J Hattori Bot. Lab. No. 94: 87- 106 (Aug. 2003) PRELIMINARY PHYLOGENETIC ANALYSIS OF PYLAISIA (HYPNACEAE, MUSCI) AND ITS RELATIVES BASED ON rbcL GENE SEQUENCES 1 1 TOMOTSUGU ARlKAWA AND MASANOBU HIGUCHI ,2 ABSTRACT. Phylogenetic relationships among the species of Pylaisia and its relatives are investigat­ ed using nucleotide sequences of the chloroplast gene, rbeL. Nucleotide sequences of rbeL were de­ termined in fourteen samples (six species) of the genus Pylaisia, one sample of Giraldiella levieri, one sample of Platygyrium repens, and two samples of the Hookeriales as out groups. Phylogenetic trees were constructed by the maximum-parsimony (MP) method, neighbor-joining (NJ) method, and maximum-likelihood (ML) method; and these topologies were compared with each other depending on the ML criteria. The sequence comparison reveals that the sequence reported as Pylaisia polyan­ tha in our previous study is not that of P polyantha, but that of Platygyrium repens. The result of the present study indicates the following: (I) the genus Pylaisia looks homogeneous including Giraldiel­ la levieri, although it could not be concluded whether the genus Pylaisia is monophyletic because of insufficient information, (2) Platygyrium is placed in the Sematophyllaceae, and then the subfamily Pylaisioideae M.Fleisch. is polyphyletic, (3) P selwynii is phylogenetically distinguished from P brotheri, and (4) the populations of P polyantha from the Russian Far East are phylogenetically dis­ tinguished from North European ones. KEY WORDS: Pylaisia, Giraldiella, Platygyrium, Hypnaceae, rbeL, molecular phylogeny INTRODUCTION The Hypnaceae are one of the most diversified moss groups. There are many concepts for the delimitation, subdivision, and infrafamilial relationships of the family. Vitt (1984) suggested that the "pleurocarpous diplolepideae" are relatively young, and that the mosses with double costae, such as the Hypnaceae and the Sematophyllaceae, were derived from a group with single costa as represented by the Brachytheciaceae and Amblystegiaceae. The family Hypnaceae can be regarded as the most recent group evolutionarily. For instance, Nishimura et al. (1984) proposed the delimitation of the family based on a combination of the following characters: (1) paraphyllia absent, (2) costa double or ab­ sent, (3) alar cells of the leaf sometimes differentiated, but not vesiculate-inflated in a trans­ verse row except in a few species of Hypnum, (4) capsules inclined to horizontal or cernu­ ous, occasionary erect, (5) exothecial cells not collenchymatous, (6) operculum conic­ obutse to rostrate, not subulate with a few exceptions, and (7) exosxtome teeth usually with a zig-zag center line, and well-developed projecting lamellae and rather broadly bordered. I Department of Biological Sciences, Graduate School of Science, University of Tokyo, 7-3- 1 Hongo, Bunkyo-ku, Tokyo 113--0033, Japan. Present address of T. Arikawa: Department of Biologi­ cal Science, Graduate School of Science, Hiroshima University, Kagamiyama 1- 3- 1, Higashi­ hiroshima-shi, Hiroshima 739- 8526, Japan. 2 Department of Botany, National Science Museum, 4- 1- 1 Amakubo, Tsukuba, Ibaraki 305--0005, Japan. 88 J. Hattori Bot. Lab. No. 94 2 0 0 3 Nishimura et al. (1984) also provided a subdivision of the family as the following: Cl) Pleu­ rozioideae, (2) Ctenidioideae, (3) Hypnoideae, (4) Plagiothecioideae, and (5) Pylaisioideae ("Pylaisielloideae"). However, recent molecular studies suggest that the Hypnaceae as well as its type genus Hypnum itself are polyphyletic (cf. Arikawa & Higuchi 1999; Tsubota et al. 1999). Pylaisia Schimp. is the core member of the Hypnaceae subfamily Pylaisioideae. It is distributed mainly in temperate regions of the Northern Hemisphere, and many species are distributed in East Asia. Although the name Pylaisiella Kindb. ex Grout has been used as the correct name for this genus, the name Pylaisia became a nomina generica conservanda in the Saint Louis Code (Greuter et al. 2000). This genus has not been revised monographi­ cally since its establishment, though the Japanese taxa were reviewed by Toyama (1938a). Table 1 shows the various concepts of the composition of the Pylaisioideae. Accord­ ing to Nishimura et al. (1984), the Pylaisioideae are characterized by the well-differentiated clathrate alar cells and the erect capsule, and it exhibits a close relationship to the family Entodontaceae. Although the genus Orthothecium was transferred from the Entodontaceae to the Hypnaceae by Buck (1980), Nishimura et al. (1984) reserved judgement on its famil­ ial position, and Homomallium, Callicladium and Eurohypnum were placed in the subfami­ ly Hypnoideae. All genera in Table I were placed in the Hypnaceae by Vitt (1984) and Buck & Goffinet (2000), although Palisadula was placed in the Sematophyllaceae by Vitt (1984) or in the Myuriaceae by Buck & Goffinet (2000). Buck (1984) considered the genus Bryosedgwickia as a synonym of the Platygyriella. Buck & Ireland (1985) transferred the genus Entodontella to the Pylaisioideae through a reclassification of the Plagiotheciaceae. Tsubota et al. (200 I a) suggested that the Entodontaceae (s. str.) were sister to the Semato­ phyllaceae (sensu Tsubota et al. 2000, 2001 a), and supported the treatment by Buck (1980), which suggested the exclusion of the genus Orthothecium from the Entodontaceae. In the previous study (Arikawa & Higuchi 1999), we analyzed the chloroplast coded protein coding ribulose 1,5-bisphosphate carboxylase/oxigenase large subunit (rbcL) gene Table I. Summary of various classification of the subfamily Pylaisioideae. Nishimura et al. (1984) Fleischer (1923) Walther (1983) (' Py laisie 11 oideae ') Bryosedgwickia Homomallium Bryosedgwickia Giraldiella Callicladium Giraldiella P latygyriell a Tripterocladium Platygyriella Platygyrium Pylaisia Platygyrium Pylaisia Orthothecium Pylaisia Homomallium Palisadula Giraldiella Eurohypnum Bryosedgwickia Platygirium Platygyriella T. ARlKAWA & M. HIGUCHI: Phylogenetic analysis of Pylaisia and its relatives 89 sequence of two samples recognized as the members of the genus Pylaisia ("Pylaisiella"). Nucleotide sequences of the chloroplast-encoded gene rbcL is one of the most widely used genes in phylogeny of green plants, and also used most frequently for the phylogenetic analysis of pleurocarpous mosses (e.g., De Luna et al. 1999; Tsubota et al. 1999, 2000, 200 I a, 2001 b; Maeda et al. 2000; Arikawa & Higuchi 2002). We showed that Pylaisia polyantha and P intricata did not form a monophyletic clade, and mentioned, "molecular data suggests that the genus is heterogeneous." Pylaisia polyantha formed a clade with Brotherella recurvans, Pylaisiadelpha tenuirostris, and Hypnurn tristo-viride. Tsubota et al. (2000) referred to these data, and showed that P polyantha was included in the 'Semato­ phyllaceae (present sense) clade'. They concluded that the Sematophyllaceae (present sense) were monophyletic, although P polyantha would need to be transferred to this fami­ ly. For the purpose of reconsidering the classification of the Hypnales, further study of the circumscription of the Pylaisioideae should be carried out. A molecular phylogenetic study should be effective for reconsidering the complicated classification. Some representa­ tives of other genera of the subfamily and other species of Pylaisia should be added to the molecular phylogenetic analysis. There are several morphologically based taxonomic arguments in the genus Pylaisia. The peristome structures of P brotheri, P subcircinata, and P selwynii resemble each other closely. Pylaisia subcircinata can be distinguished from the related taxa by the long, subu­ late leaves and the small alar regions. Noguchi (1994) mentioned that P brotheri is closely allied to P selwynii and slightly distinguished by its broader leaves, broader alar regions and broader capsule; and that it is questionable whether these species could specifically distinguished. Ignatov et al. (2000) reported Pylaisia polyantha (,Pylaisiella polyantha') from the upper Bureya River, Russian Far East. Ignatov et al. (2000) mentioned that some popula­ tions of P polyantha have acute to shortly acuminate branch leaves, which make the branch foliation terete and the overall appearance quite distinct. However, peristome and spores were identical with those of P polyantha and in some shoots leaves are intermediate, so they referred these collections to P polyantha. The purpose of the present study is to investigate the phylogenetic relationships of Pylaisia and its purported allies inferred from rbcL gene, especially (1) whether the genus Pylaisia is paraphyletic, (2) whether the genera Platygyriurn, Giraldiella, and Orthotheci­ urn, which have been placed in the Pylaisioideae, are closely related to the species of Py­ laisia, (3) whether P brotheri is phylogenetically distinguished from P selwynii, and (4) whether the populations of P polyantha from the Russian Far East are phylogenetically dis­ tinguishable from North Europian ones. M ATERIALS AND METHODS This study consists of two steps: Cl) obtaining sequence data (DNA extraction, peR amplification, DNA sequencing, and download from DNA database), and (2) data analysis (sequence comparison and construction ofphylogenetic trees). 90 1. Hattori Bot. Lab. No. 94 2 003 Obtaining Sequence Data A total of fourteen samples (six species) of the genus Pyla isia , one sample of Giraldiella levieri, one sample of Platygyrium repens, and two Hookerialean samples (Hookeria acutifolia and Hypopterygiumfiavolimbatum)