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

ABSTRACT

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 confirm 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 field-collected material were subjected to standard methods for light and scanning electron microscopy. Four different forms of nectaries were observed: urceolate, patelliform, verruciform and cupuliform. The 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 differed. Moreover, the glands are indeed nectaries and are similar to those observed in other species of the genus Chamaecrista. These data hold potential taxonomic usefulness for the studied sections.

Keywords
foliar glands; histochemistry; leaf glands; secretory structures; taxonomy

Introduction

Nectaries are specialized structures for the secretion of a sugary solution called nectar, which contains sucrose, glucose and fructose as primary solutes (Fahn 1979Fahn A. 1979. Secretory tissues in plants. London, Academic Press.; Nicolson et al. 2007Nicolson SW, Nepi M, Pacini E. 2007. Nectaries and nectar. Dordrecht, Springer .). According to their location, nectaries are classified as reproductive (i.e. when found on the inflorescence axis, bracts, sepals, ovary, stamens, etc.) and extra-reproductive (i.e. when found on the petiole, rachis, leaf blade, stems, etc.) (Schmid 1988Schmid R. 1988. Reproductive versus extra-reproductive nectarines - historical perspective and terminological recommendations. Botanical Review 54: 179-227. ). 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 1988Schmid R. 1988. Reproductive versus extra-reproductive nectarines - historical perspective and terminological recommendations. Botanical Review 54: 179-227. ; Madureira & Sobrinho 2002Madureira M, Sobrinho TG. 2002. Evidência de mutualismo entre Qualea cordata (Vochysiaceae) e Cephalotes sp. (Hymenoptera: Formicidae). Academia Insecta 2: 1-4.; Rutter & Rausher 2004Rutter MT, Rausher MD. 2004. Natural selection on extrafloral nectar production in Chamaecrista fasciculata: the costs and benefits of a mutualism trait. Evolution 58: 2657-2668.; Fernandes et al. 2005Fernandes GW, Fagundes M, Greco MKB, Barbeitos MS, Santos JC. 2005. Ants and their effects on an insect herbivore community associated with the inflorescences of Byrsonima crassifolia (Linneaus) H.B.K. (Malpighiaceae). Revista Brasileira de Entomologia 49: 264-269.; Nascimento & Del-Claro 2010Nascimento EA, Del-Claro K. 2010. Ant visitation to extrafloral nectaries decreases herbivory and increases fruit set in Chamaecrista debilis (Fabaceae) in a Neotropical savanna. Flora 205: 754-756.; Del-Claro et al. 2016Del-Claro K, Rico-Gray V, Torezan-Silingardi, HM et al. 2016. Loss and gains in ant-plant interactions mediated by extrafloral nectar: fidelity, cheats, and lies. Insectes Sociaux 63: 207-221.).

The structural diversity and topography of nectaries are used in taxonomic and evolutionary studies (Bhattacharyya & Mareshwari 1971Bhattacharyya B, Maheshwari JK. 1971. Studies on extrafloral nectaries of the Leguminales - II. The genus Cassia Linn. (Caesalpiniaceae). Proceedings of the Indian National Science Academy 37: 74-90.; Metcalfe & Chalk 1979Metcalfe CR, Chalk L. 1979. Anatomy of the Dicotyledons. Vol. 1. Oxford, Clarendon Press.; Coutinho et al. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.; Dalvi et al. 2013Dalvi VC, Meira RMSA, Azevedo AA. 2013. Extrafloral nectaries in neotropical Gentianaceae: Occurrence, distribution patterns, and Anatomical characterization. American Journal of Botany 100: 1-11.; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.). In Leguminosae, leaf nectaries are most common in the subfamily Mimosoideae, followed by Caesalpinioideae and less frequently in Papilionoideae (Polhill et al. 1981Polhill RM, Raven PH, Stirton CH. 1981. Evolution and systematics of the Leguminosae. In: Polhill RM, Raven PH. (eds.) Advances in Legumes systematic Part 1. Kew, Royal Botanic Gardens. p 1-26.). 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. 1981Polhill RM, Raven PH, Stirton CH. 1981. Evolution and systematics of the Leguminosae. In: Polhill RM, Raven PH. (eds.) Advances in Legumes systematic Part 1. Kew, Royal Botanic Gardens. p 1-26.).

As suggested by Conceição et al. (2009Conceição AS, Queiroz LP, Lewis GP, et al. 2009. Phylogeny of Chamaecrista Moench (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168-1180.), 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 1982Irwin HS, Barneby RC. 1982. The American Cassiinae, a synoptical revision of Leguminosae tribe Cassieae subtribe Cassiinae in the New World. Memoirs of the New York Botanical Garden 35: 1-918.).

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. 2009Conceição AS, Queiroz LP, Lewis GP, et al. 2009. Phylogeny of Chamaecrista Moench (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168-1180.). 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 1982Irwin HS, Barneby RC. 1982. The American Cassiinae, a synoptical revision of Leguminosae tribe Cassieae subtribe Cassiinae in the New World. Memoirs of the New York Botanical Garden 35: 1-918.; Rando & Pirani 2012Rando JG, Pirani JR. 2012. A new species of Chamaecrista sect. Chamaecrista ser. Flexuosae (Leguminosae, Caesalpinioideae) from Serra do Cipó, Minas Gerais, Brazil. Brittonia 64: 241-245. ; Rando et al. 2013Rando JG, Loeuille B, Pirani JR. 2013. Taxonomic novelties in Chamaecrista (Leguminosae: Caesalpinioideae) from Brazil. Phytotaxa 97: 17-25.).

The morphoanatomy of leaf nectaries has been studied in Chamaecrista sect. Apoucouita (Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.) and sect. Absus subsect. Baseophyllum (Coutinho et al. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.) and subsect. Otophyllum (Francino et al. 2015Francino DMT, Coutinho IAC, Dalvi VC, Azevedo AA, Conceição AS, Meira RMSA. 2015. Anatomical interpretations of the taxonomy of Chamaecrista section Absus (Leguminosae - Caesalpinioideae s.l.). Plant Systematics and Evolution. 301: 2087-2103.). 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. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.; Francino et al. 2015Francino DMT, Coutinho IAC, Dalvi VC, Azevedo AA, Conceição AS, Meira RMSA. 2015. Anatomical interpretations of the taxonomy of Chamaecrista section Absus (Leguminosae - Caesalpinioideae s.l.). Plant Systematics and Evolution. 301: 2087-2103.).

Regarding sect. Chamaecrista, only C. trichopoda, C. rotundata and C. mucronata of ser. Coriaceae have been investigated (Francino et al. 2006Francino DMT, Sant’Anna-Santos BF, Silva KLF. 2006. Anatomia foliar e caulinar de Chamaecrista trichopoda (Caesalpinioideae) e histoquímica do nectário extrafloral. Planta Daninha 2: 695-705.; 2015Francino DMT, Coutinho IAC, Dalvi VC, Azevedo AA, Conceição AS, Meira RMSA. 2015. Anatomical interpretations of the taxonomy of Chamaecrista section Absus (Leguminosae - Caesalpinioideae s.l.). Plant Systematics and Evolution. 301: 2087-2103.), 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.e. Coriaceae (Benth.) H.S.Irwin & Barneby, Flexuosae H.S.Irwin & Barneby, Prostratae (Benth.) H.S.Irwin & Barneby, Greggiianae H.S.Irwin & Barneby and Chamaecrista) two species of sect. Caliciopsis Irwin & Barneby and three species of sect. Xerocalyx (Bentham) Irwin & Barneby (Tab. 1). 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 2016Thiers B. 2016. Index Herbariorum: A global directory of public herbaria and associated staff. New York, New York Botanical Garden’s Virtual Herbarium. ). 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 1942Smith FH, Smith EC. 1942. Anatomy of the inferior ovary of Darbya. American Journal of Botany 29: 464-471.) 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 1940Johansen DA. 1940. Plant microtechnique. New York, McGraw-Hill. ). Detection of phenolic compounds was performed through fixation with FSF (ferrous sulphate in formalin) (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York, McGraw-Hill. ).

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 1981O`Brien TPE, Mccully ME. 1981. The study of plant structure principles and select methods. Melbourne, Termarcarphi Pty.) 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 1940Johansen DA. 1940. Plant microtechnique. New York, McGraw-Hill. ) 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 1940Johansen DA. 1940. Plant microtechnique. New York, McGraw-Hill. ).

The presence of glucose in gland exudates was tested using urinetest strips (Alamar Tecno Científica Ltda., São Paulo, Brazil) for the following species: C. rotundata (Vogel) H.S.Irwin & Barneby, C. mucronata (Spreng.) H.S.Irwin & Barneby, C. latifolia (Benth.) Rando, C. potentilla (Mart. ex Benth.) H.S.Irwin & Barneby, C. simplifacta H.S.Irwin & Barneby, C. cinerascens (Vogel) H.S.Irwin & Barneby, C. choriophylla (Vogel) Irwin & Barneby, C. aristata (Benth.) H.S.Irwin & Barneby, C. papillata H.S.Irwin & Barneby and C. flexuosa Greene. The following histochemical tests were performed: neutral red (under fluorescence) and Sudan IV for total lipids (Pearse 1980Pearse AGE. 1980. Histochemistry theoretical and applied. Vol 2. Edinburgh, Churchill Livingston.); periodic acid-Schiff reagent for total polysaccharides (Maia 1979Maia V. 1979. Técnica histológica. São Paulo, Atheneu.); ruthenium red for pectins/mucilage (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York, McGraw-Hill. ); alcian blue for acid mucopolysaccharides (Pearse 1980Pearse AGE. 1980. Histochemistry theoretical and applied. Vol 2. Edinburgh, Churchill Livingston.); xylidine Ponceau for total proteins (O’Brien & McCully 1981O`Brien TPE, Mccully ME. 1981. The study of plant structure principles and select methods. Melbourne, Termarcarphi Pty.); phloroglucinol for lignin; and ferrous sulphate in formalin for phenolic compounds (Johansen 1940Johansen DA. 1940. Plant microtechnique. New York, McGraw-Hill. ). All observations and image captures were obtained using a light microscope (model AX70TRF; Olympus Optical, Tokyo, Japan) equipped with U-Photo and a digital camera (AxioCam HRc; Carl Zeiss, Gottingen, Germany).

For scanning electron microscopy (SEM) analysis, samples of glands stored in 70 % ethanol were critical-point dried with CO2 (CPD 030, Bal-Tec, Balzers, Liechtenstein), mounted on stubs and sputter coated with gold (Modular Balzers Union FDU 010, SCA 010) (Bozzola & Russel 1991Bozzola JJ, Russel LD. 1991. Electron microscopy: principles and techniques for biologists. New York, Jones and Bartlett Publishers.). 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. (1974Radford AE, Dickison WC, Massey JR, Bell CR. 1974. Vascular plant systematics. New York, Harper & Row.).

Results

Due to the presence of glucose in the secretion of the petiole/rachis glands of all the species tested with urinetest strips (i.e. C. rotundata, C. mucronata, C. latifolia, C. potentilla, C. simplifacta, C. cinerascens, C. choriophylla, C. aristata, C. papillata and C. flexuosa), the glands are hereafter considered nectaries. Nectaries occured in a variety of positions on the petioles of the studied species (Fig. 1, Tab. 1). 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).

Figure 1
Nectaries on leaves of species of Chamaecrista sect. Chamaecrista and Xerocalyx. A. C. aristata: nectary on petiole and raquis. B. C. lagotois: sessile patelliform. C-D. C. venturiana and C. nictitans var. paraguariensis: sessile and short-stalked patelliform respectivelly. E. C. simplifacta: verruciform. F. C. desvauxii var. desvauxii: short-stalked cupuliform. G. C. flexuosa var. flexuosa: stalked cupuliform. H. C. pascuorum: long-stalked cupuliform. I. C. vestita: long-stalked cupuliform. Scale bars: A: 5000 µm; B: 2000 µm; C-I: 1000 µm.

Table 1
Nectaries in Chamaecrista sections Chamaecrista, Caliciopsis and Xerocalyx.

Four forms of nectaries were recorded: urceolate (Fig. 2A, B), patelliform (Fig. 2C, D), verruciform (Fig. 2E, F) and cupuliform (Figs. 2G, H, 3A-E). Secretory surfaces were found to be concave (Fig. 2D, H, 3B), convex (Fig. 2F) or flat (Fig. 3D). Some nectaries were stalked (i.e. a cylindrical structure that is vascularized and nonsecretory, but which bears a secretory portion at the top) while others were sessile (Tab. 1). Urceolate nectaries were sessile; patelliform were sessile (Fig. 2D) or short stalked; verruciform were short-stalked (2F) and cupuliform were short-stalked, stalked (Fig. 3B) or long-stalked (Fig. 3C-E).

Figure 2
SEM images and anatomical sections (stained with toluidine blue) of nectaries of Chamaecrista sect. Chamaecrista. A-B. C. repens var. repens: sessile urceolate (note detail of intercellular spaces). C-D. C. lagotois: sessile patelliform. E-F. C. simplifacta: short-stalked verruciform. G-H. C. potentilla: short-stalked cupuliform. Note the cuticle distended (arrow) and intercellular spaces (asterisk). (Ep: epidermis; Sc: sclereids; NP: nectary parenchyma; PA: subnectary parenchyma; Va: vascular tissue). Scale bars: A: 200 µm; B, G: 100 µm; C, D: 400 µm; H: 50 µm; E, F: 300 µm.

Figure 3
SEM images and anatomicals sections (stained with toluidine blue) of nectaries of Chamaecrista sect. Chamaecrista. A-B. C. roraimae: stalked cupuliform. C-E. C. vestita and C. pedicellaris var. pedicellaris, respectivelly: long-stalked cupuliform (Note the fibers: arrow). F. C. mucronata. Note the cuticle distended (arrow) and intercellular spaces (asterisk). (NP: nectary parenchyma; Va: vascular tissue; Pa: subnectary parenchyma; Ph: phloem; Xy: xylem; Sc: esclereides; TT: non-glandular trichomes). Scale bars: A, C, E, F: 200 µm; B, D: 300 µm.

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).

Figure 4
Histochemical tests in nectaries of Chamaecrista sect. Chamaecrista. A-B. Totals polysaccharides (periodic acid-Schiff reagent). A. C. desvauxii var. glauca. B. C. tragacanthoides var. tragacanthoides. C. Acid mucopolysaccharides (alcian blue). C.desvauxii var. glauca. D. Pectins (ruthenium red). C. roraimae. E. Totals proteins (xylidine Ponceau). C. mucronata. F. Lignin (phloroglucinol). C. simplifacta. G. General phenolic compounds (ferrous sulfate in formalin). C. simplifacta. H-J. Totals lipids (Sudan IV/ neutral red). H. C. roraimae (fluorescence). I. C. potentilla. J. C. lagotois. (Ep: epidermis; Ct: cuticle; Ph: phloem; Sc: sclereids; NP: nectary parenchyma) Scale bars: A, C: 300 μm; B: 25 μm; D, I: 100 μm; E, G, H, J: 50 μm; F: 200 μm.

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 parenchyma of the vascular region (Figs. 2F, 3B, F, 4F) in C. anceps, C. aristata, C. cardiostegia, C. cinerascens, C. multinervia, C. rotundata var. rotundata, C. ulmea, C. roraimae, C. mucronata, C. latifolia, C. potentilla and C. simplifacta (series Coriaceae).

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.

Table 2
Histochemical tests in Chamaecrista studied.

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 1971Bhattacharyya B, Maheshwari JK. 1971. Studies on extrafloral nectaries of the Leguminales - II. The genus Cassia Linn. (Caesalpiniaceae). Proceedings of the Indian National Science Academy 37: 74-90.; Elias 1983Elias TS. 1983. Extrafloral nectaries: their structure and distribution. In: Bentley BL, Elias TS. (eds.) The Biology of Nectaries. New York, Columbia University Press . p. 174-203. ; Francino et al. 2006Francino DMT, Sant’Anna-Santos BF, Silva KLF. 2006. Anatomia foliar e caulinar de Chamaecrista trichopoda (Caesalpinioideae) e histoquímica do nectário extrafloral. Planta Daninha 2: 695-705.; 2015Francino DMT, Coutinho IAC, Dalvi VC, Azevedo AA, Conceição AS, Meira RMSA. 2015. Anatomical interpretations of the taxonomy of Chamaecrista section Absus (Leguminosae - Caesalpinioideae s.l.). Plant Systematics and Evolution. 301: 2087-2103.; Paiva & Machado 2006Paiva EAS, Machado RS. 2006. Ontogênese, anatomia e ultra-estrutura dos nectários extraflorais de Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae-Caesalpinioideae). Acta Botanica Brasilica 20: 471-482. ; Melo et al. 2010Melo Y, Machado SR, Alves M. 2010. Anatomy of extrafloral nectaries in Fabaceae from dry-seasonal forest in Brazil. Botanical Journal of the Linnean Society 163: 87-98.; Coutinho et al. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.).

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. 2015Francino DMT, Coutinho IAC, Dalvi VC, Azevedo AA, Conceição AS, Meira RMSA. 2015. Anatomical interpretations of the taxonomy of Chamaecrista section Absus (Leguminosae - Caesalpinioideae s.l.). Plant Systematics and Evolution. 301: 2087-2103.). 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. (2009Conceição AS, Queiroz LP, Lewis GP, et al. 2009. Phylogeny of Chamaecrista Moench (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168-1180.) and Rando et al. (2016Rando JG, Zuntini AR, Conceição AS, Berg C, Pirani JR, Queiroz LP. 2016. Phylogeny of Chamaecrista ser. Coriaceae (Leguminosae) unveils a lineage recently diversified in Brazilian Campo Rupestre vegetation. International Journal of Plant Sciences 177: 3-17.), 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. 2016Rando JG, Zuntini AR, Conceição AS, Berg C, Pirani JR, Queiroz LP. 2016. Phylogeny of Chamaecrista ser. Coriaceae (Leguminosae) unveils a lineage recently diversified in Brazilian Campo Rupestre vegetation. International Journal of Plant Sciences 177: 3-17.). 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. 2009Conceição AS, Queiroz LP, Lewis GP, et al. 2009. Phylogeny of Chamaecrista Moench (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168-1180.; Rando et al. 2016Rando JG, Zuntini AR, Conceição AS, Berg C, Pirani JR, Queiroz LP. 2016. Phylogeny of Chamaecrista ser. Coriaceae (Leguminosae) unveils a lineage recently diversified in Brazilian Campo Rupestre vegetation. International Journal of Plant Sciences 177: 3-17.).

Nectaries are an effective tool for taxonomy because of variation in their shape and position on the plant body (Keeler & Kaul 1979Keeler KH, Kaul RB. 1979. Morphology and distribution of petiolar nectarines in Ipomoea (Convolvulaceae). American Journal of Botany 66: 946-952.; Bentley & Elias 1983Elias TS. 1983. Extrafloral nectaries: their structure and distribution. In: Bentley BL, Elias TS. (eds.) The Biology of Nectaries. New York, Columbia University Press . p. 174-203. ). In species of Chamaecrista sect. Apoucouita,Coutinho & Meira (2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.) and Coutinho et al. (2016Coutinho IAC, Rando JG, Conceição AS, Meira RMSA. 2016. A study of the morphoanatomical characters of Chamaecrista sect. Apoucouita. Acta Botanica Brasilica 30: 205-221.) 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. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.), as proposed by Conceição et al. (2009Conceição AS, Queiroz LP, Lewis GP, et al. 2009. Phylogeny of Chamaecrista Moench (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168-1180.) with studies based on molecular data. Although morphologically different, our study found anatomical similarities among the extra-reproductive 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. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.; Francino et al. 2015Francino DMT, Coutinho IAC, Dalvi VC, Azevedo AA, Conceição AS, Meira RMSA. 2015. Anatomical interpretations of the taxonomy of Chamaecrista section Absus (Leguminosae - Caesalpinioideae s.l.). Plant Systematics and Evolution. 301: 2087-2103.).

Extra-reproductive nectaries vascularized by xylem and phloem are common in species of the genus Chamaecrista (Coutinho et al. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.), and the vascularization often originates from the main vascular system of the petiole and/or rachis (Francino et al. 2006Francino DMT, Sant’Anna-Santos BF, Silva KLF. 2006. Anatomia foliar e caulinar de Chamaecrista trichopoda (Caesalpinioideae) e histoquímica do nectário extrafloral. Planta Daninha 2: 695-705.; Coutinho et al. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.), 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. (2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.) in species of Chamaecrista sect. Absus subsect. Baseophyllum and may serve to provide mechanical support for nectaries. Paiva & Machado (2006Paiva EAS, Machado RS. 2006. Ontogênese, anatomia e ultra-estrutura dos nectários extraflorais de Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae-Caesalpinioideae). Acta Botanica Brasilica 20: 471-482. ) 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 1984Contreras LS, Lersten NR. 1984. Extrafloral nectaries in Ebenaceae: anatomy, morphology and distribution. American Journal of Botany 71: 865-872.; Francino et al. 2006Francino DMT, Sant’Anna-Santos BF, Silva KLF. 2006. Anatomia foliar e caulinar de Chamaecrista trichopoda (Caesalpinioideae) e histoquímica do nectário extrafloral. Planta Daninha 2: 695-705.; Paiva & Machado 2006Paiva EAS, Machado RS. 2006. Ontogênese, anatomia e ultra-estrutura dos nectários extraflorais de Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae-Caesalpinioideae). Acta Botanica Brasilica 20: 471-482. ; Melo et al. 2010Melo Y, Machado SR, Alves M. 2010. Anatomy of extrafloral nectaries in Fabaceae from dry-seasonal forest in Brazil. Botanical Journal of the Linnean Society 163: 87-98.). 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. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.), 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 2010Vassilyev AE. 2010. On the mechanisms of nectar secretion: revisited. Annals of Botanany 105: 349-54. ).

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 1979Fahn A. 1979. Secretory tissues in plants. London, Academic Press.; Elias 1983Elias TS. 1983. Extrafloral nectaries: their structure and distribution. In: Bentley BL, Elias TS. (eds.) The Biology of Nectaries. New York, Columbia University Press . p. 174-203. ; Paiva & Machado 2006Paiva EAS, Machado RS. 2006. Ontogênese, anatomia e ultra-estrutura dos nectários extraflorais de Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae-Caesalpinioideae). Acta Botanica Brasilica 20: 471-482. ; Nepi 2007Nepi M. 2007. Nectary structure and ultrastructure. In: Nicolson SW, Nepi M, Pacini E. (eds.) Nectaries and nectar. Dordrecht, Springer. p. 129-166.; Thadeo et al. 2008Thadeo M, Cassino MF, Vitarelli NC, et al. 2008. Anatomical and histochemical characterization of extrafloral nectaries of Prockia.crucis (Salicaceae). American Journal of Botany 95: 1515-1522. ; Rocha et al. 2009Rocha DI, Silva LC, Valente VMM, Francino DMT, Meira RMSA. 2009. Morphoanatomy and development of leaf secretory structures in Passiflora amethystina Mikan (Passifloraceae). Australian Journal of Botany 57: 619-626. ; Paiva 2016Paiva EAS. 2016. How do secretory products cross the plant cell wall to be released? A new hypothesis involving cyclic mechanical actions of the protoplast. Annals of Botany 117: 533-540.).

Conceição et al. (2009Conceição AS, Queiroz LP, Lewis GP, et al. 2009. Phylogeny of Chamaecrista Moench (Leguminosae-Caesalpinioideae) based on nuclear and chloroplast DNA regions. Taxon 58: 1168-1180.) 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 extra-reproductive 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 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.). In species of sect. Absus subsect. Baseophyllum, lipids were not detected, but phenolic compounds were (Coutinho et al. 2012Coutinho IAC, Francino DMT, Azevedo AA, Meira RMSA. 2012. Anatomy of the extrafloral nectaries in species of Chamaecrista section Absus subsection Baseophyllum (Leguminosae, Caesalpinioideae). Flora 207: 427-435.). 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. (1993Lanza J, Vargo E, Pulim S, Chang Yu Z. 1993. Preferences of the fire ants Solenopsis invicta and S. geminata (Hymenoptera: Formicidae) for amino acid and sugar components of extrafloral nectars. Environmental Entomology 22: 411-417. ) 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. 1993Lanza J, Vargo E, Pulim S, Chang Yu Z. 1993. Preferences of the fire ants Solenopsis invicta and S. geminata (Hymenoptera: Formicidae) for amino acid and sugar components of extrafloral nectars. Environmental Entomology 22: 411-417. ). 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. 1978Baker DA, Hall JL, Thorpe JRA. 1978. Study of the extrafloral nectaries of Ricinus communis. New Phytologist 81: 129-137.; Fahn 1979Fahn A. 1979. Secretory tissues in plants. London, Academic Press.; 1988Fahn A. 1988. Secretory tissues in vascular plants. New phytologist 108: 229-257.; 2000Fahn A. 2000. Structure and function of secretory cells. Advances in Botanical Research 31: 37-75. ; Coutinho & Meira 2015Coutinho IAC, Meira RMSA. 2015. Structural diversity of extrafloral nectaries in Chamaecrista sect. Apoucouita. Botany 93: 379-388.). As stated by Paiva & Machado (2006Paiva EAS, Machado RS. 2006. Ontogênese, anatomia e ultra-estrutura dos nectários extraflorais de Hymenaea stigonocarpa Mart. ex Hayne (Fabaceae-Caesalpinioideae). Acta Botanica Brasilica 20: 471-482. ), the presence of lipids in the intercellular spaces of the secretory parenchyma indicates that these compounds are part 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. (1997Forcone A, Galetto L, Bernardello L. 1997. Floral nectar chemical composition of some species from Patagonia. Biochemical Systematics and Ecology 25: 395-402.) and Bernardello et al. (1999Bernardello L, Galetto L, Forcone A. 1999. Floral nectar chemical composition of some species from Patagonia. II. Biochemical Systematics and Ecology 27: 779-790.). 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 2007Nicolson SW, Nepi M, Pacini E. 2007. Nectaries and nectar. Dordrecht, Springer .). 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 1991Lanza J. 1991. Response of fire ants (Formicidae: Solenopsis invicta and S. geminata) to artificial nectars with amino acids. Ecological Entomology 16: 203-210.; Wagner & Kay 2002Wagner D, Kay A. 2002. Do extrafloral nectaries distract ants from visiting flowers? An experimental test of an overlooked hypothesis. Evolutionary Ecology Research 4: 293-305.; Wilder & Eubanks 2009Wilder SM, Eubanks MD. 2009. Extrafloral nectar content alters foraging preferences of a predatory ant. Biology Letters. 6: 177-179.).

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.

Acknowledgements

We thank CNPq, CAPES, FAPEMIG and Floresta Escola (SECTES/UNESCO/HidroEX/FAPEMIG) for financial support. We also thank CNPq for providing a research scholarship to RMSA Meira and CAPES for the PhD scholarship to MS Silva. We are grateful to the herbaria HUEFS, SPF, NY, RB and VIC for kindly allowing the sampling of their voucher specimens. We thank the Centro de Microscopia e Microanálise of the Universidade Federal de Viçosa, as well as Patricia Fonseca and Aurora Dias for technical assistance.

References

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Publication Dates

  • Publication in this collection
    Jul-Sep 2017

History

  • Received
    16 Mar 2017
  • Accepted
    29 May 2017
Sociedade Botânica do Brasil SCLN 307 - Bloco B - Sala 218 - Ed. Constrol Center Asa Norte CEP: 70746-520 Brasília/DF. - Alta Floresta - MT - Brazil
E-mail: acta@botanica.org.br