Evaluating the Phylogenetic Status of the Extinct Japanese Otter on the Basis of Mitochondrial Genome Analysis

Abstract

The Japanese otter lived throughout four main Japanese islands, but it has not been observed in the wild since 1979 and was declared extinct in 2012. Although recent taxonomic and molecular phylogenetic studies suggest that it should be treated as an independent species, International Union for Conservation of Nature Red List considers it as subspecies of Lutra lutra. Therefore, the taxonomic status of this species needs to be resolved. Here we determined the complete mitochondrial genome of two Japanese otters caught in Kanagawa and Kochi prefectures and five Eurasian otters (L. lutra). We reconstructed a molecular phylogenetic tree to estimate the phylogenetic position of the Japanese otter in Lutrinae using the Japanese otters and the other 11 Lutrinae species on the basis of ND5 (692 bp) and cytochrome b (1,140 bp) sequences. We observed that the two Japanese otters had close relationships with Eurasian otters, forming a monophyletic group (100% bootstrap probability). To elucidate detailed phylogenetic relationships among the Japanese and Eurasian otters, we reconstructed a maximum likelihood tree according to mitochondrial genome sequences (14,740 bp). The Japanese otter (JO1) collected in Kanagawa was deeply nested in the Eurasian otter clade, whereas the Japanese otter (JO2) collected in Kochi formed a distinct independent lineage in the Lutra clade. The estimated molecular divergences time for the ancestral lineages of the Japanese otters was 0.10 Ma (95%: 0.06–0.16 Ma) and 1.27 Ma (95%: 0.98–1.59 Ma) for JO1 and JO2 lineages, respectively. Thus, JO1 was identified as a member of L. lutra; JO2 represented the old Japanese otter lineage, which may be a distinct new species or subspecies of Lutra. We suggest that the ancestral population of the JO2 lineage migrated to Japan via the land bridge that existed between western Japanese islands and Asian continent at 1.27 Ma.

Evolutionary History of the Extinct Otter Lived in Japanese Islands

“…In the course of time, each lineage evolves to a different species. JO2 diverged from the ancestral lineage of L. lutra, which includes an ancestor of JO1 and the divergence time was calculated at approximately 1.27 Mega annum (Ma) during the Calabrian stage (1.80-0.78 Ma) of the Early Pleistocene (Figure 3, node1) [10]. During this stage, Japanese islands and the Eurasian continent were connected by a land bridge, the presence of which is supported by geological study [14].…”

“…JO1 formed a monophyletic group with six individuals of L. lutra, indicating that the JO1 lineage evolved from a common ancestor of L. lutra ( Figure 3, node 2) [10]. In particular, the phylogenetic tree suggests that JO1 lineage has a close genetic relationship to Chinese populations of L. lutra, and JO1 was identical to the species of L. lutra.…”

“…In particular, the phylogenetic tree suggests that JO1 lineage has a close genetic relationship to Chinese populations of L. lutra, and JO1 was identical to the species of L. lutra. In contrast, JO2 showed a distinct and independent lineage, which diverged from the monophyletic group of L. lutra and JO1 (Figure 3, node 1) [10]. This result suggests that the JO2 lineage should be treated as an independent species (L. nippon) or subspecies (L. l. nippon).…”

“…One of the individuals was captured in the Kanagawa prefecture in 1915 or 1916 (JO1) and the other was captured in the Kochi prefecture in 1977 (JO2; Figure 2). Subsequently, we used NGS to determine the mitochondrial genome (mtgenome) sequence (approximately 16,400 bp) of our samples [10]. Recently, various genetic studies for estimating the evolution of extinct organisms have been reported; however, these results were obtained from a limited number of study groups [11][12][13].…”

“…This result suggests that the JO2 lineage should be treated as an independent species (L. nippon) or subspecies (L. l. nippon). Waku et al [10] suggested that the Japanese otter was divided into Honshu and Shikoku lineages based on a molecular phylogenetic analysis. The molecular divergence time, which is estimated based on mtgenome sequences, indicates the start of a unique evolutionary history of each lineage.…”