Plethodontid Receptivity Factor Research Articles

Pheromone Gene Diversification and the Evolution of Courtship Glands in Plethodontid Salamanders.

Proteinaceous pheromones that diversify through gene duplication can result in shifts in courtship cocktails that may serve as a mechanism for reproductive isolation. The molecular evolution of pheromones has been extensively studied in salamanders, but how these genes and associated novel courtship glands have codiversified has not been evaluated. In this study we used transcriptional analyses to examine the relationship between pheromone diversification and gland type in three divergent lineages of plethodontid salamanders. Our results revealed that plethodontid salamanders express up to eight divergent Sodefrin Precursor-like Factor genes (spf, representing both alpha and beta subfamilies) along with Plethodontid Modulating Factor (pmf) and Plethodontid Receptivity Factor (prf). Expression of pheromone genes is tissue specific with pmf, prf, and some spf genes restricted to the mental gland. In contrast, the caudal gland shows strong expression of the other spf genes. We found evidence for punctuated changes in pheromone cocktail composition related to the loss of metamorphosis, and subsequent extreme reduction of the mental gland, in a paedomorphic lineage. Our study provides insight into how pheromone diversification can be partitioned into unique glands, which may lead to cocktail specificity in behavioral modules during courtship.

Proteomic analyses of courtship pheromones in the redback salamander, Plethodon cinereus.

The evolutionary success of plethodontid salamanders for ~100 MY is due partly to the use of courtship pheromones that regulate female receptivity. In ~90 % of plethodontid species, males deliver pheromones by "scratching" a female's dorsum, where pheromones diffuse transdermally into the bloodstream. However, in a single clade, representing ~10 % of Plethodon spp., males apply pheromones to the female's nares for olfactory delivery. Molecular studies have identified three major pheromone families: Plethodontid Receptivity Factor (PRF), Plethodontid Modulating Factor (PMF), and Sodefrin Precursor-like Factor (SPF). SPF and PMF genes are relatively ancient and found in all plethodontid species; however, PRF is found exclusively in the genus Plethodon - which includes species with transdermal, olfactory, and intermediate delivery behaviors. While previous proteomic analyses suggested PRF and PMF are dominant in slapping species and SPF is dominant in non-Plethodon scratching species, it was unclear how protein expression of different pheromone components may vary across delivery modes within Plethodon. Therefore, the aim of this study was to proteomically characterize the pheromones of a key scratching species in this evolutionary transition, Plethodon cinereus. Using mass spectrometry-based techniques, our data support the functional replacement of SPF by PRF in Plethodon spp. and an increase in PMF gene duplication events in both lineage-dependent and delivery-dependent manners. Novel glycosylation was observed on P. cinereus PRFs, which may modulate the metabolism and/or mechanism of action for PRF in scratching species. Cumulatively, these molecular data suggest that the replacement of pheromone components (e.g., SPF by PRF) preceded the evolutionary transition of the functional complex from transdermal to olfactory delivery.

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.

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 female's 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 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

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 male's 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.

In situ localization of plethodontid courtship pheromone mRNA in formalin-fixed tissue

Male plethodontid salamanders produce courtship pheromones that increase female receptivity. Three protein components of the courtship pheromone cocktail have been characterized in the mental gland of Plethodon shermani, the red-legged salamander: plethodontid receptivity factor (PRF), plethodontid modulating factor (PMF), and sodefrin precursor-like factor (SPF). In this study, a streamlined in situ hybridization (ISH) protocol, employing a biotinylated oligonucleotide probe, is used to visualize the sites of pheromone expression in formalin-fixed paraffin-embedded P. shermani mental gland and post-cloacal tail tissue. Results corroborate previous RT-PCR studies on pheromone expression. PRF and PMF are highly expressed in P. shermani mental gland, while SPF expression is more variable. None of the tested pheromones is expressed in dorsal or ventral tail glands. The reported protocol is simple, rapid, and effective, allowing visualization of high-copy mRNA transcript in formalin-fixed tissue.

Disulfide bonding pattern in a structurally constrained family of 7 kDa vertebrate pheromones

During courtship, a male P. shermani salamander delivers pheromones from a submandibular mental gland to the female's nares. Following binding of pheromones to receptors in the vomeronasal organ, female receptivity increases. The pheromone mixture consists of two major families, each of which contains multiple isoforms. The 22 kDa family (Plethodontid Receptivity Factor) is related to the IL-6 family of cytokines, while the 7 kDa family (Plethodontid Modulating Factor; PMF) is related to xenoxins and snake toxins. Recently, we purified and performed peptide mass fingerprinting of 16 PMF isoforms, of which 8 matched our current database of 32 unique DNA sequences. The 7 kDa pheromones have 8 conserved Cys residues (4 disulfide bonds; ~60 residues) with spacing similar to that for xenoxins and snake toxins, but with limited sequence homology. Conditions for the determination of the disulfide bonding pattern were developed for a single purified isoform which resulted in the average reduction and alkylation of 1, 2 or 3 disulfide bonds. Aliquots of each were then subjected to complete digestion with trypsin, chymotrypsin or Glu-C prior to direct MALDI-TOF analysis. All of the MS data sets are consistent with a sequential disulfide bonding pattern of 1–2, 3–4, 5–6 and 7–8, with the 3–4 bond being more susceptible to reduction and alkylation than the 1–2 bond. This pattern will dictate a 3-dimensional structure different from that for xenoxins and snake toxins (1–3, 2–4, 5–6, 7–8) and may represent a subfamily for courtship pheromones or a new family of 8 Cys/7 kDa proteins. Supported in part by NSF Grant # IBN 0110666

Lineage-Specific Differences in Evolutionary Mode in a Salamander Courtship Pheromone

Functionally equivalent genes may evolve heterogeneously across closely related taxa as a consequence of lineage-specific selective pressures. Such disparate evolutionary modes are especially prevalent in genes that encode postcopulatory reproductive proteins, presumably as a result of sexual selection. We might therefore expect genes that mediate reproduction prior to insemination to evolve in a similar manner. Plethodontid receptivity factor (PRF), a proteinaceous salamander pheromone produced by the male, increases female receptivity during courtship interactions. To test for lineage-specific differences in PRF's evolution, we intensively sampled PRF genes across the eastern Plethodon phylogeny (27 spp.; 34 populations) to compare gene diversification, rates of evolution, modes of selection, and types of amino acid substitution. Our analyses indicate that PRF evolutionary dynamics vary considerably from lineage to lineage. Underlying this heterogeneity, however, are two well-defined transitions in evolutionary mode. The first mode is representative of a typical protein profile, wherein neutral divergence and purifying selection are the dominant features. The second mode is characterized by incessant, cyclical evolution driven by positive selection. In this mode, the positively selected sites are bound by a limited assortment of acceptable amino acids that appear to evolve independently of other sites, resulting in a tremendous number of unique PRF alleles. Several of these selected sites are implicated in receptor binding. These sites are apparently involved in a molecular tango in which the male signal and female receptors coevolve within a confined molecular space. PRF's lineage-specific evolutionary dynamics, in combination with evidence of a molecular tango, highlight the molecular action of sexual selection on a chemical signal that is used during courtship.

Population variation in salamander courtship pheromones

We examined intraand interspecific chemical variation in salamander courtship pheromones. The extent to which plethodontid receptivity factor (PRF) and other glandular components vary among populations of Plethodon jordani was analyzed by reverse-phase HPLC. Gland extracts from individuals revealed that males within a population differ in the relative amounts of the glandular components as well as in the presence and absence of components, including PRF. Comparisons between populations showed that each population had a unique elution profile and differed significantly in the relative amounts of PRF and the other extract components. Knowledge about the nature of geographic variation in the courtship pheromone, combined with behavioral tests of female response, will contribute towards understanding how the pheromone evolved.

Proteinaceous pheromone affecting female receptivity in a terrestrial salamander.

A 22-kilodalton protein was isolated from the submandibular (mental) gland of the male terrestrial salamander, Plethodon jordani (family: Plethodontidae). This proteinaceous pheromone, termed plethodontid receptivity factor (PRF), was experimentally delivered to the female during courtship and shown to increase female receptivity. In most plethodontid salamanders, ovulation occurs weeks or months after insemination, so the pheromone-induced change in receptivity is the only known function of PRF. The messenger RNAs corresponding to isoforms of PRF were transcribed into complementary DNA, cloned, sequenced, and shown to have homology with cytokines of the interleukin-6 family. Pheromone activity would represent a previously unrecognized function for cytokines.