Classification of common oysters from North China.
KEY WORDS: oyster, taxonomy, cytochrome oxidase I, 16S rRNA, 28S rRNA, Crassostrea gigas, C. ariakensis, Alectryonella plicatula, Saccostrea cucullata, Suminoe oyster
There are several unresolved issues in oyster classification. The problems are caused by classification based on shell morphology, which is highly variable in oysters. One of the problems is the taxonomic status of oysters found along China's north coast. Oysters are common and abundant on the rocky shores of North China. They are found on rocks, concrete and other hard surfaces in almost all intertidal zones and harbors. These oysters are typically small in size and highly variable in shell morphology. There is considerable confusion and disagreement about the taxonomic identity of these oysters.
According to existing literature, eight oyster species occur in North China: the Pacific oyster (Crassostrea gigas), zhe or folded oyster (C. plicatula [Gmelin, 1791]), Sengmao or monkhat oyster (Saccostrea cucullata [Born, 1778]), Dalianwan oyster (C. talienwhanensis), jinjiang or near-river oyster (C. rivularis, now C. ariakensis), Portuguese oyster (C. angulata [Lamark, 1819]), milin or dense-scale oyster (Ostrea denselamellosa [Lischke, 1869]), and maozhua or cat's paw oyster (Talonostrea talonata [Hanley, 1864]) (Zhang & Lou 1956, Qi 1989, Li & Qi 1994, Xu & Huang 1993, Guo et al. 1999, Yu et al. 2003, Wang, et al. 2004, Lapegue et al. 2004). The latter two species are well defined, and there is no dispute about their taxonomic status. The dense-scale oysters are characterized by large (up to 15 cm), round and flat shells with densely populated layers of scales along the edge. The cat's paw oyster is small in size (2-3 cm), and its left shell has 5-8 ribs protruding out in the shape of cat's paws. These two species are relatively rare, but they can be easily identified and separated from other species.
Zhang & Lou (1956) classified the common oysters from North China as the following: (1) the monk-hat oyster (O. cucullata), a small sized oyster attached to rocks in shallow water between high and low tidal lines; (2) the Pacific oyster (C. gigas), a large sized and long shaped oyster found below the low tidal line in moderate and high salinity waters; (3) Dalianwan oyster (C. talienwhanensis), a large, triangular shaped oyster with purple spots on shells found throughout north China; (4) the near-river oyster (C. rivularis) with large and highly variable shells, found mostly in low and intermediate (10 [per thousand] to 25 [per thousand]) salinity waters. According to Zhang & Lou (1956), the oysters that are commonly found on intertidal rocks should be the monk-hat oyster.
Zhao et al. (1982) named the oyster commonly found on the rocks of Dalian coast as the folded oyster (C. plicatula). Since then, there has been no agreement whether the common intertidal oysters from north China is O. cucullata or C. plicatula, or both (Zhang & Lou 1956, Qi 1989, Guo et al. 1999, Yu et al. 2003, Xu & Huang 1993). Li & Qi (1994) considered that the O. cucullata, C. rivularis, and C. talienwhanensis of Zhang & Lou (1956) and C. plicatula of Zhao et al. (1982) were all C. gigas. They identified the monk-hat and folded oysters as Saccostrea cucullata and Alectryonella plicatula, respectively, two species of different genus found in southern China.
However, after analyzing genetic variation revealed by random amplified polymorphic DNA (RAPD), Liu & Dai (1998) concluded that C. talienwhanensis, C. plicatula, and C. rivularis were three different species. An analysis of 16S and COI sequences by Yu et al. (2003) indicated that C. gigas and C. talienwhanensis were the same species, and C. plicatula might be a morph of C. ariakensis. Further, Lapegue et al. (2004) reanalyzed the sequences of Yu et al. (2003) and showed that some of the C. talienwhanensis samples might be C. angulata.
The confusion about the taxonomic status of common oysters from north China calls for further analysis. In this study, we collected and sequenced oysters from 9 locations along China's northern coast for fragments of three genes: the mitochondrial 16S rRNA gene and cytochrome oxidase I (COI), and the nuclear 28S rRNA. Here we provide molecular evidence that the small oysters commonly found on rocky shore belong to one species--C, gigas, and the large oysters (referred to as the near-river oysters) collected from Weifang are the same species as C. ariakensis (Wakiya, 1929).
MATERIALS AND METHODS
Sample Collection and Morphological Analyses
Oysters were collected from nine typical habitats along China's coast north of Yangtze River: Zhuanghe (ZH), Zhangzidao (ZZD), Dalian (DL) in Liaoning Province; Dongying (DY), Weifang (WF), Rongcheng (RC), Rushan (RS) and Qingdao (QD) in Shandong Province; and Lianyungang (LYG) in Jiangsu Province (Fig. 1). At Zhuanghe, Zhangzidao, Dongying, Rongcheng, Qingdao, and Lianyungang oysters were collected from rocks in intertidal zones. At Weifang, oysters were collected by divers from a depth of about eight meters. Oysters from Dalian and Rushan were cultured oysters derived from local wild seed.
The following shell characteristics were recorded: shell morphology, shell wall rigidity or thickness, valve lamellae, radial ribs, shell color, ligament channel, umbonal cavity volume, interior shell color, adductor muscle scar color and shape. Shell parameters were identified following definitions of Moore et al. (1971).
DNA Extraction, PCR Amplification and Sequencing
Six oysters from each site were selected for sequencing (Table 1). Oysters with variable shell morphology were chosen to cover all possible species. DNA was extracted from fresh adductor muscle tissue using phenol/chloroform extraction as described by Moore (1993). Primers (Invitrogen, USA), 16sar and 16sbr, were used to amplify a segment of the mitochondrial 16S ribosomal RNA gene (Palumbi et al. 1991). A segment of cytochrome oxidase subunit I (COI) was amplified using LCO1490 and HCO2198 primers (Folmer et al. 1994). A 28S rRNA fragment was amplified using primers D1F and D6R as described by Park and O Foighil (2000). PCR amplification was performed using a Biometra T1 thermal cycler. Reactions were performed in 50 [micro]L with final concentrations of: 2.0 mM Mg[Cl.sub.2], 150 [micro]M of each dNTP, 0.2 [micro]M each primer, 20 ng of template DNA, and 2 units of Taq polymerase (Promega, USA) in 5 [micro]L of x 10 buffer. COI and 16S were amplified using the following protocol: initial denature at 95[degrees]C for 2 min, 30 cycles of 95[degrees]C for 1 min, 51[degrees]C (COI) or 57[degrees]C (16S) for 1 min, and 72[degrees]C for 1 min with a final extension at 72[degrees]C for 5 min. The 28S fragment was amplified by an initial denaturing at 94[degrees]C for 4 min, addition of Taq polymerase; 30 cycles of denaturing at 94[degrees]C for 40 s, annealing at 60[degrees]C for 40 s and extension at 72[degrees]C for 1.25 min; plus a final extension at 72[degrees]C for 10 min. A negative control (no template) was included during each PCR run. PCR products were purified using EZ Spin Column PCR Product Purification Kit (BBI, Canada). Sequencing was performed in both directions on an ABI PRISM 377XL DNA sequencer using the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer, USA).
[FIGURE 1 OMITTED]
The COI, 16S, and 28S sequences obtained in this study and those of other Crassostrea, Ostrea and Saccostrea species available from GenBank were used for phylogenetic analysis. Initial alignments were performed using CLUSTALW (Thompson et al. 1994). The sequences were trimmed to the same length as other published sequences after alignment. Maximum parsimony (MP) and neighbor-joining (NJ) analyses were performed using PHYLIP (Vet. 3.56C, Felsenstein 1989). Bootstrap analysis with 1,000 replications was performed using the SEQBOOT and CONSENSE programs. Consensus phylogenetic trees were drawn with DRAWGRAM program in the PHYLIP package. Pair-wise sequence divergences among haplotypes and reference species were calculated using the DNADIST program of PHYLIP according to Kimura's two-parameter model (Kimura 1980).
Distribution and Morphology of Oysters from North China
Oysters are common and abundant at all nine sites sampled in this study (Table 1). Shell morphology varies greatly within and among sites. In general, oysters collected below or at the low tidal line are larger than oysters collected high above the low tidal line, so are the cultured oysters obtained from farms.
[FIGURE 2 OMITTED]
At most sites (ZH, ZZD, DY, RC, QD and LYG), oysters were collected from rocks at or above the low tidal line. These oysters are typically small (30-60 mm), flat and irregular in shape (Fig. 2). The color of the shells is variable ranging from white to yellow to brown. Some oysters especially those from Rongcheng have purple strips on the right valve. The shell's edge is highly variable: some smooth and some with waving plates protruding. Internally, the shells are milky white, sometimes with areas of gray, yellow, or brown. Muscle scars range from pink to dark purple. Ligament channels are mostly short with some exceptions.
Oysters collected from deep waters at Weifang are noticeably different from oysters from other sites. They are large, up to 200 mm, and some are round and horse-shoe shaped. The shells are thick and relatively smooth with multiple layers of overlapping and concentric plates on the right valve. Radiant ribs are absent or weak. Shell color is variable, ranging from yellow to brown (Fig. 2E). Ligament channels are short and shallow. The umbo cavity is shallow to medium. The internal side of shells is milky white with areas chalky or lacking of the nacre layer. The adductor muscle scar has no coloration, which is unique for the subtidal oysters from Weifang.
The cultured oysters from Dalian and Rushan are large (100-200 mm), elongated or ovate. The left valve is convex with obvious radial ribs. The right valve is flat. Shell color ranges from white, yellow to brown, and often with purple strips. The internal sides of the shells are white with pink to purple muscle scars. Ligament channels are shallow, and the umbo cavity is deep (Fig. 2C, G).
Mitochondrial 16S rRNA Sequence
A 497 bp fragment of the mitochondrial 16S rRNA gene was sequenced for 54 individuals collected from nine sites (Table 1). Five haplotypes (Haplotype 1, 2, 3, 4, 5) were identified among the 54 sequences. Oysters from Weifang had two haplotypes (Haplotype 4, 5), with five oysters having Haplotype 4 and one oyster having Haplotype 5 (Table 2). Oysters from the other eight sites had 3 haplotypes (Haplotype 1, 2, 3). Haplotype 1 is the common haplotype and shared by 46 of the 48 oysters. One oyster from Dongying has Haplotype 2, and one oyster from Qingdao has Haplotype 3 (Table 2).
[FIGURE 3 OMITTED]
Phylogenetic analysis was conducted with the five 16S haplotypes identified in this study and the following reference sequences from GenBank: C. gigas (S66183), C. ariakensis (Kim AY007427), S. cucullata (AF498507), C. virginica (AF092285), C. rhizophorae (AJ312938), C. gasar (AJ312937), C. hongkongensis (AY160756), and C. sikamea (AY632551). S. cucullata was used as an outgroup. Including the outgroup, 26 nucleotide positions were variable in the 16S data set. Phylogenetic analysis using NJ and MP procedures produced almost identical results (Fig. 3A, B). As expected, all five haplotypes identified in this study were clustered with members of Crassostrea, away from S. cucullata. The two haplotypes from Weifang were closely clustered with C. ariakensis, and formed one clade. The two haplotypes differed from the reference sequence by only one base. A closer examination indicated that Haplotype 4 was the same sequence as that of C. ariakensis from Korea (Kim et al. 2000). The three haplotypes from all other eight sites were closely clustered with C. gigas. Haplotype 1 is identical with the reference sequence from C. gigas. Haplotype 2 and Haplotype 3 differed from the C. gigas sequence by one base. Sequence divergence between Haplotypes 1-3 and Haplotypes 4-5 was about 4.65% to 5.10%, which was higher than that between C. gigas and C. sikamea (1.73%) (Table 3). No divergence was observed between Haplotype 1 and C. gigas, the divergence between Haplotype 2/3 and C. gigas was only 0.21%. There was no divergence between Haplotype 4 and C. ariakensis from Korea, and the divergence between Haplotype 5 and C. ariakensis was only 0.36%, which is much lower than that between C. gigas and C. sikamea (1.73%). These data show that oysters from Weifang are C. ariakensis, and oysters from all other sites are C. gigas. The haplotypes obtained from this study are neither S. cucullata nor A. plicatula, as evidenced by the high divergences, 17.44% to 21.95% (Table 3).
[FIGURE 4 OMITTED]
Mitochondrial COI DNA Sequence
A 607 bp fragment of the COI sequences was sequenced for the 54 oysters selected in this study, generating 13 haplotypes (Table 4). Three (Haplotype 11, 12, 13) of these haplotypes occurred in Weifang oysters, four oysters had Haplotype 11, and the other two oysters had Haplotype 12 and 13, respectively. The majority of oysters from the other eight sites (38 of 48) shared a common haplotype--Haplotype 1. Haplotype 2 was shared by two oysters from Dalian. Haplotypes 3-10 were represented by one oyster each from variable sites (Table 4).
The partial COI sequences from the 13 haplotypes obtained in this study were subjected to phylogenetic analysis along with the following reference sequences from GenBank: C. gigas (AF152565), C. iredalei (AY038078), C. belcheri (AY038077), C. nippona (AF300616), C. hongkongensis (AY160746), C. virginica (AF152566), S. cucullata (AY038076), and C. sikamea (AY632568). S. cucullata was used as an outgroup. Including the outgroup, 121 nucleotide positions were variable in the COI data set. The NJ and MP trees were nearly identical, and both supported the same general patterns: (1) the oysters in Weifang clustered together with C. ariakensis; (2) the oysters from other sites clustered together with C. gigas (Fig. 4). Haplotype 1 is identical to the C. gigas sequence, and Haplotypel 1 is identical to C. ariakensis.
Sequence divergence between Haplotypes 1-10 and Haplotypes 11-13 ranged from 16% to 16.7%. There was no divergence between Haplotype 1 and C. gigas, and between Haplotype 11 and C. ariakensis. The divergence between Haplotypes 2 ~ 10 and C. gigas was about 0.2% ~ 0.4%. The divergence between Haplotype 12 ~ 13 and C. ariakensis was 0.2%. These levels of divergence were consistent with intraspecific variation and considerably smaller than that between C. gigas and C. sikamea (9.74%) and that between C. gigas and C. angulata (2.46%). Divergences between the 13 haplotypes of this study and that of C. sikamea, C. ariakensis, S. cucullata were too high (9.74% to 31.70%) to be conspecific (Table 5).
Nuclear 28S rRNA Sequence
A 945 bp fragment of the nuclear 28S rRNA gene was sequenced for all 54 oysters collected in this study. Only two haplotypes were identified: one (Haplotype 1) for the 48 oysters from ZH, ZZD, DL, DY, RC, RS, QD, and LYG and the other (Haplotype 2) for the six oysters from Weifang.
Phylogenetic analysis was conducted with the two haplotypes identified in this study and reference sequences from GenBank: C. gigas (AF 137051), C. ariakensis (AF137052), C. virginica (AF137050), C. rhizophorae (AF137049), C. belcheri (Z29545), S. cucullata (Z29553), S. commercialis (Z29552), and C. sikamea (AY632554). S. cucullata was used as an outgroup. Including the outgroup, 33 nucleotide positions were variable in the 28S data set. In NJ and MP trees, oysters from Weifang formed one clade with C. ariakensis, and oysters from other sites were identified with C. gigas (Fig. 5).
No divergence was observed between Haplotype 1 and C. gigas and between Haplotype 2 and C. ariakensis, and the oysters collected in this study were clearly different from C. sikamea, S. cucullata, and A. plicatula (Table 6).
Classification of the Oysters in North China
The taxonomic status of the oysters in north China has been the subject of much confusion. Because the oysters are typically found on intertidal rocks, their morphological characteristics vary greatly because of the stressful and variable environment they live in. Consequently, classification based on shell characteristics has been difficult, and different studies have reached different conclusions (Zhang & Lou 1956, Qi 1989, Li & Qi 1994, Xu & Huang 1993). The existence of C. gigas and C. rivularis in north China is well accepted, although the two species have often been misidentified by different authors (Zhang & Lou 1956, Li & Qi 1994). It has been shown that the C. rivularis from Bohai Sea in north China is the same species as C. ariakensis (Wang et al. 2004). Two questions remain unresolved: (1) what species are the small oysters that are commonly found in intertidal zones along the northern coast? and (2) is C. talienwhanensis an independent species?
[FIGURE 5 OMITTED]
The Small Intertidal Oysters
The small intertidal oysters from north China were first named the monk-hat oyster (O. cucullata) by Zhang & Lou (1956) and then the folded oyster C. plicatula by Zhao et al. (1982). After analyzing anatomic characteristics, Li & Qi (1994) concluded that neither classification was correct, and the small intertidal oysters were actually C. gigas. On the other hand, a genetic analysis suggested that C. plicatula was an independent species (Liu & Dai 1998).
In this study, we collected the common oysters from nine sites and sequenced three gene fragments for phylogenetic analysis. Our data support Li & Qi's (1994) classification that the small intertidal oysters from all sites (except Weifang) are C. gigas. This is clear from all three genes studied. At the mtl6S gene, the common haplotype shared by 96% of the oysters from the eight sites (excluding Weifang) are identical to that of C. gigas. The remaining two haplotypes are minor variants of C. gigas haplotypes, as shown by the small sequence divergences (Table 3). For the mtCOI gene, although many haplotypes are observed, the common haplotypes are identical with that of C. gigas, and all other haplotypes are minor variants and closely clustered with C. gigas (Fig. 4). The highly conserved 28S rRNA genes show that all intertidal oysters have the same haplotypes as C. gigas.
The small intertidal oysters are clearly not O. cucullata or C. plicatula, two species now known as S. cucullata and A. plicatula, respectively (Li & Qi 1994). This is evident from both morphological and molecular data. The molecular data show that the divergence between the oysters sampled in this study and S. cucullata is larger than that between any sister-species within Crassostrea. Similarly, oysters from this study are clearly not the folded oyster (A. plicatula) as indicated by large divergences in mtl6S and 28S sequences (Table 3 and 6).
Except for the small size, the intertidal oysters collected in this study have similar characteristics as C. gigas (Li & Qi 1994, Torigoe 1981, Okutani 2000), albeit more variable. The small size is probably caused by limitations in food as a consequence of living in the intertidal zone. The strong wave action may also force oysters to stick close to the substrate, causing the shells to be flat and irregular in shape.
The Large Subtidal Oysters from Weifang
In shell morphology and DNA sequences, oysters collected from deep waters at Weifang are different from oysters from other sites. In morphology, the Weifang oysters have distinctive smooth shells with layered concentric plates and a white adductor muscle scar. In DNA sequence, all three genes show that the Weifang oysters are different from oysters from other sites (shared no haplotypes). Phylogenetic analysis clearly indicates that the Weifang oysters, which are referred to as jinjiang or near-river oyster C. rivularis locally, are indeed C. ariakensis. This finding is in agreement with our previous studies (Wang et al. 2004, Wang 2004). The shell morphology of the Weifang oyster is almost identical to that of C. ariakensis from Ariake Bay in Japan (Okutani 2000).
Status of the Dalianwan Oyster (C. talenwhanesis)
Some believe C. talienwhanensis is an independent species (Zhang & Lou 1956, Liu & Dai 1988), whereas others consider it a synonym of C. gigas (Li & Qi 1994). We consciously collected oysters from three sites in and around Dalian. Some of the oysters we collected, especially those from Dalian and Dongying had obvious radial ribs and purple strips that are characteristic of C. talienwhanesis. However, the molecular data indicate that they are the same species as C. gigas. It is possible that our sampling missed true C talienwhanensis, but available data from this study do not support the existence of the Dalianwan oyster (C. talienwhanensis) as an independent species.
Lapegue et al. (2004) reanalyzed the sequences of Yu et al. (2003) and showed that some of the C. talienwhanensis sequences are possibly C. angulata. Our analysis finds no C. angulata from Dalian or any of the nine sites in north China. The COI haplotypes obtained from this study are clearly different from that of C. angulata (Table 5). In another analysis of thousands of oysters from China, we observed C. angulata in southern China but never north of Yangtze River (Guo et al. 2006). It is possible that our sampling missed C. angulata from north China. It is also possible that the C. angulata samples of Yu et al. (2003) may not be native to Dalian as there is considerable cross-regional transportation of oyster seed in China. Anyway, further research is needed to determine if there is C. angulata in north China.
In summary, this study provides clear molecular evidence that the common oysters from north China belong to two species: the small intertidal oysters are C. gigas and the large subtidal oysters from Weifang are C. ariakensis. We see no evidence for the existence of C. angulata and C. talienwhanensis in Dalian, and the latter is probably synonymous with C. gigas.
The authors thank Fengshan Xu, Suping Zhang, and Xiaoxu Li for advice on oyster taxonomy. This work is supported by grants from National Science Foundation of China (No.40406032 to Wang, No. 39825121 to Guo and No. 40730845 to Zhang), SEPAC (species 07-2-9), and the US NOAA CBO Non-native Oyster Research Program (NA04NMF4570424). This is contribution No. IMCS-2008-7 and NJSG-08-688.
Felsenstein, J. 1989. PHYLIP--Phylogeny Inference Package (Version 3.2). Cladistics 5:164-166.
Folmer, O., M. Black, W. Hoeh, R. Lutz & R. Vrijenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrate. Mol. Mar. Biol. Biotechnol. 3:294-299.
Guo, X., S. Ford & F. Zhang. 1999. Molluscan aquaculture in China. J. Shellfish Res. 18:19-31.
Guo, X., G. Zhang, L. Qian, H. Wang, X. Liu & A. Wang. 2006. Oysters and oyster farming in China: a review. J. Shellfish Res. 25:734.
Kim, S., S. Lee, D. Park & H. An. 2000. Phylogenetic relationship among four species of Korean oysters based on mitochondrial 16S rDNA and COl gene. Korean J. Biol. Sci. 16:203-211.
Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111-120.
Lapegue, S., M. Frederico, S. Heurtebise, Z. Yu & P. Boudry. 2004. Evidence for the presence of the Portuguese oyster, Crassostrea angulata, in Northern China. J. Shellfish Res. 23:759-763.
Li, X. & Z. Qi. 1994. Studies on the comparative anatomy, systematic classification and evolution of Chinese oysters. Studia Marina Sinica 35:143-173. (In Chinese)
Liu, B. & J. Dai. 1998. Studies on genetic diversity of oysters of genus Crassostrea. J. Fish. China 22:193-198. (In Chinese)
Moore, D. 1993. Preparation of genomic DNA from mammalian tissue. In: F. M. Ausubel, editors. Current protocols in molecular biology. Vol. 1. New York: John Wiley. pp. 1-2.
Moore, R.C., C. Teichert, L. Mccormick & R.B. Williams. 1971. Treatise on invertebrate paleontology, Part N, 3(3). Lawrence: The Geological Society of America, Inc. and the University of Kansas.
Okutani, T. 2000. Marine mollusks in Japan. Shizuoka: Tokai University. pp. 923-927.
Palumbi, S. R., A. Martin & S. Romano. 1991. The simple fool's guide to PCR. Honolulu: University of Hawaii Press.
Park, J. K. & D. O. Foighil. 2000. Sphaeriid and corbiculid clam represent separate heterodont bivalve radiations into freshwater environments. Mol. Phylogenet. Evol. 14:75-88.
Qi, Z. 1989. Mollusk of Yellow Sea and Bohai Sea. Beijing: Agricultural Publishing House. pp. 176-180. (In Chinese).
Thompson, J. D., D. G. Higgins & T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4672-4680.
Torigoe, K. 1981. Oysters in Japan. J. Sci. Hiroshima Univ. Ser. B. Div. 129:291-481.
Wang, H., X. Guo, G. Zhang & F. Zhang. 2004. Classification of jinjiang oysters Crassostrea rivularis (Gould, 1861) from China based on morphology and phylogenetic analysis. Aquaculture 242:137-155.
Wang, H. 2004. Studies on the molecular phylogeny and taxonomy of common oysters in China seas. Doctoral dissertation, Institute of Oceanology, Chinese Academy of Sciences, Qingdao.
Xu, F. & X. Huang. 1993. The new record of Ostreacea in China sea. Studia Marina Sinica 34:175-179. (in Chinese)
Yu, Z., X. Kong, L. Zhang, X. Guo & J. Xiang. 2003. Taxonomic status of four Crassostrea oysters from China as inferred from mitochondriai DNA sequences. J. Shellfish Res. 22:31-38.
Zhang, X. & Z. Lou. 1956. Studies on oysters in China. Acta. Zool. Sinica 8:65-94. (In Chinese)
Zhao, R., J. Cheng & D. Zhao. 1982. The marine invertebrate from Dalian, China. China Ocean Press 167:1-11. (In Chinese)
HAIYAN WANG, (1,2) GUOFAN ZHANG, (1) XIAO LIU (1) AND XIMING GUO (2) *
(1) Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, People's Republic of China," (2) Haskin Shellfish Research Laboratory, Institute of Marine and Coastal Sciences, Rutgers University, 6959 Miller Avenue, New Jersey 08349
* Corresponding author. E-mail: email@example.com
TABLE 1. Location and number of oysters sequenced. Location Code No. Sequenced Zhuanghe, Liaoning Province ZH 6 Zhangzidao, Liaoning Province ZZD 6 Dalian, Liaoning Province DL 6 Dongying, Shandong Province DY 6 Weifang, Shandong Province WF 6 Rongcheng, Shandong Province RC 6 Rushan, Shandong Province RS 6 Qingdao, Shandong Province QD 6 Lianyungang, Jiangsu Province LYG 6 TABLE 2. Distribution of the five 16S rRNA haplotypes obtained in this study. Haplotype Site 1 2 3 4 5 ZH 6 0 0 0 0 ZZD 6 0 0 0 0 DL 6 0 0 0 0 DY 5 1 0 0 0 WF 0 0 0 5 1 RC 6 0 0 0 0 RS 6 0 0 0 0 QD 5 0 1 0 0 LYG 6 0 0 0 0 TABLE 3. Pair-wise divergence among five mt16S rRNA haplotypes obtained in this study (1-5) and reference species. 1 2 3 4 5 1 (all) 0 2 (DY) 0.0021 0 3 (QD) 0.0021 0.0043 0 C. gigas 0.0000 0.0021 0.0021 0 C. sikamea 0.0173 0.0195 0.0195 0.0173 0 4 (WF) 0.0488 0.0510 0.0510 0.0488 0.0442 5 (WF) 0.0465 0.0488 0.0488 0.0465 0.0419 C. ariakensis 0.0465 0.0488 0.0488 0.0465 0.0419 C. hongkongensis 0.0239 0.0261 0.0261 0.0239 0.0239 C. gasar 0.1967 0.1967 0.1995 0.1967 0.1908 C. rhizophorae 0.1859 0.1859 0.1831 0.1859 0.1884 C. virginica 0.2027 0.206 0.1993 0.2027 0.2156 S. cucullata 0.1772 0.1799 0.1744 0.1772 0.1747 Alectryonella plicatula 0.2165 0.2195 0.2135 0.2165 0.2139 6 7 8 9 10 1 (all) 2 (DY) 3 (QD) C. gigas C. sikamea 4 (WF) 0 5 (WF) 0.0021 0 C. ariakensis 0.0000 0.0036 0 C. hongkongensis 0.0285 0.0263 0.0307 0 C. gasar 0.1995 0.1995 0.1967 0.1967 0 C. rhizophorae 0.2034 0.2034 0.2005 0.1943 0.1181 C. virginica 0.2305 0.2271 0.2271 0.2194 0.2349 S. cucullata 0.1744 0.1717 0.1717 0.1690 0.2567 Alectryonella plicatula 0.1980 0.1980 0.1951 0.2046 0.2960 11 12 13 14 1 (all) 2 (DY) 3 (QD) C. gigas C. sikamea 4 (WF) 5 (WF) C. ariakensis C. hongkongensis C. gasar C. rhizophorae 0 C. virginica 0.1446 0 S. cucullata 0.2335 0.2913 0 Alectryonella plicatula 0.2576 0.3308 0.1744 0 TABLE 4. Distribution of 13 mt COI haplotypes obtained in this study. Haplotype Site 1 2 3 4 5 6 7 ZH 4 0 0 0 1 1 0 ZZD 4 0 0 0 0 0 0 DL 4 2 0 0 0 0 0 DY 5 0 1 0 0 0 0 WF 0 0 0 0 0 0 0 RC 5 0 0 0 0 0 1 RS 5 0 0 0 0 0 0 QD 5 0 0 1 0 0 0 LYG 6 0 0 0 0 0 0 Haplotype Site 8 9 10 11 12 13 ZH 0 0 0 0 0 0 ZZD 0 1 1 0 0 0 DL 0 0 0 0 0 0 DY 0 0 0 0 0 0 WF 0 0 0 4 1 1 RC 0 0 0 0 0 0 RS 1 0 0 0 0 0 QD 0 0 0 0 0 0 LYG 0 0 0 0 0 0 TABLE 5. Pair-wise divergence among 13 COI sequences obtained in this study and reference species. 1 2 3 4 5 1 (all) 0 2(DL) 0.0017 0 3(DY) 0.0017 0.0035 0 4(QD) 0.0035 0.0052 0.0052 0 5(ZH) 0.0017 0.0035 0.0035 0.0052 0 6(ZH) 0.0035 0.0052 0.0052 0.0070 0.0052 7(RC) 0.0017 0.0035 0.0035 0.0052 0.0035 8(RS) 0.0017 0.0035 0.0035 0.0052 0.0035 9(ZZD) 0.0017 0.0035 0.0035 0.0017 0.0035 10(ZZD) 0.0017 0.0035 0.0035 0.0052 0.0035 C. gigas 0.0000 0.0017 0.0017 0.0035 0.0017 C. angulata 0.0246 0.0246 0.0263 0.0281 0.0263 C. sikamea 0.0974 0.0995 0.0994 0.0974 0.0994 11(WF) 0.1622 0.162 0.1643 0.1622 0.1601 12(WF) 0.1643 0.1641 0.1664 0.1643 0.1622 13(WF) 0.1645 0.1643 0.1666 0.1645 0.1624 C. ariakens 0.1622 0.162 0.1643 0.1622 0.1601 C. hongkong 0.1327 0.1349 0.1347 0.1327 0.1347 C. nippona 0.1618 0.1616 0.1639 0.1639 0.1639 C. belcheri 0.1777 0.1775 0.1799 0.1777 0.1756 C. iredalei 0.1817 0.1793 0.1839 0.1861 0.1795 C. virginica 0.2677 0.2705 0.2702 0.2726 0.2652 S. cucullata 0.3143 0.3139 0.3170 0.3117 0.3170 6 7 8 9 10 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 0 7(RC) 0.0052 0 8(RS) 0.0052 0.0035 0 9(ZZD) 0.0052 0.0035 0.0035 0 10(ZZD) 0.0052 0.0035 0.0035 0.0035 0 C. gigas 0.0035 0.0017 0.0017 0.0017 0.0017 C. angulata 0.0281 0.0263 0.0263 0.0263 0.0228 C. sikamea 0.1013 0.0994 0.0994 0.0955 0.0955 11(WF) 0.1622 0.1643 0.1643 0.1601 0.1622 12(WF) 0.1643 0.1664 0.1664 0.1622 0.1643 13(WF) 0.1645 0.1666 0.1666 0.1624 0.1645 C. ariakens 0.1622 0.1643 0.1643 0.1601 0.1622 C. hongkong 0.1368 0.1347 0.1347 0.1306 0.1347 C. nippona 0.1660 0.1639 0.1618 0.1618 0.1639 C. belcheri 0.1777 0.1756 0.1777 0.1799 0.1756 C. iredalei 0.1817 0.1839 0.1839 0.1839 0.1795 C. virginica 0.2677 0.2652 0.2677 0.2702 0.2677 S. cucullata 0.3143 0.3170 0.3117 0.3117 0.3143 11 12 13 14 15 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 7(RC) 8(RS) 9(ZZD) 10(ZZD) C. gigas 0 C. angulata 0.0246 0 C. sikamea 0.0974 0.0960 0 11(WF) 0.1622 0.1680 0.1660 0 12(WF) 0.1643 0.170 0.1680 0.0020 0 13(WF) 0.1645 0.1710 0.1660 0.0020 0.0040 C. ariakens 0.1622 0.1680 0.1660 0.0000 0.0020 C. hongkong 0.1327 0.1390 0.1480 0.1400 0.1380 C. nippona 0.1618 0.170 0.1630 0.1500 0.1480 C. belcheri 0.1777 0.1750 0.1660 0.1740 0.1760 C. iredalei 0.1817 0.1790 0.1900 0.1780 0.1800 C. virginica 0.2677 0.2670 0.2740 0.2970 0.2990 S. cucullata 0.3143 0.3140 0.3350 0.3350 0.3380 16 17 18 19 20 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 7(RC) 8(RS) 9(ZZD) 10(ZZD) C. gigas C. angulata C. sikamea 11(WF) 12(WF) 13(WF) 0 C. ariakens 0.0020 0 C. hongkong 0.1420 0.1400 0 C. nippona 0.1520 0.1500 0.1280 0 C. belcheri 0.1740 0.1740 0.1790 0.1790 0 C. iredalei 0.1780 0.1780 0.1750 0.1650 0.1770 C. virginica 0.2940 0.2970 0.2820 0.270 0.2760 S. cucullata 0.3350 0.3350 0.3320 0.3170 0.3280 21 22 23 1 (all) 2(DL) 3(DY) 4(QD) 5(ZH) 6(ZH) 7(RC) 8(RS) 9(ZZD) 10(ZZD) C. gigas C. angulata C. sikamea 11(WF) 12(WF) 13(WF) C. ariakens C. hongkong C. nippona C. belcheri C. iredalei 0 C. virginica 0.2810 0 S. cucullata 0.2970 0.4000 0 TABLE 6. Pair-wise divergence among two 28S rRNA sequences obtained from this study and reference species. 1 2 3 4 1 (all) 0 C. gigas 0.0000 0 2 (WF) 0.0111 0.0111 0 C. ariakensis 0.0111 0.0111 0.0000 0 C. sikameal 0.0033 0.0033 0.0100 0.0100 C.sikamea2 0.0022 0.0022 0.0089 0.0089 C. belcheri 0.0271 0.0271 0.0294 0.0294 C. rhizophorae 0.0798 0.0798 0.0797 0.0797 C. virginica 0.0684 0.0684 0.0707 0.0707 S. cucullata 0.0685 0.0685 0.0709 0.0709 Alectryone plicatula 0.0957 0.0957 0.0983 0.0983 5 6 7 8 1 (all) C. gigas 2 (WF) C. ariakensis C. sikameal 0 C.sikamea2 0.0000 0 C. belcheri 0.0260 0.0249 0 C. rhizophorae 0.0835 0.0824 0.0853 0 C. virginica 0.0709 0.0697 0.0774 0.0848 S. cucullata 0.0711 0.0699 0.0776 0.0876 Alectryone plicatula 0.0970 0.0959 0.1130 0.1222 9 10 11 1 (all) C. gigas 2 (WF) C. ariakensis C. sikameal C.sikamea2 C. belcheri C. rhizophorae C. virginica 0 S. cucullata 0.0090 0 Alectryone plicatula 0.0807 0.0835 0