Evaluation of three mitochondrial DNA markers for species identification, genetic diversity assessment, and phylogenetic positioning of five Hyalomma tick species from Tunisia.

Hyalomma ticks are important vectors of pathogens affecting human and animal health. This study aimed to assess the outputs of three molecular markers (16S, 12S rRNA, and COI) for accurate tick species molecular identification, genetic diversity assessment, and phylogenetic positioning of Hyalomma tick specimens from Tunisia. A total of 20 tickspecimens were collected from different hosts including cattle, camels, and turtles in nine Tunisian governorates. Morphological diagnosis confirmed five Hyalomma species: H. scupense, H. marginatum, H. excavatum, H. aegyptium, and H. dromedarii. Molecular analysis confirmed species' identification and revealed varying levels of genetic diversity within each species. Within the H. scupense and H. aegyptium species, low genetic diversity was observed, with no variable sites detected in the 16 S and 12 S rRNA markers, except for a single site in H. aegyptium's COI marker. H. marginatum isolates showed moderate diversity, with no variable sites in the 16S and 12S rRNA markers, and four variable sites in the COI marker. In contrast, H. excavatum and H. dromedarii isolates exhibited higher diversity, with variable sites identified in all three markers (16S, 12S rRNA, and COI). Phylogenetic analysis revealed that, with the exception of H. scupense, the COI marker exhibited superior discriminatory power compared to the 16 S and 12 S rRNA markers. This enabled the discernment of distinct genetic clusters within each species. These findings underscore the importance of utilizing multiple molecular markers for confirming morphological diagnosis and specifically highlight the significance of the COI marker in assessing the genetic diversity and phylogenetic relationships of Hyalomma ticks. By employing these markers, particularly COI, valuable insights into the evolutionary dynamics and epidemiological significance of Hyalomma ticks can be gained, facilitating targeted control measures against tick-borne diseases.