Pollen of Bauhinia L. and Phanera Lour. (Leguminosae-Caesalpinioideae) from the Brazilian Caatinga

A study of pollen morphology, in species of the genera Bauhinia and Phanera (Leguminosae-Caesalpinioideae) which occur in the Brazilian caatinga, is presented. The pollen was examined using light and scanning electron microscopy. Samples were prepared by the KOH method; non-treated pollen grains were examined by scanning electron microscopy. Pollen grains in Bauhinia species (11) are colpate, but porate in B. cheilantha and B. subclavata. All four species of Phanera present colporate pollen grains; P. outimouta is the only to have syncolporate pollen grains. Species of both genera present heteromorphism in the number of apertures. Excepting P. outimouta whose pollen grains are psilate, all species in the two genera present supratectal processes (gemmae/clavae), however the exine in the two genera are dif-ferent-semitectate, reticulate in Bauhinia species, and tectate, rugulate (or psilate) in Phanera ones. Pollen characters support the segregation of Phanera from Bauhinia .


Introduction
The family Leguminosae includes about 727 genera and 19,325 species, with cosmopolitan distribution and enormous economic importance [1].It is one of the most diverse families in most neotropical vegetation types, especially in the seasonally dry tropical forests [2].It presents great morphological variation in both macro-and micro-morphological traits, including characters derived from pollen grains [1].The subfamily Caesalpinioideae is a paraphyletic grade of 171 genera and approximately 2250 species from which derived the monophyletic subfamilies Mimosoideae and Papilionoideae.The Caesapinioideae are currently divided into four tribes: Cercideae, Detarieae, Cassieae, and Caesalpinieae [1].
Pollen of the most Leguminosae species is characterised as monad, tricolporate and reticulate; this is a very common pollen type among more derived angiosperms [9].However, dyads, tetrads, and polyads with varied numbers of pollen grains can be found among different genera of the family [9,10].Caesalpinioideae pollen grains follow the general pattern of pollen morphology in the family, with single pollen grains [9].
The pollen of Bauhinia species has a tectate exine and is usually shed in monads.The tectum may present a (micro-)reticulate, striate, verrucate, or rugulate surface or a combination of these ornamentation features [11].According to some authors [11][12][13], the more notable pollen characters of the genus Bauhinia are the gemmae and verrucae in several sizes and patterns of distribution on the tectum, in addition to the organisation of the reticulum.
Ferguson and Banks [13] cite the occurrence of tetrahedral tetrads in Bauhinia, with perforate tectum and small verrucae in the contact walls of the pollen grains.Such tetrads occur in only two species of the genus, B. phoenicea G. Don and B. pottsii Heyne; tetrads can be considered to be a derived character in the genus, because of their limited occurrence [13].
As described above, previous pollen studies in the genus Bauhinia (s.l.) disclose great variation in the pollen morphological features: ornamentation, apertures, and units of dispersal.Thus, Bauhinia is a eurypalynous taxon.This morphological diversity makes it possible to utilise pollen characters in phylogenetic and taxonomic studies of the genus.
This paper presents a survey of the pollen of the species of Bauhinia and Phanera native to the caatinga biome in northeastern Brazil, aiming to assess the implications of pollen morphology for the taxonomy of the group.

Material and Methods
The inventory of Bauhinia and Phanera species that occur in the caatinga was based on the work of [8].All species cited by that author were surveyed, except for B. dubia G. Don, for which suitable material was not available.Specimens were taken from the collections of the ALCB, CEPEC, HRB, and HUEFS herbaria, acronyms according [14] (Table 1).Whenever possible, the polliniferous material was taken from specimens collected in caatinga vegetation.
Polliniferous material (floral buds and flowers) was prepared for light microscopy (LM) according to the KOH method of Faegri and Iversen [15].The traditional acetolysis method of Erdtman [16] was not used, due to the fragility of the exine found in some species.Pollen slide preparations have been incorporated into the Palynotheca of the Universidade Estadual de Feira de Santana (PUEFS).
Whenever possible, three specimens of each species were studied.For each specimen, measurements of polar and equatorial diameter (in equatorial view) or equatorial diameter (in polar view) were taken from 25 pollen grains.In addition, the thickness of the exine and the height and width of ten gemmae were measured from ten pollen grains in each specimen.The ratio between sexine and nexine thickness was assessed qualitatively.
Untreated pollen grains for scanning electron microscopy (SEM) were taken from the anthers and put on sticky carbon tape on a stub.The prepared stubs were coated with gold using an SCD 050 sputter coater.The pollen grains were examined with a LEO 1430 VP JSM microscope, and images were digitally recorded.
Descriptions of pollen grains follow the terminology of Punt et al. [17].Pollen size classes follow Erdtman [18].

General Pollen Morphology
The pollen grains of the caatinga species of Bauhinia and Phanera are medium to very large in size (medium = 25 -50 µm; large = 50 -100 µm; very large ≥ 100 µm) and peroblate to subprolate in shape.The amb is circular (Figure 6 Pollen grains of Phanera species are smaller than those of Bauhinia, not exceeding 80 µm in P. trichosepala, whose pollen grains are the largest in the genus.Among Bauhinia species, pollen grain diameter can reach more than 160 µm in B. pentandra (Table 2).Most species have angulaperturate pollen grains (e.g.Figures 1(B),  (F  Heteromorphism in pollen aperture number is apparent in some species.The ornamentation of the exine surface presents several patterns: psilate, rugulate, and (micro-) reticulate, with gemmiform and/or claviform supratectal processes (Figures 1-6).

Apertures
The basic number of apertures varies widely, from three (B.In pollen grains of P. outimouta, the columellae layer is not seen under LM (Figure 6 Ectexine surfaces display in three basic ornamentation patterns among Bauhinia and Phanera species: 1) (micro-)reticulate, with supratectal processes (mainly gemmae and clavae)-this pattern is present on pollen grains of Bauhinia species (e.g.Figures 1(A), (C), (G

Exine Structure and Surface
3) rugulate and gemmate-this pattern is restricted to other species of Phanera (P.flexuosa, P. microstachya, and P. trichosepala), in which the tectum is irregularly rugulate with gemmae in different sizes (Table 2
Phanera trichosepala L.P. Queiroz (Figures 6(H)-(L)) -Pollen grains large, 3(4)-syncolporate, angulaperturate, suboblate to oblate-spheroidal; amb circular to subtriangular; ectoapertures long, with diffuse outline, with a slightly granulate membrane; endoapertures circular, costate, narrower than the ectoapertures; exine rugulate, with gemmae bordering the ectoapertures; columellae diffuse; exine a little thicker than the nexine.The size range of pollen grains recorded here is consistent with that published by previous authors who have studied Bauhinia s.l.pollen grains [19][20][21][22][23]. Size differences among pollen grains are common within individuals of the same species (see Table 2).In both Bauhinia and Phanera, size is not a conservative pollen character at the species level.Aperture type is a good character to unite species within both genera; apertures are colporate in Phanera species and colpate or porate in Bauhinia species.

Apertures
Variation in the number of apertures in different species of Bauhinia and Phanera was first recorded by Vishnu-Mittre and Sharma [24], who described pollen types in Indian Bauhinia s.l.species ranging from inaperturate to 3-aperturate.Larsen [25] described pollen grains of Thai species ranging from inaperturate to 3-5-aperturate, and Ferguson and Pearce [21] found 3-7-zonoaperturate pollen grains in Neotropical species.Heteromorphism in aperture number has also been recorded in Bauhinia s.l.

Exine
The presence of verrucae, gemmae, and clavae as elements of the ornamentation of the exine is a noticeable characteristic of the pollen morphology of the studied species of Bauhinia and Phanera.Reticulation of the exine supporting gemmiform or claviform processes has been recorded by authors who have studied the pollen grains of Bauhinia s.l.species [12,19,20,22,23].However, these pollen features are not exclusive to Bauhinia.Thus, this ornamentation pattern is of limited utility at higher taxonomic levels among the Caesalpinioideae, within which it has appeared several times [11,26].
According to [12], the general features of the of the pollen exine of Bauhinia s.l., together with the aperture patterns, place the genus as one of the most specialised within the subfamily Caesalpinioideae.However, most phylogenetic studies of the Leguminosae support the tribe Cercideae as the first diverging clade within the family [e.g., 3,27].Hence, such specialised features of the exine should be regarded as recent acquisitions of Bauhinia s.l.within the Cercideae, rather than as ancient features of Leguminosae pollen grains.
All species of Bauhinia from caatinga present large flowers with white petals, nocturnal anthesis, and nectar produced inside a cylindrical-tubular hypanthium [8].These features match the criteria presented by Faegri and van der Pijl [28] for the chiropterophily syndrome.Indeed, Arroyo [29] has reported that species of Bauhinia with such features are largely pollinated by bats.This has been subsequently confirmed by other pollination studies [30,31].On the other hand, the caatinga species of Phanera have much smaller flowers with matutinal anthesis and a wider, campanulate hypanthium [8], and are probably pollinated by bees.Large supratectal processes in caesalpinioid legumes have been reported for the Amazonian genera Dicymbe Spruce ex Benth., Eperua Aubl., and Paloue Aubl.(tribe Detarieae) [11,32].Species of these genera are largely pollinated by bats [29], which may suggest an association between this kind of ornamentation and adaptation to bat pollination.A corollary of this hypothesis is that the bee-pollinated genus Phanera, which largely presents the same supratectal processes, is derived from bat-pollinated ancestors and has retained this kind of ornamentation due to phylogenetic constraint.This intuitive hypothesis could be tested by a phylogenetic study of the Cercideae with a wide sampling of bee-and bat-pollinated species.

Taxonomic Implications
According to Vaz's classification [6], the Bauhinia species from caatinga are classified in the section Pauletia Series Pentandrae is characterised by 3-7-colporate pollen [4].However, the results presented here show 5(4, 6)-colpate pollen grains, which suggests a closer relationship with B. bauhinioides (series Perlebia).A close relationship between B. bauhinioides and B. pentandra has been proposed previously by [21], who grouped these species in a taxonomic complex called the "aculeata/ perlebia/pentandra-Alliance".According to these authors, this group is formed of species with 5-7-colpate pollen grains, which would include B. cacovia and B. catingae as reported here.The authors, however, included in this group Bauhinia cheilantha, which is characterised here by 3-colpate pollen grains.
Another group formed by Ferguson and Pearce [21] on the basis of pollen morphology is called the "Cansenia-Alliance"; it includes the species B. acuruana, B. dubia, and B. brevipes.All species in this group were described as having pollen morphology consistent with the results presented here: pollen grains large, 3-colpate, microreticulate and with gemmae and/or clavae.
Vaz and Tozzi [33,34] proposed the new Aculeatae, segregated from the series Cansenia sensu [4].This series includes the caatinga species B. cacovia, B. catingae, and B. forficata.These species form a homogeneous group based on pollen features.B. catingae pollen differs from that of the other two species mainly by of aperture number heteromorphism.
The surveyed species of the genus Phanera from caatinga belong to the sections Caulotretus (P.outimuota) and Schnella (P.flexuosa, P. microstachya, and P. trichosepala) in the classification of [4] for Bauhinia s.l.Species of this genus present exines with different ornamentation features.In the species formerly included in Bauhinia sect.Schnella, now placed in Phanera, the exine is rugulate with gemmae on the surface.However, the patterns of distribution of the supratectal processes differ between P. trichosepala and the group formed by P. flexuosa and P. microstachya.In the former species, gemmae are distributed only around the edges of the ectoapertures; in the other two, they are distributed heterogeneously on the surface.Ferguson and Pearce [21], studying New World species of Phanera (as Bauhinia s.l.), recorded pollen grains similar to those of sect.Schnella.Those authors also analyzed Phanera microstachya (as Bauhinia s.l.).
Phanera outimouta differs from the remaining caatinga species of Phanera by its psilate exine without supratectal processes.This species was classified in Bauhinia sect.Caulotretus by [4].There are no previous records for P. outimouta (= B. outimouta) in the pollen literature.The pollen features presented by P. outimouta match those reported by [21] for the "Caulotetrus-group": pollen grains that vary from prolate-spheroidal to subprolate, ectoapertures with length generally about twothirds of the polar axis, and characteristically circular endoapertures.The non-observation of the columellae layer under LM may indicate the presence of a granular infratectum, also cited by [12,21] as characteristic of the group.

Conclusions
Many taxa of tribe Cercideae (subfamily Caesalpinioideae), including Bauhinia and Phanera, have been the subject of pollen studies in relation to taxonomy and systematics.Their characteristically gemmate exine has assumed an important role to pollen feature of both genera-even when they were merged into Bauhinia.We believe that pollen studies for the complete set of species of Bauhinia, including related genera such as Phanera, would provide secure data from which to assess the taxonomic relationship among genera and, furthermore, the relationship of pollen morphology and the pattern of pollination which has a great range of vectors, including bees, butterflies, birds and bats [35,36].
Different positions about the taxonomy of Bauhinia s.l. are summarised in the revisionary works of [4] and [5].Wunderlin et al. [4] adopt a wider circumscription of Bauhinia and divide this genus into 4 subgenera and 37 subsections and series.On the other hand, Lewis and Forest [5] split Bauhinia into nine different genera, of which Bauhinia and Phanera occur in the New World.Our results reinforce the distinction between Bauhinia and Phanera by the different aperture patterns found in the two groups: all surveyed species of Phanera present colporate apertures, while Bauhinia species have porate or colpate apertures.
Besides pollen data from Bauhinia and Phanera species will be helpful to studies on pollen dispersion by vectors including bats and hummingbirds in caatinga areas, where that scope of study is rare.