Evolutionary Process of Deep-Sea Bathymodiolus
Mussels
Jun-Ichi Miyazaki
1
*, Leonardo de Oliveira Martins
2
, Yuko Fujita
3
, Hiroto Matsumoto
3
, Yoshihiro
Fujiwara
4
1 Faculty of Education and Human Sciences, University of Yamanashi, Kofu, Yamanashi, Japan, 2 Department of Biochemistry, Genetics and Immunology, University of
Vigo, Vigo, Spain, 3 Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan, 4 Research Program for Marine Biology and Ecology, Japan Agency for
Marine-Earth Science and Technology (JAMSTEC), Yokosuka, Kanagawa, Japan
Abstract
Background: Since the discovery of deep-sea chemosynthesis-based communities, much work has been done to clarify
their organismal and environmental aspects. However, major topics remain to be resolved, including when and how
organisms invade and adapt to deep-sea environments; whether strategies for invasion and adaptation are shared by
different taxa or unique to each taxon; how organisms extend their distribution and diversity; and how they become
isolated to speciate in continuous waters. Deep-sea mussels are one of the dominant organisms in chemosynthesis-based
communities, thus investigations of their origin and evolution contribute to resolving questions about life in those
communities.
Methodology/Principal Finding: We investigated worldwide phylogenetic relationships of deep-sea Bathymodiolus mussels
and their mytilid relatives by analyzing nucleotide sequences of the mitochondrial cytochrome c oxidase subunit I (COI) and
NADH dehydrogenase subunit 4 (ND4) genes. Phylogenetic analysis of the concatenated sequence data showed that
mussels of the subfamily Bathymodiolinae from vents and seeps were divided into four groups, and that mussels of the
subfamily Modiolinae from sunken wood and whale carcasses assumed the outgroup position and shallow-water modioline
mussels were positioned more distantly to the bathymodioline mussels. We provisionally hypothesized the evolutionary
history of Bathymodilolus mussels by estimating evolutionary time under a relaxed molecular clock model. Diversification of
bathymodioline mussels was initiated in the early Miocene, and subsequently diversification of the groups occurred in the
early to middle Miocene.
Conclusions/Significance: The phylogenetic relationships support the ‘‘Evolutionary stepping stone hypothesis,’’ in which
mytilid ancestors exploited sunken wood and whale carcasses in their progressive adaptation to deep-sea environments.
This hypothesis is also supported by the evolutionary transition of symbiosis in that nutritional adaptation to the deep sea
proceeded from extracellular to intracellular symbiotic states in whale carcasses. The estimated evolutionary time suggests
that the mytilid ancestors were able to exploit whales during adaptation to the deep sea.
Citation: Miyazaki J-I, Martins LdO, Fujita Y, Matsumoto H, Fujiwara Y (2010) Evolutionary Process of Deep-Sea Bathymodiolus Mussels. PLoS ONE 5(4): e10363.
doi:10.1371/journal.pone.0010363
Editor: Richard Kazimierz Frank Unsworth, Northern Fisheries Centre, Australia
Received October 7, 2009; Accepted February 27, 2010; Published April 27, 2010
Copyright:  2010 Miyazaki et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan (no. 21570093, http://www.jsps.go.jp/). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: miyazaki@yamanashi.ac.jp
Introduction
Deep-sea mussels of the genus Bathymodiolus (Mytilidae, Bath-
ymodiolinae) are one of the dominant macroorganisms in
chemosynthesis-based communities in hydrothermal vents on
spreading ridges and back-arc basins and in cold-water seeps
along subduction zones. Since the original description of the genus
[1], 22 Bathymodiolus species have been described [2–13], and their
biogeographic distributions are as follows. There are: 1) 14 Pacific
species, B. japonicus Hashimoto & Okutani 1994, B. platifrons
Hashimoto & Okutani 1994, B. septemdierum Hashimoto & Okutani
1994, B. hirtus Okutani et al. 2004, B. securiformis Okutani et al.
2004, B. aduloides Hashimoto & Okutani 1994, B. taiwanensis Cosel
2008, B. brevior Cosel et al. 1994, B. elongates Cosel et al. 1994, B.
tangaroa Cosel & Marshall 2003, B. manusensis Hashimoto & Furuta
2007, B. edisonensis Cosel and Janssen 2008, and B. anteumbonatus
Cosel and Janssen 2008 from the West Pacific and B. thermophilus
Kenk & Wilson, 1985 from the East Pacific; 2) seven Atlantic
species, B. childressi Gustafson et al. 1998, B. heckerae Gustafson et
al. 1998, and B. brooksi Gustafson et al. 1998 from the West
Atlantic, B. azoricus Cosel & Comtet 1999 and B. puteoserpentis Cosel
et al. 1994 from the Mid-Atlantic Ridge, and the trans-Atlantic B.
mauritanicus Cosel 2002 and B. boomerang Cosel & Ole 1998; and 3)
one Indian Ocean species, B. marisindicus Hashimoto 2001. Two
species of the genus Gigantidas from the West Pacific, G. horikoshii
Hashimoto &Yamane 2005 and G. gladius Cosel & Marshall 2003,
and one species of the genus Tamu from the Atlantic, T. fisheri
Gustafson et al. 1998, belong to the subfamily Bathymodiolinae
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[5,9,14]. Active exploration of new localities and careful surveys of
known localities suggests the existence of many cryptic species.
The species diversity is very high in the West Pacific compared
with other areas, and thus the origin of the bathymodioline
mussels seems to be located in the West Pacific. However, the
mismatch distributions of the West Pacific B. septemdierum and B.
brevior and the Indian Ocean B. marisindicus suggest that the
Southern Central Indian Ridge of the Indian Ocean might be the
more ancient residence rather than the Izu-Ogasawara Island-arc
and the North Fuji Basin of the West Pacific, if periods from
formation to expansion of their populations were not significantly
different among them [15].
In Japanese waters (Figs. 1 and 2), six Bathymodiolus and one
Gigantidas species have steady residences as evidenced by a stable,
constant supply of their propagules [3, 10. 14]. Some species
possibly have transient residences through incident, leaky supply of
propagules as mentioned below. Bathymodiolus japonicus and B.
platifrons are distributed in seeps in Sagami Bay and vents of the
Okinawa Trough, which are separated by approximately
1,500 km. Bathymodiolus aduloides is distributed in seeps in Sagami
Bay, the Nankai Trough, and the subduction zone of the Nansei-
shoto Trench and vents of the Okinawa Trough. However, our
genetic analyses have not confirmed its existence in the Nankai
Trough. The Nankai Trough is situated between Sagami Bay and
the Okinawa Trough. There appear to be some barriers to gene
flow between the Nankai Trough and Sagami Bay and between
the Nankai Trough and Okinawa Trough. Only one specimen,
identified genetically as B. aduloides, has been obtained so far from
vents in the Izu-Ogasawara Island-arc. The three species of
Bathymodiolus mussels can exploit both seeps and vents as habitats.
No significant genetic differentiation was discernible between seep
and vent populations of B. platifrons [15]. Our studies also
suggested a genetic similarity between seep and vent populations
of B. japonicus [16], indicating the high adaptability of these species
to deep-sea environments, albeit the seemingly large environmen-
tal differences between seeps and vents. Bathymodiolus septemdierum is
distributed in vents in the Izu-Ogasawara Island-arc, but not in
Sagami Bay, which is approximately 500 km from the Myojin
Knoll and Suiyo Seamount in the Izu-Ogasawara Island-arc. Only
one specimen, identified genetically as B. septemdierum, has been
obtained so far from the Okinawa Trough. There are relatively
large obstacles to gene flow between Sagami Bay and the Izu-
Ogasawara Island-arc. Our genetic studies suggested that this
species was conspecific to B. brevior and might possibly be
conspecific to B. marisindicus [15]. If this is the case, this species
has the widest habitat range among Bathymodiolus species from
Japanese waters southeastward to the North Fuji Basin in the West
Pacific Ocean and southwestward to the Kairei Field in the Indian
Ocean. The two remaining species, B. hirtus and B. securiformis, are
distributed in seeps of the subduction zone of the Nansei-shoto
Trench. The latter is also distributed in seeps in the Nankai
Trough. Gigantidas horikoshii is distributed in vents in the Izu-
Ogasawara Island-arc. One specimen from Sagami Bay has been
identified as Sissano B. sp. 1, which resides mainly in Sissano in the
West Pacific Ocean.
Organisms initially invading the deep sea encounter serious
difficulties and must alter their feeding strategies to overcome poor
nutrition and acquire tolerance to high pressure and cold seawater.
Furthermore, organisms in vents and seeps must establish
symbiosis with chemosynthetic bacteria as an effective feeding
strategy and tolerance to toxic H
2
S. The ‘‘Evolutionary stepping
stone hypothesis’’ has been proposed, in which the ancestors of
bathymodioline mussels exploited sunken wood and whale
carcasses in their progressive adaptation to deep-sea environments
[17,18]. Further studies are required to elucidate the origin and
adaptive process of bathymodioline mussels as a representative of
organisms in chemosynthesis-based communities. Our previous
research suggested an evolutionary transition from shallow water
to vent/seep sites via sunken wood/whale carcass sites and
supported the hypothesis [15,19], although deeper branching was
poorly supported. However, the process did not occur in a single,
unidirectional manner. Our research also suggested independent
invasion into vents and seeps and reversion into whale carcass sites
from vent or seep sites in the mytilid lineages.
Only some Bathymodiolus species from limited areas were the
subjects of earlier molecular phylogenetic studies [18,20–22].
Subsequently, using updated databases, molecular phylogenetics
searched for the phylogeny of about 10 species [16,23,24], and
mytilid relatives from whale carcasses and wood were included to
trace the origins of Bathymodiolus mussels [25,26]. In our previous
studies [15,19], we sequenced the mitochondrial genes of more
than 15 nominal and cryptic species, and showed that mussels in
Figure 1. The sampling sites for deep-sea Bathymodiolus mussels and their relatives used in this study. Refer to Table 2 for details of the
sampling sites. #, hydrothermal vent;
N
, cold-water seep; &, wood/whale bone; m, shallow.
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the subfamily Bathymodiolinae comprised four groups. The first
group (Group1) includes seven West Pacific and Atlantic Bath-
ymodiolus species (Group 1-1) and two West Pacific Gigantidas
species (Group 1-2). The group includes members of the B.
childressi clade (B. childressi, B. mauritanicus, B. platifrons, B. hirtus, B.
anteumbonatus, B. japonicus, B. tangaroa. B. securiformis, B. edisonensis,
and two Gigantidas species) based on morphological traits [13]. The
second group (Group 2) includes six or seven Bathymodiolus species,
which are subdivided into three subclusters consisting of the Indo-
West Pacific, Atlantic, and East Pacific species, respectively, with
the exception of B. brooksi that diverges basally to the subclusters.
Group 2 includes members of Cosel’s B. thermophilus clade (B.
thermophilus and East Pacific B. sp., B. brevior, B. azoricus, B. elongates,
B. puteoserpentis, B. septemdierum, B. boomerang, B. heckerae, and B.
brooksi). The third group (Group 3) includes two West Pacific
Bathymodiolus species, and the fourth group includes only one
species, T. fisheri. The group includes members of Cosel’s B.
aduloides clade (B. aduloides and B. manusensis). Our studies also
showed that the subfamily Bathymodiolinae and the genus
Bathymodiolus were not monophyletic, suggesting the need to
reevaluate the classification.
In the present study, we investigated worldwide phylogenetic
relationships of Bathymodiolus mussels and their mytilid relatives by
analyzing concatenated sequences of the mitochondrial cyto-
chrome c oxidase subunit I (COI) and NADH dehydrogenase
subunit 4 (ND4) genes. We also investigated the evolutionary
processes of Bathymodiolus mussels by estimating evolutionary
divergence times with variable rates over time.
Results
Phylogenetic relationships of Bathymodiolus mussels and
their relatives
Mussels of the subfamily Bathymodiolinae were divided into
four groups (Fig. 3). The first group (Group 1) was subdivided into
two subgroups. One subgroup (Group 1-1) consisted of seven
Figure 2. The sampling sites for deep-sea Bathymodiolus mussels and their relatives in Japanese waters. The boxed region in Fig. 1 is
enlarged. Refer to Table 2 for details of the sampling sites. #, hydrothermal vent;
N
, cold-water seep; &, wood/whale bone; m, shallow.
doi:10.1371/journal.pone.0010363.g002
Figure 3. Phylogenetic relationships of deep-sea Bathymodiolus mussels and their relatives based on the 401-bp COI and 423-bp ND4
sequences. The NJ tree was constructed based on the genetic distances calculated according to Kimura’s two-parameter method using Modiolus
nipponicus as an outgroup species. The MP and Bayesian trees presented essentially the same topology as the NJ tree. Only the NJ (left) and MP (middle)
bootstrap values .50% and Bayesian posterior probabilities (right) .0.50 are specified. The scale bar indicates 0.01 substitutions per site. See Table 1 for
abbreviations of Bathymodiolus mussels and their relatives. #, hydrothermal vent;
N
, cold-water seep; &, wood/whale bone; m, shallow.
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nominal species, B. hirtus, B. japonicus, B. platifrons, and B. securiformis
from Japanese waters, B. tangaroa from the West Pacific, B.
mauritanicus and B. childressi from the Atlantic, and five unidentified
(not morphologically examined) Bathymodiolus-related 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 West Pacific. All the members so far examined in
Group 1-1 (6 nominal species and Sissano B. sp.) have
methanotrophic endosymbionts [27–28]. Group 1-2 included
two nominal species, G. horikoshii and G. gladius, and four
unidentified Gigantidas-related mussels from the Nikko Seamount
(Nikko G. sp.), Sumisu Caldera (Sumisu G. sp.), Aitape (Aitape G.
sp.), and off Ashizuri Cape (Ashizuri G. sp.) in the West Pacific.
The former two unidentified mussels are likely to be conspecific
with G. horikoshii because of their genetic similarity. The species has
thioautotrophic endosymbionts (the data will be published
elsewhere).
The second group (Group 2) consisted of eight nominal and one
undescribed (morphologically examined but not yet described)
Bathymodiolus species. This group was subdivided into three
subclusters including the Indo-West Pacific B. septemdierum, B.
brevior, and B. marisindicus, Atlantic B. azoricus, B. puteoserpentis, and
B. heckerae, and East Pacific B. thermophilus and undescribed species
(East Pacific B. sp.), with the exception of the Atlantic B. brooksi,
which diverged basally to the three clusters. Bathymodiolus
septemdierum, B. brevior, and B. marisindicus comprised the closely
related species group (Cluster A [16]). We showed in our previous
study that a high gene flow occurred between B. septemdierum and
B. brevior and that the gene flow between B. marisindicus and B.
septemdierum or B. brevior was low but not negligible, although their
habitats are approximately 5,000–10,000 km apart [15]. Mussels
from the Lau Basin (Lau B. sp.1) and Eifuku Seamount (Eifuku B.
sp.) included in the cluster are likely to be conspecific to B.
septemdierum because of their genetic similarity. Four species in
Group 2, B. septemdierum, B. brevior, B. marisindicus, and B.
thermophilus, contain solely thioautotrophs, and B. puteoserpentis, B.
azoricus, B. heckerae, and B. brooksi harbor both thioautotrophs and
methanotrophs [27,29–35].
The third group (Group 3) consisted of two nominal species, B.
aduloides and B. manusensis, restricted to the West Pacific. Mussels
from the Lau Basin (Lau B. sp. 2) and off New Zealand (NZ B. sp.)
are likely to be conspecific with B. manusensis because of their
genetic similarity. Species in this group contain thioautotrophic
endosymbionts [36]. The above three groups, two subgroups, and
three subclusters were well supported. The fourth group (Group 4)
consisted only of T. fisheri. Groups 3 and 4 were allied and in turn
they were allied with Group 1, but the relationships were poorly
supported.
Mussels of the subfamily Modiolinae from sunken wood and
whale carcasses assumed the outgroup position to the bath-
ymodioline mussels from vents and seeps, with the exception of
Adipicola crypta (Dall et al. 1938) from whale carcasses. The species
was allied with Group 1 with marginal support. The cluster
including vent/seep bathymodioline mussels and wood/whale
modioline mussels was well supported. Mytilid mussels flourishing
in shallow water were positioned more distantly to the vent/seep
bathymodioline mussels. Although only the modioline Modiolus
nipponicus (Oyama 1950) was included in the present tree,
modioline Modiolus modiolus (Linnaeous 1758) and three species
of the subfamily Mytilinae were also positioned, as was M.
nipponicus in our previous studies [15]. The unity of Bathymodiolus
mussels was not supported, because two species of Gigantidas in the
Bathymodiolinae and A. crypta in the Modiolinae perturbed the
unity.
Estimation of divergence time
Estimating divergence time is useful to reconstruct evolutionary
history. The mean evolutionary rate of mitochondrial DNA has
been estimated to be 1,2% per million years [37]. However, the
application of a molecular clock is problematic in some cases,
because the rate constancy of molecular evolution is a prerequisite
[38,39]. Our preliminary study showed that the rate of molecular
evolution varied among lineages of Bathymodiolus mussels, and thus
we adopted Thorne and Kishino’s approach (see [40] for details of
the application of this approach). We show estimates of
evolutionary time on the ML tree (Fig. 4). For calibration, we
used reference time associated with the split between the Atlantic
and East Pacific subclusters (12 to 10 MYA) in Group 2. Our
results (Table 1) showed that diversification of bathymodioline
mussels initiated in the early Miocene (about 20 MYA).
Subsequently, Groups 1 to 3 started differentiating in the early
to middle Miocene (about 19 to 14 MYA).
Discussion
Phylogenetic relationships of Bathymodiolus mussels and
their relatives
Bathymodioline species from vents and seeps were divided into
four well-supported groups (Fig. 3). Together they comprised the
poorly-supported bathymodioline cluster. Concatenated sequence
data, however, provided better resolution of the phylogeny of
Bathymodiolus mussels and their relatives than those derived from
single COI [19] or ND4 [15] sequence data, although some OTUs
could not be used because of the lack of sequence data on either
gene. Modioline species from sunken wood and whale carcasses
assumed the outgroup position to the bathymodioline mussels,
with the exception of A. crypta from whale carcasses. Mytilid species
from shallow water such as M. nipponicus were positioned more
distantly to the bathymodioline mussels. The results support the
‘‘Evolutionary stepping stone hypothesis,’’ which advocates
adaptive progress of deep-sea organisms from shallow-water to
vent/seep sites via wood/whale carcass sites [17,18].
Three modioline species, Benthomodiolus geikotsucola Okutani &
Miyazaki 2007, A. pacifica (Dall et al. 1938), and A. crypta, were
obtained from whale carcasses in Japanese waters, and the
epidermal cells of their gills harbored thioautotrophic bacterial
symbionts [15], although no mytilid mussels haboring symbionts
have previously been reported from shallow water. As shown
schematically in Fig. 5, Benthomodiolus geikotsucola from naturally
sunken Bryde’s whale carcasses at the Torishima Seamount
(approximately 4,000 m in depth) had extracellular symbionts
trapped by microvilli of the host cells (the data will be published
elsewhere). Adipicola pacifica and A. crypta inhabit artificially settled
sperm whale carcasses off Noma Cape (approximately 250 m in
depth). The former species had extracellular symbionts enclosed
by the protrudent host cell membrane (the data will be published
elsewhere). Enclosure by the cell membrane appears more
effective to maintain extracellular symbionts than microvilli
trapping. The symbionts of the latter species existed inside the
host cells, as in Bathymodiolus mussels. These findings suggest that
nutritional adaptation to the deep sea proceeded from the
extracellular symbiotic state to the intracellular symbiotic state in
whale carcasses. The evolutionary transition of symbiosis also
supports the ‘‘Evolutionary stepping stone hypothesis’’. Benthomo-
diolus geikotsucola is one of mytilid mussels that can live in the
deepest sea and thus is more adaptive to abyssal waters than vent/
seep bathymodioline mussels (their habitats are up to 4,000 m in
depth), but maintains primitive state of symbiosis. However, some
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species live in sunken wood in shallower water and trap symbionts
using microvilli [41].
Modioline A. pacifica, A. iwaotakii (Habe 1958), and Idasola
japonica Habe 1976 comprised the most closely related outgroup to
the bathymodioline cluster, while A. crypta was included in it.
Adipicola crypta does not differ from bathymodioline mussels in the
phylogenetic position and symbiotic status. Since our results
indicated that monophylies of the subfamily Bathymodiolinae and
the genus Bathymodiolus were not supported, the classification
should be reevaluated. Moreover, our results showed the existence
Figure 4. Posterior distribution of evolutionary divergence times. Phylogenetic relationships of deep-sea Bathymodiolus mussels based the
concatenated 401-bp COI and 423-bp ND4 sequences. The ML tree was constructed using Modiolus nipponicus as an outgroup species. The red lines
represent 95% credibility intervals of sampled values. See Table 2 for abbreviations of Bathymodiolus mussels.
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of several cryptic species, and thus more extensive morphological
investigation is indispensable.
Our studies suggested that B. brevior was conspecific to B.
septemdierum and might possibly be conspecific to B. marisindicus.
Similar situations are discernible in other species. Bathymodiolus
childressi was clearly distinguished from B. mauritanicus by the
mitochondrial COI gene, but the two species did not form a
separate clade based on the nuclear rDNA spacer ITS2 [24].
Table 1. Estimated evolutionary time (t).
t (MYA)
Onset of diversification of the subfmaily Bathumodiolinae 21.163.6
Split of Groups 2 and 3 19.463.4
Split of Groups 1-1 and 1-2 14.163.0
Split of B. brooksi and the other members in Group2 13.361.7
Split of East Pacific subcluster from the common ancestor of Indo-West Pacfic and Atlantic subclusters in Group 2 (12,10)
a
Onset of diversification in Bathymodiolus spp. of Group 1-1 11.262.5
Onset of diversification in Gigantidas spp. of Group 1-2 10.462.5
Split of B. hirtus from the common ancestor of Sissano B. spp. 1 and 2, Kikaijima B. sp., and Chamorro B. sp. in Group 1-1 9.862.3
Split between Indo-West Pacfic and Atlantic subclusters in Group 2 9.761.0
Split of B. aduloides and B. manusensis in Group 3 8.462.6
Split of B. japonicus from the common ancestor of B. securiformis, B. tangaroa, and Sissano B. sp. 3 in Group 1-1 6.661.8
Onset of diversification of Atlantic species in Group 2 6.261.2
Split of B. platifrons from the common ancestor of B. mauritanicus and B. childressi in Group 1-1 3.861.2
Split of B. securiformis from the common ancestor of B. tangaroa and Sissano B. sp. 3 in Group 1-1 3.561.5
Split of B. thermophilus and East Pacific sp. in Group 2 2.660.9
Onset of diversification in Cluster A of Group 2 0.960.4
a
reference time.
doi:10.1371/journal.pone.0010363.t001
Figure 5. Schematic representation of evolutionary symbiostic transition. Mytilid mussels from shallow water with no symbionts;
Benthomodiolus geikotsucola from whale carcasses haboring extracellular symbionts trapped by microvilli of the host cells; Adipicola pacifica from
whale carcasses haboring extracellular symbionts enclosed by the protrudent host cell membrane; A. crypta from whale carcasses haboring
intracellular symbionts; Bathymodiolus mussels with intracellular symbionts.
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Bathymodiolus boomerang was not genetically discriminated from B.
heckerae by either the COI gene or nuclear rDNA spacer ITS2 [24].
Systematics including possible synonyms should be revised, while
cryptic species must be formally described.
Evolutionary process of Bathymodiolus mussels
After basal trichotomous divergence of the three groups, the
East Pacific subcluster diverged in Group 2. Next the Atlantic and
Indo-West Pacific subclusters split (Fig. 3). However, the
divergence of the three subclusters also appears trichotomous,
because alliance of the latter subclusters was marginally supported.
Divergence of the Atlantic and East Pacific subclusters may have
been caused by the closure of the Isthmus of Panama. The rise of
the Isthmus of Panama began in the middle Miocene (15.5 to 12
MYA), an island chain emerged 13 to 12 MYA, and in the late
Miocene (11.5,8 MYA) terrestrial species were able to move
between North and South America [42]. Although the final
closure was only accomplished by 3 to 3.5 MYA, faunal changes
between the East Pacific Ocean and the Caribbean Sea had begun
long before. The formation of the Isthmus of Panama has exerted
profound effects on shallow-water animals since the late Miocene.
Diversification of reef corals in the Caribbean, followed by an
increase in carbonate-associated benthic foraminiferans, began in
the late Miocene [43]. Snapping shrimps, living at depths of less
than 20 m in mangrove stands and shallow waters, diverged
between the East Pacific and the Caribbean 9 to 3 MYA [44].
Transisthmian divergence of shallow-water gastropods occurred
8.5 to 5.3 MYA [45]. It seems reasonable to consider that the
transisthmian isolation of deep-sea animals preceded that of
shallow-sea animals, although this depends on larval behavior and
physiology. It is not known whether larvae of Bathymodiolus mussels
are transported by bottom currents, but the blocking of larval
transport through the closing transisthmian seaway is more likely
for deep-sea animals than for shallow-water animals. The isotope
composition of hydrogenous ferromanganese crusts from the
western North Atlantic and the central Pacific Oceans suggests
that the compositional shift around 12 MYA might have been
related to the initial shallowing of the Central American Isthmus,
which prevented the access of deep water from the North Atlantic
[46].
It is very difficult to set unequivocal reference times.
Nevertheless, we tentatively set reference times of 12 to 10 MYA
for the split between the Atlantic and East Pacific subclusters. We
provisionally propose here a hypothesis on the evolutionary
process of Bathymodiolus mussels to provide a basis for discussions of
the evolution of deep-sea animals.
According to the evolutionary time estimated by Thorne and
Kishino’s approach (Table 2), the three groups are estimated to
have diverged in the Miocene, when climates changed markedly.
It is generally accepted that transgression and regression
concomitant with global warming and cooling lead to worldwide
dispersal and diversification of sea animals. If this is the case for
deep-sea animals, the ancestor of Bathymodiolus established
worldwide distribution in the sea enlarged by transgression in
the early Miocene. Subsequently, in the early to middle Miocene,
diversification was caused by vicariance due to regression and
plate tectonic events. Our estimate of the onset of bathymodiolin
diversification (ca. 21 MYA) is roughly consistent with the younger
estimate based on 18S rRNA data that showed that the common
ancestor of modern bathymodioline vent and seep species might
have lived as early as 22 MYA [47,48]. If the ancestor of the
Bathymodiolinae originated in the Miocene, it is possible that it
used whale carcasses as evolutionary stepping stones for progres-
sive adaptation from shallow to deep waters, because whale
carcasses have been available since the late Eocene (ca. 39 MYA
[49]).
Species in Group 3 could be relics of cosmopolitans, because
there are only two species, the distribution of which is restricted to
the West Pacific although they have a long history (since ca. 8.4
MYA). Species in Group 1-1 started differentiating ca. 11 MYA,
and the four species in the Okinawa Trough, B. hirtus, B. japonicus,
B. platifrons, and B. securiformis, speciated 3.5 to 9.8 MYA, long
before the formation of the Okinawa Trough (ca. 2 MYA). Thus,
members of Group 1-1 living in the Okinawa Trough speciated
elsewhere and thereafter migrated to the Okinawa Trough [15]. It
is unlikely that species in Group 1-1 are also relics of
cosmopolitans. They are distributed in highly isolated localities,
like Japanese waters and the East and West Atlantic, without any
species in intervening areas. However, our results showed that B.
platifrons from Japanese waters was very closely related to B.
childressi from the West Atlantic and trans-Atlantic B. mauritanicus
(Fig. 3). It is evident that further surveys of novel vent and seep
sites and genetic examination of deep-sea mussels are needed to
discover cryptic members of Groups 3 and 1-1 and to elucidate the
evolutionary history of Bathymodiolus mussels.
Materials and Methods
Materials and sequencing of mitochondrial genes
Specimens used in this study are listed in Table 2 and collection
sites are mapped in Figs. 1 and 2. Sequencing was performed as
described previously [15,18,20]. Since the doubly uniparental
inheritance of mitochondrial DNA is known in some mytilid
mussels, we have included at least five specimens, if available, of
each nominal and cryptic species in our previous analyses to detect
divergent and highly heterogeneous DNA sequences [15,16,19].
However, we used one specimen of each species, because we have
not seen any evidence of doubly uniparental inheritance so far.
Nevertheless, it is not plausible to represent each species by a single
sequence, especially for mytilid mussels from sunken whale
carcasses and wood and shallow water, although many phyloge-
netic studies did so. Heteroplasmy of mitochondrial DNA was
shown even in Bathymodiolus [50].
Phylogenetic analysis
DNA sequences were edited and aligned using DNASIS
(Hitachi Software Engineering Co., Ltd., Tokyo, Japan) and
MEGA 3.1 software [51], and the alignments were corrected by
visual inspection for phylogenetic analysis. We used 401-bp COI
and 423-bp ND4 sequences, excluding ambiguous sites. Dendro-
grams were constructed using the neighbor-joining (NJ) and
maximum parsimony (MP) methods using PAUP*4.0 beta 10
software [52]. Genetic distances were computed using the
Kimura’s two-parameter method [53]. The reliability of trees
was evaluated by producing 1,000 bootstrap replicates. 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 software [54] based on the model evaluated
by the Mrmodel test 2.2 [55]. The Monte Carlo Markov chain
(MCMC) length was 5610
6
generations, and we sampled the
chain after every 100 generations. MCMC convergence was
assessed by calculating the potential scale reduction factor, and the
first 1610
4
generations were discarded. We used Modiolus nipponicus
(Mytilidae, Modiolinae) as an outgroup species.
Evolutionary divergence times were estimated using the relaxed
molecular clock model implemented in the software Multidivtime
[56,57]. This model depends on the Maximum Likelihood
Evolution of Deep-Sea Mussels
PLoS ONE | www.plosone.org 8 April 2010 | Volume 5 | Issue 4 | e10363

Table 2. Sample list.
Species Sample abbreviation Sampling site (locality number in Fig. 1) Depth (m) Habitat type
Bathymodiolinae
Bathymodiolus aduloides AK1 Off Kikaijima Island (1) 1 451 Seep
B. azoricus AZL1 Lucky Strike, Mid-Atlantic Ridge (2) Unknown Vent
B. brevior NF BN Mussele Valley, North Fiji Basin (3) Unknown Vent
B. childressi ChiG1 Gulf of Mexico(4) 1 859 Seep
B. hirtus HK1 Kuroshima Knoll, Off Yaeyama Islands (5) 637 Seep
B. japonicus JH1 Off Hatsushima, Sagami Bay (6) 1 170–1 180 Seep
B. marisindicus MK1 Kairei Field, Southern Central Indian Ridge (7) 2 443–2 454 Vent
B. platifrons PH1 Off Hatsushima, Sagami Bay (6) 1 170–1 180 Seep
B. puteoserpentis PUS1 Snake Pit, Mid-Atlantic Ridge (8) 3 023–3 510 Vent
B. securiformis LK1 Kuroshima Knoll, Off Yaeyama Islands (5) 641 Seep
B. septemdierum SM1 Myojin Knoll, Izu-Ogasawara Island-arc (9) 1 288–1 290 Vent
B. thermophilus ThE1 9N East Pacific Rise (10) 2 524 Vent
Chamorro B. sp. C1 South Chamorro Seamount, Mariana (11) 2 899 Seep
Eifuku B. sp. EF1 Northwest Eifuku Seamount (12) 1 625 Vent
Kikaijima B. sp. Kikaijima Off Kikaijima Island (1) 1 430 Seep
Lau B. sp. 1 Lau1 Hine Hina, Lau Basin (13) 1 818 vent
Lau B. sp. 2 BR1 Hine Hina, Lau Basin (13) 1 818 Vent
B. manusensis BE1 PACKMANUS Field E, Manus Basin (14) 1 627–1 629 Vent
NZ B. sp. Ne1 Off New Zea land (unknown) Unknown Vent
Sissano B. sp. 1 Si2-1 Sissano, Papua New Guinea (15) 1 646 Seep
Sissano B. sp. 2 Si1-1 Sissano, Papua New Guinea (15) 1 881 Seep
Sissano B. sp. 3 Si3-3 Sissano, Papua New Guinea (15) 1 881 Seep
Gigantidas horikoshii Kaikata Kaikata Seamount (16) 486 Vent
Aitape G. sp. Aitape1 Aitape, Papua New Guinea (17) 470 Seep
Ashizuri G. sp. Ashizuri Off Ashizuri Cape (18) 575 Seep
Nikko G. sp. NK1 Nikko Seamount (19) 485 Vent
Sumisu G. sp. Su1 Sumisu Caldera (20) 676–686 Vent
Database
B. brooksi B. brooksiWFE(DB) West Florida Escarpment (21) 3 314 Seep
B. heckerae B. heckeraeWFE(DB) West Florida Escarpment (21) 3 314 Seep
B. mauritanicus B. mauritanicus(DB) West Africa (22) 1 000–1 267 Seep
B. sp. East Pacific B. aff. thermophilus(DB) 32S East Pacific Rise (23) 2 331 Vent
B. sp. NZ3 B. sp. NZ3(DB) Macauley Cone (24) 200 Vent
B. tangaroa B. tangaroa(DB) Off Turnagain Cape, New Zea land (25) 920–1 205 Seep
B. brevior MT B. breviorMT(DB) Mariana Trough (26) 3 589 Vent
Gigantidas gladius Gigantidas gladius(DB) Rumble III (27) 300–460 Vent
Tamu fisheri Tamu fisheri(DB) Garden Banks (28) 546–650 Seep
Modiolinae & Mytilinae
Adipicola crypta ACN1 Off Noma Cape, Kagoshima (29) 225–229 Whale bone
Adipicola iwaotakii AIH1 Off Nakaminato, Ibaraki (30) 490 Wood
Adipicola pacifica APN1 Off Noma Cape, Kagoshima (29) 225–229 Whale bone
Idasola japonica IJN1 Off Noma Cape, Kagoshima (29) 400,425 Wood
Benthomodiolus geikotsucola Tori1-1 Torishima Seamount (31) 4 051 Whale bone
Modiolus nipponicus Modiolus nipponicus Off Oura Harbor, Shizuika - Shallow
Database
Benthomodiolus lignicola Benthomodiolus lignicola(DB) Chatham Rise (32) 826–1 174 Whale bone, Wood
Idas macdonaldi Idas macdonaldi(DB) Garden Banks (28) 650 Seep
Idas washingtonia Idas washingtonia(DB) Monterey Bay (33) 960–1 910 Whale bone, Wood,
Vent
doi:10.1371/journal.pone.0010363.t002
Evolution of Deep-Sea Mussels
PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10363

topology, which was inferred with PAUP [52] using the
Hasegawa-Kishino-Yano evolutionary model [58] assuming that
rates across sites vary according to a discretized gamma
distribution [59]. Under this relaxed molecular clock model the
proportion of times from internal nodes to the ingroup root node –
the root when we exclude the outgroup and then reroot the tree –
are robust to time scaling [57]. Thus a preliminary run of
Multidivtime without any calibration point was conducted to find
an initial guess for the divergence time of the ingroup root node.
The Multidivtime analysis was conducted assuming a gamma
distribution with mean and standard deviation of 30MYA for the
divergence time of the ingroup root node. Furthermore the
splitting time between the Atlantic and East Pacific subclusters was
calibrated to be between 10 and 12MYA. We then sampled 10
3
posterior estimates of divergence times and other parameters at
every 10
4
iterations after discarding the first 10
5
samples.
Acknowledgments
We wish to express our thanks to Drs. Takashi Okutani, Shigeaki Kojima,
Hiromi Watanabe, 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.
Author Contributions
Conceived and designed the experiments: JIM. Performed the experi-
ments: JIM YF HM YF. Analyzed the data: JIM LdOM YF HM YF.
Contributed reagents/materials/analysis tools: JIM LdOM YF. Wrote the
paper: JIM LdOM.
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