Molecular parallelism in signaling function across different sexually selected ornaments in a warbler.

Extravagant ornaments are thought to signal male quality to females choosing mates, but the evidence linking ornament size to male quality is controversial, particularly in cases in which females prefer different ornaments in different populations. Here, we use whole-genome sequencing and transcriptomics to determine the genetic basis of ornament size in two populations of a widespread warbler, the common yellowthroat (Geothlypis trichas). Within a single subspecies, females in a Wisconsin population prefer males with larger black masks as mates, while females in a New York population prefer males with larger yellow bibs. Despite being produced by different pigments in different patches on the body, the size of the ornament preferred by females in each population was linked to numerous genes that function in many of the same core aspects of male quality (e.g., immunity and oxidative balance). These relationships confirm recent hypotheses linking the signaling function of ornaments to male quality. Furthermore, the parallelism in signaling function provides the flexibility for different types of ornaments to be used as signals of similar aspects of male quality. This could facilitate switches in female preference for different ornaments, a potentially important step in the early stages of divergence among populations.

Sexually selected differences in warbler plumage are related to a putative inversion on the Z chromosome.

Large structural variants in the genome, such as inversions, may play an important role in producing population structure and local adaptation to the environment through suppression of recombination. However, relatively few studies have linked inversions to phenotypic traits that are sexually selected and may play a role in reproductive isolation. Here, we found that geographic differences in the sexually selected plumage of a warbler, the common yellowthroat (Geothlypis trichas), are largely due to differences in the Z (sex) chromosome (males are ZZ), which contains at least one putative inversion spanning 40% (31/77 Mb) of its length. The inversions on the Z chromosome vary dramatically east and west of the Appalachian Mountains, which provides evidence of cryptic population structure within the range of the most widespread eastern subspecies (G. t. trichas). In an eastern (New York) and western (Wisconsin) population of this subspecies, female prefer different male ornaments; larger black facial masks are preferred in Wisconsin and larger yellow breasts are preferred in New York. The putative inversion also contains genes related to vision, which could influence mating preferences. Thus, structural variants on the Z chromosome are associated with geographic differences in male ornaments and female choice, which may provide a mechanism for maintaining different patterns of sexual selection in spite of gene flow between populations of the same subspecies.

High MHC diversity confers no advantage for phenotypic quality and reproductive performance in a wild bird.

Genes of the major histocompatibility complex (MHC) encode antigen-binding molecules and are an integral part of the acquired immune response of vertebrates. In general, high individual MHC diversity is expected to increase fitness by broadening the spectrum of pathogens recognized by the immune system, in accordance with the heterozygote advantage mechanism. On the other hand, the optimality hypothesis assumes that individuals with optimal (intermediate), rather than maximum, diversity of the MHC will achieve the highest fitness because of inherent costs associated with expressing diverse MHC alleles. Here, we tested for associations between individual diversity of the MHC class I and class II genes (binding antigens of intra- and extracellular pathogens respectively) and a range of fitness-related traits (condition, ornament expression and reproduction) in an urban population of the Eurasian coot Fulica atra. Contrary to our expectation, we found that high within-individual allelic diversity of MHC genes (both class I and II) was associated with poorer condition (lower blood haemoglobin concentrations), weaker expression of the putative ornament (smaller frontal shield), later onset of breeding and smaller clutches. An analysis of functional MHC allele clusters (supertypes) provided further support for negative associations of MHC diversity with phenotypic quality and reproductive performance, but most of these relationships could not be explained by the presence of specific maladaptive supertypes. Finally, we found little empirical support for the optimality hypothesis in the Eurasian coot. Our results suggest that the costs of high MHC diversity outweighed any benefits associated with broad MHC repertoire, which could be driven by depauperate pathogen diversity in an urban landscape. To the best of our knowledge, this is one of the first studies providing consistent evidence for negative associations of MHC diversity with a range of fitness-related traits in a natural avian population.

Polymorphism and varying selection within the MHC class I of four Anas species.

Ducks (Anatidae) are often vectors for the spread of pathogens because of their long-distance migrations. These migrations also expose ducks to a wide variety of pathogens in their wintering and breeding grounds, and, as a consequence, we might expect strong selection on their immune genes. Here, we studied exons 2 and 3 of the MHC class I in four species of Anas ducks (A. platyrhynchos, A. poecilorhyncha, A. formosa, and A. querquedula) using Illumina-sequencing. Both exons 2 and 3 code for the peptide-binding region of class I molecules; however, most previous studies of birds have only focused on exon 3. Here, we found stronger positive selection on exon 2 than exon 3, as indicated by more species with dN/dS > 1 and higher Wu-Kabat values. There was little evidence that divergence time influenced polymorphism, the numbers of identical alleles (partial α1 or α2 regions) among four Anas, or selection, suggesting that these widespread species might share similar levels of selection from pathogens. The high similarity of allele numbers, positively selected sites (PSS), conserved motifs, and variable protein sites (VPS) supported the persistence of trans-species polymorphism in Anas for at least 10 million years. Our study revealed exon 2 as a relatively unexplored source of variation in avian MHC class I, which should be considered in future studies.

Immunocompetent birds choose larger breeding colonies.

Optimal size of social groups may vary between individuals, depending on their phenotypic traits, such as dominance status, age or personality. Larger social groups often enhance transmission rates of pathogens and should be avoided by individuals with poor immune defences. In contrast, more immunocompetent individuals are expected to take advantage of larger group sizes (e.g. better protection, information transfer) with smaller extra costs from pathogen or parasite pressure. Here, we hypothesized that immunocompetence may be a key determinant of group size choice and tested this hypothesis in a colonial waterbird, the common tern Sterna hirundo. We used a unique experimental framework, where formation of breeding colonies of different sizes was induced under uniform environmental conditions. For this purpose, different-size patches of attractive nesting substrate (artificial floating rafts) were provided at a single site with limited availability of natural nesting habitat. Colony size was identified as the only significant predictor of both innate (natural antibody-mediated complement activation) and adaptive (immunoglobulin concentrations) immunological traits in the common terns, as more immunocompetent birds settled in larger experimental colonies. In contrast, we found no significant associations between colony size and genetic diversity of key pathogen-recognition receptors, toll-like receptors (TLRs) and the Major Histocompatibility Complex (MHC) or genome-wide heterozygosity. We conclude that settlement decisions may be flexible within individuals and, thus, are likely to be primarily determined by the current immunological status, rather than fixed immunogenetic traits. Our study sheds new light on the complex interface between immunity and sociality in animals.

Avian major histocompatibility complex copy number variation is associated with helminth richness.

Genes of the major histocompatibility complex (MHC) play a key role in the adaptive immunity of vertebrates, as they encode receptors responsible for antigen recognition. Evolutionary history of the MHC proceeded through numerous gene duplications, which increase the spectrum of pathogens recognized by individuals. Although pathogen-mediated selection is believed to be a primary driver of MHC expansion over evolutionary times, empirical evidence for this association is virtually lacking. Here, we used an extensive dataset on MHC class II copy number variation in non-passerine birds to test for an evolutionary correlation with helminth parasite richness. As expected, our phylogenetically-informed modelling revealed a positive association between MHC copy number and total helminth richness, even after controlling for a broad spectrum of ecological and life-history traits. Thus, total helminth richness appears to be the most important correlate of MHC copy number, supporting a leading role of pathogen-mediated selection in the evolution of MHC in birds. Our results provide some of the first, although correlative, evidence linking parasitism to interspecific variation in MHC copy number among birds.

A range-wide domino effect and resetting of the annual cycle in a migratory songbird.

Latitudinal differences in timing of breeding are well documented but how such differences carry over to influence timing of events in the annual cycle of migratory birds is not well understood. We examined geographical variation in timing of events throughout the year using light-level geolocator tracking data from 133 migratory tree swallows ( Tachycineta bicolor) originating from 12 North American breeding populations. A swallow's breeding latitude influenced timing of breeding, which then carried over to affect breeding ground departure. This resulted in subsequent effects on the arrival and departure schedules at autumn stopover locations and timing of arrival at non-breeding locations. This 'domino effect' between timing events was no longer apparent by the time individuals departed for spring migration. Our range-wide analysis demonstrates the lasting impact breeding latitude can have on migration schedules but also highlights how such timing relationships can reset when individuals reside at non-breeding sites for extended periods of time.

Extensive shared polymorphism at non-MHC immune genes in recently diverged North American prairie grouse.

Gene polymorphisms shared between recently diverged species are thought to be widespread and most commonly reflect introgression from hybridization or retention of ancestral polymorphism through incomplete lineage sorting. Shared genetic diversity resulting from incomplete lineage sorting is usually maintained for a relatively short period of time, but under strong balancing selection it may persist for millions of years beyond species divergence (balanced trans-species polymorphism), as in the case of the major histocompatibility complex (MHC) genes. However, balancing selection is much less likely to act on non-MHC immune genes. The aim of this study was to investigate the patterns of shared polymorphism and selection at non-MHC immune genes in five grouse species from Centrocercus and Tympanuchus genera. For this purpose, we genotyped five non-MHC immune genes that do not interact directly with pathogens, but are involved in signaling and regulate immune cell growth. In contrast to previous studies with MHC, we found no evidence for balancing selection or balanced trans-species polymorphism among the non-MHC immune genes. No haplotypes were shared between genera and in most cases more similar allelic variants sorted by genus. Between species within genera, however, we found extensive shared polymorphism, which was most likely attributable to introgression or incomplete lineage sorting following recent divergence and large ancestral effective population size (i.e., weak genetic drift). Our study suggests that North American prairie grouse may have attained relatively low degree of reciprocal monophyly at nuclear loci and reinforces the rarity of balancing selection in non-MHC immune genes.

Major histocompatibility complex variation and blood parasites in resident and migratory populations of the common yellowthroat.

Genes of the major histocompatibility complex (MHC) are a critical part of the adaptive immune response, and the most polymorphic genes in the vertebrate genome, especially in passerine birds. This diversity is thought to be influenced by exposure to pathogens which can vary in relation to numerous factors. Migratory behaviour may be a particularly important trait to consider because migratory birds are exposed to a greater number of different pathogens and parasites at both breeding (i.e. temperate) and overwintering (i.e. tropical and subtropical) areas, as well as at stopover sites during migration. Thus, migrants are predicted to have greater MHC diversity than residents. We compared MHC variation, at both class I and II, and levels of haemosporidian infection between one resident and two migratory populations of the common yellowthroat (Geothlypis trichas). We found that residents were less likely to be infected with haemosporidian parasites and had lower MHC diversity at class I; however, variation at MHC class II was greater in residents than migrants, contrary to our prediction. These patterns were not likely to be caused by differences in population demography as genomewide heterozygosity (based on 9225 single nucleotide polymorphisms) was high in all three populations and not correlated with MHC variation. Our different results for MHC class I and II suggest that studies of immune gene variation in relation to life history need to consider that there could be different selection pressures arising from intracellular (class I) and extracellular (class II) pathogens in different populations.

Experimental evidence that brighter males sire more extra-pair young in tree swallows.

Across taxa, extra-pair mating is widespread among socially monogamous species, but few studies have identified male ornamental traits associated with extra-pair mating success, and even fewer studies have experimentally manipulated male traits to determine whether they are related directly to paternity. As a consequence, there is little experimental evidence to support the widespread hypothesis that females choose more ornamented males as extra-pair mates. Here, we conducted an experimental study of the relationship between male plumage colour and fertilization success in tree swallows (Tachycineta bicolor), which have one of the highest levels of extra-pair mating in birds. In this study, we experimentally dulled the bright blue plumage on the back of males (with nontoxic ink markers) early in the breeding season prior to most mating. Compared with control males, dulled males sired fewer extra-pair young, and, as a result, fewer young overall. Among untreated males, brighter blue males also sired more extra-pair young, and in paired comparisons, extra-pair sires had brighter blue plumage than the within-pair male they cuckolded. These results, together with previous work on tree swallows, suggest that extra-pair mating behaviour is driven by benefits to both males and females.

Specific alleles at immune genes, rather than genome-wide heterozygosity, are related to immunity and survival in the critically endangered Attwater's prairie-chicken.

The negative effects of inbreeding on fitness are serious concerns for populations of endangered species. Reduced fitness has been associated with lower genome-wide heterozygosity and immune gene diversity in the wild; however, it is rare that both types of genetic measures are included in the same study. Thus, it is often unclear whether the variation in fitness is due to the general effects of inbreeding, immunity-related genes or both. Here, we tested whether genome-wide heterozygosity (20 990 SNPs) and diversity at nine immune genes were better predictors of two measures of fitness (immune response and survival) in the endangered Attwater's prairie-chicken (Tympanuchus cupido attwateri). We found that postrelease survival of captive-bred birds was related to alleles of the innate (Toll-like receptors, TLRs) and adaptive (major histocompatibility complex, MHC) immune systems, but not to genome-wide heterozygosity. Likewise, we found that the immune response at the time of release was related to TLR and MHC alleles, and not to genome-wide heterozygosity. Overall, this study demonstrates that immune genes may serve as important genetic markers when monitoring fitness in inbred populations and that in some populations specific functional genes may be better predictors of fitness than genome-wide heterozygosity.

Contrasting patterns of selection and drift between two categories of immune genes in prairie-chickens.

Immune-receptor genes of the adaptive immune system, such as the major histocompatibility complex (MHC), are involved in recognizing specific pathogens and are known to have high rates of adaptive evolution, presumably as a consequence of rapid co-evolution between hosts and pathogens. In contrast, many 'mediating' genes of the immune system do not interact directly with specific pathogens and are involved in signalling (e.g. cytokines) or controlling immune cell growth. As a consequence, we might expect stronger selection at immune-receptor than mediating genes, but these two types of genes have not been compared directly in wild populations. Here, we tested the hypothesis that selection differs between MHC (class I and II) and mediating genes by comparing levels of population differentiation across the range of greater prairie-chickens (Tympanuchus cupido). As predicted, there was stronger population differentiation and isolation by distance at immune receptor (MHC) than at either mediating genes or neutral microsatellites, suggesting a stronger role of local adaptation at the MHC. In contrast, mediating genes displayed weaker differentiation between populations than neutral microsatellites, consistent with selection favouring similar alleles across populations for mediating genes. In addition to selection, drift also had a stronger effect on immune receptor (MHC) than mediating genes as indicated by the stronger decline of MHC variation in relation to population size. This is the first study in the wild to show that the effects of selection and drift on immune genes vary across populations depending on their functional role.

Different ornaments signal male health and MHC variation in two populations of a warbler.

Male traits that signal health and vigour are used by females to choose better quality mates, but in some cases the male trait selected by females differs among populations. Multiple male traits can be maintained through female mate choice if both traits are equally honest indicators of male quality, but tests of this prediction are rare. By choosing males based on such traits, females could gain direct benefits from males (assistance with parental care), but when females choose extra-pair mates based on these traits, females gain only male sperm, and potentially indirect genetic benefits for their offspring. In common yellowthroats (Geothylpis trichas), female choice of extra-pair mates targets two different plumage ornaments: the black mask in a Wisconsin population and the yellow bib in a New York population. Previously, we found that the black mask in Wisconsin is related to greater major histocompatibility complex (MHC) class II variation, which in turn signals better survival and disease resistance. In this study, we examined the signalling function of the yellow bib in New York to test whether it signals the same aspects of male quality as the black mask in Wisconsin. As predicted, we found that the yellow bib in New York is most closely associated with MHC variation, which also signals survival and resistance to blood parasites. Thus, the ornament preferred by females differs between the two populations, but the different ornaments signal similar aspects of male health and genetic quality, specifically information regarding MHC variation and potential indirect genetic benefits to females.

Changes in breeding phenology and population size of birds.

Although the phenology of numerous organisms has advanced significantly in response to recent climate change, the life-history and population consequences of earlier reproduction remain poorly understood. We analysed extensive data on temporal change in laying date and clutch size of birds from Europe and North America to test whether these changes were related to recent trends in population size. Across studies, laying date advanced significantly, while clutch size did not change. However, within populations, changes in laying date and clutch size were positively correlated, implying that species which advanced their laying date the most were also those that increased their clutch size the most. Greater advances in laying date were associated with species that had multiple broods per season, lived in nonagricultural habitats and were herbivorous or predatory. The duration of the breeding season increased for multibrooded species and decreased for single-brooded species. Changes in laying date and clutch size were not related to changes in population size (for resident or migratory species). This suggests that, across a wide variety of species, mismatches in the timing of egg laying or numbers of offspring have had relatively little influence on population size compared with other aspects of phenology and life history.

Sexual selection accelerates signal evolution during speciation in birds.

Sexual selection is proposed to be an important driver of diversification in animal systems, yet previous tests of this hypothesis have produced mixed results and the mechanisms involved remain unclear. Here, we use a novel phylogenetic approach to assess the influence of sexual selection on patterns of evolutionary change during 84 recent speciation events across 23 passerine bird families. We show that elevated levels of sexual selection are associated with more rapid phenotypic divergence between related lineages, and that this effect is restricted to male plumage traits proposed to function in mate choice and species recognition. Conversely, we found no evidence that sexual selection promoted divergence in female plumage traits, or in male traits related to foraging and locomotion. These results provide strong evidence that female choice and male-male competition are dominant mechanisms driving divergence during speciation in birds, potentially linking sexual selection to the accelerated evolution of pre-mating reproductive isolation.

Extensive regional variation in the phenology of insects and their response to temperature across North America.

Climate change models often assume similar responses to temperatures across the range of a species, but local adaptation or phenotypic plasticity can lead plants and animals to respond differently to temperature in different parts of their range. To date, there have been few tests of this assumption at the scale of continents, so it is unclear if this is a large-scale problem. Here, we examined the assumption that insect taxa show similar responses to temperature at 96 sites in grassy habitats across North America. We sampled insects with Malaise traps during 2019-2021 (N = 1041 samples) and examined the biomass of insects in relation to temperature and time of season. Our samples mostly contained Diptera (33%), Lepidoptera (19%), Hymenoptera (18%), and Coleoptera (10%). We found strong regional differences in the phenology of insects and their response to temperature, even within the same taxonomic group, habitat type, and time of season. For example, the biomass of nematoceran flies increased across the season in the central part of the continent, but it only showed a small increase in the Northeast and a seasonal decline in the Southeast and West. At a smaller scale, insect biomass at different traps operating on the same days was correlated up to ~75 km apart. Large-scale geographic and phenological variation in insect biomass and abundance has not been studied well, and it is a major source of controversy in previous analyses of insect declines that have aggregated studies from different locations and time periods. Our study illustrates that large-scale predictions about changes in insect populations, and their causes, will need to incorporate regional and taxonomic differences in the response to temperature.

Social and extra-pair mating in relation to major histocompatibility complex variation in common yellowthroats.

Females are thought to gain better-quality genes for their offspring by mating with particular males. Genes of the major histocompatibility complex (MHC) play a critical role in adaptive immunity, and several studies have examined female mate choice in relation to MHC variation. In common yellowthroats, females prefer males that have larger black facial masks, an ornament associated with MHC variation, immune function and condition. Here we also tested whether mating patterns are directly correlated with MHC diversity or similarity. Using pyrosequencing, we found that the presence of extra-pair young in the brood was not related to male MHC diversity or similarity between the female and her within-pair mate. Furthermore, extra-pair sires did not differ in overall diversity from males they cuckolded, or in their similarity to the female. MHC diversity is extremely high in this species, and it may limit the ability of females to assess MHC variation in males. Thus, mating may be based on ornaments, such as mask size, which are better indicators of overall male health and genetic quality.

Drift and selection influence geographic variation at immune loci of prairie-chickens.

Previous studies of immunity in wild populations have focused primarily on genes of the major histocompatibility complex (MHC); however, studies of model species have identified additional immune-related genes that also affect fitness. In this study, we sequenced five non-MHC immune genes in six greater prairie-chicken (Tympanuchus cupido) populations that have experienced varying degrees of genetic drift as a consequence of population bottlenecks and fragmentation. We compared patterns of geographic variation at the immune genes with six neutral microsatellite markers to investigate the relative effects of selection and genetic drift. Global F(ST) outlier tests identified positive selection on just one of five immune genes (IAP-1) in one population. In contrast, at other immune genes, standardized G'(ST) values were lower than those at microsatellites for a majority of pairwise population comparisons, consistent with balancing selection or with species-wide positive or purifying selection resulting in similar haplotype frequencies across populations. The effects of genetic drift were also evident as summary statistics (e.g., Tajima's D) did not differ from neutrality for the majority of cases, and immune gene diversity (number of haplotypes per gene) was correlated positively with population size. In summary, we found that both genetic drift and selection shaped variation at the five immune genes, and the strength and type of selection varied among genes. Our results caution that neutral forces, such as drift, can make it difficult to detect current selection on genes.

A test of the mismatch hypothesis: How is timing of reproduction related to food abundance in an aerial insectivore?

In seasonal environments, vertebrates are generally thought to time their reproduction so offspring are raised during the peak of food abundance. The mismatch hypothesis predicts that reproductive success is maximized when animals synchronize their reproduction with the food supply. Understanding the mechanisms influencing the timing of reproduction has taken on new urgency as climate change is altering environmental conditions during reproduction, and there is concern that species will not be able to synchronize their reproduction with changing food supplies. Using data from five sites over 24 years (37 site-years), we tested the assumptions of the mismatch hypothesis in the Tree Swallow (Tachycineta bicolor), a widespread aerial insectivore, whose timing of egg-laying has shifted earlier by nine days since the 1950s. Contrary to the mismatch hypothesis, the start of egg-laying was strongly related to food abundance (flying insect biomass) during the laying period and not to timing of the seasonal peak in food supply. In fact, food abundance generally continued to increase throughout the breeding season, and there was no evidence of selection based on the mistiming of laying with the seasonal peak of food abundance. In contrast, there was selection for laying earlier, because birds that lay earlier generally have larger clutches and fledge more young. Overall, initial reproductive decisions in this insectivore appear to be based on the food supply during egg formation and not the nestling period. Thus, the mismatch hypothesis may not apply in environments with relatively constant or abundant food throughout the breeding season. Although climate change is often associated with earlier reproduction, our results caution that it is not necessarily driven by selection for synchronized reproduction.

Long-Read Genome Assemblies Reveal Extraordinary Variation in the Number and Structure of MHC Loci in Birds.

Our knowledge of the Major Histocompatibility Complex (MHC) in birds is limited because it often consists of numerous duplicated genes within individuals that are difficult to assemble with short read sequencing technology. Long-read sequencing provides an opportunity to overcome this limitation because it allows the assembly of long regions with repetitive elements. In this study, we used genomes based on long-read sequencing to predict the number and location of MHC loci in a broad range of bird taxa. From the long-read-based genomes of 34 species, we found that there was extremely large variation in the number of MHC loci between species. Overall, there were greater numbers of both class I and II loci in passerines than nonpasserines. The highest numbers of loci (up to 193 class II loci) were found in manakins (Pipridae), which had previously not been studied at the MHC. Our results provide the first direct evidence from passerine genomes of this high level of duplication. We also found different duplication patterns between species. In some species, both MHC class I and II genes were duplicated together, whereas in most species they were duplicated independently. Our study shows that the analysis of long-read-based genomes can dramatically improve our knowledge of MHC structure, although further improvements in chromosome level assembly are needed to understand the evolutionary mechanisms producing the extraordinary interspecific variation in the architecture of the MHC region.