Glands on the foliar surfaces of tribe Cercideae ( Caesapiniodeae , Leguminosae ) : distribution and taxonomic significance

Large elongated glands occur on Cercideae leaf surfaces. Leaves of Bauhinia (55 taxa, 53 species), Cercis (1 species), Phanera (1 species), Piliostigma (2 species), Schnella (19 species) and Tylosema (1 species) were observed to determine location and relative number of glands. They were only observed on the abaxial leaf surface of 42 Bauhinia taxa. The glands were analyzed by light stereomicroscope and scanning electron microscopy. They are large (up to 270 μm long and 115 μm wide) and multicellular, containing lipophilic substances, probably volatile oils. Presence or absence and density of the glands in species of Bauhinia may be useful to determine species delimitation or distinction among infraspecific taxa. Higher density of glands is more common in species from “cerrado” (a savanna ecosystem) and “caatinga” (a semiarid ecosystem from northeast Brazil) areas. Bauhinia species devoid of foliar glands are frequently from humid forests.


INTRODUCTION
Studies about secretory structures are valuable for many fields of plant biology.Some examples of such studies and corresponding objectives are: 1) identification or assignment of classes of substances produced and accumulated (Roshchina and Roshchina 1993); 2) test of hypotheses about their adaptive and physiological roles (Farrell et al. 1991); and 3) evaluation of their taxonomic significance (Sartori and Tozzi 2002).Secretory structures consist of highly specialized cells involved in the synthesis and secretion, and in some cases reabsorption of substances, often corresponding to secondary metabolites (Esau 1977, Fahn 1979, Roshchina and Roshchina 1993).Secretory structures may occur on foliar surfaces of some taxa of Fabaceae, such as species of the tribe Caesalpinieae (Lersten and Curtis 1996) and of the genus Chamaecrista (Conceição et al. 2009).JOAQUIM M. DUARTE-ALMEIDA et al.
In Brazil, there are approximately 100 species of Cercideae (Rodrigues and Vaz 2008).They correspond mostly to Bauhinia and Schnella, plus a few species of Bauhinia sect Benthamia (Fortunato and Wunderlin 1985), here included under Piliostigma (see footnote in Table SI) (Supplementary Material).The species are distributed in nearly all ecosystems, including forests (Amazonian, Atlantic, gallery forests), savannas (cerrados, campos rupestres) and caatinga (dry deciduous forest of the semi-arid Brazilian Northeast) (Vaz andTozzi 2003, 2005).Species of Bauhinia and Piliostigma are trees and shrubs lacking tendrils, whereas species of Schnella are lianas and vines with tendrils.
An unusual kind of multicellular secretory structure, which is known as either a "boat-shaped" gland (Solereder 1908, Metcalfe andChalk 1950) or an "inflated" trichome (Tucker et al. 1984), was observed on leaf surfaces of Bauhinia.
We hypothesize that the presence of such glands may be a morphological character of taxonomic use for distinguishing amongst some taxa of Cercideae.Therefore, the present work aims to show the foliar glands of species of Cercideae and investigate FOLIAR GLANDS IN CERCIDEAE their distribution and density on leaves in order to evaluate their distribution and possible taxonomic significance.In an attempt to detect differences in the density of glands among Brazilian species from distinct ecosystems, attention was given to habitat preferences of species.SI.

SCANNING ELECTRON MICROSCOPY (SEM)
For scanning electron microscopy, fixed leaves were dehydrated in graded ethanol series, submitted to critical point drying with CO 2 (Leica EM CPD-030, Leica Microsystems, Heerbrugg, Switzerland), mounted on stubs, and coated with gold-palladium (Berlyn and Miksche 1976).The preparations were examined with a Zeiss DSM-940 SEM (Karl Zeiss, Oberkochen, Germany).

IMAGE PROCESSING
Glands on leaves of herbarium specimens were observed and photographed with a Canon Rebel XT digital camera (Canon Inc., Tokyo, Japan), equipped with a 100 mm close-up lens and 68 mm extension tubes.
Leaves of herbarium specimens of all material listed in Table SI were observed with light reflected from abaxial leaf surfaces, using a stereomicroscope Leica DMLB (Leica Microsystems, Heerbrugg, Switzerland).IM50 software (Leica) was used for direct observation and image processing.For each taxon, the presence and density of glands were examined on the surface of three fully expanded leaves of each specimen.The number of glands was counted in five squares of 2.3 mm x 1.8 mm randomly selected on the lower third of the abaxial face of one of the leaves, paying particular attention to veins, intervenal spaces and margins.

LEAF GLANDS: MORPHOLOGY AND CONTENT
Leaf glands of Bauhinia are elongated and emerge from the abaxial surface on a short multicellular stalk (Fig. 2A).A large internal space is visible in transversal and longitudinal sections of the glands (Fig. 2A).The glands are formed by a single layer of cells surrounding a cavity that accumulates oil (Figs.2A, C).
Gland contents are usually lost after fixation and by procedures to prepare histological sections in Paraplast ® or historesin.Hence, in this investigation, hand sections fixed in CRAF III (Johansen 1940) were used for histological observation.Reflecting strong staining by Sudan IV, the secretion inside the glands is oily.Glands of clarified leaves treated with Sudan IV exhibit a spherical lipophilic content (Fig. 2B).A similar content is observed in non-sectioned glands (Fig. 2C).Viewed by scanning electron microscopy, the cells delimiting the glands have no surface ornamentation and are elongated along the longer axis of the glands (Figs.3C-E).Glands are visible near the margin of the lamina in B. aculeata (Fig. 2D).Glands are variable in size in Bauhinia.
In some species, such as B. variegata, they are long and slender, up to 200 µm long and 30 µm wide.In B. pentandra, however, they are much shorter, 95 µm long and 35 µm wide.The largest glands were observed on leaves of B. subclavata, reaching up to 270 µm in length and 115 µm in width; the smallest were on leaves of B. campestris, 50 µm long and 27 µm wide.Leaves of several species of Bauhinia have thick, profusely fibrous veins, which protrude from the abaxial surface forming deep ridges, delimiting intervenous regions where cells, frequently papillate, constitute the epidermis (Figs.2A, 3A-C).Quite often these intervenous regions have many non-glandular trichomes (Figs.2B, 3A-C), among which glands may be nested (Figs.2A,  D, 3B).In some species, such as B. brevipes, the glands are elliptical (Fig. 3B).In other species, such as B. forficata, they are long and slender (Fig. 3C).In B. aculeata, the glands are wider at one end and narrower at the other (Figs.3A, E), while other species (e.g., B. longifolia) have broad cylindrical glands (Fig. 3D).Glands of B. bombaciflora are spread uniformly on the leaf lamina (Fig. 4A), while the glands are restricted to the leaf margin on leaves of B. galpinii (Fig. 4B). Figure 4C shows the abaxial surface of a B. ovata leaf obtained with reflected light by stereomicroscopy; glands are seen to be spread evenly on the foliar lamina.A foliar gland of B. subclavata observed with higher magnification is seen in Fig. 4D.B. cinnamomea, B. pentandra, B. pulchella, B. purpurea, B. rufa, B. subrotundifolia, B. ungulata var. ungulata and B. variegata).Margins and veins appear to have more glands than other leaf parts in some species with high gland density (e. g., B. cheilantha and B. membranacea).
Lersten and Curtis (1996) described foliar surface glands of species of Caesalpinieae.types of glands that may appear on the surface of reproductive and vegetative organs of Indigofera (Leguminosae, Papilionoideae).Type 3 resembles the glands described in the present work: they have a short peduncule and a curved and oval head.But unlike the glands of Bauhinia, Indigofera type 3 glands have massive head.In addition, Bauhinia glands are longer than Indigofera type 3.

GLANDS AND ENVIRONMENTAL CONDITION
The glands described in the present work are confined to the abaxial leaf surface of the majority of species of Bauhinia.The material contained in the glands is probably volatile oil.Several lines of evidence lead to this assumption.First, the secretion is lipophilic, as shown by Sudan staining.Second, Duarte-Almeida et al. ( 2004) reported the content and composition of volatile oils from leaves of B.  SI, the leaves of all these species possessed glands.Third, two species examined by Duarte-Almeida et al. (2004) failed to yield volatile oils: Schnella alata and S. outimouta (syn.Bauhinia alata and B. outimouta, respectively).Neither of the species exhibited foliar surface glands in the present investigation (Table SI).

IMPLICATIONS FOR TAXONOMY
Our results indicate that foliar surface glands are useful to distinguish the genus Bauhinia from Cercis, Piliostigma, Schnella and Tylosema.Cercis is basal in the Cercideae phylogeny (Sinou et al. 2009).Cercis, plus Adenolobus, Griffonia and the large clade embracing Gigasiphon, Lysiphyllum, High density of glands was observed on leaves of species of Bauhinia from the cerrado and caatinga vegetation, as described above.The highest density, 55 glands/mm 2 , was found in B. tenella, a species typical of the cerrado (Table SI).Values up to 24, 26 and 36 glands/mm 2 were registered for B. curvula, B. bombaciflora and B. campestris, all species from the cerrado (Table SI).Leaves of B. subclavata, a species occurring in the cerrado and the caatinga, may have 15 glands/mm 2 (Table SI).Leaves of species typical of the caatinga, such as B. cacovia, B. cheilantha and B. corifolia, also have relatively high gland density on the leaf lamina (13, 15, and 10 glands/ mm 2 , respectively; Table SI).Among species with leaves devoid of glands, only B. dubia occurs in the cerrado and the caatinga, but also in humid forests.Species of Bauhinia without glands seem to occur mostly in humid forests.Among the 13 species and one subspecies of Bauhinia studied with leaves on which no glands were found, four are not native to Brazil (B. acuminate, B. dipetala, B. monandra and B. tomentosa), and eight occur in humid forests.Some species of Bauhinia from humid forests do have leaves with glands (e.g., B. aureopunctata, B. cinnamomea, B. corniculata, B. fusconervis and B. ovata), although these are mostly species with low gland density (Table SI).

GLANDS: POTENTIAL TAXONOMIC IMPLICATIONS
Presence of glands in Cercideae may be taxonomically useful at the genus level.Within Bauhinia, the gland density seems to be ecologically related.Vaz and Tozzi (2003) (Vaz and Tozzi 2005).These observations indicate that presence and density of glands on leaves of Bauhinia may be useful for distinguishing among species or infraspecific taxa.Palavras-chave: Bauhinia, floresta brasileira, Leguminosae, estruturas secretoras, taxonomia, ecossistema tropical.

Figure 1
Figure 1 presents the affinity relationships within Cercideae and indicates which groups of the tribe are represented in the present work.TableSI lists

Figure 1 -
Figure 1 -Simplified phylogenetic relationships among groups of Cercideae, based on Sinou et al. (2009).NR and R: taxa not represented and represented, respectively, in the present work; s. str.: sensu stricto.

Figure 2 -
Figure 2 -Glands seen in light microscopy.(A) Transversal section of the leaf blade of Bauhina rufa (SPF 79418), showing a gland inserted in an intervenal region, scale bar =50 µm.(B) Segment of cleared leaf showing lipid content inside a gland of B. brevipes (SPF 142960) and non-glandular trichome (arrow), scale bar = 100 µm.(C) Transversal section of leaf blade of B. brevipes, showing a gland in lateral view inserted in a small depression on the abaxial surface with a lipid drop visible inside, scale bar = 100 µm.(D) Segment of a cleared leaf of B. aculeata (RB 94867) showing glands (arrows) distributed in intervenal regions, scale bar = 400 µm.

Figure 3 -
Figure 3 -Scanning Electron Microscopy of leaf surface of subgenus Bauhinia.(A) Abaxial side of leaf of B. aculeata (RB 94867) with glands (arrows) and non-glandular trichomes, scale bar = 150 µm.(B) Glands (arrows) at the base of a depression formed by protruding veins (left) of B. brevipes (SPF 142960), and papillate epidermal cells also can be seen, scale bar = 50 µm.(C) Detail of a gland on the abaxial surface of a leaf of B. forficata¸ scale bar = 50 µm.(D) Secretory gland of B. longifolia (SPF 122376), scale bar = 50 µm.(E) Detail of a single gland of B. aculeata (RB 94867), scale bar = 50 µm.

Table SI
recognized B. holophylla, B. longifolia and B. rufa as a species complex sharing several morphological characters.Leaves of both B. holophylla and B. rufa have a relatively high density of glands on the leaf lamina and margin (Table SI).They differ, however, from leaves of B. longifolia which has low gland density, restricted to the margins or veins.B. holophylla and B. rufa are frequent in dry savannas and rarely occur in humid forests, while the opposite applies to B. longifolia.Presence or absence of glands in Bauhinia may also be taxonomically useful for distinguishing among infraspecific taxa.Two specimens of B. forficata were analyzed in the present work: SPF 34660, corresponding to a specimen of the type subspecies, and SPF 105974, belonging to B. forficata subsp.pruinosa.On the margin of the leaves of subspecies forficata were detected 3-5 glands/mm 2 , while no glands were detected on leaves of subspecies pruinosa.B. forficata subsp.pruinosa now includes B. candicans Benth. in synonymy