Mitochondrial ribosomal genes as novel genetic markers for discrimination of closely related species in the Angiostrongylus cantonensis lineage

The Angiostrongylus cantonensis lineage (Nematoda: Metastrongyloidea) consists of the closely related species A. cantonensis, Angiostrongylus malaysiensis, and Angiostrongylus mackerrasae. Various genetic markers have been used for species discrimination in molecular phylogenetic studies of this lineage. However, despite showing potential in other organisms, mitochondrial 12S and 16S ribosomal RNA (rRNA) genes have not been used for Angiostrongylus species discrimination. Therefore, this study assessed these genes' suitability for inter- and intraspecies discrimination in the A. cantonensis lineage. The ultimate aim was to provide a novel genetic marker to support existing phylogenies. Sixty adult Angiostrongylus spp. worms from four geographic locations in Thailand were identified morphologically before molecular identification with 12S and 16S rRNA genes. Neighbor-joining and maximum likelihood algorithms were used for phylogenetic analyzes, and sequence variation was calculated to determine whether the genes could be used to discriminate among species. Furthermore, sequence variation was compared among previously used genetic markers to evaluate the robustness of the 12S and 16S rRNA genes as markers. Using both markers, the A. cantonensis lineage formed a monophyletic clade with a clear separation between A. cantonensis, A. malaysiensis, and A. mackerrasae. From our representative A. cantonensis and A. malaysiensis specimens, the genetic distance between the two clades was 6.8% -7.9% and 7.9% -10.0% for 12S and 16S rRNA genes, respectively, which is sufficient interspecific genetic variation for species discrimination. Higher levels of genetic variation were observed for the 16S rRNA gene, with 12 haplotypes and an intraspecific variation ≤2.2%. Thus, as a genetic marker, the 16S rRNA gene is comparable to mitochondrial protein-coding genes, which are commonly used in intra-level Angiostrongylus spp. studies. In conclusion, mitochondrial 12S and 16S rRNA genes can discriminate among closely related species in the A. cantonensis lineage, and they represent novel genetic markers for supporting existing phylogenies and verifying the phylogenetic position of A. mackerrasae.