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Spitting cobras protect themselves by shooting jets of venom into the eyes of their attackers. A new study suggests that over the course of several million years, all three groups of spitters independently tailored the chemistry of their toxins in the same way to cause pain to a would-be predator. The work provides a novel example of convergent evolution that “deepens our understanding of this unique system” for delivering venom, says Timothy Jackson, an evolutionary toxinologist at the University of Melbourne.

Like other cobras, spitting cobras will bite attackers in self-defense. Spitting is their signature move, however, and the snakes are crack shots. They can direct a stream of venom into an attacker’s face from more than 2 meters away, aiming for the eyes. The behavior is such a formidable defense that it evolved independently three times: in Asian cobras, African cobras, and a cobra cousin called the rinkhals (Hemachatus haemachatus) that lives in southern Africa.

Scientists previously found the venom of some other snakes evolved to better subdue their prey. By analyzing the venoms of 17 spitting and nonspitting species—and measuring their effects—venom biologist Nicholas Casewell of the Liverpool School of Tropical Medicine and colleagues tested whether the makeup of spitting cobra venom had also changed over time to become a more effective defense.

The most common compounds in cobra venom are the so-called three-finger toxins—proteins named for their 3D chemical shape, not the number of digits you can expect to lose if a snake bites your hand. Three-finger toxins are equally abundant in the venom of spitters and nonspitters, Casewell and colleagues found, constituting about 60% of the toxic molecules. However, the spitting species’ venom contained higher levels of another group of proteins known as phospholipase A₂ toxins, which nonspitters produce only in small quantities, or not at all.

To probe the effects of the extra phospholipase A₂ proteins, the scientists dabbed different combinations of toxins from the snakes onto isolated mouse nerves that are sensitive to pain. The more neurons a toxin stimulates, the more pain would result. The researchers determined the three-finger toxins triggered more pain when combined with phospholipase A₂ toxins than alone. For instance, when the researchers applied both kinds of toxins from rinkhals venom to mouse nerves, the mix stimulated about twice as many nerve cells as the rinkhals’ three-finger toxins alone, they report today in Science.

The work suggests natural selection fine-tuned the composition of the snakes’ venom to make it a better defense, Casewell says. That the three groups of spitters independently derived the same solution—increased abundance of phospholipase A₂ toxins—is an example of convergent evolution, in which species that aren’t closely related but face similar survival challenges acquire similar adaptations. “Evolution can be highly repeatable,” Casewell says.

The study’s evolutionary logic makes sense, says toxinologist Stephen Mackessy of the University of Northern Colorado, Greeley, who wasn’t connected to the research. Increasing the venom’s agony-inducing power would help the snakes ward off predators because “one of the best learning tools is production of pain,” he says. But Joe Alcock, an evolutionary medicine researcher at the University of New Mexico, Albuquerque, says it’s possible that damaging an attacker’s eyes was the driving force to evolve unique chemistry. “If you can blind a predator, that would prevent an attack independent of pain,” he says.

Why some cobras began to spray venom rather than just deliver it through bites remains unclear. Some researchers argue the behavior protects the snakes from being stomped on by hoofed mammals. But the side-facing eyes of buffalos, zebras, and other heavy-footed mammals would be hard to hit with a single jet of venom, Casewell notes. Instead, he and his colleagues postulate that early humans motivated the origin of spitting behavior. Our ancestors would have been a menace to the snakes, and they conveniently had forward-facing eyes that would make good targets for a stream of noxious venom.