Varying Intensities of Introgression Obscure Incipient Venom-Associated Speciation in the Timber Rattlesnake (Crotalus horridus).

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.

Phenotypic integration in the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus).

Selection can vary geographically across environments and temporally over the lifetime of an individual. Unlike geographic contexts, where different selective regimes can act on different alleles, age-specific selection is constrained to act on the same genome by altering age-specific expression. Snake venoms are exceptional traits for studying ontogeny because toxin expression variation directly changes the phenotype; relative amounts of venom components determine, in part, venom efficacy. Phenotypic integration is the dependent relationship between different traits that collectively produce a complex phenotype and, in venomous snakes, may include traits as diverse as venom, head shape and fang length. We examined the feeding system of the eastern diamondback rattlesnake (Crotalus adamanteus) across environments and over the lifetime of individuals and used a genotype-phenotype map approach, protein expression data and morphological data to demonstrate that: (i) ontogenetic effects explained more of the variation in toxin expression variation than geographic effects, (ii) both juveniles and adults varied geographically, (iii) toxin expression variation was a result of directional selection and (iv) different venom phenotypes covaried with morphological traits also associated with feeding in temporal (ontogenetic) and geographic (functional) contexts. These data are the first to demonstrate, to our knowledge, phenotypic integration between multiple morphological characters and a biochemical phenotype across populations and age classes. We identified copy number variation as the mechanism driving the difference in the venom phenotype associated with these morphological differences, and the parallel mitochondrial, venom and morphological divergence between northern and southern clades suggests that each clade may warrant classification as a separate evolutionarily significant unit.