J. Hattori Bot. lab. No. 88: 79- 99 (Aug. 2000) PRELIMINARY PHYLOGENETIC RELATIONSHIPS OF THE GENUS BROTHERELLA AND ITS ALLIED GENERA (HYPNALES, MUSCI) BASED ON CHLOROPLAST rbcL SEQUENCE DATA 1 2 2 2 HIROMI TSUBOTA , NARUM! NAKA0 , TOMIO YAMAGUCHI , TAROW SEKI3 AND HlRONORI DEGUCHI2 ABSTRACT: Distinction between the Hypnaceae and the Sematophyllaceae is controversial. Taxonom­ ic position of the genera Brotherella, Wijkia and its allies is still fluctuating and has been repeatedly discussed. A phylogenetic study was carried out to investigate the phylogenetic position of the genera Brotherella, Wijkia and their allies, based on phylogenetic analyses of the chloroplast ribulose 1,5- bisphosphate carboxylase/oxygenase large subunit (rbcL) gene data. Chloroplast rbcL genes from eight mosses were newly sequenced. Phylogenetic trees were constructed by neighbor-joining (NJ), minimum-evolution (ME), maximum-parsimony (MP) and maximum-likelihood (ML) methods for 30 rbcL sequences. The results suggest that the genus Brotherella and its allies (Pylaisiadelpha tenuirostris, Wijkia hornschuchii and Heterophy/lium nematosum) are monophyletic with high boot­ strap support. Hypnum tristo-viride is also included in the Brotherella clade. The Sematophyllaceae (sensu Seki 1968) are paraphyletic to the Brotherella clade. These results support the conclusion that the Sematophyllaceae (sensu present), including Brotherella and its allies, are monophyletic. The present study also suggests that the genus Entodon should be placed in a clade sister to the Semato­ phyllaceae (sensu present). The results show the need to reconsider the familial circumscription of the Hypnaceae and Sematophyllaceae. KEY WORDS : Brotherella, Wijkia , Hypnum, Sematophyllaceae, Hypnaceae, rbcL, molecular phylogeny INTRODUCTION Distinction between the Hypnaceae and the Sematophyllaceae has been controversial, especially concerning the phylogenetic position of the genera Brotherella, Pylaisiadelpha, Wijkia and Heterophyllium. The families Hypnaceae (with ea 40 genera: Vitt 1984) and the Sematophyllaceae (with ea 50: Vitt 1984), are among the most diversified taxonomic groups in the mosses. The taxonomic relationship of the genera in the Sematophyllaceae and the Hypnaceae is still fluctuating, and has been repeatedly discussed together with cir­ cumscriptions of the families. Vitt ( 1984) provided a new system for classification of pleurocarpous Bryopsida. That system suggested that a "double costa group" as represented by the Hypnaceae, the Se­ matophyllaceae and the Entodontaceae, is divergent from a group with a single costa, 1 Contribution from the Laboratory of Plant Taxonomy and Ecology, Hiroshima University. N. Ser. No. 503. 2 Department of Biologica.I Science, Graduate School of Science, Hiroshima University, Kagarniyama, l-3-1 Higashi-hiroshima-shi, Hiroshima 739- 8526, Japan. 3 Hera-yamate 3- 26, Hatsukaichi-shi, Hiroshima 738- 0027, Japan. 80 J. Hattori Bot. Lab. No. 88 2 0 0 0 which can be regarded as the most recent evolutionary stage of the order Hypnales ( = Hyp­ nobryales). The Sematophyllaceae were placed close to the Entodontaceae on the basis of morphological and ecological characters. Later, Buck & Vitt ( 1986) proposed another new classification for pleurocarpous mosses, following the familial arrangement of Vitt ( 1984). There still remain a variety of opinions on the taxonomic treatments of the Hypnaceae and the Sematophyllaceae (cf. Nishimura et al. 1984; Ando, Seki & Schofield 1989; Iwatsuki 1991; Hedenas 1995; Tan & Jia 1999). Horikawa & Seki (1960) reviewed the genus Brotherella, and taxonomically revised B. complanata and its relatives on the basis of statistical and ecological discussions of the species. Seki ( 1968), thereafter, published a taxonomic treatment of the Japanese Semato­ phyllaceae, introducing numerical taxonomy to Japanese bryology. He regarded the sporo­ phytic characters such as peristome and exothecial cells as significant for taxonomic recog­ nition, and treated taxa with Jess well-developed inner peristome teeth as members of the family Sematophyllaceae. He defined the Sematophyllaceae in a narrower sense, excluding the genera Brotherella and Heterophyllium from the family, and placed them in the Hyp­ naceae. Subsequently, Nishimura et al. (1984) and Ando et al. (1989) discussed the relation­ ships between the Hypnaceae and their relatives including the Sematophyllaceae. Nishimura et al. (1984) considered the well-developed alar cells a remarkable character to recognize the family Sematophyllaceae. Though they supported Seki 's (1968) treatment of Heterophyllium as a member of the Hypnaceae, they suggested that Brotherella and Wijkia should be placed in the Sematophyllaceae (s. lat.). They also included the genus Plagiothe­ cium in subfamily Plagiothecioideae of the Hypnaceae. Buck ( 1986) reviewed the genus Wijkia, and placed it in the Sematophyllaceae, em­ phasizing characters such as golden-green plant color, frequently turning a purer gold on aging, leaf cells thick-walled, and alar cells colored enlarged and inflated. He also pointed to the habitat preference for growing on twigs or logs. Buck (1984) treated Brotherella as a taxonomic synonym of Pylaisiadelpha. Ando et al. (1989), however, recognized the two genera as taxonomically distinct, providing diagnostic characters to distinguish Brotherella from Pylaisiadelpha. In turn, Hedenas (1996) suggested that a large number of the species of the Sematophyllaceae form a single clade, though the clade patterns were not consistent. Recently, Tan & Jia ( 1998) made a cladistic study on the Sematophyllaceae using the genus Hypnum as outgroup, and regarded the family as paraphyletic. Hedenas & Buck ( 1999) also reevaluated the Sematophyllaceae based on morphological and anatomical characters with a cladistic method. They suggested that the family could be subdivided into two sub­ families (Sematophylloideae and Wijkioideae ), and the 14 genera, such as Brotherella, Py­ laisiadelpha, lsocladiella and Heterophyllium, could be included in "Hypnaceae" or Hook­ eriaceae. Tan & Jia ( 1999) monographed the Chinese Sematophyllaceae, in which they also treated the genera Brotherella and Pylaisiadelpha as separate and included in the Semato­ phyllaceae following Vitt (1984). They treated Pylaisiadelpha tenuirostris and P yoko­ hamae (=Brotherella yokohamae) as independent species from one another although they were considered as conspecific by Ando et al. ( 1989). H. TsusoTA et al. : Phylogenetic relationships of the genus Brotherel/a and its allied genera 81 The family Sematophyllaceae is defined variously below with the following indica­ tion: the familial definition by Seki ( 1968), "(sensu Seki)"; by Nishimura et al. (1984), "(sensu Nishimura et al.)"; by Vitt ( 1984) and Buck & Vitt ( 1986), "(sensu Buck & Vitt)"; and by the present study including some species and genera treated as members of the Hypnaceae, "(sensu present)". The definition without the word "sensu" means the tradi­ tional conceptual sense or the Sematophyllaceae sensu Jato, including some species and genera whose position have been discussed with the family. Phylogenetic analyses of the Hypnales with molecular data have been attempted by Tsubota et al. (1999), Arikawa & Higuchi (1999), De Luna et al. ( 1999) and De Luna et al. (2000). The relationships among taxa in the Hypnales, however, have not been discussed comprehensively. The purpose of this study is to make clear the phylogenetic position of the genera Brotherella, Pylaisiadelpha, Wijkia and Heterophy/lium, and to approach a clas­ sification of the Sematophyllaceae. We also discuss the phylogenetic relationship among the Hypnaceae, the Sematophyllaceae and the Entodontaceae, as inferred from the molecu­ lar data. MATERIALS AND M ETHODS This study consists of two steps: (I) obtaining sequence data (DNA extraction, PCR amplification, and DNA sequencing), and (2) data analyses. Total DNA was extracted by two methods: phenol-chloroform method and CTAB method. The former method follows Tsubota et al. (I 999) and the latter is shown below. Thirty species of mosses were examined as shown in Table I and in Appendix A. The latter contains voucher information and DDBJ Accession Nos., including 19 species of Brothere/la and others in the Sematophyllaceae and the Hypnaceae, and 8 species of other families in the Hypnales and their allies, and 3 species of other orders as outgroups. The rbcL gene sequences of 8 species were newly obtained, with voucher specimens deposited in HIRO. DNA Extraction Total DNAs were extracted from fresh materials or dried herbarium specimens follow­ ing modifications of Murray & Thompson (I 980) and Arikawa & Higuchi ( 1999). Green parts of shoots were obtained from fresh material (100-500 mg fresh weight) or dried specimens (50-300mg dry weight) for DNA extraction. Samples were washed sever­ al times with distilled water, and water was removed from the sample with paper towels. Frozen samples in liquid nitrogen were ground to yield a fine powder using a mortar and pestle. This powder was suspended in extraction buffer composed of 2% cetyltrimethyl­ ammonium bromide (CTAB), 1.4 M NaCl, 50 mM Tris-HCI (pH 8.0), 10 mM EDTA. Ex­ traction solution was incubated at 65°C for 20- 30 min. The samples were kept at more than l 5°C in order to prevent re-crystallization of CTAB in the following step. An equal volume of chloroform/isoamylalcohol (24: I, CIA) was added to the digestion mixture. The sus­ pension was mixed gently for 10 min, and centrifuged