Hikobia 16: 221–228. 2012

Phylogenetic note on Pachyneuropsis miyagii T.Yamag. (Pottiaceae, Bryophyta)

YUYA INOUE, HIROMI TSUBOTA, HIROYUKI SATO AND TOMIO YAMAGUCHI

INOUE, Y., TSUBOTA, H., SATO, H. & YAMAGUCHI, T. 2012. Phylogenetic note on Pachyneuropsis miyagii T.Yamag. (Pottiaceae, Bryophyta) Hikobia 16: 221–228. The Japanese endemic Pachyneuropsis miyagii (Pottiaceae, Bryophyta) is a distinct species immediately distinguished from others of the genus by its long moniliform axillary hairs. The phylogenetic position of this species was investigated based on chloroplast rps4 gene sequences. The resultant tree supported the taxonomic treat-ment that Pachyneuropsis be placed in the subfamily Trichostomoideae. Our result also suggested P. miyagii was phylogenetically close to Chionoloma, Pseudosym-blepharis and Oxystegus.

Yuya Inoue and Hiromi Tsubota, Miyajima Natural Botanical Garden, Graduate School of Science, Hiroshima University, Mitsumaruko-yama 1156–2, Miyajima-cho, Hatsukaichi-shi, Hiroshima 739–0543, Japan.Hiroyuki Sato, Hikino-cho, 3-chome 17–29–203, Fukuyama-shi, Hiroshima 721–0942, Japan.Tomio Yamaguchi, Department of Biological Science, Graduate school of Science, Hiroshima University, Kagamiyama 1–3–1, Higashi-hiroshima-shi, Hiroshima 739–8526, Japan.

Introduction

　Pachyneuropsis H.A.Mill. (Pottiaceae; Tricho-stomoideae) is a genus comprising two species, with a distribution range in the Philippines and Japan (Bartram 1939, as Pachyneurum; Miller 1970; Yamaguchi 2006). The genus is character-ized by a very narrow or absent upper lamina with bi- to tristratose margins, and a thick costa (Zander 1993). Pachyneuropsis was placed in the subfamily Chionolomoideae, together with the genera Chionoloma and Pseudosymblepharis, based on morphological data (Zander 1993).　P. bartlettii (E.B.Bartram) H.A.Mill., the type species of the genus, was synonymized with Pseudosymblepharis bombayensis (Müll.Hal.) P.Sollman (Sollman 2000). Later, all Asian spe-cies of Pseudosymblepharis were transferred to Chionoloma and treated under the name Chionoloma bombayense (Müll.Hal.) P.Sollman (Sollman 2001). However, Yamaguchi (2006) resurrected Pachyneuropsis. Zander (2006) also adopted the genus and placed in it all species of

Chionolomoideae that had been transferred to Trichostomoideae, reinterpreting the molecular results of Werner et al. (2004).　P. miyagii T.Yamag. is considered endemic to Okinawa Isl., Japan (Yamaguchi 2006), and is characterized by: (1) linear leaves with distinct sheathing bases; (2) plane leaf margins except in the apical portions; (3) well-developed stereid bands of the costa, especially on the adaxial side; (4) bi- to tristratose leaf borders; (5) long monili-form axillary hairs; and (6) minutely papillose rhizoids. This species is similar to P. bartlettii in leaf shape: linear lanceolate with distinct sheath-ing bases and with bi- to tristratose borders. However, morphology of the axiallary hairs is quite different. Those of P. bartlettii consist of 6–15 oblong swollen cells with somewhat thick walls, while those of P. miyagii consist of 20–40 swollen quadrate cells which show a moniliform appearance, with 2–4 elongate cells in the basal and apical portions. Furthermore, the quadrate cells are colored (brownish in harbarium speci-mens), somewhat inflated with outer thin walls

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which are often porose. This character is quite different from those known in the Pottiaceae (Ya-maguchi 2006).　Although in a preliminary phylogenetic study based on sequences of chloroplast r ibulose 1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) and ribosomal protein S4 (rps4) genes, P. miyagii was placed in the family Pottia-ceae (data not shown), the phylogenetic position within the family remained to be clarified (Yama-guchi 2006).　In the present study, a phylogenetic analysis is presented based on sequences of chloroplast rps4 gene to clarify the phylogenetic relationship of P. miyagii with other members of Pottiaceae.

Materials and methods

Taxon sampling　Newly obtained rps4 sequences for Pachyneu-ropsis miyagii and Pottia intermedia (Turner) Fürnr. were included in this analysis. In addition, members of the Pottiaceae sensu Werner et al. (2004) and outgroup species were also sampled from the DNA database based on the previous studies (Tsubota et al. 2004; Werner et al. 2004; Cano et al. 2010). Reference or voucher informa-tion, DDBJ/EMBL/GenBank accession numbers are summarized in Table 1. The data set of rps4 gene sequences includes 39 OTUs of Pottiaceae and two OTUs for outgroups.

DNA extraction, PCR amplification and DNA se-quencing　Total DNA was extracted using the simplified chloroform method (Tsubota et al. 2009). Stems were placed under a stereo microscope and the green part of the shoot dissected. A single shoot was used for the DNA extraction procedure. Several segments of chloroplast rps4 genes were amplified by standard polymerase chain reaction (PCR) with a KOD-Plus-Neo polymerase PCR amplification kit (Toyobo Life Science, Osaka), following the manufacturer’s protocols using a DNA thermal cycler (Mastercycler gradient, Ep-pendorf Co., Ltd., Tokyo) with synthetic primers (Table 2). Direct DNA sequence analyses of the PCR products were performed by the dideoxy chain termination method using the ABI BigDye Terminator Cycle Sequencing Kit v.3.1 (Life

Technologies Japan, Tokyo) with the additional internal primers (Table 2) using an automated sequencer (ABI 3130 Genetic Analyzers, Life Technologies Japan, Tokyo). PCR amplification was performed using the program set to denature at 98°C for 10 sec., anneal at 58°C for 35 sec., and extend at 65°C for 10 min. for total 45 cycles. Conditions of sequencing were based on Tsubota et al. (1999 & 2000) and Handa et al. (2003). The sequences obtained in the present study have been submitted to DDBJ/EMBL/GenBank Internation-al Nucleotide Sequence Database (Appendix 1).

Sequence alignment, and phylogenetic analysis　The sequences were aligned using the program MAFFT var. 6 (Katoh & Toh 2007) with some manual adjustment on sequence editor of MEGA5 (Tamura et al. 2011).　Phylogenetic analysis using the rps4 gene se-quences was performed based on maximum like-lihood criteria as previously described (Tsubota et al. 2004; Ozeki et al. 2007; Masuzaki et al. 2010), with some differences as follows: Prior to the phylogenetic reconstruction, Kakusan4 (Tanabe 2011) was implemented in AICc (Sugiura 1978) to make a rational decision regarding the nucleotide-based substitution model that best fitted our data, and AU test in the final stage of the analysis scheme. Phylogenetic trees were constructed using the following four program packages to obtain the candidate topologies: (1) RAxML ver. 7.3.2 (Stamatakis 2006) with maximum likeli-hood (ML) method (Felsenstein 1981) using GTR + gamma model; (2) PAUPRat (Sikes & Lewis 2001) over PAUP* ver. 4.0b10 (Swofford 2002) with maximum parsimony (MP) method (Fitch 1971) to implement Parsimony Ratchet searches (Nixon 1999) using the Parsimony Ratchet search strategy with random weighting of each character in fifty 200 iteration runs; (3) MrBayes ver. 3.1.2 (Ronquist & Huelsenbeck 2003) with Bayesian inference (BI) method using GTR + gamma mod-el with 1,000,000 generations and (4) MEGA5 (Tamura et al. 2011) with neighbor-joining (NJ) method (Saitou & Nei 1987).　Based on the ML criteria, re-calculation of likelihood values for each tree topology was per-formed with the GTR + gamma model by baseml in PAML ver. 4.5 (Yang 2007), with the set of candidate topologies being evaluated by the ap-

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Table 1. List of species investigated for rps4 gene sequence with the accession number and voucher or reference of the sequences. Further information for the sequences obtained in the present study is shown in Appendix 1.

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proximate unbiased test (AU; Shimodaira 2002, 2004) and Bayesian posterior probability (PP) cal-culated by the BIC approximation (Schwarz 1978; Hasegawa & Kishino 1989) using CONSEL ver. 0.20 (Shimodaira & Hasegawa 2001). A 50% ma-jority-rule condensed tree for the topologies with best ranking log-likelihood values that passed the AU test was also computed by MEGA5. Support-ing values more than 50% were overlaid to assess the robustness of each branch of the condensed topology: AU test (AU), bootstrap probabilities (NP), and Bayesian posterior probabilities (PP) are shown on or near each branch (AU/NP/PP; in%).

Results

　The GTR + gamma model provided the best fitted model for the dataset. A total of 1187 dis-tinct topologies were obtained in the ML, MP, BI and NJ analyses, of which 729 topologies passed the AU test. Fig. 1 shows the condensed topol-ogy of the 729 topologies with best ranking log-likelihood values (Fig. 1). Within the Pottiaceae clade, two subclades were resolved based on rps4 gene sequences: one corresponds to the subfam-ily Trichostomoideae (84/82/0.99), and the other to the subfamily Pottioideae (95/96/1.00) (sensu Werner et al. 2004). P. miyagii was confirmed in the Trichostomoideae clade, and P. miyagii, Chionoloma bombayense, Pseudosymblepha-ris guatemalensis, P. richardsii, P. schimperi-

ana and Oxystegus tenuirostris formed a clade (97/94/1.00), being sister to Tortella.

Discussion

　In the revision of the family Pottiaceae, Zander (1993) recognized seven subfamilies: Timmiell-oideae, Erythrophyllopsoideae, Gertrudielloideae, Chionolomoideae, Trichostomoideae, Mercey-oideae and Pottioideae, and placed Pachyneurop-sis in Chionolomoideae together with the genera Chionoloma and Pseudosymblepharis, based on morphological data. The genera of this subfamily share two advanced character states: leaves be-ing long-linear and in the costa the ventral stereid band is larger than the dorsal. Werner et al. (2004) accepted three subamilies: Merceyoideae, Trichostomoideae (including Chionolomoideae) and Pottioideae (including Erythrophyllopsoi-deae) based on rps4 gene sequence data. Reinter-preting the result of Werner et al. (2004), Zander (2006) recognized five subfamilies: Merceyoideae, Barbuloideae, Trichostomoideae, Pottioideae and Timmielloideae, and synonymized the subfamily Chionolomoideae in Trichostomoideae. In the present study, two subclades were resolved: one corresponds to the Trichostomoideae and another corresponds to the Pottioideae (sensu Werner et al. 2004). P. miyagii was placed in the former clade, and this phylogenetic position supports the taxonomic treatment of Zander (2006).　Zander (1993) indicated Pachyneuropsis is

Table 2. Primers used for PCR amplification and sequencing of the rps4 regions. The primers numbered from the 5’ end correspond to approximate positions in the sequence of Marchantia polymorpha (X04465; Ohyama et al. 1986).

Y. INOUE, H. TSUBOTA, H. SATO AND T. YAMAGUCHI 225

morphologically similar to Pseudosymblepha-ris in having the linear leaf shape with abruptly broadened base and a tendency to extreme length, and its thick costa with the ventral stereid band being larger than the dorsal, but it differed in the very narrow or absent upper lamina with bi- to tristratose margins, and the costa even thicker than is usual in the latter genus.　In his taxonomic revision of the genus Pseudo-

symblepharis in Asia, Sollman (2000) synonymized Pachyneuropsis bartlettii, the type species of Pachyneuropsis, with Pseudosymblepharis bom-bayensis. He suggested that some collections of Pseudosymblepharis bombayensis had bistratose leaf margins mostly near the apex, and in the col-lection of Pachyneuropsis bartlettii, the border thickening is rather variable, viz. of one to two or three layers, mostly only in the apex region. Lat-

Fig. 1. Phylogenetic tree based on analysis of the chloroplast rps4 sequence, depicted by a 50% majority-rule condensed tree for the 729 topologies with the best ranking log-likelihood value. Supporting values more than 50% obtained by the program CONSEL were overlaid: the values by the AU test (AU), bootstrap probabilities (NP), and Bayesian posterior prob-abilities (PP) are shown on or near each branch (AU/NP/PP; in %). The root is arbitrarily placed on the branch leading to the clade which includes members of the Dicranaceae and Encalyptaceae following Tsubota et al. (2004), Werner et al. (2004) and Cano et al. (2010).

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er, Sollman (2001) transferred all Asian species of Pseudosymblepharis to Chionoloma.　However, Yamaguchi (2006) indicated the dis-tinct bi- to tristratose leaf borders of Pachyneu-ropsis are a remarkable character which distin-guishes the genus from Pseudosymblepharis, and described Pachyneuropsis miyagii as the second species of the genus.　In the resultant tree based on rps4 sequences, P. miyagii, Chionoloma bombayense, three species of Pseudosymblepharis and Oxystegus tenuiros-tris form a clade. In previous phylogenetic stud-ies based on nrITS and/or cpDNA sequences by Werner et al. (2005), Grundmann et al. (2006) and Köckinger et al. (2010), the genera Chionoloma, Pseudosymblepharis and Oxystegus also formed a clade. Our result suggests P. miyagii is phyloge-netically close to these three genera. Synapomor-phies of these four genera include caducous leaf apices, papillae extending over the entire lumen except at the sheathing leaf bases, and the ventral stereid band in the costa being larger than the dorsal (Werner et al. 2005). The long moniliform axillary hairs of P. miyagii are a distinctive char-acter of these genera. P. miyagii grows on Paleo-zoic limestone outcrops at sunny sites on ridges or tops of hills in subtropical evergreen broad-leaved forest (Yamaguchi 2006). This limited habitat in the isolated island environment may have led to the evolution of the distinct character of this species. More materials are needed, including P. bartlettii and its related species, to clarify the in-dependence of the genus Pachyneuropsis.

Acknowledgments

　We thank Prof. R. D. Seppelt for checking the English text and for useful suggestion. This study is partly supported by KAKENHI 23770089 to HT. Sequencing for this study was carried out at the Analysis Center of Life Science, Natural Sci-ence Center for Basic Research and Development, Hiroshima University.

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Accepted 12. XI. 2012

井上侑哉・坪田博美・佐藤裕幸・山口富美夫：フチドリコゴケの系統学的位置づけ

　フチドリコゴケPachyneuropsis miyagii T.Yamag.はセンボンゴケ科 Pottiaceaeの日本固有種として沖縄本島の標本にもとづいて新種記載された．本種は，葉腋毛が基部と先端部の 2–4個の長い細胞をのぞいて，20–40個の膨らんだ方形細胞が数珠状に連なり，さらに数珠状に連なる膨らんだ方形細胞は，外側の表面にしばしば小孔をもつという，センボンゴケ科の中では非常に特異的な形態をもつ．本研究では，葉緑体 rps4遺伝子の塩基配列にもとづいて，本種の系統解析をおこなった．系統解析の結果，本種はセンボンゴケ科のクチヒゲゴケ亜科 Trichostomoideaeに相当するクレードに含まれ，Chionolomaや Pseudosym-blepharis，Oxystegusと系統的に近い関係にあることが示唆された．

Appendix 1. Taxa whose rps4 sequences were utilized in the present study.　The list includes accession numbers, their source and voucher specimen information. Voucher specimens are kept in HIRO.　The sequence of information is: the name of taxon, accession number, locality and specimen number. Pottiaceae: Pachyneuropsis miyagii T.Yamag., AB759969, Japan, Ryukyu, Okinawa-ken, Kunigami-gun, Ogimison, Mt. Boujimui, 9 ix 2011, T. Yamaguchi 34243 (HIRO); Pottia interme-dia (Turner) Fürnr., AB759970, Shikoku, Ehime-ken, Imabari-shi, Ohmishima-cho, Ohmishima Isl., 20 ii 2011, Y. Inoue 56 (HIRO).