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.