AbstractDeciding which offspring to feed is one of the most critical decisions parents make for both parental and offspring fitness. Despite knowing much about what choices parents make, we know little about how parents choose. What we do know about how the brain integrates sensory evidence when choosing between options comes from laboratory studies and models. However, such studies may not adequately reflect decisions made in nature-with real-world complexity and consequences. Our naturalistic experiment on decision-making in 62 wild Parus major parents addresses this issue. Decision speed was impacted by whether parents chose to feed a typically preferred chick, offspring starvation risk, decision complexity, and parental sex. Parents regularly moved food between chicks before committing, suggesting that parents perhaps were not confident in their initial decision, had made a mistake, were continuing to collect evidence, or could not execute their initial decision. Such decision changes were predicted by similar factors as speed. After moving food, parents were more likely to continue gathering evidence after their decision, and their next decision was slower. These results demonstrate several factors impacting cognition, and perhaps metacognition, in wild birds. More broadly, our study demonstrates how crucial evolutionarily relevant experiments in natural settings are.

Understanding the causal effects of genetic mutations is essential for explaining fitness variation, forecasting evolutionary trajectories and assessing extinction risk, yet remains a fundamental challenge, particularly in natural populations. While amino acid substitutions can alter protein structure and function, mutations affecting gene regulation can also have significant fitness consequences. In this Opinion Piece, we argue that epigenetic mechanisms, given their central role in gene regulation, probably modulate the deleteriousness of mutations. Drawing on evidence from humans and model organisms, we identify three ways in which epigenetic mechanisms might interact with deleterious mutations. Specifically, we hypothesize that epigenetic regulation may: (i) be disrupted by deleterious mutations in non-coding regions and epigenetic regulator genes; (ii) buffer the expression of deleterious mutations; and (iii) contribute to the repair and purging of deleterious mutations. Advances in next- and third-generation sequencing and bioinformatics now allow these hypotheses to be empirically tested in wild populations. As many species face ongoing population declines, unravelling how epigenetic mechanisms influence the functional effects of mutations is vital for understanding fitness variation, guiding evolutionary predictions and informing conservation strategies. This article is part of the theme issue 'Ecological epigenetics at the intersection of behaviour and life history variation in non-model animals'.

Individual variation in reactions to novel aposematic prey is common in avian predators. In wild adults, this variation may be caused by differences among individuals in experience with various prey, but similar variation exists in naive juveniles, and this is linked to personality-a complex of correlated, partly heritable behavioral traits that are consistent across time. Along the extremes on an axis of early exploratory behavior in great tits (Parus major), fast explorers are bold, aggressive, and routine-forming, whereas slow explorers are shy, less aggressive, and more innovative. We tested the effect of personality on innate wariness toward aposematic prey in adult hand-reared great tits from 2 lines selected for opposite levels of early exploratory behavior (fast vs. slow). The birds were offered aposematic firebugs (Pyrrhocoris apterus) over 2 d. Birds from both selection lines showed a similar degree of innate wariness toward the firebugs on the first day, but on the second day, fast explorers approached the firebugs significantly faster and more frequently than slow birds. Whether the birds attacked the firebugs was also dependent on their personality. Thus, personality-related individual differences in reactions of great tits toward the aposematic prey were maintained in the adult life stage. Personality affects avian responses to aposematic prey. In great tits, fast explorers are bolder, slow explorers more cautious. We tested hand-reared adults from lines selected for fast versus slow exploration. Both fast and slow birds were initially wary of aposematic firebugs, but fast explorers approached and attacked firebugs more frequently in repeated test. Although adults were less likely to attack aposematic prey than juveniles tested in a previous study, personality-related differences were maintained over time.

Abstract Not all odors influencing mating behavior evolve as sex pheromones. Female butterflies’ post-mating odors have been considered species-specific anti-aphrodisiac pheromones shaped by sexual selection but may also serve broader ecological roles shaped by natural selection. Males transfer odors to females that repel rivals, yet the widespread use of these compounds across phyla makes them targets for eavesdropping, such as by phoretic egg parasitoids. We show that in cabbage white butterflies ( Pieris spp.), these odors are highly variable and attract parasitoids, deter predators, and influence oviposition. Using gas chromatography and electroantennography, we demonstrate that odor emission and perception lack species-specificity: compounds once thought unique to P. brassicae and P. rapae are shared across Pieridae. In P. napi , odor variation among populations correlates with parasitoid pressure, but not with latitude, genetic distance, or mating frequency, suggesting ecological rather than sexual drivers. In P. brassicae , CRISPR/Cas9 disruption of odor perception alters oviposition and increases susceptibility to parasitism. Moreover, these odors render females unpalatable to birds. Together, our results show that post-mating odors in Pieris butterflies may act as aposematic signals. We provide evidence that these signals evolve under multiple selective pressures, balancing deterrence of mates and predators, parasitoid avoidance, and host-plant interactions. These findings suggest that chemical signals should be viewed as integrating ecological and reproductive pressures, rather than being interpreted solely through the lens of sexual communication.

Abstract Introgression redistributes genetic variation among diverging lineages, shaping evolution-ary trajectories and contributing to phenotypic evolution. Yet how localized introgressed genomic regions persist despite extensive genomic homogenization remains poorly under-stood. Here, we investigate the evolutionary history of the northeastern Iranian great tit ( Parus major intermedius ), a grey-plumaged member of the great tit complex occurring at the eastern range margin of the green- and yellow-plumaged major lineage, adjacent to the grey-plumaged Central Asian bokharensis lineage, and long regarded as a putative hybrid. We find that P. m. intermedius retains predominantly major -derived genomic ancestry despite its grey plumage, revealing extensive genomic homogenization across the genome. Surprisingly, a single localized introgressed haplotype on chromosome 24 retains bokharensis -derived ancestry, exhibits elevated genomic differentiation relative to the genomic background, and overlaps the carotenoid-processing gene BCO2 , a strong candidate underly-ing plumage pigmentation. Our findings provide a genomic explanation for the discordance between phenotype and genome-wide ancestry, demonstrating how localized introgression can preserve genomic regions associated with phenotypic divergence despite extensive genomic homogenization. This system illustrates how individual genomic regions can retain distinct evolutionary histories long after the surrounding genome has largely homogenized.

Information on individual age is a fundamental aspect in many ecological and evolutionary studies. However, accurate and non-lethal methods that can be applied to estimate the age of wild animals are often absent. Furthermore, since the process of ageing is accompanied by a physical decline and the deterioration of biological functions, the biological age often deviates from the chronological age. Epigenetic marks are widely suggested to be associated with this age-related physical decline, and especially changes in DNA methylation are suggested to be reliable age-predictive biomarkers. Here, we developed separate epigenetic clocks for ageing and development in a small passerine bird, the great tit (Parus major). The ageing clock was constructed and evaluated using erythrocyte DNA methylation data of 122 post-fledging individuals, and the developmental clock using 67 pre-fledging individuals from a wild population. Using a leave-one-out cross-validation approach, we were able to accurately predict the ages of individuals with median absolute deviations of 0.40 years for the ageing and 1.06 days for the development clock. Moreover, using existing data from a brood-size manipulation, we show that nestlings from reduced broods are estimated to be biologically older compared to control nestlings, while they are expected to have higher fitness. These epigenetic clocks provide further evidence that, as observed in mammals, changes in DNA methylation of certain CpG sites are highly correlated with chronological age in birds and this opens up new avenues for broad applications in behavioural and evolutionary ecology.

Life-history trade-offs are a central concept in evolutionary biology, yet their underlying molecular mechanisms are not yet fully understood. Whilst much research has focused on genetic variation, epigenetic mechanisms, which regulate gene regulation, may be equally important. To investigate this, we collected blood samples from 50 male black grouse (Lyrurus tetrix) before and after the breeding (lekking) period and quantified genome-wide DNA methylation changes using reduced representation bisulphite sequencing. We identified 1,026 CpG sites that changed significantly in methylation across the breeding period, many residing within genes involved in the regulation of RNA biosynthesis. We tested whether these DNA methylation changes were associated with reproductive investment and future fitness-relevant traits: survival and the expression of post-breeding sexual ornaments, which reflect body condition after the strenuous lekking period. Dozens of CpG sites showed significant associations, often clustering within the same genes, suggesting that epigenetic changes associated with reproduction and survival are localized rather than widespread. Moreover, changes at three CpG sites exhibited opposite relationships between current reproductive investment and future fitness-relevant traits, suggesting that epigenetic mechanisms might contribute to shaping life-history trade-offs. Our study demonstrates that DNA methylation changes are associated with the expression of costly reproductive traits, highlighting the importance of epigenetic mechanisms in shaping life-history traits and fitness.

Unpredictable environmental conditions due to climate change affect the availability and quality of food resources, a limiting factor for survival. Behavioral plasticity is one of the most important mechanisms that help individuals to face rapid environmental changes. However, we still lack experimental information on how foraging decisions are modulated by other behavioral traits when facing changes in food resources. In this study we aimed to explore how individual foraging decisions regarding food quality are related to exploratory behavior, cognitive flexibility, and local dominance in wild great tits (Parus major). We offered different food types varying in quality at supplementary feeders and subsequently changed the availability and location of these food types. We then assessed whether the proportion of high-quality food individuals consumed was influenced by their exploratory tendency, reversal learning performance, and local dominance. We found that local dominance (ie distance from territory to the feeder), but not exploratory tendency or reversal learning performance affected the fraction of high-quality food in an individual's diet. Locally dominant individuals were constrained in their response to changes in the quality of available resources, leading them to stay at their preferred feeder even when this was providing low-quality food alone. In conclusion, we show the importance of the social context when studying the behavioral mechanisms of adaptation to changing environments in wild animals. With climate change, the predictability of food is decreasing, with consequences for survival. We expect individuals that are dominant, more exploratory and more flexible in their behavior to cope better with these changes. In a great tit population, we show that when manipulating the availability of high- and low-quality supplemented food, dominant birds with territories close to the feeders were constrained in changing their foraging behavior. They were less likely to leave their preferred feeder, resulting in them getting food of lower quality. Exploratory tendency and behavioral flexibility did not have an effect. This indicates that social factors are important in determining how well individuals cope with changes in their environment.

Neophobia, or aversion to novelty, is important for adaptability and survival as it influences the ways in which animals navigate risk and interact with their environments. Across individuals, species and other taxonomic levels, neophobia is known to vary considerably, but our understanding of the wider ecological drivers of neophobia is hampered by a lack of comparative multispecies studies using standardized methods. Here, we utilized the ManyBirds Project, a Big Team Science large-scale collaborative open science framework, to pool efforts and resources of 129 collaborators at 77 institutions from 24 countries worldwide across six continents. We examined both difference scores (between novel object test and control conditions) and raw data of latency to touch familiar food in the presence (test) and absence (control) of a novel object among 1,439 subjects from 136 bird species across 25 taxonomic orders incorporating lab, field, and zoo sites. We first demonstrated that consistent differences in neophobia existed among individuals, among species, and among other taxonomic levels in our dataset, rejecting the null hypothesis that neophobia is highly plastic at all taxonomic levels with no evidence for evolutionary divergence. We then tested for effects of ecological factors on neophobia, including diet, sociality, habitat, and range, while accounting for phylogeny. We found that (i) species with more specialist diets were more neophobic than those with more generalist diets, providing support for the Neophobia Threshold Hypothesis; (ii) migratory species were also more neophobic than nonmigratory species, which supports the Dangerous Niche Hypothesis. Our study shows that the evolution of avian neophobia has been shaped by ecological drivers and demonstrates the potential of Big Team Science to advance our understanding of animal behavior.

Variation in age structure influences population dynamics, yet we have limited understanding of the spatial scale at which its fluctuations are synchronised between populations. Using 32 great tit populations, spanning 4° W-33° E and 35°-65° N involving > 130,000 birds across 67 years, we quantify spatial synchrony in breeding demographic structure (subadult vs. adult breeders) and its drivers. We show that larger clutch sizes, colder winters, and larger beech crops lead to younger populations. We report distance-dependent synchrony of demographic structure, maintained at approximately 650 km. Despite covariation with demographic structure, we do not find evidence for environmental variables influencing the scale of synchrony, except for beech masting. We suggest that local ecological and density-dependent dynamics impact how environmental variation interacts with demographic structure, influencing estimates of the environment's effect on synchrony. Our analyses demonstrate the operation of synchrony in demographic structure over large scales, with implications for age-dependent demography in populations.

Birds differ in their parent-offspring interactions, and these differences may be caused by environmental variation. When food is plentiful, chicks that are begging more are fed more. When food is scarce, parents instead feed larger offspring. This change could be due to offspring adjusting their behavior, or to confounding factors not directly related to current food availability, such as brood size. Alternatively, it could be due to parents responding to signals differently based on their experience of food availability in the recent past. We tested these competing explanations experimentally by manipulating food availability in wild great tits, Parus major. We then standardized food availability, and manipulated offspring size and behavior by creating mixed cross-fostered broods just before filming. This isolated the effect of parental strategies while holding food availability and offspring begging and size constant across treatments. We found that when parents received supplemented food prior to filming, they were: (1) more likely to preferentially feed chicks that were begging more; and (2) less likely to preferentially feed larger chicks. Chicks, conversely, did not differ in their begging in relation to prior environmental conditions, but instead begged in relation to their immediate feeding history and their nestmates' begging intensity. Overall, our results suggest that parents have more control over food distribution than suggested by scramble competition models, and that parents can flexibly adjust how they respond to offspring signals and cues in response to food availability. Consequently, different signaling systems and parental plasticity are favored depending on environmental conditions. Parents control food distribution in a flexible manner, even when it seems like offspring run the show. New experimental research in great tits reveals widespread plasticity in parental response to offspring signals, and that who and what parents pay attention to depends on environmental quality.

Maternal hormones can profoundly impact offspring physiology and behaviour in sex-dependent ways. Yet little is known about the molecular mechanisms linking these maternal effects to offspring phenotypes. DNA methylation, an epigenetic mechanism, is suggested to facilitate maternal androgens' effects. To assess whether phenotypic changes induced by maternal androgens associate with DNA methylation changes, we experimentally manipulated yolk testosterone levels in wild great tit eggs (Parus major) and quantified phenotypic and DNA methylation changes in the hatched offspring. While we found no effect on the handing stress response, increased yolk testosterone levels decreased the begging probability, emphasised sex differences in fledging mass, and affected methylation at 763 CpG sites, but always in a sex-specific way. These sites are associated with genes involved in growth, oxidative stress, and reproduction, suggesting sex-specific trade-offs to balance the costs and benefits of exposure to high yolk testosterone levels. Future studies should assess if these effects extend beyond the nestling stage and impact fitness.

To forecast how fast populations can adapt to climate change, it is essential to determine the evolutionary potential of different life-cycle stages under selection. In birds, timing of gonadal development and moult are primarily regulated by photoperiod, while laying date is highly phenotypically plastic to temperature. We tested whether geographic variation in phenology of these life-cycle events between populations of great tits (Parus major) has a genetic basis, indicating that contemporary genetic adaptation is possible. We carried out a common garden experiment in which we bred first- and second-generation pairs in captivity originating from eggs from Gotland (Sweden) and Hoge Veluwe (The Netherlands), two populations that showed different temperature sensitivity of laying date in a recent meta-analysis. We recorded the phenology of egg-laying, moult and gonadal size in early spring. We found no significant differences in laying date between the populations, but they did differ in moult timing and testis size. This implies that under climate change the timing of gonadal development and moult, which are mainly regulated by photoperiod, will not respond to increased temperature but can respond by genetic adaptation in response to selection, while the opposite holds for laying date, perhaps indicating that plasticity is constraining genetic adaptation.

Animals living in cities are smaller than their conspecifics from rural areas but whether such differences are caused by genetic differences or food constraints remains untested. We performed a multi-generation common garden study where we raised great tits (Parus major), originating from eggs collected from multiple Dutch cities and forests under the same conditions for two generations. Offspring from city birds had a smaller tarsus than forest birds in both generations, demonstrating that these morphological differences are genetic. Next, we tested whether size differences are an adaptation to the low food abundance when offspring are raised in the city. Third-generation birds of both origins were given food amounts mimicking being raised in forests or cities during the second part of their nestling development. While the treatment resulted in birds in the lower feeding frequency treatment to be smaller, city and forest birds responded the same way, suggesting that city birds do not cope better with reduced food availability. Our study shows that the smaller size of urban birds has a genetic basis and is not only caused by a plastic response to restricted resources in the urban environment. Our experiment does not provide evidence that these genetic differences have evolved as an adaptive response to a reduced food availability in cities.

Anthropomorphic activities have a large impact on ecosystems in many ways, one of which is how animals behave. Non‐motorised nature recreation is a popular human activity of which the impacts on nature are largely unknown. These activities, which include hiking, biking, pet walking and horseback riding, tend to increase during the commencement of the breeding activity for most passerine forest birds in temperate zones. We here investigated whether variation in recreational activity associates with patterns of nest box occupation and reproductive success in a long‐term study of personality‐typed great tits Parus major. We measured human disturbance in the area by recording the frequency of non‐motorised recreational activities by observations. We were particularly interested in the relationship between disturbance levels and nest box occupancy as well as the relationship between disturbance levels of occupied nest boxes and exploratory scores of the great tits that occupied them. We also investigated whether reproductive characteristics such as fledging success, clutch size, chick weight and tarsus length varied with disturbance levels at occupied nest boxes. We did not find a direct association between nest box occupation and disturbance. Habitat quality rather than disturbance explained the nest occupation. Furthermore, more exploratory individuals occupied boxes in less disturbed areas, independent of habitat quality. Fitness decreased with increasing disturbance independent of habitat quality. Chicks were heavier and had longer tarsi, and clutch sizes were bigger in less disturbed areas. In conclusion, we found breeding site choice of great tits to be independent of human activity, although there are clear fitness effects of human disturbance.

Behavioural traits are under both genetic and environmental influence during early life stages. Early environmental conditions related to the amount and type of food have been found to alter behaviour in many organisms. However, how early life diet affects the variation in and the correlation between behavioural traits is largely unknown. Using a multivariate approach, we investigated how variation in parental prey selection is related to three repeatable nestling personality traits, and explored the within and between-individual covariation between these behaviours in a wild passerine, the great tit (Parus major). Our results confirm that breath rate, docility and handling aggression (HA) in great tit nestlings are repeatable traits. Contrary to our expectation, the three nestling personality traits did not form a behavioural 'syndrome' on the phenotypic level in the study population, but we found two of three expected phenotypic correlations, mostly at the within-individual level. Moreover, we found that breath rate significantly decreased with a higher number of spiders in the diet, and docility and handling aggression were significantly and inversely related to higher numbers of noctuids and tortricids in the diets of individuals within broods. Thus, our findings suggest that provisioning quantity and quality during the early life, affects variation in behavioural phenotypes, which occurs mainly at the within-individual level. In nature, animal behaviour is both determined by the genes and the environment. The environmental conditions animals face in their early development, such as quality and quantity of food is known to alter behaviour. However, is diet composition related to the expression and relationship between behaviours? We here investigated how three nestling behaviours covaried and showed that different types of prey might determine the expression of particular behaviours in early life-stages.image

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.

The recognition that climate change is occurring at an unprecedented rate means that there is increased urgency in understanding how organisms can adapt to a changing environment. Wild great tit (Parus major) populations represent an attractive ecological model system to understand the genomics of climate adaptation. They are widely distributed across Eurasia and they have been documented to respond to climate change. We performed a Bayesian genome-environment analysis, by combining local climate data with single nucleotide polymorphisms genotype data from 20 European populations (broadly spanning the species’ continental range). We found 36 genes putatively linked to adaptation to climate. Following an enrichment analysis of biological process Gene Ontology (GO) terms, we identified over-represented terms and pathways among the candidate genes. Because many different genes and GO terms are associated with climate variables, it seems likely that climate adaptation is polygenic and genetically complex. Our findings also suggest that geographical climate adaptation has been occurring since great tits left their Southern European refugia at the end of the last ice age. Finally, we show that substantial climate-associated genetic variation remains, which will be essential for adaptation to future changes.