J. Hattori Bot. lab. No. 88: 79- 99 (Aug. 2000)

PRELIMINARY PHYLOGENETIC RELATIONSHIPS OF THE GENUS BROTHERELLA AND ITS ALLIED GENERA (, 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 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 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 . 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 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 for I 0 min at 6,000 X g. Then, the upper phase was transferred to a new microtube. An equal volume of 1110 CTAB buffer and 1/50 5 M NaCl was added. The solution was incubated at 65°C for 10 min, and mixed 82 J. Hattori Bot. Lab. No. 88 2 0 0 0 ------atpB - rbcL - - - efpZ I - KH 1 kbp ------Fig. 1. Relative position of the PCR amplification and sequencing primers used in this study (horizontal arrows). The empty boxes represent coding regions in the data of Marchantia polymorpha and Zea mays and connecting lines spacer regions. gently several times. An equal volume of CIA was then added to the mixture. The suspen­ sion was mixed gently for 10 min once again. The solution was centrifuged for 10 min at 6,000 X g. After transfer of the upper layer to a new tube, 2.5-volume cold absolute ethanol was then added, and the solution was mixed gently. The tubes were placed at - 20°C for 2 hr, and centrifuged for 5 min at 20,000 X g. The DNA pellet was rinsed with cold 70% ethanol by inverting the tube several times. After centrifugation for 2 min, the supernatant was discarded and the pellet was dried under vacuum. The DNA was then dissolved in 10- 20 µ1 TE buffer. DNA concentration was estimated by ethidium bromide staining after 1% gel electrophoresis. The DNA was stored at - 20°C or - 80°C. PCR Amplification We amplified rbcL gene segments for direct sequencing by polymerase chain reaction (PCR). PCR amplifications were performed using a DNA thermal cycler (Thermal Cycler 9600, Perkin Elmer) with PCR amplification kit (TaKaRa) following Tsubota et al. (1999). The synthetic primers for PCR were 16- 33 nucleotides in length (Table 2). The amplifica­ tions were performed using the program set to denature at 94°C for 10-60 sec, anneal at 42- 52°C for 20-30 sec, and extend at 68- 72°C for 1- 3 min for a total of 25- 30 cycles. The primers for PCR amplifications were shown in Fig. 1. Most of them were designed by Tsubota et al. (1999) and Manhart ( 1994 ). Several primers were also additionally designed for Brotherella based on the obtained sequencing data. After amplification, products were checked, and purified on SUPREC-02 concentrating filters (TaKaRa) or by eluting nucleic acids from agarose gels with SUPREC-01 concentrating filters (TaKaRa). DNA Sequencing Direct DNA sequence analysis of the PCR products was performed by dideoxy chain termination method using the ABI (Perkin-Elmer) or Amasham (Pharmacia biotech) kits according to manufacturer's instructions following Tsubota et al. (1999). The reactions were performed in the thermal cycler. The primers for sequencing were the same ones for the PCR amplifications. The sequences were electrophoresed on an automated sequencer (ABI 373S, Perkin Elmer), and analyzed on the data-analysis system. Alignment of the rbcL sequence data was straightforward because of the presence of codon structure. The sequences were deposited in the DNA Data, Bank of Japan (DDBJ) (Tateno & Gojobori 1997), a part of the DDBJ/EMBL/GenBank International Nucleotide Sequence Database Collaboration. H. TsusoTA et al. : Phylogenetic relationships of the genus Brotherel/a and its allied genera 83

Table 1. List of species and families investigated for rbcL gene with accession num­ bers of the sequence.

Family Species Accession number

Thuidiaceae A bietinella abietina AF005519 Amblystegiaceae Calliergonella cuspidata AB024678 Myuroc/ada maximowiczii AB029389 Platyhypnidium riparioides AB029385 Rhynchostegium pallidifolium AB024944 Hylocomiaceae Rhytidiadelphus japonicus AB039788 Entodontaceae Entodon rubicundus AB029386 Sematophyllaceae Brotherella complanata AB039785 Brotherella fauriei AB039786 Brotherella henonii AB029167 Brotherella herbacea AB039787 Brotherella recurvans Ll3475 Heterophyllium nematosum AB029391 Isoc/adiella surcu/aris AB039784 Pylaisiadelpha tenuirostris AB039789 Sematophyl/um subhumile AB039675 Wijkia hornschuchii AB029383 Plagiotheciaceae Plagiothecium neckeroideum AB024630 Plagiothecium nemorale AB029387 Hypnaceae Hypnum cupressiforme AB039674 Hypnum lindbergii AB029390 Hypnum p/umaeforme AB029384 Hypnum tristo-viride AB024656 Pylaisiella intricata AB024642 Pylaisiella polyantha AB024645 Leskeaceae Bou/aya mittenii AB024963 Hedwigiaceae Hedwigia cilia/a AF005517 Mniaceae Mnium thomsonii AF0055 l 8 Plagiomnium cuspidatum U87082 Orthotrichaceae Ulota lutea AF005540

Data Analysis Taxa Examined- The exemplar taxa treated in this study are shown in Table 1 with DDBJ accession numbers. The taxa examined include Abietinella abietina, Calliergonella cuspidata, Myuroclada maximowiczii, Platyhypnidium riparioides, Rhynchostegium pallidifolium, Rhytidiadelphus japonicus, Entodon rubicundus, Brotherella complanata, B. fauriei, B. henonii, B. herbacea, B. recurvans, Heterophyllium nematosum, !socladiella surcularis, Pylaisiadelpha tenuirostris, Sematophyllum subhumile, Wijkia hornschuchii, Plagiothecium neckeroideum, P nemorale, Hypnum cupressiforme, H. lindbergii, H. plumaeforme, H. tristo-viride, Pylaisiella intricata and P polyantha. Boulaya mittenii and Hedwigia ciliata were treated as outgroups. Sequences of other taxa used as outgroups for 84 J. Hattori Bot. Lab. No. 88 2 0 0 0

Table 2. Sequences (5'- 3') of synthetic primers used for PCR amplification and se­ quencing of the rbcL gene. The primer numbers from 5' end correspond to rough positions in the sequence of Marchantia polymorpha (Ohyama et al. 1986).

Forward primers Sequence atpB!Ol l *** GATCTACTAC TTTAATTCCT GT atpB370*** AGTTGAATCT AAAGGATCTA c atbBl75R* TGTTGAACTT CACAAGTAAC A rbcL-130*** ACAATGATAC TGTTTGTTAT AG rbcL-73*** ATACCAAAGA TGTI'ITITTA TAAG rbcL7*** TGGATTTAAA GCTGGTGTTA AAG rbcL152*** GAATCCTCCA CTGGTACATG rbcL218h GACGATGCTA TGATCTCGAA GC rbcL256 GCTATGATCT TGAAGCAGTT CCTGGAGAAG rbcL265*** ATTGCTTATG TTGCTTATCC rbcL549*** TGTCTTCGTG GTGGAC rbcL557 GGTAGAGCTG TATATGAATG TCTTCGTGGT GG rbcL862*** CAATGCATGC AGTTATTGAC rbcL 1117*** GGTATTCATG TTTGGC rbcL 1220*** GGAGCAGTTG CTAATAGAG rbcL1276*** GGACGTGATC TTGCTCG Reverse primers rbcL199R*** GGTAAGTCCA TCAGTCCAAA c rbcL270R *** GCAATATATT GATTTTCTTC TCCAG rbcL600R*** GTGAAATCAA GTCCACCACG rbcL804hR*** TGCAGTAAAA CCACCTG rbcL957R*** GCGTGAATAT GGTCTCCACC rbcL989R CTACGGTACC AGCGTGAATA TGGTCTCCAC CGG rbcLl098R*** AACACCTGGT AAAGAAACC rbcLI 145R*** TTAATGCTGG CATATGCCAA AC rbcLl346hR*** GCAGCTAATT CAGGACTCC trnR24R*** CTCTAATCCA CTGAGCTACA trnR66R*** GAAGGGATTC GAACCCTTG trnRn** GGGTTAGAAG GGATTCGAAC CCTTGAC zfpA546R *** ATGATATCCA CATTCTTCAC A

* Marks primers designed by Manhart (1994). ** Marks primers designed by Manhart (1994) and shortened by Tsubota et al. ( 1999). *** Marks primers designed by Tsubota et al. ( 1999); others were additionally designed by au- thors. the analyses were obtained from the DNA database. Alignment- Manual alignment was done for 30 sequences against those of Marchan­ tia polymorpha and the other hypnalean mosses such as Rhynchostegium pallidifolium with an editor program and our original program, Sclean 2.53 (by Tsubota 1998- 2000, in our http site, see Appendix B), to reverse sequences. Our original Perl and C programs were applied for the data processing. The undetermined sites were removed from the alignment H. TSUBOTA et al.: Phylogenetic relationships of the genus Brothere/la and its allied genera 85

before the subsequent analyses. Analysis Methods- Phylogenetic analysis was performed following Tsubota et al. ( 1999). Trees were constructed by the following four methods: the neighbor-joining (NJ) (Saitou & Nei 1987), the minimum-evolution (ME) method (Rzhetsky & Nei 1992, 1993), the maximum-parsimony (MP) method (Fitch 1971), and the maximum-likelihood (ML) method (Felsenstein 1981 ). In distance methods (NJ and ME), the simplest model of Kimura's (1980) two-parameter model for estimating the distances (Table 3) was used, be­ cause of transition bias for the base substitutions and negligible bias for the base composi­ tion. NJ and ME trees were constructed by using MEBoot 2.00 (by Tamura 1997- 2000, downloaded from his FTP site, see Appendix 8). Standard bootstrap analysis (Felsenstein 1985) was performed with 10,000 bootstrap replicates with the same data and option sets in effect. MP trees were constructed with a program Parsimony 1.00 (by Tamura 1998- 2000, downloaded from his FTP site, see Appendix 8). MP trees were searched by Heuristic search algorithm, in which weight for transversion (Tv) relative to transition (Ts) was 1, 2 or 3. The search level was set to six for extent of local branch rearrangement, and the num­ ber of search paths was set to 40 for independent initial trees constructed under random branch addition. ML analysis was carried out with the following three programs: NucML in MOLPHY 2.3b3 package programs (Adachi & Hasegawa 1996), PUZZLE 4.0.1 (Strimmer & von Haeseler 1996, updated 1999), and fastDNAml I. I. I a (Olsen et al. 1994). In analysis with NucML, ML trees were searched with the local rearrangement method from the NJ tree (Adachi & Hasegawa 1996). In analysis by fas tDNAml, ML trees were searched with the global rearrangements option. In analysis by PUZZLE, ML trees were searched using tree reconstruction. For the ML analysis, HKY85 model (Hasegawa, Kishino & Yano 1985) was used as the estimate model. Best-conformed Ts/Tv parameter was estimated based on the calculations by NucML with "-topt" option employed the parameter as 4.26 (8.51 as NucML notational system), and by PUZZLE approximately employed as 4.14 using para­ meter estimation based on NJ tree for substitution process and rate variation. The Ts/Tv pa­ rameter was then used as 4.14 for the analysis by fastDNAml. Several measures were used to evaluate the resulting trees, including the log-likeli­ hood, standard errors of the difference in log-likelihood (Kishino & Hasegawa 1989), and the bootstrap probability estimated by the RELL (resampling of the estimated log-likeli­ hood) method (Kishino, Miyata & Hasegawa 1990; Hasegawa & Kishino 1994). Local bootstrap probabilities (LBPs; in % ) (Adachi & Hasegawa 1996) by NucML were also calculated respectively for the best ML topology. LBP is a relative bootstrap fre­ quency obtained from topology search by local rearrangements of NucML. The value was comparable with a value ofFelsenstein's (1985) bootstrap probabi lity, and it is a little larger than the standard bootstrap probability. A majority rule consensus tree also calculated for the highest log-likelihood trees by CONSENSE in PHYLIP 3.573c package programs by Felsenstein (1980- 2000, in his http site, see Appendix 8). Table 3. Pairwise distances of 1,228 bp for 30 rbcL sequences. G + C contents(%) (left column), Kimura 2-parameter dis- 00 tance (upper-right matrix) and homology(%) (lower-left matrix). °'

G+ C% 1 2 3 4 5 6 7 8 9 10 11 12

1 Brotherella complanata 39.8 1 0.002 0.011 0.022 0.017 0.012 0.012 0.017 0.027 0.045 0.029 0.046 2 Brotherella fauriei 40.5 99.8 2 0.012 0.022 0.017 0.013 0.013 0.018 0.028 0.047 0.032 0.048 3 Brotherella henonii 39.6 98.9 98.9 3 0.019 0.017 0.017 0.012 0.017 0.03 1 0.044 0.030 0.047 4 Brotherella herbacea 40.6 97.9 97.8 98.1 4 0.027 0.028 0.021 0.023 0.035 0.048 0.038 0.053 5 Brotherella recurvans 39.8 98.4 98.3 98.3 97.4 5 0.023 0.009 0.014 0.030 0.047 0.033 0.044 6 Heterophyllium nematosum 39.8 98 .8 98.7 98.4 97.3 97.8 6 0.017 0.022 0.035 0.051 0.037 0.050 7 Hypnum tristo-viride 39.6 98 .8 98.7 98.9 98.0 99.1 98.4 7 0.007 0.024 0.039 0.027 0.043 8 Wijkia hornschuchii 39.4 98.3 98.2 98.4 97.7 98 .6 97.9 99.3 8 0.030 0.045 0.031 0.046 9 Pylaisiadelpha tenuirostris 40.1 97.4 97.3 97.0 96.6 97.1 96.7 97.6 97 .1 9 0.054 0.043 0.056 c- 10 lsocladiella surcularis 40.3 95 .7 95.4 95 .8 95.4 95 .5 95 .1 96.3 95 .7 94.9 10 0.039 0.060 :c 11 Pylaisiella polyantha 39.2 97.1 96.9 97.1 96.3 96.8 96.4 97.4 97 .0 95 .8 96.2 11 0.040 "' 12 Sematophyllum subhumile 39.3 95.6 95.4 95.5 95.0 95 .8 95 .2 95 .8 95 .6 94.6 94.3 96.1 12 §. 13 Entodon rubicundus 38.2 96.8 96.7 96.9 96.3 96.7 96.6 97.2 96.7 95.8 96.1 97.5 96.3 Ol 14 Hypnum cupressiforme ~ 38.7 96.2 96.1 96.3 95.4 96.2 95 .9 96.6 96.0 95 .0 95.0 96.7 95 .7 r 15 Calliergonella cuspidata 38.5 96.2 96.1 96.4 95 .5 95 .8 95 .9 96.6 96.2 95 .2 95.0 96.7 95 .8 ,,.."' 16 Hypnum lindbergii 38.2 96.0 95.9 96.3 95.4 95 .7 95 .8 96.4 96.0 95 .2 94.8 96.4 95.7 zp 17 Hypnum plumaeforme 38.7 95 .9 95.8 96.0 95.4 95 .7 95.4 96.2 95 .6 94.6 95.0 96.3 95.8 00 18 Pylaisiella intricata 38.6 96.7 96.6 96.9 96.3 96.7 96.4 97.2 96.7 95 .7 95 .9 97 .2 96.3 00 19 Bou/aya mittenii 38.8 96.7 96.6 96.7 96.2 96.3 96.3 96.9 96.3 95 .7 95 .6 96.7 95 .8 20 A bietinella abietina 38.5 96.5 96.4 96.7 96.0 96.5 96.3 97.1 96.5 95 .7 95.8 97 .3 96.5 21 Plagiothecium neckeroideum 39.0 96.0 95 .9 96.1 95 .3 96.0 95.8 96.6 96.0 95.1 95.0 96.4 95 .6 22 Plagiothecium nemorale 38.7 95 .2 95 . l 95.3 95 .0 95.2 95.3 95.9 95.4 94.5 94.5 95 .8 94.9 23 Platyhypnidium riparioides 38.5 95.4 95 .1 95.3 94.7 95 .l 94.8 95.8 95 .2 94.4 95 .0 96.2 95 .6 24 Rhynchostegium pallidifolium 38.5 95 .5 95.4 95 .6 94.9 95 .3 95 .l 95.9 95.4 94.7 95 .0 96.0 95.4 25 Myuroclada maximowiczii 39.7 95.8 95.7 95 .8 95 .3 95.4 95.5 96.2 95 .6 94.8 95 .0 96.4 95.1 26 Rhytidiadelphus japonicus 38.2 96.3 96.3 96.4 95 .8 96.3 96.3 96.7 96.2 95.4 95 .l 96.8 96.0 27 Ulota /utea 36.8 93 .5 93.2 93.2 92.4 93.4 92.9 93.6 93 .3 92.5 92 .2 93. 7 93 .3 28 Hedwigia ciliata 38.4 94.5 94.5 94.3 93 .7 94.5 94.1 95 .0 94.4 93 .7 93.2 94.6 94.0 29 Plagiomnium cuspidatum 40.0 94.5 94.4 93.9 93 .5 94.0 93.9 94.4 93 .8 93.6 92.8 94.2 93.6 30 Mnium thomsonii 40.5 93.3 93.2 93.4 92.8 93.6 93.2 93.9 93.3 93 .0 92 .6 93.6 93.4 N 0 0 0 Table 3. Continued.

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 ;::c I 0.033 0.040 0.040 0.041 0.042 0.034 0.034 0.036 0.041 0.050 0.048 0.047 0.044 0.038 0.069 0.057 0.058 0.071 ..., 2 0.034 0.040 0.040 0.042 0.043 0.035 0.035 0.037 0.042 0.051 0.051 0.048 0.045 0.039 0.071 0.058 0.059 0.072 c 3 0.032 0.039 0.03 7 0.039 0.04 1 0.032 0.034 0.034 0.040 0.049 0.049 0.046 0.043 0.037 0.072 0.060 0.064 0.070 "' 4 0.038 0.048 0.047 0.048 0.047 0.038 0.040 0.041 0.049 0.052 0.055 0.054 0.049 0.043 0.081 0.066 0.069 0.076 ~ ~ 5 0.034 0.040 0.043 0.045 0.045 0.034 0.038 0.036 0.041 0.050 0.051 0.049 0.048 0.038 0.070 0.057 0.063 0.067 ~ 6 0.035 0.042 0.042 0.044 0.048 0.037 0.039 0.039 0.044 0.049 0.055 0.05 1 0.047 0.039 0.075 0.062 0.064 0.072 ..,, ::r 7 0.028 0.035 0.035 0.037 0.040 0.028 0.032 0.030 0.035 0.042 0.044 0.042 0.040 0.034 0.068 0.053 0.059 0.064 '< 0 8 0.034 0.041 0.040 0.041 0.046 0.034 0.038 0.036 0.04 1 0.048 0.050 0.048 0.046 0.040 0.07 1 0.059 0.065 0.07 1 °":::> 9 0.043 0.052 0.050 0.050 0.056 0.045 0.045 0.045 0.051 0.057 0.059 0.055 0.055 0.048 0.080 0.066 0.067 0.074 "' ;:;·~ 10 0.040 0.053 0.053 0.054 0.053 0.042 0.046 0.044 0.052 0.058 0.052 0.053 0.052 0.051 0.083 0.07 1 0.076 0.079 iil 11 0.026 0.033 0.033 0.037 0.038 0.028 0.034 0.027 0.037 0.043 0.040 0.041 0.037 0.033 0.066 0.056 0.061 0.067 c;~· 12 0.038 0.045 0.043 0.045 0.044 0.038 0.043 0.036 0.046 0.054 0.046 0.047 0.051 0.04 l 0.070 0.063 0.068 0.070 ;;; 13 13 0.026 0.026 0.027 0.028 0.015 0.021 0.016 0.027 0.033 0.034 0.033 0.035 0.022 0.056 0.049 0.049 0.056 ::r 14 97.5 14 0.028 0.028 0.031 0.022 0.027 0.020 0.026 0.034 0.034 0.034 0.033 0.027 0.061 0.048 0.054 0.057 ~ - 0-, 15 97.5 97.2 15 0.005 0.022 0.0 15 0.022 0.0 19 0.028 0.034 0.031 0.029 0.032 0.024 0.052 0.043 0.048 0.052 ;;. 16 97.3 97 .2 99.5 16 0.026 0.017 0.024 0.021 0.028 0.034 0.034 0.03 l 0.032 0.026 0.051 0.045 0.047 0.051 "' 17 97.2 97.0 97 .8 97.5 17 0.0 16 0.020 0.020 0.027 0.033 0.027 0.027 0.033 0.028 0.058 0.045 0.050 0.060 °""':::> c 18 98.5 97 .9 98 .5 98.4 98.5 18 0.01 l 0.007 0.018 0.026 0.026 0.024 0.025 0.017 0.054 0.040 0.046 0.052 "'~ 19 98.0 97.4 97.8 97.6 98.0 98.9 19 0.012 0.021 0.028 0.027 0.025 0.026 0.020 0.055 0.043 0.046 0.056 Cl ;;. 20 98.5 98 .0 98 .l 98.0 98 .0 99.3 98.9 20 0.017 0.025 0.025 0.023 0.024 0.0 18 0.053 0.039 0.044 0.05 1 ~"' 21 97.4 97 .5 97.3 97.2 97.3 98 .2 98.0 98 .3 21 0.0 12 0.029 0.028 0.028 0.02 l 0.057 0.042 0.048 0.052 ~ 22 96.8 96.7 96.7 96.7 96.7 97.5 97.2 97.6 98.8 22 0.032 0.032 0.033 0.028 0.065 0.048 0.053 0.056 "':::> 23 96.7 96.7 97 .0 96.7 97.4 97.5 97.4 97 .6 97.1 96.9 23 0.0 l 0 0.029 0.028 0.057 0.045 0.052 0.052 0- 24 96.8 96.7 97.l 97.0 97.4 97.6 97.6 97.7 97.3 96.9 99.0 24 0.026 0.026 0.057 0.043 0.052 0.054 "' ~ 25 96.6 96.8 96.9 96.9 96.8 97 .6 97.5 97 .6 97.2 96.8 97. l 97.5 25 0.029 0.065 0.047 0.056 0.057 ;;; · 0- 26 97.8 97 .4 97 .6 97.5 97.2 98 .3 98.0 98 .2 98.0 97 .2 97 .3 97 .5 97.1 26 0.057 0.040 0.046 0.054 °""':::> 27 94.6 94.2 95.0 95.l 94.5 94.8 94.7 94.9 94.5 93 .8 94.5 94.5 93 .8 94.5 27 0.055 0.068 0.077 "' 28 95 .3 95.4 95 .8 95.7 95.7 96.1 95 .8 96.2 95 .9 95.4 95 .7 95 .8 95.4 96.1 94.7 28 0.049 0.063 Ol 29 95.3 94.9 95.4 95.4 95.2 95.6 95 .6 95 .8 95.4 95.0 95.0 95 .0 94.6 95.6 93.6 95 .3 29 0.036 30 94.6 94.5 95 .0 95.1 94.3 95 .0 94.6 95. 1 95 .0 94.6 95 .0 94.9 94.6 94.9 92 .8 94.1 96.5 30 00 -i 88 J. Hattori Bot. Lab. No. 88 2 0 0 0

RESULTS Structure of Sequence Data and Sequence Alignment New rbcL sequences were obtained for eight species: *Rhytidiade/phus japonicus, *Brothere//a complanata, B.fauriei, *B. herbacea, lsocladiella surcularis, *Pylaisiadelpha tenuirostris, Sematophy//um subhumi/e and *Hypnum cupressiforme, for which five species with asterisks are shown as entire sequences. Supplemental sequence data were also ob­ tained for Hypnum plumaeforme and its entire sequence was newly determined in this study. The sequences were deposited in the DDBJ under serial accession numbers (Table l ). The G + C contents of these nucleotide sequences were investigated for all known sites, and they ranged from 36.8 to 40.6% (Table 3). These proportions proved to have no signifi­ cant differences in GC contents from those previously reported for mosses: Brothere//a henonii (39.6%), Heterophyllium nematosum (39.9%) and Hypnum lindbergii (38.2%) (Tsubota et al. 1999). The data set had a 5% chi-square test and p-value by PUZZLE, and all the data passed these tests. The data set can exert no bad influence on the analyses judg­ ing from the comparatively compact distribution ofG+C content and the results of the test. Nucleotide frequencies estimated from data set were A= 29.0%, C = 17.7%, G = 21.7% and T = 31.7%, which indi cate little bias of base composition. Homologies among se­ quence data ranged from 92.2 to 99.8% (Table 3). The full length of the entire rbcL sequences newly obtained for the six species investi­ gated was 1,428 bp long. The length was proved to be the same as those of other bryophyte species previously reported, but shorter than those of tracheophytes by 3 or 6 bp. Each nu­ cleotide sequence could be translated respectively into a 475-amino acid sequence and one stop codon assuming that the genetic code is universal. The sequences also have the gap common to those of tracheophytes, but had no stop codon that is commonly found in horn­ wort species (see Malek et al. 1996; Lewis et al. 1997). The data matrix was prepared for analyses of phylogenetic relationships of the Hyp­ naceae, the Sematophyllaceae, and their allies by manual alignment of the sequences. These analyses of all 30 OTUs were based on the alignment of 1,228 rbcL sites. The nu­ cleotide substitutions for each position were estimated by translating them into amino acid sequences. Most were third position but synonymous nucleotide substitutions, and most of the others with amino acid replacement were conservative substitutions. The number of constant sites was 963 of 1,228 ( = 78.4% of all sites). No insertion or deletion sites were found. In the 1,241 aligned rbcL data set for 13 sematophyllacean and their al lied mosses, 164 sites (= 13.2%) were variable; namely those total 127 sites (= 77.4%) were binary transitions, 34 sites ( = 20. 7%) were binary transversions, 3 sites ( = 1.8%) were multi state sites. Phylogenetic Analysis A total of 66 topologies were obtained from four analyses: 1 ML topology by NucML, 1 ML by fastDNAml, 1 NJ and 21 ME by MEBoot, and 42 MP by Parsimony. The program PUZZLE, however, made a multifurcating tree, and the quartet puzzling tree was not com­ pletely resolved. The best ML topology was searched through the obtained trees (Table 4). They were compared with each other using log-likelihood measure and Pr indicates boot- H. TsuBOTA et al. : Phylogenetic relationships of the genus Brotherella and its allied genera 89

Table 4. NucML analyses for 1,228 bp of 30 rbcL sequences. The 42 high-ranking trees are shown. The ML tree is indicated as 'MC. The differences in log-likelihood of al- temative trees from that of the ML tree (l;-IMJ are shown with their SE following :±: esti- mated by Kishino & Hasegawa's (1989) formula. 'ME' means tree with the least sum of edge length optimized by ML with NucML. Pr indicates bootstrap probability of a tree among 66 alternatives estimated by the RELL method (Kishino et al. 1990) with I 0,000 replications. Term 'tv' means weight for transversions in parsimony method; 'tv = I' is un- weighted analyses, ' tv = 2' or 'tv = 3' are weighted.

Tree ranking l;-/Ml Pr Method Program

ML,ME 0.243 ML fastDNAml 2 - 6.0 :±: 12.7 0.019 MP Parsimony (tv = 2) 3 -7.3:±:14.3 0.017 MP Parsimony (tv = 2) 4 -7.5 :±: 14.0 0.091 ML NucML 5 -7.8 :±: 10.7 0.016 MP Parsimony (tv = I) 6 -8.1 :±: 16.2 0.015 MP Parsimony (tv = 2) 7 - 8.1 :±: 16.1 0.024 MP Parsimony (tv = 3) 8 - 8.5:±:15.3 0.013 MP Parsimony (tv = 2) 9 - 8.6 :±: 13.1 0.007 MP Parsimony (tv = 2) 10 - 9.0 :±: 12.5 0.016 MP Parsimony (tv = I) 11 - 9.3 :±: 17.4 0.019 MP Parsimony (tv = 3) 12 - 9.4 :±: 15.2 0.011 MP Parsimony (tv = 2) 13 - 9.8 :±: 16.6 0.013 MP Parsimony (tv = 2) 14 - 10.0 :±: 14.7 0.008 MP Parsimony (tv = 2) 15 - 10.2 :±: 17.0 0.017 MP Parsimony (tv = 2) 16 - 10.4 :±: 11.2 0.009 MP Parsimony (tv = I) 17 -10.6 :±: 16.4 0.007 MP Parsimony (tv = 2, 3) 19 - 10.9 :±: 14.6 0.020 MP Parsimony (tv = I) 20 - 10.9 :±: 16.5 0.007 MP Parsimony (tv = 2) 21 - 10.9:±:19.4 0.014 MP Parsimony (tv = 3) 22 - 11.1:±:15.5 0.007 MP Parsimony (tv = 2) 23 -1 1.4 :±: 18.2 0.008 MP Parsimony (tv = 2) 24 -11.4 :±: 18.0 0.011 MP Parsimony (tv = 3) 25 -11.5 :±: 21.3 0.046 MP Parsimony (tv = 3) 26 -11.8 :±: 13.0 0.008 MP Parsimony (tv = 1) 27 -12.1 :±:20.5 0.014 MP Parsimony (tv = 3) 28 -12.l :±: 17.7 0.011 MP Parsimony (tv = 3) 29 -12.3 :±: 13 .5 0.015 MP Parsimony (tv = I) 30 -12.6 :±: 16.9 0.007 MP Parsimony (tv = 2) 31 - 12.6 :±: 19.1 0.013 MP Parsi mony (tv = 3) 32 - 12.7 :±: 22.4 0.039 MP Parsimony (tv = 3) 33 - 13 .5 :±: 19.6 0.007 MP Parsimony (tv = 3) 34 - 13.7 :±: 15.0 0.009 MP Parsimony (tv = I) 35 - 13 .9 :±: 18 .2 0.006 MP Parsimony (tv = 3) 36 - 14.0 :±: 21.5 0.025 MP Parsimony (tv = 3) 37 - 14.2 :±: 18.5 0.006 MP Parsimony (tv = 2) 38 - 14.4 :±: 17.6 0.038 ME ME Boot 90 J. Hattori Bot. Lab. No. 88 2 0 0 0

Table 4. Continued.

Tree ranking Pr Method Program

39 -14.9 ::!:: 20.7 0.006 MP Parsimony (tv = 3) 40 - 15 .5 ::!::22.6 0.020 MP Parsimony (tv = 3)

46 - 18.4 ::!:: 18.9 0.007 MP Parsimony (tv = 2) 57 - 22.4 ::!:: 22.6 0.007 MP Parsimony (tv = 3) strap probability by NucML with HKY85 model. Each topology with high log-likelihood value was very close to one another. Consequently, the best topology was obtained by fastDNAml. Tree-1 in Table 4 is the best tree by the likelihood criterion, and the sum of edge length for the tree is also the least. The ML tree supported by the most log-likelihood and Pr value is presented in Fig. 2. Log-likelihood value for the ML tree was -4879.7. Pr indicated by RELL method for the ML tree was 0.243, of which value is appearing a little low but the highest of the 66 topologies. Topology of the ML tree was approximately consistent with the other high-ranking topologies, especially on the Brotherella clade and the Sematophyllaceae (sensu present) clade. Definition of the clades is introduced in Fig. 3. The LBPs for the clades were of comparatively low va lues ( 61 - 100% ). The bootstrap values by NJ and ME methods were comparatively lower than LBPs. The lineage of the genus Brothere/la and its allies [the Brotherella clade] and the Sematophyllaceae (sensu present) [the Sematophyllaceae (sensu present) clade] was supported by high LBPs in ML method. The tree supported the monophyly of the Sematophyllaceae (sensu present) (75% LBP value in ML tree). The genus Brotherella, Hypnum tristo-viride, Heterophyllium nematosum and Wijkia hornschuchii form a clade [the Brotherella clade] in the most ML tree (68% LBP). The other high-ranking log-likeli hood trees also showed that the Brotherella clade can be distin­ guished as a resolved monophyletic group by both high bootstrap values and LBPs. [n all high-ranking topologies, Hypnum tristo-viride and Heterophyllium nematosum were placed in the Brotherella clade. Both the Brotherella complanata-B. fauriei-Heterophyllium ne­ matosum-B. henonii clade (clade A in Fig. 3) and the Brotherella recurvans-Hypnum tristo­ viride-Wijkia hornschuchii clade ( clade B in Fig. 3) were also supported by all trees with high log-likelihood score. The Brotherella-Pylaisiadelpha clade form as a single group (99% LBP in ML tree). The Sematophyllaceae (sensu Seki) were paraphyletic to the Brotherella-Pylaisiadel­ pha clade. Meanwhile, the Sematophyllaceae (sensu present) including Brotherella, its al ­ lies and Pylaisie/la polyantha appear as a monophyletic group (75% LBP in ML tree). These results support the hypothesis that the Sematophyllaceae (sensu present) are mono­ phyletic. In addition, the genus Hypnum including Hypnum cupressiforme, H. plumaeforme, H. lindbergii and H. tristo-viride, never formed a monophyletic group in any of the analyses. The phylogenetic trees also showed polyphyly of the fami ly Hypnaceae. Monophyly for the H. Tsu BOTA et al.: Phylogenetic relationships of the genus Brotherel/a and its allied genera 91

Brotherel/a complanata Brotherel/a fauriei '---- Heterophyllium nematosum Brotherella henonii ....______Brotherel/a herhacea 7 ~ Brotherella recurvans 99 67{ Hypnum tristo-viride 91 Wijkia hornschuchii '------Pylaisiadelpha tenuirostris 81 '------/socladiel/a surcu/aris ..____ Pylaisiella polyantha '------Sematophyllum suhhumile Entodon ruhicundus ----- Hypnum cupressiforme 100 Hypnum lindhergii .-----1 Calliergone/la cuspidata 66 ---- Hypnum plumaeforme Pylaisie/la intricata .-8.-5t--- Boulaya mittenii Ahietine/la abietina Plagiothecium neckeroideum ---- Plagiothecium nemorale 67 100 Platyhypnidium riparioides 100 92 Rhynchostegium pallidifolium ----- Myuroclada maximowiczii Rhytidiade/phus japonicus 61.------U/ota lutea '------Hedwigia ciliata i----- Plagiomnium cuspidatum ------Mnium thomsonii

0.01 substitutions/site

Fig. 2. The highest likelihood tree of the aligned rbcL gene sequences (HKY85 model; a/[3 = 8.37; In l = -4879.7 by NucML). The horizontal length of each branch is pro­ portional to the estimated number of nucleotide substitutions. The root is arbitrarily placed on the branch leading to P/agiomnium cuspidatum and Mnium thomsonii. Local bootstrap probabilities(%) are shown above branches.

Hypnum lindbergii-Calliergonella cuspidata clade was supported by high bootstrap value (100% LBP in ML tree). This result was suggested by Hedeniis (1990) based on morpholo­ gy and Tsubota et al. ( 1999) based on molecular data, and confirmed again in this study. 92 J. Hattori Bot. Lab. No. 88 2 0 0 0

~ ~ "O "Cl ~ ~ Brotherella complanata ~ -C.J ~ ~ Brotherel/a fauriei "Cl -:: ~ < ~ - ~ ..s. "'c Heterophyllium nematosum "Cl ~ ~ ~ ~ ~ "' Brotherella henonii 0 ::::::: ~ c ~ .~ -~ Brotherella herbacea ·- ~ ~ .s i."' Brotherella recurvans '5 ~ Q., ~ I ~ .s -~ .... ~ Wijkia hornschuchii ]j ~ ~ C.J Pylaisiadelpha tenuirostris ~ ~ '5 -...... l socladiella surcularis l: -.c ~ Q., Pylaisiella polyantha 0 Sematophyllum subhumile -e~ Entodon rubicundus ~ Hypnum cupressiforme l'1 Fi g. 3. Clades in the Sematophyllaceae. Clade A means the Brotherella complanata­ B. fauriei-Heterophyllium nematosum-B. henonii clade, clade B the Brotherella recurvans­ Hypnum tristo-viride- Wijkia hornschuchii clade. Broth erella clade unites with clade A, clade B and Brotherel/a herbacea. Brotherella-Pylaisiadelpha clade combines the Brotherella clade and Pylaisiadelpha tenuirostris. Sematophyllaceae (sensu present) clade unites with the Brotherella-Pylaisiadelpha clade, the Sematophyllaceae (sensu Seki) and Pylaisie/la polyantha.

The tree also suggests that the genus Entodon be placed in a clade near the Semato­ phyllaceae (sensu present). The clade consisting of the Sematophyllaceae (sensu present) and the Entodontaceae was supported with high LBP (98% LBP in ML tree). Hypnum cu­ pressiforme, which is the type species of Hypnum, appeared as a sister clade of the Semato­ phyllaceae (sensu present) and the Entodontaceae (89% LBP in ML tree). The tree also suggests that the phylogenetic relationship among the taxa was not so far remote fro m each other as expected formerly. Because each topology did not gain a decided advantage over other topologies, a 50% majority-rule consensus tree of high ranking trees was also obtained, and presented in Fig. 4. This tree also has a tendency toward the ML tree. The consensus tree also shows that the Hypnaceae are polyphyletic, and the Sematophyllaceae (sensu present) including some species so far treated as member of the Hypnaceae are monophyletic.

D lSCU SS!ON Monophyly of the fami ly Sematophyllaceae (sensu present) was revealed by both the most ML tree and the consensus tree. The length of the gene rbcL was the same fo r the six species obtained in the present study, and this fact suggests a monophyletic origin of these mosses. Manhart ( 1994) showed the stop codons in the hornwort rbcL gene. Meantime, H. TsuBOTA et al. : Phylogenetic relationships of the genus Brotherella and its allied genera 93

Brotherella complanata Brotherella fauriei Heterophyllium nematosum Brotherella henonii Brotherella recurvans Hypnum tristo-viride Sematophyllaceae Wijkia hornschuchii (present se nse) 98 Brotherella herbacea

100 Pylaisiadelpha tenuirostris lsocladiella surcularis Pylaisiella polyantha Sematophyllum subhumile Entodon rubicundus J Entodontaceae Hypnum cupressiforme Hypnum lindbergii 86 Calliergonella cuspidata Hypnum plumaeforme Pylaisiella intricata 69 Boulaya mittenii Abietinella abietina Plagiothecium neckeroideum] 100 ,. . h . Plagiotheciaceae r,1 agwt ectum nemora /e Rhytidiadelphus japonicus

79 Platyhypnidium riparioides ~ Rhynchostegium pallidifolium Brachytheciaceae Myuroclada maximowiczii Viola lutea Hedwigia ciliata Plagiomnium cuspidatum Mnium thomsonii

Fig. 4. The 50% majority-rule consensus tree for 42 trees with high-ranking log-like­ lihood values. The root is arbitrarily placed on the branch leading to Plagiomnium cuspida­ tum and Mnium thomsonii. The numbers for interior branches indicate the percentage of the group consisting of the times which are to the right of that fork occurred, and the branches with a frequency less than 50% were not shown.

Tsubota et al. ( 1999) reported no evidence of the presence of stop codon in rbcL gene for mosses. The present study also shows no evidence for it in the six species sequenced. This result also suggests the monophyletic origin of these mosses. The genus Brotherel/a can be regarded as belonging to a monophyletic group, togeth­ er with Hypnum tristo-viride, Heterophy/lium nematosum and Wijkia hornschuchii. This clade is supported by both ML tree and 50% majori ty-rule consensus tree. The relation­ ships among Brotherella, Wijkia, Heterophyl/ium and Hypnum were already indicated by Tsubota et al. ( 1999). Previous analyses on the karyotypes of Hypnum tristo-viride sug­ gested that it be closely related to the genus Brotherella (Sannomiya 1957; Yano 1960; 94 J. Hattori Bot. Lab. No. 88 2 0 0 0

Araki 1963). Taxonomic discussions have been made on the species complex involving Brotherella yokohamae, Clastobryella kusatsuensis and Pylaisiadelpha tenuirostris (Noguchi & Inoue 1981; Buck 1984; Ando et al. 1989). Noguchi and Inoue (1981) cytotaxonomically investi­ gated Brotherella yokohamae and Clastobryella kusatsuensis, and reduced C. kusatsuensis to the synonymy of B. yokohamae. However, they did not treat Pylaisiadelpha tenuirostris. Ando et al. ( 1989) considered that Brotherella yokohamae is conspecific with Py/aisiade/­ pha tenuirostris. Meanwhile, Tan & Jia (1999) regarded the two species as independent: Pylaisiadelpha tenuirostris and P. yokohamae ( = Brotherella yokohamae). They also re­ garded Brotherella integrifolia as synonymous with Hypnum tristo-viride. These results suggest that the genus Pylaisiadelpha is closely related to the genus Brotherella as suggest­ ed by Buck (1984). The present study based on molecular data support Buck (1984) [the Brotherella-Pylaisiadelpha clade in Fig. 3]. Tan & Jia (1998) showed that the family Sematophyllaceae (sensu Buck & Vitt) were paraphyletic, and recognized three clades in the family Sematophyllaceae: Sematophyllum clade, Brotherella-Wijkia-A canthorrhynchium-Warburgiella clade and Heterophyllium­ Clastobryopsis-Gammiella clade. Our present results show a clade, including the genera Brotherella and Wijkia and the species Heterophyllium nematosum and Hypnum tristo-viri­ de [the Brotherella clade], that is distinct as a resolved monophyletic group in all obtained trees. These results disagree with the Hedenas & Buck's ( 1999) treatment, in which Wijkia was placed in subfamily Wijkioideae of the Sematophyllaceae and Brotherella in the Hyp­ naceae. Robinson & Reed (1966) revised the genus Heterophyllium and concluded that the genus consists of only a single species: H. nemorosum (= H. affine). They also considered H. nematosum from Japan and H. piligerum from Vietnam (Tonkin), in which they found a similar morphology to Acanthocladiella. Although they synonymized the latter species with lsopterygium longitheca (Mitt.) Jaeg., the former species has been left without any taxonomic treatment. Tan & Jia (1998) made a cladistic study of the family Sematophyl­ laceae in China and treated the genus Heterophyllium. The genus Heterophyllium discussed in Tan & Jia (1998) should not be directly compared with the genus Heterophyllium repre­ sented by H. nematosum in our present tree, since Tan & Jia (1999) treated the genus as monotypic, consisting of H. affine. Additional phylogenetic analyses incorporating H. affine are needed for resolution of the phylogenetic position of Heterophyllium. Pylaisiella polyantha should also be considered in the Sematophyllaceae (sensu pres­ ent) clade. Our results confirmed Arikawa & Higuchi's (1999) suggestion that Pylaisiella polyantha and P. intricata do not form a monophyletic clade on the basis of rbcL analysis, showing the genus as heterogeneous. The Sematophyllaceae (sensu Seki) appears as paraphyletic to the Brothere/la-lineage. The fami ly Sematophyllaceae (sensu present) as a monophyletic group, also includes some taxa previously treated as members of the Hypnaceae. Thus the present circumscription is wider than those of both Nishimura et al. ( 1984) and Vitt ( 1984). Our results also showed that Brotherella and its allies are a specialized group of the Sematophyllaceae in the tem­ perate zone. This isolation might be correlated to the ecological requirements of these taxa in the lineage showing a habitat preference for growing on tree trunks. Further investiga- H. TsuBOTA et al. : Phylogenetic re lationships of the genus Brotherella and its allied genera 95 tion should be made with more species from a variety of genera of the Sematophyllaceae in the tropics: for example Acroporium, Taxith elium, Trismegistia and Meiothecium. The present study reveals at least that the characters of well-developed alar cells and their habi­ tat are significant for taxonomic recognition of the family Sematophyllaceae. The families Hypnaceae and Sematophyllaceae are large taxonomic groups including numerous species. The familial recognition of the Hypnaceae and the Sematophyllaceae varies among taxonomists. Any tree obtained from the present investigation shows the fam­ ily Hypnaceae to be polyphyletic regardless of the inclusion of the genus Brotherella in the family. Moreover, the species of the genus Hypnum treated here are dispersed in both the ML and consensus trees. Tsubota et al. (1999) reported that the family Hypnaceae as well as its type genus Hypnum itself proved polyphyletic, and this is corroborated by the present study supplemented by additional taxa. This fact suggests that the genus Hypnum needs further revision, although the genus has been carefully revised by taxonomic authorities (e. g., Ando 1986, 1995). Discussion on the relationship between the families Sematophyl­ laceae and Hypnaceae becomes more complicated, because the Hypnaceae is polyphyletic and Hypnum cupressiforme, type species of the genus, positions as a sister to the group with the Sematophyllaceae (sensu present) and the Entodontaceae. De Luna et al. (2000) showed the rough concept of the Sematophyllaceae, although lsopterygium would need to be transferred from the Hypnaceae. The present result also supported that the Sematophyl­ laceae is a monophyletic group in the same situation for Py/aisiella po/yantha. The present study also shows that the Entodontaceae is the sister group of the Semato­ phyllaceae (sensu present). Little discussion has been made on the relationship of the Entodontaceae to the Hypnaceae and the Sematophyllaceae from molecular viewpoints. Mizushima (1960) and Buck (1980) published monographic works on the Entodontaceae, and they have different views with regard to the systematic position of the genus Orthothe­ cium. Mizushima (1960) placed it in Entodontaceae, while Buck (1980) in the Hypnaceae. For further discussion, more molecular information is needed for the taxa of Orthothecium. The current treatment of the systematic position of Entodon should be revised, since the Sematophyllaceae (sensu present) has been proven to be closely related to the genus Entodon with high supporting values in all of the analyses, and the position of Entodon and Sematophyllum sometimes changes to each other. The results of the present study based on the rbcL gene do not concur with the classi­ fication and phylogeny of the order Hypnales proposed by Vitt (1984) and Buck & Vitt ( 1986) and the circumscription of the Hypnaceae by Nishimura et al. (1984), since some species currently treated as members of the Hypnaceae were placed in the Sematophyl­ laceae (sensu present). In general, the present concept of Sematophyllaceae based on a larger sample of exemplars agree with that of De Luna et al. (2000), which had fewer ex­ emplars, but based on a wider character set taken from three genes trnL-F, rps4 and rbcL.

ACKNOWL EDG EMENTS Particular thanks to W. R. Buck and E. De Luna for kindly reading the manuscript and making helpful comments with suggestions. We are also grateful to S. Takio and T. Arikawa for giving us valuable advice and guidance for the extraction of DNA, K. Suzuki 96 J. Hattori Bot. Lab. No. 88 2 0 0 0 for technique of PCR amplification and DNA sequencing, M. Hasegawa for advice about phylogenetic analysis, K. Tamura for advice and MEBoot. We would like to thank the DNA database members for keeping the system, as well as the PDS and GNU software program­ mers and other members of useful programs used in this project. Moreover, we would also like to thank several anonymous reviewers for their useful comments. We thank the Infor­ mation Processing Center in Hiroshima University for keeping the computer system and the Cryogenic Center in Hiroshima University for supplying cryogen. This study would not have been possible without their collaboration. Research on molecular systematics of mosses was funded by grants: MES SC (Japan) 11640698 to H. Deguchi and 10640683 to T. Yamaguchi.

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Appendix A. Information on specimens from which DNA was extracted for this study Here is a list of taxa whose rbcL sequences were utilized in this study with their ac­ cession numbers, showing their sources and voucher specimen information. Voucher speci­ mens are kept in HIRO. Treatment of families follows Vitt (1984) and Buck & Vitt (1986). The sequences of information are: the name of taxon, accession number, locality and specimen number, or reference source. Thuidiaceae Abietinella abietina (Hedw.) Fleisch., AF0055 l 9, Goffinet et al. (l 998); Leskeaceae Boulaya mittenii (Broth.) Card., AB024963, Tsubota et al. (1999); Amblystegiaceae Calliergonella cuspidata (Hedw.) Loeske, AB024678, Arikawa and Higuchi (1999); Brachytheciaceae Myuroclada maximowiczii (Borszcz.) Steere & Schof., AB029389, Tsubota et al. (1999); Platyhypnidium riparioides (Hedw.) Dix., AB029385, Tsubota et al. (1999); Rhynchostegium pallidifolium (Mitt.) Jaeg., AB024944, Tsubota et al. ( 1999); Hylocomiaceae Rhytidiadelphus japonicus (Reim.) T. Kop ., AB039788, Japan, Shikoku, Ehime-ken, Mt. Myojin, H. Deguchi 33244, this study; Entodontaceae Entodon rubicundus (Mitt.) Jaeg. & Sauerb., AB029386, Tsubo­ ta et al. ( 1999); Sematophyllaceae Brotherella complanata Reim. & Sak., AB039785, H. TSUBOTA et al.: Phylogenetic relationships of the genus Brotherel/a and its allied genera 99

Japan, Honshu, Hiroshima-ken, Miyajima Is., H. Tsubota 2295, this study; Brotherellafau­ riei (Card.) Broth., AB039786, Japan, Honshu, Gifu-ken, Mt. Kinkazan, N. Nakao 25, this study; Brotherella henonii (Duby) Fleisch., AB029 I 67, Tsubota et al. (1999); Brotherella herbacea Sak. ex Oti, AB039787, Japan, Shikoku, Ehime-ken, Mt. Myojin, H. Deguchi 33243, this study; Brotherella recurvans (Michx.) Fleisch, LI 3475, Manhart (1994); Het­ erophyllium nematosum (Bruch & Schimp. ex Sull.) Buck, AB02939 I, Tsubota et al. (1999); Pylaisiadelpha tenuirostris (Bruch & Schimp. ex Sull.) Buck (sensu Ando et al. 1989), AB039789, Japan, Honshu, Hatsukaichi-shi, H. Tsubota 2291, this study; Iso­ cladiella surcularis (Dix.) Tan & Mohamed, AB039784 (as Neacroporium jiagelliferum (Sak.) Iwats. & Nog.), Japan, Shikoku, Kochi-ken, Kuroson Gorge, H. Deguchi 33075, this study. Sematophyllum subhumile (C. Miill.) Fleisch., AB039675 (as Sematophyllum subhu­ mile ssp. japonicum), Japan, Honshu, Hiroshima-ken, Nishi-nomi-jima Is., H. Tsubota 2558, this study; Wijkia hornschuchii (Dozy & Molk.) Crum, AB029383, Tsubota et al. ( 1999); Plagiotheciaceae Plagiothecium neckeroideum Bruch & Schimp., AB024630, Arikawa and Higuchi (1999); Plagiothecium nemorale (Mitt.) Jaeg., AB029387, Tsubota et al. ( 1999); Hypnaceae Hypnum cupressiforme Hedw., AB039674, Japan, Honshu, Shi­ mane-ken, Hikimi-cho, H. Tsubota 2793, this study; Hypnum lindbergii Mitt., AB029390, Tsubota et al. ( 1999); Hypnum plumaeforme Wils., AB029384, Tsubota et al. ( 1999); Hyp­ num tristo-viride (Broth.) Par., AB024656, Tsubota et al. (1999); Pylaisiella intricata (Hedw.) Grout, AB024642, Arikawa and Higuchi (1999); Pylaisiella polyantha (Hedw.) Grout, AB024645, Arikawa and Higuchi (1999); Hedwigiaceae Hedwigia ciliata (Hedw.) P. Beauv., AF0055 l 7, Goffinet et al. ( 1998); Mniaceae Mnium thomsonii Schimp., AF005518, Goffinet et al. (1998); Plagiomnium cuspidatum (Hedw.) T. Kop., U87082 (as Mnium cuspidatum), Lewis et al. (1997); Orthotrichaceae Ulota lutea (Hook. f. & Wils.) Mitt., AF005540, Goffinet et al. (1998);

Appendix B. Homepage URLs and FTP sites of software for phylogenetic analysis and database fastDNAml download site (ftp://rdp.l ife .uiuc.edu/-gary/) MEBoot download site (ftp://evolgen.biol.metro-u.ac.jp/) MEGA download site (http://evolgen.biol.metro-u.ac.j p/MEGA/) MOLPHY download site (ftp://ftp.ism.ac.jp/pub/molphy/) PHYLIP download site (http://evolution.genetics.washington.edu/phylip.html) PUZZLE download site (http://members.tripod.de/korbi/puzzle/) or (http://www.tree-puzzle.de/) Sclean and our programs download site (http://home.hiroshima-u.ac.jp/chubo/) or linked with (http://home.hiroshima-u.ac.jp/miyajima/) Sequence data download site (http://ftp2.ddbj .nig.ac.jp:8000/cgi-bin/get_entry.pl? Accession number) (For example, a link to an entry with accession number AB029390 is (http://ftp2 .ddbj .nig.ac.jp: 8000/cgi-bin/get_entry.pl? AB029390))