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Oxidative metabolism is a key component of organismal physiology and it is primarily determined by aerobic capacity, which depends on the capacity of blood to carry oxygen. However, experimental manipulations of blood oxygen-carrying capacity are rarely implemented to test ecophysiological hypotheses in vertebrate populations. Here, we combined an experimental manipulation of blood oxygen-carrying capacity with GPS tracking to test whether suboptimal (reduced) haematological performance affects foraging behaviour in a colonial waterbird, the black-headed gull, Chroicocephalus ridibundus. First, a validation of phenylhydrazine (PHZ) treatment in gulls revealed a 9–18% reduction in haematocrit and blood haemoglobin concentration (via oxidative denaturation and haemolysis of erythrocytes). Then, GPS tracking of experimental (PHZ-treated) and control (saline-treated) gulls during the incubation period provided no support for reduced or suspended engagement in energetically costly activities (long-distance foraging trips) by experimental birds. Instead, we found evidence for fine-scale alterations in foraging behaviour of PHZ-treated individuals, which resulted in fewer foraging trips per unit time, but trips that were longer in duration and distance compared with those of control birds. This suggests reduced foraging performance of experimental birds (e.g. lower capacity to find and collect food during trips) or evasion of social competition, although no differences in the total investment in foraging may also suggest compensatory physiological responses to haemolytic anaemia. Our study contributes to a better understanding of the physio-ecological nexus in non-diving colonial avian species. Whether behavioural effects of reduced aerobic capacity have any implications for gull condition and reproductive performance should be the subject of further investigation.

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.

The hypervariable major histocompatibility complex (MHC) is a crucial component of vertebrate adaptive immunity, but large-scale studies on MHC macroevolution in nonmodel vertebrates have long been constrained by methodological limitations. Here, we used rapidly accumulating genomic data to reconstruct macroevolution of the MHC region in amphibians. We retrieved contigs containing the MHC region from genome assemblies of 32 amphibian species and examined major structural rearrangements, duplication patterns, and gene structure across the amphibian phylogeny. Based on the few available caecilian and urodele genomes, we showed that the structure of ancestral MHC region in amphibians was probably relatively simple and compact, with a close physical linkage between MHC-I and MHC-II regions. This ancestral MHC architecture was generally conserved in anurans, although the evolution of class I subregion proceeded toward more extensive duplication and rapid expansion of gene copy number, providing evidence for dynamic evolutionary trajectories. Although, in anurans, we recorded tandems of duplicated MHC-I genes outside the core subregion, our phylogenetic analyses of MHC-I sequences provided little support for an expansion of nonclassical MHC-Ib genes across amphibian families. Finally, we found that intronic regions of amphibian classical MHC genes were much longer when compared with other tetrapod lineages (birds and mammals), which could partly be driven by the expansion of genome size. Our study reveals novel evolutionary patterns of the MHC region in amphibians and provides a comprehensive framework for further studies on the MHC macroevolution across vertebrates.

We investigate the role of black-headed gulls (Chroicocephalus ridibundus), an omnivorous species that is among the most likely wild bird candidates for transmission of zoonotic agents, as a potential reservoir ofCampylobacterspp. Colonies with different anthropogenic pressures were studied to examine differences in exposure to sources ofCampylobacterbetween rural and urban birds. We recordedCampylobacterspp. in 4.87% of adult black-headed gulls and 2.22% of their chicks after analysing 1036 cloacal swabs collected over two breeding seasons in three colonies in northern Poland.Campylobacter jejuniwas found most frequently (85.72%), andCampylobacter lariandCampylobacter coliwere much scarcer. Prevalence ofCampylobacterdid not differ significantly between black-headed gulls breeding in urban (4.27%) and rural (3.80%) habitats. Almost all isolates from chicks and adults were susceptible to azithromycin (97.62%) and erythromycin (95.24%), but fewer to tetracycline (50.00%) and ciprofloxacin (47.62%).Campylobacterprevalence was unrelated to the date of sampling. Our study indicates that black-headed gulls are carriers of resistant to antibioticsCampylobacterand they can contaminate natural waterbodies with their faeces, which poses a threat to human and farm animal health.

Landscape conversions induced by human activities can affect dispersal patterns of various bird species and, as a result, affect genetic structure of their populations. Genetic differentiation of bird populations may be enhanced by habitat variation, especially in urban-non-urban systems. The majority of population genetic studies focus on single species, which inflicts limitations for direct comparisons of genetic responses of avian populations to urbanization. Here, we used a set of microsatellite markers to examine genetic diversity, gene flow and population structure in two common parid species, great tits Parus major and blue tits Cyanistes caeruleus occupying three sites in habitats with contrasting urbanization level in central Poland. We found low but significant divergence of urban park population with both suburban and non-urban forest great tit populations, while no differentiation was found between suburban forest and non-urban forest populations. In contrast, no evidence for genetic differentiation was found between blue tit populations from the urban park, suburban forest and non-urban forest sites. We conclude that great tits and blue tits respond to urbanization-related changes in a different way, which may be a result of different rates of migration and/or dispersal, likely higher in blue tits. Some impact may be also induced by interspecific competition. We suggest that changing the focus of urban genetic research from single to multiple species may provide novel insights into how natural populations respond to the processes of urbanization.

Major histocompatibility complex (MHC) genes code for key proteins of the adaptive immune system, which present antigens from intra-cellular (MHC class I) and extra-cellular (MHC class II) pathogens. Because of their unprecedented diversity, MHC genes have long been an object of scientific interest, but due to methodological difficulties in genotyping of duplicated loci, our knowledge on the evolution of the MHC across different vertebrate lineages is still limited. Here, we compared the evolution of MHC class I and class II genes in three sister clades of common passerine birds, finches (Fringillinae and Carduelinae) and buntings (Emberizidae) using a uniform methodological (genotyping and data processing) approach and uniform sample sizes. Our analyses revealed contrasting evolutionary trajectories of the two MHC classes. We found a stronger signature of pervasive positive selection and higher allele diversity (allele numbers) at the MHC class I than class II. In contrast, MHC class II genes showed greater allele divergence (in terms of nucleotide diversity) and a much stronger recombination (gene conversion) signal. Gene copy numbers at both MHC class I and class II evolved via fluctuating selection and drift (Brownian Motion evolution), but the evolutionary rate was higher at class I. Our study constitutes one of few existing examples, where evolution of MHC class I and class II genes was directly compared using a multi-species approach. We recommend that re-focusing MHC research from single-species and single-class approaches towards multi-species analyses of both MHC classes can substantially increase our understanding MHC evolution in a broad phylogenetic context.

Background Nesting in large aggregations provides several important advantages for colonially breeding birds. However, it also imposes certain costs, associated with facilitated pathogen transmission and social stress. The cost-benefit ratio is not similar for all the birds in a colony and it might be mediated by nest density. To investigate the influence of nest density on cell-mediated immune function and on physiological condition of nestlings, we arranged a cross-fostering experiment in three breeding colonies of black-headed gulls Chroicocephalus ridibundus. First, we exchanged eggs between plots of high and low nest density. Afterwards, we performed phytohaemagglutinin (PHA) skin test and we measured blood haemoglobin concentration in nearly 350 nestlings from experimental (exchanged) and control (non-exchanged) groups. Results We found that PHA response was lowest in high nest density control group, indicating that depressed immune function of offspring, likely caused by social stress, can be considered as a cost of colonial breeding. Contrastingly, body condition of nestlings was the poorest in low density control group. Conclusion Nestlings hatched and raised in high nest density plots did not have higher blood haemoglobin concentration in comparison to other study groups. Furthermore, they were affected with depressed cell mediated immune function, which is possibly driven by combined maternal (corticosteroid hormones deposited in yolk) and environmental (elevated social stress) effects. These results indicate that breeders from high nest densities do not benefit by rising offspring in better quality, in terms of immune function and body condition, although, in the light of previous studies, high nest densities are occupied by birds of higher individual quality, than low density areas. Our study provides a novel insight into the mechanisms of density-dependence that govern fitness of colonially nesting birds.

Haemoparasites represent a diverse group of vector-borne parasites that infect a wide range of vertebrate hosts. In birds, haemoparasite infection rates may be associated with various ecological and life history traits, including habitat choice, colony size and migration distance. Here, we molecularly assessed the prevalence of 3 main haemoparasite genera (Plasmodium, Haemoproteus and Leucocytozoon) in 2 bird species with different habitat preferences and migratory behaviour: black-headed gulls (Chroicocephalus ridibundus) and common terns (Sterna hirundo). We found that gulls showed a much higher prevalence and diversity of Plasmodium or Haemoproteus (ca. 60% of individuals infected) than terns (zero prevalence). The prevalence of Leucocytozoon was low in both species (Haemoproteus than Plasmodium. Our results suggest that the preference for coastal areas and less vegetated habitats in terns may reduce haemoparasite infection rates compared to other larids, regardless of their migratory distance, emphasizing the role of ecological niches in parasite exposure.

Balancing selection is a classic mechanism for maintaining variability in immune genes involved in host–pathogen interactions. However, it remains unclear how widespread the mechanism is across immune genes other than the major histocompatibility complex (MHC). Although occasional reports suggest that balancing selection (heterozygote advantage, negative frequency-dependent selection, and fluctuating selection) may act on other immune genes, the current understanding of the phenomenon in non-MHC immune genes is far from solid. In this review, we focus on Toll-like receptors (TLRs), innate immune genes directly involved in pathogen recognition and immune response activation, as there is a growing body of research testing the assumptions of balancing selection in these genes. After reviewing infection- and fitness-based evidence, along with evidence based on population allelic frequencies and heterozygosity levels, we conclude that balancing selection maintains variation in TLRs, though it tends to occur under specific conditions in certain evolutionary lineages rather than being universal and ubiquitous. Our review also identifies key gaps in current knowledge and proposes promising areas for future research. Improving our understanding of host–pathogen interactions and balancing selection in innate immune genes are increasingly important, particularly regarding threats from emerging zoonotic diseases.

Nonspecific innate immune response is activated by toll-like receptors (TLRs), which recognize conserved molecular motifs characteristic for a broad spectrum of pathogens. In this study, we examined nucleotide substitution patterns and allelic diversity at five TLR genes in a wild nonpasserine bird, the black-headed gullChroicocephalus ridibundus. We hypothesized that balancing selection can maintain high allelic diversity of TLR genes in the black-headed gull because of its ecological characteristics, coloniality, and migratoriness, which are associated with increased exposure and transmission of pathogens. Although we found moderately high levels of sequence polymorphism (8–49 haplotypes retrieved per locus within a sample of 60 individuals), most of these haplotypes were recorded at low frequencies within our study population. At the same time, we found no convincing evidence for the role of balancing selection in the maintenance of this variation (Tajima’s D 1) were recorded only at two loci (TLR5 and TLR7). This pattern is consistent with relaxation of selective constraints, where most mutations are slightly deleterious and usually removed by purifying selection. No differences in the diversity and nucleotide substitution rates were found between endosomal loci responsible for viral RNA sensing and loci responsible for the recognition of extracellular pathogens. Our study provides the first information on evolutionary mechanisms shaping polymorphism of TLRs in a species from Lari suborder (gulls and allies) and suggests that TLR genes may be poorly responsive to ecological and life-history characteristics of hosts.

Genes of the major histocompatibility complex (MHC) code for immune proteins that are crucial for pathogen recognition in vertebrates. MHC research in nonmodel taxa has long been hampered by its genomic complexity that makes the locus-specific genotyping challenging. The recent progress in sequencing and genotyping methodologies allows an extensive phylogenetic coverage in studies of MHC evolution. Here, we analyzed the peptide-binding region of MHC class I (MHC-I) in 30 species of salamanders from six families representative of Urodela phylogeny. This extensive dataset revealed an extreme diversity of MHC-I in salamanders, both in terms of sequence diversity (about 3000 variants) and architecture (2-22 gene copies per species). The signal of positive selection was moderate and consistent between both peptide-binding domains, but varied greatly between genera. Positions of positively selected sites mostly coincided with human peptide-binding sites, suggesting similar structural properties of MHC-I molecules across distant vertebrate lineages. Finally, we provided evidence for the common intraexonic recombination at MHC-I and for the role of life history traits in the processes of MHC-I expansion/contraction. Our study revealed novel evolutionary trajectories of amphibian MHC and it contributes to the understanding of the mechanisms that generated extraordinary MHC diversity throughout vertebrate evolution.

Thriving under high population density is considered a major feature of urban exploiter species. Nevertheless, population density appears to be a surprisingly overlooked factor in urban ecology studies. High population numbers observed in urban species might promote pathogen transmission and negatively affect health or condition, thus requiring investments in immunocompetence. The feral pigeon Columba livia domestica is an example of a successful city-dweller, found in great abundance in large cities across the globe. We investigated the effects of population density on induced immune response (phytohaemagglutinin skin test) and body condition (blood haemoglobin concentration and size-corrected body mass) in 120 feral pigeons, captured along population density gradient in Łódź (central Poland). We found that stronger immune response was associated with higher population density, but was not related to physiological condition and physiological stress (heterophil/lymphocyte ratio). Moreover, condition indices were not associated with population density. However, since pigeon population density was highly correlated with the level of habitat urbanization, we cannot exclude that any density-dependent effects may be mediated by habitat variation. Our results indicate that urban environment, via population density, might exert different selective pressures on immunocompetence and body condition in this successful urban exploiter.

The size and shape of red blood cells (erythrocytes) is determined by key life history strategies in vertebrates. They have a fundamental role to deliver oxygen to tissues, and their ability to do so is shaped by the tissue's need and their shape. Despite considerable interest in how other components of blood are shaped by ecology and life history, few studies have considered erythrocytes themselves. We tested how erythrocyte size and shape varied in relation to energetically demanding activities using a dataset of 631 bird species. We found that in general, birds undergoing greater activities such as long distance migration had smaller and more elongated cells, while those with greater male-male competition had smaller and rounder cells. Smaller, more elongated erythrocytes allow more rapid oxygenation/deoxygenation and support greater aerobic activity. The rounder erythrocytes found in species with strong male–male competition may stem from younger erythrocytes deriving from androgen-induced erythropoiesis rates. Finally, diving species of bird had larger erythrocytes, indicating that erythrocytes are acting as a vital oxygen store. In summary, erythrocyte size and shape in birds are driven by the need to deliver oxygen during energetically costly activities.

Toll-like receptors (TLRs) are a crucial component of vertebrate innate immune response. Despite their importance, associations of TLR diversity with fitness-related traits have rarely been examined in wild animal populations. Here, we tested for associations of TLR polymorphism with physiological condition in a colonial waterbird, the Black-headed Gull (Chroicocephalus ridibundus). Physiological condition and polymorphism at 4 TLR loci were assessed in 60 gulls from a breeding colony in northern Poland. We found that blood hemoglobin and plasma albumin concentrations were positively associated with TLR diversity across all genotyped loci. Plasma concentrations of albumin and triglycerides were also associated with the presence of specific TLR variants and locus-specific diversity. All significant associations between physiological condition and TLRs were primarily apparent at the level of nucleotide, rather than amino acid allelic variants. Although the exact molecular mechanisms responsible for these associations require further investigation, our study provides strong correlational support for links between TLR diversity and physiological condition in a wild avian population, and it adds to the growing, but still modest, body of evidence for the fitness-related consequences of individual TLR repertoire in wild birds. LAY SUMMARY Toll-like receptors (TLRs) initiate innate immune response by recognition of conserved pathogen-associated molecular patterns. We tested for associations between TLR diversity and fitness-related traits (physiological condition) in the Black-headed Gull. Physiological condition was associated with both TLR diversity and presence of specific TLR variants, but these associations were mostly prevalent at the nucleotide, rather than amino acid, level. Our results raise the question on the adaptive significance of synonymous variation at TLR genes.

Weather conditions are recognized as one of key determinants of animal reproductive performance; however, the effect of weather on breeding success can be modulated by different features of breeding habitat. Constantly expanding urban areas cause significant changes in land cover and environmental conditions, but whether and how urban landscape mitigates weather impact on animal fitness remains little explored. The aim of this study was to investigate the association between weather parameters and reproductive performance in a reed-nesting waterbird species, the Eurasian coot Fulica atra. For this purpose, we performed a long-term monitoring of an urban coot population from central Poland, collecting data for over 400 breeding events. The results indicated that temperature may have contrasting effects on coot reproductive output at different stages of chick-rearing period (positive at early chick-rearing and negative at late chick-rearing). Also, contrary to our expectation, we found a positive relationship between mean daily precipitation in early chick-rearing period and reproductive output in our study population. Our study constitutes one of few examples showing how weather may affect fitness in urban wildlife and provides evidence for high complexity of associations between weather conditions and animal reproductive performance.

In species with sexual dimorphism, raising female or male offspring may be associated with different costs and benefits, resulting in a skewed nestling sex ratio. We examined the influence of: weather conditions, hatching date and brood size on nestling sex ratio in the black stork Ciconia nigra. We used molecular methods to determine the sex of 284 nestlings. Samples were collected during a 12-years study in central Poland. The overall nestling sex ratio was skewed towards females (61%), which are smaller, and presumably easier to raise than males. Delayed hatching date significantly increased the proportion of female nestlings. Warmer temperatures in the pre-breeding season were correlated with lower proportions of males. This is probably mediated by the influence of weather on water levels in black stork foraging sites. The second most important weather trait was total rainfall in May, the month in which the majority of nestlings hatch. Total May rainfall was negatively correlated with the percentage of male offspring. We used blood haemoglobin concentration as an indicator of body condition in a subsample of 122 nestlings. The males from the study population had lower blood haemoglobin concentrations, indicating their poorer body condition and supporting the hypothesis that they are the more vulnerable sex. We also observed that blood haemoglobin concentration of nestlings is lower in late broods. Deteriorating body condition of late offspring can explain the observed increase in female nestling proportions in delayed broods.

There is increasing evidence that melanin-based plumage ornaments play a role in the sexual selection of birds, although there seems to be little consensus on the mechanisms underlying the signalling function of melanin-based plumage. The aim of this study was to assess the potential of a melanin-based plumage ornament (brown hood) to reflect components of individual quality (condition and physiological stress) in a common larid species, the Black-headed Gull Chroicocephalus ridibundus. For this purpose, we measured the size of the hood in over 500 Black-headed Gulls captured in several breeding colonies distributed across Poland. We found that hood size correlated positively with blood haemoglobin concentration, although we found no evidence for a relationship with blood glucose concentration or body mass. There was also a negative relationship between hood size and physiological stress, as assessed by leucocyte profiles (heterophil/lymphocyte ratio). We found this correlation in both sexes, suggesting that hood size may be an honest signal of individual quality in males and females, which implies a mutual mate choice in the Black-headed Gull. Finally, the relationship between hood size and blood haemoglobin concentration was primarily attributed to the parallel variation of these traits among the colonies, suggesting that Black-headed Gulls may settle in colonies in a despotic manner. As far as we are aware, our study is one of the first to show an honest signalling role of melanin-based ornaments in the gull family, Laridae. It remains to be tested whether different melanin-based plumage ornaments in gulls are developmentally and functionally integrated with each other and with carotenoid-based integument coloration.

Elevated transmission rate of pathogens and parasites is considered one of the major costs of sociality in birds. However, greater risk of infection in colonial birds might be compensated by specific immune adaptations. Here, we predicted that nestlings raised in larger colonies should invest more in their immune function. To test this hypothesis, we manipulated colony size and conduced cross-fostering experiment in a colonial waterbird, the common tern Sterna hirundo. Establishment of different size colonies under uniform environmental conditions was induced by providing large and small patches of attractive nesting area for terns (floating rafts). Then, pairs of clutches were swapped between large and small tern colonies, and skin-swelling response to phytohaemagglutinin (PHA) was assessed for nestlings from experimental and control broods. Contrary to our expectations, we found a negative effect of foster colony size on nestling PHA response (nestlings raised in the larger colony had lower PHA response). In addition, nestling PHA response correlated negatively with heterophil/lymphocyte ratio used as a measure of physiological stress. This suggested that low PHA response of nestlings raised in the larger colony could be mediated by an elevated level of social stress. We suggest that depression of immune function via social stress may constitute a strong selective pressure against large colony size in the common tern, and possibly in other colonial species. We also recommend that this largely overlooked cost of sociality should be considered in the further studies on the evolution and ecology of avian coloniality. Electronic supplementary material The online version of this article (10.1007/s00442-019-04402-3) contains supplementary material, which is available to authorized users.

Background Genes of the Major Histocompatibility Complex (MHC) are essential for adaptive immune response in vertebrates, as they encode receptors that recognize peptides derived from the processing of intracellular (MHC class I) and extracellular (MHC class II) pathogens. High MHC diversity in natural populations is primarily generated and maintained by pathogen-mediated diversifying and balancing selection. It is, however, debated whether selection at the MHC can counterbalance the effects of drift in bottlenecked populations. The aim of this study was to assess allelic diversity of MHC genes in a recently bottlenecked bird of prey, the White-tailed Eagle Haliaeetus albicilla, as well as to compare mechanisms that shaped the evolution of MHC class I and class II in this species. Results We showed that significant levels of MHC diversity were retained in the core Central European (Polish) population of White-tailed Eagles. Ten MHC class I and 17 MHC class II alleles were recovered in total and individual birds showed high average MHC diversity (3.80 and 6.48 MHC class I and class II alleles per individual, respectively). Distribution of alleles within individuals provided evidence for the presence of at least three class I and five class II loci the White-tailed Eagle, which suggests recent duplication events. MHC class II showed greater sequence polymorphism than MHC class I and there was much stronger signature of diversifying selection acting on MHC class II than class I. Phylogenetic analysis provided evidence for trans-species similarity of class II, but not class I, sequences, which is likely consistent with stronger balancing selection at MHC class II. Conclusions Relatively high MHC diversity retained in the White-tailed Eagles from northern Poland reinforces high conservation value of local eagle populations. At the same time, our study is the first to demonstrate contrasting patterns of allelic diversity and selection at MHC class I and class II in an accipitrid species, supporting the hypothesis that different mechanisms can shape evolutionary trajectories of MHC class I and class II genes. Electronic supplementary material The online version of this article (10.1186/s12862-018-1338-3) contains supplementary material, which is available to authorized users.

Toll-like receptors (TLRs) and the Major Histocompatibility Complex (MHC) are the key pathogen-recognition genes of vertebrate immune system and they have a crucial role in the initiation of innate and adaptive immune response, respectively. Recent advancements in sequencing technology sparked research on highly duplicated MHC genes in non-model species, but TLR variation in natural vertebrate populations has remained little studied and comparisons of polymorphism across both TLRs and MHC are scarce. Here, we aimed to compare variation across innate (four TLR loci) and adaptive (MHC class I and class II) immune genes in a non-model avian species, the common tern Sterna hirundo. We detected relatively high allelic richness at TLR genes (9-48 alleles per locus), which was similar to or even higher than the estimated per locus allelic richness at the MHC (24-30 alleles at class I and 13-16 alleles at class II under uniform sample sizes). Despite this, the total number of MHC alleles across all duplicated loci (four class I and three class II) was much higher and MHC alleles showed greater sequence divergence than TLRs. Positive selection targeted relatively more sites at the MHC than TLRs, but the strength of selection (dN/dS ratios) at TLRs was higher when compared to MHC class I. There were also differences in the signature of positive selection and recombination (gene conversion) between MHC class I and II (stronger signature at class II), suggesting that mechanisms maintaining variation at the MHC may vary between both classes. Our study indicates that allelic richness of both innate and adaptive immune receptors may be maintained at relatively high levels in viable avian populations and we recommend a transition from the traditional gene-specific to multi-gene approach in studying molecular evolution of vertebrate immune system.

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.

Rapid urbanization has a great impact on avian distribution, ecology, habitat selection, and behavior. Recent avian studies indicated that individuals remain consistent in their behavioral responses to human disturbance across short periods of time. However, there is still little information about keeping consistent behaviors in distinct locations across different stages of the annual cycle. In this study, we aimed to test for long-term consistency in habitat selection with respect to urbanization in a migratory waterbird species, the Eurasian coot Fulica atra. For this purpose, we individually marked ca. 300 coots from four populations that varied in urbanization level and tracked their habitat preferences during the non-breeding season. We found that individuals from urban breeding populations selected habitats with a higher share of artificial areas during the non-breeding season, when compared to non-urban individuals. Also, a comparison of non-breeding sites selected by birds from our study populations with random sites showed that urban birds selected sites with higher urbanization level than resulting from random availability. Finally, we found a seasonal variation in habitat preferences in coots—individuals from all study populations selected more urbanized areas as the non-breeding season progressed. The results indicate that birds are able to remain consistent in habitat preferences not only at a large geographical scale, but also across different seasons. Marked between-population variation in habitat selection across the annual cycle may reflect personality differences of coots from urban and non-urban populations, and it stays in line with the personality-matching habitat choice hypothesis.

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.

Eggshell pigmentation is generated by 2 major pigments, biliverdin and protoporphyrin. The latter is mostly deposited in red, brown, and black egg spots and it has been hypothesized that greater expression of egg spottiness (as measured by the number, area, and coloration of spots) may act as an honest signal of female quality for males (sexual signaling hypothesis, SSH). The important assumption of the SSH is that eggshell pigmentation correlates with phenotypic and genetic components of female quality, although phenotypic quality of females may also be under environmental control. The aim of this study was to test for the associations of protoporphyrin-based egg pigmentation with both phenotypic and genetic female traits and environmental variables (microhabitat and urbanization) in a common rallid species, the Eurasian Coot (Fulica atra). We found that the total number of egg spots was positively associated with female condition (size-corrected body mass) and expression of a putative bare-part ornament (frontal shield). The same measure of spottiness negatively correlated with the level of physiological stress in females. No evidence was found for associations between egg spottiness and genetic traits in females (neutral heterozygosity and polymorphism of pathogen recognition receptors, the Major Histocompatibility Complex), but there was a linear increase in the expression of egg spottiness over the breeding season, which may suggest that it is regulated by food availability. Our study indicates that protoporphyrin-based pigmentation of eggs reflects female phenotypic traits (condition, stress, and ornament expression) in the Eurasian Coot, although it remains to be established whether it plays any signaling role and whether it is driven by sexual selection in this species.LAY SUMMARY There is equivocal support for direct associations between maternal quality and deposition of protoporphyrin pigmentation (dark spots and blotches) in avian eggshells. Research on protoporphyrin eggshell pigmentation has primarily focused on a single avian order (Passeriformes). We examined associations of protoporphyrin-based eggshell pigmentation with female phenotypic and genetic traits in a non-passerine species, the Eurasian Coot. Deposition of protoporphyrin in eggshells (total number per area of egg spots) positively correlated with female condition and expression of a putative bare-part ornament (frontal shield), while it was negatively associated with the level of physiological stress. Protoporphyrin-based eggshell pigmentation acts as a reliable signal of female phenotypic (but not genetic) traits in the Eurasian Coot. Signaling properties of protoporphyrin-based egg coloration are likely to largely differ between different evolutionary lineages of birds.