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Comparative genetic characterization of ark shell Scapharca broughtonii in northeast Asia.

ABSTRACT Ark shell Scapharca broughtonii is one of the most commercially important bivalve resources and is widely distributed in northeastern Asia. The aim of this study was to determine its genetic structure in Japan, China, and Korea by means of nucleotide sequence analysis of a 507-bp portion of the mitochondrial DNA cytochrome c oxidase subunit I gene. Of 180 individuals collected from three localities in Japan and one locality each from China and Korea, a total of 66 haplotypes were found on the basis of 56 variable nucleotides. Although Japan and Korea populations shared six common haplotypes, there was no common haplotype between Japan/Korea and China. The pairwise [F.sub.ST] and genetic distance values were comprehensively high, with significant differences between Japan/Korea and China, indicating that the divergence had reached subspecies level. In conclusion, haphazard transplantation and import of seedlings and/or adults of S. broughtonii and its related species should be absolutely avoided as S. broughtonii had previously shown distinct genetic differences between Japan/Korea and China.

KEY WORDS: ark shell, genetic characterization, mitochondrial DNA, COI gene, Scapharca broughtonii

INTRODUCTION

Ark shell Scapharca broughtonii belongs to the family Arcidae inhabiting the muddy sediments of shallow coastal waters. This species is widely distributed along the northwestern Pacific Coast and is one of the most commercially important bivalves in northeast Asia, particularly in Japan, China, and Korea (Qi 1998, Liu et al. 2013). In Japan, S. broughtonii has been a target species of the dredge fishery and for aquaculture and stock enhancement via release of hatchery-reared juveniles (Sugiura et al. 2014). In recent decades, the catch yields of ark shell declined in most local fishing grounds in Japan including Miyagi and Ishikawa (Sugiura et al. 2014). In China, this species is one of the dominant species along the coast of Shandong and Liaoning provinces and has also been a fishery target species, particularly in the Yellow Sea. Its average density in Shandong Province, however, decreased by approximately 98% from 1980 to 2010 (Wang & Wang 2008). In Korea, this species is generally found along the eastern and southern coasts and is one of the major aquaculture species (An & Park 2005). Its production rate, however, declined due to repeated annual cultivation and environmental pollution (Park et al. 1998). In Japan, China, and Korea, the natural resources of S. broughtonii have precipitously declined across its main habitats in the past several decades due to overexploitation and deterioration of environmental conditions (Yu et al. 2015). The decline of ark shell has prompted several restoration strategies, such as artificial breeding programs and fishing area protection (Yu et al. 2015). In addition, in an effort to promote catch and stock increases, ark shell and its seedlings were imported from China and released into the fishing areas in Japan (Yokogawa 1997). It is noteworthy that ark shell imported from China and/ or Korea occupied more than 90% of its total amount of production in Japan. In Korea, to enhance production, ark shell seedlings were also imported from China and Japan. Thus, this gave rise to a mixture of domestic and foreign populations (An & Park 2005).

The potential for genetic exchange has recently increased among northeast Asian ark shell populations due to commercial purposes and the uncontrolled bulk release of nonnative genetic variants. This could significantly alter the genetic and ecological variety of wild populations by displacement and/or interbreeding (An & Park 2005). Therefore, a basic understanding of genetic differences among the geographically distinct populations is required. Moreover, studies using genetic markers of population genetics may play an important role in conservation of the native genetic pool and selection for genetic improvement in the future (Kitada et al. 1998, Aritaki 2013). A few preceding studies of Scapharca broughtonii using genetic markers have been reported (Cho et al. 2007, Yu et al. 2015). Cho et al. (2007) examined its population genetic structure based on the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I gene (COT) among Korea, China, and Russia, and reported a significantly different genetic structure between Korea and China/ Russia populations. Yu et al. (2015) studied genetic diversity along the coast of China based on microsatellite DNA and reported a significant differentiation between the north and south populations of the Shandong Peninsula. Unfortunately, the general genetic relationship and variability among Japan, China, and Korea remain poorly understood.

With the advent of advanced molecular methods, genetic data now play an important role in stock management and assessment of marine organisms (Avise 1994). Data on the genetic relationships and genetic variability are of particular interest because Scapharca broughtonii may reveal molecular evidence of gene flow and/or genetic isolation. The mtDNA COI gene has often been adopted as a tool for determining the genetic relationships and variability of bivalves valuable to fisheries in Japan (Mito & Aranishi 2010, Tanaka & Aranishi 2013, 2014). The aim of the current study was to evaluate the genetic diversity and genetic structure among five wild populations of ark shell collected from three localities in Japan and one locality each from Korea and China.

MATERIALS AND METHODS

Sample Collection

A total of 180 individuals were collected at the following five localities from 2011 to 2012: Miyagi Prefecture in Japan (n = 37); Ishikawa Prefecture in Japan (n = 34); and Aichi Prefecture in Japan (n = 35), Korea (n = 35), and China (n = 39). Sampling locations and sample sizes were shown in Figure 1 and listed in Table 1. All specimens were identified to be Scapharca broughtonii using phylogenetic analysis (Tanaka & Aranishi 2013). The adductor muscle of each individual was removed and immediately stored at -20[degrees]C until required.

DNA Extraction

High-quality total genomic DNA was prepared from small scraps of frozen adductor muscle of foot according to the modified urea-SDS-proteinase K method (Aranishi & Okimoto 2004, 2005, Aranishi 2006). Samples were incubated in the extraction buffer (10 mM Tris-HCl, pH 7.5, 20 mM EDTA, pH 8.0, 1% SDS, and 4 M urea) containing 25 [micro]g proteinase K at 55[degrees]C for 60 min, following which 5 M NaCl was added and mixed. DNA was isolated with phenol-chloroform-isoamyl alcohol and subsequent chloroform-isoamyl alcohol, followed by precipitation with ethanol. DNA pellets were washed with ethanol, dried, and resuspended in 10T0.1E (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, pH 8.0).

Polymerase Chain Reaction Amplification and Sequencing

Polymerase chain reaction (PCR) amplification of mtDNA CO I gene was performed in GoTaq Green PCR Master Mix (Promega, Madison, WI), containing 2 mM Mg[Cl.sub.2], 0.5 [micro]M of each primer, and 20 ng of template DNA in a Techgene thermal cycler (Techne). The primers used for PCR were L 5'-GGTGT GTGTT TAAGA TTTCA CA-3' (Lee & Kim 2003) and HC02198 5'-TAAAC TTCAG GGTGA CCAAA AAATC A-3' (Folmer et al. 1994). The PCR protocol consisted of an initial denaturation at 95[degrees]C for 2 min, followed by 40 cycles of 10 sec at 95[degrees]C, 20 sec at 50[degrees]C, and 40 sec at 72[degrees]C, and a final extension at 72[degrees]C for 5 min. The PCR products were analyzed using a DNA-1000 Reagent Kit (Shimadzu) containing a SYBR Gold Nucleic Acid Gel Stain (Invitrogen) in an MCE-202 MultiNA microchip electrophoresis system (Shimadzu). Nucleotide sequencing of double strands of PCR product was obtained using a BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) in an automated 3730x/ DNA Analyzer (Applied Biosystems).

Data Analysis

Sequences were edited with Sequence Scanner v1.0 (Applied Biosystems) and then aligned with the CLUSTAL W (Thompson et al. 1994) using MEGA 5.5 (Tamura et al. 2011). Haplotype diversity (h) measured the probability that two randomly chosen haplotypes were different, and nucleotide diversity ([pi]) was the mean number of nucleotide differences between all pairs of haplotypes. The degree of genetic diversity was estimated for each population using Arlequin 3.5.1.3 (Excoffier & Lischer 2010).

The following phylogenetic analysis, analysis of molecular variance (AMOVA), and pairwise [F.sub.ST] analysis were conducted to better understand population structure. Phylogenetic analyses among congeneric species and haplotypes were reconstructed using the neighbor-joining (NJ) method based on the Kimura two-parameter model (K2P; Kimura 1980) using bootstrap analysis with 10,000 replicates implemented in MEGA 5.5. The homologous sequences of two species such as Scapharca broughtonii (AB729113, AY040551, and HQ258854) and Scapharca satowi (AB050898, AB690347, and AY040552) were available from the GenBank database. The genetic structure was further investigated using AMOVA to define groups of proximal populations using Arlequin 3.5.1.3. The purpose of an AMOVA is to infer population genetic structure between predefined groups of individuals. This is accomplished by partitioning the total genetic variance into multiple components: the amount of variation that can be explained by (1) differences within individuals, (2) differences among individuals within a group, and (3) differences among groups. Although the approaches are different, the phylogenetic analysis and AMOYA address the same question. Genetic differentiation between populations was assessed by comparing the pairwise [F.sub.ST] and the genetic distances with K2P using Arlequin 3.5.1.3 and MEGA 5.5, respectively. These are different approaches to answer the question of genetic structure in S. broughtonii.

The null hypothesis of neutral evolution of the CO I gene was tested using Fu's [F.sub.S] (Fu 1997) with Arlequin 3.5.1.3. Mismatch distribution can be used to test the hypotheses regarding the population demographic history and selection (Rogers & Harpending 1992). The observed distribution was tested for goodness of fit to simulated values of sudden population expansion using parametric bootstrapping with 10,000 replicates in Arlequin 3.5.1.3. The significances of the sum of squared differences and Harpending's raggedness index, which measures the smoothness of the mismatch distribution, were also calculated in Arlequin 3.5.1.3.

RESULTS

After editing, nucleotide sequences of PCR product encoding the mtDNA COI gene of 507-bp sequence from 180 individuals across 5 populations were aligned, and 66 haplotypes were detected. The h values ranged from 0.7664 [+ or -] 0.0767 in Korea to 0.8889 [+ or -] 0.0379 in Miyagi, and the [pi] values ranged from 0.3351% [+ or -] 0.2234% in Ishikawa to 0.4727% [+ or -] 0.2921% in Miyagi. These results suggested relatively high levels of genetic diversity. In particular, Miyagi in Japan showed the highest estimates of maximum h and [pi] values compared with other populations (Table 1). Among haplotypes, 56 variable sites were presents and no insertions or deletions were obtained. Of the 66 detected haplotypes identified to be Scapharca broughtonii (Fig. 2), 59 haplotypes were unique to individual populations, and the remaining 7 haplotypes were common to Japan and Korea. No haplotypes were shared between Japan/Korea and China (Table 2).

The NJ tree constructed using the complete data set of 66 haplotypes, identified two distinct lineages A and B supported by high bootstrap values. It is clear from Figure 2 that there are two distinct lineages within Scapharca broughtonii. Lineage A consisted of HT01 through HT49 and A. broughtonii sequences from Japan and Korea. Lineage B consisted of HT50-66, all haplotypes unique to China, as well as S. broughtonii from China and Scapharca satowi from Korea. Lee and Kim (2003) reported that the nucleotide sequences of unidentified ark shell imported from China were identical to 5. satowi of Korea. Therefore, ark shell classification in China and/or Korea remains problematic, and taxonomic statuses might not have been adequately resolved.

The AMOVA of the data when no structure was specified a priori showed that the majority of the genetic variance (84.22%, P = 0.0000) can be explained by differences between populations, whereas only 15.78% of the variance can be explained by differences among individuals within populations. When the populations were subdivided into two groups representing lineages A and B, the AMOVA showed that the majority of variation in the data (91.92%, P < 0.0000) was explained by differences between the two groups (Table 3). The amount of genetic diversity among four populations from Japan and Korea was 10.67%. Pairwise [F.sub.ST] values between five populations ranged from -0.00652 to 0.2123, little genetic differentiation ([F.sub.ST] < 0.05) was detected among Japan and Korea populations. Great differentiation was, however, observed between Japan/Korea and China. The highest differentiation was observed between Korea and China populations (Table 4). These results indicated that all analyses (NJ tree, AMOVA, and [F.sub.ST]) detected the same pattern of genetic structure, which was realistic and robust.

None of the mismatch distributions for the two lineages A and B deviated significantly from expectations under the sudden expansion model (Fig. 3), therefore, the analysis of demographic patterns was suitable (Table 5). The mismatch distributions for all individuals were multimodal, suggesting that these populations have not remained stationary since the time of coalescence of the haplotypes (Fig. 3). Among the different methods used, the Fu's [F.sub.S] test based on h showed deviation from neutral expectation in ark shell populations (P < 0.01). Fu's [F.sub.S] values were all negative and significantly deviated from a natural state (P <0.01) as well as were negative and significant in all populations (P < 0.01). The significance of Fu's [F.sub.S] values in ark shell could be explained by a recent population expansion (positive selection) (Fu 1997).

DISCUSSION

The present study thoroughly compared genetic diversity within and genetic differentiation among five wild populations of Scapharca broughtonii collected from three localities in Japan and one locality from Korea and China, respectively.

A total of 66 haplotypes from 180 individuals were observed, with no haplotype shared between Japan/Korea and China (Table 2). Based on both haplotype and nucleotide diversities, all populations showed a comparable level of genetic diversity (Table 1). No significant genetic differentiation was detected between Japan and Korea populations through phylogenetic analysis, AMOVA, and pairwise [F.sub.ST] analysis. This suggested that Japan and Korea were to some degree genetically homogeneous. In addition, genetic differences between Japan/Korea and China were confirmed in all three analyses, thereby strengthening the suggestion that Japan/Korea and China were genetically distinct.

Cho et al. (2007) determined that the all Korea populations were not indistinguishable among one another but from China and Russia populations, thereby suggesting that geographic barriers to larval dispersal separated Korea from China/Russia. Yu et al. (2015) noted that the Shandong Peninsula was a putative barrier separating the northern and southern population of Scapharca broughtonii and, furthermore, that larval dispersal driven by ocean currents might block gene flow between the northern and southern populations. These results illustrated the strong barrier to gene flow between Japan/Korea and China, and indicated that the Korean peninsula and both the Kuroshio and Tsushima Currents might act as a restriction for the gene flow. During 23 million years (Myr) ago and 5 Myr during the Miocene, the Korean peninsula was separated from China by the opening of the Bohai Sea and was connected with Japan (Omori & Isozaki 2011). Korea subsequently attained its present position due to crustal movement (Liu et al. 2014). Moreover, the coasts around Japan, China, and Korea are strongly affected by the Kuroshio and Tsushima warm currents (Fig. 1).

Net average genetic distances, based on K2P, between Japan/Korea and China, ranged between 4.75% and 4.68% (Table 4). By applying both the mtDNA COI sequence mutation rate of 0.6%/Myr (Baldwin et al. 1996) and Nei's formula (Nei 1975), it was indicated that Japan/Korea and China would have split approximately 4 Myr during the Pliocene. In addition, the Tsushima Current flowed into the Japan Sea at 2.4-1.72 Myr, and the southern channel opened (Kitamura & Kimoto 2004). Therefore, it was estimated that genetic differentiation of Scapharca broughtonii occurred at approximately 4 Myr, with the species dispersing around Japan, China, and Korea via the Tsushima Current after 2.4-1.72 Myr. Consequently, these results guessed that China had a different geographic origin from Japan and Korea.

The mismatch distributions for lineages A and B were clearly unimodal, none of the goodness fit test deviated significantly from expectations under the sudden expansion model, and the Fu's [F.sub.S] values were significantly negative (P < 0.02; Fig. 3, Table 5). These results demonstrated that all populations fit with the population expansion model, indicating the maintenance of the independent genetic structures in Japan/Korea and China after population expansion.

According to the genetic distance, the values between Japan/Korea and China ranged from 0.047 to 0.048 (Table 4). Feng et al. (2011) found that the mean genetic distance between Japan and China was 0.042 based on the mtDNA COI gene; these observations demonstrated that genetic divergence existed. In addition, the genetic distance values among related Scapharca species were smaller than the value obtained in the present study, such as 0.032 between Scapharca kagoshimensis and Scapharca inaequivalvis, 0.043 between S. kagoshimensis and Scapharca globosa ursus, and 0.045 between S. globosa ursus and S. inaequivalvis (Tanaka & Aranishi 2013). Therefore, the genetic difference between Japan/Korea and China indicated that the divergence reached subspecies or species level.

In general, it is very important to assess the genetic variability and stock structure of wild populations before starting largescale stock enhancement programs because released individuals can negatively affect wild populations through genetic contamination and induction of premature migration (Nohara et al. 2010). All the results from the present study definitely indicate little effect of past transplantation to the current genetic diversity and maintenance of the independent genetic structures of Scapharca broughtonii. A long dispersal and larval duration may lower the chances of survival and efficiency. Furthermore, when the larvae passively drift over a long distance, they must overcome environmental differences to successfully become mature ark shells. Therefore, it was proposed that S. broughtonii populations should be managed and conserved separately. Moreover, special attention must be paid to habitat protection and pollution control as this species is susceptible to changes in their appropriate habitat.

CONCLUSION

The present study compared population genetic structure of Scapharca broughtonii among five wild populations collected from three localities in Japan and one locality from Korea and China, respectively, by means of nucleotide sequence analysis of a 507-bp portion of the mtDNA COI gene. Its genetic structure has arisen specifically between Japan/Korea and China examined in the present study. Besides ark shell, however, many other kinds of fisheries resources have been vigorously introduced into Japan from foreign countries for fisheries and aquaculture (Yokogawa 1997). Haphazard transplantation and import of seedlings and/or adults of Scapharca broughtonii and its related species should be absolutely avoided not only to conserve S. broughtonii as a genetic resource but also to manage them as a fisheries stock.

ACKNOWLEDGMENTS

We would like to thank Dr. Tadahide Kurokawa (Tohoku National Fisheries Research Institute, Fisheries Research Agency) and Dr. Kei Senbokuya (Ishikawa Prefecture Fisheries Research Center) for providing ark shell individuals. This research was supported in part by Grant-in-Aid for JSPS Fellows (grant number 24-2801), to TT and Strategic Research Development Grant from Shimane University to FA.

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TOMOMI TANAKA (1,2) AND FUTOSHI ARANISHI (1,2) *

(1) Coastal Lagoon Research Center, Shimane University, 1060 Nishikawatsu, Matsue 690-8504, Japan;

(2) Fisheries Resources Management Center, Shimane University, 1060 Nishikawatsu, Matsue 690-8504 Japan

* Corresponding author. E-mail: aranishi@soc.shimane-u.ac.jp

DOI: 10.2983/035.035.0216

TABLE 1.
Collection sites, sample sizes, and measures of genetic diversity
for each Scapharca broughtonii population.

Population   Collection date    Number of     Haplotype diversity
                               individuals

Japan
  Miyagi      November 2012        37        0.8889 [+ or -] 0.0379
  Ishikawa    November 2012        34        0.8396 [+ or -] 0.0558
  Aichi       December 2012        35        0.8605 [+ or -] 0.0423
Korea         November 2008        35        0.7664 [+ or -] 0.0767
China         December 2012        39        0.8084 [+ or -] 0.0569

Population   Nucleotide diversity (%)

Japan
  Miyagi      0.4727 [+ or -] 0.2921
  Ishikawa    0.3351 [+ or -] 0.2234
  Aichi       0.4057 [+ or -] 0.2590
Korea         0.3361 [+ or -] 0.2237
China         0.3434 [+ or -] 0.2267

TABLE 2.
The distribution of shared and unique haplotypes
across Scapharca broughtonii populations.

                                    Number of individuals

                                      Shared haplotype
              Number of
Population    haplotypes  HT01  HT02  HT03  HT04  HT05  HT06  HT07

Japan
  Miyagi          16       11          3     4                 1
  Ishikawa        15       13    4     3           1     1
Aichi             15       11    7     4           1     1
Korea             16       17    2     1     2     1           1
China             17

                               Number of individuals

                                 Unique haplotype

Population    HT18-29   HT08-17   HT30-39   HT40-49   HT50-66

Japan
  Miyagi        18
  Ishikawa                12
Aichi                               11
Korea                                         11
China                                                   39

TABLE 3.
Hierarchical AMOVA for phylogenetic
distance Scapharca broughtonii populations.

                                    Percentage
Source of variation     Variance       of         F/[phi]       P
                       components    variance    statistics
One gene pool
  Among populations      5.1507       84.22        0.8422     0.0000
  Within populations     0.9653       15.78
Two groups
(lineage A/B)
  Between groups        11.8513       91.62        0.9162     0.0000
  Among populations      0.1190        0.92        0.1098     0.0000
    within groups
  Within populations     0.9653        7.46        0.9254     0.1945
Lineage A
(Japan and Korea)
  Among populations      0.1183       10.67        0.1067     0.0000
  Within populations     0.9906       89.33

TABLE 4.
Pairwise population differences for mtDNA COI sequences of
Scapharca broughtonii populations.

           Miyagi    Ishikawa   Aichi    Korea     China

Mivagi                0.0017    0.0019   0.0004    0.0475#
Ishikawa   0.01691              0.0004   0.0006    0.4750#
Aichi      0.02515   -0.0065             0.0007    0.0473#
Korea      0.02193    0.0016    0.0221             0.0468#
China      0.15158#   0.1763#   0.1659#  0.2123#

Above diagonal: average genetic distance based on K2P.
Below diagonal: pairwise [F.sub.ST]. Bold values represent
significance at P < 0.01 level.

Note: Significance at P < 0.01 level are
indicated with #.

TABLE 5.
Neutrality tests and mismatch distributions
for each Scapharca broughtonii population.

                           Neutrality
                             tests
                              Fu's
Locality                   [F.sub.s]       [tau]

Japan        Miyagi         -8.6069#        1.6797
             Ishikawa      -10.7440#        1.7344
             Aich           -8.8034#        2.0430
Korea                      -12.3312#        1.7988
Japan and    Lineage A *   -27.2120#        1.3848
  Korea
China        Lineage B *   -13.1324#        2.0117

                               Mismatch analysis

                            [[theta]     [[theta]
Locality                    .sub.0]       .sub.1]

Japan        Miyagi          0.7928        79.2188
             Ishikawa        0.0000     99999.0000
             Aich            0.0492        28.2422
Korea                        0.0264         7.7106
Japan and    Lineage A *     0.8631        30.4297
  Korea
China        Lineage B *     0.0000        12.1930

                               Goodness of fit test

Locality                      SSD       [H.sub.rag]

Japan        Miyagi           0.0012        0.0292
             Ishikawa         0.0015        0.0570
             Aich             0.0015        0.0370
Korea                         0.0060        0.0415
Japan and    Lineage A *      0.0011        0.0267
  Korea
China        Lineage B *      0.0021        0.0315

SSD, the sum of squared differences; [H.sub.rag]. Harpending's
raggedness index. Bold values represent significance at P < 0.01
level. * Lineages A and B refer to Figure 2.

Note: Significance at P < 0.01 level are indicated with #.


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Author:Tanaka, Tomomi; Aranishi, Futoshi
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Date:Aug 1, 2016
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