Evolutionary shifts in courtship pheromone composition revealed by EST analysis of plethodontid salamander mental glands

Courtship behavior in salamanders of the family Plethodontidae can last more than an hour. During courtship, males use stereotyped behaviors to repeatedly deliver a variety of proteinaceous pheromones to the female. These pheromones are produced and released from a specialized gland on the male's chin (the mental gland). Several pheromone components are well characterized and represented by high frequency transcripts in cDNA pools derived from plethodontid mental glands. However, evolutionary trends in the overall composition of the pheromonal signal are poorly understood. To address this issue, we used random sequencing to survey the pheromone composition of the mental gland in a representative species from each of three distantly related plethodontid genera. We analyzed 856 high-quality expressed sequence tags (ESTs) derived from unamplified primary cDNA libraries constructed from mental glands of Desmognathus ocoee, Eurycea guttolineata, and Plethodon shermani. We found marked differences among these species in the transcript frequency for three previously identified, functional pheromone components: Plethodontid Receptivity Factor (PRF), Sodefrin Precursor-Like Factor (SPF), and Plethodontid Modulating Factor (PMF). In P. shermani mental glands, transcripts predominately encoded PMF (45% of all ESTs) and PRF (15%), with less than 0.5% SPF. In contrast, in D. ocoee and E. guttolineata the proportions were ∼ 20% SPF, 5% PMF, and PRF was absent. For both D. ocoee and E. guttolineata, peptide hormone-like transcripts occur at high frequency and may encode peptides that change the physiological state of the female, influencing the female's likelihood to complete courtship. These and previous results indicate that the evolution of courtship pheromones in the Plethodontidae is dynamic, contrasting with the predominant mode of evolutionary stasis for courtship behavior and morphology.