Recurrent hybridization shapes the diversification of Western Palearctic common toads (Bufo bufo complex).

Abstract

Glacial cycles repeatedly fragmented temperate species into refugial populations, fostering divergence as a first stage towards speciation. Yet, interglacial expansions often reconnected these lineages, allowing gene flow to erode differentiation and reshape phylogeographic trajectories. We investigate these dynamics in Western Palearctic common toads (Bufo bufo complex), integrating genome-wide ddRAD-seq loci with an extensive mitochondrial dataset. Phylogenomic analyses resolved the three recognized species B. eichwaldi, B. spinosus and B. bufo, and within the latter, four major lineages distributed across the Apennine Peninsula, the Balkans, the Caucasus, and northern Europe. Mitochondrial and nuclear patterns were deeply discordant, and different approaches of historical gene flow inferences all supported past hybridization. In particular, our analyses suggest that the Caucasian population, previously attributed to the disputed species "B. verrucosissimus" based on its deeply divergent mtDNA, represents a shallow nuclear lineage within B. bufo and forms a broad intergradation zone with the Balkan lineage in Anatolia, arguing against a species status. Altogether, these results highlight a recurrent process in which refugial lineages do not diverge in strict isolation but repeatedly experience gene flow, thus reducing opportunities to speciate, and blurring tree-based phylogeographic and systematic hypotheses.