Mitogenomic diversity and genetic population structure of the pinewood nematode (PWN) Bursaphelenchus xylophilus inhabiting Kyushu, Japan were analyzed. A method for performing long PCR using single nematodes and sequencing nematode mitochondrial genomes individually is presented here. About 8 kb (∼55%) of the complete mitochondrial genome was successfully obtained from 285 individuals collected from 12 populations. The 158 single nucleotide polymorphisms detected corresponded to 30 haplotypes, clearly classified into two clades. Haplotype diversity was 0.83, evidencing a remarkable high diversity within Kyushu. The high genetic differentiation among the 12 populations (0.331) might be due to past invasion and expansion routes of PWN in northeastern and southeastern Kyushu. The distinct genetic composition of populations within the northwestern, central western, and southwestern Kyushu seems to be mostly related to the extinction of pine forests and long-range migration of PWN due to human activity. Overall, direct long PCR and sequencing of single nematode individuals are effective methods for investigating mitochondrial polymorphisms, and these are effective tools for PWN population genetics and other intraspecific studies.
