Abstract
Speciation, i.e., the formation of new species, implies that diverging populations evolve genetic, phenotypic or ecological factors that promote reproductive isolation (RI), but the relative contributions of these factors remain elusive. Here we test which of genomic, bioacoustic, morphological, and environmental differences best predicts RI across a continuum of divergence in the midwife toads (genus Alytes), a group of Western Mediterranean amphibians, using a total evidence approach. We found that, without strong geographic barriers to dispersal, the extent of introgression across hybrid zones between phylogeographic lineages, which should reflect the strength of RI, predominantly covaries with genomic divergence. Overall phenotypic differentiation becomes substantial only between well established, fully isolated species. These results suggest that speciation in midwife toads initially involve cryptic lineages, which probably evolve RI through intrinsic (genetic) hybrid incompatibilities. As they continue to diverge, these nascent species eventually differentiate externally, which potentially enforces pre-mating barriers and facilitates sympatry. This speciation scenario has practical implications for species delimitation, notably when using hybrid zones and divergence thresholds as proxies for reproductive isolation.