Molecular insights into spined loaches (Cobitidae: <i>Cobitis</i> ) reveal the complex evolutionary history of freshwater fishes in the Iberian Peninsula and North Africa

Abstract Palaeogeographic and tectonic rearrangements of rivers have significantly influenced the phylogeography of Iberian and North African freshwater fishes, but their impact remains unclear. We revisit the phylogeographic relationships and diversification timelines of Cobitis (spined loaches, family Cobitidae) on both sides of the Strait of Gibraltar. Sampling 230 sites across 41 river basins, we analysed genetic variability using haplotype networks of two mitochondrial (Cytb and COI) and two nuclear (S7 and RAG-1) genes. A time-calibrated multilocus phylogeny was constructed and compared with the evolution timeline of the Iberian hydrographic network. Our results indicate that Iberian and North African Cobitis are monophyletic at the mitochondrial, but not at the nuclear level. Three new mitochondrial lineages, potentially new species, were identified in Iberia (2) and Morocco (1). Most Iberian Cobitis show wide distributions and frequent sympatry in major basins. Diversification started in the Mid-Late Miocene (7.9–8.9 Mya), with major speciation during the Late Miocene–Pliocene transition (6.5–5.7 Mya). Palaeographic changes in river basins, from endorheic to exorheic regimes, probably affected Cobitis speciation. Tectonic strike-slip faults probably influenced isolation, genetic mixing, and incomplete lineage sorting, particularly along Atlantic margins. These results highlight the interplay of geological and evolutionary processes in shaping freshwater fish diversity.