Abstract
This study investigates the phylogenetic relationships, genetic diversity, and biogeographic structure of Pipistrellus species in Türkiye using mitochondrial cytochrome b (Cytb) sequences from 156 specimens collected across 26 localities. Our primary aim was to clarify taxonomic boundaries of morphologically cryptic species and elucidate the evolutionary role of Anatolia in the Western Palearctic. Analyses strongly confirmed that molecular data are mandatory for defining taxonomic boundaries. Crucially, all individuals morphologically identified as P. pygmaeus were genetically determined to be P. pipistrellus, highlighting the inadequacy of external traits for cryptic species. We resolved deep intraspecific divergence across the genus. In P. pipistrellus, two major lineages (Eastern and Western) were identified, partially separated by the Anatolian Diagonal. Their co-occurrence in multiple localities confirms Anatolia's function as a secondary contact zone. Similarly, P. kuhlii populations represent a transition zone where two distinct lineages, one of Asiatic origin (P. k. lepidus) and one Mediterranean-Levantine (P. k. kuhlii), meet. Furthermore, while P. nathusii is largely associated with migratory European lineages; a genetically distinct, potentially resident lineage was revealed in southwestern Anatolia. Divergence time estimations indicate that this diversification was shaped by major climatic events from the Miocene to the Pleistocene. This study demonstrates that Anatolia is more than just a geographic bridge; it is a dynamic center of evolution, functioning critically as both a glacial refugium and a secondary contact zone for Palearctic bat fauna.