Importance of palynology in the taxonomy of Piptolepis Sch.Bip. (Asteraceae: Lychnophorinae), a genus endemic to Brazil

: Lychnophorinae comprises 19 genera and 117 species. Its representatives are found mostly in the rocky grasslands (“campos rupestres”) of Minas Gerais, Bahia, and Goiás States, Brazil. This study presents a palynological investigation of 10 species belonging to the genus Piptolepis , endemic to the rocky grasslands of the Espinhaço Mountains, in Minas Gerais State. The plant material used in this investigation was obtained from specimens deposited in Brazilian herbaria. Pollen grains were processed by the acetolysis method, measured, described, and photomicrographed in light microscopy and scanning electron microscopy. Morphological observations revealed Piptolepis pollen to be large, prolate spheroidal in most species, 3-colporate, ectoapertures long, sexine subechinolophate or sublophate. Endoapertures vary from almost circular, lalongate or lalongate, with a median constriction observed only in three species. Three types of spines were identifi ed: prostrate and disorganized, elongated and narrow, and conical, erect, apex acute in the other species. This fi rst palynological study of Piptolepis species allowed the separation of representatives of the genus using pollen keys. The most signifi cant characters were pollen shape, ornamentation, endoaperture, and presence of median constriction in the endoaperture. The fi ndings presented here do not support the infrageneric classifi cation of Piptolepis , as pollen characters were not unique to any section.


INTRODUCTION
The Vernonieae is one of the largest tribes of the family Asteraceae, consisting of about 129 genera and 1,100 species distributed in all tropical regions of the world.The tribe has two centers of diversifi cation: southeastern Brazil and central Africa (Robinson 1999, 2007, Keeley & Robinson 2009).Its 129 recognized genera were grouped into 21 subtribes mainly based on infl orescence pattern, pollen morphology, chemical composition, and chromosome number (Keeley & Robinson 2009), but newly Loeuille et al. (2015b) in their study on the systematics and evolution of Syncephaly in American Vernonieae synonimized Centratherinae and Sipolisiinae under Lychnophorinae, reducing the number of subtribes to 19.
The importance of palynology in the taxonomy of Vernonieae is evidenced by the study of Robinson (1999), in which pollen characters were fundamental for the separation of several genera from Vernonia Schreb.Many other studies provided valuable contributions to the classifi cation of groups and species of Vernonieae, such as, for instance, Mendonça et al. (2007aMendonça et al. ( , b, 2009Mendonça et al. ( , 2010)), Peçanha et al. (2001), Souza-Souza et al. (2016) and Siniscalchi et al. (2017), who examined the palynology of several representatives of the tribe, reinforcing consistency among genera and distinction between species.
Lychnophorinae, an important subtribe of the Vernonieae, comprises 19 genera and about 120 species.Most species are distributed in the Brazilian Cerrado, predominantly in rocky grasslands ("campos rupestres"), with the exception of one species distributed to Bolivia and a weedy species that shows pantropical distribution (Loeuille et al. 2015a, 2019, Bringel Jr. et al 2019).Taxonomic considerations based on molecular and morphological data resulted in the expansion of the subtribe to make it monophyletic.Piptolepis Sch.Bip. is one of the genera subordinate to Lychnophorinae.
The genus Piptolepis, represented by 13 species, is endemic to rocky grasslands ("campos rupestres") of the Espinhaço Mountains in Minas Gerais State and the Central Plateau of Goiás State, Brazil.Its taxa are characteristically shrubby with a solitary terminal capitulum or few capitula forming a terminal synflorescence.Padlike leaf sheath and weakly imbricate caducous phyllaries.The pappus is biseriate, with bristles flattened, the outer series sometimes shorter than the inner series and usually deciduous (Hind 2003, Robinson 2006, Loeuille et al. 2019).Due to the high degree of endemism, low number of analyzed specimens and anthropic pressures, 40% of the Piptolepis taxa are included in the Red List of Endangered Species (CNCflora 2020).
Given the importance of palynology for Asteraceae, particularly Vernonieae, the lack of palynological studies on Piptolepis, and the several taxonomic changes in the genus, this study aimed to analyze the pollen types of 10 Piptolepis species and contribute to their delimitation.

MATERIALS AND METHODS
The plant material examined in this study was obtained from specimens deposited in the following herbaria: Herbarium of the National Museum of Rio de Janeiro (R), Herbarium Urbelandense (HUFU), Herbarium of the Rio de Janeiro Botanical Garden (RB), and Herbarium Bradeanum (HB).The material examined is listed in Appendix 1.
Three of the 13 species that constitute the genus Piptolepis (P.pabstii, P. riparia, and P. rosmarinifolia), other closely related genera of Piptolepis have been sampled and analyzed.All microscope slides were deposited in the botanical collection of the Álvaro Xavier Moreira Laboratory of Palynology (Department of Botany, National Museum of Rio de Janeiro, Federal University of Rio de Janeiro, Brazil).
Pollen grains were prepared for light microscopic (LM) examination by the acetolysis method of Erdtman (1960), with the modifications proposed by Melhem et al. (2003).Acetolyzed pollen specimens were analyzed within 7 days of preparation, in accordance with the recommendations of Salgado-Labouriau (1973).Twenty-five measurements were taken of the polar (PD) and equatorial (ED) diameters of pollen grains in equatorial view on a minimum of three slides (Salgado-Labouriau et al. 1965).The data were treated statistically to obtain the arithmetic mean, sample standard deviation, mean standard deviation, and 95% confidence interval (CI 95%).Other pollen parameters: equatorial diameter in polar view (EDPV), apocolpium size (SA), apertures, and exine thickness were determined in 10 pollen grains on a minimum of three slides; the results are presented as arithmetic mean.The terminology used in the description of pollen size, shape, aperture number, and sexine ornamentation follows the glossary of Punt et al. (2007).Description of the polar area and the aperture size follows the classification established by Faegri & Iversen (1966) for the polar area index.
Non-acetolyzed pollen samples were used for scanning electron microscope (SEM) analysis.First, two or three anthers were removed from the flowers or flower buds of each specimen.
Pollen grains were released from the anthers and then mounted on metal stubs with doublesided carbon tape.Samples were sputter-coated with gold for about 3 min and examined under a Jeol JSM-6510 scanning electron microscope at the Laboratory of Optical and Scanning Microscopy of the Federal University of Rio de Janeiro or sputter-coated with platinum for about 3 min and examined under a FEI Helios NanoLab DualBeam G3 CX at the National Institute of Technology (Rio de Janeiro, Brazil).

DISCUSSION
This is the first palynological study of the genus Piptolepis.Ten of the 13 species were investigated and found to have similar palynological characteristics.Within subtribe Lychnophorinae, only the genera Paralychnophora MacLeish (Souza-Souza et al. 2016) and Eremanthus Less.(Loeuille et al. 2012b) and one species of the genus Lychnophora Mart.(Marques et al. 2018) have been the focus of palynological studies.
It was possible to distinguish Piptolepis species on the basis of their pollen shape, sexine ornamentation, endoaperture shape, and presence or absence of endoapertures with median constriction.These characteristics were used to develop an identification key.
Homogeneity in exine ornamentation was observed (subechinolophate in most taxa, sublophate only in P. imbricata).Only P. campestris showed a Y-shaped structure in the apocolpium.Other important characters to distinguish between species were spine size and shape (disorganized and prostrate in P. monticola, elongated and narrow in P. oleaster, and conical in the other species).
Schultz-Bipontinus (1863) grouped species with narrow leaves and solitary capitula into Table I.Measurements (in µm) of pollen grains in equatorial view of Piptolepis species.x --arithmetic mean; s x -standard deviation; CI -confidence interval; P/E -relationship between the polar and equatorial diameters.
Sect.Macrophyllum comprises species with broad leaves and grouped capitula.All species of this section have pollen grains with lalongate endoapertures.P. imbricata and P. ericoides, however, which are part of sect.Microphyllum, also have lalongate endoapertures.In this section, only P. monticola shows pollen grains with disorganized and prostrate spines and endoapertures with median contraction.P. oleaster pollen shows elongated and narrow spines, whereas P. campestris pollen has conical spines.
One of the three species described by Loeuille et al. (2012a), P. schultziana, could not be placed in the sections proposed by Schultz-Bipontinus (1863).The pollen grains of P. schultziana are similar to those of P. campestris, which belongs to sect.Macrophyllum.The species differ only in that P. schultziana pollen grains have sexine with disorganized muri that do not form a Y-shaped structure in the apocolpium.The pollen characters described here do not corroborate the infrageneric classification of Schultz-Bipontinus (1863), as pollen attributes were not unique to the proposed sections.
Piptolepis and Eremanthus have many p a l y n o l o g i c a l s i m i l a r i t i e s re g a rd i n g ornamentation.Loeuille et al. (2012b) investigated the palynotaxonomy of 20 of the 23 Eremanthus species and characterized pollen grains as oblate-spheroidal in most taxa, rarely prolatespheroidal or suboblate, amb subtriangular, tricolporate, and subechinolophate.The authors emphasized that variations in quantitative characteristics are not related to the macromorphological subdivision of the genus or to generic or specific limits.In the present study, it was observed that Piptolepis pollen grains are spheroidal in P. leptospermoides and P. oleaster, oblate-spheroidal in P. buxoides, P. ericoides, Paralychnophora pollen grains, according to Souza-Souza et al. (2016), are medium to large, oblate-spheroidal, amb subcircular in most species, polar area small and very small in a single taxon, and sexine subechinolophate.These characteristics are very similar to those observed in Piptolepis, especially regarding sexine ornamentation and its variations.The authors observed endoapertures with median constriction in six Paralychnophora species, whereas we observed median constriction only in three species.Souza-Souza et al. (2016) emphasized the importance of pollen grains to describe the taxonomy of Paralychnophora and developed an identification key based on pollen morphology.We underscore the importance of pollen grains in the taxonomy of Lychnophorinae, as palynological analyses revealed a set of consistent information for the diagnosis of Piptolepis.
This study demonstrated the palynotaxonomic potential of Piptolepis pollen grains.Piptolepis pollen are quite similar to each other, but it is possible to distinguish taxa using qualitative and quantitative data.The results expand the knowledge of Piptolepis, providing elements that can assist in species placement and guide future studies aimed at the evolution of Lychnophorinae.

Table III .
Measurements (in µm) of the apertures and layers of exine and spines of pollen grains of Piptolepis species (n=10).sexine* -tetum + columella.DBS -distance between the spines.