PCR-RFLP typing reveals a new invasion of Taiwanese Meretrix (Bivalvia: Veneridae) to Japan

Samples of three hard clam Meretrix spp. (M. lusoria, M. petechialis, and Taiwanese Meretrix) were collected from 12 localities in Japan, China, Korea, and Taiwan between 2004 and 2013. PCR-RFLP analysis and nucleotide sequence analysis for the mtDNA COI region was performed on these samples. HincII and AseI restriction assay discriminated M. lusoria, M. petechialis, and Taiwanese Meretrix, corresponding to the relatively large nucleotide substitution (6.35–8.20 %). In Taiwan, M. lusoria was introduced from Japan in the 1920s; however, our results suggest that Taiwanese Meretrix is genetically differentiated from M. lusoria. As well, the exotic Taiwanese Meretrix was found from Nishinagisa on the north coast of Tokyo Bay, demonstrating the Taiwanese Meretrix now occurs with M. lusoria in its native habitat. Meretrix seedlings (Kumamoto origin) from a Taiwanese aquaculture facility has been released into Nishinagisa since 2008 suggesting the Taiwanese Meretrix is inadvertently mixed with the M. lusoria produced in the aquaculture facility. In contrast, all samples from Kisarazu, on the east coast of Tokyo Bay, were identified as M. lusoria despite there being mass releases of M. lusoria (Kumamoto origin) cultured in Taiwan since 2007. Quality control procedures are needed for future Meretrix spp. releases to prevent further spread of the Taiwanese Meretrix.

Because of the economic importance of Meretrix, previous research has mainly focused on its production in aquaculture (Yoshida 1941;Wu and Liu 1992;Tuan and Phung 1998), effects of organotin compounds (Midorikawa et al. 2004;Harino et al. 2006), and incidences of shellfish poisoning (Nguyen et al. 2006).Seedlings of a few Meretrix species are mass-produced in several countries, and nearly all hard clams sold in Taiwanese markets originate from aquaculture (Wu and Liu 1989).Past taxonomic studies on Meretrix considered only shell morphology (Fischer-Piette andFischer 1940-1941;Yoosukh and Matsukuma 2001), although shell shape and color patterns often show marked intraspecific variability (Hamai 1934(Hamai , 1935;;Kosuge 2003).
Two species of Meretrix, M. lusoria and M. lamarckii, occur in tidal flats and shallow waters in Japan.M. lusoria is distributed in sheltered sandy tidal flats in Japan (except for Hokkaido and the Ryukyu Archipelago) and along the southern coast of Korea (Yamashita et al. 2004).M. lusoria once provided an important commercial and recreational fishery resource; however, landings have decreased significantly since the mid-1960s, and the commercial catch remains low (Higano 2004;Fishbase 2013).M. lusoria was designated an endangered species by the Japanese Ministry of the Environment in 2012 (Ministry of the Environment 2012) and is now considered to be locally extinct in Chiba Prefecture in Tokyo Bay (Chiba Prefecture 2011).In response to a decrease in the supply of M. lusoria, M. petechialis was recently imported from China and the Korean Peninsula.
In 2008, media reports indicated that the commercial harvest of M. lusoria, under the brand name of Edomae hamaguri, resumed on the east coast of Tokyo Bay after a 40-year closure (Japanese Daily, The Asahi Shimbun 2008;Yomiuri Shimbun 2008;Tokyo Shimbun 2008;Minato Daily 2008).Since 2007, local fishery cooperatives have released large quantities of M. lusoria seedlings (Kumamoto, Japan origin) along the east coast of Tokyo Bay (Minato Daily 2008) and, since 2008, on the Nishinagisa artificial tidal flat near Tokyo Disneyland on the north coast of Tokyo Bay (NPO Executive Committee of Furusato, Tokyo 2011).These Meretrix seedlings (Kumamoto origin) were mass-cultured in an aquaculture facility in Changhua Prefecture in Taiwan (Minato Daily 2008).Meretrix aquaculture in Taiwan has a long history, starting in the 1930s (Chen 1990).The dominant Meretrix species in aquaculture was generally regarded to be M. lusoria that originated from Japan (Chen 1984;Chen 1990;Shao and Chiu 2003).Yamakawa et al. (2008) suggested that cultured Taiwanese Meretrix might be a distinct species from M. lusoria due to a high degree of genetic differentiation.Moreover, Taiwanese Meretrix is indistinguishable from Japanese M. lusoria by shell appearance (Yamakawa unpublished data) and, therefore, can occur undetected with M. lusoria on shallow tidal flats in Tokyo Bay.
In the current study, we examined genetic variation within M. lusoria, M. petechialis, and Taiwanese Meretrix populations in Japan, China, Korea, and Taiwan.We conducted species identification and detected introduced specimens in Japan using genetic markers developed from mitochondrial DNA.

Specimens and DNA samples
Meretrix samples were collected from 12 localities in Japan, China, Korea, and Taiwan between 2004 and 2013 (Table 1).Foot muscle tissue was dissected from fresh specimens for DNA analysis.A small amount of muscle tissue was maintained in 500 µl of TNES [10 mM Tris-HCl, 0.3 M NaCl, 10 mM EDTA, 2% sodium dodecyl sulfate (SDS)]/8M urea buffer for DNA extraction (Asahida et al. 1996).For tissue digestion, 10 µl of proteinase K (Wako Chemicals, Osaka, Japan) was added to the TNES mixture, which was then incubated for 2 h at 37°C.Total DNA was extracted using a phenol-chloroformisoamyl alcohol method (Imai et al. 2004).
Nucleotide sequence data were examined to identify restriction site differences between M. lusoria, M. petechialis, and Taiwanese Meretrix (GENETYX-MAC Ver.8.0).We selected two restriction enzymes, HincII and AseI (Toyobo, Tokyo, Japan), which provided diagnostic restriction patterns among three Meretrix spp.. RFLP analysis was performed in a 10 µl volume containing 1 µl of buffer H (Toyobo), 3-5 µl of PCR product, and 5 units of restriction enzyme (HincII and AseI) at 37°C for 2 h.A 10-µl sample of the reactant was examined using electrophoresis on a 1% agarose gel (Trevi Gel TM 500; Trevigen, Gaithersburg, MD, USA) in TAE buffer at 100 V.After electrophoresis, gels were stained with ethidium bromide, visualized under ultraviolet (UV) light, and photographed.2), and six composite haplotypes were found for 12 local samples (Table 3 and Figure 3).No composite haplotype was shared by the different species, except for the Nishinagisa samples, indicating that the three Meretrix spp.examined in this study could be readily discriminated using HincII and AseI.Six individuals from Nishinagisa, on the north coast of Tokyo Bay, showed Taiwanese Meretrix haplotypes (Table 3 and Figures 3-4).

Discussion
This study revealed that both natural and cultured Taiwanese Meretrix samples were genetically distinct, in terms of mtDNA COI, from M. lusoria and M. petechialis.M. lusoria seedlings were introduced to the Tanshui River in Taiwan from Midorikawa, Kumamoto Prefecture and Saga Prefecture, Japan, in 1920s (Ministry of Agriculture and Forestry, Fisheries Research Institute 1931, Shao and Chiu 2003).Since then, Meretrix aquaculture has been very successful and spread rapidly around the west coast of Taiwan, specifically in Changhua and Yunlin prefectures (Chen 1984;Kuo 2005;Chien and Hsu 2006).
The percentage of aquaculture-produced clams on the market reached 98.8% in 1986 (Wu and Liu 1989).The dominant species in aquaculture is generally regarded as M. lusoria (Chen 1990).Introduced Japanese M. lusoria are assumed to have become established as a cultured stock in Taiwan, and both cultured and natural Meretrix clams are thought to be artificially introduced species (Shao and Chiu 2003).However, our PCR-RFLP results indicate Taiwanese Meretrix to be genetically different from M. lusoria and M. petechialis.Our results suggest that a native population of Taiwanese Meretrix has existed since before the introduction of Japanese M. lusoria in the 1920s.Our previous allozyme analysis on 12 loci (Yamakawa et al. 2008) discovered that genetic distance between the Taiwanese Meretrix (aquacultured) and Japanese M. lusoria populations showed a high degree of genetic differentiation (D > 0.386), and genetic distance was large enough indicate separate species (based on Nei 1975).It is difficult to conclude that Taiwanese Meretrix is a different species from M. lusoria on the basis of RFLP analysis; however, we have a stronger case to suggest this by combining our RFLP and allozyme results.Further study using multiple genetic markers and suitable phylogenetic and morphological analysis would be needed to confirm whether Taiwanese Meretrix is a distinct species or not.Six of 62 individuals collected in the artificial tidal flat at Nishinagisa on the north coast of Tokyo Bay possessed genetic characteristics of Taiwanese Meretrix (Table 3 and Figure 3) presumably were introduced from Taiwan.This is the first study to demonstrate that Taiwanese Meretrix individuals co-occur with local M. lusoria   samples (48) was too small to detect Taiwanese Meretrix in Kisarazu.A larger sample from other sites should be tested to see whether the Taiwanese Meretrix was simply missed due to chance and/or the small sample size.
A molecular-based study reported that M. petechialis that originated from China and Korea were observed in Japan as introduced species (Yamakawa and Imai 2012), and hybrids between M. lusoria and M. petechialis were found in natural habitats.The spawning season of M. lusoria in Tokyo Bay is from June to September (Taki 1950;Nakamura et al. 2010) while the spawning season of Taiwanese Meretrix is from April to November (Chen 1990;Wu and Liu 1992).Thus at the Nishinagisa site where the two co-occur, it is possible that hybridization could occur, if it has not already done so.
The impacts of introduced species can be diverse, including genetic disturbance through hybridization, exclusion and predation on native species, and economic damage to fisheries (Iwasaki 2006).For example, the Manila clam Ruditapes philippinarum (Adams and Reeve, 1850) is one of the most important commercial clams in Japan.Since the mid-1980s, the abundance of the Manila clam has declined considerably in Japan and large quantities of clams have been imported from China and Korea to provide seed for stocking and recreational shellfish gathering (Okoshi 2004).Kitada et al. (2013) reported that mass introductions of Chinese Ruditapes, which are genetically and morphologically different from Japanese R. philippinarum, were repeated in Japan.The Ruditapes population in the Ariake Sea includes hybrids between the alien Chinese Ruditapes and the native R. philippinarum.Furthermore, infections of the protozoan Perkinsus on R. philippinarum have been reported from various localities in Japan.Hamaguchi et al. (2002) reported that imported and introduced clams from China and Korea were infected with Perkinsus sp., that caused high mortality of R. philippinarum juveniles (Shimokawa et al. 2010) and contributed to drastic decreases in Japanese Manila clam resources (Waki et al. 2012).In Taiwan, several viruses have been isolated from cultured hard clams (Meretrix spp.); one birnavirus is associated with high juvenile mortality (Chou et al. 1994(Chou et al. , 1998)).Birnavirus-infected hard clams experience a higher predation pressure than noninfected clams (Liao et al. 2008).Avoiding clam-seedling releases from foreign countries is advisable until they can be given careful consideration because they can result in unexpected hybridization, new diseases, and high mortality in Japan.
Approximately 12 alien marine bivalve species have been introduced to Japan, either intentionally or accidentally (Iwasaki 2006)

Figure 3 .
Figure 3. Haplotype frequencies for samples of Meretrix spp.collected from 12 locations in Japan, China, Korea, and Taiwan.

Table 1 .
Collection information of 12 locations where samples of Meretrix spp.were collected in Japan, China, Korea, and Taiwan.

Table 2 .
Number of substitutions and substitution percentage (bracket) between three Meretrix spp.from Japan, China and Taiwan (Length 634 bp).
COI partial region indicated the Taiwanese Meretrix was genetically differentiated from M. lusoria and M. petechialis.HincII and AseI digestions detected two and four restriction types, respectively (Figure

Table 3 .
Composite haplotype frequencies for samples of Meretrix spp.collected from 12 locations in Japan, China, Korea, and Taiwan.A-D indicates restriction types refer to Figure 2.
. Most have successfully emigrated and undergone rapid nationwide expansion.Examples include Mytilus galloprovincialis Lamarck, 1819, Perna viridis (Linnaeus, 1758), Xenostrobus securis (Lamarck, 1819), and Corbicula fluminea (Müller, 1774).Our results suggest that Taiwanese Meretrix has genetically different characteristics from M. lusoria and M. petechialis, although further study would be needed to evaluate whether the Taiwanese Meretrix is a discrete species.The occurrence of exotic Taiwanese Meretrix is currently very limited in the north of Tokyo Bay.If Taiwanese Meretrix becomes established and expands its range within Tokyo Bay, it may cause the exclusion of native species and genetic disturbance via hybridization with M. lusoria.To conserve the local, endangered, M. lusoria resources, the identification of introduced Taiwanese Meretrix and M. petechialis by PCR-RFLP analysis is very effective because this method is much easier and cheaper than DNA sequencing methods.Rapid species identification at the local research institute is essential for monitoring introduced Meretrix species.While the Japanese Meretrix resource has almost collapsed in Tokyo Bay (Chiba Prefecture 2011), mass releases of M. lusoria seedlings from Taiwan are to be discouraged because of the possibility of introducing alien species.