Pollen morphology and its systematic value to southern South American species of Lepidaploa (Vernonieae: Asteraceae)

Palynological data have been used in Vernonieae for generic and specific delimitations, particularly in the Lepidaploinae subtribe. For this reason, pollen studies in the genus Lepidaploa are important to solve taxonomic conflicts. We characterized 23 species of Lepidaploa searching for morphological differences among themselves. We then compared the data obtained with other genera of the Lepidaploinae. The results show that the species have pollen type “C” (with polar lacuna) or “G” (without polar lacuna), oblate-spheroidal, subprolate or prolate-spheroidal, 3-colporate and equinolophate. The pollen types and shape of pollen grain are taxonomically useful. Pollen morphology is useful to distinguish species and genera of Lepidaploinae. Together with macromorphological data it is possible to delimit Lepidaploa and the species studied here. polar), são oblato-esferoidal, subprolato o prolato-esferoidal, 3-colporado e equinolofado. O tipo de grão de pólen e sua forma foram taxonômicamente úteis. Ambos os caracteres foram utilizados para distinguir gêneros da subtribo e as espécies de Lepidaploa . Juntamente com a macromorfologia os dados dos grãos de pólen permitem delimitar os gêneros e espécies estudados.


Introduction
The palynological studies are a very important tool in taxonomy allowing the differentiation of the taxonomically complex groups through the shape of pollen grains (Fazal et al. 2013). The size of pollen grains, types of aperture and structure of the exine have contributed with important differences in the classification of families, tribes, genera and species (Fazal et al. 2013).
Throughout the decades several palynological studies have been used in the Asteraceae family (Compositae) due to the taxonomic importance that pollen has for the taxa of this family (Stix 1960;Besold 1971;Kingham 1976;Skvarla et al. 1977; Rodriguésia 72: e01412019. 2021 Keeley & Jones 1979;Blackmore 1982;Robinson & Marticorena 1986;Bremer 1994). Despite the taxonomic importance of pollen grains for Asteraceae as a whole, the type of pollen present is more important in certain tribes than in others. For example, pollen grains of the Eupatorieae tribe are very uniform and do not provide palynological data for classification at the generic level (Payne & Skvarla 1970;Sullivan 1975). In contrast, the palynological data found in the Vernonieae tribe have been successfully used in the infratribal classification (Keeley & Robinson 2009).
Currently, the pollen classification most used in Vernonieae was based on the studies of Keeley & Jones (1977, 1979 and Robinson (1990Robinson ( , 1992Robinson ( , 1999 being that in these studies, 10 pollen types (A-H, J and Aynia-type) were recognized for the Vernonieae tribe. The different types of pollen grain of Vernonieae and their correlation with morphological and chromosomal characters provide important taxonomic data, especially for the subtribe Lepidaploinae which includes 12 genera and about 320 species, which occur mainly in the Western Hemisphere (Keeley & Robinson 2009). Since these genera were previously part of Vernonia sensu lato, they are morphologically closely related, and palynology is necessary for their distinctions.
The genus Lepidaploa Cass. (Cass.) belongs to the subtribe Lepidaploinae and it was reestablished by Robinson (1990) from Vernonia s.l. using the type of indumentum, inflorescence, the number of phyllaries over number of flowers, and lophate pollen grains of the types "C", "D" and "G". The pollen types found so far in few species of Lepidaploa can differentiate it from most other genera found in the subtribe Lepidaploinae (Robinson 1999).
Other authors have studied the pollen types of Lepidaploa (Dematteis & Pire 2008, Mendonça & Gonçalves-Esteves 2000Mendonça et al. 2007a). However, many species of the genus have not been studied and the pollen type of more than half of these species is unknown. In addition, in recent years several genera of the subtribe Lepidaploinae was palynologically studied (Robinson 1990(Robinson , 1992(Robinson , 1999Carrijo et al. 2005;Dematteis 2005;Mendonça et al. 2007a, b;Bunwong & Chantaranothai 2008;Angulo & Dematteis 2010;Via do Pico & Dematteis 2013), and there is no recent study that presents the relationship of the Lepidaploa pollen types with other genera of this subtribe.
Thus, we evaluate the taxonomic value of the pollen morphology of the Lepidaploa species occurring in Southern South America, allowing us to compare it to other members of Lepidaploinae.

Material and Methods
We analyzed 23 species of Lepidaploa from Southern South America (Argentina, Bolivia, Brazil and Paraguay). Pollen preparations were made from anthers of floral buds in pre-anthesis obtained from herbarium specimens from the Instituto de Botánica del Nordeste (CTES). The source and herbarium data of the specimens analyzed are detailed in Table 1.
The pollen grains were acetolyzed according to the methodology of Erdtman (1960). For light microscopy (LM), pollen grains were mounted on glass slides using glycerine jelly and subsequently examined with a Zeiss Axioplan light microscope. Permanent preparations were deposited at the Palynological Laboratory of the Universidad Nacional del Nordeste (PAL-CTES). The polar axis (P), the equatorial diameter (E), the ratio between polar axis and equatorial diameter (P/E), the exine thickness, the pore diameter, the spine length, and the diameter of the polar lacuna were measurement in at least 30 grains per sample.
For studies with the scanning electron microscope (SEM), acetolyzed pollen grains were first washed in alcohol 96% and absolute alcohol (100%), then plated with gold-palladium and examined with a JEOL 5801 LV microscope.
To show the palynological patterns between species, the qualitative characters were coded in binary (shape of pollen grain = 1: prolatespheroidal 2: oblate-spheroidal and type of pollen grain = 1: type C 2: type G), and for the quantitative characters (polar axis and equatorial axis) the averages were considered. The values of the exine thickness, spine length, diameter of polar lacuna and pore were not considered because they showed little variability among the analyzed specimens. A UPGMA cluster analysis (Unweighted Pair Group Method with Arithmetic Mean) was applied, based on the Gower distance index. The InfoStat program version 2018 was used for this analysis (Di Rienzo et al. 2018).

Results
The analyzed measurements of pollen grain of the Lepidaploa species are listed in Table 3.

General pollen morphology (Figs. 1-6)
The pollen grains are radially symmetric, isopolar, small to medium, oblate-spheroidal, subprolate or prolate-spheroidal (P/E = 0.96-1.1), 3-colporate and equinolophate (Figs. 1-6). The colpi are long and visible in polar view, with the lophae separating or not the polar lacuna from the abporal lacunae (Fig. 1c,f,i,l,o;Fig. 2c,f,i,l,o;Fig. 3c,f,i,l,o;Fig. 4b,d,g;Fig. 5d,g,l,o;Fig. 6a,d,f,g,l,o). The polar axis ranged between 13.6 and 50.43 μm and the equatorial diameter between 14.96 and 50.56 μm. The thickness of the exine, excluding the spines, varied between 1.36 and 6.8 μm. The tectum is discontinuous and formed by lacunae surrounded by lophae. The number of lacunae may be 27, 29 or 30. The diameter of the poral lacuna oscillated between 2.72 and 13.8 μm and its shape varied between more or less regular and irregular. The tectum has a microperforate surface with spines (Figs. 1-6). The spines oscillated between 1.36 and 5.44 μm length and they have a linear distribution along the walls of the lophae. The diameter of the endoaperture (pore) ranged between 2.04 and 16.32 μm. Observations with SEM showed an exine formed by columella joined at the base by a transversal structure that separates the tectum from the floor (Figs. 5h; 6e).
Cluster Analysis: the phenogram resulting from the UPGMA analysis is shown in (Fig. 7). The value of the cophenetic correlation (r = 0.86) coefficient indicates that the technique used is a good estimator of the relationship between the characters analyzed. The 23 species analyzed (OTUs) are grouped into two main clusters (group 1 and group 2). Group 1 is represented only by L. novarae, which is the only species that presented an average value of polar and equatorial axis greater than 40 μm. Group 2 is formed by species that presented an average value of polar and equatorial axis lower than 40 μm. The species in group 2 were grouped into two subgroups (2A and 2B) depending on the type of pollen. Subgroup 2A is formed by species that have pollen type "C", while subgroup 2B includes species that possess pollen type "G". Subgroup 2A is sub-divided into 2Aa, including species with prolate-spheroidal pollen grains and 2Ab with oblate-spheroidal pollen grains. Subgroup 2B is also divided according to the shape of the In the terminals of the phenogram it can be seen that most of the species can be differentiated from each other based on the values of the averages of the polar axis and the equatorial axis. The only species that could not be differentiated from each other by cluster analysis were L. eriolepis and L. fournetii, which have the same type and shape of pollen, in addition to the same values of polar and equatorial axis.

Pollen morphology
All 23 species of Lepidaploa studied have echinolophate and tricolporate pollen grains. According to the morphological analysis, the species have variations in the aperture of the colpus and number of lacunae.
Nine species present a pollen type that coincides with the "Vernonia cognata" pollen type designated by Stix (1960) or with pollen type "C" designated by Keeley & Jones (1977). Pollen type "C" was also designated as characteristic of the genera Chrysolaena (Robinson 1988), Stenocephalum (Robinson 1987a), and some species of Lepidaploa (Robinson 1990). The characteristics that differentiate this type of pollen from the other types of pollen found in the Vernonieae tribe are the presence of polar lacuna in both poles of the pollen grain, equinolophate sexine and tricolporate colpus (Robinson 1988(Robinson , 1990(Robinson , 1999. The other 14 species present a pollen morphology that coincides with pollen type "G" described by Robinson (1990). This pollen type is very similar to the "Vernonia arenaria" pollen type described by Stix (1960) and pollen type "D" described by Keeley & Jones (1979), but both are triporate instead tricolporate, which is a characteristic of pollen type "G (Keeley & Jones 1979;Robinson 1990).
The atypical pollen type "G" found in the species studied here was reported by Robinson (1990) for L. psilostachya. In this species, Robinson (1990), observed the presence of an equatorial lacuna (typical of type "B" pollen grains) in a few pollen grains. In the species studied in our work, a few pollen grains of L. psilostachya display a well-developed equatorial lacuna, whereas in L. amambaia this lacuna is reduced. Robinson (1990) suggests that the presence of equatorial lacuna in pollen type "G" may occur, but it is not a common feature in most Lepidaploa species. Unusually, the presence of a reduced polar lacuna (typical of pollen type "C") was seen in some pollen type "G" of L. remotiflora.
The variations mentioned above, although not common, were reported in other genera and species of the Vernonieae tribe. Intermediate states between pollen type "B" and "C" were found in Lepidaploa pluvialis (Gleason) H. Rob. and Vernonia trinitatis Ekman (Keeley & Jones 1977). In some populations of Chrysolaena, or  in the genus Echinocoryne H. Rob. a variation in pollen type "G" was found that presented two equatorial lacunae (Robinson 1987b(Robinson , 1990. The results obtained in the analyzed species of Lepidaploa demonstrate that in the same species some pollen grains may present small variations (polar and equatorial lacuna in pollen type "G") which may be results of the position occupied by the lacunae in the tetrad during the formation of the pollen grain as mentioned by Robinson (1990).

Taxonomic implications
Pollen grains are one of the most important microcharacters for the segregation of genera belonging to the genus Vernonia s.l. (Robinson 1999) and the palynological studies made it possible to differentiate genera in the Lepidaploinae subtribe (Robinson 1999). In recent years several studies described many pollen types for the genera of the Lepidaploinae subtribe (Robinson 1987a(Robinson , b, 1988(Robinson , 1990(Robinson , 1992(Robinson , 1999Carrijo et al. 2005;Mendonça et al. 2007a, b;Bunwong & Chantaranothai 2008;Angulo & Dematteis 2010;Via do Pico & Dematteis 2013). In Table 4, we highlight the importance of knowledge about the pollen types found in Lepidaploinae and how these pollen types contribute to the segregation of genera.
The pollen "Aynia-type" is present in three genera of the subtribe: Aynia H. Rob., Harleya H. Rob. and Pseudopiptocarpha H. Rob (Robinson 1999). This pollen type is similar to pollen type "C" described for Lepidaploa in our work and for other genera of Lepidaploinae, but in the "Aynia-type" there are three polar lacunae at each pole of the pollen grain, while a typical pollen grain type "C" possesses only one polar lacuna per pole (Robinson 1987a(Robinson , 1988(Robinson , 1990(Robinson , 1999Mendonça et al. 2007a, b;Via do Pico & Dematteis 2013).
Lessingianthus and Mattfeldanthus have pollen type "B" which is characterized by the presence of equatorial lacunae (Angulo & Dematteis 2010). Lessingianthus is the sister group of Chrysolaena and Lepidaploa (Keeley et al. 2007), and since these three genera are morphologically very similar, the pollinic type is very important for their distinction (Robinson 1999).
Pollen type "C" is present in all species of Chrysolaena, Stenocephalum, Strophopappus, and in Lepidaploa this pollen type is present only in some species (Robinson 1999). Although these genera and species present the same pollen type, the morphological characteristics of Stenocephalum and Strophopappus differentiate these genera from the other two. The cylindrical heads with few flowers are exclusive of Stenocephalum (Robinson 1987a), while the paleaceous pappus occurs only in Strophopappus (Esteves et al. 2017).
C h r y s o l a e n a a n d L e p i d a p l o a a r e phylogenetically related (Keeley et al. 2007) and the differentiation between both genera is very complex, since there is an overlap of pollen types, although the presence of pollen type "D" or "G" is more common in Lepidaploa (Robinson 1999;Mendonça et al. 2007a). Until now, the only feature that distinguished the species of Lepidaploa that present pollen type "C" from Chrysolaena is their basic chromosome numbers, since Chrysolaena presents basic chromosomal number x = 10 (Via do Pico & Dematteis 2012Dematteis , 2014Dematteis , 2019, while the basic chromosomal number of Lepidaploa is x = 14, 15, 16 or 17 (Dematteis 2002;Oliveira et al. 2007Oliveira et al. , 2012. Pollen type "D" and "G" that occur in Lepidaploa can also be found in other genera of Lepidaploinae. The morphological characteristics of the pappus, phyllaries and habit differentiate the genus Lepidaploa from the genera Echinocoryne H. Rob., Stilpnopappus Mart. ex DC., Struchium (L.) Kuntze and Xipochaeta Poepp. (Robinson 1999;Esteves & Gonçalves-Esteves 2003;Bunwong et al. 2014;Lorencini et al. 2017).
Finally, pollen type "F" found in Caatinganthus H. Rob. (Robinson 1999) is similar to pollen type "D" found in Lepidaploa. However, pollen type "F" has very short spines with a rounded apex, whereas the spines of pollen type "D" are large and have an acute apex.
In the species of Lepidaploa analyzed in our study there is a great similarity between pollen grains, such as the size of the polar axis, equatorial axis, thickness of the tectum, length of the spines and the diameter of the lacunae and pore. In this way, it was not possible to separate all the species studied in a taxonomic key.
Some closely related species can be differentiated by the type or form of the pollen grain that they present. Lepidaploa argyrotricha, is a morphologically similar species to L. chamissonis and L. salzmannii, but it can be differentiated from these other two species by the pollen type, since L. argyrotricha presents type "G" pollen while the other species present type "C" pollen. In turn, the species L. amambaia is morphologically related to the species L. remotiflora and L. setososquamosa. However, the first species differs from the others because it presents oblate-spheroidal pollen while the others possess prolate-spheroidal pollen.
Through the UPGMA analysis it is possible to infer that the shape of the pollen grain and the type of pollen grain are the most important variables for the grouping of the taxa. However, although most species are separated in the phenogram resulting from this analysis, it should be noted that the species appear isolated from each other only because the analysis was based on the averages of the polar and equatorial axis. In Table 3, it is possible to identify the superposition that exists between the values of these two variables (polar and equatorial axis) that make it difficult to separate these species only by quantitative data.
All species studied possess pollen type "C" or "G". According to our results, the information with taxonomic value are pollen type and shape. This information is important to identify morphologically related species as in the case of L. amambaia / L. remotiflora / L. setososquamosa and L. argyrotricha / L. chamissonis / L. salzmanii. Finally, pollen type and macromorphological characteristics, when used together, can be useful  a These studies do not use the alphabetical terminology to pollen-type adopted by Keeley & Jones (1977, 1979 and Robinson (1990Robinson ( , 1999. However, when analyzing pollen descriptions of Carrijo et al. (2005) and Mendonça et al. (2007a,b), we noticed correspondence between the pollen descriptions of these studies and those of Keeley & Jones (1977, 1979 and Robinson (1990Robinson ( , 1999.