Phytotaxa 400 (3): 123–144 https://www.mapress.com/j/pt/ Copyright © 2019 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) https://doi.org/10.11646/phytotaxa.400.3.2 The differentiation and speciation of Scapania javanica and S. undulata complexes in the Eastern Sino-Himalayas and perimeters for Scapania Sect. Stephania (Scapaniaceae, Hepaticae) VADIM BAKALIN1, ANNA VILNET2, WEN ZHANG MA3 & KSENIA KLIMOVA1 Running title: Differentiation and speciation in Scapania Sect. Stephania 1 Botanical Garden-Institute, Makovskogo Street, 142, Vladivostok, 690024, Russia, vabakalin@gmail.com 2 Polar-Alpine Botanical Garden-Institute, Fersmana Street, 18A, Apatity, Murmansk Province, 184209, Russia 3 Herbarium, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China Corresponding author: Vadim Bakalin (vabakalin@gmail.com) Abstract Three taxa of Scapania with Sino-Himalayan and meta-Himalayan distribution are described as new to science: S. pseudojavanica from the southern portion of the eastern spur of the Tibetan Plateau in southern China and North Vietnam, S. metahimalayana with a disjunct distribution that ranges from Nepal (where its distribution is questionable) to North Vietnam, and S. sichuanica from high elevation in Sichuan in SW China. Each species is morphologically similar to taxa distributed in the more humid and warmer climates of East and South-East amphi-oceanic Asia. The differentiation is presumably the result of recent speciation in the Sino-Himalayas and adjacent lands and the observed differentiation and distribution patterns are in support of the geographic approach in splitting hepatic species. The initial differentiation of these Scapania in sect. Stephania was the specialization into two groups: with brown and colorless gemmae, both being predominantly unicellular. Keywords: Geographic species concept, molecular phylogenetic, Scapania, speciation, taxonomy Introduction This work was inspired by the collections in the genus Scapania (Dumortier 1831: 38) Dumortier (1835: 14) by the first author in Guizhou Province of China in 2013. The collections yielded plants somewhat similar to South-East Asian S. javanica Gottsche (1853: 575), a species not included in the Scapania treatment of the Chinese Flora (Cao & Sun 2007). The similarity was based on distinctive brown, unicellular gemmae, and long decurrent and relatively narrow ventral leaf lobes. The record was not included in a previous account on Guizhou liverworts by Bakalin et al. (2015) in order to further explore the possibility of speciation in the group of meta-Himalayan taxa (cf. Bakalin et al. 2018a, 2018b). Genetic analysis at KPABG yielded unexpected results: the Guizhou specimen joined the clade with Scapania griffithii Schiffner (1899: 204) in Heinrichs et al. (2012). However, S. griffithii is characterized by colorless bicellular bacilliform gemmae (Müller 1905, Potemkin 2001) that are quite different from the unicellular brown gemmae observed in the Guizhou specimen. It is worth noting here that the original description of S. griffithii was too concise and lacked necessary detail. The sister subclade for ‘S. griffithii’ in Heinrichs et al. (2012) was represented by S. javanica, another taxon with unicellular brown gemmae. We studied available specimens of S. griffithii, S. javanica and some morphologically similar taxa to determine limits of variability and to look for morphological features of potential differentiation within the group. We studied types, our own collections and those subsequently gathered in Sichuan Province of China and North Vietnam-an area belonging to the Eastern meta-Himalayas. This work eventually revealed new Sino-Himalayan Scapania species as described in the present paper. Accepted by Christine Cargill: 21 Mar. 2019; published: 5 Apr. 2019 123 Material and methods Specimen collecting and processing We conducted a detailed morphological study of the type specimens of Scapania javanica, S. griffithii, S. spathulata Stephani (1899: 48) and S. spathulifolia Stephani ex Warnstorf (1921: 86) in G and PC. We also studied specimens from JE, including those used for DNA analysis in Heinrichs et al. 2012, and our own collections from China and North Vietnam (deposited at VBGI and KPABG) as detailed below. The focal geography of all specimens studied is within a large spur that descends southeastward from the Tibetan Plateau. Where the Tibetan Plateau is characterized by dry and cold conditions, the climate in the focal area is milder with precipitation concentrated in summer months and thus possessing monsoon characteristics. Climate data from the nearest town is provided below but the actual collection sites were in higher elevations with colder and milder conditions. The vegetation of all actual collection localities included more mesophytic elements than is typical at low elevations. Guizhou. The Duyun municipality in Guizhou Province receives an average of 1224 mm of precipitation per year, with a mean monthly temperature ranging between 6.6°C to 25.8°C (Climate-data.org: https://en.climate-data. org/location/2269/). The Scapania collections were gathered in mostly evergreen, broadleaved forest at an elevation of 1300 m a.s.l. in November 2013. Kangding. Kangding in Sichuan Province receives an average of 824 mm of precipitation per year, with a mean monthly temperature ranging from–1.3°C to 15.5°C (Climate-data.org: https://en.climate-data.org/location/1062473/). The general collection locality was at 3165 m a.s.l. in Abies Miller (1754: 1)-Betula Linnaeus (1753: 982)-Rhododendron Linnaeus (1753: 392) dominated forest along a stream in a narrow valley. The Scapania specimens were collected at a nominally higher altitude where climate should be colder and wetter. One additional specimen in the Kangding area was collected above the timberline at an elevation of 4187 m a.s.l. with highland semi-desert cold communities that suggest the climate similar to that in the inner part of southern Tibetan Plateau. Hoang Lien Range. Sapa Town in Lao Cai Province, North Vietnam receives an average of 2223 mm of precipitation per year, with a mean monthly temperature ranging between 9.3°C to 21.2°C at an elevation near 1500 m a.s.l. (Climate-data.org: https://en.climate-data.org/location/36229/). The Scapania specimens were collected near 2900 m a.s.l. in Rhododendron ‘mossy’ forest on a steep mountain slope in 2017. Specimens were transferred to VBGI where the morphological study was conducted. After morphological investigation, our collections were split and duplicates were sent to KPABG for molecular-genetic analysis. The ITS1-2 nrDNA and trnL-F cpDNA loci of 23 specimens from China and Vietnam were sequenced to clarify their phylogenetic affinity. The resultant data were incorporated in the dataset ITS1-2+trnL-F for family Scapaniaceae from Mamontov et al. (2018). We also added the nucleotide sequence data from 20 specimens obtained by Heinrichs et al. (2012). Lophozia silvicoloides Kitagawa (1965: 276) was chosen as the outgroup taxon according to its phylogenetic relations (Vilnet et al. 2010). The voucher details and GenBank accession numbers for sequenced specimens are shown in Table 1. DNA isolation, amplification and sequencing DNA was extracted from dried liverwort tissue using the NucleoSpin Plant Kit (Macherey-Nagel, Germany). Amplification and sequencing were performed using primers suggested by White et al. (1990) for ITS1-2 and Taberlet et al. (1991) for trnL-F. PCR was carried out in 20 μl volumes with the following amplification cycles: 3 min at 94°C, 30 cycles (30 s 94°C, 40 s 56°C, 60 s 72°C) and 2 min of final extension time at 72°C. Amplified fragments were visualized on 1% agarose TAE gels by EthBr staining, purified using the GFX PCR DNA and Gel Band Purification Kit (Amersham Biosciences, U.S.A.), and then used as a template in sequencing reactions with the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction Kit (Applied Biosystems, U.S.A.) following the standard protocol provided for 3100 Avant Genetic Analyzer (Applied Biosystems, USA). Phylogenetic analyses The final combined ITS1-2+trnL-F alignments were corrected in BioEdit v. 7.0.1 (Hall 1999). All positions of alignment were included in the phylogenetic analysis, absent data was coded as missing. The dataset was analyzed by three analytical procedures: the maximum parsimony method (MP) with TNT v. 1.5 (Goloboff & Catalano 2016), the maximum likelihood method (ML) with PhyML v. 3.0 (Guindon et al. 2010) and the Bayesian method (BA) with MrBayes v. 3.2.1 (Ronquist et al. 2012). The parsimony analysis with TNT involved a 124 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. TABLE 1. The list of taxa, specimen vouchers and GenBank accession numbers, obtained in current study. Taxon GenBank accession number Specimen voucher ITS1-2 nrDNA trnL-F cpDNA China: Sichuan Prov., V. Bakalin, China40-14-17, 37321 (VBGI), 1 MH930853 MH931440 China: Sichuan Prov., V. Bakalin, China41-1-17, 37329 (VBGI), 2 MH930854 MH931441 China: Sichuan Prov., V. Bakalin, China40-7-17, 37311 (VBGI) MH930852 MH931439 Vietnam: Lao Cai Prov., V. Bakalin, V-837a-17 (VBGI), 1 MH930855 MH931442 Vietnam: Lao Cai Prov., V. Bakalin, V-850-17 (VBGI), 2 MH930856 MH931443 Vietnam: Lao Cai Prov., V. Bakalin, V-942-17, 36464 (VBGI), 3 MH930857 MH931444 China: Sichuan Prov., V. Bakalin, China36-6-17, 37243 (VBGI), 1 MH930847 MH931434 China: Sichuan Prov., V. Bakalin, China36-18-17, 37263 (VBGI), 2 MH930848 MH931435 China: Sichuan Prov., V. Bakalin, China42-1-17, 37339 (VBGI), 3 MH930849 MH931436 Scapania aff. cuspiduligera (Nees) Muell. Frib. China: Sichuan Prov., V. Bakalin, China40-8-17, 37312 (VBGI) MH930846 MH931433 Scapania griffithii Schiffn. China: Sichuan Prov., V. Bakalin, China35-1-17, 37432 (VBGI) MH930835 MH931422 Vietnam: Lao Cai Prov., V. Bakalin, V-1018-17, 36567 (VBGI), 1 MH930850 MH931437 Vietnam: Lao Cai Prov., V. Bakalin, V-124-17 (VBGI), 2 MH930851 MH931438 Scapania metahimalayana Vilnet et Bakalin sp. nov. Vietnam: Lao Cai Prov., V. Bakalin, V-917a-17 (VBGI) MH930836 MH931423 Scapania parvitexta Steph. Vietnam: Lao Cai Prov., V. Bakalin, V-721-17 (VBGI) MH930841 MH931428 Vietnam: Lao Cai Prov., V. Bakalin, V-727-17 (VBGI), 3 MH930837 MH931424 Vietnam: Lao Cai Prov., V. Bakalin, V-717-17 (VBGI), 1 MH930838 MH931425 Vietnam: Lao Cai Prov., V. Bakalin, V-719-17 (VBGI), 2 MH930839 MH931426 China: Guizhou Prov., V. Bakalin, China56-37-13 (VBGI) MH930840 MH931427 China: Sichuan Prov., V. Bakalin, China42-3-17, 37342 (VBGI) MH930845 MH931432 China: Sichuan Prov., V. Bakalin, China32-9-17, 37411 (VBGI), 1 MH930842 MH931429 China: Sichuan Prov., V. Bakalin, China36-5-17, 37457 (VBGI), 2 MH930843 MH931430 China: Sichuan Prov., V. Bakalin, China36-19-17, 37264 (VBGI), 3 MH930844 MH931431 Scapania bhutanensis Amakawa Scapania cf. ornithopodioides (With.) Waddell Scapania contorta Mitt. Scapania cuspiduligera (Nees) Muell. Frib. Scapania maxima Horik. Scapania pseudojavanica Vilnet et Bakalin sp. nov. Scapania sichuanica Bakalin et Vilnet sp. nov. Scapania verrucosa Heeg DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 125 New Technology Search with search minimal length tree by five iterations and 1000 bootstrap replicates, the default settings were used for other parameters, gaps were coded as missing. The GTR+I+G model was selected as the best-fit evolutionary model of nucleotide substitutions for alignment using the ModelGenerator software (Keane et al. 2004). In ML analysis this model was used and the rate heterogeneity among sites was modeled using a gamma distribution with four rate categories. Bootstrap support (BS) for individual nodes was assessed using a resampling procedure with 300 replicates. According to the stopping frequency criterion (FC) for the bootstrapping procedure (Pattengale et al. 2010) for our dataset, 150 replicates were enough to reach BS convergence with Pearson average ρ100 = 0.992960 as estimated by RAxML v. 7.2.6 (Stamatakis 2006). For the Bayesian analysis each of the partitions of combined alignment (ITS1-2, trnL-F) was separately assigned the GTR+I+G model, and gamma distributions were approximated using four categories. Two independent runs of the Metropolis-coupled MCMC were used to sample parameter values in proportion to their posterior probability. Each run included three heated chains and one unheated, and two starting trees were chosen randomly. The number of generations was one million, and trees were saved once every 10th generation. The average standard deviation of split frequencies between two runs was 0.007536. The software tool Tracer (Rambaut & Drummond 2007) revealed effective sample size (ESS) as 958.5681 and auto-correlation time (ACT) as 1877.8217 for our data. The first 10,000 trees as determined by Tracer were discarded in each run, and 180,000 trees from both runs were sampled after burning. Bayesian posterior probabilities (PP) were calculated as branch support values from trees sampled after burn-in. The infraspecific and infrageneric variability of ITS1-2 and trnL-F sequences for some Scapania species was estimated as the value of the p-distances, as calculated in Mega 5.1 (Tamura et al. 2011) using the pairwise deletion option for counting gaps. Results The combined ITS1-2+trnL-F alignment for 101 specimens of family Scapaniaceae and outgroup taxon consists of 1469 character sites; among them 933 sites belong to ITS1-2 and 536 sites to trnL-F. The number of invariable sites in ITS1-2 and trnL-F are 425 (45.55%) and 353 (65.86%) respectively, variable positions are 483 (51.77%) and 175 (32.65%), parsimony informative positions are 348 (37.30%) and 108 (20.15%) respectively. In the combined alignment there are 778 (52.96%) invariable sites, 658 (44.79%) variable and 456 (31.04%) parsimony informative positions. The MP analysis with TNT yielded 29 equally parsimonious trees at different runs with a length of 2049 steps, with CI = 0.471958 and RI = 0.779257 calculated in Mega 5.1. The ML calculation resulted in a tree, the arithmetic means of Log likelihood was-13634.033865. Arithmetic means of Log likelihoods in BA analysis for both runs sampled were-13350.17 and-13351.58. The tree topologies achieved by the three methods are highly congruent. Thus, on Fig. 1 we provide the BA tree with bootstrap support values calculated by MP and ML analyses and posterior probabilities calculated by Bayesian approach. The obtained tree agreed with the previously published phylogeny for the family Scapaniaceae (cf. Heinrichs et al. 2012: Figs 1–2). The 23 specimens from China and Vietnam were located in different clades and marked by asterisks in Fig. 1. The specimens morphologically identified as S. cuspiduligera (Nees 1833: 180) Müller (1915: 472), S. verrucosa Heeg (1893: 81) and S. contorta Mitten (1860: 101) were found near previously sequenced accessions downloaded from GenBank. Specimens identified as S. maxima Horikawa (1934: 223) and S. bhutanensis Amakawa (Hattori 1971: 230)-species, which earlier were not studied by molecular methods-placed in the clade with species from Section Planifoliae (Müller 1905: 286) Potemkin (1998: 56). Here, we paid special attention to eight specimens situated in clades of Sections Curtae (Müller 1905: 245) Buch (1928: 55), Stephania Potemkin (1998: 57) and Scapania. The Chinese specimen of S. griffithii #China-35-1-17 formed a basal group in the clade of Section Curtae with BS=50% in MP, BS=87% in ML and PP= 1.00 in BA (or 50/87/1.00). The level of sequence divergence of this specimen from other species of the clade varied from 5.7 to 6.7 % in ITS1-2 and from 2.1 to 3.1% in trnL-F (Table 2a). The other five specimens we tested placed in the clade of Section Stephania in relation to heterogeneous samples cited in Heinrichs et al. (2012) as “S. griffithii”. The specimen from Vietnam # V-9-17a-17 placed in the subclade with S. “griffithii” from Nepal (-/69/96). The level of p-distances for their ITS1-2 is 1.4% (Table 2b), p-distances for trnL-F were not calculated due to absent data for the specimen from Nepal. Possibly, both specimens belong to a 126 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. TABLE 2. The value of infraspecific and infrageneric p-distances for the genus Scapania: a) Section Curtae, b) Section Stephania, c) Section Scapania. “n/c”-non calculated value due to single DNA locus, “-“non calculated value due to unsequenced DNA locus. a) section Curtae Infrageneric p-distances, ITS1-2/trnL-F, % Infraspecific p-distances, ITS12/trnL-F, % Taxon S. curta S. obcordata S. helvetica S. irrigua S. scandicaS. griffithii S. lingulata S.curta China S. curta n/c/n/c S. obcordata n/c/n/c 1.6/0.8 S. helvetica n/c/n/c 2.7/0.2 2.4/0.6 S. irrigua n/c/n/c 2.2/0.2 1.9/0.6 0.7/0 S. scandica-S.curta 0.1/0 2.5/0.2 2.2/0.9 1.3/0.4 0.8/0.4 S. lingulata n/c/- 5.0/- 4.5/- 5.5/- 4.9/- 5.0/- S. griffithii China n/c/n/c 5.9/2.1 5.7/3.1 6.4/2.3 5.9/2.4 6.3/2.4 b) 6.7/- section Stephania Infrageneric p-distances, ITS1-2/trnL-F, % Infraspecific pdistances, ITS12/trnL-F, % Taxon S. “griffithii” Nepal S. metahimalayana S. javanica S. pseudojavanica S. “griffithii” Nepal n/c/n/c S. metahimalayana n/c/n/c 1.4/- S. javanica 0.3/0 2.9/- 2.2/0.5 S. pseudojavanica 1.5/0.3 2.2/- 2.1/0.4 2.3/0.7 S. ligulata China 0.7/0.2 4.4/- 4.7/1.7 5.2/1.7 4.3/1.9 S. ligulata Nepal, South Korea + S. parvitexta Vietnam 1.2/0.3 4.5/- 4.9/1.1 5.3/1.3 5.3/1.3 c) S. ligulata China S. ligulata Nepal, South Korea + S. parvitexta Vietnam 4.9/1.8 section Scapania Taxon Infraspecific p-distances, ITS1-2/trnL-F, % Infrageneric p-distances, ITS1-2/trnL-F, % S. undulata S. paludosa S. subalpina S. undulata 0.2/0 S. paludosa 2.7/n/c 4.4/1.6 S. subalpina 3.8/0 4.7/2.1 3.8/0.6 S. rufidula n/c/n/c 3.2/2.3 4.2/0.6 4.8/1.8 S. sichuanica n/c/n/c 5.6/3.4 6.9/1.5 6.7/2.6 DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA S. rufidula S. sichuanica 4.5/2.9 Phytotaxa 400 (3) © 2019 Magnolia Press • 127 single species, but the sequence and morphological data were too incomplete to draw such a conclusion. Three other Vietnamese and Chinese specimens were located in a subclade with two Chinese “S. griffithii” samples (-/99/1.00). The level of ITS1-2 sequence divergence within this subclade is 1.5% in ITS1-2 and 0.3% in trnL-F (Table 2b). We propose to treat these specimens as belonging to a single species. Both S. javanica specimens from Indonesia compose a subclade (100/100/1.00) and reveal a low level of sequence divergence–0.3% in ITS1-2 and 0% in trnLF. The sequence divergence between three discussed subclades is 2.1–2.9% in ITS1-2 and 0.4–0.7% in trnL-F that exceed variability within subclades. Thus, taking into account morphological features and their probable recent divergence we refer these two species as new to science: Scapania metahimalayana Vilnet et Bakalin sp. nov. and S. pseudojavanica Vilnet et Bakalin sp. nov. The specimen identified as S. parvitexta Stephani (1897: 107) from Vietnam came out in the subclade composed of two specimens of S. ligulata Stephani (1904: 14) from Nepal and South Korea (100/100/1.00) in a sister relationship (66/85/0.86) to a subclade of two Chinese specimens of S. lingulata Buch (1916: 92) (100/100/1.00). The level of sequence divergence within the listed subclades counted 1.2% and 0.7% in ITS1-2 and 0.3% and 0.2% in trnL-F. Both subclades had diverged by 4.9% in ITS1-2 and 1.8% in trnL-F that corresponded to a level of species differentiation in Scapania. We suppose that morphological examination of tested accessions allow clarifying diagnostic characters in the S. parvitexta-S. lingulata-complex. The single Chinese specimen # China-42-3-17 with putative morphology of S. undulata (Linnaeus 1753: 1132) Dumortier (1835: 14) was tested together with multiple samples of S. undulata from Europe and North America (100/100/1.00) and resolved in a separate lineage (100/100/1.00) from S. rufidula Warnstorf (1921: 94) and the subclade of sister related S. subalpina (Nees et Lindenberg in Lindenberg 1829: 55) Dumortier (1835: 14) and S. paludosa (Müller 1901: 220) Müller (1902: 287). The level of p-distances between the Chinese specimen and other species of Section Scapania varied from 4.5 to 6.9% in ITS1-2 and from 1.5 to 3.4% in trnL-F (Table 2c). The reevaluation of morphological features allowed us to describe a third species as new for science: Scapania sichuanica Bakalin et Vilnet sp. nov. Discussion The morphological characters of Scapania griffithii Scapania griffithii was described by Schiffner (1899) from a specimen collected by L. Durel 12 April 1898 in “British Bhutan, zwischen Maria Basti und Labar in 5000–6000 Fuss Seehohe Gemaht”, under no. 183b (as in l.c.). However, the specimen with the same label data is numbered in PC (PC 0103800) as 193b. We here assume that the discrepancy between the number on the original description in Schiffner (1899) and the number on the voucher envelope is the result of a miswriting or misprinting. The voucher specimen is characterized by: 1) narrow and acute lobes, coarsely (short-) dentate along margin of the apical third of the ventral lobe, and entire dorsal lobe margin, 2) long decurrent ventral leaf lobe and not or barely decurrent dorsal leaf lobe, 3) gemmae colorless, long ellipsoidal (bacilliform), 2celled, 32–35 × 10–14 μm, 4) very small leaf cells 12.5–22.5 × 10–12.5 μm in mid-ventral lobe, with trigones small, concave, cuticle smooth, cells along margin unequally to subequally thickened, 7–12 μm, with trigones small to indistinct, concave (Fig. 2). It worth noting there is further confusion in the understanding of this species due to the ‘lectotypification’ of S. griffithii by the type of Scapania spathulata (G 00064180/8172) in Potemkin (2001), then cited by Potemkin et al. (2004). Indeed, on the label of this specimen it is written ‘Scapania griffithii’. However, the specimen represents the syntype of Scapania spathulata collected by L. Steevens in Darjeeling (Sikkim Himalaya) and therefore cannot be used as lectotype of S. griffithii. The plants in the isotype of S. griffithii in PC and both syntypes of S. spathulata present in G (G 00043851/8173, G 00064180/8172) are morphologically identical to one another and may be regarded as conspecific. This then results in the synonymy of both names, beginning with Müller (1905) who treated the latter as the variety under the former, which was widely accepted (cf. Potemkin, 2001). To fix the understanding of the taxon we provide the new lectotypification of the species by the specimen from PC, since types are absent in FH, where most of Schiffner’s originals are kept. Scapania griffithii Schiffner Lectotypus designated here: L. Durel 193b, 12 April 1898 in “British Bhutan, zwischen Maria Basti und Labar in 5000–6000 Fuss Seehohe Gemaht” (PC 0103800). 128 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. FIGURE 1. Phylogram obtained in a Bayesian analysis for the family Scapaniaceae based on combined nucleotide sequences dataset of ITS1-2+trnL-F. Bootstrap support values of maximum parsimony, maximum likelihood analyses and Bayesian posterior probabilities more than 50% (0.50) are indicated. The values of length for cut branches are shown. The taxon names, country and GenBank accessions numbers for ITS1-2 and trnL-F are provided. Accessions started with EU/AF/AY were taken from Vilnet et al. (2010), accessions starting with JN-from Heinrichs et al. (2012), data for Scapania marsupelloides-from Mamontov et al. (2018). Newly studied specimens are marked by asterisk. DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 129 FIGURE 1. (Continued) ‘Scapania griffithii’ specimens in Heinrichs et al. (2012) Three specimens named as Scapania griffithii by Heinrichs et al. (2012) were found in the same clade with our collection from Guizhou Province (China-56-37-13). One of the specimens at JE was available for study, the others at HSNU were unavailable. The specimen at JE, Long 17552, was identified as S. griffithii by D.G. Long with the identification confirmed by J. Váňa in 2011. The description based on the plants from the specimen (Long 17552, JE) is as follows: “Plants relatively rigid, 2.2–3.2 mm wide, 10–15 mm long, yellowish greenish, strongly dorsiventrally compressed. Stem brownish to blackish brown, cross-section 250–300 μm wide, 150–200 μm high, hyaloderm absent, scleroderm with brownish colored thick cell walls in (1)2–3-layers in older part of the shoot. Rhizoids sparse, colorless to grayish, restricted to lower portion of the shoot. Leaf lobes strongly unequal, dorsal lobe subtransversely inserted, not or barely decurrent, 1.0–1.1 × 0.7–0.8 mm, nearly plane; ventral lobe nearly plane to slightly convex, not turned to ventral side, decurrent for ca. 1.0 of stem width or more, 1.6–1.7 × 1.0–1.1 mm, keel at 70–80° with axis, nearly straight, not or obscurely winged. Cells along ventral lobe outer margin (6)7–10(12) μm, thick-walled, with small 130 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. concave trigones, cuticle smooth; cells in mid-ventral lobe 12–20 × 10–15 μm, nearly thin-walled, to somewhat thickened, trigones small, concave, cuticle obscurely to more or less distinctly papillose. Gemmae in brown masses in shoot apices, brown, unicellular, ellipsoidal, 13–18 × 7–8 μm”. At least, because of the copious and distinctively bright brown colored unicellular gemmae, the plant is not Scapania griffithii. FIGURE 2. Scapania griffithii Schiffn.: 1—plant habit, fragment, dorsal view; 2—plant habit, fragment, ventral view; 3, 4—leaves; 5—leaf margin cells of ventral lobe; 6—gemmae. Scales: a—1 mm, for 1–4; b—100 μm, for 5, 6. All from PC 0103800 (PC). The infrageneric position of Scapania griffithii Potemkin (2001, 2002) placed S. griffithii into Sect. Nemorosae (Buch 1928: 152) Müller (1905: 155) that included taxa with leaves more or less “arcuately inserted, ovate to oblong reniform dorsal lobes short to long decurrent lingulate to subrotund and lanceolate ventral lobes, with marginal teeth occurring mostly in their distal and often median portions; midleaf cells thick-walled, with mostly 3–7 ± large oil bodies occluding mainly over half of cell lumen. Perianth mouth not lobulate, short to long and dentate, normally with no additional basal teeth. Gemmae 1–2-celled, mostly ± ovoid to (rarely) subspheric, bacilliform to ± angulate, brown to/or colorless” (Potemkin 2002: 313). Heinrichs et al. (2012: 983) did not provide data on the relationships of S. griffithii, although the authors wrote that “Potemkin et al. (2004) doubted the occurrence of S. griffithii outside the Himalayan region and adjacent territories. However, in our present study, we recognized two specimens with the morphology of S. griffithii from Fujian, China. In our phylogeny, they form a polytomy with S. griffithii from Nepal, and S. javanica Gottsche”. Heinrichs et al. (2012: 983) suggestion is based on the placement of the specimens from Nepal (Long 17552) into the same clade as two specimens from China (which we were not able to study) with Scapania javanica. Since the cited specimens have nothing to do with true S. griffithii, there are no grounds to conclude that ‘true’ S. griffithii occurs outside of the Himalayan Region. Among our collections, we found one specimen that corresponds well both to the lectotype of Scapania griffithii (PC 0103800), the subsequent morphological descriptions in Müller (1905), and the differentiation note in Potemkin (2001). The specimen was collected in the southeastern Tibetan spur with characteristic Sino-Himalayan flora: “Sichuan Province of China, Zheduotangcum Area (29,991°N 101,888°E), 3165 m alt., the valley of small stream with evergreen Quercus Linnaeus (1753: 994), Betula and Rhododendron tree forest, in open moist clayish soil on path side in steep slope, leg. Bakalin V.A. & K.G. Klimova 13 October 2017, China-35-1-17” (VBGI-27432, duplicate in KPABG, MH930835, MH931422 cf. also Table 1, for GenBank accession numbers). The specimen forms the basal clade to Section Curtae in our phylogenetic tree (Fig. 1). The latter is not surprising. The close morphological similarity of Sect. Curtae and Sect. Nemorosae was noted and exhaustively discussed by Potemkin (2001, 2002) to which Potemkin placed both S. griffithii and S. javanica (!). Phylogenetic reconstructions show (Heinrichs et al. 2012; Fig. 1) the absence of affinity between taxa with ‘unicellular brown gemmae’, e.g. Scapania nemorea (Linnaeus 1759: 1337) Grolle (1963: 160) and S. crassiretis Bryhn (1892: 7) were found to be distinct from the S. javanica-complex, and therefore unicellular gemmae could be regarded here as the result of convergence or plesiomorphy. Features such as small cells along leaf margin, bicellular gemmae, crispate to remotely dentate perianth mouth and relatively small size also suggest Sect. Curtae for S. griffithii. Although the relatively long-decurrent ventral lobe and DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 131 restricted distribution put it aside of the bulk of other taxa of the Sect. Curtae that is also reflected in the basal position of the species within the Section. Below is the description based on plants from Sichuan Province: Scapania griffithii Schiffn. Plants prostrate to ascending, over fine soil or mixed with Solenostoma flagellare (Amakawa 1966: 258) Váňa and Long (2009: 501), dorsiventrally compressed, pale greenish to greenish brownish when fresh, 1.3–2.0 mm wide, 4–6 mm long. Rhizoids few, colorless, separated or united into indistinct fascicles, erect spreading. Stem brownish and greenish brownish to blackish brown in basal portion, rarely ventrally branched, but commonly with 1–2 subfloral innovations. Leaves merely distant to contiguous, keel 1/4–1/3 of ventral lobe length, rarely winged, slightly arched, at the distal sector at 70–80° with stem axis; dorsal lobe appressed to the stem, obliquely cordate, subtransversely to arcuately inserted, not or barely decurrent, margin entire, 0.6–0.7 × 0.5–0.7 mm, apex obtusely rounded; ventral lobe slightly convex to obcanaliculate, decurrent for 0.5–1.9 of stem width, margin nearly entire to remotely toothed (with low teeth), ovate to obovate, 0.8–1.2 × 0.5–0.7 mm. Cells in mid-lobe sub-isodiametric to slightly oblong, 12–23 × 10–16 μm, thin-walled, trigones moderate, concave, cuticle smooth or very loosely papillose; cells along ventral lobe margin thin-walled, 7–13 μm, with moderate to small trigones, teeth (if present) remote, obtuse, 1-celled, present in upper half. Gemmae bicellular, bacilliform, colorless. Dioicous. Perianth obtrapezoidal, loosely dorsiventrally compressed, somewhat plicate in dorsal side, 0.7–0.8 × 0.7 mm, mouth crispate to remotely dentate, with obtuse teeth 1–3 cells in the base. Speciation in the Scapania javanica-complex and Sect. Stephania The molecular-genetic analysis combined with observation on morphology (where available) showed: 1) close relationships of ‘S. griffithii’ named specimens in Henrichs et al. (2012), the specimen from Guizhou (China-56-3713) and three specimens from Vietnam (V-7-27-17, V-7-17-17 and V-7-19-17), 2) rich infraspecific variation within this clade, 3) remote position of the specimen named as S. griffithii from Nepal (Long 17552) in Heinrichs et al. (2012) study that was united into the same clade with the specimen initially named by us as S. undulata (V-9-17a-17) and 4) close relationships of all mentioned specimens with S. javanica s. str. from Indonesia. The genetic distances between three subclades observed are sufficiently large to treat each subclade as a separate species. We were not able to find existing names for both subclades allied to ‘true’ Scapania javanica and describe them here as taxa new to science. Additionally, we provide the description prepared from the type and other Javanese material of S. javanica s. str. for the purpose of comparison. Scapania pseudojavanica Vilnet et Bakalin sp. nov. Description. Plants rigid, in pure loose patches, deep green, green brown, brownish yellowish to brownish, without traces of red or purple pigmentation, (1.8-) 3.2–4.5 mm wide and 15–40 mm long. Rhizoids virtually absent. Stem brownish to blackish brown in lower portions, branching not seen (probably due to regular regeneration of population from gemmae); cross-section of well-developed shoots 225–320 × 300–440 μm, slightly transversely elliptic, outer wall of the stem cross-section commonly papillose, scleroderm with very thick-walled cells, sometimes with lumens absent and middle lamina visible, cells 10–20 μm in diameter, in 3–4 layers, inward thinner and larger, to 25 μm in diameter. Leaves distant to contiguous near apex, keel not winged, short, 1/8–1/4 of ventral lobe length, slightly arcuate; dorsal lobe nearly plane to slightly convex, appressed to stem, obliquely cordate to obliquely ovate, arcuately inserted, decurrent for ca. 1/5–1/2 of stem width, toothed along margin (teeth short, triangular), with exception of the base, 0.75–1.8 × 0.75–1.3 mm; ventral lobe obcanaliculate, more or less erect spreading, turned in ventral direction, decurrent for (1.0-)1.5–2.0 of stem width, dentate in upper half, entire in lower half and decurrencies, rarely somewhat crispate and without teeth, when flattened on the slide ellipsoidal to obovate, 1.4–2.5 × 1.0–1.6 mm. Cells in midventral lobe thin-walled, shortly oblong, with prominently nodulose, large to moderate (comparative trigones size is given after Bakalin, 2014) in size trigones, 20–38 × 12–20 μm, cuticle obscurely and finely to distinctly papillose; cells along lobe margin very strongly thick-walled, with moderate in size to large, concave trigones, (8-)12–18 μm along margin, teeth triangular to narrowly triangular, 1–2(–3) cells wide and 1–5 cells long, apical cell with thickened apical wall, 9–15 μm thick, toothed apical cells with finely verruculose cuticle. Dioicous. Androecia virtually intercalary, 3–5-androus, antheridium body obovate, male bracts ventricose with subequal lobes, brownish to reddish brownish in inflated area. Perianth 4–5 mm long, strongly dorsiventrally compressed, mouth densely spinosely dentate, with teeth 2–4 cells wide and 2–5 cell long, with narrowly triangular apical cell. Gemmae rare to copious, brown to rusty-brown, 10–17 × 7–9 μm, 1-celled, ellipsoidal, sometimes with mamillose projection at one end, in masses at the shoot apices (Fig. 3, 4). 132 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. FIGURE 3. Scapania pseudojavanica Vilnet & Bakalin: 1—plant habit, dorsal view; 2—perianthous plant habit, fragment, ventral view; 3—plant habit, fragment, ventral view; 4–7—leaves; 8—midleaf cells of ventral lobe, showed papilla; 9—ventral lobe; 10—ventral lobe, fragment, increased with cells showed; 11—perianth mouth, fragment; 12—perianth mouth, fragment, increased with cells showed; 13— stem cross-section, fragment. Scales: a—1 mm, for 1; b—1 mm, for 2; c—1 mm, for 3; d—1 mm, for 4–7; e—100 μm, for 8; f—1 mm, for 9; g—100 μm, for 10; h—1 mm, for 11; i—100 μm, for 12; j—100 μm, for 13. 1, 3–13—from V-7-27-17; 2 from—V-7-17-17 (VBGI). DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 133 FIGURE 4. Scapania pseudojavanica Vilnet & Bakalin: 1—plant habit, fragment, dorsal view; 2—plant habit, fragment, ventral view; 3—vegetative proliferation from inside of perianth; 4–8—leaves; 9—leaf margin cells of ventral lobe; 10—stem cross Section with leaf initial zone. Scales: a—1 mm, for 1, 2; b—1 mm, for 3; c—1 mm, for 4–8; d—50 μm, for 9, 10. All from China-56-37-13 (VBGI). HOLOTYPE:—Vietnam. Lao Cai Province, SaPa District, Hoang Lien National Park (22.340°N 103.776°E), 1995 m alt., evergreen mountain south-subtropical forest in the narrow stream valley, open cliffs near stream, leg. Bakalin V.A. & K.G. Klimova, 19 April 2017 (V-7-27-17, VBGI-36336, duplicate in KPABG, MH930837, MH931424). PARATYPES:—Vietnam. Lao Cai Province, SaPa District, Hoang Lien National Park (22.340°N 103.776°E), 1995 m alt., evergreen mountain south-subtropical forest in the narrow stream valley, open cliffs near stream, leg. Bakalin V.A. & K.G. Klimova, 19 April 2017 (V-7-19-17, VBGI-36329, duplicate in KPABG; V-7-17-17, VBGI36327, MH930838, MH931425, duplicate in KPABG). China. Guizhou Province. Duyun Municipality, Doupeng 134 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. Mountains, Xiniu Waterfall area (26°22’23.0”N 107°21’21.0”E), 1300 m alt., broadleaved (mostly evergreen) forest, moist boulder in open to partly shaded place, leg. Bakalin V.A. 22 November 2013. (China-56-37-13, MH930840, MH931427, duplicate in KPABG). Other specimens that are possibly the same species, where the decision was based on genetic study only: China (I), Fujian, Dehua Co. Zhu et al. 20100403-21 (HSNU) JN631419 JN631557 JN631684; Zhu et al. 20100403-25 (HSNU) JN631420 JN631558 JN631685 (Heinrichs et al. 2012). Distribution. The species is characterized by a southern Sino-Himalayan distribution; all known localities (including those known for specimens not studied morphologically) are at the southern part of spur SE from Tibetan Plateau. All localities lie in moist middle-to south-subtropical forests in mountainous areas of North Vietnam and Chinese Fujian and Guizhou Provinces. We may expect to find the species from Yunnan Province in China. The taxon prefers moist partially shaded places on neutral to slightly acidic boulders and cliffs near streams. Scapania metahimalayana Vilnet et Bakalin sp. nov. Description. Plants rigid, in loose patches, 3–4 mm wide, 20–40 mm long, greenish brownish with common purple tint when fresh to green-brown-purple when dry. Rhizoids virtually absent or a few in basal part of the shoot. Stem blackish brown, branching seen as subfloral innovations only; cross-section of well-developed shoots ca 225 × 280 μm, slightly transversely elliptic, scleroderm in 3–4 layers of cells, with such thick-walled cells as to sometimes render the lumen absent, 7–10 μm in diameter (between median laminas), inward with thin-walled cells 10–15 μm in diameter, with small concave trigones. Leaves contiguous, dorsal lobe subtransversely to loosely arcuately inserted, appressed to the stem dorsiventrally, when flattened on the slide widely obliquely ovate, 0.8–1.3 × 0.9–1.2 mm; ventral lobe ventrally spreading to almost at right angle with stem, convex-obcanaliculate, decurrent for 1.0–1.5 of stem width, when flattened on the slide obovate, 1.5–2.0 × 1.0–1.3 mm, dentate throughout, although teeth become smaller in ventral basal third, or nearly absent there (basal part of leaves slightly purplish), teeth 1–4 cells high and 1–2(–3) cells wide in the base, longer than wide; leaf keel not winged, ca 1.3 of ventral lobe length. Cells in mid-ventral lobe subisodiametric to shortly oblong, 17–25 × 12–20 μm, more or less thin-walled, trigones moderate to large in size, concave to convex, cuticle densely papillose to papillose-verrucose throughout; cells along margin 10–12 μm, thickwalled, with moderate in size, concave trigones, cuticle slightly verruculose to virtually smooth. Gemmae not seen. Dioicous? (Androecia not seen) Perianth strongly dorsiventrally compressed, ca 3 mm long, curved to ventral side, roughly dentate along mouth, teeth 3–4 cells high and 2–4 cells wide, nearly triangular (not cilia-like) (Fig. 5). HOLOTYPE:—Vietnam. Lao Cai Province, SaPa District, Hoang Lien National Park (22.308°N 103.776°E), 2900 m alt., Rhododendron dominated forest with Sinobambusa sat (Balansa 1890: 28) Chao & Renvoize (1989: 366) thickets, open moist cliffs, leg. Bakalin V.A. & K.G. Klimova, 19 April 2017 (V-9-17a-17, VBGI-36336, MH930836, MH931423, duplicate in KPABG) Comment. The specimen was initially named Scapania aff. undulata but differs from S. undulata in smaller cells both in the mid-ventral lobe and in the leaf margin: 17–25 × 12–20 and 10–12 μm, versus 30–50 × 16–25 and (12–)14–25 μm correspondingly. The new species differs from taxa of Section Stephania (taxa of the same super-clade with the S. javanica-complex in Fig. 1) in the long decurrent ventral lobe. Moreover, although we did not find the gemmae in the present species they tentatively should be brown and one-celled as in other taxa of the clade. Scapania metahimalayana differs from other taxa of S. javanica-complex in the possession of purple pigmentation that is not known in other taxa of the complex. The more problematic question is the relationships of Scapania metahimalayana with the specimen collected by Long in Nepal (Long 17552, JE). It is united in the same clade as S. metahimalayana, but the genetic distance is quite high. Moreover, the relationship of both taxa were measured only using ITS (trnL-F was not obtained in Henrichs et al. 2012). Whether the specimen named as S. griffithii from Nepal belongs to S. metahimalayana but as a subspecies (the point of view we are inclined to accept) or represents another, also undescribed species is unclear to us. The morphology of both specimens (V-9-17a-17 and Long 17552) is somewhat similar with the exception of a purple tint in pigmentation in the S. metahimalayana holotype that is completely absent in Long 17552. Scapania javanica Gottsche (Description based on isotypes, holotype is probably lost, and an additional specimen from Indonesia). Description. Plants rigid, yellowish brownish to somewhat rusty brownish, without red or purple tint, 2–3 mm wide and 15–20 mm long. Rhizoids virtually absent or a few, colorless, if present–in basal portions of the stem. Stem brownish to rusty brown, with somewhat copper tint, rarely laterally branched, but with common ventral subfloral innovations; the cross-section with scleroderm in 2 layers, cells very thick-walled, lumen sometimes absent, DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 135 FIGURE 5. Scapania metahimalayana Vilnet & Bakalin: 1–plant habit, dorsal view; 2–perianthous plant habit, ventral view; 3–6–leaves; 7–ventral lobe, fragment, increased with cells showed; 8–stem cross-section, fragment. Scales: a–1 mm, for 1; b–1 mm, for 2; c–1 mm, for 3–6; d–100 μm, for 8. All from V-9-17a-17 (VBGI). 136 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. 10–15 μm in diameter, inner walls of cells become thinner, inner tissue, 12–20 μm in diameter, with thin to thickened walls and small to large, concave to convex trigones. Leaves mostly contiguous, rarely distant, keel straight, erect spreading (80–90° with axis), not winged; dorsal lobe erect spreading to loosely appressed to the stem, somewhat convex, margin entire to scarcely dentate, obliquely cordate, 1.0–1.3 × 1.0–1.3 mm, not or barely decurrent; ventral lobe slightly convex, appressed to ventral side of the stem or curved to the ventral direction, shortly decurrent or up to 0.5–1.0 of stem width, remotely and shortly dentate, with teeth 2(–3) cells long, in lower third of the ventral lobe margins and the decurrencies entire, when flattened on the slide obliquely ovate, 1.7–2.0 × 1.2–1.3 mm. Cells in the mid-ventral lobe shortly oblong, 20–40 × 12–25 μm, thin-walled, trigones large, convex, nodulose, cuticle minutely verrucose along ventral lobe margin and finely to distinctly papillose in mid-leaf; cells along margin slightly thickened to almost thin, 10–20 μm, trigones large, concave, external wall noticeably thickened, with verruculose cuticle. Dioicous. Perianth loosely compressed, crispate and additionally shortly dentate along mouth edge, with teeth 2–4 cells long (Fig. 6). Specimens examined, are those upon which the description is based (we avoided the use of many specimens for the description because of the variability in the Scapania javanica complex which is poorly understood and the description may acquire alien traits due to ‘admixtures’ of the material belonging to another taxa):—Indonesia. Java Island, montis Prabakti leg. Zollinger n. 3502 (PC 0103796, G 00115740, isotypes); Java occidentalis Res. Priangan, G. Gedeh, in decliv. G. Pangerano, eiusque in cacumine, alt. 2700–3060 m a.s.l., leg. F. Verdoorn, August 1930 (VBGI); Philippines, Canlaon Volcano, 100 m a.s.l., leg. E.D. Merril, n. 6858, April 1910 (G 00067941/11619, holotype of Scapania spathulifolia). Comment. The species is morphologically most similar to S. pseudojavanica described above. The differences are minor and include: 1) scleroderm in stem cross-section in two layers of thick-walled cells in S. javanica, but with 3–4 cell rows in S. pseudojavanica, 2) cells along ventral lobe margin are strongly thick-walled in S. pseudojavanica while thin-walled or nearly so in S. javanica, 3) plants of S. pseudojavanica are more robust than those of S. javanica (commonly wider than 3 mm versus narrower than 3 mm wide). Aside from the mentioned morphological differences, the two taxa are strikingly different in distribution. Whereas S. javanica is presumably restricted to Malesia, S. pseudojavanica may be regarded as a Sino-to meta-Himalayan taxon distributed on the spurs of the Tibetan Plateau and as far as mountainous northern Indochina. Due to similar ecological niches of both taxa we do not think they have sympatric distribution even in Indochina where both taxa may potentially meet one another. The characters of Sect. Stephania The last entirely morphological classification of Scapania was proposed by Potemkin (2001) who treated Sect. Nemorosae very broadly by the inclusion of such taxa as Scapania nemorea, S. griffithii and S. integerrima Stephani (1910: 148) (in total 8 species, including S. crassiretis as a subspecies under S. nemorea) within one Section. This point of view was not supported by molecular-genetic investigations by Heinrichs et al. (2012) where the general topology is somewhat similar to that in the present account. Three taxa were referred to Sect. Stephania: S. ligulata, S. javanica and S. griffithii. This circumscription should be clarified in the light of the results obtained here by excluding S. griffithii due to its transfer to Sect. Curtae. Sect. Stephania, as identified above may be circumscribed as following: Plants medium sized, ventral lobes ellipsoidal to obovate, distinctly longer than wide, distinctly although in many cases shortly decurrent, leaf margins toothed, leaf cells pachydermous, relatively small, with marginal cells as a rule with distinctly thickened walls, trigones conspicuous although never nodulose, perianth mouth dentate to laciniate-dentate, strongly compressed, gemmae predominantly unicellular. Two subclades occur within the Section, that may conditionally be called browngemmiferous (S. javanica and relatives) and green-gemmiferous (S. ligulata and relatives). The first group, aside from having brown gemmae, is characterized by longer decurrencies of the ventral leaf lobes and also pronouncedly with a meta-Himalayan-Southeast Asian distribution. On the contrary, the second group tend to be more species-rich in the areas with more wet suboceanic to oceanic climates and stretches from the northern edge of the East Asian floristic region (as far as from the south of the Russian Far East in hemiboreal zone) to Southeast Asia, hardly spreading to the southeastern spur of Tibetan Upland. The only species of the group that may develop somewhat brownish gemmae is S. parvidens Stephani (1904: 15), a poorly known North Japanese species that has not yet been investigated molecularly. Moreover, it is worth noting the sampling of the second group uniting colorless-to green-gemmiferous taxa of the Section is hardly appropriate, and many taxa of East-Asian distribution (also including those of unclear status, like S. parvidens and S. stephanii Müller (1905: 273)) that need to be included in the analysis. Although even in the present topology, the molecular-genetic variation within the group suggested robust differences between different populations named as S. ligulata. DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 137 FIGURE 6. Scapania javanica Gottsche: 1—plant habit, fragment, dorsal view; 2—perianthous plant habit, fragment, ventral view; 7—perianthous plant habit, fragment, dorsal view; 8—plant habit, fragment, ventral view; 3, 4, 10—leaves; 5—ventral lobe, fragment, increased with cells showed; 6—stem cross-section, fragment; 9—gemmae; 11—midleaf cells of ventral lobe; 12—leaf margin cells of ventral lobe. Scales: a—1 mm, for 1, 2; b—1 mm, for 3, 4; c—100 μm, for 5; d—100 μm, for 6; e—1 mm, for 7, 8; f—1 mm, for 9, 10; g—100 μm, for 11, 12. 1–6 from F. Verdoorn, 1930 (VBGI), 7–11 from PC 0103796 (PC), 12 from Isotype G 00115740 (G). 138 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. Considering the date of the present study, unicellular gemmae in Scapania largely but not uniquely characterize Sect. Stephania, Sect. Nemorosae, partly so Sect. Apiculatae and Sect. Scapania. Sect. Nemorosae differs from Sect. Stephania by its markedly robust plants with wide ventral leaf lobes and mostly distinctly decurrent dorsal leaf lobes. Sect. Apiculatae generally differs from Sect. Stephania by its smaller sized plants and where unicellular gemmae are observed in epixylous S. apiculata Spruce (1847: no. 15). Sect. Scapania differs from Sect. Stephania by large sized plants and includes taxa with occasionally or predominantly unicellular gemmae (e.g. S. gigantea Horikawa (1931: 15)) where the ventral leaf base is widely and long decurrent. These four sections are distanced one from another and therefore the unicellular gemmae cannot be regarded as an apomorphic feature of species or Section complexes. The unicellular gemmae therefore may constitute evidence of convergence that appeared in several groups independently. The peculiar Scapania of the S. undulata complex from Sichuan Province of China Scapania metahimalayana described here is somewhat similar to S. undulata; and was the reason to include additional ‘Scapania undulata-like’ specimens to identify their relationships using molecular-genetic methods. One such specimen was ‘Scapania aff. undulata’ (China-42-3-17, VBGI-37342) that was found to be a well-defined species that formed a basal clade to other taxa of Sect. Scapania, as delimited in the present account and that of Potemkin (2002) and Heinrich et al. (2012). The description of the new species is as follows. Scapania sichuanica Bakalin et Vilnet sp. nov. Description. Plants brownish to deep brown, without traces of red or purple pigmentation, relatively rigid, in pure patches, submerged in ponds or sluggishly flowing watercourses, 3.5–4.5 mm wide and 25–50 mm long. Rhizoids nearly absent to very few in loose fascicles along ventral side of stem. Stem light to deep brown, rarely laterally branched, occurred once with depauperate ventral branch; cross-section of well-developed plants ca. 450 μm wide and 320 μm high, scleroderm 3(–4) cells thick, cells 7–10 μm in diameter, cell walls brown, thick, inner cells with thinner cell walls, with concave trigones, 10–25 μm in diameter. Leaves contiguous, dorsal lobe convex, with apex commonly incurved, arcuately inserted, short decurrent (to 1/3(–1/2) of stem width), when flattened on the slide, cordate, 1.3–1.5 × 1.4–1.7 mm, apex obtuse, rounded, margin entire or with sparse (2–4 in number) obtuse teeth in upper half; ventral lobe concave, with apices commonly incurved, decurrent for 0.5–1.0 of stem width, when flattened on the slide 1.7–2.3 × 1.3–1.5 mm, apex rounded, margin entire or with 1-several obtuse teeth in upper part; keel ca. 1/4–1/3 of ventral lobe length, not winged. Cells in mid-ventral lobe thick-walled, with somewhat visible median lamina, walls brown (median lamina bright rusty colored), oblong to subisodiametric, trigones small, 25–38 × 17–25 μm, cuticle finely and loosely papillose; cells along leaf margin 12–25 μm, with thick walls and vestigial trigones, external wall thickened, teeth unicellular, cuticle verruculose along margin. All other structures unknown (Fig. 7). HOLOTYPE:—China. Sichuan province, Kangding Airport area, (30.107°N 101.766°E), 4187 m a.s.l., SWfacing gentle slope of the range, covered with communities like dry hummocky tundra with low Rhododendron and Pentaphylloides Duhamel du Monceau (1755: 99) shrubs, over stone submerged in sluggishly flowing stream, in open, leg. Bakalin V.A. & K.G. Klimova 14 October 2017 (China-42-3-17, VBGI-37342, MH930845, MH931432, duplicate in KPABG). Comment. Among other taxa of the Section the species is the most morphologically similar to Scapania gigantea— a poorly understood species, nevertheless well-discussed by Potemkin (2001, 2002). Scapania gigantea was described from Japan (Honshu) and then recorded in Yunnan by Potemkin (2002) an unexpected record. Scapania gigantea differs from S. sichuanica in: 1) purple to purple-brown pigmentation (versus purple pigmentation absent, even as traces), 2) thin-walled cell walls in the middle part of leaf ventral lobe (versus thick-walled, with visible median lamina, despite submerged habitat), 3) finely dentate leaves, with teeth developed even on the decurrency, where they are sometimes larger than above (versus leaves entire or with 1-several remote and obtuse teeth in upper 1/3–1/2 of ventral lobe length). In addition to morphology there is the difference in the distribution. Scapania sichuanica was collected in habitat surrounded by alpine steppe, and is one of few reports for Scapania above 4000 m a.s.l. The collections of S. gigantea (whose range is poorly understood since the taxon was accepted as distinct only recently, (cf. Potemkin 2001) belong to an oro-temperate belt of mountains with lower elevation in the middle of Japan (800 m a.s.l.) to 2800 m a.s.l. in Yunnan. Concerning the latter record we suggest the possible misidentification of S. sichuanica for S. gigantea in Potemkin (2001). DIFFERENTIATION IN SCAPANIA SECT. STEPHANIA Phytotaxa 400 (3) © 2019 Magnolia Press • 139 FIGURE 7. Scapania sichuanica Bakalin & Vilnet: 1—plant habit, fragment, dorsal view; 2—plant habit, fragment, ventral view; 3–5— leaves; 6—leaf margin cells of ventral lobe; 7—stem cross-section, fragment. Scales: a—1 mm, for 1; b—1 mm, for 2; c—1 mm, for 3–5; d—100 μm, for 6, 7. All from China-42-3-17 (VBGI). Phytogeographic speculations Sino-Himalayas and the adjacent ‘meta-Himalayan’ mountains represent a rich taxonomic center of Scapania distribution with a number of morphologically distinct and geographically restricted taxa, e.g. S. bhutanensis Amakawa, S. harae Amakawa (1964: 5), S. pseudocontorta Potemkin (2000: 115). The area appears to have been a refuge for Scapania and the source from which their subsequent dissemination occurred after a period of unfavorable environmental conditions in the past. Along the way, some species apparently diverged. The results of the present work suggest that unicellular gemmae may have been the result of convergent evolution within Scapania and therefore is a feature that may be helpful in morphology-based identification at the Section level but not higher (e.g. subgenus). Additionally, the color of gemmae in ‘unicellular’ groups may have identification value at the sub-Section level of Scapania but not higher. The Sect. Stephania houses two groups of taxa that conditionally may be regarded as green gemmae-bearing and brown gemmae-bearing groups. These groups (possible sub-Sections) are different in distribution and may illustrate speciation at a later stage of the evolutionary process. In general Sect. Stephania is the East Asian Section with some taxa spreading to South-East Asia. Taking into account the moderate size of plants, relatively small-sized leaf cells and pachydermous leaf cell structure we may estimate its origin in the relatively dry monsoon climate of eastern Sino-Himalaya from where these taxa have spread East-, Northeast-and Southeast-ward. This spreading process was accompanied by speciation that provided diversity within the group that is still poorly understood. 140 • Phytotaxa 400 (3) © 2019 Magnolia Press BAKALIN ET AL. Resulting from the present work, the S. javanica complex comprises two or three taxa in Sino-Himalaya that are similar to another and to S. javanica s. str. Scapania javanica may be regarded as possibly the end result of the speciation process when it reached Malesia, whereas the number of taxa has been retained (and probably restricted) by the Sino-Himalayas. In a very general way we suggest the speciation process was taking place across the Himalayas into the southeastern spur of the Tibetan Plateau and subsequently into Malesia (if not farther). The variability of ecological niches (and climates) across this range promoted speciation and resulted in sets of related taxa, where the distribution of some taxa may be very narrow. A similar situation was observed in the Scapania undulata complex, where S. sichuanica was observed and described. The above pattern is certainly not limited to Scapania, but occurs in other groups at least those tending to mountainous distribution, e.g. Marsupella Dumortier (1822: 114), Solenostoma Mitten (1864a: 51), Plectocolea (Mitten 1864b: 156) Mitten (1871: 405) and some Plagiochila (Dumortier 1835: 14) Dumortier (1831: 42). A similar situation was recently observed in Liochlaena Nees (Gottsche et al. 1845: 150) (Bakalin et al. 2018b). Considering that these dispersal and speciation processes are probably common in the mountainous terrain of Sino-and meta-Himalayan regions, formal identification of morphologically similar plants at the edges of the region may remain doubtful without careful comparison using an integrative approach such as described in the present study. Acknowledgements The authors are sincerely grateful to Mr. M. Bakalin for his line drawings and to Dr. M. Hutten for English correction in the manuscript. We are very thankful for the specimen collection and transfer permits that were facilitated by the Institute of Ecology and Biological Resources of the Vietnam Academy of Science (Dr. V.S. Nguyen) and Guizhou University (Prof. Y. Xiong). This study was partially supported by grants from the Russian Foundation for Basic Research, including: 12-04-91150-ГФЕН_а for international Russian-Chinese collaboration work and specimen collecting and 17-04-00018 for molecular-genetic analysis; no. 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