Pollen morphology of the early branching papilionoid legume genera Harleyodendron, Holocalyx and Uribea

ABSTRACT In this study, pollen grains of Harleyodendron unifoliolatum, Holocalyx balansae, and Uribea tamarindoides, three monospecific genera with Neotropical distribution belonging to the clade Exostyleae, one of the basal lineages of the family Leguminosae, subfamily Papilionoideae, were analyzed. The palynological material was acetolyzed and analyzed under light microscopy (LM) and scanning electron microscopy (SEM). Under LM, small and medium-sized pollen grains were observed, which showed a prolate-spheroidal to prolate shape, 3-colporate, microreticulate and reticulate exine, sexine and nexine of equal thickness in Harleyodendron and Holocalyx, while the sexine was thicker than nexine in Uribea. Analysis under SEM showed finely granulate, psilate, and granulate apertural membrane, while the exine ornamentation varied from perforate-microechinate in Harleyodendron, to perforate in Holocalyx, while in Uribea showed an irregular relief and granular projections on the perforate tectum. The pollen morphology of these species is similar to each other, varying in sexine/nexine ratio, apertural membrane, and exine ornamentation. Harleyodendron has a finely granulate apertural membrane, sexine and nexine of equal thickness, and a perforate-microreticulate exine ornamentation, Holocalyx shows a psilated apertural membrane, sexine and nexine of equal thickness, and exine ornamentation perforate, and Uribea has a granulate apertural membrane, sexine thicker than nexine, and exine ornamentation with granular projections.

The monospecific genera Harleyodendron, Holocalyx, and Uribea are trees distributed in humid and seasonally dry forests through South and Central America (Cardoso et al. 2012) that exhibit a very different floral morphology (Fig. 1).
Several studies have indicated that the Exostyleae clade is monophyletic, based on molecular and macromorphological character analysis, which has allowed the precise delimitation of each of its genera (Cardoso et al. 2012;2013;LPWG 2017;Zhang et al. 2020;Zhao et al. 2021).Nonetheless, there is still a gap in the knowledge of the intergeneric relationships in the clade, whose doubts arise as a consequence of the divergent vegetative and reproductive morphology within each genus, their distant pattern of distribution, and the lack of phylogenetic resolution regarding the intergeneric relationships within the clade.For that reason, further research to provide additional information is needed, since a broader and more comprehensive analysis of the clade and the relationships between its members would be carried out (Cardoso et al. 2012).
Palynological studies involving representatives of the Exostyleae clade (Ferguson & Skvarla 1981;Skvarla & Ferguson 1988;Ferguson & Schrire 1994;Ferguson et al. 1994) have shown variation in pollen morphology, mainly regarding their exine ornamentation.Therefore, the palynological study of the genera Harleyodendron, Holocalyx, and Uribea allows us to understand part of the micromorphological diversity that exists in the clade and can be useful as a basis for further and more comprehensive research to clarify the intergeneric and interspecific relationships in the clade Exostyleae.
Thus, this work aims to describe the pollen morphology of the monospecific genera Harleyodendron, Holocalyx, and Uribea, aiming to increase the knowledge about them and other related genera.Pollen grains were subjected to the acetolysis method (Erdtman 1960), mounted between slides and coverslips with glycerin gelatin, and sealed with paraffin for observation under Light Microscopy (LM).Under LM, pollen grains were measured and microphotographed using a Leica ICC50 W light microscope.All slides were deposited in the Palinoteca of the Laboratory of Plant Micromorphology at the Universidade Estadual de Feira de Santana (PUEFS).

Materials and methods
Under light microscopy (LM), the main morphometric parameters (equatorial diameter, polar diameter, and equatorial diameter in polar view) were measured, when possible, for 25 pollen grains randomly distributed among at least three slides in order to standardize the sampling.Other parameters (aperture diameter, and thickness of the exine, sexine, and nexine) were measured for ten pollen grains, also taken at random.
For analysis under Scanning Electron Microscopy (SEM), the acetolyzed pollen grains were washed in 70% acetone and dripped directly onto the specimen holder of the SEM, after total drying, they were metalized by evaporation of gold in high vacuum and electromicrographed in the JEOL 6390LV microscope from the Electronic Microscopy Platform of the Oswaldo Cruz Foundation -Gonçalo Moniz Research Center.
All pollen descriptions followed the palynological nomenclature proposed by Punt et al. (2007) and Halbritter et al. (2018).
Regarding the sexine/nexine ratio, Harleyodendron and Holocalyx showed pollen grains with sexine and nexine of equal thickness, while in Uribea the sexine is thicker than the nexine.Under LM, a finely microreticulate exine was observed in Harleyodendron (Fig. 2C).In Holocalyx, the exine ranged from finely microreticulate to reticulate (Fig. 2H), whereas in Uribea the exine is microreticulate (Fig. 2M).Furthermore, when analyzed under SEM, pollen grains of Harleyodendron are perforate-microechinate (Fig. 2E), while those from Holocalyx are perforate with perforations of different sizes (Fig. 2J).On the other hand, the exine ornamentation in Uribea showed an irregular relief with granular projections on the perforate tectum and slightly rugulate areas (Fig. 2O).The presence of Ubisch bodies in the three genera analyzed was registered (Fig. 2D, I, O).Pollen morphology of the early branching papilionoid legume genera Harleyodendron, Holocalyx and Uribea

Pollen morphology and taxonomy
Our results regarding the pollen morphology of the analyzed taxa are supported by extensive literature (Cowan 1979;Ferguson & Skvarla 1981;Skvarla & Ferguson 1988;Ferguson & Schrire 1994;Ferguson et al. 1994).Concerning the apertural type, Skvarla & Ferguson (1988) described Harleyodendron and Holocalyx as showing a lolongate endoaperture.However, Ferguson & Skvarla (1991) reported that there was confusion regarding the endoapertures and clarified that the pollen grains of Harleyodendron and Holocalyx present lalongate endoapertures.Our results also reported a lalongate endoaperture, which is sometimes difficult to visualize since the constriction of the ectoaperture usually is positioned upon the endoaperture.Some differences were observed between the results found here and data from the literature.Regarding the apertural membrane, Skvarla & Ferguson (1988) described a finely granulate apertural membrane in Holocalyx, while a psilate apertural membrane is reported in this research.
Under SEM, the exine of the pollen grains of Harleyodendron is perforate-microechinate, which coincides with the description presented by Skvarla & Ferguson (1988), and it is different from that reported by Cowan (1979), who described the exine as finely reticulate with numerous rounded projections on the murus.Skvarla & Ferguson (1988) also described the exine of pollen grains of Holocalyx as sparsely scrobiculate/fossulate (scrobiculate=punctate in Punt et al. 2007), whereas Ferguson et al. (1994) characterized the pollen grains of this species as foveolate and slightly perforate.The specimens included in this study presented exine ornamentation with perforations of different sizes.Regarding the exine of Uribea, our results coincide with those published by Ferguson et al. (1994).
The pollen grains of these species can be differentiated from each other under LM in terms of size, shape, and sexine/nexine ratio.Harleyodendron has small to medium pollen grains, shapes ranging from prolate-spheroidal to subprolate, and a finely microreticulate exine, Holocalyx has small pollen grains, shapes ranging from subprolate to prolate, and exine ranging from finely microreticulate to reticulate, and Uribea shows medium pollen grains with prolate-spheroidal shape, microreticulate exine, and sexine thicker than nexine.Under SEM analysis, the difference in pollen morphology among these species is remarkable when their exine ornamentation is compared.While in Holocalyx was found a less complex exine ornamentation without supratectal structures, being perforate, the other genera showed a more specialized exine which were classified as perforatemicroechinate in Harleyodendron and as having an irregular relief and granular projections on the perforate tectum with slightly rugulate areas in Uribea.
Morphological characteristics such as shape, apertures, and perforate exine with heterogeneous perforations exhibited by Holocalyx coincide with those that are commonly found in Leguminosae since the family and most genera of the Papilionoideae subfamily present monad, tricolporate, and finely reticulate pollen grains (Erdtman 1952;Guinet 1981;Ferguson & Skvarla 1981).
The exine ornamentation here observed in Holocalyx has also been reported by Skvarla & Ferguson (1988) for Exostyles glabra Vogel and for some species of Zollernia, for which Ferguson & Schrire (1994) also reported the same exine ornamentation.As well as Holocalyx, these taxa are included in the Exostyleae clade (Mansano et al. 2002;Cardoso et al. 2012Cardoso et al. , 2013)).
The exine ornamentation with the presence of microspines observed in Harleyodendron unifoliolatum as well as the exine with granular projections on the perforate tectum shown in Uribea tamarindoides are uncommon exine ornamentations among the species of Leguminosae (Skvarla & Ferguson 1988;Ferguson et al. 1994).Nonetheless, these traits have been reported in other species of the Exostyleae clade such as Exostyles venusta Schott and Lecointea amazonica Ducke (Skvarla & Ferguson 1988;Ferguson & Schrire 1994), and other genera of the Papilionoideae subfamily such as Macrotyloma (Wight & Arn.) Verdc.(Ferguson 1981).
The variation in exine ornamentation among related genera of the clade Exostyleae have raised the question about whether all genera of the clade show variation in exine ornamentation and which is included in this clade share the same type of ornamentation.

Implications of pollen morphology on pollination
Among the species included in this study, Holocalyx balansae is pollinated by bees and other small insects (Carvalho 2003), its flowers are small (2-3 x 0.5-0.9mm), greenish, actinomorphic, with five petals, ten stamens (Mansano & Viana-Filho 2010) and their pollen grains are perforate.These characteristics were associated with the pollination by small insects by Ferguson (1984).This author reported Templetonia egena Benth.and Camoensia brevicalyx Benth.(Papilionoideae), whose pollen grains show a finely perforate exine and are pollinated by insects.Additionally, several plant species with microreticulate pollen grains have been reported as pollinated by different species of bees (Braga et al. 2012;Bastos et al. 2020;2021;Dias et al. 2022).
Regarding Harleyodendron unifoliolatum, Cowan (1979) stated that this species may be pollinated by small insects that are attracted to its whitish, fragrant flowers, or that they are self-fertile.However, this species presents a unique and unusual floral morphology among the papilionoid legumes, as they are radially symmetrical with the five fleshy petals becoming reflexed backward its open flowers, and the ten big and rigid anthers are kept coherent by intermixed long hairs making a dome an apical opening like a pore (Fig. 1A).The anthers dehisce through two longitudinal somewhat introrse slits making pollen available within the dome.Unfortunately, there is no empirical data that could suggest what flower visitors could act as pollinators of this interesting plant.Pollen grains with echinate exine have been linked to pollination by animals (Wodehouse 1935;Hesse 2000;Tanaka et al. 2004), an idea that is shared by Ferguson & Skvarla (1982), who report that species in the genera Ambrosia L. and Artemisia L. (Asteraceae) generally have psilate pollen grains and are wind-pollinated, while other genera with ornate pollen grains (commonly echinate) are pollinated by insects.
Jones & Jones (2001) reported that butterflies and moths pollinate species that usually have reticulate, striate, and echinate pollen grains.Butterfly-pollinated flowers usually display showy colors such as red, yellow, blue, and orange, are erect, and exhibit a landing platform, nectaries hid in spurs or narrow tubes, and simple nectar guides.On the other hand, moth-pollinated flowers are white, light pink or light yellow, zygomorphic, pendulous or horizontal without a landing platform, with nectaries very hidden in long tubes or spurs, and without nectar guides (Jones & Jones 2001;Rech et al. 2014).
As Harleyodendron unifoliolatum has flowers with large and rigid anthers and perforate-microechinate pollen grains, it could be assumed that this species is pollinated by large bees.Nonetheless, because of the distinct floral morphology, as well as the exine ornamentation, and the lack of information about the pollinator, further research focused on this species is needed to elucidate the pollination of H. unifoliolatum.
Finally, information regarding the pollination of Uribea tamarindoides was not found.Its flowers are zygomorphic, purple or light purple with five petals, the adaxial being differentiated into a banner and ten stamens (Dugand 1962).Although there is no information about the U. tamarindoides pollinator, it can be suggested that this species is pollinated by bees, taking into account that the specialized bilateral flowers of Papilionoideae facilitate bee-flower interaction, as they have a standard petal, which is larger and more external, serving as an attraction, two wings that act as a landing area for visiting insects and a keel that surrounds the androecium and gynoecium (Lewis et al. 2005).

Conclusion
The pollen data of the species of Harleyodendron, Holocalyx, and Uribea showed that their pollen morphology is, in general, similar to each other, varying in the form under LM (prolate-spheroidal to prolate) and the ornamentation of the exine under SEM, with the grains of pollen from Holocalyx resembling the pollen type commonly found in Leguminosae.On the other hand, the exine ornamentation found in Harleyodendron (perforate-microechinate), and Uribea (granular projections on the perforate tectum) are unusual among the representatives of Leguminosae, requiring further studies focused on the relationship between these species and others included in the clade Exostyleae.
Although these monospecific genera were previously published and described, there is still a considerable lack of information related to the pollination of Harleyodendron, Holocalyx, and Uribea.Thus, research focused on the floral biology of these species is necessary, which will contribute to expanding knowledge about them and their ecological relationships with their pollinators, and will allow the establishment of strategies for their conservation.
Note: PD= Polar diameter; ED= Equatorial diameter; EDp= Equatorial diameter in polar view; x= Arithmetic mean; Sx= Standard deviation of the mean; P/E= Polar diameter-Equatorial diameter ratio; Ecto= Length x Width of the ectoaperture; Endo= Length x Width of the endoaperture; Sex= sexine; Nex= nexine; measurements in μm and indices in absolute numbers.