Richard A. Watts

A new vertebrate courtship pheromone, PMF, affects female receptivity in a terrestrial salamander

Vertebrate pheromones that affect female receptivity have been documented only in salamanders. These courtship pheromones have been investigated most intensively in plethodontid salamanders. The source of the plethodontid courtship pheromone is the male’s submandibular (mental) gland, which produces a multiprotein secretion. In earlier work with our main study species, Plethodon shermani (the red-legged salamander), an extract of protein secretions obtained from male mental glands acted to increase sexual receptivity in females. In addition, one particular protein in the gland secretion, plethodontid receptivity factor (PRF), could act alone to increase female receptivity. We now report that a second protein, termed ‘plethodontid modulating factor’ (PMF), acts oppositely to reduce female receptivity. The natural courtship pheromone blend thus includes two proteins that separately produce opposing messages, even though the combined effect of both proteins is to increase female receptivity.

EVOLUTIONARY REPLACEMENT OF COMPONENTS IN A SALAMANDER PHEROMONE SIGNALING COMPLEX: MORE EVIDENCE FOR PHENOTYPIC-MOLECULAR DECOUPLING

Abstract In this article we explore the evolutionary history of a functional complex at the molecular level in plethodontid salamanders. The complex consists of a proteinaceous courtship pheromone, a pheromone-producing gland on the males chin, and a set of behaviors for delivering the pheromone to the female. Long-term evolutionary stasis is the defining feature of this complex at both the morphological and behavioral levels. However, our previous assessment of the pheromone gene, plethodontid receptivity factor (PRF), revealed rapid evolution at the molecular level despite stasis at higher levels of organization. Analysis of a second pheromone gene, sodefrin precursor-like factor (SPF), now indicates that evolutionary decoupling in this complex is pervasive. The evolutionary profiles of SPF and PRF are remarkably similar in that: (a) both genes exhibit high levels of sequence diversity both within and across taxa, (b) genetic diversity has been driven by strong positive selection, and (c) the genes have evolved heterogeneously in different salamander lineages. The composition of the pheromone signal as a whole, however, has experienced an extraordinary evolutionary transition. Whereas SPF has been retained throughout the 100 MY radiation of salamanders, PRF has only recently been recruited to a pheromone function (27 million years ago). When SPF and PRF coexist in the same clade, they show contrasting patterns of evolution. When one shows rapid evolution driven by positive selection, the other shows neutral divergence restrained by purifying selection. In one clade, the origin and subsequent rapid evolution of PRF appear to have interfered with the evolution and persistence of SPF, leading to a pattern of evolutionary replacement. Overall, these two pheromone genes provide a revealing window on the dynamics that drive the evolution of multiple traits in a signaling complex.

Plethodontid modulating factor, a hypervariable salamander courtship pheromone in the three-finger protein superfamily

The soluble members of the three‐finger protein superfamily all share a relatively simple ‘three‐finger’ structure, yet perform radically different functions. Plethodontid modulating factor (PMF), a pheromone protein produced by the lungless salamander, Plethodon shermani, is a new and unusual member of this group. It affects female receptivity when delivered to the females nares during courtship. As with other plethodontid pheromone genes, PMF is hyperexpressed in a specialized male mental (chin) gland. Unlike other plethodontid pheromone genes, however, PMF is also expressed at low levels in the skin, liver, intestine and kidneys of both sexes. The PMF sequences obtained from all tissue types were highly variable, with 103 unique haplotypes identified which averaged 35% sequence dissimilarity (range 1–60%) at the protein level. Despite this variation, however, all PMF sequences contained a conserved ≈ 20‐amino‐acid secretion signal sequence and a pattern of eight cysteines that is also found in cytotoxins and short neurotoxins from snake venoms, as well as xenoxins from Xenopus. Although they share a common cysteine pattern, PMF isoforms differ from other three‐finger proteins in: (a) amino‐acid composition outside of the conserved motif; (b) length of the three distinguishing ‘fingers’; (c) net charge at neutral pH. Whereas most three‐finger proteins have a net positive charge at pH 7.0, PMF has a high net negative charge at neutral pH (pI range of most PMFs 3.5–4.0). Sequence comparisons suggest that PMF belongs to a distinct multigene subfamily within the three‐finger protein superfamily.

A Recombinant Courtship Pheromone Affects Sexual Receptivity in a Plethodontid Salamander

Pheromones are important chemical signals for many vertebrates, particularly during reproductive interactions. In the terrestrial salamander Plethodon shermani, a male delivers proteinaceous pheromones to the female as part of their ritualistic courtship behavior. These pheromones increase the females receptivity to mating, as shown by a reduction in courtship duration. One pheromone component in particular is plethodontid receptivity factor (PRF), a 22-kDa protein with multiple isoforms. This protein alone can act as a courtship pheromone that causes the female to be more receptive. We used a bacterial expression system to synthesize a single recombinant isoform of PRF. The recombinant protein was identical to the native PRF, based on mass spectrometry, circular dichroism spectra, and a behavioral bioassay that tested the effects of recombinant PRF (rPRF) on female receptivity (21% reduction in courtship duration). The rPRF appears to mimic the activity of a mixture of PRF isoforms, as well as a mixture of multiple different proteins that comprise the male courtship gland extract. Pheromones that are peptides have been characterized for some vertebrates; to date, however, rPRF is one of only 2 synthesized vertebrate proteins to retain full biological activity.

A Candidate Vertebrate Pheromone, SPF, Increases Female Receptivity in a Salamander

Plethodontid (lungless) salamanders have evolved an unusual pheromone delivery system in which the male courtship pheromone is applied to the skin of the female, apparently diffusing through the mucosal-rich epithelia into her superficial capillary system. In Desmognathus ocoee, a plethodontid salamander that uses the diffusion mode of pheromone delivery, we conducted a behavioural bioassay to test a 20–25 kDa molecular weight fraction of the male courtship pheromone: this fraction was effective in increasing female receptivity. The principal component of the D. ocoee pheromone fraction was identified as a 25 kDa protein that had significant sequence similarity with the precursor of a newt reproductive pheromone (a decapeptide termed sodefrin). We termed the principal protein component in the D. ocoee pheromone “Sodefrin Precursor-like Factor” (SPF). SPF also occurs in other plethodontid salamanders, including species of Plethodon, Aneides and Eurycea. Across these species, SPF is a highly variable protein that bears the signature of positive selection. The presence of SPF in distantly related genera suggests that the sodefrin precursor gene has been retained as a courtship signal throughout the evolutionary radiation of plethodontid salamanders.

Rapid evolution of plethodontid modulating factor, a hypervariable salamander courtship pheromone, is driven by positive selection.

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.

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 males 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 approximately 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 females 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.

Expression of Vomeronasal Receptors and Related Signaling Molecules in the Nasal Cavity of a Caudate Amphibian (Plethodon shermani)

G-protein-coupled receptors are responsible for binding to chemosensory cues and initiating responses in vertebrate olfactory neurons. We investigated the genetic diversity and expression of one family of G-protein-coupled receptors in a terrestrial caudate amphibian (the red-legged salamander, Plethodon shermani). We used degenerate RT-PCR to isolate vomeronasal type 2 receptors (V2Rs)--including full-length sequences--and compared them with other vertebrate V2Rs with phylogenetic analyses. We also amplified a salamander Golf, a G-protein usually expressed in the main olfactory epithelium (MOE) of vertebrates, and an ion channel expressed in the rodent vomeronasal organ: trpc2. We then localized mRNA expression of V2Rs, trpc2, and Golf in the olfactory and vomeronasal epithelia with in situ hybridization. The mRNA transcripts of V2Rs and trpc2 were detected solely in the vomeronasal epithelium of P. shermani. Furthermore, there were differences in the density of cells that expressed particular subclasses of V2Rs: 2 probes showed sexually dimorphic expression, whereas a third did not. Although Golf mRNA was expressed primarily in the MOE, Golf transcripts also were found in the vomeronasal epithelium. Thus, some aspects of mRNA expression of vomeronasal receptors and related molecules differ between salamanders and frogs, and between salamanders and mice.

Concurrent speciation in the eastern woodland salamanders (Genus Plethodon): DNA sequences of the complete albumin nuclear and partial mitochondrial 12s genes

Salamanders of the North American plethodontid genus Plethodon are important model organisms in a variety of studies that depend on a phylogenetic framework (e.g., chemical communication, ecological competition, life histories, hybridization, and speciation), and consequently their systematics has been intensively investigated over several decades. Nevertheless, we lack a synthesis of relationships among the species. In the analyses reported here we use new DNA sequence data from the complete nuclear albumin gene (1818 bp) and the 12s mitochondrial gene (355 bp), as well as published data for four other genes (Wiens et al., 2006), up to a total of 6989 bp, to infer relationships. We relate these results to past systematic work based on morphology, allozymes, and DNA sequences. Although basal relationships show a strong consensus across studies, many terminal relationships remain in flux despite substantial sequencing and other molecular and morphological studies. This systematic instability appears to be a consequence of contemporaneous bursts of speciation in the late Miocene and Pliocene, yielding many closely related extant species in each of the four eastern species groups. Therefore we conclude that many relationships are likely to remain poorly resolved in the face of additional sequencing efforts. On the other hand, the current classification of the 45 eastern species into four species groups is supported. The Plethodon cinereus group (10 species) is the sister group to the clade comprising the other three groups, but these latter groups (Plethodon glutinosus [28 species], Plethodon welleri [5 species], and Plethodon wehrlei [2 species]) probably diverged from each other at approximately the same time.