Pollen morphology of Brazilian species of Verbesina L . ( Heliantheae-Asteraceae ) 1

There are nine species of the plant genus Verbesina in Brazil, which are distributed in the Northeast, Center-West, Southeast and South regions of the country. The objective of the present study was to describe the pollinic morphology of eight of these species to better characterize them and evaluate potential species-level taxonomic characters. Acetolysed pollen material was measured within seven days under light microscopy, while non-acetolyzsed pollen grains were used for scanning electronic microscopy. The pollen grains were found to be oblate-spheroidal, mediumsized, isopolar, monads that are 3-colporate with a subtriangular amb, a small polar area, a long colpus, a lalongate endoaperture, a caveate exine and an echinate sexine. Although the shape of the pollen grains of these Brazilian species of Verbesina is homogeneous, some attributes were observed to be useful for characterizing the species, such as exine thickness, distance between spines and side of the apocolpus.


Introduction
The family Asteraceae, order Asterales (APG IV 2016) has a holistic distribution but is common in the dry and open tropical montane climate zone (Anderberg et al. 2007).The family includes 13 subfamilies, 44 tribes, about 1,700 genera and proximately 27,000 species, and represents about 10 % of all Angiosperms (Funk et al. 2009;Panero et al. 2014;Panero & Crozier 2016).There are about 2,097 species of Asteraceae grouped among 289 genera in Brazil (Flora do Brasil 2020).
The genera Verbesina belongs to the tribe Heliantheae (subtribe Verbesininae) and possesses around 300 species distributed throughout the Americas, with the most occurring in Mexico and the Andes (Panero 2007).Nine species have been recorded in Brazil, which are distributed in the Northeast, Southeast, Center-West and South regions of the country, especially in forest environments.
Some studies that have addressed the pollen morphology of Verbesina have indicated the existence of characters with potential use for characterizing and differentiating species of the genus.Harker & Jiménez-Reyes (2002) found that some pollen grain characters, such as shape, size, number of spicules, transverse size colpus and polar area index, were useful for separating Verbesina barrancae and V. crocata, two morphologically close Mexican species.
The objective of the present study was to describe the pollen morphology of eight Brazilian species of Verbesina to better characterize the species of the genus and to evaluate potential species-level taxonomic characters.
Pollen material was prepared for light microscopy using acetolysis following the method of Erdtman (1952), with the modifications proposed by Melhem et al. (2003).Acetolysed pollen grains were measured within seven days of their preparation, in accordance with Salgado-Labouriau (1973).Twenty-five measurements of polar diameter (PD) and equatorial diameter (ED) in equatorial view, and 10 measurements of the equatorial diameter in polar view (EDPV) and apocolpium side (AS), were made on standard material distributed among at least three slides.For other dimensions, such as those of apertures, exine layers and diameters of comparison material, 10 pollen grains were measured on at least in three slides and the arithmetic mean calculated.Description of polar area and aperture size followed the classification established by Faegri & Iversen (1966) for the polar area index.Pollen grain size classes follow Erdtman (1952).
Permanent slides of pollen material generated for this study are deposited in the Laboratory of Palynology of the National Museum of the Federal University of Rio de Janeiro.
For scanning electron photomicrography, two to three anthers were removed from flowers or flower buds extracted from herbarium specimens.The anthers were macerated using properly flamed forceps and stylus to release nonacetolysed pollen grains over a metallic stub previously covered with double-sided carbon tape.The material was spatter-coated with gold for approximately three minutes and then analyzed and photomicrographed using a JSM-5310 scanning electron microscope at the Optical and Scanning Microscopy Laboratory, Federal University of Rio de Janeiro.
To assess whether pollen characteristics discriminated the studied species of Verbesina, a principal component analysis (PCA) was performed using eleven metric variables.The results were biplotted on a graph with axes 1 and 2 of the PCA.
In all species the exine was found to be caveate and echinate (Figs. 1, 2).The sexine and nexine were almost always of the same thickness, but when they did differ the sexine was thicker than the nexine.Mean width exine ranged 2.2-3.4 µm (Tab.3).The sexine and nexine are very close to the cavea, making it difficult to see.
Four to six pairs of spines were observed around the aperture in equatorial view (Figs.1D, H, L, P, 2D, H, L, P), which were longer than wide and with perforations at the base.The shortest spines were found in Verbesina macrophylla (4.0 µm) while the longest were for V. baccharifolia (5.9 µm).The distance between spines varied (6.1-8.6 µm), with it being the greatest in V. floribunda (8.6 µm) and least in V. sordescens (6.1 µm) (Tab.3).10.4 4.9 3.9 10.4 2.9 1.9 1.0 5.2 3.4 6.1 Verbesina glabrata was the only species to exhibit variation in pollen grain shape, ranging from oblatespheroidal to prolate-spheroidal (Tabs.2, 4).The mean PD and ED of the comparison material of V. macrophylla were 31.9µm and 32.6 µm, respectively, which differed from that of the standard material (25.8 µm and 27.2 µm, respectively), however, pollen grain shape did not differ.The comparison material for the other species had means that fell within the range found for the standard material.

Principal component analysis (PCA)
The first two axes of the PCA explained 83.6 % of the variability of the analyzed data.The first axes explained 64.8 % of the data, with Verbesina bipinnatifida, V. macrophylla and V. nicotianifolia having, in general, the lowest values for PD, ED, EDPV, colpus length, endoaperture length and spine length, while Verbesina baccharifolia and V. luetzelburgii had  the highest values for these attributes.These attributes were closely correlated, as shown in Figure 3.The second axis explained 18.8 % of the data, with Verbesina floribunda having the highest values for distance between spines (DBS), exine thickness and endoaperture width, and the lowest values for the apocolpus side (AS) index and colpus width.

Discussion
Pollen grains of Verbesina can be classified as the "Aspilia" type described by Salgado-Labouriau (1973), because they are medium to large in size, 3-colporate, and lalongate, with an endoaperture with a medium constriction, an echinate exine and conical spines with perforations at the base.
Previous studies of Verbesina have shown that pollen grain shape can vary from suboblate to prolate-spheroidal (PD / ED 0.75-1.14),with oblate-spheroidal to prolatespheroidal being most common, as was reported by Gonçalves (1976) who analyzed 17 species of Verbesina, including three Brazilian species (V.diversifolia DC = V. macrophylla, V. glabrata and V. sordescens).These differences in pollen grain shape (Gonçalves 1976;Jesus & Lima 2013;Radaeski et al. 2016) (Tab.5) are  consistent with the high coefficient of variation found, which in the present study ranged 4.6-10.4% for PD and 4.7-11.6% for ED, showing tha the shape of the pollen grain is variable.
The presence of a cavea has been reported to be very common in the tribe Heliantheae (Cancelli et al. 2007;Stanski et al. 2013;Radaeski et al. 2016).In some genera the cavea is evident and its thickness easily measured, as observed by Magenta et al. (2010) with Viguiera, for which it ranged 0.8-1.5µm.
Due to the proximity of the sexine and nexine, the cavea in Verbesina is difficult to visualize, which can lead to the false impression of its non-existence, as pointed out by Cancelli et al. (2010) for Verbesina glabrata.Some studies of species of Verbesina did not indicate the presence of a cavea, while others indicated that only the exine is of the cavea; only Radaeski et al. (2016) reported measurements of the cavea, which was 1µm for V. sordescens (Tab.5).
Although the pollen grains of the studied Brazilian species of Verbesina exhibited homogeneity of shape, aperture type, and ornamentation of the sexine, some attributes show potential usefulness for characterizing species, such as the distance between spines, and dimensions of the apocolpus side and aperture.Thus, these characters of pollen grains have potential taxonomic value for distinguishing species.

Table 3 .
Measurements of the aperture and layers of exine pollen grains in Brazilian Verbesina L. species (Heliantheae-Asteraceae); n=10; DBS = distance between spines; * measured without the spines.

Table 5 .
Shape of pollen grains and presence of cavea reported in previous studies that dealt with Brazilian taxa of the genus Verbesina (Heliantheae-Asteraceae).