Genomic incompatibilities are persistent barriers when speciation happens with gene flow in Formica ants.

A current goal of speciation research is identifying loci underlying reproductive barriers between species. Locating barrier loci in population genomic data is difficult due to the often-complex demographic history of diverged taxa and heterogeneity in evolutionary forces across the genome. We take advantage of natural hybridization between 2 wood ant species (Formica aquilonia and Formica polyctena) to identify regions of reduced long-term gene flow using demographically explicit scans of nonadmixed genomes. In addition, we identify candidate Bateson-Dobzhansky-Muller incompatibilities (BDMIs) through an imbalanced recombinant haplotype frequency analysis using a large sample of natural F. aquilonia × F. polyctena hybrid genomes. These approaches find barriers and BDMIs scattered across the genome. Furthermore, BDMIs significantly overlap with long-term barriers, indicating that some BDMIs have persisted despite divergence with gene flow. Intriguingly, the number of pairwise interactions a BDMI has correlates with its long-term barrier strength: hub-like BDMIs with many interactions reduce gene flow more effectively. Finally, we find that long-term barriers are depleted for both coding sequences (CDS) and transposable elements (TEs), while candidate BDMIs are associated with snRNAs and LTR transposons, specifically Ty1-copia. In contrast, regions where long-term barriers and BDMIs co-locate are significantly associated with introns but not CDS or TEs, implying a potential role of alternative splicing or gene regulation in long-term incompatibilities. Our results highlight the underappreciated impact of BDMI connectivity on the persistence of reproductive barriers over time.