Sucupira-branca ( Pterodon - Fabaceae): does wood anatomy support the distinction among species and hybrid?

,


Introduction
Pterodon is a genus of arboreal species distributed in South America in Brazil and Bolivia (Pinto et al. 2014;Tropicos.org 2021).The natural high resistance of Pterodon wood to xylophagous organisms makes this genus a good candidate for being used in civil construction and fences (Mainieri & Chimelo 1989).The phytoextracts are employed by traditional communities as anti-inflammatory (Hansen et al. 2010) and the ethanolic fruit extracts used as larvicides against Aedes aegypti (De Omena et al. 2007).
Pterodon is monophyletic, well-supported, and related to Dipteryx, Taralea and Monopteryx (Cardoso et al. 2015;Leite et al. 2015;Silva et al. 2018).However, there is no phylogeny for Pterodon genus as a whole; and according to Cardoso et al. (2015) P. emarginatus and P. pubescens are closer phylogenetically to each other than to P. abruptus.
The circumscription of Pterodon species differs and varies among published databases and the exact number of species remains uncertain.For example, in ILDIS (2021) P. apparicioi, P. pubescens and P. polygalaeflorus are considered synonyms of P. emarginatus.In Tropicos.org(2021), P. polygalaeflorus and P. apparicioi are legitimate species, but P. pubescens is considered a synonym of P. emarginatus.In Flora do Brasil 2020 (2021), P. polygalaeflorus is considered a synonym of P. emarginatus, and P. pubescens and P. apparicioi are accepted.Pterodon abruptus is accepted in all databases.Five species are assigned to Pterodon: P. abruptus, P. apparicioi, P. emarginatus, P. pubescens and P. polygaliflorus.(P.polygalaeflorus variation in writing), according to IPNI (2021).In addition, P. macrophylla was cited in IPNI ( 2021), but should be considered a nomen nudum as it was only cited in Reisen in British-Guiana in den Jahren 1840-1844, volume 3 (page 1103), without description or type assigned and not reported again.
Part of this meandering history regarding the number of species in Pterodon dates back to Legumes of Bahia (Lewis 1987), in which the author indicated that P. polygaliflorus and P. pubescens are synonyms of P. emarginatus.Pterodon emarginatus as proposed by Lewis is a dimorphic species with individuals with pink flowers, pubescent leaves and retuse folioles apex as well as plants with purple flowers, glabrate leaves with emarginate to truncate folioles apex.These two phenotypes do not occur side by side, the populations of each form are parapatric (Rocha 2006).The same author employing RAPD (random polymorphic DNA) and morphological data recognized two distinct taxa.Comparison with phototypes and literature of the tree taxa (P.emarginatus, P. polygaliflorus, P. pubescens) allowed to conclude that P. polygaliflorus is a synonym of P. emarginatus (with purple flowers) and P. pubescens (with pink flowers) is a separate valid species.Rocha (2006), based on RAPD, also recognized some hybrids between P. emarginatus and P. pubescens, forming a narrow range in the Federal District, which suggests that the diverging time of these two species should be recent.The flower color of hybrid individuals has pink sepals and purple petals.Baretta-Kuipers (1981) found that the different patterns of parenchyma and ray structure were the most significant anatomical features to identify Fabaceae genera.Gasson (1999), when comparing the genera within Dipterygeae, found that Dipteryx and Pterodon are anatomically more similar to each other than they are to Taralea.Despite having remarkably similar wood anatomy, Dipteryx and Pterodon differed due to the presence of irregularly or non-storied rays and axial parenchyma, as well as differences in intervessel pitting diameter.
Secondary xylem studies have proved useful in the taxonomy and highly significant in the elucidation of plant phylogeny (Baretta-Kuipers 1981;Gasson 1999;Nisgoski et al. 1998), by providing evidence for assigning a definite position to taxa of uncertain affinity (Sharma 2009).
According to Dickison (2000), in no other plant tissue the trends of structural evolution are so clearly defined, since the wood structure tends to be more conservative in many features than the external morphology (Baretta-Kuipers 1981).However, it is noteworthy that the quantitative characteristics will show more dissimilarities than the qualitative ones (see Loureiro et al. 1984;Nisgoski et al. 1998;Oliveira et al. 2001), which varies in amplitude within each species (Sonsin et al. 2012).
The aim of this study was to characterize the wood anatomy of Pterodon species, including the hybrid, to verify which anatomical features vary among species, and mapping for the first time the most important wood anatomical characters using multivariate data analysis, providing informative characters to help circumscribe the Pterodon species.

Material and Methods
Wood samples were collected at diameter at breast high (DBH) = 1,30 m with a hand-held drill (BT45-Still®), adapted with a hollow drill bit (non-destructive sampling method), in all Pterodon Vogel species (Tab.1).All were deposited in the UBw wood collection accompanied by vouchers deposited in UB herbarium (acronym according to Thiers 2018).Pterodon species were always collected in flower, except for P. abruptus.The flowering period is especially important to identify hybrid trees.The plants were identified by DMS Rocha.Pterodon abruptus was collected in the Caatinga because it is endemic from this biome (Shimizu & Semir 2016).
Climate data is classified according to Köppen and Geiger and is given for each location: P. apparicioi collection site climate classification is Cwa, has an annual mean temperature (AMT) of 20.7 ºC, annual mean precipitation (AMP) of 1149 mm, with a dry season of five months with precipitation below 45 mm; P. abruptus climate classification is BSh, has an AMT of 26 ºC, AMP of 747 mm, with precipitation below 25 mm for five months; P. pubescens climate classification is AW, has an AMT of 21.9 ºC, AMP of 1443 mm, with precipitation below 45 mm for five months; P. emarginatus and hybrid climate classification is AW, has an AMT of 22.3 ºC, AMP of 1223 mm, with precipitation below 40 mm for five months.Climate data were obtained from the website Climate-Data.org (https://pt.climate-data.org,weather data between 1999 to 2019 and refreshed from time to time).
Wood samples were stored in 70 % alcohol with 50 ml of glycerin to soften the wood.Additionally, samples were placed in a pressure cooker for about two hours (separate periods of 30 minutes), since boiling them was not enough.We cut the wood into cubes (ca. 2 cm²) and microtome sectioned into transverse (TRS), tangential (TLS) and radial longitudinal (RLS) sections, with 15 to 20 μm, and mounted according to Kraus & Arduim (1997).Histological slides were permanently embedded in synthetic resin (Entellan®).
The wood qualitative and quantitative anatomical descriptions were based on IAWA Committee (1989), and COPANT (1974) was used only for ray width category (microscopy).All measurements were made with Image-Pro Plus ® (6.0).Thirty measurements per feature per individual were made, including height and ray width, vessel and axial parenchyma area.This last one was measured in 0.25 mm², by dividing 1mm² into four; using a Surface Microsoft notebook with a digital pen we were able to draw the area.Olympus light microscope was used, and the images were recorded with a Leica photomicroscope associated with a microcomputer with LAS EZ image capture system.
Statistical analyzes were performed with PAST v3 and Microsoft Excel software.The mean, standard deviation and coefficient of variation (CV) were calculated.A normality test was done, and the Log values of the anatomical characteristics were used to reduce the large differences in values of each measured structure.One-way ANOVA (several samples) and Tukey tests were performed on each anatomical feature to test if anatomical variables were significantly different among the Pterodon species.
We performed an UPGMA cluster analysis based on a binary matrix representing the presence (1) or absence (0) of a character state and employing Jaccard similarity coefficient for the qualitative and quantitative anatomical features, the latter transformed into classes given by IAWA variables.The characters employed in this matrix were: growth rings; intervessel pits shape and size; axial parenchyma strand; ray frequency, width and storied; presence of prismatic crystals; vessel diameter and density; fiber length.The axial parenchyma area was not included on cluster analysis because it did not have IAWA (1989) variable available, and we were not able to transform it into classes, since we do not have enough data to use as a parameter.
Principal Component Analysis (PCA) with correlation matrix was employed to quantitative characters in order to verify the differences among species, using average values of the IAWA List and Gasson et al. (2010) parameters (maximum and minimum vessel lumen diameter (μm), maximum and minimum ray height (cells)), in addition to maximum and minimum axial parenchyma area.All data were log transformed prior to PCA to standardize the dataset.
A dichotomous identification key was made to help separate taxa.

Results
In macroscopic analysis we found that all woods were hard to cut, with fine texture, irregular to straight grain, with no odor or distinct luster.Heartwood color varied from yellow brown to dark brown.Pterodon abruptus may be easily distinguished in macroscopic analyzes by highest vessel density (40-100/mm²), smaller vessels (< 0.05 mm) and predominance of the unilateral long confluent parenchyma forming bands.All other species had clearly wood characteristics of Pterodon, and could not be distinguished with macroscopy.Therefore, we made a description for the genus as follows (Figs.1-3, S1).
A description of the P. apparicioi wood anatomy and hybrid are presented here for the first time (Text S2).
The axial parenchyma predominant type was short to long confluent (oblique), lozenge aliform, vasicentric, unilateral and in marginal lines, but some differences were observed (Fig. S1), such as: in P. pubescens (Fig. 1A) there was a predominance of short confluent (mostly up to three vessels) and lozenge unilateral; hybrids had long and short confluent (oblique), with the cells being conspicuously larger when compared to the other species; in P. abruptus (Fig. 1E) the long confluent (oblique) forming bands exclusively unilateral was observed; in addition, we have noted a variation in the amount of axial parenchyma becoming only unilateral and lozenge and few short confluent near the growth ring (Fig. 1B).
Except for P. abruptus, all other Pterodon species and hybrids (Fig. 2B) had some individuals with irregular storied rays.
Major differences of qualitative and few quantitative analyzes were added according to IAWA and COPANT (for ray width) category in Table 2, for better comparison.Yet, some anatomical features which were associated remained in the Table.
The quantitative data are shown in Table 3, with some overlap among them.Of the 14 anatomical features analyzed, the hybrid was similar to P. emarginatus in seven, to P. apparicioi in five, to P. pubescens in five, and to P. abruptus in eight (Tab.3).In addition, the hybrid shared three anatomical characteristics (statistically similar) with both P. emarginatus and P. pubescens (intervessel pit diameter and fiber diameter and thickness), while was similar to P. emarginatus and P. apparicioi in four anatomical features (vessel ray pit diameter, fiber diameter and wall thickness and ray width) (Tab.3).
PCA, made only with quantitative anatomical features, showed 64 % of the total variance explained by component 1 and 2 (Fig. 4; Tab.S3).Component 1 explained 41 % of the variance, separated P. abruptus from the other species and was influenced mostly by vessel density, area, diameter.Component 2 explained 23 % but is not sufficient to separate the remaining taxa.Pterodon abruptus was the only species that remained totally separated from the other Pterodon species.Cluster analyses (Fig. 5) clearly separated P. abruptus.Pterodon apparicioi although clustered together with the other species and hybrid, seems to consist of a separated group inside this major one.An identification key using wood anatomy characteristics permitted the separation of P. abruptus and P. apparicioi (Tab.4).However, wood characteristics were not enough to distinguish P. pubescens, P. emarginatus and hybrid between these species.Despite that, we could observe some wood anatomical details that may help to distinguish P. pubescens, P. emarginatus and hybrid, as follows: P. pubescens have over 70 % of solitary vessels/mm², while the other two have below 62 %; large vessels (100-200 μm) were present in 59 % of P. pubescens and 57 % in P. emarginatus, while the hybrid had 62 % of vessels of medium diameter (51-100 μm); 68% of rays density predominantly few (4-12/mm) in P. pubescens while P. emarginatus and hybrid had below 48 %; vessel area average was statistically different in the three species (Tab.3); axial parenchyma area average of hybrid was significant higher than the other two (Tab.3); prismatic crystals in chambered axial parenchyma cells were only observed in P. emarginatus and the hybrid.

Discussion
In general, the qualitative anatomical description of secondary xylem made here for the genus Pterodon is similar to the bibliography consulted (Mainieri et al. 1983;Gonçalez et al. 1985;Mainieri & Chimelo 1989;Paula & Cardoso 1995;Gasson 1999;InsideWood 2004).For P. abruptus there were only available images from three sections in "InsideWood" website (https://insidewood.lib.ncsu.edu/),and a description for the Pterodon genus based on this species and P. pubescens.When comparing the image from Sucupira-branca (Pterodon -Fabaceae): does wood anatomy support the distinction among species and hybrid?InsideWood with P. abruptus from our study, the wood anatomy in general is quite similar, differentiating only regarding the quantity of multiple vessels, that was clearly lower than ours, but there were only two imagens from the TRS (transverse section).Despite the observed differences this species was the same as P. abruptus collected by us.
In both PCA and cluster analyses, P. abruptus was easily separated from the other species either in macroscopy and/ or microscopy, because it showed a high density of narrow vessels in multiple radial parenchyma, and the unilateral long confluent axial parenchyma forming bands.Despite the presence of axial parenchyma in long bands found only in this species, P. abruptus had the lowest axial parenchyma area when compared with the other taxa.Also, it had the lowest maximum number of ray height cells (up to 20), an important feature indicated by Gasson (1999) for identification.
Please note that quantitative features such as vessels and axial parenchyma abundancy may be influenced by both environmental (Baas & Wheeler 1991;Alves & Angyalossy-Alfonso, 2002) and genetic characteristics (Schweingruber et al. 2008).For example, small vessels are related to drier environments (Wheeler & Baas 1993;Carlquist 2001), here reflected in P. abruptus collected in the drier caatinga.However, parenchyma type is one of the most relevant anatomical features when identifying a family, genus or species as it is considered a conservative characteristic within a taxon.Nevertheless, the quantitative data of the species vary within the range for each taxon (Sonsin et al. 2012); so, those anatomical features were very useful to separate the species.
When analyzing P. apparicioi we observed that while using only quantitative anatomical features, even the ones proposed by Gasson et al. (2010), in PCA, it remains strongly related to P. pubescens and P. emarginatus, but not much with the hybrid.However, in cluster analysis, when qualitative data was also used, P. apparicioi was distinct from all other taxa (Fig. 5).Probably, if we were able to put the axial parenchyma into classes to do the analyzes, as we did with vessels, fiber and ray features, the hybrid distinction would be more evident.
In InsideWood, P. pubescens is considered a synonym of P. emarginatus and they are probably genetically similar because where their distributions overlap, they form a zone of hybridization (Rocha 2006) and are phylogenetically related (Cardoso et al. 2015).Therefore, we here compare P. pubescens InsideWood descriptions with anatomical data of P. pubescens, P. emarginatus and the hybrid in our study.
These species and the hybrid are all similar regarding quantitative and qualitative features, except for: presence of growth rings observed in both species studied by us, which are absent in the InsideWood description, however it is possible to observe in the TRS image; the axial parenchyma is two seriate in InsideWood, while the two studied species and the hybrid had a variation from 2 to 5 cells per strand; exclusively uniseriate rays is mentioned in InsideWood, but in both species studied by us they were predominantly uniseriate, with some biseriate rays, however the hybrid was predominantly multiseriate; only for P. pubescens and the hybrid the intervessel pit diameter were smaller than <10 μm, while in InsideWood, it is reported to be larger than 10 μm; also we observed irregular storied rays in some individuals of both species and hybrid, and prismatic crystals only in P. emarginatus and hybrid, all features not observed in InsideWood website description.Gasson (1999) described intervessel pitting fine (up to 10 μm), clearly vestured for Pterodon genera, based on P. abruptus and P. emarginatus.Here, only P. emarginatus, P. pubescens and hybrids had intervessel pit diameters varying from 9.6 and 10 μm.The other two species had smaller pits, about 7.2 μm (P.abruptus) and 8.4 μm (P.apparicioi).Therefore, for the genus intervessel pit diameter should be of medium size category, 7-10 μm.
Fiber walls were statistically similar in all studied species, similar to Gasson (1999) for Pterodon genus.Notwithstanding the significant differences between precipitation rates of species collection sites, the thickness of fibers in this genus could be genetically intrinsic and not variable with climatic conditions (see Schweingruber et al. 2008), as seen in other studies for different Fabaceae species (e.g.Alves & Angyalossy-Alfonso 2002;Sonsin et al. 2012).
According to Gasson (1999), the axial parenchyma was mainly aliform and confluent, strands storied and 2-4-celled.The axial parenchyma type is consistent for the species studies here (predominantly short to long confluent (oblique), lozenge aliform and 2-5 celled), except for P. abruptus which had a distinctive axial parenchyma