Cladistics

Cladistics (2014) 1–18 10.1111/cla.12094

A global plastid phylogeny of the brake fern genus Pteris (Pteridaceae) and related genera in the Pteridoideae

Liang Zhanga, Carl J. Rothfelsb, Atsushi Ebiharac, Eric Schuettpelzd, Timothee Le Pechona, Peris Kamaue, Hai Hef, Xin-Mao Zhoua, Jefferson Pradog, Ashley Fieldh,i, George Yatskievychj, Xin-Fen Gaoa,* and Li-Bing Zhangj,* aChengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu, Sichuan, 610041, China; bDepartment of Zoology, University of British Columbia, #4200-6270 University Blvd., Vancouver, BC, V6T 1Z4, Canada; cDepartment of Botany, National Museum of Nature and Science, Tsukuba-shi, Ibaraki, 305-0005, Japan; dDepartment of Botany (MRC 166), National Museum of Natural History, Smithsonian Institution, P.O. Box 37012, Washington, DC, 20013-7012, USA; eDepartment of Botany, National Museum of Kenya, P.O. Box 45166-00100, Nairobi, Kenya; fDepartment of Biology, Chongqing Normal University, Shapingba, Chongqing, 400047, China; gInstituto de Botanica^ Herbario SP, Avenida Miguel Estefano 3687, CEP 04301-012, Sao~ Paulo, Brazil; hAustralian Tropical Herbarium, James Cook University, Smithfield, Qld, 4878, Australia; iQueensland Herbarium, Department of Science, Information Technology, Innovation and the Arts, Toowong, Qld, 4066, Australia; jMissouri Botanical Garden, P.O. Box 299, St Louis, MO, 63166-0299, USA Accepted 30 June 2014

Abstract

The brake fern genus Pteris belongs to the Pteridaceae subfamily Pteridoideae. It contains 200–250 species distributed on all continents except Antarctica, with its highest species diversity in tropical and subtropical regions. The monophyly of Pteris has long been in question because of its great morphological diversity and because of the controversial relationships of the Austra- lian endemic monospecific genus Platyzoma. The circumscription of the Pteridoideae has likewise been uncertain. Previous stud- ies typically had sparse sampling of Pteris species and related genera and used limited DNA sequence data. In the present study, DNA sequences of six plastid loci of 146 accessions representing 119 species of Pteris (including the type of the genus) and 18 related genera were used to infer a phylogeny using maximum-likelihood, Bayesian-inference and maximum-parsimony methods. Our major results include: (i) the previous uncertain relationships of Platyzoma were due to long-branch attraction; (ii) Afropter- is, Neurocallis, Ochropteris and Platyzoma are all embedded within a well-supported Pteris sensu lato; (iii) the traditionally cir- cumscribed Jamesonia is paraphyletic in relation to a monophyletic Eriosorus; (iv) Pteridoideae contains 15 genera: Actiniopteris, Anogramma, Austrogramme, Cerosora, Cosentinia, Eriosorus, Jamesonia, Nephopteris (no molecular data), Onychium, Pityrogram- ma, Pteris, Pterozonium, Syngramma, Taenitis and Tryonia; and (v) 15 well-supported clades within Pteris are identified, which differ from one another on molecular, morphological and geographical grounds, and represent 15 major evolutionary lineages. © The Willi Hennig Society 2014.

Introduction one of the largest fern genera, Pteris has been estimated to contain ca. 200 (Tryon and Tryon, The fern genus Pteris L. (Pteridaceae: Pteridoideae; 1982) or 250 species (Tryon et al., 1990) distributed Tryon et al., 1990) is characterized by having sporan- throughout the tropical, subtropical, and temperate gia borne continuously along most of the length of areas of all continents except Antarctica, from the pinnae from commissural veins, and having Australia, New Zealand and South Africa northward pinnae that are entire or pectinately divided into to Japan and North America. From open slopes to segments (with these sometimes asymmetrical). As dense forests and from acid soils to limestone rock, the habitats of Pteris are diverse, and the genus *Corresponding authors: includes considerable morphological variation (Liao E-mail addresses: [email protected]; [email protected] et al., 2013).

© The Willi Hennig Society 2014 2 L. Zhang et al. / Cladistics (2014) 1–18

The circumscription of Pteris has been unstable 2004b; Prado et al., 2007; Schuettpelz et al., 2007; since its establishment by Linnaeus (1753). A broad Bouma et al., 2010; Schneider et al., 2013; Cochran concept of the genus initially included most ferns with et al., 2014), or three or four (Schuettpelz et al., 2007; continuous sori along pinna margins, even those now Cochran et al., 2014) plastid genes, there have been recognized to fall in other families (e.g. Pteridium very few studies focusing on the relationships within Gled. ex Scop.). Some later concepts, relying heavily Pteris. Li et al. (2004) used trnL-F intergenic spacer on venation patterns (Presl, 1836; Moore, 1857), were sequences (< 400 base pairs) from 16 Chinese species very narrow and placed most species in segregated to reconstruct the first phylogeny of Pteris, and con- genera (e.g. Campteria C. Presl and Litobrochia C. cluded that Pteris was strongly supported as mono- Presl). Since the twentieth century, it has generally phyletic and that P. vittata L. was resolved as the been agreed that: (i) species of Doryopteris J. Sm., His- earliest diverging lineage. However, Platyzoma R. Br. tiopteris (J. Agardh) J. Sm., Paesia J. St.-Hil. and was not sampled and only one distantly related out- Pteridium should be excluded from Pteris; (ii) the gen- group, Adiantum capillus-veneris L., was included in eric recognition of Campteria,“Eupteris” and Litobro- their analysis. Using rbcL gene sequences of 10 species chia should be abandoned; and (iii) the names of Pteris and related genera in Pteridaceae, Prado corresponding to the distinct venation patterns should et al. (2007) found that Pteris was paraphyletic in rela- be used for infrageneric classification. tion to Platyzoma, a monospecific Australian endemic. In comparison with the instability of the definition of Based on atpA, atpB and rbcL gene data from nine Pteris, the infrageneric relationships of Pteris are far species of Pteris and a broad sampling of related gen- clearer (Presl, 1836; Smith, 1841; Moore, 1857; Hooker era, Schuettpelz et al. (2007) discovered that Pteris and Baker, 1868). The latest global classification of was also paraphyletic with respect to Neurocallis Fee Pteris is that published by Christensen (1906). Based on (a Neotropical monospecific genus), Ochropteris J. Sm. different venation patterns, three existing sections, (a Malagasy and Mascarene bispecific genus), and “P. sect. Eupteris” (nom. inval. = P. sect. Pteris), Platyzoma. Most interestingly, Schuettpelz et al. (2007) P. sect. Heterophlebium (Fee) Hook. and P. sect. Lito- found that P. vittata, a species morphologically similar brochia (C. Presl) Hook. were accepted, and later three to the type of the genus (P. longifolia L., not sampled subgenera “P. subg. Eupteris”(= P. subg. Pteris), in their study), was not resolved as closely allied to the P. subg. Campteria (C. Presl) C. Chr., and P. subg. remainder of the genus. They suggested that the defini- Litobrochia (C. Presl) C. Chr., were proposed. In spite tion of Pteris would need to be expanded to include of the obvious artificiality of Christensen’s (1906) infra- their entire “pteridoid clade” (Rothfels, 2008) or generic classification (Walker, 1962), many more recent reduced to the small clade of P. longifolia, P. vittata taxonomic studies have adopted it to a large extent (e.g. and their close allies. More recent studies of the Pte- Wu, 1990; Yang, 2011; Liao et al., 2013). The earliest ridaceae (e.g. Bouma et al., 2010; Chao et al., 2012a; competitor to Christensen’s system was that of Shieh Jaruwattanaphan et al., 2013; Schneider et al., 2013) (1966), who emphasized the importance of patterns of have provided only limited information concerning the leaf architecture and proposed a reclassification into phylogeny of Pteris. To date, there have not been any two subgenera, P. subg. Pteris (leaves pinnate or bipin- large-scale multilocus molecular phylogenetic studies nate) and P. subg. Tripedipteris W. C. Shieh (leaves of Pteris. tripartite), with the two subgenera each divided into Pteris is normally placed in the subfamily Pteridoi- two sections: P. subg. Pteris into P. sect. Pteris and deae. The circumscription of the latter, however, has P. sect. Campteria (C. Presl) Hook., and P. subg. Trip- been controversial: Tryon et al. (1990) placed five edipteris into P. sect. Hypsopodium W. C. Shieh and genera in the Pteridoideae and 13 genera in the P. sect. Tripedipteris W. C. Shieh. Combining the pat- Taenitidoideae; Sanchez-Baracaldo (2004b) rejected terns of frond architecture and venation type, Tryon the monophyly of the Taenitidoideae sensu Tryon and Tryon (1982) divided the Neotropical species of et al. (1990) using rps4-trnS data; Smith et al. (2006) Pteris into six unranked groups. Finally, in classifying combined the two subfamilies; Schuettpelz et al. (2007) species of Pteris of China, Ching and Wu (1983) recog- suggested that the Taenitidoideae lineage was nested nized three sections, P. sect. Pteris, P. sect. Campteria within the Pteridoideae. The sampling of taxa and/or and P. sect. Quadriauricula Ching, with the last two characters so far has been limited. each further divided into two series. Ching and Wu’s The objectives of this study were: (i) to resolve the classification was adopted in a recent taxonomic treat- relationships within the Pteridoideae; (ii) to test the ment in Flora of China (Liao et al., 2013). monophyly of Pteris with the largest taxon and Although the generic relationships within the Pterid- character sampling so far utilized, including the type aceae subfamily Pteridoideae have frequently been of the genus, P. longifolia; (iii) to better resolve the explored based on DNA sequences of one (Nakazato relationships of the enigmatic genus Platyzoma; (iv) to and Gastony, 2003; Li et al., 2004; Sanchez-Baracaldo, identify major evolutionary lineages within Pteris and L. Zhang et al. / Cladistics (2014) 1–18 3 the position of its type species; (v) to evaluate previous South China Botanical Garden, Chinese Academy of morphological hypotheses about the relationships Sciences (IBSC), Kunming Institute of Botany, Chinese within Pteridaceae subfamily Pteridoideae; and (vi) to Academy of Sciences (KUN), Missouri Botanical understand morphological features for the resolved lin- Garden (MO), Yunnan University (PYU). eages. DNA extraction, amplification and sequencing

Materials and methods Total genomic DNA was extracted from silica-dried material or sometimes from herbarium specimens Taxon sampling using a Tiangen Biotech plant genomic DNA extrac- tion kit (Beijing, China) or DNeasy Plant Mini Kits The taxa were sampled to include representatives of (Qiagen, Dusseldorf,€ Germany) following the manu- each of the four sections recognized by Hooker and facturers’ protocols. Baker (1874) and Christensen (1906), the four sections Six plastid regions (the atpA gene, the atpB gene, and seven subsections proposed by Shieh (1966), the the rbcL gene, the rps4-trnS intergenic spacer, the trnL six unranked groups defined by Tryon and Tryon intron and the trnL-F intergenic spacer) were selected (1982), and the three sections and four series circum- based on their use in earlier studies of the Pteridaceae scribed by Ching and Wu (1983). The generic type, (Nakazato and Gastony, 2003; Prado et al., 2007; P. longifolia, is included for the first time. In total, Schuettpelz et al., 2007; Rothfels et al., 2008; Bouma Pteris sensu lato was represented by 105 accessions of et al., 2010; Lu et al., 2012; Sigel et al., 2011). The 84 species from four continents, including one acces- atpA gene was amplified with primers ESATPF412F sion of Afropteris barklyae (Baker) Alston, two of and ESTRNR46F (Schuettpelz et al., 2006) and the Neurocallis praestantissima Bory ex Fee and one of atpB gene with primers ESATB672F and ES- Ochropteris pallens (Sw.) J. Sm. To test the monophyly ATPE384R (Pryer et al., 2004). Most rbcL sequences of Pteris and its relationships within the Pteridoideae, were amplified with primers F1 (Fay et al., 1997) and 38 accessions of 15 additional genera in Pteridoideae 1379R, originally designed by Zurawski et al. (1984) and Taenitidoideae sensu Tryon et al. (1990), including and modified by Wolf et al. (1999). For some a newly described genus, Tryonia Schuettp., J. Prado herbarium specimens with degraded DNA, newly & A. T. Cochran, segregated from Eriosorus Fee and designed internal primers of rbcL gene 595F (50-AAT Jamesonia Hook. & Grev. (Cochran et al., 2014), were TCYCARCCRTTCATGCGT-30), 650R (50-AGA- included based largely on the results of Schuettpelz GCTTCYGCYACRAATA-30) and 819R (50-AGCTA et al. (2007). Eriosorus and Platyzoma were treated as AGCTGGTRTTKGCRGT -30) were used when two independent genera. In total, our ingroup com- amplification of the larger region was unsuccessful. prised 142 accessions representing 116 species in 16 The rps4-trnS intergenic spacer was amplified with genera of the Pteridoideae. primers TRNS (Souza-Chies et al., 1997) and an One species each of Adiantum L., Ceratopteris Bron- anonymous primer derived from Li and Lu (2006). gn. and Acrostichum L. were used as outgroups, given The trnL intron and trnL-F intergenic spacer were that the Ceratopteridoideae (comprising Acrostichum amplified together using the primers FERN1 (Trewick and Ceratopteris) was resolved as sister to the Pteridoi- et al., 2002) and F (Taberlet et al., 1991). The PCR deae and that Adiantum is in a clade that is a further sis- conditions followed Zhang et al. (2001). Amplified ter to the clade containing the Ceratopteridoideae and fragments were purified with TIANquick Mini Purifi- the Pteridoideae (Prado et al., 2007; Schuettpelz et al., cation Kits (Tiangen Biotech) and purified polymerase 2007). chain reaction (PCR) products were sequenced by Voucher information and GenBank accession num- Invitrogen (Shanghai, China). bers for each sampled taxon are provided in Appen- dix 1. Sequence alignment and phylogenetic analysis

Morphology Sequencher 4.1 (Gene Codes Corp., Ann Arbor, MI, USA) was used to assemble and edit complementary Morphological data were obtained from field strands. Sequences obtained for each locus were aligned observations, herbarium investigations and literature individually using Clustal X 1.81 (Thompson et al., study (see ‘Discussion’ below for references), field 1997) followed by manual adjustments using BioEdit observations were mainly conducted by the first author (Hall, 1999). Partial regions of the rps4-trnS spacer and in China, Myanmar and Vietnam, and herbarium inves- trnL-F spacer of several genera (such as Pityrogramma tigations were carried out at herbaria Chengdu Institute Link) were removed prior to analysis, because they of Biology, Chinese Academy of Sciences (CDBI), were highly divergent and difficult to align with Pteris. 4 L. Zhang et al. / Cladistics (2014) 1–18

Equally weighted maximum-parsimony (MP) analy- ses were conducted for each locus using 1000 tree-bisec- tion-reconnection (TBR) searches in PAUP* version 4.0b10 (Swofford, 2002) with MAXTREES set to increase without limit. Insertions and deletions were coded as missing data. Parsimony jackknife (JK) analy- IG 0 0.3220 0 1.0850 0 1.7710 0 0.9170 ses (Farris et al., 1996) were conducted using PAUP* 00.59700.5500 0.9650 0 0.2920 0.8570 0.6110 with the removal probability set to approximately 37%, and “jac” resampling emulated. Two thousand repli- – – – – cates were performed with ten TBR searches per repli- – – – – cate and a maximum of 100 trees held per TBR search.

jModelTest 0.1.1 (Guindon and Gascuel, 2003; T Ti/tv – Posada, 2008) was used to select the best-fitting likeli- hood model for maximum likelihood (ML; Felsenstein,

1973) analyses. The Akaike information criterion TG – (Akaike, 1974) was used to select among models instead of the hierarchical likelihood ratio test, following Pol

(2004) and Posada and Buckley (2004). The best-fitting GC – models and parameter values are provided in Table 1. For each locus and the simultaneous analysis TC –

(Kluge, 1989; Nixon and Carpenter, 1996) of all ) and simultaneous plastid datasets in this study nucleotide characters, ML tree searches and ML boot-

strapping were conducted using the web server trnL-F GA &

RAxML-HPC2 on TG version 7.2.8 (Stamatakis et al., – 2008; Miller et al., 2010) with 5000 rapid bootstrap e, 1986); I, proportion of invariable sites; Ti/Tv, transition/transversion ratio. analyses followed by a search for the best-scoring tree trnL CA in a single run (Stamatakis et al., 2008). – , and Well-supported (≥ 70% JK or BS support; Zhang and Simmons, 2006; Zhang et al., 2012) clades that con- flicted with one another in the parsimony JK and likeli- hood BS trees were then tested for long-branch trnL, trnL-F , attraction (Felsenstein, 1978) by alternatively removing the terminals in question. If the terminal(s) remaining in

the parsimony analysis moved to a different part of the rps4-trnS tree (with ≥ 70% JK support) when the potentially , attracting terminal(s) were removed, the result was con- sistent with the explanation of long-branch attraction (Siddall and Whiting, 1999; Zhang and Simmons, 2006). Bayesian inference (BI) was conducted using MrBayes Base frequencies Substitution model (rate matrix) ACGTA 3.1.2 (Huelsenbeck and Ronquist, 2001; Ronquist and atpA, atpB, rbcL Huelsenbeck, 2003) on Cipres (Miller et al., 2010). Four GG 0.2907G 0.2714 0.1959 0.3618 0.2312 0.2201 0.1575 0.2211 0.2934 0.1805 0.2764 1.6928 0.3001 0.7584 6.2772 0.9610 4.3946 0.6786 2.5069 0.8251 0.7985 0.3072 0.4392 5.6368 0.6261 3.8994 1.0000 2.4720 1.0000 1.0000

Markov chain Monte Carlo chains were run, each + + + G 0.3151 0.1886 0.2012 0.2951 1.0281 3.4344 0.5937 0.5464 3.5402 1.0000 G 0.3523 0.1636 0.1712 0.3129 0.8775 2.0827 0.4835 0.5172 1.8974 1.0000 G 0.3507 0.1721 0.1622 0.3150 0.8258 2.0845 0.4123 0.6506 1.8990 1.0000 I I I G 0.3531 0.1558 0.1762 0.3150 0.9324 2.1279 0.5364 0.4233 1.9001 1.0000 G 0.3217 0.1775 0.1964 0.3045 1.0765 4.0668 0.4339 0.6820 6.8240 1.0000 + + + + + + + beginning with a random tree and sampling one tree + every 1000 generations of 10 000 000 generations. Convergence among generations was checked using Tracer (Rambaut and Drummond, 2007) and the first 25% was discarded as burn-in to ensure that stationarity in log-likelihood had been reached. The remaining trees spacer GTR were used to calculate a 50% majority-rule consensus

topology. trnL-F spacer GTR

Results spacer GTR gene GTR gene GTR gene GTR intron and intron GTR Simultaneous GTR trnL trnL-F atpB rbcL rps4-trnS trnL atpA Region Selected model This study generated 573 new sequences (Appen- G, gamma-distribution-shape parameter (Yang, 1994); GTR, general-time-reversible model (Tavar

dix 1). The dataset characteristics and tree statistics Table 1 Best-fitting models and parameter values for separate ( L. Zhang et al. / Cladistics (2014) 1–18 5 for the analyses are presented in Table 2. Comparisons 15 clades were named for descriptive convenience. Su- of tree topologies from the MP JK analyses of the perclade A (BS = 96%, PP = 100%, JK = 78%) con- individual markers identified no well-supported con- tains clades I–III; Superclade B (BS = 100%, flicts (JK ≥ 70%; Mason-Gamer and Kellogg, 1996; PP = 100%, JK = 100%) comprises clades V–IX; and Zhang and Simmons, 2006; Zhang et al., 2012). Thus, Superclade C (well supported only by BS = 72%; the six plastid datasets were concatenated. The topol- PP = 77%,) contains clades X–XV. ogy of the ML tree based on the combined dataset (Fig. 1a, b) was mostly identical to those based on each individual marker, but with generally increased Discussion support values. The topologies and support values from ML and MP analyses were similar except for the Circumscription of Pteridoideae position of Platyzoma. The MP analysis resolved Platyzoma as sister to the rest of Pteridoideae, with Pteridaceae subfamily Pteridoideae sensu Tryon maximum support values and a long branch. In con- et al. (1990) contains Acrostichum, Anopteris (Prantl) trast, the BI and ML analyses resolved Platyzoma as Diels, Neurocallis, Ochropteris and Pteris. Acrostichum sister to the rest of Pteris superclade A (Fig. 1b), but should be excluded from Pteridoideae based on the support values were low. This well-supported conflict findings by Prado et al. (2007) and Schuettpelz et al. could point to long-branch attraction (Bergsten, 2005; (2007). The monospecific genus Anopteris has not been Zhang and Simmons, 2006). We then conducted tests included in any molecular studies so far. It resembles of long-branch attraction (Siddall and Whiting, 1999) Pteris morphologically in having sporangia grouped by repeating BI, ML BS and MP JK analyses with the into submarginal sori (Tryon et al., 1990). Whereas it following six removals, because these groups had the might well be a member of Pteris, Neurocallis and longest branches in the ML analysis (Schuettpelz Ochropteris undoubtedly are (see ‘Discussion’ below; et al., 2007; Fig. 1a): Acrostichum, Adiantum, Ceratop- Fig. 1b: clades VII, XI). The Pteridoideae sensu Tryon teris, Acrostichum + Ceratopteris, Acrostichum + Adi- et al. (1990) excluding Acrostichum is basically Pteris antum and Acrostichum + Adiantum + Ceratopteris. in our definition below. Our tests showed that when these three genera were all The subfamily Taenitidoideae sensu Tryon et al. removed, all BI, ML BS and MP JK analyses resolved (1990) contains 13 genera: Actiniopteris, Afropteris Al- Platyzoma as sister to clades II and III with relatively ston, Anogramma Link, Austrogramme E. Fourn., Ce- strong BI PP (100%), ML BS (96%) and MP JK sup- rosora Baker, Eriosorus, Jamesonia, Nephopteris port (78%), consistent with the result of BI and ML Lellinger, Onychium Kaulf., Pityrogramma, Pterozoni- analyses of the full taxon sample. um Fee, Syngramma J. Sm. and Taenitis Willd. ex The monophyly of the Pteridoideae was maximally Schkuhr. Our study shows that the Taenitidoideae sensu supported in all BI, ML and MP analyses, which Tryon et al. (1990) is paraphyletic in relation to Pteris helped confirm the Pteridoideae as a natural group sensu lato because Afropteris is resolved as part of Pteris (Fig. 1a). Pteris plus Platyzoma (when the long-branch (Fig. 1b). One genus included in the Taenitidoideae attraction artefact was avoided) was resolved as mono- sensu Tryon et al. (1990), Nephopteris, was not sampled phyletic with strong support (ML BS 100%, PP 100%, in our study. The Taenitidoideae, if recognized, should MP JK 97%) and as sister to the rest of the ingroup. exclude Afropteris but include Cosentinia Tod. and the The phylogenetic relationships among the remaining newly described Tryonia (Fig. 1a). Cosentinia was syn- genera were well resolved (Fig. 1a, b). Species of Eri- onymized with Cheilanthes Sw., which was included in osorus were nested within Jamesonia (Fig. 1a). Ceros- the Cheilanthoideae by Tryon et al. (1990). ora microphylla (Hook.) R.M.Tryon [Anogramma Our results (Fig. 1a, b) indicate that a monophyletic microphylla (Hook.) Diels] was more closely related to Pteridoideae sensu stricto comprising only Pteris could Pityrogramma than to A. guatemalensis (Domin) C. be recognized, with the Taenitidoideae containing the Chr. and A. leptophylla (L.) Link. rest of our ingroup. Here, following the broader cir- Based on our reconstructed phylogeny (Fig. 1a, b) cumscription sensu Smith et al. (2006) we adopt the and in consideration of morphological characters and Pteridoideae sensu lato to include both of these clades. distribution information, 15 clades representing 15 The Pteridoideae in our definition contains 15 genera: major evolutionary lineages of Pteris were identified Actiniopteris Link, Anogramma, Austrogramme, Ceros- (Fig. 1b). All 15 clades were well supported (14 were ora, Cosentinia, Eriosorus, Jamesonia, Nephopteris, supported by ML BS ≥ 98, PP = 100% and MP Onychium, Pityrogramma, Pteris, Pterozonium, Syn- JK ≥ 99%, and one by ML BS = 79%, PP = 100% gramma, Taenitis and Tryonia. In comparison with its and MP JK = 84%). The relationships among most of sister group the Ceratopteridoideae (Schuettpelz et al., the 15 clades were also well supported. Three addi- 2007), which has sporangia distantly on veins (Tryon tional supported monophyletic superclades within the et al., 1990), members of the Pteridoideae have spo- 6 L. Zhang et al. / Cladistics (2014) 1–18 rangia approximate and in sori or soral lines on veins studies (Schuettpelz et al., 2007; Schneider et al., or on a marginal commissure (Tryon et al., 1990). 2013); our study is the first to provide strong support for this relationship. Marginal commissural veins also Phylogeny of Pteridoideae appear in Pteris. Based on our phylogeny, it is equally parsimonious to hypothesize that this charac- The monophyly of the Pteridoideae as defined above ter state either evolved once in the common ancestor is confirmed by our study with strong support of the Pteridoideae or independently evolved twice in (Fig. 1a, b). Pteris together with Afropteris, Neurocal- Pteris and in the Actiniopteris + Onychium clade. lis, Ochropteris and Platyzoma (see ‘Discussion’ below) Our data resolved the recently described genus was resolved as sister to the remaining genera of the Tryonia as sister to a clade containing Austrogramme, Pteridoideae. Although the overall relationships Syngramma and Taenitis, and these three together are determined are not in conflict with those inferred by sister to Pterozonium (Fig. 1a, b), a resolution consis- Schuettpelz et al. (2007) or Cochran et al. (2014), our tent with findings by Cochran et al. (2014). greater taxon and character sampling resulted in better Nakazato and Gastony (2003) provided the first support. molecular evidence that Anogramma, as traditionally The sister relationship between Actiniopteris and circumscribed, is highly paraphyletic, with everything Onychium (Prado et al., 2007; Schuettpelz et al., 2007; except A. leptophylla (and A. guatemalensis) moved to Schneider et al., 2013) is also recognized in our study other genera, including Eriosorus, Pityrogramma, etc. (BS = 100%, PP = 100%, JK = 100%). The two gen- Our results confirm the polyphyly of Anogramma. era share the morphological features of sporangia in Schneider et al. (2013) transferred a portion of a soral line in a marginal commissure covered by a Anogramma to Cerosora. Previous studies were uncer- well-developed marginal indusium. The Actiniopter- tain about the distinctiveness of A. guatemalensis is + Onychium clade is strongly supported as sister to from A. leptophylla (Tryon, 1962; Gastony and the remaining non-Pteris genera in Pteridoideae Baroutsis, 1975; Smith, 1981; Mickel and Smith, (Fig. 1a, b), a relationship consistent with earlier 2004), however, there was an amplified fragment

(a) Pteris (see Fig. 1b)

Pteridoideae

Onychium contiguum Onychium japonicum Actiniopteris dimorpha Actiniopteris semiflabella Cosentinia vellea Anogramma leptophylla Anogramma guatemalensis Cerosora (Anogramma) microphylla Pityrogramma ebenea Pityrogramma jamesonii Pityrogramma sp. Pityrogramma austroamericana Pityrogramma presliana Pityrogramma calomelanos Pityrogramma calomelanos Pityrogramma ochracea Pityrogramma trifoliata Eriosorus flexuosus Eriosorus flexuosus Eriosorus cheilanthoides Eriosorus elongatus Eriosorus hirtus Jamesonia scammaniae Jamesonia goudotii Jamesonia verticalis Jamesonia verticalis Pterozonium brevifrons Pterozonium brevifrons Pterozonium reniforme Austrogramme decipiens Austrogramme marginata Syngramma quinata Taenitis blechnoide Taenitis interrupta Tryonia myriophylla Tryonia myriophylla Acrostichum danaeifolium Acrostichum danaeifolium Ceratopteris richardii Adiantum capillusveneris

0.04 substitutions/site Fig. 1. (a) Maximum likelihood phylogeny of the Pteridoideae based on six plastid regions (atpA, atpB, rbcL, rps4-trnS, trnL and trnL-F) (ln = 45 767.2102). Thickest lines indicate strong support [maximum parsimony jackknife support (MP JK) ≥ 75%, maximum likelihood boot- strap support (ML BS) ≥ 75% and Bayesian inference posterior probability (BI PP) ≥ 95%)], medium lines indicate moderate support (either ML BS ≥ 75% or BI PP ≥ 95%), and thin lines indicate weak support (ML BS ≤ 75% and BI PP ≤ 95%). Dashed branches indicate those that have been truncated. The 15 major clades of Pteris resolved in this study are indicated. In (b) the major diagnostic morphological features are shown in green and geographical provenances of samples of Pteris are indicated in blue. (Colour online) L. Zhang et al. / Cladistics (2014) 1–18 7 length polymorphism (AFLP) study of populations in sample from Mexico (Nakazato and Gastony, 2003). the complex showing a high genetic identity of Our data suggest that the two taxa are distinct (23 A. guatemalensis with A. leptophylla, especially the nucleotide differences in rbcL and rps4-trnS) and that

(b) P. nanlingensis Guizhou, China P. japonica Taiwan, China P. esquirolii Guizhou, China P. cretica Yunnan, China P. umbrosa Atherton, Australia P. umbrosa cult. in Atherton, Australia P. deltodon Sichuan, China P. xiaoyingiae Guangxi, China Veins free, frond often dimorphic, P. actiniopteroides Sichuan, China P. gallinopes Guizhou, China XV lamina pedate, pinnatifid or pinnate, P. henryi Guizhou, China P. dactylina Sichuan, China pinnae never pectinate P. multifida Selangor, Malaysia P. multifida Guayas, Ecuador P. multifida Jiangxi, China P. morii Hainan, China P. morii Haina n, China P. pseudopellucida Louangphrabang, Laos P. sp. Sulawesi Tenggara, Indonesia P. mutilata Puerto Rico P. pungens Heredia, Costa Rica Terminal or lateral pinna conspicuously P. usambarensis Taita-Taveta, Kenya XIV P. commutata Buliisa, Uganda decurrent P. burtonii Buliisa, Uganda P. navarrensis San José, Costa Rica P. livida Napo, Ecuador P. decurrens São Paulo, Brazil P. deflexa Rio de Janeiro, Brazil P. muricata Pichincha, Ecuador P. deflexa Rio Grande do Sul., Brazil Native to South America, veins areolate P. altissima Alajuela, Costa Rica XIII P. chiapensis Chiapas, Mexico (except P. deflexa and P. muricata) P. altissima cult. in UC Berkeley Bot. Gard., USA P. speciosa Alajuela, Costa Rica P. haenkeana Pasco, Peru P. podophylla Pichincha, Ecuador P. orizabae Chiapas, Mexico Lamina herbaceous, pinnae pectinate, length-width P. dentata La Réunion, France XII P. finotii Hainan, China ratio of pinnules of middle part of pinnae ca. 5:1 P. wallichiana var. wallichiana Guangdong, China P. wallichiana var. yunnanensis Yunnan, China Superclade C P. arborea Guadeloupe P. tripartita cult. in Atherton, Australia P. tripartita cult. in Bot. Gart. Berlin-Dahlem, Germany Large habit, veins areolate P. buchananii Chogoria, Kenya XI (except P. barklyae and P. saxatilis New Zealand P. comans New Zealand P. pallens ) P. macilenta New Zealand P. microptera Lord Howe Island, Australia P. (Ochropteris) pallens La Réunion, France P. (Afropteris) barklyae Seychelles P. lechleri Paraná, Brazil P. lechleri São Paulo, Brazil P. propinqua Zamora-Chinchipe, Brazil P. splendens São Paulo, Brazil X P. brasiliensis São Paulo, Brazil Native to South America, veins areolate P. denticulata São Paulo, Brazil P. denticulata Rio de Janeiro, Brazil P. leptophylla São Paulo, Brazil P. linearis Hainan, China P. linearis La Réunion, France P. setulosocostulata Sichuan, China P. catoptera Samburu, Kenya P. preussii Masindi, Uganda Veins free (except P. biaurita), pinnae P. quadriaurita cult. in Alter Bot. Gart., Germany P. friesii Kagera, Tanzania IX pectinate, basal pairs of pinnae with P. griffithii Kachin, Myanmar 1–3 (or 4) pinnules near base at P. biaurita Nan, Thailand P. biaurita Hainan, China basiscopic side P. splendida Guangxi, China P. pacifica cult. in Atherton, Australia P. viridissima Guizhou, China P. puberula Yunnan, China P. heteromorpha Louangphrabang, Laos P. pellucida Kachin, Myanmar P. decrescens Guangxi, China P. decrescens Guizhou, China VIII Veins free, lamina 1-pinnate P. cadieri Guangdong, China to 2-pinnatifid P. grevilleana Hainan, China P. quadristipitis Guizhou, China P. (Neurocallis) praestantissima Guadeloupe, France P. (Neurocallis) praestantissima Heredia, Costa Rica P. fraseri Napo, Ecuador Length-width ratio of lateral pinnae P. terminalis Sichuan, China VII Superclade B P. insignis Guangdong, China or pinnules ca. 5:1 or larger P. ensiformis cult. in Australia P. ensiformis Yunnan, China P. bella Hainan, China P. bella Guangdong, China P. amoena Tibet, China VI Native to Asia, pinnae pectinate, P. mcclurei Guangdong, China P. mcclurei P. longipes Hainan, China veins free (except ) P. longipes Yunnan, China P. semipinnata Sichuan, China P. dimidiata Guangdong, China V Veins free, pinnae pectinate, acroscopic P. dimidiata Pahang, Malaysia pinnae entire, subentire or lobed P. chilensis Limache, Chile P. tremula cult. in Australia IV Veins free, lamina 3-pinnate to P. tremula cult. in Australia 4-pinnatifid Superclade A P. vittata Florida, USA P. vittata cult. in Australia P. vittata Sichuan, China III Veins free, lamina 1-pinnate, P. longifolia Guerrero, Mexico lanceolate P. bahamensis Florida, USA P. grandifolia Huehuetenango, Guatemala II Length-width ratio of costal areolae ca. 5:1 or larger Platyzoma microphyllum I Heterosporous condition

rest of Pteridoideae (see Fig. 1a)

0.04 substitutions/site Fig. 1b. Continued 8 L. Zhang et al. / Cladistics (2014) 1–18

A. guatemalensis might be recognized taxonomically Resolution of Neurocallis at some rank. Cerosora, Platyzoma and all members of the lower Neurocallis comprises only N. praestantissima,an clade of Fig. 1a (Austrogramme, Eriosorus, Jamesonia, uncommonly collected species with a discontinuous Pterozonium, Syngramma, Taenitis and Tryonia) bear distribution in the Neotropics (Christensen, 1934; trichomes or bristles instead of scales on rhizome or Copeland, 1947; Wagner, 1980; Tryon et al., 1990). stem (Tryon et al., 1990). Based on our phylogeny Leaves of N. praestantissima are dimorphic, singly (Fig. 1a, b), trichomes or bristles appear to have pinnate and have anastomosing veins. Based on the evolved from scales three times independently in these nearly acrostichoid fertile fronds, some authors treated three clades. the genus as a synonym of Acrostichum (e.g. Christen- Additional important relationships in the Pteridoi- sen, 1906). Wagner (1980) found that the chromosome deae will be discussed below. number (n=58) of Neurocallis is consistent with Pteris and the leaves have some similarities to those of Resolution of Pterozonium P. grandifolia L., and he suggested a closer relation- ship between Neurocallis and Pteris. Schuettpelz et al. Copeland (1947) suggested that the morphologically (2007) showed that N. praestantissima should be variable South American genus Pterozonium might be included in Pteris and was sister to a clade containing related to Eriosorus and Jamesonia. Our study found P. argyraea T. Moore, P. fauriei Hieron. and P. qua- instead that Pterozonium is sister to a clade contain- driaurita Retz. Our study supports the inclusion of ing Austrogramme, Syngramma, Taenitis and Tryonia. Neurocallis in Pteris but found that it is more closely The sister genera Austrogramme and Syngramma are related with P. fraseri Mett. ex Kuhn than with together sister to Taenitis, which is consistent with P. grandifolia or the P. quadriaurita complex (Fig. 1b: the findings of Sanchez-Baracaldo (2004b), who did clade VII). Pteris fraseri is distributed in tropical mon- not sample species of Tryonia. In comparison with tane forests of Ecuador and Peru. The remarkable Taenitis (which has veins anastomosing from costa to morphological characters of P. fraseri include the tall margins or rarely only costal areolae present), both habit, large sterile fronds of more than 2 m long and Austrogramme and Syngramma have veins joined only the ultimate pinnae ternate—long, entire and remotely at the margin or in one or two series of marginal spaced. However, the species has dimorphic fronds, areolae. Biogeographically, these five genera are dis- sterile fronds much wider than fertile fronds, anasto- tributed pantropically from India (to Fiji; Taenitis), mosing veins, and entire pinna margins, which are Malesia to the Pacific islands (Austrogramme and comparable to N. praestantissima. Syngramma) and to South America (Pterozonium and Tryonia). Resolution of Afropteris and Ochropteris

Resolution of Eriosorus and Jamesonia According to Alston (1956), Afropteris consists of two species: A. repens (C. Chr.) Alston in tropical Austrogramme, Pterozonium, Syngramma, Taenitis West Africa and A. barklyae, which is endemic to the and Tryonia together are sister to a clade comprising Seychelles. Tryon et al. (1990) placed Afropteris in the Eriosorus (five species sampled) and Jamesonia (four Pteridaceae subfamily Taenitidoideae. Ochropteris is a species sampled) (Fig. 1a, b). Notably, Jamesonia as small tropical genus occurring in Madagascar and the currently circumscribed is paraphyletic in relation to a nearby Seychelles and Mascarenes; O. bosseri Tardieu, monophyletic Eriosorus, a resolution different from the O. pallens and O. peltigera Fee are typically recog- rps4 phylogeny of Sanchez-Baracaldo (2004a) and the nized, although some authors have treated O. bosseri rbcL phylogeny of Schuettpelz et al. (2007), where and O. peltigera as synonyms of O. pallens (e.g. Tar- both genera were found to be paraphyletic in relation dieu-Blot, 1958; Autrey et al., 2008). Before the genus to each other. However, our resolution is consistent was described, O. pallens was placed in Adiantum, with that found by Cochran et al. (2014), who placed Cheilanthes Sw. or Cryptogramma R. Br. When Smith species of Eriosorus in Jamesonia. Based on our result, (1841) published Ochropteris, he recognized that the simply transferring J. scammaniae A. F. Tryon to Eri- morphology of the sori was similar to that of Pteris, osorus would make both genera monophyletic but that the habit was inconsistent with any species of (Fig. 1a) in our current sampling. However, our taxon Pteris. Tryon and Tryon (1982) noted that Afropteris sampling of the two genera is much smaller than that and Ochropteris were doubtfully distinct from Pteris of Sanchez-Baracaldo (2004a) but we have much larger and Tryon et al. (1990) placed Ochropteris in the Pte- character sampling. In order to resolve the relation- ridaceae subfamily Pteridoideae. Using single plastid ships between Eriosorus and Jamesonia it is desirable sequence data rps4-trnS Sanchez-Baracaldo (2004b) to have larger sampling of both taxa and characters. found that A. barklyae was nested within Pteris—simi- L. Zhang et al. / Cladistics (2014) 1–18 9 lar results to those found by Schuettpelz et al. (2007) et al., 2010) or combined with data of two additional using rbcL data. Our study confirms their findings and plastid genes, atpA and atpB (Schuettpelz et al., 2007), further reveals the sister relationship between Ochrop- the inclusion of Platyzoma in Pteridaceae was con- teris and Afopteris (Fig. 1b: clade XI). Although the firmed, but its precise phylogenetic relationships habits of the two taxa are quite different from other remained unresolved. In our analysis, the inclusion of species of Pteris, they are morphologically similar to Platyzoma in Pteridoideae is strongly supported. With each other in having the long-creeping rhizome, lamina the three long-branched attracting genera removed, our herbaceous and three-pinnate to four-pinnatifid, and BI, ML and MP analyses of six-gene data resolved fine ultimate pinnules. Platyzoma as being nested within Pteris and as sister to clade II (P. grandifolia) + clade III [P. longifolia/ Resolution of Platyzoma P. bahamensis (J. Agardh) Fee and P. vittata; Fig. 1b]. This resolution received relatively high BS (96%), The genus Platyzoma contains only the striking spe- PP (100%) and JK (78%) support in ML, BI and cies P. microphyllum, which is endemic to northern MP analyses, respectively. Notably, the effect of long- Australia (Brown, 1810). Due to its anomalous branch attraction was neglected by all previous studies morphology (narrowly linear single-pinnate fronds, when Platyzoma was involved (Hasebe et al., 1994; presence of abundant farina, and incipient hetero- Prado et al., 2007; Schuettpelz et al., 2007; Schneider spory), the systematic position of the species has long et al., 2013). Interestingly, all species of superclade A been controversial. The initial studies tended to treat it (clades I–III; Fig. 1b) have single-pinnate lanceolate/ as a genus of Gleicheniaceae allied to Gleichenia Link linear lamina, which might be interpreted as a morpho- (Brown, 1810; Presl, 1836; Moore, 1857; Hooker and logical synapomorphy of superclade A. Baker, 1874), or as a species of Gleichenia (Christ, Platyzoma microphyllum is the only non-aquatic fern 1897; Christensen, 1906). Thompson (1917) provided with a heterosporous condition. In about 11 000 species detailed data on anatomy of the species but still treated of ferns, heterospory occurs in only six genera: Azolla it in Gleicheniaceae. However, he suggested that its Lam., Marsilea L., Pilularia L., Regnellidium Lindm., close affinity with Gleichenia was dubious. Nakai Salvinia Seg. (aquatic ferns) and Platyzoma. Similar to (1950) proposed a monospecific family Platyzomata- habitats of other heterosporous ferns, P. microphyllum ceae, which was adopted by Bostock et al. (1998). primarily grows in deep granitic sand that overlies heavy Tryon (1961, 1964) placed Platyzoma in the Polypodia- clay and is often flooded in the rainy season (Tryon ceae subfamily Platyzomatoideae and later (Tryon and et al., 1990). Adaption to wet conditions might have dri- Tryon, 1982) in the Pteridaceae tribe Platyzomateae or ven the evolution of heterospory in all these ferns. the Pteridaceae subfamily Platyzomatoideae (Tryon et al., 1990). Hasebe et al. (1994), who sampled 12 Monophyly of Pteris species in 11 genera of Pteridaceae, first resolved Platy- zoma as a member of Pteridaceae based on a single- Our analyses showed that Pteris in its current locus dataset of rbcL. In their study, Platyzoma and circumscription is paraphyletic in relation to Afropteris, Taenitis were resolved as sister to each other. Based on Neurocallis, Ochropteris and Platyzoma (Fig. 1b). This the same rbcL sequence (Prado et al., 2007; Bouma resolution is consistent with the results of earlier stud-

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

Number of Number of Average Number of Number of PI characters MPT Number of MP JK/ML MP JK/ML Matrix accessions characters (%)* length MPTs BS clades BS support (%) CI RI

rbcL gene 139 1286 338 (26.2) 1334 1 791 800 93/105 85/86 0.4303 0.8177 atpB gene 127 1189 297 (25.0) 1104 1 899 400 76/86 85/87 0.4457 0.8234 atpA gene 129 1819 528 (29.0) 1816 1 971 100 92/96 89/91 0.5061 0.8447 rps4-trnS spacer 127 1018 402 (39.5) 1277 1 864 600 100/100 87/88 0.5732 0.8612 trnL intron 113 755 319 (42.3) 1006 2 015 100 75/85 85/87 0.5895 0.8693 trnL-F spacer 113 466 236 (50.6) 831 1 989 500 78/80 81/85 0.5620 0.8436 trnL intron and 114 1221 555 (45.5) 1846 2 016 200 91/98 89/89 0.5742 0.8568 trnL-F spacer Simultaneous 146 6534 2199 (33.7) 7442 1 676 700 129/136 93/94 0.5075 0.8394

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. 10 L. Zhang et al. / Cladistics (2014) 1–18 ies using limited character and taxon sampling above), Platyzoma microphyllum is clearly different (Sanchez-Baracaldo, 2004a; Prado et al., 2007; Schuett- from all the other species of Pteris by having two sizes pelz et al., 2007; Schneider et al., 2013). Our study pro- of spores, a dioecious condition of the gametophytes vides the first strong molecular evidence that the and chromosome number of 2n = 76 (Tryon and Vida, Australian endemic Platyzoma, along with Afropteris, 1967). However, it shares single-pinnate lamina with Neurocallis and Ochropteris, is embedded in Pteris other members of superclade A (see above). sensu lato. At this stage we advocate a broad circum- scription of Pteris that includes these smaller genera Clade II—the P. grandifolia clade (Afropteris, Neurocallis, Ochropteris and Platyzoma each contain only one to three species; names for all This clade contains only one species, the remark- the taxa are already available in Pteris (Zhang et al., able P. grandifolia. Plants of the species are large, 2014)) in order to maintain maximum nomenclatural with leaves 1–4 m, and singly pinnate (rarely with stability (Pteris comprises about 200–250 species, most basal pinna pinnate-pinnatifid). Veins of P. grandifolia of which would require new names were Pteris sensu are copiously anastomosing, the costal areolae are lato to be divided into smaller genera). Under this defi- very long, and their long axis is nearly at a right nition—a broadly defined Pteris—Pteris is resolved as angle to the costa (Fee, 1852). Because of the unusual monophyletic (Fig. 1a) with high support (BS = 100%, morphological characters, Fee (1852) proposed a PP = 100%, MP JK = 77%—provided that the long- monospecific genus Heterophlebium Fee. Later some branch attraction artefact was accounted for). With the authors treated this as a section, Litobrochia sect. type Pteris, P. longifolia, included, our analyses are the Heterophlebia (Fee) T. Moore (Moore, 1857) or first to show strong support for the monophyly of the P. sect. Heterophlebium (Fee) Hook. (Hooker, 1858; expanded Pteris. The morphological synapomorphies Hooker and Baker, 1874; Christensen, 1906). In of the newly defined Pteris are rhizome erect or ascend- Tryon and Tyron’s (1982) six groups of tropical ing (but long-creeping in Pteris buchananii Sim, Afrop- American species of Pteris, it was placed within the teris and Ochropteris), sporangia continuous along “P. haenkeana Group” together with several species commissural veins of pinna margins (but along mar- that have anastomosing veins and large ultimate pin- ginal portions of the veins and not on a commissure in nae, for example, P. haenkeana C. Presl. Our study Platyzoma), and pinna apex and pinna base often ster- provides the first evidence that P. grandifolia is an ile (but not in Afropteris or Platyzoma, and often not isolated species, most allied to P. vittata and related in Ochropteris; acrostichoid in Neurocallis). Placing species. Morphologically, the two clades share single- these genera into Pteris appears to destroy the morpho- pinnate lamina and the terminal pinna larger than the logical cohesiveness of Pteris, but apparently, the adjacent lateral pinnae. In addition, Copeland (1947) inconsistent morphological character states in some found that the annuli of P. vittata are usually taxa can most parsimoniously be interpreted as composed of more than 30 thickened cells, a number autapomorphic based on our phylogeny (Fig. 1b). closer to that of P. grandifolia (26–31) than to those of other species of Pteris (16–20). Major evolutionary lineages of Pteris Clade III—the P. longifolia clade Within the newly defined Pteris (including Afropter- is, Neurocallis, Ochropteris and Platyzoma), the 86 This clade is consistent with the P. longifolia Group species included in the current study are resolved into determined by Tryon and Tryon (1982). Pteris vittata the following 15 well-supported major clades (Fig. 1b). and P. longifolia were the first species included in Pter- Most of these major clades are also supported by is when Linnaeus (1753) published the genus, and the morphological characters. The 15 major clades listed latter is designated as lectotype of Pteris (Smith, represent major evolutionary lineages in Pteris at the 1875). Pteris vittata is one of most widely distributed global level. Before making any taxonomic and species in the tropics and subtropics of the Old World, nomenclatural decision on infrageneric classification, and is commonly naturalized in the New World. Mor- more samples (especially from the Neotropics, Africa phologically, P. vittata is similar to P. longifolia; the and Malesia) and molecular data (especially nuclear latter is a polyploid complex that in the broad sense is genes) are needed to further test the monophyly of the limited to Mexico and the Caribbean islands (Mickel 15 clades defined. and Smith, 2004). The two species share nearly the same morphology as a whole, for example, lamina sin- Clade I—the Pteris (Platyzoma) microphylla clade gly pinnate with independent terminal pinna, stipe base densely scaly. Hieronymus (1914) pointed out Although the inclusion of Platyzoma in Pteris was that P. longifolia has articulate pinna bases and strongly supported by our plastid data analysis (see spreading pinnae, which can be used to distinguish it L. Zhang et al. / Cladistics (2014) 1–18 11 from P. vittata. Our results confirmed the close affinity Clade VII—the P. ensiformis clade of the two species, however, the genetic divergence of the two species is distinct (Fig. 1b). The Bahamian This clade contains species occurring in America, endemic P. bahamensis was once treated as a variety Asia and Oceania, and encompasses considerable mor- of P. longifolia (Hieronymus, 1914), but the former phological disparity. Our results suggest a close rela- has a glabrous rachis and its pinna bases are never tionship among those species, which has not been cordate. The sister relationship of the two species is suggested by earlier studies due to the lack of diagnos- well supported in our analysis. tic characters. There are obvious divergences among each species. Two South American species, P. fraseri Clade IV—the P. chilensis clade and P. praestantissimam, together are resolved as monophyletic with strong support. Pteris terminalis In our sample, this clade contains two species, Wall. ex J. Agardh (synonym: P. excelsa Gaudich.; P. tremula R. Br. and P. chilensis Desv, which were Liao et al., 2013) is commonly distributed in Asia, both included in “P. sect. Pteris” by Hooker and reaching the Hawaiian Islands and Fiji. It is resolved Baker (1874) or in the “P. chilensis Group” by Tryon as sister to the monophyletic lineage formed by P. ens- and Tryon (1982). Pteris tremula is distributed in the iformis and P. insignis Mett. ex Kuhn. Further studies South Pacific and is often locally naturalized in the are needed to unravel the evolutionary history of the Northern Hemisphere. The Chilean endemic P. chilen- species in clade VII. sis is sister to our two P. tremula samples, but the sequence variation between the two species is low. The Clade VIII—the P. decrescens clade two species occur naturally far away from each other, but they are consistent morphologically in their three- All species of this clade occur in Asia. Our results to four-pinnatifid lamina, ultimate pinnules (lobes) for the first time disclosed the close relationships much smaller and veins free. among the species in this clade. The morphological cir- cumscription of this clade is difficult; potential diag- Clade V—the P. semipinnata clade nostic features include veins free and lamina one- pinnate to two-pinnatifid. Three species, P. cadieri, This clade contains only two endemic Asian species P. decrescens and P. grevilleana, of clade VIII have —Pteris dimidiata Willd. and P. semipinnata L.—in been observed to have silica bodies on their leaves our sampling. The position of the two species has not (Wagner, 1978; Kao et al., 2008; Sundue, 2009). This been determined in previous classifications (Shieh, feature appears to have evolved independently in 1966; Wu, 1990; Yang, 2011; Liao et al., 2013). Actu- P. multifida Poir. (clade XV). Three deeply divergent ally, the two species differ from most species of Pteris lineages can be recognized within this clade. Forming in having fronds that are singly pinnate, but with the the first diverging lineage, the Southeast Asian P. het- basiscopic side of at least the proximal pinnae pinnati- eromorpha Fee and P. pellucida C. Presl are resolved fid or pinnate, but the acroscopic side undivided and as sister to each other. They together are sister to poorly developed. P. decrescens Christ + a clade containing P. cadieri Christ, P. grevilleana Wall. ex J. Agardh and P. quad- Clade VI—the P. longipes clade ristipitis X.Y.Wang & P.S.Wang. Pteris cadieri was resolved as sister to a clade composed of P. grevilleana Species of this clade are endemic to subtropical and and P. quadristipitis with maximum support. Except tropical Asia. The included four species of this clade for P. heteromorpha and P. pellucida, all remaining were placed in P. sect. Quadriauricula (including species of clade VIII previously were placed in P. sect. P. amoena Blume, P. bella Tagawa and P. longipes D. Quadriauricula (Wu, 1990; Liao et al., 2013). Don) and P. sect. Campteria (including P. mcclurei Ching) (Liao et al., 2013). None of the previous stud- Clade IX—the P. quadriaurita clade ies proposed a close affinity among these species. Mor- phologically, species of this clade have thinner This clade’s composition is similar to P. sect. Qua- laminae, petioles brown or reddish brown (except driauricula sensu Wu (1990) and Liao et al. (2013). P. longipes), and leaves two-pinnate to three-pinnatifid Species of this clade are widespread on four conti- with pectinate pinnae. Pteris longipes differs from nents. Morphologically they share the lamina two-pin- other species in this clade in its ternate fronds. Pteris natifid to two-pinnate, fronds not ternate or pedate, mcclurei has one row of areoles along the costa and basal pairs of pinnae often with one to three (or four) was resolved as closely allied to P. amoena, which has pinnules near the base on the basiscopic side, and free veins. These two species form a well-supported veins free (except P. biaurita L.). Two Asian endemics, clade. P. puberula Ching and P. viridissima Ching, and the 12 L. Zhang et al. / Cladistics (2014) 1–18

Malesian–Australian–South Pacific species P. pacifica sometimes treated as a complex (Bostock et al., 1998). Hieron. form the basal grade, followed by an unre- Our data resolved the Lord Howe Island endemic solved polytomy. This poorly resolved clade is taxo- P. microptera as sister to a clade of the remaining nomically complex and is beyond the scope of the three taxa. present study. Two samples of P. biaurita from China and Thailand together, respectively, are resolved as sis- Clade XII—the P. dentata clade ter to the Asian endemic P. griffithii Hook. Two sam- ples of P. linearis Poir. from China and Reunion, This clade contains one species only, P. dentata For- respectively, formed a monophyletic clade but ssk. It formed an unresolved trichotomy with clade sequence divergence between these two samples is pro- XIII and clades XIV + XV. Pteris dentata is widely nounced. Our plastid data well resolved the maternal distributed in the Arabian Peninsula, Sub-Saharan relationships between P. biaurita and P. linearis, while Africa, northwestern Africa and the Mascarenes. This relationships based on morphology seem complicated, species has the lamina herbaceous and two-pinnate to as suggested previously by some authors (Walker, three-pinnatifid, pinnules (lobes) long and narrow and 1962; Shieh, 1966). The three African endemics P. ca- with toothed distal margins, veins free and lacking toptera Kunze, P. friesii Hieron. and P. preussii Hier- anastomosing veins below the sinus. The uncommon on. form a monophyletic group with the widespread morphology and distributional pattern are concordant species P. quadriaurita, suggestive of an African origin with the isolated phylogenetic position in the genus of the latter. (Fig. 1b).

Clade X—the P. splendens clade Clade XIII—the P. navarrens clade

All species of this clade sampled are distributed in All species of this clade are distributed from Central Brazil and were included in Pteris subg. Litobrochia by to South America. The sampled species of this clade Christensen (1906). In consideration of the diversity of largely correspond to Tryon and Tryon’s (1982) Pteris frond architecture, six species of this clade were deflexa group when P. propinqua J. Agardh. and included in five groups by Tryon and Tryon (1982). P. tripartita are excluded and P. chiapensis A.R. Sm., The morphological synapomorphy of this clade could P. decurrens C. Presl, P. haenkeana and P. speciosa be the anastomosing veins. Except for P. leptophylla Mett. ex Kuhn are added to the group. This clade can Sw., which has only several areolae near costae and be divided into three subclades. The first subclade con- costules, all other species in this clade have multiple tains P. livida Mett. and P. navarrensis Christ, which rows of areolae (Prado and Windisch, 2000). share large habit, ternate or pedate fronds, and anas- tomosing veins. The second subclade contains P. de- Clade XI—the P. tripartita clade currens, P. deflexa Link, and P. muricata Hook. The first species has reticulate veins and the other two have All species of this clade are distributed in the Old free venation. Notably, two samples of P. deflexa did World (including Oceania) and have large leaves. not form a monophyletic clade; more studies on this Some species can grow to 3 m tall, for example, P. ar- species are needed. The third subclade contains P. al- borea L. Except Afropteris barklyae and Ochropteris tissima Poir., P. chiapensis, P. haenkeana, P. orizabae pallens, all other species have anastomosing veins. This M. Martens & Galeotti, P. podophylla Sw. and clade can be divided into four subclades. The first con- P. speciosa. The relationships of this subclade are well tains P. arborea, P. finotii Christ, P. tripartita Sw. and resolved. Pteris orizabae is sister to the rest of this P. wallichiana J. Agardh.; species of this subclade have subclade, followed by P. haenkeana and P. podophylla. largely pedate fronds. The Old World widespread trop- Pteris orizabae and P. podophylla both have ternate ical species P. tripartite, together with P. arborea,is fronds. The resolution of the morphologically anoma- sister to the Asian endemic P. finotii plus P. wallichi- lous P. haenkeana in this subclade is surprising. This ana. The second subclade contains only the African species has a single pinnate lamina (two pinnate at endemic P. buchananii. The third subclade contains Af- base), large ultimate pinnae, and is often considered ropteris barklyae and Ochropteris pallens, two endemics close to P. grandifolia (Tryon and Tryon, 1982; Tryon of Indian Ocean islands, all with free veins. The mor- et al., 1989). Its affinity with P. speciosa was strongly phology of the sporangia of both is similar to that of supported. Two samples of P. altissima did not form a Pteris. The fourth subclade contains four Oceanian monophyletic group because P. chiapensis was placed species, P. comans G. Forst., P. macilenta A. Rich., between the two samples. Generally, species of clade P. microptera Mett. ex Kuhn and P. saxatilis Carse, XIII have significant morphological variability: frond all with anastomosing veins. These four species are pinnate to pedate or ternate, lamina one-pinnate to similar to one another in morphology and thus are three- or four-pinnatifid, veins free or anastomosing, L. Zhang et al. / Cladistics (2014) 1–18 13 etc. However, most species of this clade have toothed cies. The third subclade contains eight species mostly distal pinna margins and anastomosing veins (except distributed in limestone areas: P. actiniopteroides P. deflexa and P. muricata). Christ, P. dactylina Hook., P. gallinopes Ching, P. henryi Christ, P. multifida, P. deltodon Baker and Clade XIV—the P. mutilata clade P. xiaoyingiae H. He & Li Bing Zhang. The latter two (P. deltodon and P. xiaoyingiae) together are strongly This clade contains five species, P. burtonii Baker, supported as sister to a clade containing the other P. commutata Kuhn, P. mutilata L., P. pungens Willd. seven species. Interestingly, these two have ovate to and P. usambarensis Hieron., and can be divided into elliptic pinnae, whereas the other seven have linear to two well-supported subclades. Two American endem- lanceolate pinnae. Pteris multifida has conspicuously ics, P. mutilata and P. pungens, constitute the first decurrent pinnae. Pteris deltodon and P. xiaoyingiae subclade. Pteris pungens is a relatively common spe- often occur on limestone cliffs and the close relation- cies ranging from Mesoamerica to South America. It ship between them has been recognized morphologi- is morphologically similar to members of the P. qua- cally (He and Zhang, 2010) as well as with molecular driaurita complex and was placed in the “P. quadriau- data (Fig. 1b). rita Group” by Tryon and Tryon (1982). However, Previous studies found discrepancies in basic chro- this species has one or two veinlets arising from the mosome numbers from x = 29 in Pteris deltodon to costa between adjacent ultimate segments, as well as x = 55 in all other species of Pteris (Walker, 1960, reddish brown stipe with sparse spines and often much 1962; Punetha and Sen, 1989; Wang, 1989; Kato et al., longer than the lamina, making it distinct from other 1992; Lin et al., 1996; Chao et al., 2012b; Jaruwat- species of the “P. quadriaurita Group”. Pteris mutilata tanaphan et al., 2013). However, a clear divergence is one of eight taxa first recognized by Linnaeus (1753) between P. deltodon and other species of the genus is in the genus Pteris and is distributed only in parts of not confirmed in our phylogeny. the Caribbean. This species has dimorphic fronds, sin- gle-pinnate lamina and basal pinnae often pinnate or pinnatifid, which is similar to P. ensiformis, but P. mu- Acknowledgements tilata has slender cartilaginous margins of the sterile pinnules (Hooker, 1858). The close phylogenetic rela- We thank Holly Forbes, Barbara Keller, Anders tionship between P. mutilata and P. pungens is unex- Larsson, Joel Nitta, Hank Oppenheimer, Tom Ranker, pected given their morphological differences. Three James Solomon, Michael Sundue, Pei-Shan Wang, Su- African endemics form the second subclade. 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Actiniopteris dimorpha Pic. Serm., Schneider s.n. (GOET): rbcL KM008027, trnL-F KM007915, rps4-trnS KM007800; Zhang Liang EF452130 (Schuettpelz et al., 2007), atpB EF452009 (Schuettpelz 1186 (CDBI): rbcL KM008140, atpB KM007688, atpA KM007571, et al., 2007), atpA EF452066 (Schuettpelz et al., 2007), trnL trnL KM008028, trnL-F KM007916, rps4-trnS KM007801. Pityro- KM008014, trnL-F KM007902, rps4-trnS KM007786. Actiniopteris gramma ebenea (L.) Proctor, Rothfels et al. 08-19 (DUKE): rbcL semiflabellata Pic. Serm., Smith s.n. (UC): rbcL KM008127, atpB KM008141, atpB KM007689, atpA KM007572, rps4-trnS KM007674, atpA KM007557, trnL KM008015, trnL-F KM007903, KM007802. Pityrogramma jamesonii (Baker) Domin, Moran 7592 rps4-trnS KM007787. (NY): rbcL EF452167 (Schuettpelz et al., 2007), atpB EF463519 Adiantum capillusveneris L., Wen 10360 (US): rbcL JF935345 (Lu (Schuettpelz et al., 2007), atpA EF463857 (Schuettpelz et al., et al., 2012), atpB JF935427 (Lu et al., 2012), atpA JF937300 (Lu 2007). Pityrogramma ochracea (C. Presl) Domin, Araujo 4253 et al., 2012). (MO): rbcL KM008142, atpB KM007690, atpA KM007573, trnL KM008029, trnL-F KM007917, rps4-trnS KM007803. Pityrogramma Anogramma guatemalensis (Domin) C. Chr., Gastony 1037D presliana Domin, Rothfels et al. 3664 (DUKE): rbcL KM008143, atpB (IND) & Smith 2586 (UC): rbcL AY168716 (Nakazato and Gastony, KM007691, atpA KM007574, trnL KM008030, trnL-F KM007918, 2003), rps4-trnS AF321699 (Sanchez-Baracaldo, 2004a). Anogramma rps4-trnS KM007804. Pityrogramma trifoliata (L.) R.M. Tryon, Roth- leptophylla (L.) Link, Zylinski s.n. (DUKE): rbcL KM008128, atpB fels 3658 (DUKE): rbcL KM008145, atpB KM007693, atpA KM007675, atpA KM007558, trnL KM008016, trnL-F KM007904, KM007576, trnL KM008032, trnL-F KM007920, rps4-trnS rps4-trnS KM007788. KM007806. Pityrogramma sp., Rothfels 3608 (DUKE): rbcL Austrogramme decipiens (Mett.) Hennipman, H. van der Werff KM008144, atpB KM007692, atpA KM007575, trnL KM008031, 16114 (UC): rps4-trnS AF321702 (Sanchez-Baracaldo, 2004a). trnL-F KM007919, rps4-trnS KM007805. Austrogramme marginata (Mett.) E. Fourn., D. Hodel 1454 (UC): Platyzoma microphyllum R. Br., Bostock s.n. (IND): rbcL rps4-trnS AY357704 (Sanchez-Baracaldo, 2004a). AY168721 (Nakazato and Gastony, 2003), atpB KM007694, atpA Ceratopteris richardii Brongn., Killip 44595(GH): rbcL EU352297 KM007577, trnL KM008033, trnL-F KM007921, rps4-trnS (Rai and Graham, 2010), atpB AY612691 (Pryer et al., 2004), atpA KM007811. DQ390550 (Schuettpelz et al., 2006), rps4-trnS AY612653 (Pryer Pteris actiniopteroides Christ, Zhang Liang 1384 (CDBI): rbcL et al., 2004). KM008146, atpB KM007696, atpA KM007580, trnL KM008036, Cerosora microphylla (Hook.) R.M. Tryon, Jin 11264 (CDBI): trnL-F KM007924, rps4-trnS KM007812. Pteris altissima Poir., atpB KM007676, atpA KM007559, rps4-trnS KM007789. 1002w (cult.): atpB KM007697, atpA KM007581, trnL KM008037, Cosentinia vellea (Aiton) Tod., Larsson 55 (UPS, DUKE): rbcL trnL-F KM007925, rps4-trnS KM007813; Nitta 863 (UC): rbcL KM008129, atpB KM007677, atpA KM007560, trnL KM008017, KM008147, atpB KM007698, atpA KM007582, trnL KM008038, trnL-F KM007905, rps4-trnS KM007790. trnL-F KM007926. Pteris amoena Blume, Qingzang 74-5078 Eriosorus cheilanthoides (Sw.) A.F. Tryon, Moran 7579 (NY): (KUN): rbcL KM008148, atpA KM007583, trnL KM008039, trnL- rbcL EF452152 (Schuettpelz et al., 2007), atpB EF452034 (Schuett- F KM007927, rps4-trnS KM007814. Pteris arborea L., Christenhusz pelz et al., 2007), atpA EF452095 (Schuettpelz et al., 2007). Eriosorus 4050 (TUR): rbcL KM008149, atpB KM007699, atpA KM007584. elongatus (Grev. & Hook.) Copel., Rothfels 3602 (DUKE): rbcL Pteris bahamensis (J. Agardh) Fee, Rothfels 4024 (DUKE): rbcL KM008130, atpB KM007678, atpA KM007561, trnL KM008018, KM008150, atpB KM007700, atpA KM007585, trnL KM008040, trnL-F KM007906, rps4-trnS KM007791. Eriosorus flexuosus (Ku- trnL-F KM007928, rps4-trnS KM007815. Pteris barklyae “Afropteris nth) Copel., Rothfels 08-042 (DUKE): rbcL KM008132, atpB barklyae (Baker) Alston”, Kramer 11086 (Z): rps4-trnS AF544984 KM007680, atpA KM007562, trnL KM008019, trnL-F KM007908, (Sanchez-Baracaldo, 2004b). Pteris bella Tagawa, Wu s.n. (KUN): rps4-trnS KM007793; Rothfels et al. 3552 (DUKE): rbcL rbcL KM008151, atpB KM007701, atpA KM007586, trnL KM008131, atpB KM007679, atpA KM007563, trnL KM008020, KM008041, trnL-F KM007929, rps4-trnS KM007816; Zhang Liang trnL-F KM007907, rps4-trnS KM007792. Eriosorus hirtus (Kunth) 1300 (CDBI): rbcL KM008152, atpB KM007702, atpA KM007587, Copel., Rothfels 3668 (DUKE): rbcL KM008133, atpB KM007681, trnL KM008042, trnL-F KM007930, rps4-trnS KM007817. Pteris atpA KM007564, trnL KM008021, trnL-F KM007909, rps4-trnS biaurita L., Larsen 44455 (MO): rbcL KM008153, atpB KM007703, KM007794. atpA KM007588, trnL KM008043, trnL-F KM007931, rps4-trnS Jamesonia goudotii (Hieron.) C. Chr., Rothfels 3694 (DUKE): KM007818; Zhang Liang 1310 (CDBI): rbcL KM008154, atpB rbcL KM008134, atpB KM007682, atpA KM007565, trnL KM007704, atpA KM007589, trnL KM008044, trnL-F KM007932, KM008022, trnL-F KM007910, rps4-trnS KM007795. Jamesonia rps4-trnS KM007819. Pteris brasiliensis Raddi, Prado 2049 (SP): scammaniae A.F. Tryon, Rothfels 2631 (DUKE): rbcL KM008135, rbcL KM008155, atpB KM007705, atpA KM007590, trnL atpB KM007683, atpA KM007566, trnL KM008023, trnL-F KM008045, trnL-F KM007933, rps4-trnS KM007820. Pteris bu- KM007911, rps4-trnS KM007796. Jamesonia verticalis Kunze, chananii Sim, Kamau 382 (EA): rbcL KM008156, atpB KM007706, Moran 7593 (NY): rbcL EF452155 (Schuettpelz et al., 2007), atpB atpA KM007591, trnL KM008046, trnL-F KM007934, rps4-trnS EF452038 (Schuettpelz et al., 2007), atpA EF452099 (Schuettpelz KM007821. Pteris burtonii Baker, Kamau 227 (EA): rbcL et al., 2007); Rothfels 3638 (DUKE): rbcL KM008136, atpB KM008157, atpB KM007707, atpA KM007592, trnL KM008047, KM007684, atpA KM007567, trnL KM008024, trnL-F KM007912, trnL-F KM007935, rps4-trnS KM007822. rps4-trnS KM007797. Pteris cadieri Christ, Zhang Liang 1240 (CDBI): rbcL Onychium contiguum Wall. ex C. Hope, Gao 13378 (CDBI): rbcL KM008158, atpB KM007708, atpA KM007593, trnL KM008048, KM008137, atpB KM007685, atpA KM007568, trnL KM008025, trnL-F KM007936, rps4-trnS KM007823. Pteris catoptera Kunze, trnL-F KM007913, rps4-trnS KM007798. Onychium japonicum Kamau 463 (EA): rbcL KM008159, atpB KM007709, atpA (Thunb.) Kunze, Zhang et al. 5997 (CDBI): rbcL KM008138, atpB KM007594, trnL KM008049, rps4-trnS KM007824. Pteris chiapen- KM007686, atpA KM007569, trnL KM008026, trnL-F KM007914, sis A.R. Sm., Perez-Farrera 2978 (MO): rbcL KM008160, atpB rps4-trnS KM007799. KM007710, atpA KM007595, trnL KM008050, trnL-F KM007937, Pityrogramma austroamericana Domin, E. Schuettpelz 301 rps4-trnS KM007825. Pteris chilensis Desv., Zollner€ 17416 (MO): (DUKE): rbcL EF452166 (Schuettpelz et al., 2007), atpB rbcL KM008161, atpB KM007711, atpA KM007596, trnL EF452050 (Schuettpelz et al., 2007), atpA EF452112 (Schuettpelz KM008051, trnL-F KM007938, rps4-trnS KM007826. Pteris co- et al., 2007), rps4-trnS AF321698 (Schuettpelz et al., 2007). Pityro- mans G. Forst., Welt P20796 (WELT): rbcL EF469954, atpB gramma calomelanos (L.) Link, Rothfels et al. 08-107 (DUKE): GU136777. Pteris commutata Kuhn, Kamau 331 (EA): rbcL rbcL KM008139, atpB KM007687, atpA KM007570, trnL KM008162, atpB KM007712, atpA KM007597, trnL KM008052, L. Zhang et al. / Cladistics (2014) 1–18 17 trnL-F KM007939, rps4-trnS KM007827. Pteris cretica L., Jin Pteris japonica (Thunb.) Mett., E. Schuettpelz 1070 (DUKE): rbcL 11016 (CDBI): rbcL KM008163, atpB KM007713, atpA KM008187, atpB KM007736, atpA KM007622, trnL KM008077, KM007598, trnL KM008053, trnL-F KM007940, rps4-trnS trnL-F KM007964, rps4-trnS KM007852. KM007828. Pteris lechleri Mett., Prado 2061 (SP): rbcL KM008188, atpB Pteris dactylina Hook., Zhang Liang 1391 (CDBI): rbcL KM007737, atpA KM007623, trnL KM008078, trnL-F KM007965, KM008164, atpB KM007714, atpA KM007599, trnL KM008054, rps4-trnS KM007853; Prado 2190 (SP): rbcL KM008189, trnL trnL-F KM007941, rps4-trnS KM007829. Pteris decrescens Christ, KM008079, trnL-F KM007966, rps4-trnS KM007854. Pteris lepto- Pan 053 (GZTM): rbcL KM008166, atpB KM007716, atpA phylla Sw., Boldrin 160 (SP): rbcL EF473707 (Prado et al., 2007). KM007601, trnL KM008056, trnL-F KM007943, rps4-trnS Pteris linearis Poir., Grangaud s.n. (MO): rbcL KM008190, atpB KM007831; Zhang et al. 5474 (CDBI): rbcL KM008165, atpB KM007738, atpA KM007624, trnL KM008080, trnL-F KM007967, KM007715, atpA KM007600, trnL KM008055, trnL-F KM007942, rps4-trnS KM007855; Zhang Liang 1309 (CDBI): rbcL KM008191, rps4-trnS KM007830. Pteris decurrens C. Presl, Prado 1082 (SP): atpB KM007739, atpA KM007625, trnL KM008081, trnL-F rbcL EF473703 (Prado et al., 2007). Pteris deflexa Link, Prado KM007968, rps4-trnS KM007856. Pteris livida Mett., E. Schuett- 1089 (SP): rbcL EF473704 (Prado et al., 2007); Prado 2124 (SP): pelz 936 (DUKE): rbcL KM008192, atpB KM007740, atpA rbcL KM008167, atpB KM007717, atpA KM007602, trnL KM007626, trnL KM008082, trnL-F KM007969, rps4-trnS KM008057, trnL-F KM007944, rps4-trnS KM007832. Pteris delto- KM007857. Pteris longifolia L., Rothfels 3276 (DUKE): rbcL don Baker, Zhang Liang 1356 (CDBI): rbcL KM008168, atpB KM008193, atpB KM007741, atpA KM007627, trnL KM008083, KM007718, atpA KM007603, trnL KM008058, trnL-F KM007945 trnL-F KM007970, rps4-trnS KM007858. Pteris longipes D. Don, rps4-trnS KM007833. Pteris dentata Forssk., Tamon s.n.: rbcL Jin 11507 (CDBI): rbcL KM008194, atpB KM007742, atpA KM008169, atpB KM007719, atpA KM007604, trnL KM008059, KM007628, trnL KM008084, trnL-F KM007971, rps4-trnS trnL-F KM007946, rps4-trnS KM007834. Pteris denticulata Sw., KM007859; Zhang Liang 1321 (CDBI): rbcL KM008195, atpB Prado 2159 (SP): rbcL KM008170, atpB KM007720, atpA KM007743, atpA KM007629, trnL KM008085, trnL-F KM007972, KM007605, trnL KM008060, trnL-F KM007947, rps4-trnS rps4-trnS KM007860. KM007835; Prado 1084 (SP): rbcL EF473705. Pteris dimidiata Pteris macilenta A. Rich., Welt P021006 (WELT): rbcL Willd., E. Schuettpelz 893 (DUKE): rbcL KM008171, atpB GU136797 (Bouma et al., 2010), atpB GU136778 (Bouma et al., KM007721, atpA KM007606, trnL KM008061, trnL-F KM007948, 2010). Pteris mcclurei Ching, Zhang Liang 1289 (CDBI): rbcL rps4-trnS KM007836; Zhang Liang 1287 (CDBI): rbcL KM008172, KM008196, atpB KM007744, atpA KM007630, trnL KM008086, atpB KM007722, atpA KM007607, trnL KM008062, trnL-F trnL-F KM007973, rps4-trnS KM007861. Pteris microptera Mett. ex KM007949, rps4-trnS KM007837. Kuhn, Papadopulos AP960: rbcL JF950814 (Papadopulos et al., Pteris ensiformis Burm., ARF3539 (cult.): rbcL KM008173, atpB 2011). Pteris morii Masam., Zhang Liang 1314 (CDBI): rbcL KM007723, atpA KM007608, trnL KM008063, trnL-F KM007950, KM008197, atpB KM007745, atpA KM007631, trnL KM008087, rps4-trnS KM007838; Zhang Liang 1312 (CDBI): rbcL KM008174, trnL-F KM007974 rps4-trnS KM007862; Zhang Liang 1328 (CDBI): atpB KM007724, atpA KM007609, trnL KM008064, trnL-F rbcL KM008198, atpB KM007746, atpA KM007632, trnL KM007951, rps4-trnS KM007839. Pteris esquirolii Christ, Pan 033 KM008088, trnL-F KM007975, rps4-trnS KM007863. Pteris multifi- (GZTM): rbcL KM008175, atpB KM007725, atpA KM007610, trnL da Poir., Rothfels 3960 (DUKE): rbcL KM008199, atpB KM007747, KM008065, trnL-F KM007952, rps4-trnS KM007840. atpA KM007633, trnL KM008089, trnL-F KM007976, rps4-trnS Pteris finotii Christ, Zhang Liang 1323 (CDBI): rbcL KM008176, KM007864; E. Schuettpelz 710 (DUKE): rbcL KM008229, atpB atpB KM007726, atpA KM007611, trnL KM008066, trnL-F KM007776, atpA KM007663, trnL KM008117, trnL-F KM008005, KM007953, rps4-trnS KM007841. Pteris fraseri Mett. ex Kuhn, rps4-trnS KM007894; Zhang Liang 1167 (CDBI): rbcL KM008200, Rothfels 3712 (DUKE): rbcL KM008177, atpB KM007727, atpA atpB KM007748, atpA KM007634, trnL KM008090, trnL-F KM007612, trnL KM008067, trnL-F KM007954, rps4-trnS KM007977, rps4-trnS KM007865. Pteris muricata Hook., Rothfels KM007842. Pteris friesii Hieron., Festo & Kayombo 430 (MO): rbcL 3745 (DUKE): rbcL KM008201, atpB KM007749, atpA KM007635, KM008178, atpA KM007613, trnL KM008068, trnL-F KM007955, trnL KM008091, trnL-F KM007978, rps4-trnS KM007866. Pteris rps4-trnS KM007843. mutilata L., Sundue 2096 (MO): rbcL KM008202, atpB KM007750, Pteris gallinopes Ching, He 1378 (CTC): rbcL KM008179, atpA KM007636, trnL KM008092, trnL-F KM007979, rps4-trnS atpB KM007728, atpA KM007614, trnL KM008069, trnL-F KM007867. KM007956, rps4-trnS KM007844. Pteris grandifolia L., Rodas Pteris nanlingensis R. H. Miau, Zhang et al. 6003 (CDBI): rbcL 296 (MO): rbcL KM008180, atpB KM007729, atpA KM007615, KM008203, atpB KM007751, atpA KM007637, trnL KM008093, trnL KM008070, trnL-F KM007957, rps4-trnS KM007845. Pteris trnL-F KM007980, rps4-trnS KM007868. Pteris navarrensis Christ, grevilleana Wall. ex J. Agardh, Zhang Liang 1319 (CDBI): rbcL Rothfels 2640 (DUKE): rbcL KM008204, atpB KM007752, atpA KM008181, atpB KM007730, atpA KM007616, trnL KM008071, KM007638, trnL KM008094, trnL-F KM007981, rps4-trnS trnL-F KM007958, rps4-trnS KM007846. Pteris griffithii Hook., KM007869. Deng 3818 (CDBI): rbcL KM008182, atpB KM007731, atpA Pteris orizabae M. Martens & Galeotti, Reyes-Garcıa 7311 (MO): KM007617, trnL KM008072, trnL-F KM007959, rps4-trnS rbcL KM008205, atpB KM007753, atpA KM007639, trnL-F KM007847. KM007982, rps4-trnS KM007870. Pteris haenkeana C. Presl, H. van der Werff 17869 (MO): rbcL Pteris pacifica Hieron., ARF3536 (cult.): rbcL KM008206, atpB KM008183, atpB KM007732, atpA KM007618, trnL KM008073, KM007754, atpA KM007640, trnL KM008095, trnL-F KM007983, trnL-F KM007960, rps4-trnS KM007848. Pteris henryi Christ, Zhang rps4-trnS KM007871. Pteris pallens (Sw.) Mett., Janssen 2677 (P): et al. 6038 (CDBI): rbcL KM008184, atpB KM007733, atpA rbcL KM008207, atpB KM007755, atpA KM007641, trnL KM007619, trnL KM008074, trnL-F KM007961, rps4-trnS KM008096, trnL-F KM007984, rps4-trnS KM007872. Pteris pellu- KM007849. Pteris heteromorpha Fee, Wu ws-2622 (MO): rbcL cida C. Presl, Xia 172 (CDBI): rbcL KM008208, atpB KM007756, KM008185, atpB KM007734, atpA KM007620, trnL KM008075, atpA KM007642, trnL KM008097, trnL-F KM007985, rps4-trnS trnL-F KM007962, rps4-trnS KM007850. KM007873. Pteris podophylla Sw., Rothfels 3746 (DUKE): rbcL Pteris insignis Mett. ex Kuhn, Zhang Liang 1274 (CDBI): rbcL KM008209, atpB KM007757, atpA KM007643, trnL KM008098, KM008186, atpB KM007735, atpA KM007621, trnL KM008076, trnL-F KM007986, rps4-trnS KM007874. Pteris praestantissima trnL-F KM007963, rps4-trnS KM007851. (Bory ex Fee) Christenh., Rothfels 2680 (DUKE): rbcL 18 L. Zhang et al. / Cladistics (2014) 1–18

KM008210, atpB KM007758, atpA KM007644, trnL KM008099, rps4-trnS KM007891; Field s.n. (BRI): rbcL KM008227, atpA trnL-F KM007987, rps4-trnS KM007875; Christenhusz 3997 KM007661, trnL KM008115, trnL-F KM008003, rps4-trnS (TUR): rbcL EF452158 (Schuettpelz et al., 2007), atpB EF452042 KM007892. Pteris usambarensis Hieron., Kamau 388 (EA): rbcL (Schuettpelz et al., 2007), atpA EF452104 (Schuettpelz et al., KM008228, atpB KM007775, atpA KM007662, trnL KM008116, 2007). Pteris preussii Hieron., Kamau 212 (EA): rbcL KM008211, trnL-F KM008004, rps4-trnS KM007893. atpB KM007759, atpA KM007645, rps4-trnS KM007876. Pteris Pteris viridissima Ching, Zhang & He 5944 (CDBI): rbcL propinqua J.Agardh, E. Schuettpelz 268 (DUKE): rbcL KM008212, KM008230, atpB KM007777, atpA KM007664, trnL KM008118, atpB KM007760, atpA KM007646, trnL KM008100, trnL-F trnL-F KM008006, rps4-trnS KM007895. Pteris vittata L., KM007988, rps4-trnS KM007877. Pteris pseudopellucida Ching, ARF3534 (cult.): rbcL KM008231, atpB KM007778, atpA Wu ws-2580 (MO): rbcL KM008213, atpB KM007761, atpA KM007665, trnL KM008119, trnL-F KM008007, rps4-trnS KM007647, trnL KM008101, trnL-F KM007989, rps4-trnS KM007896; Rothfels 4016 (DUKE): rbcL KM008232, atpB KM007878. Pteris puberula Ching, Jin 11305 (CDBI): rbcL KM007779, atpA KM007666, trnL KM008120, trnL-F KM008008, KM008214, atpB KM007762, atpA KM007648, trnL KM008102, rps4-trnS KM007897; Zhang Liang 1466 (CDBI): rbcL KM008233, trnL-F KM007990, rps4-trnS KM007879. Pteris pungens Willd., atpB KM007780, atpA KM007667, trnL KM008121, trnL-F Rothfels 08-165 (DUKE): rbcL KM008215, atpB KM007763, atpA KM008009, rps4-trnS KM007898. KM007649, trnL KM008103, trnL-F KM007991, rps4-trnS Pteris wallichiana J. Agardh, Zhang Liang 1284 (CDBI): rbcL KM007880. KM008235, atpB KM007782, atpA KM007669, trnL KM008123, Pteris quadriaurita Retz., E. Schuettpelz 546 (GOET): rbcL trnL-F KM008011, rps4-trnS KM007900. Pteris wallichiana var. yun- EF452173 (Schuettpelz et al., 2007), atpB EF452058 (Schuettpelz nanensis (Christ) Ching & S.H. Wu, Jin 11183 (CDBI): rbcL et al., 2007), atpA EF452121 (Schuettpelz et al., 2007). Pteris quad- KM008234, atpB KM007781, atpA KM007668, trnL KM008122, ristipitis X.Y. Wang & P.S. Wang, Sun s.n. (CDBI): rbcL trnL-F KM008010, rps4-trnS KM007899. KM008216, atpB KM007764, atpA KM007650, trnL KM008104, Pteris xiaoyingiae H. He & Li Bing Zhang, Zhang & He 5326 trnL-F KM007992, rps4-trnS KM007881. (CDBI): rbcL KM008236, atpB KM007783, atpA KM007670, trnL Pteris saxatilis Carse, Welt P022567 (WELT): rbcL GU136798 KM008124, trnL-F KM008012, rps4-trnS KM007901. (Bouma et al., 2010), atpB GU136779 (Bouma et al., 2010). Pteris Pteris sp., Wen 10179 (US): rbcL JF935342 (Lu et al., 2012), semipinnata L., Zhang Liang 1463 (CDBI): rbcL KM008217, atpB atpB JF935424 (Lu et al., 2012), atpA JF937297 (Lu et al., 2012), KM007765, atpA KM007651, trnL KM008105, trnL-F KM007993, trnL-F JF980687 (Lu et al., 2012), rps4-trnS JF980608 (Lu et al., rps4-trnS KM007882. Pteris setulosocostulata Hayata, Zhang Liang 2012). 1379 (CDBI): rbcL KM008218, atpB KM007766, atpA KM007652, Pterozonium brevifrons (A.C.Sm.) Lellinger, E. Schuettpelz 285 trnL KM008106, trnL-F KM007994, rps4-trnS KM007883. Pteris (DUKE): rbcL EF452175 (Schuettpelz et al., 2007), atpB EF452061 speciosa Mett. ex Kuhn, Nitta 777 (UC): rbcL KM008219, atpB (Schuettpelz et al., 2007), atpA EF452124 (Schuettpelz et al., 2007); KM007767, atpA KM007653, trnL KM008107, trnL-F KM007995, Neill 15671 (MO): rbcL KM008237, atpA KM007578, trnL rps4-trnS KM007884. Pteris splendens Kaulf., Prado 1131a: rbcL KM008034, trnL-F KM007922, rps4-trnS KM007807. Pterozonium EF473708 (Prado et al., 2007). Pteris splendida Ching, Zhang et al. reniforme (Mart.) Fee, van der Werff 16216 (MO): rbcL KM008238, 5632 (CDBI): rbcL KM008220, atpB KM007768, atpA KM007654, atpB KM007695, atpA KM007579, trnL KM008035, trnL-F trnL KM008108, trnL-F KM007996, rps4-trnS KM007885. KM007923, rps4-trnS KM007808. Pteris terminalis Wall. ex J. Agardh, Zhang Liang 1341 (CDBI): Syngramma quinata (Hook.) Carruth., M. Kessler 2273 (L): rps4- rbcL KM008221, atpB KM007769, atpA KM007655, trnL trnS AF321701 (Sanchez-Baracaldo, 2004a). KM008109, trnL-F KM007997, rps4-trnS KM007886. Pteris tremula R. Br., ARF3535 (cult.): rbcL KM008222, atpB KM007770, atpA Taenitis blechnoides (Willd.) Sw., E. Schuettpelz 689 (DUKE): KM007656, trnL KM008110, trnL-F KM007998, rps4-trnS rbcL KM008239, atpB KM007784, atpA KM007671, trnL KM007887; E. Schuettpelz 620 (B): rbcL KM008223, atpB KM008125, trnL-F KM008013 rps4-trnS KM007809. Taenitis inter- KM007771, atpA KM007657, trnL KM008111, trnL-F KM007999, rupta Hook. & Grev., E. Schuettpelz 851 (DUKE): rbcL KM008240, rps4-trnS KM007888. Pteris tripartita Sw., ARF3538 (cult.): rbcL atpA KM007672, rps4-trnS KM007810. KM008224, atpB KM007772, atpA KM007658, trnL KM008112, Tryonia myriophylla (Sw.) Schuettp., Prado & Yano 1033 (SP): trnL-F KM008000, rps4-trnS KM007889; E. Schuettpelz 621(B): rbcL rbcL EF473710 (Prado et al., 2007); Prado 896: rps4-trnS AF321706 KM008225, atpB KM007773, atpA KM007659, trnL KM008113, (Sanchez-Baracaldo, 2004a); Prado 999: rps4-trnS AF321707 trnL-F KM008001, rps4-trnS KM007890. (Sanchez-Baracaldo, 2004a). Pteris umbrosa R. Br., ARF3537 (cult.): rbcL KM008226, atpB KM007774, atpA KM007660, trnL KM008114, trnL-F KM008002,