Are stem nectaries common in Gentianaceae Juss . ?

Extrafl oral nectaries (EFNs) are specialized structures that produce and release nectar and are located on leaves, cotyledons and, more seldomly, stems. Peculiar leaf nectaries have been described for thirty-three Neotropical species of Gentianaceae, while stem nectaries have been reported for only nine. Th e aim of this study was to verify the occurrence of stem EFNs within Gentianaceae and investigate the existence of a correlation between their occurrence and the geographical distribution of species. Samples of internodal regions from fi eld and herbarium specimens were submitted to standard light microscopy techniques. Data regarding the geographical distribution of species were acquired from herbarium specimens and the literature. A total of 37 species were investigated, representing 25 genera distributed among fi ve tribes. Nectaries, composed of modifi ed epidermal cells, were observed in 16 species restricted to the Neotropical tribes Helieae, Saccifolieae, Potalieae and Coutoubeinae; exceptions were Cicendia quadrangularis and Zygostigma australe, which both occur in the Neotropics but do not possess EFNs. Th ese results demonstrate that stem EFNs are common among Neotropical taxa of Gentianaceae, and are typically absent from taxa in temperate regions.


Introduction
Nectaries are structures that release nectar, a solution composed mainly of mono-and disaccharides, amino acids, proteins and trace amounts of other compounds (Elias 1983;Nicolson & Th ornburg 2007).Extrafl oral nectaries (EFNs) play an important ecological role in plant protection, since nectar acts as a carbohydrate-rich liquid reward for ants in exchange for protection against herbivores (Heil 2015;Del-Claro et al. 2016).
EFNs occur in 110 families of vascular tracheophytes, but are unknown in gymnosperms, early angiosperms and magnoliids (Weber & Keeler 2013;Weber et al. 2015).Although EFNs occur in monocots, the bulk of their occurrence is widespread among eudicots, especially in the rosids clade (Weber & Keeler 2013).Evidence suggests that EFNs originated a remarkable number of times in the evolution of tracheophytes (Weber & Keeler 2013) and played a key evolutionary role in the diversifi cation of certain plant clades (Marazzi & Sanderson 2010;Nogueira et al. 2012).
Although EFNs have been reported to be absent in Gentianaceae Juss.(Elias 1983) peculiar nectaries composed of only modifi ed epidermal cells were described for 33 species of Gentianaceae, including 13 Neotropical genera (Delgado et al. 2011a;b;Dalvi et al. 2013;2014;Guimarães et al. 2013).Such nectaries are microscopic structures and occur along the leaf blade in single units or aggregates (Dalvi et al. 2013).Anatomically similar nectaries were also observed on the stem of seven species of Curtia, in Hockinia Acta Botanica Brasilica -31(3): 403-410.July-September 2017 montana (Dalvi et al. 2014), and in Schultesia pachyphylla (Guimarães et al. 2013).However, a profuse secretion and the presence of ants were observed only in the aggregated leaf nectaries of Calolisianthus speciosus (Delgado et al. 2011a) and Chelonanthus viridiflorus (Dalvi et al. 2013).In both species the nectaries occur at the base of the leaf blade are recognized as yellow areas.As usually described, the nectar of Calolisianthus speciosus is composed of glucose, fructose, and sucrose (Delgado et al. 2011a).
EFNs have been considered more abundant in species from tropical communities than in those from temperate regions (Oliveira & Freitas 2004;Rico-Gray & Oliveira 2007).However, information about the possible influences of geographic regions over the occurrence of EFNs is still scarce (Pemberton 1998).In addition, data about stem EFNs are limited to just a few families, and detailed structural descriptions are lacking (Machado et al. 2008;Weber & Keeler 2013).
Considering the widespread distribution of Gentianaceae and the usual occurrence of leaf nectaries in Neotropical species, the aim of this study was to investigate how common is the occurrence of stem nectaries in Neotropical and temperate species of Gentianaceae.

Materials and methods
Samples from the third to fifth internodes were obtained from exsiccates of national and international herbaria (Tab.1).The material was rehydrated by microwaving in distilled water for 5 min and left to rest overnight.Rehydrated samples were then treated with 2 % potassium hydroxide for 1-2 h, dehydrated in an ethanol series and stored in 70 % ethanol (Smith & Smith 1942).According to the occurrence of species, different locations in the states of Minas Gerais and Bahia (Brazil) were selected for field expeditions.In the same way, samples from the third to fifth internodes of field-collected material were fixed in FAA (formalin, acetic acid, 50 % ethanol, 1:1:18 by volume) and stored in 70 % ethanol (Johansen 1940).Voucher materials were deposited in the VIC Herbarium of Universidade Federal de Viçosa (UFV), Minas Gerais, Brazil.Fragments of young branches were sampled only from Potalia resinifera Mart., the unique tree species analyzed here.
For the anatomical studies, samples stored in 70 % ethanol were submitted to an ethanol dehydration series and subsequently embedded in methacrylate (Historesin, Leica Microsystems Nussloch GmbH, Heidelberg, Germany).The material was sectioned using a rotary microtome (model RM2155, Leica Microsystems Inc., Deerfield, USA).Cross and paradermal sections (7 µm thick) were stained with toluidine blue, pH 4.7 (O' Brien et al. 1964), and permanent slides were mounted with synthetic resin (Permount, Fisher Scientific, New Jersey, USA).To observe epidermal cells from frontal view, paradermal hand-sections were obtained, clarified in sodium hypochlorite (20 %), stained with 0.001 % basic alcoholic fuchsin and mounted in gelatin (Johansen 1940).
Analyses and image captures were conducted using an Olympus Optical AX70TRF (Tokyo, Japan) with a U-Photo system and coupled digital camera (AxioCam Zeiss, Göttingen, Germany) in the Laboratory of Plant Anatomy of UFV.
The data for the geographical distribution of species and tribes/subtribes/genera/species were obtained from herbarium data and literature (Tab.1).

Results
We analyzed 37 species (25 genera), comprising representatives of the tribes Chironieae, Gentianeae, Helieae, Potalieae and Saccifolieae (Tab.1).The stem EFNs were not visible to the naked eye neither in materials collected in the field nor in those from herbaria.Conversely, secretion or other macro-morphological evidence of the presence of these nectaries was not detected as well.However nectaries were observed under light microscopy on internodes of the stems in 16 species of 11 genera comprising almost 50 % of taxa studied (Tab.1).
The stem nectaries are non-vascularized glands comprised of a single central epidermal cell surrounded by concentrically arranged secretory cells, as shown in Chelonanthus purpurascens (Fig. 1A, B) and Tetrapollinia caerulescens (Fig. 1C, D).The region of contact between secretory cells and the central cell stained intensely with toluidine blue (Fig. 1E, F).Stem nectaries have a scattered distribution and are interspersed among stomata, except in Potalia resinifera, where stomata were not detected.
In the tribe Helieae, stem nectaries were observed in all analyzed species (Tab.1).Voyriella parviflora was the only species of tribe Saccifolieae analyzed that bore stem nectaries.The presence of stem nectaries was ubiquitous in Potalieae.On the other hand, stem nectaries were absent in seven analyzed taxa of the tribe Gentianeae.In Chironieae, stem nectaries were present in three species from different genera.In Coutoubeinae, Canscorinae and Chironiinae the pattern of presence or absence of stem nectaries remained constant.Two species of Coutoubeinae, Symphyllophytton caprifolioides (Fig. 1E) and Xestaea lisianthoides (Fig. 1F), bore stem nectaries, while they were not detected in Canscorinae (Canscora diffusa) and in Chironiinae (Tab.1).
Regarding the correlation between presence/absence of stem nectaries and pattern of geographic distribution of species, tribes or subtribes, the presence of these nectaries was constant in species restricted to the Neotropics, including all species of tribes Helieae, Saccifolieae, subtribe Coutoubeinae (Chironieae) and Potalia resinifera (Potalieae -Potaliinae) (Tab.1).Besides occurring in the Neotropics, Neurotheca loeselioides (Potalieae-Faroinae) is also found in Africa and bears stem nectaries.Exceptions could be seen in Chironieae, in which Cicendia quadrangularis  and Zygostigma australe (Chironieae -Chironiinae), both occurring in South America, do not possess stem nectaries.In contrast, nectaries are absent in all species restricted to temperate-alpine regions (Gentianinae), as well as in species restricted to the Paleotropics (Chironieae -Canscorinae) and in those distributed in the Pantropics to temperate regions (Chironieae -Chironiinae) (Tab.1).

Discussion
The present work demonstrates that in Gentianaceae stem nectaries are common, occurring in 50 % of the studied species, and mainly in Neotropical taxa.These results are interesting since the presence of stem nectaries is considered an uncommon character in eudicotyledon families (Elias 1983;Machado et al. 2008).Nectaries of the Gentianaceae were described by Vogel (1998) as unusual nectaries on the sepals of species of Irlbachia and were termed nectarioles by the author.Dalvi et al. (2013) named such structures present on leaves as epidermal nectaries, following the classification proposed by Bernadello (2007).
The lack of evidences regarding the occurrence of stem nectaries in Gentianaceae is due to the absence of microscopic analyses, as also stated by Dalvi et al. (2013) for leaf nectaries.Additionally, anatomical studies of the stem in the species of Gentianaceae are even scarcer than those performed with leaves, and are restricted to a few species of Deianira, Schultesia (Delgado et al. 2009;Guimarães et al. 2013), Curtia and Hockinia (Dalvi et al. 2014).In these genera, stem nectaries were reported for only seven species of Curtia, Hockinia montana and Schultesia pachyphylla.Besides demonstrating that stem nectaries are common in Gentianaceae, the present study highlights the importance of anatomical analysis to confirm the presence of these structures in this family.
Anatomically, the stem nectaries of Gentianaceae are similar to most of the leaf nectaries found in the family (Delgado et al. 2011a;b;Dalvi et al. 2013;2014;Guimarães et al. 2013), with the exception of those on the leaf base of Calolisianthus speciosus, which are vascularized (Delgado et al. 2011a).Stem nectaries are randomly distributed and do not form aggregates, as observed for leaf nectaries of certain species of Gentianaceae (Dalvi et al. 2013).This pattern of distribution may explain why the exudate is not observed in the field.Abundant nectar was reported only for leaf nectaries of species of Calolisianthus, which occur aggregated at the leaf base (Delgado et al. 2011a).In other species of Gentianaceae, which have isolated nectaries dispersed along the leaf blade, an apparent secretion was also not detected (Delgado et al. 2011a;Dalvi et al. 2013;2014).
The presence of visible secretion in the field may also be related to the stage of development of the organ, to the season and to the time of day in which the plant was collected.Delgado et al. (2011a) noted that in species of Calolisianthus, it is impossible to collect nectar during the dry season because the secretory cells are not active.Therefore, field studies are necessary to investigate the patterns of nectar secretion related to the environmental factors and to clarify the role played by both leaf and stem nectaries.
In the present study, a correlation between the presence of stem nectaries and geographic distribution of species, subtribes or tribes were indeed observed in Gentianaceae, since among all studied species only those restricted to the Neotropics bore stem nectaries.The Neotropics is a center of diversification of Gentianaceae and comprises the lineages of the most basal nodes of the clade, represented by Saccifolieae (Albert & Struwe 2002).The presence of units of stem nectaries could represent an ancestral condition in Gentianaceae.On the other hand, the non-occurrence of stem nectaries in the studied species of temperate and paleotropical regions suggests the loss of this structure in the species of these regions.However, futures studies focusing on combinations of morphoanatomical and molecular data are necessary to elucidated how nectaries evolved in Gentianaceae.
Zygostigma australis and Cicendia quadrangularis, both of the subtribe Chironiinae (Chironieae), represent the exceptions to the correlation described above since they occur in Neotropical regions but do not bear stem nectaries.Cicendia quadrangularis is pantropical, while Zygostigma australis is restricted to the South Region of Brazil.This variation represents an interesting model to test the hypothesis proposed by Pemberton (1998) that the incidence of plants bearing EFNs increases along the latitudinal gradient (cold temperate to warm subtropical regions).
The presence of stem nectaries corresponds with the presence of leaf nectaries in Gentianaceae, as reported by Dalvi et al. (2013).The stem nectaries in Gentianaceae are peculiar and originate exclusively by protoderm activity.The underlying premise that the presence of stem nectaries is common in Neotropical, and their absence typical among the temperate, Gentianaceae taxa is confirmed here.

Table 1 .
Sampled Gentianaceae species, organized by tribes, with their collectors (herbaria), collection sites and geographical distribution of tribes, subtribes and species.