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.

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.

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.

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.

The strength of selection is consistent across both domains of the MHC class I peptide-binding groove in birds.

BACKGROUND: The Major Histocompatibility Complex (MHC) codes for the key vertebrate immune receptors responsible for pathogen recognition. Foreign antigens are recognized via their compatibility to hyper-variable region of the peptide-binding groove (PBR), which consists of two separate protein domains. Specifically, the PBR of the MHC class I receptors, which recognize intra-cellular pathogens, has two α domains encoded by exon 2 (α1) and exon 3 (α2) of the same gene. Most research on avian MHC class I polymorphism has traditionally focused exclusively on exon 3 and comparisons of selection between the two domains have been hampered by the scarcity of molecular data for exon 2. Thus, it is not clear whether the two domains vary in their specificity towards different antigens and whether they are subject to different selective pressure. RESULTS: Here, we took advantage of rapidly accumulating genomic resources to test for the differences in selection patterns between both MHC class I domains of the peptide-binding groove in birds. For this purpose, we compiled a dataset of MHC class I exon 2 and 3 sequences for 120 avian species from 46 families. Our phylogenetically-robust approach provided strong evidence for highly consistent levels of selection on the α1 and α2 domains. There were strong correlations in all selection measures (number of positively/negatively selected residues and dN/dS ratios) between both PBR exons. Similar positive associations were found for the level of amino acid polymorphism across the two domains. CONCLUSIONS: We conclude that the strength of selection and the level of polymorphism are highly consistent between both peptide-binding domains (α1 and α2) of the avian MHC class I.

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.

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.

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.

Evaluation of a Chicken 600K SNP genotyping array in non-model species of grouse.

The use of single nucleotide polymorphism (SNP) arrays to generate large SNP datasets for comparison purposes have recently become an attractive alternative to other genotyping methods. Although most SNP arrays were originally developed for domestic organisms, they can be effectively applied to wild relatives to obtain large panels of SNPs. In this study, we tested the cross-species application of the Affymetrix 600K Chicken SNP array in five species of North American prairie grouse (Centrocercus and Tympanuchus genera). Two individuals were genotyped per species for a total of ten samples. A high proportion (91%) of the total 580 961 SNPs were genotyped in at least one individual (73-76% SNPs genotyped per species). Principal component analysis with autosomal SNPs separated the two genera, but failed to clearly distinguish species within genera. Gene ontology analysis identified a set of genes related to morphogenesis and development (including genes involved in feather development), which may be primarily responsible for large phenotypic differences between Centrocercus and Tympanuchus grouse. Our study provided evidence for successful cross-species application of the chicken SNP array in grouse which diverged ca. 37 mya from the chicken lineage. As far as we are aware, this is the first reported application of a SNP array in non-passerine birds, and it demonstrates the feasibility of using commercial SNP arrays in research on non-model bird species.

Evolution of Copy Number at the MHC Varies across the Avian Tree of Life.

The evolution of the major histocompatibility complex (MHC) is shaped by frequent gene duplications and deletions, which generate extensive variation in the number of loci (gene copies) between different taxa. Here, we collected estimates of copy number at the MHC for over 250 bird species from 68 families. We found contrasting patterns of copy number evolution between MHC class I and class IIB, which encode receptors for intra- and extracellular pathogens, respectively. Across the avian evolutionary tree, there was evidence of accelerated evolution and stabilizing selection acting on copy number at class I, while copy number at class IIB was primarily influenced by fluctuating selection and drift. Reconstruction of MHC copy number variation showed ancestrally low numbers of MHC loci in nonpasserines and evolution toward larger numbers of loci in passerines. Different passerine lineages had the highest duplication rates for MHC class I (Sylvioidea) and class IIB (Muscicapoidea and Passeroidea). We also found support for the correlated evolution of MHC copy number and life-history traits such as lifespan and migratory behavior. These results suggest that MHC copy number evolution in birds has been driven by life histories and differences in exposure to intra- and extracellular pathogens.

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.

A global analysis of selection at the avian MHC.

Recent advancements in sequencing technology have resulted in rapid progress in the study of the major histocompatibility complex (MHC) in non-model avian species. Here, we analyze a global dataset of avian MHC class I and class II sequences (ca. 11,000 sequences from over 250 species) to gain insight into the processes that govern macroevolution of MHC genes in birds. Analysis of substitution rates revealed striking differences in the patterns of diversifying selection between passerine and non-passerine birds. Non-passerines showed stronger selection at MHC class II, which is primarily involved in recognition of extracellular pathogens, while passerines showed stronger selection at MHC class I, which is involved in recognition of intracellular pathogens. Positions of positively selected amino-acid residues showed marked discrepancies with peptide-binding residues (PBRs) of human MHC molecules, suggesting that using a human classification of PBRs to assess selection patterns at the avian MHC may be unjustified. Finally, our analysis provided evidence that indel mutations can make a substantial contribution to adaptive variation at the avian MHC.

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.

Coloniality and migration are related to selection on MHC genes in birds.

The major histocompatibility complex (MHC) plays a key role in pathogen recognition as a part of the vertebrate adaptive immune system. The great diversity of MHC genes in natural populations is maintained by different forms of balancing selection and its strength should correlate with the diversity of pathogens to which a population is exposed and the rate of exposure. Despite this prediction, little is known about how life-history characteristics affect selection at the MHC. Here, we examined whether the strength of balancing selection on MHC class II genes in birds (as measured with nonsynonymous nucleotide substitutions, dN) was related to their social or migratory behavior, two life-history characteristics correlated with pathogen exposure. Our comparative analysis indicated that the rate of nonsynonymous substitutions was higher in colonial and migratory species than solitary and resident species, suggesting that the strength of balancing selection increases with coloniality and migratory status. These patterns could be attributed to: (1) elevated transmission rates of pathogens in species that breed in dense aggregations, or (2) exposure to a more diverse fauna of pathogens and parasites in migratory species. Our study suggests that differences in social structure and basic ecological traits influence MHC diversity in natural vertebrate populations.

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.

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.

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.

Natural and sexual selection act on different axes of variation in avian plumage color.

The bright colors of birds are often attributed to sexual selection on males, but in many species both sexes are colorful and it has been long debated whether sexual selection can also explain this variation. We show that most evolutionary transitions in color have been toward similar plumage in both sexes, and the color of both sexes (for example, bright or dull) was associated with indices of natural selection (for example, habitat type), whereas sexual differences in color were primarily associated with indices of sexual selection on males (for example, polygyny and large testes size). Debate about the evolution of bird coloration can be resolved by recognizing that both natural and sexual selection have been influential, but they have generally acted on two different axes: sexual selection on an axis of sexual differences and natural selection on both sexes for the type of color (for example, bright or dull).

Assessing costs of carrying geolocators using feather corticosterone in two species of aerial insectivore.

Despite benefits of using light-sensitive geolocators to track animal movements and describe patterns of migratory connectivity, concerns have been raised about negative effects of these devices, particularly in small species of aerial insectivore. Geolocators may act as handicaps that increase energetic expenditure, which could explain reported effects of geolocators on survival. We tested this 'Energetic Expenditure Hypothesis' in 12 populations of tree swallows (Tachycineta bicolor) and barn swallows (Hirundo rustica) from North America and Europe, using measurements of corticosterone from feathers (CORTf) grown after deployment of geolocators as a measure of physiology relevant to energetics. Contrary to predictions, neither among- (both species) nor within-individual (tree swallows only) levels of CORTf differed with respect to instrumentation. Thus, to the extent that CORTf reflects energetic expenditure, geolocators apparently were not a strong handicap for birds that returned post-deployment. While this physiological evidence suggests that information about migration obtained from returning geolocator-equipped swallows is unbiased with regard to levels of stress, we cannot discount the possibility that corticosterone played a role in reported effects of geolocators on survival in birds, and suggest that future studies relate corticosterone to antecedent factors, such as reproductive history, and to downstream fitness costs.