Abstract
Stomoxys calcitrans (Diptera: Muscidae) is a significant insect in the veterinary and medical fields. Previous studies have found a pronounced genetic divergence between populations from the Oriental region and those from other zoogeographical zones. Understanding the morphological and genetic variation within S. calcitrans populations is essential for clarifying the evolutionary processes underlying their structure across biogeographical regions. This study conducted geometric morphometrics on a total of 120 wings (30 per group: Thailand males, Thailand females, Spain males, and Spain females) to assess wing size and shape variations between S. calcitrans populations from Thailand (Oriental region) and Spain (Palaearctic region). Molecular analyses utilized two mitochondrial markers, cox1 and cytb, the nuclear marker ITS2, and a concatenated dataset of all three. Geometric morphometric analysis revealed statistically significant differences in wing size and shape (P < 0.05), although the classification accuracy based on wing shape was moderate (70%), indicating phenotypic plasticity rather than species-level differentiation. Phylogenetic reconstruction identified two well-supported genetic lineages. However, the results from the species delimitation methods (Assemble Species by Automatic Partitioning, Automated Barcode Gap Discovery, and multi-rate Poisson Tree Processes), low interpopulation divergence, and a shared haplotype all verify that these lineages represent a single, globally distributed species. Further neutrality tests and mismatch distribution analyses revealed that the Oriental population has a deeper evolutionary history, while the European population likely arose from a more recent colonization event. These findings demonstrate the influence of historical biogeographical processes in shaping the global genetic structure of S. calcitrans and underscore the importance of broader geographical sampling to fully elucidate its evolutionary history.