Morphological and genetic polymorphisms in Rhipicephalus sanguineus (Latreille 1806) from the Iberian Peninsula.

Rhipicephalus sanguineus is a tick species characterized by high intraspecific variability, even among populations occupying the same habitats. This study investigated whether two distinct phenotypes of R. sanguineus, collected from four geographically separated regions, are associated with specific nucleotide polymorphisms in the mitochondrial cox1 and 16S rRNA gene regions-markers commonly employed in phylogenetic analyses. Morphometric analysis based on established criteria clearly distinguished two morphotypes-Morphotype 1 and Morphotype 2-with statistically significant differences. This morphological differentiation was supported by phylogenetic and genetic divergence analyses, primarily based on mitochondrial cox1 sequences, which revealed six polymorphic sites within the analyzed fragment. Among the identified haplotypes, two-Haplotype 2a and Haplotype 2b-were consistently associated with Morphotype 2. This genetic segregation was confirmed in extended analyses including reference isolates (morphologically and/or genetically defined either as R. sanguineus or R. hibericus). These analyses also demonstrated a close phylogenetic relationship between Morphotype 1 and the R. sanguineus neotype, as well as between R. hibericus and both morphotypes. Although the 16S rRNA marker exhibited lower resolution, it still effectively distinguished the two morphotypes and corroborated the haplotype assignments established by the cox1 phylogeny. The observed genetic divergence (p-distance of 1% in cox1 gene) and the phylogenetic relationship between R. sanguineus and R. hibericus confirmed that R. sanguineus is a polymorphic species, with well-defined haplo-morphotypes, and that R. hibericus is not genetically distinguishable from R. sanguineus.