A New Perspective on Female-to-Male Communication in Salamander Courtship.

Courtship behavior in salamanders is often complex and involves well-documented communication from males to females in multiple sensory modalities. Historically, behaviors exhibited during the major stages of courtship have been predominately framed as a male acting and signaling to "persuade" a passive female to participate in courtship and remain with him until sperm release is completed. In this review, we use courtship descriptions for lungless salamanders (Plethodontidae) as a case study to illustrate this historical bias of a male-centered perspective. We then re-examine the literature and summarize the many ways females are active participants during plethodontid courtships. We also relate female behaviors to the types of female-to-male communication that may occur. For example, females have been documented to approach a male and initiate courtship, participate in mutual head rubbing, and step astride the male's tail to begin the tail-straddling walk (a key courtship behavior observed in all plethodontids). Additionally, females have glands that may produce chemical signals that males respond to during courtship. We conclude that communication during courtship is more accurately described as a two-way interaction where each partner's behavior is coordinated with the other's via multi-modal signaling. Shifting the lens through which we view courtship and behavior provides insight into which female behaviors and anatomical features are most likely to be used for communication with males.

Genetic variation and selection of MHC class I loci differ in two congeneric frogs.

Major histocompatibility complex (MHC) genes encode proteins in the acquired immune response pathway that often show distinctive selection-driven patterns in wild vertebrate populations. We examined genetic variation and signatures of selection in the MHC class I alpha 1 (A1)- and alpha 2 (A2)-domain encoding exons of two frog congeners [Agalychnis callidryas (n = 20) and A. lemur (n = 20)] from a single locality in Panama. We also investigated how historical demographic processes may have impacted MHC genetic diversity by analyzing a neutral mitochondrial marker. We found that both MHC domains were highly variable in both species, with both species likely expressing three loci. Our analyses revealed different signatures of selection between the two species, most notably that the A. callidryas A2 domain had experienced positive selection while the A2 domain of A. lemur had not. Diversifying selection acted on the same number of A1 and A2 allelic lineages, but on a higher percentage of A1 sites compared to A2 sites. Neutrality tests of mitochondrial haplotypes predominately indicated that the two species were at genetic equilibrium when the samples were collected. In addition, two historical tests of demography indicated both species have had relatively stable population sizes over the past 100,000 years; thus large population size changes are unlikely to have greatly influenced MHC diversity in either species during this time period. In conclusion, our results suggest that the impact of selection on MHC diversity varied between these two closely related species, likely due to a combination of distinct ecological conditions and past pathogenic pressures.

Positive selection drives the evolution of a major histocompatibility complex gene in an endangered Mexican salamander species complex.

Immune gene evolution can be critical to species survival in the face of infectious disease. In particular, polymorphism in the genes of the major histocompatibility complex (MHC) helps vertebrates combat novel and diverse pathogens by increasing the number of pathogen-derived proteins that can initiate the host's acquired immune response. In this study, we used a combination of presumably adaptive and neutral markers to investigate MHC evolution in populations of five salamander species within the Ambystoma velasci complex, a group consisting of 15 recently diverged species, several of which are endangered. We isolated 31 unique MHC class II ß alleles from 75 total individuals from five species in this complex. MHC heterozygosity was significantly lower than expected for all five species, and we found no clear relationship between number of MHC alleles and species range, life history, or level of heterozygosity. We inferred a phylogeny representing the evolutionary history of Ambystoma MHC, with which we found signatures of positive selection on the overall gene, putative peptide-binding residues, and allelic lineages. We identified several instances of trans-species polymorphism, a hallmark of balancing selection observed in other groups of closely related species. In contrast, we did not detect comparable allelic diversity or signatures of selection on neutral loci. Additionally, we identified 17 supertypes among the 44 unique Ambystoma alleles, indicating that these sequences may encode functionally distinct MHC variants. We therefore have strong evidence that positive selection is a major evolutionary force driving patterns of MHC polymorphism in this recently radiated species complex.

Conservation and divergence in the frog immunome: pyrosequencing and de novo assembly of immune tissue transcriptomes.

BACKGROUND: Frogs are a diverse group of vertebrates for which limited genomic resources are available. Natural frog populations face a multitude of threats, including habitat degradation, infectious disease, and environmental change. Characterizing the functional genomics of anuran tissues in general - and the immune system in particular - will enhance our knowledge of genetic and epigenetic responses to environmental threats and inform conservation and recovery efforts. RESULTS: To increase the number of species with genomic datasets and characterize gene expression in immune-related tissues, we sequenced the transcriptomes of three tissues from two frogs (Espadarana prosoblepon and Lithobates yavapaiensis) on the Roche 454 GS FLX platform. Our sequencing produced 8881 E. prosoblepon and 5428 L. yavapaiensis annotated gene products after de novo assembly and Gene Ontology classification. Transcripts of the innate and acquired immune system were expressed in all three tissues. Inflammatory response and acquired immunity transcripts were significantly more diverged between E. prosoblepon and L. yavapaiensis compared to innate immunity and immune system development transcripts. Immune-related transcripts did not show an overall elevated rate of functional evolution, with the exception of glycosyl proteases, which include lysozymes, central bacterial and fungal-killing enzymes of the innate immune system. CONCLUSIONS: The three frog transcriptomes provide more than 600 Mbp of new genomic data, and will serve as a valuable framework for future comparative studies of non-model anurans. Additionally, we show that immune gene divergence varies by functional group and that transcriptome studies can be useful in comparing rates of evolutionary change across gene families.

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.

Characterization of two putative cytokine receptors, gp130 and ciliary neurotrophic factor receptor, from terrestrial salamanders.

Cytokines of the gp130 family are fundamental regulators of immune responses and signal through multimeric receptors to initiate intracellular second-messenger cascades. Here, we provide the first characterization of two full-length gp130 cytokine receptors from the cDNA of the red-legged salamander (Plethodon shermani). The first, gp130 (2745 bp), is a common signaling receptor for several multi-functional cytokines in vertebrates. We also isolated the full-length (1104 bp) sequence of the ciliary neurotrophic factor receptor (CNTFR), which forms a heteromeric signaling complex with gp130. The open reading frames of both receptors were predicted to contain many of the conserved features found in mammalian gp130s, such as cytokine binding homology regions and residues known to form disulfide bonds. Finally, we used RT-PCR to show that gp130 and CNTFR were expressed in most P. shermani tissues, including brain, intestine and muscle. The expression profiles, along with the structural predictions, show that gp130, CNTFR, and their cytokine ligands are parts of the immune system of P. shermani and other caudate amphibians.

Selection, trans-species polymorphism, and locus identification of major histocompatibility complex class IIß alleles of New World ranid frogs.

Genes encoded by the major histocompatibility complex (MHC) play key roles in the vertebrate immune system. However, our understanding of the evolutionary processes and underlying genetic mechanisms shaping these genes is limited in many taxa, including amphibians, a group currently impacted by emerging infectious diseases. To further elucidate the evolution of the MHC in frogs (anurans) and develop tools for population genetics, we surveyed allelic diversity of the MHC class II ß1 domain in both genomic and complementary DNA of seven New World species in the genus Rana (Lithobates). To assign locus affiliation to our alleles, we used a "gene walking" technique to obtain intron 2 sequences that flanked MHC class IIß exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIß loci in Rana. We designed a primer pair that successfully amplified an orthologous locus from all seven Rana species. In total, we recovered 13 alleles and documented trans-species polymorphism for four of the alleles. We also found quantitative evidence of selection acting on amino acid residues that are putatively involved in peptide binding and structural stability of the ß1 domain of anurans. Our results indicated that primer mismatch can result in polymerase chain reaction (PCR) bias, which influences the number of alleles that are recovered. Using a single locus may minimize PCR bias caused by primer mismatch, and the gene walking technique was an effective approach for generating single-copy orthologous markers necessary for future studies of MHC allelic variation in natural amphibian populations.

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 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 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.