Proteomic analyses of courtship pheromones in the redback salamander, Plethodon cinereus.

The evolutionary success of plethodontid salamanders for ~100 MY is due partly to the use of courtship pheromones that regulate female receptivity. In ~90 % of plethodontid species, males deliver pheromones by "scratching" a female's dorsum, where pheromones diffuse transdermally into the bloodstream. However, in a single clade, representing ~10 % of Plethodon spp., males apply pheromones to the female's nares for olfactory delivery. Molecular studies have identified three major pheromone families: Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). SPF and PMF genes are relatively ancient and found in all plethodontid species; however, PRF is found exclusively in the genus Plethodon - which includes species with transdermal, olfactory, and intermediate delivery behaviors. While previous proteomic analyses suggested PRF and PMF are dominant in slapping species and SPF is dominant in non-Plethodon scratching species, it was unclear how protein expression of different pheromone components may vary across delivery modes within Plethodon. Therefore, the aim of this study was to proteomically characterize the pheromones of a key scratching species in this evolutionary transition, Plethodon cinereus. Using mass spectrometry-based techniques, our data support the functional replacement of SPF by PRF in Plethodon spp. and an increase in PMF gene duplication events in both lineage-dependent and delivery-dependent manners. Novel glycosylation was observed on P. cinereus PRFs, which may modulate the metabolism and/or mechanism of action for PRF in scratching species. Cumulatively, these molecular data suggest that the replacement of pheromone components (e.g., SPF by PRF) preceded the evolutionary transition of the functional complex from transdermal to olfactory delivery.