Phylogeographic history of the Japanese Alpine Ringlet Erebia niphonica (Lepidoptera, Nymphalidae) : Fragmentation and secondary contact

2007·2.6 MB·

TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
uettigk7lransI.epidS.oc. 58(3)2:53-275, Jun 2007
,lapan ¢
Phylogeographic history of the Japanese Alpine Ringlet Erebia niphonica
(Lepidopt Neymrpah,alidae): Fragmentation and secondary contact
[Ibkatosh iNAKATANii' ,Shin-ich UisAMii ]and [fate IoToH3)
i )Graduat esehool of Science and Tcchnology, Shinsh uUniversity,
3-1-1Asahi,Matsumoto,Nagane, 390-8621Jupan

]i Departmcnt of Otorhinolar}ingol Sohgiyn,sh uUniversi tSychoo lof Medicine,
3-1-1Asahi,Matsumoto, Nagano, 390-8621Japan

i' Depeitment of Bielogy, Shinsh uUniversit 3y-,1- 1Asahi, Matsumoto, Nagano, 390-S621 Japan
Abstrac tThere have been some previous studies about when a]d through which route the alpine
organisms ranging in the Japanes eisland smigrated frem the continent, and how thei rdistribution
range has changed within the islands whi]e adapting to the climate changes ln the Quaternary.
There are studics about alpine plant bsascd on recent molecular information .A scenario hus becn
propose dthat therc are two lineages .One is the lineag ethat had relict distrjbut idounring inter-
glacial s.The other is the lineage that rnigruted into the Japancse island sduring latc rglaci apleri-
ods. Oll the other hand, anirnals have mobility and are able to move around followin egnvi renmen-
tal changes such as climate changes. Therefore ,therc is a possibili toyf having a differe snctenario
from that of plants .We chose Erebia niphonica, and studied the distribntio cnhange of alpine or-
ganisn) sin the Japanes eislan ddsurin gthe Quaterna ryWe. studied lhe haplotypes of base se-
quences of 942 bp in total using ND5 aiid COL Samples arc 427 individual sfrom the Japanese is-
lands M,ongolia and Russia .We inferrc dpast historic aelvents by analyzing the haplotypes using
the Nested Clade Phylugeographical Analysis (NCPA) .As a result it has been suggested that the
Erebi .naiphonica group wellt through isolatio nin more than one refugium in Hokkaido and more
than one in Honshu durin gwarm interglaci aalnds ,tbat thcy fragmented into differen tLineages
within the Japanes cisland s,Also we specificd the secondary conLact points which support the
NCPA which inferre dthat rangc fragmentati oannd dispersa lwere repeated.
Key words Phylogeograph yh,iogeegraph yE,rebia niphonica, ncsted clade phylogcographieal
analysis, haplotype, IVD5, COJ, Quaternary.
1.Introduction

1.1 Phylogeographi chistory of the Japanes eislands
There were perieds vv'hen the Japanes eislands i the Japancs eArchipelago wlere connected
with the Asian continent during the glacia pleriod sand served as refugia fbr animals and
plant sof continental origin. During the g]acia lage, many of the arctic and subarctic ani-
mals and plant smigrated into the Japanes eisland s.During the interglac iaagel, distribu-
tions of those animals and plant sdiminishe dor disappeare d(Yasud aand Miyoshi (Eds),
1998; Machida et at., 2003). There have been many studies of fauna based on fossils
(Kawatnu ert aal., 1989; Kawamura, 1998), and of flor abased on polle nand wood fossils
(Sohm aand Tsaji ,l988; Suzuki and [[lakeut i1,989; Morita, 2000).
The major islands that constitute the Japanes eisland sare Sakhalin, Hokkaido, Honshu,
Shikoku ,and Kyushu, They stretch in a row firo mnorth to south (Fi g1.) .There is the
Tarta rStrai t(=th eMamiya Strai tb)etween the Amur region and Sakhalin, and the Soya
Strai tbetween Sakhali nand Hokkaido, and the Tsugaru Strai tbetween Hoklcaid oand
Honshu, and the Tsushima Strai btetween the Korean Peninsul aand Honshu-Kyushu. Of
these, the rkLrtar Strai atnd the Soya Strai atre sha]low, and were fonned later than 1 1,OOO
NNIII-IE-leEcltreoncitcronic LMbirabrryary Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
254 Takatoshi NAKAi'ANi S,hin-ich UisAMi and Tateo ITo}i
]-gaf

:/
Me
f'
=-Z[ l.--.- "
Fig. 1, The japanese islands .Shows rnajor island asndstraits.
years ago. On the other hand, the Tsugaru Strai tand the Tsushima Strai atre deep. Honshu
separated from the Korean Peninsula and Holclcaid oand became an islan dduring the inter-
glacia alge after the Riss glaciati oabnout 150,OOO years ago. It is believe dthat Honshu has
not bcen connected with the continent since then (Ohshim a1,990, 2000; Matsui et al,,
1998). Sakhali nand Hokkaido had been connected with the continent almost continuous]y
during the period of 65,OOO year sfrom about 75,OOO years ago up to abeut 1O,OOO years ago
(Ono ,1990) .They had been parts of the continent for longer period sof time than the peri-
ods when they were isolate disland s.Thus, during the glacia plerio dthe geographic condi-
tion allowed organisms to migrate southward from the continent to Sakhali nand Hokkaido.
On the other hand, alpine organisms that now havc isolate ddistribut iion nthe alpine belt of
Honshu had expanded their distribut ipornio rto about 150,OOO years ago from the conti-
nent, and they are the species that were aliected by climate changes since then during the
interglaci apleriod and the last glacia pleriod,
The high altitude areas of central Honshu and Hokkajdo in the Japanes eislttn hdasve had at
leas ttwo period swhen glaci.e rdseveloped ,and there is rnountainous permafi'o sitn some
limite dareas in the high mountainous areas in Hokkaido and central Honshu even now
(Iwat a2,003). Reconstruction studies of ancient flor aby pollen analyses (Sakaguc 1h9i8,9;
Igurashi et al,, 1993; Yamada, l998; Hoshino ,1998) suggest that the Iow altitude areas in
Hoklcaido were the southern limit of taiga during the las tglacia pleriod ,and that there were
forest sricher with Larix gmelini tihan those in the present northern Sakhalin .In the eastern
and northern part sof Hokkaido, the distribut ioofn taiga with Larix gmetinii as it smain
plant was divide din such a way that each of coniferous forests ,grassland swe,t vegetation
and alpine tundra formed segregated niches according to altitude andlor the geographical
feature s.Thus the areas functione das refugia fbr many organisms in the Far East (Ono,
1990).
NII-Electronic Library Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
Phylogeographic Histor vof Erebia ti iphonica 2)U5
In Honshu there is a larg erift zone called Fossa Magna that runs across the islan dfrom
NNW to SSE at around 138 degree sof east longitud eT,he effect of the rift zone on the dis-
tribution of organisms has been noted biogeographicall y(Su et at., 1998) .However, there
has been no study that suggests releyance of the rift zone to phylogeography of alpine or-
ganisms. The time of it stectoni cformatio n(i wtas under the sea up until several millions
years ago) was much earlier than the time when the alpine organisms came to the Japanese
islands.
Regarding the understanding of the immigration routes of alpine butterfi ieinsto the
Japanes eisland sand varying pattern sof distributi oonn the island st,he limitatio nofs re-
giona lbiogeographi acpproaches or of studies based on the degree of specialization of ex-
ternal characters have been pointed out (Hiur a1,971, 1977). There were, however ,no alter-
native means ef approach. Now, using the new viewpoint of molecular phylogenetic tsh,ere
have been studies on l)kedicul acrhaimsissonis (Fuj eit ial., 1997) and on Primula czaneijiolia
(Fuj ieti aZ,, 1999). There are also phylogeographi sctudies on the whole Eurasian popula-
tions of Japanes eRock Ptarmigan Lagqpus mutusjaponicus (Bab aet al., 2001) and on the
Small Tbrtoiseshe blultterfl yAgtais "rticae (Vandewoesti ejtn aeg., 2004).
The proces sof range expansion and contraction of erganisms due to climate changes in the
Quaternariys known in Eurasia(Hewitt1,996), But
well as an east-west migration western
as the geographi cchaTacteristics of the north and south extension ef the Japanes eislands
may suggest, the proces sin the Japanese island sis composed of north-south migration.
Each species of alpine plant shas two lineage sin Honshu, northern and southern, and the
boundary is in the northeast of the island (Fuj ieti at., 1997, 1999) ,As the primary cause of
such a distribut icohanracter, a scenario has been suggested whereby the original population
remained in the mountainous areas of central Honshu as refugia during the interglac ipaelri-
od and late rbecame the southern lineage a,nd a new lineag emigrated int othe Japanes eis-
lands during the glacia lperiod after the interglacia pleriod and formed the northern lineage
(sing rlefeugium model). However, it could be considered to be possibl ethat more than one
refugium existed in the Japanes eisland sbecause of it scomplex mountain topography and
the micrometeorological environments caused by it ,That is to say, there eould be a new
scenario. As a result of isolatio nby taking refuge in more than one refugium during the
wann periods, geneticall ydifferentia ptoepdulation hgave survived til lnow in more than one
lineag eby expanding and contracting their distributio anreas adapting to late rglacia clycles
(multi prelfuegia model). In order to study such a possibili tiyt i,s necessary that the subject
organisms of the study must have thc following characteristics (Schmi tett at., 2006;
DeChaine and Martin ,2004) ,(1 )Adapting to environmental changes, they should be able
to moye to suitable surroundings, (2 )Each individua ldoes not usual]y move around and
tends to stay in the maternal population ,and they inheri tphylogeographic ailnfbrmatio ans
a populatio n.C3 )They are able to maintain continuous population esven when the areas
with inhabitab lenevironment are geographical lsmyali. (4 )They are currently distribut iend
wide areas even though sporadic,
Body sizes of insect sare relatively small, and they are capable of reproducing themse]ves
as stable population isn relatively small habitat sB.utterfii heasve more adyantageous capa-
biliti tehsan plant sfbr maintaining population usnder the infiuenc oef glacia clycles, such as
migrating to more suitable surroundings on thei rown. Therefore i,t is highly possibl ethat
there is a historica lscenario for alpin ebutterfl ideistfere nftrom that of alpine plants.
12 Systematic sand ecology of the subject species
We havc chosen Eiebia niphonica as the subject species of this study, and we will verify
that this species satisfies the aforementioned requirements (1 to 4) .The species is fOund in
NII-Electronic Library Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgleitycal Society oofJfap anJapan
256 Tdkatosh iNAK4T4Ni Shin ichi Us4Mi andTdteo [Toii
ts N
HAooo

ges#igsg
:...v"g HAool

t ny:4 ･w HAO02
cr
HA021
sc'
c) E#liil$l$l
HBool

[=1
di-}igl..2if HCOOI
[= ]HCO1O
aj".
Il: - HDOOO
t.pt. EiZ i
HDoe3

M
HFool

es
HFO02

[ ]HFO03

ge'g"HFiOS0e4

2a
Fig 2 Erebta niphom(a distnbutio nranges and sample siteg, (a )Hokka]do (b )Honbhu Pale gieen
dot denotes the distnbuti oiannges, ied dot denetes the sample yte Indicatio nunit is 75
rongitud ×e 5 O' Iatitu dePie charts sho- the proportio nofs haplotlrpe swithm cdch re.oion
Whtte is pnvate hdplotypes restnLted m one region For abbreviattons refer to Table 1
Sakhahn, the gouthern Kuril Iglands (Kunats Ihsilrand )H,okl(aido, and the alpine areag of
Honshu, and can be claggified mto geveral subspecies (FuJio k1a981 Inomata, 1986) In
northeabtern Euragi athe Llosely related ErebLa nerieite is ditstribut edThere it smuch con-
trovergy about the taxonomic relationship of niphonica and nenene Some taxonomistt sre-
gard them as geographical xdrianons of the game species (Filjio ka1,981, Asahi et at,
1999) ,some ag ddieren tgpecies (Warre n19,36, Inomdta ,1986, Gorbunov, 2001), and some
regard only the Honshu one ag an mdependent species (Kogur e19,79) In the monograph
on the genut sErebta (Warre n19,36) ,these taxa belong to the aethtops group together with
EJ aethtqps which rdngeg firo mEurope to near Lake Bdika lin southern Siberi aa,nd E al-
cmena which is fbund in the alpine drea of ccntral south China The monophyly of this
group has been venfied by molecular phylogeny (Sekigu ceth ail 2002) There wag a view
,
thdt Eiebia vidlen, which is found m northwestern Nerth Amenca and Ei ebia niphontca are
cLogely reldted species (Laybe retr aly 1998) ,but they die quit efar apart bdtse don recent
,
molccular phylogeny (Nakata etm at in pieparation)
,
Eiebia mphontca inhabits grassland galong streamg in subalpine comferous foregt asbove
1,OOO m above sed leve lin northeabtern Honghu and above 1,500 m in central Honshu,
grasgland stn spdrge forets tofs BetLtl aeimamt near the tmiberhne and dlpine herbaceous
and scree grass ldnd above the timberhne (3,OOO-3, m05 0mdximum) (Nakdta nanid
Kitdgawa, 2001) In Hoklcaido it ranges widely from near the seaghore to the alpine herba-
ceous 7one dround 1,600 m above sea leve l(Takeuc h20i0,3) In Sakhali nit occurg in the
south of the central part (N51-N52 degree) a,nd ranges from near the sedshore to grdsslands
m the torest s(Agah eit at,1999 Asahi and Kohara, 2004)
NII-Electronic Library Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
Phylogcographi Hcistor yof Erebia niphonica 257
oge-1 -CHIIO
"".tce.t
--l-, NcH12o
/.t
Y/lliglilCH121
es:lg?

-CH140
i:lii
[ i
-iet-- ee
,
-NSIOO

aNSIII
t.tt
MNs121

2b
t t t t
tt/
As for the larva lfood plants of Erebia niphonica, about 10 species of Gramineae and
Cyperaceae have been liste d(Fukud eat at., 1984) .In Hokl<aido l,arva ehave also becn
found from Pba prcttens iansd Fkrstuc arubra which were brought in and plante don the
slopes ofwood]and trail s(Kawat aand Kitahara, 1990). This species is a generalis tfbr lar-
val food plants and appeurs once a year.
The habitat sin Honshu consist of rclatively small field issolate bdy forcst ssh,rubs, or bam-
boo gras slands, ,and populatio nsize is small (Nakata anndi Kitagawa ,2000) .There has
been no detaile sdtudy on the home range size. We have observed that thc insect rsemained
still in the thickct sunder strong wind conditions, and that they had a tendency to land
quickl yand remain stiH when forccd to fly .It seems that usually they seldom move firem
the natal site. However, a case has been reported in which a new populatio nstarted to brecd
along a new trai lwhich had been opened in a subalpine coniferous fores t(Kawat aand
Kitahara ,1990; Nakatan iand Hosoya, 2003). Therefore ,females seem to have some mi-
gratin ghabit .In other words, that the process repeats itsel wfhereby a new colony comes
int oexistence at the peripher yof distribut iroannges, and then becomes extinct as grassland
shifts to shrub or bamboo grassland.
NII-Electronic Library Service
TThhee　 LLeepipdiopdteorpoltoegiroaollogical　 SSoooiceityety 　ooff　 JJaapapnan
258 Takutosh　iINAKATANI ．　Shin−ich　iUsAMJ　aild　Tateo　IToH
　　　　　O℃一旨邸口訂 個刺順て［一凶oロhのH＞oQ ひトO【OひOひりO料．． 寸り寸OO寸【卜αO爿σ．． 咽ひN【OOO爿O，O．O ひトウOOα寸OいO爿り5、． OぴOOσ嶋〇一O刊5N．． OOOO刊OOO．．OOO （）〔）QOOO刊OO．O．O 寸O卜Q○q〔）nいO刊「．． ゆト祠【ON円Q一O爿．． 〔Q寸OOσ【O【｝刊oaう馬．． O寸O【O【卜OO爿O．， 一寸D四OαN寸ゆOo刊．． Nり円因Oρめ酷【O刑り｝．． （OOOO｝爿OOO．O．O OOOO爿QOO．．OCO O〔）〔）OOOO料O，．OO O寸門QQ【α一O判6o〇覧．． O○Q〔）○〔）O爿OO．．O mnめα○（〕ぴ寸O引D．．O
の ℃
　1田0↓ P尸一Ht −（団 　℃ 卜N 甑− 　個 　OO　　 N一 卜円 卜 c5　 Dり  　D 卜　 頃　 w　o 卩一噌 o尸司 一寸
oユeA ！J〔IZglHOOgr∩lH 　 Pt N 　 Pt
⊃
1⊆［H：）
0⊆IH：）
0セIH：）
1［IH：）
09 31ZIH
田 り
．
（頃 ：）
≧ 0 lH
o 乙 ⊃
ご。OlIH
つ
oじb
乃 1 ISNHlSNO
舅 乙 姶
ζ
℃
o
覧 乙τSNO oH
目oの ∫ISNOE 頃 　　一
‘Qoo 【SNOO 頃 ト い
正SNVOOtlHfOOEHZOOtlH OQ
霞p 一 − e7）
二（ ｛　
の N
蝦
巳 ¢ t丶
三
）〇Q τ00と丑｛ m N
q fooqHOOOGHOIO 【
ミ 卜 H
窺o 鬥　 σ　「 O咽G　　c尸o　　61　　　QO　　り　　o一
爵
嵐ミ． ：）HtoO卜　
國 ：〕HI 寸
』oOO9 乙0∀HZOOVHIOO．v「V冫HOOO
個 　
の 　 　【

五hゴQ ∀H 寸　 頃　刷 　り
）＞・［oogHO n
り。 一 一 （） 【 【 【
五‘鋒o〇轄q 　で層日一のコ邸 つ二」⇒じ口Oo NNり寸N．．O笛邑O一寸一．．卜OO岶寸O．．o寸い【N寸Ho．．寸 m［n寸．．OσOいq寸一5．．【O口O寸寸「丶．．O寸ゆOn寸一．．鴇q一〇【寸5「「．，邑Hめ【σ寸一う，．卜戸【O寸寸「，，ゆ一りO哺寸【．．Oσ寸りO「．．ひ〔鬥佃寸鬥り）．．℃OいN寸ト．，寸姶N寸N寸H ．．o めd寸oN．．寸一頃N寸【．．OO寸寸Om，，卜四【り［寸【勹．，鱈姶りd寸、，りNN尸寸冖O．，α寸寸⊃寸鬥σり，，OO［O寸冖d．．O個斡凹5Qe「．．卜n卜N【寸【．．り冒寸笛aρ「．．α一嶋寸O寸一「．．寸OOα帆O．．O寸一〇O寸【．．eo〇一ひ劇「「「．．りO寸一5一1．．寸寸燈o笛【o．．ひひ寸【りm【腎．．cd寸［り卜「．，mひm含斡ひ一c．．頃Nい℃の．，O寸卜嶋Dσ冖c「．．
ヨ廟ヨゴ唱呵撰oo口o 喇ひ一 冖＝訂口〇勹 　コ唄謂帽名』餡硫目づO． 　　　喟＝G⇔o」冨（OリoΩ 　一順）［ブHΣ留一の⊆目O 芝哨起〉剛≧の輯のd［［邸 　　　Σ＝図喟謂一一Q而龜ワ〇 　図｝‘の一b H〇〇」O．の 　　　薯刷』卜一切』冂O 　Σ図℃蝋H宀乞一り¢5り 　の一λ【曽弓祠【宀一の訂 　　　Σ嘱4」［一りO 0り0 唱7目o自謁日0．Z 　潤躍Oの＝o邸口のq， 刷』＝芝個 O口9邸〉邸 　　Σ州謂署一9戸國帽謦 　　　個韻謂Qロ雨o‘Oう 　［‘望溜ン輌0』ΦO． 　　　Σ〜』＜一而脇の 　　　　　Σ＝H鈴o 　　円Z一b屆昌の〇〇　コゆO
∩
， 。
一【ρ〇 爵。＝o N8E 蘯 留 d一a ヌ f　if　i 8 δ 6 ＝ハ ≧r邸 　σ［　　　　 顔　　　　 　く頃 　　一冒 Zき，
」「帽 puelSI op ！e羽OH 　邸 m 　H
一
NNI工I工-EElleoetcrotniroonic　 LLiibrbarryary 　Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society oofJfap anJapan
Phylogeographi Hcistory of Erebia n.iphonica 2S9
gg88 M8?E1e1? $1us9m$e dFd¢8 M8" o,1
88g8 -. .Y t- h .
dddd oooo " T.
g+1s g+1 +] g+1 g+1 R+I o+1 r g+1e

g ce gg 96g:

ddd6 dd6d
ge/"9pt"wnFpt-,ev#rcv[)/nt9'e:Gptpt!wnsFtsstwopt'e2n
ve g R wn X9E :KT- tvpt

- cu v- -pttr Esv-gEt7goEHvTog-gsvgofEv
vgvgoEffv zgvgoEEv
EEt]90EffV isvgo£gv
wnb ZEV90£ffV 09V90EflV
IEtr90EEV 6Lvgo£av
ll oEvgoEav 8LV90EEV
thee 6ZV90EflV LLV90EEV
: 8ZV90EEV gLvgo£gv
xt LZV90EHV gLt]goEflv
za pt st 9 gzvgoEav vLvgoggv

- 9 um glvgoEav ELvgofgv
trzvgoEgv zLvgoEav
t [Ztr90fEV ILV90EEV
zzvgogEv oLtrgogHv
IzvgoEgv 6gvgoEgv
OZV90fEV ggvgofuv
9, - 6tvgogfiv L9V90EaV
8Itr90£EV 99ts90EEV
LItr90fEV ggvgoEffv
gLvgo£av trgvgoEgv
gLvgoEav S9V90EEV
VIV90EEV Z9V90fflV
EIvgo£gv Lgvgogav
uvgoEav ogtrgoEgv
1Ltr90EgV 6gvgoEEv

O!V90EffV sgvgoEEv
6ovgoggv LgV90EHV
sovgogv ggvgogsv
£
LOV90EEV ggvgoEav
govgoEgv vgvgoEav
/
Ra8g8ag89 ks:eg eeggggvt xxdi
¢ueclfcio eti¢foitc etsposto g rptdmtsit- tnt-hq spnt nt"h rnt.-n ht"t. rdttXi tRctEXt eztdta cwtgoi mJpt2 o･"S"e-, dL2d go-g sQnazja
e- ---- rl -eeElr-.9.a<opc'PM<
gli i, ･ { eggi･Oca3asDEptZ8's'C89<gvv8eE8･tz8v<
/S,
/L,

mR}z m tc ->- =Z
ebpE E hZ hr' utt" ys f i zi z1.,8

NII-Electronic Library Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society oofJfap anJapan
260 Takatesh iN,xK,NTANI,Shin-ich Uis,"t iand Tateo I'ioH
Contir/enLalIlneag ieA muHforkka/dio ie/nea'ge sll'RT-'lE/lt;litZnern-gien rre uara -g- -e-g 'M -' 1e Hokkalbo
l NortihEm lineege / - Hn
Reg,Russie EE i Hs[=:1
l'E' t'pm
K"ll
KsEtw
o6wa
On-si-Som
lil
y.
i 2･.5r--
'--'
.s[Honsh Lli:neEge Honshv111111N-sNAk-NEX]As=]He"Hi-SMH[-NM[;"JGtwJv-S-Jy-NenKLetuypaYkggkiglyt
Fig. 3. Statistic aplarsirno nnyetwork. Circle sdenote haplotypes, size of the circle reflects the
wideness of distribut riuongnes in 3 levels ,small b]ack circles denot emissing or theoretical
haptetype sso,lid lines connccting each pair of haplotype resprcsent a single niutational step,
regardlcss of their length .Pie charts shew the proportions of regions within each haplotype.
Uncolored haplotypc was detected in one region only. The haplutype fsroTn the ¢ontinenr
are from Khrebe tKhekhtsir ,Amur region, Russia (N48015'21 "E,l35e03'57") and Tsenkher
Jiguu rSPA, Hangai Mts, Mongolia (N47"19'1 6EI"O,I039'25"), Geneti cdistanc e(s%) are
calculated in Kimura's 2 paramcte rmethod based on COI (51 Obp).
VV' ecan see that the ecological characteristjcs of Erebia niphonica well satisfy the afOre-
mentioned necessary conditions for the study of phylogeographi hcistor yS.ince the goal of
this study is to know how the glacia clycles of the Pleistocene affected the geneti cdiversity
of alpine butterflie si,t is desirab lteo collect samples at many sites covering a wide range of
geographica dlistribut i(oVni leta al., 2005).
2.Materials
and methods
2.1 Specimens and collection of molecular information
We have collected 423 individua lisn 289 populations in tota], including 147 individua lisn
119 population sin Hokkaido and 276 individua lisn 170 population sin Honshu. Fig. 2
shows the distrihut iaondn sampl.ing sites of Erehia niphonica jn the Japanes eisland swith
7.5' longitud ex 5.0 'latitud eas a unit, and the sampling sites cover appreximately 70% of
the distribut iroannge. Samples were col]ected betwee nyears 2003 and 2005. Although
many samples including males and females are required fbr reliable statistical analyses,
from the standpoint of nature conservation, we have collected only males as samples. In
samples from some areas, specimens were include dthat had been collected, drie dand pre-
served since 1996. We also analyzed 2 individua losf Erebia neriene from the Khangai
NII-Electronic Library Service
TThheLee piLdeoppteirdoloopgitcaelrSooclieotygical Society oofJfap anJapan
Phylogeograph iHcistory of Erebia niphonica 261
4-1
1-12
HFO03 HFO043-2
3-1
2-1 HFOOI
Hcoe2
1-11HFD02
1-1
2-4
HCOOI
2-31-1
1-2HCOIO
HEOOO1-9
1-3HAO02HAOOI1-5
1-S
HAOOO HDeoeHDO03
1-HA023HAe21 HBeOl
HAO031-6HAOe4
HbOOIHDO02
HAOIe
HA022
HA020
2-2
4-2
3-3
2-61-15
1-16NS17e
NSI12NSIII
2-5
1-13NS130NS131
NSI13 NSI14
1-14
NS150NS121 Nsleo
1-17
2-9CHUT1-23CH14e 1-IS 2-7 3-4
1-19
1-22
CHIIO CH151CH152
CH131CH130
1-21 1-2D
CH121CH12e CH160
en161CH162
CH122CH123CH150
2-8
F ig-. 4, Haplotyp enetwork design,AllhapLetypesare nested .tn4t hlevel
clade in both islands.andassociatednested
NII-Electronic Library Service
TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society oofJfap anJapan
262 Takatogb iNAKATANi ,Shin-ic hUisAMi and Tateo IToH
Table 2. AMOVA results for tes tof geneti scubclivision betwcen populations,
(a)Holtkaide

Variance
Source
of variation of variation
components%
Among mountain regions O.746*O.133O.4679.34*8
Among populuti owinthin mountain regions 8.4544.07
Within o ulation
(b)Honshu

Variance
Source
of variation of variation
components%
Among mountain regions 1,492*O.165*O.27779*.08
Among populatien within mountain regions 8.5114.41
Within o ulation
*P<O.OO1.
Mountains in Mongolia and 2 from the Amur region in Russia fbr reference. Each sample
was caught with a net, wrapped in parathn paper ,and brought back within two days .Thc
body of the sample without wings was preserve din 99% ethanol and kept at 40C until DNA
was extracted,
For genetic markers, we used a part of the NADH dehydrogenas esubunit 5 (ND5 )and cy-
tochrome oxidase subunit I (COD genes in mitochondrial DNA. DNA was extracted from
the body and legs by DNeasy Tissue Extractio Knit (Qiage nP)C.R was conducted in a 10
"t reaction system by adding DNA (c a40 ng), as recommended by Applied Biosystem sre-
action constitution. Thermal cycler profil ewas one cycle at 950C for 10 min; 25 cycles at
950C for 30 sec, 45eC for 30 sec, 720C for 1 min 30 sec, one extension cycle at 720C fbr 8
min 30 sec. PCR product swere processe dafter Exo-SAP treatment .The nucleotide se-
quences of the amplified DNA fragment swere determined by direc tsequencing method
with Big Dye [Ilermina tvol.r1 Cycle Sequence Kit. As primers for PCR and direc tse-
quencing ,we used Vl (5'-CC GTTT TCT GCT TTA GTT (]A-3') C,2 (5LMC YTT WGA
MTA AAA YCC AGC-3') (Yag eit al., 1999) for ND5, and mtD6 (Cl-J-17 15'8-;GGA GGA
TTT GGA AM TGA TTA GTT CC-3'),Nancy (C1-N-2191; 5LCCC GGT AAA ATT AAA

MA [IAA ACT TC-3') (Simo net al., 1994) for COL We aligned the nucleotide sequence
data with Clustal X (Thompson et al,, 1997) ,and conducted the fragment, comparisen of
432 bp fOr ND5, 51O bp for COI, and 942 bp in total.
2.2 Analysis of geneti ccharacteristics of populations
Tn order to test isolati obny distanc ew,e divide dthe Hokkaido population into 12 groups
and the Honshu population into 17 groups ([fab l1)e .We then computed the geneti cvari-
ance at each hierarchic alleve lwithin a populatio nb,etween populations ,and between
groups based on AMOVA (Excodi eet ral., 1992) by software Arlequi n2.0 (Schneid eetr al.,
2000). The number of permutation wsas set at 10,OOO times when computing. Genetic di-
versity of a population is considered to increase by repeating the proces sof range fragmen-
tation int oloca lgroups and re-merging under climate changes (DeChain eand Martin,
2004) ,We calculated standard diyersi tiyndice sby Arlequin 2.0 as the indicat oorf genetic
diversi tofy the population,
2,3 Nested clade phylogeographical analysis
Nested cEade phylogeographic aalnalysis (NCPA) ([Ibmple teotn al,, 19951 [fempleton,
1998, 2004) can infer past events that aifected the populatio nconstituents of a species such
as past fragmentation ,restrjcted gene flQw and range expansion by combining haplotype
NII-Electronic Library Service