Comparative leaf anatomy and morphology of some Brazilian species of Crotalaria L . ( Leguminosae : Papilionoideae : Crotalarieae )

Anatomical characters often provide strong taxonomic evidence and many times help define species whose morphological limits are in question. This study aimed to survey characters of the leaf anatomy of 16 species of Crotalaria L. occurring mostly in the cerrado (savanna) of central Brazil and in areas of restinga (coastal woodland, or sandy forest) along the coast of the country, in order to improve the current taxonomic circumscription of sections and species. Leaf samples were collected in the field and obtained from herbarium specimens. Standard techniques in plant anatomy were employed, including analyses under light and scanning electron microscopy. Many of the characters analyzed are relatively uniform, but some are diagnostic for species. At the section level, only trichomes with a base composed of radially distributed cells proved useful in the diagnosis of C. sect. Calycinae. The other characters analyzed showed interspecific variation, but no diagnostic value for recognition of the sections. At the species level, unlike the results found for sections, various characters have diagnostic value. Among the characters with diagnostic value at the species level, those related to texture, the venation pattern (such as the formation of areolas and insertion of the ribs secondary to midrib), and, in particular, the epidermis, showed potential for aiding the circumscription of some species.


Introduction
Crotalaria L. (Leguminosae: Papilionoideae) is the only genus of the tribe Crotalarieae with native representatives in South America (Wyk 1991;Wyk & Schutte 1995).The genus is one of the largest in the family Leguminosae (Lewis et al. 2005) and comprises some 720 species distributed mainly in tropical and subtropical areas of the southern hemisphere (Wyk 2005) with the principal center of diversity in the central-south and eastern regions of Tropical Africa (Polhill 1982).A phylogenetic study of the tribe Crotalarieae (Boatwright et al. 2008) confirmed the tribe as monophyletic and revealed Bolusia as a sister genus to Crotalaria.In a recent molecular systematic study based on nuclear internal transcribed spacer and the plastid markers matK, psbA-trnH and rbcLa, Roux et al. (2013) showed that Crotalaria is monophyletic (99% bootstrap support; posterior probability of 1.0).Systematic studies of the Crotalarieae have mainly covered external morphology and anatomy (Roux et al. 2011), cytogenetics (Windler 1974;Oliveira & Aguiar-Perecin 1999), or chemotaxonomy (Flores 2004).
Although infrageneric relationships are not well understood, it would appear that Crotalaria originated in Africa but diversified more recently into other regions of the world (Lewis et al. 2005).Some taxonomists (Senn 1939;Bisby 1970) have highlighted the difficulties of proposing an infrageneric classification within the group, owing to the large, highly reticulate and complex interspecific variation.In general, the most widely accepted infrageneric classification of the group is that proposed by Bisby & Polhill (1973), which is based mainly on the morphological characters of the flowers of African species.Roux et al. (2013) proposed a new infrageneric classification for the entire genus, based on molecular and morphological evidence, resulting in 11 recognized sections.To date, 42 species of Crotalaria have been recorded for Brazil (Flores 2014) Those Crotalaria species include shrubs, subshrubs, and herbaceous perennials that grow in open areas, ranging from high hills to semi-deserts.The plants of Crotalaria can be easily recognized by the presence of simple leaves or leaves containing one to three leaflets, a rostrate keel, a style with trichomes in a single or double line toward the stigma, a monadelphous androecium (5 + 5-anther configuration), paired callosities on the standard petal and highly inflated fruit (Polhill 1982;Wyk 2005).
Many of the species represent economically important sources of fiber, animal forage, or green manure, and some have medicinal or ornamental value (Lewis et al. 2005).
Anatomical studies of the subfamily Papilionoideae have provided significant support for the taxonomic classification of the group (Metcalfe & Chalk 1950;Krishnamurthy & Kannabiram 1970;Kannabiram & Krishnamurthy 1974;Kothari & Shah 1975;Lackey 1978;Crow et al. 1997;Teixeira & Gabrielli 2006;Fortuna-Perez et al. 2012).Some of these studies (e.g., Kothari & Shah 1975) have focused on the morphological diversity of specific characters, such as the types of stomata and trichomes in the tribe Hedysareae, and these have proven to quite useful in generic grouping, despite their heterogeneity.In addition, studies of leaf anatomy have made significant contributions to the systematic classification of the Leguminosae.In the case of the Phaseoleae, detailed information concerning the leaflet anatomy within the tribe not only confirmed and expanded previous knowledge, but also highlighted the usefulness of the data in taxonomic classification (Lackey 1978).
Despite the importance of anatomical detail in determining the taxonomy of some plant groups, the anatomical characteristics of the Leguminosae have not been fully explored.This is particularly so in the case of Crotalaria, a genus that requires better infrageneric delimitation.Among the few anatomical studies that are available for this genus, some were restricted to the leaf epidermis (Krishnamurthy & Kannabiram 1970;Kannabiram & Krishnamurthy 1974;Gill et al. 1982), whereas others focused on the anatomy of the cell wall of the fruits (Roux et al. 2011).In the present study, we examined the anatomical characters of the leaves of 16 species of Crotalaria occurring mostly in the cerrado (savanna) of central Brazil and in areas of restinga (coastal woodland, or sandy forest) along the coast of the country, with the objective of increasing knowledge regarding this group, specifically its current sectional classification and species circumscriptions.

Materials
We sampled leaves from a total of 48 specimens, belonging to 16 species of Crotalaria, occurring in open habitats: most are from the cerrado and campo rupestre (dry rocky grassland) formations, both in the Cerrado Biome in central Brazil, although two specimens were collected from the sandy restingas of the southeastern Atlantic coast of Brazil.The choice of species of each section was made according to the representativeness, selecting those with occurrence in Brazil.Samples of three mature leaves of each species were collected in the field or were obtained from herbarium material.A list of voucher specimens is given in Tab. 1.We compared species according to their morphological and anatomical characteristics, using a presence/absence table (Tab.2).

Light microscopy
Samples were collected from the leaves of the second to fifth nodes of the stem, and the leaflets obtained from herbarium specimens were rehydrated by heating in aqueous glycerin (50%) for 3 min.All material was fixed in 50% formalin/acetic acid-alcohol, dehydrated in an ethanol series, and stored in 70% ethanol (Johansen 1940).The middle region of the leaf blade was studied from freehand sections and subsequently stained with 1% Astra Blue in 50% ethanol and 1% Safranin in 50% ethanol, as modified from Kraus et al. (1998).
To analyze the epidermis and stomata type, 100 mm 2 samples were subjected to diaphanization and staining with 1% Safranin in 50% ethanol (Franklin 1945).Leaf blades were clarified with a technique modified from Shobe & Lersten (1967), and the venation pattern of the leaf blade was classified according to the guidelines established by the Leaf Architecture Working Group (1999), Ellis et al. (2009) and Hickey & Wolfe (1975).Trichomes were classified according to their overall morphology (Theobald et al. 1979).

Scanning electron microscopy
A scanning electron microscopy study was conducted to visualize the cuticle and trichomes.Small pieces of leaves were fixed on aluminum stubs using double-sided adhesive, and were coated with a thin layer (ca. 25 nm) of gold-palladium, as described by Silveira (1989).The scanning electron microscopy micrographs were taken through a Zeiss DSM 940 (Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 10 kV.

Venation
The primary venation pattern in all of the studied species of Crotalaria is pinnate, whereas the secondary venation pattern is brochidodromous and characterized by secondary veins that do not terminate at the margin but join to form arches (Fig. 1A-P).
The insertion of secondary veins along the principal vein varies considerably (Tab.2).In Crotalaria lanceolata, C. martiana, C. micans, C. pallida, C. rufipila and C. vitellina, all secondary veins are decurrent to the primary vein (Fig. 2A), whereas in C. breviflora, C. flavicoma, C. juncea, C. stipularia, C. velutina, C. maypurensis and C. miottoae only the proximal secondary veins are so.Less frequently, uniformly spaced excurrent veins appear distributed across the leaf blade (Fig. 2B) in C. incana, C. paulina and C. spectabilis.Intersecondary veins (Fig. 2C) are present in all species, and their proximal courses are always parallel to the main secondary veins.The tertiary veins (Fig. 2C) of the intercostal region are percurrent in all species (Fig. 2C), and the external tertiary veins (i.e.those outside the arches formed by the secondary veins) join to form loops (Fig. 2C).
The areoles can be classified into two groups according to the stage of development, i.e. well developed as in C. miottoae (Fig. 2H) and moderately developed as in the other species studied (Fig. 2I).Within the areoles, when present, it is possible to identify three types of freely ending veinlets (FEVs): those without branches (Fig. 2D); those with a single branch (Fig. 2E); and those with dendritic ramifications (Fig. 2F).In some species, FEVs are absent (Fig. 2G).
In the majority of species, the marginal terminal veins are discontinuous (Fig. 2J), although some species exhibit loopforming veins (Fig. 2K) along almost the entire length of the margin.

Trichomes
Trichomes are present on both surfaces of the leaves of all of the species studied, except for Crotalaria lanceolata, C. maypurensis, C. micans and C. paulina, in which they appear only on the abaxial surface.Although C. miottoae and C. pallida present trichomes on both surfaces, the structures are rare on the adaxial surface and are distributed predominantly near the midrib (Fig. 3A).The trichomes are non-glandular, uniseriate and filiform in all of the studied species, although the structures can be short or long, and the cuticular layer of the body can be smooth (Fig. 3F) or granular (Fig. 3G).The structures comprise a cellular base (Fig. 3B), with a body consisting of a short subterminal cell with a thickened wall and a further elongated terminal cell (Fig. 3C).In some cases, the trichome base is comprised of radially distributed cells (Fig. 3D) or not (Fig. 3E).

Leaf blade
Transversal sections revealed that, in all of the studied species, the leaf epidermis is uniseriate on both surfaces.In most of the species, cells of the adaxial surface presented walls with contours that were ≥ 2× higher than were those of cells on the abaxial surface (Fig. 4A), although in Crotalaria maypurensis, C. micans, C. spectabilis, C. stipularia and C. velutina, the cell walls of the abaxial and adaxial surfaces showed similar contours.It is possible to observe thickening of the external periclinal walls of epidermal cells on the abaxial and adaxial surfaces (Fig. 4A), except in C. lanceolata, C. micans, C. miottoae, C. pallida and C. paulina, in which such thickening is observed only on the adaxial surfaces, as well as in C. incana, in which no such thickening can be detected.Epidermal cells on the adaxial surfaces of leaves of C. breviflora exhibit projections (small papilla) that are restricted to the region of the midrib (Fig. 4B).
Frontal views of the dissociated epidermis reveal the presence of two types of cell contour.Therefore, whereas most species present anticlinal cell walls with straight contours (Fig. 4C) on both surfaces of the leaves, Crotalaria incana, C. miottoae, C. pallida and C. vitellina exhibit sinuous cell walls (Fig. 4D) on both surfaces, and C. rufipila show sinuous cell walls only on the adaxial surface.
All of the studied species present amphistomatic leaves with the exception of Crotalaria breviflora, the leaves of which were hypostomatic.However, the distribution of stomata is sparse on the adaxial surfaces of C. miottoae, C. spectabilis and C. vitellina, and was generally limited to areas near to the main veins (Fig. 3A).In all species, the stomata are located at the same level as the epidermal cells.
The leaves of 14 of the 16 species studied presented a dorsiventral mesophyll (Fig. 5A), a homogeneous mesophyll being observed only in Crotalaria spectabilis and C. velutina (Fig. 5B).In the species with a dorsiventral mesophyll, the palisade parenchyma frequently consists of two layers (Fig. 5A), although it appears sometimes as a single layer (Fig. 5C), as in C. flavicoma, C. incana, C. miottoae and C. vitellina.
The midrib region shows a concave-convex contour in all of the studied species (Fig. 5E).The vascular system exhibits a half-moon shape with the xylem and phloem polarized in the adaxial and abaxial surfaces, respectively.Lignified cells associated with vascular tissues (perivascular cells) are observed in all species (Fig. 5F) except Crotalaria maypurensis.Lignification of the perivascular cells occurs initially in the abaxial flanks lateral to the phloem, and in some cases they form a continuous cylinder (Fig. 5E).In the region of the central vein, it is possible to observe collenchyma on both surfaces or just on the abaxial surface, as in C. spectabilis.The vascular tissues in the lateral veins are also polarized, although no lignified perivascular cells similar to those of the central vein were observed here.The central and lateral veins are externally limited by a bundle sheath (endodermis; Fig. 5D), which is more visible in the smaller veins.

Discussion
Among the characters studied, the most valuable in helping delimitate the sections of Crotalaria was the presence of trichomes with bases comprising radially distributed cells.Apart from two exceptions, this feature is observed only in species classified in C. sect.Calycinae.Although interspecific variation is observed in all of the other characters studied, those have no diagnostic value with respect to the recognition of sections.
In contrast to the results obtained for sections, a number of characters show diagnostic value for the circumscription of Crotalaria at the specific level.Among these characters are those related to texture, venation patterns (areoles and type of insertion of secondary veins adjoining the main vein) and, most particularly, the characteristics of the epidermal cells (Tab.2).
Kannabiram & Krishnamurthy (1974) investigated leaves of 10 species of Crotalaria and described the consistent presence of epidermal cells with straight walls (in front view) on the adaxial surfaces and sinuous walls on the abaxial surfaces.However, the present study demonstrates that this pattern is variable, because, in some species, cells with similar wall contours (predominantly straight) can be observed on both surfaces of the leaves.Therefore, C. juncea and C. micans present epidermal cells with straight walls on the abaxial and adaxial surfaces, whereas C. rufipila exhibits epidermal cell walls with sinuous and straight contours on the adaxial and abaxial surfaces, respectively.Two trichomes Acta bot.bras.28(4): 583-593.2014.types-unicellular (Procópio et al. 2003) and multicellular uniseriate (Metcalfe & Chalk 1950)-have been described in some Crotalaria species.Among the Crotalaria species studied here, including C. incana, in which Procópio et al. (2003) described unicellular trichomes, we observed only multicellular uniseriate trichomes.Procópio et al. (2003) also observed that leaves of Crotalaria incana showed two-layer palisade parenchyma, although that feature not was confirmed in the present study, in which all analyzed samples showed a single-layered palisade parenchyma.Amphistomatic leaves predominate in all of the species investigated, with the exception of C. breviflora, which has hypostomatic leaves.Variation regarding the occurrence of stomata on just one or both surfaces of the leaves of Cro-talaria species has been described previously by Metcalfe & Chalk (1950).
A dorsiventral mesophyll is observed in all of the species studied, except for Crotalaria spectabilis and C. velutina, in which the mesophyll is homogeneous, a pattern that has been described previously in the Leguminosae (Cowan 1981;Crow et al. 1997;Metcalfe & Chalk 1950).The presence of lignified cells in association with the vascular tissues of the central vein, which is observed in most of the studied species, has been reported by Alvarez et al. (2001) for Swartzia brachyrhachis Harms and S. laurifolia Benth.The distribution of these lignified cells, differentiating initially in the lateral flanks and sometimes forming a continuous cylinder, is the most common pattern observed in the studied species.This Acta bot.bras.28(4): 583-593.2014.feature has been described previously by Metcalfe & Chalk (1950), although it is somewhat variable in the Papilionoideae.
In some cases, anatomical characteristics taken in combination can be quite valuable in the recognition of different taxa.An example in which the consideration of morphological and anatomical characters has aided the circumscription of morphologically similar taxa is provided by Crotalaria miottoae.Flores & Tozzi (2005) reported that, despite the significant morphological similarity to C. vitellina and C. micans, the delimitation of C. miottoae could be successfully achieved through distinctive anatomical leaf characters (Tab.2).Another example involves C. maypurensis and C. micans, species that are morphologically quite similar and difficult to distinguish (Lewis 1987;Gómez-Sosa 2000) because of significant intraspecific variation.Although these species do share some similarities in leaf anatomy, a number of characters present distinctive states (Tab.2).
The characters of the leaves analyzed in this study are of potential value for the circumscription of a number of Crotalaria species.This is particularly valuable in the cases of species that are share similar external morphological features.Consequently, characters such as the degree of development of areoles and the presence of a coating of epicuticular wax on the abaxial surface corroborate the circumscription of C. miottoae, whereas the presence of perivascular fibers in the central and secondary veins, with fibers adjacent to the phloem, allow a distinction to be drawn between C. micans and C. maypurensis.Further anatomical studies regarding other species of this large genus are needed in order to improve the taxonomy of Crotalaria.

Figure 2 .
Figure 2. Details of leaf architecture: A. decurrent insertion of secondary veins in Crotalaria flavicoma Benth.; B. excurrent insertion of secondary veins in C. incana L.; C. intersecondary veins (In) with their proximal courses parallel to the main secondary veins, tertiary veins percurrent (Pe), external tertiary veins joined to form loops (*) in C. rufipila Benth.; D-G.Freely ending veinlets (FEVs)-D.FEVs without branches (arrow) in C. velutina Benth., E. FEVs with a single branch in C. miottoae A.S. Flores & A.M.G.Azevedo, F. FEVs with dendritic ramifications in C. incana L. and G. FEVs absent in C. flavicoma; H. areoles well developed in C. miottoae; I. areoles moderately developed, with FEV bearing a tracheoid idioblast in the terminal portion (arrow) in C. flavicoma; J. marginal terminal veins discontinuous in C. flavicoma; and K. marginal terminal veins loop-forming in C. maypurensis Kunth.

Figure 3 .
Figure 3. Types of trichomes in Crotalaria spp.: A. frontal view of the adaxial surface with trichomes (Tr) and stomata (St) distributed predominantly near the midrib in C. miottoae A.S. Flores & A.M.G.Azevedo; B. trichome comprising a basal cell (Bc), body consisting of a short subterminal cell with a thickened wall and an elongate terminal cell in C. martiana Benth.; C. trichome with a short subterminal cell (Sc) and an elongate terminal cell (Tc) in C. vitellina Ker Gawl.; D. scanning electron microscopy (SEM) micrograph of adaxial surface of C. stipularia Desv., showing trichome with cells distributed radially in the base; E. SEM micrograph of abaxial surface showing trichomes without cells distributed radially in the base; F. SEM micrograph showing details of the trichome with a smooth surface in C. juncea L.; and G. SEM micrograph showing details of the trichome with a granular surface in C. flavicoma Benth.

Figure 4 .
Figure 4. Leaf epidermis in Crotalaria spp.: A. uniseriate epidermis with cell contours that are ≥ 2× higher on the adaxial surface than on the abaxial surface in C. breviflora DC., details showing stomata on abaxial surface; B. epidermal cells showing projections (papillae), restricted to the region of midrib on the adaxial surface (arrow) in C. breviflora; C. epidermal cells in frontal view with straight anticlinal walls in C. breviflora; and D. epidermal cells in frontal view with sinuous anticlinal walls in C. vitellina Ker Gawl.St -stomata; Aw -anticlinal wall.

Figure 5 .
Figure 5. Leaf blade characters: A. dorsiventral mesophyll with two layers of palisade parenchyma on the adaxial surface in C. paulina Aiton; B. homogeneous mesophyll in C. velutina Benth.; C. palisade parenchyma with a single layer in C. vitellina Ker Gawl.; D. vascular bundles with perivascular fibers and endodermis in C. spectabilis Roth, details showing smaller vascular bundles limited by endodermis and no vascular bundles; E. midrib region showing concave-convex petiole outline, in transverse section, with lignified cells associated (perivascular cells) in C. breviflora DC.; and F. details of the midrib region of C. velutina, showing the perivascular cells forming a continuous cylinder.Pp -palisade parenchyma; Xy -xylem; Ph -phloem; Vf -vascular fibers; En -endodermis.

Table 1 .
List of the analyzed species, with voucher, collection site and herbarium (codes according toThiers 2009).BRA Brazil, (including state abbreviation).