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

<div><p>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 <i>Lutra lutra</i>. 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 (<i>L</i>. <i>lutra</i>). 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 <i>ND5</i> (692 bp) and cytochrome <i>b</i> (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 <i>Lutra</i> 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 <i>L</i>. <i>lutra</i>; JO2 represented the old Japanese otter lineage, which may be a distinct new species or subspecies of <i>Lutra</i>. 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.</p></div

Phylogenetic tree of Eurasian and Japanese otters based on the mtGenome.

<p>The ML tree based on the mtGenome comprised 14,740 bp (tRNAs = 1,488 bp, rRNAs = 2,532 bp, and protein-coding genes = 10,720 bp). This tree was estimated using the GTR+Γ+I model. Numbered boxes denote nodes. The nodal number indicates the BP value. BP was estimated on the basis of 1,000 bootstrap replicates. The evolutionary constraints on nucleotide substitutions must differ between the first, second, and third codon positions as well as between tRNA and rRNA. Therefore, we specified partitions for each region. Data for the <i>E</i>. <i>lutris</i> and <i>L</i>. <i>lutra</i> (South Korea) were reported previously NC_009692 and FJ236015, respectively.</p

Phylogenetic tree for Lutrinae based on the partial mtDNA together with the <i>L</i>. <i>nippon</i> (Ehime).

<p>This ML tree was based on the partial <i>ND5</i> gene (692 bp) and complete <i>cytb</i> gene (1,134 bp) dataset, which included the <i>L</i>. <i>nippon</i> (Ehime) sequence (224 bp) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149341#pone.0149341.ref010" target="_blank">10</a>]. This tree was estimated using the GTR+Γ+I model. Numbered boxes denote nodes. The nodal number indicates the BP value. BP was estimated on the basis of 1,000 bootstrap replicates. The evolutionary constraints on the nucleotide substitutions must differ between the first, second, and third codon positions; therefore, we specified partitions for each region. OTUs without localities are previously reported data (Koepfli and Wayne [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149341#pone.0149341.ref001" target="_blank">1</a>]; Koepfli et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149341#pone.0149341.ref012" target="_blank">12</a>]). Data for <i>L</i>. <i>lutra</i> (South Korea) is FJ236015.</p

Origins of the Eurasian and Japanese otters used in this study.

<p>Origins of the Eurasian and Japanese otters used in this study.</p

Map showing the locations of Japanese and Eurasian otters.

<p>(A) Map of East Asia. (B) Map of Shikoku Island. The capture locations of the individual Japanese otters used in this study are indicated by filled circles (JO1, JO2, and JO3). The capture location of the individual Japanese otter used by Suzuki et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0149341#pone.0149341.ref010" target="_blank">10</a>] is indicated by an open circle. EO indicates the locations from where Eurasian otter samples were obtained (EO3, EO5). Reprinted from PLOS ONE under a CC BY license, with permission from Environmental Systems Research Institute, Inc. (Esri), original copyright 2015.</p