Species of the nematode genus Heterorhabditis are important biological control agents against agricultural pests. The taxonomy of this group is still unclear as it currently relies on phylogenetic reconstructions based on a few genetic markers with little resolutive power, specially of closely related species. To fill this knowledge gap, we sequenced several phylogenetically relevant genetic loci and used them to reconstruct phylogenetic trees, to calculate sequence similarity scores, and to determine signatures of species- and population-specific genetic polymorphism. In addition, we revisited the current literature related to the description, synonymisation, and declaration as species inquirendae of Heterorhabditis species to compile taxonomically relevant morphological and morphometric characters, characterized new nematode isolates at the morphological and morphometrical level, and conducted self-crossing and cross-hybridization experiments. The results of this study show that the sequences of the mitochondrial cytochrome C oxidase subunit I (COI) gene provide better phylogenetic resolutive power than the sequences of nuclear rRNA genes and that this gene marker can phylogenetically resolve closely related species and even populations of the same species with high precision. Using this gene marker, we found two new species, Heterorhabditis ruandica n. sp. and Heterorhabditis zacatecana n. sp. A detailed characterization of these species at the morphological and morphometric levels and nematode reproduction assays revealed that the threshold for species delimitation in this genus, using COI sequences, is 97% to 98%. Our study illustrates the importance of rigorous morphological and morphometric characterization and multi-locus sequencing for the description of new species within the genus Heterorhabditis, serves to clarify the phylogenetic relationships of this important group of biological control agents, and can inform future species descriptions to advance our efforts towards developing more tools for sustainable and environmentally friendly agriculture.
