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Studies of MHC-based mate choice in wild populations often test hypotheses on species exhibiting female choice and male-male competition, which reflects the general prevalence of females as the choosy sex in natural systems. Here, we examined mutual mate-choice patterns in a small burrow-nesting seabird, the Leach's storm-petrel (Oceanodroma leucorhoa), using the major histocompatibility complex (MHC). The life history and ecology of this species are extreme: both partners work together to fledge a single chick during the breeding season, a task that requires regularly travelling hundreds of kilometres to and from foraging grounds over a 6- to 8-week provisioning period. Using a 5-year data set unprecedented for this species (n = 1078 adults and 925 chicks), we found a positive relationship between variation in the likelihood of female reproductive success and heterozygosity at Ocle-DAB2, a MHC class IIB locus. Contrary to previous reports rejecting disassortative mating as a mechanism for maintaining genetic polymorphism in this species, here we show that males make significant disassortative mate-choice decisions. Variability in female reproductive success suggests that the most common homozygous females (Ocle-DAB2*01/Ocle-DAB2*01) may be physiologically disadvantaged and, therefore, less preferred as lifelong partners for choosy males. The results from this study support the role of mate choice in maintaining high levels of MHC variability in a wild seabird species and highlight the need to incorporate a broader ecological framework and sufficient sample sizes into studies of MHC-based mating patterns in wild populations in general.

The Atlantic Forest (AF) harbours one of the most diverse vertebrate faunas of the world, including 199 endemic species of birds. Understanding the evolutionary processes behind such diversity has become the focus of many recent, primarily single locus, phylogeographic studies. These studies suggest that isolation in forest refugia may have been a major mechanism promoting diversification, although there is also support for a role of riverine and geotectonic barriers, two sets of hypotheses that can best be tested with multilocus data. Here we combined multilocus data (one mtDNA marker and eight anonymous nuclear loci) from two species of parapatric antbirds, Myrmeciza loricata and M. squamosa, and Approximate Bayesian Computation to determine whether isolation in refugia explains current patterns of genetic variation and their status as independent evolutionary units. Patterns of population structure, differences in intraspecific levels of divergence and coalescent estimates of historical demography fit the predictions of a recently proposed model of refuge isolation in which climatic stability in the northern AF sustains higher diversity and demographic stability than in the southern AF. However, a pre-Pleistocene divergence associated with their abutting range limits in a region of past tectonic activity also suggests a role for rivers or geotectonic barriers. Little or no gene flow between these species suggests the development of reproductive barriers or competitive exclusion. Our results suggests that limited marker sampling in recent AF studies may compromise estimates of divergence times and historical demography, and we discuss the effects of such sampling on this and other studies.

Understanding the mechanisms that produce variation in thermal performance is a key component to investigating climatic effects on evolution and adaptation. However, disentangling the effects of local adaptation and phenotypic plasticity in shaping patterns of geographic variation in natural populations can prove challenging. Additionally, the physiological mechanisms that cause organismal dysfunction at extreme temperatures are still largely under debate. Using the green anole, Anolis carolinensis, we integrate measures of cold tolerance (CTmin ), standard metabolic rate, heart size, blood lactate concentration and RNAseq data from liver tissue to investigate geographic variation in cold tolerance and its underlying mechanisms along a latitudinal cline. We found significant effects of thermal acclimation and latitude of origin on variation in cold tolerance. Increased cold tolerance correlates with decreased rates of oxygen consumption and blood lactate concentration (a proxy for oxygen limitation), suggesting elevated performance is associated with improved oxygen economy during cold exposure. Consistent with these results, co-expression modules associated with blood lactate concentration are enriched for functions associated with blood circulation, coagulation and clotting. Expression of these modules correlates with thermal acclimation and latitude of origin. Our findings support the oxygen and capacity-limited thermal tolerance hypothesis as a potential contributor to variation in reptilian cold tolerance. Moreover, differences in gene expression suggest regulation of the blood coagulation cascade may play an important role in reptilian cold tolerance and may be the target of natural selection in populations inhabiting colder environments.

Identifying the genetic basis of phenotypic variation and its relationship with the environment is key to understanding how local adaptations evolve. Such patterns are especially interesting among populations distributed across habitat gradients, where genetic structure can be driven by isolation by distance (IBD) and/or isolation by environment (IBE). Here, we used variation in ~1600 high-quality SNPs derived from paired-end sequencing of double-digest restriction site-associated DNA (ddRAD-Seq) to test hypotheses related to IBD and IBE in the Yucatan jay (Cyanocorax yucatanicus), a tropical bird endemic to the Yucatan Peninsula. This peninsula is characterized by a precipitation and vegetation gradient—from dry to evergreen tropical forests—that is associated with morphological variation in this species. We found a moderate level of nucleotide diversity (π = 0.008) and little evidence for genetic differentiation among vegetation types. Analyses of neutral and putatively adaptive SNPs (identified by complementary genome-scan approaches) indicate that IBD is the most reliable explanation to account for frequency distribution of the former, while IBE has to be invoked to explain those of the later. These results suggest that selective factors acting along a vegetation gradient can promote local adaptation in the presence of gene flow in a vagile, non-migratory and geographically restricted species. The putative candidate SNPs identified here are located within or linked to a variety of genes that represent ideal targets for future genomic surveys. This article is protected by copyright. All rights reserved.

Innate immunity is expected to play a primary role in conferring resistance to novel infectious diseases, but few studies have attempted to examine its role in the evolution of resistance to emerging pathogens in wild vertebrate populations. Here, we used experimental infections and cDNA microarrays to examine whether changes in the innate and ⁄or acquired immune responses likely accompanied the emergence of resistance in house finches (Carpodacus mexicanus) in the eastern United States subject to a recent outbreak of conjunctivitis-causing bacterium (Mycoplasma gallisepticum—MG). Three days following experimental infection with MG, we observed differences in the splenic transcriptional responses between house finches from eastern U.S. populations, with a 12year history of MG exposure, versus western U.S. populations, with no history of exposure to MG. In particular, western birds down-regulated gene expression, while eastern finches showed no expression change relative to controls. Studies involving poultry have shown that MG can manipulate host immunity, and our observations suggest that pathogen manipulation occurred only in finches from the western populations, outside the range of MG. Fourteen days after infection, eastern finches, but not western finches, up-regulated genes associated with acquired immunity (cell-mediated immunity) relative to controls. These observations suggest population differences in the temporal course of the response to infection with MG and imply that innate immune processes were targets of selection in response to MG in the eastern U.S. population.

Next-generation sequencing technology provides an attractive means to obtain large-scale sequence data necessary for comparative genomic analysis. To analyse the patterns of mutation rate variation and selection intensity across the avian genome, we performed brain transcriptome sequencing using Roche 454 technology of 10 different non-model avian species. Contigs from de novo assemblies were aligned to the two available avian reference genomes, chicken and zebra finch. In total, we identified 6499 different genes across all 10 species, with approximately 1000 genes found in each full run per species. We found evidence for a higher mutation rate of the Z chromosome than of autosomes (male-biased mutation) and a negative correlation between the neutral substitution rate (d(S)) and chromosome size. Analyses of the mean d(N)/d(S) ratio (omega) of genes across chromosomes supported the Hill-Robertson effect (the effect of selection at linked loci) and point at stochastic problems with omega as an independent measure of selection. Overall, this study demonstrates the usefulness of next-generation sequencing for obtaining genomic resources for comparative genomic analysis of non-model organisms.

Understanding the selective forces that shape genetic variation in natural populations remains a high priority in evolutionary biology. Genes at the major histocompatibility complex (MHC) have become excellent models for the investigation of adaptive variation and natural selection because of their crucial role in fighting off pathogens. Here we present one of the first data sets examining patterns of MHC variation in wild populations of a bird of prey, the lesser kestrel, Falco naumanni. We report extensive polymorphism at the second exon of a putatively functional MHC class II gene, Fana-DAB*1. Overall, 103 alleles were isolated from 121 individuals sampled from Spain to Kazakhstan. Bayesian inference of diversifying selection suggests that several amino acid sites may have experienced strong positive selection (omega = 4.02 per codon). The analysis also suggests a prominent role of recombination in generating and maintaining MHC diversity (rho = 4Nc = 0.389 per codon, theta = 0.017 per codon). Both the Fana-DAB*1 locus and a set of eight polymorphic microsatellite markers revealed an isolation-by-distance pattern across the Western Palaearctic (r = 0.67; P = 0.01 and r = 0.50; P = 0.04, respectively). Nonetheless, geographical variation at the MHC contrasts with relatively uniform distributions in the frequencies of microsatellite alleles. In addition, we found lower fixation rates in the MHC than those predicted by genetic drift after controlling for neutral mitochondrial sequences. Our results therefore underscore the role of balancing selection as well as spatial variations in parasite-mediated selection regimes in shaping MHC diversity when gene flow is limited.

In 1994, the bacterial parasite Mycoplasma gallisepticum expanded its host range and swept through populations of a novel host — eastern US populations of the house finch ( Carpodacus mexicanus ). This epizootic caused a dramatic decline in finch population numbers, has been shown to have caused strong selection on house finch morphology, and presumably caused evolutionary change at the molecular level as finches evolved enhanced resistance. As a first step toward identifying finch genes that respond to infection by Mycoplasma and which may have experienced natural selection by this parasite, we used suppression subtractive hybridization (SSH) and cDNA macroarray approaches to identify differentially expressed genes regulated by the Mycoplasma parasite. Two subtractive cDNA libraries consisting of 16 512 clones were developed from spleen using an experimentally uninfected bird as the ‘tester’ and an infected bird as ‘driver’, and vice versa. Two hundred and twenty cDNA clones corresponding 34 genes with known vertebrate homologues and a large number of novel transcripts were found to be qualitatively up- or down-regulated genes by high-density filter hybridization. These gene expression changes were further confirmed by a high throughout reverse Northern blot approach and in specific cases by targeted Northern analysis. BLAST searches show that heat shock protein (HSP) 90, MHC II-associated invariant chain (CD74), T-cell immunoglobulin mucin 1 (TIM1), as well as numerous novel expressed genes not found in the databases were up- or down-regulated by the host in response to this parasite. Our results and macroarray resources provide a foundation for molecular co-evolutionary studies of the Mycoplasma parasite and its recently colonized avian host.

For many molecular ecologists, the mantra and mission of the field of ecological genomics could be encapsulated by the phrase ‘to find the genes that matter’ (Mitchell-Olds 2001; Rockman 2012). This phrase of course refers to the early hope and current increasing success in the search for genes whose variation underlies phenotypic variation and fitness in natural populations. In the years since the modern incarnation of the field of ecological genomics, many would agree that the low-hanging fruit has, at least in principle, been plucked: we now have several elegant examples of genes whose variation influences key adaptive traits in natural populations, and these examples have revealed important insights into the architecture of adaptive variation (Hoekstra et al. 2006; Shapiro et al. 2009; Chan et al. 2010). But how well will these early examples, often involving single genes of large effect on discrete or near-discrete phenotypes, represent the dynamics of adaptive change for the totality of phenotypes in nature? Will traits exhibiting continuous rather than discrete variation in natural populations have as simple a genetic basis as these early examples suggest (Prasad et al. 2012; Rockman 2012)? Two papers in this issue (Robinson et al. 2013; Santure et al. 2013) not only suggest answers to these questions but also provide useful extensions of statistical approaches for ecological geneticists to study the genetics of continuous variation in nature. Together these papers, by the same research groups studying evolution in a natural population of Great Tits (Parus major), provide a glimpse of what we should expect as the field begins to dissect the genetic basis of what is arguably the most common type of variation in nature, and how genome-wide surveys of variation can be applied to natural populations without pedigrees.

Innate immunity is expected to play a primary role in conferring resistance to novel infectious diseases, but few studies have attempted to examine its role in the evolution of resistance to emerging pathogens in wild vertebrate populations. Here, we used experimental infections and cDNA microarrays to examine whether changes in the innate and/or acquired immune responses likely accompanied the emergence of resistance in house finches (Carpodacus mexicanus) in the eastern United States subject to a recent outbreak of conjunctivitis-causing bacterium (Mycoplasma gallisepticum-MG). Three days following experimental infection with MG, we observed differences in the splenic transcriptional responses between house finches from eastern U.S. populations, with a 12-year history of MG exposure, versus western U.S. populations, with no history of exposure to MG. In particular, western birds down-regulated gene expression, while eastern finches showed no expression change relative to controls. Studies involving poultry have shown that MG can manipulate host immunity, and our observations suggest that pathogen manipulation occurred only in finches from the western populations, outside the range of MG. Fourteen days after infection, eastern finches, but not western finches, up-regulated genes associated with acquired immunity (cell-mediated immunity) relative to controls. These observations suggest population differences in the temporal course of the response to infection with MG and imply that innate immune processes were targets of selection in response to MG in the eastern U.S. population.

Understanding the selective forces that shape genetic variation in natural populations remains a high priority in evolutionary biology. Genes at the major histocompatibility complex (MHC) have become excellent models for the investigation of adaptive variation and natural selection because of their crucial role in fighting off pathogens. Here we present one of the first data sets examining patterns of MHC variation in wild populations of a bird of prey, the lesser kestrel, Falco naumanni. We report extensive polymorphism at the second exon of a putatively functional MHC class II gene, Fana-DAB*1. Overall, 103 alleles were isolated from 121 individuals sampled from Spain to Kazakhstan. Bayesian inference of diversifying selection suggests that several amino acid sites may have experienced strong positive selection (omega = 4.02 per codon). The analysis also suggests a prominent role of recombination in generating and maintaining MHC diversity (rho = 4Nc = 0.389 per codon, theta = 0.017 per codon). Both the Fana-DAB*1 locus and a set of eight polymorphic microsatellite markers revealed an isolation-by-distance pattern across the Western Palaearctic (r = 0.67; P = 0.01 and r = 0.50; P = 0.04, respectively). Nonetheless, geographical variation at the MHC contrasts with relatively uniform distributions in the frequencies of microsatellite alleles. In addition, we found lower fixation rates in the MHC than those predicted by genetic drift after controlling for neutral mitochondrial sequences. Our results therefore underscore the role of balancing selection as well as spatial variations in parasite-mediated selection regimes in shaping MHC diversity when gene flow is limited.

In 1994, the bacterial parasite Mycoplasma gallisepticum expanded its host range and swept through populations of a novel host--eastern US populations of the house finch (Carpodacus mexicanus). This epizootic caused a dramatic decline in finch population numbers, has been shown to have caused strong selection on house finch morphology, and presumably caused evolutionary change at the molecular level as finches evolved enhanced resistance. As a first step toward identifying finch genes that respond to infection by Mycoplasma and which may have experienced natural selection by this parasite, we used suppression subtractive hybridization (SSH) and cDNA macroarray approaches to identify differentially expressed genes regulated by the Mycoplasma parasite. Two subtractive cDNA libraries consisting of 16,512 clones were developed from spleen using an experimentally uninfected bird as the 'tester' and an infected bird as 'driver', and vice versa. Two hundred and twenty cDNA clones corresponding 34 genes with known vertebrate homologues and a large number of novel transcripts were found to be qualitatively up- or down-regulated genes by high-density filter hybridization. These gene expression changes were further confirmed by a high throughout reverse Northern blot approach and in specific cases by targeted Northern analysis. blast searches show that heat shock protein (HSP) 90, MHC II-associated invariant chain (CD74), T-cell immunoglobulin mucin 1 (TIM1), as well as numerous novel expressed genes not found in the databases were up- or down-regulated by the host in response to this parasite. Our results and macroarray resources provide a foundation for molecular co-evolutionary studies of the Mycoplasma parasite and its recently colonized avian host.

The polymerase chain reaction has been a boon to the study of molecular ecology and population genetics of birds. But the nagging truth is that for many bird species, the number of polymerase chain reaction (PCR) primer pairs that one can pick off the shelf and expect to amplify their target loci with ease is frustratingly small. Now, studying DNA sequence variation in natural populations of birds just got a whole lot easier. This issue of Molecular Ecology reports a large-scale bioinformatics search for exonic sequences conserved between the chicken and zebra finch genomes and flanking polymorphic introns that has generated a staggering 242 PCR primer pairs that readily amplify their single-copy target loci in five avian species spanning approximately 100 million years of avian evolution (Backstrom et al. 2008). As proof of principle, these primers have also been used to survey the genomic landscape in over 110 kb of intronic sequence in the collared flycatcher, a model species in ecology and evolution. These resources pave the way for easy multilocus study of evolving populations and lineages of birds, and bring the goal of quickly turning nonmodel species in to ecological genomic models tantalizingly close.