Multilocus basis of incipient reproductive isolation in hybridizing populations is revealed by pangenomic and epigenetic divergence.

Incipient reproductive isolation in the presence of gene flow has traditionally been attributed to a small number of major-effect loci under strong selection. Here, using the Heliconius erato adaptive radiation, we apply a pangenome framework to examine how mutational divergence, regulatory variation, and structural variants contribute to genome-wide divergence. In contrast to earlier studies, our high-resolution analyses reveal widespread divergence across the genome, consistent with a multilocus barrier to gene flow. Our findings support a model in which selection acts on regulatory phenotypes under migration-selection balance, with genetic differentiation becoming more pronounced as gene flow declines. By integrating population-level sampling, we show that apparent population-specific structural and regulatory variation inferred from single-reference genomes is overestimated, reflecting pervasive reference bias. While structural variants contribute to genomic variation, in our system, most are shared or polymorphic rather than fixed differences between populations. Together, our results show that the genomic landscape of H. erato divergence reflects the combined contributions of regulatory variation and mutational change, while highlighting the importance of accounting for reference bias when interpreting structural and regulatory divergence. This multilocus framework provides a more accurate view of how reproductive barriers emerge and strengthen under ongoing gene flow.