Evolution of pollen grain morphology in Amorimia and allies evidences the importance of palynological apomorphies and homoplasies in Malpighiaceae systematics

Background and aims – Pollen grain morphology is an important morphological character for aiding the systematics of flowering plants. For Malpighiaceae, only a single unpublished palynological study has comprehensively sampled ca 60 of this family’s 75 currently accepted genera. To test the systematic relevance of pollen morphology in Amorimia and allies, we characterised the pollen morphology of these lineages. We scored, coded, and mapped 12 characters onto the most recent molecular phylogeny of Amorimia and allies. Material and methods – We sampled 13 species of Amorimia as ingroup and two species of Mascagnia and Ectopopterys soejartoi as outgroup. Pollen grains were acetolised, characterised, and measured using light microscopy and scanning electron microscopy. Pollen quantitative measurements were submitted to a PCA multivariate analysis. Additionally, quantitative and qualitative characters were scored and coded into 12 characters and mapped onto the molecular phylogeny of Amorimia and allies.


INTRODUCTION
Pollen grains (i.e.male gametophytes) are one of the key innovations that allowed seed plants to successfully colonise terrestrial habitats (Wallace et al. 2011).These male gametophytes comprise an inner triploid reproductive cell and an outer protective wall (i.e. the exine) made mainly of sporopollenin, which is incredibly resistant to degradation (Wallace et al. 2011;Williams et al. 2014).The pollen wall and other pollen morphological traits show different layers, structures, and ornamentations used over the past two centuries to improve the taxonomic classification of different ranks of flowering plants (Melhem 1978;Bahadur et al. 2022).The discovery of pollen grains was made by Marcello Malpighi in 1670, but studies detailing their morphology in several groups of plants have only arisen about two centuries later (i.e.19 th and 20 th centuries) due to innovations in light microscopy (Melhem 1978;Melhem et al. 2003).Since then, pollen morphology has been widely used to aid plant taxonomic studies for the past two centuries (Lindley 1830).
Nonetheless, its central role in plant systematics was only established three decades ago by the first molecular phylogenetic studies of flowering plants.These studies found that the traditional division of angiosperms in dicots/monocots was artificial, with only monocots representing a natural group (Chase et al. 1993).The dicots represented, in fact, several early diverging or derived lineages in flowering plants, with its largest clade, the eudicots (i.e. the new dicots), being solely differentiated by their tricolpate pollen grains from the remaining angiosperms (i.e.basal angiosperms and monocots) showing monosulcate pollen grains (APG 1998(APG , 2016)).Since then, several studies have been published to explore the morphological characterisation and phylogenetic relevance of pollen in several major lineages of angiosperms (i.e.basal angiosperms - Lu et al. 2015;monocots -Furness and Rudall 2001;Lu et al. 2015;and eudicots -Yu et al. 2018), orders (e.g.Myrtales - Kriebel et al. 2017), and families (e.g.Amaranthaceae -Müller and Borsch 2005; Annonaceae -Doyle and Thomas 2012; Euphorbiaceae -Cardinal-McTeague and Gillespie 2016; Loranthaceae - Grímsson et al. 2019;Myrtaceae -Thornhill and Crisp 2012;Rubiaceae -Dessein et al. 2005;Zingiberaceae -Zou et al. 2022).
Malpighiaceae is a medium-sized family of flowering plants comprising 75 genera and ca 1,400 species, mostly endemic to the Neotropics (Almeida and van den Berg 2021;POWO 2023).In Brazil, 56 genera and 592 species of this family are recorded (Flora e Funga do Brasil 2023).Its species are characterised by a conspicuous floral conservatism represented by calyx oil glands, unguiculate petals, and Malpighiaceous pollen type (Anderson 1979(Anderson , 1981)).Due to molecular phylogenetic studies, this family has undergone unprecedented changes in its traditional classification in the past few years (Cameron et al. 2001;Davis et al. 2001;Davis and Anderson 2010).The recognition of new lineages brought to light deep taxonomic problems regarding the monophyly of subfamilies, tribes, and genera (Cameron et al. 2001;Davis et al. 2001;Davis and Anderson 2010;Almeida et al. 2017a;Almeida and van den Berg 2021).Since then, different authors have gradually proposed new genera and combinations (Anderson 2006;Davis and Anderson 2010) to accommodate these newly identified lineages.Some studies describe the palynology of Malpighiaceae, but most of these only present details of a few genera or isolated species.In Erdtman (1952), Lobreau (1967), Anderson (1982), Lobreau-Callen (1983), Makino (1986), Makino-Watanabe et al. (1993a, 1993b, 1998), Gonçalves-Esteves et al. (2007), Sebastiani et al. (2014), Belonsi and Gasparino (2015), and Chaisongkram et al. (2022), we can observe the characterisation of pollen diversity in the family, and sometimes, its use in the taxonomy of some groups.As occurs in most eudicot families, it is common to observe stenopalynous genera in eurypalynous families (Harley 1991;Luz et al. 2013;Teixeira et al. 2013;Gasparino et al. 2021).For Malpighiaceae, Belonsi and Gasparino (2015) highlighted the taxonomic importance of pollen characters, such as polarity, exine ornamentation, and the type of apertures, in distinguishing genera (especially stenopalynous ones).In some cases, such as in Banisteriopsis, the amb, details of the apertures (presence of aspides), and thickness of the exine can help distinguish species (Belonsi and Gasparino 2015).
Amorimia W.R.Anderson is one of the several new lineages identified on those previous molecular phylogenies (Anderson 2006;Davis and Anderson 2010), representing one of the eight genera segregated from the polyphyletic Mascagnia (Bertero ex DC.) Bertero, but remaining closely related to this genus.Amorimia was described by Anderson (2006) to accommodate ten species of lianas and shrubs mostly confined to Seasonally Dry Tropical Forests of South America.It is currently distinguished from other Malpighiaceae by the presence of glands on the abaxial side of inflorescence bracts, petals pubescent on both sides, and straight styles (Almeida et al. 2016;Almeida 2018).The monophyly of Amorimia was corroborated by Almeida et al. (2017a) and Almeida (2018), with two subgenera being proposed for their currently 15 accepted species.The same authors recovered several macro-and micromorphological synapomorphies supporting the recognition of both subgenera, including pollen amb (Almeida et al. 2017a).
In this study, we describe in detail the pollen morphology of 13 (out of 15) accepted species of Amorimia and allies (Ectopopterys W.R. Anderson and Mascagnia) and use the phylogenetic framework presented by Almeida (2018) as the basis for further understanding the patterns of pollen morphology evolution in the genus.We scored and coded 12 micromorphological characters to test for secondary homologies.

Sampling
A total of 13 species of Amorimia were sampled (out of 15), comprising both subgenera currently recognised by Almeida et al. (2017a) (Supplementary material 1).Only two species of Amorimia could not be sampled (A. andersonii R.F.Almeida and A. tumida R.F.Almeida & A.C.Marques) due to the lack of flower buds or flowering specimens.Ectopopterys soejartoi W.R.Anderson and Mascagnia cordifolia (A.Juss.)Griseb.were sampled as outgroup (Supplementary material 1).

Pollen analysis
For light microscopy (LM), the pollen grains were treated with the acetolysis method (Erdtman 1960), with modifications cited by Melhem et al. (2003).The obtained microscope slides were incorporated into the pollen slide collection from the Plant Morphology and Palynology Lab, São Paulo State University, Campus Jaboticabal.Pollen micrographs were taken using an optic microscope Leica IM50 coupled with a video camera and computer, and digitally treated and edited using Photoshop.For scanning electron microscopy (SEM), the material was prepared following Melhem et al. (2003) for nonacetolised pollen grains.The pollen electron micrographs were generated using a JEOL JSM5410 scanning electron microscope.

Pollen descriptions and measurements
Pollen descriptions follow the terminology by Barth and Melhem (1988), Punt et al. (2007), and Bellonzi et al. (2020) for LM.The pollen grains of the analysed Amorimia species using SEM are described following Halbritter et al. (2018).Shape and size terminology follows Erdtman (1952), and exine thickness follows the classification (i.e.very thin, thin, or thick) proposed by Faegri and Iversen (1950).Diameter measurements (DI and DII) were taken within seven days of acetolysis to avoid pollen grain alterations related to the method (Melhem and Matos 1972).All measurements were randomly taken for 25 pollen grains.In contrast, other pollen characters (i.e.aperture, total exine, sexine, nexine, and tectum) were randomly measured for only ten pollen grains (Melhem and Matos 1972;Salgado-Laboriau 1973).The measurements referring to the tectum are included in the value of the sexine, as this structure is part of the sexine.

Character mapping analyses
The consensus phylogenetic tree by Almeida (2018) was pruned with Mesquite v.2.73 (Maddison and Maddison 2006) to show only the outgroup sampled in our study and used for the character mapping of the pollen morphology.Character coding followed the recommendations of Sereno (2007) for morphological analyses.Primary homology hypotheses (De Pinna 1991) were proposed for pollen shape, size, ornamentation, exine structure, and apertures.A total of 12 pollen characters were scored for Amorimia and outgroups (Supplementary material 3).In addition to Mascagnia cordifolia, we sampled M. sepium from the palynological literature (Makino 1986) for the character mapping analyses.All characters were optimised on the concatenated tree using the Maximum Likelihood function (mk1 model) using Mesquite v.2.73 (Maddison and Maddison 2006) and visualised with Winclada (beta) v.0.9 (Nixon 1999).

Quantitative data
The quantitative data analyses used pollen grain diameters and their respective averages and confidence intervals (Supplementary materials 4-5).We observed three distinct groups when analysing the metric values of the diameters (Fig. 5 The PCA summarised 78.82% of the total variability of the data, in which axis 1 was more informative to the PCA since it summarised 43.47% of the variability (Fig. 6).The analysed Mascagnia and Ectopopterys were recovered far from those of Amorimia (Fig. 6).The species of A. subg.Uncinae showed lower values for all metric variables analysed (negative side of axis 1), except for A. camporum and A. kariniana, which were positioned with the species of A. subg.Amorimia.For axis 1, the most significant variable was ECLEN (Supplementary material 2), which distinguished Ectopopterys from other species by the colporate ectoaperture.Axis 2 summarised 31.34 % of the variability in our data; the most significant variables for this axis were SEXI and ECWID (Supplementary material 2).Note that the species of Mascagnia and Ectopopterys had lower values for these variables and were close to A. amazonica.In general, the species of A. subg.Uncinae are positioned on the positive side of axis 2, except for A. camporum, which appears alongside the species of A. subg.Amorimia with higher values for the variables that stand out in the ordination of axis 2.

Palynology of Amorimia and allies
The genus Amorimia was recently segregated from Mascagnia, and, unfortunately, no palynological evidence was included in its original description (Anderson 2006).Lowrie (1982) performed a comprehensive study of the pollen morphology of 60 out of the 75 currently accepted genera of Malpighiaceae, describing three main morphological types and a few subtypes for this family.Radially symmetric pollen grains divided into 1.3-colporate, 2. parasyntricolporate, 3. syntricolporate, 4. 4-colporate, or 5. polycolporate genera.Globally symmetric pollen grains lacking ectoapertures divided into 1.Aspidopteroid, 2. Bunchosioid, and 3. Ryssopteroid types, and globally symmetric pollen grains with ectoapertures divided into 1.Banisterioid, 2. Clonodioid, 3. Mascagnioid, and 4. Tetrapteroid types (Lowrie 1982).This author described the pollen grains of some species of Mascagnia, now treated in Amorimia, as of the Mascagnioid subtype, characterised as pollen grains with branched exine ornamentation with fused rugae and more than six pores randomly dispersed by the intersection of two rugae.Our results demonstrated clypeate pollen grains with fossulate or psilate-perforate exine for all species of Amorimia, different from those presented by Lowrie (1982).Amorimia pollen grains are similar to the pollen grains of the genus Mascagnia but differ concerning the number of apertures, corroborating the taxonomic changes proposed by Anderson (2006).In general, our data agree with the denomination of Mascagnioid pollen grains, as previously done by Lowrie (1982), for Amorimia species, since the pollen grains present patterns of aperture and ornamentation somewhat similar to those observed by Lowrie (1982), varying however in the number of apertures and ornamentation type, which allows to use them as diagnostic traits to easily distinguish Amorimia from Mascagnia.Makino (1986) and Belonsi and Gasparino (2015) also found 8-porate pollen grains in Mascagnia cordifolia and M. sepium.Still, they observed nexine thicker than sexine, different from the data found in this study (i.e.sexine thicker than nexine).Therefore, the number and type of apertures corroborate the pollen pattern found for the species of Mascagnia, even with slight differences observed in the present study.The pollen grains of Ectopopterys soejartoi were briefly described by Anderson (1980) as 3-colporate, with the present study also corroborating the description presented by this author.Pollen grain type and number of apertures are also useful to differentiate Amorimia species from their allied genera (Ectopopterys and Mascagnia).
Another factor to be highlighted is the corroboration obtained from the ancestral pollen character reconstructions that placed colporate pollen grains (as in Ectopopterys soejartoi) as the probable ancestral state and porate pollen grains (Amorimia and Mascagnia) as a derived character in the Malpighioid clade.We corroborated that the pollen morphology of Amorimia regarding qualitative characters is constant for the species analysed, but showed that quantitative characters are very informative for their taxonomy.According to traditional palynological classification, Amorimia can be considered stenopalynous (i.e. with minor discrete morphological variations).As abovementioned, the type and number of apertures allow the distinction of Amorimia from the closely related Mascagnia and Ectopopterys.In the PCA analysis, the metric variables of the pollen grains confirm the qualitative data and help to distinguish the analysed genera since the differences in the measurements of the ectoapertures and the exine layers allowed Mascagnia and Ectopopterys to be separated from Amorimia.
In LM, the pollen grains of the species analysed here present sexine rugulate with areolate or psilate areas, which was also verified in previous studies for Malpighiaceae (Makino 1986;Makino-Watanabe et al. 1993a, 1993b, 1998;Belonsi and Gasparino 2015).For the description of pollen ornamentation in SEM, clypeate pollen grains were observed (as described by Halbritter et al. 2018), and the sexine is fossulate or psilate-perforate, details not previously described for species of Amorimia.
Evolution of pollen grains in Amorimia and allies Cameron et al. (2001) tested the phylogenetic relevance of Lowrie's (1982) three main micromorphological pollen groups in Malpighiaceae.These authors found that radially symmetrical pollen grains are probably plesiomorphic in Malpighiaceae, occurring in the Byrsonimoid, Acridocarpoid, Mcvaughioid, and Ptilochaetoid clades.Since this author did not sample any outgroups outside Malpighiaceae in his study, it is impossible to confidently state the plesiomorphic nature of this pollen grain morphology in Malpighiaceae.Nonetheless, Perveen and Quaiser (1995) show that the pollen grains of two species of Bergia L. (Elatinaceae) are indeed radially symmetrical, corroborating this character state as probably plesiomorphic for the Elatinaceae + Malpighiaceae clade.The same pattern of pollen morphology was also reported for two species of Elatine L. (Ramayya and Rajagopal 1971), increasing the chances of radially symmetrical pollen grains being, indeed, a synapomorphy for the clade formed by both families.Additional studies for the remaining 54 accepted species in Elatinaceae are needed for an in-depth assessment of this hypothesis.
In contrast, globally symmetrical pollen grains with ectoapertures were recovered as a synapomorphy for the Bunchosioid + Hiraeoid + Tetrapteroid + Malpighioid + Stigmaphylloid clade by Cameron et al. (2001).Globally symmetrical pollen grains without ectoapertures were recovered as homoplastic, having independently arisen at least seven times mostly in Old World genera of Malpighiaceae (except for the New World Barnebya W.R. Anderson & B.Gates, Bunchosia Rich. ex Kunth, and Heladena A.Juss.;Cameron et al. 2001).Amorimia and allies have constantly been placed by several molecular phylogenetic studies (Cameron et al. 2001;Davis et al. 2001Davis et al. , 2014;;Davis and Anderson 2010;Almeida et al. 2017a;Almeida 2018) as early diverging lineages in the Malpighioid clade, showing globally symmetrical pollen grains with ectoapertures, as described by Lowrie (1982).The remaining lineages of the Malpighioid clade are endemic to the Old World (i.e.Africa + Asia) and show globally symmetrical pollen grains without ectoapertures as an adaptation to the generalist pollination syndrome arisen in Old World lineages due to the lack of oil collecting bees in this region of the planet (Lobreau 1968;Lowrie 1982;Cameron et al. 2001;Davis et al. 2014).
Our results recovered two evolutionary patterns regarding homoplastic and apomorphic pollen micromorphological characters.Regarding synapomorphies or autapomorphies (i.e.apomorphies), few qualitative characters, such as the type of pollen aperture (colporate or porate) and the number of apertures (3, 6, or 8), were very informative in distinguishing lineages at the generic rank.A few quantitative characters, such as exine thickness and aperture width, were informative at the generic level for the species sampled in this study.On the other hand, a few quantitative characters such as exine, nexine, sexine, and tectum thickness, and aperture length and width were informative to distinguish lineages at an infrageneric rank.Only the ornamentation type was informative to distinguish species at an infrageneric level.Regarding homoplasies, all quantitative and almost all qualitative micromorphological pollen characters analysed were informative both at the generic and infrageneric levels in Amorimia and allies.It is also worth mentioning that most of the homoplastic characters recovered as informative in our results were related to quantitative pollen characters, which are frequently underexplored in evolutionary studies of pollen grains.
Finally, Almeida et al. (2017a) proposed two subgenera within Amorimia: A. subg.Amorimia, or Atlantic clade, and A. subg.Uncinae, or Amazonian clade.As the clade names suggest, these species are already geographically separated.Molecular parsimony and bootstrap analyses showed that this separation is supported, and some morphological characters also support both subgenera.The authors suggest a differentiation of both subgenera based on pollen characters such as pollen amb and size (Almeida et al. 2017b).Nonetheless, the data from the present study do not corroborate Almeida et al. (2017b), since their species sampling for Amorimia was incomplete.In fact, when this classification was proposed, Amorimia tumida was still unknown to science and was not included in the molecular phylogeny published by these authors (Almeida et al. 2017b).This species is sister to the remaining species of the A. subg.Uncinae and was described only based on molecular data, and vegetative and fruit morphology, with its flowers still being unknown to science (Almeida et al. 2017b).Consequently, it was impossible to analyse this species' pollen morphology in the present study.Since A. tumida is a crucial species placed at the base of the A. subg.Uncinae clade, this taxon's missing data directly impacts the phylogenetic reconstruction of the analysed pollen micromorphological characters.Thus, only future studies focusing on the pollen morphology of A. tumida will be able to shed light on the relevance of pollen morphology to corroborate the classification system of Amorimia.

CONCLUSIONS
According to palynological standards of pollen morphology variation, Amorimia can be categorised as stenopalynous since all species show the same pollen type, with some subtle differences between the pollen grains, such as ornamentation, shape, size, and thickness of the exine.The micromorphological patterns of pollen grain evolution found by Lowrie (1982) showed several qualitative and only a few quantitative pollen characters informative at suprageneric levels (i.e.phylogenetic clades).On the other hand, the patterns of pollen grain evolution demonstrated by our results showed few qualitative characters informative at intergeneric levels.Still, almost all quantitative characters analysed were informative at infrageneric levels.The quantitative and qualitative analyses do not corroborate the currently recognised subgenera of Amorimia due to not sampling A. tumida, a critical species in the phylogenetic backbone known only by fruiting specimens.Sampling A. tumida should be a future priority to shed light on the evolutionary patterns of pollen micromorphology in Amorimia.

Figure 7 .
Figure 7. Molecular phylogeny and pollen character mapping of Amorimia and allies (Malpighiaceae) pruned from Almeida (2018).A. Phylogenetic tree -numbers above and below branches represent posterior probability and bootstrap values, respectively.B.Character mapping tree -red circles represent apomorphies (synapomorphies and autapomorphies); white circles represent homoplasies; numbers above circles represent the number of the pollen character; numbers below circles represent the number of the pollen character state reconstructed.

Table 1 .
List of homoplasies and apomorphies (including synapomorphies and autapomorphies) recovered for all lineages in this study.

Table 1 (
continued).List of homoplasies and apomorphies (including synapomorphies and autapomorphies) recovered for all lineages in this study.