The Comparison of Shell Morphology and Genetic Relationship Between Meretrix Lusoria and M. Petechialis in Japan and Korea

Plankton Benthos Res 5(Suppl.): 231–241, 2010 Plankton & Benthos Research © The Japanese Association of Benthology

The comparison of shell morphology and genetic relationship between Meretrix lusoria and M. petechialis in Japan and Korea

HIROSHI TORII1, SHIN’ICHI SATO2,*, MASAMI HAMAGUCHI3, YASUHISA HENMI4 & HIROYOSHI YAMASHITA5

1 Department of Earth Sciences, Graduate School of Science, Tohoku University, Aoba 6–3, Aramaki, Aoba-ku, Sendai 980–8578, Japan 2 The Tohoku University Museum, Aoba 6–3, Aramaki, Aoba-ku, Sendai 980–8578, Japan 3 Fishery Research Agency, Research Institute of Seto Inland Sea, Tidal Zone Environment Section, Maruishi 2–17–5, Hatsukaichi, Hiroshima 739–0452, Japan 4 Center for Marine Environment Studies, Kumamoto University, Aidu 6061, Matsushima, Kami-amakusa 861–6102, Japan 5 Association of Conservation Malacology, Mastugaoka 3–1–26–03, Kugenuma, Fujisawa 251–0038, Japan Received 17 May 2010; Accepted 8 August 2010

Abstract: Morphological and genetic traits of Meretrix lusoria and M. petechialis were compared among individuals from Japan and Korea. Multivariate analysis of shell morphology revealed that M. lusoria from all localities of Japan (Aomori to Kyushu) and from the southern and southwestern coasts of Korea (Sacheon Bay and Gangjin Bay) have some common characters, namely more linear shape in posterior-dorsal margin, smaller width of socket and larger shell breadth rather than M. petechialis from the western coasts of Korea (Baeksu and Saemangeum). Among M. lusoria, individuals from the Japan/East Sea coasts (Yuya Bay, Aso Sea and Mutsu Bay) have more linear shape in posterior-dorsal margin than those from other localities. The distribution border between M. lusoria and M. petechialis is located around the southwestern coasts of Korea (from Gangjin Bay to Baeksu). Analyses of mitochondrial COI and nucleus ITS also revealed that individuals from Japan and the southern coasts of Korea (Sacheon Bay) were classified as M. lusoria, and those from the western coasts of Korea (Baeksu and Saemangeum) as M. petechialis. However, all individuals from Gangjin Bay were classified as M. petechialis based on the analysis of mitochondrial COI, although most individuals were classified as M. lusoria by the analysis of nucleus ITS. These results suggest that hybridization between M. lusoria and M. petechialis occurs around Gangjin Bay. Further, we established a method to identify M. lusoria and M. petechialis from shell morphology. The modified discriminant score using the 5 selected characters, i.e. shell length (L), shell breadth (B), width of socket (SW), length of posterior-dorsal margin (LPM) and height of posterior-dorsal margin (HPM), is D110.2661.61(log B/log L)10.90(log SW/log L)81.72(log LPM/log L)27.27(log HPM/log L). Using this discriminant score, we can identify M. petechialis and M. lusoria with 98.89% correct percentage. Key words: Japan, Korea, Meretrix lusoria, Meretrix petechialis, mitochondrial COI gene, shell morphology

et al. 2000). Then, many benthic animals such as M. lusoria Introduction are drastically decreasing (Wada et al. 1996, Tsutsumi Meretrix lusoria (Röding) is one of the most popular 2006). In Korea, the Saemangeum area, which is the most clams around the Japanese coasts. However, after the productive site of Meretrix petechialis Lamarck in that 1970’s, most habitats and populations of this species de- country, has also been isolated by the reclamation dike of creased drastically due to reclamation and ocean pollution 33 km in length (the longest in the world) since April 2006 (Yamashita et al. 2004, Henmi 2009). In Japan, more than (Sato 2006, Hong et al. 2007, Sato et al. 2007). 800 km2 of tidal ﬂats formerly existed, but more than 40% Furthermore, M. petechialis is now artiﬁcially introduced of them have been lost during the past 50 years (Tsutsumi from China and Korea to Japan, and it has been pointed out that the hybrid between M. lusoria and M. petechialis may * Corresponding author: Shin’ichi Sato; E-mail, [email protected] have occurred in Japan (e.g. Kosuge 1995, Wada et al. 232 H. TORII et al.

1996). However, there are few studies that make clear the shell were numbered in each individual, so that genetic and relationship between these two species using morphological morphological data could be collated and related. and genetic analysis, and a method to identify M. lusoria Morphological analysis and M. petechialis has not been established (Yamakawa et 10 shell characters (in mm): shell length (L), shell height al. 2008). Thus, the objectives of the present study are 1) to (H), shell breadth (B), pallial sinus length (PL), ligament distinguish clearly between M. lusoria and M. petechialis based on morphological and genetic analyses, 2) to con- sider the phylogenic relationship between these species, and 3) to discuss possibility of hybridization and fear an artiﬁ- cial mating between them. Using these results, we established the method to identify M. lusoria and M. petechialis from shell morphology.

Materials and Methods Individuals of M. lusoria and M. petechialis were collected from 11 localities: i.e. Mutsu Bay (MT), Sendai Bay (SN), Aso Sea (AS), Ise Bay (IS), Yuya Bay (YY), Kafuri Bay (KF) and Ariake Bay (AR) in Japan, and Sacheon Bay (SC), Gangjin Bay (GJ), Baeksu (BK) and Saemangeum (SM) in Korea (Fig. 1). All individuals are stored at the To- hoku University Museum (TUMC111000-111010, Table 1). Living animals from the intertidal zone were dug at low Fig. 1. Sampling localities of Meretrix lusoria and M. pe- tide, and those from the subtidal zone were collected from techialis in Japan and Korea. MT: Mutsu Bay, SN: Sendai Bay, commercial port landings. In order to examine the genetic AS: Aso Sea, IS: Ise Bay, YY: Yuya Bay, KF: Kafuri Bay, AR: traits, the soft tissue of living animal was removed from its Ariake Bay, SC: Sacheon Bay, GJ: Gangjin Bay, BK: Baeksu, SM: shell and preserved in 99.5% ethanol. The soft tissue and Saemangeum.

Table 1. Information of locality, number of individuals, sample date and catalogue number at the Tohoku Univresity Musuem (TUMC) for Meretrix lusoria and M. petechialis from Japan and Korea.

Latitude and No. of Locality Sample point Sample date Catalogue No. Longitude indiv.

The mouth of Sin-Tanabe River, Mutsu City, 41°1621N, Mutsu Bay (MT) 44 May 10, 2005 TUMC111000 Aomori Pref., Japan 141°1146E The mouth of Nanakita River, Sendai City, 38°1513N, Sendai Bay (SN) 83 Feb.–Sep. 2007 TUMC111001 Miyagi Pref., Japan 141°0037E The mouth of Noda River, Yosano Town, Kyoto Pref., 35°3339N, Aso Sea (AS) 34 June 8, 2008 TUMC111002 Japan 135°0930E The mouth of Sakauchi River, Matsuzaka City, 34°3630N, Ise Bay (IS) 28 Apr. 23, 2005 TUMC111003 Mie Pref., Japan 136°3254E Yuya-igami, Nagato City, Yamaguchi Pref., Japan 34°2217N, Yuya Bay (YY) 30 June 28, 2004 TUMC111004 131°0142E The mouth of Izumi River, Itoshima City, 33°3310N, Apr. 2005– Kafuri Bay (KF) 72 TUMC111005 Fukuoka Pref., Japan 130°0940E Aug. 2006 The mouth of Shirakawa River, Kumamoto City, 32°4654N, Jan. 2005– Ariake Bay (AR) 59 TUMC111006 Kumamoto Pref., Japan 130°3517E Jul. 2006

35°0226N, Sacheon Bay (SC) Seonjin-ri, Sacheon City, Gyeongsangnam-do, Korea 31 Apr. 26, 2003 TUMC111007 128°0216E 34°3528N, Apr. 5, 2008, Gangjin Bay (GJ) Guro, Sonro-ri, Gangjin-gun, Jeonranam-do, Korea 110 TUMC111008 126°4650E Apr. 30, 2003 Baekpawi, Duu-ri, Yeonggwang-gun, Jeonranam-do, 35°1423N, Apr. 9, 2008, Baeksu (BK) 60 TUMC111009 Korea 126°1830E May 7, 2003

35°5543N, Saemangeum (SM) Sura, Okbong-ri, Gunsan City, Jeonrabuk-do, Korea 49 Aug. 25, 2007 TUMC111010 126°3605E Relationship between Meretrix lusoria and M. petechialis 233 length (LL), socket width (SW), anterior shell length (AL), valve was photographed with a digital camera, and then upper shell height (UH), length of posterior-dorsal margin each character was measured using image analysis software (LPM) and height of posterior-dorsal margin (HPM) were (Scion Image ver. 1.63). Further, L, H, B, PL, LL and SW measured on each individual (Fig. 2). For measurement of were measured using a digital slide caliper (accuracy0.01 L, H, AL, UH, LPM and HPM, the outside of the right shell mm). L and H were measured using both methods, but there were no significant differences (p0.05) between them. Measured characters were analyzed with reduced major axis regression (RMA) against shell length and we compared their slopes using the method of significance test at 95% confidence level (Hayami & Matsukuma 1971). Then, to standardize the variability for size, all characters were log-transformed (using base 10 logs), and the 9 characters excluding shell length were divided by the log-transformed shell length (Table 2). Canonical discriminant analysis (CDA) was tested with the standardized 9 characters using SPSS (ver. 16.0). The method of multivariate Fig. 2. 9 characters of right shell valve and shell breadth mea- analysis was partly borrowed from Takada (1992), Mat- sured on Meretrix shell. L: shell length, H: shell height, B: shell sumasa et al. (1999) and Sato & Matsushima (2000). breadth, PL: pallial sinus length, LL: ligament length, SW: socket width, AL: anterior shell length, UH: upper shell height, LPM: Genetic analysis length of posterior-dorsal margin, HPM: height of posterior-dorsal Genomic DNA was isolated from adductor muscle of the margin. clam by the DNeasy Blood & Tissue kit (Qiagen) following

Table 2. Standardized 9 characters against log-transformed shell length (log L) calculated in individuals of Meretrix lusoria and M. petechialis from the 11 localities. N in parentheses is number of individuals from each locality, and SD is standard deviation.

Locality (N) Characters MT SN AS IS YY KF AR SC GJ BK SM (44) (83) (34) (28) (30) (72) (59) (31) (110) (60) (49)

log H/log L mean 0.946 0.948 0.946 0.942 0.950 0.944 0.951 0.943 0.945 0.948 0.947 SD 0.005 0.007 0.007 0.006 0.007 0.008 0.008 0.006 0.006 0.005 0.006 log B/log L mean 0.811 0.803 0.828 0.831 0.820 0.825 0.827 0.832 0.827 0.822 0.802 SD 0.011 0.024 0.009 0.010 0.013 0.013 0.021 0.009 0.010 0.008 0.022 log PL/log L mean 0.717 0.690 0.747 0.738 0.700 0.724 0.726 0.740 0.721 0.739 0.721 SD 0.021 0.039 0.010 0.014 0.029 0.017 0.018 0.013 0.015 0.008 0.018 log LL/log L mean 0.650 0.600 0.681 0.683 0.617 0.653 0.667 0.699 0.657 0.684 0.639 SD 0.034 0.062 0.015 0.023 0.040 0.030 0.034 0.016 0.025 0.017 0.055 log SW/log L mean 0.315 0.263 0.347 0.360 0.263 0.303 0.310 0.374 0.331 0.399 0.329 SD 0.042 0.072 0.019 0.034 0.051 0.038 0.072 0.026 0.035 0.019 0.052 log AL/log L mean 0.395 0.023 0.503 0.430 0.285 0.329 0.335 0.494 0.392 0.438 0.342 SD 0.103 0.913 0.031 0.065 0.131 0.086 0.114 0.047 0.074 0.085 0.138 log UH/log L mean 0.519 0.213 0.578 0.532 0.439 0.489 0.461 0.577 0.501 0.549 0.418 SD 0.068 0.733 0.031 0.054 0.091 0.068 0.110 0.040 0.054 0.065 0.141 log LPM/log L mean 0.936 0.922 0.932 0.936 0.932 0.938 0.932 0.936 0.931 0.933 0.920 SD 0.007 0.010 0.006 0.008 0.009 0.008 0.010 0.007 0.008 0.006 0.011 log HPM/log L mean 0.401 0.342 0.394 0.405 0.380 0.393 0.409 0.438 0.411 0.483 0.433 SD 0.034 0.063 0.029 0.037 0.025 0.031 0.038 0.019 0.033 0.020 0.051 234 H. TORII et al. the manufacture’s protocol. from each locality (Table 4). The mitochondrial cytochrome C oxidase subunit 1 In individuals from Ariake Bay and Saemangeum, the (COI) was amplified from the genomic DNA using the slopes in H-L and UH-L were significantly larger (K1.96, primers reported by Folmer et al. (1994). Internally tran- p0.05) than those from the other localities (Table 4A and scribed spacer-1 (ITS-1) gene was amplified using our orig- 4G, Fig. 3A and 3G). Shell morphology of these individu- inal primers, PEF-10 (5-TAG AGG AAG GAG AAG TCG als looks more isosceles triangular than individuals from TAA CAA GG) and 5.8R (5-CAA KRT GCG TTC RAR other localities. Further, the slopes in B-L were signifi- RTG TCG ATG WTC A). PCR was performed in a total cantly larger in individuals from Ariake Bay and Sae- volume of 25 mL containing 1.25 U of Ex-Taq polymerase mangeum (Table 4B), and the shell breadth especially be- (TaKaRa), 2.5 mL of 10 Ex-Taq buffer, 2 mM MgCl2, comes larger with increase of the shell size in individuals 200 mM each of dNTPs, 50 pmol each of oligonucleotide from Ariake Bay (Fig. 3B). primers, and 10 to 50 ng of the genomic DNA. The mixture The slopes in HPM-L were significantly smaller in indi- was subjected to 35 cycles of amplification in a thermal cy- viduals from Mutsu Bay and Yuya Bay but significantly cler (BioRad). The first cycle was preceded by an initial de- larger in those from Aso Sea and Saemangeum than the naturation for 1 min at 94°C. Each cycle consisted of denat- other locality individuals (Table 4I). However, the coeffi- uration for 30 sec at 94°C, annealing for 30 sec at 40°C, cient b of RMA in HPM-L was much smaller in individuals and extension for 60 sec at 72°C. The last cycle was fol- from Aso Sea (Table 3), therefore the shape of posterior- lowed by a final extension step for 10 min at 72°C. dorsal margin is usually straighter than those from Sae- PCR products of COI were directly sequenced by their mangeum and Baeksu through ontogeny (Fig. 4I). As a re- PCR primers. Each PCR products was treated with Ex- sult, individuals from Mutsu Bay, Yuya Bay and Aso Sea, oSAP-IT (Amersham Bioscience) for inactivation of resid- which are located around or near the Japan/East Sea coasts, ual primers and dNTPs. Nucleotide sequences were deter- have more linear shape of posterior-dorsal margin, and mined by ABI PRISM 3100 Genetic Analyzer (Applied those from Baeksu and Saemangeum, located around the Biosystems) using a DYEnamic ET Terminator Cycle Se- Yellow Sea coasts, have more rounded shape of posterior- quencing Kit and DYEnamic Terminator Dilution Buffer dorsal margin compared to those from other localities. In (Amersham Bioscience). the same manner, the slopes in PL-L were also significantly For cloning, the PCR product of ITS genes was loaded larger in individuals from Yuya Bay but smaller in those onto an Agarose S agarose gel (Nippon gene) and elec- from Baeksu and Saemangeum (Table 4C, Fig. 3C). trophoresed in TBE (89 mM Tris-borate, 89 mM boric acid, The slopes in LL-L were significantly larger in the Sae- 2 mM EDTA) buffer. The gel portion containing the ampli- mangeum but smaller in the Sacheon Bay individuals, and fied DNA fragment was removed, and the DNA was ex- those in SW-L were significantly larger in the Ariake Bay tracted with Quiaquick spin kit (Qiagen). The extracted but smaller in the Sendai Bay individuals than those from DNA was directly cloned into the pGEM-T vector in the the other localities (Table 4D, 4E, Fig. 3D, 3E). In AL-L Original TA cloning kit (Invitrogen) following the recom- and LPM-L, there were no or few significant differences mended protocol. After cloning, isolation of plasmid DNA among all the individuals (Table 4F, 4H, Fig. 3F, 3H). was performed by the Qiaprep mini kit (Qiagen), and DNA was sequenced by the dideoxy-chain termination method of Multivariate analysis among the individuals of Meretrix Sanger et al. (1977), using an automatic DNA sequencer, spp. model 3100 (Applied Biosystems). According to the results of CDA, individuals from Homology searches of the nucleotide sequences were Baeksu and Saemangeum, western coasts of Korea, can be performed by BLAST and FASTA program supplied by divided clearly from those from all the other localities, i.e. GenBank for identification of the M. lusoria and M. pete- southwestern and southern coasts of Korea and Japan, by cialis. the standardized 9 morphological characters (Fig. 4). The proportions of canonical variate 1 and 2 were 60.9% and 13.0% respectively (Table 5). The centroid of canonical Results variate 1 in individuals from Baeksu and Saemangeum Morphological comparison among individuals from were both more than 3.5, but those from the other 9 locali- each locality ties were all less than 0 (Table 6). The shell characters that Among the 9 shell characters, the correlation coefficients largely affect canonical variate 1 were length and height of (r2) of RMA in H, B, PL, LL, AL, UH and LPM against L posterior-dorsal margin (log LPM/log L: 1.372, log HPM/ were usually 0.9, but those in SW and HPM against L log L: 1.137), socket width (log SW/log L: 1.070), and shell were relatively low in some localities although all regres- breadth (log B/log L: 0.974) against shell length (Table 5). sions were significant at 95% confidence level (Table 3). Namely, individuals from Baeksu and Saemangeum have 1) The slopes of RMA (coefficient a) in the 9 characters more rounded shape in posterior-dorsal margin, 2) larger ranged between 0.7 and 1.4 (Table 3), and there were sig- width of socket and 3) smaller shell breadth rather than nificant differences in some characters among individuals those from the other 9 localities. Relationship between Meretrix lusoria and M. petechialis 235

Table 3. Regression coefﬁcients of reduced major axis (log ya log xlog b) of 9 shell characters (y) against shell length (x) and correlation coefﬁcients (r2) calculated in individuals of Meretrix lusoria and M. petechialis from the 11 localities. N in parentheses is number of individuals from each locality.

Locality (N) MT SN AS IS YY KF AR SC GJ BK SM (44) (83) (34) (28) (30) (72) (59) (31) (110) (60) (49)

H-L a 0.943 0.928 0.970 0.909 0.957 0.915 1.012 0.965 0.921 0.941 0.991 b 1.026 1.086 0.936 1.152 0.985 1.121 0.794 0.930 1.121 1.057 0.859 r2 0.994 0.997 0.925 0.985 0.988 0.981 0.989 0.972 0.984 0.990 0.996 B-L a 0.954 0.990 1.008 0.990 0.967 0.981 1.115 0.917 0.940 1.011 1.037 b 0.575 0.526 0.477 0.521 0.586 0.549 0.340 0.699 0.647 0.469 0.423 r2 0.989 0.993 0.866 0.979 0.985 0.959 0.988 0.934 0.961 0.963 0.992 PL-L a 1.022 1.009 1.034 0.973 1.101 1.010 0.989 1.033 0.979 0.920 0.900 b 0.309 0.335 0.306 0.383 0.230 0.333 0.372 0.059 0.369 0.483 0.519 r2 0.983 0.990 0.875 0.965 0.978 0.965 0.990 0.951 0.956 0.952 0.987 LL-L a 1.140 1.113 1.174 1.136 1.182 1.189 1.165 1.002 1.132 1.108 1.242 b 0.150 0.172 0.130 0.157 0.128 0.127 0.154 0.280 0.160 0.186 0.110 r2 0.971 0.975 0.770 0.981 0.961 0.954 0.987 0.890 0.935 0.944 0.975 SW-L a 0.951 0.865 1.102 1.002 0.972 0.961 1.376 0.985 1.061 1.013 0.921 b 0.085 0.126 0.044 0.072 0.075 0.080 0.018 0.076 0.060 0.086 0.115 r2 0.964 0.971 0.682 0.853 0.926 0.837 0.966 0.788 0.897 0.869 0.969 AL-L a 1.020 0.990 0.993 0.943 0.995 0.954 0.973 0.994 0.972 0.959 0.955 b 0.338 0.416 0.412 0.457 0.377 0.426 0.421 0.386 0.434 0.471 0.494 r2 0.957 0.977 0.817 0.887 0.935 0.937 0.971 0.833 0.918 0.885 0.985 UH-L a 0.950 0.908 1.035 0.948 0.961 0.950 1.068 0.999 0.952 0.940 1.045 b 0.548 0.654 0.398 0.537 0.530 0.552 0.353 0.444 0.555 0.608 0.393 r2 0.987 0.987 0.797 0.941 0.969 0.925 0.945 0.904 0.938 0.908 0.992 LPM-L a 0.985 0.977 1.042 1.023 1.004 1.013 1.024 1.019 1.001 0.997 1.037 b 0.824 0.825 0.634 0.700 0.765 0.748 0.706 0.703 0.763 0.777 0.650 r2 0.992 0.995 0.940 0.985 0.987 0.983 0.991 0.976 0.972 0.987 0.997 HPM-L a 0.759 0.872 1.437 1.020 0.710 0.908 0.918 0.858 0.949 0.941 1.003 b 0.242 0.160 0.013 0.081 0.298 0.137 0.148 0.169 0.125 0.155 0.124 r2 0.735 0.924 0.442 0.693 0.823 0.778 0.910 0.533 0.583 0.766 0.944

Among individuals from each locality, percent of correct Genetic analysis of Meretrix spp. cases in multivariate discriminant analysis ranged between The 710 bp of the COI and 823–837 bp of ITS-1 nu- 97.1% and 48.2% (Table 7). However, most cases of mis- cleotide sequences were determined from each population classification were found among individuals from all locali- collected in the present study. The matching scores between ties in Japan and southern and southwestern coasts in Korea M. lusoria and M. petechialis were 93% per 710 bp of COI (MT-GJ in Table 7) or between those from the western nucleotide sequences and 97% per 823–837 bp of ITS-1 nu- coasts of Korea (BK & SM in Table 7). Most individuals cleotide sequences, respectively. Insertions or deletions of from Baeksu and Saemangeum (81 indiv./82 indiv.; 98.8%) some nucleotides were observed in ITS-1 genes compared were classified into those from the 2 localities (BK & SM), from M. lusoria and M. petechialis. The nucleotide se- and almost all individuals from the other 9 localities (447 quences of ITS-1 of M. lusoria and M. petechialis are de- indiv./448 indiv.; 99.8%) were also classified into these in- posited in DDBJ/EMBL/Gen Bank (accession numbers dividuals (MT-GJ) (Table 7). AB499129 and AB499729, respectively). Individuals from the Japanese coasts, i.e. Mutsu Bay, Sendai Bay, Aso Sea, 236 H. TORII et al. S S — —— p 0.05) than that ——S : The slope of RMA — ——— ————— ———— S S Locality 2 Locality 2 Locality 2 S S —S —S ———— —S —S —S S ——— —S ——————S —S ———— S S —S —S —S —S S S S S S S MT SN AS IS YY KF AR SC GJ BK MT SN AS IS YY KF AR SC GJ BK MT SN AS IS YY KF AR SC GJ BK

IS——— IS——— IS S GJ S GJ——————S GJ———————— SC — — S SC——————— SC——————— SNAS S — — KF————— SNAS — — — KF — S SNAS — S KF — — S AR S AR S AR S BK————————— BK S BK——————S YY———— YY———— YY — S SM S SM S SM S

Locality 1 Locality 1 Locality UH-L 1 Locality LPM-L HPM-L G. upper shell height (UH)—L H. (LPM)—L margin length of posterior-dorsal I. (HPM)—L margin height of posterior-dorsal — S S —S — —— S S S S ——S Locality 2 Locality 2 Locality 2 S S : The slope of RMA in individuals from locality 1 is signiﬁcantly smaller ( from locality 1 is signiﬁcantly : The slope of RMA in individuals S ——— — —— —S ———— ————S ————S S — ——S 0.05) in slope of RMA between the pair of individuals from localities 1 and 2. S the pair of individuals p 0.05) in slope of RMA between S S S for the slopes of reduced major axis regressionshell length among each (RMA) in the 9 characters against MT SN AS IS YY KF AR SC GJ BK MT SN AS IS YY KF AR SC GJ BK MT SN AS IS YY KF AR SC GJ BK

IS——— IS — S IS——— GJ———————— GJ S GJ———————S SC——————S SC——————— SC S SNAS — — — KF — S SNAS S S KF————— SNAS — — — KF————— AR — S AR S AR—————— BK————————— BK — S BK————————— YY———— YY — S YY———— SM S SM——————S SM——————————

Locality 1 Locality 1 Locality LL-L 1 Locality AL-L SW-L D. ligament length (LL)—L E. width (SW)—L socket F anterior shell length (AL)—L M. petechialis and — — S p 0.05) than that from locality 2. S — S S — — —— —— S S S ——S S S S — ——— S S S S S —S Locality 2 Locality 2 Locality 2 S S —S ——S —S —S ——S —S —S ————S S S S S S S S Meretrix lusoria Results of signiﬁcance test Meretrix MT SN AS IS YY KF AR SC GJ BK MT SN AS IS YY KF AR SC GJ BK MT SN AS IS YY KF AR SC GJ BK IS——— IS——— IS——— GJ————S GJ——————S GJ — S SC——————— SC——————— SC——————S SNAS — — — KF————S SNAS — — — KF————— SN AS — — KF————— BK S AR————S AR S AR S BK——————S BK——————S YY — S YY———— YY———— SM S SM S SM S

B-L H-L Locality 1 Locality 1 Locality Locality 1 Locality PL-L C. pallial sinus length (PL)—L pair of individuals from 2 localities. —: There are no significant differences ( from 2 localities. —: There are no significant differences pair of individuals Table 4. A. shell height (H)—shell length (L) B. shell breadth (B)—L in individuals from locality 1 is significantly larger ( larger from locality 1 is significantly in individuals BK: Baeksu, SM: Sae- GJ: Gangjin Bay, SC: Sacheon Bay, Bay, AR: Ariake KF: Kafuri Bay, Bay, Yuya YY: AS: Aso Sea, IS: Ise Bay, SN: Sendai Bay, Mutsu Bay, from locality 2. MT: mangeum. Relationship between Meretrix lusoria and M. petechialis 237

Fig. 3. 2 dimension scattergrams of log-transformed 9 shell characters against log-transformed shell length (L: mm) with reduced major axis regression (RMA) for 11 groups of Meretrix lusoria and M. petechialis. The regression lines of groups have the smallest and largest slopes (coefﬁcient a) in RMA were shown with their locality symbols. MT: Mutsu Bay, SN: Sendai Bay, AS: Aso Sea, IS: Ise Bay, YY: Yuya Bay, KF: Kafuri Bay, AR: Ariake Bay, SC: Sacheon Bay, GJ: Gangjin Bay, BK: Baeksu, SM: Sae- mangeum.

Fig. 4. 2 dimension scattergrams of Meretrix lusoria and M. petechialis from 11 localities obtained with canonical discriminant analysis (CDA). The values of canonical variate 1 and 2 for each individual and centroid for individuals from each locality were shown. 238 H. TORII et al.

Table 5. Standardized coefﬁcients of 9 characters, eigenvalue Table 6. Centroids of canonical variate 1 and 2 for individuals and proportion of canonical variate 1 and 2 for 11 groups of Mere- of Meretrix lusoria and M. petechialis from each locality. trix lusoria and M. petechialis. Centroid of canonical variate Standardized coefﬁcients Country Locality Characters 12 12 log H/log L 0.558 0.076 Mutsu Bay 0.322 0.256 log B/log L 0.974 0.206 Sendai Bay 0.101 1.695 log PL/log L 0.007 0.665 Aso Sea 0.289 0.675 log LL/log L 0.177 0.397 Japan Ise Bay 1.276 0.872 log SW/log L 1.070 0.287 Yuya Bay 1.512 0.738 log AL/log L 0.275 0.102 Kafuri Bay 2.329 0.426 log UH/log L 0.210 0.306 Ariake Bay 0.617 0.414 log LPM/log L 1.372 0.439 log HPM/log L 1.137 0.295 Sacheon Bay 0.049 1.124 Gangjin Bay 0.031 0.034 Korea Eigenvalue 3.180 0.680 Baeksu 3.689 0.843 Proportion 60.90 13.00 Saemangeum 3.728 0.117

Table 7. Discrimination score using multivariate discriminant analysis among individuals of Meretrix lusoria and M. petechialis from each locality (center column) and those from all localities in Japan and southwestern coasts in Korea (MT–GJ; right column) and from the western coasts of Korea (BK & SM; right column).

No. of No. of Percent Number of classiﬁed MT– BK & total correct of correct GJ SM cases cases cases MT SN AS IS YY KF AR SC GJ BK SM

Mutsu Bay (MT) 26 15 57.7 150431011100 Sendai Bay (SN) 78 45 57.7 2 45446227501 Aso Sea (AS) 34 33 97.1 0 0 3300010000 Ise Bay (IS) 28 18 64.3 0 0 0 181314100 Yuya Bay (YY) 25 16 64.0 0120162103004471 Kafuri Bay (KF) 72 55 76.4 301535550000 Ariake Bay (AR) 53 35 66.0 103130351900 Sacheon Bay (SC) 22 15 68.2 001400015200 Gangjin Bay (GJ) 110 53 48.2 9195134412530

Baeksu (BK) 34 32 94.1 000000000322 Saemangeum (SM) 48 36 75.0 0010000001136181

Total 530 353 66.6 30 47 58 40 43 66 50 40 74 43 39 448 82

Ise Bay, Yuya Bay, Kafuri Bay and Ariake Bay, were mostly techialis, and 1 individual has both M. lusoria and M. pe- classified as M. lusoria by COI and ITS-1 genes, except for techialis genes. 3 individuals from Ariake Bay (3.1% among 96 individuals examined) have ITS-1 of M. petechialis genes (Fig. 5). Fur- Discussion ther, all individuals from Sacheon Bay, southern coast of Korea, were classified as M. lusoria by both COI and ITS-1 Discrimination of M. lusoria and M. petechialis based on genes. By contrast, the analyses of mitochondrial COI and morphological and genetic analyses nucleus ITS revealed that individuals from the western Multivariate analysis of shell morphology revealed that coasts of Korea, Baeksu and Saemangeum, were almost all individuals from all localities in Japan (Mutsu Bay to Ari- classified as M. petechialis, except that 1 individual from ake Bay) and from the southern and southwestern coasts of Baeksu (10.0% among 10 individuals examined) has ITS-1 Korea (Sacheon Bay and Gangjin Bay) have some common of both M. lusoria and M. petechialis genes. All individuals characters, namely more linear shape in posterior-dorsal from Gangjin Bay, southwestern coast of Korea, were clas- margin, smaller width of socket and larger shell breadth sified as M. petechialis by COI genes (Fig. 5). However, in than those from the western coasts of Korea (Baeksu and the analysis of nucleus ITS-1, 40 individuals among them Saemangeum) (Fig. 4, Table 5). Only 2 individuals from were classified as M. lusoria, 2 individuals as M. pe- Sendai Bay and Saemangeum were misclassified into the Relationship between Meretrix lusoria and M. petechialis 239

individuals have similar shell morphology to those of M. petechialis and ITS-1 genes of this species. These results may be concerned with their faunal similarities between Ariake Bay and the Yellow Sea (Sato & Takita 2000). The distribution border between M. lusoria and M. petechialis is located around the southwestern coasts of Korea (from Gangjin Bay to Baeksu), as Yamashita et al. (2004) reported. According to mitochondrial COI, all individuals from Gangjin Bay were classified as M. petechialis, but in nucleus ITS-1, most individuals were classified as M. lusoria (Fig. 5) and 1 individual has ITS-1 genes both M. lusoria and M. petechialis. However, the result of CDA discrim- inated all individuals from Gangjin Bay as M. lusoria (Fig. 4), and the slopes of RMA in most shell characters were significantly different between individuals from Gangjin Bay and those from Saemangeum (Table 4). Therefore, most individuals from Gangjin Bay can be identified as M. lusoria, and hybridization between M. lusoria and M. petechialis may occur around Gangjin Bay. If individuals from Gangjin Bay are assumed as native ones without the artificial introduction, M. lusoria and M. Fig. 5. Meretrix lusoria and M. petechialis classified by the petechialis are considered as a subspecies producing weak analyses of (A) mitochondrial COI and (B) nucleus ITS-1 in 11 geographical isolation. In Japan, many individuals of M. groups from Japan and Korea. In nucleus ITS-1 (B), 2 individuals petechialis are artificially introduced from China and from each Gangjin Bay and Baeksu have both M. lusoria and M. Korea, because native individuals of M. lusoria decrease petechialis genes. MT: Mutsu Bay, SN: Sendai Bay, AS: Aso Sea, drastically. In such cases, there is fear that an artificial mat- IS: Ise Bay, YY: Yuya Bay, KF: Kafuri Bay, AR: Ariake Bay, SC: ing may occur among M. lusoria and M. petechialis (Ko- Sacheon Bay, GJ: Gangjin Bay, BK: Baeksu, SM: Saemangeum. suge 1995, Wada et al. 1996, Yamashita et al. 2004). The present study found that 3 individuals from Ariake Bay opposite group among 530 individuals (Table 7). The have ITS-1 genes of M. petechialis (Fig. 5), and the results height of posterior-dorsal margin (HPM) and pallial sinus of AFLP analysis also showed that individuals from Ariake length (PL) against shell length (L) were also significantly Bay are genetically similar to those of M. petechialis (Ham- different (|K|1.96, p0.05) through ontogeny in individu- aguchi et al. unpublished data). These facts suggest that in- als from Baeksu and Saemangeum than those from the dividuals from Ariake Bay might have been influenced by other localities (Table 4C, I). the recent artificial hybrid in addition to the ancient rela- The genetic analyses also revealed that individuals from tionship with M. petechialis from the Yellow Sea. In any the Japanese coasts and Sacheon Bay were almost classified case, the artificial introduction of M. petechialis to the as M. lusoria, and those from Baeksu and Saemangeum Japanese coasts should be stopped. were almost all classified as M. petechialis (Fig. 5). There- fore, these results suggested that individuals from Baeksu Method to identify M. lusoria and M. petechialis from and Saemangeum, western coasts of Korea, can be identi- shell morphology fied as M. petechialis, and those from Sacheon Bay, south- Genus Meretrix comprises 9 recognized species at the ern coast of Korea and those from the Japanese coasts can present day; however, systematic descriptions of these be identified as M. lusoria based on morphological and ge- species are often confusing because of their morphological netic analyses (Fig. 5, Table 7). similarities (Yamakawa et al. 2008). M. lusoria and M. pe- In individuals from Ariake Bay, the slopes of RMA in H- techialis are especially similar in their shell morphology, L, B-L, SW-L and UH-L were significantly different and there are many erroneous identifications and notations (|K|1.96, p0.05) from those of M. lusoria from any in many books, reports and articles (Yamakawa et al. 2008). other localities (Table 4). However, all individuals from Pan et al. (2006) analyzed 16S rRNA and ITS-1 sequences Ariake Bay were classified as M. lusoria according to the of Meretrix spp. including M. lusoria and M. petechialis, results of CDA (Table 7). The analyses of mitochondrial and Chen et al. (2009) also studied COI of these species. COI and nucleus ITS revealed that individuals from Ariake However, they did not explain how to identify the two Bay were also classified as M. lusoria by COI and ITS-1 species from shell morphology. The present study suc- genes, except that 3 individuals have ITS-1 of M. pe- ceeded in discriminating clearly between the two species techialis genes (Fig. 5). Therefore, almost all individuals using analyses of shell morphology and genetics. Then, we from Ariake Bay can be identified as M. lusoria, but some establish the method to identify M. lusoria and M. pe- 240 H. TORII et al.

Table 8. Modified discriminant score using the selected 5 characters (in mm), shell length (L), shell breadth (B), width of socket (SW), length of posterior-dorsal margin (LPM) and height of posterior-dorsal margin (HPM) for identification of Meretrix lusoria and M. petechialis. A: The raw and standardized coefficients of canonical variate 1, B: Discrimination score of CDA for the 5 characters.

A. The raw and standardized coefﬁcients of canonical variate 1

Characters Raw coefﬁcients Standardized coefﬁcients

log B/log L 61.61 1.145 log SW/log L 10.90 0.637 log LPM/log L 81.72 0.838 log HPM/log L 27.27 1.293 Fig. 6. Distribution of the modified discriminant score, D110.2661.61(log B/log L)10.90(log SW/log L)81.72(log Constant 110.26 LPM/log L)27.27(log HPM/log L), for individuals of Meretrix lusoria and M. petechialis from 11 localities in Japan and Korea. B. Discrimination score of CDA The horizontal line in the box is the mean value of individuals from each locality, and the edges of the box are the upper and Actual Group Predicted Group Correct cases lower quartile. Error bar means the 95% confidence level, and open circle is discriminant score for each individual outside the Meretrix Meretrix Species N N percent 95% confidence level. lusoria petechialis

M. lusoria 458 456 2 456 99.56% centage of correct cases in this discriminant score is M. petechialis 82 4 78 78 95.12% 98.89% (Table 8B). Using this discriminant score, we can identify M. lusoria and M. petechialis clearly based on the Total 540 460 80 534 98.89% 5 shell characters, i.e. shell length (L), shell breadth (B), width of socket (SW), length of posterior-dorsal margin (LPM) and height of posterior-dorsal margin (HPM) (Fig. techialis from shell morphology. 6). In the past studies, shell color patterns, shell height, shell breadth, position of umbo (represented by AL and UH) and the shape of posterior-dorsal margin have been usually used Acknowledgments as remarkable characters to identify M. petechialis and M. We thank Mr. Takeshi Igarashi, Mr. Morio Takarada, Ms. lusoria (e.g. Habe 1983, Matsukuma 2000, Min et al. Yuki Chikuchi, Dr. Akiko Ikeguchi, Mr. Shigeo Yamamoto, 2004). In the present study, the results of CDA revealed that Ms. Hisayo Kihara and Dr. Hiroshi Fukuda for their help in the shape of posterior-dorsal margin, shell breadth and collecting samples in Japan. We also thank Dr. Jae-Sang width of socket are more important to discriminate the two Hong, Mr. Kyung-Won Kim, Mr. Dong-Pil Oh, Mr. Kil- species than shell height and the other characters (Table 5). Wook Yoe, Mr. Yung-Ki Ju, Mr. Gyung-Wan Kim, Mr. It was also impossible to discriminate the two species based Dong-Min Kwak, Mr. Jong-Myoung Lee and members of on only shell color patterns (Torii, unpublished data). the Japan/Korea Tidal-flats Joint Survey Group for their Then, in order to simplify the method to identify M. pe- help in collecting samples in Korea. This work was partly techialis and M. lusoria from shell morphology, we modify supported by the Japanese Association of Benthology (for the discriminant score (D) using the 5 selected characters as H.T.), the Toyota Foundation, Takagi Fund for Citizen Sci- follows (Table 8A). ence and Grant-in-Aids for Scientific Research from the D110.2661.61(log B/log L)10.90(log SW/log L) Japan Society for the Promotion of Science (No. 15740308 81.72(log LPM/log L)27.27(log HPM/log L) . and 1965021 for S.S. and No. 19580210 for Y.H.). This discriminant score can indicate that when D value 0 that individual is identified as M. petechialis, and when References D 0 that individual is identified as M. lusoria. The D val- Chen AH, Li ZX, Feng GN (2009) Phylogenetic relationships of ues for almost all individuals of M. lusoria were less than 0 the genus Meretrix (Mollusca: Veneridae) based on mitochondr- (456 indiv./458 indiv., 99.6%), and those of M. petechialis ial COI gene sequences. Zool Res 30: 233–239. were more than 0 (78 indiv./82indiv., 95.1%) among the all Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA individuals studied in the present study (Fig. 6). The per- primers for amplification of mitochondrial cytochrome c oxi- Relationship between Meretrix lusoria and M. petechialis 241

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