High Mitochondrial Genome Diversity and Intricate Population Structure of Bursaphelenchus xylophilus in Kyushu, Japan

Abstract

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.

Keywords: Bursaphelenchus xylophilus; Genomics; Haplotype diversity; Mitochondrial polymorphism; Pinewood nematode; Population structure; Sequence polymorphism..

Figure 1

Location of the studied populations of Bursaphelenchus xylophilus (black dots) in Kyushu and the group of haplotype distribution of each population. The names of populations are written in code as indicated in Table 1. The classification of haplotypes is presented as in Figure 2.

Figure 2

Phylogentic tree for 33 mitochondrial haplotypes of Bursaphelenchus xylophilus containing 30 from Kyushu and 3 from previous studies was inferred by using the Maximum Likelihood (ML) method based on the Kimura 2-parameter model. Numbers under the branches represent support values from bootstrap replications of 1,000. Evolutionary distances greater than 0.0001 are shown above branches. Wide bars indicate the major clades clustering.

Figure 3

Neighbor-joining (NJ) tree diagram showing the genetic relationship that was determined using genetic distances of 12 populations. Numbers next to corresponding nodes indicate the branch lengths in the same units of the genetic distances that are computed based on the haplotype frequency for each population.