The Paraphyly of Osmunda is Confirmed by Phylogenetic Analyses of Seven Plastid Loci

To resolve phylogenetic relationships among all genera and subgenera in Osmundaceae, we analyzed over 8,500 characters of DNA sequence data from seven plastid loci (atpA, rbcL, rbcL–accD, rbcL–atpB, rps4–trnS, trnG–trnR, and trnL–trnF). Our results confirm those from earlier anatomical and single-gene (rbcL) studies that suggested Osmunda s.l. is paraphyletic. Osmunda cinnamomea is sister to the remainder of Osmundaceae (Leptopteris, Todea, and Osmunda s.s.). We support the recognition of a monotypic fourth genus, Osmundastrum, to reflect these results. We also resolve subgeneric relationships within Osmunda s.s. and find that subg. Claytosmunda is strongly supported as sister to the rest of Osmunda. A stable, well-supported classification for extant Osmundaceae is proposed, along with a key to all genera and subgenera. Keywords—ferns, Osmunda, Osmundaceae, Osmundastrum, paraphyly, plastid DNA. Osmundales is the smallest but most ancient order of leptosporangiate ferns and occupies an important phylogenetic position as sister to all other extant leptosporangiates (Hasebe et al. 1995; Pryer et al. 2004; Schuettpelz and Pryer 2007). Numerous fossil representatives are known from the Permian onwards (Tidwell and Ash 1994) and fossil representatives of Osmunda L. are known since the Triassic (Phipps et al. 1998; Vavrek et al. 2006). The single extant family Osmundaceae is characterized by rhizomes with a highly distinctive anatomy in transverse section that is consistent across the family and unique for ferns (Hewitson 1962; see Fig. 13-20 on pg. 267 in Gifford and Foster 1989), sporangia that are not organized into sori, green spores, and a unique suite of reproductive characters that appears intermediate between eusporangiate and leptosporangiate ferns (Kramer 1990). Osmundaceae sporangia develop from multiple initial cells and produce hundreds of spores, both traits associated with eusporangiate fern lineages (Bierhorst 1971; Ogura 1972). The sporangia also have a rudimentary patchlike annulus that causes longitudinal dehiscence, distinct from all other annulus morphologies present in leptosporangiate ferns (Bierhorst 1971). Osmundaceae is commonly thought to comprise three extant genera: Osmunda, Leptopteris C. Presl, and Todea Willd. ex Bernh. (Kramer 1990; Smith et al. 2006). Leptopteris and Todea share many characters, including monomorphic leaves and sporangia that follow veins on uncontracted fertile pinnae (Kramer 1990), but the two genera are readily distinguished. Leptopteris, with about six species, has filmy leaves that lack stomata and sporangia sparsely arranged on the abaxial surface; Todea, with two species, has coriaceous leaves with stomata and sporangia densely covering the abaxial surface (Hennipman 1968; Brownsey 1981). Osmunda has been distinguished from these two genera by its contracted fertile pinnae and contains eight to nine species that have been recognized in three subgenera (Kramer 1990): subg. Osmunda L. with three species, subg. Osmundastrum (C. Presl) C. Presl with two species, and subg. Plenasium (C. Presl) J. Smith with three to four species. Although most authors define Osmunda in this manner, there have been indications that the genus may not be monophyletic. Anatomical studies of extant and fossil species of subg. Osmundastrum by Miller (1967, 1971) led him to conclude that O. cinnamomea was not closely related to the rest of Osmunda and that O. claytoniana should be transferred to subg. Osmunda. Miller (1967, 1971) also recommended that all three subgenera be elevated to generic level, as previously suggested by other authors (Tagawa 1941; Bobrov 1967). Using this taxonomic approach, the genus Osmundastrum C. Presl would include one extant and one fossil species (Miller 1967). Miller’s suggestions were not widely accepted and most subsequent studies did not adopt Osmundastrum sensu Miller (e.g. Stein and Thompson 1975; Sobel and Whalen 1983; Li and Haufler 1994). An rbcL study of Osmundaceae found O. cinnamomea to be sister to the rest of Osmundaceae, including Leptopteris and Todea (Yatabe et al. 1999). This single gene study also found O. claytoniana sister to a clade containing subg. Plenasium and subg. Osmunda. Although this relationship was not well supported, Yatabe et al. (2005) proposed a new subgenus, Claytosmunda Yatabe, Murakami & Iwatsuki, to accommodate the putative phylogenetic position of O. claytoniana. The most recent classification for Osmundaceae recognizes four genera (Yatabe et al. 2005): Osmundastrum, Todea, Leptopteris, and Osmunda with its three subgenera (Claytosmunda, Plenasium, and Osmunda). In the current study, we reconstruct the first multilocus phylogeny for Osmundaceae, assess relationships within and among all genera and subgenera, and seek to settle the taxonomic and nomenclatural uncertainty that surrounds Osmunda. Our highly resolved phylogeny allows us to evaluate previous classifications and recommend which, if any, should be followed. MATERIALS AND METHODS Taxon Sampling—We sampled 24 accessions representing 13 ingroup and four outgroup species (Appendix 1). Ingroup sampling included all described extant genera and subgenera of Osmundaceae, with multiple accessions for four species to assess intraspecific and geographic variation. We selected outgroup taxa from the gleichenioid lineage based on its phylogenetic proximity to Osmundaceae (Pryer et al. 2004). DNA Isolation, Amplification, and Sequencing—DNA extraction, amplification, sequencing and assembly for seven plastid loci (atpA, atpB– rbcL, rbcL–accD, rbcL, rps4–trnS, trnG–trnR, and trnL–trnF) followed esSystematic Botany (2008), 33(1): pp. 31–36 © Copyright 2008 by the American Society of Plant Taxonomists