Comparative studies of major histocompatibility complex (MHC) genes across vertebrate species can reveal the evolutionary processes that shape the structure and function of immune regulatory proteins. In this study, we characterized MHC class I sequences from six frog species representing three anuran families (Hylidae, Centrolenidae and Ranidae). Using cDNA from our focal species, we amplified a total of 79 unique sequences spanning exons 2-4 that encode the extracellular domains of the functional alpha chain protein. We compared intra-and interspecific nucleotide and amino-acid divergence, tested for recombination, and identified codon sites under selection by estimating the rate of non-synonymous to synonymous substitutions with multiple codon-based maximum likelihood methods. We determined that positive (diversifying) selection was acting on specific amino-acid sites located within the domains that bind pathogen-derived peptides. We also found significant signals of recombination across the physical distance of the genes. Finally, we determined that all the six species expressed two or three putative classical class I loci, in contrast to the single locus condition of Xenopus laevis. Our results suggest that MHC evolution in anurans is a dynamic process and that variation in numbers of loci and genetic diversity can exist among taxa. Thus, the accumulation of genetic data for more species will be useful in further characterizing the relative importance of processes such as selection, recombination and gene duplication in shaping MHC loci among amphibian lineages.

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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 β1 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 IIβ exon 2. Two distinct intron sequences were recovered, suggesting the presence of at least two class IIβ 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 β1 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.

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.

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