Molecular Phylogeny of the Fern Family Dryopteridaceae Inferred

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Molecular Phylogeny of the Fern Family Dryopteridaceae Inferred from Chloroplast rbc L and atp B Genes Author(s): Hong‐Mei Liu, Xian‐Chun Zhang, Wei Wang, Yin‐Long Qiu, and Zhi‐Duan Chen Source: International Journal of Plant Sciences, Vol. 168, No. 9 (November/December 2007), pp. 1311-1323 Published by: The University of Chicago Press Stable URL: http://www.jstor.org/stable/10.1086/521710 . Accessed: 02/04/2015 02:30

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This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions Int. J. Plant Sci. 168(9):1311–1323. 2007. Ó 2007 by The University of Chicago. All rights reserved. 1058-5893/2007/16809-0007$15.00 DOI: 10.1086/521710

MOLECULAR PHYLOGENY OF THE FERN FAMILY DRYOPTERIDACEAE INFERRED FROM CHLOROPLAST rbcL AND atpB GENES

Hong-Mei Liu,*,y Xian-Chun Zhang,1,* Wei Wang,* Yin-Long Qiu,z and Zhi-Duan Chen2,*

*State Key Laboratory of Systematic and Evolutionary Botany, Institute of Botany, Chinese Academy of Sciences, Xiangshan, Beijing 100093, People’s Republic of China; yGraduate University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China; and zDepartment of Ecology and Evolutionary Biology, University of Michigan, Ann Arbor, Michigan 48109-1048, U.S.A.

Dryopteridaceae are a large family of leptosporangiate ferns exhibiting a rich diversity of morphological characteristics and generating many taxonomical controversies. Previous efforts to systematize this large group without a cladistic phylogenetic framework have not reached a consensus on circumscription of the family. In order to circumscribe the family and identify major groups within it, a broadscale phylogenetic analysis was conducted on these ferns plus representatives of putatively related families within the Eupolypods I clade. Two chloroplast loci, rbcL and atpB, were used in this study. A combined analysis of the two genes generated a well-resolved and strongly supported overall phylogeny of the Dryopteridaceae. The results indicate that the Dryopteridaceae form a monophyletic group with the exception of Didymochlaena, Hypodematium, and Leucostegia and are sister to a large clade comprising Lomariopsidaceae, Tectariaceae, Polypodiaceae, Davalliaceae, and Oleandraceae. Of the three previously unincorporated genera, Leptorumohra and Phanero- phlebiopsis are nested within the genus Arachniodes, and Diacalpe is closely related to Acrophorus. The genus Pleocnemia, previously classified in Tectariaceae, clearly belongs in Dryopteridaceae and shows a close affinity to Lastreopsis. This study identifies four major lineages within the family, each strongly supported as a monophyletic group. Two large genera, Dryopteris and Polystichum, as traditionally defined, are shown to be polyphyletic and need to be revised in the future.

Keywords: atpB, chloroplast loci, Dryopteridaceae, ferns, molecular phylogeny, rbcL.

Introduction these studies were based mainly on morphological characteristics, some of which were used as diagnostic characters, Dryopteridaceae are a large and diverse family of lepto- such as indusial characters (indusiate condition vs. exindusi- sporangiate ferns that have a pantropical distribution and ate condition), dimorphism of the fertile and sterile leaves, also many temperate representatives. Members of the family and growth habit, which have been shown to be homoplastic are usually terrestrial, medium-sized plants. The plants usu- (Smith 1995). ally have creeping rhizomes, ascending or erect, with non- Recent advances in molecular systematics of ferns make it clathrate scales at apices; petioles with numerous round, possible to address long-standing questions about the classi- vascular bundles arranged in a ring; veins pinnate or forking, fication of different fern groups. In the first insight of the in- free to variously anastomosing, with or without included vein- terfamilial relationships of ferns based on rbcL sequences, lets; spores reniform, monolete, perine winged; and the basic Hasebe et al. (1995) showed that Dryopteridaceae sensu chromosome number of x¼41, rarely 40. Kramer et al. (1990) were not monophyletic, which was sub- The circumscription of Dryopteridaceae has historically sequently confirmed by a combined molecular and morpho- been controversial (Pichi Sermolli 1977; Ching 1978; Tryon logical analysis (Pryer et al. 1995). and Tryon 1982; Kramer et al. 1990). Ching (1978) treated Recently, Li and Lu (2006a) performed a molecular analy- Dryopteridaceae in a strict sense with 14 genera, 13 of which sis of 12 recognized genera and other putatively related taxa are distributed in China, while other authors defined it in to reconstruct the phylogeny of Dryopteridaceae sensu Ching a broader sense with 32–52 genera (Pichi Sermolli 1977; (1978). Their study indicated that Dryopteridaceae sensu Tryon and Tryon 1982; Kramer et al. 1990). The major con- Ching (1978), except for Cyclopeltis J. Smith, were mono- troversy among different classifications centers on the issue phyletic but should be expanded to include Acrophorus C. of whether athyrioid, tectarioid, and onocleoid as well as lo- Presl, Diacalpe Blume, Peranema D. Don, and Ctenitis C. mariopsidoid ferns should be included in the family (table 1). Chr (including Dryopsis Holttum and Edwards). Two mono- However, no previous efforts to systematize this large group phyletic groups were also identified in their study, which cor- were conducted in a phylogenetic framework. Furthermore, responded largely to two tribes recognized by Ching (1978). Although it was very useful and the earliest scheme of molec- 1 Author for correspondence; e-mail [email protected]. ular phylogeny for this group, Li and Lu’s study (2006a) was 2 Author for correspondence; e-mail [email protected]. based on a narrowly defined Dryopteridaceae (sensu Ching Manuscript received May 2007; revised manuscript received July 2007. 1978), and only a single gene was used. Other molecular

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This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions Table 1 Genera Included in Different Classification Systems of Dryopteridaceae Ching 1978 Smith et al. 2006b Pichi Sermolli 1977 Tryon and Tryon 1982 Kramer et al. 1990 Acrorumohra Acrorumohra Acrorumohra Acrorumohraa Acrorumohraa Arachniodes Arachniodes Arachniodes Arachniodesa Arachniodes Cyrtogonellum Cyrtogonellum Cyrtogonellum Cyrtogonellum Cyrtogonelluma Cyrtomidictyum Cyrtomidictyum Cyrtomidictyum Cyrtomidictyum Cyrtomidictyuma Cyrtomium Cyrtomium Cyrtomium Cyrtomium Cyrtomiuma Dryopteris Dryopteris Dryopteris Dryopteris Dryopteris Leptorumohra ? Leptorumohra Leptorumohraa Leptorumohraa Lithostegia Lithostegia Lithostegia Lithostegia Lithostegia Nothoperanema Nothoperanemaa Nothoperanema Nothoperanema Nothoperanema Phanerophlebia Phanerophlebia Phanerophlebia Phanerophlebiaa Phanerophlebiaa Phanerophlebiopsis ? Phanerophlebiopsis Phanerophlebiopsisa Phanerophlebiopsisa Polystichum Polystichum Polystichum Polystichum Polystichum Sorolepidium ? Sorolepidiuma Sorolepidiuma Sorolepidiuma Cyclopeltis Cyclopeltis Cyclopeltis Cyclopeltis Acrophorus Acrophorus Acrophorus Acrophorus Ctenitis Ctenitis Ctenitis Ctenitis Didymochlaena Didymochlaena Didymochlaena Didymochlaena Dryopolystichum Dryopolystichum Dryopolystichum Dryopolystichum Lastreopsis Lastreopsis Lastreopsis Lastreopsis Maxonia Maxonia Maxonia Maxonia Olfersia Olfersia Olfersiaa Olfersia Peranema Peranema Peranema Peranema Plecosorusa Plecosorus Plecosorusa Plecosorusa Polybotrya Polybotrya Polybotrya Polybotrya Polystichopsis Polystichopsis Polystichopsisa Polystichopsisa Rumohra Rumohra Rumohra Rumohra Stenolepia Stenolepia Stenolepia Stenolepia Stigmatopteris Stigmatopteris Stigmatopteris Stigmatopteris Cyclodium Cyclodium Cyclodium Hypodematium Hypodematium Hypodematium Papuapterisa Papuapterisa Papuapterisa Adenoderris Adenoderris Bolbitis Bolbitis Egenolfiaa Egenolfiaa Elaphoglossum Elaphoglossum Lomagramma Lomagramma Teratophyllum Teratophyllum Coveniella Coveniella Dryopsis Dryopsis Megalastrum Megalastruma Ataxipteris Leucostegia Acropelta Acropeltaa Acropeltaa Amphiblastra Amphiblastraa Amphiblastraa Atalopteris Atalopteris Atalopterisa Camptodium Camptodiuma Camptodiuma Cionidium Cionidiuma Cionidiuma Ctenitopsis Ctenitopsisa Ctenitopsisa ? Diacalpe Diacalpea Diacalpea Dictyoxiphium Dictyoxiphiuma Dictyoxiphiuma Fadyenia Fadyeniaa Fadyeniaa Hemigramma Hemigramma Hemigrammaa Heterogonium Heterogonium Heterogonium Hypoderris Hypoderris Hypoderris Luerssenia Luerssenia Luersseniaa Pleocnemia Pleocnemia Pleocnemia Pleuroderris Pleuroderrisa Pleuroderrisa Pseudotectaria Pseudotectariaa Pseudotectariaa Psomiocarpa Psomiocarpa Psomiocarpa Pteridrys Pteridrys Pteridrys Quercifilix Quercifilixa Quercifilixa

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Table 1 (Continued) Ching 1978 Smith et al. 2006b Pichi Sermolli 1977 Tryon and Tryon 1982 Kramer et al. 1990 Stenosemia Stenosemia Stenosemiaa Tectaria Tectaria Tectaria Tectaridium Tectaridium Tectaridiuma Soromanes Soromanesa Papuapteris Acystopterisa Acystopteris Arcypteris Arcypterisa Athyrium Athyrium Cornopteris Cornopteris Curraniaa Curraniaa Cystopteris Cystopteris Diplazium Diplazium Gymnocarpium Gymnocarpium Hemidictyum Hemidictyum Matteuccia Matteuccia Onoclea Onoclea Onocleopsis Onocleopsis Neoathyriuma Neoathyriuma Pseudoathyriuma Pseudoathyriuma Pseudocystopterisa Pseudocystopterisa Woodsia Woodsia Arthropteris Lomariopsis Oleandra Psammiosorus Trichoneuron Aneigmopteris Anisocarpiuma Athyriopsisa Callipterisa Cystoathyriuma Deparia Dictyodroma Diplaziopsis Dryoathyriuma Kuniwatsukiaa Lunathyriuma Parathyriuma Rhachidosorusa Triblemmaa Triplophyllum Note. Question marks indicate genera on which Smith et al. (2006b) did not comment. a Genera that were not accepted but treated as synonyms in different classification systems of Dryopteridaceae. analyses have focused on either a taxonomic subset of the family controversial genera, Didymochlaena Desvaux, Hypodematium (Gastony and Ungerer 1997; Sano et al. 2000; Little and Barring- Kunze, and Leucostegia C. Presl, were also tentatively included ton 2003; Li et al. 2004a; Lu et al. 2005, 2007; Li and Lu in Dryopteridaceae. However, other previously included groups, 2006b; Liu et al. 2007) or a geographic area (Geiger and such as athyrioid, tectarioid, and onocleoid ferns, were segre- Ranker 2005). gated as distinct families. Based on consensus of a variety of morphological and mo- Unraveling the phylogeny of major derived fern groups, lecular evidence, Smith et al. (2006b) recently proposed a sum- such as asplenioids, dryopteroids, and polygrammoids, is of mary and new familial classification for extant ferns, among great interest because these ferns are important components which Dryopteridaceae were recircumscribed as almost cer- of modern fern floras. Dryopteroids are not only one of the tainly a monophyletic group of 40–45 genera with ca. 1700 most species-rich groups of ferns but they also exhibit a species. Most genera of Lomariopsidaceae sensu Kramer (1990b), rich diversity of morphological characters and have gener- including Bolbitis Schott (including Egenolfia Schott), Elapho- ated many taxonomical controversies. In contrast to the ex- glossum J. Smith, Lomagramma J. Smith, and Teratophyllum tensively studied polygrammoids (e.g., Schneider et al. 2002, Mettenius ex Kuhn, were moved into Dryopteridaceae. Three 2004c, 2006; Ranker et al. 2004; Smith et al. 2006a) and

This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions Table 2 Materials Used in This Study GenBank accession number Family and species Voucher Locality rbcL atpB Dryopteridaceae: Acrophorus emeiensis DQ054522a Acrophorus stipellatus Liu H-M YN274 Kunming, Yunnan EF463106 EF450509 Acrorumohra diffracta Zhang X-C 2344 Jinping, Yunnan EF463107 EF450504 Acrorumohra hasseltii Zhang X-C 3518 Diaoluoshan, Hainan EF463108 EF450505 Arachniodes amoena Liu H-M A237 Shangyou, Jiangxi EF463109 EF450481 Arachniodes cavalerii Wu-zhishan Exp. HN122 Wuzhishan, Hainan EF463111 EF450483 Arachniodes festina Liu H-M A298 Shangyou, Jiangxi EF463114 EF450486 Arachniodes rhomboidea Zhang X-C 2611 Nanchuan, Chongqing EF463115 EF450487 Ataxipteris sinii D43898a Bolbitis appendiculata Wu-zhishan Exp. HN022 Wuzhishan, Hainan EF463118 EF450513 Bolbitis repanda AB232399a Ctenitis decurrenti-pinnata Dong S-Y 54 Changjiang, Hainan EF460676 EF450519 Ctenitis rhodolepis Dong S-Y 1056 Changjiang, Hainan EF460678 EF450517 Cyrtogonellum caducum Liu H-M GX273 Huanjiang, Guangxi EF463120 EF450476 Cyrtogonellum fraxinellum Zhang X-C 2596 Nanchuan, Chongqing EF463121 EF450477 Cyrtomidictyum conjunctum Zhang X-C 1895 Jiulianshan, Jiangxi EF463123 Cyrtomidictyum lepidocaulon Wang X-X BY04 Changting, Fujian DQ508767a EF450480 Cyrtomium balansae AY694799a Cyrtomium falcatum Zhang X-C 2397 Korea EF394238b EF450470 Cyrtomium fortunei Zhang X-C 2700 Nanchuan, Chongqing EF394237b EF450471 Cyrtomium hookerianum Liu H-M BJ003 Kunming, Yunnan EF540720 EF540726 Diacalpe annamensis Wu-zhishan Exp. HN138 Wuzhishan, Hainan EF463125 EF450510 Diacalpe aspidiodes Wu-zhishan Exp. HN118 Wuzhishan, Hainan EF463126 EF450511 Didymochlaena truncatula AF425105a Dryopsis heterolaena Liu Z-Y 2022718 Shuifu, Yunnan EF460682 EF450516 Dryopsis mariformis Zhang X-C 2673 Nanchuan, Chongqing EF460683 EF450518 Dryopteris cycadina Liu H-M A353 Shangyou, Jiangxi EF463127 EF450492 Dryopteris labordei Liu H-M A360 Shangyou, Jiangxi EF463128 EF450493 Dryopteris paciﬁca Zhang X-C 3616 Korea AY268879a EF450495 Dryopteris sieboldii Liu H-M A376 Shangyou, Jiangxi EF463130 EF450498 Dryopteris sparsa Liu H-M A338 Shangyou, Jiangxi EF463131 EF450497 Elaphoglossum sartorii AY818705a Elaphoglossum wacketii AY818710a Elaphoglossum mcclurei Wu-zhishan Exp. HN133 Wuzhishan, Hainan EF450514 Hypodematium crenatum D43916a H. crenatum var. fauriei AB232414a Lastreopsis effusa AF537230a Lastreopsis subrecedens Dong S-Y 606 Wuzhishan, Hainan EF460685 EF450531 Leptorumohra miqueliana Zhang X-C 3469 Korea EF463132 EF450506 Leptorumohra quadripinnata Liu Z-Y 2022723 Shuifu, Yunnan EF463133 EF450507 Leucostegia immersa AB232388a Leucostegia pallida AB232389a Lithostegia foeniculacea LuSG/DL33 Dali, Yunnan DQ054516a EF450501 Lomagramma matthewii Wu-zhishan Exp. HN202 Wuzhishan, Hainan EF463134 EF450530 Megalastrum atrogriseum 1 AF537263a M. atrogriseum 2 AF537264a Nothoperanema hendersonii Zhang X-C 2981 Tengchong, Yunnan EF463135 EF450499 Nothoperanema shikokianum Liu H-M GX241 Wuming, Guangxi EF463136 EF450500 Nothoperanema squamisetum DQ054512a Peranema cyatheoides DQ054513a Peranema luzonium Zhu D-H 1572 Nanchuan, Chongqing EF450512 Phanerophlebia nobilis var. nobilis AF537231a Phanerophlebia umbonata AF537233a Phanerophlebiopsis bipinnata Yan Y-H HN002 Cult., Hunan EF463137 EF450502 Phanerophlebiopsis duplicato-serrata Zhang X-C 2624 Nanchuan, Chongqing EF463138 EF450503 Polybotrya caudata AB232393a Polystichopsis chaerophylloides AF537234a Polystichum acutidens Zhang X-C 3404 Shennongjia, Hubei EF394244b EF450459

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This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions Table 2 (Continued) GenBank accession number Family and species Voucher Locality rbcL atpB Polystichum anomalum Liu H-M A379 Shangyou, Jiangxi EF394248b EF450460 Polystichum luctuosum Zhang X-C 2421 Miyi, Sichuan EF394245b EF450466 Polystichum makinoi Zhang X-C 3365 Shennongjia, Hubei EF394247b EF450467 Polystichum omeiense Li D EMLD3 Emeishan, Sichuan EF540723 EF450469 Polystichum tonkinense Li L-Q et al. 272 Vietnam EF540724 EF540728 Polystichum tripteron U30832a Rumohra adiantiformis 1 AY818678a R. adiantiformis 2 U05648a Teratophyllum wilkesianum AB232402a Lomariopsidaceae: Cyclopeltis crenata Zhang X-C 2312 Wuzhishan, Hainan EF463119 EF450534 Lomariopsis marginata AY818677a Lomariopsis spectabilis Dong S-Y 834 Limushan, Hainan AB232401a EF450533 Nephrolepis acuminata AB232403a Nephrolepis auriculata Wu-zhishan Exp. HN274 Wuzhishan, Hainan EF450535 Nephrolepis hirsutula U05638a Tectariaceae: Arthropteris backleri U05605a Ctenitopsis devexa Dong S-Y 180 EF460679 EF450520 Ctenitopsis fuscipes Dong S-Y 832 Limushan, Hainan EF460680 EF450521 Hemigramma decurrens Dong S-Y 734 Dongfang, Hainan EF460684 EF450529 Pleocnemia winitii Dong S-Y 1025 Jiangfengling, Hainan EF460686 EF450528 Pteridrys lofouensis Liu H-M BJ01 Cult., Beijing EF460687 EF450527 Querciﬁlix zeylanica Dong S-Y 182 Changjiang, Hainan EF460688 EF450532 Tectaria decurrens Dong S-Y 1026 Changjiang, Hainan EF460689 EF450523 Tectaria variolosa Dong S-Y 1024 Changjiang, Hainan EF460690 EF450525 Polypodiaceae: Grammitis tenella AF468198a AF469773a Microgramma squamulosa AY362579a AY459517a Polypodium glycyrrhiza U21146a AY459518a Terpsichore senilis AY096208a AY459510a Davalliaceae: Araiostegia pulchra AB212692a Davallia solida AY096193a Davallia fejeensis DQ646107a Davallodes hirsutum AY096196a Oleandraceae: Oleandra cumingii AY093597a Oleandra hainanensis Dong S-Y 136 Yuedong, Hainan EF450536 Oleandra wallichii AB212687a Woodsiaceae: Athyrium niponicum D43891a Diplazium wichurae AB042742a Blechnaceae: Blechunum occidentale AB040565a U93838a Onocleaceae: Matteuccia struthiopteris U05930a Thelypteridaceae: Thelypteris palustris U05947a AY612713a Aspleniaceae: Asplenium nidus AF525270a AY612688a Diella mannii AY549739a Note. Circumscription of family and genera follows Smith et al. (2006b; some genera are tentatively accepted to incorporate our phylogenetic results). Accession numbers for newly generated sequences begin with ‘‘EF.’’ a Sequences from GenBank. b rbcL sequences from Liu et al. (2007).

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Table 3 Statistics for the Four Data Sets Analyzed in This Study Data set Included taxa Alignment length Variable characters Best-fitting modela rbcL 95 1230 501 (41%) GTR þ I þ G rbcLb 62 1230 447 (36%) GTR þ I þ G atpB 62 1182 420 (36%) GTR þ I þ G rbcL þ atpB 62 2414 868 (36%) GTR þ I þ G a As identified with the Akaike Information Criterion in Modeltest. b Reduced rbcL data set for sequence combination. asplenioids (e.g., Murakami et al. 1999; Van Den Heede et al. on previously published molecular systematic studies of all lep- 2003; Schneider et al. 2004a; Perrie and Brownsey 2005), a tosporangiate ferns (Hasebe et al. 1995), we selected multiple global phylogeny for Dryopteridaceae has not been reconstructed taxa (Athyrium, Diplazium, Thelypteris, Matteuccia, Blechnum, with large molecular data sets. Asplenium,andDiella) belonging to the Eupolypods II clade Here, we present a phylogenetic analysis of Dryopterida- (Schneider et al. 2004b; Smith et al. 2006b)asoutgroups. ceae based on nucleotide sequence data from two chloroplast loci (rbcLandatpB). Our primary objective is to recover a global DNA Extraction, Gene Amplification, and Sequencing phylogeny of Dryopteridaceae by a wide sampling throughout Total genomic DNA was extracted from silica gel–dried the family and other putatively related taxa. A further goal of leaves using the modified CTAB procedure of Doyle and our study is to identify major lineages within the family and also Doyle (1987). The selected DNA regions were amplified with to investigate phylogenetic placement of several Asian genera standard polymerase chain reaction (PCR). The protocols used (Diacalpe, Leptorumohra H. Ito, and Phanerophlebiopsis Ching) to amplify rbcLandatpB genes were identical and followed Li about which no comments were made in the recent classification et al. (2004b). The rbcLgenewasamplifiedusingprimers1F of Dryopteridaceae (Smith et al. 2006b). (Chen et al. 1998) and 1351R (Gastony and Rollo 1995) and an additional forward-sequencing primer, 440F (Gastony and Material and Methods Rollo 1995). Primers for amplifying and sequencing the atpB gene were newly designed: the forward primer F1 (59-GGT Taxon Sampling CAG CAG ATT AAT GTT AC-39) and the reverse primer R1 (59-GCT TCG TCG ATA TTG CCA AC-39). The PCR products We assembled two data matrices, both of which included were purified using a GFX PCR DNA and Gel Band Purifica- newly generated sequences and sequences obtained from Gen- tion Kit and were directly sequenced. Sequencing reactions Bank. Taxa, sources, vouchers, and accession numbers are were conducted using the DYEnamic ET Dye Terminator Cycle provided in table 2. The first data matrix consisted of 95 rbcL Sequencing Kit. Sequences were analyzed using MegaBACE1000 sequences, of which 41 were newly generated. The second ma- DNA Analysis Systems, following the manufacture’s protocols. trix comprised rbcL and atpB sequences of 62 taxa, which in- Nucleotide sequences were aligned using ClustalX (Thompson cluded 41 rbcL sequences from the former single rbcL data set et al. 1997). and 54 atpB sequences newly generated in this study plus additional sequences from GenBank. Our sampling included 27 of 36 recognized genera plus Phylogenetic Analyses one synonym in Dryopteridaceae according to the concept of The preliminary analysis using only rbcL sequences, de- Smith et al. (2006b). Three previously unincorporated Asian signed to verify the boundary of the Dryopteridaceae and to genera, Diacalpe, Leptorumohra, and Phanerophlebiopsis, find their closest and progressively more distant relatives, were also included. Two or more representatives of each genus was conducted on a large 165-sequence data set comprising (as well as different samples of the same species) were sampled, all major groups of the core leptosporangiate ferns (results except for several small genera. In addition, we examined spe- not shown). Phylogenetic relationships of Dryopteridaceae cies of Lomariopsidaceae, Polypodiaceae, Davalliaceae, and were investigated by maximum parsimony (MP), maximum Oleandraceae, which are comembers of the Eupolypods I clade likelihood (ML), and Bayesian inference (BI) methods in (Schneider et al. 2004b; Smith et al. 2006b). We also examined PAUP* 4.0b10 (Swofford 2003), PHYML 2.4.3 (Guindon species of Tectariaceae, which have been placed near or included and Gascuel 2003), and MrBayes 3.0b4 (Ronquist and Huel- in Dryopteridaceae (Ching 1978; Kramer et al. 1990). Relying senbeck 2003), respectively. All three analyses were performed

Fig. 1 Fifty-percent majority-rule consensus tree from Bayesian inference (BI) of the 95-taxon phylogeny based on rbcL sequence data. Values above branches are posterior probability support 0.90 from BI; values below branches are bootstrap percentage 50% from maximum likelihood analysis. Rumohra adiantiformis and Megalastrum atrogriseum are represented by two different samples each. The arrow indicates the Eupolypods I clade. Four clades within Dryopteridaceae are marked in the phylogenetic tree. Taxon names in boldface type represent species of Dryopteridaceae; four genera suggested to be included within Dryopteridaceae based on the current analyses are shown in lighter type. Family circumscription follows Smith et al. (2006b).

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This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions 1318 INTERNATIONAL JOURNAL OF PLANT SCIENCES for the 62-taxon combined two-gene (plastid rbcL and atpB) majority-rule consensus tree resulting from Bayesian analysis data set and the two 62-taxon single-gene data sets that to- of the 95-taxon rbcL data set is shown in figure 1. gether composed the combined data set, and only ML and BI The aligned matrix of the 62-taxon rbcL gene had 1230 were performed for the 95-taxon rbcL data set. For MP analy- characters, of which 447 were variable (36%) and 312 (25%) sis, unweighted analyses were performed by heuristic searches were parsimony informative. The 62-taxon atpB gene was with tree-bisection-reconnection (TBR) branch swapping, the 1182 nucleotides in length, of which 420 (36%) were variable MulTrees in effect, steepest descent off using 1000 random characters and 294 (25%) were parsimony informative. The taxon-addition replicates, and one tree held at each step dur- topologies of the 62-taxon single rbcL and atpB matrices were ing stepwise addition. Bootstrap analyses (Felsenstein 1985) highly congruent, although the strict atpB tree was not re- were conducted to examine the relative level of support for in- solved well and had lower support values (tree not shown). dividual clades on the cladograms of each search, using 1000 The combined rbcL and atpB data matrix included 62 taxa bootstrap replicates and the same tree search procedure as de- and 2414 characters, with 606 (25%) characters that were scribed above. parsimony informative. The results from the combined rbcL For each data set, the best-fitting model of sequence evolu- and atpB sequences showed finely resolved and well-supported tion was identified with the Akaike Information Criterion in inter- and intrafamilial relationships. Modeltest, version 3.7 (Posada and Crandall 1998). Once the According to our results, Dryopteridaceae, excluding Hy- best sequence evolution model was determined (table 3), an podematium, Leucostegia, and Didymochlaena, are mono- ML analysis was performed for each data set. Nodal robustness phyletic and sister to a clade comprising Lomariopsidaceae, on the ML tree was estimated by the nonparametric bootstrap Tectariaceae, Polypodiaceae, Davalliaceae, and Oleandraceae (1000 replicates). (fig. 1). Hypodematium and Leucostegia are sister to each BI was conducted using MrBayes 3.0b4 (Ronquist and other, and the monotypic genus Didymochlaena forms an Huelsenbeck 2003) with appropriate evolutionary models de- isolated lineage as a sister group to the rest of the Eupoly- termined as described above. We ran four chains of the Markov pods I clade. The inclusion of Diacalpe, Leptorumohra, and chain Monte Carlo, sampling one tree every 1000 generations Phanerophlebiopsis as well as Pleocnemia C. Presl within the for 1,000,000 generations, starting with a random tree. The Dryopteridaceae is clearly justified (figs. 1–3). first 50 trees were the ‘‘burn-in’’ of the chain, and phylogenetic Four major clades are recovered within Dryopteridaceae inferences were based on those trees sampled after generation (clades I–IV in figs. 1–3). Clade I comprises the dryopteroid 50,000. The posterior probability (PP) was used to estimate ferns, within which are two strongly supported subclades. nodal robustness. In our Bayesian analyses, we considered Three Asian genera, Diacalpe, Leptorumohra, and Phanero- branches with a PP between 1.00 and 0.95 as strongly supported, phlebiopsis, are clearly nested in this group. Clade II consists those with a PP between 0.95 and 0.90 as moderately supported, of Cyrtogonellum Ching, Polystichum Roth, Cyrtomidictyum and those with a PP of <0.90 as having weak support. For ML Ching, and Cyrtomium C. Presl, which can be named as the and MP analyses, we considered branches with a bootstrap per- polystichoid clade. Phanerophlebia and Polystichopsis are centage (MLBS, MPBS) of 90% or higher as well (or strongly) also strongly supported as members of the polystichoid clade supported, 89%–70% as moderately supported, and 70% or in the rbcL-based trees (fig. 1). Clade III includes two closely lower as having weak (or low) support (Korall et al. 2006). related genera, Ctenitis and Ataxipteris, although the latter was included only in the rbcL data set. A sister group relationship between clade III and the clade made of clades I and Results II is resolved in this study, but support for this relationship is low (PP < 0:90, MLBS ¼ 53%, MPBS < 50%; figs. 2, 3). The rbcL and atpB sequence alignments were straightfor- Bolbitis, Lomagramma, Elaphoglossum, Pleocnemia, and ward; there were no insertions or deletions. The alignment Lastreopsis constitute the last of the four major lineages within length and the number of included characters for the two single- the family (clade IV in figs. 2, 3). Megalastrum, Rumohra,and gene data sets, as well as for the combined data set, are pre- Teratophyllum also belong to this group, according to the rbcL sented in table 3. sequence (fig. 1), although there were no atpB data for these The aligned rbcL matrix contained 95 taxa and 1230 char- taxa. This clade was always recovered with good bootstrap acters, of which 501 were variable. The GTR þ I þ G model support as the sister group to the remaining Dryopteridaceae. and gamma rates were selected for the 95-taxon rbcL data set. In our phylogenetic analyses, Pleocnemia is clearly allied with ML and BI analyses resulted in nearly identical topologies, members of Dryopteridaceae rather than with Tectariaceae with several minor differences at the genus level. Topological (figs. 1–3). The genus is part of a polytomy together with Las- incongruences between two analytical methods were not sup- treopsis, Megalastrum, Rumohra, and the related taxa in the ported by high bootstrap values (results not shown). The 50% rbcL-based phylogenetic trees (fig. 1). In the combined trees,

Fig. 2 Fifty-percent majority-rule phylogram from Bayesian inference (BI) of the combined rbcL and atpB sequence data. Thick lines indicate branches with posterior probability support of 0.90 from BI and maximum likelihood bootstrap percentage values 50%. Asterisks indicate clades with Bayesian posterior probability support <0.90 but maximum likelihood bootstrap values 50%. The arrow indicates the Eupolypods I clade. Vertical lines on the right indicate four clades within Dryopteridaceae. Taxon names in boldface type represent species of Dryopteridaceae; four genera suggested to be included within Dryopteridaceae based on the current analyses are shown in lighter type.

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This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions Fig. 3 Strict consensus tree of 308 most parsimonious trees obtained in maximum parsimony analysis of the combined rbcL and atpB sequence data. Bootstrap values 50% are given above branches. Boldface type, lighter type, arrow, and vertical lines are as in ﬁg. 2.

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Pleocnemia is found as a sister to the clade comprising Bolbitis, relatively isolated position of clade III is consistent with its Lomagramma,andElaphoglossum, followed by Lastreopsis (figs. rather distinct morphological and anatomical characters in 2, 3). comparison to other members within the family, with its cte- nitoid type of rachis and epidermal appendages being articu- late hairs rather than scales. Discussion Bolbitis, Lomagramma, Elaphoglossum and Teratophyl- lum have been assigned to a distinct family (Lomariopsida- Our molecular phylogenetic analyses based on both single- ceae; Kramer 1990b), two families (Lomariopsidaceae and gene data sets and the combined data set of rbcL and atpB Elaphoglossaceae; Pichi Sermolli 1977), and even three dis- identified a monophyletic Dryopteridaceae. This result gener- tinct families (Bolbitidaceae, Elaphoglossaceae, and Lomari- ally agrees with the family concept of Smith et al. (2006b) opsidaceae; Ching 1978). However, our molecular data but differs in the following two aspects: Hypodematium, Leu- suggest otherwise; these four genera and related taxa form a costegia, and Didymochlaena should be excluded from the well-resolved and strongly supported monophyletic group family, and Diacalpe, Leptorumohra, Phanerophlebiopsis, and and are clearly members of the well-supported Dryopterida- Pleocnemia should be included in the family. In this study, ceae (clade IV in figs. 1–3). On the other hand, it is difficult several taxa that have previously been proposed as separate at present to identify synapomorphic morphological charac- families, Bolbitis (Bolbitidaceae), Elaphoglossum (Elapho- ters to define such a clade. As for the large and diverse genus glossaceae), and Peranemataceae (Pichi Sermolli 1977; Ching Elaphoglossum, two detailed molecular studies were carried 1978), are nested within Dryopteridaceae. Our sequence data out (Rouhan et al. 2004; Skog et al. 2004), both of which support a single origin of the Dryopteridaceae, even though showed that Elaphoglossum was recognized as a monophy- there are evident diverse morphological characters for mem- letic group. bers of the family. Genera to Be Excluded from Dryopteridaceae Major Lineages within Dryopteridaceae Based on our phylogenetic analyses, Hypodematium, Leu- In our molecular phylogenetic analyses, four major clades costegia, and Didymochlaena should be excluded from are recovered within the Dryopteridaceae, each strongly sup- Dryopteridaceae, although they have tentatively been in- ported as a monophyletic group (clades I–IV in figs. 1–3). cluded in the family by Smith et al. (2006b). Historically, the The first clade includes dryopteroid ferns (Dryopteris, Arach- placement of Hypodematium and Leucostegia has been prob- niodes, and related taxa). This result was almost identical to lematic. Leucostegia was always assigned to the family Da- that of Li and Lu (2006a) except for Polybotrya, which was valliaceae based on morphological characters (e.g., Kramer not sampled by them. The dryopteroid clade largely corre- 1990a; Nooteboom 1992). In contrast, Hypodematium has sponds to the tribe Dryopterideae sensu Ching (1978) but been placed in various families, such as Athyriaceae, Dryop- with a broader sense than its original concept, which includes teridaceae, and Thelypteridaceae. Most recently, Hypodematium Dryopterideae sensu Ching (1978), Peranemataceae sensu was referred to an athyrioid group (Tryon and Tryon 1982; Ching (1978), Dryopsis, and Polybotrya. With respect to the Kramer et al. 1990). Based on molecular data from two gene large genus Dryopteris, the polyphyly of the genus is clear, sequences (rbcLandaccD), Tsutsumi and Kato (2006) found which is consistent with two recent studies that used more that Hypodematium wassistertoLeucostegia. In contrast to rapidly evolving sequences (trnL-F and rps4-trnS) and sam- these diverse treatments, Smith et al. (2006b) tentatively in- pled more taxa (Geiger and Ranker 2005; Li and Lu 2006b). cluded both genera within Dryopteridaceae and suggested that Within the arachnioid subclade, the inclusion of Lithostegia further studies are needed to address their precise phylogenetic renders Arachniodes paraphyletic. Therefore, proper delimi- affinity. tations of Arachniodes and Lithostegia are needed to fully The molecular data in this study showed that Hypodematium address the generic boundaries of these two groups. and Leucostegia should be excluded from the family Dry- The polystichoid clade (clade II in figs. 1–3) is comparable opteridaceae (fig. 1). Hypodematium and Leucostegia are sister to the tribe Polysticheae sensu Ching (1978) except for Cy- to each other, with strong support in terms of bootstrap and clopeltis, which has recently been moved into the family Lo- posterior probability (fig. 1), and together they form a sister mariopsidaceae (Smith et al. 2006b). Based on an rbcL data group to the large clade of Dryopteridaceae, Lomariopsidaceae, set, Little and Barrington (2003) showed that Phanerophlebia Tectariaceae, Polypodiaceae, Davalliaceae, and Oleandraceae. and Polystichopsis were sister to one another and that together The sister group relationship between Hypodematium and they were sister to the remaining polystichoid ferns. Our phylo- Leucostegia was consistent with well-documented similar mor- genetic analyses obtained well-resolved and strongly supported phological characters and molecular evidence presented by relationships for these taxa. The placement of Polystichum and Tsutsumi and Kato (2006). Cyrtomium in our rbcL/atpB-based phylogeny is in agreement The monotypic genus Didymochlaena shows a remote af- with several more detailed studies focusing on the polystichoids finity to Dryopteridaceae and is sister to the rest of the Eu- (Little and Barrington 2003; Li et al. 2004a; Lu et al. 2005, polypods I taxa (fig. 1). In three traditional classifications, 2007; Li and Lu 2006a; Liu et al. 2007). Didymochlaena was assigned to the family Dryopteridaceae The third major lineage within the family includes Ataxip- (Pichi Sermolli 1977; Tryon and Tryon 1982; Kramer et al. teris and Ctenitis, and together they form a distinct group 1990). That assignment was based on morphological charac- with moderate to high support (clade III in figs. 1–3). The ters, such as the erect stem bearing scales, petiole adaxially

This content downloaded from 159.226.100.224 on Thu, 2 Apr 2015 02:30:39 AM All use subject to JSTOR Terms and Conditions 1322 INTERNATIONAL JOURNAL OF PLANT SCIENCES grooved, scales covering the rachis and petiole, monolete moved to Dryopteridaceae (Smith et al. 2006b) and is re- spores with prominent folds, and the basic chromosome solved clearly as a member of Dryopteridaceae in this study. number (x¼41). In a phylogenetic analysis (Schneider et al. Therefore, Pleocnemia should also be treated as a member of 2004b), Didymochlaena and Hypodematium formed a clade Dryopteridaceae, and a relative close relationship with Las- and together were sister to the remainder of the Eupolypods I treopsis is suggested although not clearly and strongly sup- clade, although support for these relationships was poor. The ported. isolated position of Didymochlaena in our trees is consistent with the treatment of segregating the genus in its own family, Didymochlaenaceae (Ching 1978). However, in consideration Conclusions of the poorly supported relationship as resolved in this study, additional data are needed in future studies before a firm con- Our analyses have shown that Dryopteridaceae (sensu clusion on the position of Didymochlaena can be drawn. Smith et al. 2006b) are monophyletic except for three tentatively included genera (Hypodematium, Leucostegia,andDidy- mochlaena). Two eastern Asian endemic genera, Leptorumohra Genera to Be Included in Dryopteridaceae and Phanerophlebiopsis, are members of Dryopteridaceae and Based on our molecular data, the three Asian genera Diacalpe, should be treated as synonyms of Arachniodes. The traditional Leptorumohra,andPhanerophlebiopsis and the traditional tec- tectarioid genus Pleocnemia clearly belongs in Dryopteridaceae tarioid genus Pleocnemia clearly belong to Dryopteridaceae. Di- and shows a relatively close affinity to Lastreopsis. Within the acalpe is a traditional member of dryopteroid ferns that has been family, we have identified four distinct monophyletic groups, segregated to the family Peranemataceae (e.g., Ching 1978; Wu some of which correspond to earlier classifications and some of 1983). In a recent classification of Dryopteridaceae, however, whichneednewnomenclaturaltreatment. Smith et al. (2006b) did not make any comment on Diacalpe. This study indicates that some uncertain relationships re- According to our DNA sequence data, Diacalpe was nested quire further analysis in the future. More data, including both within the family Dryopteridaceae (figs. 1–3). This result corrob- more characters and more taxa, are needed before a well- orates previous suggestions made from both morphology (Pichi resolved phylogenetic hypothesis for the dryopteroid ferns can Sermolli 1977; Kramer et al. 1990) and rbcL sequence data (Li be offered. Also, there is indication that some genera (e.g., and Lu 2006a). Arachniodes and Polystichum) are not monophyletic, and it is Leptorumohra and Phanerophlebiopsis are two eastern clear that more species belonging to these genera need to be Asian endemic genera that have close affinities to (Pichi Ser- sampled. molli 1977; Ching 1978; Li and Lu 2006a) or are even treated as synonyms of Arachniodes (Tryon and Tryon 1982; Kramer Acknowledgments et al. 1990). Our chloroplast DNA sequences showed that both genera are nested within the genus Arachniodes. Given We thank Shi-Yong Dong, Zhen-Dong Fang, Zheng-Yu the position of Leptorumohra and Phanerophlebiopsis in our Liu, and Yue-Hong Yan for providing plant materials, Cha- phylogenetic trees, it appears that these two genera should be Cha Huang for lab assistance, and Yang Liu and Jin Pan for treated as synonyms of Arachniodes, as has been suggested by help and valuable discussion. This study was supported by Tryon and Tryon (1982) and Kramer et al. (1990). the National Natural Science Foundation of China (grants Pleocnemia is traditionally treated as tectarioids in most 30121003 to Song Ge and 30228004 to Yin-Long Qiu and classifications (Pichi Sermolli 1977; Holttum 1987; Kramer Zhi-Duan Chen), the National Basic Research Program of et al. 1990; Smith et al. 2006b). However, in no analysis of China (973 program 2007CB411601), and a grant from the any data set did we find this genus clustered with other tec- Wong Kuan Chung Education Foundation of Hong Kong to tarioids except for Lastreopsis, which has recently been Yin-Long Qiu.

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