The systematic value of pollen morphology in trees and shrubs species of Bauhinia L . ( Caesalpinioideae-subg . Bauhinia-sect . Pauletia ) occurring in Brazil 1

We analyzed the pollen grains of 30 taxa of the genus Bauhinia L., within five series: Aculeatae (9 species); Acuminatae (1 species); Cansenia (17 species); Pentandrae (2 species); and Perlebia (1 species). The pollen grains were acetolysed, after which they were photographed and analyzed under light microscopy. Non-acetolysed pollen grains were analyzed under scanning electronic microscopy. Characters such as shape, size, exine and aperture constitution were studied. The species were separated by a dichotomous key, which showed pollen grains that were classified as very large; isopolar or apolar; oblate, suboblate, oblate spheroidal or spherical; large or small in terms of the polar area; inaperturate, 3-porate or (3-7)-colpate; and microreticulate or reticulate, with or without supratectal elements (gemmae, bacula, clavae). The variation in pollen morphology confirms the eurypalynous status of the genus Bauhinia.

The infrageneric taxonomic organization of Bauhinia proposed by Wunderlin et al. (1987) consists of a group of species represented by trees, shrubs or subshrubs, multicaules, sometimes semi-scandent, although never with tendrils.The branches may have pairs of adpetiolar spines or are unarmed, and have extrafloral nectaries and well-developed intrastipular trichomes on the inner surface of the stipules.The hypanthium is short-turbinate or, in general, long and tubular, the calyx is spathaceous or irregularly adnate at the apex and opens at the base in 2-5 lobes.The fruit is woody or coriaceous and dehiscent.The taxonomic arrangement of Wunderlin et al. (1987) is used in this study.
Few other studies have examined the pollen grains of the species of Bauhinia, especially the native Brazilian taxa.Vaz (2001) noted that the pollen morphology is known for only a few species of section Pauletia and emphasized the need for studies on the external morphology of pollen.
In the present study, we analyzed the pollen of 30 tree and shrub species of Bauhinia, subg.Bauhinia sect.Pauletia series Aculeatae, Acuminatae, Cansenia, Pentandrae and Perlebia occurring in Brazil, in order to support an eventual revision of the genus in the neotropics.Our objective was to expand knowledge of the attributes of the pollen of this group.

Material and methods
Pollen grains of 30 Bauhinia species were examined.The samples were obtained from the anthers of flower buds of specimens held in the following Brazilian herbaria: the Herbarium of the Ecological Reserve of the Brazilian Institute of Geography and Statistics (code, IBGE); the Herbarium of the Botany Department of the National Museum in Rio de Janeiro (code, R); and the Herbarium of the Rio de Janeiro Botanical Garden (code, RB).Acronyms are according to Thiers (continuously updated).
Whenever possible, four specimens of each species were analyzed and compared in order to obtain accurate results (Appendix 1).The permanent slides from this study are stored in the Álvaro Xavier Moreira Laboratory of Palynology of the Botany Department at the Federal University of Rio de Janeiro/National Museum.
Pollen grains were prepared by acetolysis with the method developed by Erdtman (1952), as modified by Melhem et al. (2003) for light microscopy.Pollen grains for scanning electron microscopy were placed on stubs covered with carbon tape and sputter-coated with gold.Samples were then examined using a scanning electron microscope (JSM-5310; JEOL, Tokyo, Japan) in the Hertha Meyer Laboratory of Cell Ultrastructure at the Biophysics Institute of the Federal University of Rio de Janeiro.
One specimen of each species was chosen for statistical treatment and illustrations, and is indicated by an asterisk (*) after the collector's name (Appendix 1).Measurements in equatorial view (polar diameter and equatorial diameter) were taken for 25 pollen grains per sample.Means, standard deviations, and 95% confidence intervals (CIs) were calculated.For measurements of equatorial diameter in polar view (EDPV), apocolpium side, apertures and exine thickness, the arithmetic means of 10 measurements were used.Ten similar measurements of pollen grains were made on additional material, from another collection, to check the stability of the data (treated further as comparison material).
The number of supratectal elements was established from a 50 μm 2 area on the surface of the pollen in polar view.
The terminology used for pollen descriptions follows Punt et al. (2007), which takes into consideration elements of size, shape, number of apertures, and sexine ornamentation.The descriptions of the polar area and aperture size were made according to the classification system established by Faegri & Iversen (1966) for the polar area index.

Results
We evaluated the pollen of 30 tree and shrub species of Bauhinia L. sect.Pauletia, within five series:

Size and polarity
In the group evaluated, the pollen grains were classified as very large in polar view (Tab.2): 77.5-137.5 μm in polar diameter; and 102.5-170.0μm in equatorial diameter.Isopolar pollen grains were found in members of the series Aculeatae, Cansenia, Pentandrae, and Perlebia.Apolar pollen grains were found only in the series Acuminatae (B.acuminata).

Dispersal unit and shape
All species examined had pollen grains arranged in monads (Tab.2).Oblate pollen grains were found in B. aculeata (ser.Aculeatae), B. acuruana, B. brevipes, B. cheilantha, B. curvula, B. longifolia, B. rufa, B. subclavata  In B. membranacea, the form could not be established, because the pollen grains were found only in polar view.

Polar area and apertures
A small polar area was found in all species of the series Aculeatae, Pentandrae, and Perlebia, and in B. angulicaulis (ser.Cansenia).The polar area was large in most of the species of the series Cansenia (Tab.1).
In most species within the series Cansenia, the colpi were short, the margo being pronounced only in B. cupulata (Fig. 35,36) and long only in B. angulicaulis.The apex was rounded in most species and acute in B. curvula, covered by a thin membrane, transparent, with gemmae and/or verrucae, and rarely granulate.Bauhinia cheilantha (Fig. 33) was the only species to show slightly elongated pores (Tab. 3)     In the series Pentandrae, the colpi were long with a slightly rounded apex, covered by a thin membrane, transparent, and ornamented (Fig. 71).The colpus was longest and widest in B. vespertilio (ca.78.0 × 8.8 μm) (Tab.3).In B. bauhinioides (ser.Perlebia) the colpi were long and covered by a thin membrane, transparent, and lightly ornamented (Fig. 76, 77), with a rounded apex (Fig. 75) (Tab.3).
In the series Cansenia, the sexine was reticulate in most species, microreticulate in B. cheilantha (Fig. 34) and B. ungulata (Fig. 62, 63); and perforate in B. cupulata (Fig. 87), in which the mesocolpium was conspicuously undulate, resembling rugulae, and the apocolpium was slightly undulate.The muri of the sexine were simplicolumellate in all species; reticulate in most species (Figs. 32,39,44,50,56,59,65); microreticulate in B. cheilantha (Fig. 34) and B. ungulata (Fig. 62, 63); and perforate with the surface in the mesocolpium, conspicuously undulate, resembling rugulae, and slightly undulate in the apocolpium only in B. cupulata (Fig. 35,36).The muri were straight or sinuous, low or high in the intersections, with or without perforations, some species showing interruptions in some areas that nevertheless did not close the lacunae; the lumina were ornamented or not, with small lumina surrounding the larger lumina.In most species, there were five types of supratectal elements (scabrae, gemmae, clavae, bacula and verrucae) with or without a predominance of any given type (Tab.4).The nexine was thicker than the sexine in most species; approximately the same thickness as the sexine in B. angulicaulis and B. dubia; and thinner than the sexine of B. cupulata (Tab.3).
In the series Pentandrae, the sexine was microreticulate, with supratectal elements in the shape of scabrae and gemmae (Fig. 69, 73) the muri were straight, smooth and Th e systematic value of pollen morphology in trees and shrubs species of Bauhinia L.
Dichotomous key to Bauhinia species, based on pollen characters

Discussion
Bauhinia acuminata (ser.Acuminatae) differs from all other species by presenting inaperturate pollen grains separated in the key based on this characteristic.The species B. bauhinioides was the only one belonging to the series Perlebia and had pollen grains 4(5)-colpate with a microreticulate sexine.These features placed it next to B. albicans (ser.Aculeatae) in the pollen key.The species of the series Pentandrae could not be separated in the key because their pollen was similar.The series Aculeatae formed two groups in the key, based on the type of sexine, which was microreticulate (B. affinis, B. forficata, and B. platypetala) or reticulate (B. acreana, B. aculeata, B. catingae, B. mollis, and B. ovata).Other features such as the number and type of supratectal elements were similar among the species of this series; i.e., some heterogeneity exists within this group.The taxa B. cheilantha and B. ungulata fell rather far from each other in the key, closer to representatives of other series.A large part of the species of the series Cansenia were united in the pollen key, based on the number and type of aperture (3(4)-colpate).
The species studied here all showed very large pollen grains.However, they differed primarily with respect to shape; amb; polar area; presence or absence of aperture (number and type); and sexine ornamentation.The members of the genus Bauhinia analyzed here showed predominantly reticulate pollen grains; only one species (B.cupulata) differed from the pattern, with the sexine perforate, although the mesocolpium was conspicuously undulate, with rugulae, and the apocolpium was slightly undulate.Some species differed from the others in the type of muri, which were low in B. dubia; with a smaller lumina around the larger lumina in B. angulicaulis, B. brevipes, and B. membranacea; sinuous in B. curvula and B. viscidula; and with elevations at the intersections in B. holophylla and B. ungulata.Other features were also important, and the key effectively differentiated the species by the shape of the pollen grain; aperture dimensions; and number and type of supratectal elements (scabrae, gemmae, clavae or bacula).
According to Salgado-Labouriau (1973), all Caesalpinioideae have pollen grains in monads.However, pollen grains in tetrads were found in representatives of the subgenus Bauhinia by Wunderlin et al. (1987), although those authors did not mention which species have this characteristic.In the present study, we found pollen grains only in monads, in agreement with Salgado-Labouriau (1973).Melhem & Salgado-Labouriau (1963) described the pollen grains of species of the Leguminosae of the Cerrado.Among the taxa studied, the authors analyzed two of the species discussed in the present study: B. holophylla and B. rufa.Our results corroborate those described for B. rufa and differ with respect to B. holophylla in terms of the shape (oblate) and the sexine/nexine ratio.Barth & Bousada (1964) studied the pollen grains of three species of Bauhinia, including B. forficata, which was analyzed here.The authors described the pollen grains of B. forficata as having a pore-type aperture and supratectal elements of the spine type, differing from our observation that this species has supratectal elements of the clava type and aperture of the colpus type.
The variability of supratectal elements has been demonstrated by various authors, showing that Bauhinia species can have supratectal elements ranging from scabrae to bacula.The presence of verrucae, bacula, gemmae and scabrae was previously reported by Palacios-Chávez (1970).(Caesalpinioideae-subg.Bauhinia -sect.Pauletia) occurring in Brazil Acta bot.bras.27(2): 400-417.2013.This wide variation was observed in our study; the absence of supratectal elements of the scabra type was characteristic of the pollen grains of the representatives of the series Aculeatae.The similarity between some supratectal elements might explain the lack of specificity in the descriptions of pollen grains in certain studies.Schmitz (1973) examined the pollen of 100 species of Bauhinia, with simplified descriptions based mainly on the number and type of aperture, shape and sexine ornamentation.Among the species treated by that author and also studied here are the following: Perlebia acuminata  Schmitz (1973) with those of our study, the pollen descriptions generally agree for the species B. acuruana, B. curvula, B. rufa, B. ungulata and B. viscidula.Note that Bauhinia curvula was characterized by Schmitz (1973) as having pollen grains with supratectal processes in the form of verrucae.This characteristic corroborates our results, albeit found only in three of the taxa studied here.Our observations regarding B. acuminata diverged from those of Schmitz (1973), who classified the pollen as 3-4(5)-aperturate, whereas we classified it as inaperturate.The variations in the number of apertures indicated by Schmitz (1973) for B. bauhinioides and B. longifolia were not observed in this study.Schmitz (1973) classified B. platypetala as 3-colporate, rugulate to striated; whereas we observed six colpi and a reticulate sexine.The classification of the number of apertures of B. forficata (3(4)-colpate), does not agree with our observation of 6-colpate pollen grains.Wunderlin (1976) considered B. affinis a synonym of B. aculeata, along with numerous other binomials, including B. catingae and B. albicans.The proximity of the species mentioned by Wunderlin (1976) was confirmed by the pollen characters observed in this study.These taxa belong to the series Aculeatae and have pollen grains with supratectal processes such as clavae or bacula.This type of ornamentation, together with other characters, places these species in the series Aculeatae with regard to pollen characters.Guinet (1981) reported that the vast majority of the representatives of the subfamily Caesalpinioideae have 3-colporate pollen grains, with some colpate and a few porate.Despite that author's assertion regarding the predominance of colpori in the subfamily, our results for the pollen grains of species of the genus Bauhinia showed that practically all taxa had colpate pollen, which probably indicates that the section Pauletia does not follow the aperture type pattern found in Caesalpinioideae.The only exception, B. cheilantha, had a pore-type aperture.Porate pollen grains were observed in the genus Bauhinia, in B. seleriana, by Palacios-Chávez (1970).The species B. acuminata showed no apertures, differing from all other species examined here.Inaperturate pollen grains were observed in B. tomentosa by Vishnu-Mitre & Sharma (1962).Wunderlin et al. (1987) established five series under the section Pauletia: Acuminatae, Ariaria, Cansenia, Pentandrae and Perlebia, making use of macro-and micromorphological characters, including those of pollen.Of those series, only Ariaria was not analyzed here.The palynological characters used by the authors in the formulation of the series were as follows: for the series Cansenia, including ca.50 species-pollen grains (3-7)-colpate or (3-7)-colporoidate, sexine reticulate, usually with supratectal processes; for the series Perlebia, with only one species-pollen grains 3-7-colpate, sexine reticulate with supratectal processes; and for the series Pentandrae, comprising ca.seven species-pollen grains 3-7 colporate, sexine reticulate with supratectal processes.Comparing the results obtained here with those of Wunderlin et al. (1987), the observations for the series Cansenia and Perlebia were similar.However, descriptions for the series Pentandra differ, because Wunderlin et al. (1987) reported colporate pollen grains, whereas we found that the species B. pentandra and B. vespertilio both have apertures of the colpus type.Vaz (2001) carried out a taxonomic study of 58 species of Bauhinia sect.Pauletia, native to or cultivated in Brazil.Among those taxa, the author analyzed the pollen grains of seven species, six of which were also analyzed here.The author divided the species into five pollen types (type B. acuminata, type B. catingae, type B. pentandra, type B. ungulata, and type B. tarapotensis).Vaz (2001) defined the pollen types mainly on the basis of the classification of the aperture, amb and shape of pollen grains.Unfortunately, we could not accept the pollen types established by Vaz (2001), due to the presence of important characters related to muri that were not examined by those authors.Vaz & Tozzi (2003) considered the series Cansenia to be well-defined and recognizable by the type of terminal aphyllous inflorescence, called pseudo-raceme with foliaceous bracts, as well as the floral bracts and bracteoles associated with pollen grains of type 3-(4)-colpate, angulaperturate.The pollen morphology described in the present study differs significantly for some species, especially as regards the type of supratectal elements, shape of the reticulum, and type of the muri.We note that those authors included B. cheilantha in the series Cansenia, although that species was previously assigned to another group by Bentham (1870).Our results show that the pollen of B. cheilantha shares many features with other species of the series Cansenia, although it was the only member of the series that had apertures of the pore type.Also according to Vaz & Tozzi (2003), B. cupulata is often confused with B. longifolia, especially when they occur sympatrically.Our results show a large difference between those two taxa in terms of the pollen morphology.Bauhinia cupulata has, among other characters, pollen grains with a perforate sexine and without supratectal Acta bot.bras.27(2): 400-417.2013.elements; whereas B. longifolia has reticulate pollen grains with supratectal elements of the scabra and gemma types.These respective differences in pollen were also observed by Ferguson (1986).We believe that these characters can help discriminate between the two species.Santos et al. (2012) examined the pollen grains of 15 species of Bauhinia occurring in Caatinga areas of Bahia, some of them also analyzed here: B. aculeata, B. acuruana, B. bauhinioides, B. brevipes, B. cheilantha, B. dumosa, B. forficata, B. pentandra and B. subclavata.Comparing their results with our own, we found differences in the size of pollen grains of B. acuruana, B. bauhinioides, B. cheilantha and B. dumosa which those authors described as large and we classified as very large.The differences in the size of pollen grains might be related to the technique of preparation for light microscopy (10% KOH) used by Santos et al. (2012).The exine measurements, as well as the shape of the muri, also showed a large discrepancy because that author described the species with sinuous muri and the exine thicker than or the same as the nexine; whereas we observed that the pollen grains of the same species have straight muri, with the sexine thicker than the nexine only in B. aculeata.According to Santos et al. (2012), the number of apertures varies among B. aculeata, B. bauhinioides and B. brevipes.Although we did not observe such variation among those species, we did see similar variations among other species.This heteromorphism in the number of apertures in Bauhinia species has been reported by other investigators (Schmitz 1973;Gamerro & Fortunato 2001).Also according to Santos et al. (2012), the pollen grains of B. cheilantha, B. dumosa and B. subclavata have microreticulate exine, whereas we found the exines to be reticulate in those species.We obtained similar results in the classification of supratectal elements (gemmae, clavae and verrucae), although Santos et al. (2012) made no mention of the presence of bacula in any species, which were observed here in B. brevipes and B. subclavata.Regarding the type of aperture of the latter species, Santos et al. (2012) considered it to be the pore type but did not provide measurements.Vaz & Tozzi (2005), in making a synopsis of Bauhinia sect.Pauletia in Brazil, used palynological characters for the composition of key species.Our results differ from theirs for the series Cansenia, because we found no colpori in the species examined here.These authors indicated that B. acreana belongs to the B. forficata complex and is accepted by some authors as a synonym of this.Considering our observations of pollen reported here, we can conclude that these taxa are actually quite similar, confirming the taxonomic observations.The species can be separated, mainly by the fact that the sexine is microreticulate in B. forficata and reticulate in B. acreana, although they share other characters.In their taxonomic analysis of populations of B. affinis, B. catingae, B. albicans and B. aculeata, considering that these species occur in geographical areas that are not connected, Vaz & Tozzi (2005) decided to retain these as separate species, based on the characters of leaves and branches.Although the palynological characters indicate a close similarity between these species, it was possible to separate them based mainly on the shape, aperture number and sexine ornamentation.Vaz & Tozzi (2005) considered B. mollis a distinct species, isolated in the series Aculeatae by the shape of the bud, which has a clavate outline and a median-region constriction that divides the calyx into two parts.However, considering the pollen characters, the species is located well within the series Aculeatae in that it shares many characters with the other representatives, differing mainly with respect to heteromorphism of the number of apertures (6(7)-colpate).
Across palynological studies of B. forficata, there are discrepancies regarding the classification of the type of aperture: Leonhardt & Lorscheitter (2008) termed the aperture of B. forficata a colporus, whereas Barth & Bouzada (1964) stated that B. forficata has apertures of the pore type.The observations made in the present study indicate that the apertures of B. forficata should be classified as colpi.
The present study has established that representatives of the series Aculeatae, Cansenia, Pentandrae and Perlebia differ from those of the series Acuminatae mainly in that Acuminatae representatives are inaperturate, whereas the taxa within the series Cansenia were heterogeneous (B.cheilantha was the only species that had apertures of the pore type).Knowledge of pollen characters is important to improving the taxonomic organization of the genus Bauhinia.

Table 3 .
Mean (μm)of the measurements of aperture, exine layers and ornamentation elements of pollen grains of Bauhinia species (n = 10).

Table 4 .
Number of elements in an area of 50 μm 2 of the surface of the pollen grains in polar view and sexine ornamentation of Bauhinia species, by series.