Pollen Morphological Peculiarities of Selected Mimosoideae Taxa of Hainan Island and Their Taxonomic Relevance

: Mimosoideae is one of the taxonomically complex subfamilies of Fabaceae. Several studies have reported the pollen morphology of Mimosoideae taxa and their taxonomic relevance, but no such study was found speciﬁcally for Hainan Island in southern China. Therefore, the present study was designed to investigate the selected Mimosoideae taxa and explore the new palynological traits to support and strengthen the systematics of Mimosoideae using multiple microscopic techniques. The polar axis, equatorial diameter of the pollen grains, colpus length and width were measured. The smallest pollen grain size was found in Mimosa pudica (7.8 × 7.75 µ m), while the largest pollen size was found in Albizia lebback (87.54 × 77.97 µ m). Similarly, signiﬁcant variation was found in the exine and colpus surface patterns. The subfamily Mimosoideae is considered eurypalynous because of the variation in pollen traits. In addition, variation was also found in the quantitative traits. Compara-tively, the pollen features were found to be helpful at the genus and species levels, as well as in the correct identiﬁcation and discrimination of the taxa. Hence, this study gives a detailed account of the pollen morphologies of certain selected taxa of Mimosoideae collected from different geographical regions on Hainan Island. The pollen morphological traits were proven to have signiﬁcant taxonomic potential and can be used as additional tools for the correct identiﬁcation and discrimination of Mimosoideae taxa. These results will provide the basis for further systematic studies.


Introduction
Fabaceae is one of the most diverse families, being found in most ecosystems around the globe. It has been divided into three subfamilies-Papilionoideae, Caesalpinioideae and Mimosoideae [1]. In terms of modern Fabaceae classifications, the Legume Phylogeny Working Group (LPWG 2017) [2] recognized six subfamilies, whereby the traditional subfamily Mimosoideae was presented as a clade nested in the Caesalpinioideae family with a new level of discrimination. Hence, our study is based on the classifications made by Lewis et al. [1]. Furthermore, the recent classifications, which only propose changes at the subfamily level and have not yet solved the taxonomic problem of the tribes in the Mimosoid clad, do not affect the interpretation of our results. Mimosoideae is a subfamily of Fabaceae, although is sometimes treated as a separate family [3]. Fabaceae contains about 18,000 species belonging to 697 genera [4]. Fabaceae is the second largest family among the angiosperms after Asteraceae [5] and contains three subfamilies, i.e., Mimosoideae, Papilionoideae and Caesalpinioideae [6]. Mimosoideae comprises 3100 species and 58 genera around the world [7]. About 79 genera are distributed worldwide, including in tropical, subtropical, arid and semiarid regions, as well as in rocky soil [8]. The species has numerous filaments and stamens and a freely downwards-extending base [9]. The group was on Hainan Island have been documented yet. Hence, to fill this gap, further palynological studies are needed.
It is hoped that this palynological study will not only add to the systematic information about certain selected Mimosaceae taxa, but will also provide the basis for further phylogenetic studies. The present study aims (1) to provide descriptions of the pollen types of selected taxa of Mimosoideae and outline their taxonomic relevance and (2) to strengthen and support the taxonomy of Mimosoideae and define species boundaries using both light microscopy and scanning electron microscopy.

Collection and Identification
For the collection of Mimosoideae taxa, various field trips were arranged in the year 2021 to various geographical regions on Hainan Island ( Table 1). The island is characterized by a tropical monsoon climate with a wet season ranging from May to October and a dry season ranging from November to April. The annual average precipitation ranges from 2350 to 2651 mm [50][51][52]. The species were collected and identified with the help of the available literature Flora of China efloras.org World Flora Online.

Light Microscopy
For the light microscopic study, the filaments were first separated from the flowers and crushed on a glass slide by adding 2-3 drops of acetic acid. The debris was removed through a needle and then a cover slip was placed on it. The prepared slide was then observed under a light microscope equipped with a digital camera. The pollen micrograph was taken and its various taxonomic features were observed. For pollen descriptions, we mostly followed the terminology used by Punt et al. [53].

Scanning Electron Microscopy
The anthers were separated from the mature flower as performed by Ali et al. [23]. The anthers were then crushed on the glass slide by adding a few drops of acetic acid and transferred into Eppendorf tubes. With the help of a micropipette, pollen samples from the tubes were taken and placed on a metallic stub attached with double-sided sticky tape. The prepared samples were sputtered with platinum for twenty-five minutes in a Leica Mikrosystem made in Austria with a high-vacuum coater (ACE600) and observed under SEM (Thermo Scientific, Model: verios g 4 uc) Manufacturer Seimer Technology was installed in the analytical and testing center of Hainan University, Haikou, China.

Quantitative Analysis
For quantitative analysis, we measured about ten pollen grains in each sample. The polar axis and equatorial diameter of the polyads and colpus length and width were measured using Image J software.

Results and Discussion
The Mimosaceae taxa were analyzed using both light and scanning electron microscopic techniques. LM and SEM micrographs are illustrated in Figures 1-5. Details of pollen traits of each species are given below. through a needle and then a cover slip was placed on it. The prepared slide was then observed under a light microscope equipped with a digital camera. The pollen micrograph was taken and its various taxonomic features were observed. For pollen descriptions, we mostly followed the terminology used by Punt et al. [53].

Scanning Electron Microscopy
The anthers were separated from the mature flower as performed by Ali et al. [23]. The anthers were then crushed on the glass slide by adding a few drops of acetic acid and transferred into Eppendorf tubes. With the help of a micropipette, pollen samples from the tubes were taken and placed on a metallic stub attached with double-sided sticky tape. The prepared samples were sputtered with platinum for twenty-five minutes in a Leica Mikrosystem made in Austria with a high-vacuum coater (ACE600) and observed under SEM (Thermo Scientific, Model: verios g 4 uc) Manufacturer Seimer Technology was installed in the analytical and testing center of Hainan University, Haikou, China.

Quantitative Analysis
For quantitative analysis, we measured about ten pollen grains in each sample. The polar axis and equatorial diameter of the polyads and colpus length and width were measured using Image J software.

Results and Discussion
The Mimosaceae taxa were analyzed using both light and scanning electron microscopic techniques. LM and SEM micrographs are illustrated in

Acacia Auricifoliformis
The pollen of A. auricifoliformis was a polyad comprising 16 grains. Eight grains are arranged in the center, which is surrounded by eight periphery cells. Each grain was to be found inaperturate, while the exine sculpturing was found to be psilate. The average diameter of the polyads was noted as 32 µm. Similarly, some other species of Acacia

Acacia Auricifoliformis
The pollen of A. auricifoliformis was a polyad comprising 16 grains. Eight grains are arranged in the center, which is surrounded by eight periphery cells. Each grain was to be found inaperturate, while the exine sculpturing was found to be psilate. The average diameter of the polyads was noted as 32 µm. Similarly, some other species of Acacia

Acacia auricifoliformis
The pollen of A. auricifoliformis was a polyad comprising 16 grains. Eight grains are arranged in the center, which is surrounded by eight periphery cells. Each grain was to be found inaperturate, while the exine sculpturing was found to be psilate. The average diameter of the polyads was noted as 32 µm. Similarly, some other species of Acacia have been described previously and the polyad type was also reported to be pollen (16-32-celled), with or without distinct apertures [54][55][56]. The general description of polyads and the interrelationships of the grains in Acacia have also been discussed previously [57].

Acacia confusa
The pollen was shed in polyads (12 grains), the outline in the polar view was rhomboidal, the symmetry was bilateral and the polarity was heteropolar. The aperture condition was noted as inaperturate. Exine sculpturing was found as mega-reticulate and rarely scabrate. The reticula were thick and flattened, having a rarely scabrate structure, while the lumina was filled with a somewhat micro-scabrate structure (Figure 1a-c). The pollen size was noted as 23.19 × 13.88 µm, while the individual grain diameter was 5.745 µm.

Acacia mangium
The pollen of A. mangium was polyad of 12 grains (Figure 4a). The four grains were arranged in the center, surrounded by eight periphery grains. The exine sculpturing was found to be megareticulate and heterobrochate. An operculum was observed in the central grains of the polyads. The average diameter of the polyad was noted as 35.61 µm (Figure 1a). To the best of our knowledge, no descriptive palynological studies have been reported yet. However, Wang et al. [58] documented only the isolation and characterization of flower-specific transcripts in Acacia mangium.

Albizia julibrissin
The pollen type was polyad, comprising 12 grains. The outline in polar view was elliptical. The central 4 grains were arranged in tetragonal form. The exine sculpturing was psilate (Figure 3a-c). The pollen size was noted as 71.81 × 81.01 µm, while the individual grain diameter was 25.13 µm (Figure 4c). The pollen of A. julibrissin was closely related to that of A. lebback and A. procera. For example, Khan et al. [41] documented the pollen diversity and its implications in Mimosaceous taxa using light and scanning electron microscopic techniques, and reported psilate-poveolate and sparsely psilate-scabrate exine sculpturing in A. procera pollen. Hence, these traits were found to be useful in delimiting and defining species boundaries within the genus Albizia.

Albizia lebback
The pollen type was polyad (12-grains), while the outline in polar view was spheroidal. The central 4 grains were arranged in tetragonal form. The exine sculpturing was psilate to rugulate (Figure 3a-c). The pollen size was 87.54 × 77.97 µm, while the individual grain diameter was 24.35 µm. However, in comparison with the previous results, the 16-celled polyad-type pollen grains reported by Agashe and Caulton [59] were not corroborated by our findings. Moreover, a minimum length of 28 µm and maximum length of 57.4 µm and a minimum width of 30.5 µm and maximum width of 43.05 µm were reported by Parveen and Qaiser [44] in the pollen grains of A.lebback. Similarly, the aperture and exine sculpturing of Albizia was found to be smooth or almost smooth and different from Calliandra, Cylindrokelupha and Zygia, due to the presence of tubercles [48].

Calliandra haematocephala
Pollen was shed as octad polyads (8 grains), while the outline in the polar view was a pyramid. The two central large-sized grains are in the center and the other grains lie in the periphery. As found in the previous study by Guinet [39], individual pollen grains from the same polyads showed a marked heteromorphic structure, with peripheral grains differing from the central grains in shape, size and less obviously ornamentation. The exine sculpturing was found to be verrucate, aerolate and somewhat reticulate towards the periphery. The pollen tip acute and the base was constricted. The surface of pollen was found to be undulate. According to Van Campo and Guinet [60], the pollen dispersal units of Calliandra are bi-tetrads and polyads. Similarly, Chen [61] mentioned that the bi-tetrad pollen of the Calliandra is family is rare in Angiosperms. Some other species of this genus also have polyad-type pollen [47]. The pollen grain octads, shed as polyads, were flattened and oval-shaped, while the longest axes ranged from 164 to 173 µm and the shortest axes ranged from 82 to 93 µm. Similarly, the tectate and perforate exine was found Agronomy 2022, 12, 1122 8 of 13 in the Philippines in Calliandra haematocephala. However, to clarify the differences in exine sculpturing of the appendix in detail, transmission electron microscopy will be needed.

Entada phaseoloides
The pollen type was monad, the outline in polar view was ovate and was elliptical in equatorial view, the symmetry was bilateral and the polarity was heteropolar. The aperture condition was noted as trizonocolpate. The orientation of the colpus was sunken and wide, having an obtuse tip, while the sculpturing was scabrate to verrucate and sometimes psilate. The exine sculpturing was perforate and rarely scabrate, with ubisch bodies found on its surface. The polar area was found to be very small. The pollen size was 34.61 × 26.76 µm. The colpus length was noted as 21.31 µm and the width was 6.72 µm. The polar area was 5.45 µm and the meso-colpium diameter was 25.33. Furthermore, Rao and Lee [62] documented the pollen flora of Singapore and Malaya and observed a tricolporate and subprolate structure, a size of 30 × 35µm and a faintly reticulate exine pattern of the Entada spiralis pollen. Similarly, the shape of Entada scandens pollen grains and other pollen morphological features of Entada phaseoloides were reported in a previous study [54].

Leucaena leucocephala
The pollen type was monad, the outline in polar view was triangular, the symmetry was bilateral and the polarity was isopolar. The aperture condition was noted as tricolpate. The orientation of the colpus was sunken and wide, having an obtuse tip, while the sculpturing was scabrate to rugulate. The exine sculpturing was perforate and rarely scabrate. The polar area was found to be very small. The pollen size was 21.48 × 21.35 µm. The colpus length was 9.27 µm and the width was 3.4 µm. In comparison with a previous study [45], where the exine sculpturing was found to be subpsilate, this was not corroborated by our findings, while the polar axis was 42.42 µm and the equatorial diameter was 31.5 µm. The colpus length was 17.01 µm and the width was 8.61 µm. The mesocolpium area was 23.31 µm, while the apocolpium was 39.95 µm. The sexine was thicker than the nexine. Hence, such variation may be due to variation in the pollen acetolysis techniques and the different geographical regions, i.e., the tropical rain forest in Hainan Island and subtropical regions in Pakistan having different altitudes and different environmental filters.

Mimosa bimucronata
The pollen was small in size and shed in polyad bitetrads containing 8 grains, i.e., four pollen grains in two planes (4 + 4). The prolate was spheroidal and tetragonal, the polarity was heteropolar and the symmetry was bilateral. Aperture condition was found as tri-to tetracolporate. The exine sculpturing was found to be rugulate and rarely scabrate (Figure 5b). The pollen size was noted as 16.7 × 12.6 µm. Similarly, 14 species of the Mimosoideae family of the Atlantic Forest in Brazil were investigated, and it was reported that pollen grains of Mimosa bimucronata are bitetrad with exine sculpturing that is rugulate, which supports our findings. However, bitetrad-type pollen is also found in Mimosa elliptica and Mimosa pellita. Hence, further palynological studies are needed to explore new palynological traits, which will help to solve the taxonomic problem within the Mimosa genus.

Mimosa diplotricha
The first sample of this species was collected from a tropical rainforest in Beihualing. The pollen was shed as tetrad polyads, the shape was tetrahedral, the symmetry was bilateral and the polarity was heteropolar. The aperture condition was noted as inaperturate. The uniplanar tetrad with the proximal sides of two individual units is directly connected, while the other two units are separated. The pollen size was noted as 19.1 × 22.39 µm, while the individual grain diameter was 14.28 µm. The exine sculpturing was predominantly areolate and verrucate, rarely being scabrate and gemmate. Ubisch bodies were found as supra-tectal elements (Figure 2d-g). The second sample was collected from Haikou, and an additional perforate, scabrate and densely verrucate exine sculpturing was found, while the center of the polyad was observed as concave, which was found to be different from the former species. The pollen size was noted as 22.21 × 22.11 µm, while the individual grain diameter was 13.47 µm (Figure 2h,i). In conclusion, variation was found in these two populations of Mimosa diplotricha. This may have been due to the variations in altitude and environmental factors such as temperature and humidity. The previous study reported that the number of grains per polyad and their shape are useful taxonomic traits in the characterization of Mimosa taxa [63]. Hence, our study confirmed that the number of grains per polyad and their shape are valuable taxonomic characteristics for the characterization of Mimosaceae taxa. Additionally, the structure of the polyads may protect the pollen grains from dehydration in dry habitats.

Mimosa pudica
The first sample was collected from Wanning. The pollen was shed as tetrad polyads, the shape was tetrahedral, the symmetry was bilateral and the polarity was heteropolar. The aperture condition was noted as inaperturate. The uniplanar tetrad with the proximal sides of two individual units is directly connected, while the other two units are separated. The exine sculpturing was predominantly areolate and psilate, with various sized verrucae having an isodiametric shape (Figure 1h,i, Figure 2a-c). The pollen size was noted as 7.8 × 7.75 µm. The individual grain diameter was 3.85 µm and the diameter of each verruca was 0.64 µm. In comparison with previous studies, the exine sculpturing was characterized by tubercles that were inconsistent with our results, except for the tetrads type of pollen noted by Agashe and Caulton [59]. Inaperturate tetrahedral-and tetrad-type pollen with an average diameter of 9 µm in M. pudica samples in Singapore has also been reported previously [62].
In Fabaceae, the pollen evolution ranges from simple to tetrads and polyads. The simple grains were confirmed in our study, for example for the pollen from Entada phaseoloides and Leucaena leucocephala. The pollen morphology of the Mimosa genus has been extensively studied. For instance, pollen grains of about 255 species have been described, of which 247 were studied analyzed by light microscopy, 216 by scanning electron microscopy and 19 by transmission electron microscopy [37,41,[63][64][65][66][67][68]. These studies documented detailed descriptions of the pollen morphologies of many Mimosa taxa, showing the variation in the different types of pollen within the genus, i.e., polyads, tetrads and octads in various shapes and sizes, with verrucate to microverrucate sculpturing, porate apertures in the subdistal position and the presence of an operculum and annulus [69]. However, in the present study, this was not noted in Mimosa bimucronata, where the bitetrad 8-grain polyads were arranged in two planes (4 + 4) with a rugulate exine structure.
In Acacia, polymorphism has been found in the grains of each polyad (numbering 4, 8, 12, or 16 grains) in Australian species, while 16 to 32 grains were found in polyads in African species [71]. Five species of Pakistani Acacia were studied by Perveen and Qaiser [44], who concluded that morphological differences in pollen of the Mimosoideae species are significant at the tribe and genera levels. Caccavari and Dome [9] analyzed the pollen morphologies of 77 American Acacia species and suggested that pollen features can be used as a distinguishing factor for the generic limitation of Acacia. Similarly, Rajurkar et al. [72], reported 16 grains per polyad in Acacia species and variations in their morphological traits, i.e., size, shape and exine pattern, which were found to be helpful in genera-and species-level identification. However, in comparison with these findings, 16 grains per polyad were found in the present study for Acacia auricifoliformis and 12 grains per polyad were found in A. confusa and A. mangium. The latter species can be delimited from the former due to the presence of operculum on the central grain surface of the polyad. Hence, our study also confirmed that morphological traits of the pollen of Acacia species are useful at the generic and specific levels.
The pollen morphology of Leuceana leucocephala species was noted as a perforate and scabrate exine pattern, while pollen units of monad and tricolpate types were found in the present study. In comparison with a previously published study [46], the variation in pollen morphology of the Leucaena species was found to be helpful in showing the relationship and defining generic boundaries within Mimosoideae. For instance, they reported perforate and punctate exine sculpturing with pollen units of monad and tricolporate types in Leucaena leucocephala. However, psilate to scabrate exine sculpturing in Pakistani L. leucocephala was found by Khan et al. [41]. These findings mostly support our study results.
The pollen morphologies of 11 Calliandra taxa were investigated by De Assis-Ribeiro dos Santos and De Oliveira-Romão [47], who concluded that some of the polyad characteristics are useful in corroborating previous infrageneric classifications of the genus. Several palynological studies included Calliandra taxa. Sorsa [37], gathered data from nine species. Similarly, Guinet and Hernández [73] made a comparison between Calliandra and Zapoteca, the former of which has eight grains per polyad, while the latter has 16 grains per polyad. Our study corroborated these findings (8-grain polyads) in Calliandra haematocephala. Other studies [74][75][76] recorded the pollen morphology of one or species of the genus.

Conclusions
Taxa from the subfamily Mimosoideae collected from different geographical regions on Hainan Island were analyzed using both light microscopy and scanning electron microscopy. Both LM and SEM morphological descriptions of the pollen can be helpful for plant taxonomists to correctly identify and discriminate the Mimosoideae taxa at generic and specific levels. Significant variation was found in both the qualitative and quantitative traits of the pollen, confirming the Mimosoideae species as eurypalynous. The pollen traits were significantly proven to have taxonomic potential that will support and strengthen the systematics of this subfamily. This study will also provide a basis for further phylogenetic and molecular studies to strengthen the systematics of the Mimosoideae subfamily. Heteromorphism was found in the shape of the monads in the pollen association (polyads, bitetrad, octads). Additionally, exines exhibited heteromorphism in the colpus surface membrane ornamentation of the studied taxa. Interspecies variation was present in the exine ultrastructure of the pollen grains of the studied Mimosoideae taxa. Furthermore, palynological, anatomical, molecular and phylogenetic studies are needed to support and strengthen the systematics of the Mimosoideae subfamily.
Author Contributions: All authors contributed to the study conception and design. Funding acquisition, provision of resources, study design, supervision of the project and critical review of the manuscript were performed by W.L. Material preparation, data collection and analyses were performed by S.B. Material preparation and analysis and interpretation of the data were performed by S.T. The first draft of the manuscript was written by S.B.,S.T. and U.H. wrote and revised the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research project was supported by the National Natural Science Foundation of China (31870508, 31660163, 32171772).

Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available within the article or on request from Saraj Bahadur.