RESEARCH ARTICLE

Hiromi Iwasaki Æ Akiko Kyuno Æ Mifue Shintaku

Yuko Fujita Æ Yoshihiro Fujiwara Æ Katsunori FujikuraJun Hashimoto Æ Leonardo de Oliveira Martins

Andrey Gebruk Æ Jun-Ichi Miyazaki

Evolutionary relationships of deep-sea mussels inferred by mitochondrialDNA sequences

Received: 17 August 2005 / Accepted: 1 February 2006 / Published online: 9 March 2006� Springer-Verlag 2006

Abstract In order to elucidate the evolutionary processof deep-sea Bathymodiolus mussels, we investigated thephylogenetic relationships of 16 species worldwide byanalyzing nucleotide sequences of the mitochondrialCOI and ND4 genes. Deep-sea mussels were clusteredinto three groups by basal trichotomous divergence. Thefirst was composed of four species found in Japanesewaters and one species from the Gulf of Mexico, whichcontain methanotrophic endosymbiotic bacteria. Thesecond included nine species distributed in the West andEast Pacific, Indian, and Atlantic Oceans. Members ofthe second group were trichotomously divided into theIndo-West Pacific, Atlantic, and East Pacific subclusters.The Indo-West Pacific subcluster was composed of three

very closely related species with mutual genetic distancesat the intraspecific level (av. 0.019 in COI and 0.009 inND4 relative to av. 0.156 in COI and 0.265 in ND4among Bathymodiolus species other than Cluster Aspecies), suggesting some gene flow among these species.The third consisted of two West Pacific species. Speciesin the second and third groups contain mainly thio-autotrophic endosymbionts, including some speciesharboring both methanotrophs and thioautotrophs.

Introduction

In hydrothermal vents on spreading ridges and back-arcbasins and cold-water seeps along subduction zones,there exists a unique biological world where primaryproduction is not based on photosynthesis but on bac-terial chemosynthesis. By emitting water heated byunderlying magmatic chambers, vents that persist foronly a few decades supply inorganic nutrients such assulfide and methane to chemosynthetic bacteria (Jollivet1996). Seeps, where water as cold as the ambient deep-sea water exudes, provide a relatively stable source ofmaterials. In these deep-sea reductive environments,dense biological assemblages including many endemicspecies, i.e. chemosynthesis-based communities, areflourishing. Since their patchy and ephemeral habitatsare separated from one another by various distances, itseems easy for organisms in chemosynthesis-basedcommunities to be genetically isolated. However, geneticdifferentiation and consequent speciation are notdependent simply on geographical distances among thediscontinuous localities, but are caused by a combina-tion of factors shared by diverse taxa (topography,geological histories, and oceanic currents) and thoseunique to their respective taxa (dispersal ability, physi-ology, and settlement cues). Comparative studies onevolutionary processes of different lineages, based onrefined worldwide phylogenetic studies in each lineage,can provide useful information to specify factors driving

Communicated by S. Nishida, Tokyo

H. Iwasaki Æ A. Kyuno Æ M. Shintaku Æ Y. FujitaInstitute of Biological Sciences, University of Tsukuba,Tsukuba, Ibaraki, 305-8572, Japan

Y. Fujiwara Æ K. FujikuraResearch Program for Marine Biology and Ecology,Japan Agency for Marine-Earth Science andTechnology (JAMSTEC), Natsushima, Yokosuka,Kanagawa, 237-0061, Japan

J. HashimotoFaculty of Fisheries, Nagasaki University,Bunkyo1-14, Nagasaki, 852-8521, Japan

L. de O. MartinsGraduate School of Agriculture and Life Sciences,University of Tokyo, Yayoi 1-1-1, Bunkyo-ku,Tokyo, 113-8657, Japan

A. GebrukLaboratory of Ocean Benthic Fauna, P. P. Shirshov Instituteof Oceanology, Russian Academy of Sciences,Nakhimovsky Pr., 36, Moscow, 117991, Russia

J.-I. Miyazaki (&)Faculty of Education and Human Sciences,University of Yamanashi, Kofu, Yamanashi, 400-8510, JapanE-mail: miyazaki@yamanashi.ac.jpTel.: +81-55-2208149Fax: +81-55-2208211

Marine Biology (2006) 149: 1111–1122DOI 10.1007/s00227-006-0268-6

dispersion, isolation, and speciation of organisms inchemosynthesis-based communities. In this study, wefocused our interest on deep-sea mussels to reconstructtheir evolutionary processes.

Mussels of the genus Bathymodiolus are one ofdominant macroorganisms in chemosynthesis-basedcommunities. Deep-sea mussels rely primarily on che-moautotrophic endosymbionts for their nutrition asfound with vesicomyid clams and vestimentiferan tube-worms, although the mussels do not abandon filterfeeding. Since the genus Bathymodiolus (the subfamilyBathymodiolinae of Mytilidae) was first described(Kenk and Wilson 1985), a total of 18 species have beendescribed: 9 species, including 6 of Japanese waters,from the West Pacific Ocean (Cosel et al. 1994; Coseland Marshall 2003; Hashimoto and Okutani 1994;Okutani et al. 2004b), 1 species from the Indian Ocean(Hashimoto 2001), 7 species, including 4 of the Gulf ofMexico, from the Atlantic Ocean (Cosel et al. 1994,1999; Cosel and Olu 1998; Cosel 2002; Gustafson et al.1998), 1 species from the East Pacific Ocean (Kenk andWilson 1985). Several undescribed species, e.g. thosefrom the West and East Pacific (Manus Bathymodiolussp. and East Pacific Bathymodiolus sp., respectively inthis study), have also been found (Cosel 2002). A singlespecies belonging to the genus of the subfamily Bathy-modiolinae, Tamu fisheri, has been described from seepsof the Gulf of Mexico (Gustafson et al. 1998). Twoother consubfamilial species, Gigantidas gladius andG. horikoshii, have been described from off northernNew Zealand and southwest of the Ogasawara Islands,respectively (Cosel and Marshall 2003; Hashimoto andYamane 2005). Most of these species are restricted intheir habitats to either hydrothermal vents or cold-waterseeps, but the three species (B. japonicus, B. platifrons,and B. aduloides) endemic to Japanese waters exploitboth types of habitats.

Our previous study, using two-dimensional gel elec-trophoresis of foot proteins and nucleotide sequencingof the mitochondrial COI gene (Miyazaki et al. 2004),showed that small genetic distances and common COIhaplotypes (possessed by four of the five specimens inB. japonicus and by four of the six specimens in B. plat-ifrons, respectively) were found between specimens fromhydrothermal vents of the Okinawa Trough and cold-water seeps of the Sagami Bay in the two species fromJapanese waters, indicating intraspecific genetic ex-changes between them. This suggests that larval dis-persal ability is relatively high, which is favorable forcolonization of patchy and ephemeral habitats, andenvironmental types (vent vs. seep) are not responsiblefor habitat segregation and speciation in these Bathy-modiolus species. The East Pacific species B. thermophi-lus, as well as the vesicomyid clam Calyptogenamagnifica, are thought to travel thousands of kilometers(Vrijenhoek 1997). While gene flow was expected be-tween populations of the two species from Sagami Bayand the Okinawa Trough (ca. 1,500 km apart) in Japa-nese waters, no species are shared by the Sagami Bay

and the Izu-Ogasawara Island-arc (ca. 500 km apart),which shows that colonization and speciation are notdependent on geographical distances. Genetic homoge-neity over long distances has been shown in someputative species of vestimentiferan tubeworms (Kojimaet al. 2001a, 2002, 2003).

Our study (Miyazaki et al. 2004) also showed thatbathymetrical zonation was not likely responsible forhabitat segregation and speciation in the three speciesfrom Japanese waters, because their vertical distributionranges broadly overlapped. On the other hand, Maaset al. (1999) showed that two Mid-Atlantic speciesinhabited different bathymetrical zones separated byapproximately 1,000 m and proposed that depth mightprovide fundamental barriers to dispersal in these spe-cies. It has been shown that vesicomyid clams havevertical stratification of their habitats, and habitation isnot constrained by environmental types and geographi-cal distances, but by bathymetry probably due to theirdifferential physiological tolerance to pressure (Olu et al.1996; Fujikura et al. 2000; Goffredi et al. 2003). Habitatsegregation of provannid gastropods (Kojima et al.2001b) and neoverrucid barnacles (Watanabe et al.2005) in chemosynthesis-based communities has beensuggested to depend on some differences in sea areas, butnot on bathymetry.

In this study, we compared sequences of the mito-chondrial COI and ND4 genes among the 16 speciessampled and from a database in order to infer theworldwide phylogeny of deep-sea Bathymodiolus mus-sels.

Materials and methods

Materials

Specimens used in this study are listed in Table 1 andcollection sites are mapped in Fig. 1. All the deep-seamussels of the genus Bathymodiolus (Mytilidae, Bathy-modiolinae) except for two Mid-Atlantic species werecollected during dives of submersibles from JapanAgency for Marine-Earth Science and Technology(JAMSTEC). B. puteoserpentis and B. azoricus werecollected by the scientific research vessel ‘‘AkademikMstislav Keldysh’’ belonging to the Institute of Ocean-ology at the Russian Academy of Sciences. The out-group species, Adipicola pacifica (Mytilidae,Modiolinae), was collected from sunken whale carcassesduring dives of the remotely operated vehicle ‘‘HyperDolphin’’ from JAMSTEC (Okutani et al. 2004a). TheBathymodiolus sp. from the Manus Basin is definitelydifferent from B. brevior in conchological traits and willbe described elsewhere. Specimens obtained from theOkinawa Trough and the Marian Back-Arc Basin weretentatively identified as B. septemdierum, but extensivemorphological examination is necessary for strict iden-tification. These mussels were frozen and preserved at�80�C or preserved in 100% ethanol.

1112

Table

1Listofsamples

Species

Sample

no.

Accession

no.COI(D

DBJ)

Accessionno.

ND4(D

DBJ)

Samplingsite

Depth

(m)

Location

Habitat

type

Dive

no.

Bathymodiolinae

Bathymodiolus

aduloides

AI1

AB170054

AB175323

IheyaRidge,

Mid-O

kinawaTrough

1,378

2732.835N;12658.400E

Vent

3K#375

AK1-5

AB170055-059

AB175324-326

OffKikaijim

aIsland

1,451

2826.44N;13019.08E

Vent

2K#1022

B.azoricus

AZL1

AB170060

AB175319

LuckyStrike,

Mid-A

tlanticRidge

Unknown

Unknown

Vent

Cruise47

AZL2

AB170061

bMaaset

al.(1999)

LuckyStrike,

Mid-A

tlanticRidge

Unknown

Unknown

Vent

Cruise47

B.japonicus

JH1,2

aAB101423

AB175281,282

OffHatsushim

a,SagamiBay

1,170

3500.00N;139.13.50E

Seep

2K#715

JH4

AB175285

OffHatsushim

a,SagamiBay

1,180

3559.99N;13913.68E

Seep

2K#792

JM1-3

aAB101422,423

AB175281,283,284

Minami-enseiKnoll,

Mid

OkinawaTrough

718

2823.50N;12738.50E

Vent

2K#618

B.hirtus

HK1-5

AB170047

AB175299-302

Kuroshim

aKnoll,

OffYaeyamaIslands

637

2407.813N;12411.541E

Seep

2K#1370

B.marisindicus

MK1-5

AB170042-045

AB175310-312

Kairei

Field,Southern

CentralIndianRidge

2,454

2519.22S;7002.39E

Vent

6K#659

B.platifrons

PH1-3

aAB101419-421

AB175286,287

OffHatsushim

a,SagamiBay

1,180

3559.99N;13913.68E

Seep

2K#792

PH4

aAB101421

AB175291

OffHatsushim

a,SagamiBay

1,170

3500.00N;139.13.50E

Seep

2K#715

PH5-7

AB175287,288,292

OffHatsushim

a,SagamiBay

1,170

3500.00N;139.13.50E

Seep

2K#715

PH8-10

AB175286,289

OffHatsushim

a,SagamiBay

1,170

3459.99N;13913.69E

Seep

2K#831

PI1,2

aAB101421

AB175286

NorthIheyaRidge,

Mid-O

kinawaTrough

1,028

2747.181N;12653.985E

Vent

2K#863

PI3,4

AB175286

NorthIheyaRidge,

Mid-O

kinawaTrough

1,028

2747.181N;12653.985E

Vent

2K#863

PT1,2

AB175286

HatomaKnoll,OkinawaTrough

1,523

2451.454N;12350.351E

Vent

2K#1270

PT3-10

AB175286,289,290,293

HatomaKnoll,OkinawaTrough

1,523

2451.465N;12350.3517

Vent

2K#1269

PY1,2

AB175286

Dai-yon(N

o.4)YonaguniKnoll,

southernOkinawaTrough

1,336

2450.935N;12242.013E

Vent

2K#1267

B.puteoserpentis

PUS1,2

AB170062

bMaaset

al.(1999)

SnakePit,Mid-A

tlanticRidge

Unknown

Unknown

Vent

Cruise47

B.securiform

isLK1-5

AB170048-051

AB175294-296

Kuroshim

aKnoll,

OffYaeyamaIslands

641

2487.813N;12411.558E

Seep

2K#1370

LA1-2

AB170052,053

AB175297,298

Dai-ni(N

o.2)AtsumiKnoll,

NankaiTrough

1,042

3351.636N;13722.706E

Seep

2K#1378

B.septemdierum

SM1

aAB101424

AB175303

MyojinKnoll,

Izu-O

gasawara

Island-arc

1,288

3206.27N;13952.18E

Vent

2K#1009

SM2

aAB101425

AB175313

MyojinKnoll,

Izu-O

gasawara

Island-arc

1,290

3206.271N;13952.123E

Vent

2K#1115

SM3-4

aAB101426,427

AB175314

MyojinKnoll,

Izu-O

gasawara

Island-arc

1,346

3206.301N;13952.043E

Vent

2K#1112

SM5-10

AB175303,308,315,318

MyojinKnoll,

Izu-O

gasawara

Island-arc

1,346

3206.301N;13952.043E

Vent

2K#1112

SS1

aAB101428

AB175304

SuiyoSeamount,

Izu-O

gasawara

Island-arc

1,375

2834.05N;14038.65E

Vent

2K#627

SS2,3

aAB101429,430

AB175303,317

SuiyoSeamount,

Izu-O

gasawara

Island-arc

1,373

2834.046N;14038.773E

Vent

2K#889

SS4

AB170041

AB175303

SuiyoSeamount,

Izu-O

gasawara

Island-arc

1,373

2834.046N;14038.773E

Vent

2K#889

SS5-7

AB175303,309,316

SuiyoSeamount,

Izu-O

gasawara

Island-arc

1,373

2834.046N;14038.773E

Vent

2K#889

1113

Table

1(C

ontd.)

Species

Sample

no.

Accession

no.COI(D

DBJ)

Accessionno.

ND4(D

DBJ)

Samplingsite

Depth

(m)

Location

Habitat

type

Dive

no.

SS8-11

AB175305,306,313,

SuiyoSeamount,

Izu-O

gasawara

Island-arc

1,382

2834.268N;14038.621E

Vent

2K#1388

AB242561

ST1

aAB101425

AB175304

HatomaKnoll,

OkinawaTrough

1,523

2451.465N;12350.357E

Vent

2K#1269

SA1

AB170046

AB175307

MarianaBack-A

rcBasin

3,600

1812.8N;14442.4E

Vent

6K#357

Bathymodiolussp.

BSP(M

anu)1

aAB101431

AB175320

PACKMANUSField

E,

ManusBasin

1,629

0343.728S;15140.183E

Vent

2K#1075

BSP(M

anu)2-5

aAB101432-434

AB175320-322

PACKMANUSField

E,

ManusBasin

1,627

0343.731S;15140.188E

Vent

2K#913

B.azoricus

Baz1(D

B)

bAF128534

Baz2(D

B)

bMaaset

al.(1999)

Baz3(D

B)

bMaaset

al.(1999)

B.brevior

B.brevior-Lau(D

B)

bAY046277

B.brooksi

B.brooksi(D

B)

bAY130247

B.childressi

B.childressi(D

B)

bAY130248

B.heckerae

B.heckerae(DB)

bAY130246

Bathymodiolus

sp.cf.heckerae

(DB)

bAY130245

B.marisindicus

B.marisindicus—

Edomond(D

B)

bAY046279

B.marisindicus—

Kairei

(DB)

bAY046278

B.puteoserpentis

Bpu1,2(D

B)

bAF128533

Bpu3(D

B)

bMaaset

al.(1999)

B.thermophilus

BTH-Bt1-3(D

B)

bAF456282-284

BTH-Bt5,6(D

B)

bAF456286,287

Bathymodiolussp.

BSP-Bt28-32(D

B)

bAF456309-313

Modiolinae

Adipicola

pacifica

A.pacifica

(outgroup)

AB170040

AB175280

OffNomaCape,

Kagoshim

a229

3120.72N;12959.29E

Whale

bone

HD#189

Each

specim

enwasnumbered

withaprefixincorporatinganabbreviationofitsscientificnameandsamplingsite

aCOIsequenceswerereported

inourpreviouspaper

(Miyazakiet

al.2004)

bCOIandND4sequencesare

obtained

from

DDBJ,

EMBL,andGenBankdatebasesandMaaset

al.(1999)

1114

Sequencing of the mitochondrial genes

Total DNA was prepared from the frozen foot muscletissue as described previously (Miyazaki et al. 2004). Asmall piece of foot muscle was homogenized in 400 ll ofthe T10E100 buffer (10 mM Tris–HCl, pH 7.2 and100 mM EDTA). The cellular membrane was dissolvedby addition of 20% SDS (20 ll) to the homogenate.DNA was purified, mixed with 1/10 volume of 5 MNaCland 2 volumes of 95% ethanol, left on ice for 10 min, and

centrifuged at 16,000g for 15 min at 4�C with a TMA-30rotor (TOMY SEIKO Co., Tokyo). After washing with70% ethanol, the pellet was dissolved in 500 ll of theT10E100 buffer and mixed well with 5 ll of RNase A(10 lg/ll), 106 ll of 5 M NaCl, and 151.5 ll of 5% ce-tyltrimethylammonium bromide (CTAB) in 0.7 MNaCl.After incubation for 30 min at 65�C, DNA was precipi-tated as described above. We also used a DNeasy TissueKit (QIAGEN GmbH, Hilden) to prepare total DNAefficiently from the small foot muscle of A. pacifica.

Fig. 1 The sampling sites for the deep-sea Bathymodiolus musselsand Adipicola pacifica used in this study. 1 Off Hatsushima (seep),Sagami Bay, 2Dai-ni (No. 2) Atsumi Knoll (seep), Nankai Trough,3 Off Noma Cape (whale bone), Kagoshima, 4 Minami-ensei Knoll(vent), Okinawa Trough, 5 Iheya Ridge (vent), Okinawa Trough, 6Off Kikaijima Island (seep), 7 Hatoma Knoll (vent), OkinawaTrough, 8 Dai-yon (No. 4) Yonaguni Knoll (vent), OkinawaTrough, 9 Kuroshima Knoll (seep), Off Yaeyama Islands, 10Myojin Knoll (vent), Izu-Ogasawara Island-arc, 11 Suiyo Se-

amount (vent), Izu-Ogasawara Island-arc, 12 Mariana Back-ArcBasin (vent), 13 PACMANUS Field E (vent), Manus Basin, 14Kairei Field (vent), Southern Central Indian Ridge, 15 LuckyStrike (vent), Mid-Atlantic Ridge, 16 Snake Pit (vent), Mid-Atlantic Ridge. The symbols (X) denote representative samplinglocalities of East Pacific B. thermophilus (upper) and Bathymodiolussp. (lower) for reference. open circle, hydrothermal vent; filled circle,cold-water seep; open triangle, whale bone. The region aroundJapan is magnified

1115

To amplify the approximately 710 bp partial frag-ment of mitochondrial cytochrome c oxidase subunit I(COI), PCR was performed in a reaction solution con-taining template DNA and KOD dash (TOYOBO Co.,Osaka) with 30 cycles of 30 s denaturation at 94�C, 5 sannealing at 42 or 49�C (for B. septemdierum usinguniversal primers) or 55�C (for the others), and 30 sextension at 74�C. We used universal primers (senseLCO1490 and antisense HCO2198 in Table 2) for theCOI gene of metazoan invertebrates (Folmer et al.1994), and also primers (sense SMANU and antisenseASMANU in Table 2) designed from the sequence ofBathymodiolus sp. from the Manus Basin and primers(sense COI-S4 and antisense COI-A4 in Table 2) de-signed from the sequence of B. septemdierum.

For amplification of the approximately 710 bp frag-ment including mitochondrial NADH dehydrogenasesubunit 4 (ND4), PCR was performed with 30 cycles of30 s denaturation at 94�C, 10 s annealing at 56.5�C, and40 s extension at 74�C. We used primers (sense ArgBLand antisense NAP2H in Table 2) designed for amplifi-cation of fish ND4 (Arevalo et al. 1994; Bielawski andGold 1996), and also primers (sense ND46S and anti-sense ND47A in Table 2) designed from the compositefrom sequences of B. platifrons, B. japonicus, andB. septemdierum.

Direct sequencing of the purified double-strand PCRproducts was performed using an ABI PRISM Big-DyeTM Terminator Cycle Sequencing Ready ReactionKit (Applied Biosystems Inc., California, USA) and theprimers used for PCR on a Model 377 DNA sequencer(Applied Biosystems Inc.) according to the manufac-turer’s directions.

Phylogenetic analyses

DNA sequences were edited and aligned with DNASIS(Hitachi Software Engineering Co., Ltd., Tokyo) andcorrected by visual inspection for the phylogeneticanalyses. We used 431 bp for COI and 423 bp for ND4,excluding ambiguous sites. Dendrograms were con-structed by the neighbor-joining (NJ) and maximumparsimony (MP) methods using PAUP*4.0 beta10(Swofford 2002). Genetic distances were computed byKimura’s two-parameter method (Kimura 1980). Thereliability of trees was evaluated by producing 1,000bootstrap replicates. We used A. pacifica as the outgroupspecies in this study instead of Modiolus modiolus whichwe used in our previous study (Miyazaki et al. 2004),because Distel et al. (2000) reported that Adipicola wasmore closely related to Bathymodiolus than Modiolus.Five COI sequences from each East Pacific speciesB. thermophilus (AF456282 to 456284, 456286, 456287)and Bathymodiolus sp. (AF456309 to 4563), three ND4sequences from each Atlantic species B. puteoserpentis(AF128533 and Maas et al. 1999) and B. azoricus(AF128534 and Maas et al. 1999), two ND4 sequencesfrom Bathymodiolus (cf.) heckerae (AY130245, 130246),

and one ND4 sequence from each of B. childressi(AY130248), B. brooksi (AY130247), and B. brevior(AY046277) were obtained from a database. We alsoused two ND4 sequences from Bathymodiolus musselsfrom the Edmond (AY046279) and Kairei (AY046278)vent fields, which were registered as B. brevior in adatabase. However, to the best of our knowledge, onlyone species, B. marisindicus, not B. brevior, has beenreported in the Southern Central Indian Ridge, and thuswe labeled the sequences as B. marisidincus in this study.

Results

Phylogenetic relationships of Bathymodiolus mussels

The partial DNA fragments of the mitochondrial COIand ND4 genes were sequenced from more than fivespecimens, if available, of each Bathymodiolus speciesand A. pacifica. Sequence data were deposited in DDBJ,EMBL, and GenBank databases under accession num-bers AB170040–170062 for COI and AB175280–175326and 242561 for ND4. A part of the COI sequence datawas previously reported (AB101419–101434, Miyazakiet al. 2004). No deletions or insertions were found in431 bp COI and 423 bp ND4 sequences and, 145 out of431 sites (19 at the first position and 4 at the secondposition in the codon) in COI and 213 out of 423 sites(66 at the first and 26 at the second) in ND4 were var-iable. Amino acid substitutions were found at 10 of 143residues with two fixed replacements (not intraspecificvariations but interspecific differences) in COI; Leu intwo Mid-Atlantic species against Met in the others at thefifth position, and Ser in two Mid-Atlantic species,B. hirtus, B. septemdierum, and B. marisindicus againstAla in the others at the 92nd position. Amino acidsubstitutions in ND4 were found at 63 of 141 residueswith 51 fixed replacements. The genetic distances ob-tained by Kimura’s two-parameter method (Table 3)were 0.005–0.209 (av. 0.152) in COI and 0.002–0.349(av. 0.253) in ND4 among Bathymodiolus species and0.179–0.228 (av. 0.197) in COI and 0.318–0.385 (av.0.356) in ND4 between Bathymodiolus species(Bathymodiolinae) and A. pacifica (Modiolinae).

Dendrograms were constructed by the NJ method forCOI (Fig. 2) and ND4 (Fig. 3). The COI tree includedEast Pacific species B. thermophilus and Bathymodiolussp. from a database. The ND4 tree included three speciesB. childressi, B. heckerae, and B. brooksi from the Gulfof Mexico and also West Pacific species B. brevior from adatabase. Except for the species from a database, boththe COI and ND4 trees gave fundamentally the sametopology with some minor changes as mentioned below.Bathymodiolus species were clustered into three groups.The monophyly of each cluster was supported with rel-atively high bootstrap probabilities (95 and 97% in NJand 83 and 97% in MP for COI, 98–99% in NJ and 91–99% in MP for ND4), but the first cluster of the COItree was supported marginally (60% in NJ and 78% in

1116

MP). The first cluster (Group 1) consisted of B. japoni-cus, B. platifrons, B. securiformis, and B. hirtus, thesecond (Group 2-1) of B. septemdierum, B. marisindicus,B. puteoserpentis, and B. azoricus, and the third (Group2-2) of B. aduloides and Manus Bathymodiolus sp. EastPacific B. thermophilus and Bathymodiolus sp. were in-cluded in Group 2-1 of the COI tree. Bathymodioluschildressi was included in Group 1, and B. heckerae,B. brooksi and B. brevior in Group 2-1 of the ND4 tree.Group 2-1 was more closely related to Group 2-2 than toGroup 1 in both the COI and ND4 trees. However, therelatedness was not supported with high bootstrap val-ues (<50% in NJ for COI, 78% in NJ and 55% in MPfor ND4) and the majority-rule consensus tree of COIdepicted by the MP method based on 1,000 bootstrapreplicates showed that Group 2-2 was allied with Group1 rather than with Group 2-1 (data not shown), showingtrichotomous nature in divergence of the three clusters.Species in Group 1 are distributed mainly in Japanesewaters and only one species in the Gulf of Mexico.Species in Group 2-1 are distributed in a wide rangeincluding the West and East Pacific, Indian, and AtlanticOceans. Species in Group 2-2 are restricted to the WestPacific.

The topology of Group 1 was not relatively solid. Inthe COI tree, B. securiformis was more closely related toB. platifrons than to B. japonicus. The three species wereclustered with high bootstrap probabilities (99% in NJand 93% in MP) and subsequently allied with B. hirtus.On the other hand, the ND4 tree depicted by the NJmethod showed that B. securiformis was more closelyrelated to the cluster including B. japonicus, B. platifrons,and B. childressi, where B. platifrons and B. childressiwas the most closely related.

Bathymodiolus mussels of Group 2-1 were dividedinto three subclusters in the COI tree. The subclusterswere comprised of East Pacific species (B. thermophilusand Bathymodiolus sp.), Mid-Atlantic species (B. puteo-serpentis and B. azoricus), and Indo-West Pacific species(B. septemdierum and B. marisindicus), respectively. TheND4 tree included two species (B. heckerae andB. brooksi) living in the Gulf of Mexico and West PacificB. brevior. Bathymodiolus brooksi differentiated basallyand B. heckerae was allied with the Mid-Atlantic species.Indo-West Pacific species formed a complex subcluster(Cluster A). Neither B. septemdierum nor B. marisindicusin Cluster A formed a well supported clade, although themonophyly of each Bathymodiolus species except thetwo species was supported with high bootstrap values.Interspecific genetic distances in Cluster A ranged from0.005 to 0.040 (av. 0.019) in COI and 0.002 to 0.019 (av.0.009) in ND4 (Table 3). Those excluding the values inCluster A ranged from 0.045 to 0.209 (av. 0.156) in COIand 0.070 to 0.349 (av. 0.265) in ND4. Intraspecific ge-netic distances in Bathymodiolus species were 0.000–0.041 (av. 0.009) in COI and 0.000–0.017 (av. 0.004) inND4. Therefore, interspecific genetic distances in Clus-ter A were significantly smaller (P<0.01 by Student’st test) than those of Bathymodiolus species other than

Cluster A species, and overlapped greatly with intra-specific genetic distances.

Group 2-2 consisted of two West Pacific species. Oneof the species, B. aduloides can live both in vents andseeps as well as B. japonicus and B. platifrons in Group 1.Habitats of the versatile species are restricted to Japa-nese waters.

Discussion

Dispersal and speciation of Bathymodiolus mussels

Our previous phylogenetic study using the COI gene(Miyazaki et al. 2004) suggested intraspecific geneticexchanges between populations from hydrothermalvents of the Okinawa Trough and cold-water seeps ofthe Sagami Bay (over 1,500 km apart) in two species ofGroup 1, B. japonicus and B. platifrons. The presentstudy using another mitochondrial gene and an in-creased number of specimens also showed that the samehaplotypes were held in common by specimens obtainedfrom distant localities (Okinawa Trough vs. SagamiBay) and highly differentiated environments (vent vs.seep), and that their genetic distances (B. japonicus,0.001 in COI and 0.006 in ND4; B. platifrons, 0.005 inCOI and 0.003 in ND4) were approximate with theaverage intraspecific values (0.009 in COI and 0.004 inND4). Our preliminary estimation based on ND4 se-quences gave values of FST 0.054 and Nm 8.81 betweenspecimens (N=16) from the Okinawa Trough and those(N=10) from the Sagami Bay in B. platifrons, suggestinga significant gene flow between them. These resultssupport the capability for relatively high larval dispersaland show that environmental types (vent vs. seep) arenot the primary factor responsible for habitat segrega-tion and speciation in these Bathymodiolus species.

It seems surprising that B. childressi from the Gulf ofMexico was the most closely related to B. platifrons fromJapanese waters in Group 1 of the ND4 tree. There is anextremely long distance and many geological obstaclesbetween the Gulf of Mexico and Japanese waters sug-gesting high dispersal ability of species in Group 1.

Our results showed, moreover, that interspecific ge-netic distances in Cluster A were significantly smaller

Table 2 Primers used for amplification of COI and ND4

COISense LCO1490 5¢-ggtcaacaaatcataaagatattgg-3¢Antisense HCO2198 5¢-taaacttcagggtgaccaaaaaatca-3¢Sense SMANU 5¢-ggtttgtgatcgggaataattgggac-3¢Antisense ASMANU 5¢-ctattcgctcccctcgcatactttc-3¢Sense COI-S4 5¢-ggtttgtggtctggaataattgggac-3¢Antisense COI-A4 5¢-actattcgttcaccccgcattctttc-3¢ND4Sense ArgBL 5¢-caagacccttgatttcggctca-3¢Antisense NAP2H 5¢-tggagcttctacgtgrgcttt-3¢Sense ND46S 5¢-gctcatgccccgaatatgtct-3¢Antisense ND47A 5¢-caacctaaacaaattatctctccc-3¢

1117

Table

3Averagepairwisegenetic

distancescalculatedbyKim

ura’stw

o-parameter

methodbetweenandwithin

BathymodiolusspeciesbasedonCOI(abovediagonal)andND4(below

diagonal)

sequences

Bathy-

modiolus

japonicus

B.plati-

frons

B.securi-

form

isB.hir-

tus

B.child-

ressi

B.septem-

dierum

B.mari-

sindicus

B.bre-

vior

B.puteoser-

pentis

B.azo-

ricus

B.thermo-

philus

East

Pacific

Bathy-

modiolussp.

B.hec-

kerae

B.broo-

ksi

B.adu-

loides

Manu

Bathy-

modiolussp.

Bathymodiolusjaponicus

0.001

0.006

0.089

0.097

0.131

–0.195

0.188

–0.191

0.177

0.174

0.180

––

0.179

0.186

B.platifrons

0.132

0.005

0.003

0.068

0.127

–0.189

0.186

–0.182

0.171

0.187

0.191

––

0.177

0.178

B.securiform

is0.139

0.142

0.008

0.003

0.132

–0.177

0.174

–0.169

0.175

0.164

0.171

––

0.167

0.172

B.hirtus

0.166

0.179

0.173

0.000

0.004

–0.157

0.152

–0.165

0.169

0.159

0.160

––

0.152

0.178

B.childressi

0.126

0.078

0.140

0.191

– 0.000

––

––

––

––

––

–

B.septemdierum

0.336

0.306

0.309

0.315

0.296

0.019

0.006

0.019

–0.125

0.125

0.123

0.117

––

0.181

0.166

B.marisindicus

0.330

0.295

0.300

0.306

0.288

0.009

0.007

0.006

–0.122

0.118

0.119

0.108

––

0.185

0.173

B.brevior

0.335

0.304

0.308

0.314

0.295

0.008

0.007

– 0.000

––

––

––

––

B.puteoserpentis

0.301

0.280

0.255

0.303

0.289

0.211

0.210

0.210

0.000

0.001

0.072

0.139

0.149

––

0.190

0.193

B.azoricus

0.323

0.295

0.279

0.300

0.285

0.168

0.172

0.174

0.130

0.002

0.002

0.127

0.133

––

0.198

0.170

B.thermophilus

––

––

––

––

––

0.004

–0.050

––

0.157

0.157

East

PacificBathymodiolussp.–

––

––

––

––

––

0.005

––

–0.164

0.171

B.heckerae

0.309

0.307

0.289

0.293

0.292

0.202

0.198

0.201

0.138

0.108

––

– 0.012

––

–

B.brooksi

0.303

0.257

0.267

0.303

0.260

0.205

0.200

0.209

0.231

0.209

––

0.224

– 0.000

––

B.aduloides

0.320

0.298

0.290

0.312

0.297

0.313

0.310

0.310

0.284

0.286

––

0.315

0.308

0.005

0.005

0.111

ManusBathymodiolussp.

0.325

0.318

0.317

0.338

0.333

0.266

0.272

0.271

0.273

0.283

––

0.307

0.317

0.144

0.004

0.003

1118

Fig. 2 Neighbor-joining (NJ) tree showing phylogenetic relation-ships of deep-sea Bathymodiolus mussels based on the 431 bp COIsequences. The dendrogram was constructed based on geneticdistances calculated by Kimura’s two-parameter method usingAdipicola pacifica as the outgroup species. The maximum-parsi-mony tree presented essentially the same topology as the NJ treeexcept that Group 2-2 was allied with Group 1 rather than withGroup 2-1 in the MP tree. Bootstrap probabilities of 1,000

replicates are presented for the NJ (above branches) and MP (belowbranches) trees, when they exceeded 50%. Scale bar indicates 0.01substitutions per site. See Table 1 for abbreviations of Bathymod-iolus mussels. Sequence data of East Pacific species B. thermophilusand Bathymodiolus. sp. were derived from a database (DB).Diamond Atlantic species, star West Pacific species, circle Indianspecies, triangle East Pacific species

1119

than those among Bathymodiolus species other thanCluster A species and highly overlapped with intraspe-cific genetic distances. Our preliminary estimation basedon ND4 sequences gave values of FST 0.288 and Nm 1.23between specimens (N=11) of B. septemdierum from theSuiyo Seamount and those (N=7) of B. marisindicus

from the Kairei and Edmond Fields, showing significantgene flow between them. Our results suggested thatmembers in Cluster A were conspecific or diverged veryrecently. Further morphological and genetic studies areneeded to assess the synonymy of species in Cluster A.Since the Suiyo Seamount (Izu-Ogasawara Island-arc) is

Fig. 3 Neighbor-joining (NJ) tree showing phylogenetic relation-ships of deep-sea Bathymodiolus mussels based on the 423 bp ND4sequences. The dendrogram was constructed based on geneticdistances calculated by Kimura’s two-parameter method usingAdipicola pacifica as the outgroup species. The maximum-parsi-mony tree presented essentially the same topology as the NJ tree.Bootstrap probabilities of 1,000 replicates are presented for the NJ

(above branches) and MP (below branches) trees, when theyexceeded 50%. Scale bar indicates 0.01 substitutions per site. SeeTable 1 for abbreviations of Bathymodiolus mussels. Sequence dataof B. childressi, B. heckerae, and B. brooksi in the Gulf of Mexicoand West Pacific B. brevior were derived from a database (DB).Diamond Atlantic species, star West Pacific species, circle Indianspecies, triangle East Pacific species

1120

about 10,000 km from the Kairei Field (Southern Cen-tral Indian Ridge), our results also suggested a strikingdispersal ability for larvae, which may allow species inGroup 2-1 to be distributed widely over the West andEast Pacific, Indian, and Atlantic Oceans (Van Doveret al. 2001). This indicates that speciation of Bathy-modiolus cannot be ascribed to poor larval dispersalability. The high dispersal capability of Bathymodiolusmussels may result from their planktotrophic (activelyfeeding planktonic larval) development as deduced fromlarval shell morphologies (Lutz et al. 1986). Develop-mental arrest at cold temperature also seems to play animportant role in extending the planktonic stage andincreasing dispersal distances (Lutz et al. 1980; Pradillonet al. 2001). However, it is unlikely that Bathymodiolusmussels traversed the Pacific Ocean because no closelyrelated species are discernible between the East and WestPacific, although multiple trans-Pacific migrations havebeen reported for vesicomyid bivalves (Kojima et al.2004).

Coevolution of mussels and endosymbionts

Bathymodiolus mussels were clustered into three groups.The grouping was related to types of their endos-ymbionts. Species within Group 1 contain methano-trophs (Fujiwara et al. 2000; Childress et al. 1986; ourunpublished data). Species within Group 2 (no data forEast Pacific Bathymodiolus sp.) contain mainly thio-autotrophs (Cavanaugh et al. 1987; Distel et al. 1988,1995; Dubilier et al. 1998; Fiala-Medioni et al. 2002;Fisher et al. 1993; Fujiwara et al. 2000; Yamanaka et al.2003; our unpublished data). Four species in Group 2-1,B. septemdierum, B. brevior, B. marisindicus, andB. thermophilus, contain solely thioautotrophs, and theother species harbor both of thioautotrophs and met-hanotrophs, although the proportion of methanotrophsto thioautotrophs is considerably low (Distel et al.1995). Species in Group 2-2 solely contain thioauto-trophs (Yamanaka et al. 2000; our unpublished data).The correlation of the mussel groups with their endo-symbiotic types indicates coevolution of hosts andsymbionts, although the way of transmission of endo-symbiotic bacteria is still controversial. Cary and Gio-vannoni (1993) suggested vertical transmission frommothers to offspring through eggs, but Won et al. (2003)claimed that bacteria were acquired from externalenvironment. Comprehensive phylogenetic studies ofendosymbionts are needed to answer the question.

Phylogeny of Bathymodiolus mussels

Cosel (2002) tentatively classified Bathymodiolus musselsinto four groups by morphological traits; (1) theB. thermophilus group including East Pacific Bathy-modiolus sp., (2) the B. brevior group including B. sep-temdierum, B. elongates, B. marisindicus, and

B. puteoserpentis, (3) the B. heckerae group includingB. boomerang, (4) the B. childressi group includingB. platifrons, B. mauritanicus, and undescribed speciesfrom the Barbados Accretionary Prism. The other spe-cies were not unequivocally assigned to any group. Thegrouping does not conflict with the phylogenetic rela-tionships presented in this study, and suggest possibleadditional members of Group 1, such as B. mauritanicusfrom the East Atlantic and Barbados Bathymodiolus sp.from the West Atlantic, thus indicating that members ofGroup 1 are likely distributed worldwide. It is evidentthat further surveys of novel vent and seep sites andgenetic examination of deep-sea mussels to be found areneeded to find cryptic members and to elucidate theevolutionary process of Bathymodiolus mussels.

Acknowledgements We wish to express our thanks to Drs. ShigeakiKojima, Takashi Okutani, Chitoshi Mizota, and Motoo Utsumifor their useful advice and support throughout this work. We arealso grateful to Drs. Nobuhiro Minaka and Hirohisa Kishino forhelpful advice on phylogenetic analyses. Thanks are also extendedto the operation teams of the submersibles ‘‘Shinkai 2000’’,‘‘Shinkai 6500’’, ‘‘Dolphin 3K’’, ‘‘Hyper Dolphin’’ and ‘‘Kaiko’’and the officers and crew of the support vessels ‘‘Natsushima’’,‘‘Yokosuka’’ and ‘‘ Kairei’’ for their help in collecting samples.This study is supported in part by a grant from the ResearchInstitute of Marine Invertebrates and grants from the Ministry ofEducation, Culture, Sports, Science and Technology of Japan (nos.12NP0201 and 80229830).

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