Of all mutational mechanisms contributing to phenotypic variation, structural variants are both among the most capable of causing major effects as well as the most technically challenging to identify. Intraspecific variation in snake venoms is widely reported, and one of the most dramatic patterns described is the parallel evolution of streamlined neurotoxic rattlesnake venoms from hemorrhagic ancestors by means of deletion of snake venom metalloproteinase (SVMP) toxins and recruitment of neurotoxic dimeric phospholipase A2 (PLA2) toxins. While generating a haplotype-resolved, chromosome-level genome assembly for the eastern diamondback rattlesnake (Crotalus adamanteus), we discovered that our genome animal was heterozygous for a ∼225 Kb deletion containing six SVMP genes, paralleling one of the two steps involved in the origin of neurotoxic rattlesnake venoms. Range-wide population-genomic analysis revealed that, although this deletion is rare overall, it is the dominant homozygous genotype near the northwestern periphery of the species’ range, where this species is vulnerable to extirpation. Although major SVMP deletions have been described in at least five other rattlesnake species, C. adamanteus is unique in not additionally gaining neurotoxic PLA2s. Previous work established a superficially complementary north–south gradient in myotoxin (MYO) expression based on copy number variation with high expression in the north and low in the south, yet we found that the SVMP and MYO genotypes vary independently, giving rise to an array of diverse, novel venom phenotypes across the range. Structural variation, therefore, forms the basis for the major axes of geographic venom variation for C. adamanteus.

The evolutionary interplay between predator and prey has significantly shaped the development of snake venom, a critical adaptation for subduing prey. This arms race has spurred the diversification of the components of venom and the corresponding emergence of resistance mechanisms in the prey and predators of venomous snakes. Our study investigates the molecular basis of venom resistance in pythons, focusing on electrostatic charge repulsion as a defense against α-neurotoxins binding to the alpha-1 subunit of the postsynaptic nicotinic acetylcholine receptor. Through phylogenetic and bioactivity analyses of orthosteric site sequences from various python species, we explore the prevalence and evolution of amino acid substitutions that confer resistance by electrostatic repulsion, which initially evolved in response to predatory pressure by Naja (cobra) species (which occurs across Africa and Asia). The small African species Python regius retains the two resistance-conferring lysines (positions 189 and 191) of the ancestral Python genus, conferring resistance to sympatric Naja venoms. This differed from the giant African species Python sebae, which has secondarily lost one of these lysines, potentially due to its rapid growth out of the prey size range of sympatric Naja species. In contrast, the two Asian species Python brongersmai (small) and Python bivittatus (giant) share an identical orthosteric site, which exhibits the highest degree of resistance, attributed to three lysine residues in the orthosteric sites. One of these lysines (at orthosteric position 195) evolved in the last common ancestor of these two species, which may reflect an adaptive response to increased predation pressures from the sympatric α-neurotoxic snake-eating genus Ophiophagus (King Cobras) in Asia. All these terrestrial Python species, however, were less neurotoxin-susceptible than pythons in other genera which have evolved under different predatory pressure as: the Asian species Malayopython reticulatus which is arboreal as neonates and juveniles before rapidly reaching sizes as terrestrial adults too large for sympatric Ophiophagus species to consider as prey; and the terrestrial Australian species Aspidites melanocephalus which occupies a niche, devoid of selection pressure from α-neurotoxic predatory snakes. Our findings underline the importance of positive selection in the evolution of venom resistance and suggest a complex evolutionary history involving both conserved traits and secondary evolution. This study enhances our understanding of the molecular adaptations that enable pythons to survive in environments laden with venomous threats and offers insights into the ongoing co-evolution between venomous snakes and their prey.