Endemic Papilionoideae of the Caatinga: a contribution to the palynological knowledge of Leguminosae

Papilionoideae is the most diverse subfamily of Leguminosae, representing approximately 39 % of its species. Although it is particularly species rich and diverse in the Caatinga, the main phytophysiognomy of the semiarid Northeast Region of Brazil, little is known about the pollen morphology of the species that occur there. This study analyzes and describes the pollen morphology of the 27 species of Papilionoideae endemic to the Caatinga. Pollen grains were acetolyzed, mounted on permanent slides, measured, described and photomicrographed using light and scanning electron microscopy (LM and SEM, respectively). The main variable morphological features were pollen grain size, shape, amb, apertural type and exine ornamentation under LM (microreticulate, reticulate, psilate or finely scabrate) and SEM (microreticulate, reticulate, rugulate, granulate, fossulate and perforate). High intergeneric morphological heterogeneity was observed while morphological variation within each genus was subtle. Nonetheless, size, endoaperture shape, fastigium presence/absence and exine ornamentation were found to be important characteristics for the identification of some species.

Papilionoideae is the most species rich and diverse subfamily within Leguminosae with approximately 14,000 species and 500 genera distributed among 28 tribes (Lewis et al. 2005;LPWG 2017). The subfamily is distributed worldwide and includes representatives of varied habits. Flower morphology is the most specialized among Leguminosae and is mainly related to pollination by bees (Judd et al. 2009). Papilionoideae accounts for a significant portion of the taxonomic diversity of the Caatinga biome in Brazil, representing approximately 39 % of the species of Leguminosae found there (Queiroz 2009 The Caatinga is an exclusively Brazilian biome that covers most of the Northeast Region of the country and has a rich diversity of plants, fungi and animals Forzza et al. 2010). The vegetation has peculiar characteristics that help them adapt to the physical characteristics of the environment such as high temperatures and lack of water (Costa et al. 2010). Physiognomies of the Caatinga include subshrubs, shrubs and short deciduous trees, with crooked trunks, thorns and wax, in addition to some succulent species and annual herbs (Leal et al. 2003). There is also significant flower diversity and a high number of endemic species in the biome (Giulietti et al. 2002).
The family Leguminosae is known for its richness and diversity and is the angiosperm family with the greatest number of species in the Caatinga (Forzza et al. 2010). Papilionoideae is the most species rich subfamily of Leguminosae (Queiroz 2009), however, little is known about the pollen morphology of the endemic species of the subfamily.
Palynological studies of legumes describe the family as generally a eurypalynous group (Salgado-Labouriau 1973;Miranda & Andrade 1990;Roubik & Moreno 1991;Silvestre-Capelato & Melhem 1997;Souza et al. 2004;2014;Buril et al. 2011;Silva et al. 2016) and that it may provide the basis for important investigations within the area of applied palynology, thus confirming the need to better understand its morphopalynous characteristics.
In general, the pollen grains of Papilionoideae are dispersed in monads that vary from small to large and among 3-colpate, 3-colporate or rarely porate with a microreticulate, reticulate, psilate or scabrate exine and the presence/absence of a fastigium or costa (Salgado-Labouriau 1973;Miranda & Andrade 1990;Buril et al. 2011). However, detailed palynological research is scarce for species of Leguminosae, especially those that are endemic to the Caatinga. Silva et al. (2016) analyzed and described the pollen morphology of 144 species in an area of caatinga in Canudos, state of Bahia, out of which 30 belong to Leguminosae and 11 to the subfamily Papilionoideae. Buril et al. (2011) studied palynous typification in an area of caatinga in the state of Pernambuco and provided palynological descriptions for 35 species of Papilionoideae. These authors reported that the subfamily is palynologically homogenous but pointed out some variation with respect to apertures, shape, scope and exine stratification. Nonetheless, systemic studies of endemic taxa of the Caatinga are still insufficient. Therefore, this study aimed to reduce this knowledge gap by analyzing and describing the pollen morphology of species of Papilionoideae that are endemic to the Caatinga.
The acetolysis method of Erdtman (1960) was used to prepare pollen grains of all species for light microscopy (LM). After chemical preparation, the pollen grains were mounted in glycerinated gelatin on slides with a coverslip, sealed with paraffin, and measured, described and photomicrographed using a Zeiss Axioskop microscope. The slides were deposited in the pollen collection of the Laboratory of Palynological Studies of Universidade do Estado Bahia, Senhor do Bonfim -BA. For analysis by scanning electron microscopy (SEM), fresh (with little polyniferous material) and acetolyzed pollen grains were rinsed in an ethanol series up to 100%, pipetted onto specimen stubs. After ethanol evaporation, the stubs were coated with gold by vacuum evaporation and photographed using either a Zeiss LEO 1430 VP microscope (SEMLab, Biological Sciences Department, Universidade Estadual de Feira de Santana) or a Jeol JSM-6390LV microscope (Instituto Gonçalo Muniz -IGM / Fiocruz, Salvador, Bahia).
Pollen grains were described based on the glossaries of Punt et al. (2007) and Hesse et al. (2009). Measurements of the main morphometric parameters (equatorial and polar diameters) were made, whenever possible, on 25 pollen grains within eight days after mounting (Salgado-Labouriau 1973). Other parameters (diameter of apertures and thickness of exine) were measured on 10 randomlychosen pollen grains. Quantitative data were submitted to statistical analyses adequate for the sample size. The arithmetic mean (χ), standard deviation (Sχ), standard error (σ), 95 % confidence interval (CI) and coefficient of variation (CV) were calculated for all of the diameters of the pollen grains with a sample size of 25, while only the arithmetic mean was calculated for parameters with a sample size of less than 25.
Pollen grains small and medium in size, subprolate to spheroidal prolate, amb circular, subcircular in Z. echinocarpa and Z. tenuifolia; 3-colpate, colpi long and large with fine extremities, margins finely scabrate, apertural membrane granulate-scabrate, however, the margins in Z. echinocarpa are finely rugulate and the apertural membranes are with conspicuous rugulae, with lateral fusions mainly in the central areas and sparse granules in the peripheral areas; exine microreticulate, heterobrochate with smaller lumina around the apertures in Z. gardneriana and Z. harmsiana, and exine reticulate, heterobrochate, also with reduced lumina around the apertures in Z. echinocarpa and Z. harmsiana. Under SEM, exine microreticulate or reticulate; sexine thicker than nexine.

Discussion
The palynological characteristics of the analyzed species of the genus Aeschynomene were homogeneous. However, the species, A. sabulicola and A. soniae can be separated from the others due to their possessing a reticulate exine. In general, the species of Aeschynomene endemic to the Caatinga exhibited morphopalynous characteristics similar to those reported in the literature for other species of the genus regarding size, aperture and exine stratification (Salgado-Labouriau 1973;Carreira et al. 1996;Buril et al. 2011;Silva et al. 2016). However, with the exception of Buril et al. (2011), other authors did not mention the presence of a fastigium for species of this genus.
The results here for Cratyllia mollis pollen grains were similar to those of Carreira et al. (1996) for C. argentea, differing only with regards to the amb, which they described as triangular. Miranda & Andrade (1990) also investigated species of Cratyllia and registered the presence of a psilate exine. Silva et al. (2016) studied the pollen of species that occur in the vegetation of Canudos, Bahia, and described Cratyllia mollis, which was corroborated by the present study.
Pollen grains of the genus Crotalaria, characterized as medium-sized 3-colporate, were relatively homogeneous, making it difficult to delimit species. However, C. bahiensis, C. harleyi and C. holosericea diverged from the others due to the presence of a fastigium, and C. brachycarpa due to a reticulate exine. The results obtained here for Crotalaria are generally in accordance with the literature for other species of the genus (Salgado-Labouriau 1973; Carreira et al. 1996;Melhem et al. 2003;Silva et al. 2010;Buril et al. 2011;Mouga & Dec 2012). However, Silva et al. (2010) described the exine of C. micans as perforated and Melhem et al. (2003) described the pollen grains of C. bracystachia as having a margin.
The pollen grains of Dioclea were large and with the sexine being twice as thick as the nexine. The results for Dioclea grandiflora were very similar to those found by Miranda & Andrade (1990) and Buril et al. (2011) for the same species. However, Miranda & Andrade (1990) considered the exine ornamentation as granular or psilate, whereas the present study found it psilate or finely scabrate. Acta Botanica Brasilica, 2022, 36: e2021abb0150