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Many studies have reported the complete mitochondrial genome sequences of Chinese Megascolecidae earthworms, however, there have been no reports on sequences originating from Japanese Megascolecidae earthworms. In this study, we determined complete mitochondrial genome sequences of two Japanese earthworms belonging to the Pheretima complex within the Megascolecidae family. Metaphire hilgendorfi is one of the most common earthworms in Japan and Amynthas yunoshimensis was found to be morphologically similar to M. hilgendorfi. The complete mitochondrial genomes of M. hilgendorfi (15,186 bp; LC573968) and A. yunoshimensis (15,109 bp; LC573969) contained typical 13 protein coding genes, 22 tRNA genes, and 2 rRNA genes. The phylogenetic analysis revealed that these two species were sister species. Therefore, our findings will further contribute to phylogenetic and genetic diversity analyses of Megascolecidae earthworms.

Although earthworms are hermaphroditic animals with biparental sexual reproduction, some parthenogenetic species have been found. Evolutionary trends in parthenogenetic earthworms revealed a reduction in the reproductive organs. To clarify the phylogenetic relationships of parthenogenetic earthworms with different degree of degraded reproductive organs, we conducted a morphological analysis of the reproductive organs and molecular phylogenetic analyses of Amynthas vittatus which usually degraded a part of reproductive organs. Morphological analysis revealed that almost all individuals collected around Mt. Aobayama, Sendai city of northeastern Japan, possessed male pores, while individuals collected from areas located across Hirose River did not. Phylogenetic analysis using mitochondrial DNA sequences of 48 individuals representing 20 populations indicated that almost all individuals collected around Mt. Aobayama belonged to a different lineage from the other populations collected around Sendai, and that almost all individuals collected from across Japan belonged to the latter lineage. We suggest that the difference in the male pore possession rate was caused by histories of each population, but the A. vittatus population found on Mt. Aobayama belongs to a different lineage as compared to the other Japanese populations and not the primitive population. Thus, the parthenogenetic earthworm A. vittatus has undergone at least two morphological evolutionary processes.

To clarify the historical phylogeography of a Japanese earthworm, nucleotide sequence variations of Metaphaire sieboldi were investigated with respect to its geographic distribution. Sequencing of mitochondrial DNA (mtDNA), including the cytochrome oxidase subunit I (COI) region and the 16S rDNA region, of 72 samples including 4 outgroup taxa, and phylogenetic analyses using neighbour-joining (NJ) and Bayesian inference (BI) methods indicated that M. sieboldi forms a monophyletic group. This monophyletic group was divided into seven subgroups; most subgroups comprised individuals from several areas, except for southern Shikoku and southern Kyushu. The phylogenetic tree demonstrated that the Shikoku and Kinki populations were paraphyletic at the basal node, and individuals from Kyushu and western Shikoku were positioned at the advanced group. Estimated phylogeographic events based on mtDNA analyses include (1) M. sieboldi originated in Shikoku and/or Kinki, (2) individuals from Chugoku and Kyushu have a common ancestor from Shikoku, and (3) individuals from western Shikoku originated from Kyushu progenitors (about 0.4–0.5 mya) after separation by rising sea levels.

"Supplemental Table 1" in the 23rd line from the top in the right column on the 32nd page should be replaced with "Table 5".