Pollen morphology of species of Graphistylis B . Nord . ( Asteraceae ) of Brazil

This paper presents the results of a palynological study of the species of Graphistylis that were segregated from Senecio (s.l.) (Senecio sect. Dichroa) and that are all endemic of Brazil. These species are found in mountainous regions of the states of Espirito Santo, Minas Gerais, Paraná, Rio de Janeiro, São Paulo and Santa Catarina. The pollen grains of eight species were acetolyzed and analyzed and photographed using light microscopy. Unacetolyzed pollen grains were analyzed and photomicrographed using scanning electron microscopy. The current study undertook a palynologically analysis of Graphistylis in order to obtain information useful for making a better ranking of these genus. The results show medium-sized pollen grains that were oblate spheroidal or prolate spheroidal and subtriangular amb and tricolporate. The pollen grains possessed lalongate endoapertures with a median constriction and the presence of costa in only two species. The exine is echinate with a varying number of spines in the apocolpium region around the central spine in polar view. The spines are short with basal perforations at varying distances. Although the morphology of the pollen of species of Graphistylis is very similar, multivariate analysis highlights the importance of quantitative traits in distinguishing species.


Introduction
The genus Graphistylis is endemic to Brazil where it is found in the mountainous regions of the states of Espírito Santo, Minas Gerais, Rio de Janeiro, São Paulo, Paraná and Santa Catarina (Nordenstam 1978;Jeffrey 1992).It is in the tribe Senecioneae (Cassini 1818), which is considered the largest tribe of Asteraceae with approximately 3500 species distributed among 150 genera (Nordenstam 2007).
The forms of the style-arm apices are useful for distinguishing species within the genera of Senecioneae.In Senecio, the style-arm apices may be truncated, convex, circular or rhombic, with papillae varying in length, form, and distribution (Jeffrey et al. 1975).
It was mainly based on the characteristics of the style arms that Graphistylis was segregated from Senecio s.l.In Graphistylis, species are easily recognized by having style arms that have a conspicuous tuft of trichomes emerging from the apex of the central arm, surrounded by a crown of shorter trichomes.
The morphology and ultrastructure of pollen grains have been useful in the systematics of Asteraceae, and although part of the family has been well studied palyno-logically, there have been few such studies of Brazilian species (Mendonça & Gonçalves-Esteves 2000).
Therefore, the present study aims to fill this gap in knowledge by palynologically analyzing the species of Graphistylis and, consequently, provide information that will aid in better classifying this genus.

Materials and methods
Pollen grains of all species of Graphistylis B. Nord.were analyzed.Pollen material was obtained from fertile anthers of flowers in anthesis and/or from buds in pre-anthesis, derived from samples deposited in herbaria in southeastern and southern Brazil: MBM, R, RB and RFA.Acronyms are in accordance with Thiers (continuously updated).For light microscopy, pollen material was processed according to the method of acetolysis established by Erdtman (1952) with the modifications proposed by Melhem et al. (2003).
Slides were deposited in the Palynology Collection of Alvaro Xavier Moreira Palynology Laboratory, in the Botany Department at National Museum of Federal University of Rio de Janeiro.
For scanning electron microscopy (SEM), anthers were macerated and the pollen grains, non-acetolyzed, were pulverized over stubs covered in carbon tape (Melhem et al. 2003).The pollen was metallized with a layer of pure gold for approximately 3 minutes and subsequently analyzed using a JSM-5310 SEM of the Hertha Meyer Ultrastructure Laboratory of the Biophysics Institute at the Federal University of Rio de Janeiro and a JEOL JSM 6390 LV SEM at the Electronic Microscopy Laboratory of Invertebrates of the National Museum, Federal University of Rio de Janeiro.
For descriptions and illustrations a specimen considered as "standard" was selected for each studied species and is indicated by an asterisk (*) after the name of the collector.In order to document morphometric variation we studied, when possible, up to four specimens for each species as "material for comparison" (Abreu et al. 2014;Mezzonato-Pires et al. 2015;Vignoli-Silva et al. 2015).
The polar and equatorial views of pollen grains from the standard material were measured.Random measurments included twenty-five measurements of polar (PD) and equatorial (ED) diameters in equatorial view, 10 meas-urements of equatorial diameter in polar view (EDPV) and 10 measurments of the apocolpium side (AS).
Additionally, for each species 10 measurements of the aperture and endoaperture (length and width) were taken, as well as exine thickness (sexine and nexine), length, and width and the distance between the spines.
The arithmetic mean (x ) , standard deviation (s), the standard deviation of the mean (s x ) and the confidence interval of 95% (CI) were calculated for each sample.The statistical results are shown in tables.The standard deviation and the coefficient of variation of the sample were calculated to confirm the results, but are not included in the tables.
Terminology for size, shape, number of apertures and the pattern of sexine ornamentation follows Punt et al. (2007).Descriptions of the polar area and the aperture size follow the classification established by Faegri & Iversen (1966) for the polar area index.
Electron micrographs and photomicrographs were transformed into shades of gray and modified for contrast and brightness using Corel Draw X5.
Exploratory data analysis was carried out using the PC-ORD version 5.31 (McCune & Mefford 2011) program.Pollen features of the analyzed species were organized in a matrix with 17 variables.In the graphs species names were abbreviated with the first three letters and pollen features (variables) were expressed as numbers.
Measured data used in the multivariate analyses (PCA and Cluster Analysis) were standardize by ½ power square root transformation.
Principal component analysis (PCA) was conducted to determine whether the pollen features could cluster species.The variance and covariance matrix (var-cov) was obtained from the means of the morphometric data of the palynological analysis, and the coordinates in the biplot graph are based on Euclidian distances and show the first and second principal components.The matrix of characters, the values of the vectors in each axis and the total of accumulative variance are presented in tables.
The cluster analysis (AHC Clustering) was conducted with the aim of classifying the analyzed species into groups based on shared pollen variables (similarity).Two factors were considered in forming the groups from the set of analyzed variables: the percentage of information (variables) necessary to arrive at the groups related to the final number of groups formed.A dendrogram was made using Euclidian distances (Caccavari et al. 2008) and using Ward's linkage method.

Polarity, shape and polar area
All the species examined have isopolar pollen grains that are radially symmetrical, oblate spheroidal or prolate spheroidal, and subtriangular in shape.The polar area ranges from very small in G. argyrotricha, G. itatiaiae and G. serrana, to small in the other species (Tab.2).

Pollen grain apertures
The pollen grains are tricolporate; the ectoaperture is restricted to an ornamented aperture area (Fig. 2L, bracket) of varying dimensions and whose limits are usually poorly define their limits within the ectoaperture.The ectoaperture range from being long to very long, and are narrow with acute ends and lalongate endoaperture (Tab.3), and possess a median constriction (Figs.1B, 2F).
The longest aperture area was found in G. dichroa (ca.28.8 m) and the shortest was in G. toledoi (ca.23.7 m).The longest colpus was found in G. organensis (ca.19.6 m) and the shortest in G. toledoi (ca.13.4 m); the longest endoaperture was found in G. serrana (7.7 m), and the shortest in G. argyrotricha (3.1 m); the smallest width was found in G. toledoi (ca.14.7 m) and the largest in G. dichroa (18.2 m).When seen in equatorial view, a row with five to seven pairs of spines can be seen bordering the aperture (Figs.1F, G, 2B, L).Graphistylis oreophila (Fig. 2A, arrow) and G. organensis (Fig. 2D, arrow) were the only two species with costa.

Species
The spines possess projections formed by the layers of columellae at their base, with the width of the base of the spines being less than the area between spines (Figs.1D,  J, 2A, D).G. cuneifolia had the widest (ca.6.6 m) and furthest apart spines (ca.12.6 m).G. argyrotricha, G. serrana and G. toledoi had the shortest spines (ca.3.0 m), while G. argyrotricha, G. dichroa and G. oreophila had the most narrow spines (ca.3,4 m).The smallest distance between spines was found in G. dichroa (ca.7.6 m).
The species of Graphistylis vary in the number of spines, in a polar view, in the apocolpium region and in their arrangement around the central spines: G. argyrotricha, G. dichroa, G. oreophila, G. serrana and G. toledoi have six spines (Fig, 1H); G. cuneifolia has seven spines (Fig. 1E); G. organensis (Fig. 2E) and G. itatiaiae have a single central spine surrounded by five spines.

Hierarchical cluster analysis (HCA)
The cluster analysis of species of Graphistylis produced a dendrogram with a linkage value of 9.09 for explaining the data.Taking into account the percentage of information (variables) and the final number of groups, three groups were recognized when 50% of the information was analyzed.The species of Group 1 were G. argyrotricha, G. dichroa, G. oreophila, G. serrana and G. toledoi.Group 2 included only G. cuneifolia, and Group 3 contained G. itatiaiae and G. organensis.When 75% of the information was analyzed, four groups were produced consisting of the previous three groups, but with G. serrana and G. toledoi being removed from Group 1 to form a new group (Fig. 3).

Principal component analysis (PCA)
The first two axes of the PCA explain 64.64% of the total variance, with first and second axes explaining 43.46% and 21.18% of the variance, respectively.The most significant variables of the first principal component are: polar diameter/PD (1), EDPV (8), equatorial diameter/ED   (2), and colpus width (7).The most significant variables of the second axis are: presence of costa ( 14), number of spines on the apocolpium (15,16,17), index of polar area (10) and nexine thickness (13).
The species of Graphistylis are quite dispersed between the positive and negative poles of the first two axes.G. organensis, G. cuneifolia, G. oreophila, G. serrana and G. toledoi are polarized with G. oreophila and G. organensis isolated from the other species towards the positive pole of the second axis due to possessing costa; G. organensis separates from G. oreophila because it has one of the longest colpi (4) and P/E (3), and from G. cuneifolia at the negative pole of the scond axis because it has the greatest values for PD (1) and ED (2), as well as the thickest exine (11).Variables 6 and 16, corresponding to aperture length and number of spines on the apocolpium, were most significant in grouping G. serrana and G. toledoi, with the latter distancing itself due to smaller values of PD (1) and ED (2).
Although morphologically the pollen grains of species of Graphistylis are very similar, the analyses conducted here highlight the importance of quantitative characters in distinguishing species.

Discussion
Pollen grain morphology is quite homogeneous among the species of Graphistylis (medium, isopolar, tricolporate, lalongate endoaperture with mean constriction, presence of cavea and echinate sexine).However, they do differ in shape (oblate spheroidal or prolate spheroidal), polar area (small or very small), presence of costa (only in G. oreophila and G. organensis), and size of aperture area.Graphistylis toledoi differed from all other species by having the smallest pollen grains.
Using transmission electron microscopy, Skvarla & Turner (1966), analyzed the pollen grains of Blennosperma and Crocidium, subordinate genera to the tribes Helenieae and Senecioneae (Asteroideae), respectively.They reached the conclusion that the ultrastructure of the pollen grain wall is a diagnostic characteristic since these genera are closely related.The three large groups of pollen types created by Skvarla & Larson (1965), and confirmed in Skvarla & Turner (1966), are: helianthoid, anthemoid and senecioid, the last being characteristic of Senecioneae and Ambrosiineae.Senecioid type of pollen is described as lacking an internal foramen but having a thick basal layer and an interrupted nexine.The pollen grains of Graphistylis are of the senecioid type.Moore & Webb (1978) succinctly analyzed Senecio pollen grains and regarded them as not possessing a visible columella below the structure of the echinate tectum.The species of Graphistylis of the present study, formerly regarded as subordinate to Senecio, differ from what was reported by Moore & Webb (1978) for Senecio, specifically their observation of bacula crossing the tectum in LOanalysis and in optical sectioning.
Using scanning electron microscopy, Vincent & Norris (1989) analyzed 95 species of Senecio that occur predominantly in South Africa, plus 11 species of genera of the subtribe Senecionineae.These authors described the pollen grains as having a high density of spines that they classified with respect to length/height (very large to moderately large).The authors concluded by affirming that the genus Senecio is closely related to the subtribe Senecionineae, in agreement with the studies of Jeffrey et al. (1978).The results presented herein show that the spines of Graphistylis range from 3m to 4.8m, while those of Senecio, according to Vincent & Norris (1989), only reach up to 3m in length.Therefore, Graphistylis spines would be in the range of "very large" and of high density in the opinion Vincent & Norris (1989); however, the present study considered these spines as short, thereby differing from these authors.Bolick (1991) analyzed the size of the pollen grains and exine thickness in species belonging to 14 tribes of the subfamily Asteroideae, and concluded that pollen grains are small and have a proportionately thinner exine.These characteristics were correlated with the type of exine ultrastructure, which may be caveate (helianthoid and senecioid types) or non-caveate (anthemoid and arctoid types).According to the present study, the pollen grains of Graphistylis can be considered senecioid due to the presence of cavea.
In a study of pollen morphology of the family Asteraceae in Rio Grande do Sul, Cancelli et al. (2007) analyzed Senecio cisplatinus Cabrera, S. conyzaefolius Baker, and S. platensis Arechav and found their pollen grains to be medium, suboblate and prolate spheroidal in shape, tricolporate, with lalongate endoaperture, long colpia bordered by four to five pairs of spines, and caveate exine with large columellae at the base of the spines.In the polar view, there were 12-15 large, conic, columellate spines with large bases and tapering apices.The results reported herein for the species of Graphistylis are similar, differing only in shape, and in the number of pairs of spines in the aperture and the apocolpium.The differences in the findings of these two studies underscore the diversity of pollen grains among the species of these two genera.

Conclusion
According to the literature, the pollen grains of Senecio and Graphistylis are similar, differing mainly in the number of spines and in the appearance of the cavea, which is more apparent in Graphistylis.The pollen grains of the species of Graphistylis are quite homogenous, which was confirmed by the multivariate analysis presented herein.