Cytochrome c oxydase I phylogenetic analysis of Haemogregarina parasites (Apicomplexa, Coccidia, Eucoccidiorida, Haemogregarinidae) confirms the presence of three distinct species within the freshwater turtles of Tunisia.

Species of Haemogregarina are apicomplexan blood parasites that use vertebrates as intermediate hosts. Due to limited interspecific morphological characters within the genus during the last decade, 18S rRNA gene sequences were widely used for species identification. As coinfection patterns were recently reported from nuclear molecular data for two sympatric freshwater turtles Mauremys leprosa and Emys orbicularis from Tunisia, our objectives were to design COI specific primers to confirm the presence of three distinct species in both host species. Blood samples were collected from 22 turtles, from which DNAs were extracted and used as templates for amplification. Following different rounds of PCR and nested PCR, we designed specific Haemogregarina COI primers that allowed the sequencing of nine distinct haplotypes. Phylogenetic Bayesian analysis revealed the occurrence of three well-differentiated sublineages that clustered together into a single clade. Based on pairwise genetic distances (p-distance), we confirmed the occurrence of three distinct but phylogenetically closely related species coinfecting M. leprosa and E. orbicularis in the same aquatic environments. Our results demonstrate that the use of fast evolving genes within Haemogregarina will help to investigate the parasite diversity within both intermediate vertebrate and definitive invertebrate hosts, and to assess the evolution, historical biogeography and specificity of haemogregarines.