We used Random amplified polymorphic DNA (RAPDs), partial ribosomal DNA (rDNA), the mitochondrial gene cytochrome oxidase subunit I (cox1) and major sperm protein (msp) sequence analysis to investigate genetic diversity and population structure in Heterorhabditis bacteriophora, H. indica, H. marelata, H. megidis, H. downesi and H. zealandica comprising 18 isolates collected from different parts of the world. Blastn similarity search performed for the partial rDNA sequences confirmed the identity of Heterorhabditis species and suggested that all the unknown isolates belonged to H. bacteriophora. Blastn e-values for the species and isolates ranged between 0 to 9e-145. Genetic diversity and phylogenetic analysis produced dendrograms that showed high degrees of genetic variations among Heterorhabditis species with the overall average pairwise distance values of 0.3217, 0.6391, 0.7963 and 0.0572 for RAPD, partial rDNA, cox1 and msp, respectively. Although we expected low genetic diversity among H. bacteriophora isolates due to alternate automictic and amphimictic lifecycle and lack of long distance movement capability restricting gene flow, our results demonstrate highly structured genetic variation among the isolates. Phylogenetic analysis showed that H. bacteriophora isolates is a species complex that contain at least two two new species KMD10 and GPS5. Further H. bacteriophora sensu Pionar populations can be divided into two major groups: “HP88” and “Oswego”. We conclude that strictly relying on ITS sequences based blastn for Heterorhabditis species identification is misleading.OARD
