Cave-Dwelling Planariidae in Dinaric Karst Exhibit Differing Levels of Cave Trait Evolution

The Dinaric karst encompasses a network of over 10,000 caves and represents one of the world's most important subterranean biodiversity hotspots. It is inhabited by remarkably diverse and often endemic species, including planarian flatworms. Although cave planarians have long been known from the region, they have not yet been investigated using an integrative approach, and their evolutionary relationships with surface-dwelling relatives remain unresolved. To address these gaps, we combined field sampling, phylogenetic analyses based on the COI and 18S genes, and phenotypic characterisation. Our results show that cave planariids in the Dinaric karst belong to at least three genera and are more widespread and diverse than previously assumed. We increased the number of cave records in the Dinaric karst from 26 to 37 and documented cf. Atrioplanaria and Phagocata in Croatian caves for the first time. Phylogenetic reconstructions are consistent with independent cave colonisation events, including multiple instances within the genera Crenobia and cf. Atrioplanaria. Variation in pigmentation and eye reduction, both within and between populations, further reveals heterogeneous evolutionary trajectories of cave-associated phenotypes. Our molecular phylogeny and documentation of trait variability constitute a first step towards establishing Planariidae as a model for investigating the mechanisms underlying the convergent evolution of pigment loss and eye reduction in cave environments.