Details of pollen diversity of Euphorbiaceae sensu stricto and Phyllanthaceae native from forest fragments of Cerrado under light microscopy and scanning electron microscopy

Details of pollen diversity of Euphorbiaceae sensu stricto and Phyllanthaceae native from forest fragments of Cerrado under light microscopy and scanning electron microscopy | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 18 February 2025 V1 Latest version Share on Details of pollen diversity of Euphorbiaceae sensu stricto and Phyllanthaceae native from forest fragments of Cerrado under light microscopy and scanning electron microscopy Authors : Carolina Prandi da Silva , Eduardo Lopes Soares , Cintia Neves de Souza , Isaura de Paula Cerdan , Ana Carolina Venancio Lopes , Letícia Vieira Basílio , and Eduardo Gasparino 0000-0001-6078-7341 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173984612.29162895/v1 301 views 147 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract The pollen morphology of 13 Euphorbiaceae species and one Phyllanthaceae species native to forest fragments of Cerrado was investigated to contribute to expanding the morphological knowledge and characterizing species for the conservation of degraded areas. The pollen grains were acetolyzed, measured, and photographed using light microscopy (LM). To obtain more details of exine ornamentation, non-acetolyzed pollen grains of some species were observed using scanning electron microscopy (SEM). The qualitative data were described, a pollen key was presented, and descriptive and multivariate statistics analyzed the quantitative data. The pollen grains of Euphorbiaceae differ in polarity, size, amb, shape, number, and type of apertures (inaperturate, 3-colporate, 4-porate, or pantoporate), type of endoapertures (circular, lalongate, lolongate or endocingulate) and exine ornamentation (psilate, microreticulate, reticulate, or with Croton-pattern). Phyllanthus acuminatus Vahl. (Phyllanthaceae) presents pollen grains in monads, isopolar, medium, circular amb, oblate spheroidal, 3-diploporate, and exine pilate. The number and type of apertures, the morphology of the endoapertures, and the ornamentation of the exine were the essential data for distinguishing the studied species, in addition to the diameter measurements and the variations in the thickness of the exine layers being quantitative characters that characterize the analyzed taxa. 1. Introduction Phylogenetic studies, primarily based on molecular data, have provided new insights into the classification of taxa within Euphorbiaceae. Research conducted by Chase et al. (1993, 2002), Savolainen et al. (2000), Soltis et al. (2000), Davis and Chase (2004), Wurdack et al . (2005), and Tokuoka (2007) demonstrated that the family, according to its traditional and historical classification, was polyphyletic, corroborating Hutchison’s (1969) findings that the family likely originated from orders exhibiting hypogynous flowers, such as Tiliales, Malvales, Rhamnales, and Celastrales. Chase et al. (2002) proposed that the Euphorbiaceae family be separated into Euphorbiaceae sensu stricto ( s.s. ), Phyllanthaceae, and Picrodendraceae. In APG II (2003), based on morphological and molecular data, Euphorbiaceae sensu lato ( s.l. ) is divided into four families: Euphorbiaceae s.s. , Picrodendraceae, Phyllanthaceae, and Putranjivaceae. This classification was maintained in APG III (2009). Later, Wurdack and Davis (2009) suggested that Peroideae be recognized as a family to preserve the monophyly of Euphorbiaceae s.s. Thus, in APG IV (2016), Peraceae is considered a distinct family, as all families derived from Euphorbiaceae are included in the Order Malpighiales. The Euphorbiaceae family comprises approximately 6,462 species across 218 genera globally (WCSP 2016). Their representatives are primarily found in tropical and subtropical regions, especially on the American and African continents, with a few genera extending into extratropical areas (Webster 1994, 2014). This family is one of the most morphologically complex and diverse taxonomic groups within the Eudicots (Soltis et al. 2005) and is currently classified under Malpighiales, alongside 36 other families, within the Rosids clade and Fabids subclade (APG IV 2016, Stevens 2017). In Brazil, 983 species and 68 genera are occurring throughout the national territory, represented in all types of vegetation (Flora e Funga do Brasil 2024). Euphorbiaceae (as defined by APG IV 2016) is a monophyletic family featuring uniovulate plants and is divided into four subfamilies: Cheilosoideae, Acalyphoideae, Crotonoideae, and Euphorbioideae (Wurdack et al. 2005, Tokuoka 2007, Webster 2014). The Phyllanthaceae family comprises 59 genera and approximately 2,000 species, with Phyllanthus L. being the largest genus, containing 1,269 species (Chase et al. 2002, Samuel et al. 2005). Its distribution is broad but more diverse in tropical regions, represented by a range of forms from small herbs and aquatic plants to xeromorphic subshrubs and large trees (Kathriarachchi et al. 2005). Webster (2002) reported about 135 species in Brazil, out of 200 natives to neotropical regions; however, currently, 15 genera and 141 species are documented, of which 85 are endemic (Flora e Funga do Brasil 2024). Phylogenetic studies indicate that Phyllanthaceae is a monophyletic family (Wurdack et al. 2004; Kathriarachchi et al. 2005; Samuel et al . 2005), supported by morphological data such as unisexual flowers, bifid styles, and schizocarpic fruits (Webster 1994). Morphologically, the presence of biovulate ovaries and the absence of laticiferous channels and arils in the seeds set Phyllanthaceae apart from Euphorbiaceae s.s. (Martins and Lima 2011). This group can be divided into two distinct clades: one includes plants that are generally monoecious, featuring fasciculate inflorescences and lacking taniferous epidermal cells, along with fruits that typically exhibit explosive dehiscence; the other clade consists of mostly dioecious plants, characterized by racemose and elongated inflorescences, possessing tan-bearing epidermal cells and indehiscent fruits or those that decay late or incompletely (Kathriarachchi et al. 2005, Martins and Lima 2011). The palynology of Euphorbiaceae sensu lato has been addressed in several studies that illustrate the significant morphological diversity of the family’s pollen grains. Webster (1975), when proposing the five subfamilies that are still part of the original Euphorbiaceae classification, utilized morphological characteristics, particularly pollen morphology data from Erdtman (1952) and Punt (1962), to infer relationships among the genera within the subfamilies. Lobreau-Callen et al. (2000) examined the pollen and macromorphological traits of 28 genera from the five original subfamilies of Euphorbiaceae and, based on this analysis, concluded that four of these constitute monophyletic groups, with only Phyllanthoideae (Phyllanthaceae sensu APG IV 2016) identified as a polyphyletic group. A wide morphological variation is noted for the species in Euphorbiaceae sensu lato , and the family serves as an excellent example of a eurypalynous taxon (see Erdtman 1952, Punt 1962, 1980, 1987, Webster and Webster 1972, Bor 1979, Meewis and Punt 1983, El Ghazaly and Chaudhary 1993, Nowicke 1994, Takahashi et al. 1995, Carreira et al. 1996, Nowicke et al. 1998, Nowicke and Takahashi 2002, Sagun and Van der Han 2003, Santiago et al. 2004, Sagun et al. 2006, Lima et al. 2007, Souza et al. 2016, Souza et al. 2017, Sakugawa et al. 2019, Santos et al. 2019, Souza et al. 2019, Souza, Carneiro-Torres, et al. 2020, Souza et al. 2020, and Sakugawa et al. 2021). Within the literature, the pollen grains of Euphorbiaceae s.l. are typically dispersed in monads. They can be apolar or isopolar, varying in size and ambient conditions, featuring a very small to small polar area, and are sometimes syncolporate, with variations in the number and type of apertures (inaperturate, porate, colpate, colporate, or diploporate). Furthermore, the exine ornamentation exhibits considerable variation, as does the relationship of the exine layer thickness. Other details, such as the presence or absence of operculum, margo, bridge, fastigium, and ornamented membrane in the apertures, have also been documented for the pollen of Euphorbiaceae s.l. . Thus, understanding the palynological diversity of Euphorbiaceae s.l. and aiming to extend pollen morphology studies to the remaining forest fragments, this study seeks to characterize the pollen morphology of Euphorbiaceae s.s. and Phyllanthaceae species ( sensu APG IV 2016) found in degraded regions (using LM and SEM) to provide insights for better morphological knowledge of these taxa, thereby aiding in their conservation. This study complements the series of palynological studies conducted on the remaining forest fragments of the Northwest Region of the State of São Paulo (Souza and Gasparino 2014; Belonsi and Gasparino 2015; Dutra and Gasparino 2018; Landi and Gasparino 2018; Souza et al. 2019; Bellonzi et al. 2020; Dutra et al. 2020; Landi et al. 2021; Soares et al. 2021; Landi et al. 2022; Lopes et al. 2022; Soares et al. 2022; Torati-Guioti et al. 2023; Cerdan et al. 2024; Soares et al. 2024; Gusman et al. 2024). 2. Material and Methods For this study, pollen grains from 14 species were analyzed: 13 from Euphorbiaceae s.s. and one species from Phyllanthaceae (Table 1). These species are distributed across 18 fragments sampled by the project “Fauna and Flora of Forest Fragments Remaining in the Northwest Region of the State of São Paulo” (Necchi 2012). The species Alchornea glandulosa Poepp. & Endl., Croton floribundus Spreng., Manihot tripartita (Spreng.) Müll.Arg., Margaritaria nobilis L.f., and Savia dictyocarpa Müll.Arg. mentioned by Ranga et al. (2012) for the area in question were not analyzed due to a lack of pollen material. Sebastiania commersoniana (Baill.) L.B.Sm. & Downs, cited in the project, is currently regarded as a synonym of Gymnanthes klotzschiana Müll.Arg. The materials used for pollen analysis (Table 1) were obtained from specimens held in the JABU and SJRP herbarium (acronyms according to Thiers [2024]). Three flower buds from each specimen were selected for light microscopy (LM) analysis. In the laboratory, anthers removed from the flower buds were prepared using the acetolysis method outlined by Erdtman (1960), following the modifications made by Melhem et al. (2003). After assembling the slides to record the diameters, 25 measurements were taken on pollen grains randomly distributed across at least four slides within a maximum period of one week (Salgado-Labouriau 1973). Ten measurements were made using different pollen grains for data on the aperture and thickness of the exine layers. The permanent slides were deposited in the pollen reference collection of the Laboratory of Plant Morphology and Palynology, Department of Biology, UNESP, Jaboticabal, SP, Brazil. The non-acetolyzed pollen grains were prepared according to Melhem et al. (2003) for scanning electron microscopy (SEM) analysis to elucidate in greater detail the exine ornamentation of some species ( Actinostemon concepcionis (Chodat & Hassl) Hochr, Croton urucurana Baill, Dalechampia pentaphylla Lam, Mabea fistulifera Mart, and Sapium glandulosum (L.) Morong). To compare the measurements of pollen grain diameters, a descriptive statistical analysis was performed, yielding the means (x), standard deviation (sx), standard error (s), 95% confidence intervals (CI), coefficient of variation (V), and range (R) according to Zar (2010) and Vieira (2011). The arithmetic mean is presented for the other measures. To determine whether the pollen data on diameter and exine thickness allowed for species grouping, a principal component analysis (PCA) was conducted using FITOPAC 1 (Shepherd 1996) and PC-ORD version 5.15 (McCune and Mefford 2011), with comparisons made using the Pearson and Kendall correlation coefficients. Seven metric variables were utilized for the PCA: D1/PD (diameter 1 or polar diameter in equatorial view), D2/ED (diameter 2 or equatorial diameter in equatorial view), SHAP (pollen shape), EXIN (exine thickness), SEXI (sexine thickness), TECT (tectum thickness), and NEX (nexine thickness). The photomicrographs of pollen grains were captured using an optical microscope (Leica IM50) along with a video camera connected to a microcomputer at the Laboratory of Plant Morphology and Palynology, Department of Biology, UNESP, Jaboticabal, SP, Brazil. The electron micrographs were taken with a scanning electron microscope (ZEISS EVO MA10) at the Electronic Microscopy Laboratory, UNESP, Jaboticabal, Brazil. Plates were created from these light microscope (LM) and scanning electron microscope (SEM) images to illustrate the analyzed pollen grains. Pollen terminology was based on Punt et al. (2007) and Halbritter et al. (2018), alongside the pollen descriptions from Bellonzi et al. (2020). The polar area and colpus width indices were calculated using Faegri and Iversen (1966) and Gasparino et al. (2013), respectively. We adopted the definitions from Erdtman (1952) and Walker and Doyle (1975) for the aperture positions in the polar view; to determine the colpus lengths, we employed the colpus length index established by Dutra et al. (2023); and Soares et al. (2021) were referenced to characterize the types of endoapertures by Erdtman (1952), taking into account the standard deviation of the measurements. 3. Results 3.1. General pollen description Pollen grains in monads, apolar or isopolar; small to giant size (Tables 2-3); circular, spheroidal, subcircular or subtriangular amb; very small polar area, syncolporate or parasyncolporate; oblate spheroidal to prolate; inaperturate, (2)(3)-4-(5)-porate, 3-colporate, 3-diplomate, pantoporate; angulaperturate, circulaperturate, planaperturate; short to very long and narrow colpi; tapered or rounded in the ends; with or without margo, fastigium, vestibulum or constriction; endocingulate, circular, lalongate or lolongate endoapertures, with or without costa. Exine atectate pilate, tectate psilate, or semitectate microreticulate, reticulate, or Croton pattern ornamentation; very thin to very thick exine and sexine thicker than nexine (Table 3). 3.2. Species pollen description Euphorbiaceae s.s. (Figures 1-5; Tables 2-3): Acalypha diversifolia Jacq. (Figure 1 a-h; Tables 2-3) Pollen grains isopolar, small size (Tables 2-3), circular (Figure 1a, c) or subcircular amb (Figure 1b), subprolate, 4-porate (Figure 1a), with some pollen grains, 2-porate (Figure 1e), 3-porate (Figure 1b) or 5-porate (Figure 1c); circulaperturate, with margo (Figure 1f) and vestibulum (Figures 1a-c). Exine tectate, psilate (Figure 1g-h), thick exine and sexine thicker than nexine (Table 3). Actinostemon concepcionis (Chadat & Hassl.) Hochr. (Figures 1i-m; 5a-c; Tables 2-3) Pollen grains isopolar, medium size (Tables 2-3), circular amb (Figure 1i; Figure 5a), prolate spheroidal (Figure 5b), 3-colporate (Figure 1i, k), circulaperturate, short and narrow colpi, rounded in the ends, with margo (Figure 1j), lolongate endoapertures (Figure 1k; Table 3). Exine semitectate, microreticulate (Figures 1l-m; 5c), very thick exine and sexine thicker than nexine (Table 3). Croton urucurana Baill. (Figures 1n-r; 5d; Tables 2-3) Pollen grains apolar, large size (Tables 2-3), spheroidal (Figure 1n), inaperturate (Figure 1n). Exine semitectate, croton pattern, rosettes with 6-7 subunits, subtriangular and psilate (Figures 1o-r; 5d), very thin exine and sexine thicker than nexine (Table 3). Dalechampia pentaphylla Lam. (Figures 2a-e; 5e; Tables 2-3) Pollen grains isopolar, large size (Tables 2-3), subtriangular amb (Figure 2a), subprolate, 3-colporate (Figure 2a, c), planaperturate, very long and narrow colpi, rounded in the ends, with margo (Figure 2c), endocingulate (Figure 2c; Table 3). Exine semitectate, microreticulate (Figures 2d-e; 5e), thin exine and sexine thicker than nexine (Table 3). Dalechampia scandens L. (Figure 2f-j; Table 2-3) Pollen grains isopolar, large size (Tables 2-3), subtriangular amb (Figure 2f), subprolate, 3-colporate (Figure 2f, h), angulaperturate, very long and narrow colpi, rounded in the ends, without margo, endocingulate (Figure 2h; Table 3). Exine semitectate, reticulate (Figure 2i-j), thin exine, and sexine thicker than nexine (Table 3). Dalechampia triphylla Lam. (Figure 2k-o; Tables 2-3) Pollen grains isopolar, large size (Tables 2-3), subtriangular amb (Figure 2k), prolate spheroidal, 3-colporate (Figure 2k, m), angulaperturate, long and narrow colpi, rounded in the ends, without margo, endocingulate (Figure 2m; Table 3). Exine semitectate, reticulate (Figure 2n-o), thin exine, and sexine thicker than nexine (Table 3). Gymnanthes klotzchiana Müll. Arg. (Figure 2p-v; Tables 2-3) Pollen grains isopolar, medium size (Tables 2-3), subtriangular amb (Figure 2p), very small polar area (Figure 2q), presenting some syncolporate pollen grains (Figure 2r), subprolate, 3-colporate (Figure 2p, s), planaperturate, very long and narrow colpi, rounded in the ends, with margo (Figure 2q-r) and fastigium (Figure 2s), circular endoapertures (Figure 2s; Table 3). Exine semitectate, microreticulate (Figure 2t-v), very thin exine, and sexine thicker than nexine (Table 3). Mabea fistulifera Mart. (Figures 3a-e; 5f-g; Tables 2-3) Pollen grains isopolar, medium to large size (Tables 2-3), circular amb (Figures 3a; 5f), very small polar area, subprolate, 3-colporate (Figures 3a-b), circulaperturate, very long and narrow colpi (Figures 3b; 5g), rounded in the ends, with margo (Figure 3b), lalongate endoapertures (Figure 3b; Table 3). Exine semitectate, microreticulate (Figure 3c-e, 5f-g), very thin exine and sexine thicker than nexine (Table 3). Manihot pilosa Pohl. (Figure 3f-n; Tables 2-3) Pollen grains apolar, very large to giant size (Tables 2-3), spheroidal (Figure 3f-h), pantoporate (Figure 3f, i). Exine semitectate, croton pattern, rosettes with 6-7 subunits, triangular and psilate (Figure 3i-n), thick exine and sexine thicker than nexine (Table 3). Sapium glandulosum (L.) Morong (Figures 3o-r; 5h-i; Tables 2-3) Pollen grains isopolar, medium to large size (Tables 2-3), subcircular amb (Figure 3o), very small polar area, prolate (Figure 5h), 3-colporate (Figure 3o-p), circulaperturate, long and narrow colpi, tapered in the ends, without margo, endocingulate (Figure 3p; Table 3). Exine semitectate, microreticulate (Figures 3q-r; 5i), thin exine and sexine thicker than nexine (Table 3). Sapium haematospermum Müll. Arg. (Figure 4a-d; Tables 2-3) Pollen grains isopolar, medium size (Tables 2-3), subcircular amb (Figure 4a), very small polar area, prolate spheroidal, 3-colporate (Figure 4a-b), circulaperturate, long and narrow colpi, tapered in the ends, without margo, lolongate endoapertures and with costa (Figure 4b; Table 3). Exine semitectate, microreticulate (Figure 4c-d), thin exine and sexine thicker than nexine (Table 3). Sebastiania brasiliensis Spreng. (Figure 4e-k; Tables 2-3) Pollen grains isopolar, large size (Tables 2-3), circular amb (Figure 4e), very small polar area, parasyncolporate (Figure 4f-g), prolate, 3-colporate (Figure 4e, i), circulaperturate, very long and narrow colpi, constricted (Figure 4i), lalongate endoapertures (Figure 4i; Table 3). Exine semitectate, microreticulate (Figure 4j-k), very thin exine and sexine thicker than nexine (Table 3). Tragia volubilis L. (Figure 4l-n; Tables 2-3) Pollen grains apolar, large size (Tables 2-3), spheroidal (Figure 4l), inaperturate (Figure 4l). Exine semitectate, microreticulate (Figure 4m-n), thick exine and nexine thicker than sexine (Table 3). Phyllanthaceae: Phyllanthus acuminatus Vahl. (Figure 4o-r; Tables 2-3) Pollen grains isopolar, medium size (Tables 2-3), circular amb (Figure 4o), oblate spheroidal, 3-diploporate (Figures 4o-p) circulaperturate, long and narrow colpi, circular endoapertures (Figure 4p; Table 3). Exine atectate, pilate (Figure 4q-r), thin exine and nexine thicker than sexine (Table 3). 3.3. Artificial pollen key to pollen grains of Euphorbiaceae s.s. and Phyllanthaceae species from forest fragments 1. Apolar pollen grains ……………….…………….…………………………………. 2 1’. Polar pollen grains ………………….………………………………………………. 4 2. Microreticulate ornamentation ………….……….……………….…. Tragia volubilis 2’. Croton pattern ornamentation …………………………………………………………………………. 3 3. Large pollen grains, inaperturate; exine 20.0 μm ..………. Manihot pilosa 4. Porate pollen grains, small size ……….………………………… Acalypha diversifolia 4’. Colporate pollen grains, medium/large size ………….…….………………….……. 5 5. Ectocolpus with two endoapertures (diploporate) …….….… Phyllanthus acuminatus 5’. Ectocolpus with one endoaperture ………………………….………………………. 6 6. Distinct and not ununited endoapertures …………………….………………………. 7 6’. Endocingulate endoapertures ……………………….….…………………………. 11 7. Short colpi ………………….……………………………. Actinostemon concepcionis 7’. Long or very long colpi …………………………………….………………………. 8 8. Lalongate endoapertures …………………………………….……………….……… 9 8’. Lolongate endoapertures ……………………………….…………………………. 10 9. Subprolate pollen grains; exine ≥ 2.50 μm ……………….….………. Mabea fistulifera 9’. Prolate pollen grains; exine < 2.50 μm ………………………. Sebastiania brasiliensis 10. Subtriangular amb, planaperturate pollen grains …….……. Gymnanthes klotzchiana 10’. Subcircular amb, circulaperturate pollen grains ……….…. Sapium haematospermum 11. Prolate pollen grains ……………………….…………………. Sapium glandulosum 11’. Subprolate or prolate spheroidal pollen grains ……………………………………. 12 12. Microreticulate pollen grains ……………………………… Dalechampia pentaphylla 12’. Reticulate pollen grains ………………………………….………………………. 13 13. Very long colpi; exine > 5.00 μm …………………….………. Dalechampia scandens 13’. Long colpi; exine ≤ 5.00 μm ……………………….………. Dalechampia triphylla 3.4. Principal component analysis (PCA) The multivariate analysis of principal components was conducted using seven metric variables (Table 4) to emphasize the groups formed by the quantitative data in Euphorbiaceae s.s. and Phyllanthaceae pollen grains examined here. The analysis accounted for 97.36% of the total data variability across its first two axes, with the first axis representing 87.73% of the data variability and the second axis 9.63% (Figure 1). Despite the significant contribution of axis 1 of the PCA in explaining data variability, most metric variables showed a low correlation with the axis (Table 4). Conversely, pollen grain diameters exhibited a moderate correlation with axis 2 of the PCA. Regarding axis 1, Manihot pilosa stood apart from the other species on the negative side of the axis, having the highest averages of all measured pollen metric variables, particularly in diameter measurements (D1/PD and D2/ED), total exine (EXIN), and sexine (SEXI). Conversely, at the other end of axis 1 (the positive side), Acalypha diversifolia had the lowest averages of the analyzed variables and was also isolated from the other species. A group formed by the other species analyzed displayed generally intermediate measurements in pollen grains (Figure 1). Even with a low contribution to the analysis (9.63%), the relationship of the variables with axis 2, especially diameters, was more pronounced than in axis 1 (Table 4). When we examine the distribution of taxa in axis 2 of the PCA (Figure 1), only two groups are visible: the first emphasizing Acalypha diversifolia , located on the positive side of the axis, and the other species grouped in the central region on the negative side of axis 2. Among the metric variables associated with axis 2, we should highlight the contribution of nexine (NEXI), which likely influenced the positioning of Croton urucurana at the extreme of axis 2 (negative side). 4. Discussion Euphorbiaceae s.l. is well-known for its notable variation in pollen grain morphology, with key characteristics including polarity, amb, size, shape, number and type of apertures, and exine ornamentation. The literature indicates that most studies classify the family into pollen types primarily based on the number and type of apertures: inaperturate, pantoporate, colpate, colporate, or diploporate; and based on the ornamentation of the exine: areolate, areolate-rugulate, baculate, bireticulate, clavate, striate, fossulate, foveolate, gemmate, microechinate, microgranulate, microreticulate, microrugulate-scabrate, microverrucate, croton pattern, pilate, psilate, psilate-perforate, reticulate, rugulate, or verrucate (Erdtman 1952; Punt 1962, 1980, 1987; Webster and Burch 1967; Webster and Webster 1972; Punt and Rentrop 1973; Salgado-Labouriau 1973; Barth et al. 1976; Bor 1979; Meewis and Punt 1983; El-Ghobary 1985; Park and Lee 1988; El Ghazaly 1989; El Ghazaly and Chaudhary 1993; Gillespie 1994; Nowicke 1994; Takahashi et al. 1995; Carreira et al. 1996; Nowicke et al. 1998; Saad and El-Ghazaly 1998; Carneiro-Torres et al. 2002; Nowicke and Takahashi 2002; Webster and Carpenter 2002; Melhem et al. 2003; Sagun and Van der Han 2003; Santiago et al. 2004; Perveen and Qaiser 2005; Cruz-Barros et al. 2006; Sagun et al. 2006; Lima et al. 2007; Silva 2008; Chen et al. 2009; Corrêa et al. 2010; Sales et al. 2011; Vieira et al. 2012; Cardinal-McTeague and Gillespie 2016; Cassino et al. 2016; Souza et al. 2016; Wu et al. 2016; Lorente et al. 2017; Souza et al. 2017; Ybert et al. 2017; Sakugawa et al. 2019; Santos et al. 2019; Souza et al. 2019; Souza, Carneiro-Torres, et al. 2020; Souza, Silva, et al. 2020; Yang et al. 2020; Bomfim et al. 2021; Sakugawa et al. 2021). Qualitative data on the pollen grains of Euphorbiaceae s.l. , including the number and type of apertures as well as exine ornamentation, are extensively utilized to define pollen types, as they represent valuable and distinctive characteristics in applied palynology. Various proposals for pollen types in Euphorbiaceae have been reported in studies by Punt (1962, 1980, 1987), Punt and Rentrop (1973), Salgado-Labouriau (1973), Bor (1979), Meewis and Punt (1983), El Ghazaly and Chaudhary (1993), Nowicke (1994), Takahashi et al. (1995), Carneiro-Torres et al. (2002), Nowicke and Takahashi (2002), Webster and Carpenter (2002), Sagun and Van der Han (2003), Santiago et al. (2004), Perveen and Qaiser (2005), Sagun et al. (2006), Silva (2008), Chen et al. (2009), Cardinal-McTeague and Gillespie (2016), Souza et al. (2016), Wu et al. (2016), Souza et al. (2017), and Sakugawa et al. (2021). On the other hand, the size of pollen grains and the thickness values of exine and sexine are distinctive quantitative traits that can group species and genera, as demonstrated by the multivariate analysis here. For Microstachys A. Juss., Sakugawa et al. (2019) identified a moderate correlation between the principal components and the length of the endoaperture as well as the width of the margo. Considering the metric data of pollen from the tribe Hippomaneae, the discriminant variables are the length and width of the endoaperture (Sakugawa et al. 2021). Silva (2008) found that for Phyllanthus , the width of the colpus and the length of the endoaperture are correlated with the principal components when examining Brazilian species of Phyllanthaceae. Therefore, it is important to characterize quantitative pollen data to better understand the role of metric variables in the palynology of Euphorbiaceae s.l. The pollen of Acalypha species primarily varies in the number and type of apertures, as well as in exine ornamentation. Pollen grains can have between two to eight apertures, and variations may be present within the same species; however, colporate pollen is typical for the genus (see Punt 1962; Webster and Burch 1967; Nowicke and Takahashi 2002; Sagun et al. 2006; Yang et al. 2020; Bomfim et al. 2021). Porate pollen was noted here (for Acalypha diversifolia ) and also observed by Webster and Burch (1967) in nine species of the genus found in Panama (pollen grains 3-5 pseudoporate). Most species in the genus exhibit psilate pollen ornamentation, as confirmed here for the pollen grains of A. diversifolia ; however, microreticulate, microrugulate, microverrucate, rugulate, areolate, microrugulate-scabrate, areolate-rugulate, or microequinate pollen grains have also been reported for Acalypha (Nowicke, Takahashi, 2002; Sagun, Levin, Van der Ham, 2006; Bomfim, Santos, Carneiro-Torres, 2021). Therefore, our data on pollen ornamentation supports the findings of Punt (1962) and Webster and Burch (1967). Within Euphorbiaceae s.s. , one of the most distinctive pollen characteristics is the presence of Croton-pattern ornamentation, which features reticulate ornamentation with generally triangular supratectal elements (rosettes) arranged in a circular pattern (Erdtman 1952; Punt et al. 2007; Halbritter et al. 2018). This ornamentation pattern serves as a synapomorphy for Crotonoideae species (Erdtman 1952; Lima and Pirani 2008). Furthermore, the subfamily is the primary group within the eudicots that predominantly presents inaperturate pollen grains (Nowicke 1994), with approximately 1,500 species distributed across Baliospermum Blume, Croton , Jatropha L., Joannesia Vell., Neoboutonia Müll.Arg., and Tritaxis Baillon (Furness 2007). The pollen grains of Croton and Manihot examined here are apolar, ranging from medium to very large in size, and spheroidal. We confirm the presence of inaperturate pollen grains in Croton urucurana , supporting previous findings (Webster and Burch 1967; Carreira et al. 1996; Melhem et al. 2003; Cruz-Barros et al. 2006; Lima et al. 2007; Corrêa et al. 2010; Souza et al. 2016; Ybert et al. 2017; Souza, Silva, et al. 2020; Yang et al. 2020), and pantoporate pollen grains in Manihot pilosa, as noted by Webster and Burch 1967, Nowicke 1994, Vieira et al. 2012, Souza et al. 2016, and Ybert et al. 2017. Pollen grains with Croton-pattern ornamentation may exhibit variations in the size of the rosettes, the number and shape of rosette subunits, and the ornamentation covering each subunit, which can range from psilate to granulate, striate, or reticulate. Furthermore, other ornamental elements (such as clavae, pila, granules, bacula, or gemmae) may appear within the spaces of the rosettes (Souza et al. 2016). This morphological variation in the Croton-pattern may facilitate the differentiation of species or species groups (see Webster and Burch 1967; Nowicke 1994; Carreira et al. 1996; Melhem et al. 2003; Cruz-Barros et al. 2006; Lima et al. 2007; Corrêa et al. 2010; Vieira et al. 2012; Ybert et al. 2017; Souza et al. 2016; Souza, Silva, et al. 2020; and Yang et al. 2020). For the species analyzed here featuring Croton-pattern ornamentation, we observed subtriangular rosette subunits in Croton urucurana and triangular subunits in Manihot pilosa , both of which exhibited psilate ornamentation. In colporate pollen grains of Euphorbiaceae s.l. , the endoapertures can vary among circular, lalongate, or lolongate; however, species of Dalechampia exhibit endocingulate endoapertures, a characteristic unique to the family (Souza et al. 2017). Aside from Dalechampia species, endocingulate pollen grains have only been described for one other species, Sapium glandulosum , as noted here and by Ybert et al. (2017). Nowicke and Takahashi (2002) identified the endocingulate endoaperture and other distinctive features of Dalechampia pollen grains, indicating that the genus can be classified into a monogeneric tribe. The pollen grains of the species examined here belong to Euphorboideae ( Actinostemon , Gymnanthes , Mabea , Sapium, and Sebastiania ). They constitute a characteristic pollen type that ranges from small to large, exhibiting an oblate spheroidal to prolate shape, with 3-colporate and microreticulate or psilate-perforate ornamentation (Webster and Burch 1967; Melhem et al. 2003; Cruz-Barros et al. 2006; Corrêa et al. 2010; Sales et al. 2011; Cassino et al. 2016; Lorente et al. 2017; Souza et al. 2017; Ybert et al. 2017; Yang et al. 2020; Sakugawa et al. 2021). In this study, Actinostemon pollen grains showed short colpi with lolongate endoapertures for the first time, while the literature describes colpi ranging from long to very long, with various endoaperture shapes (circular, lolongate, or lalongate) for the genus (Cruz-Barros et al. 2006; Corrêa et al. 2010; Sales et al. 2011; Ybert et al. 2017; Sakugawa et al. 2021). Therefore, the data on the endoapertures of A. concepcionis align with prior studies (Cruz-Barros et al. 2006; Corrêa et al. 2010). Regarding colpus length, a more detailed analysis may unveil another distinguishing characteristic within the genus; thus, utilizing indices that assist in defining these traits is recommended, such as the one applied here by Dutra et al. (2023). Our data confirm previous observations concerning the pollen grains of the other species and genera of Euphorboideae analyzed here. Thus, the results obtained for the pollen of Gymnanthes klotzschiana support the study by Sakugawa et al. (2021); those of Mabea fistulifera support Webster and Burch (1967) and Sakugawa et al. (2021); those of Sapium species align with Webster and Burch (1967), Melhem et al. (2003), Sales et al. (2011), Cassino et al. (2016), Souza et al. (2017), Ybert et al. (2017), Yang et al. (2020), and Sakugawa et al. (2021). The results for Sebastiania brasiliensis corroborate Webster and Burch (1967), Melhem et al. (2003), Cruz-Barros et al. (2006), Corrêa et al. (2010), Sales et al. (2011), Cassino et al. (2016), Lorente et al. (2017), Souza et al. (2017), Ybert et al. (2017), and Sakugawa et al. (2021). We identified some differences when we compared our observations of Tragia volubilis pollen with the descriptions presented in previous studies by Ybert et al. (2017) and Santos et al. (2019). Ybert et al. (2017) and Santos et al. (2019) characterize the pollen grains of this species as isopolar, small to medium, prolate spheroidal to prolate, 3-colporate, and featuring pilate or microreticulate ornamentation. Thus, our results support the authors regarding the ornamentation of the pollen grains, while there are differences concerning polarity, size, and apertures; the two specimens analyzed here show apolar, large, and inaperturate pollen grains. The sole species analyzed here for Phyllanthaceae, Phyllanthus acuminatus , features pollen grains that are medium-sized, 3-diploporate, and possess pilate ornamentation. These morphological data have also been noted in other studies, including Punt (1962, 1987), Webster and Burch (1967), Webster and Carpenter (2002), Sagun and Van der Han (2003), and Silva (2008). According to the literature, diploporate apertures were not recorded for any other species of the genus. Phyllanthus pollen is distinguished by a broad diversity of numbers and types of apertures, ranging from pantoporate, pantocolpate, colpate, colporate, to syncolporate (Webster and Burch 1967; Punt and Rentrop 1973; Bor 1979; Punt 1980; Meewia and Punt 1983; Saad and El-Ghazaly 1998; Santiago et al. 2003; Perveen and Qaiser 2005; Silva 2008; Chen et al. 2009; Corrêa et al. 2010; Wu et al. 2016; Ybert et al. 2017; Yang et al. 2020). The results obtained here, as expected, confirm the morphological diversity of these taxa. Additionally, the data presented enhance our understanding of pollen morphology for these species, particularly Acalypha diversifolia , Actinostemon concepcionis , and Dalechampia pentaphylla, which were not previously analyzed. 5. Conclusion The characteristics of pollen grains in Euphorbiaceae s.l. (Euphorbiaceae s.s. and Phyllanthaceae) are essential for distinguishing and characterizing species and genera. The most significant features include pollen size, number, type of apertures, and the ornamentation of the exine, which serves as a basis for defining pollen types in future studies. Quantitative attributes of pollen grains, such as diameters and thickness of the exine layers, also play a crucial role in differentiating species, which should be taken into account in pollen studies. The diverse characteristics highlighted here, along with those mentioned in other studies, confirm the eurypalynous nature of the family. Author Contributions Carolina Prandi da Silva : investigation, formal analysis, methodology, writing – original draft. Eduardo Lopes Soares : formal analysis, methodology, software, writing – review and editing. Cintia Neves de Souza : formal analysis, project administration, writing – review and editing. Isaura de Paula Cerdan : formal analysis, methodology, software, writing – review and editing. Ana Carolina Venancio Lopes : formal analysis, methodology, software, writing – review and editing. Letícia Vieira Basílio : conceptualization, investigation, formal analysis. Eduardo Custódio Gasparino : conceptualization, supervision, resources, data curation, project administration, writing – review and editing. Acknowledgments The authors are grateful for grants from FAPESP 2016/19246-8 for C.P. Silva and from CNPq- 309555/2021-3 for E.C. Gasparino; to the curators of the herbaria for pollen sampling and the Laboratório de Microscopia Eletrônica, FCAV, for providing technical assistance with scanning electron microscopy. Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References APG II. 2003. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG II. Botanical Journal of the Linnean Society. 141: 399-436. APG III. 2009. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG III. Botanical Journal of the Linnean Society. 161: 105-121. APG IV. 2016. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Botanical Journal of the Linnean Society. 181: 1-20. 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Molecular phylogenetic analysis of Phyllanthaceae (Phyllanthoideae pro parte, Euphorbiaceae sensu lato) using plastid rbcL DNA sequences. American Journal of Botany. 91:1882–1900. Yang S, Maod L, Zheng Z, Chene B, Li J. 2020. Pollen atlas for the selected subfamilies of Euphorbiaceae from southern China: a complementary contribution to Quaternary pollen analysis. Palynology. 44(4):659–673. doi: 10.1080/01916122.2019.1658235 Ybert JP, Carvalho MA, Scheel-Ybert R. 2017. Grãos de pólen de plantas vasculares do Estado do Rio de Janeiro, Brasil: volume II. Rio de Janeiro (RJ): Museu Nacional, Universidade Federal do Rio de Janeiro. ISBN: 978-85- 7427-065- 41 Zar JH. 2010. Biostatistical analysis. Upper Saddle: Pearson Prentice-Hall. Tables and Figures TABLE 1. Voucher specimens of Euphorbiaceae s.s. and Phyllanthaceae from Brazilian forest fragments. TABLE 2. Quantitative data of Euphorbiaceae s.s and Phyllanthaceae pollen grains, n = 25. R = range (µm); x= mean (µm); s x = standard deviation (µm); s = standard error (µm); CI = confidence interval in 95% (µm); V = coefficient of variation (%). *Diameter 1 and diameter 2 for apolar pollen grains. TABLE 3 . Morphological characterization and measures (μm) of apertures and exine of Euphorbiaceae s.s. and Phyllanthaceae pollen grains. - = absence of measures or structure. S = small; M = medium; L = large; VL = very large; GG = giant; P/E = ratio between polar and equatorial diameter; PAI = polar area index; SP = subprolate; PS = prolate spheroidal; S = spheroidal; OS= oblate spheroidal; PR= prolate; WCI = width colpus index; OR= ornamentation; PSI= psilate; MR= microreticulate; CP= Croton pattern; RE= reticulate; PI = pilate. *Apolar pollen grains. ** endocingulate. ° caput of pilum. n = 10 TABLE 4. Pearson and Kendall correlation coefficients for pollen grain metric variables of the first and second axes of PCA ordination in Euphorbiaceae s.s. and Phyllanthaceae species. * caput of pilum for atectate pollen grains FIGURE 1. Photomicrographs of pollen grains from species of Acalypha L., Actinostemon Mart. ex Klotzsch and Croton L. (Euphorbiaceae s.s. ). (a-h) Acalypha diversifolia Jacq. (a-c) Polar view. (a) 4 apertures; (b) 3 apertures; (c) 5 apertures. (d-f) Equatorial view. (d) 3 apertures; (e) 2 apertures; (f) emphasis on the aperture. (g-h) Ornamentation in high and low focus. (i-m) Actinostemon concepcionis (Chodat & Hassl.) Hochr. (i) Polar view; (j) Equatorial view with an emphasis on the apertures; (k) Equatorial view with emphasis on the endoaperture; (l-m) Ornamentation in high and low focus. (n-r) Croton urucurana Baill. (n) General view; (o) Ornamentation; (p-r) Ornamentation in high and low focus. Scales: 3 µm = g-h; 5 µm = other figures. FIGURE 2. Photomicrographs of pollen grains from Dalechampia L. and Gymnanthes Sw. species (Euphorbiaceae s.s. ). (a-e) Dalechampia pentaphylla Lam. (a) Polar view; (b) Equatorial view; (c) Equatorial view with emphasis on the apertures; (d-e) Ornamentation in high and low focus. (f-j) Dalechampia scandens L. (f) Polar view; (g) Equatorial view; (h) Equatorial view with emphasis on the apertures; (i-j) Ornamentation in high and low focus. (k-o) Dalechampia triphylla Lam. (k) Polar view; (l) Equatorial view; (m) Equatorial view; (n-o) Ornamentation in high and low focus. (p-v) Gymnanthes klotzchiana Müll. Arg. (p) Polar view outline; (q-r) Polar view ornamentation. (s) Equatorial view. (t-v) Ornamentation in high and low focus. Scales: 5 µm = p-v; 10 µm = d-e, i-j, n-o; 20 µm = other figures. FIGURE 3. Photomicrographs of pollen grains from species of Mabea Aubl., Manihot Tourn. ex Adans. and Sapium Jacq. (a-e) Mabea fistulifera Mart. (a) Polar view; (b) Equatorial view, aperture details; (c-e) Ornamentation in high and low focus. (f-n) Manihot pilosa Pohl. (f) General view outline; (g) General view high focus; (h) General view low focus; (i-n) Details on ornamentation in high and low focus. (o-r) Sapium glandulosum (L.) Morong. (o) Polar view; (p) Equatorial view with emphasis on the apertures; (q-r) Ornamentation in high and low focus . Scales: 5 µm = c-e, q-r; 10 µm = a-b, i-p; 20 µm = f-h. FIGURE 4. Photomicrographs of pollen grains from Euphorbiaceae (Sapium Jacq., Sebastiania Spreng. and Tragia L.) and Phyllanthaceae species (Phyllanthus L.). (a-d) Sapium haematospermum Müll. Arg. (a) Polar view; (b) Equatorial view with emphasis on the apertures; (c-d) Ornamentation in high and low focus . (e-k) Sebastiania brasiliensis Spreng. (e-g) Polar view; (h) Equatorial view; (i) Emphasis on the apertures; (j-k) Ornamentation in high and low focus. (l-n) Tragia volubilis L. (l) Overview; (m-n) Ornamentation in high and low focus. (o-r) Phyllanthus acuminatus Vahl. (o) Polar view; (p) Equatorial view with emphasis on the apertures; (q-r) Ornamentation in high and low focus. Scales: 5 µm = a-d, f-g, i, j-k, m-n, o-r; 10 µm = other figures. FIGURE 5. Scanning electron micrographs of pollen grains from Euphorbiaceae . (a-c) Actinostemon concepcionis (Chodat & Hassl.) Hochr. (a) Polar view; (b) General view; (c) Details of ornamentation. (d) Croton urucurana Baill., details of ornamentation. (e) Dalechampia pentaphylla Lam., details of ornamentation; (f-g) Mabea fistulifera Mart. (f) Polar view; (g) Equatorial view; (h-i) Sapium glandulosum (L.) Morong. (h) Equatorial view; (i) Details of ornamentation. Scales: 5 µm = c, i; 10 µm = a, d-e; 20 µm = other figures. FIGURE 6. Principal component analysis performed with Euphorbiaceae s.s. and Phyllanthaceae pollen metric variables. (D1/PD = diameter 1 or polar diameter in equatorial view; D2/ED = diameter 2 or equatorial diameter in equatorial view; EXIN = exine thickness; SEXI = sexine thickness; NEXI = nexine thickness; TECT = tectum thickness; SHAP = shape – P/E or D1/D2; Adiv = Acalypha diversifolia ; Acon = Actinostemon concepcionis ; Dpen = Dalechampia pentaphylla ; Dsca = Dalechampia scandens ; Dtri = Dalechampia triphylla ; Gklo = Gymnanthes klotzchiana ; Mfis = Mabea fistulifera ; Sgla = Sapium glandulosum ; Shae = Sapium haematospermum ; Sbra = Sebastiania brasiliensis ; Curu = Croton urucurana ; Mpil = Manihot pilosa ; Tvol = Tragia volubilis ; Pacu = Phyllanthus acuminatus ). 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Keywords brazil eurypalynous malpighiales pollen grains pollen morphology Authors Affiliations Carolina Prandi da Silva Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Ciencias Agrarias e Veterinarias View all articles by this author Eduardo Lopes Soares Universidade de Sao Paulo Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto View all articles by this author Cintia Neves de Souza Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Ciencias Agrarias e Veterinarias View all articles by this author Isaura de Paula Cerdan Universidade de Sao Paulo Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto View all articles by this author Ana Carolina Venancio Lopes Universidade de Sao Paulo Faculdade de Filosofia Ciencias e Letras de Ribeirao Preto View all articles by this author Letícia Vieira Basílio Colégio Poligenes Jaboticabal SP Brazil View all articles by this author Eduardo Gasparino 0000-0001-6078-7341 [email protected] Universidade Estadual Paulista Julio de Mesquita Filho Faculdade de Ciencias Agrarias e Veterinarias View all articles by this author Metrics & Citations Metrics Article Usage 301 views 147 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Carolina Prandi da Silva, Eduardo Lopes Soares, Cintia Neves de Souza, et al. 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