Palynological investigation of some Euphorbia L. (Euphorbiaceae) taxa from Turkey using light and scanning electron microscopy

Euphorbia L., one of the largest genera of angiosperms, is represented by the subgenera Esula Pers. and Chamaesyce Raf. in Turkey. Subg. Esula is confined mainly to Eurasia, especially in the Mediterranean region, and has not yet been studied in detail palynologically. In the present study, the pollen morphology of 14 taxa belonging to the sections Helioscopia Dumort., Myrsiniteae (Boiss.) Lojac., Pithyusa (Raf.) Lázaro, Patellares (Prokh.) Frajman, Exiguae (Geltman) Riina & Molero, Paralias Dumort., and Esula (Pers) Dumort. of Euphorbia L. subg. Esula Pers. from European Turkey was examined using light and scanning electron microscopy. The pollen grains were found to be trizonocolporate, isopolar, radially symmetrical, medium in size, and oblate spheroidal and suboblate in shape. Ectoapertures were elongated colpi with distinct margins, widened at the equatorial region, and tapered at the ends; margo was present in all samples as a smooth exine surrounding the colpi. The endoapertures were lolongate pori with distinct costae. The exine was tectate‐perforate. The ornamentation was perforate, microreticulate‐perforate, microreticulate‐perforate with rugulose, or microreticulate‐perforate with a heterobrochate pattern. The sexine was generally thicker than the nexine, with some exceptions where the sexine was almost equal to the nexine. Pollen size, pollen shape, and exine ornamentation have taxonomic importance in distinguishing species; however, these features do not provide strong support for grouping or delimitating the taxa at the sectional level.


| INTRODUCTION
Euphorbia L. is one of the largest recognized genera of flowering plants with approximately 2000 species.It has a worldwide distribution but is especially diverse in arid and semi-arid regions of the tropics and subtropics (Dorsey et al., 2013).Based on a phylogenetic investigation, Euphorbia was divided into four major clades (A-D) by Steinmann and Porter (2002).Later, Bruyns et al. (2006) recognized these clades as the following subgenera: subg.Chamaesyce Raf., subg.
The leafy spurges, viz.Euphorbia subg.Esula, make up one of the four main lineages of Euphorbia.The subgenus comprises approximately 480 species, most of which are annual or perennial herbs, with a small number of dendroid shrubs and nearly leafless, pencilstemmed succulents.The subgenus has a nearly worldwide distribution; however, it is most diverse in temperate Eurasia, particularly in the Mediterranean and Irano-Turanian regions (Riina et al., 2013).
Recent studies pertaining to this "giant" and diverse genus Euphorbia have mostly focused on its molecular phylogenetic systematics (Bruyns et al., 2006;Dorsey et al., 2013;Horn et al., 2012;Peirson et al., 2013Peirson et al., , 2014;;Riina et al., 2013;Steinmann & Porter, 2002).However, Geltman (2015) emphasized the need to integrate data from traditional fields of botany with new classification systems for the genus and its subgenera to fill the new Euphorbia system with morphological, geographical, and other content as the next step in understanding the evolution and systematics of Euphorbia.
Only a few previous studies on the pollen morphology of Euphorbia have focused on its taxonomic significance.El-Ghazaly and Chaudhary (1993) examined the pollen morphology of 60 species of Euphorbia and presented taxonomic suggestions based on their data.
The pollen morphology of Euphorbia subg.Agaloma (Raf.)House section Tithymalopsis and related species was used to evaluate the sectional boundaries by Park (1997).Oh et al. (2002) have reported that the pollen size and surface sculpturing were comparable at the subgenus and section levels.Perveen and Qaiser (2005) considered the pollen morphology of the family to be significantly helpful at the generic, specific, and subfamily levels.In a palynotaxonomic study based on sculpturing patterns and numerical analyses, Lee and Park (2006) determined the sectional boundaries and relationships among 13 species of Euphorbia.Studies pertaining to the pollen morphology were conducted to determine the subsectional boundaries and relationships among the species of subsect.Meleuphorbia and related taxa by Noh and Park (2008).Nobarinezhad et al. (2018) presented the pollen morphology of nine species of Euphorbia in a biosystematic study of the subgenus Chamaesyce in Iran.Alyas et al. (2021) conducted a study on palynological features that provided useful taxonomic data for the identification of Euphorbiaceae (nine species of Euphorbia) in Pakistan.
Several studies have investigated the morphology and palynology of this genus in Turkey.Genç and Kültür (2017) determined the morphological and karyological features of Euphorbia smirnovii Geltman.
Seed, capsule, leaf, and cyathial gland morphology of the Turkish perennial taxa of Euphorbia sect.Pithyusa (Raf.)Lázaro was studied by Genç and Kültür (2018) and Genç et al. (2018).Can and Küçüker (2015) reported the seed morphology and surface microstructure of some Euphorbia taxa distributed in the European region of Turkey.
Only a few Euphorbia species belonging to the subg.Esula, including E. kotschyana Fenzl, E. stricta L., E. peplus L., E. macroclada Boiss., and E. erythrodon Boiss.& Heldr.from Turkey have been palynologically investigated (Gökçen et al., 2018;Gökçen & Koyuncu, 2019;Koyuncu et al., 2019).This paper presents a detailed palynological investigation using both light and scanning electron microscopy of 14 taxa from seven sections of Euphorbia subg.Esula, which is naturally distributed in European Turkey.The aim of the present study was to determine the palynological characteristics that are of systematic importance and contribute to the delimitation of the sections and species.

| Plant material
The pollen samples were obtained from dried herbarium specimens collected by the author (NŞO) from European Turkey (Appendix A) and were deposited in the Tekirda g Namık Kemal University Herbarium (NAKU).The following sections and taxa were studied: sect.

| Light microscopy
For light microscopy (LM), glycerin jelly stained with fuchsin was used to prepare the pollen slides (Wodehouse, 1935).Pollen grains were observed under 100Â magnification using an Olympus CX41 light microscope, which was compatible with an SC30 Olympus digital camera and Cell* imaging software.Ten morphometric characteristics, including the polar axis (P), equatorial diameter (E), colpi length (Clg), colpi width (Clt), pori length (Plg), pori width (Plt), distance between the colpi apices (t), sexine (Sex), nexine (Nex), and intine (In), were measured.The pollen shape was determined as observed in the equatorial view using the P/E ratio according to the classification given by Erdtman (1943).For each characteristic, 40 measurements were recorded, and the arithmetic mean and standard deviation were calculated (Table 1).Morphological features, such as the shape, amb, and ornamentation of the exine, are presented in Table 2.In addition, the pollen dimensions, polar area index, and ratios of a number of relevant characters are given comparatively in Table 3.

| Scanning electron microscopy
For scanning electron microscopy (SEM), pollen grains were mounted onto metal stubs using double-sided adhesive tape.Micrographs of the general morphology and detailed exine ornamentation were obtained using an FEI QUANTA FEG 250 SEM at a magnification of 5000-15,000Â.
The terminology follows that used by Punt et al. (2007).

| General pollen remarks
The pollen grains of all the studied Euphorbia taxa from all sections were trizonocolporate, isopolar, and radially symmetrical.The shapes were oblate spheroidal or suboblate.Pollen dimensions were medium; the largest one belonged to E. rigida (P Â E = 43.70Â 49.38 μm) and the smallest one belonged to E. illirica (P Â E = 29.25 Â 31.98 μm).
Two aperture types were observed: ectoaperture (colpus) and endoaperture (porus).The colpi were observed to be widened in the middle with conspicuous or inconspicuous tapering ends, and a smooth margo was present in all samples.Pori were lolongate and costate.
The exine had a perforated tectum.The sexine was generally thicker than the nexine; however, in some cases, it was almost equal to the nexine.The ornamentation was perforate, microreticulate-perforate, microreticulate-perforate with rugulose, or microreticulate-perforate with a heterobrochate pattern.

| Statistical analysis
Principal component analysis (PCA) was employed to assess the pollen characteristics that expressed the greatest proportion of variability, and whether the species and sections could be grouped based on these pollen characteristics.The first two components, PC1 and PC2, accounted for 64.4% and 13.9% of the variance, respectively (Table 4).The PCA biplot of the individuals and explanatory variables as vectors is shown in Figure 7a E. amygdaloides subsp.amygdaloides, and E. characias subsp.wulfenii) have higher values of Plt and Clt, whereas those in the lower left quadrant (E.helioscopia, E. illirica, E. platyphyllos subsp.platyphyllos, and E. taurinensis) have lower values of these contributors (Figure 7a).
The pollen grains of Euphorbiaceae species have been determined to be very diverse in previous studies.The family was found to be distinctly eurypalynous by Saad and El-Ghazaly (1988), with pollen grains being either colpate, colporate, porate, or inaperturate; the sexine pattern was found to vary considerably and being either tectate, semitectate, reticulate, or microreticulate, and provided with spinules or a crotonoid pattern.Similarly, evidence from a study based on nine species of Euphorbia from the Euphorbioideae subfamily suggests that the subfamily is heterogeneous (El-Ghazaly, 1989).In addition, the genus has been found to be remarkably eurypalynous by El-Ghazaly and Chaudhary (1993).Earlier, Punt (1962) has noted when considering the family pollen grains, their dimensions and proportions may differ, but, in general, they are identical.In addition, Punt (1987) has claimed that the pollens of Euphorbioideae subfamily members are consistent in shape, ornamentation, and size.In the present study, it was evident that the pollen grains of different taxa, although from different sections, were generally similar.The most striking differences were observed in terms of pollen dimensions, pollen shape, and the ornamentation of exine (Tables 1 and 2; Figures 2c,f ,i,l,o, 4c,f,i,l, and 6c,f,i,l,o).
The pollen size of the studied species was medium with the larg-  7a), the species and sections outside of Myrsiniteae were grouped closely because most of the variables overlapped (Figure 7b).In addition to the pollen size, sect.Myrsiniteae was characterized by large ecto-and endoapertures.Another section with large pollen grains and endoapertures was Patellares.E. agraria from sect.Esula also showed large pollen grains with large ecto-and endoapertures, and was observed to be close to E. amygdaloides subsp.
amygdaloides and E. characias subsp.wulfenii from sect.Patellares (Table 1, Figure 7a).Pithyusa was the section with the largest endoa-  in exine structure and ornamentation make it difficult to establish taxonomic boundaries within the species belonging to sect.Anisophyllum.
Nevertheless, these characteristics can be useful for comparing species from different sections or subgenera within Euphorbia.El-Ghazaly and Chaudhary (1993) examined the pollen of 60 species of Euphorbia and presented a key by which all taxa could be grouped into seven types based on sexine ornamentation.In a palynotaxonomical study of Euphorbia based on sculpturing patterns and results of a numerical analysis using 10 pollen characters, 13 species from sect.Zygophyllidium were divided into four pollen types: microreticulate, reticulate, microreticulate pollen with platten muri, and foveolate pollen (Lee & Park, 2006).In the present study, exine ornamentation was another feature that differed among the species; however, it alone could not be used to distinguish between the species and discriminate the sections.This is because species in the same section may have the same or different exine ornamentation types and patterns.For example, in sect.Myrsiniteae, the exine ornamentation of both E. myrsinites subsp.
myrsinites and E. rigida was observed to be microreticulate-perforate with a rugulose pattern.In sect.Helioscopia, the exine of E. helioscopia was microreticulate-perforate, whereas that of the other four species in this section was perforate.Additionally, although the ornamentation of E. amygdaloides subsp.amygdaloides and E. characias subsp.
wulfenii from sect.Patellares was microreticulate-perforate in both cases, the pattern of the latter was observed to be rugulose.
The pollen grains of the studied species from subg.Esula were more or less similar in terms of the exine sculpture.
The ornamentation was perforate, microreticulate-perforate, microreticulate-perforate with rugulose, or microreticulate-perforate with a heterobrochate pattern.These were regarded as different patterns of the perforate exine.El-Ghazaly and Chaudhary (1993) examined the pollen of 60 species of Euphorbia, presented a key in which all taxa were grouped into seven types based on the exine ornamentation, and proposed a putative evolutionary relationship between the pollen types.They interpreted the pertectate perforate sexine as the basic ancestral type (type 1a), and the other type that forms a transition between the perforate and ±rugulose exine with pertectate pollen grains as having minute perforations which fuse in groups of two, three, or four, and finally differentiate into rugulae of various shapes and grooves (type 1b).Another type of pollen that they described is type 4, which diverges from the rugulose pattern of type 1b by widening the area of grooves, giving a general reticulate pattern with perforated lumina.In this study, the basic type of ornamentation was considered to be perforate, with a transition towards perforate ornamentation forming microreticula.In some cases, these microreticula formed a rugulate (perforations solitary or 2-4 in lumina) pattern, and in others, a heterobrochate (perforations solitary or 2-14 or more in the lumina) pattern was observed.Based on the results of this study (for subgen.Esula) and evidence from previous studies on the other subgenera of Euphorbia (El-Ghazaly, 1989;El-Ghazaly & Chaudhary, 1993;Lee & Park, 2006;Nobarinezhad et al., 2018), it can be said that not only the sections but also the subgenera cannot be discriminated based solely on the exine ornamentation.

| CONCLUSION
In conclusion, the studied species of subg.Esula show a more or less homogenous structure in terms of general pollen morphological characteristics, such as being tricolporate and having colpi surrounded by smooth margo, costate endoapertures, and tectate perforate exine.
The most important characteristics that differ among species are pollen size, pollen shape, and exine ornamentation.These characteristics are useful for discriminating between individual species; however, they are not significant for determining the relationships at recently recognized sectional levels.Palynological studies with more specimen sampling, particularly focusing on exine sculpture evolution, would contribute to the future classification systems of Euphorbia.
. Variable vectors have been presented based on their contribution to the principal components (gradient colors and transparency of vectors) as follows: black and longer represent high contributions, brown or light brown are intermediate, and yellow and shorter constitute very low contributions.P, E, and Clg were the most significant and positively correlated contributors to the first principal component and are shown as black and long vectors.E. rigida and E. myrsinites subsp.myrsinites from sect.Myrsiniteae, with high values of P, E, Clg, were grouped in the lower-right quadrant.The remaining 12 species with lower P, E, and Clg values were clustered on the opposite side.Plt and Clt were the most significant and positively correlated contributors to the second principal component and are shown as black and long vectors.Taxa in the upper right quadrant (E.oblongata, E. agraria, E. falcata subsp.falcata, F I G U R E 6 Scanning electron microscope (SEM) micrographs of pollen grains of the studied Euphorbia taxa.(a-c) E. amygdaloides subsp.amygdaloides, (d-f) E. characias subsp.wulfenii, (g-i) E. exigua subsp.exigua, (j-l) E. taurinensis, and (m-o) E. agraria.(a, d, g, j, m) Equatorial view, (b, e, h, k, n) polar view, and (c, f, i, l, o) exine close-up.Scale bars = 10 μm (a, b, d, e, g, h, j, k, m, n) and 5 μm (c, f, i, l, o).

F
I G U R E 7 (a) Principal component analysis (PCA) biplot of 14 Euphorbia species based on the variance observed for 10 morphometric characters.The first two components (PC1 and PC2) accounted for 64.4% and 13.9% of the variances, respectively.The different color intensities and lengths of the vectors denote the contribution of the characteristic to the first two components in the PCA.The variable vectors are presented based on their contributions to the principal components (gradient colors and transparency of vectors) with black and long representing high contributions, brown or light brown representing intermediate contribution, and yellow and short representing very low contributions.E. helioscopia (hel), E. illirica (ill), E. oblongata (obl), E. platyphyllos subsp.platyphyllos (pla), E. stricta (str), E. myrsinites subsp.myrsinites (myr), E. rigida (rig), E. falcata subsp.falcata (fal), E. niciciana (nic), E. amygdaloides subsp.amygdaloides (amy), E. characias subsp.wulfenii (cha), E. exigua subsp.exigua (exi), E. taurinensis (tau), and E. agraria (agr).(b) Principal component analysis (PCA) plot for the studied sections of subg.Esula.
Morphological data of the studied Euphorbia pollen grains.Morphometric comparison of the pollen grains of the studied Euphorbia taxa.