Mitochondrial DNA Markers for PCR-Based Phylogenetic Analysis of Ark Shells

Tomomi Tanaka, Futoshi Aranishi

United Graduate School of Agricultural Sciences, Tottori University, Tottori, Japan; Coastal Lagoon Research Center, Shimane University, Matsue, Japan.

DOI: 10.4236/ojms.2013.34021                

Abstract

Arcidae species are commercially important bivalves in Japan and are commonly referred to as bloody ark due to their red blood. They have thick shells with distinct radiating ribs, and the numbers of these ribs are important morphological features for species discrimination. However, some Arcidae species are morphologically indistinguishable, with a similar number of the ribs in adults and deficient rib formation, particularly among juveniles. Thus, we developed a reliable molecular marker to genetically discriminate between 7 Arcidae species belonging to Scapharca, Anadara, and Tegillarca based on species-specific polymorphic segments of mitochondrial DNA. PCR amplification of partial COI, 16S rRNA, 12S rRNA, and Cyt b genes was performed on 7 species using 8 primer sets. Only the set of Scapharca-specific forward primer and universal reverse primer for the partial COI gene successfully yielded single PCR products from all 7 species examined. Thus, nucleotide sequences of 481 bp portion of these PCR products were determined, and the degrees of nucleotide substitutions ranged from 0.4% between S. broughtonii and T. granosa to 20.2% between S. satowi and A. antiquata. In addition, a phylogenetic tree showed significant differences between 7 species, with higher bootstrap support than 69.

Keywords

Ark Shell; Arcidae; Mitochondrial DNA; COI Gene; Phylogenetic Analysis

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	J. E. Morton, “Molluscs, an Introduction to Their Form and Function,” Harper and Brothers, New York, 1960.
[2]	S. Y. Lee and S. H. Kim, “Genetic Variation and Discrimination of Korean Arkshell Scapharca Species (Bivalvia, Arcoida) Based on Mitochondrial COI Gene Sequences and PCR-RFLP,” Korean Journal of Genetics, Vol. 25, No. 4, 2003, pp. 309-315.
[3]	Geological Survey, “Geological Survey Professional Paper Volume 1228,” Government Printing Office, Washington DC, 1983.
[4]	J.D. Taylor, J. Kennedy, M. Nei and S. Kumar, “The Shell Structure and Mineralogy of Bivalvia. Introduction. Nuculacea-Trigonacea,” British Museum Natural History, London, 1969.
[5]	Y. Feng, Q. Li, L. Kong and X. Zheng, “COI-Based DNA Barcoding of Arcoida Species (Bivalvia: Pteriomorphia) along the Coast of China,” Molecular Ecology Resources, Vol. 11, No. 3, 2011, pp. 435-441. http://dx.doi.org/10.1111/j.1755-0998.2010.02975.x
[6]	T. Okutani, “Class BIVALVIA/Subclass PTERIOMORPHIA/Orde ARCOIDA,” In: T. Okutani, Ed., Marine Mollusks in Japan, Tokai University press, Kanagawa, 2001, pp. 845-855. (in Japanese)
[7]	G. R. Carvalho and T. J. Pitcher, “Molecular Genetics in Fisheries,” Chapman & Hall, London, 1995. http://dx.doi.org/10.1007/978-94-011-1218-5
[8]	Y. Iidzuka and F. Aranishi, “DNA Fingerprinting for Ostreidae Oysters in Kyushu,” LAGUNA, Vol. 15, 2008, pp. 69-76. (in Japanese)
[9]	T. D. Kocher, W. K. Thomas, A. Meyer, S. V. Edwards, S. Paabo, F. X. Villablanca and A. C. Wilson, “Dynamics of Mitochondrial DNA Evolution in Animals: Amplification and Sequencing with Conserved Primers,” Proceedings of the National Academy of Sciences of the United States of America, Vol. 86, No. 16, 1989, pp. 6196-6200. http://dx.doi.org/10.1073/pnas.86.16.6196
[10]	F. Aranishi and T. Okimoto, “Genetic Relationship between Cultured Populations of Pacific Oyster Revealed by RAPD Analysis,” Journal of Applied Genetics, Vol. 45, No. 4, 2004, pp. 435-443.
[11]	F. Aranishi and T. Okimoto, “Sequence Polymorphism in a Novel Noncoding Region of Pacific Oyster Mitochondrial DNA,” Journal of Applied Genetics, Vol. 46, No. 2, 2005, pp. 201-206.
[12]	F. Aranishi, “A Novel Mitochondrial Intergenic Spacer Reflecting Population Structure of Pacific Oyster,” Journal of Applied Genetics, Vol. 47, No. 2, 2006, pp. 119-123. http://dx.doi.org/10.1007/BF03194610
[13]	O. Folmer, M. Black, W. Hoeh, R. Lutz and R. Vrijenhoek, “DNA Primers for Amplification of Mitochondrial Cytochrome c Oxidase Subunit I from Diverse Metazoan Invertebrates,” Molecular Marine Biology and Biotechnology, Vol. 3, No. 5, 1994, pp. 294-299.
[14]	M. Matsumoto and I. Hayami, “Phylogenetic Analysis of the Family Pectinidae (Bivalvia) Based on Mitochondrial Cytochrome C Oxidase Subunit I,” Journal of Molluscan Studies, Vol. 66, No. 4, 2000, pp. 477-488. http://dx.doi.org/10.1093/mollus/66.4.477
[15]	S. R. Palumbi, A. P. Martin, W. O. McMillan, S. Romano, L. Stice and G. Grabowsky, “The Simple Fool’s Guide to PCR,” 2nd Edition, University of Hawaii, Honolulu, 1991.
[16]	B. Kessing, H. Croom, A. Martin, C. McIntosh, W. O. McMillan and S. Palumbi, “The Simple Fool’s Guide to PCR,” Department of Zoology, University of Hawaii, Honolulu, 1989.
[17]	T. J. S. Merritt, L. Shi, M. C. Chase, M. A. Rex, R. J. Etter and J. M. Quattro, “Universal Cytochrome b Primers Facilitate Intraspecific Studies in Molluscan Taxa,” Molecular Marine Biology and Biotechnology, Vol. 7, No. 1, 1998, pp. 7-11.
[18]	P. Berschick, “One Primer Pair Amplifies Small Subunit Ribosomal DNA from Mitochondria, Plastids and Bacteria,” Biotechniques, Vol. 23, No. 3, 1997, pp. 494-498.
[19]	J. D. Thompson, D. G. Higgins and T. J. Gibson, “CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice,” Nucleic Acids Research, Vol. 22, No. 22, 1994, pp. 4673-4680. http://dx.doi.org/10.1093/nar/22.22.4673
[20]	K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei and S. Kumar, “MEGA5: Molecular Evolutionary Genetics Analysis Using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods,” Molecular Biology and Evolution, Vol. 28, No. 10, 2011, pp. 2731-2739. http://dx.doi.org/10.1093/nar/22.22.4673
[21]	M. Kimura, “A Simple Method for Estimating Evolutionary Rates of Base Substitutions through Comparative Studies of Nucleotide Sequences,” Journal of Molecular Evolution, Vol. 16, No. 2, 1980, pp. 111-120. http://dx.doi.org/10.1007/BF01731581
[22]	Ministry of Agriculture, Forestry and Fisheries, “Annual Statistics of Fishery and Aquaculture Production,” Association of Agriculture & Forestry Statistics, Tokyo, 2009. (in Japanese)
[23]	K. Okoshi, “Alien Species Introduced with Imported Clams: The Clam-Eating Moon Snail Euspira fortunei and Other Unintentionally Introduced Species,” Japanese Journal of Benthology, Vol. 59, 2004, pp. 74-82. (in Japanese)
[24]	M. Matsumoto and I. Hayami, “Molecular Phylogenetics and Taxonomic Characters of the Anadarinae (Bivalvia),” FOSSILS, Vol. 69, 2001, pp. 25-34. (in Japanese)
[25]	M. J. Lee, J. W. Park, M. S. Yoo and Y. K. Hong, “Morphological Characteristics and Genetic Diversity Using the RAPD Technique in the Ark Shell, Scapharca broughtonii (Schrenck) from Korea and China,” Journal of the Korean Fisheries Society, Vol. 30, No. 2, 1997, pp. 297-304.
[26]	E. S. Cho, C. G. Jung, C. W. Kim and S. G. Sohn, “Development of Molecular Detection Marks Using PCRRFLP Technique for Arkshell (Scapharca broughtonii Schrenck),” Korean Journal of Life Science, Vol. 15, No. 6, 2005, pp. 879-883. http://dx.doi.org/10.5352/JLS.2005.15.6.879
[27]	H. Wang and X. Guo, “Identification of Crassostrea ariakensis and Related Oysters by Multiplex Species-Specific PCR,” Journal of Shellfish Research, Vol. 27, No. 3, 2008, pp. 489-493. http://dx.doi.org/10.5352/JLS.2005.15.6.879