Ancient pheromone blend as an alternative for copulation in internally fertilizing salamanders

Throughout the animal kingdom, internal fertilization - the merging of sperm and egg inside the female body - nearly invariably relies on the use of a copulatory organ. In contrast, males of advanced salamanders (Salamandroidea) attain internal fertilization by depositing a spermatophore on the substrate in the environment, which females subsequently take up with their cloaca. The aquatically reproducing modern Eurasian newts (Salamandridae) have taken this to extremes, since the majority does not display physical contact between the sexes and largely rely on females following the male track at spermatophore deposition. Although the use of pheromones has been widely assumed during their courtship, molecules able to induce the female following behaviour that culminates in insemination have not been identified. Here we show that uncleaved glycosylated SPF protein pheromones, secreted during courtship, are sufficient to elicit such behaviour in palmate newts (Lissotriton h. helveticus), indicating that these molecules obviate the need for copulation in these salamanders. Surprisingly, our finding of side-by-side secretion of Late Palaeozoic diverged proteins in a single species suggests that these molecules already had a courtship function in stem salamanders about 300 million years ago, rendering them one of the oldest vertebrate pheromone systems.