Plant Syst Evol (2012) 298:1437–1453 DOI 10.1007/s00606-012-0648-y ORIGINAL ARTICLE Variation of pollen morphology, and its implications in the phylogeny of Clematis (Ranunculaceae) Lei Xie • Liang-Qian Li Received: 13 February 2012 / Accepted: 25 April 2012 / Published online: 26 May 2012 Ó Springer-Verlag 2012 Abstract Clematis s.l. (including Archiclematis and supported. Though pollen morphology may contribute to Naravelia) is a genus of approximately 300 species with investigation of problematic taxa, the taxonomic value of cosmopolitan distribution. The diversity of its pollen was pollen morphology is limited at the species level. surveyed in 162 taxa belonging to all infrageneric groups of Clematis s.l. Pollen morphology was investigated by Keywords Archiclematis Á Clematis Á Naravelia Á use of scanning electron microscopy to identify useful Phylogeny Á Pollen morphology Á Ranunculaceae Á characters, test taxonomic and systematic hypotheses, Scanning electron microscopy and elucidate pollen character evolution on the basis of the molecular phylogeny. Clematis pollen is small to medium (14.8–32.1 lm 9 14.2–28.7 lm), oblate to pro- Introduction late (P/E = 0.9–1.4) in shape. The apertures may be tricolpate and pantoporate sometimes with 4-zonocolpate The genus Clematis L., with approximately 280–350 spe- and pantocolpate pollen grains as transitional forms. The cies, is one of the largest genera in Ranunculaceae. This tricolpate pollen grains are predominant and occur in all cosmopolitan genus comprises climbing lianas, small the sections of the genus, whereas pantoporate pollen shrubs, and erect sub-shrubs distributed predominantly in grains can be found in sect. Tubulosae, sect. Viorna, sect. the temperate zone of both hemispheres but with some Viticella, and Naravelia only. Phylogenetic mapping of species distributed in tropical areas (Tamura 1995; Johnson aperture types reveals that the pantoporate pollen type may 1997; Grey-Wilson 2000; Wang and Li 2005). The genus is be the apomorphy in the genus and evolved several times. extremely diverse in temperate and subtropical regions of The surface ornamentation in all taxa studied is similar and the Northern Hemisphere, especially in eastern Asia, with characterized by microechinae evenly distributed on the 147 species reported in China, 93 of which are endemic to microperforate tectum. The size and density of spinules on the country (Wang and Bartholomew 2001). Many Cle- the tectum vary greatly but successive in the whole genus. matis species are of horticultural interest (e.g. C. montana, According to the character syndromes of the ornamenta- C. patens, and C. viorna), and some others are regarded as tion, separating sect. Brachiata from sect. Meclatis is pharmaceutically important (e.g. C. chinensis, C. henryi, and C. armandii). According to Tamura (1991), Clematis belongs to sub- L. Xie tribe Clematidinae Lotsy (including Clematis, Naravelia, College of Biological Sciences and Biotechnology, and Archiclematis) in the tribe Anemoneae DC. Molecular Beijing Forestry University, Beijing 100083, China e-mail: [email protected] phylogenetic studies support the monophyly of both the tribe Anemoneae and the subtribe Clematidinae (Johansson L.-Q. Li (&) and Jansen 1993; Johansson 1995; Hoot 1995; Ro et al. State Key Laboratory of Systematic and Evolutionary Botany, 1997; Wang et al. 2009). However, the genus Clematis had Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, China been defined differently by different authors. For example, e-mail: [email protected] the genus Archiclematis was recognized by Tamura (1987, 123 1438 L. Xie, L.-Q. Li 1991, 1995), whereas Johnson (1997), Grey-Wilson (2000), Recent molecular phylogenetic studies (Miikeda et al. and Wang and Li (2005) reduced it into Clematis. Another 2006; Xie et al. 2011) showed that the previously recog- example is Naravelia, which was often accepted as a nized genera Archiclematis and Naravelia are nested separate genus (Tamura 1995; Wang and Li 2005), but within Clematis and most subgenera and sections defined Johnson (1997) merged it into Clematis as sect. Naravelia. by morphological characters (Tamura 1995; Wang and Li Recent molecular phylogenetic studies (Miikeda et al. 2005) are not supported. The newly established phyloge- 2006; Xie et al. 2011) support the widest circumscription netic framework enables the examination of pollen char- (Clematis s.l.) of the genus defined by Johnson (1997), acters from an evolutionary perspective. The purpose of which including Archiclematis and Naravelia in Clematis. this study was to: The taxonomy of Clematis is regarded as difficult 1. survey the diversity of pollen morphology across the because Clematis species are morphologically highly var- genus by use of SEM; iable (Tamura 1995; Johnson 1997; Brandenburg 2000; 2. place palynologic variation into an evolutionary con- Grey-Wilson 2000; Wang and Li 2005). Classifications of text; and the genus differ from each other, mainly because of dif- 3. assess the usefulness of pollen morphology for ferent morphological characters emphasized in each sys- systematic study of Clematis. tem. In addition to recent taxonomic revisions (Tamura 1995; Johnson 1997; Brandenburg 2000; Grey-Wilson For Clematis s.str., the classification by Tamura (1995) 2000; Wang and Li 2005), systematic studies based on was followed in this study. anatomy, seedling morphology, palynology, cytology, and molecular phylogeny have also been conducted (Tobe 1974, 1980a, b; Essig 1991; Zhang 1991; Yano 1993; Materials and methods Miikeda et al. 1999, 2006; Yang and Moore 1999; Shi and Li 2003; Xie et al. 2011). Among these, pollen morphology In total, 162 taxa, including 145 species and 13 varieties had potential systematic value within the genus, because of from Clematis, one species from Archiclematis, and three wide variation in aperture size, configuration, and number from Naravelia, were examined (Appendix). Dried her- (Tobe 1974; Nowicke and Skvarla 1995). barium material was used for all the sampled species. The Pollen morphology of a small number of Clematis spe- sampling covered all the currently recognized subgenera cies has been reported in some studies of the palynologic and sections (sensu Tamura 1995) throughout all the dis- characters of Ranunculaceae (Wodehouse 1935, 1936; tribution areas of Clematis. Efforts were made to sample Kumazawa 1936; Erdtman 1952; Petrov and Ivanova 1975; multiple accessions, particularly for those taxa spanning Nowicke and Skvarla 1995; Santisuk 1979; Al-Eisawi large biogeographical ranges or having a diverse mor- 1986; Clarke et al. 1991; Blackmore et al. 1995). Several phology, to observe potential infraspecific variation. palynologic studies specifically on Clematis have also been Pollen samples of some Clematis species, e.g. C. ake- conducted, mainly using light microscopy (LM) (Ikuse bioides, C. pinnata, and C. henryi, are fragile and the 1956; Vishnu-Mittre and Sharma 1963; Nair 1965; Tobe acetolysis method for pollen preparation (Erdtman 1960)is 1974; Petrov and Ivanova 1975; Kapoor et al. 1989; Yang too drastic and damages the pollen grains for SEM obser- and Huang 1992; Yano 1993; Yang and Moore 1999). vation (Hesse et al. 2009). Thus, in this study mature Kumazawa (1936) reported pollen morphology of 28 taxa anthers were fragmented and mounted directly on the stubs of Clematis by use of LM. Three pollen types were rec- with double-sided adhesive tape without acetolysis to ognized, summarized, and illustrated in the study. Kapoor preserve the exine and intine. This treatment for SEM et al. (1989) observed the pollen grains of 32 taxa of observation was applied by Liu et al. (2010, 2011), Clematis and one species of Naravelia representing the Polevova et al. (2010), Welsh et al. (2010), among others. South Asian species of the genus by use of LM. Three- The samples were gold-coated and examined by use of an zono-colpate pollen grains were observed in most species Hitachi S-800 SEM. and only C. cadmia was reported to have panporate pollen Morphological features of pollen grains were observed grains. Nowicke and Skvarla (1995) sampled 13 Clematis for each species and were analyzed to describe and cate- species and examined pollen morphology by use of scan- gorize pollen types. Measurements were based on 20 pollen ning electronic microscopy (SEM). They observed three grains, the values of P (polar axis length), E (equatorial aperture types within Clematis, tricolpate, pantoporate, and diameter), spinule height, and density were measured and pantocolpate. However, until now, there has been no the P/E ratio was calculated. Statistics of the palynologic comprehensive, systematic survey of Clematis pollen, and characters were calculated by use of PAST 2.14 (Hammer there is still a lack of knowledge about pollen morphology et al. 2001). Descriptive terminology follows Clarke et al. of most species of Clematis, especially using SEM. (1991), Hesse et al. (2009), and Punt et al. (2007). 123 Variation of pollen morphology, and its implications in the phylogeny of Clematis (Ranunculaceae) 1439 To evaluate character evolution, aperture types were Evolution of aperture types mapped on to a simplified strict consensus tree of Clematis built in Mesquite 2.7 (Maddison and Maddison 2011), Although a molecular phylogenetic study by Xie et al. resulting from the phylogenetic analyses based on three (2011) did not generate a robust phylogenetic framework plastid (atpB-rbcL spacer, psbA-trnH-trnQ spacer, and for Clematis,