Molecular Phylogeny of the Lady Fern Genus Athyrium in Japan Based on Chloroplast rbcL and trnL-trnF Sequences

2008·1.3 MB·English

by  Adjie, Bayu

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ISSN1346-7565 Acta Phytotax. Gcobot. 59 (2)7:9-95 (2008)

Molecular Phylogeny of the LadyrFbecrLn Genus Athyrium inJapan

Based on Chloroplast and trnL-trnF Sequences
BAYU ADJIEi, MASAYUKI TAKAMIYA2, MICHIHITO OHTA3,

TAKESHI A. OHSAWA` and YASUYUKI WATAN04
fDepartment qfGeosystem andBiosystem Stiences ,Graduate Sc'hoo otfSt'ie anndce 7Lichnotog yC,Vzib aU}iivensi41
1-33 IZtyo ii,nage ,Chiba 263-8522, Jopan; 2Department qfBiotogical Science ,Graduate Schooi (lf'Stience and
7bchnologu Kumamoto LJhiversi4 n2-39-1 Kurokami, 1(izimamoto 860-8555, Jtu)an iS7byama Science Museum,
Mshinakano-mac'hi, 7oj)ama 939-8084,Jopan;4Department ofBiologFyacuio,ofSciencCehi,baUniversiijV-33

}Zu,oi ,Inage, Chiba 263-8522, .iopan
Phy]ogenetic relationships in Ath],rium and Cornopteris were deduccd from two chloroplast DNA
fragments ,rbcL and trnL 5'exon-trnF ,of 32 specics, 2 varieties, 3 putativ ehybrid sofAtbyrium,
three taxa of Cornopteris ,and five outgroups. Atbyrium is paraphyletic, and the Atlo,rium-
Cornopteris cornplex comprises fiv eclades. Clade I, the most basal ,comprises A. niponicum, A. (-:
Anisocampium) sheareri, and A. (= Kuniwatsukia )cuspiclatuni. Clade II include sA. distentijblium
and Cornopteri Asl.l species ofclades Il tand IV are diploids ,while most species of clade V
are polyploid sT.he parentage of the putativ ehybrids and of specics of hybrid origin were also
suggested. The results were compared to previou smajor classifications based on morphology.
Key words: Atltyrium h,ybrid ,melecular phylogeny ,rbcL, trnL-trnF
Atlu]rium Roth is a worldwide genus of about femin garoups (Tagaw a1933, Kurata 1961, Seri-
200 species. Most of the species are distributedzawa 1981) .Wang (199 71,999) revised the Chi-
in the northem hemisphere, especially in eastem nese species ofAthyrium, classifying them into 14
and southeastern Asia as well as in the Himalaya sections. Among the species recognized in those
and adjacent mountain chains; cemparatively few studies, 20 taxa are cemmon in Japan .However,
species are found in tropical and southern Africa the monophyly ofeach group and the phylogenet-
or in South America, and very few are in Europe ic relationships among the groups have not been
(Kramer& Kato 1990).Approximately37 The large interspccific
spe- examined. very number of
cies, 1 subspecies, 6 varieties, 9 forms, and 74 hybrid salso poses a problem in understanding the
putative hybrid soccur in Japan. Most taxa are in taxonomy of the genus, because the hybrid scan
the southwestern part of the country (Iwats uekt i obscure the circumscription of species. Unfor-
aL 1992) .Severa lJapanes especies groups within tunately, only two geneti cstudies on putative
the genus have been recognized and revised based hybrids or hybrid species have been undertaken
on morphology, includin gthe A, yokoscense ,A. (Kuriha reta al. 1996, Terada & Takamiya 2006).
otophorum, A. viclaiii, A. iseanum, and A. .filix- In addition to the paucit yof knowledge on
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intragener siucbdivisions, the generi ccircumscrip-
tion ofAthyrium is stil lcontroversial. The genus Materials and Methods
was recently revised (Krame r& Kato 1990) to
includ esmall allied genera such as Anisocampium Tczxon sampting
C. Presl ,Pseudocystopteri sChing, Clvstoathy- A total of l37 plant sfrom 43 taxa was col-
rium Ching, and Kuniwatsuki aPich iSermolii I,n lected for DNA extraction. For about half of the
molecular phylogenetic studies of the lady fern taxa, we deterrnined chloroplast DNA sequences
group jncludin Agtl"?riu mand a]lied genera (Sano from multiple samples. GenBank sequcnces of
et ai. 2000, Wang et al. 2003) ,Athyrium (Kramer non-Japanese and uncollected Japanese taxa were
& Kato 1990) was suggested te be paraphyletic,also added to the DNA sequence dataset ,The
becaus ethe species of Cornopteri sNakai were sources of the materials and sequences are listed
nested within the AtbyTium clade. in Appendix 1. The ingrou ptaxa of the rbeL data-
The goa] of the present study was to eluci- set comprised 32 species, two varieties, and three
date the phylogenetic relationships between spe- putative hybrids ofAtiryrium, as well as two spe-
Athyrium Kramer & Kato (1990) hybrid Cornqpteris,
cies of sensu cies and one of representing
mainly from Japan, and also to examine the all previously recognized major groups in Japan,
intergeneri crelationship between Attryrium and Three species ofDeparia Hooker & Grevill eand
Cornopteris. Although the molecular studies of two ofDiptazium Swart were chosen to serve
Sano et aL (2000 a)nd Wang et aL (2003 )pro- as an outgroup, based on previou sphylogenetic
posed to resolve the intergeneric relationships analyses (San oet al, 2000, Wang et al. 2003).
in the lady fern group, the numbeT of species of The freshl ycollected materials were preserve din
Atfp?rium sampled in those studies was insuffi- silica gel until DNA extraction.
cient to deterrni ntehe intragener isucbdivisions of
Atlp,rium ,Our ingroup sample set comprised 37 DAC4 extraction, PCR amplipcation, andsequenc-
taxa ofAthyrium and three taxa of Cornopteris, lng
whieh represent about 64% of the taxa ofAtlry- The total DNA was extracted from dried
rium, except hybrids ,in Japan. The phylogenetic leaf samples using the method of Doyle & Doyle
analysis was based on a combined datase tof two (1987 )al,though some samples were extracted
chloroplast regions, the rbcL gene and the region using DNeasy Plant Mini Kit (Qiage nT,okyo,
fi;o mtrnL 5' exon to trnF. The rbcL gene alone Japan). The PCR primers aF and cR of Hasebe et
has been wjdely used to analyze many fern ]jne- al. (1994 )were used to amp]ify rbcL fragments,
ages (Hase beet aL 1993, 1994, 1995, Pryer et al. and specific primers rbcL2F (5'CCCCCTGCT-
1995,Wolf 1994,Gastony& Ungerer1997, TATTCCAAAAC3') (5'TTCCG-
et al. and rbcL2R
Sano et aL 2000, Littl e& Barringte n2003, Lu GCGTGTZATATGMCC3 ') were designed as inter-
et al, 2007), but the combination with trnL-trnF nal primers fbr sequencing. The region from trnL
was expected to irnprov eresolution, particularly (UAA)5'ex otno trnF (GAA), late rcalled trnL-
fbr studying intragener ireclationships (Schnei- F, was amplified with forward primer trnLF2F
der 2004, Geiger & Ranker 2005, Lu (5'ATGAATTTCGGGCGATGAG3'),
et aL et aL which
2005, Driscoll & Barrington 2007), Our findings was designed fbr this study, and with primerfof
fi ta revised taxonomy of the genus Atiu,riu mthat Taberle tet ai. (1991 )T,he PCR products were
cornbines elements ofall previous systems into a purifie dusing the Geneclean III kit (Qbiogene,
phylogenetical mleayningfu1 classification. Irvine ,CA, USA) after electrophoresis in 1% aga-
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rose gel and used as templates for direc tsequenc- perfbrmed to estimate incongruent length differ-
ing. Sequencing reactions were carried out with ences between the two single sequence datasets.
a BigDye Terminator v3.1 cycle sequencing kit The NJ tree was constructed with genetic
(Appli eBidosystems, Foster CitM CA, USA). All distanc eset according to Kimura's two-parameter
sequencing reactions were processed using either method (Kimur a1980) and with bootstrapp{ nogf
ABI 310 or ABI 377XL automated sequencers 1,OOO replicates, MP trees were calculated with
(Appli eBdiosystems) .A total of 91 new DNA the following options: heuristic search mode,
sequences were deposite din GenBank as part of tTee bisection-reconnect i(oTnBR) branch swap-
this study (Appendi x1). ping, MULtrees option on, and collapse zero-
Sequence fragment swere analyzed using the lengt hbranch off. Branch support was estimated
Sequencing Analysi sv5.2 (Appli eBdiosystems) by bootstra panalysis (Felsenst e1i98n5) with fu1]
and assembled by use of SeqScape v2,5 (Applied heuristi csearches, 1,OOO replicates. In Bayesian
Biosystems) .The corrected consensus sequences analysis, each region was assigned it sown model
were then automatically aligned on ClustalX of nucleotide substitution as determined by the
1.81(Thompson 1997) fbllowedby Akaikeinformation (AIC)inMrModel-
et aL manual criterion
acljustment using BioEdit (Hal 1l999), test 2,O (Nyland 2e0r04) ,For the combined data-
set, we ran a mixed-model analysis, allewing each
PCLR-SUSC Panalysis region to evolve under it sown best-f imtodel.
Parentage and hybridit ywere tested on the Posterior probabiliti eofs generated trees were
putative hybrids or hybrid species incorporated approximated using a Markov chain Monte Carlo
into phylogenetic analyses. The partia lnuclear (MCMC) algorithm with four incremental lhyeat-
RgiC gene was amplified with primer set l5F and ed chains fbr 1 million generatio nasnd sampling
16R ofIshikawa et aL (2002 f)brputati vheybrids, trees every 100 generations .A 5096 majority-rule
hybrid species, and their hypothesize dparents. consensus tree was calculated to obtain topology
To survey intraspeci fgiencetic variation, multiple with average branch lengths as well as posterior
samples were examined for hypothesized parental probabiliti efsbr all resolved nodes. We consid-
species in this analysis. The PCR product swere ered values greater than 85% to indicat estrong
analyzed using the single-strand confbrmation support for common ancestry.
polymorphism (SSCP )method fbllowing the pro-
cedure of Watano et aL (2004). Results
Phylogenetic Seguencecharacteristics
analysis
A separate phylogenetic analysis was con- We determined DNA sequences of both
ducted fbr each datase t(rbcL t,rnL=F, and the cpDNA regions from multiple samples (tw oto
combined dataset )N,eighborjoinin g(NJ )analy- six per taxon) fbr about half of the taxa. There
sis and maximum parsimony (MP) anatysis were was no intraspecif iscequence variation, except in
perfbrmed with PAUP' 4.0blO (Swoffb 2r0d02), Cornopteri schristenseniana and Athyrium tashi-
and Bayesian phylogenetic analysis was per- roi (on esubstitution in trnL-Ii). The a]ignment
fbrrne dwith MrBayes ver,3,1,2 (Huelsenb e&ck of the 47 rbcL sequences (thr efreom GenBank)
Ronquist 2001). All of the phylogenetic analyses ofAtlp?rium and allied genera consisted of 1200
were conducted with indels excluded. A partitioncharacters, ofwhich 140 (11.7% w)ere variable
homogeneit tyest as implemente idn PAUP* was and 100 (8,39 wier6e) parsimony-infbrrnativ eT.he
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sequence of the trnL-F region ofA, .fi"angulum and Bayesian analyses, and was not supported
f ,viride could not be determined ,nor could the by MP. The larg ec}ade Vl a divers eevolutionary
trnL intro nsequences of the three taxa from Gen- group, was supported only jn Bayesian analysis
Bank used in the rbcL dataset (A .distentijblium, (PP = 67%). The relationships between clades III,
A. and A. spinuiosum) .Therefbre, IM and V were not resolved.
.filixzfemina,
43 sequences were included in the trnLTF data- All phylogeneti chypotheses of the trnL-
set. The trnL.F sequences varied from 694 base F dataset recovered the same five clades with
pairs (bp )in Cornopteri schristenseniana to 761 the same memberships as a result of the rbcL
bp in A. niponicum. The alignment is available datase t(Fig 2.). Each clade was supported as
upon request from the corresponding author, The monophyletic in all three analyses with moder-
aligned sequence of trnL=F resulted in a matrix of ate or strong support. Support of the five clades
816 characters, efwhich 245 (309 ,we6re) variable was higher in trnL=F than in rbcL. The ILD test
and 190 (23,39 wter6e) parsimony-informative. indicate dno significant eonflict between the two
The aligned combined rbcL and trnL-F matrix of dataset stp = O,243) ,indicati ntghe combinability
43 taxa consisted ef2016 characters; 379 (18.8%)of the datasets.
of these were variable and 289 (14,3 %we)re par-
simony-infbrmative. The best-f imtodel fbr each Combinedpbyiogenetic analysis
dat apartitio nis SYM+I+G fbr rbcL and GTR+G The parsimony analysis of the combined
fbr trnL-LF, rbcL and trnL-F data recovered 102 shortest trees
of 568 steps (CI = O.717; RI = O,875) .The tree
Separat ephylogenetic analysis obtained from the Bayesian analysis (show nin
Phylogenetic analysis was conducted fbr Fig. 3) resulted in nearly the same topology as the
each dataset employing NJ, MP, and Bayesian NJ and MP analyses of the combined datase t(not
methods. The MP method recovered 1557 short- shown), Consisten twith both individua dlataset
est trees of246 steps (CI - O.736; RI - O.871) fbr topologies ,the combined analysis recovered five
the rbcL dataset a,nd 1185 shortest trees of 380 major clades (clad Ie-sV), all of which were mod-
steps (CI = O.700 ;RI = O.869) for trnL-F data- erately or strongly supported in all analyses. The
set. Thesc tree reconstruction methods generated relationships among c]ades were the same as those
mostly congruent topologies for each dataset. in the separate datase tanalyses, and clades III,
Thus, Bayesian trees with the support ofNJ boot- IVI and V were not resolved even in the combined
strap, MP bootstrap ,and Bayesian posterio prrob- dataset ,Using the combined dataset ,we recog-
abilities are shown in Figs. 1 and 2 as examples. nized two subclades (Va and Vb) within a large
In the rbcL analysis, there are five major clade V with strong support. The relationships of
clades, each with differe nsttatistical support (Fig,Atbyrium atkinsonii and A, rupestne to clades III,
1) .Clade I was positione adt the most basal posi- IY and V were varied in each analysis, and thus
tion ofthe tree and comprised three species: Atby- were not included in the clades defined here. The
rium niponicum, A. sheareri, and A. cuspidatum. result from the combined analysis is taken here as
Clade II comprised A. distentCfoliu mand Corno- the best estimate of the phylogenetic relationships
pteris ,and was sister to the group of Clades III, ofthe genus, because it is the best summary of the
IV, V, with some species not {ncluded in any data to date .The discussio ins therefbre based on
clades. Clade III received strong support in all the phylogeny obtained from this analysis unless
analyses. Clade IV was weakly supported by NJ otherwise noted,
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D>
A. oblitescens
A. x akiense
A. otophorum
eremicola
A, wardii
A. tseanum
A. x hisatsuanum >ovtuo
A. iseanum var. angustisectum
A,
strigillosum
A, frangulum f ,viride
A. neglectum subsp. australe
A. vidalii g
A.deltoidofrons

A. arisanense
nakanoi
AB021714 A. spinulosum
tozanense
A.tashiroi

A,kirisimaense >ovasdo-oUtuo

A. yokoscense var.
A, yokoscense
A,
nikkoense
melanolepis
A. brevifrons

A. filix-fem 'ifrenzealliae'
A. atkinsonii
A. rupestre
Cornopterischristenseniana

Cornopterisdecurrentia-alata

divNo
Cornopteriscrenulatoserrulata
Cornopterischristenseniana
distentifolium

A. sheareri
ovtuo
A. niponicum
78 i 73 i 94 A. cuspidatum
"OO/100ilOO Diplazium nipponicum
Diplaziumhachijoense a=o1..o-=o

Deparialobatecrenata

Deparia petersonii
Deparia conilii
O.1

expected changes per site
FiG. 1. Phylogenctic tree based on the rbcL datase tusing a Bayesian analysis. Measures of support are given at the nodes: NJ
bootstra p(BS)tM Pbootstra p(BS)fBayes pioastnerio rpro b a bi l i ti e( sP P .)Suppor tvalues underSO are shown as hyphens (-).
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A. oblitescens
A. otophorum
A, x akiense
A. viridescentipes
A. subrigescens
62t-t91 A.kuratae
D>
A. clivicola
A. setuligerum
A. x tokashikli
A. wardii
>ovtu-o
A,iseanum

A. x hisatsuanum
A iseanum var, angustisectum
A, strigillosum
A. negiectum subsp. australe
g
A, vidalii
A.deltoidofrons

A. nakanoi
A. atkinsonii
A. yokoscense var. dilatatum
A. tashiroi >'ovcoo
A. kirisimaense
A. yokoscense
A, nikkoense
A. melanolepis
A. brevifrons mvtuo
A. filix-fem i'frnezelaliae'
A.rupestre

Cornopterischri$tenseniana
Cornopteri$decurrentia-alata ¢
vcuo
Cornopteriscrenulatoserrulata
Cornopterischristenseniana
A, cuspidatum
ovos6
A, sheareri
A, niponicum
Diplaziumnipponioum
Diplaziumhachijoense a=oLa-=o

Deparia petersonii
Deparia conilii
Deparia]obatocrenata
expected changes peF site
FtG, 2. Phylogeneti ctree based on the trnL-F datase tusing a Bayesiar ianalysis. Measures of support are given at the nodes: NJ
bootstra p(BS)IMP bootstra p(BS)IBayes pioastnerio rprobabiliti e(sPP) S.upport va]ues under 50 are shown as hyphens (-),
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94n3i9284/86MOOA.
D>
A. x tokashikii
A. setuligerum
A. wardii
A. eremicoia >ovtuo
75173ilOO
-t-117-t-ttoo - iA-.i lisOeOanum

A. x hisatsuanum
A.iseanumvar,angusti$ectum

A. strigillosum
A, ansanense
A, tozanense
A. neglectum subsp, australe
A. vidalii g
A,deltoidofrons

A. atkinson"
A. tashiroi
A. kirisimaense >ovpmo
A. yokoscense var. dilatatum
A.yokoscense

go/solaoo A. nikkoense
A. melanolepis
9Sf971100
A.brevifrons ovtuo

A. filix-femin 'afrezelliae
A. rupestre
Cornopterischristenseniana
Cornopterisdecurrentia-alata
ovtuo
Cornopteriscrenulatoserrulata
Cornopterischristen$eniana
A. cuspidatum ovo6
nlponicum
Diplaziu mnipponicum
Diplazium hachijoense
Q)oLen-=o
Deparia conilii
Depariapetersonii
Deparialobatocrenata
O.1
expected chenges per site
FIG ,3. Phylogeneti ctree based on the combined datas eutsinga Bayesian analysis, Measures ofsupport are given at the nodes: NJ
bootstra p{BS)/M Pbootstra p(BS)/Bayes pioastnerio rprobabiliti (esPP) S.upport values under 50 are shown as hyphens (-).
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Analysi sof'puta thi)v,ebr iandd s iijpbr sipdecies The results show that Athyrium is paraphyletic
We include the fo11owing three putative and that the Atfp,rium-Cornopteris complex com-
hybrids and two hybrid species ofAthyrium in prises fiy emajor clades (I I,I ,Ill ,IM and V).
the dataset of chloroplast phylogeneti canalysis: Clade I consists of three species in which
A. Xakiense (A .eremicola × A.otophorum), Athyrium niponicum is the sister taxon to A,
A, × tokashikii (A .wardii × A. ciivicola), A. sheareri andA. cuspiclatum. The placement ofthis
× hisatsuanum (A .iseanum × A. clivicola), A. clade as the most basal in the Athyrium phyloge-
obliteseens (A .otophorum × A. waidii and A. netic tree agrees with the results of previous stud-
otophororm × A. clivicola), andA. setuligerurn (A. ies (Sano et al. 2000, Wting et al. 2003). Atto,rium
clivicola × A, iseanum). Thc chloroplast DNA sheareri and A, cuspidatum were formerl ytreated
sequence of each plant was identica lto that of as members of distinc tgenera ,Anisocampium
either of hypothesize dparents. PCR-SSCP of the and Kuniwatsukia, respectively, showing that the
nuclear single-copy gene (Ilgi fQor these hybrids clade is a morphologically divers egroup. In com-
and taxa of hybrid origin also supported their parison with other species ofAtltyrium, Atlryrium
hypothesized (Fig4.).For has
parentage example, sheareri an unusual combination ofmorpho-
A. × akiense (lan e11, Fig, 4) showed overlap- logical characters, includin ga long creeping rhi-
ping band pattem ofA, eremicola (la n1e) and A. zome, pinnat efronds with a chartaceous texture,
otophorum (lan 2e), suggesting that the hypoth- and orbicular sori (San oet al, 2000). A creeping
is inA, Atltyrium
esized parentage was correct, rhizome also present niponicum.
however,has
cuspiclatum, an erect or ascending
Discussion rhizome, Although this clade is strongly support-
ed, no synapomorphic morphological characters
Phylogenetic re]ationships in Atlryrium and are known.
Cornopteri swere deduced from two chloroplast Our rbcL tree shows that Athyrium dis-
DNA fragments ,rbcL and trnL 5'exon to trnF. tentijblium and Cornopteri sare monophyletic,
valt///utee
m ･- ,,,.".....,..mu asremW e c ･･ ･･ ･--epte･-

･
.･v.- ,/,ww."
m,-
i' .
g,w' ee
w urwW '" tw /-n .pt
ma maW ec} w . w w
･

/tt ,
1ss 23456 78910 111213 14 t5

FIG. 4. PCR-SSCP band patter nofpanial IlgiC gene. Lane (1 )Atdyrium eremicola, (2 ,3) A. otophorum, (4 )A, wardii, (S ,6)
A. elivicola, (7 )A, iseanum, (8 ,9) A, viclalii, (10 )A. cielteidojrrons, (11 )A. Xakiense [A .e,emicola × A. otophorum], (12)
A. Xtokasikii [A .ivaniii × A, clivicola], (13 )A. ><hisatsuanum [A ,iseanum × A. clivicota], (14 )A. obtitescens [A,otopho-
rum x A. clivicola or A. watdii],(15) A. setuligenim [A .ctivicota × A. iseanum].
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although the phylogeneti crelationship is not yet nopteris is a small Asian genus with nine species,
understood (Fi g1.). Atbyrium distentijbl ihuansi define dby the comiculate base of the pinnae and
a worldwide disnibutio annd is characterized by pinnule sand the exindusiate seri (Kat o1979),
circular to elliptic sori, with irregula frilaments while it sother morphological characters are simi-
making up a rudimentary indusium when young, lar to these ofAtlryrium, Clade II ,therefore ,can
but soon obscured as the sori grow (Iwatsu ekt i be recognized as a group with by the synapomor-
aL 1992, McHaffie 2005). In comparison, Cor- phic characteristic of exindusiate sori on mature
Kurata ,Serizawa and lagawa's Present study Wang's (t997 c)lassification
classification
Athyrium otophorumgroup (Kura t19a61) clade Vb section Otophora
A. otophotum A. otophorum subsec. Otophone
AAA... sveuriberrmiiidgceeoslscaceen nsti pa s - AAA･.. sveuitbr?timeedSseccsoeclnseantlpes - AAA... scouftbiorvipgihe'oscrcoevnlmsa
A. cfiw'cola A. warttii
A. -ferdii A. kuratae
A. oblitescens A. arisenense
A. setuligerum A. kenzo-satakei
A. A, kuratae

subsec. Strigiitosa
clade Ve A. iseanum
A. videtii A. strigiliosum
A. AA.. ncetgeiletcotludmbhans subsp. austtaie A . Ai.s ecatnyputmog rvaamrm.oi adnegsustisectum ±
A, sitvicoia
other taxa in cladeV
A. iseanum section Polystichoides
A. iseanum van angustisectu A. nakanoj
A. sntgi"osum
A. tVengulum s ctien Mackinnoniana
Atozanense A. deitoldottons
A. arisenense A. videM
A. A. nakanei A. refiexipinnum

clade IV section Niponica
AA.. yyookkoosscceennssee van dilatatum / AA.. yniokkkoosecnseense
A. tashiroi A. nlponicum
A. ldrisimeense
A. A. nikeeense seetionAthyn'um
A. brevN?vns
clade 11l A. melanolapis
A. bevifrons
A. pinetorum A. melanelepls
A. palust re A. tilix-femina
ciadel
A. nlponicum
A. cuspidatum
A. sheareri
unsettled taxa
FIG. 5, Comparison ofthe previou cslassification (speci ecosmmonly found in Japan were sc]ected) with the one proposed in the
present study, Taxa in boldface represent species examined in this study. The lightL yshaded boxes show the correspondence
between previous taxonomic groups and clades in our phylogenetic tree. The darkl yshaded boxes show species with
uncertainpositions,
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88 ActaPhytotax,Geobot, va)1.59

leaves. intrageneric subgroups based on morphological
The nested positio nof Cornopteri sin the tree characters ([laga w19a33, Kurata 1961, Serizawa
ofAtbyrium was suggested by previous molecular 1981, Wang 1997). Figure 5 illustra ttehse cor-
analyses (San oet al, 2000, Wang et al. 2003) ,and respondence of the morphological groups with
confirmed in the presen tstudy based on a wider the clades resolved in this study. Kurata (1961)
taxon sampling and longer DNA sequences, Some considered that the A. otophorum group, the A.
hybrid taxa between Atlryriu mand Cornopteris viddlii group, and the A. iseanum group were
have been proposed (Kura t1a963, 1965, Hira- closcly related to onc another, based on the fre-
bayashi 1970, Nakaike 1992) .Serizawa (1981b)quent hybridizati oanrnong them. Our phyloge-
treated Cornopteris as a subgenus ofAthyrium netic analyses support that opinion because all
based on the occurrence of these hybrids, These members of the three groups examined in this
observations are concordant with the close alli- study are include din clade V Furthemnore ,clade
ance between Atiryrium and Cornqpteris in our V also contains A, deltoidqfro nosf the A. .fitix-
analysis, In this study we sampled two specimens group sensu Kurata (1961 )In.terestingly,
.fiimina
of C. christenseniana. Park & Kato (2003 )con- all members of clade V are polyplojd s(4 xor 6x)
firmed this taxon to be an interspecifi ctriploid except A. fhangulu fm. viride, which contrasts
hybrid of diploi dC. crenulatoserrulata and tetra- with the findin gthat A. atkinsonii, A. rupestre,
ploid C decurrenti-alata .One of our samples of and members of clades III and IV are a]1 diploids
C. christenseniana was grouped with C. crenu- (Takamiy a1996). Speciatio nat the polyploid
latoserrula taand another was grouped with C leve lappears to be unlikely due to the existence
decurrenti-at a(tFai g3.) ,confirming the recipro- ofA. .frangulum f viride, but any selective pres-
ca] occurrence of hybridizati oans suggested by sure for the success of polyploids may be related
Park (2002). to the evolution ofclade M
To prevent a paraphyletic Athyrium, Cor- Among the morphologically define dgroups
nqpteris should be include dwithin Atlpirium o,r (Fig 5.), the Atbyrium otophorum group sensu
clades I and II should be treated as genera sepa- Kurata (l961 a)nd section Otqphora subsection
rate from Atdyrium. If the latte rt,hen additional Otqphora sensu Wang (1997 c)ould be referred to
studies are desireabl eto clarify the delimitati oofn as clade Vb. Atiryrium arisanense of the A. oto-
cladesI and II .First t,he type species of the genus phorum group is include din clade Vl but it sposi-
Anisocampium, A. cumingianum C. Presl was not tion varies among DNA dataset sT,he Atbyrium
include din the presen tstudy, Because A. cuming- vidolii group sensu Kurata (1961 w)as divided
ianum is characterized by goniopteroi vdenation, into two clades: A. wardii and A. clivicola were
which is not shared by A. sheafieri, the addition include din clade Vb with the members of the A.
ofA. cumingianum to clade l should be tested, otophorum group ,and A. viclalii was include din
Secondly ,Wang (1997 r)eported that three Chi- clade Vii with A. deltoidoLfro nosfthe A. filix:femi-
nese species ofAthyTium, A. wallichianum, A. dis- na group, In cornparison, Wang (1997 c)lassified
sitij?)lium and A. exindusiatum, have exindusiate A. wan:iii and A, clivicola in sect. Otophora sub-
sori. Their phylogeneti rcelationship to A. disten- sect. Otqphora, and classified A, viclalii in sect.
tijblium and Cornopteri sis critical fbr diagnosing Mackinnoniana ,which include sA. deltoicloLfirons.
clade II. Our phylogeneti acnalysis supports Wang's (1997)
The remainder of the members ofAtbyrium system with respect to this group of species,
form a monophyletic clade and contain several The Atbyrium iseanum group sensu Serizawa
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