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Morphological and Genetic Identification of the Validity of the SpeciesAtrina chinensis(: )

ARTICLE in JOURNAL OF SHELLFISH RESEARCH · AUGUST 2012 Impact Factor: 0.79 · DOI: 10.2983/035.031.0318

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Available from: Dongxiu Xue Retrieved on: 27 October 2015 Morphological and Genetic Identification of the Validity of the Species chinensis (Bivalvia: Pinnidae) Author(s): Dongxiu Xue, Haiyan Wang, Tao Zhang, Yan Gao, Suping Zhang and Fengshan Xu Source: Journal of Shellfish Research, 31(3):739-747. 2012. Published By: National Shellfisheries Association DOI: http://dx.doi.org/10.2983/035.031.0318 URL: http://www.bioone.org/doi/full/10.2983/035.031.0318

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MORPHOLOGICAL AND GENETIC IDENTIFICATION OF THE VALIDITY OF THE SPECIES ATRINA CHINENSIS (BIVALVIA: PINNIDAE)

DONGXIU XUE,1,3 HAIYAN WANG,2 TAO ZHANG,1* YAN GAO,1,3 SUPING ZHANG2 AND FENGSHAN XU2 1Key Laboratory of Ecology and Environment Science, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, China; 2Department of Marine Organism and Phylogeny, Institute of Oceanology, Chinese Academy of Sciences, 7 Nanhai Road, Qingdao, Shandong 266071, China; 3The Graduate School of Chinese Academy of Sciences, Beijing, 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 Atrina 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. pectinata 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

INTRODUCTION MATERIALS AND METHODS

The pen shells of the family Pinnidae are commercially Sample Collection and Morphological Analyses important in a number of Indo-Pacific countries. They are economically valuable species because they have a large and Fifty-six individuals of A. chinensis were collected from 5 edible posterior adductor muscle. Pinnid species are a challeng- sites along the coast of southern China (Figs. 1 and 2). For ing group for taxonomists and phylogeneticists because of the comparative purposes, 118 individuals of A. pectinata were phenotypic plasticity of their shells. For example, the pinnid collected from 4 locations along the coast of northern China. species referred to as the ‘‘scabrous’’ pen shell in southern China Two individuals of Atrina vexillum and two individuals of Pinna has been identified by various authors as Atrina pectinata bicolor also were collected from Beibu Gulf and used in the (Winckworth 1929), Atrina chinensis (Deshayes 1841, Huber phylogenetic analyses. Table 1 shows the number of individuals 2010), Atrina lurida (Reeve 1858–1859), Atrina chemnitzii collected and sequenced. All samples were transported alive to (Hanley 1858), and Atrina lamellata (Habe 1964, Habe 1980, the laboratory for morphological measurements, and then Okutani 2000). Based on morphological characteristics and a muscle sample was taken from each individual and preserved distribution, the scabrous pen shell from southern China in absolute ethyl alcohol. matches A. chinensis (Deshayes 1841) rather than A. pectinata Shell height (SH), shell length (SL), shell width (SW) and the (Linnaeus 1767). However, this species was treated as a syno- major and minor axis (MA and MiA) lengths of the posterior nym of A. pectinata by Winckworth (1929), Rosewater (1961), adductor scar were measured to an accuracy of 0.1 mm using Wang (1964, 1997), and Liang et al. (1986). Results of recent a Vernier caliper (Liang et al. 1986, Yu et al. 2000). To eliminate studies based on morphological differences (Wang et al. 2000), the impact of body size, relative ratios for each measurement isozyme phenotypic divergence (Wang et al. 2000), and random were used for shape discrimination and analysis (SL/SH, SL/SW, amplified polymorphic DNA (Yu et al. 2000) of 4 forms of and SH/SW). The comparative size of the posterior adductor scar A. pectinata from China suggested that the scabrous pen shell (CSPAS) was also calculated as follows (Yu et al. 2000): may be a distinct species rather than a synonym of A. pectinata. p 3 MA 3 MiA A. chinensis CSPAS ¼ SW 3 In this study, we collected samples of from 5 typical 4 3 SH 3 SL sites in southern China, A. pectinata from northern China, and other related pinnid species. Two mitochondrial genes (mito- SPSS (version 13.0) was used to conduct the statistical an- chondrial cytochrome oxidase I (mtCOI) and 16S rRNA) and alyses. One-way analysis of variance (ANOVA), followed by 1 nuclear gene (28S rRNA) were analyzed, as was the morphol- TukeyÕs test for multiple comparisons, was used to compare the ogy of the specimens, and a phylogenetic analysis was con- 4 morphological parameters among samples of A. chinensis and ducted. This study is the first attempt at clarifying the taxonomic A. pectinata collected from 9 different sites. status of A. chinensis using molecular genetic information. The results may prove useful for the development of aquaculture of DNA Extraction, PCR Amplification, and Sequencing A. chinensis, and for the conservation and sustainable manage- ment of its genetic resources. DNA was isolated from ethanol-fixed adductor muscle tissue using the TIANamp marine DNA kit (Tiangen, *Corresponding author. E-mail: [email protected] Beijing, China). The PCR amplification for 3 target gene frag- DOI: 10.2983/035.031.0318 ments (nr28S, mtCOI, and 16S) used the universal nr28S

739 740 XUE ET AL.

Figure 1. A map of ChinaÕs coast showing the sites where Atrina samples were collected. : denotes the collection sites of Atrina chinensis; d denotes the collection sites of Atrina pectinata. primers D1F and D6R (Park & Foighil 2000), mtCOI primers criterion (AIC) with the program Modeltest 3.7 (Posada & LCO1490 and HCO2198 (Folmer et al. 1994), and mt16S primers Crandall 1998). Bayesian inference (BI) trees were estimated 16sar and 16sbr (Kessing et al. 1989). Reactions were performed using MrBayes 3.1 (Ronquist & Huelsenbeck 2003) for in 50 mL with final concentrations of 2.0 mM MgCl2,150mMof 10,000,000 generations, with a sample frequency of 100 gener- each dNTP, 0.2 mM of each primer, 20 ng template DNA, 2.5 U ations and an initial burn-in of 100,000. Maximum likelihood Taq polymerase (Takara, Dalian, China), and 5 mL103 buffer. (ML) analysis was performed using the Phyml 2.4.4 program Fragments were amplified under the following conditions: initial (Guindon & Gascuel 2003). Neighbor-joining (NJ) and maxi- denaturing at 95°C for 5 min; 30 cycles of 95°C for 1 min, 58°C mum parsimony (MP) analyses were conducting using PAUP (28S), 50°C(mtCOI),48°C (16S) for 1 min, and 72°C for 1 min; 4.0b1.0 (Swofford 2002). The reliability of the internal branches and a final extension at 72°C for 5 min. A negative control (no of the NJ, MP, and ML trees was estimated with bootstrap template) was included during each PCR run. values of 1,000 replicates. Pairwise sequence divergences among PCR product was purified using the TIANgel Midi Purifi- the haplotypes of A. chinensis, A. pectinata, and reference cation Kit (Tiangen Bio. Co.). All amplified DNA fragments species were calculated with MEGA 5.05 (Tamura et al. 2011) were sequenced in both directions using the same PCR primers and DnaSP 5.10 (Librado & Rozas 2009) using KimuraÕs on an ABI3730 XL DNA sequencer. 2-parameter model (K2P (Kimura 1980)). In addition, genea- logical relationships for both the mtCOI and 16S rRNA genes Phylogenetic Analysis were further assessed using a minimum spanning tree constructed by Arlequin 3.5.1.2 (Excoffier & Lischer 2010). The nucleotide sequences obtained in this study and those of other pen shell species used for phylogenetic analyses are RESULTS available from GenBank, and their accession numbers are shown in Figures 3–6. The nucleotide sequences of the 3 gene Morphology fragments of P. bicolor were used as the outgroup. Initial alignments were performed using CLUSTAL X2 (Larkin et al. To be consistent with homogeneity of variance, the values of 2007). The sequences were trimmed to the same length as other the 4 morphological parameters were logarithmically trans- published sequences after alignment. The best substitution formed, the small sample from Weizhoudao was merged into models for the sequences of the 3 genes to be used for phylo- the Beihai population (BH), and Nanaodao and Zhapo were genetic analyses were selected based on the Akaike information merged into the Dongshan population (DS). ANOVA of the THE VALIDITY OF ATRINA CHINENSIS 741

Figure 2. Shell morphology of Atrina chinensis and Atrina pectinata in this study. (A) The pinnid A. chinensis from Weizhoudao. (B) The pinnid A. chinensis from Beihai. (C) The pinnid A. chinensis from Dongshan. (D) The pinnid A. pectinata from Liugongdao. Scale bar$ 20 mm.

4 morphological parameters among the 6 populations (i.e., values of SL/SH and SL/SW for the DS population were Dongshan, Fujian (DS) and Beihai, Guangxi (BH) for A. significantly smaller than those of the BH population, which chinensis; Zhangzidao, Liaoning (ZZD); Liugongdao, Shandong suggests that the shells of samples from BH were narrower and (LGD); Hongdao, Shandong (HD); and Lianyungang, Jiangsu flatter than those from DS. (LYG) for A. pectinata; Table 2) showed significant differences < (P 0.05) for all 4 morphological parameters. Table 3 shows the Nuclear 28S rRNA Sequence Analysis result of TukeyÕs test for multiple comparisons of these param- eters. There were significant differences among the 6 samples, Four haplotypes were detected among the 85 individual pen but no significant difference between A. pectinata and A. shells sequenced. One of these haplotypes, which is a 1,059-bp- chinensis for SL/SH, SL/SW, and SH/SW. The value of CSPAS long sequence, occurred in the A. chinensis samples. Moreover, for A. chinensis was significantly lower than that of A. pectinata, all the A. pectinata samples collected from northern China which indicates that the size of the posterior adductor in A. shared one 1,060-bp-long haplotype, and the other 2 haplotypes chinensis is smaller than that of A. pectinata. Interestingly, the (both 1,058-bp long) were found in A. vexillum and P. bicolor,

TABLE 1. Sampling information and molecular diversity indices for nr28S, mtCOI, and 16S sequences.

nh (np) Date of Locality Abb. Collection N(n) nr28S mtCOI mt16S Dongshan, Fujian DS January 2009 22 (22) 1 (0) 8 (6) 3 (2) Nanaodao, Guangdong NAD March 2011 3 (3) 1 (0) 2 (1) 1 (0) Zhapo, Guangdong ZP March 2011 1 (1) 1 (0) 1 (0) 1 (1) Beihai, Guangxi BH November 2008 30 (30) 1 (0) 9 (8) 8 (7) Weizhoudao, Guangxi WZD January 2010 1 (1) 1 (0) 1 (0) 1 (0) Zhangzidao, Liaoning ZZD November 2008 23 (6) 1 (0) 5 (4) 2 (1) Liugongdao, Shandong LGD November 2008 42 (6) 1 (0) 2 (1) 3 (2) Hongdao, Shandong HD May 2009 23 (6) 1 (0) 3 (2) 2 (1) Lianyungang, Jiangsu LYG November 2008 30 (6) 1 (0) 4 (3) 3 (2) Oita, Japan* OT — 30 (30) — 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. 742 XUE ET AL.

Figure 3. Bayesian tree of 28S rDNA showing the phylogenetic placement of Atrina chinensis. The codes following the names are the GenBank accession numbers. *Sequences obtained from GenBank. The numbers at nodes represent Bayesian inference posterior probability, maximum likelihood, maximum parsimony, neighbor-joining probability support. respectively. Excluding the inferred gaps, which were treated as A. seminuda nested in a well-supported subclade. The pinnid missing data, 18 sites were variable, and all the variable sites A. chinensis appeared as a sister taxon of A. vexillum, as both were parsimony informative in the A. chinensis and A. pectinata form a robust subclade that is a sister clade to A. rigida and data set. A. seminuda. Table 4A presents the K2P genetic distance in the The 28S sequences obtained in this study and those of 28S gene fragments for these taxa. A. pectinata, A. vexillum, Atrina rigida, Atrina seminuda, and P. bicolor from GenBank were subjected to phylogenetic mtCOI Gene Sequence Analysis analysis. The program Modeltest3.7 selected GTR + I (0.4076) + G (0.4487) as the best model based on the AIC. Phylogenetic A 650-bp fragment of the mtCOI gene sequenced for the 85 analysis of the 28S data set conducted using the BI, ML, MP, and pen shells (A. chinensis, A. pectinata, A. vexillum, and P. bicolor) NJ procedures produced almost identical results (Fig. 3). The along with 30 sequences of A. chinensis from Oita, Japan, pinnids A. chinensis, A. pectinata, A. vexillum, A. rigida, and generated 43 haplotypes. Twenty-eight haplotypes were observed

Figure 4. Bayesian tree of mtCOI showing the phylogenetic placement of Atrina chinensis. Other explanations are as in Figure 3. Differences in topology of the maximum likelihood, maximum parsimony, neighbor-joining probability trees from the Bayesian inference tree are marked by #. THE VALIDITY OF ATRINA CHINENSIS 743

Figure 5. Bayesian tree of 16S rRNA showing the phylogenetic placement of Atrina chinensis. Other explanations are as in Figure 3. Differences in topology of the maximum likelihood, maximum parsimony, neighbor-joining probability trees from the Bayesian inference tree are marked by #. for A. chinensis,whereasA. pectinata, A. vexillum,andP. bicolor (2 haplotypes). The best model based on the AIC estimate using had 12, 2, and 1, respectively. Nucleotide variation was found in Modeltest3.7 corresponded with the TIM + G (0.1545) model. 129 base loci, of which 112 were parsimony informative, in the Phylogenetic analysis of the mtCOI data set using the BI, ML, A. chinensis and A. pectinata data set. MP, and NJ procedures produced almost identical results (Fig. 4). Partial mtCOI sequences from the 43 haplotypes were sub- The mtCOI sequences of A. chinensis formed 1 robust subclade jected to phylogenetic analysis along with reference sequences and were split into 2 lineages (labeled A and B). Lineage A from GenBank for A. pectinata (2 haplotypes) and Atrina fragilis includes samples from DS; Nanaodao, Guangdong (NAD);

Figure 6. Minimum spanning tree showing genetic relationship among mtCOI haplotypes in Atrina chinensis. Circles represent haplotypes with sizes proportional to their respective frequencies. The population origins of haplotypes are indicated by patterns. Tick marks represent deduced numbers of nucleotide substitutions along each branch. BH, Beihai, Guangxi; DS, Dongshan, Fujian; NAD, Nanaodao, Guangdong; OT, Oita, Japan; WZD, Weizhoudao, Guangxi; YJZP, Zhapo, Guangdong. 744 XUE ET AL.

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 SH/SW CSPAS SOV df MS F MS F MS F MS F Interpopulation 5 0.147 179.434* 0.031 26.893* 0.066 44.292* 0.010 59.061* Intrapopulation 168 0.001 0.001 0.001 0.000 Total variation 173

* Difference is extremely significant.

F0.05(5120) ¼ 2.290; F0.01(5120) ¼ 3.174. CSPAS, comparative size of the posterior adductor scar; SH, shell height; SL, shell length; SOV, source of variation; SW, shell width.

Zhapo, Guangdong (ZP); and Oita, Japan (OT); and lineage B Phylogenetic analysis was conducted using all the 16S haplo- contains samples from BH and Weizhoudao, Guangxi (WZD). types obtained in this study and other sequences from GenBank These 2 lineages form a robust clade with A. vexillum. The for A. rigida, A. seminuda,andA. fragilis. The program Mod- minimum spanning tree of haplotypes (Fig. 6) also revealed the eltest3.7 selected TVM + G as the best model based on the AIC. 2 lineages of A. chinensis, which in this case were separated from Phylogenetic analysis of the 16S data set using the BI, ML, MP, one another by 28 mutational steps. In lineage A, the dominant and BI procedures produced almost identical results (Fig. 5). haplotype (48.2%) shared by the DS, NAD, and OT locations The phylogram and the minimum spanning tree of the 16S data formed the center of a starlike network. The starlike network of (Fig. 7) showed that A. chinensis contains lineages A and B, which lineage B exhibited a dominant haplotype (73.3%) shared by is in accordance with the results for mtCOI. Both the networks of the BH and WZD sites. Table 4B presents the genetic distances lineage A and B were starlike, with 1 dominant haplotype (80.7% between and in populations based on the K2P model of the and 73.3%, respectively). Moreover, A. chinensis wasclosertax- mtCOI gene fragments for the taxa noted earlier. The net onomically to A. vexillum, A. rigida,andA. seminuda than to genetic distance (±SE) between A. chinensis and A. pectinata was A. pectinata, because all of these were nested in a terminal clade. 0.204 ± 0.023. Applying the mtCOI divergence rate (0.7–2.4%/ Table 4C presents the genetic distance based on the K2P model million years (Hellberg & Vacquier 1999, Marko 2002)), the of the 16S gene fragments for the taxa noted. evolutionary separation between A. chinensis and A. pectinata occurred about 8.5–29.1 million years ago during the Neogene DISCUSSION period. Identification of Atrina chinensis Mitochondrial 16S rRNA Sequence Analysis In different environments, the shell shapes of pinnids can Sequences of the 16S region amplicons from the 85 pen shell vary greatly, and no obvious, unified morphological character- individuals were 480–488 bp in length. Twenty-one haplotypes istic can be applied to distinguish them. Therefore, classifying were obtained, of which A. chinensis had 12 and A. pectinata pinnids based only on morphological characteristics is very had 7. The pinnids A. vexillum and P. bicolor had 2 haplotypes difficult. During the past few decades, genetic markers have and 1 haplotype, respectively. In the 16S data set, 129 sites were been developed and used to identify some bivalve species (Yu variable, of which 125 sites were parsimony informative. et al. 2000, Lam & Morton 2003, Reece et al. 2007, Wang et al.

TABLE 3. Results of TukeyÕs 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 SH/SW CSPAS DS 26 1.748 ± 0.117a 4.584 ± 0.360a 2.634 ± 0.195a 0.121 ± 0.024a BH 30 2.034 ± 0.141c 5.422 ± 0.487c 2.670 ± 0.217a 0.112 ± 0.025a ZZD 23 1.715 ± 0.106a 5.138 ± 0.442b 2.643 ± 0.161a 0.232 ± 0.026c LGD 42 1.842 ± 0.104b 5.243 ± 0.305b 2.908 ± 0.339b 0.150 ± 0.041b HD 23 1.729 ± 0.141a 4.429 ± 0.436a 3.013 ± 0.283b 0.230 ± 0.054c LYG 30 1.886 ± 0.104b 5.332 ± 0.429b 2.769 ± 0.230a 0.146 ± 0.021b

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. THE VALIDITY OF ATRINA CHINENSIS 745

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 456789 (A) 28S A. pectinata DQ343846* — A. pectinata 0.000 0.000 A. chinensis-DS 0.017 0.017 0.000 A. vexillum 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 0.001 — P. bicolor 0.073 0.073 0.077 0.082 0.082 0.077 0.078 0.000 P. bicolor AB594398* 0.075 0.075 0.078 0.083 0.083 0.078 0.079 0.001 — (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 0.014 P. bicolor 0.260 0.258 0.277 0.268 0.267 0.258 0.000 (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. fragilis 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 0.002 — P. bicolor 0.184 0.192 0.191 0.203 0.183 0.197 0.194 0.000

2010). In controversial cases such as identification of A. pectinata that of A. pectinata.AlthoughA. chinensis can be distinguished and A. chinensis, it is imperative to use a modern molecular easily from A. pectinata by the presence of foliaceous growth biology technology to solve taxonomic issues. lines all over the surface of the shell (Habe 1964), there are no In our study, morphological analysis revealed that A. pecti- quantitative morphological characteristics of shell shape that nata and A. chinensis did not differ noticeably in SH, SL, or SW, can be used to tell them apart. The description of A. chinensis although the comparative size of the CSPAS of A. chinensis was provided by Deshayes (1841) matches the description of ReeveÕs significantly smaller than that of A. pectinata. This result indi- A. lurida (1858) and HabeÕs A. lamellata (1961) and the sca- cates that the economic value of A. chinensis would be lower than brous pen shell in our study. Winckworth (1936) proposed that

Figure 7. Minimum spanning tree showing genetic relationship among mt16S rRNA haplotypes in Atrina chinensis. Other explanations are as in Figure 6. BH, Beihai, Guangxi; DS, Dongshan, Fujian; NAD, Nanaodao, Guangdong; WZD, Weizhoudao, Guangxi; YJZP, Zhapo, Guangdong. 746 XUE ET AL.

A. chinensis together with A. pectinata and A. lurida should be WZD populations) differed significantly from lineage A (the synonymized, whereas Huber (2010) synonymized A. chinensis DS, NAD, and ZP population) for the SL/SH and SL/SW with A. lurida, A. lamellata, and Atrina chemnizii and did not parameters, which indicates that the shell of members of lineage include A. pectinata. Yu et al. (2000) conducted a comparative B is narrow and relatively flat compared with that of members study of the morphological differences of 4 different types of of lineage A. The sequence divergence between lineage A and A. pectinata in China and found that the scabrous pen shell was lineage B was also significant for the mtCOI (3.6%) and 16S especially distinct from the other 3 types of A. pectinata in terms rRNA (1.4%) sequences, whereas the sequence divergences of shell morphology and anatomy, but Wang (1964, 1997) within lineage A and lineage B were 0.9% and 0.6%, respec- thought that all types of A. pectinata in China were 1 species tively, for mtCOI, and were 0.4% and 0.4%, respectively, for because of the continuity of morphological variation. Based on 16S rRNA. Based on the criteria of the DNA barcode tech- the results of our study, we agree with HuberÕs view. nique, interspecific mtCOI divergence in general exceeds 4%, In the current study, the genetic distances between A. chinensis whereas intraspecific divergence typically is less than 2% (Avise and A. pectinata, A. vexillum, and A. rigida generally were equal 2000, Hebert et al. 2003). The net genetic distance between to that between A. vexillum and A. rigida for the 28S rRNA and lineage A and lineage B based on mtCOI was 0.034 ± 0.007. 16S rRNA sequences, and the genetic distances between Assuming an mtCOI divergence rate of 0.7–2.4%/Million A. chinensis and A. pectinata, A. vexillum, and A. fragilis gener- years, lineage A and lineage B may have been separated for 1.4– ally were equal to that between A. pectinata and A. vexillum for 4.9 Million years ago during the Pleistocene, which spans the the mtCOI sequences. In all the phylograms obtained in our recent period of repeated glaciations. Within the glacial period, study based on our molecular phylogenic analysis, A. chinensis the South China Sea was a semienclosed gulf connected with the and A. vexillum formed a distinct clade, and then together with Pacific mainly through the Bashi Strait between Taiwan and A. pectinata they constituted another clade. Wang et al. (2000) Luzon (Wang 1999). Land bridges were formed between the compared EST, SOD, LDH, MDH, and ME isozymes of the Asian continent and nearby islands as a result of the lower sea kidney, the posterior adductor muscle, and the gill tissues of level. Land bridges would have acted as a barrier for A. chinensis 4 types of A. pectinata from China and found that the scabrous and may have led to its allopatric diversification. During the pen shell clearly differed from the other 3 types of A. pectinata. interglacial period, the sea level rose and the land bridges dis- Yu et al. (2004) studied the genetic heterogeneity among the appeared, thereby interconnecting previously isolated marginal 4 forms of A. pectinata using random amplified polymorphic seas and facilitating dispersal over vast geographical ranges (Liu DNA markers and suggested that the scabrous pen shell should et al. 2007). In short, the significant genetic divergence between be a species distinct from A. pectinata. Our study confirms this the 2 lineages likely was the result of long-term geographical view; the scabrous pen shell—namely, A. chinensis—is a distinct isolation and secondary contact of A. chinensis in accordance species and not a synonym of A. pectinata. with the sea-level change that occurred after the isolation and reconnection of the South China Sea and the Sea of Japan Distribution of Atrina chinensis during the Pleistocene. Thus, differentiation, long-term geo- graphical isolation, and subsequent contact may have played Our results indicate that all the pinnid samples from the 4 sites a role in the evolutionary history of A. chinensis. in northern China are A. pectinata, and all pinnid samples from In summary, the results of our study support the validity of the 5 southern China sites and Oita (Japan) are A. chinensis. The A. chinensis as its own taxonomic unit instead of as a synonym pinnids A. chinensis and A. pectinata exhibit distinct biogeog- of A. pectinata. This study also showed that morphological raphy; A. pectinata is a temperate species and A. chinensis is analysis should be accompanied by genetic descriptions to a tropical species. The pen shell A. chinensis is a sublittoral identify species, especially for taxa with high economic value species, occurring at depths of 200 m, whereas A. pectinata lives and plastic morphologies. 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). ACKNOWLEDGMENTS We thank Markus Huber for advice on pen shell taxonomy. The Genetic Population Structure of Atrina chinensis from We thank Jinxian Liu for helping with data analysis. This work Southern China was supported by grants from the National Key Technology We discovered significant genetic divergence among the R&D Program in the 12th Five-Year Plan of China (no. populations of A. chinensis studied. The phylograms and the 2011BAD13B01), the National Marine Public Welfare Re- minimum spanning trees based on both the mtCOI and 16S search Project (no. 200805069), the National Natural Science rRNA sequences showed that A. chinensis contained 2 lineages. Foundation of China (no. 40876084), and the Jiangsu Province The morphological analyses showed that lineage B (the BH and Key Technology Support Program (no. BE2008344).

LITERATURE CITED Avise, J. C. 2000. Phylogeography: the history and formation of species. compare´e, par Georges Cuvier. E´dition accompagne´e de planches Cambridge, MA: Harvard University Press. 447 pp. grave´es, repre´sentant les types de tous les genres, les caracte` res Deshayes, G. P. 1841. Planches mollusques. In: G. Cuvier, editor. Le dı´stinctifs des divers groupes et les modifications de structure sur re` gne , distribue´dÕapre` s son organisation, pour servir de base lesquelles repose cette classification; par une re´union de disciples de a` lÕhistoire naturelle des animaux et dÕintroduction a` lÕanatomie Cuvier. Fortin, Paris: Masson and Cie. pl. 85. THE VALIDITY OF ATRINA CHINENSIS 747

Excoffier, L. & H. E. L. Lischer. 2010. Arlequin suite ver 3.5: a new Marko, P. B. 2002. Fossil calibration of molecular clocks and the series of programs to perform population genetics analyses under divergence times of geminate species pairs separated by the Isthmus Linux and Windows. Mol. Ecol. Res. 10:564–567. of Panama. Mol. Biol. Evol. 19:2005–2021. Folmer, O., M. Black, W. Hoeh, R. Lutz & R. Vrijenhoek. 1994. DNA Okutani, T. 2000. Marine mollusks in Japan. Tokyo: Tokai University primers for amplification of mitochondrial cytochrome c oxidase Press. pp. 887–889. 1173 pp. subunit I from diverse metazoan invertebrate. Mol. Mar. Biol. Park, J. K. & D. O´. Foighil. 2000. Sphaerid and corbiculid clam Biotechnol. 3:294–299. represent separate heterodont bivalve radiations into freshwater Guindon, S. & O. Gascuel. 2003. A simple, fast, and accurate algorithm environments. Mol. Phyl. Evol. 14:75–88. to estimate large phylogenies by maximum likelihood. Syst. Biol. Posada, D. & K. A. Crandall. 1998. Modeltest: testing the model of 52:696–704. DNA substitution. Bioinformatics 14:817–818. Habe, T. 1964. Shells of the western Pacific in color, 2nd edition. Osaka: Reece, K. S., J. F. Cordes, J. B. Stubbs, K. L. Hudson & F. A. Francis. Hoikusha Publishing. pp. 171–172, pl. 52. 233 pp. 2007. Molecular phylogenies help resolve taxonomic confusion with Habe, T. 1980. Coloured illustrations of the shell of Japan, 2nd edition. Asian Crassostrea oyster species. Mar. Biol. 153:709–721. Osaka: Hoikusha Publishing. 117 pp., 52 pl. Reeve, L. A. 1858–1859. Monograph of genus Pinna, in Conchologia Hanley, S. C. T. 1858. Description of two species of Pinna. Proc. Zool. Iconic. London: Reeve & Co. pl. I-XXXIV. Soc. Lond. 26:136. Ronquist, F. & J. P. Huelsenbeck. 2003. MrBayes 3: Bayesian phylo- Hebert, P. D. N., S. Ratnasingham & J. R. deWard. 2003. Barcoding genetic inference under mixed models. Bioinformatics 19:1572–1574. animal life: cytochrome c oxidase subunit I divergences among Rosewater, J. 1961. The family Pinnidae in the Indo-Pacific. Indo Pacific closely related species. Proc. Biol. Sci. 270:96–99. 1:175–226. Hellberg, M. E. & V. D. Vacquier. 1999. Rapid evolution of fertilization Swofford, D. L. 2002. Paup*: phylogenetic analysis using parsimony (* and selectivity and lysin cDNA sequences in teguline gastropods. Mol. other methods). Version 4.0b 10. Sinauer Associates, Sunderland, Biol. Evol. 16:839–848. Massachusetts. 144 pp. Huber, M. 2010. Compendium of bivalves. Hackenheim: ConchBooks. Tamura, K., D. Peterson, N. Peterson, G. Stecher, M. Nei & S. Kuamr. pp.163–167, 592–598. 901 pp. 2011. MEGA 5: molecular evolutionary genetics analysis using Kessing, B., H. Croom, A. Martin, C. Mclntosh, W. Owen Mcmillan & maximum likelihood, evolutionary distance, and maximum parsi- S. Palumbi. 1989. The simple foolÕs guide to PCR. Honolulu, HI: mony methods. Mol. Biol. Evol. 28:2731–2739. Department of Zoology, University of Hawaii. 23 pp. Wang, Z. 1964. Preliminary studies on Chinese Pinnidae. Stud. Mar. Kimura, M. 1980. A simple method for estimating evolutionary rate of Sin. 5:131–141. (in Chinese). base substitutions through comparative studies of nucleotide se- Wang, Z. 1997. Phylum Mollusca, Order Mytiloida, in Peking, Fauna quences. J. Mol. Evol. 16:111–120. Sinica. Beijing: Chinese Science Press. pp. 214–237. (in Chinese). Lam, K. & B. Morton. 2003. Mitochondrial DNA and morphological Wang, P. 1999. Response of western Pacific marginal seas to glacial cycles: identification of a new species of Crassostrea (Bivalvia: Ostreidae) paleoceanographic and sedimentological features. Mar. Geol. 156:5–39. cultured for centuries in the Pearl River Delta, Hongkong, China. Wang, H., L. Qian, X. Liu, G. Zhang & X. Guo. 2010. Classification of Aquaculture 228:1–13. a common cupped oyster from southern China. J. Shellfish Res. Larkin,M.A.,G.Blackshields,N.P.Brown,R.Chenna,P.A. 29:857–866. McGettigan, H. McWilliam, F. Valentin, I. M. Wallace, A. Wilm, Wang, M., X. Yu, S. Yang & J. Gui. 2000. A comparative study on R. Lopez, J. D. Thompson, T. J. Gibson & D. G. Higgins. 2007. isozyme phenotypic divergence among four types of pen shell Atrina Clustal W and Clustal X version 2.0. Bioinformatics 23:2947– pectinata Linnaeus. Trop. Oceanol. 19:45–50. (in Chinese). 2948. Winckworth, R. A. 1929. Marine Mollusca from South India and Liang, X., H. Lin, P. Wu & Y. Liu. 1986. Comparison on morphology Ceylon. III: Pinna. With an index to the recent species of Pinna. of Pinnidae (Mollusca, Lamellibranchia) from Fujian coast. Trop. Proc. Malacol. Soc. 18:276–297. Oceanol. 5:13–19. Winckworth, R. A. 1936. Further note on Pinna. Proc. Malacol. Soc. Librado, P. & J. Rozas. 2009. DnaSP v5: a software for comprehensive 22:20–23. analysis of DNA polymorphism data. Bioinformatics 25:1451– Yu, X., Y. Mao, M. Wang, Z. Li & J. Gui. 2004. Genetic heterogeneity 1452. analysis and RAPD marker detection among four forms of Atrina Liu, J., T. Gao, S. Wu & Y. Zhang. 2007. Pleistocene isolation in the pectinata Linnaeus. J. Shellfish Res. 23:165–171. northwestern Pacific marginal seas and limited dispersal in a marine Yu, X., M. Wang, H. Li & Y. Cai. 2000. Comparison on morphological fish, Chelon haematocheilus (Temminck and Schlegel, 1845). Mol. difference inside species of pen shell Atrina pectinata. Trop. Oceanol. Ecol. 16:275–288. 19:39–44. (in Chinese).