Molecular variation across populations of a widespread North American firefly, Photinus pyralis, reveals that coding changes do not underlie flash color variation or associated visual sensitivity

Background: Genes underlying signal production and reception are expected to evolve to maximize signal detection in specific environments. Fireflies vary in their light signal color both within and between species, and thus provide an excellent system in which to study signal production and reception in the context of signaling environments. Differences in signal color have been hypothesized to be due to variation in the sequence of luciferase, the enzyme that catalyzes the light reaction. Similarly, differences in visual sensitivity, which are expected to match signal color, have been hypothesized to be due to variation in the sequence of opsins, the protein component of visual pigments. Here we investigated (1) whether sequence variation in luciferase correlates with variation in signal color and (2) whether sequence variation in opsins correlates with inferred matching visual sensitivity across populations of a widespread North American firefly species, Photinus pyralis. We further tested (3) whether selection has acted on these loci by examining their population-level differentiation relative to the distribution of differentiation derived from a genome-wide sample of loci generated by double-digest RADseq. Results: We found virtually no coding variation in luciferase or opsins. However, there was extreme divergence in non-coding variation in luciferase across populations relative to a panel of random genomic loci. Conclusions: The absence of protein variation at both loci challenges the paradigm that variation in signal color and visual sensitivity in fireflies is exclusively due to coding variation in luciferase and opsin genes. Instead, flash color variation within species must involve other mechanisms, such as abdominal pigmentation or regulation of light organ physiology. Evidence for selection at non-coding variation in luciferase suggests that selection is targeting luciferase regulation and may favor differ expression levels across populations.