ABSTRACTEnvironmental variation can shape the gut microbiome, but majority of studies use captive-bred species, while data on large-scale variation in the gut microbiome and the associated environmental factors is lacking. Furthermore, previous studies have limited taxonomical coverage, and for example knowledge about avian gut microbiomes is still scarce. We investigated large-scale environmental variation in the gut microbiome of wild adult great tits across the species’ European distribution range. Our results show that gut microbiome diversity is higher during winter and that there are compositional differences between winter and summer gut microbiomes. During winter, individuals inhabiting mixed forest habitat show higher gut microbiome diversity, whereas there was no similar association during summer. Also, temperature was found to be a small contributor to compositional differences in the gut microbiome. We did not find significant differences in the gut microbiome among populations, nor any association between latitude, rainfall, and the gut microbiome. The results suggest that there is a seasonal change in wild avian gut microbiomes, but that there are still many unknown factors that shape the gut microbiome of wild bird populations.

A major aim of evolutionary biology is to understand why patterns of genomic diversity vary within taxa and space. Large-scale genomic studies of widespread species are useful for studying how environment and demography shape patterns of genomic divergence. Here, we describe one of the most geographically comprehensive surveys of genomic variation in a wild vertebrate to date; the great tit (Parus major) HapMap project. We screened ca 500,000 SNP markers across 647 individuals from 29 populations, spanning similar to 30 degrees of latitude and 40 degrees of longitude - almost the entire geographical range of the European subspecies. Genome-wide variation was consistent with a recent colonisation across Europe from a South-East European refugium, with bottlenecks and reduced genetic diversity in island populations. Differentiation across the genome was highly heterogeneous, with clear 'islands of differentiation', even among populations with very low levels of genome-wide differentiation. Low local recombination rates were a strong predictor of high local genomic differentiation (FST), especially in island and peripheral mainland populations, suggesting that the interplay between genetic drift and recombination causes highly heterogeneous differentiation landscapes. We also detected genomic outlier regions that were confined to one or more peripheral great tit populations, probably as a result of recent directional selection at the species' range edges. Haplotype-based measures of selection were related to recombination rate, albeit less strongly, and highlighted population-specific sweeps that likely resulted from positive selection. Our study highlights how comprehensive screens of genomic variation in wild organisms can provide unique insights into spatio-temporal evolutionary dynamics.

Agricultural intensification has a profound negative impact on farmland biodiversity worldwide. Birds closely associated with agricultural landscapes are a key indicator group for evaluating sustainability in land use. We identified key determinants of hatching success in northern lapwings (Vanellus vanellus) in agricultural landscapes of Estonia. In total, we monitored 133 nests to record the causes of nest failures on 85 field plots from three farming management types over two years. Hatching success as indicated by daily survival rate of nests was significantly lower in organic (94%) when compared to conventionally managed (98%) cultivated fields. We showed that this was mainly caused by increased nest predation by mammalian and avian predators in organic fields. Landscape elements, such as drainage ditches with tall vegetation decreased nest predation by mammalian predators. Depredation of nests by avian species was negatively related to the breeding density of lapwings, suggesting the effectiveness of their collective defense against raptors and corvids. We conclude that the hatching success for ground-breeding birds in arable fields is mainly affected by differences in nest predation. Agri-environment schemes, such as providing funding for organic farming, effectively increase the diversity and abundance of wild animals, while also increasing the number of nest predators as a side effect. The challenge is to control the abundance of mammalian and avian nest predators using different methods. Regional agricultural policy should support the retention of ditches with tall vegetation in agricultural landscapes that can effectively reduce nest predation by mammals.

Noise pollution is one of the leading environmental risks to human health. However, noise effects on the reproduction and behaviour of wild animals have been ambiguous. The reason can be that most studies focus on the effect of one type of noise, while structurally distinct noise types with the same loudness, but with various frequencies may cause different responses due to masking effect. Hence, comparing spectrally different noises in a single study can provide valuable insight into those noise differences, as all other ecological conditions for different experiments are nearly the same. Here, we exposed free-living Great Tits Parus major breeding in nest boxes to three different noise types, such as car traffic, lawnmower, and chainsaw noise, which are common in the breeding habitats of passerines, and investigated these effects on parental provisioning behaviour. We found that noise as such had a negative effect on nest visits but the effect was not detected when analysing noise types separately. These findings do not support the acoustic masking hypothesis, given that structurally different noise types had similar effects on parental provisioning. We suggest that different anthropogenic noises may alter avian reproductive behaviour through disruption and disturbance, potentially having cumulative and negative effects on reproduction.

Anthropogenic noise is increasing at an alarming rate in urban as well as rural ecosystems and detrimental effects have been reported on many animals relying on acoustic communication. Little is known regarding the impact of different types of noise on community level predator perception and breeding behaviour. Here, we presented a predator model alone or a predator combined with a specific noise type, such as traffic, a lawnmower or a chainsaw, to breeding pairs of great tits, Parus major, giving them the opportunity to perform mobbing behaviour that could provide public information to their neighbours. We then measured the provisioning behaviour of adjacent tit pairs, which were 50–150 m away from the focal pairs' nests. The adjacent breeding pairs decreased nest visits when focal birds were exposed to the sparrowhawk model or to the sparrowhawk model combined with the lawnmower noise. Neighbouring pairs did not reduce the rate of nest visits when focal birds were exposed to the hawk model combined with the traffic or the chainsaw noise. The distance from focal nests did not alter the noise effect on neighbouring pairs. We suggest that auditory masking is the most likely mechanism to explain why noise compromises public information. The chainsaw and traffic noise overlap with the frequency of mobbing calls and disrupt the transmission of auditory signals in the community. Further, when the hawk presentation was combined with the chainsaw noise, which was the type of noise that had the highest frequencies, the latency of mobbing by the focal birds was strongly increased, and the number of species involved in mobbing was reduced. This study demonstrates that the signalling function of mobbing is ineffective in noisy environments which ultimately impacts fitness via increased predation to adults or their offspring.

Laboratory animal models have shown that blood serotonin levels reflect consistent individual differences in behavioral decision-making and maternal behavior. Serotonin could also help to understand intraspecific variation in reproductive strategies, although the mechanisms are poorly understood. In this study, the relationships of plasma serotonin with breeding parameters and parental behavior were examined in wild great tits (Parus major). Females who laid eggs earlier had higher levels of serotonin in the second half of the nestling period, while no significant relationship of serotonin with clutch size, brood size, and body size was detected. In males, serotonin levels were negatively related to clutch size and brood size and positively related to body size. The association of serotonin with provisioning behavior was sex specific, and acute fear stress induced by a predator presentation did not change this relationship. Food provisioning was positively related to size-corrected serotonin levels in females and negatively related to size-corrected serotonin levels in males. These results suggest that peripheral serotonin is a sensitive marker of parental behavior and reproductive effort in wild birds, while the mechanisms linking this neurotransmitter to reproduction are probably mediated by interplay between the serotonergic system, sex hormones, and other neurotransmitters.

The effects of anthropogenic noise on the incubation behaviour of birds are poorly studied. The available studies often focus on the effect of one type of noise, but recent work has demonstrated that structurally different noises with the same loudness may cause different responses depending on the masking effect on birds’ acoustic communication. Therefore, including different noise types in the same study can best elucidate the effects of noise properties on bird behaviour and reproduction. In this study, we used two structurally different noise types to explore noise effects on the incubation behaviour of great tits (Parus major). Female great tits incubate, and males feed them. We filmed 25 pairs of great tits in the control phase (no additional noise) and the experimental phase for two hours using the continuous playback of a chainsaw or the lawnmower noise with the same amplitude. The chainsaw noise significantly decreased the number of female nest visits but did not affect nest attendance. The lawnmower noise, meanwhile, did not influence any aspect of incubation behaviour. As chainsaw noise is higher in frequency (0.5–8 kHz vs. 0.2–2 kHz for lawnmower), there is a greater degree of overlap with passerines’ hearing range (1–8 kHz), and it probably hinders their ability to receive acoustic signals and cues. The chainsaw noise likely alters the incubating behaviour of passerines via increased vigilance (the incubating female does not hear partner’s calls) or decreased foraging as well. The impact of noise on birds’ behaviour can also be explained by possible association with human activity and the match between acoustic and visual cues.

Telomere length and shortening rate are increasingly being used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between-population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the nonbreeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north-south gradient (Finland, Estonia, England and Spain) predicting a negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from midlatitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level.

The integration and synthesis of the data in different areas of science is drastically slowed and hindered by a lack of standards and networking programmes. Long-term studies of individually marked animals are not an exception. These studies are especially important as instrumental for understanding evolutionary and ecological processes in the wild. Further, their number and global distribution provides a unique opportunity to assess the generality of patterns and to address broad-scale global issues (e.g. climate change). To solve data integration issues and enable a new scale of ecological and evolutionary research based on long-terms studies of birds, we have created the SPI-Birds Network and Database (www.spibirds.org) – a large-scale initiative that connects data from, and researchers working on, studies of wild populations of individually recognizable (usually ringed) birds. Within a year of the establishment, SPI-Birds counts 120 members working on more than 80 populations, with data concerning breeding attempts of almost a million individual birds over a 1700 cumulative years, and counting. SPI-Birds acts as a data hub and a catalogue of studied populations. It prevents data loss, secures easy data finding, use and integration, and thus facilitates collaboration and synthesis. We provide community-derived data and meta-data standards and improve data integrity guided by of Findable, Accessible, Interoperable, and Reusable (FAIR), and aligned with the existing metadata languages (e.g. ecological meta-data language). The encouraging community involvement stems from SPI-Bird's decentralized approach: research groups retain full control over data use and their way of data management, while SPI-Birds creates tailored pipelines to convert each unique data format into a standard format. We outline the lessons learned, so that other communities (e.g. those working on other taxa) can adapt our successful model. Creating community-specific hubs (such as ours, COMADRE for animal demography, etc.) will aid much-needed large-scale ecological data integration.

Studies have provided inconsistent evidence regarding the effects on behaviour of nucleotide polymorphism at the DRD4 gene. For instance, the DRD4 gene affects exploratory behaviour in some bird species and populations but not in others. Our earlier experiment with a wild Great Tit Parus major population revealed an important impact of the DRD4 gene on behavioural variation during the breeding season. The aim of this study was to clarify these results by measuring individual behavioural differences (exploration and anxiety) in Great Tits under controlled conditions before the beginning of egg-laying in order to exclude the potential impacts of breeding (egg-laying, motivation to feed nestlings, the potential effect of a partner) and the natural environment (e.g. the presence of predators). Birds were caught from their natural habitat and their behaviour was tested in a trial room the following morning. Individuals with the CT heterozygous genotype (SNP830) performed more actively in different contexts (visiting a novel object in the trial room and vocal behaviour during handling) compared with homozygotes, and the relationships were consistent in males and females. In light of the results from our previous study (heterozygous males, but not females, had a shorter latency to feed nestlings than CC homozygotes), we conclude that the same DRD4 gene variants affect behavioural responses in a controlled environment and wild contexts in the same directions, but that these relationships are not sex-specific under standardized conditions.

Prolonged stress can have long-lasting effects on an individual physiology and growth. However, the impact of chronically elevated glucocorticoids on the expression of early antipredator responses is still poorly documented. In this study I simulated the effect of repeated acute stress on offspring phenotype in free-living pied flycatchers (Ficedula hypoleuca) by administering adrenocorticotropic hormone (ACTH) to nestlings for 6 days. The results showed that frequent induction of stress responses by ACTH injections, independently of parental care, adversely affected offspring final body size, wing length and baseline corticosterone levels. Nestling behavioural activity did not differ between ACTH- and saline-treated groups during exposure to control sounds while behavioural activity during exposure to alarm calls was reduced in manipulated offspring only. I conclude that prolonged physiological stress may have short-term benefits to nest-bound offspring, such as more effective antipredator behaviour, but at the expense of negative effects on body size and developmental speed.

Elucidating associations between behavioural variation and genes may help to understand the co-existence of different behavioural phenotypes in natural populations. In birds, two main candidate genes are known to affect exploratory and risk-taking behaviour through the neurotransmitter system: the dopamine receptor D4 gene (DRD4) and the serotonin transporter gene (SERT/SLC6A4). In a small passerine bird, the great tit (Parus major), anti-predator responses (hissing or non-hissing behavioural type) towards a nest predator differ consistently between individuals. As defensive anti-predator behaviours are likely related to variations in neurotransmitter systems, we tested the associations between polymorphisms of the two candidate genes (SERT and DRD4) and the propensity to give hissing calls towards a nest predator in incubating female great tits. We found that acoustic anti-predator responses in wild birds were linked to a single-nucleotide polymorphism in the SERT gene (SNP187 in exon 1), but not in the DRD4 gene. The polymorphism in the SERT gene explains about 16% of the total variance in hissing behaviour. However, as the sample size in this study is quite small, these results are preliminary and should be replicated in future studies.

Antipredator strategies vary remarkably between individuals within populations. Parents tend to take greater risks when brood value is increased. Moreover, individuals consistently differ in a whole suite of correlated behaviours that may cause distinctive responses to predators. It is likely that individual differences in antipredator behaviour may co-vary with proxies for fitness such as reproductive success. We used a 4-year data from wild great tits (Parus major) to test whether passive and active antipredator strategies (females with no response vs. those giving hissing calls towards a nest predator) during the incubation stage can reflect variation in breeding success. Although clutch size did not depend on hissing behaviour, the number of surviving offspring from eggs and neonates to fledglings was higher for non-hissing than hissing birds. We conclude that females with distinct antipredator strategies can prioritize different fitness components.

Individual differences in coping with potentially dangerous situations are affected by a combination of genetic and environmental factors. How genetic polymorphisms and behavioural variations are related to fitness is unknown. One of the candidate genes affecting a variety of behavioural processes, including impulsivity, anxiety and mood fluctuations in both humans and other vertebrates, is the serotonin transporter gene (SERT/SLC6A). The aim of this study was to assess an association between SERT genotypes and novelty-seeking and risk-taking behaviours as well as breeding parameters of great tits (Parus major) in a natural environment. We associated polymorphisms in the promoter exonic regions of the SERT gene with parental risk-taking-related behaviour and fitness traits. Our results show that: (1) risk-taking behaviour in our great tit population is linked to single nucleotide polymorphisms in the SERT gene exon 3 and exon 8; (2) the genotype-behaviour associations are consistent with the presence of different stressors; and (3) polymorphisms in exon 8 could be associated with fitness-related traits, such as the start of egg-laying and hatching success. We showed for the first time that genetic variability of SERT plays an important role in shaping individual decision-making that affects fitness in a wild population. However, the results are based on one population and on the polymorphisms that are in a single gene. Therefore, replication studies are needed in order to confirm these preliminary results.

The environment can affect individual performance directly via resource availability or indirectly through resource allocation among competing fitness components, such as body growth and maintenance activities related to short-term survival. Corticosterone (CORT), the main glucocorticoid in birds, may be an important mediator of energy allocation to different organismal functions, but its effect on the plasticity of fitness-related traits has rarely been investigated at different ontogenetic stages. Here, we evaluated the role of baseline and stress-induced CORT on nestling development of wild great tits (Parus major) under different growth conditions and at different developmental stages. We found that nestling mass and condition were positively related to stress-induced levels of total CORT and free CORT in the middle of nestling stage but negatively related at the prefledging stage. Our results also showed that stress-induced levels of total CORT can be used as a proxy for bioavailable free CORT. No significant relationship of body mass or condition in either stage was shown with levels of baseline CORT or stress-induced CORT-binding globulin capacity. None of these age-specific relationships depend on brood size manipulation. We conclude that the effects of glucocorticoids on nestling performance are stage specific. The ability to secrete CORT may have beneficial effects on nestling development in the middle of the nestling stage, while it can be adaptive to keep CORT levels at optimal ranges before fledging to give priority to growth-related processes that are important for long-term fitness.

Environmental change associated with urbanization is considered one of the major threats to biodiversity. Some species nevertheless seem to thrive in the urban areas, probably associated with selection for phenotypes that match urban habitats. Previous research defined different "copying styles" in distress behavior during the handling of birds. These behaviors vary along a continuum from "proactive" to "reactive" copers. By studying avian distress behaviors we aimed to broaden our understanding of the relationship between coping styles and urbanization. Using a large-scale comparative study of seven paired rural and urban sites across Europe, we assayed distress behaviors during handling of urban and rural-dwelling populations of the great tit Parus major. We detected no consistent pairwise differences in breath rate between urban and rural habitats. However, urban great tits displayed more distress calling (fear screams) and higher pecking rate (handling aggression) than rural birds. These findings suggest that urban great tits have a more proactive coping strategy when dealing with stressful conditions. This finding is in line with previous studies implying that urban great tits are more explorative, less neophobic and display shorter flight distances than their rural counterparts, representing further aspects of the same "proactive", coping strategy. Future research should investigate whether reported differences in distress behavior are due to local adaption caused by natural selection or due to phenotypic plasticity.

Maternally-derived glucocorticoids can modify the normal development of young animals. To date, little is known about maternal effects that are mediated by acute embryonic exposure to glucocorticoids. In birds, elevated maternal transmission of corticosterone (CORT) to egg albumen is mainly dependent on acute stress. In this study, we increased CORT levels in the egg albumen of a wild passerine, the great tit (Parus major), breeding in favourable deciduous and less suitable coniferous habitat. Subsequently we measured the somatic growth, baseline and acute glucocorticoid responses, immunity and behaviour of prenatally manipulated offspring with respect to control siblings. We found that prenatally CORT-exposed nestlings had lower baseline CORT levels, a more rapid decline in CORT during recovery from a standardized stressor, and a reduced heterophil/lymphocyte ratio compared with controls. Although stress-induced total CORT levels remained unchanged, free CORT levels were significantly lower and the levels of corticosteroid binding globulins (CBG) significantly higher in experimental offspring. Prenatally CORT-exposed offspring begged longer after hatching than controls. Stress-induced behavioural activity of fledglings did not differ between treatments, while its association with baseline CORT levels was significant in the control group only. The body mass and tarsus length of fledglings was positively affected by manipulation in unfavourable coniferous habitat only. We conclude that maternal effects related to elevated levels of albumen CORT modify diverse aspects of offspring phenotype and potentially increase offspring performance in resource poor environments. Moreover, our results indicate that maternal glucocorticoids may suppress the effect of hormones on behavioural responses.

Anti-predator behaviour of breeding animals is a complex trait that depends on current reproductive investment as well as individual differences in risk-taking propensities. In response to nest predation, many bird species produce specific sounds, such as the hissing calls in incubating great tits (Parus major), that may provoke an acoustic startle response in the predator. However, it is still unclear whether the propensity of incubating females to produce hissing calls towards nest predators depends on the reproductive investment. With our 3-year study, we show that response type (females that do not hiss versus females giving hissing calls) to a potential nest predator, the woodpecker, is a repeatable trait. We found no differences in the studied reproductive traits between hissing and non-hissing birds. Interestingly, among hissing birds, fast-responding females started egg-laying earlier than slow-responding ones. Among non-hissing birds, heavier birds initiated clutches earlier. We also revealed that hissing birds breed in areas with decreased nest-box occupancy, suggesting either that they potentially select different areas to breed or that territory size is larger as a result of hissing birds being more aggressive. These findings demonstrate that response type is not related to the early reproductive value of the brood across distinct behavioural groups. However, our results do suggest that non-hissing and hissing females differ in terms of individual quality or dominance or personality related aspects.

Predation risk has negative indirect effects on prey fitness, partly mediated through changes in behaviour. Evidence that individuals gather social information from other members of the population suggests that events in a community may impact the behaviour of distant individuals. However, spatially wide-ranging impacts on individual behaviour caused by a predator encounter elsewhere in a community have not been documented before. We investigated the effect of a predator encounter (hawk model presented at a focal nest) on the parental behaviour of pied flycatchers (Ficedula hypoleuca), both at the focal nest and at nearby nests different distances from the predator encounter. We show that nest visitation of both focal pairs and nearby pairs were affected, up to 3 h and 1 h, respectively. Parents also appeared to compensate initial disrupted feeding by later increasing nest visitation rates. This is the first evidence showing that the behaviour of nearby pairs was affected away from an immediate source of risk. Our results indicate that the impacts of short-term predator encounters may immediately extend spatially to the broader community, affecting the behaviour of distant individuals. Information about predators is probably quickly spread by cues such as intra- and heterospecific alarm calls, in communities of different taxa.