Where the ruber meets the road: Using the genome of the red diamond rattlesnake (Crotalus ruber) to unravel the neutral and adaptive processes driving venom evolution

Phenotypic variation in natural populations forms a fundamental aspect of evolutionary research as it provides the substrate for natural selection to act upon. Although such variation can arise due to selection, the importance of neutral evolutionary processes when investigating intraspecific variation are often ignored. To adequately investigate phenotypic diversity, evolutionary studies must use a more integrative approach to determine how adaptive and neutral processes potentially drive trait variation. In this study, we presented the the first reference-quality genome of the red diamond rattlesnake (Crotalus ruber) and generated additional, range-wide genomic, transcriptomic, and proteomic data to estimate the degree to which neutral evolutionary processes and selective pressures drive venom variation. We characterized population structure and found substantial genetic differentiation between two geographically separated populations. We determined that venom expression was significantly variable and that discrete ontogenetic shifts underlie the majority of variation observed in the venom phenotype with reduced but discernible differentiation between the two populations. Although positive selection has been detected in venom across species, we found that sequence divergence has contributed little to venom evolution within C. ruber. We incorporated multiple evolutionary factors, neutral and adaptive, into a single model and tested whether venom phenotypic variation was best predicted by neutral evolutionary processes or geographically variable selective abiotic or biotic pressures. Snake size and neutral sequence variation were found to be the best predictors of venom phenotypic variation across the range, indicating that 1) venom variation is most strongly predicted by ontogeny and 2) venom is potentially evolving, at least in part, under neutral evolutionary processes. Our results emphasize the importance of accounting for all potential evolutionary processes, neutral and adaptive, when investigating factors driving trait variation.