Mental Gland Research Articles

Species-specific variation in mental gland secretions of turtles revealed by proteomic and lipidomic profiling

Chemical signaling through pheromones is an ancient and widespread mode of communication. Turtles and tortoises (chelonians) secrete pheromones via mental (chin) glands and have superior olfactory capacities rendering them a promising group to study the evolution and function of chemical communication in vertebrates. Here, we use state-of-the art proteomics and lipidomics techniques to identify and explore the possible functions of proteins and lipids secreted by mental glands in turtles. We examined four turtle species all from the family Geoemydidae, to understand among-species as well as sexual variation in the composition of mental gland secretions. Differential expression of a relatively small number (ca. 65) of proteins explained a large portion of the proteome variation across species, highlighting the existence of specific signals evolving even in closely related species. Lipidomic analysis revealed high inter-individual variation, but species differences could be attributed to five different lipid classes. The lipids found in mental glands could have a dietary origin or be related to individual condition, but may nonetheless be used in communication. We also examined sex-specific differences in the proteome of a single species (Mauremys leprosa) and found that males expressed a much larger array of proteins than females. Our findings establish a group of candidate proteins potentially involved in chemical signaling in freshwater geoemydid turtles. Alternatively, differently expressed proteins in mental glands could have an indirect link to chemical communication, being involved in pheromone transport and/or antioxidant protection.

Presence of sodefrin precursor-like factor pheromone candidates in mental and dorsal tail base glands in the plethodontid salamander, Karsenia koreana.

Plethodontid salamanders are well known for their distinct courtship rituals and the associated pheromonal signaling. However, little is known about pheromones produced in the lone Asian plethodontid species Karsenia koreana. Here, we examined the localization patterns of proteins of the sodefrin precursor-like factor (SPF) pheromone system in K. koreana. Using an antibody generated against SPF proteins from another plethodontid, Desmognathus ocoee, we tested three types of skin glands in K. koreana males via immunohistochemistry: the mental gland and two types of dorsal tail base glands-caudal courtship glands and dorsal granular glands. SPF immunoreactivity was detected in the known courtship gland, the mental gland, as well as granular glands, but not in caudal courtship glands. Due to immunoreaction specificity, we hypothesize the proteins of the SPF system in K. koreana and D. ocoee are structurally and functionally related and are used as courtship pheromones in K. koreana. Also, we hypothesize that K. koreana males transmit SPF to the female during the tail-straddling walk via dorsal granular glands. Finally, K. koreana male caudal courtship glands may be producing SPF proteins that are not recognized by our SPF antibody or these glands may play a different role in courtship than anticipated.

Male gopher tortoise (Gopherus polyphemus) concentration-dependent social responses to diluted mental gland pheromones

In complex terrestrial environments, chemical signals can be the most important sensory modality for locating conspecifics for potential mating opportunities, especially in spatially segregated populations or habitats. Organisms must evolve chemical signals to maximize the efficacy of conveying information, particularly in creating trails or mate-choice cues. Long-distance transmission of chemical signals may be an increasingly important management concern for small and fractured populations or potentially threatened species, such as gopher tortoises in the southeastern U.S. Mental gland secretions have been shown to have pheromonal function in gopher tortoises, suggesting a potential role as trail or marking pheromones, allowing males to track females or other males to find females. In this study, male gopher tortoises were given paired presentations of a negative control (distilled water) with serial dilutions (1:4, 1:20, 1:100, and 1:500) of male mental gland secretions. Male tortoises were able to discern treatment differences up to 1:20 diluted secretions, responding with an array of social behaviors (e.g. for the 1:20 dilution trial, carapace alignment and head bobbing occurred more frequently for the mental gland secretion relative to the control; p < 0.01). Multivariate principal components analysis yielded PC1 (including, approach, carapace alignment, head bobbing, tasting air, sniffing, and doubleback) that differed by treatment (p = 0.0007) and also was higher for the 1:20 diluted presentation relative to the 1:500 diluted presentation (p = 0.04). This study provides insight into gopher tortoise ecology, mate-choice, and the utility of environmentally diluted mental gland secretions in the external environment when seeking mating opportunities.

External nasal gland morphology of Eurycea bislineata (Amphibia, Urodela, Plethodontidae).

Plethodontid salamanders possess numerous courtship glands. Previous studies have shown that the glands are more prominent in male individuals than females, and often experience periods of atrophy and hypertrophy throughout the year that correlate to the nonmating and mating seasons, respectively. We sampled male and female Eurycea bislineata throughout the year to test the hypothesis that external nasal glands are courtship glands. External nasal glands are paired, branched tubular glands that extend from excretory ducts dorsal to the nares to terminal secretory units posterior to the eyes. We found that the glands hypertrophy and stain/react more intensely with histochemical procedures during the mating season. Hypertrophy of the glands is more pronounced in males, and seasonal variation in epithelial height of external nasal glands of males is significantly correlated to that of seasonal variation in mental gland epithelial height, a known courtship gland found in males, when compared throughout the year. This correlation was not as strong in females, confirming sexual dimorphism of external nasal glands in terms of seasonal variation. We found no ultrastructural differences between male and female external nasal glands. In all specimens, the glandular tubules were lined by a simple, columnar epithelium that was packed with secretory granules that often obscured other cytoplasmic contents.

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.

Behavioural discrimination of male mental gland secretions of the gopher tortoise (Gopherus polyphemus) by both sexes

Chemical communication is important for mate choice, especially at long distances in fragmented populations. The gopher tortoise is a social species that is threatened in the southeast U.S. due to habitat fragmentation and decline. One consequence of habitat loss is reduced mating opportunities, yet chemical signalling in gopher tortoises is relatively under-studied. Here, we investigated chemoreception of tortoise discrimination of chin secretions, or mental gland (MG) secretions. To assess conspecific recognition of male MG secretions, we conducted two paired-choice experiments: one with a neutral odorant control (NC; distilled water) and one with a pungent odorant control (PC; acetone) vs. male MG secretions. Behaviours were defined a priori, and their durations were quantified relative to treatments. Each sex spent significantly more time with MG secretions vs. acetone control during the PC study (p= 0.001). Each sex also sniffed MG swabs more frequently in both studies (PC study: p=0.0003; NC study: p=0.001). A principal components analysis of behavioural durations from the PC study identified one component with a significant treatment effect performed to MG secretions (p=0.0003), including the behaviours sniffing, head bobbing, biting, and eating near a swab. Our study provides the first chemical-behavioural bioassay of MG secretions from male gopher tortoises, suggesting MG secretions may be a source of pheromones.

Mental gland secretions as a social cue in gopher tortoises (Gopherus polyphemus): tortoise presence stimulates and maintains social behaviour with chemical cues

Multimodal signalling reinforces specific messages in communication. In gopher tortoises, similar to other reptilian species, visualization of conspecific and chemical exudates from the skin may serve as a multimodal display advertising information about conspecific species, sex, or individual qualities, but this has not been fully elucidated. For gopher tortoises, one such possible source of chemical cues could be secretions from seasonally enlarged mental glands (MG). Here, we used both sexes of gopher tortoises in a paired choice presentation of MG secretions vs. distilled (DI) water on resin tortoise models to assess visual presence with tortoise-specific secretions. We examined behaviours to treatments to examine if MG secretions are recognizable olfactory cues and if visual cues alone are sufficient to maintain social interactions using a simple visual presentation vs. a complex visual and olfactory presentation. Tortoises of both sexes spent more total time (p < 0.001) and performed a greater number of behaviours (p < 0.001) towards the MG-treated model, relative to the neutral control (DI-treated model), suggesting that olfactory MG secretions are also required, along with visual presence of a tortoise, to engage in social behaviours. Our results are among the first for this species suggesting that pheromone usage may drive social interactions in social behaviours.

The chemistry and histology of sexually dimorphic mental glands in the freshwater turtle, Mauremys leprosa.

Despite evidence from anatomy, behavior and genomics indicating that the sense of smell in turtles is important, our understanding of chemical communication in this group is still rudimentary. Our aim was to describe the microanatomy of mental glands (MGs) in a freshwater turtle, Mauremys leprosa (Geoemydidae), and to assess the chemical composition of their secretions with respect to variation among individuals and between sexes. MGs are paired sac-like organs on the gular region of the neck and are dimorphic in this species with males having fully functional holocrine glands while those of females appear non-secretory and vestigial. In adult males, the glandular epithelium of the inner portion of the gland provides exocytotic products as well as cellular debris into the lumen of the gland. The contents of the lumen can be secreted through the narrow duct portion of the gland ending in an orifice on the surface of the skin. Females have invaginated structures similar in general outline to male glands, but lack a glandular epithelium. Using gas chromatography coupled to mass spectrometry, we identified a total of 61 compounds in mental gland secretions, the most numerous being carboxylic acids, carbohydrates, alkanes, steroids and alcohols. The number of compounds per individual varied widely (mean (median) ± SD = 14.54 (13) ± 8.44; min = 3; max = 40), but only cholesterol was found in all samples. We found that the relative abundances of only six chemicals were different between the sexes, although males tended to have larger amounts of particular compounds. Although the lipid fraction of mental gland secretions is rich in chemical compounds, most occur in both sexes suggesting that they are metabolic byproducts with no role in chemical signaling. However, the relative amounts of some compounds tended to be higher in males, with significantly larger amounts of two carboxylic acids and one steroid, suggesting their putative involvement in chemical communication.

Museum Specimens Reveal Life History Characteristics in Plethodon montanus

Amphibian life history strategies can vary across broad geographic patterns, which have been explained, at least in part, by how development, growth, maintenance, and reproduction respond to temperature. However, there is a dearth of information regarding life history traits and their variation among populations for many amphibian species, which prevents assessment of these broad patterns among species. Therefore, we used museum specimens to assess the variation in reproductive condition of Plethodon montanus across its range and used skeletochronology at five populations along an elevational gradient to determine the elevational variation in age, growth rate, and asymptotic size. We found that body size and temperature seasonality best explained the variation in the presence of eggs and a mental gland, whereas body size alone best explained the presence of enlarged pigmented testes. We also found that longevity in P. montanus is at least 12 years with higher elevations having a higher proportion of older individuals compared to lower elevations and that size was a better explanation of reproductive condition than age across the elevation. Our results further demonstrate the importance of body size for amphibian life history traits and the utility of museum specimens to estimate these traits. Variation in reproductive condition across the range of P. montanus is likely the result of variation in growing season length. Lastly, we found evidence suggesting asymptotic size and growth rates may have changed over the last half century, which would ultimately affect fertility and other life history traits and warrants future studies.

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

BackgroundCell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be regulated to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~ 2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to the synthesis of rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history: following gene duplication, the protein coding sequence diversified from positive sexual selection while the untranslated regions have been conserved by purifying selection. The molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and conservation of the untranslated regions remain to be determined.ResultsUsing Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3′ UTR. CIRBP, along with other proteins associated with stress response, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis.ConclusionsThe P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of coding and noncoding segments in rapid gene evolution, and necessitate the study of co-evolution between pheromone gene products and their transcriptional/translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated tissue proliferation and subsequent resorption within the dermis and share molecular links to skin cancer biology.

A new species of Hyloscirtus (Anura, Hylidae) from the Colombian and Venezuelan slopes of Sierra de Perijá, and the phylogenetic position of Hyloscirtus jahni (Rivero, 1961).

A new species of Hyloscirtus, belonging to the H. bogotensis species Group, is described from the Venezuelan and Colombian slopes of the Sierra de Perijá. The new species can be readily distinguished from its congeners by the combination of the following characters: mental gland present, disc-shaped and small; ulnar, outer, and inner tarsal folds present; calcar tubercle absent; whitish stripes on external border of upper eyelids and supratympanic folds, longitudinally on the mid-dorsum, on supracloacal fold, outer ulnar folds, inner and outer tarsal folds, and also on dorsal internal surface of shanks. We estimate phylogenetic relationships based on mtDNA (spanning fragments of 12S rRNA, tRNA-Val and 16S rRNA), of all Hyloscirtus species available in Genbank, as well as the new species described herein, H. callipeza, H. jahni, and H. platydactylus, all of which have not been previously sequenced. Our molecular data support the hypothesis of the new species as sister species of H. callipeza and indicates that H. jahni does not belong to the H. bogotensis species Group, but rather is sister species of all other Hyloscirtus (sensu Faivovich et al. 2005). Based on this last result we propose a new species group for H. jahni and the synonymy of Colomascirtus in Hyloscirtus. We also provide the first description of the advertisement call of H. callipeza. With the new species described herein, the number of Hyloscirtus species increases to 37.

Gene Duplication, Co-option, Structural Evolution, and Phenotypic Tango in the Courtship Pheromones of Plethodontid Salamanders

Abstract Rapid evolution is a hallmark of proteins involved in reproduction. The protein courtship pheromones in plethodontid salamanders are classic examples of such rapidly evolving reproductive proteins, with male pheromones likely coevolving with female receptors to improve reproductive success. Over the past 66 million years of plethodontid evolution, the structure and composition of the male mental gland has evolved with changes in courtship timing and behavior. More than 20 yr of biochemical and molecular studies have provided insight into how multiple gene families have been duplicated, mutated, and co-opted for pheromone roles. Sequencing and mass spectral proteomic analyses have enabled identification and characterization of multiple pheromone families, some with lineage-specific expression. In this review, we provide a phenotypic tango model to better understand male pheromone and female receptor coevolution that has driven the rapid evolution of multiple diverse pheromone families. To offer su...

Phylogeny of Mental Glands, Revisited.

Mental glands and their associated delivery behaviors during courtship are unique to the plethodontid salamanders. Because previous interpretations of the evolution of these features were conducted using older phylogenetic hypotheses, we reanalyzed these traits with newer courtship descriptions and contemporary phylogenetic methods. Using Bayesian ancestral state reconstruction methods that have been developed since the first phylogenetic analyses were conducted in the mid-1990s, we reconstructed mental gland and courtship behavior evolution on a Bayesian phylogeny of the nuclear gene Rag1. The most probable ancestral condition for plethodontids was resolved as presence of a mental gland. Loss of a mental gland occurred in each subfamily and was recovered as the most probable ancestral condition for the Spelerpinae. In contrast, parsimony reconstruction recovered the presence of a mental gland in the ancestor to Spelerpinae with multiple secondary losses. We hypothesize that that absence of a mental gland is possibly ancestral in some clades (i.e., Spelerpinae) and secondary in others (e.g., paedomorphic Eurycea). The most probable ancestral form of the mental gland is likely to be the large pad-type distributed extensively in Plethodontinae and Bolitoglossinae. Desmognathans have the most unique mental glands, occurring in an anterior protrusion or bifurcated form (in Desmognathus wrighti). Fan-shaped mental glands evolved independently in Eurycea and Oedipina. Small pads arose independently in Bolitoglossinae, Plethodontinae, and Spelerpinae. Head-rubbing behavior for mental gland delivery mode was recovered as the most probable and parsimonious ancestral state for the Plethodontidae, with independent losses of this behavior in Plethodontinae and Spelerpinae. Because head-rubbing was observed in outgroups, we hypothesize that head-rubbing behavior predated mental gland evolution. Pulling, snapping, slapping, and biting behaviors evolved independently in the Plethodontinae and Spelerpinae and are not homologous with head-rubbing. All hypotheses of mental gland and courtship evolution invoke homoplasy.

Ultrastructure of the mental gland of the Red‐Backed Salamander, Plethodon cinereus (Amphibia: Plethodontidae)

AbstractMental glands are sexually dimorphic glands found in the skin of the submandibular region in males of many species in the salamander family Plethodontidae. These glands have been demonstrated to secrete pheromones that increase female receptivity to courtship and mating. A number of studies exist on the morphology of mental glands at the light microscopy level, but ultrastructural studies on the glands exist for only two species, Eurycea quadridigitata and Hydromantes italicus. In this study, ultrastructure of the mental gland is described from seasonal samples of the eastern North American plethodontid Plethodon cinereus. The mental glands are most hypertrophied in males examined in mating condition (i.e. sperm in their Wolffian ducts) in October and April. In these specimens, 6‐ to 8‐μm electron‐dense secretory vacuoles are found in the apical cytoplasm (October) or throughout the entire epithelial cells (April). Mental glands from June and August contain only condensing vacuoles, and the cytoplasm is otherwise packed with stacks of rough endoplasmic reticulum. The ultrastructure of the mental gland of P. cinereus agrees with earlier descriptions for E. quadridigitata and H. italicus. Two other sexually dimorphic skin glands, the caudal courtship gland and the cloacal vent gland, were previously examined from males in this sample of P. cinereus. Although all three glands show similarities in histology and histochemistry at the light microscopy level, they are dissimilar in ultrastructure. Whereas the secretory vacuoles of mental glands are uniformly electron dense, those of caudal courtship glands are smaller (1.2–1.5 μm), oblong and biphasic, and vent glands have spherical biphasic secretory vacuoles (1.8–2.1 μm). This indicates a difference in secretory product and function for the three glands and needs further investigation. Whether other plethodontids show similar variation among sexually dimorphic glands is another topic for future research.

High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens (Salamandridae).

BackgroundMale salamanders (Urodela) often make use of pheromones that are produced in sexually dimorphic glands to persuade the female into courtship and mating. The mental gland of lungless salamanders (Plethodontidae) and dorsal cloacal glands (or abdominal glands) of newts (Salamandridae) have been particularly well studied in that respect. In both families, sodefrin precursor-like factor (SPF) proteins have been identified as major components of the courtship pheromone system. However, similar to plethodontids, some newts also make use of subtle head glands during courtship, but few pheromones have been characterized from such structures. Males of red-spotted newts (Notophthalmus viridescens, Salamandridae) have both cloacal and cheek (genial) glands, and are known to apply secretions to the female’s nose by both tail-fanning and cheek-rubbing. Here we combined transcriptomic and phylogenetic analyses to investigate the presence, diversity and evolution of SPF proteins in the cloacal and cheek glands of this species.ResultsOur analyses indicate that the cheek glands of male N. viridescens produce a similar amount and diversity of SPF isoforms as the cloacal glands in this species. Expression in other tissues was much lower, suggesting that both male-specific courtship glands secrete SPF pheromones during courtship. Our phylogenetic analyses show that N. viridescens expresses a combination of isoforms that stem from four highly diverged evolutionary lineages of SPF variants, that together form a basis for the broad diversity of SPF precursors in the breeding glands.ConclusionsThe similar SPF expression of cheek and cloacal glands suggests that this protein family is used for pheromone signalling through cheek rubbing in the red-spotted newt. Since several male salamandrids in other genera have comparable head glands, SPF application via other glands than the cloacal glands may be more widespread than currently appreciated in salamandrids.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0333-1) contains supplementary material, which is available to authorized users.

The Presence of Sexually Dimorphic Submandibular Glands inTaricha granulosa, the Rough-Skinned Newt (Salamandridae)

We investigate whether Taricha granulosa, the rough-skinned newt, possesses pheromone-producing courtship glands in the submandibular dermis. The hypothesis that these newts have a pheromone-producing mental gland repeatedly appears in the literature based on a small histological study and on courtship behavior in which males press their chins on females' nares. We identify a sexually dimorphic granular gland in the submandibular region of male newts. Its secretion is granular and PAS (periodic acid-Schiff)-positive, in contrast to the PAS-negative secretion of typical granular glands. This gland type is found only in males and only in the anterior submandibular region. Compared to glands evaluated from non-breeding males, the glands from animals collected in the breeding season are hypertrophied. This sexually dimorphic granular gland is distinct from the mental gland found in plethodontid salamanders; the plethodontid mental gland is an aggregate of individual glands, whereas the granular glands in T. granulosa are not clustered together to form a distinct unit. Our results support the hypothesis that males are secreting pheromones from submandibular glands during courtship in T. granulosa.

PROTEOMIC AND UTR ANALYSES OF A RAPIDLY EVOLVING HYPERVARIABLE FAMILY OF VERTEBRATE PHEROMONES

During the annual mating season, the mental gland of male plethodontid salamanders diverts its protein synthesizing capacity to the production of courtship pheromones that increase female receptivity. Plethodontid modulating factor (PMF), a highly disulfide-bonded 7-kDa pheromone, shows unusual hypervariability with each male expressing >30 isoforms. Twenty-eight PMFs were purified and matched by proteomic analyses to cDNA sequences. In contrast to coding sequence hypervariability, the untranslated regions (UTRs) show extraordinary conservation, no predicted microRNA binding sites, and an overlapping triplet polyadenylation signal. Full-length cDNA sequencing revealed three PMF gene classes containing subclasses of clustered sequences that support ≥ 13 PMF gene duplications. The unusual phenomena of hypervariable coding regions embedded within extremely conserved UTRs is proposed to occur by a disjunctive evolutionary process. During the short courtship season, the UTRs are hypothesized to subsume and coordinate the transcriptional and translational regulatory mechanisms of the mental gland. PMF, as a secreted protein with limited metabolic feedback in the male, is under minimal mutational restraint and thus has experienced highly accelerated rates of evolution. Consequently, plethodontid salamanders may provide a unique model for furthering our understanding of the selective forces that determine differential rates of gene duplication and evolution in protein families.

Consistency of Male Courtship Behavior in a Plethodontid Salamander

Abstract Flexibility can be important in male–female behavioral interactions, particularly during courtship when a male typically adjusts his behavior in response to the female. Red-legged Salamanders (Plethodon shermani) have highly stereotyped courtship behaviors but show flexibility in the repetition of particular behaviors. We tested one measure of flexibility in a specific male courtship behavior: pheromone delivery. We compared the number of male pheromone delivery attempts that occurred when a male “slapped” his pheromone-producing mental gland on the female's nares. We compiled a multiyear data set from earlier experiments on salamander behavior to test the consistency in the number of slaps each male delivered in two different courtships. The consistency level was 26% (26% of the total deviance in slapping behavior could be explained by previous slapping behavior), a value within the range of measures for other vertebrate behavioral traits. We interpret this consistency as a possible advantage to...

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.

A New Species of the Bokermannohyla martinsi Group from Central Bahia, Brazil with Comments on Bokermannohyla (Anura: Hylidae)

A new species of the Bokermannohyla martinsi group is described from Chapada Diamantina, State of Bahia, northeastern Brazil. It is differentiated from the other two species in the group by the combination of slender body (quite robust in B. langei and B. martinsi), non-bifid distal prepollex (bifid in B. langei and B. martinsi), squared shape of the posterior portion of the distal prepollex (pointed process in B. langei and B. martinsi), large size and lateral inclination of the tympanum (quite reduced in B. langei, almost parallel to the longitudinal axis in both B. langei and B. martinsi), absence of dark bars in hidden areas of thighs (dark bars present on anterior, dorsal and posterior surfaces of thighs in B. martinsi), and shape of the supratympanic fold. We report for the first time the occurrence of a mental gland in 16 species of Bokermannohyla.