Aquilegia formosa and Aquilegia pubescens are two closely–related species belonging to the columbine genus. Despite their morphological and ecological differences, previous studies have revealed a large degree of intercompatibility as well as little sequence divergence between these two taxa [25, 81], and the genetic mechanisms underpinning reproductive isolation remain unknown. In order to assess the feasibility of a full genome scan for speciation genes, inter– and intraspecific patterns of variation were compared for 9 nuclear loci; it was concluded that the two species were practically indistinguishable at the level of DNA sequence polymorphism, indicating either very recent speciation or continued gene flow. As a comparison, the variation at two loci was analyzed across 30 other Aquilegia species, revealing slightly more differentiation among taxa and evidence for isolation by geographic distance (which was not the case on a more local geographic scale).

The extremely low levels of genetic variation found between A. formosa and A. pubescens at neutral loci was deemed ideal for a genome-wide scan for allele frequency differences, so this was done using Solexa deep sequencing of pooled samples from each species. Polymorphisms were identified and annotated based on alignment with the A. coerulea reference genome, and SNPs with extreme values of allele frequency differentiation (≥93.5%) were selected as candidate speciation genes. Two of these extreme SNPs caused amino acid changes in the MYB and UGT proteins, both of which are known components and regulators of the anthocyanin (pigmentation) pathway.