Genus Zacco specimens collected in this study were classified genetically as five species, Zacco platypus, Z. temminckii, Z. koreanus and two unidentified species, using DNA barcoding analysis based on 655 bp of mitochondrial cytochrome c oxidase subunit I (COI) gene. Two of unidentified species (Z. sp.1 and Z. sp.2) were considered to be unrecorded or new species of genus Zacco according to genetic distances between Zacco species. In addition, we determined a natural hybrid based on polymorphic base at the diagnostic positions displayed on nuclear recombination activating gene 1 (RAG1) gene (965 bp), and estimated paternal and maternal species of natural hybrid comparing phylogenetic tree between COI and RAG1, and Z. sp.1♀ × Z. koreanus♂, Z. sp.2♀ × Z. koreanus♂ and Z. koreanus♀ × Z. sp.1♂ individuals were confirmed. The habitat of natural hybrids of Z. koreanus between Z. sp.1 and Z. sp.2 was identified as Geum and Yeongsan River, respectively. In our data, only F1 hybrid generation was identified; however, generations after F1 hybrid or backcross were not demonstrated.
Keywords: COI; RAG1; DNA barcoding; Natural hybrid; genus Zacco.