ARTICLE IN PRESS Flora 205 (2010) 221–228 Contents lists available at ScienceDirect Flora journal homepage: www.elsevier.de/flora Palynological data on Illiciaceae and Schisandraceae 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 Austrobaileyales 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 Illiciales the current study, pollen grains of 21 taxa from Illiciaceae and Schisandraceae were investigated. These Illicium Linn. data, together with palynological data for taxa previously studied, were mapped into recent molecular Schisandra Michx. phylogenetic trees to re-evaluate the existing classification and phylogenetic relationships in the two Kadsura 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 species 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 tree 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, Trimeniaceae 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 Austrobaileya 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 magnoliids, 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  objective lens (total magni- and Kapil (1964), Huang (1972), Walker (1974), Vijayaraghavan fication: 100  ), 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,