Morphoanatomy of nectaries of Chamaecrista ( L . ) Moench sections Chamaecrista , Caliciopsis and Xerocalyx ( Leguminosae : Caesalpinioideae )

Nectaries are specialized structures that secrete nectar. Several species of Chamaecrista possess nectaries on the petiole, which have been shown to vary widely in morphology and the chemical nature of their secretion. However, a comprehensive investigation of the nectaries of the clade formed by sect. Chamaecrista, Caliciopsis and Xerocalyx has yet to be performed. Our study aimed to confi rm whether or not the leaf glands of species of this clade are in fact nectaries, determine the chemical nature of their secretion and expand the morphoanatomical database on leaf nectaries in Chamaecrista with the intention of contributing to the taxonomy and phylogeny of the genus. Samples from herbarium and fi eld-collected material were subjected to standard methods for light and scanning electron microscopy. Four diff erent forms of nectaries were observed: urceolate, patelliform, verruciform and cupuliform. Th e nectaries were found to comprise a single-layered epidermis, nectary parenchyma, subnectary parenchyma and vascularization. Polysaccharides, lipids, phenolic compounds and proteins were detected in secretions. Although anatomical similarities were observed among the studied species, their morphology diff ered. Moreover, the glands are indeed nectaries and are similar to those observed in other species of the genus Chamaecrista. Th ese data hold potential taxonomic usefulness for the studied sections.


Introduction
Nectaries are specialized structures for the secretion of a sugary solution called nectar, which contains sucrose, glucose and fructose as primary solutes (Fahn 1979;Nicolson et al. 2007).According to their location, nectaries are classifi ed as reproductive (i.e. when found on the infl orescence axis, bracts, sepals, ovary, stamens, etc.) and extra-reproductive (i.e. when found on the petiole, rachis, leaf blade, stems, etc.) (Schmid 1988).Reproductive nectaries are usually involved in pollination strategies while extra-reproductive nectaries are related to the protection of plants from the attack of herbivores and pathogens through mutualistic interactions with ants (Schmid 1988;Madureira & Sobrinho 2002;Rutter & Rausher 2004;Fernandes et al. 2005;Nascimento & Del-Claro 2010;Del-Claro et al. 2016).
The structural diversity and topography of nectaries are used in taxonomic and evolutionary studies (Bhattacharyya & Mareshwari 1971;Metcalfe & Chalk 1979;Coutinho et al. 2012;Dalvi et al. 2013;Coutinho & Meira 2015).In Leguminosae, leaf nectaries are most common in the subfamily Mimosoideae, followed by Caesalpinioideae and less frequently in Papilionoideae (Polhill et al. 1981).The association with ants is a common defense system in Mimosoideae and Caesalpinioideae, while species of Papilionoideae are more dependent on chemical defenses (Polhill et al. 1981).
As suggested by Conceição et al. (2009), the leaf nectaries of Chamaecrista may have a single evolutionary origin.The species of Chamaecrista that bear leaf nectaries are placed in the sect.Apoucouita, Caliciopsis, Xerocalyx and Chamaecrista (except in the ser.Bauhinianae) and Absus subsect.Baseophyllum and Otophyllum (Irwin & Barneby 1982).
Molecular phylogenetic studies group sect.Caliciopsis, Chamaecrista and Xerocalyx in a single clade of species possessing inflorescences with axillary racemes and a reduced number of chromosomes as common characters (Conceição et al. 2009).Section Chamaecrista is the second most representative of the genus, with about 75 species (~55 in Americas) while Caliciopsis has only two species and Xerocalyx three, but a high number of varieties (Irwin & Barneby 1982;Rando & Pirani 2012;Rando et al. 2013).
The morphoanatomy of leaf nectaries has been studied in Chamaecrista sect.Apoucouita (Coutinho & Meira 2015) and sect.Absus subsect.Baseophyllum (Coutinho et al. 2012) and subsect.Otophyllum (Francino et al. 2015).These studies have demonstrated how different extra-reprodutive nectaries can be even among species of the same genus, since the chemical nature of the secretion of extra-reproductive nectaries in Chamaecrista can differ and some species develop a wound-healing periderm during senescence.The extra-reproductive nectaries of the species of Chamaecrista may be stalked or sessile, and are anatomically composed of four distinct regions (Coutinho et al. 2012;Coutinho & Meira 2015;Francino et al. 2015).
Regarding sect.Chamaecrista, only C. trichopoda, C. rotundata and C. mucronata of ser.Coriaceae have been investigated (Francino et al. 2006;2015), while in sect.Xerocalyx only one taxon has been evaluated, Chamaecrista desvauxii var.langsdorfii.Moreover, although sect.Caliciopsis has been reported to bear leaf nectaries on the petiole, as far as we are concerned, there have been no morphoanatomical studies on the petiole nectaries of this section.
This paper aims to expand the database on the morphoanatomy of leaf nectaries in the sect.Chamaecrista, Caliciopsis and Xerocalyx, with the intention of contributing to the taxonomy and phylogeny of the genus Chamaecrista.We address the following questions: Are the leaf glands of sect.Chamaecrista, Caliciopsis and Xerocalyx indeed nectaries?What is the chemical nature of the secreted compounds?Are there morphoanatomical differences that may indicate distinct patterns between sections or between species?Do morphoanatomical characteristics and the secreted products have any taxonomic implications at the level of section in the genus Chamaecrista?

Materials and methods
We studied 49 species of Chamaecrista (L.) Moench sect.Chamaecrista including species of all its series (i.A list of all sources of material used and authorities for sections, subsections and species names are given in the supplementary data.Field-collected samples and voucher specimens used are deposited in the following herbaria: HUNEB, HUEFS, NY, RB, SPF and VIC (acronyms according to Thiers 2016).When available, three specimens of each taxon were analyzed (List S1 in supplementary material).
Samples of fully developed leaves from herbarium material were rehydrated (Smith & Smith 1942) and stored in 70 % ethanol.Samples from species collected in the field were fixed in FAA (formalin:acetic acid:50 % ethanol, 1:1:18 by volume) and NBF (neutral buffered formalin) (Johansen 1940).Detection of phenolic compounds was performed through fixation with FSF (ferrous sulphate in formalin) (Johansen 1940).
Samples of petioles/rachises possessing glands were dehydrated through an ethanol series and embedded in methacrylate (Historesin Leica; Leica Microsystems Nussloch, Heidelberg, Germany).Cross and longitudinal sections 5 μm thick were made using an automatic rotary microtome (Leica RM2155, Deerfield, IL, USA) and subsequently stained with toluidine blue at pH 4.4 (O`Brien & McCully 1981) for structural characterization.The slides were mounted in synthetic resin (Permount, Fisher Scientific, New Jersey, USA).Some samples were dehydrated through a tert-butanol series, embedded in histological paraffin enriched with dimethyl sulfoxide (Histosec ® , Merck, Germany) (Johansen 1940) and cross and longitudinally sectioned at 7-μm thick using a rotary microtome (Spencer 820 American optical Corporation, Buffalo, NY, USA).The sections were deparaffinized in xylene, rehydrated through a decreasing ethanol series and used in histochemical tests (Johansen 1940).
For scanning electron microscopy (SEM) analysis, samples of glands stored in 70 % ethanol were critical-point dried with CO 2 (CPD 030, Bal-Tec, Balzers, Liechtenstein), mounted on stubs and sputter coated with gold (Modular Balzers Union FDU 010, SCA 010) (Bozzola & Russel 1991).Observations and image captures were obtained using a LEO model 1430 VP SEM (Cambridge, England).Morphological descriptions of glands are in accordance with Radford et al. (1974).

Results
Due to the presence of glucose in the secretion of the petiole/rachis glands of all the species tested with urinetest strips (i.In 11 taxa (Tab.1), nectaries were also present between the pairs of leaflets (Fig. 1A).These nectaries were located mainly at the apex of the petiole in ser.Coriaceae and sect.Caliciopsis and Xerocalyx (Tab.1), while in the ser.Prostratae and Greggiianae they were located predominantly in the median region.In ser.Flexuosae (Fig. 1G) and Chamaecrista, the nectaries were found in both the basal and median regions (Tab.1).
Although most species had one to two nectaries, some had a variable number, such as C. aristata (Fig. 1A).
The distribution of nectary forms is as follows: urceolate nectaries occured in three taxa of sect.Chamaecrista ser.Chamaecrista; patelliform nectaries occurred in 17 taxa of ser.Coriaceae, two of ser.Flexuosae, three of ser.Chamaecrista and in C. desvauxii var.glauca (sect.Xerocalyx); verruciform nectaties were exclusive to C. simplifacta (sect.Chamaecrista ser.Coriaceae); and cupuliform nectaries occured in 43 taxa (Tab.1).Although there was no pattern to the distribution of nectary forms with regard to sections, in ser.Coriaceae, nectaries were mostly patelliform, while in the ser.Prostratae and Greggiianae the nectaries were exclusively cupuliform.The secretory surface was concave in most of species studied, but convex in C. simplifacta and flat in C. vestita, C. pedicellaris var.pedicellaris and C. nictitans var.paraguariensis.
The nectaries were mostly short-stalked in ser.Coriaceae (Fig. 2F; Tab. 1).In ser.Flexuosae and in the sect.Xerocalyx the nectaries were short-stalked or stalked.On the other hand, the species of the ser.Prostratae and those of sect.Caliciopsis were found to have long-stalked nectaries.In the ser.Greggiianae, nectaries were stalked or long-stalked.As for ser.Chamaecrista, nectaries were found to be sessile, short-stalked, stalked or long-stalked (Fig. 3D).
In all species studied, the nectaries were characterized by having four distinct regions: a single-layered epidermis, a nectary parenchyma, a subnectary parenchyma and vascularization (Figs.2B, D, F, H, 3B, D, F).The epidermis was uniseriate, deprived of stomata, and with more columnar shaped cells at the edges of the nectary and smaller and more cuboidal, sometimes papillary, cells in the center (Figs.2B,  D, 4).Throughout the nectary, the cuticle was thick except at the center of the secretory epidermis (Fig. 4I).In most samples studied, a distended cuticle was observed in the central area (Figs.2A, H, 3E).Very prominent intercellular spaces were oobserved at the edges of the nectary (Fig. 2B, H, 3B).Non-glandular trichomes were observed, especially in the epidermal cells of the stalk (Fig. 3A).
The nectary parenchyma was typically formed of small polyhedral cells with dense cytoplasm (Fig. 4E).The number of cell layers in the secretory parenchyma was variable, from five, as observed in C. kunthiana, to more than 20, as was the case for C. repens (Fig. 2B).The subnectary parenchyma possessed highly vacuolated cells, which are larger than those of the secretory parenchyma (Fig. 3B).The number of cell layers in the subnectary parenchyma was also found to vary (Fig. 3F).
The nectaries of all species studied were vascularized predominantly by phloem (Figs.3F).Vascularization originates from the main vascular bundles in most taxa (Figs.2F, 4D; Tab. 1).In nine taxa, only the accessory bundles contributed to nectary vascularization (Tab.1), while in nine other taxa both the main and accessory vascular bundles contributed to nectary vascularization (Tab.1).
Fibers adjacent to the vascularization that reaches the nectary were noticed in 56 taxa (Figs.2F, 3D, Tab. 1).A layer of sclereids was found to clearly separate the secretory Note: A: Apex; M: Middle; B: Base.1: vascularization originates from the main vascular bundles; 2: vascularization originates from the accessory bundles; 3: Vascularization originates from both vascular accessory bundles and main vascular bundles.The histochenical results are summarizated in table 2. Total polysaccharides (Fig. 4A, B), acid muco-polysaccharides (Fig. 4C) and pectin (4D) were detected in the secretory parenchyma as well as in the intercellular spaces; proteins (Fig. 4E) were detected only in the protoplast of cells of the secretory parenchyma; general phenolic compounds (Fig. 4G) were detected in secretory parenchyma, subnectary parenchyma and epidermis; and lipids (Fig. 4H-J) were detected in the intercellular spaces and the cuticle.

Discussion
The glandular structures present on the petioles/rachises of the studied species are classified as extra-reproductive nectaries based on their topography, morphology, anatomical structure and presence of glucose.The structure of the nectaries studied here is similar to that of nectaries described for other species of Chamaecrista and for other genera of the subfamily Caesalpinioideae (Bhattacharyya & Maheshwari 1971;Elias 1983;Francino et al. 2006;2015;Paiva & Machado 2006;Melo et al. 2010;Coutinho et al. 2012;Coutinho & Meira 2015).
Cupuliform nectaries were the most common form recorded for the three sections studied.Patelliform nectaries are almost exclusive to species of the sect.Chamaecrista ser.Coriaceae.Our observations confirmed the occurrence of patelliform nectaries in three previously studied species of ser.Coriaceae and in C. desvauxii var.langsdorfii (section Xerocalyx) (Francino et al. 2015).Verruciform and urceolate nectaries were uncommon.The presence of cupuliform nectaries in C. flexuosa and C. swainsoni (ser.Flexuosae) and in the eight taxa of ser.Coriaceae studied is a morphological similarity that reinforces the hypothesis made by Conceição et al. (2009) and Rando et al. (2016), that ser.Flexuosae is the sister group to ser.Coriaceae.Additionally, C. caribaea, C. venulosa and C. roraimae, which all have the same type of nectary, were separated from the other species of ser.Coriaceae and considered related to species of ser.Chamaecrista, ser.Prostratae and sect.Caliciopsis, respectively (Rando et al. 2016).The species of sect.Caliciopsis, and most of the species of the sect.Xerocalyx studied, exhibited cupuliform nectaries, the same as observed in 37 taxa of sect.Chamaecrista.These data demonstrate affinities between the three sections, a relationship that had already been proposed in studies of molecular phylogeny (Conceição et al. 2009;Rando et al. 2016).
Nectaries are an effective tool for taxonomy because of variation in their shape and position on the plant body (Keeler & Kaul 1979;Bentley & Elias 1983).In species of Chamaecrista sect.Apoucouita, Coutinho & Meira (2015) and Coutinho et al. (2016) observed 13 different types of extra-reproductive nectaries and demonstrated their important role in the taxonomy of the genus.On the other hand, morphoanatomical similarities among the extra-reproductive nectaries of sect.Absus subsect.Baseophyllum seems to support the elevation of this subsection to sectional level (Coutinho et al. 2012), as proposed by Conceição et al. (2009) with studies based on molecular data.Although morphologically different, our study found anatomical similarities among the extrareproductive nectaries of Chamaecrista in that all of them comprise a single layered epidermis, several layers of nectary parenchyma with underlying layers of subnectary parenchyma and vascularization.Such anatomical similarity is also shared with other species of Chamaecrista that bear extra-reproductive nectaries (Coutinho et al. 2012;Coutinho & Meira 2015;Francino et al. 2015).
Extra-reproductive nectaries vascularized by xylem and phloem are common in species of the genus Chamaecrista (Coutinho et al. 2012;Coutinho & Meira 2015), and the vascularization often originates from the main vascular system of the petiole and/or rachis (Francino et al. 2006;Coutinho et al. 2012), as observed for most species in our study.The layer of sclereids that has been observed only in species of the ser.Coriaceae is similar to that found by Coutinho et al. (2012) in species of Chamaecrista sect.Absus subsect.Baseophyllum and may serve to provide mechanical support for nectaries.Paiva & Machado (2006) reported the presence of an endoderm with lignified and suberized cells in the nectary of Hymenaea stigonocarpa, and according to them the endoderm may prevent the reflux of nectar and direct its release extenally.A similar function can be attributed to the boundary layer of sclereids observed in the nectaries studied herein.The non-secreting parenchyma has also been considered a barrier to apoplastic transport, preventing the reflux of nectar to internal tissues (Contreras & Lersten 1984;Francino et al. 2006;Paiva & Machado 2006;Melo et al. 2010).The conspicuous intercellular spaces present in the secretory tissue of the nectaries of the species studied here have already been observed in other species of Chamaecrista (Coutinho et al. 2012;Coutinho & Meira 2015), and is considered the likely location for the accumulation of nectar prior to its being released to the exterior, as well as contributing to apoplastic transport of nectar (Vassilyev 2010).
The distended cuticle in the center of the secreting portion of the nectaries leads us to conclude that nectar is accumulated below the cuticle and that it is later secreted to the outside through cuticular burst.This manner of secretion release is in accordance with several authors (Fahn 1979;Elias 1983;Paiva & Machado 2006;Nepi 2007;Thadeo et al. 2008;Rocha et al. 2009;Paiva 2016).Conceição et al. (2009) hypothesized a single origin for extra-reproductive nectaries in Chamaecrista, which is supported, thus far, by the anatomical similarity among these structures.However, despite their singular origin, the extrareproductive nectaries of Chamaecrista may have followed different evolutionary trends, which may be supported by the type of secretion released.The extra-reproductive nectaries of the species of sect.Apoucouita secrete lipids in addition to carbohydrates and sugars (Coutinho & Meira 2015).In species of sect.Absus subsect.Baseophyllum, lipids were not detected, but phenolic compounds were (Coutinho et al. 2012).Similar to other species of Chamaecrista already studied, the nectar released by species of sect.Chamaecrista, Caliciopsis and Xerocalyx studied herein are complex.This nectar is made up of a mixture of lipids, phenolic compounds and proteins, which may be important in plant-animal interactions.Lanza et al. (1993) showed that Solenopsis geminata and S. invicta exhibited different preferences regarding nectar composition; S. gemitala workers preferred extra-reproductive nectars that were richer in aminoacids, while S. invicta did not discrinnate between the two types of nectar offered.Nectar richer in aminoacids is produced by Impatiens sultani when plants are subjected to simulated herbivory (Lanza et al. 1993).Therefore, if a mutualistic ant, such as S. geminata, which prefers aminoacid-rich nectars, was involved, such behavior could favor the survival of species with aminoacid-rich extra-reproductive nectars.Such preferential behavior may also exist for other substances, such as the presence/absence of lipids, cardohydrates and so on.Comprehensive evolutionary and ecological studies are needed for the species of the genus Chamaecrista in order to better understand the role of secretions in mediating interactions with visitors and promoting the evolution and diversification of the group.
The lipid content found in the intercellular spaces of the species studied here is in accordance with reports for other genera, including Chamaecrista (Baker et al. 1978;Fahn 1979;1988;2000;Coutinho & Meira 2015).As stated by Paiva & Machado (2006), the presence of lipids in the intercellular spaces of the secretory parenchyma indicates that these compounds are of the secretion and that the plant offers a reward in a more energetic form.Additionally, extreme environmental conditions may require high-energy food resource for visitors, a hypothesis proposed by Forcone et al. (1997) and Bernardello et al. (1999).The phenolic compounds found within idioblasts may act in defense against herbivores, as their content renders a plants organs/ structures unpalatable, in adidiiton to providing protection from pathogens (Nicolson & Thornburg 2007).Some studies have suggested that plants with high levels of amino acids in their nectar attract more ants, and therefore may suffer less herbivory (Lanza 1991;Wagner & Kay 2002;Wilder & Eubanks 2009).
Although anatomical similarities were observed among the species studied, the morphology differed.The form of the nectaries of ser.Prostratae and Greggiianae were found to be well-defined, representing important data for the taxonomy of these series.The similarity among the nectaries of the three studied sections (i.e.Chamaecrista, Caliciopsis and Xerocalyx) may also be of taxonomic value in justifying the grouping of these three sections into a single clade.
Our study also provides unprecedented data regarding the anatomy of the extra-reproductive nectaries of the species of the sect.Caliciopsis.The database provided by this work will likely be important for future studies into the taxonomy and phylogeny of these plants.