Comparative analyses of mitochondrial and nuclear genetic markers for the molecular identification of Rhipicephalus spp.

- Molecular and phylogenetic analyses of five Rhipicephalus species are presented.
- Mitochondrial (mt) and nuclear (ITS-2) genes are examined.
- Mean interspecific divergence up to 15.2% for mt and 2.8% for ITS-2, respectively, were recorded.
- Conflicting results were obtained when comparing mt and ITS-2 markers.
- ITS-2 did not allow the identification of ticks of the R. sanguineus complex.

The genus Rhipicephalus (Acari: Ixodidae) comprises a large number of vectors of pathogens of substantial medical and veterinary concern; however, species identification based solely on morphological features is often challenging. In the present study, genetic distance within selected Rhipicephalus species (i.e., Rhipicephalus bursa, Rhipicephalus guilhoni, Rhipicephalus muhsamae, Rhipicephalus sanguineus sensu lato and Rhipicephalus turanicus), were investigated based on molecular and phylogenetic analyses of fragments of the mitochondrial 16S, 12S and cytochrome c oxidase subunit 1 (cox1) genes, as well as of the whole sequences of the ribosomal internal transcribed spacer-2 (ITS-2) region. Mean values of inter-specific genetic distance (e.g., up to 12.6%, 11.1% and 15.2%), as well as of intra-specific genetic distance (e.g., 0.9%, 0.9% and 1%), calculated using the Kimura-2 parameter substitution model with uniform rates among sites for 16S, 12S and cox1 genes, respectively, confirmed the differentiation of the rhipicephaline species herein examined. The molecular identification was also supported by the distinct separation of species-specific clades inferred from the phylogenetic analyses of all mitochondrial sequences. Conversely, little interspecific divergence was detected amongst ribosomal ITS-2 sequences (i.e., up to 2.8%) for species belonging to the R. sanguineus complex, which resulted in the ambiguous placement of selected R. sanguineus s.l. and R. turanicus sequences in the corresponding phylogenetic tree. Results from this study confirm the suitability of mtDNA markers for the reliable identification of ticks within the Rhipicephalus genus and provide a framework for future studies of taxonomy, speciation history and evolution of this group of ticks.