Botanical Journal of the Linnean Society, 2010, 162, 28–38. With 1 figure A molecular phylogeny and a revised classification of tribe Lepisoreae (Polypodiaceae) based on an analysis of four plastid DNA regionsboj_1018 28..38 LI WANG1,2,3,4, ZHI-QIANG WU1,4, QIAO-PING XIANG1, JOCHEN HEINRICHS2, HARALD SCHNEIDER FLS2,3* and XIAN-CHUN ZHANG1* 1State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, the Chinese Academy of Sciences, Beijing 100093, China 2Albrecht-von-Haller Institute of Plant Sciences, Georg-August University Göttingen, Untere Karspüle 2, 37073 Göttingen, Germany 3Department of Botany, The Natural History Museum, London SW7 5BD, UK 4Graduate University of Chinese Academy of Sciences, Beijing 100049, China Received 12 September 2009; accept for publication 9 November 2009 Phylogenetic relationships within the palaeotropical tribe Lepisoroideae (Polypodiaceae) were investigated by studying sequence variation of four plastid DNA regions: rbcL, rps4 plus rps4-trnS IGS, trnL intron plus trnL-F IGS, rbcL-atpB IGS plus part of atpB. In total, over 4000 nucleotides were sequenced for 39 species. Seven well-supported clades were found in the analyses of the combined data set. We provide a new classification of Lepisoroideae by integrating phylogenetic results and known variation of morphological characters. The two small genera Neocheiropteris and Tricholepidium are supported as monophyletic, the genus Paragramma is resurrected and the genera Lepisorus, Neolepisorus, Lemmaphyllum and Lepidomicrosorium are re-circumscribed. We proposed 14 new combinations, among which Caobangia is treated as a synonym of Lemmaphyllum. A key for identifying the recognized genera is presented. © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 28–38. ADDITIONAL KEYWORDS: Caobangia – Lemmaphyllum – Lepidomicrosorium – Lepisorus – Neolepisorus – Paragramma – south-east Asia – taxonomic treatment. INTRODUCTION loxogrammoids (Kreier & Schneider, 2006b), micro- soroids (Schneider et al., 2006a; Kreier et al., 2008b), The application of DNA sequence data to the analysis platycerioids (Kreier & Schneider, 2006a) and various of phylogenetic relationships has led to major neotropical genera (Smith et al., 2006a; Schneider improvements in our understanding of intrafamilial et al., 2006b; Kreier et al., 2007, 2008a; Salino et al., relationships of the most species-rich fern family, 2008; Otto et al., 2009). Less attention has been paid Polypodiaceae. Schneider et al. (2004) resolved the to poorly understood, mainly south-east Asian lin- broad relationships within the family by reporting eages such as selligueoids and lepisoroids. To date, evidence for four main lineages. Successive studies lepisoroids have been studied only in the context focused on the relationships within selected lineages of deeper phylogenetic relationships such as the of Polypodiaceae, for example, drynaroids (Janssen & family Polypodiaceae (Schneider et al., 2004) and Schneider, 2005), grammitids (Ranker et al., 2004), the microsoroid clade (Kreier et al., 2008b). In both studies, the lepisoroids were found to be monophyletic *Corresponding authors. E-mail: [email protected]; and nested within the paraphyletic microsoroids. This [email protected] clade appeared to be nearly identical in its taxonomic 28 © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 28–38 PHYLOGENY WITHIN THE TRIBE LEPISOROIDEAE 29 breadth to tribe Lepisoreae as defined by Hennipman, Lemmaphyllum lineage are based on the occurrence Veldhoen & Kramer (1990), but with the inclusion of coenosori and leaf dimorphisms. However, these of several species treated by Nooteboom (1997, 1998) characters are prone to convergent evolution in Poly- as part of the microsoroid genus Microsorum Link podiaceae (Hovenkamp & Franken, 1993; Janssen & (Kreier et al., 2008b). Schneider, 2005). Considering conflicting interpreta- The lepisoroid ferns are distributed throughout con- tions of generic limits, the application of DNA tinental Asia, Australasia and Afromadagascar and sequences in a phylogenetic context was needed to constitute one of the most abundant and species-rich elucidate a more natural classification of this lineage. fern lineages in south-east Asia. The circumscription and classification of genera within this lineage are still poorly understood. Each author studying this group MATERIAL AND METHODS has suggested a different number of genera, although TAXONOMIC SAMPLING these various circumscriptions were based on the same A total of 34 described species, representing all cur- morphological evidence (Ching, 1978a, b; Hennipman rently recognized genera of lepisoroids, was included et al., 1990; Shi, 1999; Shi and Zhang, 1999; Smith in this study (Ching, 1978a, b; Ching & Wu, 1980; et al., 2006b). Hennipman et al. (1990) accepted only Ching & Shing, 1983a, b; Hennipman et al., 1990; four genera (Lepisorus (J.Sm.) Ching, Belvisia Mirb., Smith & Zhang, 2002). Five representatives belonging Drymotaenium Makino, and Lemmaphyllum C.Presl), to the genera Microsorum, Leptochilus C.Presl and whereas Ching and his students (Ching, 1978c; Ching Lecanopteris Reinw. were included as outgroups; & Wu, 1980; Ching & Shing, 1983a, b) accepted several these were selected on the basis of previous phyloge- small genera, including Lepidogrammitis Ching, Neol- netic studies (Kreier et al., 2008b; Schneider et al., episorus Ching, Tricholepidium Ching, Lepidomicroso- 2004). Voucher information for all included samples is rium Ching & K.H.Shing, and Platygyria Ching & given in the Supporting Information (Appendix). S.K.Wu. Ching treated Neocheiropteris Christ as belonging to the lepisoroids, whereas Nooteboom (1997, 1998) reduced the genus to a synonym of the DNA ISOLATION, AMPLIFICATION, AND SEQUENCING microsoroid genus Microsorum. Total genomic DNA was extracted from silica-gel- Recent phylogenetic studies (Schneider et al., 2004, dried leaves using the modified cetyl trimethyl ammo- Kreier et al., 2008b), however, rejected Nooteboom’s nium bromide (CTAB) procedure of Doyle & Doyle concept of Microsorum and found several species (1987). For each taxon, four plastid genome regions of Microsorum, for example, Microsorum fortunei (rbcL, rbcL-atpB, rps4+rps4-trnS, trnL-trnF) were (T.Moore) Ching, M. zippelii (Blume) Ching, M. amplified separately with standard polymerase chain pappei (Mett ex Kuhn) Tardieu and M. superficiale reaction (PCR) by using published primer sets: rbcL- (Blume) Ching, to have close relationships with Neol- atpB intergenic spacer (IGS) plus part of the atpB episorus or Neocheiropteris. These findings echoed coding region (http://www.pryerlab.net/), the rbcL Bosman’s interpretation of the relationships (1991) gene (Olmstead et al., 1992; Gastony & Rollo, 1995), but rejected Nooteboom’s treatment (1997). At the rps4+rps4-trnS IGS (Nadot et al., 1995; Smith & same time, these findings emphasized the need to Cranfill, 2002) and the trnL-trnF region including the redefine lepisoroid genera. trnL intron and the trnL-trnF IGS (Taberlet et al., The taxonomic ambiguity also extends to the genus 1991; Trewick et al., 2002). To simplify discussion, the Lemmaphyllum and its putative segregates. Hennip- following terms will be used: trnL-F for the trnL-trnF man et al. (1990) treated the monotypic genus region, rbcL-atpB IGS for rbcL-atpB IGS + parts of Weatherbya Copel. as a synonym of Lemmaphyllum. atpB, and rps4-trnS for rps4+rps4-trnS. Weatherbya accedens (Blume) Copel. (= Lemmaphyl- GFX™ PCR DNA and the Gel Band Purification lum accedens (Blume) Donk) is distinguished from Kit (Amersham Pharmacia Biotech, Piscataway, NJ, other species of Lemmaphyllum by the distinctive USA) were used to prepare the PCR products for shape of the fertile leaves (Copeland, 1947; Rahaman direct sequencing using the DYEnamic™ ETDye Ter- & Sen, 2000). Most recently, Smith & Zhang (2002) minator Cycle Sequencing Kit (Amersham Pharmacia described the monotypic genus Caobangia A.R.Sm. & Biotech) and the MegaBACE™1000 DNA Analysis X.C.Zhang, which is undoubtedly closely related to Systems, following the manufacturer’s protocols. species belonging to Lemmaphyllum, as sharing many Sequence data were edited and assembled in Con- morphological similarities, although differing in its tigExpress program from the Vector NTI Suite 6.0 distinctive dense indumentum on the leaves. The (Informax Inc., North Bethesda, MD, USA). The status of Ching’s genus Lepidogrammitis (Ching, resulting sequences were aligned using CLUSTAL X 1940) is another problematic issue. The segregation of with default settings (Thompson et al., 1997) and Lemmaphyllum and other entities belonging to the further adjusted manually in MacClade 4.0 (Maddi- © 2010 The Linnean Society of London, Botanical Journal of the Linnean Society, 2010, 162, 28–38 30 L. WANG ET AL. son & Maddison, 2002). Ambiguous positions were PHYLOGENETIC ANALYSES identified visually and excluded from all phylogenetic Maximum parsimony (MP) analyses of the four analyses. All sequences have been deposited in plastid DNA regions were conducted separately with GenBank (see Table 1 for accession numbers). the same settings as for the combined data matrix Table 1. Information regarding taxon names, collecting localities, collector, voucher deposition and GenBank accession numbers for sequences included in the phylogenetic analyses Taxon Voucher rbcL rbcL-atpB rps4-trnS trnL-F Leptochilus henryi (Baker) Ching China, Sichuan; Zhang