The LepidopterologicalSocietyLepidopterological Society of Japan
ectva Trans. Iqpid. Soc. ,Japan 57 (2): 137-147, March 2006
Phylogeny, biogeography and wing pattern convergence of the subgenus Achillides (Lepidoptera, Papilionidae, llapilio) reyealed by a mitochondrial DNA sequence analysis
TakashiYAGii)*. Go SAsAKT2)** and Keiichi OMoTo]'*']:*
]' Laboratory of Environmcntal Genetics, Frontier Scjence Innovatioii Centcr, Osaka Prefecture University, 1-2 Gakuen-cho, Sakai, Osaka, 599-8570 Japan 2) Department of Biophysics, Graduate Scliool of Sciencc, Kyoto University Kitashirakawa-oiwake-cho, Sakyo-ku. Kyoto, 606-8502 Japan ]" International Research Center for Japanese Studies, Goryo-oeyama-cho, Nishikyo-ku, Kyoto, 61 O- 1 192 Japan
Abstract Achitlides is a subgenus of the genus Pcy)ilio commonly called swallowtail hutterflies, which inhabits East Asia ineluding Japan. Achillideh' consists of many similar species, of which subspecies are distributed in many East Asian islands. Here, we analyzed phylogenetic relation- ships among these spccies and their subspccies with nuc]eotide sequences of thc mitochodrial ND5 gene. The ana]ysis revealed that Papitio polyctor and P. bianor bianor are an identica] specics, and P. s)tiitnius and P, maackii are also an identica] species duc to the identity of their nucleotlde sc- quences, Thcsc butterfiies have been considered taxonomically different species based on their dis- tjnct sving patterns. The analysis also rcvcaled that Achitiides are separated into two clades, which, however, do not agree with the grouping bascd on wing patt,ern similarity. One clade is composed of P. bianor, I'. polyctor, P, hernteli, and P, dialis, while the other clade is compesed of IJ. maackii, P, s)ifanius. P. krishna, P, hqmionis, P, arcturus, P. karna and P. paris. Several pairs pf species having a similar wing pattern are present in both cladcs. These findings may be evidence of paral- lel evolution, indicating that rnorphologieal cons,ergence occurred between the species belonging to different clades after their ancestors had divergcd. Speciation due to the wing pattern changes may have occun'ed in a relatively short period during the evolution ofAchiltides. Finally, we also dis- cuss the revision of classification of the P, bianer subspccies and P. ,fitscus based on DNA sequence ana]ysis.
Key words Achittides, P. bianot', 1', maackii, phylogeny, biogeography, convergence, rntDNA.
Introduction
Molecular phylogeny of papilionid butterflies, commonly called swallowtails, has been ex- tensively studied recently (Sperling, 1993a, b; Caterino & Sperling, 1999; Yagi et al,, 1999; Kato and Yagi, 2004; Zakharov et al., 2004), because their taxa and werldwide distributions were well elucidated. Achitlides is a subgenus of the genus lhpilio, which mainly inhabits east Asia, AchiUides consists of at least twenty morphologically similar species all of which have black wings covered with beautifu1 bright green scales (Shimogori, 1997:. Bauer & Frankenbach, 1998). Some species of Achillides are distributed in the far east Asian is- lands, where the species are classified into subspecies,
One of the issues of the subgenus Achiltides is the presence of taxonomically ambiguous
"Corresponding Author: E-maii: [email protected],ac.jp, Phone: +81-72-254-9862, Fax: +81- 72-2S4-9938 "":Present Address: Biohistory Research Hall, 1-1 Murasaki-cho, Takatsuki, Osaka, 569-1 125 Japan. ""'Thc Graduate University for Advanced Studies, Shonan Villagc, Hayama, Kanagawa, 240-O193 Japan.
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138 Takashi YAGi, Go SAsAK] and Keiichi OMoTo
greups. The taxonomic relationships between l'kepitio polyctor and P, bianor and those be- tween P. s)lfLinius and P. maackii have been disputed for a ]ong time, because a cline of wing patterns between the species was found in some areas (Harada, 1992; FLijioka, 1997; Ybshimoto, 1998). Most taKonomists classified these groups to different species because their typical wing patterns are obviously distinct (Igarashi, 1979; Shimogori, 1997; Bauer & Frankenbach, 1998).
The classification of P. fLtscus is also ambiguous, The adult P. .fttscus has the wing pattern that is a typical merphological feature of the subgenus Menelaides but its larva ttnd pupa have morphological features of the subgenus Achitlides. Igarashi (1979) classified P, ,fas- ctts as belonging to Achiltides.
Another unresolved issue is the classification of P. bianor, which has a latitudinally wide distribution in east Asia. P. hianor is distributed from Russian Sakhalin Island, a subfrigid region, to Vietnam and Thailand, subtropical regions, through Korea, China, Japan and the Ryukyu Archipelago, temperate regions. P. bianor is classified into many subspecies (qfl Table 1), and some of them are suggested to be genetically distinct as species (Kawazoe and Wakabayashi, 1976). These observations prompted our interest in phylogenetic rela- tionships among the subspecies.
Table 1.Taxa and locality of specimens ofthc subgenera AchiUides and Menelaides used in this studyi'.
speclessubspecjeslocaLity database acccssion number Achillides bianofr'ihianor Fujian,China AB212916 Siehuan,China AB2i2917 Guangxi, China AB2I2918 dehaanii Sakhalin, Russia AB212919
Myohyangsan, North Korea AB212920 Chunchon, South Korea AB212921 Biei, Hokkaido, Japan AB212922
Ishinomaki, Miyagi, Honshu, Japan AB212923 Tohi-cho, Shizuoka, Honshu, Japan AB212924
Shirahama-cho, Wakayama, Honshu, Japan AB212925 Kyoto, Honshu, Japan ABO13156
Kamiyama-cho, Tokushima, Shikoku, Japan AB212926 Kasuya-cho, Fukuoka, Kyushu, Japan AB212927 Tsushima Is., Japan AB212928
tokaraensis Tokara-Nakanoshima Is., Japan AB212929 Tokara-AkusekijimaIs.,Japan AB2t2931
hachijonis Hachijo Is. 1, Japan AB212930 Hachijo Is. 2, Japan AB223163 Miyakejima 1's. 1, Japan AB223164 Miyakc.'lima Is, 2, Japan AB223165 Mikurajimals.,Japun AB223166 thrasymedes Nantou,Taiwan AB223167 Taoyuan, Taiwan AB223168
kotoensis Lanyu Is.. Taiwan AB223169 Huoshao Is., Taiwan AB223170
okinawensis Iriomotejima Is., Japan AB231690 lshigakijima Is., Japan AB231691 TaketomijimaIs.,Japan AB231692 Yonagunijima Is. 1. Japan AB231693
Yonagunijima Is, 2, Japan AB231694
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139 MoLecular Phylogenetic Analysis of Subgenus Achillides
Table1 (cDntinued),
amamlensls Amami-oshima Is. 1, Japan AB239775
Amami-oshima Is. 2, Japan AB239776
Tokunoshima Is. 1, Japaii AB239777
Tokunoshima Is. 2, Japan AB239778
ryuk),uensiti Okinawa.iima Is. 1, Japan ABO13157 Okinaw ajima Is. 2, Japan AB239779 po4>'ctor pol.t,ctorstockleFiKashmir. India AB239780 West Thailand AB239781 triuitrphator Laksao,Laos AB2397S2
Nonhet, Laos AB239783 ganesamaackii Tibet, China AB239784 maackii Primorskyi,Russia AB239808
Hciloajjiang, China AB239809
Jilin, China AB239810
Zhejian, China AB239811
Sichuan, China AB239812 Solaksan, Seuth Korea AB239813
tutamcs Sakhalin, Russia AB239814 HokkaidQ,Japan AB23981S
Scndai, Miyagi, Honshu, Japan AB239816
Yuzawa-cho, Niigata, Honshu, Japan AB239817
Kyoto, Honshu, Japan AB239818
Yamashiro-cho, Tokushima, Shikoku, Japan AB239819
Kasuya-cho, Fukuoka, Kyushu, japan ABO13158
Kikuchi, Kumamoto, Kyushu, Japan AB239820
Tsushima Is., Japan AB239821 TanegashimaIs.,Japan AB239822
Yakushima Is., Japan AB239823
s)lfanius albos)ll? nius Yunnan, China AB239824 kitawakii East Tibet. China AB239825
hermelidiatiskrishnahclprponisMindero ls,, Philippine AB239826 Nantou, Taiwan AB239827 NepalNantou, AB239828
Taiwan AB239829
arcturusPans Sichuan, China AB239830 parlsnakoharai Guangdong, China AB239831 Nantou, Taiwan AB239832
hefmosan"s Nantou, Tuiwan AB239833 tamilanakamalrauanacamatusSouth India AB239834 karna West Java, Indonesia AB239835
Palawan, Philippine AB239836 Sabah,Berneo,Malaysia AB239837 palinurus Malaya, Malaysia AB239838 peranth"s Bali Is., Indonesia AB239839 juscus Celebes, [ndonesia AB239840 Menetaides protenor Kyoto, Hunshu, Japan ABO13153
helen"s Kyoto, Honshu, Japan ABO13152 polytes IriomotejimaIs,,Japan ABO13151 i} Revision of classifictttion Df P. bianor, P. pol.yctor, P. s)eftinius and P..iiiscus is proposed in the text ef thispaper, ?) The names of P. bianor subspecies in this paper tentatively fo1]ow the nomenclature used in the books of Hidaka et al. (1984), Inomata and Matsumoto (1995) and Fujioka (1997). It should be noted that the other nomenclature used by Shir6zu (1966) and Kawazee and Wakabayashi (1976) is also present.
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140 Takashi YAGi, Go SAsAKT and Keiichi OMoTo
Tb reveal taxonomic or phylogenetic relationships among these rather complicated species and their subspecies of the subgenus Achitlides, nucleotide sequences of mitochondrial DNA should be very useful, The mitochondrial NADH dehydrogenase subunit 5 (ND5) gene is one of the fastest-evolving genes CGaresse, 1988) and has been used for the molecu- lar phylogenetic analysis of insects (Su, Tbminaga et at., 1996; Su, Ohama et al., 1996; Su et al., ]998). We previously showed that ND5 sequences could be used to reveal the phylo- genetic relationships among lower categories of I]epidoptera (Yhgi et al,, 1999).
In this paper, we show phylogenetic trees of mogt species and their subspecies ofAchiltides based on IVD5 nucleotide sequences, and discuss the relationship between phylogenetic di- vergence and wing pattern evolution, We also show iVD5 phylogenetic trees of P. bianor collected at various localities in east Asia, and discuss the classification and biogeography of the species relative to the paleogeography of the Ryukyus and Japan, the far east Asian islands.
Materials and Methods
Most species of the subgenus AchiUides as l-5-year-old dried specimens were kindly sup- plied by various Japanese entomologists (Table 1). Most butterfiies collected by the au- thors, mainly in Japan, had been stored in ethanol until use, DNA was extracted from mus- cles of the thorax and legs by thc phenol-chlorofbrm extraction method described previous- ly (Yagi et al., 1999), In case of the mounted $pecimens, a few legs were removed forDNA extractlon.
A section of the mitochondrial ND5 gene (81O bases) was annplified from the DNA by poly- merase chain reaction (PCR) using primers V1 and Al, also described previously (Yagi et al., 1999). Nucleotide sequen ¢ es of the PCR products were directly determined with the BigDye[Ibrminator Cycle Sequencing FS Kit by an automated DNA scquencer 373A or 377 (Applied Biosystems). The primers Vl ancl Al were usually used for the nucleotide se- quencing, and internal primers A3 (5LTTCGAfrrTTAGCTTTATGTGG-3') and C2 (5'-M- CYTTWGAATAAAAYCCAGC-3') were also used when Vl and A1 did not producc clear results.
ND5 nucleotide sequences (789 bp) of the samples were edited and aligned using the YboEdit software (version 1.63). Phylogenetic trees were constructcd with the neighbor- joining (NJ) methed and the unweighted pair-group method using an arithmetic average (UPGMA) by the PHYLIP software package version 3.572c (Felsenstein, 1993), or with the maximum parsimony (MP) method by the PAUP software version 4,0 beta (Swoffbrd, 1998), For NJ and MP methods, P. memnon, P. protenor and P, helenus belonging to an- other subgenus Menelaides were taken as outgroup species. The confidence level of branching in the phylogenetic trees (NJ and UPGMA) was evaluatcd with the beotstrap test based on 100 rcsamplings (Felscnstein, 1985), The nucleotide sequences of the ND5 gene of these butterflies were registered in the DDBJ/GenBank!EMBL database.
Results
Alignment of the ND5 nucleotide sequences from Achitlides species and their subspecies revealed that DNA length variation was not present and the maximum pairwise base differ- ences were 6.29b, suggesting that ND5 sequences are valid for the phylogenetic analygis of Achiltides, The phylogenetic analyses ofAchilZides species and their subspecies were car- ried out with the NJ, MP and UPGMA methods. As all these methods gave comparative phylogenetic trees, only the NJ tree is shown in Fig, L Fig. 1 indicates that P, peranthus
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]41 Melecular Phylogenetic Analysis of Subgenus Achillides
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Fig, 1. The phylogenetic tree of Aehillides butterfiies constructed with the NJ method using the PHYLIP software package. P. helenus be]onging to another gubgenus Menelaides was taken as an outgroup. The confidence ]evel ofbranching in the ph.ylogenetic trees was eval- uated with the bootstrap test based on 100 resamplings, and the values higher than 40 are shown at branching points of the ti'ee, InAchiilides, P, peranihus and P. palinulrts diverge early, and the so-called real Achillides group rhen diverges to two groups, which are namcd clades 1 and 2 in the tree.
and P, palinuhts diverged earlier than the so-called genuine AchilXides greup in the sub- genus Achillides. The genuine Achillides then diverged to two groups (clades 1 and 2 in Fig. 1). Clade 1 consists of P. diatis, P. hermeti, P. hianor and P. polyctor, while clade 2 consists of P, paris, P. kama, P. cuvt"rtts, P, hopponis, P. krishna, P, maackii and P. s.y,fa- nius, P. fitscus was not included inAchillides but in the Menelaides group. rlb clarify the phylogenetic relationships among the subspecies of P, bianor, butterfiics col- lected at various localities wcre analyzed with the NJ and UPGMA methodsl These meth- ods gave phylegenetic trees with a similar topotogy except fer the djvergences with low
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l42 Takashi YAGT, Go SAsAKi and Keiichi Ots{oTo
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Fig. 2.The phylogenetic trees of P, bianor and P. potyctor collected at various regions. The trees were constructed with the NJ (a) and UPGMA (b) methods by the PHYLIP software pack- age. P. hopl)onis, P. krishna and P. maackii were used as outgroup spccics in the NJ tree. Thc confidence level of branching in the phy]ogenetic trees was evaluated with the boot- strap test based on tOO resamplings, and the values higher than 40 are shown at branching points of the trees.
bootstrap (Fig, 2), As the evolution rate of individuals would be almost constant within a svaluespecies, the UPGMA tree is used further to discuss the biogeography o'f P. bianor.
Discussion
Revision of classification by DNA sequences
Figs 1 and 2 unexpectedly show that nucleotide sequences of P, pot.yctor including three subspecies polyctor, stockteyi and triunrphator coincide with those of P. bianor bianor, and the sequences ofP. s)tfanius coincide with those of P. maackii. P. polyctor has a large sky- blue spot on the apical area of the hindwings but P. bianor does not (Fig. 3). Il sptinius
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MolecularPhylogeneticAnalysisofSubgenusAchiUides 143
Sakh21in, Russia
Myohyangsan, N, Kgrea
Cnunchan, S. Korea Siei,Hokkaida,Japan Ishinamaki,Henshu,Japan
Tohi, ilonshu, Japan A bianor Shirahame,Honshu,Japan deheent' Kyoto,Honshu,Japan Kamiy3ma,Shjkoku,J2p2n
Kasuya, Kyushu, Japafi
Tsushima ls, Japan A,g,'.a"."..,. ;::g::I::il:::･-jtt egfi-b;,ag,a"l HzchijD-jima ls, 1, Japan
Hachljo-jima Is. 2, Japin A bibncrhechijbmS Miyake-jima ls. 1 , Jepan
Miyake-j ima ls. 2, Jepan Mlkurariirnais.,Japan Fojlan,China P･ bianorbiar7ar" China 1 Sichuan, - Laksao, Laos A PatyCtof' tnictmphator - W. Thaitanti A patyc tor stockbyi' - Kashm[r, lndia nPoe(cterpoetctor #aUnOySuh9sO.IS"'aTEweai:en I a biener kotoensts Guangxi,China -abienorbjbncr
Nonhet, Laos -a pobtctor tn'upmetor Tibet,China -Apaetctorgsnese Nantou,Taiwan a bianar thrasynreties Taoyuan,Teiwan l lriomate-limals.,Japan t lshLgakiSlmals,,Japan A biendi- TeketomEri+m2ls.j2p2n 1 okv"avensts Yanagunj{ima ls. 2, JaFan 1 Yonaguni-jimals. 1,Japan t
Amami-oshime ls. 1, Japan
Amami-oshimals.2,Japan Abianor Tokunashime ls. 1,Japan smanntensis Tokunfishlma ls. 2, Japan A b)el ::I::::Il'IMm:::I lll:::: l Ztluensis n dieh'sn
hopoonts Pt masckti
a knstue o,al
Fig. 2 (continued).
has a large white spot on the postmedian area of the hindwings but P, maackii does not (Fig. 3), Therefore, these butterflies have been considered taxonomically dilferent species due to their distinct wing patterns. Here, our molecular phylogenetic analysis revealed that P. pelyctor and P. hianor bianor should be an identical species, and P, s)ifinnius and P. maackii should also be an identical species, These findings are reinfbrced by the evidence
that distribution of the two species is roughly allopatric in the Eurasian Continent and a cline of their wing patterns has been observed in the small area where their distributions overlap (Fejioka, 1997; Shimogori, 1997),
P, fascus was fbund to be a species of the subgenus Menetaides from mitochondrial DNA sequences. P. fascus has been included in the subgenus Achiliides since the description by Igarashi (1979), because the morphological features of the larva and pupa are simi!ar to those of Achillides species, Our result shows that the wing pattern of P. fitsct{s reflects its phylogenetic features.
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144 Takashi YAq Go SAsAKi and Keiichi OMo'ro
't x.'
' -
a b c
K ,x {s'J
d e f
Fig. 3."ring pattern convergence between phylogenetically distinct gpecies. a: P. bianor bian.or (Sichuan, China); b: P, pol.vctor pol),c'tor (North India); c: P. s.)rfttnitts aibos}tiinnius (Yunnan, China); d: P. maackii maackii (Sichan, China); e: P. paris paris (North India); f: Parides nevilli (Yunnan, China). Upper and lower individuals in each verticul row inhabit the same region and have similar wing patterns but are phylogenetically distinct. P. hianor bicmor (a) and P. pol.},ctorpol.vcton (b), and also P. s)ifafzius (c) anct P. maackii In Fig. 1, boetstrap values higher than 40 were shown at branching points of the phyloge- netic tree. It shouldbe noted that the branching with the value lower than approximately 60 is unreliable. For instance, connection of P. pari.s hermosanus to the P, kama group, and inclusion of P. hermeli and P. diaZis in the P. bianor group are not trustworthy. Other di- vergences at the points with low bootstrap values in the phylo.oenetic trees also could not be discussed, P. hianor was divided into four groups (Figs 1 and 2), whose pair-wisc Kimura's two-pa- rameter distances were larger than O,O19, The first group includes ssp. dehaanii, tokanaen- sis, hachij'onis, which inhabits the Japan Archipelago and the Korean Peninsula. The sec- ond group includes ssp. amamiens'is and o,ttkyttensis, which inhabit the middle Ryukyu lslands, The third group includes ssp. hianor, thrasymedes, kotoensis and P, pol.v･ctor, which inhabit continental China and the Taiwan Islands. The fourth group consists of ssp. okinawensis, which inhabit the Yacyama Islands in south Ryukyu. These four groups should not be regarded as subspecies but four indepcndent species, because their pair-wise genetic distances are as 1arge as the distances between other distinct species, for instance, between P. paris and P. karna, and between P. hqp4)onis and P. a}Ttttrus (Fig 1). In addi- tion, the divergence of four P. hianor groups took place at almost the same time as those of other species P. dialis and P. hermeli (Fig, 1). This consideration is supported by failure of normal development of pair-wise hybrids of these four group individuals (Ae, 1995; NII-Electronic Library Service The LepidopterologicalSocietyLepidopterological Society of Japan ]45 Molecular Phylogenetic Analysis of Subgenus Achillides Kawazo6, pers, comm.). The divergence order of the four groups was not clarified in this study, because the bootstrap values were low, and therefore, the order is yariable among the analytical methods (Fig. 2), Parallel evolution and convergence of wing pattern Similarity of butterfly wing patterns contradicts the mitochondrial DNA phylogeny in the subgenus Achillides. Pairs of species with a similar wing pattern are present between clades 1 and 2, fbr example; P, bianor bianor and P. maackii maackii; P. polyctorpolyctor and P. paris paris (Fig, 1). Fig. 3 (a and d; b and e) shows pairs of species of clades 1 and 2 with a similar wing pattern. These pairs of species are distributed in identical or neigh- boring areas (Fajioka, 1997; Shimogori, 1997). The phylogenetic tree (Fig. 1) indicates that phylogenetic divergences occurred earlier than wing pattern changes, and the wing patterns of the different species then converged independent of the phylogeny. The species inhabit- ing a geographically iselated region may be liable to develop a ditferent wing pattern from the original one, and some ditferent species inhabiting the same region or a similar environ- ment may evolve a similar pattern on thejr wings, These findings are similar to the phe- nomenon, parallel evolution, found in races of South American long wing butterflies Hleliconius erato (Brower, 1994a7 b), Japanese ground beetles Carabus (Su, [Ibminaga et at., 1996; Su, Ohama et al,, 1996) and African lake fish cichlid (Meyer et al., 1990), sug- gesting that morphologically similar species inhabiting different areas are phylogenetically distinct, and that morphologically different species inhabiting the same area are phylogenet- ically proximate. In any case, these studies revealed that morphological changes of species occurred independently of their phylogenetic divergences. ND5 evolutionary rate and biogeography of P. bianor As the UPGMA tree is thought to be correlated with the past phylogenetic divergence time in a lower taxonomic greup, it would be reasonable to hypothesize that the branching points of the four P. bianor groups (Fig. 2) were settled at the time when the groups were isolated in the Japan and Ryukyu Archipelagoes. According to the paleogeography of the Ryukyu Archipelago, the Ryukyu and the Sakishima Super-islands formed by the end of the Pliocene (1.65 MY]A), as a result of subsidence of the east periphery of the Eurasian Continent during the Pliocene (1.65-5,3 MYA) (Kizaki & Oshiro, l977; Ujiie, l990; Ota, 1998), The Ryukyu and Sakishima Super-islands separated far later to the present Okinawa and Amami Islands, and the present Yaeyama and Miyako Islands, respectively (Kizaki & Oshiro, 1977; Ota, 1998). From this assumption, Kimura's two-parameter distance O.OID would be estimated to be about O.7-O.8 million years (MY). This rough estimation can also reasonably explain that tbe divergence points of the continental and [[hiwan populations, of the Iriornote and Ybnaguni populations, and of the Okinawajjma and Amami-Oshima popu- lations in the UPGMA tree are about O.17-O.35 MYA, which roughly corresponds to the time the present Ryukyu Islands fbrmed in the late Pleistocene (Kizaki & Oshiro, 1977; Ujii6, 1990; Ota, 1998). This ND5 evolutionary rate of P. bianor agrees with that of Ptarnassius butterfiies (0.01D=O.75 MY) (Yagi et at., 2001). The distinct wing pattem difference between P. polyctor and P. hianor bianor, and between P, sMfanius and P. tnaackii without ND5 sequence change indicates that the wing pattern change occurred in the period while one base change mutation had been fixed in the ND5 gene (shorter than O.07 MY), The wing pattern of P, polyctor pol.vctor resembles that of the phylogenetical]y distinct P. paris paris, and P. s)0Ziniu,g may mimic the poisonous papil- ionid species Plarides nevilli, Pkir. hedist"s and Fitr. polyeblctes (tribe Troidini) which occur NII-Electronic Library Service TheTheLepidopterologicalSociety Lepidopterological Society of Japan 146 Takashi YAGT, Go SAsAKT and Keiichi Opa]oTo sympatrically (Figs 3c and D. These results that wing pattern convergence in Achillides may occur in a relatively short period.indicate Acknowledgements We thank many entomologists who kindly supplied the butterfly specimens. We are espe- cially gratefu1 to Drs H. Ftijii, T. Fqjioka, H. Fukuda, S. Koiwaya, T. Shinkawa and O. Yata for supplying foreign specimens. We also thank Ms C. Shimohara for her excellent techni- cal assistance. This study was partly supported by a grant from the Institute of Applied Optics. References Ae, S. A., 1995. Ecological and evolutionary aspects ef hybridization in some Ptrpilio butterflics. In Scriber, J.ptC., Tsubaki, Y, and R. C, Ledcrhousc CEds), Svt,alloivtait Burtetfiies: their Ecotogy･ and ei,olutionary Biotogy: 229-235. Scientific Publishers, Inc., Gainesville, FL. Bauer, E, and T. Frankenbach, 1998. Papilionidae I. Buttectlies ofthe Wbrld (1): 1-12. Goecke & Hvers, Keltern. Brower, A. V. Z., 1994a. Rapid morphological radiation and convergence aniung races ot' the butterfly Heticonius erato inferred from patterns of mitochondrial DNA ex,olution, Proc. natl. Acad, Sci, UEh`L 91] 6491-6495. l994b. Phylogeny of Hkliconittsbutterfiiesinferred , from mitochondriaE DNA sequences (Lepidoptera: Nymphalidae). Molec,. Ph.vlogenet. Evol. 3: 159-174. Caterino, M. S. and F. A. H. Sperling, 1999. Papilio phylogciiy bascd on mitochondrial cytochrome oxidase I and II genes. Molec. Ph}'togenet. Evot. 11: 122-137. Felsenstein, J., 1985. Confidence limits on phylogenies: an approachusing the bootstrap. Evotution 39: 783- 791. , 1993. PHYLJP (Ph}/logeny l4ference Package). Ver. 3.5c. Dcpartmcnt of Genetics, University of Washi ngton, Seatt]e. Fujioka, T,, 1997. Papilionidae, In Fu.jioka, T, Tsukiyama, H. and H. Chiba, Jag)anese Buttetlflies and their Relatives in the l4i?)rld 1. Shuppai]-Geijutu, Tokyo. (In Japanese), Garesse, R., 1988. Drosophilta melanogastef' mitochondrial DNA: gene organization and evolutionary con- siderations. Genetics 118: 649-663, Harada, M., 1992. The status of Achiiiides hianor (Cramer) in Sichuan, China, Butteij7ies (2): 19-25 (in Japanese). Hidaka, T., Fojii, H., Unno, K. and M. lmamori, 1984. Butte,jlies. Tokai University Press, Tokyo, (In Japanese). Igarashi, S., 1979. Papilionidae and their earty Stages. 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NII-Electronic Library Service The LepidopterologioalLepidopterological SooietySociety of Japan 147 Moleoular Phylogenetic Analysis of Subgenus AchiUides ’ − species group． Heredit） 71 ：227 233 ． Su Z ．−H ．Ohama T ．Okada T ．S．Nakamura K ．［ − ， ， ， ， ， ， ， ，shikawa ， R ． and S ． Osawa ，1996． PhyLogenetic reletion ships and evolution of the Japal1巳 se Carabinae ground beeIles l〕ased on lnitochodrial M ）5 gene se− qucnces．」． MotCiC ． Evol，，42 ： 124−129． − Su， Z ．H ．， Tonlinaga，0 ．， Ohama ， T ．， Kajiwara， E ， lshikawa， R ．， Okada， T ， S，，Nakamura， K ． and S． Osawa， 1996．Parallcl ev lution in radiatiDn of Ohmopteru，g ground beetles lnf巳rred 丘om mitochondrial ？＞D5 − 9en巳 sequel 〕ces ・ノ． molec ． Evo ’．43 ： 662 671 ， − Su， Z ．H ．， Tominaga，0 ．， Okamoto， M and S． Osawa，1998，0rigin and diversification of hindwingless Damaster ground beetles within Japanese Islands as deduccd from mitochondrial 八の 5 gene sequences − （α ｝leoptera， Carabidae）， Molec． Biol． Evot．15： 1026 1039． ＊ ） ＊ Swoff rd， D ， L ，1998． PA UP ．1 1〜yJogθπ 顔 c Artal｝，sis ttsing Parsimony 〔and other Jl4ethods ）， Vcr 4，0． Sinauer Associates， Sunderland， Massachusetts． 「 Ujii6． H ．．1990． Ged 【〕gical history of the Ryukyu Island Arc ， ln Ujii6， H ．（Ed，），ハatt｛re ρプOkifta｝va ： ， − Geomorphotog ．〉 an ‘i Geolo8y：251 255． Hirugisha， Naha ．（ln 亅apanese ）． Yagi T ．Katoh A T and K Omoto 2001 Molecular ， ， ，T ．，Chichvarkhin， ，，Shinkawa， ． ． ， ， phylogeny of butter且主es Parna ∬ ius glaciatis and P ．ytubben． （10壌’at 、町 ious localities in East Asia． Genes Genet ， Syst．761 229−234． − Yagi， T ．， Sasaki， G ， and H 、 Takebe ，1999． Phylogeny of Japanese papilionid butter且ies inferred from nu − cleotide sequences of thc mitechondria1 NI ）5 gene．ノ． moiec 、 Evol．48 ： 42 48． − Yoshimoto ， Y ．，1998． Papilio biano厂 and Papilio dehaanii， two distinct species ． Butterflies（20）： 45 49 （in Japanese with English summary ）． Zakharov ， E V ，， Catcrino， M S． alld F， A ． Sper且ing，2004． MQlccular phylogeny ， historica］biogeography， and divergence time estimales for swal1Qwtai 且 butter且ics of the genus Ptrpiiio （Lepidoptera： Papili｛〕nidae ）， Syst． Biot．53： 193−215， 摘 要 ミ ト コ ン ドリ ア DNA 解析 に よ っ て 明 ら か に な っ た カ ラ ス ア ゲ ハ 亜 属 （ア ゲ ハ チ ョ ウ科 ア ゲ ハ ョ ・ 々 ・ チ ウ 属 ）の 系統 ， 生物 地 理 ， 斑紋 の 収斂 現象 （八 木孝司 佐 木剛 尾 本 惠市 ） 円本 を含 む東 ア ジ ア に 主 に 分布 す る カ ラ ス ア ゲ ハ 亜 属各種 の 系統関係 を ミ ト コ ン ドリ ア DNA の 1＞D5 ー 遺伝子 部分 配 列 に よ っ て 解 析 した ．そ の 結 果，カ ラ ス ア ゲ ハ 亜属 は 2 つ の グ ル プ に 大 き く分か れ る 一 ー こ と が わか っ た．第 の グ ル プ に は カ ラ ス ア ゲ ハ ，ク ジ ャ ク ア ゲ ハ ，ミ ン ド ロ カ ラ ス ア ゲ ハ ，タ イ ワ ン ー カ ラ ス ア ゲ ハ が 含ま れ る ．第 二 の グ ル プ に は ．ミ ヤ マ カ ラ ス ア ゲ ハ ，シ ナ カ ラ ス ア ゲ ハ ，タ カ ネ ク ジ ャ ク ア ゲ ハ ，オ オ ク ジ ャ ク ア ゲ ハ ，ポ ッ ポ ア ゲ ハ ，ル リモ ン ア ゲ ハ ，カ ル ナ ル リ モ ン ア ゲ ハ が含 まれ る 、 ー ー 各グ ル プ の 種 問 に は 斑 紋 の 共通 性 が あ る わけ で はな く，グ ル プ間 に い くつ か の 斑 紋が似 た種 の 組 み 合 わ せ が存 在す る ．た と え ば 中 国 四 川 省 の カ ラ ス ア ゲ ハ と ミヤ マ カ ラ ス ア ゲ ハ ，北 イ ン ド の ク ジ ャ ク ー ー ア ゲ ハ と ル リ モ ン ア ゲ ハ な どで あ る ．こ の こ と は 2 グ ル プ の 分 岐 後 に グ ル プ間 の 種 ど う しで 翅 の 一 斑紋 の 収斂 が 起 こ っ た と考 え ら れ ．平 行進化 の 例 と い える か も しk な い ．各地 産 の カ ラ ス ア ゲハ は4 つ ー 一 ・ の グ ル プ に大 き く分 か れ る こ とがわ か っ た．す なわ ち第 は トカ ラ 列島以 北 の 日 本列 島 サ ハ リ ン ・ 二 ・ ・ ・ 朝鮮半島産， 第 は奄美大島 徳之島 沖縄 島産，第 三 は 八 重 山諸 島産，第四 は 中国 大陸 南部 ー 台湾 諸 島産 で あ る ．こ れ ら 4 グ ル プ の DNA 配 列 の 違い は ，各 々 が 種 で あ る と して も妥 当 な ほ ど 大 き い ．ま た ，カ ラ ス ア ゲ ハ 原 名亜 種 と ク ジ ャ ク ア ゲ ハ の DNA 配 列，ミヤ マ カ ラ ス ア ゲ ハ と シ ナ カ ラ ス ア ゲ ハ の DNA 配列 は 同 じか ほ とん ど違 い が な く，こ れ ら は そ れ ぞ れ 同 種 で あ る こ と を強 く示 唆す る ．ネ モ ン ハ ハ ロ ッ タ イ キ ア ゲ は 幼 虫 と 蛹 の 形 態 か ら カ ラ ス ア ゲ 亜 属 と 分 類 さ れ る こ と が あ っ た が，シ オ ビ ア ゲ ハ 亜 属 と す べ き で あ る こ と が わか っ た， （Accepted December 15，2005） Published by the Lepidopterological Society of 亅apall ， 5−20 Motoyokoyama 2 Hachi〔’ Tokyo − ， ， ji， ，1920063Japan 一 NII-ElectronicN 工 工 Eleotronio Library Service