How genetic information is modified to generate phenotypic variation within a species is one of the central questions in evolutionary biology. Here we focus on the striking intraspecific diversity of >200 aposematic elytral (forewing) colour patterns of the multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo genome assemblies, and gene expression data reveal that the GATA transcription factor gene pannier is the major regulatory gene located at the h locus, and suggest that repeated inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern variation in H. axyridis. Moreover, we show that the colour-patterning function of pannier is conserved in the seven-spotted ladybird beetle, Coccinella septempunctata, suggesting that H. axyridis’ extraordinary intraspecific variation may have arisen from ancient modifications in conserved elytral colour-patterning mechanisms in ladybird beetles.

1How genetic information is modified to generate phenotypic variation within a 2 species is one of the central questions in evolutionary biology. Here we focus on the striking 3 intraspecific diversity of more than 200 aposematic elytral (forewing) colour patterns of the 4 multicoloured Asian ladybird beetle, Harmonia axyridis, which is regulated by a tightly 5 linked genetic locus h. Our loss-of-function analyses, genetic association studies, de novo 6 genome assemblies, and gene expression data reveal that the GATA transcription factor gene 7 pannier is the major regulatory gene located at the h locus, and suggest that repeated 8 inversions and cis-regulatory modifications at pannier led to the expansion of colour pattern 9 variation in H. axyridis. Moreover, we show that the colour patterning function of pannier is 1 0 conserved in the seven spotted ladybird beetle, Coccinella septempunctata, suggesting that H. 1 1