Novelties in secretory structures and anatomy of Rhynchosia ( Fabaceae )

A comparative anatomical study was carried out on the secretory structures of leaflets from taxa belonging to the genus Rhynchosia taxa difficult to delimit because of uncertain interspecific relations in order to evaluate the potential diagnostic value of these anatomical traits for taxonomic assignment. A further objective was to establish consensual denomination for these secretory structures. The new anatomical features found in these taxa were sufficiently consistent to separate the species evaluated. The presence and localization of glandular-punctate structures bulbous-based trichomes, the number of layers in the palisade parenchyma and the arrangement of vascular units distinguish the taxa investigated and these characteristics can be extended to other species of Papilionoideae. The trichomes analyzed were described and classified into five types. Depicted in diagrams, photomicrographs, and by scanning electron microscopy, and listed for the first time at the genus and species levels. The information obtained served to effectively distinguish the taxa investigated among species of Papilonoideae.


INTRODUCTION
Investigated in several studies, secretory structures have contributed to the taxonomy of Fabaceae at various taxonomic levels (Lackey 1978, Lersten and Curtis 1994, 1995, Teixeira et al. 2000, Fortuna-Perez et al. 2012).Despite a consensus among these researchers on the importance of these secretory structures in Papilionoideae, only Solereder (1908) has pointed out that, of the 28 tribes in this subfamily, six exhibit these structures, which substantially facilitates genus separation.Nonetheless, no detailed micromorphological studies have been published addressing this feature, and appropriate terminology for these structures is lacking.
Macroscopic examination of leaflets reveals glandular-punctate and bulbous-based trichomes (Polhill et al. 1981, Fortunato 2000, Cristaldo et al. 2012).Drawing on anatomical features, these structures were confirmed as secretory organs by Lackey (1978) and assigned diagnostic value for certain species.To date, however, few species in this genus have been evaluated in comparative anatomical studies.
No phylogenetic studies on the genus have been conducted and few anatomy reports on the Rhynchosia leaflets are available -e.g.Solereder (1908) and Metcalfe and Chalk (1950, 1979, 1983), along with a single investigation (Lackey 1978) of secretory structures of R. densiflora (Roth.)DC, R. phaseoloides (Sw.)DC, and R. reticulata (Sw.)DC.
The purpose of this study was to evaluate the secretory structures and anatomy of Rhynchosia, so as to establish taxon separation by addressing circumscription problems.To this end, sampling included one taxon from each series in the genus.

MATERIALS AND METHODS
Rhynchosia is a genus found in savannas, prairies, scrublands, rocky highlands, pastures, forest interiors and edges, and humid slopes (Grear 1978).Leaflets from seven taxa of Rhynchosia were obtained from herborized materials (see Appendix).Three apical leaflets were evaluated in each taxon sampling.
The samples were rehydrated in glycerin, washed several times in distilled water (Smith and Smith 1942), serially dehydrated in ethanol, and stored in 0.7 ethanol (Kraus and Arduin 1997).
Sections were cut across the leaf blade of the terminal leaflet.Cross-sections of the leaflets in the apical, mid-and basal portions of the midrib were obtained by freehand sectioning using a steel blade and were stained with Astra blue and safranin, based on Kraus and Arduin (1997).
The documentation was prepared with the aid of a photonic microscope with a camera lucida attachment, using projected micrometric scales.Five replicates of measurements were made for each trichome type in each taxon using an appropriate program and a Leica photomicrography optical microscope.
For scanning electron microscopy, 1 cm 2 samples in 0.7 ethanol were critical-point dried using carbon dioxide, fixed on the sample holder, and coated with a fine layer of gold (Denton Vacuum Desk IV, LLC).The samples were analyzed and photomicrographs were obtained using a device (JEOL JSM-6380LV) from the Multiuser Scanning Electron Microscopy Laboratory of the Universidade Federal de Mato Grosso do Sul at Campo Grande.

RESULTS
The results obtained indicate that several anatomical traits are variable to all taxa (Table I) and thus of diagnostic value down to the generic or specific level.Observed in cross-section, the epidermis is uniseriate and variable in all taxa evaluated (Table I).The outline of epidermal cells in all species is sinuous on the abaxial surface and irregular in the adaxial portion of the leaflet blade (Figs. 1 -2).
Leaflet midrib varies across taxa (Figs. 3 -9), eg. the shape and number of the vascular bundle and the collenchymas and sclerenchymas distribution   (Table I).Papillose epidermal cells are seen at midrib level, except in R. corylifolia.
Non-glandular trichomes with one to three cells (Figs. 17, 18, and 33-36) were homogenously distributed on the blade in all species, and measured from 64 to 296 µm (139.7 ± 54.7).Their apical cell is consistently larger than basal cells.When inserted at the epidermis level, the apical cell is larger than and distinct from adjacent cells.

DISCUSSION
Papillose epidermal cells are evident in the midrib region of the leaf epidermis, except in R. corylifolia, and are present on the abaxial leaflet surface in all taxa, representing an important taxonomic trait for the group of species investigated.Papillose cells are also an additional useful trait for separating species of Dalbergia L. (Farooqui et al. 1989) and from those of Myrocarpus Allemão, Myroxylon L.f. or Myrospermum Jacq.(Sartori and Tozzi 2002).
Marked vascular system variation, particularly in the sclerenchymatous sheath, differentiates Rhynchosia species.Variable arrangement of the sclerenchymatous sheath in species of Tephrosia, Alysicarpus, and Rhynchosia has been reported only by Metcalfe and Chalk (1950), particularly for tribe Phaseoleae.
Trichomes of spherical and ellipsoid types were termed capitate by Abu-Asab and Cantino (1987), because these trichome heads are twice higher than stalk length.The dimensions of capitate trichomes (15 to 42 µm in length and 14 to 18 µm in width) and bulky capitate (20 to 67 µm in length and 18 to 57 µm in width) allow these two structures to be set apart.Both the in-depth evaluation of these trichomes and the denomination suggested herein are novel for the taxa investigated.
Secretory trichomes, usually termed glandularpunctate trichomes or vesicular glands (Lackey 1978), feature a head size twice as large that of stalk, are located in depressions in the epidermis, and have been reported as having diagnostic value for the tribe.In the present study, these glands were named bulky capitate trichomes, as their former denomination applied to a vast range of secretory structures.In a detailed anatomical study of species of Indigofera L. (Marquiafavel et al. 2009), seven types of secretory trichomes, generically referred to as punctiform glands at the time, were identified and named.
Capitate trichomes, bulbous-based trichomes, and non-glandular trichomes, found in all seven taxa investigated, are being reported for genus Rhynchosia for the first time.Lackey (1978) reported bulky capitates trichomes and bulbous-based trichomes only for R. densiflora, R. phaseoloides, and R. reticulata.Glandular trichomes have taxonomic value for Cajaninae (Polhill et al. 1981) and represent a unifying characteristic for members of this subtribe (Lackey 1978, Solereder 1908).
According to Lackey (1978), bulbous-based trichomes are not a trait common to every genus of Phaseoleae, being absent, for instance, from genera Baukea, Flemingia, Carissoa, Eriosema and Paracalyx), as well as from Rhynchosia densiflora.The present study revealed an absence of this type of trichome from R. balansae var.balansae, R. balansae var.psilantha, and from the abaxial surface of R. leucophylla, making bulbous-based trichome a useful taxonomic characteristic that sets R. leucophylla apart from R. corylifolia.Metcalfe and Chalk (1950) reported that glandular trichomes with large, spherical heads, whether uni-or biseriate and featuring a bulbous base, are common only in genera such as Desmodium, Eleiotis, Hallia, Onomis, Rhynchosia, and Tephrosia, of tribe Phaseoleae.
In the taxonomic revision of the genus Rhynchosia, Grear (1978) reported pubescence as a variable trait across species, confirmed in the present study by the observation of non-glandular trichomes of differing lengths.According to Metcalfe and Chalk (1950) and Solereder (1908), non-glandular trichomes, including uniseriate structures with a basal cell accompanied by a long terminal cell, can exhibit wide variations -traits corroborated by the present investigation.
The taxonomic importance of trichomes in Leguminosae, and particularly of secretory structures, has been pointed out in a number of studies, as particular types of trichomes are often useful for delimiting species (Fortuna-Perez et al. 2012, Teixeira and Gabrielli 2000, Teixeira et al. 2000, Teixeira and Rocha 2009), genera (Lersten and Curtis 1993, 1994, 1995), and tribes (Lackey 1978).
None of the secretory structures found in the present study had previously been detailed micromorphologically in the genus investigated.The research literature (Solereder 1908, Metcalfe and Chalk 1950, Lackey 1978, Polhill et al. 1981, Fanh 1979, Pyykko 1966) highlights the usefulness of these structures in taxonomic studies, and the present study provides morphological, anatomical, and ultrastructural details for group separation, which can further support phylogenetic studies.

LEAFLETS OF RHYNCHOSIA SPECIES
The set of anatomical data addressed in the present study effectively separates the Rhynchosia taxa investigated.The shape of the epidermal cells and vascular bundles, sclerenchyma discontinuity, absence of collenchymas and ellipsoid capitated trichomes in R. balansae var.psilantha differentiates this variety from R. balansae var.balansae.
Rhynchosia edulis, R. melanocarpa, and R. minima are considered morphologically similar, but WANDERLEIA DE VARGAS, ÂNGELA L.B. SARTORI and EDNA S. DIAS cross-sectional epidermal cells, shape vascular budles, organization of collenchymas and sclerenchyma separate these species.Presence of bulky capitate trichomes on the adaxial surface is relevant to distinguish R. minima from the other species investigated.Rhynchosia minima was the only taxon to exhibit, on both leaflet surfaces, all the epidermal indumenta investigated in the present study, a feature that distinguishes it from the other species.
Orbicular shape of the main vascular bundle, presence of epidermal papillae, absence of bulbous-based trichomes on the abaxial surface and continuity sclerenchima in R. leucophylla consistently differentiates this species from R. corylifolia, which has bulbous-based trichomes on the abaxial surface, discontinuous sclerenchyma and an irregularly elliptical main vascular bundle, but lacks epidermal papillae.
The results of the present study can be generalized to other species in tribes of the same family, as no changes in the types proposed are found in other genera of this tribe.Moreover, the presence or absence of these structures is a strong differential feature among species of Papilionoideae.
Recognition of secretory structures associated with midrib shape has diagnostic value for the species investigated, contributing towards elucidating taxonomic problems generally encountered by experts.These results indicate that studies of this nature should be encouraged, yielding responses to issues on species complexes or even allowing these species to be grouped into higher taxonomic categories or specific clades.
Figures 1 -2 -Photomicrographs with details of the leaflet blade of Rhynchosia, superficial view, evidencing abaxial and adaxial surfaces, respectively, of the leaflets.Fig. 1: Epidermal cells with shape slightly sinuous in R. minima.Fig. 2: Epidermal cells with shape irregular in R. edulis.Non-glandular trichomes (White arrow), secretory trichomes (asterisk) and base of non-glandular trichomes (black arrow).
10-18).Non-glandular trichomes are single, uni-to multicellular, and non-branched, consisting of one to three cells, with cubic base cells and elongated apical cells (Figs 17 and 18).Secretory trichomes (Table I; Figs.10-16) can be differentiated according to shape and size, and can be classified into four types.Capitate types (Figs.10-14) have multicellular heads and uni-or multicellular stalks, bulbous base type (Figs. 15 and 16) has a rounded base consisting of apically elongated cells.