Extensive haplotype diversity in a butterfly colour pattern supergene is fuelled by incomplete recombination suppression

Supergenes can evolve when recombination-suppressing mechanisms like inversions promote co-inheritance of alleles at two or more polymorphic loci that affect a complex trait. Theory shows that such genetic architectures can be favoured under balancing selection or local adaptation in the face of gene flow, but they can also bring costs associated with reduced opportunities for recombination. These costs may in turn be offset by rare ‘gene flux’ between inverted and ancestral haplotypes, with a range of possible outcomes. We aimed to shed light on these processes by investigating the BC supergene, which underlies three distinct wing colour morphs in Danaus chrysippus , a butterfly known as the African monarch, African queen and plain tiger. Using whole-genome resequencing data from 174 individuals, we first confirm the effects of BC on wing colour pattern: background coloration is associated with SNPs in the promoter region of yellow , within an inversionted part of the supergene, while forewing tip pattern is most likely associated with a copy-number-variable part of the same supergene. We then show that haplotype diversity within the supergene is surprisingly extensive: there are at least six divergent haplotype groups that experience suppressed recombination with respect to each other. Despite high divergence between these haplotype groups, we identify an unexpectedly large number of natural recombinant haplotypes. These evidently arose through crossovers between adjacent inversion ‘modules’ as well as through double crossovers within inversions. Furthermore, we show that at least one of the established haplotype groups probably arose through recombination between two pre-existing ones. Moreover, on at least two occasions, double crossovers within an inversion have led to the transfer of alleles for dark colouration in the promoter of yellow onto a different haplotype background. Overall, our findings paint a picture of dynamic evolution of supergene haplotypes, fuelled by incomplete recombination suppression.