Evolution of a supergene in Linum

Distyly is a multi-trait balanced floral polymorphism that promotes outcrossing, and that has evolved convergently in multiple flowering plant lineages. While recent genomic studies have revealed striking similarities in the type of structural variation present at independently evolved distyly S-locus supergenes, it remains unclear whether such similarities extend to the molecular mechanisms regulating distyly. Here, we build on our recent identification of the distyly supergene in Linum tenue to study the origin, evolution, and mechanisms underlying distyly and stylar polymorphism in Linum, a classic system for the study of distyly. For this purpose, we generate haplotype-resolved genome assemblies of two Linumspecies which diverged from L. tenue ~33 Mya: the distylous Linum perenne and the style height dimorphic Linum grandiflorum, and use population genetic analyses to identify and characterize their S-loci. We find that in all three Linum species, hemizygosity in short-styled floral morph individuals is a common feature of the S-locus supergene, but that the size and gene content of the supergene differs greatly among species. However, all three species share two conserved key candidate genes at their distyly S-loci; the style length candidate gene TSS1 and the anther height/male self-incompatibility candidate gene WDR-44. Consistent with expectations if TSS1 in thrums regulates style length via the brassinosteroid pathway, we find that brassinosteroid treatment specifically elongates styles only in thrums but not in pins in both L. perenne and L. tenue. Taken together, our results show that while the S-locus supergene has diverged greatly in terms of size, gene and repeat content, key candidate genes and mechanisms regulating distyly remain conserved across these widely diverged Linum species. In combination with previous findings in Primula and Turnera, our results suggest that patterns of molecular parallelism at distyly supergenes extend to mechanisms regulating distyly, implying the presence of functional constraints to the evolution of distyly.