In this paper we examine pollen types from four representative subfamilies of Euphorbiaceae in southern China, including 34 species (21 genera) of Crotonoideae, Euphorbioideae, Acalyphoideae and Phyllanthoideae. The morphology of the investigated species is described and illustrated with high-resolution photographs observed by transmitted light microscope. These descriptions and illustrations are presented at the species level, and pollen types and identifying features are also noted. This study is a complementary contribution to Quaternary pollen analysis, and should aid in the identification of pollen types assigned to Euphorbiaceae, especially in Southern China, as well as elsewhere in the tropical and subtropical regions of East Asia.
As one of the largest and most diversified families of angiosperms, the Euphorbiaceae has about 8000 species in over 300 genera (Webster 1975, 1987, 1994a, 1994b; Radcliffe-Smith 2001). Jensen et al. (1994) suggested that on the basis of serological data there appear to be two main groups of Euphorbiaceae: Phyllanthoideae and Oldfieldioideae (Picrodendraceae); and Acalyphoideae, Crotonoideae and Euphorbioideae. However, molecular phylogenetic analysis assessed the systematic position of Picrodendraceae based on a comparative study of floral structure of the recently established family Picrodendraceae (part of Euphorbiaceae sensu lato) in Malpighiales (Sutter et al. 2006); and Wurdack et al. (2004) analysed plastid rbcL DNA sequence data of the pantropical family Phyllanthaceae (Malpighiales) and related biovulate lineages of Euphorbiaceae sensu lato. These phylogenic works suggest the dismemberment of Phyllanthoideae, considering a family other than Euphorbiaceae or Phyllanthaceae (Radcliffe-Smith 2001; APG 2016). The classification of Euphorbiaceae sensu lato has a long history (e.g. Webster 1975, 1994a, 1994b; Jensen et al. 1994; Radcliffe-Smith 2001; APG 2003, 2009, 2016), which is complicated beyond the scope of this paper. In this study, we follow the most fundamental division in Euphorbiaceae sensu lato with a grouping of two biovulate subfamilies (Phyllanthoideae and Oldfieldioideae) and three uniovulate ones (Acalyphoideae, Crotonoideae and Euphorbioideae) (Webster 1975, 1987, 1994a, 1994b).
The Euphorbiaceae sensu lato includes great diversity in growth form, from tall rain forest trees to lianas, shrubs, perennial and annual herbs, geophytes, succulents and floating aquatics (Webster 1987). Euphorbia, for example – one of the largest genera in the subfamily Euphorbioideae – has around 2000 species from tiny annual plants to large and long-lived trees, and their geographical distribution is cosmopolitan and largely in tropical and subtropical regions. So, palaeoecological interpretation based on pollen records of Euphorbiaceae is a significant challenge, and improving pollen identification is an effective way to refine pollen signals from a palaeoecological point of view. However, pollen types of Euphorbiaceae show high diversity according to previous studies; for example, early investigations of Phyllanthus (Phyllanthoideae) show much variation in pollen types based on a large collection of specimens from worldwide reported by Punt (1967, 1972, 1980, 1987) and Meewis and Punt (1983); and later the pollen morphology and taxonomy of the Phyllanthus species native to New Caledonia were studied by Loberau-Callen et al. (1988). Webster and Carpenter (2002) examined pollen morphology and provided new insights into phylogenetic relationships among the neotropical taxa of Phyllanthus. Moreover, Santiago et al. (2004) examined eight South American species of the genus Phyllanthus, subgenus Phyllanthus, section Choretropsis, endemic to Brazil. Chen et al. (2009) carried out a palynological study of Malesian Phyllanthus, and recently Wu et al. (2016) investigated the pollen morphology of 89 of the 127 Malesian Phyllanthus species, presented a pollen key for the pollen classes and types, and discussed evolutionary trends in pollen morphology and infrageneric classification in Phyllanthus. Yao and Zhang (2016) examined the pollen morphology of Chinese Glochidion (Phyllanthaceae), and its taxonomic implications. El-Ghazaly and Chaudhary (1993) distinguished seven pollen types, with a detailed diagnosis of each type based on the pollen morphology of 60 species from Euphorbia, and discussed the geographical distribution of the types, as well as putative evolutionary relationships between them. Park (1997) re-evaluated a sectional boundary and previous taxonomic treatments, based on pollen morphology of Euphorbia (Euphorbioideae), section Tithymalopsis, and related species. Pollen morphology, exine structure and systematics of the subfamily Acalyphoideae have been intensively investigated in many representative tribes (Nowicke et al. 1998, 1999; Takahashi et al. 2000; Nowicke and Takahashi 2002). Sagun et al. (2006) reported on the pollen morphology and ultrastructure of Acalypha (Acalyphoideae), and evaluated pollen exine structure variation, phylogeny and evolutionary trends. de Souza et al. (2017) found the pollen morphological characteristics more variable in the Acalyphoideae compared to the relatively homogeneous Euphorbioideae, according to their selected species from the Caatinga ecoregion in Brazil. Sagun and van der Ham (2003) investigated the pollen morphology of the Flueggeinae (a subtribe of Phyllantheae) and evaluated the relationships between the eight constituent genera. An early study by Lobreau-Callen and Cervera (1997) focused on pollen exine ultrastructure of the apetalous Crotonoideae, with five tribes and one subtribe. High diversity in pollen morphology of the subfamily Crotonoideae from north-eastern Brazil has been documented, and three pollen types and seven subtypes were recognised based on aperture type, polarity, shape, size and details of exine sculpture (de Souza et al. 2016). A new investigation by Yu et al. (2018) on the pollen of Trigonostemon and the related genera Dimorphocalyx, Ostodes, Tritaxis and Jatropha (outgroup) shows that the two major pollen types within Trigonostemon correlate well with macromorphological characters, and species belonging to the Trigonostemon reidioides type have pollen with ‘Croton pattern’ ornamentation (Yu et al. 2018).
Most of these previous studies on Euphorbiaceae pollen morphology mainly focused on taxonomic and evolutionary significance; however, the distinguishing features of Euphorbiaceae remain poorly documented regarding an identification guide or pollen atlas for Quaternary palynology. An illustrated and descriptive atlas of pollen, particularly for those families (such as Euphorbiaceae) with a high diversity of pollen types, is an indispensable reference for Quaternary pollen analysis and palaeoecological interpretation. As useful references for Quaternary pollen identification, many pollen atlases from different parts of the world were published before 1998, as listed by Hooghiemstra and Van Geel (1998), followed by many new pollen atlases published since 2000 (so far, no new world pollen atlas has been published since 1998). In China, there are 67 genera of Euphorbiaceae with some 406 species (Editorial Committee of Flora of China 1994); however, only a limited number of pollen types of Euphorbiaceae have been examined so far as an indispensable reference to Quaternary palynological studies (e.g. Huang 1972; IBCAS 1982; Wang et al. 1995; Wei 2003). Considering diverse pollen types of Euphorbiaceae frequently occur in Quaternary sediments, especially from southern China, it is necessary to investigate more pollen types covering representative subfamilies of Euphorbiaceae. In this paper, we present pollen descriptions and transmitted light photographs for 34 species from 21 genera of four subfamilies (Acalyphoideae, Crotonoideae, Euphorbioideae, Phyllanthoideae) from southern China. This study is a complementary contribution to Quaternary pollen analysis, and our results should aid Quaternary pollen identification and subsequent interpretation of past vegetation history and palaeoecology based on fossil pollen data.
2. Material and methods
To examine pollen morphological characters, pollen grains of 34 plant species belonging to 21 genera were extracted from specimens collected from the Herbarium of Sun Yat-sen University (SYSU) and Nanjing Institute of Geology and Palaeontology, Chinese Academy of Sciences (NIGPAS). All pollen specimens were collected from South China (Figure 1). The samples were prepared in the Quaternary Environmental Laboratory of SYSU and NIGPAS, and a list of voucher specimens used in this study as well as notes on the pollen plates are summarised in Table 1. Slides were prepared by mounting the pollen grains in glycerine jelly after acetolysis (treatment with an acetolytic mixture of nine parts acetic anhydride and one part sulphuric acid). All pollen grains were observed and photographed in both polar and equatorial views at a magnification of ×600 or ×1000 using a Nikon E200 microscope equipped with a COOLSNAP 5.0 camera, and an Olympus BX51 microscope equipped with a DP72 Charge Coupled Device (CCD) camera. The pollen dimensions obtained and mentioned in the description of each species are the average of measurements of at least 20 pollen grains. Pollen terms used in our descriptions are standard (for definitions, see Kremp 1965; Punt et al. 2007; Hesse et al. 2009).
List of pollen species examined in this study,a with voucher information and plate numbers if illustrated here.
3. Descriptions of pollen morphology
Five genera were investigated in the subfamily Crotonoideae, including Aleurites J. R. et G. Forst., Vernicia Lour., Croton Linn., Codiaeum A. Juss. and Jatropha Linn. Six species of these genera are examined for pollen description, namely Aleurites moluccanus (L.) Willd, Vernicia fordii (Hemsl.) Airy-Shaw, Croton tiglium Linn., C. lachnocarpus Benth., Codiaeum variegatum (L.) Rumph. ex A. Juss. and Jatropha curcas Linn.
Aleurites J.R. & G. Forst.
Aleurites moluccanus (L.) Willd
Plate 1, figures 1, 2
Pollen grains spheroidal, inaperturate, 50 (55) 70 µm in diameter; sexine thicker than nexine, sculpture presents a pattern covered by triangular prism baculate processes, clavae with acute ends, a set of (4)-5-7-(-8) triangular verrucae forms a visible circle and radiated pattern, but some circles not clear.
Vernicia fordii (Hemsl.) Airy-Shaw
Plate 1, figures 3, 4, 4a)
Pollen grains spheroidal, inaperturate, 55 (66.5) 75 µm in diameter; sexine thicker than nexine, sculpture coarsely reticulate (Plate 1, figures 3, 4a); muri with verrucae forming triangular prism shape (Plate 1, figure 4); the triangular prism was formed by three protuberances. As the focus moves downwards, the verrucae appeared to join into muri. A set of 5–8 verrucae form a visible circle.
Croton tiglium Linn.
Plate 1, figures 5, 6
Pollen grains spheroidal, inaperturate, 27 (30.5) 47 µm in diameter; sexine thicker than nexine, sculpture coarsely reticulate, muri with bead-like grains, usually 6–7 such grains arranged in a circle, and linked at the base of the exine (Yang et al. 2015).
Croton lachnocarpus Benth.
Plate 1, figures 7, 8
Pollen grains spheroidal, inaperturate, 30 (38) 47 µm in diameter, sculpture coarsely reticulate. The exine ornamentation resembles that of C. tiglium except in size.
Codiaeum A. Juss.
Codiaeum variegatum (L.) Rumph. ex A. Juss.
Plate 1, figures 9, 10
Pollen grains ellipsoidal, subcircular in polar view, inaperturate, 30 (34.5) 40 × 35 (38) 43 µm in diameter, sculpture with different sizes of strumae, 5–7 verrucae arranged in a circle and formed coarse reticulum, verrucae smaller than those of Croton and Vernicia, similar to coarse granule; exine ca. 2–3 µm thick with a wavy contour.
Jatropha curcas Linn.
Plate 1, figures 11, 12
Pollen grains subspheroidal, inaperturate, 50 (55) 75 µm in diameter, exine ca. 5 µm thick, sculpture verrucate, the protruding verrucae irregular in size and shape, relatively larger than those of its close relatives; each verruca has 4–6 bead-like structures; pollen wall with clearly clavate columellae, with indistinct stratification (Yang et al. 2015).
In this subfamily, five representative species of three genera are examined for pollen description: Euphorbia characias subsp. wulfenii (Hoppe ex W.D.J.Koch) Radcl.-Sm., E. heterophylla Linn., E. milii Des Moul., Sapium sebiferum (L.) Roxb., Excoecaria formosana (Hayata) Hayata & Kawak, from Euphorbia Linn., Sapium Jacq., and Excoecaria Linn., respectively.
Euphorbia characias subsp. wulfenii (Hoppe ex W.D.J.Koch)
Plate 2, figures 1–4
Pollen grains oblate spheroidal, subcircular in polar view, 50 (52) 54 × 48 (50) 52 µm in diameter, tricolporate, colpus slender and long, pore ellipsoidal and lolongate, ca. 12 µm, sexine thicker than nexine, exine gradually thinner around pores, exine ca. 3 µm thick, sculpture reticulate but appears finely granulate as the focus moves downwards.
Euphorbia heterophylla Murray
Plate 2, figures 5–8
Pollen grains oblate spheroidal, subcircular in polar view, 50 (53) 54 × 46 (47) 50 µm in diameter, tricolporate, colpus slender and long, pore lolongate and ellipsoidal, ca. (12 × 8) µm, exine ca. 5-6 µm thick, sculpture coarsely reticulate, texture coarsely granulate forming spines, contour fluctuating and not psilate.
Euphorbia milii Des Moul.
Plate 2, figures 9–12
Pollen grains prolate, trilobed circular in polar view, 35 (38) 40 × 38 (40) 44 µm in diameter, tricolporate, colpus slender and long, exine ca. 3 µm, exine around pore slightly thickened to 4–5 µm, sculpture finely reticulate, muri finely granulate.
Sapium sebiferum (L.) Roxb.
Plate 3, figures 1–3, 3a
Pollen grains prolate, trilobed circular in polar view, 30 (35) 45 × 34 (40) 46 µm in diameter, tricolporate, colpus wide at the poles and constricted in the middle, pore lalongate and ellipsoidal, ca. 5 × 11 µm in diameter, sculpture mimics reticulate pattern, muri with fine granulate ornamentation.
Excoecaria formosana (Hayata) Hayata & Kawak
Plate 3, figures 4, 5
Pollen grains subprolate, subcircular in polar view, 50 (52) 54 × 59 (50) 53 µm in diameter, tricolporate, colpus slender and long, pore circular, nexine and sexine similar in thickness, sexine with pilum, sculpture granulate-reticulate, contour of optical section irregular.
Six genera are selected in the subfamily Acalyphoideae: Mallotus Lour., Macaranga Thou., Alchornea Sw., Cleidion Bl., Acalypha Linn. and Ricinus Linn. Eight species of these genera are investigated for pollen description, namely Mallotus apelta (Lour.) Müll. Arg., M. japonicus (Thunb.) Müll. Arg., M. paniculatus (Lam.) Müll. Arg., Macaranga denticulata (Blume) Müll. Arg., Alchornea trewioides (Benth.) Müll. Arg., Cleidion brevipetiolatum Pax & K. Hoffm., Acalypha hispida Burm.f. and Ricinus communis Linn.
Mallotus apelta (Lour.) Müll. Arg.
Plate 3, figures 6–9
Pollen grains spheroidal, subcircular in polar view, 22 (22) 28 × 24 (24) 30 µm in diameter, tricolporate, colpus slender and short, pore wide and lalongate, sexine and nexine similar in thickness but thicker around aperture, ca. 2 µm, sculpture coarsely granulate.
Mallotus japonicus (Thunb.) Müll. Arg.
Plate 3, figures 10–13
Pollen grains 22 (22) 28 × 24 (26) 30 µm, morphological characteristics similar to those of M. apelta.
Mallotus paniculatus (Lam.) Müll. Arg.
Plate 3, figures 14–17
Pollen grains 16 (18) 20 × 16 (18) 20 µm in diameter, Morphological characteristics similar to those of the above species except in size.
Macaranga denticulata (Blume) Müll. Arg.
Plate 3, figures 18–21
Pollen grains spheroidal, subcircular in polar view, 13 (17) 19 × 15 (18) 20 µm in diameter; tricolporate, colpi slender and short, pore lalongate; exine ca. 1.5–3 µm thick, sexine and nexine similar in thickness but thicker around aperture, sculpture finely reticulate, different from that of Mallotus (Yang et al. 2015).
Cleidion brevipetiolatum Pax & K. Hoffm.
Plate 3, figures 22–25
Pollen grains oblate spheroidal, trilobed circular in polar view, 16 (17) 20 × 16 (18) 21 µm in diameter, tricolporate, colpus slender and short, pore lalongate, sexine thicker than nexine, nexine thicker around aperture, sculpture faintly coarsely granulate.
Alchornea trewioides (Benth.) Müll. Arg.
Plate 4, figures 1–4
Pollen grains spheroidal, semi-circular in polar view, 13 (21) 25 × 17 (22) 27 µm in diameter, tricolporate, colpi with colpus membrane, colpus membrane edge sags, with ligulate protrusions in polar view, pore lalongate, sculpture unclearly granulate or mimic reticulate and more coarsely granulate around colpus and aperture.
Acalypha hispida Burm.f.
Plate 4, figures 5–9
Pollen grains spheroidal, trilobed circular in polar view, 24 (26) 28 × 25 (25) 33 µm in diameter, tricolporate, colpus wide but narrowing to the poles, pore rhombic, exine and nexine similar in thickness, sculpture reticulate, muri with pilum and stripe array ornamentation.
Ricinus communis Linn.
Plate 4, figures 10–14
Pollen grains spheroidal, semi-circular in polar view, 23 (25) 27 × 24 (27) 31 µm in diameter, tricolporate, colpi long and almost extending to the poles, colpus slender and narrow at ends, pore lalongate and rectangle, exine protruding around pore, sculpture faint reticulate, lumina irregular in shape and size, exine ca. 2–2.5 µm thick.
In this subfamily, seven genera (Phyllanthus Linn., Glochidion T.R. & G. Forst., nom. cons., Flueggea Willd., Aporosa Bl., Baccaurea Lour., Bridelia Willd. and Antidesma Linn.) with 15 representative species are observed for pollen description: Phyllanthus embilica Linn., P. flexuosus (Siebold & Zucc.) Müll. Arg., P. rheophyticus Gilbert & Li, Glochidion puberum (L.) Hutch., G. wilsonii Hutch, G. lanceolarium (Roxb.) Voigt, G. zeylanicum (Gaertn.) A. Juss., Flueggea virosa (Roxb. ex Willd.) Royle, F. suffruticosa (Pall.) Baill., Aporosa yunnanensis (Pax & K. Hoffm.) F.P. Metcalf, A. chinensis (Roxb. Ex Benth.) Merr., Baccaurea ramiflora Lour., Bridelia tomentosa Bl., B. balansae Tutcher, Antidesma japonicum Siebold & Zucc.
Aporosa yunnanensis (Pax & K. Hoffm.) F.P. Metcalf
Plate 4, figures 15–18
Pollen grains subprolate, semi-circular in view polar, 15 (17) 20 × 16 (18) 22 µm in diameter, tricolporate, colpus slender and long extending almost to the poles, pore lalongate, nexine around pore slightly thickened, sculpture reticulate, lumia regular in size and shape, but slightly smaller towards edge of colpus.
Aporosa chinensis (Roxb.) Müll. Arg.
Plate 4, figures 19–22
Pollen grains 15 (17) 20 × 16 (18) 22 µm in diameter, Morphological characteristics similar to those of A. yunnanensis except in size.
Pollen grains spherical, 4–6-lobate circular in polar view, 18 (19) 23 × 17 (18) 19 µm in diameter, 5-colporate dominant, rarely 4- or 6-colporate, colpus short, pore circular, 1.5–2 µm × 2–3 µm, sculpture finely reticulate.
Phyllanthus flexuosus (Siebold & Zucc.) Müll. Arg.
Plate 5, figures 4–7
Pollen grains spheroidal, trilobed circular in polar view, 18 (20) 23 × 17 (18) 19 µm in diameter, tricolporate, colpus slender and long, pore circular, sculpture finely reticulate.
Phyllanthus rheophyticus Gilbert & Li
Plate 5, figures 8–11
Pollen grains spheroidal, 4-lobed circular in polar view, 12 (15) 20 × 11 (13.5) 19 µm in diameter. Pollen grains normally 4-colporate, a few penta-colporate; colpus slender and short, pore circular; exine ca. 1 µm thick, sexine and nexine similar in thickness; nexine around pore slightly thickened, sculpture finely reticulate, morphological characteristics similar to those of P. embilca except pore number and size.
Flueggea virosa (Roxb. ex Willd.) Royle
Plate 5, figures 12–15
Pollen grains subprolate, trilobed circular in polar view, 13 (17) 22 × 11 (15.5) 18 µm in diameter, tricolporate, colpus slender and long, pore circular, ca. 2.5–3.5 µm in diameter, exine two layered, ca. 1–1.5 µm in thickness, sculpture clearly reticulate, lumina regular.
Flueggea suffruticosa (Pall.) Baill.
Plate 5, figures 16, 17
Pollen grains subprolate, trilobed circular in polar view, 28 (30) 32 × 28 (30.5) 32 µm in diameter, tricolporate, colpus slender and long, pore lalongate, sexine thicker than nexine, sculpture reticulate, lumina reticulate and irregular.
Glochidion T.R. & G. Forst., nom. cons.
Glochidion puberum (L.) Hutch.
Plate 5, figures 18–21
Pollen grains spheroidal, trilobed circular in polar view, 12 (14) 18 × 16 (18) 22 µm in diameter, tricolporate, rarely tetracolporate, colpus slender and long, pore subcircular, exine ca. 2–3 µm in thickness, sculpture coarsely reticulate.
Glochidion wilsonii Hutch.
Plate 5, figures 22–25
Pollen grains spheroidal, 4-lobed circular in polar view, 12 (17) 18 × 16 (22) 24 µm in diameter, mostly tetracolporate, colpus slender and short, pore circular, sculpture coarsely reticulate, exine with pilum, contour scabrate and wavy.
Glochidion lanceolarium (Roxb.) Voigt
Plate 6, figures 1–4
Pollen grains spheroidal, 5-lobed circular in polar view, 14 (20) 22 × 16 (21) 24 µm in diameter, tetracolporate, colpus slender and long, pore subcircular, sculpture coarsely reticulate.
Glochidion zeylanicum (Gaertn.) A. Juss.
Plate 6, figures 5–8
Pollen grains spheroidal, 4-lobed circular in polar view, 20 (24) 27 × 22 (27) 32 µm in diameter, mostly tetracolporate, pore outline faint, sculpture coarsely reticulate.
Baccaurea ramiflora Lour.
Plate 6, figures 9–12
Pollen grains ellipsoidal, trilobed or 4-lobed circular in polar view, 17 (23) 30 × 18 (23) 28 µm in diameter. tricolporate to tetracolporate, colpus slender, pore lalongate with unclear outline, exine with pilum, sculpture coarsely reticulate.
Bridelia tomentosa Bl.
Plate 6, figures 13–17
Pollen grains spheroidal or subsphaeroidal, semi-angular in polar view, 22 (23) 24 × 19 (21) 24 µm in diameter, angulaperturate, tricolporate, pore lalongate and ellipsoidal, sculpture clearly reticulate, lumina in various sizes and shapes.
Bridelia balansae Tutcher
Plate 6, figures 18–22
Pollen grains spheroidal or subspheroidal, trilobed circular in polar view, 20.5 (21) 32 × 20.5 (22) 23.5 µm in diameter; tricolporate, pore lalongate; sexine and nexine similar in thickness, sculpture clearly striate-reticulate, lumina heterobrochate; muri near colpus striate and becoming reticulate. In mesocolpium, ornamentation is striato-reticulate and contour is wavy (Yang et al. 2015).
Antidesma japonicum Siebold & Zucc.
Plate 6, figures 23–26
Pollen grains perprolate, trilobed circular in polar view, 22 (25) 28 × 10 (13) 18 µm in diameter, tricolporate, colpus slender and long, pore lalongate and thickened around aperture forming cross with colpus, exine faintly layered, sculpture faintly granulate.
4. Notes on pollen types and distinguishing features
The binary division in Euphorbiaceae sensu lato provided by de Jussieu (1823, 1824) is based on ovule number, with a grouping of two biovulate subfamilies (Phyllanthoideae and Oldfieldioideae) and three uniovulate ones (Acalyphoideae, Crotonoideae, and Euphorbioideae) (Webster 1975, 1987, 1994a, 1994b). As one of five subfamilies of Euphorbiaceae sensu lato, Oldfieldioideae (Picrodendraceae) is an apparently diverse assemblage of mostly southern hemisphere trees and shrubs that traditionally have been allied with genera of Phyllanthoideae and Porantheroideae (Hayden 1994), while China in the northern hemisphere is home to another four subfamilies of Euphorbiaceae sensu lato, according to the Flora of China (Editorial Committee of Flora of China 1994) and the updated online eflora of China ( www.efloras.org: vol. 11), namely Acalyphoideae, Crotonoideae, Euphorbioideae and Phyllanthoideae. Based on the pollen morphological characteristics investigated here, we found morphological categories of Euphorbiaceae in support of the subfamily taxonomy. Previous studies suggested that each subfamily has its own unique morphological characteristics distinguishable from those of the other subfamilies (Nowicke et al. 1998, 1999; Takahashi et al. 2000; Nowicke and Takahashi 2002; de Souza et al. 2016, 2017). According to previous palynological studies and our new observations, we outlined pollen morphological characteristics for each subfamily investigated in this study (Table 2).
Comparative characteristics of pollen morphology for the investigated subfamilies of Euphorbiaceae.
The pollen morphology and size of Euphorbiaceae display notable differences at the level of species or genera: diameter ranges from 13.5µm (Flueggea virosa) to 66.5µm (Aleurites fordii) and the longest axis is generally less than 50µm (Wang et al. 1995). However, both processing and mounting media can influence pollen size (e.g. Andersen 1960; Cushing 1961; Reitsma 1969); for instance, acetolysis and glycerine jelly are thought to increase size (e.g. Cushing 1961; Reitsma 1969). This issue is also discussed by Faegri and Iversen (1989). In our study, we used glycerine jelly after acetolysis, thus pollen size could be larger than normal, but not significantly so according to our comparison with the published pollen descriptions for some species. Pollen morphology of this family mostly includes colporate types, and rarely inaperturate types. For colporate pollen, tricolporate is the dominant type; however 4–5-colpo-rate or stephanocolpate forms are also found in some genera and species. Exine ornamentation varies from reticulate to granulate, striate, tuberculate and so on. We can identify fossil pollen grains from Quaternary deposits to the genus level in some cases; however, it remains difficult to distinguish some species and genera accurately. Therefore, we examined practical classification for Euphorbiaceae pollen grains according to morphological characteristics which are summarised in Table 2.
The subfamily Crotonoideae, sensu Webster, consists of 13 tribes and approximately 73 genera, of which three genera – Croton, Jatropha and Manihot – account for half of the 2000 species (Nowicke 1994). Crotonoideae now appear to be an exception to pollen diversity and have surprisingly uniform pollen morphology, united by the Croton pattern (Erdtman 1952; Nowicke 1994; Webster 1994a; Lobreau-Callen and Suarez-Cervera 1997; de Souza et al. 2016): triangular supratectal elements attached to a network of muri having short or irregular columellae. In this paper, we observed Aleurites, Croton, Vernicia, Jatropha and Codiaeum, which are of the inaperturate type. Pollen morphological characteristics of these genera can be summarised as follows: pollen grains spherical, 30–80 µm in diameter, inaperaturate; exine ca. 1.5–8 µm in thickness; sexine far thicker than nexine; sculpture with Croton pattern, clavate or tuberculate; each design rosette with 5–8 short bars or verrucae and the design densely or sparsely arranged. However, this study dealt with a limited number of species from Crotonoideae, so further investigation to cover more species is needed prior to making an overall assessment of pollen morphological characteristics in this subfamily. According to previous studies (Nowicke 1994; Webster 1994a; Lobreau-Callen and Suarez-Cervera 1997; de Souza et al. 2016), this subfamily mainly has three pollen morphological types: inaperturate with plicate pila, pantoporate, and colpate. However, the triangular supratectal elements, muri, and modified columellae feature the pollen type of Croton, representing the key pollen morphology of most species in the subfamily Crotonoideae.
Previous studies on the pollen morphology of Euphorbioideae suggested the uniformity of pollen characters of this family, with tricolporate with margo and tectate perforate types (Punt 1987; Park and Lee 1988; Nowicke 1994). However, most pollen morphological studies have focused mainly on the tribe Euphorbieae (El-Ghazaly 1989; Suárez-Cervera et al. 2001; Lee and Park 2006; Noh and Park 2008). Punt (1962) intensively studied the pollen morphology of this subfamily using light microscopy. The results suggested a close relationship between Euphorbieae and Hippomaneae (Punt 1962). These two tribes show a similarity in endexine structure, but they display different intine thickness along each side of the aperture. However, these subtle characters are only identifiable under scanning electron microscopy and transmission electron microscopy (Suárez-Cervera et al. 2001; Park and Lee 2013). In China, the Euphorbioideae include two tribes, Euphorbieae and Hippomaneae. Our materials also include these tribes, including Sapium, Euphorbia and Excoecaria. From observation and description under a light microscope, the morphological characteristics of the above genera in this subfamily can be summarised as follows: tricolporate, pollen diameter mostly more than 30µm, pore spherical or ellipsoidal and lalongate, exine with two layers having the same thickness, sculpture finely reticulate, muri with granules.
Pollen morphological comparison for the investigated genera of Acalyphoideae.
Acalyphoideae has 115 genera in 20 tribes, with the largest aggregation of taxa within the Euphorbiaceae (Webster 1994a). This subfamily is remarkably diverse in terms of exine structure and morphology (e.g. Takahashi et al. 1995; Fernández-González and Lobreau-Callen 1996). Almost all species of this subfamily are 3-colporate, and the endoaperture frequently has well-defined polar margins but diffuse lateral ones. The structure of the tectum and exine is more variable (Nowicke et al. 1998; Nowicke and Takahashi 2002). Lobreau-Callen and Suarez-Cervera (1994) concluded that some species from Acalyphoideae are suboblate to subprolate, mostly 3-colporate, and tectate with a wide range of sculpture/ornamentation. To better understand the diverse pollen morphology of this subfamily, we examined pollen morphological characteristics in detail, focusing on pollen size, aperture and ornamentation for some genera in Acalyphoideae, namely Mallotus, Macaranga, Alchornea, Cleidion, Acalypha and Ricinus. We provide a pollen morphological comparison for the investigated genera of Acalyphoideae, summarised in Table 3. Pollen grains are mainly tricolporate, colpus slim and long, pore mostly lalongate, and size relatively smaller. Some pollen types of this subfamily are frequently found in subtropical Quaternary sediments. Some species can be identified to the genus level – for example, Alchornea, Mallotus and Macaranga. The main characteristics of Mallotus are its slender colpus, lalongate pore that is nearly connected to the neighboring aperture, and with coarsely reticulate sculpture (Table 3). The pollen morphological characteristics of Mallotus are similar to those of Cleidion, although the pore thickens close to the aperture for Cleidion, which is different from Mallotus. Macaranga pollen grains are relatively smaller in size, generally less than 20 µm, the sexine and nexine are layered clearly and the same thickness, and the sculpture is granulate-reticulate different from Mallotus. Alchornea pollen has a wider colpus and sharpens towards the poles. In polar view, the pore has ligulate protrusions, and it is easy to identify fossil pollen grains in strata. Acalypha pollen grains show a wider colpus whcih gradually narrows to the poles, a rhomboid pore, and reticulate sculpture formed by fine granules with a striate pattern. As Ricinus is a cultivated plant, its pollen grains scarcely occur in Quaternary sediments. Ricinus pollen grains are ca. 26–28 µm in diameter, relatively bigger than those of the five genera discussed above, with an elongate, rectangular pore, a slender and narrow colpus, extending to the poles, and microreticulate sculpture with irregular lumina.
The subfamily Phyllanthoideae consists of 54–60 genera and ca. 2000, species mostly from pantropical areas, but many occur in southern temperate zones and some northern temperate zones as well (Kathriarachchi et al. 2006; Byng 2014) There are some 18 genera with ca. 164 species and 14 varieties in China (Li 1994). The largest genera and the approximate number of species in each are the following: Phyllanthus (1270), Acalypha (450), Glochidion (300), Cleistanthus (140), Antidesma (100), Aporosa (90), Uapaca (60), Baccaurea (50), Bridelia (50) and Flueggea (15) (Webster 1994a; Radcliffe-Smith 2001; Sagun and van der Ham 2003; Stevens 2010; Yao and Zhang 2016). This paper focuses on Phyllanthus, Glochidion, Flueggea, Aporosa, Baccaurea, Bridelia and Antidesma, and we found that key pollen types are tricolporate to stephanocolporate, with slim colpi, circular or lalongate pore crossed with colpus forming decussation, relatively smaller pollen, and clearly reticulate sculpture. Pollen grains of Phyllanthus are spherical, 3–6-colporate, colpus slender and short, pore circular, sculpture clearly reticulate. Santiago et al. (2004) found that the pollen shape and number of colpi varied within and between some species. Wu et al. (2016) identified five pollen classes (colporate pollen, incomplete synaperturate pollen, synaperturate pollen, clypeate pollen and porate pollen) based on a pollen morphological study of 89 species. In general, the pollen morphology of Phyllanthus is close to that of Glochidion; its exine ornamentation is more finely reticulate compared with that of Glochidion, but it is difficult to differentiate between these pollen types. Pollen grains of Flueggea are usually tricolporate, and the colpus width is different from that of Phyllanthus. Aporosa pollen grains are very similar to those of Anacardiaceae and Rutaceae in morphology, but the pore is lalongate, forming a crisscross with the colpus, which is different from the morphology of the other two subfamilies. The sexine of Baccaurea pollen grains has a pilum and muri and is composed of granulae. Bridelia pollen grains have lalongate pores, an elliptical or egg-shaped aperture and striate reticulate sculpture. Antidesma pollen has similar morphological features to Castanopsis and Lithocarpus in Fagaceae; however, the colpus crosses with the pore forming a decussation and the pollen grains have a longer polar axis. In these aspects, Antidesma is different from Castanopsis and Lithocarpus.
According to our new investigation of selected taxa, pollen types of four subfamilies (Acalyphoideae, Crotonoideae, Euphorbioideae, Phyllanthoideae) from Euphorbiaceae show moderately high diversity in general (Table 2). Their pollen grains show diverse shapes, with a large variation in size – ranging from 13.5 µm to ∼75 µm in diameter, but mostly less than 30 µm. Pollen grains are mostly prolate, with a few spherical or ellipsoid and colporate or inaperturate. Of the colporate pollen grains, most are tricolporate, but tetracolporate, 5-colpo-rate or stephanocolporate forms also occur. Exine sculpture is reticulate, granulate, striate or verrucate (such as the Croton type). At the subfamily level, Crotonoideae pollen grains are inaperturate, large in size and with regularly arranged verrucate sculpture. Euphorbioideae pollen grains are tricolporate, more than 30 µm in size, with pores of various shapes, and finely reticulate sculpture on muri with granules. Acalyphoideae pollen grains are relatively small and tricolporate, with a slender and long colpus (Table 3). Phyllanthoideae pollen grains are also usually relatively small, tricolporate to stephanocolporate, with slender colpi and a small and circular pore; individual species may have an elongated pore crossing the colpi, and clearly reticulate sculpture. Some pollen types of Euphorbiaceae can be identified to the genus level, such as Euphorbia, Mallotus, Macaranga, Alchornea, Phyllanthus, Glochidion, Aporosa, Bridelia and Antidesma. There is a potential to interpret the palaecological significance of these genera in terms of their diverse habitats.
5. Concluding remarks
In this study, we have examined four selected subfamilies (Crotonoideae, Euphorbioideae, Acalyphoideae and Phyllanthoideae), including 21 genera and 34 species whose fossil pollen grains are frequently found in Quaternary sediments. Our illustrated and descriptive pollen atlas will aid Quaternary pollen analysis in southern China, as well as in the surrounding subtropical and tropical areas. The pollen morphology of some species overlaps, especially within the same genus, which may make it difficult, if not impossible, to identify some grains. This is inevitable for closely related pollen types. Further investigation can be aimed at widening our examination to cover many more genera and species of Euphorbiaceae that have yet to be investigated palynologically, to create a robust basis for pollen analysis and palaeoecological interpretation of Quaternary sediments. Pollen identification of the selected subfamilies of Euphorbiaceae can improve palaeoecological and palaeobiogeographical research based on Quaternary fossil pollen.
This study was jointly funded by the National Natural Science Foundation of China (NSFC, grant numbers 41876057, 41877437, 41606053), the Natural Science Foundation of Guangdong Province, China (grant number 2016A030310364), the Key Program for International S & T Cooperation Projects of China (grant number 2016yfe0109600), China geological survey projects (grant numbers DD20160144, DD20189503) and the Taishan Scholars Program of Shandong Province, China (grant number ts201511077). We deeply appreciate Prof. Henry Hooghiemstra and anonymous reviewer, and Editors Prof. James Riding and Dr Matthew Pound for their critical comments and constructive suggestions, all of which helped us improve the manuscript significantly.