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A large-scale phylogeny of the lycophyte genus Selaginella (Selaginellaceae: Lycopodiopsida) based on plastid and nuclear loci

ARTICLE in CLADISTICS · AUGUST 2015 Impact Factor: 6.22 · DOI: 10.1111/cla.12136

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Cladistics (2015) 1–30 10.1111/cla.12136

A large-scale phylogeny of the lycophyte genus Selaginella (Selaginellaceae: Lycopodiopsida) based on plastid and nuclear loci

Xin-Mao Zhoua,b, Carl J. Rothfelsc,d, Liang Zhanga, Zhao-Rong Hee, Timothee Le Pechona,f, Hai Heg, Ngan Thi Luh, Ralf Knappi, David Lorencej, Xing-Jin Heb, Xin-Fen Gaoa,* and Li-Bing Zhangk,*

aChengdu Institute of Biology, Chinese Academy of Sciences, PO Box 416, Chengdu, Sichuan 610041, China; bSchool of Life Sciences, Sichuan University, Chengdu, Sichuan 610064, China; cDepartment of Zoology, University of British Columbia, #4200-6270 University Blvd, Vancouver, BC V6T 1Z4, Canada; dUniversity Herbarium and Department of Integrative Biology, University of California, Berkeley, CA 94720-2465, USA; eSchool of Life Science, Yunnan University, Kunming, Yunnan 650091, China; fSchool of Life Sciences, University of KwaZulu-Natal, Private Bag X01 Scottsville, Pietermaritzburg 3209, South Africa; gDepartment of Biology, Chongqing Normal University, Shapingba, Chongqing 400047, China; hDepartment of Botany, Vietnam National Museum of Nature, Vietnam Academy of Science and Technology, 18th Hoang Quoc Viet Road, Cau Giay, Ha Noi, Viet Nam; iCorrespondent of the Museum national d’Histoire naturelle (MNHN, Paris, France), Steigestrasse 78, 69412 Eberbach, Germany; jNational Tropical Botanical Garden, 3530 Papalina Road, Kalaheo, HI 96741, USA; kMissouri Botanical Garden, PO Box 299, St Louis, MO 63166-0299, USA Accepted 28 June 2015

Abstract

The lycophyte genus Selaginella alone constitutes the family Selaginellaceae, the largest of the lycophyte families. The genus is estimated to contain 700–800 species distributed on all continents except Antarctica, with highest species diversity in tropical and subtropical regions. The monophyly of Selaginella in this broad sense has rarely been doubted, whereas its intrageneric clas- sification has been notoriously contentious. Previous molecular studies were based on very sparse sampling of Selaginella (up to 62 species) and often used DNA sequence data from one genome. In the present study, DNA sequences of one plastid (rbcL) and one nuclear (ITS) locus from 394 accessions representing approximately 200 species of Selaginella worldwide were used to infer a phylogeny using maximum likelihood, Bayesian inference and maximum parsimony methods. The study identifies strongly supported major clades and well resolves relationships among them. Major results include: (i) six deep-level clades are discovered representing the deep splits of Selaginella; and (ii) 20 major clades representing 20 major evolutionary lineages are identified, which differ from one another in molecular, macro-morphological, ecological and spore features, and/or geographical distribution. © The Willi Hennig Society 2015.

The lycophyte family Selaginellaceae contains only climbing, creeping, prostrate and special rosetting one genus, Selaginella P. Beauv. The largest lycophyte forms. Many species of Selaginella are medicinally genus, Selaginella is cosmopolitan and is estimated to important and are reported as sources of bioactive com- contain 700 (Tryon and Lugardon, 1991), 750 (Jermy, pounds (Banks, 2009; Setyawan, 2011; da Silva Almeida 1990) or ca. 800 species (X.-M. Zhou and L.-B. Zhang, et al., 2013). Recently, the nuclear genome of one spe- unpublished data), which inhabit an impressive range of cies of the genus, Selaginella moellendorffii Hieron., has habitats, including desert, tropical rain forest, and been sequenced. This makes the genus enormously alpine and arctic habitats. Members of the genus also important in studying the evolution of vascular plants, have highly diverse growth forms, including erect, as the species is the only seed-free vascular plant whose nuclear genome is known so far (Banks et al., 2011; *Corresponding authors: Sessa et al., 2014). E-mail addresses: [email protected]; [email protected]

© The Willi Hennig Society 2015 2 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

Selaginella was established by Palisot-Beauvois (1804) tional genera in the family. Rothmaler (1944) divided based on S. selaginoides (L.) P. Beauv. ex Mart. & Selaginella into three genera: Didiclis P. Beauv., Schrank. The genus was characterized by usually having Lycopodioides Boehm. and Selaginella sensu stricto (s.s.). distinct-sized and dorsiventral leaves in four rows, pre- The last two were accepted by some later authors (e.g. senting a ligule at the base of each leaf and sporophyll, Kung, 1988; Sojak, 1993; Tzvelev, 2004). Sojak (1993) and having heterospores (microspores and megaspores) and Weakley (2012) instead recognized Selaginella, and rhizophores (Thomas, 1997). Before Spring’s (1850) Lycopodiodes and Bryodesma Sojak in the family. major monograph of the genus, Selaginella was usually Based on plastid rbcL sequences of 18 species of Se- treated as a synonym of another lycophyte genus, Ly- laginella, Korall et al. (1999) conducted the first copodium L. (Lycopodiaceae) (e.g. Greville and Hooker, molecular analysis of the family, and recovered the 1831). Although the delimitation of Selaginella has monophyly of the genus and S. subg. Selaginella,and rarely been controversial [but see Rothmaler’s (1944), resolved the latter as sister to the rest of species sam- Sojak’s (1993), Kung’s (1988) and Tzvelev’s (2004) clas- pled. Later Korall and Kenrick (2002) increased the sifications below], the intrageneric classification within taxon sampling to 62 species and found support for Selaginella has been notoriously contentious (Spring, the monophyly of “S. subg. Tetragonostachys” too, 1850; Braun, 1857; Hieronymus and Sadebeck, 1901; and identified a so-called “dorsal rhizophoric” clade Walton and Alston, 1938; Rothmaler, 1944; Tryon and that included all species having a distinctive and Tryon, 1982; Jermy, 1986, 1990; Kung, 1988), with taxo- unique root-like structure (the rhizophore) emerging nomic treatments differing dramatically in the number from dorsal surfaces. This later study further discov- of subgenera/sections recognized. In general, previous ered the polyphyly of S. subg. Stachygynandrum and authors tended to divide Selaginella into two primary S. subg. Heterostachys, but their analysis was uncer- subdivisions, including the isophyllous versus aniso- tain about the monophyly of S. subg. Ericetorum. phyllous species, respectively. For example, Spring Using nuclear 26S and rbcL data of 23 species, Korall (1840) divided the genus into three sections: S. sect. Se- and Kenrick (2004) resolved the dorsal rhizophoric laginella (autonym, implied), S. sect. Homoeophyllae clade into two major subclades, although neither of Spring and S. sect. Heterophyllae Spring and similarly which is characterized by a clear morphological Braun (1857) also recognized three sections but named synapomorphy. Most recently, based on nuclear ITS them S. sect. Selaginella, S. sect. Dichotropae (internal transcribed spacer) and rbcL data of 40 A. Braun and S. sect. Homotropae A. Braun, while mainly North American species, Arrigo et al. (2013) Moore (1857) instead used S. sect. Diplostachyum confirmed the monophyly of “S. subg. Tetragonos- (P. Beauv.) T. Moore and S. sect. Stachygynandrum (P. tachys” and resolved it as sister to S. lepidophylla Beauv. ex Mirb.) T. Moore for the latter two. Hierony- (Hook. & Grev.) Spring, a species native to the Chi- mus and Sadebeck (1901) also recognized two groups, huahuan Desert of the USA and Mexico and known but at the rank of subgenus: S. subg. Homoeophyllum for its ability to survive almost complete desiccation. Hieron. & Sadeb. and S. subg. Heterophyllum Hieron. Arrigo et al. (2013) showed that the “dorsal rhi- & Sadeb. Based on morphology (especially that of stro- zophoric’’ clade defined by Korall and Kenrick (2002) bili) and habit forms, Warburg (1900), Walton and is paraphyletic or polyphyletic. Alston (1938) and Jermy (1986, 1990) subdivided the Notably, previous molecular studies focused mainly genus further. Warburg (1900) recognized five subgen- on the two species-poor subgenera only, Selaginella era in the genus: S. subg. Boreoselaginella Warb., subg. Selaginella (2 spp.) and “S. subg. Tetragonos- “S. subg. Euselaginella” (nom. inval., = S. subg. tachys” (ca. 50 spp.), while S. subg. Heterostachys and Selaginella), S. subg. Heterostachys Baker, S. subg. S. subg. Stachygynandrum, which contain a total of Homostachys Baker and S. subg. Stachygynandrum (P. ca. 660 species (Tryon and Lugardon, 1991), were Beauv. ex Mirb.) Baker. Walton and Alston (1938) weakly sampled yet nonetheless were resolved as poly- accepted Warburg’s (1900) subgenera except S. subg. phyletic (Korall et al., 1999; Korall and Kenrick, Boreoselaginella. Jermy (1986) divided the genus into 2002, 2004; Arrigo et al., 2013), and S. subg. Ericeto- five subgenera: S. subg. Selaginella, S. subg. Ericetorum rum (ca. 8 spp., see below) remains poorly known. In Jermy, S. subg. Heterostachys, S. subg. Tetragonos- addition, very few Asian species were included in pre- tachya (Hook. & Grev.) Jermy and S. subg. Stachygy- vious molecular studies, and these studies mainly nandrum. In addition to the different number of tested the five-subgenus classification of Jermy (1986, intrageneric subdivisions recognized by different 1990). Alternative morphological hypotheses of rela- authors, the delimitations of these subdivisions have tionship, e.g. the monophyly of S. subg. Homostachys been controversial and morphological synapomorphies by Baker (1883, 1887), Warburg (1900), and Walton of most subgenera have remained ambiguous. and Alston (1938), have rarely been tested. Previous While most authors agreed that Selaginellaceae authors nicely associated various macro-morphologi- contain only one genus, a few authors recognized addi- cal, ecological, physiological and spore features with Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 3 the monophyletic clades found in their molecular stud- represent geographical ranges and intraspecific varia- ies (Korall et al., 1999; Korall and Kenrick, 2002, tions and to test the monophyly of individual species. 2004; Arrigo et al., 2013). Still, a large number of In total, 394 accessions representing ca. 200 species monophyletic clades were supported by molecular (ca. 30% of the extant diversity of the genus) consti- synapomorphies only, mainly because non-molecular tuted our ingroup. features, e.g. spore morphology, are missing for most The following species were excluded from current species of Selaginella in spite of tremendous efforts so sampling: Selaginella australiensis Baker, S. chuweimin- far (e.g. Hellwig, 1969; Tryon and Lugardon, 1991; gii Xin M. Zhou, Z. R. He, Liang Zhang & Li Bing Korall and Taylor, 2006; Zhou et al., 2015b). Zhang (Zhou et al., 2015a) S. fruticulosa (Bory ex The objectives of this study included: (i) to test the Willd.) Spring, S. obtusa Spring, S. sinensis (Desv.) monophyly of Selaginella using the largest taxon sam- Spring and S. viridula Spring. These species formed a pling so far and both plastid and nuclear data; (ii) to well-supported clade with long branches in the prelimi- resolve the deep-level relationships within Selaginella; nary analysis but there are no clear morphological (iii) to identify major evolutionary lineages within synapomorphies supporting this clade. Korall and Selaginella by including more Asian species; (iv) to Kenrick (2004) already found this clade but with only evaluate the monophyly of S. subg. Heterostachys, S. australiensis and S. sinensis included. Korall and S. subg. Homostachys, S. ser. Rosulatae (A. Braun) Kenrick (2002) postulated possible existence of a pseu- Baker and S. subg. Stachygynandrum and other previ- dogene of rbcL in this clade. More study on these spe- ous morphology-based hypotheses of relationship cies are needed. within Selaginella; and (v) to understand morphologi- Five species of Isoetes€ (Iso€etaceae), I. kersii Wan- cal synapomorphies for the major lineages resolved by ntorp, I. kirkii A. Braun, I. nuttallii A. Braun ex incorporating newly obtained data, especially those Engelm., I. sinensis Palmer and I. taiwanensis De Vol, from spore morphology. were chosen as outgroups based on the previous find- ings that Iso€etaceae are sister to Selaginellaceae (Wik- strom€ and Kenrick, 1997; Korall et al., 1999) and the Materials and methods observations that this sister relationship is supported by the heterospory and ligules in both families (Zhang Taxon sampling and Taylor, 2013; Zhang et al., 2013). Voucher information and GenBank accession To test the morphological hypotheses about the sub- numbers for each sampled taxon are provided in the genera, sections, series, subseries and groups previ- Appendix. ously recognized by various authors (Spring, 1850; Braun, 1857; Baker, 1883, 1887; Warburg, 1900; Morphology Hieronymus and Sadebeck, 1901; Walton and Alston, 1938; Tryon and Tryon, 1982; Jermy, 1986, 1990; Morphological data were obtained from field obser- Kung, 1988; Sojak, 1993; Tzvelev, 2004), two or more vations, herbarium investigations and literature study. species of each of these intrageneric taxa were sam- Field observations were mainly conducted by the first pled. We included as many data as possible from Gen- author in China and other co-authors in various parts Bank to maximize our taxon sampling and of the world (C.J.R. in the New World, by L.Z., additionally to draw attention to potential problems L.-B.Z. and N.T.L. in Vietnam, T.L.P. in La Reunion, (misidentifications) in the GenBank data, and to invite D.L. in the Pacific islands, etc.). Herbarium investiga- further investigations on shallow-level relationships, tions were carried out at herbaria CDBI, KUN, MO, cryptic speciation and potential hybridization. We PYU and SZ (herbarium acronyms follow Index Her- think this sampling strategy is justified because our bariorum by Holmgren and Holmgren, 1998). major interest was to identify major evolutionary lin- eages in the genus and resolve the deep-level relation- Spore morphology ships among these lineages. All newly included accessions for which the vouchers were examined by The microspore and megaspore morphology of ca. us or determined by another authority (e.g. Chinese 65 Asian species of Selaginella was observed using a material by Wei-Ming Chu or Pei-Shan Wang, Ameri- QUANTA 200 scanning electron microscope (FEI can material by Michael Windham and/or Alan R. Co., USA), and the gold–palladium plating was per- Smith) are indicated in red and those from GenBank formed using a BAL-TEC SCD 005 cool sputter are indicated in black in our figures. Information on coater (BAL-TEC AG., Liechtenstein) at Yunnan the geographical provenances of the GenBank acces- University, Kunming, China. Additional spore mor- sions are given in the Appendix as much as possible. phology data were taken from the literature (see refer- Multiple accessions of ca. 130 species were included to ences below). 4 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

DNA extraction, amplification and sequencing

Total genomic DNA was extracted from silica-dried transition/

material or sometimes from herbarium specimens = using a TIANGEN plant genomic DNA extraction kit (TIANGEN Biotech., Beijing, China) or DNeasy Plant 0.29800.0000 1.0890 0.1480 0.3310 0.5230 Mini Kits (Qiagen, Germany) following the manufac- IG turers’ protocols. One plastid region (the rbcL gene) and one nuclear region [the internal transcribed spacer (ITS)] were selected based on their use in earlier studies of Se- – – – laginella and allies (Korall et al., 1999; Korall and

Kenrick, 2002, 2004; Arrigo et al., 2013; Gu et al., T Ti/Tv – 2013; Saha et al., 2013). Most rbcL sequences were amplified with primers F1 (Fay et al., 1997) and

1379R originally designed by Zurawski et al. (1984) TG – and modified by Wolf et al. (1999). Internal primers proportion of invariable sites; “Ti/Tv” rbcL-406F, rbcL-770R (Korall et al., 1999) and newly = designed primer rbcL-449F (50TTGGATACCRTGAG GC GYGGRCC30) were used when amplification was not – successful. The PCR conditions of rbcL regions fol- e, 1986); “I” lowed Zhang et al. (2001) and those of the ITS region TC followed Arrigo et al. (2013). Sometimes the reverse – primer ITS-S3R (Gu et al., 2013) was used in ITS amplification. Amplified fragments were purified with GA

TIANquick Mini Purification kits (TIANGEN). Puri- – fied PCR products were sequenced by Invitrogen (Shanghai, China). CA – Sequence alignment and phylogenetic analysis

Sequencher 4.1 (Gene Codes Corp., Ann Arbor, MI,

USA) was used to assemble and edit complementary general-time-reversible model (Tavar strands. Sequences obtained for each locus were = aligned using Clustal X 1.81 (Thompson et al., 1997) followed by manual adjustments using BioEdit (Hall, 1999). The full length of the ITS region was sequenced in our study. However, as previous studies have and ITS) and simultaneous plastid and nuclear datasets in this study demonstrated (Arrigo et al., 2013; Gu et al., 2013), ITS1 and ITS2 have a large number of inser- rbcL tions and deletions that could not be aligned with confidence. Therefore only 5.8S and part of the unam- ACGTA biguously aligned ITS2 region were used; the ambigu- Base frequencies Substitution model (rate matrix) ous regions were excluded prior to analysis. Equally weighted maximum parsimony (MP) analy- ses were conducted for each locus using 1000 tree- * bisection-reconnection (TBR) searches in PAUP ver. GG 0.2657 0.2533 0.2532 0.2405 0.2417 0.2405 0.2565 0.5475 0.2486 4.6936 0.7070 0.5020 4.7193 0.5300 0.6373 2.2570 0.6157 1.0000 3.2664 1.0000 + + I GI 0.1813 0.2889 0.2702 0.2596 1.0251 3.6505 2.0448 0.7545 4.8722 1.0000 + 4.0b10 (Swofford, 2002) with MAXTREES set to + + increase without limit. Insertions and deletions were coded as missing data. Parsimony jackknife (JK) anal- yses (Farris et al., 1996) were conducted using PAUP* with the removal probability set to approximately

37%, and “jac” resampling emulated. Two hundred gamma distribution shape parameter (Yang, 1994); “GTR” gene GTR replicates were performed with 100 TBR searches per = rbcL ITSSimultaneous GTR “G” GTR replicate and a maximum of 300 trees held per TBR Region AIC selected model transversion ratio. search. Table 1 Best-fitting models and parameter values for separate ( Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 5

Table 2 Data matrices and tree statistics for each of the analyses

No. of PI No. of MP Average MP No. of No. of characters MPT JK/ML BS JK/ML BS Matrix accessions characters (%)* length clades support (%) CI RI

rbcL gene 399 1279 666 4016 228/262 81/81 0.2903 0.9277 ITS 278 217 91 980 90/126 76/79 0.2051 0.8597 Simultaneous 399 1496 757 5611 228/265 82/83 0.2436 0.9056

PI, parsimony-informative; MPT, most parsimonious trees; MP, maximum parsimony; ML, maximum likelihood; JK, jackknife; BS, boot- strap; CI, consistency index; RI, retention index. *Inclusive of outgroups.

jModelTest 0.1.1 (Guindon and Gascuel, 2003; the ML tree based on the concatenated dataset (Fig. 1) Posada, 2008) was used to select the best-fitting likeli- is mostly identical to those based on each individual hood model for maximum likelihood (ML; Felsenstein, marker, but with generally increased support values. 1973) and Bayesian analyses. The Akaike information Based on our reconstructed phylogeny (Figs 1 and criterion (Akaike, 1974) was used to select among 2a–e) and in consideration of macro-morphological, models instead of the hierarchical likelihood ratio test, ecological, and spore features and distribution infor- following Pol (2004) and Posada and Buckley (2004). mation, 17 major evolutionary lineages of Selaginella The best-fitting models and parameter values are pro- were identified (Fig. 1). Fourteen of the 17 clades were vided in Table 1. well supported (ML BS ≥ 91%, MP JK ≥ 96%, PP ≥ For each locus and the combined analysis or the 0.99, except for clade XV with MP JK = 88% and simultaneous analysis (Kluge, 1989; Nixon and Car- PP = 0.89), while one was weakly supported (clade penter, 1996) of all nucleotide characters, ML tree XIV: ML BS = 45%, MP JK < 50%, PP = 0.74), and searches and ML bootstrapping were conducted using two were monospecific (clades IV and V). The rela- the web server RAxML-HPC2 on TG ver. 7.2.8 (Sta- tionships among the 17 clades except those among matakis et al., 2008; Miller et al., 2010) with 5000 clades III–VI and those among XIV–XVII were well rapid bootstrap analyses followed by a search for the resolved and strongly supported. Three additional best-scoring tree in a single run (Stamatakis et al., strongly supported monophyletic superclades within 2008). the genus were identified for descriptive convenience Bayesian inference (BI) was conducted using and for future consideration of taxonomy of the MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001; genus. Superclade A (BS = 100%, JK = 98%, PP = Ronquist and Huelsenbeck, 2003) on Cipres (Miller 1.00) contains clades III–VIII, superclade B (BS = et al., 2010). Four Markov chain Monte Carlo chains 97%, JK = 97%, PP = 0.99) comprises clades X–XIII were run, each beginning with a random tree and sam- and superclade C (BS = 94%, JK = 97%, PP = 1.00) pling one tree every 1000 generations of 10 000 000 is composed of clades XIV–XVII. generations. Convergence among generations was The ML and BI analyses based on the concatenated checked using Tracer (Rambaut and Drummond, dataset resolved the Selaginella sanguinolenta clade (II) 2007) and the first 25% of samples were discarded as as the second earliest diverging lineage and is strongly burnin. The remaining trees were used to calculate a supported as sister to the rest of the genus except the 50% majority-rule consensus topology and posterior S. selaginoides clade (I) (ML BS = 99%, PP = 0.99). probability (PP) values. However, in our MP analysis based on the concate- nated dataset, the S. sanguinolenta clade (II) was weakly supported (MP JK = 66%) as sister to the Results Rosulatae clade and superclades B+C. Examples of spore morphology are illustrated in This study generated 506 new sequences (Appendix). Fig. 3. The dataset characteristics and tree statistics for the analyses are presented in Table 2. Comparisons of tree topologies from the MP JK analyses of the individual Discussion markers did not identify any well-supported conflicts (JK ≥ 70%; Mason-Gamer and Kellogg, 1996; Zhang The monophyly of Selaginellaceae and Simmons, 2006; Zhang et al., 2012b, 2015). Thus, the two datasets were concatenated using MS Excel As the largest lycophyte family, Selaginellaceae 2010 (Microsoft, Seattle, WA, USA). The topology of have long been recognized as sister to Iso€etaceae 6 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

99/100/1.00 XX. S. doederleinii clade (Asia+Australia) 100/100/1.00 55/61/0.99 XVI. S. oaxacana clade (America) OPHA clade 60/55/*

45/*/0.74 XVII. S. pallescens clade (America) */78/1.00 XVIII. S. hartwegiana clade (South America) 100/100/1.00 Superclade C 62/61/* XIX. S. anceps clade (America) Stachygynandrum */53/0.99 superclade 94/97/1.00 XV. S. involvens clade (Asia) 91/88/0.89

91/96/0.99 XIV. S. biformis clade (Asia)

80/73/0.99 79/69/0.97 S. heterostachys subclade

100/100/0.99 XIII. Heterostachys clade

81/85/1.00 subclade s. ciliaris Pacific

69/82/0.99 Asia 95/87/1.00

97/97/0.99 99/99/0.99 XII. Homostachys clade 82/74/0.97 Heterostachys XI. S. douglasii clade Superclade B 100/100/1.00 superclade

94/96/1.00 100/99/0.99 S. braunii subclade 100/91/0.99 X. Oligomacrosporangiatae 90/93/0.95 S. willdenowii subclade 100/100/1.00

73/51/0.78 S. delicatula subclade

S. siamensis subclade 0.05

clade S. pervillei subclade

S. pennata subclade 100/100/1.00 89/*/0.99 81/82/1.00 S. imbricata subclade 100/100/1.00 IX. Rosulatae clade S. stauntoniana subclade 90/97/1.00

100/100/1.00 VIII. S. lepidophylla clade

98/99/0.99

100/100/0.99 VII. Homoeophyllae clade VI. 99/*/0.99 100/98/1.00 Articulatae

99/98/1.00 S. kraussiana Ericetorum subclade 100/100/1.00 superclade 100/100/100 S. fragilis clade Superclade A subclade

55/81/0.94 V. S. exaltata clade

100/100/1.00 IV. S. myosurus clade

100/100/1.00 III. Lyallia clade 100/100/1.00 II. S. sanguinolenta clade

100/100/1.00 I. S. selaginoides clade

Outgroup Fig. 1. Simplified maximum likelihood phylogeny of Selaginella based on plastid rbcL and nuclear ITS sequences. The sizes of black triangles are in proportion to the sampled sizes of individual clades. Support values (maximum likelihood bootstrap support, maximum parsimony jack- knife support and posterior probability) are shown along the branches. The 17 major clades and three superclades of Selaginella resolved in this study are indicated. Green vertical bars indicate superclades or deep-level clades. Blue vertical bars indicate major clades. Black vertical bars indicate subclades. Red bar indicates outgroups. Purple bar indicates the major clade from America. Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 7

(a) Superclade C Figure 2e Stachygynandrum Megaspore surfaces reticulate; superclade Sporophylls usually monomorphic Figure 2d Heterostachys Megaspore surfaces tuberculate or verrucose; Figure 2c superclade sporophylls usually dimorphic Superclade S. imbricata 1 Dhofar, Oman B S. imbricata 2 Unknown S. pilifera Unknown S. imbricata subclade S. helioclada d'Andohahela, Madagascar IX. Rosulatae S. digitata d'Andohahela, Madagascar S. stauntoniana 1 Unknown clade 2 S. stauntoniana Henan, China Plants xerophytic, rosette, tufted or erect; branches 3 S. stauntoniana Beijing, China curled up during drought, megaspore surfaces coarse, 1 S. tamariscina Okinawa, Japan S. stauntoniana sometimes covered irregularly sized and spaced verrucae S. tamariscina 2 Unknown subclade S. tamariscina 3 Unknown S. pulvinata 1 Sichuan, China S. pulvinata 2 Xizang, China Homoeophyllae S. lepidophylla Figure 2b clade + clade

S. kraussiana 1 Cult. S. kraussiana 2 Cult. S. kraussiana 3 Cult. S. remotifolia 1 Unknown Dorsal rhizophoric S. remotifolia 2 Shizuoka, Japan S. remotifolia 3 Sichuan, China S. kraussiana (except Ericetorum S. remotifolia 4 Yunnan, China subclade clade uncertain) S. fragilis Unknown

S. articulata Unknown VI. Stems articulate or with 1 S. sericea Unknown Articulatae swollen joints; only one S. sericea 2 Pichincha, Ecuador megasporangium per S. suavis Unknown strobilus on the base; Superclade A S. sulcata Unknown megaspore surfaces Ericetorum

S. eurynota Heredia, Costa Rica clade reticulate and microspore S. sertata Jalisco, Mexico surfaces spiny superclade S. lingulata Unknown S. arthritica 1 Heredia, Costa Rica S. fragilis subclade S. arthritica 2 Heredia, Costa Rica S. silvestris Heredia, Costa Rica Plants erect and large-sized; S. kunzeana Puntarenas, Costa Rica stems action-plectostely S. diffusa 1 Puntarenas, Costa Rica S. diffusa 2 Cult. S. exaltata Unknown V. S. exaltata clade S. myosurus Unknown IV. S. myosurus clade

S. pygmaea Unknown clade III. S. lyallii Fianarantsoa, Madagascar S. moratii Fianarantsoa, Madagascar Lyallia S. polymorpha d'Antsiranana, Madagascar S. gracillima Tasmania, Australia S. uliginosa Tasmania, Australia S. sanguinolenta 1 Yunnan, China 2 S. sanguinolenta Sichuan, China II. S. sanguinolenta clade Megaspore surfaces reticulate; 3 Plants creeping; megaspore S. sanguinolenta Sichuan, China Plants xerophytic, creeping; sterile laesurae of megaspores wing-like 4 surfaces reticulate and with S. sanguinolenta Sichuan, China leaves nearly monomorphic and highly convoluted close to 5 wide and high muri S. sanguinolenta Unknown the pole forming a complex mass S. numularifolia Xizang, China S. deflexa Hawaii I. S. selaginoides clade S. selaginoides 1 Nagano, Japan 0.05 S. selaginoides 2 Ontario, Canada Plants erect; uniform sterile leaves and sporophylls spirally arranged S. selaginoides 3 Unknown Isoëtes nuttallii Isoëtes kersii Isoëtes sinensis Isoëtes taiwanensis Outgroups Isoëtes kirkii Fig. 2. Maximum likelihood phylogeny of Selaginella based on plastid rbcL and nuclear ITS sequences. Thick lines indicate strong support (maximum parsimony jackknife support ≥ 75%, maximum likelihood bootstrap support ≥ 75% and posterior probability ≥ 95%), and thin lines indicate moderate or weak support (either ML BS < 75% or MP JK < 75%). Major diagnostic features are shown in dark blue. Geographical provenances of newly included samples of Selaginella are indicated in red and those from GenBank are indicated in black. Green vertical bars indicate superclades or deep-level clades. Blue vertical bars indicate major clades. Black vertical bars indicate subclades. Red vertical bar indi- cates outgroups. Brown vertical bars in (d) indicate groupings under a subclade. Asterisks in (e) indicate New World species with dimorphic sporophylls. using molecular data but based on small taxon sam- The monophyly of species, hybridization and cryptic pling (e.g. Wikstrom€ and Kenrick, 1997; Korall et al., speciation in Selaginella 1999; Pryer et al., 2001). The sister relationship between these two families is further corroborated by Within several clades resolved in our study, such as the shared features of heterospory and the presence the Homoeophyllae clade (VII), the Homostachys clade of ligules (Zhang and Taylor, 2013; Zhang et al., (XII) and the S. pallescens clade (XVI), some species 2013). appeared in multiple clades, such as S. densa R. Sim, The monophyly of Selaginellaceae is supported by S. vardei H. Lev., S. wallacei Hieron. and S. wrightii morphology. In comparison with Iso€etaceae, Selaginel- Hieron (Fig. 2b). These patterns could be due to laceae species usually have distinctly sized and dor- misidentifications, laboratory errors or contamination, siventral leaves in four rows and rhizophores and/or misunderstood species boundaries. As Tryon (Thomas, 1997; Zhang et al., 2013). Based on our rela- (1955) pointed out, there is plasticity in several of the tively large sampling and combined analysis of plastid morphological characters (including those of vegetative and nuclear data Selaginellaceae are strongly sup- parts, strobili, sporophylls and spores) present in some ported as monophyletic (100% ML bootstrap support sections. Another reason for these seemingly non- (BS), 84% MP JK) (Figs 1 and 2a). monophyletic species could be the occurrence of 8 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

S. lepidophylla Unknown (b) S. lepidophylla Texas, USA VIII. S. lepidophylla clade S. phillipsiana Isiolo District, Kenya S. wightii Matale District, Sri-Lanka With similiar dorsal and ventral S. landii 1 Jalisco, Mexico leaves; megaspore surfaces S. asprella California, USA S. leucobryoides 1 California, USA ridged and reticulate S. leucobryoides 2 Arizona, USA S. utahensis Utah, USA S. watsonii 1 Idnho, USA S. densa 1 British Columbia, Canada S. scopulorum 1 Washington, USA S. scopulorum 2 British Columbia, Canada S. acanthonota North Carolina, USA S. rupestris 1 North Carolina, USA S. rupestris 2 Ontario, Canada S. sibirica 1 Hokkaido，Japan S. sibirica 2 Alaska, USA Spirally arranged monomorphic S. watsonii 2 Utah to Arizona, USA S. rupestris 3 Illinois, USA sterile leaves; decussate S. wrightii 1 Texas, USA sporophylls; megaspore surfaces S. arsenei 1 Hidalgo, Mexico S. sheldonii Texas, USA with interwoven reticulation S. peruviana1 Oaxaca, Mexico S. mutica Colorado, USA S. dregei Isiolo District, Kenya S. echinata Fianarantsoa, Madagascar S. grisea Unknown S. nivea Tulear, Madagascar S. arsenei 2 San Luis Potosi, Mexico S. sellowii 1 Carchi, Ecuador S. sellowii 2 Florída, Bolivia S. sellowii 3 Unknown S. sartorii 1 Boyocá Dept., Colombia S. cinerascens 1 California, USA S. cinerascens 2 California, USA

S. arenicola Louisiana, USA. VII. S. extensa Jalisco, Mexico S. weatherbiana Colorado, USA Homoeophyllae S. oregana Washington, USA S. balansae Tamegdoult, Morocco S. tortipila 1 South carolina, USA S. tortipila 2 North Carolina, USA S. caffrorum Pretoria, South Africa S. indica 1 Hassan District, India S. indica 2 Cult. S. indica 3 Yunnan, China S. vardei 1 Sichuan, China S. vardei 2 Yunnan, China clade S. vardei 3 Yunnan, China S. vardei 4 Yunnan, China S. steyermarkii Quezaltenango, Guatemala S. hansenii California, USA S. wallacei 1 California, USA S. wallacei 2 Oregon, USA S. wallacei 3 British Columbia, Canada S. wallacei 4 Oregon, USA S. bigelovii 1 California, USA S. bigelovii 2 California, USA S. bigelovii 3 California, USA S. bigelovii 4 California, USA S. mutica var. limitanea 1 New Mexico, USA S. mutica var. limitanea 2 Texas, USA S. × neomexicana 1 New Mexico, USA S. × neomexicana 2 New Mexico, USA S. sartorii 2 San Luis PotosÌ, Mexico S. rupincola 1 New Mexico, USA S. rupincola 2 Arizona, USA S. rupincola 3 Unknown S. underwoodii Colorado, USA S. njamnjamensis Kwale District, Kenya S. vardei 5 Yunnan, China S. shakotanensis 1 Nagano, Japan S. shakotanensis 2 Nagano, Japan S. densa 2 Colorado, USA S. wallacei 5 Idaho, USA S. wallacei 6 Washington, USA S. wallacei 7 Idaho, USA S. peruviana 2 Texas, USA S. landii 2 Moreles, Mexico S. wrightii 2 Texas, USA S. arizonica 1 Arizona, USA S. arizonica 2 Unknown S. arizonica 3 Arizona, USA. S. eremophila 1 California, USA S. eremophila 2 California, USA S. eremophila 3 California, USA

Fig. 2. Continued. Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 9

S. tama-montana 1 Mie, Japan (c) S. helvetica 1 Unknown S. prostrata 1 Sichuan, China S. prostrata 2 Sichuan, China S. jugorum Yunnan, China S. pallidissima Sichuan, China S. laxistrobila 1 Yunnan, China S. laxistrobila 2 Yunnan, China S. laxistrobila 3 Yunnan, China S. helvetica 2 Sichuan, China S. helvetica 3 Akita, Japan S. denticulata Unknown S. tama-montana 2 Taiwan, China S. pseudonipponica 1 Taiwan, China XII. Homostachys clade S. pseudonipponica 2 Taiwan, China S. pseudonipponica 3 Taiwan, China Plants creeping; fertile S. pseudonipponica 4 Taiwan, China branches erect; sporophylls S. pseudonipponica 5 Taiwan, China dimorphic and loose; strobili S. pseudonipponica 6 Taiwan, China non-resupinate S. sp_A Taiwan, China S. nipponica 1 Sichuan, China S. nipponica 2 Sichuan, China S. nipponica 3 Sichuan, China S. nipponica 4 Taiwan, China S. nipponica 5 Sichuan, China S. matsuensis Taiwan, China S. nipponica 6 Tokyo, Japan S. pseudonipponica 7 Taiwan, China S. pseudonipponica 8 Taiwan, China S. douglasii 1 Unknown XI. S. douglasii clade Native to west of USA; plant creeping; strobili S. douglasii 2 Oregon, USA uniform and megaspore surfaces verrucate S. mairei 1 Yunnan, China S. mairei 2 Yunnan, China

S. mairei 3 Yunnan, China S. mairei 4 Sichuan, China Plants xerophytic, erect; stems with 2 steles, leaves S. braunii 1 Cult. involute when dry

S. braunii 2 Yunnan, China S. braunii subclade S. braunii 3 Hainan, China S. braunii 4 Hubei, China S. pseudopaleifera Hoa Binh, Vietnam S. limbata Kagoshima, Japan S. helferi 1 Yunnan, China clade S. helferi 2 Ha Giang, Vietnam Plants scandent and large; megaspore S. helferi 3 Yunnan, China surfaces tuberculate- subclade Plants erect, scandent or creeping; S. willdenowii 1 Cult. iatae reticulate; microspore g S. willdenowii 2 Guizhou, China sporophylls monomorphic, except distal surfaces twisted S. willdenowii 3 Guangxi, China lamellate or with S. bisulcata and S. pennata S. uncinata 1 Peninsula Malaysia widely blunt spine S. uncinata cf. Puntarenas, Costa Rica

S. uncinata 2 Tokyo, Japan S. willdenowii S. uncinata 3 Sichuan, China S. mayeri Peninsula Malaysia S. plana 1 Cult. omacrosporan g S. plana 2 Cult.

S. hoffmannii 1 Puntarenas, Costa Rica Plants erect; megaspore Oli S. hoffmannii 2 Puntarenas, Costa Rica surfaces tuberculate; subclade microspore surfaces X. S. wallichii Bac Kan, Vietnam usually blunt-spiny S. delicatula 1 Sichuan, China S. delicatula 2 Sichuan, China Plants xerophytic; megaspore S. picta 1 Phu Tho, Vietnam surfaces regularly verrucose; S. picta 2 Yunnan, China S. delicatula microspore surfaces regular S. picta 3 Yunnan, China and twisted lamellate S. siamensis Yunnan, China S. siamensis subclade S. pervillei Fianarantsoa, Madagascar S. pervillei subclade S. pennata 1 Yunnan, China Plants erect; leaves on stem

S. pennata 2 Yunnan, China monomorphic, megaspore surfaces S.bisulcata 1 Yunnan, China sparsely papillate or tuberculate S. bisulcata 2 Yunnan, China S. pennata subclade S. bisulcata 3 Yunnan, China Sporophylls dimorphic; S. bisulcata 4 Yunnan, China megaspore exospores smooth S. bisulcata 5 Yunnan, China

Fig. 2. Continued. i.2 Continued. 2. Fig. 10 (d) strobili resupinate Sporophylls dimorphic; i-a hue l ldsis0(05 1–30 (2015) 0 Cladistics / al. et Zhou Xin-Mao .kanehirae S. .kanehirae S. .chrysocaulos S. .lutchuensis S. .albociliata S. S.repanda S.repanda repanda S. S.repanda repanda S. .sp_ S. .labordei S. .sp_ S. S. leptophylla .labordei S. labordei S. .sp_ S. sp_ S. .arbuscula S. S. sp_ .labordei S. .arbuscula S. .drepanophylla S. .decipens S. decipiens S. .drepanophylla S. .amblyphylla S. amblyphylla S. .boninensis S. S. heterostachys .boninensis S. .sichuanica S. S. monospora monospora S. S. sp_ S.heterostachys S.heterostachys .heterostachys S. .sp_ S. .effusa S. effusa S. .kurzii S. .leptophylla S. leptophylla S. S.leptophylla S. heterostachys S.heterostachys S.heterostachys S.heterostachys .sp_ S. .xichouensis S. .effusa S. .trichophylla S. trichophylla S. .kurzii S. .leptophylla S. .chaetoloma S. .boninensis S. S.heterostachys .effusa S. S.heterostachys S.heterostachys S. effusa .amblyphylla S. .monospora S. .trichophylla S. trichophylla S. .heterostachys S. .bodinieri S. S.bodinieri S.bodinieri S.bodinieri .bodinieri S. .megaphylla S. megaphylla S. megaphylla S. S. megaphylla C .bodinieri S. C S.tenuifolia C .oopr S.monospora .megaphylla S. B B S.heterostachys S.heterostachys .megaphylla S. .sichuanica S. .chaetoloma S. .chaetoloma S. 3 2 .sichuanica S. 2 1 D F 1 S. daozhenensis S. heterostachys E .ciliaris S. S. ciliaris 4 2 3 1 2 1 4 3 1 1 .compta S. 3 2 2 .compta S. 4 2 var. 2 1 1 1 2 S. compta Caroline Is.,Kosrae,F.S.M. Guizhou, China Guizhou, China 1

Huahine, SocietyIs. 2 1 Caroline Is.,Kosrae,F.S.M. cf. S.compta S.compta S.compta S.compta Yunnan, China Phu Tho,Vietnam 1 Ha Giang, Vietnam Guizhou, China 2 3 3 Guizhou, China 3 2 Yunnan, China 4 5 Yunnan, China 2 Yunnan, China 1 dulongjiangensis 3 4 3 Caroline Is.,Pohnpei,F.S.M. 6 Caroline Is.,Pohnpei,F.S.M. Sichuan, China 1 5 Sichuan, China Sichuan, China Sichuan, China 9 Guizhou, China 2 1 Sichuan, China 2 4 3 Guangdong, China Yunnan, China 1 Guizhou, China Okinawa, Japan 7 5 4 3 1 8 6 5 Bac Kan, Vietnam 1 Yunnan, China 12 2 1 Guizhou, China 2 2 cf. 2 Kauai, HawaiianIs. Bac Kan, Vietnam 4 Maui, HawaiianIs. 1 2 6 3 Tokyo, Japan .xipholepis S. .xipholepis S. 3 2 1 Sichuan, China Lang Son, Vietnam 10

5 Yunnan, China Sichuan, China 4 Yunnan, China Yunnan, China Sichuan, China Sichuan, China Guizhou, China Sichuan, China Guizhou, China Sichuan, China Lang Son, Vietnam Yunnan, China Vinh Phuc, Vietnam Mie, Japan Guizhou, China Sichuan, China Bac Kan,Vietnam Guizhou, China Hainan, China Cao Bang, Vietnam Yunnan, China Sichuan, China Guizhou, China Guizhou, China 11 Yunnan, China Guizhou, China Guizhou, China Hainan, China Thai Nguyen, Vietnam Yunnan, China Lang Son, Vietnam Guizhou, China Guizhou, China Guizhou, China 1 Guizhou, China Guizhou, China Guizhou, China Hainan, China 3 Ha Giang, Vietnam Guizhou, China 2 Lang Son, Vietnam Hoa Binh, Vietnam Sichuan, China Peninsular Malaysia Hainan, China Hoa Binh, Vietnam Yunnan, China Yunnan, China Yunnan, China Sichuan, China Yunnan, China Sichuan, China Guizhou, China Sichuan, China Sichuan, China Sichuan, China Guizhou, China Sichuan, China 1 2 Guangdong, China Bac Kan, Vietnam Yunnan, China

Asia Pacific subclade S. heterostachys subclade S. ciliaris tuberculate orverrucose megaspore surfaces Native to Asia; usually fine-reticulate Megaspores surfaces

XIII. Heterostachys clade Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 11

S. biformis 1 Ha Giang, Vietnam (e) S. biformis 2 Bac Kan, Vietnam S. biformis 3 Hainan, China XIV. S. biformis clade (Asia) S. biformis 4 Hainan, China S. biformis 5 Bac Kan,Vietnam Plants flagelliform and erect; S. biformis 6 Hainan, China erect plants pubescent S. biformis 7 Bac Kan,Vietnam S. biformis 8 Okinawa, Japan S. involvens 1 Cult. S.involvens 2 Phu Tho, Vietnam S.involvens 3 Yunnan, China S.involvens 4 Sichuan, China S. involvens 5 Kagoshima, Japan S.involvens 6 Yunnan, China S.moellendorffii 1 Sichuan, China S.moellendorffii 2 Sichuan, China XV. S. involvens clade (Asia) S.moellendorffii 3 Sichuan, China S. moellendorffii 4 Introduced to Costa Rica Plants erect or creeping; S.moellendorffii 5 Cult. on veins of ventral leaves S. davidii Beijing, China with two light color bands S.gebaueriana 1 Guizhou, China S. gebaueriana 2 Yunnan, China S. gebaueriana 3 Guizhou, China S. gebaueriana 4 Yunnan, China S. gebaueriana 5 Guizhou, China Plants erect, suberect or creeping; S.gebaueriana 6 Yunnan, China S. novae-hollandiae 1* Venezuela sterile leaves dimorphic; S. oaxacana 1 Oaxaca, Mexico megaspore surfaces typically S. oaxacana 2 Oaxaca, Mexico XVI. S. oaxacana clade S. oaxacana 3 Heredia, Costa Rica reticulate, microspore S. martensii 1 Cult. (America) S. martensii 2 Oaxaca, Mexico surfaces usually baculate S. reflexa San Luis Potosi, Mexico S. apoda 1 Unknown S. apoda 2 Virginia, USA S. apoda 3 Tennessee, USA S. acanthostachys Unknown S. harrisii 1 San Luis PotosÌ, Mexico S. pallescens 1 Cult. S. harrisii 2 Hidalgo, Mexico XVII. S. pallescens clade S. pulcherrima Cult. (America) S. pallescens 2 Unknown S. pallescens 3 Unknown S. pallescens 4 San Jose, Costa Rica S. pallescens 5 Guanacaste, Costa Rica S. pallescens 6 Hidalgo, Mexico S. pallescens 7 Guanacaste, Costa Rica OPHA clade S. pallescens 8 Jalisco, Mexico Native to America; S. pallescens 9 Heredia, Costa Rica S. nothohybrida San Luis PotosÌ, Mexico plants suberect or S. hartwegiana * Loja, Ecuador XVIII. S. hartwegiana clade creeping; megaspore S. novae-hollandiae 2 * Ecuador S. flexuosa Zamora-Chinchipe, Ecuador (South America) surfaces reticulate S. anceps 1 Guanacaste, Costa Rica S. anceps 2 Heredia, Costa Rica S. microphylla Carchi, Ecuador S. haematodes Unknown S. erythropus Cult. S. bombycina 1 Heredia, Costa Rica S.bombycina 2 Unknown S. radiata * French guiana XIX. S. anceps clade S. attirense cf. 1 Guanacaste, Costa Rica S. umbrosa 1 Heredia, Costa Rica (America) S. umbrosa 2 Cult. S. flabellata Lesser Antilles, Grenada S. flagellata 1 * Heredia, Costa Rica S. flagellata 2 * Unknown S. simplex * Unknown S. moritziana * Unknown S. attirense cf. 2 San José, Costa Rica S. porphyrospora * San José, Costa Rica S. alopecuroides Borneo S. intermedia Peninsular Malaysia S. firmuloides Unknown S. longipinna Unknown S. kerstingii Cult. S. doederleinii 1 Hainan, China S. doederleinii 2 Kagoshima, Japan S. doederleinii 3 Yunnan, China XX. S. doederleinii clade S. commutata Guangxi, China (Asia+Australia) S. scabrifolia Hainan, China S. trachyphylla Yunnan, China Plants suberect; megaspore S. frondosa 1 Unknown S. frondosa 2 Yunnan, China proximal surfaces with a pronounced S. superba Yunnan, China zona at the equator S. roxburghii Peninsular Malaysia S. brooksii Borneo S. longiaristata Unknown

Fig. 2. Continued. 12 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

(a) (b) (c) (d)

(e) (f) (g) (h)

(i) (j) (k) (l)

(m) (n) (o) (p)

(q) (r) (s) (t)

(u) (v) (w) (x) Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 13 hybridization (and possibly allopolyploidy), which has Jermy (1986) and three other Malagasy species [S. lyallii been reported and postulated earlier in Selaginellaceae (Hook. & Grev.) Spring, S. moratii Rauh & W. Hage- (e.g. Graustein, 1930; Somers and Buck, 1975). Fur- mann, S. polymorpha Badre] from their “dorsal rhi- ther examples of suspected hybrids in Selaginella zophoric clade”, although these species together were include S. 9 neomexicana Maxon—a possible sterile resolved as sister to that clade. Our analysis, with ca. 88 hybrid between S. rupincola Underw. and S. mutica species included, shows that the “dorsal rhizophoric D.C. Eaton (Tryon, 1955; Valdespino, 1993)—and an clade’’ defined by Korall and Kenrick (2002) is para- unnamed hybrid between S. arizonica Maxon and phyletic in relation to S. subg. Ericetorum sensu Jermy S. eremophila (Yatskievych and Windham, 2009). (1986) and several other species (Figs 1 and 2b). Our More studies are needed to better understand the results are consistent with those of Arrigo et al. (2013) species-level relationships within these clades with who sampled 51 species of this clade. We discover a non-monophyletic species. strongly supported clade (superclade A) containing S. exaltata, S. lepidophylla, S. myosurus, S. subg. Erice- Deep-level relationships in Selaginella torum sensu Jermy (1986), S. ser. Articulatae sensu Hieronymus and Sadebeck (1901), “S. subg. Te- Our combined plastid and nuclear data resolve the tragonostachys” sensu Jermy (1986), and three Malagasy deep-level relationships in Selaginella with strong sup- species (S. lyalii, S. moratii, S. polymorpha). The most port (Figs 1, 2a and 4). We recognize here six major parsimonious interpretation would be that dorsal rhi- deep-level clades (ML BS ≥ 94%, MP JK ≥ 97%, zophores evolved once in the most recent common PP ≥ 0.99): clades I and II, superclades A–C and clade ancestor of superclade A and this character state was IX. Clade I (the S. selaginoides clade, see below), con- lost in the Lyallia clade (III). Here we advocate recogni- taining only two species, was resolved as sister to the tion of this expanded “S. subg. Tetragonostachys”asa rest of the genus (ML BS = 100%, MP JK = 100%, subgenus of Selaginella (Fig. 4). Based on our estimate PP = 1.00). Clade II (the S. sanguinolenta clade, see superclade A contains ca. 200 species, while “S. subg. below) is the second earliest diverging lineage (ML Tetragonostachys” sensu Jermy (1986) contains ca. 50 BS = 99%, PP = 0.99), and is a novel deep-level lin- species only. Such an expanded “S. subg. Tetragonos- eage not found in previous molecular studies. Clade tachys” differs from the rest of the genus by the dorsal IX (the Rosulatae clade, see below) was found to be rhizophoric (except clade III), and includes the xero- sister to superclades B+C (ML BS = 100%, MP JK = morphic isophyllous species, the articulate species, the 91%, PP = 0.99), and was not previously recognized species with monomorphic and decussately arranged as a deep-level lineage by studies based on either leaves, and a rosette-forming species [S. lepidophylla morphology or molecular data. Below is our detailed (Hook. & Grev.) Spring]. discussion of superclades A–C. Within superclade A, the S. lepidophylla clade (VIII) is resolved as sister to the Homoeophyllae clade (VII), Superclade A—the Ericetorum superclade and they together are sister to a clade containing clades III–VI. Our study resolves superclade A as sis- The Ericetorum superclade contains clades III–VIII in ter to a clade containing clade IX (the Rosulatae clade) our study (Figs 1, 2a and 4). Based on rbcL data alone and superclades B and C with strong support (ML Korall and Kenrick (2002) identified a weakly sup- BS = 89%, PP = 1.00). ported so-called “dorsal rhizophoric clade’’ including S. myosurus Alston, and members of S. ser. Articulatae Superclade B—the Heterostachys superclade (Spring) Hieronymus and Sadebeck (1901) and “S. subg. Tetragonostachys” sensu Jermy (1986, 1990). The Heterostachys superclade contains clades They excluded members of S. subg. Ericetorum sensu X–XIII in our study (Figs 1 and 2c,d). In the previous

Fig. 3. Scanning electron micrographs of megaspores and microspores of selected species of Selaginella (see Zhou et al., 2015b for more details). (a–q) megaspore morphology of the major clades and subclades; (r–x) microspore morphology of clade XIII, the Heterostachys clade. (a) S. sanguinolenta (L.) Spring (clade II), Chu & Wu 68 (PYU). (b) S. remotifolia Spring (clade VI), Chu & Zhang 20188 (PYU). (c) S. vardei H. Lev. (clade VII), Jin & Cao s.n. (PYU). (d) S. stauntoniana Spring (clade IX), PYU 870080 (PYU). (e) S. nipponica Franch. & Sav. (clade XII), Chu 4753 (PYU). (f) R, S. braunii Baker (clade X: the S. braunii subclade), Chu & Wu 3043 (PYU). (g) S, S. willdenowii (Desv. ex Poir.) Baker (clade X: the S. willdenowii subclade), Chu et al. 18474 (PYU). (h) T, S. picta A. Braun ex Baker (clade X: the S. delicatula subclade), Lu & Zou 18748 (PYU). (i) U, S. siamensis Hieron. (clade X: the S. siamensis subclade), Chu et al. 15741 (PYU). (j) V. S. pennata (D. Don) Spring (clade X: the S. pennata subclade), Chu & Zhang 24587 (PYU). (k) W, S. kurzii Baker (clade XIII: the S. heterostachys subclade), Chu 3833 (PYU). (l) X, S. ciliaris (Retz.) Spring (clade XIII: the S. ciliaris subclade), Chu et al. 15426 (PYU). (m) S. biformis A. Braun ex Kuhn (clade XIV), Chu et al. 23504 (PYU). (n) S. involvens (Sw.) Spring (clade XV), Chu & Feng 51 (PYU). (o) S. gebaueriana Hand.-Mazz. (clade XV), Chu & Feng 89 (PYU). (p,q) S. commutata Alderw. (clade XVII), Zhou 2441 (PYU). (A) The obviously disconnected laesurae at the pole in the Homostachys clade. (B) The extremely smooth exospore in the S. pennata subclade. (C) The equatorial flange in S. involvens. (D) The zona at equator in the S. doederleinii clade. 14 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

(a)(b) (c)(d)(e) classifications of Selaginella, S. subg. Heterostachys comprised the species with dimorphic, resupinate XX sporophylls (Baker, 1884, 1887; Warburg, 1900; Wal- ton and Alston, 1938; Jermy, 1986, 1990). Previous molecular studies (e.g. Korall and Kenrick, 2002) sam- XVII XVI pled only a few species (all from the Americas), but S. subg. Heterostachys sensu Baker contains ca. 60 XVIII species (Jermy, 1990) and is distributed globally in the XIX tropics (Tryon and Lugardon, 1991). Asia is the major

Stachygynandrum distribution area with more than 28 species in China XV C: Stachygynandrum (Zhang et al., 2013) and more than 25 species in India (Alston, 1945). In our study, 167 accessions respresent- XIV ing ca. 80 species form the strongly supported mono- phyletic superclade B (which we advocate recognizing as the expanded S. subg. Heterostachys), which con- tains members of S. subg. Heterostachys sensu Baker (1883, 1887), S. subg. Homostachys sensu Baker (1883, 1887), Warburg (1900), and Walton and Alston (1938), S. ser. Suberosae (Baker) Warb., the tropical and subtropical species with uniform sporophylls, and S. douglasii (Hook. & Grev.) Spring. In other words, superclade B contains all species with dimorphic ventral rhizophores Lycopodiodes sporophylls from Asia and the Pacific islands and some species with uniform sporophylls from tropical

XII and subtropical regions (e.g. S. braunii, S. delicatula,

B: Heterostachys S. hoffmannii, S. pervillei, S. siamensis, S. uncinata)

XI XIII and temperate S. douglasii. Species of the Heterostachys superclade appear highly variable in morphology and geographical distri- bution. They can have various habits (erect, scandent, X

Stachygynandrum creeping or prostrate) and strobili (with uniform sporophylls, or dimorphic non-resupinate or resupinate sporophylls) and grow in various habitats. However, species of this superclade share some megaspore fea- He Heterostachys tures. Their megaspore surfaces are tuberculate, some- IX

Sta times papillate, verrucate and rugulate. In some cases, these megaspore ornamentations combine with each other and even form irregular reticulate ornamenta- tions although completely different from the reticulate ornamentations in S. subg. Stachygynandrum (Fig. – – VIII 3e l vs. m q). Our study resolves superclade B as sister to super- Bryodesma clade C with relatively strong support (ML BS = 80%, Tetragonostachys

Fig. 4. Simplified maximum likelihood phylogeny of Selaginella based on plastid rbcL and nuclear ITS sequences. (a) 20 major lin- eages. (b) three superclades. (c) five-subgenus classification of Jermy dorsal rhizophores

A: Ericetorum = = VI (1986). Clade III, marked as Er ( S. subg. Ericetorum the Lyallia

Sta clade), contains three species (S. lyallii, S. moratii, S. polymorpha) classified in Stachygynandrum. Clade XVI, marked as Stachygynan-

V drum, contains some species classified in Heterostachys as well.

IV s = S. subg. Selaginella; Sta = S. subg. Stachygynandrum;He= Lycopodiodes III ? Er S. subg. Heterostachys. (d) the three-genus classification of Sojak (1993). (e) rhizophores. vr = ventral rhizophores. nr = no rhi- II vr Sta zophores. ? = the Lyallia clade, where rhizophore position is difficult IVII S S nr to determine. Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 15

MP JK = 75%, PP = 0.99). Within superclade B, the S. selaginoides (L.) P. Beauv. ex Schrank & Mart. S. douglasii clade (XI) is resolved as sister to the (Jermy, 1990) and the Hawaiian endemic S. deflexa Homostachys clade (XII), together they are sister to Brack. (Jermy, 1986). The former is also the type of the Oligomacrosporangiatae clade (X) and the three the genus. In earlier classifications, although the close together are sister to the Heterostachys clade (XIII). relationship between the two species had long been recognized, the subgenus was often lumped with other Superclade C—the Stachygynandrum superclade isophyllous species, e.g. in S. sect. Homoeophyllae Spring by Spring (1850), in S. sect. Homotropae by The Stachygynandrum superclade contains clades Braun (1857), in S. sect. Cylindrostachyae by Hierony- XIV–XX (Fig. 2e). Traditionally all species of Se- mus and Sadebeck (1901) and in “S. subg. Euse- laginella with dimorphic leaves and uniform strobili laginella” (nom. inval.) by Walton and Alston (1938). were included in S. subg. Stachygynandrum (P. Beauv.) Conversely, Baker (1883, 1887) accepted S. subg. Se- Baker (e.g. Baker, 1887; Warburg, 1900; Walton and laginella, but his concept of the subgenus included all Alston, 1938; Jermy, 1986, 1990). However, a molecu- isophyllous species. Also, S. subg. Selaginella sensu lar phylogenetic study has demonstrated that S. subg. Tryon and Tryon (1982) contains about 50 species but Stachygynandrum is not monophyletic (Korall and our study shows that it contains only two species. Kenrick, 2002), a result confirmed in our study We sampled both species in our analysis, which (Fig. 4). We find a narrowly defined S. subg. shows that S. subg. Selaginella is strongly supported Stachygynandrum s.s. that is supported as mono- as monophyletic (ML BS = 100%, MP JK = 100%, phyletic (ML BS = 94%, MP JK = 97%, PP = 1.00), PP = 1.00). For the first time multiple samples of and which corresponds to our superclade C. This sub- S. selaginoides are included, which supports the mono- genus in our definition includes the members of phyly of this species. Selaginella subg. Selaginella is S. subg. Stachygynandrum sensu (P. Beauv.) Baker robustly supported as sister to the rest of the genus (1883) excluding the species of S. ser. Articulatae (ML BS = 100%, MP JK = 100%, PP = 1.00), which (Spring) Hieron. & Sadeb., S. ser. Rosulatae sensu (A. is consistent with the findings of Korall and Kenrick Braun) Baker and S. ser. Suberosae sensu (Baker) (2002) and Arrigo et al. (2013). This subgenus is char- Warb. This newly circumscribed subgenus may contain acterized by plants erect, uniform leaves and sporo- 200 species and is characterized by plants that are phylls spirally arranged, and the chromosome number erect, suberect or creeping, megaspore surfaces that are (of S. selaginoides)of2n = 18 (Heidel and Handley, reticulate (Fig. 3m–q), and microspore surfaces usually 2006). baculate (Liu et al., 1989; Korall and Taylor, 2006; Xia Two characteristics of S. subg. Selaginella (determi- et al., 2013; Singh et al., 2014; our own observations). nate growth and rootstock) have been interpreted as Within Selaginella subg. Stachygynandrum s.s.,88 support for a closer relationship to Iso€etales than to accessions representing ca. 50 species in our analysis other species of Selaginellaceae (Bateman, 1992; see are resolved into four clades (clades XIV–XVII; alternative interpretation in Kenrick and Crane, 1997, Fig. 2e), three of which are well supported. The S. bi- pp. 212–213). formis clade (XIV) is sister to the remaining three, fol- lowed by the S. involvens clade (XV) which is sister to Clade II—the Selaginella sanguinolenta clade a clade containing the OPHA clade (clades XVI–XIX) and the S. doederleinii clade (XX) (see discussion The Selaginella sanguinolenta clade (II) contains Se- below). laginella sanguinolenta (L.) Spring and its allies (an additional three or four species based on our estimate). Major evolutionary lineages in Selaginella Because of the nearly monomorphic sterile leaves, the relationships of this subgenus had been controversial. The ca. 200 species of the genus sampled in our Spring (1850) placed it in S. sect. Homoeophyllae with study are resolved into the following 20 well-supported the other isophyllous species. Baker (1883, 1887) and (except one weakly supported) major clades including Warburg (1900) also placed it together with isophyl- two currently monospecific clades (Fig. 1). All of these lous species in S. subg. Selaginella. Hieronymus and major clades are also supported by macro-morphologi- Sadebeck (1901) placed in it S. subg. Heterophyllum cal, ecological, and/or spore features and/or distribu- sect. Pleiomacrosporangiatae ser. Monostelicae with tion information. dimorphic sterile leaves and thought that the nearly monomorphic sterile leaves may be intermediate Clade I—the Selaginella selaginoides clade between S. subg. Heterophyllum and S. subg. Homoeo- phyllum. Walton and Alston (1938) placed in it Clade I corresponds to Selaginella subg. Selaginella S. subg. Stachygynandrum ser. Decumbentes with and contains only two species: the circumboreal dimorphic sterile leaves and hypothesized that it is an 16 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 intermediate stage between S. subg. Stachygynandrum and spore features: (i) the megaspore surfaces are retic- ser. Decumbents and “S. subg. Euselaginella”. In addi- ulate and have wing-like and highly convoluted lae- tion to the nearly monomorphic sterile leaves, mem- surae forming a complex mass at the pole (Stefanovic bers of this clade have megaspore surfaces with et al., 1997; Korall and Taylor, 2006; Schulz et al., densely contiguous tubercles (Fig. 3a) and microspore 2013); (ii) microspores are globose-triangular, and surfaces rugulate and tuberculate (for spore morphol- their proximal surfaces are coarse, sometimes covered ogy of S. sanguinolenta also see Minaki, 1984; Liu with fine spines and the distal surfaces are usually reg- et al., 1989; for that of S. nummularifolia see Liu and ulate (for microspore morphology of S. pygmaea, see Yan, 2005). Schulz et al., 2013; for that of S. moratii and S. poly- Members of the Selaginella sanguinolenta clade were morpha, see Stefanovic et al., 1997); (iii) although the not previously included in a phylogenetic study. We Malagasy species have dimorphic sterile branch leaves, included two species in our analysis, S. sanguinolenta their monomorphic and decussately arranged sterile (L.) Spring with a broad Asian distribution and stem leaves are consistent with members of S. subg. S. nummularifolia Ching endemic to Xizang, China. Ericetorum sensu Jermy (1986); (iv) rhizophores are The monophyly of this clade is strongly supported strictly restricted to the base of erect stems within all (ML BS = 100%, MP JK = 100%, PP = 1.00). members of the Lyallia clade; and (v) these species Based on our ML and BI analyses, the Selaginella have the same chromosome number 2n = 18 as far as sanguinolenta clade (II) is the second earliest diverging known [(S. uliginosa and S. lyallii were examined by lineage and is strongly supported as sister to the rest Jermy et al. (1967)]. of the genus except the S. selaginoides clade (I) (ML Note that Schulz et al. (2013) segregated Selaginella BS = 99%, PP = 0.99). However, in our MP analysis, pygmaea into two species, S. aboriginalis C. Schulz & the S. sanguinolenta clade (II) was weakly supported Homberg (from Australia) and S. pygmaea (from (MP JK = 66%) as sister to the Rosulatae clade and South Africa), and added S. royenii Alston from superclades B+C. This conflict was first found by Kor- southern New Guinea, Indonesia, to this clade all and Kenrick (2004) in Selaginella. Potentially, this (S. subg. Ericetorum). So, the Lyallia clade probably could be caused by GC-rich plastid DNA in Se- includes eight species: four Australasian ones (S. abo- laginella (Smith, 2009). As Eyre-Walker (1998) riginalis, S. gracillima, S. uliginosa, S. royenii) and showed, parsimony analyses may encounter problems four Afro-Madagascar ones (S. lyallii, S. moratii, when resolving the phylogenies which contain biased S. polymorpha, S. pygmaea). base composition. In other words, MP may be extre- Korall and Kenrick (2002) excluded members of Se- mely sensitive to nucleotide composition (Smith, 2009). laginella subg. Ericetorum sensu Jermy (1986) from Based on spore morphology (Liu et al., 2002) and their “dorsal rhizophoric clade”. However, because habit, Selaginella kashminiana Dixit from Pakistan these three species have erect stems and the rhi- may be a member of this clade. zophores are strictly at the base, it is not easy to assess accurately if these three species have dorsal Clade III—the Lyallia clade rhizophores (Fig. 4).

The Lyallia clade (III) contains six species. In our Clade IV—the Selaginella myosurus clade analysis, two Australian species [Selaginella gracillima (Kuntze) Alston and S. uliginosa (Labill.) Spring] and The Selaginella myosurus clade (IV) contains only four Afro-Madagascar species (S. lyallii, S. moratii, the western African S. myosurus in our sampling. S. polymorpha and S. pygmaea Alston) form a Judging from morphology, S. scandens (P. Beauv.) strongly supported monophyletic clade (ML BS = Spring from Gabon might belong to this clade. In our 100%, MP JK = 100%, PP = 1.00), and the Aus- analysis, S. myosurus is resolved as sister to the Lyallia tralian species together are sister to the Afro-Madagas- clade but with weak support. Hieronymus and Sade- car species, consistent with the results of Korall and beck (1901) placed S. scandens and S. puberula Kenrick (2002) that. Selaginella subg. Ericetorum sensu Klotzsch from Guiana together in the S. scandens Jermy (1986) containing S. gracillima, S. pygmaea and group, so S. puberula may also be in this clade. S. uliginosa is paraphyletic in relation to the four Based on its creeping habit, a single large megaspo- Afro-Madagascar species (Fig. 2a). rangium on the base of strobili, dorsal rhizophores Although the three species of Selaginella subg. Erice- (our observations), megaspore surfaces reticulate, and torum sensu Jermy (1986, 1990) are different from the wide and high muri (Korall and Taylor, 2006), three Malagasy species (S. lyallii, S. moratii, S. poly- S. myosurus should be close to the Articulatae clade, morpha) in morphology and habit (Korall and Ken- but our study resolves this species outside of that rick, 2002), these six species share some morphological clade, but without support. Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 17

Clade V—the Selaginella exaltata clade S. kraussiana has one vascular bundle while S. remoti- folia has two (Zhang, 2004; Chu, 2006; our own obser- The Selaginella exaltata clade (V) contains only vations). The members of the S. fragilis subclade have Central and South American species in our sampling. from one to more vascular bundles. Hieronymus and Sadebeck (1901) established the monospecific S. exaltata group, one of the four groups Clade VII—the Homoeophyllae clade under their S. subser. Monostelicae. This subseries also included S. kraussiana (Kunze) A. Braun, S. remotifo- The Homoeophyllae clade (VII) approximates “Se- lia Spring, etc. Our study resolves S. exaltata (clade V) laginella subg. Tetragonostachys” sensu Jermy (1986), away from S. kraussiana and S. remotifolia, etc. (clade which contains ca. 45–60 species distributed across the VI), but without support (Fig. 2a); it is possible that Old and New Worlds except Australia (Tryon and additional data will demonstrate that S. exaltata is sis- Lugardon, 1991), and most species occupy xeric habits ter to S. kraussiana and its allies. Morphologically and (Tryon, 1955; Arrigo et al., 2013). Previous studies anatomically, S. exaltata has large erect plants (up to (Korall and Kenrick, 2002, 2004; Arrigo et al., 2013) 1 m) and has several steles in a special actino-plectos- pointed out that there is little doubt about the mono- tele (a sort of three-lobed plectostele) and extremely phyly of “S. subg. Tetragonostachys” sensu Jermy, and large megaspores (ca. 1.5 mm) (Mickel and Hellwig, its sister relationship with the resurrection plant 1969), which are different from members of the Articu- S. lepidophylla has been strongly supported. Our study latae clade. With denser future samples, possibly more further corroborates these results. Morphologically, members of the S. exaltata clade will be identified. members of “S. subg. Tetragonostachys” share spirally arranged monomorphic leaves and decussate sporo- Clade VI—the Articulatae clade phylls (Jermy, 1986; Sojak, 1993). Megaspore surfaces of members of this clade have an interwoven, closed, The Articulatae clade (VI) approximates Selaginella irregular and fine reticulum (Fig. 3c), and sometimes ser. Articulatae (Spring) Hieronymus and Sadebeck the muri are not obvious (e.g. Tryon, 1949; Minaki, (1901). Morphologically, members of the series share 1984; Liu et al., 1989; Chu, 2006; Korall and Taylor, the following features: (i) below bifurcation of the 2006; Zhu et al., 2006; Xia et al., 2013). The megas- stem there are articulations or swollen joints; (ii) rhi- pore morphology is stable and unique to the Homoeo- zophores are borne on the dorsal side of the stems; phyllae clade. The microspore proximal surfaces have (iii) a single large megasporangium is located at the rough and irregular rugulate ornamentation and distal base of a strobilus; (iv) megaspore surfaces have irreg- surfaces are verrucate and rugulate (Liu et al., 1989; ular or regular (Fig. 3b) and coarse reticulum usually Chu, 2006; Zhu et al., 2006; Xia et al., 2013). with high and wide muri (e.g. Minaki, 1984; Liu et al., Within the Homoeophyllae clade the relationships 1989, 2006; Giorgi et al., 1997; Korall and Taylor, are poorly resolved. 2006; Xia et al., 2013); (v) megaspores are extremely large; and (vi) microspore surfaces usually have spines Clade VIII—the Selaginella lepidophylla clade (e.g. Hellwig, 1969; Tryon and Lugardon, 1991; Giorgi et al., 1997; Xia et al., 2013). In our study, the 22 The Selaginella lepidophylla clade (VIII) contains ca. accessions included in the Articulatae clade are four species including the American species S. lepido- strongly supported as monophyletic. Previous classifi- phylla (Hook. & Grev.) Spring. The clade is resolved cations placed members of this clade among other as sister to the Homoeophyllae clade with strong sup- anisophyllous groups. Our study shows, instead, that port. This sister relationship of the two clades is sup- they have closer relationships with isophyllous taxa. ported by their shared xerophytic ecologies. Based on Within the clade the S. kraussiana subclade is the rosulate and xerophytic habit, S. lepidophylla was strongly supported as sister to the S. fragilis subclade. usually placed in the resurrection group, the Rosulatae Members of the first subclade occur in Asia and clade (see below), but our study shows that the S. lepi- Africa, while those of the second subclade occur in the dophylla clade and the Rosulatae clade are not closely Americas. These two subclades have no significant dif- related (Fig. 2b). ferences in morphology, habit or number of chromo- Morphologically, the S. lepidophylla clade also has somes. Although the number of vascular bundles is some particular features, such as plants having similar mainly one in Selaginella, it also shows great variation dorsal leaves and ventral leaves (Mickel and Smith, in some groups (such as the Oligomacrosporangiatae 2004), distal surfaces of the megaspores having high clade, see below) (Chu, 2006). In the Articulatae clade, ridges and proximal surfaces being reticulate forming the number of vascular bundles ranges from one to connected ridges (Korall and Taylor, 2006), and five, and usually two (Walton and Alston, 1938; Korall microspores scattering in tetrads surrounded and orna- and Kenrick, 2002). Within the S. kraussiana subclade, mented by several ridges (Hellwig, 1969; Giorgi et al., 18 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

1997; Mickel and Smith, 2004). Therefore, the S. lepi- variations [chromosome number, habit, habitat, num- dophylla clade is morphologically distinct from both ber of stele, sporophylls, and megaspore (Fig. 3f–j) the Rosulatae clade (IX) and the Homoeophyllae clade and microspore (Fig. 3r–v) morphology]; understand- (VII). Mickel and Smith (2004) also thought that ing relationships within this clade is difficult because S. lepidophylla was different from the Rosulatae clade. of the complexity of features exhibited and the poorly Based on their judgment two species (S. novoleonensis resolved phylogeny we obtained (Fig. 2c). Hieron. and S. ribae Valdespino) from Mexico might Within the Oligomacrosporangiatae clade, the 41 belong to the S. lepidophylla clade. Morphologically, accessions representing ca. 30 species in our sampling their plants all have similar dorsal and ventral leaves are resolved into six subclades: the S. braunii subclade, and share the rosette-forming habit. the S. delicatula subclade, the S. pennata subclade, the S. pervillei subclade, the S. siamensis subclade and the Clade IX—the Rosulatae clade S. willdenowii subclade. All of these subclades are strongly supported as monophyletic except the The Rosulatae clade (IX) approximates Selaginella S. delicatula subclade whose monophyly is moderately ser. Rosulatae (A. Braun) Baker (1883), and contains at supported, and the S. siamensis subclade and the least 12 species. Members of this clade occur in season- S. pervillei subclade, which are monospecific in our ally dry areas and are rosette, tufted or erect plants. sampling. Their branches curl up during drought (“resurrection plants”) and the ventral leaves usually have a membra- The Selaginella braunii subclade nous and lacerate margin. Their megaspores have a coarse surface and irregularly sized and spaced verrucae This subclade contains two xerophytic species, (Fig. 3d) (e.g. Minaki, 1984; Liu et al., 1989; Korall and S. braunii Baker and S. mairei H. Lev. The first spe- Taylor, 2006; our own observations) and their micro- cies was assigned to S. ser. Caulescentes Baker (1887) spores are globose (Liu et al., 1989; our own observa- and both were assigned to the same series by Walton tions). Although the rosette form is also present in other and Alston (1938). Morphologically, the two species species, such as S. lepidophylla (see above) and are erect plants with monomorphic sterile leaves on S. pallescens (C. Presl) Spring, the megaspore surfaces the main stem and dimorphic leaves on branches, of these species are entirely different from those of the involute leaves when dry, and two-banded steles Rosulatae clade. The megaspores of S. pallescens have (although the stem has only one “X”- or “V”-shaped very fine reticulate ornamentation, while the proximal vascular bundle in S. mairei, its rhizome clearly has surfaces of the megaspores of S. lepidophylla are reticu- two vascular bundles) (Chu, 2006). These morphologi- late and the distal surfaces have coarse, sparse, hamu- cal features are not present in other subclades in the late, and high and wide ridges (Korall and Taylor, Oligomacrosporangiatae clade. 2006). The rosette form appears to have independently Megaspore surfaces are tuberculate, papillate and evolved at least three times, once in the S. lepidophylla verrrucate, and these elements are usually combined clade, in S. pallescens and in the Rosulatae clade. (Fig. 3f), and microspores have broadly blunt Our study, with eight Old World species and one spines, sometimes lamellate (Fig. 3r) (e.g. Liu et al., New World species included, shows that Selaginella 1989, 2006; Zhou et al., 2012; Xia et al., 2013). subg. Rosulatae is strongly supported as monophyletic, Walton and Alston (1938) thought that those species consistent with the result of Korall and Kenrick (such as S. biformis A. Braun ex Kuhn, S. moellen- (2002). This clade is strongly supported as sister to dorffii) with monomorphic sterile stem leaves were clo- superclades A+B, which also have dimorphic leaves. sely related, a hypothesis rejected by our study that Within the Rosulatae clade, the Asian species (the shows that these species are scattered in different S. stauntoniana subclade) are resolved as sister to those major clades. from the Arabian Peninsula, Africa/Madagascar, and North America (the S. imbricata subclade) (ML The Selaginella delicatula subclade BS = 100%, MP JK = 100%, PP = 1.00). Baker (1884) included Selaginella convoluta (Arn.) This subclade contains six Asian species and a Cen- Spring (America), S. bryopteris (L.) Baker (India), tral American species (S. hoffmannii Hieron.) in our S. pringlei Baker (Mexico), etc., in this clade. Further current sampling. The subclade is divided into two data will be necessary to test these hypotheses. strongly supported clades—the S. plana grouping [in- cluding S. hoffmannii Hieron., S. mayeri Hieron., Clade X—the Oligomacrosporangiatae clade S. plana (Desv. ex Poir.) Hieron.] and the S. delicatula grouping [including S. delicatula, S. picta A. Braun ex The Oligomacrosporangiatae clade (X) presents a Baker and S. wallichii (Hook. & Grev.) Spring] complex group. Members of this clade show numerous (Fig. 2c). Having three vascular bundles (the number Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 19 of vascular bundles in S. hoffmannii and S. mayeri is Sadebeck (1901) also placed S. braunii with the unknown) and entire or subentire leaves, this subclade S. pervillei group under their S. sect. Pleiomacrospo- is similar to the S. willdenowii subclade (see below). rangiatae ser. Monostelicae. This group in their defini- The members of this subclade are erect or suberect tion also includes three other species [S. eublepharis (except S. mayeri), with megaspore surfaces typically AI. Br., S. fulcrata (Ham.) Spring and S. vogelii tuberculate (Fig. 3h), sometimes combined (e.g. Min- Spring], which are probably part of this clade. aki, 1984; Chang et al., 2002a; Korall and Taylor, Megaspore surfaces of S. pervillei are sparsely papil- 2006; Xia et al., 2013; Singh et al., 2014), and micro- late or tuberculate (Stefanovic et al., 1997; Korall and spore surfaces usually bluntly spiny (Fig. 3t) (e.g. Hell- Taylor, 2006) and microspore surfaces are tuberculate wig, 1969; Chang et al., 2002b; Xia et al., 2013), (Stefanovic et al., 1997). It is similar to the S. delicat- different from the members of the S. willdenowii sub- ula subclade. clade. Note that three of the members of the S. deli- catula subclade, S. delicatula, S. plana and S. wallichii, The Selaginella siamensis subclade have the chromosome number 2n = 20 (Kuriachan, 1963; Jermy et al., 1967), while the S. willdenowii This subclade contains only Selaginella siamensis subclade has 2n = 18 (see below). These features all Hieron. from Southeast Asia in our sampling. This corroborate our result that the two subclades are not species is similar to the members of the S. willdenowii closely related. subclade. However, this species occurs in drier habitats Within the Selaginella delicatula subclade, S. hoff- than members of the S. willdenowii subclade. Walton mannii is unique in terms of morphology and geogra- and Alston (1938) placed S. siamensis in a xerophytic phy: it is distributed in the Americas and its leaves series and thought this species may be an intermediate have ciliate margins. stage between taxa inhabiting xeromorphic versus other habitats. This species has only one stele, its The Selaginella pennata subclade megaspore surfaces are regularly verrucose (Fig. 3i), and microspores have regular and twisted lamellate This subclade is strongly supported as monophyletic ornamentation (Fig. 3u) (Liu et al., 2003). These mor- (ML BS = 100%, MP JK = 100%, PP = 1.00) and phological features are different from other members contains two species [S. bisulcata Spring and S. pen- of the Heterostachys clade. nata (D. Don) Spring] in our sampling. These two spe- cies are very similar to each other morphologically, The Selaginella willdenowii subclade but their megaspore ornamentations differ. These two species are the only ones with dimorphic sporophylls This subclade contains ca. six tropical Asian species in the Oligomacrosporangiatae clade, and all the in our sampling. These species share the following fea- remaining Asian/Pacific islands species of the genus tures: (i) stems have three-banded vascular bundles [ex- with dimorphic sporophylls are resolved as members cept that of S. uncinata]; (ii) plants are scandent and of the Heterostachys clade (XIII). Morphologically, usually 1 m long or longer; (iii) megaspore surfaces the distinguishing features of the S. pennata subclade have tuberculate–reticulate composite ornamentation include dimorphic sporophylls, obovate dorsal leaves (Fig. 3g) (e.g. Minaki, 1984; Liu et al., 2003; Chu, and a swelling vein apex of ventral leaves. Addition- 2006; Zhu et al., 2006; Korall and Taylor, 2006; Singh ally, the exospores of the megaspores of this subclade et al., 2014; our own observations) and microspore are smooth (Fig. 3j, ‘B’), which is characteristic of this distal surfaces have long and twisted lamellate or subclade (Chang et al., 2002a; Liu et al., 2003). Our widely blunt–spiny ornamentation (Fig. 3s) (e.g. Liu study shows that the dimorphic sporophylls in Se- et al., 1989, 2003; Li et al., 2003); (iv) all leaves (dorsal laginella evolved at least twice independently, in the leaves, ventral leaves, axillary leaves and sporophylls) S. pennata subclade and in the Heterostachys clade. are entire; and (v) the chromosome number is 2n = 18 [three species, S. uncinata, S. willdenowii (Desv. ex The Selaginella pervillei subclade Poir.) Baker and S. limbata Alston, have been exam- ined] (Jermy et al., 1967; Takamiya, 1993). In addi- This subclade contains only Selaginella pervillei tion, the leaves of some species (such as S. uncinata Spring in our sampling. Plants of this species are erect and S. willdenowii) have iridescent schemochromic and have monomorphic stem leaves, and pubescent tydall-blue colour (Setyawan, 2005). Two species of stems and branches. These morphological features are this subclade, S. uncinata and S. willdenowii, have similar to those of members of the S. brunii subclade, been widely introduced to the Americas. especially those of S. braunii. Our study shows, how- Based on the long stems, entire leaves, and similar ever, that they are not closely related, although they megaspores and microspores to those of the S. are in the same larger clade (Fig. 2c). Hieronymus and willdenowii subclade (Zhang et al., 2005), the Hainan 20 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 endemic S. hainanensis X. C. Zhang & Noot. is proba- S. ciliaris and S. heterostachys from the Old World, and bly a member of this subclade. S. flagellata and S. hartwegiana from the New World) on the basis of their non-resupinate sporophylls and Clade XI—the Selaginella douglasii clade loose strobili. Minaki (1984) studied the megaspore morphology of four species of this clade (S. denticulata, This clade contains only the North American species S. nipponica Franch. & Sav., S. helvetica, S. tama- Selaginella douglasii (Hook. & Grev.) Spring in our montana Seriz.) and showed that they had similar tuber- sampling. This species has a restricted geographical culate and/or vermiculate megaspore surfaces (Fig. 3e) distribution and is found only in California, Idaho, and wall structures. This sort of megaspore morphology Oregon, Washington and British Columbia (Jones, is not otherwise known in Selaginella. In fact members 1964; Valdespino, 1993). Hieronymus and Sadebeck of the Homostachys clade have obviously disconnected (1901) placed it in their S. douglasii group, one of their laesurae at the pole of the megaspore (Fig. 3e, ‘A’), 28 groups under their S. sect. Pleiomacrosporangiatae which have long been ignored but earlier established by ser. Monostelicae, with three other species: S. delicatis- several studies (e.g. Minaki, 1984; Liu et al., 1989, 2003; sima Linden ex A. Braun, S. saccharata A. Braun and Korall and Taylor, 2006). S. reflexa Underw. The last species is a member of Although Selaginella longistrobilina P.S. Wang, X.Y. clade I, but the other two may not be members of this Wang & Li Bing Zhang (endemic to China) (Zhang clade. The S. douglasii clade was resolved as sister to et al., 2012a) was not sampled in our analysis, it is prob- the Homostachys clade in our study (ML BS = 82%, ably a member of this clade based on its morphology. MP JK = 74%, PP = 0.97). This sister relationship is supported by the short and creeping habit and verru- Clade XIII—the Heterostachys clade cate ornamentation on megaspore surface (Tryon, 1949). However, this species has uniform strobili and This clade contains species occurring mainly in Asia auriculate leaf bases, distinguishing it from members and a small number of species in the Pacific islands, of the Homostachys clade, but similar to members of and includes nearly all Asian species with resupinate the Oligomacrosporangiatae clade. strobili (except S. bisulcata and S. pennata; see above). Morphologically, members of this clade have dimor- Clade XII—the Homostachys clade phic sporophylls and resupinate strobili. However, none of the American species with dimorphic sporo- This clade approximates Selaginella subg. Ho- phylls belongs in the Heterostachys clade, which shows mostachys Baker (1883), which was recognized by that dimorphic sporophylls evolved independently in Baker (1887), Warburg (1900) and Walton and Alston Asian and American species. In our study, species of (1938), but its members were included in either the Heterostachys clade were resolved into two moder- S. subg. Homoeophyllae (Spring, 1850), S. sect. Di- ately supported subclades: the S. heterostachys sub- chotropae (Braun, 1857), S. subg. Heterophyllum clade (from Asia) and the S. ciliaris subclade (from (Hieronymus and Sadebeck, 1901) or S. subg. Stachyg- Asia and the Pacific islands). The S. heterostachys sub- ynandrum (Jermy, 1986, 1990). Previous molecular clade contains most Asian species with tuberculate and analyses based on sparse sampling included them in verrucose megaspore surfaces (Fig. 3k) (e.g. Liu et al., S. subg. Stachygynandrum (Korall et al., 1999; Korall 1989, 2003, 2006; Zhou et al., 2012; Xia et al., 2013) and Kenrick, 2002). In our study, 29 accessions repre- including the newly described S. daozhenensis Li Bing senting ca. 14 Asian and one Mediterranean species Zhang, Q.W. Sun & Jun H. Zhao (Sun et al., 2015), (S. denticulata Spring) were sampled. The monophyly while the S. ciliaris subclade contains species of Asia of this clade was well established. Morphologically, and the Pacific islands with megaspore surfaces that species of this clade share dimorphic leaves, creeping are mainly finely reticulate (Fig. 3l). stems and strobili non-resupinate (i.e. with dorsal In the S. ciliaris subclade, the Asian species were sporophylls smaller than ventral ones), except for resolved as sister to the Pacific island species (moder- S. denticulata Spring and S. helvetica (L.) Link, which ately supported: ML BS = 69%, MP JK = 82%, PP = have nearly isomorphic strobili. Based on the mor- 0.99). The megaspores of the six Asian species [S. phology of the strobili, the Homostachys clade can be albociliata P.S. Wang, S. ciliaris (Retz.) Spring, divided into two groups: (1) those with dimorphic stro- S. lutchensis Koidz., S. repanda (Desv. ex Poir.) bili that are loose and non-resupinate; and (2) those Spring, S. compta Spring, S. xipholepis Baker] and with strobili that are approximately monomorphic and Hawaiian species (S. arbuscula Spring) share an obvi- loose (S. denticulata and S. helvetica). ously open and fine reticulation (Fig. 3l) (except Although species of the Homostachys clade have S. compta) (e.g. Minaki, 1984; Liu et al., 2003; Liu dimorphic sporophylls, they are obviously different and Yan, 2004; Zhu et al., 2006; Singh et al., 2014). from other species with dimorphic sporophylls (e.g. This megaspore feature occurs only in this subclade. Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 21

Based on the features of the megaspore morphology The Selaginella davidii subclade (Singh et al., 2014) two Indian species, S. panchghani- ana Dixit and S. proniflora (L.) Baker, are probably This subclade contains two species in our sampling: members of the S. ciliaris subclade. Also, most other S. davidii and S. gebaueriana. Plants of this subclade Asian species with resupinate strobili probably fall are creeping and their chromosome numbers are here. unknown. The two species are difficult to distinguish from each other and were treated as one species (e.g. Clade XIV—the Selaginella biformis clade Alston, 1934; Zhang et al., 2013), two subspecies (Zhang, 2004) or two species (Kung, 1988; Wang, This clade contains only one Asian species, Se- 1990; Chu, 2006). In our phylogenetic analysis S. da- laginella biformis A. Braun ex Kuhn, in our sampling. vidii was resolved as sister to S. gebaueriana with This species has two forms of habit: creeping flagelli- strong support. form plants and erect plants. Its flagelliform plants are glabrous and have no strobili, while its erect plants are The Selaginella involvens subclade pubescent and often have strobili (Zhang et al., 2013; our own observations). The flagelliform habit is also This subclade contains two species in our sampling: present in some species of the S. pallescens clade S. involvens and S. moellendorffii. Plants of this sub- (XVI), such as S. porphyrospora A. Braun and S. flag- clade are erect and have a chromosome number of ellata Spring, and the Articulatae clade (VI), such as 2n = 20 (Takamiya, 1993), and S. involvens also has a S. sertata Spring, and these species all develop a flagel- varied number of 2n = 18–20 (Jermy et al., 1967). In liform structure from the apex of the stem or branch addition, S. involvens often grows on rocks and trees, (Mickel and Smith, 2004). In contrast, the flagelliform while S. moellendorffii can usually be found on the structure of S. biformis is developed from the base of ground. Because of their similar morphology, S. invol- erect plants. vens and S. moellendorffii are often confused (Gu Selaginella biformis is similar to two species of the et al., 2013), but the former has an equatorial flange S. involvens clade (XV), S. involvens (Sw.) Spring and on its megaspore surfaces (Fig. 3n, ‘C’). S. moellendorffii Hieron., in megaspore morphology An equatorial flange similar to that of S. involvens is (Fig. 3m,o) and the morphology of leaves on the erect also present in S. anceps A. Braun (Giorgi et al., stems. These species also share the chromosome num- 1997), a member of our S. pallescens clade (XVI). ber of 2n = 20 (Takamiya, 1993). Meanwhile, the sepa- These two species also share erect plant habit and rate ITS analysis resolved the clade XIV and clade XV microspore morphology, but the two were resolved in as sister clades, with moderate support (ML BS = different clades in the Stachygynandrum superclade, 80%, MP JK = 84%) (tree not shown). The megas- suggesting that the equatorial flange may have evolved pores of S. biformis are reticulate (Fig. 3m) (Minaki, twice independently. 1984; Liu et al., 1989) and the microspores are usually Clades XVI–XIX—the OPHA clade (the S. oaxacana sterile (our own observations). clade, S. pallescens clade, S. hartwegiana clade and S. anceps clade). Clade XV—the Selaginella involvens clade These four clades together (the OPHA clade) were This clade contains ca. four Asian species (Se- weakly supported (ML BS = 45%, MP JK < 50%, laginella davidii Franch., S. gebaueriana Hand.-Mazz., PP = 0.74), and are composed of only New World spe- S. involvens (Sw.) Spring and S. moellendorffii cies. Also these species share consistent megaspore Hieron.). Ventral leaves of the four species usually morphology, which are typically reticulate (Tryon, have two light-coloured bands on the sides of veins 1949; Hellwig, 1969; Korall and Taylor, 2006). They (Chu, 2006) and their megaspore surfaces are reticu- are suberect, rarely creeping or erect, and have dimor- late (Fig. 3n,o) (e.g. Minaki, 1984; Liu et al., 1989; phic leaves. These clades present various chromosome Sun et al., 2002; Xia et al., 2013). Selaginella invol- numbers: 2n = 18 [S. apoda (L.) Fernald and S. sim- vens has a unique equatorial flange (Fig. 3n, ‘C’) plex Baker] (Graustein, 1930; Marcon et al., 2005), (Minaki, 1984; Liu et al., 1989; Xia et al., 2013). 2n = 20 [S. erythropus Spring, S. flabellata (L.) Spring Their microspores are baculate (S. davidii, S. gebaue- and S. pulcherrima Liebm.] (Jermy et al., 1967). Mean- riana and S. moellendorffii) or tuberculate and blunt– while 2n = 20 (Jermy et al., 1967) and 22 (Tschermak- spiny (S. involvens) (our own observations). This Woess and Dolezal-Janish, 1959) both occur in clade was further resolved into two subclades: a S. pallescens (C. Presl) Spring. Species of these clades weakly supported S. involvens/S. moellendorffii sub- with monomorphic or dimorphic sporophylls were clade and a strongly supported S. davidii/S. gebaueri- assigned to S. subg. Heterostachys and S. subg. ana subclade. Stachygynandrum, respectively. The treatment was 22 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 adopted by Walton and Alston (1938) and Jermy monomorphic sporophylls and reticulate megaspore (1986). Our results, consistent with those of Korall surfaces (Reeve, 1935; Tryon and Lugardon, 1991; and Kenrick (2002), show that neither S. subg. Hetero- Korall and Taylor, 2006). However, members of this stachys nor S. subg. Stachygynandrum is mono- clade present variable numbers of chromosomes phyletic. (S. apoda:2n = 18; S. pallescens:2n = 20, 22). Within In addition to the species with dimorphic sporo- the clade, S. pallescens presents great variation in phylls sampled by Korall and Kenrick (2002), we sam- habit (rosette to erect) and number of chromosomes pled three species with dimorphic sporophylls: (2n = 20, 22). In our study, we added seven samples of S. flagellata, S. hartwegiana Spring and S. porphy- S. pallescens to the existing data, revealing a series of rospora. Morphologically, these species are usually complicated phylogenetic relationships. More morpho- small and creeping in habit and have resupinate stro- logical observations and cytological study are neces- bili and dorsal sporophylls with sporophyll-pteryx sary to understand the species delimitations in this (Quansah and Thomas, 1985). The New World species complex. with dimorphic sporophylls are similar to the Asian species in the Heterostachys clade (XIII), but they have Clade XVIII—the Selaginella hartwegiana clade very different megaspore morphologies. Megaspore surfaces of the species of clade XIII are tuberculate, This clade contains species occurring in Central to verrucose or papillate. According to Quansah and South America including Selaginella hartwegiana and Thomas (1985), about 14 species with dimorphic S. novae-hollandiae. They have stem nearly erect, sporophylls are distributed in West Africa and about different from those species with dimorphic sporo- 20 species in the Americas. Up to now, only seven phylls from America that often have creeping stems. American species with dimorphic sporophylls have They have dimorphic sterile leaves on the stem, differ- been sampled in molecular studies, but no African spe- ent from those species with erect stems and monomor- cies have been sampled. To understand the evolution phic sporophyll species from America that have of dimorphic sporophylls well, more American and monomorphic sterile leaves on the stem (Valdespino, African species need to be included in the phylogenetic 1993). framework of Selaginella. The relationships among 45 accessions representing Clade XIX—the Selaginella anceps clade ca. 28 species in our sampling within the OPHA clade are poorly resolved, but four strongly/weakly sup- This clade was weakly supported as monophyletic ported monophyletic clades are identified in our study: (Fig. 2e) and is composed of only New World (South the S. oaxacana clade (XVI), the S. pallescens clade & Central America) species. These species share typi- (XVII), the S. hartwegiana clade (XVIII) and the cally reticulate megaspore surfaces (Tryon, 1949; Hell- S. anceps clade (XIX). wig, 1969; Korall and Taylor, 2006). They are suberect, rarely creeping or erect (if plants are erect, Clade XVI—the Selaginella oaxacana clade main stems have monomorphic leaves), and have dimorphic leaves and various chromosome numbers: This clade contains ca. three species in our sampling. 2n = 18 (S. simplex Baker) (Graustein, 1930; Marcon All species have reticulate megaspore surfaces et al., 2005), 2n = 20 [S. erythropus Spring and S. [S. martensii Spring: Giorgi et al., 1997; Korall and flabellata (L.) Spring] (Jermy et al., 1967). Taylor, 2006; S. novae-hollandiae (Sw.) Spring: Korall and Taylor, 2006; S. oaxacana Spring: Hellwig, 1969]. Clade XX—the Selaginella doederleinii clade Although S. novae-hollandiae has dimorphic sporo- phylls, the megaspore morphology and our molecular This clade was first found by Korall and Taylor data all show that the three species in this clade are (2006). They observed megaspore morphology of five closely related with one another. However, another species from Asia and Australia and found that their accession of S. novae-hollandiae from Ecuador has ver- megaspores have a surface with reticulum (Fig. 3p,q) rucate and spiny megaspore surfaces (Korall and Tay- and a unique zona at the equator (Fig. 3p, ‘D’). In our lor, 2006). The two accessions were resolved as being analysis, 18 accessions representing ca. 13 species across far away on our tree (Fig. 2e) and apparently repre- China, Japan, Vietnam and Australia are sampled. Our sent different species (see below). study further supports this clade as monophyletic. The megaspore and microspore morphology of more species Clade XVII—the Selaginella pallescens clade have been observed, such as that of S. doederleinii (Liu et al., 1989), S. frondosa Warb. (Liu et al., 2001), This clade contains ca. nine species in our sampling. S. commutata Alderw. (Zhao et al., 2006b; Fig. 3p,q), Morphologically, all species of the clade have and S. trichophylla K.H. Shing and S. scabrifolia Ching Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 23

& Chu H. Wang (our own observations). These results Harholt, J., Hattori, M., Heyl, A., Hirai, T., Hiwatashi, Y., all support the monophyly of this clade. Also, consistent Ishikawa, M., Iwata, M., Karol, K.G., Koehler, B., Kolukisaoglu, U., Kubo, M., Kurata, T., Lalonde, S., Li, K., Li, baculate microspores support the monophyly of the Y., Litt, A., Lyons, E., Manning, G., Maruyama, T., Michael, clade (Zhao et al., 2006a; our own observations). Based T.P., Mikami, K., Miyazaki, S., Morinaga, S., Murata, T., on the megaspore and microspore morphology (Liu Mueller–Roeber, B., Nelson, D.R., Obara, M., Oguri, Y., Olmstead, R.G., Onodera, N., Petersen, B.L., Pils, B., Prigge, et al., 2001; our own observations), S. rolandi-principis M., Rensing, S.A., Riano–Pachon, D.M., Roberts, A.W., Sato, Alston, a species distributed Asia and with extremely Y., Scheller, H.V., Schulz, B., Schulz, C., Shakirov, E.V., large ventral leaves (~7 mm), appears to be a member of Shibagaki, N., Shinohara, N., Shippen, D.E., Sorensen, I., this clade. Sotooka, R., Sugimoto, N., Sugita, M., Sumikawa, N., Tanurdzic, M., Theissen, G., Ulvskov, P., Wakazuki, S., Weng, J.K., Willats, W.W., Wipf, D., Wolf, P.G., Yang, L., Zimmer, A.D., Zhu, Q., Mitros, T., Hellsten, U., Loque, D., Otillar, R., Conclusions Salamov, A., Schmutz, J., Shapiro, H., Lindquist, E.,Lucas, S., Rokhsar, D., Grigoriev, I.V., 2011. 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Zhu, Y.Q., Wang, G.Z., Liu, X.R., Zhang, Q.Y., Sun, X.H., Liu, Zhang & al. 6923 (CDBI, MO, VNMN, PYU), Vietnam (Ha Giang): J.X., 2006. Spore morphology of Selaginellaceae from torrid and rbcL KT161398, ITS KT161668; (2) Zhang & al. 6729 (CDBI, MO, subtorrid zone. J. Jishou Univ. Nat. Sci. 27, 89–94, 12, 8. VNMN, PYU), Vietnam (Bac Kan): rbcL KT161399, ITS Zurawski, G., Clegg, M.T., Brown, A.H.D., 1984. The nature of KT161667; (3) Jiang 316 (PYU, CDBI), China (Hainan): rbcL nucleotide sequence divergence between barley and maize KT161394, ITS KT161665; (4) He & Zhou 112 (PYU, CDBI), China – chloroplast DNA. Genetics 106, 735 749. (Hainan): rbcL KT161395, ITS KT161663; (5) Zhang & al. 6687 (CDBI, MO, VNMN, PYU), Vietnam (Bac Kan): rbcL KT161400; (6) Jiang 309 (PYU, CDBI), China (Hainan): rbcL KT161396, ITS Appendix KT161664; (7) Zhang & al. 6685 (CDBI, MO, VNMN, PYU), Viet- nam (Bac Kan): rbcL KT161397, ITS KT161666; (8) TNS777848 List of taxa sampled with information related to taxonomy, Gen- (TNS), Japan (Okinawa): rbcL AB574641 (Ebihara et al., 2010); Bank accession numbers, references and voucher information. Her- Selaginella bigelovii Underw., (1) Therrien s.n. (KANU), USA (Cali- barium acronyms follow Index Herbariorum (Holmgren and fornia): rbcL AF419082 (Arrigo et al., 2013), ITS AF419004 (Arrigo Holmgren, 1998). et al., 2013); (2) Rothfels & al. 2555 (DUKE), USA (California): Isoetes€ kersii Wanntorp, Kers 3130 (S), South Africa: rbcL rbcL KT161401, ITS KT161669; (3) Rothfels & al. 2556 (DUKE), AF404498 (Rydin & Wikstrom,€ 2002); Isoetes€ kirkii A. Braun, Chin- USA (California): rbcL KT161402, ITS KT161670; (4) Rothfels & al. nock 447 (BM), New Zealand: rbcL AF404499 (Rydin & Wikstrom,€ 2559 (DUKE), USA (California): rbcL KT161403, ITS KT161671; 2002); Isoetes€ nuttallii A. Braun ex Engelm., Unknown, rbcL Selaginella bisulcata Spring, (1) Chu & al. 31292 (PYU), China (Yun- DQ294246 (Schuettpelz & Hoot, 2006); Isoetes€ sinensis Palmer, nan): rbcL KT161404, ITS KT161673; (2) Chu & al. 29311 (PYU), Unknown, rbcL GU564272 (X.Z. Kan, Z.C. Guo, and J. Wu, China (Yunnan): rbcL KT161405, ITS KT161672; (3) Chu & al. unpublished); Isoetes€ taiwanensis De Vol., Unknown, rbcL 21737 (PYU), China (Yunnan): rbcL KT161408; (4) He & al. 71201 DQ294250 (Schuettpelz & Hoot, 2006). (PYU), China (Yunnan): rbcL KT161406, ITS KT161675; (5) He & Selaginella acanthonota Underw., Rothfels & Rushworth 4132 Zhou 122 (PYU, CDBI), China (Yunnan): rbcL KT161407, ITS (DUKE), USA (North Carolina): rbcL KT161378, ITS KT161647; KT161674; Selaginella bodinieri Hieron., (1) Zhou & al. HGX11073 Selaginella acanthostachys Baker, Korall 14a (S), Unknown: rbcL (CDBI), China (Sichuan): rbcL KT161412, ITS KT161676; (2) Ju & AJ295884 (Korall and Kenrick, 2002); Selaginella albociliata P.S. Deng HGX13005 (CDBI), China (Sichuan): rbcL KT161413, ITS Wang, Zhang 5302 (CDBI), China (Guizhou): rbcL KT161379, ITS KT161678; (3) Ju & Deng HGX12828 (CDBI), China (Sichuan): rbcL KT161648; Selaginella alopecuroides Baker, Wikstrom€ & Wanntorp KT161414, ITS KT161677; (4) Zhang 5193 (CDBI), China (Guiz- nr146 (S), Borneo: rbcL AJ295875 (Korall and Kenrick, 2002); Se- hou): rbcL KT161410, ITS KT161681; (5) Zhang 5913 (CDBI), laginella amblyphylla Alston, (1) Zhang 5572 (CDBI), China (Guiz- China (Guizhou): rbcL KT161409, ITS KT161680; (6) Zhang 5177 hou): rbcL KT161380, ITS KT161651; (2) Gao & al. DJY04053 (CDBI), China (Guizhou): rbcL KT161411, ITS KT161679; (CDBI), China (Sichuan): rbcL KT161381, ITS KT161649; (3) Gao Selaginella bombycina Spring, (1) Rothfels & al. 08-204 (DUKE), & al. DJY06425 (CDBI), China (Sichuan): rbcL KT161382, ITS Costa Rica (Heredia): rbcL KT161415, ITS KT161682; (2) Korall 31 KT161650; Selaginella anceps (C. Presl) C. Presl, (1) Rothfels & al. (S), Unknown: rbcL AJ010848 (Korall et al., 1999); Selaginella 08-095 (DUKE), Costa Rica (Guanacaste): rbcL KT161383, ITS boninensis Baker., (1) TNS766618 (TNS), Japan (Tokyo): rbcL KT161652; (2) Rothfels & al. 08-156 (DUKE), Costa Rica (Heredia): AB574642 (Ebihara et al., 2010); (2) Zhang & al. 6594 (CDBI, MO, rbcL KT161384, ITS KT161653; Selaginella apoda (L.) C. Morren, VNMN, PYU), Vietnam (Lang Son): rbcL KT161416, ITS (1) Murrell 6484 (S), Unknown: rbcL AJ010854 (Korall et al., 1999); KT161868; (3) Zhang & al. 6587 (CDBI, MO, VNMN, PYU), (2) Rothfels & al. 2884 (DUKE), USA (Virginia): rbcL KT161386, Vietnam (Lang Son): rbcL KT161417, ITS KT161683; Selaginella ITS KT161655; (3) Rothfels & al. 2795 (DUKE), USA (Tennessee): braunii Baker, (1) Tiew 12 (CDBI), cult.: rbcL KT161421, ITS rbcL KT161385, ITS KT161654; Selaginella arbuscula Spring, (1) KT161869; (2) Chu & al. s.n. (PYU), China (Yunnan): rbcL Wood 13746 (PTBG), Hawaii (Maui, Kipahulu): rbcL KT161388, KT161419, ITS KT161684; (3) Zhang 1332 (PYU, CDBI), China ITS KT161657; (2) Wood 13447 (PTBG), Hawaii (Kauai, Wainiha): (Hainan): rbcL KT161420, ITS KT161686; (4) Zhao 272 (CDBI), rbcL KT161387, ITS KT161656; Selaginella arenicola Underw., Ter- China (Hubei): rbcL KT161418, ITS KT161685; Selaginella brooksii rel 19 (MO), USA (Louisiana): rbcL AF419084 (Arrigo et al., 2013), Hieron., Wikstrom€ & Wanntorp nr141 (S), Borneo: rbcL AJ295876 ITS AF419008 (Arrigo et al., 2013); Selaginella arizonica Maxon, (1) (Korall and Kenrick, 2002). Rothfels & al. 2539 (DUKE), USA (Arizona): rbcL KT161389, ITS Selaginella caffrorum (Milde) Hieron., Ventor and Vorster s.n. KT161658; (2) Eriksson 10/13 (S) Unknown: rbcL AJ010851 (Korall (US), South Africa (Pretoria): rbcL AF419070 (Arrigo et al., 2013), et al., 1999); (3) Therrien and Windham s.n. (KANU), USA (Ari- ITS AF419017 (Arrigo et al., 2013); Selaginella chaetoloma Alston, zona): rbcL AF419078 (Arrigo et al., 2013), ITS AF419009 (Arrigo (1) He & al. 29625 (PYU), China (Yunnan): rbcL KT161424, ITS et al., 2013); Selaginella arsenei Weath., (1) Rothfels & al. 2991 KT161689; (2) Chu & al. 29610 (PYU), China (Yunnan): rbcL (DUKE), Mexico (Hidalgo): rbcL KT161390, ITS KT161659; (2) KT161425, ITS KT161688; (3) Chu & al. 24404 (PYU), China (Yun- Ferguson 14 (MO), Mexico (San Luis Potosı): rbcL AF419056 (Ar- nan): rbcL KT161426, ITS KT161687; Selaginella chrysocaulos rigo et al., 2013), ITS AF419029 (Arrigo et al., 2013); Selaginella (Hook. & Grev.) Spring, Xu & al. 2014-156 (CDBI), China arthritica Alston (1) Rothfels & al. 2669 (DUKE), Costa Rica (Here- (Sichuan): rbcL KT161427, ITS KT161690; Selaginella ciliaris (Retz.) dia): rbcL KT161391, ITS KT161661; (2) Rothfels & al. 08-185 Spring, (1) Jiang 310 (PYU, CDBI), China (Hainan): rbcL (DUKE), Costa Rica (Heredia): rbcL KT161392, ITS KT161660; KT161428, ITS KT161691; (2) Unknown, Peninsula Malaysia: rbcL Selaginella articulata (Kunze) Spring, Eriksson s.n. (S), Unknown: EU126658 (Y. Yi, Y. He, and B. Tan, unpublished); Selaginella cin- rbcL AJ295894 (Korall and Kenrick, 2002); Selaginella asprella erascens A.A. Eaton, (1) Rothfels & al. 2557 (DUKE), USA (Califor- Maxon, Therrien s.n. (KANU), USA (California): rbcL AF419064 nia): rbcL KT161429, ITS KT161692; (2) Palmer 2651 (KANU), (Arrigo et al., 2013), ITS AF419022 (Arrigo et al., 2013); Selaginella USA (California): rbcL AF419063 (Arrigo et al., 2013), Therrien s.n. cf. attirense, (1) Rothfels & al. 2657 (DUKE), Costa Rica (Gua- (KANU), USA (California):ITS AF419027 (Arrigo et al., 2013); Se- nacaste): rbcL KT161393; (2) Rothfels & al. 2611 (DUKE), Costa laginella commutata Alderw., Zhou 2441 (PYU), China (Guangxi): Rica (San Jose): rbcL KT161422, ITS KT161662. rbcL KT161430, ITS KT161693. Selaginella compta Hand-Mazz. (1) Selaginella balansae (A. Br.) Hieron., Gattefosse s.n. (S), Morocco Zhou & al. DJY07488 (CDBI), China (Sichuan): rbcL KT161432, (Tamegdoult): rbcL AF419080 (Arrigo et al., 2013), ITS AF419005 ITS KT161694; (2) Zhou 012 (CDBI), China (Sichuan): rbcL (Arrigo et al., 2013); Selaginella biformis A. Braun ex Kuhn, (1) KT161434; (3) Luo 1378 (CDBI), China (Guizhou): rbcL KT161431, Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 27

ITS KT161695; (4) Sun 2001 (CDBI), China (Guizhou): rbcL Korall 1996:3 (S), Unknown: rbcL AJ295872 (Korall and Kenrick, KT161433; (5) He H3384 (CTC) China (Sichuan): rbcL KT161435. 2002); Selaginella frondosa Warb., (1) Wikstrom€ & Wanntorp nr126 Selaginella daozhenensis Li Bing Zhang, Q.W. Sun & Jun H. (S), Unknown: rbcL AJ295874 (Korall and Kenrick, 2002); (2) Chu Zhao, Sun, Zhao & Pan 201435 (CDBI, GZTM), China (Guizhou): & al. 29274A (PYU), China (Yunnan): rbcL KT161459, ITS rbcL KT161436; Selaginella davidii Franch., Chu & al. 23308 (PYU), KT161713. China (Beijing): rbcL KT161437, ITS KT161696; Selaginella decipi- Selaginella gebaueriana Hand.-Mazz., (1) Deng 102 (CDBI), China ens Warb., (1) Zhang & al. 6764 (CDBI, MO, VNMN, PYU), Viet- (Guizhou): rbcL KT161460, ITS KT161716; (2) Chu & al.089 nam (Bac Kan): rbcL KT161438, ITS KT161698; (2) Zhang & al. (PYU), China (Yunnan): rbcL KT161463, ITS KT161715; (3) Zhang 6761 (CDBI, MO, VNMN, PYU), Vietnam (Bac Kan): rbcL 5804 (CDBI), China (Guizhou): rbcL KT161461, ITS KT161718; (4) KT161439, ITS KT161697; Selaginella deflexa Brack., Palmer 2651 Shui 40568 (PYU), China (Yunnan): rbcL KT161464, ITS (KANU), Hawaii (Honolulu): rbcL AF093253 (Arrigo et al., 2013), KT161719; (5) Zhang 5735 (CDBI), China (Guizhou): rbcL ITS AF418999 (Arrigo et al., 2013); Selaginella delicatula (Desv. ex KT161462, ITS KT161717; (6) Chu & He 29611 (PYU), China (Yun- Poir.) Alston, (1) Ju & Deng HGX12938 (CDBI), China (Sichuan): nan): rbcL KT161465, ITS KT161714; Selaginella gracillima rbcL KT161440, ITS KT161700; (2) Gao & al. HGX10734 (CDBI), (Kuntze) Alston, Nordenstam & Anderberg 1124 (S), Australia (Tas- China (Sichuan): rbcL KT161441, ITS KT161699; Selaginella densa mania): rbcL AJ010844 (Korall et al., 1999); Selaginella grisea Rydb., (1) Bjork€ 25055 (UBC), Canada (British Columbia): rbcL Alston, Unknown, rbcL AF419072 (Arrigo et al., 2013). KT161442, ITS KT161701; (2) Therrien s.n. (KANU), USA (Color- Selaginella haematodes (Kunze) Spring, Korall 1996:15b (S), ado): rbcL AF419069 (Arrigo et al., 2013), ITS AF419036 (Arrigo Unknown: rbcL AJ010846 (Korall et al., 1999); Selaginella hansenii et al., 2013); Selaginella denticulata Spring, Korall & Eriksson 715 Hieron., Weber 15108 (COLO), USA (California): rbcL AF419057 (S), Unknown: rbcL AJ010853 (Korall et al., 1999); Selaginella dif- (Arrigo et al., 2013), ITS AF419033 (Arrigo et al., 2013); Selaginella fusa (C. Presl) Spring, (1) Rothfels & al. 08-087 (DUKE), Costa Rica harrisii Underw. & Hieron., (1) Beck 1124 (DUKE), Mexico (San (Puntarenas): rbcL KT161443, ITS KT161702; (2) Korall 33 (S), Luis Potosı): rbcL KT161466, ITS KT161720; (2) Beck 1112 cult.: rbcL AJ010852 (Korall et al., 1999); Selaginella digitata Spring, (DUKE), Mexico (Hidalgo): rbcL KT161467; Selaginella hartwegiana Rakotondrainibe 3255 (P), Madagascar (d’Andohahela): rbcL Spring, Rothfels & al. 3605 (DUKE), Ecuador (Loja): rbcL AJ295895 (Korall and Kenrick, 2002); Selaginella doederleinii KT161468, ITS KT161721; Selaginella helferi Warb., (1) He & Jiang Hieron., (1) Jiang 313 (PYU, CDBI), China (Hainan): rbcL 302 (PYU, CDBI), China (Yunnan): rbcL KT161469, ITS KT161444; (2) TNS763070 (TNS), Japan (Kagoshima): rbcL KT161722; (2) Zhang & al. 6919 (CDBI, MO, VNMN, PYU), Viet- AB574643 (Ebihara et al., 2010); (3) Zhang & al. 6447 (CDBI, MO, nam (Ha Giang): rbcL KT161471, ITS KT161724; (3) He & Jiang VNMN, PYU), Vietnam (Vinh Phuc): rbcL KT161445; Selaginella 406 (PYU, CDBI), China (Yunnan): rbcL KT161470, ITS douglasii (Hook. & Grev.) Spring, (1) Unknown, rbcL AF419049 KT161723; Selaginella helioclada Alston ex Alston, Rakotondrainibe (J.P. Therrien and C.H. Haufler, unpublished); (2) Rothfels & al. 3262 (P), Madagascar (d’Andohahela): rbcL AJ295896 (Korall and 3863 (DUKE), USA (Oregon): rbcL KT161446, ITS KT161870; Se- Kenrick, 2002); Selaginella helvetica (L.) Link, (1) Klackenberg laginella dregei (C. Presl) Hieron., Faden 9-85 (US), Kenya (Isiolo): 820617-12 (S), Unknown: rbcL AJ295891 (Korall and Kenrick, rbcL AF419055 (Arrigo et al., 2013), ITS AF419015 (Arrigo et al., 2002); (2) Zhou 093 (CDBI), China (Sichuan): rbcL KT161472, ITS 2013); Selaginella drepanophylla Alston, (1) Zhang 5117 (CDBI), KT161871; (3) TNS765114 (TNS), Japan (Akita): rbcL AB574644 China (Guizhou): rbcL KT161447, ITS KT161703; (2) Zhang 5862 (Ebihara et al., 2010); Selaginella heterostachys Baker, (1) Jiang 317 (CDBI), China (Guizhou): rbcL KT161448. (PYU, CDBI), China (Hainan): rbcL KT161478, ITS KT161732; (2) Selaginella echinata Baker, Fosberg 52393 (US), Madagascar (Fia- Jiang 315 (PYU, CDBI), China (Hainan): rbcL KT161479; (3) narantsoa): rbcL AF419071 (Arrigo et al., 2013), ITS AF419021 (Ar- Zhang 5455 (CDBI), China (Guizhou): rbcL KT161473, ITS rigo et al., 2013); Selaginella effusa var. dulongjiangensis W. M. Chu, KT161733; (4) Zhang & al. 6641 (CDBI, MO, VNMN, PYU), Viet- Chu & al. 31299 (PYU), China (Yunnan): rbcL KT161449, ITS nam (Thai Nguyen to Kim Hy): rbcL KT161483, ITS KT161731; (5) KT161704; Selaginella effusa Alston, (1) Zhang 5438 (CDBI), China Zhang & al. 6608 (CDBI, MO, VNMN, PYU), Vietnam (Lang Son): (Guizhou): rbcL KT161450, ITS KT161706; (2) Dong 2470 (PYU), rbcL KT161481, ITS KT161729; (6) Zhang 5550 (CDBI), China China (Guangdong): rbcL KT161453, ITS KT161705; (3) Zhang (Guizhou): rbcL KT161474, ITS KT161727; (7) Zhang & al. 6617 5442 (CDBI), China (Guizhou): rbcL KT161451, ITS KT161707; (4) (CDBI, MO, VNMN, PYU), Vietnam (Lang Son): rbcL KT161482, Wang 20051 (CDBI), China (Guizhou): rbcL KT161452, ITS ITS KT161730; (8) Zhang 5655 (CDBI), China (Guizhou): rbcL KT161708; Selaginella eremophila Maxon, (1) Therrien s.n. (KANU), KT161475, ITS KT161728; (9) TNS769193 (TNS), Japan (Mie): USA (California): rbcL AF419079 (Arrigo et al., 2013), ITS rbcL AB574645 (Ebihara et al., 2010); (10) Gao & al. DJY05340 AF419010 (Arrigo et al., 2013); (2) Rothfels & al. 2549 (DUKE), (CDBI), China (Sichuan): rbcL KT161477, ITS KT161725; (11) USA (California): rbcL KT161454, ITS KT161710; (3) Rothfels & al. Zhang 5180 (CDBI), China (Guizhou): rbcL KT161476, ITS 2561 (DUKE), USA (California): rbcL KT161455, ITS KT161709; KT161726; (12) Zhang & al. 6390 (CDBI, MO, VNMN, PYU), Viet- Selaginella erythropus (Mart.) Spring, Korall 1998:2 (S), cult.: rbcL nam (Hoa Binh): rbcL KT161480, ITS KT161734; Selaginella hoff- AJ295877 (Korall and Kenrick, 2002); Selaginella eurynota A. mannii Hieron., (1) Rothfels & al. 08-088 (DUKE), Costa Rica Braun, Rothfels & al. 08-183 (DUKE), Costa Rica (Heredia): rbcL (Puntarenas): rbcL KT161485, ITS KT161735; (2) Rothfels & al. 08- KT161456; Selaginella exaltata (Kunze) Spring, Korall 1996:1 (S), 138 (DUKE), Costa Rica (Puntarenas): rbcL KT161484, ITS Unknown: rbcL AJ010849 (Korall et al., 1999); Selaginella extensa KT161736. Underw., McVaugh 11829 (US), Mexico (Jalisco): rbcL AF419085 Selaginella imbricata (Forssk.) Spring ex Decne., (1) Rothfels & al. (Arrigo et al., 2013), ITS AF419026 (Arrigo et al., 2013). 4275 (DUKE), Oman (Dhofar, Governorate) rbcL KT161486, ITS Selaginella firmuloides Warb., Wikstrom€ 233 (S), New Caledonia: KT161737; (2) Parris & Croxall 83/19 (P), Unknown: rbcL AJ295897 rbcL AJ295870 (Korall and Kenrick, 2002); Selaginella flabellata (L.) (Korall and Kenrick, 2002); Selaginella indica (Milde) R.M. Tryon, (1) Spring, Webster & al. 9553 (S), Grenada (Lesser Antilles): rbcL Saldanha 15198 (US), India (Hassan): rbcL AF419052 (Arrigo et al., AJ295885 (Korall and Kenrick, 2002); Selaginella flagellata Spring, 2013), ITS AF419020 (Arrigo et al., 2013); (2) Tiew 01 (CDBI), cult.: (1) Rothfels & al. 08-157 (DUKE), Costa Rica (Heredia): rbcL rbcL KT161488, ITS KT161739; (3) He & al. 29779 (PYU), China KT161457, ITS KT161711; (2) Ortega & al. 3090 (BM), Unknown: (Yunnan): rbcL KT161487, ITS KT161738; Selaginella intermedia rbcL AJ295866 (Korall and Kenrick, 2002); Selaginella flexuosa Spring Unknown, Peninsula Malaysia: rbcL EU086853 (Y. Yi, Y. He, Spring, Rothfels & Sigel 3627 (DUKE), Ecuador (Zamora-Chinch- and B. Tan, unpublished); Selaginella involvens (Sw.) Spring (1) Tiew ipe): rbcL KT161458, ITS KT161712; Selaginella fragilis A. Braun, 13 (CDBI), cult.: rbcL KT161492, ITS KT161743; (2) Zhang & al. 28 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

6576 (CDBI, MO, VNMN, PYU), Vietnam (Phu Tho): rbcL KT161521, ITS KT161766; Selaginella mayeri Hieron., Unknown, KT161493, ITS KT161742; (3) He & Zhou 103 (PYU, CDBI), China Peninsula Malaysia: rbcL EU197125 (Y. Yi, Y. He, and B. Tan, (Yunnan): rbcL KT161491; (4) Zhou & al. DJY03558 (CDBI), China unpublished); Selaginella megaphylla Baker, (1) Zhang & al. 6330 (Sichuan): rbcL KT161489, ITS KT161740; (5) TNS762549 (TNS), (CDBI, MO, VNMN, PYU), Vietnam (Hoa Binh): rbcL KT161527, Japan (Kagoshima): rbcL AB574646 (Ebihara et al., 2010); (6) He & ITS KT161771; (2) Zhang & al. 6935 (CDBI, MO, VNMN, PYU), Zhou 114 (PYU, CDBI), China (Yunnan): rbcL KT161490, ITS Vietnam (Ha Giang): rbcL KT161526, ITS KT161772; (3) Zhang KT161741.Selaginella jugorum Hand.-Mazz., Meili Exped. 25060 4958 (CDBI), China (Guizhou): rbcL KT161522, ITS KT161768; (4) (PYU), China (Yunnan): rbcL KT161494. Zhang 5185 (CDBI), China (Guizhou): rbcL KT161523, ITS Selaginella kanehirae Alston, (1) Wood 13568 (PTBG), F.S.M. KT161769; (5) Zhang 5200 (CDBI), China (Guizhou): rbcL (Caroline Is., Pohnpei): rbcL KT161495, ITS KT161745; (2) Wood KT161524, ITS KT161770; (6) Chu & al. 8226 (PYU), China (Yun- 13567 (PTBG), F.S.M. (Caroline Is., Pohnpei): rbcL KT161496, ITS nan): rbcL KT161525, ITS KT161767; Selaginella microphylla KT161744; Selaginella kerstingii Hieron., Korall 1998:8 (S), cult.: (Kunth) Spring, Rothfels & al. 3586 (DUKE), Ecuador (Carchi): rbcL AJ295881 (Korall and Kenrick, 2002); Selaginella kraussiana rbcL KT161528, ITS KT161773; Selaginella moellendorffii Hieron., (Kunze) A. Braun, (1) Chu & al. s.n. (PYU), cult.,: rbcL KT161497; (1) Ju & Deng HGX12307 (CDBI), China (Sichuan): rbcL (2) Zhou 062 (CDBI), cult.,: rbcL KT161498, ITS KT161746; (3) KT161529, ITS KT161775; (2) Ju & Deng HGX12475 (CDBI), China Korall 1997:30 (S), cult.: rbcL AJ010845 (Korall et al., 1999); (Sichuan): rbcL KT161530, ITS KT161776; (3) Ju & Deng Selaginella kunzeana A. Braun, Rothfels & al. 08-096 (DUKE), Costa HGX12295 (CDBI), China (Sichuan): rbcL KT161531, ITS Rica (Puntarenas): rbcL KT161499, ITS KT161747; Selaginella KT161774; (4) Rothfels & al. 08-090 (DUKE), introduced to Costa kurzii Baker, (1) He & Jiang 405 (PYU, CDBI), China (Yunnan): Rica (Puntarenas): rbcL KT161533, ITS KT161777; (5) Tiew 14 rbcL KT161500, ITS KT161749; (2) He & Zhou 117 (PYU, CDBI), (CDBI), cult.: rbcL KT161532, ITS KT161778; Selaginella mono- China (Yunnan): rbcL KT161501, ITS KT161748. spora Spring, (1) Zhang & al. 6430 (CDBI, MO, VNMN, PYU), Selaginella labordei Hieron. ex Christ, (1) Zhou & al. DJY03560 Vietnam (Vinh Phuc): rbcL KT161537, ITS KT161782; (2) Chu & al. (CDBI), China (Sichuan): rbcL KT161504; (2) Gao & al. DJY03894 31293 (PYU), China (Yunnan): rbcL KT161535, ITS KT161780; (3) (CDBI), China (Sichuan): rbcL KT161502, ITS KT161750; (3) Gao Jiao s.n. (PYU), China (Yunnan): rbcL KT161536, ITS KT161781; & al. DJY04311 (CDBI), China (Sichuan): rbcL KT161503, ITS Selaginella cf. monospora, Chu & al. 14863 (PYU), China (Yunnan): KT161751; (4) Zhou 008 (CDBI), China (Sichuan): rbcL KT161505; rbcL KT161534, ITS KT161779; Selaginella moratii Rauh & W. Selaginella landii Greenm. & N. Pfeiff., (1) Rothfels & al. 3111 Hagemann, Mabberley 913 (P), Madagascar (Fianarantsoa): rbcL (DUKE), Mexico (Jalisco): rbcL KT161506, ITS KT161752; (2) AJ295899 (Korall and Kenrick, 2002); Selaginella moritziana Spring Matuda 8-52 (US), Mexico (Moreles): rbcL AF419086 (Arrigo et al., ex Klotzsch, Korall 1996:12 (S), Unknown: rbcL AJ010856 (Korall 2013), ITS AF419012 (Arrigo et al., 2013); Selaginella laxistrobila et al., 1999); Selaginella mutica D.C.Eaton, Therrien s.n. (KANU), K.H. Shing, (1) He & al. 29624 (PYU), China (Yunnan): rbcL USA (Colorado): rbcL AF419058 (Arrigo et al., 2013), ITS KT161507, ITS KT161754; (2) He & al. 81841 (PYU), China (Yun- AF419025 (Arrigo et al., 2013); Selaginella mutica var. limitanea nan): rbcL KT161508, ITS KT161755; (3) Chu & al. 24449 (PYU), Weath., (1) Rothfels & al. 2505 (DUKE), USA (New Mexico): rbcL China (Yunnan): rbcL KT161509, ITS KT161753; Selaginella lepido- KT161538, ITS KT161872; (2) Rothfels & al. 2495 (DUKE), USA € phylla (Hook. & Grev.) Spring, (1) Therrien 1996:s.n. (KANU), (Texas): rbcL KT161539; Selaginella myosurus (Sw.) Alston, Gosta Unknown: rbcL AF093254 (Arrigo et al., 2013); (2) Worthington s.n. Lindeberg s.n. (S), Unknown: rbcL AJ295863 (Korall and Kenrick, (US), USA (Texas): rbcL AF419051 (Arrigo et al., 2013); Selaginella 2002). leptophylla Baker, (1) He & Zhou 101 (PYU, CDBI), China (Yun- Selaginella 9neomexicana Maxon, (1) Rothfels & al. 2506 nan): rbcL KT161514, ITS KT161757; (2) Zhou 011 (CDBI), China (DUKE), USA (New Mexico): rbcL KT161642, ITS KT161883; (2) (Sichuan): rbcL KT161512, ITS KT161760; (3) Zhou & al. Beck & al. 1055 (DUKE), USA (New Mexico): rbcL KT161643, ITS DJY05380 (CDBI), China (Sichuan): rbcL KT161513, ITS KT161882; Selaginella nipponica Franch. & Sav., (1) Zhou & al. KT161756; (4) Zhang 5853 (CDBI), China (Guizhou): rbcL DJY05391 (CDBI), China (Sichuan): rbcL KT161540, ITS KT161510, ITS KT161759; (5) Zhang 5199 (CDBI), China (Guiz- KT161873; (2) Zhou & al. DJY05495 (CDBI), China (Sichuan): hou): rbcL KT161511, ITS KT161758; Selaginella leucobryoides rbcL KT161541, ITS KT161783; (3) Zhou & al. DJY07479 (CDBI), Maxon, (1) Rothfels & al. 2565 (DUKE), USA (California): rbcL China (Sichuan): rbcL KT161542, ITS KT161784; (4) Knapp 2650 KT161515, ITS KT161761; (2) Windham 97-055 (DUKE), USA (HAST), China (Taiwan): rbcL KT161544, ITS KT161785; (5) Zhou (Arizona): rbcL AF419068 (Arrigo et al., 2013), ITS AF419023 (Ar- 098 (CDBI), China (Sichuan): rbcL KT161543, ITS KT161786; (6) rigo et al., 2013); Selaginella limbata Alston, TNS764147 (TNS), TNS738139 (TNS), Japan (Tokyo): rbcL AB574649 (Ebihara et al., Japan (Kagoshima): rbcL AB574647 (Ebihara et al., 2010); Se- 2010); Selaginella nivea Alston, Fosberg 52583 (US), Madagascar laginella lingulata Spring, Korall 1996:4 (S), Unknown: rbcL (Tulear): rbcL AF419073 (Arrigo et al., 2013), ITS AF419014 AJ295882 (Korall and Kenrick, 2002); Selaginella longiaristata (Arrigo et al., 2013); Selaginella njamnjamensis Hieron., Faden 344- Hieron., Wikstrom€ & Wanntorp nr139(S), Unknown: rbcL 85 (MO), Kenya (Kwale): rbcL AF419074 (Arrigo et al., 2013), ITS AJ295873 (Korall and Kenrick, 2002); Selaginella longipinna Warb., AF419016 (Arrigo et al., 2013); Selaginella nothohybrida Valdespino, Lundberg s.n. (S), Unknown: rbcL AJ295860 (Korall and Kenrick, Rothfels & al. 3069 (DUKE), Mexico (San Luis Potosı): rbcL 2002); Selaginella lutchuensis Koidz., TNS759343 (TNS), Japan (Oki- KT161545; Selaginella novae-hollandiae (Sw.) Spring, (1) Ortega & nawa): rbcL AB574648 (Ebihara et al., 2010); Selaginella lyallii Smith 3136 (BM), Venezuela: rbcL AJ295865 (Korall and Kenrick, (Hook. & Grev.) Spring, Malcomber & al. 1383 (P), Madagascar 2002); (2) Korall 1996:8 (S), Ecuador: rbcL AJ295883 (Korall and (Fianarantsoa): rbcL AJ295898 (Korall and Kenrick, 2002). Kenrick, 2002); Selaginella nummularifolia Ching, Ludlow & al. 4233 Selaginella mairei H. Lev., (1) He & al. 81906 (PYU), China (PE), China (Xizang): rbcL KT161546, ITS KT161787. (Yunnan): rbcL KT161517, ITS KT161765; (2) He & Zhou 118 Selaginella oaxacana Spring, (1) Rothfels & al. 3345 (DUKE), (PYU, CDBI), China (Yunnan): rbcL KT161519, ITS KT161764; (3) Mexico (Oaxaca): rbcL KT161548, ITS KT161789; (2) Rothfels & Chu & He s.n. (PYU), China (Yunnan): rbcL KT161518, ITS al. 3344 (DUKE), Mexico (Oaxaca): rbcL KT161549, ITS KT161762; (4) He & Zhao 3348 (CTC) China (Sichuan): rbcL KT161788; (3) Rothfels & al. 08-186 (DUKE), Costa Rica (Heredia): KT161516, ITS KT161763; Selaginella martensii Spring, (1) Korall rbcL KT161547; Selaginella oregana D.C. Eaton, Therrien and Bost- 1998:3 (S), cult.: rbcL AJ295878 (Korall and Kenrick, 2002); (2) wick s.n. (KANU), USA (Washington): rbcL AF419066 (Arrigo Beck 1222 (DUKE), Mexico (Oaxaca): rbcL KT161520; Selaginella et al., 2013), ITS AF419030 (Arrigo et al., 2013). matsuensis C. M. Kuo, Knapp 2642 (HAST), China (Taiwan): rbcL Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30 29

Selaginella pallescens (C. Presl) Spring, (1) Korall 1998:7 (S), cult.: (Yunnan): rbcL KT161580, ITS KT161814; Selaginella repanda rbcL AJ295858 (Korall and Kenrick, 2002); (2) Unknown: rbcL (Desv. ex Poir.) Spring, (1) He & Jiang 405-1 (CDBI), China (Yun- AJ295859 (Korall and Kenrick, 2002); (3) Unknown, rbcL nan): rbcL KT161584; (2) He & Zhou 115 (PYU, CDBI), China AF419050 (J.P. Therrien and C.H. Haufler, unpublished); (4) Roth- (Yunnan): rbcL KT161582, ITS KT161815; (3) He & al. s.n. (PYU), fels & al. 2701 (DUKE), Costa Rica (San Jose): rbcL KT161553, China (Yunnan): rbcL KT161581, ITS KT161817; (4) He & Zhou ITS KT161794; (5) Rothfels & al. 2644 (DUKE), Costa Rica (Gua- 119 (PYU, CDBI), China (Yunnan): rbcL KT161583, ITS nacaste): rbcL KT161550, ITS KT161792; (6) Beck 1120 (DUKE), KT161816; Selaginella roxburghii (Hook. & Grev.) Spring, Mexico (Hidalgo): rbcL KT161555, ITS KT161790; (7) Rothfels & Unknown, Peninsula Malaysia: rbcL EU140945 (Y. Yi, Y. He, and al. 2656 (DUKE), Costa Rica (Guanacaste): rbcL KT161551, ITS B. Tan, unpublished); Selaginella rupestris (L.) Spring, (1) Rothfels & KT161793; (8) Rothfels & al. 3279 (DUKE), Mexico (Oaxaca): rbcL Rushworth 4172 (DUKE), USA (North Carolina): rbcL KT161586, KT161554, ITS KT161795; (9) Grusz & al. 08-002 (DUKE), Costa ITS KT161819; (2) Rothfels & al. 4493 (UBC), Canada (Ontario): Rica (Heredia): rbcL KT161552, ITS KT161791; Selaginella pallidis- rbcL KT161585, ITS KT161818; (3) Therrien 1996:356 (KANU), sima Spring, He & Zhao 3386 (CTC) China (Sichuan): rbcL USA (Illinois): rbcL AF093255 (Korall et al., 1999), ITS AF419038 KT161556, ITS KT161796; Selaginella pennata (D. Don) Spring (1) (Arrigo et al., 2013); Selaginella rupincola Underw., (1) Rothfels & He & Jiang 404 (PYU, CDBI), China (Yunnan): rbcL KT161558, al. 2508 (DUKE), USA (New Mexico): rbcL KT161587, ITS ITS KT161798; (2) Chu & al. 24587 (PYU), China (Yunnan): rbcL KT161820; (2) Therrien and Windham s.n. (KANU), USA (Arizona): KT161557, ITS KT161797; Selaginella peruviana (Milde) Hieron., (1) rbcL AF419083 (Arrigo et al., 2013), ITS AF419003 (Arrigo et al., Rothfels & al. 3280 (MEXU) Mexico (Oaxaca): rbcL KT161559, ITS 2013); (3) s.n. (S), S. USA: rbcL AJ010850 (Korall et al., 1999). KT161799; (2) Therrien and Bostwick s.n. (KANU), USA (Texas): Selaginella sanguinolenta (L.) Spring, (1) Zhou 003 (PYU, CDBI), rbcL AF419087 (Arrigo et al., 2013), ITS AF419013 (Arrigo et al., China (Yunnan): rbcL KT161591; (2) He & Zhao 3514 (CTC) China 2013); Selaginella pervillei Spring, Rakotondrainibe 3355 (P), Mada- (Sichuan): rbcL KT161589, ITS KT161821; (3) He & Zhao 3399 gascar (d’Antsiranana): rbcL AJ295901 (Korall and Kenrick, 2002); (CTC) China (Sichuan): rbcL KT161590, ITS KT161822; (4) Zhou Selaginella phillipsiana (Hieron.) Alston, Faden 74-935 (F), Kenya 092 (CDBI), China (Sichuan): rbcL KT161588, ITS KT161823; (5) (Isiolo): rbcL AF419061 (Arrigo et al., 2013), ITS AF419019 (Arrigo Unknown, rbcL EU197124 (Y. Yi, Y. He, and B. Tan, unpublished); et al., 2013); Selaginella picta A. Braun ex Baker, (1) Zhang & al. Selaginella sartorii Hieron., (1) Fosberg 22205 (US), Colombia (Boy- 6525 (CDBI, MO, VNMN, PYU), Vietnam (Phu Tho): rbcL oca): rbcL AF419054 (Arrigo et al., 2013), ITS AF419039 (Arrigo KT161562, ITS KT161802; (2) Chu & al. 21911 (PYU), China (Yun- et al., 2013); (2) Rothfels & al. 3067 (DUKE), Mexico (San Luis nan): rbcL KT161560, ITS KT161800; (3) He & Zhou 110 (PYU, Potosı): rbcL KT161592, ITS KT161877; Selaginella scabrifolia CDBI), China (Yunnan): rbcL KT161561, ITS KT161801; Ching & Chu H. Wang, He & Zhou 109 (PYU, CDBI), China (Hai- Selaginella pilifera A. Braun, Pringle 13959 (S), Unknown: rbcL nan): rbcL KT161593, ITS KT161824; Selaginella scopulorum AJ295862 (Korall and Kenrick, 2002); Selaginella plana (Desv. ex Maxon, (1) Rothfels & al. 4507 (UBC), USA (Washington): rbcL Poir.) Hieron., (1) Korall 1998:5 (S), cult.: rbcL AJ295880 (Korall KT161595, ITS KT161825; (2) Rothfels & al. 4473 (UBC), Canada and Kenrick, 2002); (2) Tiew 08 (CDBI), cult.:rbcL KT161563, ITS (British Columbia): rbcL KT161594, ITS KT161826; Selaginella KT161803; Selaginella polymorpha Badre, Rakotondrainibe 3533 (P), selaginoides (L.) P. Beauv. ex Mart. & Schrank, (1) TNS766485 Madagascar (d’Antsiranana): rbcL AJ295900 (Korall and Kenrick, (TNS), Japan (Nagano): rbcL AB574651 (Ebihara et al., 2010); (2) 2002); Selaginella porphyrospora A. Braun, Rothfels & al. 08-001 Therrien s.n. (KANU), Canada (Ontario): rbcL AF419048 (Arrigo (DUKE), Costa Rica (Heredia): rbcL KT161564, ITS KT161804; Se- et al., 2013), ITS AF419000; (3) Eriksson s.n. (S), Unknown: rbcL laginella prostrata (H.S. Kung) Li Bing Zhang, (1) Gao & al. Y07940 (Wikstrom€ and Kenrick, 1997); Selaginella sellowii Hieron., DJY03769 (CDBI), China (Sichuan): rbcL KT161566; (2) Zhu & (1) Rothfels & al. 3604 (DUKE), Ecuador (Carchi): rbcL KT161596, Zhou DJY03956 (CDBI), China (Sichuan): rbcL KT161565, ITS ITS KT161827; (2) Acevedo 4571 (US), Bolivia (Florıda): rbcL KT161874; Selaginella pseudonipponica Tagawa, (1) Knapp 3375, AF419053 (Arrigo et al., 2013), ITS AF419028 (Arrigo et al., 2013); Knapp’s private herbarium, China (Taiwan): rbcL KT161567, ITS (3) Wanntorp & Holmgren 557 (S), Unknown: rbcL AJ295889 (Ko- KT161806; (2) Knapp 3381 (P), China (Taiwan): rbcL KT161569, rall and Kenrick, 2002); Selaginella sericea A. Braun, (1) Korall ITS KT161808; (3) Knapp 3291 (P), China (Taiwan): rbcL 1996:13 (S), Unknown: rbcL AJ295871 (Korall and Kenrick, 2002); KT161570, ITS KT161805; (4) Moore 17161 (PYU), China (Tai- (2) Rothfels & al. 3747 (DUKE), Ecuador (Pichincha): rbcL wan): rbcL KT161574; (5) Knapp 3378 (P), China (Taiwan): rbcL KT161597, ITS KT161828; Selaginella sertata Spring, Rothfels & al. KT161571, ITS KT161876; (6) Knapp 3377, Knapp’s private herbar- 3192 (DUKE), Mexico (Jalisco): rbcL KT161598, ITS KT161829; ium, China (Taiwan): rbcL KT161568, ITS KT161875; (7) Knapp Selaginella shakotanensis (Franch. ex Takeda) Miyabe & Kudo^ (1) 3379 (P), China (Taiwan): rbcL KT161572, ITS KT161807; (8) Iwatsuki 53 (US), Japan (Nagano): rbcL AF419059 (Arrigo et al., Knapp 3380 (P), China (Taiwan): rbcL KT161573; Selaginella 2013), ITS AF419040 (Arrigo et al., 2013); (2) TNS776371 (TNS), pseudopaleifera Hand.-Mazz., Zhang & al. 6324 (CDBI, MO, Japan (Nagano): rbcL AB574652 (Ebihara et al., 2010); Selaginella VNMN, PYU), Vietnam (Hoa Binh): rbcL KT161575, ITS sheldonii Maxon, Rothfels & al. 2487 (DUKE), USA (Texas): rbcL KT161809; Selaginella pulcherrima Liebm., Korall 1997:32 (S), cult.: KT161599, ITS KT161830; Selaginella siamensis Hieron., He & Zhou rbcL AJ010847 (Korall et al., 1999); Selaginella pulvinata (Hook. & 102 (PYU, CDBI), China (Yunnan): rbcL KT161600, ITS Grev.) Maxim., (1) Zhou 066 (CDBI), China (Sichuan): rbcL KT161831; Selaginella sibirica (Milde) Hieron., (1) TNS743691 KT161576, ITS KT161810; (2) Zhou 501 (PYU, CDBI), China (TNS), Japan (Hokkaido): rbcL AB574653 (Ebihara et al., 2010); (2) (Xizang): rbcL KT161577, ITS KT161811; Selaginella pygmaea Kelso 83-222. (COLO), USA (Alaska): rbcL AF419076 (Arrigo et al., Alston, Esterhuysen 34078a (BOL) Unknown: rbcL AJ295892 2013), ITS AF419032 (Arrigo et al., 2013); Selaginella sichuanica (Korall and Kenrick, 2002). H.S. Kung (1) Chu & al. 7385 (PYU), China (Sichuan): rbcL Selaginella radiata Baker, Cremers 10068 (BM), French guiana: KT161602; (2) Luo 1349 (CDBI), China (Guizhou): rbcL KT161601; rbcL AJ295867 (Korall and Kenrick, 2002); Selaginella reflexa Selaginella cf. sichuanica, Zhang & al. DJY04930 (CDBI), China Underw., Beck 1126 (DUKE), Mexico (San Luis Potosı): rbcL (Sichuan): rbcL KT161603, ITS KT161832; Selaginella silvestris KT161578, ITS KT161812; Selaginella remotifolia Spring, (1) Hsu Aspl. Rothfels & al. 08-202 (DUKE), Costa Rica (Heredia): rbcL 5194 (S), Unknown: rbcL AJ295864 (Korall and Kenrick, 2002); (2) KT161604, ITS KT161833; Selaginella simplex Baker, Tehler 8038 TNS765133 (TNS), Japan (Shizuoka): rbcL AB574650 (Ebihara (S), Unknown: rbcL AJ295888 (Korall and Kenrick, 2002); Se- et al., 2010); (3) Ju & Deng HGX13191 (CDBI), China (Sichuan): laginella sp_A, Knapp 3260 (P), China (Taiwan): rbcL KT161605, rbcL KT161579, ITS KT161813; (4) Zhou 005 (PYU, CDBI), China ITS KT161834; Selaginella sp_B, (1) Zhang & al. 6570 (CDBI, MO, 30 Xin-Mao Zhou et al. / Cladistics 0 (2015) 1–30

VNMN, PYU), Vietnam (Phu Tho): rbcL KT161607, ITS TNS9508552 (TNS), Japan (Tokyo): rbcL AB574656 (Ebihara et al., KT161835; (2) Zhang & al. 6925 (CDBI, MO, VNMN, PYU), Viet- 2010); (3) Zhang & Zhou DJY04101 (CDBI), China (Sichuan): rbcL nam (Ha Giang): rbcL KT161606, ITS KT161878; Selaginella sp_C KT161626, ITS KT161852; Selaginella cf. uncinata, Rothfels & al. (1) Zhang 5888 (CDBI), China (Guizhou): rbcL KT161608, ITS 08-137 (DUKE), introduced to Costa Rica (Puntarenas): rbcL KT161879; (2) Zhang 5909 (CDBI), China (Guizhou): rbcL KT161423, ITS KT161851; Selaginella underwoodii Hieron., Therrien KT161609, ITS KT161836; (3) Zhang 6055 (CDBI), China (Guiz- s.n. (KANU), USA (Colorado): rbcL AF419077 (Arrigo et al., hou): rbcL KT161610, ITS KT161837; Selaginella sp_D, Wood 11034 2013), ITS AF419031 (Arrigo et al., 2013); Selaginella utahensis (PTBG), Society Islands (Huahine): rbcL KT161611, ITS KT161838; Flowers, Windham 96-052 (UT), USA (Utah): rbcL AF419067 (Ar- Selaginella sp_E,Wood 13942 (PTBG), F.S.M. (Caroline_Is., Kos- rigo et al., 2013), ITS AF419024 (Arrigo et al., 2013). rae): rbcL KT161612, ITS KT161839; Selaginella sp_F, Wood 13673 Selaginella vardei H. Lev., (1) Zhou 091 (CDBI), China (Sichuan): (PTBG), F.S.M. (Caroline_Is., Kosrae): rbcL KT161613, ITS rbcL KT161627, ITS KT161856; (2) Chu & al. 29607 (PYU), China KT161840; Selaginella stauntoniana Spring, (1) Kenrick s.n. (S), (Yunnan): rbcL KT161628, ITS KT161853; (3) He & al. 81904 Unknown: rbcL AJ295869 (Korall and Kenrick, 2002); (2) Quan 1 (PYU), China (Yunnan): rbcL KT161629, ITS KT161855; (4) Jin (PYU), China (Henan): rbcL KT161615, ITS KT161880; (3) Zhao s.n. (PYU), China (Yunnan): rbcL KT161630, ITS KT161854; (5) 169 (CDBI), China (Beijing): rbcL KT161614, ITS KT161841; Se- Wu s.n. (MO), China (Yunnan): rbcL AF419060 (Arrigo et al., laginella steyermarkii Alston, Williams 23005 (US), Guatemala 2013), ITS AF419041 (Arrigo et al., 2013). (Quezaltenango): rbcL AF419088 (Arrigo et al., 2013), ITS Selaginella wallacei Hieron., (1) Rothfels & al. 2933 (DUKE), AF419034 (Arrigo et al., 2013); Selaginella suavis Klotzsch, Tehler USA (California): rbcL KT161631, ITS KT161858; (2) Rothfels & al. 7929 (S), Unknown: rbcL AJ295886 (Korall and Kenrick, 2002); Se- 3852 (DUKE), USA (Oregon): rbcL KT161635, ITS KT161859; (3) laginella sulcata (Desv. ex Poir.) Spring ex Mart., Tehler 7939 (S), Rothfels & Zylinski 4061 (DUKE), Canada (British Columbia): rbcL Unknown: rbcL AJ295887 (Korall and Kenrick, 2002); Selaginella KT161632, ITS KT161860; (4) Therrien s.n. (KANU), USA (Ore- superba Alston, Chu & al. 19425 (PYU), China (Yunnan): rbcL gon): rbcL AF419065 (Arrigo et al., 2013), ITS AF419035 (Arrigo KT161616, ITS KT161842. et al., 2013); (5) Rothfels & Park 4191 (DUKE), USA (Idaho): rbcL Selaginella tama-montana Seriz., (1) TNS769195 (TNS), Japan KT161633, ITS KT161861; (6) Rothfels & al. 4514 (UBC), USA (Mie): rbcL AB574654 (Ebihara et al., 2010); (2) Knapp 3257 (P), (Washington): rbcL KT161636, ITS KT161857; (7) Rothfels & Rush- China (Taiwan): rbcL KT161617, ITS KT161843; Selaginella tamar- worth 4196 (DUKE), USA (Idaho): rbcL KT161634, ITS KT161862; iscina (P. Beauv.) Spring, (1) TNS759348 (TNS), Japan (Okinawa): Selaginella wallichii (Hook. & Grev.) Spring, Zhang & al. 6744 rbcL AB574655 (Ebihara et al., 2010); (2) Shurba 5157 (S), (CDBI, MO, VNMN, PYU), Vietnam (Bac Kan): rbcL KT161637; Unknown: rbcL AJ295861 (Korall and Kenrick, 2002); (3) Unknown Selaginella watsonii Underw., (1) Li 1601 (DUKE), USA (Idnho): Peninsula Malaysia: rbcL JF950018 (Herrmann et al., 2011); rbcL KT161638, ITS KT161863; (2) Windham 91-137 (UT), USA Selaginella tenuifolia spring, Zhang & al. 6780 (CDBI, MO, VNMN, (Utah & Arizona): rbcL AF419090 (Arrigo et al., 2013), ITS PYU), Vietnam (Bac Kan): rbcL KT161618, ITS KT161844; AF419037 (Arrigo et al., 2013); Selaginella weatherbiana R.M. Tryon Selaginella tortipila A. Braun, (1) Hill 20496 (KANU), USA (South Therrien s.n. (KANU), USA (Colorado): rbcL AF419075 (Arrigo carolina): rbcL AF419081 (Arrigo et al., 2013), ITS AF419007 et al., 2013), ITS AF419006 (Arrigo et al., 2013); Selaginella wightii (Arrigo et al., 2013); (2) Rothfels & al. 2718 (DUKE), USA (North Hieron., Faden 76-537 (US), Sri-Lanka (Matale): rbcL AF419062 Carolina): rbcL KT161619, ITS KT161845; Selaginella trachyphylla (Arrigo et al., 2013), ITS AF419018 (Arrigo et al., 2013); Selaginella A. Braun ex Hieron., Chu & al. 5841 (PYU, CDBI), China (Yun- willdenowii (Desv. ex Poir.) Baker (1) Korall 1998:11 (S), cult.: rbcL nan): rbcL KT161620; Selaginella trichophylla K.H. Shing, (1) Chu & AJ295893 (Korall and Kenrick, 2002), (2) Wang 90408 (PYU), al. 31925 (PYU), China (Yunnan): rbcL KT161621, ITS KT161847; China (Guizhou): rbcL KT161639, ITS KT161881; (3) Chu & al. (2) Chu & al. 29310 (PYU), China (Yunnan): rbcL KT161622, ITS 18474 (PYU), China (Guangxi): rbcL KT161640, ITS KT161864; KT161846; (3) Zhang & al. 6784 (CDBI, MO, VNMN, PYU), Viet- Selaginella wrightii Hieron., (1) Rothfels & al. 2480 (DUKE), USA nam (Cao Bang): rbcL KT161624, ITS KT161849; (4) Jiang 318 (Texas): rbcL KT161641, ITS KT161865; (2) Worthington 21748 (PYU, CDBI), China (Hainan): rbcL KT161623, ITS KT161848. (KANU), USA (Texas): rbcL AF419089 (Arrigo et al., 2013), ITS Selaginella uliginosa (Labill.) Spring, Gray & al. 523 (S), Australia AF419011 (Arrigo et al., 2013). (Tasmania): rbcL AJ010843 (Korall et al., 1999); Selaginella umbrosa Selaginella xichouensis W.M. Chu, Chu & al. 13149 (PYU, CDBI), Lem. ex Hieron., (1) Rothfels & al. 08-155 (DUKE), Costa Rica China (Yunnan): rbcL KT161644, ITS KT161866; Selaginella (Heredia): rbcL KT161625, ITS KT161850; (2) Korall 1998:4 (S), xipholepis Baker, (1) Dong 2377 (PYU), China (Guangdong): rbcL cult.: rbcL AJ295879 (Korall and Kenrick, 2002); Selaginella unci- KT161645; (2) Zhang & al. 6668 (CDBI, MO, VNMN, PYU), nata (Desv. ex Poir.) Spring (1) Unknown Peninsula Malaysia: rbcL Vietnam (Bac Kan): rbcL KT161646, ITS KT161867. EU197126 (Y. Yi, Y. He, and B. Tan, unpublished); (2)