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ISSN 1346-756S Acta Phytotax .Geebot.59 (2):165-1 (724008)
Originof the Endangered TetraploidAdonis ramosa
(RanunculaceAasese)ssed Chloroplast NuclearDNA
with and
Sequence Data
SHINGO KANEKOi', NOBUKAZU NAKAGOSHIiand YUJIISAGI2
['Graduate Schoolfor lnternational Development and Cooperation, Ifiroshima Uitivensit )1L-5-1 Kbgamtyama,
Higah'hi-Hiroshima 739-8529, .lapan ;2LahoratoT:v ofFore sBitology Division oflq?] anndBriomsatterials St'ience,
GraduateSt]hooolj'Agricul1t<uyorteoC,lniversiKoiltashirakawaOiwake-cho.SkekJ.'o-1k<uv,oto606-8502,lapan
Polyploidizati oins an impertan tevolutionary force in plant speciation, and elucidating the erigin
and evolutionary histor yof a particular polyploid is important for understanding the evolution
and distributi oofn plants .We investigat etdhe origin of the endangered tetraploid Adonis ramosa
Franch. ,which is endemic to Japan .Tb clarify the origin and phylogeneti crelationships among A.
ramosa and diploi dspecies, we conducted a phylogeneti canalysis of the species ofAdbnis in Japan
and Korea using chloroplast trnL- trnF spacer and nuclear ribosomal interna ltranscribed spacer
(ITS s)equences. The sequences of the ITS region of showed that the sequences ofA. ramosa were
completely consistent with or quite similar to those ofA. amunensis Regel & Radde of Hokkaido,
Japan ,and clearLy differe dfrom those ofA. amurensis in Korea and three diploi dspecies from
Japan and Korea. The resuLts suggest the possibility that A. ramosa is an autotetraploid, and the
diploi dprogenito ris A. amunensis from Hokkaido. The distributi poantter nof the ITS haplotyp eef
A. ramosa also indicate rsange expansion ofA. ramosa from northern to southern Japan Ecological
divergenc eand adaptation to new habitats after polyploidizati aorne likely to increase the survival of
A. ramosa and enable it to widely colonize in new environments in Japan,
Key words: Adonis ramosa, divergence F,ar East Asia,intern atlranscribed spacer (ITS),
polyploid yt,rnL-trnF spacerecological
Polyploidizatio nis an importan tevolutionary origin and evolutionary histor yof a particular
fbrce in plant speciation (Solt &is Solti s1999). polyploid is importan tfbr understanding the evo-
Polyploids often diffe frrom their diploi dprogeni- lutio nand distributi oofn the species,
tors in morphological and ecological aspects, and Adonis rainosa Franch. is a tetraploid (2 n=
neopolyploids may replace their diploi dprogeni- 32) endemic to Japan .It is distribut eacdross the
tors and colonize ecological niches unsuitable island sof Hokkaido, Honshu, and Shikoku (Fig,
for their diploi dprogenitors because of superior 1) and is the most common species ofAdonis
fitnes s(Levi n2002). Therefore ,elucidating the in Japan, Although A. ramosa was believed to
'Present address: Laboratory of Forest Biology, Diyision of Forest and Biornateria lSseience ,Graduate School ofAgriculture,
Kyoto UniversityK,itashirakawaOiwake-cho,Sakyo-ku,Kyoto 606-8502,Japan
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166 Acta Phytotax ,Geobot. Nbl. S9
ts Hokkaido b 11
o
2
4g'N
t
`
v
sis
ts ts
a v
or;a sis
FiG. 1, Distributio onf four species ofAdonis in Japan (modifi ferdom Kawano & Hayashi 2004). The numbers
indicat csampling location sshown in Table 1 .
be the only species of Adonis native to Japan kawa & KQji Ito (Nishika w19a89a) ,and A, shi-
(Sata keet al. 1982, Ohwi & Kitagawa 1983), kokuensis Nishikawa & Koji Ito (Nishjka w&a
Ito2001),Aclonis isfbundin
recent morphological studies and chromosome amurensis northern
analysis (Nishika w19a88, 1989a, b, Nishikawa and eastern Hokkaido, and is widely distributed
& Ito 2001) have shown that Adonis in Japan around the Japanese Archipelago, for example in
consists of four species: A. ramosa Franch., A, Far East Russia ,includin Sgakhali nand the Kurile
amunensis Regel & Radde, A. muttijiora Nishi- islands ,northeastern China, and the Korean pen-
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insula (Hoffman n1998) ,Adonis multtfibra occurs Gold (Appli eBdiosystems ,Foster City ,Califor-
on Honshu and Kyushu in Japan and on Cheju nia, USA). The PCR amplification was perfbrmed
Island in Korea (Kawan o& Hayashi 2004, Suh et in a thermal cycler (GeneAm pPCR System 2700,
al. 2002) .Adbnis shikokuensis has been reported Applied Biosystems) under the fo11owin gcondi-
from Shikoku and Kyushu in Japan (Nishika &wa tions: fbr the trnL-trnF region, initi adlenatur-
Ito 2001). ation at 940C for 9 min was fo11owed by 30 cycles
Four of five species ofAdbnis in Far East of denaturatio nat 940C fbr 1 min, annealing at
Asia, includin Ag. amunensis. A, multijlora, A, shi- 600C fbr 1 min, and extension at 720C for 2 min,
kokuensi sa,nd A. pseudoamurensis Wang (Wlingand fina lextension at 720C fbr 7 min; and for
1980) from China and Korea, are diploi d(2n = the ITS region, initi adlenaturat iato n940C fbr 9
16; Nishikawa 1988, Ahn et al. 1999, Nishikawa min was fbllowe dby 25 cycles of denaturation at
& Ito 2001); Adonis ramosa is tetraploid (2n = 940C for1 min, annealing at 55eC for 30 s, and
32). Adonis ramosa is therefore likel yto have extension at 720C fbr 1 min, and fina lextension at
oTiginated from one or two of the dipleid spe- 720C fbr 7 min. The PCR products were purified
cies. Even though A, ramosa has been known fbr using a High Pure PCR Product Purificati oKnit
several decades to be a tetrap)oi d(Nishika &wa (Roche Applied Science ,Penzberg, Germany),
Ito 1978), litt lienformation about the origin of The purifie dproduct swere sequenced directly
A, ramosa has been reported, and there has been with an ABI BigDye Tbrrninato rCycle Sequenc-
insuflici eenvtidence to pinpoin tit sorigin. In this ing Kit ver, 3,1 (Appli eBidosysterns) on the ABI
report, we presen tinfbrrnati oonn the origin ofA, PRISM 31OO Geneti cAna]yzer (Applie Bdiosys-
ramosa using the trnL-trnF chleroplast spacer tems), Electropherogram swere assembled with
and the interna ]transcribed spacer (ITS )of the Sequencher 3,1 software (GeneCode Asnn, Arbor,
nuclear DNA sequence data ofAdonis in Japan, Michigan, USA), and sequences were aligned
and publis hITS sequence data fbr the species of using CLUSTAL X (Thompso ent aL 1997).
Atloni sin Korea. We determine dthe trnL-trnF and ITS haplo-
types based on site change data and indels I.n the
Materials and Methods trnL-trn]F' region, we compared results between
haplotypes ofAdonis ramosa and three other spe-
We collected leaf samples from 14 popula- cies ofAdonis (A .amurensis, A. multijlora, and
tions of four species ofAdonis in Japan (TabteA, shikokuensis) from Japan. For the ITS region,
1). Plant materials were kept fresh on ice after the sequence data of three species ofAdonis (A,
collection in the fiel dT.he samples were frozen amurensis, A. mblltij7bra, and A, pseudoamurensis
to -300C in the laborator uyntil DNA extraction. Wang) from Korea and A, vernalis L, from Hun-
Genomic DNA was extracted from leave susing a gary (DDBJ DNA data base accession numbers
modified CTAB method (Milli g1a99n2). AF454924 to AF454936, Suh et al. 2002) were
The non-coding regions of the chloroplast also used for analysis. Based on the sequence
DNA between the trnL(UAA) 3 'exon and the trnF data, phylogenetic analyses were performed
(GAA) (Taber lete atl. 1991) and the interna tlran- using maximum parsimony methods. The mest
scribed spacer (ITS )of nuclear ribosomal DNA parsimonious trees were obtained with PAUP 4.0
between ITS5 and ITS4 (Whit eet at. 1990) were (Swoffb r2d00i) using heuristi csearches with
amplified by PCR and then sequenced. We ampli- 100 random taxon addition replicates and TBR
fied the ITS and trnL-trnF regions with impliTaq swapping. Gaps were excluded and site changes
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TABLE 1.ITS and cpDNA variation ofAdonis in Japan and Kerea.
Haplotypea AccessionNo.
Species ,No. and locality
trnL-FITS trnL-F ITS
Adonis ramosa
1 Urakawa,HoltkaidoPrefecture,japan AAAAAAAAAAAAAAAB AB361596 AB361610
2 Furano, Holclcai dPorefectur eJ,apan AB361597 AB361611
3 Hachinohe,Aomori Prefecture,Japan AB361598 AB361612
4 Fajisato,AkitaPrefecture,Japan AB361599 AB361613
5 Ybnczawa,YamagataPrefecture,Japan AB361600 AB361614
6 Tadami,FukushimaPrefecture,Japan AB361601 AB361615
7 Ina,NaganoPrefecture,Japan (IID)B(IID)B AB361602 AB361616
8 Asahi,GifuPrefecture,Japan AB361603 AB361617
9 Mt.Fuiiwara,MiePrefecture,Japan (IID) AB361604 AB361618
Adonis
amurensis
1O Nemuro, Hokkaido Prefectur eJ,apan AA AAC AB361605 AB361619
11 Shari,HokkaidoPrefecture,Japan AB361606 AB361620
12 Mt. Jeoksang, Chonbuk Province, Korea (10NS 2,1D) AF454927
13 Mt.Chookryung,KyunggiProvince,Korea D(1ONS) AF454928
14 Daekwanryuung , Kyungwon Province ,KDrca C (1 ONS, 2ID) AF454929
Adonis multCt7bra
15 Kawai,GifuPrefecture,Japan B (2NS)I (15NS I,ID) AB361607 AB361621
16 Shoubara,HireshimaPrefecture,Japan B (2NS)I (15NS ,IID) AB361608 AB361622
17 Mt.Halla,ChejuIsland,Korea J(15NS) AF454924
18 Mt,Halla,ChejuIsland,Korea E (14NS) AF454925
19 Sangumbri, Cheju Island, Korea E (14NS) AF454926
Adonis shikokuensis
20 Ootoyo,KouchiPrefecture,Japan B (2NS)E(14NS) AB361609 AB361623
Adbnispseudoamunensis
21 Euyung,KyungamProvinee,Korea F(15NS) AF454930
22 Kwanchon,ChunbukProvince,Korea E(14NS) AF454931
23 Mt.Palgong,KyungbukProvince,Korea E(14NS) AF454932
24 Mt. Kyeryong, Choongnam Province ,Korea G ( 15NS) AF454933
25 Chollipo, Choongnam Province ,Korea G ( 15NS) AF454934
26 JangbongIsland,KyunggiProvince,Korea H(14NS) AF454935
Adonis vernalis (outgroup)
27 Mt.Cserhat,Hungary Out (20N S) AF454936
a The number ofvariable sites cempared to haplotype A is shown in parentheses ;NS, Nuclcotide substitution; ID, Insertienl
Deletion. A dash ( -) lndicat tehsa tthe nucleotide sequence of the region has not determined.
were weighted equally in the analysis. The phylo- Results
geneti ctree was rooted by the outgroup compari-
sen. A strict consensus tree was constmcted from In the trnL-trnF region (29 9bp), sequences
the most parsimonjous trees ,and bootstra vpalues of Adonis ramosa were completely consistent
were estimated with l,OOO replicates using the with those ofA. amurensis in Japan ,but difTered
heurist isecarch option, from A, multijlora and A. shikokuensis (Tabl e1).
In the ITS region (603bp )we, detected 36 variable
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( TypeA(AA'.a.M."re,,",SSSpJNPN)
TYpeB(A.ramosa JPN)
TypeC <A, KOR)
amuiensis
TypeD (Aa,murensis KOR)
T y p e E IUA:,pgse?uchlo"am iKuOrxRe X'ntsits6(IP,e,5i25pN
TYpeF (Ap,seudoamurenKsOiRs)
TYpeG (Ap.seudbamurensKiOsR)
"fype
H (A, KOR)
pseudoamurensis
TYpe1(Am,uttifiorJaPN>
TYpeJ<A.muftifioraKOR)
OUT(A,
verr7alis)
(
ti
doIsland
a
Hon$hulsland
n
lsland
o e
.'
500km
'
V
""D
F[G. 2, (A) Thc strict conscnsus tree ofthe four most parsimonio utsrees arnong the ITS haplotype sof species ofAdonis using
site changes, Numbers below branches are bootstra pvaLues in percentages based on 1,eOO replicates. Solid and open bars
represent site changes and indels ,respectively, (B) Distribution ofhaplotypcs cornprising Clade I .Letter sindicat ehaplotypes
(solid (open
ofA. amurensis circle) andA. ramosa circle),
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sites includin g17 phylogenetically infbrmative polyploid origin based only on ITS sequence data,
sites. In the phylogeny often haplotypes based on ITS sequence analysis in many allopolyploid
ITS sequences of five species ofAdonis, we iden- taxa, however, have shown that both parental ITS
tified two distinc tclades (Fi g2,A). Clade I was sequence or chimeric (mosai lcike) ITS repeat
composed ofA. amurensis and A. ramosa; Clade types of parental motifs as overlapping double
Il was composed ofA. multij7bra, A, shikokuensis, peaks (Kim & Jansen 1994, O'Kane et aL l996,
and A. pseudbamurensis .ITS Sequences fbr the Franzke & Mummenhoff 1999, Vargas et at,
nine populations ofA. ramosa were completely 1999, Gaut et al. 2000, Hughes et aL 2002, Wich-
consistent or quit esimilar to those ofA. amunen- man et at, 2002, Koch et al. 2003, Lihova et aL
sis in Japan ,but c]early differe fdrom those ofA. 2004, Marhold et ai. 2004, Rauscher et al. 2004,
{nKorea (Tabl1e,Fig.2A).In Devos 2006).The lack intraindividual
amurensis addi- et at. ef
tion, intraindividu asilngle-site polyrnorphisms polymorphisms in the A. ramosa sequences there-
as overlapping double peaks were not detecte din fbre suggests the pessibilit ythat A. raTnosa is an
the ITS region for all A, ramosa samples. Adonis autotetraploid, and that the diploid progenito ris
ramosa from Hokkaido and northern Honshu A. amurensis from Hokkaido.
(Nos ,1-6) shewed identica lsequences with those Autopolyploi dplant swere once considered
ofA. amurensis from Hokkaido (Typ eA), A. extremely rare in nature. They usually repro-
from Honshu(Nos7,-9) duce by
ramosa central showed a asexually, either vegetative propagation
derivati vaend specific haplotyp e(rl} JB)p, ewhich or apomixis, because of difficult iiens meiosis
was distinguish efdrom Type A haplotyp eby one (Futuym a1986). Recent studies from various
inde l(Tabl e1, Fig, 2B) These results showed that angiosperm lineages ,however, have indicated
A, amurensis is phylogenetical lthye closest dip- that autopolyploidy is much more common than
loi dspecies toA. ramosa, and furthermor et,hat A. once considered (Solt eit saL 2004), and sexual
amurensis from Hokkaido is closer to A. ramosa autotetraploidy has been reported in several taxa,
than to A. amurensis from Korea with haplotypes includin gChamerion angustijblium (repor tased
C and D. EPilobiu mangustijblium; Onagraceae; Husband
& Schemske 1997),Asterkantoensis(Asteraceae;
Discussion Inoue 1998), Ranunculus
et al. cassubic4folius
(RanunculaceHa6era;ndl & Greilhuber2002),
In the trnL-trnF region of the chloroplast and Campanula americana (Campanulaceae;
DNA, the sequences ofAdonis ramosa were Galloway et al. 2003), Adonis rcrmosa produces
completely consistent with those ofA. amurensis ferti lseeeds. Hand-pollination experiments have
in Japan, but differe dfrom A, multijiora and A. shown that seed-set is increase dby outcrossing
shikokuensis, indicatin tghat the maternal parent (Kudo 1995). These observations indicat esexual
(cytopla sdmoinocr) ofA. ramosa is A. amurensis. reproduction in A. ramosa, which is likel yto be a
In the ITS region of the nuclear DNA, sequences sexual autotetraploid.
ofA. ramosa were identic aolr quite similar to ITS sequences ofAdonis amurensis in Japan
those ofA. amurensis in Japan .In the ITS region, and Korea showed high intraspeci fviarciability.
examples of concerted evolution which has com- The ITS sequences ofA. amurensis from Hok-
pletel yerased traces of other possibl eparents kaido were identic aorl quite similar to those ofA.
were reported (Wende let al, 1995, Truyens et aL ramosa, suggesting that A. ramosa was derived
2005). It is therefore undesirable to determine from A. amurensis, now restricted to Hokkaido,
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July 2008 KANEKe & AL.: Origin of fetraploi Acdlonis ramosa 171
by autopolyploidization. The high similarity ofthe the result of ecological divergence after poly-
ITS sequences amongA. ramosa andA. amurensis ploidization .For example, ecological differences
in Japan suggests that speciation occurred recent- between Adonis ratnosa and A. atnurensis are
ly .Although intraspeci fyairciability in A. ramosa eyident in their seed production and habitats.
was low and showed only two ITS hapletypes, Because of the larger number of flowers on A.
distribut ipoantterns of the haplotyp eare likel yto ramosa compared to A. amurensis (Nishikaw &a
indicat erange expansion after polyploidization,Ito 2001), seed prQductio nin A, ramosa is greater
The ITS sequences ofA. ramosa from Hokkaido than in A. amurensis (Nishika 1w9a88, Kaneko et
and northern Honshu were consistent with those aL 2005), With regard to habitats ,A. amurensis
inHokkaido (TypeA),butA, inbroadleaveddecid-
ofA. amttrensis grows on grassy slopes and
from Honshu derivative forest(sNishika&waKadota2006,Kaneko
ramosa central showed a uous
and specific haplotype ofA. ramosa ([IY pBe; Fig. pers .obs.). Adbnis ramosa also occurs in these
2B). It is therefbre likel ythat the range expansion habita tons Hoklcaido ,but on Honshu the char-
ofA. ramosa was as fbllows A,donis ratnosa with acteristi¢ habitat osfA. ramosa are grassy banks
the [[Yp eA haplotyp ecolonized southern Hokkai- of terrace fields m,eadows, and clearcut slopes
de and northern Honshu firs tf,bllowed by a one around houses. Populations occurring in broad-
indel mutation in the proces sof range expansion leaved deciduous fbrest sare uncommon (Kaneko
int onorthern and central Honshu. Adonis ramosa pers .ebs.). It appears that adaptation te environ-
with the Type B haplotyp ethen spread in central ments created by humans is a distin cfteatur oefA.
Honshu. ramosa.
Tetraploid Adonis ramosa clearly ranges The human impact of grassland manage-
more widely than does it sdiploi dprogenitor A, ment in rural environments affects the survival of
amurensis in Japan (Fig. 1L)ik.e many allotetra- Adonis ramosa, especially on Honshu. On Hon-
ploids a,utopQlyploids are not always more vigor- shu, grassland management has been conducted
ous than their progenito r(sRieseb e&r Egllstrand fbr at leas t10,OOO years (Yamano 1i996, Ogura
1993, Rieseberg l995, Solti s& Solti s1995, 20e2), and grassland smanaged by humans are
Song et al. 1995). However, cornbined analysis likel yto provide habitat fbr A, ramosa. Changes
in however,have
of ecological niche modelling and molecular methods of agricultural, caused
phylogeography fOr autotetraploid and diploid a massive reduction in traditionally managed
Hordeum gussoneanum (Poacea esh)ows that grassland ssince the 1960s, In recent years, popu-
autopolyploids dominate ecological niches that lation sofA. ramosa have declined through habitat
are unsuitable for their diploi dprogenitors (Jakobloss because of the abandonment of traditionally
et al. 2007). A similar differenc ien distributionmanaged grasslands .Acloni sramosa is now c]as-
among autopolyploids and diploi dprogenitors sified as "vulnerable" in the Japanese Red Data
foundin diploids Bis- Book (EnvironmeAgnetncy 2000),
was autotetraploids and of ofJapan
cuteUa laevigat a(Brassicace aien )the western Our analysis using molecular matkers shows
Alps (Tremetsbe r20g0e2)r, and the autotetraploid that Adonis ramosa is derived from one lineage
7lrraxacu mvenustum (Asterace aaend) diploi dZ of A. amurensis, now distribut eodn Hokkaido,
platycai:pum subsp. hondoense in Far East Asia by autopolyploidization, Ecological divergence
(Mori t1a976, Akhter et aL 1993). and adaptation to new habitat asfter polyploidiza-
These difference sin distributi obnetween tion likely increased the survival ofA. ramosa in
polyploid sand their djploi dprogenitors may be japan and enabled it to colonize rural enyiron-
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172 Acta Phytotax, Geobot. Vbl. 59
ments. Rapid environmental change and habitat geneti cdivcrsit aymong mernbers of the filestuca-
alteration in recent years may have endangered Lolium complcx (Poacea bea)scd on ITS sequence
data.PlantSyst,EvoJ.224:33-53.
A. ramosa. The present finding sare importan tfbr
Hoffman,H. M. 1998.Ecogeographicaldifferentiation
understanding the evolutionary history ofAdonis, patt crn sin Adoni s sect, Consilig o (Ranuncula-
basicknowledge long-terrn
and provide on eco- ceae), Pl .Syst .and Evol. 2ll: 85-100,
logica clonservation ofA, ramosa. H6randl, E, & J .Greilhube r2.002. Diploid and autotet-
raploid sexuals and their relationships to apomicts
We thank S. Gotq H. Ida ,K. Ishida, and A, Uchida for in the Ranunculus cassubicus group: insight fsrom
their support of this research, This study was partly DNA content and isozyme variation, Pl. Syst.
supported by a Grant-in-Ai dfbr scientific research from Evol, 234: 85-100.
the Ministr yof Education ,Culture ,Sports ,Science and Hughes, C. E., C. D. Bailey & S. A. Harris .2002.
Technology ofJapan, the Ministry ofAgriculture, For- Divergent and reticulate species reLationships
estry and Fisherie osf Japan ,and the 2 1 S` Century Center in Leucaena (Fabaceae i)nferre dfrem multiple
data insightisnto
ofExcel]ence Program at Hiroshima University. sources: polyploidy origins and
nrDNA polymorphism. Arner ,J. Bot. 89i 1057-
1073,
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