Pollen Grain Morphology of Fabaceae in the Special Protection Area

The presented paper considered the pollen morphology of thirteen species belonging to seven genera of the Fabaceae family occurring in the Pau-de-Fruta Special Protection Area (SPA), Diamantina, state of Minas Gerais, Brazil. The pollen grains of six species of Chamaecrista [C. cathartica] have a similar morphology, characterized by three long colporated apertures with a central constriction. The species share specific morphological features regarding pollen size, endoaperture type (circular, lalongate or lolongate) and SEM ornamentation patterns of the exine (rugulate with perforations or perforate). Barneby and Zornia diphylla (L.) Pers showed different pollen types in small to large size; oblate spheroidal to prolate form; colpus or colporus apertures; circular, lalongate or lolongate endoapertures and distinctive SEM ornamentation patterns of the exine (perforate, microreticulate, reticulate or rugulate with perforations). Only Stryphnodendron adstringens (Mart.) Coville presents polyads. The pollen morphology variation of these species allowed the Fabaceae family to be characterized as eurypalynous in the SPA Pau-de-Fruta.


INTRODUCTION
The family Fabaceae Lindl.(sensu APG II 2003, APG III 2009) is the third largest family of Angiospermae, second in economic importance of agriculture and widespread in the world, comprising about 727 genera and 19,325 species (Lewis et al. 2005, Wojciechowski 2003).In Brazil, an estimated number of 200 genera and 1,500 species occur in almost all vegetation formations from the Amazon region to the south of the country.In most natural ecosystems in Brazil, Fabaceae is included among the main families (Barroso et al. 1991, Souza andLorenzi 2005).Many species are pioneers and heliophytes, while others occur in more humid and darker environments and can be selective hygrophytes, or xerophytes aphilas.The Fabaceae have distinct well-developed floral traits that result in a diversity of pollination mechanisms (Stirton 1981).
The Fabaceae is commonly found in the Special Protection Area (SPA) Pau-de-Fruta, totaling thirteen species, with herbaceous, shrubs or arboreal representatives (Mendonça Filho 2005, Silva et al. 2005).Inserted in the Espinhaço Southern Mountain Range, the SPA Pau-de-Fruta belongs to Companhia de Saneamento de Minas Gerais (COPASA -MG), located 6 km from the city of Diamantina.It stretches over 1,700 ha located in the watershed of the Córrego das Pedras, a source used to supply water to the population of the Diamantina city.It consists of flat surfaces interspersed with outcrops of quartz rocks at an average altitude of around 1,366 m a.s.l with mild temperatures in most part of the year (annual average of 19.0°C), the monthly average of the coldest month is 16.1°C (July) and the hottest, 21.3°C (January).The average annual precipitation is 1,351 mm (Neves et al. 2005).The SPA Pau-de-Fruta has unique geo-morphological features of approximately 81.75 ha occupied by Quaternary peatlands (Campos et al. 2010).The predominant phytophyisiognomy in the area is a humid grassland due to the upwelling of groundwater.It is characterized by the predominance of herbaceous species, and rare shrubs or trees.However, the occurrence of by grassland surrounded "capões" is common, as well as small forest fragments usually associated with wetlands (Ribeiro and Walter 1998, Mendonça Filho 2005, Horak 2009).
The morphology of the Fabaceae pollen is relatively well known.In Brazil, taxonomic studies based on the palynology of the family were developed by Barth (1964) for the arboreal flora of Santa Catarina state; by Melhem (1966Melhem ( , 1968Melhem ( , 1971) ) and Salgado-Labouriau (1973) for species of the Cerrado; by Silvestre-Capelato and Melhem (1997) for the flora of a preserved area in São Paulo state; by Souza et al. (2004) for a "Restinga" vegetation (coastal tropical forest) of Rio de Janeiro state and by Carreira et al. (1996) for the Brazilian Amazon, in addition to the compendium on the Fabaceae pollen aimed at the apiculture sector presented by Moreti et al. (2007a, b).
The present study aims to attain the palynological characterization of thirteen species belonging to seven genera of Fabaceae occurring in the SPA of Pau-de-Fruta, Diamantina, Minas Gerais State, Brazil.

MATERIALS AND METHODS
Fertile individuals of Fabaceae were collected in the Special Protection Area (SPA) Pau-de-Fruta and processed within the usual methods in botany (Mori et al. 1989).Three samples of each species were pressed separately in a newspaper and dried in a drying kiln for three days.Exsiccate specimens were prepared and incorporated into the Herbarium of the Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM, Herbário DIAM).Only for the Dalbergia miscolobium Benth, which showed insufficient pollen material, the flower buds were removed from exsiccate specimens from the Herbarium Científico do Estado "Maria Eneyda P. Kauffmann Fidalgo" (SP) of the Botanical Institute of São Paulo -Brazil.
The following species were investigated: Pollen grains were acetolysed following Erdtman (1960).Pollen dimension averages were derived from measurements of 25 pollen grains per sample.Measurements of morphological structures were based upon ten pollen grains per sample.Scanning electron microscope images of the non acetolysed pollen grains were obtained using a PHILIPS XL 20 microscope of the Instituto de Botânica.The pollen grains were spread directly on the microscope stubs, and sputtered with a 20 nm thick gold layer.Terminology follows that of Barth and Melhem (1988) and Punt et al. (2007).

RESULTS
The description of pollen grains and polyads morphology of the thirteen species of Fabaceae occurring in SPA Pau-de-Fruta, Diamantina, Minas Gerais, Brazil, is presented.The size of pollen grains and polyads is presented in Tables 2, 3, 4, 5 and Figures 1 and 2 Comments: The pollen grains of C. flexuosa crumple and wrinkle easily and are difficult to observe in polar view.

DISCUSSION
Pollen morphology of Andira fraxinifolia has not been studied previously.However, other Andira species have been the subject of palynological studies developed by Barth (1964), Melhem (1968), Salgado-Labouriau (1973), Roubik and Moreno (1991), Silvestre-Capelato and Melhem (1997) and Carreira et al. (1996).The species studied by Barth (1964) and Silvestre-Capelato and Melhem (1997), A. anthelmia differs from the species described in the present study due to the presence of a equatorial constriction of the colpi, absent in the species analyzed here.Barth (1964) also noted 2-colpate pollen grains, which was not observed in the present study, moreover, the measurements of the diameters were larger than those obtained here.Salgado-Labouriau (1973) studied the pollen morphology of four species of Andira.The author obtained different results in terms of a larger size of pollen grains, a pilate or reticulate ornamentation, the constant presence of psilate margos, and a decrease of the brochi diameter towards the apertures.Carreira et al. (1996) studied pollen grain morphology of Andira multistipula Ducke from the Amazon region, characterized being psilate and not microreticulate.The pollen material from Andira inermis (W.Wright) HBK studied in Panama by Roubik and Moreno (1991) was characterized as psilate also, and its pollen grains were much smaller than those analyzed here.Melhem (1968) studied four species from the Cerrado (A. humilis, A. laurifolia, A. vermifuga and A. paniculata) and concluded that the genus is stenopalynous.It was noted that the six taxa of Chamaecrista analyzed showed homogeneity of pollen characteristics, mainly related to the apertures that are very long, 3-colporate and with a central constriction, pointed apices, sometimes giving the impression of binding at the poles, ascertaining the truly syncolporate pollen grains in C. glandulosa only.The main pollen intra-genetic characteristics observed were: shape (subprolate to prolate); amb (slightly sinu-aperturate or not), size of the polar and equatorial diameters (from 20.0 to 61.5μm) and endoapertures ranging from circular, lalongate to lolongate, but not always clearly visible.C. hedysaroides stands out as the only slightly sinuaperturate species, with pollen grain size smaller   12) and low (13) focus.14.Polar view, SEM. 15.Equatorial view, SEM.16.Detail of aperture, SEM.Scale bar = 2 μm (9, 16); 5 μm (6,7,12,13); 10 μm (3,4,5,8,10,11,14,15).(17,18,19,20,22,23,25,26,27).CYNTHIA F.P. DA LUZ et al.
than 25 μm.However, shape, amb and size of pollen grains are of lower diagnostic value, as they can vary within the same species and be affected by the preparation method and the environmental conditions of the plants.Pollen grains of C. debilis, C. flexuosa and C. glandulosa, can not be distinguished.Its rugula are smaller than of the remaining species and little developed.C. cathartica and C. papillata differ in terms of the polar and equatorial diameters only.It is noteworthy that the exine ornamentation variations may be observed only using ultrastructural magnification.Therefore, Chamaecrista can be considered as a stenopalynous genus in the SPA Pau-of-Fruta area.Other species of Chamaecrista were analysed by Carreira et al. (1996) and Silvestre-Capelato and Melhem (1997), the latter authors agree with our results, characterizing the genus as stenopalynous.Among the studied species by these authors, two specimens of C. glandulosa showed 3-colporate pollen grains, microreticulate sculpture and smaller diameters.Their microreticulate feature may be attributed to the lack of SEM observation.Syncolporate apertures occurring in the present sample showed, therefore, a variability of the aperture structure in pollen grains of this species.It should be noted, however, the need for further studies involving more specimens of C. glandulosa collected in the SPA Pau-de-Fruta to assess the confluence of the colpi.
The Dalbergia miscolobium pollen grains has been studied by Melhem (1968), presenting a similar description, differing in terms of the presence of a margo and lalongate endoapertures, unlike the circular and lolongate ones observed in the present study.Two more species from the Brazilian Cerrado region were there studied also, concluding that the species of Dalbergia could only be distinguished by their diameters.Salgado-Labouriau (1973) studied the same species from the Cerrado previously studied by Melhem (1968), grouping them by their similar pollen morphology into the Andira pollen type, joining the species of Andira and Machaerium.SPA Pau-de-Fruta Dalbergia miscolobium pollen grains differ from that of Andira by the shape, amb, type of endoapertures and exine ornamentation.
The pollen morphology of Galactia martii has not been studied previously.Pollen grains of other species of Galactia were examined by Melhem (1971) and Silvestre-Capelato and Melhem (1997); the palynological description matches, except by the oblate spheroidal form of G. martii pollen grains.Melhem (1971) noted a polymorphism of the pollen grain shape of G. macrophylla, considering the genus as a stenopalinous one.Carreira et al. (1996) indicated a microreticulate ornamentation of Galactia jussiaeana from the Amazon region, which does not match with the reticulate pattern of G. martii.Studying the bee pollen morphology of three species of Galactia, Moreti et al. (2007a) characterized them as small to medium sized, microreticulate and subprolate.As in Carreira et al. (1996), there was no analysis in SEM and in Moreti et al. (2007a) the increase of ultrastructure photos was small, the microreticulate within the brochi of the reticulum were not disclosed, as described in the present analysis.
Pollen morphology of six Brazilian species of Periandra was studied by Funch and Watanabe (1994), among them P. mediterranea, previously studied by Melhem (1971) and Carreira et al. (1996).The data obtained differ from those in terms of smaller sizes of polar and equatorial diameters, and a thinner aperture margo.The microreticulate exine corroborates with Carreira et al. (1996), differing from the reticulate described by Funch and Watanabe (1994).These authors also indicated that the Periandra genus is eurypalynous.
Guinet and Caccavari (1992) considered the pollen morphology of 27 South American Stryphnodendron species, grouped according to pollen types.The basic S. adstringens group comprised 12 species, presenting polyads ranging of up to 40 μm, always with 16 pollen grains covered with very small verrucae.Although the dimension of the major axis of the polyads was higher in our specimen, all the features presented by the authors agree with ours, demonstrating the constancy of the pollen characteristics of these species.

Figure 1 -
Figure 1 -Representation of confidence interval of mean in 95% of Polar diameter (µm) in equatorial view of the pollen grains of the Fabaceae family from the Special Protection Area (SPA) Pau-de-Fruta, Diamantina, Minas Gerais.The higher and lower boundaries showing the confidence interval; the average circle showing the arithmetic mean.

Figure 2 -
Figure 2 -Representation of confidence interval of mean in 95% of Equatorial diameter (µm) in equatorial view of the pollen grains of the Fabaceae family from the Special Protection Area (SPA) Pau-de-Fruta, Diamantina, Minas Gerais.The higher and lower boundaries showing the confidence interval; the average circle showing the arithmetic mean.

TABLE II Arithmetic average of measures (µm) of colpus, presence or not of margo and thickness of the exine layers of the pollen grains of the Fabaceae family from the Special Protection Area (SPA) Pau-de-Fruta, Diamantina, Minas Gerais (n=10).
The pollen grains of S. rugosa were observed as 3-aperturate (isopolar 3-colporate, CYNTHIA F.P. DA LUZ et al.