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

Molecular Phylogenies of Leeches and Haemoparasites Infecting Freshwater Turtles in Aquatic Ecosystems of Northern Africa Suggest Phylogenetic Congruence between Placobdella costata Sensu Lato and Haemogregarina stepanowi Sensu Lato

“…Based on genetic data, one of these haemogregarine species could be considered with certainty to be H. stepanowi . All of these results were soon confirmed from an analysis of target COI sequences obtained from the same isolates of blood samples [ 16 ]. Nine distinct COI haplotypes were revealed corresponding to three molecular lineages, each corresponding to a distinct Haemogregarina species, as previously advocated from an analysis of nuclear markers [ 15 ].…”

“…The COI of haemogregarines was amplified using the forward HemoFor4 5′-TGGACATTATACCCACCTTTAAG-3′ and reverse HemoRev4 5′-ATACAACCCATAGCTAGTATCAT-3′ primers which were designed for Haemogregarina spp. [ 16 ]. PCR assays of COI were conducted in a final volume of 25 μL comprising 1X buffer, 1.5 mM MgCl 2 , 0.2 mM dNTPs, 0.4 mM primers, 0.75 units GoTaq Polymerase (Promega, Charbonnières-les-Bains, France), and DNA (3 µL) under the following conditions: an initial step of 5 min at 95 °C for long denaturation; followed by 35 cycles of 30 s at 95 °C for denaturation, 30 s at 48 °C for annealing, and 1 min at 72 °C for elongation; with a final step of 10 min at 72 °C for terminal extension.…”

“…At this stage, P. costata was considered as a single species and referred as P. costata sensu lato. Conversely, the final COI protozoan data set included 86 COI sequences corresponding to seven species of Piroplasmida, 17 species of Haemosporida, and 30 species of Eucoccidiorida, including nine putative Haemogregarina species from two South American turtles, and Scrippsiella sweeneyae Balech 1965 used for outgroup comparisons [ 16 ]. At this stage, H. stepanowi was considered as a single species and referred as H. stepanowi sensu lato.…”

“…The interspecific average pairwise COI p-distance was estimated to about 2.1% between two closely related valid distinct species, namely Eimeria lancasterensis Joseph 1969 and Eimeria sciurorum Galli-Valerio 1922, and further considered as the molecular threshold within Haemogregarina [ 16 ]. Therefore, pairwise p-distances were calculated within and between haploclades of H. stepanowi sensu lato to explore the species diversity based on the molecular threshold.…”

“…Due to the low host specificity of H. stepanowi against its intermediate host on the one hand [ 12 , 13 , 14 ] and the high diversity of Haemogregarina spp. in certain turtle species on the other [ 16 , 17 , 18 , 19 ], the host specificity of Haemogregarina could act against leeches—namely Placobdella as the definitive host—as claimed earlier by Attia El Hili et al [ 16 ].…”