Multiplex Polymerase Chain Reaction Method with Species-specific Primers for Differentiation of Two Closely Related Fish Species, Beryx splendens and B. mollis (Actinopterygii: Beryciformes)

Distinguishing between Beryx splendens and Beryx mollis (Actinopterygii: Beryciformes) on the basis of external morphology is difficult, and a reliable method of differentiation must be established. In this study, we developed a multiplex polymerase chain reaction (PCR) method with species-specific primers for distinguishing B. splendens from B. mollis. In total, 146 specimens were collected in the North Pacific Ocean, East China Sea, and Southwest Indian Ocean. Provisionally, 115 specimens were identified on the basis of the number of pyloric caeca. Phylogenetic analysis was also performed by using the 146 partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene and sequences from a DNA database. Identification by the number of pyloric caeca was consistent with the result of the COI phylogeny, and we surmised that the use of the COI sequences was effective for the differentiation of the two species. Multiplex PCR with speciesspecific primers was subsequently developed on the basis of the partial COI sequences. All of the specimens used in the molecular analyses were successfully identified as B. splendens or B. mollis via electrophoresis of the PCR products amplified using the species-specific PCR primers.