) for the second one, u is expressed as the sum of solutions of these two problems and satisﬁes (iii), too. Thus, let us assume that F(t) ≡ξ ∈X. Due to Proposition 1.2 (ii) [28], (4.9) has the solution v = S(ξ + Aϕ) ∈V . This implies the existence assertion of (iii). Due to the strong continuity of S(t) [28], ∥S(t)∥L(X) ≤C3, t ∈[0, T], where C3 is a constant. Thus, ∥v∥C([0,T ],X) ≤C3 (∥ξ∥X + ∥Aϕ∥X). Extracting the term A(M∗v) from (4.9) and estimating it we obtain ∥A(M∗v)∥C0([0,T ],X) ≤ (C3 + 1)(∥ξ∥X + ∥Aϕ∥X). Consequently, ∥u∥C{k}([0,T ];X) T C([0,T ];XA) = ∥v∥V + ∥ϕ∥XA ≤C4(∥ξ∥X + ∥ϕ∥XA) with a constant C4. This implies (4.8). ⊓⊔ ∥u∥C{k}([0,T ];X) T C([0,T ];XA) = ∥v∥V + ∥ϕ∥XA ≤C4(∥ξ∥X + ∥ϕ∥XA) with a constant C4. This implies (4.8). ⊓⊔ 4.2 Abstract Cauchy problem This solution satisﬁes the estimate ∥u∥C{k},α 0 ([0,T ];X) T Cα 0 ([0,T ];XA) ≤C1∥F∥Cα 0 ([0,T ];X). (4.7) (4.7) (iii) Let F ∈Cα([0, T]; X) and ϕ ∈XA. Then (4.6) has a solution u in the space C{k}([0, T]; X) T C([0, T]; XA). This solution satisﬁes the estimate ∥u∥C{k}([0,T ];X) T C([0,T ];XA) ≤C2(∥F∥Cα([0,T ];X) + ∥ϕ∥XA). (4.8) ∥u∥C{k}([0,T ];X) T C([0,T ];XA) ≤C2(∥F∥Cα([0,T ];X) + ∥ϕ∥XA). (4.8 (4.8) The constants C1 and C2 depend on M and A. The constants C1 and C2 depend on M and A. The constants C1 and C2 depend on M and A. Proof. The change of variable v = D{k} t (u −ϕ) ⇔u = M ∗v + ϕ reduces (4.6) of the integral equation v(t) = A(M ∗v)(t) + F(t) + Aϕ, t ∈[0, T]. (4.9) (4.9) Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. 244 N. Kinash and J. Janno Provided F ∈C([0, T]; X), ϕ ∈XA, the function u ∈C{k}([0, T]; X) T C([0, T]; XA) solves (4.6) if and only if v ∈V := {v ∈C([0, T]; X) : M ∗ v ∈C0([0, T]; XA)} solves (4.9). Similar one-to-one correspondence holds for u ∈C{k},α 0 ([0, T]; X) T Cα 0 ([0, T]; XA) and v ∈V α := {v ∈Cα 0 ([0, T]; X) : M ∗v ∈Cα 0 ([0, T]; XA)} in the particular case F ∈Cα 0 ([0, T]; X), ϕ = 0. Since M satisﬁes the conditions (3.1) and A ∈S(η, π 2 ), we can apply results of Ch. 3 of [28] to (4.9). Since M satisﬁes the conditions (3.1) and A ∈S(η, π 2 ), we can apply results of Ch. 3 of [28] to (4.9). (i) Theorem 3.2 with Corollary 1.1 and Proposition 1.2 in [28] implies that there exists a family of operators S : [0, ∞) →L(X) (called resolvent of (4.9)) so that a solution v ∈V (if it exists) is represented by the formula v = d dtS ∗F. By assumptions of (i), (4.9) has a solution v ∈V . Since F = 0, we have v = 0. Thus, u = 0. (ii) Theorem 3.3 (i) [28] implies that for F ∈Cα 0 ([0, T]; X) there exists a solution v ∈V α of (4.9). 4.3 Statements on direct problem Finally, let and satisﬁes the smoothness conditions u ∈C([0, T] × Ω), uxj ∈C((0, T] × Ω), u ∈C((0, T]; W 2 p (Ω)) for some p > n, L1u ∈C((0, T] × Ω), D{K} t (u −ϕ) ∈ C((0, T] × Ω). Finally, let lim ϵ→0+ 1 ϵ Z ϵ 0 K(τ)dτ sup 0≤s≤ϵ \|u(t−s, x)−u(t, x)\| = 0, ∀t ∈(0, T], x ∈Ω. (4.10) If ϕ ≥0, F ≥0 and Bu\|∂Ω≥0 then the following assertions are valid. (i) u ≥0; (ii) if u(t0, x0) = 0 in some point (t0, x0) ∈(0, T] × ΩN, where If ϕ ≥0, F ≥0 and Bu\|∂Ω≥0 then the following assertions are valid. (i) u ≥0; (ii) if u(t0, x0) = 0 in some point (t0, x0) ∈(0, T] × ΩN, where \| (i) u ≥0; (ii) if u(t0, x0) = 0 in some point (t0, x0) ∈(0, T] × ΩN, where (i) u ≥0; (ii) if u(t0, x0) = 0 in some point (t0, x0) ∈(0, T] × ΩN, where ΩN = Ω in case B = I Ω in case B = ω · ∇, then u(t, x0) = 0 for any t ∈[0, t0]. then u(t, x0) = 0 for any t ∈[0, t0]. This lemma is a slight modiﬁcation of a positivity principle that was proved in [15] for a semilinear equation in case of a more smooth solution u ∈ C((0, T]; C2(Ω)) and strictly decreasing in (0, T) kernel K. To prove Lemma 4, we need the following auxiliary result. It is proved in Appendix of the paper. Lemma 5. Let w ∈W 2 p (Ω) for some p>n, L1w ∈C(Ω) and x∗=argmin x∈Ω w(x). In case x∗∈∂Ωwe also assume that (ω · ∇w)(x∗) ≥0. Then L1w(x∗) ≥0. Lemma 5. Let w ∈W 2 p (Ω) for some p>n, L1w ∈C(Ω) and x∗=argmin x∈Ω w(x). In case x∗∈∂Ωwe also assume that (ω · ∇w)(x∗) ≥0. Then L1w(x∗) ≥0. Lemma 5. Let w ∈W 2 p (Ω) for some p>n, L1w ∈C(Ω) and x∗=argmin x∈Ω w(x). In case x∗∈∂Ωwe also assume that (ω · ∇w)(x∗) ≥0. Then L1w(x∗) ≥0. Proof of Lemma 4. Without a restriction of generality we assume that r ≤0. Otherwise it is possible to deﬁne ˜u = e−σtu as in [15] and to consider the corresponding problem for ˜u. 4.3 Statements on direct problem In order to apply Lemma 3 to the direct problem (2.6), we must introduce appropriate Banach spaces of x-dependent functions and deﬁne realizations of the operator L in these spaces so that they belong to S η, π 2 . Let us introduce the following spaces and operators: 1. Xp = Lp(Ω), 1 < p < ∞, Ap : XAp →Xp with XAp = {z ∈W 2 p (Ω) : Bz\|∂Ω= 0} and Apz = Lz, z ∈XAp. 1. Xp = Lp(Ω), 1 < p < ∞, Ap : XAp →Xp with XAp = {z ∈W 2 p (Ω) : Bz\|∂Ω= 0} and Apz = Lz, z ∈XAp. 2. X0 = ( C0(Ω) = {z ∈C(Ω) : z\|∂Ω= 0} in case B = I, C(Ω) in case B = ω · ∇, A0 : XA0 →X0 with XA0 = {z ∈ T 1<p<∞ W 2 p (Ω) : Bz\|∂Ω= 0, L and A0z = Lz, z ∈XA0. 2. X0 = ( C0(Ω) = {z ∈C(Ω) : z\|∂Ω= 0} in case B = I, C(Ω) in case B = ω · ∇, A0 : XA0 →X0 with XA0 = {z ∈ T 1<p<∞ W 2 p (Ω) : Bz\|∂Ω= 0, Lz ∈X0} and A0z = Lz, z ∈XA0. 245 Inverse Problems for a Generalized Subdiﬀusion Equation Corollary 1. Operators Ap, p ∈{0} S(1, ∞), are sectorial. Thus, Lemma 3 holds in cases X = Xp, A = Ap, p ∈{0} S(1, ∞) and applies to problem (2.6). Corollary 1. Operators Ap, p ∈{0} S(1, ∞), are sectorial. Thus, Lemma 3 holds in cases X = Xp, A = Ap, p ∈{0} S(1, ∞) and applies to problem (2.6). Proof. It follows from Theorems 3.1.2, 3.1.3 and Corollary 3.1.24 (ii) in [24]. ⊓⊔ Lemma 4. Let K ∈L1(0, T) T C1(0, T), lim t→0+K(t) = ∞, K > 0, K be non- increasing and ∃tK > 0 : K is strictly decreasing in (0, tK). Moreover, let F ∈C([0, T] × Ω). Assume that u solves the problem D{K} t (u −ϕ)(t, x) = Lu(t, x) + F(t, x), t ∈(0, T), x ∈Ω, u(0, x) = ϕ, x ∈Ω and satisﬁes the smoothness conditions u ∈C([0, T] × Ω), uxj ∈C((0, T] × Ω), u ∈C((0, T]; W 2 p (Ω)) for some p > n, L1u ∈C((0, T] × Ω), D{K} t (u −ϕ) ∈ C((0, T] × Ω). 4.3 Statements on direct problem Such a problem also satisﬁes the assumptions of Lemma 4 and has the coeﬃcient ˜r = r −σ R T 0 e−σsK(s)ds in place of r. Since lim t→0+ K(t) = ∞, for suﬃciently large σ, ˜r ≤0. Let us suppose that (i) does not hold. Then there exists (t1, x1) ∈(0, T]×Ω such that u(t1, x1) < 0 and (t1, x1) = argmin x∈Ω,t∈[0,T ] u(t, x). It was shown in [15] (formula (37)) that the assumptions D{K} t (u −ϕ) ∈C((0, T] × Ω), (4.10), Math. Model. Anal., 24(2):236–262, 2019. 246 N. Kinash and J. Janno K > 0 and K – nonincreasing together with the relations u(t, x1) ≥u(t1, x1) and u(t1, x1) < 0 imply D{K} t (u −ϕ)(t1, x1) < 0. On the other hand, Lemma 5 applies to the function w = u(t1, ·) at x∗= x1. We obtain L1u(t1, x1) ≥0. Also r(x1)u(t1, x1) ≥0 and F ≥0. Thus, the left-hand side of the equation D{K} t (u −ϕ)(t1, x1) = [Lu + F](t1, x1) is negative, but the right-hand side is nonnegative. We have reached a contradiction. The assertion (i) is valid. Let us prove (ii). Let u(t0, x0) = 0 at (t0, x0) ∈(0, T] × ΩN. Deﬁne ˆt0 = inf t : t ≤t0, u(τ, x0) = 0 for τ ∈[t, t0] . If (ii) is not valid, then ˆt0 > 0 and u(t, x0) ≥δ, t ∈(t2, t3) for some δ > 0 and (t2, t3) ⊂(0, ˆt0) such that ˆt0 −t2 < tK. Then, similarly to the proof in [15] p.138, from the assumptions D{K} t (u −ϕ) ∈C((0, T] × Ω), (4.10), K > 0, K – nonincreasing and relations u ≥0, u(t, x0) ≥δ > 0, t ∈(t2, t3), we derive D{K} t (u −ϕ)(ˆt0, x0) ≤δ(K(ˆt0 −t2) −K(ˆt0 −t3)). (4.11) (4.11) Since 0 < ˆt0 −t3 < ˆt0 −t2 < tK and K is strictly decreasing in (0, tK), (4.11) implies D{K} t (u −ϕ)(ˆt0, x0) < 0. On the other hand, from u(ˆt0, x0) = 0 and u(t, x) ≥0, (t, x) ∈(0, T] × Ω, we conclude that (ˆt0, x0) = argmin x∈Ω u(ˆt0, x). By Lemma 5, L1u(ˆt0, x0) ≥0. Moreover, (ru)(ˆt0, x0) = 0 and F ≥0. Left- hand side of the equation D{K} t (u −ϕ)(ˆt0, x0) = [Lu + F](ˆt0, x0) is negative, but right-hand side is nonnegative. 4.3 Statements on direct problem = 0, ∀t ∈(0, T], x ∈Ω. = 0, ∀t ∈(0, T], x ∈Ω. The case (a2). Again, by Lemma 3 (ii), u ∈C{k},α 0 ([0, T]; X0) and by (4.5), u ∈Cα 0 ([0, T]; X0). The relation (4.10) follows from the estimate lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ sup 0≤s≤ϵ \|u(t −s, x) −u(t, x)\| = lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ · O(ϵα) ≤lim ϵ→0+ O(ϵα) ϵM(ϵ) Z ϵ 0 M(ϵ −τ)k(τ)dτ = lim ϵ→0+ O(ϵα−γ) = 0 ∀t ∈(0, T], x ∈Ω lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ sup 0≤s≤ϵ \|u(t −s, x) −u(t, x)\| = lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ · O(ϵα) ≤lim ϵ→0+ O(ϵα) ϵM(ϵ) Z ϵ 0 M(ϵ −τ)k(τ)dτ = lim ϵ→0+ O(ϵα−γ) = 0 ∀t ∈(0, T], x ∈Ω. The case (a3). According to Lemma 3 (ii), F ∈Cα−β 0 ([0, T]; X0) implies that u ∈C{k},α−β 0 ([0, T]; X0) = M ∗Cα−β 0 ([0, T]; X0). By Lemma 2 it holds u ∈Cα 0 ([0, T]; X0). This enables us ﬁnish the proof as in case (a2). ⊓⊔ 5 Results on IP1 We will study IP1 in context of H¨older spaces with respect to t. For the sake of generality, we will assume diﬀerent orders of spaces related to g and h: for g we use α1 and for h we use α2. Theorem 1. Let one of the following assumptions be valid: 1 4.3 Statements on direct problem Again, we have reached the contradiction. Thus, (ii) holds. 2 2 At this point we present somewhat more concrete assumptions on the input data of the direct problem (2.6) that imply the assumptions of Lemma 4 and Lemma 3. Corollary 2. Let F ≥0, ϕ = 0 and one of the assumptions (a1)–(a3) hold: (a1) F ∈C{M},α([0, T]; X0) for some 0 < α < 1 and F(0, ·) = 0; (a3) F ∈Cα−β 0 ([0, T]; X0) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤c3tβ−2 t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α < 1. (a3) F ∈Cα−β 0 ([0, T]; X0) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤c3tβ−2, t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α < 1. (a3) F ∈Cα−β 0 ([0, T]; X0) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤c3tβ−2, t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α < 1. Then assertions Lemma 4 are satisﬁed by solution of the problem (2.6). Proof. Deﬁning X = X0, Lemma 3 with Corollary 1 implies that the solution of (2.6) exists and satisﬁes the smoothness conditions of Lemma 4. It remains to show that (4.10) holds. The case (a1). The relations F ∈C{M},α([0, T]; X0), F(0, ·) = 0 mean that F = k ∗ˆF, where ˆF ∈Cα([0, T]; X0). Thus, it follows from Lemma 3 that the function ˆu that solves (2.6) with F, ϕ replaced by ˆF, ˆϕ = 0 belongs to the space C{k}([0, T]; X0). Next, after convolving equation for ˆu with k it is easy to see that u = k ∗ˆu solves (2.6) with F = k ∗ˆF. Therefore, u ∈k ∗C{k}([0, T]; X0), that is u = k ∗M ∗v = 1 ∗v, v ∈C([0, T]; X0). This allows us to conclude that Inverse Problems for a Generalized Subdiﬀusion Equation Inverse Problems for a Generalized Subdiﬀusion Equation 247 u ∈C1([0, T]; X0). Hence, u ∈C1([0, T]; X0). Hence, lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ sup 0≤s≤ϵ \|u(t −s, x) −u(t, x)\| = lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ · O(ϵ) lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ sup 0≤s≤ϵ \|u(t −s, x) −u(t, x)\| = lim ϵ→0+ 1 ϵ Z ϵ 0 k(τ)dτ · O(ϵ) = 0, ∀t ∈(0, T], x ∈Ω. Theorem 1. Let one of the following assumptions be valid: (A1) g ∈C1+α1 0 ([0, T]; C(Ω)) for some 0 < α1 < 1; { } (A1) g ∈C + 1 0 ([0, T]; C(Ω)) for some 0 < α1 < 1; (A2) g ∈C{k},α1 0 ([0, T]; C(Ω)) and M(t) ≥ctγ−1, t ∈(0, T) for some c ∈R+, 0 < γ < α1 < 1; (A2) g ∈C{k},α1 0 ([0, T]; C(Ω)) and M(t) ≥ctγ−1, t ∈(0, T) for some c ∈R+ 0 < γ < α1 < 1; 0 < γ < α1 < ; (A3) g ∈C{k},α1−β 0 ([0, T]; C(Ω)) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤ c3tβ−2, t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α1 < 1. Additionally, we assume that g ≥0, g1 := D{k} t g−Rg ≥0 where R := max x∈Ω r(x) and and a.e. x ∈Ω ∃tx ∈(0, T] : g(tx, x) > 0. (5.1) (5.1) In case B = I we also assume that ∀x ∈∂Ω, either g(T, x) > 0 or g(·, x) = 0. Finally, let (f, u) ∈C(Ω) × C{k} 0 ([0, T]; C(Ω)) T C0([0, T]; W 2 p (Ω)) for some p > 1 solve IP1 for ϕ = 0, ψ = 0, h = 0. Then (f, u) = (0, 0). In case B = I we also assume that ∀x ∈∂Ω, either g(T, x) > 0 or g(·, x) = 0 In case B = I we also assume that ∀x ∈∂Ω, either g(T, x) > 0 or g(·, x) = 0. Finally, let (f, u) ∈C(Ω) × C{k} 0 ([0, T]; C(Ω)) T C0([0, T]; W 2 p (Ω)) for In case B = I we also assume that ∀x ∈∂Ω, either g(T, x) > 0 or g(·, x) = 0. Finally, let (f, u) ∈C(Ω) × C{k} 0 ([0, T]; C(Ω)) T C0([0, T]; W 2 p (Ω)) for some p > 1 solve IP1 for ϕ = 0, ψ = 0, h = 0. Then (f, u) = (0, 0). In case B = I we also assume that ∀x ∈∂Ω, either g(T, x) > 0 or g(·, x) = 0. Theorem 1. Let one of the following assumptions be valid: Finally, let (f, u) ∈C(Ω) × C{k} 0 ([0, T]; C(Ω)) T C0([0, T]; W 2 p (Ω)) for some p > 1 solve IP1 for ϕ = 0, ψ = 0, h = 0. Then (f, u) = (0, 0). Proof. We start the proof by showing that in case B = I, for any x ∈∂Ωsuch that g(T, x) > 0, the equality f(x) = 0 is valid. To show this, we consider the equality D{k} t u(T, x) = f(x)g(T, x), x ∈Ω, that follows from equation (2.6) in view of ψ = 0. If x ∈∂Ωand B = I then the left-hand side of this equality equals zero. Thus, f(x)g(T, x) = 0 and provided g(T, x) > 0 we obtain f(x) = 0. Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. 248 N Kinash and J Janno 248 N. Kinash and J. Janno Let us introduce the functions f + = \|f\|−f 2 and f −= \|f\|+f 2 . Due to the deﬁnition, f ± ∈C(Ω) and f ± ≥0. Moreover, in case B = I, for any x ∈∂Ωsuch that g(T, x) > 0, it holds f ±(x) = 0. (5.2) Firstly, we consider the problems D{k} t u±(t, x) = Lu±(t, x) + g(t, x)f ±(x), x ∈Ω, t ∈(0, T), u±(0, x) = 0, x ∈Ω, Bu±(t, x) = 0, x ∈∂Ω, t ∈(0, T). (5.3) (5.3) By assumptions of the theorem and (5.2), g(t, ·)f ± ∈X0, t ∈[0, T]. There- fore, in cases (A1) and (A2) due to (4.5) we have gf ± ∈C{M},α1 0 ([0, T]; X0) and gf ± ∈Cα1 0 ([0, T]; X0), respectively. Similarly, in case (A3) due to (4.5) and Lemma 2 we obtain gf ± ∈Cα1 0 ([0, T]; X0). Moreover, gf ± ≥0. The assumptions of Corollary 2 are satisﬁed for the functions F = gf ±. Hence, the solutions u± of (5.3) satisfy the assertions of Lemma 4. ( ) Secondly, let us consider the problems D{k} t v±(t, x) = Lv±(t, x) + g1(t, x)f ±(x), x ∈Ω, t ∈(0, T), v±(0, x) = 0, x ∈Ω, Bv±(t, x) = 0, x ∈∂Ω, t ∈(0, T). (5.4) (5.4) In case (A1) we have g′ ∈Cα1 0 ([0, T]; C(Ω)). Thus, g1 = D{k} t g −Rg = k ∗g′ −Rg ∈C{M},α1 0 ([0, T]; C(Ω)). Theorem 1. Let one of the following assumptions be valid: From g(t, ·)f ± ∈X0, t ∈[0, T] we imme- diately get g1(t, ·)f ± ∈X0, t ∈[0, T]. Therefore, g1f ± ∈C{M},α1 0 ([0, T]; X0). Using similar reasoning, we deduce g1f ± ∈Cα1 0 ([0, T]; X0) and g1f ± ∈ Cα1−β 0 ([0, T]; X0) in cases (A2) and (A3), respectively. Moreover, g1f ± ≥0. Again, the assumptions of Corollary 2 are satisﬁed for F = g1f ±. The solutions v± of (5.4) satisfy the assertions of Lemma 4. ± Let us point out that the problem for M ∗v± is equivalent to the problem for u± −RM ∗u±. Thus, v± = D{k} t u± −Ru±. (5.5) (5.5) Moreover, since f = f + −f −, we have u = u+ −u−. Thus, ψ = u(T, ·) = 0 implies that u+(T, ·) = u−(T, ·). Let us denote x∗= argmax ∈Ω u+(T, x) = Moreover, since f = f + −f −, we have u = u+ −u−. Thus, ψ = u(T, ·) = 0 implies that u+(T, ·) = u−(T, ·). Let us denote x∗= argmax x∈Ω u+(T, x) = argmax x∈Ω u−(T, x). By deﬁnition, either f +(x∗) = 0 or f −(x∗) = 0. Let us assume that f +(x∗) = 0 (the situation when f −(x∗) = 0 can be considered in a similar manner). Let us suppose that either x∗∈Ωor B = ω · ∇(the case x∗∈∂Ωand B = I will be considered later separately). Then we can apply Lemma 5 to the function w = −u+(T, ·). We get L1u+(T, x∗) ≤0. Thus, from (5.3), (5.5) and u+ ≥0, r ≤R it follows: v+(T, x∗) = L1u+(T, x∗) + (r(x+) −R)u+(T, x∗) ≤0. (5.6) to Lemma 4 (i), (5.6) Due to Lemma 4 (i), v+(t, x) ≥0, (t, x) ∈(0, T) × Ω. (5.7) (5.7) Inverse Problems for a Generalized Subdiﬀusion Equation 249 Hence, (5.6) and (5.7) imply v+(T, x∗) = 0. Thus, by Lemma 4 (ii), v+(t, x∗) = 0, t ∈[0, T]. By formula (5.5) it means D{k} t u+(t, x∗) −Ru+(t, x∗) = 0, t ∈ [0, T]. Applying M∗to to this equality, we obtain the following homogeneous Volterra equation of the second kind: u+(t, x∗) −RM ∗u+(t, x∗) = 0, t ∈[0, T]. It has only the trivial solution u+(t, x∗) = 0, t ∈[0, T]. Hence, u+(T, x∗) = 0. Math. Model. Anal., 24(2):236–262, 2019. Theorem 1. Let one of the following assumptions be valid: Since x∗is a maximum point of u+(T, x) and u+(T, x) ≥0, we also get It has only the trivial solution u+(t, x∗) = 0, t ∈[0, T]. Hence, u+(T, x∗) = 0. Since x∗is a maximum point of u+(T, x) and u+(T, x) ≥0, we also get u+(T, x) = 0, x ∈Ω. (5.8) (5.8) Now we consider the case x∗∈∂Ω, B = I, too. Then by Bu+\|∂Ω= 0, immediately u+(T, x∗) = 0 and again we have (5.8). Now we consider the case x∗∈∂Ω, B = I, too. Then by Bu+\|∂Ω= 0, immediately u+(T, x∗) = 0 and again we have (5.8). Since u = u+−u−and ψ = u(T, ·) = 0 holds, from (5.8) we get u±(T, x) = 0, x ∈Ω. Lemma 4 (ii) implies u±(t, x) = 0, (t, x) ∈[0, T] × Ω. Therefore, u(t, x) = 0, (t, x) ∈[0, T] × Ω. From the diﬀerential equation for u we obtain f(x)g(t, x) = 0, (t, x) ∈[0, T] × Ω. Finally, (5.1) yields f = 0. ⊓⊔ Next we provide simple suﬃcient conditions that imply the assumption D{k} t g −Rg ≥0 in Theorem 1. For this we need the following lemma. Lemma 6. Let w ∈C{k}([0, T]; R) be nonnegative and nonincreasing. Then D{k} t w ≥k(T)w. Proof. The assertion follows from the estimate D{k} t w(t) = lim δ→0+ 1 δ hZ t+δ t k(τ)w(t+δ−τ)dτ + Z t 0 k(τ)(w(t+δ−τ) −w(t−τ))dτ i ≥lim δ→0+ k(T +δ)1 δ hZ t+δ t w(t+δ−τ)dτ + Z t 0 (w(t+δ−τ) −w(t−τ))dτ i = k(T)w(t), 0 < t < T. Due to that Lemma 6, D{k} t g −Rg ≥0 holds provided along with other assumptions on g in Theorem 1, g is nondecreasing in t and k(T) ≥R in case R > 0. Theorem 2. Let g, M satisfy the assumptions of Theorem 1 and the inequality g(T, x) > 0, x ∈Ω, hold. If ϕ, ψ ∈XAp and h ∈Cα2([0, T]; Xp), where p ∈{0} S(1, ∞), 0 < α2 < 1, then IP1 has a unique solution (f, u) ∈Xp × C{k}([0, T]; Xp) T C([0, T]; XAp) and the following estimate holds: ∥f∥Xp + ∥u∥C{k}([0,T ];Xp) T C([0,T ];XAp) ≤C5 ∥ϕ∥XAp + ∥ψ∥XAp + ∥h∥Cα2([0,T ];Xp) . (5.9) (5.9) Math. Model. Anal., 24(2):236–262, 2019. 250 N. Kinash and J. Theorem 1. Let one of the following assumptions be valid: Janno If additionally ϕ = h(0, ·) = 0, then u ∈C{k},α 0 ([0, T]; Xp) T Cα 0 ([0, T]; XAp) where α = α2, in case (A1), min{α1, α2}, in cases (A2), (A3) and the estimate If additionally ϕ = h(0, ·) = 0, then u ∈C{k},α 0 ([0, T]; Xp) T Cα 0 ([0, T]; XAp) where α = α2, in case (A1), min{α1, α2}, in cases (A2), (A3) and the estimate ∥f∥Xp+∥u∥C{k},α 0 ([0,T ];Xp)TCα 0 ([0,T ];XAp)≤C6 ∥ψ∥XAp+∥h∥Cα2 0 ([0,T ];Xp) (5.10) ∥f∥Xp+∥u∥C{k},α 0 ([0,T ];Xp)TCα 0 ([0,T ];XAp)≤C6 ∥ψ∥XAp+∥h∥Cα2 0 ([0,T ];Xp) (5.10 is valid. The constants C5 and C6 depend on the parameters M, L, g, p, α2. Proof. Firstly, we are going to replace the overdetermination condition (2.9) by a ﬁxed-point equation with respect to f. { } Suppose that (f, u) ∈Xp × C{k}([0, T]; Xp) T C([0, T]; XAp) solves IP1. Then, since (2.9) holds, the equation (2.6) at t = T with F = fg + h yields f(x) = D{k} t (u −ϕ) −ηu (T, x) −(Ap −η)ψ(x) −h(T, x) g(T, x) , (5.11) (5.11) where η is chosen so that 0 ∈ρ(Ap −ηI). where η is chosen so that 0 ∈ρ(Ap −ηI). where η is chosen so that 0 ∈ρ(Ap −ηI). where η is chosen so that 0 ∈ρ(Ap −ηI). Let us split u into the sum of two functions: u = u1 + u2, such that D{k} t u1 = Apu1 + fg, u1(0, ·) = 0, D{k} t (u2 −ϕ) = Apu2 + h, u2(0, ·) = ϕ. (5.12) (5.12) In the context of IP1, u2 is a known function. According to Lemma 3, the solution to (5.12) belongs to u2 ∈C{k}([0, T]; Xp). Thus, v2 := D{k} t (u2 −ϕ)− ηu2 ∈C([0, T]; Xp). Next we formulate the following problem: In the context of IP1, u2 is a known function. According to Lemma 3, the solution to (5.12) belongs to u2 ∈C{k}([0, T]; Xp). Thus, v2 := D{k} t (u2 −ϕ)− ηu2 ∈C([0, T]; Xp). Next we formulate the following problem: D{k} t v1 = Apv1 + f(D{k} t g −ηg), v1(0, ·) = 0. Theorem 1. Let one of the following assumptions be valid: To- gether with (2.6) at t = T and (2.10) it implies (Ap −η)u(T, x) = (Ap −η)ψ(x). Since (Ap −η) is injective, it yields (2.9). Consequently, IP1 is in the space Xp × C{k}([0, T]; Xp) T C([0, T]; XAp) equivalent to the problem of ﬁnding the pair of functions (f, u) that solves (2.6), (2.10), (5.15). We point out that (5.15) is an independent equation for the ﬁrst component f of the solution of IP1. Let us analyse properties of the operator F involved in this equation. By Lemma 3, v1[·] ∈L(Xp; C ˆα 0 ([0, T]; XAp)). Thus, v1[·](T, ·) ∈ L(Xp, XAp). Furthermore, XAp ,→,→Xp. In case p ∈(1, ∞) it is a direct consequence of W 2 p (Ω) ,→,→Lp(Ω). In case p = 0 it follows from the continuous embedding of XA0 in C1 B(Ω) := X0 T C1(Ω) (see Theorems 3.1.19, 3.1.22 in [24]) and C1 B(Ω) ,→,→X0. Furthermore, XAp ,→,→Xp. In case p ∈(1, ∞) it is a direct consequence of W 2 p (Ω) ,→,→Lp(Ω). In case p = 0 it follows from the continuous embedding of XA0 in C1 B(Ω) := X0 T C1(Ω) (see Theorems 3.1.19, 3.1.22 in [24]) and C1 B(Ω) ,→,→X0. Therefore, v1[·](T, ·) : Xp →Xp is compact. Since 1 g(T,·) ∈C(Ω) due to the assumptions of this theorem, F : Xp →Xp is also compact. Therefore, v1[·](T, ·) : Xp →Xp is compact. Since 1 g(T,·) ∈C(Ω) due to the assumptions of this theorem, F : Xp →Xp is also compact. Next, let us show that 1 /∈σ(F). Firstly, let us consider the case p = 0. Suppose that 1 ∈σ(F). Then the equation f = Ff has a solution f ∈X0, f ̸= 0. This means that the problem (2.6), (2.10), (5.15) with homogeneous data ϕ = 0, ψ = 0, h = 0 has the nontrivial solution (f, u1) in the space X0 × C{k} 0 ([0, T]; X0) T C0([0, T]; XA0). But due to the Theorem 1, IP1 with a homogeneous data has only the trivial solution in such a space. We came to a contradiction. Consequently, 1 /∈σ(F). Secondly, let us consider the case p ∈(1, ∞). We again suppose that 1 ∈σ(F), hence the equation f = Ff has a nontrivial solution f ∈Xp. The idea is to show that this solution actually belongs to X0. ∥f∥Xp ≤∥(I −F)−1∥L(Xp)∥G∥Xp ≤bC ∥h(T, ·)∥Xp + \|η\|∥ψ∥Xp + ∥ψ∥XAp Theorem 1. Let one of the following assumptions be valid: (5.13) (5.13) Due to the assumptions (A1)–(A3) and (4.5), it holds D{k} t g ∈C ˆα 0 ([0, T]; C(Ω)) where Due to the assumptions (A1)–(A3) and (4.5), it holds D{k} t g ∈C ˆα 0 ([0, T]; C(Ω) where ˆα = α1, in cases (A1), (A2), α1 −β, in case (A3). (5.14) (5.14) Thus, f(D{k} t g −ηg) ∈C ˆα 0 ([0, T]; Xp). According to Lemma 3, (5.13) has a solution v1 in C{k},ˆα 0 ([0, T]; Xp) T C ˆα 0 ([0, T]; XAp). It is easy to check that v1 = D{k} t u1 −ηu1. Thus, f(D{k} t g −ηg) ∈C ˆα 0 ([0, T]; Xp). According to Lemma 3, (5.13) has a solution v1 in C{k},ˆα 0 ([0, T]; Xp) T C ˆα 0 ([0, T]; XAp). It is easy to check that v1 = D{k} t u1 −ηu1. hus, f(D{k} t g −ηg) ∈C ˆα 0 ([0, T]; Xp). According to Lemma 3, (5.13) has solution v1 in C{k},ˆα 0 ([0, T]; Xp) T C ˆα 0 ([0, T]; XAp). It is easy to check that 1 = D{k} t u1 −ηu1. The notations introduced allow us to rewrite (5.11) in the form f = Ff + G, (5.15) (5.15) where where G(x) = v2(T, x) −(Ap −η)ψ(x) −h(T, x) g(T, x) , x ∈Ω, (5.16) (Ff) (x) = v1[f](T, x)/g(T, x) (5.17) G(x) = v2(T, x) −(Ap −η)ψ(x) −h(T, x) g(T, x) , x ∈Ω, (5.16) (Ff) (x) = v1[f](T, x)/g(T, x) (5.17) (5.17) Inverse Problems for a Generalized Subdiﬀusion Equation 251 and v1[·] stands for the operator that assigns to f the solution v1 of (5.13). Thus, (2.6), (2.9), (2.10) imply (5.15). On the other hand, taking into account all the substitutions performed, we can move back from (5.15) to (5.11). To- gether with (2.6) at t = T and (2.10) it implies (Ap −η)u(T, x) = (Ap −η)ψ(x). Since (Ap −η) is injective, it yields (2.9). Consequently, IP1 is in the space Xp × C{k}([0, T]; Xp) T C([0, T]; XAp) equivalent to the problem of ﬁnding the pair of functions (f, u) that solves (2.6), (2.10), (5.15). and v1[·] stands for the operator that assigns to f the solution v1 of (5.13). Thus, (2.6), (2.9), (2.10) imply (5.15). On the other hand, taking into account all the substitutions performed, we can move back from (5.15) to (5.11). Math. Model. Anal., 24(2):236–262, 2019. Theorem 1. Let one of the following assumptions be valid: In case ϕ = h(0, ·) = 0, the solution of (2.6), (2.10) belongs to the space C{k},α 0 ([0, T]; Xp) T Cα 0 ([0, T]; XAp) and can be estimated as In case ϕ = h(0, ·) = 0, the solution of (2.6), (2.10) belo C{k},α 0 ([0, T]; Xp) T Cα 0 ([0, T]; XAp) and can be estimated as ∥u∥C{k},α 0 ([0,T ];Xp)TCα 0 ([0,T ];XAp) ≤bC(∥f∥Xp∥g∥Cγ 0 ([0,T ];C(Ω))+∥h∥Cα2 0 ([0,T ];Xp)). This with (5.18) implies (5.10). ⊓⊔ This with (5.18) implies (5.10). ⊓⊔ We point out that in case p = 0 and B = I, the assumptions of Theorem 2 allow to recover f ∈X0 = C0(Ω) only. In order to ﬁx that in the following theorem we provide some additional conditions that are suﬃcient to restore f ∈C(Ω) in case B = I. Theorem 3. Let g, M satisfy the assumptions of Theorem 2. If ϕ, ψ, Lϕ ∈ XAp for some p > n 2 , Lψ ∈C(Ω), h ∈C{k},α2([0, T]; Xp) T C([0, T]; C(Ω)), where 0 < α2 < 1 and h(0, ·) ∈XAp then IP1 has a unique solution (f, u) ∈ C(Ω) × C{k}([0, T]; XAp). Moreover, Lu ∈C([0, T]; C(Ω)) and the estimate ∥f∥C(Ω)+∥u∥C{k}([0,T ];XAp)+∥Lu∥C([0,T ];C(Ω)) ≤C7 ∥ϕ∥Xp +∥Lϕ∥XAp +∥ψ∥Xp +∥Lψ∥C(Ω)+∥h∥C{k},α2([0,T ];Xp) T C([0,T ];C(Ω))+∥h(0, ·)∥XAp (5.19) holds. If additionally ϕ=h(0, ·)=D{k} t h(0, ·)=0, then u∈C{k},α′ 0 ([0, T]; XAp) and the estimate ∥f∥C(Ω)+∥u∥C{k}([0,T ];XAp)+∥Lu∥C([0,T ];C(Ω)) ≤C7 ∥ϕ∥Xp +∥Lϕ∥XAp ∥f∥C(Ω)+∥u∥C{k}([0,T ];XAp)+∥Lu∥C([0,T ];C(Ω)) ≤C7 ∥ϕ∥Xp +∥Lϕ∥XAp +∥ψ∥Xp +∥Lψ∥C(Ω)+∥h∥C{k},α2([0,T ];Xp) T C([0,T ];C(Ω))+∥h(0, ·)∥XAp (5.19) holds. If additionally ϕ=h(0, ·)=D{k} t h(0, ·)=0, then u∈C{k},α′ 0 ([0, T]; XAp) and the estimate ∥f∥C(Ω) + ∥u∥C{k},α′ 0 ([0,T ];XAp) + ∥Lu∥C0([0,T ];C(Ω)) ∥f∥C(Ω) + ∥u∥C{k},α′ 0 ([0,T ];XAp) + ∥Lu∥C0([0,T ];C(Ω)) ∥f∥C(Ω) + ∥u∥C{k},α′ 0 ([0,T ];XAp) + ∥Lu∥C0([0,T ];C(Ω)) ≤C8 ∥ψ∥Xp + ∥Lψ∥C(Ω) + ∥h∥C{k},α2 0 ([0,T ];Xp) T C0([0,T ];C(Ω)) (5.20) ≤C8 ∥ψ∥Xp + ∥Lψ∥C(Ω) + ∥h∥C{k},α2 0 ([0,T ];Xp) T C0([0,T ];C(Ω)) (5.20) (5.20) is valid where α′ = min{ˆα; α2} and ˆα is given by (5.14). The constants C7 and C8 depend on M, L, g, p, α2. is valid where α′ = min{ˆα; α2} and ˆα is given by (5.14). The constants C7 and C8 depend on M, L, g, p, α2. Proof. Theorem 1. Let one of the following assumptions be valid: Then we can apply the arguments from the previous case to show that 1 ∈σ(F) leads to a contradiction. If p > n 2 , then v1[f](T, ·) ∈XAp ,→X0. Thus, f = Ff = 1 g(T,x)v1[f](T, ·) ∈ X0. If p ≤n 2 , then according to embedding theorems, XAp ,→Xp1 = Lp1(Ω), where p1 = np n−2p > p. Therefore, v1[f](T, ·) ∈Xp1 and f = Ff = 1 g(T,x)v1[f](T, ·) ∈Xp1. After a ﬁnite number of iterations we obtain f ∈Xpi, where pi = np n−2ip > n 2 (works for i > n 2p −1). Next iteration gives f ∈X0. p p We have shown that the ﬁrst case of Fredholm alternative is satisﬁed for the equation (5.15). Consequently, the solution to (5.15) exists and is unique for any G ∈Xp and (I −F)−1 ∈L(Xp). Since F = fg + h is H¨older-continuous with values in Xp, Lemma 3 im- plies that the problem (2.6), (2.10) has unique solution u ∈C{k}([0, T]; Xp) T C([0, T]; XAp). This completes the proof of the existence and uniqueness as- sertion of the theorem. b In the rest of the proof, bC stands for a generic constant depending on the parameters M, L, g, p, α2. Let us deduce the stability estimate (5.9). We obtain ∥f∥Xp ≤∥(I −F)−1∥L(Xp)∥G∥Xp ≤bC ∥h(T, ·)∥Xp + \|η\|∥ψ∥Xp + ∥ψ∥XAp Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. 252 N. Kinash and J. Janno + ∥D{k} t (u2 −ϕ) −ηu2∥C([0,T ];Xp) ≤bC ∥h∥Cα2([0,T ];Xp) + ∥ψ∥XAp +∥ϕ∥XAp . (5.18) (5.18) Further, we note that g ∈Cγ 0 ([0, T]; C(Ω)) for any γ ∈(0, 1) in case (A1) and for γ = α1 in cases (A2), (A3). Using Lemma 3 we have Further, we note that g ∈Cγ 0 ([0, T]; C(Ω)) for any γ ∈(0, 1) in case (A1) and for γ = α1 in cases (A2), (A3). Using Lemma 3 we have ∥u∥C{k}([0,T ];Xp) T C([0,T ];XAp) = ∥u1 + u2∥C{k}([0,T ];Xp) T C([0,T ];XAp) ≤bC(∥f∥Xp∥g∥Cγ 0 ([0,T ];C(Ω)) + ∥h∥Cα2([0,T ];Xp) + ∥ϕ∥XAp ). ∥u∥C{k}([0,T ];Xp) T C([0,T ];XAp) = ∥u1 + u2∥C{k}([0,T ];Xp) T C([0,T ];XAp) ≤bC(∥f∥Xp∥g∥Cγ 0 ([0,T ];C(Ω)) + ∥h∥Cα2([0,T ];Xp) + ∥ϕ∥XAp ). Together with the estimate of f (5.18) it implies (5.9). Together with the estimate of f (5.18) it implies (5.9). Theorem 1. Let one of the following assumptions be valid: Throughout the proof, bC denotes a generic constant depending on M, L, g, p, α2 and RHS stands for the expression in brackets at the right- hand side of (5.19). By Theorem 2, IP1 has a unique solution (f, u) ∈Xp × C{k}([0, T]; Xp) T C([0, T]; XAp). Let us consider the problem D{k} t (w2−w2(0, ·)) = Apw2 + D{k} t (h−h(0, ·)), w2(0, ·) = Lϕ + h(0, ·). (5.21) 253 Inverse Problems for a Generalized Subdiﬀusion Equation Under the assumptions of this theorem, Lemma 3 implies that (5.21) has a unique solution w2 ∈C{k}([0, T]; Xp) ∩C([0, T]; XAp). Moreover, due to (4.7) and (4.8), ∥w2∥C([0,T ];XAp) ≤bC(∥h∥C{k},α2([0,T ];Xp) +∥h(0, ·)∥XAp +∥Lϕ∥XAp ). It is easy to check that w2 = D{k} t ∗(u2 −ϕ) and u2 = M ∗w2 + ϕ where u2 solves (5.12). Therefore, we have u2 ∈C{k}([0, T]; XAp) ,→C{k}([0, T]; C(Ω)) and ∥u2∥C{k}([0,T ];XAp) ≤bC ∥h∥C{k},α2([0,T ];Xp)+∥h(0, ·)∥XAp+∥Lϕ∥XAp +∥ϕ∥XAp . (5.22) ∥u2∥C{k}([0,T ];XAp) ≤bC ∥h∥C{k},α2([0,T ];Xp)+∥h(0, ·)∥XAp+∥Lϕ∥XAp +∥ϕ∥XAp . (5.22) (5.22) Let us consider the function G given by (5.16). (Recall that there v2 = w2 − ηu2.) Due the proved properties of w2 and u2 and the assumptions of the theorem, it holds G ∈C(Ω) and ∥G∥C(Ω) ≤bC RHS. Let us consider the function G given by (5.16). (Recall that there v2 = w2 − ηu2.) Due the proved properties of w2 and u2 and the assumptions of the theorem, it holds G ∈C(Ω) and ∥G∥C(Ω) ≤bC RHS. ( ) Now, let us provide an estimate for ∥f∥C(Ω) using the formulas (5.15) and (5.17). Since 1/g(T, ·) ∈C(Ω) and v1[·](T, ·) ∈L(Xp, XAp), we have ∥f∥C(Ω) ≤∥Ff∥C(Ω) + ∥G∥C(Ω) ≤bC∥v1[f](T, ·)∥C(Ω) + ∥G∥C(Ω) ≤bC∥v1[f](T, ·)∥XAp + ∥G∥C(Ω) ≤bC∥f∥Xp + ∥G∥C(Ω). Since (I −F) is invertible in Xp, the estimate holds Since (I −F) is invertible in Xp, the estimate holds ∥f∥Xp ≤∥(I −F)−1∥L(Xp)∥G∥Xp ≤bC∥G∥C(Ω). Thus, we obtain ∥f∥C(Ω) ≤bC RHS. (5.23) (5.23) Finally, let us derive an estimate for u and ﬁnish the proof of the ﬁrst part of the theorem. We have u = u1 + u2, where u1 = M ∗w1, w1 = D{k} t u1 and w1 solves the problem D{k} t w1 = Apw1 + fD{k} t g, w1(0, ·) = 0. Theorem 1. Let one of the following assumptions be valid: Since fD{k} t g ∈Cα′ 0 ([0, T]; Xp), Lemma 3 implies w1 ∈Cα′ 0 ([0, T]; XAp) and ∥u1∥C{k},α′ 0 ([0,T ];XAp) = ∥w1∥Cα′ 0 ([0,T ];XAp) ≤ bC∥f∥C(Ω)∥D{k} t g∥Cα′ 0 ([0,T ];Xp). Using here (5.23) we have ∥u1∥C{k},α′ 0 ([0,T ];XAp) ≤bC RHS. (5.24) (5.24) From (5.22) and (5.24) we obtain for u = u1 + u2 the estimate From (5.22) and (5.24) we obtain for u = u1 + u2 the estimate ∥u∥C{k}([0,T ];XAp) ≤bC RHS. (5.25) (5.25) It remains to estimate Lu in the space C([0, T]; C(Ω)). Using (5.25) we deduce ∥D{k} t (u −ϕ)∥C([0,T ];C(Ω)) ≤bC∥D{k} t (u −ϕ)∥C([0,T ];XAp) ≤bC RHS. It remains to estimate Lu in the space C([0, T]; C(Ω)). Using (5.25) we deduce ∥D{k} t (u −ϕ)∥C([0,T ];C(Ω)) ≤bC∥D{k} t (u −ϕ)∥C([0,T ];XAp) ≤bC RHS. It remains to estimate Lu in the space C([0, T]; C(Ω)). Using (5.25) we deduce ∥D{k} t (u −ϕ)∥C([0,T ];C(Ω)) ≤bC∥D{k} t (u −ϕ)∥C([0,T ];XAp) ≤bC RHS. It remains to estimate Lu in the space C([0, T]; C(Ω)). Using (5.25) we deduce ∥D{k}(u ϕ)∥ ≤bC∥D{k}(u ϕ)∥ ≤bC RHS It remains to estimate Lu in the space C([0, T]; C(Ω)). Using (5.25) we deduce It remains to estimate Lu in the space C([0, T]; C(Ω)). Using (5.25) we deduce ∥D{k} t (u −ϕ)∥C([0,T ];C(Ω)) ≤bC∥D{k} t (u −ϕ)∥C([0,T ];XAp) ≤bC RHS. Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. 4 N K h d J J 254 N. Kinash and J. Janno From the expression Lu = D{k} t (u −ϕ) −fg −h due to the proved estimates for D{k} t (u −ϕ) and f we obtain From the expression Lu = D{k} t (u −ϕ) −fg −h due to the proved estimates for D{k} t (u −ϕ) and f we obtain ∥Lu∥C([0,T ];C(Ω)) ≤bC RHS. (5.26) (5.26) Summing up, (5.23), (5.25) and (5.26) imply (5.19). Summing up, (5.23), (5.25) and (5.26) imply (5.19). Now let us focus on the second part of this theorem that is concerned with the particular case ϕ = h(0, ·) = D{k} t h(0, ·) = 0. Then RHS reduces to the ex- pression in brackets at the right-hand side of (5.20). Theorem 1. Let one of the following assumptions be valid: Lemma 3 implies that the function w2 which solves (5.21) belongs the space Cα′ 0 ([0, T]; XAp), the func- tion u2 = M ∗w2 belongs to C{k},α′ 0 ([0, T]; XAp) and ∥u2∥C{k},α′ 0 ([0,T ];XAp) ≤ bC∥h∥C{k},α2 0 ([0,T ];Xp). This relation by u = u1 + u2 and the estimates (5.23), (5.24) and (5.26) implies (5.20). ⊓⊔ Provided the assumptions of Theorem 3 hold and B = I, an explicit expres- sion of the unknown function f at the boundary can be derived. Namely, setting t = T and x ∈∂Ωin (2.6) and taking the relations F = fg + h and u(T, ·) = ψ into account we obtain f(x) = − 1 g(T, x)[Lψ(x) + h(T, x)], x ∈∂Ω. 6 Results on IP2 In the context of IP2 let us introduce the following sets for the coeﬃcient r: KR = {r ∈C(Ω) : r(x) ≤R, x ∈Ω}, where R ∈R. Theorem 4. Let R be some real number and IP2 have two solutions (r, u), (r1, u1), such that r ∈C(Ω), r1 ∈KR, u, u1 ∈C{k} 0 ([0, T]; L1(Ω)) \ C0([0, T]; W 2 1 (Ω)), u1 −u ∈C{k} 0 ([0, T]; C(Ω)) \ C0([0, T]; W 2 p (Ω)) for some p > 1 and the function U = u+b (and M) satisfy one of the following assumptions: for some p > 1 and the function U = u+b (and M) satisfy one of the following assumptions: p (A4) U ∈C1+α1 0 ([0, T]; C(Ω)) for some 0 < α1 < 1; {k} p (A4) U ∈C1+α1 0 ([0, T]; C(Ω)) for some 0 < α1 < 1; (A5) U ∈C{k},α1 0 ([0, T]; C(Ω)) and M(t) ≥ctγ−1, t ∈(0, T) for some c ∈R+, 0 < γ < α1 < 1; (A6) U ∈C{k},α1−β 0 ([0, T]; C(Ω)) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤ c3tβ−2, t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α1 < 1. (A4) U ∈C1+α1 0 ([0, T]; C(Ω)) for some 0 < α1 < 1; (A5) U ∈C{k},α1 0 ([0, T]; C(Ω)) and M(t) ≥ctγ−1, t ∈(0, T) for some c ∈R+, 0 < γ < α1 < 1; (A6) U ∈C{k},α1−β 0 ([0, T]; C(Ω)) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤ c3tβ−2, t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α1 < 1. A5) U ∈C{k},α1 0 ([0, T]; C(Ω)) and M(t) ≥ctγ−1, t ∈(0, T) for some c ∈R+, < γ < α1 < 1; (A5) U ∈C0 ([0, T]; C(Ω)) and M(t) ≥ct , t ∈(0, T) for some c ∈R+, 0 < γ < α1 < 1; (A6) U ∈C{k},α1−β 0 ([0, T]; C(Ω)) and c1tγ−1 ≤M(t) ≤c2tβ−1, \|M ′(t)\| ≤ c3tβ−2, t ∈(0, T), for some c1, c2, c3 ∈R+, 0 < β ≤γ < α1 < 1. Additionally, we assume that U ≥0, D{k} t U −RU ≥0, (6.1) a.e. x ∈Ω, ∃tx ∈(0, T] : U(tx, x) > 0. (6.1) In case B = I we also assume that ∀x ∈∂Ω, either U(T, x) > 0 or U(·, x) = 0. 6 Results on IP2 Then (r1, u1) = (r, u). In case B = I we also assume that ∀x ∈∂Ω, either U(T, x) > 0 or U(·, x) = 0. Then (r1, u1) = (r, u). Inverse Problems for a Generalized Subdiﬀusion Equation 25 Inverse Problems for a Generalized Subdiﬀusion Equation 255 Proof. The diﬀerence (ˆr, ˆu) = (r1 −r, u1 −u) ∈C(Ω) × C{k} 0 ([0, T]; C(Ω)) T C0([0, T]; W 2 p (Ω)) solves the problem Proof. The diﬀerence (ˆr, ˆu) = (r1 −r, u1 −u) ∈C(Ω) × C{k} 0 ([0, T]; C(Ω) T C0([0 T]; W 2(Ω)) solves the problem D{k} t ˆu(t, x) = (L1 + r1)ˆu(t, x) + U(t, x)ˆr(x), x ∈Ω, t ∈(0, T), ˆu(0, x) = 0, x ∈Ω, Bˆu(t, x) = 0, x ∈∂Ω, t ∈(0, T), (6.2) ˆu(T, x) = 0, x ∈Ω. (6.2) The inequalities (6.1) imply that D{k} t U −RrU ≥0, where Rr := max x∈Ω r1(x) ≤ The inequalities (6.1) imply that D{k} t U −RrU ≥0, where Rr := max x∈Ω r1(x) ≤ R Consequently the assumptions of Theorem 1 are satisﬁed for the problem The inequalities (6.1) imply that D{k} t U −RrU ≥0, where Rr := max x∈Ω r1(x) ≤ x∈Ω R. Consequently, the assumptions of Theorem 1 are satisﬁed for the problem 6.2) and we obtain ˆr = 0, ˆu = 0. ⊓⊔ Let us formulate a problem that contains approximate data: D{k} t (˜u−˜ϕ)(t, x) = L1˜u(t, x)+˜r(x)(˜u+˜b)(t, x)+ ˜F1(t, x), x ∈Ω, t ∈(0, T), ˜u(0, x) = 0, x ∈Ω, B˜u(t, x) = 0, x ∈∂Ω, t ∈(0, T), (6.3) ˜u(T, x) = ˜ψ, x ∈Ω. (6.3) We are going to prove an existence and approximation theorem for this problem in case its data vector eD = (˜b, ˜F1, ˜ψ) is close to the data vector D = (b, F1, ψ) of the exact problem IP2. Theorem 5. Assume that R ∈R and IP2 has a solution (r, u) ∈KR × C{k} 0 ([0, T]; L1(Ω)) T C0([0, T]; W 2 1 (Ω)) such that U = u + b (and M) sat- isfy one of the assumptions (A4)–(A6), the inequalities (6.1) and U(T, x) > 0, x ∈Ω. Then the following statements are valid. (i) Let p ∈{0} S n 2 , ∞ , α2 ∈(0, 1). 6 Results on IP2 There exist constants δ1 > 0 and K1 > 0 depending on M, L1, r, U, p, α2 such that if (i) Let p ∈{0} S n 2 , ∞ , α2 ∈(0, 1). There exist constants δ1 > 0 and K1 > depending on M, L1, r, U, p, α2 such that if ˜D −D ∈D1 = Cα2 0 ([0, T]; C(p)(Ω)) × Cα2 0 ([0, T]; Xp) × XAp and ∥˜D −D∥D1 ≤δ1, where C(p)(Ω) = ( C(Ω), in case p ∈ n 2 , ∞ , X0, in case p = 0, the problem (6.3) has a unique solution in the set and ∥˜D −D∥D1 ≤δ1, where C(p)(Ω) = ( C(Ω), in case p ∈ n 2 , ∞ , X0, in case p = 0, then problem (6.3) has a unique solution in the set ( problem (6.3) has a unique solution in the set n (˜r, ˜u) : (˜r −r, ˜u −u) ∈X1 := Xp × C{k},α 0 ([0, T]; Xp) \ Cα 0 ([0, T]; XAp) , ∥(˜r −r, ˜u −u)∥X1 ≤K1∥˜D −D∥D1 o , in case (A4), in case (A4), where α = α2, in case (A4), min{α1, α2}, in cases (A5), (A6). where α = 2, ( ), min{α1, α2}, in cases (A5), (A6). (ii) Let p ∈ n 2 , ∞ , α2 ∈(0, 1). There exist constants δ2 > 0 and K2 > 0 depending on M, L1, r, U, p, α2 such that if ˜D −D ∈D2 = C{k},α2 0 ([0, T]; Xp) \ Cα2 0 ([0, T]; C(Ω)) 2 × Yp Math. Model. Anal., 24(2):236–262, 2019. J. Janno Ap, Lψ ∈C(Ω)}, (˜r−r, ˜u−u)∥X2 ≤ Lv ∈C0([0, T 4), (A5), ). ator Ap and the sp omponent r of th 256 N. Kinash and J. Janno and ∥˜D−D∥D2 ≤δ2 where Yp = {ψ : ψ ∈XAp, Lψ ∈C(Ω)}, then the problem (6.3) has a unique solution in the set and ∥˜D−D∥D2 ≤δ2 where Yp = {ψ : ψ ∈XAp, Lψ ∈C(Ω)}, then the problem (6.3) has a unique solution in the set n (˜r, ˜u) : (˜r−r, ˜u−u) ∈X2 := C(Ω)× Up,α′, ∥(˜r−r, ˜u−u)∥X2 ≤K2∥˜D−D∥D2 o , where Up,α′ = {v ∈C{k},α′ 0 ([0, T]; XAp) : Lv ∈C0([0, T]; C(Ω))}, α′ = min{ˆα; α2} and ˆα = α1, in cases (A4), (A5), α1 −β, in case (A6). 6 Results on IP2 Inverse Problems for a Generalized Subdiﬀusion Equation 257 In case ∥˜D −D∥D1 ≤δ1 we have In case ∥˜D −D∥D1 ≤δ1 we have ∥F2(ˆr, ˆu)∥X1 ≤C6 (cpK2 1 + K1)δ1 + 1 + R1 ∥˜D −D∥D1. ∥F2(ˆr, ˆu)∥X1 ≤C6 (cpK2 1 + K1)δ1 + 1 + R1 ∥˜D −D∥D1. Let us deﬁne the constants as follows: K1 = C6(2 + R1), δ1 = 1 cpK2 1+K1 . Then ∥F2(ˆr, ˆu)∥X1 ≤K1∥˜D −D∥D1. Consequently, for ∥(ˆr, ˆu)∥X1 ≤ρ we have ∥F2(ˆr, ˆu)∥X1 ≤ρ. Secondly, inside the set ∥(ˆr, ˆu)∥X1 ≤ρ = K1∥˜D −D∥D1 let us consider the diﬀerence of F2 at (ˆr1, ˆu2) and (ˆr2, ˆu2). Assuming ∥˜D −D∥D1 ≤δ1, we deduce the estimate ∥F2(ˆr1, ˆu1) −F2(ˆr2, ˆu2)∥X1 ≤∥A∥∥(ˆr1 −ˆr2)ˆu1 + ˆr2(ˆu1 −ˆu2) +(ˆr1 −ˆr2)(˜b −b)∥Cα2 0 ([0,T ];Xp) ≤C6 cpρ∥ˆr1 −ˆr2∥Xp +cpρ∥ˆu1 −ˆu2∥Cα 0 ([0,T ];XAp) + δ1∥ˆr1 −ˆr2∥Xp ≤C6(cpK1δ1 + δ1) ×∥(ˆr1 −ˆr2, ˆu1 −ˆu2)∥X1 = 1 (2 + R1)∥(ˆr1 −ˆr2, ˆu1 −ˆu2)∥X1. It shows that the operator F2 is a contraction in the ball ∥(ˆr, ˆu)∥X1 ≤ρ. According to the Banach ﬁxed point theorem there exists a unique solution to the equation (6.5) in that ball. This proves the assertion (i). It shows that the operator F2 is a contraction in the ball ∥(ˆr, ˆu)∥X1 ≤ρ. According to the Banach ﬁxed point theorem there exists a unique solution to the equation (6.5) in that ball. This proves the assertion (i). ( ) ( ) (ii) The proof of (ii) repeats the proof of (i) with appropriate changes of spaces and norms. For A, the estimate (5.20) is used instead of (5.10). ⊓⊔ (ii) The proof of (ii) repeats the proof of (i) with appropriate changes of spaces and norms. For A, the estimate (5.20) is used instead of (5.10). ⊓⊔ Remark 1. In case the data of (6.3) are close to data of a process without reaction (i.e. r = 0), Theorem 5 implies the existence of the reaction coeﬃcient ˜r in small. Remark 2. Supposing the existence of a solution (r, u) of IP2, we ask: what are suﬃcient conditions on the data that guarantee the validity of inequality-type conditions (6.1) and U(T, x) > 0, x ∈Ωin Theorems 4, 5? To answer this ques- tion, we return to the problem (2.3) for U and set there Φ = H(0, ·) = 0. Let us suppose that U is suﬃciently smooth. 6 Results on IP2 We mention that in this theorem, the operator Ap and the space XAp deﬁned on the basis of L = L1 + rI depend on the component r of the solution of the exact problem IP2. Proof. Let us denote the diﬀerence (ˆr, ˆu) = (˜r −r, ˜u −u). Then the problem for the pair (ˆr, ˆu) reads D{k} t ˆu = (L1 + r)ˆu + ˆr(u + b) + h ˆrˆu + ˜F1 −F1 + (ˆr + r)(˜b −b) i , ˆu(0, ·) = 0, Bˆu\|∂Ω= 0, ˆu(T, ·) = ˜ψ −ψ. (6.4) (6.4) This problem can be treated as IP1 with f = ˆr, g = u + b, h = ˆrˆu + ˜F1 −F1 + (ˆr + r)(˜b −b). Therefore, applying the solution operator of IP1 A to (6.4), it is reduced to the operator equation (ˆr, ˆu) = F2(ˆr, ˆu), (6.5) (6.5) where F2(ˆr, ˆu) = A(ˆrˆu + ˜F1 −F1 + (ˆr + r)(˜b −b), 0, ˜ψ −ψ). where F2(ˆr, ˆu) = A(ˆrˆu + ˜F1 −F1 + (ˆr + r)(˜b −b), 0, ˜ψ −ψ). where F2(ˆr, ˆu) = A(ˆrˆu + ˜F1 −F1 + (ˆr + r)(˜b −b), 0, ˜ψ −ψ). We are going to show that F2 is a contraction in a ball ∥(ˆr, ˆu)∥X1 ≤ρ with a suitable chosen ρ > 0. Firstly, we have to prove that this ball remains invariant with respect to the operator F2. Let ∥(ˆr, ˆu)∥X1 ≤ρ. According to (5.10), ∥F2(ˆr, ˆu)∥X1 ≤C6 ∥˜ψ−ψ∥XAp +∥ˆrˆu+ ˜F1−F1+(ˆr+r)(˜b−b)∥Cα2 0 ([0,T ];Xp) . Let cp be an embedding constant such that ∥w∥C(Ω) ≤cp∥w∥XAp . Then ∥ˆrˆu∥Cα2 0 ([0,T ];Xp) ≤∥ˆr∥Xp∥ˆu∥Cα 0 ([0,T ];C(Ω)) ≤∥ˆr∥Xpcp∥ˆu∥Cα 0 ([0,T ];XAp) ≤cpρ2. Therefore, ∥ˆrˆu∥Cα2 0 ([0,T ];Xp) ≤∥ˆr∥Xp∥ˆu∥Cα 0 ([0,T ];C(Ω)) ≤∥ˆr∥Xpcp∥ˆu∥Cα 0 ([0,T ];XAp) ≤cpρ2. Therefore, ∥F2(ˆr, ˆu)∥X1 ≤C6 ∥˜ψ −ψ∥XAp + cpρ2 + ∥˜F1 −F1∥Cα2 0 ([0,T ];Xp) +(ρ + R1)∥˜b −b∥Cα2 0 ([0,T ];C(p)(Ω)) ≤C6 cpρ2 + (ρ + 1 + R1)∥˜D −D∥D1 , where R1 = ∥r∥Xp in case p ∈ n 2 , ∞ and R1 = ∥r∥C(Ω) in case p = 0. Now let us take ρ = K1∥˜D −D∥D1 with a constant K1. Then ∥F2(ˆr, ˆu)∥X1 ≤C6 (cpK2 1 + K1)∥˜D −D∥D1 + 1 + R1 ∥˜D −D∥D1. 6 Results on IP2 Then constructing a corresponding prob- lem for D{k} t U −RU and assuming D{k} t H −RH ≥0, (D{k} t Bb−RBb)\|∂Ω≥0, Lemma 4 (i) implies the inequality D{k} t U −RU ≥0. Next, we consider the conditions U ≥0 and U(T, x) > 0, x ∈Ω. Let us assume that ∃µ ∈C[0, T], µ ≥0, µ ̸= 0, µ −nondecreasing : H(t, x) ≥µ(t), x ∈Ω, t ∈[0, T], Bb(t, x) ≥µ(t), x ∈∂Ω, t ∈[0, T]. Deﬁne V = U −δ1 ∗µ with δ > 0. The function V solves the problem ∃µ ∈C[0, T], µ ≥0, µ ̸= 0, µ −nondecreasing : H(t, x) ≥µ(t), x ∈Ω, t ∈[0, T], Bb(t, x) ≥µ(t), x ∈∂Ω, t ∈[0, T]. Deﬁne V = U −δ1 ∗µ with δ > 0. The function V solves the problem D{k} t V = LV + H1, V (0, ·) = 0 , B(V −(b −δ1 ∗µ))\|∂Ω= 0, where H1 = H + δ(r1 ∗µ −D{k} t 1 ∗µ). Since D{k} t 1 ∗µ = k ∗µ, we get that for suﬃciently small δ, Deﬁne V = U −δ1 ∗µ with δ > 0. The function V solves the problem D{k} t V = LV + H1, V (0, ·) = 0 , B(V −(b −δ1 ∗µ))\|∂Ω= 0, H1(t, x) ≥µ(t)[1 −δ(max x∈Ω r(x)T + ∥k∥L1(0,T ))] ≥0, t ∈[0, T], x ∈Ω H1(t, x) ≥µ(t)[1 −δ(max x∈Ω r(x)T + ∥k∥L1(0,T ))] ≥0, t ∈[0, T], x ∈Ω Math. Model. Anal., 24(2):236–262, 2019. 258 N. Kinash and J. Janno and BV \|∂Ω= B(b −δ1 ∗µ)\|∂Ω≥0. Lemma 4 (i) yields V ≥0. Thus, U = V + δ1 ∗µ ≥0 and U(T, x) = V (T, x) + δ R T 0 µ(τ)dτ > 0, x ∈Ω. At the end of this section, we make some general remarks. We applied results on IP1 to analyze IP2. In a similar manner, results on IP1 can be applied to study inverse problems to determine other coeﬃcients of L, too. The basic set of assumptions (A1)–(A3) for g involves the restriction The basic set of assumptions (A1)–(A3) for g involves the restriction g(0, ·) = 0. This is due to the fact that in case g(0, ·) ̸= 0 we cannot ensure suﬃcient regularity of u to apply the positivity principle in the proof of Theo- rem 1. Acknowledgements The research was supported by the Estonian Research Council, Grant PUT568. Authors thank the referee whose valuable comments led to the improvement of the paper. 6 Results on IP2 In IP2, the function u + b = U works as g. For that reason, we consider the case Φ = U(0, ·) = 0 in IP2. In the beginning of the proof of Lemma 4 we showed that the direct problem with r > 0 can be reduced to a problem with r ≤0 by the change of unknown ˜u = e−σtu, where σ > 0. 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Vergara and R. Zacher. Stability, instability, and blowup for time fractional and other nonlocal in time semilinear subdiﬀusion equations. Journal of Evolu- tion Equations, 17(1):599–626, 2017. https://doi.org/10.1007/s00028-016-0370- 2. [34] R. Zacher. Weak solutions of abstract evolutionary integro-diﬀerential equations in Hilbert spaces. Funkcialaj Ekvacioj, 52(1):1–18, 2009. https://doi.org/10.1619/fesi.52.1. Appendix: Proofs of Lemmas 1, 2 and 5 Proof of Lemma 1. Theorems 3 and 4 of [9] guarantee that k is nonnegative, nonincreasing and convex. Convexity implies the continuity of k. From the equation M ∗k = 1 we easily deduce lim t→0+k(t) = +∞, because in the opposite Proof of Lemma 1. Theorems 3 and 4 of [9] guarantee that k is nonnegative, nonincreasing and convex. Convexity implies the continuity of k. From the equation M ∗k = 1 we easily deduce lim t→0+k(t) = +∞, because in the opposite case k is bounded from which it follows that lim t→0+(M ∗k)(t) = 0. 0 Let us prove k > 0. Suppose that it is not true. Then in view of proved properties of k, ∃t0 : k(t) > 0, t < t0 and k(t) = 0, t > t0. For t > t0 from M ∗k = 1 we get R t0 0 M(t −τ)k(τ)dτ = 1. Therefore, R t0 0 M ′(t −τ)k(τ)dτ = 0. Let us prove k > 0. Suppose that it is not true. Then in view of proved properties of k, ∃t0 : k(t) > 0, t < t0 and k(t) = 0, t > t0. For t > t0 from M ∗k = 1 we get R t0 0 M(t −τ)k(τ)dτ = 1. Therefore, R t0 0 M ′(t −τ)k(τ)dτ = 0. Inverse Problems for a Generalized Subdiﬀusion Equation 261 The last equality contradicts to the assumptions k(t) > 0, t ∈(0, t0) and M ′ < 0. Thus, k > 0. The last equality contradicts to the assumptions k(t) > 0, t ∈(0, t0) and M ′ < 0. Thus, k > 0. Finally, let us prove (3.3) Let us choose some t3 > 0. Since lim t→0+k(t) = +∞, there exists an interval (0, δ), δ < t3, such that k(t) > k(t3) for t ∈(0, δ). Suppose that (3.3) is not true. Then we can ﬁnd two points t1 < t2 in (0, δ) so that k(t1) = k(t2). Consequently, for t1 < t2 < t3 we have k(t1) = k(t2) > k(t3). Obviously, it contradicts to the convexity of k. Therefore, (3.3) is valid. ⊓⊔ Proof of Lemma 2 is similar to proof of Theorem 14 in [11] that is concerned with the case M(t) = tβ−1 Γ (β). Let z ∈Cα−β 0 ([0, T]; X). Then ∥M ∗z(t)∥X ≤ const tβ−1 ∗tα−β = O(tα). Appendix: Proofs of Lemmas 1, 2 and 5 Secondly, (M ∗z)(t) −(M ∗z)(t −h) = J1 + J2 + J3, where where J1 = z(t) Z t t−h M(τ)dτ, J2 = − Z h 0 [z(t) −z(t −τ)M(τ)dτ, J3 = − Z t h [z(t) −z(t −τ)] Z τ τ−h M ′(s)ds dτ. J1 = z(t) Z t t−h M(τ)dτ, J2 = − Z h 0 [z(t) −z(t −τ)M(τ)dτ, t J3 = − Z t h [z(t) −z(t −τ)] Z τ τ−h M ′(s)ds dτ. Immediately, ∥J2∥X ≤const R h 0 τ α−βτ β−1dτ = O(hα). Moreover, Immediately, ∥J2∥X ≤const R h 0 τ α−βτ β−1dτ = O(hα). Moreover, Immediately, ∥J2∥X ≤const R h 0 τ α−βτ β−1dτ = O(hα). Moreover, ∥J1∥X ≤const tα−β Z t t−h τ β−1 = const tα−β[tβ −(t −h)β], ∥J3∥X ≤const Z t h τ α−β Z τ τ−h sβ−2ds dτ = const Z t h τ α−β[(τ −h)β−1 −τ β−1]dτ ∥J3∥X ≤const Further estimation of J1 and J3 can be performed exactly as in [11]. As a result, we get ∥J1∥X, ∥J3∥X = O(hα). This completes the proof. ⊓⊔ Further estimation of J1 and J3 can be performed exactly as in [11]. As a result, we get ∥J1∥X, ∥J3∥X = O(hα). This completes the proof. ⊓⊔ Proof of Lemma 5. Firstly, we point out that the assumption w ∈W 2 p (Ω), p > n implies w ∈C1(Ω). We will use maximum principles for elliptic equations in Sobolev spaces to prove the lemma. Let us consider the case x∗∈Ω. Suppose that L1w(x∗) < 0. Then there exists a ball B(x∗, ε) ⊂Ωand δ > 0 such that L1w(x) ≤−δ < 0 for x ∈B(x∗, ε). Let us deﬁne the auxiliary function z(x) = α\|x −x∗\|2 with α > 0 (7.1) (7.1) such that L1(w + z) ≤0 in B(x∗, ε). Since w(x∗) ≤w(x) and z(x∗) < z(x) for x ∈∂B(x∗, ε), we get (w + z)(x∗) < (w + z)(x), x ∈∂B(x∗, ε). (7.2) (7.2) On the other hand, due to L1(w+z) ≤0 it follows from the Theorem 9.1 [8] that min x∈B(x∗,ε)(w + z)(x) = min x∈∂B(x∗,ε)(w + z)(x), that contradicts (7.2). Therefore, the supposition L1w(x∗) < 0 was wrong. On the other hand, due to L1(w+z) ≤0 it follows from the Theorem 9.1 [8] that min x∈B(x∗,ε)(w + z)(x) = min x∈∂B(x∗,ε)(w + z)(x), that contradicts (7.2). Therefore, the supposition L1w(x∗) < 0 was wrong. Appendix: Proofs of Lemmas 1, 2 and 5 ( ) ( ) supposition L1w(x∗) < 0 was wrong. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. Math. Model. Anal., 24(2):236–262, 2019. 262 N. Kinash and J. Janno Next let us consider the case x∗∈∂Ω. Again, suppose L1w(x∗) < 0. Then there exists B(x∗, ε) and δ > 0 such that L1w(x) ≤−δ < 0 for x ∈ B(x∗, ε) T Ω. Similarly to the previous case we deﬁne z by (7.1) so that L1(w + z) ≤0 in B(x∗, ε) T Ω. Then (w + z)(x∗) < (w + z)(x) for x ∈ B(x∗, ε) T Ω. Hence, Lemma 3.4 [8] is applicable and yields ∂w ∂ν (x∗) = ∂(w+z) ∂ν (x∗) < 0. That contradicts to ∂ ∂ν w(x∗) ≥0 following from the assump- tion ∂ ∂ωw(x∗) ≥0. Therefore, L1w(x∗) ≥0 holds. ⊓⊔
https://openalex.org/W3154795235	https://publicatt.unicatt.it/bitstream/10807/180780/1/2021%20microphytorem%20FM.pdf	English	null	Bioaugmented Phytoremediation of Metal-Contaminated Soils and Sediments by Hemp and Giant Reed	Frontiers in microbiology	2,021	cc-by	18,301	Bioaugmented Phytoremediation of Metal-Contaminated Soils and Sediments by Hemp and Giant Reed Andrea Ferrarini1, Alessandra Fracasso1, Giulia Spini2, Flavio Fornasier3,4, Eren Taskin2, Maria Chiara Fontanella2, Gian Maria Beone2, Stefano Amaducci1 and Edoardo Puglisi2 1 Department of Sustainable Crop Production, Università Cattolica del Sacro Cuore, Piacenza, Italy, 2 Department for Sustainable Food Process, Università Cattolica del Sacro Cuore, Piacenza, Italy, 3 CREA – Centro Viticoltura ed Enologia, Gorizia, Italy, 4 SOLIOMICS srl, Udine, Italy We assessed the effects of EDTA and selected plant growth-promoting rhizobacteria (PGPR) on the phytoremediation of soils and sediments historically contaminated by Cr, Ni, and Cu. A total of 42 bacterial strains resistant to these heavy metals (HMs) were isolated and screened for PGP traits and metal bioaccumulation, and two Enterobacter spp. strains were ﬁnally selected. Phytoremediation pot experiments of 2 months duration were carried out with hemp (Cannabis sativa L.) and giant reed (Arundo donax L.) grown on soils and sediments respectively, comparing in both cases the effects of bioaugmentation with a single PGPR and EDTA addition on plant and root growth, plant HM uptake, HM leaching, as well as the changes that occurred in soil microbial communities (structure, biomass, and activity). Good removal percentages on a dry mass basis of Cr (0.4%), Ni (0.6%), and Cu (0.9%) were observed in giant reed while negligible values (<100h) in hemp. In giant reed, HMs accumulated differentially in plant (rhizomes > > roots > leaves > stems) with largest quantities in rhizomes (Cr 0.6, Ni 3.7, and Cu 2.2 g plant−1). EDTA increased Ni and Cu translocation to aerial parts in both crops, despite that in sediments high HM concentrations in leachates were measured. PGPR did not impact ﬁne root diameter distribution of both crops compared with control while EDTA negatively affected root diameter class length (DCL) distribution. Under HM contamination, giant reed roots become shorter (from 5.2 to 2.3 mm cm−3) while hemp roots become shorter and thickened from 0.13 to 0.26 mm. A consistent indirect effect of HM levels on the soil microbiome (diversity and activity) mediated by plant response (root DCL distribution) was observed. Multivariate analysis of bacterial diversity and activity revealed not only signiﬁcant effects of plant and soil type (rhizosphere vs. bulk) but also a clear and similar differentiation of communities between control, EDTA, and PGPR treatments. We propose root DCL distribution as a key plant trait to understand detrimental effect of HMs on microbial communities. Positive evidence of the soil-microbe-plant interactions occurring when bioaugmentation with PGPR is associated with deep-rooting perennial crops makes this combination preferable over the one with chelating agents. Such knowledge might help to yield better bioaugmented bioremediation results in contaminated sites. ORIGINAL RESEARCH published: 20 April 2021 doi: 10.3389/fmicb.2021.645893 ORIGINAL RESEARCH Keywords: phytoremediation, bioaugmentation, heavy metals, plant-root-microbes interactions, plant uptake and accumulation, Arundo donax (L.), Cannabis sativa L., plant growth-promoting rhizobacteria Edited by: Markus Puschenreiter, University of Natural Resources and Life Sciences Vienna, Austria Reviewed by: Francesca Mapelli, University of Milan, Italy Muhammad Saleem, Alabama State University, United States Correspondence: Andrea Ferrarini andrea.ferrarini@unicatt.it Specialty section: This article was submitted to Microbiotechnology, a section of the journal Frontiers in Microbiology Received: 24 December 2020 Accepted: 10 March 2021 Published: 20 April 2021 INTRODUCTION HM uptake, for which the following crop categories have been proposed: ﬁeld crops (Vamerali et al., 2010), aromatic plants (Pandey et al., 2019), Brassica species (Marchiol et al., 2004; Mourato et al., 2015), hyperaccumulator plants (Cheng, 2003; Peer et al., 2006) and biomass crops, either annual or perennial (Shi and Cai, 2009; Pandey et al., 2016; Tripathi et al., 2016). p Here in thus study, we choose two model non-food high- yielding crop for phytoremediation to address the contamination by Cr, Zn, and Cu of two distinct environmental matrices: hemp for soil and giant reed for sediment. Hemp (Cannabis sativa L.) can tolerate high heavy metal content in soil (Angelova et al., 2004), and it can be considered a good candidate crop in phytoremediation experiments (Linger et al., 2002; Rheay et al., 2020) because of its fast growth (Struik et al., 2000), HM stress tolerance genes (Ahmad et al., 2016), and ﬁne and deep rooting systems (Amaducci et al., 2008). Phytoremediation with hemp permits to produce biomass for multipurpose bioenergy applications (Amaducci et al., 2015; Rheay et al., 2020), simultaneously with the reduction of soil contaminants (Citterio et al., 2003; Linger et al., 2005). Giant reed (Arundo donax L.) is a perennial plant with a high biomass yield in marginal land with low inputs (Amaducci et al., 2017), high belowground C storage potential (Martani et al., 2020) and is tolerant to heavy metals (Papazoglou et al., 2005, 2007; Yang et al., 2012; Barbosa et al., 2015; Cristaldi et al., 2020) thanks to its ability to store HMs in belowground organs (Fiorentino et al., 2017). Giant reed especially in wetland and sediments along riverbanks, where it naturally grows (Barney and DiTomaso, 2008; Nackley et al., 2013), is a good candidate for phytoremediation (Mirza et al., 2010; Bonanno, 2012; Truu et al., 2015). Traditional soil remediation technologies, such as contaminant immobilization, pollutant stabilization, soil washing, and vitriﬁcation are expensive and detrimental for the chemical properties of soil and for its biodiversity (Cunningham and Ow, 1996; Ali et al., 2013; Mahar et al., 2016). On the contrary, phytoremediation, the use of plant to immobilize or remove the contaminants in soils, is a green technology that improves chemical, physical, and organic soil properties, and that is cheaper than traditional remediation techniques (Cunningham et al., 1995; Salt et al., 1995; Barbosa et al., 2015). INTRODUCTION (Passatore et al., 2014; Truu et al., 2015; Schwitzguébel, 2017) but only recently for heavy metals (Abhilash et al., 2012; Truu et al., 2015; Pandey et al., 2016; Tripathi et al., 2016). Soil represents a crucial but limited resource for human activities; erosion, loss of organic matter, landslides, and contamination are critical problems that limit its utilization. Among the inorganic compounds, heavy metals (HM) have a great importance in industrial application (Lebeau et al., 2008; Rajkumar et al., 2012; Ali et al., 2013), but their release into the environment poses a serious risk to human health and other living organisms (Duruibe et al., 2007; Liu et al., 2013). Bioremediation that utilizes living organisms and/or their products to improve removal of pollutants from the environment is an emerging low-input biotechnology for ecosystem revitalization (Abhilash et al., 2012). Diﬀerent microbes with plant growth-promoting traits (Tak et al., 2013), the so-called PGPR rhizobacteria (Gullap et al., 2014), have been studied for their potential to stimulate plant nutrient uptake, alleviate metal toxicity, immobilize/mobilize heavy metals in the soil, improve plant health and regulate plant pathogens (Manoj et al., 2020; Sahib et al., 2020; Prakash, 2021). Nickel (Ni) is a heavy metal widely distributed in the environment and is released from both natural sources and anthropogenic activity (Sarwar et al., 2017). Chromium (Cr), being very resistant to corrosion is broadly utilized in various industrial applications (Emsley, 2011). Chromium is essential for living organisms, but it is toxic in excessive concentrations; in particular for humans, Cr deﬁciency could negatively aﬀect lipid and sugar metabolisms (Anderson, 1997). Copper (Cu) is an essential trace element in plants and animals, but high quantities of copper salts produce acute toxicity in humans and animals (Wuana and Okieimen, 2011), possibly due to the generation of reactive oxygen species (ROS) (Liu J. et al., 2018). Cu contamination in soils could derive from natural sources like rock phosphate, from Cu-based fungicides (Komárek et al., 2010) or from zinc fertilizer application in agricultural land (Ali et al., 2013; Sarwar et al., 2017). A crucial aspect in phytoremediation trials is the choice of the most appropriate plant species, as the tolerance to contaminants and accumulation capacity vary greatly among species and at times also within the same species (Pietrini et al., 2010; Shi et al., 2012). The success of phytoremediation depends on the combination of yield vs. Citation: Ferrarini A, Fracasso A, Spini G, Fornasier F, Taskin E, Fontanella MC, Beone GM, Amaducci S and Puglisi E (2021) Bioaugmented Phytoremediation of Metal-Contaminated Soils and Sediments by Hemp and Giant Reed. Front. Microbiol. 12:645893. doi: 10.3389/fmicb.2021.645893 April 2021 \| Volume 12 \| Article 645893 1 Frontiers in Microbiology \| www.frontiersin.org Phyto-Assisted HMs Bioremediation Ferrarini et al. Frontiers in Microbiology \| www.frontiersin.org INTRODUCTION Several methods to improve phytoremediation eﬃciency have been proposed, one is the assisted phytoremediation (Lebeau et al., 2008; Shahid et al., 2014), where the availability of the contaminants is increased by the addiction of diﬀerent chelating agents (CA). One of the most utilized CA to improve phytoextraction eﬃciency and to reduce the duration of the phytoremediation process is the ethylene diamine tetraacetic acid (EDTA), which increases the metal solubilization in soils (Shahid et al., 2014). A negative aspect of EDTA-assisted phytoremediation is the low degradability of EDTA (Lombi et al., 2001) that could be toxic for plants and animals (Lasat, 2002; Römkens et al., 2002; Evangelou et al., 2007). A promising alternative to EDTA-assisted phytoremediation can be obtained by stimulating the degradative microbial population naturally evolved in polluted soils, a process called rhizoremediation (Kuiper et al., 2004; Vergani et al., 2017; Terzaghi et al., 2019). This process can be further improved by selecting and adding to the soil selected microorganism with high degradation or biosorption abilities, an approach that can be deﬁned as bioaugmented rhizoremediation (Lebeau et al., 2008; Rajkumar et al., 2012; Truu et al., 2015; Sarathambal et al., 2017). This technique has been proposed in case of organic pollutants Contaminated soil and sediments from an industrial area of Northern Italy were used to isolate, screen, and select metal-chelating plant growth-promoting bacteria. A pot experiment on the same soil was performed to compare the phytoremediation potential of both traditional (crop alone) and assisted phytoremediation techniques (PGPR and chelating agent) in order to quantify (1) HM uptake and (2) understand the plant-soil-microbe interactions. We hypothesized that April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 2 Phyto-Assisted HMs Bioremediation Ferrarini et al. grade). Representative colonies were picked and dereplicated with random ampliﬁed polymorphic DNA (RAPD) ampliﬁcation as detailed in Spini et al. (2018). bioaugmentation with PGPR, more than with chelating agents, can alleviate HM stress on plant growth of these two high-yielding non-food crops and its combination with such deep-rooted crops can help in increasing their phytoremediation potential of HM-contaminated soils and sediments. Soil Collection Site and Plants Preparation The ability of all isolates to withstand increasing metal concentrations was quantiﬁed with a modiﬁcation of the MIC method usually applied for antibiotic. Each strain was grown overnight in tryptone soy broth (TSB), and 100 µl of a 1/10 dilutions were dispensed in 96-well microplates together with 100 µl of TSB at increasing concentrations of 0, 200, 400, 800, 1,600, and 3,200 ppm of NiCl2, CuSO4, and CrCl3. Each strain was tested in 10 replicates. p Surface soil (0–60 cm) and sediment (0–30 cm) for pot experiment were collected within the polluted area around an industrial site operating in the sector of plastic galvanization in Northern Italy. Contaminated and non-contaminated soil and sediments were collected respectively 100 m after and before a factory’s discharge point into the river. Both soil sites (dystric cambisols) are hay meadows while sediment has been colleted within the 5-m-wide sandy ﬂood bed of the river. Soil and sediment samples were air dried and then sieved at 8 mm, mixed, and homogenized, then aliquots were further sieved at 2 mm to eliminate the skeleton and analyzed for their main physiochemical properties and HM levels (Table 1). Soil samples were mainly contaminated by Cr, Ni, and Cu while sediment mainly by Cu according to the Italian legislative limits for public areas (Table 1). Giant reed (Arundo donax L.) rhizomes to be grown in sediments were collected from a 9-year ﬁeld trial (Ferrarini et al., 2020), washed, cut into 3-cm-length pieces and precultivated in peat for 7 days using a modiﬁed Hoagland solution to check for growth rate homogeneity. Hemp (Cannabis sativa L.) seeds of commercially available variety Futura (Hemp- it, France) were used in the experiment. A screening was carried out by selecting strains that had a P solubilizing ability of level 1 or higher and the ability to withstand the metal mixture with a MIC higher than 200 ppm concentration. The retained strain were then tested for their metal biosorption abilities with the method described by Ma et al. (2015). Brieﬂy, strains grown overnight in TSB were washed twice with distilled water and resuspended in 2 ml of distilled water containing 200 ppm of NiCl2, CuSO4, and CrCl3. After 8 h of incubation at 30◦C, the tubes were centrifuged and the unsorbed metals remaining in the cells free supernatant quantiﬁed as described below. Soil Collection Site and Plants Preparation The bacterial pellet was dried and weighted to measure the biomass and normalize accordingly the data obtained. Two strains showing the best biosorption abilities were ﬁnally selected from the soil and the sediment batches respectively, and were identiﬁed by means of Sanger sequencing of 16S rRNA PCR amplicons as described in Spini et al. (2018). The ampliﬁcation was carried out using the primers P0 (5′-GAG AGT TTG ATC CTG GCT-3′) and P6 (5′-CTA CGG CTA CCT TGT TAC-3′) described in Di Cello and Fani (1996) prior to their use in the microbial-assisted phytoremediation experiments; the two strains were grown overnight in TSB to an exponential phase. They were then washed twice with distilled water and ﬁnally resuspended in distilled water to an OD at 600 nm as determined by UV-VIS Spectrophotometer (AT1409001, Aurogene, Italy) corresponding to 1010 CFUs L−1. In the PGPR-treated thesis, the resuspension was added to the soil pots in order to reach a ﬁnal bacterial load of 108 CFUs kg−1 of soil. INTRODUCTION If decreasing stress on root growth and plant photosynthesis this should led to (1) a ﬁne root system more similar to those of the same crops grown on non-contaminated matrices, (2) a higher HM uptake in plant tissues, and (2) a less marked eﬀect of HMs on the microbial community structure and activity of rhizosphere soil. The resulting unique strains were then screened for their phosphate solubilization abilities by spotting them on GY/tricalcium phosphate medium containing Ca3(PO4)2 as insoluble source of phosphorus: the plates were incubated at 30◦C, and after 7 days, the P solubilization ability was quantiﬁed by measuring the halos diameter as previously described (Ambrosini and Passaglia, 2017; Guerrieri et al., 2020). Isolates without a halo were considered non-solubilizers (−); isolates with a halo between 1 and 2 cm as level 1 (++); isolates between 2 and 3 cm as level 2 (++); and isolates with > 3 cm as level 3 (+++). Frontiers in Microbiology \| www.frontiersin.org Isolation, Screening, and Selection of Metal-Chelating Plant Growth-Promoting Bacteria A sequential screening approach was carried out in order to select from the contaminated soil and sediment bacterial strains with the ability to grow under selective pressure of Zn, Cu, and Cr; the resulting strains were then screened and quantiﬁed for plant growth-promoting traits (P solubilization), minimum inhibitory concentrations (MICs), and biosorption abilities toward the three tested metals. Isolations were carried out using three replicates of the same contaminated soil and sediment used for greenhouse pot experiment. Ten grams of soil or sediment were added with 100 ml of sterile physiological solution and placed on a horizontal shaker for 24 h. The obtained slurries were then diluted decimally and plated on tryptone soy agar (TSA) plates containing 100 ppm of each metal as NiCl2·6H2O, CuSO4, and CrCl3·6H2O salts (Carlo Erba reagents, RPE, analytical reagent Greenhouse Pot Experiment In particular, sediment pots with a collecting tube for leaching were built at the bottom of the top to collect leachate solutions before and after treatments. The use of, e.g., EDTA mobilizing agents, indeed, in ﬁeld trials is sensitive and requires adequate greenhouse evaluation prior to upscaling to the ﬁeld scale. Four main treatments (n = 4 replicates) were applied to sediment and soil respectively grown with giant reed and hemp (n = 32 pots, Supplementary Figure 1): not contaminated with crop alone—control (NC), contaminated with crop alone (C), contaminated and treated with PGPR (C+PGRP), and contaminated and treated with EDTA (C+EDTA). Three additional pots for both soil and sediment (C and NC) were kept for the duration of the experiment without plant to characterize microbial diversity without plants (Supplementary Figure 1). Pots were inoculated with PGPR and irrigated with EDTA twice during the experiment: 22 and 51 days after transplanting (DAT) for the giant reed pots and 34 and 53 days after sowing (DAS) for the hemp pots. PGPR were inoculated via irrigation of pots of 1 L solution of 108 UFC ml−1 of selected bacteria strain (Section “Isolation, Screening, and Selection of Metal-Chelating Plant Growth-Promoting Bacteria”). EDTA (Carlo Erba reagents, RPE, analytical reagent grade) were applied as 1 L solutions at 0.5 g kg−1 concentration as suggested by Shahid et al. (2014) for the same HMs and C3 plants. Both solutions were inoculated at the end of the lighting period in order to allow the plants to adapt to the solutions and to show the inoculation eﬀects on photosynthetic performances the following day. At the end of the experiment (78 DAT and respectively 56 and 28 days after ﬁrst and second applications), aboveground (ABG) and belowground (BGB) biomass were harvested from all pots (n = 32). Leaves and stems were sampled separately for giant reed, while hemp samples were sampled from leaves, stem, and ﬂowers. Aboveground biomass sample were dried at 65◦C to determine dry matter content and then samples were milled and sieved at 1 mm for HM analysis. Belowground samples (roots for hemp and rhizome+root for giant reed) were carefully washed with distilled water before root analysis (Section “Fine Root System Characterization”). The rhizosphere soil (RS) was collected for each pot according to previously described methodology (Barillot et al., 2013; Marasco et al., 2018; Guerrieri et al., 2020). Greenhouse Pot Experiment Pot dry weights were respectively 6 and 7.2 kg for giant reed and hemp, respectively. Soil has been maintained at 60% of water holding capacity while sediment at 100% WHC to simulate sediment water conditions. This implies that the ﬁndings derived from this study will require ﬁeld validation. In particular, sediment pots with a collecting tube for leaching were built at the bottom of the top to collect leachate solutions before and after treatments. The use of, e.g., EDTA mobilizing agents, indeed, in ﬁeld trials is sensitive and requires adequate greenhouse evaluation prior to upscaling to the ﬁeld scale. Four main treatments (n = 4 replicates) were applied to sediment and soil respectively grown with giant reed and hemp (n = 32 pots, Supplementary Figure 1): not contaminated with crop alone—control (NC), contaminated with crop alone (C), contaminated and treated with PGPR (C+PGRP), and contaminated and treated with EDTA (C+EDTA). Three additional pots for both soil and sediment (C and NC) were kept for the duration of the experiment without plant to characterize microbial diversity without plants (Supplementary Figure 1). Pots were inoculated with PGPR and irrigated with EDTA twice during the experiment: 22 and 51 days after transplanting (DAT) for the giant reed pots and 34 and 53 days after sowing (DAS) for the hemp pots. PGPR were inoculated via irrigation of pots of 1 L solution of 108 UFC ml−1 of selected bacteria strain (Section “Isolation, Screening, and Selection of Metal-Chelating Plant Growth-Promoting Bacteria”). EDTA (Carlo Erba reagents, RPE, analytical reagent grade) were applied as 1 L solutions at 0.5 g kg−1 concentration as suggested by Shahid et al. (2014) for the same HMs and C3 plants. Both solutions were inoculated at the end of the lighting period in order to allow the plants to adapt to the solutions and to show the inoculation eﬀects on photosynthetic performances the following day. reed were transplanted into ﬁlled pot of 16 L (60 cm height, 16 cm diameter) and cultivated 60 days (25:16◦C day:night temperature, with a photoperiod of 16 h). One rhizome/seed were transplanted/sown per pot. Pot dry weights were respectively 6 and 7.2 kg for giant reed and hemp, respectively. Soil has been maintained at 60% of water holding capacity while sediment at 100% WHC to simulate sediment water conditions. This implies that the ﬁndings derived from this study will require ﬁeld validation. Greenhouse Pot Experiment The comparison of the phytoremediation potential with common practices (crop alone) as compared with assisted phytoremediation techniques (PGPR and chelating agent) was conducted through a pot experiment performed under controlled conditions. Hemp seeds and rhizome of giant April 2021 \| Volume 12 \| Article 645893 3 Phyto-Assisted HMs Bioremediation Ferrarini et al. TABLE 1 \| Main physiochemical parameters of for contaminated (C) and non-contaminated (NC) soil and sediments and their total chromium (Cr), nickel (Ni), and copper (Cu) concentrations (mg kg−1) at the beginning of the experiment. Sand Silt Clay Texture class Field capacity Wilting point SOM N tot C/N pH CaCO3 Cr* Ni* Cu* % % % % % % % % mg kg−1 NC soil 47 31 23 Loam 26 14 1.9 0.09 12.1 6.9 1.5 19.1 21.7 41.7 Contaminated soil 45 38 17 Loam 26 13 2.0 0.10 11.3 6.8 2.0 97 526 172 NC sediment 87 9 4 Sand 13 5 0.9 0.07 7.5 7.4 3.0 24.8 26.8 39 Contaminated sediment 83 12 5 Loamy sand 14 5 1.1 0.08 7.9 7.3 3.2 33.5 133.2 64.9 Bold values above the screening values are values established for residential soil use by the Italian Ministry of Environment (DM 152/2006). Legislative limits currently adopted in Italy (DM 152/2006) for HM concentration in soil for green areas/residential use are respectively 150, 120, and 120 mg kg−1 for Cr, Ni, and Cu. TABLE 1 \| Main physiochemical parameters of for contaminated (C) and non-contaminated (NC) soil and sediments and their total chromium (Cr), nickel (Ni), and copper (Cu) concentrations (mg kg−1) at the beginning of the experiment. s are values established for residential soil use by the Italian Ministry of Environment (DM 152/2006). Italy (DM 152/2006) for HM concentration in soil for green areas/residential use are respectively 150, 120, and 120 mg kg−1 Bold values above the screening values are values established for residential soil use by the Italian Ministry of Environment (DM 152/2006). Legislative limits currently adopted in Italy (DM 152/2006) for HM concentration in soil for green areas/residential use are respectively 150, 120, and 120 mg kg−1 for Cr, Ni, and Cu. solutions were immediately ﬁltered at 0.45 µm and stored at −18◦C until analysis. reed were transplanted into ﬁlled pot of 16 L (60 cm height, 16 cm diameter) and cultivated 60 days (25:16◦C day:night temperature, with a photoperiod of 16 h). One rhizome/seed were transplanted/sown per pot. Greenhouse Pot Experiment Brieﬂy, bulk soil (BS) was removed by shaking plants by hand for 10 min vigorously, paying attention to the roots’ integrity, as long as the roots’ non-adhering soil particles were completely removed. In order to collect rhizosphere soil, the root system was washed with 500 ml of 0.9% NaCl added and afterward 150 ml of bacterial suspension were incubated at 25◦C for 90 min with shaking at 180 rpm. BS and RS were immediately dried at 65◦C for HM analyses and stored at −18◦C until soil enzyme activities and DNA extraction for bacterial diversity analysis. Soil and sediment total Cr, Ni, and Cu concentrations were analyzed at beginning (Tzero) on BS samples and at the end of experiment (Tf inal) on either BS and RS samples. Soil and sediment samples were digested with a solution of aqua regia (HCl:HNO3 in a volume ratio 3:1) and heated under reﬂux, after pretreatment with H2O2; Ni, Cr, and Cu concentrations were determined in all samples by graphite furnace atomic absorption spectrometry (GFAAS) (Perkin-Elmer AA-600). Above and belowground dry samples (1 g subsamples from each individual pot sample) were analyzed for total Ni, Cr, and Cu concentrations as in Watanabe et al. (2015). The samples were digested in a solution of 6 ml of concentrated HNO3 and 1 ml of H2O2, the solution was heated at 110◦C for 2 h, and then distilled water was added to reach the volume of 50 ml, the solution was ﬁltered at 0.45 µm and then read with ICP-MS (Agilent 7900). Fine Root System Characterization Once cleaned, roots were hand recovered from the water using a 2-mm mesh sieve. Determination of root length density (RLD, cm cm−3) and root diameters was performed with the software winRHIZO Pro 2019. The images were acquired using the TWAIN interface at 600 dpi and with a scanner (model: Epson Expression 10000xl) equipped with a double light source to avoid roots overlapping. Fine roots dry biomass weight was determined gravimetrically, after taking scanned images, drying the roots at 60◦C until constant weight. The dried ﬁne root sample were then analyzed for total HM concentration as in Section “Heavy Metals Determination on Plant, Soil, and Leachate Samples.” The diameter class length (DCL, mm cm−3) was calculated for very ﬁne (0.0–0.5 mm), ﬁne (0.5–2 mm), and coarse (>2 mm) diameters for both crops. The DCL was calculated for 13- diameter classes from 0 to 3.15 mm (with a 0.15-mm increase per class). To describe crops’ DCL distribution as aﬀected by treatments, the DCL data of hemp and giant reed were ﬁtted with the non-linear regression extreme value model (Curve expert Professional 2.6.4) as suggested by Zobel et al. (2007) and successfully applied to biomass crops by Chimento and Amaducci (2015): Once cleaned, roots were hand recovered from the water using a 2-mm mesh sieve. Determination of root length density (RLD, cm cm−3) and root diameters was performed with the software winRHIZO Pro 2019. The images were acquired using the TWAIN interface at 600 dpi and with a scanner (model: Epson Expression 10000xl) equipped with a double light source to avoid roots overlapping. Fine roots dry biomass weight was determined gravimetrically, after taking scanned images, drying the roots at 60◦C until constant weight. The dried ﬁne root sample were then analyzed for total HM concentration as in Section “Heavy Metals Determination on Plant, Soil, and Leachate Samples.” Molecular Analyses of Bulk and Rhizosphere Bacterial Diversity Soil and rhizosphere samples from the hemp and the giant reed experiments were collected at the beginning and at the end of the experiments and analyzed in quadruplicates for bacterial diversity by means of high-throughput sequencing (HTS) of 16S rRNA amplicons. The procedure applied is described in detailed in Spini et al. (2018) and summarized as follows. Total microbial DNA was extracted from 0.5 g of each soil sample with the Fast DNATM SPIN Kit for Soil (MP Biomedicals, United States) with the number of modiﬁcations: homogenization in the FastPrep R⃝for 40 s at speed setting of 6.5 twice, keeping in ice between the two homogenization steps, ﬁnal centrifugation at 14,000 × g for 15 min, and the ﬁnal resuspension of the binding matrix was carried out in 50 µl−1 of nuclease-free water. The DNA quality of each sample was checked with electrophoresis on a 1% agarose gel, and QuBitTM ﬂuorometer (Invitrogen, United Kingdom) quantiﬁcation was carried out in order to dilute each DNA sample to 1 ng µl−1 concentration. PCR ampliﬁcations of the V3-V4 regions of bacterial 16S rRNA were carried out using the universal primers 343f (5′-TACGGRAGGCAGCAG-3′) and 802r (5′-TACNVGGGTWTCTAATCC-3′) (Połka et al., 2015). Ampliﬁcations were carried out in two steps, a ﬁrst with untagged primers in order to reduce the possibility of preferential primers annealing (Berry et al., 2011) and a second step using a dedicated forward primer with a 9-base extension at the 5’ end, which acts as a tag, in order to make simultaneous analyses of all samples in a single sequencing run possible. The PCR reaction mix is composed of 20.5 µl of MegaMix (Microzone Limited, The diameter class length (DCL, mm cm−3) was calculated for very ﬁne (0.0–0.5 mm), ﬁne (0.5–2 mm), and coarse (>2 mm) diameters for both crops. The DCL was calculated for 13- diameter classes from 0 to 3.15 mm (with a 0.15-mm increase per class). To describe crops’ DCL distribution as aﬀected by treatments, the DCL data of hemp and giant reed were ﬁtted with the non-linear regression extreme value model (Curve expert Professional 2.6.4) as suggested by Zobel et al. (2007) and successfully applied to biomass crops by Chimento and Amaducci (2015): DCL (mm/cm3) = a + b e−e −x−c d −x−c d +1 where x refers to diameter class (mm). Heavy Metals Determination on Plant, Soil, and Leachate Samples To assess the performance of the phytoextraction-assisted bioaugmentation with PGPR and addition of chelating agents, the following factors (Lebeau et al., 2008; Ali et al., 2013) were Leachate solution were collected in 1 L ﬂask from the bottom of the n = 16 giant reed pots 17, 26, 38, and 60 DAT April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 4 Phyto-Assisted HMs Bioremediation Ferrarini et al. Whole-Canopy Gas Exchange Measurements py g The day before the ﬁrst EDTA/PGPR application, hemp and giant reed pots were placed into a semi-automated gas exchange platform to measure whole-canopy gas exchanges for 7 days. Canopy net assimilation rate (Pn) and transpiration rate (E) were determined with a self-assembled multichamber gas exchange apparatus (fully described in Fracasso et al., 2017). In brief, in this system, air is drawn from outside and blown into the chambers while a CIRAS-DC double-channel absolute CO2/H2O infrared gas analyzer (PP-System) combined to a datalogger measures continuously, 24 h day−1, CO2 and H2O concentrations at the inlet and outlet of each chamber. Pn and E were calculated from ﬂow rates and CO2 and water vapor diﬀerentials using the formula provided by Long and Hällgren (1993). Microbial Biomass and Enzyme Activities of Bulk and Rhizosphere Soils calculated for hemp and giant reed: plant biomass (mg pot−1), concentration (mg kg−1) and amount of metal extracted by plants (µg pot−1 in each plant component), bioconcentration (BCF), and translocation (TF) factors deﬁned, respectively, as the metal in AGB to the metal in soil ratio and the metal in AGB to the metal in BGB ratio. BGB in hemp was only roots while root and rhizomes in giant reed. Twenty soil enzymatic activities (EA) involved in key steps of soil C, N, P, and S cycling were measured: (i) α-glucosidase (agluc, EC 3.2.1.20), β-glucosidase (bgluc, EC.3.2.1.21), α-galactosidase (alfaGAL, EC 3.2.1.22), β-galactosidase (betaGAL, EC 3.2.1.23), α-mannosidase (alfaMAN, EC 3.2.1.24), β-mannosidase (betaMAN, EC 3.2.1.25), β-D-glucuronidase (uroni, EC 3.2.1.31); β-1,4-glucanase (cell, EC 3.2.1.4), β-1,4-xylanase (xilo, EC 3.2.1.8) involved in C cycling; (ii) N-acetyl-b-D- glucosaminidase (chit, EC 3.2.1.14), leucine amino-peptidase (leu, EC.3.4.11.1.), trypsin-like protease (tryp, EC 3.4.21. 4) involved in N cycling; (iii) acid (acP, EC.3.1.3.2) and alkaline phosphomonoesterase (alkP, EC.3.1.3.1), phosphodiesterase (bisP, E.C.3.1.4.1.), pyrophosphodiesterase (piroP, EC.3.6.1.9.), inositol-P phosphatase (inositP, EC 3.1.3.25) involved in P cycling; (iv) arylsulfatase (aryS, EC.3.1.6.1.) involved in S cycling; and (v) non-anoate (nona) and palmitate (palmit) esterase (EC 3.1.) involved in the hydrolysis of ester bonds. EA were determined on soil extracts (Bardelli et al., 2017) using ﬂuorogenic substrates containing 4-methyl–umbelliferyl (MUF) and 7-amino-4-methyl coumarin (AMC) as ﬂuorophores. Soil enzymes were desorbed by heteromolecular exchange procedure via bead-beating according to Ferrarini et al. (2020). Soil microbial biomass was determined as double-strand DNA (dsDNA) content (Fornasier et al., 2014). Molecular Analyses of Bulk and Rhizosphere Bacterial Diversity Step 2: initial hold at 95◦C for 5 min, followed by 10 cycles of 95◦C for 30 s, 50◦C for 30 s, and 30◦C for 30 s; then, a ﬁnal extension at 72◦C for 10 min. The DNA ampliﬁcations were checked with electrophoresis on a 1% agarose gel, and then quantiﬁed using a QuBitTM ﬂuorometer (Invitrogen, United Kingdom). PCR products generated from the second step were multiplexed as a single pool using equivalent molecular weights (20 ng). The pool was then puriﬁed using the solid-phase reversible immobilization (SPRI) method with Agencourt AMPure XP kit (REF A63880, Beckman Coulter, Milan, Italy), then sequenced by Fasteris S.A. (Geneva, Switzerland). The TruSeq DNA sample preparation kit (REF 15026486, Illumina Inc., San Diego, CA, United States) was used for amplicon library preparation, whereas the sequencing was carried out with the MiSeq Illumina instrument (Illumina Inc., San Diego, CA, United States) generating 300 bp paired- end reads. sequences following the operational taxonomic units (OTUs) at 97% similarity, and the taxonomy-based approach, which was implemented using an amended version of the Greengenes database (McDonald et al., 2012). Sequence data were submitted to the National Centre for Biotechnology Information Sequence Read Archive (BioProject PRJNA687540). Soil EA and OTU from microbial sequencing were analyzed through multivariate analysis (distance-based redundancy analyses (dbRDA)) while OTU was also analyzed with hierarchical clustering. We used Mothur and R for statistical analyses on OTU and taxonomy matrixes using hierarchical clustering with the average linkage algorithm at diﬀerent taxonomic levels. Statistical Analyses Heavy metal uptake and concentration data were analyzed separately for both crops and their relative plant components using a one-way ANOVA with treatment (C, C+PGPR, C+EDTA) as main eﬀect. HM concentration in leachate and Simpson diversity index were analyzed using a two- way mixed-model ANOVA for complete a randomized design. Treatment combination and sampling time (Tz, Tf ) and their interaction were considered ﬁxed main eﬀects with replicates as a random eﬀect. Molecular Analyses of Bulk and Rhizosphere Bacterial Diversity dbRDA was run on a three step basis (Ferrarini et al., 2020) separately for soil type (BS, RS) of hemp and giant reed: (1) Bray–Curtis dissimilarity (non-linear) matrix is calculated on square root transformed data for soil EA and raw data for OTU database; (2) stepwise multiple regression was performed to select the best model (AIC) including environmental variables only for soil EA data and OTU database of RS; (3) a principal coordinate analysis (PCoA) is calculated based on the distance matrix (999 permutations) to obtain dbRDA axis coordinates for main treatments (treatment for soil EA data and Trt × sampling time for bacterial diversity data) to be plotted as multivariate centroids surrounded by 95% conﬁdence interval ellipsoids and coordinates of species (only for soil EA) and environmental variables (RLD and soil HM concentrations) respectively as points and arrows; (3) one-way permutational multivariate analysis of variance (PERMANOVA) based on Bray-Curtis matrix was conducted for 9,999 permutations was used to test for main treatment eﬀects on soil EA and sequencing data with replicate as random eﬀect. Planned contrasts of PERMANOVA, according to Bonferroni’s test (P > 0.05) were set as follows: treatment vs. soil EA grouped by element cycle (C-, N-, P-, and S-cycling, and esterases) and all contrasts for treatment × sampling time interaction terms in the case of PERMANOVA on sequencing data. A fourth dbRDA step was only run for soil EA data (Mattarozzi et al., 2020). Brieﬂy, a similarity percentage (SIMPER) was used to select the soil EA accounting for > 90% of cumulative dissimilarity between each of all planned contrasts for main treatments (NC, C, C+EDTA, C+PGPR). dbRDA, PERMANOVA, and SIMPER analysis were run by using vegan R packages (capscale, pairwise.adonis and simper functions, respectively). High-throughput sequencing data ﬁltering, multiplexing, and preparation for subsequent statistical analyses were carried out as previously detailed (Vasileiadis et al., 2015). Paired reads were assembled to reconstruct the full V3-V4 amplicons using the FLASH assembler (Magoˇc and Salzberg, 2011), and samples were demultiplexed according to their tag using SeqKit (Shen et al., 2016). Further screenings were carried out with Mothur (Schloss et al., 2009) in order to remove sequences with large homopolymers (≥10), sequences that did not align within the targeted V3-V4 region, chimeric sequences, and sequences not classiﬁed as bacterial. Sequence data were submitted to the National Centre for Biotechnology Information Sequence Read Archive (BioProject PRJNA687540). Molecular Analyses of Bulk and Rhizosphere Bacterial Diversity In general, the coeﬃcient a (baseline) is the value approached by DCL as x approaches positive or negative inﬁnity, b is the DCL peak value minus a, c is diameter class at peak value (the x-axis location of b), and d (amplitude of the curve) is related to the width across the curve at half maximum (b / 2 + a) so that width at half maximum equals 2.446 d (Zobel et al., 2007). April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org Frontiers in Microbiology \| www.frontiersin.org 5 Phyto-Assisted HMs Bioremediation Ferrarini et al. United Kingdom), 1.25 µl of each primer (10 µM), and 2 µl (1 ng µl−1 concentration) of DNA template. Thermal cycling conditions were as follows: Step 1: an initial denaturation at 94◦C for 5 min, followed by 25 cycles at 94◦C for 30 s, 50◦C for 30 s, 72◦C for 30 s, followed by a ﬁnal extension at 72◦C for 10 min. Step 2: initial hold at 95◦C for 5 min, followed by 10 cycles of 95◦C for 30 s, 50◦C for 30 s, and 30◦C for 30 s; then, a ﬁnal extension at 72◦C for 10 min. The DNA ampliﬁcations were checked with electrophoresis on a 1% agarose gel, and then quantiﬁed using a QuBitTM ﬂuorometer (Invitrogen, United Kingdom). PCR products generated from the second step were multiplexed as a single pool using equivalent molecular weights (20 ng). The pool was then puriﬁed using the solid-phase reversible immobilization (SPRI) method with Agencourt AMPure XP kit (REF A63880, Beckman Coulter, Milan, Italy), then sequenced by Fasteris S.A. (Geneva, Switzerland). The TruSeq DNA sample preparation kit (REF 15026486, Illumina Inc., San Diego, CA, United States) was used for amplicon library preparation, whereas the sequencing was carried out with the MiSeq Illumina instrument (Illumina Inc., San Diego, CA, United States) generating 300 bp paired- end reads. United Kingdom), 1.25 µl of each primer (10 µM), and 2 µl (1 ng µl−1 concentration) of DNA template. Thermal cycling conditions were as follows: Step 1: an initial denaturation at 94◦C for 5 min, followed by 25 cycles at 94◦C for 30 s, 50◦C for 30 s, 72◦C for 30 s, followed by a ﬁnal extension at 72◦C for 10 min. Frontiers in Microbiology \| www.frontiersin.org RESULTS Pn and E data were analyzed via one-way ANOVA and, when the F-test was signiﬁcant, mean separation was performed by the t-test at P < 0.05 and P < 0.01. Degree of variation around means is given as standard error (SE). All ANOVA were performed with agricolae R package while post hoc men separation via multicomp R package. Selection of Bacterial Strains for Microbial-Assisted Phytoremediation Performances for Cr, Ni, and Cu Heavy Metals Accumulation in Plant Organs of Hemp and Giant Reed Heavy metal concentration and total concentration of heavy metals (Cr, Ni, and Cu) in BS at the end of the experiment generally did not decrease (Supplementary Figure 2). Only Ni were signiﬁcantly lower with hemp treated with EDTA and PGRP (F = 12, P = 0.04). Plant yield was signiﬁcantly aﬀected by HM pollution in sediment (−19%) and soil (−16%) as shown by tolerance index (TI) values (Table 2). Bioaugmentation with PGPR signiﬁcantly alleviate HM stress on plant yield showing no diﬀerence in plant yield compared with non-contaminated sediment (NC). Giant reed and hemp treated with PGPR showed a TI of 117 and 89%, respectively. So_18 - 800 nd nd So_19 - 400 nd nd So_20 - 800 nd nd So_21 + 1600 nd nd So_22 - 800 nd Nd Se_01 ++ 800 4.64 6.62 Se_02 +++ 400 7.5 13.5 Se_03 + 800 4 5.5 Se_04 + 800 nd Nd Se_05 + 800 nd Nd Se_06 + 1600 nd Nd Heavy metal concentration and total concentration of heavy metals (Cr, Ni, and Cu) in BS at the end of the experiment generally did not decrease (Supplementary Figure 2). Only Ni were signiﬁcantly lower with hemp treated with EDTA and PGRP (F = 12, P = 0.04). Plant yield was signiﬁcantly aﬀected by HM pollution in sediment (−19%) and soil (−16%) as shown by tolerance index (TI) values (Table 2). Bioaugmentation with PGPR signiﬁcantly alleviate HM stress on plant yield showing no diﬀerence in plant yield compared with non-contaminated sediment (NC). Giant reed and hemp treated with PGPR showed a TI of 117 and 89%, respectively. y Heavy metals content (Figure 1) and uptake (Figure 2) were signiﬁcantly enhanced by bioaugmentation with PGPR and addition of EDTA chelating agent in contaminated soil and sediments. Bioconcentration (BCF) and translocation (TF) factors of chromium, nickel, and copper in the belowground and aboveground of giant reed and hemp were depicted in Table 3. The three HMs assessed in this study showed clear distant accumulation (Figure 1) and uptake (Figure 2) patterns among plant organs. All plant organs showed Cr, Ni, and Cu accumulations (Figure 1). Among organs, the concentration trend was belowground organs (rhizomes, roots) > > aboveground organs (leaves, stems) for Cr and Ni whereas Cu showed similar concentration in belowground and aboveground plant organs (Figure 1). Selection of Bacterial Strains for Microbial-Assisted Phytoremediation Isolation and molecular ﬁngerprint genotyping resulted in a total of 42 unique strains: 22 derived from the contaminated sediments and 20 from the contaminated soil. The assessment of phosphate solubilization ability and MIC for the three tested metals are reported in Table 2, together with measurement on biosorption abilities on 12 selected strains. Seven out of 22 strains from soil had P solubilization ability, with one (strain So17) having to generate a larger halo. Regarding sediment strains, 12 out of 17 had P solubilization abilities, with one as well To determine whether treatments inﬂuence ﬁne root systems, especially whether roots become thinner or thicker in response to treatments, the statistical signiﬁcance of the DCL curve parameters (a–d) were assessed through testing their standard errors using the t-statistics at P < 0.05. Relatively to 16S, Mothur and R were employed to analyze the resulting high-quality April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 6 Phyto-Assisted HMs Bioremediation Ferrarini et al. TABLE 2 \| Screening of isolated strains for P solubilization ability, minimum inhibitory concentrations (MICs) of Cr, Cu, and Ni and biosorption (BS) toward the three testes metals. having level 3. The ability to withstand high metal concentrations was conﬁrmed by MIC values, that in most cases had values of 800 ppm or more, thus much higher than the selective concentration used in the isolations. According to the results obtained for P solubilization and MIC, 12 strains (six from soil and six from sediments) were selected for the measurement of metal biosorption ability, an important trait to improve the phytoremediation potential. Data were normalized per gram of dry cells and showed values between 0.1 and 13.5 mg of metal per gram of dry cell biomass (Table 2). The highest biosorption levels were found for strains So17 among soil isolates and strain Se02 among sediment isolates: interestingly those were also among the three strains that had the highest P solubilization abilities. Strains So17 and Se02 were thus selected for the microbial-assisted phytoremediation experiments and taxonomically identiﬁed as Enterobacter spp. (So17) and Enterobacter asburiae (Se02) (GenBank submission SUB9058427). having level 3. The ability to withstand high metal concentrations was conﬁrmed by MIC values, that in most cases had values of 800 ppm or more, thus much higher than the selective concentration used in the isolations. Selection of Bacterial Strains for Microbial-Assisted Phytoremediation According to the results obtained for P solubilization and MIC, 12 strains (six from soil and six from sediments) were selected for the measurement of metal biosorption ability, an important trait to improve the phytoremediation potential. Data were normalized per gram of dry cells and showed values between 0.1 and 13.5 mg of metal per gram of dry cell biomass (Table 2). The highest biosorption levels were found for strains So17 among soil isolates and strain Se02 among sediment isolates: interestingly those were also among the three strains that had the highest P solubilization abilities. Strains So17 and Se02 were thus selected for the microbial-assisted phytoremediation experiments and taxonomically identiﬁed as Enterobacter spp. (So17) and Enterobacter asburiae (Se02) (GenBank submission SUB9058427). Phyto-Assisted Bioremediation Performances for Cr, Ni, and Cu Heavy Metals Accumulation in Plant Organs of Hemp and Giant Reed Heavy metal concentration and total concentration of heavy metals (Cr, Ni, and Cu) in BS at the end of the experiment generally did not decrease (Supplementary Figure 2). Only Ni were signiﬁcantly lower with hemp treated with EDTA and PGRP (F = 12, P = 0.04). Plant yield was signiﬁcantly aﬀected by HM pollution in sediment (−19%) and soil (−16%) as shown by tolerance index (TI) values (Table 2). Bioaugmentation with PGPR signiﬁcantly alleviate HM stress on plant yield showing no diﬀerence in plant yield compared with non-contaminated sediment (NC). Giant reed and hemp treated with PGPR showed a TI of 117 and 89%, respectively. Heavy metals content (Figure 1) and uptake (Figure 2) were signiﬁcantly enhanced by bioaugmentation with PGPR and addition of EDTA chelating agent in contaminated soil and sediments. Bioconcentration (BCF) and translocation (TF) factors of chromium, nickel, and copper in the belowground and aboveground of giant reed and hemp were depicted in Table 3. The three HMs assessed in this study showed clear distant accumulation (Figure 1) and uptake (Figure 2) patterns among plant organs. All plant organs showed Cr, Ni, and Cu accumulations (Figure 1). Among organs, the concentration trend was belowground organs (rhizomes, roots) > > aboveground organs (leaves, stems) for Cr and Ni whereas Cu showed similar concentration in belowground and aboveground plant organs (Figure 1). Hemp translocated more Cu and Ni in ABG than BGB than giant reed that instead showed the opposite for Cr (Table 3). Selection of Bacterial Strains for Microbial-Assisted Phytoremediation TF showed the following crop ranking for the three HMs: Cr (giant reed 0.15 > hemp 0.05), Cr (hemp 0.46 > giant reed 0.07), and Cu (hemp 0.44 > giant reed 0.30). BCF values for ABG (Table 3) was in general similar among phytoremediation technique with giant reed showing higher BCF than hemp for Cr (0.062 vs. 0.003), Ni (0.04 vs. 0.12), and Cu (0.32 vs. 0.22). The only exception was TABLE 2 \| Screening of isolated strains for P solubilization ability, minimum inhibitory concentrations (MICs) of Cr, Cu, and Ni and biosorption (BS) toward the three testes metals. Strain P solubilizationa MIC (mg L−1) Cr BS (mg g−1 drycells) Cu BS (mg g−1 drycells) Ni BS (mg g−1 drycells) So_01 + 800 1.14 0.9 2.3 So_02 - 400 nd nd nd So_03 + 800 5.88 8.19 2.08 So_04 - 800 nd nd nd So_05 - 400 nd nd nd So_06 - 1600 nd nd nd So_07 - 800 nd nd nd So_08 + 800 5.59 8.73 3.55 So_09 ++ 200 nd nd nd So_10 - 400 nd nd nd So_11 - 800 nd nd nd So_12 + 800 4.13 7 3.25 So_13 - 800 nd nd nd So_14 - 400 nd nd nd So_15 - 800 nd nd nd So_16 + 400 0.133 3 0.17 So_17 +++ 800 3.25 5.42 1.67 So_18 - 800 nd nd nd So_19 - 400 nd nd nd So_20 - 800 nd nd nd So_21 + 1600 nd nd nd So_22 - 800 nd Nd nd Se_01 ++ 800 4.64 6.62 2.13 Se_02 +++ 400 7.5 13.5 2.1 Se_03 + 800 4 5.5 0.9 Se_04 + 800 nd Nd nd Se_05 + 800 nd Nd nd Se_06 + 1600 nd Nd nd Se_07 + 1600 nd Nd nd Se_08 + 1600 nd Nd nd Se_09 + 800 nd Nd nd Se_10 ++ 800 4.15 5.65 0.23 Se_11 + 400 0.5 1.67 1.5 Se_12 - 400 nd Nd nd Se_13 - 400 nd Nd nd Se_14 - 400 nd Nd nd Se_15 - 200 nd Nd nd Se_16 - 200 nd Nd nd Se_17 + 800 nd Nd nd Se_18 ++ 400 1.75 2.75 0.25 Se_19 - 800 nd Nd nd Se_20 + 800 nd Nd nd The two strains ﬁnally selected for the phytoremediation experiments are highlighted in bold. Selection of Bacterial Strains for Microbial-Assisted Phytoremediation aData for P solubilization abilities are categorized in four groups: non-solubilizers (no halo on GYT medium); + level 1 solubilizers (halo between 1 and 2 cm) as level 1; ++ level 2 solubilizers (halo between 2 and 3 cm); +++ ++ level 2 solubilizers (halo > 3 cm). observed in the BCF-ABG of Ni for hemp treated with EDTA (0.29) that was signiﬁcantly higher than other treatments (0.04). EDTA increased signiﬁcantly Cu concentration in belowground Performances for Cr, Ni, and Cu Heavy Metals Accumulation in Plant Organs of Hemp and Giant Reed Hemp translocated more Cu and Ni in ABG than BGB than giant reed that instead showed the opposite for Cr (Table 3). TF showed the following crop ranking for the three HMs: Cr (giant reed 0.15 > hemp 0.05), Cr (hemp 0.46 > giant reed 0.07), and Cu (hemp 0.44 > giant reed 0.30). BCF values for ABG (Table 3) was in general similar among phytoremediation technique with giant reed showing higher BCF than hemp for Cr (0.062 vs. 0.003), Ni (0.04 vs. 0.12), and Cu (0.32 vs. 0.22). The only exception was observed in the BCF-ABG of Ni for hemp treated with EDTA (0.29) that was signiﬁcantly higher than other treatments (0.04). EDTA increased signiﬁcantly Cu concentration in belowground April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 7 Phyto-Assisted HMs Bioremediation Ferrarini et al. FIGURE 1 \| Mean values of Cr, Ni, and Cu concentration (mg kg−1) in plant components for hemp (roots, stems, leaves, and ﬂowers) and giant reed (rhizome, roots, stems, and leaves) as affected by treatments. Different letters denote statistically different (Tukey’s test, P = 0.05) concentration values among treatment for each HM/crop combination. FIGURE 1 \| Mean values of Cr, Ni, and Cu concentration (mg kg−1) in plant components for hemp (roots, stems, leaves, and ﬂowers) and giant reed (rhizome, roots, stems, and leaves) as affected by treatments. Different letters denote statistically different (Tukey’s test, P = 0.05) concentration values among treatment for each HM/crop combination. sediment (99.7, 99.5, and 96.3%) and in the soil (>99%). A maximum of 1.45% of Cu, 0.72% of Ni, and 0.5% of Cr in the sediment was removed by giant reed treated with EDTA although at the same time 7.9% of Cu and 3% of Ni was lost with leaching. Hemp showed a lower HM removal from soil mass balance than giant reed with less variation among treatments (Supplementary Figure 3). An average of 30, 26, and 37h of Cu, Ni, and Cr, respectively, in soil was removed by hemp. organs of giant reed while in hemp either aboveground and belowground organs had higher Cu concentration with EDTA addition than C and C+PGRP (Figure 1). EDTA addition increased signiﬁcantly only Ni concentration in hemp leaves and giant reed rhizomes. Cr concentration in ABG is generally less aﬀected by phytoremediation techniques. Performances for Cr, Ni, and Cu Heavy Metals Accumulation in Plant Organs of Hemp and Giant Reed Only rhizomes of giant reed showed a signiﬁcantly higher Cr concentration than C and C+EDTA. EDTA greatly enhances BCF of Ni and Cu in belowground organs of both crops especially (Table 3). HM element concentrations decreased diﬀerently in the plant organs of giant reed and hemp (Figure 1). Considering leaching in sediments cultivated with giant reed, levels of heavy metals in the leachate were diﬀerentially aﬀected by EDTA (Supplementary Table 1). With EDTA, Ni and Cu leached easily after two applications showing, at the end of the experiment, signiﬁcantly higher concentration of Ni (20.4 mg L−1) and Cu (17.9 mg L−1). Peak concentration of Cu in leachate was observed already 4 days after treatment while 16 DAT for Ni. Without EDTA, heavy metal concentration in leachates were very low on average (Cr: 2.68 ng L−1, Ni: 0.04 mg L−1, and Cu: 0.03 mg L−1). Bioaugmentation with PGPR never sustained HM leaching compared with sediment contaminated alone. Overall ABG contributed very little to HM removal either in terms of mass balance (h) (Supplementary Figure 3) and HM uptake at plant level (µg tissue−1) (Figure 2). The two plant micronutrients, Ni and Cu, showed very low whole plant uptake values (Ni: < 4 µg plant−1 and Cu: < 2 µg plant−1) for both crops grown in NC soil and sediment, respectively. In giant reed, compared with contaminated control (C), the signiﬁcantly highest uptake values were observed in rhizomes with EDTA (Ni: 4.8 mg plant−1, Ni: 3.4 mg plant−1) and with PGPR either in leaves (Cr: 51 µg plant−1, Ni: 27 µg plant−1, Cu: 130 µg plant−1) and in stems (Ni: 41 µg plant−1, Cu: 137 µg plant−1). Roots of giant reed also contributed signiﬁcantly to BGB HM mass balance (Figure 2 and Supplementary Figure 3). PGPR and EDTA signiﬁcantly increased Cr and Cu root uptake in giant reed (Figure 2). In hemp, compared with the contaminated control (C), the signiﬁcantly highest uptake values were observed for Cu in stems, leaves, and ﬂowers with EDTA Heavy Metals Mass Balance and Uptake Supplementary Figure 3 shows the HM mass balance for the hemp and giant reed experiments. At the time scale of the experiment, main Cu, Ni, and Cr remained in the April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 8 Phyto-Assisted HMs Bioremediation Ferrarini et al. FIGURE 2 \| Summary of mean values of Cr, Ni, and Cu uptake (µg HM plant−1) in plant components for hemp and giant reed as affected by treatments. Different letters denote statistically different (Tukey’s test, P = 0.05) uptake values among treatment for each HM. FIGURE 2 \| Summary of mean values of Cr, Ni, and Cu uptake (µg HM plant−1) in plant components for hemp and giant reed as affected by treatments. Different letters denote statistically different (Tukey’s test, P = 0.05) uptake values among treatment for each HM. (950, 760, and 91 µg plant−1) and for Ni in stems with EDTA (2,800 µg plant−1). EDTA inoculation, no statistically signiﬁcant diﬀerences were highlighted between the three treatments (Figures 3B,D). After 3 days from inoculation, diﬀerences in Pn become signiﬁcant between PGPR and EDTA-inoculated plants (Figure 4B) with 9 and 12 µmol m−2 s−1, respectively. Transpiration rate was heavily aﬀected by soil pollution: only hemp plants grown on non-contaminated soil reached on average E-values around 3.5 mmol m−2 i−1. No statistical diﬀerences resulted between treatments before and after the inoculation (Figure 3D). Plant Photosynthetic Performances The eﬀects on photosynthetic performances (Pn and E) of HM pollution were more evident in hemp (Figures 3B,D) than in giant reed (Figures 3A,C). In fact, hemp plants grown in non- contaminated soil (NC) showed higher and constant Pn and E-values (13.9 and 3.5 mmol m−2 s−1, respectively) than hemp plants grown in contaminated soil (Figures 3B,D, P < 0.05). On the other hand, Pn of hemp plants grown in contaminated soil slightly decreased from an initial value of 8.3 until 6.6 µmol m−2 s−1 (P < 0.05). On 53 DAS, before PGPR and In giant reed plants, soil pollution did not aﬀect Pn neither before nor after the inoculation, remaining stable around 20.4 µmol m−2 s−1. Before the inoculation, no statistically signiﬁcant diﬀerences in Pn were highlighted between PGPR April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 9 Phyto-Assisted HMs Bioremediation Ferrarini et al. TABLE 3 \| Bioconcentration (BCF) and translocation (TF) factors and tolerance index (TI) for hemp and giant reed. Crop Treatment BCF-ABG BCF-BGB* TF TI Cr Ni Cu Cr Ni Cu Cr Ni Cu % Giant Reed C 0.084 a 0.04 a 0.32 a 0.52 a 0.59 ab 0.85 a 0.16 b 0.07 a 0.38 a 59 a† C+PGPR 0.052 b 0.03 a 0.26 a 0.38 a 0.53 a 0.82 a 0.14 b 0.07 a 0.32 a 117 b C+EDTA 0.051 b 0.05 a 0.36 a 0.42 a 0.68 b 1.87 b 0.13 b 0.07 a 0.19 b 66 a† Mean 0.062 0.04 0.32 0.44 0.60 1.18 0.15 0.07 0.30 81 Hemp C 0.003 a 0.03 a 0.08 a 0.07 a 0.24 a 0.32 a 0.05 a 0.13 a 0.27 a 78 a† C+PGPR 0.004 a 0.04 a 0.08 a 0.07 a 0.21 a 0.30 a 0.06 a 0.19 a 0.28 a 89 b† C+EDTA 0.003 a 0.29 b 0.49 b 0.08 a 0.29 a 0.65 b 0.03 a 1.07 b 0.77 b 84 ab† Mean 0.003 0.12 0.22 0.07 0.25 0.42 0.05 0.46 0.44 84 Different lowercase letters indicate signiﬁcant differences (Tukey’s test P < 0.05) among treatments for each factor. ANOVA has been run separately for giant reed and hemp. ABG, aboveground biomass; BGB, belowground biomass. BGB of Giant reed is the sum roots and rhizome. Plant Photosynthetic Performances †ABG l f C t t t diff d i iﬁ tl f NC t t t ( t i t d t i ) TABLE 3 \| Bioconcentration (BCF) and translocation (TF) factors and tolerance index (TI) for hemp and giant reed. Different lowercase letters indicate signiﬁcant differences (Tukey’s test P < 0.05) among treatments for each factor. ANOVA has been run separately for giant reed and hemp. ABG, aboveground biomass; BGB, belowground biomass. BGB of Giant reed is the sum roots and rhizome. †ABG average value of C-treatment differed signiﬁcantly from NC-treatment (non-contaminated matrix). FIGURE 3 \| Whole-canopy net photosynthetic rate (Pn, µmol m−2 s−1) and transpiration rate (E, mmol m−2 s−1) of giant reed (A) and hemp (B) as affected by PGRP and EDTA application (black arrows). DAT, day after transplanting. FIGURE 3 \| Whole-canopy net photosynthetic rate (Pn, µmol m−2 s−1) and transpiration rate (E, mmol m−2 s−1) of giant reed (A) and hemp (B) as affected by PGRP and EDTA application (black arrows). DAT, day after transplanting. Transpiration rate of plants grown on non-contaminated soil was not statistically diﬀerent from E of PGPR-inoculated plants and lower than E of giant reed plant grown on contaminated soil. Pn of EDTA-inoculated plants slightly increased in response to the and EDTA treatment. The 2nd day after inoculation, Pn of giant reed plants treated with PGPR signiﬁcantly decreased Pn until 15.5 µmol m−2 s−1, whilse Pn of EDTA-treated plants increased until 25.5 µmol m−2 s−1 (Figure 3A, P < 0.05). Transpiration rate of plants grown on non-contaminated soil was not statistically diﬀerent from E of PGPR-inoculated plants and lower than E of giant reed plant grown on contaminated soil. Pn of EDTA-inoculated plants slightly increased in response to the April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 10 Phyto-Assisted HMs Bioremediation Ferrarini et al. FIGURE 4 \| Diameter class length (DCL, mm cm−3) distribution of hemp (A) and giant reed (B) whole root systems and (C,D) relative contribution (%) of the very ﬁne, ﬁne, and coarse roots to the total root length density (RLD). Coefﬁcients and statistics obtained from the regression of extreme value model are reported in the table where the statistical signiﬁcance of the DCL curve parameters (a–d) were assessed through testing their standard errors using the t-statistics at P < 0.05. Plant Photosynthetic Performances Different letters in graph (C,D) denote statistical differences (Tukey’s test, P = 0.05) among treatment for each root diameter classes. FIGURE 4 \| Diameter class length (DCL, mm cm−3) distribution of hemp (A) and giant reed (B) whole root systems and (C,D) relative contribution (%) of the very ﬁne, ﬁne, and coarse roots to the total root length density (RLD). Coefﬁcients and statistics obtained from the regression of extreme value model are reported in the table where the statistical signiﬁcance of the DCL curve parameters (a–d) were assessed through testing their standard errors using the t-statistics at P < 0.05. Different letters in graph (C,D) denote statistical differences (Tukey’s test, P = 0.05) among treatment for each root diameter classes. inoculation treatments from 3 to 4.4 mmol m−2 s−1 (Figure 3B, P < 0.05). ﬁne roots of giant reed at the expense of ﬁne roots. The extreme value model accurately described (average R2 of 0.98) the DCL distribution of the whole ﬁne root system of giant reed and hemp (Figures 4A,B). Root system of both crops responded to heavy metal contamination by becoming thicker and shorter. Coeﬃcients a, b, and c were signiﬁcantly aﬀected by NC and C treatments in both crops. Hemp roots thickened (c coeﬃcient) from 0.16 mm with NC to an average value of 0.23 mm for contaminated soil treatments (Figure 4B). EDTA and PGPR in hemp signiﬁcantly suppressed DCL at peack values (b coeﬃcient) and increase the curve amplitude (width across the curve at half maximum—d parameter). For giant reed (Figure 4B), the model estimated that DCL peak was reached at a higher root diameter size in EDTA than in other treatment herbaceous crops (0.291 vs. 0.265 mm, respectively; P < 0.001) (Figure 4A). In particular, EDTA suppressed signiﬁcantly DCL of these peak value (b parameter) to 1.2 cm cm−3 compared with 5.5 of NC and 2.7 and 2.8, respectively, of C and C+PGPR. Frontiers in Microbiology \| www.frontiersin.org Root-Microorganism Activity Interactions Samples were rareﬁed to 10,000 sequences each, which was the abundance of the lowest populated sample: an average Good’s coverage of 86.5% (standard deviation 1) was found, indicating a good coverage of total bacterial diversity. When samples were analyzed by means of hierarchical clustering of sequences taxonomically classiﬁed at the genus level, clear diﬀerences emerged for both giant reed (Supplementary Figure 4) and hemp experiments (Supplementary Figure 5). It is worth noting that not only the EDTA but also the inoculation with a single strain (PGPR theses) resulted in diﬀerent bacterial communities already at time zero. Interestingly, the relative amount of sequences was also found to be classiﬁed as belonging to the Enterobacter genus (to which both inoculated strains belong) where higher in the PGPR treatments, thus indicating a signiﬁcant enrichment due to the bacteria inoculation. Soil EA patterns in RS signiﬁcantly diﬀered among treatments more in hemp (F = 12, P = 0.002) than in giant reed (F = 8.9, P = 0.003) (Figures 5A,C). Similar but less pronounced separation along dbRDA axes were observed for BS of both crops. Relatively to RS, soil EA associated to hemp diﬀered among NC and C treatments with separation along axes 1 (F = 69.6 and P = 0.002) accounting for 49.6% of the total variance and along axes 2 (F = 6.7 and P = 0.045) accounting for 29.8% of the total variance (Figure 5A). In hemp, soil EA patterns for C+EDTA were closer to each C and C+PGPR only in BS than those in BS (Figures 5A,B). PERMANOVA analysis (Supplementary Table 3) of EA and species score plot (Figures 5A,B) showed that EDTA signiﬁcantly reduced C-, N-, and P-acquiring enzymes (alkP and leu) compared with contaminated control (C) while PGPR diﬀered from C only for lower P- and S-acquiring enzymes (leu and bisP). Multivariate analyses on the total OTU matrixes show signiﬁcant eﬀects for sample type and for their interaction time × treatment terms (Figure 6). All tested eﬀects were signiﬁcant, with a percentage of variance ranging between 21.0% (hemp bulk soil) and 61.1% (giant reed BS). In agreement with the hierarchical clustering analyses, samples were forming separate groups, especially in the rhizosphere samples (Figure 6). Root-Microorganism Activity Interactions Heavy metal contamination aﬀected both in sediment and soil ﬁne root biomass (FRB) and RLD of giant reed and hemp. Both crop yielded in non-contaminated matrices more FRB than in contaminated ones. EDTA signiﬁcantly aﬀected RLD of giant reed (F = 34, P = 0.001) showing a peak negative value on average of 0.5 cm cm−3. Signiﬁcantly, RLD higher values were observed for C and PGPR (on average 1 cm cm−3) and NC (1.7 cm cm−3). A signiﬁcant denser ﬁne root system was observed in C treatments with hemp (average 3.66 cm cm−3) with no diﬀerences among them. Diameter class length results indicate that the large majority (88.2% in giant reed and 99.2% in hemp) of the roots, expressed as RLD is composed of roots with a diameter lower than 2 mm (Figures 4C,D). Among these, very ﬁne roots (0.0–0.5 mm) were more frequent than ﬁne roots (0.5–2.0 mm), but the latter were the most aﬀected by heavy metal contamination. In particular, EDTA application signiﬁcantly decreases RLD of very The results of soil EA (Supplementary Table 2) showed that HM contamination decreased signiﬁcantly either of BS (-28% hemp and −37% giant reed) and RS (−39% hemp and −48% giant reed). In particular, RS of giant reed when cultivate on contaminated sediment showed a decrease compared with NC at April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 11 Phyto-Assisted HMs Bioremediation Ferrarini et al. 37, 38, 46, and 38%, respectively, for C-, N-, P-, and S-acquiring enzymes while esterases and microbial biomass were reduced at 34 and 12%, respectively. RS of hemp instead when cultivate on contaminated sediment showed a decrease compared with NC at 40, 32, 47, and 66%, respectively, for C-, N-, P-, and S-acquiring enzymes while esterases and microbial biomass were reduced of 55 and 32%, respectively. Highest reduction in EA and microbial biomass of RS were observed with EDTA where it reached values of −44 and −48% in hemp and −7 and −44% in giant reed. The highest reduction were observed for P- and S-acquiring enzymes in giant reed RS (−6 and −76%) and N- and P-acquiring in hemp (−55 and −56%). ﬁltering. Average number of reads per sample was 89,849, and the average length was 300 bp in paired reads. Root-Microorganism Activity Interactions In the case of giant reed, C+EDTA-treated samples were completely grouped apart, while C+PGPR were partly overlapping with C, while in hemp it was found that the C+PGPR samples formed a separate cluster from the other two C groups. q g y Similar eﬀects of treatment on EA patterns diﬀerentiation were observed in giant reed, but diﬀerent EA caused the horizontal diﬀerentiation in the dbRDA plots (Figures 5C,D). In the RS of giant reed EA diﬀered among NC and C treatments with the separation along axes 1 (F = 72.7 and P = 0.052) accounting for 53.7% of the total variance (Figure 5C) while in BS C treatments did not diﬀer from each other but only with NC with the separation along axes 1 (F = 28.1 and P = 0.015), accounting for 53% of the total variance (Figure 5D). PERMANOVA analysis (Supplementary Table 1) of EA and species score plot (Figures 5A,B) showed that EDTA in giant reed signiﬁcantly reduced only N- and P-acquiring enzymes (acP, bisP, piroP, chit, leu) compared with the contaminated control (C) while PGPR did not diﬀer from C for any EA group. dbRDA results from multivariate multiple regression on EA (arrows in Figure 5) indicate that the HM that had the highest inﬂuence on EA distribution in hemp were Ni and Cu (Figures 5A,B) and Ni and Cr in giant reed (Figures 5C,D). DCL curve parameters representative of ticker and longer root system (Figures 5C,D parameters) of hemp and giant reed were positively correlated with HM concentration and negatively correlated with EA. Denser root system represented by higher value of b parameter in NC treatment were instead positively correlated with EA. A number of signiﬁcant diﬀerences were also found for α-diversity index, as depicted in Figure 7 for Simpson’s index. Focusing on rhizosphere samples, it was worth noting that in the case of giant reed, a signiﬁcant reduction of diversity was found for C+EDTA as compared with C+PGPR, C, and NC. The same trend was observed for hemp, but with no statistical diﬀerences. Hemp and Giant Reed Phyto-Assisted Bioremediation Potential In this study, two non-food crops (giant reed and hemp) were selected as candidate crops to reduce HMs of soil and sediments characterized by high concentration of Cr, Cu, and Ni. To do that, the phytoremediation potential of both traditional (crop alone) and assisted phytoremediation techniques (PGPR and chelating agent) was assessed. HM mass balance (Supplementary Figure 2), tolerance index (Table 1), and HM accumulation in plant organs (Figure 2) together indicated a good phytostabilization performance in giant reed and a moderate phytoextraction performance in hemp. Heavy metal accumulation in hemp can be considered low for phytoextraction purposes under real ﬁeld conditions (<100h in AGB, < 1% in BGB). Hemp showed a relatively low average Ni (25.1 mg kg−1) and Cu (8.4 mg kg−1) concentration and negligible Cr (<1 mg kg−1) concentration in AGB. Similar HM concentration values were found in other pot experiments with hemp grown on contaminated soil (Citterio et al., 2003; Bulk Soil and Rhizosphere Bacterial Diversity FIGURE 6 \| Mean values of Simpson’s index (D) in BS and RS of giant reed (Left) and hemp (Right) as affected by treatments and sampling time (Tz, time zero sampling; Tf, at the end of experiment). Different letters denote statistically different (Tukey’s test, P = 0.05) D-values among treatment in BS and RS for single crops. Angelova et al., 2004; Ahmad et al., 2016). The BCF values observed for Ni (0.3) and Cr (0.003) in AGB are indeed in line with those reported by Citterio et al. (2003). The low TF of HMs can be attributed to the low bioavailability of HMs in the soil (<2% as extracted with NH4NO3 1:2.5 (w/v)—DIN 19730). Hemp yield was aﬀected by the soil co-contaminants Ni (>500 mg kg−1) and Cu (>150 mg kg−1). Despite the use of EDTA slightly improved growth performance in terms of canopy photosynthesis, as observed also by Linger et al. (2005), a signiﬁcant increase in HM uptake and translocation to aerial parts was observed. This conﬁrms a good combination of hemp with EDTA (Citterio et al., 2003; Angelova et al., 2004; Ahmad et al., 2016) in particular for Cu uptake (3 times higher) but not for Ni (0.8 times higher). The mobilizing eﬀects on soil HMs induced by EDTA (+10% in bioavailability of Ni and Cu) has to be considered, however, in view of its permanence in soil (Meers et al., 2005; Evangelou et al., 2007; Shahid et al., 2014) especially when high content of clay and soil organic matter are present, since they can both promote adsorption mechanisms of available HM. transpiration. This results is unexpected for giant reed grown on contaminated sediment (Bonanno, 2012), but this can be partly explained by solubilization from sediments of entrapped plant nutrient. The results on bioaugmented rhizoremediation of giant reed are promising for the following reasons: it tolerates from moderate to high level of a wide range of HMs (Fiorentino et al., 2017; Cristaldi et al., 2020), is a low-input perennial energy crop suitable for several marginal environment (Amaducci and Perego, 2015; Amaducci et al., 2016), and it performs equally in terms of phytoremediation as native species (Huguenot et al., 2015) such as common reed (Phragmites australis) if contaminated wastewaters are applied (Mirza et al., 2010; Kausar et al., 2012). Bulk Soil and Rhizosphere Bacterial Diversity A total of 9,021,165 raw reads were obtained for all soil/sediment analyzed, which were ﬁnally reduced to 8,086,439 after quality April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 12 Phyto-Assisted HMs Bioremediation Ferrarini et al. Distance-based redundancy analysis (dbRDA) plots showing shifts in enzyme activities of RS (A,C) and BS (B,D) of hemp (A,B) and giant reed (C,D) ments. Arrow indicates environmental variables with signiﬁcance level (* < 0.05, < 0.001, * < 0.001). Species scores corresponding to the dbRDA nates for enzymes included in model) are reported in the scatter plots on the right. Letters within ellipses denote signiﬁcant differences (Bonferroni’s test, EA similarity matrices among fertilizers as assessed by permutational multivariate analysis of variance (PERMANOVA). RE 5 \| Distance-based redundancy analysis (dbRDA) plots showing shifts in enzyme activities of RS (A,C) and BS (B,D) of hemp (A,B) and giant reed (C,D) g treatments. Arrow indicates environmental variables with signiﬁcance level (* < 0.05, < 0.001, * < 0.001). Species scores corresponding to the dbRDA coordinates for enzymes included in model) are reported in the scatter plots on the right. Letters within ellipses denote signiﬁcant differences (Bonferroni’s test, FIGURE 5 \| Distance-based redundancy analysis (dbRDA) plots showing shifts in enzyme activities of RS (A,C) and BS (B,D) of hemp (A,B) and giant reed (C,D) among treatments. Arrow indicates environmental variables with signiﬁcance level (* < 0.05, < 0.001, * < 0.001). Species scores corresponding to the dbRDA plots (coordinates for enzymes included in model) are reported in the scatter plots on the right. Letters within ellipses denote signiﬁcant differences (Bonferroni’s test, P = 0.05) in EA similarity matrices among fertilizers as assessed by permutational multivariate analysis of variance (PERMANOVA). April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 13 Phyto-Assisted HMs Bioremediation Ferrarini et al. FIGURE 6 \| Mean values of Simpson’s index (D) in BS and RS of giant reed (Left) and hemp (Right) as affected by treatments and sampling time (Tz, time zero sampling; Tf, at the end of experiment). Different letters denote statistically different (Tukey’s test, P = 0.05) D-values among treatment in BS and RS for single crops. Frontiers in Microbiology \| www.frontiersin.org Bulk Soil and Rhizosphere Bacterial Diversity From the comparison of bioaugmentation with PGPR and addition of EDTA, it emerged clearly that, considering ﬁeld application and from an environmental point of view, microbial inoculum seems preferable over chelating agents. Although EDTA application showed an increase in the uptake of Ni and Cu (two known HMs for their mobility), but not in TF, the enrichment of leachates with HMs raise concerns over EDTA application in open environment conditions, especially for potential contamination of groundwaters, as already pointed out by other authors (Evangelou et al., 2007; Yang et al., 2012; Shahid et al., 2014). In particular, other chelating agents have been proposed to treat contaminated matrices with giant reed (Yang et al., 2012) and hemp (Meers et al., 2005). Alternatively, addition of chelating agents can be successfully performed with perennial plant in small stormwater basins connected to discharge areas of industrial sites (Huguenot et al., 2015). Perennial energy crops have already been proposed by several authors as promising phytoremediation crop (Barbosa et al., 2015; Pandey et al., 2016). Our results on HM uptake and BCF indicate that giant reed in wet conditions accumulates most of the “extracted” HMs in belowground organs. The consistent BCF values observed in giant reed BGB (0.44, 0.56, and 0.84 for Cr, Ni, and Cu, respectively) conﬁrm results by Barbosa et al. (2015) for Zn and Cr on soil and by Cristaldi et al. (2020) and Bonanno (2012) for Cr, Ni, and Cu in soil and sediment, respectively. Our results on canopy photosynthesis (Figure 3) conﬁrmed what already was found by several authors (Papazoglou et al., 2005, 2007; Fiorentino et al., 2017), as follows: giant reed tolerates well HM contamination because no statistically signiﬁcant diﬀerences was observed between contaminated and not contaminated grown plants. Giant reed treated with PGPR showed contrasting results than EDTA which signiﬁcantly increase both photosynthesis and Bioaugmentation with PGPR showed interesting results when combined with giant reed. PGPR increased the accumulation of Cr and Cu in rhizome of giant reed and enhanced the TF of these metals in AGB. Although PGPR decreased net photosynthesis in giant reed, we observed an increase of Cr, Ni, and Cu uptakes in leaves and Ni and Cu in stems. Our results showed that the PGPR strains selected (Enterobacter April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 14 Phyto-Assisted HMs Bioremediation Ferrarini et al. Bulk Soil and Rhizosphere Bacterial Diversity Moreover, this Gram-negative enteric bacteria have already been successfully inoculated to alleviated HM stress in other crops: soybean (Kang et al., 2015), rice (Mitra et al., 2018), and hyperaccumulator plants (Whiting et al., 2001). In giant reed, the use of other PGPR are documented for their HM biosorption capacity such as Agrobacterium spp. (Guarino et al., 2020) or Bacillus spp. (Sarathambal et al., 2017). The use of microorganism to alleviate HM stress of hemp is more focused toward AMF (Citterio et al., 2005) while PGPR associated to hemp plant growth improvement are more common (Pagnani et al., 2018; Lyu et al., 2019). Interestingly, the two PGPR strains used in this Bulk Soil and Rhizosphere Bacterial Diversity FIGURE 7 \| Distance-based redundancy analysis (dbRDA) plots showing shifts in microbial diversity (OUT) of RS and BS of hemp and giant reed among treatments and sampling time. Arrow indicates environmental variables with signiﬁcance level (* < 0.05, < 0.001, * < 0.001). Letters within ellipses denote signiﬁcant differences (Bonferroni’s test, P = 0.05) in OUT’s similarity matrices among treatments/sampling time combination as assessed by permutational multivariate analysis of variance (PERMANOVA). FIGURE 7 \| Distance-based redundancy analysis (dbRDA) plots showing shifts in microbial diversity (OUT) of RS and BS of hemp and giant reed among treatments and sampling time. Arrow indicates environmental variables with signiﬁcance level (* < 0.05, < 0.001, * < 0.001). Letters within ellipses denote signiﬁcant differences (Bonferroni’s test, P = 0.05) in OUT’s similarity matrices among treatments/sampling time combination as assessed by permutational multivariate analysis of variance (PERMANOVA). study were selected for both high HM biosorption and P solubilization abilities. spp.) are not inhibited by HM contamination under in vitro conditions. There are several evidences that Enterobacter spp., and in particular Enterobacter asburiae shows tolerance genes to HMs (Nguyen et al., 2019). A strong resistance to heavy metals was reported for Enterobacter spp. found in contaminated soil and sediments (Neeta et al., 2016; Chen et al., 2017; Sharma et al., 2020). Moreover, this Gram-negative enteric bacteria have already been successfully inoculated to alleviated HM stress in other crops: soybean (Kang et al., 2015), rice (Mitra et al., 2018), and hyperaccumulator plants (Whiting et al., 2001). In giant reed, the use of other PGPR are documented for their HM biosorption capacity such as Agrobacterium spp. (Guarino et al., 2020) or Bacillus spp. (Sarathambal et al., 2017). The use of microorganism to alleviate HM stress of hemp is more focused toward AMF (Citterio et al., 2005) while PGPR associated to hemp plant growth improvement are more common (Pagnani et al., 2018; Lyu et al., 2019). Interestingly, the two PGPR strains used in this spp.) are not inhibited by HM contamination under in vitro conditions. There are several evidences that Enterobacter spp., and in particular Enterobacter asburiae shows tolerance genes to HMs (Nguyen et al., 2019). A strong resistance to heavy metals was reported for Enterobacter spp. found in contaminated soil and sediments (Neeta et al., 2016; Chen et al., 2017; Sharma et al., 2020). Insights From Plant-Soil-Microbe Interactions in Microbial-Assisted Phytoremediation In this work, we investigated the eﬀects of three microbial- assisted phytoremediation strategies of HM-contaminated soils and sediments on ﬁne root system morphology and bacterial community structure and activity. Although giant reed may appear suitable for phytostabilization, based on its HM tolerance, exposure to HMs drastically impairs its root distribution (Figure 4). This is even more evident in hemp grown on contaminated soil. A general reduction of RLD associated to HMs is a known fact for many crops (Keller et al., 2003; Peer et al., 2006; Ostonen et al., 2007). Our result provided for the ﬁrst-time evidence April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 15 Phyto-Assisted HMs Bioremediation Ferrarini et al. diﬀerentially microbial community either in terms of activity (Figure 5) and diversity (Figure 6). of the eﬀect of HM contamination of soil and sediment on root diameter class length distribution of hemp and giant reed. HM contamination resulted in ticker and shorter root system, as shown by data on relative contribution to total RLD and root DCL curve distribution. Fine root system morphology of these crops have been characterized under ﬁeld condition in non-contaminated soil (Amaducci et al., 2008; Chimento and Amaducci, 2015). A consistent indirect eﬀect of HM levels on the soil microbiome (diversity and activity) mediated by plant response in terms of root growth (DCL distribution) was observed for both crops. HMs aﬀected ﬁrst root architecture, with ﬁne roots thicker and longer, and a result of these changes occurred at root level the microorganism colonizing root systems have been consequentially aﬀected (EA and OTU diversity). Considering the results obtained by Illumina OTU analyses, it is worth noting that the dbRDA pattern of OTU distribution (Figure 6) was quite consistent with the ones obtained on soil EA (Figure 5), thus showing a good agreement between the response of microbial communities both in terms of structure and activity measurements. It is also worth noting that EDTA had a more detrimental eﬀect on bacterial diversity as compared with PGPR: this outcome points once again to a higher acceptability and environmental sustainability of the bioaugmentation approach as compared with the addition of chelating agent. Interestingly, RLD of very ﬁne roots of giant reed were stimulated more by PGPR than EDTA. Stimulation of root and shoot length by microbial inoculations was also observed under HMs stress by other authors (Liu L. Insights From Plant-Soil-Microbe Interactions in Microbial-Assisted Phytoremediation et al., 2018; Pagnani et al., 2018), and is in line with the role of Enterobacter spp. in improving root systems through the production of phytohormones (Naveed et al., 2014). This outcome is also in agreement with the contemporary enhancement of growth and HM uptake of giant reed under Ni and Cu contaminations in sediments. The increase in RLD of very ﬁne roots can be attributed to the plant growth-promoting traits possessed by the inoculated microorganisms. g The HM-induced alterations of ﬁne root system morphology are often reported to be metal and species speciﬁc (Lambrechts et al., 2014). Despite that the environmental matrices used in this study are contaminated by diﬀerent HMs, a larger cumulative root density/aboveground biomass ratio as suggested by Keller et al. (2003), together with similar relative proportion of ﬁne roots to contaminated control (C), are two root traits associated with PGPR addition that helped increase HM uptake by giant reed. We suggest therefore that DCL curve distribution can be used successfully as an indicator of HM phytoextraction ability of perennial crops, but this hypothesis has to be further tested under real ﬁeld conditions. Another relevant ﬁnding of our study is that DCL curve parameters representative of the thickest and shortest root system (c and d parameters) of hemp and giant reed were negatively correlated with soil EA and positively correlated with HM levels (Figure 5). The negative eﬀects of single- or multi-HM pollution on soil EA is well known (Burges et al., 2015; Xian et al., 2015). A recent meta-analysis (Aponte et al., 2020) on HM eﬀect on soil EA showed that HM contamination linearly reduce the activities of extracellular enzymes involved in S (−60%) and N (−30%) cycling two–three times more than those involved in P and C cycling (−10%). Our ﬁndings showed a general higher reduction of EA under HM contamination in RS than BS. In rhizosphere soil, C-, N-, P-, and S-acquiring enzymes were on average reduced by 38, 35, 47, and 52%, respectively. In particular, along root phosphomoestares (alkP), phosphomoestares (piroP), and arylsulfatase (aryS) were the EA most impacted by HM contamination. This conﬁrms what was observed spatially with zymography under HM stress by Ma et al. (2018). Fine roots are on hot spot for microbial activity (Spohn et al., 2013; Kuzyakov and Blagodatskaya, 2015) and more diverse and species-rich microbial community (Pervaiz et al., 2020). DATA AVAILABILITY STATEMENT The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm. nih.gov/, BioProject PRJNA687540. Insights From Plant-Soil-Microbe Interactions in Microbial-Assisted Phytoremediation Fine roots play also an important role in managing the accessibility of metal ions to plant roots (Srivastava et al., 2017). Multivariate analyses (dbRDA) of EA patterns clearly indicated how bioaugmentation with PGPR and addition of EDTA shaped Despite that results on HM removal eﬃciency are promising especially for giant reed, the detrimental eﬀect of HMs on root system morphology is the main cause for the lower activity and diversity (Figure 7) of microbial communities in RS and BS. Root DCL distribution, represented by the coeﬃcient of extreme value model proposed by Zobel et al. (2007), might be suggested as a key ecological trait to understand crop-speciﬁc eﬀect of HMs on microbial activity and diversity. 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FF: performed soil enzymatic activities analysis. GB and MF: performed HM analysis. AFe: analyzed the data. SA and EP: contributed reagents/materials. AFe, SA, and EP: wrote the manuscript. All authors contributed to the article and approved the submitted version. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2021.645893/full#supplementary-material AUTHOR CONTRIBUTIONS AFe, AFr, SA, and EP: conceived and designed the experiment. AFr and AFe: managed the pot experiment. AFe and EP: performed soil sampling. GS: performed PGPR selection. GS and ET: performed microbiological analysis. AFr: performed plant growth analyses and performed root measurements. April 2021 \| Volume 12 \| Article 645893 Frontiers in Microbiology \| www.frontiersin.org 16 Phyto-Assisted HMs Bioremediation Ferrarini et al. REFERENCES G., and Smith, D. L. (2019). 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https://openalex.org/W2041148890	https://bmcresnotes.biomedcentral.com/track/pdf/10.1186/1756-0500-4-369	English	null	Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type	BMC research notes	2,011	cc-by	11,147	http://www.biomedcentral.com/1756-0500/4/369 (26 September 2011) Guard et al. BMC Research Notes 2011, 4:369 Abstract Background: Salmonella Enteritidis is currently the world’s leading cause of salmonellosis, in part because of its ability to contaminate the internal contents of eggs. Previous analyses have shown that it is an exceptionally clonal serotype, which nonetheless generates considerable phenotypic heterogeneity. Due to its clonality, whole genome analysis is required to find genetic determinants that contribute to strain heterogeneity of Salmonella Enteritidis. Comparative whole genome mutational mapping of two PT13a strains that varied in the ability to contaminate eggs and to form biofilm was achieved using a high-density tiling platform with primers designed from a PT4 reference genome. Confirmatory Sanger sequencing was used on each putative SNP identified by mutational mapping to confirm its presence and location as compared to the reference sequence. High coverage pyrosequencing was used as a supporting technology to review results. Results: A total of 250 confirmed SNPs were detected that differentiated the PT13a strains. From these 250 SNPS, 247 were in the chromosome and 3 were in the large virulence plasmid. SNPs ranged from single base pair substitutions to a deletion of 215 bp. A total of 15 SNPs (3 in egg-contaminating PT13a 21046 and 12 in biofilm forming PT13a 21027) altered coding sequences of 16 genes. Pyrosequencing of the two PT13a subpopulations detected 8.9% fewer SNPs than were detected by high-density tiling. Deletions and ribosomal gene differences were classes of SNPs not efficiently detected by pyrosequencing. Conclusions: These results increase knowledge of evolutionary trends within Salmonella enterica that impact the safety of the food supply. Results may also facilitate designing 2nd generation vaccines, because gene targets were identified that differentiate subpopulations with variant phenotypes. High-throughput genome sequencing platforms should be assessed for the ability to detect classes of SNPs equivalently, because each platform has different advantages and limits of detection. Keywords: Evolution, Salmonella, egg, chicken, genome, epidemiology associated with eggs, egg products, poultry, the farm envir- onment, and cross contamination of other foods from eggs [1,3,4,6]. S. Gallinarum and S. Pullorum are avian-patho- genic serotypes of Salmonella enterica that are closely related to S. Enteritidis. They too contaminate eggs, but they have accumulated a number of pseudogenes that severely limit host range [7,8]. Other Salmonellae may be found in eggs, but only S. Enteritidis does so in a manner that propagates efficiently through the food chain. S. RESEARCH ARTICLE Open Access Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type Guard et al. Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type Guard et al. Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type Guard et al. Guard et al. BMC Research Notes 2011, 4:369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 © 2011 Guard et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Enteritidis is unique in part because it produces a spe- cialized LPS O-antigen capsule that contributes to long term survival in eggs [9-11]. Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type Jean Guard, Cesar A Morales, Paula Fedorka-Cray and Richard K Gast Jean Guard, Cesar A Morales, Paula Fedorka-Cray and Richard K Gast * Correspondence: jean.guard@ars.usda.gov Agricultural Research Service (ARS), U. S. Department of Agriculture (USDA), 950 College Station Road, Athens, GA, 30605, USA Biofilm formation facilitated strains selection Biofilm formation was a useful phenotypic trait for identi- fying strains for comparison, because it was a simple agar- based assay that facilitated identification of strains that varied in metabolic properties, invasion potential, and in the ability to contaminate eggs (Figure 1) [17]. To reiter- ate, PT13a 21027 formed a strong biofilm but did not con- taminate eggs (BF+ EC-), PT13a 21046 did not form biofilm but did contaminate eggs (BF- EC+), whereas PT4 22079 formed biofilm and contaminated eggs (BF+ EC+). The strong biofilm of PT13a 21027 was distinguishable from the weak one formed by PT4 22079, because the lat- ter took longer to form and was never developed as in 21027 [Figure 1]. [ ] To understand more details of the evolutionary trends that contribute to the unique ability of some strains of S. Enteritidis to contaminate eggs requires analysis of whole genome databases, in large part to select genes relevant for evaluating in biological studies. We applied three methods of genome analysis to detect and confirm single nucleotide polymorphisms (SNPs) that differenti- ate gene content of two PT13a S. Enteritidis strains. These approaches were mutational mapping using a high density tiling microarray, pyrosequencing and con- firmatory F/R Sanger sequencing of all suspect SNPs. A critical component of the research was strain selection. The strains that were compared were previously shown to vary in metabolic properties and in the ability to con- taminate eggs [12,22,23]. Strain PT13a 21046 contami- nates eggs, but does not form biofilm. In contrast, PT13a 21027 does not contaminate eggs, but does form biofilm. The phenotypes for these strains are thus BF+, EC- and BF-, EC+ respectively. Both of the PT13a strains were compared directly to strain PT4 22079, which contaminates eggs, forms biofilm (BF+, EC+) and has a metabolic profile intermediate to that of the two PT13a strains [11]. Thus, two highly clonal PT13a strains were available for comparing to a slightly more genetically distant strain of a different phage type. By triangulating three genomic databases, it became possi- ble to find only those SNPs that differentiated the two PT13a strains. By using redundant methods for proces- sing genomes, we increased the stringency of analysis and revealed some inherent limitations of each method. Results here show the chromosomal differences that dif- ferentiate two PT13a strains that vary in the ability to contaminate eggs and to form biofilm. Biofilm formation facilitated strains selection This work was made possible by the availability of the whole genome Background Salmonella enterica subspecies I serotype Enteritidis (S. Enteritidis) is a leading cause of salmonellosis world- wide [1,2]. It is the only serotype of approximately 1400 that has evolved the ability to survive in the internal con- tents of eggs produced by otherwise healthy hens and to be linked to frequent human illness [3]. S. Enteritidis con- taminates foods other than eggs and it colonizes animals other than chickens [2,4,5]. However, it is predominantly Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 2 of 14 sequence from PT4 S. Enteritidis P125109 (Refseq NC_011294) [8]. Strains of S. Enteritidis vary greatly in their ability to contaminate eggs and this virulence attribute is indepen- dent of phage type lineage [12,5,13-15,10]. The ability to metabolize a wide range of amino acids has been linked to virulence [10,11,16], as has the ability to make the O-antigen capsule. Strain heterogeneity and variant metabolic profiles may facilitate completion of the infec- tion pathway by overcoming a multitude of microenvir- onments resulting from cellular barriers, flock immunity and management practices [12]. Another reason S. Enter- itidis may generate strain heterogeneity is to facilitate colonization of the reproductive tract of hens, which is subject to hormonally-dependent cyclical changes that impact local immunity and cell function [12,17,18]. S. Enteritidis produces 3 classes of fimbriae that contri- bute to colonization and invasion [19]. S. Enteritidis has a full repertoire of virulence factors in common with other pathogenic Salmonellae such as S. Typhimurium [8,20,21]. S. Enteritidis strains chosen for analysis have commonly encountered PFGE patterns At the time of analysis, the USDA VetNet PFGE database had 676 Enteritidis isolates and 51 unique patterns. PT13a 21027 was pattern JEGX01.0003 ARS (PulseNet equivalent pattern JEGX01.0004) (Figure 2). This pattern is the most common Enteritidis pattern in the database (308 out of 676 - 45.56%). PT13a 21046 was pattern JEGX01.0013 ARS (PulseNet equivalent pattern JEGX01.0037) (Figure 2). This pattern is the 10th most common Enteritidis pattern in the database (3 out of 676 - 0.44%). PT4 22079 was pattern JEGX01.0017 ARS (Pul- seNet equivalent pattern JEGX01.0002) (Figure 2). This pattern is the 6th most common Enteritidis pattern in the database. PFGE results indicate that the two strains being compared in reference to the PT4 genome are represen- tative of strains found in outbreaks. PT13a 21027, which had the most common VN PFGE fingerprint, had a pro- minent biofilm within 48 hr at ambient temperatures on select media. Subpopulation biology of S. Enteritidis results from accumulation of SNPs, only some of which appear consequential for phenotype Subpopulation biology of S. Enteritidis results from accumulation of SNPs, only some of which appear consequential for phenotype Of the 250 confirmed polymorphisms that differentiated PT13a 21046 from PT13a 21027, 132 (52.8%) did not alter amino acid sequence (Table 1; For details, see Additional File 1). Of the 132 polymorphisms that did not alter amino acid sequences, 38 were intergenic, 12 involved ribosomal genes, and 82 were synonymous nucleotide sub- stitutions The 3 SNPs that were in virulence plasmids were intergenic. Of the 250 polymorphisms, 115 (46.0%) were non-synonymous and thus they altered primary amino acid sequence. Five of 250 SNPs (2%) introduced Page 3 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 10 mm A B C Figure 1 Prominent colony morphologies of Salmonella Enteritidis. TOP. Multiple colonies, 10 per plate, were grown for five days at ambient temperature following a 16 hr incubation at 37°C. BOTTOM. Colonies are magnified. Inset markers are 10 mm. This picture is reprinted with permission from Avian Diseases [12]. Copyright is property of the federal government. A. PT4 S. Enteritidis strain 22079, with weak slow biofilm formation; B. PT13a S. Enteritidis strain 21027, with strong rapid biofilm formation; C. PT13a S. Enteritidis strain 21046, with no discernible biofilm formation. 10 mm A B C Figure 1 Prominent colony morphologies of Salmonella Enteritidis. TOP. Multiple colonies, 10 per plate, were grown for five days at ambient temperature following a 16 hr incubation at 37°C. BOTTOM. Colonies are magnified. Inset markers are 10 mm. This picture is reprinted with permission from Avian Diseases [12]. Copyright is property of the federal government. A. PT4 S. Enteritidis strain 22079, with weak slow biofilm formation; B. PT13a S. Enteritidis strain 21027, with strong rapid biofilm formation; C. PT13a S. Enteritidis strain 21046, with no discernible biofilm formation. C B A Figure 1 Prominent colony morphologies of Salmonella Enteritidis. TOP. Multiple colonies, 10 per plate, were grown for five days at ambient temperature following a 16 hr incubation at 37°C. BOTTOM. Colonies are magnified. Inset markers are 10 mm. This picture is reprinted with permission from Avian Diseases [12]. Copyright is property of the federal government. A. PT4 S. Enteritidis strain 22079, with weak slow biofilm formation; B. PT13a S. Enteritidis strain 21027, with strong rapid biofilm formation; C. PT13a S. Subpopulation biology of S. Enteritidis results from accumulation of SNPs, only some of which appear consequential for phenotype Lanes 1 and 4, DNA fragment Subpopulation biology of S. Enteritidis results from accumulation of SNPs, only some of which appear consequential for phenotype Enteritidis strain 21046, with no discernible biofilm formation premature termination codons in putative genes. Table 1 summarizes the types of SNPs detected. Overall, less than 0.01% of the total genome content differentiated PT13a subpopulations. However, when expressed as a percentage of total genes with non-synonymous changes, 1.1% and 1.5% of genes from PT13a 21046 and PT13a 21027 varied from PT4 22079, respectively. PT13a 21046 had a higher incidence of SNPs in ribosomal genes than did PT13a 21027, but PT13a 21027 accumulated more ORF-disrupt- ing events than did PT13a 21046 (Table 1). Figure 2 PFGE patterns of Salmonella Enteritidis strains with known subpopulation characteristics. Lanes 1 and 4, DNA fragment size markers; lane 2, PT13a S. Enteritidis strain 21027, which forms biofilm but does not contaminate eggs; lane 3, PT13a S. Enteritidis strain 21046, which does not form biofilm but does contaminate eggs; lane 5, PT4 S. Enteritidis strain 22079, which forms biofilm and contaminates eggs. S. Enteritidis strains 21027, 22079 and 21046 had the 1st, 6th and 10th most common PFGE profiles in the US, respectively, according to PulseNet typing classification schemes. Relative incidence may change by publication date. Detection of rare substitutions of amino acids within proteins is important, because a dramatic change in the class of amino acid is more likely to affect function. Of the 100 amino acid substitutions, neutral non-polar (nn) and neutral polar (np) amino acids were frequently sub- stituted within the two chemical classes, but substitution between acidic and basic polar amino acids did not occur (Table 2). Between these two extremes, substitu- tion of np for acidic (ap) or basic (bp) amino acids was very rare. Further research is needed to show the impact of altered amino acid sequence on protein function. Figure 2 PFGE patterns of Salmonella Enteritidis strains with known subpopulation characteristics. Lanes 1 and 4, DNA fragment size markers; lane 2, PT13a S. Enteritidis strain 21027, which forms biofilm but does not contaminate eggs; lane 3, PT13a S. Enteritidis strain 21046, which does not form biofilm but does contaminate eggs; lane 5, PT4 S. Enteritidis strain 22079, which forms biofilm and contaminates eggs. S. Enteritidis strains 21027, 22079 and 21046 had the 1st, 6th and 10th most common PFGE profiles in the US, respectively, according to PulseNet typing classification schemes. Relative incidence may change by publication date. Figure 2 PFGE patterns of Salmonella Enteritidis strains with known subpopulation characteristics. Lysogenic bacteriophage in the reference genome but absent in test genomes was easily detected Mutational mapping of the two PT13a strains gave a prominent signal at a site in the reference genome known to include an ST64b-like bacteriophage, which defines the PT4 lineage. The absence of binding by the PT13a strains to the PT4 primers that hybridized ST64b-like sequence resulted in a run length (number Page 4 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 of consecutive events) of more than mispriming events occurring in a wi mately 47,000 bp. This large run of m has so far only been associated with lysogenic bacteriophage. A bacteriophage absent in the referenc the test strains was detected by interva The strategy used for comparison of ently biased against detection of inse omes compared to the PT4 reference no PT13a specific primers would hav Table 2 Amino acid substitutions of Sa PT13a substitution Number in 21027b ap to bp 0 bp to ap 0 np to ap 1 np to bp 1 ap to nn 2 nn to bp 2 bp to bp 2 np to np 2 ap to ap 3 ap to np 3 bp to nn 4 bp to np 4 nn to ap 4 nn to np 6 np to nn 7 nn to nn 15 Total 56 a Classes of amino acids are: nn, neutral non-polar; n polar; bp, basic polar. For greater detail about amino supplementary material (S1, because polar amino aci Table 1 Summary of 250 confirmed SN differentiate subpopulations of Salmon PT13a Type of genetic changea PT13a 210 Deletionb Change in terminationc Amino acid substitution RNA gene change Intergenic polymorphism Synonymous TOTAL 1 aSNPs composed of contiguous base pairs, ranging fr deletion, are counted as single SNPs. bA deletion in ydcZ changes one AA and deletes two DEL event). clncludes 3 SNPs in the virulence plasmid, which wer Table 1 Summary of 250 confirmed SNPs that differentiate subpopulations of Salmonella enteritidis PT13a Type of genetic changea PT13a 21027 PT13a 21046 Deletionb 8 2 Change in terminationc 4 2 Amino acid substitution 55 44 RNA gene change 4 8 Intergenic polymorphism 22 19c Synonymous 43 39 TOTAL 136 114 aSNPs composed of contiguous base pairs, ranging from doublets to a 215 bp deletion, are counted as single SNPs. bA deletion in ydcZ changes one AA and deletes two others (counted as one DEL event). A bacteriophage absent in the reference but present in the test strains was detected by interval mapping The strategy used for comparison of genomes is inher- ently biased against detection of inserts in PT13a gen- omes compared to the PT4 reference genome, because no PT13a specific primers would have been generated Table 2 Amino acid substitutions of Salmonella enteritidis PT13a substitution Number in 21027b Number in 21046 ap to bp 0 0 bp to ap 0 0 np to ap 1 0 np to bp 1 0 ap to nn 2 2 nn to bp 2 3 bp to bp 2 1 np to np 2 2 ap to ap 3 1 ap to np 3 1 bp to nn 4 3 bp to np 4 5 nn to ap 4 1 nn to np 6 9 np to nn 7 4 nn to nn 15 12 Total 56 44 a Classes of amino acids are: nn, neutral non-polar; np, neutral polar; ap, acidic polar; bp, basic polar. For greater detail about amino acid polarity refer to supplementary material (S1, because polar amino acids are not classified as weak or strong in this table. bincludes AA change in ydcZ, which also has a deletion. Table 2 Amino acid substitutions of Salmonella enteritidis Table 2 Amino acid substitutions of Salmonella enteritidis PT13a Lysogenic bacteriophage in the reference genome but absent in test genomes was easily detected clncludes 3 SNPs in the virulence plasmid, which were all intergenic. Table 1 Summary of 250 confirmed SNPs that differentiate subpopulations of Salmonella enteritidis PT13a from the PT4 genome. For example, DNA-DNA microar- ray hybridization had previously detected bacteriophage Fels2 in S. Enteritidis PT13a strains [7,11]. However, there was no possibility of detecting bacteriophage Fels2 within PT13a strains by mutational mapping in the high- density tiling approach, because there was no template in S. Enteritidis PT4 for production of primers that would hybridize Fels2 sequence. To objectively assess whole genome data for the presence of sequence unique to test strains and absent in the reference, an interval map based on the average number of genes between SNPs was con- structed from all data available in supplementary material (Additional File 2). Interval mapping of all SNPs from both strains against S. Typhimurium LT2 http://www. ncbi.nlm.nih.gov/nuccore/NC_003197) helped to locate the Fels2 bacteriophage in PT13a strains between S. Enteritidis gene (SEN) SEN2609 and SEN2624 of the PT4 genome. This location was confirmed by sequencing the beginning and end of the bacteriophage 3’ to SEN2592 and 5’ to SEN2623, respectively (data not shown); however, the complete bacteriophage sequence was not sequenced de novo. An additional benefit of interval mapping is that it appears to locate regions that have significant gaps in alignment defined by a gap of only 5 genes. This could help to locate even small inver- sions and gene transfers between serotypes. Otherwise, a strategy similar to interval mapping applied here is used elsewhere in software that depicts gene inversions and alignments in a graphing output (e. g. MAUVE); how- ever, we needed finer detail to evaluate alignments to avoid missing inserted genes or non-coding sequence between the PT13a strains. of consecutive events) of more than 5500 consecutive mispriming events occurring in a window of approxi- mately 47,000 bp. This large run of mispriming events has so far only been associated with the presence of a lysogenic bacteriophage. a Classes of amino acids are: nn, neutral non-polar; np, neutral polar; ap, acidic polar; bp, basic polar. For greater detail about amino acid polarity refer to supplementary material (S1, because polar amino acids are not classified as weak or strong in this table. bincludes AA change in ydcZ, which also has a deletion. Discussion Importance o smallest deletion was 1 run length (1 mispriming event), which was confirmed to be a 1 bp deletion in the gene sefD of PT13a 21027. In a run, each mispriming event is separated by 7 bp due to the nature of the high-density til- ing approach and the size of the primer overlap. Raw data showed that in some cases there were easily observed spectral evidence of deletions that varied between strains. This result indicates that larger deletions can be rapidly detected by observance of spectral signal alone. However, algorithms with a stringency of filtering for run length of 2 or greater will miss some deletions, and thus type II errors (false negatives) are possible. In summary, rapid filtering of data for run lengths of 2 or greater will catch most dele- tions, but there is a risk that false negative results will occur and that the smallest deletions will be missed using filtering approaches that attempt to avoid confirmatory sequencing. Importance of starting analyses with characterized strains The 3 strains chosen for this comparative approach var- ied in a number of phenotypic assays, which was used as prima facie evidence that the strains must vary in genetic content even in the absence of specific knowl- edge of the differences. Rejection of the concept that there was an identifiable difference in genomic content would have required rejecting basic tenants of evolu- tionary theory. However, this research does not claim that any one genetic difference causes any one pheno- type in the absence of further research to establish bio- logical role. It is likely that phenotypes observed with various assays require a combination of genetic events. Supplementary material in Additional File 1 provides a searchable database for investigators interested in acquiring specific information on any one gene, which includes information on KEGG pathways and gene function. Description of open reading frames of PT13a S. Enteritidis that were disrupted Both PT13a strains had multiple SNPs that disrupted open reading frames (ORFs) (Table 3). Using the inci- dence of disrupted ORFs as a measure of genetic dis- tance from PT4 22079, PT13a 21046 is more closely related to the PT4 reference strain, because only 3 ORFs were disrupted in contrast to 12 for PT13a 21027. One mutation fused two genes, which means that a total of 16 genes, 3 for PT13a 21046 and 13 for PT13a 21027, have disrupted ORFs. Linkage of genotype to phenotype requires stringent application of comparative genome approaches and phenotypic analyses Detection of SNPs that may be causally associated with phenotype of pathogenic bacteria is an inherently pro- blematic undertaking, because a change of only 1 bp out of millions has the potential to alter the biology of the bacterium [24]. If the genetic distance between two organisms is too much, the ability to link a phenotype to a genotype is complicated by the numbers of poly- morphisms that will be found. In other words, the chal- lenge of the bacterial genome is its sheer capacity to rapidly accumulate polymorphisms, either by random genetic drift, selection of specific genetic capabilities, or by lateral transfer of sets of polymorphisms through events such as homologous recombination or acquisition of genes from extrachromosal DNA [12]. By triangulat- ing 2 genomes of strains within a single phage type to that of a genome from a different phage type, genetic noise was reduced and a discrete number of non-synon- ymous polymorphisms were found that differentiated the two PT13a strains. Selection of strains was key to the success of the approach and multiple phenotypic assays were used to select stable strains with variant phenotypes [12]. Assay of raw data for deletion events can be used to detect multiple deletion events across the genome but may miss single nucleotide events A useful feature of whole genome analysis that would facilitate outbreak investigations is if raw data could be quickly gleaned for evidence of mutational events that would distinguish strains within serotypes. For this reason, we analyzed technical details of the size of window and mispriming events (run length) associated in the raw data with SNPs that were confirmed as resulting from a dele- tion. The 215 bp deletion in STM4551 of PT13a 21046 occurred within a 231 bp window as reported in raw data files. It was associated with 34 mispriming events, or a run length of 34. The 92 bp deletion in STY3762 of PT13a 21046 had a run length of 14 within a 91 bp window. The 10 bp deletion within dsdA, the 12 bp deletion in lrgB, and the 12 bp deletion in lysR (STM2912), all of which occurred in PT13a 21027, each had a run length of 4 that spanned a 21 bp window. The 11 bp deletion that intro- duced the read-through fusion of yjfK and yjfL of PT13a 21027 had a run length of 6 and a 35 bp window. The Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 5 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric Table 3 summarizes results from BLAST searches of dis- rupted ORFs across publicly available Salmonella enterica databases. The database of serotype genomes is in general heavily skewed towards mutation in mocR and sefD; in addition, fhuA has two alternative sequences that varies among serotypes. Each Salmonella serotype appears to have its own unique combination of ORFs that vary in completeness. For example, S. Typhi lacks fhuA and putP, whereas S. Kentucky lacks mocR, putP and kdgM. E. coli lacks 13 of the 16 genes listed in Table 3; thus, this subset of genes may be more likely to be linked to the biology of Salmonella enterica than it is to the biology of either E. coli and perhaps Shigella. Salmonella bongori lacks 8 of the 16 genes, which suggests that the subset of genes is more a marker of Salmonella causing illness than it is to environmental adaptation. Finally, Salmonella enterica subsp. Arizonae did not have complete ORFs for 6 of the genes in the subset, which suggests that this pathogen more associated with colonization of reptiles and amphi- bians has also evolved differently in regards to genes impacted by these SNPs. It must be noted that there are more subpopulations to be characterized. Also, only a few of the SNPs listed here are likely to have epidemiological significance and/ or a causal link to virulence attributes. Further analysis that compares mutant to complemented mutant and parent strain is required to identify markers of highest value for causing anyone phenotypic attribute. ORF dis- rupting mutations are emphasized as being especially Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 6 of 14 Table 3 SNPs of Salmonella Enteritidis that disrupt open-reading-frames (ORFs) and comparison of translated genes to NCBI reference sequences of Salmonella enterica Salmonella Enteritidis gene with SNP Type of SNP as referenced to S. Enteritidis PT4 Location of SNP in S. Enteritidis PT4 genome Salmonella Enteritidisa D1 D1 D1 D1 D1 D1 D1 A D1 PT 13a 21046 PT13a 21027 PT4 22079 (same as reference) Gallinarum Dublin Typhi Paratyphi A Javiana Typhimurium LT2 A) Polymorphisms that distinguish PT13a 21046 from PT13a 21027 and S. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric Enteritidis PT4 fhuA (SEN0196) substitution: ORF termination 226922 - + + + (78) + (78) - (23) - (23) + (78) + (78) mocR (SEN3898) deletion: 92 bp 4189388- 4189479 - + + + (100) + (99) + (98) + (98) nd - nd DGC (SEN4316) deletion: 80 bp of 5’ end + 135 bp upstream 4642252- 4642331 - + + + (99) + (99) + (99) + (99) + (99) + (99) B) Polymorphisms that distinguish PT13a 21027 from PT13a 21046 and S. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric Enteritidis PT4 foxA (SEN0347) deletion: 1 bp 393529 + - + - (99) + (99) + (98) + (99) + (99) + (99) putP (SEN0989) substitution: ORF termination 1095448 + - + + (99) + (100) - nd + (100) + (100) + (99) kdgM (SEN0992) deletion: 1 bp 1099079 + - + + (99) + (99) + (97) + (99) + (98) + (99) ydcZ (SEN1464) deletion: 6 bp 1554930 + - + + (100) + (99) + (99) + (98) + (99) + (99) ydjN (SEN1723) substitution: ORF termination 1828544 + - + + (99) + (99) + (99) + (99) + (99) + (100) ytcJ-like (SEN1576) substitution: ORF termination 1681327 + - + + (99) + (99) + (99) + (98) + (98) + (99) pgk (SEN2751) deletion: 12 bp in-frame 2939833- 2939844 + - + + (99) + (97) + (98) + (97) + (99) + (98) cysN (SEN2773) substitution: ORF termination 2957802 + - + + (99) + (99) + (99) + (99) + (99) + (99) dsdA (SEN3619) deletion: 10 bp 3880104- 3880113 + - + + (100) + (99) + (99) + (99) + (99) + (99) lrgB (SEN4042) deletion: 12 bp in-frame 4368126- 4368137 + - + + (100) + (99) + (99) + + (100) + (100) yjfK (SEN4139) shared deletion: fusion of yjfK and yjfL; ribosomal binding site of yjfL removed 4472579- 4472589 + - + + (99) + (99) + (99) + (99) + (99) + (99) yjfL (SEN4140) + - + - nd + (100) + (99) + (100) + (99) + (100) sefD (SEN4250) deletion: single bp 4574262 + - + - (99) + (100) - (99) - (99) - nd - nd a Abbreviations and symbols: na, possible error in contig assembly or database or otherwise not applicable; nd, no similar gene detected; nucleotide sequence similarity rather than amino acid sequence similarity is included for genes that are pseudogenes, which are recorded by a “-” even if percent nucleotide similarity is high. b Numbers in parentheses indicate % similarty between translated amino acid sequences of S. Enteritidis in comparison to indicated serotype, unless a pseudogene is present and reported as nucleotide similarity. c Strains and databases referenced are: Serotype designation Refseq: Genbank accession Salmonella enterica subsp. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric arizonae serovar 62:z4,z23:–, complete genome NC_010067 CP000880 Table 3 SNPs of Salmonella Enteritidis that disrupt open-reading-frames (ORFs) and comparison of translated genes to NCBI reference sequences of Salmonella enterica Table 3 SNPs of Salmonella Enteritidis that disrupt open-reading-frames (ORFs) and comparison of translated genes to NCBI reference sequences of Salmonella enterica Strains and databases referenced are: Serotype designation Refseq: Genbank accession Salmonella enterica subsp. arizonae serovar 62:z4,z23:–, complete genome NC_010067 CP000880 Salmonella enterica subsp. enterica serovar 4,[5],12:i:- str. CVM23701 NZ_ABAO00000000 ABAO00000000 Salmonella enterica subsp. enterica serovar Agona str. SL483 NZ_ABEK00000000 ABEK00000000 Salmonella enterica subsp. enterica serovar Choleraesuis str. SC-B67 NC_006905 AE017220 Refseq: Genbank accession ete genome NC_010067 CP000880 23701 NZ_ABAO00000000 ABAO00000000 NZ_ABEK00000000 ABEK00000000 C-B67 NC_006905 AE017220 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 7 of 14 Other Salmonella enterica subsp. I serotypesb,c Outlier microbes O-antigen serotype B B B B B B C1 C1 C2 C2 C3 na na na Typhimurium DT104 Typhimurium 4,[5],12:i:- Saintpaul Schwarzengrund Heidelberg Agona Choleraesuis Infantis Hadar Newport Kentucky Salmonella enterica subsp. arizonae Salmonella bongori E. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric coli 0157: H7 Sakai + (78) + (78) + (78) + (78) + (99) + (100) + (78) + (78) + (78) + (78) + (99) + (97) + (99) + (93) - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (90) + (90) - nd + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (98) + (99) + (99) - nd - nd - nd + (99) + (99) + (99) + (99) + (100) - nd - (44) + (100) + (99) + (100) - (nd) + (99) - nd - nd + (99) + (99) + (99) + (98) + (99) - nd + (99) + (99) + (99) + (99) - nd + (96) - nd - nd + (99) + (99) + (100) + (100) + (98) + (99) + (99) + (98) + (99) + (99) + (99) + (95) - nd - nd + (100) + (100) + (100) + (99) + (100) + (99) + (99) + (100) + (100) + (99) + (100) + (99) + (99) + (96) + (99) na + (98) + (98) + (99) + (99) + (98) + (99) + (99) + (99) + (99) - nd + (96) - nd + (98) + (98) + (98) + (99) + (98) + (97) - (80) + (97) + (98) + (98) + (97) + (99) + (85) - nd + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (99) + (92) + (87) + (99) + (99) + (99) + (99) + (100) + (99) + (99) + (99) + (99) + (100) + (99) + (97) - nd - nd + (100) + (100) + (100) + (100) + (100) + (100) + (100) + (100) + (100) + (100) + (100) + (99) + (97) - nd + (100) + (100) + (100) + (99) + (100) + (99) + (99) + (100) + (100) + (100) + (99) - nd - (89) - nd + (100) + (99) + (100) + (99) + (100) + (99) + (99) + (100) + (100) + (100) + (99) - (nd) + (98) - (nd) - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd - nd Guard et al. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric Finding that pyrosequencing was excellent for detection of SNPs that involve basepair substitutions suggests that it may be a preferred method for epidemiological inves- tigations, because it is amenable to being automated. However, if certain SNPs occur repeatedly within differ- ent subpopulations, then targeted sequencing may be more cost effective. For example, if epidemiological tra- cebacks could be accomplished using as few as 10 SNPs to track an outbreak strain, a baseline cost would be approximately $100 per strain ($10/SNP). It is not yet possible to complete whole genome sequencing for such a low cost, although it is required for the initial identifi- cation of SNPs useful for building analytical platforms. Application of these methods requires acknowledgement of method limitations and a strategy for overcoming deficiencies and skew in order to produce the highest quality databases. important in future research, because they introduce an obvious change in genome content. Obtaining informa- tion about which SNPs occur frequently within the environment of poultry and that can be linked to egg contaminating phenotypes is also important, because these can be genetic markers that may facilitate epide- miological investigations. Research is in progress to eval- uate if SNPs described here are commonly found in clinical and environmental strains and to determine their biological impact. It is expected that many SNPs will be inconsequential. The ability to find a discrete set of SNPs was depen- dent on extensive research completed prior to initiating genomic analyses. For example, biofilm production was selected as an indicator of potential virulence, because it is often cited as contributing to the ability of bacterial pathogens to colonize, survive and persist in hosts [25-28]. Phenotype microarray was a second assay that more specifically and quantitatively defined how strains varied in regards to metabolic properties [11]. Finally, the use of a highly relevant animal model, namely the hen infection model, increased the ability to keep ana- lyses focused on traits impacting egg contamination. In contrast, mouse models are inherently limited in their ability to assess genetic factors impacting egg contami- nation, because mice lack an oviduct and the reproduc- tive cycle of a hen. Other approaches to studying phenotype include tissue cell assays, injecting bacterial cells into egg contents and coating the shell of eggs with cells [29]. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 7 of 14 Other Salmonella enterica subsp. I serotypesb,c Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 8 of 14 Table 3 SNPs of Salmonella Enteritidis that disrupt open-reading-frames (ORFs) and comparison of translated genes to NCBI reference sequences of Salmonella enterica (Continued) Salmonella enterica subsp. enterica serovar Dublin str. CT_02021853 NZ_ABAP00000000 ABAP00000000 Salmonella enterica subsp. enterica serovar Heidelberg str. SL476 NC_011083 CP001120 Salmonella enterica subsp. enterica serovar Heidelberg str. SL486 NZ_ABEL00000000 ABEL00000000 Salmonella enterica subsp. enterica serovar Javiana str. GA_MM04042433 NZ_ABEH00000000 ABEH00000000 Salmonella enterica subsp. enterica serovar Kentucky str. CDC 191 NZ_ABEI00000000 ABEI00000000 Salmonella enterica subsp. enterica serovar Kentucky str. CVM29188 NZ_ABAK00000000 ABAK00000000 Salmonella enterica subsp. enterica serovar Newport str. SL254 NC_011080 CP000604 Salmonella enterica subsp. enterica serovar Newport str. SL317 NZ_ABEW00000000 ABEW00000000 Salmonella enterica subsp. enterica serovar Paratyphi A str. ATCC 9150 NC_006511 CP000026 Salmonella enterica subsp. enterica serovar Saintpaul str. SARA23 NZ_ABAM00000000 ABAM00000000 Salmonella enterica subsp. enterica serovar Saintpaul str. SARA29 NZ_ABAN00000000 ABAN00000000 Salmonella enterica subsp. enterica serovar Schwarzengrund str. CVM19633 NC_011094 CP001127 Salmonella enterica subsp. enterica serovar Schwarzengrund str. SL480 NZ_ABEJ00000000 ABEJ00000000 Salmonella enterica subsp. enterica serovar Typhi Ty2 NC_004631 AE014613 Salmonella enterica subsp. enterica serovar Typhi str. CT18 NC_003198 AL513382 Salmonella typhimurium LT2 NC_003197 AE006468 E. coli 0157:H7 Sakai NC_002695 BA000007 Sanger Institute databases are available at http://www.sanger.ac.uk/Projects/Salmonella/. Salmonella bongori 12419 ATCC 43975 Salmonella enterica Enteritidis PT4 NCTC 13349 Salmonella enterica Gallinarum 287/91 NCTC 13346 Table 3 SNPs of Salmonella Enteritidis that disrupt open-reading-frames (ORFs) and comparison of translated genes to NCBI reference sequences of Salmonella enterica (Continued) eritidis that disrupt open-reading-frames (ORFs) and comparison of translated genes to NCBI ella enterica (Continued) Page 9 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 and accurate method for finding true polymorphisms that differentiated PT13a strains 21027 and 21046 as confirmed by sequencing f/r strains PT13a 21027, PT13a 21046 and PT4 22079. The cost of sequencing was greatly reduced by incorporating an approach that reduced genetic noise, namely triangulation of 3 gen- omes within the same serotype for virtual subtractive hybridization. Combining mutational mapping with pyr- osequencing may eventually be the most cost effective and time efficient approach for SNP analysis of bacteria that have an exceptionally clonal population structure. Mutational analysis, development of 2nd generation vaccines and future research objectives The relative contribution of each non-synonymous SNP to the pathway that results in egg contamination will be better understood as defined mutants within a similar genomic background are characterized. Knowledge of how combinations of genes aid organ colonization and growth will help to choose strains that are suitable for development of vaccines. The possibility that there is a vaccination strategy that prevents multiple serotypes from colonizing and growing in internal organs should be explored, because serotypes other than S. Enteritidis could evolve the ability to contaminate eggs. These results support the conclusion that S. Enteritidis has multiple evolutionary trajectories, involving multiple polymorphisms throughout the genome, which impart combinatorial complexity [12]. Finding which SNPs impact the ability of S. Enteritidis to complete the infec- tion pathway to the egg and ultimately to the consumer is an objective of future research. Assay of S. Enteritidis SNPs that differentiate within phage type across Salmonella enteric While these methods help identify sets of polymorphisms and biological properties specific to a narrow niche, they are unlikely to identify the broader set of polymorphisms required for S. Enteritidis to com- plete an entire infection pathway that results in egg con- tamination. It thus appears important for a number of phenotypic assays to be used to characterize strains [18]. A change in 1 to 2% of whole gene content, or about 1 gene of every 100, is a point where niche specialization of S. Enteritidis impacting egg contamination is apparent. Application of high throughput technology is not complete without a discussion of limits It is important to assess how likely automated methods of genome analysis will generate false positive and false negative results, because high throughput methods can rapidly propagate misinformation. High-density tiling mutational mapping alone was subject to Type I (false positive) error, whereas comparative genome re-sequen- cing (CGS) was subject to Type II (false negative) error. Pyrosequencing was subject to Type II error for riboso- mal gene base pair substitutions and deletions, but was otherwise accurate for calling base pair substitutions. Mutational mapping was overall the more cost-effective Conclusions The conundrum of how Salmonella enterica serovar Enteritidis generates an exceptional degree of strain het- erogeneity while exhibiting a highly clonal population Page 10 of 14 Page 10 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 the same phenotypes within a single culture. The bio- film-forming characteristics of each strain used herein were confirmed as follows. Colonies from primary plates were transferred to brilliant green (BG) agar (Acumedia- Neogen, Lansing, MI, USA) and inoculated at 10 differ- ent spots per plate so that colonies were well spaced. Spacing is required to allow colonies to grow large enough to form a well-developed biofilm. Plates were incubated for 16 hr at 37°C and then transferred to ambient temperature (24 to 27°C). Colonies were scored for morphology at 24 hr intervals and given a final clas- sification following a total of 120 hr incubation as hav- ing: i) strong biofilm formation, indicating colonies formed a well-developed organic matrix that covered the entire colony beginning within 48 hr of incubation at ambient temperature, ii) smooth, indicating colonies formed no biofilm, or iii) weak, indicating biofilm for- mation was not apparent until after 48 hr at room tem- perature and it became more organized as incubation progressed. A strain that was classified as weak never formed a biofilm resembling one that was classified as strong. Colony images were recorded using the Molecu- lar Imager ChemiDox XRS (Biorad) with epi white light and auto-capture. Whole colony images were digitally edited to increase contrast for purposes of publication and have been previously published [17]. structure has been solved for the most part. S. Enteriti- dis appears to be undergoing evolution primarily at the level of the single nucleotide polymorphism. Even bac- teriophage lineages share most chromosomal informa- tion outside of regions of lysogeny. This result indicates that exceptionally stringent methods for analysis of genetic variation are required for characterization of strains. This research supports concepts of evolution that have been previously published [12]. Linking variant genotypes to distinctive phenotypes remains a focal area of research intended to characterize evolutionary events that enable S. Enteritidis to contaminate the internal contents of eggs produced by otherwise healthy hens. Differentiation of strains by biofilm phenotype The three strains varied in biofilm production, which facilitated confirming the presence of different genomic variants. Colony morphologies for the three strains were previously published, but are shown again here to emphasize a type of phenotypic variation commonly encountered in cultures of S. Enteritidis that cannot be detected without following culturing methodology (Fig- ure 1) [12]. A review of the literature suggests that our laboratory uses a technique not in general use [17]; thus, other laboratories may not be aware that they have Strains for analysis The PT4 22079 was an environmental isolate obtained from water downstream from a flock and traced back to a major outbreak of egg contamination that was histori- cally associated with introduction of PT4 to the United States [14]. The two PT13a strains were from the spleen of a rodent caught in a hen house located in the North- east of the United States, which is a region that had been heavily impacted by the 1980s outbreak of S. Enteritidis. PT13a 21046 was shown to contaminate eggs, but it did not form biofilm; conversely, PT13a 21027 formed biofilm, but it did not contaminate eggs [12]. PT4 22079 contaminated eggs and formed biofilm, but its biofilm was weak in comparison to PT13a 21027 [11] (Figure 1). The two PT13a strains are clonally related, because no differences in overall gene content could be detected by microarray analysis [11,12]. The summation of phenotypes for strains PT13a 21046, PT13a 21027 and PT4 22079 based upon biofilm (BF) and egg contamination (EC) is BF- EC+, BF+ EC-, and BF+ EC+, respectively. Gene nomenclature for S. Enteri- tidis is SEN plus a 4 digit number that indicates relative position within in the genome. SEN0001 is thrA and the last gene is SEN4356A, which overlaps thrA. Ribosomal genes are accessioned separately, from SEN_r001 to SEN_r022 for ribosomal RNA and SEN_t001 to SEN_t084 for transfer RNA [8]. Pulsed field gel electrophoresis (PFGE) The PFGE patterns for PT13a 21027, PT13a 21046 and PT4 22079 were determined using standardized meth- ods conducted by personnel trained in the method developed by the Centers for Disease Control as part of PulseNet http://www.cdc.gov/pulsenet/references.htm [30-32]. Briefly, bacterial genomic DNA plugs were digested using the restriction enzyme, XbaI (Promega, Madison, WI, USA). Digested DNA was separated using the CHEF-DRII PFGE system as per the manufacturer’s instructions (Bio-Rad, Hercules, CA, USA). Electrophor- esis was carried out for 19 h at 6 V, using 2.2 L of the buffer× Tris/borate/EDTA0.5 (TBE) at a temperature of 14°C, and an initial pulse time of 2.16 s followed by a final switch time of 63.8 s. BioNumerics software (Applied Maths Scientific Software Development, Bel- gium) was used to normalize the band patterns based on the molecular weight standards included on each gel. Whole genome analysis and confirmatory sequencing of putative polymorphisms Salmonella enterica has seven ribosomal regions that are highly similar and which can complicate assembly and annotation of libraries. For this reason, a high density tiling method was selected for first analysis over 454 sequencing, because it was less likely to produce assem- bly artifact due to its progressive nature. Whole genome analysis was divided into two phases as described by the provider of service (Nimblegen, Inc.; http://www.nimble- gen.com/products/cgr/index.html). Briefly, the first phase is called mutational mapping. A set of 30-mer probes is computer generated from a reference sequence. For these analyses, the database of the S. Enteritidis PT4 reference genome from the Pathogen Sequencing Group at the Sanger Institute was used for generation of primers [8] (GenBank AM933172) and DNA for the experimental protocol was from PT4 22079. The primer set is used to densely tile the test genome that has been labeled with a fluorescent signal. The probes overlap every 7 bases. For a genome that is approximately 5 million base pairs, approximately 1.42 million primers are required for tiling hybridization, with the service provider stating that chip capacity is about 1.3 million base pairs of genome and 385,000 probes. Our experiments required 4 chips to process the entire genome of S. Enteritidis, which is approximately 4.86 million base pairs. Genomic regions identified by mutational mapping are then fed into the second phase of analysis, which is referred to as targeted resequen- cing. Any location of the test DNA that has a suspected SNP is resequenced in the presence of all 4 nucleotides to see which one allows sequencing to progress. Since the nucleotides are labeled, it is possible to tell which nucleotide is incorporated and thus identity is established. Pairs of primers used to amplify DNA amplicons for confirmatory sequencing of putative polymorphisms were generated from the S. Enteritidis PT4 whole gen- ome sequence made publicly available by the Pathogen Sequencing Unit of the Sanger Institute (EMBL acces- sion no.: AM933172) [8]. Forward and reverse primers used for confirmatory sequencing are available as catalo- gued information at the National Center for Biotechnol- ogy Information (NCBI) and identified within dbSNP by Assay ID. DNA isolation l l Single colonies of S. Enteritidis strains were grown in 10 ml of Brain Heart Infusion broth (BHI) (Difco BD, Franklin Lakes, NJ) at 37°C for 16 hr. Bacterial cells were pelleted in a Sorvall RC5B Plus centrifuge at 5000 × g for 15 min in a Sorvall Super-lite SLA 600 TC rotor. For mutational mapping and associated re-sequencing services (Nimblegen, Inc.), total DNA was extracted Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 11 of 14 Page 11 of 14 Page 11 of 14 using a Qiagen Genomic-tip 500/G kit following the protocol designated for bacteria. Precipitated DNA was dissolved in 150 ml of Tris-EDTA buffer (10 mM Tris- HCl, 1 mM EDTA [pH 8]) and stored at -20°C. For con- firmatory sequencing from PCR-amplified product, total DNA was extracted using a Qiagen Genomic-tip 100/G kit following the protocol designated for bacteria (Qia- gen, Valencia, Calif.). Precipitated DNA was dissolved in 200 ml of Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA [pH 8]) and stored at -20°C. Spectrophotometric readings were performed to ensure an OD260/280 ratio greater than 1.7 and a genomic DNA concentration of 1 μg/μl as required for CGS. file format that were accessed using proprietary software (SignalMap Versions 1.8 or 1.9 by Nimblegen, Inc.). To facilitate analysis, numerical signal data available as tab- format text files and readable with Notepad (Microsoft) were opened and transferred to multiple spreadsheets in 400 kb sections. 3 SNPs in the genes rrlC, rrlA, and cyaA had previously been found by use of a modified ribotyping approach [33]. All three of these known SNPs, which served as internal controls, were detected by Phase I mutational mapping. Phase II of CGS is an array-based re-sequencing pro- cess that identifies which single base pair is substituted for another and at what position the substitution or other change occurs in the reference genome. The known polymorphisms in rrlC, rrlA and cyaA were again used as internal controls for assessing specificity and sensitivity of mutational mapping done in Phase I and confirmatory resequencing done in Phase II. All sig- nals for the control SNPs generated during Phase II fell into the category “non-called ROI”. This meant that Phase II re-sequencing technology resulted in many false negatives. Therefore, SNPs that fell into the “non- called ROI” category were analyzed by confirmatory PCR-based sequencing in forward and reverse directions. Whole genome analysis and confirmatory sequencing of putative polymorphisms The cycling conditions for an Applied Biosystems 2400 Gene Amp PCR system were determined individu- ally for each primer pair and in general included dena- turation at 95°C for 1 min, then 30 cycles of 95°C for 30 s, 60°C to 70°C for 30 s, and 72°C for 2 to 3 min. Each reaction contained 400 nM of each primer, 200 μM ACGT deoxynucleotide triphosphates (dNTPs), 1.5 mM Mg++, 2.5 U Taq enzyme (Fisher, Pittsburg, Pa.), and 1μl template DNA. Single amplicon products were con- firmed by gel electrophoresis. PCR products were puri- fied using a Qiagen QIAquick PCR Purification kit and submitted for sequencing to the Eastern Regional Research Center (Wyndmoor, PA, USA). PCR amplicons were sequenced using an Applied Biosystems BigDye Terminator 1.1 reaction mix on an Applied Biosystems 3730 DNA Analyzer. Although the two phases are meant to progress seam- lessly, we did them separately to evaluate datasets at each stage for sensitivity and stringency in regards to detection of polymorphisms. Raw data were provided in Pyrosequencing (SeqWright) was performed according to the manufacturer’s instructions following methods developed for the sequencing of bacteria with a genome Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 Page 12 of 14 Page 12 of 14 of approximately 5 MB [34]. At least 10 ug of DNA was submitted for each strain. DNA quality was confirmed to have an OD260/280 ratio > 1.8 and a minimum con- centration of 50 ng/uL in TE as measured by spectro- photometer (NanoDrop). Briefly, sample was fragmented to between 300-500 bp and ends were repaired. Adaptor ligation was used to tag fragments with a 5’-biotin tag. Library beads were emulsified with amplification reagents in an oil water mix. After amplification, library beads were layered onto a PicoTiterPlate™. Single- stranded PCR products were sequenced using a GS FLX XLR70 Titanium platform (454 Life Sciences) and pro- duct from all 3 strains was run on one full plate. Data assembly was done with commercially available software (Newbler™Assembler). according to its location in the S. Enteritidis PT4 refer- ence database and according to similarity to genes in S. Typhimurium LT2 [35]. SNPs in intergenic regions were included in the interval map by annotating with the 3’ gene flanking the polymorphism with the extension “.5” after annotation. The number of genes between S. Whole genome analysis and confirmatory sequencing of putative polymorphisms Enteritidis PT13a genes containing SNPs was deter- mined using a Microsoft Excel automated sequential function to calculate the difference between gene acces- sion numbers for the two serotypes. To do this, the gene annotations were numbered from least to greatest for S. Enteritidis PT4, and then these genes were aligned with the similar gene from S. Typhimurium LT2. It is important to note that the genes were initially selected as similar pairs by BLAST search; however, the order within respective genomes can be different due to inver- sions [7,21]. Sequence alignment, similarities, and phylogenetic analysis Disclosure of Competing interests Nucleotide and amino acid sequence alignment was done by the Clustal W method using software from DNASTAR. Default parameter settings were used. Nucleotide polymorphisms were located within the PT4 reference genome using the EditSeq and SeqMan pro- grams. The nucleotide location of interest ± 150 bp of flanking DNA was used to conduct BLAST searches against the S. Typhimurium LT2 genome available at NCBI (GenBank: AE006468). If no match with similarity greater than 90% was found between the two serotypes, the BLAST search was first extended to other Salmo- nella enterica serotypes with available whole genome databases, then to all available Salmonella databases and finally to all proteobacteria in the gamma subdivision. Additional BLAST searches were conducted on other databases available at the Sanger Institute. The authors declare that they have no competing interests. Additional material Additional file 1: Single nucleotide polymorphisms (SNPs) of Salmonella enterica subsp. I serovar Enteritidis PT13a that differentiate subpopulations within variant pathotypes. This file is a searchable spreadsheet that lists each confirmed SNP according to its location in reference strain Salmonella enterica subsp. enterica serovar str. P125109NC_011294 (GenBank AM933172). An investigator can type in a gene name or label under the “find” function under “Edit” in Word Excel to see if the gene they are interested in contains a SNP. Column headings indicate specific information about each SNP, including if it is synonymous or non-synonymous. This is the file that will be updated in the event errors or additions are reported or discovered within the database. Additional file 2: Interval map aligning SNPs of Salmonella Enteritidis PT13a (SEN) to the genome of Salmonella Typhimurium LT2 (STM). This file was generated to search for gaps between genes that might indicate an insertion or deletion that was missed by the mutational mapping approach. Primers were generated only from the reference sequence. If the test sequence has additional DNA not present in the reference sequence, it could go unnoticed. Regions offset by 5 genes or greater suggest different gene contents. See text for further information. g Once it was determined that a polymorphism was within a gene, all available sequences for that gene were downloaded to EditSeq files from completed Salmonella genomes available as public databases from NCBI http:// www.ncbi.nlm.nih.gov/sutils/genom_table.cgi and The Sanger Institute http://www.sanger.ac.uk/cgi-bin/blast/ submitblast/Salmonella. Gene sequences were translated to amino acid sequences and aligned with DNAStar MegAlign software. Results are shown in Table 2. Attempts were made to annotate genes and to identify genes flanking non-coding regions based upon available annotation from both S. Enteritidis and S. Typhimur- ium. Linkages to proteins and inclusion of some infor- mation about conservation of amino acid change, gene class and gene function is included in supplemental information (Additional File 1). List of Abbreviations aa: amino acid,; ap: acidic polar; BG: brilliant green; BHI: brain heart infusion; bp: basic polar; CGS: comparative genome sequencing; dNTPs: deoxynucleotide triphosphates; EMBL: The European Molecular Biology Laboratory; HMM LPS: high-molecular-mass LPS; LPS: lipopolysaccharide; NCBI: National Center for Biotechnology Information; nn: neutral non-polar; np: neutral polar; PCR: polymerase chain reaction; PFGE: pulsed field gel electrophoresis; PT: phage type; S. Enteritidis: Salmonella enterica serovar Enteritidis; S. Typhimurium: Salmonella enterica serovar Typhimurium; SNP: single nucleotide polymorphism Construction of a SNP interval map Acknowledgements This research was supported and conducted primarily by funds from ARS Project Number 6612-32000-004-00. Thanks to Bill Klimke and Hua Zhang at Any gene with a SNP that differentiated the two PT13a strains was annotated by gene accession number Page 13 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 NIH/NLM/NCBI for review, construction and editing of the SNP database website and for managing entries into dbSNP. Thanks to staff at the Bacterial Epidemiology and Antibiotic Resistance Unit at USDA-ARS in Athens, Ga for performing PFGE analysis through funds associated with ARS Project Number 6612-32000-002-00. Dirk Bumann at the University of Basil, Switzerland reviewed cellular pathways. The reference whole-genome database for S. Enteritidis PT4 was produced by the Pathogen Genome Sequencing Group at the Sanger Institute and is available at http://www. sanger.ac.uk/Projects/Salmonella/. Details included in supplementary information and other information on S. Enteritidis are available at http:// www.ncbi.nlm.nih.gov/genomes/static/Salmonella_SNPS.html. Animal experimentation using Leghorn hens referred to in this manuscript was completed prior to these genomic analyses and was published elsewhere [9,11,17,33]. All experiments involving animals that resulted in the current research project were reviewed by an Institutional Animal Use and Care Committee (IACUC), which is overseen by the Office of Laboratory Welfare (OLAW) under assurance number A4298-01. Any animals used in past research were treated in a humane and ethical manner and procedures for handling, treatment, and euthanasia followed agency, national and international guidelines. provides insights into evolutionary and host adaptation pathways. Genome Res 2008, 18(10):1624-1637. provides insights into evolutionary and host adaptation pathways. Genome Res 2008, 18(10):1624-1637. 9. Parker CT, Liebana E, Henzler DJ, Guard-Petter J: Lipopolysaccharide O- chain microheterogeneity of Salmonella serotypes Enteritidis and Typhimurium. Environ Microbiol 2001, 3(5):332-342. 10. Pan Z, Carter B, Nunez-Garcia J, Abuoun M, Fookes M, Ivens A, Woodward MJ, Anjum MF: Identification of genetic and phenotypic differences associated with prevalent and non-prevalent Salmonella Enteritidis phage types: analysis of variation in amino acid transport. Microbiology 2009, 155(Pt 10):3200-3213. 11. Morales CA, Porwollik S, Frye JG, Kinde H, McClelland M, Guard-Bouldin J: Correlation of phenotype with the genotype of egg-contaminating Salmonella enterica serovar Enteritidis. Appl Environ Microbiol 2005, 71(8):4388-4399. 12. Guard J, Shah DH, Morales CA, Call DR: Evolutionary trends associated with niche specialization as modeled by whole genome analysis of egg- contaminating Salmonella enterica serovar Enteritidis. In Salmonella: From Genome to Function. Edited by: Porowollik S. Caister Academic Press; 2011:91-106. 13. References 1. 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Int J Med Microbiol 2004, 294(2-3):95-102. 21. Porwollik S, McClelland M: Lateral gene transfer in Salmonella. Microbes Infect 2003, 5(11):977-989. 21. Porwollik S, McClelland M: Lateral gene transfer in Salmonella. Microbes Infect 2003, 5(11):977-989. 3. St Louis ME, Morse DL, Potter ME, DeMelfi TM, Guzewich JJ, Tauxe RV, Blake PA: The emergence of grade A eggs as a major source of Salmonella enteritidis infections. New implications for the control of salmonellosis. JAMA 1988, 259(14):2103-2107. 22. Herring CD, Palsson BO: An evaluation of Comparative Genome Sequencing (CGS) by comparing two previously-sequenced bacterial genomes. BMC Genomics 2007, 8:274. 4. Landers E, Gonzalez-Hevia MA, Mendoza MC: Molecular epidemiology of Salmonella serotype Enteritidis. Relationships between food, water and pathogenic strains. Int J Food Microbiol 1998, 43(1-2):81-90. 23. Scacheri PC, Crawford GE, Davis S: Statistics for ChIP-chip and DNase hypersensitivity experiments on NimbleGen arrays. Methods Enzymol 2006, 411:270-282. 23. Scacheri PC, Crawford GE, Davis S: Statistics for ChIP-chip and DNase hypersensitivity experiments on NimbleGen arrays. Methods Enzymol 2006, 411:270-282. 5. White PL, Naugle AL, Jackson CR, Fedorka-Cray PJ, Rose BE, Pritchard KM, Levine P, Saini PK, Schroeder CM, Dreyfuss MS, et al: Salmonella Enteritidis in meat, poultry, and pasteurized egg products regulated by the U.S. Food Safety and Inspection Service, 1998 through 2003. J Food Prot 2007, 70(3):582-591. 24. Dotsch A, Pommerenke C, Bredenbruch F, Geffers R, Haussler S: Evaluation of a microarray-hybridization based method applicable for discovery of single nucleotide polymorphisms (SNPs) in the Pseudomonas aeruginosa genome. BMC Genomics 2009, 10:29. g 25. Authors’ contributions JG conceived of, designed and implemented the approach used to conduct whole genome sequencing in a manner to minimize genetic noise. JG previously conducted animal and phenotypic experiments with technical staff. JG developed the biofilm assay. JG collated and managed all data, reviewed methods for accuracy, chose providers of services, and drafted the manuscript. JG developed strategies for animal and phenotype assays that led to selection of strains. CM designed primers, carried out confirmatory sequencing and performed phenotype microarray assays. CM participated in reviewing drafts of the manuscript and to the management of data. CM managed the laboratory on a daily basis, maintaining reagents and working stocks. PFC conducted PFGE analysis. RKG participated in related animal experimentation. All authors read and approved the final manuscript. 14. Kinde H, Read DH, Chin RP, Bickford AA, Walker RL, Ardans A, Breitmeyer RE, Willoughby D, Little HE, Kerr D, et al: Salmonella enteritidis, phase type 4 infection in a commercial layer flock in southern California: bacteriologic and epidemiologic findings. Avian Dis 1996, 40(3):665-671. 15. 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Microbiology 2011, 157(Pt 5):1428-1445. Acknowledgements Pang JC, Chiu TH, Helmuth R, Schroeter A, Guerra B, Tsen HY: A pulsed field gel electrophoresis (PFGE) study that suggests a major world-wide clone of Salmonella enterica serovar Enteritidis. Int J Food Microbiol 2007, 116(3):305-312. References Solano C, Garcia B, Valle J, Berasain C, Ghigo JM, Gamazo C, Lasa I: Genetic analysis of Salmonella enteritidis biofilm formation: critical role of cellulose. Mol Microbiol 2002, 43(3):793-808. 6. Cowden JM, Chisholm D, O’Mahony M, Lynch D, Mawer SL, Spain GE, Ward L, Rowe B: Two outbreaks of Salmonella enteritidis phage type 4 infection associated with the consumption of fresh shell-egg products. Epidemiol Infect 1989, 103(1):47-52. 26. Mangalappalli-Illathu AK, Lawrence JR, Swerhone GD, Korber DR: Architectural adaptation and protein expression patterns of Salmonella enterica serovar Enteritidis biofilms under laminar flow conditions. Int J Food Microbiol 2008, 123(1-2):109-120. 7. Porwollik S, Santiviago CA, Cheng P, Florea L, McClelland M: Differences in gene content between Salmonella enterica serovar Enteritidis isolates and comparison to closely related serovars Gallinarum and Dublin. J Bacteriol 2005, 187(18):6545-6555. 27. Costerton JW, Stewart PS, Greenberg EP: Bacterial biofilms: a common cause of persistent infections. Science 1999, 284(5418):1318-1322. 8. Thomson NR, Clayton DJ, Windhorst D, Vernikos G, Davidson S, Churcher C, Quail MA, Stevens M, Jones MA, Watson M, et al: Comparative genome analysis of Salmonella Enteritidis PT4 and Salmonella Gallinarum 287/91 28. Hall-Stoodley L, Stoodley P: Evolving concepts in biofilm infections. Cell Microbiol 2009, 11(7):1034-1043. Page 14 of 14 Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 29. Gantois I, Ducatelle R, Pasmans F, Haesebrouck F, Van Immerseel F: The Salmonella Enteritidis lipopolysaccharide biosynthesis gene rfbH is required for survival in egg albumen. Zoonoses Public Health 2009, 56(3):145-149. 30. Jackson CR, Fedorka-Cray PJ, Wineland N, Tankson JD, Barrett JB, Douris A, Gresham CP, Jackson-Hall C, McGlinchey BM, Price MV: Introduction to United States Department of Agriculture VetNet: status of Salmonella and Campylobacter databases from 2004 through 2005. Foodborne Pathog Dis 2007, 4(2):241-248. 31. Ribot EM, Fair MA, Gautom R, Cameron DN, Hunter SB, Swaminathan B, Barrett TJ: Standardization of pulsed-field gel electrophoresis protocols for the subtyping of Escherichia coli O157:H7, Salmonella, and Shigella for PulseNet. Foodborne Pathog Dis 2006, 3(1):59-67. 32. Tankson JD, Fedorka-Cray PJ, Jackson CR, Headrick M: Genetic relatedness of a rarely isolated Salmonella: Salmonella enterica serotype Niakhar from NARMS animal isolates. J Antimicrob Chemother 2006, 57(2):190-198. 33. Morales CA, Musgrove M, Humphrey TJ, Cates C, Gast R, Guard-Bouldin J: Pathotyping of Salmonella enterica by analysis of single-nucleotide polymorphisms in cyaA and flanking 23S ribosomal sequences. Environ Microbiol 2007, 9(4):1047-1059. 34. Guard et al. BMC Research Notes 2011, 4:369 http://www.biomedcentral.com/1756-0500/4/369 References Gharizadeh B, Akhras M, Unemo M, Wretlind B, Nyren P, Pourmand N: Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing. Int J Antimicrob Agents 2005, 26(6):486-490. 35. McClelland M, Sanderson KE, Spieth J, Clifton SW, Latreille P, Courtney L, Porwollik S, Ali J, Dante M, Du F, et al: Complete genome sequence of Salmonella enterica serovar Typhimurium LT2. Nature 2001, 413(6858):852-856. doi:10.1186/1756-0500-4-369 Cite this article as: Guard et al.: Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type. BMC Research Notes 2011 4:369. doi:10.1186/1756-0500-4-369 Cite this article as: Guard et al.: Single nucleotide polymorphisms that differentiate two subpopulations of Salmonella enteritidis within phage type. BMC Research Notes 2011 4:369. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4390275793	https://jurnalnew.unimus.ac.id/index.php/jodi/article/download/149/173	Indonesian	null	Clustering Untuk Menentukan Indeks Kesejahteraan Rakyat di Provinsi Jawa Tengah 2022 Menggunakan Metode Fuzzy C-Means	Journal of Data Insights	2,023	cc-by	4,351	Info Artikel Kesejahtraan rakyat merupakan salah satu tujuan negara yang tercantum pada Undang- undang Dasar 1945. Dalam meningkatakan kesejahtraan rakyat, tentunya perlu adanya pembangunan yang merata. Untuk menjalankan program pembangunan yang merata, harus dilakukan identifikasi berdsarkan karaktaeristik tingkat kesejahtraan rakyat berdasarkan variabel-variabel yang telah ditentukan agar dalam membuat strategi dan mengambil kebijakan untuk meningkatkan kesejahtraan rakyat dapat tepat sasaran dan optimal. Tujuan dari penelitian ini adalah untuk mengetahui pengelompokkan 35 Kabupatan/Kota di Provinsi Jawa Tengah dan k arakteristik dari setiap kelompok berdasarkan indeks kesejahtraan rakyat. Berdasarkan hasil analisis yang dilakukan, bahwa terdapat 35 Kabupaten/Kota di Provinsi Jawa Tengah dapat membentuk 4 kelompok (cluster), dimana pada cluster 0 beranggotakan 8 Kabupaten/Kota dengan karakteristik Jumlah peduduk miskin tinggi, Daya beli cenderung rendah, rata-rata lama sekolah rendah, angka harapan hidup sangat rendah. Pada cluster 1 terdapat 12 Kabupaten/Kota dengan karakteristik Nilai PDRB sangat tinggi, angka pengangguran relatif rendah, angka lama sekolah relatif tinggi. Pada cluster 2 terdapat 5 Kabupaten/Kota dengan karakteristik Nilai PDRB sangat rendah, jumlah penduduk miskin sangat rendah, daya beli sangat tinggi, kepemilikan rumah rendah, kepadatan penduduk tinggi, daya beli tinggi, angka melek huruf tinggi, rata-rata lama sekolah tinggi, dan yang terakhir cluster 3 terdapat 10 Kabupaten/Kota dengan karakteristik PDRB rendah, kepadatan penduduk sangat rendah, kepemilikan rumah sangat tinggi, daya beli cenderung rendah. 12345Statistika Universitas Muhammadiyah Semarang, Indonesia DOI: https://doi.org/10.26714/jodi  Alamat Korespondensi: E-mail: indrafirmansyah@gmail.com E-mail: indrafirmansyah@gmail.com Abstrak Info Artikel ___________________ Sejarah Artikel: Disubmit 17 Juli 2023 Direvisi 16 November 2023 Disetujui 12 Desember 2023 ___________________ Keywords: Fuzzy C-Means; Clustering; Data Mining; Indeks Kesejahteraan Rakyat  Alamat Korespondensi: Vol. 1 No. (2) (Desember 2023) 81-91 Vol. 1 No. (2) (Desember 2023) 81-91 PENDAHULUAN Clustering atau pengelompokan merupkan metode yang diterapkan pengelompokkan data di beberapa grup atau cluster berdasarkan persamaan-persamaan yang telah ditentukan sebelumnya. Dimana dalam satu grup atau cluster terebut memiliki kesamaan atau karateristik yang tinggi dan miliki perbedaan yang tinggi pada antar grup atau cluster. Dengan adanya perbedaan dan persamaan tersebut yang ada dalam data, dapat dijadikan sebgai perhitungan jarak (Dhuhita, 2015) (Kresna Amijaya et al., 2018), salah satu metode untuk pengelompokan yaitu Fuzzy C-Means. Tujuan dari negara salah satunya adalah kesejahtraan rakyat yang tercantum dalam UUD1945 pada Alinea ke-4 (Kholis et al., n.d.). Kesejahtraan raykat akan selalu dinamis, yang akan selalu berubah-ubah sesuai dengan kebutuhan hidup seiring perjalanannya waktu dan berkembangnya zaman. Serba berkecukupan baik secara material dan spiritual merupakan kedaan yang dapat dikatakan sejahtera. Masyarakat dapat dikatakan sejahtra juga ketika manusia sudah dapat mememnuhi kebutuhan hidup (Setiawan, 2019). Ada beberapa indeks yang menjadi pengukur kesejahtraan yaitu, PDRB kabupaten/kota, kepadatan penduduk, jumlah penduduk miskin di tiap kabupaten/kota, daya beli masyarakat, jumlah angkatan kerja, angka harapan hidup, angka melek huruf, rata-rata lama sekolah, angka harapan lama sekolah, tingkat pengangguran terbuka, dan kepemilikan rumah sendiri (Alwi & Hasrul, 2018). Provinsi Jawa Tengah memiliki 35 kabupaten/kota (Sukowati, 2020). Setiap kabupaten/kota melilki tingkat kesejahrtaan yang berbeda-beda. Jumlah penduduknya di tahun 2022 sebanyak 37.032.410 jiwa dengan angka harapan hidup pada tahun 2022 sebsesar 74,57 tahun, dimana angka harapan hidup merupakan salah satu indeks kesejahtraan rakyat (Tengah, 2022). Untuk menyamaratakan dan meningkatkan kesejahteraan rakyat ini dapat dilakukan pengelompokan atau clustering. Tujuan mengelompokan kabupaten/kota yang ada di Provinsi Jawa Tengah dengan optimal, agar strategi dan kebijakan-kebijakan yang ditetapkan oleh pemerintah dapat di implementasikan sesuai dengan kendala yang ada pada setiap cluster. Untuk mengclusterkan hal tersebut tentunya didasarkan oleh indeks-indeks kesejahtraan rakyat (Yulianto & Hidayatullah, 2016). Pengelompokkan dapat dilakukan dengan data mining. Dalam data mining, ada teknik yang biasa dikenal dengan clustering. Fuzzy C-Means (FCM) yaitu salah satu dalam metode pengelompokan yang terdapat dalam data mining (Zahrotun, 2018). Metode Fuzzy C-Means (FCM) diperkenalkan pertama kali pada tahun 1981 oleh Jim Bezdek, dimana pada suatu cluster ditentukan oleh derajat anggotanya pada keberadaan data masing-masing titik, di kondisi awalnya pusat cluster belum akurat. Metode ini dapat diterapkan dalam memecahkan persoalan mengenai clustering pada bidang astronomi, target pengenalan, segmentasi gambar, kimia, geolodi, dan diagnosis medis (Riandayani et al., 2014) (Suroso et al., 2011). Abstract People's welfare is one of the goals of the state stated in the 1945 Constitution. In improving people's welfare, of course there is a need for equitable development. In order to carry out an equitable development program, identification must be carried out based on the characteristics of the level of people's welfare based on predetermined variables so that in making strategies and adopting policies to improve people's welfare can be right on target and optimal. The purpose of this study was to determine the grouping of 35 regencies/cities in Central Java Province and the characteristics of each group based on the people's welfare index. Based on the results of the analysis carried out, that there are 35 regencies/cities in Central Java province that can form 4 groups (clusters), where in cluster 0 there are 8 regencies/cities with the characteristics of a high number of poor people, purchasing power tends to be low, the average length of schooling low, life expectancy is very low. In cluster 1 there are 12 regencies/cities with the characteristics of a very high GRDP value, a relatively low unemployment rate, a relatively high number of years of schooling. In cluster 2 there are 5 regencies/cities with the characteristics of very low GRDP, very low number of poor people, very high purchasing power, low house ownership, high population density, high purchasing power, high literacy rate, average length of schooling is high, and finally, in cluster 3, there are 10 regencies/cities with low GRDP characteristics, very low population density, very high house ownership, and low purchasing power e-ISSN: 2988 - 2109 81 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023 dengan kedua metode yang digunakan berbeda (Ramadhan et al., 2017). Selanjutnya penelitian oleh Iin Parlina denga judul Penerapan Metode Fuzzy C-Means Pada Pengelompokan Kabupaten/Kota di Pulau Kalimantan Berdasarkan Indikator Kesejahteraan Rakyat Tahun 2020, dengan hasil penelitian terdapat 3 jumlah cluster optimal, adapun pada anggota klaster 2, pemerintah dapat memperhatikan variabel angka beban ketergantungan, keluhan kesehatan, pada anggota klaster 3, pemerintah dapat memperhatikan variabel kepemilikan akta kelahiran, APS, APM, fasilitas tempat buang air besar pada anggota klaster 1, pemerintah dapat memperhatikan variabel angka beban ketergantungan, APS, APM SMP, APM SMA (Nurmin et al., 2022). dengan kedua metode yang digunakan berbeda (Ramadhan et al., 2017). Selanjutnya penelitian oleh Iin Parlina denga judul Penerapan Metode Fuzzy C-Means Pada Pengelompokan Kabupaten/Kota di Pulau Kalimantan Berdasarkan Indikator Kesejahteraan Rakyat Tahun 2020, dengan hasil penelitian terdapat 3 jumlah cluster optimal, adapun pada anggota klaster 2, pemerintah dapat memperhatikan variabel angka beban ketergantungan, keluhan kesehatan, pada anggota klaster 3, pemerintah dapat memperhatikan variabel kepemilikan akta kelahiran, APS, APM, fasilitas tempat buang air besar pada anggota klaster 1, pemerintah dapat memperhatikan variabel angka beban ketergantungan, APS, APM SMP, APM SMA (Nurmin et al., 2022). Urgensi dan tujuan dilakukannya kasus ini untuk menjelaskan n cluster paling optimal dalam pengelompokkan mengenai kabupaten/kota Jawa Tengah berdasarkan Indeks Kesejahteraan Rakyat 2022 dengan menggunakan Fuzzy C-Means. Informasi dari peelitian ini diharapkan dapat memberikan manfaat dan diharapkan sebagai bahan petimbangan dalam membuat kebijakan ataupun keputusan yang sesuai dengan kendala yang ada apa setiap cluster, sehingga solusi ataupun kebijakan yang telah direncanakan dapat diterapkan dengan baik serta sesuai dengan kondisi yang ada pada setiap cluster PENDAHULUAN Tujuan dari metode ini untuk memperoleh dan menentukan pusat cluster, digunakan untuk mengetahui data yang masuk pada tiap cluster (Simbolon et al., 2013). Penelitian ini akan didasarkan pada penggunaan algoritma Fuzzy C-Means (FCM). FCM adalah teknik pengelompokan dimana keberadaan setiap bagian data pada suatu cluster ditentukan oleh nilai keanggotaannya. Awalnya, tentukan pusat cluster terlebih dahulu yang dimana pusat cluster iniuntuk pengelompokkan titiknya di suatu kelompok. Penentuan cluster nilai keanggotaan akan dilakukan dengan cara berulang sehingga didapat pusat cluster yang nantinya akan bergerak ke lokasi yang tepat dikarenakan kondisi pada pusat cluster dan titik setiap datanya berdasarkan derajat keanggotaan belum bisa terlalu akurat (Mulyani et al., 2020). Dan peneliti juga memakai algoritma FCM karena beberapa penelitian yang memakai algoritma (FCM) telah dijelaskan bahwa Fuzzy C- Means lebih akurat dibandingkan algoritma K-Means . Beberapa Artikel sebelumnya telah berhasil mengelompokkan dengen menggunakan metode Fuzzy C-Means. Diantaranya Rahman Syarif, dkk dengan judul “Perbandingan Algoritma K-Means dengan Algoritma Fuzzy C- Means (FCM) Pada Kelompok Moda Transportasi Berbasis GPS”. Berdasarkan hasil penelitian klasifikasi moda transportasi berbasis GPS, hasil pengujian sebanyak sepuluh kali menghasilkan tingkat akurasi algoritma FCMs lebih akurat daripada algoritma K-Means dan dibutuhkan metode lanjutan khusus pada pengelompokkan data trajectory karena adanya keunikan dan kerumitan pada data tesebut (Syarif et al., 2018). Selanjutnya penelitian oleh Aditya Ramadhan, dkk dengan judul Perbandingan K-Means dan Fuzzy C-Means untuk Pengelompokan Data User Knowledge Modeling, dengan hasil penelitian metode Fuzzy ini metode yang lebih akurat untuk melakukan clustering pada data user knowledge modelling disebabkan validitasnya yang menuju satu. hasil pengelompokan 82 1. Masukkan data yang mau dilakukan pengelompokan, X merupakan matrix ukuran n x m (dengan ‘n’ yaitu total sample dan ‘m’ atribut di setiap datanya). Xij merupakan data di sampel ke-I (I=1,2,..,dst sampai n), serta atribut ke-j(j=1,2,…, dst sampai m) Data Data penelitian yang dipakai untuk artikel kali ini yaitu data sekunder berasal dari Website BPS Provinsi Jawa Tengah. Metode yang diterapkan yaitu clustering menggunakan algoritma Fuzzy C-Means dengan tujuan pengelompokkan Kab/Kota Jawa Tengah dalam beberapa cluster menggunakan variabel yang sudah ditentukan oleh peneliti. Variabel yang digunakan yaitu PDRB pada tiap kab/kota (X1), Kepadatan penduduk (X2), Jumlah Penduduk Miskin (X3), Daya Beli (X4), Jumlah Angkatan Kerja (X5), Tingkat Pengangguran Terbuka (X6), Angka Melek Huruf (X7), Rata-rata Lama Sekolah (X8), Angka Harapan Lama Sekolah (X9), Angka Harapan Hidup (X10), Kepemilikan Rumah Sendiri (X11). Tabel 1. Data Indeks Kesejahteraan Rakyat Tabel 1. Data Indeks Kesejahteraan Rakyat . Kab/Kota X 1 X 2 X 3 X 4 X 5 X 6 X 7 X 8 X 9 X 10 X 11 Kab. Cilacap 𝑋 1,1 𝑋 1,2 𝑋 1,3 𝑋 1,4 𝑋 1,5 𝑋 1,6 𝑋 1,7 𝑋 1,8 𝑋 1,9 𝑋 1,10 𝑋 1,11 Kab. Banyumas 𝑋 2,1 𝑋 2,2 𝑋 2,3 𝑋 2,4 𝑋 2,5 𝑋 2,6 𝑋 2,7 𝑋 2,8 𝑋 2,9 𝑋 2,10 𝑋 2,11 …. …. …. …. …. …. …. …. …. …. …. …. Kota Tegal 𝑋 35,1 𝑋 35,2 𝑋 35,3 𝑋 35,4 𝑋 35,5 𝑋 35,6 𝑋 35,7 𝑋 35,8 𝑋 35,9 𝑋 35,10 𝑋 35,11  Langkah-langkah Penelitian Fuzzy C-Means (Yani et al., 2014): 1. Masukkan data yang mau dilakukan pengelompokan, X merupakan matrix ukuran n x m (dengan ‘n’ yaitu total sample dan ‘m’ atribut di setiap datanya). Xij merupakan data di sampel ke-I (I=1,2,..,dst sampai n), serta atribut ke-j(j=1,2,…, dst sampai m) 2. Menentukan : Banyaknya h cluster ( c ), pangkat (w), max iterasi (masxlter), error terkecil ( e), fungsi objektif awal(po=0), iterasi awal(t=1) ( ), g j (p ), ( ) 3. Membangkitkan bilangan random μik, I=1,2,..,n; k=1,2,…,c; yang menjadi elemen matrix partisi awal U. Hitunglah n(jumlah) per kolomnya k=1 Hitung : Qi = ∑c μik , dgn j = 1,2, . . , n μik = μik Qi μik = μik Qi μik = μik Qi 4. Menghitung pusatnya di cluster ke-k, Vkj dengan k=1,2,…, dst samapi c dan j=1,2,…, sampai m 83 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) ∑n ((μik)w ∗ X ij) Vkj = i=1 ∑n ((μik)w i=1 5. 7. Melakukan pengecekan saat posisi stop(berhenti) -jika (\|Pt-Pt-1\|<e) atau (t> maxlter) makaberhenti; -jika tidak:t=t+1, ulangi langkah no empat 1. Analisis Deskriptif Analisa deskriptif dipakai guna memperoleh gambaran yang general mengenai masing-masing variabel penelitian dalam mengukur indeks kesejahteraan masyarakat, meliputi mean(rataan), standar deviasi (stdev), nilai max, dan nilai min. Tabel 2. Analisis Deskriptif Vari abel Minu mum Maximu m Rata- rata Std Deviasi X2 495 11878 2148, 45 2506,7 X3 8,65 290,66 109,4 7 61,13 X4 8994 16351 11533 ,6 1813,53 X5 6,68 1,07 5,56 2,28 X6 69,74 77,82 75,11 1,82 X7 96,06 100 97,52 1,52 X8 6,35 10,95 8,14 1,27 X9 11,78 15,54 13,01 0,91 X10 1,76 9,64 5,34 1,97 X11 60,65 97,83 87,97 87,97 Dapat dilihat pada tabel diatas, nilai minimum variabel X1 yaitu PDRB 0.65 sementara nilai maksimumnya sebesar 14.58 dan mean-nya sebesar 2.85 juga nilai st.deviasinya sebesar 2.53. Untuk variabel lain dapat dilihat di tabel diatas. HASIL DAN PEMBAHASAN 1. Analisis Deskriptif Dapat dilihat pada Heatmap di bawah ini, semakin terang warnanya, maka memiliki korelasi yang semakin kuat, sedangkan semakin gelap warnanya, maka semakin tidak memiliki korelasi yang kuat. 2. Korelasi Data Hitunglah fungsi objektif di iterasi ke-t, Pt n c m Pt = ∑ ∑ ([∑(Xij − Vkj)2] (μik)w) i=1 k=1 j=1 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) ∑n ((μik)w ∗ X ij) Vkj = i=1 ∑n ((μik)w i=1 5. Hitunglah fungsi objektif di iterasi ke-t, Pt n c m Pt = ∑ ∑ ([∑(Xij − Vkj)2] (μik)w) i=1 k=1 j=1 5. Hitunglah fungsi objektif di iterasi ke-t, Pt 5. Hitunglah fungsi objektif di iterasi ke-t, Pt n c m Pt = ∑ ∑ ([∑(Xij − Vkj)2] (μik)w) i=1 k=1 j=1 6. Hitung adanya perubahan di matriks partisi 6. Hitung adanya perubahan di matriks partisi j=1 μik = −1 [∑m (Xij − Vkj)2]w−1 −1 , dengan i = 1,2, . . . , n dan k = 1,2, . . . , c 84 84 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) ∑c [∑m (Xij − Vkj)2]w−1 k=1 j=1 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) ∑c [∑m (Xij − Vkj)2]w−1 k=1 j=1 7. Melakukan pengecekan saat posisi stop(berhenti) 7. Melakukan pengecekan saat posisi stop(berhenti) Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Gambar 1. Heatmap Korelasi Berdasarkan hasil output diatas dapat diketahui nilai korelasi paling tinggi yaitu pada variabel Rata-rata Lamanya Sekolah (X8) dengan variabel Angka Harapan Lama Sekolah (X9) sebesar 0.92, sementara yang memiliki korelasi paling rendah yaitu pada variabel Kepemilikan Rumah (X11) dengan variabel Kepadatan Penduduk (X2) sebesar -0.89. 3 Pe e t cluster Gambar 1. Heatmap Korelasi Gambar 1. Heatmap Korelasi Gambar 1. Heatmap Korelasi Berdasarkan hasil output diatas dapat diketahui nilai korelasi paling tinggi yaitu pada variabel Rata-rata Lamanya Sekolah (X8) dengan variabel Angka Harapan Lama Sekolah (X9) sebesar 0.92, sementara yang memiliki korelasi paling rendah yaitu pada variabel Kepemilikan Rumah (X11) dengan variabel Kepadatan Penduduk (X2) sebesar -0.89. Berdasarkan hasil output diatas dapat diketahui nilai korelasi paling tinggi yaitu pada variabel Rata-rata Lamanya Sekolah (X8) dengan variabel Angka Harapan Lama Sekolah (X9) sebesar 0.92, sementara yang memiliki korelasi paling rendah yaitu pada variabel Kepemilikan Rumah (X11) dengan variabel Kepadatan Penduduk (X2) sebesar -0.89. 2. Korelasi Dapat dilihat pada Heatmap di bawah ini, semakin terang warnanya, maka memiliki korelasi yang semakin kuat, sedangkan semakin gelap warnanya, maka semakin tidak memiliki korelasi yang kuat. 85 3. Penentuan cluster Untuk menentukan tingkat cluster pada Indeks Kesejahteraan Rakyat di provinsi Jawa Tengah dapat menggunakan Elbow Method ditentukan. Dalam analisis cluster, metode siku merupakan heuristik yang diterapkan dalam menetukan jumlah cluster dalam suatu kumpulan data yang akan dipakai. Metode ini terdiri dari melakukan plot variasi yang digunakan sebagai fungsi dari jumlah cluster serta peemilihan siku kurva sebagai jumlah cluster yang akan digunakan. Asumsinya titik batas heuristik umum dalam pengoptimalan matematis memilih titik di mana hasil yang menurun tidak lagi sepadan dengan biaya tambahan. Gambar 2. Elbow Method Gambar 2. Elbow Method Gambar 2. Elbow Method Dalam gambar diatas, tingkat cluster pada Indeks Kesejahteraan Rakyat Prov. Jawa Tengah, dilihat di nilai grafik bahwa terlihat seperti siku pada cluster ke-4. Sementara untuk cluster ke-5 dan seterusnya tidak terlihat seperti siku (lurus). Untuk memperkuat penentuan jumlah cluster perlu dilakukan validasi cluster. Pada validasi ini kita menggunakan Silhouette Score. Pendekatan silhouette dengan memakai mean cluster untuk 86 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) melakukan pendugaan nilai k optimum. Jika semakin keatas nilai silhoutte maka cluster dikatakan semakin baik(akurat). Gambar 3. Silhouett Score Gambar 3. Silhouett Score 4. Berdasarkan grafik diatas, terdapat cluster optimal sebanyak 4. Ini sama dengan hasil penentuan k optimal menggunakan Elbow Method. Gambar diatas, menunjukkan angka 4 sebagai angka teringgi, sehingga dapat disimpulkan 4 merupakan k optimal. Clustering Langkah pertama kita lakukan standarisasi data untuk menghapus rata-rata dan menskalakan 4. Berdasarkan grafik diatas, terdapat cluster optimal sebanyak 4. Ini sama dengan hasil penentuan k optimal menggunakan Elbow Method. Gambar diatas, menunjukkan angka 4 sebagai angka teringgi, sehingga dapat disimpulkan 4 merupakan k optimal. Clustering penentuan k optimal menggunakan Elbow Method. Gambar diatas, menunjukkan angka 4 sebagai angka teringgi, sehingga dapat disimpulkan 4 merupakan k optimal. Clustering Langkah pertama kita lakukan standarisasi data untuk menghapus rata-rata dan menskalakan it varian. Berikut hasil yang sudah di standarisasi datanya. Langkah pertama kita lakukan standarisasi data untuk menghapus rata-rata dan menskalakan unit varian. Berikut hasil yang sudah di standarisasi datanya. Tabel 3. Data Standarisasi Kab/Kota X1 X2 …. X11 Kab. Cilacap 1,9 57 - 0,493 …. 0,8 05 Kab.Bany umas 0,4 68 - 0,319 …. 0,4 05 …. …. …. …. …. Kota Tegal 0,0 37 - 0,352 …. 0,3 4 Tabel diatas, menunjukkan 35 kab dan kota di Prov Jawa Tengah data yang sudah terstandarisasi dengan masing masing memiliki 11 variabel X, (X1,X2,…,X11). Tabel diatas, menunjukkan 35 kab dan kota di Prov Jawa Tengah data yang sudah terstandarisasi dengan masing masing memiliki 11 variabel X, (X1,X2,…,X11). Setelah dilakukan standarisasi selanjutnya kita tentukan nilai tengah dari setiap variabel untuk Setelah dilakukan standarisasi selanjutnya kita tentukan nilai tengah dari setiap variabel untuk k optimal. Di dapat nilai tengah setiap variabel pada k optimal. Tabel 4. Nilai Tengah Cluster Vari abel Cluster X1 0.0 595 0.17 29 - 0.4315 - 0.2278 X2 - 0.3212 - 0.2764 1.96 66 - 0.4267 … …. …. …. …. 87 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) X11 0.3 124 0.22 80 - 2.0210 0.46 93 Jika sudah didapat nilai tengah, selanjutnya kita lakukan pengelompokan berdasarkan algoritma yang sudah kita buat. Hailnya tertera di gambar bawah ini. Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) X11 0.3 124 0.22 80 - 2.0210 0.46 93 Jika sudah didapat nilai tengah, selanjutnya kita lakukan pengelompokan berdasarkan algoritma yang sudah kita buat. Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) 1. Karakteristiktik Untuk melihat karakteristik dari tiap cluster yang kita dapatkan, kita dapat melihat pada nilai rata-rata setiap variabel dalam cluster. Seperti tabel dibawah. Tabel 6. Rata-rata cluster per variabel V ariabel Cluster 0 1 2 3 X 1 3,28 5 4,08 9 1,43 2 1, 751 X 2 12.3 20,25 1.57 3,08 7.44 6,6 92 2,700 X 3 189, 53 97,9 7 21,1 26 10 3,386 X 4 10.2 80 12.2 40,2 14.2 86,6 10 .331 X 5 789. 309,4 605. 767,3 154. 835,4 51 1.708 X 6 72,3 2 76,3 42 76,2 44 74 ,565 X 7 97,7 97,4 94 98,8 28 96 ,771 X 8 7,24 8,6 10,2 02 7, 275 X 9 12,6 6 13,4 27 14,1 14 12 ,257 X 10 7,08 5,00 5 5,95 6 4, 054 X 11 91,1 3 89,4 62 66,1 86 94 ,529 Berdasarkan hasil perhitungan, kami membandingkan nilai rata-rata keseluruhan dengan rata- rata setiap cluster untuk menentukan karakteristik per clusternya. Berdasarkan hasil perhitungan, kami membandingkan nilai rata-rata keseluruhan dengan rata- rata setiap cluster untuk menentukan karakteristik per clusternya. Berdasarkan hasil perhitungan, kami membandingkan nilai rata-rata keseluruhan dengan rat rata setiap cluster untuk menentukan karakteristik per clusternya. Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Hailnya tertera di gambar bawah ini. Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Jika sudah didapat nilai tengah, selanjutnya kita lakukan pengelompokan berdasarkan algoritma yang sudah kita buat. Hailnya tertera di gambar bawah ini. Jika sudah didapat nilai tengah, selanjutnya kita lakukan pengelompokan berdasarkan algoritma yang sudah kita buat. Hailnya tertera di gambar bawah ini. Gambar 4 Clustering plot Indeks Kesejahtraan Rakyat di Prov. Jawa Tengah Gambar 4 Clustering plot Indeks Kesejahtraan Rakyat di Prov. Jawa Tengah Dari hasi polt diatas diatas, terlihat terdapat tanda "+" yang merupakan pusat cluster dari 4 cluster yang terbentuk. Sedangkan 4 titik berwarna merupakan sebaran cluster yang ada dari Indeks Kesejahtraan Rakyat di Prov. Jawa Tengah. Tabel 5.Hasil cluster dan karakterisik Tabel 5.Hasil cluster dan karakterisik Clu ster Hasil Karakteristik 0 Kab. Cilacap, Kab. Banyumas, Kab. Kebumen, Kab. Magelang, Kab. Grobogan, Kab. Pemalang, Kab. Tegal, Kab. Brebes Cluster 0 memiliki Jumlah peduduk miskin tinggi, Daya beli cenderung rendah, lama sekolah rendah, AHH sangat rendah mengindikasi daerah perlu perhatian khusus. 1 Kab. Purworejo, Kab. Boyolali, Kab. Klaten, Kab. Sukoharjo, Kab. Karanganyar, Kab. Sragen, Kab. Pati, Kab. Kudus, Kab. Demak, Kab. Semarang, Kab. Kendal, Kota Semarang. Cluster 1 memiliki Nilai PDRB sangat tinggi, angka pengangguran relatif rendah, angka lama sekolah relatif tinggi mengindikasi wilayah industri. 2 Kota Magelang, Kota Surakarta, Kota Salatiga, Kota Pekalongan, Kota Tegal. Cluster 2 mendapati Nilai PDRB sangat rendah, jumlah penduduk miskin sangat rendah, daya beli sangat tinggi, kepemilikan rumah rendah, kepadatan penduduk tinggi, daya beli tinggi, angka melek huruf tinggi, rata-rata lama sekolah tinggi dimana mengindikasi wilayah perkotaan. 3 Kab.Purbalingga, Kab. Banjarnegara, Kab. Wonosobo, Kab. Wonogiri, Kab. Blora, Kab. Rembang, Kab. Jepara, Kab. Temanggung, Kab. Batang, Kab. Pekalongan. Cluster 3 memiliki PDRB rendah, kepadatan penduduk sangat rendah, kepemilikan rumah sangat tinggi, daya beli cenderung rendah indikasi wilayah perdesaan. 88 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) perhatian khusus. Cluster 1 memiliki Nilai PDRB sangat tinggi, angka pengangguran relatif rendah, angka lama sekolah relatif tinggi mengindikasi wilayah industri. Cluster 2 mendapati Nilai PDRB sangat rendah, jumlah penduduk miskin sangat rendah, daya beli sangat tinggi, kepemilikan rumah rendah, kepadatan penduduknya tinggi, tingkat daya beli tinggi, angka melek huruf masih tinggi, rata- rata lama sekolah(RLS) tinggi dimana mengindikasi wilayah perkotaan. Cluster 3 memiliki PDRB rendah, kepadatan penduduk sangat rendah, kepemilikan rumah sangat tinggi, daya beli cenderung rendah indikasi wilayah perdesaan. KESIMPULAN Berdasarkan hasil validitas pada metode FCM, jumlah cluster optimal dalam pengelompokkan kab dan kota di provinsi Jateng berdasar indeks kesejahteraan masyarakat yaitu 4 cluster. Untuk cluster 0 beranggotakan Kab Cilacap, Kab Banyumas, Kab Kebumen, Kab Magelang, Kab Grobogan, Kab Pemalang, Kab Tegal, Kab Brebes. Anggota cluster 1 Kab Purworejo, Kab Boyolali, Kab Klaten, Kab Sukoharjo, Kab Karanganyar, Kab Sragen, Kab Pati, Kab Kudus, Kab Demak, Kab Semarang, Kab Kendal, Kota Semarang. Anggota cluster 2 Kota Magelang, Kota Surakarta, Kota Salatiga, Kota Pekalongan, Kota Tegal. Sedangkan cluster 3 beranggotakan Kab Purbalingga, Kab Banjarnegara, Kab Wonosobo, Kab Wonogiri, Kab Blora, Kab Rembang, Kab Jepara, Kab Temanggung, Kab Batang, Kab Pekalongan. Adapun Cluster 0 memiliki Jumlah peduduk miskin tinggi, Daya beli cenderung rendah, rata- rata lama sekolahnya rendah, angka harapan hidupnya sangat rendah mengindikasi wilayah perlu 89 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) DAFTAR PUSTAKA Alwi, W., & Hasrul, M. (2018). Analisis Klaster Untuk Pengelompokkan Kabupaten/Kota Di Provinsi Sulawesi Selatan Berdasarkan Indikator Kesejahteraan Rakyat. Jurnal MSA ( Matematika Dan Statistika Serta Aplikasinya ), 6(1), 35.https://doi.org/10.24252/msa.v6i1.4782 Kholis, N., Kunci, K., Kesejahteraan, :, Islam, J., & Sosial, D. E. (n.d.). Kesejahteraan Sosial Di Indonesia Perspektif Ekonomi Islam. Mulyani, S., Sari, B. N., & Ridha, A. A. (2020). Clustering Productivity of Rice in Karawang Regency Using the Fuzzy C-Means Method. Indonesian Journal of Artificial Intelligence and Data Mining, 3(2), 103–112. https://ejournal.uin- suska.ac.id/index.php/IJAIDM/article/view/10415 Nurmin, D., Hayati, M. N., & Goejantoro, R. (2022). Penerapan Metode Fuzzy C-Means Pada Pengelompokan Kabupaten/Kota di Pulau Kalimantan Berdasarkan Indikator Kesejahteraan Rakyat Tahun 2020. Eksponensial, 13(2), 189–196. Ramadhan, A., Efendi, Z., & Mustakim. (2017). Perbandingan K-Means dan Fuzzy C- Means untuk Pengelompokan Data User Knowledge Modeling. Seminar Nasional Teknologi Informasi, Komunikasi Dan Industri (SNTIKI) 9, 219–226. Riandayani, D. A., Darma Putra, I. K. G., & Buana, P. W. (2014). Comparing Fuzzy Logic and Fuzzy C-Means (FCM) on summarizing indonesian language document. Journal of Theoretical and Applied Information Technology, 59(3), 718–724. Setiawan, H. H. (2019). Merumuskan Indeks Kesejahteraan Sosial (Iks) Di Indonesia. Sosio Informa, 5(3). https://doi.org/10.33007/inf.v5i3.1786. Simbolon, C. L., Kusumastuti, N., & Irawan, B. (2013). Clustering lulusan mahasiswa matematika fmipa untan pontianak menggunakan algoritma fuzzy c - means. Buletin Ilmiah Mat. Stat. Dan Terapannya (Bimaster), 02(1), 21–26. Sukowati, J. L. (2020). Klasterisasi Kabupaten Di Provjawa Tengah. 3(2), 136–149. Sukowati, J. L. (2020). Klasterisasi Kabupaten Di Provjawa Tengah. 3(2), 136–149. Suroso, D. J., Cherntanomwong, P., Sooraksa, P., & Takada, J. I. (2011). Location fingerprint technique using Fuzzy C-Means clustering algorithm for indoor localization. IEEE Region 10 Annual International Conference, Proceedings/TENCON, November, 88–92. https://doi.org/10.1109/TENCON.2011.6129069 Syarif, R., Furqon, M. T., & Adinugroho, S. (2018). Perbandingan Algoritme K-Means Dengan Algoritme Fuzzy C Means ( FCM ) Dalam Clustering Moda Transportasi Berbasis GPS. Jurnal Pengembangan Teknologi Informasi Dan Ilmu Komputer (J-PTIIK) Universitas Brawijaya, 2(10), 4107–4115. http://j- ptiik.ub.ac.id/index.php/j- ptiik/article/view/2852 Tengah, B. P. S. P. J. (2022). No Title. Bps Provinsi Jawa Tengah. Tengah, B. P. S. P. J. (2022). No Title. Bps Provinsi Jawa Tengah. Yani, J. A., Banjarbaru, K., & Selatan, K. (2014). Implementasi Metode Fuzzy C-Means Pada Sistem Clustering Data Varietas Padi. 01(01), 23–32. Yulianto, S., & Hidayatullah, K. H. (2016). Analisis Klaster Untuk Pengelompokan Kabupaten/Kota Di Provinsi Jawa Tengah Berdasarkan Indikator Kesejahteraan Rakyat. Statistika, 2(1), 56–63. DAFTAR PUSTAKA https://jurnal.unimus.ac.id/index.php/statistik/article/view/1115 90 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Zahrotun, L. (2018). Implementation of data mining technique for customer relationship management (CRM) on online shop tokodiapers.com with fuzzy c-means clustering. Proceedings - 2017 2nd International Conferences on Information Technology, Information Systems and Electrical Engineering, ICITISEE 2017, 2018-Janua, 299–303. https://doi.org/10.1109/ICITISEE.2017.8285515 Journal of Data Insights e-ISSN: 2988 - 2109 vol.1 (2) (Desember 2023) Zahrotun, L. (2018). Implementation of data mining technique for customer relationship management (CRM) on online shop tokodiapers.com with fuzzy c-means clustering. Proceedings - 2017 2nd International Conferences on Information Technology, Information Systems and Electrical Engineering, ICITISEE 2017, 2018-Janua, 299–303. https://doi.org/10.1109/ICITISEE.2017.8285515 Zahrotun, L. (2018). Implementation of data mining technique for customer relationship management (CRM) on online shop tokodiapers.com with fuzzy c-means clustering. Proceedings - 2017 2nd International Conferences on Information Technology, Information Systems and Electrical Engineering, ICITISEE 2017, 2018-Janua, 299–303. https://doi.org/10.1109/ICITISEE.2017.8285515 91 91
https://openalex.org/W3033592537	https://europepmc.org/articles/pmc7296197?pdf=render	English	null	Synthesis, antiinflammatory activity, and molecular docking studies of bisphosphonic esters as potential MMP-8 and MMP-9 inhibitors	Beilstein journal of organic chemistry	2,020	cc-by	7,593	Full Research Paper Open Access Beilstein J. Org. Chem. 2020, 16, 1277–1287. doi:10.3762/bjoc.16.108 Received: 12 March 2020 Accepted: 25 May 2020 Published: 08 June 2020 Associate Editor: I. Baxendale © 2020 Cortes-Pacheco et al.; licensee Beilstein-Institut. License and terms: see end of document. Address: 1Instituto de Química Aplicada, Universidad del Papaloapan. Tuxtepec, 68301, Mexico, 2Unidad de Investigación Médica (UIM) en Farmacología, UMAE Hospital de Especialidades, Centro Médico Nacional Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS). Av. Cuauhtémoc 330, Col. Doctores 06720, Ciudad de México (CdMx), Mexico and 3Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos Avenida Universidad 1001, Chamilpa, 62210 Cuernavaca, Morelos, Mexico © 2020 Cortes-Pacheco et al.; licensee Beilstein-Institut. License and terms: see end of document. Synthesis, antiinflammatory activity, and molecular docking studies of bisphosphonic esters as potential MMP-8 and MMP-9 inhibitors Abimelek Cortes-Pacheco1,2, María Adelina Jiménez-Arellanes2, Francisco José Palacios-Can3, José Antonio Valcarcel-Gamiño3, Rodrigo Said Razo-Hernández3, María del Carmen Juárez-Vázquez2, Adolfo López-Torres1 and Oscar Abelardo Ramírez-Marroquín1 Full Research Paper Open Access Address: 1Instituto de Química Aplicada, Universidad del Papaloapan. Tuxtepec, 68301, Mexico, 2Unidad de Investigación Médica (UIM) en Farmacología, UMAE Hospital de Especialidades, Centro Médico Nacional Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS). Av. Cuauhtémoc 330, Col. Doctores 06720, Ciudad de México (CdMx), Mexico and 3Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos Avenida Universidad 1001, Chamilpa, 62210 Cuernavaca, Morelos, Mexico Email: Oscar Abelardo Ramírez-Marroquín - oramirez@unpa.edu.mx * Corresponding author Keywords: inflammation; molecular docking; organophosphorus compounds Beilstein J. Org. Chem. 2020, 16, 1277–1287. doi:10.3762/bjoc.16.108 Received: 12 March 2020 Accepted: 25 May 2020 Published: 08 June 2020 Associate Editor: I. Baxendale © 2020 Cortes-Pacheco et al.; licensee Beilstein-Institut. License and terms: see end of document. Full Research Paper Open Access Address: 1Instituto de Química Aplicada, Universidad del Papaloapan. Tuxtepec, 68301, Mexico, 2Unidad de Investigación Médica (UIM) en Farmacología, UMAE Hospital de Especialidades, Centro Médico Nacional Siglo XXI (CMN-SXXI), Instituto Mexicano del Seguro Social (IMSS). Av. Cuauhtémoc 330, Col. Doctores 06720, Ciudad de México (CdMx), Mexico and 3Centro de Investigación en Dinámica Celular, Universidad Autónoma del Estado de Morelos Avenida Universidad 1001, Chamilpa, 62210 Cuernavaca, Morelos, Mexico Email: Oscar Abelardo Ramírez-Marroquín* - oramirez@unpa.edu.mx * Corresponding author Keywords: inflammation; molecular docking; organophosphorus compounds Beilstein J. Org. Chem. 2020, 16, 1277–1287. doi:10.3762/bjoc.16.108 Received: 12 March 2020 Accepted: 25 May 2020 Published: 08 June 2020 Associate Editor: I. Baxendale © 2020 Cortes-Pacheco et al.; licensee Beilstein-Institut. License and terms: see end of document. Introduction Figure 1: Previously reported antiinflammatory bisphosphonates 1 and 2. edema inhibition (in %, carrageenan model, 50 mg/kg) for 1: 7.0; for 2: 22.2. Bisphosphonic acids (or bisphosphonates) are organophos- phorus compounds characterized by a P–C–P moiety. These organic compounds are valuable drugs for the treatment of bone diseases as osteoporosis, Paget’s disease, and malignant hyper- calcemia [1-3]. Specifically, bisphosphonates act as osteoclast resorption inhibitors, augmenting the bone density and preventing osteoporosis [4]. Moreover, some bisphosphonates have gained attention as potential antiinflammatory agents by in vitro and in vivo assays [5-8]. Additionally, bisphosphonates have been reported as inhibition and downregulation matrix metalloproteinase (MMP) agents [9-11]. In this regard, MMPs are a family of extracellular proteinases (24 isoenzymes in human) involved in tissue regeneration and are closely related to physiologic and physiopathological processes, such as inflammation, angiogenesis, and metastasis in cancer [12-15], pointing at bisphosphonates as potential treatments for cancer and other inflammation-related diseases. In this respect, MMP inhibition by phosphonates or bisphosphonates has been previ- ously studied through computational or X-ray diffraction analyses to describe the enzyme inhibitor site binding modes [16-18]. Figure 1: Previously reported antiinflammatory bisphosphonates 1 and 2. edema inhibition (in %, carrageenan model, 50 mg/kg) for 1: 7.0; for 2: 22.2. Furthermore, in the search of more potent and low-toxicity de- rivatives, in this work, we have focussed our attention on the molecular modification of the derivatives 1 and 2 through the bioisosteric replacement [20] of the amide functional group by an aliphatic or aromatic ester. The potential antiinflammatory activity of the new bisphosphonates was predicted using the Prediction of Activity Spectra for Substances (PASSOnline) database, which compares the molecular structure of test com- pounds vs a large training set of experimental bioactive or inac- tive compounds [21]. The results of the prediction are summa- rized as probability of activity (Pa) and probability of inactivity (Pi) values, both ranging from 0 to 1 (Figure 2), where a higher Pa value is desired. Thus, a Pa value of new bisphosphonates ≥ the Pa value of 1 and 2 was the applied inclusion criterion in this study. As can be seen in Figure 2, the Pa is greater for the new derivatives 3–6 (0.63–0.77) compared to the previous bio- active compounds 1 and 2 (0.51). Introduction In order to explore the SAR in the proposed compounds 3–6, we evaluated the effect of the volume of the ester group by the inclusion of ethyl or tert-butyl substituents in the aliphatic derivatives 3 and 4. On the other hand, the replacement of the aliphatic chains by an aromatic portion led us to the derivatives 5 and 6 where the effect of benzyl or 4-methoxybenzyl substituents was assessed (Figure 2). Thus, in this work, the bisphosphonates 3–6 were synthesized by a two-step method and then evaluated through two in vivo acute inflammation models in BALB/c mice. Furthermore, the acute toxicity was determined for these derivatives, and molec- ular docking studies were performed to account for a possible action mechanism as MMP-8 and MMP-9 inhibitors. Abstract Bisphosphonic acids (or bisphosphonates) have been successfully used in the clinic treatment of bone diseases for over decades. Additionally, the antiinflammatory activity of these compounds has been gaining attention. In our previous work, we synthesized and in vivo evaluated the bisphosphonic esters 1 and 2, finding a moderate edema inhibition upon oral and topical administration on BALB/c mice. Thus, in this work, the bioisosteric replacement of an amide functional group for an ester afforded the new bisphos- phonates 3–6, which had a moderate oral edema inhibition (25 mg/kg dose) and a significant topical antiinflammatory activity (2 mg/ear) on BALB/c mice, with 6 being the most active hit (55.9% edema inhibition), comparable to the positive control (55.5% edema inhibition) on a TPA topical model. Next, to assess the acute toxicity of the synthesized derivatives, test animals were administered with 50–100 mg/kg of 3–6, respectively, by an oral route, and after 14 days, neither lethality nor a significative weight loss were observed. Finally, a structure–activity relationship (SAR) and a molecular docking analysis of 3–6 helped us to explain the trend observed in biological tests. Considering all these aspects, we propose the inhibition of MMP-8 and MMP-9 as a possible action mechanism of the synthesized derivatives. 1277 1277 Beilstein J. Org. Chem. 2020, 16, 1277–1287. Results and Discussion Chemistry As part of our ongoing interest in the discovery of new antiin- flammatory agents, our research group have previously addressed the synthesis and in vivo antiinflammatory activity evaluation of the bisphosphonic esters 1 and 2, observing activi- ty by oral (carrageenan model) and topical administration (TPA model) in BALB/c mice (Figure 1) [19]. Furthermore, the bisphosphonic esters 3–6 passed the Lipinski’s rules [22] as criteria for drugs for an oral administration as we Figure 2: Designed bisphosphonic esters as antiinflammatory agents. Figure 2: Designed bisphosphonic esters as antiinflammatory agents. Figure 2: Designed bisphosphonic esters as antiinflammatory agents. 1278 Beilstein J. Org. Chem. 2020, 16, 1277–1287. canoylphorbol-13-acetate (TPA) through topical administration [25] and b) a carrageenan model, through oral administration [25]. When the bisphosphonates 3–6 (2 mg/ear) were assayed with a TPA model, the derivatives 5 (40.7% edema inhibition) and 6 (55.9% edema inhibition) were the most active ones, with 6 having a comparable edema inhibition to the positive control (indomethacin, 55.5% edema inhibition, Table 1). Nevertheless, the derivatives 3 and 4 exhibited a moderate antiinflammatory activity (25.5% and 23.9% edema inhibition, respectively). Thus, the inclusion of an aromatic ring in the derivatives 5 and 6 clearly potentiated the desired pharmacological effect on mice. The rationale behind this could be the difference in lipophilicity between the aliphatic esters 3 and 4, which were less lipophilic (by means of the clogP value of 0.58 and 1.39, respectively) compared to the aromatic derivatives 5 and 6 (clogP = 1.80 and 1.85, respectively), with the last being the most permeable one through mice skin (Table 1). Nevertheless, it is important to note that the aliphatic derivatives 3 and 4 considerably differed in the clogP value between each other (0.58 and 1.39) but had a comparable edema inhibition, indicat- ing that for this study, the volume of the ester group had little importance for the pharmacological activity. More important was the replacement of the aliphatic for an aromatic residue in wanted to test these derivatives through in vivo acute inflamma- tion models [23]. Then, the above-named derivatives were syn- thesized in a first stage by the esterification of bromoacetyl bro- mide and the corresponding alcohol. The reaction of ethyl or tert-butyl alcohol and bromoacetyl bromide in the presence of triethylamine in CH2Cl2 yielded the bromoaceto esters 7 and 8 in 42 and 71% yield (Scheme 1b, method A). Results and Discussion Chemistry Nonetheless, when benzyl or 4-methoxybenzyl alcohol were used under the same reaction conditions, 9 and 10 were obtained in poor yields. Thus, the subsequent use of NaHCO3 as a base in CH3CN [24] afforded the bromoaceto esters 9 and 10 in 71 and 91% yield (Scheme 1b, method B). It is important to note that 4-methoxy- benzyl alcohol was prepared by the reduction of 4-methoxy- benzaldehyde (Scheme 1a). Next, the treatment of tetraethyl methylenediphosphonate with NaHMDS under an anhydrous atmosphere, followed by the ad- dition of 7–10, respectively, afforded the final products 3–6, re- spectively, in 41–73% yield (Scheme 2). Pharmacological activity Next, the DE50 value was assessed for the more interesting targets 5 (DE50 = 1.4 mg/ear) and 6 (DE50 = 0.9 mg/ear), with the methoxy derivative 6 having a higher po- tency and efficacy (55.9% edema inhibition) in the series (Table 1). Our results were in good accordance with the previous observation of the antiinflammatory activity of the few bisphosphonic esters [26-30]. were predicted to be more bioactive than 5 and 6 (Table 2). Ad- ditionally, this tendency was strongly connected to the clogP value, where the edema inhibition was inversely proportional to the clogP value (Table 2). Thus, the higher the predicted Pa and the lower the clogP, the higher the observed activity. It is worth to mention that the oral efficacy of the tested compounds was opposed to that observed with the TPA topical model. This may be a consequence of the lower lipophilicity of 3 (clogP = 0.58) and 4 (clogP = 1.39) compared to 5 (clogP = 1.80) and 6 (clogP = 1.85), influencing the better dissolution of 3 and 4 in an aqueous medium prior to its absorption through gut mice (Table 2). Finally, the synthesized bisphosphonates 3 and 4 have proven to be more active (24.6% and 20.9% edema inhibi- tion, respectively, at a 25 mg/kg dose) by oral administration than the parent compounds 1 and 2 (7.0% and 22.2% edema inhibition, respectively, at a 50 mg/kg dose, Figure 1) [19]. In addition, 3 and 4 had a comparable activity than what was re- ported for other bisphosphonic esters [30]. Next, the antiinflammatory activity of the bisphosphonates 3–6 was assayed with a carrageenan model by intragastric adminis- tration. As can be seen in Table 2, the derivatives 3 and 4 were the more active ones this time (24.6% and 20.9% edema inhibi- tion, respectively). A remarkable difference was observed for the derivatives 5 (13.8% edema inhibition) and 6 (9.1% edema inhibition) where a low antiinflammatory activity was observed. In this regard, a clear correlation between the experimental and predicted activity was observed. Thus, the compounds 3 and 4 Following this, the acute toxicity of 3–6 was determined through one oral administration of 50 or 100 mg/kg in BALB/c mice, and after 14 days, no significant weight loss or lethality was observed in the individuals. Additionally, the post-mortem Table 2: Antiinflammatory activity of 3–6 with a carrageenan oral model. Pharmacological activity With the target compounds on hand, we proceed to evaluate them with two acute inflammation models: a) 12-O-tetrade- S h 1 S h i f h i di b 7 10 Scheme 1: Synthesis of the intermediate bromoaceto esters 7–10. Scheme 1: Synthesis of the intermediate bromoaceto esters 7–10. Scheme 2: Synthesis of the bisphosphonates 3–6. Scheme 2: Synthesis of the bisphosphonates 3–6. 1279 Beilstein J. Org. Chem. 2020, 16, 1277–1287. Table 1: Antiinflammatory activity of 3–6 using a TPA topical model. 2 mg/ear of the test compounds was used.a treatment auricular edema (mg) % inhibition clogPb DE50 (mg/ear) TPA 8.80 mg ± 0.46 – – – indomethacin (2 mg/ear) 3.92 mg ± 0.37c 55.5 – – 3 6.56 mg ± 0.19c,d 25.5 0.58 n.d. 4 6.70 mg ± 0.39c,d 23.9 1.39 n.d. 5 5.22 mg ± 0.37c,d 40.7 1.80 1.4 6 3.88 mg ± 0.21c 55.9 1.85 0.9 aThe data is presented as mean ± standard error (s.e.). The percentage of inhibition of the edema is given in respect to the TPA group. Statistical analysis one-way ANOVA, post hoc SNK test (p ≤ 0.05). bCalculated using the Molinspiration property engine v2018.10b [31]. cVs TPA control. dVs indomethacin; n = 5. Table 1: Antiinflammatory activity of 3–6 using a TPA topical model. 2 mg/ear of the test compounds was used.a aThe data is presented as mean ± standard error (s.e.). The percentage of inhibition of the edema is given in respect to the TPA group. Statistical analysis one-way ANOVA, post hoc SNK test (p ≤ 0.05). bCalculated using the Molinspiration property engine v2018.10b [31]. cVs TPA control. dVs indomethacin; n = 5. as mean ± standard error (s.e.). The percentage of inhibition of the edema is given in respect to the TPA group. Statistical VA, post hoc SNK test (p ≤ 0.05). bCalculated using the Molinspiration property engine v2018.10b [31]. cVs TPA control. dVs the ester group, leading to the more active derivatives 5 and 6. The clogP value of 5 and 6 was more similar between them, but a remarked difference in the edema inhibition was observed (40.7 vs 55.9%), indicating that the introduction of an electron- donating 4-methoxy substituent on the phenyl ring of 6 potenti- ated the antiinflammatory activity compared to the nonsubsti- tuted derivative 5. Pharmacological activity Test compounds: 25 mg/kg.a treatment paw edema (mm)b % inhibition Pa clogPc carrageenan 0.830 ± 0.017d – – – indomethacin (20 mg/kg) 0.530 ± 0.04d 36.1 – – 3 0.626 ± 0.03d,e 24.6 0.77 0.58 4 0.657 ± 0.02d,e 20.9 0.71 1.39 5 0.716 ± 0.03d,e 13.8 0.66 1.80 6 0.755 ± 0.06e 9.1 0.63 1.85 aThe data is presented as mean ± standard error (s.e.). The percentage of inhibition of the edema is in respect to the carrageenan group. Statistical analysis one-way ANOVA, post hoc SNK test (p ≤ 0.05). bAt 5 h. cCalculated using Molinspiration property engine v2018.10b [31]. dVs control carrageenan. eVs indomethacin; n = 5. Table 2: Antiinflammatory activity of 3–6 with a carrageenan oral model. Test compounds: 25 mg/kg.a aThe data is presented as mean ± standard error (s.e.). The percentage of inhibition of the edema is in respect to the carrageenan group. Statistical analysis one-way ANOVA, post hoc SNK test (p ≤ 0.05). bAt 5 h. cCalculated using Molinspiration property engine v2018.10b [31]. dVs control carrageenan. eVs indomethacin; n = 5. 1280 Beilstein J. Org. Chem. 2020, 16, 1277–1287. From Table 3, we observed that a correlation between the anti- inflammatory activity of the compounds and the molecular properties can be established. The structural modifications are directly correlated to the molecular weight (MW) of the com- pounds, and the MW can be correlated to the molecular volume. We can appreciate that 3 and 4 have the lowest volume com- pared to 5 and 6. This fact can help us to explain the greater antiinflammatory activity of 3 and 4 with a carrageenan model; a small molecular volume increases the pharmacokinetic abili- ties of the compounds. In addition, 3 and 4 have a greater dipole moment compared to 5 and 6. The hydrogen-bond formation and the noncovalent interactions are influenced by the dipole moment. This means that an increased dipole moment can improve the binding properties of a molecule. Besides the dipole moment, 3 and 4 have the greatest chemical hardness of the bisphosphonates. This descriptor is related to the chemical susceptibility to an external potential. Therefore, the antiinflam- matory activity of these compounds can be related to the size (volume), solvation (dipole moment), and chemical reactivity (hardness; probably related to a minor metabolic biotransforma- tion). Pharmacological activity These descriptors can be associated with the better phar- macokinetic profile of the derivatives 3 and 4 by oral adminis- tration. inspection of the kidneys, heart, and bowel of the experimental mice did not show any significant weight differences to the control group (Supporting Information File 1, Table 1, and Table 2). Lastly, in order to acknowledge a potential mechanism of action of the bisphosphonates 3–6, we propose that the tested deriva- tives are acting as MMP inhibitors. In this respect, MMP-8 and MMP-9 isoenzymes are related to inflammatory processes in different tissues [32-35]. Furthermore, for MMP-8 and MMP-9, enzyme–inhibitor interaction modes are well known. For exam- ple, the coordination of the P=O oxygen atom in bisphospho- nates with a zinc cation in the catalytic site of the MMPs has been characterized, both through X-ray diffraction and molecu- lar docking studies [11,36,37]. Consequently, we propose MMP-8 and MMP-9 as potential biological targets of 3–6. Computational and theoretical analysis Ligands structure–activity relationship As a first approximation, we studied the structural and physico- chemical features of the compounds to explain the antiinflam- matory activity. The structure geometry used to obtain the mo- lecular properties of each bisphosphonate represented a minimum in the potential energy surface since all vibrational frequency values were positive. In Table 3, all the molecular properties obtained for the compounds are displayed. The chemical hardness (η) and softness (S) were calculated using Equation 1 and Equation 2, which are based in the Koopman’s theorem for the determination of the global chemical reactivity descriptors. Molecular docking His201 was far apart from the catalytic site, and the basic nitrogen atom in this residue was pointing away from the zinc ion [39]. The calculated distances from the Zn2+ ion and the different sites at the ligand and the protein are summarized in Table 5. 3, with the most potent inhibition observed with the carrageenan model (Table 2). The predicted LE value for 4 in MMP-9 was comparable to 3 but showed less activity than 3. For both dock- ings, 6 showed some of the highest MolDock Score values, but nevertheless, its inhibition activity was the lowest (Table 2). It is important to note that a clear correlation between the pre- dicted interaction energy of 3–6 with MMP-8 and the topical antiinflammatory activity was observed (TPA model), with the derivative 6 being the most active one, followed by 5, 3, and 4 in that order (Table 1). Thus, the efficacy of the tested com- pounds 3–6 was well correlated with the lipophilicity and the predicted interaction energy with MMP-8 (Table 4). Next, we searched for other energy interaction features from the docking calculation. Table 5: Calculated distances (Å) for the coordination of the zinc(II) ion at MMP-8. molecule 3 4 5 6 His207 2.28 3.30 2.36 2.26 His197 2.47 2.46 2.45 2.44 Glu198 3.84 3.85 4.16 4.13 oxygen (ligand) 1.99 1.99 1.99 1.99 Table 5: Calculated distances (Å) for the coordination of the zinc(II) ion at MMP-8. As expected, 4–6 could bind the Zn2+ ion in a monodentate fashion through the oxygen atom double bonded to the phos- phorus atom (P=O) of one of the phosphonate moieties, as re- ported in the literature for other structures [38,39]. Only for 3, zinc chelation was observed through the oxygen atom double bonded to the carbon atom (C=O) of the ester group (Figure 3). The zinc chelation in MMP-9 occurred in a different way. The predicted orientation of the histidine residues at the catalytic site were not aiming directly at the metal ion. However, it can be assumed that because of the flexibility of the protein in solution, the coordination to the metal ion would be possible. The targets 3 and 4 could coordinate the Zn2+ ion through the oxygen atom double bonded to the phosphorus atom (P=O) of one of the phosphonate moieties, while 5 and 6 had interactions through the C=O oxygen atom of the ester group. Molecular docking As a second approximation, to study the effect of these struc- tural modification on the pharmacodynamics, we performed a molecular docking over two MPPs. In Table 4, the interaction energy value (MolDock Score) [38] of each compound with the two different MMPs obtained from the docking calculation is displayed. Also, the ligand efficiency (LE) of each bisphospho- nate is shown; the ligand efficiency stands for the coefficient of the interaction energy by the number of atoms in the molecule (excluding hydrogen atoms). (1) (2) (1) (2) By the inspection of the results above, there is a correlation be- tween the LE parameter in MMP-8 and the experimental activi- ty of the bisphosphonates, resulting in the highest LE value for Table 3: Molecular properties of the compounds 3–6. Table 3: Molecular properties of the compounds 3–6. molecular properties 3 4 5 6 molecular weight (amu) 374.307 402.361 436.378 466.404 dipole moment (Debye) 3.4 3.4 2.43 2.4 EHOMO (eV) −10.57 −10.44 −9.41 −8.83 ELUMO (eV) 0.82 0.72 0.25 0.19 volume (Å3) 363.78 401.16 430 455.41 PSA (Å2) 71.532 73.573 70.905 74.198 hardness (η) 5.69 5.58 4.83 4.51 softness (S) 0.1756 0.1792 0.207 0.2217 1281 Beilstein J. Org. Chem. 2020, 16, 1277–1287. Table 4: MolDock Score and LE1 values (kcal/mol) for the docking experiments of the molecules 3–6 with MMP-8 and MMP-9 enzymes and the cor- responding inhibition values.a Table 4: MolDock Score and LE1 values (kcal/mol) for the docking experiments of the molecules 3–6 with MMP-8 and MMP-9 enzymes and the cor- responding inhibition values.a ligand MMP-8 MMP-9 % inhibition MolDock Score LE MolDock Score LE 3 −146.72 −6.38 −134.16 −5.83 24.57 4 −137.92 −5.52 −147.76 −5.91 20.86 5 −150.42 −5.37 −147.24 −5.26 13.80 6 −168.58 −5.62 −146.50 −4.88 9.06 aIndomethacin was set as the reference (with 36.1272%). In all cases, a distorted tetrahedral coordination geometry was observed for the zinc ion, caused by the chelation with His197, Glu198, and His207 residues. His201 was far apart from the catalytic site, and the basic nitrogen atom in this residue was pointing away from the zinc ion [39]. The calculated distances from the Zn2+ ion and the different sites at the ligand and the protein are summarized in Table 5. In all cases, a distorted tetrahedral coordination geometry was observed for the zinc ion, caused by the chelation with His197, Glu198, and His207 residues. Molecular docking Because the interac- tions of the benzyl group of 5 and 6, respectively, with the Phe110 residue present at the catalytic site through π–π interac- tions are possible, the orientation of the molecules inside the catalytic site allowed the coordination through the ester groups rather than through the phosphonate moieties. The calculated distances from the Zn2+ ion and the different sites at the ligand and the protein are summarized in Table 6. Figure 3: Coordination of the Zn2+ ion by residues and by the carbon- yl ester oxygen atom of molecule 3. The basic coordinating nitrogen and oxygen atoms are marked in light blue. In Figure 4, a schematic representation of the interactions of the ligands in MMP-8 is shown. As expected, the oxygen atoms that are coordinating the zinc ion have the greatest contribution. For example, 3 in MMP-8 has a greater contribution energy Figure 3: Coordination of the Zn2+ ion by residues and by the carbon- yl ester oxygen atom of molecule 3. The basic coordinating nitrogen and oxygen atoms are marked in light blue. Figure 3: Coordination of the Zn2+ ion by residues and by the carbon- yl ester oxygen atom of molecule 3. The basic coordinating nitrogen and oxygen atoms are marked in light blue. 1282 Beilstein J. Org. Chem. 2020, 16, 1277–1287. Table 7: Calculated total (ETotal) and electrostatic energy (EElec) for the most contributing oxygen atoms to the interaction energy of the complex. Energies in kcal/mol. energy MMP-8 3 4 5 6 ETotal (O1) −22.03 −32.22 −35.880 −34.11 EElec (O1) −24.20 −39.97 −38.200 −38.71 EElec (Zn) −13.63 −22.83 −20.4092 −19.51 Compound 3 displayed two hydrogen bonds. The first one was formed between a hydrogen atom of His162 with an oxygen atom from the ester moiety (the oxygen atom bound to the ethyl Table 6: Calculated distances (Å) for the coordination of the zinc(II) ion at MMP-9. molecule 3 4 5 6 His411 2.35 2.67 2.80 2.72 His401 2.72 3.33 3.30 3.17 Glu402 4.53 4.51 4.12 4.48 oxygen (ligand) 2.00 2.00 2.00 2.00 from oxygen O1, with an electrostatic energy (EElec) of −24.20 kcal/mol and a total energy (ETotal) of −22.03 kcal/mol; the observed energy values are shown in Table 7. Table 6: Calculated distances (Å) for the coordination of the zinc(II) ion at MMP-9. Molecular docking Table 7: Calculated total (ETotal) and electrostatic energy (EElec) for the most contributing oxygen atoms to the interaction energy of the complex. Energies in kcal/mol. Table 6: Calculated distances (Å) for the coordination of the zinc(II) ion at MMP-9. molecule 3 4 5 6 His411 2.35 2.67 2.80 2.72 His401 2.72 3.33 3.30 3.17 Glu402 4.53 4.51 4.12 4.48 oxygen (ligand) 2.00 2.00 2.00 2.00 energy MMP-8 3 4 5 6 ETotal (O1) −22.03 −32.22 −35.880 −34.11 EElec (O1) −24.20 −39.97 −38.200 −38.71 EElec (Zn) −13.63 −22.83 −20.4092 −19.51 Compound 3 displayed two hydrogen bonds. The first one was formed between a hydrogen atom of His162 with an oxygen atom from the ester moiety (the oxygen atom bound to the ethyl Compound 3 displayed two hydrogen bonds. The first one was formed between a hydrogen atom of His162 with an oxygen atom from the ester moiety (the oxygen atom bound to the ethyl from oxygen O1, with an electrostatic energy (EElec) of −24.20 kcal/mol and a total energy (ETotal) of −22.03 kcal/mol; the observed energy values are shown in Table 7. from oxygen O1, with an electrostatic energy (EElec) of −24.20 kcal/mol and a total energy (ETotal) of −22.03 kcal/mol; the observed energy values are shown in Table 7. Figure 4: 2D schematic representations of the MMP-8 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions. Figure 4: 2D schematic representations of the MMP-8 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions. 1283 Beilstein J. Org. Chem. 2020, 16, 1277–1287. (Figure 4b). Also, for compound 5, only one hydrogen bond interaction was seen. Indeed, the oxygen atom double bonded to the phosphorus atom of the phosphonate group was strongly bonded to an amide hydrogen atom localized between Ile159 and Leu160, with a 3.08 Å O–N distance and an energy of −2.5 kcal/mol (Figure 4c). Compound 6 showed four hydrogen bonds that varied in force. Two were observed between an oxygen atom from an OEt moiety of a phosphonate group to amide hydrogen atoms localized between Ile159, Leu160, and Ala161 (Figure 4d). Molecular docking Table 8: Calculated total (ETotal) and electrostatic energy (EElec) for the most contributing oxygen atoms to the system. Energies in kcal/mol. energy MMP-9 3 4 5 6 ETotal (O1) −38.4103 −39.9732 −23.9506 −23.8484 EElec (O1) −34.1810 −32.8937 −17.2600 −7.5905 EElec (Zn) −20.9699 −19.8742 −20.2706 −19.022 As can be seen in Figure 4, the hydrophobic zones of the cata- lytic site in MMP-8 were formed by Ile159, Leu160, and Ala161, as well for Gly158 and Val194. However, for 3, the ethyl groups were not entirely pointing at this zone. The calcu- lated C–C distance from a methylene group to a Leu160 methyl group was 3.35 Å. The other methyl groups from the ligand had interactions with His162 (2.96 Å), His197 (2.98 Å), and His207 (3.20 Å). Indeed, the rest of the molecules also presented this type of interactions between an ethyl fragment of the ligand with Leu160, and less with His207 and Val194. Weak hydro- phobic interactions were also observed from Tyr219 and the ar- omatic ring of 6 as well as π–π interactions to His197. The mol- ecules were not adequately occupying the cavity of the enzyme in MMP-8, as shown in Figure 4. For the compounds 4 and 5, the tert-butyl and benzyl groups at the ester moiety were pointing out to the solvent, without any important interactions with the hydrophobic zone inside the cavity (Figure 4b and Figure 4c). The only molecule that was occupying the entire pocket was compound 6 due to the formation of four hydrogen bonds and a favourable π–π interaction between the methoxy- benzyl group and His197. actions from an oxygen atom (P=O) of the phosphonate group to amide hydrogen atoms located between Ala189, Leu188, and Leu187, with energy values of −2.36 and −1.60 kcal/mol (Figure 5b). Compound 5 exhibited two weak hydrogen bonds from an OEt oxygen atom to an amide hydrogen atom of Ala189 and Leu188 (Figure 5c). The energy value was −0.89 kcal/mol, and the O–N distance was 3.24 Å. The second interaction was very weak, with a value of −0.15 kcal/mol from a P=O oxygen atom to the His401 amine hydrogen atom. Finally, for 6, the hydrogen bond interactions were lacking. From the energy contribution profile seen in Figure 5d, most of the interaction energy was due to the coordination of the ligand to the zinc ion, with the same applying to 5. Molecular docking This could explain why no predicted hydrogen bonding was present in 6 and just very weak ones in 5. For MMP-9, the hydrophobic zones were formed by Ile159, Leu160, and Ala161, as was the case for Gly158 and Val194. As stated before, π–π interactions from the aromatic ring of the ligands 5 and 6 with the Phe110 residue were displayed (Figure 5c and 5d). Furthermore, CH–π interactions from the tert-butyl group with Phe110 were also seen, with a 3.12 Å dis- tance (Figure 5b). All molecules had hydrophobic interactions from an ethyl group of the ligands to a methyl moiety of Val398 and with Leu187, with distances below 3.5 Å. Only for 3 and 4, one of the oxygen atoms of the phosphonate units was the most contributing atom to the interaction energy. However, for 5 and 6, the electrostatic energy was almost equally distributed in the three oxygen atoms of the two phos- phonate and ester groups. For example, in 5, the three above- mentioned oxygen atoms displayed a total energy-per-atom value of −17.26 (C=O), −15.62 (P1=O), and −14.07 (P2=O) kcal/mol, although the electrostatic energy (EElec) was higher at the ester oxygen atom (C=O), with a value of −23.95 kcal/mol, while the other two oxygen atoms from the phosphonate units had energies of −13.65 and −11.53 kcal/mol, respectively. The same energy trend was observed for 6, which correlated well with the predicted interactions of the molecule (the C=O and P=O units binding Zn2+) at the catalytic site (Table 8). For MMP-9, the ligands were even more exposed to the solvent, although many more noncovalent interactions were seen be- tween the catalytic site and the molecules (Figure 5). Molecular docking These interactions had energy values of −1.94 and −2.5 kcal/mol, which is regarded as characteristic for strong hydrogen bonds. The measured O–N distances had group of the phosphonate unit), with a calculated distance (O–H) of 1.86 Å (or a 2.60 Å O–N distance) and an energy of −1.51 kcal/mol. The second interaction, stronger than the first one, was observed from an oxygen atom (P=O) of the phos- phonate unit to an amide hydrogen atom located between Ile159 and Leu160. It possesses an energy of −2.5 kcal/mol and a 1.86 Å distance from the oxygen atom to the amide hydrogen atom (or a 3.09 Å O–N distance, Figure 4a). Compound 4 only displayed one hydrogen bond of the double bonded carbonyl oxygen atom to an amide hydrogen atom located between Asn218 and Tyr219. This interaction was regarded as weak, with an energy of −0.47 kcal/mol and a 3.51 Å O–N distance Figure 5: 2D schematic representations of the MMP-9 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions; green = CH–π interactions. Figure 5: 2D schematic representations of the MMP-9 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions; green Figure 5: 2D schematic representations of the MMP-9 catalytic site, with 3–6 and the most relevant interactions. Red = hydrogen bonds; dotted bonds = zinc complexation; highlighted in blue = most relevant residues; dashed pink lines = π–π interactions; grey = hydrophobic interactions; green = CH–π interactions. 1284 Beilstein J. Org. Chem. 2020, 16, 1277–1287. Table 8: Calculated total (ETotal) and electrostatic energy (EElec) for the most contributing oxygen atoms to the system. Energies in kcal/mol. energy MMP-9 3 4 5 6 ETotal (O1) −38.4103 −39.9732 −23.9506 −23.8484 EElec (O1) −34.1810 −32.8937 −17.2600 −7.5905 EElec (Zn) −20.9699 −19.8742 −20.2706 −19.022 values of 3.15 and 2.67 Å. Another hydrogen-bond interaction was seen from an OEt moiety (from the phosphonate group coordinating the zinc ion), with a weak energy of −0.95 kcal/mol and O–N distance of 3.26 Å. Finally, the me- thoxy group at the ligand had a weak energy of −1.0 kcal/mol and a 3.12 Å distance (Figure 4d). Funding 7.Pochetti, G.; Gavuzzo, E.; Campestre, C.; Agamennone, M.; 17.Pochetti, G.; Gavuzzo, E.; Campestre, C.; Agamennone, M.; Tortorella, P.; Consalvi, V.; Gallina, C.; Hiller, O.; Tschesche, H.; Tucker, P. A.; Mazza, F. J. Med. Chem. 2006, 49, 923–931. doi:10.1021/jm050787+ The authors thank CONACyT of Mexico for financial support via project CB-2017-2018 A1-S-10787, INFRA-2015-01- 252013, and INFRA-2019-301144. Abimelek Cortes thanks CONACyT for the graduate grant 942820. Antonio Valcarcel wants to thank SEP-PRODEP for the postdoctoral fellowship. On behalf of all authors, the corresponding author states that there is no conflict of interest. 18.Tochowicz, A.; Maskos, K.; Huber, R.; Oltenfreiter, R.; Dive, V.; Yiotakis, A.; Zanda, M.; Bode, W.; Goettig, P. J. Mol. Biol. 2007, 371, 989–1006. doi:10.1016/j.jmb.2007.05.068 19.Ramírez-Marroquín, O. A.; Jiménez-Arellanes, M. A.; Cortés-Pacheco, A.; Zambrano-Vásquez, O. R.; López-Torres, A. Monatsh. Chem. 2019, 150, 267–274. doi:10.1007/s00706-018-2328- Monatsh. Chem. 2019, 150, 267–274. doi:10.1007/s00706-018-23 Conclusion In this work, we reported the two-step synthesis of the bisphos- phonic esters 3–6. For the first time, the antiinflammatory activ- ity of the compounds was assessed by oral (carrageenan model) and topical administration (TPA model) to mice. Among these, the derivative 6 had an excellent edema inhibition, comparable to the positive control with the TPA model. On the other hand, the bioisosteric replacement of an amide for an ester group in the parent compounds 1 and 2 afforded the more potent deriva- tives 3 and 4, which had a higher antiinflammatory activity than As before, in Figure 5, the schematic representations of the interaction of the ligands in MMP-9 are displayed. Compound 3 displayed one hydrogen bond, from the carbonyl oxygen atom to an amide hydrogen atom located between Ala189 and Leu188, with this interaction being regarded as very weak, having an energy of −0.36 kcal/mol and a 3.53 Å O–N distance (Figure 5a). On the other hand, 4 had two hydrogen bond inter- 1285 Beilstein J. Org. Chem. 2020, 16, 1277–1287. Oscar Abelardo Ramírez-Marroquín - https://orcid.org/0000-0003-4354-5468 Oscar Abelardo Ramírez-Marroquín - https://orcid.org/0000-0003-4354-5468 Oscar Abelardo Ramírez-Marroquín - https://orcid.org/0000-0003-4354-5468 the parent bisphosphonates 1 and 2 using a carrageenan model. Moreover, a lipophilicity–activity relationship was observed for the two acute inflammation models: the aliphatic hydrophilic compounds 3 and 4 were the more potent ones by oral adminis- tration, and the aromatic lipophilic bisphosphonates 5 and 6 had a better antiinflammatory activity by topical administration. In addition, the safety of the test compounds 3–6 was evaluated by an acute toxicity determination where no significant weight loss or lethality was observed in individuals at a 50 and 100 mg/kg dose (the two- or four-fold dose as used in the original study). Finally, a ligand structure–activity relationship and molecular docking analysis led us to propose MMP-8 and MMP-9 inhibi- tion as the possible action mechanism of 3–6 due to the good correlation between the antiinflammatory activity of the bispho- sphonic esters and the interaction energy with these enzymes (especially MMP-8). Also, a good correlation between the bio- logical effects and interaction of the compounds with the Zn2+ cofactor of these enzymes was observed. Supporting Information File 1 Supporting Information File 1 General procedures of the synthesis, characterization of the compounds, the biological activity methodology, computational details, and NMR/HRMS spectra of the final products. Supporting Information File 1 General procedures of the synthesis, characterization of the compounds, the biological activity methodology, computational details, and NMR/HRMS spectra of the final products. 10.Tauro, M.; Laghezza, A.; Loiodice, F.; Agamennone, M.; Campestre, C.; Tortorella, P. Bioorg. Med. Chem. 2013, 21, 6456–6465. doi:10.1016/j.bmc.2013.08.054 11.Valleala, H.; Hanemaaijer, R.; Mandelin, J.; Salminen, A.; Teronen, O.; Mönkkönen, J.; Konttinen, Y. T. Life Sci. 2003, 73, 2413–2420. doi:10.1016/s0024-3205(03)00657-x [https://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-16-108-S1.pdf] [https://www.beilstein-journals.org/bjoc/content/ supplementary/1860-5397-16-108-S1.pdf] 12.Jabłońska-Trypuć, A.; Matejczyk, M.; Rosochacki, S. J. Enzyme Inhib. Med. Chem. 2016, 31 (Suppl. 1), 177–183. 12.Jabłońska-Trypuć, A.; Matejczyk, M.; Rosochacki, S. J E I hib M d Ch 20 6 (S l ) doi:10.3109/14756366.2016.1161620 13.Li, K.; Tay, F. R.; Yiu, C. K. Y. Pharmacol. 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https://openalex.org/W4285728345	https://air.unimi.it/bitstream/2434/934370/2/ijerph-19-08718%20%281%29.pdf	English	null	Protein Intake and Sarcopenia in Older Adults: A Systematic Review and Meta-Analysis	International journal of environmental research and public health/International journal of environmental research and public health	2,022	cc-by	8,011	Citation: Coelho-Junior, H.J.; Calvani, R.; Azzolino, D.; Picca, A.; Tosato, M.; Landi, F.; Cesari, M.; Marzetti, E. Protein Intake and Sarcopenia in Older Adults: A Systematic Review and Meta-Analysis. Int. J. Environ. Res. Public Health 2022, 19, 8718. https://doi.org/10.3390/ ijerph19148718 Keywords: nutrition; anorexia; physical function; walking speed; muscle strength; dynapenia; frailty; elderly International Journal of Environmental Research and Public Health International Journal of Environmental Research and Public Health Int. J. Environ. Res. Public Health 2022, 19, 8718. https://doi.org/10.3390/ijerph19148718 Review Hélio José Coelho-Junior 1,2,* , Riccardo Calvani 2 , Domenico Azzolino 3,4,* , Anna Picca 2 , Matteo Tosato 2 Francesco Landi 1,2, Matteo Cesari 3,4 and Emanuele Marzetti 1,2 Hélio José Coelho-Junior 1,2,* , Riccardo Calvani 2 , Domenico Azzolino 3,4,* , Anna Picca Francesco Landi 1,2, Matteo Cesari 3,4 and Emanuele Marzetti 1,2 1 Department of Geriatrics and Orthopedics, Università Cattolica del Sacro Cuore, L.go F. Vito 1, 00168 Rome, Italy; francesco.landi@unicatt.it (F.L.); emanuele.marzetti@policlinicogemelli.it (E.M.) y p g 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, L.go A. Gemelli 8, 00168 Rome, Italy; riccardo.calvani@policlinicogemelli.it (R.C.); anna.picca@policlinicogemelli.it (A.P.); matteo.tosato@policlinicogemelli.it (M.T.) 2 Fondazione Policlinico Universitario “Agostino Gemelli” IRCCS, L.go A. Gemelli 8, 00168 Rome, Italy; riccardo.calvani@policlinicogemelli.it (R.C.); anna.picca@policlinicogemelli.it (A.P.); matteo.tosato@policlinicogemelli.it (M.T.) 3 Department of Clinical and Community Sciences, Università di Milano, Via Festa del Perdono 7, 20122 Milan, Italy; matteo.cesari@unimi.it y 4 Geriatric Unit, IRCCS Istituti Clinici Scientiﬁci Maugeri, Via Camaldoli 64, 20138 Milan, Italy * Correspondence: coelhojunior@hotmail.com.br (H.J.C.-J.); domenico.azzolino@unimi.it (D.A.); Tel.: +39-06-3015-5559 (H.J.C.-J.); +39-02-5072-5218 (D.A.) 4 Geriatric Unit, IRCCS Istituti Clinici Scientiﬁci Maugeri, Via Camaldoli 64, 20138 Milan, Italy 4 Geriatric Unit, IRCCS Istituti Clinici Scientiﬁci Maugeri, Via Camaldoli 64, 20138 Milan, Italy * Correspondence: coelhojunior@hotmail.com.br (H.J.C.-J.); domenico.azzolino@unimi.it (D.A.); Tel.: +39-06-3015-5559 (H.J.C.-J.); +39-02-5072-5218 (D.A.) Geriatric Unit, IRCCS Istituti Clinici Scientiﬁci Maugeri, Via Camaldoli 64, 20138 Milan, Italy * Correspondence: coelhojunior@hotmail.com.br (H.J.C.-J.); domenico.azzolino@unimi.it (D.A.); Tel.: +39-06-3015-5559 (H.J.C.-J.); +39-02-5072-5218 (D.A.) Abstract: Background: The present systematic review and meta-analysis investigated the cross- sectional and longitudinal associations between protein intake and sarcopenia in older adults. Meth- ods: Observational studies that investigated the association between protein intake and sarcopenia as the primary or secondary outcome in people aged 60 years and older were included. Studies published in languages other than English, Italian, Portuguese, and Spanish were excluded. Studies were retrieved from MEDLINE, SCOPUS, EMBASE, CINAHL, AgeLine, and Food Science Source databases through January 31, 2022. A pooled effect size was calculated based on standard mean dif- ferences. Results: Five cross-sectional studies, one longitudinal study, and one case-control study that investigated 3353 community-dwelling older adults with a mean age of approximately 73 years were included. The meta-analysis of four studies indicated that older adults with sarcopenia consumed signiﬁcantly less protein than their peers with no sarcopenia. Conclusions: Results of the present study suggest that an inadequate protein intake might be associated with sarcopenia in older adults. 1. Introduction Sarcopenia is a neuromuscular disease characterized by muscle atrophy, dynapenia, and loss of physical function [1–5]. The overall prevalence of sarcopenia might reach up to 86.5% in adults depending on the deﬁnition used and the setting of evaluation, and is especially high in the older population [6]. This scenario deserves concern, given that the progression of sarcopenia is associated with the incidence of numerous negative events, including malnutrition, anorexia, physical inactivity, metabolic and osteoarticular disorders, cognitive impairment, falls, depressive symptoms, and death [7,8]. As such, sarcopenia is recognized as a public health problem and the identiﬁcation of potential strategies to prevent its development and progression is a priority [1,2]. Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- iations. Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). Nutrition is a modiﬁable lifestyle factor that may be harnessed to foster active and healthy aging [9,10]. In particular, a protein consumption higher than the current recom- mended dietary allowance (RDA, 0.8 g/kg/day) is proposed as a strategy to preserve muscle mass and physical function in advanced age [11–14]. This recommendation is based on the fact that the aged muscle requires a greater amount of amino acids (AAs) to maximally stimulate muscle protein synthesis (MPS) in response to hyperaminoacidemia, Int. J. Environ. Res. Public Health 2022, 19, 8718. https://doi.org/10.3390/ijerph19148718 https://www.mdpi.com/journal/ijerph Int. J. Environ. Res. Public Health 2022, 19, 8718 2 of 10 2 of 10 a phenomenon known as anabolic resistance [15–20]. The failure to properly stimulate MPS predisposes to gradual loss of muscle mass, mainly of type II muscle ﬁbers, which impacts muscle strength generation and physical function [21,22]. Although it is commonly believed that an adequate protein intake could prevent the development of sarcopenia or at least attenuate its progression [23–25], ﬁndings on the matter are inconclusive. To provide an up-to-date and comprehensive appraisal of the topic, we conducted a systematic review and meta-analysis of cross-sectional and longitudinal studies that explored the relationship between protein intake and sarcopenia in older adults. 2. Materials and Methods This is a systematic review and meta-analysis of observational studies that investi- gated cross-sectional and longitudinal associations between protein intake and sarcopenia. The study was fully performed by investigators and no librarian was part of the team. The study complies with the criteria of the Meta-analysis of Observational Studies in Epidemi- ology (MOOSE) guidelines [26] and the Cochrane Handbook for Systematic Reviews and Interventions [27]. 2.1. Eligibility Criteria Inclusion criteria were: (1) observational studies (e.g., case-control, cross-sectional, cohort longitudinal studies) that investigated the association between protein intake and sarcopenia; (2) participants aged 60 years or older; (3) sarcopenia identiﬁed according to the presence of muscle atrophy plus dynapenia, low muscle power, physical dysfunction, reduced mobility, and/or low scores on batteries of physical performance tests (e.g., Short Physical Performance Battery (SPPB)); (4) published studies in English, Italian, Portuguese, or Spanish language. p g g To be included in the meta-analysis of cross-sectional studies, investigations should provide mean and standard deviation (SD) of case (i.e., high protein intake (HPI)) and control groups (i.e., low protein intake, (LPI)), or at least two groups divided according to protein consumption and the sample size of each group, or Pearson’s correlation coefﬁ- cient (r)/Betas (β)/odds ratio (OR) values for the association between protein intake and sarcopenia. For the meta-analysis of longitudinal studies, investigations should provide the number of participants, β, OR, hazard ratio, and/or risk ratio for the development of sarcopenia according to protein consumption levels. We excluded randomized controlled trials, quasi-experimental, cross-over, and preclinical studies, as well as investigations that examined the effects of nutritional interventions alone or combined with other interventions (e.g., physical exercise) on sarcopenia. Studies that enrolled participants with gastroin- testinal and/or renal diseases, anorexia, cancer, or any condition that may directly impair protein metabolism (e.g., maple syrup urine disease, tyrosinemia) were also excluded. 2.2. Search Strategy and Selection Criteria Studies published on or before 31 January 2022 were retrieved from the following six electronic databases by one investigator: (1) MEDLINE (PubMed interface); (2) SCO- PUS (Elsevier interface); (3) EMBASE (OVID interface), (4) CINAHL (EBSCO interface); (5) AgeLine (EBSCO interface); and (6) Food Science Source (EBSCO interface). Further eligible articles were identiﬁed by checking the reference lists of retrieved articles. In addition, citation searches on key articles were performed in Google Scholar and Research- Gate. Initially, a search strategy was designed using keywords, MeSH terms, and free text words, such as "protein intake", "sarcopenia", and "older adults". Afterwards, keywords and subject headings were exhaustively combined using Boolean operators. The complete search strategy is shown in Supplementary Material S1. 2.3. Data Extraction, Quality Assessment, and Risk of Bias Titles and abstracts of retrieved articles were screened for eligibility by two researchers (HJCJ, RC). The full text was consulted if the abstract did not provide enough informa- Int. J. Environ. Res. Public Health 2022, 19, 8718 3 of 10 3 of 10 tion for ﬁnal evaluation. Two reviewers (HJCJ, RC) extracted the coded variables (i.e., methodological quality, risk of bias, and characteristics of the studies) using a standardized coding form. A third researcher was consulted to solve disagreements (EM), if necessary. The quality of reporting for each study was performed by two researchers (HJCJ, RC) using the Quality Assessment Tool for Observational Cohort and Cross-Sectional of the National Institutes of Health [28]. This tool contains 14 questions that assess several aspects associated with the risk of bias, type I and type II errors, transparency, and confounding factors. The studies were positive for item 8 if they investigated protein sources and/or distribution. Items 6, 7, and 13 do not refer to cross-sectional studies and were removed from the quality analysis. The maximum scores for cross-sectional and prospective studies were 11 and 14, respectively. The agreement rate for quality assessment between reviewers was 98%. 3.1. Literature Search Six-thousand and twenty-nine records were identiﬁed through database and hand searches. Of these, 2011 were excluded based on duplicated data, and 4018 titles and abstracts were evaluated. Eleven articles were fully assessed for eligibility and four studies were excluded based on selection criteria (Supplementary Material S2). Seven articles were included in the investigation. The ﬂowchart of the study is shown in Figure 1. 3.2. Characteristics of the Included Studies 2.4. Statistical Analysis The meta-analysis was conducted using Revman 5.4.1 (Cochrane Collaboration, Copen- hagen, Denmark). Effect sizes (ESs) were measured using means and SDs. Central and dispersion values were obtained from included studies or were calculated according to the Cochrane guidelines [27]. Speciﬁcally, medians were assumed as means when studies presented symmetrical data. SDs were calculated from conﬁdence intervals (CIs) and standard errors (SEs), according to the following formulas: SD1 = √N × (Upper limit −Lower limit)/3.92 (1) SD2 = SE × √N (2) SD1 = √N × (Upper limit −Lower limit)/3.92 (1) SD2 = SE × √N (2) (1) (Upper limit −Lower limit)/3.92 (1) SD2 = SE × √N (2) SD2 = SE × √N (2) (2) From the interquartile range, SDs were obtained according to the formulas proposed by Luo [29] and Shi [30]. A single pairwise comparison was created when multiple studies referred to the same database, using the formulas proposed by the Cochrane guidelines [27]. The pooled ES was calculated based on standard mean differences (SMDs), because studies used different tests and/or protocols to operationalize sarcopenia. Due to the variability of sample characteristics, a random-effect model was used to calculate the pooled ES. Addi- tionally, the I2 index was classiﬁed as “might not be important”(0–40%), “may represent moderate heterogeneity” (30–60%), “may represent substantial heterogeneity” (50–90%), or “may represent considerable heterogeneity” (75–100%) [27]. Forest plots were used to illustrate the summary statistics and the variation (heterogeneity) across studies. 3.2. Characteristics of the Included Studies The main characteristics of the included studies are shown in Table 1. Five cross- sectional studies [31–35], one longitudinal study [36], and one case-control study [37] that investigated 3353 community-dwelling older adults with a mean age of approximately 73 years from Australia, Belgium, Finland, India, and the Netherlands were included. One study [32] included participants from Italy, Poland, the Netherlands, and the United Kingdom. Nutritional habits were assessed using 24-h dietary recall, 3- and 7-day food records, diet history, and food frequency questionaries. Sarcopenia was operationalized according to the European Working Group on Sarcopenia in Older People (EWGSOP) [1], EWGSOP2 [2], and the Foundation for the National Institutes of Health (FNIH) sarcopenia project [5]. One study compared all three sarcopenia frameworks [34], and one study [32] Int. J. Environ. Res. Public Health 2022, 19, 8718 4 of 10 diagnosed sarcopenia according to the presence of low skeletal muscle index (SMI) and SPPB score. es. Public Health 2022, 19, x 4 of 11 Figure 1. Flowchart of the study. Figure 1. Flowchart of the study. diagnosed sarcopenia according to the presence of low skeletal muscle index (SMI) and SPPB score. 4 of 11 diagnosed sarcopenia according to the presence of low skeletal muscle index (SMI) and SPPB score. 4 of 11 wchart of the study. Figure 1. Flowchart of the study. eristics of the Included Studies Table 1. Characteristics of the included studies. teristics of the Included Studies Table 1. Characteristics of the included studies. 3.2. Characteristics of the Included Studies The main characteristics of the included studies are shown in Table 1. Five cross-sectional studies [31–35], one longitudinal study [36], and one case-control study [37] that investigated 3353 community-dwelling older adults with a mean age of ap- proximately 73 years from Australia, Belgium, Finland, India, and the Netherlands were included. One study [32] included participants from Italy, Poland, the Netherlands, and the United Kingdom. Nutritional habits were assessed using 24-h dietary recall, 3- and 7-day food records, diet history, and food frequency questionaries. Sarcopenia was op- erationalized according to the European Working Group on Sarcopenia in Older People (EWGSOP) [1], EWGSOP2 [2], and the Foundation for the National Institutes of Health (FNIH) sarcopenia project [5]. One study compared all three sarcopenia frameworks [34], and one study [32] diagnosed sarcopenia according to the presence of low skeletal muscle index (SMI) and SPPB score. Table 1. Characteristics of the included studies. 3.2. Characteristics of the Included Studies Year Author Study Type Follow- Up (Years) Country Sample Characteristics Sample Size Mean Age (Years) Mean Daily Protein Intake Dietary Intake Assessment Method Sarcopenia Assessment Method 2017 Veerlan et al. [37] Case- Control — Netherland Community-dwelling older adults 132 ~71 ~73.9 g 3-d food record (a) SMI and (b) SPPB 2019 Beaudart et al. [35] Cross- sectional — Belgium Community-dwelling older adults 331 74.8 ~82.7 g Food frequency questionnaire EWGSOP 2020 Das et al. [34] Cross- sectional — Australia Community-dwelling older men 794 81.1 — Diet history questionnaire FNIH, EWGSOP, and EWGSOP2 2020 Granic et al. [36] Longitudinal 3 United King- dom Community-dwelling older adults 757 85+ — 24-h dietary recall EWGSOP 2020 Jyväkorpi et al. [33] Cross- sectional — Finland Community-dwelling older adults 126 ~87.4 ~0.93 g/kg BW 3-d food record EWGSOP2 2020 Montiel- Rojas et al. [32] Cross- sectional — Europe Community-dwelling women 986 ~71 — 7-d food record EWGSOP2 2021 Rahman et al. [31] Cross- sectional — Indian Community-dwelling women 227 65.1 ~52.2 g Diet history EWGSOP BW= body weight; EWGSOP = European Working Group on Sarcopenia in Older People; FNIH, Foundation for the National Institutes of Health; SMI = Skeletal muscle index; SPPB = Short Physical Performance Battery. BW= body weight; EWGSOP = European Working Group on Sarcopenia in Older People; FNIH, Foundation for the National Institutes of Health; SMI = Skeletal muscle index; SPPB = Short Physical Performance Battery. 3.3. Quality Assessment Quality assessment scores are shown in Supplementary Material S3. The overall score of cross-sectional studies [31–35] ranged from six to seven. All of the studies clearly stated the research question (item 1), speciﬁed the study population (item 2), recruited participants from the same or a similar population (item 4), clearly deﬁned and used valid and reliable exposure (item 9) and outcome (item 11) measures. Four investigations reported a participation rate of eligible persons of at least 50% (item 3), two studies investigated different levels of exposure (item 8), and three investigations adjusted their results according to confounding parameters (item 14). No studies justiﬁed the sample size (item 5) or reported whether investigators were blinded to the exposure of participants (item 12). Int. J. Environ. Res. Public Health 2022, 19, 8718 5 of 10 5 of 10 The longitudinal study [36] had an overall score of 10. The study established the research question (item 1), speciﬁed the study population (item 2), investigated a study population with a participation rate of eligible persons of at least 50% (item 3), recruited participants from the same or a similar population (item 4), justiﬁed the sample size (item 5), measured the exposure of interest before the outcome being measured (item 6), used a timeframe sufﬁcient to expect to see an association between exposure and outcome (item 7), clearly deﬁned and used valid and reliable exposure (item 9) and outcome measures (item 11), and adjusted their results according to confounding parameters (item 14). The study did not investigate different levels of exposure (item 8), did not assess the exposure more than once (item 10), and did not report whether investigators were blinded to the exposure of participants (item 12). The case-control study [37] had an overall score of eight. The study clearly stated the research question (item 1), speciﬁed the study population (item 2), recruited control and case participants from the same or a similar population (item 4), clearly deﬁned the inclusion and exclusion criteria (item 5), clearly deﬁned and differentiated cases from controls (item 6), selected the participants randomly from eligible candidates (item 7), used concurrent control (item 8), and clearly deﬁned and used valid and reliable exposure (item 10). 3.4. Cross-Sectional Association between Protein Intake and Sarcopenia 3.4. Cross-Sectional Association between Protein Intake and Sarcopenia The cross sectional association between protein intake and sa The cross-sectional association between protein intake and sarcopenia is shown in Figure 2. Four studies were included in the pooled analysis [31,33,35,37]. Older adults with sarcopenia consumed signiﬁcantly less protein than their non-sarcopenic counterparts (SMD = 0.37, 95% CI = 0.19–0.55, p < 0.0001). Heterogeneity was classiﬁed as “might not be important” (I2 = 18%, p = 0.30). The cross-sectional association between protein intake and sarcopenia is shown in Figure 2. Four studies were included in the pooled analysis [31,33,35,37]. Older adults with sarcopenia consumed significantly less protein than their non-sarcopenic counter- parts (SMD = 0.37, 95% CI = 0.19–0.55, p < 0.0001). Heterogeneity was classified as "might not be important" (I2 = 18%, p = 0.30). Figure 2. Standard mean differences of protein intake between older adults with and without sar- copenia [31,33,35,37]. Figure 2. Standard mean differences of protein intake between older adults with and without sarcopenia [31,33,35,37]. Figure 2. Standard mean differences of protein intake between older adults with and without sar- copenia [31,33,35,37]. Figure 2. Standard mean differences of protein intake between older adults with and without sarcopenia [31,33,35,37]. Figure 2. Standard mean differences of protein intake between older adults with and without sar- copenia [31,33,35,37]. Figure 2. Standard mean differences of protein intake between older adults with and without sarcopenia [31,33,35,37]. 3.5. Cross-Sectional Association between Protein Sources and Sarcopenia 3.5. Cross-Sectional Association between Protein Sources and Sarcopenia 3.5. Cross-Sectional Association between Protein Sources and Sarcopenia 3.5. Cross-Sectional Association between Protein Sources and Sarcopenia p One study investigated the association between protein sources and sarcopenia [32]. Montiel-Rojas et al. [32] enrolled 986 older European adults and explored the association between protein sources and sarcopenia, diagnosed according to the presence of low SMI plus reduced handgrip strength. The authors found that the risk of sarcopenia was lower in those with greater protein consumption. In addition, the replacement of ani- mal-derived proteins with an equal amount of plant-derived proteins was associated with a reduced risk of sarcopenia One study investigated the association between protein sources and sarcopenia [32]. Montiel-Rojas et al. [32] enrolled 986 older European adults and explored the association between protein sources and sarcopenia, diagnosed according to the presence of low SMI plus reduced handgrip strength. The authors found that the risk of sarcopenia was lower in those with greater protein consumption. 3.4. Cross-Sectional Association between Protein Intake and Sarcopenia 3.4. Cross-Sectional Association between Protein Intake and Sarcopenia The cross sectional association between protein intake and sa In addition, the replacement of animal-derived proteins with an equal amount of plant-derived proteins was associated with a reduced risk of sarcopenia. 3.3. Quality Assessment The study did not justify the sample size (item 3), did not conﬁrm whether the exposure occurred before the development of sarcopenia (item 9), did not report whether assessors were blinded to case or control participants (item 11), and did not adjust results according to potential covariates (item 12). , 19, x 6 of 11 3.4. Cross-Sectional Association between Protein Intake and Sarcopenia 3.4. Cross-Sectional Association between Protein Intake and Sarcopenia The cross sectional association between protein intake and sa 4. Discussion The present systematic review and meta-analysis investigated the association between protein intake and sarcopenia in older adults. The pooled analysis of cross-sectional studies indicated that older adults with sarcopenia have a lower intake of proteins compared with non-sarcopenic peers. Two additional potentially important results were observed. First, the consumption of plant-based protein was cross-sectionally associated with a low prevalence of sarcopenia. Second, older adults on a high-fat/high-energy diet may be at high risk of sarcopenia even if their protein intake is greater than the RDA. g p p g HPI has long been considered to be a modiﬁable lifestyle factor that might potentially counteract sarcopenia [23–25]. This assumption is based on the effects of AAs on muscle protein metabolism. Muscle mass is regulated by a dynamic equilibrium between MPS and muscle protein breakdown (MPB) [38–42]. Adequate protein ingestion is expected to increase AA availability and stimulate sarcoplasmic and myoﬁbrillar protein synthesis by activating the mammalian target of rapamycin (mTOR) and its downstream targets [38–42]. However, the aged muscle frequently shows anabolic resistance, a state of submaximal MPS in response to hyperaminoacidemia, suggesting that greater amounts of protein are required to properly stimulate muscle anabolism in older adults [15–20]. If the anabolic resistance is not overcome through the diet, an imbalance in muscle metabolism in favor of MPB might occur, promoting muscle loss [43]. Muscle atrophy occurs preferably in type II muscle ﬁbers [21,22,44], those that contract faster and have a greater capacity to generate tension [21,22]. Hence, it may be expected that older adults with HPI might experience less muscle atrophy and neuromuscular dysfunction. However, such a view is not supported by the only longitudinal investigation included in the present study. Granic et al. [36] observed that older adults on a traditional British diet and with a protein intake ≥1 g/kg of BW/d had an increased risk of developing sarcopenia compared with those on a low-butter diet during three years of follow-up. These ﬁndings have some possible explanations. Protein quality refers to the anabolic response elicited by protein sources [45]. Numer- ous studies found that animal-based proteins produced greater muscular anabolism than plant-based proteins [40,46,47]. These divergent anabolic responses are attributed to differ- ences in digestion and absorption rates, and branched-chain AA (BCAA) content [45,48]. Indeed, animal proteins are characterized by digestibility rates higher than 90%, whereas digestibility rates barely reache 50% with plant proteins [45,48]. 3 6 Longitudinal Associations between Protein Intake and Sarcopenia 3.6. Longitudinal Associations between Protein Intake and Sarcopenia 3.6. Longitudinal Associations between Protein Intake and Sarcopenia One study investigated the longitudinal association between protein intake and in- cident sarcopenia [36]. Compared with older adults on a low-butter diet, those eating a traditional British diet (i.e., rich in butter, red meat, gravy, and potato) had an increased risk of sarcopenia over a 3-year follow-up even if protein intake was ≥1 g/kg of body weight (BW)/d Results were similar when the HPI threshold was set at ≥0 8 g/kg of One study investigated the longitudinal association between protein intake and in- cident sarcopenia [36]. Compared with older adults on a low-butter diet, those eating a traditional British diet (i.e., rich in butter, red meat, gravy, and potato) had an increased risk of sarcopenia over a 3-year follow-up even if protein intake was ≥1 g/kg of body weight (BW)/d. Results were similar when the HPI threshold was set at ≥0.8 g/kg of Int. J. Environ. Res. Public Health 2022, 19, 8718 6 of 10 BW/d. However, the risk of incident sarcopenia at three years was no longer signiﬁcant in the fully adjusted model. 4. Discussion Furthermore, animal proteins have a greater content of BCAAs in comparison to plant-based proteins [14,45]. Such data are important because BCAAs, and mainly leucine, are considered to be major stimulators of MPS [42,49,50]. Hence, it is possible that older adults with HPI who developed sarcopenia had a protein consumption mostly based on plant sources, providing an insufﬁcient supply of AAs to properly stimulate MPS. Although this hypothesis offers a reasonable explanation for the report by Granic et al. [36], other investigations found that a high intake of plant- based proteins was associated with faster walking speed [51] and lower prevalence of frailty [52]. Experts in the ﬁeld interpreted these ﬁndings as the indication that an adequate intake of vegetable proteins may also properly stimulate muscle anabolism [14]. Another possible explanation to the ﬁndings by Granic et al. [36] is that the traditional British diet is characterized by a high intake of fat and energy. Such dietary regimes are associated with an increased risk of obesity which, in turn, promotes the development of insulin resistance, oxidative stress, low-grade systemic inﬂammation, and hormonal changes [53]. All of these factors play a role in the pathophysiology of sarcopenia. Finally, results by Granic et al. [36] were not controlled for many covariables that might impact the association between protein intake and sarcopenia, including the practice of physical exercise [54,55], the presence of frailty [56], and oral health [57]. 7 of 10 7 of 10 Int. J. Environ. Res. Public Health 2022, 19, 8718 Only one study investigated the association between protein sources and sarcope- nia [32]. Montiel-Rojas et al. [32] observed that the consumption of plant protein was negatively associated with the presence of sarcopenia. Additional studies investigating the potential role of protein sources on the development of sarcopenia are warranted. Our study has limitations that deserve discussion. First, all of the investigations included examined community-dwelling older adults, and extrapolations to hospitalized patients and people living in long-term institutions should be made with caution. Second, our pooled analysis was conducted to identify differences in means and SDs, given the limited number of studies that performed regression analyses. This indicates that results were not adjusted for numerous covariables. Third, substantial heterogeneity was observed in the way protein consumption data were presented (e.g., absolute, adjusted according to BW, percentage of calories). 4. Discussion Fourth, the limited number of included studies did not allow meta-regression, dose-response, risk of bias, or “trim and ﬁll” analysis to be conducted. Fifth, the ﬁndings on the longitudinal association between protein intake and those on protein sources with sarcopenia were based on one study each. Sixth, different studies were included in the cross-sectional and longitudinal analyses, which might produce divergent results. Seventh, although most studies used EWGSOP criteria to identify people with sarcopenia, different instruments, cutoff points, and other operational deﬁnitions of sarcopenia were also utilized. This aspect deserves concern because protein intake might be associated with each one of those variables, therefore inﬂuencing our results. In fact, vegetal protein has been associated with walking speed, but not with muscle strength [51,58]. Eighth, most of the investigations were conducted in Europe. Finally, no studies took into account the severity of sarcopenia. Notwithstanding, our study provides directions for future investigations. The ﬁnding that sarcopenic older adults consumed signiﬁcantly less protein than their non-sarcopenic counterparts partially supports the assumption that protein intake is associated with sar- copenia and encourages the conduct of large multicentric, cross-sectional and longitudinal studies to better explore the subject. Future investigations should take into consideration several nutritional and sarcopenia-related aspects that are still lacking in the literature, including differences between protein sources, diagnostic criteria for sarcopenia, and socio- cultural factors. The impact of relevant covariables should also be explored. The lack of this information still limits extrapolations of the current ﬁndings to clinical practice. Author Contributions: Conceptualization, H.J.C.-J. and E.M.; methodology, H.J.C.-J., D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; formal analysis, H.J.C.-J., D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; investigation, H.J.C.-J., R.C. and D.A.; resources, H.J.C.-J., D.A., M.C. and E.M.; data curation, H.J.C.-J., D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; writing—original draft preparation, H.J.C.-J.; writing— review and editing, D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; supervision, M.C. and E.M. All authors have read and agreed to the published version of the manuscript. References 1. Cruz-Jentoft, A.J.; Baeyens, J.P.; Bauer, J.M.; Boirie, Y.; Cederholm, T.; Landi, F.; Martin, F.C.; Michel, J.-P.; Rolland, Y.; Schneider, S.M.; et al. Sarcopenia: European Consensus on Deﬁnition and Diagnosis: Report of the European Working Group on Sarcopenia in Older People. Age Ageing 2010, 39, 412–423. [CrossRef] [PubMed] 2. 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Bauer, J.; Biolo, G.; Cederholm, T.; Cesari, M.; Cruz-Jentoft, A.J.; Morley, J.E.; Phillips, S.; Sieber, C.; Stehle, P.; Teta, D.; et al. Evidence-Based Recommendations for Optimal Dietary Protein Intake in Older People: A Position Paper from the Prot-Age Study Group. J. Am. Med. Dir. Assoc. 2013, 14, 542–559. [CrossRef] y p J [ ] 12. Coelho-Junior, H.J.; Marzetti, E.; Picca, A.; Cesari, M.; Uchida, M.C.; Calvani, R. Protein Intake and Frailty: A Matter of Quantity, Quality, and Timing. Nutrients 2020, 12, 2915. [CrossRef] [PubMed] 13. Deer, R.R.; Volpi, E. Protein Intake and Muscle Function in Older Adults. Curr. Opin. Clin. Nutr. Metab. Care 2015, 18, 248–253. [CrossRef] [PubMed] 13. Deer, R.R.; Volpi, E. Protein Intake and Muscle Function in Older Adults. Curr. Opin. Clin. Nutr. Metab. Care 2015, 18, 248–253. [CrossRef] [PubMed] 14. Landi, F.; Calvani, R.; Tosato, M.; Martone, A.M.; Ortolani, E.; Savera, G.; D’Angelo, E.; Sisto, A.; Marzetti, E. Protein Intake and Muscle Health in Old Age: From Biological Plausibility to Clinical Evidence Nutrients 2016 8 295 [CrossRef] [PubMed] , ; p , p , , [CrossRef] [PubMed] 14. Landi, F.; Calvani, R.; Tosato, M.; Martone, A.M.; Ortolani, E.; Savera, G.; D’Angelo, E.; Sisto, A.; Marzetti, E. Protein Intake and Muscle Health in Old Age: From Biological Plausibility to Clinical Evidence. Nutrients 2016, 8, 295. [CrossRef] [PubMed] [ ] [ ] 14. Landi, F.; Calvani, R.; Tosato, M.; Martone, A.M.; Ortolani, E.; Savera, G.; D’Angelo, E.; Sisto, A.; Marzetti, E. Protein Intake and Muscle Health in Old Age: From Biological Plausibility to Clinical Evidence. Nutrients 2016, 8, 295. [CrossRef] [PubMed] 14. Landi, F.; Calvani, R.; Tosato, M.; Martone, A.M.; Ortolani, E.; Savera, G.; D Angelo, E.; Sisto, A.; Marzetti, E. Protein Intake and Muscle Health in Old Age: From Biological Plausibility to Clinical Evidence. Nutrients 2016, 8, 295. [CrossRef] [PubMed] 15. Data Availability Statement: Data are available in the manuscript. Data Availability Statement: Data are available in the manuscript. 18. Wall, B.T.; Gorissen, S.H.; Pennings, B.; Koopman, R.; Groen, B.B.L.; Verdijk, L.B.; van Loon, L.J.C. Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion. PLoS ONE 2015, 10, e0140903. [CrossRef] Informed Consent Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data are available in the manuscript. 5. Conclusions Our pooled analysis indicate that older adults with sarcopenia consumed signiﬁcantly less protein than their non-sarcopenic counterparts. These results were based on differences in means and SDs, given the lack of investigations that conducted regression analyses. One cross-sectional study noted that plant-based protein might be negatively associated with the prevalence of sarcopenia. On the other hand, a longitudinal study observed that older adults following a traditional British dietary pattern had an increased risk of sarcopenia even if protein intake was high. These ﬁndings suggest that more cross-sectional and longitudinal studies, with deeper statistical approaches and more comprehensive analyses of protein-related parameters are required to conﬁrm and expand the current results. Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19148718/s1, Supplementary Material S1: Search strategy, Supplementary Material S2: Reasons for study exclusion, Supplementary Material S3: Quality analysis. Author Contributions: Conceptualization, H.J.C.-J. and E.M.; methodology, H.J.C.-J., D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; formal analysis, H.J.C.-J., D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; investigation, H.J.C.-J., R.C. and D.A.; resources, H.J.C.-J., D.A., M.C. and E.M.; data curation, H.J.C.-J., D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; writing—original draft preparation, H.J.C.-J.; writing— review and editing, D.A., R.C., A.P., M.T., F.L., M.C. and E.M.; supervision, M.C. and E.M. All authors have read and agreed to the published version of the manuscript. Int. J. Environ. Res. Public Health 2022, 19, 8718 8 of 10 8 of 10 Funding: This work was partially funded by an Intramural Research Grant from the Università Cattolica del Sacro Cuore (D1.2020; E.M.) and the nonproﬁt research foundation Centro Studi Achille e Linda Lorenzon (A.P., E.M., H.J.C.-J. and R.C.). The APC was funded by Ministero della Salute— Ricerca Corrente 2022. 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https://openalex.org/W2951860936	https://www.frontiersin.org/articles/10.3389/fendo.2018.00792/pdf	English	null	Plasticity in the glucagon interactome reveals novel proteins that regulate glucagon secretion in αTC1-6 cells	bioRxiv (Cold Spring Harbor Laboratory)	2,018	cc-by	12,753	Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells Farzad Asadi 1 and Savita Dhanvantari 1,2,3* Keywords: glucagon, alpha cell, proteomics, co-immunoprecipitation, confocal microscopy, glucagon interactome, glucagon secretion We suggest that variations in the alpha cell secretory response to stimuli may be governed by plasticity in the glucagon “interactome.” Correspondence: Savita Dhanvantari sdhanvan@lawsonimaging.ca Specialty section: This article was submitted to Diabetes, a section of the journal Frontiers in Endocrinology Received: 17 October 2018 Accepted: 17 December 2018 Published: 18 January 2019 1 Department of Pathology and Laboratory Medicine, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada, 2 Department of Medical Biophysics, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada, 3 Metabolism, Diabetes and Imaging Programs, Lawson Health Research Institute, London, ON, Canada Edited by: Åke Sjöholm, Gävle Hospital, Sweden Reviewed by: Gabriela Da Silva Xavier, University of Birmingham, United Kingdom Céline Cruciani-Guglielmacci, Paris Diderot University, France Yukihiro Fujita, Shiga University of Medical Science, Japan Alex Rafacho, Federal University of Santa Catarina, Brazil Daniel Hesselson, Garvan Institute of Medical Research, Australia Reviewed by: Gabriela Da Silva Xavier, University of Birmingham, United Kingdom Céline Cruciani-Guglielmacci, Paris Diderot University, France Yukihiro Fujita, Shiga University of Medical Science, Japan Alex Rafacho, Federal University of Santa Catarina, Brazil Daniel Hesselson, Garvan Institute of Medical Research, Australia Glucagon is stored within the secretory granules of pancreatic alpha cells until stimuli trigger its release. The alpha cell secretory responses to the stimuli vary widely, possibly due to differences in experimental models or microenvironmental conditions. We hypothesized that the response of the alpha cell to various stimuli could be due to plasticity in the network of proteins that interact with glucagon within alpha cell secretory granules. We used tagged glucagon with Fc to pull out glucagon from the enriched preparation of secretory granules in α-TC1-6 cells. Isolation of secretory granules was validated by immunoisolation with Fc-glucagon and immunoblotting for organelle-speciﬁc proteins. Isolated enriched secretory granules were then used for afﬁnity puriﬁcation with Fc-glucagon followed by liquid chromatography/tandem mass spectrometry to identify secretory granule proteins that interact with glucagon. Proteomic analyses revealed a network of proteins containing glucose regulated protein 78 KDa (GRP78) and histone H4. The interaction between glucagon and the ER stress protein GRP78 and histone H4 was conﬁrmed through co-immunoprecipitation of secretory granule lysates, and colocalization immunoﬂuorescence confocal microscopy. Composition of the protein networks was altered at different glucose levels (25 vs. 5.5 mM) and in response to the paracrine inhibitors of glucagon secretion, GABA and insulin. siRNA-mediated silencing of a subset of these proteins revealed their involvement in glucagon secretion in α-TC1-6 cells. Therefore, our results show a novel and dynamic glucagon interactome within α-TC1-6 cell secretory granules. ORIGINAL RESEARCH published: 18 January 2019 doi: 10.3389/fendo.2018.00792 INTRODUCTION and somatostatin and nutritional eﬀectors (12, 17). Our work has revealed a novel glucagon “interactome” that exhibits plasticity in response to glucose, insulin and GABA, and contains some novel glucagon-interacting proteins that may regulate glucagon secretion in α-TC1-6 cells. Glucagon is the major glucose counter-regulatory hormone, and maintains euglycemia by enhancing hepatic gluconeogenesis and glycogenolysis (1). However, both type 1 and type 2 diabetes are characterized by varying levels of hyperglucagonemia (2), which paradoxically exacerbates the hyperglycemia of diabetes (3, 4). More recently, it has been shown that glucagon may be an amino acid regulatory hormone, suggesting a link between hepatic amino acid metabolism and hyperglucagonemia (5). In pancreatic alpha cells, glucagon secretion is tightly regulated by nutritional, hormonal, and neural eﬀectors to maintain normal glucose homeostasis. However, in diabetes, this tight coupling is disrupted (6), resulting in dysfunctional glucagon secretion, which may be a factor in the development of type 2 diabetes (7). This abnormal glucagon secretion has led to strategies (8) to control glucagon action to ameliorate the hyperglycemia of diabetes, such as administering glucagon receptor antagonists or neutralizing antibodies against the glucagon receptor (9, 10). Although eﬀective in the short term, this strategy tends to increase alpha cell mass and worsen alpha cell dysfunction over the long term (6). Therefore, a preferable strategy may be to control the secretion, rather than the action, of glucagon for improved glycemic control in diabetes. Citation: Asadi F and Dhanvantari S (2019) Plasticity in the Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion in α-TC1-6 Cells. Front. Endocrinol. 9:792. doi: 10.3389/fendo.2018.00792 Keywords: glucagon, alpha cell, proteomics, co-immunoprecipitation, confocal microscopy, glucagon interactome, glucagon secretion January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 1 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari Frontiers in Endocrinology \| www.frontiersin.org Gene Construct and Plasmid Preparation Ge e Co s uc a d as d epa a o We designed a glucagon fusion construct [Fc-glucagon – pcDNA3.1(+)] as follows: the amino acid sequence of glucagon derived from human proglucagon (GenScript, USA; http://www. genscript.com) was fused to the 3′ end of cDNA encoding the CH2/CH3 domain of mouse IgG-2b (Fc), preceded by a 28 amino acid signal peptide as described previously (19). As a negative control for all transfections, proteomics, immunoﬂuorescence microscopy, and co-immunoprecipitation experiments, we also designed a Fc-pcDNA3.1(+) construct. DNA sequences were conﬁrmed at the London Regional Genomics Facility, Western University. In the context of the pancreatic islet, there is some debate as to whether glucagon secretion is primarily regulated by the paracrine inﬂuence of the beta cell, or through intrinsic factors (11, 12). Both insulin and GABA secreted from the beta cell strongly inhibit glucagon secretion, as does somatostatin (13, 14). However, these actions are dependent on prevailing glucose concentrations; at 5 mM glucose, both glucagon and insulin secretion are maximally suppressed (11), suggesting that intrinsic factors may exert an equally prominent inﬂuence on glucagon secretion. Some proposed mechanisms of intrinsic regulation of glucagon secretion include glucose metabolic- induced changes in Ca2+ and K+ membrane conductances or intracellular Ca2+ oscillations (15, 16). Intrinsic factors can also include proteins involved in the intracellular traﬃcking of glucagon. We have previously shown that prolonged culture of α- TC1-6 cells in medium containing 25 mM glucose resulted in the up-regulation of components of the regulated secretory pathway (17), notably proteins associated with secretory granules, such as SNARE exocytotic proteins and granins. There may be direct interactions between granule proteins, such as chromogranin A and carboxypeptidase E, to ensure proper traﬃcking of glucagon into secretory granules (18), and distinct sorting signals within glucagon may mediate these interactions (19). Therefore, proteins within the alpha cell secretory granules that directly interact with glucagon may provide additional clues for the regulation of glucagon secretion. MATERIALS AND METHODS Sources for all reagents, assays, and software packages are listed in Supplementary Table 1. Nano-Flow Analysis again 10 times through a 25G needle. The resulting lysates were centrifuged to obtain a post-nuclear supernatant (PNS). The nuclear fraction was washed seven times in ice-cold homogenization buﬀer and stored at −80◦C. The post-nuclear supernatant (PNS) was centrifuged at 5,400 × g for 15 min at 4◦C to obtain a post-mitochondrial supernatant, which then was spun at 25,000 ×g for 20 min, and the resultant pellet was washed ﬁve times at 4◦C. Enrichment was conﬁrmed through immunoblotting for organelle-speciﬁc markers as described below. Nano-Flow Analysis To count the numbers of Fc-glucagon+ granules, ﬂuorescence of FITC excitation (L488) was gated and the numbers of Fc- glucagon+ granules were counted at 110, 179, 235, 304, 585, and 880 nm within the LALS vs. L488 plot. To get the LALS vs. L488 plot, its gate was normalized for the following isotypes: secretory granules of non-transfected cells, secretory granules of Fc-transfected cells, FITC-IgG and diluent. This method resulted in size distributions of the granules that were positive for Fc-glucagon, speciﬁcally. All experiments were done in three biological samples and values were expressed as percent distribution of gated granules. Afﬁnity Puriﬁcation Fc or Fc-glucagon was puriﬁed from the granule lysate by immunoprecipitation as we have done previously (19). Brieﬂy, a slurry of Protein A-Sepharose beads (Supplementary Table 1) was mixed 1:1 with the granule lysate and rotated overnight at 4◦C. The mixture was then centrifuged at 500 × g for 2 min at 4◦C and the pellet was washed twice with 50 mM Tris (pH 7.5) and once with pre-urea wash buﬀer (50 mM Tris pH 8.5, 1 mM EGTA, 75 mM KCl). Fc or Fc-glucagon was eluted with two volumes of urea elution buﬀer (7 M urea in 20 mM Tris buﬀer pH 7.5 plus 100 mM NaCl). This step was repeated twice more and the supernatants were collected and pooled. The pooled supernatant was mixed with acetone in a 1:4 ratio and kept at −20◦C overnight, then centrifuged at 16,000 × g for 15 min at 4◦C. The pellet was air-dried for proteomic analysis. Proteomic Analysis y Protein identiﬁcation was conducted using LC-MS/MS according to the protocols of the Western University Mass Spectrometry Laboratory (https://www.schulich.uwo.ca/lrpc/ bmsl/protocols/index.html). Brieﬂy, the air-dried pellet was reconstituted in 50 mM NH4CO3, and proteins were reduced in 200 mM dithiothreitol (DTT), alkylated in freshly prepared 1M iodoacetamide and digested with trypsin for 18 h at 37◦C with occasional shaking. Tryptic peptides were acidiﬁed using formic acid (0.25; v/v), loaded onto a Hypersep C18 column, washed, and eluted in 50% acetonitrile. The eluent was dried down in a speed vacuum and reconstituted in acetonitrile. Each experimental condition was done in three biological replicates. Peptide sequences were identiﬁed using the mouse database and further analyzed for protein categorization through PANTHER GO (www.Pantherdb.org), functional protein-protein interaction clustering through http://string-db.org and determination of subcellular locations and activity using www.uniport.org. Immunoblotting for Organelle-Speciﬁc Markers Proteomic Analysis of Secretory Granule Proteins Associated With Glucagon Granule Lysate Preparation α-TC1-6 cells were transfected by Fc-glucagon and treated with eﬀectors (GABA, insulin and GABA plus insulin) in media containing 25 or 5.5 mM glucose as described above. To identify non-speciﬁc interactors, we used the Fc construct in untreated conditions. Secretory granules were extracted as described above, and lysed in a non-ionic lysis buﬀer. Nanoscale Flow Cytometry Secretory Granule Preparation We used nano-scale ﬂow cytometry (A50-Micro nanoscale ﬂow cytometer; Apogee FlowSystems Inc.) to conﬁrm enrichment of the secretory granules and to determine the size distribution of the granules. α-TC1-6 cells were transfected with Fc-glucagon or Fc alone, and secretory granules were extracted as described above. Granules were ﬁxed in freshly prepared 2% PFA (pH 7.4), permeabilized with 0.5% saponin at room temperature, centrifuged at 25,000 × g for 20 min at 4◦C and washed three times in 0.1% saponin in PBS. Fc-containing granules were labeled with FITC-IgG (1:250 dilution in 0.1% saponin in 1% BSA/PBS) in the dark for 1 h, and diluted 200X in 0.1% saponin. Proteomic Analysis of Secretory Granule Proteins Associated With Glucagon Granule Lysate Preparation a e s The enriched preparations of secretory granules from α- TC1-6 cells were lysed using non-ionic lysis buﬀer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100 plus cOmplete Mini Protease Inhibitor Cocktail, and 5 µg/mL Aprotinin). Proteins were resolved by 4–12% NuPAGE, transferred to a PVDF membrane and probed with the following antibodies (Supplementary Table 1): vesicle-associated membrane protein 2 (VAMP2) for mature secretory granules; calreticulin for the endoplasmic reticulum; TGN46 for the trans-Golgi network; and Lamin B1 for the nuclear envelope. Immunoreactive bands were visualized using HRP-conjugated goat anti-rabbit secondary antibody and Clarity Western ECL substrate. Images were acquired on a BioRad ChemiDoc Imaging System. Total cell extracts were used as positive controls. y p α-TC1-6 cells were transfected by Fc-glucagon and treated with eﬀectors (GABA, insulin and GABA plus insulin) in media containing 25 or 5.5 mM glucose as described above. To identify non-speciﬁc interactors, we used the Fc construct in untreated conditions. Secretory granules were extracted as described above, and lysed in a non-ionic lysis buﬀer. Extraction and Enrichment of Secretory Granules Wild type α-TC1-6 cells (a kind gift from C. Bruce Verchere, Vancouver, BC) were cultured in DMEM containing 25 mM glucose, L-glutamine, 15% horse serum, and 2.5% fetal bovine serum, as described previously (17, 23). Based on the ATCC product sheet, the base cell culture medium for α-TC1- 6 cells is low glucose (5.5 mM) Dulbecco’s Modiﬁed Eagle’s Medium (DMEM); however, for glucagon secretion (glucagon hypersecretion) studies, high glucose DMEM (16.7 or 25 mM) has been traditionally used to prepare α-TC-6 cells for downstream experiments (17, 24). Cells were grown to 90% conﬂuency and transfected with Fc alone or Fc-glucagon using Lipofectamine 2000. To determine changes in granule size, mass, and proteome, cells were incubated with or without GABA (25 µM), insulin (100 pM), or GABA (25 µM) plus insulin (100 pM) in either 25 or 5.5 mM glucose prior to the granule enrichment procedure. To account for all potential modulators of glucagon secretion, including the possibility of autocrine regulation of glucagon secretion (25, 26) we chose long-term cumulative incubation that has previously been used by our team (17) and other investigators (23) for secretion studies in α-TC1-6 cells. At the end of the incubation period, granules were extracted as previously published (27) with some modiﬁcations. Brieﬂy, cells were detached using 5 mM EDTA in PBS (pH 7.4) containing cOmplete Mini Protease Inhibitor Cocktail (Supplementary Table 1) on ice, centrifuged and resuspended in ice-cold homogenization buﬀer (20 mM Tris-HCl pH7.4, 0.5 mM EDTA, 0.5 mM EGTA, 250 mM sucrose, 1 mM DTT, cOmplete Mini Protease Inhibitor Cocktail, and 5 µg/mL Aprotinin). The cells were passed 10 times through a 21G needle and In order to identify networks of secretory granule proteins that interact with glucagon, we have continued to use the α-TC1-6 cell line, as this is a well-established cell line in which to study the intrinsic regulation of glucagon secretion (20). This cell line has been extensively used to study glucagon secretory pathway (17, 21) due to its resemblance to the normal pancreatic α-cell in terms of proglucagon processing (22) and response to insulin January 2019 \| Volume 9 \| Article 792 2 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari Frontiers in Endocrinology \| www.frontiersin.org Immunoﬂuorescence Microscopy Immunoﬂuorescence Microscopy To validate the presence of GRP78 and histone H4 in glucagon- positive secretory granules, α-TC1-6 cells were cultured on collagen1-coated coverslips (three per experiment), and processed for immunoﬂuorescence microscopy as described previously (18). Brieﬂy, cells were ﬁxed in 4% paraformaldehyde and permeabilized in 0.1% saponin in 0.5% BSA for 1 h. After blocking in 10% goat serum, cells were incubated with primary antibodies (mouse anti-glucagon and rabbit anti-GRP78 or rabbit anti-histone H4) overnight. Coverslips were washed in PBS and incubated with goat anti-mouse Alexa Fluor IgG 488 and goat anti-rabbit Alexa Fluor 594 (Supplementary Table 1) for 3 h in the dark at room temperature, then mounted using ProLong Gold Antifade Mountant. Images were acquired on a Nikon A1R Confocal microscope with a 60x Nikon Plan-Apochromat oil diﬀerential interference contrast objective lens using NIS- Elements, software. To show secretory granule co-localization, images were post-processed by 2D deconvolution. To measure the degree of co-localization, regions of interest were manually drawn around distinct single or multicell bodies, positive for Fc-glucagon and either GRP78 or histone H4 and cropped for analysis. Co-localization of the pixels from each pseudo-colored image were used to calculate Pearson’s correlation coeﬃcient (PCC), as we described previously (19). siRNA-Mediated Depletion of Targeted Proteins Proteins that were pulled down using Fc alone were subtracted from proteins pulled down by Fc-glucagon to obtain the proﬁle of proteins that speciﬁcally interact with glucagon. Proteins After treatment of α-TC1-6 cells with GABA and/or insulin in media containing 25 mM glucose as described above, the proteomes were tabulated, and Venn diagram analysis revealed 27 metabolic/regulatory/secretory proteins and 36 histone/cytoskeletal/ribosomal proteins that were common between the groups treated with GABA and insulin. We selected 11 of these proteins (based on availability of the pre-designed siRNA) for siRNA-mediated depletion: Peroxiredoxin-2 (PRDX2), Malate dehydrogenase 1 (MDH1), Aconitate hydratase, mitochondrial (ACO2), 14-3-3 protein zeta/delta (KCIP-1), ELKS/Rab6-interacting/CAST family member 1 (ERC1), Alpha-tubulin 2 (AT2), ATP synthase F1 subunit alpha (ATP5F1A), Histone H4, GRP78, FXYD domain-containing ion transport regulator 2 (FXYD2), and Protein disulﬁde-isomerase (PDI), (Silencer siRNA, Thermo Fisher Scientiﬁc Inc. MA, USA). Gene silencing was based on a published protocol (28). Brieﬂy, α-TC1-6 cells were cultured to 60% conﬂuency and transfected with ﬁnal concentrations of 50 nM of pooled siRNAs (three siRNAs for each target) or control scrambled siRNA using Lipofectamine2000. Cells were incubated for 48 h, after which media were removed and replaced. After 24 h, expression levels of the targeted proteins were evaluated by immunoblotting using primary antibodies against each protein (Supplementary Table 1). Meanwhile, siRNA mediated knockdown of the proglucagon gene was shown as a positive control using real-time PCR (Quant Studio Design and Analysis Real-Time PCR Detection System) (Supplementary Figure 4). After treatment of α-TC1-6 cells with GABA and/or insulin in media containing 25 mM glucose as described above, the proteomes were tabulated, and Venn diagram analysis revealed 27 metabolic/regulatory/secretory proteins and 36 histone/cytoskeletal/ribosomal proteins that were common between the groups treated with GABA and insulin. We selected 11 of these proteins (based on availability of the pre-designed siRNA) for siRNA-mediated depletion: Peroxiredoxin-2 (PRDX2), Malate dehydrogenase 1 (MDH1), Aconitate hydratase, mitochondrial (ACO2), 14-3-3 protein zeta/delta (KCIP-1), ELKS/Rab6-interacting/CAST family member 1 (ERC1), Alpha-tubulin 2 (AT2), ATP synthase F1 subunit alpha (ATP5F1A), Histone H4, GRP78, FXYD domain-containing ion transport regulator 2 (FXYD2), and Protein disulﬁde-isomerase (PDI), (Silencer siRNA, Thermo Fisher Scientiﬁc Inc. MA, USA). Size Calibration Secretory granules of non-transfected cells were used for size calibration. ApogeeMix beads were used to establish sizing gates along the Y axis—large angle light scattering (LALS) vs. X- axis- small angle light scattering (SALS) plot. The microparticle mixture contained plastic spheres with diameters of 180, 240, 300, 590, 880, and 1,300 nm with refractive indexes of 1.43 and 110 nm, and 500 nm green ﬂuorescent beads with refractive index of 1.59. Based on the manufacturer’s default settings, the calibrated gates of the size distribution were 110, 179, 235, 304, 585, and 880 nm, which were used to categorize subpopulations of the enriched secretory granules. January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 3 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari Immunoprecipitation-Immunoblotting of Proteins Associated With Glucagon To validate the interaction of glucagon with either GRP78 or histone H4 within secretory granules we ﬁrst puriﬁed Fc-glucagon or Fc (as control) from the secretory granule preparation by incubating the secretory granule lysate with Protein A-Sepharose beads overnight at 4◦C with rotation. The Fc or Fc-glucagon complex was eluted from the beads with 0.1 M glycine buﬀer (pH 2.8). The eluate was concentrated 50 times using a speed vac, run on a 10% Bis-Tris NuPAGE gel (Supplementary Table 1) and proteins were transferred onto a PVDF membrane. After an overnight incubation with primary antibodies against GRP78 or histone H4, bands were visualized with HRP-conjugated goat anti-rabbit secondary antibody and Clarity Western ECL substrate (Supplementary Table 1). Images were acquired on a BioRad ChemiDoc Imaging System. Gene silencing was based on a published protocol (28). Brieﬂy, α-TC1-6 cells were cultured to 60% conﬂuency and transfected with ﬁnal concentrations of 50 nM of pooled siRNAs (three siRNAs for each target) or control scrambled siRNA using Lipofectamine2000. Cells were incubated for 48 h, after which media were removed and replaced. After 24 h, expression levels of the targeted proteins were evaluated by immunoblotting using primary antibodies against each protein (Supplementary Table 1). Meanwhile, siRNA mediated knockdown of the proglucagon gene was shown as a positive control using real-time PCR (Quant Studio Design and Analysis Real-Time PCR Detection System) (Supplementary Figure 4). Histone H4 Assay Enriched secretory granule fractions were prepared, resuspended in 0.2 N HCl, passed 10 times through a 30G needle, and kept at 4◦C overnight. The reaction was stopped by addition of 0.2 volumes of 1N NaOH. The supernatant was collected after centrifugation at 6,500 × g at 4◦C for 10 min. Protein levels were determined by BCA assay, and 100 ng of protein was used for measuring total histone H4 (Histone H4 Modiﬁcation Multiplex ELISA-like format Kit, Supplementary Table 1), as per the manufacturer’s instructions. The nuclear fraction was also assayed for histone H4 as a positive control. Glucagon Measurement To measure cellular and secreted glucagon levels after siRNA- mediated gene silencing, cell lysates or media were acidiﬁed in HCl-ethanol (92:2 v/v) in a 1:3 ratio, kept at −20◦C overnight, then centrifuged at 13,000 × g for 15 min at 4◦C. The supernatant was then mixed 1:1 with 20 mM Tris, pH 7.5 26 and glucagon levels were measured by ELISA (Thermo Fisher Scientiﬁc, Supplementary Table 1) according to the manufacturer’s instructions. To measure Fc-glucagon, samples were diluted to reach an OD at the linear part of the standard curve. Frontiers in Endocrinology \| www.frontiersin.org RESULTS Our method for puriﬁcation of proteins that associate with glucagon within the α cell secretory granules consisted of two sequential steps. First, we modiﬁed and used a previously published method (27) for enrichment of the secretory granule fraction. Second, we used Fc-glucagon for aﬃnity puriﬁcation to pull down proteins associated with glucagon within the secretory granules. Secretory Granule Enrichment y Immunoblotting for organelle-speciﬁc markers conﬁrmed enrichment of secretory granules (Supplementary Figures 1A–D). The ﬁnal granule fraction was positive for the secretory granule marker, VAMP2. In contrast, the granule fraction did not contain the trans-Golgi marker TGN46, the nuclear envelope marker LaminB1, or the endoplasmic reticulum marker Calreticulin. As a positive control, the general cell lysate contained all four markers. Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors α-TC1-6 cells cultured and kept under chronic exposure to 25 mM glucose and at conﬂuency rate of ∼70% were plated out into six-well plates. After 24 h, two sets of experiments were designed. In one set, medium was replaced by fresh 25 mM glucose-containing medium and in the other set medium was replaced by fresh medium containing 5.5 mM glucose. In both sets, cells were treated by GABA (25 µM), insulin (100 pM), or GABA (25 µM) + insulin (100 pM), and incubated for 24 h. At the end of incubation, plates were placed on ice and media were collected, centrifuged at 16,000 × g for 5 min, and supernatant was removed for glucagon measurement. The cells were washed January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 4 Asadi and Dhanvantari Glucagon Interactome and Glucagon Secretion Statistical Analysis Experiments were done in three biological replicates, each of which had two technical replicates. Values were compared among treatment groups by one-way ANOVA using Sigma Stat 3.5 software (α = 0.05). For image analysis, co-localization of channels in the merged images was calculated by PCC using NIS-Elements software (Nikon, Canada). In media containing 25 mM glucose, there was a predicted direct interaction of glucagon with glucose regulated protein 78 kDa (GRP78 or Hspa5), and ATPase copper transporting alpha polypeptide (Atp7a) (Figure 1A), while in media containing 5.5 mM glucose, GRP78, Stathmin1 (Stmn1), and Heat shock protein 90- alpha (Hsp90aa1) were predicted to directly interact with glucagon (Figure 1B). Under conditions of either 25 or 5.5 mM glucose, one common predicted interaction was that between glucagon and GRP78. Proteomic Analysis of Proteins That Are Associated With Glucagon Within Alpha Cell Secretory Granules three times with ice-cold PBS and scraped in Glycine-BSA buﬀer (100 mM glycine, 0.25% BSA, cOmplete Mini Protease Inhibitor Cocktail, 5 µg/mL Aprotinin, pH 8.8). The scraped cells were lysed by sonication (12 s at 30% amplitude on ice), and centrifuged at 16,000 × g for 45 min, from which the supernatant was collected for analysis. The protein concentration of the cell lysate was measured using BCA assay. To measure glucagon levels, the cell lysate or medium was mixed in an ethanol-acid solution (96% ethanol containing 0.18 M HCl) in a 1:3 ratio, kept at −20◦C overnight, then centrifuged at 16,000 × g for 15 min at 4◦C. The supernatant was then mixed with 20 mM Tris buﬀer, pH 7.5, and glucagon measurements were conducted by ELISA. y Fc or Fc-glucagon was puriﬁed from the granule lysate by aﬃnity puriﬁcation, and proteins that interact with either Fc alone or Fc-glucagon were identiﬁed with LC-MS/MS. Proteins that were pulled down by Fc alone in both 25 mM glucose (Supplementary Table 2) and 5.5 mM glucose (Supplementary Table 3) conditions were subtracted from the list of proteins identiﬁed using Fc-glucagon, thus identifying proteins that speciﬁcally interact with glucagon, which we term the glucagon interactome. Proteins were assigned the following categories: metabolic-secretory-regulatory, histones, cytoskeletal, and ribosomal. We identiﬁed 42 and 96 glucagon-interacting proteins within the category of metabolic-regulatory-secretory proteins when the cells were cultured in media containing 25 mM (Figure 1A) and 5.5 mM glucose (Figure 1B), respectively. GRP78 Interacts With Glucagon and Co-localizes to Glucagon-Positive Secretory Granules Aﬃnity puriﬁcation of Fc-glucagon or Fc alone from the secretory granule lysate was followed by immunoblotting for GRP78. The presence of GRP78 immunoreactivity with Fc- glucagon, and not Fc alone, demonstrates a direct interaction with glucagon in the enriched secretory granules (Figure 2A). Immunoﬂuorescence microscopy showed co-localization of GRP78 and endogenous glucagon within the secretory granules in α-TC1-6 cells (Figure 2B). There was a strong positive correlation between glucagon and GRP78 immunoreactivities (PCC = 0.85 ± 0.08), indicating signiﬁcant co-localization of GRP78 and glucagon. GABA Induces Histone H4 Interaction and Co-localization With Glucagon Cells were transfected with Fc-glucagon or Fc alone, and cultured in DMEM containing 25 or 5.5 mM glucose for 24 h. Fc-glucagon was puriﬁed from enriched secretory granules and associated proteins were identiﬁed by LC-MS/MS. (A) Proteomic map of the metabolic-regulatory-secretory proteins that are predicted to associate with glucagon in the context of 25 mM glucose. Network clustering predicts direct interactions between glucagon and glucose regulated protein 78 KDa (Hspa5, also known as Grp78), and ATPase copper transporting alpha polypeptide (Atp7). (B) Proteomic map of the metabolic-regulatory-secretory proteins that are predicted to associate with glucagon in the context of 5.5 mM glucose. Network clustering predicts direct interactions between glucagon and GRP78, stathmin1 (Stmn1), and heat shock protein 90-alpha (Hsp90aa1). The thickness of the lines indicate the strength of the predicted protein-protein interaction. presence of histone H4 in secretory granules of α-TC1-6 cells after treatment with GABA (Figure 3). Aﬃnity puriﬁcation of Fc-glucagon or Fc alone from the secretory granule lysate was followed by immunoblotting for histone H4 (Figure 3A). The presence of histone H4 immunoreactivity with Fc-glucagon, and not Fc alone, demonstrates a direct interaction with glucagon in the enriched secretory granules (Figure 3A). We then conﬁrmed the presence of histone H4 in the enriched secretory granules of α-TC1-6 cells by ELISA (Figure 3B). In cells treated with GABA in 25 mM glucose, there was a detectable amount of histone H4 in the granules. That this result was not due to contamination from the nuclear fraction was shown by the ﬁnding that histone H4 levels were undetectable in the secretory granules of cells not treated with GABA. As a positive control, the nuclear fraction showed high levels of histone H4. Finally, immunoﬂuorescence microscopy showed the presence of histone H4 in glucagon-containing secretory granules (Figure 3C), and there was signiﬁcant co-localization with glucagon as assessed by Pearson’s correlation coeﬃcient (PCC = 0.78 ± 0.08). treatment with GABA, insulin or GABA + insulin, respectively, when α-TC1-6 cells were cultured in medium containing 25 mM glucose (Figure 4) and in 5.5 mM glucose (Figure 5). Additionally, we tabulated the proﬁles of histone, cytoskeletal, and ribosomal proteins in response to GABA, insulin and GABA + insulin in 25 mM glucose (Supplementary Tables 5A–C) or 5.5 mM glucose (Supplementary Tables 6A–C). The glucagon interactomes were functionally classiﬁed into the following groups: Binding, Structural molecule, Catalytic, Receptor, Translation regulator, Transporter, Signal transducer, Antioxidant. GABA Induces Histone H4 Interaction and Co-localization With Glucagon The proportion of proteins in each category is shown in the context of 25 mM glucose (Supplementary Table 7) and 5.5 mM glucose (Supplementary Table 8). g The protein networks that are predicted to interact with glucagon within the secretory granules under conditions of 25 mM glucose are illustrated in Figure 4. In cells treated with GABA, glucagon is predicted to directly interact with GRP78, HSP1B, HSP90, and vimentin (Figure 4A); however, in cells treated with insulin and GABA + insulin, glucagon interacts directly with only GRP78 (Figures 4B,C). The clusters of metabolic-secretory-regulatory proteins that make up the rest of the glucagon interactomes change in composition in response to the diﬀerent treatments. The numbers of proteins categorized as “structural molecule activities” decreased in response to GABA (∼45%) or insulin (∼38%) and increased in the GABA + insulin group (∼16%) compared to the control (Supplementary Table 7). The numbers of cytoskeletal proteins increased in the GABA (29%), insulin (12%), and GABA + GABA Induces Histone H4 Interaction and Co-localization With Glucagon Secretory granules in alpha cells have been previously studied using transmission electron microscopy and their average sizes have been reported to be in the range of 180–240 nm (29– 31). Accordingly, we conﬁrmed the presence of secretory granules using nano-scale ﬂow cytometry with Fc-glucagon as an exclusive marker for alpha cell secretory granules (32, 33). We used beads in the range of 110–880 nm for calibration in the range of the reported sizes for secretory granules (Supplementary Figure 2A). Fc-glucagon+ secretory granules distributed mostly to the gated regions of 179 and 235 nm (Supplementary Figures 2B,C), conﬁrming enrichment of secretory granules from α-TC1-6 cells. Interestingly, proteomic analysis also revealed the presence of histone proteins, along with structural proteins and ribosomal proteins, within the secretory granules in α-TC1-6 cells (Supplementary Table 4). Histone H4 was predicted to interact with glucagon in cells incubated in medium containing 5.5 mM glucose. Therefore, we reasoned that this interaction was responsive to external eﬀectors. We treated α-TC1-6 cells with GABA, a well-known modulator of glucagon secretion (21) and examined the interaction between histone and glucagon. Co-immunoprecipitation of granule lysates, histone H4 ELISA of granule lysates, and immunoﬂuorescence microscopy all validated the interaction of histone H4 with glucagon and January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org Frontiers in Endocrinology \| www.frontiersin.org 5 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari FIGURE 1 \| The glucagon interactome in secretory granules of α-TC1-6 cells. Cells were transfected with Fc-glucagon or Fc alone, and cultured in DMEM containing 25 or 5.5 mM glucose for 24 h. Fc-glucagon was puriﬁed from enriched secretory granules and associated proteins were identiﬁed by LC-MS/MS. (A) Proteomic map of the metabolic-regulatory-secretory proteins that are predicted to associate with glucagon in the context of 25 mM glucose. Network clustering predicts direct interactions between glucagon and glucose regulated protein 78 KDa (Hspa5, also known as Grp78), and ATPase copper transporting alpha polypeptide (Atp7). (B) Proteomic map of the metabolic-regulatory-secretory proteins that are predicted to associate with glucagon in the context of 5.5 mM glucose. Network clustering predicts direct interactions between glucagon and GRP78, stathmin1 (Stmn1), and heat shock protein 90-alpha (Hsp90aa1). The thickness of the lines indicate the strength of the predicted protein-protein interaction. FIGURE 1 \| The glucagon interactome in secretory granules of α-TC1-6 cells. The Glucagon Interactome Changes in Response to Glucose, GABA and Insulin Since the interaction between histone H4 and glucagon was dependent on glucose levels and GABA, we determined the eﬀects of the major alpha cell paracrine eﬀectors, GABA and insulin, on the glucagon interactome. The proﬁles of the metabolic-regulatory-secretory proteins that associate with glucagon within secretory granules were altered upon January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 6 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari FIGURE 2 \| Glucagon and GRP78 directly interact and are localized within secretory granules in α-TC1-6 cells. (A) Western blot showing GRP78 immunoreactivity in: total cell extracts from untransfected (lane 2) and transfected (lane 3) cells; afﬁnity-puriﬁed Fc-glucagon from isolated secretory granules (lane 4); and afﬁnity-puriﬁed Fc alone from isolated secretory granules (lane 5). GRP78 binds to Fc-glucagon, but not Fc alone. (B) Immunoﬂuorescence microscopy of glucagon (green), GRP78 (red) and both images merged. Cells were cultured on collagen-coated coverslips for 24 h in DMEM containing 25 mM glucose. Images were acquired, 2D deconvoluted and analyzed with NIS-Elements, software (Nikon, Canada). Pearson correlation coefﬁcient (PCC) indicates strong correlation between GRP78 and glucagon (PCC = 0.85 ± 0.08). ROI shows areas of colocalization of GRP78 and glucagon within secretory granules. FIGURE 2 \| Glucagon and GRP78 directly interact and are localized within secretory granules in α-TC1-6 cells. (A) Western blot showing GRP78 immunoreactivity in: total cell extracts from untransfected (lane 2) and transfected (lane 3) cells; afﬁnity-puriﬁed Fc-glucagon from isolated secretory granules (lane 4); and afﬁnity-puriﬁed Fc alone from isolated secretory granules (lane 5). GRP78 binds to Fc-glucagon, but not Fc alone. (B) Immunoﬂuorescence microscopy of glucagon (green), GRP78 (red) and both images merged. Cells were cultured on collagen-coated coverslips for 24 h in DMEM containing 25 mM glucose. Images were acquired, 2D deconvoluted and analyzed with NIS-Elements, software (Nikon, Canada). Pearson correlation coefﬁcient (PCC) indicates strong correlation between GRP78 and glucagon (PCC = 0.85 ± 0.08). ROI shows areas of colocalization of GRP78 and glucagon within secretory granules. FIGURE 2 \| Glucagon and GRP78 directly interact and are localized within secretory granules in α-TC1-6 cells. (A) Western blot showing GRP78 immunoreactivity in: total cell extracts from untransfected (lane 2) and transfected (lane 3) cells; afﬁnity-puriﬁed Fc-glucagon from isolated secretory granules (lane 4); and afﬁnity-puriﬁed Fc alone from isolated secretory granules (lane 5). GRP78 binds to Fc-glucagon, but not Fc alone. The Glucagon Interactome Changes in Response to Glucose, GABA and Insulin (B) Immunoﬂuorescence microscopy of glucagon (green), GRP78 (red) and both images merged. Cells were cultured on collagen-coated coverslips for 24 h in DMEM containing 25 mM glucose. Images were acquired, 2D deconvoluted and analyzed with NIS-Elements, software (Nikon, Canada). Pearson correlation coefﬁcient (PCC) indicates strong correlation between GRP78 and glucagon (PCC = 0.85 ± 0.08). ROI shows areas of colocalization of GRP78 and glucagon within secretory granules. insulin (35%) groups, while the numbers of ribosomal proteins decreased in those groups by 51, 14, and 66%, respectively (Table 1A). GRP78 and PCSK2, were predicted to directly interact with glucagon after treatment with GABA + insulin (Figure 5C). In the context of 5.5 mM glucose, the number of cytoskeletal proteins decreased, and the number of ribosomal proteins increased compared to cells treated with GABA, insulin and GABA + insulin in 25 mM glucose (Table 1B). Interestingly, the total numbers of proteins classiﬁed as “structural molecule activities” did not change appreciably across treatments (Supplementary Table 8). However, diﬀerences became apparent when cytoskeletal and ribosomal proteins were compared separately. When compared to 5.5 mM glucose alone, there were decreases of ∼24 and ∼35%, respectively, in the numbers of cytoskeletal proteins when cells were treated with GABA or insulin alone, but a ∼71% increase in response to GABA + Insulin. Conversely, the numbers of ribosomal proteins increased by ∼26 and ∼43% in response to GABA and insulin, respectively, and decreased by ∼69% in response to GABA + Insulin (Table 1B). Compared to cells incubated in medium containing 25 mM glucose, there were dramatic increases in the numbers of metabolic-regulatory-secretory proteins associated with glucagon after treatment with GABA, insulin or GABA + insulin in cells incubated in media containing 5.5 mM glucose (Figure 5). In cells treated with GABA, glucagon is predicted to directly interact with the following proteins: GRP78 (Hspa5), HSP 90alpha (Hsp90aa1), proprotein convertase subtilisin/kexin type 2 (PCSK2), heat shock 70 kDa protein 1B (Hsp1b), calmodulin 1(Calm1), and guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-7 (Gng7) (Figure 5A). The Glucagon Interactome Changes in Response to Glucose, GABA and Insulin Pearson correlation coefﬁcient (PCC) indicates strong correlation between histone H4 and glucagon (PCC = 0.78 ± 0.08). ROI shows areas of colocalization of histone H4 and glucagon within secretory granules. FIGURE 3 \| GABA induces direct interaction between glucagon and histone H4 within secretory granules in α-TC1-6 cells. (A) Western blot shows histone H4 immunoreactivity in: total cell extracts from untransfected (lane 2) and transfected (lane 3) cells; afﬁnity-puriﬁed Fc-glucagon from isolated secretory granules (lane 4); and afﬁnity-puriﬁed Fc alone from isolated secretory granules (lane 5). Histone H4 binds to Fc-glucagon, but not Fc alone. (B) Quantitative ELISA measurement of histone H4 (left Y axis) and glucagon (right Y axis) within the secretory granules (control GABA, insulin) and the nuclear fraction of α-TC1-6 cells. Values are expressed as mean±SD and compared with 1-way ANOVA (α = 0.05). p < 0.05; *p < 0.001. (C) Immunoﬂuorescence microscopy of glucagon (green), histone H4 (red), and both images merged. Cells were cultured on collagen-coated coverslips for 24 h in DMEM containing 25 mM glucose. Images were acquired, 2D deconvoluted and analyzed with NIS-Elements, software (Nikon, Canada). Pearson correlation coefﬁcient (PCC) indicates strong correlation between histone H4 and glucagon (PCC = 0.78 ± 0.08). ROI shows areas of colocalization of histone H4 and glucagon within secretory granules. The Glucagon Interactome Changes in Response to Glucose, GABA and Insulin Under insulin treatment, the following proteins were predicted to directly interact with glucagon: GRP78, HSP 90-alpha, annexin A5 (Anxa5), stathmin1 (Stmn1), fatty acid synthase (Fasn), and chromogranin A (Chga) (Figure 5B); and only two proteins, January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 7 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari FIGURE 3 \| GABA induces direct interaction between glucagon and histone H4 within secretory granules in α-TC1-6 cells. (A) Western blot shows histone H4 immunoreactivity in: total cell extracts from untransfected (lane 2) and transfected (lane 3) cells; afﬁnity-puriﬁed Fc-glucagon from isolated secretory granules (lane 4); and afﬁnity-puriﬁed Fc alone from isolated secretory granules (lane 5). Histone H4 binds to Fc-glucagon, but not Fc alone. (B) Quantitative ELISA measurement of histone H4 (left Y axis) and glucagon (right Y axis) within the secretory granules (control GABA, insulin) and the nuclear fraction of α-TC1-6 cells. Values are expressed as mean±SD and compared with 1-way ANOVA (α = 0.05). p < 0.05; *p < 0.001. (C) Immunoﬂuorescence microscopy of glucagon (green), histone H4 (red), and both images merged. Cells were cultured on collagen-coated coverslips for 24 h in DMEM containing 25 mM glucose. Images were acquired, 2D deconvoluted and analyzed with NIS-Elements, software (Nikon, Canada). Pearson correlation coefﬁcient (PCC) indicates strong correlation between histone H4 and glucagon (PCC = 0.78 ± 0.08). ROI shows areas of colocalization of histone H4 and glucagon within secretory granules. FIGURE 3 \| GABA induces direct interaction between glucagon and histone H4 within secretory granules in α-TC1-6 cells. (A) Western blot shows histone H4 immunoreactivity in: total cell extracts from untransfected (lane 2) and transfected (lane 3) cells; afﬁnity-puriﬁed Fc-glucagon from isolated secretory granules (lane 4); and afﬁnity-puriﬁed Fc alone from isolated secretory granules (lane 5). Histone H4 binds to Fc-glucagon, but not Fc alone. (B) Quantitative ELISA measurement of histone H4 (left Y axis) and glucagon (right Y axis) within the secretory granules (control GABA, insulin) and the nuclear fraction of α-TC1-6 cells. Values are expressed as mean±SD and compared with 1-way ANOVA (α = 0.05). p < 0.05; *p < 0.001. (C) Immunoﬂuorescence microscopy of glucagon (green), histone H4 (red), and both images merged. Cells were cultured on collagen-coated coverslips for 24 h in DMEM containing 25 mM glucose. Images were acquired, 2D deconvoluted and analyzed with NIS-Elements, software (Nikon, Canada). The Dynamic Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion statistically signiﬁcant level (p < 0.001). As well, knockdown of peroxiredoxin-2 (PRDX2), ATP synthase F1 subunit alpha (ATP5F1A), histone H4, and aconitate hydratase mitochondrial (ACO2) reduced glucagon secretion (p < 0.01), as did knockdown of alpha-tubulin 2 (AT2) (p < 0.05) (Figure 6A). Gene silencing of MDH1, PRDX2, ATP5F1A, and FXYD2 reduced cellular glucagon content to a signiﬁcance level of p < 0.001. Gene silencing of KCIP-1, ACO2, Histone H4 and PDI all reduced the levels of cellular glucagon content to a signiﬁcance level of p < 0.01 and that for ERC1 at p < 0.05 (Figure 6B). Gene silencing of GRP78 had no eﬀect on glucagon secretion, and reduced cellular glucagon content (p < 0.05). From our glucagon interactomes, we identiﬁed 11 proteins that interact with glucagon after treatment of α-TC1-6 cells with either GABA or insulin in media containing 25 mM glucose. To determine their eﬀects on glucagon secretion, these proteins were depleted with siRNAs (Supplementary Figure 3) and glucagon secretion and cell content were measured. Of these 11 proteins, knockdown of ELKS/Rab6-interacting/CAST family member 1 (ERC1) increased glucagon secretion (p < 0.001), while gene silencing of 14-3-3 zeta/delta (KCIP-1), cytosolic malate dehydrogenase (MDH1), FXYD domain-containing ion transport regulator 2 (FXYD2) and protein disulﬁde- isomerase (PDI) reduced glucagon secretion to the same In the context of 5.5 mM glucose, signiﬁcant reduction of glucagon secretion occurred by depletion of MDH1 (p < 0.05), PDI(p < 0.05), ERC1(p < 0.01), and ACO2 (p < 0.01). However, January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 8 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari FIGURE 4 \| The glucagon interactome is altered in response to paracrine effectors in 25 mM glucose. α-TC1-6 cells were transfected with Fc-glucagon or Fc alone, and treated with GABA (25 µM), insulin (100 pM) or GABA (25 µM) plus insulin (100 pM) for 24 h in DMEM containing 25 mM glucose. Fc-glucagon was puriﬁed from isolated secretory granules and associated proteins were identiﬁed by LC-MS/MS. (A) Proteomic map of metabolic-regulatory-secretory proteins that are associated with glucagon after treatment of α-TC1-6 cells with GABA shows direct interactions with 4 proteins: GRP78, Heat shock 70 kDa protein 1B (Hspa1b) Heat shock protein 90- alpha (Hsp90aa1), and Vimentin (Vim). (B) After treatment with insulin or (C) GABA + Insulin, glucagon is predicted to interact only with GRP78. The Dynamic Glucagon Interactome Reveals Novel Proteins That Regulate Glucagon Secretion Line thickness indicates the strength of data support. FIGURE 4 \| The glucagon interactome is altered in response to paracrine effectors in 25 mM glucose. α-TC1-6 cells were transfected with Fc-glucagon or Fc alone, and treated with GABA (25 µM), insulin (100 pM) or GABA (25 µM) plus insulin (100 pM) for 24 h in DMEM containing 25 mM glucose. Fc-glucagon was puriﬁed from isolated secretory granules and associated proteins were identiﬁed by LC-MS/MS. (A) Proteomic map of metabolic-regulatory-secretory proteins that are associated with glucagon after treatment of α-TC1-6 cells with GABA shows direct interactions with 4 proteins: GRP78, Heat shock 70 kDa protein 1B (Hspa1b) Heat shock protein 90- alpha (Hsp90aa1), and Vimentin (Vim). (B) After treatment with insulin or (C) GABA + Insulin, glucagon is predicted to interact only with GRP78. Line thickness indicates the strength of data support. silencing of the other abovementioned genes did not signiﬁcantly alter glucagon secretion (Figure 6C). Cellular glucagon content was signiﬁcantly decreased by silencing of ATP5F1A (p < 0.05), AT2, PDI, ERC1, FXYD2, KCIP-1, histone H4, GRP78 (p < 0.01), ACO2, and PRDX2 (p < 0.001) (Figure 6D). increased cellular glucagon content (Supplementary Figure 5B); in contrast, neither GABA nor insulin alone aﬀected cellular glucagon content, but in combination, they decreased cellular glucagon content. Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors Structural constituent of cytoskeleton Structural constituent of ribosome Extracellular matrix structural constituent Control 50 45.5 4.5 GABA 38.1 57.1 4.8 Insulin 32.4 64.9 2.7 GABA + Insulin 85.7 14.3 – Panther GO-Slim Molecular Function analysis resulted in 3 sub-categories. The values represent protein hits as a percentage of the total number of hits within each sub-category when α-TC1-6 cells were cultured in media containing 5.5 mM glucose. ysis resulted in 3 sub-categories. The values represent protein hits as a percentage of the total number of hits within each sub-category when taining 5.5 mM glucose. interactome in the regulation of glucagon secretion in α-TC1-6 cells. lysosomes. Our data suggest that GRP78 may be a novel sorting receptor for glucagon in the regulated secretory pathway of alpha cells. We have previously shown a potential role of chromogranin A as a sorting receptor for glucagon in both α-TC1-6 cells and PC12 cells (19), but unlike GRP78, we did not demonstrate any direct interactions with glucagon. While knockdown of GRP78 did not reduce glucagon secretion, it did reduce cell content, indicating a potential role in intracellular traﬃcking, but not exocytosis, of glucagon. We have previously shown that α-TC1-6 cells have elevated levels of both proglucagon mRNA and glucagon secretion in response to 25 mM glucose (17), and other groups have shown the same eﬀect in isolated mouse islets (3), clonal hamster InR1G9 glucagon-releasing cells (3, 34), and perfused rat pancreas (35). We also showed that this paradoxical glucagon release is accompanied by an up-regulation of components of the regulated secretory pathway, particularly in the active forms of PC1/3 and PC2 that post-translationally process proglucagon to glucagon, and in SNARE proteins that mediate vesicle exocytosis (17). Under conditions of 5.5 mM glucose, the up-regulation in RNA-binding proteins that modulate biosynthesis of islet secretory granule proteins, along with chaperonins, may indicate an increase in protein synthesis (36, 37). Chaperonins, as key components of the cellular chaperone machinery, are involved in maturation of newly-synthesized proteins in an ATP dependent manner (36). As ATP-generating proteins, such as ATP5F1A, MDH1, and glucose metabolic proteins, were also increased, we speculate that 5.5 mM glucose induced a stress response that resulted in increased protein translation. Frontiers in Endocrinology \| www.frontiersin.org Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors We have identiﬁed a dynamic “glucagon interactome” within secretory granules of alpha cells that is altered in response to glucose levels and the paracrine eﬀectors GABA and insulin. We used a tagged glucagon construct, Fc-glucagon, to bring down proteins within secretory granules. We validated enrichment of the secretory granules by nano-scale ﬂow cytometry and immunoblotting with compartment-speciﬁc markers. We identiﬁed a network of 392 proteins within the secretory granules that interact with glucagon and showed a direct interaction with GRP78 and Histone H4. Components of the interactome played a role in glucagon secretion, thus revealing a role for the α-TC1-6 cells were cultured under high glucose conditions (25 mM) and then treated with paracrine eﬀectors (GABA, insulin or GABA + insulin). The proﬁles of cumulative glucagon secretion and cellular glucagon content in 25 mM glucose was diﬀerent from that in 5.5 mM glucose. While neither GABA nor insulin aﬀected glucagon secretion in 5.5 mM glucose, they suppressed glucagon secretion in 25 mM glucose (Supplementary Figure 5A). In the context of 25 mM glucose, GABA reduced cellular glucagon content, while insulin January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 9 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari TABLE 1A \| Sub-groups of proteins categorized as “structural molecules” in the glucagon interactome under conditions of 25 mM glucose. Structural constituent of cytoskeleton Structural constituent of ribosome Extracellular matrix structural constituent Control 66.7 29.2 4.2 GABA 85.7 14.3 – Insulin 75 25 – GABA + Insulin 90 10 – Panther GO-Slim Molecular Function analysis resulted in 3 sub-categories. The values represent protein hits as a percentage of the total number of hits within each sub-category when α-TC1-6 cells were cultured in media containing 25 mM glucose. TABLE 1B \| Sub-groups of proteins categorized as “structural molecules” in the glucagon interactome under conditions of 5.5 mM glucose. Structural constituent of cytoskeleton Structural constituent of ribosome Extracellular matrix structural constituent Control 50 45.5 4.5 GABA 38.1 57.1 4.8 Insulin 32.4 64.9 2.7 GABA + Insulin 85.7 14.3 – Panther GO-Slim Molecular Function analysis resulted in 3 sub-categories. The values represent protein hits as a percentage of the total number of hits within each sub-category when α-TC1-6 cells were cultured in media containing 5.5 mM glucose. TABLE 1B \| Sub-groups of proteins categorized as “structural molecules” in the glucagon interactome under conditions of 5.5 mM glucose. Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors This hypothesis is strengthened by the identiﬁcation of cold shock protein, peroxiredoxin, thiol-disulﬁde isomerase and thioredoxin within the glucagon interactome at 5.5 mM glucose, all of which are up-regulated in pancreatic islets in response to stress (37). y g g Interestingly, we identiﬁed histone proteins as a functional part of the glucagon interactome. The discovery of histone proteins within alpha cell secretory granules is novel, and supported by the ﬁndings that the cytosolic fraction of pooled islets from multiple human donors had abundant amounts of the histone H2A (40). As well, quantitative proteomics of both αTC1 and ßTC3 cells revealed the presence of histones H4, H3, H2A, H2B, and H1 (41). Our data indicate that one of these histones, H4, may directly bind to glucagon and regulate its basal level of secretion, perhaps under conditions of stress. Oxidative stress contributes to the pathogenesis of diabetes by disrupting the balance between reactive oxygen species and antioxidant proteins (42). Such an imbalance could target chromatin and globally alter proﬁles of gene expression, especially those encoding histone and DNA-binding proteins (42, 43). Thus, we speculate that the presence of histone H4 in the secretory granules could reﬂect a response to microenvironmental stress. Furthermore, it has been suggested that histones contained within secretory granules in neutrophils could function as a defense mechanism, interacting with the plasma membrane to generate extracellular traps in response to bacterial infections (44). Thus, it is possible that histone proteins in the glucagon interactome take a role in the fusion step of granule exocytosis. Additionally, secretion One protein that was consistently predicted as interacting directly with glucagon was the ER stress protein and molecular chaperone GRP78. Previous proteomic studies have identiﬁed GRP78 in islets and beta cells (38, 39). Its presence in alpha cell secretory granules may not be surprising, as it has previously been found in non-ER compartments such as the nucleus and January 2019 \| Volume 9 \| Article 792 10 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari FIGURE 5 \| The glucagon interactome is altered in response to paracrine effectors in 5.5 mM glucose. α-TC1-6 cells were transfected with Fc-glucagon or Fc alone, and treated with GABA (25 µM), insulin (100 pM) or GABA (25 µM) plus insulin (100 pM) for 24 h in DMEM containing 5.5 mM glucose. Fc-glucagon was puriﬁed from isolated secretory granules and associated proteins were identiﬁed by LC-MS/MS. Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors (A) Proteomic map of metabolic-regulatory-secretory proteins that are associated with glucagon after treatment of α-TC1-6 cells with GABA shows direct interactions with 6 proteins: GRP78, Heat shock protein 90- alpha (Hsp90aa1), Protein convertase subtilisin/kexin type2 (PCSK2), Heat shock 70 kDa protein 1B (Hspa1b), Calmodulin 1 (Calm1), Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-7 (Gng7). (B) After treatment with insulin, glucagon is predicted to directly interact with 7 proteins: GRP78, Heat shock protein 90-alpha, Annexin A5 (Anxa5), Stathmin1 (Stmn1), PCSK2, Fatty acid synthase (Fasn), and Chromogranin A (Chga). (C) After treatment with GABA + Insulin, glucagon is predicted to directly interact with GRP78 and PCSK2. Line thickness indicates the strength of data support. FIGURE 5 \| The glucagon interactome is altered in response to paracrine effectors in 5.5 mM glucose. α-TC1-6 cells were transfected with Fc-glucagon or Fc alone, and treated with GABA (25 µM), insulin (100 pM) or GABA (25 µM) plus insulin (100 pM) for 24 h in DMEM containing 5.5 mM glucose. Fc-glucagon was puriﬁed from isolated secretory granules and associated proteins were identiﬁed by LC-MS/MS. (A) Proteomic map of metabolic-regulatory-secretory proteins that are associated with glucagon after treatment of α-TC1-6 cells with GABA shows direct interactions with 6 proteins: GRP78, Heat shock protein 90- alpha (Hsp90aa1), Protein convertase subtilisin/kexin type2 (PCSK2), Heat shock 70 kDa protein 1B (Hspa1b), Calmodulin 1 (Calm1), Guanine nucleotide-binding protein G(I)/G(S)/G(O) subunit gamma-7 (Gng7). (B) After treatment with insulin, glucagon is predicted to directly interact with 7 proteins: GRP78, Heat shock protein 90-alpha, Annexin A5 (Anxa5), Stathmin1 (Stmn1), PCSK2, Fatty acid synthase (Fasn), and Chromogranin A (Chga). (C) After treatment with GABA + Insulin, glucagon is predicted to directly interact with GRP78 and PCSK2. Line thickness indicates the strength of data support. on GABA. Ohara-Imaizumi et al. showed that ERC1 depletion in MIN6 cells and rat pancreatic β-cells suppressed glucose stimulated insulin secretion (47). When pancreatic β-cells were exposed to high glucose conditions, ERC1 takes a role in the process of granule docking and fusion toward insulin exocytosis. Here, by showing that depletion of ERC1 increased glucagon secretion at 25 mM glucose and reduced it at 5.5 mM glucose, it is tempting to speculate that ERC1 is a part of the granule exocytosis machinery in alpha cells and plays a potential role in controlling glucagon exocytosis under diabetic conditions. Frontiers in Endocrinology \| www.frontiersin.org Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors (A) Glucagon secretion and (B) cell content in the context of 25 mM glucose, and (C) glucagon secretion and (D) cell content in the context of 5.5 mM glucose were assessed after siRNA-mediated gene silencing of the following proteins: Alpha-tubulin 2 (AT2), ATP synthase F1 subunit alpha (ATP5F1A), Malate dehydrogenase 1 (MDH1), Protein disulﬁde-isomerase (PDI), ELKS/Rab6-interacting/CAST family member 1 (ERC1), Aconitate hydratase mitochondrial (ACO2), Peroxiredoxin-2 (PRDX2), 14-3-3 protein zeta/delta (KCIP-1), FXYD domain-containing ion transport regulator 2 (FXYD2), histone H4, and GRP78 using pre-designed siRNAs for the mouse genome. After siRNA transfection, α-TC1-6 cells were cultured in DMEM containing 25 or 5.5 mM glucose for 24 h and glucagon levels were measured using ELISA. Values are expressed as mean ± SD (α = 0.05; n = 3–4). p < 0.05; p < 0.01; p < 0.001. regulators of insulin secretion (51). We also identiﬁed the small G proteins SAR1, Rab2A, and RhoA, present in INS-1 cell secretory granules (52); however, their functions are not known. to promote glucose uptake in beta cells (49), and thus may be a new paracrine, or even autocrine, regulator of alpha cell function. Interestingly, in the context of 5.5 mM glucose, GABA recruited PCSK2 and secretogranin 2, known alpha cell granule proteins that function in proglucagon processing (19). Although our previous work showed no changes in PCSK2 in response to 5.5 mM glucose (17), we now show that plasticity in PCSK2 expression may be due to GABA under these glucose concentrations. Interestingly, treatment of the α-TC1-6 cells with GABA + insulin in 25 mM glucose caused a dramatic decrease in the overall numbers of proteins within the glucagon interactome. Interaction with GRP78 remained preserved, while a new protein, microtubule-associated protein 2, appeared in the glucagon interactome. This protein may have a potential role in glucose homeostasis, as it is down-regulated in isolated diabetic rat islets exposed to low glucose conditions (53). In the context of 5.5 mM glucose, the combination of GABA and insulin again predicted the presence of PCSK2 in the glucagon interactome, as seen with GABA treatment alone and invites revisiting the question of PCSK2 acting as a sorting receptor for glucagon (19). In the context of 25 mM glucose, insulin treatment increased the number of biosynthetic proteins, consistent with its role in cellular growth. Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors Another potentially novel player in GABA-regulated glucagon secretion is KCIP-1, associated with beta cell survival (48). Furthermore, our proteomics ﬁndings suggest that GABA may enhance glucose uptake and glucose tolerance through leucine- rich repeat proteins. These proteins bind to the insulin receptor of histones and other nuclear proteins has been associated with an inﬂammatory or senescent secretory phenotype (45, 46). The alpha cell paracrine eﬀectors, GABA and insulin, remodeled the glucagon interactome in α-TC1-6 cells in a manner that was dependent on glucose levels. Compared to the respective control groups, GABA altered >70 and >80% of the metabolic-regulatory-secretory proteins within the glucagon interactome in the context of 25 and 5.5 mM glucose, respectively. One potentially novel GABA-regulated protein that may function in glucagon secretion in 25 mM glucose is ERC1, which has a role in the formation of the cytomatrix active zone and insulin exocytosis from beta cells (47), and we show for the ﬁrst time a potential inhibitory eﬀect of ERC1 on glucagon secretion that may be dependent January 2019 \| Volume 9 \| Article 792 11 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari FIGURE 6 \| Glucagon secretion and cell content are regulated by a subset of interactome proteins. (A) Glucagon secretion and (B) cell content in the context of 25 mM glucose, and (C) glucagon secretion and (D) cell content in the context of 5.5 mM glucose were assessed after siRNA-mediated gene silencing of the following proteins: Alpha-tubulin 2 (AT2), ATP synthase F1 subunit alpha (ATP5F1A), Malate dehydrogenase 1 (MDH1), Protein disulﬁde-isomerase (PDI), ELKS/Rab6-interacting/CAST family member 1 (ERC1), Aconitate hydratase mitochondrial (ACO2), Peroxiredoxin-2 (PRDX2), 14-3-3 protein zeta/delta (KCIP-1), FXYD domain-containing ion transport regulator 2 (FXYD2), histone H4, and GRP78 using pre-designed siRNAs for the mouse genome. After siRNA transfection, α-TC1-6 cells were cultured in DMEM containing 25 or 5.5 mM glucose for 24 h and glucagon levels were measured using ELISA. Values are expressed as mean ± SD (α = 0.05; n = 3–4). p < 0.05; p < 0.01; *p < 0.001. FIGURE 6 \| Glucagon secretion and cell content are regulated by a subset of interactome proteins. Frontiers in Endocrinology \| www.frontiersin.org Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors Kinesin-like proteins also increased, suggesting a potential role in alpha cell secretory granule synthesis and glucose homeostasis, as has been documented in beta cells (50). In the context of 5.5 mM glucose, insulin up- regulated nucleoside diphosphate kinases A and B, proposed January 2019 \| Volume 9 \| Article 792 12 Glucagon Interactome and Glucagon Secretion Asadi and Dhanvantari signaling is required for suppression of glucagon secretion. Here, we show that treatment with insulin suppresses the long-term cumulative secretion of glucagon when α-TC1-6 cells were cultured and chronically kept in 25 mM glucose. Interestingly, under these conditions, cellular glucagon content increased, perhaps due to excess glucagon in the medium and its potential abolishing eﬀect on insulin action (63). In addition, this increase could be due to an autocrine eﬀect of glucagon on proglucagon gene expression, a notion that has been argued by Leibiger et al. for short-term eﬀect of glucagon on proglucagon gene expression in non-cumulative culturing (26). As well, it is known that GABA inhibits glucagon secretion under high glucose conditions (64). Importantly, our ﬁndings show reductions in both glucagon secretion and content. Surprisingly, the combination of GABA and insulin did not suppress glucagon secretion, leading to questions on the mechanism of the interactions between these two signaling pathways. The design of our experiments was to mimic blood glucose volatility in diabetes in particular, and not in normal physiology, to investigate potential dynamic alterations in the glucagon interactome (54). Here, we have identiﬁed the glucagon interactome in α-TC1-6 cells after chronic exposure to extremely high glucose (25 mM), which, in diabetes, paradoxically increases glucagon secretion from pancreatic alpha cells (3, 5). We further showed remodeling of this interactome by replacing that extremely high glucose condition (25 mM) with a relatively low glucose (5.5 mM) medium, which mimic conditions that represent glucose volatility in diabetes. However, we did not examine changes in the glucagon interactome throughout a range of high and low glucose conditions, which could be a limitation for the current study. Also, we used our negative control, Fc alone, only in the two glucose conditions and not in treatments with GABA and insulin, which may aﬀect the interpretation of the interactome under these conditions. It is well-established that, under normal physiological conditions, glucagon secretion is suppressed by high glucose (21). However, chronic hyperglycemia disrupts this ﬁne regulation and results in elevated glucagon secretion (34, 55). FUNDING This work has been ﬁnancially supported by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada to SD, and by a Dean’s Award Scholarship to FA. ACKNOWLEDGMENTS We would like to thank Paula Pittock at the Siebens-Drake Research Institute, University of Western Ontario for assistance in LC-MS/MS analysis and Dr. Hon Leong lab members for assistance with nanoﬂow cytometry. This manuscript has been released by the preprint server for biology, bioRXiv (https://www. biorxiv.org/content/early/2018/07/20/373118) (65). Under normal physiological conditions, GABA and insulin suppress glucagon secretion in pancreatic α-cells (21, 60). This response to GABA and insulin may diﬀer depending on the cell line and experimental conditions used. Piro et al. (61) showed that with short-term treatment, insulin signiﬁcantly suppressed glucagon secretion in α-TC1-6 cells without aﬀecting cellular glucagon content. In INR1G cells, Kawamori et al. (62) showed that silencing of the insulin receptor signiﬁcantly increased glucagon secretion, indicating that insulin receptor Frontiers in Endocrinology \| www.frontiersin.org Alterations in Glucagon Secretion and Cell Glucagon Content in Response to Nutritional and Paracrine Effectors It has been documented that chronic exposure to 25 mM glucose stimulates glucagon secretion in α-TC1-6 cells (17), thus mimicking the alpha cell response to glucose in the diabetic, and not normal, condition. In conclusion, we have described a novel and dynamic glucagon interactome in α-TC1-6 cells that is remodeled in response to glucose and the alpha cell paracrine eﬀectors, GABA and insulin. Our proteomics approach has revealed a number of novel secretory granule proteins that function in the regulation of glucagon secretion and illustrates the plasticity in the protein components of the alpha cell secretory granules. These ﬁndings provide an important proteomics resource for further data mining of the alpha cell secretory granules and targeting diabetes treatment. While we presented a novel glucagon interactome within enriched secretory granules of α-TC1-6 cells and its alterations due to nutritional or paracrine eﬀectors, direct comparisons to primary alpha cells may be limited. When we compared our described glucagon interactome with the transcriptomic proﬁle of mouse alpha cells (56), and human α-cells (57), there were some diﬀerences in the protein proﬁles. Additionally, Lawlor et al. (58) compared gene expression proﬁles of α-TC1 cells with their primary mouse and human counterparts and showed a high level of discrepancy between them. One possibility for this discrepancy may be changes in gene expression in primary cells while cultured in vitro (17, 59). However, we feel that the ﬁndings we are reporting generally show that: (1) networks of proteins can interact with glucagon within the secretory granule compartment of the pancreatic α-cell; (2) this interactome is remodeled according to the micro-environmental milieu; and (3) some proteins within the interactome can regulate glucagon secretion. The next step will be to use our data to guide the identiﬁcation of glucagon-interacting proteins that may regulate glucagon secretion within primary alpha cells. AUTHOR CONTRIBUTIONS FA and SD designed the experiments, wrote, and prepared the manuscript text and ﬁgures, and reviewed the manuscript prior to submission. REFERENCES The role of dysregulated glucagon secretion in type 2 diabetes. Diabetes Obes Metab. (2011) 13:126–32. doi: 10.1111/j.1463-1326.2011.01449.x 7. D’alessio D. 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Mol Cell Proteomics (2007) 6:1007–17. doi: 10.1074/mcp.M600443-MCP200 Conﬂict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 53. Ghanaat-Pour H, Huang Z, Lehtihet M, Sjöholm A. Global expression proﬁling of glucose-regulated genes in pancreatic islets of spontaneously diabetic Goto-Kakizaki rats. J Mol Endocrinol. (2007) 39:135–50. doi: 10.1677/JME-07-0002 Copyright © 2019 Asadi and Dhanvantari. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2019 Asadi and Dhanvantari. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 54. Whitelaw BC, Choudhary P HD. Evaluating rate of change as an index of glycemic variability, using continuous glucose monitoring data. Diabetes Technol Ther. (2011) 13:631–6. doi: 10.1089/dia.2010.0215 January 2019 \| Volume 9 \| Article 792 Frontiers in Endocrinology \| www.frontiersin.org 15
https://openalex.org/W2074201374	https://europepmc.org/articles/pmc4042319?pdf=render	English	null	PReS-FINAL-2197: Teenage boy suffering from diabetes mellitus type 1 and heterozygous Familial Mediterranean Fever: a case report	Pediatric rheumatology online journal	2,013	cc-by	780	© 2013 Olivieri et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Objectives Our aim is to emphasize that FMF must be taken into consideration as a possible disease associated with type 1 DM in the presence of suggestive findings. Introduction The molecular analysis of the nucleotide sequence of exon 2 of MEFV gene, performed in genomic DNA extracted from peripheral blood leukocytes, showed the mutation c.442G> C (p.E148Q) in the heterozygous state. The colchicine therapy, started 20 months ago at a dose of 1 mg/day, is well tolerated and has determined the immediate disappearance of the symptoms so far. Renal biopsy, performed at 14 years and 6 months of age because of persistent proteinuria, showed the absence of amyloidosis but a slight and irregular thickening of the lamina densa of some glomerular capillaries presumably due to diabetes. The serological tests have ruled out so far CD, AD, ATD and connective tissue diseases (CTD). Until now, the eye examination did not detect the presence of iridocyclitis. It is known that type 1 diabetes mellitus (type 1 DM) maybe associated with other autoimmune diseases, such as autoimmune thyroid disease (ATD), celiac disease (CD) and Addison’s disease (AD). We report the case of a patient with type 1 DM associated with familial medi- terranean fever (FMF). It is the third association of these two diseases described in the medical literature to our knowledge so far. Methods A 13 year old boy already suffering from diabetes mellitus type 1 since the age of 4 years and 3 months, came to our attention because of the presence of periodic fever associated with abdominal pain, oral ulcers, chest pain and diffuse arthralgia. The fever appeared every 15-30 days with peaks that reached 40°C and lasted 24-48 hours. Blood tests (complete blood count, blood culture, serum immunoglobulins, viral serology, biochemical profile), instrumental examinations (ultrasound of the abdomen and chest x-ray) and the rest of laboratory investigations (culture of throat swab, stool examination and urinalysis) were normal in the interval between febrile episodes, but during the attacks revealed an increase in inflammatory markers (ESR 60 mm/1 h, CRP 3.7 mg/dl). For the clinical suspicion of FMF we requested that the genetic investiga- tion was performed. Olivieri et al. Pediatric Rheumatology 2013, 11(Suppl 2):P187 http://www.ped-rheum.com/content/11/S2/P187 Olivieri et al. Pediatric Rheumatology 2013, 11(Suppl 2):P187 http://www.ped-rheum.com/content/11/S2/P187 Open Access Conclusion The coexistence of FMF and type 1 DM is a very rare finding. FMF heterozygotes tend to have a milder course of the disease and are less prone to experience new clinical manifestations than homozygotes. Moreover, at puberty, their symptomatology could disappear allowing to cease colchicine without relapses or an increase of inflammatory markers. Recently, a case of simultaneous protracted febrile myalgia syndrome (PFMS) preceded by diabetic ketoacidosis (DKA) has been described for which the authors have suggested that DKA-associated cytokine release could be a predisposing factor or a trigger for FMF-associated PFMS. Conversely, it was hypothesized that the immune dysregulation in FMF could be involved in the autoimmune mechanism that leads to type 1 DM. Finally, a prolonged follow up is needed to verify the Pediatrics, Second University of Naples, Naples, Italy Page 2 of 2 Page 2 of 2 Olivieri et al. Pediatric Rheumatology 2013, 11(Suppl 2):P187 http://www.ped-rheum.com/content/11/S2/P187 long-term necessity of colchicine for our patient and further studies are required to reveal any possible shared molecular mechanisms that are responsible for these two diseases. Disclosure of interest None declared. None declared. Published: 5 December 2013 Published: 5 December 2013 Published: 5 December 2013 doi:10.1186/1546-0096-11-S2-P187 Cite this article as: Olivieri et al.: PReS-FINAL-2197: Teenage boy suffering from diabetes mellitus type 1 and heterozygous Familial Mediterranean Fever: a case report. Pediatric Rheumatology 2013 11(Suppl 2):P187. doi:10.1186/1546-0096-11-S2-P187 Cite this article as: Olivieri et al.: PReS-FINAL-2197: Teenage boy suffering from diabetes mellitus type 1 and heterozygous Familial Mediterranean Fever: a case report. Pediatric Rheumatology 2013 11(Suppl 2):P187. doi:10.1186/1546-0096-11-S2-P187 Cite this article as: Olivieri et al.: PReS-FINAL-2197: Teenage boy suffering from diabetes mellitus type 1 and heterozygous Familial Mediterranean Fever: a case report. Pediatric Rheumatology 2013 11(Suppl 2):P187. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit
https://openalex.org/W4291584776	https://zenodo.org/records/3837426/files/STRPDUS_2015_3_402-406.pdf	Ukrainian	null	Соціальні мережі, як спосіб деструктивного впливу спецслужбами Російської Федерації на свідомість молоді	Zenodo (CERN European Organization for Nuclear Research)	2,015	cc-by	1,377	ЧЕРНИШ Р.Ф. к.ю.н., доцент кафедри правознавства ЖНАЕУ ЧЕРНИШ Р.Ф. к.ю.н., доцент кафедри правознавства ЖНАЕУ ЧЕРНИШ Р.Ф. к.ю.н., доцент кафе СОЦІАЛЬНІ МЕРЕЖІ, ЯК СПОСІБ ДЕСТРУКТИВНОГО ВПЛИВУ СПЕЦСЛУЖБАМИ РОСІЙСЬКОЇ ФЕДЕРАЦІЇ НА СВІДОМІСТЬ МОЛОДІ В даний час соціальні мережі набули великої популярності як на території України, так і в усьому світі. В них майже щодня реєструються тисячі нових користувачів мережі Інтернет. Соціальна мережа - це спільнота людей, об’єднаних однаковими інтересами, уподобаннями, або тих, що мають інші причини для безпосереднього спілкування між собою. Вказаний термін було введено в 1954 році соціологом Джеймсом Барнсом. Перша соціальна мережа в Інтернеті з’явилася в 1995 році. Це був американський портал classmates.com (соціальна спільнота “Однокласники” є аналогом вказаного сайту). 402 Однак, офіційним початком життя соціальних мереж прийнято вважати 2003-2004 роки. Саме в цей період було запущено такі ресурси як: Linkedln, MySpace і Facebook. Сучасні Інтернет-сервіси забезпечують користувачів усіма можливими інструментами для спілкування одне з одним - відео, чати, зображення, музика, блоги, форуми тощо. Для бізнесу ж соціальні мережі виступають новим каналом комунікації із споживачем та інструментом дослідження уподобань аудиторії. Як відомо, метою створення соціальних мереж є, передусім, спілкування людей, але сьогодні ми спостерігаємо зовсім іншу картину: мережі наповнені непотрібною рекламою та непристойним матеріалом. Засновники мереж створюють їх, перш за все, з комерційною метою (рекламодавці платять гроші за показ контекстної реклами), а вже потім для спілкування людей. Однак, окрім видимої шкоди, соціальні мережі містять невидиму, яка особливо активізувалася в ході проведення АТО на сході України - це накопичення та протиправне використання інформації про користувачів представниками спецслужб РФ. Зазначена діяльність здійснюється з метою впливу на свідомість і підсвідомість громадян (насамперед молоді) та формування у них антигромадської позиції (недовіри до чинної влади, правоохоронних органів тощо), а також спонукання до вчинення актів непокори чи інших протиправних дій, які можуть призвести як до дестабілізації суспільно-політичної ситуації всередині країни, так і до нанесення шкоди іміджу держави у сфері зовнішньополітичних відносин. В умовах сьогодення значної актуальності набуло питання виявлення та блокування сепаратистських інтернет-ресурсів, які використовуються представниками терористичних організацій ДНР/ЛНР та особами, які їх підтримують, з метою поширення соціально небезпечної інформації. Вказані ресурси виступають інструментом пропаганди ідеології, що закликає до повалення конституційного ладу в Україні, відокремлення її окремих територій, екстремізму та тероризму. Основну небезпеку становить те, що вони негативно впливають на свідомість громадян та поступово її змінюють. Зазначене призводить до того, що особи, які піддалися впливу, у випадку інтервенції з боку держави-агресора (на прикладі анексії РФ території АР Крим та участі громадян України в бойових діях на Сході України в складі терористичних підрозділів) не дивлячись на необхідність виконання конституційного обов’язку (захист територіальної цілісності), виступають на стороні противника. СОЦІАЛЬНІ МЕРЕЖІ, ЯК СПОСІБ ДЕСТРУКТИВНОГО ВПЛИВУ СПЕЦСЛУЖБАМИ РОСІЙСЬКОЇ ФЕДЕРАЦІЇ НА СВІДОМІСТЬ МОЛОДІ До поширення інформації сепаратистського змісту представниками спецслужб РФ залучаються окремі громадяни, телевізійні телеканали (“Россия 1”, “Life News” тощо) та ін., журналісти яких націлені на здобування інформації деструктивного характеру або її відображення в необ’єктивному ракурсі. Однак, зважаючи на стрімке збільшення кількості активних інтернет- 403 користувачів, найбільш поширеним способом донесення протиправної інформації вищевказаного характеру є використання Інтернет-ресурсів: веб- сторінок та соціальних спільнот (є найбільш небезпечними, так як їх аудиторію складає переважно молодь, яка швидше піддається негативному впливу). користувачів, найбільш поширеним способом донесення протиправної інформації вищевказаного характеру є використання Інтернет-ресурсів: веб- сторінок та соціальних спільнот (є найбільш небезпечними, так як їх аудиторію складає переважно молодь, яка швидше піддається негативному впливу). На Сході країни існують два центри сепаратистського руху. Це, так звані, незаконні та невизнанні псевдореспубліки: “Донецька народна республіка” (надалі ДНР) та “Луганська народна республіка” (надалі ЛНР). Нижче наведена орієнтовна схема розповсюдження інформації сепаратистського призначення за допомогою веб-сторінок. д р р д ф р ц сепаратистського призначення за допомогою веб-сторінок. Сепаратистська інформація rusvesna.su “lugansk-online.info” - офіційний сайт нелегітимного уряду “ЛНР”. Сторінка представляє собою портал, на якому розміщуються новини регіону, в основному, про успіхи терористичних дій представників псевдо уряду. Також, на порталі публікується неправдиві новини про ситуацію в країні. На веб- сторінці відсутня можливість користувачів прокоментувати новину чи додати свою інформацію. “antimaydan. info” - сторінка, яка є найбільшим осередком пропаганди суспільно небезпечної інформації. Була створена у період початку Євромайдану в м. Києві і за цей час змогла сформувати значну аудиторію користувачів. Увесь масив інформації направлений на приниження та глузування з української державності. Ресурс небезпечний тим, що має значну кількість читачів, з числа яких ведеться набір до лав терористів. Для цього на 404 Сепаратистська інформація rusvesna.su “lugansk-online.info” - офіційний сайт нелегітимного уряду “ЛНР”. Сторінка представляє собою портал, на якому розміщуються новини регіону, в основному, про успіхи терористичних дій представників псевдо уряду. Також, на порталі публікується неправдиві новини про ситуацію в країні. На веб- сторінці відсутня можливість користувачів прокоментувати новину чи додати свою інформацію. “antimaydan. info” - сторінка, яка є найбільшим осередком пропаганди суспільно небезпечної інформації. Була створена у період початку Євромайдану в м. Києві і за цей час змогла сформувати значну аудиторію користувачів. Увесь масив інформації направлений на приниження та глузування з української державності. Ресурс небезпечний тим, що має значну кількість читачів, з числа яких ведеться набір до лав терористів. Для цього на 404 сторінці функціонує розділ “Как вступить в ополчение Новороссии, Донбасса, ДНР, ЛНР”. сторінці функціонує розділ “Как вступить в ополчение Новороссии, Донбасса, ДНР, ЛНР”. СОЦІАЛЬНІ МЕРЕЖІ, ЯК СПОСІБ ДЕСТРУКТИВНОГО ВПЛИВУ СПЕЦСЛУЖБАМИ РОСІЙСЬКОЇ ФЕДЕРАЦІЇ НА СВІДОМІСТЬ МОЛОДІ “dnr-news.com” - портал, на якому активно висвітлюються лише негативні або спотворенні новини. Головною особливістю даного ресурсу є те, що окрім загального функціоналу сайту, тут знаходяться посилання на он-лайн телебачення та радіо-канал сепаратистів. Коментування новин та статей на сайті не передбачено, але є функція їх перепосту в соціальні мережі. “dnr.today” - Інтернет-ресурс, який представляє офіційний сайт “ДНР”. Новини, що публікуються, в більшості випадків стосуються розпоряджень, рішень та указів нелегітимного уряду “ДНР”. Зокрема, його можна розцінювати, як інструмент “легітимізації” псевдоуряду. Основна мета - продемонструвати населенню захоплених терористами територій, що вказаний “уряд” повноцінно діє та виконує державні функції. “dnr.today” - Інтернет-ресурс, який представляє офіційний сайт “ДНР”. Новини, що публікуються, в більшості випадків стосуються розпоряджень, рішень та указів нелегітимного уряду “ДНР”. Зокрема, його можна розцінювати, як інструмент “легітимізації” псевдоуряду. Основна мета - продемонструвати населенню захоплених терористами територій, що вказаний “уряд” повноцінно діє та виконує державні функції. За результатами аналізу встановлено, що найбільшого розмаху пропаганда сепаратистської інформації досягла на сторінках соціальної мережі “Вконтакте”. Зважаючи на те, що зазначена мережа заснована громадянами РФ і фактично підконтрольна спеціальним службам Російської Федерації (зокрема, ФСБ), її власники безперешкодно та ефективно реалізують проросійську політику. Оскільки адміністрація мережі жодним чином не протидіє розповсюдженню антидержавних закликів, сторінки окремих сепаратистськи налаштованих Інтернет-груп (“ДЫР МЫ РУССКИЕ ЛНР”, “Безлер Инфо”, “ANNA-News \| NewsFront \| ДНР \| ЛНР” тощо), набули значної популярності (загальна кількість учасників становить понад 500 тис. осіб; 90 % аудиторії складає переважно молодь, яка швидше піддається негативному впливу). За наявними даними, другою, і не менш значущою за розмірами спільнотою сепаратистського спрямування, є соціальна мережа “Одноклассники”. Три її найбільші спільноти сепаратистського спрямування об’єднують навколо себе майже 200 тисяч користувачів (“Новости Мира. Славянск, Мариуполь ДНР, ЛНР”, “Сводки от ополчения Новороссии”, “Последние новости!”). Загальна аудиторія користувачів зазначених груп в соціальній мережі складає орієнтовно понад 500 тис. осіб. Також, в серпні місяця 2014 року Прем’єр-міністр Росії Дмитро Медведев підписав постанову про контроль за діяльністю організаторів поширення інформації в мережі “Інтернет”. Зокрема, Роскомнадзор тепер має право перевіряти особисте листування користувачів соціальних мереж, серед яких - “Однокласники” і “ВКонтакте”. Крім того, відомство може встановлювати контроль за електронною поштою, месенджерами Агент.МаіІ.т, ICQ, а також блог-платформами. Відомство має право встановлювати факт прийому, передачі, доставки та обробки повідомлень, переглядати і аналізувати ресурси організатора поширення інформації, а також записувати і фіксувати дії, доступні 405 користувачам. Зважаючи на викладене, особливої актуальності набуває питання можливості блокування сепаратистських ресурсів. Наприклад, аналогічна процедура вже майже рік діє на території РФ. СОЦІАЛЬНІ МЕРЕЖІ, ЯК СПОСІБ ДЕСТРУКТИВНОГО ВПЛИВУ СПЕЦСЛУЖБАМИ РОСІЙСЬКОЇ ФЕДЕРАЦІЇ НА СВІДОМІСТЬ МОЛОДІ Роскомнадзор негайно на підставі відповідного запиту генпрокурора або його заступників (без відповідного рішення суду) може блокувати інтернет- ресурси, на яких розміщено заклики до тероризму та порушення територіальної цілісності тощо. Процедура ініціюється на підставі моніторингу інтернету, а також в ході реагування на повідомлення, які надійшли від органів державної влади, організацій та громадян. Отже, підсумовуючи вищевикладене, керуючись необхідністю організації ефективної та дієвої протидії агресору (в т.ч. й в інформаційному просторі), вважається за доцільне, на рівні відповідних органів виконавчої влади (зокрема Інтернет-асоціації України) ініціювати прийняття законопроекту, який дозволить на законних підставах на час проведення АТО за спрощеною процедурою (без обов’язкового отримання рішення суду) блокувати можливість перегляду на території України інтернет-ресурсів, діяльність яких загрожує територіальній цілісності України та безпеці її громадян (не зважаючи на місце їх реєстрації чи фізичне розташування технічного обладнання).
https://openalex.org/W2162615325	https://europepmc.org/articles/pmc3578208?pdf=render	English	null	PANDA: a pipeline toolbox for analyzing brain diffusion images	Frontiers in human neuroscience	2,013	cc-by	10,088	Edited by: Reviewed by: R. Matthew Hutchison, Western University, Canada Christopher J. Steele, Max Planck Institute for Human Cognitive and Brain Sciences, Germany Correspondence: Gaolang Gong, State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, #19 Xinjiekouwai Street, Beijing 100875, China. e-mail: gaolang.gong@bnu.edu.cn INTRODUCTION or subject. In addition, this pattern requires a large amount of manual operation, which potentially increases the possibility of processing errors caused by manual mistakes. To date, a tool- box supporting fully automated processing of raw dMRI datasets to diffusion metrics that are ready for statistical analysis is still lacking. Diffusion magnetic resonance imaging (dMRI) has become one of the most popular MRI techniques for brain research. dMRI can be used to quantify white matter (WM) property and to virtually reconstruct WM pathways in the living brain (Le Bihan, 2003). Given its unique merits, dMRI has been extensively applied to the study of WM connectivity in both normal and abnormal condi- tions, leading to a substantial enhancement in our understanding of the role of WM, particularly in brain diseases (Johansen-Berg and Rushworth, 2009). Another popular application of dMRI is to virtually recon- struct WM tracts, referred to as diffusion tractography (Mori et al., 1999; Behrens et al., 2007). Previous studies using dif- fusion tractography mainly focus on a few speciﬁc WM tracts. Recently, accurately constructed entire brain anatomical networks (i.e., the connectome) based on diffusion tractography have attracted a lot of attention (Behrens and Sporns, 2012) and are the key target of the ongoing human connectome project (http:// humanconnectome.org/). While the framework for constructing anatomical networks of the human brain (i.e., deﬁnition of net- work nodes and edges) has been established (Hagmann et al., 2008; Gong et al., 2009a,b), it is mainly implemented in-house. The community is in urgent need of a fully automated public tool that can construct anatomical brain networks using dMRI datasets. One popular application of dMRI is to extract various diffu- sion metrics [e.g., fractional anisotropy (FA) and mean diffusivity (MD)] that putatively reﬂect WM integrity (Basser and Pierpaoli, 1996; Pierpaoli and Basser, 1996; Beaulieu, 2002). These metrics can be further applied to identify differences in WM integrity across subjects. To perform this type of analysis, multiple sequen- tial image-processing steps (e.g., eddy-current correction, tensor calculation, metric calculation, and normalization) are required. ORIGINAL RESEARCH ARTICLE bli h d 21 F b 2013 ORIGINAL RESEARCH ARTICLE bli h d 21 F b 2013 Frontiers in Human Neuroscience Keywords: PANDA, diffusion MRI, DTI, pipeline, diffusion metrics, structural connectivity, network, connectome PANDA: a pipeline toolbox for analyzing brain diffusion i Zaixu Cui, Suyu Zhong , Pengfei Xu, Yong He and Gaolang Gong State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, Beijing, China Diffusion magnetic resonance imaging (dMRI) is widely used in both scientiﬁc research and clinical practice in in-vivo studies of the human brain. While a number of post-processing packages have been developed, fully automated processing of dMRI datasets remains challenging. Here, we developed a MATLAB toolbox named “Pipeline for Analyzing braiN Diffusion imAges” (PANDA) for fully automated processing of brain diffusion images. The processing modules of a few established packages, including FMRIB Software Library (FSL), Pipeline System for Octave and Matlab (PSOM), Diffusion Toolkit and MRIcron, were employed in PANDA. Using any number of raw dMRI datasets from different subjects, in either DICOM or NIfTI format, PANDA can automatically perform a series of steps to process DICOM/NIfTI to diffusion metrics [e.g., fractional anisotropy (FA) and mean diffusivity (MD)] that are ready for statistical analysis at the voxel-level, the atlas-level and the Tract-Based Spatial Statistics (TBSS)-level and can ﬁnish the construction of anatomical brain networks for all subjects. In particular, PANDA can process different subjects in parallel, using multiple cores either in a single computer or in a distributed computing environment, thus greatly reducing the time cost when dealing with a large number of datasets. In addition, PANDA has a friendly graphical user interface (GUI), allowing the user to be interactive and to adjust the input/output settings, as well as the processing parameters. As an open-source package, PANDA is freely available at http:// www.nitrc.org/projects/panda/. This novel toolbox is expected to substantially simplify the image processing of dMRI datasets and facilitate human structural connectome studies. Edited by: Hauke R. Heekeren, Freie Universität Berlin, Germany Reviewed by: R. Matthew Hutchison, Western University, Canada Christopher J. Steele, Max Planck Institute for Human Cognitive and Brain Sciences, Germany Correspondence: Gaolang Gong, State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, #19 Xinjiekouwai Street, Beijing 100875, China. e-mail: gaolang.gong@bnu.edu.cn HUMAN NEUROSCIENCE published: 21 February 2013 doi: 10.3389/fnhum.2013.00042 published: 21 February 2013 doi: 10.3389/fnhum.2013.00042 Edited by: Hauke R. Heekeren, Freie Universität Berlin, Germany Reviewed by: R. Matthew Hutchison, Western University, Canada Christopher J. Steele, Max Planck Institute for Human Cognitive and Brain Sciences, Germany Correspondence: Gaolang Gong, State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, #19 Xinjiekouwai Street, Beijing 100875, China. e-mail: gaolang.gong@bnu.edu.cn Correspondence: Gaolang Gong, State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University, #19 Xinjiekouwai Street, Beijing 100875, China. e-mail: gaolang.gong@bnu.edu.cn Preprocessing Converting DICOM ﬁles into NIfTI images. The input ﬁles of PANDA can be in either DICOM or NIfTI format. If the input ﬁles are in NIfTI format, this conversion step will be skipped. Otherwise, DICOM ﬁles will be converted into NIfTI format during this step. The dcm2nii tool embedded in MRIcron accom- plished this task. Estimating the brain mask. This step yields the brain mask by using the bet command of FSL (Smith, 2002). The brain mask is required for the subsequent processing steps. Here, the b0 image without diffusion weighting was used for the estimation. Here, we present a MATLAB toolbox named PANDA (a Pipeline for Analyzing braiN Diffusion imAges) for a com- prehensive pipeline processing of dMRI dataset, aiming to facilitate image processing for the across-subject analysis of diffusion metrics and brain network constructions. Of note, the processing pipelines in this toolbox have been com- pletely set up, allowing the end-users of dMRI to process the data immediately. Moreover, the processing procedures within this pipeline were carefully designed to follow the recom- mended practice as possible (Jones et al., 2012). After the user sets the input/output and processing parameters through the friendly graphical user interface (GUI), PANDA fully auto- mates all processing steps for datasets of any number of sub- jects, and results in data pertaining to many diffusion metrics that are ready for statistical analysis at three levels (Voxel- level, ROI-level, and TBSS-level). Additionally, anatomical brain networks can be automatically generated using either deter- ministic or probabilistic tractography techniques. Particularly, PANDA can run processing jobs in parallel with multiple cores either in a single computer or within a distributed com- puting environment using a Sun Grid Engine (SGE) system, thus allowing for maximum usage of the available computing resources. Cropping the raw images. To reduce the memory cost and speed up the processing in subsequent steps we cut off the non-brain space in the raw images, leading to a reduced image size. The acquired brain mask was used to determine the borders of the brain along the three dimensions. The fslroi command of FSL was then applied to remove the non-brain spaces. Correcting for the eddy-current effect. Eddy-current induced distortion of diffusion weighted images (DWI), as well as sim- ple head-motion during scanning, can be corrected by registering the DW images to the b0 image with an afﬁne transformation. PANDA PROCESSING PROCEDURES Essentially, these packages provide environments for constructing analysis workﬂows with a number of pre-included processing modules from existing tools (e.g., Camino, FSL, AFNI, FreeSurfer, and SPM), and therefore various automated processing pipelines (e.g., a dMRI processing pipeline) can be developed within these environments. In order to construct pipelines with these packages, users need to choose processing modules and deﬁne dependencies and parameters themselves. It is noted that, if particular processing modules are not encapsulated [e.g., JIST does not include Tract-Based Spatial Statistics (TBSS) analysis], users have to develop their own modules and further incor- porate them into the environment. While these powerful and sophisticated packages make it possible to generate a dMRI processing pipeline, they are favored by developers, and not end users without programming skills. A ready-for-use pipeline tool for dMRI processing is highly desired, particularly for end users. The main procedure of PANDA is shown in Figure 1 and includes three steps: (1) preprocessing; (2) producing diffusion metrics (ready for statistical analysis); and (3) constructing networks. Edited by: Currently, a number of packages, such as FMRIB Software Library (FSL) (Smith et al., 2004) and DTI-Studio (Jiang et al., 2006), provide a set of functions that can carry out these jobs. However, these packages typically perform the processing step-by-step and subject-by-subject. Obviously, this processing pattern is inefﬁ- cient, as users have to wait until the preceding steps or until each subject is completely ﬁnished before initiating the next step Currently, there have been a few packages such as MIPAV (McAuliffe et al., 2001), JIST (Lucas et al., 2010), Nipype (Gorgolewski et al., 2011), and LONI (Dinov et al., 2009), which aim to facilitate automated processing of neuroimaging dataset. February 2013 \| Volume 7 \| Article 42 \| 1 www.frontiersin.org www.frontiersin.org www.frontiersin.org A pipeline for diffusion MRI Cui et al. Preprocessing To achieve this, the ﬂirt command of FSL was used. Notably, this registering procedure was applied to all images, with the b0 image of ﬁrst acquisition used as the target if multiple DWI acquisitions were scanned. It is worth mentioning that while the eddy_correct command of FSL is not called here, the result of this step is exactly the same as the output of eddy_correct. Basically, PANDA just splits the 4D ﬁle (the input ﬁle of eddy_correct) into a number of 3D ﬁles and then performed the afﬁne-registration exactly like eddy_correct. The purpose of this implementation is to avoid the large memory demand when the 4D ﬁle size is huge. Finally, the gradient direction of each DWI volume was rotated according to the resultant afﬁne transformations (Leemans and Jones, 2009). To assess the usability and validity of PANDA, we apply PANDA to study the age effect (i.e., old vs. young) on the dif- fusion metrics of WM as well as the topological properties of the WM network. According to previous ﬁndings, decreased WM anisotropy and weakened network efﬁciency are expected in old individuals. Averaging multiple acquisitions. This step will be skipped if there is only one DWI acquisition. Otherwise, after eddy-current correction, the aligned multiple DWI was averaged by calling the fslmaths command of FSL. Averaging multiple acquisitions. This step will be skipped if there is only one DWI acquisition. Otherwise, after eddy-current correction, the aligned multiple DWI was averaged by calling the fslmaths command of FSL. Calculating diffusion tensor (DT) metrics. This step involves a voxel-wise calculation of the tensor matrix and the DT metrics, including FA, MD, axial diffusivity (AD), and radial diffusivity (RD) (Pierpaoli and Basser, 1996; Song et al., 2002). The dtiﬁt command of FSL was applied. Frontiers in Human Neuroscience MATERIALS AND METHODS PANDA was developed by using MATLAB under an Ubuntu Operating System. A number of processing functions from FSL (Smith et al., 2004), Pipeline System for Octave and Matlab (PSOM) (Bellec et al., 2012), Diffusion Toolkit (Wang et al., 2007), and MRIcron (http://www.mccauslandcenter. sc.edu/mricro/mricron/) were called by PANDA. Here, we will describe the procedures of pipeline processing in PANDA, followed by an introduction to the realization of pipelines. Producing diffusion metrics that are ready for statistical analysis Producing diffusion metrics that are ready for statistical analysis Normalizing. To allow for comparison across subjects, location correspondence has to be established. To end this, registra- tion of all the individual images to a standardized template is always applied. Here, PANDA non-linearly registered individual Producing diffusion metrics that are ready for statistical analysis Normalizing. To allow for comparison across subjects, location correspondence has to be established. To end this, registra- tion of all the individual images to a standardized template is always applied. Here, PANDA non-linearly registered individual February 2013 \| Volume 7 \| Article 42 \| 2 Frontiers in Human Neuroscience www.frontiersin.org Cui et al. A pipeline for diffusion MRI FIGURE 1 \| Main procedure for pipeline processing of dMRI datasets in PANDA. The procedure includes three parts: (1) preprocessing; (2) producing diffusion metrics that are ready for statistical analysis; and (3) constructing networks. FIGURE 1 \| Main procedure for pipeline processing of dMRI datasets in PANDA. The procedure includes three parts: (1) preprocessing; (2) producing diffusion metrics that are ready for statistical analysis; and (3) constructing networks. a given Gaussian kernel, which was realized by calling the fslmaths command of FSL. The smoothed diffusion metric images can then be directly used for voxel-based statistical analysis with any preferred tools, e.g., FSL (http://www.fmrib.ox.ac.uk/fsl/), SPM (http://www.ﬁl.ion.ucl.ac.uk/spm/), or AFNI (http://afni.nimh. nih.gov/afni/). FA images of native space to the FA template in the MNI space by calling the fnirt command of FSL. The resultant warping transfor- mations were then used to resample the images of the diffusion metrics (i.e., FA, MD, AD, and RD) into the MNI space with a customized spatial resolution (e.g., 1 × 1 × 1 mm or 2 × 2 × 2 mm). This resampling step was implemented by the applywarp command of FSL. Output for atlas-based analysis. In addition to the popular voxel-based method of analysis, diffusion metrics can be ana- lyzed at the level of region of interests (ROI), which may provide better statistical sensitivity in some cases (Faria et al., 2010). Recently, a few WM atlases (e.g., the ICBM-DTI-81 WM labels atlas and the JHU WM tractography atlas) have been proposed (Mori et al., 2008). These WM atlases in the standard space Output for voxel-based analysis. The resultant images of the dif- fusion metrics in the standard space are ready for voxel-based statistical analysis. Producing diffusion metrics that are ready for statistical analysis However, in the framework of voxel-based analysis, these images are typically smoothed to some degree, which can reduce the effect of image noise and misalignment between subjects. Accordingly, PANDA smoothed the images with February 2013 \| Volume 7 \| Article 42 \| 3 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Cui et al. A pipeline for diffusion MRI allow for parcellation of the entire WM into multiple ROIs, each representing a labeled region in the atlas. To support ROI- based analysis, PANDA calculates the regional diffusion metrics (i.e., FA, MD, AD, and RD) by averaging the values within each region of the WM atlases. These resultant ROI-based data (saved as text ﬁles) can be statistically analyzed with SPSS (http:// www-01.ibm.com/software/analytics/spss/) and other statistical packages. individual images with data on the skeleton were created. The resultant images can be directly used for voxel-wise statistical analysis on the skeleton. Here, the fslmaths and tbss_skeleton commands of FSL were employed. Constructing networks Two basic elements are required for a network: a node and a con- nection. Thus, the central tasks for constructing brain networks are: (1) deﬁning network nodes and (2) deﬁning connections between nodes. The schematic ﬂowchart of network construction is demonstrated in Figure 2. Output for TBSS-based analysis. The TBSS framework avoids the necessity of choosing a spatial smoothing procedure during voxel-based analysis and may provide better sensitivity and inter- pretability when it is applied to multi-subjects dMRI datasets (Smith et al., 2006). To support this type of analysis, PANDA follows the standard TBSS framework. Firstly, the mean of all the aligned FA images was created and skeletonized, resulting in a mean FA skeleton. Secondly, the diffusion metric data from individual subjects were projected onto the skeleton. Finally, Deﬁning network nodes. Typically, the entire brain is divided into multiple regions using a prior gray matter (GM) atlas, where each region represents a network node (Bullmore and Sporns, 2009). However, the prior atlases are generally deﬁned in the stan- dard space and need to be transformed to the native dMRI space of each individual. To address this, PANDA uses the framework FIGURE 2 \| Flowchart for constructing anatomical brain networks using diffusion tractography in PANDA. (A) White matter tracts reconstructed using deterministic tractography. (B) Parcellation of gray matter in diffusion space. Each color represents a node in a brain network. (C) White matter connectivity maps using FSL probabilistic tractography. (D) Three resultant network matrices weighted by ﬁber number, averaged length, and averaged FA. (E) The network matrix weighted by connectivity probability. FIGURE 2 \| Flowchart for constructing anatomical brain networks using diffusion tractography in PANDA. (A) White matter tracts reconstructed using deterministic tractography. (B) Parcellation of gray matter in diffusion space. Each color represents a node in a brain network. (C) White matter connectivity maps using FSL probabilistic tractography. (D) Three resultant network matrices weighted by ﬁber number, averaged length, and averaged FA. (E) The network matrix weighted by connectivity probability. FIGURE 2 \| Flowchart for constructing anatomical brain networks using diffusion tractography in PANDA. (A) White matter tracts reconstructed using deterministic tractography. (B) Parcellation of gray matter in diffusion space. Each color represents a node in a brain February 2013 \| Volume 7 \| Article 42 \| 4 Frontiers in Human Neuroscience Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org A pipeline for diffusion MRI Cui et al. Frontiers in Human Neuroscience REALIZATION OF PIPELINES PSOM is a ﬂexible framework for the implementation of pipelines in the form of Octave or Matlab scripts (Bellec et al., 2012), and was employed to build up the processing pipeline in our study. Here, a pipeline refers to a collection of jobs with a well identiﬁed set of options that use ﬁles for inputs and outputs. The entire processing ﬂow of PANDA includes 41 steps, each of which is a job within the PANDA pipeline. Notably, more steps can be added if new functions or processing steps are included. The workﬂow of the current PANDA pipeline show- ing all the jobs and their associated dependencies is illustrated in Appendix A. Constructing networks using deterministic tractography. In gen- eral, deterministic tractography assumes a deterministic ﬁber orientation at every location during tracking, typically ending up with 3D trajectories for reconstructed WM tracts. Here, the dti_recon and dti_tracker commands of the Diffusion Toolkit (http://trackvis.org/dtk/) were called to reconstruct all possible ﬁbers within the brain by seeding from all the WM voxels. For every pair of brain nodes/regions deﬁned above, ﬁbers with two end-points located in their respective masks were considered to link the two nodes. Based on the linking ﬁbers, PANDA calculated three basic weighted matrices: number-weighted matrix (MN), FA-weighted matrix (MFA), and length-weighted matrix (ML). In the matrices, each row or column represents a brain region/node. The values of the elements M(i, j)N, M(i, j)FA, and M(i, j)L represent the number, averaged FA and averaged length of link- ing ﬁbers between node i and node j, respectively. The resultant matrices were saved as a MATLAB data ﬁle and can be directly used for topological analysis with graph theoretic approaches (Bullmore and Sporns, 2009; Bullmore and Bassett, 2011). In particular, PANDA was designed to allow for jobs running in parallel either on a single computer with multiple cores or on a computing cluster. Notably, the PANDA processing steps are parallelizable at multiple levels. For example, the same pro- cessing steps (i.e., preprocessing) for a group of subjects can be parallelized, since the steps are independent across subjects. In addition, for the same subject, different processing steps without between-dependency such as producing diffusion met- rics and brain parcellation can be parallelized as well. Finally, a few very time-consuming steps (i.e., BedpostX and Probabilistic Tractography and Network Construction) have been internally par- allelized. The parallelizing strategies in PANDA are demonstrated in Figure 3. TESTING THE AGE EFFECT ON WM CONNECTIVITY BY USING PANDA Subjects Constructing networks using probabilistic tractography. In contrast, probabilistic tractography typically runs the tracking procedure many times, and ﬁber orientation is determined probabilistically. This type of tractography may improve tracking sensitivity, particularly for non-dominant ﬁbers. The probabilis- tic tractography proposed by Behrens et al. (2003, 2007) has been implemented in FSL and is called by PANDA for network construction. This process involves two steps as follows: The test included data from 23 young adults (males, 11; females, 12; age, 17–24 years) and 17 elderly individuals (males, 8; females, 9; age, 54–77 years). All subjects were recruited from the campus and the local community. Subjects with a history of neuro- logical or psychiatric disorders were excluded from this study. Written informed consent was obtained from each subject, and the protocol was approved by the Ethics Committee of the State Key Laboratory of Cognitive Neuroscience and Learning, Beijing Normal University. BedpostX. Using the Markov Chain Monte Carlo sampling technique, this module estimated the local probability distribu- tion of ﬁber direction at each voxel, a prerequisite for running subsequent probabilistic tractography (Behrens et al., 2003). In PANDA, bedpostX was realized by calling the xﬁbres command of FSL. Constructing networks proposed by Gong et al. (2009a). Speciﬁcally, the individual FA image in native space was co-registered to its corresponding struc- tural image (i.e., T1-weighted) using an afﬁne transformation. The individual structural image was then non-linearly registered to the ICBM152 template. Based on the resultant transforma- tions in these two steps, an inverse warping transformation from the standard space to the native dMRI space can be obtained. Prior atlases in the standard space were then inversely warped back to individual native space by applying this inverse transfor- mation. Currently, PANDA provides two well-deﬁned atlases: the Automated Anatomical Labeling (AAL) (Tzourio-Mazoyer et al., 2002) atlas and the Harvard-Oxford atlas (HOA) (http://www. cma.mgh.harvard.edu/fslatlas.html). Notably, users can import customized atlases into PANDA to deﬁne the network nodes. During this step, the ﬂirt, fnirt, inwarp, and applywarp commands of FSL were used. was called. The connectivity probability from the seed region i to another region j was deﬁned by the number of ﬁbers passing through region j divided by the total number of ﬁbers sampled from region i. The connectivity probability of each node to the other nodes within the brain network can be calculated by repeat- ing the tractography procedure for all nodes. This leads to an individual-speciﬁc weighted matrix, whose rows and columns represent the brain nodes and whose elements represent the con- nectivity probability between nodes. This matrix can also be directly used for various network analyses. MRI acquisition All scans were performed using the 3-T Siemens Tim Trio MRI scanner in the Imaging Center for Brain Research, Beijing Normal University. Diffusion MRI was acquired using a single-shot echo- planar imaging-based sequence with following parameters: cov- erage of the whole brain; slice thickness, 2 mm; no gap; 68 axial slices; repetition time (TR), 9000 ms; echo time (TE), 92 ms; ﬂip angle, 90◦; 66 non-linear diffusion weighting directions with Probabilistic Tractography and Network Construction. Network construction using FSL-based probabilistic tractography has been previously described (Gong et al., 2009b). Brieﬂy, for each deﬁned brain region/node, probabilistic tractography was performed by seeding from all voxels of this region. For each voxel, 5000 ﬁbers were sampled. To achieve this, the probtrackx command of FSL February 2013 \| Volume 7 \| Article 42 \| 5 www.frontiersin.org www.frontiersin.org www.frontiersin.org Cui et al. A pipeline for diffusion MRI FIGURE 3 \| The schematic parallelizing strategy of PANDA. For example, pre-processing steps in Stage 1 are parallelizable across subjects. Independent processing steps from the same subject or across subjects in Stage 2 and Stage 3 can be parallelized as well. In addition, BedpostX and Probabilistic Network Construction have been internally parallelized, as indicated by orange boxes. Stage 2 and Stage 3 can be parallelized as well. In addition, BedpostX and Probabilistic Network Construction have been internally parallelized, as indicated by orange boxes. FIGURE 3 \| The schematic parallelizing strategy of PANDA. For example, pre-processing steps in Stage 1 are parallelizable across subjects. Independent processing steps from the same subject or across subjects in topological network parameters: global efﬁciency and local efﬁ- ciency. Global efﬁciency was deﬁned as the average of the inverse of the “harmonic mean” of the characteristic path length, which represents global information transferring ability within the net- work (Latora and Marchiori, 2001). Local efﬁciency quantiﬁes the ability of the network fault tolerant, corresponding to the efﬁciency of the information ﬂow between nodal neighbors. Speciﬁcally, local efﬁciency was deﬁned as the average of nodal local efﬁciency that is computed as the global efﬁciency of the sub- graph composed by its nearest neighbors (Latora and Marchiori, 2001). b = 1000 s/mm2 and one image without diffusion weighting (i.e., b = 0 s/mm2); 4 repetitive acquisitions; acquisition matrix, 128 × 124; ﬁeld of view (FOV), 256 × 248 mm2; resolution, 2 × 2 × 2 mm. Statistical analysis For diffusion metric, we tested the group difference on FA across the entire WM. Speciﬁcally, normalized and smoothed (6 mm Gaussian kernel) FA images produced by PANDA were employed for this voxel-based analysis. A general linear model (GLM) with gender being taken as a covariate was applied to each WM voxel. For multiple comparison correction, false discovery ratio (FDR) was applied, and p < 0.01 was considered as signiﬁcant. Image processing The whole pipeline procedure of PANDA was run on all dMRI datasets with an in-house computing cluster of 6 nodes, each with 30GB of memory and 12 Intel Xeon E5649 2.53 GHz cores. For each pipeline step, default parameters were chosen. MRI acquisition Three-dimensional T1-weighted images with high reso- lution were obtained using a three-dimensional magnetization prepared rapid gradient echo (MP-RAGE) sequence with the fol- lowing parameters: 1 mm slice thickness without gap; 176 sagittal slices; TR, 1900 ms; TE, 3.44 ms; ﬂip angle, 9◦; acquisition matrix, 256 × 256; FOV, 256 × 256 mm2; resolution, 1 × 1 × 1 mm. Network topology Graph theoretical approaches have been applied to characterize the topology of brain networks that are derived from neuroimag- ing data (Bullmore and Sporns, 2009). Here, we focus on two February 2013 \| Volume 7 \| Article 42 \| 6 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Cui et al. A pipeline for diffusion MRI pipeline processing for any number of subjects, after raw dMRI datasets are loaded into the program. This running mode will ﬁn- ish all processing steps and end up with all outputs as described in “Materials and Methods.” In contrast, the utilities can be used separately for speciﬁc processing steps (e.g., DICOM conversion, TBSS, and brain parcellation). Particularly, PANDA has a very friendly GUI (Figure 4), with which users can perform various interactions with the embedded functions, e.g., setting inputs or outputs and conﬁguring the processing parameters. In addition, PANDA can provide the status of the ongoing pipeline process- ing in real-time, allowing users to monitor progress through the GUI. The detailed descriptions for GUIs of PANDA are included in Appendix B. For each subject, the FA-weighted matrix generated from PANDA was selected for topological analysis. Each matrix is 78 × 78 and represents the WM network of cerebral cortex. Each row or column of the matrix represents a cortical region of the AAL template (Gong et al., 2009a,b). The global efﬁciency and local efﬁciency were then calculated. To test the group effect on the global and local efﬁciency, a GLM with gender and brain size as covariates was applied, and p < 0.05 was chosen as the signiﬁcant level. AN INTEGRATED MATLAB TOOLBOX: PANDA An integrated MATLAB toolbox named PANDA has been devel- oped for fully automated processing of dMRI datasets, which is an open-source package and is freely available at http://www. nitrc.org/projects/panda. An online discussion forum (http:// www.nitrc.org/forum/forum.php?forumid=2731) and a mail- ing list (http://www.nitrc.org/mailman/listinfo/panda-commits) have been registered for PANDA, and technical supports and updates will be constantly provided by the developers. Notably, PANDA has been packaged with PSOM, MRIcron, and Diffusion Toolkit. Only FSL is required to be installed separately. As provided by PSOM (Bellec et al., 2012), PANDA has a number of advantages as follows: (1) it can run jobs in parallel either in a single computer with multiple cores or in a comput- ing cluster; (2) it can generate log ﬁles and keep track of the pipeline execution; (3) if the program terminates before ﬁnish- ing, users can load a conﬁguration ﬁle, click “RUN,” and PANDA will restart from the termination point; (4) if users re-run the pipelines after changing some options, PANDA will only restart the procedures related to these options; and (5) the jobs will run in the background and PANDA & MATLAB can be closed after clicking the “RUN” button. Speciﬁcally, PANDA includes a main function and a set of sep- arate modules/utilities. Using the main function, PANDA can run FIGURE 4 \| A snapshot of the GUIs of PANDA. (A) The main GUI for loading dataset and monitoring job status. (B) The GUI for initiating separate utilities. FIGURE 4 \| A snapshot of the GUIs of PANDA. (A) The main GUI for loading dataset and moni the GUIs of PANDA. (A) The main GUI for loading dataset and monitoring job status. (B) The GUI for initiating separate utilitie February 2013 \| Volume 7 \| Article 42 \| 7 Frontiers in Human Neuroscience www.frontiersin.org A pipeline for diffusion MRI Cui et al. RESULTANT FILES OF PANDA for multiple subjects with multiple cores in PANDA can be effec- tively saved, due to the parallelized processing. For example, ﬁnishing the pre-processing steps for two subjects costs almost the same time as for one subject (Table 2). In addition, since the bedpostX has been parallelized internally, ﬁnishing bedpostX with eight cores cost only half of time as cost with four cores (Table 2). For each subject, PANDA generates six folders containing resul- tant ﬁles, as listed in Table 1. Speciﬁcally, the native_space folder consists of all images and ﬁles in the native space. The ﬁles in the quality_control folder include 2D snapshot pictures of FA, T1, normalized FA, and normalized T1, which can be quickly viewed to check the quality of the data and related registrations (Figure 5). All ﬁles of the diffusion metrics that are ready for sta- tistical analysis are stored in the folder named standard_space. The trackvis folder consists of resultant ﬁles generated by the “Diffusion Toolkit” for deterministic tractography, which can be opened with Trackvis. The native_space.bedpostx folder contains the resultant ﬁles of bedpostX that are required for FSL prob- abilistic tractography. Finally, the MATLAB ﬁles containing the network matrices with different weighting (i.e., ﬁber number, averaged FA, averaged length, and connectivity probability) are stored in the folder named network. THE AGE EFFECT ON WM CONNECTIVITY USING PANDA As expected, voxel-based comparison revealed a distributed FA decreases (p < 0.01, FDR corrected) throughout the brain in the old group. Speciﬁcally, FA was mainly affected in the bilat- eral superior longitudinal fasciculus, uncinate fasciculus, inter- nal capsules, external capsules, fornices, and corpus callosum (Figure 6). Moreover, we observed group differences in topological efﬁ- ciencies of WM network of cerebral cortex. As demonstrated in Figure 7, the global efﬁciency of the WM network showed a sig- niﬁcant reduction in the old group (p = 0.03) after controlling for gender and brain size, and the local efﬁciency exhibited only a trend of reduction (p = 0.16). TIME COST To provide information about the time cost of PANDA pro- cedures, a few baseline running-time tests were conducted. Speciﬁcally, two dMRI datasets with different acquisition schemes (dataset I: 64 directions, 4 repetitive acquisitions, resolution: 2 × 2 × 2 mm; dataset II: 30 directions, 2 repetitive acquisitions, resolution: 2.2 × 2.2 × 2.2 mm) were tested under four condi- tions (one subject with four cores; one subject with eight cores; two subjects with four cores; two subjects with eight cores). The results are listed in Table 2. Frontiers in Human Neuroscience DISCUSSION hhhhhhhhhhhhhhhhhhhhh Dataset II Dataset I Time cost (h) One subject One subject Two subjects Two subjects Four cores Eight cores Four cores Eight cores Preprocessing and producing data that is ready for statistical analysis PPPPPPP 0.167 0.883 PPPPPPP 0.150 0.900 PPPPPPP 0.217 0.917 PPPPPPP 0.183 0.900 Brain parcellation (for network node deﬁnition) PPPPPPP 0.133 0.167 PPPPPPP 0.133 0.167 PPPPPPP 0.183 0.167 PPPPPPP 0.183 0.150 Deterministic tractography and network construction (AAL template 90) PPPPPPP 0.017 0.067 PPPPPPP 0.017 0.050 PPPPPPP 0.033 0.050 PPPPPPP 0.033 0.067 BedpostX PPPPPPP 1.467 3.667 PPPPPPP 0.883 2.317 PPPPPPP 2.933 7.117 PPPPPPP 1.650 4.233 Probabilistic tractography and network construction (AAL template 90) PPPPPPP 3.283 6.017 PPPPPPP 1.917 3.683 PPPPPPP 6.583 11.883 PPPPPPP 3.633 6.750 The processing was performed using a local workstation with 30 GB of memory and Intel Xeon E5649 2.53 GHz cores. Four conditions were tested: one subject with four cores; two subjects with four cores; one subject with eight cores; two subjects with eight cores. Table 2 \| Baseline time cost of pipeline processing on dataset I (64 DWI directions, 4 repetitive acquisitions, resolution: 2 × 2 × 2 mm) and dataset II (30 DWI directions, 2 repetitive acquisitions, resolution: 2.2 × 2.2 × 2.2mm) with PANDA. Table 2 \| Baseline time cost of pipeline processing on dataset I (64 DWI directions, 4 repetitive acquisitions, resolution: 2 × 2 × 2 mm) and dataset II (30 DWI directions, 2 repetitive acquisitions, resolution: 2.2 × 2.2 × 2.2mm) with PANDA. FIGURE 6 \| The statistical map showing signiﬁcant FA decreases in old group (p < 0.01, FDR corrected). The hot color represents t values for the age effect. FIGURE 6 \| The statistical map showing signiﬁcant FA decreases in old group (p < 0.01, FDR corrected). The hot color represents t values for the age effect. FIGURE 6 \| The statistical map showing signiﬁcant FA decreases in old group (p < 0.01, FDR corrected). The hot color represents t values for the age effect. p showing signiﬁcant FA decreases in old group (p < 0.01, FDR corrected). The hot color represents t values for the age FIGURE 6 \| The statistical map showing signiﬁcant FA decreases in old group (p < 0.01, FDR corrected). The hot color effect. A fully automated pipeline naturally makes the data processing efﬁcient, at the same time reducing potential mistakes by avoid- ing manual processing of individual steps. DISCUSSION hhhhhhhhhhhhhhhhhhhhh Dataset II Dataset I Time cost (h) One subject One subject Two subjects Two subjects Four cores Eight cores Four cores Eight cores Preprocessing and producing data that is ready for statistical analysis PPPPPPP 0.167 0.883 PPPPPPP 0.150 0.900 PPPPPPP 0.217 0.917 PPPPPPP 0.183 0.900 Brain parcellation (for network node deﬁnition) PPPPPPP 0.133 0.167 PPPPPPP 0.133 0.167 PPPPPPP 0.183 0.167 PPPPPPP 0.183 0.150 Deterministic tractography and network construction (AAL template 90) PPPPPPP 0.017 0.067 PPPPPPP 0.017 0.050 PPPPPPP 0.033 0.050 PPPPPPP 0.033 0.067 BedpostX PPPPPPP 1.467 3.667 PPPPPPP 0.883 2.317 PPPPPPP 2.933 7.117 PPPPPPP 1.650 4.233 Probabilistic tractography and network construction (AAL template 90) PPPPPPP 3.283 6.017 PPPPPPP 1.917 3.683 PPPPPPP 6.583 11.883 PPPPPPP 3.633 6.750 The processing was performed using a local workstation with 30 GB of memory and Intel Xeon E5649 2.53 GHz cores. Four conditions were tested: one subject with four cores; two subjects with four cores; one subject with eight cores; two subjects with eight cores. FIGURE 6 \| The statistical map showing signiﬁcant FA decreases in old group (p < 0.01, FDR corrected). The hot color represents t values for the age effect. A fully automated pipeline naturally makes the data processing efﬁcient, at the same time reducing potential mistakes by avoid- ing manual processing of individual steps While constructing a Notably, there exist differences in the processing procedures across existing dMRI packages, and some important process- ing steps might be overlooked (Jones et al 2012) These issues Table 2 \| Baseline time cost of pipeline processing on dataset I (64 DWI directions, 4 repetitive acquisitions, resolution: 2 × 2 × 2 mm) and dataset II (30 DWI directions, 2 repetitive acquisitions, resolution: 2.2 × 2.2 × 2.2mm) with PANDA. DISCUSSION In this study, we have developed a MATLAB toolbox named PANDA for comprehensively processing dMRI datasets. The key advantage of PANDA is that it fully automates all the pro- cessing steps of dMRI datasets for any number of subjects. PANDA can yield diffusion metric data that is ready for statis- tical analysis at three levels (voxel-level, atlas-level, and TBSS- level), and can generate anatomical networks/matrices of the entire brain using either deterministic or probabilistic diffusion tractography. Obviously, the running time depends on dMRI scanning schemes. More DWI directions and more repetitive acquisitions will result in longer running time of preprocessing and bed- postX. Our results further demonstrated that the running-time FIGURE 5 \| Snapshot pictures for quality control of FA normalization. The normalized FA is overlaid with image edges that were derived from the FA template. These pictures can be quickly viewed to check the quality of normalization. Table 1 \| Folders produced by PANDA. Folder name Files native_space Text ﬁles of bvals and bvecs Native-space images of DWI, b0, brain mask, FA, MD, AD, RD, and parcellation mask quality_control Snapshot pictures of native FA, native T1, normalized FA, and normalized T1 standard_space Normalized images of FA, MD, AD, and RD (ready for voxel-based analysis) Text ﬁles of regional FA, MD, AD, and RD (ready for ROI-based analysis) Images of skeletonized FA, MD, AD, and RD (ready for TBSS analysis) trackvis Trackvis-related resultant ﬁles (for deterministic tractography) native_space bedpostx BedpostX-related resultant ﬁles (for probabilistic tractography) network MATLAB ﬁles containing network matrices weighted by ﬁber number, averaged FA, averaged length (from deterministic tractography), and connectivity probability (from probabilistic tractography) Table 1 \| Folders produced by PANDA. FIGURE 5 \| Snapshot pictures for quality control of FA normalization. The normalized FA is overlaid with image edges that were derived from the FA template. These pictures can be quickly viewed to check the quality of normalization. February 2013 \| Volume 7 \| Article 42 \| 8 Frontiers in Human Neuroscience Frontiers in Human Neuroscience www.frontiersin.org A pipeline for diffusion MRI Cui et al. Table 2 \| Baseline time cost of pipeline processing on dataset I (64 DWI directions, 4 repetitive acquisitions, resolution: 2 × 2 × 2 mm) and dataset II (30 DWI directions, 2 repetitive acquisitions, resolution: 2.2 × 2.2 × 2.2mm) with PANDA. Frontiers in Human Neuroscience DISCUSSION While constructing a dMRI processing pipeline with MIPAV (McAuliffe et al., 2001), JIST (Lucas et al., 2010), Nipype (Gorgolewski et al., 2011), or LONI (Dinov et al., 2009) is possible, it requires prior knowl- edge on pipeline design and programming skills related to these packages. In addition, knowledge on the details of all steps for processing dMRI dataset is required, which might be another challenge for end users. To provide a ready-for-use pipeline tool for end users, PANDA was developed, making it possible to process dMRI datasets immediately with established pipelines. Notably, there exist differences in the processing procedures across existing dMRI packages, and some important process- ing steps might be overlooked (Jones et al., 2012). These issues have been well discussed by a few recent articles (Jones and Cercignani, 2010; Jones et al., 2012). The processing pipelines of PANDA have tried to follow the best practice as possible. For example, the adjustment of diffusion gradient directions after eddy-current correction, which has been frequently missed (Leemans and Jones, 2009; Jones et al., 2012), has been included in the PANDA pipeline. In future versions, PANDA will keep being updated to include processing steps of the best practice at the moment. February 2013 \| Volume 7 \| Article 42 \| 9 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org www.frontiersin.org Cui et al. A pipeline for diffusion MRI FIGURE 7 \| The group comparison of network efﬁciency. The old group showed a signiﬁcant reduction of global efﬁciency and a trend of reduction in local efﬁciency. superior longitudinal fasciculus, external capsules, fornices and corpus callosum, which are highly consistent with previous ﬁnd- ings (Bennett et al., 2010; Michielse et al., 2010). In addition, signiﬁcant reduction of global efﬁciency and a trend of reduction of local efﬁciency were observed in the old group. These topolog- ical changes are largely compatible with our previous results that are based on a larger dataset (Gong et al., 2009b). The declined WM connectivity and topology may underlie various patterns of cognitive decline during normal aging. The results for this speciﬁc study prove the usability and validity of the PANDA processing. y p y y p g PANDA is of great applicability in the area of connectivity neuroscience. For example, this tool can be applied to dMRI datasets that are collected to study various connectivity hypothe- ses. engine for scientiﬁc workﬂows. Front. Neuroinform. 6:7. doi: 10.3389/fninf.2012.00007 DISCUSSION Also, the effects of dMRI processing parameters or steps on the ﬁnal connectivity results can be easily tested by using PANDA. Recently, the term “connectome” has been proposed to advocate efforts for comprehensively mapping and analyzing brain connectivity and networks (Sporns et al., 2005), and dMRI has been taken as a primary technique for structural macro- connectome (Behrens and Sporns, 2012). This will lead to a large number of dMRI datasets in the foreseeable future (http:// humanconnectome.org/). To process these connectome dataset, PANDA has unique advantages, as it can handle the large number of datasets very efﬁciently because of its parallelizing strategies. Meanwhile, it can automatically provide important metrics of interest (e.g., diffusion metrics of brain connectivity and brain network matrices) for connectome studies. Therefore, PANDA can potentially make contributions to the study of the human connectome in the near future. FIGURE 7 \| The group comparison of network efﬁciency. The old group showed a signiﬁcant reduction of global efﬁciency and a trend of reduction in local efﬁciency. Another advantage of PANDA is that both sequential and par- allel processing modes are supported, which makes it possible to take full advantage of available computing resources. The paral- lel environment can be either a single computer with multiple cores or a computing cluster, which increasingly enters into labs around the world. As shown in Figure 3, the PANDA processing have been parallelized as much as possible, and can thus reduce the time cost substantially under a parallel processing mode. For instance, the running time for pre-processing two subjects is almost the same as for one subject by using a workstation with four cores. In summary, PANDA can substantially facilitate/simplify image processing in a dMRI-related study, and can provide mea- sures for WM connectivity and network analysis. It has an extend- able design framework, and new functions or utilities can and will be added in the future. Finally, PANDA has a very friendly GUI (Figure 4), allowing the advanced users to select the desired options for each process- ing step. Depending on the datasets, users may change the options of some processing steps to optimize the processing quality. The reference data, e.g., image templates for normalization or prior atlases for node deﬁnition, can also be replaced by customized data, making it possible for processing dMRI data of non-human (e.g., primate) brains. ACKNOWLEDGMENTS The authors sincerely thank all the developers of FSL, PSOM, Diffusion Toolkit, and MRIcron, whose functions are called by PANDA. In addition, the authors thank Dr. Pierre Bellec for his support when implementing PSOM to PANDA, and thank Dr. Yanchao Bi for English editing. This work was supported by the National Science Foundation of China (No. 31000499, 81271649, 81030028), the Beijing Nova Program (No. Z121110002512032), the 973 program (No. 2013CB837300), and Open Research Fund of the State Key Laboratory of Cognitive Neuroscience and Learning. In the present study, we applied PANDA to produce results for testing the age effect on WM diffusion metrics as well as topological properties of the WM network. 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This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in other forums, provided the original authors and source are credited and subject to any copyright notices concerning any third-party graphics etc. Song, S. K., Sun, S. W., Ramsbottom, M. J., Chang, C., Russell, J., and Cross, A. H. (2002). Dysmyelination revealed through MRI as increased McAuliffe, M. J., Lalonde, F. Frontiers in Human Neuroscience APPENDIX B: GUIs OF PANDA Main function and (3) a preﬁx. The IDs and preﬁxes are used to name the resultant subfolder or ﬁles for each subject. In addition, users may change the pipeline options (Figure B2A), diffusion options (Figure B2B), and tracking options (Figure B2C). The default setting for these options will be used if no changes are made. The main GUI of PANDA is shown in Figure B1. Users are required to set up inputs and conﬁgure outputs through this GUI. Speciﬁcally, the data inputs are folders, each containing ﬁles in either DICOM or NIfTI format, for each subject. The output conﬁguration includes: (1) a main output folder that con- tains subject-speciﬁc subfolders of results; (2) digital subject IDs; Once all required settings are established, users simply click the “RUN” button to start the processing. PANDA will auto- matically ﬁnish all the sequential jobs and yield ﬁles containing February 2013 \| Volume 7 \| Article 42 \| 12 REFERENCES M., McGarry, D., Gandler, W., Csaky, K., and Trus, B. L. (2001). “Medical Hagmann, P., Cammoun, L., Gigandet, X., Meuli, R., Honey, C. J., Wedeen, V. J., et al. (2008). Mapping the February 2013 \| Volume 7 \| Article 42 \| 11 Frontiers in Human Neuroscience www.frontiersin.org A pipeline for diffusion MRI Cui et al. FIGURE A1 \| Implementation of the PANDA pipeline. The entire process of the PANDA pipeline was divided into 41 steps. Arrows indicate dependencies: A→B means that B cannot start until A is ﬁnished. FIGURE A1 \| Implementation of the PANDA pipeline. The entire process of the PANDA pipeline was divided into 41 steps. Arrows indicate dependencies: A→B means that B cannot start until A is ﬁnished. Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org A pipeline for diffusion MRI Cui et al. Bedpostx. As shown in Figure B4A, this utility allows for the estimation of voxel-wise local probability distributions of ﬁber orientation for a set of subjects, which is typically very time- consuming. The input for each subject should be a folder con- taining four ﬁles as listed: (1) a 4D image named data.nii.gz containing diffusion-weighted volumes and volumes without dif- fusion weighting; (2) a 3D binary brain mask volume named nodif_brain_mask.nii.gz; (3) a text ﬁle named bvecs containing gradient directions for diffusion weighted volumes; and (4) a text ﬁle named bvals containing the b-values that were applied to each volume acquisition. This module will generate a sepa- rate folder containing all the ﬁles that are required for subsequent probabilistic tractography. FIGURE B1 \| A snapshot of the main GUI while PANDA is running. The GUI allows for (1) inputting raw dMRI datasets and conﬁguring processing parameters and (2) monitoring the progress of data processing in real-time. Bedpostx. As shown in Figure B4A, this utility allows for the estimation of voxel-wise local probability distributions of ﬁber orientation for a set of subjects, which is typically very time- consuming. The input for each subject should be a folder con- taining four ﬁles as listed: (1) a 4D image named data.nii.gz containing diffusion-weighted volumes and volumes without dif- fusion weighting; (2) a 3D binary brain mask volume named nodif_brain_mask.nii.gz; (3) a text ﬁle named bvecs containing gradient directions for diffusion weighted volumes; and (4) a text ﬁle named bvals containing the b-values that were applied to each volume acquisition. This module will generate a sepa- rate folder containing all the ﬁles that are required for subsequent probabilistic tractography. Tracking & Network. This utility can separately construct anatomical brain networks based on tractography. The sub- GUI is shown in Figure B4B. For a deterministic tractography- based network, a folder with four ﬁles described in the section “Bedpostx” together with an individual-speciﬁc atlas image gen- erated by the utility “Brain Parcellation” are required. For a prob- abilistic tractography-based network, the resultant folder of the utility “Bedpostx” and the individual-speciﬁc atlas image should be the inputs. As described in the “Materials and Methods,” this module will generate network matrices that are saved in a MATLAB data ﬁle. FIGURE B1 \| A snapshot of the main GUI while PANDA is running. Frontiers in Human Neuroscience The GUI allows for (1) inputting raw dMRI datasets and conﬁguring processing parameters and (2) monitoring the progress of data processing in real-time. DICOM sorter. This handy utility, as shown in Figure B5A, can automatically sort multiple DICOM ﬁles in the same folder into sequence-speciﬁc or subject-speciﬁc sub-folders, based on the header information of the DICOM ﬁles. This is particu- larly useful when the DICOM ﬁles from different sequences or subjects are saved in the same folder, which happens very often. diffusion metrics and anatomical brain networks, as described in the “Materials and Methods.” During processing, the status of jobs can be checked in the monitor table of the GUI (Figure B1). Frontiers in Human Neuroscience Separate utilities Image converter. The NIfTI format can be a pair of ﬁles (hdr/img), a single ﬁle (nii), or a compressed ﬁle (nii.gz). A NIfTI ﬁle may be required in a certain ﬁle type, e.g., .nii or *.hdr/img. As shown in Figure B5B, this utility can convert NIfTI pair for- mat (hdr/img), NIfTI format (nii), and NIfTI GZ format (nii.gz) ﬁle types. TBSS. As shown in Figure B3A, this utility is for separate TBSS procedures, which require all images of FA and other diffusion metrics to be aligned in the MNI space. With correct input set- tings, this module will automatically generate individual images with data on the skeleton for all subjects. Statistical analyses can be directly applied to the resultant images. File copier. This utility can copy a large number of ﬁles located in different source folders into the same target folder. The sub- GUI is shown in Figure B5C. After PANDA processing, each subject will have unique folders containing the resultant ﬁles. “File Copier” can easily copy the same types of resultant ﬁles (e.g., aligned FA images) of all the subjects to one target folder, which might be helpful for further statistical analysis or other purposes. Brain parcellation (node deﬁnition). This utility is used to sep- arately deﬁne the brain network nodes. The sub-GUI is shown in Figure B3B. This module requires FA images of native space and skull-stripped T1 images as inputs. A prior atlas in the MNI space should also be speciﬁed. The results of this utility are individual atlas images in the dMRI native space for all subjects. These images can be directly loaded by the utility “Tracking & Network.” February 2013 \| Volume 7 \| Article 42 \| 13 Frontiers in Human Neuroscience www.frontiersin.org Cui et al. A pipeline for diffusion MRI FIGURE B2 \| Snapshots of the GUI for conﬁguring the processing parameters. (A) A snapshot of the GUI for changing the preprocessing parameters and for producing diffusion metrics that are ready for statistical analysis. (B) A snapshot of the GUI for changing the computing modes of PANDA. (C) A snapshot of the GUI for changing the parameters used to construct anatomical brain networks. analysis. (B) A snapshot of the GUI for changing the computing modes of PANDA. (C) A snapshot of the GUI for changing the parameters used to construct anatomical brain networks. analysis. Frontiers in Human Neuroscience February 2013 \| Volume 7 \| Article 42 \| 15 Frontiers in Human Neuroscience February 2013 \| Volume 7 \| Article 42 \| 16 Separate utilities February 2013 \| Volume 7 \| Article 42 \| 16 Frontiers in Human Neuroscience Separate utilities (B) A snapshot of the GUI for changing the computing modes of PANDA. (C) A snapshot of the GUI for changing the parameters used to construct anatomical brain networks. FIGURE B2 \| Snapshots of the GUI for conﬁguring the processing parameters. (A) A snapshot of the GUI for changing the preprocessing parameters and for producing diffusion metrics that are ready for statistical FIGURE B3 \| GUIs for the utilities “TBSS” and “brain parcellation” in PANDA. (A) The utility for TBSS analysis. (B) The utility for brain parcellation. FIGURE B3 \| GUIs for the utilities “TBSS” and “brain parcellation” in PANDA. (A) The utility for TBSS analysis. (B) The utility for brain parcellation. Frontiers in Human Neuroscience February 2013 \| Volume 7 \| Article 42 \| 14 Frontiers in Human Neuroscience www.frontiersin.org Cui et al. A pipeline for diffusion MRI FIGURE B4 \| GUIs for the utilities “Bedpostx” and “Tracking & Network” in PANDA. (A) The utility for Bedpostx. (B) The utility for tractography and network construction. FIGURE B4 \| GUIs for the utilities “Bedpostx” and “Tracking & Network” in PANDA. (A) The utility for Bedpostx. (B) The utility for tractography and network construction FIGURE B4 \| GUIs for the utilities “Bedpostx” and “Tracking & Network” in PANDA. (A) The utility for Bedpostx. (B) The utility for tractography and network construction. utilities “Bedpostx” and “Tracking & Network” in PANDA. (A) The utility for Bedpostx. (B) The utility for tractography and GURE B4 \| GUIs for the utilities “Bedpostx” and “Tracking & Network” in PANDA. (A) The utility for Bedpostx. (B) The ut etwork construction. February 2013 \| Volume 7 \| Article 42 \| 15 Frontiers in Human Neuroscience www.frontiersin.org www.frontiersin.org Cui et al. A pipeline for diffusion MRI FIGURE B5 \| GUIs for the utilities “DICOM Sorter,” “Image Converter,” and “File Copier” in PANDA. (A) The utility for DICOM Sorter. (B) The utility for Image Converter. (C) The utility for File Copier. FIGURE B5 \| GUIs for the utilities “DICOM Sorter,” “Image Converter,” and “File Copier” in PANDA. (A) The utility for DICOM Sorter. (B) The utility for Image Converter. (C) The utility for File Copier. FIGURE B5 \| GUIs for the utilities “DICOM Sorter,” “Image Converter,” and “File Copier” in PANDA. (A) The utility for DICOM Sorter. (B) The utility for Image Converter. (C) The utility for File Copier. www.frontiersin.org www.frontiersin.org
https://openalex.org/W4318696318	https://bjocs.site/index.php/bjocs/article/download/225/236	English	null	Opinions toward the War in Ukraine among Global Chinese Communities: Diversities, Connections, and New Research Opportunities	null	2,023	cc-by	2,700	British Journal of Chinese Studies, Vol. 13.1, January 2023 ISSN 2048-0601 © British Association for Chinese Studies British Journal of Chinese Studies, Vol. 13.1, January 2023 ISSN 2048-0601 © British Association for Chinese Studies Opinions Toward the War in Ukraine among Global Chinese Communities: Diversities, Connections, and New Research Opportunities Yu Tao, The University of Western Australia Racheline Tantular, The University of Western Australia Yu Tao, The University of Western Australia Racheline Tantular, The University of Western Australia Abstract The war in Ukraine has triggered varying responses from people with Chinese heritage worldwide. These responses to the war highlight the diversities within global Chinese communities. They also serve as a lens through which we can observe how different Chinese communities intersect and interact through the internet and social media in a globalised world. This paper creates a few snapshots of juxtaposing opinions on the war in Ukraine among global Chinese communities. It identifies three preliminary patterns underlying these diverse opinions, pointing to directions for subsequent empirical studies. It also discusses how opinions toward the war in Ukraine expose the complex and complicated diversities and connections among global Chinese communities, calling for more scholarly efforts to situate Chinese studies within the global context. Keywords: public opinion, global Chinese communities, the war in Ukraine, global Chinese studies, diversity in Chinese communities, connections between Chinese communities The war in Ukraine has been one of the most significant international affairs in 2022. It has triggered varying responses from people with Chinese heritage worldwide. These responses to the war, sometimes in sharp contrast with one another, highlight the diversities within global Chinese communities. They also serve as a lens through which we can observe how different Chinese communities intersect and interact via the internet and social media in a globalised world. As the war remains ongoing, people’s opinions toward it can change rapidly as new developments unfold and information surfaces. Therefore, this paper does not intend to attempt the methodologically and practically impossible mission of comprehensively assessing the public opinion on the war across global Chinese communities. Instead, it creates a few snapshots of various opinions on the war among global Chinese communities. This paper then identifies three preliminary patterns underlying these diverse opinions, pointing to directions for subsequent empirical studies. Finally, it discusses how opinions toward the war in Ukraine expose the complex and complicated diversities and connections among global Chinese communities, calling for more scholarly efforts to situate Chinese studies within the global context. At the time of the final editing of this article (mid-October 2022), Beijing has continued to refrain from condemning Russia’s use of force in Ukraine. Formal announcements by the 111 \| British Journal of Chinese Studies PRC government regarding the war in Ukraine maintain a neutral position. Abstract However, some PRC officials have framed the war as “reverberating from military escalation triggered by the United States” (Repnikova & Zhou, 2022) and have portrayed the Ukrainian military as overrun by neo-Nazis (Kuo & Shepherd, 2022). Moreover, it has been revealed that “major state-run news media outlets … have been largely echoing Russian state media stories or information from Russian officials” (McCarthy et al., 2022a). Social media platforms in China have also amplified official voices, “prioritis[ing] posts sent by state outlets in users’ interface” (Luo & Li, 2022). For example, on Weibo, arguably the most influential Chinese-language microblogging website, a hashtag related to the conspiracy theory that Ukraine houses American-funded biolabs had been viewed 180 million times by 10 March 2022, while a hashtag counteracting this claim had only been viewed 3,500 times (Kuo & Shepherd, 2022). This pro-Russia information environment is generally effective in influencing public opinion in mainland China. For example, 75% of the respondents to an online survey conducted between 28 March and 5 April 2022 believed that supporting Russia in Ukraine is in the PRC’s national interest (UCPM, 2022). Similarly, a systematic analysis of half a million Weibo posts in February and March 2022 found that 50% of the posts attributed the cause of war to the West (Buckley 2022). However, public opinion on the war in Ukraine is far from uniform in mainland China. For example, five Chinese historians from the country’s top universities issued an open letter in late February condemning Russia’s invasion of Ukraine (Qiao et al., 2022). Likewise, Hu Wei 胡伟, an established political researcher in Shanghai, published an influential article in early March urging China to cut ties with Putin’s Russia (Hu, 2022). Even though these statements were quickly censored (Luo & Li, 2022), they demonstrate that not all Chinese citizens think and speak according to the official lines. In addition to fact-checking posts made by the official social media accounts of foreign organisations like the European Union and the French Embassy, Chinese university students and volunteers also provide resources and regular updates to combat disinformation surrounding the war (Kuo & Shepherd, 2022). Public opinion towards the war in Ukraine differs significantly between Hong Kong and mainland China. As previously discussed, on the mainland, only a small number of primarily well-educated citizens have openly expressed their support for Ukraine’s resistance. Abstract Meanwhile, since the war started, a Ukrainian-owned restaurant in Hong Kong has seen new customers pouring in with donations and messages of support because many Hong Kong residents see “echoes of their pro-democracy struggle in Ukraine’s resistance against Russia” (Jakubec, 2022). Diverse opinions on the war in Ukraine can also be found in overseas Chinese communities, including the 6,000 Chinese nationals living in Ukraine when the war started (Zhou & Zhou, 2022). Although many have since fled the country, some were stranded, and others have chosen to stay. For example, Wang Jixian 王吉贤, a programmer originally from Beijing who decided to remain in Odesa, regularly broadcasts his experiences in war-torn Ukraine via social media to show people back in China “what the real battlefield is” (Yeung & Xiong, 2022). Other Chinese citizens in Ukraine have voiced worries about “identify[ing] 112 Yu Tao, Racheline Tantular \| 1 themselves as Chinese” due to increasing local hostility, calling for people back in China to “show greater empathy” for the Ukrainian people (McCarthy et al., 2022b). Many ethnic Chinese who are not PRC citizens have also expressed support for Ukraine. For example, on 10 March 2022, a group of Chinese Americans gathered in Manhattan’s Chinatown to show solidarity with the Ukrainian people (Chow, 2022). More remarkably, Ix Shen 沈倾, a Chinese-Singaporean former actor who emigrated to Ukraine, decided to return as a humanitarian volunteer after initially escaping to Poland with his Ukrainian wife. Shen related the killing of civilians in the Ukrainian town of Bucha to the Sook Ching massacre, an anti-Chinese operation carried out by the Japanese invaders eighty years ago in Singapore (Leo, 2022). However, it should not be assumed that all overseas Chinese hold similar views. For example, it is reported that the war has divided Australia’s Chinese community with heated discussions in chat groups and on social media platforms (Xing, 2022). China’s official position has subtly shifted since the outbreak of war in February, especially as Ukraine took back Russia-occupied territories in September 2022. For example, in February and June 2022, Chinese President Xi Jinping 习近平 made statements on reshaping the US-led international order with a Sino-Russian strategic partnership. However, observers have pointed out that while Beijing still wishes to reshape the world order, Xi refrained from explicitly mentioning its cooperation with Moscow to do so during the Shanghai Cooperation Organisation summit in September (Tiezzi, 2022). Abstract Likewise, people’s opinions toward the war in Ukraine among the global Chinese communities may change as the situation continues to evolve. For example, China’s backing away from its already nuanced support for Russia in late September appears to be accompanied by the emergence of anti-Russian narratives on Chinese social media in late September (Tiezzi, 2022). Notwithstanding the constant dynamics in public opinion toward the war in Ukraine among global Chinese communities, three preliminary patterns can be identified from the existing observations by journalists and researchers. First, despite the shift mentioned above, the public opinion toward the war in Ukraine remains significantly more pro-Russia in mainland China than in Hong Kong and overseas Chinese communities. Second, although public opinion in mainland China is generally pro-Russia, there exists a “critical minority… includ[ing] academics and professionals whose views carry more weight” (Buckley, 2022). Third, across global Chinese communities, many pro-Russia opinions during the war are linked to or driven by anti-Western sentiment rather than substantive support for Russia (Kuo, 2022; Repnikova & Zhou, 2022; Xing, 2022). These preliminary patterns should inspire empirical studies that place the relevant hypotheses under rigorous examination. On the macro or aggregated level, the war in Ukraine could bring new opportunities to observe, examine, and analyse how public opinion is formed among Chinese communities under different sociopolitical circumstances. In particular, statistical analysis based on representative samples could reveal the extent to which public opinion towards the war differs between mainland China, Hong Kong, Taiwan, and overseas Chinese communities. Moreover, comparative list experiments could be applied to measure whether social desirability effects associated with the expression of views on the war differ significantly between mainland China 113 \| British Journal of Chinese Studies and other Chinese communities, providing insight into how censorship may shape public opinion. On the micro or individual level, existing research has shown that personal characteristics such as age, gender, occupational sector, and religious belief may affect people’s political efficacy (Liu et al., 2022). Likewise, empirical investigations could reveal how personal characteristics impact people’s opinions on the war. Three sets of variables are worth particular attention. First, in mainland China, although a small group of well-educated intellectuals has formed a “critical minority” vocal in opposing the war (Buckley, 2022), a preliminary survey found that higher-educated respondents seemed to hold more pro-Russia views (UCPM 2022). Abstract Therefore, further systematic empirical studies are needed to examine how education impacts people’s opinions toward the war in Ukraine. Second, several observers have suggested that the pro-Russia opinions among global Chinese communities are driven primarily by anti- Western sentiments rather than any substantive sense of affection for Russia (Kuo, 2022; Repnikova & Zhou, 2022). Subsequent empirical studies could examine this testable proposition by testing whether and how people’s opinions toward the war in Ukraine correlate with their attitudes toward the United States and Russia. Finally, it has been argued that opinions toward the war in Ukraine among global Chinese communities are diverse “because people [have] different ideologies” (Xing, 2022). Therefore, subsequent empirical studies exploring whether and how people’s opinion toward the war aligns with their positions in the ideological spectra of liberalism and nationalism could yield illuminating results. Beyond inspiring the specific research agendas mentioned above, the war in Ukraine also invites researchers to reflect on the substance and extent of Chinese studies as an academic field. For example, existing research has demonstrated that the Chinese community in Australia is highly diverse, even though the Australian media and general public often neglect this group’s internal complexities and nuances (Tao & Stapleton, 2018; Tao & Loo, 2022). Likewise, the significantly varying opinions toward the war in Ukraine within and between various Chinese communities across the globe should further remind Chinese studies researchers to notice the in-group and between-group diversities among global Chinese communities. Furthermore, the formation and evolution of opinions toward the war in Ukraine highlights the connections between contemporary Chinese society in the PRC and overseas Chinese communities. For example, in the early days of the war, over 130 alumni of Chinese universities signed a petition condemning Russia, involving people residing in and outside China (Buckley, 2022). Meanwhile, some Chinese citizens in war-torn Ukraine experienced local hostility after the circulation of jokes insulting Ukraine on Chinese social media had negatively impacted many Ukrainians’ perception of China (Hille, 2022). It has also been suggested that “the propaganda from Beijing was influencing Chinese Australian migrants” and their views on the war in Ukraine (Xing, 2022). To understand these recent phenomena and many other aspects of the opinions toward the war in Ukraine held by people with Chinese heritage around the world, researchers need to consider how various Chinese communities across the globe are interconnected and impact one another. Abstract In today’s globalised world, geographic and political boundaries should no longer Yu Tao, Racheline Tantular \| 114 divide subfields of Chinese studies. Instead, we need to situate Chinese studies within the global context, look beyond a few specific communities of people with Chinese heritage, and pay more attention to the connections and interactions between these communities. The analysis of opinions towards the war in Ukraine is one example that offers a unique lens into the complexities of Chinese heritage groups within and beyond mainland China – complexities that all scholars of Chinese studies should consider. References Buckley, Chris (2022), “Defying China’s Censors to Urge Beijing to Denounce Russia’s War”, The New York Times, 18 March, available at: https://www.nytimes.com/2022/03/18/world/asia/china-ukraine-russia-dissent.html (accessed 17.08.2022). Chow, Mantai (2022), “Chinese-Americans in New York City gather to show solidarity with war-torn Ukraine”, The South China Morning Post, 21 March, available at: https://www.scmp.com/video/world/3171211/chinese-americans-new-york-city- gather-show-solidarity-war-torn-ukraine (accessed 18.08.2022). Hille, Kathrin (2022), “Chinese people stranded in Kyiv become target of Ukrainian anger”, Financial Times, 2 March, available at: https://www.ft.com/content/af0dbe39-e31a- 4db0-8ea9-6e0cb842f7b4 (accessed 13.07.2022). Hu, Wei (2022), “Possible Outcomes of the Russo-Ukrainian War and China’s Choice”, U.S.–China Perception Monitor, 12 March, available at: https://uscnpm.org/2022/03/12/hu-wei-russia-ukraine-war-china-choice/ (accessed 16.08.2022). Jakubec, Marco (2022), “In Hong Kong, a Ukrainian eatery inspires solidarity, resistance”, Al Jazeera, 2 March, available at: https://www.aljazeera.com/economy/2022/3/2/over- ukrainian-food-hong-kongers-express-solidarity-resistance (accessed 18.08.2022). Kuo, Kaiser (2022), “What do Chinese people think about the war in Ukraine?”, Sinica Podcast, 5 May, available at: https://supchina.com/2022/05/05/what-do-chinese- people-think-about-the-war-in-ukraine/ (accessed 16.08.2022). Kuo, Lily, and Christian Shepherd (2022), “In China, a battle for public opinion over Ukraine pits facts against propaganda”, The Washington Post, 10 March, available at: https://www.washingtonpost.com/world/2022/03/10/china-russia-ukraine-media-eu/ (accessed 16.06.2022). Leo, Lakeisha (2022), “Singaporean Ix Shen returns to Ukraine, joins volunteer group to provide humanitarian aid”, Channel News Asia, 9 April, available at: https://www.channelnewsasia.com/singapore/ix-shen-singaporean-returns-ukraine- joins-volunteer-group-humanitarian-aid-2617681 (accessed 18.08.2022). Liu, Caiyun, Yu Tao, & Chengzhi Yi (2022), “How Does Workplace Affect Employee Political Efficacy in China?”, Journal of Chinese Political Science, 1-29. 115 \| British Journal of Chinese Studies Luo, Zhifan, and Muyang Li (2022), “Online posts may not reflect Chinese opinion when it comes to the Russian invasion of Ukraine”, The Conversation, 30 March, available at: https://theconversation.com/online-posts-may-not-reflect-chinese-opinion-when- it-comes-to-the-russian-invasion-of-ukraine-179136 (accessed 16.06.2022). McCarthy, Simone, and CNN’s Beijing Bureau (2022a), “China’s promotion of Russian disinformation indicates where its loyalties lie”, CNN, 10 March, available at: https://edition.cnn.com/2022/03/10/china/china-russia-disinformation-campaign- ukraine-intl-dst-hnk/index.html (accessed 17.08.2022). —— (2022b), “China’s reaction to Ukraine could be putting its citizens in danger”, CNN, 28 February, available at: https://edition.cnn.com/2022/02/28/china/chinese-citizens- ukraine-danger-intl-hnk-mic/index.html (accessed 19.08.2022). Qiao, Long, Jia Ao, Chen Meihua & Liu Aoran (2022), “Chinese historians break ranks with party line in condemning Russia’s Ukraine war”, Free Radio Asia, 28 March, available at https://www.rfa.org/english/news/china/historians- 02282022102049.html (accessed 17.08.2022). Repnikova, Maria, and Wendy Zhou (2022), “What China’s Social Media Is Saying About Ukraine,” The Atlantic, 11 March, available at: https://www.theatlantic.com/ideas/archive/2022/03/china-xi-ukraine-war- america/627028/ (accessed 17.08.2022). Tao, Yu, and Cheng Yen Loo (2022), “Chinese Identities in Australia amid the COVID-19 Pandemic.” British Journal of Chinese Studies, 12(1), 129-133. References Tao, Yu, and Theo Stapleton (2018), “Religious affiliations of the Chinese community in Australia: findings from 2016 census data,” Religions, 9(4), 93. Tiezzi, Shannon (2022), “Is China Breaking With Russia Over Ukraine?”, The Diplomat, 17 September, available at: https://thediplomat.com/2022/09/is-china-breaking-with- russia-over-ukraine/ (accessed 06.10.22). UCPM (U.S.-China Perception Monitor) (2022), “Chinese Public Opinion on the War in Ukraine”, The Carter Center, 19 April, available at: https://uscnpm.org/2022/04/19/chinese-public-opinion-war-in-ukraine/ (accessed 16.06.2022). Xing, Dong (2022), “Why the Ukraine-Russia war is dividing the Australian Chinese community”, ABC News, 11 April, available at: https://www.abc.net.au/news/2022- 04-11/ukraine-russia-war-dividing-chinese-australians/100954590 (accessed 18.08.2022). Yeung, Jessica, and Yong Xiong (2022), “A Chinese vlogger shared videos of war-torn Ukraine. He’s been labeled a national traitor”, CNN, 19 March, available at: https://edition.cnn.com/2022/03/18/asia/wang-jixian-chinese-vlogger-ukraine-intl- hnk/index.html (accessed 16.06.2022). Yu Tao, Racheline Tantular \| 116 Zhou, Marrian, and Cissy Zhou (2022), “The Chinese nationals who chose to stay in Ukraine”, Nikkei Asia, 14 April, available at: https://asia.nikkei.com/Politics/Ukraine-war/The-Chinese-nationals-who-chose-to- stay-in-Ukraine (accessed 13.07.2022). Yu Tao, Racheline Tantular \| 116 Zhou, Marrian, and Cissy Zhou (2022), “The Chinese nationals who chose to stay in Ukraine”, Nikkei Asia, 14 April, available at: https://asia.nikkei.com/Politics/Ukraine-war/The-Chinese-nationals-who-chose-to- stay-in-Ukraine (accessed 13.07.2022). Yu Tao, Racheline Tantular \| 116
https://openalex.org/W4385761506	https://www.nature.com/articles/s41598-023-39820-0.pdf	English	null	A GIS-based framework to assess heatwave vulnerability and impact scenarios in urban systems	Scientific reports	2,023	cc-by	13,108	Valeria D’Ambrosio 1, Ferdinando Di Martino 1,2* & Vittorio Miraglia 1 In this work, we propose a GIS-based platform aimed at the analysis of heatwave scenarios risks produced in urbanised environments, applied to assess vulnerability and impact heatwave scenarios. Our framework implements a hierarchical model that represents a good trade-off between forecast accuracy and portability in different urban fabrics, apart from the spatial scale of the data, using topographic and remote sensing spatial data provided by institutional agencies. The framework has been applied to two study areas: the dense city of Naples (Italy) and the intermediately populated city of Avellino (Italy) in order to evaluate its accuracy performances and portability in different urban fabrics. Our framework can be used by urban planners and decision makers as a tool to locate potential risk zones where it is necessary to implement climate-resilient solutions. The increasing need to address climate hazards in urban tissues encouraged researchers to develop decision-sup- port tools to assess risk levels and identify critical urban areas where climate-resilient solutions must be adopted. The increasing need to address climate hazards in urban tissues encouraged researchers to develop decision-sup- port tools to assess risk levels and identify critical urban areas where climate-resilient solutions must be adopted. In recent years, some researchers have developed studies aimed at understanding the relationships between the built environment and climate phenomena in order to define adaptation strategies and programs that aim h The increasing need to address climate hazards in urban tissues encouraged researchers to develop decision-sup- port tools to assess risk levels and identify critical urban areas where climate-resilient solutions must be adopted. In recent years, some researchers have developed studies aimed at understanding the relationships between the built environment and climate phenomena in order to define adaptation strategies and programs that aim to reduce the risks caused by climate change. These studies have resulted in the development of methodologies, procedures, guidelines, web services, and tools to increase awareness of climate vulnerabilities and risks and 1 7 In recent years, some researchers have developed studies aimed at understanding the relationships between the built environment and climate phenomena in order to define adaptation strategies and programs that aim to reduce the risks caused by climate change. These studies have resulted in the development of methodologies, procedures, guidelines, web services, and tools to increase awareness of climate vulnerabilities and risks and support the development of local adaptation strategies and plans1–7. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Valeria D’Ambrosio 1, Ferdinando Di Martino 1,2* & Vittorio Miraglia 1 pp p p g p Many of these contributions, in particular those focused on assessing the climate vulnerability of the urban system, base the starting analysis on a synthesized set of characteristics of the study applying statistical methods and approaches, such as the Principal Component Analysis (PCA). pp p p y PCA is a consolidated multivariate statistical method aimed at aggregating information and reducing the number of variables; it is used to assess the spatial distribution of the vulnerability indicator based on a reduced set of characteristics of the urban model. However, although this method is used in various studies that estimate the heatwave vulnerability of urban systems, it has limitations and shortcomings mainly due to poor model port- ability and usability in different urban contexts and on different scales. Its limitation is the inability to identify peculiarities specific to the study’s geographical context; this implies that the same characteristics may not be attributable to urban systems located in different territorial contexts. yf Wolf and McGregor8 create a heatwave vulnerability index for a region stretching over Greater London by census area by using the PCA approach to represent a collection of relevant characteristics. In our opinion, due to the identification of traits that are intimately tied to the territorial environment, the procedure cannot be replicated in a different urban design. pf g A study of the impact of the heatwave on urban patterns is proposed in9; the authors apply their method to the city of Paris, taking into consideration five scenarios of urban expansion over time, depending on the climatic conditions. The results of the study show that in urban areas with a high concentration of inhabitants the impact of the phenomenon is greater. p g In4 ana analysis of the relationship between heatwave vulnerability and mortality is carried out in the New York City study area. The results show that mortality during heatwave periods is higher in zones with the highest mean surface temperature and lower in areas with the highest density of green spaces.it In10 the authors propose a study conducted in fifteen urban patterns in Europe and the United States aimed at assessing the heatwave vulnerability of urban fabrics. A GIS‑based framework to assess heatwave vulnerability and impact scenarios in urban systems OPEN Valeria D’Ambrosio 1, Ferdinando Di Martino 1,2* & Vittorio Miraglia 1 www.nature.com/scientificreports/ relation to different local situations. A local study is therefore necessary to evaluate which are the specific char- acteristics of an urban pattern that affect heatwave vulnerability.f relation to different local situations. A local study is therefore necessary to evaluate which are the specific char- acteristics of an urban pattern that affect heatwave vulnerability.f In3 the PCA algorithm is applied to eight variables that affect the heatwave vulnerability of an urban pattern. The experimentation is carried out in a study area that includes the city of Osaka in Japan. The eight variables are extracted from population data by census area and from land use data. The variables that have the greatest impact on vulnerability are the age of the population, the education and social condition of the population, and the density and size of green areas. y g Several additional research studies11–14 support the continued evolution of the vulnerability concept, which places a strong emphasis on local assessment to better understand how the vulnerability features are connected to losses and damages during and after the occurrence of climatic events. g gt A critical point of these models is the use of local data at a detailed scale, which limits their portability on different urban models; in addition, the use of too many parameters makes these models unsuitable to be applied in different contexts. f To overcome these limitations, we propose a framework implemented in a GIS environment that can ensure both the portability and reusability of the model for different urban patterns; to achieve this goal, we propose the use of a hierarchical model for assessing vulnerability and heatwave impacts on urban systems that does not require a large number of parameters and can be replicated on different urban fabrics. Furthermore, the accuracy of vulnerability and impact assessments. By replicable model, we suggest a vulnerability and impact assessment model that incorporates measurable parameters through the collection of data available and tested by institutional bodies and available in different urban fabrics. Coming from different institutional sources, these datasets are not homogeneous, and a reconcili- ation activity was necessary to bring them back to the same coordinate system and reproduce the correct coding of the fields to be able to acquire and use them in the algorithms developed in a GIS platform for the generation of impact scenarios. www.nature.com/scientificreports/ For this purpose, the census section is considered as the atomic space unit, representing a unit recognized as homogenous for urban characteristics and on which the censuses of the characteristics of the population, build- ings, etc. are carried out by the institutional bodies in charge. This choice represents a trade-off between the need to use information on a large scale, which allows greater accuracy in estimates but reduces the portability of the model, and the use of too small scale of data, which could affect the accuracy of the results. f Our framework is based on the model proposed in15 to assess vulnerability and heatwave impact scenarios on urban systems. The decision to use this model is mainly linked to the hierarchical structure adopted to subdivide the urban system, to the use of a small number of parameters that are easily measurable at the spatial scale of the census sections, and above all to its accuracy, which was measured in the experimentation carried out in the METROPOLIS research project15.h This model was tested on two districts in the east and west of the city of Naples (Italy). It breaks down the system into three subsystems: the residential buildings, the open spaces, and the residential population. The model assesses the vulnerability of each subsystem and uses the IPCC AR5 model described in16 to evaluate the impacts generated by heatwave hazard scenarios. We use this model to obtain subsystems vulnerability estimates and of the impact scenarios attributed to the individual census areas. Our research was conducted as part of the PLANNER project17,18, in which a geocomputational platform was tested, breaking down the urban system into atomic units made up of the individual census sections. In order to evaluate the performances and the portability of our framework, it was developed and tested on two study areas: the city of Naples, a complex urban system with a very high build density, and the city of Avellino, a less dense context both for building and housing density.h The prerequisite for making our framework portable is the choice to consider the census section as an atomic spatial entity. The primary factor is the census section’s ability to reflect the smallest spatial unit with uniformly homogenous urban and territorial features. For this reason, the indicators used for evaluating the vulnerability and impacts of the heatwave phenomenon have been aggregated for census sections. www.nature.com/scientificreports/ In the following section, we introduce the model used for our studies to better explain the meaning of the proposal; it is possible to read about the structure of the hierarchical model and how it was built. In the third section, we present our framework, and in "Results and discussion" section are shown the results of the applica- tion of our framework on two different urban fabrics given by the cities of Naples and Avellino. The conclusions about our work are given in "Conclusions" section. Valeria D’Ambrosio 1, Ferdinando Di Martino 1,2* & Vittorio Miraglia 1 This study highlights that the spatial distributions of vulnerability in these urban patterns are different from each other, showing that different urban fabrics are in 1Dipartimento di Architettura, Università degli Studi di Napoli Federico II, Via Toledo 402, 80134 Naples, Italy. 2Centro Interdipartimentale di Ricerca “A. Calza Bini”, Università degli Studi di Napoli Federico II, Via Toledo 402, 80134 Naples, Italy. *email: fdimarti@unina.it Scientific Reports \| (2023) 13:13073 \| https://doi.org/10.1038/s41598-023-39820-0 www.nature.com/scientificreports/ Preliminaries The hierarchical vulnerability and impact model. The model chosen to develop our framework is the one defined in15 for the generation of the impact scenarios; it is based on the general model proposed in the AR5 report of the IPPC—Intergovernmental Panel on Climate Change16, which assesses the impacts of climatic phenomena as a result of the combination of vulnerability, hazard and exposure.i An impact scenario is generated by referring to a specific hazard scenario caused by a climatic phenomenon that acts on an urban system whose vulnerability to the phenomenon has been assessed. The AR5 report evalu- ates the effects of climate change by taking into account the subsystems’ vulnerabilities whereby the exposure interacts. In15 the combination of exposure and the vulnerabilities of each subsystem (called Intrinsic Vulnerabilities) is measured with a specific indicator labelled Combined Vulnerability. The impact indicator is measured as a merger of the Combined Vulnerability and the hazard scenario (Fig. 1). The exposure is valuated in two phases: in the former phase (phase 1) the elements exposed to the climate risk are defined, and in the latter phase (phase 2) the exposure of those elements is measured. Scientific Reports \| (2023) 13:13073 \| https://doi.org/10.1038/s41598-023-39820-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 1. Scheme of the process applied in15 to evaluate the impact scenarios to climate phenomena. Figure 1. Scheme of the process applied in15 to evaluate the impact scenarios to climate phenomena. The partitioning of the urban pattern into subsystems is done to separate and recognize the elements and components that make it up. Since each subsystem is taken into consideration regardless of its relationship with the other urban subsystems, this partitioning simplifies the assessment of the vulnerability of the urban system. The vulnerability of each subsystem is measured by considering only the characteristics of the subsystem affected by the heatwave phenomenon.hh This model is applied in15 to evaluate the impact heatwave scenarios. The urban system is partitioned into three subsystems: residential buildings, open spaces, and population; the model was implemented and tested to evaluate the vulnerability of the three subsystems to the heatwave phenomenon and the impact scenarios. Preliminaries The model was evaluated in accordance with the research reported in17,18, considering the resident population as an exposure component and assessing the combined vulnerability by combining the intrinsic vulnerabilities of the subsystems with the exposure.hf The characteristics of the subsystem elements that might affect a subsystem’s response to heatwave events are assessed in order to evaluate the intrinsic vulnerability of the subsystems. They refer to the construction, morphological, technological, and environmental aspects of residential buildings and open spaces (eras and construction techniques, volume and solar exposure of the building envelope for the residential buildings, and, Sky View Factor, solar exposure, Albedo and NDVI for the open space). In19 are applied recent remote sensing techniques aimed at the acquisition and synthesis of the NDVI. In similar ways, the same techniques are used to calculate the characteristics of the physical system that have as a source given satellite images (Albedo, Sky View Factor, etc.).h The assessment of heatwave impact scenarios takes account of the major risk factors for residents who are most inclined to suffer heat exhaustion and other health effects because of the phenomenon. The exposure is measured by computing the number of residents living in each residential building. The hazard scenarios are computed from the spatial distribution of the raster satellite data of the surface temperature at both daily and nightly times measured during heatwave periods. The impact scenarios are calculated as a combination of a hazard scenario and a combined vulnerability. Process employed to assess heatwave vulnerability and impact scenarios. This section briefly presents the model proposed in15, chosen as the starting point for the elaboration of our framework. The overall process is detailed in15 and it was implemented on a GIS platform. Scientific Reports \| (2023) 13:13073 \| Process employed to assess heatwave vulnerability and impact scenarios. This section briefly presents the model proposed in15, chosen as the starting point for the elaboration of our framework. The overall process is detailed in15 and it was implemented on a GIS platform. Indicators of physical subsystem. Subsystem Indicator Measure Measure domain Weight Buildings Thermal lag Thermal lag (s) 5 Thermal decrement factor Thermal decrement factor [0, 1] 4 Building volume Building volume (m3) 2 Solar exposure of building envelope Façades and roofs Hillshade [0, 255] 5 Open spaces SVF Sky View Factor [0, 2π] 5 Albedo Albedo [0, 1] 2 Solar exposure of open spaces Open space Hillshade [0, 255] 4 NDVI Normalized Difference Vegetation Index [− 1, 1] 3 Table 1. Indicators of physical subsystem. Subsystem Indicator Measure Measure domain Weight Buildings Thermal lag Thermal lag (s) 5 Thermal decrement factor Thermal decrement factor [0, 1] 4 Building volume Building volume (m3) 2 Solar exposure of building envelope Façades and roofs Hillshade [0, 255] 5 Open spaces SVF Sky View Factor [0, 2π] 5 Albedo Albedo [0, 1] 2 Solar exposure of open spaces Open space Hillshade [0, 255] 4 NDVI Normalized Difference Vegetation Index [− 1, 1] 3 Table 1. Indicators of physical subsystem. Table 2. Classification of indicators. Class Label 1 High 2 Medium–high 3 Medium 4 Medium–low 5 Low Table 2. Classification of indicators. portable. In fact, regardless of the spatial accuracy and amount of input data available in the research region, it may be used on any sort of geographical scale. The hazard scenarios are constructed by considering the pro- jected variations in maximum and minimum temperatures, Heat Index and surface temperature, according to two IPCC emission scenarios: portable. In fact, regardless of the spatial accuracy and amount of input data available in the research region, it may be used on any sort of geographical scale. The hazard scenarios are constructed by considering the pro- jected variations in maximum and minimum temperatures, Heat Index and surface temperature, according to two IPCC emission scenarios: • RCP 4.5 scenario which provides for a constant concentration of greenhouse gases until 2100; h C h h d f f h • RCP 4.5 scenario which provides for a constant concentration of greenhouse gases until 2100; • the RCP 8.5 scenario which provides for a constant increase in concentration of greenhouse gases up to 2100. Process employed to assess heatwave vulnerability and impact scenarios. This section briefly presents the model proposed in15, chosen as the starting point for the elaboration of our framework. The overall process is detailed in15 and it was implemented on a GIS platform. To evaluate the vulnerability of the residential building and open space subsystems, a set of indicators was computed that recognizes the main characteristics of the settlements in the type-morphological and technological aspects, as well as in the presence and intensity of greenery and urban elements capable of affecting the aspects of temperature, ventilation, and relative humidity during intense climatic phenomena20. These indicators are shown in Fig. 2. g Each indicator is obtained by a measure of a characteristic of buildings and open spaces; to each indicator is assigned a weight based on how much it affects the intrinsic vulnerability of the subsystem. The weights are assigned in15 following a specific calibration (Table 1).hi i The indicators are partitioned into five classes from 1 to 5, making them comparable to each other to normal- ize the impact assessment operations. All indicators foresee a partition into five classes (Table 2). The partition rules are obtained through calibrations on specific samples15.h g pi p The combined vulnerability is computed by merging the contribution of the exposure, given by the resident population living in a residential building, the intrinsic vulnerability of the residential buildings, and the mean intrinsic vulnerability of the surrounding open spaces.h The hazard features that constitute the hazard scenario are evaluated and distributed over the study region in a collection of raster data. The spatial model used to realize the scenarios was designed to be inter-scalable and Scientific Reports \| (2023) 13:13073 \| https://doi.org/10.1038/s41598-023-39820-0 www.nature.com/scientificreports/ Figure 2. Schema of the indicators used in15 to evaluate the residential buildings and open spaces heatwave vulnerability. Table 1. Indicators of physical subsystem. Subsystem Indicator Measure Measure domain Weight Buildings Thermal lag Thermal lag (s) 5 Thermal decrement factor Thermal decrement factor [0, 1] 4 Building volume Building volume (m3) 2 Solar exposure of building envelope Façades and roofs Hillshade [0, 255] 5 Open spaces SVF Sky View Factor [0, 2π] 5 Albedo Albedo [0, 1] 2 Solar exposure of open spaces Open space Hillshade [0, 255] 4 NDVI Normalized Difference Vegetation Index [− 1, 1] 3 Figure 2. Schema of the indicators used in15 to evaluate the residential buildings and open spaces heatwave vulnerability. Figure 2. Schema of the indicators used in15 to evaluate the residential buildings and open spaces heatwave vulnerability. Figure 2. Schema of the indicators used in15 to evaluate the residential buildings and open spaces heatwave vulnerability. Table 1. Materials and methods Less compact buildings provide the benefit of having distributed open areas. The core of the building itself is surrounded by a predominantly natural territory with a strong mountainous character. This directly affects the climate, which offers lower temperatures than the city of Naples but nevertheless remains subject to extreme weather events such as heatwaves (on average, in the summer months, the max air temperature is 28.7 °C; in July and August, it is about 30 °C). Methodological framework. Our framework was developed starting from the model15 and it was inte- grated with an approach aimed at achieving the reusability capabilities of the model in order to be applied in different urban fabrics. f In order to better analyze vulnerabilities and impacts, it was chosen to employ a smaller spatial scale than that specified in the model of15, which set up building and open space polygonal entities as atomic units. We have cho- sen to use the census section as an atomic entity since it represents the smallest area recognized as homogenous regarding urban characteristics and is frequently updated by the national census bodies. This choice was made to guarantee the usability of our framework even in urban fabrics where the data at this scale are not accessible and to provide a trade-off between the assessment accuracy and the portability of the model. pf y p y Figure 3 shows the entire structure of the framework and the connections between its parts to determine the heatwave impact scenarios.h The intrinsic vulnerability of the urban physical subsystems, the exposure, and the hazard scenarios generated by heatwave phenomena are computed using input data from certified sources, where for intrinsic vulnerability we mean the vulnerability of each subsystem. The combined vulnerability is then calculated by combining the Exposure with the building and open spaces intrinsic vulnerabilities. Finally, an Impact scenario is created by merging the effects of a hazard scenario with the combined vulnerability.h g gf y The buildings and open spaces Intrinsic Vulnerabilities are computed by using the indicators in Fig. 2 and applying the processes described and synthetized in "Preliminaries" section. To calculate exposure, we focus on the population density and identify categories of residents who are most vulnerable to the risks linked to the heatwave phenomena. In fact, the population is subject to discomfort and possible damage to health caused by the occurrence of the phenomenon25–27. Materials and methods The study areas. Our framework was tested on the study area of the dense city of Naples (Italy) and sub- sequently, in order to verify its portability in other urban fabrics, it was tested on the city of Avellino (Italy), a different urban fabric less populated and characterized by different climatic conditions than Naples.h ff The municipality of Naples is populated by a little less than a million inhabitants, it represents a complex urban fabric with a high population density. It possesses a multiplicity of different urban forms, which connote a certain level of homogeneity. In fact, it is possible to identify cohesive areas with high construction density, such as those present in the historic center, areas with a lower concentration of buildings and a clear prevalence of forested areas, such as the peripheral areas to the north, areas with greater extension and a prevalence of manufacturing building artefacts, such as the areas to the east with a strongly industrial vocation. g g y A study about climate change carried out in21 predicts that all of Italy’s coastal areas will be marked by a temperature increase in the period 2021–2050, compared to 1981–2010, and that this increase equates to 1.3 °C in the Central and Western Mediterranean.i Naples is among the Italian cities with the highest percentage of artificial surface compared to administrative thresholds, at around 63%22; in addition, the city of Naples, comprising about one million inhabitants, is clas- sified as the Italian urban area with the highest density of inhabitants per square kilometer. These factors make the city of Naples particularly vulnerable to heatwave impact scenarios.h The maximum temperature anomalies observed in the city in 2018 were about 0.9 °C lower than those recorded in the period 1971–200023. In contrast, despite the drop in maximum temperature anomalies, the number of tropical nights has grown dramatically, with an increase of about 37 days over the course of the cur- rent season compared to the median of the reference period24.f p p In order to test the performance of our framework on different urban fabrics, we replicated the processes used to create the resident building vulnerability in the city of Avellino. Avellino, in contrast to Naples, is characterized by a less complex urban fabric, a substantially smaller overall population (about 52,000), and a population density that is five times lower. www.nature.com/scientificreports/ the RCP4.5 model has a standard deviation from the projected average summer temperature up to 2050 of about 1 °C and then increases to 1.5 °C by 2100.hf the RCP4.5 model has a standard deviation from the projected average summer temperature up to 2050 of about 1 °C and then increases to 1.5 °C by 2100.hf The hazard is analyzed based on three different scenarios for each of the two RCP models: a short-term, a medium-term, and a long-term scenario. The impact of the heatwave phenomenon is assessed for each hazard scenario by combining the hazard and Combined Vulnerability (on a scale from one, more serious, to five, less severe).h The impact scenarios were evaluated by combing on one side with the Combined Vulnerability, which is used as a fixed, non-mutable part that represents the conditions that lead to the urban environment, and on the other side with all the hazard scenarios, which possess a greater dynamism as they represent the evolution of the danger during a given period. Process employed to assess heatwave vulnerability and impact scenarios. This section briefly presents the model proposed in15, chosen as the starting point for the elaboration of our framework. The overall process is detailed in15 and it was implemented on a GIS platform. • the RCP 8.5 scenario which provides for a constant increase in concentration of gr • the RCP 8.5 scenario which provides for a constant increase in concentration of greenhouse gases up to 2 The IPCC RCP 4.5 model predicts that greenhouse gas emissions will slow down over the next 50 years but that their concentrations in the atmosphere will continue to increase. The choice of this model is related to its degree of likelihood, as the current trend suggests a decrease in greenhouse gas emissions from human activities, so that the radiative forcing may reach the value of 4.5 W/m2 with a consequent increase in average temperature of less than 2 °C. The RCP4.5 model assumes an almost linear trend with a consequent constant temperature growth until reaching 2 °C in 50 years. From the study carried out by Euro-Mediterranean Center on Climate Change (CMCC) on the city of Naples (https://www.cmcc.it/it/report-napoli), the projections of the two models have a similar trend until 2050 and then vary more and more until 2100, with a final average change of about 2 °C. The Root Mean Square Error (RMSE) value obtained by comparing the temperature difference between the models’ projection and the actual measurement recorded over the past 5 years is below 0.1 °C. Furthermore, https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ Scientific Reports \| (2023) 13:13073 Materials and methods The exposure is computed start- ing from the population census data for census sections provided and updated by a national statistical institute (e.g., the Italian statistical institute of ISTAT in Italy, the Office for National Statistics in UK, the French national institute of statistics and economic studies INSEE in France, the Spanish statistical office INE in Spain, etc.). It was allowed to individually assess the impact scenarios produced by different categories of inhabitants exposed to risk since exposure, in the model created for calculating the impacts, is an independent variable from the other entities of the model. We have considered three types of exposure: Scientific Reports \| (2023) 13:13073 \| https://doi.org/10.1038/s41598-023-39820-0 www.nature.com/scientificreports/ Figure 3. Model for assessing impact scenarios for climatic phenomena. Figure 3. Model for assessing impact scenarios for climatic phenomena. • Population density (PD); aimed at identifying the spread of the population in the study area and highlighting the degree of crowding in that area; • Population density (PD); aimed at identifying the spread of the population in the study area and highlighting the degree of crowding in that area; g g • Disadvantaged population (DP); aimed at identifying the spread in the territory of the population belonging to the weaker groups in relation to age (children and the elderly) and the relative crowding in certain sections of the census; • Disadvantaged population (DP); aimed at identifying the spread in the territory of the population belonging to the weaker groups in relation to age (children and the elderly) and the relative crowding in certain sections of the census; ; • Fuel Poverty (FP); aimed at identifying the spread over the territory of the population in particularly condi- tions disadvantaged people (households > 5 members, non-income earners, unemployed) and the relative crowding in certain sections of the census. ; • Fuel Poverty (FP); aimed at identifying the spread over the territory of the population in particularly condi- tions disadvantaged people (households > 5 members, non-income earners, unemployed) and the relative crowding in certain sections of the census. The PD is expressed in inhabitants per square kilometer, and it is determined using the following formula: The PD is expressed in inhabitants per square kilometer, and it is determined using the following formula: (1) PD = P SS (1) where P is the number of residents in the census section and SS is the area of census section in km2. www.nature.com/scientificreports/ www.nature.com/scientificreports/ Table 4. Classification of the disadvantaged population indicator. Data representation Class Label Classification method DP ≥ 5000 1 High Manual 5300 ≤ DP < 5000 2 Medium–high Manual 2000 ≤ DP < 5300 3 Medium Manual 500 ≤ DP < 2000 4 Medium–low Manual DP < 500 5 Low Manual Table 4. Classification of the disadvantaged population indicator. Table 4. Classification of the disadvantaged population indicator. The FP is expressed in inhabitants per square kilometer, and it is computesd using the following formula: The FP is expressed in inhabitants per square kilometer, and it is computesd using the following formula: The FP is expressed in inhabitants per square kilometer, and it is computesd using the following formul (3) FP = NF + N + D SS (3) where NF is the number of households whit more than five members, N is the number of non-recipients of income, D is the number of unemployed and SS is the area of census section.hi where NF is the number of households whit more than five members, N is the number of non-recipients of income, D is the number of unemployed and SS is the area of census section.hi The fuel poverty indicator is obtained by using the partitioning of the FP index in five classes, following the ne used in15. That partitioning is shown in Table 5 h Since it has been considered that residents interact primarily with the material components of the urban sys- tem, residential buildings and open spaces constitute all the subsystems with which exposure interacts13. In our framework, in which the atomic entity is the census section, the values of the two physical subsystems indicators are aggregated considering all the components of the subsystem located in the census section.h The combined vulnerability merges the exposure with the intrinsic vulnerability of the subsystems. A set of calibration processes were performed in15 to extract the combined vulnerability as a function of the exposure and intrinsic vulnerabilities of the two physical subsystems. Table 6 shows the calibration used to obtain the combined vulnerability values.h The features that connote a hazard scenario can be divided into spatial and time features; the time features are given by the following daily climatic parameters: the maximum and minimum air temperatures and the relative humidity. These three parameters are used to compute the Heat Index (HI) measure defined by the USA National Weather Service. Table 6. Combined vulnerability indicator. Materials and methods The indicator is obtained by using the partitioning of the PD index in five classes, following the one used in15. That partitioning is shown in Table 3 p g The DP is expressed in inhabitants per square kilometer and it is computed using the following formula: (2) DP = PD SS (2) DP = PD SS (2) where PD is the number of residents in the census section with younger than 6 years and older than 65 years, and SS is the area of census section.hi S The disadvantaged population indicator is obtained by using the partitioning of the DP index in five classes ollowing the one used in in15. That partitioning is shown in Table 4 Rule Class Label PD ≥ 20,000 1 High 15,000 PD < 20,000 2 Medium–high 10,000 ≤ PD < 15,000 3 Medium 5000 ≤ PD < 10,000 4 Medium–low PD < 5000 5 Low Table 3. Classification of the population density indicator. Table 3. Classification of the population density indicator. Table 3. Classification of the population density indicator. Table 3. Classification of the population density indicator. https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ The hazard spatial features are given by the remote sensed surface temperature raster data collected during a heatwave and monitored both daily and at night. The most dangerous areas are detected measuring the difference between the day and overnight temperatures. The hazard increases as this difference decreases.h y ghf The assessment of the hazard is obtained by combining time and spatial features, following the processes described in15,17,18 in which are considered three types of hazard scenarios: short-term (2020–2040), medium- term (2041–2070), and long-term (2071–2100). g To each scenario is assigned a mean number of heatwave consecutive days: short-term 6 days, medium-term 0 days, and long-term 60 days. y g y In a short-term scenario, in which the duration of heatwave consecutive days ranges between 3 and 8 days, the hazard indicator obtained using Table 715,18:h The variations of the impact classes are evaluated with the worsening of the phenomenon, using the same calibration criteria proposed in15. Table 8 shows the respective variations for each hazard class as the scenario worsens. For example, an urban area classified with a Medium–low hazard (4), in a short-term scenario, will be assigned to the hazard Medium (3) class in a medium-term scenario, and to High class (1) in a long-term sce- nario. This implies that areas with Low hazard values, determined in a short-term scenario without appropriate mitigation and/or adaptation interventions, are transformed into High hazard areas in a long-term scenario.h g p g g This assessment considers adaptive and mitigation changes to the urban setting do not occur until 2100 h Impact scenarios are computed by combining hazard scenarios with exposure. A different impact scenario is generated depending on the hazard discharge and the type of population exposed. Combining the two indicators of hazard and combined vulnerability has resulted in the creation of three impact scenarios—one for each of the obtained thematic maps of integrated vulnerability—for each hazard scenario. Overall, nine impact scenario maps are produced: three short-term, three medium-term and three long-term (Fig. 4). p p g ( g ) To evaluate the functional dependence of the impact class on both hazard and vulnerability indicators, a specific calibrations rule set proposed in15 is used. It is schematized in Table 9. i Following this calibration rule set, if both hazard and combined vulnerability are High or Medium–high the impact class is set to High. www.nature.com/scientificreports/ Otherwise, if the combined vulnerability is at least Medium (Medium, Medium–low or Low), and if also the hazard is at least Medium–high, the impact class is identical to the combined vulnerability class; finally, it is given by the combined vulnerability class values − 1. www.nature.com/scientificreports/ A heatwave scenario is detected by the presence of a HI higher than 32 °C for at least three consecutive days28. Table 5. Classification of the fuel poverty indicator. Data representation Class Label Classification method FP ≥ 5000 1 High Manual 5300 ≤ FP < 5000 2 Medium–high Manual 2000 ≤ FP < 3500 3 Medium Manual 500 ≤ FP < 2000 4 Medium–low Manual FP < 500 5 Low Manual Table 5. Classification of the fuel poverty indicator. Residential buildings intrinsic vulnerability Open spaces intrinsic vulnerability Exposure Combined vulnerability < 3 < 3 < 3 1 < 3 ≥ 3 < 3 2 < 3 < 3 ≥ 3 2 < 3 ≥ 3 ≥ 3 3 ≥ 3 < 3 < 3 3 > 3 ≥ 3 < 3 3 = 3 ≥ 3 < 3 3 = 3 < 3 ≥ 3 3 > 3 < 3 ≥ 3 4 = 3 ≥ 3 ≥ 3 4 > 3 ≥ 3 ≥ 3 5 https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| Results and discussionh Risk/Impact scenarios evaluating by different exposition. Table 9. Impact class according with combined vulnerability and hazard classes. Hazard class Combined vulnerability class Impact class < 3 < 3 1 ≥ 3 < 3 2 < 3 ≥ 3 Combined vulnerability class—1 ≥ 2 ≥ 3 Combined vulnerability class Table 9. Impact class according with combined vulnerability and hazard classes. Hazard class Combined vulnerability class Impact class < 3 < 3 1 ≥ 3 < 3 2 < 3 ≥ 3 Combined vulnerability class—1 ≥ 2 ≥ 3 Combined vulnerability class Table 9. Impact class according with combined vulnerability and hazard classes. Table 9. Impact class according with combined vulnerability and hazard classes. Table 9. Impact class according with combined vulnerability and hazard classes. • Digital Terrain Model (DTM) and Digital Surface Model (DSM) obtained from LIDAR surveys provided by the Italian Ministry and provided by the Italian Ministry of the Environment and Protection of Land and Sea in raster format, with a resolution of 1 m × 1 m;i • Digital Terrain Model (DTM) and Digital Surface Model (DSM) obtained from LIDAR surveys provided by the Italian Ministry and provided by the Italian Ministry of the Environment and Protection of Land and Sea in raster format, with a resolution of 1 m × 1 m;i g g p ; • National Census Data in shapefile format with a scale of 1:10,000 processed and supplied by the Natio Institute of Statistics (ISTA) and constantly updated; y p Raster Albedo processed using the remote sensed Sentinel2 multi-band images, with a resolution of 7 m × 9 m Raster Sky View Factor processed using the remote sensed Sentinel2 multi-band images, with a resolution of 1 m × 1 m; • Raster NDVI, processed using the remote sensed Sentinel2 red and near infrared bands, with a resolutio 7 m × 7 m; ; • Satellite data of daytime and night-time Land Surface Temperature (LST) processed using the remote sensed Landsat8 multi-band images, with a resolution of 30 m × 30 m. • Satellite data of daytime and night-time Land Surface Temperature (LST) processed using the remote sensed Landsat8 multi-band images, with a resolution of 30 m × 30 m. Results and discussionh Results. The model has been structured and implemented in a GIS platform using the suite GIS ESRI Arc- GIS Desktop. The spatial analysis processes were implemented using the ArcGIS tool ModelBuilder, a visual programming language for building geoprocessing workflows that generate geoprocessing models that automate and document spatial analysis and data management processes. Vulnerabilities and impact scenarios on the municipality of Naples (Italy). To execute our framework the follow- ing datasets have been imported: Table 7. Hazard classification in a heatwave short-term scenario. where ΔT is the surface temperature difference between day and night. Surface temperature difference Hazard class Label ΔT ≤ 7 1 High 7 < ΔT ≤ 10 2 Medium–high 10 < ΔT ≤ 15 3 Medium 15 < ΔT ≤ 20 4 Medium–low ΔT > 20 5 Low Table 7. Hazard classification in a heatwave short-term scenario. where ΔT is the surface temperature difference between day and night. Table 8. Change of the hazard classes on varying the hazard scenario. Short-term Medium-term Long-term Class Label Class Label Class Label 1 High 1 High 1 High 2 Medium–high 1 High 1 High 3 Medium 2 Medium–high 1 High 4 Medium–low 3 Medium 1 High 5 Low 4 Medium–low 2 Medium–high Table 8. Change of the hazard classes on varying the hazard scenario. Short-term Medium-term Long-term Class Label Class Label Class Label 1 High 1 High 1 High 2 Medium–high 1 High 1 High 3 Medium 2 Medium–high 1 High 4 Medium–low 3 Medium 1 High 5 Low 4 Medium–low 2 Medium–high Table 8. Change of the hazard classes on varying the hazard scenario. Table 8. Change of the hazard classes on varying the hazard scenario. https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 4. Risk/Impact scenarios evaluating by different exposition. Table 9. Impact class according with combined vulnerability and hazard classes. Hazard class Combined vulnerability class Impact class < 3 < 3 1 ≥ 3 < 3 2 < 3 ≥ 3 Combined vulnerability class—1 ≥ 2 ≥ 3 Combined vulnerability class Figure 4. Risk/Impact scenarios evaluating by different exposition. Table 9. Impact class according with combined vulnerability and hazard classes. Hazard class Combined vulnerability class Impact class < 3 < 3 1 ≥ 3 < 3 2 < 3 ≥ 3 Combined vulnerability class—1 ≥ 2 ≥ 3 Combined vulnerability class Figure 4. Risk/Impact scenarios evaluating by different exposition. Figure 4. Results and discussionh The satellite raster data are necessary to extract the spatial distribution of some parameters, as Albedo, NDVI, and surface temperatures, to compute intermediate indicators; all are single images and were processed during the occurrence of the heatwave phenomenon in Naples on 14/07/2020.hh p p Figure 5 shows the thematic maps of the residential building subsystem indicators: Thermal lag, Thermal ecrement factor, Building volume and Solar exposure of building envelope.hh The Thermal lag map shows the presence of census sections with Medium–high value in some areas to the north and mainly in the districts of the central-western areas of the municipality of Naples. Similarly, in these zones the thematic map of the Thermal decrement factor shows a Medium–high criticality. The Building volume indicator thematic map shows that the census areas containing the most voluminous residential buildings are mainly located in the peripheral areas of the city, especially in the north-western area. The Solar exposure of building envelope thematic map presents a uniform distribution throughout the municipal area with over 90% of the sections classified as Medium. Scientific Reports \| (2023) 13:13073 https://doi.org/10.1038/s41598-023-39820-0 www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 5. Residential building subsystem vulnerability intermediate indicators: Thermal lag (a); Thermal decrement factor (b); Building volume (c); Solar exposure of building envelope (d). Figure 5. Residential building subsystem vulnerability intermediate indicators: Thermal lag (a); Thermal decrement factor (b); Building volume (c); Solar exposure of building envelope (d). The thematic maps shown in Fig. 6 are the outcome of the process required for computing the intermediat vulnerability indicators of the open space subsystem.h The thematic maps shown in Fig. 6 are the outcome of the process required for computing the intermediate vulnerability indicators of the open space subsystem.h The Albedo thematic map shows a distribution almost uniform in which most of the census sections are classified as Medium–high or High, with a distribution of greater criticality located in the historic center. In the Sky view factor thematic map, most of the census sections are classified as Low or Medium–low; this is mainly due to the prevalence of census sections in which the portion of the visible sky in the open space is reduced by the presence of neighboring buildings and other vertical structures. In the NDVI map, most of the densely Figure 6. Open spaces subsystem vulnerability indicators: Albedo (a); Sky view Factor (b); NDVI (c); Solar exposure of open space (d). Figure 6. www.nature.com/scientificreports/ inhabited census sections and the historic center, which are poor in living vegetation, are classified as High or Medium–high; conversely, the north-western census areas, which enjoy the greatest presence of plant species, are classified as Medium–low or Low. In the Solar exposure thematic map, the census sections located in the city center are prevalently classified as Medium, in contrast to the census sections of the peripheral areas, which are classified as Medium–high.h inhabited census sections and the historic center, which are poor in living vegetation, are classified as High or Medium–high; conversely, the north-western census areas, which enjoy the greatest presence of plant species, are classified as Medium–low or Low. In the Solar exposure thematic map, the census sections located in the city center are prevalently classified as Medium, in contrast to the census sections of the peripheral areas, which are classified as Medium–high.h i g The thematic maps presented in Fig. 7 are the outcome of the process for calculating the intrinsic vulner- abilities of the urban system. Th d l b ld l b l h h l d f d d l y The residential building vulnerability thematic map shows a prevailing condition of Medium or Medium–low vulnerability throughout the area of the municipality of Naples (91% of the census sections). In particular, in the historic center there is an extended area covered by census sections with Low or Medium–low vulnerability. In the open spaces subsystem vulnerability map the east side of the city is prevalently covered by census sec-ii The residential building vulnerability thematic map shows a prevailing condition of Medium or Medium–low vulnerability throughout the area of the municipality of Naples (91% of the census sections). In particular, in the historic center there is an extended area covered by census sections with Low or Medium–low vulnerability. In the open spaces subsystem vulnerability map the east side of the city is prevalently covered by census sec- tions classified as Medium–high. In the western and central-western areas, census sections classified as Medium vulnerability class prevail, whereas only a few census sections, mainly covered by vegetated areas, are classified with Medium–low vulnerability. y In Fig. Results and discussionh Open spaces subsystem vulnerability indicators: Albedo (a); Sky view Factor (b); NDVI (c); Solar exposure of open space (d). https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ The spatial distribution of the disadvantaged population exposure is similar to Population density distribu- tion; the areas most affected are mainly located in the historic center of the city and some peripheral districts; about 49% of the census sections are classified as High or Medium–high.h i g g The fuel poverty thematic map presents more critical issues than the two other exposure thematic maps. In fact, it shows larger areas with high exposure value in the historic center and in some peripheral areas: 72% of the census sections are classified as High or Medium–high. Only 23% of the census sections are classified as Low or Medium–low. To develop the impact scenarios is considered the IPCC RCP 4.5 model which predicts that greenhouse gas emissions would slow down over the next 50 years but that their atmospheric concentrations will continue to rise. the reason for choosing this model is that the RCP 4.5 model is the one that seems more probable today as the current trend suggests a decrease in greenhouse gas emissions produced by human activities, so that the radioactive forcing will not be able to reach the value of 8.5 W m2 as in the RCP 8.5 model, even if it will not be such as to avoid global warming of the planet with an average temperature increase of less than 2 °C. For this reason, we decided to focus the heatwave impact scenarios on relation to hazard scenarios, taking into account the RCP 4.5 model, in which the average duration of a heatwave is 6 days in the short-term, 30 days in the medium-term and 60 days in the long-term. y g Figure 9 shows the thematic maps corresponding to the three-time scenarios: short-term, medium-term, and long-term. As seen in the study of the three hazard scenario maps, the threat of the phenomenon increases over time, becoming highly significant in the long run with High and Medium–high hazard over the study area. Th h d h h l f h h d h h f h h i The histogram in Fig. 9d shows the evolution of the hazard in the three scenarios. Starting from the short- term scenario, about 58% of the census sections are classified as Medium–low or Medium. www.nature.com/scientificreports/ 8 are shown the thematic maps obtained for the three types of exposure analyzed correspond to the three categories of population: Population density, Fuel poverty and Disadvantaged population.h The population density map shows that the areas of the historic center and some suburban districts are more exposed, justified by a greater population density. About 43% of the census sections have High or Medium–high exposure, while only 30% of them is classified as Medium–low or Low. Figure 7. Urban system intrinsic vulnerabilities: residential building vulnerability (a); open spaces vulnerability (b). igure 7. Urban system intrinsic vulnerabilities: residential building vulnerability (a); open spaces vulnerability b). Figure 7. Urban system intrinsic vulnerabilities: residential building vulnerability (a); open spaces vulnerability (b). Figure 8. Exposure to heatwave phenomena: Population density (PD) (a); Disadvantaged population (DP) (b); Fuel poverty (FP) (c); frequency histogram (d). Figure 8. Exposure to heatwave phenomena: Population density (PD) (a); Disadvantaged population (DP) (b); Fuel poverty (FP) (c); frequency histogram (d). https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ In the medium-term scenario the distribution worsens by one class and the percentage of census sections classified as Medium is reduced to 38%; conversely, the percentage of High and Medium–high which increases to 62%. In the long-term scenario, there are no longer sections belonging to the Medium or higher classes, 100% of the sections reach High or Medium–high hazard levels. This conclusion shows that, unless adaptation measures are planned, the phenomena will reach a very critical level by 2100, affecting the entire study area, independent of the diverse typologies of the portions that make it up.h yp g p p The thematic maps of the three impact scenarios have been drawn up for each hazard scenario by each cat- gory of exposure. In Fig. 10 it is illustrated the impacts maps by Population density.h The short-term impact scenario map shows a higher percentage of census sections belonging to Medium class (39%), the census sections that have the most crucial difficulties is also the 33% classified as High or Medium–high, only the 28% of sections has a good level of impact, classified as Medium–low or Low.f i In the medium-term scenario map the distribution is different, there is a worsening of the condition of the impact levels with the census sections percentage belonging to the highest levels classified as High or Medium–High reachings 43%, while the census sections percentage classified as Medium decreases to 34% and that classified as Medium–low or Low decreases to 23%. Figure 9. Heatwave hazard scenarios: short-term hazard (S) (a); medium-term hazard (M) (b); long-term hazard (L) (c); frequency histogram (d). Figure 9. Heatwave hazard scenarios: short-term hazard (S) (a); medium-term hazard (M) (b); long-term hazard (L) (c); frequency histogram (d). https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ Figure 10. Impact scenarios by population density: short-term impact scenario (S) (a); medium-term impact scenario (M) (b); long-term impact scenario (L) (c); frequency histogram (d). Figure 10. Impact scenarios by population density: short-term impact scenario (S) (a); medium-term impa scenario (M) (b); long-term impact scenario (L) (c); frequency histogram (d). In the long-term scenario maps the impact levels worsen dramatically, compared to the initial conditions, the census sections classified as High or Medium–high reach 66%, sections percentage classified as Medium decreasse to 20% and that classified as Medium–low is reduced to 14%, there is no census section belonging to the Low level. www.nature.com/scientificreports/ www.nature.com/scientificreports/ The short-term impact scenario map shows a higher percentage of census sections belonging to Medium class (48%), the census sections that have the most crucial difficulties is also the 38% classified as High or Medium–high, only the 14% of sections has a good level of impact, classified as Medium–low or Low.f g y g pi In the medium-term scenario map the distribution is different, there is a worsening of the condition of the impact levels with the census sections belonging to the highest levels classified as High or Medium–high reach- ing 52%, while the census sections classified as Medium decrease to 36% and those classified as Medium–low or Low decrease to 12%. In the long-term scenario maps the impact levels worsen dramatically, compared to the initial conditions, the census sections classified as High or Medium–high reach 84%, the sections percentage classified as Medium decreases to 9% and that classified as Medium–low is reduced to 7%, there is no census section belonging to the Low level.h The most critical areas are mainly distributed in the historic center and in the northern and eastern peripheral areas. The north–western zones in the longer scenario maintain conditions of Medium or Medium–low levels. Figure 12 shows the distribution of impact classes in each hazard scenario by the fuel poverty population. h Figure 12 shows the distribution of impact classes in each hazard scenario by the fuel poverty populatio Th h h h h f b l M d g p y p y p p The short-term impact scenario map shows a higher percentage of census sections belonging to Medium class (41%), the census sections that have the most crucial difficulties is also the 31% classified as High or Medium–high; only the 28% of them all has a good level of impact, classified as Medium–low or Low.f i In the medium-term scenario map the distribution is different, there is a worsening of the condition of the impact levels with the census sections belonging to the highest levels classified as High or Medium–High reach- ing 41%, while the census sections classified as Medium decrease to 36% and those classified as Medium–low or Low decrease to 23%. www.nature.com/scientificreports/ In the long-term scenario maps the impact levels worsen dramatically, compared to the initial conditions, the census sections classified as High or Medium–high reach 65%, those classified as Medium decrease to 21%, and those classified as Medium–low are reduced to 14%; there is no census section belonging to the Low level.h the census sections classified as High or Medium–high reach 65%, those classified as Medium decrease to 21%, and those classified as Medium–low are reduced to 14%; there is no census section belonging to the Low level. The most critical areas are mainly distributed in the historic center and in the northern and eastern peripheral areas. The north–western zones in the longer scenario maintain conditions of Medium or Medium–low levels. B i i h i i i i f ibl h i h di d l i i g g The most critical areas are mainly distributed in the historic center and in the northern and eastern periph areas. The north–western zones in the longer scenario maintain conditions of Medium or Medium–low lev h By examining the various impact maps, it is feasible to see that in the medium-term and long-term scenarios, over 40% of the census sections already experience significant criticalities. These percentages reach 52% in the medium-term scenario and 84% in the long-term scenario, specifically in the impact scenarios by people living in fuel poverty.hf y The results also reveals that the fuel poverty population is highly exposed to the effects of climate change; in fact, already in medium-term scenarios the impacts on the fuel poverty population take on Medium High and High values over more than 50% of the municipal extension; this value exceeds 80% in the long-term scenario. Portability test: residential bulding vulnerability on municipality of Avellino (Italy). In order to evaluate the port- ability of our framework, we’ve tested it in the study area of the municipality of Avellino. As a demonstration, we show the findings obtained from the assessment of the building system heatwave vulnerability. As evidence of this, the results of the experimentation in the form of thematic maps are represented in Fig. 13. Figure 12. Impact scenarios by fuel poverty: short-term impact scenario (S) (a); medium-term impact scenario (M) (b); long-term impact scenario (L) (c); frequency histogram (d). Figure 12. www.nature.com/scientificreports/ The most critical areas are mainly distributed in the historic center and in the northern and eastern sur- rounding areas. The western zones in the longer scenario maintain conditions of Medium or Medium–low levels. In Fig. 11, it is possible to see the impacts maps for each hazard scenario by disadvantaged population. In the long-term scenario maps the impact levels worsen dramatically, compared to the initial conditions, the census sections classified as High or Medium–high reach 66%, sections percentage classified as Medium decreasse to 20% and that classified as Medium–low is reduced to 14%, there is no census section belonging to the Low level. Low level. The most critical areas are mainly distributed in the historic center and in the northern and eastern sur- ding areas. The western zones in the longer scenario maintain conditions of Medium or Medium–low levels. n Fig. 11, it is possible to see the impacts maps for each hazard scenario by disadvantaged population. the Low level. The most critical areas are mainly distributed in the historic center and in the northern and eastern sur- rounding areas. The western zones in the longer scenario maintain conditions of Medium or Medium–low levels. In Fig. 11, it is possible to see the impacts maps for each hazard scenario by disadvantaged population. e ow eve . The most critical areas are mainly distributed in the historic center and in the northern and eastern sur- ounding areas. The western zones in the longer scenario maintain conditions of Medium or Medium–low levels In Fig 11 it is possible to see the impacts maps for each hazard scenario by disadvantaged population The most critical areas are mainly distributed in the historic center and in the northern and eastern sur- rounding areas. The western zones in the longer scenario maintain conditions of Medium or Medium–low levels. I F bl h f h h d b d d d l Figure 11. Impact scenarios by disadvantaged population: short-term impact scenario (S) (a); medium-term impact scenario (M) (b); long-term impact scenario (L) (c); frequency histogram (d). Figure 11. Impact scenarios by disadvantaged population: short-term impact scenario (S) (a); medium-term impact scenario (M) (b); long-term impact scenario (L) (c); frequency histogram (d). https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ In Table 10, for each vulnerability class, the mean value of the four intermediate indicators is shown:ii , y , Based on Table 4’s findings, the vulnerability class is most significantly impacted by three indicators: Solar exposure Thermal lag and Thermal decrement factor, the former remains constant on average as the vulner- ability class varies; its value is Medium–low, when the vulnerability class is Medium–high, and it becomes Low, for all other vulnerability classes. Instead, the average values of the Thermal lag and Thermal decrement factor rindicators are High and Medium–high, respectively when the vulnerability class is Medium–high and Medium. This fact highlights that these indicators are the ones that most determine the vulnerability class of residential buildings. In fact, the most vulnerable census areas of Avellino, classified with Medium–high and Medium vulnerability, are those in which a more recent construction in reinforced concrete with more than two blocks is more frequent; in them, the contribution of the vertical closures of buildings prevails in the evaluation of the Thermal lag and Thermal decrement factor indicators. www.nature.com/scientificreports/ Impact scenarios by fuel poverty: short-term impact scenario (S) (a); medium-term impact scenario (M) (b); long-term impact scenario (L) (c); frequency histogram (d). https://doi.org/10.1038/s41598-023-39820-0 Scientific Reports \| (2023) 13:13073 \| www.nature.com/scientificreports/ www.nature.com/scientificreports/ Figure 13. Buildings subsystem vulnerability of Avellino (a); frequency histogram (b). Figure 13. Buildings subsystem vulnerability of Avellino (a); frequency histogram (b). The thematic map in Fig. 13a shows a prevailing condition of Medium–low vulnerability throughout the municipality (62%). In particular, the census sections in the city center areas are mainly classifies with Low vulnerability and the census sections in the surrounding areas, are mainly classified whit a medium–low vul- nerability. The census section classified with Medium or Medium–high vulnerability are those with the highest built and population densities.i The thematic map in Fig. 13a shows a prevailing condition of Medium–low vulnerability throughout the municipality (62%). In particular, the census sections in the city center areas are mainly classifies with Low vulnerability and the census sections in the surrounding areas, are mainly classified whit a medium–low vul- nerability. The census section classified with Medium or Medium–high vulnerability are those with the highest built and population densities.i As can be seen from the histogram (Fig. 13b), overall, sections classified with Low and Medium–low residen- tial buildings vulnerability prevail (69%); 28% of sections are classified with Medium vulnerability and only 3% are classified with Medium–high vulnerability. This distribution of the residential building heatwave vulnerability by census zone in the municipality of Avellino is consistent with the type of urban pattern characterized by a low population density, perfectly, in contrast to the city of Naples where the population density is much higher.h i g yh g y by census zone in the municipality of Avellino is consistent with the type of urban pattern characterized by a low population density, perfectly, in contrast to the city of Naples where the population density is much higher. The fact that the results obtained are in line with the typology of the two urban patterns demonstrates the adaptability of our framework to model the assessment of heatwave vulnerability and impact scenarios of dif- ferent urban fabrics. y y y y g The fact that the results obtained are in line with the typology of the two urban patterns demonstrates the adaptability of our framework to model the assessment of heatwave vulnerability and impact scenarios of dif- ferent urban fabrics. Data availability Th d d y The datasets generated during and/or analysed during the current study are available from the corresponding uthor on reasonable request. Received: 28 April 2023; Accepted: 31 July 2023 Received: 28 April 2023; Accepted: 31 July 2023 Conclusions In this paper, we propose a GIS-based framework, developed in the PLANNER research project, for the assess- ment of vulnerability and impact scenarios to heatwaves in different urban fabrics.h The framework implements the model of heatwave vulnerability and impacts in urban systems proposed in15, using the census zone as an atomic spatial entity, which represents the smallest area of the urban fabric with homogeneous urban characteristics; periodic censuses are provided on various characteristics referring to the resident population, residential buildings, families, and buildings. This was done in order to ensure, in addition to the good accuracy of the results, the portability of the framework in different urban fabrics, in which it is always possible to use basic cartographic, satellite data and aggregated census data. In order to evaluate the spatial distribution of vulnerability and impacts on a study area characterized by a multitude of different urban forms and inhabited by a high population density characterized by different social classes, which represent different types of subjects exposed to risk, it was decided to test the model on a complex urban fabric, such as the city of Naples. Thematic maps of vulnerability and impact scenarios demonstrate that our approach produces findings that are compatible with the geographical distribution of the many building types, urban forms, and social fabrics that are present. As a consequence, it provides a good compromise between the mobility of the model and the findings’ correctness. i It was tested for the assessment of the vulnerability of residential buildings in the study area of Avellino (Italy) in order to confirm the portability of the framework in various urban fabrics; the most vulnerable areas are those of new construction, in which the contribution of the vertical component of the building is relevant. This finding indicates how the framework may easily be modified to accommodate urban fabrics with various physical and morphological properties. To improve the accuracy and portability of the model, we intend to conduct in the future a lot of framework tests on further urban fabrics on an international scale, in order to analyze territorial contexts characterized by different urban forms and social fabrics. In this approach, it is possible to evaluate the real requirement for morphological and technical-constructive characteristics to be added to or modified in connection to the system’s components (buildings and open spaces) in order improve the assessments of vulnerability and impacts on the heatwave phenomena. References References 1. Disher, B., Edwards, B., Lawler, T., Radford, D. Measuring heatwaves and their impacts. In: Air Pollution Sources, Statistics and Health Effects. Encyclopedia of Sustainability Science and Technology Series (eds Goodsite, M. E., Johnson, M. S. & Hertel, O.). (Springer, New York, NY, 2021). https://doi.org/10.1007/978-1-0716-0596-7_1102 References 1. Disher, B., Edwards, B., Lawler, T., Radford, D. Measuring heatwaves and their impacts. In: Air Pollution Sources, Statistics and Health Effects. Encyclopedia of Sustainability Science and Technology Series (eds Goodsite, M. E., Johnson, M. S. & Hertel, O.). (Springer, New York, NY, 2021). https://doi.org/10.1007/978-1-0716-0596-7_1102 2. Kumar, D., Alam, M. & Sanjayan, J. Building adaptation to extreme heatwaves. In: Engineering for Extremes. Springer Tracts in Civil Engineering, (eds Stewart, M. G. & Rosowsky, D. V.) (Springer, Cham, 2022). https://doi.org/10.1007/978-3-030-85018-0_9 3. Macnee, R. G. D. & Tokai, A. Heatwave vulnerability and exposure mapping for Osaka City. Environ. Syst. Decis. Jpn. 36, 368–376. https://doi.org/10.1007/s10669-016-9607-4 (2016). 2. Kumar, D., Alam, M. & Sanjayan, J. Building adaptation to extreme heatwaves. In: Engineering for Extremes. Springer Tracts in Civil Engineering, (eds Stewart, M. G. & Rosowsky, D. V.) (Springer, Cham, 2022). https://doi.org/10.1007/978-3-030-85018-0_9 3. Macnee, R. G. D. & Tokai, A. Heatwave vulnerability and exposure mapping for Osaka City. Environ. Syst. Decis. Jpn. 36, 368–376. https://doi.org/10.1007/s10669-016-9607-4 (2016). p g 4. Madrigano, J., Ito, K., Johnson, S., Kinney, P. L. & Matte, T. A case-only study of vulnerability to heatwave-related mortality in New York City (2000–2011). Environ. Health Perspect. 13(7), 672–678. https://doi.org/10.1289/ehp.1408178 (2015). y p p g p 5. Swart, R., Fons, J., Geertsema, W., van Hove, B., Gregor, M., Havranek, M., Jacobs, C., Kazmierczak, A., Krellenberg, K., Kuhlicke, C., Peltonen, L. Urban Vulnerability Indicators. A joint report of ETC-CCA and ETC-SIA, ETC-CCA and ETC-SIA Technical Report 933, 01/2012, ETC/CCA and ETC/SIA (2012). y p p g p 5. Swart, R., Fons, J., Geertsema, W., van Hove, B., Gregor, M., Havranek, M., Jacobs, C., Kazmierczak, A., Krellenberg, K., Kuhlicke, C., Peltonen, L. Urban Vulnerability Indicators. A joint report of ETC-CCA and ETC-SIA, ETC-CCA and ETC-SIA Technical Report 933, 01/2012, ETC/CCA and ETC/SIA (2012). p , , ( ) 6. Timmerman, J., Breil, M., Bacciu, V., Coninx, I. Fons, J., Gregor, M., Havranek, M., Jacobs, C., Loehnertz, M., Pelton, L., Sainz, M., Swart, R. Map book urban vulnerability to climate change—Factsheets, EEA, ETC/CCA, ETC/SIA (2017).i p 6. Timmerman, J., Breil, M., Bacciu, V., Coninx, I. www.nature.com/scientificreports/ www.nature.com/scientificreports/ A crucial decision that needs to be made in order to model the entire study area reflecting the conditions of the constituent elements while maintaining an average detail scale, is the use of census sections as the spatial atomic information for the maps. The use of a higher detail scale, in which each single building or open space is used as an atomic unit, does not guarantee the fundamental characteristic of portability necessary to replicate the analysis on different urban fabrics, due to the difficulties of finding certified data at that scale of detail. The information sources at this level of detail are often not homogeneous, not limited, not very reliable, or almost out of date. t To evaluate the portability of our framework on other urban fabrics, the GIS-based framework has been used to create the residential buildings heatwave vulnerability maps of the municipality of Avelino (Italy). To produce this map, the same types of spatial datasets used to obtain the Naples thematic map of vulnerability were used, in order to analyze the portability level of the framework by exploring a different urban fabric starting from the same type of source data. This map shows that the most vulnerable census sections (classified with Medium–high and Medium vulnerability) are those of recent construction in which the contribution of the vertical component of the building prevails, which affects the Thermal Lag and Thermal decrement factor indicators. Because of this, even if the urban fabric of Avellino and Naples are very different, the conclusions derived using the framework are still valid. This demonstrates how the suggested GIS-based methodology may be used to analyze heatwave susceptibil- ity and effect scenarios in different urban patterns characterized by specific building fabrics and urban shapes. Discussion Th ’ l The test’s results on the urban fabric of Naples (Italy), show that the aggregation by census zone represents a good trade-off between the accuracy of the resulting maps and the portability of the model. In fact, the vulnerability maps of the two subsystems and the resulting impact scenarios show spatial distributions consistent with the vari- ous types of urban forms present in the city. The vulnerability analysis exactly reflects the technical-constructive buildings characteristics and the formation of open spaces. From checks carried out with expert decision-makers, it has emerged that the most vulnerable areas are really the most critical in terms of the vulnerability for its mor- phological and technical-constructive characteristics. This mainly relates to the basic characteristics that were identified for defining the indicators, which are sufficient to assess the urban system’s vulnerability. Analyzing the short-, medium-, and long-term impact scenario maps, it is possible to see that the worsening risk is directly proportional to the duration of the heatwave. The census sections classified with Medium–low vulnerability being subjected to a High impact in future scenarios. This implies the need to intervene with mitigation and adaptation solutions in order to prevent the deterioration of the conditions of the urban system components. Table 10. Mean class value of the four Residential building intermediate indicators, computed for each vulnerability class. 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Linee guida per preparare piani di sorveglianza e risposta verso gli effetti sulla salute di ondate di calore anomalo. Direzione Generale Prevenzione Sanitaria del Ministero della Salute, Centro Nazionale Prevenzione e Controllo Malattie, Roma. Fundingh g The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. Author contributions All authors contributed to the study conception and design, to the material preparation, data collection, analysis and writing the document. All authors read and approved the final manuscript. All authors contributed to the study conception and design, to the material preparation, data collection, analysis and writing the document. All authors read and approved the final manuscript. Competing interests h The authors declare no competing interests. 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https://openalex.org/W2921407445	https://discovery.ucl.ac.uk/id/eprint/10069486/1/Rowcliffe_Chausson2019_Article_UnderstandingTheSocioculturalD.pdf	English	null	Understanding the Sociocultural Drivers of Urban Bushmeat Consumption for Behavior Change Interventions in Pointe Noire, Republic of Congo	Human ecology	2,019	cc-by	9,876	* Alexandre M. Chausson alexandre.chausson@zoo.ox.ac.uk Abstract Interventions targeting consumer behavior may help to reduce demand for bushmeat in urban areas. Understanding the drivers of urban bushmeat consumption is crucial to guide such interventions; however the cultural and socio-psychological factors driving consumer behavior remain understudied. Through qualitative interviews with urban bushmeat consumers in Pointe Noire, Republic of Congo, we investigated perceptions of bushmeat and other animal proteins, and social norms regulating urban demand for bushmeat. The perception of bushmeat as natural, tasty and healthy, and a rare luxury product functioning as a symbol of social status, underpins social norms to provide bushmeat. The main barriers to purchasing were cost and availability. Locally produced fish, meat, and poultry were positively perceived as organic and healthy, whereas frozen imported animal proteins were perceived negatively as transformed, of poor quality and taste, and unhealthy. Our findings provide an initial baseline understanding of social-psychological drivers shaping consumption that can inform the design of bushmeat demand reduction campaigns. Keywords Central Africa . Perceptions . Social norms . Demand reduction . Behavior change urban bushmeat consumption, Pointe Noire hunting is considered a major threat to biodiversity (Nasi et al. 2011) with implications for ecosystem function (Effiom et al. 2013) and associated ecosystem services, such as carbon sequestration (Peres et al. 2016). Demand for bushmeat in ur- ban areas in Central Africa plays a substantial role in driving bushmeat hunting and trade (de Merode and Cowlishaw 2006; van Vliet and Mbazza 2011), with urban populations consum- ing a substantial proportion of the total harvest in the Congo Basin. Yet wildlife depletion caused by overhunting can threat- en the food and livelihood security of rural communities (Nasi et al. 2011). Bushmeat is a vital source of protein in many rural areas and reduced availability can have negative health and nutrition impacts (Golden et al. 2011). https://doi.org/10.1007/s10745-019-0061-z Human Ecology (2019) 47:179–191 https://doi.org/10.1007/s10745-019-0061-z Human Ecology (2019) 47:179–191 Understanding the Sociocultural Drivers of Urban Bushmeat Consumption for Behavior Change Interventions in Pointe Noire, Republic of Congo ausson1,2 & J. Marcus Rowcliffe2,3 & Lucie Escouflaire4 & Michelle Wieland4 & Juliet H. Wright2, Alexandre M. Chausson1,2 & J. Marcus Rowcliffe2,3 & Lucie Escouflaire4 & Michelle Wieland4 # The Author(s) 2019 Published online: 18 March 2019 # The Author(s) 2019 Published online: 18 March 2019 Introduction Central Africa harbors the second largest extent of tropical rainforest in the world, and its integrity is essential for biodi- versity and livelihoods (Somorin et al. 2012). Bushmeat 2 Imperial College London, Silwood Park Campus, Buckhurst Road, Ascot SL5 7PY, UK 4 Wildlife Conservation Society, Africa Program, 2300 Southern Blvd, Bronx, NY 10460, USA Data Collection We chose a qualitative approach involving guided semi- structured interviews. Qualitative approaches focus on obtaining a small set of high-quality in-depth interviews rather than the large sample sizes characteristic of quantitative sur- veys aimed at producing generalizable observations (Crouch and McKenzie 2006; Moon and Blackman 2014). We chose this approach to explore the complex interplay of perceptions and social influences underpinning bushmeat consumption, factors not easily captured through structured survey ap- proaches yet essential to provide rich contextually explicit knowledge for the development of behavior change cam- paigns. Unstructured key informant interviews, informal dis- cussions, and two focus group discussions informed the core structure and content of the semi-structured interview guide (Supplementary Materials). Key informants consisted of long- term Pointe Noire residents, including NGO personnel. These individuals were willing to share in-depth perspectives on the consumption of bushmeat in Pointe Noire and later helped to identify people to interview. Informal discussions were held with owners of bushmeat restaurants, and separate focus groups were held with five men and five women of a range of ages and from different districts within the city to get an initial feel for the context of the study. Interventions aimed at changing consumer behavior should be guided by an evidence-informed theory of change (Olmedo et al. 2017). This requires developing an in-depth understanding of why people consume bushmeat when other protein choices are available in urban environments. We address this knowledge gap through an in-depth qualitative approach to investigate socio-cultural drivers of bushmeat demand in Pointe Noire, Republic of Congo. Qualitative research is essential for expanding our understanding of the social aspects of conserva- tion (Drury et al. 2011) as it enables investigation of perceptions and social influences underpinning bushmeat consumption, fac- tors not easily captured through structured survey approaches. We identify: 1) perceptions of bushmeat and other animal proteins, 2) social norms and the social context associated with bushmeat consumption, and 3) factors limiting or encouraging bushmeat consumption. The role of social norms in influencing bushmeat consumption has rarely been considered (Morsello et al. 2015) yet understanding the perception of norms is essen- tial to devise effective interventions and promote sustainable behavior (McKenzie-Mohr 2011). Here we define social norms as customary rules shaped by how individuals are expected to behave, and how individuals observe others to behave (Cialdini 2003; Bicchieri et al. 2018). Methods We used convenience and snowball sampling to select re- spondents (Newing 2010). This involved asking key infor- mants, personal contacts, and respondents to suggest individ- uals from a variety of different backgrounds who would be willing to participate. We consciously attempted to ensure het- erogeneity for age, education, profession, wealth, district of residence, and department of origin to capture individuals from a range of strata in Pointe Noire in order to capture a diversity of Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10745-019-0061-z) contains supplementary material, which is available to authorized users. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10745-019-0061-z) contains supplementary material, which is available to authorized users. * Alexandre M. Chausson alexandre.chausson@zoo.ox.ac.uk Reduction of urban demand for bushmeat is therefore essen- tial for biodiversity conservation and rural communities’ well- being. In situations of ineffective regulations and weak law enforcement, interventions targeting consumer preferences of- fer a possible avenue to achieve such a reduction (Rowcliffe et al. 2004). However, to be effective, behavior change inter- ventions must be grounded in an understanding of the socio- cultural drivers underpinning consumer demand patterns. While previous studies have investigated economic factors 1 Present address: Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK 1 Present address: Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK 2 Imperial College London, Silwood Park Campus, Buckhurst Road, Ascot SL5 7PY, UK 3 Institute of Zoology, Zoological Society of London, Regent’s Park, London NW1 4RY, UK 4 Wildlife Conservation Society, Africa Program, 2300 Southern Blvd, Bronx, NY 10460, USA 180 Hum Ecol (2019) 47:179–191 Conservation-focused non-governmental organizations (NGOs) based in Pointe Noire have raised concerns about the extent of bushmeat consumption in the city, which prompted this study. driving bushmeat consumption (East et al. 2005; Wilkie et al. 2005), research on the cultural, social, and psychological di- mensions of urban bushmeat demand is still lacking (van Vliet and Mbazza 2011; Randolph 2016). Although bushmeat consumption in rural areas is often driven by a lack of available and affordable alternatives, in urban centers, bushmeat can be more expensive than other animal proteins and is regarded as a luxury item (Cowlishaw et al. 2005; Shairp et al. 2016). Research has shown bushmeat consumption to be influenced by taste preferences in addition to health, cultural, and spiritual reasons (Schenck et al. 2006; van Vliet and Mbazza 2011). Data Collection Preliminary field-work to inform the construction of the interview guide highlighted that in Pointe Noire bushmeat is perceived as a distinct category of meat. Therefore, we aimed to understand the diversity and complex interplay among social, cultural, and psychological factors un- derpinning the consumption of bushmeat in general. The in- sights gained from this study will be useful for conservation practitioners working on the development of behavior change interventions aimed at reducing the consumption of bushmeat among urban residents in Central Africa. From May to July 2016, the lead author, initially accompa- nied by a local research assistant, conducted 30 semi- structured interviews with Congolese respondents aged 18 years and over residing in Pointe Noire (Supporting Information). Free Prior Informed Consent (FPIC) was obtain- ed prior to each interview, and respondents were informed that they could stop the interview process at any point. The inter- view protocol was reviewed and approved by the ethics com- mittee of the Faculty of Natural Sciences, Imperial College London. We obtained a research permit (#031) from the Institut National de Recherche Forestière (IRF) of the Republic of Congo. Interviews were conducted in French, spoken fluently by all respondents and the research team. We applied care to remain neutral and avoid leading questions and biasing interviews. Follow-up questions were asked dur- ing interviews to explore emerging avenues of discussion. Study Location Interviews ranged in length from 21 minutes to two hours and were concluded when a sense of data richness emerged or due to time restric- tions for the respondent. For interviews that were ended prema- turely, we arranged a follow-up interview in-person or via tele- phone. Detailed notes were taken immediately after each inter- view describing the interview setting, context, and the interviewer-interviewee relationship. Many participants felt un- comfortable being audio recorded, so only eight interviews were recorded with participant consent and later transcribed. For the remaining interviews, the lead author and his assistant took detailed notes with direct quotations. The consumption of bushmeat is not actively controlled in the Republic of Congo, and most respondents appeared comfortable discussing openly their bushmeat consumption. However, in addition to the 30 interviews mentioned, three others were conducted but not an- alyzed due to reliability concerns since these respondents ap- peared uncomfortable talking about bushmeat. Study Location Pointe Noire, a city with a population of 969,000 (UN DESA 2015; Fig. 1), is the economic hub of the Republic of Congo and center of the country’s petroleum industry. It has seen strong population growth in recent decades (Dorier and Joncheray 2013). Hum Ecol (2019) 47:179–191 181 Fig. 1 Map of the Republic of Congo showing Pointe Noire in red, other major cities, protected areas in green, and administrative divisions (departments) f Congo showing Pointe Noire in red, other major cities, protected areas in green, and administrative divisions (departments) Fig. 1 Map of the Republic of Congo showing Pointe Noire in red, other major cities, protected areas in green, and adm (Harding 2013). Broad-brush open coding was applied to the first 10 interviews to categorize responses relating to per- ceptions of bushmeat and other proteins, normative influences on bushmeat consumption, and factors limiting or encourag- ing bushmeat consumption. The lead author subsequently re- fined the coding framework to ensure consistency and used this version to code the remaining interviews, remaining flex- ible for topics and themes not captured in the first 10 inter- views. The coding framework was reviewed again post- coding to ensure clear and identifiable distinctions between each code (Braun and Clarke 2006) and that coded data ex- tracts aligned with code definitions. Themes discussed in this paper were mentioned in two or more interviews, following the data reduction approach for qualitative data (Namey et al. 2008). To facilitate analyzing relationships between themes both within and across interviews, the lead author summarized coded data extracts for each respondent using framework ma- trixes in NVivo. To complement the qualitative analysis, we looked for associations at the individual level between coded perceptions. We tabulated whether each respondent men- tioned each perception and calculated the phi coefficient φ to measure associations for each pairwise combination of perceptions in R (R Development Core Team 2018). The coefficient ranges from −1 to 1, where ±1 indicates perfect association, and 0 indicates no association (Allen 2017). All pairwise combinations of perceptions not reported in the results are available in the Supplementary Materials. We views across socioeconomic backgrounds. A deliberate attempt was also made to balance the gender ratio. We arranged all interviews, apart from one, in advance and conducted inter- views in various settings, including respondent households and urban venues with minimal distractions. Data Coding and Analysis Interview transcripts were qualitatively analyzed in NVivo 11.2 (QSR International 2016) through thematic analysis 182 Hum Ecol (2019) 47:179–191 also tabulated the number of respondents expressing each per- ception to explore within-sample associations among socio- demographic characteristics, bushmeat consumption frequen- cy, and perceptions. However, we intentionally refrain from specifying the number or proportion of respondents express- ing particular views in the narrative synthesis as this is not the objective of qualitative inquiry and doing so may be mislead- ing given the low sample size and purposive sampling ap- proach. Importantly, this can distract from the substantive in- sight that in-depth analyses aim to generate (Patton 2015). To test for bias introduced by including both audio and non-audio transcribed interview transcripts, the lead author plotted the coding output between the two sets (n = 8 and n = 22 respec- tively). The output was proportionally similar suggesting that non-recorded transcripts were reliable (Supplementary Materials). Our approach prioritizes depth of detail from indi- vidual respondents over number of respondents and therefore may not provide an exhaustive list of all perceptions, social norms, and other factors influencing bushmeat consumption in Pointe Noire. However, we feel the data still provide new insight into the main drivers of bushmeat demand in urban centers of the Republic of Congo. of consumption, and most respondents who reported high to very high level of bushmeat consumption were men (at least once a week to several times a week). Just over half of respon- dents ate bushmeat primarily in the household and the remain- der ate primarily in restaurants (formal or informal). We did not aim to determine consumption behavior at the species level, but respondents mentioned consuming a variety of different spe- cies, including pangolin, porcupine, cane rat, monkey, wild pig, crocodile, duiker, bushbuck, and tortoise. Some respondents also differentiated between categories of bushmeat such as smoked versus fresh, or ‘red’ meat (sometimes also re- ferred to as ‘black’ meat) vs. ‘white’ meat (i.e., the meat of reptiles and birds). However, bushmeat was salient as a category of its own: BIn general, it’s important to know that we are Bantu, we are in the great equatorial forest, bushmeat was our sta- ple food...banana, tarot, and bushmeat^ (Respondent 1, 50-year-old male, unemployed engineer). Sample Characterization The importance of organic, natural, fresh, healthy and local in relation to animal proteins was a prevalent theme (Supplementary Materials). Most respondents perceived bushmeat as meeting these criteria, while also highlighting that it is tasty or appetizing - overall the most prevalent char- acteristic attributed to bushmeat (Fig. 3a): The sample was well balanced with respect to age and educa- tion, although most respondents were male (Fig. 2). All but two respondents spent the greater part of their youth in Pointe Noire but traced their roots to eight different regions of the Republic of Congo - Lékoumou, Bouenza, Cuvette, Kouilou, Likouala, Niari, Plateaux, and Pool (Supplementary Materials). In our sample, frequency of bushmeat consump- tion varied from no consumption in 2016, to several times a week (Fig. 2). Because our sampling approach specifically targeted bushmeat consumers, we expect our sample to reflect higher bushmeat consumption frequencies than found at the city-level. BBushmeat has a different taste to other meats, it’s nat- ural, isn’t kept in freezers for long periods, and contains a lot of vitamins because it eats all that is natural^ (Respondent 13, 36-year-old male, security agent). The strong taste of bushmeat (smoked or fresh) appears im- portant in part because it removes the need for condiments. Consumers often mentioned taste or health first, but frequent- ly in parallel with perceptions of bushmeat as natural, fresh, or organic and not farmed. Perceptions of bushmeat as tasty were most strongly associated with perceptions of bushmeat as fresh (φ = 0.26) and local (φ = 0.20). In turn, the perception of bushmeat as fresh was most strongly associated with the perception of bushmeat as healthy (φ = 0.25), which was most strongly associated with the perception of bushmeat as natural (φ = 0.33). This supports the notion that these dimensions are interlinked. Wild animals are said to feed on natural, healthy foods that produce nutritious meat high in vitamins that can be eaten fresh locally. These were key characteristics that distin- guished bushmeat from human reared meat. Bushmeat Consumption There was no significant association within the sample be- tween bushmeat consumption frequency and gender, educa- tion level, age group, region of origin, or profession (Supplementary Materials). Analyzing the relationship be- tween household wealth and bushmeat consumption frequen- cy was beyond the scope of our study. However, most women reported either not consuming bushmeat, or low levels of con- sumption (Supplementary Materials; Fig. S2a). Of the five respondents not reporting consumption in 2016, four were women. Most men however reported mid to very high levels Hum Ecol (2019) 47:179–191 183 When specifically asked why they consumed bushmeat respondents most frequently cited attributes of The Village Environment, Region of Origin, and Cultural Identity Fig. 2 Socio-economic characteristics of respondents (n = 30) and reported bushmeat consumption frequency: a Age category, b Employment category as reported by the respondent (BC = blue-collar, SW = service worker, U = unemployed, WCN = white-collar non- governmental organization, WCG = white-collar government sector, WCP = white-collar private sector), c Education level (highest level com- pleted), and d Bushmeat consumption frequency, see Supporting Information for a detailed respondent summary Hum Ecol (2019) 47:179 191 183 Fig 2 Socio economic characteristics of respondents (n = 30) and governmental organization WCG = white collar government sector governmental organization, WCG = white-collar government sector, WCP = white-collar private sector), c Education level (highest level com- Fig. 2 Socio-economic characteristics of respondents (n = 30) and reported bushmeat consumption frequency: a Age category, b Employment category as reported by the respondent (BC = blue-collar, SW = service worker, U = unemployed, WCN = white-collar non- governmental organization, WCG = white-collar government sector, WCP = white-collar private sector), c Education level (highest level com- pleted), and d Bushmeat consumption frequency, see Supporting Information for a detailed respondent summary The Village Environment, Region of Origin, and Cultural Identity When specifically asked why they consumed bushmeat, respondents most frequently cited attributes of natural and fresh (often mentioned together) after taste and cultural influences (i.e., tradition, region of origin, or the influence of village life) (Fig. 3b). Interestingly, younger respondents emphasized the natural and fresh as- pects of bushmeat as reasons for their consumption of bushmeat, whereas older respondents emphasized cultural drivers (i.e., tradition, region of origin, or the influence of village life) (Supplementary Materials; Fig. S3b). Only young to middle-aged respondents cited the perception of bushmeat as healthy as a reason for consuming it. Interestingly, only men reported the perception of bushmeat as organic. In parallel with the emphasis on the natural properties and source of bushmeat, the continuing influence of rural village life (i.e., cultural influences) emerged as an important theme and was cited by respondents as a prime driver of bushmeat consumption (Fig. 3b). When explaining why they ate bushmeat, respondents often highlighted growing up eating bushmeat in their village, coming from forested zones where bushmeat is the main staple, or how eating bushmeat is part of their tradition or culture as BBantu^ or BAfricans.^ The link between specific regions of the country and bushmeat con- sumption, in comparison with the local coastal ethnic group 184 Hum Ecol (2019) 47:179–191 Fig. 3 a Perceptions of bushmeat, b reasons given for consuming bushmeat, c perceptions of frozen imported animals, d factors limiting bushmeat consumption; bars represent the number of respondents reporting each perception. Refer to code book (Appendix S2) for coding definitions Hum Ecol (2019) 47:179–191 184 Fig. 3 a Perceptions of bushmeat, b reasons given for consuming bushmeat, c perceptions of frozen imported animals, d factors limiting bushmeat consumption; bars represent the number of respondents reporting each perception. Refer to code book (Appendix S2) for coding definitions Fig. 3 a Perceptions of bushmeat, b reasons given for consuming bushmeat, c perceptions of frozen imported animals, d factors limiting bushmeat consumption; bars represent the number of respondents reporting each perception. Refer to code book (Appendix S2) for coding definitions environment in which someone grew up, as highlighted by respondent 18 (31 years old, saleswoman), who stated: ‘Vili’ who traditionally consume fish, was even emphasized by respondents born in the city. Associations between the traditional consumption of certain species and region were sometimes emphasized. The Village Environment, Region of Origin, and Cultural Identity For example, Respondent 6 (small business owner, 53 year-old male), who used to traffic bushmeat to Pointe Noire, stated that: Bin the Lékoumou re- gion, monkey meat is particularly appreciated.^ A few respon- dents associated the consumption of crocodile meat with the forested districts of the north, such as the Niari, Cuvette, and Plateaux departments. This association holds importance for traditional weddings, for which it is customary for the family of the bride to prepare species traditionally consumed in their region as a gift to the future husband and his family. Some respondents also discussed the pleasure of consuming bushmeat when returning to the village to visit relatives. The taste of bushmeat can bring back memories of the BI like the smoked taste, it reminds me of when I was with my grandmother, it gives it an African touch.^ Cultural identity therefore appears to be a driver of bushmeat consumption in Pointe Noire. BI like the smoked taste, it reminds me of when I was with my grandmother, it gives it an African touch.^ Cultural identity therefore appears to be a driver of bushmeat consumption in Pointe Noire. Family Influence Of respondents who consumed bushmeat over the last year, half recalled developing the taste for bushmeat during childhood, often stating that their parents influenced their consumption. Some respondents continued to feed, recommend, or want their children to eat bushmeat, either because of its good taste and healthy properties in comparison to frozen animal proteins, or to maintain tradition. However, in light of its high prices, sev- eral parents explained that they often feed cheaper frozen meats to their children. Nearly half of respondents, all 36 years or older, mentioned generational variation in food preferences and eating habits with the older generation having a strong preference for bushmeat or fish, while young people growing up in the city do not eat or even like bushmeat: BThe young do not try bushmeat, it’s a problem in light of the issues with frozen meats. The young here, they don’t want to try [bushmeat] they’re not used to it^ (Respondent 10, 40-year old, salesman). The perception of bushmeat as a luxury item appears to confer a special status to those who can afford to provide or consume it, thus influencing both purchasing and consumption. Respondent 7 stated that: Bin restaurants, when you arrive and ask for a bushmeat dish people look at you. It’s exceptional to order bushmeat at a restaurant.^ In Pointe Noire, bushmeat is often eaten during weekend social gatherings with friends and family members. Just as bushmeat can be used as a gift to obtain favors, it also appears to play an important social func- tion in forging and maintaining relationships; for example, Respondent 18 primarily ate bushmeat in various restaurants where she would meet friends after work. In fact, most young respondents (18–35 years old) ate bushmeat primarily outside of the household, whereas only a few respondents 36 years and They have become accustomed to eating and have developed a taste for frozen meats, including imported chicken, which has become widely available. Both men and women respondents emphasized that household bushmeat consumption was driven by the male household head, for whom bushmeat was preferentially reserved as a more exclusive or luxurious meat. Of those who can afford to consume bushmeat on a day-to-day basis, it tends to be the male household head requesting it. Respondent 2, a 57-year-old government employee ex- plained: B…I send my wife. Bushmeat to Please, Honor, Show Appreciation, and Obtain Favors Bushmeat to Please, Honor, Show Appreciation, and Obtain Favors BAmong our forefathers, women could not eat certain bushmeat, such as monkey or python…However, with the world that is developing, women now eat it^ (Respondent 19, 31-year-old stay-at-home mother). The drive to provide and offer bushmeat to important individ- uals or for special ceremonies appears to be a predominant influence on bushmeat purchasing in Pointe Noire. Bushmeat was perceived as a status symbol and reported to give recogni- tion to the provider and the receiver. BWe feel pressured to serve bushmeat because it’s precious, …bushmeat is special and it’s not to be eaten everyday^ (Respondent 14). BI am proud of eating bushmeat…it’s not anyone that eats bushmeat here in Pointe Noire…it’s rare in households^ (Respondent 30, 43- year-old male, unemployed). Serving bushmeat was associ- ated with a desire to please, reward, honor, show appre- ciation, or obtain favors from important individuals such as partners, parents, close family members or friends, au- thorities, or influential private or public-sector employees. It was often mentioned that small amounts of bushmeat are reserved for important individuals due to its high price. Respondent 24 (bushmeat vendor, 40 years old) stated that: Bmany clients buy bushmeat for traditional weddings, fu- nerals, or birthdays.^ And the important role of bushmeat at such events, including impressing important guests, was reiterated by many respondents. However, other re- spondents reported not being used to seeing bushmeat at festivities, citing as a reason its high cost. Bushmeat as Rare, Prized, Luxurious, and Exclusive Bushmeat is considered a luxury food, often reported as rare, prized, luxurious, and exclusive (Fig. 3a). Some respondents as- sociated the reported rarity and luxury of bushmeat directly with its perceived natural, organic, and fresh characteristics. Respondent 7 (36–45 years old male, city government official) Hum Ecol (2019) 47:179–191 185 stated that: Bpeople know its importance, its value, because it [bushmeat] is pure, it’s natural.^ This perception of luxury alone also appears to attract people to bushmeat. Some specifically stated that they consume bushmeat Bbecause it is rare^ (Respondent 3, 56+ year-old woman, retired). Respondent 7 in- dicated that bushmeat is not something that everyone gets the opportunity to eat regularly, but Bwhen it’s on the table during a festivity, people jump on it.^ Bushmeat Badds value to an event^ (Respondent 14, 34-year-old male, logistician), which reflects social norms influencing bushmeat demand in Pointe Noire. older ate primarily in restaurants (Supplementary Materials; Fig. S6). Younger respondents, most of whom did not live with a partner at home, emphasized the social purpose of consuming bushmeat outside of the household to forge relationships. Older respondents are more likely to be married and settled in a household, and therefore more likely to consume bushmeat at home. A few respondents explained that bushmeat is consumed primarily by men while women are more often responsible for preparation. This was sometimes attributed to the legacy of traditional norms that restrict women from the consumption of certain bushmeat species. However, this appears to be changing in Pointe-Noire: Social Norms Associated with Bushmeat Demand Bushmeat to Please, Honor, Show Appreciation, and Obtain Favors Factors Limiting Bushmeat Consumption with bushmeat consumption, including that excessive bushmeat consumption can lead to gout, although they asso- ciated this exclusively with ‘red’ bushmeat. Several reported being aware of recommendations to avoid bushmeat due to Ebola outbreaks, but only a few listed concerns over Ebola as a limiting factor, in part because they did not perceive Ebola to be a threat in the Republic of Congo. For example, Respondent 12, a 25-year-old male mechanic who consumes bushmeat at least once a week, reported that people have avoided bushmeat for concerns over Ebola, but stated: Bwe’re lucky that here in Congo [Republic] we didn’t really have the epidemic, unlike in neighboring countries where there were many cases.^ Family Influence It’s her that often buys it at my request and sometimes she suggests buying bushmeat knowing that I like to eat bushmeat.^ Hum Ecol (2019) 47:179–191 186 Law Enforcement No respondents were asked about knowledge of the law. However, half expressed awareness of hunting restrictions (seasonal- or species-based), protected areas, or the control of bushmeat transportation: BWhen the hunting season is closed it [bushmeat] is more expensive. The transport is exposed to more sur- veillance by the authorities^ (Respondent 23, 36-year- old male, event organizer). Reduced availability and increasing prices were mentioned by nearly half of respondents, with several attributing this directly to overhunting or animals being Brarer and rarer in the bush^ (Respondent 8, 32-year-old male, unemployed). Others attributed lower availability and increased prices to hunting regulations, law enforcement, or conservation measures. However, only one respondent reported reduc- ing their bushmeat consumption because of concern about overhunting. Nearly all these respondents perceived that restrictions in- creased prices or decreased availability. Eight respondents suggested the regulatory environment is ineffective as bushmeat is still available during the closed hunting season (November 1 to April 30), albeit at a higher price due to bribes reportedly paid to authorities to facilitate transportation. Importantly, no one mentioned legal restrictions associated with bushmeat purchasing. Despite the protected status of some species according to national law, respondents were still happy to discuss consuming species such as pangolin (see Republic of Congo Law No. 37–2008 on Wildlife and Protected Areas). Price and Availability All respondents, regardless of age or gender, emphasized the high price of bushmeat while explaining that it is reserved for special occasions or important individuals. Price was the most frequently cited barrier to purchasing and consuming bushmeat, followed by low availability, quality, disease, and dietary concerns (Fig. 3d). Four of the five respondents reporting no bushmeat consumption during 2016 cited high prices as the reason. There was no apparent relationship be- tween bushmeat consumption frequency and reported limiting factors (Supplementary Materials). Respondents reported circumventing price barriers in various ways, including buy- ing bushmeat from wholesalers in Pointe Noire or obtaining it outside the city, and eating at cheaper restaurants in lower- income neighborhoods. Respondents also mentioned that it is possible to buy bushmeat in smaller chopped-up portions according to what one can afford. Price and availability are linked, but price seems to be the main perceived barrier, as one responded commented: Bif you have financial means, you can find bushmeat any day^ (Respondent 28, 50-year-old male, football coach). Disease Risks and Food Safety Half of our respondents expressed concerns about contagious diseases and the lack of food safety relating to bushmeat, suggesting that negative perceptions coexist alongside posi- tive perceptions of bushmeat, although the former do not nec- essarily limit levels of consumption. These concerns were often dependent on the state or source of the bushmeat. For example, several respondents expressed concerns about smoked bushmeat, which they believed to be an inefficient means of preservation, and suspected it was often sold long after slaughter. Yet the same individuals did not express such concerns about fresh bushmeat. Others had concerns about how bushmeat is sold, preferring certain sellers, such as wholesalers at the edge of town with whom they have an established relationship, or specific locations to obtain fresher bushmeat, such as villages outside the city rather than the open-air markets in Pointe Noire. A few, generally older, re- spondents mentioned concerns about side-effects associated Perceptions of Other Animal Proteins For example: B… one portion of bushmeat for 5000 FCFA [8.65 USD] costs the same as buying five days’ worth of fro- zen meat supplies at the market^ (Respondent 13, 36- year-old male, security agent). B… one portion of bushmeat for 5000 FCFA [8.65 USD] costs the same as buying five days’ worth of fro- zen meat supplies at the market^ (Respondent 13, 36- year-old male, security agent). BEven on television sometimes, we see how things [fro- zen imported meats] were thrown away because they were expired… Therefore, it’s not good. We must not consume it anymore.^ However, every respondent but one held negative perceptions of frozen animal proteins (Fig. 3c). There were no apparent associations between reported perceptions and gender, educa- tion level, or age, although older (56+) respondents did not mention concerns associated with the high fat content of fro- zen imported proteins (Supplementary Materials; Fig. S4b). A customs officer confirmed that imports of past sell by-date frozen meats had been intercepted by customs, and that im- porters regularly try to place spoiled meats for sale on the market (Respondent 29, 47-year-old female). A dietician re- ported that in medical establishments people are also advised to eat fresh and local products rather than frozen if they can afford to (Respondent 22, 41-year-old female). Respondent 7 stated BIn the hospital they recommended she [his sister] eat natural things, and not frozen imported meats in which prod- ucts are injected and make you sick,^ subsequently clarifying that Bit’s necessary to eat bushmeat because it’s from the bush, the blood is fresh.^ The dietician stated that: Bfresh meat con- serves its nutritional value whereas freezing leads to its degradation.^ In fact, several respondents emphasized bushmeat as a healthier alternative to frozen animal proteins, and many reported either giving or receiving advice to con- sume bushmeat for this reason, including Respondent 13 (36- year-old male, security agent) who recommended bushmeat consumption to others because: B…when you eat a lot of bushmeat you age more slowly and rarely fall ill.^ Frozen animal proteins were said to cause illness and short- en life spans (Fig. 3c). Respondents complained about not knowing where or how the meat was raised and slaughtered, and when and how it was transported and stored, which af- fected perceptions of quality. Perceptions of Other Animal Proteins The lack of taste of frozen imported meats was a common complaint and was associated with concerns over the quality of frozen imported proteins (φ = 0.34) and health consequences stemming from their con- sumption (φ = 0.24). Respondents shared their reservations about intensive livestock rearing, with animals subjected to Binjections^ and Bchemicals^ creating Bartificial^ meat. They also mentioned lengthy transport and storage periods, and lack of quality control and safety regulations. In contrast, fresh animal proteins are seen as subjected to less intensive production methods. Many respondents expressed a sense of both hopelessness and helplessness when discussing the real- ity that households are forced to resort to poor quality meats not subject to rigorous controls to feed themselves because they cannot afford healthier alternatives: Perceptions of Other Animal Proteins The theme of organic, natural, fresh, and local extended to other animal proteins locally caught or reared and butchered (hereafter referred to as fresh animal proteins). Locally butch- ered livestock and fish were perceived positively by most respondents who emphasized their Bfresh^ and Borganic^ characteristics, with fish often also perceived, like bushmeat, as Bnatural.^ Bushmeat consumers perceived fresh animal proteins as healthier and more nutritious because they are not subject to freezing and or other human interference. It is this perception of minimal interference that respondents termed Borganic^, or Bbio^ in French, which does not directly map onto the standard definition of Borganic^ as produced Hum Ecol (2019) 47:179–191 187 perceptions of frozen imported meats. Respondents stated lack of quality and taste, often in conjunction with negative health impacts in contrast to the positive qualities of fresh animal proteins, including bushmeat. Quality concerns of frozen imported meats were most strongly associated with the per- ception of bushmeat as natural (φ = 0.36), followed by healthy (φ = 0.29). Concerns over lack of taste were most strongly associated with perceptions of bushmeat as fresh (φ = 0.35), healthy (φ = 0.33), and natural (φ = 0.27). without use of artificial chemical fertilizers, pesticides, or oth- er non-organic inputs. The most frequently mentioned Bfresh^ non-fish animal proteins were locally reared and butchered beef, chicken, and pork. The bushmeat vendor (Respondent 24) explained that some clients state they do not eat frozen meats, and instead look for organic products such as bushmeat, fresh beef, or fresh fish. However, a few respon- dents expressed concerns over specific production and selling practices, including fish rotting on market stalls having been caught using dynamite, or poor conditions for pigs and other livestock around Pointe Noire. All respondents reported receiving advice to avoid frozen animal proteins due to the poor quality and negative health impacts, and to eat fresh products. This information came from a variety of sources including the media, schools, med- ical professionals practicing western medicine, and the public health department through public notices, as well as through friends, relatives, and general word of mouth. Respondent 20, a 45-year-old stay-at-home mother recalled: Respondents reported that the most abundant, affordable, and consumed animal proteins were frozen and imported (hereafter referred to as frozen animal proteins). Discussion BWe are forced to fall back on the frozen [imported meats] to have a reasonable quantity. Otherwise, every- body would prefer to eat fresh meat^ (Respondent 26, 51-year-old male, unemployed). The perceptions of bushmeat as a natural, organic, healthy, tasty, luxury product, associated with rarity and high prices, appear to drive its consumption and reinforce the social norm of offering bushmeat to important individuals and on impor- tant ceremonial occasions. Our findings corroborate previous studies that have shown bushmeat in central African urban Interestingly, respondents reporting infrequent bushmeat consumption were more vocal about their negative 188 Hum Ecol (2019) 47:179–191 areas is considered to be a luxury good (Cowlishaw et al. 2005; Randolph 2016) as well as a healthy, nutritious, and natural food (Kümpel 2006). In Yaoundé, Cameroon, and in Ho Chi Minh City, Vietnam, bushmeat has been shown to signal wealth (Randolph 2016; Shairp et al. 2016). While the luxury perception may be taxon-dependent, as it was in the Yaoundé study, our results suggest that bushmeat in gen- eral is considered a luxury. In addition, bushmeat in Pointe Noire contributes to building and maintaining social relation- ships, as was also shown to be the case in Brazil and Vietnam (Morsello et al. 2015; Shairp et al. 2016). Bushmeat con- sumption appears to provide strong cultural ties connecting urbanites to their villages and regions of origin (see also van Vliet and Mbazza 2011). Bushmeat consumption forms a part of the cultural identity of those who trace their origins to forested regions. Cultural associations have also been shown to drive bushmeat consumption among urban Amazonians (Morsello et al. 2015), as well as in Bata, Equatorial Guinea, where ethnicity and nationality were found to be key determi- nants of consumption (East et al. 2005). Deconstructing behavioral complexity is a prerequisite to identifying high-leverage behavior change intervention strat- egies. Our results show that bushmeat demand is associated with multiple social norms, perceptions, and other drivers. Specific drivers rooted in perceptions and norms should be targeted individually by tailoring messages and identifying the most appropriate channels to communicate these messages to a specific segment of the population (Michie et al. 2011). For example, a strategy aimed at reducing bushmeat provi- sioning during special occasions, a practice reinforced by the perception of luxury and the desire to please or gain favor, would require a different approach than reducing day-to-day consumption of bushmeat. Developing Effective Messages Messages must be culturally sensitive to resonate with bushmeat purchasers. Highlighting the negative impacts of a behavior risks reinforcing it if this message clashes with pre- vailing norms and culture (Schultz 2011; Lertzman and Baragona 2016). Though a substantial proportion of con- sumers are aware of overhunting, respondents did not indicate any intention to reduce consumption. Raising environmental awareness alone is unlikely to affect de- mand. High environmental awareness levels and support for environmental protection are often not matched by behavioral changes, usually due to factors perceived to be beyond the individual’s control (Kühl et al. 2009; Heberlein 2012). Positive social messages, rather than negative environmental messages, often align better with the priorities of the target audience. For instance, com- munication targeted at climate change deniers was more effective when the emphasis was placed on improved social welfare outcomes of climate mitigation efforts rather than risks and realities of climate change (Bain et al. 2012). A positive messaging strategy in Pointe Noire could focus on the importance consumers place on the natural sourcing of bushmeat, the forest, to em- phasize the benefits provided by forests to people, build- ing pride in this national resource. Discussion Our results also suggest that drivers of consumption can vary according to socio- demographic characteristics. For example, younger respon- dents emphasized the natural, fresh, and healthy aspects of bushmeat, whereas older respondents emphasized cultural connections. City-wide surveys should be conducted to con- firm these relationships and tailor messaging to specific seg- ments of the population. East et al. (2005) found that consumers differentiated ani- mal proteins first and foremost by state, with a strong preference for fresh over frozen, as Starkey (2004) also found in Libreville, Gabon. In stark contrast to the positive percep- tions of bushmeat, our study also found a widespread disdain towards frozen animal proteins due to negative health out- comes, quality concerns, as well as taste. In Pointe Noire, it appears that positive perceptions associated with the notions of fresh and organic extend beyond bushmeat to other animal proteins, including locally butchered livestock and locally caught fish. We found the health and taste dimensions are interlinked determinants of consumer preference for protein types. Animal proteins subject to limited human interference are considered Borganic^ and are perceived as fresher, tastier, and healthier. Fish sold fresh and any form of locally butch- ered meat, including bushmeat, meet this criterion. In contrast, frozen animal proteins are seen as transformed by intensive husbandry, the freezing process, and overly lengthy storage. The disdain towards frozen animal proteins, reinforced by medical, media, and social pressures, coupled with percep- tions of bushmeat as tasty and healthy, emerged in this study as key drivers of bushmeat demand. Harnessing the Support of Local Actors and Institutions The reach and uptake of a message is heavily dependent on who presents it (McKenzie-Mohr 2011), so that harnessing the support of locally influential figures and institutions as mes- sengers to change perceptions and beliefs could be an effec- tive way to bring about behavior change. For example, in Central Africa, including in the Republic of Congo, faith leaders are socially and politically very influential, particularly with the growing influence of Protestant groups (Fath and Mayrargue 2014). Since bushmeat consumption is customary on weekends, messages disseminated during Sunday worship could be effective. Our findings do not quantify the extent to which medical professionals or the media specifically promote bushmeat consumption when advising people to avoid frozen animal proteins. However, these actors clearly exert an impor- tant influence on dietary choices and may indirectly incite consumers to turn towards bushmeat through their advice. Interventions could potentially involve these actors to ensure they avoid promoting bushmeat demand. Respondent statements highlighted a potential Veblen ef- fect driving bushmeat consumption, whereby the conspicuous consumption of bushmeat acts as a status symbol because of its high price (Bagwell and Bernheim 1996). Therefore, con- sumers are more likely to switch to prized local fresh meats such as beef or expensive fish. Positive perceptions attributed to fresh fish among bushmeat consumers and evidence for economic substitution of fish and bushmeat in similar urban coastal contexts would suggest that fish protein might be a promising alternative (Brashares 2004). However, perceptions attributed to bushmeat and other available animal proteins may not be static. That younger people are reported to be more accepting of frozen animal proteins is potentially promising for reduced bushmeat consumption in future. Yet it is unclear how this will play out, since general distain for frozen meat in parallel with bushmeat being perceived positively could influence the dietary habits of younger generations as they exit the household. Introducing environmental education activities in schools to raise awareness of urban bushmeat consumption impacts could be crafted to preemptively counter social pressures promoting bushmeat consumption. We found bushmeat consumption is a customary practice in Pointe Noire. Structural elements and systemic influences that maintain the practice, such as informal and formal restaurants selling bushmeat, should also be targeted (Shove 2010). Without complementary actions tackling systemic elements driving bushmeat consumption, behavior change interven- tions may fall short of their goals. Facilitating a Product Switch frozen animal proteins show that consumers are cognizant of their eating habits in relation to health, or more importantly what they perceive to be healthy. Interventions could highlight the negative health impacts of bushmeat overconsumption (e.g., gout), which some respondents and the dietician (Respondent 22), were concerned about. In Yaoundé, this was the reason most frequently given for stopping bushmeat consumption (Randolph 2016). Campaign strategies should not use zoonotic disease threats from bushmeat consumption for conservation purposes as this could have unintended re- percussions, such as eradication of disease vectors or undermining confidence in the campaign among consumers who do not perceive disease to be a threat in the region (Pooley et al. 2015). Changing bushmeat consumption behavior implies con- sumers will turn towards other protein sources. Interventions should combine messages about bushmeat with a careful at- tempt to harness positive perceptions associated with other protein sources to encourage consumers to switch. Understanding perceptions and social norms associated with other animal proteins can provide insight into necessary attri- butes of substitutable alternatives. Because they are afford- able, frozen animal proteins are already consumed in large quantities in Central Africa (Nasi et al. 2011), but there is also widespread disdain towards frozen animal proteins, to a large extent driven by what appear to be serious product quality issues that would need to be addressed. The well-being of people targeted by behavior change interventions should re- main a core focus of any strategy. Therefore, any promotion of alternatives to bushmeat would likely be more effective if it concentrated on local fish, livestock, and poultry, which con- sumers perceive more positively. 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Harnessing the Support of Local Actors and Institutions Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appro- priate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References Data Availability Statement The data that support the findings of this study are available from the Wildlife Conservation Society but restric- tions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of the Wildlife Conservation Society. de Merode, E., and Cowlishaw, G. (2006). 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Determinants of urban bushmeat consumption in Rio muni, Equatorial Guinea. Biological Conservation 126(2): 206–215. Harnessing the Support of Local Actors and Institutions Therefore, in addition to intervention strategies designed to target individual bushmeat consumers, infrastructural elements that sustain bushmeat consumption also need to be targeted where possible. For example, restaurants could be targeted to promote non- bushmeat dishes, thus supporting messaging aimed at consumers themselves. Similarly, establishments, such as hotels or private companies that facilitate consumption by providing bushmeat to their guests and employees for special events, as reported by Respondent 27, a hotel em- ployee, could be targeted. In addition to institutional and economic barriers to the intensification of livestock production (McDermott et al. 2010), the impacts of substituting bushmeat with local live- stock, poultry, or fish can present new problems. 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https://openalex.org/W4283031062	https://www.frontiersin.org/articles/10.3389/fsurg.2021.771785/pdf	English	null	SENP1 Aberrance and Its Linkage to Clinical Features, Adjuvant Regimen, and Prognosis in Patients With Surgical Non-small Cell Lung Cancer Receiving Adjuvant Chemotherapy	Frontiers in surgery	2,022	cc-by	7,030	Keywords: small ubiquitin-like modiﬁer-speciﬁc protease 1, surgical non-small cell lung cancer, clinical features, adjuvant chemotherapy regimen, prognosis ORIGINAL RESEARCH published: 17 June 2022 doi: 10.3389/fsurg.2021.771785 Reviewed by: Reviewed by: Xiao-Ming Hou, First Hospital of Lanzhou University, China Antonio Mazzella, European Institute of Oncology (IEO), Italy Francesco Zaraca, Ospedale di Bolzano, Italy Reviewed by: Xiao-Ming Hou, First Hospital of Lanzhou University, China Antonio Mazzella, European Institute of Oncology (IEO), Italy Francesco Zaraca, Ospedale di Bolzano, Italy Correspondence: Yuquan Ma qunaishi3028407@163.com Methods: Tumor and adjacent tissues were collected from 157 patients with surgical NSCLC receiving adjuvant chemotherapy. Meanwhile, tumor tissue and paired adjacent tissue specimens were obtained to evaluate SENP1 protein expression by immunohistochemistry (IHC) assay; among which, 102 pairs were used to detect SENP1 messenger RNA (mRNA) by reverse transcription quantitative PCR. Results: SENP1 IHC score and SENP1 mRNA expression were increased in tumor tissue than adjacent tissue (p < 0.001). Besides, elevated SENP1 IHC score was correlated with > 5 cm tumor size (p = 0.045), lymph node metastasis occurrence (p = 0.003), and advanced tumor-node-metastasis (TNM) stage (p = 0.012); meanwhile, increased SENP1 mRNA expression was associated with histopathological subtype (p = 0.011), lymph node metastasis occurrence (p = 0.008), and higher TNM stage (p = 0.015). Besides, no correlation was found in SENP1 IHC score (p = 0.424) or mRNA expression (p = 0.927) with speciﬁc adjuvant chemotherapy regimen. Additionally, both the SENP1 protein (high) (p = 0.003) and mRNA high (p = 0.028) were correlated with poor disease-free survival (DFS), while SENP1 protein high was also associated with shorter overall survival (OS) (p = 0.029). Furthermore, SENP1 protein (high vs. low) was independently associated with unsatisfying DFS [p = 0.009, hazard ratio (HR) = 1.798] and OS (p = 0.049, HR = 1.735). Correspondence: Yuquan Ma qunaishi3028407@163.com Specialty section: This article was submitted to Thoracic Surgery, a section of the journal Frontiers in Surgery Specialty section: This article was submitted to Thoracic Surgery, a section of the journal Frontiers in Surgery Received: 07 September 2021 Accepted: 26 November 2021 Published: 17 June 2022 Received: 07 September 2021 Accepted: 26 November 2021 Published: 17 June 2022 Background: Small ubiquitin-like modiﬁer-speciﬁc protease 1 (SENP1) plays vital roles in cancer progression and chemoresistance, but its prognostic value in non-small cell lung cancer (NSCLC) is vague. This study aimed to explore the correlation of SENP1 with clinical features, adjuvant chemotherapy regimen, and prognosis in patients with surgical NSCLC receiving adjuvant chemotherapy. Edited by: Luca Bertolaccini, European Institute of Oncology (IEO), Italy Citation: Yang Q, Yang M, Zhang J and Ma Y (2022) SENP1 Aberrance and Its Linkage to Clinical Features, Adjuvant Regimen, and Prognosis in Patients With Surgical Non-small Cell Lung Cancer Receiving Adjuvant Chemotherapy. Conclusion: SENP1 may serve as a potential biomarker to improve the management of patients with surgical NSCLC receiving adjuvant chemotherapy. Front. Surg. 8:771785. doi: 10.3389/fsurg.2021.771785 June 2022 \| Volume 8 \| Article 771785 1 Frontiers in Surgery \| www.frontiersin.org SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. Items Therefore, this study measured the expression of SENP1 by immunohistochemistry (IHC) assay and reverse transcription quantitative PCR (RT-qPCR) detection, with the objective to explore the correlation of SENP1 expression with clinical features, chemotherapy regimen, and prognosis in patients with surgical NSCLC receiving adjuvant chemotherapy. INTRODUCTION scores 0–1; (v) underwent NSCLC surgical resection and adjuvant chemotherapy; and (vi) had available specimens to perform IHC assay. The exclusion criteria were: (i) had history of other cancers or malignancies at diagnosis; (ii) underwent chemotherapy or radiotherapy before surgical resection; and (iii) without complete clinical characteristics and survival data for analysis. This study was approved by the Institutional Review Board. Lung cancer is a common human cancer with an incidence of nearly two million cases per year and a death number of 1,796,144 in 2020 worldwide (1, 2). Accounting for the majority (80% to 85%) of all the cases of lung cancer, non-small cell lung cancer (NSCLC) is often caused by environmental and genetic factors with cigarette smoking being the major one (3, 4). Over the past two decades, advancements in treating NSCLC have been achieved and adjuvant chemotherapy is recommended for patients with NSCLC at stage II and stage IIIA following surgery (5–7). Although adjuvant chemotherapy improves their survival outcome to some extent, their prognosis remains unsatisfactory due to the high incidence of recurrence (8–10). Therefore, to improve the survival outcome and the management of patients with NSCLC, it is necessary to ﬁnd out new biomarkers. Collection of Clinical Data The following clinical characteristics of all the patients were recorded in this study: age, gender, smoking, drinking, TABLE 1 \| Clinical characteristics. Items NSCLC patients (N = 157) Age (years), mean ± SD 61.4 ± 10.7 Gender, No. (%) Female 29 (18.5) Male 128 (81.5) Smoking, No. (%) 90 (57.3) Drinking, No. (%) 60 (38.2) Hypertension, No. (%) 47 (29.9) Hyperlipidemia, No. (%) 48 (30.6) Diabetes, No. (%) 22 (14.0) Histopathological subtype, No. (%) ADC 84 (53.5) SCC 54 (34.4) ASC 19 (12.1) Differentiation, No. (%) Well 37 (23.6) Moderate 68 (43.3) Poor 52 (33.1) Tumor size (cm), median (IQR) 6.0 (4.0–8.0) Lymph node metastasis, No. (%) 70 (44.6) TNM stage, No. (%) II 90 (57.3) III 67 (42.7) ECOG PS score, No. (%) 0 122 (77.7) 1 35 (22.3) CEA (ng/mL), median (IQR) 5.6 (2.8–33.4) CA125 (U/mL), median (IQR) 34.5 (14.0–77.9) Adjuvant chemotherapy regimen, No. (%) NP 79 (50.3) TP 24 (15.3) GP 25 (15.9) DP 29 (18.5) NSCLC, non-small cell lung cancer; ADC, adenocarcinoma; SCC, squamous cell carcinoma; ASC, adenosquamous carcinoma; IQR, interquartile range; TNM, tumor- y Small ubiquitin-like modiﬁer-speciﬁc protease 1 (SENP1) is a nuclear protease, which deconjugates small ubiquitin-like modiﬁer (SUMO) ylated proteins (11). It is reported that SENP1 participates in the progression of various cancers. For instance, by mediating deSUMOylation of ubiquitin-conjugating enzyme E2T (UBE2T) and the subsequent protein kinase B (Akt) pathway, SENP1 promotes tumor progression in hepatocellular carcinoma (HCC) (12); meanwhile, by modulating epithelial–mesenchymal transition (EMT), SENP1 plays a vital role in invasion and migration of HCC cells (13); by regulating phosphatase and tensin homolog (PTEN) stability, SENP1 facilitates prostate cancer progression (14); SENP1 also involves in irinotecan resistance in colon cancer (15). Besides, in terms of NSCLC, it is suggested that overexpression of SENP1 in NSCLC relates to chemotherapy resistance. For example, one study suggests that SENP1 expression in tumor is negatively correlated with treatment response in patients with NSCLC (16); another study reports that SENP1 is a potential predictive factor for chemosensitivity in patients with NSCLC (17). Based on the above information, we hypothesized that SENP1 might be a potential biomarker for patients with surgical NSCLC receiving adjuvant chemotherapy. However, no previous study investigated this issue. Assay y Among 157 patients with NSCLC, a total of 102 tumor tissue and paired adjacent tissue specimens, which were frozen at −80◦C, were accessible for RT-qPCR assay to detect the expression of SENP1 messenger RNA (mRNA). The sample was treated by TRIzolTM Reagent (Thermo Fisher Scientiﬁc, Waltham, Massachusetts, USA) to extract total RNA, which was then submitted to perform reverse transcription using the iScriptTM cDNA Synthesis Kit (with random primer) (Bio-Rad, Hercules, California, USA). After that, qPCR was carried out with the QuantiNova SYBR Green PCR Kit (Qiagen, Duesseldorf, Nordrhein-Westfalen, Germany). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was served as reference gene. The quantitative analysis of SENP1 mRNA expression was conducted with the use of 2−11Ct method. Primers were designed referring to a previous study (21). According to the median value (2.593) of SENP1 mRNA in NSCLC tumor, the expression of SENP1 mRNA was classiﬁed as high (>2.593) and low (≤2.593). Patients This retrospective study reviewed 157 patients with NSCLC who received surgical resection and adjuvant chemotherapy in our hospital between January 2016 and December 2019. The screening criteria were: (i) diagnosed as primary NSCLC according to the European Society for Medical Oncology (ESMO) clinical recommendation (18); (ii) aged over 18 years; (iii) tumor-node-metastasis (TNM) stages II-III; (iv) the Eastern Cooperative Oncology Group Performance Status (ECOG PS) NSCLC, non-small cell lung cancer; ADC, adenocarcinoma; SCC, squamous cell carcinoma; ASC, adenosquamous carcinoma; IQR, interquartile range; TNM, tumor- node-metastasis; ECOG PS, Eastern Cooperative Oncology Group Performance Status; CEA, carcinoembryonic antigen; CA125, cancer antigen 125; NP, vinorelbine + cisplatin; TP, taxol + cisplatin or carboplatin; GP, gemcitabine + cisplatin or carboplatin; DP, docetaxel+ cisplatin or carboplatin. Frontiers in Surgery \| www.frontiersin.org June 2022 \| Volume 8 \| Article 771785 2 SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. Immunohistochemistry Assay y y Tumor tissue specimens and paired adjacent tissue specimens of all the patients were used to assess the expression of SENP1 protein by IHC assay. The rabbit monoclonal anti- SENP1 antibody (1:250; Abcam, Waltham, USA) was applied as primary antibody and the goat anti-rabbit immunoglobulin G (IgG) (H&L) (1:2,000; Abcam, Waltham, USA) was applied as secondary antibody (20). Staining images were taken with a light microscope and IHC results were graded according to intensity and density of staining cells: (i) staining intensity: 0 (negative), 1 (weak), 2 (moderate), and 3 (strong) and (ii) staining density: 0 (0%), 1 (1–25%), 2 (26–50%), 3 (51–75%), and 4 (76–100%). The IHC score was calculated by the product of the staining intensity score and staining density score. Based on the IHC score, SENP1 protein expression was classiﬁed as high (IHC score > 3) and low (IHC score ≤3). Reverse Transcription Quantitative PCR Assay hypertension, hyperlipidemia, diabetes, histopathological subtype, diﬀerentiation, tumor size, lymph node metastasis, TNM stage, the ECOG PS score, carcinoembryonic antigen (CEA), and cancer antigen 125 (CA125). Besides, the adjuvant chemotherapy regimen was also recorded, which included vinorelbine + cisplatin (NP), taxol + cisplatin or carboplatin (TP), gemcitabine + cisplatin or carboplatin (GP), and docetaxel + cisplatin or carboplatin (DP). The follow-up was performed by clinic visit or telephone and the ﬁnal date of follow-up was June 30, 2021. The median follow-up duration was 4.2 years with a 95% CI of 3.8 to 4.6 years, which was estimated using the reverse Kaplan–Meier (KM) method (19). Survival data were collected to assess disease-free survival (DFS) and overall survival (OS). hypertension, hyperlipidemia, diabetes, histopathological subtype, diﬀerentiation, tumor size, lymph node metastasis, TNM stage, the ECOG PS score, carcinoembryonic antigen (CEA), and cancer antigen 125 (CA125). Besides, the adjuvant chemotherapy regimen was also recorded, which included vinorelbine + cisplatin (NP), taxol + cisplatin or carboplatin (TP), gemcitabine + cisplatin or carboplatin (GP), and docetaxel + cisplatin or carboplatin (DP). The follow-up was performed by clinic visit or telephone and the ﬁnal date of follow-up was June 30, 2021. The median follow-up duration was 4.2 years with a 95% CI of 3.8 to 4.6 years, which was estimated using the reverse Kaplan–Meier (KM) method (19). Survival data were collected to assess disease-free survival (DFS) and overall survival (OS). Clinical Characteristics A total of 157 patients with NSCLC who received surgical resection and adjuvant chemotherapy were included for analyses; the mean age of these patients was 61.4 ± 10.7 years including 29 (18.5%) female patients and 128 (81.5%) male patients. Their clinical characteristics are shown in Table 1. With respect to the histopathological subtype, there were 84 (53.5%), 54 (34.4%), and 19 (12.1%) patients with adenocarcinoma, squamous cell carcinoma, and adenosquamous carcinoma, respectively. Meanwhile, 37 (23.6%) patients had well diﬀerentiation, 68 (43.3%) patients had moderate diﬀerentiation, and 52 (33.1%) patients had poor diﬀerentiation. Besides, there were 90 (57.3%) and 67 (42.7%) at TNM stages II and III, respectively. Concerning adjuvant chemotherapy regimen, the number of patients who received NP, TP, GP, and DP was 79 (50.3%), 24 (15.3%), 25 (15.9%), and 29 (18.5%), respectively. Small Ubiquitin-Like Modiﬁer-Speciﬁc Protease 1 Expression in the Tumor Tissue and Adjacent Tissue The expression of SENP1 protein was assessed in the tumor tissue and paired adjacent tissue by IHC assay (Figure 1A). Meanwhile, SENP1 IHC score was higher in the tumor tissue than in the adjacent tissue (n = 157) (p < 0.001) (Figure 1B); SENP1 mRNA expression in the tumor tissue was also elevated than that in the adjacent tissue (n = 102) (p < 0.001) (Figure 1C). Statistical Analysis SENP1 IHC score SENP1 mRNA expression Mean ± SD P-value Median (IQR) P-value 0.231 0.272 5.3 ± 2.8 2.492 (1.281–4.122) 5.9 ± 2.7 2.669 (1.871–4.171) 0.337 0.338 6.1 ± 3.1 2.846 (1.836–5.429) 5.6 ± 2.7 2.566 (1.756–3.878) 0.104 0.768 5.2 ± 2.5 2.570 (1.315–4.437) 6.0 ± 2.9 2.649 (1.879–3.392) 0.108 0.407 5.4 ± 2.5 2.680 (1.815–4.189) 6.1 ± 3.1 2.435 (1.713–3.480) 0.282 0.298 5.5 ± 2.6 2.629 (1.794–3.641) 6.0 ± 3.1 2.553 (1.814–5.796) 0.732 0.971 5.6 ± 2.7 2.600 (1.845–3.831) 5.8 ± 2.9 2.565 (1.648–4.276) 0.061 0.355 5.5 ± 2.7 2.577 (1.835–3.880) 6.7 ± 3.0 3.722 (1.521–5.737) 0.785 0.011 5.8 ± 2.8 2.698 (2.021–4.694) 5.4 ± 2.9 1.672 (1.095–2.933) 5.7 ± 2.4 2.697 (2.118–3.891) 0.190 0.189 5.4 ± 2.8 1.958 (1.283–2.663) 5.5 ± 2.6 3.101 (1.820–5.442) 6.1 ± 2.9 2.566 (1.962–3.445) 0.045 0.437 5.1 ± 2.2 2.582 (1.310–4.119) 6.0 ± 3.0 2.608 (1.896–4.189) 0.003 0.008 5.1 ± 2.5 2.318 (1.519–3.053) 6.4 ± 3.0 3.055 (2.167–5.349) 0.012 0.015 5.2 ± 2.3 2.318 (1.376–3.300) 6.3 ± 3.2 2.704 (2.251–4.920) 0.269 0.056 5.5 ± 2.7 2.442 (1.693–4.052) 6.1 ± 3.0 2.894 (2.560–5.460) 0.852 0.976 5.6 ± 2.6 2.598 (1.574–4.243) l) 5.7 ± 2.9 2.553 (1.871–3.885) 0.732 0.954 5.6 ± 2.6 2.548 (1.862–4.501) 5.7 ± 2.9 2.684 (1.765–3.325) -like modiﬁer (SUMO)-speciﬁc protease 1; IQR, interquartile noma; SCC squamous cell carcinoma; ASC adenosquamous TABLE 2 \| Correlation between SENP1 expression and clinical characteristics. Statistical Analysis Items SENP1 IHC score SENP1 mRNA expression Mean ± SD P-value Median (IQR) P-value Age 0.231 0.272 ≤60 years 5.3 ± 2.8 2.492 (1.281–4.122) > 60 years 5.9 ± 2.7 2.669 (1.871–4.171) Gender 0.337 0.338 Female 6.1 ± 3.1 2.846 (1.836–5.429) Male 5.6 ± 2.7 2.566 (1.756–3.878) Smoking 0.104 0.768 No 5.2 ± 2.5 2.570 (1.315–4.437) Yes 6.0 ± 2.9 2.649 (1.879–3.392) Drinking 0.108 0.407 No 5.4 ± 2.5 2.680 (1.815–4.189) Yes 6.1 ± 3.1 2.435 (1.713–3.480) Hypertension 0.282 0.298 No 5.5 ± 2.6 2.629 (1.794–3.641) Yes 6.0 ± 3.1 2.553 (1.814–5.796) Hyperlipidemia 0.732 0.971 No 5.6 ± 2.7 2.600 (1.845–3.831) Yes 5.8 ± 2.9 2.565 (1.648–4.276) Diabetes 0.061 0.355 No 5.5 ± 2.7 2.577 (1.835–3.880) Yes 6.7 ± 3.0 3.722 (1.521–5.737) Histopathological subtype 0.785 0.011 ADC 5.8 ± 2.8 2.698 (2.021–4.694) SCC 5.4 ± 2.9 1.672 (1.095–2.933) ASC 5.7 ± 2.4 2.697 (2.118–3.891) Differentiation 0.190 0.189 Well 5.4 ± 2.8 1.958 (1.283–2.663) Moderate 5.5 ± 2.6 3.101 (1.820–5.442) Poor 6.1 ± 2.9 2.566 (1.962–3.445) Tumor size 0.045 0.437 ≤5 cm 5.1 ± 2.2 2.582 (1.310–4.119) > 5 cm 6.0 ± 3.0 2.608 (1.896–4.189) Lymph node metastasis 0.003 0.008 No 5.1 ± 2.5 2.318 (1.519–3.053) Yes 6.4 ± 3.0 3.055 (2.167–5.349) TNM stage 0.012 0.015 II 5.2 ± 2.3 2.318 (1.376–3.300) III 6.3 ± 3.2 2.704 (2.251–4.920) ECOG PS score 0.269 0.056 0 5.5 ± 2.7 2.442 (1.693–4.052) 1 6.1 ± 3.0 2.894 (2.560–5.460) CEA 0.852 0.976 Normal (≤5 ng/ml) 5.6 ± 2.6 2.598 (1.574–4.243) Abnormal (> 5 ng/ml) 5.7 ± 2.9 2.553 (1.871–3.885) CA125 0.732 0.954 Normal (≤35 U/ml) 5.6 ± 2.6 2.548 (1.862–4.501) Abnormal (> 35 U/ml) 5.7 ± 2.9 2.684 (1.765–3.325) TABLE 2 \| Correlation between SENP1 expression and clinical characteristics. rrelation between SENP1 expression and clinical characteristics. test, the Mann–Whitney U test, or the Kruskal–Wallis H rank- sum test. The KM curve was plotted to display survival proﬁle and log-rank test was used to determine accumulating DFS and OS diﬀerences between patients. The Cox proportional hazards regression analysis was used for prognostic analysis. Statistical signiﬁcance was deﬁned as p < 0.05. Correlation Between SENP1 Expression and Clinical Features Elevated SENP1 IHC score was correlated with > 5 cm tumor size (p = 0.045), the occurrence of lymph node metastasis (p = 0.003), and more advanced TNM stage (p = 0.012). However, no correlation was found in the SENP1 IHC score with other clinical features (p > 0.05). In addition, increased SENP1 mRNA expression was associated with histopathological subtype (p = 0.011), the occurrence of lymph node metastasis (p = 0.008), and higher TNM stage (p = 0.015). However, no association of SENP1 mRNA expression with other clinical features was observed (p > 0.05) (Table 2). SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; IQR, interquartile range; ADC, adenocarcinoma; SCC, squamous cell carcinoma; ASC, adenosquamous carcinoma; TNM, tumor-node-metastasis; ECOG PS, Eastern Cooperative Oncology Group Performance Status; CEA, carcinoembryonic antigen; CA125, cancer antigen 125. The bold value indicates statistical signiﬁcance. Statistical Analysis The SPSS software version 24.0 (IBM Corporation, Armonk, New York, USA) and the GraphPad Prism version 7.01 (GraphPad Software Incorporation, San Diego, California, USA) were used for data analysis and graph plotting, respectively. SENP1 expression between tumor tissue specimens and paired adjacent tissue specimens was compared using the paired samples t-test and the Wilcoxon signed-rank test. Variance equality was assessed by F-test. Comparison of SENP1 expression between/among patients with diﬀerent clinical characteristics was analyzed using the Student’s t-test, the one-way ANOVA FIGURE 1 \| SENP1 expression in patients with surgical NSCLC receiving adjuvant chemotherapy. Examples of SENP1 IHC staining in negative control, adjacent tissues, and tumor tissues (A) comparison of SENP1 IHC score (B) and SENP1 mRNA expression (C) between the tumor tissues and the adjacent tissues. SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer. FIGURE 1 \| SENP1 expression in patients with surgical NSCLC receiving adjuvant chemotherapy. Examples of SENP1 IHC staining in negative control, adjacent tissues, and tumor tissues (A) comparison of SENP1 IHC score (B) and SENP1 mRNA expression (C) between the tumor tissues and the adjacent tissues. SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; IHC, immunohistochemistry; NSCLC, non-small cell lung cancer. June 2022 \| Volume 8 \| Article 771785 3 Frontiers in Surgery \| www.frontiersin.org SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. SE TABLE 2 \| Correlation betwe Items S M Age ≤60 years 5 > 60 years 5 Gender Female 6 Male 5 Smoking No 5 Yes 6 Drinking No 5 Yes 6 Hypertension No 5 Yes 6 Hyperlipidemia No 5 Yes 5 Diabetes No 5 Yes 6 Histopathological subtype ADC 5 SCC 5 ASC 5 Differentiation Well 5 Moderate 5 Poor 6 Tumor size ≤5 cm 5 > 5 cm 6 Lymph node metastasis No 5 Yes 6 TNM stage II 5 III 6 ECOG PS score 0 5 1 6 CEA Normal (≤5 ng/ml) 5 Abnormal (> 5 ng/ml) 5 CA125 Normal (≤35 U/ml) 5 Abnormal (> 35 U/ml) 5 SENP1, small ubiquitin-like m range; ADC, adenocarcinoma; carcinoma; TNM, tumor-node Group Performance Status; CE The bold value indicates statist SENP1 in Surgical NSCLC and Adjuvant Chemotherapy between SENP1 expression and clinical characteristics. Association of SENP1 Expression With 5-year DFS rate in patients with SENP1 mRNA high were 92.2, 33.1, and 13.8%, respectively, while those in patients with SENP1 mRNA low were 96.1, 58.6, and 11.4%, respectively (Figure 3B). Association of SENP1 Expression With j py g Adjuvant chemotherapy regimen was recorded in this study and analysis of the correlation of SENP1 expression with adjuvant chemotherapy regimen was conducted, which discovered that June 2022 \| Volume 8 \| Article 771785 Frontiers in Surgery \| www.frontiersin.org 4 SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. FIGURE 2 \| Comparison of SENP1 expression among patients with surgical NSCLC receiving different adjuvant chemotherapy regimens. Comparison of SENP1 IHC score (A) and SENP1 mRNA expression (B) among patients with NSCLC who received NP, TP, GP, and DP. SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; IHC, immunohistochemistry; NP, vinorelbine + cisplatin; TP, taxol + cisplatin or carboplatin; GP, gemcitabine + cisplatin or carboplatin; DP, docetaxel + cisplatin or carboplatin; NSCLC, non-small cell lung cancer. FIGURE 2 \| Comparison of SENP1 expression among patients with surgical NSCLC receiving different adjuvant chemotherapy regimens. Comparison of SENP1 IHC score (A) and SENP1 mRNA expression (B) among patients with NSCLC who received NP, TP, GP, and DP. SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; IHC, immunohistochemistry; NP, vinorelbine + cisplatin; TP, taxol + cisplatin or carboplatin; GP, gemcitabine + cisplatin or carboplatin; DP, docetaxel + cisplatin or carboplatin; NSCLC, non-small cell lung cancer. FIGURE 3 \| Comparison of accumulating DFS between patients with surgical NSCLC with different SENP1 expressions. Comparison of accumulating DFS between patients with surgical NSCLC with SENP1 protein high and SENP1 protein low (A) and comparison of accumulating DFS between patients with surgical NSCLC with SENP1 mRNA high and SENP1 mRNA low (B). SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; DFS, disease-free survival; NSCLC, non-small cell lung cancer. FIGURE 3 \| Comparison of accumulating DFS between patients with surgical NSCLC with different SENP1 expressions. Comparison of accumulating DFS between patients with surgical NSCLC with SENP1 protein high and SENP1 protein low (A) and comparison of accumulating DFS between patients with surgical NSCLC with SENP1 mRNA high and SENP1 mRNA low (B). SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; DFS, disease-free survival; NSCLC, non-small cell lung cancer. both the SENP1 IHC score (p = 0.424) and SENP1 mRNA expression (p = 0.927) showed no correlation with adjuvant chemotherapy regimen (Figures 2A,B). 5-year DFS rate in patients with SENP1 mRNA high were 92.2, 33.1, and 13.8%, respectively, while those in patients with SENP1 mRNA low were 96.1, 58.6, and 11.4%, respectively (Figure 3B). Correlation of SENP1 Expression With Accumulating DFS g Additionally, according to the univariate Cox proportional hazards regression analysis, SENP1 protein (high vs. low) [p = 0.004, hazard ratio (HR) = 1.889], SENP1 mRNA (high vs. low) (p = 0.032, HR = 1.676), poor diﬀerentiation (p = 0.002, HR = 1.524), tumor size (>5 vs. ≤5 cm) (p = 0.020, HR = 1.603), lymph node metastasis (yes vs. no) (p = 0.007, HR = 1.679), TNM stage (III vs. II) (p = 0.001, HR = 1.874), and CA125 (>35 vs. ≤35 U/ml) (p = 0.034, HR = 1.500) were correlated with shorter accumulating DFS. Furthermore, the multivariate Cox proportional hazards Small ubiquitin-like modiﬁer-speciﬁc protease 1 protein high was correlated with poor accumulating DFS (p = 0.003). Meanwhile, the 1-year, 3-year, and 5-year DFS rates of patients with SENP1 protein high were 93.7, 37.8, and 8.2%, respectively, while those of patients with SENP1 protein low were 97.8, 66.5, and 25.0%, respectively (Figure 3A). Besides, SENP1 mRNA high was also associated with worse accumulating DFS (p = 0.028) and the 1-year, 3-year, and Frontiers in Surgery \| www.frontiersin.org June 2022 \| Volume 8 \| Article 771785 5 SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. TABLE 3 \| The Cox proportional hazards regression analysis for DFS. Items P-value HR 95% CI Lower Upper Univariate Cox proportional hazards regression analysis SENP1 protein (high vs. low) 0.004 1.889 1.222 2.921 SENP1 mRNA (high vs. low) 0.032 1.676 1.044 2.691 Age (> 60 vs. ≤60 years) 0.052 1.478 0.996 2.195 Gender (male vs. female) 0.412 1.231 0.750 2.022 Smoking (yes vs. no) 0.281 1.233 0.843 1.805 Drinking (yes vs. no) 0.414 0.851 0.577 1.254 Hypertension (yes vs. no) 0.762 0.939 0.623 1.414 Hyperlipidemia (yes vs. no) 0.839 1.043 0.694 1.568 Diabetes (yes vs. no) 0.813 1.064 0.635 1.785 Histopathological subtype ASC Reference ADC 0.710 0.903 0.527 1.548 SCC 0.104 0.613 0.339 1.106 Poor differentiation 0.002 1.524 1.169 1.987 Tumor size (> 5 vs. ≤5 cm) 0.020 1.603 1.077 2.387 Lymph node metastasis (yes vs. no) 0.007 1.679 1.152 2.446 TNM stage (III vs, II) 0.001 1.874 1.286 2.730 ECOG PS score (1 vs. 0) 0.193 1.337 0.864 2.070 CEA (> 5 vs. ≤5 ng/ml) 0.155 1.317 0.901 1.924 CA125 (> 35 vs. DISCUSSION With respect to the SENP1 expression in cancer tissues and adjacent tissues, it is suggested that SENP1 upregulates in pancreatic ductal adenocarcinoma tissues than in adjacent tissues (22). Additionally, the expression of SENP1 is higher in tumor tissues than paracarcinoma tissues in patients with HCC (12). This study found that SENP1 expression was increased in NSCLC tumor tissues than adjacent tissues in patients with surgical NSCLC receiving adjuvant chemotherapy. A possible reason could be that: SENP1 reﬂected the higher proliferation rate of cells; meanwhile, the proliferation rate in NSCLC cells in the tumor tissue was increased than that in the adjacent tissue cells. Thus, SENP1 expression was higher in NSCLC tumor tissues compared with adjacent tissues in patients with surgical NSCLC receiving adjuvant chemotherapy. In terms of the correlation of SENP1 with clinical features, a previous study shows that SENP1 expression positively correlates with lymph node metastasis and TNM stage in patients with pancreatic cancer (22). Another study suggests that plasma exosome-derived SENP1 associates with higher tumor diameter and tumor stage in patients with osteosarcoma (23). Besides, SENP1 overexpression is correlated with moderate and low diﬀerentiation of NSCLC tumors (11). In this study, we observed that in patients with surgical NSCLC receiving adjuvant chemotherapy, SENP1 expression was correlated with larger tumor size, histopathological subtype, the occurrence of lymph node metastasis, and higher TNM stage. Possible explanations could be that: (1) through the hypoxia-induced factor-1α (HIF- 1α) signaling pathway, SENP1 could promote the proliferation of NSCLC cancer cells, resulting in larger tumor size; (2) SENP1 could regulate matrix metalloproteinase-9 (MMP-9) to The bold value indicates statistical signiﬁcance. regression analysis showed that SENP1 protein (high vs. low) (p = 0.009, HR = 1.798), age (>60 vs. ≤60 years) (p = 0.019, HR = 1.617), poor diﬀerentiation (p = 0.011, HR = 1.422), and TNM stage (III vs. II) (p = 0.002, HR = 1.811) were independently associated with unsatisfying accumulating DFS (Table 3). Correlation of SENP1 Expression With Accumulating DFS 9 1.043 0.694 1.56 3 1.064 0.635 1.78 nce 0 0.903 0.527 1.54 4 0.613 0.339 1.10 2 1.524 1.169 1.98 0 1.603 1.077 2.38 7 1.679 1.152 2.44 1.874 1.286 2.73 3 1.337 0.864 2.07 5 1.317 0.901 1.92 4 1.500 1.031 2.18 nce 0 0.848 0.506 1.42 5 1.019 0.528 1.96 1.305 0.710 2.39 oportional hazards regression 9 1.798 1.161 2.78 9 1.617 1.081 2.41 1.422 1.085 1.86 2 1.811 1.235 2.65 SENP1, small ubiquitin-like modiﬁer (SU carcinoma; ADC, adenocarcinoma; S 0 0.903 0.527 1. 4 0.613 0.339 1. 2 1.524 1.169 1. 0 1.603 1.077 2. 7 1.679 1.152 2. 1 1.874 1.286 2. 3 1.337 0.864 2. 5 1.317 0.901 1. 4 1.500 1.031 2. nce 0 0.848 0.506 1. 5 1.019 0.528 1. 1 1.305 0.710 2. roportional hazards regression 9 1.798 1.161 2. 9 1.617 1.081 2. 1 1.422 1.085 1. 2 1.811 1.235 2. SENP1, small ubiquitin-like modiﬁer (S carcinoma; ADC, adenocarcinoma -metastasis; ECOG PS, Eastern Coop carcinoembryonic antigen; CA125, ce. Correlation of SENP1 Expression With Accumulating DFS ≤35 U/ml) 0.034 1.500 1.031 2.182 Adjuvant chemotherapy regimen DP Reference NP 0.530 0.848 0.506 1.420 TP 0.955 1.019 0.528 1.969 GP 0.391 1.305 0.710 2.399 Forward stepwise multivariate Cox proportional hazards regression analysis SENP1 protein (high vs. low) 0.009 1.798 1.161 2.783 Age (> 60 vs. ≤60 years) 0.019 1.617 1.081 2.419 Poor differentiation 0.011 1.422 1.085 1.864 TNM stage (III vs, II) 0.002 1.811 1.235 2.654 DFS, disease-free survival; HR, hazard ratio; SENP1, small ubiquitin-like modiﬁer (SUMO)- speciﬁc protease 1; ASC, adenosquamous carcinoma; ADC, adenocarcinoma; SCC, squamous cell carcinoma; TNM, tumor-node-metastasis; ECOG PS, Eastern Cooperative Oncology Group Performance Status; CEA, carcinoembryonic antigen; CA125, cancer antigen 125. The bold value indicates statistical signiﬁcance. 5-year OS rates were 99.1, 70.5, and 18.8%, respectively, while those in patients with SENP1 protein low were 100.0, 82.8, and 43.7%, respectively (Figure 4A). However, no correlation was found in SENP1 mRNA with accumulating OS (p = 0.132). Additionally, the 1-year, 3-year, and 5-year OS rates of patients with SENP1 mRNA high were 98.0, 69.9, and 23.8%, respectively, while those of patients with SENP1 mRNA low were 100.0, 79.1, and 26.1%, respectively (Figure 4B). Moreover, the univariate Cox proportional hazards regression analysis suggested that SENP1 protein (high vs. low) (p = 0.033, HR = 1.816), age (>60 vs. ≤60 years) (p = 0.014, HR = 1.887), poor diﬀerentiation (p < 0.001, HR = 1.868), tumor size (>5 vs. ≤5 cm) (p = 0.002, HR = 2.285), lymph node metastasis (yes vs. no) (p < 0.001, HR = 2.412), TNM stage (III vs. II) (p < 0.001, HR = 2.542), and CA125 (>35 vs. ≤35 U/ml) (p = 0.032, HR = 1.671) were correlated with unsatisfying accumulating OS. Furthermore, the multivariate Cox proportional hazards regression analysis revealed that SENP1 protein (high vs. low) (p = 0.049, HR = 1.735), age (>60 vs. ≤60 years) (p = 0.005, HR = 2.119), poor diﬀerentiation (p = 0.001, HR = 1.843), and TNM stage (III vs. II) (p < 0.001, HR = 2.415) were independently associated with poor accumulating OS (Table 4). Association of SENP1 Expression With Accumulating OS Small ubiquitin-like modiﬁer-speciﬁc protease 1 protein high was associated with shorter accumulating OS (p = 0.029). In patients with SENP1 protein high, the 1-year, 3-year, and June 2022 \| Volume 8 \| Article 771785 Frontiers in Surgery \| www.frontiersin.org 6 SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. FIGURE 4 \| Comparison of accumulating OS between patients with surgical NSCLC with different SENP1 expressions. Comparison of accumulating OS between patients with surgical NSCLC with SENP1 protein high and SENP1 protein low (A) and comparison of accumulating OS between patients with surgical NSCLC with SENP1 mRNA high and SENP1 mRNA low (B). SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; OS, overall survival; NSCLC, non-small cell lung cancer. FIGURE 4 \| Comparison of accumulating OS between patients with surgical NSCLC with different SENP1 expressions. Comparison of accumulating OS between patients with surgical NSCLC with SENP1 protein high and SENP1 protein low (A) and comparison of accumulating OS between patients with surgical NSCLC with SENP1 mRNA high and SENP1 mRNA low (B). SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; OS, overall survival; NSCLC, non-small cell lung cancer. TABLE 4 \| The Cox proportional hazards regression analysis for OS. Items P-value HR 95% CI Lower Upper Univariate Cox proportional hazards regression analysis SENP1 protein (high vs. low) 0.033 1.816 1.049 3.141 SENP1 mRNA (high vs. low) 0.138 1.593 0.861 2.947 Age (> 60 vs. ≤60 years) 0.014 1.887 1.138 3.128 Gender (male vs. female) 0.510 1.231 0.663 2.288 Smoking (yes vs. no) 0.506 1.173 0.733 1.880 Drinking (yes vs. no) 0.834 1.052 0.656 1.687 Hypertension (yes vs. no) 0.495 0.833 0.493 1.408 Hyperlipidemia (yes vs. no) 0.144 1.432 0.885 2.317 Diabetes (yes vs. no) 0.533 0.801 0.398 1.610 Histopathological subtype ASC Reference ADC 0.523 0.809 0.422 1.551 SCC 0.373 0.727 0.361 1.465 Poor differentiation <0.001 1.868 1.318 2.646 Tumor size (> 5 vs. ≤5 cm) 0.002 2.285 1.351 3.864 Lymph node metastasis (yes vs. no) <0.001 2.412 1.489 3.907 TNM stage (III vs, II) <0.001 2.542 1.592 4.059 ECOG PS score (1 vs. 0) 0.504 1.193 0.712 1.999 CEA (> 5 vs. ≤5 ng/ml) 0.215 1.345 0.841 2.151 CA125 (> 35 vs. ≤35 U/ml) 0.032 1.671 1.045 2.674 Adjuvant chemotherapy regimen DP Reference NP 0.834 1.075 0.547 2.112 TP 0.553 1.281 0.565 2.906 GP 0.658 1.200 0.535 2.694 Forward stepwise multivariate Cox proportional hazards regression analysis SENP1 protein (high vs. Association of SENP1 Expression With Accumulating OS low) 0.049 1.735 1.003 3.000 Age (> 60 vs. ≤60 years) 0.005 2.119 1.262 3.557 Poor differentiation 0.001 1.843 1.289 2.635 TNM stage (III vs, II) <0.001 2.415 1.504 3.879 OS, overall survival; HR, hazard ratio; SENP1, small ubiquitin-like modiﬁer (SUMO)-speciﬁc protease 1; ASC, adenosquamous carcinoma; ADC, adenocarcinoma; SCC, squamous cell carcinoma; TNM, tumor-node-metastasis; ECOG PS, Eastern Cooperative Oncology Group Performance Status; CEA, carcinoembryonic antigen; CA125, cancer antigen 125. The bold value indicates statistical signiﬁcance. TABLE 4 \| The Cox proportional hazards regression analysis for OS. promote NSCLC cancer metastasis; meanwhile, SENP1 might enhance NSCLC cell invasive ability via modulating epithelial– mesenchymal transition marked genes, which contributed to the occurrence of lymph node metastasis (22, 24). Thus, SENP1 expression was associated with larger tumor size and occurrence of lymph node metastasis (17); and (3) SENP1 expression was correlated with larger tumor size and lymph node metastasis, which were features of more advanced TNM stage. Therefore, SENP1 expression was associated with higher TNM stage (25). Moreover, no correlation was found in SENP1 expression with adjuvant chemotherapy regimen in this study, which could be explained by that: adjuvant chemotherapy regimen was applied after surgical resection and the sample for analyzing was collected before but not during or after adjuvant chemotherapy, thus no correlation in SENP1 expression with adjuvant chemotherapy regimen was found. 1.052 0.656 1.6 0.833 0.493 1.4 1.432 0.885 2.3 0.801 0.398 1.6 e 0.809 0.422 1.5 0.727 0.361 1.4 1.868 1.318 2.6 2.285 1.351 3.8 2.412 1.489 3.9 2.542 1.592 4.0 1.193 0.712 1.9 1.345 0.841 2.1 1.671 1.045 2.6 e 1.075 0.547 2.1 1.281 0.565 2.9 1.200 0.535 2.6 portional hazards regression 1.735 1.003 3.0 2.119 1.262 3.5 1.843 1.289 2.6 2.415 1.504 3.8 mall ubiquitin-like modiﬁer (SUMO)-s ADC, adenocarcinoma; SCC, squa ECOG PS Eastern Cooperative On Concerning the association of SENP1 expression with prognosis in patients with cancer, higher plasma exosome-derived SENP1 correlates with worse DFS and OS in patients with osteosarcoma (23); additionally, SENP1 overexpression independently correlates with poor prognosis in patients with NSCLC (11). This study discovered that both the SENP1 protein high and SENP1 mRNA high were correlated with poor accumulating DFS, while SENP1 protein high was also associated with shorter accumulating OS in patients with surgical NSCLC receiving adjuvant chemotherapy. Meanwhile, SENP1 protein (high vs. low) was an independent risk factor for unsatisfying accumulating DFS and OS. REFERENCES 10. Mulherkar R, Grewal AS, Berman AT. Emerging role of immunotherapy in locally advanced non-small cell lung cancer. Clin Adv Hematol Oncol. (2020) 18:212–17. 1. Suster DI, Mino-Kenudson M. Molecular pathology of primary non-small cell lung cancer. Arch Med Res. (2020) 51:784–98. doi: 10.1016/j.arcmed.2020.08.004 1. Suster DI, Mino-Kenudson M. Molecular pathology of primary non-small cell lung cancer. Arch Med Res. (2020) 51:784–98. doi: 10.1016/j.arcmed.2020.08.004 11. Zuo Y, Cheng JK. Small ubiquitin-like modiﬁer protein-speciﬁc protease 1 and prostate cancer. Asian J Androl. (2009) 11:36–8. doi: 10.1038/aja.2008.45 12. Tao Y, Li R, Shen C, Li J, Zhang Q, Ma Z, et al. SENP1 is a crucial promotor for hepatocellular carcinoma through deSUMOylation of UBE2T. Aging. (2020) 12:1563–76. doi: 10.18632/aging.102700 2. Thai AA, Solomon BJ, Sequist LV, Gainor JF, Heist RS. Lung cancer. Lancet. (2021) 398:535–54. doi: 10.1016/S0140-6736(21)00312-3 3. Zappa C, Mousa SA. Non-small cell lung cancer: current treatment and future advances. Transl Lung Cancer Res. (2016) 5:288–300. doi: 10.21037/tlcr.2016.06.07 13. Zhang W, Sun H, Shi X, Wang H, Cui C, Xiao F, et al. SENP1 regulates hepatocyte growth factor-induced migration and epithelial-mesenchymal transition of hepatocellular carcinoma. Tumour Biol. (2016) 37:7741–8. doi: 10.1007/s13277-015-4406-y 4. Khan S, Ali S, Muhammad. Exhaustive review on lung cancers: novel technologies. Curr Med Imaging Rev. (2019) 15:873–83. doi: 10.2174/1573405615666181128124528 14. Bawa-Khalfe T, Yang FM, Ritho J, Lin HK, Cheng J, Yeh ET. SENP1 regulates PTEN stability to dictate prostate cancer development. Oncotarget. (2017) 8:17651–64. doi: 10.18632/oncotarget.13283 5. Kris MG, Gaspar LE, Chaft JE, Kennedy EB, Azzoli CG, Ellis PM, et al. Adjuvant systemic therapy and adjuvant radiation therapy for stage I to IIIA completely resected non-small-cell lung cancers: American Society of Clinical Oncology/Cancer Care Ontario clinical practice guideline update. J Clin Oncol. (2017) 35:2960–74. doi: 10.1200/JCO.2017.72.4401 15. Chen MC, Nhan DC, Hsu CH, Wang TF, Li CC, Ho TJ, et al. SENP1 participates in Irinotecan resistance in human colon cancer cells. J Cell Biochem. (2021) 122:1277–94. doi: 10.1002/jcb.29946 6. Herbst RS, Morgensztern D, BoshoﬀC. The biology and management of non- small cell lung cancer. Nature. (2018) 553:446–54. doi: 10.1038/nature25183 16. Liu K, Zhang J, Wang H. Small ubiquitin-like modiﬁer/sentrin-speciﬁc peptidase 1 associates with chemotherapy and is a risk factor for poor prognosis of non-small cell lung cancer. J Clin Lab Anal. (2018) 32:e22611. doi: 10.1002/jcla.22611 7. Valladares BT, Crespo PC, Herranz UA, Caamano AG. Adjuvant treatment in lung cancer. J Clin Transl Res. (2021) 7:175–84. doi: 10.18053/jctres.07.202102.012 17. AUTHOR CONTRIBUTIONS QY and YM contribute to the conception, design, data analysis, and interpretation. QY contributes to the administrative support. QY, MY, JZ, and YM contribute to the provision of study materials or patients. QY, MY, and JZ contribute to the collection and assembly of data. All the authors involved in writing of manuscript and submitted the ﬁnal approval of the manuscript. CONCLUSION Except for the above discussion and explanation, detection of SENP1 might have the following clinical implication: SENP1 might serve as an indicator for tumor characteristics and prognosis in NSCLC, which could further improve the management of patients with NSCLC. Furthermore, the detection of SENP1 might inﬂuence the choice of diﬀerent therapies and serve as a decision- making factor in the choice or the change of therapy. However, these descriptions needed a number of multicenter prospective studies with larger sample size to validate further ﬁndings. Small ubiquitin-like modiﬁer-speciﬁc protease 1 overexpression correlates with larger tumor size, lymph node metastasis, higher TNM stage, as well as shorter DFS and OS in patients with surgical NSCLC receiving adjuvant chemotherapy. Small ubiquitin-like modiﬁer-speciﬁc protease 1 overexpression correlates with larger tumor size, lymph node metastasis, higher TNM stage, as well as shorter DFS and OS in patients with surgical NSCLC receiving adjuvant chemotherapy. Association of SENP1 Expression With Accumulating OS The explanation could be that: (1) as mentioned earlier, SENP1 was related to chemotherapy resistance in patients with surgical NSCLC receiving adjuvant chemotherapy (11), further causing unsatisfying DFS and OS and (2) SENP1 expression was correlated with the occurrence of lymph node metastasis and higher TNM stage (as mentioned above), which could indirectly cause poor prognosis. June 2022 \| Volume 8 \| Article 771785 Frontiers in Surgery \| www.frontiersin.org Frontiers in Surgery \| www.frontiersin.org 7 SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. ETHICS STATEMENT The studies involving human participants were reviewed and approved by HanDan Central Hospital. The patients/participants provided their written informed consent to participate in this study. DATA AVAILABILITY STATEMENT The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s. Although a lot of ﬁndings were identiﬁed, there were still some limitations in this study. First, this study had a relatively small sample size, which might cause low statistical power; secondly, this study did not investigate the underlying mechanism of SENP1 in NSCLC progression and chemoresistance. Therefore, further in-vivo and in-vitro experiments were needed; third, although this study had a 5-year follow-up duration, longer follow-up could be conducted in the future to investigate the long-term prognostic eﬀect of SENP1 in patients with surgical NSCLC receiving adjuvant chemotherapy; fourth, blood samples of patients with surgical NSCLC receiving adjuvant chemotherapy might be collected in the future study to compare and analyze the changes of SENP1 protein before and after adjuvant chemotherapy; ﬁfth, since this was a retrospective study, its evidence-based medicine was of low value, thus a prospective study might be further performed to validate the ﬁndings. REFERENCES Mu J, Zuo Y, Yang W, Chen Z, Liu Z, Tu J, et al. Over-expression of small ubiquitin-like modiﬁer proteases 1 predicts chemo-sensitivity and poor survival in non-small cell lung cancer. Chin Med J. (2014) 127:4060–5. doi: 10.3760/cma.j.issn.0366-6999.20141013 8. Santarpia M, Rolfo C, Peters GJ, Leon LG, Giovannetti E. On the pharmacogenetics of non-small cell lung cancer treatment. Expert Opin Drug Metab Toxicol. (2016) 12:307–17. doi: 10.1517/17425255.2016.1141894 9. Arbour KC, Riely GJ. Systemic therapy for locally advanced and metastatic non-small cell lung cancer: a review. JAMA. (2019) 322:764–74. doi: 10.1001/jama.2019.11058 18. D’Addario G, Felip E, Group EGW. Non-small-cell lung cancer: ESMO clinical recommendations for diagnosis, treatment and follow-up. Ann Oncol. (2009) 20 Suppl 4:68–70. doi: 10.1093/annonc/mdp132 June 2022 \| Volume 8 \| Article 771785 Frontiers in Surgery \| www.frontiersin.org 8 SENP1 in Surgical NSCLC and Adjuvant Chemotherapy Yang et al. June 2022 \| Volume 8 \| Article 771785 Frontiers in Surgery \| www.frontiersin.org 25. Woodard GA, Jones KD, Jablons DM. Lung cancer staging and prognosis. Cancer Treat Res. (2016) 170:47–75. doi: 10.1007/978-3-319-40389-2_3 25. Woodard GA, Jones KD, Jablons DM. Lung cancer staging and prognosis. Cancer Treat Res. (2016) 170:47–75. doi: 10.1007/978-3-319-40389-2_3 19. Schemper M, Smith TL. A note on quantifying follow-up in studies of failure time. Control Clin Trials. (1996) 17:343–6. doi: 10.1016/0197-2456(96)00075-X 20. Lin X, Wang Y, Jiang Y, Xu M, Pang Q, Sun J, et al. Sumoylation enhances the activity of the TGF-beta/SMAD and HIF-1 signaling pathways in keloids. Life Sci. (2020) 255:117859. doi: 10.1016/j.lfs.2020.117859 Conﬂict of Interest: The authors declare that the research was conducted in the absence of any commercial or ﬁnancial relationships that could be construed as a potential conﬂict of interest. 21. Li T, Huang S, Dong M, Gui Y, Wu D. Prognostic impact of SUMO- speciﬁc protease 1 (SENP1) in prostate cancer patients undergoing radical prostatectomy. Urol Oncol. (2013) 31:1539–45. doi: 10.1016/j.urolonc.2012. 03.007 Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their aﬃliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. 22. Ma C, Wu B, Huang X, Yuan Z, Nong K, Dong B, et al. SUMO- speciﬁc protease 1 regulates pancreatic cancer cell proliferation and invasion by targeting MMP-9. Tumour Biol. (2014) 35:12729–35. doi: 10.1007/s13277-014-2598-1 23. Wang L, Wu J, Song S, Chen H, Hu Y, Xu B, et al. Plasma exosome- derived sentrin SUMO-speciﬁc protease 1: a prognostic biomarker in patients with osteosarcoma. Front Oncol. (2021) 11:625109. doi: 10.3389/fonc.2021.6 25109 Copyright © 2022 Yang, Yang, Zhang and Ma. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. 24. Wang X, Liang X, Liang H, Wang B. SENP1/HIF-1alpha feedback loop modulates hypoxia-induced cell proliferation, invasion, and EMT in human osteosarcoma cells. J Cell Biochem. (2018) 119:1819–26. doi: 10.1002/jcb.26342 June 2022 \| Volume 8 \| Article 771785 Frontiers in Surgery \| www.frontiersin.org 9
https://openalex.org/W4388490963	https://journals.open.tudelft.nl/abe/article/download/3525/3668	English	null	The Global Financial Crisis and neighborhood decline	A+BE	2,018	cc-by	8,492	2 The Global Financial Crisis and neighborhood decline Merle Zwiers, Gideon Bolt, Maarten van Ham, Ronald van Kempen Published in Urban Geography (2016), 37(5): 664-684. Merle Zwiers, Gideon Bolt, Maarten van Ham, Ronald van Kempen Published in Urban Geography (2016), 37(5): 664-684. § 2.1 Introduction In some countries, investors in real estate became more selective, avoiding projects with too much risk; in the United States, in contrast, investors of another ilk have bought large numbers of foreclosed, real estate owned (REO) properties with the main goal of making a profit (e.g. Mallach, 2010b). Regeneration and restructuring initiatives have been put on hold throughout Western Europe (Boelhouwer & Priemus, 2014; Raco & Tasan-Kok, 2009; Schwartz, 2011). While some governments, such as the United Kingdom and the Netherlands, implemented stimulus programs to generate more (affordable) housing in the years after the crisis, recent budget cuts have put an end to these programs (Scanlon & Elsinga, 2014; Schwartz, 2011). be a persistent problem in many countries (OECD, 2014; Shierholz, 2014). Similarly, despite graudual stock market recoveries and some modest increases in house prices, repercussions from the GFC and economic recession persist in all countries. In many countries, the GFC has had predictable effects on the supply side of the housing market - the willingness of banks to lend money to prospective owners has generally declined. In some countries, investors in real estate became more selective, avoiding projects with too much risk; in the United States, in contrast, investors of another ilk have bought large numbers of foreclosed, real estate owned (REO) properties with the main goal of making a profit (e.g. Mallach, 2010b). Regeneration and restructuring initiatives have been put on hold throughout Western Europe (Boelhouwer & Priemus, 2014; Raco & Tasan-Kok, 2009; Schwartz, 2011). While some governments, such as the United Kingdom and the Netherlands, implemented stimulus programs to generate more (affordable) housing in the years after the crisis, recent budget cuts have put an end to these programs (Scanlon & Elsinga, 2014; Schwartz, 2011). The demand side of the housing market has also changed. Banks have tightened lending terms, making it more difficult for many households to obtain a mortgage (Goodman et al., 2015). As a result, there is more demand for private rentals and social or public housing. The GFC has affected employment on both sides of the Atlantic, in terms of either high unemployment levels or a shift toward more casualized labor contracts such as zero hour or temporary employment contracts (Aalbers, 2015; Puno & Thomas, 2010). This has led to financial strain and housing affordability problems for many households (JCHS, 2015). § 2.1 Introduction The Global Financial Crisis (GFC), which started in 2008, has had a major impact on many Western European and North American countries. In the years preceding the crisis, many countries in the Global North experienced rising house prices, accompanied by an expansion of mortgage financing (Wachter, 2015). As the financial market has become increasingly global, the collapse of the subprime mortgage market and house price bubble in the United States (US) has had repercussions on a global scale (Martin, 2011). While there were significant differences between impacted countries in the timing and macroeconomic processes underlying the GFC, the characteristics of the subsequent economic recession have been similar: stagnating economic growth, a sovereign debt crisis, and rising unemployment (Aalbers, 2015). Many governments have responded to the declining economy and growing unemployment levels with the implementation of major budget cuts for social provisions (Peck, 2012). This has contributed to both relative and absolute growth in the number of economically disadvantaged households and has exacerbated poverty on both sides of the Atlantic. While the average income of the top 10% of the populations of OECD countries was essentially unaffected by the crisis, the average income of households in the lowest income decile experienced an annual decline of 2% between 2007 and 2010 (OECD, 2013a). In many countries, the GFC has also had a major impact on the housing market, evidenced by a large drop in home prices and declining sales of both existing and new-build housing (Van Der Heijden et al., 2011). Today, many countries are slowly recovering from the first shocks of the GFC and the economic recession that followed. However, in many Southern European countries, unemployment rates continue to be very high and, although unemployment is declining in places like the United States and Germany, long-term unemployment appears to 43 The Global Financial Crisis and neighborhood decline TOC be a persistent problem in many countries (OECD, 2014; Shierholz, 2014). Similarly, despite graudual stock market recoveries and some modest increases in house prices, repercussions from the GFC and economic recession persist in all countries. In many countries, the GFC has had predictable effects on the supply side of the housing market - the willingness of banks to lend money to prospective owners has generally declined. 44 Trajectories of neighborhood change § 2.1 Introduction In the United States, households that are behind on their mortgage payments, and that are unable to obtain a mortgage modification with their lender, are faced with displacement due to foreclosure. This results in a large group of residents with badly damaged credit ratings who are in search of post-foreclosure housing in nearby areas (Martin, 2012). In other countries where the option of foreclosure is often not available, households that are unable to pay their rent or mortgage often have to move to cheaper dwellings and less attractive neighborhoods, while others have to stay put, because moving is too expensive or alternatives are not available, or because negative equity makes it impossible for them to move. All of these developments have contributed to rising inequality in the Global North, particularly in terms of income and housing (e.g. Immervoll et al., 2011; Bellman & Gerner, 2011). The GFC therefore raises questions about the future development of neighborhoods, especially because inequality tends to have specific spatial outcomes including increased segregation, increased spatial concentrations of low-income groups, and negative neighborhood effects (e.g., European Commission, 2010; Glaeser et al., 2009; Van Eijk, 2010; Zwiers & Koster, 2015). While there has been little research on the effects of the GFC at the neighborhood level, the evidence described above suggests that the effects are distributed unevenly across urban areas (Foster & Kleit, 2015; 44 Trajectories of neighborhood change TOC Batson & Monnat, 2015). As households in the bottom income decile have experienced the sharpest drop in income, the effects of the GFC are likely to be felt most acutely in the most disadvantaged neighborhoods (see also Rugh & Massey, 2010; Thomas, 2013). In view of these concerns, this article sets out to identify factors that affect neighborhood decline in the aftermath of the GFC. Many economists have pointed to structural changes in national housing markets and to the changing role of states as important consequences of the GFC (e.g. Wachter, 2015), yet, few researchers analyze how these changes play out at the neighborhood level. Similarly, housing researchers have identified multiple drivers behind neighborhood decline, but many of them focus on within-neighborhood processes at the expense of developments at higher scale levels (Van Beckhoven et al., 2009). In this paper, we aim to bridge this gap by presenting 10 hypotheses on how changes at different geographical scales affect neighborhood decline. § 2.2 Defining neighborhoods and neighborhood decline 6 Trajectories of neighborhood change Neighborhoods are defined in various ways. Some definitions are the neighborhood covers the area within which one can reach im (schools, shops, and friends) within walking distance (e.g. Mo Other definitions are based on social networks and refer explicitl social bonds in the area (e.g. Warren, 1981). However, these defin neighborhood’ is different for each individual, which makes resea outcomes extremely complicated. Galster (2001) defines neighbor of spatially based attributes associated with clusters of reside conjunction with other land uses” (p. 2112). The ‘spatially based for example, the characteristics of buildings, and infrastructural, status, social interactive, and sentimental characteristics. Def based on spatial similarities (such as housing type or populat difficult, especially in mixed-housing areas. All definitions of neighborhoods have their advantages and disadvan ideal neighborhood definition. The choice of definition depends on and should be substantiated by the researcher, bearing in mind that of neighborhoods may lead to different outcomes. For our purposes a rather general and pragmatic definition of neighborhood: a neighb small spatial subdivision of a city or town for which a number of ph and socioeconomic characteristics can be measured. The size of a ne by city. Neighborhoods play an important role in shaping the lives of individ in relation to their social contacts, identity, health, and happiness (se Moreover, neighborhoods have become increasingly important a economic entities, with many governments focusing on neighborh array of social and economic problems (Martin, 2003). This highligh neighborhoods in a post-crisis society: with declining national gove in many countries, there may be an even stronger need to deal w locally, on, for example, the level of cities or neighborhoods. Neighborhoods can develop in different directions: a nei demographically stable for years or even decades. Neighborho gentrification, indicated by, for example, rising house prices, an ou households and an inflow of more affluent households. The ex this topic documents such processes in great detail (e.g. Doucet, Neighborhoods are defined in various ways. Some definitions are related to distance: the neighborhood covers the area within which one can reach important destinations (schools, shops, and friends) within walking distance (e.g. Morris & Hess, 1976). Other definitions are based on social networks and refer explicitly to the existence of social bonds in the area (e.g. Warren, 1981). However, these definitions imply that ‘the neighborhood’ is different for each individual, which makes research on neighborhood outcomes extremely complicated. § 2.1 Introduction Our goal is not to create the next ideal-type model of neighborhood decline processes; rather, we seek to further the intellectual debate on neighborhood decline call for more research on the spatial consequences of the GFC, specifically on neighborhoods as an important territorial dimension of increasing inequality. In view of these concerns, this article sets out to identify factors that affect neighborhood decline in the aftermath of the GFC. Many economists have pointed to structural changes in national housing markets and to the changing role of states as important consequences of the GFC (e.g. Wachter, 2015), yet, few researchers analyze how these changes play out at the neighborhood level. Similarly, housing researchers have identified multiple drivers behind neighborhood decline, but many of them focus on within-neighborhood processes at the expense of developments at higher scale levels (Van Beckhoven et al., 2009). In this paper, we aim to bridge this gap by presenting 10 hypotheses on how changes at different geographical scales affect neighborhood decline. Our goal is not to create the next ideal-type model of neighborhood decline processes; rather, we seek to further the intellectual debate on neighborhood decline call for more research on the spatial consequences of the GFC, specifically on neighborhoods as an important territorial dimension of increasing inequality. Our hypotheses mainly pertain to the Global North. Although these countries have very different political, economic, and social structures, research on neighborhood change in different contexts in the Global North has often led to broadly similar findings. This suggests that many of the underlying processes of neighborhood change are comparable across countries. In the same vein, the increasingly global nature of financial and housing markets (Aalbers, 2015) yields similarities in the effects of the GFC and the economic recession between countries. However, the effects of the GFC are mediated by national policies, local (housing market) circumstances, and intra-neighborhood processes, meaning that the GFC has different outcomes in different places. The next section of this article presents a short discussion of definitions of neighborhoods and neighborhood decline. We then highlight important elements from existing studies to formulate 10 hypotheses about the effects of the GFC and the economic recession on neighborhood decline. These hypotheses are divided over three sections, each with a different geographical focus. The conclusion brings our arguments together and calls for more contextualized longitudinal research. 45 The Global Financial Crisis and neighborhood decline TOC 46 Trajectories of neighborhood change § 2.2 Defining neighborhoods and neighborhood decline Galster (2001) defines neighborhoods as “… bundles of spatially based attributes associated with clusters of residences, sometimes in conjunction with other land uses” (p. 2112). The ‘spatially based attributes’ refer to, for example, the characteristics of buildings, and infrastructural, demographic, class, status, social interactive, and sentimental characteristics. Defining neighborhoods based on spatial similarities (such as housing type or population composition) is difficult, especially in mixed-housing areas. All definitions of neighborhoods have their advantages and disadvantages and there is no ideal neighborhood definition. The choice of definition depends on the type of research and should be substantiated by the researcher, bearing in mind that different definitions of neighborhoods may lead to different outcomes. For our purposes, it is sufficient to use a rather general and pragmatic definition of neighborhood: a neighborhood is a relatively small spatial subdivision of a city or town for which a number of physical, demographic, and socioeconomic characteristics can be measured. The size of a neighborhood may vary by city. Neighborhoods play an important role in shaping the lives of individuals and households, in relation to their social contacts, identity, health, and happiness (see also Martin, 2003). Moreover, neighborhoods have become increasingly important as local political and economic entities, with many governments focusing on neighborhoods to solve a wide array of social and economic problems (Martin, 2003). This highlights the importance of neighborhoods in a post-crisis society: with declining national government involvement in many countries, there may be an even stronger need to deal with many problems locally, on, for example, the level of cities or neighborhoods. Neighborhoods can develop in different directions: a neighborhood can be demographically stable for years or even decades. Neighborhoods can experience gentrification, indicated by, for example, rising house prices, an outflow of low-income households and an inflow of more affluent households. The extensive literature on this topic documents such processes in great detail (e.g. Doucet, 2014; Lees, 2008). TOC Neighborhoods can also show a process of decline, indicated by falling house prices, an inflow of low-income households and an outflow of more affluent households. In this article, we assume that the long-lasting effects of the GFC and the economic recession will fuel neighborhood decline. We use a broad definition of neighborhood decline: any negative development in the physical, demographic, or socioeconomic conditions of a neighborhood as experienced by its residents or other stakeholders. § 2.3 Ten hypotheses on the GFC and neighborhoods The remainder of this article consists of ten hypotheses about the ways in which the GFC might influence neighborhood decline. They are intended as a challenge to researchers to test whether these hypotheses can be confirmed or rejected in different national and urban contexts. The hypotheses are divided into three sections. The first part focuses on how the GFC plays out in different national housing and welfare systems. The next part zooms in on the local context as a mediating variable in processes of neighborhood decline, while the final part concentrates on residents as drivers of neighborhood change. § 2.3.1 The role of national housing and welfare systems Differences in welfare state regimes are an important explanatory factor in the wide range of national differences in housing systems (Priemus & Whitehead, 2014). In countries where the government has historically been strongly involved in the development of affordable (social) housing, such as Denmark, Sweden, and the Netherlands, the quality and the size of the social housing stock was originally very high (Van Kempen & Priemus, 2002; Tsenkova & Turner, 2004). This high initial quality has mitigated processes of neighborhood decline and has led to relatively low levels of income segregation in these countries. However, over the past few decades, severe cuts in housing subsidies took place in these countries, and they have moved toward a more market-based housing system, where the responsibility for social housing shifted from public authorities to housing associations or NGO landlords. Housing associations are now increasingly dependent on their own revenue to construct new social housing (Van Kempen & Priemus, 2002; Schwartz, 2011). To generate revenue, many housing associations have been selling off the better parts of their social housing stock over the past decade, significantly reducing 47 The Global Financial Crisis and neighborhood decline TOC the share and average quality of the social housing stock (Kleinhans & Van Ham, 2013; Schwartz, 2011). In many countries, the GFC has led to the implementation of budget cuts and austerity programs. In combination with cuts in (social) housing subsidies before the GFC, these austerity programs have had an important impact on the opportunities for households on the housing market. Firstly, especially in times of economic recession, austerity programs and budget cuts directly affect the financial resources of households (cf. Lindbeck, 2006; Swank, 1998). Secondly, austerity programs and budget cuts have restricted the resources available for the maintenance and construction of affordable social housing, although these processes have been more dramatic in some countries than in others (Van der Heijden et al., 2011; Priemus & Whitehead, 2014). In the United States, for example, Low-Income Housing Tax Credit (LIHTC) programs were implemented in the 1980s and these programs were extended during the mortgage crisis and the years after to stimulate the development of low-income housing (Schwartz, 2011). § 2.3.1 The role of national housing and welfare systems However, because of the unstable market for tax credits, the LIHTC program tends to be more successful in the more robust housing markets in major metropolitan areas where banks are still dependent on the Community Reinvestment Act (Schwartz, 2011; Belsky & Nipson, 2010). Next to showing geographical differences in the effectiveness of tax credit programs, it is unlikely that they will generate as much equity for housing as it did before the GFC (Schwartz, 2011). We can thus see that the GFC has affected the production of affordable housing in many countries in different ways. In countries where housing associations are dependent on private investors, we can expect to see the production of social housing to increase in those areas where there is a more robust housing market and where there is potential for financial gain. In other countries, we can generally expect a declining production of affordable housing. Together with more financial restrictions for households as a direct effect of the crisis, these processes can reduce residential mobility and force low-income groups to concentrate in neighborhoods where affordable housing options are still available. This can easily lead to increasing concentrations of low-income groups in the most deprived areas. Hypothesis 1 Austerity programs and budget cuts lead to a smaller social safety net for vulnerable groups on the one hand, and to more limited options on the social housing market on the other, leading to increasing concentrations of low-income groups in particular neighborhoods. The extent of the impact of the GFC on the housing market depends on the volatility and structure of local and regional housing markets in different countries (Van der Heijden et 48 Trajectories of neighborhood change TOC al., 2011). In countries with highly regulated housing finance systems, such as Germany, Switzerland, and Austria, the housing market was barely affected by the crisis (Whitehead et al., 2014). The most important explanations for housing market stability in these countries are the well-developed rental markets and the low homeownership rates, together with conservative lending policies (Schneider and Wagner, 2015; Whitehead et al., 2014). In countries with more open finance markets, of which Ireland and Iceland are the main examples, house prices fell considerably due to the rapid expansion of mortgage debt in the years before the crisis (Whitehead et al., 2014). In countries with high mortgage indebtedness, states and financial institutions deliberately stimulated homeownership over the past few decades. § 2.3.1 The role of national housing and welfare systems First, many low- to middle-income groups and first-time buyers were allowed to obtain a mortgage by engaging in high loan-to-value lending (Schelke, 2012). Second, direct subsidies or tax allowances were implemented to support low- to middle-income groups’ entry into homeownership (though in some countries, subsidies such as mortgage interested deductions tend to benefit high-income groups the most) (Hanson et al., 2014; Schelkle, 2012). Low- to middle-income groups have generally been hit the hardest by the GFC and the subsequent economic recession in terms of underwater mortgages, unemployment, and declining incomes (Dreier et al., 2014). In the United States, subprime and predatory lending practices have disproportionally targeted disadvantaged groups in disadvantaged neighborhoods (Aalbers, 2009; Martin, 2011; Mayer & Pence, 2008). Subprime and predatory lending generally refer to high loan-to-value lending, compensating for higher credit risks with unfavorable terms such as higher fees and interest rates that are not beneficial to the borrower (Crossney, 2010; Aalbers, 2013). These practices increase the debt of the borrower beyond the collateral property and reduce the value of the underlying asset and accumulated equity (Crossney, 2010; Schloemer et al., 2006). Subprime and predatory lending tended to be spatially clustered in particular disadvantaged and segregated parts of US cities, resulting in high numbers of foreclosures in these areas (e.g., Anacker & Carr, 2011; Batson & Monnat, 2015; Crossney, 2010; Hyra & Rugh, 2016; Immergluck, 2008; Mallach, 2010a; Rugh & Massey, 2010). Concentrations of foreclosures and vacancies in particular areas may lead to declining housing values of nearby properties (Immergluck, 2009; Immergluck & Smith, 2006) and fuel neighborhood decline through vandalism and increasing crime rates (Aalbers, 2013; Jones & Pridemore, 2016; Martin, 2011; Newman, 2009; Ojeda, 2009). In general, declining house prices have disproportionally affected low- to middle-income groups, often leaving them with a very unstable financial situation and negative equity (e.g., Crossney, 2010; Dreier et al., 2014; Thomas, 2013). In the United States, this has resulted in high concentrations of foreclosures in disadvantaged neighborhoods, 49 The Global Financial Crisis and neighborhood decline TOC displacing large numbers of people who are in need of (affordable) housing and have lost the ability to obtain a mortgage due to badly damaged credit (Goodman et al., 2015; Martin, 2012). These post-foreclosure households tend to relocate in other hard-hit foreclosure areas, contributing to declining average household income and neighborhood instability (Martin, 2012). § 2.3.1 The role of national housing and welfare systems Hypothesis 2 The neighborhood effects of the GFC on neighborhoods are stronger in countries that have actively stimulated homeownership at high loan-to-value rates. Vulnerable groups such as racial or ethnic minorities, low- to middle-income households, and first-time buyers are especially affected by the GFC. When these groups are overrepresented in particular neighborhoods, these neighborhoods are often affected by rapid processes of decline. Hypothesis 2 In countries where there has been a deliberate policy to expand homeownership over the past few decades, it has become more difficult for low- to middle-income groups and first-time buyers to obtain a mortgage than in the years preceding the crisis (Boelhouwer & Priemus, 2014; Clark, 2013; Goodman et al., 2015). The mortgage systems that have emerged from the crisis generally favor higher income groups, leading to increasing disparities between financially stable and financially unstable households (Forrest & Hirayama, 2015). This ultimately means that particular groups and areas are excluded from the mortgage housing market (Clark, 2013; Forrest & Hirayama, 2015; Martin, 2011; Watson, 2009). When it is more difficult for low- to middle-income groups to obtain a mortgage, they are forced to turn to the rental sector. Because renters spend a significantly higher share of their income on housing costs than homeowners (e.g. Haffner & Boumeester, 2014) and because they are not able to accumulate housing equity, this will ultimately contribute to increasing inequality between renters and owners. Hypothesis 3 After the GFC, low- to middle-income groups and first-time buyers are increasingly excluded from the mortgage market, which creates a large group in need of affordable rental housing. At the same time, these changes will lead to a declining homeownership rate in particular areas, creating a spatial divide based on different tenures, and ultimately leading to increasing inequality. Housing opportunities typically differ between generations. The GFC and subsequent recession is likely to further increase intra-generational differences in terms of housing opportunities (e.g., Forrest & Hirayama, 2015). There is already a clear difference between older generations and younger generations - the former have been more able to transform their housing investments into assets over time. High student debts, Housing opportunities typically differ between generations. The GFC and subsequent recession is likely to further increase intra-generational differences in terms of housing opportunities (e.g., Forrest & Hirayama, 2015). There is already a clear difference between older generations and younger generations - the former have been more able to transform their housing investments into assets over time. High student debts, 50 Trajectories of neighborhood change TOC long-term unemployment, a shift towards a more casualized workforce, and stricter mortgage eligibility criteria make it more difficult for the millennial generation to pursue homeownership (JCHS, 2015). 51 The Global Financial Crisis and neighborhood decline Hypothesis 2 The older members of this cohort are just entering the housing market and studies have shown that only a small percentage has been able to become homeowners; this is even more difficult for minority groups (Clark, 2013; JCHS, 2015). In many countries, there has been a decline in homeownership rates among younger households as they postpone marriage and childbirth and tend to prolong their stay in the parental home (Aalbers, 2015; JCHS, 2015; Lennartz et al., 2016). Although many young people might have always been dependent on family financial support to some extent (in the sense of receiving down payments), in recent times, the dependence on family resources to achieve homeownership is becoming more pronounced (Forrest & Hirayama, 2015). However, as many parents have also been subjected to the effects of the GFC and the recession (in terms of unemployment, declining incomes, foreclosures, and negative equity), parents are not equally able to transfer wealth to their children. This is especially true for the younger, lower educated, and minority groups that have accumulated only modest equity (Clark, 2013). In the long run, children from more privileged families will be able to maintain their relatively privileged status by investing in homeownership and accumulate wealth through mortgage amortization and housing appreciation (Forrest & Hirayama, 2015; Rohe et al., 2002). Children from more economically deprived backgrounds, however, will be more dependent on the rental market, thereby increasing their housing costs and reducing their ability to use homeownership as a way to accumulate wealth. These developments will ultimately lead to strong inter- and intra-generational disparities on the housing market (see also Clark, 2013; Forrest & Hirayama, 2015). Hypothesis 4 Hypothesis 4 The GFC has fueled intra-generational differences in terms of housing opportunities. This will increase the influence of social class and the inter-generational transmission of resources as stratifying factors. Countries like Japan, England, the United States, and Australia witness an increase in the proportion of households (often young people) who enter the private rental sector (Forrest & Hirayama, 2015). There is much concern amongst scholars that the rise of the private rental sector has negative consequences for both the renters and the neighborhoods in which these houses are concentrated. In the United States, for example, the number of foreclosed properties owned by banks and other mortgage lenders has spiked the post-crisis period. These REO properties are often acquired by private investors with the main goal of making their investment profitable (Mallach, 2010b). Scholars and activists fear that investors in private housing have little interest 51 The Global Financial Crisis and neighborhood decline TOC in maintaining these dwellings and that practices of ‘milking’ and speculation will spur the process of neighborhood decline (Aalbers, 2013; Ellen et al., 2014; Fields & Uffer, 2016; Forrest & Hirayama, 2015). Although the US federal government has invested billions into the Neighborhood Stabilization Program targeting REO and other vacant properties, the majority of these properties are purchased by private investors rather than owner-occupiers (Ellen et al., 2014). Researchers have argued that private investors play an important role in reducing concentrations of REO properties in particular neighborhoods and that they have been successful in reducing vacancy periods (Ellen et al., 2014; Immergluck, 2010; Pfeiffer & Molina, 2013). Despite the widespread assumption that the sales of REO properties to private investors accelerates neighborhood decline in the most hard-hit neighborhoods due to a lack of maintenance (e.g. Mallach, 2010a), recent studies show that not all private investors adopt business models that negatively affect neighborhoods, (Ellen et al. 2014; Immergluck & Law, 2014; Mallach, 2010b). Though corporate investment does not necessarily harm neighborhoods, the conversion of REO properties into rental units might still fuel processes of neighborhood decline. First of all, renting out properties can contribute to neighborhood instability because of high turnover rates (Kleinhans & Van Ham, 2013; Mallach, 2010a). Second, research has shown that properties sold to private investors and converted into rental units negatively affect the value of surrounding properties (Ihlanfeldt & Mayock, 2016). 52 Trajectories of neighborhood change Hypothesis 4 Hypothesis 5 The crisis has led to an increase of corporate investment in the private rental sector. Converting properties into rental units might lead to neighborhood instability and might negatively impact surrounding property values. These effects will be the strongest in the most hard-hit neighborhoods and are likely to have negative spillover effects on surrounding areas. § 2.3.2 The mediating role of the local context The effects of the GFC and recession, and the austerity programs and budget cuts that followed, are unevenly distributed within countries (cf. Peck, 2012). Cities have been hit hardest, because housing markets are essentially localized and public services and social housing generally tend to be concentrated in city areas (Blank, 1988; Borjas, 1999; Peck, 2012). Yet, the effects of the crisis differ between cities. Although most scholars 52 Trajectories of neighborhood change TOC have focused mainly on neighborhood-level characteristics to explain neighborhood decline, Jun (2013) argues that metropolitan and municipal factors significantly affect neighborhood change. Jun (2013) finds that the neighborhood economic status trends in a positive direction in smaller and more homogeneous cities (in terms of race/ ethnicity), while the reverse applies to larger heterogeneous cities. Her explanation is that smaller cities are less bureaucratic, that there is more room for citizen participation, and that the spending on public goods is lower in ethnically and racially diverse cities, possibly because there are more dissenting views than in homogeneous cities (Jun, 2013). At the metropolitan level, economic strength is obviously an important factor associated with neighborhood change. Lauria and Baxter (1999) showed how the economic shock in New Orleans in the 1980s (caused by falling oil prices) led to the racial transition of neighborhoods, through the mechanisms of foreclosures. It intensified White flight from neighborhoods with relatively small but increasing Black populations. While Lauria and Baxter (1999) focused on the effect of a regional economic downturn, Hyra and Rugh (2016, this issue) look at the effects of the Great Recession that followed the GFC. They compare three gentrifying African American neighborhoods in Chicago, New York, and Washington, DC. The Chicago neighborhood suffered more than the other two from foreclosure and house price decline, whereas the home values in the other two neighborhoods recovered to pre-recession levels. This may be related to the fact that the recession hit Chicago relatively hard, which led to a higher unemployment and vacancy rate than in the other two cities. At the metropolitan level, economic strength is obviously an important factor associated with neighborhood change. Lauria and Baxter (1999) showed how the economic shock in New Orleans in the 1980s (caused by falling oil prices) led to the racial transition of neighborhoods, through the mechanisms of foreclosures. It intensified White flight from neighborhoods with relatively small but increasing Black populations. § 2.3.2 The mediating role of the local context While Lauria and Baxter (1999) focused on the effect of a regional economic downturn, Hyra and Rugh (2016, this issue) look at the effects of the Great Recession that followed the GFC. They compare three gentrifying African American neighborhoods in Chicago, New York, and Washington, DC. The Chicago neighborhood suffered more than the other two from foreclosure and house price decline, whereas the home values in the other two neighborhoods recovered to pre-recession levels. This may be related to the fact that the recession hit Chicago relatively hard, which led to a higher unemployment and vacancy rate than in the other two cities. Hypothesis 6 Hypothesis 6 The crisis has the strongest negative effect on neighborhoods in metropolitan areas with a weak economy and their recovery (if any) will also take longer than in neighborhoods that are situated in a strong regional economy. In addition to exogenous factors that can set off processes of neighborhood decline, some of which we have identified above, characteristics of the neighborhood itself may fuel or mitigate these processes. The initial economic status of a neighborhood is a very strong predictor of its course of development in the long run. Meen and colleagues (2013) have shown how some areas have always had a natural advantage over others because of their location and/or access to particular resources, such as a proximity to ports or transportation centers, and that they maintain their high-quality status and position in the neighborhood hierarchy over longer periods of time. The importance of the relative ‘starting position’ of a neighborhood also relates to the physical quality of the dwellings. Some authors take an almost deterministic stance 53 The Global Financial Crisis and neighborhood decline TOC regarding the relevance of this ‘hard’ variable (e.g. Newman, 1972; Coleman, 1985; and to a lesser extent Power, 1997). In the European context, there is much research on neighborhoods with a high share of post-war, high-rise residential buildings which are prone to processes of neighborhood decline due to the low quality of, and technical problems with, these buildings (e.g., Dekker & Van Kempen, 2004; Kearns et al., 2012; Kleinhans, 2004; Prak & Priemus, 1986; Van Beckhoven et al., 2009). But also in the US context, high foreclosure rates and predatory lending practices cannot only be attributed to the socioeconomic profile of residents (Strom & Reader, 2013). Neighborhoods characterized by a marginal housing stock and poor residents are often explicitly targeted by investors looking to make a profit (Aalbers, 2006; Strom & Reader, 2013). However, the position of neighborhoods in the neighborhood hierarchy is not only a question of location or physical quality, but also a consequence of social processes. Similar types of housing (in physical terms) can acquire a vastly different social status dependent on the identity of a neighborhood. This identity can be very long-lasting (see also Tunstall, 2016, this issue). 54 Trajectories of neighborhood change Hypothesis 6 Comparing three neighborhoods in Stirling, Scotland, Robertson and colleagues (2010) show that the social positioning in terms of class (poor, ‘respectable’ working-class, and middle-class) did not significantly change since the time they were built (1920s and 1930s). This reveals that neighborhood reputations are sticky, which is partly due to the one-sided way in which neighborhoods are covered in the local media (Kearns et al., 2013; see also Tunstall, 2016). Similarly, Wacquant (2008) has shown how political and academic debates on the American ghetto reinforce divisions in society based on race and class, thereby contributing to collective processes of stigmatization and exclusion. The stigmatizing perception of neighborhoods with concentrations of poor and/or racial/ethnic minorities as disordered environments leads to a reinforcement of segregation as middle-class residents and especially Whites are moving (or staying) away from these kinds of neighborhoods (Sampson, 2009). Hypothesis 7 Areas that are characterized by a low-quality housing stock and a negative reputation are particularly prone to processes of neighborhood decline. Hypothesis 7 Areas that are characterized by a low-quality housing stock and a negative reputation are particularly prone to processes of neighborhood decline. yp Areas that are characterized by a low-quality housing stock and a negative reputation are particularly prone to processes of neighborhood decline. Over the past decades, many countries have implemented neighborhood regeneration programs. The general goal of these programs was to reduce relative inequality between the most disadvantaged neighborhoods and the city or the national average (Jivraj, 2012). The ways in which these urban restructuring programs are pursued in practice differs between countries (Skifter Andersen, 1999). However, in general, policies were implemented to stimulate a socioeconomic residential mix in deprived neighborhoods. Examples are the HOPE VI program in the United States, the Urban Restructuring TOC Program in the Netherlands and the New Deal for Communities in the United Kingdom (e.g. Bolt & Van Kempen, 2011; Goetz, 2010; Phillips & Harrison, 2010). Many policymakers believe that the mixing of different socioeconomic groups in disadvantaged areas will lead to neighborhood upgrading (Andersson & Musterd, 2005; Van Gent et al., 2009). In many cases, urban restructuring meant the demolition of low-quality rental dwellings, replacing them with more upmarket owner-occupied and luxury rental dwellings (Kleinhans, 2004). § 2.3.3 Behavioral responses: Exit and voice The dynamics of a neighborhood are highly affected by the decisions of its residents. Following Hirschman’s (1970) ‘Exit, voice and loyalty’ framework, Permentier and colleagues (2007) argue that residents who are dissatisfied with their neighborhood can either choose to move out (exit) or adopt problem-solving strategies (voice). Loyalty (the attachment to neighborhood and its residents) increases the likelihood of the voice option and reduces the probability of residential mobility (Permentier et al., 2007) Residential mobility is the central explanatory variable in the neighborhood decline model of Grigsby and others (1987). Neighborhoods can change rapidly as a result of selective mobility where the demographic and socioeconomic characteristics of those households leaving are different from the characteristics of the newly arriving households. Declining housing and neighborhood quality can spur residential mobility: middle- and high- income groups move away from declining neighborhoods as a result of the decreasing attraction of dwellings and neighborhoods and the creation of new dwellings elsewhere - a process also known as relative depreciation (Grigsby et al., 1987; Hoyt, 1939). The likelihood of a household moving depends on whether household preferences can be realized by the resources available to the household within the opportunities (available dwellings) and restrictions (ability to obtain a mortgage) of the desired housing market (Clark & Dieleman, 1996; Mulder & Hooimeijer, 1999). Generally speaking, more affluent households have a larger choice set of dwellings and neighborhoods. The GFC and subsequent recession is likely to have major impacts on residential mobility. On the one hand, we have argued that people tend to be more limited in their options due to financial restrictions and stricter mortgage eligibility criteria. Households might want to move, but are not able to move because they cannot obtain a mortgage or do not find a suitable rental dwelling. In the European context, many low-income households are dependent on the availability of social or public housing and waiting lists are long, making it difficult for these households to move from one to another rented dwelling. Similarly, many homeowners in Western Europe might be forced to stay in a particular dwelling and neighborhood, because they cannot sell their current home without taking a large financial loss. In the US context, foreclosures force people to move and thus lead to a wave of residential moves at first. Hypothesis 6 In this way, spatial concentrations of low-cost rental dwellings were reduced and the residents of the demolished dwellings were forced to relocate to other (often nearby, often also disadvantaged) neighborhoods where affordable housing was still available (Bolt et al., 2008; Crump, 2002; Posthumus et al., 2013; Van Kempen & Priemus, 2002). Most of these residents did not have the means to move back to the more expensive, newly created housing in the regeneration area (Kleinhans & Varady, 2011). It has thus been argued that restructuring programs may lead to the downgrading of other (surrounding) neighborhoods, because the previous spatially concentrated deprivation becomes dispersed over a larger geographical area (Andersson et al., 2010; Bråmå, 2013; Posthumus et al., 2013). While these mixing policies can be successful in improving the economic statistics of a neighborhood, most of these policies have, however, been heavily criticized for failing to really improve the lives of the original residents (Doff & Kleinhans, 2011; Goetz, 2010; Van Ham & Manley, 2012). Nevertheless, policymakers often frame such programs as successful, and these programs have contributed to some extent of segregation decrease (Feins & Shroder, 2005; Frey, 2010; Musterd & Ostendorf, 2005b). A well-known argument is that countries such as the Netherlands and Sweden do not have ghetto-like neighborhoods because of a strong government involvement and mixing policies. This raises the question whether the retreat of governments from deprived neighborhoods as a result of the crisis will fuel processes of socioeconomic segregation and neighborhood decline. On the basis of Tunstall’s (2016) conclusion that neighborhood renewal policies have not made a significant change in the neighborhood hierarchy, one might speculate that government retreat does not make much of a difference. On the other hand, it can be argued that whether a neighborhood is at the bottom rung of the ladder is not the only important factor; stratification between neighborhoods also contributes to their various fates. Hypothesis 8 The crisis will have the largest effect on processes of neighborhood decline in neighborhoods where there has been a strong government involvement in urban regeneration and other neighborhood policies. 55 The Global Financial Crisis and neighborhood decline TOC § 2.3.3 Behavioral responses: Exit and voice However, the unstable financial situation of many foreclosed households, together with tight credit standards, make it nearly impossible for these households to obtain a mortgage in the future (Goodman et al., 2015; Martin, 2012). Residential mobility can therefore also be expected to decrease in the United States, although a recent study by Pfeiffer and Molina (2013) shows how the foreclosure crisis offers an 56 Trajectories of neighborhood change TOC opportunity for Latino households in terms of socioeconomic mobility; however, they also argue that Latino households are more likely to purchase properties in Latino- concentrated areas, thereby exacerbating existing patterns of spatial segregation (Pfeiffer & Molina, 2013). Similarly, research has shown how many foreclosed households tend to end up in other hard-hit foreclosure areas (Martin, 2012), after which they are more or less stuck in these neighborhoods because they are unable to obtain a mortgage and move to a different area. The unstable financial situation of many households, combined with stricter mortgage eligibility, complicates residential mobility on both sides of the Atlantic. Even though residential mobility has decreased on both continents, the outcomes may be very different. In the United States, we can expect that limited residential mobility has further contributed to existing socioeconomic and racial segregation, while in Europe, it can be expected that the process of segregation has slowed down. Hypothesis 9 In these neighborhoods, where many residents are unable to move, people may feel close to each other because of a common fate, actually increasing social cohesion. This can lead to a strengthening of solidarity networks and a deepening attachment to place, even in very stigmatized areas like the French banlieues (Kirkness, 2014). However, it is also possible that neighborhoods experiencing an inflow of lower-income groups are prone to increasing social disorganization. A change of population composition might lead to residential stress as people tend to prefer a neighborhood population that matches their own characteristics (Feijten & Van Ham, 2009; McPherson et al., 2001). Hypothesis 10 In times of crisis, social cohesion may be reinforced in areas where there has been a reasonable level of social interaction in the past, while it is likely to crumble in areas that experience increasing tensions because of a diversification of the population, or in areas that are experiencing significant declines in population density. Hypothesis 9 Hypothesis 9 Decreases in residential mobility rates can have different outcomes in different contexts. In many Western European countries, we expect a lower likelihood of an increase in residential segregation, while in the United States, foreclosures have led to a small short- term upsurge in residential mobility patterns, exacerbating existing segregation. If residents are not satisfied with their neighborhood, they can (instead of moving out) also opt to organize themselves to address neighborhood problems. Whether that is a feasible strategy depends on the level of social cohesion in the neighborhood. It is often assumed that disadvantaged neighborhoods suffer from the lack of strong social ties and the advantages these ties bring along (Forrest and Kearns, 2001). Without a strong social fabric, neighborhoods are more prone to disorder in terms of vandalism, nuisance, and crime (Kleinhans & Bolt, 2014). Social disorganization theory, which originated from the Chicago School of Sociology, stated that disorganization in neighborhoods is caused by incapability of the local community in terms of a lack of (access) to resources, residential instability, or a weak social network (Shaw & McKay, 1942). Physical and social problems arise because residents are not able to enforce certain norms and to maintain social control. As a result, governments tend to retreat from public space and residents lose their trust in each other and ‘hunker down’ (Putnam, 2007; Ross et al., 2001). Some researchers have argued that small levels of disorder (such as graffiti or broken windows) give rise to more serious crime offenses. The broken windows theory states that potential criminals interpret these levels of disorder as a sign of a lack of social control or involvement of the residents, and as such, feel free to engage in criminal behavior (Wilson & Kelling, 1982). 57 The Global Financial Crisis and neighborhood decline TOC Recent research by Jones & Pridemore (2016) on the effect of vacancies on crime rates after the GFC concludes that population loss and vacant homes complicate neighborhood social organization. In line with social disorganization theory and the broken windows theory, they argue that the lack of collective efficacy as a result of low levels of population density makes those areas more attractive to criminals (Jones & Pridemore, 2016). In times of crisis, social cohesion in (disadvantaged) neighborhoods can develop in different directions. With many governments retreating, an increasing level of responsibility for the neighborhood has shifted to its residents. § 2.4 Conclusions In this article, we have argued that contemporary neighborhood decline is a multidimensional process fuelled by several macroeconomic processes related to the GFC and the recession that followed. However, we have also argued that there are several local and internal factors that might function as a mediating factor in processes of neighborhood decline. The interaction of micro-, meso- and macro-level factors heavily depends on the context in space and time. In this article, we have argued that contemporary neighborhood decline is a multidimensional process fuelled by several macroeconomic processes related to the GFC and the recession that followed. However, we have also argued that there are several local and internal factors that might function as a mediating factor in processes of neighborhood decline. The interaction of micro-, meso- and macro-level factors heavily depends on the context in space and time. There is a lack of empirical studies that focuses on the effects of the GFC on neighborhoods and their residents. In an attempt to push the debate forward, we have formulated 10 hypotheses on how the GFC might interplay with processes of neighborhood decline. We submit these hypotheses as a guide for future empirical research. Research is necessary 58 Trajectories of neighborhood change TOC because differences in the local effects of the GFC are likely to lead to a widening of the gap between wealthy and disadvantaged neighborhoods, between high-income mortgage borrowers and low-income borrowers, between privileged and less privileged households, and between renters and homeowners (Forrest & Hirayama, 2015). In combination with severe budget cuts and the implementation of austerity programs, this raises concerns about increasing spatial segregation based on social class (see also Tammaru et al., 2016). We have identified several factors from the literature that influence neighborhood change. However, little is actually known about the ways in which these factors interact in different contexts. We therefore call for more longitudinal research of neighborhoods and households that focuses on the drivers of neighborhood decline and disinvestment, and more generally, neighborhood change. Without longitudinal data on the residential and social mobility of households, it is difficult to disentangle the relative weight of residential sorting and incumbent processes in explaining neighborhood change. Incumbent upgrading and downgrading refers to the changing socioeconomic profile of the resident population within an area (e.g. Teernstra, 2014). 60 Trajectories of neighborhood change § 2.4 Conclusions It is an empirical question regarding how important external forces and internal developments are to neighborhoods; this can differ by country, city, or even by neighborhood. This question is crucial, especially because in countries where the crisis has reduced residential mobility, incumbent processes may become relatively more important in explaining neighborhood decline through processes of rising unemployment and declining incomes (Andersson & Hedman, 2016, this issue). Individual-level data over long periods of time are needed to address this question. Such data are not available in all countries; however, as better data becomes available, researchers should aim to take a richer array of longitudinal individual and spatial variables into account (Van Ham & Manley, 2012). This is not only an academic question, but also relevant in the evaluation of neighborhood restructuring programs. Is there, for instance, an improvement in the livability and social status of neighborhoods due to the empowerment of the sitting population or due to the replacement of vulnerable groups by middle-class households? Most studies that focus on neighborhood change tend to concentrate on case studies of specific cities, or specific gentrifying or declining neighborhoods. This focus can be largely attributed to the complexity of the subject, a lack of detailed (comparable) longitudinal data, and a bulk of statistical problems with which researchers are confronted; it nevertheless constitutes a large gap in research on neighborhood dynamics. Neighborhoods do not operate in a vacuum and while a particular neighborhood may experience absolute change, the picture may be completely different when we look at the relative change in a city or a country. Moreover, in a globalizing world, with growing 59 The Global Financial Crisis and neighborhood decline TOC internationally connected economies and housing markets, it will become increasingly important to understand neighborhood change from a more global perspective. The GFC has had different economic, physical, social, and health-related outcomes, most of which we are only now beginning to grasp. Researchers have argued that the GFC has had different local outcomes between and within countries (Aalbers, 2009), but we have little insight in the long-term effects of the GFC on neighborhoods and its residents. It is important to understand how the crisis has affected spatial patterns of increasing inequality, and neighborhood trajectories. A deeper understanding of the drivers behind neighborhood decline can contribute to the development of effective policymaking in the aftermath of the GFC and the economic recession. § 2.4 Conclusions 60 Trajectories of neighborhood change TOC
https://openalex.org/W2668840119	https://cdr.lib.unc.edu/downloads/9880w185p	English	null	Heterotrophic Extracellular Enzymatic Activities in the Atlantic Ocean Follow Patterns Across Spatial and Depth Regimes	Frontiers in marine science	2,017	cc-by	8,991	ORIGINAL RESEARCH published: 23 June 2017 doi: 10.3389/fmars.2017.00200 Heterotrophic Extracellular Enzymatic Activities in the Atlantic Ocean Follow Patterns Across Spatial and Depth Regimes Adrienne Hoarfrost* and Carol Arnosti Department of Marine Sciences, University of North Carolina at Chapel Hill, Chapel Hill, NC, United States Heterotrophic microbial communities use extracellular enzymes to initialize degradation of high molecular weight organic matter in the ocean. The potential of microbial communities to access organic matter, and the resultant rates of hydrolysis, affect the efﬁciency of the biological pump as well as the rate and location of organic carbon cycling in surface and deep waters. In order to investigate spatial- and depth-related patterns in microbial enzymatic capacities in the ocean, we measured hydrolysis rates of six high-molecular-weight polysaccharides and two low-molecular-weight substrate proxies at sites spanning 38◦S to 10◦N in the Atlantic Ocean, and at six depths ranging from surface to bottom water. In surface to upper mesopelagic waters, the spectrum of substrates hydrolyzed followed distinct patterns, with hydrolytic assemblages more similar vertically within a single station than at similar depths across multiple stations. Additionally, the proportion of total hydrolysis occurring above the pycnocline, and the spectrum of substrates hydrolyzed in mesopelagic and deep waters, was positively related to the strength of stratiﬁcation at a site, while other physichochemical parameters were generally poor predictors of the measured hydrolysis rates. Spatial as well as depth-driven constraints on heterotrophic hydrolytic capacities result in broad variations in potential carbon-degrading activity in the ocean. The spectrum of enzymatic capabilities and rates of hydrolysis in the ocean, and the proportion of organic carbon hydrolyzed above the permanent thermocline, may inﬂuence the efﬁciency of the biological pump and net carbon export across distinct latitudinal and depth regions. Keywords: carbon cycling, extracellular enzymes, heterotrophy, functional biogeography, deep ocean, microbial activity, biogeochemistry Edited by: Edited by: Maria Montserrat Sala, Consejo Superior de Investigaciones Cientíﬁcas(CSIC),Spain Reviewed by: Hila Elifantz, Bar-Ilan University, Israel Zhanfei Liu, University of Texas at Austin, United States *Correspondence: Adrienne Hoarfrost adrienne.l.hoarfrost@unc.edu Specialty section: This article was submitted to Aquatic Microbiology, a section of the journal Frontiers in Marine Science Received: 12 April 2017 Accepted: 12 June 2017 Published: 23 June 2017 Seawater Sampling Seawater was collected via Niskin rosette equipped with a conductivity-temperature-depth sensor (CTD) at nine stations spanning 38◦S to 10◦N in the subtropical to equatorial Atlantic Ocean (Figure 1). Hydrolysis rates of polysaccharides and of monomeric substrates were measured at 6 stations, ranging from 38◦S to 3.5◦N; only activities of monomeric substrates could be measured at the northernmost three stations due to lack of time for extended substrate incubations. The enzymatic capacity of a microbial community is a critical determinant of the breadth of organic compounds which may be recycled. Most organic carbon is biosynthesized as high molecular weight compounds, which are typically hydrolyzed by both endo-acting (mid-chain cleaving) and exo-acting enzymes (Warren, 1996). In order for heterotrophs to access natural organic matter, they must produce the appropriate enzymes to hydrolyze a particular substrate into sizes small enough to be transported into the cell. These enzymes have highly targeted structural speciﬁcities and are very diverse (Aspeborg et al., 2012; Teeling et al., 2012), reﬂecting both the complexity of natural organic matter and of the microbial communities that access it. At each station (stations 2, 4, 7, 10, 15, 18, 21, 22, and 23; part of the DeepDOM cruise, Kujawinski, 2013), seawater was collected from six depths: surface (SuW, 5 m), deep chlorophyll maximum (DCM, ∼50–100 m), mesopelagic (meso, 250 m), Antarctic Intermediate Water (AAIW, ∼750–850 m), North Atlantic Deep Water (NADW, 2,500 m), and bottom water (bot, ∼3,700–4,600 m). Speciﬁc depths of DCM, AAIW, and bottom water were chosen according to a maximum in ﬂuorescence (DCM), a minimum in salinity and peak in oxygen (AAIW), and a few meters above bottom (bot), respectively (Supplementary Figure 1), and thus varied by station. Diﬀerences in microbial enzymatic capacities thus may result in functional biogeographical patterns in carbon export and recycling. Field studies measuring activities of extracellular enzymes that degrade organic carbon have demonstrated that the types of substrates hydrolyzed and their rates of hydrolysis vary along latitudinal gradients (Arnosti et al., 2011, 2012), a pattern that parallels biogeographical patterns in microbial community composition (Fuhrman et al., 2008). Beyond community composition, the genetic capacity to hydrolyze individual substrates may also follow biogeographical patterns. Frontiers in Marine Science \| www.frontiersin.org Citation: Microbial communities are major drivers of organic carbon cycling in the ocean. The carbon cycling capacities of these communities ultimately aﬀect the inventories of oxygen and CO2 in the atmosphere, the magnitude and composition of organic carbon export from the surface to the deep ocean, and resource availability to higher trophic levels (Azam and Malfatti, 2007; Jiao et al., 2010). Although 99.9% of autochthonous organic carbon is remineralized before it reaches sediments, a large standing pool of dissolved organic carbon (DOC) persists in the water column (Hedges, 1992), Hoarfrost A and Arnosti C (2017) Heterotrophic Extracellular Enzymatic Activities in the Atlantic Ocean Follow Patterns Across Spatial and Depth Regimes. Front. Mar. Sci. 4:200. doi: 10.3389/fmars.2017.00200 June 2017 \| Volume 4 \| Article 200 Frontiers in Marine Science \| www.frontiersin.org Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti rates to stratiﬁcation and physicochemical parameters. These factors may shape biogeographical patterns in heterotrophy, and ultimately aﬀect the location and magnitude of organic carbon remineralization, and thus carbon sequestration, by the biological pump. demonstrating that some fraction of marine organic carbon is not readily amenable to microbial degradation. The microbial enzymatic capacities to access organic carbon is a potentially important driver shaping the ocean carbon reservoir, but the factors that determine whether and how much organic matter is remineralized are poorly understood (Arnosti, 2011). Biogeographical patterns in microbial communities and their enzymatic capacities (Arnosti et al., 2012; Gomez-Pereira et al., 2012; Sunagawa et al., 2015), and their net eﬀect on organic carbon transformations in the ocean, may in turn be shaped by a poorly constrained set of factors (Hanson et al., 2012). Seawater Sampling Although, most of the enzymes involved in the extracellular breakdown of organic carbon have not yet been annotated, a targeted study of the biogeography of enzymes in glycosyl hydrolase family 5 revealed wide diversity and variation in relative abundance across the North Atlantic (Elifantz et al., 2008). Genetic distributions of polysaccharide-degrading enzymes in common heterotrophic marine microbial clades also vary considerably across oceanic provinces (Gomez-Pereira et al., 2012), as do the activities of polysaccharide hydrolyzing enzymes (Arnosti et al., 2012). At each depth, 1 L glass Duran bottles were rinsed three times with seawater from the corresponding depth, then ﬁlled without using tubing from a single Niskin bottle. 200 mL of seawater from each depth was autoclaved in a separate glass Duran bottle for use in killed control incubations. FIGURE 1 \| Map of DeepDOM cruise track from 38S to 9N in the South Atlantic. Figure made in Ocean Data View Schlitzer, 2015. Organic carbon cycling capabilities are thus heterogeneously distributed across both microbial phylogenies (Zimmerman et al., 2013) and the natural environment. In order to examine latitudinal and depth-related patterns in hydrolytic capabilities of heterotrophic microbial communities, we measured extracellular enzyme activities across a broad range of latitude and depth, identiﬁed geospatial patterns in those activities, and investigated potential environmental factors aﬀecting heterotrophic enzymatic activity. The hydrolysis of six high-molecular- weight and two low-molecular-weight organic substrates was measured across 48 degrees of latitude and from surface to bottom waters in the South and Equatorial Atlantic Ocean. These data enable us to explore the connectivity of hydrolytic capacities between stations and depths, and the relationship of hydrolysis FIGURE 1 \| Map of DeepDOM cruise track from 38S to 9N in the South Atlantic. Figure made in Ocean Data View Schlitzer, 2015. June 2017 \| Volume 4 \| Article 200 2 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti Physical and Chemical Parameters Temperature, salinity, oxygen, and ﬂuorescence were measured via CTD (Supplementary Figure 1), and used to calculate potential temperature (θ), potential density (σθ), and buoyancy frequency (N2) (Supplementary Figure 2). CTD data for every cast throughout the cruise, and nutrient data collected from discrete depths and analyzed by K. Longnecker, are available through the BCO-DMO database (Supplementary Figure 1, Kujawinski, 2013). α-Glucosidase and Leucine Aminopeptidase Activities p p Two substrate proxies, α- glucose linked to 4- methylumbelliferone (α-Glu; Chem Impex 21676) and leucine linked to 4-methylcoumarinyl-7-amide (L-MCA; Sigma 62480- 44-8), were used to measure the activities of α-glucosidase and leucine aminopeptidase, respectively, after the method of Hoppe (1983). The enzymes hydrolyzing these substrates act on the α-1→4-linked terminal glucose of oligo- and polysaccharides, and N-terminal leucine residues of peptides or proteins, respectively. Recent work has demonstrated that L-MCA can also be hydrolyzed by enzymes other than leucine aminopeptidase (Steen et al., 2015), but this widely-used method still provides a measure of peptidase activity in the environment. For each substrate, triplicate aliquots of 4mL of live seawater and one autoclaved seawater killed control were incubated in plastic cuvettes at as close to in situ temperature as possible. Available incubation temperatures were 3, 12, 15, 18, 25, and 28◦C. Two cuvettes with 4 mL of live or autoclaved seawater and no added substrate served as live and killed blank incubations, respectively. Fluorescently-labeled polysaccharides were prepared after the method of Arnosti (1996, 2003). Each polysaccharide was incubated in triplicate live incubations in 17 mL sterilized glass vials, and one killed control incubation using autoclaved seawater. In addition, incubations without substrate with live seawater and autoclaved seawater were used as live and killed blank controls, respectively. Seawater was sterilized for 20 min in an autoclave upon recovery and incubations initiated after autoclaved water had cooled in an ice bath. Substrate was added at concentrations suﬃcient to detect ﬂuorescence of the substrate, at 3.5 µM monomer-equivalent concentrations in all cases, except for fucoidan, which was added at a concentration of 5 µM due to its lower ﬂuorescence intensity. Substrate addition is kept to the lowest concentration that is technically feasible in order to minimize growth responses due to added substrate. All samples were incubated at as close to in situ temperature as possible. Saturating concentrations were determined at each station via a saturation curve conducted over 24 h using surface water, testing increasing concentrations of substrate. The saturating concentration was identiﬁed as the concentration of substrate at which addition of higher concentrations of substrate does not induce higher rates of activity. Seawater Sampling CTD and nutrient data used for analysis in this study are provided through the associated BCO-DMO repository (Hoarfrost and Arnosti, 2016), and can be reproduced using scripts provided at the associated Github repository (Hoarfrost, 2016). incubation was sampled later than 72 h. All incubations were sampled at 24 h to provide a common timepoint reference. Rates reported here are maximum hydrolysis rates, typically at T3 for α-glucosidase and at T1 for leucine aminopeptidase. T3 for α-glucosidase was typically sampled at 36–48 h in shallow, more active waters, or 60–72 h in deeper, less active waters. T1 for leucine aminopeptidase was typically sampled at 4–6 h in shallow waters, or 24 h in deeper waters. α-glucosidase activities sampled at later timepoints may include a growth response, whereas the shorter timecourse of leucine aminopeptidase incubations likely does not include a growth response. In all cases, rates represent potential hydrolysis rates, since added substrate competes with naturally-occurring substrate for enzyme active sites. incubation was sampled later than 72 h. All incubations were sampled at 24 h to provide a common timepoint reference. Rates reported here are maximum hydrolysis rates, typically at T3 for α-glucosidase and at T1 for leucine aminopeptidase. T3 for α-glucosidase was typically sampled at 36–48 h in shallow, more active waters, or 60–72 h in deeper, less active waters. T1 for leucine aminopeptidase was typically sampled at 4–6 h in shallow waters, or 24 h in deeper waters. α-glucosidase activities sampled at later timepoints may include a growth response, whereas the shorter timecourse of leucine aminopeptidase incubations likely does not include a growth response. In all cases, rates represent potential hydrolysis rates, since added substrate competes with naturally-occurring substrate for enzyme active sites. Polysaccharide Hydrolysis Measurements Activities of extracellular enzymes that hydrolyze six diﬀerent ﬂuorescently labeled polysaccharides were measured at all six depths between 38◦S and 3.5◦N. These substrates— arabinogalactan, chondroitin sulfate, fucoidan, laminarin, pullulan, and xylan—were chosen for their diverse monosaccharide compositions and macromolecular structures. All of these polysaccharides are found in marine environments, and/or enzymes and genes corresponding to their hydrolysis have been identiﬁed in marine prokaryotes (e.g., Alderkamp et al., 2007; Wegner et al., 2013; Xing et al., 2015). Furthermore, the activities of enzymes hydrolyzing these substrates have been detected in a wide variety of marine environments (e.g., Arnosti, 2008; Arnosti et al., 2009, 2011). Frontiers in Marine Science \| www.frontiersin.org Polysaccharide Hydrolysis Rates and Patterns Polysaccharide hydrolysis rates and patterns varied across depths as well as stations, as evident for individual substrates (Figure 2), by the summed hydrolysis rates (Supplementary Figure 5), and by the diversity of substrates hydrolyzed at a given depth (Figure 3). Some polysaccharides—such as laminarin—were hydrolyzed at nearly every station and depth, whereas fucoidan was not measurably hydrolyzed at any site, and arabinogalactan was hydrolyzed only in surface waters of station 15. Chondroitin, pullulan, and xylan were hydrolyzed only at particular stations and depths: chondroitin was the only substrate other than laminarin hydrolyzed below 250 m, but at some stations it was not hydrolyzed at any depths. Pullulan was hydrolyzed only Hydrolytic Compositional Dissimilarity among Sampling Sites Using Bray-Curtis Dissimilarity p g g y y The Bray-Curtis Dissimilarity, BC, is used to describe the compositional dissimilarity between two sites (Bray and Curtis, 1957). As applied here, “composition” is deﬁned as the hydrolytic composition, or the assemblage of substrates hydrolyzed and their relative rates of hydrolysis. BC is a unitless index between 0 and 1, with a minimum of 0 when the two sites have exactly the same composition (e.g., all the same substrates are hydrolyzed at the same rate), and a maximum of 1 when none of the same substrates are hydrolyzed at the two sites. The pairwise BC dissimilarity matrix was calculated between every site with every other site. The Bray-Curtis Dissimilarity between two sites i and j is calculated as BCij = 1 − 2Cij Si + Sj , where Cij is the sum of the lesser hydrolysis rates for only those substrates that were hydrolyzed at both sites i and j, and Si and Sj are the total hydrolysis rates at site i and site j respectively. Reproducibility from the size-separated chromatograms using the scripts hosted at the associated Github repository (Hoarfrost, 2016). Chromatograms were manually curated after processing to verify chromatographic changes and to identify incubations with zero activity or non-hydrolytic ﬂuorescence of the free ﬂuorophore label, which can produce artiﬁcially high hydrolysis rates. Those incubation sets were tagged and their calculated rates adjusted by setting the activity to zero, or recalculating the rate while neglecting the free ﬂuorophore portion of the chromatogram, respectively. The scripts to process the GPC chromatograms and calculate rates, manipulate physicochemical data, perform statistical analyses, and generate the ﬁgures in this paper were all written in the R programming language (R Core Team, 2014), and can be reproduced using the scripts hosted at the associated Github repository (Hoarfrost, 2016). The raw data is hosted on BCO-DMO (Hoarfrost and Arnosti, 2016), and instructions to download raw data and run scripts can also be found in the Readme for the Github repository. α-Glucosidase and Leucine Aminopeptidase Activities Since enzymatic activity is typically highest in surface or near-surface waters (e.g., Baltar et al., 2009; Steen et al., 2012), and leucine aminopeptidase activity is typically higher than α-glucosidase activity (Baltar et al., 2010, 2013), saturation concentrations determined for leucine-MCA in surface waters were used for all depths and substrates at each station. Substrates were added at saturating concentrations 100 µM at stations 2, 4, and 7; 75 µM at stations 10, 15, 18, 22, and 23; and 50 µM at station 21. p p Each incubation was sampled at four timepoints: 0, 5, 12, and 21 days. Kill and live blank controls were sampled at T0 and Tﬁnal only. Due to time constraints, at Station 15 the ﬁnal timepoint was taken at day 20 instead of 21, and at Station 18 only three timepoints were collected, at 0, 5, and 12 days. At each timepoint, ∼1.8 mL was withdrawn from each incubation, ﬁltered through a 0.2 µm pore-size syringe ﬁlter and stored at −20◦C until analysis. Enzyme activity was measured by tracking hydrolysis of the high-molecular-weight substrate into lower-molecular- weight hydrolysis products over time, as determined using gel permeation chromatography with ﬂuorescence detection (Arnosti, 1996, 2003). Hydrolysis rates were calculated from these shifts in molecular size distribution over time Incubations were sampled at four timepoints, and ﬂuorescence was measured in a Turner Biosystems spectrophotometer (TBS-380). Later timepoints were chosen based on the rate of activity at earlier timepoints. A typical timecourse for a rapidly-hydrolyzed substrate was 6, 12, and 24 h; for a low- to no-hydrolysis substrate, 24, 48, and 72 h. No Frontiers in Marine Science \| www.frontiersin.org June 2017 \| Volume 4 \| Article 200 3 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti Polysaccharide Hydrolytic Diversity Using Shannon Diversity Indices Polysaccharide Hydrolytic Diversity Using Shannon Diversity Indices Physical Context and Water Masses The transect covered a broad range of latitude and physicochemical conditions, as well as several distinct water masses (Supplementary Figures 1, 3). Antarctic Intermediate Water (AAIW) ﬂowing south to north was detectable as a minimum in salinity at ca. 750–850 m depth throughout the transect. North Atlantic Deep Water (NADW) ﬂowing north to south was identiﬁed as a maximum in oxygen between ca. 1500–4,000 m water depth. A large circulation-driven oxygen minimum zone encompassed stations 10–23. At the southernmost station sampled, station 2, the inﬂuence of Antarctic circulation was still apparent, with circumpolar deep waters bounding NADW above and below, and Antarctic Bottom Water (AABW, detectable as a temperature minimum) in the bottom water sample (Supplementary Figures 1, 3). At the northernmost station (station 23), the Amazon River plume was sampled in surface waters, detectable by a sharp halocline. The strength of the pycnocline generally increased from south to north, such that the southernmost stations were less stratiﬁed than the northernmost stations (Supplementary Figures 2, 4). A south to north gradient in chlorophyll a concentrations was also evident at the deep chlorophyll maximum, which increased from ca. 0.242 mg m−3 at station 2 to over 1 mg m−3 at station 15 (Kujawinski, 2013). DOC concentrations ranged from ca. 70–82 µM in surface, ca. 62–77 µM in DCM, and ca. 47–58 µM in mesopelagic depths, and did not directly track chlorophyll a concentrations (Kujawinski, 2013). Shannon indices, which reﬂect both the number of substrates hydrolyzed as well as the evenness of hydrolysis rates, were used to calculate hydrolytic diversity at all sites (Steen et al., 2010). The Shannon index is expressed as H = − nP i=1 piln(pi), where n is the total number of substrates and pi is the hydrolysis rate of the ith substrate normalized to the summed hydrolysis rate of all substrates at that site. H is equal to zero when only one substrate is hydrolyzed, and is maximal at 1.79 when all six substrates are hydrolyzed at equal rates. Multiple Regression Analysis of Environmental Parameters vs. Hydrolytic Activity Multivariate linear regression models between polysaccharide hydrolysis rates and up to ten environmental parameters— in situ potential temperature, incubation temperature, salinity, oxygen, chlorophyll a, buoyancy frequency, phosphate, total nitrogen, DOC, and silicate—were generated. By testing several permutations of models considering diﬀerent combinations of environmental parameters, the best ﬁt multiple regression model was selected by manually maximizing correlation coeﬃcient values (Supplementary Table 1). June 2017 \| Volume 4 \| Article 200 Frontiers in Marine Science \| www.frontiersin.org 4 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti FIGURE 2 \| Maximum potential hydrolysis rate (nM h−1) of the six polysaccharide substrates arabinogalactan (A), chondroitin sulfate (C), fucoidan (F), laminarin (L), pullulan (P), and xylan (Y) at each station (vertical panels) and each depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. Missing data at stn 15, mesopelagic depth for the xylan substrate indicated by asterisk. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. FIGURE 2 \| Maximum potential hydrolysis rate (nM h−1) of the six polysaccharide substrates arabinogalactan (A), chondroitin sulfate (C), fucoidan (F), laminarin (L), pullulan (P), and xylan (Y) at each station (vertical panels) and each depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. Missing data at stn 15, mesopelagic depth for the xylan substrate indicated by asterisk. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. FIGURE 2 \| Maximum potential hydrolysis rate (nM h−1) of the six polysaccharide substrates arabinogalactan (A), chondroitin sulfate (C), fucoidan (F), laminarin (L), pullulan (P), and xylan (Y) at each station (vertical panels) and each depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. Missing data at stn 15, mesopelagic depth for the xylan substrate indicated by asterisk. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. FIGURE 2 \| Maximum potential hydrolysis rate (nM h−1) of the six polysaccharide substrates arabinogalactan (A), chondroitin sulfate (C), fucoidan (F), laminarin (L), pullulan (P), and xylan (Y) at each station (vertical panels) and each depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. Missing data at stn 15, mesopelagic depth for the xylan substrate indicated by asterisk. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. Multiple Regression Analysis of Environmental Parameters vs. Hydrolytic Activity Hydrolytic diversity also decreased with depth from shallow to deeper waters (Figure 3), and sites with higher overall rates of activity also had higher hydrolytic diversity (Supplementary Figure 6). Maximum hydrolytic diversity was typically measured at the surface or DCM, although station 2 exhibited highest hydrolytic diversity at mesopelagic depths, probably because the same assemblage of substrates was hydrolyzed at surface, DCM, and mesopelagic depths at station 2, but with diﬀerent degrees of evenness. FIGURE 5 \| Non-metric multidimensional scaling (NMDS) representation of Bray-Curtis dissimilarities of hydrolytic compositions among sampling sites in the upper 250 m of the water column. Sites grouped by (A) station show signiﬁcant hydrolytic compositional differences distinct from other stations (PERMANOVA P = 0.007), whereas the hydrolytic composition of sites grouped by (B) depth are not signiﬁcantly different (PERMANOVA P = 0.399). FIGURE 4 \| Percent of hydrolysis occurring above the pycnocline at each station is positively correlated with the maximum buoyancy frequency at that station. R2 = 0.66, P = 0.048. FIGURE 5 \| Non-metric multidimensional scaling (NMDS) repr Bray-Curtis dissimilarities of hydrolytic compositions among sa the upper 250 m of the water column. Sites grouped by (A) st signiﬁcant hydrolytic compositional differences distinct from ot (PERMANOVA P = 0.007), whereas the hydrolytic compositio grouped by (B) depth are not signiﬁcantly different (PERMANO FIGURE 4 \| Percent of hydrolysis occurring above the pycnocline at each station is positively correlated with the maximum buoyancy frequency at that station. R2 = 0.66, P = 0.048. FIGURE 5 \| Non-metric multidimensional scaling (NMDS) representation of Bray-Curtis dissimilarities of hydrolytic compositions among sampling sites in the upper 250 m of the water column. Sites grouped by (A) station show signiﬁcant hydrolytic compositional differences distinct from other stations (PERMANOVA P = 0.007), whereas the hydrolytic composition of sites grouped by (B) depth are not signiﬁcantly different (PERMANOVA P = 0.399). FIGURE 5 \| Non-metric multidimensional scaling (NMDS) representation of Bray-Curtis dissimilarities of hydrolytic compositions among sampling sites in the upper 250 m of the water column. Sites grouped by (A) station show signiﬁcant hydrolytic compositional differences distinct from other stations (PERMANOVA P = 0.007), whereas the hydrolytic composition of sites grouped by (B) depth are not signiﬁcantly different (PERMANOVA P = 0.399). FIGURE 4 \| Percent of hydrolysis occurring above the pycnocline at each station is positively correlated with the maximum buoyancy frequency at that station. Multiple Regression Analysis of Environmental Parameters vs. Hydrolytic Activity The transect covered a gradient in water column productivity (as represented by chlorophyll a ﬂuorescence) and in water column stratiﬁcation. At the more northerly stations where stratiﬁcation was stronger and chlorophyll a concentrations were higher, the highest hydrolytic diversity and rates of enzymatic activity were measured. Additionally, the depth at which the highest hydrolysis rate was observed at a particular station was at shallower depths at northerly, more stratiﬁed stations than at southerly, less stratiﬁed stations (Figure 4, Figure 2, Supplementary Figure 5). FIGURE 5 \| Non-metric multidimensional scaling (NMDS) representation of Bray-Curtis dissimilarities of hydrolytic compositions among sampling sites in the upper 250 m of the water column. Sites grouped by (A) station show signiﬁcant hydrolytic compositional differences distinct from other stations (PERMANOVA P = 0.007), whereas the hydrolytic composition of sites grouped by (B) depth are not signiﬁcantly different (PERMANOVA P = 0.399). column stratiﬁcation. At the more northerly stations where stratiﬁcation was stronger and chlorophyll a concentrations were higher, the highest hydrolytic diversity and rates of enzymatic activity were measured. Additionally, the depth at which the highest hydrolysis rate was observed at a particular station was at shallower depths at northerly, more stratiﬁed stations than at southerly, less stratiﬁed stations (Figure 4, Figure 2, Supplementary Figure 5). Hydrolytic diversity also decreased with depth from shallow to deeper waters (Figure 3), and sites with higher overall rates of activity also had higher hydrolytic diversity (Supplementary Figure 6). Maximum hydrolytic diversity was typically measured at the surface or DCM, although station 2 exhibited highest hydrolytic diversity at mesopelagic depths, probably because the same assemblage of substrates was hydrolyzed at surface, DCM, and mesopelagic depths at station 2, but with diﬀerent degrees of evenness. FIGURE 4 \| Percent of hydrolysis occurring above the pycnocline at each station is positively correlated with the maximum buoyancy frequency at that station. R2 = 0.66, P = 0.048. FIGURE 5 \| Non-metric multidimensional scaling (NMDS) representation of Bray-Curtis dissimilarities of hydrolytic compositions among sampling sites in the upper 250 m of the water column. Sites grouped by (A) station show signiﬁcant hydrolytic compositional differences distinct from other stations (PERMANOVA P = 0.007), whereas the hydrolytic composition of sites grouped by (B) depth are not signiﬁcantly different (PERMANOVA P = 0.399). Multiple Regression Analysis of Environmental Parameters vs. Hydrolytic Activity FIGURE 3 \| Shannon diversity, H, at each station (vertical panels) and each depth surface to bottom (y axis). A shannon diversiy of zero is indicated by a straight line, whereas asterisks indicate H was not calculated due to no detectable hydrolysis at that site. The maximum possible value of H, 1.79, is shown as a dashed line. FIGURE 3 \| Shannon diversity, H, at each station (vertical panels) and each depth surface to bottom (y axis). A shannon diversiy of zero is indicated by a straight line, whereas asterisks indicate H was not calculated due to no detectable hydrolysis at that site. The maximum possible value of H, 1.79, is shown as a dashed line. FIGURE 3 \| Shannon diversity, H, at each station (vertical panels) and each depth surface to bottom (y axis). A shannon diversiy of zero is indicated by a straight line, whereas asterisks indicate H was not calculated due to no detectable hydrolysis at that site. The maximum possible value of H, 1.79, is shown as a dashed line. June 2017 \| Volume 4 \| Article 200 Frontiers in Marine Science \| www.frontiersin.org Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti above the pycnocline, while xylan was also hydrolyzed in shallow waters but only at some stations (Figure 2). In the upper 250 m of the water column, the assemblage of polysaccharide substrates hydrolyzed at a given station followed distinct patterns. Comparing Bray-Curtis dissimilarities among surface, DCM, and mesopelagic depths for each station, hydrolytic assemblages clustered strongly when grouped by station (Figure 5A, PERMANOVA P = 0.007). This result contrasted with grouping by depth sampled, which did not produce any distinguishable eﬀect on Bray-Curtis distances between assemblages (Figure 5B, PERMANOVA P = 0.399). A similar analysis could not be done for the full depth range due to the lack of any detectable hydrolytic activity at many of the deeper depths. Hydrolysis rates (Figure 2) and summed hydrolysis rates (Supplementary Figure 5) decreased with depth. This decrease was more abrupt and occurred at shallower depths at more stratiﬁed stations, with most hydrolytic activity occurring in the surface and DCM depths. At less stratiﬁed stations (where maximum buoyancy frequency in the water column was lower), hydrolytic activities decreased more gradually with depth (Figure 2), and a greater proportion of summed activity occurred below the pycnocline (Figure 4, R2 = 0.66, P = 0.048). Multiple Regression Analysis of Environmental Parameters vs. Hydrolytic Activity R2 = 0.66, P = 0.048. June 2017 \| Volume 4 \| Article 200 Frontiers in Marine Science \| www.frontiersin.org 6 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti FIGURE 6 \| Maximum potential hydrolysis rate (nM h−1) of the monomeric substrates leucine (Le) and α-glucose (G) at each station (vertical panels) and depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. A particularly high hydrolysis rate of α-glucose at stn 7, mesopelagic depth indicated by the number in the panel. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. FIGURE 6 \| Maximum potential hydrolysis rate (nM h−1) of the monomeric substrates leucine (Le) and α-glucose (G) at each station (vertical panels) and depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. A particularly high hydrolysis rate of α-glucose at stn 7, mesopelagic depth indicated by the number in the panel. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. FIGURE 6 \| Maximum potential hydrolysis rate (nM h−1) of the monomeric substrates leucine (Le) and α-glucose (G) at each station (vertical panels) and depth (horizontal panels). Error bars indicate standard deviation of experimental triplicates. A particularly high hydrolysis rate of α-glucose at stn 7, mesopelagic depth indicated by the number in the panel. Low, non-zero hydrolysis rates in deep waters indicated by numbers in the ﬁgure panel. Relationship between Polysaccharide Hydrolysis and Environmental Parameters The strength of the relationship between polysaccharide hydrolysis rates and up to 10 environmental parameters was investigated by ﬁtting the multiple regression model that maximized R2-values (Supplementary Table 1). Overall, environmental parameters poorly explained the observed variation in hydrolysis rates (R2 = 0.22). Since many of these parameters co-correlate with each other, one or two of these parameters generally explained as much or nearly as much of the variation in hydrolysis rates as all ten environmental variables. Temperature and chlorophyll a accounted for most of the relationship in the overall model (R2 = 0.19), while Monomeric Substrate Hydrolysis Rates the inclusion of the additional eight environmental variables only slightly improved the model (R2 = 0.22). This result is mainly due to the diﬀerence in temperature and chlorophyll a in shallow vs. deep waters corresponding with higher rates of hydrolysis in shallower waters, since models using samples from just shallow or just deep water yielded very poor ﬁts. The high frequency of zero hydrolysis rates did not appear to bias the model, however, since models using only non-zero rates yielded similar ﬁts as the overall model (Supplementary Table 1). Hydrolysis rates of monomeric substrates also varied by station, with maximal activity at the surface or DCM, and decreasing activity with depth (Figure 6), with the exception of a single replicate for α-glucose at station 7 where high activity was observed at mesopelagic depths. Depth-related decreases in α- glucosidase and leucine aminopeptidase activities, unlike the polysaccharide hydrolase activities, did not correspond to the degree of water column stratiﬁcation. Below 250 m, α-glucosidase activity was undetectable at all sites even after 72 h of incubation, whereas leucine activities were very low in deep water, but nonzero. When broken down by individual substrate, models were generally better ﬁtted than the model of aggregated hydrolysis rates (Supplementary Table 1). However, the combination of environmental variables that best ﬁt the data diﬀered by substrate: for chondroitin, temperature only; for laminarin, temperature and chlorophyll a; for pullulan, temperature and buoyancy frequency; for xylan, chlorophyll a and salinity. Arabinogalactan and fucoidan were not modeled individually due to the lack of non-zero hydrolysis rates across all sites. DISCUSSION Instead, the patterns of enzymatic activities observed along this transect may be tied to the biogeography of the underlying microbial communities (Rusch et al., 2007; Fuhrman et al., 2008; Zinger et al., 2011; Sunagawa et al., 2015) and their functional capacities (DeLong et al., 2006; Shi et al., 2011). For example, the capacity to produce three extracellular enzymes (alkaline phosphatase, chitinase, and β-N-acetyl-glucosaminidase) commonly measured in ﬁeld studies varies on very ﬁne phylogenetic scales across all annotated prokaryotic genomes (Zimmerman et al., 2013). The heterogeneous distribution of heterotrophic genetic capacities among microbial phylogenies, and a varying distribution of these capabilities among surface water and subsurface environments (DeLong et al., 2006; Elifantz et al., 2008; Shi et al., 2011; Gomez-Pereira et al., 2012) results in functional stratiﬁcation and resource partitioning along depth- and horizontal gradients. Diﬀerences in community composition and function are driven by a complex combination of factors that may include organic carbon composition and concentration (McCarren et al., 2010), distribution limitation (Follows et al., 2007; Hellweger et al., 2014), environmental selection (Ladau et al., 2013), or a conﬂuence of interacting factors that defy simple categorization (Hanson et al., 2012). Patterns in hydrolytic assemblages among deeper water masses remain to be investigated, since activities were low or not measurable over the timescale of incubation at many of the deeper depths. A lack of measurable polysaccharide hydrolysis at deep sites may indicate that the heterotrophic community had no capacity to detect or to hydrolyze the substrates tested, or that the 21-day incubation timescale was insuﬃcient to measure hydrolysis. In particular, low hydrolytic activities, or activities that require the growth of potentially slow-growing and/or rare members of the microbial community might not be detectable over a 21-day time course (Arnosti, 2008), since a suﬃcient fraction of the total added polysaccharide pool must be hydrolyzed to detect activity. The observation that leucine aminopeptidase was hydrolyzed—albeit at low rates—in bottom waters at almost all stations, however, demonstrates that an active heterotrophic community was present at these depths. Irrespective of the underlying factors, the relationship between water column stratiﬁcation and the fraction of hydrolysis occurring in the shallow surface or DCM relative to deeper mesopelagic waters potentially has implications for the location of nutrient regeneration and for carbon export in the ocean. DISCUSSION Microbial communities rely on extracellular enzymes to hydrolyze high molecular weight organic matter prior to uptake. The structural speciﬁcities of the enzymes active at a given site and depth determine which substrates are available for June 2017 \| Volume 4 \| Article 200 Frontiers in Marine Science \| www.frontiersin.org 7 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti For both leucine aminopeptidase and α-glucosidase activities, the subtraction of killed control ﬂuorescence from live incubation ﬂuorescence may have contributed to the lower rates measured in deep water in our experiments. further metabolism, while relative rates of hydrolysis reﬂect the potential speed of substrate processing. Site- and depth-related diﬀerences in hydrolysis rates and capacities imply diﬀerential remineralization of organic matter across latitude and depth in the ocean. The overall patterns of enzyme activities observed along this transect—spatial diﬀerences in hydrolytic diversity in surface waters and a decrease in the spectrum of polysaccharides hydrolyzed with depth—are consistent with studies of surface waters from other parts of the world’s oceans (e.g., Arnosti et al., 2011), and add considerably to the very few other depth proﬁles of polysaccharide hydrolase activities in the ocean (Steen et al., 2012; D’Ambrosio et al., 2014). Multiple factors may contribute to the patterns of enzyme activities we measured. Environmental parameters alone are not likely to be the principal drivers for these patterns: the environmental variables measured at these stations poorly predicted observed rates, in univariate as well as multivariate models (Supplementary Table 1). While the speciﬁc environmental variable(s) that best ﬁt each model varied by individual substrate (Supplementary Table 1), a causal explanation for the correlation strengths between hydrolysis rates of individual substrates and speciﬁc environmental factors is not obvious in most cases. Distinct functional assemblages characterized individual stations along the latitudinal gradient, such that the diversity of substrates hydrolyzed was more similar from surface to mesopelagic depths at a single station than at similar depth levels across diﬀerent stations (Figure 5). These spatial and depth-related patterns in hydrolytic diversity, hydrolysis rate, and functional similarity together suggest that the vertical transfer of enzymatic capabilities through the upper depths of the water column—whether through cells, cellular material, or active enzymes—inﬂuences the hydrolytic signature of a station, but that this vertical transfer may be more limited at more stratiﬁed stations. Frontiers in Marine Science \| www.frontiersin.org DISCUSSION This point can be illustrated with a simple conceptual box model showing hypothetical DOC generation from particles sinking through the water column at the diﬀerent stations along the transect (Figure 7). While there are many interacting factors that aﬀect carbon export (De La Rocha and Passow, 2007), for the purposes of this discussion we consider only the degree of stratiﬁcation and the spatial patterns of hydrolysis measured in this study. Assuming that particles are being hydrolyzed at the summed hydrolysis rates measured at our sites, DOC will be generated from particles during their passage through the water column at a rate related to the depth-integrated hydrolytic capacities at that location and the sinking rate of particles. For the purpose of this conceptual calculation, we assume a constant particle sinking rate of 100 m day−1 and divide the upper water column into three boxes: DCM—centered at the DCM sampling depth for that station and arbitrarily set at Measurable hydrolysis of leucine-MCA and MUF-α-glucose in deep waters has also been reported at other sites in the South and Equatorial Atlantic Ocean (Baltar et al., 2009, 2010, 2013). The leucine-aminopeptidase activities of 1–4 nM h−1 reported by Baltar and colleagues in deep water are considerably higher than the 0–0.35 nM h−1 in the present study, although the range of leucine-aminopeptidase activities measured in surface waters are similar between this study and previous studies (Baltar et al., 2009, 2010, 2013). The range of α-glucosidase activities measured in the present study in surface water (0–20 nM h−1) are much greater than reported in previous studies (∼0–0.25 nM h−1), perhaps because of a growth response during the extended timecourse of our incubations (maximum of 72 h, vs. maximum of 48 h in Baltar et al., 2009, 2010, 2013). No α-glucosidase activity was detected at any depths below 250 m in this study, even after 72 h incubation, whereas previous investigations measured low but non-zero α-glucose hydrolysis rates in deep water (∼0–0.8 nM/h, Baltar et al., 2009, 2010, 2013). June 2017 \| Volume 4 \| Article 200 June 2017 \| Volume 4 \| Article 200 8 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti FIGURE 7 \| Conceptual model of DOC generation under varying hydrolytic capacities and rates observed in this study, at each station from southernmost to northernmost (vertical panels). DISCUSSION The amount of DOC generated, in µM, at the surface (top), DCM (middle), and mesopelagic (bottom) is shown at the top of each panel, and varies according to the relative hydrolytic capacities and rates, and the degree of stratiﬁcation at that site. The total amount of DOC generated throughout the upper water column is shown below each station, and varies by station. FIGURE 7 \| Conceptual model of DOC generation under varying hydrolytic capacities and rates observed in this study, at each station from southernmost to northernmost (vertical panels). The amount of DOC generated, in µM, at the surface (top), DCM (middle), and mesopelagic (bottom) is shown at the top of each panel, and varies according to the relative hydrolytic capacities and rates, and the degree of stratiﬁcation at that site. The total amount of DOC generated throughout the upper water column is shown below each station, and varies by station. a thickness of 50 m; surface–all depths above the DCM; and upper mesopelagic—from below the DCM to 300 m. To estimate carbon remineralization, carbohydrates hydrolyzed from the particulate to the dissolved phase are then converted to DOC generated, assuming 6 C per monosaccharide produced. In this scenario, the total quantity of DOC generated in the upper 300 m of the water column, as well as the depth at which this DOC would be generated, varies greatly along the transect (Figure 7). At the productive and more strongly stratiﬁed station 15 (2.7◦S), for example, most of the DOC would be generated in the surface and DCM, and labile DOC would likely quickly be respired to CO2 which would remain in the surface ocean. The highest overall quantity of DOC, however, would be produced at station 7 (22.5◦S), where more than half of the total generated DOC would be in the mesopelagic zone, and thus below the permanent thermocline. Labile DOC that is respired to CO2 would likely remain below the thermocline, and would not exchange with surface waters or with the atmosphere on short timescales (Kheshgi, 2004). stratiﬁed stations—such as stations 2, 4, and 7—where a larger proportion of the hydrolytic capacity occurs in the mesopelagic zone, below the permanent thermocline (Figure 7). Biogeographical patterns in carbon cycling activities, and their relationship to oceanographic features, are of crucial signiﬁcance to our ability to predict future conditions. DISCUSSION For example, if increasing global temperatures result in a more stratiﬁed ocean (Capotondi et al., 2012), the quantity of organic matter sequestered by the biological pump below the thermocline may decrease. Such a decrease in turn would place greater inﬂuence on the relative hydrolytic capacities of microbial communities in the surface ocean, rather than in deeper waters, in determining the overall eﬃciency of the biological pump. The eﬀects of an increase in stratiﬁcation on carbon export will also depend on its impact on the biogeography of microbial communities and hydrolytic activities themselves. Increased stratiﬁcation may in turn have complex downstream consequences for higher trophic levels that function in both shallow and deep waters, and depend on the availability of particulate organic carbon in both depth regions. Disentangling the roles of environmental characteristics, microbial community composition, functional capacities, and activities in regulating the marine carbon cycle is a prerequisite for a better understanding of the modern ocean, and of its sensitivity to perturbations in the future. The eﬃciency with which the biological pump removes surface-derived carbon from the upper ocean thus depends in part on the quantity of carbon remineralized from a sinking particle while it is still above the permanent thermocline (De La Rocha and Passow, 2007). This calculation in turn depends on the relative lability of the organic carbon in the sinking particles (Engel et al., 2009), the hydrolytic capacities of the microbial communities acting on them as they sink, the extent of water column stratiﬁcation, and the residence time of the particle at diﬀerent depths (Prairie et al., 2015). Holding all other factors constant, the biological pump would be more eﬃcient at less Frontiers in Marine Science \| www.frontiersin.org REFERENCES D., Ziervogel, K., Ghobrial, S., and Jeﬀrey, W. H. (2011). Latitudinal gradients in degradation of marine dissolved organic carbon. PLoS ONE 6:e28900. doi: 10.1371/journal.pone.0028900 Gomez-Pereira, P. R., Schuler, B. M., Fuchs, C. 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AH conducted ﬁeld experiments, collected and processed samples, and June 2017 \| Volume 4 \| Article 200 9 Enzymatic Activity Patterns in the Ocean Hoarfrost and Arnosti work was provided by the National Science Foundation (OCE-1332881 to CA). work was provided by the National Science Foundation (OCE-1332881 to CA). processed data. AH and CA analyzed results and wrote the manuscript. FUNDING The Supplementary Material for this article can be found online at: http://journal.frontiersin.org/article/10.3389/fmars. 2017.00200/full#supplementary-material The authors would like to thank Liz Kujawinski for the invitation to join the DeepDOM cruise, and Krista Longnecker for providing nutrient measurements. Funding for this REFERENCES Prokaryotic extracellular enzymatic activity in relation to biomass production and respiration in the meso- and bathypelagic waters of the (sub)tropical Atlantic. Environ. Microbiol. 11, 1998–2014. doi: 10.1111/j.1462-2920.2009.01922.x Jiao, N., Herndl, G. J. 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https://openalex.org/W2940892137	https://link.springer.com/content/pdf/10.1007/s13659-019-0205-2.pdf	English	null	Two New C21 Steroidal Glycosides from the Roots of Cynanchum paniculatum	Natural products and bioprospecting	2,019	cc-by	3,137	Graphical Abstract Keywords Cynanchum paniculatum · Steroidal glycosides · Bioactivities · NMR data Keywords Cynanchum paniculatum · Steroidal glycosides · Bioactivities · NMR data Two New C21 Steroidal Glycosides from the Roots of Cynanchum paniculatum Received: 27 February 2019 / Accepted: 9 April 2019 / Published online: 26 April 2019 © The Author(s) 2019 Abstract Two new C21 steroidal glycosides, paniculatumosides H and I, together with four known ones were isolated from the roots of Cynanchum paniculatum (Bge.) Kitag. Their structures were identified by spectroscopic methods including extensive 1D and 2D NMR techniques. All compounds were subjected to detect the anti-tobacco mosaic virus (TMV) activities and their cytotoxities against three human tumor cell lines (SMMC-7721, MDA-MB-231 and A549). The results showed that compounds 1 and 5 exhibited potent protective activities against TMV, while 2, 4 and 6 had moderate effects on the SMMC- 7721 cancer cells viability. Natural Products and Bioprospecting (2019) 9:209–214 https://doi.org/10.1007/s13659-019-0205-2 ORIGINAL ARTICLE ORIGINAL ARTICLE 1 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China * Xiao Ding dingxiao@mail.kib.ac.cn * Shun‑Lin Li lisl@mail.kib.ac.cn 2 University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China 1 Introduction treatment of rheumatic arthralgia, epigastric pain, tooth- ache, lumbago, traumatic injuries, and eczema [1–3]. In the previous work, it has been confirmed that C. paniculatum contains C21 steroidal glycosides [4, 5]. Pregnanes and their glycosides have shown many aspect activities, such as antitu- mor, antifungal, antiviral and cytotoxic activities [6, 7]. Our previous works found that pregnanes and their glycosides could inhibit tobacco mosaic virus through suppressing the expression of viral subgenomic RNA but without affect- ing the accumulation of viral genomic RNA [6]. Therefore, in order to find the structurally unique natural products in C. paniculatum and explore their biological activities, we investigated the dichloromethane extract of C. paniculatum, and two new steroidal glycosides paniculatumosides H (1) and I (2), together with four known ones glaucogenin C (3) [8], cynatratoside A (4) [8], cynapanoside A (5) [9] and Cynanchum paniculatum (Bunge) Kitag is a vivacious herb broadly distributed in China, Japan and Korea, whose dried roots have been used as a Chinese herbal medicine for the 1 State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, People’s Republic of China (0121 3456789) 3 210 H.-L. Yu et al. The sugar carbon signals (δC 95.2, 78.2, 76.8, 67.5, 56.5, 34.5, 17.8) illustrated that the sugar belonged to 2,6-deoxy sugar because it contained four methines, one methylene, a terminal methyl group and a methoxyl group. In the sugar spin systems, the large coupling constants JH-3′/H-4′ (9.1 Hz) and JH-4′/H-5′ (9.3 Hz) disclosed their trans-diaxial relation- ship, which suggested that the sugar was oleandropyranose [8]. Moreover, the anomeric proton of d-oleandrose was α-orientation based on the small coupling constants of H-1′ (3JH-1,H-2 = 3.5 Hz) [11]. Furthermore, the sugar carbon and proton signals were identical with α-d-oleandropyranosyl unit by comparison with reported literature [12], and the ROESY correlations of H-2′b/H-4′ supported this configura- tion (Fig. 2). Thus, compound 1 could be glycosylated at the C-3 with α-linkage by d-oleandropyranosyl unit, which was also concluded from the HMBC correlation of δH 5.04 (d, J = 3.5 Hz, H-1′) to δC 75.8 (C-3) (Fig. 3). The structure of 1 was finally established as glaucogenin C 3-O-α-d-oleand ropyranoside. neocynapanogenin F 3-O-β-d-oleandropyranoside (6) [10] were isolated. Furthermore, we tested the anti-TMV activi- ties and cytotoxicity of these compounds (Fig. 1). 2.1 Structure Elucidation Paniculatumoside H (1) was obtained as a yellow powder. Its molecular formula, C28H40O8, was determined by analysis the peak at m/z 527.2629 [M + Na]+ in the positive HRESI- MS (calcd for C28H40O8Na 527.2615). The IR spectrum dis- played absorption bands for hydroxyl (3443 cm−1), carbonyl (1736 cm−1), olefinic (1635 cm−1) and carbon–oxygen bond (1081 cm−1). The 1H NMR spectrum of compound 1 showed the presence of two tertiary methyl groups at δH 0.92 (3H, s, H-19) and δH 1.53 (3H, s, H-21), one olefinic proton at δH 5.38 (d, J = 5.3 Hz, H-6), one olefinic deshielded proton at δH 6.24 (s, H-18), two oxygen-substituted methine protons at δH 3.44 (m, H-3) and δH 5.29 (td, 9.5, 7.1, H-16) and two methylene protons at δH 3.84 (t, J = 8.6 Hz, H-15a) and δH 4.15 (td, J = 8.6, 1.4 Hz, H-15b). By comparison of its 1H and 13C NMR spectra (Table 1) to those of glaucogenin C (3) indicated that the aglycone of 1 is glaucogenin C, which also confirmed by correlations of H-19/H-2b, H-19/H-8, H-1b/H-3, H-21/H-16 and H-16/H-17 in the ROESY experi- ment (Fig. 2). The main difference was an extra sugar unit present at 1, which could be further verified by the glycosi- dation shifts at C-2 (− 3.8), C-3 (+ 4.2) and C-4 (− 2.2). Paniculatumoside I (2) was obtained as a colourless powder. The molecular formula of 2, C34H50O11, was deduced by HRESI-MS (m/z 657.3258 [M + Na]+, calcd for C34H50O11Na 657.3245). The IR spectrum also sug- gested the presence of hydroxyl (3433 cm−1), carbonyl (1736 cm−1), olefinic (1632 cm−1) and carbon–oxygen bond (1070 cm−1). Compounds 2 and 1 possess the same aglycone by analyzing their 1H and 13C NMR spectra (Table 1), which was confirmed to be glaucogenin C by detailed analysis of their 2D NMR spectra. 2.1 Structure Elucidation Two anomeric proton signals δH 4.53 (d, J = 9.6 Hz, H-1′) and δH 5.02 O O O O R1O R3 H H R2 = Ra O HO MeO H H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 compound R1 R2 1 2 3 4 5 6 R3 Ra Rb H Rc Rc Rc H H H H OH H H H H H H OH O HO MeO = = Rc Rb O O MeO O HO OH Fig 1 Ch i l t t f d 1 6 Fig. 1 Chemical structures of compounds 1–6 3 Two New C21 Steroidal Glycosides from the Roots 211 correlated to the correspond- s at δC 97.8 (C-1′) and 98.5 um, respectively. It showed as made up of two monosac- and 1H, 1H-COSY experiments, the spin system of each monosaccharide could be established. Two methylenes (δC 36.5 and 38.2) and methyl groups (δH 1.30 and 1.31) suggested both monosaccharides of 2 were deoxysugars. No. 2.1 Structure Elucidation 1 2 3 δC δH δC δH δC δH 1a 1b 36.2 1.96 (m) 1.02 (td, 14.4, 3.8) 36.4 1.96 (m) 1.05 (td, 14.6, 3.9) 36.4 1.96 (m) 1.07 (td, 13.7, 3.3) 2a 2b 27.5 1.91 (m) 1.45 (m) 29.4 1.97 (m) 1.60 (m) 31.3 1.86 (m) 1.51 (m) 3 75.8 3.44 (m) 77.9 3.53 (m) 71.6 3.53 (m) 4a 4b 39.7 2.30 (m) 2.24 (m) 38.6 2.33 (m) 2.16 (m) 41.9 2.32 (dd, 13.0, 2.5), 2.17 (dd, 13.0, 11.9) 5 140.3 – 140.2 – 140.2 – 6 120.2 5.38 (d, 5.3) 120.3 5.39 (d, 5.2) 120.2 5.39 (d, 5.3) 7a 7b 28.0 2.43 (m) 2.05 (m) 28.0 2.42 (m) 2.04 (m) 27.9 2.43 (m) 2.02 (m) 8 40.4 2.42 (m) 40.4 2.42 (m) 40.4 2.43 (m) 9 52.8 1.19 (overlapped) 52.9 1.19 (overlapped) 52.8 1.20 (t, 9.7) 10 38.5 – 38.6 – 38.3 – 11a 11b 23.6 2.56 (m) 1.29 (m) 23.6 2.56 (m) 1.28 (m) 23.6 2.57 (m) 1.27 (m) 12a 12b 29.6 2.06 (m) 1.33 (m) 29.6 2.07 (m) 1.33 (m) 29.6 2.07 (m) 1.33 (m) 13 118.1 – 118.1 – 118.1 – 14 175.6 – 175.6 – 175.6 – 15a 15b 67.6 4.15 (td, 8.6, 1.4) 3.84 (t, 8.6) 67.6 4.15 (td, 7.8, 1.6), 3.84 (t, 7.8) 67.5 4.16 (td, 8.1, 1.6), 3.85 (t, 8.1) 16 75.1 5.29 (td, 9.5,7.1) 75.1 5.29 (td, 9.7, 7.3) 75.0 5.29 (td, 9.7, 7.8) 17 55.7 3.43 (overlapped) 55.7 3.43 (overlapped) 55.7 3.43 (d, 7.8) 18 143.3 6.24 (s) 143.3 6.25 (s) 143.3 6.25 (s) 19 18.0 0.92 (s) 18.0 0.91 (s) 18.0 0.91 (s) 20 114.0 – 113.9 – 113.9 – 21 24.6 1.53 (s) 24.6 1.53 (s) 24.5 1.53 (s) Sugar 1′ 95.2 5.04 (d, 3.5) 97.8 4.53 (d, 9.6) 2′a 2′b 34.5 2.22 (m) 1.50 (m) 36.5 2.26 (m) 1.53 (m) 3′a 78.2 3.52 (ddd, 9.1, 8.8, 4.9) 79.1 3.38 (m) 4′ 76.8 3.15 (dd, 9.3, 9.1) 82.5 3.19 (dd, 9.0, 8.8) 5′ 67.5 3.73 (dq, 9.3, 6.3) 71.1 3.29 (m) 6′ 17.8 1.28 (d, 6.3) 18.2 1.30 (d, 6.3) 3′-OCH3 56.5 3.39 (s) 56.4 3.40 (s) 1′′ 98.5 5.02 (d, 9.7) 2′′ 38.2 2.12 (m) 1.72 (m) 3′′ 68.3 4.11 (m) 4′′ 72.9 3.31 (m) 5′′ 69.6 3.74 (dq, 9.7, 6.3) 6′′ 18.4 1.31 (d, 6.3) (d, J = 9.7 Hz, H-1′′), which correlated to the correspond- ing anomeric carbon signals at δC 97.8 (C-1′) and 98.5 (C-1′′) in the HSQC spectrum, respectively. 2.1 Structure Elucidation It showed that the sugar moiety of 2 was made up of two monosac- charides. Meanwhile, from the HSQC, HSQC-TOCSY, 1 3 212 H.-L. Yu et al. Fig. 5 The selected HMBC and COSY correlations of 2 H O O O O H O H H CH3 H CH3 O HO MeO H H H H H H H H 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Fig. 2 The key ROESY correlations of 1 Fig. 5 The selected HMBC and COSY correlations of 2 Fig. 2 The key ROESY correlations of 1 Fig. 5 The selected HMBC and COSY correlations of 2 O O O O O O HO MeO HMBC COSY Fig. 3 The selected HMBC and COSY correlations of 1 2.2.2 Cytotoxic Activities Fig. 4 The key ROESY correlations of 2 In order to evaluate whether these compounds have any biological functions on cancer cells, we tested them on SMMC-7721, MDA-MB-231 and A549 human cancer cell lines for their impact on tumor cell growth by MTT method. All the compounds have no inhibitory effect on the MDA- MB-231 and A549 cell lines. But compounds 2, 4 and 6 have moderate effects on the SMMC-7721 cancer cells viability with the IC50 values of 27.4 ± 0.24 μM, 22.2 ± 0.11 μM, 27.2 ± 0.13 μM, respectively. Cisplatin was used as a posi- tive control and its IC50 for the SMMC-7721 cancer cells was 13.07 ± 0.27 μM. Fig. 4 The key ROESY correlations of 2 (3JH-1,H-2 = 9.6 Hz) and H-1″ (3JH-1,H-2 = 9.7 Hz) illus- trated that the anomeric protons of the two sugar were β-oriented, which was also proved by the ROESY colla- tions between δH 4.53 (d, J = 9.6 Hz, H-1′) and δH 3.29 (m, H-5′), δH 5.02 (d, J = 9.7 Hz, H-1′′) and δH 3.74 (dq, H-5′′) (Fig. 4). The oligosaccharide moiety was identified as β-d-oleandropyranoside and β-l-digitoxopytanoside by analyzing from comparing the 1H and 13C signals of 2 with those in the literatures [6, 13]. Compound 2 was glycosylated at the C-3, which was concluded from the HMBC correlation of δH 4.53 (H-1′) to δC 77.9 (C-3), and the HMBC correlation of δH 5.02 (H-1′′) of δC 82.5 (C-4′) suggested digitoxopytanose connected to C-4′ of oleandropyranose (Fig. 5). Therefore, com- pound 2 was established to be glaucogenin C 3-O-β-l- digitoxopytanosyl-(1 → 4)-β-d-oleandropyranoside. 2.2.1 Anti‑TMV Activities All compounds were tested for anti-TMV activity using the half-leaf method. The results revealed that compounds 1 and 5 exhibited protective activities at concentration of 200 μM, with the antiviral inhibition rates of 62.7% and 59.5%, respectively. Ningnanmycin, which was used as a positive control, showed inhibition rates of 57.3%. Fig. 3 The selected HMBC and COSY correlations of 1 Fig. 3 The selected HMBC and COSY correlations of 1 3.3.1 Paniculatumoside H (1) White amorphous powder; [α]D 20−21.5 (c 0.11, MeOH); IR (KBr) νmax 3443 (OH), 2919, 1736, 1635, 1384, 1081 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 527 [M + Na]+; HRESIMS m/z 527.2629 [M + Na]+ (calcd for C28H40O8Na, 527.2615). White amorphous powder; [α]D 20−21.5 (c 0.11, MeOH); IR (KBr) νmax 3443 (OH), 2919, 1736, 1635, 1384, 1081 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 527 [M + Na]+; HRESIMS m/z 527.2629 [M + Na]+ (calcd for C28H40O8Na, 527.2615). 3.4.2 Cytotoxicity Assay The cytotoxicity of each compound on three cultured human cancer cell lines was tested by MTT assay. The cell lines used were SMMC-7721 (human hepatoma cells), MDA-MB-231 (triple-negative breast cancer cells), A549 (human lung cancer cell). Cell growth inhibition assay was performed as reported literatures [15]. Cisplatin was used as a positive control. Acknowledgements This project was supported financially by grants from the National Natural Science Foundation of China (Nos. 31770389, 81703393). We thank Professor Hua Peng, Kunming Insti- tute of Botany (KIB), CAS, for identifying the plant material; Analysis and Test Center, KIB, CAS, for the technical support. 3.2 Plant Material The roots of C. paniculatum were purchased from a medici- nal market (Kunming luosiwan Chinese herbal medicine market) in August 2017 and identified by prof. Hua Peng of Kumming Institute of Botany, Chinese Acdemy of Sci- ences (CAS). 3.3.2 Paniculatumoside I (2) White amorphous powder; [α]D 20−52.0 (c 0.09, MeOH); IR (KBr) νmax 3433 (OH), 2922, 1736, 1632, 1383, 1070 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 657 [M + Na]+; HRESIMS m/z 657.3258 [M + Na]+ (calcd for C34H50O11Na, 657.3245). White amorphous powder; [α]D 20−52.0 (c 0.09, MeOH); IR (KBr) νmax 3433 (OH), 2922, 1736, 1632, 1383, 1070 cm−1; 1H and 13C NMR data, see Table 1; ESIMS m/z 657 [M + Na]+; HRESIMS m/z 657.3258 [M + Na]+ (calcd for C34H50O11Na, 657.3245). 3.3 Extraction and Isolation The half-leaf method was used to evaluate the anti-TMV activities as literatures reported [14]. Ningnanmycin, a Chinese commercial product for plant disease, obtained from Heilongjiang Qiang’er Biochemical Technology Development Company, was administered as a positive control. The roots of C. paniculatum (100.0 kg) were powdered and extracted three times with MeOH at room temperature to afford 7.2 kg of crude extract. The extract was partitioned between CH2Cl2 and aqueous solution portions which yielded 4.4 kg crude CH2Cl2 extract. This extract was sub- jected to normal-phase silica gel column chromatography eluted with a gradient of petroleum ether–acetone (from 1:0 to 1:2) and CH2Cl2–MeOH (10:1–0:1) to obtain eight major fractions (Fr.1-8). Fr.6 (182.3 g) was separated by reversed-phase separation (CH3OH–H2O, 4:6–9:1) to get twelve subfractions (Fr.6a-6l). Fr.6g (2.1 g) was purified by Sephadex LH-20 eluting with MeOH to yield three fractions. Fr.6g-2 (161.4 mg) was applied to a silica gel column using CH2Cl2–MeOH (200:1–0:1) to obtain 3 (7.1 mg). Fr.6g-3 (92.5 mg) was subject to a normal-phase column chroma- tography and further purified by semiprepative HPLC (68% CH3CN in water) to yield 6 (5.0 mg, tR = 15.0 min). Fr6I (4.3 g) was separated into four subfractions by Sephadex LH-20 eluting with CH2Cl2–MeOH (1:1). Fr.6I-2 (1.9 g) was chromatographed on a silica gel column eluting with CH2Cl2–MeOH (200:1–0:1) to get Fr.6I-2c (109.2 mg) and Fr.6I-2e (79.3 mg). Fr6I-2c was purified by semi- prepative HPLC (50% CH3CN in water) to obtain 1 (5 mg, tR = 27.5 min) and 4 (92.0 mg, tR = 29.0 min). Fr.6I-2d also used semiprepative HPLC (46% CH3CN in water) to yield 5 (10.2 mg, tR = 35.0 min). Fr.6 k (3.6 g) was fractioned by a silica gel column eluting with petroleum ether-acetone (80:1–0:1) to afford the Fr6 k-3 (39.3 mg), which was puri- fied by Sephadex LH-20 (MeOH) and semiprepative HPLC (49% CH3CN in water) in sequence to yield 2 (8.2 mg, tR = 31.5 min). 3.1 General Experimental Procedures UV spectra were measured with a Shimadzu UV-2401A spectrophotometer. Optical rotations were determined on a Jasco P-1020 polarimeter. Infrared spectroscopy (IR) spectra were measured on a Bio-Rad FTS-135 spectrom- eter with KBr pellets. HRESIMS data were collected on a triple quadrupole mass spectrometer. 1D and 2D NMR spectra were recorded on a Bruker spectrometer with tetra- methylsilane as the internal standard. Preparative HPLC separations were carried out using an Agilent 1200 liq- uid chromatograph with a Waters X-select CSH Prep RP 3 213 Two New C21 Steroidal Glycosides from the Roots 3.3.1 Paniculatumoside H (1) C18 (19 × 150 mm) column and the flowing rate is 8 mL/ min. Semipreparation HPLC separations were performed on an Agilent 1100 liquid chromatograph using a YMC- Pack CDS-A (10 × 250 mm) column with flowing rate of 3 mL/min. Sephadex LH-20 (40–70 mm, Amersham Pharmacia Biotech AB, Uppsala, Sweden) and Silica gel (100–200 mesh and 300–400 mesh, Qingdao Marine Chemical, Inc., Qingdao, P. R. China) were used for col- umn chromatography. Compliance with Ethical Standards Conflicts of interest The authors declare that they have no conflict of interest. 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https://openalex.org/W4377939901	https://research.tilburguniversity.edu/files/76828080/MCP_vd_Graaf_experiences_of_cancer_survivors_with_chemotherapy_induced_peripheral_neuropathy_JoCS_2023.pdf	English	null	Experiences of cancer survivors with chemotherapy-induced peripheral neuropathy in the Netherlands: symptoms, daily limitations, involvement of healthcare professionals, and social support	Journal of cancer survivorship	2,023	cc-by	7,473	Experiences of cancer survivors with chemotherapy induced peripheral neuropathy in the Netherlands van de Graaf, D.L.; Engelen, V.; de Boer, Aize ; Vreugdenhil, G.; Smeets, T.; van der Lee, M.L.; Trompetter, H.R.; Mols, F. Published in: Journal of Cancer Survivorship DOI: 10.1007/s11764-023-01402-4 Publication date: 2023 Document Version Publisher's PDF, also known as Version of record Link to publication in Tilburg University Research Portal Citation for published version (APA): van de Graaf, D. L., Engelen, V., de Boer, A., Vreugdenhil, G., Smeets, T., van der Lee, M. L., Trompetter, H. R., & Mols, F. (2023). Experiences of cancer survivors with chemotherapy induced peripheral neuropathy in the Netherlands: Symptoms, daily limitations, involvement of healthcare professionals, and social support. Journal of Cancer Survivorship. Advance online publication. https://doi.org/10.1007/s11764-023-01402-4 Experiences of cancer survivors with chemotherapy induced peripheral neuropathy in the Netherlands van de Graaf, D.L.; Engelen, V.; de Boer, Aize ; Vreugdenhil, G.; Smeets, T.; van der Lee, M.L.; Trompetter, H.R.; Mols, F. Published in: Journal of Cancer Survivorship Document Version Publisher's PDF, also known as Version of record Citation for published version (APA): van de Graaf, D. L., Engelen, V., de Boer, A., Vreugdenhil, G., Smeets, T., van der Lee, M. L., Trompetter, H. R., & Mols, F. (2023). Experiences of cancer survivors with chemotherapy induced peripheral neuropathy in the Netherlands: Symptoms, daily limitations, involvement of healthcare professionals, and social support. Journal of Cancer Survivorship. Advance online publication. https://doi.org/10.1007/s11764-023-01402-4 General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. Citation for published version (APA): van de Graaf, D. L., Engelen, V., de Boer, A., Vreugdenhil, G., Smeets, T., van der Lee, M. L., Trompetter, H. R., & Mols, F. (2023). Experiences of cancer survivors with chemotherapy induced peripheral neuropathy in the Netherlands: Symptoms, daily limitations, involvement of healthcare professionals, and social support. Journal of Cancer Survivorship. 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Oct. 2024 Journal of Cancer Survivorship https://doi.org/10.1007/s11764-023-01402-4 Abstract Purpose A significant proportion of cancer patients suffer from chemotherapy-induced peripheral neuropathy (CIPN). This descriptive study aimed to examine patients’ experience of CIPN symptoms, daily limitations, involvement of healthcare professionals, and social support. p pp Methods Cross-sectional data have been collected in the Netherlands via a national online questionnaire comprising closed items only (February 2021). Results Out of 3752 respondents, 1975 received chemotherapy only (i.e., without targeted therapy) and were therefore included. The majority (71.2%) reported symptoms in both hands and feet (e.g., tingling and loss of sensation or diminished sensation). Participants reported most limitations in household chores, social activities, hobbies, sports, walking, and sleeping and least in family/(taking care of) children, cycling, driving, self-care, eating and drinking, and sexuality and intimacy. Many patients indicated that their healthcare professionals informed them about the possibility of CIPN development before treat- ment (58.4%), and they paid attention to CIPN during and after treatment (53.1%). However, many patients (43%) reported a lack of information on what to do when CIPN develops. Few participants (22%) visited their general practitioner (GP) for CIPN. In general, patients’ social environments sometimes to always showed empathy to patients. Conclusions Symptoms of CIPN are frequently reported and can result in various daily limitations. Support from profession- als and peers is crucial in managing CIPN, which is sometimes lacking. Appropriate guidance and support should be provided to patients to decrease the impact of CIPN on daily life. Future research should investigate differences in chemotherapeutic agents and the resulting symptoms and consequences. Keywords Chemotherapy-induced peripheral neuropathy · Symptoms · Daily limitations · Healthcare professionals · Social support · Self-reported Daniëlle L. van de Graaf1,2 · Vivian Engelen3 · Aize de Boer3 · Gerard Vreugdenhil4 · Tom Smeets1 · Marije L. van der Lee1,5 · Hester R. Trompetter1 · Floortje Mols1,2 Daniëlle L. van de Graaf1,2 · Vivian Engelen3 · Aize de Boer3 · Gerard Vreugdenhil4 · Tom Smeets1 · Marije L. van der Lee1,5 · Hester R. Trompetter1 · Floortje Mols1,2 Received: 24 March 2023 / Accepted: 6 May 2023 © The Author(s) 2023 Extended author information available on the last page of the article Experiences of cancer survivors with chemotherapy‑induced peripheral neuropathy in the Netherlands: symptoms, daily limitations, involvement of healthcare professionals, and social support Daniëlle L. van de Graaf1,2 · Vivian Engelen3 · Aize de Boer3 · Gerard Vreugdenhil4 · Tom Smeets1 · Marije L. van der Lee1,5 · Hester R. Trompetter1 · Floortje Mols1,2 Introduction or disappear over time, in some patients, the symptoms remain, leaving them with chronic symptoms [7]. In the Netherlands, 123,672 new patients were diagnosed with cancer in 2021 [1]. The 5-year survival rate currently is 66% and increases by about 1% each year due to improved diagnostics and treatment [2]. The long-term consequences of cancer and its treatment become more prevalent [3], which means that after completion of treatment, patients often face several physical and psychosocial limitations in their daily lives [4–6]. Although symptoms usually improve A common long-term consequence of cancer treatment is chemotherapy-induced peripheral neuropathy (CIPN), which is caused by chemotherapeutic agents like taxanes, platinum compounds, and vinca alkaloids [8, 9]. Peripheral neuropathy is defined by the National Cancer Institute as: “a nerve problem that causes pain, numbness, tingling, swell- ing, or muscle weakness in different parts of the body”[10], which can be experienced as both painful and nonpainful [11]. The sensations patients experience can vary greatly depending on patient characteristics and perceptions, as (0121 3456789) 3 Journal of Cancer Survivorship of symptoms. It is important to examine the social support experienced by cancer survivors. In this study, social support is referred to as the degree of empathy shown by the social environment. well as chemotherapy type and cumulative dose [11]. One month after completion of chemotherapy, almost 80% of cancer survivors experience CIPN, which decreases to 30% after 6 months or longer [3, 8, 12–15], showing that it is still present in a significant group of patients until late after chemotherapy.fi This study aimed to report the experiences of cancer sur- vivors who suffer or had suffered from self-reported CIPN in the Netherlands regarding (1) CIPN symptoms, (2) daily limitations, (3) involvement of healthcare professionals, and (4) social support. CIPN symptoms can be difficult to deal with and bring significant limitations to patients’ daily lives [16]. Even though patients are often cured of cancer, they might still feel ill due to CIPN as it continuously reminds them of being treated for a life-threatening disease [17]. Earlier research has shown that CIPN strongly interferes with daily life activities, such as walking, hobbies, and relationships [11]. Patients’ identities can change due to CIPN, as they have to make changes in their roles because certain activities can no longer be performed (e.g., performing a job or playing an instrument) [17]. Study design A cross-sectional exploratory national online questionnaire study was performed among adult cancer survivors with CIPN in the Netherlands. The questionnaire was initiated and developed by a patient advocate and a researcher from the Dutch Federation of Cancer Patients Organizations (NFK), which is the Dutch umbrella organization that represents 19 cancer patient organizations. A researcher of the PROFILES Registry with scientific expertise in CIPN was also involved in the development of the questionnaire [30]. Furthermore, four patient advocates of two patient organizations (the Dutch breast cancer patient organization (Borstkankervereniging Nederland) and the Dutch gynaecologic cancer patient sup- port group (Stichting Olijf) were involved, three of whom experienced CIPN themselves. These people participated in a workgroup. The workgroup met three times to discuss the content of the questionnaire. In between, the workgroup pro- vided (digital) feedback on draft versions of the questionnaire. It is crucial to be aware of the symptoms and daily con- sequences these patients experience as well as the attempts they make to control or reduce these symptoms since this knowledge enables healthcare professionals to adequately support patients in their needs [11]. Healthcare professionals should assess and address the symptoms of CIPN, weigh the impact on the daily lives of their patients and, subsequently, provide appropriate support to try to preserve their quality of life (QoL) [11]. A Dutch study examined reasons of colorec- tal cancer patients to visit their GP during the first 5 years of follow-up and concluded that chemotherapy-related symp- toms, among which was CIPN, was one of the most fre- quent reasons [20]. However, to the best of our knowledge, no study has assessed how patients perceive support from healthcare professionals regarding CIPN. Introduction Furthermore, several studies showed that CIPN symptoms can negatively affect physical, social, and emotional areas of life [11, 16, 18, 19]. This shows that CIPN can be a highly relevant limiting side effect of chemo- therapy with major consequences on daily life that affects more than just physical functioning. However, to our knowl- edge, no earlier study examined the specific daily limitations of a broad patient group (e.g., multiple tumor types) with a large sample size. Data collection The questionnaire was distributed between February 1 and 15, 2021 via www.doneerjeervaring.nl, social media chan- nels and the Doneer Je Ervaring (Donate Your Experience) panel. Additionally, patient organizations have spread invita- tions for the questionnaire among their members and spon- sors via email. Finally, partner organizations like the Dutch Cancer Society and Kanker.nl (Dutch web platform with tai- lored medical information and peer-support targeted at can- cer survivors and relatives [31]) have spread the invitation. In addition to support from healthcare professionals, support from friends, family, and significant others (i.e., social support) is important [21]. Social support can allevi- ate several disease aspects, such as coping with cancer and stress [22–27], anxiety, depression, and QoL [28]. A recent study among breast cancer patients examined the effect of perceived social support on chemotherapy-related symp- toms, including CIPN symptoms [29]. Results showed that these symptoms were identified as less severe when patients reported medium to high perceived social support, compared to those with low perceived social support. However, the rel- evant CIPN symptoms examined in the study were pain and numbness, whereas CIPN involves a much broader spectrum Results (55.1%) (Table 2). In 71.2% of the participants, CIPN was present in both foot/feet and hand(s). The most prevalent symptoms in hands were tingling (59.6%) and loss of sen- sation or diminished sensation (47.7%). Most participants attempted to reduce or control these symptoms by applying self-management strategies (69.9%). In total, 3752 participants filled in the questionnaire. Par- ticipants were included if they had received “chemotherapy only” as treatment. Excluded were participants that received “targeted therapy,” “targeted therapy combined with chemo- therapy,” or neither of those. In total, 1975 participants who suffered from of had suffered from CIPN remained and were included in this study. Sociodemographic characteristics of participants are shown in Table 1. Participants had a mean age of 58.8 (SD = 11.3), and 76.1% were female. The most prevalent tumor types were breast (42.8%), blood/lymph (24.2%), colorectal (8.9%), and gynecological cancer (5.8%). In 41% of patients, cancer had been diagnosed more than 5 years ago, followed by 2–5 years ago (34.7%) and less than 2 years ago (23.6%). Participants Patients could participate if they currently suffered from CIPN or had suffered from CIPN in the past. Participants were informed about privacy regulations of the NFK, in accordance with the General Data Protection Regulation (EU). The Medical Research Involving Human Subjects Act (WMO) did not apply since the study did not include 1 3 3 Journal of Cancer Survivorship Table 1 Sociodemographic characteristics of participants Characteristics n % M SD Gender Male 469 23.7 Female 1506 76.1 Other 5 .3 Age 58.8 11.3 Cancer type Breast 848 42.8 Blood/lymph 480 24.2 Colorectal 177 8.9 Gynecological 115 5.8 Other 90 4.5 Lung 56 2.8 Prostate 44 2.2 Bladder/kidney 37 1.9 Pancreas 35 1.8 Testicle 35 1.8 Stomach/esophageal 31 1.6 Head/neck 10 .5 Sarcoma 9 .5 Brain 9 .5 Melanoma 4 .2 Time since diagnosis < 2 years 468 23.6 2–5 years 687 34.7 > 5 years 825 41.7 an intervention wherefore ethical approval by the Medical Ethical Review Board was not needed. By completing the questionnaire, patients gave implied consent. Participation was completely online and anonymous. Statistical analyses No minimum sample size was calculated prior to the study since this study was explorative in nature. Descriptive statis- tics were reported. Absolute numbers and percentages were provided for nominal variables. Means and standard devia- tions were provided for continuous variables. For both items related to satisfaction with the supervision of healthcare pro- fessionals, the numerical scores 1 to 10 were recoded to the categorical scores insufficient (1–5), satisfactory to good (6–8), and excellent (9–10). IBM SPSS Statistics version 28 was used for all analyses. Questionnaire The questionnaire started with four closed-ended questions regarding demographics. The remaining 29 closed-ended questions included the following topics: CIPN sensations, daily limitations, attention to CIPN by healthcare professionals, and social support. In questioning the daily limitations, cycling was included as a separate category since cycling is one of the most widely used means of transportation in the Netherlands, making it a daily activity for many people and not just a sports activity. Daily limitations Figure 1 shows the reported daily limitations due to CIPN. In several types of daily activities, most patients report never experiencing limitations, namely, eating and drink- ing (69.4%), self-care (58.2%), driving (46%), sexuality and intimacy (45%), cycling (40.8%), and family/(tak- ing care of) children (31.7%). In the remaining catego- ries, limitations were more common. Being “sometimes limited” was most reported by participants for household chores (41.3%), sleep (38.1%), social activities (37.8%), hobbies (35.9%), walking (34.8%), sports (29.0%), and work (25.7%). CIPN symptoms More than half of the participants reported to suffer from CIPN for more than 2 years at the time of the questionnaire 1 3 Journal of Cancer Survivorship Involvement of healthcare professionals Table 3 shows an overview of hospital healthcare profes- sionals’ and GPs’ attention to CIPN. Regarding hospital healthcare professionals, more than half of the participants (58.4%) reported being informed before treatment about the possibility of the development of CIPN. Also, most par- symptoms during or after treatment. However, many (43%) reported not being informed about what to do when CIPN develops. Satisfaction with attention to CIPN was rated as insufficient by almost a quarter of patients (23%). However, many patients rated it as satisfactory to good (50.3%), or as excellent (26.7%). Only a small proportion of the participants (22%) reported having visited their GP for CIPN. The major- ity rated the GP attention to CIPN as satisfactory to good (53.5%), followed by excellent (25.5%). Few indicated this as insufficient (21.2%). Social support Participants indicated that partners showed empathy regard- ing CIPN always (47.6%), often (22.3%), and sometimes (11%) respectively (Fig. 2). Only a small minority reported that their partner never showed empathy (1.8%). This also applied to children (30.2%, 19.8%, 12.2%, 2.1% respec- tively). In the case of family, friends, and acquaintances, participants reported that empathy was shown often (27.1%), sometimes (25.5%), and always (25.4%), respectively, fol- lowed by a small minority of participants who reported that empathy was never shown (3.2%). Most participants indicated “I don’t know/not applicable” for social support regarding colleagues and business associates (55.3%) as well as for employers (60.6%). However, the remaining par- ticipants indicated that empathy was shown never (4.2%), sometimes (14.5%), often (12.8%), and always (10.8%) by colleagues and business associates. In the case of employers, participants reported that empathy was shown never (5.5%), sometimes (9.6%), often (10.6%), and always (11%). Discussion The aim of this study was to explore patients’ experience of CIPN symptoms, daily limitations, involvement of health- care professionals, and social support. Although symptoms are mostly experienced in both hands and feet and are often non-painful (e.g., tingling and loss of sensation or dimin- ished sensation), a significant part of participants reports painful symptoms. CIPN symptoms Only a small minority reported that their partner never showed empathy (1.8%). This also applied to children (30.2%, 19.8%, 12.2%, 2.1% respec- tively). In the case of family, friends, and acquaintances, participants reported that empathy was shown often (27.1%), sometimes (25.5%), and always (25.4%), respectively, fol- lowed by a small minority of participants who reported that empathy was never shown (3.2%). Most participants indicated “I don’t know/not applicable” for social support regarding colleagues and business associates (55.3%) as well as for employers (60.6%). However, the remaining par- ticipants indicated that empathy was shown never (4.2%), sometimes (14.5%), often (12.8%), and always (10.8%) by colleagues and business associates. In the case of employers, participants reported that empathy was shown never (5.5%), sometimes (9.6%), often (10.6%), and always (11%). The aim of this study was to explore patients’ experience of CIPN symptoms, daily limitations, involvement of health- care professionals, and social support. Although symptoms are mostly experienced in both hands and feet and are often non-painful (e.g., tingling and loss of sensation or dimin- ished sensation), a significant part of participants reports painful symptoms. These symptoms can result in a variety of daily limitations, in which most patients are able to perform activities of daily life (ADL) but seem to experience prob- lems mainly in their roles and social activities. Support and empathy are not always experienced by patients. This mainly applies to support and empathy from healthcare profession- als, friends and acquaintances, and work-related peers. CIPN symptoms These symptoms can result in a variety of daily limitations, in which most patients are able to perform activities of daily life (ADL) but seem to experience prob- lems mainly in their roles and social activities. Support and empathy are not always experienced by patients. This mainly applies to support and empathy from healthcare profession- als, friends and acquaintances, and work-related peers. This study has shown that even though most patients with CIPN experience non-painful symptoms, a significant Table 2 Experienced symptoms of CIPN Number % Duration of symptoms A few weeks 82 4.1 A few months 195 9.8 About half a year 168 8.5 About 1 year 198 10.0 About 2 years 164 8.3 More than 2 years 1090 55.1 Don’t know (anymore) 83 4.2 Location of symptoms Foot/feet 423 21.4 Hand(s) 147 7.4 Foot/feet and hand(s) 1410 71.2 Type of symptoms in feet Tingling 1327 67.0 Loss of sensation or diminished sensation 1360 68.7 Changed sensation 1043 52.7 Pain 758 38.3 Pain from touch 457 23.1 Pain from temperature changes 669 33.8 Burning or stabbing pain 664 33.5 Balance disorders 592 29.9 Muscle weakness or reduced strength 552 27.9 Muscle cramp 716 36.2 Thinning of muscles 215 10.9 Type of symptoms in hands Tingling 1180 59.6 Loss of sensation or diminished sensation 944 47.7 Changed sensation 724 36.6 Pain 486 24.5 Pain from touch 293 14.8 Pain from temperature changes 587 29.6 Burning or stabbing pain 318 16.1 Balance disorders 93 4.7 Muscle weakness or reduced strength 653 33.0 Muscle cramp 334 16.9 Muscle loss 137 6.9 Attempted to reduce or cope with symptoms Yes 1384 69.9 No 532 26.9 I don’t know/not applicable 64 3.2 Table 2 Experienced symptoms of CIPN symptoms during or after treatment. However, many (43%) reported not being informed about what to do when CIPN develops. Satisfaction with attention to CIPN was rated as insufficient by almost a quarter of patients (23%). However, many patients rated it as satisfactory to good (50.3%), or as excellent (26.7%). Only a small proportion of the participants (22%) reported having visited their GP for CIPN. The major- ity rated the GP attention to CIPN as satisfactory to good (53.5%), followed by excellent (25.5%). Few indicated this as insufficient (21.2%). Participants indicated that partners showed empathy regard- ing CIPN always (47.6%), often (22.3%), and sometimes (11%) respectively (Fig. 2). Involvement of healthcare professionals Table 3 shows an overview of hospital healthcare profes- sionals’ and GPs’ attention to CIPN. Regarding hospital healthcare professionals, more than half of the participants (58.4%) reported being informed before treatment about the possibility of the development of CIPN. Also, most par- ticipants (72.7%) reported that attention was given to CIPN This study has shown that even though most patients with CIPN experience non-painful symptoms, a significant proportion of people (also) experience painful symptoms. 1 3 Journal of Cancer Survivorship 0% 10% 20% 30% 40% 50% 60% 70% 80% Never limited Somemes limited Oen limited Always limited Don’t know/not applicable Journal of Cancer Survivorship Journal of Cancer Survivorship 0% 10% 20% 30% 40% 50% 60% 70% 80% Never limited Somemes limited Oen limited Always limited Don’t know/not applicable Fig. 1 Daily limitations due to CIPN Fig. 1 Daily limitations due to CIPN 1 3 a CIPN chemotherapy-induced peripheral neuropathy b Due to none-obligatory nature of item, valid percentages were reported Hospital healthcare professional N % Before treatment: informed about possibility of CIPNa development Yes 1157 58.4 No 523 26.4 Don’t know (anymore)/not applicable 300 15.2 During or after treatment: attention to CIPNa Yes 1439 72.7 No 541 27.3 During or after treatment: informed about what to do when CIPNa develops Yes 716 36.2 No 852 43.0 Don’t know (anymore)/not applicable 412 20.8 Satisfaction with attention to CIPNab Insufficient (1–5) 417 23.0 Satisfactory-good (6–8) 914 50.3 Excellent (9–10) 485 26.7 General practitioner N % During or after treatment: visited general practitioner for CIPNa Yes 436 22.0 No 1481 74.8 Don’t know (anymore)/not applicable 63 3.2 Satisfaction with attention to CIPNab Insufficient (1–5) 89 21.2 Satisfactory-good (6–8) 224 53.3 Excellent (9–10) 107 25.5 Table 3 Attention to CIPN by healthcare professionals Table 3 Attention to CIPN by healthcare professionals Hospital healthcare professional General practitioner a CIPN chemotherapy-induced peripheral neuropathy b Due to none-obligatory nature of item, valid percentages were reported Journal of Cancer Survivorship 0% 10% 20% 30% 40% 50% 60% 70% Partner Children Family/friends/acquaintances Employer Colleagues/business associates Never empathy Somemes empathy Oen empathy Always empathy I don’t know/not applicable Fig. 2 Social support and empathy Journal of Cancer Survivorship Fig. 2 Social support and empathy general population may also experience limitations given the relatively high average age in this sample. Earlier research into colorectal cancer survivors has shown that QoL and physical, role, cognitive, and social function- ing are worse in patients with painful CIPN compared with patients with non-painful CIPN [18]. However, the effect of painful versus non-painful CIPN on the daily limitations people experience was not examined in our study. For this reason, and since painful versus non-painful CIPN may involve different impairments and coping mechanisms, research on the difference in daily limitations and appro- priate psychosocial interventions between painful and non- painful CIPN is needed. Furthermore, this study showed that patients indicate they were not informed about what to do when CIPN develops. It is not known whether information has not been provided to patients, or whether patients were informed but had dif- ferent priorities in the process of facing a life-threatening disease. This means that for many patients, a search for symptom self-management begins when CIPN symptoms arise. Research has shown that patients often lack knowledge and self-management skills to properly manage their cancer- related pain [36]. Several studies have shown that psych- oeducation for cancer-related pain can positively influence patients’ knowledge and ability to self-manage their symp- toms [37–41]. Applying symptom self-management must be supported by healthcare professionals [42], which starts with informing patients appropriately and providing advice, start- ing before treatment. Furthermore, options and wishes in dose reduction of chemotherapy should be considered dur- ing treatment to possibly limit development or worsening of CIPN [8, 43–47]. However, no evidence-based treatment recommendations can currently be provided as there are no effective treatments for non-painful CIPN [45, 46]. p Results of this study have shown that most patients expe- rience limitations in daily life, which vary in how often peo- ple experience them. For example, daily activities such as eating and drinking, self-care, cycling, intimacy and sexu- ality, and driving are not limiting for a reasonable group of patients. General practitioner Therefore, some patients seem able to perform the ADL themselves. However, there are also many ADL in which many patients often experience limitations, such as walking, sleeping, and household chores. Previous research has indeed shown that patients with CIPN might become limited in various aspects of functioning (i.e., physical, social, emotional, role, and cognitive), which in turn dete- riorates their QoL [13, 14, 16, 18, 32]. Patients should there- fore receive support from healthcare professionals and peers. This may increase the (sense of control over their) ability to perform daily activities independently, contributing to patient empowerment, which can improve QoL [33–35]. However, results should be interpreted with caution as no comparison in daily limitations between patients with CIPN and the general population has been made. Therefore, it is not clear what the CIPN-specific limitations are, as the f In addition, patients should also be supported in self-man- agement by their social environment [42]. Our study showed that most patients often feel empathy by their social environ- ment mainly by partners, family, and friends, in which the degree of empathy varies. Earlier research has shown that such support improves CIPN and coping with cancer [27, 29]. However, our study showed that empathy by colleagues and employers is often lacking. Work-related social support includes both organizational support from employers (e.g., 1 3 3 3 Journal of Cancer Survivorship Some limitations also need to be discussed. First, the questionnaire was probably mainly filled in by patients that are connected to patient organizations, which might not be representative of all cancer patients. Second, it also appears to be an unrepresentative sample in terms of distribution in tumor types. This applies, for example, to lymphoma, which involves a much lower percentage of patients in the Netherlands than in this sample. It also applies to breast cancer, which explains the high number of women in this sample. As women and men have different coping strate- gies in general [57] and relating to cancer specifically [58], which may also affect the daily limitations they face, this may have affected the results of daily limitations. Third, the questionnaire was only available in Dutch, which pre- vented non-Dutch-speaking residents from completing the questionnaire. Fourth, a non-validated questionnaire was used. Fifth, only physical, role, and social aspects of func- tioning were considered in examining daily limitations. Future research should also look at emotional and cogni- tive functioning. General practitioner Sixth, fatigue was not taken into account in the assessment of daily limitations, while fatigue is one of the most common side effects of cancer treatment [59]. Seventh, the sample is very heterogeneous and no analyses regarding differences between chemotherapeutic agents could be made since no data regarding chemotherapeutic agents was collected. Future research should examine com- parisons between chemotherapeutic agents and associated symptom and consequences. job security, flexible working hours, and sick pay) and inter- personal support from colleagues (e.g., empathy and posi- tive attitudes) [48–51]. Since work-related social support is crucial in achieving work-related goals and returning to work after cancer [48–51], more attention must be paid to social support from colleagues and employers. However, in this study, colleague and employer support was not applicable for most participants since many patients are probably retired given the high average age of the sample. Therefore, these results should be interpreted cautiously, and further research on work-related social support is needed. Furthermore, even though our research shows that many patients often or always feel empathy with respect to CIPN by family, friends, and acquaintances, there is still a sig- nificant group of patients who never or only sometimes feel empathy is shown. Earlier research has found that 52% of breast cancer patients experienced to be sometimes avoided or contact is feared by friends and family [52]. Interest- ingly, this study also examined the perspective of healthy people, which showed that 61% of them would or might avoid people with cancer. Reasons of relatives for not pro- viding social support to cancer patients appear to be diverse and can include, for example, the perception of one’s own inability to provide support, as well as not wanting to burden the cancer patient emotionally [53]. However, it has been shown that patients wish to receive social support, and they experience increased QoL when they receive helpful social support [54]. However, social support appears to diminish significantly within 1 year after diagnosis [54], which could possibly explain the lack of empathy regarding CIPN expe- rienced by some of the patients in our study, since CIPN can be present for a long time after treatment [3, 8, 12–15]. Another explanation may be that relatives often do not know what CIPN entails and do not understand the symp- toms [55]. General practitioner Because of the variety of symptoms, it can be difficult to understand and explain CIPN to family, friends, and acquaintances. Healthcare professionals should provide appropriate information to patients about CIPN even before treatment has started, so that patients can properly explain their symptoms when they arise [55], thus creating more openness and awareness about CIPN. Conclusion This exploratory study showed that patients with CIPN suf- fer from various symptoms which may result in daily limita- tions. The prevalence of these limitations differs, and, as a result, the extent to which patients are able to perform ADL also varies. The degree of attention to, and satisfaction with, this attention from healthcare professionals varies, which also applies to the level of empathy from the social environ- ment. Appropriate guidance from healthcare professionals, starting before treatment, and support from the social envi- ronment is crucial in enabling patients to feel empowered in their daily lives despite CIPN. References 1. Integraal Kankercentrum Nederland. Nederlandse Kankerregis- tratie [Internet]. 2021. www.iknl.nl/nkr-cijfers 18 Bonhof CS, Trompetter HR, Vreugdenhil G, et al. Painful and non-painful chemotherapy-induced peripheral neuropathy and quality of life in colorectal cancer survivors: results from the population-based PROFILES registry. Support Care Cancer. 2020;28:5933–41. https://doi.org/10.1007/s00520-020-05438-5. 2. Integraal Kankercentrum Nederland. 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Trompetter1 · Floortje Mols1,2 References Perceived help- fulness and impact of social support provided by family, friends, and health care providers to women newly diagnosed with breast cancer. Psychooncology. 2007;16:474–86. https://doi.org/10.1002/ PON.1084.f 38. Valenta S, Miaskowski C, Spirig R, et al. Randomized clinical trial to evaluate a cancer pain self-management intervention for outpa- tients. Asia-Pacific J Oncol Nurs. 2022;9:39–47. https://doi.org/10. 1016/j.apjon.2021.12.003. 39. Miaskowski C, Dodd M, West C, et al. Randomized clinical trial of the effectiveness of a self-care intervention to improve cancer pain management. J Clin Oncol. 2004;22:1713–20. https://doi.org/ 10.1200/JCO.2004.06.140.f 55. Tanay MAL, Robert G, Rafferty AM, et al. Clinician and patient experiences when providing and receiving information and sup- port for managing chemotherapy-induced peripheral neuropathy: a qualitative multiple methods study. Eur J Cancer Care (Engl). 2022;31:e13517. https://doi.org/10.1111/ECC.13517. 40 Park YJ, Lee MK. Effects of nurse-led nonpharmacological pain interventions for patients with cancer: a systematic review and meta- analysis. J Nurs Scholarsh. 2022;54:422–33. https://doi.org/10.1111/ JNU.12750. 56. Staff NP, Grisold A, Grisold W, et al. Chemotherapy-induced peripheral neuropathy: a current review. Ann. Neurol. John Wiley and Sons Inc.; 2017;81;6:772–781. https://doi.org/10.1002/ana. 24951.f 41 Oldenmenger WH, Geerling JI, Mostovaya I, et al. A systematic review of the effectiveness of patient-based educational interven- tions to improve cancer-related pain. Cancer Treat Rev. 2018;63:96– 103. https://doi.org/10.1016/J.CTRV.2017.12.005. 57 Tamres LK, Janicki D, Helgeson VS. Sex Differences in coping behavior: a meta-analytic review and an examination of relative coping. Personal Soc Psychol Rev. 2002;6:2–30. https://doi.org/10. 1207/S15327957PSPR0601_1. p g 42. van Dongen SI, de Nooijer K, Cramm JM, et al. Self-management of patients with advanced cancer: a systematic review of experiences 1 3 Journal of Cancer Survivorship Daniëlle L. van de Graaf1,2 · Vivian Engelen3 · Aize de Boer3 · Gerard Vreugdenhil4 · Tom Smeets1 · Marije L. van der Lee1,5 · Hester R. Trompetter1 · Floortje Mols1,2 Daniëlle L. van de Graaf D.L.vdGraaf@tilburguniversity.edu Vivian Engelen v.engelen@nfk.nl Aize de Boer a.deboer@nfk.nl Gerard Vreugdenhil G.Vreugdenhil@mmc.nl Tom Smeets t.smeets@tilburguniversity.edu Marije L. van der Lee MvanderLee@hdi.nl Hester R. Trompetter h.r.trompetter@tilburguniversity.edu Floortje Mols f.mols@tilburguniversity.edu 1 CoRPS ‑ Center of Research on Psychological disorders and Somatic diseases, Department of Medical and Clinical Psychology, Tilburg University, PO Box 90153, 5000 LE Tilburg, The Netherlands 2 Department of Research, Netherlands Comprehensive Cancer Organisation (IKNL), Utrecht, The Netherlands 3 Dutch Federation of Cancer Patient Organisations, Utrecht, The Netherlands 4 Department of Internal Medicine, Máxima Medical Centre, Veldhoven, Eindhoven, The Netherlands 5 Centre for Psycho‑Oncology, Scientific Research Department, Helen Dowling Institute, Bilthoven, The Netherlands 1 3

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