Factors Beyond Karstification Have Shaped the Population Structure of a Surface-Dwelling Minnow () Able to Disperse Underground.

The Dinaric Karst, a biodiversity hotspot, features complex surface and subterranean hydrological networks that influence aquatic species distribution. This study investigates how karst hydrology shapes the genetic structure of the surface-dwelling minnow , examining both large-scale and small-scale population patterns. Using mitochondrial DNA and genome-wide single nucleotide polymorphism (SNP) data of 827 specimens of , three hypotheses were tested: (1) karst underground water connections facilitate genetic connectivity within and across river systems, whereas non-karst rivers exhibit genetic connectivity mostly within the same system; (2) historical and occasional hydrological connections have shaped present-day population structure, leaving genetic signatures of relatedness where no contemporary hydrological links exist; and (3) genomic approaches provide additional insights into biologically relevant connections that may not be captured by classical tracing tests. The large-scale analyses confirmed three main genetic groups (1a-c), whose structure was likely shaped by Pleistocene glaciations and associated microrefugia rather than by karst hydrology. Small-scale structure analyses revealed that while karst hydrology facilitated gene flow within specific areas, connectivity was uneven and influenced by local hydrological dynamics and historical admixture events. Furthermore, some underground pathways identified by classical tracing tests lacked evidence of genetic connectivity, underscoring the limitations of traditional methods and the added value of genomic data in indirectly detecting biologically relevant hydrological connections. These findings highlight the influence of both historical processes and contemporary karst hydrology on populations, emphasizing their vulnerability in karst ecosystems and the need for targeted conservation efforts.