Co-option and evolution of non-olfactory proteinaceous pheromones in a terrestrial lungless salamander

Abstract

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.

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones

“…Clusters 4 and 5 together include the genes most highly expressed in the later phases of gland maturation (0.8% of all genes). As expected, the majority of transcripts coded for pheromone, including PRF and PMF, but also (in lower abundance) many putative pheromones that were identified in other plethodontid species (natriuretic peptide, vasoactive intestinal peptide, sodefrin precursor-like factor, cysteine rich secretory protein) [39,41,42]. Included in these sequences was a predicted protein related to the tissue inhibitor of metalloproteinase (TIMP) family that included an extraordinarily long 3' UTR (~3700 nt); by mass spectrometry, this sequence matched a protein previously termed C3 that constitutes ~10% of the pheromone extract [33].…”

From molecules to mating: Rapid evolution and biochemical studies of reproductive proteins

“…Initial pheromone characterization was performed for P. shermani using multiple types of high performance liquid chromatography (HPLC) to isolate individual proteins, which were then identified by MS analysis using a shotgun EST library as a reference [109,110]. Subsequent studies with D. ocoee and P. cinereus instead used de novo transcriptomes as reference databases, with amplification of identified candidates by RT-PCR and validation by Sanger sequencing [111,112]. Despite detection of SPF mRNA by RT-PCR in all three species, it was only observed proteomically in D. ocoee , where it comprised ∼30% of the total pheromone.…”

“…While D. ocoee may express up to ∼70 different SPF isoforms, six major classes of highly divergent isoforms shared <50% sequence identity. Protein abundance estimates by spectral counting suggest that isoform expression is relatively stable between individual male D. ocoee [112]. When applied to females during courtship, purified SPF decreased courtship time [102].…”

“…Proteomic analyses of the D. ocoee pheromone extract also led to the identification of several peptide hormone paralogs such as glucagon, insulin, leptin, and relaxin. It was hypothesized that when delivered to the female bloodstream, these hormone-like pheromones may directly influence female physiology by exploiting pre-existing endocrine receptors [112]. In contrast, both P. cinereus and P. shermani with transdermal and olfactory delivery, respectively, secrete pheromone mixtures of predominantly PRF and PMF, but with multiple notable differences.…”

A pheromone by any other gene would smell as sweet

“…Proteins that are used for communication, such as those in pheromones, have provided examples of divergence caused by single amino acid substitutions (Leary et al, 2012), changes in sequences within biosynthetic pathways (Lassance & L€ ofstedt, 2013) or species-specific alterations to gene regulation (Doty, Wilburn, Bowen, Feldhoff, & Feldhoff, 2016). The sodefrin precursor-like factor (SPF) pheromone system present in salamanders is the result of multiple gene duplications, taking place throughout their diversification (Van Bocxlaer et al, 2015).…”