The Asian shore crab Hemigrapsus sanguineus is distrib-uted widely in the western Pacific, including Hong Kong, Taiwan, Korean, Chinese, and Japanese coastal waters, and as far north as Sakhalin Island, Russia (Sakai, 1976; Fukui, 1988; Dai and Yang, 1991; Hwang et al., 1993). It is abundant in rocky intertidal habitats and is an ecologically important predator in coastal ecosystems, as are other shore crab species (Kikuchi et al., 1981; Takada and Kikuchi, 1991; McDermott, 1998a, 1998b; Lohrer et al., 2000). In Japan, the breeding season of H. sanguineus is March-October, with a main peak May-June (Fukui, 1988). It has a planktonic larval stage of more than 1 month before developing into the juvenile crab (Fukui, 1988). The larval dispersal pattern and preferred habi-tat might have caused geographically distinct regional popula-tions to become homogeneous.
Estimating genetic structure among populations using mo-lecular markers has become a common approach to determin-ing sustainable yields and genetic diversity (Dunham, 2004). The population genetic structures of some marine species are influenced by their larval dispersal pattern and behavior after spawning, which are determined by oceanographic features including sea currents, hydrological conditions, and physical barriers (Doyle et al., 1993; Hsieh et al., 2010). In general, most marine species have limited population substructures and high levels of gene flow because of the effect of sea cur-rents. Ocean structure and dynamics, including current bound-aries and hydrographic conditions, have caused reproductive and partial genetic isolation in geographically distinct regional populations (Wares et al., 2001; Bilton et al., 2002).
Although DNA markers are expected to overcome defi-ciencies in allozyme analysis by increasing the accuracy and resolution of population-structure assessments in crab species (McMillen-Jackson et al., 1994; Creasey et al., 1997), there are few reports on the use of mitochondrial DNA (mtDNA) to measure genetic variation in shore crabs (Cassone and Bould-ing, 2006). Maternally inherited mtDNA has greater sequence variability than do most nuclear genes (Brown et al., 1979). Moreover, it has a compact genome size in both conserved and
variable regions and a conserved gene content and arrange-ment (Anderson et al., 1981). Therefore, mtDNA analysis has become a key method in evolutionary and ecological studies of crabs (Pfeiler et al., 2005; Cassone and Boulding, 2006; Azuma et al., 2008; Wang et al., 2008).
This study investigated the genetic variation and popula-tion structure of H. sanguineus in Korean coastal waters using mtDNA cytochrome b (Cytb) sequences to assess phylogeo-graphic and demographic patterns.
Muscle samples were taken from 143 live crabs collected from six localities along three coastlines of Korea from 2009 to 2010 (Table 1, Fig. 1). The collected samples were stored at -20℃ or kept in 100% ethanol at room temperature until used.
Genomic DNA was extracted from about 20 mg of each specimen by a PUREGENE DNA isolation kit (Gentra Sys-tems, Minneapolis, MN, USA) following the manufacturer’s instructions. The purified DNA was dried at room temperature and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA; pH 8.0). To amplify the Cytb gene, a pair of degenerate primers (Cyt9237 5′-GTWGCHCAYATTTGYCGAGA-3′; Cyt10050 5′-ACWGGKCGWGCWCCAATTCA-3′) with expected amplicon sizes of 850 bp was designed based on mtDNA sequences of the closely related crab species available in GenBank (AB093006, AY659990, AY562127, FJ797435, FJ827758-827761). The PCR amplification was performed with a thermocycler DNA Engine (MJ Research, Tokyo, Ja-pan). in a 20-μL reaction volume containing 1-2 μL of genom-ic DNA, 2 μM of each primer, 0.25 mM of each dNTP, 1 unit of Takara LA Taq DNA polymerase (Takara Shuzo, Kyoto, Japan), and 2 μL of 10× LA Taq reaction buffer (Takara Shuzo). The PCR conditions consisted of preheating at 94℃ for 5 min, followed by 35 cycles of 94℃ for 30 s, 55℃ for 30 s, and 72℃ for 30 s, with final extensions at 72℃ for 5 min. The fragment size of the PCR product was verified using 2% aga-rose gel electrophoresis after ethidium bromide staining. The PCR product was purified with the AccuPrep PCR Purification Kit (Bioneer, Daejeon, Korea). After cycle sequencing with the ABI PRISM BigDye Terminator v3.1 Cycle Sequencing Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA), the purified PCR product was sequenced directly on an ABI 3730xl DNA Analyzer (Applied Biosystems) with two newly designed internal primers, HsCytF (5′-GGGGT-CAAATATCATTCTGG-3′) and HsCytR (5′-GCCTTTG-GAATTTTGAAGAG-3′).
The sequence fragments obtained in this study were aligned with GENETIX-WIN ver. 4.0.1 (Software Development, To-kyo, Japan) to identify sequence variants. The integrated soft-ware package DnaSP version 5 (Librado and Rozas, 2009) was used to determine the genotypes or haplotypes. A parsi-mony network connecting the observed haplotypes to resolve the genealogy was plotted with TCS version 1.21 (Clement et al., 2000). Genetic variation within the populations, expressed as haplotype diversity (h) and nucleotide diversity (π), was estimated according to Nei (1987) based on Kimura’s two-parameter distance method using K and DA in the REAP pro-gram (McElroy et al., 1992). Pairwise population FST values were calculated to estimate the genetic differentiation among the populations according to Slatkin and Hudson (1991) us-ing ARLEQUIN version 3.1 (Excoffier et al., 2005). The sig-nificance of each FST value was tested using 10,000 random permutations. Gene flow among populations was estimated with Nem, the number of migrations per generation between population pairs (Slatkin, 1993) using the following equation:
Nem = (1/FST -1)/4,
where N is the effective population size, m is the effective proportion of immigrants, and FST is the fixation index. To ex-amine the relationships among populations visually, we used a two-dimensional scaling (TDS) analysis based on the matrix of pairwise FST values from the Cytb sequence data calculated by SPSS version 14.0K (SPSS, Inc., Chicago, IL, USA).
Neutral expectation and historic demographic expansions were investigated by examining Fu’s FS and Tajima’s D and mismatch distributions with the sudden expansion model (Rogers and Harpending, 1992). A goodness-of-fit test was used to test the validity of the sudden-expansion model using a parametric bootstrap approach based on the sum of squared deviations (SSD) to compare the observed and estimated mis-match distributions (Schneider and Excoffier, 1999). Both the neutrality test and mismatch distribution analysis were per-formed in ARLEQUIN version 2000 (Schneider et al., 2000). Since the mutation rate of the H. sanguineus Cytb gene over the estimated time since expansion was unknown, a molecu-lar clock was calculated using the sequence-divergence rates of mitochondrial protein coding regions for other marine crustaceans; rates ranged from 2.2 to 3.1% per million years (Knowlton and Weigt, 1998; Schubart et al., 1998).
The degenerate primers newly designed in this study (Cyt9237 and Cyt10050) successfully amplified the mtDNA Cytb region of the 143 H. sanguineus individuals. Direct se-quencing of the PCR products with two internal primers (Hs-CytF and HsCytR) yielded a fragment with an amplicon size of 470 bp, which revealed 38 variable nucleotide sites defin-ing 37 haplotypes among the populations (Table 2). The vari-able nucleotide sites observed consisted of 33 transitions and four transversions. All substitutions were biallelic except one, which was triallelic, suggesting the occurrence of a single base substitution among sequences. The nucleotide sequences of the 37 haplotypes have been deposited in the DDBJ/GenBank database under accession numbers AB570203-AB570239.
The parsimony network of the Cytb haplotypes in H. san-guineus did not provide evidence of geographical associa-tion (Fig. 2). In the network, two focal haplotypes, HeS1 and HeS12, were abundant, whereas the others, including single-tons, were rare and radiated from these focal haplotypes. The distribution of the 37 haplotypes among the six H. sanguin-eus populations is presented in Table 3. Among them, 28 were found at single localities, and nine (HeS1, HeS4, HeS9,
HeS12, HeS21, HeS26, HeS30, HeS32, and HeS35) were ob-served in two or more localities. A number of individuals from the populations examined had the HeS12 and HeS26 haplo-types, although these were not found in JUM. HeS1 occurred in all populations.
The haplotype and nucleotide diversities of H. sanguineus demonstrated varying levels of genetic variation among populations. Variation was higher in the Guryonpo (GUR), Haenam (HAE), and Jumunjin (JUM) populations (Table 1). Reduced haplotype diversity was observed in the Taean (TAE), Buan (BUA), and Geoje (GUJ) populations.
The pairwise population FST estimates and migration rate based on the Cytb sequences are presented in Table 4. Most pairwise values were not significantly different among popu-lations. Although the pairwise FST estimates showed no clear genetic differentiation (low values among populations), the
pairwise population FST estimates were large when JUM was compared with the other populations. These findings were evi-dent in the TDS analysis and Nem values (Fig. 3). Overall, the TAE, BUA, HAE, GUJ, and GUR populations were geneti-cally close to one another, but distinct from JUM.
Mismatch distributions for all populations pooled and the northernmost population (JUM) are shown in Fig. 4. The pooled populations and JUM had no additional distribution peaks; the highest frequency occurred at one difference. The D, Fu’s FS, and SSD (Table 5) values indicated expansion in these populations. Tajima’s D and Fu’s FS were significantly negative, with markedly reduced SSD in the pooled popula-
tion group and JUM population, a parameter combination that strongly supported sudden expansion. Sudden expansion of the pooled population group was estimated to have occurred 0.042-0.060 million years ago (Ma) and that in the JUM popu-lation, 0.049-0.069 Ma (Table 5).
The genetic variation analysis of the mitochondrial Cytb gene revealed the followings: (i) haplotype and nucleotide diversity occurred at various levels of genetic variation and (ii) there was a low level of genetic differentiation in coastal areas of Korea, but there was some genetic differentiation of the northernmost population (JUM) in the East Sea from the other populations. The observed haplotypes from the Cytb region in H. sanguineus were arrayed in star-like genealogies, each with several closely related, low-frequency haplotypes around a central high-fre-quency haplotype, suggesting a shallow haplotype genealogy. The star-like pattern and shallow genealogy indicate recent appearance and rapid population growth (Slatkin and Hudson, 1991; Rogers and Harpending, 1992). Our estimates showed that H. sanguineus began expanding 42,862-60,396 years ago (Table 5). High haplotype diversity (0.616-0.880) but low nucle-otide diversity (0.0017-0.0034) within the populations indicates that the populations might have experienced historically rapid population growth from an ancestral population with a small effective population size in the Late Pleistocene (Avise, 2000).
A reduced genetic diversity in the TAE and BUA populations from the west coast in the Yellow Sea compared with the other regional populations, except the GUJ population, was observed from the haplotype and nucleotide diversities. The western Ko-rean populations showed no genetic differentiation from the southern populations as inferred from the pairwise FST values. Therefore, crabs might have been introduced continuously with high rates of gene flow to the TAE and BUA populations from other sources following a reduction in effective population size.
The estimated pairwise FST values, Nem rate, and TDS analy-sis indicate that substantial gene flow has occurred among the populations, suggesting that larval behavior and sea currents are responsible for the high rates of gene flow. The passive dispersal of planktonic larvae and sedentary lifestyle of adult marine in-vertebrates limit the formation of population substructure (Les-sios et al., 2003; Waters and Roy, 2004). Many other marine crab species reportedly have high levels of gene flow between populations or regions (Beckwitt, 1985; Merkouris et al., 1998; Azuma et al., 2008). Hwang et al. (1993) reported that the plank-tonic larval stage of H. sanguineus persists for 25 days until metamorphosis on the sea floor to form the first crab stage. In general, crustacean species with similar persistent pelagic larval stages have a high dispersal potential that produces genetic ho-mogeneity among local populations (Palumbi, 1994). The Tsu-shima Warm Current (TC) branches off the Kuroshio Current, with part of the TC running into the Yellow Sea and the main part entering the East Sea along the Korean Peninsula (Fig. 1). Therefore, the TC might transport H. sanguineus larvae to the western coast of Korea.
Despite the lack of geographically associated haplotypes and genetic structure within and among populations, our analyses indicated a degree of genetic differentiation between the north-ernmost population (JUM) in the East Sea and the other popula-tions. The sub-polar front in the East Sea is similar to the west-ern boundary current in that a polar front forms at the boundary between the low-temperature, low-salinity waters of the north-ern region and the high-temperature, high-salinity waters of the southern region (Rhein et al., 1995; Pickart et al., 1997). The sub-polar front, which extends along the coast of Japan before turning abruptly at the Noto Peninsula frontal region toward the center of the East Sea, has a close relationship to the TC and cold-water currents, including the Liman Current (LC) (Senjyu, 1999; Ichikawa and Beardsley, 2002) (Fig. 1). Restricted or se-lective gene flow from main distributions can result from such physical barriers, which restrict larval transport (Hedgecock, 1986; Scheltema, 1986; Bowen and Avise, 1990; Palumbi, 1994; Burton, 1998). The JUM population is located at the sub-polar front, where the cold and warm currents of the East Sea meet. Therefore, the balanced effects of the cold (LC) and warm (TC) water currents in the East Sea might explain the genetic differen-tiation between the JUM population and other wild populations.
The mismatch distribution in our data was unimodal, and the neutrality test gave a significantly negative value, suggesting re-cent population expansion of H. sanguineus in Korea. Estimates of other mismatch distribution parameters corroborate this evi-dence. Therefore, H. sanguineus population expansion in Ko-rean coastal waters resulted from rapid population growth and recent, sudden expansion in the Late Pleistocene. This was cor-roborated by the star-like genealogy of the haplotypes, high hap-lotype diversities, close genetic similarities among haplotypes, mismatch distribution pattern supporting a sudden expansion model, and estimated expansion time. However, this perspective remains ambiguous, and further extended sampling from Hong Kong through Japan to Russia is necessary to clarify the histori-cal influences.
Based on our results, the H. sanguineus populations might be one large panmictic population in Korean coastal waters, with the exception of the northeastern population. This genetic information about the current condition of H. sanguineus will be useful in developing a conservation strategy and subsequent ecological monitoring. H. sanguineus has colonized the eastern coast of the USA, Atlantic France, and the Netherlands in recent years. Comparative studies of genetic variation and structure in donor and invader populations using the molecular procedures employed in this study are essential for developing a greater un-derstanding of the spatiotemporal invasion-pattern mechanism. Finally, we demonstrated that genetic variation analysis using mtDNA Cytb sequences is a useful model for research on popu-lation-level studies in closely related species.