A plastid phylogeny of the Old World fern genus Leptochilus (Polypodiaceae): Implications for cryptic speciation and progressive colonization from lower to higher latitudes

Accepted Manuscript A plastid phylogeny of the Old World fern genus Leptochilus (Polypodiaceae): Implications for cryptic speciation and progressive colonization from lower to higher latitudes Liang Zhang, Ngan Thi Lu, Xin-Mao Zhou, De-Kui Chen, Ralf Knapp, Lin Zhou, Lei Guo, Thien Tam Luong, Hang Sun, Xin-Fen Gao, Li-Bing Zhang PII: DOI: Reference:

S1055-7903(18)30368-3 https://doi.org/10.1016/j.ympev.2019.01.013 YMPEV 6401

To appear in:

Molecular Phylogenetics and Evolution

Received Date: Revised Date: Accepted Date:

8 June 2018 16 December 2018 20 January 2019

Please cite this article as: Zhang, L., Thi Lu, N., Zhou, X-M., Chen, D-K., Knapp, R., Zhou, L., Guo, L., Tam Luong, T., Sun, H., Gao, X-F., Zhang, L-B., A plastid phylogeny of the Old World fern genus Leptochilus (Polypodiaceae): Implications for cryptic speciation and progressive colonization from lower to higher latitudes, Molecular Phylogenetics and Evolution (2019), doi: https://doi.org/10.1016/j.ympev.2019.01.013

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Running title: Zhang et al. — Phylogeny of Leptochilus

A plastid phylogeny of the Old World fern genus Leptochilus (Polypodiaceae): Implications for cryptic speciation and progressive colonization from lower to higher latitudes Liang Zhanga, Ngan Thi Lub, d, e, Xin-Mao Zhouc, j, De-Kui Chenf, Ralf Knappg, Lin Zhoub, Lei Guoa, Thien Tam Luongh, i, Hang Suna, Xin-Fen Gaob, *, and Li-Bing Zhangb, j, * a

Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese

Academy of Sciences, Kunming, Yunnan 650201, 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 e

University of Chinese Academy of Sciences, Beijing 100049, China;

Department of Biology, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology,

18th Hoang Quoc Viet Road, Ha Noi, Vietnam; f

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

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

Department of Biology, University of Turku, Turku, Finland;

j

Missouri Botanical Garden, 4344 Shaw Blvd., St. Louis, Missouri 63110, U.S.A.

*Authors for correspondence: Xin-Fen Gao, [email protected]; Li-Bing Zhang, [email protected]

Abstract: The newly defined fern genus Leptochilus contains about 50 species occurring in subtropical to tropical Asia and adjacent Pacific islands. The circumscription and phylogeny of the genus have been ambiguous and its species had been included in various genera such as Colysis, Dendroglossa, Kontumia, Microsorum, and Paraleptochilus. Previous molecular studies sampled only 2–4 molecular markers and 2–16 accessions of Leptochilus. In the present study, DNA sequences of six plastid markers of 105 accessions representing ca. 40 species of Leptochilus, including types of Colysis, Kontumia, Leptochilus, and Paraleptochilus, 39 species of six non-Leptochilus genera of Microsoroideae, and one species of Pyrrosia, are used to infer a phylogeny. Our major results include: (1) Leptochilus is monophyletic and resolved as nested within the microsoroid ferns, but its relationships with other members of Microsoroideae are not well

resolved; (2) Six well-supported major clades in Leptochilus are recognized, differing from one another in molecular, morphological, and geographical features; (3) Species related to L. macrophyllus representing earliest split in Leptochilus are identified; (4) The inclusion of Microsorum pteropus in Leptochilus is confirmed, whereas M. insigne is closely related to Leptochilus but not resolved as a member of the genus; (5) The species number of the genus is likely to double the most recent estimate following our study, and quite a few cryptic species should be recognized; and (6) A basal grade formed by three major clades is recovered and they are composed of species almost exclusively distributed at lower latitudes (the Malay Archipelago), whereas the shallow-level clades contain species distributed at mainly higher latitudes, suggesting that Leptochilus might have evolved at lower latitudes and progressively dispersed to and colonized higher latitudes. Key words: Colysis; fern phylogeny; latitudinal dispersals; Malay Archipelago; Microsorum; Paraleptochilus

1 Introduction Species of the Old World fern genus Leptochilus Kaulf. (Polypodiaceae) sensu Kim et al. (2012) are terrestrial or epiphytic (on tree trunks or rocks) and are characterized by having rhizome long-creeping and with clathrate or subclathrate scales, scales usually not fully covering the rhizome and not spreading, laminae simple to pinnatifid, or rarely 2-pinnate to 4-pinnatifid, and sori orbicular, elongate to linear, or sometimes acrostichoid (Fig. 1). The genus was estimated to contain 25 "indistinct" species (Zhang and Nooteboom, 2013) and they mainly occur in subtropical, tropical Asia and adjacent Pacific islands. The circumscription and the taxonomy of Leptochilus had always been ambiguous. Before the application of molecular systematics, species of Leptochilus were placed in or related to various genera with acrostichoid sori, e.g., Acrostichum L., Bolbitis Schott, Colysis C. Presl, Leptochilus, Paraleptochilus Copel., and Tectaria Cav., etc. (Christensen, 1904; Ching, 1933; Copeland, 1947; Bosman, 1991; Shi and Zhang, 1999a). Leptochilus was published based on L. axillaris (Cav.) Kaulf., the only species then included (Kaulfuss, 1824). As one of the genera segregated from Acrostichum sensu lato (s.l.), Leptochilus had been distinguished by having simple sterile leaves and much contracted fertile leaves fully covered with sori (acrostichoid sori). Before the 1870s, there were about 16 names synonymized with Leptochilus (Blume, 1828; Fée, 1844–1845), all of them having acrostichoid sori, and some of the species having been confirmed as members of other genera or families (e.g., Schneider, 2004). For a long time, Leptochilus had been overlooked by most pteridologists until Christensen’s recognition of Leptochilus as the earliest valid name of the related genera (Christensen, 1904). However, Leptochilus sensu Christensen (1904) contained Bolbitis Schott, Lomagramma J. Sm., and other small genera and is clearly non-monophyletic. Copeland (1928) noticed the problem

with Leptochilus sensu Christensen (1904) and recognized six genera. Leptochilus sensu Copeland (1928) only contained two species, L. axillaris and L. platyphyllus Copel., while the genus Campium C. Presl revived by him contained 55 species and is also highly polyphyletic. Of his later work, Copeland realized his misuse of Campium and synonymized it with Bolbitis (Copeland, 1947). Meanwhile, he published a new genus Paraleptochilus Copel. and reinstalled Dendroglossa C. Presl, which are both synonyms of Leptochilus sensu Nooteboom (1997) and Zhang and Nooteboom (2013). From then on, most taxonomists had adopted the original circumscription of Leptochilus with limited species included (Sledge, 1956) and had accepted Colysis C. Presl, Leptochilus, or sometimes Paraleptochilus as separated genera. Ching (1933) clarified the distinction between microsoroid ferns (Microsorum and allies; subfam. Microsoroideae) and Bolbitis, and indicated the intermediate states of soral conditions among species of Colysis, Leptochilus, and Microsorum Link. Ching (1933) recognized 25 species in Colysis, and treated them in two sections (i.e., "C. sect. Eucolysis C. Presl" (=C. sect. Colysis) and C. sect. Dictyogramme C. Presl) based on the leaf dissections. Bosman (1991) placed three species of Microsorum in Leptochilus, two of which, L. buergerianus (Miq.) Bosman and L. subhemionitideus (Christ) Bosman, are more similar to Microsorum than to Leptochilus in having separate sori. Nooteboom (1997) was the first who synonymized Colysis with Leptochilus, and he recognized 10 species in Leptochilus for Asia, the Solomon Islands, and northern Australia. His circumscription of Leptochilus was adopted by Flora of China, in which 13 species in China were recognized (Zhang and Nooteboom, 2013). Using three plastid markers, Schneider (2004) for the first time found that Leptochilus (two species sampled) was nested within a paraphyletic Microsorum. The DNA data of four plastid markers from Kreier et al. (2008) resolved microsoroid ferns into six major clades. As one of the largest genera in microsoroids, Microsorum with its species being resolved into four lineages was demonstrated to be highly polyphyletic. In addition, the traditional circumscriptions of a number of genera such as Belvisia Mirb., Lemmaphyllum C. Presl, Lepisorus (J. Sm.) Ching, Neocheiropteris Christ, and Neolepisorus Ching were challenged but remained ambiguous due to the largely unresolved phylogeny (Kreier et al. (2008). In contrast, 10 species of Leptochilus (including types of Colysis and Paralepptochilus) were found to form a clade, and to be well supported as sister to Microsorum pteropus (Blume) Copel. However, within Leptochilus, the relationships among the most species/clades were unresolved. Based on two plastid markers, Dong et al. (2008) also confirmed the monophyly of Leptochilus, with Paraleptochilus and 12 species of Colysis being resolved in the monophyletic Leptochilus. Using data from four plastid markers of 47 samples representing six species of Leptochilus, Kim et al. (2012) found that the monotypic genus Kontumia S.K. Wu & K.L. Phan was embedded within Leptochilus. Therefore, PPG I (2016) recognized a monophyletic Leptochilus, while treating Colysis and Kontumia as synonyms of Leptochilus. Based on three plastid markers, Testo and Sundue (2014) discovered that the Australian endemic Colysis ampla (F. Muell. ex Benth.) Copel. was sister to a clade containing species of Phymatosorus Pic. Serm. and Microsorum, but not closely related to

Leptochilus, and thus the species does not belong to Colysis (=Leptochilus). Since Colysis ampla is the only species of Colysis in Australia, the exclusion of the species from Colysis demonstrates that species of Leptochilus do not reach Australia. Therefore, molecular studies have helped re-circumscribe Leptochilus as including Colysis, Dendroglossa, Kontumia, and Paralepptochilus. Molecular studies so far have also resolved intergeneric and infrageneric relationships to some extent. However, previous studies were all based on limited taxon and molecular sampling, the monophyly of the newly defined Leptochilus has not rigorously been tested, and the infrageneric relationships within Leptochilus largely remain unresolved. Although the relationships among most lineages within microsoroids including Leptochilus remain unresolved (e.g., Dong et al., 2008; Kreier et al., 2008; Kim et al., 2012), as the earliest valid name at the genetic level among genera of microsoroids, Leptochilus will be maintained regardless of the changing relationships with the related genera. In this study, we aimed: (1) to test the monophyly of Leptochilus with the largest taxon and molecular sampling so far; (2) to better resolve relationships across Leptochilus and to identify major evolutionary lineages in the genus; (3) to determine the relationships between species from Asia and those from the Pacific islands; and (4) to assess the systematic and biogeographical implication based on the reconstructed phylogeny. 2 Materials and methods 2.1 Taxon sampling One hundred and five accessions representing ca. 40 species of the newly defined Leptochilus are included covering the major distribution range of the genus. Multiple accessions of the type species of Colysis, Leptochilus, and Paraleptochilus are sampled. Thirteen accessions of Leptochilus digitatus (Baker) Noot. with different lobe numbers and variable lobe shapes are sampled to test the monophyly of the species. Twenty-nine accessions related to L. ellipticus s.l., five accessions of L. macrophyllus s.l., five accessions of L. pedunculatus s.l., are sampled to test species delimitations of these species. Thirty-nine species of non-Leptochilus microsoroids representing eight genera paraphyletic in relation to Leptochilus are sampled based on previous studies (Dong et al., 2008; Kreier et al., 2008). One species of Pyrrosia Mirb. is used as outgroup because together with Platycerium Desv., Pyrrosia has been resolved as sister to microsoroids ferns (Kim et al., 2012; Zhou et al., 2017). 2.2 DNA extraction, amplification, and sequencing Total genomic DNA was extracted from silica-dried material or sometimes from herbarium specimens using the TIANGEN plant genomic DNA extraction kit (TIANGEN Biotech., Beijing, China) or the DNeasy Plant Mini Kit (Qiagen, Shanghai, China) following the manufacturers’ protocols. Six plastid markers (atpB, rbcL, rps4, the rps4-trnS intergenic spacer, the trnL intron, and the trnL-F intergenic spacer) were selected for amplification and sequencing based on their resolving power as demonstrated in earlier studies (Kim et al., 2012; Kreier et al., 2008;

Schuettpelz and Pryer, 2007; Zhou et al., 2017) The atpB gene was amplified with the primers ESATB672F (Wolf, 1997) and ESATPE384R (Pryer et al., 2004). The rbcL gene was amplified with the primers F1 (Fay et al., 1997) and 1379R originally designed by Zurawski et al. (1984) and modified by Wolf et al. (1999). The rps4 gene and rps4-trnS intergenic spacer were amplified with the primers TRNS (Souza-Chies et al., 1997) and an unnamed primer derived from Li and Lu (2006). The trnL intron and trnL-F intergenic spacer were amplified together using the primers FERN1 (Trewick et al., 2002) and F (Taberlet et al., 1991). The PCR conditions followed Zhang et al. (2001). Amplified fragments were purified with TIANquick Mini Purification Kits (Tiangen Biotech, Beijing, China) and purified polymerase chain reaction (PCR) products were sequenced by Invitrogen (Shanghai, China). 2.3 Sequence alignment and phylogenetic analysis Sequences obtained for each marker were initially aligned using MAFFT ver. 7 (Katoh & Standley, 2013) and then manually adjusted in BioEdit (Hall, 1999). The data were partitioned by locus. Equally weighted maximum parsimony (MP) analyses for each locus and the combined dataset were conducted in PAUP* ver. 4.0b10 (Swofford, 2002) using 1000 tree-bisection-reconnection (TBR) searches with MAXTREES set to increase without limit. Gaps 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 0.1.1 (Guindon and Gascuel, 2003; Posada, 2008) was used to select the best-fitting likelihood model for maximum likelihood (ML; Felsenstein, 1973) and Bayesian analyses. The Akaike information criterion (Akaike, 1974) was used to select among models instead of the hierarchical likelihood ratio test, following Pol (2004) and Posada and Buckley (2004). The best-fitting models and parameter values are provided in Table 1. For each marker and the combined analysis (Farris et al., 1996; Nixon and Carpenter, 1996) of all nucleotide characters, maximum likelihood tree searches and ML bootstrapping were conducted using RAxML-HPC2 on TG ver. 7.2.8 on CIPRES web server (Miller et al., 2010; Stamatakis et al., 2008), with 1000 rapid bootstrap (BS) analyses followed by a search for the best-scoring tree in a single run (Stamatakis et al., 2008). Bayesian inference (BI) was conducted for the combined dataset using MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; 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). 3 Results A total of 248 sequences are newly generated for this study (Appendix). All 146 accessions are represented by at least one plastid marker. A comparison of the trees resulting from MPJK analyses of the individual plastid markers, the combined plastid dataset, did not identify any well-supported conflicts (MPJK 70%; (Mason-Gamer and Kellogg, 1996; Zhang and Simmons, 2006)). Two suspicious sequences from GenBank: rps4 (accession # EU363255) of Leptochilus pentaphyllus Libing Zhang & Liang Zhang and a sequence of rbcL (accession # AY096203) of L. decurrens were removed. For details regarding the data sets analyzed and statistics for the resulting trees are given in Table 2. The alignments and ML tree are deposited at TreeBase with study # S 23357 (http://purl.org/phylo/treebase/phylows/study/TB2:S 23357). Leptochilus was resolved as a monophyletic group and nested within microsoroids. The monophyly of Leptochilus is maximally supported in all BI, ML and MP analyses. Within Leptochilus, six major clades can be identified based on our phylogeny, morphology, and geographic distribution. These major clades include the Colysis clade (MLBS: 100%, MPJK: 95%, BIPP: 1.0), the L. ellipticus clade (MLBS: 89%, MPJK: 89%, BIPP: 1.0), the L. macrophyllus clade (MLBS: 100%, MPJK: 100%, BIPP: 1.0), the L. pentaphyllus clade (MLBS: 100%, MPJK: 92%, BIPP: 1.0), the L. pteropus clade (MLBS: 100%, MPJK: 100%, BIPP: 1.0), and the Vietnam clade (MLBS: 100%, MPJK: 100%, BIPP: 1.0; Fig. 2). All these major clades are well supported in all three analyses [MLBS ≥ 89%, MPJK ≥ 89 %, BIPP = 1.0; Fig. 2]. Five samples representing at least three species from the Malay Archipelago (the L. macrophyllus clade) are resolved as sister to the rest of the species in the genus, consecutively followed by the L. pteropus clade, the Vietnam clade, the Colysis clade, the L. pentaphyllus clade, and the L. ellipticus clade. The relationships among the six major clades are weakly, moderately or strongly supported. Within the Colysis clade and the L. ellipticus clade, six and seven subclades, respectively, can be identified based on molecular and non-molecular information. 4 Discussion 4.1 The monophyly of Leptochilus With data from six plastid markers of 105 accessions representing ca. 40 species of Leptochilus, the largest taxon and character sampling so far, our analyses resolved Leptochilus sensu Kim et al. (2012) including Colysis, Kontumia, and Paraleptochilus as monophyletic with strong support in all analyses. This result is consistent with previous studies (Dong et al., 2008; Kreier et al., 2008; Kim et al., 2012). A morphologically aberrant member, Leptochilus pteropus (Blume) Fraser-Jenk., recently transferred from Microsorum (Fraser-Jenkins, 2008), is confirmed to belong to Leptochilus. Morphologically, species of Leptochilus have rhizome scales that are clathrate or subclathrate and not spreading, and usually do not fully covered the rhizome and, laminae simple

to pinnatifid, or rarely 2-pinnate to 4-pinnatifid, sori orbicular, elongate to linear, or sometimes acrostichoid. Leptochilus is resolved as nested within other microsoid ferns in our study, as found by previous studies (Dong et al., 2008; Kreier et al., 2008; Kim et al., 2012). It is, however, ambiguous even with our current data to which taxa Leptochilus is sister. It is not clear either morphologically. Our analyses found Leptochilus to be sister to a clade containing the Microsorum insigne complex and Phymatosorus in ML analysis, but with very low support, and this resolution is not supported in MP analysis. 4.2 Infrageneric relationships The six major clades are discussed below in an order they appear on the tree from bottom up (Fig. 2): the L. macrophyllus clade, the L. pteropus clade, the Vietnam clade, the Colysis clade, the L. pentaphyllus clade, and the L. ellipticus clade. 4.2.1 The L. macrophyllus clade This clade is characterized by having leaves simple, laminae broad-elliptic or ovate, rhizomes covered with circumvascular sheaths and scattered strands of sclerenchyma (Nooteboom, 1997). At the present all species of the clade are from the Malay Archipelago including Java, South Philippines, and Solomon Islands. Three lineages can be well recognized corresponding to their geographic distributions. Two accessions together, Wade 1962 and Wade 1772, from Java, are resolved as sister to a clade containing one accession from the Philippines and two from the Solomon Islands. All three lineages are similar to one another by being large and having leaves simple, broad-lanceolate, and monomorphic, and sori linear, and continuous or sometime discontinuous. Given the types of L. macrophyllus were collected from Java, the material from the Philippines and the Solomon Islands could be two new species pending further studies. Nooteboom (1997) adopted an enlarged concept of Leptochilus macrophyllus, and synonymized quite a few names with it. Meanwhile, he recognized three varieties in addition to the typical variety, i.e., L. macrophyllus var. fluviatilis (Lauterb.) Noot., L. macrophyllus var. pedunculatus (Hook. & Grev.) Noot., and L. macrophyllus var. wrightii (Hook. & Baker) Noot. Our phylogeny does not support his treatment. Instead samples related to L. pedunculatus (Hook. & Grev.) Fraser-Jenk. and L. wrightii (Hook. & Baker) X.C. Zhang are resolved in the L. ellipticus clade, not even closely related to the L. macrophyllus clade. Colysis fluviatila (Lauterb.) Ching from Borneo has small habit and dimorphic leaves, which should be recognized as a different species instead of a variety of L. macrophyllus. Whether it belongs to the L. macrophyllus clade needs further studies. It is highly possible that L. macrophyllus only occurs in Java and adjacent areas. The taxonomy of Leptochilus in the Malay Archipelago is far from clear. More work is needed to clarify the diversity of the genus in this region.

4.2.2 The L. pteropus clade This clade is characterized by having fronds simple and entire or irregularly lobed, petioles and costae scaly abaxially, sori orbicular or elongate, spores covered with abundant globules and irregular spines (Zhang and Nooteboom, 2013). The species number of the clade is unclear and the adoption of a broad species concept of L. pteropus is challenged by our study. Species of this clade tend to grow on moist rocks near or in streams, sometime even under water in rainy seasons. Leptochilus pteropus is often used as ornamental plants being kept in the fish tank. Species of this clade are distributed from southern China to subtropical and tropical Asia. Microsorum insigne (Blume) Copel. and M. pteropus are often treated as members of Microsorum (Zhang and Nooteboom, 2013). Having noticed the elongate sori and rounded sori both occur on fertile leaves of the two species and Colysis hemionitidea C. Presl (type of Colysis), Bosman (1991) regarded the former two as members of Colysis, i.e., C. insignis (Blume) J. Sm. and C. pteropus (Blume) Bosman. Later, the two species were transferred to Leptochilus by Fraser-Jenkins (2008). Our phylogeny supports the inclusion of Microsorum pteropus in Leptochilus. On the other hand, the relationships among Leptochilus, M. insigne, and some species of Phymatosorus and Microsorum are unresolved. Thus, the inclusion of M. insigne in Leptochilus awaits further studies. The close affinity between L. pteropus and L. hemionitideus is not supported by our study. Ching (1933) hypothesized that M. pteropus may be intermediate between Microsorum and Colysis, which is partially confirmed by our phylogeny. 4.2.3 The Vietnam clade This clade is characterized by having large plants (50–80 cm), leaves slightly dimorphic, laminae herbaceous, deeply pinnatifid and with narrow winged rachis, part of rachis wingless, pinnae (1–)2–6 pairs, 7–10 cm long, and sori usually linear but sometimes interrupted. This newly identified clade comprises three accessions representing two lineages. Species of this clade can easily be confused with Leptochilus pothifolius (D. Don) Fraser-Jenk., because of the similarities in lamina shape and broad-lanceolate pinnae, but the latter has widely winged rachises. Species of this clade grow in acidic soil developed from limestones, and are only known to occur in limestone regions of central and northern Vietnam. This clade is resolved in an unexpected systematic position on the tree (Fig. 2) considering that its morphology is consistent with that of Leptochilus ellipticus and allies. The number of species in this clade can increase after thorough investigations in the limestone areas of central to northern Vietnam which have been little explored so far. 4.2.4 The Colysis clade This clade is characterized by having simple sterile leaves, contracted fertile leaves (except Leptochilus hemionitideus), and visible venation. At least nine species can be recognized based on our phylogeny and morphology. Of the nine species, L. axillaris, L. decurrens, and L.

hemionitideus are type species of Leptochilus, Paraleptochilus, and Colysis, respectively. Leptochilus evrardii (Tardieu) Liang Zhang & Li Bing Zhang were synonymized with L. decurrens (e.g., Zhang and Nooteboom, 2013). Morphologically, the former has much contracted fertile leaves that are wider than those of L. axillaris and L. decurrens, and sori not fully covered the abaxial laminae. Our analyses with four accessions from Laos and Vietnam included support the recognition of the species. In addition to the four species mentioned above, at least five more species can be recognized based on our phylogeny. Species of this clade are epiphytic on rocks, trunks, or grow in rock crevices, occurring from the Malay Archipelago, Southeast Asia to southern China. The Colysis clade is resolved into six subclades, all well supported: the Leptochilus subclade, the Yunnan subclade, the Paraleptochilus subclade, the Laos subclade, the Tonkin subclade, and the Colysis subclade from the bottom up (Fig. 2). The Leptochilus subclade is composed of three accessions representing two species from Indonesia, Myanmar, and Vietnam, which are morphologically similar to Leptochilus axillaris, the type of the genus. However, we did not sample material from the Philippines, the type country of Leptochilus axillaris. The Yunnan subclade contains a cryptic species similar to L. decurrens. Two accessions (Douglas 28 and Wade 1769) of L. decurrens from the type locality Java, and an accession from Taiwan (Knapp 4128) constitute the Paraleptochilus subclade. Leptochilus evrardii constitutes the Laos subclade. Four accessions in our sampling from China, Laos and Vietnam representing about four species similar to L. decurrens are included in the Tonkin subclade. Leptochilus hemionitideus, type of Colysis, and allies constitute the Colysis subclade. The relationships among these six subclades are well resolved and strongly supported except those between the Tonkin subclade and the Colysis subclade, and those between the Laos subclade and the Paraleptochilus subclade (Fig. 2). 4.2.5 The L. pentaphyllus clade This clade is recognized by having laminae deeply pinnatifid to pinnate, pinnae large, pinna bases extending to rachis, rachises narrowly winged or wingless, petioles robust, the same length with or longer than laminae, and sori linear, sometimes interrupted, and half length of lateral veins. This clade contains Leptochilus pentaphyllus only, which is endemic to southern Yunnan. The species is much similar to species of the Vietnam clade, but with leaves less than 50 cm and pinnae usually less than five pairs. Leptochilus pentaphyllus was often treated as a synonym or a variety of L. ellipticus due to the deeply pinnatifid laminae (Nooteboom, 1997; Zhang and Nooteboom, 2013), which is falsified by our phylogeny. Leptochilus pentaphyllus should be recognized as a distinct species. Morphologically, L. pentaphyllus has wider pinnae than L. ellipticus and its varieties do. Our phylogeny resolved L. pentaphyllus as sister to the Colysis clade, but this sister relationship is only weakly supported in the ML analysis.

4.2.6 The L. ellipticus clade Species of this clade appear to be very different in morphology: leaves monomorphic to strongly dimorphic, laminae simple to pinnate, or even 2-pinnate to 3-pinnate, and sori elongate to linear. Seven well supported subclades can be recognized, although the relationships among them were not well resolved. These seven subclades are discussed bellow in an order they appear on the tree from bottom up (Fig. 2). 4.2.6.1 The Khanhhoa subclade This subclade is characterized by having leaves 15–30(–50) cm long, laminae deeply pinnatifid, pinnae 1–4 pairs and 1–1.5(–2) cm wide, rachises with wings of ca. 3–8 mm wide. Five accessions from Lam Dong and Khanh Hoa of southern Vietnam constitute this subclade. Morphologically, except Zhang et al. 8596 all other four accessions fit well with Leptochilus longipes (Ching) X.C. Zhang. Zhang et al. 8596 has larger habit and more pairs of pinnae which are up to 11 cm long. We included one accession of L. longipes (Lu S.G. V56) from the type locality, Hainan, and it is resolved in the L. ellipticus subclade (Fig. 2). Thus, the Khanhhoa subclade contains at least a cryptic species with similar morphology to that of L. longipes. 4.2.6.2 The Kontumia subclade This subclade is distinguishable in the genus by having leaves strongly dimorphic, sterile leaves 2-pinnate and petioles shorter than laminae, fertile leaves 2-pinnate to 3-pinnatified, petioles longer than laminae, and terminal lobes less than 2 mm long, and sori orbicular and located on distal veinlets. Due to the unique dissection of both sterile and fertile leaves, Kontumia was published as a new genus to incorporate the species (Wu et al., 2005). Kim et al. (2012) resolved Kontumia as a member of Leptochilus based on data of four plastid genes. Our study confirms the inclusion of Kontumia in Leptochilus. Kontumia is resolved as nested within the L. ellipticus clade, sister to a clade containing five of the seven subclades in the L. ellipticus clade (Fig. 2). Morphologically, the inclusion of Kontumia in Leptochilus is supported by the similar rhizome scales (ovate-lanceolate, appressed), while the much dissected leaves of Kontumia is quite diverged from the rest of Leptochilus which has simple or pinnatifid leaves. Only one species is included in the subclade, which is endemic to Kon Tom Province of southern Vietnam. 4.2.6.3 The L. henryi subclade This subclade is characterized by having leaves simple, monomorphic, or slightly dimorphic with fertile leaves smaller than the sterile leaves, and sori linear and nearly reaching the lamina margins. The subclade is composed of 11 accessions representing ca. six species from China and Vietnam in our sampling and is further resolved into two lineages. The first lineage contains Leptochilus henryi and L. leveillei (Christ) X.C. Zhang & Noot., which are endemic to China. The two species were treated as synonyms of L. macrophyllus var.

wrightii by Nooteboom (1997) but recognized as two distinct species by (Zhang and Nooteboom, 2013). Our data resolved the two species as closely related to neither L. macrophyllus nor L. wrightii, which partially confirmed the recognition of the two species. Our data resolved the lineage into two clades but we are not sure if these correspond L. henryi and L. leveillei, respectively. More material especially that from the type locality of L. henryi is needed to clarify the species diversity of this lineage in China. Five accessions from Vietnam in our sampling constitute the second lineage. Except Zhang et al. 6931 which lacks fertile leaves of collections, other four samples are similar to Leptochilus pedunculatus by having dimorphic simple leaves. The broad species concept of L. pedunculatus was widely adopted with quite a few synonyms, e.g., Colysis bonii Ching (type from Vietnam), C. fluviatila (Lauterb.) Ching (type from Boneo), C. saxicola H.G. Zhou & Hua Li (type from Guangxi, China) (Nooteboom, 1997; Zhang and Nooteboom, 2013). As noted by Ching (1933), L. pedunculatus (type from India) is very rare in China, which is not included in our analyses. Leptochilus pedunculatus has fertile leaves 1.5–2 times longer than sterile leaves, petioles the same length or longer than laminae, and laminae oblong or ovate-lanceolate. Our study detected fair amount of molecular divergence among the five accessions from northern Vietnam. 4.2.6.4 The L. oblongus subclade This subclade is characterized by having oblong lamina shape similar to that of Leptochilus ellipticus, and sori oblong and close to the lobe margins (Zhang et al., 2015). This subclade contains at least two species, i.e., L. oblongus Li Bing Zhang, Liang Zhang & N.T. Lu from Hoa Binh of Vietnam, and a potentially new species from Guangxi and Guizhou of China. Species of this subclade occur in limestone regions of southern China and northern Vietnam. 4.2.6.5 The L. cantoniensis subclade The subclade can be recognized by having leaves dimorphic, sterile leaves ovate or triangular, 2–7 cm, base cordate or cuneate, fertile leaves with petiole 12–30 cm, linear, sori acrostichoid, and paraphyses present. One species Leptochilus cantoniensis (Baker) Ching constitutes the subclade. Three accessions from the type locality, Hainan, are resolved into two lineages. Species of this subclade only occurs in limestone regions in Hainan and Guangdong of China, probably extending to northern Vietnam. Morphologically, the affinity between the species and L. decurrens is supported by the similarity in both sterile and fertile leaf shapes. However, our phylogeny resolved L. decurrens in the Colysis clade, phylogenetically quite distant from this subclade. The L. cantoniensis subclade.is resolved as sister to the Taiwan subclade + the L. ellipticus subclade, but this resolution is only supported by BI analysis (Fig. 2). 4.2.6.6 The Taiwan subclade The morphology of this subclade generally intermediates between Leptochilus ellipticus and L. pothifolius: laminae deeply pinnatifid to pinnate, pinnae 3–5 pairs, 8–10 × 1–2 cm, sori

short-linear and about half length of the lateral veins. Two accessions from Japan and one from Taiwan constitute this subclade. Wade 3681 from Japan has cristate pinna apex, which is unique in the genus. 4.2.6.7 The L. ellipticus subclade This subclade contains the largest number of species in the L. ellipticus clade and is strongly supported as monophyletic (MLBS: 89%, MPJK: 84%, BIPP: 1.0). This subclade is quite diverse in morphology: leaves monomorphic or slightly dimorphic, simple, palmately lobed, pinnatifid to pinnate, and sori linear. Species of this subclade occur in Southeast Asia, China, and Japan. Four lineages can be recognized based on molecular and/or morphological information. The first lineage is composed of four accessions including Colysis simplicifrons (Christ) Tagawa and species close to Leptochilus hemitomus and L. ellipticus. Colysis simplicifrons was synonymized with Leptochilus ×hemitomus or L. ×shintenensis (Hayata) X.C. Zhang & Noot. (Nooteboom, 1997; Zhang and Nooteboom, 2013), and resolved as sister to a small clade containing two species close to L. hemitomus and L. ellipticus. Thus, the recognition of C. simplicifrons as a different species is supported here. One accession (Lu SG/S15) from Fujian, China is similar to L. ×hemitomus, however, the identity need to be further confirmed given other two samples (HGX 13253 and Zhang X.C. 3302) from China tentatively labeled as L. ×hemitomus are resolved in the fourth lineage. Two accessions (TNS 774822 and Wade 3656) from Japan in this lineage may represent the true L. ellipticus, since the type of L. ellipticus was collected from Japan. The second lineage is also closely related to Leptochilus ellipticus and contains one accession of L. longipes (from the type locality, Hainan, China), and three from China and Vietnam representing at least one undescribed species. The third lineage is composed of 13 accessions of Leptochilus digitatus. This lineage differs from other species of the genus in having palmately lobed leaves with wingless petioles (Ching, 1933). One accession (A.R. Smith 00-036) from Vietnam is resolved as sister to a clade containing 12 accessions from China and Vietnam. Unlike species related to L. ellipticus or L. pedunculatus, very little genetic divergence among the 13 accessions was detected, though the shapes and amount of lobes are quite variable in L. digitatus. The fourth lineage contains 18 accessions in our sampling representing at least seven species. This lineage is diverse in leaf dissection, but generally has monomorphic leaves and linear sori. Species with simple or irregularly lobed leaves (i.e., L. shintenensis, L. cf. hemitomus, L. wrightii, L. saxicola (H.G. Zhou & H. Li) Liang Zhang & Li Bing Zhang) are not monophyletic, while their relationships with one another are not resolved or not well supported. Species with pinnatifid laminae, i.e., L. dissimilialatus (Bonap.) Liang Zhang & Li Bing Zhang and L. flexilobus (Christ) Liang Zhang & Li Bing Zhang formed a clade (Fig. 2). 4.3 Cryptic species with restricted distributions Cryptic species shares similar morphology with related species but genetically quite

different from the latter (Paris et al., 1989). Cryptic speciation is common in ferns and previous studies have unraveled partial cryptic speciation in ferns, e.g., Asplenium L., Botrychium Sw., Ceratopteris Brongn., Hymenasplenium Hayata, and Polystichum Roth (Yatabe and Murakami, 2003; Adjie et al., 2007; Dauphin et al., 2017; Masuyama et al., 2002; Xu et al., 2018a, b; Han et al., 2016, 2018). As discussed above, our results have recovered cryptic speciation in Leptochilus. About 25 samples initially identified as L. macrophyllus, L. pothifolius, L. ellipticus, L. decurrens, and L. pedunculatus from different geographical ranges rather than type locality are resolved to be polyphyletic or paraphyletic in our tree (Fig. 2). In addition to four species reinstalled and three varieties raised to species rank, there are still at least 10 cryptic species to be recognized. Although we failed to find distinguishing morphological characters to delimitate these cryptic species so far, besides the molecular evidence, the distributional patterns are also important to define those cryptic species. For example, Nooteboom (1997) regarded L. macrophyllus as a widely distributed species ranging from the Malay Archipelago, Southeast Asia to South China; our analyses indicate that L. macrophyllus may only occur in the type locality of Java and adjacent regions, whereas materials from the Philippines and the Solomon Islands represent two different species. Populations with similar morphology but different in habitats (e.g., acidic soils or basic soils, limestone rocks or sandstones, on rock with thick soils or in rock crevices, on trunk or near trunk base) may represent different species. Notably, the majority of the cryptic speciation in Leptochilus is found in subtropical regions except that of L. macrophyllus. Similar pattern has been found in Hymenasplenium Hayata (Aspleniaceae; Xu et al., 2018a, b), Polystichum Roth (Dryopteridaceae; Han et al., 2016, 2018) recently. This well demonstrates that attention of biodiversity conservation also needs to be paid to subtropical regions, contrasting the current efforts focusing on tropical areas (e.g., Lucus et al., 2016). 4.4 Hybridization in Leptochilus Hybridization has presumably contributed to some extent to the species diversity of Leptochilus and hybrids have been reported in previous studies (Fraser-Jenkins, 2008; Manickam et al., 1997; Nooteboom, 1997). However, these published hybrids were described based on morphology only and without any molecular evidence. Nooteboom (1997) regarded L. ×hemitomus as a hybrid between L. macrophyllus and L. ellipticus on the basis of the irregular leaf shape and abortive sporangia. Our phylogeny resolved L. ×hemitomus as closely related to neither L. macrophyllus nor L. ellipticus (Fig. 2). Judging from our phylogeny, some species in the L. ellipticus subclade of the L. ellipticus clade could be the maternal donor of the hybrid L. ×hemitomus, if the latter turns out to be a true hybrid. Manickam et al. (1997) published a hybrid Colysis ×beddomei Manickam & Irudayaraj (=Leptochilus × beddomei (Manickam & Irudayaraj) X.C. Zhang & Noot.) and assumed L. decurrens and L. hemionitideus to be the parents based on the intermediate states of stipe anatomy and shape of fronds. Our sampling does not include L. ×bedomei, but a

morphologically similar species, L. evrardii, with discontinuous sori on contracted fertile leaves, a possible indication of hybridization between species related to L. decurrens and L. hemionitideus. Our analyses resolved L. evrardii in the Colysis clade including L. hemionitideus, L. decurrens, and their allies, which partially confirmed the hybrid origin of L. evrardii. 4.5 Biogeographical implications Detailed biogeographical analysis and discussion are beyond the scope of the current study. However, some biogeographical implications inferred from our phylogeny are worth mentioning. As a genus nested within the microsoroid ferns (Polypodiaceae subfam. Microsoideae), Leptochilus is undoubtedly a young lineage (stem age: ca. 33 million years ago; Testo and Sundue, 2016). The young age of Leptochilus precludes vicariance to explain disjunctions of the genus in Asia. Our study discovered a basal grade, although not strongly supported, formed by three major clades, the L. macrophyllus clade, the L. pteropus clade, and the Vietnam clade, composed of species almost exclusively with distributions at lower latitudes (the Malay Archipelago). In contrast, the shallow-level clades, the Colysis clade, the L. pentaphyllus clade, and the L. ellipticus clade, are composed of species mainly with distributions at higher latitudes. This suggests that Leptochilus might have evolved at lower latitudes and progressively dispersed to and colonized higher latitudes. This interesting biogeographical pattern does not appear to have been documented in ferns before. Acknowledgments The research was partially supported by a grant from the National Natural Science Foundation of China (NSFC) to L.-B.Z. (#31628002), the Major Program of National Natural Science Foundation of China (31590823) to H.S., Project of Platform Construction for Plant Resources of Sichuan Province to X.-F. G. (2016TJPT0001-3), the Glory Light International Fellowship for Chinese Botanists at Missouri Botanical Garden to X.-M.Z., and the CAS-TWAS President Fellowship to N.T.L. We thank Cheng-Wei Chen and Yao-Moan Huang for kindly sharing important material, and Cheng-Wei Chen and two anonymous reviewers and editors for helpful comments. References Adjie, B., Masuyama, S., Ishikawa, H., Watano, Y., 2007. Independent origins of tetraploid cryptic species in the fern Ceratopteris thalictroides. J. Plant Res. 120, 129–138. Akaike, H., 1974. A new look at the statistical model identification. IEEE Trans. Automat. Contr. 19, 716–723. Blume, C., 1828. Enumeratio plantarum Javae et insularum adjacentium: minus cognitarum vel novarum ex herbariis Reinwardtii, Kuhlii, Hasseltii et Blumii fasc. 2. Lugduni Batavorum,Apud J.W. van Leeuwen, Leiden. Bosman, M., 1991. A monograph of the fern genus Microsorum (Polypodiaceae), including an

attempt towards a reconstruction of the phylogenetic history of the microsoroids. Leiden Bot. Ser. 14, 1–161. Ching, R.-C., 1933. Studies of Chinese ferns X. Bull. Fan Mem. Inst. Biol. Bot. Ser. 4, 293–362. Christensen, C., 1904. On the American species of Leptochilus sect. Bolbitis. Bot. Tidsskr. 26, 283–300. Copeland, E.B., 1928. Leptochilus and genera confused with it. Philipp. J. Sci. 37, 333–416. Copeland, E.B., 1947. Genera Filicum. Chronica Botanica Co., Waltham. Dauphin, B., Farrar, D.R., Maccagni, A., Grant, J.R., 2017. A worldwide molecular phylogeny provides new insight on cryptic diversity within the moonworts (Botrychium s. s., Ophioglossaceae). Syst. Bot. 42, 620–639. Dong, X.-D., Lu, S.-G., Li, C.-X., 2008. Molecular phylogeny of Colysis (Polypodiaceae) based on chloroplast rbcL and rps4-trnS sequences. Acta Phytotax. Sin. 46, 658–666. Fée, A.L.A., 1844-1845. Mém. Foug., 2. Hist. Acrostich. Impr. de Ve. Berger-Levrault, Strassbourg. Farris, J.S., Albert, V.A., Källersjö, M., Lipscomb, D., Kluge, A.G., 1996. Parsimony jackknifing outperforms neighbor‐ joining. Cladistics 12, 99–124. Fay, M.F., Swensen, S.M., Chase, M.W., 1997. Taxonomic affinities of Medusagyne oppositifolia (Medusagynaceae). Kew Bull., 111–120. Felsenstein, J., 1973. Maximum likelihood and minimum-steps methods for estimating evolutionary trees from data on discrete characters. Syst. Biol. 22, 240–249. Fraser-Jenkins, C.R., 2008. Taxonomic revision of three hundred Indian subcontinental Pteridophytes: with a revised census list; a new picture of fern-taxonomy and nomenclature in the Indian subcontinent. Bishen Singh Mahendra Pal Singh. Guindon, S., Gascuel, O., 2003. A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst. Biol. 52, 696–704. Hall, T.A., 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl. Acids Symp. Ser. 41, 95–98. Han, M.-Q., Liu, Y. & Zhang, L.-B., 2016. Seven new species of (subg. ; Dryopteridaceae) from southern China. 280, 201–221. Han, M.-Q., Liu, Y. & Zhang, L.-B. 2018. Eight new species of Polystichum (subg. Haplopolystichum; Dryopteridaceae) from limestone caves in Guangdong and Yunnan, China, with reference to species diversity in the karst terrains at high elevations in subtropical areas. Phytotaxa 365, 145–168. Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics. Bioinformatics 17. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780. Kaulfuss, G.F., 1824. Enumeratio filicum quas in itinere circa terra legit CL. Adalbertus de Chamisso adiectis in omnia harum plantarum genera permultasque species non satis

cognitas vel novas animadversionibus. sumtibus C. Cnobloch, Leipzig. Kim, C., Zha, H.-G., Deng, T., Sun, H., Wu, S.-G., 2012. Phylogenetic position of Kontumia (Polypodiaceae) inferred from four chloroplast DNA regions. J. Syst. Evol., 154–163. Kreier, H.-P., Zhang, X.-C., Muth, H., Schneider, H., 2008. The microsoroid ferns: Inferring the relationships of a highly diverse lineage of Paleotropical epiphytic ferns (Polypodiaceae, Polypodiopsida). Molec. Phylogenet. Evol. 48, 1155–1167. Li, C.-X., Lu, S.-G., 2006. Phylogenetics of Chinese Dryopteris (Dryopteridaceae) based on the chloroplast rps4-trnS sequence data. J. Plant Res. 119, 589–598. Lucas, E., Wilson, C.E., Lima, E.F., Sobral, M., Matsumoto, K., 2016. A conspectus of Myrcia sect. Aulomyrcia (Myrtaceae). Ann. Missouri Bot. Gard. 101, 648–698. Manickam, V., Irudayaraj, V., Rajkumar, S.D., 1997. Colysis× beddomei (Polypodiaceae, Pteridophyta): A new interspecific fern hybrid from India, Burma and China. Taxon, 265–268. Mason-Gamer, R.J., Kellogg, E.A., 1996. Testing for phylogenetic conflict among molecular data sets in the tribe Triticeae (Gramineae). Syst. Biol. 45, 524–545. Masuyama, S., Yatabe, Y., Murakami, N., Watano, Y., 2002. Cryptic species in the fern Ceratopteris thalictroides (L.) Brongn.(Parkeriaceae). I. Molecular analyses and crossing tests. J. Plant Res. 115, 87–97. Miller, M.A., Pfeiffer, W., Schwartz, T., 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 November 2010, New Orleans, LA, pp. 1–8. Nixon, K.C., Carpenter, J.M., 1996. On simultaneous analysis. Cladistics 12, 221–241. Nooteboom, H.P., 1997. The microsoroid ferns (Polypodiaceae). Blumea 42, 261–395. Paris, C.A., Wagner, F.S., Wagner, H.W., 1989. Cryptic species, species delimitation, and taxonomic practice in the homosporous ferns. Amer. Fern J. 79, 46–54. Pol, D., 2004. Empirical problems of the hierarchical likelihood ratio test for model selection. Syst. Biol. 53, 949–962. Posada, D., 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Posada, D., Buckley, T.R., 2004. Model selection and model averaging in phylogenetics: advantages of Akaike information criterion and Bayesian approaches over likelihood ratio tests. Syst. Biol. 53, 793–808. PPG I. 2016. A community-derived classification for extant lycophytes and ferns. J. Syst. Evol. 54, 563–603. Pryer, K.M., Schuettpelz, E., Wolf, P.G., Schneider, H., Smith, A.R., Cranfill, R., 2004. Phylogeny and evolution of ferns (monilophytes) with a focus on the early leptosporangiate divergences. Am. J. Bot. 91: 1582–1598. Rambaut, A., Drummond, A.J., 2007. Tracer 1.4. Available: http:// beast.bio.ed.ac.uk/Tracer. Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.

Schneider, H., 2004. Unraveling the phylogeny of polygrammoid ferns (Polypodiaceae and Grammitidaceae): exploring aspects of the diversification of epiphytic plants. Molec. Phylogenet. Evol. 31, 1041–1063. Schuettpelz, E., Pryer, K.M., 2007. Fern phylogeny inferred from 400 leptosporangiate species and three plastid genes. Taxon 56, 1037–1050. Shi, L., Zhang, X.-C., 1999a. Taxonomic studies of the genus Colysis C. Presl (Polypodiaceae) from China and neighboring regions. Acta Phytotax. Sin. 37, 54–80. Shi, L., Zhang, X.-C., 1999b. Taxonomy of the fern genus Leptochilus Kaulf. (Polypodiaceae). Acta Phytotax. Sin. 37, 145–152. Sledge, W.A., 1956. CXI.—The Ceylon species of Leptochilus. J. Nat. Hist. 9, 865–877. Souza-Chies, T.T., Bittar, G., Nadot, S., Carter, L., Besin, E., Lejeune, B., 1997. Phylogenetic analysis of Iridaceae with parsimony and distance methods using the plastid gene rps4. Plant Syst. Evol. 204, 109–123. Stamatakis, A., Hoover, P., Rougemont, J., 2008. A rapid bootstrap algorithm for the RAxML Web servers. Syst. Biol. 57, 758–771. Swofford, D.L., 2002. PAUP*: phylogenetic analysis using parsimony, version 4.0 b10. Sinauer, Sunderland, Massachusetts. Taberlet, P., Gielly, L., Pautou, G., Bouvet, J., 1991. Universal primers for amplification of three non-coding regions of chloroplast DNA. Pl. Molec. Biol. 17, 1105–1109. Testo, W., Sundue, M., 2014. Primary Hemiepiphytism in Colysis ampla (Polypodiaceae) Provides New Insight into the Evolution of Growth Habit in Ferns. Int. J. Plant Sci. 175, 526–536. Testo, W., Sundue, M., 2016. A 4000-species dataset provides new insight into the evolution of ferns. Molec. Phylogen. Evol. 105, 200–211. Trewick, S., Morgan-Richards, M., Russell, S., Henderson, S., Rumsey, F., Pinter, I., Barrett, J., Gibby, M., Vogel, J., 2002. Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach: evidence from chloroplast DNA. Mol. Ecol. 11, 2003–2012. Wolf, P.G., 1997. Evaluation of atpB nucleotide sequences for phylogenetic studies of ferns and other pteridophytes. Am. J. Bot. 84, 1429–1440. Wolf, P.G., Sipes, S.D., White, M.R., Martines, M.L., Pryer, K.M., Smith, A.R., Ueda, K., 1999. Phylogenetic relationships of the enigmatic fern families Hymenophyllopsidaceae and Lophosoriaceae: Evidence from rbcL nucleotide sequences. Plant Syst. Evol. 219, 263–270. Wu, S.-G., Phan Ke, L., Xiang, J.-Y., 2005. A new genus and two new species of ferns from Vietnam. Novon, 245-249. Xu, K.-W., Zhou, X.-M., Yin, Q.-Y., Zhang, L., Lu, N.T., Knapp, R., Luong, T.T., He, H., Fan, Q., Zhao, W.-Y., Gao, X.-F., Liao , W.-B., Zhang, L.-B., 2018a. A global plastid phylogeny uncovers extensive cryptic speciation in the fern genus Hymenasplenium (Aspleniaceae). Molec. Phylogenet. Evol. 127: 203–216.

Xu, K.-W., Zhang, L., Lu, T.N., Zhou, X.-M., He, H., Luong, T.T., Knapp, R., Liao W.-B., Zhang, L.-B., 2018b. Nine new species of Hymenasplenium (Aspleniaceae) from Asia. Phytotaxa 358, 1–25. Yatabe, Y., Murakami, N., 2003. Recognition of cryptic species in the Asplenium nidus complex using molecular data — a progress report. Telopea 10(1): 487–496. Zhang, L., Lu, N.T., Zhang, L.-B., 2015. Leptochilus oblongus (Polypodiaceae), a new fern species from northern Vietnam. Phytotaxa 234, 195–198. Zhang, L.-B., Simmons, M.P., 2006. Phylogeny and delimitation of the Celastrales inferred from nuclear and plastid genes. Syst. Bot. 31, 122–137. Zhang, L.-B., Comes, H.P., Kadereit, J.W., 2001. Phylogeny and quaternary history of the European montane/alpine endemic Soldanella (Primulaceae) based on ITS and AFLP variation. Am. J. Bot. 88, 2331–2345. Zhang, X.-C., Nooteboom, H.P. 2013. Leptochilus. Pp. 833–838 in Wu, Z.-Y., Raven, P.H., Hong, D.-Y. (eds.). Flora of China, vol. 2–3, Lycopodiaceae through Polypodiaceae. Science Press & Missouri Botanical Garden Press, Beijing & St. Louis. Zhou, X.-M., Zhang, L., Chen, C.-W., Li, C.-X., Huang, Y.-M., Chen, D.-K., Lu, N.T., Cicuzza, D., Knapp, R., Luong, T.T., Nitta, J.H., Gao, X.-F., Zhang, L.-B., 2017. A plastid phylogeny and character evolution of the Old World fern genus Pyrrosia (Polypodiaceae) with the description of a new genus: Hovenkampia (Polypodiaceae). Molec. Phylogenet. Evol. 114, 271–294. Zurawski, G., Clegg, M.T., Brown, A.H.D., 1984. The nature of nucleotide sequence divergence between barley and maize chloroplast DNA. Genetics 106, 735–749.

Table 1 Data matrices and tree statistics for each of the analyses. Missing data include missing sequences, uncertain bases (R, A or G; Y, C or T; S, G or C; W, A or T; K, G or T; M, A or C) and gaps (–). PI, parsimony-informative; MPT, most parsimonious trees; CI, consistency index; RI, retention index. a

Inclusive of outgroups.

matrix

# accessions

# chars.

# PI chars. (%)a

MPT length

missing data (%)

CI

RI

Plastid atpB gene

78

1230

130(10.6)

374

54.3%

0.5909

0.8541

Plastid rbcL gene

135

1327

234(17.6)

861

13.1%

0.4413

0.8106

Plastid rps4 gene + rps4-trnS spacer

130

1050

350(33.3)

1129

21.9%

0.5128

0.8538

Plastid trnL intron + trnL-F spacer

115

1017

324(31.9)

1050

54.9%

0.5171

0.8254

Simultaneous

146

4624

1038(22.4)

3514

35.2%

0.4903

0.8253

Table 2. Best-fitting models and parameter values for separate (atpB, rbcL, rps4-trnS, trnL, trnL-F, and trnL & trnL-F) and simultaneous plastid datasets in this study. “G” = gamma distribution shape parameter (Yang, 1994). “GTR” = general-time-reversible model (Tavaré, 1986). “I” = proportion of invariable sites. “Ti/Tv” = transition/transversion ratio.

Region

AIC selected

Base frequencies

model

Substitution model (rate matrix)

I

A

C

G

T

A–C

A–G

A–T

C–G

C–T

G–T

Ti/tv

G

atpB gene

TIM1+I+G

0.2702

0.2158

0.2538

0.2603

1.0000

18.6745

0.4435

0.4435

43.9578

1.0000

–

0 0.6500

rbcL gene

TIM2ef+I+G

-

-

-

-

2.0640

13.8543

2.0640

1.0000

20.3971

1.0000

–

0 0.6640

rps4 gene+rps4-trnS space

TVM+I+G

0.3053

0.1947

0.1938

0.3062

0.7075

4.2613

0.1614

0.3891

4.2613

1.0000

–

0 0.8800

trnL intron+trnL-F spacer

TPM1uf+I+G

0.2989

0.2195

0.2073

0.2744

1.0000

7.8573

0.3673

0.3673

7.8573

1.0000

–

0 1.0450

simultaneous

GTR+I+G

0.2839

0.2839

0.2296

0.2742

1.1413

7.0846

0.4833

0.5119

9.8900

1.0000

–

0 0.7450

Figure legends Fig. 1. Habit of 11 species of Leptochilus representing five of the six major clades identified in the current study. —A. Leptochilus cf. macrophyllus (the L. macrophyllus clade; photo credit: Cheng-Wei Chen). —B. L. pteropus (the L. pteropus Clade). —C. L. cf. axillaris (the Colysis Clade). —D. L. cf. decurrens (the Colysis Clade). —E. L. hemionitideus (the Colysis Clade). —F. L. pentaphyllus (the L. pentaphyllus Clade). —G. L. flexilobus (the L. ellipticus Clade). —H. L. henryi (the L. ellipticus Clade). —I. L. wrightii (the L. ellipticus Clade). —J. L. digitatus (the L. ellipticus Clade). —K. L. saxicola (the L. ellipticus Clade; photo credit: Qi Wei.). Fig. 2. Maximum likelihood phylogeny of Leptochilus based on six plastid markers (atpB, rbcL, rps4, rps4-trnS, trnL, trnL-F). Maximum likelihood bootstrap support (MLBS), maximum parsimony jackknife support (MPJK), and Bayesian inference posterior probability (BIPP) are on the left, middle, and right along the branches, Voucher information is indicated in blue. Geographical provenances are indicated in red. Black vertical bars on the rightmost indicate the six major clades in Leptochilus, while other vertical bars in green indicate the subclades. Species names with larger and bold fonts indicate the types of Colysis, Kontumia, and Paraleptochilus which are included in parentheses.

Appendix List of taxa sampled with information related to taxonomy, GenBank accession numbers, references, and voucher information. Herbarium acronyms follow Index Herbariorum (Thiers, 2015). Drymotaenium miyoshianum Makino, Liu C.C. DB06104 (PE), Sichuan, China: rbcL GQ256255 (Wang et al., 2010), trnL-F spacer GQ256172 (Wang et al., 2010), rps4-trnS GQ256327 (Wang et al., 2010). Goniophlebium argutum (Wall. ex Hook.) J. Sm. ex Hook., Wu S.K. et al. WS-2440 (KUN), Champasak Province, Laos: atpB JX103667 (Kim et al., 2013), rbcL JX103709 (Kim et al., 2013), trnL-F spacer JX103793 (Kim et al., 2013), rps4-trnS JX103751 (Kim et al., 2013). Goniophlebium formosanum (Baker) Rodl-Linder, Ranker 1998 (COLO), Taiwan Island: atpB EF463495 (Schuettpelz & Pryer, 2007), rbcL EF463249 (Schuettpelz & Pryer, 2007). Kontumia heterophylla S.K. Wu & K.L. Phan, Wu S.K. et al. WP-135 (KUN), Vietnam: atpB JX103646 (Kim et al., 2013), rbcL JX10 3688 (Kim et al., 2013), trnL-F spacer JX103772 (Kim et al., 2013), rps4-trnS JX103730 (Kim et al., 2013); Wu S.K. et al. WP-136 (KUN), Vietnam: atpB JX520931 (Kim et al., 2013), rbcL JX520933 (Kim et al., 2013), trnL-F spacer JX520937 (Kim et al., 2013), rps4-trnS JX520935 (Kim et al., 2013); Wu S.K. et al. WP-201 (KUN), Vietnam: atpB JX520932 (Kim et al., 2013), rbcL JX520934 (Kim et al., 2013), trnL-F spacer JX520938 (Kim et al., 2013), rps4-trnS JX520936 (Kim et al., 2013). Lemmaphyllum microphyllum C. Presl, Schneider s.n. (GOET), Cult. BGZ: rbcL EU482938 (Kreier et al., 2008), rps4-trnS EU482988 (Kreier et al., 2008). Lepidogrammitis diversa (Rosenst.) Ching, Zhang X.C. 1854 (PE), China: rbcL EU482939 (Kreier et al.,

2008), trnL-F spacer EU483034 (Kreier et al., 2008), rps4-trnS EU482989 (Kreier et al., 2008). Lepidomicrosorium buergerianum (Miq.) Ching & K.H. Shing, Lu S.G./QC14 (PYU), Sichuan, China: rbcL EU363247 (Dong et al., 2008), rps4-trnS EU363260 (Dong et al., 2008). Lepisorus clathratus (C.B. Clarke) Ching, Dickore 12430 (GOET), Xizang, China: rbcL DQ642154 (Janssen et al., 2007), rps4-trnS DQ642192 (Janssen et al., 2007). Lepisorus macrosphaerus (Baker) Ching, Kim 2012-3 (KUN), Cult. KBG, China: atpB JX103655 (Kim et al., 2013), rbcL JX103697 (Kim et al., 2013), trnL-F spacer JX103781 (Kim et al., 2013), rps4-trnS JX103739 (Kim et al., 2013). Lepisorus scolopendrius (Buch.-Ham. ex D.Don) Mehra & Bir, Wu S.K. et al. 2441 (KUN), Laos: atpB JX103656 (Kim et al., 2013), rbcL JX103698 (Kim et al., 2013), trnL-F spacer JX103782 (Kim et al., 2013), rps4-trnS JX103740 (Kim et al., 2013). Leptochilus × simplicifrons (Christ) Nakaike, Zhang X.C. 3800 (PE), Cult.: rbcL EU482953 (Kreier et al., 2008), trnL-F spacer EU483049 (Kreier et al., 2008), rps4-trnS EU483003 (Kreier et al., 2008). Leptochilus axillaris (Cav.) Kaulf., Walker 11557 (BM), Java, Indonesia: trnL-F spacer EU483040 (Kreier et al., 2008). Leptochilus cantoniensis (Baker) Ching, Dong S.Y. 1034 (IBSC), Hainan, China: rbcL EU363245 (Dong et al., 2008), rps4-trnS EU363258 (Dong et al., 2008); Dong S.Y. 172 (PE), Hainan, China: rbcL EU482945 (Kreier et al., 2008), trnL-F spacer EU483041 (Kreier et al., 2008), rps4-trnS EU482995 (Kreier et al., 2008); Dong S.Y. 743 (PE), Hainan, China: rbcL EU482946 (Kreier et al., 2008), trnL-F spacer EU483042 (Kreier et al., 2008), rps4-trnS EU482996 (Kreier et al., 2008). Leptochilus cf. axillaris, Deng Y.F. 3485 (CDBI), Kachin State, Myanmar: trnL intron & trnL-F spacer MH768580 (this study), rps4-trnS MH768521 (this study). Leptochilus cf. axillaris, Zhang et al. 7047 (CDBI, MO, VNMN), Thanh Hoa, Vietnam: atpB MH768377 (this study), rbcL MH768434 (this study), trnL intron & trnL-F spacer MH768562 (this study), rps4-trnS MH768499 (this study). Leptochilus cf. decurrens, Du X.Y. 1003(KUN), cult. in KBG: trnL intron & trnL-F spacer HQ597016 (Du & Chen, 2011). Leptochilus cf. decurrens, Kim 2012-12(KUN), cult. from Yunnan, China: rbcL JX103724 (Kim et al., 2013), trnL-F spacer JX103808 (Kim et al., 2013), rps4-trnS JX103766 (Kim et al., 2013). Leptochilus cf. decurrens, Knapp 4128 (P), Taiwan Island: atpB MH768402 (this study), rbcL

MH768463 (this study), trnL intron & trnL-F spacer MH768587 (this study), rps4-trnS MH768528 (this study). Leptochilus cf. decurrens, Lu S.G./J16 (PYU), Yunnan (Xichou), China: rbcL EU363246 (Dong et al., 2008), rps4-trnS EU363259 (Dong et al., 2008). Leptochilus cf. decurrens, Wu S.K. et al. WS-2344 (KUN), Salavan Province, Laos: atpB JX103657 (Kim et al., 2013), rbcL JX103699 (Kim et al., 2013), trnL-F spacer JX103783 (Kim et al., 2013), rps4-trnS JX103741 (Kim et al., 2013). Leptochilus cf. decurrens, Zhang et al. 6302 (CDBI, MO, VNMN), Hoa Binh, Vietnam: atpB MH768353 (this study), rbcL MH768413 (this study), trnL intron & trnL-F spacer MH768541 (this study), rps4-trnS MH768475 (this study). Leptochilus cf. decurrens, Zhang et al. 7986 (CDBI, MO, VNMN), Quang Nam, Vietnam: atpB MH768354 (this study), rbcL MH768414 (this study), trnL intron & trnL-F spacer MH768542(this study), rps4-trnS MH768476 (this study). Leptochilus cf. flexilobus, Zhang et al. 6710 (CDBI, MO, VNMN), Bac Kan, Vietnam: atpB MH768357 (this study), rbcL MH768417 (this study), trnL intron & trnL-F spacer MH768545 (this study), rps4-trnS MH768479 (this study). Leptochilus cf. hemitoma, Lu S.G./S15 (PYU), Fujian, China: rbcL EU363236 (Dong et al., 2008), rps4-trnS EU363252 (Dong et al., 2008). Leptochilus cf. hemitomus, Zhang et al. 6484 (CDBI, MO, VNMN), Hanoi, Vietnam: atpB

MH768367 (this study), rbcL MH768427 (this study), trnL intron & trnL-F spacer MH768555 (this study), rps4-trnS MH768489 (this study). Leptochilus cf. longipes, Wade 1594 (TAIF), Lam Dong, Vietnam: atpB MH768390 (this study), rbcL MH768448 (this study), trnL intron & trnL-F spacer

MH768574 (this study), rps4-trnS MH768512 (this study). Leptochilus cf. longipes, Wade 3697 (TAIF), Khanh Hoa, Vietnam: rbcL MH768451 (this study), trnL intron & trnL-F spacer MH768577 (this study), rps4-trnS MH768515 (this study). Leptochilus cf. longipes, Zhang et al. 8727 (CDBI), Khanh Hoa, Vietnam: atpB MH768407 (this study), rbcL MH768468 (this study), trnL intron & trnL-F spacer

MH768592 (this study), rps4-trnS MH768533 (this study). Leptochilus cf. longipes, Zhang et al. 8768 (CDBI, MO), Khanh Hoa, Vietnam: atpB MH768396 (this study), rbcL MH768456 (this study), trnL intron & trnL-F spacer MH768579 (this study), rps4-trnS MH768520 (this study). Leptochilus cf. macrophyllus, Wade 1772 (TAIF), West Java, Indonesia: atpB MH768389 (this study), rbcL MH768447 (this study), trnL intron & trnL-F spacer MH768573 (this study), rps4-trnS MH768511 (this study). Leptochilus cf. macrophyllus, Wade 2776 (TAIF), Kolombangara, Solomon Islands: rbcL MH768473 (this study), trnL intron & trnL-F spacer MH768597 (this study), rps4-trnS MH768538 (this study). Leptochilus cf. macrophyllus, Wade 3076 (TAIF), Rendova, Solomon Islands: rbcL MH768472 (this study), trnL intron & trnL-F spacer MH768596 (this study), rps4-trnS MH768537 (this study). Leptochilus cf. macrophyllus, Wade 3798 (TAIF), Negros Oriental, Philippines: atpB MH768395 (this study), rbcL MH768455 (this study), trnL intron & trnL-F spacer MH768578 (this study), rps4-trnS MH768519 (this study). Leptochilus cf. pedunculatus, Zhang et al. 6334 (CDBI, MO, VNMN), Hoa Binh, Vietnam: atpB MH768385 (this study), rbcL MH768442 (this study), trnL intron & trnL-F spacer MH768570 (this study), rps4-trnS MH768506 (this study). Leptochilus cf. pedunculatus, Zhang et al. 6378 (CDBI, MO, VNMN), Hoa Binh, Vietnam: atpB MH768370 (this study), rbcL MH768430 (this study), trnL intron & trnL-F spacer MH768558 (this study), rps4-trnS MH768492 (this study). Leptochilus cf. pedunculatus, Zhang et al. 6692 (CDBI, MO, VNMN), Bac Kan, Vietnam: atpB MH768373 (this study), rbcL MH768432 (this study), trnL intron & trnL-F spacer MH768560 (this study), rps4-trnS MH768495 (this study). Leptochilus cf. pedunculatus, Zhang et al. 6931 (CDBI, MO, VNMN), Ha Giang, Vietnam: atpB MH768376 (this study), rbcL MH768433 (this study), trnL intron & trnL-F spacer MH768561 (this study), rps4-trnS MH768498 (this study). Leptochilus chingii Li Bing Zhang & Liang Zhang, Zhang et al. 7453 (CDBI, MO, VNMN), Quang Binh, Vietnam: atpB MH768380 (this study), rbcL MH768437 (this study), trnL intron & trnL-F spacer MH768565 (this study), rps4-trnS MH768502 (this study). Leptochilus decurrens Blume, Douglas 28 (UC), Java, Indonesia: rps4-trnS AY096228 (Schneider et al., 2002), Wade 1769 (TAIF), West Java, Indonesia: atpB MH768401 (this study), rbcL MH768462 (this study), trnL intron & trnL-F spacer MH768586 (this study), rps4-trnS MH768527 (this study). Leptochilus digitatus (Baker) Noot., A.R. Smith 00-036 (UC), Vietnam: rbcL EU482948 (Kreier et al., 2008), trnL-F spacer EU483044 (Kreier et al., 2008), rps4-trnS EU482998 (Kreier et al., 2008); Lu S.G./V99 (PYU), Hainan, China: rbcL EU363232 (Dong et al., 2008), rps4-trnS EU363250 (Dong et al., 2008); Wu S.K. et al. WS-2515 (KUN), Bolikhamsai Province, Laos: atpB JX103653 (Kim et al., 2013), rbcL JX103695 (Kim et al., 2013), trnL-F spacer JX103779 (Kim et al., 2013), rps4-trnS JX103737 (Kim et al., 2013); Zhang et al. 6443 (CDBI, MO, VNMN), Vinh Phuc, Vietnam: atpB MH768355 (this study), rbcL MH768415 (this study), trnL intron & trnL-F spacer

MH768543 (this study), rps4-trnS MH768477 (this study); Zhang et al. 6487 (CDBI, MO, VNMN), Hanoi, Vietnam: atpB MH768356 (this study), rbcL MH768416 (this study), trnL intron & trnL-F spacer MH768544 (this study), rps4-trnS MH768478 (this study); Zhang et al. 6543 (CDBI, MO, VNMN), Phu Tho, Vietnam: atpB MH768372 (this study), rps4-trnS MH768494 (this study); Zhang et al. 6779 (CDBI, MO, VNMN), Bac Kan, Vietnam: atpB MH768374 (this study), rps4-trnS MH768496 (this study); Zhang

et al. 6850 (CDBI, MO, VNMN), Cao Bang, Vietnam: atpB MH768375 (this study), rps4-trnS

MH768497 (this study); Zhang et al. 7199 (CDBI, MO, VNMN), Ha Tinh, Vietnam: atpB MH768378 (this study), rbcL MH768435 (this study), trnL intron & trnL-F spacer MH768563 (this study), rps4-trnS MH768500 (this study); Zhang et al. 7250 (CDBI, MO, VNMN), Quang Binh, Vietnam: atpB MH768358 (this study), rbcL MH768418 (this study), trnL intron & trnL-F spacer MH768546 (this study), rps4-trnS MH768480 (this study); Zhang et al. 7359 (CDBI, MO, VNMN), Quang Binh, Vietnam: atpB MH768409 (this study), rbcL MH768470 (this study), trnL intron & trnL-F spacer MH768594 (this study), rps4-trnS MH768535 (this study); Zhang et al. 8010 (CDBI, MO, VNMN), Quang Nam, Vietnam: atpB MH768382 (this study), rbcL MH768439 (this study), trnL intron & trnL-F spacer MH768567 (this study), rps4-trnS MH768503 (this study). Leptochilus dissimilialatum (Bonap.) Li Bing Zhang & Liang Zhang, Zhang et al. 6362 (CDBI, MO, VNMN), Hoa Binh, Vietnam: atpB MH768359 (this study), rbcL MH768419 (this study), trnL intron & trnL-F spacer MH768547 (this study), rps4-trnS MH768481 (this study); Zhang et al. 6601 (CDBI, MO, VNMN), Lang Son, Vietnam: atpB MH768360 (this study), rbcL MH768420(this study), trnL intron & trnL-F spacer MH768548 (this study), rps4-trnS MH768482 (this study); Zhang et al. 6943 (CDBI, MO, VNMN), Ha Giang, Vietnam: atpB MH768364 (this study), rbcL MH768424 (this study), trnL intron & trnL-F spacer MH768552 (this study), rps4-trnS MH768486 (this study). Leptochilus ellipticus (Thunb. ex Murray) Noot., TNS774822 (TNS), Mie Pref., Japan: rbcL AB575243 (Ebihara et al., 2010); Wade 3656 (TAIF), Okinawa, Japan: atpB MH768387 (this study), rbcL MH768444 (this study), trnL intron & trnL-F spacer MH768572 (this study), rps4-trnS MH768508 (this study). Leptochilus evrardii (Tardieu) Li Bing Zhang & Liang Zhang, Wu S.K. et al. WS-2439 (KUN), Champasak Province, Laos: atpB JX103658 (Kim et al., 2013), rbcL JX103700 (Kim et al., 2013), trnL-F spacer JX103784 (Kim et al., 2013), rps4-trnS JX103742 (Kim et al., 2013); Wu S.K. et al. WS-2462 (KUN), Champasak Province, Laos: atpB JX103659 (Kim et al., 2013), rbcL JX103701 (Kim et al., 2013), trnL-F spacer JX103785 (Kim et al., 2013), rps4-trnS JX103743 (Kim et al., 2013); Zhang et al. 8514 (CDBI, MO), Lam Dong, Vietnam: atpB MH768399 (this study), rbcL MH768460 (this study), trnL intron & trnL-F spacer MH768584 (this study), rps4-trnS MH768525 (this study); Zhang et al. 8633 (CDBI, MO), Lam Dong, Vietnam: atpB MH768400 (this study), rbcL MH768461 (this study), trnL intron & trnL-F spacer MH768585 (this study), rps4-trnS MH768526 (this study). Leptochilus flexilobus (Christ) Li Bing Zhang & Liang Zhang, DJY04059 (CDBI), Sichuan, China: atpB MH768361 (this study), rbcL MH768421 (this study), trnL intron & trnL-F spacer MH768549 (this study), rps4-trnS MH768483 (this study); Lu S.G./EM17 (PYU), Sichuan, China: rbcL EU363234 (Dong et al., 2008); Wu S.K. et al. WS-2712 (KUN), Luang Namtha Province, Laos: atpB JX103654 (Kim et al., 2013), rbcL JX103696 (Kim et al., 2013), trnL-F spacer JX103780 (Kim et al., 2013), rps4-trnS JX103738 (Kim et al., 2013); Zhang et al. 527 (CDBI), Guizhou, China: atpB MH768397 (this study), rbcL MH768458 (this study), trnL intron & trnL-F spacer MH768582 (this study), rps4-trnS MH768523 (this study). Leptochilus hemionitideus (C. Presl) Noot., FN43 (CDBI), China: atpB MH768405 (this study), rbcL MH768466 (this study), trnL intron & trnL-F spacer MH768590 (this study), rps4-trnS MH768531 (this study); Knapp 3832 (P), Taiwan Island: rbcL MH768445 (this study), rps4-trnS MH768509 (this study); Wu S.K. et al. WS-2437 (KUN), Champasak Province, Laos: atpB JX103652 (Kim et al., 2013), rbcL JX103694 (Kim et al., 2013), trnL-F spacer JX103778 (Kim et al., 2013), rps4-trnS JX103736 (Kim et al., 2013); Zhang et al. 8038 (CDBI, MO, VNMN), Quang Nam, Vietnam: atpB MH768383 (this study), rbcL MH768440 (this study), trnL intron & trnL-F spacer MH768568 (this study), rps4-trnS MH768504 (this study); Zhang et al. 8317

(CDBI, MO), Lam Dong, Vietnam: atpB MH768403 (this study), rbcL MH768464 (this study), trnL intron & trnL-F spacer MH768588 (this study), rps4-trnS MH768529 (this study); Zhang et al. 8763 (CDBI, MO), Khanh Hoa, Vietnam: atpB MH768404 (this study), rbcL MH768465 (this study), trnL intron & trnL-F spacer MH768589 (this study), rps4-trnS MH768530 (this study); Zhang Liang 1256 (CDBI), Guangdong, China: atpB MH768365 (this study), rbcL MH768425 (this study), trnL intron & trnL-F spacer MH768553 (this study), rps4-trnS MH768487 (this study); HGX 13253 (CDBI), Sichuan, China: atpB MH768366 (this study), rbcL MH768426 (this study), trnL intron & trnL-F spacer

MH768554 (this study), rps4-trnS MH768488 (this study); Zhang X.C. 3302 (PE), China: rbcL EU482951 (Kreier et al., 2008), trnL-F spacer EU483047 (Kreier et al., 2008), rps4-trnS EU483001 (Kreier et al., 2008). Leptochilus henryi (Baker) X.C. Zhang, DJY04047 (CDBI), Sichuan, China: atpB

MH768368 (this study), rbcL MH768428 (this study), trnL intron & trnL-F spacer MH768556 (this study), rps4-trnS MH768490 (this study); Lu S.G./EM1 (PYU), Sichuan, China: rbcL EU363237 (Dong et al., 2008), rps4-trnS EU363253 (Dong et al., 2008); Lu S.G./Q8 (PYU), Zhejiang, China: rbcL EU363238 (Dong et al., 2008); Zhang et al. 9289 (CDBI), Guizhou, China: atpB MH768398 (this study), rbcL

MH768459 (this study), trnL intron & trnL-F spacer MH768583 (this study), rps4-trnS MH768524 (this study); Zhang X.C. 2541 (PE), China: rbcL EU482952 (Kreier et al., 2008), trnL-F spacer EU483048 (Kreier et al., 2008), rps4-trnS EU483002 (Kreier et al., 2008). Leptochilus leveillei (Christ) X.C. Zhang & Noot., Lu S.G./EM26 (PYU), Sichuan, China: rbcL EU363240 (Dong et al., 2008), rps4-trnS EU363254 (Dong et al., 2008). Leptochilus longipes (Ching) X.C. Zhang, Lu S.G./V56 (PYU), Hainan, China: rbcL EU363241 (Dong et al., 2008). Leptochilus macrophyllus (Blume) Noot., Wade 1962 (TAIF), West Java, Indonesia: rbcL MH768449 (this study), trnL intron & trnL-F spacer MH768575 (this study), rps4-trnS MH768513 (this study). Leptochilus oblongus Li Bing Zhang, Liang Zhang & N.T. Lu, Zhang et al. 6299 (CDBI, MO, VNMN), Hoa Binh, Vietnam: atpB MH768369 (this study), rbcL MH768429 (this study), trnL intron & trnL-F spacer MH768557 (this study), rps4-trnS MH768491 (this study). Leptochilus pentaphyllus (Baker) Li Bing Zhang & Liang Zhang, Lu S.G./SG9 (PYU), Ruili, Yunnan, China: rbcL EU363242 (Dong et al., 2008); Zhang Liang 1777 (KUN), Yingjiang, Yunnan, China: atpB MH768411 (this study), rbcL MH768474 (this study), trnL intron & trnL-F spacer MH768599 (this study), rps4-trnS MH768539 (this study). Leptochilus pteropus (Blume) Fraser-Jenk., Kreier s.n. (GOET), Cult. BGG: rbcL EU482965 (Kreier et al., 2008), trnL-F spacer EU483061 (Kreier et al., 2008), rps4-trnS EU483016 (Kreier et al., 2008); TNS759285(TNS), Okinawa Pref., Japan: rbcL AB575246 (Ebihara et al., 2010); unknown resource cult. at BGUG: rbcL AY362565 (Schneider et al., 2004); Zhang et al. 8014 (CDBI, MO, VNMN), Quang Nam, Vietnam: atpB MH768352 (this study), rbcL MH768412 (this study), trnL intron & trnL-F spacer MH768540 (this study); Zhang et al. 8287 (CDBI, MO), Dong Nai, Vietnam: trnL intron & trnL-F spacer MH768598 (this study). Leptochilus saxicola (H.G. Zhou & H. Li) Li Bing Zhang & Liang Zhang, Wei Q. 2017, Guangxi, China: atpB MH768410 (this study), rbcL MH768471 (this study), trnL intron & trnL-F spacer MH768595 (this study), rps4-trnS MH768536 (this study); Zhang et al. 6772 (CDBI, MO, VNMN), Bac Kan, Vietnam: atpB MH768384 (this study), rbcL MH768441 (this study), trnL intron & trnL-F spacer MH768569 (this study), rps4-trnS MH768505 (this study). Leptochilus shintenensis (Hayata) X.C. Zhang & Noot., Knapp 3874 (P), Taiwan Island: atpB MH768394 (this study), rbcL MH768454 (this study), rps4-trnS MH768518 (this study). Leptochilus sp., Zhang et al. 7937 (CDBI, MO, VNMN), Da Nang, Vietnam: atpB MH768381 (this study), rbcL MH768438 (this study), trnL intron & trnL-F spacer MH768566 (this study). Leptochilus sp., Knapp 3849 (P), Taiwan Island:

atpB MH768386 (this study), rbcL MH768443 (this study), trnL intron & trnL-F spacer MH768571 (this study), rps4-trnS MH768507 (this study). Leptochilus sp., Knapp 3851 (P), Taiwan Island: atpB

MH768388 (this study), rbcL MH768446 (this study), rps4-trnS MH768510 (this study). Leptochilus sp., Lu S.G./V111 (PYU), Hainan, China: rbcL EU363243 (Dong et al., 2008), rps4-trnS EU363256 (Dong et al., 2008). Leptochilus sp., TNS774819 (TNS), Tokyo Pref., Japan: rbcL AB575245 (Ebihara et al., 2010). Leptochilus sp., Wade 3681 (TAIF), Ishigaki island, Okinawa, Japan: atpB MH768408 (this study), rbcL MH768469 (this study), trnL intron & trnL-F spacer MH768593 (this study), rps4-trnS MH768534 (this study). Leptochilus sp., Zhang et al. 8596 (CDBI), Lam Dong, Vietnam: atpB MH768406 (this study), rbcL MH768467 (this study), trnL intron & trnL-F spacer MH768591 (this study), rps4-trnS MH768532 (this study). Leptochilus sp., Zhang et al. 512 (CDBI), Guizhou, China: rbcL MH768457 (this study), trnL intron & trnL-F spacer MH768581 (this study), rps4-trnS MH768522 (this study). Leptochilus sp., Zhang et al. 5569 (CDBI), Guangxi, China: atpB MH768363 (this study), rbcL MH768423 (this study), trnL intron & trnL-F spacer MH768551 (this study), rps4-trnS MH768485 (this study). Leptochilus sp., Zhang et al. 6377 (CDBI, MO, VNMN), Hoa Binh, Vietnam: atpB MH768362 (this study), rbcL MH768422 (this study), trnL intron & trnL-F spacer MH768550 (this study), rps4-trnS MH768484 (this study). Leptochilus sp., Zhang et al. 6402 (CDBI, MO, VNMN), Vinh Phuc, Vietnam: atpB MH768371 (this study), rbcL MH768431 (this study), trnL intron & trnL-F spacer MH768559 (this study), rps4-trnS MH768493 (this study). Leptochilus sp., Zhang et al. 6711 (CDBI, MO, VNMN), Bac Kan, Vietnam: atpB MH768391 (this study), rbcL MH768450 (this study), trnL intron & trnL-F spacer MH768576 (this study), rps4-trnS MH768514 (this study). Leptochilus sp., Zhang et al. 7365 (CDBI, MO, VNMN), Quang Binh, Vietnam: atpB MH768379 (this study), rbcL MH768436 (this study), trnL intron & trnL-F spacer MH768564 (this study), rps4-trnS MH768501 (this study). Leptochilus sp., Zhang X.C. 1923 (PE), Jiangxi, China: rbcL EU482949 (Kreier et al., 2008), trnL-F spacer EU483045 (Kreier et al., 2008), rps4-trnS EU482999 (Kreier et al., 2008). Leptochilus wrightii (Hook. & Baker) X.C. Zhang, Knapp 3834 (P), Taiwan Island: atpB MH768392 (this study), rbcL MH768452 (this study), rps4-trnS MH768516 (this study); Knapp 3850 (P), Taiwan Island: atpB MH768393 (this study), rbcL MH768453 (this study), rps4-trnS MH768517 (this study); Tsutsumi 1067 (CT), Okinawa, Japan: rbcL EU482954 (Kreier et al., 2008), trnL-F spacer EU483050 (Kreier et al., 2008), rps4-trnS EU483004 (Kreier et al., 2008). Microsorum commutatum Copel., A.R. Smith 2901 (UC), Cult. Whitehead: rbcL AY362571 (Schneider et al., 2004), rps4-trnS AY362644 (Schneider et al., 2004). Microsorum dilatatum (Wall. ex Bedd.) Sledge, Lu S.G./I41 (PYU), Yunnan, China: rbcL EU363248 (Dong et al., 2008). Microsorum fortunei (T. Moore) Ching, Kim 2012-5 (KUN), Cult. KBG, China: rbcL JX103702 (Kim et al., 2013), trnL-F spacer JX103786 (Kim et al., 2013), rps4-trnS JX103744 (Kim et al., 2013). Microsorum grossum (Langsd. & Fisch.) S.B.Andrews, Lorence 9155 (DL), Hawaii: rbcL EU482956 (Kreier et al., 2008), trnL-F spacer EU483053 (Kreier et al., 2008), rps4-trnS EU483007 (Kreier et al., 2008). Microsorum insigne (Blume) Copel., Liu 204 (PE), China: rbcL EU482957 (Kreier et al., 2008), trnL-F spacer EU483054 (Kreier et al., 2008), rps4-trnS EU483008 (Kreier et al., 2008); Wu S.K. et al. 2435 (KUN), Laos: atpB JX103661 (Kim et al., 2013), rbcL JX103703 (Kim et al., 2013), trnL-F spacer JX103787 (Kim et al., 2013), rps4-trnS JX103745 (Kim et al., 2013); Zhang 3510 (PE), Hainan, China: rbcL EU482959 (Kreier et al., 2008), trnL-F spacer EU483056 (Kreier et al., 2008), rps4-trnS EU483010 (Kreier et al., 2008). Microsorum lastii (Baker) Tardieu, Perier 7937 (P): rbcL EU482961 (Kreier et al., 2008), trnL-F spacer EU483058 (Kreier et al., 2008), rps4-trnS EU483012 (Kreier et al., 2008). Microsorum linguiforme Copel., Ranker

1176 (UC), New Guinea: trnL-F spacer AY083637 (Haufler et al., 2003), rps4-trnS AY362635 (Haufler et al., 2003). Microsorum membranifolium (R. Br.) Ching, Kim 2012-2 (KUN), Cult. KBG, China: atpB JX103662 (Kim et al., 2013), rbcL JX103704 (Kim et al., 2013), trnL-F spacer JX103788 (Kim et al., 2013), rps4-trnS JX103746 (Kim et al., 2013); Schneider s.n. (GOET), Cult. BGG: rbcL DQ642161 (Janssen et al., 2007), trnL-F spacer DQ642245 (Janssen et al., 2007), rps4-trnS DQ642200 (Janssen et al., 2007). Microsorum musifolium Copel., s.n. (UC), Java, Indonesia: rbcL AF470333 (Haufler et al.,2003), trnL-F spacer AY083636 (Haufler et al., 2003). Microsorum novae-zealandiae Copel., Perrie et al. P20873 (WELT), Thames, New Zealand: rbcL DQ401116 (Schneider et al., 2006), trnL intron & trnL-F spacer DQ401121 (Schneider et al., 2006), rps4-trnS DQ401126 (Schneider et al., 2006). Microsorum papuanum Parris, Schuettpelz 603 (GOET), Cult. BGB: rbcL DQ642162 (Janssen et al., 2007), trnL-F spacer DQ642246 (Janssen et al., 2007), rps4-trnS EU483015 (Kreier et al., 2008). Microsorum pustulatum (G.Forst.) Copel., Perrie et al. P20874 (WELT), New Zealand: rbcL DQ401117 (Schneider et al., 2006), trnL intron & trnL-F spacer DQ401122 (Schneider et al., 2006), rps4-trnS DQ401127 (Schneider et al., 2006); Wu S.K. et al. WS-2506 (KUN), Laos: rbcL JX103705 (Kim et al., 2013), trnL-F spacer JX103789 (Kim et al., 2013), rps4-trnS JX103747 (Kim et al., 2013). Microsorum scandens (G.Forst.) Tindale, Kreier s.n. (GOET), Cult. BGG: rbcL DQ212057 (Schneider et al., 2006), trnL-F spacer DQ179641 (Schneider et al., 2006), rps4-trnS DQ212058 (Schneider et al., 2006). Microsorum scolopendria (Burm. f.) Copel., Rakotondrainibe et al. 6601 (P), Mayotte: rbcL DQ642164 (Janssen et al., 2007), trnL-F spacer DQ642248 (Janssen et al., 2007), rps4-trnS DQ642202 (Janssen et al., 2007). Microsorum spectrum (Kaulf.) Copel., Hoshizaki 1350 (UC), Hawaii: rbcL EU482968 (Kreier et al., 2008), trnL-F spacer EU483065 (Kreier et al., 2008), rps4-trnS EU483019 (Kreier et al., 2008). Microsorum thailandicum Boonkerd & Noot., Schwertfeger s.n. (GOET), Cult. BGG: rbcL EU482969 (Kreier et al., 2008), trnL-F spacer EU483066 (Kreier et al., 2008), rps4-trnS EU483020 (Kreier et al., 2008). Microsorum vieillardii (Mett.) Copel., Schneider s.n. (GOET), Cult. BGD: rbcL DQ179635 (Schneider et al., 2006), trnL-F spacer DQ179645 (Schneider et al., 2006), rps4-trnS DQ179638 (Schneider et al., 2006). Microsorum whiteheadii A.R. Sm. & Hoshiz., Whitehead s.n. (UC), Sumatra: rbcL EU482970 (Kreier et al., 2008), trnL-F spacer EU483067 (Kreier et al., 2008), rps4-trnS EU483021 (Kreier et al., 2008). Microsorum zippelii (Blume) Ching, IN112 (TI), Gunung Gede, Indonesia: rbcL AB232411 (Tsutsumi & Masahiro 2006), trnL-F spacer DQ642249 (Janssen et al., 2007), rps4-trnS DQ642203 (Janssen et al., 2007). Neocheiropteris palmatopedata (Baker) Christ, Kim 2012-1 (KUN), Yunnan, China: atpB JX103664 (Kim et al., 2013), rbcL JX103706 (Kim et al., 2013), trnL-F spacer JX103790 (Kim et al., 2013). Neolepisorus ensatus (Thunb.) Ching, Zhang 3611 (PE), Korea: rbcL GQ256319 (Wang et al., 2010), trnL intron & trnL-F spacer GQ256247 (Wang et al., 2010), rps4-trnS GQ256397. Phymatosorus cuspidatus (D. Don) Pic. Serm., Kim 2012-6 (KUN), Cult. KBG, China: atpB JX103665 (Kim et al., 2013), trnL-F spacer JX103791 (Kim et al., 2013), rps4-trnS JX103749 (Kim et al., 2013). Phymatosorus hainanensis (Noot.) S.G. Lu, Wang 1348 (PE), Cult. SCIB: rbcL EU482960 (Kreier et al., 2008), trnL-F spacer EU483057 (Kreier et al., 2008), rps4-trnS EU483011 (Kreier et al., 2008). Platygyria soulieana (Christ) X.C. Zhang & Q.R. Liu, Zhang 5168 (PE), Sichuan: rbcL GQ256321 (Wang et al., 2010), trnL intron & trnL-F spacer GQ256249 (Wang et al., 2010), rps4-trnS GQ256399 (Wang et al., 2010). Platygyria waltonii (Ching) Ching & S.K. Wu, Zhang 4639 (PE), Xizang, China: rbcL GQ256322 (Wang et al., 2010), trnL intron & trnL-F spacer GQ256250 (Wang et al., 2010), rps4-trnS GQ256400 (Wang et al., 2010). Pyrrosia adnascens (Sw.) Ching, Zhang et al. 7064 (CDBI, MO, VNMN),

Thanh Hoa, Vietnam: rbcL KY931036 (Zhou et al., 2017), trnL intron & trnL-F spacer KY931337 (Zhou et al., 2017). Tricholepidium maculosum (Christ) Ching, Shui 80596 (PE), Yunnan, China: trnL intron & trnL-F spacer GQ256251 (Wang et al., 2010), rps4-trnS GQ256401 (Wang et al., 2010); Zhang X.C. 3100 (PE), China: rbcL EU482974 (Kreier et al., 2008).

Zhang L.B. 527 Guizhou, China Leptochilus flexilobus Wu S.K. WS-2712 Leptochilus flexilobus Luang Namtha, Laos DJY 04059 Sichuan, China Leptochilus flexilobus Lu S.G. EM17 Sichuan, China Leptochilus flexilobus 72/84/* Zhang L.B. 6362 Leptochilus dissimilialatum Hoa Binh, Vietnam 67/63/* Ha Giang, Vietnam Leptochilus dissimilialatum Zhang L.B. 6943 Zhang L.B. 6601 Lang Son, Vietnam Leptochilus dissimilialatum 57/55/* Zhang L.B. 6710 Bac Kan, Vietnam Leptochilus cf. flexilobus Zhang L.B. 6772 Bac Kan, Vietnam Leptochilus saxicola Wei Qi 2017 Leptochilus saxicola Guangxi, China 91/95/* Knapp 3874 Taiwan Island Leptochilus shintenensis 95/85/* Knapp 3834 Taiwan Island Leptochilus wrightii Knapp 3851 Taiwan Island 63/71/- Leptochilus sp. Tsutsumi 1067 Okinawa, Japan Leptochilus wrightii Knapp 3850 Taiwan Island Leptochilus wrightii 53/-/0.87 Zhang X.C. 3302 Leptochilus hemitomus Cult. SCBG, China -/-/0.99 51/57/* HGX 13253 Sichuan, China Leptochilus hemitomus Zhang L.B. 6484 Hanoi, Vietnam Leptochilus cf. hemitomus Zhang L.B. 6543 Leptochilus digitatus Phu Tho, Vietnam 64/56/0.99 */98/* Zhang L.B. 6487 Hanoi, Vietnam Leptochilus digitatus Wu S.K. WS-2515 Leptochilus digitatus Bolikhamsai, Laos Zhang L.B. 6443 Vinh Phuc, Vietnam Leptochilus digitatus Zhang L.B. 6850 Cao Bang, Vietnam Leptochilus digitatus Zhang L.B. 7199 Ha Tinh, Vietnam Leptochilus digitatus Zhang L.B. 6779 Bac Kan, Vietnam Leptochilus digitatus Zhang L.B. 8010 Quang Nam, Vietnam Leptochilus digitatus */99/* -/-/* Zhang L.B. 7359 Quang Binh, Vietnam Leptochilus digitatus 97/96/* Zhang L.B. 7250 Quang Binh, Vietnam Leptochilus digitatus 99/95/* Lu S.G. V99 Hainan, China Leptochilus digitatus Zhang L.B. 7937 Leptochilus digitatus Da Nang, Vietnam A.R. Smith 00-036 Vietnam Leptochilus cf. digitatus Leptochilus sp. Lu S.G. V111 Hainan, China 89/84/* Zhang X.C. 1923 Leptochilus sp. Jiangxi, China */*/* Lu S.G. V56 Hainan, China Leptochilus longipes Leptochilus sp. Zhang L.B. 6402 Vinh Phuc, Vietnam 76/74/0.98 Leptochilus ellipticus Wade 3656 Okinawa, Japan -/-/0.95 */91/* TNS774822 Mie Pref., Japan Leptochilus ellipticus */*/* Lu S.G. S15 Leptochilus cf. hemitomus Fujian, China Zhang X.C. 3800 Leptochilus simplicifrons Cult. JNU, China -/-/0.94 TNS774819 Tokyo Pref., Japan Leptochilus sp. */99/* Knapp 3849 Taiwan Island Leptochilus sp. Wade 3681 Okinawa, Japan Leptochilus sp. 88/90/* Dong S.Y. 172 Hainan, China Leptochilus cantoniensis -/-/* */*/* Dong S.Y. 743 Hainan, China Leptochilus cantoniensis Dong S.Y. 1034 Hainan, China Leptochilus cantoniensis */*/* Zhang L.B. 512 Guizhou, China Leptochilus sp. */*/* Leptochilus sp. Zhang L.B. 5569 Guangxi, China Zhang L.B. 6299 Hoa Binh, Vietnam Leptochilus oblongus 62/69/0.99 Leptochilus henryi Lu S.G. EM1 Sichuan, China -/-/* Zhang L.B. 9289 Leptochilus henryi Guizhou, China Lu S.G. EM26 Sichuan, China Leptochilus leveillei 99/94/* DJY04047 Sichuan, China Leptochilus henryi */99/* Lu S.G. Q8 Leptochilus henryi Zhejiang, China Zhang X.C. 2541 Leptochilus henryi China 72/74/* 99/99/* 86/86/* Zhang L.B. 6334 Hoa Binh, Vietnam Leptochilus cf. pedunculatus 67/75/0.98 Zhang L.B. 6378 Hoa Binh, Vietnam Leptochilus cf. pedunculatus */*/* Leptochilus cf. pedunculatus Zhang L.B. 6692 Bac Kan, Vietnam */*/* Zhang L.B. 6931 Ha Giang, Vietnam Leptochilus cf. pedunculatus Leptochilus chingii Zhang L.B. 7453 Quang Binh, Vietnam Wu S.K. WP-136 Kon Tum, Vietnam 89/89/* */*/* Leptochilus (Kontumia) heterophyllus */*/* Wu S.K. WP-201 Kon Tum, Vietnam Leptochilus (Kontumia) heterophyllus Wu S.K. WP-135 Leptochilus (Kontumia) heterophyllus Kon Tum, Vietnam 99/98/* Leptochilus cf. longipes Wade 1594 Lam Dong, Vietnam Zhang L.B. 8596 Lam Dong, Vietnam Leptochilus sp. 73/-/0.98 Zhang L.B. 8727 Leptochilus cf. longipes Khanh Hoa, Vietnam */*/* Zhang L.B. 8768 Leptochilus cf. longipes Khanh Hoa, Vietnam Wade 3697 Leptochilus cf. longipes Khanh Hoa, Vietnam */92/* Lu S.G. SG9 Leptochilus pentaphyllus Yunnan, China */*/* Zhang Liang 1777 Leptochilus pentaphyllus Yunnan, China Knapp 3832 Taiwan Island Leptochilus (Colysis) hemionitideus Wu S.K. WS-2437 Champasak, Laos Leptochilus (Colysis) hemionitideus Zhang L.B. 8317 Leptochilus (Colysis) hemionitideus Lam Dong, Vietnam 90/94/* Zhang L.B. 8038 Quang Nam, Vietnam Leptochilus (Colysis) hemionitideus 97/*/* Zhang L.B. 8763 Khanh Hoa, Vietnam Leptochilus (Colysis) hemionitideus 97/*/* China FN43 Leptochilus (Colysis) hemionitideus Guangdong, China Leptochilus (Colysis) hemionitideus Zhang Liang 1256 84/65/0.98 Du X.Y. 1003 Yunnan, China Leptochilus cf. decurrens 87/64/0.99 Zhang L.B. 6302 Leptochilus cf. decurrense Hoa Binh, Vietnam 67/-/96/97/* Zhang L.B. 7986 Quang Nam, Vietnam Leptochilus cf. decurrens 93/95/* Wu S.K. WS-2344 Leptochilus cf. decurrens Salavan, Laos 92/83/0.99 Zhang L.B. 8633 Lam Dong, Vietnam Leptochilus evrardii 58/51/98/98/* Zhang L.B. 8514 Lam Dong, Vietnam Leptochilus evrardii Wu S.K. WS-2439 Champasak, Laos Leptochilus evrardii 76/81/* 93/98/* Wu S.K. WS-2462 Champasak, Laos Leptochilus evrardii 94/-/88/75/0.99 Leptochilus (Paraleptochilus) decurrens Wade 1769 Java, Indonesia */*/* Douglas 28 Java, Indonesia Leptochilus (Paraleptochilus) decurrens Knapp 4128 Leptochilus cf. decurrens Taiwan Island */95/* 99/99/* Kim 2012-12 Yunnan, China Leptochilus cf. decurrens Lu S.G. J16 Yunnan, China Leptochilus cf. decurrens 78/-/97/85/0.98 Leptochilus cf. axillaris Deng Y.F. 3485 Kachin State, Myanmar 87/85/0.99 Zhang L.B. 7047 Thanh Hoa, Vietnam Leptochilus cf. axillaris Leptochilus axillaris Java, Indonesia Walker 11557 66/91/0.99 Zhang L.B. 6377 Leptochilus sp. Hoa Binh, Vietnam */*/* Zhang L.B. 7365 Leptochilus sp. Quang Binh, Vietnam Zhang L.B. 6711 Leptochilus sp. Bac Kan, Vietnam Cult. Leptochilus pteropus */*/* Zhang L.B. 8287 Dong Nai, Vietnam Leptochilus peteropus */*/* Cult. BGUG Leptochilus pteropus TNS759285 Okinawa, Japan Leptochilus pteropus Zhang L.B. 8014 Quang Nam, Vietnam Leptochilus pteropus */*/* Wade 2776 Leptochilus cf. macrophyllus Solomon Islands 81/62/0.99 Wade 3076 Leptochilus cf. macrophyllus Solomon Islands */*/* Wade 3798 Leptochilus cf. macrophyllus Negros Oriental, Philippines */*/* Wade 1772 Leptochilus cf. macrophyllus West Java, Indonesia Wade 1962 Leptochilus macrophyllus West Java, Indonesia 66/59/0.85 Microsorum dilatatum -/70/Microsorum insigne */99/* Microsorum insigne 88/79/0.93 Microsorum insigne Phymatosorus hainanensis 57/-/99/*/* Microsorum spectrum Phymatosorus cuspidatus 77/55/0.93 Microsorum membranifolium 99/93/* Microsorum commutatum */*/* Microsorum thailandicum */*/* Microsorum whiteheadii 80/98/* Microsorum punctatum 99/97/* Microsorum musifolium 83/79/0.99 Microsorum scolopendria 99/*/* Microsorum grossum Microsorum papuanum 94/95/* Microsorum zippelii 99/95/* 99/*/* Microsorum fortunei Neolepisorus ensatus 81/88/0.99 Neocheiropteris palmatopedata Lepidomicrosorium Tricholepidium maculosum 95/92/* Tricholepidium maculosum */*/* Lepidogrammitis diversa Lemmaphyllum microphyllum */*/* */87/0.97 Platygyria waltonii */*/* Platygyria soulieana Lepisorus clathratus 56/78/* Lepisorus scolopendrius 77/77/0.99 Lepisorus macrosphaerus Drymotaenium miyoshianum 99/*/* Microsorum pustulatum */99/* Microsorum vieillardii 99/97/* Microsorum novae-zealandiae 98/*/* Microsorum inguiforme Microsorum scandens 98/*/* Microsorum lastii Microsorum membranaceum Goniophlebium argutum Goniophlebium formosanum Pyrrosia adnascens 55/55/0.99

Taiwan subclade

L. cantoniensis subclade

L. ellipticus Clade

L. ellipticus subclade

L. oblongus subclade

L. henryi subclade

Kontumia subclade

Khanhhoa subclade

L. pentaphyllus Clade

Tonkin subclade

Laos subclade

Paraleptochilus subclade Yunnan subclade

Colysis Clade

Colysis subclade

Leptochilus subclade

Leptochilus

Vietnam Clade

L. pteropus Clade

L. macrophyllus Clade

mi cr

os

or

oid

s

89/93/*

75/-/-

*/*/*

0.02

Zhang L.B. 527 Guizhou, China Leptochilus flexilobus Wu S.K. WS-2712 Leptochilus flexilobus Luang Namtha, Laos DJY 04059 Sichuan, China Leptochilus flexilobus Lu S.G. EM17 Sichuan, China Leptochilus flexilobus 72/84/* Zhang L.B. 6362 Leptochilus dissimilialatum Hoa Binh, Vietnam 67/63/* Ha Giang, Vietnam Leptochilus dissimilialatum Zhang L.B. 6943 Zhang L.B. 6601 Lang Son, Vietnam Leptochilus dissimilialatum 57/55/* Zhang L.B. 6710 Bac Kan, Vietnam Leptochilus cf. flexilobus Zhang L.B. 6772 Bac Kan, Vietnam Leptochilus saxicola Wei Qi 2017 Leptochilus saxicola Guangxi, China 91/95/* Knapp 3874 Taiwan Island Leptochilus shintenensis 95/85/* Knapp 3834 Taiwan Island Leptochilus wrightii Knapp 3851 Taiwan Island 63/71/- Leptochilus sp. Tsutsumi 1067 Okinawa, Japan Leptochilus wrightii Knapp 3850 Taiwan Island Leptochilus wrightii 53/-/0.87 Zhang X.C. 3302 Leptochilus hemitomus Cult. SCBG, China -/-/0.99 51/57/* HGX 13253 Sichuan, China Leptochilus hemitomus Zhang L.B. 6484 Hanoi, Vietnam Leptochilus cf. hemitomus Zhang L.B. 6543 Leptochilus digitatus Phu Tho, Vietnam 64/56/0.99 */98/* Zhang L.B. 6487 Hanoi, Vietnam Leptochilus digitatus Wu S.K. WS-2515 Leptochilus digitatus Bolikhamsai, Laos Zhang L.B. 6443 Vinh Phuc, Vietnam Leptochilus digitatus Zhang L.B. 6850 Cao Bang, Vietnam Leptochilus digitatus Zhang L.B. 7199 Ha Tinh, Vietnam Leptochilus digitatus Zhang L.B. 6779 Bac Kan, Vietnam Leptochilus digitatus Zhang L.B. 8010 Quang Nam, Vietnam Leptochilus digitatus */99/* -/-/* Zhang L.B. 7359 Quang Binh, Vietnam Leptochilus digitatus 97/96/* Zhang L.B. 7250 Quang Binh, Vietnam Leptochilus digitatus 99/95/* Lu S.G. V99 Hainan, China Leptochilus digitatus Zhang L.B. 7937 Leptochilus digitatus Da Nang, Vietnam A.R. Smith 00-036 Vietnam Leptochilus cf. digitatus Lu S.G. V111 Leptochilus sp. Hainan, China 89/84/* Zhang X.C. 1923 Leptochilus sp. Jiangxi, China */*/* Lu S.G. V56 Hainan, China Leptochilus longipes Leptochilus sp. Zhang L.B. 6402 Vinh Phuc, Vietnam 76/74/0.98 Leptochilus ellipticus Wade 3656 Okinawa, Japan -/-/0.95 */91/* TNS774822 Mie Pref., Japan Leptochilus ellipticus */*/* Lu S.G. S15 Leptochilus cf. hemitomus Fujian, China Zhang X.C. 3800 Leptochilus simplicifrons Cult. JNU, China -/-/0.94 TNS774819 Tokyo Pref., Japan Leptochilus sp. */99/* Knapp 3849 Taiwan Island Leptochilus sp. Wade 3681 Okinawa, Japan Leptochilus sp. 88/90/* Dong S.Y. 172 Hainan, China Leptochilus cantoniensis -/-/* */*/* Dong S.Y. 743 Hainan, China Leptochilus cantoniensis Dong S.Y. 1034 Hainan, China Leptochilus cantoniensis */*/* Zhang L.B. 512 Guizhou, China Leptochilus sp. */*/* Zhang L.B. 5569 Leptochilus sp. Guangxi, China Zhang L.B. 6299 Hoa Binh, Vietnam Leptochilus oblongus 62/69/0.99 Leptochilus henryi Lu S.G. EM1 Sichuan, China -/-/* Zhang L.B. 9289 Leptochilus henryi Guizhou, China Lu S.G. EM26 Sichuan, China Leptochilus leveillei 99/94/* DJY04047 Sichuan, China Leptochilus henryi */99/* Lu S.G. Q8 Leptochilus henryi Zhejiang, China Zhang X.C. 2541 Leptochilus henryi China 72/74/* 99/99/* 86/86/* Zhang L.B. 6334 Hoa Binh, Vietnam Leptochilus cf. pedunculatus 67/75/0.98 Zhang L.B. 6378 Hoa Binh, Vietnam Leptochilus cf. pedunculatus */*/* Zhang L.B. 6692 Leptochilus cf. pedunculatus Bac Kan, Vietnam */*/* Zhang L.B. 6931 Ha Giang, Vietnam Leptochilus cf. pedunculatus Zhang L.B. 7453 Leptochilus chingii Quang Binh, Vietnam Wu S.K. WP-136 Kon Tum, Vietnam 89/89/* */*/* Leptochilus (Kontumia) heterophyllus */*/* Wu S.K. WP-201 Kon Tum, Vietnam Leptochilus (Kontumia) heterophyllus Wu S.K. WP-135 Leptochilus (Kontumia) heterophyllus Kon Tum, Vietnam 99/98/* Leptochilus cf. longipes Wade 1594 Lam Dong, Vietnam Zhang L.B. 8596 Lam Dong, Vietnam Leptochilus sp. 73/-/0.98 Zhang L.B. 8727 Leptochilus cf. longipes Khanh Hoa, Vietnam */*/* Zhang L.B. 8768 Leptochilus cf. longipes Khanh Hoa, Vietnam Wade 3697 Leptochilus cf. longipes Khanh Hoa, Vietnam */92/* Lu S.G. SG9 Leptochilus pentaphyllus Yunnan, China */*/* Zhang Liang 1777 Leptochilus pentaphyllus Yunnan, China Knapp 3832 Taiwan Island Leptochilus (Colysis) hemionitideus Wu S.K. WS-2437 Champasak, Laos Leptochilus (Colysis) hemionitideus Zhang L.B. 8317 Leptochilus (Colysis) hemionitideus Lam Dong, Vietnam 90/94/* Zhang L.B. 8038 Quang Nam, Vietnam Leptochilus (Colysis) hemionitideus 97/*/* Zhang L.B. 8763 Khanh Hoa, Vietnam Leptochilus (Colysis) hemionitideus 97/*/* China FN43 Leptochilus (Colysis) hemionitideus Guangdong, China Leptochilus (Colysis) hemionitideus Zhang Liang 1256 84/65/0.98 Du X.Y. 1003 Yunnan, China Leptochilus cf. decurrens 87/64/0.99 Zhang L.B. 6302 Leptochilus cf. decurrense Hoa Binh, Vietnam 67/-/96/97/* Zhang L.B. 7986 Quang Nam, Vietnam Leptochilus cf. decurrens 93/95/* Wu S.K. WS-2344 Leptochilus cf. decurrens Salavan, Laos 92/83/0.99 Zhang L.B. 8633 Lam Dong, Vietnam Leptochilus evrardii 98/98/* Zhang L.B. 8514 Lam Dong, Vietnam Leptochilus evrardii Wu S.K. WS-2439 Champasak, Laos Leptochilus evrardii 76/81/* 93/98/* Wu S.K. WS-2462 Champasak, Laos Leptochilus evrardii 94/-/88/75/0.99 Leptochilus (Paraleptochilus) decurrens Wade 1769 Java, Indonesia */*/* Douglas 28 Java, Indonesia Leptochilus (Paraleptochilus) decurrens Knapp 4128 Leptochilus cf. decurrens Taiwan Island */95/* 99/99/* Kim 2012-12 Yunnan, China Leptochilus cf. decurrens Lu S.G. J16 Yunnan, China Leptochilus cf. decurrens 97/85/0.98 Leptochilus cf. axillaris Deng Y.F. 3485 Kachin State, Myanmar 87/85/0.99 Zhang L.B. 7047 Thanh Hoa, Vietnam Leptochilus cf. axillaris Leptochilus axillaris Java, Indonesia Walker 11557 66/91/0.99 Zhang L.B. 6377 Leptochilus sp. Hoa Binh, Vietnam */*/* Zhang L.B. 7365 Leptochilus sp. Quang Binh, Vietnam Zhang L.B. 6711 Leptochilus sp. Bac Kan, Vietnam Cult. Leptochilus pteropus Zhang L.B. 8287 Dong Nai, Vietnam Leptochilus peteropus */*/* Cult. BGUG Leptochilus pteropus TNS759285 Okinawa, Japan Leptochilus pteropus Zhang L.B. 8014 Quang Nam, Vietnam Leptochilus pteropus */*/* Wade 2776 Leptochilus cf. macrophyllus Solomon Islands 81/62/0.99 Wade 3076 Leptochilus cf. macrophyllus Solomon Islands */*/* Wade 3798 Leptochilus cf. macrophyllus Negros Oriental, Philippines */*/* Wade 1772 Leptochilus cf. macrophyllus West Java, Indonesia Wade 1962 Leptochilus macrophyllus West Java, Indonesia 55/55/0.99

Taiwan subclade

L. cantoniensis subclade

L. ellipticus Clade

L. ellipticus subclade

L. oblongus subclade

L. henryi subclade

Kontumia subclade

Khanhhoa subclade

L. pentaphyllus Clade

Tonkin subclade

58/51/-

78/-/-

Laos subclade

Paraleptochilus subclade Yunnan subclade

Colysis Clade

Colysis subclade

Leptochilus subclade

Leptochilus */*/*

Vietnam Clade

L. pteropus Clade

L. macrophyllus Clade

1. A large sampling (105 accessions vs. 2–16 accessions in earlier studies) has been achieved; 2. The species number of the genus is likely to double the most recent estimate following our study; 3. Leptochilus is monophyletic and resolved as nested within the microsoroid ferns; 4. Major evolutionary lineages have been identified; 5. Leptochilus might have evolved at lower latitudes and progressively dispersed to and colonized higher latitudes.