Phylogeography of bivalve Meretrix petechialis in the Northwestern Pacific indicated by mitochondrial and nuclear DNA data
Phylogeography of bivalve Meretrix petechialis in the Northwestern Pacific indicated by mitochondrial and nuclear DNA data
Xiaoxuan Wang 1 2 3
Lingfeng Kong 1 2 3
Jun Chen 0 1 3
Akihiko Matsukuma 1 2 3
Qi Li 1 2 3
0 Institute of Geology and Paleontology, Linyi University , Linyi , China
1 Funding: This research was supported by National Natural Science Foundation of China (41276138) and Fundamental Research Funds for the Central Universities , 201564021
2 Key Laboratory of Mariculture, Ministry of Education, Ocean University of China , Qingdao , China
3 Editor: Tzen-Yuh Chiang, National Cheng Kung University , TAIWAN
The marine clam Meretrix petechialis is an important economic shellfish species in Northwestern Pacific, but little is known about its phylogeographical pattern. Here, we analyzed 311 samples from 22 locations along the northwestern Pacific using combined profiling of one mitochondrial gene (the first subunit of cytochrome coxidase, COI) and one nuclear DNA marker (the internal transcribed spacer region 1, ITS-1) to investigate contemporary genetic structure and reconstruct phylogenetic history of this species. The results revealed that two distinct phylogeographic lineages dominated marginal seasÐthe East China Sea (ECS) and the South China Sea (SCS) respectively. The estimation of divergence time between two lineages was 2.1±3.8 Ma, corresponding to a period of the early Pleistocene to late Pliocene. The vicariance of the two lineages was connected to the historical isolation of marginal seas and sea surface temperature (SST) gradient, pointing that SST might play an important role in maintaining phylogeographical patterns of M. petachialis. Significant overlaps between two lineages were observed in 23Ê to 29Ê N, located at the adjacent area of the ECS and SCS, which might be promoted by the connectivity of China Coast Current. However, the influence of ocean currents on mixings between two lineages was limited. In comparison, significant relationships were found between genetic distances and geographic distances if the North and South populations were analyzed separately, result of which might be due to some small reciprocal, rotating flows along coastal areas and special geographical conditions.
Competing interests: The authors have declared
that no competing interests exist.
Historical climatic changes in sea level, water temperature and ocean currents can have a
powerful impact on the distribution and genetic structure of the marine biota [
]. During the
Pleistocene, glacial-interglacial climate fluctuations resulted in falling and rising sea levels that
alternately exposed and submerged shallow seafloors , which in the end might have led to
genetic divergence and demographic expansion, respectively [
]. As an important marine
centre of origin, the Northwest Pacific , especially China Seas, have gained much attention
to understand the past global climate and marine biogeography. China Seas have a long
coastline across the temperate, subtropical and tropical climate zones. From the north to the south,
the number of species in China Seas increases distinctly [
]. China Seas were separated into
two marginal seas: the East China Sea (ECS) and the South China Sea (SCS), connected each
other by the Taiwan Strait (<100 m depth). During glacial epoch, according to Miller et al., the
major sea-level dropping 60 to 120 m started in Late Pliocene (~2.5 Ma ago) [
], so that land
bridges were formed between Asian continent and Taiwan Strait, which isolated two marginal
seas to make ECS and SCS serve as two independent glacial refugia [
]. During the
interglacial period, Taiwan Strait became flooded as the sea-level rose, and then the barrier of ECS and
SCS was disappeared. Thereafter, historical population demographic expansion might occur
in marine organisms inhabiting in the ECS and SCS.
Sea surface temperature (SST) is always an important factor governing dispersal range of
marine organisms [
]. Studies have shown that the range limits of some bivalve species were
mainly affected by SST gradients [
]. The intraspecific phylogeographic patterns
frequently correlate with interspecific biogeographic patterns in the Indo-Pacific or in other
marine regions [14±16]. As a barrier, thermal gradients in SST may limit the dispersal of
pelagic larvae, and it has been considered to be a plausible driver for the species diversification,
such as the flathead mullet Mugil cephalus in northwestern Pacific , the supralittoral isopod
Ligia occidentalis in North America [
], and the marine clam Lasaea australis in Australia
]. Furthermore, SST could be affected by sea level fluctuation. In glacial epoch, sea level
falling caused the closure of the Japan Sea, so that the warm Tsushima Current, a branch of the
Kuroshio Current was prevented from entering the Japan Sea, leading to rising temperature of
northern areas [
Ocean currents have an important influence on secondary contract by promoting larval
dispersal and enriching population connectivity [21±23]. In China Seas, two currents: the warmer
China Coastal Current (CCC) flowing from the SCS into the ECS through the Taiwan Strait,
which transports a large amount of warm-water marine species from their tropical center to
the north [
] and the colder Subei Coastal Current (SCC) flowing southward along the ECS
coast (Fig 1) in summer, which would potentially play a significant role in enhancing larval
dispersal in many marine species [24±26]. At the same time, West Korea Coastal Current
(WKCC) flowing southward along the Korean Peninsula through the Tsushima Strait enters
the Japan Sea. Generally speaking, pelagic larval stage of marine species vary from several days
to months, and an extended pelagic larval stage increases the opportunities for long-distance
dispersal in the marine realm and is often associated with little genetic differentiation over
large geographical distances [
]. However, genetic patterns are not simply related to pelagic
larval duration, but also compounded with other factors, e.g. fecundity, life histories and sea
temperature [28±30]. Furthermore, sea surface temperature can be affected by ocean currents.
Under the influence of warm currents, the water temperature is relatively high, while cold
currents make water temperature decrease [
]. To date, a small number of studies have been
conducted in shellfish to elucidate the phylogeographic patterns in the coastal areas of China,
including the bivalve Cyclina sinensis [
], Ruditapes philippinarum [
] and Tegillarca granosa
], rock shell Thais clavigera [
], the limpet Cellana toreuma [
]. However, the
phylogeographic patterns of most mollusk species have not been well characterized yet.
Meretrix petechialis is an important economic shellfish species, widely distributed along the
coastal areas of the northwestern Pacific, as far north as the Bohai Sea, and as far south as
Beibu Gulf. The water temperature in reproductive period for M. petechialis was 20±30ÊC [
This eurythermic clam inhabits the tidal flats, estuaries and sandy beaches [
] with a relative
short pelagic larvae stage duration of 5±8 days before settlement and metamorphoses. M.
petechialis was able to live in a salinity ranging from 17.3 to 28.4½ in incubation period, 9.2 to
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Fig 1. Map showing sample locations, spatial distribution of the two mitochondrial clades by pie diagrams for M. petechialis and
the coastal currents in summer. Sampling sites are labelled with numbers in Table 1. Two lineages are color-coded separately: Red,
North lineage; Yellow, South lineage. CCC, China Coastal Current; WKCC, West Korea Coastal Current; SCC, Subei Coastal Current. This
map is rendered with ODV v4.7.6 [
29.3½ in pelagic larval stage and 9.2 to 36.0½ in larval period, and was more successful in
lower salinity environments [
]. Culture of M. petechialis has been developed rapidly to meet
the huge demand of market, and consequently this clam had become one of the most
commercially maricultured bivalves in China. In order to ensure sustainable exploitation, management
and conservation must rely on population genetic structure and gene flow among populations
of the species . M. petechialis was often misidentified as M. meretrix. A previous study
reported that M. meretrix was only distributed in the South China Sea, while M. petechialis was
more widely distributed throughout the coasts of China [
]. Genetic surveys of M. petechialis
populations have been performed in China Seas [
], however, sampling locations were
limited in these previous studies and no accurate factors that affected the genetic population
structure were elucidated.
Mitochondrial DNA is well established to study population genetics and phylogeography
due to its comparatively fast rate of evolution and lack of recombination [
]. Because of ease
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of amplification using the polymerase chain reaction method and universal primers [
(the first subunit of cytochrome coxidase) is one of the most frequently used mitochondrial
genes and has been widely applied to a number of phylogeographical studies for marine species
including Ruditapes philippinarum [
], Cellana toreuma [
], the scyphozoan jellyfish Aurelia
] and the Rock Shell Thais clavigera [
]. MtDNA markers are maternally inherited,
representing only one evolutionary history [
]. Combining nuclear and mitochondrial DNA
markers can improve the power of molecular data to test phylogeographic hypotheses and
provide complementary information about population structure [
]. ITS1 gene (the internal
transcribed spacer region 1) is a common nuclear marker used with COI gene in
phylogeographic studies [
]. In the present study, we used COI and ITS1 markers to determine
phylogeographical pattern of M. petechialis populations in the northwestern Pacific.
This work aims to (1) understand the contemporary genetic structure of M. petechialis; (2)
calculate approximate divergence time between major lineages; (3) elucidate the factors that
may affect the genetic population structure.
Materials and methods
A total of 311 M. petechialis samples were collected from 22 locations along the coastal areas of
China, Korea and Vietnam (Fig 1) during 2006±2016 (Table 1), and were preserved in 95%
ethanol. Specific permit was not required to collect M. petechialis from these
locations/activities, because M. petechialis is not an endangered or protected species and were only collected
from public access areas.
DNA extraction, PCR amplification, cloning, and sequencing
Genomic DNA was extracted from the ethanol-preserved adductor muscle using an improved
phenol-chloroform procedure, which was described by Li et al [
]. A fragment of COI gene
was amplified for all samples, while a fragment of ITS gene was only amplified for partial
samples to verify the results of COI (Table 1). Polymerase chain reaction (PCR) was carried out in
a 50 μL volume containing 2 U Taq DNA polymerase, 100 ng template DNA, 1 μM each
primer, 0.2 mM of each dNTP, 1×PCR buffer, 2 mM MgCl2, and 4% DMSO. For a fragment of
COI gene amplifications, we used the primers: LCO1490 5’-ATTATTCAGAACCAATCA
TAAAGATATTGG-3’ and HCO2198 5’-TGTAGGAATAGCAATAATAAAAGTTAC-3’ [
For a fragment of ITS gene amplifications, we used the primers: PEF-10 5’-TAGAGGAAG
GAGAAGTCGTAACAAGG-3’ and 5.8R 5’-CAAKRTGCGTTCRARRTGTCGATGWTCA-3’
]. PCR was performed in a GeneAmp1 9700 PCR System (Applied Biosystems, Foster City,
CA, USA) under the following conditions: an initial denaturation for 3 min at 94ÊC, followed
by 35 cycles of 40 s at 94ÊC, 40 s at 48ÊC in COI or 40ÊC in ITS, 40s at 72ÊC, and completed
with a final extension for 5 min at 72ÊC.
For COI, PCR products were directly sequenced using respective primers on an ABI 3730
automated sequencer. For ITS, PCR products were purified by DNA gel extraction kit
UNIQ10 following the recommended protocol, then were cloned into cloning vector pEASY-T1
(TaKaRa). For each individual, we selected two to five clones to sequence by M13 primers.
Sequences of COI and ITS were assembled and checked by SeqMan in DNASTAR software.
The consensus sequences were then aligned with BioEdit [
] using ClustalW [
default settings. Sequences were cut into the same length using MEGA 6.0 [
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] was used to detect genetic recombination by the default parameter settings within
ITS sequences. Haplotypes of sequences were determined with the software program DnaSP
], and sites with gaps/missing in the ITS-1 sequences were considered. All sequences of
haplotypes were deposited in GenBank database (Accession numbers: KY318078-KY318174,
KY318176-KY318461). The program Arlequin 3.5 [
] was used to analyze molecular diversity
indices including haplotype diversity (h), nucleotide diversity (P), and mean number of
pairwise differences (k). For the net average genetic distance between major lineages, the Kimura
2-parameter (K2P) model [
], pairwise deletion of gaps data, and ªTransitions +
Transversionsº options were used to calculate in MEGA 6.0 [
The neighbor-joining (NJ) tree with bootstrap analysis (1000 pseudoreplicates) was
constructed using MEGA 6.0, and parameters setting was same as distance analyses. Bayesian
inference (BI) was performed in MrBayes 3.2.0 [
], in which the Markov-chain Monte Carlo
(MCMC) search was run with four chains for 50 million generations with a sampling
frequency of 1/1000 trees. Substitution models were inferred using jModeltest 0.1.1 [
] using the
Akaike information criterion (AIC). M. lamarckii (KY318175) was used as outgroup.
Genetic diversity and population structure
Hierarchical analyses of molecular variance (AMOVA) [
] were performed using Arlequin
3.5 on both COI and ITS data set to evaluate population structure. The variance components,
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sum of squares and F statistics were calculated between lineages, among populations within
lineages and within populations, respectively. The significance of F-statistic analogs was
evaluated with 10000 random permutations, and the Tamura-Nei model was selected to correct for
multiple substitutions because the HKY+I model is not implemented in Arlequin. Haplotype
network showing the genetic relationships was generated with PopART 1.7 [
] at the
provincial level based on COI gene.
Pairwise FST values among populations within lineages were evaluated using COI gene by
Arlequin 3.5, and the statistical significance was tested by 10000 random permutations. Mantel
tests to test the relationship between genetic distances and geographic distances were
performed on the Isolation by Distance Web Service (IBDWS at http://ibdws.sdsu.edu/~ibdws/)
] with 10,000 permutations using COI sequences.
To infer if there had been a past population expansion or selective sweep of M. petechialis,
Fu's Fs [
] and Tajima's D [
] statistics along with their statistical significance (1000
permutations) were carried out using DnaSP v5. For the purposes of eliminating the possibility of
a selective sweep, the McDonald-Kreitman test [
] was conducted in DnaSP v5 using M.
lusoria as outgroup species. Because ITS-1 was not a protein-coding gene, only COI sequences
were analyzed for the McDonald-Kreitman test. In cases where expansion was evident based
on neutrality statistics, statistics in mismatch distributions were performed in DnaSPv5 to test
whether a sudden population expansion occurred in the history. Sum of squared deviation
] and Harpending's raggedness index (RI) [
] were calculated in Arlequin 3.5.
Divergence time estimation
Divergence time between the major distinct lineages was estimated in Beast v1.7.5 software
] only on the more stable COI marker with a calibrated molecular clock and HKY+I
models. We supposed an uncorrelated lognormal distribution for the clock and speciation: Yule
Process as the tree prior. Analyses were run for 10 million iterations and sampled every 1000
iterations with a final burn-in of 10%. The lack of a fossil record for M. petechialis precludes an
estimate of a species-specific molecular clock, which will influence any estimates of divergence
time. Molecular clocks have been estimated for the COI gene in several bivalves such as Cyclina
sinensis (0.7±2.4%/Myr) [
], Ruditapes philippinarum (0.9±3.35%/Myr) [
granosa (2±2.4%/Myr) [
] and Atrina pectinata (2.4%/Myr) [
], but the rates vary widely. In our
study, we used molluscan specific mtCOI divergence rates calibrated for bivalve (1%/Myr) [
] and gastropoda (2%/Myr) [
] to estimate the divergence time between two lineages.
Although large variance might exist in the divergence rate of 1%/Myr to 2%/Myr for M.
petechialis, the rate was used to estimate a rough time for elucidating phylogeographic hypotheses.
The mean substitution rates defined in our analyses were obtained by dividing the mean
sequence divergence rates by two [
]. Convergence diagnostics were conducted in Tracer
] and the effective sample size (ESS) for each parameter exceeded 200. TreeAnnotater
v1.7.5 was used to generate the maximum credibility tree.
The aligned COI and ITS-1 gene sequences were obtained with lengths of 747 bp and 860 bp,
respectively. The obtained 305 COI gene sequences without indels contained 112 variable
sites and 68 parsimony informative sites, yielding 97 haplotypes, of which 72 (74.2%)
haplotypes were singletons, being represented by a single sequence in the sample. Wenzhou
population showed the highest values of nucleotide diversity (0.01339 ± 0.00701) and mean
number of pairwise differences (9.99900 ± 4.70302) because lineage overlap was observed in
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this population. Overall values of COI gene for haplotype and nucleotide diversities were
0.9483 ± 0.0054 and 0.03364 ± 0.01638, respectively. The obtained 345 ITS-1 gene sequences
contained 194 variable sites and 65 parsimony informative sites yielding 286 haplotypes, in
which 260 (90.9%) haplotypes were singletons. Overall values of ITS-1 gene for haplotype and
nucleotide diversities were 0.9968 ± 0.0009 and 0.00734 ± 0.00386. No genetic recombination
was detected within ITS-1 sequences. Overall, values of haplotype diversity, nucleotide
diversity and mean number of pairwise differences for ITS-1 gene were higher than those of COI
gene (Table 1).
Phylogenetic reconstruction with the COI gene based on NJ and BI methods separated M.
petechialis into two lineages with high nodal support (Fig 2), which were identified as North
and South lineages, each corresponding to one of the biogeographic areas. North lineage
included samples from Bohai Sea, Yellow Sea and East China Sea, while samples of South
lineage were from East China Sea and South China Sea. The overlap zone was observed in
Zhejiang and Fujian provinces. But no obvious difference was found between two lineages in
phylogenetic reconstruction with the ITS-1 gene. The net average genetic distance (± SD)
between lineages was 5.85 ± 0.95% for COI and 0.08 ± 0.03% for ITS-1.
Genetic diversity and population structure
When populations were grouped by sea basins, for COI gene, hierarchical analyses of
AMOVA (Table 2) indicated that the most of the total genetic variation (93.22%) was from
Fig 2. Phylogenetic hypothesis based on COI sequences represented by Bayesian inference method. Bootstrap values were
represented near the branches (BI and NJ bootstrap values, respectively).
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differences between two lineages (P < 0.01). A smaller (5.98%, P<0.01) amount of genetic
diversity occurred within populations, and the smallest (0.80%), yet significant (P < 0.01), of
the total variation was present among populations within lineages. For ITS-1 gene, the result
showed that the most of the total genetic variation (84.20%, P<0.01) was apportioned within
populations, followed by a smaller (9.00%, P<0.01) amount occurred between groups, and
only a small part (6.81%, P<0.01) among populations within groups.
Based on the COI, two haplogroups were formed in the network (Fig 3), which was
consistent with the BI and NJ trees. The topology of haplotype network was characterized by multiple
star-like type. Significant overlaps between two lineages were observed in 23Ê to 29Ê N, where
is located at the adjacent area of the ECS and SCS.
Pairwise FST values based on COI sequences among populations within lineages were
shown in Tables 3 and 4. For North lineages, Changyi and Weihai populations were found
significantly different from others except for Busan. For South lineages, Xuwen, Qishui, Qinzhou
and Haiphong populations located in Beibu Gulf, were remarkably different from Wenzhou,
Xiamen, Shantou and Zhanjiang populations. The IBD analysis displayed a significant
Fig 3. Network of M. petechialis using COI data. The pie charts refer to the haplotypes. B&YS: Bohai and Yellow Sea; ECS: East China
Sea; SCS: South China Sea.
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relationship between genetic distances (FST) and geographic distances (Fig 4. All populations:
r = 0.6928, p<0.0001; North populations: r = 0.3321, p = 0.0381; South populations: r = 0.2977,
p = 0.0395).
The McDonald and Kreitman test demonstrated no significant difference (NI = 1.329,
Fisher' s exact test P-value = 0.764), ruling out the possible of the selective sweep. Estimates of
neutral tests for two lineages conducted on both COI and ITS data indicated population
expansion by significant negative values of Fu' Fs and Tajima's D statistics (Table 5). The
observed mismatch distributions of two lineages based on COI and ITS data (except for
Southern lineage on COI) uniformly displayed unimodal distributions, which fit well with the model
of sudden expansion (Fig 5). The RI and SSD for two lineages all matched the null hypothesis
of sudden expansion model with nonsignificant values (Table 5). Via an inspection of the
network of lineage south (Fig 3), two haplogroups could lead to the bimodal distribution.
Divergence time estimation
Results of the COI divergence time estimations for M. petechialis are shown in Fig 6. The
divergence age estimate between North and South lineages was approximately 2.1±3.8 Ma,
Fig 4. Relationship between genetic vs. geographical distance (log-transformed) in M. petechialis. A,
all China populations except Wenzhou; B, the North populations; C, the South populations.
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corresponding to the early Pleistocene to late Pliocene. The time of origin of North and South
lineages were about 0.7±1.2 Ma and 0.8±1.5 Ma respectively, dating back to Pleistocene.
Based on the network relationships and phylogenetic reconstruction for COI markers,
significant population differentiation were observed between northern (ECS) and southern groups
(SCS), and overlapped at the adjacent area of the ECS and SCS (Fig 1). In addition, genetic
differentiation between two lineages accounted for a large proportion according to the AMOVA
analysis. Therefore, we concluded that phylogeographical pattern of M. petechialis was
connected to the historical isolation of marginal seas, which is consistent with the observations in
Fig 5. Mismatch distributions for two lineages. Dotted lines with circles represent the observed frequency of pairwise differences,
whereas the solid lines show the expected values under the sudden population expansion model.
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Fig 6. BEAST divergence time estimation of M. petechialis using calibrated molecular clocks method. Divergence rates: A, 1%/Myr;
B, 2%/Myr. Node ages were represented near the branches. Blue bars on branch nodes indicate 95% Highest Posterior Density age
intervals. Timescales in million years before present (Ma).
mitten crab Eriocheir sensu stricto [
], marine fish (Chelon haematocheilus [
Bostrychus sinensis [
]) and bivalves (Cyclina sinensis [
] and Tegillarca granosa [
divergence time between two lineages dated back to 2.1±3.8 Ma, generally near to early glaciations
intensified time and the start time that sea level major drops 60 to 120 m (2.5 Ma) [
Because of the sea level falling, ECS and SCS were isolated, so a physical barrier was created for
M. petechialis, giving rise to divergent lineages. The divergence time in our study is similar
with two subspecies of oyster (2.0±3.6 Ma) [
] and two species in the mitten crab (2.24 Ma)
. Above all, in the northwestern Pacific region, historical glaciation during Pleistocene
played an effective role in generating intraspecific genetic divergences [
]. In postglacial
period, sea level rising and land bridge disappearing contributed to rapid population
expansion of intertidal species and secondary contact at the adjacent areas [
]. The signal of
the demographic expansion of M. petechialis was also detected through star-like type of
network topology, mismatch distribution analysis and neutrality tests.
One major cooling period was occurred in the 2.2±1.0 Ma, which are reflected in the
increasing SST gradient in the subtropical area, where SST cooled significantly (decreased by
3.3±5.4ÊC and 1.0±2.1ÊC in winter and summer respectively), while SST showed little or no
cooling (decreased by 0.9ÊC and 0.6ÊC in winter and summer respectively) in the tropical
western Pacific . SST gradient between the ECS located in the subtropical area and the SCS
located in the tropical area might lead to genetic divergence combined with glacial isolation.
Furthermore, SST possibly plays an important role in maintaining phylogeographical pattern
of M. petechialis. Firstly, the boundary of two M. petechialis groups is approximately near to
Taiwan Strait. In the northwestern Pacific, the Tropic of Cancer is the boundary between the
warm-temperate and tropical regions, and 20ÊC isotherm of annual mean temperature
running through the Taiwan Strait, which is a major biogeographic boundary for many species
]. Secondly, analogous to two lineages of Mugil cephalus [
], the distribution range of
northern group is consistent with gradients of the cold waters from the Subei Coastal Current
(SCC) flowing southward along the ECS coast, while the southern group appeared be restricted
to the warm water of the China Coastal Current (CCC) flowing from the SCS into the ECS.
Thirdly, because of the temperature difference from the north to south, northern populations
have a later reproduction period. These phenomena indicated that SST might limit the
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dispersal of M. petechialis. Moreover, from 1982, SST have continued to rise [
will potentially promote species to migrate northward along Chinese coastline .
Similarly, Tegillarca granosa and Cyclina sinensis, plankton larval duration of M. petechialis
is about 5±8 days, however, M. petechialis migrates from mid-tidal region to low-tidal region
along with tidal currents extending from late May to late June and mid-September to
late-September. This specific feature may increase opportunity for gene flow among different
populations. During the breeding season of M. petechialis (spring and summer), the China Coast
Current with a common velocity of 20 cm/s [
] flowing northward, transported pelagic larvae
from SCS to ECS. Overlapping phenomenon was found at the adjacent area of the ECS and
SCS (23Ê to 29Ê N) because of secondary contact, which might be promoted by the
connectivity of China Coast Current [
]. Significant relationship was found between genetic distances
(FST) and geographic distances when the North and South populations were analyzed
respectively, after fitting IBD model. In addition, some pairwise FST values were significant between
populations within each lineage. For North lineage, the genetic difference between some Bohai
Sea populations (Changyi and Weihai) and East Sea populations were statistically significant.
For South lineage, some populations located in Beibu Gulf (Xuwen, Qishui, Qinzhou and
Haiphong) were significantly divergent from Wenzhou, Xiamen, Shantou and Zhanjiang
populations. This is most likely attributed to the low-dispersal ability of the clam and the existence of
small but persistent reciprocal flow and rotating flow along coastal areas, which impede the
exposure of larvae to currents and their ability to be transported effectively [
He et al.'s [
]study indicates that the South China Sea should be considered to be a
colonization origin of Periophthalmus modestus because of an older coalescence time, a putative
ancestral haplotype, higher genetic diversity, more proportion of private haplotypes and higher
species diversity of the genus Periophthalmus. Similarly, we proposed that South China Sea is
the earlier refuge for M. petechialis. First of all, north lineage originated from refugium in the
Okinawa Trough about 0.7 Ma, whereas South lineage derived from refugium in South China
Sea about 0.8 Ma. Although they are approximate times, the relative coalescence times of two
populations are reasonable. Moreover, the portion of private haplotypes from South lineage
(75.9%) was higher than that from North lineage (72.1%), which supported the South China
Sea was the colonization origin [
]. In addition, haplotype and nucleotide diversity (h:
0.8996, P: 0.00543) of South lineage had higher values compared with Northern lineage (h:
0.8939, P: 0.00274). Generally speaking, older colonized regions are expected to exhibit higher
genetic diversity due to the longer evolutionary history [
]. Finally, although the bivalve
genus Meretrix Lamarck, 1799 are broadly distributed in the West Pacific and the Indian
Ocean, M. petechialis is the only Meretrix species in the ECS regions.
The discrepancy between mitochondrial and nuclear topologies
MtCOI gene analysis separated M. petechialis into two lineages, while nrITS data showed a
slightly different topology. The net average genetic distance (± SD) between lineages was
5.85 ± 0.95% for COI, but only 0.08 ± 0.03% for ITS. Result of AMOVA also displayed that
nrITS were not significantly differentiated between groups. This inconsistent pattern has also
been found in black-throated tits Aegithalos concinnus [
] and the pen shell Atrina pectinata
]. A large proportion of cases that discordance between mitochondrial and nuclear data
likely appeared following geographic isolation and secondary contact, which attributed to
incomplete lineage sorting and post-glacial introgression, respectively [
]. However, they are
difficult to be distinguished [
]. COI gene will have a four times faster coalescence time than
that of ITS gene [
], because this rate is inversely proportional to the effective population size
] (a fourfold smaller effective population size [
]). In our study, the divergence time
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between two lineages dates back to a relatively young divergence (Pleistocene). It is reasonable
that ITS gene keep homogeneous and will have longer time to spit, while COI gene have
achieved coalescent. Furthermore, under incomplete lineage sorting, no discernible
biogeographic pattern among populations is expected to be found [
]. In our study, phylogenetic
reconstruction with the COI gene separated M. petechialis into two lineages, but no obvious
difference and discernible geographic pattern was found between the two lineages using the
ITS gene. We also found samples from different mtDNA lineages shared the same haplotype
for ITS gene, for example, samples from Busan, Beihai and Caotan shared the same haplotype.
Therefore, incomplete lineage sorting is more likely to be the reason of the discepancy between
mitochondrial and nuclear topologies.
To better elucidate phylogeographic pattern of M. petechialis, we sequenced the COI and ITS1
genes from 311 individuals sampled from 22 locations along the coastal areas of China, Korea
and Vietnam. The results provided evidence for strong genetic divergence between two
lineages, which might be attributed to variance of sea level changes and SST gradients. Here, the
combinatory use of nuclear ITS gene and the mitochondrial COI gene helped elucidate the
phylogeographical pattern of M. petechialis, such as population structure and population
expansion analyses. With the objectives of getting more detailed information about
phylogeography of M. petechialis with higher genetic resolution, analyses with multiple nuclear genes
would be conducted.
Many thanks to Dr. Tzen-Yuh Chiang, Dr. Adam TomaÏsovyÂch, Dr. Haifeng Geng, Dr. Shikai
Liu, Dr. Yuanning Li and three other anonymous reviewers for their constructive comments
which undoubtedly improved this manuscript.
Conceptualization: Lingfeng Kong, Qi Li.
Formal analysis: Xiaoxuan Wang, Jun Chen.
Funding acquisition: Lingfeng Kong.
Investigation: Xiaoxuan Wang, Jun Chen.
Project administration: Lingfeng Kong.
Data curation: Xiaoxuan Wang, Lingfeng Kong, Akihiko Matsukuma, Qi Li.
Writing ± original draft: Xiaoxuan Wang, Lingfeng Kong.
Writing ± review & editing: Xiaoxuan Wang, Lingfeng Kong, Akihiko Matsukuma, Qi Li.
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