ORIGINAL PAPER

Molecular phylogeography of two sibling species of Euremabutterflies

Satoko Narita Æ Masashi Nomura Æ Yoshiomi Kato ÆOsamu Yata Æ Daisuke Kageyama

Received: 3 November 2006 / Accepted: 12 December 2006 / Published online: 11 January 2007� Springer Science+Business Media B.V. 2007

Abstract The common yellow butterfly Eurema hec-

abe is widely distributed in East Asia, and is one of the

most burdensome species for taxonomists due to the

numerous geographic and seasonal wing colour pat-

terns. Moreover, within this species, individuals with a

yellow wing fringe that occur in temperate regions of

Japan (Y type) proved to be biologically different from

others that occur widely in subtropical regions of Japan

and all over East Asia (B type). To unveil the genetic

variation within and between the two types, a total of

50 butterflies collected at 18 geographic localities in

East Asia were examined for nucleotide sequence

variation of three mitochondrial regions: cytochrome c

oxidase subunit I (COI), cytochrome c oxidase subunit

III (COIII) and NADH dehydrogenase subunit 5

(ND5). In addition, they were also examined for

infection status with the endosymbiotic bacteria

Wolbachia. The three mitochondrial sequences con-

sistently showed that (i) Y type and B type were highly

divergent, (ii) nucleotide variation within B type was

very small although sampled from a geographically

wide range, and (iii) a weak association existed be-

tween mitochondrial DNA haplotypes and Wolbachia

infection status.

Keywords Cytochrome c oxidase subunit I (COI) �Cytochrome c oxidase subunit III (COIII) � NADH

dehydrogenase subunit 5 (ND5) � Butterfly � Eurema

hecabe � Mitochondrial DNA � Phylogeography �Sibling species � Wolbachia

Introduction

The common yellow butterfly, Eurema hecabe

(Linnaeus, 1758) (Lepidoptera: Pieridae), is distributed

almost all over the Oriental, Australian and Afro-

tropical Regions, and extends into the temperate zone

of the Eastern Palaearctic Region such as the northern

part of Honshu, Japan (up to 40�N) (Yata 1989, 1995).

Numerous seasonal and geographic variations of wing

colour pattern in E. hecabe have confounded taxono-

mists, and have resulted in many subspecies and forms

(Yata 1995). But Yata (1989), found that the black

distal border on the forewing upperside and the

markings on the underside show a continuous clinal

variation with latitude. Based on this finding, he inte-

grated these subspecies into the nominotypical E.

hecabe hecabe (Yata 1989).

However, Kato and Handa (1992) found that tem-

perate and subtropical populations of Japan differed

in expression of the polyphenism in response to

S. Narita � M. NomuraLaboratory of Applied Entomology and Zoology,Faculty of Horticulture, Chiba University, Matsudo, Chiba271-8510, Japan

Y. KatoDepartment of Biology, International Christian University,Mitaka, Tokyo 181-8585, Japan

O. YataBiosystematics Laboratory, Faculty of Social and CulturalStudies, Kyushu University, Ropponmatsu, Fukuoka 810-8560, Japan

D. Kageyama (&)Insect-Microbe Research Unit, National Institute ofAgrobiological Sciences (NIAS), Owashi 1-2, Tsukuba,Ibaraki 305-8634, Japane-mail: kagymad@affrc.go.jp

123

Genetica (2007) 131:241–253

DOI 10.1007/s10709-006-9134-1

photoperiod and temperature. Following this discov-

ery, it was demonstrated that the temperate popula-

tions and the subtropical ones of E. hecabe are distinct

from each other in a number of traits: use of host

plants (Kato et al. 1992), colouration of wing fringe

(Kato 1999), reflection pattern against ultra-violet

rays (Matsuno 1999), allelic frequencies of allo-

zymes (Nomura and Kato 1993), and nuclear locus

Tpi (Narita et al. 2006); moreover, they are repro-

ductively isolated through selective mate choice by

females, although hybridization can occur when

reared in the laboratory (Kato 2000b; Kobayashi et al.

2001). These data consistently and strongly suggested

that the temperate populations with a yellow wing

fringe (Y type) and the subtropical populations with

a brown wing fringe (B type) represent two closely

related but biologically different species (Kato and

Yata 2005).

Molecular phylogenetic analyses of Y-type and

B-type populations in Japan showed that, unlike

nuclear DNA sequences, mitochondrial DNA

(mtDNA) sequences did not represent the diver-

gence of the two types. Indeed, mtDNA clearly di-

verged into two clades, and one of them contained

only the Y type. Curiously, however, the other clade

contained both types. All the butterflies in the latter

clade were infected with a single strain of endo-

symbiont Wolbachia (wHec1), while all the butter-

flies in the former clade were uninfected. These

findings suggest that the B-type mitochondria have

introgressed into Y type through hybridization in

the past (Narita et al. 2006). Similarly, Wolbachia

and its associated mitochondria were suggested to

have experienced introgression in other insect species

such as Acraea butterflies, Drosophila fruit flies and

Solenopsis fire ants (Jiggins 2003; Dean et al. 2003;

Shoemaker et al. 2003).

Wolbachia, belonging to the alpha subdivision of

Proteobacteria, is ubiquitously found in arthropods

and filarial nematodes. Wolbachia can affect repro-

duction or sex determination of its insect hosts in

various ways; e.g. via cytoplasmic incompatibility,

parthenogenesis induction, male killing or feminiza-

tion (O’Neill et al. 1997; Werren 1997; Stouthamer

et al. 1999: Bourtzis and Miller 2003, 2006). In

general, Wolbachia are transmitted exclusively from

mothers to offspring, i.e. not from fathers. It can be

considered that these reproductive alterations are

elegant strategies adopted by Wolbachia in order to

increase their transmission efficiency through host

generations.

In a Y-type population in Okinawa, a subtropical

southwestern island of Japan, feminized genetic males

producing almost exclusively female progeny were

found (Hiroki et al. 2002). These females were infected

with two different strains of Wolbachia, wHec1 and

wHec2, while normal females were infected with only

wHec1. It has been known that wHec1 is indistin-

guishable by means of a wsp sequence from the cyto-

plasmic incompatibility-inducing Wolbachia strain

found on mainland Japan (Hiroki et al. 2004; Narita

et al. 2006).

The purpose of this study was to answer the fol-

lowing questions. (1) What is the extent of divergence

between the Y and B types? (2) What is the extent of

variability of mtDNA sequences within the B type? (3)

Is mtDNA variation within the B type associated with

biogeography? (4) Is Wolbachia infection associated

with mtDNA variation?

Keeping these questions in mind, we conducted

molecular phylogenetic analyses of Y and B type but-

terflies collected from 18 geographic localities of seven

countries in East Asia using three mitochondrial

regions. Furthermore, all samples used in this study

were diagnosed to ascertain the status of Wolbachia

infection and examined for its possible effects on the

phylogenetic relationship within and between the two

types of E. hecabe.

Materials and methods

Butterfly sampling and DNA extraction

In total, 49 adults of E. hecabe were collected from 18

geographic localities in East Asia (Table 1, Fig. 1).

Two or more butterflies were obtained from each

locality except for Japan where a single butterfly was

available per location. Field-collected adults were

stored at –20�C until DNA extraction; a small number

of dry specimens were also used. The butterflies were

divided into Y or B type based on the fringe colour

of the forewings (Kato 1999, 2000a). E. blanda from

Iriomote Island and Kanchanaburi (Table 1) was used

as outgroup. Dissected thoracic muscles were subjected

to DNA extraction by using the DNeasy Tissue Kit

(Qiagen).

PCR and sequencing

The mitochondrial COI region, encoding cytochrome

c oxidase subunit I, was amplified by the primers

C1-J-1718 (5¢-GGGGGGTTTGGAAATTGATTAGT

GCC-3¢) and TL2-N-3014 (5¢-TCCATTGCACTAA

TCTGCCATATTA-3¢) (Simon et al. 1994). The mito-

242 Genetica (2007) 131:241–253

123

Table 1 Butterfly samples used in this study

Locality Symbol Individual code Date ofcollection

Type Infectionstatus*

Accession number

COI COIII ND5

Eurema hecabeSendai, Miyagi, Japan JP1 a Jul. 1998 Y – – – AB277717Matsudo, Chiba, Japan JP2 a Jul. 2002 Y – AB277619 AB277663 AB277716Takao, Tokyo, Japan JP3 a Jul. 1994 Y – AB277616 AB277664 AB277718Tsushima Is., Nagasaki, Japan JP4 a Nov. 1996 Y – AB277617 AB277666 AB277719

b Sep. 2003 Y – AB277618 AB277665 AB277720Okinawa Is., Okinawa, Japan JP5 a Jun. 1993 B 1 AB277620 AB277667 AB277714Kume Is., Okinawa, Japan JP6 a May 1996 B 1 AB277623 AB277671 –Ishigaki Is., Okinawa, Japan JP7 a Dec. 1996 B 1 AB277622 AB277669 AB277713Hateruma Is., Okinawa, Japan JP8 a Dec. 1996 B 1 AB277621 AB277668 AB277715Yonaguni Is., Okinawa, Japan JP9 a Nov. 1996 B 1 – AB277670 –Nanjing, China CN1 a Jul. 1999 B 1 + 2 – – AB277674

CN1 b Jul. 1999 B 1 + 2 AB277584 – AB277676CN1 c Jul. 1999 B 1 + 2 – AB277626 AB277675

Hong Kong, China CN2 a May 1995 Y 1 AB277593 AB277641 –CN2 b May 1995 B 1 AB277585 AB277629 AB277677CN2 c May 1995 B 1 AB277592 AB277630 AB277678CN2 d May 1995 B 1 AB277586 AB277631 AB277679

Hainan Is., China CN3 a Mar. 2004 B 1 AB277594 AB277632 AB277680CN3 b Mar. 2004 B 1 – AB277633 AB277681CN3 c Mar. 2004 B 1 + 2 – AB277642 AB277682CN3 d Mar. 2004 B 1 + 2 – AB277643 AB277683

Cheju Is., Korea KR a Aug. 2003 B 1 AB277587 AB277634 AB277684KR b Aug. 2003 B 1 AB277588 AB277649 AB277685KR c Aug. 2003 B 1 AB277595 AB277635 AB277686KR d Aug. 2003 B 1 AB277589 AB277636 AB277700

Sarawak, Malaysia ML a Mar. 2001 B 1 AB277596 AB277650 AB277701ML b Mar. 2001 B 1 AB277597 AB277651 AB277687ML c Mar. 2001 B 1 AB277598 AB277657 AB277688ML d Mar. 2001 B 1 AB277599 AB277652 AB277689

Wetan Is., Indonesia IN a Oct. 2000 B 1 – AB277656 AB277702IN b Oct. 2000 B 1 + 2 AB277600 AB277655 AB277703

Dong Nai, Vietnam VN1 a Mar. 2005 B 1 AB277605 AB277661 AB277707VN1 b Mar. 2005 B 1 AB277606 AB277662 AB277708VN1 c Mar. 2005 B 1 + 2 AB277609 AB277644 AB277693VN1 d Mar. 2005 B 1 AB277610 AB277645 AB277712VN1 e Mar. 2005 B 1 AB277611 AB277646 AB277694VN1 f Mar. 2005 B 1 AB277612 AB277647 AB277709

Cao Bang, Vietman VN2 a Oct. 2004 B 1 AB277591 AB277628 AB277695VN2 b Oct. 2004 B 1 + 2 AB277614 AB277638 AB277696VN2 c Oct. 2004 B 1 AB277615 AB277648 AB277697VN2 d Oct. 2004 B 1 AB277613 AB277639 AB277698VN2 e Oct. 2004 B 1 – AB277640 AB277699

Kanchanaburi, Thailand TL a Sep. 2005 B 1 AB277601 AB277637 AB277690TL b Sep. 2005 B 1 AB277602 AB277653 AB277704TL c Sep. 2005 B 1 – AB277658 AB277705TL d Sep. 2005 B 1 AB277604 AB277659 AB277706TL E Sep. 2005 B – AB277590 AB277627 AB277710TL F Sep. 2005 B – AB277603 AB277654 AB277691TL G Sep. 2005 B – AB277607 AB277660 AB277711TL H Sep. 2005 B – AB277608 – AB277692

Eurema blanda (Outgroup)Kanchanaburi, Thailand Sep. 2005 – AB277624 AB277672 AB277721Iriomote Is., Okinawa, Japan Nov. 2002 – AB277625 AB277673 AB277722

* ‘1’ denotes infection of wHec1; ‘2’ denotes infection of wHec2; ‘1 + 2’ denotes double infection of wHec1 and wHec2

Underlined samples repesent those that were subjected to cloning and sequencing for characterisation of Wolbachia

Genetica (2007) 131:241–253 243

123

chondrial COIII region, encoding cytochrome c oxidase

subunit III, was amplified by the primers Cox3-F2 (5¢-TCAGCTGTTGCTATAATTCAA-3¢) and Cox3-R2

(5¢-TATGATTGGAAGTCAAATATA-3¢) (designed

by N. Haruyama). The mitochondrial ND5 region,

encoding NADH dehydrogenase subunit 5, was ampli-

fied by using the primers V1 (5¢-CCTGTTT

CTCTGCTTTAGTTTAGTTCA-3¢) and A1 (5¢-AA-

TATDA GGTATAAATCATAC-3¢) (Yagi et al.

1999).The PCR temperature profile for the mitochon-

drial genes was 94�C for 2 min, followed by 35 cycles of

94�C for 1 min, 47�C for 1.5 min and 72�C for 1.5 min,

and finally 72�C for 7 min. The PCR products were

purified using the PCR Purification Kit (Qiagen), and

directly subjected to DNA sequencing.

The nucleotide sequences of E. hecabe and E. blanda

were deposited in the DDBJ/EMBL/GenBank data-

bases. Accession numbers are listed in Table 1.

Phylogenetic analyses

Nucleotide sequences were aligned using the program

package ClustalW (Thompson et al. 1994). The se-

quence data of COI, COIII and ND5 regions was anal-

ysed separately. Phylogenetic trees were constructed by

the maximum likelihood (ML) and the maximum par-

simony (MP) methods using the program package

PAUP version 4.0b10 (Swofford 2001). For ML and MP

analyses, the F81 model was used to estimate the num-

ber of nucleotide substitutions. To visualize genealogical

relationships and possible population substructure, net-

works on the basis of sequence data were constructed by

the statistical parsimony algorithm (Templeton et al.

1992) implemented in the program package TCS version

1.13 (Clement et al. 2000). The TCS program calculates

the minimal number of mutational steps by which the

sequences can be joined with >95% confidence.

Fig. 1 Collection localities ofthe Y and B types of E.hecabe used in this study.Locality symbols representthose in Table 1. Scale barindicates 1000 km

244 Genetica (2007) 131:241–253

123

Detection and identification of Wolbachia

PCR detection of Wolbachia infection was performed

using the primers (5¢-TTGTAGCCTGCTATGGTA-

TAACT-3¢) and (5¢-GAATAGG TATGATTTT-

CATGT-3¢) for the 16S rRNA gene (O’Neill et al.

1992), and the primers wsp81F (5¢-TGGTCCAA-

TAAGTGATGAAGAAAC-3¢) and wsp691R (5¢-AA

AAATTAAACGCTACTCCA-3¢) for the wsp gene

(Zhou et al. 1998).

Infection with Wolbachia of either wHec1 or wHec2

strain was discriminated by specific PCR detection

targeting the wsp gene using the primers wsp81F and

WHecFem1 (5¢-ACTAACGTCGTTTTTGTTTAG-3¢:reverse) (designed by Y. Tagami) that amplify the 232

bp DNA fragment for wHec1, and primers WHecFem2

(5¢-TTACTCACAATTGGCTAAAGAT-3¢: forward)

(designed by Y. Tagami) and wsp691R that amplify the

398 bp DNA fragment for wHec2 under temperature

profile 35 cycles of 95�C for 1 min, 58�C for 1.5 min

and 72�C for 1.5 min, and then followed by 72�C for

7 min.

For characterization of Wolbachia strains, one or

two butterflies were used from each locality. A 0.6 kb

segment of wsp gene was amplified using the primers

wsp81F and wsp691R, and the PCR products were

cloned and subjected to DNA sequencing. In addition,

the diagnostic PCR product of the wsp gene (398 bp)

for wHec2 was ligated to the T-easy vector (Promega),

and transformed into E. coli JM109 competent cells.

Inserted plasmids were cloned and extracted from the

transformants, and subsequently subjected to DNA

sequencing using an ABI Prism Big DyeTM terminator

chemistry and ABI 3100 capillary sequencer (Applied

Biosystems).

Nucleotide sequences were aligned with published

Wolbachia sequences from other insects using the

program package ClustalW. Phylogenetic trees were

constructed by the ML methods using the PAUP

4.0b10.

Estimation of nucleotide diversity

Nucleotide diversity (P), i.e. the degree of polymor-

phism within a population, is defined as the average

number of nucleotide differences per site between any

two DNA sequences chosen randomly from the sample

population (Nei and Li 1979). In each mitochondrial

region, nucleotide diversity within double-infected,

single-infected and uninfected butterflies was calcu-

lated using the program package DnaSP (Rozas et al.

2003).

Results

COI region

Haplotype sequences

Forty butterflies (5 of the Y type and 35 of the B type)

sequenced for the COI region, 1055 bp in size, were

polymorphic in 51 nucleotide sites including 34 parsi-

moniously informative sites and constituted 22 haplo-

types.

Maximum parsimony phylogeny

On the phylogeny, 19 haplotypes constituted a mono-

phyletic group exclusively containing B-type butter-

flies, whereas 3 haplotypes of the Y type constituted a

clade although the bootstrap value was low (52%). On

the other hand, Y-type haplotypes, except for that of

CN2-a (from Hong Kong), formed a strongly sup-

ported clade (98%) (Fig. 2a).

Maximum likelihood phylogeny

On the phylogeny, 19 haplotypes constituted a mono-

phyletic group exclusively containing B type butter-

flies, whereas 3 haplotypes of the Y type formed a

clade, although the bootstrap value was low (42%).

Conversely, Y-type haplotypes, except for that of CN2-

a, formed a strongly supported clade (98%) (Fig. 2b).

Statistical parsimony network

TCS calculated a 95% parsimony connection limit of

14 steps. Three clades containing the Y type, CN2-a,

and B-type haplotypes, could not be connected within

this limit. One position in the TCS network had a

closed loop that could not unambiguously be resolved.

This loop was caused by a single homoplastic align-

ment position (Fig. 2c).

COIII region

Haplotype sequences

Forty-eight butterflies (6 of the Y type and 42 of the B

type) sequenced for the COIII region, 736 bp in size,

were polymorphic in 44 nucleotide sites, including 29

parsimoniously informative sites and constituting 21

haplotypes.

Genetica (2007) 131:241–253 245

123

E. blanda (Kanchanaburi)

E. blanda (Iriomote Is.)

Hong KongTokyo

Matsudo

Tsushima Is. Tsushima Is.

Wetan Is. Kanchanaburi

NanjinHong KongHong KongCheju Is. Cheju Is. Cheju Is. KanchanaburiCao Bang

Hong KongHainan Is. Cheju Is.

SarawakCao Bang

Hateruma Is.

Kanchanaburi

Okinawa Is. Kume Is.

Kanchanaburi

KanchanaburiKanchanaburi

Dong Nai

Dong NaiDong NaiDong NaiCao Bang

Kanchanaburi e

pyt-

Ye

pyt-

B

SarawakSarawakSarawak

Dong NaiDong Nai

Cao Bang

Ishigaki Is.

CN1-b

CN2-b

CN2-d

KR-a

KR-b

KR-d

TL-e

VN2-a

CN2-c

CN3-a

KR-c

ML-a

ML-b

ML-c

ML-d

VN2-b

VN2-c

JP8

JP7

TL-g

TL-f

TL-b

TL-a

VN1-d

VN2-d

VN1-c

IN-b

TL-h

VN1-e

VN1-f

JP5

JP6

VN1-a

VN1-b

TL-d

JP2

JP4-b

JP4-a

JP3

CN2-a

99

52

98

62

87

86

10 changes

100

E.blanda (Kanchanaburi)E.blanda (Iriomote Is.)

TokyoTsushima Is.Tsushima Is.

Hong Kong

Matsudo

0.005 substitutions/site

SarawakSarawakSarawak

Dong NaiDong Nai

Sarawak

Okinawa Is. Kume Is.

Kanchanaburi

Wetan Is. Kanchanaburi

NanjinHong Kong

Hong KongCheju Is. Cheju Is. Cheju Is. KanchanaburiCao Bang

Hateruma Is.

Ishigaki Is.

Cao Bang

Cao Bang

Hong Kong

Kanchanaburi

KanchanaburiKanchanaburi

Dong NaiDong NaiDong NaiCao Bang

Dong Nai

Hainan Is. Cheju Is.

100

99

42

98

48

48

50

64

61

89

86

ep y

t-Y

ep y

t-B

CN1-bCN2-b

CN2-dKR-aKR-bKR-d

TL-e

VN2-aCN2-cCN3-aKR-c

ML-aML-bML-c

ML-d

VN2-b

VN2-cJP8

JP7

TL-g

TL-fTL-b

TL-aVN1-d

VN2-dVN1-c

IN-bTL-h

VN1-eVN1-f

JP5JP6

VN1-aVN1-b

TL-d

JP2JP4-bJP4-a

JP3

CN2-a

Kanchanaburi

B-type

JP2JP3

JP4-aJP4-b

CN2-a

Y-type

TL-d

CN1-b

CN2-b

CN2-d

TL-a

TL-g

ML-d

TL-h

VN1-c

IN-b

CN3-a

CN2-c

KR-c

JP7JP8

VN1-d

VN1-eVN1-f

VN2-d

VN2-bVN2-c

KR-a

KR-b

KR-d

TL-e

VN2-a

Eurasian Continent

Cheju-Is.

JP5

JP6

Polynesia

Japan

TL-bTL-fML-b

ML-c

ML-a

VN1-aVN1-b

a b

c

Fig. 2 Molecular phylogenetic analyses based on COI regionsequences (1055 aligned nucleotide sites) of the Y type and Btype of E. hecabe. (A) Maximum likelihood phylogenetic tree. A50% majority-rule consensus bootstrap tree is shown, withbootstrap values over 50% at the nodes. The tree has a loglikelihood of –1919.25. (B) Maximum parsimony phylogenetictree. The tree has consistency index of 0.865 and retention indexof 0.875. The bootstrap values over 50% are shown at the nodes.

(C) TCS parsimony network. Ellipses represent individualbutterflies (white: uninfected, grey: single-infected with wHec1,black: double-infected with wHec1 and wHec2). Rectanglesrepresent haplotypes. The size of each rectangle reflects anumber of butterflies. Small black circles represent missinghaplotypes. A network with 95% connection limit is shown. Thesequences are labelled according to the locality as shown inTable 1 and Fig. 1

246 Genetica (2007) 131:241–253

123

Maximum parsimony phylogeny

On the phylogeny, 18 haplotypes, constituted a

monophyletic group, exclusively containing B-type

butterflies, whereas 3 haplotypes of the Y type con-

stituted a clade which was weakly supported (59%).

On the other hand, Y-type haplotypes, except for that

of CN2-a, formed a strongly supported clade (100%)

(Fig. 3a).

Maximum likelihood phylogeny

On the phylogeny, 18 haplotypes, constituted a

monophyletic group, exclusively containing B-type

butterflies, whereas 3 haplotypes of the Y type con-

stituted a clade which was weakly supported (58%).

On the other hand, Y-type haplotypes, except for that

of CN2-a, formed a strongly supported clade (100%)

(Fig. 3b).

Statistical parsimony network

TCS calculated a 95% parsimony connection limit of

11 steps. Each of the three clades containing Y-type,

CN2-a, and B-type haplotypes could not be connected

within this limit. One position in the TCS network had

a closed loop that could not unambiguously be re-

solved. This loop was associated with combinations of

three homoplastic alignment positions (Fig. 3c).

ND5 region

Haplotype sequences

Forty-seven butterflies (five of the Y type and 42 of the

B type) sequenced for the ND5 region, 716 bp in size,

were polymorphic in 38 nucleotide sites, including 25

parsimoniously informative sites and constituted 21

haplotypes. A sample from Hong Kong (CN2-a) could

not be contained in this phylogenetic tree because we

failed to amplify the ND5 region.

Maximum parsimony phylogeny

On the phylogeny, the haplotypes were split into two

discrete clades. One clade with five haplotypes con-

sisted exclusively of Y type butterflies from Japan

(strongly supported by the bootstrap probability of

99%), whereas the other clade with 14 haplotypes

exclusively consisted of the B type (strongly supported

with bootstrap probability of 86%) (Fig. 4a).

Maximum likelihood phylogeny

On the phylogeny, the haplotypes were split into two

discrete clades. One clade with five haplotypes con-

sisted exclusively of the Y type from Japan (strongly

supported by the bootstrap probability of 97%),

whereas the other clade with 14 haplotypes exclusively

consisted of the B type (strongly supported with

bootstrap probability of 100%) (Fig. 4b).

Statistical parsimony network

TCS calculated a 95% parsimony connection limit of

11 steps. Two clades containing the Y-type and B-type

haplotypes could not be connected within this limit.

One position in the TCS network revealed a closed

loop that could not unambiguously be resolved. This

loop was associated with combinations of four homo-

plastic alignment positions (Fig. 4c).

Wolbachia endosymbiont

Diagnostic PCR

Of 43 B-type adults examined by diagnostic PCR for

Wolbachia, 39 (90.9%) were positive. Of 6 Y-type

adults, one from Hong Kong (CN2-a) was positive

(Table 1). In addition, of all the 40 infected samples

subjected to diagnostic PCR for discriminating be-

tween wHec1 and wHec2, seven were double-infected

with wHec1 and wHec2, while 33 were single-infected

with wHec1 (Table 1).

Characterisation of Wolbachia strains

One or two Wolbachia-infected adults from each

locality, i.e. 15 adults (JP5, JP6, JP8, JP9, CN1-b, CN3-

b, CN3-c, KR-c, ML-a, IN-a, VN1-a, VN1-c, VN2-a,

VN2-c, TL-a), were subjected to PCR that amplifies

the ca. 600 bp segment of wsp gene by using primer

pair wsp81F and wsp691R followed by cloning and

DNA sequencing.

About 71 clones in total, i.e. three to eight clones

from each of the 15 Wolbachia-infected butterflies

(including 12 single-infected and three double-infected

butterflies), were identical to that of wHec1 (Hiroki

et al. 2004). Since no wHec2-specific sequence was

found from the three double-infected butterflies (24

clones in total), the wHec2-specific PCR product

of the wsp gene (398 bp) in each of the double-

infected butterflies (Table 1) was directly subjected to

DNA sequencing. The sequences were identical to

Genetica (2007) 131:241–253 247

123

10 changes

Miyagi MatsudoTokyoTsushima Is.

Hong Kong

Tsushima Is.

Hong Kong Hong Kong Hong Kong Hainan Is. Hainan Is. Cheju Is. Cheju Is. Cheju Is. Kanchanaburi Cao Bang Cao Bang Cao Bang

Hainan Is. Cheju Is. Wetan Is.

SarawakSarawak Kanchanaburi Kanchanaburi Yonaguni Is.

Ishigaki Is. Hateruma Is.

Hainan Is. Dong NaiDong NaiDong NaiDong NaiCao Bang

SarawakWetan Is.

Sarawak

KanchanaburiDong NaiOkinawa Is.

Dong Nai

Kanchanaburi Kanchanaburi

Kume Is.

Nanjing KanchanaburiCao Bang

59

100

96

62

E.blanda (Kanchanaburi)E.blanda (Iriomote Is.)

epy

t -Y

epy

t -B

CN3-d

CN2-b

CN2-d

KR-a

KR-b

KR-d

TL-eVN2-a

CN2-c

CN3-a

KR-c

ML-a

ML-b

ML-c

ML-d

VN2-b

VN2-e

JP8JP7

JP4-a

TL-fTL-b

TL-a

VN1-d

VN2-d

VN1-c

IN-bTL-g

TL-c

VN1-a

JP5

JP6

VN1-e

VN1-b

VN1-f

CN1-c

CN3-c

TL-d

CN2-c

JP9

CN3-b

IN-a

CN2-aJP1JP2

JP3JP4-b

0.005 substitutions/site

E.blanda (Kanchanaburi)E.blanda (Iriomote Is.)

Hong Kong

MigagiMatsudoTokyoTsushima Is. Tsushima Is.

100

58

Hong Kong Hong Kong Hong Kong Hainan Is. Hainan Is. Cheju Is. Cheju Is. Cheju Is. Kanchanaburi Cao Bang Cao Bang Cao Bang

Hainan Is. Dong NaiDong NaiDong NaiDong NaiCao Bang

Sarawak Wetan Is.

Sarawak

KanchanaburiDong NaiOkinawa Is.

Kanchanaburi Kanchanaburi Dong NaiKume Is.

Nanjing KanchanaburiCao Bang

Hainan Is.

Sarawak Sarawak Kanchanaburi Kanchanaburi

Cheju Is. Wetan Is.

61Yonaguni Is.

Ishigaki Is. Hateruma Is.

90

100

ep y

t -Y

epy

t -B

CN3-d

CN2-b

CN2-d

KR-a

KR-b

KR-d

TL-eVN2-a

CN2-c

CN3-a

KR-c

ML-a

ML-b

ML-c

ML-d

VN2-b

VN2-e

JP8JP7

JP4-a

TL-fTL-b

TL-a

VN1-d

VN2-d

VN1-c

IN-b

TL-g

TL-c

VN1-a

JP5

JP6

VN1-e

VN1-b

VN1-f

CN1-c

CN3-c

TL-d

CN2-c

JP9

CN3-b

IN-a

CN2-a

JP1JP2

JP3JP4-b

ML-c

JP7JP8

CN1-c

TL-eVN2-a

ML-b

ML-dTL-b

TL-f

TL-g

JP9

ML-a

IN-bIN-a

JP5

JP6

CN3-c

VN1-a

VN1-b

TL-cTL-d

CN3-d

VN1-c

VN1-d

VN1-e

VN1-f

VN2-c

B-type

JP1

JP2JP3

JP4-bJP4-a

CN2-aY-type

Japan

Polynesia

Eurasian Continent

CN2-b

CN2-c

CN2-d

CN3-a

CN3-bKR-a

KR-c

KR-d

TL-a

VN2-b

VN2-d

VN2-e

KR-b

Cheju Is.

a b

c

Fig. 3 Molecular phylogenetic analyses based on the COIIIregion (736 aligned nucleotide sites) of the Y type and B type ofE. hecabe. (A) Maximum likelihood phylogenetic tree shown asin Fig. 2. The tree has a log likelihood of -1731.86. (B) Maximum

parsimony phylogenetic tree shown as in Fig. 2. The tree hasconsistency index of 0.809 and retention index of 0.806. (C) TCSparsimony network shown as in Fig. 2

248 Genetica (2007) 131:241–253

123

Matsudo

TokyoTsushima Is.

Tsushima Is.

Miyagi

99

Nanjing Hong Kong Hong Kong Hong Kong Hainan Is. Hainan Is. Hainan Is. Hainan Is. Cheju Is. Cheju Is. Cheju Is. Sarawak Sarawak Sarawak Kanchanaburi Kanchanaburi Kanchanaburi Don NaiDon NaiCao Bang Cao Bang Cao Bang Cao Bang Cao Bang

Wetan Is. Kanchanaburi Kanchanaburi Ishigaki Is.

Okinawa Is. Hateruma Is.

86

Nanjing

Cheju Is. Kanchanaburi

Sarawak Wetan Is. Kanchanaburi Don Nai

10 changes

56

64

62

E.blanda (Kanchanaburi)E.blanda (Iriomote Is.)

55

ep y

t-Y

epy

t-B

CN3-d

CN2-b

CN2-d

KR-aKR-b

KR-d

TL-e

VN2-a

CN2-c

CN3-a

KR-c

ML-a

ML-bML-cML-d

VN2-b

VN2-e

JP8

JP7

JP4-a

TL-f

TL-b

TL-a

VN1-d

VN2-d

VN1-c

IN-b

TL-g

TL-c

VN1-a

JP5

VN1-e

VN1-bVN1-f

CN1-c

CN3-c

TL-d

VN2-c

CN3-b

IN-a

JP1JP2

JP3JP4-b

CN1-b

TL-h

CN1-a

Kanchanaburi Don NaiDon NaiDon Nai

Nanjing

100

E.blanda (Kanchanaburi)E.blanda (Iriomote Is.)

0.005 substitutions/site

Matsudo

TokyoTsushima Is.

Tsushima Is.

Miyagi

Nanjing Hong Kong Hong Kong Hong Kong Hainan Is. Hainan Is. Hainan Is. Hainan Is. Cheju Is. Cheju Is. Cheju Is. Sarawak Sarawak Sarawak Kanchanaburi Kanchanaburi Kanchanaburi Don NaiDon NaiCao Bang Cao Bang Cao Bang Cao Bang Cao Bang

Nanjing Nanjing

KanchanaburiKanchanaburi Don NaiDon NaiDon Nai

Cheju Is.

Wetan Is. Wetan Is.

KanchanaburiKanchanaburiIshigaki Is.

Okinawa Is. Hateruma Is.

Sarawak Kanchanaburi Don Nai

100

97

62

56

60

62

epy

t-Y

epy

t -B

CN3-d

CN2-b

CN2-d

KR-aKR-b

KR-d

TL-e

VN2-a

CN2-c

CN3-a

KR-c

ML-a

ML-bML-cML-d

VN2-b

VN2-e

JP8

JP7

JP4-a

TL-f

TL-b

TL-a

VN1-d

VN2-d

VN1-c

IN-b

TL-g

TL-c

VN1-a

JP5

VN1-e

VN1-bVN1-f

CN1-c

CN3-c

TL-d

VN2-c

CN3-b

IN-a

JP1

JP2

JP3JP4-b

CN1-b

TL-h

CN1-a

a b

cB-type

IN-b IN-a

TL-e

ML-a

TL-b

KR-dTL-g

CN1-aCN1-c

CN1-b

CN3-c

CN3-d

CN3-b

CN3-a

CN2-dCN2-cCN2-b

ML-b

ML-c

ML-d

TL-aTL-fTL-h

KR-a

KR-b

KR-c

VN1-cVN1-eVN2-aVN2-b

VN2-c

VN2-d

VN2-e

TL-c

TL-d

VN1-aVN1-b

VN1-f

JP8

JP7

JP5

VN1-d

JP4-b

JP3

JP4-a

JP1

JP2

Y-type

Japan

Polynesia

Eurasian Continent

. sI

uje

hC

Fig. 4 Molecular phylogenetic analyses based on the ND5region (716 aligned nucleotide sites) of the Y type and B typeof E. hecabe. (A) Maximum likelihood phylogenetic tree shownas in Fig. 2. The tree has a log likelihood of -1283.32. (B)

Maximum parsimony phylogenetic tree shown as in Fig. 2. Thetree has consistency index of 0.897 and retention index of 0.932.(C) TCS parsimony network shown as in Fig. 2

Genetica (2007) 131:241–253 249

123

wHecFem2 (Hiroki et al. 2004). Each of the wsp se-

quences of wHec1 and wHec2 formed an independent

clade in the B supergroup of the Wolbachia phylogeny

(Fig. 5). The nucleotide sequences of wsp gene of

Wolbachia from the Y type and B type of E. hecabe

were deposited in the DDBJ/EMBL/GenBank data-

bases under accession numbers AB278198-AB278220,

respectively.

Comparison of nucleotide diversities

In each mitochondrial region, nucleotide diversity (P)

was compared between double-infected, single-in-

fected and uninfected butterflies (Table 2). In COI,

COIII and ND5, nucleotide diversity of Wolbachia-

infected butterflies, both double-infected and single-

infected, were likely to be smaller compared to that of

uninfected butterflies. Within double-infected butter-

flies, compared to that of single-infected butterflies,

nucleotide diversity was smaller in COIII and ND5, but

larger in COI.

Discussion

Mitochondrial DNA strongly supports the

distinction between Y and B type

It has been suggested that, in E. hecabe, the Y type is

biologically different from B type at species level based

on morphological, ecological, physiological, reproduc-

tive and genetic lines of evidence (Kato and Yata

2005). Our data of mitochondrial regions COI, COIII

and ND5, strongly supports the distinction of the two

types (Figs. 2–4).

Genetic distance (D), i.e. average number of

nucleotide substitutions per site, of ND5 between the

two types was calculated to be 0.03000 by using the

Kimura 2-parameter method (Kimura 1980). The

molecular evolutionary rate of ND5 in Parnassius

butterflies was estimated to be 0.01D per 0.75 million

years (Yagi et al. 2001). By extrapolating the evolu-

tionary rate, the divergence of the two types was esti-

mated to have occurred around 2.25 million years ago.

The mtDNA from one butterfly (CN2-a) from Hong

Kong was located at a peculiar position in phylogeny

(Figs. 2, 3). Although morphologically classified into

the Y type, i.e. wing fringe colour was yellow, the clade

including this sample was only supported with very low

bootstrap probabilities (COI: 52% for maximum like-

lihood, 42% for maximum parsimony; COIII: 59% for

maximum likelihood, 58% for maximum parsimony).

Apparently, this individual was closely related to either

Y-type or B-type but, at the same time, significantly

differentiated from either of the two types, presumably

Junonia almana almana[AB094384]Gronotoma micromorpha[AB037895]

Drosophila simulans Noumea [AF020074]Aedes albopictus [AF02005]

Trichogramma kaykai [AF071912]Cadra cautella[AF020075]

Encarsia formosa[AF071918]Cadra cautellaB [AF020076]

0.05 substitutions/site

Tytthus chinensis [AF481171]Tribolium confusum [AF020083]

Liriomyza bryoniae [AB231469]

98

5498

100

71

98

77

100

Eurema hecabe Y-type [AB094396]Eurema hecabe B-type [AB278213~AB278220]Pseudagrion pruinosum [AY173942]Agromyza oryzae [AF481201]

Eurema hecabe Y-type [AB210826~AB210831]Eurema hecabe B-type [AB278198~AB278212]Culex pipiens [AF020061]Acraea encedon[AJ271198]Hypolimnas bolina [AJ307074]Hercinothrips femoralis [AB245521]Everes argiades[AB094390]

w Hec2

w Hec1

Fig. 5 Maximum likelihoodphylogeny on the basis of thewsp gene sequences ofWolbachia endosymbiontsdetected from E. hecabe andother insects. A 50%majority-rule consensusbootstrap tree is shown, withbootstrap values over 50% atthe nodes. Scientific names ofthe host insects are shown. Inbrackets are nucleotidesequence accession numbers

Table 2 Comparison of nucleotide diversity (p) between indi-viduals with different infection status

COI COIII ND5

Double-infected 0.00298 (7) 0.00257 (10) 0.00112 (12)Single-infected 0.00254 (24) 0.00297 (28) 0.00261 (26)Uninfected 0.00332 (4) 0.00362 (3) 0.00279 (4)

In parentheses are the numbers of samples sequenced

250 Genetica (2007) 131:241–253

123

representing an independent species. Considering that

Hong Kong is a pivotal area for global transportation,

this individual may be a stray butterfly belonging to

another species that occurs in other part of the world.

Construction of a complete global phylogeny of Eur-

ema butterflies will clarify this point.

Low level of mitochondrial DNA differentiation

within the B type despite being sampled from wide

geographic areas

E. hecabe is morphologically most variable among the

genus Eurema. Because of seasonal and geographic

variations, numerous subspecies have been described;

e.g. mandarina from Japanese mainland, and hobsoni

from Taiwan (see Yata 1995). Close inspection by Yata

(1989), however, revealed that almost all of these

variations are clinal ones according to latitude. Based

on this finding, he proposed an integration of numer-

ous subspecies into a single subspecies Eurema hecabe

hecabe.

In this study, although sampling was conducted from

a wide geographic area in East Asia (Fig. 1), the three

mitochondrial regions consistently indicated a small

genetic variation within the B type (Figs. 2–4). This is

the first molecular phylogenetic analysis that strongly

supports the notion that the B type, which occurs

widely throughout East Asia, represents one single

subspecies, as was previously proposed from morpho-

logical data (Yata 1989).

Biogeographical implications

Statistical parsimony network reflects genealogical

relationships of the mtDNA haplotypes, that is, single

mutation steps separate adjacent haplotypes in the

network, and older haplotypes are placed at internal

branching points whereas younger ones occur toward

the tip positions.

In each of the three mitochondrial regions, two

discrete parsimony networks which could not be con-

nected at statistically significant level were generated.

The two networks corresponded to the Y type and B

type, respectively. Despite the small sequence varia-

tion within the B type, the network revealed that pat-

terns of mtDNA haplotypes are more or less associated

with geographic locations. The most remarkable asso-

ciation among them comes from Japanese subtropical

populations samples, which consistently appeared at

tip positions; this finding is likely to reflect that these

haplotypes are relatively young. This may also reflect

that, in the B type, Japanese subtropical populations

are differentiated from other populations in East Asia.

Although less clear, many of the samples from the

Eurasian Continent were placed at most major and

central positions which are likely to be ancestral

(Figs. 2c, 3c, 4c).

B-type haplotypes showed a star-shaped pattern.

This pattern is commonly understood to be indicative

of a population expansion. The pattern of geographic

distribution was in agreement with the predictions

from the coalescent theory that assumes that older al-

leles would prevail in populations and be characterised

by a higher number of descending lineages and geo-

graphically wider distribution (Crandall and Temple-

ton 1993; Posada and Crandall 2001).

Two distinct strains of Wolbachia were shared by Y

type and B type

Diagnostic PCR revealed that 39 out of 43 B-type

butterflies were infected with Wolbachia. All of them

were infected with wHec1, and 12 of them were addi-

tionally infected with wHec2. It has been previously

suggested by molecular phylogenetic analysis that the

infection of wHec1 in the Y type is associated with

introgression of B-type cytoplasm through hybridiza-

tion followed by the Wolbachia sweep up to the central

Japan (Narita et al. 2006). Likewise, wHec2 in Y-type

butterflies that causes feminization had probably int-

rogressed from B-type butterflies, but somehow re-

mained in subtropical regions in Japan. Therefore, it is

not surprising that wHec1 and wHec2 are shared by

both types.

The result that no wHec2-specific sequence was

found from 24 wsp clones suggested that wHec2-posi-

tive butterflies were doubly infected with wHec1 and

wHec2 at the density ratio of at least 24. It should be

noted that other types of Wolbachia may also coexist in

a low density comparable to that of wHec2.

One point that should be kept in mind is that the

reproductive phenotype of wHec1 and wHec2 in B-

type butterflies is unknown. It has been demonstrated

in Y-type butterflies that the occurrence of wHec1 is

involved in cytoplasmic incompatibility while co-

occurrence of wHec1 and wHec2 is involved in femi-

nization (Hiroki et al. 2004, 2005). It is unknown,

however, whether these Wolbachia strains in the B

type behave exactly as in a Y-type background. More

fundamentally, there is a possibility that wHec1 and

wHec2 in B-type butterflies may not be identical as

those in Y-type butterflies, since we relied exclusively

on wsp sequence for genotyping Wolbachia. Further

studies are required to clarify the genotype and

Genetica (2007) 131:241–253 251

123

reproductive phenotype of these Wolbachia strains in a

B-type background.

Curiously, CN2-a from Hong Kong was infected

with wHec1. Having a distinct type of mtDNA, the

presence of wHec1 cannot be explained by introgres-

sion. The presence of wHec1 in this type/species would

be an important cue in revealing the infection history

of Wolbachia in E. hecabe butterflies.

Did Wolbachia affect population structure

of B-type butterflies?

A recent theory suggests that maternally-inherited

endosymbionts, such as bacteria of the genus Wolba-

chia, may affect host mtDNA genome evolution (Prout

1994; Turelli 1994; Hurst et al. 1996; Johnstone and

Hurst 1996), and several empirical studies directly

examining the effects of Wolbachia on mtDNA varia-

tion largely support this hypothesis (Turelli et al. 1991;

Hoffmann et al. 1994; Shoemaker et al. 1999; Marcade

et al. 1999; Ballard 2000; Ballard et al. 2002).

This effect of Wolbachia, the so-called Wolbachia

sweep, has also been shown in Y-type butterflies

occurring on mainland Japan (Narita et al. 2006). In B-

type butterflies examined in this study, however, such

Wolbachia effects on mtDNA variation were not as

conspicuous as in the above studies. Still, mtDNA

variation in the B type is likely to be weakly associated

with the presence of Wolbachia, although the sample

size was not large enough to draw a firm conclusion.

The nucleotide diversity of Wolbachia-infected but-

terflies, both double-infected and single-infected, was

likely to be smaller than that of uninfected butterflies

(Table 2), presumably reflecting a Wolbachia sweep.

This tendency was consistently seen in all the mito-

chondrial regions examined. Between double-infected

and single-infected butterflies, on the other hand, such

tendency was not observed consistently. A larger

sample will reveal more in detail the association of

Wolbachia-infection status with variation of mtDNA in

E. hecabe.

Acknowledgments We would like to thank Hironori Sakamotofor helpful discussion. This study was financially supported inpart by the Japan Society for the Promotion of Science (JSPS)fellowship for Young Scientists to S. N.

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