Genetic diversity and phylogeography of Hyalomma scupense, the main vector of tropical theileriosis: Global analysis with novel data from Tunisia.

Hyalomma scupense, the main vector of tropical theileriosis, presents taxonomic challenges that impede vector surveillance. This study combines targeted sampling of 20 morphologically-confirmed specimens from El Hessiene, Tunisia (36°50'N 10°12'E) with global GenBank analysis of mitochondrial markers 16S rRNA, 12S rRNA, and COI to assess genetic diversity, phylogeographic structure, and regional gene flow. All Tunisian sequences showed 99.88-100% identity to H. scupense references. Global analysis revealed moderate genetic diversity: 8 genotypes for 16S rRNA (Hd=0.551, π=0.0036), 13 for 12S rRNA (Hd=0.416, π=0.00907, highest variability), and 6 for COI (Hd=0.518, π=0.00142, most conserved). Phylogenetic analyses confirmed H. scupense monophyly, with COI resolving four well-supported geographic clusters (bootstrap 89-98%): North African (including all Tunisian isolates), European-Asian, Central Asian, and West Asian. Tunisian isolates formed a monophyletic North African cluster, indicating ongoing regional gene flow likely mediated by livestock movements. 12S rRNA/16S rRNA markers revealed finer regional structuring. These findings establish a polymorphism hierarchy (12S rRNA > 16S rRNA > COI) and validate multi-marker approaches for tick phylogeography. The North African cluster homogeneity underscores persistent gene flow, with implications for modeling Theileria annulata dispersion and One Health surveillance strategies across Maghreb. COI emerges as optimal for broad-scale phylogeography, while 12S rRNA suits population-level resolution. This baseline informs vector monitoring and control in tropical theileriosis-endemic regions.