Sex pheromones play an essential role in the modulation of courtship behavior of many sexually reproducing animals. Newts (aquatic reproducing salamanders) display a wide variety of courtship strategies, in which pheromones are a necessary prerequisite for successful reproduction. We recently used a novel two-female ethological test to show that two different male-specific proteins isolated from courtship water of the palmate newt Lissotriton helveticus are able to induce all natural female courtship responses.
Furthermore, multiple natural isoforms of both proteins are present in courtship water of mating animals, and phylogenetic analyses indicate that several isoforms originated before the newt speciation events. Here we propose to combine the two-female behavioral test with phylogenetics, transcriptomics and proteomics to investigate pheromone evolution in newts. We will identify pheromones in the cloaca, head glands and nuptial pads of six newts with diverse behaviors, and construct recombinant protein pheromones for behavioral testing. Individual variation in male pheromone production and female preferences will be tested in a single species. In one subclade of three strictly tail-fanning newts, we will identify all available transcripts of both translated (detected in courtship water) and untranslated isoforms (present only as cDNA in the abdominal gland). This will allow to understand the role of isoform evolution in maintaining species-specificity.