Identification of Three Distinct Groups of Anopheles lindesayi in Japan by Morphological and Genetic Analyses

Anopheles (Anopheles) lindesayi Giles consists of 5 subspecies. In Japan, only one subspecies, An. l. japonicus Yamada, has been reported. Its geographical populations are morphologically diverse; however, they are regarded as a single subspecies. In this study, we re-evaluated the taxonomic status of An. l. japonicus in Japan, and that of another subspecies, An. l. pleccau, distributed in Taiwan, by comparative morphological and molecular analyses based on the gene sequences of mitochondrial DNA cytochrome c oxidase I (COI) and ribosomal DNA internal transcribed spacer 2 (ITS2). Nucleotide sequence divergence was calculated using the Kimura-two-parameter (K2P) distance model. Phylogenetic trees based on COI and ITS2 sequences showed 3 distinct clades: Eastern Japan, Western Japan, and the Ryukyus. The sequences of the Ryukyu specimens were located within the same Glade as that of the sequences of the Taiwanese specimens. Regarding the COI sequences, the 3 geographical groups in Japan were genetically distinct. The following morphological characteristics distinguished the groups: larval seta 1-S, pupal setae 5 through segments IV-VII, and pupal setae 6 on segments IV-VII. Based on these results, it was revealed that An. l. japonicus included 3 genetically and morphologically distinct groups: 2 groups of An. l. japonicus and a group in the Ryukyus, which was a synonym of An. l. pleccau.