Integrative phylogenomics uncovers hidden diversity and repeated cave colonizations in Dinaric pseudoscorpions

Subterranean ecosystems harbour exceptional biodiversity shaped by adaptation to extreme environments, yet remain poorly explored. Cave organisms, restricted to small, isolated populations, are extinction-prone, making integrative species delimitation essential for biodiversity assessment and conservation. The Dinaric Karst, a global hotspot of subterranean diversity, is home to Neobisium spelaeum, the first described cave pseudoscorpion, whose evolutionary history remains unknown. We investigated diversification dynamics by integrating mitochondrial and genomic data with population genetics, species delimitation, demographic modelling and morphological analyses across N. spelaeum and its relative N. stygium. DNA barcode analyses suggest repeated subterranean colonizations across cave-dwelling Neobisium. Within this polyphyletic assemblage, N. spelaeum and N. stygium form a clade. Genome-wide data reveal eight deeply divergent lineages corresponding to karst regions shaped by Oligocene-Miocene geological barriers, plus seven additional singletons, whereas mito-nuclear discordance complicates the reconstruction of evolutionary trajectories. Morphological differentiation among lineages is subtle but statistically significant. Demographic analyses reveal bottlenecks at 270 000-470 000 years ago followed by population expansions, compatible with regional cave colonization events during Middle Pleistocene glacial cycles. Our findings highlight the Dinaric Karst as a major centre of subterranean endemism, underscoring the need to protect karst landscapes and their endemic fauna while providing a framework for taxonomic revision and conservation planning.