Pollen morphology of Rubiaceae Juss . species occurring in an area of caatinga ( dryland ) vegetation in Bahia State , Brazil

(Pollen morphology of Rubiaceae species occurring in an area of caatinga (dryland) vegetation in Bahia State, Brazil). Th e palynology of the following 16 species of Rubiaceae, from Brejinho das Amestistas, was investigated: Coccocypselum hirsutum Bartl. ex DC., Cordiera rigida Kuntze, Coutarea hexandra K.Schum., Declieuxia fruticosa Kuntze, Diodella apiculata (Willd. ex Roem. & Schult.) Delprete, D. radula (Willd. ex Roem. & Schult.) Delprete, D. teres Small., Emmeorhiza umbellata K.Schum., Leptoscela ruellioides Hook. f., Mitracarpus baturitensis Sucre., Mitracarpus villosus Cham. & Schltdl., Palicourea rigida Kunth, Psyllocarpus asparagoides Mart., Richardia grandifl ora Steud., Staelia aurea K. Schum., and Staelia galioides DC. Th e pollen grains were acetolysed to and their morphological characters were analyzed using light and scanning electron microscopy. Th ey varied in size from small to large; were suboblate to subprolate; inaperturate (P. rigida), colpate and colporate in the remaining species, with an aperture number that varied from three to several. Th e exines were microreticulate in most species, reticulate (C. hirsutum, C. rigida and P. rigida), bireticulate (D. fruticosa), microechinate-perforated (C. hexandra), echinate-granulate (R. grandifl ora), echinate-granulate-perforate (D. apiculata and D. teres), and psilate (P. asparagoides). Based on the results, palynological data can be used to distinguish these species.


Introduction
Th e family Rubiaceae, as currently circumscribed, belongs to the order Gentianales and comprises 637 genera and approximately 10,700 species (Robbrecht 1988); approximately 112 genera and 1347 species occur in Brazil (Barbosa et al. 2010).Th e family is widely distributed globally, although its diversity is principally concentrated in tropical regions (Souza & Lorenzi 2008;Judd et al. 2009).Th e individual species of this group have various habits, such as herbs, subshrubs, shrubs, and trees; lianas and epiphytes are less common.Morphologically, the family is characterized by having simple, opposite leaves, interpetiolar stipules, and epipetalous stamens equal in number to the petals (Souza & Lorenzi 2008).Recent phylogenetic studies have suggested support for only three subfamilies: Rubioideae (18 tribes), Cinchonoideae (nine tribes), and Ixoroideae (15 tribes), plus the tribe Coptosapelteae, which cannot currently be placed in any of the subfamilies (Bremer 2009).
Heterostyly is a genetically established fl oral polymorphism, and Rubiaceae comprises more heterostylous genera than all the other plant families combined (Ganders 1979).According to the same author, this implies both physiological and morphological diff erences in the reproductive verticils and pollen grains of these plants, and diff erences in pollen grain sizes in many genera of Rubiaceae can be explained by this polymorphism.Brevistylous (thrum) forms generally have larger pollen grains than longistylous (pin) forms (Ganders 1979;Jung-Mendaçolli & Melhem 1995;Dessein et al, 2000), which must be taken into account in palynological studies.
Palynological studies involving species of Rubiaceae in Brazil have been limited to pollinic catalogs or palynological surveys, such as those undertaken by Salgado-Labouriau (1973), Melhem et al. (2003), andSilva (2007).Th e principal palynological studies of the Rubiaceae have characterized the family as eurypollinic (Erdtman 1952;Salgado-Labouriau 1973;Jung-Mendaçolli & Melhem 1995;Dessein et al. 2005), and have facilitated the use of pollen morphology in classifying and identifying its taxa.According to Dessein et al. (2005), morpho-palynological data in the Rubiaceae can elucidate evolutive relationships among the diff erent taxa, and can be used to reject or corroborate taxonomic decisions.
Th e following works stand out among the published palynological studies of the family Rubiaceae: Erdtman (1952) palynologically described 230 species belonging to 120 genera; Kirkbride (1979) revised the genus Psyllocarpus in Brazil using palynological features; Puff & Robbrecth (1988) investigated the taxonomic position of the Australian genus Durringtonia; Roubik & Moreno (1991) palynologically documented the species of Rubiaceae occurring on Barro Colorado Island in Panama; Jung-Mendaçolli & Melhem (1995) analyzed 25 heterostylous species of Rubiaceae and demonstrated diff erences in pollen size and exine ornamentation among the fl oral morphs investigated; Pire (1996;1997) palynologically studied the tribe Spermacoceae; Huysmans et al. (2003) documented the pollen morphology of all of the genera of the tribe Rubieae occurring in Europe; Perveen & Qaiser (2007) described the pollen morphology of 50 species of the family occurring in Pakistan; Verellen et al. (2007) undertook palynological studies of the tribes Naucleeae and Hymenodictyeae; and Cai et al. (2008) studied the pollen grains of Asian species of this family.
In spite of these eff orts, we are still quite far from completing the palynological documentation of this family because only about 15% its species have been investigated (Dessein et al. 2005).Most of the palynological studies of the Rubiaceae have been undertaken on taxa not found in Brazil, which has left a large gap in our knowledge about the pollen of representatives of the Brazilian fl ora.For this reason, the present study examined regional species of this family in order to contribute to the taxonomic and systematic knowledge of this group.

Materials and methods
We analyzed the pollen of 16 species belonging to 11 genera of Rubiaceae.Floral buds were collected in the fi eld following the methodology recommended by Mori et al. (1989).Five specimens of each species were collected, when possible, and deposited in the herbarium at the Universidade do Estado da Bahia (HUNEB -Caetité Collection).Material was also obtained from specimens deposited in the herbaria at the Universidade Estadual de Feira de Santana (HUEFS) and the Universidade Federal da Bahia (ALCB); abbreviations follow Index Herbariorum (Th iers, B., continuously updated).
Th e fl oral morphologies of the specimens (thrum or pin) were determined using a stereo microscope.Species without annotations regarding their fl oral morphology are those that do not have published descriptions of heterostyly available in the literature.Palynological processing -Visible light microscopic analyses (LM) were performed in the Palynological Laboratory at UNEB, Campus VI.Th e pollen grains of the majority of the species were prepared using the classical method of acetolysis (Erdtman 1960), while fragile pollen grains (such as those from Palicourea rigida) were treated using the potassa method (Faegri & Iversen 1975).Whenever possible, the anthers of more than one fl ower from a given specimen were examined to guarantee a more uniform sampling (Salgado-Labouriau 1973).Th e pollen samples were then mounted on slides with glycerinated gelatin and evaluated qualitatively and quantitatively, and photomicrographs were taken using a Zeiss Axioskop 2 microscope.
Th e principal morphometric parameters of randomly chosen pollen grains were measured according to the recommendations of Salgado-Labouriau (1973).Th e polar (PD) and equatorial (ED) diameters of 25 pollen grains were measured whenever possible.Th e other parameters, such as the polar area, aperture diameter, and exine, sexine, and nexine thicknesses were measured on 10 samples.
Th e quantitative results were statistically analyzed by calculating the arithmetic average (x -), the standard error of the sample (s), and the average standard error (S x -) for samples sizes of 25; only arithmetic averages were calculated for parameters with sample sizes of 10.
Palynological analyses using a scanning electron microscope (SEM) were undertaken in the Electron Microscope Section of the Department of Biological Sciences at the Universidade Estadual de Feira de Santana.Th e pollen grains were acetolized, washed, and dehydrated in an ascending ethanol series (50, 70, 90 and 100%, remaining for approximately 10 min.at each step).A small sample of the pollen grains from the absolute alcohol step was placed directly on the specimen holder of the SEM and, aft er drying, was sputter coated with gold under high vacuum, and viewed and photographed using a LEO 1430 VP scanning electron microscope.
Th e morpho-palynological characteristics were illustrated by photographs and electron photomicrographs, and the palynological descriptions used the terminology of Punt et al. (2007).
Th e morphological and morphometric data concerning the pollen grains are presented in Tables 1 and 2, respectively.
Coutarea hexandra K. Schum.(Fig. 1G-I) Pollen grains medium; isopolar; subprolate; amb circular; 3-colporate, angulaperturate, apertures diffi cult to see using light microscopy, more easily observed in the polar view and by an SEM; microechinate-perforated, with coniform microspines with wide base and sharp apex distributed uniformly over the surface of the pollen grains, the perforations surrounded by a thicker region of the sexine; nexine thicker than the sexine.Declieuxia fruticosa Kuntze (Fig. 1J-L
Although the pollen grains of both morpho-fl oral types are medium-sized, the largest diameter grains were observed in the brevistylous form (Tab.1).

Discussion
Our observations of the pollen grains of Coccocypselum hirsutum were diff erent from those encountered in the published literature.Medium-sized pollen grains have been reported for this species by a number of authors (Colinvaux et al. 1999;Jung-Mendaçolli & Melhem 1995;Piesschaert et al. 2000;Melhem et al. 2003); amb triangular to sub-triangular has also been described (Jung-Mendaçolli & Melhem 1995;Melhem et al. 2003).Pollen grains with a diff erent type of aperture, 3-porate, have only been reported by Delprete & Cortés (2006), while Colinvaux et al. (1999) noted the occurrence of annuli in the ectoapertures.In terms of the   (1995) reported a pilate to pilate-rugulate exine.Th is latter ornamentation pattern was described by Piesschaert et al. (2000), using an SEM, as being a "complex reticulum" formed by a suprareticulum psilate with an echinate infrareticulum, with fusion of the muri of both of the reticula.Delprete & Cortés (2006) reported the occurrence of bireticulate pollen grains for the genus.
Th e pollen type described for Cordiera rigida (3-colporate with reticulate exine) is shared by Coccocypselum hirsutum, but the pollen of these species diff ers in size and shape.Th e characteristics of the pollen grains of C. rigida were also reported by Delprete & Cortés (2006) for other species of the genus.Roubik &Moreno (1991) andHuysmans et. al. (1999) described the pollen grains of Coutarea hexandra using LM and an SEM, respectively, and, according to these authors, this species has pollen grains that are medium-sized, spheroidal to subprolate, amb circular, tricolpate, with a microechinate exine.Th e aperture type described by these authors was not observed in the present work.In terms of exine ornamentation, Roubik & Moreno (1991) did not observe the perforations seen in the tectum under light microscopy that are described in the present study.
Th e pollen morphology of Declieuxia fruticosa was described by Piesschaert et al. (2000) using an SEM, and these authors reported similar results to those of the present study; they also observed a "complex reticulum" in this species.Th is type of ornamentation is very similar to the bireticulate pattern described by Punt et al. (2007) as a reticulum composed of two layers (a suprareticulum sustained by a microreticulate tectum).As such, the ornamentation types encountered in the specimens in the present study diff ered from the patterns described by Piesschaert et al. (2000) in that micro-spines were not observed in the infrareticulum.
Th e species of Diodella Small studied here were similar in terms of the large numbers of apertures seen in the equatorial region.Diodella apiculata and D. teres appeared to be palynologically very similar because they were the same size, had the same aperture type and exine ornamentation, and were distinct only in terms of the shape of the grains.Th e palynological studies of D. apiculata (= Diodia rigida Cham.& Schltdl.)undertaken by Erdtman (1952) diff ered from the results of the present study, because this author reported very large pollen grains (ca. 100 μm) that were 16-18-colporate (brevicolpate), punctate and echinate.Very large pollen grains were also encountered by Dessein et al. (2005) in D. teres (133 μm).Delprete & Cortés (2006) reported that the pollen of species of this same genus were multicolporate and commonly had foveolate-perforate exines.Of the species studied here, D. radula was the most
Table 2. Morphometric characters of the pollen grains of Rubiaceae species occurring in an area of caatinga vegetation of Bahia, Brazil.
similar to descriptions supplied by these authors because it had grains that were (8)-9(-10)-colporate, although none of the species studied here had foveolate-perforate exine ornamentations.Th e descriptions encountered in the literature demonstrate a signifi cant degree of heterogeneity within the genus in terms of palynological characteristics when compared to those of the present study.Melhem et al. (2003) described the pollen grains of Emmeorhiza umbelllata using visible light microscopy, and diff erences were restricted to the reticulate ornamentation pattern and to the apparent lack of costae on the ectoapertures (as observed in the present study).In terms of the genus, Delprete & Cortés (2006) pointed out the occurrence of multicolporate pollen grains as well as an echinate-perforate exine.
Leptoscela ruellioides is endemic to Brazil and was invest here for the fi rst time.Th is species diff ered palynologically from the others by having a microreticulate exine and a bifurcated columellae.Th is species did, however, have pollen grains with characteristics common to other taxa, such as: pollen grains with amb subcircular, 3-colporate with a fastigium (which occurs in both Coccocypselum hirsutum and Declieuxia fruticosa).
Th e species of Mitracarpus Zucc.studied here were very similar palynologically: being the same size and having the same aperture type, exine ornamentation and shape.Published descriptions of this genus indicate, however, that the size, exine ornamentation, and aperture type of its pollen grains can vary.Melhem et al. (2003) observed that the pollen grains of M. hirtus were medium sized, (3,4,6)-7(-9)-colpate and the ornamentation of the exine was granulate-perforate, while Delprete & Cortés (2006) reported 6-7-colporate pollen grains with an echinateperforate exine.However, this data does demonstrate that it is possible to separate the species of this genus by analyzing their pollen morphologies.Salgado-Labouriau (1973) studied the pollen morphology of Palicourea rigida and encountered results similar to those of the present work, although this author did not refer to the morpho-fl oral types examined.Th is author described the sexine as being much thicker than the nexine, while the exine layers observed in the present work were indistinct.Additionally, this author observed bi-or tri-columellate muri, while in the present study the walls were described as simplicolumellate.Other authors who studied the pollen of species of this genus reported that these taxa had very homogeneous palynological characteristics, with generally large pollen grains that are spheroidal and inaperturate (Erdtman 1952;Jung-Mendaçolli & Melhem 1995;Dessein et al. 2005).Jung-Mendaçolli & Melhem (1995) described the genus as stenopollinic and included it the inaperturate pollen type.Th ese authors noted, however, that diff erent ornamentation patterns were encountered in this genus and that this characteristic not only permitted distinctions to be made between species, but also allowed the separation of the pollen grains of the longistylous and brevistylous morphofl oral types (because they had distinct exine ornamentation in at least two of the three species they analyzed).In addition to the reticulate ornamentation found by this author, Palicourea Aubl.can also have retipilate, pilate, pilate-rugulate, and pilate-reticulate pollen grains.Kirkbride (1979) analyzed the pollen grains (using SEM) of species of the genus Psyllocarpus Mart.that occur in the Amazon and Pantanal regions of Brazil, and divided them into two sections: 1) an Amazon section characterized by oblate spheroidal pollen grains, 6 to 8-colporate, perforated, with micro-spines distributed over the entire pollen grain; and 2) the section Psyllocarpus, characterized by prolate spheroidal pollen grains, 5 to 7-colporate, exine psilate, with micro-spines surrounding the colpus.Th is author did not refer to the sizes of the pollen grains of the species studied.Th e micro-spines observed by this author for this species were not seen in the light microscopic examinations in the present study.
Th e pollen grains from three species of the genus Richardia L. were studied by Salgado-Labouriau (1973), including R. grandifl ora, and were found to be medium to large, oblate spheroid to peroblate, and multicolporate with an exine ornamentation that was microechinate or pilate.Erdtman (1952) analyzed the same taxa as the previous author although in contrast, described the species analyzed here.Th is author did not specify the aperture type, describing it as polycolp(or?)ate,with a peroblate to oblate shape and an echinate exine.Th e present study corroborated the results of these authors in terms of the size of the pollen grains and exine orientation, although the specimen analyzed in the present study had colpate pollen grains that were suboblate.Th is same genus can have rugulate, echinate-perforate, or echinate-reticulate pollen grains (Delprete & Cortés 2006).
Th e two species of Staelia Cham.& Schltdl.had very similar pollen, diff ering only by their shape and the presence of costa in S. aurea.According to Delprete & Cortés (2006), 7 to multiaperturate pollen, and fi nely reticulated exines occur in the genus.Salas and Cabral (2010) also described the pollen of species of Staelia from Paraguay and, like the latter authors, reported that the grains were multi-colporate.Th ese same authors provided complete descriptions of the morphopalynological characters: medium pollen grains, isopolar, prolate spheroidal, 7-10-colporate, with long ectoapertures and lolongate endoapertures, exine perforated and scabrate, with circular perforations and scabrae distributed over the entire surface of the grain.Our data generally corroborates these observations because the two species had many apertures and similar ornamentation patterns (microreticulate), although it was not possible to observe the lolongate endoapertures or the scabrate exine using light microscopy.
Th e results encountered in the present work demonstrated variations in almost all of the morpho-palynological characters investigated, especially in the number and type of aperture and exine ornamentation -characters usually utilized in taxonomic delimitations (Miranda et al. 1993, Pire 1996, 1997;Dessein et al. 2005).Considering the variability observed here, the present study is in consonance with the authors who consider this group eurypollinic (Erdtman 1952;Salgado-Labouriau 1973;Jung-Mendaçolli & Melhem 1995;Dessein et al. 2005).
Among the heterostylous species studied, Declieuxia fruticosa and Leptoscela ruelioides had pollen grains of the two morpho-fl oral types analyzed, and the data presented here corroborates information in the literature concerning heterostyly in the family; the pollen grains from the brevistylous (thrum) morpho-fl oral type had larger diameters (Ganders 1979;Jung-Mendaçolli & Melhem 1995;Dessein et al. 2000;Piesschaert et al. 2000;Melhem et al. 2003).Th ere were no diff erences between the specimens investigated in terms of aperture types or exine ornamentation.
The present study contributes to our palynological knowledge of the tribe Spermacoceae sensu lato (s.l.), because most of the genera examined were members of this group (Diodella, Emmeorhiza, Leptoscela, Mitracarpus, Psyllocarpus, Richardia and Staelia).Th is tribe is well delimited phylogenetically, although there are still some doubts concerning some of its generic and specifi c relationships, for example, between the genera Borreria G. Mey., Diodella L., Diodia L., and Galianthe Griseb.(Groeninckx et al. 2009).Additional phylogenetic studies are needed that focus on this group and include larger numbers of taxa and characters (Groeninckx et al. 2009), and pollen morphology will have an important role in these investigations.
Th e newly described palynological characteristics of the monospecifi c genus Leptoscela, presented here, will contribute to future studies of this taxon because this species had not been securely placed in one of the subfamilies of Rubiaceae.Leptoscela ruelioides was assigned to the expanded tribe Spermacoceae s.l. that includes the traditional tribes Spermacoceae sensu stricto (s.s.), Manettieae, and the Hedyotis-Oldenlandia group, to which this genus belongs (Bremer & Manen 2000).However, several monospecifi c genera of the tribe have various peculiar characteristics that make them diffi cult to relate to other members of the same tribe.In addition, a morphological investigation would be very important to improve the phylogenetic studies of this group (Groeninckx et al. 2009).
Based on the present work, it can be concluded that the species studied were palynologically distinct.However, additional studies are necessary (principally employing SEM) to provide more detailed observations of some structures (such as the apertures and the exine ornamentation), because pollen morphology has the potential to greatly aid in the understanding of the taxonomy and phylogeny of this group.

Table 1 .
Morphological characters of pollen grains of Rubiaceae species occurring in an area of caatinga vegetation of Bahia, Brazil.