Molecular Phylogeny and Color Variation of Two Large-size Octannulate Species of Orobdella, O. octonaria and O. nakahamai (Hirudinea: Orobdellidae), with Insights into Their Distribution

Understanding species distributions is essential for biogeographical and ecological studies. This study reports the geographical ranges of two large-size octannulate species of the leech genus Orobdella Oka, 1895, O. octonaria Oka, 1895, and O. nakahamai Nakano, 2016, based on newly collected specimens. In addition, intraspecific genetic variation was investigated via phylogeographic analyses using mitochondrial markers, i.e., cytochrome c oxidase subunit I, tRNA[Leu], NADH dehydrogenase subunit 1, tRNA[Cys], tRNA[Met], 12S rRNA, tRNA[Val] and 16S rRNA. The results reveal that both species show a parapatric distribution pattern in the Sanuki Mountains on the eastern Shikoku Island, and may not occur sympatrically. Furthermore, genetic analyses may reflect that these large-size species have relatively higher dispersal ability and reduced effects of major geographic barriers on diversification. Orobdella octonaria is newly recorded from Shikoku Island, previously known only from Honshu, and the coloration of these Shikoku individuals is described. Orobdella nakahamai is reported from the eastern Shikoku Island and Kyushu Island based on voucher specimens for the first time, representing a range extension from the western Shikoku Island. Furthermore, this study provides the first comprehensive definition of the color variations in O. octonaria, categorizing them into six distinct types. Our subsequent phylogenetic analysis revealed that the Chubu-Tōkai type is polyphyletic and that two distinct color types coexist at the same locality but belong to different clades, strongly suggesting the presence of potential cryptic species within this polymorphic taxon.