ARTICLE IN PRESS

Flora 205 (2010) 221–228

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Flora

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Palynological data on and confirm phylogenetic relationships within these two basally-branching angiosperm families

Hong Wang a,b,Ã, Hua-Jie He b, Jian-Qun Chen a,L.Lub a School of Life Science, Nanjing University, Nanjing 210093, China b Key Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650204, China article info abstract

Article history: Illiciaceae and Schisandraceae, together with other members of have been identified as Received 11 September 2008 one of the earliest diverging lineages of angiosperms, within the ANITA grade. The specialized Illiciaceae Accepted 26 February 2009 and Schisandraceae comprise a clade defined by apomorphic characters including pollen grains with three or six colpate apertures. In both these families, pollen apertures and exine sculpture were found to Keywords: be very informative when considered in the context of recent understanding of evolutionary patterns. In the current study, pollen grains of 21 taxa from Illiciaceae and Schisandraceae were investigated. These Linn. data, together with palynological data for taxa previously studied, were mapped into recent molecular Michx. phylogenetic to re-evaluate the existing classification and phylogenetic relationships in the two Kaemp f. ex Juss. families. Palynological data were found to be relatively congruent with recent molecular phylogenies, Palynological characters while traditional delimitations of infra-generic taxa were somewhat conflicting and did not reflect Phylogeny phylogeny and evolution. The evolution of pollen morphology in the two families, together with other members of Austrobaileyales, is discussed in comparison with the molecular phylogenies. & 2009 Elsevier GmbH. All rights reserved.

Introduction framework for 15 Illicium using ITS sequence data. Subsequently, Oh et al. (2003) mapped morphological characters, Illiciaceae and Schisandraceae, two small east Asian–eastern including floral, seed and leaf epidermal characters into the North American disjunct families, have traditionally been placed molecular of Hao et al. (2000), and called into question the in the order Illiciales (Cronquist, 1981; Takhtajan, 1969). Recent traditional subdivisions of the genus Illicium. molecular studies have identified five groups of basal angios- The family Schisandraceae includes two genera, Schisandra and perms, Amborella, Nymphaeales, Illiciales, and Kadsura, with 23 and 16 species, respectively (Saunders, 1998, Austrobaileyaceae (ANITA) as the earliest diverging lineages of 2000). The members of this family are mainly distributed in Asia, angiosperms (APGII, 2003; Bakman et al., 2000; Qiu et al., 1999, with one species of Schisandra native to the southeastern United 2000; Soltis et al., 1997; Savolainen et al., 2000; Soltis and Soltis, States (Saunders, 2000) and Mexico (Panero and Aranda, 1998). 2004; Zanis et al., 2002). Furthermore, Illiciales along with Infra-generic classifications in Schisandraceae are unstable and Trimenia and formed a well-supported clade (the vary between authors (Law, 1996; Saunders, 1998, 2000; Smith, so-called ITA grade; Qiu et al., 2001), which has been named 1947), while phylogenetic relationships and the boundaries Austrobaileyales. The family Illiciaceae consists of a single genus, among some species remain to be resolved. However, recent Illicium, with around 35 species. Most species are primarily molecular studies concluded that neither Schisandra nor Kadsura distributed in eastern Asia, but five species occur in the south- is monophyletic (Hao et al., 2001; Liu et al., 2000, 2006). Denk and eastern United States, West Indies and Mexico (Oh et al., 2003). Oh (2006) studied seed morphology and leaf epidermal characters Based on conspicuous floral characters, Smith (1947) classified of 22 species of Schisandraceae, and found that the division of the Illicium into two sections, i.e. sects. Illicium and Cymbostemon family into Schisandra and Kadsura based on fruit morphological (raised to subgenera by Law, 1996). However, the New World characters was not supported. species fell into both sections; thus the relationships among In general, palynological data can provide valuable evidence species remain obscure. Hao et al. (2000) proposed a phylogenetic for circumscribing closely related families and genera. While summarizing pollen diversity in some modern , Sampson (2000) characterized palynological features of Illiciales à Corresponding author at: School of Life Science, Nanjing University, Key and reached the conclusion that the two families Illiciaceae and Laboratory of Biodiversity and Biogeography, Kunming Institute of Botany, Nanjing210093, China. Schisandraceae were well-defined. Both families have pollen E-mail address: [email protected] (H. Wang). grains with three or six colpi, making them distinctive from other

0367-2530/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2009.02.004 ARTICLE IN PRESS

222 H. Wang et al. / Flora 205 (2010) 221–228 magnoliids. The members of the two families also share similar the trees of the two families. We also discuss the evolution of reticulate exine sculpture, but the sequences of development of pollen morphology of the two families, together with the other the exine components are distinctly different in the two families members of ITA grade, in comparison with the molecular and could provide some significant information (Gabarayeva and phylogeny. Grigorjeva, 2003; Takahashi, 1994; Ueno, 1962; Walker, 1976). Palynology of three Illicium species was studied for the first time by Wodehouse (1935), who recognized that the pollen grains Materials and methods have tricolpate apertures. Erdtman (1952) suggested that Illicium pollen was tricolpoid. Subsequently, Huang (1972), Lin (1989) and Pollen morphology of 21 taxa representing the single genus the others examined pollen grains of some species in the genus. Illicium and the two genera Schisandra and Kadsura within Previous studies showed them to be trisyncolpate and tricolpate Illiciaceae and Schisandraceae, was selected for study and pollen grains with reticulate surface sculpture in Illicium. How- illustration with LM and scanning electron microscopy (SEM). ever, Liu and Yang (1989) investigated the pollen morphology of Pollen samples were taken from specimens at the herbaria of the 16 species and suggested that some other transitional aperture Kunming Institute of Botany (KUN) and Yunnan University (HYU; types may exist in the genus. Table 1). Pollen grains of some species in Schisandraceae were earlier For LM investigation, pollen grains were treated by acetolysis described by Erdtman (1935) and Wodehouse (1936), who noted (Erdtman, 1960) and mounted on glycerine jelly. Size measure- that their heteropolar pollen grains are unique, as is the ments were based on 20 pollen grains; the length of the polar axis arrangement of their colpi, which is tri- and hexa-colpate. They (P) and equatorial axis (E) or greatest diameter (GD) was suggested that the polar area where the three longer colpi measured, and the P/E ratio (for isopolar pollen) or P/GD ratio converge is the proximal pole, while the blank area present (for heteropolar pollen) was calculated. For each sample, the exine opposite is the distal pole. The pollen of this family has attracted sculpture, represented by the lumen size (LS), and muri width the attention of other researchers, including Hayashi (1960), Jalan (MW), was measured under a 40 Â objective lens (total magni- and Kapil (1964), Huang (1972), Walker (1974), Vijayaraghavan fication: 100 Â ), and the LS/MW ratio was also calculated. For and Dhar (1975), Praglowski (1976), Huynh (1976), Lan (1984), SEM, the acetolysed samples were mounted on glass cover slips Doyle et al. (1990), Sampson (2000), Sun (2000) and others. Based and attached to aluminium stubs. After sputter coating (with an on light microscopy (LM) and transmission electron microscopy Emscope SC500), pollen grains were observed under SEM (using a (TEM), Praglowski (1976) studied 18 representative species and KYKY-10000B, Science Instrument Company, Beijing) at 30 kV. one variety of Schisandra and 12 species of Kadsura following Micrographs were digitized using a Uniscan-O-2000 scanner classification of Smith (1947), and pointed out that the longer (Tsinghua Unisplendour Co. Ltd., Beijing). Descriptive terminology colpi ended in the distal pole. This was followed by most authors follows Punt et al. (2007). All voucher specimens for pollen including Erdtman himself (1952). However conflicting reports samples are given in Table 1. still remained (e.g. Lieux, 1980). To infer the character evolution of the pollen of Illiciaceae and Further extensive investigations of pollen morphology may Schisandraceae, based on the data from 21 currently studied and help to clarify the phylogenetic relationships of these families and 16 additional taxa from previous studies (Lan, 1984; Lin, 1989; Liu genera. In the current paper, we studied the pollen morphology of and Yang, 1989; Praglowski, 1976; Sun, 2000), pollen characters 21 taxa in Illiciaceae and Schisandraceae, of which seven taxa are were optimized on a reconstruction of recent phylogenetic studied for the first time (Illicium angustisepalum, llicium arbor- hypotheses for these taxa following Hao et al. (2000) and Liu escens, llicium burmanicum, llicium cambodianum, llicium fargesii, et al. (2006). In particular, aperture and exine sculpture were llicium tsaii and Schisandra elongata). We aimed to test whether plotted onto the molecular trees, using the computer program pollen data support the molecular phylogeny when mapped on MacClade version 4.07 (Maddison and Maddison, 2005).

Table 1 Voucher information for 21 taxa studied of Illiciaceae and Schisandraceae.

Taxon Voucher specimens Locality Herbaria

Illicium angustisepalum A.C. Smith L.-G. Lin 5737 Fujian, China KUN I. anisatum L. K. Kondo et al. 2037 Kyushu, Japan KUN I. arborescens Hayata T.-Y. Yang et al. 07874 Taiwan, China KUN I. burmanicum Wils. H. Sun et al. 2065 Tibet, China KUN I. cambodianum Hance Anonymous 160 Bogor, Cambodia KUN I. fargesii Finet & Gagnep. G.-M. Feng 73-272 Yunnan, China KUN I. floridanum Ellis F.H. Utech et al. 83-039 Louisiana, USA KUN I. henryi Diels L.-H. Liu 1771 Hunan, China KUN I. majus Hook. f. et Thoms. Yunnan University 83 Yunnan, China HYU I. pachyphyllum A.C. Smith The research team 1033 Guizhou, China KUN I. petelotii A.C. Smith Yunnan University 1200 Yunnan, China HYU I. simonsii A.C. Smith Q.-T. Zhang et al.86129 Yunnan, China KUN I. tsaii A.C. Smith T.-L. Min et al.428 Yunnan, China KUN

Schisandra chinensis Baill. Y.-C. Zhu et al.280 Liaoning, China KUN S. elongata Bl. Baill. C.-N. Chen et al.03330 Taiwan, China KUN S. grandiflora Stapf T.-P. Zhu et al. 933 Guizhou, China KUN S. henryi Clarke G.M. Feng 22375 Yunnan, China KUN S. lancifolia Stapf The China-UK team 0526 Yunnan, China KUN

Kadsura coccinea A.C. Smith Q.A. Wu 8131 Yunnan, China KUN K. heteroclite Craib. The research team 3070 Yunnan, China KUN K. japonica Dunal S.-J. Chen 14268 Guangxi, China KUN ARTICLE IN PRESS

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Results margins distinct. Colpi with or without a medium linear thickening. Ornamentation coarsely to finely reticulate, with low Based on LM and SEM observation, the 21 taxa examined or high muri, including the other three types, i.e. type B present in showed variation in pollen characters; the detailed information I. arborescens, I. floridanum and I. pachyphyllum; type C present in for these investigated taxa is shown in Table 2. I. cambodianum and I. petelotii and type D present in I. henryi and I. majus.

Pollen morphology of Illiciaceae and Schisandraceae Pollen morphology of Schisandraceae (Plates 2. 15–24) Pollen morphology of Illiciaceae (Plates 1 and 2, 13–14, 20) Pollen grains shed as monads; radially symmetrical, isopolar Pollen grains shed as monads; radially symmetrical, usually only in polar view, oblate to suboblate (P/E range: 0.70–0.85), heteropolar, oblate to suboblate (P/GD range: 0.57–0.88) with medium-sized, P 18–37 mm  E 24–48 mm. Amb often circular, distal poles broadened, medium-sized, P 12–28 mm  GD 22– slightly flattened or convex, triangular. Equatorial outline circular 39 mm. Amb often circular, slightly flattened. Equatorial outline or elliptical. Apertures tricolpate or trisyncolpate, with or without circular or elliptical. Apertures usually hexacolpate, occasionally fused furrows at both poles, usually with a thick ectexinous trisyncolpate, with or without fused furrows at poles, usually with membrane, visible in SEM. Exine sculpture reticulate, with or a thick ectexinous membrane, visible in SEM. Exine sculpturing without foveolae on the muri, lumina irregular and varying in reticulate, lumina irregular and varying in shape and size, larger at shape and size, larger at or near the equator, becoming gradually or near the equator, becoming gradually smaller towards each smaller towards each pole or colpus margin. Lumen size (SL) and pole or colpus margin. Colpi with a medium linear thickening. muri width (MW) 0.75–4.50 mm. Exine ornamentation mainly Lumen size (LS) and muri width (MW): 3.60–6.25 mm. Exine falling into the following four types: (A). finely reticulate with low ornamentation of only one type: type E, coarse reticulate with and foveolate muri (LS/MW range: 0.75–2.00), (B). coarsely large lumina. reticulate with low muri (LS/MW range: 2.26–2.60), (C). coarsely reticulate with high muri (3.00–3.20 mm) and (D). finely reticulate with high muri (3.60–4.50 mm). Pollen morphology of Schisandra Two aperture types can be distinguished as follows: Tricolpate aperture type (Plates 1.1–5, 10). Pollen grains oblate (P/GD range: 0.57–0.70) with distal poles Colpi usually wide (Plates 1.1–4) or narrow (Plates 1.1–5, 10) broadened. P 12–21 mm  GD 22–39 mm. Apertures hexacolpate or near the poles. Colpus membrane covered with granular, crustate trisyncolpate with or without fused furrows at the poles. Exine elements (Plates 1.1–3) or with a medium linear thickening. sculpturing reticulate. Lumen size (LS) and muri width (MW): Ornamentation reticulate to finely reticulate, with low and 3.80–6.25 mm. foveolate muri, belonging to type A. Present in: I. anisatum, Two aperture types can be distinguished as follows: I. angustisepalum, I. burmanicum, I. fargesii, I. simonsii and I. tsaii. Heteropolar with trisyncolpate aperture type (Plates 2.17, 18). Trisyncolpate aperture type (Plates 1.6–9, 11; Plate 2.14) Colpi long, extending to fuse at the distal pole. Colpi some- Colpi usually narrow (Plates 1.6, 8, 11, Plate 2.14), smooth and times not visible at the pole (or the blank pole). Present in sunken (Plate 1.12). Colpi long, extending to fuse at the poles, S. grandiflora.

Table 2 Pollen characters of taxa studied.

Taxon PEor GD P/E or Shape Apertures CW LS MW LS/MW Exine P/GD sculpture

Illicium angustisepalum 23–(25.5)–29 35–(38.2)–42 0.75 O- SO TC 0.4–(0.9)–1.6 0.7–(1.2)–2 .1 0.5–(0.6)–0.9 2.00 A I. anisatum 24–(25.4)–28 33–(36.5)–40 0.70 O TC 0.4–(0.7)–1.0 0.6–(1.1)–2.0 0.5–(0.7)–0.8 1.57 A I. arborescens 18–(24.3)–26 24–(33.3)–38 0.73 O TS 0.3–(0.4)–0.8 0.6–(1.3)–2.1 0.5–(0.6)–0.8 2.26 B I. burmanicum 23–(27.9)–29 31–(33.4)–43 0.83 SO TC 0.5–(1.0)–1.8 0.5–(1.0)–1.8 0.3–(0.5)–0.6 2.00 A I. cambodianum 29–(30.9)–37 40–(44.2)–48 0.70 O TS 0.3–(0.5)–0.9 0.6–(1.5)–3.1 0.4–(0.5)–0.7 3.00 C I. fargesii 28–(30.9)–34 36–(37.4)–43 0.83 SO TC 0.1–(0.3)–0.4 0.3–(1.0)–1.9 0.6–(0.9)–1.1 1.11 A I. floridanum 28–(30.3)–33 34–(35.5)–37 0.85 SO-S TS 0.2–(0.5)–1.2 0.4–(1.5)–2.1 0.5–(0.6)–0.8 2.50 B I. henryi 27–(30.6)–32 37–(38.8)–40 0.79 SO TS 0.3–(0.7)–1.1 0.5–(1.8)–2.2 0.4–(0.5)–0.8 3.60 D I. majus 27–(28.0)–30 32–(35.7)–40 0.79 SO TS 0.1–(0.2)–0.3 0.7–(1.8)–3.7 0.2–(0.4)–0.7 4.50 D I. pachyphyllum 21–(22.6)–25 27–(29.5)–33 0.77 O-SO TS 0.1–(0.3)–0.4 0.7–(1.3)–1.9 0.4–(0.5)–0.7 2.60 B I. petelotii 26–(29.2)–32 33–(39.9)–44 0.73 O-SO TS 0.2–(0.4)–0.6 0.6–(1.6)–2.3 0.4–(0.5)–0.7 3.20 C I. simonsii 26–(27.0)–33 34–(37.5)–38 0.72 O TC 0.3–(0.5)–0.6 0.7–(1.2)–1.8 0.9–(1.2)–1.4 1.00 A I. tsaii 27–(28.1)–32 33–(34.8)–38 0.81 SO TC 0.1–(0.3)–0.4 0.2–(0.9)–1.8 0.5–(1.2)-3.3 0.75 A

Schisandra chinensis 21–(23.1)–26 26–(33.2)–39 0.70 O HH 1.3–(1.6)–2.0 0.8–(2.5)–3.9 0.3–(0.4)–0.6 6.25 E S. elongata 15–(18.2)–20 23–(26.2)–28 0.69 O HH 1.2–(1.4)–1.8 0.6–(1.9)–3.3 0.4–(0.5)–0.7 3.80 E S. grandiflora 18–(19.6)–22 24–(27.0)–34 0.73 O DT 0.9–(1.3)–1.8 0.9–(2.7)–4.5 0.5–(0.7)–0.8 3.86 E S. henryi 12–(14.5)–16 22–(25.1)–30 0.57 O HH 1.3–(1.5)–1.8 0.1–(2.1)–3.6 0.4–(0.5)–0.6 4.20 E S. lancifolia 18–(19.1)–21 24–(28.8)–32 0.66 O HH 0.6–(1.0)–1.3 1.5–(3.0)–5.2 0.6–(0.7)–0.8 4.29 E

Kadsura coccinea 15–(16.8)–19 22–(24.8)–25 0.68 O HH 0.9–(1.2)–1.9 0.6–(1.8)–2.5 0.4–(0.5)–0.7 3.60 E K. heteroclite 22–(26.4)–28 25–(30.0)–31 0.88 SO HH 1.0–(1.5)–2.1 0.8–(1.9)–2.6 0.4–(0.5)–0.7 3.80 E K.. japonica 15–(17.8)–19 23–(26.1)–28 0.68 O HH 1.2–(1.6)–2.6 0.6–(1.8)–2.8 0.3–(0.4)–0.6 4.50 E

Shape: O=oblate (P/E or P/GD: 0.50–0.75); SO=suboblate (P/E or P/GD: 0.75–0.88); S=spheroidal (P/E: 0.88–1.14); aperture types: TS=trisyncolpate; TC=tricolpate, DT=distal-trisyncolpate; HH=heteropolar-hexacolpate; CW=colpus width; LS=lumen size; MW=muri width; exine sculpture types: type A=finely reticulate with low and foveolate muri; type B=coarsely reticulate with low muri; type C=coarsely reticulate with high muri; type D=finely reticulate with high muri; type E=coarsely reticulate with large lumina. All measurements are in mm. ARTICLE IN PRESS

224 H. Wang et al. / Flora 205 (2010) 221–228

Plate 1. Micrographs of pollen grains in Illicium, examined by SEM; scale bar 5 mm for whole pollen grains, 1 mm for higher magnification of pollen surface. (1) Illicium fargesii polar view; (2) I. angustisepalum polar view; (3) I. burmanicum polar view; (4) I. anisatum polar view; (5) I. simonsii polar view; (6, 8) I. floridanum polar view; (7) I. pachyphyllum polar view; (9) I. cambodianum polar view; (10) I. tsaii equatorial view; (11, 12) I. majus polar view. ARTICLE IN PRESS

H. Wang et al. / Flora 205 (2010) 221–228 225

Plate 2. Micrographs of pollen grains in Illicium, Schisandra and Kadsura species (13–19) examined by SEM; scale bar 5 mm for whole pollen grains, 1 mm for higher magnification of pollen surface; (20–24) Examined by LM. (13) Illicium arborescens polar view; (14) I. henryi equatorial view; (15) Schisandra lancifolia distal view; (16) Kadsura japonica distal view; (17, 18) S. grandiflora distal view; (19) S. elongata proximal view; (20) I. petelotii equatorial view; (21) K. heteroclita distal view; (22) S. henryi proximal view; (23, 24) K. coccinea (23) proximal view; (24) distal view. ARTICLE IN PRESS

226 H. Wang et al. / Flora 205 (2010) 221–228

Heteropolar with hexacolpate aperture type (Plates 2.15, 19, 22). Colpi arranged meridionally, three long and three short (Plate 1.22). Long colpi fused at the distal pole; short colpi not fused at the proximal pole. All six colpi ending around the area at the proximal pole (Plate 2.19). Present in S. chinensis, S. elongata, S. henryi and S. lancifolia.

Pollen morphology of Kadsura (Plates 2.16, 21, 23, 24)

Pollen grains oblate to suboblate (P/GD range: 0.68–0.88) with distal poles broadened. P 15–28 mm  GD 22–31 mm. Apertures hexacolpate with or without fused furrows at the poles. Exine sculpturing reticulate. Lumen size (LS) and muri width (MW): 3.60–4.50 mm. Only heteropolar with hexacolpate aperture type found. Present in K. coccinea, K. heteroclita and K. japonica.

Mapping pollen characters on the molecular trees (Figs. 1 and 2; Appendix A and B).

The pollen characters described in the current and previous studies were optimized and mapped on the molecular trees of Hao et al. (2000) and Liu et al. (2006). In the current study, eight of the taxa examined (I. burmanicum, I. cambodianum, I. pachyphyllum,

Fig. 2. Pollen characters of Schisandra and Kadsura species optimized on the molecular trnL-F and ITS tree of Liu et al. (2006).

I. petelotii, I. simonsii, I. tsaii, S. elongata and S. lancifolia)were unable to be used in this part of the study, because they were excluded from the molecular trees used. Of the taxa studied in genus Illicium (Fig. 1), the two North American species (I. floridanum and I. parviflorum) form clade I, and both species share trisyncolpate pollen grains, with a coarse reticulate surface and low muri. Except for three species (I. angustisepalum, I. fargesii and I. anisaturn, comprising clade V), all other species from East Asia share the synapomorphy of trisyncolpate pollen. Four of these species share similar exine sculpture with the two North American species and constitute clade IV. The members of clade II are supported by a shared coarse reticulate sculpture and high muri, whereas those of clade III have finely reticulate grains with high muri. Noticeably, the three species mentioned above comprising clade V have pollen, which is tricolpate and finely reticulate with foveolate, low muri. In the family Schisandraceae (Fig. 2), clade I contains a mixture of Schisandra and Kadsura species, while clade II is entirely composed of Schisandra species. All species studied, except S. grandiflora (with trisyncolpate pollen), share the synapomorphy of hexacolpate apertures. In addition, in clade I, the size of the lumina of two Schisandra species (S. plena and S. propinqua) is somewhat smaller than most of Schisandra species in clade II. The two other species (S. glabra and S. bicolor), being disjunct in Fig. 1. Pollen characters of Illicium species optimized on the molecular ITS tree of east Asia and eastern North America, have lumina smaller in size, Hao et al. (2000). and form a subclade. ARTICLE IN PRESS

H. Wang et al. / Flora 205 (2010) 221–228 227

Discussion Acknowledgements

In the genus Illicium, the distinction between sections Illicium We would like to thank the herbaria at the Kunming Institute and Cymbostemon based on segments is not paralleled by of Botany, Chinese Academy of Sciences (KUN) and the Yunnan differences in androecial or fruit characters (Saunders, 1995). University (HYU) for allowing us to collect pollen samples. We However, pollen characters, in particular aperture type, have been also thank Dr. A.H. Wortley, the Royal Botanic Garden Edinburgh, considered as one of the important criteria for the division of the Scotland, UK for reading a draft version of this paper, Dr. Q. Lin, the two sections (Erdtman, 1952; Wodehouse, 1959; Walker, 1976). Institute of Botany, Chinese Academy of Sciences, for confirming The current study, mapping pollen characters on molecular ITS all the taxa examined, and Mr. X.-K. Fan, the Kunming Institute of trees (Hao et al., 2000), shows a major dichotomy between the Botany, Chinese Academy of Sciences for SEM technical assistance. two North American species (I. floridanum and I. parviflorum) and This study was supported by grants from National Basic Research the remaining East Asian species. However, I. floridanum, which Program of China (973 Program, no. 2007CB411603). has trisyncolpate pollen, is placed with the tricolpate pollen members of sect. Illicium. Thus, the sects. Illicium and Cymboste- mon appear to be polyphyletic, and the pollen data do not support Appendix A. Pollen characters and data matrix for Illicium the infra-generic classification. On the other hand, the two North and Kadsura species American species have coarse reticulate sculptured pollen with low muri, showing synapomorphies shared with some East Taxon Aperture Exine sculpture L/MP/E Asian species. Furthermore, the three species from sect. Illicium bearing tricolpate and finely reticulate pollen consistently form a Illicium angustisepalum 00 31 monophyletic group. This result was also backed up by the I. anisatum 00 31 evidence from floral, seed and leaf epidermal structure by Oh et al. I. arborescens 11 21 (2003). I. difengpi 12 11 Morphology, such as fruit type and arrangement of flowers, has I. dunnianum 11 21 traditionally been used to reconstruct the phylogeny and evolu- I. fargesii 00 31 tion of the family Schisandraceae. In this study, based on the I. floridanum 11 22 recent molecular framework (Liu et al., 2006), pollen aperture I. henryi 13 01 type and size of lumen are used to test the phylogeny of I. lanceolatum 13 02 Schisandraceae. Both molecular and palynological data support I. majus 13 01 the fact that two Schisandra species (S. plena and S. propinqua)are I. micranthum 12 11 related to the members of Kadsura. Most taxa studied have I. parviflorum 11 22 hexacolpate pollen, but S. grandiflora differs both in floral I. spathulatum 11 21 morphology and in having trisyncolpate pollen, which may be I. verum 11 21 an autapomorphy. The pollen characters of most species studied Kadsura coccinea 24 00 show high convergence with molecular data, and did not support the monophyly of Schisandra and Kadsura. Furthermore the pollen Aperture: 1. tri-colpate (0), trisyncolpate (1), heteropolar-hexacolpate (2); 2. Exine characters appear to support sister relationships between some sculpture: type A (0), type B (1), type C (2), type D (3), type E (4); 3. Lumen/Murus: taxa, for example S. glabra and S. bicolor, both of which have 43.5 (0), 3–3.5 (1), 2–3 (2), o2 (3) and 4. P/E: o0.50 (0), 0.75–0.88 (1), 40.88 (2). relatively small lumen size and reticulate sculpture, as do S. plena and S. propinqua. All Kadsura species studied were found to have Appendix B. Pollen characters and data matrix for Schisandra, hexacolpate pollen. This is also in agreement with the molecular Kadsura and Illicium species tree (Liu et al., 2006). It has been known that monosulcate and inaperturate pollens occur commonly in basally-branching angiosperms, and mono- Taxon Aperture LS P/GD Pollen size sulcate pollen is the first angiosperm type to appear in the early Cretaceous fossil record (Doyle, 2005). In contrast, among the Schisandra glabra 2201 groups of the ITA grade, the single species of Austrobaileya has S. bicolor 2202 anasulcate pollen, while in Trimeniaceae few species have S. chinensis 2001 dimorphic pollen (inaperturate and polyforate), and other species S. grandiflora 1001 have either disulculate or polyporate pollen. It is considered that S. pubescens 2101 evolution seems to occur from disulculate to inaperturate to S. henryi 2101 polyporate pollen (Sampson, 2007). However, there are tri- and S. glaucescens 2101 hexa-colpate pollen types displaying a combination of characters S. viridis 2002 in Schisandraceae and partly also in Illiciaceae (Denk and Oh, S. sphenanthera 2002 2006) Furthermore, the pollen of both families is superficially Kadsura coccinea 2102 similar to the tricolpate pollen of the eudicots, but the colpus K. ananosma 2101 position is different from that in eudicots, the distal apertures are S. plena 2201 not strictly comparable with equatorial apertures. In particular, it S. propinqua 2201 is interesting to note that in some species of Schisandraceae the K. scandens 2101 additional three short apertures are positioned according to the K. heteroclite 2121 Fischer’s rule (Huynh, 1976). Otherwise, the colpi are joined at the K. japonica 2121 distal pole and oriented meridianly, while three additional colpi K. longipedunculata 2101 occur alternately in Kadsura and some Schisandra species. From Illicium fargesii 0210 comparison of these characters, we consider tri- and hexa-colpate Aperture: 1. Tri-colpate (0), distal-trisyncolpate (1), heteropolar-hexacolpate (2); 2. pollen grains may be derived independently in Schisandraceae – Lumen size (mm): 43 (0), 2–3 (1), o2 (2); 3. P/GD: o0.50 (0), 0.75–0.88 (1), Illiciaceae and eudicots. 40.88 (2); 4. Pollen size (mm): 435 (0), 25–35 (1), o25 (2). ARTICLE IN PRESS

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