Palynotaxonomy of Calea sect. Meyeria (Asteraceae: Neurolaeneae)

Abstract Calea comprises 157 species that occur in the Neotropics. The genus is organized into eight subgenera and 18 sections. Calea sect. Meyeria consists of eight species occurring exclusively in south-central Brazil. In this study, pollen grains from these eight species were sampled from herborized flower buds in pre-anthesis. Pollen samples were acetolyzed, measured, and photographed under a light microscope. Unacetolyzed pollen grains were examined under a scanning electron microscope. The results were used to characterize pollen morphology and construct a palynotaxonomic key for Calea sect. Meyeria. The analyzed pollen grains were monads, isopolar, usually medium-sized, oblate spheroidal, tricolporate, with lalongate endoaperture, the sexine echinate and thicker than the nexine, and six spines in the apocolpium region. The eight species have very similar pollen grains, but some characters are informative for this section, namely pollen size, polar area, sexine ornamentation, and endoaperture characteristics. Despite the similarity of the pollen grains, palynological analysis was able to contribute to the delimitation of Calea species, providing new information for species distinction within Calea sect. Meyeria.


Introduction
Asteraceae comprises ca.24,000-30,000 species and 1600-1700 genera with a cosmopolitan distribution (Mandel et al. 2019).The family is monophyletic (Susanna et al. 2020), as supported by phylogenetic analysis of molecular data (Mandel et al. 2019) and morphological characters, such as capitulate inflorescence, synantherous anthers, inferior ovary, and cypselate fruits, commonly with pappus (modified calyx) (Roque and Bautista 2008;Funk et al. 2009;Roque et al. 2017).Members of the family are classified into 16 subfamilies and 50 tribes (Susanna et al. 2020).One of these tribes, Neurolaeneae, was re-established based on molecular studies (Panero et al. 1999;Panero and Funk 2002;Panero 2007).The tribe contains six genera and ca.185 species, with the vast majority occurring in the tropical regions of the Neotropics (Bueno et al. 2021;Bueno 2023).Bueno et al. (in press) proposed that Neurolaeneae should be divided into six genera.
Calea L. comprises 157 species generally characterized by opposite leaves, 3-8 seriate involucre, yellow corolla, and pappus scales (Bueno 2023).In Brazil, the genus is represented by 91 species, 59 of which are endemic (Bueno et al. 2022;Reis-Silva and Nakajima 2021).Calea is currently subdivided into eight subgenera and 18 sections; Calea section Meyeria Benth.& Hook.f.consists of eight species occurring exclusively in south-central Brazil (Bueno et al. 2021(Bueno et al. , 2022;;Bueno 2023).Members of Calea sect.Meyeria are characterized by shrubby habitat, ovate leaves, involucre with more than three foliaceous phyllaries, radiate capitula, and cypsela larger than the pappus (Bueno 2023).This group of eight species is also referred to in the literature as the Calea myrtifolia complex (Pruski and Urbatsch 1988;Pruski 2005;Bueno et al. 2021Bueno et al. , 2022)).Bueno et al. (in press) supported the existence of two morphological groups in this section, the C. myrtifolia group and the C. triantha group.
Palynological analysis can be used in research on pollination, pollen dispersion, paleoecology, melissopalynology and forensics.Furthermore, it has fundamental importance in the study of ecological and evolutionary characteristics and phylogenetic relationships of different groups of plants (Cancelli et al. 2005;Cui et al. 2019;Mezzonato-Pires et al. 2019;Gonc ¸alves-Esteves et al. 2022;Quamar et al. 2022;Cartaxo-Pinto et al. 2022a, 2022b, 2022c, 2023).The study of pollen grains is a useful tool for elucidating the taxonomy of several botanical families, and circumscribing genera and subgeneric categories (Gonc¸alves-Esteves et al. 2022).
This is the first study focused exclusively on describing the pollen of Neurolaeneae species with a taxonomic scope.The objective was to analyze pollen grains of the eight species of Calea sect.Meyeria to identify characters that contribute to the palynology of the section and genus and generate data to elucidate the taxonomy of this group.

Pollen material
We analyzed pollen grains from eight species of Calea sect.Meyeria, in the Calea myrtifolia group (C.marginata S.F.Blake, C. myrtifolia (DC.) Baker, C. parvifolia (DC.) Baker and C. phyllolepis Baker), and the Calea triantha group (C.funkiana Heiden and C. triantha (Vell.)Pruski).Pollen grains were collected from fertile anthers of flowers in anthesis or flower buds in pre-anthesis.The botanical material was obtained from specimens deposited in the following Brazilian herbaria: ICN, SP, SPSF, and UPCB (acronyms according to Thiers 2023).The specimens are listed in the Appendix.

Light microscopy
For observation by light microscopy, pollen samples were prepared according to the acetolysis method of Erdtman (1952), with the modifications proposed by Melhem et al. (2003).Acetolyzed pollen grains were measured up to 7 days after preparation (Salgado-Labouriau 1973).Microscope slides of pollen grains were deposited in the pollen collection of the Alvaro Xavier Moreira Laboratory of Palynology, Department of Botany, National Museum, Federal University of Rio de Janeiro, Brazil.

Scanning electron microscopy
Unacetolyzed pollen grains were mounted on stubs with carbon tape (Cartaxo-Pinto et al. 2022a) and examined using an FEI Quanta 450 field-emission scanning electron microscope at the Nanotechnology Characterisation Center (CENANO), National Institute of Technology, Brazil.

Measurement of pollen grains
A total of 25 measurements were randomly taken of polar and equatorial diameters.Additionally, 10 measurements were randomly taken of the equatorial diameter in polar view, apocolpium side, aperture length and width, exine thickness, and echinus dimensions.The results were subjected to statistical analysis to obtain the arithmetic mean (x\bar), standard deviation of the sample (s), standard deviation of the average mean (s x\bar ), coefficient of variation, and 95% confidence interval (CI).Table 1 presents the arithmetic mean (x\bar), standard deviation of the average mean (s x\bar ), and 95% CI values.The arithmetic mean of equatorial diameter in polar view, apocolpium side, aperture length and width, exine thickness, and echinus dimensions are presented in Tables 2 and 3.For each specimen, a minimum of three permanent slides of acetolyzed pollen grains were analyzed to ensure sample standardization (Salgado-Labouriau et al. 1965).

Terminology
The terminology adopted was that of Punt et al. (2007), which takes into account pollen size, shape, aperture number, and sexine ornamentation pattern.Descriptions of polar area and aperture size follow the classification of Faegri and Iversen (1966) for calculation of the polar area index.Photomicrographs of pollen grains were captured using a digital camera coupled to a Zeiss Axiostar Plus microscope.

Results
The following species were analyzed: 3.1.Dispersion units, polarity, size, amb, shape, and polar area Pollen grains from the eight analyzed species are monads, isopolar, oblate-spheroidal, medium-sized (25.0-50.0mm) in most species and large only in C. triantha (50.0-100.0mm), with subcircular or subtriangular amb.Furthermore, the polar area is very small in most species, being small only in C. parvifolia, C. subintegerrima and C. triantha (Tables 1 and 2).

Exine ornamentation and structure
The sexine is echinate, with echinae distributed in an organized way.

Discussion
Analysis of palynological measurements and characters of Calea sect.Meyeria revealed that the eight species have very similar pollen grains.However, some characters allow the differentiation of the species, namely pollen size, polar area, and sexine ornamentation, in addition to endoaperture characteristics.Bueno et al. (in press) performed a taxonomic study of the genus and investigated the sporophyte morphology of C. section Meyeria.They observed that the species (the same as those analyzed here) are similar to each other.The authors supported the existence of two morphological groups in this section, the C. myrtifolia group and the C. triantha group.Based on our results and according to the pollen key organized here, only one trait strictly agrees with such a grouping, namely echinus length smaller than 5.4 mm in species of the C. triantha group (vs.greater than 5.8 mm in species of the C. myrtifolia group).Another informative characteristic is endoaperture constriction.Species of the C. triantha group, except C. pruskiana, do not show constriction, whereas the C. myrtifolia group shows constriction, except C. phyllolepis.Species of the C. triantha group were treated as C. triantha until recently (Bueno et al. 2021(Bueno et al. , 2022)).

Conclusion
Calea species have similar pollen characters.This is the first study to focus on Calea, particularly on species of C. sect.Meyeria.The results of this palynotaxonomic approach were very positive, as different characters that allow species distinction were identified, such as pollen size, polar area, sexine ornamentation, endoaperture type, and distance between echinae, even considering the phylogenetic proximity among the studied species.The results indicate that, even though species of the section are very similar, they can be differentiated through palynological analyses, which points to palynotaxonomy as a promising tool to enhance knowledge within the tribe Neurolaeneae in general and within Calea specifically.Here, we developed a pollen key to the section.Further studies with other genera of Neurolaeneae and other infragenera of Calea may allow a better understanding of the palynology of the genus, and this information could be combined in the phylogenetic reconstruction based on molecular data obtained by Bueno (2023) to gain greater insight into the evolution of pollen traits within the tribe and the genus.