Phylogenetic Relationships of Deep-Sea Bathymodiolus Mussels to Their Mytilid Relatives from Sunken Whale Carcasses and Wood

VENUS 67 (3-4): 123-134, 2009

Review

Phylogenetic Relationships of Deep-Sea Bathymodiolus Mussels to their Mytilid Relatives from Sunken Whale Carcasses and Wood

Yuko Fujita1, Hiroto Matsumoto1, Yoshihiro Fujiwara2, Jun Hashimoto3, Sergey V. Galkin4, Rei Ueshima5 and Jun-Ichi Miyazaki6*

1Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan 2Research Program for Marine Biology and Ecology, Japan Agency for Marine-Earth Science and Technology (JAMSTEC), Natsushima, Yokosuka, Kanagawa 237-0061, Japan 3Faculty of Fisheries, Nagasaki University, 1-14 Bunkyo, Nagasaki, Nagasaki 852-8521, Japan 4P. P. Shirshov Institute of Oceanology, Russian Academy of Sciences, Nakhimovsky Pr., 36, Moscow 117218, Russia 5Graduate School of Science, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan 6Faculty of Education and Human Sciences, University of Yamanashi, Kofu, Yamanashi 400-8510, Japan: *[email protected]

Abstract: We have investigated worldwide phylogenetic relationships of deep-sea Bathymodiolus mussels and their mytilid relatives by determining mitochondrial DNA sequences. We review herein their phylogenetic relationships and the evolutionary process deduced from s tudies of described and unidentified species collected recently from vents, seeps, sunken whale carcasses and wood. Phylogenetic analysis of the sequence data together with those from a database showed that the mytilid mussels were divided into six clusters and that the mussels in the subfamily Bathymodiolinae are split into four groups. Neither the subfamily Bathymodiolinae nor the genus Bathymodiolus were monophyletic, suggesting that it is necessary to reevaluate their classification. In the evolutionary process of the conventional Bathymodiolinae, the group including only Tamu fisheri split first, and the basal trichotomous split into the remaining three groups was followed by diversification of bathymodioline mussels in each group. The first group bifurcated into two subgroups, which include Bathymodiolus and Gigantidas species, respectively. The second group was subdivided into three subclusters containing Indo-West Pacific, Atlantic and eastern Pacific species respectively. The third group included two nominal species restricted to the western Pacific. Species obtained from sunken whale carcasses and wood took the outgroup position to the vent/seep mussels with only one exception, Adipicola crypta from whale carcasses. Modiolus modiolus from shallow water was positioned more distantly to the vent/seep mussels. The findings indicate an evolutionary transition from shallow water to vent/seep sites via whale carcass/wood sites, supporting the “Evolutionary stepping stone hypothesis”.

Keywords: chemosynthesis-based community, vent, seep, mitochondrial DNA, stepping stone 124 Y. Fujita et al.

Introduction

Deep-sea mussels of the genus Bathymodiolus (Mytilidae, Bathymodiolinae) are one of the dominant megabenthos elements in chemosynthesis-based communities around hydrothermal vents on spreading ridges and back-arc basins and in cold-water seeps along subduction zones. They rely primarily on chemoautotrophic bacterial endosymbionts for their nutrition, although they have not abandoned fi lter feeding (Fiala-Médioni et al., 1986; Fisher et al., 1988). Genetic differentiation and consequent speciation of deep-sea organisms in the community are caused by a combination both of factors shared by diverse taxa (topography, geological histories, and oceanic currents) and some that are unique to their respective taxa such as dispersal ability, physiology, and settlement cues (Vrijenhoeck, 1997; Won et al., 2003). Our previous studies (Miyazaki et al., 2004; Iwasaki et al., 2006; Miyazaki et al., 2008; Miyazaki, 2008) showed intraspecific genetic exchanges between populations from the hydrothermal vents of the Okinawa Trough and the cold-water seeps of Sagami Bay (over 1,500 km apart) in two species, B. japonicus and B. platifrons, although these two species do not inhabit the Izu-Ogasa wara Island-arc (ca. 500 km from Sagami Bay). This suggests that colonization and speciation of the two species are not dependent on geographical distances and that larval dispersal ability is relatively high and favorable for colonization of patchy and ephemeral habitats. Hydrothermal vents, emitting water heated by underlying magmatic chambers, persist for only a few decades, supplying inorganic nutrients such as sulfide and methane to chemosynthetic bacteria, while cold-water seeps, exuding water as cold as the ambient deep-sea water, provide a relatively stable source of these materials (Jollivet, 1996). Nevertheless, environmental types (vent vs. seep) are not responsible for habitat segregation and speciation in the two Bathymodiolus species (Iwasaki et al., 2006). Nineteen species of Bathymodiolus mussels have been described since the genus was first proposed (Kenk ＆ Wilson, 1985). Three bathymodioline species belonging to the genera Tamu and Gigantidas have been described (Gustafson et al., 1998; Cosel ＆ Marshall, 2003; Hashimoto ＆ Yamane, 2005). By sequencing of mitochondrial genes, 16 species of Bathymodiolus mussels were clustered into three groups as follows: the first includes four species from Japanese waters and one Atlantic species, the second includes one species from Japanese waters, one western Pacific species, one Indian species, two eastern Pacific species and four Atlantic species, and the third includes one species from Japanese waters and one western Pacific species (Iwasaki et al., 2006). Thereafter, active exploration of new localities and careful surveys of known localities has led to the discovery of many unidentified mussels. Molecular phylogenetics, by analyzing DNA sequences of the unidentified mussels as well as accumulating database sequences, offers a good opportunity to uncover the evolutionary process of Bathymodiolus mussels and their relatives.

Phylogenetic relationships of Bathymodiolus mussels and their relatives

Specimens used are listed in Table 1 and the collection sites are mapped in Fig. 1. The partial DNA fragments of the mitochondrial COI gene were sequenced from more than five specimens, if available, of each mussel species. Sequence data was deposited in DDBJ, EMBL, and GenBank databases under accession numbers AB255739-AB255743, AB257513-AB257557. No deletions or insertions were found in the COI sequences. Bathymodiolus mussels and their relatives were divided into six major clusters (Fig. 2). Those clusters were marginally or poorly supported. The first cluster (C1) was marginally supported and consisted of the modioline species Benthomodiolus lignicola and Benthomodiolus geikotsucola (Okutani & Miyazaki, 2007) and one unidentified mussels from the Juan de Fuca hydrothermal vents in the eastern Pacific (JdF B. sp.). The latter was previously cited as to Relationships of Deep-Sea Mussels to their Mytilid Relatives 125 c 2K#715 2K#863 2K#792 6K#779 2K#715 2K#618 6K#659 6K#846 6K#846 6K#846 6K#846 6K#357 6K#846 3K#375 2K#627 2K#889 2K#889 HD#492 ventvent cruise 47 cruise 47 vent E vent E seep E vent EE vent vent 2K#1009 2K#1115 W vent E seep 2K#1378 WWWW vent vent vent vent E seep EW seep vent EE vent vent 2K#1269 W vent cruise 49 E seep E vent 2K#1112 E seep 2K#1370 W vent EE vent vent 2K#1022 EE vent seep 2K#1009 EW seep vent EE vent vent WE vent seep 2K#1370 W vent E vent 49´W ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ W vent cruise 47 E vent E vent W vent W vent W seep dive#4568 W seep - W W ´ ´ ´ ´ ´ ´ ´ W seep ´ ´ ´ S; 70º02 S; 112º03 S; 69º36 S; 176º43 S; 176º43 S; 176º43 S; 176º43 S; 111º56 S; 176º36 S; 178º58 S; 176º38 N; 32º17 N; 32º17 N; 126º54 N; 44º56 N; 139º14 N; 127º39 N; 139º52 N; 139º52 N; 137º23 N; 139º14 N; 146º00 N; 123º50 N; 144º42 N; 104º18 N; 139º14 N; 5º28 N; 139º52 N; 44º56 N; 124º12 N; 126º58 N; 130º19 N; 130º19 N; 144º02 N; 86º14 N; 103º47 N; 140º39 N; 140º39 N; 124º12 N; 103º57 N; 140º39 N; 91º19 N; 84º55 ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ 18 18 - - 7º25´S; 107º48 2º11 28º24 35º00 24º08 27º44 37º17 37º17 3510 23º22 2333 31º09 1725 1267 0º53 3510 23º22 1725 2747 2229 12º49 1205 40º26 ------460 0º48 - 1028 27º47 1170 35º00 2331 31º52 3289 23º53 1042 33º52 181818181818 22º32 1908 22º32 23º32 2 1818 23º32 1180 35º60 2899 13º47 15233600 24º51 18º13 2524 9º51 13781451 27º33 28º26 1430 28º26 1625 21º29 25152 11º25 1750 23º13 3314 26º02 Depth(m) Location Habitat type Dive no. 3023 2332 1622 1000 3023 1622 2746 2228 idge Sampling site eamount ff ff Hatsushima Island, Sagami Bay 535 Northwest Eifuku S - 423045 Minami-ensei Knoll, Mid Okinawa Trough Kairei Field, Southern Central Indian Ridge 2454 25º19 053 Atsumi Knoll, Nankai Trough 541 Lau Basin 421 Off Hatsushima Island, Sagami Bay 531 South Chamorro Seamount, Mariana ucky Strike, Mid-Atlantic Ridge 4 051 Islands Kuroshima Yaeyama Knoll, Off 641 24º88 059 Off Kikaijima Island 430 Suiyo Seamount, Izu-Ogasawara Island-arc 1373 28º34 533 Gulf of Mexico 310 32SEast Pacific Rise 313 31S East Pacific Rise 287 7S East Pacific Rise ------27 Myojin Knoll, Izu-Ogasawara Island-arc 1346 32º06 AF456282 11N East Pacific Rise AF456283 13N East Pacific Rise AF456284 Rose Garden, Galapagos Rift AY649796 Snake Pit, Mid-Atlantic R AY275543 Edmond, Mid-Indian Ridge AY649801Africa West AY275544 AY275544 Lau Basin AY275544 AY275544 Lau Basin AY608439 Off Turnagain Cape, New Zealand 920 AY649798 Florida West Escarpment AB101421 Iheya Ridge, Mid-Okinawa Trough AB170062 Snake Pit, Mid-Atlantic Ridge AB101421 Off Hatsushima Island, Sagami Bay AB101425 Myojin Knoll, Izu-Ogasawara Island-arc 1290 32º06 AB101424 Myojin Knoll, Izu-Ogasawara Island-arc 1288 32º06 AB170054 Iheya Ridge, Mid-Okinawa Trough AB257542AB101425 Lau Basin Lau Basin AB170061 L AB101423 O AB101425AB170046 Hatoma Knoll, Okinawa Trough Mariana Back-Arc Basin AB257557 9N East Pacific Rise AB101428 Suiyo Seamount, Izu-Ogasawara Island-arc 1375 28º34 AB170060 Lucky Strike, Mid-Atlantic Ridge AB257556 Off Kikaijima Island AB170041 Suiyo Seamount, Izu-Ogasawara Island-arc 1373 28º34 AB170047 Islands Kuroshima Yaeyama Knoll, Off Accession no. AF456309 AF456311 AF456286 AB101422 AB170042 AB101426 AB170052 AB257540 AB101419 AB257530 AB170048 AB170055 AB101429 AB257532 AB257539,541,543 Lau Basin AB101425,AB257534 a b b Sample no. A1 PI1,2 PUS1,2 B. puteoserpentis PH4 Bt28,29 JM1-3 MK1-5 B. marisindicus SM2 SM3-4 SM1 Bt30-32 S B. mauritanicus LA1,2 AI1 Lau3,4 Lau2 Lau5 Lau7 Lau1,8,9 PH1-3 AZL2 B. brevior JH1,2 C1-3 EF1-5 B. tangaroa ThE1 Bt1 SS1 LK1-5 AZL1 AK1-5 Kikaijima Bt5,6 Bt2 SS2,3 SS4 B. brooksi HK1-5 Bt3 ST1 ChiG1,2 sp. sp. Species B. B. sp . B. sp.2 sp.1 Lau B. Lau B. B. platifrons Chamorro Eifuku B. thermophilus Kikaijima B. childressi * * B. puteoserpentis # * #East Pacific B. sp. * * B. marisindicus # * * * B. septemdierum # * # B. mauritanicus * Table 1. Table List of samples. Bathymodiolinae * Bathymodiolus aduloides * # B. brevior * B. japonicus # B. tangaroa # * * * B. securiformis * B. azoricus * # # * * # B. brooksi * B. hirtus # * 126 Y. Fujita et al. c HD#84 3K#400 2K#913 2K#558 HD#496 vent wood wood wood whale carcass, E vent E vent E seep 2K#1158 E whale carcass HD#328 E seep 2K#1158 E whale carcass HD#191 E vent E seep 02DMASFPG01 E seep 2K#1158 E whale carcass HD#189 EE vent vent 2K#1017 E seep 2K#1160 EE vent vent 2K#1075 W vent E whale carcass HD#328 E whale carcass HD#192 EE vent seep W vent W E whale carcass HD#191 E whale carcass 6K#895 ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ W seep ´ ´ S; 178º30 S; 178º12 S; 142º14 S; 142º14 S; 142º14 S; 142º16 S; 151º40 S; 151º40 S; 142º14 S; 178º27 S; 177º14 N; 129º59 N; 129º59 N; 142º19 N; 133º37 N; 129º59 N; 140º04 N; 140º04 N;141º49 N; 126º06 N; 129º59 N; 129º59 N; 141º05 N; 92º10 N; 129º59 ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ ´ unknown 460 35º45 755 36º08 425 unknown 650 27º50 1174 34º41 - - - - - 225 31º21 228 31º21 485 23º05 490 575 32º29 229 31º21 686 31º28 676 31º28 225 31º21 229 31º21 470 02º55 486 26º43 200 30º13 228 31º21 1881 02º49 1881 02º49 1881 02º49 1646 02º53 16291627 03º44 03º44 40202200 30º55 47º56 300 485 400 546 unknown unknown unknown unknown tidal zoon Depth(m) Location Habitat type Dive no. 826 Sampling site ape, Kagoshima et al ., 2006). 555 Sumisu Caldera - 528 Off Noma Cape, Kagoshima 546,555 Nikko Seamount 523 Off Nakaminato, Ibaraki 550 Sissano, Papua New Guinea 434743 Manus Basin Off New Zealand 514537 Seamount Torishima Off Noma Cape, Kagoshima 518 Off Noma Cape, Kagoshima 525 Aitape, Papua New Guinea 516 Off Noma Cape, Kagoshima 427 Rumble III 429 Rumble V ------608434 Macauley Cone 56848 et al ., 2004; Iwasaki B257553 Sumisu Caldera AY AY275545 AY275545 Chatham Rise AY649803 Garden Banks AB257551 Sissano, Papua New Guinea AB257526 Off Noma Cape, Kagoshima AB257529Ashizuri Off Cape AB257552 Sissano, Papua New Guinea AB257519 Off Noma C AB101431 Manus Basin A AB257538 Kaikata Seamount DQ077892 Juan de Fuca Ridge Accession no. AY608426 AY608428 AB257520 AB257548 AB101432 AB255739 AB257513 AB257536 AB257517 AB257524 AB257515 AB257538,554 AB170040,AB257528 Off Noma Cape, Kagoshima Shinkai 6000 ; HD, Hyper Dolphin. AB257538,544 a K ; 6K, Dolphin 3 Sample no. i3-7 RIIIla,IIIsa RVa,Vb Mclong Si3-3 APN328-25,26 AB257527 Si1-1, Si3-1,2,4,6 AB257547 Sissano, Papua New Guinea APN3 Su1 NK1-5 APN1,2 AIH1-5 ACN328-1 Ashizuri B. sp. JdF ACN3,4 Su2-5 Tamu fisheri Tamu S Si2-1-4 Manus1 Manus2-5 Ne1-5 Modiolus modiolus U 895-1-895-5 IJN1,2 Benthomodiolus lignicola ACN1,2 Aitape1,2 Kaikata Shinkai 2000 ; 3K, ignicola sp. Species sp. sp. 2 sp. 1 sp. sp. B. sp. 3 G. sp. sp. B. septemdierum , but detailed morphological examination is necessary for strict identification. Sissano Sissano B. Sumisu Nikko G. Adipicola iwaotakii Ashizuri G. Benthomodiolus l Sissano B. NZ B. Benthomodiolus geikotsucola Idasola japonica Adipicola crypta Aitape G. Gigantidas horikoshii B . Specimens obtained from the Okinawa Trough and the Mariana Back-Arc Basin were tentatively identified as Each specimen was numbered with a prefix incorporating an abbreviation of its scientific name and sampling site. Abbreviations: 2K, # Gigantidas gladius # # # * Adipicola pacifica #JdF * COI sequences were reported in our previous papers (Miyazaki # COI sequences reported herein can be obtained from DDBJ, EMBL and GenBank datebases. Tamu fisheri # Tamu Table 1 Table . (continued) List of samples. * B. manusensis * # Modiolus modiolus a b c Modiolinae Relationships of Deep-Sea Mussels to their Mytilid Relatives 127 belonging to the genus Bathymodiolus from its genetic similarity to known Bathymodiolus species (McKiness et al., 2005), but is here found to be more closely related to Benthomodiolus than to Bathymodiolus. Its phylogenetic position remains to be studied. The second cluster (C2) included only the bathymodioline seep species Tamu fisheri. The third cluster (C3) contained only Adipicola iwaotakii in the subfamily Modiolinae from sunken wood samples. The fourth cluster (C4) consisted of the modioline species A. pacifica and Idasola japonica from sunken whale carcasses and wood respectively. The fifth cluster (C5) was marginally supported and comprised Bathymodiolus and unidentified mussels. The sixth cluster (C6) included the bathymodioline species Gigantidas horikoshii, G. gladius, and Bathymodiolus mussels, the modioline A. crypta and unidentified mussels. The monophyly of the clade including all the bathymodioline members (i.e. Bathymodiolus, Gigantidas, and Tamu mussels) was marginally supported, but four modioline species were included in this clade, suggesting that the subfamily Bathymodiolinae is not monophyletic. Within the Bathymodiolinae, T. fisheri was more distantly related to the other members. Bathymodioline mussels other than T. fisheri were divided into three groups (Fig. 2). Group 1 (a part of the sixth cluster) was subdivided into two clades. Group 1-1 (G1-1) was marginally supported and consisted of seven nominal species (B. hirtus, B. japonicus, B. platifrons, and B. securiformis from Japanese waters, B. tangaroa from the Western Pacific, and B. mauritanicus and B. childressi from the Atlantic) and five unidentified mussels from Sissano (Sissano B. sp.1, B. sp.2, and B. sp.3), the Chamorro Seamount (Chamorro B. sp.), and off Kikaijima Island (Kikaijima B. sp.) in the western Pacific. Group 1-2 (G1-2) was relatively well supported and included the two nominal species G. horikoshii and G. gla dius and four unidentified mussels from the Nikko Seamount (Nikko G. sp.), the Sumisu Caldera (Sumisu G. sp.), Aitape (Aitape G. sp.), and off Ashizuri Cape (Ashizuri G. sp.) in the western Pacific. Although close morphological examination is needed, we supposed that the two former unidentified mussels were conspecific with G. horikoshii, because they were very closely related to one another and the clade was well supported with a high bootstrap value. Group 2 (G2, actually equal to the fifth cluster) consisted of seven nominal species (B. septemdierum and B. brevior from the Western Pacific, B. marisindicus from the Indian Ocean, B. azoricus, B. puteoserpentis and B. brooksi from the Atlantic, and B. thermophilus from the eastern Pacific) and one undescribed (morphologically examined but not described yet) species from the eastern Pacific (East Pacific B. sp.). Bathymodiolus septemdierum, B. brevior, and B. marisindicus comprised a closely related species group (Cluster A in Iwasaki et al., 2006). Mussels from the Lau Basin and the Eifuku Seamount were included in the cluster. Group 2 was subdivided into three clades comprising Indo-West Pacific, Atlantic and eastern Pacific speci es respectively. These divisions were supported with high bootstrap probabilities. The only exception was B. brooksi, which diverged basally in this group. Group 2 was marginally supported, but the monophyly of members other than B. brooksi was relatively well supported. The phylogenetic position of B. brooksi is ambiguous at present. Group 3 (G3, the remaining part of the sixth cluster excluding A. crypta) was well supported and consisted of two nominal species, B. aduloides and B. manusensis from the western Pacific. Mussels from the Lau Basin (Lau B. sp.) and off New Zealand (NZ B. sp.1) should prove to be conspecific with B. manusensis, because they were are closely genetically related to one another. The ancestor of Bathymodiolus and Gigantidas diversified into three groups, Groups 1 to 3 (Fig. 2). The short branch length between nodes leading to the groups suggests a trichotomous divergence, as shown by our previous study (Iwasaki et al., 2006). After the basal divergence of the three groups, Groups 1 and 2 diverged into two subgroups (Groups 1-1 and 1-2) and three subclusters respectively. The Atlantic subcluster differentiated first, and next the eastern Pacific and Indo-We st Pacific subclusters split in Group 2. The divergence of the three subclusters also 128 Y. Fujita et al. 0° 30°S 30°N Lau Chatham Macauley ▲ km 180°E 180°E ★ 0 1000 2000 Off Turnagain Manus 150°E 150°E ★ Aitape ★ ▲ ★ ★ RumbleIII, RumbleV RumbleIII, Sissano ★ ▲ ★ 120°E 120°E 90°E 90°E Kairei Edmond 60°E 60°E 30°E 30°E 0° 0° ★ West Africa West Lucky Strike Lucky 30°W 30°W Snake Pit Snake 60°W 60°W West Florida West Gulf of Mexico ★ 90°W 90°W ★ ★ ▲ :whale carcass 11N East Pcific 7S East Pcific Rose Garden 31S East Pcific 32S East Pcific Garden Banks Juan de Fuca Juan 120°W 120°W 9N East Pcific 13N East Pcific :vent ★ :seep ● :vent 0° A 30°S 30°N Relationships of Deep-Sea Mussels to their Mytilid Relatives 129

120°E 130°E 140°E 150°E

40°N 40°N

★Off Hatsushima Atsumi Myojin Off Noma ▲ Off Ashizuri Sumisu Torishima 30°N ▲ 30°N Minami-ensei Off Kikaijima Suiyo Iheya ★ Hatoma Kaikata ★ Nikko Kuroshima Northwest Eifuku 20°N 20°N

Mariana

South Chamorro ★

10°N 10°N 120°E 130°E 140°E 150°E

●:vent ★:seep ▲:whale carcass 0 200 400

Fig. 1. The sampling sites for deep-sea Bathymodiolus mussels and their relatives used in this study. Refer to Table 1 for details of the sampling sites. A (opposite page). Worldwide map. B. Magnified map around Japanese waters. ● , hydrothermal vent; ★ , cold-water seep; ▲ , whale carcass. 130 Y. Fujita et al.

Modiolus modiolus(DB) Benthomodiolus lignicola ★ 62/74/0.99 Benthomodiolus lignicola(DB) B. sp. JdF(DB) JdF B. sp. ◆ 895-1,3,4,5 C1 56/-/0.98 100/100/1.00 Benthomodiolus geikotsucola ★ 895-2 Tamu fisheri(DB) Tamu fisheri ◆ C2 AIH3 86/75/1.00 AIH2 A. iwaotakii ★ AIH1,4 C3 AIH5 IJN1 64/53/1.00 100/100/1.00 Idasola japonica ★ IJN2 APN3 100/100/1.00 APN1 ★ C4 APN2,328-26 A. pacifica APN328-25 B. brooksi(DB) B. brooksi ◆ 99/96/1.00 AZL1,2 B. azoricus ◆ PUS1,2/B.puteoserpentis(DB) B. puteoserpentis ◆ 54/-/- Bt32(DB) 100/100/1.00 Bt31(DB) Bt29(DB) East Pacific B. sp. ▲ 100/99/1.00 Bt28(DB) 80/62/0.95 Bt30(DB) ThE1 Bt1(DB) 98/90/0.97 Bt6(DB) B. thermophilus ▲ Bt5(DB) Bt2(DB) Bt3(DB) SM4 SM3 100/100/1.00 SS3 SS4 G2 C5 57/-/- SS1 SS2 "Cluster A" MK2 B.septemdierum ★ MK3 B.marisindicus ● MK4 ★ MK1,5 B. brevior B. marisindicus(DB) Lau B. sp. 2 ★ Lau2/B. brevior(DB) Eifuku B. sp. ★ SM2/ST1/EF1,2/Lau7 Lau5 SA1 EF3 SM1 EF5 ACN1 100/100/1.00 ACN328-1 ACN3 A. crypta ★ ACN2 ACN4 100/100/0.99 AI1 AK2 AK3,5 B. aduloides ★ AK1 95/91/1.00 AK4 Manus1 Manus2 Manus3,4 100/100/1.00 Manus5 Ne1 B. manusensis ★ G3 Ne2 Lau B. sp. 1 ★ Ne4 NZ B. sp. ★ Lau1 Lau4,9 Lau8 Lau3 Ne3,5 RIIIla,IIIsa/Mclong(DB) 100/100/1.00 G. gladius ★ RVa,Vb(DB) 80/52/0.98 75/69/1.00 Ashizuri Ashizuri G. sp. ★ Aitape1 100/94/0.75 ★ Aitape2 Aitape G. sp. 56/-/- NK1 100/100/1.00 G1-2 NK2 G. horikoshii ★ NK3,5/Su4 Nikko G. sp. ★ C6 Su2 Sumisu G. sp. ★ -/-/0.90 Kaikata/NK4/Su3,5 Su1 HK1,2,3,4,5 B. hirtus ★ 100/100/1.00 C1,3 ★ C2 Chamorro B. sp. 56/-/- Si2-1,2 64/-/0.98 Si2-3 100/100/1.00 Sissano B. sp.1 ★ Si2-4 58/-/- Si3-7 Kikaijima Kikaijima B. sp. ★ Si1-1/3-1,2,4,6 Sissano B. sp.2 ★ JH1,2/JM2,3 100 B. japonicus ★ JM1 50/-/0.90 99/93/0.99 PH1 B. platifrons ★ 99/87/1.00 PH2,3,4/Pl1,2 G1-1 69/-/0.98 B. mauritanicus(DB) B. mauritanicus ◆ ChiG1 95/89/1.00 B. childressi ◆ 100/100/1.00 ChiG2 55/52/- 96/72/0.70 Si3-3 B. tangaroa ★ B. tangaroa(DB) Sissano B. sp.3 ★ 93/93/0.82 LK4 100/99/0.99 LK3 LA2 B. securiformis ★ LA1 LK1 LK2,5

0.01 substitutions/site ◆Atlantic ★West Pacific ●Indian ▲East Pacific Relationships of Deep-Sea Mussels to their Mytilid Relatives 131 seems trichotomous. In this review we designate the three Bathymodiolus groups as Groups 1-1, 2, and 3 instead of Groups 1, 2-1, and 2-2 as given in Iwasaki et al. (2006), because the branching order of the three groups differed between the present study and our previous ones (the incongruence was probably ascribed to much more OTUs used herein).

Evolutionary processprocess

We used mytilid relatives to Bathymodiolus obtained from vents and sunken whale carcasses and wood except for Modiolus modiolus from shallow water, because the mytilid relatives are candidates for preservation of primitive characters possessed by the ancestor of Bathymodiolus mussels. The “Evolutionary stepping stone hypothesis” was proposed, in which the ances tor of Bathymodiolus mussels exploited sunken whale carcasses and wood in a progressive adaptation to the deep-sea in terms of nutrition and tolerance to high pressure and cold seawater (Distel et al., 2000). Although the monophyly of the three Bathymodiolus groups (but Group 2 excluding B. brooksi) was relatively well supported, the relationships of Bathymodiolus mussels with the mytilid relatives were still not well resolved. It is necessary to accumulate sequence data and to search for better molecular markers for elucidating their phylogenetic relationships. Nevertheless, our results seem to support the “Evolutionary stepping stone hypothesis”. The first, third and fourth clusters comprising exclusively modioline species that were all (but JdF B. sp.) obtained from sunken whale carcass es and wood, took the outgroup position to Bathymodiolus and Gigantidas mussels from vents and cold seeps (Fig. 2). An exception was A. crypta from whale carcasses, suggesting a reversion to whale carcasses from vents or seeps. Mytilid mussels flourish in shallow water. The representative shallow water species Modiolus modiolus was positioned more distantly to the vent/seep mussels. The findings indicate an evolutionary transition from shallow water to vent/seep sites via whale carcass/wood sites. JdF B. sp. from vents might have invaded vent sites independently of the Bathymodiolus and Gigantidas lineages. The younger estimate based on 18S rDNA sequences showed that the common ancestor of modern bathymodioline vent/seep species might have lived as early as 22 MYA (Van Dover et al., 2002; L ittle ＆ Vrijenhoek, 2003). If the ancestral stocks of the Bathymodiolinae originated in the Miocene, it is possible for them to have used whale carcasses as evolutionary stepping stones for

Fig. 2. Neighbor-joining tree showing phylogenetic relationships of deep-sea Bathymodiolus mussels and their relatives. Preparation of total DNA from the foot muscle, gill, or mantle which were preserved at －80°C or in 100% ethanol, amplification of the 710 bp partial COI gene fragment, and direct sequencing were carried out as described previously (Iwasaki et al., 2006). PCR was performed using 30 cycles of 30 sec denaturation at 94°C, 5 or 10 sec annealing at 42, 49, 50, or 55°C (depending on samples), and 30 sec extension at 74°C. Editing of DNA sequences, construction of neighbor-joining (NJ) and maximum parsimony (MP) trees with 401 bp COI sequences, and computation of K2P genetic distances and bootstrap values were carried out as described previously (Iwasaki et al., 2006). The majority- rule consensus MP tree was constructed by conducting a heuristic search based on the 1,000 bootstrap replicates with an unweighted ts/tv ratio. The Bayesian tree was constructed using MrBayes version 3.1 (Huelsenbeck & Ronquist, 2003) based on the model evaluated by the Mrmodel test 2.2 (Posada & Buckley, 2004). The Monte Carlo Markov chain (MCMC) length was 1 × 106 generations, and we sampled the chain every 100 generations. MCMC convergence was assessed by calculating the potential scale reduction factor, and the first 2 × 103 generations were discarded. MP and Bayesian trees presented essentially the same topology as the NJ tree. NJ (left) and MP (middle) bootstrap values and Bayesian posterior probabilities (right) are specified, when they exceeded 50% or 0.50, respectively. We used Modiolus modiolus (Mytilidae, Modiolinae) as an outgroup species. Scale bar indicates 0.01 substitutions per site. See Table 1 for abbreviations of Bathymodiolus mussels and their relatives. 132 Y. Fujita et al. progressive adaptation from shallow to deep waters, because whale carcasses have been around since the late Eocene (ca. 39 MYA, Squires et al., 1991). If the bathymodioline ancestor stocks originated in the mid Mesozoic to early Cenozoic, as indicated by the older estimate based on 18S rDNA sequences, it is likely that they exploited sunken wood for stepping stones.

Classification

Although all the members of the genus Bathymodiolus were included in the fifth and sixth clusters, the genus is not monophyletic. The unity of Bathymodiolus mussels is disputed by the presence of two bathymodioline species in Gigantidas and even the modioline species Adipicola crypta. Our results also indicated that the subfamily Bathymodiolinae is not monophyletic as previously discussed by Jones et al. (2006). The modioline species A. crypta was included in the sixth cluster and the bathymodioline mussels of Groups 1-1, 1-2 and 3 were thus more closely related to A. crypta than to the bathymodioline mussels of Group 2. It is likely that the bathymodioline Bathymodiolus and Gigantidas and the modioline A. crypta form a closely related assembly. The bathymodioline Tamu fisheri was more distantly re lated to the assembly than the modioline A. pacifica, A. iwaotakii and Idasola japonica. Therefore, we propose that the classification of taxa including the Bathymodiolinae, Bathymodiolus, and Adipicola should be reevaluated. Moreover, more extensive morphological investigation is indispensable, because our studies showed the existence of several possible new species.

Acknowledgements

We wish to express our thanks to Drs. Shigeaki Kojima, Takashi Okutani, Katsunori Fujikura, Shinji Tsuchida, Ken Takai and Toshiyuki Yamaguchi for their useful advice and support throughout this work. We are also grateful to Drs. Nobuhiro Minaka and Hirohisa Kishino for helpful advice on phylogenetic analysis. Thanks are also extended to 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 was supported in part by a grant from the Research Institute of Marine Invertebrates and a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 16570185). The eastern Pacific species B. thermophilus was collected by the scientific research vessel “Akademik Mstislav Keldysh” belonging to the Institute of Oceanology at the Russian Academy of Sciences. The Atlantic species B. childressi was provided by courtesy of Dr. Tadashi Maruyama. A western Pacific species from off New Zealand (herein referred to as NZ B. sp.) was generously provided by Dr. Peter Smith (National Institute of Water and Atmospheric Research Ltd.). The other deep-sea mussels were collected during dives by submersibles from Japan Agency for Marine-Earth Science and Technology (JAMSTEC).

References

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シンカイヒバリガイ類とそのイガイ科近縁種の系統関係

藤田祐子・松本寛人・藤原義弘・橋本惇・S. V. ガルキン・上島励・宮崎淳一

要約

シンカイヒバリガイ類（Mytilidae, Bathymodiolinae, Bathymodiolus）は化学合成生物群集の主要なメンバーである。シンカイヒバリガイ類の進化と起源を明らかにするため，近縁なイガイ類とともにミトコンドリア COI 遺伝子の塩基配列から系統関係をレビューした。シンカイヒバリガイ亜科は 4 つのグループに分かれ，シンカイヒバリガイ亜科もシンカイヒバリガイ属も単系統群ではなかった。鯨遺骸や沈木から得られたイガイ類の多くはシンカイヒバリガイ類の外群となり，浅海のイガイ類はさらにその外側に位置した。この結果は，シンカイヒバリガイ類の祖先が海底の鯨遺骸や沈木を利用して浅海から深海に適応したとする“飛び石仮説”を支持した。