Molecular method to identify the morphologically similar four species of the Perinereis nuntia species complex (Annelida: Nereididae) based on PCR-RFLP analysis of nuclear ribosomal ITS, with new distributional records of the two forms of Perinereis shikueii

Abstract

The present study was carried out to design a simple, new and accurate method for identification of the morphologically similar four species (forms A and B of P. shikueii, P. mictodonta, and P. wilsoni) of the Perinereis nuntia species complex by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns of the internal transcribed spacer (ITS). Restriction digestion analysis in the following two steps generated a specific restriction pattern for each species: (1) by the first digestion of amplified ITS fragments with AluI, we can distinguish the two forms of P. shikueii from P. mictodonta and P. wilsoni; (2) by the subsequent digestion of ITS fragments with Cfr10I, we can distinguish between the forms A and B of P. shikueii, and by that with HinfI, we can distinguish P. mictodonta from P. wilsoni. Our result indicates that this method enable easy and accurate identification of these taxa. We additionally examined the distributions of the two forms of P. shikueii in Japan, Korea and Taiwan using this new method. The form B was distributed in a wide range in western Japan and Korea, whereas the form A was distributed in few limited sites in the inner parts of the Seto Inland Sea and the Ariake Sea in Japan, the western and southern coasts of Korea, and the western coast of Taiwan.