Colleters in Chamaecrista ( L . ) Moench sect . Chamaecrista and sect . Caliciopsis ( Leguminosae-Caesalpinioideae ) : anatomy and taxonomic implications

Th e genus Chamaecrista contains ca. 330 species organized into six sections, of which section Chamaecrista is the second largest (ca.75 species) distributed among six series, while the section Caliciopsis contains only two species. Colleters have been described in the genus Chamaecrista and they show potential taxonomic and phylogenetic signifi cance. Th ey are associated with lubrication, desiccation prevention and protection from microbial attacks of young developing organs. Although six types of colleters have been described for the genus Chamaecrista, there have been no studies focusing on the diversity of colleters in the sections Chamaecrista and Caliciopsis. Samples from developing leaves and fl owers of both sections were obtained from herbarium and fi eld collections and subjected to standard methodologies for both light and scanning electron microscopy. Histochemical tests were also performed to determine the nature of the exudates. Five types of non-vascularized colleters were found: short digitiform, long digitiform, club-shaped, pyriform and short bottle-shaped. Polysaccharides, pectins, lipids and proteins were detected in the exudates of all types of colleters. Among the fi ve types of colleters observed, pyriform is a novelty for Chamaecrista, reinforcing the signifi cant morphological diversity of these secretory structures in this genus.


Introduction
Chamaecrista is one of the largest genera of the subfamily Caesalpinioideae, with ca.330 species (Lewis 2005).According to Irwin & Barneby (1982), the genus is organized into six sections on the basis of the type of indumentum, the presence or absence of extrafl oral nectaries, the type of infl orescence and the type of venation of both sepals and leafl ets.Th e six sections (and the number of species) are: Chamaecrista sect.Absus (ca.170 spp.), Apoucouita (ca.20 spp.), Caliciopsis (two spp.), Chamaecrista (ca.75 spp.),Grimaldia (one sp.) and Xerocalyx (three spp.).
Section Chamaecrista is the second most speciose section, with 2/3 of its species occurring in the America (Irwin & Barneby 1982;Rando & Pirani 2012;Rando et al. 2013).Species grouped in sect.Chamaecrista are characterized as shrubs or herbs with infl orescences with racemes reduced to either a few-fl owered fascicles or to a solitary fl ower, several pairs of pinnately-veined leafl ets, seeds obovate to rhombic or trapezoid in outline and extrafl oral nectaries commonly present (Irwin & Barneby 1982).Six series have been described for sect.Chamaecrista: series Bauhinianae, Chamaecrista, Coriaceae, Flexuosae, Greggianae and Prostratae.Although described by Irwin & Barneby (1982) as a separate section, a phylogenetic analysis of the genus Chamaecrista, based on molecular data, has brought sect.Caliciopsis within sect.Chamaecrista (Conceição et al. 2009).
Different secretory structures with high potential for taxonomic and phylogenetic relevance have been described in Chamaecrista, including: nectaries (on leaves and/or racemes), mucilage idioblasts in the mesophyll and/or epidermis, sticky glandular hairs and colleters (Irwin & Barneby 1982;Coutinho et al. 2012;2013;2015;2016;Meira et al. 2014;Francino et al. 2015).Colleters are secretory structures that are usually found on the adaxial side of vegetative and/or reproductive structures such as stipules, bracts, sepals and petals (Fahn 1979;Thomas 1991;Mayer et al. 2013;Coutinho et al. 2015).The viscous exudates secreted by colleters are said to be involved in lubrication, desiccation prevention and protection from microbial attacks of young and developing organs (Fahn 1979;Thomas 1991;Mayer et al. 2013;Coutinho et al. 2015).
There are several types of colleters, which may originate from protodermal cells only or from all primary meristems (Lersten 1974;Thomas 1991;Appezzato-da-Glória & Estelita 2000;Rio et al. 2002;Silva et al. 2012;Coutinho et al. 2015).Therefore, to properly evaluate the presence of colleters in plants, their morphoanatomy, position and both time and composition of the secretion must be considered in order to avoid erroneous interpretations since such structures share morphoanatomical similarities with other secretory structures (Solereder 1908;Inamdar et al. 1986;Mohan & Inamdar 1986;Subramanian et al. 1989;Thomas 1991;Coutinho et al. 2015).
The presence and types of colleters are useful characters for taxonomic studies and have been reported for more than 60 families (Thomas 1991;Rio et al. 2005;Simões et al. 2006;Silva et al. 2012;Dalvi et al. 2013;Coutinho et al. 2015).In Chamaecrista, colleters have been described in vegetative and reproductive organs of species belonging to sections Absus, Apoucouita, Grimaldia, Chamaecrista and Xerocalyx (Coutinho et al. 2013;2015).Six types of colleters were morphoanatomically described: short digitiform, long digitiform, short bottle-shaped, long bottle-shaped, clubshaped and racket-shaped (Coutinho et al. 2013;2015).The type and distribution of colleters have provided new data that reinforce the sectional rearrangement indicated by the molecular phylogeny of Chamaecrista (Coutinho et al. 2015).The presence of club-shaped colleters on the margins of sepals was indicated as a synapomorphy for species of sect.Apoucouita.Most species of sect.Absus subsect.Absus had short bottle-shaped colleters, the same type as found in C. absus (sect.Grimaldia).Based on these data, the authors suggested that the sectional boundaries of sect.Grimaldia should be reconsidered in a future taxonomic revision of Chamaecrista to determine if this section should be included within sect.Absus subsect.Absus.Although short bottle-shaped colleters were the most common type for sect.Chamaecrista, short digitiform and club-shaped types were also observed.However, only five species of sect.Chamaecrista were sampled by Coutinho et al. (2015), and none of the species of section Caliciopsis have been sampled in previous studies.This gap in knowledge has limited the taxonomic usefulness of colleters in sect.Chamaecrista and Caliciopsis.
In this paper, we assess the occurrence, distribution and types of colleters among species of Chamaecrista sect.Chamaecrista (including samples of 32 species not previously studied) and Caliciopsis (including samples of all species).The composition of the exudates of the colleters, as well as the taxonomic implications for the genus Chamaecrista, were also evaluated.

Materials and methods
We studied 37 species (50 taxa) of Chamaecrista (L.) Moench sect.Chamaecrista, and two species of sect.Caliciopsis (Tab.1).Taxonomic authorities for all taxa mentioned in this paper are given in S1 in supplementary material.Samples were obtained from field collections and herbarium material.Voucher specimens of field collections were deposited in the herbarium of the Universidade Federal de Viçosa (VIC) and Universidade do Estado da Bahia (HUNEB).Herbarium material was obtained from the collections of the following herbaria: Jardim Botânico do Rio de Janeiro (RB), Universidade Estadual de Feira de Santana (HUEFS), Universidade Federal de Viçosa (VIC), Universidade de São Paulo (SPF), and The New York Botanical Garden (NY).
In order to assess the presence, position and micromorphology of the colleters we used a stereomicroscope (Zeiss Stemi 2000-C, Germany) and a scanning electron microscopy (SEM).For SEM, some samples stored in 70 % ethanol were subjected to critical-point drying using CO 2 (CPD 030, Bal-Tec, Balzers, Liechtenstein), mounted on stubs and coated with gold (Modular Balzers Union FDU 010 with a SCA 010 sputter coating attachment, Germany) (Bozzola & Russel 1991).Examination and image capture were conducted using a LEO 1430VP SEM (Zeiss, Cambridge, United Kingdom) at the Centro de Microscopia e Microanálises at the Universidade Federal de Viçosa.
Developing leaves and flowers of both dried and fieldcollected material stored in 70 % ethanol were dehydrated through an ethanol series and embedded in methacrylate Acta Botanica Brasilica -31(3): 382-391. July-September 2017   Table 1.Chamaecrista sections Chamaecrista and Caliciopsis species studied and position of the colleters.1981) and mounted in resin (Permount, Fisher Scientific, New Jersey, USA) for structural analysis.Part of the fixed samples were dehydrated through tertbutanol series, embedded in histological paraffin enriched with dimethyl sulfoxide (Histosec ® , Merck, Germany), cut into 7 μm thick cross and longitudinal sections (Spencer 820 American Optical Corporation, Buffalo, New York, USA), deparaffinized in xylene, rehydrated through an ethanol series (Johansen 1940) and used in histochemical tests.

Section
The following histochemical tests were performed on 13 taxa using material previously fixed in the field with FAA or NBF (Tab.1): for total lipids, sudan IV (Pearse 1980); for total polysaccharides, periodic acid-Schiff reagent (Maia 1979); for pectins/mucilage, ruthenium red (Johansen 1940); for acid mucopolysaccharides, alcian blue (Pearse 1980); and for total protein, xylidine Ponceau (Clark 1981).For detection of phenolic compounds, we used samples fixed in FFS (Johansen 1940).Control samples were also tested as required for each test.All observations and image captures were obtained using a light microscope (model AX70TRF; Olympus Optical, Tokyo, Japan) equipped with a U-Photo and digital camera (AxioCam HRc; Zeiss, Gottingen, Germany).The types of colleters found are described following Coutinho et al. (2015), as was the data for C. caribaea Britton and C. cinerascens (Vogel) H.S.Irwin & Barneby.

Results
Non-vascularized colleters composed of homogenous cells were found on both vegetative and reproductive organs (Tab.1).Colleters were comprised of a stalk of varying length, and a secretory head of varying shape (Figs. 1, 2).The stalk cells were non-secretory and highly vacuolated while the cells from secretory head displayed densely stained cytoplasm (Fig. 2C-E).There was no differentiation of a palisade epidermis since the epidermal cells are similar in appearance to the internal cells of the secretory head (Fig. 2H).The cuticle was thin and appeared distended or loose in some sections (Fig. 2B) or SEM images (Fig. 1I); no pores were observed in the cuticle.Older colleters exhibit a brownish color.
Colleters were located on the adaxial side of vegetative and reproductive structures including the base of stipules, bracts and bracteoles; the base of sepals and petals; on the rachis between pairs of leaflets; and on the stem at the insertion of the petiole (Tab.1).Five types of colleters were observed (Tab.

Base of stipule
Between leaflets

Base of petal
Table 1.Cont.Types I, II and III were the most common types of colleters observed among the studied species (Tab.1), with Type I being found in all taxa, while Type II occurred  The presence of total polysaccharides (Fig. 3A, B), mucopolysaccharides (Fig. 3C), pectins (Fig. 3D), proteins (Fig. 3E) and total lipids (Fig. 3F, G) was confirmed by the histochemical tests for all types of colleters described.Of all the histochemical tests performed, only the secretory head of the colleters showed positive results.

Discussion
According to the position, morphology, anatomy and the composition of the exudates, the secretory structures present on the base of stipules, between leaflets, at the petiole insertion, and among sepals, petals and bracts/ bracteoles of the studied species of Chamaecrista sect.Chamaecrista and Caliciopsis are indeed colleters.Such structures are typically related to the protection of young leaves and flowers from desiccation and attack from microorganisms since the secretion present is made of mucilage or a mixture of mucilage, resin and protein (Thomas & Dave 1989;Thomas 1991;Rocha et al. 2009).
In Chamaecrista sect.Chamaecrista, colleters produce a mixture of hydrophilic (polysaccharides and proteins) and lipophilic (lipids) compounds.Polysaccharides are able to retain water and hence their presence in colleter secretion may indicate that they play an important functional role in keeping young structures hydrated (Fahn & Cutler 1992).Lipids have been detected in the colleter secretion of species belong Apocynaceae (Appezzato-da-Glória & Estelita 2000), Leguminosae (Paiva 2009), Gentianaceae (Dalvi et al. 2013), Orchidaceae (Mayer et al. 2011) and Rubiaceae (Machado et al. 2012;Tullii et al. 2013).As claimed for polysaccharides, lipid compounds may help avoid water loss, in addition to prohibiting fungal and microorganism attack, since this hydrophobic substance lubricates the surface of young leaves and floral buds, (Fahn 1979;Thomas & Dave 1989;Thomas 1991;Evert 2006;Paiva 2009;Mayer et al. 2013).Proteins have also been reported as a component of the secretion produced by colleters from a variety of different species (Thomas & Dave 1990;Klein et al. 2004;Miguel et al. 2006;González & Tarragó 2009;Mayer et al. 2011;Dalvi et al. 2013).Some authors have suggested that such proteins may provide additional protection from fungi and parasites (Miguel et al. 2006;Vieira et al. 2006;Mayer et al. 2011).However, research aiming to unravel how exudates produced by colleters may contribute to the success of species of Chamaecrista in stressful environments is still needed.
Although cuticular pores were not observed among the colleters of the studied species, a few samples had loosely packed secretory cells at the colleter apex and a distended cuticle.This arrangement of cells and cuticle is an indication that the secretion may be released to the outside by cuticle rupture.Releasing of secretion by cuticle rupture has been suggested for colleters of Hymenaea stigonocarpa (Paiva & Machado 2006b) and demonstrated in colleters of Senna macranthera (Souza 2014), both belonging to Caesalpinioideae, as well as other families (Paiva & Machado 2006a;Tullii et al. 2013).
As observed in the species of the sect.Chamaecrista and Caliciopsis studied, non-vascularized colleters have bee previously reported for other species of Chamaecrista belonging to other sections (De-Paula & Oliveira 2007;Coutinho et al. 2013;2015), other species of Caesalpinioideae (Paiva & Machado 2006b;Paiva 2009;Souza 2014), and even species of other families (Paiva & Machado 2006a;Paiva 2009;Vitarelli & Santos 2009;Silva et al. 2012;Dalvi et al. 2013).Anatomically, the colleters described for the species of sect.Chamaecrista and Caliciopsis studied exhibit the same homogeneous pattern of cells as reported for other Chamaecrista species (Coutinho et al. 2015).
Although Lersten (1974) pointed out that the standard type of colleter is the most widespread, variation in the morphoanatomy of these structures has since been observed such that now several different types of colleters are recognized (Mayer et al. 2011;Silva et al. 2012;Coutinho et al. 2015).Standard type colleters are comprised of a secretory palisade epidermis covering a non-secretory central axis, which may or may not be vascularized with xylem and phloem (Lersten 1974).In contrast to this standard type of colleter, which has been described for other botanical families (Appezzato-da-Glória & Estelita 2000;Paiva & Machado 2006a;Vitarelli & Santos 2009), the most common type of colleter for the genus Chamaecrista, as shown by our data along with data provided by Coutinho et al. (2015), are comprised of homogenous cells.
As the colleters of the studied species of Chamaecrista age, they exhibit a brownish color and usually fall off when they stop secreting.Similar observations were reported for other species of Chamaecrista (Coutinho et al. 2015).This change in color may be the result of the oxidation of phenolic compounds accumulated within the cells, which is followed by the shrinking of the cytoplasm of the apical cells of colleters (Souza 2014).Similar descriptions were reported for colleters from a variety of botanical families (Thomas 1991;Paiva 2009;Souza 2014).
Colleters were observed in all of the analyzed taxa from both sect.Chamaecrista and Caliciopsis.Within subfamily Caesalpinioideae, the presence of colleters stands out when we take species from subtribe Cassinae (tribe Cassieae) into account, since five species of Senna (Souza 2014) (Rio 2001), Forsteronia (Rio 2006), Mandevilla pycnantha and M. tenuifolia (Simões et al. 2006), all species that belong to Apocynaceae, as well as in Curtia and Hockinia (Dalvi et al. 2013), species of Gentianaceae.We suspect that this bifurcation is the result of abnormalities during colleter development, and so do not correspond to a distinct type of colleter.
Although six distinct types of colleters have been described for species of Chamaecrista (Coutinho et al. 2015), only five species of ser.Coriaceae (sect.Chamaecrista) were sampled and nothing about sect.Caliciopsis was provided.The same authors reported short digitiform (I), club-shaped (III) and short bottle shaped (V) colleters for sect.Chamaecrista.Our more comprehensive sample of sect.Chamaecrista enabled us to observe two additional types of colleters for this section: long digitiform and pyriform.New information on the occurrence of colleters at the insertion of the petiole of C. aristata, C. caribaea, C. cinerascens, C. potentilla and C. simplifacta, which was overlooked by previous authors, is also provided here.The pyriform (VII) type of colleter is a novelty for Chamaecrista, and is exclusive to C. rufa var.exsul (ser.Chamaecrista), while racket-shaped (IV) and long bottle-shaped (VI) colleters were not detected in the sections studied.Coutinho et al. (2015) suggested that the length (short and long) of colleters Type I and II is related to the size of the structure on which they are located such that larger stipules and bracts may bear both colleter types, while in smaller ones only the short digitiform type of colleter (Type I) would develop.However, among the species studied herein, we were able to observe that both types of colleters are independent of the size of the bearing structure.
Short digitiform (I) and short bottle shaped (V) colleters have been observed only in C. tragacanthoides var.rasa, C. venulosa, the three varieties of C. caribaea, species that belong to ser.Coriaceae, and two taxa of sect.Caliciopsis.Considering that the infrageneric relationships of Chamaecrista remain unclear, colleter type is emerging as a relevant and promising character for understanding the relationships among the species of Chamaecrista.According to both molecular and morphological data, Chamaecrista ser.Coriaceae was recognized as a monophyletic group, excluding C. caribaea, C. roraimae, and C. venulosa (Rando et al. 2016).The diversity of types and different positions reported for colleters in Chamaecrista, both by us and other authors, emphasizes how important and poorly understood this structure is.Such data promise to be useful for both taxonomic and phylogenetic studies, as has been the case for other taxa (Lersten 1975;Curtis & Lersten 1980;Thomas 1991;González 1998;Simões et al. 2006;González & Tarragó 2009;Sheue et al. 2012;Silva et al. 2012;Vitarelli et al. 2015;Fernandes et al. 2016).However, further studies are necessary to thoroughly evaluate the evolutionary history of colleters in Chamaecrista.
We showed that colleters are diverse structures in Chamaecrista sect.Chamaecrista and Caliciopsis.The short digitiform and short bottle-shaped colleters found in the two species of sect.Caliciopsis were also observed in two (C.caribaea and C. venulosa) of the three species that should be excluded from ser.Coriaceae (sect.Chamaecrista), as suggested by a recent molecular phylogenetic study.Such similar characters may suggest species of sect.Caliciopsis are closer to species of ser.Coriaceae, therefore, the position of the such species of ser.Coriaceae should be revised.However, only a comprehensive study of species of sect.Chamaecrista, including representatives of sect.Caliciopsis, will be able to confirm such a hypothesis.In addition to the types of colleters already described for sect.Chamaecrista, we added two new types of colleters (long digitiform and pyriform).Besides, the pyriform type is a novelty for the genus Chamaecrista.Our results are promising as they may be useful to future analyses combining phylogenetic and evolutionary approaches, and encourage further research on other members of Caesalpinioideae.
I, III, V I, III, V I, III, V I, III, V I, III, V I a (Historesin Leica; Leica Microsystems Nussloch, Heidelberg, Germany).Cross and longitudinal sections 5-7 μm thick were made with a rotary microtome (Spencer 820 American Optical Corporation, Buffalo, NY, USA).Sections were stained with toluidine blue at pH 4.4 (O'Brien & McCully
and 55 species of Chamaecrista (De-Paula & Oliveira 2007; Coutinho et al. 2015) display these secretory structures.Bifurcated colleters, like those observed in seven of the taxa studied here, were also reported for Temnadenia violacea (Martins et al. 2010), Prestonia coalita