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Gene co-option is a major force in the evolution of novel biological functions. In plethodontid salamanders, males deliver proteinaceous courtship pheromones to the female olfactory system or transdermally to the bloodstream. Molecular studies identified three families of highly duplicated, rapidly evolving pheromones (PRF, PMF, and SPF). Analyses for Plethodon salamanders revealed pheromone mixtures of primarily PRF and PMF. The current study demonstrates that in Desmognathus ocoee--a plesiomorphic species with transdermal delivery--SPF is the major pheromone component representing >30% of total protein. Chromatographic profiles of D. ocoee pheromones were consistent from May through October. LC/MS-MS analysis suggested uniform SPF isoform expression between individual male D. ocoee. A gene ancestry for SPF with the Three-Finger Protein superfamily was supported by intron-exon boundaries, but not by the disulfide bonding pattern. Further analysis of the pheromone mixture revealed paralogs to peptide hormones that contained mutations in receptor binding regions, such that these novel molecules may alter female physiology by acting as hormone agonists/antagonists. Cumulatively, gene co-option, duplication, and neofunctionalization have permitted recruitment of additional gene families for pheromone activity. Such independent co-option events may be playing a key role in salamander speciation by altering male traits that influence reproductive success.

In many vertebrate mating systems, mate choice evolves when signalling via visual, chemical or auditory traits is an energetically costly process. Selection may favour individuals that can discriminate among potential mates and invest in signalling to mates with particular characteristics. Most commonly, females with costly gametes are thought to be the more selective sex; however, runaway sexual selection can produce elaborate male ornaments and behaviours that are similarly costly to produce, which can lead to male mate choice. In this study, we used behavioural trials to experimentally test male mate choice in a terrestrial salamander, Plethodon shermani. We investigated whether males altered the proportion of time they spent performing a potentially costly courtship display, ‘foot dancing’, in the presence of females. Specifically, we explored male mate choice in two experiments: (1) measuring how males modified the time they invested in courtship based solely on female reproductive value, and (2) determining whether males varied the amount of time they invested in courting females of varying sizes but similar reproductive value. In the first experiment, we quantified the duration of male courtship displays when males were paired with females of differing levels of fecundity (nongravid, weakly gravid and strongly gravid). Males displayed longest for females of high reproductive value (strongly gravid females) and less for weakly gravid and nongravid females. In the second experiment, we showed that males paired sequentially with different-sized females of similar reproductive values displayed significantly more often to larger females (i.e. male effort positively correlated with female size). In conclusion, male P. shermani are one of the few vertebrates known to modify their display behaviour based on female mate quality, and visual signs, such as size, may provide honest indicators of fitness.

Chemical communication via chemosensory signaling is an essential process for promoting and modifying reproductive behavior in many species. During courtship in plethodontid salamanders, males deliver a mixture of non-volatile proteinaceous pheromones that activate chemosensory neurons in the vomeronasal epithelium (VNE) and increase female receptivity. One component of this mixture, Plethodontid Modulating Factor (PMF), is a hypervariable pheromone expressed as more than 30 unique isoforms that differ between individual males-likely driven by co-evolution with female receptors to promote gene duplication and positive selection of the PMF gene complex. Courtship trials with females receiving different PMF isoform mixtures had variable effects on female mating receptivity, with only the most complex mixtures increasing receptivity, such that we believe that sufficient isoform diversity allows males to improve their reproductive success with any female in the mating population. The aim of this study was to test the effects of isoform variability on VNE neuron activation using the agmatine uptake assay. All isoform mixtures activated a similar number of neurons (>200% over background) except for a single purified PMF isoform (+17%). These data further support the hypothesis that PMF isoforms act synergistically in order to regulate female receptivity, and different putative mechanisms are discussed.