Molecular identification of maternal species of natural hybrids between Parnassius citrinarius and P. stubbendorfii (Lepidoptera, Papilionidae) in Hokkaido

5 Pages·2001·0.48 MB·

TThhee LLeepipdiopdteorpoltoegircaollSoocgieitycal Society ooff JJaapapnan
utLwh T)ans. Iepi[Stoc.Japan 52C3)i ]63-167, June 2001

Molecular identificati oofn maternal species of natural hyi)rid sbetween
Ptiitnassii ecsiM'nan'us and R stubbendoij7i (Lepidopt Pearpial,ionidae)
inHokkaido

TakashiYAGii)' KeiichiQMoTo2)"'
and
i)Research Institut efbr Advanced Science and Technology, Osaka Prefbcture University,
1-2Gakuen ho,Sakai,Osaka,599-8570Japan
£
2}International Researc]iCenter forJapanese Studjes,

3-2Oeyama-cho,Goryo,Nishikyo-ku,Kyoto,610-1192Japan

Abstract A syrnpatric distributi oonf similar butterfli Res ,stubbendoifZl and R citrina"ius, is
formed in central Hokkaido. In these areas, butterfl iwietsh yellow body hairs a,n intermediate
character between the two species, are frequentl fyound in the butterf ipyopulatio nand thought
to be natural hybrids between the two spocies. Here, we analyzed mitochondriai DNA, which
i smaternally inherite din eukaryotes, of butterfi i-e'sith yellow body hair sto clarify their
maternal species. The mitochondrial ACD5 gene sequences from all fiv ebutterfl iceorsresponded
to those from P: citrinarius, indicating that their mothers were R citrinaritLs. This finding
supports the earlier finding sfrom artificial hybridizati osntudjes, which suggested that butterfiies
with yellow body hairs from the habitat fsor both species must be natural hybrids between female
R citrinarius and male R stubbendoi:fii,
Key Hybrid,Parnassius, IVD5, inheritance.
words mtDNA, maternal
Introduction
Ptimassius stubbendolzlZi M6netries inhabit sa 1arg earea to the nomb of the Huang River and
the east of the Altai Mountains in the East Asian subcontinent, which include snorthern
China, the Korean Peninsula ,Mongolia and Siberia .R cimnarius Motschoulsky (=R
glaciali yButler ;(zlCI Okano (2000 i)n)habit sa larg earea to the south of the Huang River in
China on the continent, In the Japan islands P,L stubbendodi inhabit sHokkaidQ only, and
R cimnarizas inhabit smountainous areas of Shikoku, Honshu, Sado Island and southern
Hokkaido. At the northern and eastern areas of the Ishikar Pilain in Hokkaido, the habitats
of the two species overlap (se reeviews Nemoto & Inomata, 1994; Weiss, 1999).
R stubbendodi and R citrinan'us have grey and orange hair son their bodies ,respoctively, but
their other morphological characteristics includin gthe wing pattem are quite similar
(Nemot o& Inomata, 1994 ;Weiss ,l999) ,In the laborator yt,he two species can be crossbred
easily by handpairing (Kitaha r&a Kawata 1991 ,1993). The copulation by handpairing
was successfu1 only between female .PL eitrinarius and rnale R stubhendocfZi, depending on the
structure of their genita lorgans, Their resultant F, hybrid sgrow up normally, and have
yellow hair son theiT bodies ,which show intermediat heair color of their parent s(Kitahara
& Kawata,1991,1993).

'Corresponding author: e-mail: yagi-t@riast,osakafu-u.ac.jp; phone: t8]-722-54-9862; fax:
[phone].
*'Present address: Faculty ofLiterature, St ,Andrew's Universit y1,-1 Manabino, Izumi, Osaka ,S94-
1198 Japan.
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164 Takashi YAGi and Keiich iOMoTo
In the overlapped habi'ta tofs the two species in Hokkaido, butterfi iweitsh yellow hair son
their bodjes are frequentl fybund in populations of R stuhbendotM and R citn'narius (Kita-
hara & Kawata, l988 ,1991 ,1993 ;Kitahara ,1990) . These butterfi iweesre suggested to be
natural hybrid sbetween the two species. If this supposition is correct, thejr maternal species
should be R citmbarius as shown previousl yin the laborator y(Kitaha &ra Kawata, 1991,
1993). To ¢onfirm this, we examined their mitochondria lDNA (mtDNA) sequenoes,
because the mtDNA is maternally inherite idn eukaryotes includin gLepjdoptera (Lansman
1983).
et al,,
Materials and methods
All butterfl iweesre collected at Hobetsu £ ho, Yufutsu County, Hokkaido, and have been
stored in ethanol, One R stubbendbcM, one .il citrinarius and fiv ebutterfl iweisth yellow
hair son their bodies (Hybri d1s-5 in Figs 1 & 2) were used fbr analyses, DNA was extracted
from the museles of the thorax of each butterfl yby the method described previousl y(Yag iet
aL, ]999).
A part of the mitochondria lDNA sequences C54 2base pairs )coding for NADH dehy-
drogenase subunit 5 (ND5) was amplified with primers Vl (5'-CCTGTTTCTGCT-
TTAGTTCA-3') and A4U (5'-GTATAAAACATTGTTAAACCTGTAG-3') and the
ExTaq DNA polymerase (Takar aKy,oto) by the polymerase chain reaction (PCR) with
experimental conditions supplied by the company. The PCR products were purjfie dwith
the Quantum Prep Spin Columns (BIO-RAD ,Hercules), The nucleotide sequences of the
PCR produets were directl ydetenmined with the primers and the DyeTerminator Cycle
Sequencing FS Kit by an automated DNA sequencer 373A (PE Applied Biosystems F,oster
City) (Yag iet aL, l999).
Since the determined DNA sequenoes showed that two and one endonuclease sspl recogni-
tion sites (AATATT) were presen tin the R citrinarius and R stubhendonjZi sequences,
respectively, the restriction fragrnen tlength polymorphism (RFLP) thereafter was used to
distingui sthhe two sequences. The PCR products were digeste dwith the endonuclease sspl
(Toyobo O,saka) at 370C fbr 2hr, and the digeste dproducts were separated with 1%
polyacrylamid egel electrophoresis at 1oo V fbr 40 min in TAE buffe rsolution, The DNA
fragrne nitn sthe gel were stained with 10 (g/m lethidium bromide solution for visualization
(Sambroo ket aL, 1989).
Results and discussion
Nucleotid esequences ofa part ofthe mitochendrial IVD5 gene (54 2base pairs )are shown in
Fig. 1. The differen ceefthe nuc]eotide numbers in the sequences between R eitmbarius and
R stubbendocM was 2.78% (1 4bases) ,and, therefbre, the two species can be distinguished
clearly by the ND5 sequences. Most of the difiere nntucleotides were found at the codon
silent position s.The sequences from a butterf lwyith yellow body hair s(Hybri d1) were
identic atlo those from R eimbiarius.
As the ND5 sequences ofR citrinarius and R stubbendoijZi had two and one sspl recognition
sjtes ,respectively (Fig s1 and 2A), other butterfi i(eHsybri d2-s5) were analyzed with the more
rapid RFLP method. Electrophoresis pattern sof sspl-digested PCR products are shown in
Fig. 2B. DNA fragment sappeared at the positjon sexpected from the sspl restriction map
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Hybrids between R citrinan'us and P: stubbendbiIEi 165
Vl primer
Gl'SlC"CTGTITCTGCTrrAGrrCATTCTTCTACTI-rAGTAACTGCTGGAGTTTAmpAAIT
CCTGMCTGCrrrAG"CATTCTTCTACTTTAGTAACTGCTGGAGTTTATnbTAATT
*
G61'S6C1G"ATnAATATA"ATTAATTGATATATTTTTnTTAAptrrTArreTATTATCMGGA
CGATTTAATATArrATTAATTGATATATrTTnTTApmTAmpATTATCkiGGA
ss p: * * *
G121,STITZAIA"pmTATAGCTGG[l]AptCTGCTAAITATGAAT'l-T'GA'TTTAAAAAAAATTA'rr
TTAAdyTATTTATAGCTGG@TptCTGCTAATTATGAATTTGAnTAAAAAAAArrATT

* **
G181TSG1C8T1T"TATCAACMAAGACAATTAGGTTTAATAATAAGAAT"[-ITAAGAATAGGAITACCA
GCTTTATCAACMAAGACAATTAGGTTTAATAATAAGAATTTTAAGAATAGGATTACCA
G241'SZ41"GAArrAGCTTTnTTCATTTATTAACTCATGCTATArrTAAAGCTTTATTATrrATATGT
GAATTAGCTTnTrrCAnTATTAACTCATGCTATATTTAAAGCnTAnATTTATATGT
G3el, GCguGGGGTAATTATTCATATAATAAATGATAATCAAGATATTCGITITATAGGkiGGTATT
S3elT,GCSGGGGTAATTAI-rCATATAATAAATGATAATCAAGATATTCGTTTTATAGGfiaGGTATr

* sspr *
G361, AGATTATATArrCCAATAACTTCTTTATGmiAAATATrrCTAAnTAGC"TATGTGGT
S361it AGATTATATA-Fl'CCAATAACIHI'CTTTATGavAAATAI-T-1'CTAAIUT"TAGCTIUI'ATGTGGG
* ssp:
G4ZIT ATTCCrmTTAGCTGGptTTATTCTAAGGAITrAATFrTAGAklATAGTAAGAA@GA
S421t, AnCCTITMAGCTGGNTnATTCTAAGGATTTAArrrrAGApmTAGTAAGAAIAAGA

* * *
G481i AAnTAAATATATTAavTTTTTTTrrATAVAMTrCTACAGGTrrAACAATGMTAT
S481' ･AAIHT'TAAATATATTAptTTTT"[Tl'TATA'FT'AT'FITT'CTACAGG'FtHT'AACAATG'[HT'ITAT
' A4U primer
G541T AC

S541" AC

Fig, 1. A part of the nucleotide seqaences of the mitochondrial ND5 gene from R citrinarius
(uppe rand) R stubbendbjyZf (lower ),Double underlines show Vl and A4U primer sfbr
PCR and sequencing. Underline sshow endonuclease sspl recQgnition sequences.
Letter swith asterisks and those surTeunded with boxes show diffbre nntucleotides
between the two species.
(Fi g2,A), in addition to a few incompletel ydigested bands (41 4and 542 base pairs ).The
position sof the bands from butterfl iweitsh yellow body hair s(Hybri d2s-5) were identical
to those from Rciit:inan't cs(128,1 4a6nd 268 base pairs ),but not to those from R
stubbencloee (14 6and 396 base pairs) .These finding sindicat ethat mtDNA of Hybrids l-
5 was introduce dfrom R citrinarius, and in Qther words, the mothers of Hybrids 1-5 were
R
citrinan'us,
The presen tfilldin gagsree with the laborator ystudy done by Kitahara and Kawata (1991,
1993) , Most butterfl iweisth yellow hair son their bodies in the everlapped habita tof the
two spocies in Hokkaido must be natural hybrids between female R citmbiarius and male R
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166 Takashi YAGi and Keiich iOMoTo
A
128 268 146
m e r-----=
sspl Sspl sspl
Vl
A4u
SSPI
- -
396 146
B
asLOR8gtajn-+
g

.9 N ; tr
-Ig vB 5- p.ts.x

aj
=)ts .x
n n n-+ n-+

- -
ssp] + +

542 542396
sgg
sg 146
Fig. 2. The sspl restriction map of the iVD5 gene from PL citn'narius Cupper )and R
stubbendocM (lowe r()A) ,and an electrophoresis pattern of the sspl-digested 2VD5 gene
(B) .The414 and 542 base pair bands ofthe sspl-treated lanes are ascribed to incomplete
digestio nof the restriction sites,
stubbendbeM. As Kitahara and Kawata (199 3s)uggested that copulation by handpairing
was rarely suecessfu1 between small female osf R stuhbendoizM and male R citrinarius, a small
populatjon of F, hybrids of this combinatien may also be present in the overlapped habitat.
More analyses are required to confirm this.
In the overlapped habitat softhe two species in Hokkaido, a high proportio n(IO-25 %of)the
hybrids was fbund in the parnassia npopulation (Kitaha &r aKawata, 1993) . This observa-
tion suggested that the two species with the same wing patter nmay not recognize each other
as a differe nstpecies and hybridi zveery often, However, neither nuclear nor mitochondrial
genes would be introgress itvoe the other species because the Fi hybrids are sterile (Kitahara
& Kawata, 1991). The diversificati ontirne of R citmbiarius and RstubbendoifZ ffrom a
common ar)cestor is estimated to be several million years ago.(Omoto et aL, in press) . These
finding sindicat ethat the speciation of R cininan'us and R stubbendoi M has been completely
established despite their frequen tnatural hybridization.
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Hybrids　between　P．　citrinarius 　and 　P．　stubbendorfO 167
Acknowledgement
We 　are 　most 　gratefu　lto　H ．　Fujii　M ．　Kawata　and 　Samuzawa 　f（）r　supplying 　the　butterfiy
，
samples ．
Refere
皿ces
Kitahara，　H ．，1990．　 Comparison　of　male 　genital　ioaf　natural 　hybrid8　in　sympatric 　habita　tof　Parna∬ius
　　g如cぬ趣　Butler　and 　R　hoenei　Schweitzエe （Lepidoptera：Papilionida）e．　乃の　Gα　41：45−49　（in
　　Japanese 輌 th　English　abstract ）．
Kitahara，　H ．＆ MKawata ，1988．　 On　the　hybrid乙iation 　betw  n　Parna∬ius　glaciagt　Sand 　P．　hoenei．
　　Natuノ’e　Inseca　s23 （6）：20−24 （in　Japanese），
　　　　．　ho　en，ei　1S9c9hLw　eiFtezretri（Plai　ptoiyfl　iarotnifiidcai）ae．　l　an7dン　∂na（tPuar　a4l4　：hy1b2r0−i1d　2sb6e（itnw　eJeanp　aPneαs用e　αw∬itihu　s　Eglglagcliia　slah　ibBsSturtaclte　）r．and 　P．
　　　　，1993，　Some 　results 　of　the　observation 　on　the　interspeci　frielcationship 　between　Pamassius
　　giaciali　sButler　and 、P．乃oenei 　Schweitzer．　乃∂σα 42： 53−62 （in　Japanese　with　Ellgli　sahbstract）．
　Lan　sm‘aしpnar，e　nRt．　a　All．e，　akAavgiese”，of」．　mCit．oc＆honMdrDial ．　H　uDeNtAtel，．1」9P8r3o．　c．　nCartnit．i！cla　cleaxdp．　erSicmetntaひl S　．t．e4s．t　8o0f：　t1h9e6　9p−o1s9s7i1bi．lit　yof
Nemoto，　T，＆ T．　Inomata，1994，　 Geographica　land 　individua　lvariations 　of　the　genus　Pα〃la∬泌
　　　　LMautsrhelil−slhe，a1，　8T0o4ky（8o）．　，（Ilnn　　OJhaypaane，　sAe，）＆．T．　Inomata 　T，（Eds），」伽 義selected　lnsects  （A）（8）： n7 −132．
Okano，　K ．，2000．　 On　Parna ∬’ ∫ citrinarius　Motschulsky　1866　and 　Pα厂刀α∬嬬 glaciali　SButle　r1866
　　（Lepidopter：aRhopalocera，　Papilionida）e，　Tokura’謬α，　MtShima （25）： 1−5（in　Japanese　w 量th　English
　　abstract ）．
Omotb ，　K ．，　Kato，　T．。　Chichvarkhin ，　A ，＆ T．　Yagi （in　press）．　 Molecular　systematics 　and 　evolution 　of
　　butterfl　iocfs　the　genus　Pα切 α5鋤 5，　Papilionidae，　based　on　mitochondrial 　DNA 　sequence 　data．　 Proc．
　　21st　int．　Congn　Ent，
Sambrook，　J，，　Fritsc，　hE．　F，＆ T，　Maniatis，1989．　 M ∂lecul厂a Cloning．　 A 五aboratoi ア Manual ．（2nd
　　Edn ），　Cold　Spfing　Harbor　Laboratory　Press，　Cold　Spring　Harbor ．
Weiss，　J．−C．，1999．　 The　Pq厂ηα∬ ’ ∫ qブ読e 肋 r観，　Part　3．　 Hillside　Books ，　CanteTberr．y
Yagi，　T．，　Sasaki，　G ．＆ H ．　Takebe ，1999．　 Phylogeny　of　Japanese　papilionid　butte伍res　inferred丘om
　　nucleotide 　sequences 　of　the　mitochondrial 　ND5 　gene、　 J．　mo 毳〜c．　EvoL　48142 −48．
摘 要
・
北海道にお けるウスバ シ ロ チョ ウ ヒ メ ウスバ シ ロ チョ ウ種問雑種の母親種の分子生物学的方法
・ 一
に よる同定 （八木孝司 尾本惠 ）
・
北海道中央部の ウスバ シ ロ チ ョ ウ ヒメウスバ シ ロ チョ ウ混棲地に は両種の 中間的な淡黄色 （ウスバ
−
シ ロ チョ ウ橙色，ヒメ ウスバ シ ロ チ ョ ウ灰色）の体毛 を持つ 個体が 10 25％存在するこ とが知られる．
北原 ・川田の精力的な研究によれば，両種のハ ン ドペ ア リン グに よる人工交雑はメス の ウスバ シロ
一一・
チョ ウとオス の ヒメ ウスバ シロ チ ョ ウとの間でのみ容易に成立 し，その雑種第 代 は正常に発育 して，
淡黄色の体毛 を持つ 成虫となる，この こ とか ら混棲地における淡黄色体毛を持つ個体は両種の 自然交
雑体である と推測 され て きた．我々 は ミ トコ ン ドリアの母系遺伝を利用 して，混棲地で採集された淡
黄色体毛を持っ 個体の母親種が ウス バ シ ロ チ ョ ウか ヒメウスバ シロ チョ ウか どちらで あるか知る こ と
を試みた．その結果，調べ た 5個体全ての ミトコ ン ドリア DNA の塩基配列は ウスバ シ ロ チョ ウの も
の と一致し，それらの母親はウス バ シロ チ ョ ウで あることが わか っ た．この結果 と北原 ・川田 らの結
果によ り，混棲地にお ける淡黄色体毛を持つ 個体はメスの ウスバ シロ チ ョ ウ とオ ス の ヒ メウスバ シ U
チョ ウとの 自然交雑体で ある と結論で きる．
（Accepted　February　26，2001）
Publishe　dby　the　Lepidopterologic　aSlociet　yof　Japan，
5−20，Motoyokoyama 2，Hachioji Tokyo ，192−0063Japan

，
一
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