A cryptic hybrid zone reveals the genomic basis of flower colour variation in a plant with a large and complex genome

Flower colour is known to be an automatic magic trait, undergoing divergent selection pressures exerted by distinct pollinators and involved in reproductive isolation. However, variation at this trait may also results from other eco-evolutionary factors whose interpretation requires throughout context-specific analyses. Here, we investigated the eco-evolutionary causes of pink- and yellow-flowered morphs in Pedicularis comosa where their parapatric ranges form a contact zone: in the East of the Pyrenees Mountains. First, we generated a near-chromosome-scale reference genome assembly for this species. We then used a Genotyping By Sequencing approach to infer patterns of genetic diversity and differentiation between populations and morphs at a total of 158 individuals from 11 localities. We found that neutral genetic structure is primarily consistent with geography rather than with colour. However, admixture analyses and clines suggest the existence of a cryptic hybrid zone between morphs. Outlier detection methods and examination of locus-by-locus cline features, then allowed to pinpoint candidate loci to explain colour variation. To gain insight into the functional aspects of these loci, we finally analysed floral transcriptomes and quantified pigments using Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and confirmed the involvement of key genes in the anthocyanin metabolic pathway (e.g. DFR, FLS) and associated pigments (e.g. cyanidin and delphinidin). Our results show that implementing a highly integrative multi-omic approach can allow unraveling the genetic basis and the eco-evolutionary significance of adaptive traits with even very limited previous knowledge, on species with large and complex genomes.