A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae)

Accepted Manuscript A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae) Ke-Wang Xu, Xin-Mao Zhou, Qian-Yi Yin, Liang Zhang, Ngan Thi Lu, Ralf Knapp, Thien Tam Luong, Hai He, Qiang Fan, Wan-Yi Zhao, Xin-Fen Gao, Wen-Bo Liao, Li-Bing Zhang PII: DOI: Reference:

S1055-7903(18)30050-2 https://doi.org/10.1016/j.ympev.2018.05.021 YMPEV 6172

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

27 January 2018 2 May 2018 17 May 2018

Please cite this article as: Xu, K-W., Zhou, X-M., Yin, Q-Y., Zhang, L., Thi Lu, N., Knapp, R., Tam Luong, T., He, H., Fan, Q., Zhao, W-Y., Gao, X-F., Liao, W-B., Zhang, L-B., A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae), Molecular Phylogenetics and Evolution (2018), doi: https://doi.org/10.1016/j.ympev.2018.05.021

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Running title: Xu et al. - Phylogeny and cryptic speciation in Hymenasplenium

A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae) Ke-Wang Xua, i , Xin-Mao Zhoub, c, i, Qian-Yi Yina, Liang Zhangd, Ngan Thi Lub, e, f, Ralf Knappg, Thien Tam Luongh, Hai Hei, Qiang Fana, Wan-Yi Zhaoa, Xin-Fen Gaob, Wen-Bo Liao a, *, and Li-Bing Zhangb, j, * a

State Key Laboratory of Biocontrol and Guangdong Provincial Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-sen University, Guangzhou, Guangdong 510275, China; b CAS Key Laboratory of Mountain Ecological Restoration and Bioresource Utilization, Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu, Sichuan 610041, China; c Laboratory of Ecology and Evolutionary Biology, State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, Yunnan University, Kunming 650091, China. d Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, Yunnan 650201, China; e University of Chinese Academy of Sciences, Beijing 100049, China; f Department of Biology, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18th Hoang Quoc Viet Road, Ha Noi, Vietnam; g Correspondent of the Muséum national d'Histoire naturelle (MNHN, Paris, France), Steigestrasse 78, 69412 Eberbach, Germany; h Department of Ecology - Evolutionary Biology, Vietnam National University Ho Chi Minh City (VNUHCM) - University of Science, 227 Nguyen Van Cu Street, District 5, Ho Chi Minh City, Vietnam; i

College of Life Sciences, Chongqing Normal University, Shapingba, Chongqing 400047, China;

j

Missouri Botanical Garden, P.O. Box 299, St. Louis, Missouri 63166-0299, U.S.A. *Authors for correspondence: Wen-Bo Liao, [email protected]; Li-Bing Zhang, [email protected]

Abstract The fern genus Hymenasplenium (Aspleniaceae) is one of the two genera in the family. It is generally recognized among modern pteridologists. However, its infrageneric relationships and species diversity have been unclear and controversial. The molecular studies so far have had small taxon and character sampling. In the present study, DNA sequences of six plastid markers of 158 accessions representing ca. 40 out of ca. 50 known species of Hymenasplenium, and 16 species of Asplenium were used to infer a phylogeny with maximum likelihood, Bayesian inference, and maximum parsimony approaches. Our major results include: (1) Hymenasplenium as currently defined is strongly supported as monophyletic; (2) three major clades representing early splits in Hymenasplenium are identified, with the Old World species being strongly supported as monophyletic; it is ambiguous if the New World species are monophyletic; (3) extensive cryptic speciation in the

Old World is discovered demonstrating the complexity of evolution of the genus; and (4) six strongly or moderately supported subclades in the Old World clade are revealed, differing from one another in molecular, morphological, and geographical features. Key words: Asplenium; cryptic species; eupolypods II; fern phylogeny; pantropical distribution

1 Introduction Species of the fern genus Hymenasplenium Hayata (Aspleniaceae) are morphologically characterized by having rhizomes long-creeping and with very few scales except near apex, fronds remote, laminae rarely simple and usually 1-pinnate, and costa usually with several (rarely one) basal basiscopic veins absent (Murakami and Moran, 1993; Lin and Viane, 2013; Xu et al., 2018; our Figs. 1, 2). They are epilithic, epiphytic or rarely terrestrial herbs distributed worldwide, of which more than 65% occur in Southwest China, with some outliers extending to the tropical regions of Afro-Madagascar, tropical Asia, and the New World (Ching, 1965; Smith, 1976; Murakami and Hatanaka, 1988a; Wu, 1989; Murakami and Moran, 1993; Lin and Viane, 2013; Gabancho and Prada, 2011; Hong and Chang, 2017; Xu et al., 2018). Hymenasplenium was established mainly based on anatomical features. Hayata (1927) studied the anatomical structure of stelar organization of Asplenium unilaterale Lam. and briefly described Hymenasplenium as a new genus based on the this species. Hayata (1927, 1928) defined the genus as having peculiar stele structure of the dorsiventrally long-creeping rhizome. After Hayata’s evaluation of anatomical structure of stelar organization in ferns, more and more anatomical observations in Aspleniaceae were conducted by pteridologists, such as those of the dorsiventral steles of H. unilaterale and H. cheilosorum (Kunze ex Mett.) Tagawa by Tardieu-Blot (1932), those of the perforated dictyostele of A. ensiforme Wall. ex Hook. & Grev., those of the radial dictyosteles with two leaf traces at each leaf gap of some other species of Aspleniaceae by Bir (1957, 1970), and those of the peculiar bud traces which originate apparently continuously from root traces of A. decrescens Kunze by Chandra and Nayar (1975). Before the advent of molecular phylogenetics, various other characters of Hymenasplenium have been observed and analyzed to elaborate the systematic status of this particular group of Aspleniaceae (Mehra and Bir, 1960; Iwatsuki and Kato, 1975; Murakami and Iwatsuki, 1983; Murakami and Hatanaka, 1988a, 1988b; Watano and Iwatsuki, 1988; Murakami and Moran, 1993), but Hymenasplenium was generally not recognized. Manton and Sledge (1954) counted the chromosomes numbers of H. unilaterale and H. cheilosorum (Kunze ex Mett.) Tagawa, both placed under Asplenium, to be 2n = 80 and +80, respectively. Iwatsuki and Kato (1975) studied the stelar structure of H. unilaterale (Lam.) Hayata and its allied species and concluded that the dorsiventral stele anatomy of this group might have evolved from the radial dictyostele typical of Asplenium in order to adapt to the petrophytic habitat. Thus, they believed that there was no sufficient evidence to segregate Hymenasplenium from Asplenium, and referred them to A. sect. Hymenasplenium (Hayata) K. Iwatsuki as a monophyletic group within Asplenium (Iwatsuki 1975). Mitui et al. (1989)

studied the chromosome numbers of seven species of Hymenasplenium and showed that the basic chromosome number of Hymenasplenium was x = 39, with only one being x = 38 (Mutui et al., 1989), different from x = 36 in Asplenium (Manton, 1950; Manton and Sledge, 1954; Bir, 1960; Bellefroid et al., 2010; Lin and Viane, 2013). Even so, they still regarded this group as a section of Asplenium. In 1992, the stelar structure of 10 New World species of Hymenasplenium was observed and compared to that of Hymenasplenium Old World species and it was concluded that the New and Old World groups are closely related (Murakami, 1992). Still, Murakami and Moran (1993) treated all members of Hymenasplenium in A. sect. Hymenasplenium in their later monograph (Murakami and Moran, 1993). Based on chloroplast restriction enzyme data of one Asian species and nine New World species of Hymenasplenium, the first molecular phylogeny of Hymenasplenium revealed that the New World species were sister to the Asian species (Murakami and Schaal, 1994). Based on plastid rbcL data, Murakami (1995) and Murakami et al. (1999) demonstrated that species with dorsiventral creeping rhizomes, including both Old and New World species of Hymenasplenium, together with Boniniella Hayata, formed a monophyletic group sister to the other members of Aspleniaceae sampled. Based on these findings, the recognition of Hymenasplenium as a genus was advocated (Murakami, 1995; Murakami et al., 1999). The sister relationship between Hymenasplenium and Asplenium s.s. was confirmed by subsequent studies (Murakami et al., 1999; Gastony and Johnson, 2001; Schneider et al., 2004, 2017; Schuettpelz and Pryer, 2007; Rothfels et al., 2012a; Ohlsen et al., 2015; Lóriga et al., 2016; Sessa et al., 2018). Therefore, Hymenasplenium as a genus is well defined by molecular, anatomical, morphological, and chromosome evidence, and its extrageneric relationships are well understood because it is one of the only two genera in Aspleniaceae (Smith et al., 2006; Rothfels et al., 2012b). However, the infrageneric relationships and species diversity within Hymenasplenium have been unclear and controversial. For example, in studying Chinese material of H. unilaterale and allies Ching (1965) described eight new members of Hymenasplenium from China. In contrast, Lin and Viane (2013) believed that the true H. unilaterale was not found in China and it was erroneously used for H. apogamum (N. Murak. & Hatan.) Nakaike, H. hondoense (N. Murak. & Hatan.) Nakaike, and H. murakami-hatanakae Nakaike in Asia. Murakami (1995) gave an estimate of 50–60 species for the genus, while Lin and Viane (2013) thought that the genus contained 30+ species. Molecular studies so far with species of Hymenasplenium included have generally had small taxon and character sampling (Murakami and Schaal 1994; Murakami, 1995; Murakami et al., 1995, 1999; Gastony and Johnson, 2001; Pinter, 2002; Schneider et al., 2004, 2005, 2017; Schuettpelz and Pryer, 2007; Bellefroid, 2010; Rothfels et al., 2012a; Ohlsen et al., 2015; Lóriga et al., 2016; Sessa et al., 2018). No comprehensive species-level phylogenies of Hymenasplenium have been available. In this study, we aim: (1) to test the monophyly of Hymenasplenium with broad taxon (including ca. 76% of global diversity) and character (six plastid markers) sampling; (2) to determine the relationships between the New World and the Old World species of Hymenasplenium and test the monophyly of the Old World species; (3) to resolve relationships across Hymenasplenium and identify major evolutionary lineages; (4) to test the monophyly of relatively widely distributed species with multiple-accession sampling across

their distribution ranges. 2 Materials and methods 2.1 Taxon sampling Material was mainly obtained from field collections and the herbarium of Missouri Botanical Garden. We also included DNA data of Hymenasplenium available in GenBank. As for the ingroup, we sampled 141 accessions representing ca. 24 (19 Old World spp. and 5 New world spp.) out of the total 34 generally accepted species of Hymenasplenium (ca. 71% of the species diversity of the genus) and 16 seemingly undescribed species. In total, ca. 40 species (ca. 80%) out of the 50 species known to us were included as our ingroup. Of the 141 accessions of the ingroup, seven were from the New World, while 135 were from the Old World. Our sampling covered most of the geographical range of Hymenasplenium (Fig. 3). For outgroups, we used 16 accessions of Asplenium representing 12 major clades of Asplenium defined by previous studies (Schneider et al., 2004; Ohlsen et al., 2015). In total, 158 accessions representing ca. 56 species of Aspleniaceae were included in the present study. Voucher information and GenBank accession numbers for each sampled taxon are provided in Appendix I. 2.2 DNA extraction, amplification, and sequencing Total genomic DNA was extracted from silica-dried material or sometimes from herbarium fragments using the TIANGEN plant genomic DNA extraction kit (TIANGEN Biotech., Beijing, China) following the manufacturer’s protocol. Six plastid markers (the atpB gene, the rbcL gene, the rps4 gene, the rps4-trnS intergenic spacer, the trnL intron, and the trnL-F intergenic spacer) were selected for the phylogenetic study. The atpB gene was amplified with primers ESATPB172F and ESATPE45R (Schuettpelz & Pryer 2007), and the newly designed internal primers atpB-20F, atpB-249F, atpB-592R, and atpB-1228R. The rbcL gene was amplified with primers ESRBCL1F and ESRBCL1361R (Schuettpelz and Pryer, 2007), and the internal primers 595F (Le Péchon et al., 2016a, b) and 819R (Zhang et al., 2015). The rps4 gene and the rps4-trnS intergenic spacer were amplified using the primers F and R (Schneider et al., 2005), and the newly designed internal primers rps4-118F, rps4-911R, and rps4-532R. The trnL intron and trnL-F intergenic spacer were amplified together using the primers FERN1 (Trewick et al., 2002), E, and F (Taberlet et al., 1991), and the newly designed primers trnL-F-43F and trnL-F-430R. The primer sequences and references are listed in Table 1. PCR conditions of the atpB gene followed Schuettpelz and Pryer (2007) and others followed Ohlsen et al. (2015). PCR products were purified and sequenced by TSINGKE Biological Technology (Guangzhou, China). 2.3 Sequence alignment and phylogenetic analysis Contiguous sequences were assembled and edited using Sequencher 4.1 (Gene Codes Corp., Ann Arbor, MI, USA). Sequences obtained for each marker were initially aligned with MAFFT ver. 7 (Katoh and Standley, 2013) and manually adjusted in BioEdit (Hall, 1999). Equally weighted maximum parsimony (MP) analyses for each locus were conducted in PAUP* ver. 4. 0b10 (Swofford, 2002) using 1000 tree-bisection-reconnection (TBR) searches

with MAXTREES set to increase without limit. Insertions and deletions were coded as missing data. Parsimony jackknife (JK) analyses (Farris et al., 1996) were conducted using PAUP* with the removal probability set to approximately 37%, and “jac” resampling emulated. One thousand replicates were performed with 10 TBR searches per replicate and a maximum of 100 trees held per TBR search. jModelTest 2 (Guindon & Gascuel, 2003; Posada, 2008; Darriba et al., 2012) was used to select the best-fitting likelihood model. The Akaike information criterion (Akaike, 1974) was used to select among models (Table 2), following Pol (2004) and Posada & Buckley (2004). For each marker and the concatenated analysis of all nucleotide characters, maximum likelihood (ML; Felsenstein, 1973) tree searches and ML bootstrapping (BS) were conducted using the web server RAxML-HPC2 on TG ver. 7. 2. 8 on the Cipres web server (Miller et al., 2010), with 5000 rapid bootstrap analyses followed by a search for the best-scoring tree in a single run (Stamatakis et al., 2008). Bayesian inference (BI) was conducted using MrBayes 3.1.2 (Ronquist and Huelsenbeck, 2003) on Cipres (Miller et al., 2010). Two runs of four Markov chain Monte Carlo chains were conducted, each beginning with a random tree and sampling one tree every 1000 generations of 10,000,000 generations. Convergence among chains was checked using Tracer ver. 1.4 (Rambaut and Drummond, 2007), and the first 25% was discarded as burnin to ensure that stationarity in log-likelihood had been reached. The remaining trees were used to calculate a 50% majority-rule consensus topology and posterior probabilities (PP). 2.4 Morphology Morphological data were obtained from field observations (e.g., Figs. 1, 2), herbarium investigations including online images available at JSTOR (plants.jstor.org) and CVH (www.cvh.ac.cn), and literature studies (e.g., Ching, 1965; Murakami and Moran, 1993; Wu, 1999; Knapp, 2011, 2013; Lin & Viane, 2013; Ebihara, 2016; Xu et al., 2018). 3 Results A total of 310 (with trnL and trnL-F combined and rps4 and rps4-trnS combined) sequences were newly generated for this study (Appendix I). The total length of the aligned sequences was 4,996 bp (details concerning the datasets analyzed and statistics for the resulting trees are shown in Table 3). A comparison of the trees resulting from MPJK analyses of the individual plastid markers did not identify any well-supported conflicts in MP analyses (MPJK > 70%; Mason-Gamer & Kellogg, 1996; Zhang & Simmons, 2006; Zhang et al., 2015; Zhou et al., 2016). The six plastid markers were therefore concatenated and analyzed in unison. The tree topologies by the MP, ML, and BI analyses were generally concordant when using the concatenated dataset. The ML phylogeny of Hymenasplenium and outgroups based on the concatenated data is presented in Fig. 4, while the same phylogeny with detailed support values (>50%) is presented in Fig. S1. Our data resolved our 141 accessions of Hymenasplenium into three strongly supported clades: the H. laetum clade (MLBS: 99%; MPJK: 92%; BIPP: 1.00), the H. riparium clade (MLBS: 100%; MPJK: 97%; BIPP: 1.00), and the Old World clade (MLBS: 100%; MPJK: 98%; BIPP: 1.00). The H. laetum clade and the H. riparium clade are endemic to the New World and they together were resolved as monophyletic but with strong support only in ML

analysis (MLBS: 90%; MPJK: 63%; BIPP: 0.7; Fig. S1). Within the Old World clade, six strongly or moderately supported subclades were identified: the H. wildii subclade (MLBS: 97%; MPJK: –; BIPP: 1), the H. hondoense subclade (MLBS: 90%; MPJK: 63%; BIPP: 0.7), the H. excisum subclade (MLBS: 73%; MPJK: 78%; BIPP: 1), the H. unilaterale subclade (MLBS: 98%; MPJK: 97%; BIPP: 1), the H. cheilosorum subclade (MLBS: 100%; MPJK: 95%; BIPP: 1), and the H. obliquissimum subclade (MLBS: 72%; MPJK: 51%; BIPP: 0.99). A sampling/distribution map of the six subclades of the Old World calde in six different colors is shown in Fig. 5. The alignments and ML tree are deposited at TreeBase with study # S22477 (http://purl.org/phylo/treebase/phylows/study/TB2:S22477). 4 Discussion 4.1 Monophyly of Hymenasplenium Hymenasplenium was established by Hayata (1927) based on its unusual dorsiventral system in long-creeping rhizomes. The characteristics of the stelar structure of H. unilaterale and its allied species made Iwatsuki and Kato (1975) conclude that this group of ferns is monophyletic. Murakami (1992) and Murakami and Moran (1993) reported that the stelar structure of the New World species of Hymenasplenium is identical to that of the Old World species. In contrast, information on chromosome numbers provided controversial results regarding the monophyly of Hymenasplenium. Asian members of Hymenasplenium studied were reported to have x =39 or rarely 38 (Mitui et al., 1989), different from x = 36 in Asplenium (Manton, 1950; Manton and Sledge, 1954; Bir, 1960; Bellefroid et al., 2010; Lin and Viane, 2013). However, Walker (1966) reported a sexual diploid cytotype of H. laetum (Sw.) L. Regalado & C. Prada from John Crow Mountains, Jamaica with x = 36, while Gabancho and Prada (2011) showed that H. delitescens (Maxon) L. Regalado & C. Prada from the New World had x = 39. Previous molecular studies confirmed the monophyly of Hymenasplenium (Murakami and Schaal, 1994; Murakami, 1995; Murakami et al., 1995; Murakami et al., 1999; Gastony and Johnson, 2001; Pinter, 2002; Schneider et al., 2004, 2005, 2017; Schuettpelz and Pryer, 2007; Bellefroid, 2010; Rothfels et al., 2012; Ohlsen et al., 2015; Lóriga et al., 2016; Sessa et al., 2018), but these studies generally sampled only limited number of species and limited number of molecular markers. Our analysis with the largest taxon and character sampling so far confirmed that Hymenasplenium is monophyletic and sister to Asplenium with strong support (Fig. 4). Morphologically, Hymenasplenium is characterized by its long-creeping rhizomes, dorsiventrally symmetrical steles, swollen petiole bases, and chromosome base numbers of x = (?36) 38 or 39 which can be easily distinguished from erect or ascending rhizomes, radially symmetrical steles, nonswollen petiole bases, and x = 36 in Asplenium (Murakami and Moran; 1993) 4.2 Cryptic speciation in Hymenasplenium One of the most surprising and interesting findings based on our large sampling is that four "species" of Hymenasplenium as currently defined are found to be highly polyphyletic or paraphyletic (Fig. 4), illustrating the complexity of the taxonomy and evolution in the genus.

These four "species" include H. excisum, H. obliquissimum, H. obscurum, and H. unilaterale. In detail, samples of H. excisum are resolved into at least four lineages, those of H. obscurum into four lineages, those of H. obliquissimum into at least seven lineages, and those of H. unilaterale into 13 lineages. In addition, although resolved as monophyletic, H. cheilosorum s.l. obviously contains two species based on our study because 17 samples included are resolved into two well supported clades sister to each other (Fig. 4). If all these additional lineages identified here are recognized as species, our study will add at least 16 species to this small fern genus with 34 currently generally accepted species, an increase of species number by nearly 50%. Our morphlogical study showed that most of these lineages can be distinguished from one another using combinations of some morphological characters. For example, species #7 (Fig. 4) is different from Hymenasplenium murakami-hatanakae in having trapeziform or triangular pinnae (vs. lanceolate or falcate pinnae in the latter); species #10 differs from H. excisum in having grayish green petioles (vs. castaneous petioles in the latter); species #11 is distinguishable from H. excisum in having dull green rachises (vs. dark brown rachises in the latter); species #12 is distinct from H. excisum in having pinna teeth retuse, veins ending below marginal notches (vs. pinna teeth entire, veins ending below marginal teeth in the latter); species #14 is different from species #15 and #16 in having pinna teeth entire, veins terminating in the marginal teeth and reaching nearly apex of teeth (vs. pinna teeth retuse, veins ending below marginal notches in the latter two); and species #15 and #16 are distinguishable from each other in costa color (dull green in the former vs. dark brown in the latter). One of the common features of these four Old World "species" are that they were all thought to be "widely" distributed (e.g., Ching, 1965; Murakami, 1988; Wu, 1999; Lin and Viane, 2013), but samples from different geographical ranges fall in different areas in our tree (Fig. 4). Since our molecular markers are all from the chloroplast genome, hybridization/polypodization alone can not account for all this discordance. The best hypothesis is that these "species" each contain more than one undescribed (cryptic) species as cryptic speciation is common in ferns such as in Asplenium L. (e.g., Chang et al., 2018), Ctenitis (C. Chr.) C. Chr. (e.g., Duan et al., 2017), Polystichum Roth (e.g., Han et al., 2016). This hypothesis is partially supported by the fact that there are limited number of morphological characters available for taxonomy in Hymenasplenium (Murakami, 1988; Wu, 1999; Lin and Viane, 2013). Cryptic species appeared to be common in groups that generally lack many morphological characters (e.g., Polystichum subg. Haplopolystichum (Tagawa) Li Bing Zhang). Murakami and Moran (1993) also reported that the New World Hymenasplenium obtusifolium (L.) L. Regalado & C. Prada has two races: one with 32 spores per sporangium occurring only in the western portion of the range, the other with 64 spores occurring only in the eastern portion, but these two races could not be distinguished by their Principal Component Analysis of 20 quantitative morphological characters. Lin and Viane (2013) thought that the Old World clade is still in an active state of diversification based on the intermediate morphology and the existence of different cytotypes and that it is diffcult to discribe them clearly in morphology. To test this hypothesis it would entail analysis of diversification rates through times which is beyond the scope of our current study. However, our current study well demonstrates the decoupling of morphological and

molecular evolution in Hymenasplenium largely pointing to extensive cryptic speciation. Interestingly, higher species diversity in Hymenasplenium is found in subtropical areas than in tropical areas, contrasting general patterns that tropical forest supports higher plant diversity in general (e.g., Aedo 2016, 2017, Alejandro et al. 2016, Faria & Araujo 2016, Lucas et al. 2016, Mello-Silva & Sasaki 2016, O'Leary & Thode 2016, O'Leary et al. 2016, Duan et al. 2017, Kolanowska & Szlachetko 2017, Morales et al. 2017, Szlachetko & Kolanowska 2017). 4.3 Infrageneric relationships within Hymenasplenium The 141 accessions of Hymenasplenium are resolved into an Old World clade and two New World clades: the H. laetum clade and the H. riparium clade. 4.3.1 Monophyly of the New World Hymenasplenium Few studies have been conducted on the taxonomy of the New World Hymenasplenium. Smith (1976) treated the five New World species, H. delitescens, H. purpurascens (Mett. ex Kuhn) L. Regalado & C. Prada, H. laetum, H. hoffmannii (Hieron.) L. Regalado & C. Prada, and H. repandulum (Kunze) L. Regalado & C. Prada, in Asplenium sect. Hymenasplenium. Murakami and Moran (1993) added four species, H. obtusifolium, H. ortegae (N. Murak. & R. C. Moran) L. Regalado & C. Prada, H. triquetrum (N. Murak. & R. C. Moran) L. Regalado & C. Prada, and H. volubile (N. Murak. & R. C. Moran) L. Regalado & C. Prada, and three hybrids from the New World to A. sect. Hymenasplenium. Moran and Sundue (2004) added one species: H. basiscopicum (R. C. Moran & M. A. Sundue) L. Regalado & C. Prada. Recently, Gabancho and Prada (2011) transferred 11 species in the New World from Asplenium to Hymenasplenium. In total, there are 11 species and three hybrids documented for the New World. Paradoxically, the New World species have been reported to have chromosome numbers x = 36 (chracteristic for Asplenium) and 39 (chracteristic for Hymenasplenium) (Walker, 1966; Gabancho and Prada, 2011). Based on chloroplast restriction enzyme data Murakami and Schaal (1994) showed that the New World species of Hymenasplenium were monophyletic, but they included one Old World species only in addition to the New World species in their analysis. Later based on rbcL data, they found New World Hymenasplenium to be paraphyletic, but only two species of the New World Hymenasplenium were sampled (Murakami et al., 1999). Our data resolved the New World species of Hymenasplenium (ca. 7 spp. sampled) as monophyletic but with strong support from ML analysis only. However, our analysis is not in conflict with that of Murakami et al. (1999) because one GenBank accession of H. laetum used by Murakami et al. (1999), which made the New World species paraphyletic, was excluded from our analysis for our suspicion of the data reliability. Therefore, the monophyly of the New World Hymenasplenium remains ambiguous. Morphologically, the monophyly of the New World Hymenasplenium is supported by having proliferous roots in the New World species (Murakami & Moran, 1993), which are lacking in the Old World species. 4.3.2 The H. laetum clade The Hymenasplenium laetum clade is resolved as sister to the H. riparium clade in our

analyses (Fig. 4). The H. laetum clade is characterized by nonconform and pinnatifid leaf apex, cristate spores, and brown or blackish rhizomes in living plants (Murakami and Moran, 1993). It contains about six species, three of which are included in our study. The clade is endemic to the New World. 4.3.3 The H. riparium clade The Hymenasplenium riparium clade is resolved sister to the H. laetum clade (Fig. 4). This clade is characterized by conform, subconform, or hastate leaf apex, spiny or papillose spores, greenish rhizomes when living (Murakami and Moran, 1993). It contains about five species and two speies are included in this study. These clade is also endemic to New World. 4.3.4 The Old World clade The Old World clade is strongly supported as monophyletic. This is consistent with results from the previous studies (e.g., Murakami et al., 1999). This clade contains most species of Hymenasplenium mainly distributed in Southeast Asian (Fig. 4), especially in Southwest China which is the center of diversity of Hymenasplenium (Lin and Viane, 2013). This genus contains two species with simple fronds and both of them are in this clade. Species of the Old World clade have x = 39, with one exception being x = 38 in H. subnormale (Murakami & Iwatsuki, 1989; Kato et al., 1990). Apomictic reproduction has evolved at least three times in the Asian group (Murakami, 1995; Lin &Viane, 2013). The Old World clade can be divided into six subclades. 4.3.4.1 The H. wildii subclade The Hymenasplenium wildii subclade is resolved as sister to the H. hondoense subclade (Fig. 4). It only contains two species: H. subnormale and H. wildii. This subclade is morphologically distinct from the rest of the Old World clade in having smaller plants with fewer pinnae in general, less cut away on the lower side, and color on the rachis of the mature plants with a transition from purple at the base to green at the apex (Holttum, 1954; Jones and Clemesha, 1981; Lin and Viane, 2013). The type of H. subnormale is collected from the Philippines and this species is also found in Taiwan and Yunnan of China, Indonesia, Japan, and Malaysia (Holttum, 1954; Lin & Viane, 2013). Our samples of this species are collected from Taiwan (Figs. 2, 3). Hymenasplenium wildii is narrowly endemic to northeastern Queensland of Australia (Jones & Clemesha, 1981). 4.3.4.2 The H. hondoense subclade The Hymenasplenium hondoense subclade is resolved as sister to the H. wildii subclade (Fig. 4). About 12 species are included in our study. Because of the previous morphological confusion with the H. unilaterale s.l. group, most sepcies in this subclade are not well known. Only four species have been named and described in the previous studies. It is interesting that the only two species in the genus with simple fronds—H. cardiophyllum (Hance) Nakaike and H. hastifolium Ke Wang Xu, Li Bing Zhang & W. B. Liao—are resolved as members of this subclade. Hymenasplenium cardiophyllum is widely distributed in China, Japan, Laos (new record in this study), Thailand (Boonkerd and Pollawatn, 2012), and Vietnam, while H. hastifolium is endemic to Guangxi, southern China (Xu et al., 2018). Kato et al. (1990)

hypothesized that H. cardiophyllum (= H. ikenoi) is closely related with H. apogamum, H. excisum, and H. obscurum based on evidence of the stelar anatomy, the epidermal cells of fronds, chromosome numbers, and perispore morphology. However, our data resolved H. cardiophyllum as a membeer of the H. hondoense subclade. No any regular morphological characteristics are known to well define this subclade. Species of this subclade appear to occur in higher elevations of eastern/southern Asia than other subclades (Fig. 4). 4.3.4.3 The H. excisum subclade The Hymenasplenium excisum subclade is resolved as sister to the H. unilaterale subclade (Fig. 4). This subclade contains three described species and about six undescribed species based on our results, of which H. obscurum (Blume) Tagawa and H. pseudobscurum Viane with dull green to grayish green stipes when dry form a well-supported clade, while H. sp 10 and H. sp 11, also with dull green to grayish green stipes when dry, group with H. excisum (which has shiny, dark purple to black stipes). The members of this subclade from Indonesia and Hawaii should not be H. excisum based on our study (Fig. 4). The distribution of H. excisum is believed to be across the Old World, including Southeast Asia and tropical African (Lin and Viane, 2013). We did not have material from Africa and it remains to test if the African material is conspecific with Asian-Pacific material. The morphology of H. excisum is variable but the basiscopic side of basal pinnae of this member is excavate (Lin and Viane, 2013). 4.3.4.4 The H. unilaterale subclade The Hymenasplenium unilaterale subclade is resolved as sister to the H. excisum subclade. Only one species, H. unilaterale, in this subclade is described. Lin and Viane (2013) thought that several taxa such as H. apogamum, H. hondoense, and H. murakami-hatanakae in Southeast Asia should be treated as distinct from H. unilaterale (contrary to Wu (1999)). This conclusion is therefore confirmed by our phylogenetic analyses. The type of H. unilaterale is collected from the Reunion Island. It has strongly dimidiate pinnae, strongly cut away on the basiscopic side, regular teeth never retuse, and blackish rachis that distinctly continues onto the costa abaxially (Figs. 1A, 2A). The true H. unilaterale is distributed in southern Africa and adjacent Islands, Indian Ocean islands, and Malaysia, and absent from China (see our Fig. 4). Three undetermined taxa from Australia and adjacent islands and Vietnam together are resolved as sister to H. unilaterale. 4.3.4.5 The H. cheilosorum subclade The Hymenasplenium cheilosorum subclade is resolved as sister to the H. obliquissimum subclade (Fig. 4). Two groups which might represent distinct taxa, respecively, are shown in our phylogeneic tree. These two groups have sori terminal on subtending vein and situated in marginal teeth, which allows member of this subclade to be easily distinguished from other subclades of the Old World. Though a lot of studies on chromosomes have been conducted (Mitui et al., 1989; Cheng & Murakami, 1998), the taxonomy of this subclade still needs further attention. 4.3.4.6 The H. obliquissimum subclade

The Hymenasplenium obliquissimum subclade is resolved as sister to the H. cheilosorum subclade (Fig. 4). Murakami (1998) studied some members of this subclade and roughly treated all of them in the H. obliquissimum complex including H. changputungense (Ching) Viane & S. Y. Dong, H. furfuraceum (Ching) Viane & S. Y. Dong, H. latedens (Ching) Viane & S. Y. Dong, H. quercicola (Ching) Viane & S. Y. Dong, H. retusulum (Ching) Viane & S. Y. Dong, H. szechuanense (Ching) Viane & S. Y. Dong, H. wuliangshanense (Ching) Viane & S. Y. Dong, and H. filipes (Copeland) Sugimoto. However, these species are different from H. obliquissimum. Hymenasplenium obliquissimum has teeth obtuse and never retuse, veins forking and terminating in marginal teeth, which is similar to those of H. filipes, while the others have teeth retuse, veins distinct, forking, and free, and each vein ending below a marginal notch. Our identification of H. latidens (Ching) Viane & S. Y. Dong followed Knapp (2013) and some other accessions from Taiwan followed Knapp and Hsu (2017). 4.4 Hybridization and polyploidization in Hymenasplenium Hybridization and auto- and allopolyploidization are common in Aspleniaceae (Wagner, 1954; Lovis, 1977; Reichstein, 1981). A number of potential hybrids in Hymenasplenium have been proposed based chromosome numbers (e.g., Bir, 1960, 1963; Kramer & Viane, 1990) and morpholgy (Murakami & Moran, 1993; Ebihara, 2016), and Schneider (2017) estimated that 37% of Hymenasplenium species are tetraploids. Hybridization and polyploidy often obscure boundaries between species (Yatabe et al., 2001, 2009; Van den Heede et al., 2003; Perrie & Brownsey, 2005; Dyer & al., 2012; Chang et al., 2013), such as H. apogamum, H. hondonense, and H. murakami-hatanakae, which had been confused with the H. unilaterale s.l. group. Limited morphological characteristics can be used in species delimitation in Hymenasplenium, which should largely accounts for extensive cryptic speciation (see above). However, our phylogeny of Hymenasplenium is only based on six plastid markers. Further studies including using nuclear markers and chromosome data are needed to evaluate our plastid phylogeny and assess the role of hybridization and polyploidy in the diversity and evolution in Hymenasplenium. Acknowledgments This research was partially supported by grants from the Basic Work Special Project of the National Ministry of Science and Technology of China (#2013FY111500) and from Pilot Work of the fourth National survey on Chinese Materia Medica Resources (#2017-2019) to W.-B.L., the National Natural Science Foundation of China (#31628002) to L.-B.Z., the scholarships from the China Scholarship Council and the Zhang Hong-Da (Chang Hung-Ta) Science Foundation at Sun Yat-sen University to K.-W.X, the National Natural Science Foundation of China (#31400196) and from Kunming Institute of Botany, Chinese Academy of Sciences to L.Z., the Glory Light International Fellowship for Chinese Botanists at Missouri Botanical Garden to X.-M.Z., and the CAS-TWAS President's Fellowship for International PhD Students to N.T.L. James Ryan Allen, Paul Goetghebeur, Daniel Ohlsen, Alison Paul, Leon Perrie, Ranil Rajapaksha, Subhani Ranasinghe, and Erin Tripp helped obtain images of vouchers. Daniele Cicuzza shared one sample. We thank Robbin C. Moran and an anonymous reviewer for helpful comments.

Appendix 1. List of taxa sampled in this study with voucher information, GenBank accession numbers and references, taxon, collector name and number, and herbarium code in that order (– indicates accessions with missing data). Asplenium aegaeum Lovis, Reichstein et Zaffran, Jermy 9181 (BM), Crete: rbcL AY300103, trnL-F AY300050, rps4 & rps4-trnS AY549774, atpB − (Schneider et al., 2005). Asplenium aureum Cav., Hughes 64 (BM), Belize: rbcL AF240651, trnL-F AF240667, rps4 & rps4-trnS AY549759, atpB − (Pinter et al., 2002). Asplenium cuspidatum Lam. Grantham & Parsons 0233090 (UC), Costa Rica: rbcL AY300111, trnL-F AY300058, rps4 & rps4-trnS AY549760, atpB − (Schneider et al., 2004). Asplenium dielfalcatum Viane, Wood 7826 (PTBG), Hawaii: rbcL AY549738, trnL-F AY549841, rps4 & rps4-trnS AY549787, atpB − (Schneider et al., 2005). Asplenium erosum Maxon: rbcL KX397706, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Unpublished). Asplenium flabellifolium Cav., Holmes 4/4/99 (BM), Australia: rbcL AY300115, trnL-F AY300062, rps4 & rps4-trnS AY549779, atpB − (Schneider et al., 2005). Asplenium hastatum Klotzsch ex Kunze, Viane 10182C, Merida, Venezuela: rbcL GU929869, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Leroux et al., 2011). Asplenium juglandifolium Lam. Viane 10667, Puerto Rico: rbcL GU929870, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Leroux et al., 2011). Asplenium normale D. Don, Ohlsen 296 (MELU), Queensland, Australia: rbcL KP774926, trnL-F KP851904, rps4-trnS KP835419, atpB − (Ohlsen et al. 2015). Asplenium polyodon G. Forst., Perrie NC 124 (WELT), New Caledonia: rbcL KP774900, trnL-F KP835397, rps4-trnS KP835433, atpB − (Ohlsen et al. 2015). Asplenium subglandulosum subsp. papaverifolium (Kunze) Salvo, Prada & Consuelo Díaz Ohlsen 442 (MELU), Victoria, Australia: rbcL KP774929, trnL-F KP851909, rps4-trnS KP835456, atpB − (Ohlsen et al. 2015). Asplenium tenerum G. Forst., Ohlsen 265 (MELU), Queensland, Australia: rbcL KP774858, trnL-F KP835346, rps4-trnS KP835437, atpB − (Ohlsen et al. 2015). Asplenium trichomanes subsp. inexpectans Lovis, Vogel I-46-B04, France: rbcL AY549743, trnL-F AY549846, rps4 & rps4-trnS AY549792, atpB − (Schneider et al., 2005). Asplenium varians Wall. ex Hook. & Grev., Fraser-Jenkins 10046-10047 (BM), China: rbcL AY300147, trnL-F AY300094, rps4 & rps4-trnS AY549802, atpB − (Schneider et al., 2005). Asplenium wrightii D. C. Eaton ex Hook. Cranfill TW040 (UC), Taiwan: rbcL AY549730, trnL-F AY549833, rps4 & rps4-trnS AY549766, atpB − (Schneider et al., 2005). Asplenium yunnanense Franch., Fraser-Jenkins 10044-10045 (BM), China: rbcL AY300149, trnL-F AY300096, rps4 & rps4-trnS AY549803, atpB − (Schneider et al., 2005). Hymenasplenium adiantifrons (Hayata) Viane & S. Y. Dong (Hayata), Knapp 3346 (KNAPP), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065528, rps4 & rps4-trnS −, atpB −. Hymenasplenium adiantifrons (Hayata) Viane & S. Y. Dong, Knapp 3609 (P), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065559, rps4 & rps4-trnS MH065322, atpB −. Hymenasplenium adiantifrons (Hayata) Viane & S. Y. Dong, Knapp 3904 (P), Taiwan Island: rbcL−, trnL intron & trnL-F spacer MH065560, rps4 & rps4-trnS MH065323, atpB −. Hymenasplenium apogamum (N. Murakami & Hatanaka) Nakaike, TNS: 1107829 (TNS), Okinawa, Japan: rbcL AB574852, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium apogamum (N. Murakami & Hatanaka) Nakaike, Xu 001 (SYS), Hong Kong, China: rbcL −, trnL intron & trnL-F spacer MH065544, rps4 & rps4-trnS MH065307, atpB −. Hymenasplenium apogamum (N. Murakami & Hatanaka) Nakaike, Xu 101 (SYS), Hainan, China: rbcL MH065382, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065305, atpB MH065449.

Hymenasplenium apogamum (N. Murakami & Hatanaka) Nakaike, Xu 273 (SYS), Hainan, China: rbcL MH065392, trnL intron & trnL-F spacer MH065540, rps4 & rps4-trnS −, atpB MH065460. Hymenasplenium apogamum (N. Murakami & Hatanaka) Nakaike, Zhang et al. 7750 (CDBI, MO, VNMN), Thua Thien-Hue, Vietnam: rbcL MH065437, trnL intron & trnL-F spacer MH065604, rps4 & rps4-trnS MH065376, atpB MH065518. Hymenasplenium cardiophyllum (Hance) Nakaike, Kun 1, Cult. in KUN: rbcL−, trnL intron & trnL-F spacer MH065542, rps4 & rps4-trnS −, atpB MH065463. Hymenasplenium cardiophyllum (Hance) Nakaike, Kun 2, Cult. in KUN: rbcL MH065395, trnL intron & trnL-F spacer MH065543, rps4 & rps4-trnS−, atpB MH065464. Hymenasplenium cardiophyllum (Hance) Nakaike, N. Murakami 596921 (KYO), Cult., Bot. Gard., Univ. of Tokyo: rbcL AB014706, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1999b). Hymenasplenium cardiophyllum (Hance) Nakaike, Sysu 2, Cult. in Sun-yat Sen University: rbcL MH065387, trnL intron & trnL-F spacer MH065534, rps4 & rps4-trnS MH065306, atpB MH065454. Hymenasplenium cardiophyllum (Hance) Nakaike, TNS: 774848 (TNS), Okinawa, Japan: rbcL AB574886, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium cardiophyllum (Hance) Nakaike, Xu 293 (SYS), Guangxi, China: rbcL MH065401, trnL intron & trnL-F spacer MH065550, rps4 & rps4-trnS MH065316, atpB MH065472. Hymenasplenium cardiophyllum (Hance) Nakaike, Xu HN1 (SYS), Hainan, China: rbc −, trnL intron & trnL-F spacer MH065545, rps4 & rps4-trnS MH065311, atpB −. Hymenasplenium cardiophyllum (Hance) Nakaike, Zhang et al. 6588 (CDBI, MO, VNMN), Lang Son, Vietnam: rbcL −, trnL intron & trnL-F spacer MH065584, rps4 & rps4-trnS −, atpB MH065501. Hymenasplenium cardiophyllum (Hance) Nakaike, Zhang LK-135 (CDBI), Laos: rbcL MH065445, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065340, atpB MH065524. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Cicuzza 2035 (CDBI), Yunnan, China: rbcL −, trnL intron & trnL-F spacer MH065611, rps4 & rps4-trnS −, atpB MH065525. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Jin & Zhang 11348 (CDBI), Yunnan, China: rbcL −, trnL intron & trnL-F spacer MH065571, rps4 & rps4-trnS MH065359, atpB MH065489. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Jin & Zhang 11348 (CDBI), Yunnan, China: rbcL MH065421, trnL intron & trnL-F spacer MH065575, rps4 & rps4-trnS MH065362, atpB MH065492. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, N. Murakami 92-J013 Kagoshima, Japan: rbcL AB016188, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Schater 55 (GOET) Yunnan, China: rbcL JF832071, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB EF463350 (Rothfel et al., 2012). Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Xu 102 (SYS), Hainan, China: rbcL MH065385, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065346, atpB MH065452. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Xu 276 (SYS), Guangdong, China: rbcL MH065393, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB MH065461. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Xu 299 (SYS), Guangxi, China: rbcL MH065402, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065350, atpB MH065473. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Xu 322 (SYS), Yunnan, China: rbcL MH065405, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065352, atpB MH065476. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Xu GX022 (SYS), Guangxi, China: rbcL MH065381, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065343, atpB MH065448. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Zhang et al. 6473 (CDBI, MO, VNMN), Vinh Phuc, Vietnam: rbcL MH065433, trnL intron & trnL-F spacer MH065593, rps4 & rps4-trnSMH065367, atpB −. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Zhang et al. 6776 (CDBI, MO, VNMN), Bac Kan, Vietnam: rbcL MH065440, trnL intron & trnL-F spacer MH065606,

rps4 & rps4-trnS MH065377, atpB MH065520. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Zhang et al. 7109 (CDBI, MO, VNMN), Thanh Hoa, Vietnam: rbcL MH065438, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB−. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Zhang et al. 7712 (CDBI, MO, VNMN), Thua Thien-Hue, Vietnam: rbcL MH065435, trnL intron & trnL-F spacer MH065601, rps4 & rps4-trnS MH065373, atpB MH065515. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Zhang et al. 8523 (CDBI, MO, PHH), Lam Dong, Vietnam: rbcL MH065416, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065358, atpB MH065488. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Zhang et al. 8629 (CDBI, MO, PHH), Lam Dong, Vietnam: rbcL MH065415, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065357, atpB MH065487. Hymenasplenium delitescens (Maxon) L. Regalado & Prada, Jones 1078 (MO), Panama: rbcL MH065443, trnL intron & trnL-F spacer MH065609, rps4 & rps4-trnS MH065338, atpB MH065522. Hymenasplenium excisum (C. Presl) S. Lindsay, Brownsey & Perrie FIJI 190 (WELT), Fiji: rbcL KP774884, trnL-F KP851914, rps4-trnS KP851882, atpB − (Ohlsen et al. 2015). Hymenasplenium excisum (C. Presl) S. Lindsay, Knapp 4129 (P), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065564, rps4 & rps4-trnS −, atpB −. Hymenasplenium excisum (C. Presl) S. Lindsay, TNS: 764169 (TNS), Kagoshima, Japan: rbcL AB574888, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium excisum (C. Presl) S. Lindsay, Xu 100 (SYS), Hainan, China: rbcL MH065383, trnL intron & trnL-F spacer MH065531, rps4 & rps4-trnS MH065344, atpB MH065450. Hymenasplenium excisum (C. Presl) S. Lindsay, Xu 196 (SYS), Hainan, China: rbcL MH065390, trnL intron & trnL-F spacer MH065538, rps4 & rps4-trnS −, atpB MH065458. Hymenasplenium excisum (C. Presl) S. Lindsay, Xu 197 (SYS), Hainan, China: rbcL MH065391, trnL intron & trnL-F spacer MH065539, rps4 & rps4-trnS −, atpB MH065459. Hymenasplenium excisum (C. Presl) S. Lindsay, Xu 268 (SYS), Hainan, China: rbcL −, trnL intron & trnL-F spacer MH065536, rps4 & rps4-trnS −, atpB MH065456. Hymenasplenium excisum (C. Presl) S. Lindsay, Xu 274 (SYS), Guangdong, China: rbcL MH065389, trnL intron & trnL-F spacer MH065537, rps4 & rps4-trnS −, atpB MH065457. Hymenasplenium excisum (C. Presl) S. Lindsay, Zhang et al. 6529 (CDBI, MO, VNMN), Phu Tho, Vietnam: rbcL −, trnL intron & trnL-F spacer MH065581, rps4 & rps4-trnS MH065363, atpB MH065498. Hymenasplenium excisum (C. Presl) S. Lindsay, Zhang et al. 6658 (CDBI, MO, VNMN), Bac Kan, Vietnam: rbcL −, trnL intron & trnL-F spacer MH065602, rps4 & rps4-trnS MH065374, atpB MH065516. Hymenasplenium excisum (C. Presl) S. Lindsay, Zhang et al. 6714 (CDBI, MO, VNMN), Bac Kan, Vietnam: rbcL MH065436, trnL intron & trnL-F spacer MH065603, rps4 & rps4-trnS MH065375, atpB MH065517. Hymenasplenium excisum (C. Presl) S. Lindsay, Zhang et al. 8039 (CDBI, MO, VNMN), Quang Nam, Vietnam: rbcL MH065419, trnL intron & trnL-F spacer MH065573, rps4 & rps4-trnS MH065361, atpB −. Hymenasplenium excisum (C. Presl) S. Lindsay, Ohlsen 305 Queensland Australia, Ohlsen 305 (MELU), Queensland, Australia: rbcL KP774930, trnL-F KP851913, rps4-trnS KP851883, atpB − (Ohlsen et al. 2015). Hymenasplenium filipes (Copeland) Sugimoto, Murakami 596902 (TI), Kagoshima, Japan: rbcL U30605, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Hasebe et al., 1995). Hymenasplenium filipes (Copeland) Sugimoto, N. Murakami 92-J012, Kagoshima, Japan: AB016176, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium furfuraceum (Ching) Viane & S. Y. Dong, Xu 307 (SYS), Yunnan, China: rbcL MH065409, trnL intron & trnL-F spacer MH065557, rps4 & rps4-trnS MH065320, atpB MH065481. Hymenasplenium hastifolium Ke Wang Xu, Li Bing Zhang & W.B.Liao, Xu 282-1 (SYS), Guangxi, China: rbcL MH065398, trnL intron & trnL-F spacer MH065547, rps4 & rps4-trnS MH065313, atpB MH065469. Hymenasplenium hastifolium Ke Wang Xu, Li Bing Zhang & W.B.Liao, Xu 282-2 (SYS), Guangxi, China: rbcL MH065397,

trnL intron & trnL-F spacer MH065546, rps4 & rps4-trnS MH065312, atpB MH065468. Hymenasplenium hoffmannii (Hieron.) L. Regalado & Prada, Croat & Zhu 76985 (MO), Canal, Panama: rbcL MH065441, trnL intron & trnL-F spacer MH065607, rps4 & rps4-trnS MH065378, atpB −. Hymenasplenium hoffmannii (Hieron.) L. Regalado & Prada, Monro & Douglas 3463 (MO), Santa Ana, El Salvador: rbcL MH065442, trnL intron & trnL-F spacer MH065608, rps4 & rps4-trnS MH065337, atpB MH065521. Hymenasplenium hondoense (N. Murakami & Hatanaka) Nakaike, N. Murakami 596920 (KYO), Kouchi, Japan: rbcL AB014705, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1999b). Hymenasplenium laetum (Sw.) L. Regalado & Prada, Jones 1155 (TUR), unknown: rbcL −, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB KM114105 (Unpublished). Hymenasplenium laetum (Sw.) L. Regalado & Prada, N. Murakami N293 (KYO), Rio Palenque, Ecuador: rbcL AB014707, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1999b). Hymenasplenium latidens (Ching) Viane & S. Y. Dong, Knapp 3672 (KNAPP), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065566, rps4 & rps4-trnS −, atpB −. Hymenasplenium murakami-hatanakae Nakaike ("H. mitanii"), TNS: 736793 (TNS), Kagoshima, Japan: rbcL AB574890, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium murakami-hatanakae Nakaike (H. unilaterale), Ranker 2072 (COLO), Taiwan Island: rbcL EF452140, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB EF452020, (Schuettpelz et al., 2007). Hymenasplenium murakami-hatanakae Nakaike, TNS: 763888 (TNS), Mie, Japan: rbcL AB574891, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and J. Yokoyama 94-T609, Chiang Mai, Thailand: rbcL AB016177, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and J. Yokoyama 94-T622, Chiang Mai, Thailand: rbcL AB016185, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and J. Yokoyama 94-T627, Chiang Mai, Thailand: rbcL AB016180, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and J. Yokoyarna 94-T628, Chiang Mai, Thailand: rbcL AB016178, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and X. Cheng 92-C1, Yunnan, China: rbcL AB016181, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and X. Cheng 92-C2, Yunnan, China: rbcL AB016182, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and X. Cheng 92-C3, Yunnan, China: rbcL AB016183, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and X. Cheng 92-C46, Yunnan, China: rbcL AB016184, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and X. Cheng 92-C7, Yunnan, China: AB016179, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, N. Murakami and X. Cheng 94-M 1811, Yunnan, China: rbcL AB016186, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, TNS: 763123 (TNS), Kagoshima, Japan: rbcL AB574892, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium obliquissimum (Hayata) Sugimoto, Jin & Zhang 11346 (CDBI), Yunnan, China: rbcL MH065413, trnL intron & trnL-F spacer MH065569, rps4 & rps4-trnS MH065356, atpB MH065485. Hymenasplenium obliquissimum (Hayata) Sugimoto, Ju & Deng 12689 (CDBI), Sichuan, China: rbcL −, trnL intron & trnL-F spacer MH065597, rps4 & rps4-trnS

MH065370, atpB MH065511. Hymenasplenium obliquissimum (Hayata) Sugimoto, K. lwatsuki et al. 94-V302, Hoang Lien Son, Vietnam: rbcL AB016187, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1998). Hymenasplenium obliquissimum (Hayata) Sugimoto, Knapp 3228 (HAST), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065561, rps4 & rps4-trnS −, atpB −. Hymenasplenium obliquissimum (Hayata) Sugimoto, Knapp 3233 (HAST), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065562, rps4 & rps4-trnS −, atpB −. Hymenasplenium obliquissimum (Hayata) Sugimoto, Knapp 3462 (KNAPP), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065563, rps4 & rps4-trnS −, atpB −. Hymenasplenium obliquissimum (Hayata) Sugimoto, Xu 134 (SYS), Jiangxi, China: rbcL MH065388, trnL intron & trnL-F spacer MH065535, rps4 & rps4-trnS MH065348, atpB MH065455. Hymenasplenium obliquissimum (Hayata) Sugimoto, Xu GX018 (SYS), Guangxi, China: rbcL −, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065349, atpB MH065465. Hymenasplenium obliquissimum (Hayata) Sugimoto, Xu PB001 (SYS), Yunnan, China: rbcL MH065386, trnL intron & trnL-F spacer MH065533, rps4 & rps4-trnS MH065347, atpB MH065453. Hymenasplenium obscurum (Blume) Tagawa, Xu 004 (SYS), Hong Kong, China: rbcL MH065380, trnL intron & trnL-F spacer MH065530, rps4 & rps4-trnS MH065342, atpB MH065447. Hymenasplenium obscurum (Blume) Tagawa, Zhang et al. 7715 (CDBI, MO, VNMN), Thanh Hoa, Vietnam: rbcL MH065411, trnL intron & trnL-F spacer MH065567, rps4 & rps4-trnS MH065354, atpB MH065483. Hymenasplenium pseudobscurum Viane, Knapp 4133 (P), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065565, rps4 & rps4-trnS −, atpB −. Hymenasplenium retusulum (Ching) Viane & S. Y. Dong, Jiang 00371 (SYS), Yunnan, China: rbcL MH065379, trnL intron & trnL-F spacer MH065529, rps4 & rps4-trnS MH065304, atpB MH065446. Hymenasplenium retusulum (Ching) Viane & S. Y. Dong, Xu 304 (SYS), Yunnan, China: rbcL MH065406, trnL intron & trnL-F spacer MH065553, rps4 & rps4-trnS MH065353, atpB MH065477. Hymenasplenium retusulum (Ching) Viane & S. Y. Dong, Xu 311 (SYS), Yunnan, China: rbcL MH065408, trnL intron & trnL-F spacer MH065556, rps4 & rps4-trnS MH065319, atpB MH065480. Hymenasplenium riparium (Liebm.) L. Regalado & Prada, N. Murakami & Grayum 281 (KYO), Virgen del Socorro, Costa Rica: rbcL AB014708, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Murakami et al., 1999b). Hymenasplenium subnormale (Copeland) Nakaike, Knapp 4509 (KNAPP), Taiwan Island: rbcL−, trnL intron & trnL-F spacer MH065613, rps4 & rps4-trnS −, atpB MH065527. Hymenasplenium subnormale (Copeland) Nakaike, Knapp 4525 (P), Taiwan Island: rbcL −, trnL intron & trnL-F spacer MH065612, rps4 & rps4-trnS −, atpB MH065526. Hymenasplenium triquetrum (N. Murak. & R.C. Moran) L. Regalado & Prada, Jimenez 5153 (MO), La Paz, Bolivia: rbcL MH065444, trnL intron & trnL-F spacer MH065610, rps4 & rps4-trnS MH065339, atpB MH065523. Hymenasplenium triquetrum (N. Murak. & R.C. Moran) L. Regalado & Prada, L.Sylvestre 2208 (RB), Brazil: rbcL KT329398, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Mynssen et al., 2016). Hymenasplenium unilaterale (Lam.) Hayata, Hemp A. 18 (BM), Kenya: rbcL AF240652, trnL-F AF525232, rps4 & rps4-trnS −, atpB − (Pinter et al., 2002). Hymenasplenium unilaterale (Lam.) Hayata, Siti Khadijah Rambe KH165 (GENT), Pahang state Tioman Island, Malaysia: rbcL GU586829, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Bellefroid et al., 2010). Hymenasplenium unilaterale (Lam.) Hayata, Viane 8344, Reunion: rbcL GU929873, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Leroux et al., 2011). Hymenasplenium wildii (F.M.Bailey) D.Ohlsen, Ohlsen 246 (MELU), Queensland, Australia: rbcL KP774927, trnL-F KP851919, rps4-trnS KP851877, atpB − (Ohlsen et al., 2015). Hymenasplenium wuliangshanense (Ching) Viane & S. Y. Dong, Xu 309 (SYS), Yunnan, China: rbcL MH065407, trnL intron & trnL-F spacer MH065554, rps4 & rps4-trnS MH065318, atpB MH065478. Hymenasplenium wuliangshanense (Ching) Viane & S. Y. Dong, Xu 310 (SYS), Yunnan, China: rbcL −, trnL intron & trnL-F

spacer MH065555, rps4 & rps4-trnS −, atpB MH065479. Hymenasplenium cheilosorum (Kunze ex Mettenius) Tagawa, Jin & Zhang 11532 (CDBI), Yunnan, China: rbcLMH065417, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB −. Hymenasplenium sp 1, Zhang et al. 6296 (CDBI, MO, VNMN), Hoa Binh, Vietnam: rbcL −, trnL intron & trnL-F spacer MH065591, rps4 & rps4-trnS MH065335, atpB MH065507. Hymenasplenium sp 2, Zhang et al. 6532 (CDBI, MO, VNMN), Phu Tho, Vietnam: rbcL MH065426, trnL intron & trnL-F spacer MH065583, rps4 & rps4-trnS MH065331, atpB MH065500. Hymenasplenium sp 2, Zhang et al. 7163 (CDBI, MO, VNMN), Nghe An, Vietnam: rbcL MH065439, trnL intron & trnL-F spacer MH065605, rps4 & rps4-trnS MH065336, atpB MH065519. Hymenasplenium sp 3, Jin & Zhang 11347 (CDBI), Yunnan, China: rbcL MH065428, trnL intron & trnL-F spacer MH065586, rps4 & rps4-trnS MH065364, atpB MH065502. Hymenasplenium sp 3, Xu YN030 (SYS), Yunnan, China: rbc −, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065310, atpB MH065467. Hymenasplenium sp 3, Zhang et al. 9455 (CDBI), Guizhou, China: rbcL MH065430, trnL intron & trnL-F spacer MH065588, rps4 & rps4-trnS MH065333, atpB MH065504. Hymenasplenium sp 3, Zhang et al. 9780 (CDBI), Guizhou, China: rbcL −, trnL intron & trnL-F spacer MH065582, rps4 & rps4-trnS MH065330, atpB MH065499. Hymenasplenium sp 4, Zhang et al. 5494 (CDBI, MO), Guangxi, China: rbcL −, trnL intron & trnL-F spacer MH065592, rps4 & rps4-trnS MH065366, atpB MH065508. Hymenasplenium sp 4, Zhang et al. 6035 (CDBI), Guizhou, China: rbcL −, trnL intron & trnL-F spacer MH065594, rps4 & rps4-trnS MH065368, atpB MH065509. Hymenasplenium sp 5, Gao et al. 11165 (CDBI), Sichuan, China: rbcL −, trnL intron & trnL-F spacer MH065599, rps4 & rps4-trnS MH065371, atpB MH065513. Hymenasplenium sp 5, Gao et al. 11687 (CDBI), Sichuan, China: rbcL −, trnL intron & trnL-F spacer MH065600, rps4 & rps4-trnS MH065372, atpB MH065514. Hymenasplenium sp 5, Ju & Deng 12495 (CDBI), Sichuan, China: rbcL −, trnL intron & trnL-F spacer MH065598, rps4 & rps4-trnS −, atpB MH065512. Hymenasplenium sp 5, Xu 289 (SYS), Guangxi, China: rbcL MH065399, trnL intron & trnL-F spacer MH065548, rps4 & rps4-trnS MH065314, atpB MH065470. Hymenasplenium sp 5, Xu 292 (SYS), Guangxi, China: rbcL MH065400, trnL intron & trnL-F spacer MH065549, rps4 & rps4-trnS MH065315, atpB MH065471. Hymenasplenium sp 5, Xu 296 (SYS), Guangxi, China: rbcL MH065403, trnL intron & trnL-F spacer MH065551, rps4 & rps4-trnS MH065317, atpB MH065474. Hymenasplenium sp 5, Xu GZ003 (SYS), Guizhou, China: rbcL MH065396, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065309, atpB MH065466. Hymenasplenium sp 5, Xu HN 009 (SYS), Hunan, China: rbcL −, trnL intron & trnL-F spacer −, rps4 & rps4-trnS MH065308, atpB −. Hymenasplenium sp 5, Zhang et al. 5863 (CDBI), Guizhou, China: rbcL MH065422, trnL intron & trnL-F spacer MH065576, rps4 & rps4-trnS MH065326, atpB MH065493. Hymenasplenium sp 5, Zhang et al. 9482 (CDBI), Guizhou, China: rbcL −, trnL intron & trnL-F spacer MH065580, rps4 & rps4-trnS MH065329, atpB MH065497. Hymenasplenium sp 5, Zhang et al. 9557 (CDBI), Guizhou, China: rbcL MH065425, trnL intron & trnL-F spacer MH065579, rps4 & rps4-trnS MH065328, atpB MH065496. Hymenasplenium sp 5, Zhang et al. 9579 (CDBI), Guizhou, China: rbcL MH065424, trnL intron & trnL-F spacer MH065578, rps4 & rps4-trnS MH065327, atpB MH065495. Hymenasplenium sp 5, Zhang et al. 9595 (CDBI), Guizhou, China: rbcL MH065434, trnL intron & trnL-F spacer MH065595, rps4 & rps4-trnS MH065369, atpB MH065510. Hymenasplenium sp 5, Zhang et al. 9604 (CDBI), Guizhou, China: rbcL MH065418, trnL intron & trnL-F spacer MH065572, rps4 & rps4-trnS MH065360, atpB MH065490. Hymenasplenium sp 5, Zhang et al. 9610 (CDBI), Guizhou, China: rbcL MH065414, trnL intron & trnL-F spacer MH065570, rps4 & rps4-trnS MH065324, atpB MH065486. Hymenasplenium sp 5, Zhang et al. 9640 (CDBI), Guizhou, China: rbcL MH065431, trnL intron & trnL-F spacer MH065589, rps4 & rps4-trnS −, atpB MH065505. Hymenasplenium sp 5, Zhang et al. 9650 (CDBI), Guizhou, China: rbcL MH065420, trnL intron & trnL-F spacer MH065574, rps4 & rps4-trnS

MH065325, atpB MH065491. Hymenasplenium sp 5, Zhang et al. 9658 (CDBI), Guizhou, China: rbcL MH065427, trnL intron & trnL-F spacer MH065585, rps4 & rps4-trnS MH065332, atpB −. Hymenasplenium sp 5, Zhang et al. 9665 (CDBI), Guizhou, China: rbcL MH065423, trnL intron & trnL-F spacer MH065577, rps4 & rps4-trnS −, atpB MH065494. Hymenasplenium sp 5, Zhang et al. EM03 (CDBI), Sichuan, China: rbcL MH065410, trnL intron & trnL-F spacer MH065558, rps4 & rps4-trnS MH065321, atpB MH065482. Hymenasplenium sp 6, Ju & Deng 13414 (CDBI), Sichuan, China: rbcL −, trnL intron & trnL-F spacer MH065596, rps4 & rps4-trnS −, atpB−. Hymenasplenium sp 6, Zhang et al. 7884 (CDBI, MO, VNMN), Quang Nam, Vietnam: rbcL MH065412, trnL intron & trnL-F spacer MH065568, rps4 & rps4-trnS MH065355, atpB MH065484. Hymenasplenium sp 7 (H. hondoense), TNS: 766457 (TNS), Tokyo, Japan: rbcL AB574889, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Ebihara et al., 2010). Hymenasplenium sp 8, Xu 324 (SYS), Yunnan, China: rbcL MH065404, trnL intron & trnL-F spacer MH065552, rps4 & rps4-trnS MH065351, atpB MH065475. Hymenasplenium sp 9, Jin & Zhang 11523 (CDBI), Yunnan, China: rbcL MH065429, trnL intron & trnL-F spacer MH065587, rps4 & rps4-trnS MH065365, atpB MH065503. Hymenasplenium sp 10, Xu 110 (SYS), Hainan, China: rbcL MH065384, trnL intron & trnL-F spacer MH065532, rps4 & rps4-trnS MH065345, atpB MH065451. Hymenasplenium sp 11, Xu 198 (SYS), Hainan, China: rbcL MH065394, trnL intron & trnL-F spacer MH065541, rps4 & rps4-trnS MH065341, atpB MH065462. Hymenasplenium sp 12 (H. excisum), Siti Khadijah Rambe KH17 (GENT), Jambi privince, Indonesia: rbcL GU586828, trnL intron & trnL-F spacer −, rps4 & rps4-trnS −, atpB − (Bellefroid et al., 2010). Hymenasplenium sp 13 (H. excisum), Ranker 1786 (COLO), Hawaii: rbcL AY549728, trnL-F AY549831, rps4 & rps4-trnS AY549758, atpB − (Schneider et al., 2005). Hymenasplenium sp 14, Zhang et al. 8819 (CDBI, MO, PHH), Khanh Hoa, Vietnam: rbcL MH065432, trnL intron & trnL-F spacer MH065590, rps4 & rps4-trnS MH065334, atpB MH065506. Hymenasplenium sp 15 (H. unilaterale), Brownsey & Perrie FIJI 13 (WELT), Fiji: rbcL KP774885, trnL-F KP851918, rps4-trnS KP851880, atpB − (Ohlsen et al. 2015). Hymenasplenium sp 15 (H. unilaterale), PerrieNC177 (WELT), New Caledonia: rbcL KP774896, trnL-F KP851915, rps4-trnS KP851878, atpB − (Ohlsen et al. 2015). Hymenasplenium sp 16 (H. unilaterale), Ohlsen 252 (MELU), Queensland, Australia: rbcL KP774849, trnL-F KP851916, rps4–trnS KP851879, atpB − (Ohlsen et al. 2015). Hymenasplenium sp 16 (H. unilaterale), Ohlsen 392 (MELU), Tanna, Vanuatu: rbcL KP774898, trnL-F KP851917, rps4–trnS KP851881, atpB − (Ohlsen et al. 2015).

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rps4-118F rps4-911R rps4-532R trnL intron & trnL-F spacer

fern1 F E trnL-F-43F trnL-F-430R

GGAGGCCAAACAGAGA TTACG GGTAATCCCTTGGTTRA TGRGA GCTTTGCGGGAGTAGT ACTC GGCAGCCCCCARATTC AGGGRAACC ATTTGAACTGGTGACA CGAG GGTTCAAGTCCCTCTAT CCC CCGACTGAGCTATCTCG GC CTATCCCTAACGAGACA C

This study This study This study Trewick et al. (2002) Taberlet et al. (1991) Taberlet et al. (1991) This study This study

Table 2. Best-fitting models and parameter values for separate (atpB, rbcL, rps4 & rps4-trnS, and trnL & trnL-F) and simultaneous plastid datasets in this study. ‘‘G” = gamma distribution shape parameter. ‘‘GTR” = general-time-reversible model. ‘‘I” = proportion of invariable sites. Region Plastid atpB gene Plastid rbcL gene Plastid rps4 gene & rps4-trnS spacer Plastid trnL intron & trnL-F spacer Plastid markers combined

Selected model

Base frequencies

Substitution model (rate matrix)

I

G

A

C

G

T

A–C

A–G

A–T

C–G

C–T

G–T

GTR+I+G

0.2746

0.2027

0.2585

0.2642

2.6993

13.7203

0.0749

1.4907

30.6899

1.0000

0.4880

0.4880

GTR+I+G

0.2691

0.2179

0.2479

0.2650

3.4700

11.1825

1.1546

1.4345

19.5803

1.0000

0.4380

0.7740

TVM+I+G

0.3120

0.1750

0.1846

0.3284

1.4487

5.0210

0.4670

0.8540

5.0210

1.0000

0.1410

1.1310

GTR+I+G

0.3169

0.1943

0.1865

0.3024

0.8656

3.0982

0.2089

0.6282

3.0863

1.0000

0.0000

1.4630

GTR+G

0.2961

0.1961

0.2192

0.2885

1.5218

4.9902

0.4792

0.8095

6.8482

1.0000

–

0.4210

Table 3. Data matrices and tree statistics for each of the analyses. Missing data include missing sequences, uncertain bases (N, R, Y, V, etc.) and gaps (-). Locus # accessions # missing # chars. # Parsimony-informative chars. (%) Plastid atpB gene 86 52 1227 106 (8.6) Plastid rbcL gene 123 33 1379 247 (17.9) Plastid rps4 gene & rps4-trnS 98 52 1118 329 (29.4) spacer Plastid trnL intron & trnL-F 108 74 1225 301 (24.6) spacer Simultaneous 158 52 4996 983 (19.7) Figure legends: Fig. 1. Habit and leaf dissections in Hymenasplenium. A, C, E–J. The Old World clade. —A. The H. unilaterale subclade (H. unilaterale). —C. The H. wildii subclade (H. subnormale). —E. The H. excisum subclade (H. excisum). —F. The H. obliquissimum subclade (H. retusulum). —H. The H. cheilosorum subclade (H. cheilosorum). —G, I, J. The H. hondoense subclade (H. sp 5, H. apogamum, H. cardiophyllum, respectively). B, D. The New World clades. —B. The H. riparium clade (H. delitescens). —D. The H. laetum clade (H. laetum). Fig. 2. Pinna shapes and sorus distributions in Hymenasplenium. A, C, E–J. The Old World clade. —A. The H. unilaterale subclade (H. unilaterale). —C. The H. wildii subclade (H. subnormale). —E. The H. excisum subclade (H. excisum). —F. The H. obliquissimum subclade (H. retusulum). —H. The H. cheilosorum subclade (H. cheilosorum). —G, I, J. The H. hondoense subclade (H. sp 5, H. apogamum, H. cardiophyllum, respectively). B, D. The New World clades. —B. The H. riparium clade (H. delitescens). —D. The H. laetum clade (H. laetum).. Fig. 3. Sampling localities of Hymenasplenium in both Old and New Worlds. Fig. 4. Maximum likelihood phylogeny of Hymenasplenium based on six plastid markers (atpB, rbcL, trnL, trnL-F, rps4, and rps4-trnS). Thick lines indicate those clades with strong support in at least two analyses [maximum likelihood bootstrap support (MLBS) ≥ 80%, maximum parsimony jackknife support (MPJK) ≥ 70%, and Bayesian inference posterior probability (BIPP) ≥ 90%], medium thickness lines indicate clades with strong support in one analysis (MLBS ≥ 80% or MPJK ≥ 70% or BIPP ≥ 90%), and thin lines indicate those clades with strong support in none of the analyses. Voucher information is indicated in dull green. Geographical provenances are indicated in blue. Black vertical bars on the rightmost indicate three major clades in Hymenasplenium, while other vertical bars indicate the six subclades of the Old World clade. Black arrows indicate the ingroup and outgroups. Species names in parentheses with quotation marks denote

species that were misidentified in previous works Fig. 5. Sampling localities and the phylogeny of each Old World subclade of Hymenasplenium. Colors in the topology and map correspond to clade membership.

Highlights 1. It deals with one of the little known fern genera with global distribution; 2. A large sampling (141 accessions) across the world has been achieved; 3. Extensive cryptic speciation in the genus is uncovered; 4. Major evolutionary lineages have been identified; 5. The monophyly of the Old World species is confirmed.

Graphical abstract