Morphological and genetic identification of the validity of the species Atrina chinensis (Bivalvia: pinnidae).
|Subject:||Noble pen shell (Genetic aspects)|
|Publication:||Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2012 National Shellfisheries Association, Inc. ISSN: 0730-8000|
|Issue:||Date: August, 2012 Source Volume: 31 Source Issue: 3|
|Geographic:||Geographic Scope: China Geographic Code: 9CHIN China|
ABSTRACT Identification of pinnid species is based largely on
morphological characteristics that are highly plastic; thus,
classification of pinnids remains controversial. We identified a species
of Atrina, found along the southern China coast, as A trina chinensis
Deshayes, 1841, but other authors have treated it as a synonym of Atrina
pectinata Linnaeus, 1767. The objective of this study was to clarify the
taxonomic status of this species by comparing both morphological and
genetic data with data from other Atrina species. Of the 4 shell
parameters analyzed, only 1 (size of the posterior adductor) differed
significantly between A. peetinata and A. chinensis. However, these
species did not form a clade on the phylogenetic trees constructed based
on nuclear 28S rRNA or the mitochondrial cytochrome oxidase I (mtCOI)
and 16S rRNA genes. Moreover, A. chinensis is, genetically, is a sister
taxon to Atrina vexillum instead of A. pectinata. We suggest that A.
chinensis is a valid taxon and not a synonym of A. pectinata.
KEY WORDS: pen shell, Atrina chinensis, Atrina pectinata, morphological variance, 28S rRNA, mtCOI, 16S rRNA
The pen shells of the family Pinnidae are commercially important in a number of Indo-Pacific countries. They are economically valuable species because they have a large and edible posterior adductor muscle. Pinnid species are a challenging group for taxonomists and phylogeneticists because of the phenotypic plasticity of their shells. For example, the pinnid species referred to as the "scabrous" pen shell in southern China has been identified by various authors as Atrina pectinata (Winckworth 1929), Atrina chinensis (Deshayes 1841, Huber 2010), Atrina lurida (Reeve 1858-1859), Atrina chemnitzii (Hanley 1858), and Atrina lamellata (Habe 1964, Habe 1980, Okutani 2000). Based on morphological characteristics and distribution, the scabrous pen shell from southern China matches A. chinensis (Deshayes 1841) rather than A. pectinata (Linnaeus 1767). However, this species was treated as a synonym of A. pectinata by Winckworth (1929), Rosewater (1961), Wang (1964, 1997), and Liang et al. (1986). Results of recent studies based on morphological differences (Wang et al. 2000), isozyme phenotypic divergence (Wang et al. 2000), and random amplified polymorphic DNA (Yu et al. 2000) of 4 forms of A. pectinata from China suggested that the scabrous pen shell may be a distinct species rather than a synonym of A. pectinata. In this study, we collected samples of A. chinensis from 5 typical sites in southern China, A. pectinata from northern China, and other related pinnid species. Two mitochondrial genes (mitochondrial cytochrome oxidase I (mtCOI) and 16S rRNA) and 1 nuclear gene (28S rRNA) were analyzed, as was the morphology of the specimens, and a phylogenetic analysis was conducted. This study is the first attempt at clarifying the taxonomic status of A. chinensis using molecular genetic information. The results may prove useful for the development of aquaculture of A. chinensis, and for the conservation and sustainable management of its genetic resources.
MATERIALS AND METHODS
Sample Collection and Morphological Analyses
Fifty-six individuals of A. chinensis were collected from 5 sites along the coast of southern China (Figs. 1 and 2). For comparative purposes, 118 individuals of A. pectinata were collected from 4 locations along the coast of northern China. Two individuals of A trina vexillum and two individuals of Pinna bicolor also were collected from Beibu Gulf and used in the phylogenetic analyses. Table 1 shows the number of individuals collected and sequenced. All samples were transported alive to the laboratory for morphological measurements, and then a muscle sample was taken from each individual and preserved in absolute ethyl alcohol.
Shell height (SH), shell length (SL), shell width (SW) and the major and minor axis (MA and MiA) lengths of the posterior adductor scar were measured to an accuracy of 0.1 mm using a Verifier caliper (Liang et al. 1986, Yu et al. 2000). To eliminate the impact of body size, relative ratios for each measurement were used for shape discrimination and analysis (SL/SH, SL/SW, and SH/SW). The comparative size of the posterior adductor scar (CSPAS) was also calculated as follows (Yu et al. 2000):
CSPAS = SW X [[pi] x MA X MiA]/[4 X SH X SL]
SPSS (version 13.0) was used to conduct the statistical analyses. One-way analysis of variance (ANOVA), followed by Tukey's test for multiple comparisons, was used to compare the 4 morphological parameters among samples of A. chinensis and A. pectinata collected from 9 different sites.
DNA was isolated from ethanol-fixed adductor muscle tissue using the TIANamp marine animals DNA kit (Tiangen, Beijing, China). The PCR amplification for 3 target gene fragments (nr28S, mtCOI, and 16S) used the universal nr28S primers D1F and D6R (Park & Foighil 2000), mtCOI primers LCO 1490 and HCO2198 (Folmer et al. 1994), and mt 16S primers 16sar and 16sbr (Kessing et al. 1989). 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 of each primer, 20 ng template DNA, 2.5 U Taq polymerase (Takara, Dalian, China), and 5 [micro]L 10 x buffer. Fragments were amplified under the following conditions: initial denaturing at 95[degrees]C for 5 min; 30 cycles of 95[degrees]C for 1 min, 58[degrees]C (28S), 50[degrees]C (mtCOI), 48[degrees]C (16S) for 1 min, and 72[degrees]C for 1 min; and a final extension at 72[degrees]C for 5 min. A negative control (no template) was included during each PCR run.
[FIGURE 1 OMITTED]
PCR product was purified using the TIANgel Midi Purification Kit (Tiangen Bio. Co.). All amplified DNA fragments were sequenced in both directions using the same PCR primers on an ABI3730 XL DNA sequencer.
The nucleotide sequences obtained in this study and those of other pen shell species used for phylogenetic analyses are available from GenBank, and their accession numbers are shown in Figures 3-6. The nucleotide sequences of the 3 gene fragments of P. bicolor were used as the outgroup. Initial alignments were performed using CLUSTAL X2 (Larkin et al. 2007). The sequences were trimmed to the same length as other published sequences after alignment. The best substitution models for the sequences of the 3 genes to be used for phylogenetic analyses were selected based on the Akaike information criterion (AIC) with the program Modeltest 3.7 (Posada & Crandall 1998). Bayesian inference (BI) trees were estimated using MrBayes 3.1 (Ronquist & Huelsenbeck 2003) for 10,000,000 generations, with a sample frequency of 100 generations and an initial burn-in of 100,000. Maximum likelihood (ML) analysis was performed using the Phyml 2.4.4 program (Guindon & Gascuel 2003). Neighbor-joining (NJ) and maximum parsimony (MP) analyses were conducting using PAUP 4.0b 1.0 (Swofford 2002). The reliability of the internal branches of the NJ, MP, and ML trees was estimated with bootstrap values of 1,000 replicates. Pairwise sequence divergences among the haplotypes of A. chinensis, A. peetinata, and reference species were calculated with MEGA 5.05 (Tamura et al. 2011) and DnaSP 5.10 (Librado & Rozas 2009) using Kimura's 2-parameter model (K2P (Kimura 1980)). In addition, genealogical relationships for both the mtCOI and 16S rRNA genes were further assessed using a minimum spanning tree constructed by Arlequin 22.214.171.124 (Excoffier & Lischer 2010).
To be consistent with homogeneity of variance, the values of the 4 morphological parameters were logarithmically transformed, the small sample from Weizhoudao was merged into the Beihai population (BH), and Nanaodao and Zhapo were merged into the Dongshan population (DS). ANOVA of the 4 morphological parameters among the 6 populations (i.e., Dongshan, Fujian (DS) and Beihai, Guangxi (BH) for A. chinensis; Zhangzidao, Liaoning (ZZD); Liugongdao, Shandong (LGD); Hongdao, Shandong (HD); and Lianyungang, Jiangsu (LYG) for A. pectinata; Table 2) showed significant differences (P < 0.05) for all 4 morphological parameters. Table 3 shows the result of Tukey's test for multiple comparisons of these parameters. There were significant differences among the 6 samples, but no significant difference between A. pectinata and A. chinensis for SL/SH, SL/SW, and SH/SW. The value of CSPAS for A. chinensis was significantly lower than that of A. pectinata, which indicates that the size of the posterior adductor in A. chinensis is smaller than that of A. pectinata. Interestingly, the values of SL/SH and SL/SW for the DS population were significantly smaller than those of the BH population, which suggests that the shells of samples from BH were narrower and flatter than those from DS.
[FIGURE 2 OMITTED]
Nuclear 28S rRNA Sequence Analysis
Four haplotypes were detected among the 85 individual pen shells sequenced. One of these haplotypes, which is a 1,059-bp-long sequence, occurred in the A. chinensis samples. Moreover, all the A. pectinata samples collected from northern China shared one 1,060-bp-long haplotype, and the other 2 haplotypes (both 1,058-bp long) were found in A. vexillum and P. bicolor, respectively. Excluding the inferred gaps, which were treated as missing data, 18 sites were variable, and all the variable sites were parsimony informative in the A. chinensis and A. pectinata data set.
[FIGURE 3 OMITTED]
The 28S sequences obtained in this study and those of A. pectinata, A. vexillum, Atrina rigida, Atrina seminuda, and P. bicolor from GenBank were subjected to phylogenetic analysis. The program Modeltest3.7 selected GTR + I (0.4076) + G (0.4487) as the best model based on the AIC. Phylogenetic analysis of the 28S data set conducted using the BI, ML, MP, and NJ procedures produced almost identical results (Fig. 3). The pinnids A. chinensis, A. pectinata, A. vexillum, A. rigida, and A. seminuda nested in a well-supported subclade. The pinnid A. chinensis appeared as a sister taxon of A. vexillum, as both form a robust subclade that is a sister clade to A. rigida and A. seminuda. Table 4A presents the K2P genetic distance in the 28S gene fragments for these taxa.
[FIGURE 4 OMITTED]
mtCOI Gene Sequence Analysis
A 650-bp fragment of the mtCOI gene sequenced for the 85 pen shells (A. chinensis, A. pectinata, A. vexillum, and P. bicolor) along with 30 sequences of A. chinensis from Oita, Japan, generated 43 haplotypes. Twenty-eight haplotypes were observed for A. chinensis, whereas A. pectinata, A. vexillum, and P. bicolor had 12, 2, and 1, respectively. Nucleotide variation was found in 129 base loci, of which 112 were parsimony informative, in the A. chinensis and A. pectinata data set.
[FIGURE 5 OMITTED]
Partial mtCOI sequences from the 43 haplotypes were subjected to phylogenetic analysis along with reference sequences from GenBank for A. pectinata (2 haplotypes) and A trinafragilis (2 haplotypes). The best model based on the AIC estimate using Modeltest3.7 corresponded with the TIM + G (0.1545) model. Phylogenetic analysis of the mtCOI data set using the BI, ML, MP, and NJ procedures produced almost identical results (Fig. 4). The mtCOI sequences of A. chinensis formed 1 robust subclade and were split into 2 lineages (labeled A and B). Lineage A includes samples from DS; Nanaodao, Guangdong (NAD); Zhapo, Guangdong (ZP); and Oita, Japan (OT); and lineage B contains samples from BH and Weizhoudao, Guangxi (WZD). These 2 lineages form a robust clade with A. vexillum. The minimum spanning tree of haplotypes (Fig. 6) also revealed the 2 lineages of A. chinensis, which in this case were separated from one another by 28 mutational steps. In lineage A, the dominant haplotype (48.2%) shared by the DS, NAD, and OT locations formed the center of a starlike network. The starlike network of lineage B exhibited a dominant haplotype (73.3%) shared by the BH and WZD sites. Table 4B presents the genetic distances between and in populations based on the K2P model of the mtCOI gene fragments for the taxa noted earlier. The net genetic distance ([+ or -] SE) between A. chinensis and A. pectinata was 0.204 [+ or -] 0.023. Applying the mtCOI divergence rate (0.7-2.4%/million years (Hellberg & Vacquier 1999, Marko 2002)), the evolutionary separation between A. chinensis and A. pectinata occurred about 8.5-29.1 million years ago during the Neogene period.
[FIGURE 6 OMITTED]
Mitochondria1 16S rRNA Sequence Analysis
Sequences of the 16S region amplicons from the 85 pen shell individuals were 480-488 bp in length. Twenty-one haplotypes were obtained, of which A. chinensis had 12 and A. pectinata had 7. The pinnids A. vexillum and P. bicolor had 2 haplotypes and 1 haplotype, respectively. In the 16S data set, 129 sites were variable, of which 125 sites were parsimony informative.
Phylogenetic analysis was conducted using all the 16S haplotypes obtained in this study and other sequences from GenBank for A. rigida, A. seminuda, and A. fragilis. The program Modeltest3.7 selected TVM + G as the best model based on the AIC. Phylogenetic analysis of the 16S data set using the BI, ML, MP, and BI procedures produced almost identical results (Fig. 5). The phylogram and the minimum spanning tree of the 16S data (Fig. 7) showed that A. chinensis contains lineages A and B, which is in accordance with the results for mtCOI. Both the networks of lineage A and B were starlike, with 1 dominant haplotype (80.7% and 73.3%, respectively). Moreover, A. chinensis was closer taxonomically to A. vexillum, A. rigida, and A. seminuda than to A. pectinata, because all of these were nested in a terminal clade. Table 4C presents the genetic distance based on the K2P model of the 16S gene fragments for the taxa noted.
Identification of Atrina chinensis
In different environments, the shell shapes of pinnids can vary greatly, and no obvious, unified morphological characteristic can be applied to distinguish them. Therefore, classifying pinnids based only on morphological characteristics is very difficult. During the past few decades, genetic markers have been developed and used to identify some bivalve species (Yu et al. 2000, Lam & Morton 2003, Reece et al. 2007, Wang et al. 2010). In controversial cases such as identification of A. pectinata and A. chinensis, it is imperative to use a modern molecular biology technology to solve taxonomic issues.
In our study, morphological analysis revealed that A. pectinata and A. chinensis did not differ noticeably in SH, SL, or SW, although the comparative size of the CSPAS of A. chinensis was significantly smaller than that of A. pectinata. This result indicates that the economic value of A. chinensis would be lower than that of A. pectinata. Although A. chinensis can be distinguished easily from A. pectinata by the presence of foliaceous growth lines all over the surface of the shell (Habe 1964), there are no quantitative morphological characteristics of shell shape that can be used to tell them apart. The description of A. ehinensis provided by Deshayes (1841) matches the description of Reeve's A. lurida (1858) and Habe's A. lamellata (1961) and the scabrous pen shell in our study. Winckworth (1936) proposed that A. chinensis together with A. pectinata and A. lurida should be synonymized, whereas Huber (2010) synonymized A. chinensis with A. lurida, A. lamellata, and Atrina chemnizii and did not include A. pectinata. Yu et al. (2000) conducted a comparative study of the morphological differences of 4 different types of A. pectinata in China and found that the scabrous pen shell was especially distinct from the other 3 types of A. pectinata in terms of shell morphology and anatomy, but Wang (1964, 1997) thought that all types of A. pectinata in China were 1 species because of the continuity of morphological variation. Based on the results of our study, we agree with Huber's view.
[FIGURE 7 OMITTED]
In the current study, the genetic distances between A. chinensis and A. pectinata, A. vexillum, and A. rigida generally were equal to that between A. vexillum and A. rigida for the 28S rRNA and 16S rRNA sequences, and the genetic distances between A. chinensis and A. pectinata, A. vexillum, and A. fragilis generally were equal to that between A. pectinata and A. vexillum for the mtCOI sequences. In all the phylograms obtained in our study based on our molecular phylogenic analysis, A. chinensis and A. vexillum formed a distinct clade, and then together with A. pectinata they constituted another clade. Wang et al. (2000) compared EST, SOD, LDH, MDH, and ME isozymes of the kidney, the posterior adductor muscle, and the gill tissues of 4 types of A. pectinata from China and found that the scabrous pen shell clearly differed from the other 3 types of A. pectinata. Yu et al. (2004) studied the genetic heterogeneity among the 4 forms of A. pectinata using random amplified polymorphic DNA markers and suggested that the scabrous pen shell should be a species distinct from A. pectinata. Our study confirms this view; the scabrous pen shell--namely, A. chinensis--is a distinct species and not a synonym of A. pectinata.
Distribution of Atrina ehinensis
Our results indicate that all the pinnid samples from the 4 sites in northern China are A. pectinata, and all pinnid samples from the 5 southern China sites and Oita (Japan) are A. chinensis. The pinnids A. chinensis and A. pectinata exhibit distinct biogeography; A. pectinata is a temperate species and A. chinensis is a tropical species. The pen shell A. chinensis is a sublittoral species, occurring at depths of 200 m, whereas A. pectinata lives subtidally, down to depth of 30 m. In addition, the distribution range of A. chinensis in the world includes Singapore, Malaysia, Thailand, Borneo, the Philippines, Vietnam, Beibu Gulf to Taiwan Island, Okinawa, and Japan-Ki (Huber 2010).
The Genetic Population Structure of Atrina chinensis from Southern China
We discovered significant genetic divergence among the populations of A. chinensis studied. The phylograms and the minimum spanning trees based on both the mtCOI and 16S rRNA sequences showed that A. chinensis contained 2 lineages. The morphological analyses showed that lineage B (the BH and WZD populations) differed significantly from lineage A (the DS, NAD, and ZP population) for the SL/SH and SL/SW parameters, which indicates that the shell of members of lineage B is narrow and relatively flat compared with that of members of lineage A. The sequence divergence between lineage A and lineage B was also significant for the mtCOI (3.6%) and 16S rRNA (1.4%) sequences, whereas the sequence divergences within lineage A and lineage B were 0.9% and 0.6%, respectively, for mtCOI, and were 0.4% and 0.4%, respectively, for 16S rRNA. Based on the criteria of the DNA barcode technique, interspecific mtCOI divergence in general exceeds 4%, whereas intraspecific divergence typically is less than 2% (Avise 2000, Hebert et al. 2003). The net genetic distance between lineage A and lineage B based on mtCOI was 0.034 [+ or -] 0.007.
Assuming an mtCOI divergence rate of 0.7-2.4%/Million years, lineage A and lineage B may have been separated for 1.4-4.9 Million years ago during the Pleistocene, which spans the recent period of repeated glaciations. Within the glacial period, the South China Sea was a semienclosed gulf connected with the Pacific mainly through the Bashi Strait between Taiwan and Luzon (Wang 1999). Land bridges were formed between the Asian continent and nearby islands as a result of the lower sea level. Land bridges would have acted as a barrier for A. chinensis and may have led to its allopatric diversification. During the interglacial period, the sea level rose and the land bridges disappeared, thereby interconnecting previously isolated marginal seas and facilitating dispersal over vast geographical ranges (Liu et al. 2007). In short, the significant genetic divergence between the 2 lineages likely was the result of long-term geographical isolation and secondary contact of A. chinensis in accordance with the sea-level change that occurred after the isolation and reconnection of the South China Sea and the Sea of Japan during the Pleistocene. Thus, differentiation, long-term geographical isolation, and subsequent contact may have played a role in the evolutionary history of A. chinensis.
In summary, the results of our study support the validity of A. chinensis as its own taxonomic unit instead of as a synonym of A. pectinata. This study also showed that morphological analysis should be accompanied by genetic descriptions to identify species, especially for taxa with high economic value and plastic morphologies.
We thank Markus Huber for advice on pen shell taxonomy. We thank Jinxian Liu for helping with data analysis. This work was supported by grants from the National Key Technology R&D Program in the 12th Five-Year Plan of China (no. 2011BAD13B01), the National Marine Public Welfare Research Project (no. 200805069), the National Natural Science Foundation of China (no. 40876084), and the Jiangsu Province Key Technology Support Program (no. BE2008344).
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DONGXIU XUE, (1,3) HAIYAN WANG, (2) TAO ZHANG, (1) * YAN GAO, (1,3) SUPING ZHANG (2) AND FENGSHAN XU (2)
(1) Key Laboratory of Ecology and Environment Science, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, China; (2) Department of Marine Organism Taxonomy and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, China; (3) The Graduate School of Chinese Academy of Sciences, Beijing, China
* Corresponding author. E-mail: firstname.lastname@example.org
TABLE 1. Sampling information and molecular diversity indices for nr28S, mtC01, and 16S sequences. Date of Locality Abb. Collection N(n) Dongshan, Fujian DS January 2009 22 (22) Nanaodao, Guangdong NAD March 2011 3 (3) Zhapo, Guangdong ZP March 2011 1 (1) Beihai, Guangxi BH November 2008 30 (30) Weizhoudao, Guangxi WZD January 2010 1 (1) Zhangzidao, Liaoning ZZD November 2008 23 (6) Liugongdao, Shandong LGD November 2008 42 (6) Hongdao, Shandong HD May 2009 23 (6) Lianyungang, Jiangsu LYG November 2008 30 (6) Oita, Japan * OT -- 30 (30) nh (np) Locality nr28S mtCOI mt16S Dongshan, Fujian 1 (0) 8 (6) 3 (2) Nanaodao, Guangdong 1 (0) 2 (1) 1 (0) Zhapo, Guangdong 1 (0) 1 (0) 1 (1) Beihai, Guangxi 1 (0) 9 (8) 8 (7) Weizhoudao, Guangxi 1 (0) 1 (0) 1 (0) Zhangzidao, Liaoning 1 (0) 5 (4) 2 (1) Liugongdao, Shandong 1 (0) 2 (l) 3 (2) Hongdao, Shandong 1 (0) 3 (2) 2 (l) Lianyungang, Jiangsu 1 (0) 4 (3) 3 (2) Oita, Japan * -- 12 (9) -- Abbreviation of populations (Abb.), date of collection, number of specimens (N) and number of specimens sequenced (n), and number of haplotypes (nh) and private haplotypes (np) are shown for each population. * Sequences of Oita (Japan) were retrieved from GenBank. TABLE 2. ANOVA results for 4 morphological parameters compared among samples of Atrina chinensis and Atrina pectinata collected from 6 different sites. SL/SH SL/SW SOV df MS F MS F Interpopulation 5 0.147 179.434 * 0.031 26.893 * Intrapopulation 168 0.001 0.001 Total variation 173 SH/SW CSPAS SOV MS F MS F Interpopulation 0.066 44.292 * 0.010 59.061 * Intrapopulation 0.001 0.000 Total variation * Difference is extremely significant. [F.sub.0.05](5120) = 2.290; [F.sub.0.01](5120) = 3.174. CSPAS, comparative size of the posterior adductor scar; SH, shell height; SL, shell length; SOV, source of variation; SW, shell width. TABLE 3. Results of Tukey's tests for multiple comparisons of 4 morphological parameters among samples of Atrina chinensis and Atrina pectinata collected from 6ix different sites. Morphological Difference (5%) Site Samples SL/SH SL/SW DS 26 1.748 [+ or -] 0.117 (a) 4.584 [+ or -] 0.360 (a) BH 30 2.034 [+ or -] 0.141 (c) 5.422 [+ or -] 0.487 (c) ZZD 23 1.715 [+ or -] 0.106 (a) 5.138 [+ or -] 0.442 (b) LGD 42 1.842 [+ or -] 0.104 (b) 5.243 [+ or -] 0.305 (b) HD 23 1.729 [+ or -] 0.141 (a) 4.429 [+ or -] 0.436 (a) LYG 30 1.886 [+ or -] 0.104 (b) 5.332 [+ or -] 0.429 (b) Morphological Difference (5%) Site SH/SW CSPAS DS 2.634 [+ or -] 0.195 (a) 0.121 [+ or -] 0.024 (a) BH 2.670 [+ or -] 0.217 (a) 0.112 [+ or -] 0.025 (a) ZZD 2.643 [+ or -] 0.161 (a) 0.232 [+ or -] 0.026 (a) LGD 2.908 [+ or -] 0.339 (b) 0.150 [+ or -] 0.04 (b) HD 3.013 [+ or -] 0.283 (b) 0.230 [+ or -] 0.054 (a) LYG 2.769 [+ or -] 0.230 (a) 0.146 [+ or -] 0.021 (b) The same letter in the same column shows that the difference is not significant (P > 0.05). A different letter indicates that the difference is significant (P < 0.05). BH, Beihai, Guangxi; CSPAS, comparative size of the posterior adductor scar; DS, Dongshan, Fujian; HD, Hongdao, Shandong; LGD, Liugongdao, Shandong; LYG, Lianyungang, Jiangsu; SH, shell height; SL, shell length; SW, shell width; ZZD, Zhangzidao, Liaoning. TABLE 4. Distance matrix of 28S (A), mtCOI (B), and 16S (C) DNA within (on the diagonal) and between (below the diagonal) various Atrina species as assessed using the Kimura 2-parameter model. Type 1 2 3 4 5 (A) 28S A. pectinata DQ343846 * -- A. pectinata 0.000 0.000 A. chinensis-DS 0.017 0.017 0.000 A. wxillum 0.022 0.022 0.011 0.000 A. vexillum AB594404 * 0.019 0.019 0.008 0.005 -- A. rigida HQ329438 * 0.016 0.016 0.013 0.013 0.011 A. seminuda HQ329439 * 0.017 0.017 0.013 0.014 0.012 P. bicolor 0.073 0.073 0.077 0.082 0.082 P. bicolor AB594398 * 0.075 0.075 0.078 0.083 0.083 (B) COI A. pectinata * 0.008 A. pectinata 0.013 0.007 A. chinensis, lineage A 0.209 0.198 0.009 A. chinensis, lineage B 0.204 0.194 0.036 0.006 A. vexillum 0.211 0.203 0.179 0.179 0.008 A. fragilis EF536851 * 0.221 0.212 0.244 0.227 0.259 P. bicolor 0.260 0.258 0.277 0.268 0.267 (C) 16S A. pectinata 0.004 A. chinensis, lineage A 0.125 0.004 A. chinensis, lineage B 0.133 0.014 0.004 A. vexillum 0.142 0.091 0.100 0.006 A. fagilis DQ663474 * 0.146 0.143 0.137 0.159 -- A. rigida HQ329397 * 0.136 0.110 0.112 0.110 0.131 A. seminuda HQ329398 * 0.134 0.107 0.110 0.110 0.128 P. bicolor 0.184 0.192 0.191 0.203 0.183 Type 6 7 8 9 (A) 28S A. pectinata DQ343846 * A. pectinata A. chinensis-DS A. wxillum A. vexillum AB594404 * A. rigida HQ329438 * -- A. seminuda HQ329439 * 0.001 -- P. bicolor 0.077 0.078 0.000 P. bicolor AB594398 * 0.078 0.079 0.001 -- (B) COI A. pectinata * A. pectinata A. chinensis, lineage A A. chinensis, lineage B A. vexillum A. fragilis EF536851 * 0.014 P. bicolor 0.258 0.000 (C) 16S A. pectinata A. chinensis, lineage A A. chinensis, lineage B A. vexillum A. fagilis DQ663474 * A. rigida HQ329397 * -- A. seminuda HQ329398 * 0.002 -- P. bicolor 0.197 0.194 0.000
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