Climatic swings triggered incipient postglacial speciation of flightless Calosoma beetles in Western Balkan sky islands

Climatic change drastically shapes the ecology and evolution of lineages across landscapes. In mountainous regions, climatic oscillations drive fine-scale biome shifts and modulate habitat availability along elevation gradients, thereby governing population connectivity. Cold-adapted highland lineages with low dispersal capacity are ideal systems for studying the impact of past climates on geographic range shifts and genomic signatures. Here, we focus on the flightless highland species Calosoma (Callisthenes) pentheri endemic to the Western Balkan mountains, which exhibits a disjunct geographic distribution. We predict that glacial and interglacial periods drove allopatry through cycles of isolation and secondary contact, leaving genomic signatures among populations. Using hyRAD museomics combined with a newly sequenced reference genome we generated 10,794 single nucleotide polymorphisms and 2,278 loci to reconstruct the evolutionary history of this lineage. Admixture and phylogenomic analyses demonstrate clear delineation between the two lineages reflecting their distribution across the Western Balkans. Genetic signatures and divergence-time estimates suggest that these two lineages represent distinct species that diverged in the Late Miocene with little interspecific, but occasional intraspecific, admixture since the Pliocene. The diversification of these species in the Balkans was likely driven by isolation in sky island glacial refugia during the Miocene to Pliocene. Subsequent secondary contact during Pleistocene glacial periods may have occurred but did not result in introgression. Climatic oscillations during Quaternary ice ages similarly triggered admixture between populations of each massif. These species display intermediate stages along the speciation continuum, representing a model system for in-depth studies of climate-driven evolution in sky islands. Genome-wide data generated from across the range of the Western Balkan highland endemic beetle Calosoma pentheri support the recognition of two distinct species. Divergence between these two species predates Quaternary glaciations and was shaped by Late Miocene to Pliocene climatic swings. Repeated cycles of isolation and connectivity across mountain reliefs have left a distinctive genomic signature among populations. Natural history collection specimens provide robust genomic data for phylogeographic analyses. Western Balkan sky islands exemplify how past climatic oscillations fostered lineage diversification, with current global warming trends threatening these unique ecosystems.