Generic Lineages in Deparia (Athyriaceae: Polypodiales)

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, petiole 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 rhizome 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/lamellate ornamentation (i.e. tuberculate series) and glandular surface, and (e) the perine of Deparia bonincola with echinate ornamentation (i.e. tuberculate 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 inferred ML bootstrap trees using nized two characters for perine morphology in Deparia each of four single-region matrices in order to under- and other athyrioid outgroups: perine ornamentation stand whether there were possible phylogenetic incon- and perine surface. For perine ornamentation, two dis- gruences between different pDNA regions. The tinct series could be distinguished: the folded and substitution model and setting of ML bootstrap tuberculate series (Fig. 1c–e). For the folded series, we searches were the same as those applied in the analysis defined those perines as being dominated by folds or of the four-region combined matrix. cristate projections extending into curved forks, which The maximum-parsimony (MP) phylogeny was sometimes form into reticulations. For the tuberculate inferred using PAUP* 4.0 (Swofford, 2003) under the series, we defined those perines as being dominated setting of random-taxon-addition, TBR swapping, with tube-like or lamellate projections, which are never gaps as missing data, and equal weighting. Heuristic forked at the base. In fact, the tuberculate series exhib- bootstrap analysis was performed with 500 bootstrap ited continuous variation among tuberculate, lamellate, replicates, ten random addition cycles per bootstrap and echinate perine projections, which even coexist on replicate, TBR swapping, and equal weighting. a single spore (Tryon and Lugardon, 1990; Liu et al., The alignment, and the phylogram and chronogram 2000; Wang et al., 2006; Wang and Dai, 2010). For of the most likely ML tree were submitted to Tree- perine surface, we recognized two character states: BASE. smooth and glandular (Fig. 1c–e). The details of taxon-states can be seen in Table S2. Morphological characters Character evolution analyses A total of 14 morphological characters were examined in this study: rachis-costa groove morphology, Mesquite v2.75 (Maddison and Madison, 2006) was leaf venation, the basal pinna morphology, rhizome used to infer ancestral states of all 14 characters in this type, the presence or absence of pneumatophores at study. The input phylogenies included the chronogram the stipe base, stipe base shape, the presence or and phylogram based on the most likely ML tree and absence of diplazioid sori, the presence or absence of the phylograms of 500 ML bootstrap trees, but with vein-crossing sori (i.e. either J-shaped, U-shaped, or Onocleaceae and Blechnaceae removed. The time cali- reniform), indusial margin, hairy or non-hairy abaxial bration method used to generate the chronogram used sides of costae, perine surface, perine ornamentation, penalized rate smoothing (implanted in r8s; Sanderson, minimum leaf dissection, and maximum leaf dissec- 2003) with two fixed constraints (“Athyriaceae” = tion. 83.32 Myr and genus “Deparia” = 27.68 Myr; follow- A tabletop scanning electron microscope (TM-3000; ing Kuo et al., 2016). With the exception of leaf Hitachi, Ibaraki, Japan) was used to examine the per- dissection, the ancestral character-state reconstruc- ine characters. The spores on herbarium specimens tions were performed under maximum-likelihood were transferred with a pipette tip to aluminium scan- models [“Markov k-state 1 parameter model (Mk1)” ning electron microscopy stubs. These spore samples or “AsymmMk (A2P)” (Lewis, 2001; Maddison and Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 81

Madison, 2006)], and their character states were binary topological conflicts (Fig. S1–S4). The genus Deparia coded (i.e. as either “0” or “1”). The leaf dissection formed a highly supported clade (MLBS = 100, character states were coded as a discrete number series PP = 1.00, MPBS = 100; Fig. 2). Within Deparia, from 0 to 4, which corresponded to the degree of leaf seven highly supported clades (MLBS ≥ 99, PP = 1.00, dissection, from low to high, respectively. Due to the MPBS = 100) were identified, which we labelled DR, unavailability of an ML model for ancestral state LU, ER, DI, CA, DE, and AT, moving up the tree, reconstruction that can use continuous characters or respectively (Fig. 2). The inferred chronogram is also discrete and ordered character states in Mesquite, the shown in Fig. 2, and the inferred divergence times ancestral state reconstructions of leaf dissection char- within Deparia are similar to those in Kuo et al. acters were based on the MP model, and we applied (2016). the ordered model and the topology of the most likely ML tree for analysis. The “Trace Character Over Character evolution analyses Trees” analysis based on all 500 MLBS phylograms was applied under the same models, ignoring the state Estimated ancestral state probabilities were different of “node absent”. To examine if there was any direc- under different models and input phylogenies tional bias in morphological transitions in Deparia,we (Table S3). Large differences of ancestral state proba- used an approach comparing the likelihood between a bilities between the Mk1 and A2P models were gener- one-rate model (i.e. Mk1 model: forward and reverse ally not found, although there were distinct differences change have a same rate) and a two-rate model (i.e. in ancestral state probabilities between the analyses A2P model: forward and reverse change have two dif- based on chronograms and on phylograms (indicated ferent rates; Maddison and Madison, 2006). If the like- as states i and ii in Figs 4–6; Table S3). In the lihood ratio test shows the two-rate model as being Deparia-only chronogram, except for perine surface, significantly more likely, then we would infer that the we found no significant evolutionary trend (i.e. either transition was biased in one direction (i.e. either 1 > 0 forward or backward change) for the characters based or 0 > 1 is biased). In this analysis we only focused on on the likelihood ratio test inferred from the Mk1 and Deparia taxa and thus removed the outgroups from A2P models (Table S4). the chronogram.

Discussion Results Phylogeny and systematics of Deparia Phylogenetic analyses Our results support the monophyly of Deparia, For the ML and Bayesian analyses, the sequence which is in agreement with previous phylogenetic stud- length, variability, and substitution model of each par- ies (Wang et al., 2003; Rothfels et al., 2012a; Wei titioned genetic region are summarized in Table 1. In et al., 2015; Kuo et al., 2016). As indicated by Kuo the Bayesian search, both runs became convergent et al. (2016), none of the infra-generic sections/subsec- after around 75 000 (initial 7.5%) generations, and the tions proposed by Kato (1984) were supported as post-burnin effective population sizes of all parameters being monophyletic. The DR, LU, CA, DE, and AT were larger than 300, except for the “TL” (effective clades mostly correspond to sect. Dryoathyrium, sect. population size = 181). The inferred four-region phylo- Lunathyrium, subsect. Caespites, sect. Deparia,and genies based on the different analyses (ML, Bayesian, subsect. Athyriopsis, respectively (Fig. 2). The DI clade and MP) resulted in similar topologies within Deparia, consists of all four species previously regarded as the and no strong conflicting relationships were found genus Dictyodroma. The ER clade contains only one among these phylogenies. Across single-region ML species, D. erecta, whose generic and infra-generic clas- phylogenies, we also found no highly supported sification has been controversial. This species was first

Table 1 Length of alignment, sequence variability, and substitution model of each pDNA region

rbcL trnL-L-F matK rps16-matK IGS

Length of alignment (bp) 1234 1002 1319 762 Variability (%) 16.53 42.81 42.84 47.24 Bayesian substitution model SYM+I+G GTR+G GTR+G GTR+G ML substitution model TIM2ef+I+G TVM+G GTR+G TIM1 + G 82 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92

0.01 per substituition per site 66/0.96/– Deparia petersenii subsp. petersenii var. petersenii Deparia biserialis Deparia confluens 57/0.94/–

Deparia pseudoconilii subsect. Deparia dimorphophylla Deparia petersenii subsp. petersenii var. yakusimensis 88/+/79 81/0.98/- Deparia lobato-crenata Athyriopsis 61/0.94/– Deparia japonica

88/+/51 Deparia tenuifolia subsp. +/+/+ Deparia petersenii deflexa Deparia longipes AT 99/+/95 Deparia timetensis AT +/+/+ Deparia kiusiana Deparia conilii

Deparia kaalaana sect. 99/0.99/97 +/+/+ Deparia marginalis Deparia +/+/+ Deparia prolifera 96/+/87 DE Deparia cataracticola Deparia fenzliana DE +/+/+ +/+/+ Deparia tomitaroana +/+/+ 65/0.99/85 Deparia lancea Deparia otomasui 74/0.97/61 Deparia bonincola

Deparia concinna Caespites subsect. CA 74/0.90/71 Deparia dickasonii CA 99/+/91 +/+/+ Deparia minamitanii Deparia omeiensis DI +/+/+ Deparia formosana DI +/+/+ Deparia hainanensis +/+/+ +/+/+ Deparia yunnanensis ER Deparia heterophlebia ER Deparia erecta +/+/98 83/+/68 Deparia macdonellii 56/0.87/- Deparia medogensis Deparia emeiensis

Deparia sichuanensis sect. Deparia wilsonii

51/0.59/- Lunathyrium +/+/98 Deparia auriculata +/+/+ Deparia jiulungensis LU +/+/+ Deparia acuta LU +/+/+ Deparia dolosa 93/+/99 Deparia pycnosora Deparia aff. jiulungensis Deparia +/+/+ Deparia acrostichoides Deparia +/+/+ 90/+/86 Deparia parvisora +/+/+ Deparia forsythii-majoris Deparia sp. sect. 63/0.59/- Deparia aff. glabrata +/+/+ Dryoathyrium Deparia boryana 77/0.99/- Deparia glabrata

+/+/+ +/+/+ Deparia subfluvialis Deparia edentula +/+/+ Deparia gordonii Athyriaceae +/+/+ Deparia unifurcata 99/+/+ Deparia coreana 68/0.99/71 99/+/+ Deparia henryi +/+/+ Deparia okuboana DR Deparia viridifrons DR Deparia pterorachis 86/+/66 Athyrium otophorum +/+/+ Athyrium atkinsonii Athyrium s. l. 98/+/91 Athyrium filix-femina Athyrium s. l. +/+/+ Cornopteris opaca +/+/+ Athyrium niponicum +/+/+ Athyrium skinneri +/+/+ Diplazium proliferum Diplazium 60/0.86/94 Diplazium dilatatum Diplazium +/+/+ Diplazium squamigerum +/+/+ Diplazium bombonasae Diplazium wichurae

Woodwardia japonica Q. P. Miocene Oligocene Eocene Paleocene Cretaceous Matteuccia struthiopteris 0 Ma 10 20 30 40 50 60 70 80 Fig. 2. The phylogram (left) and chronogram (right) of Deparia of the most likely ML tree based on rps16-matK IGS + trnL-L- F + matK + rbcL. ML bootstrap support (MLBS) values, posterior probabilities of Bayesian phylogenetic inference (PP), and MP bootstrap support (MPBS) values are indicated on each branch of the phylogram, as MLBS/PP/MPBS. The plus (+) sign represents MLBS = 100, PP = 1.00, or MPBS = 100; the minus (À) sign represents MLBS < 50 or MPBS < 50. The thickened branch indicates MLBS ≥ 70 and PP ≥ 0.95. The grey boxes indicate the section/subsection under Deparia recognized by Kato (1984). The seven clades under Deparia are indicated with black arrows. “Q.” and “P.” represent Quaternary and Pliocene, respectively. placed under sect. Caespites in Athyriopsis due to its being composed of sect. Deparia, one species of sub- erect rhizomes (Wang, 1982). Kato (1984) followed sect. Athyriopsis, and the genus Triblemma (i.e. this treatment, but moved it to sect. Athyriopsis sub- D. lancea and D. tomitaroana; Fig. 2.) sect. Caespites under Deparia. Later, it was moved to Dryoathyrium by Chu et al. (1999). The phylogenetic Different ancestral state inferences between the position of D. erecta was first revealed by Wang et al. chronogram and phylograms (2003), and, combined with evidence from morphology, Wang et al. (2006) further suggested that it is dis- There were differences of ancestral state probabilities tantly related to Dryoathyrium but closely related to in the chronogram versus the phylograms (e.g. the Athyriopsis. The taxon sampling of their phylogenetic ancestral state probabilities just on the node vs. i or ii study, however, lacked several Deparia lineages, espe- in Figs 4–6; all details about their differences can be cially from the DI and CA clades. That inadequate seen in Table S3). Such differences were conspicuous sampling led them to conclude that D. erecta is a in the ancestral state reconstruction of highly homo- member of Athyriopsis. Instead, agreeing with Kuo plastic characters (e.g. soral types and indusial margin, et al. (2016), our inferred phylogeny supports Fig. 5a,b). Although it is impossible to be certain D. erecta as belonging to a unique lineage sister to the about the causes of these differences, it may be par- DI clade (Fig. 2). Among the seven clades, the DE tially attributed to the phylogenetic uncertainty in clade comprises the greatest taxonomic complexity, both branch lengths and topologies. We found that Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 83 the ancestral state probabilities from the chronogram are either discontinuous (i.e. diplazioid sori) or continu- showed less deviation from those averaged probabili- ous (i.e. either J-shaped, U-shaped, or reniform; termed ties from the 500 MLBS phylograms than those from vein-crossing sori here). Indeed, most Deparia species the most likely ML phylogram (Figs 4–6). Theoreti- produce back-to-back sori except for a few taxa (see cally, the use of chronograms rather than phylograms below). Kato (1977) defined diplazioid sori as linear sori could better avoid effects of nucleotide substitution on the acroscopic sides of the basal/lower veins and/or rate heterogeneity among phylogenetic branches the midvein in the pinna lobes and linear sori on the because branch lengths of a chronogram reflect units in acroscopic sides of the same veins on the costa (=the a calibrated time scale while those in a phylogram reflect basiscopic side to the midvein). Based on this definition, substitution amounts of analysed genetic region(s). it seems that diplazioid sori are a form of asplenioid sori Different plant species and genetic regions may have dif- (i.e. linear and restricted to acroscopic sides of veins) ferent rates in their nucleotide substitutions (e.g. Gaut and not derived from vein-crossing sori. In our recon- et al., 2011; Lanfear et al., 2013; Li et al., 2016). On the struction, it is more likely that the ancestral state of other hand, it could be argued that a phylogram rather Deparia is the presence of diplazioid sori (Fig. 5a). than a chronogram could better present evolutionary Nonetheless, at least one Deparia species (e.g. rate, which might be positively correlated with character D. emeiensis) seemingly produces only asplenioid sori evolution rate (Litsios and Salamin, 2012). Notably, for and lacks both diplazioid and vein-crossing sori analyses employing either chronograms or phylograms, (Fig. 5a,b). In D. emeiensis, the reversal to produce only some methodological causes could exist and be even asplenioid sori is a consequence of first losing diplazioid more critical: for example, how to reconstruct these sori and then losing vein-crossing sori (Fig. 5a,b). phylogenies (e.g. time-calibrated approach and selection Among characters examined in this study, the discon- of analysed genetic regions) and whether the method tinuity of the rachis–costa grooves, the presence of used to reconstruct ancestral states allows flexible char- pneumatophores, the presence of swollen stipe bases, acter transition rates across phylogenetic branches. In and glandular perines were revealed as autapomorphies this study, we revealed a certain degree of deviation for Deparia (Figs 3a, 4b,c, and 6c); the first three were between ancestral state probabilities resulting from also identified by Sundue and Rothfels (2014). Within chronograms and phylograms. These differences imply Deparia, no characters were biased towards a particular that studies should consider the results of both phylo- directional change except for the character of perine grams and chronograms for ancestral state reconstruc- surface (Table S4), and the majority of characters were tion based on likelihood-based models, which has also homoplastic, showing no certain evolutionary trend. been recommended in recent studies (Litsios and Sala- Except for the rachis–costa groove, stipe base, and min, 2012; Cusimano and Renner, 2014). venation, the other characters switched multiple times in Deparia evolution (Figs 3–6). For leaf dissection Overview of character evolution of Deparia characters, convergences were also evident with an increase and decrease of pinnatifid order (Fig. 7). Only Athyriaceae is composed of three major lineages: the character of venation provides a unique synapomor- Athyrium s.l., Diplazium, and Deparia (also called phy at the clade level. Thus, anastomosing venation athyriid, diplaziid/diplazioid, and depariid, respectively; evolved only in the ancestor of the DI clade. Within Sano et al., 2000a; Tzeng, 2002; Wang et al., 2003; Deparia, the DR and LU clades seem to best represent Rothfels et al., 2012a,b). Among the three lineages, the ancestral characteristics of Deparia (Figs 3–7). On Deparia is sister to the two others, and it apparently the other hand, the DI, ER, CA, DE, and AT clades diverged from the ancestral Athyrium + Diplazium lin- were accompanied by the loss of pneumatophores, a eage relatively shortly after Athyriaceae diverged from transition from having a swollen stipe base to a slender the sister Eupolypods II lineage (ca. 83–89 Ma; Kuo one as well as a change in perine ornamentation from et al., 2016) while it has a relatively young divergence folded to tuberculate series (Figs 4b,c and 6b). (i.e. the crown age ≤ 32 Ma; Fig. 2; also see in Schuett- Interesting evolutionary relationships among certain pelz and Pryer, 2009; Wei et al., 2015; Kuo et al., 2016; morphological characters were also revealed. We Testo and Sundue, 2016). found that the diplazioid sori and vein-crossing sori Morphologically, Deparia is characterized as having evolved independently in Deparia while never evolving more autapomorphic than plesiomorphic characters in other athyrioids (Fig. 5a,b). Unlike Deparia, other (Sundue and Rothfels, 2014). The base chromosome athyrioids have never evolved to have both types of number of 40 is plesiomorphic for Deparia since it is sori (Rothfels et al., 2012b). The diplazioid sori are synapomorphic for Athyriaceae + Blechnaceae + Ono- present and vein-crossing sori are completely absent in cleaceae in Eupolypods II (Sundue and Rothfels, 2014). Diplazium, while this pattern is reversed in Athyrium The best-known synapomorphy of Athyriaceae is the s.l. Moreover, a seemingly correlated evolutionary pat- back-to-back sori (Sundue and Rothfels, 2014), which tern between the presence of pneumatophores and 84 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92

b Diplazium bombonasae a c Diplazium wichurae Diplazium squamigerum Diplazium proliferum Diplazium dilatatum Athyrium niponicum Athyrium skinneri Cornopteris opaca Athyrium filix-femina Athyrium otophorum Athyrium atkinsonii Deparia pterorachis DR clade Deparia viridifrons Deparia okuboana Deparia coreana Deparia henryi Deparia gordonii Deparia unifurcata Deparia subfluvialis Deparia edentula Deparia glabrata Athyriaceae Athyriaceae Athyriaceae Deparia boryana Deparia aff. glabrata Deparia sp. Deparia parvisora Deparia forsythii-majoris Deparia acrostichoides LU clade Deparia pycnosora Deparia aff. jiulungensis Deparia Deparia Deparia Deparia dolosa Deparia jiulungensis Deparia acuta Deparia emeiensis Deparia macdonellii Deparia medogensis Deparia auriculata Deparia sichuanensis Deparia wilsonii Deparia erecta ER clade Deparia formosana DI clade Deparia hainanensis Deparia yunnanensis Deparia heterophlebia Deparia omeiensis CA clade Deparia minamitanii Deparia concinna Deparia dickasonii Deparia bonincola DE clade Deparia otomasui Deparia tomitaroana Deparia lancea Deparia fenzliana Deparia cataracticola Deparia prolifera Deparia kaalaana Deparia marginalis Deparia conilii AT clade Deparia kiusiana Deparia timetensis Deparia longipes Deparia tenuifolia Deparia petersenii subsp. deflexa Deparia japonica Deparia lobato-crenata Deparia petersenii subsp. petersenii var. yakus im ens is Rachis-costa groove Venation Basal pinnae Deparia dimorphophylla Connected Anastomosing Auricled Deparia pseudoconilii Disconneced Deparia confluens Free Non-auricled Deparia petersenii subsp. petersenii var. petersenii Data not availible Data not available Deparia biserialis Fig. 3. The inferred ancestral states for characters (a) rachis–costae groove, (b) venation, and (c) basal pinnae, based on the A2P model. Black and white in pie charts on each branch indicate the probabilities of ancestral states inferred by the analysis based on the chronogram of the most likely ML tree, and black and white of branches indicate the ancestral state of highest probability. swollen stipe bases was revealed (Fig. 4a,b), which was however, are very rare in frequency and accompany also found by Sundue and Rothfels (2014). Future the more frequent vein-crossing sori (Kato, 1984). In examinations of such putative evolutionary correla- addition, there are some plesiomorphic characters tions between characters should be based on a more shared by the LU clade (see below). comprehensive sampling across Athyriaceae. Characterization of different lineages in the Deparia LU Characterization of different lineages in the Deparia DR clade clade The synapomorphy for the LU clade is auricled The creeping rhizome was inferred as a synapomor- basal pinnae, but this trait is also the synapomorphy phy of the DR clade (Fig. 4a), with one inferred rever- of the AT clade (Fig. 3c). Only one species in the LU sal to erect. The presence of diplazioid sori seems to clade reversed to non-auricled basal pinnae (Fig. 3c). have evolved independently twice within this clade Both LU and DR clades share some characters that (Fig. 5a). Diplazioid sori, in species where they exist, are absent in the other clades, including the presence Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 85

ii i Deparia lancea Deparia fenzliana Deparia cataracticola Deparia prolifera Deparia kaalaana Deparia marginalis Deparia conilii AT clade Deparia kiusiana Deparia timetensis Deparia longipes Deparia tenuifolia Deparia petersenii subsp. deflexa Deparia japonica Deparia lobato-crenata Deparia petersenii subsp. petersenii var. yakusimensis Rhizome Pneumatophore Stipe base Deparia dimorphophylla Erect Present Swollen Deparia pseudoconilii Creeping Absent Slender Deparia confluens Deparia petersenii subsp. petersenii var. petersenii Data not availible Data not availible Data not availible Deparia biserialis Fig. 4. The inferred ancestral states for characters (a) rhizome, (b) pneumatophore, and (c) stipe base, based on the A2P model. Black and white in pie charts on each branch indicate the probabilities of ancestral states inferred by the analysis based on the chronogram of the most likely ML tree, and black and white of branches indicate the ancestral state of highest probability. For a node with the ancestral state probabilities showing a difference larger than 0.25 compared to the analysis either based on the most likely phylogram or based on 500 MLBS phylograms, the pie charts present these distinct probabilities on the branches: “i” and “ii” indicate the probabilities of the results of the most likely phylogram and 500 MLBS phylograms, respectively. of pneumatophores and the swollen stipe bases clades are quite different in morphology. In contrast (Fig. 4a,b). Folded perines exist only in the LU and to the DI clade, the ER clade exhibits free venation, DR clades, with the exception of DI clade (Fig. 6b). an absence of diplazioid sori, the presence of vein- crossing sori, non-hairy abaxial costae, smooth and Characterization of different lineages in the Deparia ER tuberculate perines, and bipinnatiﬁd leaves (Figs 3b, clade 5a,b, 6, and 7).

Given that there is only one taxon, Deparia erecta, Characterization of different lineages in the Deparia DI by definition we could not find synapomorphies for clade the ER clade. Although the ER and DI clades were sister to each other, we could find no morphological Anastomosing venation and pinnatifid–pinnate synapomorphy for the combined clade, and these two leaves are synapomorphies of this clade (Figs 3b and 86 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92

ii i Deparia okuboana Deparia coreana ii i Deparia henryi i Deparia gordonii Deparia unifurcata

i Deparia subfluvialis Deparia edentula Deparia glabrata Athyriaceae Athyriaceae Athyriaceae Deparia boryana Deparia aff. glabrata Deparia sp. Deparia parvisora Deparia forsythii-majoris Deparia acrostichoides LU clade i Deparia pycnosora Deparia aff. jiulungensis Deparia Deparia Deparia Deparia dolosa Deparia jiulungensis ii i Deparia acuta Deparia emeiensis i ii Deparia macdonellii Deparia medogensis ii i ii i Deparia auriculata i Deparia sichuanensis Deparia wilsonii Deparia erecta ER clade Deparia formosana DI clade Deparia hainanensis i Deparia yunnanensis Deparia heterophlebia Deparia omeiensis CA clade i Deparia minamitanii Deparia concinna ii i i Deparia dickasonii Deparia bonincola i DE clade ii Deparia otomasui Deparia tomitaroana

ii i Deparia lancea i Deparia fenzliana Deparia cataracticola ii i i Deparia prolifera i ii i Deparia kaalaana Deparia marginalis Deparia conilii AT clade Deparia kiusiana Deparia timetensis i Deparia longipes Deparia tenuifolia Deparia petersenii subsp. deflexa Deparia japonica Deparia lobato-crenata Deparia petersenii subsp. petersenii var. yakusimensis Diplazioid sori Vein-crossing sori Indusial margin Deparia dimorphophylla Present Present Entire Deparia pseudoconilii Absent Absent Toothed Deparia confluens Deparia petersenii subsp. petersenii var. petersenii Data not availible Data not availible Data not availible Deparia biserialis Fig. 5. The inferred ancestral states for characters (a) diplazioid sori, (b) vein-crossing sori, and (c) indusial margin, based on the A2P model. Black and white in pie charts on each branch indicate the probabilities of the ancestral states inferred by the analysis based on the chronogram of the most likely ML tree, and black and white of branches indicate the ancestral state of highest probability. For a node with the ancestral state probabilities showing a difference larger than 0.25 compared to the analysis either based on the most likely phylogram or based on 500 MLBS phylograms, the pie charts present these distinct probabilities on the branches: “i” and “ii” indicate the probabilities of the results of the most likely phylogram and 500 MLBS phylograms, respectively.

7), and the former trait is absent in other Deparia Characterization of different lineages in the Deparia CA members. The hairy abaxial sides of costae are also clade a synapomorphy of this clade, but this character seems homoplastic in Deparia. This character was Hairy abaxial sides of costae are inferred as also revealed as a synapomorphy for the CA clade synapomorphies of the CA clade (Fig. 6a). Morpho- and for the AT clade. The DI clade has no pneu- logically, the CA, AT, and DE clades are difﬁcult to matophores and has slender stipe bases (Fig. 4a,b). distinguish from each other. In addition, only poly- This clade corresponds to the genus Dictyodroma, ploids are known in this clade, which Kuo et al. and the current study includes all four species recog- (2016) inferred to be due to a palaeopolyploidization nized in this genus (He et al., 2013). event. Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 87

Diplazium bombonasae a b c Diplazium wichurae Diplazium squamigerum Diplazium proliferum Diplazium dilatatum Athyrium niponicum Athyrium skinneri Cornopteris opaca Athyrium filix-femina Athyrium otophorum Athyrium atkinsonii Deparia pterorachis DR clade Deparia viridifrons Deparia okuboana Deparia coreana Deparia henryi Deparia gordonii Deparia unifurcata Deparia subfluvialis Deparia edentula Deparia glabrata Athyriaceae Athyriaceae Athyriaceae Deparia boryana Deparia aff. glabrata Deparia sp. Deparia parvisora Deparia forsythii-majoris Deparia acrostichoides LU clade Deparia pycnosora Deparia aff. jiulungensis Deparia Deparia Deparia Deparia dolosa Deparia jiulungensis Deparia acuta ii i ii i Deparia emeiensis Deparia macdonellii Deparia medogensis Deparia auriculata Deparia sichuanensis Deparia wilsonii Deparia erecta ER clade Deparia formosana DI clade Deparia hainanensis Deparia yunnanensis Deparia heterophlebia Deparia omeiensis CA clade Deparia minamitanii Deparia concinna Deparia dickasonii Deparia bonincola DE clade Deparia otomasui Deparia tomitaroana Deparia lancea i Deparia fenzliana Deparia cataracticola ii i ii i Deparia prolifera ii i Deparia kaalaana ii i Deparia marginalis Deparia conilii AT clade Deparia kiusiana Deparia timetensis Deparia longipes Deparia tenuifolia Deparia petersenii subsp. deflexa Deparia japonica Deparia lobato-crenata Deparia petersenii subsp. petersenii var. yakusimensis Abaxial sides of costa Perine ornamentation Perine surface Deparia dimorphophylla Hairy Tubercule series Glandular Deparia pseudoconilii Non-hairy Deparia confluens Fold series Smooth Deparia petersenii subsp. petersenii var. petersenii Data not availible Data not availible Data not availible Deparia biserialis Fig. 6. The reconstructed ancestral states for characters (a) abaxial sides of costae, (b) perine ornamentation, and (c) perine surface, based on the A2P model. Black and white in pie charts on each branch indicate the probabilities of the ancestral states inferred by the analysis based on the chronogram of the most likely ML tree, and black and white of branches indicates the ancestral state of highest probability. For a node with one of the ancestral state probabilities showing a difference larger than 0.25 compared to the analysis either based on the most likely phylogram or based on 500 MLBS phylograms, the pie charts present these distinct probabilities on the branches: “i” and “ii” indicate the probabilities of the results of the most likely phylogram and 500 MLBS phylograms, respectively.

Characterization of different lineages in the Deparia DE sporangia (Kato, 2001; Kuo et al., 2016; L.-Y. Kuo, clade personal observation in other non-type collections in herbarium US). Whether it is a hybrid between a spe- We inferred no synapomorphies for the DE clade. cies of the DE clade and a species of another clade This clade displays high morphological heterogeneity, needs further investigation. which spans nearly all of the character variation among the CA, DE, and AT clades. With the excep- Characterization of different lineages in the Deparia AT tion of D. cataracticola, the species in this clade have clade non-hairy abaxial sides of costae whereas the abaxial sides of costae are hairy in the CA and AT clades Auricled basal pinnae, creeping rhizomes, and (Fig. 6a). Deparia cataracticola is possibly a hybrid toothed indusial margins were revealed as synapomor- species because it produces only abortive spores and phies of the AT clade (Figs 3a, 4a, and 5c). These 88 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92

a b Diplazium bombonasae - – Diplazium wichurae Diplazium squamigerum Diplazium proliferum Diplazium dilatatum + + Athyrium niponicum - + Athyrium skinneri Cornopteris opaca + Athyrium filix-femina Athyrium otophorum Athyrium atkinsonii + + Deparia pterorachis + + DR clade Deparia viridifrons Deparia okuboana Deparia coreana Deparia henryi Deparia gordonii Deparia unifurcata Deparia subfluvialis Deparia edentula + + Deparia glabrata Athyriaceae Athyriaceae Deparia boryana – Deparia aff. glabrata Deparia sp. - Deparia parvisora – – Deparia forsythii-majoris Deparia acrostichoides LU clade Deparia pycnosora Deparia aff. jiulungensis Deparia Deparia Deparia dolosa Deparia jiulungensis Deparia acuta Deparia emeiensis Deparia macdonellii Deparia medogensis + Deparia auriculata Deparia sichuanensis Deparia wilsonii Deparia erecta ER clade Deparia formosana DI clade Deparia hainanensis – – Deparia yunnanensis Deparia heterophlebia Deparia omeiensis CA clade Deparia minamitanii Deparia concinna Deparia dickasonii Deparia bonincola DE clade Deparia otomasui - - - Deparia tomitaroana - Deparia lancea Deparia fenzliana Deparia cataracticola + Deparia prolifera Deparia kaalaana - – Deparia marginalis Deparia conilii AT clade - Deparia kiusiana Deparia timetensis Deparia longipes + + Deparia tenuifolia Mininum leaf Maximum leaf dissection Deparia petersenii subsp. deflexa dissection – Deparia japonica Simple – Deparia lobato-crenata Simple – Deparia petersenii subsp.peterseniivar. yakusimensis Pinnatifid-pinnate Pinnatifid-pinnate Deparia dimorphophylla Bipinnatifid-bipinnate + Bipinnatifid-bipinnate Deparia pseudoconilii – Tripinnatifid-tripinnate Deparia confluens Tripinnatifid-tripinnate Deparia petersenii subsp. petersenii var. petersenii Quadripinnatifid + Deparia biserialis Fig. 7. The reconstructed ancestral states for characters (a) minimum and (b) maximum leaf dissection, based on the ordered MP model. The colours in pie charts on each branch indicate the probabilities of the ancestral states inferred by the analysis based on 500 MLBS phylograms. The colours of branches indicate the ancestral state of highest probability. An increase or decrease in leaf dissection order is indicated as “+” and “–”, respectively. characters were also revealed to be homoplastic among in the AT clade (Fig. 3a). The character state of hairy all Deparia, and switched states in other clades abaxial sides of costae was also indicated as a synapo- (Figs 4a and 5c). Auricled basal pinnae reversed once morphy but with weak support (i.e. the ancestral state Table 2 Summary of the divergence time and ancestral status of Deparia lineages

Lineage iYugKoe l ldsis3 21)78–92 (2018) 34 Cladistics / al. et Kuo Li-Yaung † Deparia DR LU ER DI CA DE AT

Stem age (Ma) 84.34 28.70 23.77 16.48 16.48 14.4 13.38 13.38 ‡ (95% HPD) (83.32–89.63) (27.68–33.99) (19.59–29.56) (12.4–23.23) (12.4–23.23) (10.76–20.96) (10.07–19.75) (10.07–19.75) Crown age (Ma) 28.70 20.02 13.31 – 7.29 1.23 10.58 5.15 ‡ (95% HPD) (27.68–33.99) (15.34–26.38) (8.82–19.3) (27.68–33.99) (27.68–33.99) (7.02–17.35) (2.33–10.99) Rachis–costa Disconnected*** Disconnected*** Disconnected*** Disconnected*** Disconnected*** Disconnected*** Disconnected*** Disconnected*** groove Venation Free*** Free*** Free*** Free*** Anastomosing*** Free*** Free*** Free*** Basal pinnae Non-auricled*** Non-auricled*** Auricled*** Non-auricled*** Non-auricled*** Non-auricled*** Non-auricled*** Auricled*** Rhizome Ambiguous Creeping* Erect** Erect*** Erect*** Erect*** Erect* Creeping*** Pneumatophore Present* Present** Present*** Absent*** Absent*** Absent*** Absent*** Absent*** Stipe base Swollen* Swollen*** Swollen*** Slender*** Slender*** Slender*** Slender*** Slender*** Diplazioid sori Present* Ambiguous Present* Absent*** Present** Present*** Present** Present** Vein-crossing sori Present* Present* Present* Present*** Ambiguous Present*** Ambiguous Absent* Perine Fold series** Fold series*** Fold series*** Tubercule series*** Fold series*** Tubercule Tubercule Tubercule ornamentation series*** series*** series*** Perine surface Glandular*** Glandular*** Glandular*** Smooth*** Glandular*** Glandular*** Glandular* Glandular***

HPD, highest posterior density. *P > 0.50; **P > 0.75; ***P > 0.90. † Based on character status of Deparia erecta. ‡ Inferred by Kuo et al. (2016). 89 90 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 of the next derived node received only low support; TAIF, TI, TNS, UC, and US for making their collec- Fig. 6a). tions available for study. Finally, we express thanks to the Interchange Association (Japan), Taiwan Society Inter-clade hybrids of Plant Systematics, and Mr. Shann-Jye Moore Memorial Scholarship for providing financial support Several putative hybrid species derived from crosses for herbarium collection surveys. This work was partly between different lineages have been described, includ- supported by the Ministry of Science and Technology, ing Deparia 9 kiyozumiana, D. 9 togakushiensis, Taiwan (Grant Number 103-2313-B-002-004-MY3). D. 9 lobato-crenata, D. 9 tomitaroana, and D. 9 nakaikeana. Deparia 9 kiyozumiana is presumably a References hybrid cross between D. coreana and D. jiulungensis (syn. D. orientalis) from the DR and LU Akaike, H., 1974. A new look at the statistical model identification. clades, respectively (Kato, 1984; Nakaike, 2013). IEEE Trans. Automat. Contr. 19, 716–723. Deparia 9 togakushiensis is presumably a hybrid cross Chu, W.-M., Wang, Z.-R., Zhang, X.-C., He, Z.-R., Hsieh, Y.-T., 1999. between D. jiulungensis (syn. D. orientalis = D. pyc- Athyriaceae. In: Chu, W.-M. (Ed.), Flora of Reipublicae Popularis Sinicae, Tomus 3 (2). Science Press, Beijing, pp. 32–511. nosora var. albosquamata) and D. conilii from the LU Cusimano, N., Renner, S.S., 2014. Ultrametric trees or phylograms and AT clades, respectively (Otsuka and Fujiwara, for ancestral state reconstruction: does it matter? Taxon 63, 721– 1999). The latter three species were regarded as hybrid 726. Ebihara, A., 2011. RbcL phylogeny of Japanese pteridophyte flora descendants of D. lancea in the DE clade and species and implications on infrafamilial systematics. Bull. Natl. Mus. in the AT clade (Matsumoto and Nakaike, 1990; Nat. Sci. Ser. B Bot. 37, 63–74. Nakaike, 1992, 2013; Fraser-Jenkins, 2008). Their Fraser-Jenkins, C.R., 1997. New Species Syndrome in Indian hybrid origins still need to be confirmed, however, Pteridophyte and the Ferns of Nepal. International Book Distributors, Dehra Dun. using nuclear markers. Our pDNA phylogeny reveals Fraser-Jenkins, C.R., 2008. Taxonomic Revision of Three Hundred only the maternal parentages of D. 9 lobato-crenata Indian Subcontinental Pteridophytes With a Revised Census-List: and D. 9 tomitaroana (Fig. 2). Inclusion of these A new Picture of Fern-Taxonomy and Nomenclature in the putative inter-clade hybrids did not affect our infer- Indian Subcontinent. Bishen Singh Mahendra Pal Singh, Dehra Dun. ences of the ancestral character states of each clade Gaut, B., Yang, L., Takuno, S., Eguiarte, L.E., 2011. The patterns (data not shown). and causes of variation in plant nucleotide substitution rates. Annu. Rev. Ecol. Evol. Syst. 42, 245–266. He, Z.-R., Wang, Z.-R., Kato, M., 2013. Deparia (Athyriaceae). In: Future perspectives Wu, Z.-Y., Raven, P.H., Hong, D.-Y. (Eds.), Flora of China Vol. 2-3. Science Press, Beijing, and Missouri Botanical Garden In this study, based on a well-resolved Deparia phy- Press, St. Louis, MO, pp. 418–442. logeny, we further identified and morphologically char- Huelsenbeck,J.P.,Ronquis,F.,2001.MRBAYES:Bayesian inference of phylogenetic trees. Bioinformatics 3, 754–755. acterized seven clades within Deparia. These results Karafit, S., Rothwell, G., Stockey, R.A., Nishida, H., 2006. will provide the basis for future systematic and taxo- Evidence for sympodial vascular architecture in a filicalean fern nomic revisions of Deparia. Considering the results of rhizome: Dickwhitea allenbyensis gen. et sp. nov. (Athyriaceae). – the time-calibrated phylogeny and inferred ancestral Int. J. Plant Sci. 167, 721 727. Kato, M., 1977. Classification of Athyrium and allied genera of states together, we also summarized a reference Japan. Bot. Mag. 90, 23–40. (Table 2) for future study of the systematics of Kato, M., 1984. A taxonomic study of the athyrioid fern genus Deparia-like or athyrioid-like fossil taxa (e.g. Mako- Deparia with main reference to the Pacific species. J. Fac. Sci. Sect. 13, 371–430. topteris and Dickwhitea; Stockey et al., 1999; Karafit Kato, M., 1993a. Biogeography of ferns: dispersal and vicariance. J. et al., 2006). In addition, we provided estimates of Biogeogr. 20, 265–274. character evolution across Deparia species (Table S4). Kato, M., 1993b. Deparia and Athyrium. In: Flora of North America Editorial Committee, Morin, N.R. (Eds.), Flora of North America, Vol. 2. Oxford University Press, New York, pp. 254–258. Acknowledgements Kato, M., 2001. Deparia cataracticola (Woodsiaceae), a new species from Hawaii. Acta Phytotaxon. Geobot. 52, 1–9. Kuo, L.-Y., Li, F.-W., Chiou, W.-L., Wang, C.-N., 2011. First We thank Hank Oppenheimer for providing the insights into fern matK phylogeny. Mol. Phylogenet. Evol. 59, DNA material of Deparia kaalaana, Xin-Ping Qi for 556–566. providing the DNA material of Deparia henryi, and Kuo, L.-Y., Ebihara, A., Shinohara, W., Rouhan, G., Wood, K.R., Dedy Darnaedi for providing the DNA material of Wang, C.-N., Chiou, W.-L., 2016. Historical biogeography of the fern genus Deparia (Athyriaceae) and its relation with Deparia edentula. We thank Travis Schoneman for polyploidy. Mol. Phylogenet. Evol. 104, 123–134. manuscript editing, and Michael Sundue, Fay-Wei Li, Lanfear R, Ho S.Y.W., Jonathan Davies T, Moles A.T., Aarssen L., and two anonymous reviewers for providing comments Swenson N.G., Warman L., Zanne A.E., Allen A.P. (2013) Taller and suggestions on the draft. We also greatly appreci- plants have lower rates of molecular evolution. Nat. Commun. 4, 1879. ate the herbaria BO, KYO, MICH, MO, P, PE, PYU, Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92 91

Lewis, P.O., 2001. A likelihood approach to estimating phylogeny Tzeng, Y.-H., 2002. Phylogenetic relationships of athyrioid ferns from discrete morphological character data. Syst. Biol. 50, 913–925. inferred from chloroplast DNA sequences. Masters thesis, Li, F.-W., Kuo, L.-Y., Rothfels, C.J., Ebihara, A., Chiou, W.-L., National Sun Yat-Sen University. Windham, M.D., Pryer, K.M., 2011. RbcL and matK earn two Wang, Z.-R., 1982. Four new species of Athyriaceae from Emei thumbs up as the core DNA barcode for ferns. PLoS ONE 6, e26597. Shan, Sichuan. Acta Phytotaxon. Sin. 20, 236–240. Li, F.-W., Kuo, L.-Y., Pryer, K.M., Rothfels, C.J., 2016. Genes Wang, Q.-X., Dai, X.-L., 2010. Athyriaceae. In: Jhu, L., Chen, L. translocated into the plastid inverted repeat show decelerated (Eds.), Spores of Polypodiales (Filicales) From China. Science substitution rates and elevated GC content. Genome Biol. Evol. Press, Beijing, pp. 49–66, 178–193. 8, 2452–2458. Wang, M.-L., Chen, Z.-D., Zhang, X.-C., Lu, S.-G., Zhao, G.-F., Litsios, G., Salamin, N., 2012. Effects of phylogenetic signal on 2003. Phylogeny of the Athyriaceae: evidence from chloroplast ancestral state reconstruction. Syst. Biol. 61, 533–538. trnL-F region sequences. Acta Phytotaxon. Sin. 41, 416–426. Liu, Y.-C., Kuo, C.-M., Liu, H.-Y., 2000. SEM studies on spore in Wang, M.-L., Xu, H., Zheng, L., 2006. Dryoathyrium erectum is a Taiwanese fern genera I. Athyrioids. Taiwania 45, 181–200. member of the genus Athyriopsis. Acta Phytotaxon. Sin. 44, 204–210. Maddison, W.P., Madison, D.R., 2006. Mesquite: a modular system Wei, R., Xiang, Q., Schneider, H., Sundue, M.A., Kessler, M., Kamau, for evolutionary analysis. mesquiteproject.org. P.W., Hidayat, A., Zhang, X., 2015. Eurasian origin, boreotropical Matsumoto, S., Nakaike, T., 1990. Cytological observations of some migration and transoceanic dispersal in the pantropical fern genus ferns in Nepal (1). On the related taxa in Japan. Cryptogam. Diplazium (Athyriaceae). J. Biogeogr. 42, 1809–1819. Himal. 2, 163–178. Zwickl, D.J., 2006. Genetic algorithm approaches for the Nakaike, T., 1992. New Flora of Japan Pteridophyta. Shibundo, phylogenetic analysis of large biological sequence datasets under Tokyo. the maximum likelihood criterion. PhD thesis, The University of Nakaike, T., 2013. A Checklist of Lycophytes and Ferns of Japan. Texas, Austin. Youshi Press, Shizuoka. Otsuka, K., Fujiwara, R., 1999. A new hybrid of Deparia (Woodsiaceae) from Nagano Prefecture, central Japan. J. Supporting Information Phytogeogr. Taxon. 47, 107–110. Posada, D., 2008. jModelTest: phylogenetic model averaging. Mol. Biol. Evol. 25, 1253–1256. Additional Supporting Information may be found in Rambaut, A., Drummond, A.J., 2013. Tracer v1.6. Available from the online version of this article: http://tree.bio.ed.ac.uk/software/tracer/. Figure S1. The majority-rule consensus cladogram Ronquist, F., Huelsenbeck, J.P., 2003. MrBayes 3: Bayesian of rbcL ML phylogeny based on 500 bootstrap repli- phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574. cates. Rothfels, C.J., Larsson, A., Kuo, L.-Y., Korall, P., Chiou, W.-L., Figure S2. The majority-rule consensus cladogram Pryer, K.M., 2012a. Overcoming deep roots, fast rates, and short of matK ML phylogeny based on 500 bootstrap repli- internodes to resolve the ancient rapid radiation of eupolypod II ferns. Syst. Biol. 61, 490–509. cates. Rothfels, C.J., Sundue, M.A., Kuo, L.-Y., Larsson, A., Kato, M., Figure S3. The majority-rule consensus cladogram Schuettpelz, E., Pryer, K.M., 2012b. A revised family-level of trnL-L-F ML phylogeny based on 500 bootstrap classiﬁcation for eupolypod II ferns (Polypodiidae: Polypodiales). replicates. Taxon 61, 515–533. Sanderson, M.J., 2003. r8s: inferring absolute rates of molecular Figure S4. The majority-rule consensus cladogram evolution and divergence times in the absence of a molecular of rps16-matK IGS ML phylogeny based on 500 boot- clock. Bioinformatics 19, 301–302. strap replicates. Sano, R., Takamiya, M., Ito, M., Kurita, S., Hasebe, M., 2000a. Phylogeny of the lady fern group, tribe Physematieae Figure S5. The cladogram indicating node number (Dryopteridaceae), based on chloroplast rbcL gene sequences. as in Table S3. Mol. Phylogenet. Evol. 15, 403–413. Table S1. The voucher information for perine exam- Sano, R., Takamiya, M., Kurita, S., Ito, M., Hasebe, M., 2000b. ination and for perine character coding in this study. Diplazium subsinuatum and Di. tomitaroanum should be moved to Deparia according to molecular, morphological, and cytological Table S2. The character coding in this study. characters. J. Plant. Res. 113, 157–163. Table S3. Maximum-likelihood reconstruction ances- Schuettpelz, E., Pryer, K.M., 2009. Evidence for a Cenozoic tral states of each node. radiation of ferns in an angiosperm-dominated canopy. Proc. Table S4. The evolutionary rates and tendencies of Natl Acad. Sci. USA 106, 11200–11205. Stockey, R.A., Nishida, H., Rothwell, G.W., Columbia, B., Group, the characters inferred by the Deparia-only chrono- P., Baadsgaard, H., 1999. Permineralized ferns from the middle gram. Eocene Princeton chert. I. Makotopteris princetonensis gen. et sp. nov. (Athyriaceae). Int. J. Plant Sci. 160, 1047–1055. Sundue, M.A., Rothfels, C.J., 2014. Stasis and convergence characterize morphological evolution in eupolypod II ferns. Ann. Appendix Bot. 113, 35–54. Swofford, D.L., 2003. PAUP*. Phylogenetic Analysis Using Voucher specimens and GenBank accession numbers for Deparia Parsimony (* and Other Methods). Version 4.0b10. Sinauer pDNA sequences used in this study. Information is presented in the Associates, Sunderland, MA. following order: taxon name, collection number (deposited herbar- Testo, W., Sundue, M., 2016. A 4000-species dataset provides new ium), locality (country), matK, rbcL, trnL-L-F, rps16-matK IGS. In insight into the evolution of ferns. Mol. Phylogenet. Evol. 105, addition to 37 newly generated sequences, the other sequences are 200–211. from four previous studies: Kato (2001), Li et al. (2011), Rothfels Tryon, A.F., Lugardon, B., 1990. Spores of the Pteridophyta. et al. (2012a), and Kuo et al. (2016). “–” indicates the sequences or Springer, New York. voucher specimens not available. 92 Li-Yaung Kuo et al. / Cladistics 34 (2018) 78–92

Deparia acrostichoides (Sw.) M. Kato, Kuo120 (TAIF), Mas- (Koidz.) M. Kato, TNS764364 (TNS), Nara Pref. (Japan), sachusetts (USA), JN673820, JN673929, JN673904, KX656083. JN673832, AB574946, AB575584, KX656108. Deparia lancea Deparia acuta (Ching) Fraser-Jenk., Kuo2274 (TAIF), Sichuan (Thunb. ex Murray) Fras.- Jenk., Kuo1914 (TAIF), Taipei (Taiwan), (China), KY296505, KY296514, KY296532, KY296523. Deparia aff. JN673839, JN673941, JN673916, KX656109. Deparia lobato-crenata glabrata, MO6262952 (MO), Kakamega (Kenya), KX656032, (Tagawa) M. Kato, Kuo3953 (TAIF), Cultivated (Japan), KX656062, KX656144, KX656084. Deparia aff. jiulungensis, KY296508, KY296517, KY296535, KY296526, Deparia longipes TNS763886 (TNS), Saitama Pref. (Japan), JN673855, AB574957, (Ching) Shinohara, Liu9352 (TAIF), Yunnan (China), KX656046, AB575595, KX656085. Deparia auriculata (W. M. Chu & Z. R. KX656074, KX656158, KX656130. Deparia marginalis (Hilleb.) M. Wang) Z. R. Wang, Kuo1300 (TAIF), Yunnan (China), KX656033, Kato, OppenheimerH20917 (TAIF), Hawaii (USA), KX656047, –, KX656063, KX656145, KX656086. Deparia biserialis (Baker) M. KX656159, KX656110; Kato H-21 (TI), Hawaii (USA), –, Kato, Shinohara0810501 (KYO), Kinabalu (Indonesia), JN673822, AB046981, –, –. Deparia macdonellii (Bedd.) M. Kato, TNS731529 JN673931, JN673906, KX656087. Deparia bonincola (Nakai) M. (TNS), Sikyan (Pakistan), KY296509, KY296518, KY296536, Kato, TNS774841 (TNS), Bonin Is. (Japan), JN673823, AB574940, KY296527, Deparia medogensis (Ching & S. K. Wu) Z. R. Wang, AB575578, KX656088. Deparia boryana (Willd.) M. Kato, Liu9453 (TAIF), Yunnan (China), KX656048, KX656075, P02432539 (P), Saint-Louis (La Reunion), KX656034, KX656064, KX656160, KX656111. Deparia minamitanii S. Serizawa, TNS774852 KX656146, KX656089. Deparia cataracticola M. Kato, Wood12767 (TNS), Miyazaki Pref. (Japan), JN673847, AB574948, AB575586, (PTBG), Hawaii (USA), KX656035, –, KX656147, KX656090; Kato KX656112. Deparia okuboana (Makino) M. Kato, TNS764345 H-105 (TI), Hawaii (USA), –, AB046982, –, –. Deparia concinna (TNS), Shizuoka Pref. (Japan), JN673848, AB574949, AB575587, (Z.R.Wang) M. Kato, Kuo2268 (TAIF), Sichuan (China), KX656113. Deparia omeiensis (Z. R. Wang) M. Kato, Liu9671 KX656036, KX656065, KX656148, KX656091. Deparia confluens (TAIF), Sichuan (China), JN673849, JN673949, JN673924, (Kunze) M. Kato, – (Cultivated in Koshikawa Botanical Garden), KX656114. Deparia otomasui (Kurata) S. Serizawa, TNS764339 Surnbawa (Indonesia), JN673824, JN673932, JN673907, KX656092. (TNS), Kumamoto Pref. (Japan), JN673851, AB574950, AB575588, Deparia conilii (Fr. & Sav.) M. Kato, TNS768165 (TNS), Hachijo Is. KX656116. Deparia parvisora (C. Chr.) M. Kato, P00243925 (P), (Japan), JN673825, AB574941, AB575579, KX656093. Deparia core- Marojejy (Madagascar), KX656049, KX656076, KX656161, ana (H. Christ) M. Kato, TNS776382 (TNS), Aomori Pref. (Japan), KX656117. Deparia petersenii subsp. deflexa (Kunze) M. Kato, JN673826, AB574942, AB575580, KX656094. Deparia dickasonii M. Wade1096 (TAIF), Java (Indonesia), JN673846, JN673948, Kato, Liu9433 (TAIF), Yunnan (China), JN673827, JN673933, JN673923, KX656118. Deparia petersenii subsp. petersenii var. peter- JN673908, KX656095. Deparia dimorphophylla (Koidz.) M. Kato, senii (Kuntze) M. Kato, Kuo1922 (TAIF), Taipei (Taiwan), TNS764256 (TNS), Kagoshima Pref. (Japan), JN673828, AB574943, JN673852, JN673950, JN673925, KX656119. Deparia petersenii AB575581, KX656096. Deparia dolosa (Christ) M. Kato, Kuo1315 subsp. petersenii var. yakusimensis (H. Ito) M. Kato, Kuo1084 (TAIF), Yunnan (China), JN673829, JN673934, JN673909, (TAIF), Yakushima (Japan), KY296510, KY296519, KY296537, KX656097. Deparia edentula (Kunze) X. C. Zhang, TNS1112754 KY296528, Deparia prolifera (Kaulf.) Hook. & Grev., Wood13449 (TNS), Java (Indonesia), KX656037, KX656066, KX656149, (PTBG), Hawaii (USA), KX656050, –, KX656162, KX656120; Kato KX656098. Deparia emeiensis (Z. R. Wang) Z. R. Wang, Liu9703 H-65 (TI), Hawaii (USA), –, D43906, –, –. Deparia pseudoconilii (S. (TAIF), Sichuan (China), KX656038, KX656067, KX656150, Serizawa) S. Serizawa, TNS764016 (TNS), Okinawa Pref. (Japan), KX656099. Deparia erecta (Z. R. Wang) M. Kato, Kuo2219 (TAIF), JN673853, AB574952, AB575590, KX656121. Deparia pterorachis Sichuan (China), KX656039, KX656068, KX656151, KX656100. (H. Christ) M. Kato, TNS766637 (TNS), Nagano Pref. (Japan), Deparia fenzliana (Luers.) M. Kato, OppenheimerH20920 (TAIF), JN673854, AB574954, AB575592, KX656122. Deparia pycnosora Hawaii (USA), KX656040, –, KX656152, KX656101; Hasebe s.n. (Christ) M. Kato, Lu14388 (TAIF), Changbai Mt. (China), (TI), Hawaii (USA), –, D43900, –, –. Deparia formosana (Rosenst.) KX656051, KX656077, KX656163, KX656123. Deparia sichuanensis R. Sano, Kuo2306 (TAIF), Yilan (Taiwan), KX656041, KX656069, (Z. R. Wang) Z. R. Wang, Kuo2243 (TAIF), Sichuan (China), KX656153, KX656102. Deparia forsythii-majoris (C. Chr.) M. Kato, KX656052, KX656078, KX656164, KX656124. Deparia sp., P02432852 (P), Anjanaharibe Sud (Madagascar), KX656042, MO5596368 (MO), Fianarantsoa (Madagascar), KY296511, KX656070, KX656154, KX656103. Deparia glabrata (Mett. ex KY296520, KY296538, KY296529, Deparia subfluvialis (Hayata) M. Kuhn) M. Kato, P01515373 (P), Oloitia (Equatorial Guinea), Kato, Kuo168 (TAIF), Nantu (Taiwan), JN673857, JN673951, KX656043, KX656071, KX656155, KX656104. Deparia gordonii JN673926, KX656126. Deparia tenuifolia (Kirk) M. Kato, Perrie6488 (Baker) M. Kato, TNA9527761 (TNS), Vitilevu (Fijii), –, –, (WELT), North Island (New Zealand), –, KJ400018, KJ400019, –. KY296541, –. Deparia hainanensis (Ching) R. Sano, PE01385619 Deparia timetensis (E. Brown) M. Kato, Wood10048 (PTBG), Mar- (PE), Hainan (China), KX656044, KX656072, KX656156, quesas (French Polynesia), KX656054, KX656080, KX656166, –. KX656105. Deparia henryi (Baker) M. Kato, Kuo3953 (TAIF), Deparia tomitaroana (Masam.) R. Sano, TNS771521 (TNS), Kochi Hubei (China), KY296506, KY296515, KY296533, KY296524, Pref. (Japan), KY296512, KY296521, KY296539, KY296530, Deparia heterophlebia (Mett.) R. Sano, Liu9426 (TAIF), Yunnan Deparia unifurcata (Baker) M. Kato, Kuo2197 (TAIF), Sichuan (China), KX656045, KX656073, KX656157, KX656106. Deparia (China), KX656055, KX656081, KX656167, KX656127. Deparia japonica (Thunb. ex Murray) M. Kato, TNS763869 (TNS), Ibaraki viridifrons (Makino) M. Kato, TNS766472 (TNS), Nara Pref. Pref. (Japan), JN673831, AB574945, AB575583, KX656107. Deparia (Japan), JN673861, AB574959, AB575597, KX656128. Deparia wilso- jiulungensis (Ching) Z. R.Wang, TNS763932 (TNS), Tokyo Metro- nii (Christ) X. C. Zhang, Kuo2087 (TAIF), Sichuan (China), polis (Japan), JN673850, AB574956, AB575594, KX656115. Deparia KX656056, KX656082, KX656168, KX656129. Deparia yunnanensis kaalaana (Copel.) M. Kato, TNS1191621 (TNS), Hawaii (USA), (Ching) R. Sano, Kuo3283 (TAIF), Yunnan (China), KY296513, KY296507, KY296516, KY296534, KY296525, Deparia kiusiana KY296522, KY296540, KY296531.