Leaf morphoanatomy of "mororó" (Bauhinia and Schnella, Fabaceae)

Abstract

Bauhinia L. and Schnella (Raddi.) Wund. are popularly known in Brazil as "mororó". The leaves and stem bark are used in folk medicine for various purposes, especially against diabetes. Morphoanatomical studies of the leaves of Bauhinia cheilantha (Bong.) Steud., B. pentandra (Bong.) Steud., B. ungulata L. and Schnella outimouta (Aublet) Wund., tribe Cercidae, subtribe Bauhiniinae (Benth.) Walp., were carried out as subsidies to the quality control of their etnodrugs and their derivatives, as well as an additional support to their taxonomy. The morphological and anatomical studies employed traditional techniques of stereo- and light microscopy. All species showed bifoliate leaves, a dorsiventral mesophyll, epidermis with a papillose abaxial surface, anomocytic stomata at the level of the epidermis, and tector trichomes. Schnella outimouta showed leaf characters distinctive from the three species of Bauhinia: indument puberulous on the abaxial surface, leaves hypostomatic, midrib with two collateral bundles, and a cylindrical petiole. The species of Bauhinia have a sericeous-pubescent indument, amphistomatic leaves with boat-shaped glands, midrib with a single bundle, and a canaliculate petiole with lateral projections. Our results provide leaf morphological and anatomical parameters, useful to distinguish the four species studied, which support the quality control of its ethnodrugs.

Keywords:
Bauhiniinae; Cercideae; Caesalpinioideae; Leguminosae; Pharmacobotany; Plant anatomy

1. Introduction

Bauhinia L. and Schnella (Raddi.) Wund. both belong to the subtribe Bauhiniinae (Benth.) Walp. of the tribe Cercideae Bronn (Caesalpinioideae), with pantropical and neotropical distributions respectively. They grow as trees, shrubs, and lianas with simple tendrils, with or without intrastipular spines, having seeds with a crescentic hilum and funicular aril lobes (Wunderlin, 2010a). Bauhinia and Schnella demonstrate morphological uniformity of their vegetative organs, making it difficult to identify sterile specimens.

Bauhinia and Schnella species are popularly known in Brasil, as "mororó", "miroró", and "pata-de-vaca" (Agra et al., 2007). The leaves and stem bark of Bauhina species are used in folk medicine for various purposes, especially against diabetes (Agra et al., 2007, 2008).

Bauhinia is considered the most complex genus of the tribe Cercideae (Legume Phylogeny Working Group [LPWG], 2013), with approximately 160 species distributed in the tropical and subtropical regions of Asia, Africa, Australia, and Central and South America (Duarte-Almeida et al., 2015). A total of 61 species occur in Brazil, of which 39 are endemic (Vaz, 2015). The distinctive characteristics of the genus are its tree or shrub habit (rarely semi-scandent), sometimes with intrastipular spines, rarely with thorns, never with tendrils, calyx spathaceous or dividing the hypanthium into 2–5 lobes (Wunderlin, 2010a, 2010b). The genus shows great phenotypic plasticity, and different taxonomic treatments have been proposed: Bentham (1865) and Wunderlin (1976, 1983, 2010a, 2010b).

Schnella was proposed by Raddi (1820), which was revalidated as a generic status by Wunderlin (2010a, 2010b) based on the molecular analyses of Hao et al. (2003) and Sinou et al. (2009). Schnella is Neotropical, with about 47 species distributed from Mexico to Argentina (Trethowan et al., 2015), with its center of diversity in Brazil (35 species, of which fourteen are endemic), according to Vaz (2015).

Interest in Bauhinia has intensified due to its reported anti-diabetic activity, especially in light of studies of B. forficata Link by Cechinel-Filho (2009), Menezes et al. (2007), and Silva and Cechinel-Filho (2002). Other potential medical uses of Bauhinia have been reported, including four treating ulcers (Silva and Cechinel-Filho, 2002), and its utility as an anti-oxidant (Braca et al.,2001; Pandey et al., 2011), anti-inflammatory, analgesic, and anti-pyretic (Gupta et al., 2005).

Metcalfe and Chalk (1979) noted that anatomical studies have great value in establishing the identities of herbaria specimens, especially with sterile material. Anatomical data have been shown to lend additional support to systematics in many taxonomic groups, acting as important criteria for interspecific and infrageneric delimitations in Solanum L. (Araújo et al., 2010; Nurit-Silva and Agra, 2011; Nurit-Silva et al., 2012; Sampaio et al., 2014), Ficus L. (Araújo et al., 2014), and Bauhinia (Rezende and Cardoso, 1994; Duarte and Debur, 2003; Lusa and Bona, 2009; Albert and Sharma, 2013), and alsco can contribute to the quality control of medicinal plants (Araújo et al., 2014; Porto et al., 2016).

This study therefore sought to characterize the leaf morphological anatomy of Bauhinia cheilantha (Bong.) Steud., B. pentandra (Bong.) Steud., B. ungulata L. and Schnella outimouta (Aublet) Wund., commonly confused in the Brazilian Northeast region, to identify distinctive characters among these species, which can provide additional support to their taxonomy, as well as to the quality control of their etnodrugs and their derivatives.

2. Materials and methods

Morphological studies, identifications, collections, and field work

The identifications of the Bauhinia species were made through analyses of their reproductive and vegetative organs based on the specialized literature (Fortunato, 1986; Vaz and Tozzi, 2003a, 2005; Wunderlin, 2010a, 2010b). Additionally, comparative studies were carried out with specimens identified by specialists from collections at the Prof. Jayme de Moraes Coelho (EAN) and Prof. Lauro Pires Xavier (JPB) herbaria, both at the Federal University of Paraíba.

Botanical expeditions and field observations were conducted in Paraiba State, Brazil, to collect samples of Bauhinia cheilantha and Schnella outimouta (see Material examined). Fertile reference specimens were herborized following Bridson and Forman (1999) and were deposited in the EAN herbarium, with duplicates assigned to the JPB herbarium. Other materials were fixed in percent FAA (formalin-acetic acid-alcohol) for 48 h, and subsequently preserved in 70% alcohol (Johansen, 1940). Additional samples of dried materials of Bauhinia pentandra and B. ungulata (see Material examined) were rehydrated and used in anatomical studies. Leaf terminolo-gy was based on Van der Pijl (1952) and Lin et al. (2015). Indumentum classification follows Harris and Harris (2001).

Material examined

Bauhinia cheilantha: Brazil, Ceará: Poranga, 04°46′04″ S – 40°52′58″ W, Félix 14960 (EAN); Paraíba: Alagoa Grande, Rua Nova, 25-VIII-2015, Pereira 06 (EAN, JPB); Cabaceiras, Sítio Maniçoba, VI-2015, Pereira 04 (EAN, JPB); Campina Grande, INSA, 13-III-2012, Albuquerque & Ferraz sn (EAN, JPB); Esperança, Lagoa de Pedra, 17-VI-2003, Pitrez & Trajano 274 (EAN); Fagundes, Estrada para a Pedra de Santo Antônio, 21-V-15, Pereira et al., 03 (EAN, JPB); Itapororoca, Fazenda Macacos, 25-VI-2011, Félix 13600 (EAN, JPB); Mulungu, 25-VIII-2015, Pereira et al., 07 (EAN, JPB); Santa Terezinha, 18-IV-2006, Pegado & Félix 16 (EAN, JPB); São João Tigre, 24-II-2011, Félix 13477 (EAN, JPB); Sossego, Sítio São Miguel, 24-VI-2015, Pereira et al., 05 (EAN, JPB); Sousa, Sítio Lamarão, Estrada de acesso a São José da Lagoa Tapada, 27-V-1995, Moreira 25 (EAN, JPB).

Bauhinia pentandra: Brazil. Paraíba: Itaporanga, Caminho para a Serra Água Branca, 1993, Rocha et al., 1695 (JPB); Pombal, Fazenda Nova Canaã, 18-I-1952, Carneiro 1650 (JPB); Sousa Sítio Lamarão, 27-V-1995, Moreira 23 (JPB); Sousa, Fazenda Jangada, 17-IV-98, Gadelha Neto 424 (JPB); Sousa, Fazenda Jangada, IX-93, Gadelha Neto 61 (JPB); Sousa, Sítio Lamarão, Estrada de acesso São José da Lagoa Tapada, 06-IX-1994, Moreira 7 (JPB).

Bauhinia ungulata: Brazil, Ceará: Poranga, 04°44′49″S – 40°52′11″W, Félix 14947 (EAN).

Schnella outimouta: Brazil, Paraíba: Areia, Estrada para o Sítio Mineiro, 16-I-2015, Pereira 01 (EAN); Areia, Estrada para Pilões, 01-XII-2015, Félix et al. sn (EAN).

Anatomical studies

Leaf samples from the second to fifth nodes were used in the anatomical studies. Paradermic sections of the adaxial and abaxial surfaces, and transverse sections were performed on leaves by free hand using commercial razor blades. Transverse sections were made with adult leaves of the leaf blades, petioles, and pulvini.

All sections were cleared using 2% sodium hypochlorite, rinsed in distilled water, and neutralized with 1% acetic acid. The paradermic sections were stained with Safranin with 1% solution in 50% alcohol, according to Franklin (1945). The transverse cross sections were stained with Astra blue and Safranin, modified by Bukatsch (1972).

The sections were mounted under coverslips with glycerol (50%) and subsequently analyzed and photomicrographed using a Qwin system and video camera (Leica ICC50 HD) coupled to an optical microscope (Leica DM 750) for capturing images.

Characterizations of the cell walls of the epidermis and mesophyll are based on Fahn (1990). Classifications of the stomata follow Metcalfe and Chalk (1979), while leaf venation patterns follow Hickey (1973).

3. Results

Leaf morphology

All of the species of Bauhinia (B. cheilantha, B. pentandra, B. ungulata) and S. outimouta studied showed alternate and bilobed leaves with fused lobes and entire margins. The leaves of all of the Bauhinia species observed had chartaceous consistencies, while those of S. outimouta were coriaceous. Oval-oblong leaf blades were predominant, except in B. pentandra, which showed somewhat hastate leaves, with open and acute divaricate lobes.

All of the species showed symmetrical or slightly asymmetrical bilobed laminas. The apex is bifid to ¼ in B. cheilantha, ½ in B. pentandra, ¼ B. ungulata, and ¾ in S. outimouta. The base is cordate in B. cheilanta and S. outimouta, truncate in B. ungulata, and somewhat cordate-hastate in B. pentandra.

The adaxial leaf surface is glabrous in B. cheilantha, B. pentandra, and S. outimouta (Fig. 1A, C, G 3), and glabrescent in B. ungulata, with small trichomes on the midrib (Fig. 1E). All Bauhinia species show navicular glands occur on the abaxial surface, and the indument is sericeous-pubescent with simple and multicelled trichomes (Fig. 1B, D, F). The indument is puberulous-ferruginous with short, simple, eglandular trichomes; navicular glands are absent in S. outimouta (Fig. 1H).

Figure 1
In frontal view. (A and B) Bauhinia cheilantha, adaxial and abaxial surfaces; (C and D) Bauhinia pentandra: adaxial and abaxial surfaces; (E and F) Bauhinia ungulata: adaxial and abaxial surfaces; Schnella outimouta: (G and H) adaxial and abaxial surfaces.

Figure 2
Insertion of the proximal and distal pulvinus, and motile region. (A–C) Bauhinia cheilantha; (D–F) Bauhinia pentandra; (G–I) Bauhinia ungulata. (J–L) Schnella outimouta. Legend: (arrow) axillary bud. (st) stipule. (en) extrafloral nectary.

Figure 3
Epidermis on the adaxial (left side) and abaxial (right side) surfaces. (A and B) Bauhinia cheilantha; (C and D) Bauhinia pentandra. (E and F) Bauhinia ungulata. (G and H) Schnella outimouta. Legends: (am) anomocytic; (an) anisocytic; (pa) paracytic; (tsc) trichome scar.

The petiole is canaliculate and pubescent, with both simple, unicellular and multicellular trichomes, with navicular glands in B. cheilantha, B. pentandra, and B. ungulata. However, S. outimouta has a cylindrical and puberulent petiole, with short, ferruginous trichomes.

Two pulvini were observed on the petioles of all of species: one proximal and inserted on the stem, and the other distal and inserted at the base of the leaf blade. A motile cushion is present, from which emerge 9 to 13 main veins that are palminervous in Bauhinia, and acrodromous in S. outimouta. B. cheilantha showed axillary gemma at the base of the proximal pulvinus (Fig. 2A). The pulvinus of B. pentandra is inserted on the stem, between the geminate aculeus and the axillary gemma (Fig. 2D). A linear stipule and an extrafloral glandular nectary were observed at the base of the pulvinus in B. ungulata (Fig. 2G). The proximal pulvinus of S. outimouta is inserted between a pair of rounded stipules (Fig. 2J).

All species showed a motile cushion at the apex of the distal pulvinus, this being prominent and somewhat rounded on the adaxial surface of B. cheilantha (Fig. 2B), with a central groove on the abaxial surface (Fig. 2C). The motile cushion of B. pentandra is semicircular on the adaxial surface (Fig. 2E), but inconspicuous on the abaxial face (Fig. 2F). The motile region of B. ungulata is large-elliptical on the adaxial (Fig. 2H) and inflated on the abaxial surface (Fig. 2I). S. outimouta demonstrated circular motile cushions on the adaxial surface (Fig. 2K) that were inconspicuous on the abaxial surface (Fig. 2L).

Leaf anatomy

Bauhinia cheilantha (Fig. 3A), B. pentandra (Fig. 3C), and B. ungulata (Fig. 3E) showed straight, polygonal, anticlinal epidermal walls, slightly curved on the adaxial surface in front view, but sinuous in S. outimouta (Fig. 3G). The epidermis on the abaxial surface of B. cheilantha and B. pentandra showed sinuous anticlinal walls (Fig. 3B, D), but curved and somewhat papillose walls in B. ungulata (Fig. 3F), with elongated papillae in S. outimouta (Fig. 3H). All species showed a uniseriate and papillose leaf epidermis on the abaxial surface in cross-section (Fig. 4A, C, E, G), with a thick cuticle in B. cheilantha and B. ungulata, but a thin cuticle in B. pentandra and S. outimouta.

Figure 4
Mesophyll and leaf blade margins: (A and B) Bauhinia cheilantha; (C and D) Bauhinia pentandra; (E and F) Bauhinia ungulata; (G and H) Schnella outimouta. Legends: (arrow) Druse, (*) prismatic crystals, (ep) epidermis, (pp) palisade parenchyma, (sp) spongy parenchyma, (sc) sclerenchyma.

The species of Bauhinia studied showed amphistomatic leaves with both anomocytic and anisocytic stomata occurring on both surfaces (Fig. 3A– F). Paracitic type stomata were also observed on both surfaces of B. pentandra (Fig. 3C) and B. ungulata (Fig. 3F). S. outimouta demonstrated a hypostomatic pattern, with anisocytic and anomocytic stomata (Fig. 3H). The stomata of the four species were at the level of epidermis.

The mesophyll of B. ungulata is dorsiventral in cross section, with unisseriate palisade parenchyma in (Fig. 4E), being biseriate in the other species (Fig. 4A, C, G), with drusiferous idioblasts often being observed (Fig. 4C). The spongy parenchyma showed 2–4-layers in B. cheilantha and B. pentandra (Fig. 4A–C), with smaller cells than the other species and small intercellular spaces. B. ungulata and S. outimouta show 4–5-seriate spongy parenchyma, with large intercellular spaces and more elongated cells, tending toward braciform (Fig. 4E, G). Idioblasts of prismatic crystals were observed in the vascular systems of secondary bundles, mainly in B. pentandra (Fig. 4D).

The leaf margins appeared as very distinctive characteristics among the four species, being: obtuse and slightly reflexed in B. cheilantha, (Fig. 4B); obtuse with a collateral vascular bundle with a sclerenchymal sheath in B. pentandra (Fig. 4D); acute in B. ungulata (Fig. 4F); rounded in S. outimouta (Fig. 4H); filled by sclerenchyma in B. pentandra and B. ungulata.

The midrib of Bahuinia is plane-convex in cross section, with collateral vascular bundles that are delimited by two strands of sclerenchyma, with a crystalipherous sheath and a collenchymatous cortex with sparse drusiferous idioblasts. The median portion of the midrib of B. cheilantha has a central arc-shaped vascular bundle (Fig. 5A), V-shaped in B. pentandra (Fig. 5B), and U-shaped in B. ungulata (Fig. 5C). The median portion of the midrib of S. outimouta is concave-convex, with a cortex of angular collenchyma and drusiferous idioblasts; two semicircular vascular bundles were observed in the central portion (the main vascular bundle being amphicrival while the accessory bundle is collateral), with sclerenchymal sheaths surrounding both (Fig. 5D).

Figure 5
(A) Bauhinia cheilantha; (B) Bauhinia pentandra; (C) Bauhinia ungulata; (D) Schnella outimouta. Legends: (cl) angular collenchyma; (sc) schlerenchyma; (ph) phloem, (xy) xylem.

The proximal pulvinus was circular to subcircular in cross section in all of the species studied (Fig. 6A, D, G, J), with a conspicuous cortex and a sheath of drusiferous idioblasts surrounding the collenchyma, external to the vascular system (Fig. 7A). There were large numbers of cells near the petiole in the process of lignification. B. cheilantha has two vascular bundle systems: one larger and partially concentric cortical bundle, and a smaller medullar bundle, both collateral (Fig. 6A). The vascular system of B. pentandra showed only one vascular bundle, rounded to semicircular, bicollateral, surrounded by 3–5 thin layers of collenchyma (Figs. 6D and 7A). Two central vascular bundles were observed in B. ungulata, a larger amphicribal bundle surrounded by 3–5 layers of collenchyma, and a minor medullar collateral bundle (Fig. 6G). The vascular system of the proximal pulvinus in S. outimouta showed a discontinuous ring of colateral bundles surrounded by a collenchyma sheath, while the medullar region showed a bicollateral bundle (Fig. 6J).

Figure 6
Vascularization of the middle portion of the proximal (left) and distal (middle) pulvinus, and petiole (right). (A–C) Bauhinia cheilantha; (D–F) Bauhinia pentandra; (G–I) Bauhinia ungulata; (J–L) Schnella outimouta.

Figure 7
Details of the pulvinus and petiole: (A) Proximal pulvinus of Bauhinia pentandra; (B) distal pulvinus of Schnella outimouta, with druse idioblasts; (C) petiole of B. pentandra; (D) petiole of S. outimouta. Legends: (cl) collenchyma; (xy) xylem; (ph) phloem; (sc).Sclerenchyma: (*) druse; (arrow) crystals.

The distal pulvinus was circular in cross-section in all of the species studied, although sometimes slightly compressed on one surface. The parenchymatic cortex is conspicuous, with a sheath of drusiferous idioblasts surrounding the vascular system (Fig. 7B). The proximal pulvinus showed four parallel collateral bundles, and was wrapped with collenchyma (Fig. 6B, E, H, K). Smaller, central bundles have phloem facing to the cortex of the major bundles.

The median portion of the petiole is somewhat U-shaped in cross-section in all Bauhinia species (B. cheilantha [Fig. 6C], B. pentandra [Fig. 6F], and B. ungulata [Fig. 6I]) due to two lateral projections on the adaxial surface; the petiole of S. outimouta is rounded (Fig. 6L). The cortex of all of the species is filled with parenchyma, with areas of collenchymatic tissue (Fig. 7D) and drusiferous idioblasts (Fig. 7A).

The vascular system is surrounded by a sclerenchymatic ring, which is delimited by a crystaliferous sheath (Fig. 7C). The vascular system in B. cheilantha is formed by four bundles, two of them being central with a larger amphicrival bundle in the cortical portion; a smaller amphivasal bundle is present in the medullary portion. There are also two accessory collateral bundles that can be seen in lateral projections (Fig. 6C). B. pentandra has three bundles, two collateral in lateral projections, with a large amphicrival bundle occupying most of the cortex (Fig. 6F). B. ungulata showed four bundles, two of which are amphicrival, parallel to the central region of the cortex, and two are collateral in lateral projections (Fig. 6I). The arrangements of the central vascular bundles of S. outimouta are similar to those observed in B. cheilantha (Fig. 6L). The distinctive morphological and anatomical characters of B. cheilantha, B. pentandra, B. ungulata and S. outimouta are presented in Boxes 1 and 2.

Box 1
Morphological characters of Bauhinia and Schnella species.

Leaf characters		Species
		B. cheilantha	B. pentandra	B. ungulata	S. outimouta
Leaf blade	Consistency	Chartaceous	Chartaceous	Chartaceous	Leathery
	Shape	Oval-oblong	Cordate-hastate	Oval-oblong	Oval-oblong
	Level of bifidness of the leaf blade	1/4	1/2	1/4	3/4
	Base of blade	Cordate	Cordate-hastate	Truncate	Cordate
	Apex of blade	Rotund	Acute	Acute	Acute
	Abaxial indument	Sericeous-pubescent	Sericeous-pubescent	Sericeous-pubescent	Puberulous
	Glands on the abaxial surface	Present	Present	Present	Absent
	Venation	Palminervous	Palminervous	Palminervous	Acrodromous
Petiole and pulvinus	Petiole shape	Canaliculate	Canaliculate	Canaliculate	Cylindrical
	Insertion in the proximal pulvinus	Axilary gemma	Between geminate aculeos	Between extrafloral nectaries	Between rounded stipules
	Indument of motile region	Pubescent	Glabrous	Glabrous	Puberulous

Box 2
Anatomical characters of Bauhinia and Schnella species.

Character		Species
			B. cheilantha	B. pentandra	B. ungulata	S. outimouta
Leaf blade	Anticlinal walls	Adaxial	Straight-curved	Straight-curved	Straight-curved	Sinuous
		Abaxial	Sinuous	Sinuous	Curved	Curved
	Stomata	Distribution	Amphistomatic	Amphistomatic	Amphistomatic	Hypostomatic
		Type	Anomocytic Anisocytic	Anomocytic Anisocytic Paracytic	Anomocytic Anisocytic Paracytic	Anomocytic Anisocytic
	Margin	Shape	Obtuse and reflexed	Obtuse	Acute	Rounded
		Vascular bundle	1-collateral	1-collateral	Absent	Absent
		Presence of schlerenchyma	In the vascular bundle	In the vascular bundle	Filling the entire margin	Filling the entire margin
	Midrib	Shape	Plane-convex	Plane-convex	Plane-convex	Concave-convex
		Vascular bundles	1-collateral	1-collateral	1-collateral	2-amphicribal
Petiole and pulvinus	Proximal pulvinus	Vascular system	2-collateral	1-bicollateral	1-amphicribal + 1-collateral	colateral + 1-bicollateral
	Petiole	Shape	U-shaped	U-shaped	U-shaped	Circular
		Lateral bundles	2-lateral	2-lateral	2-lateral	Absent
		Collateral bundle	2-collateral	2-collateral	2-collateral	Absent

4. Discussion

Species of Bauhinia constitute a difficult group to identify considering only leaf morphology and, in the absence of fertile material, their morphologies are usually considered insufficient to support the identification of taxa (as was observed in the present work with fresh samples and herbarium specimens of B. cheilantha and B. ungulata). Their leaf shapes overlap and may exhibit transitional forms of mature and young leaves – so that collections of those species have become mixed, corroborating the observations of Wunderlin (1983).

Bauhinia cheilantha, B. pentandra, B. ungulata, and S. outimouta showed leaf patterns characteristic of tribe Cercideae (Chen and Zhang, 2005), although B. pentandra differed from the others by the shape of its leaves and by having navicular glands restricted to the leaf margins (corroborating the findings of Duarte-Almeida et al., 2015). The presence of navicular glands was a common character among the studied species of Bauhinia (B. cheilantha, B. pentandra and B. ungulata), and was noted by Metcalfe and Chalk (1979), differentiating them in relation to the genus Schnella. These observations are likewise consistent with those of Duarte-Almeida et al. (2015) who found that type of gland only in species of Bauhinia (and their absence in Schnella) in their analysis of 79 species of the Cercideae tribe using that character as a subsidy for the taxonomy of the group.

Pulvini are structures that aid in leaf flexibility and foliar movements (Rodrigues and Machado, 2006), especially in the motile region, when the articulation between the base of the leaf and the apex of the distal pulvinus is responsible for nictinastic movements (Vaz and Tozzi, 2003b, 2005; Rodrigues and Machado, 2006; Lusa and Bona, 2009). The shape of the distal pulvinus and the motile region were important characters that supported the delimitations of the four species studied here.

In terms of anatomical studies, analyses of the epidermis revealed a straight to curved pattern of the epidermises of species of Bauhinia, as was likewise reported for other species of the genus by Albert and Sharma (2013), Lusa and Bona (2009), and Rezende and Cardoso (1994). As that curved pattern is a common feature in species of that group, it was not useful as a diagnostic feature to separate the Bauhinia species studied here. It did differ from the pattern observed in S. outimouta (sinuous type) and that recorded for S. microstachya Raddy by Duarte and Debur (2003), thus serving as a distinctive feature for separating those genera.

An amphistomatic pattern of stomata distribution was common to the species of Bauhinia studied here, corroborating Metcalfe and Chalk (1979) who reported the stomata on the adaxial face as being sparse or restricted to the ribs; this same situation was recorded for B. blakeana Dunn by Albert and Sharma (2013), and for B. forficata Link. and B. variegata L. by Lusa and Bona (2009). The hypostomatic pattern reported by Albert and Sharma (2013) for B. tomentosa L., B. purpurea L., B. racemosa Lam., and B. malabarica Roxb. was not observed by us. The pattern of stomata distribution was hypostomatic in S. outimouta, and has also been reported for S. microstachya by Duarte and Debur (2003).

Anomocytic, anisocytic, and paracytic stomata were observed in all of the species studied. Those features were likewise previously reported for other species of Bauhinia and Schnella by Albert and Sharma (2013), Duarte and Debur (2003), Lusa and Bona (2009), and Rezende and Cardoso (1994), and corroborated by Metcalfe and Chalk (1979) who noted that different arrangements of subsidiary cells can be seen within the same species, or even a single leaf.

The dorsiventral mesophyll pattern observed in all of the species studied corroborated the dorsiventral pattern observed in other genera and species of the subfamily Caesalpinioideae (Meltcalfe and Chalk, 1979). The anatomy of the leaf margins, however, proved to be a distinctive character among the four species studied here, similar to reports for other species of Bauhinia (Lusa and Bona, 2009).

Regarding the anatomy of the pulvinus, the patterns observed here were similar to those reported by Rodrigues and Machado (2006) for the structures of the proximal pulvini of some species of the Leguminosae. Those structures are different in terms of the distributions of the vascular tissue observed in the Bauhinia species and S. outimouta studied here, but are not taxonomically informative – as the proximal pulvinus is consistently positioned in a transitional area between the stem and leaf tissues.

The canaliculate petiole shape, in cross-section, showed two vascularized lateral adaxial projections in species of Bauhinia, which was also reported for B. forficata and B. variegata (Lusa and Bona, 2009), and for B. curvula Benth (Rezende and Cardoso, 1994). The rounded shape observed in S. outimouta, on the other hand, is similar to that of S. microstachya (Duarte and Debur, 2003). The presence of a sclerenchymatous ring and a crystaliferous sheath surrounding the central vascular system in Bauhinia, also reported by Meltcalfe and Chalk (1979) was likewise observed in S. outimouta – and therefore does not constitute a differential character between the genera or species in the present work.

5. Conclusions

The morphology of the petiole, motile region, and the structures present in the insertion area of the pulvinus were one of most relevant to the separation of Bauhinia species from Schnella. The anatomy of the leaf epidermis and its auxiliary structures (stomata and navicular glands), as well as the petiole vascularization, were diagnostic for separating the species of Bauhinia and Schnella, characterizing them as distinct species.

Acknowledgements

We thank the curators of the EAN and JPB herbaria for the loans of herbarium specimens and for their support during our visits. We also thank Roy Funch for reviewing the English; the Coordenação de Aperfeiçoamento do Ensino Superior (CAPES) for the Master's scholarship awarded to L.B.S.P., and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for the doctoral scholarship awarded to R.C.S. and the PQ fellowship awarded to M.F.A.

References

• Agra, M.F., Baracho, G.S., Nurit, K., Basílio, I.J.L.D., Coelho, V.P.M., 2007. Medicinal and poisonous diversity of the flora of "Cariri Paraibano", Brazil. J. Ethnopharmacol. 111, 383-395.
• Agra, M.F., Silva, K.N., Basílio, I.J.L.D., Freitas, P.F., Barbosa-Filho, J.M., 2008. Survey of medicinal plants used in the region Northeast of Brazil. Rev. Bras. Farmacogn. 18, 472-508.
• Albert, S., Sharma, B., 2013. Comparative foliar micromorphological studies of some Bauhinia (Leguminosae) species. Turk. J. Bot. 37, 276-281.
• Araújo, N.D., Coelho, V.P.M., Agra, M.F., 2010. The pharmacobotanical comparative study of leaves of Solanum crinitum Lam., Solanum gomphodes Dunal and Solanum lycocarpum A. St-Hil, (Solanaceae). Rev. Bras. Farmacogn. 20, 666-674.
• Araújo, N.D., Coelho, V.P.M., Ventrella, M.C., Agra, M.F., 2014. Leaf anatomy and histochemistry of three species of Ficus sect. Americanae supported by light and electron microscopy. Microsc. Microanal. 20, 296-304.
• Bentham, G., 1865. Leguminnosae. In: Bentham, G., Hooker, J.D. (Eds.), Genera Plantarum. V. 1 pt 2. Reeve & Co., London, pp. 575–577, 460 pp.
• Braca, A., Tommasi, N., Bari, L., Pizza, C., Politi, M., Morelli, I., 2001. Antioxidant principles from Bauhinia tarapotensis J. Nat. Prod. 64, 892-895.
• Bridson, D., Forman, L., 1999. The Herbarium Handbook, 3rd ed. Royal Botanic Gardens, Kew Includes, Specimen storage.
• Bukatsch, F., 1972. Azul de Astra e Safranina. In: Kraus, J., Arduin, M. (Eds.), Manual Básico de Métodos em Morfologia Vegetal. Edur, Seropédica, Rio de Janeiro, p. 26.
• Cechinel Filho, V., 2009. Chemical composition and biological potential of plants from the genus Bauhinia Phytother. Res. 23, 1347-1354.
• Chen, Y.F., Zhang, D.X., 2005. Bauhinia larsenii, a fossil legume from Guangxi, China. Bot. J. Linn. Soc. 147, 437-440.
• Duarte, M., Debur, M., 2003. Caracteres morfo-anatômicos de folha e caule de Bauhinia microstachya (Raddi) JF Macbr. (Fabaceae). Rev. Bras. Farmacogn. 13, 7-15.
• Duarte-Almeida, J.M., Clemente, M.S., Arruda, R.C., Vaz, A.M., Salatino, A., 2015. Glands on the foliar surfaces of tribe Cercideae (Caesapiniodeae, Leguminosae): distribution and taxonomic significance. An. Acad. Bras. Cienc. 87, 787-796.
• Fahn, A., 1990. Plant Anatomy. Pergamon Press, Oxford.
• Fortunato, R.H., 1986. Revision del genero Bauhinia (Cercideae, Caesalpinioidea, Fabaceae) para La Argentina. Darwiniana 4, 527-557.
• Franklin, G.L., 1945. Preparation of thin sections of synthetic resins and wood-resin composites, and a new macerating method for wood. Nature 155, 51.
• Gupta, M., Mazumder, U.K., Kumar, R.S., Gomathi, P., Rajeshwar, Y., Kakoti, B.B.Y., Selven, V.T., 2005. Anti-inflammatory, analgesic and antipyretic effects of methanol extract from Bauhinia racemosa stem bark in animal models. J. Ethnopharmacol. 98, 267-273.
• Hao, G., Zhang, D.X., Zhang, M.Y., Guo, L.X., Li, S.J., 2003. Phylogenetics of Bauhinia subgenus Phanera (Leguminosae: Caesalpinioideae) based on ITS sequences of nuclear ribosomal DNA. Bot. Bull. Acad. Sin. 44, 223–228 https://ejournal.sinica.edu.tw/bbas/content/2003/3/bot443-07.pdf
  » https://ejournal.sinica.edu.tw/bbas/content/2003/3/bot443-07.pdf
• Harris, J.G., Harris, M.W., 2001. Plant Identification Terminology: An Illustrated Glossary, 2nd ed. Spring Lake Publishing, Spring Lake, UT.
• Hickey, L.J., 1973. Classification of the architecture of dicotyledonous leaves. Am. J. Bot. 60, 17-33.
• Johansen, D.A., 1940. Plant Microtechnique. McGraw-Hill, New York.
• Lin, Y., Wong, W.O., Shi, G., Shen, S., Li, Z., 2015. Bilobate leaves of Bauhinia (Leguminosae, Caesalpinioideae, Cercideae) from the middle Miocene of Fujian Province, southeastern China and their biogeographic implications. BMC Evol. Biol. 15, http://dx.doi.org/10.1186/s12862-015-0540-9
  » http://dx.doi.org/10.1186/s12862-015-0540-9
• Legume Phylogeny Working Group [LPWG], 2013. Legume phylogeny and classification in the 21st century: progress, prospects and lessons for other species-rich clades Legume phylogeny and classification in the 21st century: progress, prospects and lessons for other species-rich clades. Taxon 62, 217-248, http://www.jstor.org/stable/taxon.62.2.217
  » http://www.jstor.org/stable/taxon.62.2.217
• Lusa, M.G., Bona, C., 2009. Análise morfoanatômica comparativa da folha de Bauhinia forficata Link e B variegata Linn. (Leguminosae, Caesalpinioideae). Act. Bot. Bras. 23, 196-211.
• Menezes, F.S., Minto, A.B.M., Ruela, H.S., Kuster, R.M., Sheridan, H., Frankish, N., 2007. Hypoglycemic activity of two Brazilian Bauhinia species: Bauhinia forficata L. and Bauhinia monandra Kurz. Rev. Bras. Farmacogn. 17, 8-13.
• Metcalfe, C.R., Chalk, L., 1979. Anatomy of the Dicotyledons, 2nd ed. Claredon Press, Oxford, UK.
• Nurit-Silva, K., Agra, M.F., 2011. Leaf epidermal characters of Solanum sect. Polytrichum (Solanaceae) as taxonomic evidence. Microsc. Res. Tech. 74, 1186-1191.
• Nurit-Silva, K., Costa-Silva, R., Basílio, I.J., Agra, M.F., 2012. Leaf epidermal characters of Brazilian species of Solanum section Torva as taxonomic evidence. Botany 90, 806-814.
• Pandey, A.K., Ojha, V., Yadav, S., Sahu, S.K., 2011. Phytochemical evaluation and radical scavenging activity of Bauhinia variegata. Saraca asoca and Terminalia arjuna Barks. Res. J. Phytochem. 5, 89-97.
• Porto, N.M., Barros, Y.L., Basílio, I.J.L.D., Agra, M.F., 2016. Microscopic and UV/Vis spectrophotometric characterization of Cissampelos pareira of Brazil and Africa. Rev. Bras. Farmacogn. 26, 135-146.
• Raddi, G., 1820. Quaranta piante nuove del Brasile. Presso la Societá Tipografica, Modena, pp. 1–35.
• Rezende, M.H., Cardoso, L.A., 1994. Morfologia e anatomia foliar de Bauhinia curvula Benth. (Leguminosae – Caesalpinioideae). Act. Bot. Bras. 8, 19-34.
• Rodrigues, T.M., Machado, S.R., 2006. Anatomia comparada do pulvino primário de leguminosas com diferentes velocidades de movimento foliar. Rev. Bras. Bot. 29, 709-720.
• Sampaio, V.S., Araújo, N.D., Agra, M.F., 2014. Characters of leaf epidermis in Solanum (clade Brevantherum) species from Atlantic Forest of Northeastern Brazil. S. Afr. J. Bot. 94, 108-113.
• Silva, K.L., Cechinel Filho, V., 2002. Plantas do Gênero Bauhinia: composicão química e potencial farmacológico. Quim. Nova 25, 449-454.
• Sinou, C., Forest, F., Lewis, G.P., Bruneau, A., 2009. The genus Bauhinia s.l. (Leguminosae): a phylogeny based on the plastid trn L– trn F region. Botany 87, 947-960.
• Trethowan, L.A., Clark, R.P., Mackinder, B.A., 2015. A synopsis of the neotropical genus Schnella (Cercideae: Caesalpinioideae: Leguminosae) including 12 new combinations. Phytotaxa 204, 237-252.
• Van der Pijl, L., 1952. The leaf of Bauhinia Act. Bot. Neerl. 1, 287-309.
• Vaz, A.M.S.F., 2015. Bauhinia in: Lista de Espécies da Flora do Brasil. Jardim Botânico do Rio de Janeiro. BFG, http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB22811 (acessed 12.11.17).
  » http://floradobrasil.jbrj.gov.br/jabot/floradobrasil/FB22811
• Vaz, A.M.S.F., Tozzi, A.M.G.A., 2003. Bauhinia ser. Cansenia (Leguminosae: Caesalpinioideae) no Brasil. Rodriguésia 54, 55-143.
• Vaz, A.M.S.F., Tozzi, A.M.G.A., 2003. Aculeatae, a new series in Bauhinia section Pauletia (Leguminosae, Caesalpinioideae, Cercideae). Novon 13, 141-144.
• Vaz, A.M.S.F., Tozzi, A.M.G.A., 2005. Sinopse de Bauhinia sect. Pauletia (Cav.) DC. (Leguminosae: Caesalpinioideae: Cercideae) no Brasil. Rev. Bras. Bot. 28, 477-491.
• Wunderlin, R.P., 1976. The Panamanian species of Bauhinia (Leguminosae). Ann. Missouri Bot. Gard. 63, 346-354.
• Wunderlin, R.P., 1983. Revision of the arborescent Bauhinias (Fabaceae: Caesalpinioideae: Cercideae) native to Middle America. Ann. Missouri Bot. Gard. 70, 95-127.
• Wunderlin, R.P., 2010a. Reorganization of the Cercideae (Fabaceae: Caesalpinioideae). Phytoneuron 48, 1–5 http://www.phytoneuron.net/PhytoN-Cercideae.pdf
  » http://www.phytoneuron.net/PhytoN-Cercideae.pdf
• Wunderlin, R.P., 2010b. New combinations in Schnella (Fabaceae: Caesalpiniodeae: Cercideae). Phytoneuron 49, 1–5 http://www.phytoneuron.net/PhytoN-Schnella.pdf
  » http://www.phytoneuron.net/PhytoN-Schnella.pdf

Publication Dates

• Publication in this collection
  Jul-Aug 2018

History

• Received
  1 Feb 2018
• Accepted
  25 Apr 2018