Ecologically divergent selection can lead to the evolution of reproductive isolation through the process of ecological speciation, but the balance of responsible evolutionary forces is often obscured by an inadequate assessment of demographic history and the genetics of traits under selection. Snake venoms have emerged as a system for studying the genetic basis of adaptation because of their genetic tractability and contributions to fitness, and speciation in venomous snakes can be associated with ecological diversification such as dietary shifts and corresponding venom changes. Here, we explored the neurotoxic (type A)–hemotoxic (type B) venom dichotomy and the potential for ecological speciation among Timber Rattlesnake (Crotalus horridus) populations. Previous work identified the genetic basis of this phenotypic difference, enabling us to characterize the roles geography, history, ecology, selection, and chance play in determining when and why new species emerge or are absorbed. We identified significant genetic, proteomic, morphological, and ecological/environmental differences at smaller spatial scales, suggestive of incipient ecological speciation between type A and type B C. horridus. Range-wide analyses, however, rejected the reciprocal monophyly of venom type, indicative of varying intensities of introgression and a lack of reproductive isolation across the range. Given that we have now established the phenotypic distributions and ecological niche models of type A and B populations, genome-wide data are needed and capable of determining whether type A and type B C. horridus represent distinct, reproductively isolated lineages due to incipient ecological speciation or differentiated populations within a single species.

Understanding the proximate and ultimate causes of phenotypic variation is fundamental in evolutionary research, as such variation provides the substrate for selection to act upon. Although trait variation can arise due to selection, the importance of neutral processes is sometimes understudied. We presented the first reference-quality genome of the Red Diamond Rattlesnake (Crotalus ruber) and used range-wide ‘omic data to estimate the degree to which neutral and adaptive evolutionary processes shaped venom evolution. We characterized population structure and found substantial genetic differentiation across two populations, each with distinct demographic histories. We identified significant differentiation in venom expression across age classes with substantially reduced but discernible differentiation across populations. We then used conditional redundancy analysis to test whether venom expression variation was best predicted by neutral divergence patterns or geographically-variable (a)biotic factors. Snake size was the most significant predictor of venom variation, with environment, prey availability, and neutral sequence variation also identified as significant factors, though to a lesser degree. By directly including neutrality in the model, our results confidently highlight the predominant, yet not singular, role of life history in shaping venom evolution.

Rapid adaptive evolution may be more likely to occur not only through standing genetic variation but via existing axes of genetic variation that have previously been exposed to selection. Ontogenetic variation represents one such axis and often evolves under strong selection in snake venoms. Snake venoms are complex cocktails of proteinaceous toxins, and ontogenetic shifts in venom expression are frequent and reflect dietary shifts across life history. Here, we used morphological, proteomic, transcriptomic, epigenomic, and optical genome mapping data to investigate a well-studied island-mainland population pair of eastern diamondback rattlesnakes (Crotalus adamanteus) to determine whether rapid adaptive expression divergence across populations occurred through the co-option of the ontogenetic regulatory network, population-specific changes independent of ontogeny, or a combination thereof. We found that island snakes were significantly smaller than mainland individuals, and venom proteomic data showed that the continuous ontogenetic shift in venom expression in the mainland population was truncated in island snakes. Venom-gland RNA-seq showed that island adults exhibited juvenile-like expression patterns at key transcription factors, and chromatin accessibility was predictive of venom gene differential expression for ontogenetically co-opted venom loci. Overall, rapid adaptation in the island population appears to have predominantly occurred through the co-option and truncation of the ontogenetic venom shift, with spatial differentiation playing a secondary role. Comparative tests in other systems are needed to determine whether rapid adaptation in general is not only biased toward standing genetic variation but toward large pre-existing axes of variation that have or continue to evolve under strong selection.