Intraspecific Chloroplast DNA Variations of the Alpine Plants in Japan

2005·0.85 MB·English

by  Senni, Kei

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ISSN1346-7565 Acta Phytotax. Geobet, 56 (3) :265-275 (2005)
ShortCommunication

Intraspecifi cChloroplas tDNA Variationsof the Alpine Plants
inJapan

KEI SENNIi,NORIYUKI FUJIIi,HIDEKI TAKAHASHI2,

TAKASHI SUGAWARAi and MICHIO WAKABAYASHIi
'imkino Hbrbarium,GraduateSchoolofScienc7bed,yo MettopolitanUriivenyiJc-}1lMlnami-Osawa, Hachicv'i,

7blyo JP2-039Z .ku)an; 2The Hblakaid oUhiversit Myuseum, Sapporo 060-08iO, .k4pan
[I bobtain more inforrnati oonn the geneti cdi fferentiat iofoJanpanese alpine plant si,ntraspeci fvairic-
ations ofchloroplast DNA (cpDNA o)f23 species were investigat Weed, examined the samples frern the
populations in Centra lHonshu, Tohoku, and Hokkaido, Japan; and in Sakhalin ,Russia. In thc
nucleotide sequence of two non-coding Tegions (trnl:trnL re-gitonrn aFnd t7?l16 intron) of cpDNA, the
intraspec ivfariiactions were detecte idn all species, except Cbuua scita. The species with a relatively high
(includiinndgels) Anemone (O.909A/r6e)n,aria
proportio nof mutational events were nanrissijiora
aretica (O.74% G)e,um calthijblium (O,86% I)(,J/peric ukmamtsehaticu m(1.04% P)l,edicularisyezoen-
sis (1 , 1 8%), and 7lolli ursieclerianus (O.77%).
Key words: chloroplast DNA, haplotype i,ntraspeci vfairication, Japanese alpinc plants p,hylogeog]'a-
phy, rpII6 intron ,tm7LtrnL-trnFregion
!n recent years, the intraspeci fviacriations of of the phylogenetic analysis of the haplotype sin
cpDNA have been frequent lusyed fbr inferri nthge Rediculari schamissonis Steven (Foj eit ial, 1997)
evolutionary histor oyr the past migratien ofplants and Primula cuneijblia Ledeb. (FLi jeti ail, 1995,
(Avi s2e000) .In case of Japanese plant ss,everal 1999) ,Furthermore ,these clades have clearly shown
phylegeographic satuldies using cpDNA vanations the phylogeographica sltructure, and the clade
have been perfbrmed (Abie sTs,umura & Ohba endemic to the Centra lHonshu was observed in
1994; Iblygonum, Inamura et al. 2000; Ilagu sF,iijiieach species. The structure was discusse dto be
et aL 2002; Aucuba and Stachyurus, Ohi et al. formed due to the glacia ladvances and retreats
2003a, b; Michetia ,Lu et aL 2002; llemerocallis,during the Pleistocene .In order to assess the gen-
Noguchi et al, 2004; the plant sof broad-leaved erality of the distribut ipoantter onf these clades, we
evergreen fbrest sAo,ki et al. 2003, 2004; euereus,must accumulate more data on cpDNA variations
Kanno et al, 2004), In Japanese alpine plant sg,eo- using other species. Fiij ieti al. (1996 i)nvestigated
graphic cpDNA variation among population hsas the intraspeci fcipDcNA variation of the other
been reported fbr seyeral species (Foj eit ial. 1995, Japanes ealpine plant susing 40 taxa; however ,the
1996, 1997, 1999), Two or three major cpDNA study has been limite dwith regard to the lengt hof
clades were identifi ien dJapan based on the results the nucleotide sequences of cpDNA (approximate-
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ly 300-400 bp) and the number ofpopulations ana-
lyzed .Therefbre ,in the present study, in order to
obtain furthe irnforrnati oonn the geneti cdifferen-
tiatio nof the alpine plant samong populations in
Japan, the intraspeci fcipDcNA variations were
examined,
We selected 23 species of the subalpine or
alpine plant stha tare commonly distribu tien dJapan
(Appendi Txh)e. uncommon or rare species were
avoided in order to obtain as many populatio nass
possible .The species includ ethe seven species on
which a prelimina ranyalysis was conducted in Ft!iii
et al. (1996 a)s, fo11ows A:renaria aretica (Caryo-
phyllaceae)C,Zimpanula lasiocailp(aCampanula-

ceae), Gentiana aigidu (Gentiana Gceeuam eca)lt,h-
ijbli u(mRosaceae )Pe,dicularis yezoensis
(ScrophulariacePaoete)n,tilla
matsumurae
(Rosace aaend) ,7}olliu sriederianus (Ranuncula-
ceae). A total of five to thineen populations from
Centra lHonshu, Ibheku, and Hokkaido districts
ofJqpan; and the Sakha]i nIslan dofRussia (Fi g1.,
and Appendix) were examined fbr all the 23 species.
During sampling, we collected relatively more pop-
ulations from Central Honshu because high haplo-
type diversi wtays reported in previous studies (Fiijii
et al. 1997, 1999) .A single individu aflrom each
populatio wnas analyzed. The taxonomy ofall these
species fbllowe dShimizu (1982 1,983), though
intraspeci tfaixca were not adopted in this study,
The leaves ofthe samples were dried and pre-
served in silica gel. The leave swere powdered
using a mixer mill (Vibrat Miiolnl type MM300, FiG. 1. The sample Iocation sfbr the presen tstudy.
Retsch ,Haan, Germany), and tota lgenomic DNA
was extracted from O,O1 g of the powder by using accessions. In Ap7iic aunalaschcensis (Asteraceae),
the slightly modified C[[AB method of Doyle & the non-coding regions between the trnT (UGU)
Dickson (198 o7r )a DNeasy Plant Mini Kit (Qiagenand trnL 3' exon (faber lete atl. 1991) ,and the
Inc. ,Hilden, Germany). The non-coding regions rpl16 intro nwere used. The non-coding regions
of qpDNA between the trnL (UAA) 5' exon and the between the trxT and trnF includin tghe trnL intron
trnF (GAA) ,includin tghe trnL intron ([fabe ertlet([labe retl ael,t 1991) and the rpl16 intro nwere
al. 1991) ,and the rpll6 intro n(Jord eatn aL 1996, used in the followin fgbur species: Cassiop elycopo-
Klechner & Clark 1997) were amplified using the dioide s(Ericace Gaeen)ti,ana aigido, Geum calth-
porymeras echain reaction (PCR )method in most 4fbli aunmd, fledicular iysezoensi (sAppendi Txh)e.
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PCR product sfbr direc tsequencing were excised from 09i 6(CZii sucirta (Cyperace aDei)ap,ensia iap-
from 1% agarose gels and purified using a ponica, and Mingia cv'anensis (Apiace atoe 1) ,)59%
Geneclea nIII Kit (Qbioge Innec. ,Carlsbad, CA, (Ilyper ihcamutmschaticu mi)n the tnnL-trnP r"egion,
USA), Sequencin greactions were canied out using and from O% (Artemi ssiinaanensis, Capmmine
aii ABI Prism BigDye terminato rcycle sequencing nipponica (Brassicac eCaateex) ,scita, Cassiope
ready reaction kit ver. 3.1 (App] iBeiodsystems lycopodio Liudzeuisa ,ancuata (Juncace aande),
Inc. F,oste rCity ,CA, USA). The sequencing reac- Rumex montanus (Polygonac teo a1e.)83)9 '(67itingta
tion preducts were purified c,oncentrated by EtOH ojanens iisn )the rpl16 intron region, The value of
precipitati oannd, then apptied to an ABI Prism the trnTLtrnL 3' exon in Arnica unalaschcensis was
3100-Avant automated DNA sequencer (AppliedO.489, 6T.he values of the trn T-trnF region varied
Biosystems). The nucleotide sequence data report- from O,46% (Geum caithijblium) to 1,489'6
ed in this paper will appear in the DDBJ, EMBL, (fedicul yaerzoiesnsis) ,We did not observe any
and GenBank DNA datal)a sunedser accession num- correlation between the proportio nofsmutational
bers AB219580 to AB219763. Alignment of the events ofthe trnL-trnF region and the ilpll6 intron.
sequences was done manually using the DNASIS- For example, in Cassiop elycopodio ialdtheousgh,
Mac program ver. 3.0 (Hita cSheiftwar eEngineer- the value of the trnL-trnF region was relatively
ing ,[[bkyo J,apan) T,he insertionsfdele (tiionndselsh)igh ,no intraspecif ivacriation was observed in the
were generaI Ipylace dso as to maximize the number ";pl16 intron ,On the other hand, in 7ilingi aqfa-
of matching nucleotides in the corresponding nensis, we observed the opposite patter nth;e varia-
sequences. Subsequent lwye, determine tdhe cpDNA tion in the rpl16 intro nwas relatively high, while
haplotype osfall the samples from site change and that of the trnL-F region was O%.
inde lcharacteristics using the combined cpDNA The proporti eofn mutational events combining
regions. Additional lweM estimated the proportio nof the sequences of trn7iL-F regions and the rpt16
mutational events of each region in the fbllowing intro nranged from O% to 1. 1 8% (Appendi Txh)e.
manner: the proportio onfmutational events = (NS species with relatively high values were Anemone
+ ID)IL, where NS = number of nucleotide substi- naFirissij7ora (O.90% )Ar,enari aatz]tica (O.749t6),
tutions ,ID = number of observed indel sa,nd L = Geum calthi.f'olium (O.86%) ,Ilypericum
sequence lengt h(O'Donn e1l9l92). kamtschaticum (1.04% )Pe,dicularis yezoensis
The non-coding region between the trnL 5' (1.18% )an,d 7lolliu sriederianus (O,889 Orn6 )th.c
exon and trnF varied from 628 bp Cllypericuomther hand, the species Artemisia sinanensis, Carex
kamtschaticu m(Clusiace taoe 1)0)16 bp (Carexscita, and Lttzul aarcuata indicat ead value of O. 1%
stenantha (Cyperace aien )l)ength ,whereas the or lower,
rpll6 intro nvaried from 752 bp (Gentia anigaido) Through the presen tinvestigation st,hree or
to 1338 bp (Diapen slaipaponic a(Diapensiacemoaree) h)aplotype swere observed in most species
in lengt h(Appendi xT)he. trn7LtrnL 3' exon in (Appendix F)o.r example, in Cassiop elycopodi-
Arnica unalaschcensis was 1039 bp in length T,he oides and Geum calthijbiium, alt populations ana-
trn7LtrnF region varied from }446 bp (Gentian alyzed in this study possessed differen ctpDNA hap-
aigida) to 1747 bp (Cassiq pleycqpodio iinde slo)type sA.dditional lay r,elatively greate nrumber of
length. haplotype wsere detecte idn the fo11owin sgpecies:
The (includinAngemone (9haplotypes)A,renaria
proponion ofmutational events narcissijlora
inde lcharacters) of each region was also variable arctica (7 )C,a,ziamin eniprpoinca (7 )D,iapensia
depending on taxa (Appendi Txh)e. values varied iapponic a(7 )G,entiana aigidu (8) L,oiseleuria
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proeumbens (8 )I,ledicula yreizsoensis (8 )i],btentilla References
matsumuvae (8) a,nd 7ilingi atu'anensis (7) O.n the
other hand, only one or two cpDNA haplotypes Aeki, K,, T, Suzuki & N. Murakami. 2003. Intraspecific
were observed in Artemisi sainanensis, C2ite srcita, sequence variation ofchloroplast DNA among the
and Luzula areuata, component species of evergreen broad-lcav efdorests
cu l a risAc cckaomrisdsionnisg to (thFe tpi reetj vaiil,io u1s9 9st7u)d itehse, p rion pPoerdtii-on iP, nh yJlaopgaen, o.JTg .,rP-alWpa.nh t yo fRHes tush. e 1c&o1mp6 o:n Ne3.nt3 7M-u3r 4askp4ea.cmicis, of 2b0r0o4a,d-
ofmutational events was 2.1% and 17 haplotypes leaved cvergreen fores tisn Japan, based on chloro-
were detecte dan;d in Pri'mul acuneij2)lia (Foj eit ail, plas tDNA variation. J. PIant Res. 1 17: 77-94.
1995, 1999) the proportion was 1,9% and eight Ayise ,J .C. 1994. Molecular markers, natural history
haplotyp ewesre recognized. ln the present studM the and evolution, Chapman & Hall ,New Ybrk.
values obtained fbr all these species were lower L. o2n0d0o0n. .Phylogeography H.arvard Universit Pyress,

than those of the two above-mentioned species. Doyle, J .J. & E. E. Dickson ,1987, Preservati oofnplant
Pedicularis yezoensis showed the highes tyalue samples fo rDNA restriction endonuclease analy-
(1,18 9an(d h t)he eight haplotype swere detected. sis. faxon 36: 715-722.
Howeveg we may be al)l teo clarify the phylogeneticFiiji iN,., K. Ueda, & T, Shimizu. 1996. Intraspecific
relationships among the cpDNA haplotype sby sequcnce variation ofchloroplastrlbx DeNnA in Japanese
alpine plants J.. Phytogeogr, 44: 72-8 1 .
adding appropriate outgroups in the above-men- Y INleita&noT,Shimizu,1995.Intraspecific
tioned species that have a high pToportion ofmuta- s, equence, variation i n c hloroplast DNA ofPrimula
tional events and many haplotypes, cuneijblia Ledeb. (Primulace aJ. eP)h.ytogeogt
As mentioned above, the geneti cdifferentiatio n Taxon43: I5-24,
between the populations of Central Honshu and , , & . 1997. Intraspeci fsieqcuence
nienrtPhterendi caurelaa r(i[s[bh oHokkulc,(aiFduo ,jetic.i) were19 i9n7f)erred vsoanriisat ioSnt eofvchelno r(opSlasctrop DhNAul ainr 1i la aadcnidec augelea)o rgcihrasamipsh-ic
chamissonis et aL and structuring ofthe Japanes e"alpinc" plants. J. Plant
Primula cuneijZ)lia (FLi ejt iali. 1995, 1999) .In the Res. 110:195-207.

presen tanalyses, among the populations of central , , & .1999. Further analysis of
Honshu and northern area, most species possessed intraspecif iscequence variation ofchloroplast DNA
differe ncptDNA haplotype se,xcluding Artemisia in Primuta cuneij2)lia Ledeb. (Primulac eiamepl)i:-
sinanensis, Ctitex scita, U)/peri chaummtschaticum, cations for biogeography of the Japanese alpine
floraJ.,PlantRes,112:87-95,
Luzula 1>rollius (Appen-
areuata, and riederianus N.Tbmaru,K.Okuyama, T,Koike,T.Mikami& K.
dix) ,A]though the present results are preliminary, ,U ed a. 2e02 . Ch loroplas DtN A phy lo geogra ph yof
these results might imply a similar situation as Flagu screnata (Fagace iane J)apan .Plant Syst .Evol.
observed with Pbdicuiari schamissonis and Primula 232:21-33.
Inamura,A.,YlOhashi,E.Sato,Y.YOda, T.Masuzawa,
cuneijbtia.
M. Ito ,& K. Ybshinaga 2.000. Intraspccif iscequence
We exprcss eur sincere thanks to A. Kitada and Y, variation ofchloroplast DNA reflecting varicty and
Koshizuka fbr their assistance with the laborator wyork. g(ePooglryagpohinca acdleiasitenr)Jiabpuat nio.ofJn P.bPllyagnotnRuems .cus1pild3ot:um419-
This study was partl ysupported by a Grant-in-Ai fdor 426.

Ybung Scientist(sA)G,rantNo. 15687003,The
collect-
Jordan,W, C,,M. W, Courtney& J.E,Neigel.1996.
ing ofsamples ofthe Sakhali nIslan din Russia was sup-
porte dby a Grant-in-Ai dfbr Scientif Riecsearch, Grant Low level sof intraspeci fgiecneti cvariation at a
No. 13575008(toH. Takahashi). rapidly evolving chloroplast DNA locus in North
Americandnckweeds(LemmaceaAeme)r..J.Bot.83:

430-439.
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Kanno, M., J .Ybkoyama, Y Suyama, M. Ohyama, T. ascomycete Fuasarium sambucinum (Gibberellaputi-
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of two haplotype sof chloroplast DNA in fbur oak Ohi, T, ,T, Kajita, & J. Murata. 2003a. Distinc tgeo-
species (2uereu isn )Japan. J. Plant Res. 117: 311- graphi cstructure as evidenced by chloroplast DNA
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and phylogenetic utility of the chloroplast rpl16 , M. Wakabayashi, S, Wu, & J. Murata. 2003b.
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Phy]ogenet E.vol. 8i 385-397, ceae) in the Japanes eArchipelago based on chloro-
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Wu, & T.-Yl Chiang .2002. Genetic variation and Shimizu ,T. 1982, The new alpine flor aofJapan in color
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(Magnoliace baaes)ed on cpDNA variation and . 19g3. 1[he new alpinc flor aofJapan in color vol. 2.
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Received Mtirz r2h8, 2005, 'aecepted October 24, 2005
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AppENDTx. Intraspeci fcipDcNA variation of Japanes aelpine plants.
Haplotypei Haplotype'
Species and locality (b'nL-F) (rpl16introhna)pClootmybpeined voucheP
AnemonenarcissijloraL.(RANUNCULACEAE)
Russia :Sakhaiin ,Mt. Zhdanko [2(lNS, 12 ABCDEDEFGDHI N, FzijiFiOKI.0S6e0Snln2i2N6.FO1703FO1787FO1744S2617FO1652S874SS96S361S3225S1818S2334
JJaappaann:: Hlowkaktae iPrdeof ,P rMet£., YMatkse, iTslhaiidsaektesuzan 3(INS, 2SIIDD)) (INS)13 FLijiiN.FLljiiN.
Japan Y:arnagata Pref, M,ts. Iidesan 4 (3NS ,3ID) (INS)333333333
Japan: Gunma Pref. ,Mt. fanigawadake FLijiiK.
Japan/ Nagano Pref. M,t. Shjroumadake 5 (4NS ,3ID) SenniN.FLljiiK,
Japan/Toyama Pref,,Mts, Tbteyama 4567(3NS,51D)

Japan: Ishikaw Paref iM,ts. Hakusan SennjK,
Japan: Nagano Prei, Mts. Ontakesan SenniK.
Japan:Nagano PreE,Mt.Kisokomagadake SenniK.

JJaappaann ::YSahmiaznuaoskha iP rPerfe,£ M, tM. tA. kKaiitsahdiadkaeke 48(3NS,4ID) SSeennnniiKK..
Japan :Nagano Prefi, Mts. }latsugadake 9 (4NS ,4ID) Senni
Length]Tbtalpolymorphiccharacters 950bp8NS, 929bp2NSO.22l,879bplONS,

7ID1.58 7ID

P4 (%) O.90
"renaria aretica Steven cx Ser .(CAI(YOPHYLLACEAE)
Russia :Sakhalin ,Mt. Zhdanko 12(INS,3ID) 12 ABCDECFG N. Ftij iFiOI070
japan: Hokkaido Prefi, Mts. Thisetsuzan (INS, 2ID) K. Senni S15S2
Japan: Nagano Prefi M,t. Shiroumadake 23(1NS,4ID) 3(IID)4 K. Scnni S2818
Japan:Toyama PreC,Mts.Tlateyama (2NS,IID) N.FajjiFO1657

Japan: Nagano Pref iM,t, Kisokomagadake 4{1NS, 4ID) 5 (INS ,IID) K. Senni S243
Japan :Yhmanashi Pref, Mt. Kitadake 225 36 K.Senni S3626
Japani Shizuoka Pref, Mt. Akaishidake (2NS ,31D) K.Senni S1998
Japan :Nagano Pref, Mts. Ylatsugadake (2NS, 41D) 2 K. Senni S2354
Length'Tbtalpolymorphiccharacters 931bp3NS, 970bp5NS, 1,901bp8NS,

4IDO.75 21DO.72 6IDO.74

P4(%)

Arnica unalaschcensis Less. (ASTERACEAE)
Japan: Hekkaido Prcf, Mts, Thisetsuzan 12 12 ABCDEFFF K. SenniSN1.392FOI705FO17g6FO1740S3023FO1664S157S3267S1878
Japan: Iwate Pref, Mt. Yakeishidake (2NS ,2ID) (3NS ,IID) FLijiiN,
Japan] Yamagata Pref. M,ts. Iidesan 3 (INS, IID) 3 (2NS ,1ID) FtijiiN.
Japan: Gunma Pref, M,t. Tanigawadake 4 (INS ,21D) 4(INS)5 FLijiiK.
Japan: Nagano Pren, Mt. Shiroumadake 5 (2NS ,IID) (4NS ,IID) ScnniN.
Japan: Toyama Pref, Mts. tateyama 5555 6(3NS)66 FiijiiK.
Japan N:agano Pref, Mt. Kisokomagadake SenniK.
Japan: Yatnanash iPref, Mt, Kitadake SenniK.
Japan: Shizuoka Pref, ,Mt, Akaishidake Scnni
Length3Tbtal 1,e39 bp (ad)5984 bp6NS, 2,023 bp9NS,
pelymorphic charactcrs 3NS, 2IDO.48 2IDO.81 4IDO.64
P4(%)

Artemisi asinanensis Y. Yabe (ASTERACEAE)
Japan [Yamagata Pref, Mts. Iidesan 12(INS)222222 11111111 ABBBBBBB N. FiijiFiOK1.782S2940FO]656Sl141SS57SI89S3366S17t8
Japan /Nagano PreE, Mt. Shiroumadake SenniN.
Japan :Ibyama Pref ,M,ts, fateyama FojjiK.SenniK.
Japan: Ishikawa Pre£, Mts. Hakusan
Japan: Nagano Pref, Mts. Ontakesan SenniK.
Japan: Nagano Prei, Mt. Kisokomagadake SenniK.
Japan: Yamanashi Pref, Mt. Kitadake SenniK.
Japani Shizuoka Pref, Mt. Akaishidake Senni
LengthiTotal 841bpINSO.12972bpoo 1,813bpINSO.06

polymorphic characters
P4 (%)
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AppENDIx.Continued
Haplotype] Haplotypei
Speciesandlocality Nbuchee
(trnL-E} (rpU6intronh)aCploomtbypiened
Campanula lasioca,;pCaham. (CAMPANULACEAE)

Russia /Sakhalin ,Mt. Chamginskiy 112(INS)22222 12 ABCCCCCC N. FLijiFiOKI.183S1471S2656FO1655Sl149S780S3607S2170
Japan :Hokkaid oPrefi M,ts. Taisetsuzan (INS, IID) SenniK,
Japan :Nagano Pref. M,t, Shirournadake 3 (INS ,IID) SenniN.
Japan :Toyama Prefi M,ts. Tateyama 33333 FLljnK.
Japan :Ishikawa Prefi M,ts. Hakusan SenniK.
Japan: Nagano Pref, Mts. Ontakesan SenmK.
Japan: Yamanashi Pref, Mt. Kitadake SenniK.
Japan: Shizuoka Pref, Mt. Akaishidake Senni
Length3Total 913bpINSO.I1 1,021bp2NS, 1,934bp3NS,

polymorphic characters 1IDO.29 IIDO.21
p4(ya)

Caidamine n41ponica Franch. et Sav. (BRASSICACEAE)
Japan/ Hokkaido Prefi, Mts, faisetsuzan 12 ABCDEFCCCG K, SenniNS,13S2FO]765S2778FO1673SI020S535S42S3547S1738S2262
Japan: Ytimagata Pref, Mts. Iidesan (2NS)3 111111111 Fng11K,
Japan: Nagano Prefi M,t. Shiroumadake (4NS)4(3NS)5(5NS)6 SenniN.
Japani foyama Prefl M,ts, Thteyama Fng11K.
Japan :Ishikawa Pref. ,Mts. Hakusan SenniK.SenniK.SenmK.
Japan:Nagano Pref,M,ts.Ontakesan (5NS)3337

Japan:NaganoPref,Mt.Kisokornagadake

Japan :Yamanashi Prcf, Mt. Kitadake SenniK.
Japan iShizuoka Pref, Mt. Akaishidake SenniK.
Japan :Nagano Pref. ,Mts, Yatsugadake (3NS ,IID) Senni
Lcngth3Totalpolymerphiccharactcrs 872 bpgNS, 694+375 bpfi l,941 bp8NS,
IID1.03 oo IIDO,47

P4 (%)
CZirexhakkodensFirsanch. (CYPERACEAE)

JJaappaann:: HYaorknkaagiatdae PPrreef£,, MMttss.. TIaiidseestasnuzan 1 2(5NS)3(5NS1) 2(42(14DN)S3)(4I4I4D4A)BC3D3DD3D3D3 K. SFclnunlilNKS.,1694FOI771g3043FO1666SI098S800S452S210S
Japan: Nagano Pref. M,t. Shiroumadake SenniN.
Japan :Teyama Pref, Mts. Tateyama FiijiiK.
Japan :Ishikawa Pref. M,ts. Hakusan SenniK.
Japan :Nagano Pref, ,Mts. Ontakesan SenniK.
Japan :Nagano Pref, Mt. Kisokomagadake SenniK.
Japan: Shizuoka Pref. M,t, Akaishidake Senni
Length]fotal 989 bp7NSO.71 g49 bp3IDO,35 1,838 bp7NS,
polymorphic characters 3IDO.54
P4(%)

Carex Maxirn.(CYPERACEAE)
scita
Japan H:okkaid oPref, Mts. Tbisetsuzan 111111 11111l AAAAAA K. SenmK.S1674S2637FO1668SII33S3489S2548
Japan: Nagano Pref, Mt. Shiroumadake SenniN.
JJaappaann:: TIosyhaimkaaw aP rPere£f, .M, tMst.s .1 lHaaLktuesyaanma FSuejniniiKK..
Japan: Yarnanash iPref. M,t. Kitadake SenniK,
Japan: Nagano Prefl .Mts. Yatsugadake Senni
LengthiTotalpolymorphiccharacters 990bpoo 848bpoo 1,838bpoo

P4 (%)
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272 APG va)15.6

AppENDIx.Continued

Species and locality H(atprlontLy-peF') H(arpplottiy6peiintrohna)pClootmybpeined xlouchee
Cla,u xstenantha Franch. et Sav .(CYPERACEAE)
Russia: Sakhalin ,Mt, Zhdanko 12(IID)3(1ID)33A4BCCCDEEEF N. FiijiFiOIK0.86S1652S2837FO1646Sll13S720S266S3327S2128S2508
Japan: Hoklcaido Pref, Mts, Taisetsuzan 12(2NS)223(INS)334 SenniK.
Japan: Nagano Pref ,Mt. Shiroumadake SenniN.
Japan T:byama Pref, Mts. fateyama FiljiiK.
Japan: Ishikawa Pref, Mts. Hakusan SenniK.
Japan: Nagano Pref, Mts. Ontakesan (INS ,IID) SenniK.
Japan: Nagano Pref ,Mt. Kisokomagadake 3333 SenniK.
Japan :Yamanashi Pref. ,Mt, Kitadake SenniK.
Japan :Shizuoka Prefi, Mt. Akaishidake SenniK.
Japan :Nagano Pref., Mts. Yatsugadake (INS ,IID) Senni
Lengthifotalpolymorphiccharacters 1,O16bp2NS, 8l8bp2NS, 1,834bp4NS,

IIDO.30 IIDO,37 2IDO.33
P4 (%)
CZissiopelycopedio(iPdaelslD..)Don (ERICACEAE)

Japan: Hokkaido Prefl, Mts. faisetsuzan 12(8NS, 11111111 ABCDEFGH K. SenniSK1.208S2704Sl170SS76S318S3567S2176S2528
JJaappaann: NI:asghainkoaw Pa rPer£e,f M, tM.t sS. hHiarkouusmaandake 3 (6NS 22,IIDD)) SSeennnniiKK,.
JJaappaann:: NNaaggaannoo PPrTee£f,l MM,tt.s .K iOsnotkaokmaegsaadnake 4S ((56NSN S,, 221IDD)) SSeennnniiKK..
japan: YZimanash iPref. M,t. Kitadakc 6 (6NS ,2ID) SenniK.
Japan: Shizuoka Pref, Mt. Akaishidake 7 (7NS ,2ID) SenniK.
Japan: Nagano Pref, Mts. Yhtsugadake 8 (6NS ,1ID) Senni
Length3Tbtal 1,747 bp (aDS 884 bpoo 2,63I bp12NS,
polymorphic characters 12NS, 3ID 31D
P4 (%) O,86 O.S7
Diapensiai`pponicLa,(DIAPENSIACEAE)

Russia :Sakhalin ,Mt. Zhdanko (S-4) 11l11111111 12(2ID)3(11D)24ABCBDDEFDDG N. Faji iFOI061
Japan :Hokkaido Pref, Mts. waisetsuzan K. Senni S1311
Japan :Iwate Pref, Mt, Yitkeishidake N. Filj 'FiOi1700
JJaappaann; YN:aatgnaangoa Ptra Perfe, £M,t M.t sS,h iIrioducrsnaandake (INS ,2ID) NK.. FSuejnlnl i FSO31076638
Japan T:byarna Prcf, Mts. Tbteyama 45(3NS,2ID) N. Fu.j iFiO1651
Japan: Ishikawa Pref, Mts, Hakusan K. Scnni Sl186
Japan: Nagano Prefi M,ts, Ontakesan 6(INS,2ID) K, Senni S515
Japan: Nagano Prefi M,t. Kisokomagadake 447 K. Senni Sl
Japan: Yatnanash iPref., Mt. Kitadake K, Senn iS3528
Japan: Shizuoka Pref, Mt. Akaishidake (2NS ,2[D) K, Sermi S2088
Length3Tbtal 920 bpoo 1,338bp3NS,2IDO2,,32578 bp3NS,
pelymorphic characters 2IDO,22
P4 (%)
Gentiana aigida Pall .(GENTIANACEAE)
Japan :Hokkaido Pref, Mts. 1lajsetsuzan 12(3NS,2ID) 12(INS)3(2NS)4ABCDEFGH K. SenniKS.1512S2798FO1665S474S218S3466S2038S2468
Japan :Nagane Pref, Mt. Shireumadake SenniN.FLijiiK.
Japan: Toyama Pre£, Mts. [[hteyama 23(4NS,2ID)
Japa] :Nagano Pre£, Mts. Ontakesan (2NS)25 SenniK.
Japan :Nagano Pref, Mt. Kisokomagadake 4 (4NS ,IID) SenniK.
Japan :Yatnanash iPref, Mt. Kitadake 5 (3NS ,IID) (2NS)22 SenniK.
Japani Shizuoka Pref, Mt. Akaishidake 56 SenniK.
Japan: Nagano Prefi M,ts, Yatsugadake (3NS, 2ID) Senni
Length3Ibta1polymorphiccharacters 1,446bp(afiS 752bp4NSO,532,198bp8NS,

4NS,3IDO,48 3IDO.50

P4 (%)
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December 2005 SENNI et aL: Intraspecif cipcDNA variation in Japancse alpine plants 273
AppErvDix,Continued
Haplotypei Hapletypei
Speciesandlocality vouchee
(t,'nL-F) (rpl16introhna)pClootmybpeined
Geraniumyeseense Franch. et Sav. (GERANIACEAE)
Japan :Jwate Prei', M,t. Yhkeishidake 112 AABcCC N.FLijii FO1707
Japan :Yamagata Pref, Mts. Iidesan N. Fu]' iFiO1779
Japan: Nagano Pref. M,t, Shiroumadake (INS ,11D) K. Senn iS2960
Japan: lshikawa Prei, Mts. Hakusan 3(IID)33 K. Senn iS896
Japan: Yamanashi Pre£, Mt. Kitadake K. Senni S3247
Japan Sihizuok aPret, Mt. Akaishidake K. Senni S1758
Length3Total 926 bpINS, 926 bpINS,
polymorph iccharacters IIDO.22 IIDO.22
P4(%)

Ceum ealthijbliutn Sm. (ROSACEAE)
JJaappaann: :IYwaamtea gParet£ aP, rMte.f .YM,ttask, cIiisdheisdaanke 12(4NS, IID) 1 2C4NS ,5ID) ABCDEFGHIJK N. FFlilijj1iF1iONN1,.702Fe1784FO1748S28S7FO1650S916S659S136S3347Slg38S2426
Japan /Gunma Pref, Mt. Tbnigawadake 13(IID)4(IID)S(3 ICNS5)I1D6()44NS)67 F-nK.
Japan :Nagano Pref, Mt. Shiroumadake (41D)S SenniN.
Japan :Toyama Pref, Mts, fateyama (4NS ,5ID) F-11K.
Japan :Ishikawa Pref, Mts. Hakusan 56(4ID)7 SenniK,
Japan :Nagano PreE, Mts. Ontakesan ScnniK.
Japan: Nagano Pref. M,t. Kisokomagadake (lNS ,4ID) SenniK.
Japan: Yhmanashi Pref, Mt. Kitadake 8 (4NS, 5ID) ScnniK.
Japanl Shjzuok Paref ,Mt. Akaishidake 9(4NS,5ID) SenniK.
Japan: Nagano Pref, M,ts. Yatsugadake (4NS ,1ID) 2 Senni
Length3Total 1,738bp(afiS 1,063bp9NS, 2,801bp15NS,

polymorphic characters 6NS, 2IDO.46 7ID1.5] 9ID
P4 (%) O.86
HzpericumkamtschaticunLvedeb.(CLUSIACEAE)

Japan: Heklcaido Pref, Mts. Taisetsuzan 12(4NS,5ID) 12 ABCCDBBEFB K. ScnmN.Sl187FO1715FO1777FO1742S2558FO1677SI040S740S381S3187S1898S2412
Japan: Iwate Pret, Mt. Ylakeishidake (2NS ,2ID) FLijiiN.
Japan: Yamagata PreE, Mts. Iidesan 3 (5NS ,4ID) 223{2NS,4ID) FLijiiN
Japan: Gunma Pref, Mt. Tanigawadake 34 FojiiK.
JJaappaann:: TNoaygaamnao PPrree£f, . M,Mtt.s .S 'hi[rtoautrenyaadiankca 2 2(242N2S ,3ID) 224 SFeininjiiNi.K.
Japan: lshikawa Pref, M,ts. Hakusan SenniK.
Japan :Nagano Pref, M,ts. Ontakcsan (3NS ,2ID) SenniK,
Japan :Nagano Pref. M,t. Kisokomagadake 45(2NS)22 ScnniK,SenniK.
Japan:YhmanashiPref,Mt.Kitadake

Japan :Shizuoka Prcf. M,t. Akaishidake SenniK,
Japan :Nagano Prefi M,ts. Yhtsugadake Scnni
LengthiTotalpolymorphiccharacters 628 bp5NS, 1P13 bp3NS, 1,641 bp8NS,
5IDi.59 41DO,70 9ID1.04

P4 (%)
Lo iseleur iparocumbens (L. )Desv, (ERICACEAE)
Russia :Sakhalin ,30km South ofOkha 12(IID)3(IID)412(1ID)3 ABCDEEFGH N.FngllKF.OS15e0n7nSi1N24.8FO1790S2706FO1674S454S64S1918S2223
Japan:HoklcaidoPrefiM,ts.Taisetsuzan

Japan:YhmagataPref,Mts.Iidesan (2ID)2224(2ID)22 FL"11K.

JJaappaann ::NToaygaarnnoa PPrree£fi, , MMtts.. S Thaitreeyuammaadake 5 ((I2NSN,S1 ,2IIDD)) SFeLninjiiNi.K.
Japan :Nagana Prei, Mts, Ontakesan 56 ScnniK.
Japan :Nagano Prcf, Mt. Kisokemagadake (2NS ,IID) SenniK.
Japan :Shizueka Pref, Mt. Akaishidake 67 SenniK.
Japan: Nagano Pref, ,Mts. Yatsugadake (2NS, llD) Scnni
Length3Total 854bp3NS, 956bp2IDO.2I 1,810bp3NS,4IDe.39

polymorphic characters 2IDO.59
P4 (%)
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274 APG VOI.56

AppENDTx, Continued
Haplotype] Haplotypet
Species and locality (trnL-n (rptli6ntron)Cheampbliotnyepde xicuchee

Luzuia areuata (Wahlen bSw..) (JUNCACEAE)
Japan /Hokkaide Pref. M,ts. Taisetsuzan 12(INS)222212 l111111i ABBBBBAB K. Senni S1573
JJaappaann NT::aogyaanmoa PPrree£f,i MM,tt.s .S Ihibrtoeuymaardankae KN..SFeLninjii iFOS12677359
Japan :Nagano Pref. ,Mts. Ontakesan K.Senni S639
Japan :Nagano PreE, Mt. Kisokomagadake K, Senn iSIOO
Japan: Ybmanashi Prefi M,t. Kitadake K. Senni S3386
Japani Shizuok Paref, Mt. Akaishidake K. Senni S2048
Japan: Nagano Pref, Mts. Yatsugadake K.Senni S22g2
Length3Tota1 84SbpINSO.12 762+377bp6 1,987bpINSO.05

polyrnorphi ccharacters oe
P4(%)

RedicularisyezoensiMsaxim, (SCROPHULARIACEAE)

Japan I:wat ePref. ,Mt. Yhkeishidake 12 112(3NS)3(INS)4A<B4CNDSE)F3G5G(C4HNS)55n5o voucher speeimen
Japan :Yamagata Pref, Mts. Iidesan (4NS ,21D) N. Foji iFOI788
Japan :Gunma Pref, Mt. Tanigawadake 3 (4NS ,5ID) N. FLij iFiO1750
JJJaaapppaaannn :::NTIaosgyhaainrkonaaw PPa Prrree££ef,, M,M Mttt.s s.S ,T hHliatrktuoesuyaamnmaadake 4S6 (((781NN0SSN ,6,9S [6I,DID)D)) KNK... SFSeeenjnnin iii FSSO2l1816976771
Japan /Nagano Pref, Mt. Kisokomagadake 7(7NS,9FD) K. Senni S178
Japan: Ybmana$hi Pref, Mt. Kitadake 778 K. Senni S3123
Japan: Shizuok Paref, Mt. Akaishidake K. Senni S1978
Japan: Nagano Pref, Mts. Yatsugadake (6NS, 7ID) K.Senni S2183
Length3Total 1,627 bp (ab5 g31 bp5NSO.60 2,458 bp20NS,
polymorphic characters ]5NS, 9ID 9JD
P4 (%) 1.4g L18
llotenti tmaltsaumurae Th. Wblf(ROSACEAE)
Japan :Hoklcai dPoref ,Mts. faisetsuzan 12(2ID)23 12 ABCDEFGDHDF K. Senni S1452
Japan :Iwate Pre£, Mt. Yakeishidake (INS ,IID) N, Filj iFiOI706
Japan :Yamagata Pref, Mts. Iidesan 13 N. FLij iFiOI794
Japan :Nagano Pref, Mt. Shiroumadake (2ID)33334( (12NNSS, ,23IIDD)) K. Senni S2980
Japan :Toyama Pref, Mts. fateyaina 4 (21D)5 N. Fiiji iFO1659
Japan :Ishikawa Pref, Mts. Hakusan (2NS ,IID) K. Senni SIOOO
Japan :Nagano Pref, Mts, Ontakesan 6 (2NS ,2ID) K. Senni S70I
Japan: Nagano Pretl M,t. Kisokomagadake 37 K. Senni Sl17
Japan Y:hrnanash iPref. M,t. Kitadake (2NS ,4[D) K. Senni S3307
Japan: Shizttok aPref, Mt. Alcaishidake 33 35 K.Senni S1938
Japan: Nagane Pref, Mts. Yatsugadake K. Senni S2448
Length3Totalpolymorphiccharacters 978bpINS,31DO,49168bp2NS, 1,946bp3NS,

5IDO.72 8IDO.57

P4(%)

Rumex montanzag Desf (POLYGONACEAE)
Japan: Hekkaido Pref, Mts, Thisetsuzan 12{IID)23 1111 ABBCD K. Senn iS1413
JJaappaann:: NYaamgaagnaot la 'Prree ffM,i tM,. tSsh, iIrioduemsaadnake NK,. FSaejnni ii FSO21577875
Japan T:oyama Prefl M,ts. lateyarna (INS, IID) N. FLij iiFO1648
Japan: Nagano Pref, Mt. Kisokomagadake 4(IID) K,Senni S3S7
Length3Tbtal 864 bpINS, 691 bp (part)61,564 bpINS,
P4(% p)olymorphic characters IIDO,23 oo IIDO.13

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