McLaughlin, G. C.; Baquero-Mendez, V.; Sudak, S.; Hollis, B.

Evolutionary theory predicts that when hybridization between divergent populations is costly, natural selection should favor traits that reduce further interbreeding, a process known as reinforcement. Direct experimental evidence of reinforcement is rare, however, and no study has demonstrated that reinforcing selection can strengthen reproductive isolation between naive populations while allowing continuous gene flow. Here we show that >100 generations of experimental selection against hybridization -- imposed by removing a controlled fraction of GFP-marked hybrids -- led to the evolution of increased reproductive isolation between two naive populations of Drosophila melanogaster. Selection strengthened both sexual isolation (via reduced female remating with migrant males) and postmating-prezygotic isolation, although the specific barriers that evolved depended on the strength of selection against hybridization. Sexual isolation was evident only when females encountered both within-population and migrant males, revealing an important role for social mating context. Notably, intermediate selection against hybridization produced the strongest overall increase in reproductive isolation, suggesting that reinforcement is most effective when maladaptive hybridization with both newly arrived immigrants and early-generation hybrids remains sufficiently common to generate persistent selection for avoidance. Whole-genome sequencing revealed that divergence was concentrated in regions linked to the GFP barrier locus, where introgression was reduced. These regions were also enriched for loci showing signatures consistent with an adaptive response to selection against hybridization. Together, these results provide direct experimental evidence that reinforcement can increase reproductive isolation under ongoing gene flow and reveal how selection against hybridization shapes genomic divergence during the early stages of speciation.