Evolution towards species specificity in a protein sex pheromone system

The ability to discriminate potential mates of conspecifics from related species is crucial for successful reproduction. In aquatically breeding European newts (Salamandridae), different species often coexist in the same pond during the reproductive season. Male newts use proteins from the Sodefrin Precursor-like Factor (SPF) family to enhance female receptivity during courtship, and these molecules form an important element of prezygotic reproductive isolation. Recent research highlighted the long and dynamic evolution of the SPF pheromone system, but mechanisms that govern interspecific pheromone divergence and the role they play in speciation remain elusive. We combined proteomics and transcriptomics to investigate how speciesspecificity of SPF pheromones evolved by comparing the male repertoire of the alpine newt (Ichthyosaura alpestris) and the palmate newt (Lissotriton helveticus). Both species are relatively closely related, but the females do not show reproductive behaviour when exposed to pheromones of the heterospecific males. Our analyses demonstrate that, despite the availability of multiple SPF transcripts that diverged through gene duplications before the speciation, both species largely express orthologs from the same two clades of SPF proteins. Our findings thus indicate that there have been no dramatic, saltational shifts in expression of duplicated SPF genes during or after their speciation, and demonstrate that species specificity of protein pheromones can be obtained by sequence divergence alone. Still, the continuous presence of less expressed SPF transcripts indicates the potential for rapid recruitment of other SPFs into the pheromone blend, rendering other scenarios of protein pheromone divergence in salamandrid evolution possible.