Genetic diversity of Phlebotomus perniciosuspopulations between insular and mainland regionsin the leishmaniasis-endemic westernMediterranean area

Phlebotomus perniciosus is the primary vector of Leishmania infantum in Spain, occurring in both continental and insular regions. This study investigates the genetic structure of P. perniciosus populations from Majorca (island) and Barcelona (mainland), two geographically close but ecologically distinct regions in the western Mediterranean. Methods: Mitochondrial cytochrome c oxidase subunit I (COI) gene sequences were analyzed from 167 P. perniciosus specimens, including 100 morphologically identified field-collected specimens from Majorca and Barcelona, supplemented with reference data from the South and West Iberian Peninsula and North Africa. Population differentiation was assessed using genetic diversity indices, Bayesian phylogenetic inference, analysis of molecular variance (AMOVA), pairwise Fst values and Nm estimates, haplotype networks, and a Mantel test. Results: Phylogenetic analysis confirmed the morphological identification of all P. perniciosus specimens, grouping them into a single clade, with distinct subclades corresponding to the geographical origin. Haplotype analysis revealed 56 genetic variants, with the predominant haplotype represented by 37 specimens in Majorca and 40 in Barcelona. Significant genetic differentiation was observed between populations from Majorca and Barcelona (Fst = 0.78262; p < 0.00001), indicating limited gene flow. Nucleotide diversity was higher in Majorca (π ± SD = 0.0037 ± 0.00090) than in Barcelona (π ± SD = 0.0006 ± 0.00021). Majorcan specimens showed close genetic affinity to the Algerian and Tunisian populations (Fst = 0.02470; p > 0.05), while Barcelona specimens were more closely related to those of the South and West Iberian Peninsula (Fst = 0.51225; p < 0.00001). Conclusions: These findings indicate that geographic isolation and historical dispersal may have shaped the P. perniciosus genetic structure. The Balearic Sea appears to act as a significant barrier, restricting gene flow between island and Iberian mainland populations. The study supports the utility of COI in phylogeographic research and demonstrates how island-mainland comparisons can help reveal evolutionary processes in vector species.