Bacterial symbionts are crucial for the infectivity and success of entomopathogenic nematodes as biological control agents. However, the current understanding of the symbiotic relationships is limited by taxonomic uncertainties. Here, we used whole-genome sequencing in combination with traditional techniques to reconstruct the phylogenetic relationships between all described Photorhabdus species and subspecies as well as eleven newly isolated symbiotic bacteria of Heterorhabditis nematodes, including the unreported bacterial partner of H. beicherriana. In silico DNA-DNA hybridization scores, orthologous average nucleotide identity (OrthoANI) and nucleotide sequence identity scores of concatenated housekeeping genes (NSI) were calculated and set into relation with current cutoff values for species delimitation in bacteria. Sequence data were complemented with biochemical and chemotaxonomic markers as well as with DNA and ribosomal protein fingerprinting profiles. This polyphasic approach resolved the ambiguous taxonomy of Photorhabdus, especially at the subspecies level and lead to the proposal for reclassification of most of them into a higher taxon. The analyses of the newly isolated symbionts suggested the occurrence of two new taxa, proposed here as Photorhabdus bodei sp. nov. (type strain LJ24-63T=CCOS 1159T) and Photorhabdus laumondii subsp. clarkei subsp. nov. (type strain BOJ-47T= CCOS 1160T). Our study highlights the power of phylogenomic methods to reconstruct phylogenetic relationships and provides a high-resolution taxonomy to improve the understanding of the symbiotic interaction between Photorhabdus and Heterorhabditis entomopathogenic nematodes.

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