Rapid Evolution of Plethodontid Modulating Factor, a Hypervariable Salamander Courtship Pheromone, is Driven by Positive Selection

Abstract

Sexual communication in plethodontid salamanders is mediated by a proteinaceous pheromone that a male delivers to a female during courtship, boosting her receptivity. The pheromone consists of three proteins from three unrelated protein families. These proteins are among a small group of pheromones known to affect female receptivity in vertebrates. Previously, we showed that the genes of two of these proteins (PRF and SPF) are prone to incessant evolution driven by positive selection, presumably as a consequence of coevolution with female receptors. In this report, we focus on the evolution of the third pheromone protein gene family, plethodontid modulating factor (PMF), to determine whether it shows the same pattern of diversification. We used RT-PCR in mental gland cDNA to survey PMF sequences from three genera of plethodontid salamanders (27 spp.) to measure rates of evolution, level of gene diversification, modes of selection, and types of amino acid substitution. Like PRF and SPF, PMF is produced by a multigene family characterized by gene duplication and high levels of polymorphism. PMF evolution is rapid, incessant, and driven by positive selection. PMF is more extreme in these dimensions than both PRF and SPF. Nestled within this extraordinary variation, however, is a signature of purifying selection, acting to preserve important structural and biochemical features of the PMF protein (i.e., secretion signal, cysteine residues, and pI). Although a pattern of persistent diversification exists at the molecular level, the morphological and behavioral aspects of the pheromone delivery system show evolutionary stasis over millions of years.

Mechanisms of action and co-optive evolution for hypervariable courtship pheromones in plethodontid salamanders.

“…In a parallel analysis, PAML models for only translated PMF sequences predict similar substitution rates to those of all P. shermani PMFs for positively selected sites (ω2 = 3.99 in M2a; Table 4). The calculated rates for both analyses are faster than those predicted by Palmer et al [96] for the larger clade within which P. shermani is found (P. glutinosus complex; ω2 = 2.75 in M2a). Substitution rates and models of molecular evolution could not be determined for individual clusters due to some clusters containing as few as 2 sequences.…”

“…At the same time, these genes have been under strong sexual selective pressure to differentiate and adopt potentially novel signaling roles in order to affect female behavior and physiology [96,Chapter II]. When whole mental gland extract was applied to female salamanders, courtship time decreased by ~20% [83].…”

“…Both proteins individually activated neurons in the VNE and in the brain [86,92], but in behavioral assays, only PRF decreased courtship time similar to whole pheromone extract while PMF surprisingly increased it [83,93]. Comparison of cDNA sequences of PMF, PRF, and SPF across multiple plethodontid species revealed all three proteins are rapidly evolving and under pervasive positive selection [94][95][96][97]. The remaining 19 kDa component in P. shermani pheromone extract was unidentified until recently when it was shown to belong to the TIMP superfamily (see Chapter VI), however, its biological functions are still unknown.…”

“…Palmer et al [90] designed primers based on the conserved UTRs to obtain 27 additional mental gland PMF coding sequences with ~37% average amino acid dissimilarity. In a subsequent study comparing PMF cDNAs from multiple species in a larger phylogenetic context, the observed amino acid dissimilarity placed PMF under strong positive selection [96]. While this work serves as an important preliminary investigation, it is largely dependent on the assumption that each mRNA equates to an expressed pheromone that is inevitably delivered to a female.…”

“…Structurally, PMF contains 8 disulfide-bonded cysteine residues that form a stable molecular scaffold upon which sequence hypervariability may be mounted [96], but it is still unclear how much of this variation is functionally expressed and exposed to sexual selection in P. shermani females during courtship.…”