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ue8wt 7}ans. mpid Soc. lapan 54 (2): 91-110, March 2003

Phylogeny of the thaphium butterflies inferred from nuclear 28S rDNA and mitochondrial ND5 gene sequences

Hiromichi MAKiTAi)', Tsutomu SHiNKAwA2), Kiyotaro KoNDo2}, Lianxi XiNG3} and Tohru NAKAzAwA2)

i)Department of Biology, Faculty of Science, Toho University,

2-2-1 Miyama, Funabashi, 274-8SIO, Japan 2)Biologicar Laboratory, The University of the Air, 2-11 Wakaba, Mihama-ku, Chiba, 261-8586, japan 3)School of Life science, Northwest University, No. 229, Taibai Road, Xi'am, 710069, China

Abstract We examined the phylogenetic relationships of 40 speeies of the genus Grmphium (Papilionidae: Leptocircini) with a few related genera belonging te the tribe Leptocircini. The analysis is based on comparisons of morphological classification and about 700 base pairs of nuclear 28S ribosomal DNA and 793 base pairs of the mitochondrial NADH dehydrogenase subunit 5 (ND5) gene. In both 2gS rDNA, the NDS and the 28S rDNA+'ND5 phylogenetic trees of Graphium butterfiies, the genus Grophium was monophyletic, and diversilication occurred at almost the sarne time. Although the subgenera Pazala, Pathysa and Arisbe were monophyletic clusters, the subgenus Graphium was not. The28S rDNA sequence lengths ofthe eurp{pyltzs group belonging to this subgenus (represented in this study by G, doson, G. bathycles, G chiron, G evemon, C, leechi, G, eu,:yevins) and G! aktkoae (=phidias) to the subgenus Pathysa are 713 bases, and difiered from those ofthe other Craphium butterfiies which are 717 bases. The phylogenetic tree positions ofthe eurptpJdins group+ G, akM oae did not cluster with other subgenera, but rather independently. Therefore, we assumed that the eumpylas group did not belong in the subgenus Graphium. The current elassification leayes a certain ambiguity in

the classification ofthe African Graphium species, either all the African Graphium species being classified into the subgenus Arisbe (Miller, 1987), or the African species being classified into two groups, the non-swordtailed species in the subgenus Arisbe and the swordtailed species in the subgenus Graphium (Haneock, 1983, 1993). The prescnt study assumes that the African species should be assigned to two groups represented by Hancock, one being the non-swordtailed species in the subgenus Artsbe (represented in this study by GL ridlevanus, G. iatreillianus, G, an- golanum, G. tynderaeus, G, leonidas, G, adomastor and G schubotsi) and the other being the swordtailed species in the subgenus Graphium (represented in this study by G. poticenes>.

Key words 28S ribosornal DNA, mitoehondrial ND5 gene, phylogeny, Graphium, Leptocjrcini

Introduction

The genus Graphium Scopoli, 1777 (type species: I'apillo sarpeclon Linnaeus, 1758), which belongs to the tribe Leptocircini (Papilionidae, Papilioninae), is widely distributed in the Oriental, Australian and Ethiopian regions, and has also spread into the southern area ofthe eastern Palaearctic region. This genus is not fbund in the New World, but the Leptocircini genus EuT:yticles H'u'bner, 1821 (type species: Eui:}'tides iphitas HUbner, 1821), which contains the subgenus Rrotesdous Swainson, 1832 (type species: PapiZio protesilZxus Linnaeus, 1758), is distributed in the Neotropical regions instead ofthe genus Graphium (rocently, M6hn (2002) separated the new genus IVbagvaphium from the genus Eurytides). Other related genera

* Corresponding author: H. Makita, c/o Biological Laboratory, The University of the Air,2-li Wakaba, Mihama-ku, Chiba 261-8586 Japan; vmail: [email protected]

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92 Hiromichi MA-TA, Tsutomu SHTNKAwA, Kiyotaro KoNDo, Lianxi XINa and Tohru NAKAzAwA

belonging to this tribe, Lamptzzptera Gray, 1832 (type species: Papido eun'us Fabricius, 1787), Rrotqgraphium Munroe, I961 (Pmpillo ieasthenes Doubleday, 1846) and lphicfidds HUbner, 1819 (PapiZib podolbius Linnaeus, I758) are distributed in the Oriental, Australian and Palaearctic regions, respectively.

The relationships of the genus Graphium NN'ithin the tribe Leptocircini and the interspecific relationships within Graphium have been previously discussed by many investigators includ- ing Ehrlich (1958), Munroe & Ehrlich (1960), Munroe (1961), Saigusa et al. (1977, 1982), Hancock (1983, 1993), Miller (1987) and Smith & Vane-Wright (2001), The current classification suggests that Lamproptera is most proximate to the genus Grcrphium (Hancock, 1983; Miller, 1987), while Ehrlich (1958) included Lamproptera in the genus Grcrphium based on the high tentorial crest, Munroe & Ehrlich (1960), on the other hand, state that the closest relative of (]naphium is the genus lphiclides

The genus Graphium has been tentatively c]assified into fbur subgenera (Hancock, 1983): the eastern Palaearctic subgenus Pazala Moore, ]888 (type species: Papillo glycerion Gray, 1831), the Indo-Australian subgenus PathJtya Reakirt, 1865 (type species: Papillo antiphates Cramer, 1775), the Aftican subgenus flrdsbe HUbner, 1819 (type species: Paptilb ieonidos Fabricius, 1793) and the Asian subgenus Grcrphium Scopoli (Table 1).

In the current classification of the subgenus thaphium based on Saigusa et al. (1977, 1982), the subgenus Graphium consists of the sarpedon group, the agamemnon group and the eumpylas group. However, Saigusa et al. (1977, 1982) did not specifically state that the subgenus Graphium is monophyletic. Miller (1987) also suggested that he could not find autapomorphic characteristics to support the subgenus Graphium

Munroe (1961) classified all African species into the subgenus Artsbe, based on the rim ofthe valva of the male genitalia having stout setae concentrated on the disto-ventral half of the dorsal process, but not modified into spines in either region, Miller (1987) also classified all African species into Aiisbe, based on the lateral lobes ofthe female ostium, which are covered

with a mat of short setae and have a fuzzy appearance throughout the subgenus Anlsbe. However, Hancock (1983, 1993) classified the African species into the Afrotropical swor- dtailed species in Graphium, and the non-swerdtailed species in Arisbe, despite the assenions of Munroe (1961) and Miller (1987) that all African Graphium buttertlies should be classified into the subgenus An'sbe. The recent Smith & Vane-Wright (2oo1) classification of the Afrotropical Graphium butterflies with a few Indo-Australian butterfiies based on the

morphological characters did not involve four subgenera, and it showed that varieus species formed smal1 groups and had complicated phylogenetic relationships.

Munroe (I961) further suggested that the eurous group, placed by later authors in the subgenus Pczzala, belongs to PathJnsa, based on the complexity of the male genitalia. He does not recommend separating this group as the fourth subgenus because of significant affinities between Pazala and Path.psa. Hancock (1983) pointed out that the most primitive subgenus, Pczzala, retains the primitive banded pattern, complex male clasper and blue hindwing scales, while in the slightly more advanced Path:vasa, the pattern is either primitive or mimetic, the clasper less complex and blue scales absent. Miller (1987) also agreed that the subgenus Pazala is plesiomorphic to the rest of the genus Grophium

In spite of this seeming agreement, lgarashi's study of immature stages (1984) indicates that the genus Lampmptever is more closely related to both the subgenera Graphium and Arisbe than to either Pacala or Path.vasa. Igarashi treated Pczzala and Patlrysa as two genera, and other researchers (e. g. D'Abrera, 1982) have also recognized Pazala as a separate genus,

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Phylogeny of Graphium lnferred from 28S rDNA and ND5 Gene 93

Moreover, a cladistic study by Tsukada & Nishiyama (1980) suggested that Graphium akthoae Merita & Shinkai* (= G phidias (OberthUr)) would belong in a new subgenus, not belonging to Pathysa shown by Hancock (1983) or Collins & Morris (1985). Niculescu (1977) proposed a new genus, KZingide (a replacement name, Klingiziana Niculescu,･ 1989) for (]raphium wetwei (Ribbe), but this proposal has not been accepted by the subsequent authors (Parsons, 1999). As mentioned abeve, subgeneric classifications or grouping of species within the genus (iraphium are not yet clear.

In the present study, we examined the nucleotide sequences of a part of the nuclear 28S ribosomal DNA (about 700 base pairs) and a part ofthe mitochondrial gene encoding ND5 (793 base pairs) of thaphium butterflies. The ND5 gene shows a fairly rapid evolution and is one ofthe most usefu1 mitochondrial genes for investigating the phylogenetic relationships among related groups (Su et al., 1996a, b; Yagi et al., 1999; Kim et al., 2000; Makita et al., 2000). Although the 28S rDNA shows a slower evolution than the ND5 gene, the 28S rDNA phylogeny is mostly consistent with the ND5 gene phylogeny of the LeptocarabtLs ground beetles (Kim et al., 2000). Therefbre, we used 28S rDNA fbr the tribe Leptocircini groups and ND5 fbr tbe genus (lraphium The molecular phylogeny indicated the evolution and phylogeny of the genus (lraphium.

Materials and methods

Samples and DNA extra ¢ tion

The names and localities of the specirnens used in this study are shown in Table 1, including

their accession numbers assigned by the DDBJ/EMBL/GenBank; representative species are

shown in Fig. 1. A total of 40 ([]naphium butterfly species were analyzed, including several species of each subgenus of the genus (]raphium, as well as species from three related genera belonging to the tribe Leptocircini: the genus Eui:ytidles (EL hellos Rothschild & Jordan), lphieZides (L podoiblus (Linnaeus)), and Lampmptera (L. meges (Zinken-Sommer) and L. curitLs (Fabricius)). One species of the genus 7leinopaipus Hope (T intperiads Hope) was

used as an outgroup,

All butterfiies were collected by the authors or supplied by entomologists (see acknowledg- ments), The butterfiies collected in Japan, Kerea and Myanmar were immersed in 95% -20DC ethanol and stored at before use, and those collected in other areas, China, Malaysia, Indonesia and se on, or rare or precious samples were kept as mounted dry specimens. DNA was extracted from muscles ofthe thorax or Iegs, using a QIAamp DNeasy Kit (Qiagen, Inc,, Valencia CA, USA). (Note; many of the rare or precious samples used in this study were purchased from a dealer who obtained permission, and were collected before 1996),

PCR amplification

Extracted total DNA was used as a template fbr 28S rDNA and ND5 DNA amplification

' Papilio phidias OberthUr, 1906 is a primary homonym of Paptho phidias Linnaeus, 1758, and cannot be used as the scientific name for the well-known Graphiuni species in question. We use Graphium akikoae Morita & Shinkai, 1996 as its valid name. Although akikoae was described as a subspecies of G. phidias from Lak Sao, Laos, Masui and Uehara (1996) suggested that there is no big diflerence between the specimens from Laos and Vietnam, from which phidias OberthUr, 1906 was originally described.

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Table 1.List of species ofthe genus Graphium and other related genera used in this study. gerlussubgenus DDBJ!EMBLfGenBank 28S ND5locality species group acoession no. (28S!ND5) specles GraphiumPathysa

anmphates group agetes (Westwood) ++ +++++++++LaosMalaysiaMalaysiaSabah, ABOS96S4!AB059486 ABOS968SIAB0594g5 -IAB059487 atiyteus (Crarner) -fAB059491 stratibtes CGroscrSrnith) Borneo and?oclas {Boisduval) + Sulawesi, Indonesia AB088638fAB088644 -!AB088645 dorciLs (de Haan) Sulawesi, Indonesia antiphates (Cramer) LaosCameron AB059690fAB059494 Highland, Malaysia -fAB059495 -1. Borneo ABOS969]IABOS9496

macareus group -,/AB059488 delasserti (Guerin-Menevilie) +++++ BorneoLaosBorneoTimukn, tnacareus (Godart) ++++. ABe596g6fAB059489 AB059687/'AB059490

thule (Wal]ace) W. Irian, Indonesia AB059688fAB059492 xenoclas (Doubleday) Tam Dao, Hanoi. Vietnam AB059689!AB059493 Purala

mandarinuFn (OberthUr)i) ++."+･F･+Mt Victoria, Chin, Myanmar 1 AB059725!AB059533

Mt Victoria, Chin, Myanmar 2 AB05V726fAB059534

incertum (Bang-Haas) Yunnan, China AB059727fABe59535 hoenei CMell)2) Sichuan, China AB059728!AB059536 -++ tamehanum (OberthUr) Gansu, China AB0597Z9!ABe59537 eurovts (Leeeh) Taiwan AB059730/ABe59538 Craphium akikoae Morita & Shinkai3) + + Laos ABOS97ellAB059S05 euDvrytus group euemon (Boisduval) +'r' +++++++++Cameron Highland, Malaysia 1 AB059692!AB059497 `- Cameron Highland, Malaysia 2 AB059693fAB059497 ddsen

1 AB059696!AB059SOO -tttt Taiwan 2Zi AB059697!AB059501

chiron (Wallaee) ya dum, Myanmar ABe59698/AB059S02 bathyclas (Zinken-Sommer) Cameron Highland, Malaysia AB059699!AB059503 laechi CRothschild) Jingning, Zhejiang, China AB088639!AB088646 eunyvey'his {Linnaeus} Putuo, Kachin, Myanmur AB0597001- agamemnon group agamemnon (Linnaeus) ++++++ ++++ttBali Is., Tndonesia ABOS9702!ABOS9S06 Carneron Highland Malaysia l AB059703!AB059S07

Carneron Highland Malaysia 2 AB059704!AB059508 LaosTaiwanBorneo AB05970S!AB059S09 ABOS97e6!ABOS9SIO ABOS97071AB059Sll lvadocei group wahaeei CHewitson) + t New Britain ls. PNG AB059708!AB059SI2 sarpedbn group baijanense Okano ++ Bachan Js,, Jndonesia AIro59709/ABe59513 "eiskei (Ribbe) PNGDungog, AB0597101AB059514

macleayanum (Leaeh) t++ Australia ABOS8643!- codimLs (Cramer) +++++ Malaita Js, Solomons AB0597111AB059515 empedovanum {Cerbet) Java. Indonesia 1 AB0597121ABe59516 -IABOS9517 Java, Indonesia 2 sarpedbn (Linnaeus) ++ Bali Ts., Indonesiu ABO.S9713fABOS9518 Taiwan 1 ABOS97141ABOS9519

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Phylogeny of Graphium Inferred from 28S rDNA and ND5 Gene 95

++++++++++++++++++++Taiwan 2 AB05971S!AB059520

Chiba, Japan ABOS97I6!AB059521

Yokosuka, Japan AB060636/AB059522

Cumeron Highlalld, Mataysia 1 ABOS97171AB059523

Cameron Highland, Malaysia 2 ABOS97181ABOS9524

Cameron Highland, Malaysia 3 ABOS97191AB059525 LaosSuiawesi, AB059720fABOS9526

mfilon C, & R. Felder Indonesia ABOS97211ABOS9527 -fAB059528 Buru Is., Indonesia

eloanthus Westwood ++ TaiwanLaosSichuan, AB059722fAB059529 ABOS9723fAB059530 -IAB059531 China poficenes group poficenes (Cramer) + + Congo AB0597241ABe59532 Arisbe

ridlayanas group ridoanus (White) t t Nola. RCA ABOS97311ABOS9539 tyndoaeus group latremaantts CGodart) t+ t Carnerun ABOS9732fAB059540 tynderaeets (Fubricius) + Bungui,RCA AB08S642fAB088648 angolanum group angolanum (Goeze)d) + Bungui,RCA AB088640fAB088647 leoniclas group -!AB059541 leonidas

tneges (Zinken-Sommer} ++++ ++++ Cameron Highland, Malaysia 1 ABOS9733fABOS9542

Cnmeron Highland, Malaysia 2 ABOS9734fAB059S43

Cumeron Highland, Ma[aysia 3 ABOS9735fAB059544

curius (Fabricius) Ziya dum, Myanmur ABOS9736fABOS9545 iphiclides podobeius {Linnaeus) + + CzeckRepublic ABOS9737fAB059546 Eu-,tidesProtestit:us

protasdaus group helias (Rothschild & jordan) +'f + Minas Gerais, Braz{] AB059738fAB059547 71?inopaipus ･+ impen'ads Hope N.Yietnam AB059739fABOS9S48

Arrangement ofspecies fo11ows Hancoek (1983) except fbr G, akthoae (=phidlas). 1) G. glycerion (Gray, 183I) is a junior primary homonym of Papillo glycen'on Borkhausen, 1788. 2) G sichoranica (Koiwaya, 1993) is ajunior subjectiye synonym. 3) G phidias (Oberthttr, I906) is a junior primary homonym of Papthb phidtas Linnaeus, 1758. 4) G. Iu,lacies (Fabricius, 1793) is a junior primary homonyrn of Papillo lu,lades Stoll, 1782. 5) G, odin (Strand, 1910) is ajunior primary homonym of PapiZlo odin Fabricius, 1793.

by polymerase chain reaction (PCR), fo11owing the method described by Saiki et al, (1988). The PCR primers fbr the 28S rDNA gene were employed fbllowing the method described by Kim et aZ (2000). Other PCR primers for 28S rDNA were based on (Iraphium sequences; these were the fbllowing: 28rSF (5'-GAC TAC CCC TGA WTT WAA GCA T-3'), 28rSR (5'-GAC TCC TTG GTC CGT GTT TC-3'), and the sequence primers 28sFl (5'-AGT AGC GGC GAG CGA ACA GGA A-3'), 28sF2 (5'-GCG AAA CTC GAA TGA ACG AAC GG-3'), 28sRl (5'-CCC GAA ACT GAA TCA TCG CCG AC-3'), and 28sR2 (5'-CSG ACR TCG AAC GGG TCG CGA TG-3'). The PCR primers fbr the ND5 gene

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96 Hiromichi MAKITA, Tsutomu SHIrNKAwA, KiyotaroKoNDo, Lianxi XiNG and Tohru NAKAzAwA

!es･

1 ' f y t

A

6 7 8 9 10

' ' '

it'x '

11 12 13 14 15 tt A'

' " L'' Xx e " ,e' kxE x.

x. 16 17 18 19 20

Fig. 1, Representative species of the genus Graphium and the tribe Leptocircini used in this study. 1. Grcrphium agetes. 2. a.antiphates. 3. amacareus. 4. GL mandarinum 5. G eurous. 6. G. akikoae (=phidins). 7. G. dbson. 8. G. agamemnon. 9. G wallLzcei. IO. G. weiskei. 11. G. codeus. I2, G sarpedbn. 13. G. cloanthus. 14. G, poticenes. 15. G. riddyanus. 16. G latreilUanus. 17. G, leonidas. 18. Lam- proptera meges. 19. iphiclidbs po`laiZrius 20. Eu,lytidbs helias.

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Phylogeny of Graphium Inferred from 28S rDNA and ND5 Gene 97

were employed fbllowing the method described by Su et al. (l996a, b) and Yagi et al, (1999), Other PCR primers fbr the ND5 gefie were based on Graphium sequences: C2GR (5S-KCN GGR TTT TAT TCW AAR GAT-3t), A4G (5t-GTR TAA WAT ATA GTY ARH CCW GTW G-3'), and DR2G (5'-GTW GAT AAA TTA GGT ATA AAT CAY A-3r). PCR amplification was carried out in 50 ptl ofa selution containing 1 ptg oftemplate DNA, 1 pmol/pe1 of each primer, and 1,25 U of 7kzKaRa ]Ex Tltq (Takara, Shiga, Japan), ¢ The amplification comprised 35 cycles of denaturation at 94 C for 30 sec, primer annealing at 520C fbr 30 sec and primer extension at 720C for 1 min, or was conducted according to the method described by Taylor et al. (1993). Taylor's PCR method was used primarily fbrdry spec]mens.

Direct sequencing of PCR-amplified single stranded DNAs

The PCR products were purified using a QIAquick Gel Extraction Kit or QIAquick PCR Purification Kit (Qiagen). Direct sequencing was perfbrmed by the dideoxy chain termina- tion method (Sanger et al,, l977) using a BigDye Terrninator Cycle Sequencing Ready Reaction Kit (Applied Biosystems Japan, Ltd, Tokyo, Japan). The nucleotide sequences were determined using an ABI PRISM 377 Sequencer (Applied Biosystems).

Phylogenetic analysis

The 28S rDNA and ND5 DNA sequences were aligned using the CIustal X program (Thompson et al., 1997), and were then manually adjusted by eye to minimize gaps. Phylogenetic analysis was perfbrmed by two methods: the neighborjoining (NJ) method (Saitou & Nei, 1987) and the maximum parsimony (MP) method. Both methods with a bootstrap test (Felsenstein, 1985) were carried out with the PAUP* 4,OblO program (Swotibrd, 2000) and the heuristic parsjmony analysis used both with all characters having equal weight, stepwise sequence addition, addition sequence of 1,OOO random replicates, tree-bisection-reconnection (TBR) branch-swapping, and the MULTREES option. The bootstrap values are based on 1,ooe rep]icates. Gaps in the aiignments were excluded from the phylogenetic analysis. The evolutionary distances (D) were computed by Kimura's two-parameter method (Kimura, 1980). The gene sequences of the genus 7leinopaiptas (T imperiaiZs) were used as an outgroup,

Results

Nucleotide sequences

Partial sequences of the nuclear 28S rDNA (about 7oo base pairs) and the mitochondrial ND5 (793 base pairs) were determined, although not all taxa were sequenced successfu11y (e g,, (ir(rphium eumpyfus (Linnaeus) fbr ND5), A few deletions and length variations were fbund in each genus as well as in the Graphium 28S rDNA sequences, Certain length variations ofthe 28S rDNA were found within the genus (IFrmphium, specifically, Grophium doson (C. and R. Felder), G bat]lyelas (Zinken-Sommer), (]! chimn (Wallace), G, evemon (Boisduval), G leechi (Rothschild), and GL eut:yrwlas, which were classified into the eumpJ,las group in the subgenus Graphium and G akikoae, showed 713 bases, but other Grophium species including Patlrysa, Pazala and Ansbe showed 717. The genera ofthe tribe Leptocir- cini showed the fo11owing numbers of bases: Eui:ytides, 724; lphicLides, 718; LaTmptq]tera, 718; and 7leinopaipus, 728. There were no insertions/deletions and no length variations fbr

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Table 2. Pairwise Sequence Divergence (by Kirnura's Two-Parameters Methods) fbr 28S rDNA examined in this study l234567E" ]O TI 11 13 14 IS 16 17 IS IY 20 2T 22

r234567Bp]oTTi2t3]4EIG]TTSIP20212?!]242S2G]12S2Psu3T323Sj43i.1fi373S19an4T424],ag45 ' n.e rLa (1,oo n.e, O,]4 O,cr) O.]4 n.c, U42 1.S6 J,SS 1,S6 1,1] 1,S6 4,la 4,lg 4,]R 4,03 4,eS 4.IS 4.31 S.69 - n" n.e ].e. -.e. n,e, ].e ne, -.e n.e, ne llS, fle dl.e, n.e CLe, n.e n.e, n.e, n.e. -.e,

10.[1 9.S6 - tl.e. n.e. n.e, ]].e n,e, ]],c n,e, ]],e, n,e, ]],c, n,e, n,e, n,e, n,e, n.e, nc, n.e n.e. n.e. 9.Sl D.L? i6t - n.e. e,14 noo O,14 n.e, U41 LS6 [,SS LS6 [,13 L56 4,IS 41S 41S 4.0] 4.01 4.IS 433 - n.e, 6.)'9 S.69 9A3 10.eO n.e- n.a n.e n.q n.e n.e. n.e n.c n.e. n.e. n.e n=, n,e, n.e n.e, n.e 8,69 S,tl 9.2S S.il 9.21 O14 O,2S n.a O,S6 ;,70 1,99 170 1,?1 iTe 4,13 4]1 4]3 4,IR 41S 4.r,G 4.4S CO.OO 9.99 4,09 i,OS tO,oo 8,ss - O,14 n.e, O,4! T,S6 l,g5 i,S6 l,11 1,S6 4.IS 4,IS 4,IS 4.D3 4D3 4.]S 4.3]

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14.97 13J4 141.0 11PT ]r,43 1].!E 14.51 IS.73 1].27 ]?.11 1].59 ]:,jl ]2S? 12,j! 1]]I 1],SS 1].76 12S4 ]2,Ol M22 T2,OT' n,e. [2.e6 Tl.O] 11.3] leJ? 11.91 12.el 11.12 11.61 12.S7 12.SI 12.70 10,S7･ ]1.3! ]1.t7･ 10,2S ]2,S6 9,9S ]1.02 ]O.2S ]02S 10.12 n,a ["Og 14.6T 14,2] 1],PO T3,PO ]S,3e ]5.t4 IS,15 IS,29 13,74 IS93 14.P" 14.8Z IS.IS 14.?1 1].77 13.34 ]4.2B 12.7I 11.02 ]S.Ol n,e -The Note. numbers above the diagonal are fbr 28S rDNA, and those below the diagonal are for 1, G agetes Laes, 2. Gt artsteus Malaysia, 3. G ctelesserti Borneo, 4. GL macareus Laos, 5. G,

dorcus Sulaweshi, 10. G, antiphates Laos, 11. G mandarinum Myanmarl, 12, G. incertum Yun-

Laos, l7. G. evemon Cameron, 18. G, doson Laosl, 19. G, chiron Myanmar, 20. G. bathycles

lacei PNG, 25. G. baijanensis Bachan, 26. G wetskei PNG, 27. G. macleayanunz Australia, 28. a cloanthus Taiwan, 33. G. poi?benes Congo, 34. G. ridlq},anus Noia RCA, 3S. G. latneilUanus gui RCA, 39. G. adamastor Bungui RCA, 40. G schubotti Bungui RCA, 41. L. meges Camer-

the ND5 gene in any of the species examined. The G+C content was 59.1-60.4% fbr 28S rDNA and 16.5-18.6% fbr the ND5 gene in the analyzed Grophium species. The maximum sequence divergences of the 28S rDNA and the ND5 genes were 5.23% and 15.46% fbr the Graphium species (Table 2). The 28S rDNA was fbund to have evolved about one third as fast as the ND5 gene.

Phylogenetic tree of the thaphium butterflies

Figs 2, 3 and 4 show the NJ and MP phylogenetic trees of 28S rDNA, the ND5 gene and 28S+ND5, respectively. One 7leinopaipus species was used as an outgroup. Gaps in the alignments were excluded frem phylogenetic analysis, and both gap-excluded and unexcluded trees showed almost identical phylogenetic relationships (gap-unexcluded trees are not shown). Each phylogenetic tree indicates that the genus Graphium including Pathysa, Pazala and Arisbe is monophy]etie, however, the relationships between Grcrphium and other

NII-Electronic Library Service The LepidopterologicalSocietyLepidopterological Society of Japan

PhyLogeny of Grcrphium Inferred from 28S rDNA and ND5 Gene 99

and ND5 of representative Graphium species and related genera of the tribe Graphiini that were

2j 24 25 26 27 ?S 29 30 ]1 ]2 ]] ]4 3S 36 11 ]S 39 op 4] 42 4] 44 4S

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SS2 1[,31 ]1.27 11.31 n.e, 9A4 t].1] I].46 IO.57 T].O! IL3t - U7F e.42 n.e, O.SS n,t 4.4S 4AX 3.74 3,74 5,S4 n.c, e2S n.L 4.e4 4.04 3,5P 3,3U S.3S 10,13 10S7 JO.6V 1131 n.e F!.3fi T4.]5 f057 11,32 1116 N,3t 3.S4 O.42- 8,GS 10,gT g.91 D.9S n.e TO.11 1!.9S i].76 ll,47 IDA2 10,jt, 4.3S 7.6S n.t U4Z n.e 4.4S 4.4S 3.89 S.75 S.fiS 4.77 S.S4 4.9L n.c. n.a ii,e n.e. ".e. n.e. n,e. 7,s4 9.S4 10.4] P.69 ne, IO,42 12,eS le,87 TO,S7 IO.7! 11.02 - E,S4 ICI,ST' 9,?7 10,!S n,e, 10,42 13,60 11."T ltJ6 9S4 10.I3 6.16 S,S4 S.SS 6.]U n.e 4.3] 4.J] 3.74 3,hO j.53 2.75 - n.c. n.e. n.e. ne. ne. S,[t DPS 10!G 10SS ].e, JO,41･ 1].jO Ji.6i 11.32 1[).14 10.4S S,31 B.tt S74 6.t6 - 026 ]O,?S 10gS 10J! n.e, Jl.31 B.4S 11.01 111fi P.B4 9.99 11.3I TI.9e tO.42 le,S7 9.5S 9.9S O.oo 4an 1.15 4.93 Te,]S tOJ2 li,U6 1176 ".e 1?.5T 1].4]' 1!.il ]!S2 T].Ol T],lb 11,9T 12.S2 II.16 ll.46 LO.5T i02S 2.74 4.ca StS 4,Y3 i4i:X 2Ar' l;lg :i/:i l;ll I:/:l :,:? ll/i: :/:l ::1': i;'/?f, ,.,i 2/-i.i, l6i:llli6?lii:S';･i:i::,i:liifili,:S::II,l:ill - 13,i2 13,S9 l5,S] IS,fi2 nc, 14,]6 16,43 14.67 14.Sj J4.66 J4.9S 14,21 T5,Pl T4,!5 I4.1] 13.74 1390 [2.07 11.9! i5.14 ]4M5

ND5. n.e.--not examined. stratiotes Borneo, 6, G. thule Indonesia, Z G! xenocles Vietnam, 8, C, antbocles Sula-'eshi, 9. G, nan, 13. G, hoenei Sichuan, 14. G tamerlanum Gansu, 15. G/ eurous Taiwan, 16. G akthoae Cameron, 21. G. leechi Zhejiang, 22. G. etrt:zpyitrs Myanmar, 23. G, agantemnon Bali, 24. G. waP

G conhus Solomons, 29. G. etnpedbvanum Javal, 30. G. sarpedbn Bali, 31. G mdon Sulawesi, 32. Camerun, 36. G angoinnum Bungui RCA, 37. G. tyncleraeus Bungui RCA, 38. G leonidas Bun- onl, 42. L. curius Myanmar, 43. EL hefibs Brazil, 44. L podotZrius Czech, 45. 71 impen'ads Vietnam

genera were not clear, The 28S NJ tree (Fig. 2a) shows that Lampmptera is most closely related to the genus thaphium, however, the 28S MP tree (Fig. 2b), the ND5 tree (Fig. 3) and the 28S+ND5 tree (Fig. 4) indicate that Eui:ytidles and iphiciides are more closely related to the genus Graphium than Lanzproptera. Moreover, the bootstrap values ofthese divergences were low.

In the28S tree (Fig. 2), the relationships among each species in the genus Graphium were not clear, because they were more closely related to one another and the bootstrap values were low. However, the clusters of the subgenus Pazala (bootstrap values NJ/MP=94/87), the sarpedbn group (NJ/MP=86/69), the cluster of codrus!empedovana (NJfMP=74/76) and the eut:yavlas group and G akikoae group (NJ/MP=94/ 100) were recognized as monophyly with high bootstrap values, respectively. However, G. antiphates was not clustered with

other species of the subgenus Pathysa.

In the ND5 tree (Fig. 3), the relationships amDng the groups were not clear because of low

NII-Electronic Library Service The LepidopterologicalSocietyLepidopterological Society of Japan

100 Hiromichi MAKITA, Tsutomu SHINKAwA, Kiyotaro KoNDo, Lianxi XING and Tohru NAKAzAwA

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Fig. 2. Phylogenetic tree based on about 700 bases of the 28S rDNA sequences. Gaps in alignments were excluded. a: NJ tree; b: MP tree. (for Figs 2-4, a: Phylogenetic tree constructed using the neighborjoining (NJ) method (Saitou & Nei, 1987, using PAUP' 4. 0blO, Swofford, 2000). The evolutionary distances (D) were determined by Kimura's two-parameter evolutionary distanees (Kimura, 1980); b: Phylogenetic tree constructed using the maximum parsimony (MP) method (PAUP* 4,OblO, Swofford, 2000), Heuristic parsimony ana]ysis, all characters having egual weight, stepwise sequence

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Phylogeny of Graphium Inferred 28SornrDNA and ND5 Gene 101

〔 ［ 訂 N 〔 〔 」」 く ＜ 〔（ 厘 〔 ＜ H剖 蹙 瓢 〔U り 唱 口 匡 ∪ （ ロ口 〔 ｛〔〔 自 E A 召 ζ 属 〔 〔 〔 〔 U 幽 劉 〔 H 〔 O Oh （ （一国の 冖 唱匚 昌 昌 H5己 h 冂 ＝ 弓 P 【 冒 厨 D 輔 〔 の＝ の国 σ ヨ 屑霞 N 〔【 （ 毎 Φ 冊 （ 戸 富 富 9 自昏 囗 冖 （ （ O 四βω 雪 τ囗 口 n◎恐肥コ督＝ 日 惆 宙 診 の冂 O ｝OhU 口 （ （ 吩冖（ 曳 騨【 （＝ 』 oo 〔（（目』 匡 日 一日 冫 口 目 〔 〔 一閃め〔舉 君 富 鞄 ≧ ｛ ロ Oh W 2 口 吋 房 臨 9b 謂 房 匚 Σ Σ 口置 口 口》 ｝コ 鏨 ロ 皀 目N 団 円 司 弱 籌 再 調 彭 田 庸 厨 島 口 口儒 口 冨 E 甥 理 【）） 瓢Q OZ 雪 コ U り Q 塁 畠 q 『 〔讐 ≧ 鍔 O 厨 詔 り 鼻 h O O 日 閃 〕冂〔 U コ 甲 〔〔 h 口 （国 ← ｝（ 屑呂 日 E 日 O 畧 O 雷 P QH9 5 〔 Ψコ 雪畄 羣 P 口 巳 巴 ｝巴 日 N 自 尾 口梟 のV〕〕一一一∈ OZ 鳶 冨 罵 O 霜 口 咽 呵日 畠 口 咽 鬢 霞 ヌ 二 ｝OZ ロ 【 蠶Σ O 唱 耳O 帽 冨閥 奮 含§冨 の O 巴 げΨ 凶 E ヨ 冒 晦 冒昭鑒の円諱 誹 【｝ ∪ 口 qO 宣 口 qO qO O 冨← ← り 鈎 り きり 卜 ヨ り ひ 胴 口 〉 05 筍 V 甘 冫 伊昌ロ一筍 り 電 寄 隔 O O q丿 只） O O O ［ し ｝｝ N 日 【日 ） 〕 芻 「昌 h 目曁 臼 目」U U 驚 召 恥 ヨ O 山〕｝｝））｝）〕 扇 〕｝ 口 呉 Σ 的めロ 田 匂 ‘』 脳戟 ＝ U O ［｝ 』 Σ 固 口団窟 口【 り 二 鴇防呂匪麈 d 昭慮 り 寄圃暇鴇匂 「 日 ｝乙 自 昌 匂 毋 ヨ 卜 臼 旨 O 「巴 曇 口 窟 q9 qO 唱 qQ 口 コ 碍 ［一O ［ 帽帽 吋 毎 撰 電 qO ．巴 〕 O E O 詈 q q q 暑Q う d コ国り りりΣ 司｝ミ ℃ 起 岡 可 詮 遼 掃 配 鴨 ▽▽7 勾 § § 巳 罸 可 8 冒曙 qM 自 ロ一［▼鴨 物 」 O 4 q ε む ＝ 9 国 U 鴨 O 「粕 ¢ Q §叩匂吭 、 蚰 ｝ O む 弼 鞄 し 嵐 気 鳴 § ＝ 「 尾口 ＝MQ 丶 ＝它 し葛 唱 の b ”MO q 」 q 途 霜 ロ』罨蔭 訂 Q 句 ℃ 建 霞 目 【O 幵 8 E 日 冒 匹 自 已 O 旨 遠 ヨ り 寰 唱 ＝ 「 P OO O で 「 渓 § 」へ O 莓 恥 り 噌肖 ｝ U ＝ 帽財 ℃ 蘭 ヒ 卍 む 渦 吻さ §「5 碍 財帽 讙電 誕η 、 、 逗 笥 蘭 o Ok Q ◎ 鼕O 3 曇 馨 電 O 蓑 諍 諍 月 口 匿 じ 電 》 〉 q 塁輪 ⇔o qo ⇒o 60 目 O 田 藝 黔 琶 日 匏 龕 匿暑 己 葭 §琶 諱 曜畧 6 粳 9 6 【 【自 鳴 蓮 制 「咽 5 箆「 叫引叫眉 ℃ 唱 対 ミ り」零 町弼黜 照彫噂剛りU 霎 駐国 醤饕套寧 暴蜃畏刀 ℃ 凧 覧 、 「 潤儲 旦 贄 Ulh ・・．，，．．・．．．．．．．．，・，．、 ・・．、．．．・．・− ．．，．，．，，．．．．．．，．，．，．．．．軋 ． O O O O O O O じ O O や 始 O O 物 O 贐 O O O O 6 嘘 口 O O O O O 口 O ¢ り O O 唱 O O O O O 巳 O O O O り O O 嘘 O 娼 O O じ Q 崎晒 』 日 目 』

addition ， addition sequence of 1，000 random replicates ， tree −bis tion −reconnect ．ion （TBR ） − branch swapping ， and the MULTREES option ． The bootstrap values and majurity −rule values are based on 1，000 replicates and expressed as percentages （a ， b ））． 2b shows one of the 217most parsimonious trees（1eft）．and a consensus tree of all 217most parsimoni − − ・ ous trees with 50 ％ majorit y．rule consensus va 且ues （right ）． A11217t1ees had a length of l62 steps ； consistency index，0 ．675 ； rescaled consistency index，0 ．613； retention index， 0．909．

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102 Hirornichi MAK]TA, Tsutomu SHiNKAwA, Kiyotaro KoNDo, Lianxi XiNG and Tohru NAKAzAwA

Genus Graphium :.s Q.Q vts q e tu geg ¢ :-qtu 9 g ggv+

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Fig, 3, PhyLogenetic tree ofthe genus Graphium based on 793 bases ofthe mitochondrial ND5 sequences. a: NJ tree; b: MP tree, 3b shows one of the 60 most parsimonious trees (left) and a consensus tree of all 60 most parsimonious trees with 50% rnajority-rule consensus values (right). All 60 trees had a length of 1401 steps; consistency index, O.344; rescaled consistency index, O.242; retention index, O.704.

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Phylogeny of Graphium Inferred 28SomrDNA and ND5 Gene 103

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Genus Graphium e. ¢

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Fig. 4. Phylogenetic tree ofthe genus Graphiunz based on 28S rDNA and ND5 genes. Gaps in alignments were excluded. a: NJ tree; b: MP tree. 4b shows one of the 12 most parsimonious trees (left) and a consensus tree of all 12 most parsimonious trees with 50% majority-rule consensus v'alues (right). .Art 12 trees had a length of 1419 steps; consis- tency index, O.402; rescaled consistency index, O.296; retention index, O.735.

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’ Phylogeny of （］iaph ／zピ〃 r In飴 rred om 28S rDNA and NDs Gene 105

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罫 n 欝 （ 含 町 〔〔 く ［N り 膚 冖U 白 〔 目 目 〔 〔〔 〔 ＜ 国 ￥ 日 国 日 O 〔 （N 一 〔 〔 り． ご ． 着 ρ 言 （ 〔 【 言 言 ∩ 目 曾 9 【 ＝ 召 目 一 鴛 N 腸 〔 O 匿ヨ 冠 石 ヨ 〔 鬘 可 宙 日 【57 N 畧 δ 冖 〔（ 宕 コ弩 再 ロ 〔 ロ斈 の 口 ⊆ 9 つ 口 〔 目 口 巳 円 言 N m 【言 〔Ψ 目 〔 ．◎o 90 h あ（ 日 05 賽 翁 易 （ o 冂 誹 署 雷 雋 ＝ 尸 π囂（憩 据ヨ 冨 O 目驅 緜 鬘 霎 劼∩mQ P 曾 聾衝 爿 日 扈口 5 層 窮 ロ 萠 臣 唱 ヨ Σ Σ 口 O ∩≧ 窪ニ 需 甥 葛 邑 O o σ 覓 q O ◎昌 8 戸｝冒 昌 O 巴 り リ ← 巴 【導 ｝「 昆 冒 鬲目 〔【＝ 目 雷 P 営 Q 駈 コ冨 釁 〕諺 ｝｝腎｝ ｛目 司 冨 閥OQV国 已 N 願 日 爵 誕 缶 ヨ 8 尸驫 2 巴 篳 窪 日 5 QZ 巴 UZ 自コ 「 O 月 嵳 篝婁 一． 円 馗 q ヨ ＞ 富 ヨ βq 昌 冖冒巳 ｝ 巳 晴 ロ〜賢qO 口胃ロ蜜 忌 謬 ヨ 】U § ） 暫一璋 頃の三 瑳 ∪ り U 蚤巴 冒冒 q 日 冨 ヨ 巨 日 ど 毎 O O O O O ）じ 巳 ｝己 V ）｝9 δ q P5 靄 可電 V 目 何 ヒ 」 彎 ヨ 「 巳 舅 O ∪ 国 罵 』 函 諺 耐藁藁 防 q 量 旨 ゴ ロ口 閉 剛 訂日 口 、 日 』 曼口 富口 口 悶 口 廻 冒詈 ヨ 融 旨 Ψ冒ヨ ← Σ 8 り § Σ 卯ヨ ヨ 理 輕 ε 田 Φ鵞 窟 V 日 じ ロ▽匹 O 冒 O 己 〕 N 旨 G 9 ∪ 量驀 〕〕 3 ［ 竇 『 閉ヒ 卍 目 日 目 「句 耻 り qq 配〕甘 〕 ワ 巴 げ ▽》゜ U 毀 日 亀 轟 ψ 9 前 司 覧 ε 昌 晒 罠 眉 「月 「態 可 踏 討 り り 凵口 ヨ qO 蜀 口 』 曷 学夥 駈 芻 毎 浬 超器 05 Φ 臣 罫君 哥旬 臀ロ § 買 目 口＝ B 9 UO しU 含 「 ℃ 「ロ 8 羇 疊 葡 司 轟 暑 ◎c Φ ヨ 蜀 5 鼻 勧 口 彊 靼 纛 耳高 竃閥 臼 愚 琶馨 審 § O 自 舘 町 円 目 臼 口 § ◎島閃 馳軸 § 書 書 豊 冫畧 蠶 莓暑 藺 【 日 τ 磁 眇知 書℃ 」岩 垂 爿 認 閥葺毒 「肖鬢 現 瓢揖 彊§店 b 【日 耄鬟酒 曽導 轟可岡町町葦諺 婁 U 司 眄、薯．養．書．書嘆．固．篝．田．冒． ＆，り．、．、蓴． 、，．蕁．豊詣 署．日 日．》 ． ．．．． ． ．．．．．．．．．．．、． 、 ． 、． O 省 〇 O O q O ご O O O じ O 哨 ．司 日 H O 噂 O Q 匂 噂 奄 O 噂 O O 壱9 O 9 O O り O 噛 ゆ q O 轡 U 6 9 囁 O O ご 匂 O O O O 亀 匂 O 蟾 O 絹 一 国

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106 Hiromichi MAKITA, Tsutomu SHINKAwA, Kiyotaro KoNDo, Lianxi XING and Tohru NAKAzAwA

beotstrap values. The divergence of the subgenera Pazala (NJ/MP=100/100), Pathysa (NJ/MP=:71/70), AFisbe (NJ/MP=:85/85), the cluster groups of saipedon and poheenes (NJ/MP= 1oo/100), the agamemnon and the wallacei groups and the cluster of wetskei/ baijtinense (NJfMP=:63f59), the eu,Jiwlas group and akikoae (NJ/MP=62/58) and the cluster of codxus/empedovana (NJ/MP:= 1001100) were recognized as menophyly with high bootstrap yalues, respectively. The sarpedbn group, the crgamemnon group, the cluster of weiskei/baijanenEe and the cluster of codnts/empedbvana, which were classified into the subgenus thqphium, fbrmed a monophyletic cluster, However, the eutzpyins group was not clustered with other species of the subgenus (]iraphiuni.

In the 28S+ND5 tree (Fig, 4), the relationships among the groups were not clear, The divergence of the subgenera Pazala (NJIMP=100/1oo), PathJisa (NJ/MP=97/63), and Arisbe (NJ/MP= 98/82), the cluster groups of sa,;pedon and polYicenes (NJ/MP= 1oo/98) and the eut:Jpyfus group and akrkoae (NJ/MP= 1oo/ 100), and the cluster of codrtcsfemlJedbvana (NJ/MP=1oo/100) were recognized independently. The subgenera Ptzaala, PathJnsa and ATishe were monophyletic clusters. The cluster groups of sarpedon and agamemnon, and the cluster of codras/empedbvana, which were classified inte the subgenus Grqpkium, formed a monephyletic cluster: however, the euTp/lzylas group was not clustered with other species of the subgenus Graphium.

Discussion

Phylogenetic relationships of the tribe Leptocircini

Monophyly of the genus Graphium including Pathysa, Pazala and Aiisbe is strongly supported in this study (Figs 2, 4). The bootstrap values in the 28S tree are 96 in both the NJ and MP methods, and it is also quite certain that Pczzala and Pathysa are placed under the genus Graphium. If Pczzala and Pathysa are treated as separate genera as Igarashi (1984), both Arisbe and the euopyins group with akikoae should be upgraded to a generic level. However, the relationships between Lamproptera and the genus Graphium were determined to be unstable, since both the ND5 tree (Fig.,3) and the 28S+ND5 tree (Fig. 4) showed the cluster of Eui:ytides and iphicZ?des tQ be more closely related to the genus Graphium than Lamproptera. The current classification suggests that Lampmptera is most proximate to the genus Graphium (Hancock, 1983; Miller, 1987). We upheld the other generic separations advocated by Munroe & Ehrlich (1960), though noting that lphiclinres and (]raphium were ciosely related. However, the djvergences between the genus (Iraphium and other genera were not definitively identified in this study, and further analysis of the genus thaphium is .required.

Phylogenetic relationships of the genus thapbium

The present classification of the genus Grcrpkium includes four subgenera: Pazala, Patltysa, Aiisbe and Graphium (Hancock, 1983, 1993; Miller, 1987, [Table 1]): however, our study showed no tree in which the phylogenetic relationships of the four subgenera were clear, and their diversification occurred almost at the same time (Figs 2, 3, and 4). The subgenera Pazalb, Ratltysa and Artsbe are shown as separate monophyletic clusters, but the monophyly of the subgenus thophium is not supported as discussed below.

Munroe (1961) argues that the eurous group, placed by the later authors in the subgenus Pazala, belongs to the subgenus Pathysa based on the complexity of the male genitalia.

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Phylogeny of Grophium lnferred from 28S rDNA and ND5 Gene 107

Hancock (1983) and Miller (1987) suggest that Putala is the plesiomorphic sister group to the rest of the genus Graphium, and that Patltysa is slightly more advanced than Pcxxala, Jgarashi (1984), on the other hand, raised Pazala and Patirysa to the generic level based on their immature stages. The present study shows strong monophyly of Pazala in the ND5 tree with high bootstrap values (NJ/MP= Ioo/1oo), but its relation to the subgenus RathJtsa, or to the subgenus Gnaphium, is not clearly shown. It is not suggested that the eurous group belengs to the subgenus Patitysa, nor is it indicated that Pczzala is the sister group to the rest of the genus (irophium, In this background, it is technical!y probable that Pazain and Patirysu are treated as different genera if Arisbe is upgraded to generic level, because these subgenera were rather unstable (see Figs 2-4).

Miller (1987) proposed that all the African (]raphium species be classified into the subgenus Atisbe, and that ATisbe fbrmed acluster with Pathysa. Hancock (1983, 1993), on the other hand, classified the African species into two groups, the non-swordtailed species in the subgenus Anisbe and the swordtailed species in the subgenus (Iraphium, and suggested that Attshe is clustered with the subgenus Graphium. Smith & Vane-Wright (2oo1) classified the Afrotropical Graphium butterflies with a few Indo-Australian butterfiies into the various smal1 groups and showed complicated phylogenetic relationships, and they did not support fbur subgenera. The present study (see Figs2-4) indicates that the African species are divided into two groups, one being the species belonging to Artsbe represented by Hancock, and the other GL poiicenes. The subgenus Artsbe (the non-swordtailed African species) and Pat]rysa was monophyletic with high bootstrap values in Fig.4 (NJ/MP=70/54) as proposed by Miller (1987), and GpotZtrenas clusters with G cloanthus of the subgenus Graphium with high bootstrap values (NJ/MP=86/83 in Fig. 2; NJIMP==97/97 in Fig. 3; NJIMP=1oo!100 in Fig, 4) as proposed by Hancock (1983, 1993). Although we assume that the African species can be classified into the subgenera Arisbe and Graphium as in the

Hancock's classification, further study of more African species may change this interpretation.

The subgenus Patlrysa is sometimes divided into two species groups, viz. the antiphates group and the macareus group (Hanceck, 1983). The fbrmer consists ofsome iphicfides-like kite butterflies with banded fbrewings and long-tailed hindwings, and the latter is comprised of some mimetic butterflies with striated fbrewings and roundish hindwings. The present study of the ND5 gene shows that G. cielasserti, G xenocles and G macaieus in the maeafleus group are clustered with high bootstrap values (NJIMP= 1'OO/100), but G thule is not (Fig, 3). As mentioned below, G. akikoae classified into the macaretLs group by Hancock (1983) forms a cluster wjth the eui:Jtp?ylas group of Grcrphium outside the subgenus Grcrphium.

The antiphates group is shown to roughly be split into two clusters, Ostensibly similar pairs of species, sueh as G dorcus/G antfroclas and G antiphates/G, aristeus, are placed in different clusters with high bootstrap values in Fig. 3 (NJ/MP=97/97 in dorct4s/antiphates; NJ/MP=100/1oo in androclay/artsteus), and the cluster of the,macareus group excluding thule fbrms a higher cluster with one of two clusters of the antiphates group. The present study of 28S rDNA shows that (}L antiphates is not clustered with other species of the subgenus Padysa (Fig. 2a). Hewever, as the 28S rDNA can not be used to determine clese relationships clearly, the position of G antiphates is ambiguous. The grouping of Patdysa by Hancock (1983) is not clearty supported in this study.

In the current classification based on Saigusa et al. (1977, 1982), the subgenus (]raphium consists of the saTpedon group, the agamenznon group and the eui:zg?yhts group But, Saigusa et aZ (1977, 1982) did not specifically state that the subgenus Graphium is rnonophyletic, Miller (1987) also suggested that he could not find autapomorphic characteristics to support

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I08 Hiromichi MAKITA, Tsutomu SHiNKAwA. Kiyotaro KoNDo, Lianxi XING and Tohru NAKAzAwA

the subgenus Grophium. The present study shows that the 28S rDNA sequence lengths of the eut:zpytas group are difTerent from those of the other Graphium butterfiies, and the phylogenetic tree positions of the euurvins group do not cluster with these groups, but rather fbrm a eui ylp,ins cluster independently (Figs 2-4). Therefbre, we assume that the eur:zpylas group does not belong in the subgenus (]rmphium, An introduction ofa new subgenus will be acceptable fbr accQmmodation of this group.

Some justification of the placement of G, akikoae in the euT:ynvlas group is required. Munroe (1961) and Hancock (1983) argued that G akthoae (=phidias) belongs to the subgenus Pathysa, but G akikoae is distinguished from other Pathysa species by various

morphological structures including wing tails, forewing veins, etc. Tsukada & Nishiyama (1980), on the other hand, suggested that a new subgenus would be required for G akikoae. Our results based on the sequence lengths of 28S rDNA and on our phylogenetic trees (Figs 2-4) indicate that G akthoae is more closely related to the euilmpyfus group than to Pathjvasa, or to other groups ofthe subgenus Graphiutn. The monophyly of the cluster consisting of the eur:mpylchs group and akikoae is supported by high bootstrap value of more than 50% in the 28S rDNA tree (NJ/MP=1oo/100), in the ND5 tree (NJ/MP=62/58) and in the 28s rDNA+ND5 tree (NJ/MP= IOO!100),

In conclusion, the present phylogenetic investigation based on molecular analysis confirmed the monophyly ofthe genus Graphium: however the phylogenetic relationships ofeach ofthe genera ef the tribe Leptocircini were not made clear. These results generally agree with the traditional classification based on morphological characteristics, except for the eui:zpyins group in the subgenus thaphium and Gl akiltoae in the subgenus Patdysa. Further study of more Leptocircini butterfiies, especially by adding Pnottrgraphium and the subgenus Eui;yticies to the present material, may elucidate the relationships of each genus of the tribe Leptocircini and the subgenus Gvaphium.

Acknowledgments

We are especially gratefu1 to Dr Kazumasa Ohta, Tokyo Dental College, Dr Takashi Yagi, Osaka Prefecture University, and Mr Go Sasaki, Kyoto University, fbr their comments and

discussion, We also thank the fo"owing entomologists for supplying samples: Haruo

Fukuda, Takashi Matsuka, Sadaharu Morinaka, Mineko Nishiyarna, Youichi Obara, Michitaka Sakakibara, Akio Shinkai, Ftijio Tsukui, and Yasuyuki Watanabe. We thank Mr Toshio Inomata and Mr Hiroshi Yeshimoto fbr valuable advice of the scientific narne.

We thank MrTamio Hosokawa and MrKenichi Odagiri fbr their kind help, We would like to offer our heartfelt thanks to many amateur bUtterfly specialists in China, Malaysia, Indonesia, Myanmar and Japan for providjng samples of various species, This study was supported by the Sasakawa Scientific Research Grant from The Japan Science Society,

References

Celiins, N.S. and M.G.Morris, 1985, 7kreatened Swailowtail Butteijlies of the rvbridl The IUCN red data book viii. IUCN, Gland. D'Abrera, B.L., 1982. Buttei:17ies qf' the Oriental Region. PartI. Papilionidae, Pieridae, Danaidae. Australia: Ferry Creek. Ehrlich, P. R., 1958. The compurative morphology, phylogeny and higher ciassification ofthe butterfiies (Lepidoptera: Papilionoidea). Kans. Uhi- Sct Buza 39: 30S-370. Felsenstein, J,, l985. Confidence lim{ts on phylogenies: an approach using bootstrap. Evokition 39:

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783-791. Hanceck, D.L., 1983. Classification of the Papilionidae (Lepidoptera): a phylogenetic approach. Smithersia 2: 1-48. , 1993. 0rigins and evolution of the Afrotropical Papilionidae (Lepidoptera). Arnotdla Zimbabwe 9: 557-583. Igarashi, S., l984. The classification of the Papilionidae mainly based on the morphology of their immature stages. 7)FO Ga 34: 41-96. Kirn, C,G., Zhou, H.Z., Irnura, Y., Tominaga, O,, Su, Z.H., and S.Osawa, 2000. Pattern of mor- phological diversification in the Leptocarabus ground beetles (Coleoptera: Carabidae) as deduced from mitochondrial ND5 gene and nuctear 28S rDNA sequences. Mblec. Biod Evot 17: 137-l45. Kirnura, M,, 1980. A simple method for estimating evolutionary rate of base substitutions through comparative studies ofnucleotide sequences, X molea EvoL 32: 128-144, Makita, H., Shinkawa, T., Ohta, K., Kondo, A. and T.Nakazawa, 2ooO. Phylogeny of Luehdorfa butterflies inferred from mitochondrial ND5 gene sequences. Ent, Sci. 3: 321･-329. Masui, A. and J. Uehara, 1999. Butterfiies recently collected from Laos P, D. R. (5). Gekkan-Mushi (338): 18-23

MUhn, E., 2oo2. Papilionidae: Baronia, Eui:Fades, Protqgrqphium, IVbeographium und Euny,tidles. frt Bauer, E. and T. Frunkenbach (Eds), Schmetterfinge der Erdle 14: 1-16, pls 1-36. Morita, S. and A, Shinkai, 1996. Descfiption ofa new subspecies of thaphium phidias (OberthUr, t906) from Laos. Futao 23i 9-11. Munroe, E., 196]. The classification of the Papilionidae (Lepidoptera). Can, Ent (Suppl.) 17: l-51. Munroe, E. and P.R.Ehrlich, 1960, Harmonization of concepts of higher classification of the Papilionidae. J LepilrZ Soc. 14: 169-175. Niculescu, E, V., 1977. Un nouveau genre de Graphiini: Klinxigia n. gen. (Lep. Papilionidae). Buza Soc. ent. Muthouse (Oet.-Dec,): 51-52. male des Papilionidae BuU , 1989. L'armaturegenitale (Lepidoptera-Aparasterina),Clercle Lepid Berg 18 (l/2): 10-24. Parsens, M,, 1999. Butte,:f7ies of Papua IVb", Guinea. 711ieir Systematies and Biology. Academic Press, London. Saigusa, T., Nakanishi, A., Shima, H. and O. Yata, 1977. Phylogeny and biogeography of the subgenus Graphium Scopoli (Lepidoptera: Papilionidae, Graphium). Acta rhopaloeerot 1: 2-32. (In Japanese.) 1982. Phylogeny and , geographical distribution of the Swallow-tail subgenus Graphiuni (LepidQptera: Pap{lionidae). Entomologia gen, 8: 59-69. Saiki, R, K., Gelfand, D. H., Stotfe1, S., Schaft, S. T., Hjguchi, R., Horn, G. T., MuJ]is, K. B., and H. A. Erlich, 1988. Primer-directed enzymatic amplification ofDNA with a thermostable DNA polymer- ase. Seience 239: 487-491.

Saitou, N,, and M.Nei, I987. The neighbortl'oining method: a new method for reconstructing phylogenetic trees. Molee BioL EvoZ 4: 406-425. Sanger, F., Nicklen, S., and A.R. CouLson, 1977. DNA sequencing with chain-terminating inhibitors. Proc, natn, Acadl Sci. UL SL A. 74: 5463-5467. Smith, C. R. and R. I, Vane-Wright, 2oo1. A review of the afrotropical species of the genus Grmphiunz (Lepidoptera: Rhopaiocera: Papilienidae). ButL nat, Hist. Mus. Lonal (Ent.) 70: 503-719. Su, Z. H., Ohama, T., Okada, T. S., Nakamura, K., Ishikawa, R., and S. Osawa, 1996a. Phylogenetie relationships and evolution of the Japanese Carabinae ground beetles based on mitochondrial ND5 gene sequences. .IL melee EvoL 42: 124-129. 1996b. Geography-linked ofthe Damaster , phylogeny ground beetlesinferredfrom mitochon- drial ND5 gene sequences. X molec. EvoL 42: 130-134. Swofford, D.L., 2000. PAUZP'. Phytbgenetic Analysis using Pat:simony ('and other methods), Version 4.0blO, Sinauer Associates, Sunderland, Massachusetts.

Taylor, M. F, J., McKechnie, S. W,, Pierce. N. and M. Kreitman, 1993. The Lepidopteran mitochondrial

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110Hiromichi MAKqTA ， Tsutomu SHINKAwA ， Kiyotaro KoNDo ， Lianxi XING and Tohru NAKAzAwA

− control regionl structure and evolution ． Mole（／t BioL EvoL ll： 513 522 ． Thompson ， J． D ．， Gibson， T ． J，， Plewniak ， F、， Jeanmougin ， F 、 and D ． G ， Higgins，1997， The clusta 星X windows interface： flexible strategies for multiple sequence alignrnent aided by quality artalysis tools． − ハiucleic A cids Res ．24 ： 4876 4882 ． ’ Tsukada， E ， and Y ． Nishiyama，1980． Papilionidae． In Tsukada， E ．（£ d．）， Butteffaes of the South East ．4sian Istands 1． Plapac， Tokyo ．（ln Japanese．） Y 且gi， T ．， Sasaki， G ， and H ， Takebe，1999． Phylogeny of Japanese papilionid butterflies inferred from − nucleotide sequences of the mitochondrial ND5 gene． J． molec ． Evol．48： ．42 48．

摘 要 ・ 核 28S rDNA と ミ ト コ ン ド リア ND5 遺伝子 に基 づ く Gr αphium 属の 系 統解析 （牧 田裕 道 新川 勉 ・近藤喜代太郎 ・Lianxi Xing ・中澤透 ）

我 々 は ，Leptocircini族 （Graphiini 族 と 呼 ばれ る こ と もあ る が， こ の 名 称 の 使 用 に つ い て は Smith ＆ − Vane Wright （200 D 参 照 ）また は Graphium 属 の 既 存 の 形 態分 類 と ，遺 伝子 配 列 か ら得 られ た 分 子 系

統樹 の 比 較検 討 を行 っ た ．分 子 系統解 析 で は ，上 位 の 系統 関係 は核 な ど の 進化速 度 の 遅 い 遺 伝子 を用 い ，下 位 の 系統 関係 は ミ ト コ ン ド リア な ど の 進化速度 の 速 い 遺 伝子 を用 い る こ と が 報告 さ れ て お りt ー 本研究 で は ，Leptocircini族 の 解析 に は 進化速度 の 遅 い 核 28S リ ボ ソ ム DNA （28S 丁DN A ）約 700 塩 い ． Graphium が い コ ン NADH ヒ ロ 基 を 用 た ま た ， M 内 の 解析 に は 進化 速度 速 ミ ト ド リ ア デ ド ゲ ー ナ ゼ サ ブ ユ ニ ッ ト 5 （ND5 ）遺 伝 子 793 塩 基 を用 い た ．分 子 系 統樹 は ， NJ 法 〔近 隣 結合 法 ） も し く は MP 法 （最節 約 法 ） に よ っ て ，28S rDNA （Fig．2）， ND5 （Fig．3）な らび に 28S rDNA ＋ ND5 （Fig．4） 一 の 分子 系統樹 を作成 し ，Graphium 属 が 単 系統 で あ る こ と が 示 さ れ た ． ま た 属 内 の 各種 の 分 岐 は 斉 一 に 行 わ れ た こ と が 示 唆 さ れ た ．ま た ，各亜 属 の 系統 関係 は，形 態分 類 を支持 す る も の で あ っ た が ， 部異 な る結果 も得 られ た ．

形 態分 類 で は Graphium 属 に は Pazala， Pathysa， Arisbe，（］raphium の 4 亜 属 に 分 類 され て い る （Smith ー ＆ Vane−Wright （2002）は 4 亜 属 を支持 せ ず，複 数 の 小 グ ル プ か らな る と し て い る）．本研 究 で 得 ら ー れ た ND5 や 28S rDNA 十 ND5 の 系統樹 で は， Pazala， Pathysa， AriSbeは それ ぞ れ 単 独 の ク ラ ス タ を ー 形成 し た ． し か し Graphium 亜 属 に お け る eurypylUS グ ル プ （本研 究 で は 以 下 の 種 を解 析 し た ； G． doson， G ． bathyctes，（｝． ch iron， G ． evemon ，（7． teechi， G eurypylus ） と Hancock （1983） が Pathys α 亜 属 に 分類 し た G． akikoae Merita ＆ Shinkai（＝ phidias OberthUr） は ，本研 究 で 使用 し た 28S rDNA 領 域 の ー ー 塩 基 数 が 他 の 亜 属 や グ ル プ と 比 較 し て も 少 な く （eurypylus グ ル プ と G ． akikoae は 713 塩 基， ー Graphium E の 他 の 種 は 717 塩 基 ）， Graphium 亜 属 の 他 の 種 と は ク ラ ス タ を形 成 し な い こ と か ら， 別 の グ ル ープ で あ る こ とが 分子 系 統樹 に よ り示 され ，eurypylus グ ル ープ は Graphiuni亜 属 に は含 まれ な い こ とが 示 唆 され た ．

また ，形 態 分類 で は ア フ リカ の Graphium 属 を全 て AriSbe亜 属 とす る 説 （Munroe ，1961； Miller； 1987）

ー − と，尾 状突 起 の 無 い グ ル プ を ArtSbe亜 属 （本研究 で は以 下 の 種 を解析 した ； G． ridleyanus ， G ．iatreil

ー一 lianus， G ． angotanum ，（1． tynderaeus ， G ． leonidas， G ．adamastor ， G ．schubotzi ），尾 状突起 の あ る グ ル プ を （lraphium di属 （（1． policenes） と す る説 （Hancock ，1983，1993） に 分 か れ るが ，本研 究 の 分 子 系 統 樹 で は Hancock の 分 類 で あ る Arisbe 亜 属 と Graphium 亜 属 の 2 亜 属 に 分 か れ る こ とを支 持 し て い る．

（Accepted November 5，2002）

Published by the Lepidopterological Society of Japan， 5−20，Motoyokoyama 2， Hachioji， Tokyo ，192−0063 Japan

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