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) were obtained from the field, and the voucher specimens are deposited in the herbarium of the National Science Museum, Tokyo (TNS). As all specimens used in the present study of Pylaisia have capsules, we could confirm peristome character, which are one of the most important taxonomic charac ters for the genus. Two Hookerialean samples were used as an outgroups, because De Luna et al. (2000) revealed that the Hookeriales was a sister group of the Hypnales sensu lato which includes taxa in the traditional Leucodontales and Hypnales (see also Buck et al. 2000). Total DNAs were extracted from fresh material or dried specimens by modifications of the CTAB method following our previous study (Arikawa & Higuchi 1999). Methods of amplification of rbcL gene segments and direct sequencing were also essentially the same as our previous study (Arikawa & Higuchi 1999, 2002). In addition to the eighteen se quences newly obtained, 80 sequences of rbcL were obtained from the DNA database of the DDBJ/EMBLlGenBank International Nucleotide Sequence Database Collaboration. All taxa treated in this study are shown in Table 2 with DDBJINCBIIGenBank accession numbers.
Data Analysis A total of 98 sequences treated in this study was aligned manually. The undetermined sites were cut to fit in codon position and the determined parts were concatenated to a sin gle data matrix. Methods of phylogenetic analyses were essentially the same as Tsubota et al. (1999, 2001 b) and Arikawa & Higuchi (2002). Maximum-parsimony (MP) trees were searched with PAUP* 4.0blO (Swofford 2002) using heuristic search strategy with the settings fol lowing Tsubota et al. (2001b), but random addition sequence was only with 10 replicates and zero-length branches not collapsed. A strict consensus tree also computed for all MP trees. In order to compensate less replication, MP trees were also searched with PAUPRat (Sikes & Lewis 200 I) through 10 independent Parsimony Ratchet searches of 200 itera tions each, following the manual. Log-likelihood of all MP trees was computed using NucML 2.3b3 contained in the MOLPHY version 2.3b3 package (Adachi & Hasegawa 1996), following Tsubota et al. (200 I b) and Arikawa & Higuchi (2002). ML trees were searched with the local rearrange ment method from the neighbor-joining (NJ) tree generated by the MOLPHY package (NucML and NJdist 1.2.5) and several high log-likelihood trees of MP trees. Tree compari son depending on the ML criteria by NucML was carried out to evaluate the resulting trees. Computer programs included in the PHYLIP (phylogeny inference package) ver. T. ARlKAWA & M. HIGUCHI: Phylogenetic analysis of Pylaisia and its relatives 91
Table 2. List of species and families investigated for rbcL gene with accession num bers of DDBJ/EMBLlGenBank international nucleotide database and origin of sequences. Treatment offamilies in this Table follows Buck & Goffinet (2000).
Taxon Accession No. Voucher or Reference
Pylaisioideae Giraldiel/a levieri Miill.Hal. AB091270 CHINA. Sichuan, Higuchi 35654. Platygyrium repens (Brid.) Schimp. AB091271 JAPAN. Nagano, Tanaka 3154. Pylaisia brotheri Besch. 1 AB091272 JAPAN. Ehime, Higuchi 34292. P. brotheri 2 AB091273 JAPAN. Nagano, Arikawa 2886. P. brotheri 3 AB091274 JAPAN. Yamaguchi, Arikawa 2923. P. brotheri 4 AB091275 JAPAN. Tottori, Arikawa 2502. P. brotheri 5 AB091276 JAPAN. Okayama, Arikawa 2499. P. brotheri 6 AB091277 JAPAN. Hokkaido, Arikawa 2680. P. brotheri 7 AB091278 JAPAN. Ohita, Higuchi 39332. P. cristata Cardot AB09l279 JAPAN. Tokushima, Arikawa 3219. P. Jalcata Schimp. AB09l280 CHINA. Sichuan, Arikawa 2181. P. intricata (Hedw.) Bruch & Schimp. AB024642 Arikawa & Higuchi (1999) P. polyantha (Hedw.) Bruch & Schimp. 1 AB091281 RUSSIA. Chegdomyn, Tan 97- 101. P. polyantha 2 AB09l282 FINLAND, Higuchi 34706. P. polyantha 3 AB091283 LATVIA. Riga, Stevenson, 1999. 'P. polyantha' 4 AB024645 Arikawa & Higuchi (1999) P. selwynii Kindb. AB091284 JAPAN. Hokkaido, Arikawa 2606. P. subcircinata Cardot AB091285 JAPAN. Fukushima, Arikawa 2479. Hypnaceae (excluding Pylaisioideae) Calliergonel/a cuspidata (Hedw.) Loeske AB024678 Arikawa & Higuchi (1999) Ctenidium molluscum (Hedw.) Mitt. AB024657 Arikawa & Higuchi (1999) Glossadelphus ogatae Broth. & Yasuda AB050950 Tsubota et al. (2001a) Herzogiel/a perrobusta (Broth. & AB034944 Arikawa & Higuchi (2002) Cardot) Z.Iwats. Hypnum cupressiforme Hedw. AB039674 Tsubota et al. (2000) H. lindbergii Mitt. AB029390 Tsubota et al. (1999) H. plumaeforme Wils. AB029384 Tsubota et al. (1999) H. tristo-viride (Broth.) Paris AB05099I Tsubota et al. (200Ia) Isopterygiopsis muelleriana AB034942 Arikawa & Higuchi (2002) (Schimp.) Z.Iwats. Isopterygium tenerum (Sw.) Mitt. AF233569 De Luna et al. (2000) I. vineale E.B.Bartram AB024650 Arikawa & Higuchi (1999) Orthothecium ruJescens AB050951 Tsubota et al. (200Ia) (Dicks. ex Brid.) Schimp. Taxiphyllum aomoriense AB024648 Arikawa & Higuchi (1999) (Besch.) Z.Iwats. Amblystegiaceae Hygroamblystegium tenax AF233565 De Luna et al. (2000) (Hedw.) C.Jensen Sciaromium tricostatum (Sull.) Mitt. AB024677 Arikawa & Higuchi (1999) 92 J. Hattori Bot. Lab. No. 94 2 003
Table 2. (Continued)
Taxon Accession No. Voucher or Reference
Hylocomiaceae Hylocomiastrum pyrenaicum (Spruce) AB024660 Arikawa & Higuchi (1999) M.Fleisch. ex Broth. Hylocomium splendens (Hedw.) AB024662 Arikawa & Higuchi (\999) Bruch & Schimp. Loeskeobryum cavifolium AB024658 Arikawa & Higuchi (1999) (Sande Lac.) M.Fleisch. Neodolichomitra yunnanensis AB024671 Arikawa & Higuchi (\999) (Besch.) T.J.Kop. Pleurozium schreberi (Brid.) Mitt. AB024664 Arikawa & Higuchi (1999) Rhytidiadelphus japonicus AB039788 Tsubota et al. (2000) (Reimers) T.J.Kop. R. loreus (Hedw.) Warnst. AB024666 Arikawa & Higuchi ( 1999) R. triquetrus (Hedw.) Warnst. AB024670 Arikawa & Higuchi (1999) Leskeaceae Miyabeafruticella (Mitt.) Broth. AB019475 Maeda et al. (2000) Okamuraea hakoniensis (Mitt.) Besch. ABOI9477 Maeda et al. (2000) Thuidiaceae Boulaya mittenii (Broth.) Cardot AB024963 Tsubota et al. (\999) Thuidium recognitum (Hedw.) Lindb. AB019476 Tsubota et al. (200\a) Campyliaceae Anacamptodon splachnoides (Brid.) Brid. AF23 1077 De Luna et al. (2000) Drepanoc/adus aduncus (Hedw.) Warnst. AB02468I Arikawa & Higuchi (\ 999) Tomentypnum nitens (Hedw.) Loeske AB024676 Arikawa & Higuchi (1999) Brachytheciaceae Brachythecium plumosum AF233566 De Luna et al. (2000) (Hedw.) Schimp. B. rivulare Bruch & Schimp. AB024674 Arikawa & Higuchi (1999) Myuroc/ada maximowiczii (Borszcz.) AB029389 Tsubota et al. (1999) Steere & Schof. Platyhypnidium riparioides AB029385 T subota et al. ( 1999) (Hedw.) Dixon Rhynchostegium pallidifolium AB024944 Tsubota et al. ( 1999) (Mitt.) A.Jaeger Stereophyllaceae Entodontopsis leucostega (Brid.) AB024635 Arikawa & Higuchi ( 1999) W.R.Buck & Ireland Stereophyllum radiculosum AB024637 Arikawa & Higuchi (1999) (Hook.) Mitt. Myriniaceae Helicodontium capillare AF233571 De Luna et al. (2000) (Hedw.) A.Jaeger T. ARIKAWA & M. HIGUCH I: Phylogenetic analysis of Pylaisia and its relatives 93
Table 2. (Continued)
Taxon Accession No. Voucher or Reference
Meteoriaceae Trachypodopsis auriculata AB024682 Arikawa & Higuchi (1999) (Mitt.) M.F1eisch. Trachypus hicolor Reinw. & Hornsch. AF233577 De Luna et al. (2000) Plagiotheciaceae Plagiothecium denticulatum (Hedw.) AB024624 Arikawa & Higuchi (1999) Bruch & Schimp. P. euryphyllum (Cardot & Ther.) Z.Iwats. AB024626 Arikawa & Higuchi (1999) P. neckeroideum Bruch & Schimp. AB024629 Arikawa & Higuchi (1999) P. nemorale (Mitt.) AJaeger AB024632 Arikawa & Higuchi (1999) P. undulatum (Hedw.) Bruch & Schimp. AB024634 Arikawa & Higuchi (1999) Fontinalaceae Fontinalis antipyretica Hedw. AB050949 Tsubota et al. (2001a) Entodontaceae Entodon challengeri (Paris) Cardot AB050993 Tsubota et al. (2001a) E. luridus (Griff.) AJaeger AB050994 Tsubota et al. (2001a) E. myurus (Hook.) Hampe AB024640 Tsubota et al. (1999) Pleuroziopsidaceae Pleuroziopsis ruthenica (Weinm.) AB024683 Arikawa & Higuchi (1999) Kindb. ex E.Britton Sematophyllaceae Acanthorrhynchium papillatum AB051224 Tsubota et al. (2001a) (Harv.) M.F1eisch. Acroporium stramineum (Reinw. & AB051225 Tsubota et al. (2001a) Hornsch.) M.Fleisch. Aptychella glomeratopropagulifera AB051217 Tsubota et al. (2001a) (Toyama) Seki Brotherella complanata AB039785 Tsubota et al. (2000) Reimers & Sakurai B. henonii (Duby) M. F1eisch. AB029 167 Tsubota et al. (1999) B. herbacea Sakurai ex Oti AB039787 Tsubota et al. (2000) Heterophyllium nematosum Broth. AB029391 Tsubota et al. (1999) ex P.de la Varde & Ther. Meiothecium microcarpum (Hook.) Mitt. AB051223 Tsubota et al. (2001a) Pseudotrismegistia undulata (Broth. & AB051229 Tsubota et al. (200Ia) Yasuda) H.Akiyama & Tsubota Pylaisiadelpha tenuirostris (Bruch & AB051219 Tsubota et al. (200Ia) Schimp. ex Sull.) W.R.Buck Rhaphidostichum macrostictum AB051220 Tsubota et al. (2001a) (Broth. & Pari s) Broth. Sematophyllum subhumile subsp. AB039675 Tsubota et al. (2000) japonicum (Broth.) Seki Taxithelium planum (Brid.) Mitt. AF233573 De Luna et al. (2000) 94 1. Hattori Bot. Lab. No. 94 2 0 0 3
Table 2. (Continued)
Taxon Accession No. Voucher or Reference
Trismegistia korthalsii (Dozy & AB051227 Tsubota et al. (200Ia) Molk.) Broth. Wijkia defiexi/olia (Mitt. ex Renauld & AB051221 Tsubota et al. (200 Ia) Cardot) H.A.Crum W hornschuchii (Dozy & Molk.) AB029383 Tsubota et al. ( 1999) H.A.Crum Cryphaeaceae Cryphaea sinensis E.B.Bartram ABO l9457 Maeda et al. (2000) Leucodontaceae Dozyajaponica Sande Lac. ABOl9446 Maeda et al. (2000) Felipponea esquirolii (Ther.) H.Akiyama ABOl9447 Maeda et al. (2000) Leucodon nipponicus Nog. ABO l9451 Maeda et al. (2000) L. sohayakiensis H.Akiyama ABOl9455 Maeda et al. (2000) L. temperatus H.Akiyama ABOl9456 Maeda et at. (2000) Leptodontaceae Forsstroemiajaponica (Besch.) Paris AB0 19450 Maeda et al. (2000) Anomodontaceae Anomodon abbreviatus Mitt. ABOl9468 Maeda et al. (2000) A. giraldii Miill.Hal. ABO l9469 Maeda et al. (2000) A. rugelii (Mi.iIl.Hal.) Keissl. ABOl9470 Maeda et al. (2000) Haplohymenium longinerve ABOI9472 Maeda et al. (2000) (Broth.) Broth. Herpetineuron toccoae (Sull . & ABOl9474 Maeda et al. (2000) Lesq.) Cardot
Hypopterygiaceae (out group) Hypopterygiumffavolimbatum AB091286 JAPAN. Chiba, Arikawa 3521. Mi.iII.Hal Hookeriaceae (out group) Hookeria acuti{olia Hook. & Grev. AB09 1287 JAPAN. Chiba, Arikawa 3520.
3.572c (Felsenstein 1995) were also used for the bootstrap test fo llowing Arikawa & Higuchi (2002).
RESULTS Sequence Comparison The rbcL gene was successfully sequenced for 19 samples (Table 2). Their sequences have not been used in previously published phylogenetic constructions. No insertion or deletion event was found. Table 3 shows the differences within the 1,239 bp of the rbcL gene among 19 sequences of Py laisia and its presumed related taxa. The upper-right corner of this table shows the number of nucleotide differences. The lower-left corner indicates the Table 3. Uncorrected pairwise differences among rbcL sequences. Above diagonal: total number of differences of the nucleotide sequences within 1,239bp. Below diagonal: differences of estimated amino acid sequences within 413aa. Boxes indicate pairs which have no difference.
:-l Taxon 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 > ------'"~ 1 P. brotheri 1 o 0 5 8 10 11 11 13 14 14 14 24 41 40 ~ 2 P. brotheri 2 o 0 5 8 10 11 11 13 14 14 14 24 41 40 p,o 3 P. brotheri 3 o 0 5 8 10 11 11 13 14 14 14 24 41 40 ~ :r:: 4 P. brotheri 4 o 0 0 2 2 2 6 9 11 12 12 14 15 15 15 25 42 41 is c: 5 P. brotheri 5 o 0 0 o 6 7 9 10 10 12 13 13 13 23 40 39 (") 6 P. brotheri 6 o o o o 1-o 0 0 6 7 9 10 \0 12 13 13 13 23 40 39 ~ 7 P. brotheri 7 o o o o o 0 6 7 9 \0 \0 12 13 13 13 23 40 39 ~'"" 8 P. subcircinata 2 2 2 2 2 2 2 5 7 8 8 \0 11 11 13 21 42 41 c2 (1) 9 P. Jauriei 4 4 4 4 4 4 4 2 4 7 7 9 8 8 12 18 39 38 ~ 10 P. intricata 4 4 4 4 4 4 4 2 2 3 7 7 6 6 11 20 37 36 ";' § 11 P. cristata 3 3 3 3 3 3 3 3 1 6 4 7 7 \0 23 39 38 oo 12 P. Jalcata 3 3 3 3 3 3 3 2 2 2 6 9 9 7 22 41 40 ~ 13 P. polyantha 1 4 4 4 4 4 4 4 2 3 3 3 5 5 \0 25 40 39 o...., 14 P. polyantha 2 4 4 4 4 4 4 4 2 2 2 3 2 22 37 36 1- 0 11 ~;s 15 P. polyantha 3 4 4 4 4 4 4 4 2 2 2 3 2 o 11 22 37 36 <:;. is' 16 Giraldiella levieri 4 4 4 4 4 4 4 2 3 3 3 2 :.; 3 25 42 41 oo 5. 17 Hypnum lindbergii 4 4 4 4 4 4 4 2 2 2 3 2 3 2 2 3 47 46 @ a:;;:. 18 Platygyrium 11 11 11 11 11 11 11 9 9 7 8 9 8 7 7 10 9 ~ repens 19 Pylaisia 11 11 11 11 11 11 11 9 9 7 8 9 8 7 7 10 9 o polyantha 4
'-0 V> 96 1. Hattori Bot. Lab. No. 94 200 3 number of differences of estimated amino acid sequences. Sequences of P brotheri 1, 2, and 3 were identical to each other, and those of P broth eri 5, 6, and 7 were also identical to each other. There is one base difference between P brotheri 1- 3 and P brotheri 4, and there is also one base difference between P brotheri 1- 3 and P brotheri 5- 7. There are two base differences between P brotheri 4 and P brotheri 5- 7. Then, three types were found within the rbcL sequences of P brotheri. All differences within the sequences of P brotheri were transitional and synonymous, thus not changing the estimated amino acid sequences. There were 7- 9 nucleotide differences (4 amino acid differences) between P brotheri and P selwynii. There were 5- 6 nucleotide differences (2 amino acid differences) between P brotheri and P subcircinata. The sequences of P polyantha 2 and 3 were identical to each other, and there were five base differences between them and that of P polyantha 1, which was collected in the upper Bureya River and had acute to shortly acuminate branch leaves. There is one difference be tween the estimated amino acid sequences of them. These differences are larger than the difference between P intricata and P cristata (three nucleotide, one amino acid). There were significant differences of 36- 39 nucleotide substitutions between P polyantha 1- 3 and that of P polyantha 4, which is the culture material labeled as Pylaisia polyantha. Pylaisia polyantha 4 was obtained from the Culture Collection of Autotrophic Organisms at Trebon, Institute of Botany, Academy of Sciences of the Czech Republic: no. 667, strain KEIL 1949/752. By contrast, there was only one transitional and synonymous nucleotide difference between the sequence of P polyantha 4 and that of Platygyrium repens, while there were at most two transitional and synonymous nucleotide differences among the samples of P brotheri. The culture material labeled as Pylaisia polyantha is not P polyantha, but rather is surely Platygyrium repens. There were 7 differences between the nucleotide sequence of Giraldiella levieli and that of P falcata . There is only one difference between the estimated amino acid sequence of them. The range of nucleotide differences was from three to fifteen within the genus Py laisia (including Giraldiella, excluding P polyantha 4). These differences caused 1--4 dif ferences of estimated amino acid sequences. The sequence of Hypnum lindbergii is one of the most similar sequences as that of Pylaisia. There were 18 or more differences between the sequence of H. lindbergii and that of Pylaisia. The sequence comparison of the present study suggested that the genus Pylaisia including Giraldiella levieri is homogeneous.
Phylogenetic Analysis A total length of 1,224 bp (the region from the position 73 to 1,296) was used for the
Fig. l. The hi ghest log-likelihood tree of the aligned rbcL gene sequences (1 ,224 bp; HKY85 model; 2alj3 =8.489; InL =-9,484.04 ::':: 477.27 by NucML; 1,211 steps by MP crite ria). The horizontal length of each branch is proportional to the estimated number of nu cleotide substitutions. The root is arbitrarily placed on the branch leadin g to Hookeria aculi fO lia and Hvpopterygium ffavolimbatum. Local bootstrap probabilities (LBPs; in % ) are shown above branches. Bootstrap values (%) 10% or higher are plotted below the branch (by NJ /by MP). T. ARIKAWA & M. HIGUCHI : Phylogenetic analysis of Pylaisia and its relatives 97
===:~~~::::::::::::::::::~~~;;;;~::::::::::::~H~~~.~n.~OW~"~~~i.34 ______Hypopterygium ft avolimbaum ~ Fonlinalissntipyretica Tanentypnum nitens
1---;i~--~~;rl=~~~Orepa'loolaciJs Anecamptodoo aduncus splechnades Hygroam~ystegum tanax ..------HerzO!iella perrotuSa
• 33 ."....._2.".....poIrIntIIol,. ~."....._ I"Pylaisia c1ade" 45/36- PytIIUI cr-... ~ PJt.WI~nkIII"l.vtert """"-'10 Galliergonella cuspdata 100 100 Hypnum lindbergii
" 77/9 "..br'cltMri1-3 90·:--==.:..·1 Hylocomiastrum pyr",""m Pleurozium sc:hreberi I ..----:;!i*;--{__ ==-::Rh~r=:~~!~klJS ...------HylOCOOliurn spendens Hylocomiaceae ,----- Loaskeobryum cavifobum ..------RhybdiadelJt1us tri~etrus ctenidium molluscum 88 Sciaromium Incoetatum Pleuroziopeis ruthenica ..------Cryphaea !linenss
5452 86 7 m 8 _l~~~:S 70/72 Loucodal 9OI1aya."".' , ,ILeucodontaceae ~ 3 6 Dozya Japoruca - /33 Fellpporlaa ~irolil
Orthothecium ruleecens ISOptel'ygiop9iS muelleri8l18 l::~~~~:=~~~~~,~~~gi~um~.~'~~~.~PlagioChecium eoryphyllurn Plagiothecium neckeroideum r:;:::==:..':PI&QiotheciUm undulal1Jm 1..£ Plagiatlecium denticulatum ,'---- Plagiothecium nernor8le ,----- Anomodal rugalii --:-:---;::::::::::::::~Hy~pr1~U~m~CU~p FOf'SStroemia japooica _~:~~~~~~~~~~~A:,~omodongiraldiAncmodoo abbreviatus i r Haplohymenium long nerve L. ____--;~too-----C===-:::E'Iod""Io"..,"'- 100 100 steraophyllum radiculO9Jm I Stereophyllaceae ...------Neodoiidlomilra yunnmensis L Hypnum plumaeforme --~~~~====Bw~Ia~ya~m;,i~:=ii====:{::~~::10 0 Rhynchostagium pallidfoiium o 10 0 Platyhypnidium ripariddes Helicodontium capillare - • 96 "',achy1h ,"",um ",um"",m IBrachytheClaceae ,:~::~::~~~~~~~~~~u~r~~h~.~k,,"~i"'~siS63 47 Brachythecium nvulare _ " 2 74 Myuroc\ada ma»mowicZII 94 ~ TradlypodopSs auricolata I ' Tri~egistiakorltlalsii ' ~ ___ I(~~~~~~~~~T~~y~~~.~.~' c~~~~~~~~~~e~t~e~o~n~aceaePgeudOOi~8!jstia 1J1ci..data Sematophyllum subhumile subsp. japooicum ..------Meiolheci.Jm micro::arpxn Acroporium straonineum y')1rrC======;;;;;;;;;;;;;;;;r-; 4 41 fflaphidostichum m~rostictum Thuidium recognitum Herpetineural toccoae I '-lomrt,H-r£:==:-:Enlodoo Challengel1 E,.-.Iundu. IEntodontaceae 63 84 79 Entodon myurus 1---;::::::====:::::;T:"';"~~.~iU::'':'''yr1Ch.m pa"Ualum Sematophyllaceae --_~,,--c==::::::::::===~I!qtory~~,,::::um tenerum r 77 79 Aptychella glaneratopropagulifera ...---,;fA;r--C::::=-:::::::::::::- Bfotherella herbacea 100 100 /100 0.Q1 substtturions/stte ..------Brotherella henonii Brotherellacompla'lata Hetero~yllium nemat09Jm 98 1. Hattori Bot. Lab. No. 94 2 003 following phylogenetic analyses. Among the 1,224 bp over 98 taxa, 335 sites (27.4%) were variable and among these 224 sites (18.3%) were phylogenetically informative. The MP analysis using PAUP* resulted in 1,931 MP trees of length 1,211 . As 449 trees were of a duplicate topology, there were 1,482 unique topologies. When zero-length branches were collapsed and duplicate topologies were eliminated, 228 topologies sur vived. The search using PAUPRat resulted in 96 topologies of length 1,211, all of which were included in the 228 topologies generated by PAUP*. No shorter tree was found by the search using PAUPRat. The highest log-likelihood within 1,482 topologies was -9484.04±477.27. Three topologies have the same log-likelihood, and they were identical when zero-length branches were collapsed. Following these three topologies, the log-likelihood of twelve topologies is -9484.05±477.27. ML trees were searched with the local rearrangement method using these fifteen topologies as seed trees, but the result of this search is identical with one of the highest log-likelihood topologies within the 1,482 MP topologies (Fig. I). The log-like lihood of NJ tree is -9582.19, and that of the tree searched with the local rearrangement method using the NJ tree was -9516.09±481.88 (Fig. 2). A total of 1,485 topologies were compared with each other using log-likelihood value (Table 4). Consequently, the best is the highest log-likelihood tree of MP trees (Fig. I). All the MP topology did not gain a de cided advantage over other topologies. A strict consensus tree of all MP trees was also ob tained (Fig. 3). The best tree (Fig. I) did not gain a decided advantage over the tree on Fig. 2; the differences in log-likelihood estimated by Kishino & Hasegawa's (1989) formula is -32.0±47.0. Three types of Pylaisia brotheri form a monophyletic clade (92% LBP in Fig. I, 64% LBP in Fig. 2, 90% bootstrap values by NJ and 84% by MP). This clade forms a clade with P subcircinata in Figs. I and 3 (64% LBP, 80% by NJ and 71 % by MP). Hypnum plumae forme is also included in the clade in Fig. 2 (96% LBP). Figs. I and 3 indicate the affinity with Hypnum plumaeforme and the Brachytheciaceae, though bootstrap values were low. Calliergonella cuspidata and Hypnum lindbergii form a clade (100% LBP in Fig. I, 96% LBP in Fig. 2, 100% by NJ and MP) and, indeed, H. lindbergii has been placed in Callier gonella (Hedenas 1990). These two clades form a clade with P selwynii (90% LBP in Fig. I, but bootstrap values were lower than 20% by NJ and MP). Pylaisia falcata and Giraldiella levieri form a clade (89% LBP in Fig. I, 73% LBP in Fig. 2, 63% by NJ and 65% by MP). Pylaisia polyantha I (Far East Russia) and P polyan tha 2, 3 (North Europe) do not form a monophyletic clade in all trees. All Pylaisia (exclud- Fig. 2. The tree searched with the local rearrangement method from the neighbor joining (NJ) tree of the aligned rbcL gene sequences (1,224 bp; HKY85 model; 2al{3=8 .364; In L= -9516.09±481.88 by NucML). The horizontal length of each branch is proportional to the estimated number of nucleotide substitutions. The root is arbitrarily placed on the branch leading to Hookeria acutifolia and Hypopterygium fiavolimbatum . Local bootstrap probabilities (LBPs ; in %) are shown above branches. The highest log-likeli hood topology (Fig. I) did not gain a decided advantage over this tree; the differences in log likelihood estimated by Kishino and Hasegawa's (1989) formula is - 32.0±47.0. T. ARlKAWA & M. HIGUCHI: Phylogenetic analysis of Pyiaisia and its relatives 99 ~------H~ru~l~ Entoooo challmgert I " " Enlal",kJ,.." Entodontaceae EnlodCllITl)\lRlS _>L-I----- Sdarornum trlCoslatum Pleurozlq>Slsrullenlca 61 Toment)pnum nltens l--...!.2lL--lIe~;::==:;;HllIroarIllysIEgkJm M~tooCll ~lr:hncides I""", OrepmcxDjus oouncus I"Pyla"" cl.. ," ~------:!.!...------.,--- C8Merplella cuspldata Hypnumllndbergll I L..::;'-l. ...------=-- Hy",um ~umaBorme I {:=r=~=~~::~==::~~~m~ru~~~~~ormeMycbea lrutk:ella _ c:======~G~loosoIeIp~T8ldph~lumaCtOOriense husogsae RagIOll E __ ~~~::::::::::::~o~~::r:~~h~a~rommssH9icodOntll1l ~.a'9:_ . f BrO:hylledumpilrnoarm IBrachytheClaceae " L_ !9~O _ _.1"'"------Bra::hylledum~",Jlare T""hjJlUSblcdorTractlypOOql9S aniculala I Meteonaceae. ThJldlurn rocognltlm ::~::'i~~~~~~~~~~~~~~~~a~m;~~:~T~gneghila konhaIsIl { PseudOtri9Tl9gISlaundulata .------Sanalqlllyllum 9Jbhurrile~ . JlI!DlbJm [~~::::::::::::~~~;;OOl;;m~~~~m54 AcqK:rIumstrllTineum 11 Rh~umma::rosIictUm Sematophyllaceae .------_orrh)fdllum paplllalOOl 98 Isq)IEII')glumlEllerum 69 {~~==:;====c:::~T~~~."~m~~~m~===~~~ A!>1jd1".g __IH ... 0.01 substiturionslsite L..______.....!:10!.!O ______--l"cu=:=~.' 4 ~--....!!.---I===-;\\!;;;i~:a:de:n.::.;;::-Ia Erotherellaherba::ea Hypnumlnsto-\4ri1e l'j1oliolophallllulrostn, W~klahoms;huchH ~------BrOllerel~ h"''''1I _laOOl11llillala 1-______Heterqlh)1llum nElTlaiOfllm 100 J. Hattori Bot. Lab. No. 94 2 003 Table 4. NucML analyses for 1,224 bp of 93 rbeL sequences. All resulting trees of the present study were evaluated by the comparison depending on the ML criteria by NucML. The MP trees from 142nd to 1,337th (1,196 trees) are omitted from this Table. The best tree is the highest log-likelihood MP tree. The differences in log-likelihood of al ternative trees from that of the best tree (In Li- In LM1) are shown with their SE following ± estimated by Kishino & Hasegawa's (1989) formula. Tree ranking 1nL InLi-lnLM1. -9484.0 Best best MP, ML 2- 15 -9484.0 O.O±O.O MP 16- 30 - 9484.3 -0.3± 12.9 MP 31-45 -9484.5 - 0.5±7.4 MP 46- 51 - 9484.6 -0.6±0.9 MP 52- 75 -9484.7 -0.6± 1.2 MP 76- 81 - 9484.7 - 0.6 ± 1.1 MP 82- 96 - 9484.8 - 0.8± 14.8 MP 97- 132 - 9484.9 - 0.9 ± 13 .0 MP 133-135 - 9485.1 - 1.0± 11.5 MP 136- 141 - 9484.1 - 1.1 ± 7.5 MP 1338- 1364 - 9508.0 - 24.1 ± 29.5 MP 1365- 1418 - 9508.1 - 24.1 ±29.6 MP 1419- 1424 -9508.1 - 24.1 ± 26.1 MP 1425- 1428 - 9508.3 - 24.2 ± 26.1 MP 1429- 1434 - 9508.3 - 24.3 ± 27 .5 MP 1435- 1446 - 9508.4 - 24.4 ± 27 .6 MP 1447- 1452 -9510.5 - 26.5 ± 30.2 MP 1453- 1464 -9510.6 - 26.6±30.3 MP 1465- 1470 -9511.6 -27.6±3 1.0 MP 1471 - 1482 -9511.7 -27.7± 31.1 MP 1483 - 9516.1 - 32.0±47.0 ML from NJ 1484 - 9582.2 -98.1 ± 47.4 NJ ing P polyantha 4), and Giraldiella levieri form a "Pylaisia clade" in Fig. I and Fig. 2 (67% LBP in Fig. I, 64% LBP in Fig. 2, lower than 20% by NJ and MP), although P polyantha 2, 3 are not included in the definite "Pylaisia clade" in Fig. 3. The "Pylaisia clade" includes the clade of Calliergonella euspidata and Hypnum lindbergii in Fig. I, al- Fig. 3. The strict consensus tree for 1,482 MP trees (1 ,211 steps) deduced from rbeL nucleotide sequence data (1,224 bp). The root is arbitrarily placed on the branch leading to Hookeria aeutifolia and Hypopterygiumjfavolimbatum. Bootstrap values (%) 20% or higher are plotted below the branch (by NJ/MP). T. ARIKAWA & M. HIGUCHI: Phylogenetic analysis of Pyiaisia and its relatives 101 HOOkeria aculildia Hypopterygium tlavdimbatum Fartinalisanlipyretica Tanent'@1omnillrls Anacamptodal gpa::hnoides 9890 Orepwloolal1Js atincus 8768 HygrwnblyS8gil,m tenax Hen:ogiella perrobua... 54 / 33 -_--,-,-_.- GlhIId .... ~ -1OIwyn' "Pylaisia clade" --Caliergooella cuspdata Hypnum_ou_ lindbergi I PytIIIAIbnJthert5-7 -_.PyIiIIAIbratherl 1 -S Hytooomiastrum pyRlf'laicom I 48 47 Pleurozium schraberi Ally'lidadelptlJsjapc1licus -/- , 92 RhytidiadelptlJs lOfaJS Hylocomium gpendens Hylocomiaceae L08!keobryum ccMfoIium Rhytidiadelj1lustriquetrus 263S Ctenidium moilu9ClJm I Sciaromium tricoSatum Pleuroziopas rutheniCa Crnil8E18 Sinensis leucoclc.rl nippmiaJs 7 .. Leucodon temperaluS Leucodon 90hayakienlis Oozyaj8pXlica Leucodon taceae - 33 Flllippcl'l88891lJirolii lsopterygium Yineele 20 - OrIhothwum ru1eaoens Isopterygiop!lismueller18J18 4831 PlaglOIhecium euryJilyllum Plagiothecium necl C,*~m_... '_ poIynN" 100/100 Brollerella hernacea Sematophyllaceae 91/96 IMjkiadeftexildia HYp1umtri9lo-virlde Pylaisiadalr;tla tenuirostris 99/90 Wjlda homSChuchii BroIherelia henonii BrOllereUacanpl.-.ata 15/68 Heterop"lyllium nematoaum 102 1. Hattori Bot. Lab. No. 94 200 3 though the branch leading to the clade of C. cuspidata and H. lindbergii is longer than other branches in the "Pylaisia c1ade". The "Pylaisia c1ade" in the tree of Fig. 2 contains also Hypnum plumaeforme, although the branch is also longer than other branches. Orthothecium rufescens forms a c1ade with !sopterygium vineale (62% LBP in Fig. I, 52% LBP in Fig. 2, 20% by NJ and 17% by MP). Three species of Entodon form a mono phyletic c1ade (98% LBP in Fig. I, 100% LBP in Fig. 2, 100% by NJ and 99% by MP), and this clade shows close affinity with the Sematophyllaceae in Fig. 1 and Fig. 3. Platygyrium repens and P polyantha 4 (culture material labelled as P polyantha) form a c1ade (100% LBP in Fig. 1 and Fig. 2, 100% by NJ and MP) which is included in the Sematophyllaceae c1ade. DISCUSSION (I) Whether the genus Pylaisia is paraphyletic According to the sequence comparison, the culture material labeled as Pylaisia polyantha proved to be not of P polyantha, but of Platygyrium repens. Although we had observed only a few leaves of the culture material in our previous study (Arikawa & Higuchi 1999), we were unable to discover the misidentification. In culture conditions, morphological changes are often observed. When bryophytes are cultured, voucher speci mens should be preserved for checking the identification. Here, we rectify the conclusion of our previous study (Arikawa & Higuchi 1999) that the genus Pylaisia is heterogeneous. Nucleotide sequences of rbcL indicate that the genus Pylaisia is homogeneous includ ing Giraldiella levieri, although phylogenetic trees did not indicate that they are a mono phyletic group. Calliergonella cuspidata, Hypnum lindbergii, and H. plumaeforme were contained in the clade of Pylaisia in some trees, although their branches are very long and branches around there are not supported with high bootstrap values. These three species are usually placed in the Hypnoideae, which usually have a perfect peristome of Hypnoid type, while the Pylaisioideae show a tendency toward remarkable reduction in peristome struc ture. Although the genus Pylaisia is probably a phylogenetically monophyletic group, in cluding Giraldiella, we could not conclude whether the genus Pylaisia is monophyletic be cause of insufficient information. for further discussions, additions of other gene sequence are needed. (2) The subfamily Pylaisioideae Platygyrium has been placed in the Hypnaceae subfamily Pylaisioideae (see Table I). Platygyrium repens is widely distributed in the Northern Hemisphere. There was one tran sitional and synonymous nucleotide substitution between the sequence of the culture mate rial labelled as Pylaisia polyantha and that of Japanese Platygyrium repens. Arikawa & Higuchi (1999) reported that one to five transitional and synonymous differences of the nu cleotide sequence of rbcL were found in five moss species, such as Plagiothecium denticu lalum, P euryphyllum, P neckeroideum, P nemorale and Rhytidiadelphus triquetrus, in which two samples from different localities were examined. A close comparison of Japan ese Platygyrium repens with European plants proved that there is no morphologically dif ference between them (Higuchi & Nishimura 1984). The genus Platygyrium is easily dis- T. ARIKAWA & M. HIGUCHI: Phylogenetic analysis of Pyiaisia and its relatives 103 tinguished from Pylaisia by the structure of the peristome: the basal membrane of the en dostome is very poorly developed and is not exserted above the mouth of the capsule, the endostome teeth are narrowly linear, and cilia are absent (cf., Warburg & Perry 1965). Moreover, Platygyrium can be differentiated from Pylaisia by the mode of spiral twisting of the seta; the seta of Platygyrium is sinistrorsely twisted throughout, but the seta of Py laisia is sinistrorsely twisted at the lower portion of seta and dextrorsely twisted above. A better developed annulus, presence of propaguliferous branchlets, recurved leaf margins, and filamentous pseudoparaphyllia are also distinctive features of Platygyrium (Higuchi & Nishimura 1984). The present study suggests that Platygyrium is not closely related to Py laisia and should be transferred to the Sematophyllaceae (sensu Tsubota et al. 2000, 200Ia). Giraldiella is monotypic and endemic to the Chinese mainland and Taiwan. Fleisher (1923) placed it in the Pylaisioideae, although Muller (1898) described it in the Entodon taceae because of the erect capsule. Giraldiella levieri can be distinguished from the species of Pylaisia and related taxa by the hyaline alar cells and the much longer endos tome segments than the exostome teeth. Reimers (1931) and Toyama (l938b) transferred the genus to the Sematophyllaceae subfamily Macrohymenioideae because of the peris tome structure. Seki (1968) suggested that Giraldiella levieri did not belong to the Semato phyllaceae, but to the Pylaisioideae, because Giraldiella levieri and Pylaisia brotheri were closely associated with each other in his factor analysis. Giraldiella is currently placed in the Hypnaceae (e.g., Walther 1983; Vitt 1984; Buck & Goffinet 2000). The present study shows a close affinity between Giraldiella levieri and Pylaisia falcata. Further examination based on morphological characters is needed for the relationship between Giraldiella levieri and the genus Pylaisia. Although Orthothecium has ever been placed in the Entodontaceae (e.g., Fleischer 1923; Brotherus 1925; Noguchi 1994), it is currently placed in the Hypnaceae (e.g., Walther 1983 ; Vitt 1984; Buck & Goffinet 2000) since the revision of the Entodontaceae by Buck (1980); moreover, Tsubota et al. (200 I a) supported the exclusion of the genus from the Entodontaceae. Walther (1983) placed Orthothecium in the subfamily Py laisioideae of the Hypnaceae. On the other hand, Hedeniis (1987) placed Orthothecium in the Plagiotheciaceae. The phylogenetic tree of the present study suggests that Orthotheci um does not have a close relationship to either the Entodontaceae, or to Pylaisia. Although Nishumura et al. (1984) mentioned the close relationship between Pylaisia and Entodon taceae, the phylogenetic tree of the present study does not indicate it. The present study shows that the subfamily Pylaisioideae M.Fleisch. is polyphyletic, although we could not add to the genera assigned to the subfamily in present analysis. Fur ther phylogenetic analyses are needed for resolution of the phylogenetic relationship of the Hypnaceae and its subfamilies. (3) The relationship between P. brotheri and P. selwynii The peristomes of Pylaisia brotheri, P. subcircinata, and P. selwynii resemble each other closely in general structure. Although P. subcircinata can be distinguished from the related taxa by the long, subulate leaves and the small alar regions, P. brotheri is only 104 1. Hattori Bot. Lab. No. 94 2 003 slightly distinguishable from P selwynii by its broader leaves, larger alar regions and larger capsules. Noguchi (1994) mentioned that it was questionable whether these species could be specifically distinguished from each other. However, the result of the present study re veals that these three taxa can be recognizable as distinct species. It was not P selwynii but P subcircinata, with which P brotheri forms a monophyletic c1ade. Three types were found within the rbcL sequences of seven samples of P brotheri. These three types seem not to correspond to either geographical patterns or morphological differences. The rbcL sequences of P brotheri 6 (Hokkaido, northeastern Japan) and P brotheri 7 (Ohita, southwestern Japan) were identical. The distribution range of each type overlapped each other. For further discussions, detailed observation of morphological char acters, further exhaustive additions of samples, and employment of more suitable molecu lar marker which evolves faster are needed. (4) Populations of P polyantha from Russian Far East The differences in the sequences between northern European P polyantha and the dis tinct population collected from the Russian Far East are larger than those between P infri cata and P crisfata. Northern European P polyantha and the Russian population did not form a monophyletic c1ade. Although the peristome and spores of the Russian population are identical with those of P polyantha, the plants have acute to shortly acuminate branch leaves, which make the branch foliation terete and the overall appearance quite distinct from ordinary P polyantha (Ignatov et al. 2000). The Russian Far East population was also phylogenetically distinct from northern European P polyantha. As many infraspecific taxa have been described in P polyantha, further detailed examination and discussion on the morphological features are needed for resolution of the taxonomic treatment of the popula tion. ACKNOWLEDGEM ENTS We wish to thank Or. B. C. Tan, Mr. A. Tanaka and Mr. C. R. Stevenson for providing material, Dr. 1. Lukavsky for kindly providing culture strain and information, Dr. H. Tsubo ta for useful comments and advice during the experiments and the data analyses, and Dr. W. R. Buck for reading the manuscript and valuable comments. This study was partly sup ported by a Grant-in-Aid (nos. 10041186, 13640707) from the Ministry of Education, Cul ture, Sports, Science and Technology, Japan. LiTERATURE CiTED Adachi, 1. & M. Hasegawa. 1996. Computer Science Monographs, 28. MOLPHY version 2.3 . Pro grams for molecular phylogenetics based on maximum likelihood. 150 pp. The Institute of Sta tistical Mathematics, Tokyo. Arikawa, T. & M. Higuchi. 1999. Phylogenetic analysis of the Plagiotheciaceae (Musci) and its rela tives based on rbeL gene sequences. Cryptogamie, Bryol. 20: 231 - 245. Arikawa, T. & M. Higuchi. 2002. Phylogenetic position of the genera Isopterygiopsis and Herzogiella (Musci) based on rbeL gene sequences. Bryol. 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