AbstractUnderstanding how new species arise through the progressive establishment of reproductive isolation barriers between diverging populations is a major goal in Evolutionary Biology. One important result of speciation genomics studies is that the genomic regions involved in reproductive isolation frequently harbor anciently diverged haplotypes that predate the reconstructed history of species divergence. The possible origins of these old alleles remain highly debated, since they relate to contrasted mechanisms of speciation that are not fully understood yet. In the European sea bass (Dicentrarchus labrax), the genomic regions involved in reproductive isolation between Atlantic and Mediterranean lineages are enriched for anciently diverged alleles of unknown origin. Here, we used haplotype-resolved whole-genome sequences to test whether divergent haplotypes could have originated from a closely related species, the spotted sea bass (Dicentrarchus punctatus). We found that an ancient admixture event between D. labrax and D. punctatus is responsible for the presence of shared derived alleles that segregate at low frequencies in both lineages of D. labrax. An exception to this was found within regions involved in reproductive isolation between the two D. labrax lineages. In those regions, archaic tracts originating from D. punctatus locally reached high frequencies or even fixation in Atlantic genomes but were almost absent in the Mediterranean. We showed that the ancient admixture event most likely occurred between D. punctatus and the D. labrax Atlantic lineage, while Atlantic and Mediterranean D. labrax lineages were experiencing allopatric isolation. Our results suggest that local adaptive introgression and/or the resolution of genomic conflicts provoked by ancient admixture have probably participated to the establishment of reproductive isolation between the two D. labrax lineages.Author summarySpeciation is often viewed as a progressive accumulation of reproductive isolation barriers between two diverging lineages through the time. When initiated, the speciation process may however take different routes, sometimes leading to the erosion of an established species barrier or to the acquisition of new speciation genes transferred from another species boundary. Here, we describe such a case in the European sea bass. This marine fish species has split 300,000 years ago into an Atlantic and a Mediterranean lineage, which remained partially reproductively isolated after experiencing postglacial secondary contact. For unknown reasons, genomic regions involved in reproductive isolation between lineages have started to diverge well before the split. We here show that diverged alleles were acquired by the Atlantic lineage from an ancient event of admixture with a parapatric sister species about 80,000 years ago. Introgressed foreign alleles that were locally driven to high frequencies in the Atlantic have subsequently resisted to introgression within the Mediterranean during the postglacial secondary contact, thus contributing to increased reproductive isolation between two sea bass lineages. These results support the view that reproductive isolation barriers can evolve via reticulate gene flow across multiple species boundaries.