bs_bs_banner Botanical Journal of the Linnean Society, 2012, 169, 461–492. With 2 figures Molecular phylogenetic analysis of Podostemaceae: implications for taxonomy of major groups SATOSHI KOI1*, YOKO KITA2, YUMIKO HIRAYAMA1, ROLF RUTISHAUSER3, KONRAD A. HUBER3 and MASAHIRO KATO1 1Department of Botany, National Museum of Nature and Science, Tsukuba 305-0005, Japan 2Department of Biological Sciences, University of Tokyo, Hongo, Tokyo 113-0033, Japan 3Institute for Systematic Botany, University of Zurich, CH-8008 Zurich, Switzerland Received 23 August 2011; revised 15 January 2012; accepted for publication 27 March 2012 The river-weed family Podostemaceae (c. 300 species in c. 54 genera) shows a number of morphological innovations to be adapted to its unusual aquatic habitat, and its unique or rare bauplan features have been reflected in the traditional (i.e. non-molecular) classification recognizing numerous monotypic or oligospecific genera. The infra- subfamilial relationships of many genera remained unclear. The present study used molecular phylogenetic analysis of matK sequences for 657 samples (c. 132 species/c. 43 genera). The family was traditionally divided into three subfamilies (Podostemoideae, Tristichoideae and Weddellinoideae). American Podostemoideae were shown to be polyphyletic and divided into four clades, i.e. Ceratolacis, Diamantina, Podostemum and all other genera. Among the podostemoid clades, Diamantina was the first branching clade and a clade comprising Mourera and the Apinagia subclade was then sister to the remainder of the New World and Old World Podostemoideae with low statistic supports. The Old World Podostemoideae comprised four monophyletic clades, i.e. two African clades, one Madagascan clade and one Asian clade, although the relationships among these clades and American Ceratolacis and Podostemum were poorly resolved. African Podostemoideae were polyphyletic, with Saxicolella pro parte being weakly supported as sister to the remaining Old World Podostemoideae plus Ceratolacis and Podostemum.In contrast to the American and African clades, monophyly of four Asian subclades was well supported. Plants of Tristicha (Tristichoideae) and of Weddellina (Weddellinoideae), which are currently treated as monospecific, had great matK differentiation equivalent to at least interspecific variation. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 461–492. ADDITIONAL KEYWORDS: biogeography – clusioid Malpighiales – matK – molecular evolution. INTRODUCTION The body plans of Podostemaceae exhibit great diver- sity. For example, root morphology ranges from sub- Podostemaceae (river-weeds) are an ecologically and cylindrical to flattened ribbon-like and to crustose morphologically unusual aquatic angiosperm family. (foliose), and shoots are highly diverse with respect The plants live in rapids and waterfalls in the Tropics to size, proportion of stem and leaf and branching and the Subtropics. The vegetative plants of most (Rutishauser, 1997). Some species are devoid of roots species are composed of shoot with roots firmly adher- (Rutishauser & Grubert, 1994, 1999). Podostemaceae ing to water-worn rock surfaces. The plants grow comprise c. 280 species classified in 49 genera, of vegetatively underwater in the rainy season. The which 26 genera are monospecific, many genera plants flower and fruit during the dry season when contain < 10 species, and only a few genera consist of the water level drops and, finally, they wither and die. ten species or more (Cook & Rutishauser, 2007). Thus, the obvious morphological differences between *Corresponding author. Current address: Department the taxa were traditionally taken as significant of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0192, enough for creating many small (especially mono- Japan. E-mail: [email protected] typic) genera instead of accepting few large ones. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 461–492 461 462 S. KOI ET AL. The phylogenetic position of Podostemaceae among deduced from matK sequences of all available angiosperms was controversial until recently and has taxa, including some undescribed taxa (in total 657 been resolved now by several molecular phylogenetic samples). Based on the phylogenetic tree obtained, analyses. Studies with large data sets show that we discuss the infrasubfamilial classification of Podostemaceae are members of the clusioid Malpighi- Podostemaceae. Regional (e.g. African and Asian) ales (within fabids = eurosids I), with Hypericaceae taxonomic revisions and biogeography will be pub- as their sister family (Savolainen et al., 2000; Soltis lished in separate papers. et al., 2000, 2011; Gustafsson, Bittrich & Stevens, 2002; Davis et al., 2005; Tokuoka & Tobe, 2006; APG III, 2009; Korotkova et al., 2009; Wurdack & Davis, MATERIAL AND METHODS 2009; Ruhfel et al., 2011). PLANT SAMPLES The relationships in Podostemaceae have been A total of 365 samples (c. 82 species/c. 33 genera) of analysed for various groups and geographical regions. Podostemaceae, two samples (two species/one genus) They are useful to understand the phylogeny, classi- of Hypericaceae and one sample of Calophyllaceae fication, biogeography and morphological evolution of were collected from the field (Appendix 1). They were Podostemaceae as a whole (Kita & Kato, 2001, 2004a, dried in silica gel. Vouchers are deposited in: the b; Kato, Kita & Koi, 2003; Moline et al., 2006, 2007; Herbarium (TNS), Department of Botany, National Koi, Kita & Kato, 2008; Koi et al., 2009; Pfeifer et al., Museum of Nature and Science (Tsukuba, Japan); 2009; Thiv et al., 2009; Kelly, Ameka & Chase, 2010; Forest Herbarium (BKF), Department of National Koi & Kato, 2010a; Ruhfel et al., 2011; Tippery et al., Parks, Wildlife and Plant Conservation (Bangkok, 2011). In the earliest molecular phylogenetic study, Thailand); Herbarium (TI), University of Tokyo with plastid matK sequences of 31 worldwide samples (Tokyo, Japan); and Herbarium (TAIF), Taiwan For- of 29 species assigned to 20 genera except African estry Research Institute. Duplicates of many vouch- species, Kita & Kato (2001) suggested that the family ers from Africa and America will be deposited in the is divided into three subfamilies, Podostemoideae, combined herbaria of the University and ETH Zürich Weddellinoideae and Tristichoideae, as proposed by (Z/ZT). Engler (1930), the former two being sister to each other. Podostemoideae, which were the largest sub- family in their tree, comprised paraphyletic American DNA EXTRACTION, AMPLIFICATION AND SEQUENCING clades and a monophyletic Madagascan and a mono- Extraction of total DNA from dried material was phyletic Asian clade. Moline et al. (2007) analysed performed with the DNeasy Plant Mini Kit (Qiagen, eight species of three African genera and combined Valencia, CA, USA). The plastid matK region was their analyses with the data of Kita & Kato (2001), amplified via polymerase chain reaction (PCR) using showing the monophyly of the taxa from continental Ampdirect plus (Shimadzu, Kyoto, Japan) and Africa. Tippery et al. (2011) analysed internal tran- TaKaRa Ex Taq polymerase (TaKaRa, Tokyo, Japan) scribed spacer (ITS), rbcL and trnL regions from 38 under the following conditions: 3 min at 94 °C; 35 taxa of 15 genera, with the result that the Neotropical cycles of 30 s at 94 °C, 30 s at 55 °C, 90 s at 72 °C; and Podostemoideae, except Podostemum, are divided into 7 min at 72 °C. The primers used for the DNA ampli- ten clades. A recent large-scale analysis by Ruhfel fication and the cycle sequencing are listed in Appen- et al. (2011), using plastid matK, ndhF and rbcL and dix 2. The PCR products were treated with ExoSap-IT mitochondrial matR genes of 49 species of 35 genera, (GE Healthcare, Cleveland, OH, USA) to remove the showed the monophyly of the African, Madagascan extra primers. Sequencing was conducted using the and Asian Podostemoideae, and the polyphyly of the BigDye Terminator v3.1 Cycle Sequencing Kit American Podostemoideae. Most molecular phyloge- (Applied Biosystems, Foster City, CA, USA) and the netic trees suggested that some large genera, for ABI 3130xl Genetic Analyser (Applied Biosystems). example, American Apinagia Tul. and African Leder- manniella Engl. sensu Cusset (1983, 1984), are poly- phyletic. Based on those phylogenetic relationships, PHYLOGENETIC ANALYSES studies of taxonomic reclassification are in progress, Phylogenetic analysis was performed with the resulting in the recognition of c. 300 species assigned sequences obtained in this study (365 samples) and to c. 54 genera (Table 1; e.g. Kato, 2004, 2006a; Kato from GenBank (292 samples), in total 657 samples & Koi, 2009; Bove & Philbrick, 2010; Koi & Kato, from c. 132 species of c. 43 genera of Podostemaceae 2010a; Philbrick, Bove & Stevens, 2010; Ruhfel et al., and five samples (five species/four genera/three fami- 2011; Tippery et al., 2011). lies) of Malpighiales (Appendix 1; Kita & Kato, 2001, This paper describes phylogenetic relationships of 2004a, b; Kato et al., 2003; Davis & Wurdack, 2004; major lineages in the subfamilies of Podostemaceae, Moline et al., 2007; Koi et al., 2008, 2009; Thiv et al., © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 461–492 © 2012 The Linnean Society of London, Table 1. Current taxonomy of the genera and number of species of Podostemaceae, based on Cook & Rutishauser (2007) and others listed below