Genetic divergence and phylogenetic independence of Far Eastern species in subfamily Leuciscinae (Pisces: Cyprinidae) inferred from mitochondrial DNA analyses.

Classification of freshwater fish in the subfamily Leuciscinae, Cyprinidae is hampered by complexity or lack of morphological diversity. In this study, analyses based on mtDNA sequences were undertaken to clarify phylogenetic relationships among Far Eastern, North American and European species in the Leuciscinae. Evolutionary rate in cytochrome b gene (Cyt-b) and D-loop sequences appear to be almost constant in Leuciscinae. The topology of phylogenetic trees generated by neighbor-joining (NJ) and maximum likelihood (ML) methods based on Cyt-b gene and D-loop sequences was similar. Five major clades, designated clades 1-5, and a minor clade were discriminated. Most of the Far Eastern, North American and European species were included in the major clades. Clade 1, comprised almost entirely of Far Eastern phoxinins, is monophyletic and greatly diverged from the other species of Leuciscinae. From the present phylogenetic relationships and the previous studies, we present the following hypotheses with respect to the evolutionary history of the Far Eastern phoxinins. The Far Eastern species should be classified into Far Eastern-specific genera, although ichthyologists have still insisted that the species should be included in the European genera. The Far Eastern clade 1 consists of two subclades, including genera Pseudaspius-Tribolodon and Far Eastern Phoxinus species. According to our phylogenetic analyses, Pseudaspius leptocephalus and Tribolodon species should be reclassified into the same genus. On the basis of evolutionary rate in Cyt-b gene in Cyprinidae, it is estimated that the Far Eastern lineage diverged approximately 10-14 million years ago (mya) from the common ancestor of Leuciscinae. It is deduced that speciation of the Far Eastern species occurred until approximately 4 mya, in relation to the formation of the Sea of Japan and the Japanese Islands.