Cladistics Cladistics 34 (2018) 78–92 10.1111/cla.12192 Morphological characterization of infra-generic lineages in Deparia (Athyriaceae: Polypodiales) Li-Yaung Kuoa , Atsushi Ebiharab, Masahiro Katob, Germinal Rouhanc, Tom A. Rankerd, Chun-Neng Wanga,e,* and Wen-Liang Chiouf,g aInstitute of Ecology and Evolutionary Biology, National Taiwan University, Taipei 10617, Taiwan; bDepartment of Botany, National Museum of Nature and Science, Amakubo 4-1-1, Tsukuba, Ibaraki 305-0005, Japan; cMuseum National d’Histoire Naturelle, Institut de Systematique, Evolution, Biodiversite (UMR 7205 CNRS, MNHN, UPMC, EPHE), Herbier national, 16 rue Buffon CP39, Paris F-75005, France; dDepartment of Botany, University of Hawai'i at Manoa, Honolulu, HI 96822, USA; eDepartment of Life Science, National Taiwan University, Taipei 10617, Taiwan; fTaiwan Forestry Research Institute, Taipei 10066, Taiwan; gDr. Cecilia Koo Botanic Conservation Center, Pingtung County 906, Taiwan Accepted 6 January 2017 Abstract Deparia, including the previously recognized genera Lunathyrium, Dryoathyrium (=Parathyrium), Athyriopsis, Triblemma, and Dictyodroma, is a fern genus comprising about 70 species in Athyriaceae. In this study, we inferred a robust Deparia phylogeny based on a comprehensive taxon sampling (~81% of species) that captures the morphological diversity displayed in the genus. All Deparia species formed a highly supported monophyletic group. Within Deparia, seven major clades were identified, and most of them were characterized by inferring synapomorphies using 14 morphological characters including leaf architecture, peti- ole base, rhizome type, soral characters, spore perine, and leaf indument. These results provided the morphological basis for an infra-generic taxonomic revision of Deparia. © The Willi Hennig Society 2017. Introduction synapomorphies of this genus in Athyriaceae (Sundue and Rothfels, 2014). Deparia Hook. & Grev. is a fern genus comprising As currently circumscribed, Deparia includes several about 70 species (excluding hybrids) belonging to previously recognized genera, which vary morphologi- Athyriaceae (Polypodiales: Eupolypods II). This genus cally. Kato (1977, 1984) first outlined the generic con- is most diverse in Asia, but is also found in Africa, cept of Deparia, in which Parathyrium Holttum (nom. Madagascar and surrounding islands, Australia, north- illeg.), Lunathyrium Koidz., Dryoathyrium Ching, and east North America, the Hawaiian Islands, and south Athyriopsis Ching were combined. Based on soral Pacific islands (Kato, 1984, 1993a,b; Kuo et al., 2016). shape, characters of the petiole base, rhizome type, Deparia can be distinguished from its close relatives in and leaf indument, he further recognized four sections Athyriaceae (i.e. Athyrium s.l. and Diplazium Sw.) by a and two subsections within Deparia. Later, Triblemma disconnection of the grooves between rachises and (J.Sm.) Ching and Dictyodroma Ching were included costae, hair-like scales on the leaves, and a basic chro- as part of Deparia based on the similarities in indu- mosome number of x = 40 (Kato, 1977, 1984; Rothfels ment, basic chromosome number, rachis groove shape, et al., 2012b), with the first two characters being and molecular phylogenetic evidence (Fraser-Jenkins, 1997; Sano et al., 2000a,b). Recently, He et al. (2013) reassigned most of the Chinese endemic taxa of *Corresponding author. Lunathyrium, Dryoathyrium, and Athyriopsis to the E-mail address: [email protected] © The Willi Hennig Society 2017 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 79 genus Deparia. Due to the unification of so many mor- Materials and methods phologically distinct genera, currently circumscribed Deparia displays great morphological heterogeneity. Taxon sampling, DNA extraction, PCR amplification, For example, Athyriopsis has long-creeping rhizomes, and sequencing which are rarely found in other members now classi- fied in Deparia. Reniform sori exist only in Dryoathyr- We sampled 57 taxa in Deparia covering about 81% ium. Triblemma is unique in having simple fronds, and of the accepted species and subspecies (Appendix). Dictyodroma has anastomosing leaf venation while all Outgroup taxa included 11 species representing the other Deparia have free venation. other genera in Athyriaceae and two species from Deparia has been supported as a monophyletic Onocleaceae and Blechnaceae (Appendix; Rothfels group in all previous phylogenetic studies (Sano et al., 2012a). We obtained sequences of four pDNA et al., 2000a,b; Tzeng, 2002; Wang et al., 2003; Ebi- regions, including the rps16-matK intergenic spacer hara, 2011; Rothfels et al., 2012a; Kuo et al., 2016). (IGS) and trnL-L-F region (i.e. trnL intron + trnL-F Some characters, such as the indument and perine IGS), matK gene, and rbcL gene for phylogenetic anal- morphology (Fig. 1), could provide strong phyloge- yses. In addition to previously published sequences netic signals, as indicated by previous studies (Sano (Kato, 2001; Li et al., 2011; Rothfels et al., 2012a; et al., 2000b; Tzeng, 2002; Wang et al., 2006), but Kuo et al., 2016), 37 new sequences of Deparia were the association of phylogeny and morphology in generated in this study. The sequences of the outgroup Deparia remains to be examined. More extensive taxa were the same as in Kuo et al. (2016). The DNA investigation of character evolution within a phyloge- extraction procedure, PCR conditions and PCR primer netic context is needed to characterize infra-generic sets followed Li et al. (2011), Rothfels et al. (2012a), lineages morphologically. and Kuo et al. (2011, 2016). In this study we estimated a Deparia phylogeny based on a multiple plastid DNA (pDNA) region Phylogenetic analyses dataset and a comprehensive taxon sampling covering the infra-generic morphological diversity. Second, we To infer the appropriate nucleotide substitution examined morphological characters, including features model for phylogenetic analysis of each pDNA region, of leaf architecture, soral characters, petiole base, rhi- jModelTest (Posada, 2008) was used, and the appro- zome type, perine morphology, and indument on priate substitution models were selected based on the costae (Fig. 1). Finally, by analysing character evolu- Akaike information criterion (Akaike, 1974). Garli 2.0 tion, we inferred the evolutionary trends of these mor- (Zwickl, 2006) was used to infer the maximum-likeli- phological characters and characterized the lineages hood (ML) phylogeny. The four-region combined within Deparia. We hope these results provide the matrix was partitioned corresponding to different basis of future taxonomic revision of Deparia at the pDNA regions, and each region was assigned with its infra-generic level. own appropriate substitution model. The program Fig. 1. Morphology of Deparia. (a) The abaxial sides of fertile pinna of Deparia petersenii with diplazioid sori and hairy-costa. (b) The erect rhi- zome of Deparia subfluvialis with swollen stipe bases and pneumatophores. Scanning electron micrographs of Deparia spores: (c) the perine of Deparia pterorachis with folded ornamentation (i.e. folded series) and glandular surface, (d) the perine of Deparia lancea with tuberculate/lamel- late ornamentation (i.e. tuberculate series) and glandular surface, and (e) the perine of Deparia bonincola with echinate ornamentation (i.e. tuber- culate series) and smooth surface. Scale bars = 5 lm. 80 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 estimated the proportion of invariant sites and state were pretreated with gold coating with a sputter-coater frequencies. The “genthreshfortopoterm” option was for 1–3 min. Voucher information for all spore sam- set to 20 000. Ten independent searches were carried ples is provided in Table S1. Perine character data for out, and the tree with the highest likelihood was a few outgroup taxa were obtained from the literature selected. To calculate ML bootstrap support (BS) val- (Tryon and Lugardon, 1990; Liu et al., 2000; Wang ues, 500 replicates were run under the same criteria as and Dai, 2010) and using the scanning electron micro- described above except for the setting that only one graphs of spores generated by Robbin Moran and independent search was conducted in bootstrap tree Garrison Hanks through the website http://www.Pla searches. Bayesian phylogenetic inferences were per- ntSystematics.org (see details in Table S1). Other mor- formed in MrBayes v3.1.2, with support values esti- phological characters (i.e. rhizome types, petiole base mated as posterior probability (PP) (Huelsenbeck and characters, soral characters, leaf architecture, and Ronquis, 2001; Ronquist and Huelsenbeck, 2003). indument on costae) were recorded based on speci- Two simultaneous runs were carried out with four mens from herbaria BO, KYO, MO, P, PE, PYU, chains (106 generations each), with each chain sampled TAIF, TI, and TNS, and/or based on Kato (1984). every 1000 generations. The first 25% of the samples We scored soral types differently than Sundue and were conservatively discarded as burn-in, and the rest Rothfels (2014), which included both diplazioid and were used to calculate the 50% majority-rule consen- vein-crossing sori as a single character. In the current sus tree. Log likelihoods of Markov chain Monte study, because of greater variation of soral types in Carlo runs were inspected in Tracer v1.6 (Rambaut the Deparia species included, we separated diplazioid and Drummond, 2013) to determine convergence. In and vein-crossing sori as two characters. We recog- addition, we also