Abstract Convergent evolution provides unparalleled insights into the selective drivers underlying evolutionary change. While snakes use venom primarily for predation, and venom composition often reflects diet specificity, three lineages of spitting cobras have independently evolved the ability to use venom as a defensive projectile. Using gene, protein and functional analyses, we show that the three spitting lineages possess venom characterized by an upregulation of PLA 2 toxins, which potentiate the action of venom cytotoxins to activate mammalian sensory neurons and cause enhanced pain. These repeated independent changes provide a fascinating example of convergent evolution across multiple phenotypic levels driven by exaptations. Notably, the timing of their origins suggests that defensive venom spitting may have evolved in response to the emergence of bipedal hominids in Africa and Asia. One Sentence Summary Venom spitting by snakes coincides with the emergence of hominins and is underpinned by convergent increases in pain-enhancing toxins

Convergent Evolution of Pain-Inducing Defensive Venom Components in Spitting Cobras