Org Divers Evol DOI 10.1007/s13127-010-0017-z ORIGINAL ARTICLE Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) Sanna Olsson & Volker Buchbender & Johannes Enroth & Lars Hedenäs & Sanna Huttunen & Dietmar Quandt Received: 16 December 2008 / Accepted: 25 June 2009 # Gesellschaft für Biologische Systematik 2010 Abstract The family Neckeraceae is composed of three distinct clades, of which two, i.e. the Neckera and Thamnobryum clades, are well defined. The third clade, consisting of species belonging to Caduciella, Curvicladium, Handeliobryum, Himantocladium, Homaliodendron, Hydrocryphaea, Neckera, Neckeropsis, Pinnatella, Shevockia and Taiwanobryum, is the focus of this study. Based on sequence data from the trnS-rps4-trnT-trnL-trnF plastid cluster and the rpl16 intron as well as from nuclear ITS1&2, the phylogenetic relationships of these genera are reconstructed. The nearest relatives of this clade are resolved shedding more light on the evolution of the family. The generic composition of the clade and its individual genera are discussed; polyphyly requires redefinition of Pinnatella, Neckeropsis and Homaliodendron. The positions of Touwia and Homalia within the family are addressed in an additional analysis based on more extensive sequence data, and the corresponding new combinations are made. Several further taxonomic changes are proposed, including Circulifolium gen. nov., comprising the former Homaliodendron exiguum and H. microdendron. Keywords Pleurocarpous mosses . Circulifolium gen. nov. . Molecular systematics . Pinnatella section Tenuinervia Introduction S. Olsson : V. Buchbender : D. Quandt Plant Phylogenetics and Phylogenomics Group, Institute of Botany, Dresden University of Technology, 01062 Dresden, Germany S. Olsson : V. Buchbender : D. Quandt (*) Nees Institute for Biodiversity of Plants, University of Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany e-mail: quandt@uni-bonn.de S. Olsson : J. Enroth Department of Sciences, University of Helsinki, P.O. Box 7, FI-00014 Helsinki, Finland L. Hedenäs Department of Cryptogamic Botany, Swedish Museum of Natural History, Box 50007, SE-104 05 Stockholm, Sweden S. Huttunen Laboratory of Genetics, Department of Biology, University of Turku, FI-20014 Turku, Finland With around 5,000 species the pleurocarpous mosses represent one of the major groups of first branching landplants. The plant habit typically is creeping, branching; in contrast to most other mosses sporophyte development is restricted to the apices of short, lateral branches. According to the latest studies, the pleurocarpous mosses as defined by Bell et al. (2007) form a monophylum (“core pleurocarps”) that can be divided in four orders: Hypnodendrales, Ptychomniales, Hookeriales and Hypnales, the latter including the Neckeraceae. This family contains mainly temperate and tropical species; the species number is estimated at around 200 (Enroth 1994a; Olsson et al. 2009a). The species are mostly epiphytic or epilithic, but there are also some aquatic (rheophytic) ones. A morphological characterization of the Neckeraceae is provided by Olsson et al. (2009a). Olsson et al. (2009b) resolved the backbone relationships of the Neckeraceae, its sister-group relation to the Lembophyllaceae, and revealed that the Neckeraceae can be divided in three distinct clades. The three resolved S. Olsson et al. clades were named: Neckera clade, Thamnobryum clade and ‘Pinnatella’ clade, the first two after the respective most species-rich genus included. In addition, morphological definitions of, and evolutionary trends in, the clades were discussed (Olsson et al. 2009a). The ‘Pinnatella’ clade is mainly tropical (except for Handeliobryum) and Asiatic, only Pinnatella minuta occurs in Africa and South America. The clade is not clearly characterized by only a single dominant genus; instead, three major genera, Pinnatella, Homaliodendron and Neckeropsis, are located in this group. The remaining genera belonging here are Caduciella, Curvicladium, Handeliobryum, Himantocladium, Hydrocryphaea, Shevockia, and Taiwanobryum; in addition, several Asian species currently placed in Neckera belong here as well, thus rendering polyphyletic the genus as defined so far. The estimated total number of species in this clade is 70–80, but the exact number cannot be known before analyses with a more thorough sampling of Neckera and also Neckeropsis are carried out. The members of this clade usually have a strong costa and a long seta; a weak costa and immersed capsules are found only in some species of Neckeropsis, Hydrocryphaea and one species of Homaliodendron. The seta is often mammillose in its upper part, a character state shared by all Pinnatella species for which the sporophytes are known, by Taiwanobryum, Neckeropsis calcutensis, Neckera crenulata, and Neckera himalayana. In Homaliodendron flabellatum the seta is occasionally mammillose above, and in Himantocladium it is consistently smooth. The position of the Homalia clade (H. trichomanoides, H. lusitanica and Anomodon giraldii) remained controversial in our earlier phylogenetic analyses of the Neckeraceae. It was resolved as sister either to the ‘Pinnatella’ clade (Olsson et al. 2009a) or to the Thamnobryum clade, but with low support (Olsson et al. 2010). Morphologically the Homalia clade is heterogenic, and does not clearly belong to any of the bigger clades. Therefore, the purpose of the present study is to analyse in more detail the composition of the ‘Pinnatella’ clade. In addition, we address the relationships of the Homalia clade within the Neckeraceae with additional analyses including five marker sequences. Material and methods Taxon sampling and molecular markers The material used was taken from herbarium specimens; the taxon names (with citation of authorities), specimen voucher numbers and herbaria are listed in Tables 1 and 2. The analyses included 71 taxa from 29 genera. For the initial analysis (see Table 1), the ingroup species were selected based on previous classifications (e.g. Buck and Goffinet 2000; Goffinet and Buck 2004), our earlier molecular analyses of a wider taxon sampling (Olsson et al. 2009a, 2009b), as well as on the distribution of morphological characters, to cover the morphological variation within the study group as completely as possible. The outgroup species were selected from the other Neckeraceae clades that were resolved as sister-groups in our previous analyses (Olsson et al. 2009a), and from Lembophyllaceae, the sister group to Neckeraceae (Olsson et al. 2009a; Quandt et al. 2009). Homalia webbiana, Heterocladium dimorphum and Heterocladium procurrens are the most distant outgroups in this analysis. Since the sequence variation within the family turned out to be low, we chose for the phylogenetic reconstructions markers that are known to evolve fast: internally transcribed spacers 1 & 2 of nuclear ribosomal DNA, the plastid rpl16 intron, as well as the plastid trnT-trnL and trnL-trnF intergenic spacers (IGS) and the trnL intron. To resolve the broader relationships of the ‘Pinnatella’ clade and to pinpoint the positions of Homalia (H. trichomanoides, H. lusitanica and Anomodon giraldii) and Touwia, an additional analysis with a reduced taxon sampling was conducted (see Table 2). This second data set was based on data from our previous study resolving the backbone phylogeny of the Neckeraceae (Olsson et al. 2009a; Quandt et al. 2009), but modified by adding taxa relevant to the present study. Compared to the first data set, the second includes two additional markers (rps4 and nad5). Since some of the material was used in both analyses, Tables 1 and 2 are partly redundant. DNA isolation, PCR amplification and sequencing DNA was extracted using the DNeasy® Plant Mini Kit from Qiagen (Qiagen GmbH, Germany) following the manufacturer’s protocol. For details of the DNA extraction, PCR amplification of the ITS1-5.8S-ITS2 and the rps4-trnTtrnL-trnF cluster, purification protocols and sequencing strategies employed, see Olsson et al. (2009b). The amplification protocols for rpl16 are described in Olsson et al. (2009a, 2009b), whereas sequencing and amplification of nad5 followed Buchbender and Quandt (in press). The cleaned PCR products were sequenced by Macrogen Inc., South Korea (www.macrogen.com). Primer sequences were deleted before the final sequences were deposited in EMBL; the corresponding accession numbers are listed in Tables 1 and 2. Alignment and phylogenetic analyses Nucleotide sequences were edited manually and aligned to existing data sets of Olsson et al. (2009a, 2009b) using PhyDE® v0.995 (Müller et al. 2005), based on the criteria Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) Table 1 Taxa and specimens in the initial data set, voucher information, and EMBL or GenBank accession numbers for the generated or downloaded sequences Taxon Herbarium Anomodon giraldii Müll. Hala Caduciella guangdongensis Enrotha H H Caduciella mariei (Besch.) Enroth H Camptochaete arbuscula var. tumida (Sm.) Reichardt H Chileobryon callicostelloides (Broth. ex Thér.) Enroth Curvicladium kurzii (Kindb.) Enroth H NYBG Dolichomitriopsis diversiformis (Mitt.) Nog. H, MHA Echinodium hispidum (Hook. f. & Wilson) Reichardt Buchbender Forsstroemia producta (Hornsch.) Paris Handeliobryum sikkimense (Paris) Ochyra H H Heterocladium dimorphum (Brid.) Schimp. Heterocladium procurrens (Mitt.) A. Jaeger Himantocladium cyclophyllum (Müll. Hal.) M. Fleisch.a H H NYBG Himantocladium implanum (Mitt.) M. Fleisch. NYBG Himantocladium plumula (Nees) M. Fleisch. Homalia lusitanica Schimp. Voucher ID H B H3194078 Koponen et al. 57241 Koponen 28035 Streimann 51408 H3107865 Akiyama Th-85 Nedoluzhko s.n. Downing s.n., 29.10.2000 Koponen 46545 Redfearn et al. 33981 H3212307 H3212289 Redfearn Jr. 36081 De Sloover 21124 Tan et al. 92–232 B275202 Homalia trichomanoides (Hedw.) Schimp. Homalia webbiana (Mont.) Schimp. Homaliodendron exiguum (Bosch & Sande Lac.) M. Fleischa Homaliodendron flabellatum (Sm.) M. Fleisch. Homaliodendron flabellatum (Sm.) M. Fleisch. Homaliodendron microdendron (Mont.) M. Fleisch.a Quandt H B H Enroth H Homaliodendron neckeroides Broth. Homaliodendron scalpellifolium (Mitt.) M. Fleisch. Hydrocryphaea wardii Dix. Lembophyllum clandestinum (Hook. f. & Wilson) Lindb. in Par. Neckera complanata (Hedw.) Huebener Neckera crenulata Harv.a Neckera crispa Hedw. Neckera himalayana Mitt. Neckera pennata Hedw. Neckera polyclada Müll. Hal. Neckera warburgii Broth. Neckeropsis calcicola Nog. Neckeropsis calcutensis (M. Fleisch.) Enroth Neckeropsis disticha (Hedw.) Kindb. Neckeropsis fimbriata (Harv.) M. Fleisch. GenBank accession rps4-trnT-trnL-trnF b rpl16 AM990342 FM210281 FM210763 FM161075 FM160952 FM161083 FM210282 FM160953 FM161084 AM990353 FM160955 FM161087 FM210283 FM210285 FM200841 FM161088 FM160959 FM161093 ITS1&2 AM990362; AF397777b FM160963 FM161098 FM210286 FM160964 FM161099 FM201504 FM210287 FM160967 FM161102 FM160969 FM161110 AM990376 AM990379 FM210288 FM160970 FM161115 FM160973 FM161118 FM160974 FM161120 FM210289 FM160975 FM161121 AM990381 AM990383 FM160976 FM161122 FM160978 FM161124 Olsson 105 Müller K68 B263509 AM990385 AM990387 AM990389 FM160980 FM161126 FM160982 FM161127 FM160984 FM161130 FM210290 FM210291 AM990390 FM160985 FM161132 FM160986 FM161131 FM160987 FM161133 H H H H H3071675 Schwarz 3801 Redfearn, Jr. 35901 H3071953 H3071976 Shevock 23460 Vitt 29644 FM210306 FM210292 FM210293 AM990401; AF397823b FM161015 FM160989 FM160992 FM160996 FM161168 FM161135 FM161139 FM161145 Buchbender H Buchbender B H Buchbender 204 Long 33980 Buchbender 385 B253876 H3203794 AM990413 FM210297 FM210298 FM210301 AM990414 FM161005 FM161006 FM161007 FM161010 FM161016 FM161158 FM161159 FM161160 FM161163 FM161169 H B H H NYBG Enroth Koponen 45441 Bryo 253855 Enroth 64632 H3212832 Heras 901/93 Schäfer-Verwimp 16212 B105716 B105713 FM210307 FM210311 AM990417 AM990418 FM210313 FM210314 FM161017 FM161023 FM161025 FM161026 FM161027 FM161028 FM161170 FM161176 FM161178 FM161179 FM161180 FM161181 FM210315 AM990419 FM161029 FM161182 FM161030 FM161183 Neckeropsis gracilenta (Bosch & Sande Lac.) M. Fleisch. S Neckeropsis nitidula (Mitt.) M. Fleisch. S S. Olsson et al. Table 1 (continued) Taxon Herbarium Voucher ID GenBank accession rps4-trnT-trnL-trnF b rpl16 ITS1&2 Neckeropsis undulata (Hedw.) Reichardt Pendulothecium punctatum (Hook. f. & Wilson) Enroth & S. He Pinnatella alopecuroides (Mitt.) M. Fleisch. B S B238406 FM210316 Streimann 53845 AM990421 FM161031 FM161184 FM161033 FM161187 Enroth FM161034 FM161188 Pinnatella ambigua (Bosch & Sande Lac.) M. Fleisch. Enroth Pinnatella anacamptolepis (Müll. Hal.) Broth.a S Schäfer-Verwimp AM990423 16824 Schäfer-Verwimp FM210317 16252 B104516 FM210318 Linis 757–03 Müller S116 Deguchi 36762 Rikkinen et al. 32 Pinnatella mucronata (Bosch & Sande Lac.) M. Fleisch.a S Hedenäs MY9222 Pinnatella taiwanensis Nog. H Koponen et al. 54169 Porotrichodendron superbum (Taylor) Broth. H H3121100 H Shevock 28269 Porotrichum fruticosum (Mitt.) A. Jaegera Rigodium pseudothuidium Dusén H H3134254 Shevockia inunctocarpa Enroth & M.C.Ji H Shevock 25325 Taiwanobryum robustum Veloira H Taiwan 1544 Taiwanobryum speciosum Nog. H Enroth 64877 Thamnobryum alopecurum (Hedw.) Nieuwl. ex Gangulee Buchbender Buchbender s.n. 11.7.2003 Thamnobryum ellipticum (Bosch & Sande Lac.) Nieuwl.a Enroth Müller S114 Thamnobryum maderense (Kindb.) Hedenäs S B44108 Thamnobryum negrosense (E.B. Bartram) Z. Iwats. & H Schäfer-Verwimp B.C. Tana & Verwimp 16852 Thamnobryum pumilum (Hook. & Wilson) B.C. Tan B B268163 Touwia laticostata Ochyra Quandt Cairns 27.8. 2005 Weymouthia mollis (Hedw.) Broth. CHR, Quandt 99-Mo2 Pinnatella Pinnatella Pinnatella Pinnatella a foreauana Thér. & P. de la Varde kuehliana (Bosch & Sande Lac.) M. Fleisch. makinoi (Broth.) Broth. minuta (Mitt.) Broth. H Enroth HIRO H FM161035 FM161189 FM161036 FM161190 FM210319 FM20150 FM210320 AM990424 FM161037 FM161038 FM161039 FM161040 FM161191 FM161192 FM161193 FM161194 AM990425 FM161041 FM161195 FM210321 FM161042 FM161196 AM990427 AM990430 AM990438; AF543547b FM210323 AM990441 AM990442 AM990444 FM161043 FM161047 FM161051 FM161052 FM864218 FM161055 FM161056 FM210325 AM990445 FM210327 FM161058 FM161220 FM161061 FM161223 FM161063 FM161225 FM210329 FM210330 FM200843 FM161227 FM161070 FM161233 AM990452 FM161072 FM161237 FM161198 FM161202 – FM161212 FM161215 FM161216 FM161218 Denotes taxa for which nomenclatural changes are suggested b In three cases, sequences had been submitted to GenBank in a previous study, thus there are two accession numbers in the “rps4-trnT; trnL-trnF” column laid out in Kelchner (2000) and Quandt and Stech (2005). The alignment process was straightforward due to low sequence length variation. The reported hairpin-associated inversion in the trnL-F intergenic spacer (IGS) (Quandt and Stech 2005; Quandt et al. 2004) was positionally isolated in the alignment and included in the analysis as reverse complement in order to gain information from substitutions within the detected inversion, as discussed in Quandt et al. (2003). Indels were incorporated as binary data using a simple indel coding (SIC) strategy (Simmons and Ochoterena 2000) as implemented in SeqState (Müller 2005). Command files for using the parsimony ratchet (Nixon 1999) were generated using PRAP2 (Müller 2007) and executed in PAUP 4.0b10 (Swofford 2002). Ratchet settings were as follows: 10 random addition cycles of 200 iterations each, with 25% upweighting of the characters in the iterations. Heuristic Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) bootstrap searches under parsimony were performed with 1,000 replicates and 10 random addition cycles per bootstrap replicate. Bayesian analyses were performed with MrBayes v3.1.2 (Huelsenbeck and Ronquist 2001), applying the GTR+Γ+I model for the sequence data and the restriction site model for the binary indel partition. To allow for possible deviating substitution models for the different regions, the first data set was divided in four partitions (partition 1: rps4-trnF; partition 2: nuclear DNA; partition 3: rpl16; partition 4: indels) whereas the five-region data set with reduced taxon sampling was divided in five partitions (partition 1: trnS-trnF; partition 2: nuclear DNA; partition 3: rpl16; partition 4: mitochondrial DNA; partition 5: indels). The a priori probabilities supplied were those specified in the default settings of the program. Posterior probability (PP) distributions of trees were calculated using the Metropolis-coupled Markov chain Monte Carlo (MCMCMC) method and the search strategies suggested by Huelsenbeck et al. (2001, 2002). In each analysis ten runs with four chains (2.5×106 generations each) were run simultaneously, with the temperature of the heated chains set to 0.1. Chains were sampled every 10 generations and the respective trees written to a tree file. Calculations of the consensus tree and of the posterior probabilities of clades were performed based upon the trees sampled after the chains converged (<generation 500,000). Consensus topologies and support values from the different methodological approaches were compiled and drawn using TreeGraph (Müller and Müller 2004). The alignments and trees are available on request from the authors. Results Alignment and sequence analyses Before analysing the matrix, 14 hotspots with polyhomonucleotide repeats were recognized and excluded from the analyses following Olsson et al. (2009b). Hotspots (regions of ambiguous alignment (see Borsch and Quandt 2009; Borsch et al. 2003), were regularly distributed among the partitions: six hotspots were located in the plastid rps4trnF region (H1–H6), the remainder in the nrDNA and the rpl16 intron, with four in each region. The resulting alignment contained 3,891 positions of which 1,429 belonged to the (rps4)-trnT-trnL-trnF partition, 1,554 positions to the nuclear ribosomal partition, and 908 positions to the rpl16 partition. There were 3,142 constant characters; 434 characters were parsimony-informative. In the data matrix where the information based on indel coding was included, a total of 4,416 positions were available. This raised the number of parsimony-informative characters to 677, but the constant characters remained the same. The second data set, after exclusion of 11 hotspots, contained 5,222 positions of which 1,916 belonged to the rps4-trnT-trnL-trnF partition, 865 positions to the rpl16 partition, 1,281 to the nad5 region, and 1,160 to the ITS. Of the characters 4,477 were constant and 407 parsimonyinformative. When the information based on indel coding was included, the data matrix included 5,568 positions (4,485 constant and 549 parsimony informative). Phylogenetic analyses The parsimony analysis with indel coding resulted in 566 most parsimonious trees (length 2,548, CI=0.558, RI= 0.701), whereas analysis without indel coding returned 1,440 most parsimonious trees (length 1,595, CI=0.562, RI=0.720). The strict consensus trees of these analyses showed no conflict with the results from Bayesian inference (BI), but less resolution. Therefore, only the BI tree is illustrated in Fig. 1, with posterior probabilities (PP) and, where applicable, bootstrap values (BS) from the parsimony analysis. Values resulting from analyses with an indelcoding approach precede the values from analyses with the SIC matrix excluded. Thus, in the text below support values from the various analyses will be referred to in the following format: (PPsic / PP / BSsic / BS). The outgroup species belonging to the Neckeraceae form the following clades (Fig. 1): a fully supported Neckera clade (PP 100, BS 100), a clade containing Thamnobryum among other genera (PP 100 / 100, BS 98 / 90), and the genus Touwia, the latter well supported (PP 100 / 100, BS 100 / 100) but in an unresolved position. The ingroup is well supported in the Bayesian analyses (PP 100), including species from Caduciella, Curvicladium, Handeliobryum, Himantocladium, Homaliodendron, Hydrocryphaea, Neckera, Neckeropsis, Pinnatella, the recently described genus Shevockia, and Taiwanobryum. The ingroup is divided in three clades (A–C), but Curvicladium kurzii and three Neckera species (N. himalayana, N. polyclada and N. warburgii) are located outside of these clades. The first clade (A in Fig. 1) is well supported (PP 100 / 100, BS 88 / 91) and includes Homaliodendron species (H. neckeroides, H. scalpellifolium and H. flabellatum) together with Porotrichum fruticosum. This grouping shows Homaliodendron to be polyphyletic, since some ‘Homaliodendron’ species are found in the next clade. The second clade (B) gets high support in both the Bayesian and parsimony analyses (PP 100 / 100, BS 98 / 99) and includes the unispecific genera Hydrocryphaea and Handeliobryum, as well as Neckeropsis, the two remaining Homaliodendron species, Caduciella and Himantocladium. Even though all Table 2 Taxa and specimens in the reduced data set, voucher information, and EMBL or GenBank accession numbers for the generated or downloaded sequences DNA no. B141 SH10 B617 B423 B223 SH146 B258 B196 B349 B352 B310 B422 B111 B218 B474 B110 B230 SH249 SH103 B131 B193 B128 B347 B250 B307 Herbarium Voucher ID GenBank accession rps4-trnT-trnL-trnF a rps4 rpl16 nad5 ITS Anomodon giraldii Müll. Hal.b Camptochaete arbuscula var. tumida (Sm.) Reichardt Chileobryon callicostelloides (Broth. ex Thér.) Enroth Cryptoleptodon longisetus (Mont.) Enroth Curvicladium kurzii (Kindb.) Enroth Dolichomitriopsis diversiformis (Mitt.) Nog. Echinodium umbrosum var. glaucoviride Forsstroemia trichomitria (Hedw.) Lindb. H H H3194078 Streimann 51408 AM990342 AM990353 AY908330b FM210763 FM160955 FM161240 FM161249 FM161075 FM161087 H H 3107865 FM210283 FM882222 FM200841 FM882226 FM161088 H NYBG H, MHA Schäfer-Verwimp Buchbender AM990356 FM210285 AM990362; AF397777a EU434010 AM990365 AY908260b AY908266b AY908329b AY908269b FM160957 FM160959 FM160963 FM160965 FM160968 FM161252 AY908670b FM161257 AY908680b FM161260 FM161091 FM161093 FM161098 EU477602 FM161103 Heterocladium dimorphum (Brid.) Schimp. Heterocladium procurrens (Mitt.) A. Jaeger Himantocladium plumula (Nees) M. Fleisch. Homalia glabella (Hedw.) Schimp. Homalia lusitanica Schimp. Homalia trichomanoides (Hedw.) Schimp. Homalia webbiana (Mont.) Schimp. Homaliodendron exiguum (Bosch & Sande Lac.) M. Fleisch Homaliodendron flabellatum (Sm.) M. Fleisch. Homaliodendron microdendron (Mont.) M. Fleisch. Lembophyllum clandestinum (Hook. f. & Wilson) Lindb. in Par. Leptodon smithii (Hedw.) F. Weber & D. Mohr Neckera complanata (Hedw.) Huebener Neckera himalayana Mitt. Neckera pennata Hedw. Neckera polyclada Müll. Hal. Neckera remota Bruch & Schimp. ex Müll. Hal. Neckera urnigera Müll. Hal. Neckeropsis calcicola Nog. Neckeropsis calcutensis (M. Fleisch.) Enroth Neckeropsis nitidula (Mitt.) M. Fleisch. H H H H B Quandt H B H3038483 Akiyama Th-85 Nedoluzhko s.n. Streimann 49634 Streimann & Pocs 65120A H3212307 H3212289 Tan et al. 92–232 Townsend 93/291 B275202 Olsson 105 Müller K68 B263509 AM990376 AM990379 AM990381 AM990382 AM990383 AM990385 AM990387 AM990389 FM160970 FM160973 FM160976 FM160977 FM160978 FM160980 FM160982 FM160984 FM161271 FM161274 FM161276 FM161277 FM161278 FM161280 FM161282 FM161284 FM161115 FM161118 FM161122 FM161123 FM161124 FM161126 FM161127 FM161130 H H3071675 FM210290 FM160985 AY908671b FM161132 H Redfearn, Jr. 35901 AM990390 FM160987 FM161285 FM161133 H Vitt 29644 AM990401; AF397823a FM160996 FM161295 FM161145 B B268385 AM990403 AY908261b FM160997 FM161297 FM161147 Buchbender B H H S Buchbender 204 B253876 H3203794 Koponen 45441 B29895 AM990413 FM210301 AM990414 FM210307 AM990415 FM882219 AY908265b FM882220 FM161005 FM161010 FM161016 FM161017 FM161018 FM161305 FM882223 – FM882224 FM161307 FM161158 FM161163 FM161169 FM161170 FM161171 S H H S B15194 Enroth 64632 H3212832 B105713 AM990416 AM990417 AM990418 AM990419 FM161021 FM161025 FM161026 FM161030 FM161308 FM161309 FM161310 FM161311 FM161174 FM161178 FM161179 FM161183 AY908276b AY908271b S. Olsson et al. SH301 B247 B138 B313 Taxon DNA no. Taxon Herbarium Voucher ID GenBank accession rps4-trnT-trnL-trnF a B476 B242 B150 B309 B294 B098 B244 B369 B559 Rp47 B149 B238 B546 SH300 B148 B429 B261 SH15 DQ Pendulothecium punctatum (Hook. f. & Wilson) Enroth & S. He Pinnatella alopecuroides (Mitt.) M. Fleisch. Pinnatella minuta (Mitt.) Broth. Pinnatella mucronata (Bosch & Sande Lac.) M. Fleisch. Porotrichodendron superbum (Taylor) Broth. Porotrichum bigelovii (Sull.) Kindb. Porotrichum bigelovii (Sull.) Kindb. Porotrichum fruticosum (Mitt.) A. Jaeger Rigodium pseudothuidium Dusén Rigodium pseudothuidium Dusén Taiwanobryum speciosum Nog. Thamnobryum alopecurum (Hedw.) Nieuwl. ex Gangulee Thamnobryum ellipticum (Bosch & Sande Lac.) Nieuwl Thamnobryum maderense (Kindb.) Hedenäs Thamnobryum subserratum (Hook. ex Harv.) Nog. & Z. Iwats. Thamnobryum tumidicaule (K.A. Wagner) F.D. Bowers Touwia laticostata Ochyra Weymouthia mollis (Hedw.) Broth. Weymouthia mollis (Hedw.) Broth. rps4 rpl16 nad5 ITS S Streimann 53845 AM990421 FM161033 FM161314 FM161187 Enroth H S Schäfer-Verwimp 16824 Rikkinen et al. 32 Hedenäs MY92-22 AM990423 AM990424 AM990425 FM161034 FM161040 FM161041 FM161315 FM161316 FM161317 FM161188 FM161194 FM161195 H B H AM990427 – AM990428 FM161043 – FM161045 FM161319 FM161320 – FM161198 – FM161200 Enroth H3121100 B230549 Shevock & Kellman 27467 Shevock 28269 NYBG 00892248 H3134254 Enroth 64877 Buchbender s.n. 11.7.2003 Müller S114 S H B44108 Enroth 64595 H Quandt H CHR, Quandt H NYBG H H Buchbender AM990430 – AM990438; AF543547 AM990442 AM990444 AM990437 – AY908272b AF023834b FM161047 – FM161051 FM161055 FM161056 FM161322 FM161328 – FM161332 FM161334 FM161202 FM161210 – FM161216 FM161218 FM210325 AY908270b FM161058 AY908674b FM161220 AM990445 AM990446 FM161061 FM161067 FM161335 FM161336 FM161223 FM161230 H3141850 AM990447 FM161068 FM161337 FM161231 Cairns 27.8. 2005 Streimann 58249 99-Mo2 FM210330 – AM990452 FM161070 – FM161072 FM882225 FM161341 – FM161233 – FM161237 FM882221 – AY307014b a In two cases, sequences had been submitted to GenBank in a previous study, thus there are two accession numbers in the “rps4-trnT; trnL-trnF” column b Denotes sequences obtained from GenBank, i.e. from specimens other than those listed in unmarked cells of the respective line Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) Table 2 (continued) S. Olsson et al. Fig. 1 Phylogenetic relationships among selected Neckeraceae taxa based on rps4-trnT-trnL-trnF, rpl16, and ITS1&2 sequences. Posterior probability values from Bayesian inference indicated above branches; bootstrap values from parsimony analysis below branches, where applicable (respective left value, with indel coding; right value, without indel coding) Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) Himantocladium and Neckeropsis species are situated within this clade, the relationships within the clade render these genera non-monophyletic. Caduciella, with just two species, also turns out to be polyphyletic, because one of the species referred to this genus is found in the next clade. The third clade (C) is highly supported in the analyses (PP 100 / 100, BS 100 / 99). It includes all Pinnatella species as well as Caduciella guangdongensis, Neckera crenulata, Shevockia inunctocarpa, Taiwanobryum speciosum and T. robustum. Shevockia inunctocarpa is resolved as the sister-group to the remaining species in the clade. Pinnatella is paraphyletic, since the Pinnatella species are divided among two clades. The bigger clade (Pinnatella sensu stricto) receives good statistical support (PP 100 / 100, BS 99 / 94). Two species (P. mucronata, P. anacamptolepis) as well as Neckera crenulata and Caduciella guangdongensis group with Taiwanobryum, with lower support. The results from the second set of analyses, which were performed to resolve the relationships within the Neckeraceae in a wider framework and focused on the problematic placements of Homalia and Touwia, are illustrated in Fig. 2. In the Bayesian analyses without indel coding Homalia lusitanica forms a clade together with Homalia trichomanoides and Anomodon giraldii, but the support remains low (PP 74). Furthermore, this analysis suggests that both Touwia and the Homalia-Anomodon clade belong to the ‘Pinnatella’ clade (i.e. the ingroup species treated in the current study), even if the position does not receive statistical support. The exact position of these taxa is a particularly difficult problem to solve, since even the five markers used do not provide enough information to resolve their position reliably. The Bayesian analysis without an indel-coding approach had the highest resolution, thus is shown in Fig. 2, the latter also including support values from the other analyses. Discussion Phylogenetic analyses and taxonomic relationships The analyses by Olsson et al. (2009a, 2009b) resulted in a robust backbone structure for the Neckeraceae. This was used to guide the taxon sampling for further analyses and showed that more detailed analyses with additional molecular data were needed to resolve the circumscriptions of the genera belonging to the ‘Pinnatella’ clade, since some genera (e.g. Pinnatella and Neckeropsis) appeared to be polyphyletic. In the present study the outgroup species form groups that are congruent with earlier studies (Olsson et al. 2009a, 2009b). The inclusion of Anomodon giraldii and the genus Touwia in the backbone data set clarified the branching order and the relationships of the sister-groups of the ‘Pinnatella’ clade. In the more detailed study, taxon sampling for the ‘Pinnatella’ clade was increased and the phylogenetic relationships turned out to be more complicated than they had appeared at first glance, resulting in the loss of resolution in some branches. This is a natural consequence of adding more taxa and using fewer markers. However, no true conflicts exist between the results of our different analyses, since the apparently conflicting branches are not statistically supported, except for the position of the Homalia clade. Our present results contradict previous results by Olsson et al. (2009a) regarding the placement of Homalia lusitanica, since in the previous study this species formed a clade with H. trichomanoides with maximum support. The analysis based on more extensive sequence data resolves this incongruence and supports, at least weakly, a clade including the two Homalia species together with Anomodon giraldii. This demonstrates that even when a laborious sequencing effort was undertaken, resulting in an alignment including almost 4,000 positions, additional sequence data were needed to resolve a few remaining questions regarding the phylogenetic relationships within the family. Based on these results we suggest that Anomodon giraldii be included in the genus Homalia. Ingroup relationships and previous major treatments of the genera in the Pinnatella group According to the results of the present study, Homaliodendron exiguum and H. microdendron are close to each other but not to the other Homaliodendron species, thus should be placed in a separate genus (see the chapter on “Taxonomic and nomenclatural changes” below). Due to the polyphyly of Caduciella, Himantocladium, Homaliodendron, Neckeropsis, Pinnatella, Shevockia and Taiwanobryum, we also suggest some changes to the respective generic delimitations. As Thamnobryum ellipticum and T. negrosense are clearly resolved inside the genus Touwia, new combinations for both taxa will be provided in the section on “Taxonomic and nomenclatural changes” below. Clade A Homaliodendron Homaliodendron is a tropical genus, with the centre of diversity in Asia. The type of the genus is H. flabellatum. Ninh (1984) revised the Indochinese taxa and recognized ten species, but Enroth (1989b) found that some of them could not be distinguished from the highly variable and wide-ranging H. flabellatum. The genus was divided in the sections Homaliodendron and Circulifolia by Fleischer (1905–1906), and Ninh (1984) followed that arrangement. Given the apparent high variability of H. flabellatum S. Olsson et al. (Enroth 1989b), the species number in Homaliodendron was estimated at about 12 by Enroth (1994a), with two species in section Circulifolia and the remainder in section Homaliodendron. All species of Homaliodendron are stipitate-frondose (H. exiguum not distinctly so) and have appressed, usually overlapping stipe leaves. In the stems a central strand is not differentiated. The leaf dentation is very coarse in section Homaliodendron, in which there are large, multicellular teeth in the apical parts of the leaves, whereas in section Circulifolia the marginal teeth are small and unicellular. All species are dioicous. The seta is 1.5–4.5 mm long (Ninh 1984), smooth or in the upper part mammillose, and yellow. The capsules are exserted, orthotropous and symmetric and have 5–12 stomata in the apophysis (Ninh 1984). The peristome is of the reduced, i.e. neckeroid type. The lower dorsal plates of the exostome teeth are often somewhat cross-striolate, and their upper parts, as well as the endostome segments, are variably papillose. Homaliodendron piniforme (see Enroth 1990b), which occurs in Africa and South America, has been shown not to belong within the Neckeraceae (Olsson et al. 2009a). Due to its demonstrated polyphyly, Homaliodendron has to be divided in two genera. In Homaliodendron sensu stricto the type, H. flabellatum, is joined by Homaliodendron scalpellifolium, H. neckeroides and Porotrichum fruticosum. Our analyses included one exemplar of H. flabellatum from the Philippines and one from Honduras. Earlier, the Central and South American specimens had been called H. decompositum and the Asian ones H. flabellatum, but the two names were synonymised by Enroth (1989b), which has been followed by others (e.g. Buck 1998). The American plants display much more homogeneous morphology than the Asian ones (J. Enroth, pers. obs.). They resemble specimens from the Pacific island groups (such as Hawaii); the species may have dispersed via those islands to tropical America. It is noteworthy that, as reported by Buck (1998), sporophytes are unknown from the American plants, although they are not rare in Asia. Thus H. flabellatum probably reproduces only by asexual propagulae in tropical America. Homaliodendron neckeroides was so named by Brotherus (1929), but treated as Neckera neckeroides by Enroth and Tan (1994) based mainly on the sporophyte and perichaetial leaf characters, especially the immersed capsule typical in Neckera but not encountered in any other species of Homaliodendron. Our results indicate that the original placement and name, Homaliodendron neckeroides Broth., are correct and should be reinstated. Porotrichum fruticosum is resolved as the sister group to the four Homaliodendron species in this clade, but differs from them mainly in the spreading rather than appressed stipe leaves and a much longer seta (>1.5 cm, whereas in Fig. 2 Results of analysis to clarify relationships of Homalia lusitanica based on combined data for rps4-trnT-trnL-trnF, rpl16, ITS1&2, and nad5 intron sequences. Posterior probability values from Bayesian inference indicated above branches; bootstrap values from parsimony analysis below branches, where applicable (respective left value, with indel coding; right value, without indel coding) the other species it does not exceed c. 4.5 mm). In addition, it has a higher (c. 130 μm) endostome basal membrane with reduced cilia between the segments. Cilia are lacking in the other Homaliodendron species. However, P. fruticosum differs much more from the rest of the Porotrichum species. It occurs only in the Himalayan general region, while no other species of Porotrichum is known from Asia. Furthermore, the lack of a central strand in the stem (also lacking in Homaliodendron), the very thick-walled and porose laminal cells (also found in Homaliodendron sensu stricto), and the large composite marginal teeth in the leaves (present in some species of Porotrichum but much more pronounced in and typical of Homaliodendron sensu stricto) all suggest a close relationship with Homaliodendron sensu stricto. Since it is clearly not justified to keep this species in Porotrichum or to establish a new genus for it, we transfer it to Homaliodendron. Clade B The members of the second group in Homaliodendron sensu lato, H. microdendron and H. exiguum, belong to clade B; thus, a new genus is warranted to accommodate them. Fleischer (1905–1906) placed them in his Homaliodendron sect. Circulifolia, therefore we propose to elevate the section to generic rank (see chapter on “Taxonomic and nomenclatural changes”). They differ from H. flabellatum and its allies (cf. Enroth 1989b; Ninh 1984) in typically being smaller, having more strongly complanate leaves, in the minute, crenulate leaf dentation, in the filiform rather than leaf-like pseudoprapaphyllia, and in the relatively thinwalled, non-porose laminal cells. The sporophytes do not markedly differ. Handeliobryum and Hydrocryphaea In a detailed taxonomic analysis of Handeliobryum, Ochyra (1986) recognized only one species and placed it in the Thamnobryaceae. Handeliobryum sikkimense is a rheophytic moss growing in fast-flowing streams in the Himalayan region, including Yunnan in China. It is a very stout, rigid plant, with a dendroid habit, well-differentiated stipe leaves, a very strong costa, and a bistratose leaf lamina with multistratose margins. Hydrocryphaea was originally (Dixon 1931) placed in the Cryphaeaceae, as the generic name reflects. Manuel (1975) thought it was related to the ‘thamnobryoid’ b Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) S. Olsson et al. Neckeraceae, a view agreed with by Enroth (1999). The single species, H. wardii, is known from North India, China (Yunnan), North Vietnam and North Laos, and recently several new locations have been spotted especially in Yunnan (Shevock et al. 2006). It grows at least periodically submerged in flowing water. It is a rigid plant with a strong, subpercurrent costa in the weakly limbate leaves. The seta is just up to 0.2 mm long, rendering the erect capsule deeply immersed among the perichaetial leaves. The peristome is reduced, basically of the ‘neckeroid’ type, but there is no basal membrane in the endostome (Shevock et al. 2006). Handeliobryum and Hydrocryphaea are both Asian taxa growing in flowing water and in the same general area. Even if some of the characters that the species share may have evolved independently due to the similar habitats, the molecular data support them being closely related. Yet their gametophytes differ (cf. Ochyra 1986; Shevock et al. 2006); thus, there is no justification for uniting the species in one genus, particularly since the sporophytes of Handeliobryum remain undescribed. Neckeropsis As currently defined, Neckeropsis is a pantropical genus with 27 species. The majority of the taxa are Asian (Ochyra and Enroth 1989; Touw 1962, 1972; Touw and Ochyra 1987), but there are four species in South America (Enroth 1995; Sastre-De Jesús 1987) and eight in Africa (Enroth 1993b; Enroth and Magill 1994). The section Pseudo-Paraphysanthus of Neckeropsis consists of rheophytic taxa with several morphological adaptations to the harsh environment (Enroth 1999; Higuchi et al. 1989; Ochyra and Enroth 1989). In the papers cited above, the genus has been revised separately for South America, Africa and Asia-Oceania, but it has not been subjected to rigorous phylogenetic analysis. Neckeropsis consists of non-stipitate (except Himantocladium cyclophyllum), typically remotely and irregularly branched plants with a complanate, “pseudotetrastichous” (Touw 1962) foliation and lacking a central strand in the stem. The leaves can be undulate or not, and the leaf apex is mostly obtuse, rounded or truncate. The sexual condition varies with the species. Post-fertilization growth of the perichaetial leaves is common and often considerable. In some species the perichaetial paraphyses become leaf-like and multiseriate; they have been called “ramenta” (e.g. Buck 1998; SastreDe Jesús 1987). The seta is short, rendering the sporophytes immersed in most species. The capsules are orthotropous and symmetrical, and the peristome is of the reduced neckeroid type with spiculose-papillose exostome teeth and endostome segments, and lacking cilia. The type is Neckeropsis undulata. According to our results, Neckeropsis sensu lato is polyphyletic and divided in two genera. To Neckeropsis sensu stricto belong N. undulata, N. disticha, N. fimbriata, and Himantocladium cyclophyllum. While N. disticha and N. undulata are synoicous, H. cyclophyllum and N. fimbriata are dioicous. All species in this group have a fairly strong costa, but the leaves can be either distinctly undulate (N. fimbriata, N. undulata) or not. A synapomorphy shared by N. disticha, N. undulata and N. fimbriata is the presence of ramenta, or modified, leaf-like paraphyses. Such paraphyses are absent in H. cyclophyllum and in all species in the other ‘Neckeropsis’ clade. There are, however, three more species in Asia (not included in the current study) that also have ramenta: N. andamana, N. crinita and N. nano-disticha (Touw 1962). It remains to be determined if those three also belong in Neckeropsis sensu stricto. One feature that seems to be common to all species of Neckeropsis sensu lato is the absence of apophysal stomata (Touw 1962), but this needs to be confirmed. The basal Himantocladium cyclophyllum is somewhat anomalous in this genus, since it is stipitate, has nonauriculate leaves and an exserted capsule with apophysal stomata, and lacks ramenta. However, the support for the clade is maximal. In the other, still undefined genus, which includes N. calcicola, N. gracilenta and N. calcutensis, all species are dioicous. The last of these species was treated in Neckeropsis by Enroth (1990a), but due to some morphological characters (especially the leaf areolation strongly reminiscent of Pinnatella alopecuroides) it was later placed in Pinnatella (Enroth 1994c). Although Neckeropsis as currently circumscribed is clearly polyphyletic, we do not feel it justified to make any taxonomic rearrangements yet, mainly because our analysis contains only seven of the 27 species. Also, the genus containing N. calcicola, N. gracilenta and N. calcutensis appears as very heterogeneous morphologically; therefore, more taxa must be sampled in it. Furthermore, Neckeropsis nitidula is closely related to the other Neckeropsis species but remains in an unresolved position. Himantocladium The tropical genus Himantocladium was established by Fleischer (1906–1908) and revised by Enroth (1992), who recognized eight species. The latter author subdivided the genus in the two sections Himantocladium with five synoicous species, and Cyclophyllum with three dioicous species. Enroth (1994b) transferred one of the dioicous species (H. warburgii) back to its original genus Neckera, leaving Himantocladium with seven species. In our analysis Neckera warburgii forms a clade with N. polyclada, but the clade is in an unresolved position and weakly supported; consequently no taxonomic changes are made here. Himantocladium is an Asian-Oceanian genus, with just one species present in the Seychelles. A close relationship Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) between Himantocladium and Neckeropis was emphasized by Touw (1962) as well as by Enroth (1989a); these authors also discussed the generic distinctions. Himantocladium is characterized by the following combination of character states: stipitate-frondose plants, with the fronds usually branching sub-pinnately or pinnately; absence of a central strand in the stem; appressed, overlapping stipe leaves; fairly strong, single costa; absence of post-fertilization growth of the perichaetial leaves; a straw-yellow seta usually up to 2.0 (rarely 2.5) mm long; orthotropous, symmetrical capsules that have 2–3 apophysal stomata; and a reduced, spiculose-papillose ‘neckeroid’ peristome. The type is Himantocladium implanum. In the present paper we transfer H. cyclophyllum to Neckeropsis. This leaves Himantocladium with six species, only one of which (H. formosicum, endemic to Taiwan) is dioicous. The relationships of H. formosicum require further study. Caduciella Caduciella was described and placed in the Leptodontaceae by Enroth (1991) to accommodate a single species, Caduciella mariei, previously known as Pinnatella mariei. A second species (C. guangdongensis) from SE China was described as new 2 years later (Enroth 1993a). The total distribution area (of C. mariei) ranges from Tanzania to India and SE China, Thailand and Vietnam through Indonesia and New Guinea to Queensland in Australia; the species is also known from Micronesia. The two species of Caduciella are small, stipitate-frondose plants, with overlapping and appressed stipe leaves. There is no central strand in the stem. The costa is single and reaches to midleaf or above; the leaf margins are entire or serrulate near the leaf apex. The leaf cells are in distinct rows and the pseudoparaphyllia are numerous and leaf-like. The species are also connected by the presence of caducous distal branch leaves, often leaving the branch tips naked. This type of vegetative propagation is uncommon in the Neckeraceae as a whole. Sporophytes are unknown from both species. According to the current analyses, Caduciella mariei is closely related to Himantocladium implanum and H. plumula. Due to the much smaller size, entire leaf margins, leaf areolation, numerous leaf-like pseudoparaphyllia, and caducous leaves we recognize Caduciella as a genus distinct from Himantocladium and encompassing only C. mariei; Caduciella guangdongensis is now excluded. species being mainly Asian-Oceanic. Enroth subdivided the genus in the subgenera Urocladium with three species, and Pinnatella with 12 species. The subdivision resulted from a cladistic analysis based on 44 morphological characters (see also Hyvönen and Enroth 1994). That analysis did not support an earlier subdivision by Enroth (1989a), in which he had proposed a section Tenuinervia for two species (P. anacamptolepis and P. mucronata) which, in contrast to the remainder of Pinnatella, share a relatively weak costa and median laminal cells distinctly longer than the apical ones. The current number of species in Pinnatella is 13, since P. calcutensis actually belongs to Neckeropsis, a placement advocated by Enroth (1990a) before the monographic study. Pinnatella anacamptolepis was transferred to the recently described genus Shevockia by Enroth and Ji (2006), but our current analysis does not support that placement. In general terms Pinnatella consists of stipitate-frondose plants with usually pinnately to bi-pinnately branched fronds. The stipe leaves are distinctly differentiated, not overlapping and spreading. The laminal cells are short and the marginal cells quadrate to short-elongate in a few to several rows; the cell corners often have small papillae. The costa is single and strong, often reaching near the leaf apex. All species for which gametangia are known are dioicous and there is no post-fertilization growth of the perichaetial leaves. The seta is straw-yellow, 2.0–4.5 mm long, straight and mammillose in the upper part. The capsule is orthotropous and symmetric, with up to five phaneroporous stomata in the apophysis. The peristome is double, reduced (‘neckeroid-type’), with densely spiculose papillose exostome teeth and endostome segments. There are no cilia in the endostome. Vegetative propagation takes place through flagelliform, microphyllous branches produced in the leaf axils. The genus Pinnatella sensu lato results as polyphyletic from the current study, requiring restriction of the name (Pinnatella sensu stricto) to the species grouping with the type, P. kuehliana. Shevockia inunctocarpa remains as the only representative of its genus, since S. anacamptolepis (= Pinnatella anacamptolepis) groups with Taiwanobryum. This well-supported Taiwanobryum clade also includes Pinnatella mucronata, Neckera crenulata, Taiwanobryum speciosum, T. robustum and Caduciella guangdongensis. Since the genus name Caduciella must be applied to the clade including the type, C. mariei, Caduciella guangdongensis needs to be renamed. We suggest to include it in the genus Taiwanobryum (see the chapter on “Taxonomic and nomenclatural changes”), along with all species in its clade. Clade C Pinnatella and Shevockia The pantropical genus Pinnatella was established by Fleischer (1905–1906) and monographed by Enroth (1994c). The latter author recognized 15 species, of which only P. minuta is pantropical, the other Taiwanobryum Taiwanobryum in its previous circumscription, with two species (T. speciosum being the type), occurs in East Asia, from Japan through Taiwan and SE China to the Philippines and Borneo. It has usually been placed in the Prionodontaceae (e.g. Lai and Koponen 1981), but more S. Olsson et al. recently in the Leptodontaceae by Buck and Goffinet (2000), who included only Prionodon in the Prionodontaceae. In the phylogenetic analysis by Tsubota et al. (2002), Taiwanobryum speciosum appeared in the Neckeraceae, close to Pinnatella ambigua. Lai and Koponen (1981) suggested a close relationship between Taiwanobryum robustum and Neolindbergia (brassii), based mainly on the peculiar gemmate-tipped, axillary rhizoids. However, Neolindbergia is currently placed in the heterogeneous Pterobryaceae (Buck and Goffinet 2000) and was not included in the current study. The gametophytic characters of the two species thus far constituting Taiwanobryum are very similar; the sporophyte of T. robustum remains unknown. The plants are relatively robust, sparsely branched, with a poorly defined stipe, have crowded, ovate-lanceolate leaves with coarsely toothed margins in the upper parts, a strong, single costa, strongly incrassate and, especially in T. robustum, porose walls of the laminal cells, an elongate seta that is mammillose in its upper part, an orthotropous, symmetrical capsule, and a reduced peristome with papillose exostome teeth and no endostome. Adding the four species T. crenulatum, T. mucronatum, T. anacamptolepis and T. guangdongense renders Taiwanobryum far more heterogeneous and difficult to define morphologically, especially relative to Pinnatella. The robust T. crenulatum fits relatively well with T. speciosum and T. robustum, but the three other taxa pose problems in this grouping. Among themselves, they form a morphologically ‘acceptable’ group, being relatively small, often densely branched, with a relatively weak costa mostly ending near midleaf, and slightly asymmetric leaves with mucronate apices. However, at the same time they differ markedly from each other. For example, the stipe leaves of T. mucronatum are spreading and not overlapping, while in the two other species they are overlapping, squarrose in T. anacamptolepis and appressed in T. guangdongense. Taiwanobryum mucronatum has a stem central strand, while the two other species do not. The leaf cell walls are incrassate and porose in T. anacamptolepis, but thinner and non-porose in the other two species. The pseudoparaphyllia of T. anacamptolepis and T. guangdongense are numerous, but T. mucronatum has much fewer of them. The sporophyte is known only for T. mucronatum, and closely resembles that in Pinnatella, but has a clearly more strongly mammillose seta (Enroth 1994c). Taxonomic and nomenclatural changes Circulifolium S. Olsson, Enroth & D. Quandt, gen. nov. Type Circulifolium microdendron (Mont.) S. Olsson, Enroth & D. Quandt. Diagnosis Genus hoc ab Homaliodendron praecipue statura plantae minore, foliis valde complanatis, cellulis foliorum non porosis, dentibus unicellularis foliorum, apicibus foliorum rotundatis vel truncatis, apicibus obtusis foliorum perichaetialium et pseudoparaphylliis filiformibus differt. Circulifolium exiguum (Bosch & Sande Lac.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Homalia exigua Bosch & Sande Lac. in Dozy & Molk., Bryol. Jav. 2: 55. 1862; Thamnium exiguum (Bosch & Sande Lac.) Kindb., Hedwigia 41: 240. 1902; Homaliodendron exiguum (Bosch & Sande Lac.) M. Fleisch., Musci Fl. Buitenzorg 3: 897. 1908. Circulifolium microdendron (Mont.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Hookeria microdendron Mont., Ann. Sci. Nat. Bot. sér. 2(19): 240. 1843; Hypnum microdendron (Mont.) Müll. Hal., Syn. Musc. Frond. 2: 231. 1851; Homaliodendron microdendron (Mont.) M. Fleisch., Hedwigia 45: 78. 1906. Homalia giraldii (Müll. Hal.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Anomodon giraldii Müll. Hal., Nuov. Giorn. Bot. Ital. n. ser. 3: 117. 1896. Homaliodendron fruticosum (Mitt.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Porotrichum fruticosum (Mitt.) A. Jaeger, Ber. Thätigk. St. Gallischen Naturwiss. Ges. 1875–76: 306, Sp. Musc. 2. 1877. Neckeropsis cyclophylla (Müll. Hal.) S. Olsson, Enroth & Quandt, comb. nov. Basionym Neckera cyclophylla Müll. Hal., Syn. Musc. Frond. 2: 664. 1851; Thamnium cyclophyllum (Müll. Hal.) Kindb., Hedwigia 41: 224. 1902; Himantocladium cyclophyllum (Müll. Hal.) M. Fleisch., Musci Fl. Buitenzorg 3: 887. 1908. Taiwanobryum anacamptolepis (Müll. Hal.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Neckera anacamptolepis Müll. Hal., Syn. Musc. Frond. 2: 663. 1851; Thamnium anacamptolepis (Müll. Hal.) Kindb., Hedwigia 41: 251. 1902; Pinnatella anacamptolepis (Müll. Hal.) Broth., Nat. Pflanzenfam. 1(3): Phylogenetic relationships in the ‘Pinnatella’ clade of the moss family Neckeraceae (Bryophyta) 857. 1906; Shevockia anacamptolepis (Müll. Hal.) Enroth, J. Hattori Bot. Lab. 100: 74. 2006. Taiwanobryum crenulatum (Harv.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Neckera crenulata Harv. in Hook., Icon. Pl. Rar. 1: 21. f. 6. 1836. Taiwanobryum guangdongense (Enroth) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Caduciella guangdongensis Enroth, Bryologist 96: 471. 1994. Taiwanobryum mucronatum (Bosch & Sande Lac.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Neckera mucronata Bosch & Sande Lac. in Dozy & Molk., Bryol. Jav. 2: 68. 1863; Porotrichum mucronatum (Bosch & Sande Lac.) Broth., Monsunia 1: 49. 1899; Thamnium mucronatum (Bosch & Sande Lac.) Kindb., Hedwigia 41: 249. 1902; Pinnatella mucronata (Bosch & Sande Lac.) M. Fleisch., Hedwigia 45: 80. 1906. Touwia elliptica (Bosch & Sande Lac.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Porotrichum ellipticum Bosch & Sande Lac., Bryol. Jav. 2: 70. 1863; Thamnium ellipticum (Bosch & Sande Lac.) Kindb., Hedwigia 41: 247. 1902; Thamnobryum ellipticum (Bosch & Sande Lac.) Nog. & Z. Iwats., J. Hattori Bot. Lab. 36: 470. 1972; Parathamnium ellipticum (Bosch & Sande Lac.) Ochyra, Fragm. Flor. Geobot. 36(1): 77. 1991. Touwia negrosensis (E.B. Bartr.) S. Olsson, Enroth & D. Quandt, comb. nov. Basionym Thamnium negrosense E.B. Bartr., Philipp. J. Sci. 68: 251. 1939; Thamnobryum negrosense (E.B. Bartr.) Z. Iwats. & B.C. Tan, Miscell. Bryol. Lichenol. 7(7): 152. 1977; Parathamnium negrosense (E.B. Bartr.) Ochyra, Fragm. Flor. Geobot. 36(1): 77. 1991. Acknowledgements SO acknowledges financial support by the Helsingin Sanomat Centennial Foundation and the Research Foundation of the University of Helsinki. 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