Molecular analysis of the distributions of the invasive Asian clam , Corbicula fluminea ( O . F . Müller , 1774 ) , and threatened native clam , C . leana Prime , 1867 , on Kyushu Island , Japan

The invasion of the Asian clam, Corbicula fluminea (O.F. Müller, 1774), has caused serious ecological and economic problems worldwide. In the Japanese archipelago, C. fluminea has been identified using morphological characteristics in most distribution surveys. However, a recent study has shown that morphology alone cannot be used to identify C. fluminea due to its superficial similarity to the related native species C. leana Prime, 1867. Consequently, the present distribution of C. fluminea is still poorly understood. We surveyed the distribution of C. fluminea on Kyushu Island, Japan, by sequencing the cytochrome b region of the mitochondrial DNA, a region that can be used to distinguish the two clam species, and found that C. fluminea was already established throughout the entire island, except at a single site where Corbicula clams comprised solely of C. leana. In response to this serious situation, we suggest using PCR-RFLP method, which was employed here, to distinguish the two clam species for more effective and rapid surveys.


Introduction
Clams of the genus Corbicula Megerle von Mühlfeld, 1811 are native to Africa, Australia, Asia, and the Middle East; however, as invasive species, they are nowadays distributed worldwide (Araujo et al. 1993).Corbicula clams possess high fecundity and great adaptability allowing them to establish in new environments.For example, the genus was introduced into North America in the 1920s (Counts 1981) and spread rapidly throughout the continent, reaching South America in the 1970s (Ituarte 1994).The clams invaded Europe around 1980 (Mouthon 2001).These clams have caused serious economic and ecological problems worldwide.In Europe and America, Corbicula clams have reached high densities, competing with native bivalves (Fuller and Imlay 1976;Karatayev et al. 2003), reducing phytoplankton concentrations (Descy et al. 2003;Pigneur et al. 2014b), and damaging industrial cooling systems (Rosa et al. 2011).
In Japan, there are two native freshwater Corbicula clams.Corbicula leana Prime, 1867 is distributed in rivers and ponds throughout the Japanese archipelago, except within the Ryukyu Chain and Hokkaido Islands (Yamada et al. 2010).Corbicula sandai Reinhardt, 1878 has a more restricted distribution; it is endemic to Lake Biwa in Honshu (Ishibashi and Komaru 2003).The populations of both these native species have been decreasing, and they are now treated as threatened species (VU) in the Red List of the Japan Ministry of the Environment (Japan Ministry of the Environment 2012).One of the causes of native clam population decline is the introduction of the alien clam Corbicula fluminea (O.F.Müller, 1774).C. fluminea was imported into Japan for food trade from Korea and China around the 1980s (Tamai et al. 2008).Owing to C. fluminea brooding its larvae in the inner demibranch, larvae probably accidentally escaped and/or were discarded into drainage ditches in the process of preparation or washing such as removing sand from their body (Japan Wild Research Center 2008).In addition, C. fluminea were also released as prey for a freshwater snail, Semisulcospira libertine (Gould, 1851), in Japan (Japan Wild Research Center 2008).Thus, it is now established in several freshwater environments in Japan.Invasions of C. fluminea brought risks of modification of abiotic conditions such as water clarity and dissolved oxygen levels (Ilarri and Sousa 2012), and resource competition with the native Corbicula clams (Japan Wild Research Center 2008).Therefore, the distribution of C. fluminea must be determined in order to evaluate ecological damage, and to construct a management program for this invasive clam.
Corbicula fluminea has been reported from several areas in Japan (Masuda and Habe 1988;Ishibashi and Komaru 2003;Sonohara et al. 2005;Yasuki 2014), in which species identification has been based on shell morphology and colors.However, recent studies suggested that it is impossible to distinguish C. fluminea and C. leana morphologically (Sousa et al. 2007;Hedtke et al. 2008;Yamada et al. 2010).Another biological aspect of C. fluminea and C. leana is that these species are hermaphrodites and have a very peculiar mode of reproduction-androgenesis (Komaru et al. 1998;Houki et al. 2011;Pigneur et al. 2012).At fertilization, DNA from an unreduced spermatozoon is incorporated into the egg, while during oogenesis all the chromosomes (maternal nuclear DNA) are extruded from the egg as two polar bodies.As a consequence, only the paternal pronucleus is kept in the zygote of androgenetic Corbicula, while the mitochondria of the egg are retained (Komaru et al. 1998;Pigneur et al. 2014a).Several studies intensively investigated the evolutionary origin of this unique reproductive mode in Corbicula clams using molecular phylogenetic approaches (e.g., Hedtke et al. 2008;Houki et al. 2011;Pigneur et al. 2014a).Yamada et al. (2010) found that the cytochrome b region in the mitochondrial DNA includes mutations that nevertheless may be used to distinguish C. fluminea and C. leana.Although Yamada et al. (2010)  In this study, we investigated the distributions of C. fluminea and C. leana on Kyushu, which is the third largest island in the Japanese archipelago, using DNA sequencing of the cytochrome b region.Since Komaru et al. (1997) published a collection record of C. fluminea from northwestern Kyushu, we focused on this geographical region.Furthermore, we used a method based on cytochrome b sequences that can distinguish C. fluminea and C. leana by using PCR-restriction fragment length polymorphism (RFLP).

Materials and methods
Sampling was conducted between May 2014 and January 2015.Corbicula fluminea and C. leana were collected from 21 sites on Kyushu and the surrounding area by hand-net and hand (Figure 1).The specimens were kept in 100% ethyl alcohol after boiling for 1 min in the laboratory.Clam DNA was extracted from a piece of mantle or foot using SimplePrep™ DNA reagent (Takara, Japan).Yamada et al. (2010) revealed the haplotypes of mitochondrial cytochrome b (cyt-b) of C. leana and C. fluminea, collected form several part of Japan, and suggested the cyt-b region includes mutations which can distinguish between two species.Therefore, we used the cyt-b region for this survey.Cyt-b DNA was PCR amplified using the primer sets and protocol described by Houki et al. (2011).DNA was sequenced using an outsourced service (Fasmac, Japan).
The reference cyt-b sequences of C. fluminea and C. leana were analyzed using GENETYX-MAC ver.14.0.6 to identify specific restriction sites for each species.The PCR product was digested in a solution of 0.5 µL of restriction enzyme (10 U), 0.5 µL of buffer (New England Biolabs, Japan), and 4 µL of PCR product.The solution was incubated at 37ºC for 120 min.The DNA fragments were separated on a 1% agarose gel at 100 V for 25 min.Fragment size was determined using a 2-log DNA ladder (New England Biolabs, Japan).
Regarding distribution, C. fluminea and C. leana occurred at 20 and 18 sites, respectively (Figure 1, Table S1).Three sites (sites 4, 10, and 20 A major problem for mapping the distribution of Corbicula has been the difficulty of species identification using morphology (Sousa et al. 2007;Hedtke et al. 2008;Yamada et al. 2010).
used molecular data to clarify the distribution of C. fluminea in central Honshu and some remote islands, the distribution of C. fluminea in many other regions of the Japanese archipelago remains unclear.A more complete survey using molecular data is required to fully assess the current distributions of C. fluminea and C. leana.

Figure 1 .
Figure 1.Sampling sites and composition of C. leana and C. fluminea at each site.For details see supplementary material TableS1.
) were occupied only by C. fluminea and three other sites (sites 9, 16, and 17) were more than 80% occupied by C. fluminea.Site 19 consisted only of C. leana and site 18 was more than 80% C. leana.For the restriction site assessment, we used 12 haplotype sequences of partial cyt-b DNA of C. fluminea and C. leana (7 haplotypes found in this study, 5 haplotypes reported in Yamada et al. 2010).This showed that MspI cleaves the fragment into two fragments (436 and 185 bp) in C. fluminea, and into three fragments (436, 114, and 71 bp) in C. leana.Using MspI, we performed restriction enzyme digests of six haplotype sequences to confirm the lengths of the restricted fragments, which were as expected for the two species (Figure 2).This study is the first distribution study of Corbicula clams on Kyushu to use molecular methods to identify clam species.Our study has shed light on the serious state of the invasion by C. fluminea, demonstrating that almost all of Kyushu has already been invaded by C. fluminea (20/21 sites).We identified only one site, the Fukushimagawa River, entirely occupied by C. leana; since the Oyodogawa River is also occupied by a high proportion of C. leana, this suggests that southeast Kyushu has experienced only minor effects of the C. fluminea invasion.

Figure 2 .
Figure 2. Digestion patterns for each haplotype of C. leana and C. fluminea using the restriction enzyme MspI.M, the 2-log ladder marker; BD, before digestion; AD, after digestion.