Next-generation genome sequencing and assembly provides tools for phylogenetics and identification of closely related species of Spathius, parasitoids of Agrilus planipennis (emerald ash borer).

摘要

The ability to identify natural enemy species unequivocally is a crucial part of biological control by introductions. Such identification is often difficult for insect parasitoids, where cryptic species are often involved. However, recent advances in next-generation DNA sequencing allow whole-genome analysis of non-model species for the discovery of molecular polymorphisms that provide ample evidence to distinguish populations and species. We sequenced the genomes of four closely related species in the genus Spathius that are parasitoids of the invasive emerald ash borer, Agrilus planipennis. These include Spathius agrili, which has already been introduced in to the USA from China against A. planipennis, Spathius galinae, a recently discovered species from the Russian Far East that is being evaluated for introduction against A. planipennis, and Spathius floridanus and Spathius sp. (undescribed), which are native to North America. Using the Illumina HiSeq technology, we sequenced the whole genomes of each species and made draft de novo assemblies of the genomes which were 147-213 million nucleotides long with 58-96 fold coverage. A random sample of 1000 contigs from the assembled genomes harbored 150-550 thousand single nucleotide polymorphisms (SNPs) or 1-2.8 SNPs per kilobase that were fixed differences between these species. Using these SNPs, we developed a molecular phylogeny for the four species, which showed that the undescribed Spathius species from the USA is most closely related to S. floridanus, and S. galinae is most closely related to S. agrili. Taking advantage of insertion/deletions that differed among species, we developed primers for 10 loci that differed in amplicon length on 10 different contigs. All loci with putative indels amplified for all species with the primers we designed, although amplicon fragment lengths for about half the species/locus combinations did not match the expected lengths. Nevertheless, four of loci gave amplicons with fixed length differences between these species. One primer pair alone can distinguish all four species, providing a simple, rapid, inexpensive PCR/gel-based identification of these Spathius species as larvae and adults.