Are Acila divaricata and Acila mirabilis One Species or Two Distinct Species? Evidence from COI Mitochondrial DNA

ZHANG Junlong, SHI Huafeng, XU Fengshan, and SHA Zhongli

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China

Are Acila divaricata and Acila mirabilis One Species or Two Distinct Species? Evidence from COI Mitochondrial DNA

ZHANG Junlong, SHI Huafeng, XU Fengshan, and SHA Zhongli*

Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China

Acila divaricata (Hinds, 1843) and A. mirabilis (Adams and Reeve, 1850) are common benthic bivalves in China. A number of researchers have proposed that the latter species is a junior synonym of the former species. Because of morphological similarities, it is difficult to distinguish these two species based on visual examination only. For better understanding of their taxonomy, the mitochondrial COI gene fragments of five individuals of A. divaricata from the East China Sea and six individuals of A. mirabilis from the Yellow Sea were sequenced in this study. The phylogenetic relationships of the obtained COI sequences, together with nineteen sequences of three species of the genus Nucula, were analyzed. The pairwise intra- and inter-specific distances for the COI sequences ranged from 0.002 to 0.017 and from 0.128 to 0.134, respectively, and no overlap was found. Phylogenetically, A. divaricata and A. mirabilis form distinct clades and cluster into a sister to all other Nucula species. The results indicated that A. divaricata and A. mirabilis are two distinct species. The differences in the morphology and distribution between the two species were briefly discussed.

cytochrome oxidase I; molecular taxonomy; Bivalvia; Acila; distribution

1 Introduction

To date, approximately 150 species have been described in the family Nuculidae (Bivalvia: Nuculoida) (Huber, 2010). Of these, 22 species are reported in China (Xu, 2008). Despite being a relatively small family of protobranch bivalves, the Nuculidae is associated with considerable debate in classification primarily due to the less useful taxonomic information dervied from morphological characteristics (Kilburn, 1999). Whereas most nuculids are deposit feeders, the members of Nuculidae are characterized by a number of chevron-shaped taxodont teeth separated by a resilifer, a well-defined pallial line, an obscure or absent pallial sinus, and a lack of a siphon.

The species of the genus Acila in the family Nuculidae have divaricate riblets on the surface of the shells. A. divaricata (Hinds, 1843) and A. mirabilis (Adams and Reeve, 1850) are two common benthic bivalves in the China seas. The original description of A. divaricata is brief and includes no figures (Hinds, 1843). To our knowledge, the earliest figures of A. divaricata came from Hanley (1860: pl. 230, fig.151). A. mirabilis was first described by Adams and Reeve (1850). Hanley (1860) and Sowerby II (1871) described and illustrated these two species. A number of researchers have suggested that A. divaricata is a juvenile form of A. mirabilis and considered the latter as a synonym species of the former (Dall, 1898; Habe, 1958; Habe, 1977; Knudsen, 1967; Kuroda and Habe, 1981; Lutaenko and Noseworthy, 2012; Schenck, 1934, 1935, 1936). Conversely, other researchers believed that A. divaricata and A. mirabilis are two distinct species (Bernard et al., 1993; Hanley, 1860; Huber, 2010; Qi, 2004; Smith, 1892; Sowerby II, 1871; Xu, 1984, 1999; Xu and Zhang, 2008).

Based primarily on morphological characteristics, scientists workinig in conventional taxonomy (or descriptive taxonomy) nominate a species following a set of rules and referring to a type specimen (Godfray, 2002). To achieve accurate identification, the taxonomist must be well-trained and experience rich. However, it remains difficult to accurately identify a species with significant phenotypic variation or to distinguish two species with certain morphological similarities. Even well-trained and experienced experts often disagree with species identification and/or assignment.

Previous research assumed that interspecific sequence divergence is greater than intraspecific sequence divergence (Avise and Johns, 1999; Hebert et al., 2003; Hebert et al., 2004; Moore, 1995). Accordingly, DNA barcoding has become a reliable tool widely used for species identification (Blaxter, 2003, 2004; Hebert and Gregory, 2005; Pennisi, 2003; Stoeckle, 2003; Tautz et al., 2002; Tautz et al., 2003), even for partial specimens or those from early life stages (Radulovici et al., 2010). Despite the confounding factors involved in DNA barcoding(Hickerson et al., 2006; Lipscomb et al., 2003; Moritz and Cicero, 2004; Rubinoff et al., 2006; Will et al., 2005; Will and Rubinoff, 2004), the molecular taxonomic method is considered superior to morphology-based identification and serves as a complementary approach to addressing complex taxonomic issues.

More recently, the mitochondrial cytochrome oxidase I (COI) gene has been successfully used in molecular identification of bivalves (Gérard et al., 2008; Ladhar-Chaabouni et al., 2010; Mikkelsen et al., 2007; Wood et al., 2007). In the present study, we investigated the divergence between A. divaricata and A. mirabilis by sequencing the mitochondrial COI gene. The obtained COI sequances, together with those of three species of the genus Nucula, were compared and analyzed to determine whether A. divaricata and A. mirabilis are two distinct species.

2 Materials and Methods

2.1 Sample Collection, DNA Extraction, and COI Gene Amplification

Six specimens of A. mirabilis were collected at depths of 40–80 m in the Yellow Sea and five specimens of A. divaricata were collected at depths of 78–84 m in the East China Sea using an Agassiz trawl (Fig.1). Prior to DNA extraction, all specimens were identified based on morphological characteristics as were described early (Hinds, 1843; Hanley, 1860; Adams and Reeve, 1850 ; Sowerby II, 1871; Xu and Zhang, 2008). Voucher specimens were deposited at the Institute of Oceanology, Chinese Academy of Sciences (IOCAS, Qingdao, China) (Table 1). Total genomic DNA was extracted with phenol/chloroform and precipitated with ethanol as described by Sambrook and Russell (2006).

Fig.1 The locations of sampling sites in the Yellow Sea and East China Sea.

Table 1 Background information of the specimens used in this study

The DNA was used as the template for the amplification of the mitochondrial COI gene with universal primers LCO 1490 (5’-GGT CAA ATC ATA AAG ATA TTG G-3’) and HCO 2198 (5’-TAA ACTTCA GGG TGA CCA AAA AT CA-3’) (Folmer et al., 1994). The 25-µL reaction contained 0.125 µL of ExTaq polymerase (5 U µL−1, Takara), 2.5 µL of 10× ExTaq buffer (Mg2+plus, TaKaRa), 2.0 µL of dNTP (2.5 mmol L−1each), 0.5 µL of primers (10 µmol L−1), 1.0 µL of template DNA, and 18.5 µL of dH2O. The PCR program involved 35 cycles of degeneration at 94℃ for 30 s, annealing at 50℃ for 45 s, and extension at 72℃ for 1 min. The PCR products were purified using Minipore protocols (Qiagen) and sequenced with ABI Big Dye protocol.

An additional 19 sequences of three species, Nucula tenuis (Montagu, 1808) (= Ennucula tenuis), Nucula tumidula Maim, 1861, and Nucula nucleus (Linnaeus, 1758) of the family Nuculidae were obtained from the GenBank and included in phylogenetic analysis (Table 1). 2011) using the Kimura 2-parameter model (Kimura, 1980). The standard error estimates were obtained following a bootstrap procedure (500 replicates) (Felsenstein, 1985), and phylogenetic trees were constructed based on the distances and neighbor-joining method (Saitou and Nei, 1987) in MEGA 5.05.

2.2 Sequence Analysis

Sequences were aligned using Clustal X with default parameters and then manually checked in BioEdit (Hall, 1999). Pairwise and mean intra- and inter-specific distances of the two Alica species and three Nucula species were calculated with MEGA 5.05 (Tamura et al.,

3 Results

3.1 Nucleotide Composition of Mitocontrial COI Gene Sequences

The mitocontrial COI gene sequences of A. divaricata and A. mirabilis were 658 bp in length and directly aligned without insertions or deletions. These COI sequences contain 94 (14%) variable sites, of them 87 (13%) were potentially informative for parsimonious analysis. The average frequency of A, C, G, and T was 34.0%, 16.0%, 19.8% and 30.2%, respectively. The COI sequences exhibited a strong adenine and thymine (AT) bias of 64.1%. Compared with the first and second positions, the third position had, as expected, a stronger AT bias of 84%. All sequences were translated into amino acids using the standard invertebrate mitochondrial code.

3.2 Pairwise Interspecific and Intraspecific Distances

The existing research revealed that DNA barcode from the COI gene is useful in distinguishing bivalves. Thus, we used the mitochondrial COI sequence to investigate the divergence between A. divaricata and A. mirabilis. Results showed that there was no overlap between the pairwise intra- and inter-specific distances of the two Acila species (Fig.2). Pairwise intraspecific distance of A. mirabilis ranged from 0.002 to 0.009 and those of A. divaricata ranged from 0.002 to 0.025. The pairwise interspecific distances of these two species ranged from 0.128 to 0.134. The mean pairwise interspecific distances of the five species in the family Nuculidae ranged from 0.135 to0.311 (Table 2). The lowest interspecific divergence was between A. mirablis and A. divaricata, and the highest divergence was between A. divaricata and N. tumidula. However, the divergences of species within a genus are not always higher than that of species of different genera (Table 2). The intraspecific variation was higher in A. divaricata than in other species (Table 3).

Table 2 Mean interspecific distances among 2 Acila and 3 Nucula species

Fig.2 Distribution of intraspecific and interspecific distances of the two Acila and three Nucula species.

Table 3 Mean intraspecific distances of 2 Acila and 3 Nucula species

3.3 Phylogenetic Relationship

Phylogenetic analysis showed the relationship of all species tested, which was in agreement with their morphology-based classification. The three species of genusNucula formed percentages as were obtained in bootstrap test (100%) (Fig.3). The sister clades of A. divaricata and A. mirabilis formed separate clades. The three species of genus Nucula also formed different clades. This scenario may attribute to the divergence among the two Acila species, N. tenuis and N. nucleus, which was lower than that in genus Nucula and higher than that in genus Acila (Table 2).

Fig.3 Phylogenetic tree constructed using the Neighbor-Joining method based on the mitochondrial COI gene sequences of Alica spp. obtained in this study and those of the three species of Nucula retrieved from GenBank.

4 Discussion

Lipscomb et al. (2003) have proposed that DNA identification will not work unless the variations are much less within a species than between species. In the present study, the results suggested that A. divaricata and A. mirabilis were two distinct species. Based on examination of a large number of specimens in IOCAS, the two Alica species can be distinguished by the following morphological characteristics (Fig.4): the rather distinct ridge of A. mirabilis from the umbone to postero-ventral margin and the indistinct postero-ventral corner of A. divaricata. Additionally, A. divaricata and A. mirabilis were different in size. The former is smaller, no more than 15 mm in length, while the latter can be 30 mm in length. For a shell of the same size, A. divaricata has more teeth and stronger teeth. A. divaricata and A. mirabilis also have different distributions.

According to Salvador A (pers. comm., 26 October 2012), the type materials of N. divaricata Hinds, 1843 and N. mirabilis Adams & Reeve, 1850, which should be in the Natural History Museum, London, missed in a thorough search. The original description ambiguously indicated that the holotype of A. divaricata came from China Seas at a depth of 154 m (Hinds, 1843). In China, only the East and South China Seas exceed 100 m in depth, which overlaps with the distribution depth of A. divaricata. A. mirabilis, which was first reported in Nangasaki Bay, Japan (Adams and Reeve, 1850), is one of the dominant species in the Yellow Sea Cold Water Mass, a water body that provides conditions suitable for the survival and reproduction of cold water species below a depth of 40–50 m in the Yellow Sea (Liu, 2008; Xu, 1997; Zhang et al., 2012). A. mirabilis is commonly found in the Yellow Sea, the Northern Sea of Japan, and the Russian Far Eastern Seas. The distribution of A. divaricata and A. mirabilis in the Yellow Sea and East China Sea is separated by the Changjiang (Yangtze) River Estuary.

According to Huber (2010), the distribution of A. divaricata includes the Philippines. The type figures of A. divaricata balabacensis (see Schenck, 1936: pl. 18, figs.10–12; and the website of the National Museum of Natural History, Smithsonian Institution) is more likely A. divaricata. Thus, we proposed that A. divaricata balabacensis in the Philippines is A. divaricata, which is in agreement with the conclusion of Huber (2010).

Fig.4 Acila divaricata (Hinds, 1843) (a-d, KIII105DHM6-1, 9.11 mm in length) and Acila mirabilis (Adams and Reeve, 1850) (e-h, KI09096B-M4-1, 16.10 mm in length). a and e, exterior of right valve; b and f, exterior of left valve; c and g, interior of right valve; d and h, interior of left valve.

In accordance with the illustrations, specimens of A. divaricata reported in Japan (Habe, 1958: pl. XII, fig.7; Habe, 1977: pl. 2, fig.5; Okutani, 2000: pl. 415 fig.12) and Taiwan (Lan, 2001: fig.3) as well as A. schencki archibenthalis described in deep water of Japan (Vitousek et al., 1997: pl. 6, fig.1) are actually A. mirabilis. The large size (45 mm) of the species in Taiwan enhances our certainty that the specimen is A. mirabilis, which entered the East China Sea during the most recent ice age. As the glaciers receded and water temperatures increased, the distribution range of A. mirabilis expanded northward to the Yellow Sea, and southward to northeast Taiwan along a low temperature band at 250–300 m depth (Xu and Zhang, 2011). Huber (2010) reported that both A. mirabilis and A. divaricata exist in Taiwan. But only A. mirabilis is currently found in Japanese waters. Japan is located at the confluence of cold and warm currents, with a steeply sloping continental shelf and warm near-shore temperatures. These environmental factors provide favorable conditions for the occurrence of real A. divaricata in this region.

The largest species of the genus Alica, i.e., A. divaricata vigilia Schenck, 1936, is almost twice the size of A. mirabilis and mainly occur in north Japan and Russia. This species differs from the two species in the present study by its rather oblong shape, flat umbone, and strong black periostracum (see Schenck, 1936: pl. 17, figs.1–6; and the website of the National Museum of Natural History, Smithsonian Institution). Considering the shape, size and distribution characteristics, A. vigilia should be a valid species.

Tchang et al. (1963) studied the molluscan fauna in China and described the Yellow Sea as a warm temperate fauna region rather than subtropic. The fauna of the Yellow Sea shares a high level of similarity with that of the northern Japanese regions, and is part of the Far East Subregion of the North Pacific Temperate Region. The East China Sea belongs to the China-Japan Subtropical Biotic Subregion, which is part of the Indo-West-Pacific Warmwater Region. These assignments have been confirmed based on benthic faunal characteristics (Liu, 2008; Liu and Xu, 1963). Although a biogeographic assumption requires quantitative distribution analysis of a large number of organisms, our results provided supporting evidence for these assignment. For example, A. mirabilis is a representative of cold-water fauna in the Yellow Sea. Conversely, A. divaricata forms a subtropic element in the East China Sea.

COI sequences serve as an effective ‘barcode’ for distinguishing a number of species (Hebert et al., 2003; Stoeckle, 2003). Because of the high rate of molecular evolution, COI gene can be used to distinguish closely related taxa (Blaxter, 2003; Galtier et al., 2009; Hebert et al., 2003). The applicability of the universal DNA primers makes it easy to amplify target fragments from the COI gene of diverse taxa (Folmer et al., 1994; Hebert et al., 2003). Furthermore, the COI gene is ubiquitous among animals and has a high copy number each cell (Blaxter, 2003; Galtier et al., 2009). Together, these attributes guarantee the COI gene a useful target for DNA barcoding of animals of interest.

Molecular identification methods are advantageous in distinguishing morphologically similar species such as those in the present study and even species without significant differences such as Indoaustriella dalli and I. scarlatoi (Glover et al., 2008). However, we emphasize that molecular methods rely on a foundation of morphologically based taxonomy and provide a tool for testing hypotheses regarding morphology-based classification. Given this, we recommend the combined use of both molecular and morphological techniques to solve taxonomy-related issues.

Acknowledgements

This work was supported by the Knowledge Innovation Program of the Chinese Academy of Sciences (No. KSCX2-YW-N-0807); the External Cooperation Program of the Chinese Academy of Sciences (No. GJHZ200808); the Special Program for Key Basic Research of the Ministry of Science and Technology of China (No. 2006 FY110500); and IOCAS funding (Nos. 2012IO060102, 2012IO060104). We thank Lutaenko K. A. (Institute of Marine Biology, Far East Branch of the Russian Academyof Sciences) for critical advice on the manuscript. The authors would also like to thank Open Cruise of Chinese Offshore Oceanography Research by IOCAS to supply the data.

Adams, A., and Reeve, L., 1850. Mollusca. In: The Zoology of the Voyage of H.M.S. Samarang: under the Command of Captain Sir Edward Belcher, C. B., F. R. A. S., F. G. S., During the Years 1843–1846. Adams, A., ed., Reeve and Benham, London, 1-87.

Avise, J. C., and Johns, G. C., 1999. Proposal for a standardized temporal scheme of biological classification for extant species. Proceedings of the National Academy of Sciences, 96 (13): 7358-7363.

Bernard, F. R., Cai, Y. Y., and Morton, B., 1993. Catalogue of the Living Marine Bivalve Molluscs of China. Hong Kong University Press, Hong Kong, 146pp.

Blaxter, M., 2003. Molecular systematics: Counting angels with DNA. Nature, 421 (6919): 122-124.

Blaxter, M., 2004. The promise of a DNA taxonomy. Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences, 359 (1444): 669.

Dall, W. H., 1898. Contributions to the Tertiary fauna of Florida, with especial reference to the silex-beds of Tampa, and the Pliocene beds of the Caloosahatchie River, indluding in many cases a complete revision of the generic groups treated of and their American Tertiary species. Part IV. 1. Prionodesmacea: Nucula to Julia. 2. Teleodesmacea: Teredo to Ervila. Transactions of the Wagner Free Institute of Science of Philadelphia, 3 (4): 571-947.

Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution, 39 (4): 783-791.

Folmer, O., Black, M., Hoeh, W., Lutz, R., and Vrijenhoek, R., 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology, 3 (5): 294-299.

Galtier, N., Nabholz, B., Glémin, S., and Hurst, G. D. D., 2009. Mitochondrial DNA as a marker of molecular diversity: A reappraisal. Molecular Ecology, 18 (22): 4541-4550.

Gérard, K., Bierne, N., Borsa, P., Chenuil, A., and Féral, J.-P., 2008. Pleistocene separation of mitochondrial lineages of Mytilus spp. mussels from Northern and Southern Hemispheres and strong genetic differentiation among southern populations. Molecular Phylogenetics and Evolution, 49 (1): 84-91.

Glover, E. A., Taylor, J. D., and Williams, S. T., 2008. Mangrove associated lucinid bivalves of the central Indo-West Pacific: Review of the ‘Austriella’ group with a new genus and species (Mollusca: Bivalvia: Lucinidae). Raffles Bulletin of Zoology, Supplement, 18: 25-40.

Godfray, H. C. J., 2002. Challenges for taxonomy. Nature, 417 (6884): 17-19.

Habe, T., 1958. Report on the Mollusca chiefly collected by the S.S. Soyo-Maru of the Imperial Fisheries Experimental Station on the continental shelf bordering Japan during the years 1922–1930. Part 3. Lamellibranchia (1). Publications of the Seto Marine Biological Laboratory, 6 (3): 241-280.

Habe, T., 1977. Systematics of Mollusca in Japan: Bivalvia and Scaphopoda. Hokuryukan Publishing Co., Tokyo, 372pp.

Hall, T. A., 1999. BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series, 41: 95-98.

Hanley, S. C. T., 1860. Monograph of the family Nuculidae, forming the Lamarckian genus Nucula. In: Thesaurus Conchyliorum or Monographs of Genera of Shells, Vol. 3. Sowerby, II G. B. ed., London, 105-168.

Hebert, P. D. N., Cywinska, A., Ball, S. L., and deWaard, J. R., 2003. Biological identifications through DNA barcodes. Proceedings of the Royal Society of London. Series B: Biological Sciences, 270 (1512): 313-321.

Hebert, P. D. N., and Gregory, T. R., 2005. The promise of DNA barcoding for taxonomy. Systematic Biology, 54 (5): 852-859.

Hebert, P. D. N., Penton, E. H., Burns, J. M., Janzen, D. H., and Hallwachs, W., 2004. Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proceedings of the National Academy of Sciences of the United States of America, 101 (41): 14812-14817.

Hickerson, M. J., Meyer, C. P., and Moritz, C., 2006. DNA barcoding will often fail to discover new animal species over broad parameter space. Systematic Biology, 55 (5): 729-739.

Hinds, R. B., 1843. Descriptions of new species of Nucula, from the collections of Sir Edward Belcher, C. B., and Hugh Cuming, Esq. Proceedings of the Zoological Society of London, 11: 97-101.

Huber, M., 2010. Compendium of Bivalves. ConchBooks, Hackenheim, 901pp.

Kilburn, R. N., 1999. The family Nuculidae (Bivalvia: Protobranchia) in South Africa and Mozambique. Annals of the Natal Museum, 40: 245-268.

Kimura, M., 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution, 16 (2): 111-120.

Knudsen, J., 1967. The deep-sea Bivalvia. Scientific Reports of The John Murray Expedition, 1933-34, 11 (3): 237-343.

Kuroda, T., and Habe, T., 1981. A Catalogue of Molluscs of Wakayama Prefecture, the Province of Kii: I. Bivalvia, Scaphopoda and Cephalopoda. Nakanishi Printing Co., Ltd, Kyoto, 301pp.

Ladhar-Chaabouni, R., Hamza-Chaffai, A., Hardivillier, Y., Chénais, B., and Denis, F., 2010. A pilot study of genetic differentiation between two phenotypes of a Mediterranean population of the bivalve Cerastoderma glaucum and genetic discrimination with other Cerastoderma glaucum and Cerastoderma edule populations outside the Mediterranean. Marine Ecology, 31 (2): 355-363.

Lan, T. C., 2001. Report on Taiwan Protobranchia. The Pei-yo, 27: 37-49.

Lipscomb, D., Platnick, N., and Wheeler, Q., 2003. The intellectual content of taxonomy: A comment on DNA taxonomy. Trends in Ecology and Evolution, 18 (2): 65-66.

Liu, R. Y., 2008. Checklist of Marine Biota of China Sea. Science Press, Beijing, 1267pp.

Liu, R. Y., and Xu, F. S., 1963. Preliminary study on the benthic fauna of the Yellow Sea and the East China Sea. Oceanologia et Limnologia Sinica, 5 (4): 306-321.

Lutaenko, K. A., and Noseworthy, R. G., 2012. Catalogue of the. Living Bivalvia of the Continental Coast of the Sea of Japan (East Sea). Dalnauka, Vladivostok, 247pp.

Mikkelsen, N. T., Schander, C., and Willassen, E., 2007. Local scale DNA barcoding of bivalves (Mollusca): A case study. Zoologica Scripta, 36 (5): 455-463.

Moore, W. S., 1995. Inferring phylogenies from mtDNA variation: mitochondrial-gene trees versus nuclear-gene trees. Evolution,49 (4): 718-726.

Moritz, C., and Cicero, C., 2004. DNA barcoding: Promise and pitfalls. Plos Biology, 2 (10): 1529-1531.

Okutani, T., 2000. Marine Mollusks in Japan. Tokai University Press, Tokyo, 1173pp.

Pennisi, E., 2003. Modernizing the Tree of Life. Science, 300 (5626): 1692-1697.

Qi, Z. Y., 2004. Seashells of China. China Ocean Press, Beijing, 418pp.

Radulovici, A. E., Archambault, P., and Dufresne, F., 2010. DNA barcodes for marine biodiversity: Moving fast forward? Diversity, 2 (4): 450-472.

Rubinoff, D., Cameron, S., and Will, K., 2006. A genomic perspective on the shortcomings of mitochondrial DNA for‘Barcoding’ identification. Journal of Heredity, 97 (6): 581-594.

Saitou, N., and Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, 4 (4): 406-425.

Sambrook, J., and Russell, D. W., 2006. The Condensed Protocols from Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, 800pp.

Schenck, H. G., 1934. Classification of nuculid pelecypods. Bulletin du Musée royal d'Histoire Naturelle de Belgique, 10 (20): 1-78.

Schenck, H. G., 1935. Valid Species of the Nuculid Pelecypod Acila. Bulletin du Musée Royal d'Histoire Naturelle de Belgique, 11 (14): 1-5.

Schenck, H. G., 1936. Nuculid bivalves of the genus Acila. Geological Society of America Special Papers, 4: 1-149.

Smith, E. A., 1892. Descritpions of new species of Nucula, and a list of the species belonging to the subgenus Acila. Journal of Conchology, 7: 110-112.

Sowerby II, G. B., 1871. Monograph of the genus Nucula. In: Conchologia Iconica: Or Illustrations of the Molluscous Animals, Vol. 18. Reeve, B., ed., London.

Stoeckle, M., 2003. Taxonomy, DNA, and the bar code of life. Bioscience, 53 (9): 796-797.

Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S., 2011. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution, 28 (10): 2731-2739.

Tautz, D., Arctander, P., Minelli, A., Thomas, R., and Vogler, A., 2002. DNA points the way ahead in taxonomy. Nature, 418 (6897): 479-479.

Tautz, D., Arctander, P., Minelli, A., Thomas, R. H., and Vogler, A. P., 2003. A plea for DNA taxonomy. Trends in Ecology and Evolution, 18 (2): 70-74.

Tchang, S., Tsi, C. Y., Zhang, F. S., and Ma, S. T., 1963. A preliminary study of the demarcation of marine molluscan faunal regions of China and its adjacent waters. Oceanologia et Limnologia Sinica, 5 (2): 124-138.

Vitousek, P. M., Mooney, H. A., Lubchenco, J., and Melillo, J. M., 1997. Human domination of earth’s ecosystems. Science, 277 (5325): 494-499.

Will, K. W., and Rubinoff, D., 2004. Myth of the molecule: DNA barcodes for species cannot replace morphology for identification and classification. Cladistics, 20 (1): 47-55.

Will, K. W., Mishler, B. D., and Wheeler, Q. D., 2005. The perils of DNA barcoding and the need for integrative taxonomy. Systematic Biology, 54 (5): 844-851.

Wood, A. R., Apte, S., MacAvoy, E. S., and Gardner, J. P. A., 2007. A molecular phylogeny of the marine mussel genus Perna (Bivalvia: Mytilidae) based on nuclear (ITS1& 2) and mitochondrial (COI) DNA sequences. Molecular Phylogenetics and Evolution, 44 (2): 685-698.

Xu, F. S., 1984. Preliminary study on the Protobranchia (Mollusca) from the shallow waters of China. II. Nuculidae. Studia Marina Sinica, 22: 179-188.

Xu, F. S., 1997. Bivalve Mollusca of China sea. Science Press, Beijing, 333pp.

Xu, F. S., 1999. Fauna Sinica Phylum Mollusca Class Bivalvia Subclass Protobranchia and Anomalodesmata. Science Press, Beijing, 244pp.

Xu, F. S., 2008. Family Nuculidae Gray, 1824. In: Checklist of Marine Biota of China Sea. Liu, R. Y., ed., Science Press, Beijing, 548pp.

Xu, F. S., and Zhang, J. L., 2011. Characteristics of bivalve diversity in typical habitats of China seas. Biodiversity Science, 19 (6): 716-722.

Xu, F. S., and Zhang, S. P., 2008. An Illustrated Bivalvia Mollusca Fauna of China Seas. Science Press, Beijing, 336pp.

Zhang, J. L., Xu, F. S., and Liu, R. Y., 2012. Community structure changes of macrobenthos in the South Yellow Sea. Chinese Journal of Oceanology and Limnology, 30 (2): 248-255.

(Edited by Qiu Yantao)

(Received August 22, 2012; revised October 15, 2012; accepted July 17, 2013)

© Ocean University of China, Science Press and Spring-Verlag Berlin Heidelberg 2014

*

. E-mail: shazl@qdio.ac.cn