Sex pheromones form an important facet of reproductive strategies in many organisms throughout the Animal Kingdom. One of the oldest known sex pheromones in vertebrates are proteins of the Sodefrin Precursor-like Factor (SPF) system, which already had a courtship function in early salamanders. The subsequent evolution of salamanders is characterized by a diversification in courtship and reproduction, but little is known on how the SPF pheromone system diversified in relation to changing courtship strategies. Here, we combined transcriptomic, genomic, and phylogenetic analyses to investigate the evolution of the SPF pheromone system in nine salamandrid species with distinct courtship displays. First, we show that SPF originated from vertebrate three-finger proteins and diversified through multiple gene duplications in salamanders, while remaining a single copy in frogs. Next, we demonstrate that tail-fanning newts have retained a high phylogenetic diversity of SPFs, whereas loss of tail-fanning has been associated with a reduced importance or loss of SPF expression in the cloacal region. Finally, we show that the attractant decapeptide sodefrin is cleaved from larger SPF precursors that originated by a 62 bp insertion and consequent frameshift in an ancestral Cynops lineage. This led to the birth of a new decapeptide that rapidly evolved a pheromone function independently from uncleaved proteins.

Males of the advanced salamanders (Salamandroidea) attain internal fertilization without a copulatory organ 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, because most species do not display close physical contact during courtship, but instead largely rely on females following the male track at spermatophore deposition. Although pheromones have been widely assumed to represent an important aspect of male courtship, molecules able to induce the female following behaviour that is the prelude for successful insemination have not yet been identified. Here, we show that uncleaved sodefrin precursor-like factor (SPF) protein pheromones are sufficient to elicit such behaviour in female palmate newts (Lissotriton helveticus). Combined transcriptomic and proteomic evidence shows that males simultaneously tail-fan multiple ca 20 kDa glycosylated SPF proteins during courtship. Notably, molecular dating estimates show that the diversification of these proteins already started in the late Palaeozoic, about 300 million years ago. Our study thus not only extends the use of uncleaved SPF proteins outside terrestrially reproducing plethodontid salamanders, but also reveals one of the oldest vertebrate pheromone systems.

Internal fertilization without copulation or prolonged physical contact is a rare reproductive mode among vertebrates. In many newts (Salamandridae), the male deposits a spermatophore on the substrate in the water, which the female subsequently takes up with her cloaca. Because such an insemination requires intense coordination of both sexes, male newts have evolved a courtship display, essentially consisting of sending pheromones under water by tail-fanning towards their potential partner. Behavioral experiments until now mostly focused on an attractant function, i.e. showing that olfactory cues are able to bring both sexes together. However, since males start their display only after an initial contact phase, courtship pheromones are expected to have an alternative function. Here we developed a series of intraspecific and interspecific two-female experiments with alpine newt (Ichthyosaura alpestris) and palmate newt (Lissotriton helveticus) females, comparing behavior in male courtship water and control water. We show that male olfactory cues emitted during tail-fanning are pheromones that can induce all typical features of natural female mating behavior. Interestingly, females exposed to male pheromones of their own species show indiscriminate mating responses to conspecific and heterospecific females, indicating that visual cues are subordinate to olfactory cues during courtship.

SUMMARYMales of many frog species develop spiny nuptial pads with underlying glands on their thumbs during the mating period. We used 3D visualization on the European common frog Rana temporaria to show that the morphology of these glands allows the channelling of secreted molecules to the pad's surface during amplexus. Combined transcriptome and proteome analyses show that proteins of the Ly-6/uPAR family, here termed amplexins, are highly expressed in the nuptial glands during the mating season, but are totally absent outside that period. The function of amplexins remains unknown, but it is interesting to note that they share structural similarities with plethodontid modulating factors, proteins that influence courtship duration in salamanders. Supplementary material available online at

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