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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 ~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 (F ST ), 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., (© 2024 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Trees release Herbivore-Induced Plant Volatiles (HIPVs) into the air in response to damage inflicted by insects. It is known that songbirds use those compounds to locate their prey, but more recently the idea emerged that songbirds could also use those odours as cues in their reproductive decisions, as early spring HIPVs may contain information about the seasonal timing and abundance of insects. We exposed pre-breeding great tits (Parus major) to the odours of caterpillar-infested trees under controlled conditions, and monitored reproduction (timing of egg laying, number of eggs, egg size) and two of its main hormonal drivers (testosterone and 17β-estradiol in males and females, respectively). We found that females exposed to HIPVs did not advance their laying dates, nor laid larger clutches, or larger eggs compared to control females. 17β-estradiol concentrations in females were also similar between experimental and control birds. However, males exposed to HIPVs had higher testosterone concentrations during the egg-laying period. Our study supports the hypothesis that insectivorous songbirds are able to detect minute amounts of plant odours. The sole manipulation of plant scents was not sufficient to lure females into a higher reproductive investment, but males increased their reproductive effort in response to a novel source of information for seasonal breeding birds., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024. Published by Elsevier Inc.)

Disentangling the interaction between the genetic basis and environmental context underlying phenotypic variation is critical for understanding organismal evolution. Environmental change, such as increased rates of urbanization, can induce shifts in phenotypic plasticity with some individuals adapting to city life while others are displaced. A key trait that can facilitate adaptation is the degree at which animals respond to stressors. This stress response, which includes elevation of baseline circulating concentrations of glucocorticoids, has a heritable component and exhibits intra- and inter-individual variation. However, the mechanisms behind this variability and whether they might be responsible for adaptation to different environments are not known. Variation in DNA methylation can be a potential mechanism that mediates environmental effects on the stress response, as early-life stressors increase glucocorticoid concentrations and change adult phenotype. We used an inter- and intra-environmental cross-foster experiment to analyse the contribution of DNA methylation to early-life phenotypic variation. We found that at hatching, urban house wren (Troglodytes aedon) offspring had higher methylation frequencies compared with their rural counterparts. We also observed age-related patterns in offspring methylation, indicating the developmental effects of the rearing environment on methylation. At fledgling, differential methylation analyses showed that cellular respiration genes were differentially methylated in broods of different origins and behavioural and metabolism genes were differentially methylated in broods of different rearing environments. Lastly, hyper-methylation of a single gene (CNTNAP2) is associated with decreased glucocorticoid levels and the rearing environment. These differential methylation patterns linked to a specific physiological phenotype suggest that DNA methylation may be a mechanism by which individuals adjust to novel environments during their lifespan. Characterizing genetic and environmental influences on methylation is critical for understanding the role of epigenetic mechanisms in evolutionary adaptation., (© 2022 The Authors. Journal of Animal Ecology © 2022 British Ecological Society.)

Identifying the environmental drivers of variation in fitness-related traits is a central objective in ecology and evolutionary biology. Temporal fluctuations of these environmental drivers are often synchronized at large spatial scales. Yet, whether synchronous environmental conditions can generate spatial synchrony in fitness-related trait values (i.e., correlated temporal trait fluctuations across populations) is poorly understood. Using data from long-term monitored populations of blue tits (Cyanistes caeruleus, n = 31), great tits (Parus major, n = 35), and pied flycatchers (Ficedula hypoleuca, n = 20) across Europe, we assessed the influence of two local climatic variables (mean temperature and mean precipitation in February-May) on spatial synchrony in three fitness-related traits: laying date, clutch size, and fledgling number. We found a high degree of spatial synchrony in laying date but a lower degree in clutch size and fledgling number for each species. Temperature strongly influenced spatial synchrony in laying date for resident blue tits and great tits but not for migratory pied flycatchers. This is a relevant finding in the context of environmental impacts on populations because spatial synchrony in fitness-related trait values among populations may influence fluctuations in vital rates or population abundances. If environmentally induced spatial synchrony in fitness-related traits increases the spatial synchrony in vital rates or population abundances, this will ultimately increase the risk of extinction for populations and species. Assessing how environmental conditions influence spatiotemporal variation in trait values improves our mechanistic understanding of environmental impacts on populations., (© 2022 The Authors. Ecology published by Wiley Periodicals LLC on behalf of The Ecological Society of America.)

The phenology of many species shows strong sensitivity to climate change; however, with few large scale intra-specific studies it is unclear how such sensitivity varies over a species' range. We document large intra-specific variation in phenological sensitivity to temperature using laying date information from 67 populations of two co-familial European songbirds, the great tit (Parus major) and blue tit (Cyanistes caeruleus), covering a large part of their breeding range. Populations inhabiting deciduous habitats showed stronger phenological sensitivity than those in evergreen and mixed habitats. However, populations with higher sensitivity tended to have experienced less rapid change in climate over the past decades, such that populations with high phenological sensitivity will not necessarily exhibit the strongest phenological advancement. Our results show that to effectively assess the impact of climate change on phenology across a species' range it will be necessary to account for intra-specific variation in phenological sensitivity, climate change exposure, and the ecological characteristics of a population., (© 2022. The Author(s).)

The profiling of epigenetic marks like DNA methylation has become a central aspect of studies in evolution and ecology. Bisulphite sequencing is commonly used for assessing genome-wide DNA methylation at single nucleotide resolution but these data can also provide information on genetic variants like single nucleotide polymorphisms (SNPs). However, bisulphite conversion causes unmethylated cytosines to appear as thymines, complicating the alignment and subsequent SNP calling. Several tools have been developed to overcome this challenge, but there is no independent evaluation of such tools for non-model species, which often lack genomic references. Here, we used whole-genome bisulphite sequencing (WGBS) data from four female great tits (Parus major) to evaluate the performance of seven tools for SNP calling from bisulphite sequencing data. We used SNPs from whole-genome resequencing data of the same samples as baseline SNPs to assess common performance metrics like sensitivity, precision, and the number of true positive, false positive, and false negative SNPs for the full range of variant and genotype quality values. We found clear differences between the tools in either optimizing precision (Bis-SNP), sensitivity (biscuit), or a compromise between both (all other tools). Overall, the choice of SNP caller strongly depends on which performance parameter should be maximized and whether ascertainment bias should be minimized to optimize downstream analysis, highlighting the need for studies that assess such differences., (© 2021 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

Species with a circannual life cycle need to match the timing of their life history events to the environment to maximize fitness. However, our understanding of how circannual traits such as timing of reproduction are regulated on a molecular level remains limited. Recent studies have implicated that epigenetic mechanisms can be an important part in the processes that regulate circannual traits. Here, we explore the role of DNA methylation in mediating reproductive timing in a seasonally breeding bird species, the great tit (Parus major), using genome-wide DNA methylation data from individual females that were blood sampled repeatedly throughout the breeding season. We demonstrate rapid and directional changes in DNA methylation within the promoter region of several genes, including a key transcription factor (NR5A1) known from earlier studies to be involved in the initiation of timing of reproduction. Interestingly, the observed changes in DNA methylation at NR5A1 identified here are in line with earlier gene expression studies of reproduction in chicken, indicating that the observed shifts in DNA methylation at this gene can have a regulatory role. Our findings provide an important step towards elucidating the genomic mechanism that mediates seasonal timing of a key life history traits and provide support for the idea that epigenetic mechanisms may play an important role in circannual traits., (© 2021 The Authors. Molecular Ecology published by John Wiley & Sons Ltd.)

Background: DNA methylation is likely a key mechanism regulating changes in gene transcription in traits that show temporal fluctuations in response to environmental conditions. To understand the transcriptional role of DNA methylation we need simultaneous within-individual assessment of methylation changes and gene expression changes over time. Within-individual repeated sampling of tissues, which are essential for trait expression is, however, unfeasible (e.g. specific brain regions, liver and ovary for reproductive timing). Here, we explore to what extend between-individual changes in DNA methylation in a tissue accessible for repeated sampling (red blood cells (RBCs)) reflect such patterns in a tissue unavailable for repeated sampling (liver) and how these DNA methylation patterns are associated with gene expression in such inaccessible tissues (hypothalamus, ovary and liver). For this, 18 great tit (Parus major) females were sacrificed at three time points (n = 6 per time point) throughout the pre-laying and egg-laying period and their blood, hypothalamus, ovary and liver were sampled., Results: We simultaneously assessed DNA methylation changes (via reduced representation bisulfite sequencing) and changes in gene expression (via RNA-seq and qPCR) over time. In general, we found a positive correlation between changes in CpG site methylation in RBCs and liver across timepoints. For CpG sites in close proximity to the transcription start site, an increase in RBC methylation over time was associated with a decrease in the expression of the associated gene in the ovary. In contrast, no such association with gene expression was found for CpG site methylation within the gene body or the 10 kb up- and downstream regions adjacent to the gene body., Conclusion: Temporal changes in DNA methylation are largely tissue-general, indicating that changes in RBC methylation can reflect changes in DNA methylation in other, often less accessible, tissues such as the liver in our case. However, associations between temporal changes in DNA methylation with changes in gene expression are mostly tissue- and genomic location-dependent. The observation that temporal changes in DNA methylation within RBCs can relate to changes in gene expression in less accessible tissues is important for a better understanding of how environmental conditions shape traits that temporally change in expression in wild populations.

The search for the hereditary mechanisms underlying quantitative traits traditionally focused on the identification of underlying genomic polymorphisms such as single-nucleotide polymorphisms. It has now become clear that epigenetic mechanisms, such as DNA methylation, can consistently alter gene expression over multiple generations. It is unclear, however, if and how DNA methylation can stably be transferred from one generation to the next and can thereby be a component of the heritable variation of a trait. In this study, we explore whether DNA methylation responds to phenotypic selection using whole-genome and genome-wide bisulfite approaches. We assessed differential erythrocyte DNA methylation patterns between extreme personality types in the Great Tit (Parus major). For this, we used individuals from a four-generation artificial bi-directional selection experiment and siblings from eight F2 inter-cross families. We find no differentially methylated sites when comparing the selected personality lines, providing no evidence for the so-called epialleles associated with exploratory behavior. Using a pair-wise sibling design in the F2 intercrosses, we show that the genome-wide DNA methylation profiles of individuals are mainly explained by family structure, indicating that the majority of variation in DNA methylation in CpG sites between individuals can be explained by genetic differences. Although we found some candidates explaining behavioral differences between F2 siblings, we could not confirm this with a whole-genome approach, thereby confirming the absence of epialleles in these F2 intercrosses. We conclude that while epigenetic variation may underlie phenotypic variation in behavioral traits, we were not able to find evidence that DNA methylation can explain heritable variation in personality traits in Great Tits., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

The timing of breeding is under selection in wild populations as a result of climate change, and understanding the underlying physiological processes mediating this timing provides insight into the potential rate of adaptation. Current knowledge on this variation in physiology is, however, mostly limited to males. We assessed whether individual differences in the timing of breeding in females are reflected in differences in candidate gene expression and, if so, whether these differences occur in the upstream (hypothalamus) or downstream (ovary and liver) parts of the neuroendocrine system. We used 72 female great tits from two generations of lines artificially selected for early and late egg laying, which were housed in climate-controlled aviaries and went through two breeding cycles within 1 year. In the first breeding season we obtained individual egg-laying dates, while in the second breeding season, using the same individuals, we sampled several tissues at three time points based on the timing of the first breeding attempt. For each tissue, mRNA expression levels were measured using qPCR for a set of candidate genes associated with the timing of reproduction and subsequently analysed for differences between generations, time points and individual timing of breeding. We found differences in gene expression between generations in all tissues, with the most pronounced differences in the hypothalamus. Differences between time points, and early- and late-laying females, were found exclusively in the ovary and liver. Altogether, we show that fine-tuning of the seasonal timing of breeding, and thereby the opportunity for adaptation in the neuroendocrine system, is regulated mostly downstream in the neuro-endocrine system., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

Background: Seasonal timing of breeding is a life history trait with major fitness consequences but the genetic basis of the physiological mechanism underlying it, and how gene expression is affected by date and temperature, is not well known. In order to study this, we measured patterns of gene expression over different time points in three different tissues of the hypothalamic-pituitary-gonadal-liver axis, and investigated specifically how temperature affects this axis during breeding. We studied female great tits (Parus major) from lines artificially selected for early and late timing of breeding that were housed in two contrasting temperature environments in climate-controlled aviaries. We collected hypothalamus, liver and ovary samples at three different time points (before and after onset of egg-laying). For each tissue, we sequenced whole transcriptomes of 12 pools (n = 3 females) to analyse gene expression., Results: Birds from the selection lines differed in expression especially for one gene with clear reproductive functions, zona pellucida glycoprotein 4 (ZP4), which has also been shown to be under selection in these lines. Genes were differentially expressed at different time points in all tissues and most of the differentially expressed genes between the two temperature treatments were found in the liver. We identified a set of hub genes from all the tissues which showed high association to hormonal functions, suggesting that they have a core function in timing of breeding. We also found ample differentially expressed genes with largely unknown functions in birds., Conclusions: We found differentially expressed genes associated with selection line and temperature treatment. Interestingly, the latter mainly in the liver suggesting that temperature effects on egg-laying date may happen down-stream in the physiological pathway. These findings, as well as our datasets, will further the knowledge of the mechanisms of tissue-specific avian seasonality in the future.

Chromosome inversions have clear effects on genome evolution and have been associated with speciation, adaptation, and the evolution of the sex chromosomes. In birds, these inversions may play an important role in hybridization of species and disassortative mating. We identified a large (≈64 Mb) inversion polymorphism in the great tit (Parus major) that encompasses almost 1,000 genes and more than 90% of Chromosome 1A. The inversion occurs at a low frequency in a set of over 2,300 genotyped great tits in the Netherlands with only 5% of the birds being heterozygous for the inversion. In an additional analysis of 29 resequenced birds from across Europe, we found two heterozygotes. The likely inversion breakpoints show considerable genomic complexity, including multiple copy number variable segments. We identified different haplotypes for the inversion, which differ in the degree of recombination in the center of the chromosome. Overall, this remarkable genetic variant is widespread among distinct great tit populations and future studies of the inversion haplotype, including how it affects the fitness of carriers, may help to understand the mechanisms that maintain it., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Seasonal timing of reproduction is a key life-history trait, but we know little about the mechanisms underlying individual variation in female endocrine profiles associated with reproduction. In birds, 17β-oestradiol is a key reproductive hormone that links brain neuroendocrine mechanisms, involved in information processing and decision-making, to downstream mechanisms in the liver, where egg-yolk is produced. Here, we test, using a simulated induction of the reproductive system through a Gonadotropin-Releasing Hormone (GnRH) challenge, whether the ovary of pre-breeding female great tits responds to brain stimulation by increasing oestradiol. We also assess how this response is modified by individual-specific traits like age, ovarian follicle size, and personality, using females from lines artificially selected for divergent levels of exploratory behaviour. We show that a GnRH injection leads to a rapid increase in circulating concentrations of oestradiol, but responses varied among individuals. Females with more developed ovarian follicles showed stronger responses and females from lines selected for fast exploratory behaviour showed stronger increases compared to females from the slow line, indicating a heritable component. This study shows that the response of the ovary to reproductive stimulation from the brain greatly varies among individuals and that this variation can be attributed to several commonly measured individual traits, which sheds light on the mechanisms shaping heritable endocrine phenotypes.

In seasonal environments, timing of reproduction is a trait with important fitness consequences, but we know little about the molecular mechanisms that underlie the variation in this trait. Recently, several studies put forward DNA methylation as a mechanism regulating seasonal timing of reproduction in both plants and animals. To understand the involvement of DNA methylation in seasonal timing of reproduction, it is necessary to examine within-individual temporal changes in DNA methylation, but such studies are very rare. Here, we use a temporal sampling approach to examine changes in DNA methylation throughout the breeding season in female great tits (Parus major) that were artificially selected for early timing of breeding. These females were housed in climate-controlled aviaries and subjected to two contrasting temperature treatments. Reduced representation bisulfite sequencing on red blood cell derived DNA showed genome-wide temporal changes in more than 40,000 out of the 522,643 CpG sites examined. Although most of these changes were relatively small (mean within-individual change of 6%), the sites that showed a temporal and treatment-specific response in DNA methylation are candidate sites of interest for future studies trying to understand the link between DNA methylation patterns and timing of reproduction., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Background: A widely used approach in next-generation sequencing projects is the alignment of reads to a reference genome. Despite methodological and hardware improvements which have enhanced the efficiency and accuracy of alignments, a significant percentage of reads frequently remain unmapped. Usually, unmapped reads are discarded from the analysis process, but significant biological information and insights can be uncovered from these data. We explored the unmapped DNA (normal and bisulfite treated) and RNA sequence reads of the great tit (Parus major) reference genome individual. From the unmapped reads we generated de novo assemblies, after which the generated sequence contigs were aligned to the NCBI non-redundant nucleotide database using BLAST, identifying the closest known matching sequence., Results: Many of the aligned contigs showed sequence similarity to different bird species and genes that were absent in the great tit reference assembly. Furthermore, there were also contigs that represented known P. major pathogenic species. Most interesting were several species of blood parasites such as Plasmodium and Trypanosoma., Conclusions: Our analyses revealed that meaningful biological information can be found when further exploring unmapped reads. For instance, it is possible to discover sequences that are either absent or misassembled in the reference genome, and sequences that indicate infection or sample contamination. In this study we also propose strategies to aid the capture and interpretation of this information from unmapped reads.

Wind is an important abiotic factor that influences an array of biological processes, but it is rarely considered in studies on plant-herbivore interactions. Here, we tested whether wind exposure could directly or indirectly affect the performance of two insect herbivores, Plutella xylostella and Pieris brassicae, feeding on Brassica nigra plants. In a greenhouse study using a factorial design, B. nigra plants were exposed to different wind regimes generated by fans before and after caterpillars were introduced on plants in an attempt to separate the effects of direct and indirect wind exposure on herbivores. Wind exposure delayed flowering, decreased plant height and increased leaf concentrations of amino acids and glucosinolates. Plant-mediated effects of wind on herbivores, that is effects of exposure of plants to wind prior to herbivore feeding, were generally small. However, development time of both herbivores was extended and adult body mass of P. xylostella was reduced when they were directly exposed to wind. By contrast, wind-exposed adult P. brassicae butterflies were significantly larger, revealing a trade-off between development time and adult size. Based on these results, we conducted a behavioural experiment to study preference by an avian predator, the great tit (Parus major) for last instar P. brassicae caterpillars on plants that were exposed to either control (no wind) or wind (fan-exposed) treatments. Tits captured significantly more caterpillars on still than on wind-exposed plants. Our results suggest that P. brassicae caterpillars are able to perceive the abiotic environment and to trade off the costs of extended development time against the benefits of increased size depending on the perceived risk of predation mediated by wind exposure. Such adaptive phenotypic plasticity in insects has not yet been described in response to wind exposure., (© 2018 The Author. Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.)

Background: Understanding variation in genome structure is essential to understand phenotypic differences within populations and the evolutionary history of species. A promising form of this structural variation is copy number variation (CNV). CNVs can be generated by different recombination mechanisms, such as non-allelic homologous recombination, that rely on specific characteristics of the genome architecture. These structural variants can therefore be more abundant at particular genes ultimately leading to variation in phenotypes under selection. Detailed characterization of CNVs therefore can reveal evolutionary footprints of selection and provide insight in their contribution to phenotypic variation in wild populations., Results: Here we use genotypic data from a long-term population of great tits (Parus major), a widely studied passerine bird in ecology and evolution, to detect CNVs and identify genomic features prevailing within these regions. We used allele intensities and frequencies from high-density SNP array data from 2,175 birds. We detected 41,029 CNVs concatenated into 8,008 distinct CNV regions (CNVRs). We successfully validated 93.75% of the CNVs tested by qPCR, which were sampled at different frequencies and sizes. A mother-daughter family structure allowed for the evaluation of the inheritance of a number of these CNVs. Thereby, only CNVs with 40 probes or more display segregation in accordance with Mendelian inheritance, suggesting a high rate of false negative calls for smaller CNVs. As CNVRs are a coarse-grained map of CNV loci, we also inferred the frequency of coincident CNV start and end breakpoints. We observed frequency-dependent enrichment of these breakpoints at homologous regions, CpG sites and AT-rich intervals. A gene ontology enrichment analyses showed that CNVs are enriched in genes underpinning neural, cardiac and ion transport pathways., Conclusion: Great tit CNVs are present in almost half of the genes and prominent at repetitive-homologous and regulatory regions. Although overlapping genes under selection, the high number of false negatives make neutrality or association tests on CNVs detected here difficult. Therefore, CNVs should be further addressed in the light of their false negative rate and architecture to improve the comprehension of their association with phenotypes and evolutionary history.

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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Variation in the reactivity of the endocrine stress axis is thought to underlie aspects of persistent individual differences in behavior (i.e. animal personality). Previous studies, however, have focused largely on estimating baseline or peak levels of glucocorticoids (CORT), often in captive animals. In contrast, the temporal dynamics of the HPA axis-how quickly it turns on and off, for example-may better indicate how an individual copes with stressors. Moreover, these HPA components might be correlated, thereby representing endocrine suites. Using wild-caught great tits (Parus major) we tested birds for exploratory behavior using a standardized novel environment assay that serves as a validated proxy for personality. We then re-captured a subset of these birds (n=85) and characterized four components of HPA physiology: baseline, endogenous stress response, a dexamethasone (DEX) challenge to estimate the strength of negative feedback, and an adrenocorticotropic hormone (ACTH) challenge to estimate adrenal capacity. We predicted that these four HPA responses would be positively correlated and that less exploratory birds would have a more rapid onset of the stress response (a CORT elevation during the baseline bleed) and weaker negative feedback (higher CORT after DEX). We found support for the first two predictions but not the third. All four components were positively correlated with each other and less exploratory birds exhibited an elevation in CORT during the baseline bleed (<3min from capture). Less exploratory birds, however, did not exhibit weaker negative feedback following the DEX challenge, but did exhibit weaker adrenal capacity. Together, our findings provide partial support for the hypothesis that the temporal reactivity of the HPA axis is linked with consistent individual differences in behavior, with more cautious (slower exploring) individuals exhibiting a faster CORT response., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The spirochete bacterium Borrelia afzelii is the most common cause of Lyme borreliosis in Europe. This tick-borne pathogen can establish systemic infections in rodents but not in birds. However, several field studies have recovered larval Ixodes ricinus ticks infected with B. afzelii from songbirds suggesting successful transmission of B. afzelii. We reviewed the literature to determine which songbird species were the most frequent carriers of B. afzelii-infected I. ricinus larvae and nymphs. We tested experimentally whether B. afzelii is capable of co-feeding transmission on two common European bird species, the blackbird (Turdus merula) and the great tit (Parus major). For each bird species, four naïve individuals were infested with B. afzelii-infected I. ricinus nymphal ticks and pathogen-free larval ticks. None of the co-feeding larvae tested positive for B. afzelii in blackbirds, but a low percentage of infected larvae (3.33%) was observed in great tits. Transstadial transmission of B. afzelii DNA from the engorged nymphs to the adult ticks was observed in both bird species. However, BSK culture found that these spirochetes were not viable. Our study suggests that co-feeding transmission of B. afzelii is not efficient in these two songbird species.

In many seasonally breeding birds, female and male testosterone (T) levels peak at the start of the breeding season, coinciding with pair bonding and nesting activities. Shortly after the onset of egg laying, T levels slowly decline to baseline levels in both sexes, but more rapidly so in females. During this period, T in males may still function to facilitate territorial behaviour, mate guarding and extra pair copulations, either via short lasting peaks or elevated basal levels of the hormone. In some species, however, males become insensitive to increased T after the onset of egg laying. It has been postulated that in these species bi-parental care is essential for offspring survival, as T is known to inhibit paternal care. However, only very few studies have analysed this for females. As females are heavily involved in parental care, they too might become insensitive to T after egg laying. Alternatively, because territorial defence, mate guarding and extra pair copulations are expected to be less important for females than for males, they may not have had the need to evolve a mechanism to become insensitive to T during the period of maternal care, because their natural T levels are never elevated during this part of the breeding season anyway. We tested these alternative hypotheses in female great tits (Parus major). Male great tits have previously been shown to be insensitive to T after egg laying with regard to nestling feeding behaviour (but not song rate). When females had started nest building, we experimentally elevated their T levels up to the nestling feeding phase, and measured incubation behaviour (only females incubate) and reproductive success. T did not significantly affect nest building or egg laying behaviour, although egg laying tended to be delayed in T females. Females with experimentally enhanced T maintained lower temperature during incubation but did not spend less time incubating. This might explain the reduced hatching success of their eggs, smaller brood size and lower number of fledglings we found in this study. As in this species T-dependent behaviour by females during the phase of parental care is not needed, the results support the hypothesis that in this species the need for selection in favour of T-insensitivity did not occur., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recent studies have shown that animals are affected by night-time light exposure. Light is a continuous variable, but our knowledge on how individuals react to different light intensities during the night is limited. We therefore determined the relationship between night light intensity and the behaviour and physiology of great tits (Parus major). We measured daily activity patterns and melatonin levels in 35 males exposed to five different light intensities and found strong, dose-dependent effects. Activity onset was increasingly advanced, and activity offset delayed with higher light intensities. Furthermore, night-time activity increased and melatonin levels measured at midnight decreased with higher intensities. In this experimental study, we demonstrate for the first time dose-dependent effects of artificial light at night on birds' daily activity patterns and melatonin levels. Our results imply that these effects are not limited to a certain threshold, but emerge even when nocturnal light levels are slightly increased. However, in a natural area, these effects may be limited as artificial light levels are commonly low; light intensities drop rapidly with distance from a light source and birds can avoid exposure to light at night. Future studies should thus focus on examining the impact of different intensities of light at night in the wild., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Our study tested whether two European bird-specialized ticks, Ixodes arboricola and I. frontalis, can act as vectors in the transmission cycles of Borrelia burgdorferi s.l. The ticks have contrasting ecologies but share songbird hosts (such as the great tit, Parus major) with the generalist I. ricinus which may therefore act as a bridging vector. In the first phase of the experiment, we obtained Borrelia-infected ornithophilic nymphs by exposing larvae to great tits that had previously been exposed to I. ricinus nymphs carrying a community of genospecies (Borrelia garinii, valaisiana, afzelii, burgdorferi s.s., spielmanii). Skin samples showed that birds selectively amplified B. garinii and B. valaisiana. The spirochetes were transmitted to the ornithophilic ticks and survived moulting, leading to infection rates of 16% and 27% in nymphs of I. arboricola and I. frontalis respectively. In the second phase, pathogen-free great tits were exposed to the Borrelia-infected ornithophilic nymphs. None of these ticks were able to infect the birds, as indicated by the tissue samples. Analysis of xenodiagnostic I. ricinus larvae found no evidence for co-feeding or systemic transmission of B. burgdorferi s.l. These outcomes do not support the occurrence of enzootic cycles of Borrelia burgdorferi s.l. involving songbirds and their specialized ornithophilic ticks., (© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The relationship between the encounter rate of predators with prey and the density of this prey is fundamental to models of predator-prey interactions. The relationship determines, among other variables, the rate at which prey patches are depleted, and hence the impact of predator populations on their prey, and the optimal spatial distribution of foraging effort. Two central assumptions that are made in many models are that encounter rate is directly proportional to prey density and that it is independent of the proportion of prey already removed, other than via the decreased density. We show here, using captive great tits searching for winter moth caterpillars in their natural hiding positions, that neither of these assumptions hold. Encounter rate increased less than directly in proportion to prey density, and it depended not only on the current density of prey, but also on the proportion of prey already removed by previous foragers. Both of these effects are likely to have major consequences for the outcome of predator-prey interactions.

Personalities are general properties of humans and other animals. Different personality traits are phenotypically correlated, and heritabilities of personality traits have been reported in humans and various animals. In great tits, consistent heritable differences have been found in relation to exploration, which is correlated with various other personality traits. In this paper, we investigate whether or not risk-taking behaviour is part of these avian personalities. We found that (i) risk-taking behaviour is repeatable and correlated with exploratory behaviour in wild-caught hand-reared birds, (ii) in a bi-directional selection experiment on 'fast' and 'slow' early exploratory behaviour, bird lines tend to differ in risk-taking behaviour, and (iii) within-nest variation of risk-taking behaviour is smaller than between-nest variation. To show that risk-taking behaviour has a genetic component in a natural bird population, we bred great tits in the laboratory and artificially selected 'high' and 'low' risk-taking behaviour for two generations. Here, we report a realized heritability of 19.3 +/- 3.3% (s.e.m.) for risk-taking behaviour. With these results we show in several ways that risk-taking behaviour is linked to exploratory behaviour, and we therefore have evidence for the existence of avian personalities. Moreover, we prove that there is heritable variation in more than one correlated personality trait in a natural population, which demonstrates the potential for correlated evolution.

Behaviour under conditions of mild stress shows consistent patterns in all vertebrates: exploratory behaviour, boldness, aggressiveness covary in the same way. The existence of highly consistent individual variation in these behavioural strategies, also referred to as personalities or coping styles, allows us to measure the behaviour under standardized conditions on birds bred in captivity, link the standardized measurements to the behaviour under natural conditions and measure natural selection in the field. We have bred the great tit (Parus major), a classical model species for the study of behaviour under natural conditions, in captivity. Here, we report a realized heritability of 54 +/- 5% for early exploratory behaviour, based on four generations of bi-directional artificial selection. In addition to this, we measured hand-reared juveniles and their wild-caught parents in the laboratory. The heritability found in the mid-offspring-mid-parent regression was significantly different from zero. We have thus established the presence of considerable amounts of genetic variation for personality types in a wild bird.

The physiological mechanisms underlying avian seasonal timing of reproduction, a life‐history trait with major fitness consequences, are not well understood. Comparing individuals that have been selected to differ in their timing of breeding may prove to be a promising in studying these mechanisms, making selection lines a valuable tool.We created selection lines for early and late timing of breeding in great tits (Parus major) using genomic selection, that is selection based on multi‐marker genotypes rather than on the phenotype. We took in nestlings (F1 generation) from wild broods of which the mother was either an extremely early ("early line") or extremely late ("late line") breeder. These chicks were then genotyped and, based on their "genomic breeding values" (GEBVs), we selected individuals for early and late line breeding pairs to produce the F2 generation in captivity. The F2 offspring was hand‐reared, genotyped and selected to produce an F3 generation, which were then again genotyped and selected. This way we obtained laying dates in aviaries for F1, F2 and F3 birds.We studied the genetic response to the artificial selection and found increased genetic differentiation between the early and late reproducing selection lines over generations (F1–F3), indicated by both diverging GEBVs and increased fixation indices (FST).We studied the phenotypic response to selection for birds breeding in outdoor breeding aviaries. We found that early line birds laid earlier than late line birds, and this difference increased over the generations (F1–F3), with non‐significant line effects for the F1 and F2, but highly significant line differences for the F3.We also assessed whether there was correlated selection on two traits that are potentially part of the mechanisms underlying seasonal timing: the endogenous free‐running period of the day/night clock (tau) and basal metabolic rate, but found no correlated selection.We have successfully created selection lines on seasonal timing in a wild bird species and obtained an instrument for future studies to investigate the physiological mechanisms underlying timing of breeding, and the genetic variation in these mechanisms, an essential component for evolutionary change in timing of reproduction. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]

Circadian (i.e. daily) regulation of behaviours is thought to provide fitness benefits to organisms by enabling them to anticipate diel changes in the environment, such as sunrise., A common behaviour among socially monogamous songbirds that usually takes place in the early mornings is extra-pair mating, that is copulating with partners outside of the social pair bond., Thus, variation in when individuals begin their daily activity may influence their reproductive success; early risers may be better able to gain copulations and guard their partners, thus minimizing their risk of being cuckolded compared with late risers. Sexual selection may thus play an important role in shaping circadian behaviours, but this assumption has yet to be tested in free-living animals., Here, we experimentally weakened endogenous circadian rhythmicity, and thus, anticipation of dawn in male great tits ( Parus major) in the wild through the subcutaneous administration of implants filled with melatonin shortly before egg-laying began in this population. Melatonin is a hormone released during the dark phase at night and is one important cue animals use to entrain their circadian clock., Experimental individuals delayed the onset of daily activity compared with controls and were more likely to be cuckolded compared with control males. Manipulation did not alter other behavioural traits observed; no difference between treatments was observed in activity levels during the day or in the end time of daily activity., These results strongly support the assumption that selection, particularly sexual selection, shapes the circadian phenotypes of wild vertebrates which enable anticipation of important and predictive diel changes in an individual's biotic and abiotic environment. [ABSTRACT FROM AUTHOR]

Organisms use environmental cues to time their life-cycles and among these cues, photoperiod is the main trigger of reproductive behaviours such as territory defence or song activity. Whether photoperiod is also important for another behaviour closely associated with reproduction, mate choice, is unknown. In many bird species, mate choice occurs at two different times during the annual cycle that strongly differ in daylength: in late winter when photoperiod is short and social mates are chosen, and again around egg-laying when photoperiod is longer and extra-pair mates are chosen. This duality makes the role that photoperiod plays on mate choice behaviours intriguing. We investigated the effect of photoperiod on mate choice using three experimental photoperiodic treatments (9 L:15 D, 14 L:10 D, 18 L:6 D), using blue tits (Cyanistes caeruleus) as a biological model. We show that female choice was stronger under long photoperiods. In addition, female blue tits spent significantly more time near males with long tarsi and long wings. This latter preference was only expressed under long photoperiods, suggesting that some indices of male quality only become significant to females when they are strongly photostimulated, and therefore that females could select their social and extra-pair mates based on different phenotypic traits. These results shed light on the roles that photoperiod may play in stimulating pair-bonding and in refining female selectivity for male traits. [ABSTRACT FROM AUTHOR]

Avian malaria can affect survival and reproduction of their hosts. Two patterns commonly observed in birds are that females have a higher prevalence of malaria than do males and that prevalence decreases with age. The mechanisms behind these patterns remain unclear. However, most studies on blood parasite infections are based on cross-sectional analyses of prevalence, ignoring malaria related mortality and individual changes in infection. Here, we analyse both within-individual changes in malaria prevalence and long-term survival consequences of infection in the Seychelles Warbler ( Acrocephalus sechellensis). Adults were less likely to be infected than juveniles but, contrary to broad patterns previously reported in birds, females were less likely to be infected than males. We show by screening individual birds in two subsequent years that the decline with age is a result both of individual suppression of infection and selective mortality. Birds that were infected early in life had a lower survival rate compared to uninfected birds, but among those that survived to be screened twice the proportion of infected birds had also decreased. Uninfected birds did not become infected later in life. Males were found to be more infected than females in this species possibly because, unlike most birds, males are the dispersing sex and the cost of dispersal may have to be traded against immunity. Infected males took longer to suppress their infection than did females. We conclude that these infections are indeed costly, and that age-related patterns in blood parasite prevalence are influenced both by suppression and selective mortality. [ABSTRACT FROM AUTHOR]

Stress physiology is thought to contribute to individual differences in behaviour. In part this reflects the fact that canonical personality measures consist of responses to challenges, including novel objects and environments. Exposure to novelty is typically assumed to induce a moderate increase in glucocorticoids (CORT), although this has rarely been tested. We tested this assumption using great tits, Parus major , selected for divergent personalities (bold-fast and shy-slow explorers), predicting that the shy birds would exhibit higher CORT following exposure to a novel object. We also scored behavioural responses to the novel object, predicting that bold birds would more frequently approach the novel object and exhibit more abnormal repetitive behaviours. We found that the presence of a novel object did induce a moderate CORT response, but selection lines did not differ in the magnitude of this response. Furthermore, although both selection lines showed a robust CORT elevation to a subsequent restraint stressor, the CORT response was stronger in bold birds and this effect was specific to novel object exposure. Shy birds showed a strong positive phenotypic correlation between CORT concentrations following the novel object exposure and the subsequent restraint stress. Behaviourally, the selection lines differed in their response during novel object exposure: as predicted, bold birds more frequently approached the novel object and shy birds more strongly decreased overall locomotion during the novel object trial, but birds from both selection lines showed significant and similar frequencies of abnormal repetitive behaviours during novel object exposure. Our findings support the hypothesis that personality emerges as a result of correlated selection on behaviour and underlying endocrine mechanisms and suggest that the relationship between endocrine stress physiology and personality is context dependent. [ABSTRACT FROM AUTHOR]

Hormonal pleiotropy—the simultaneous influence of a single hormone on multiple traits—has been hypothesized as an important mechanism underlying personality, and circulating glucocorticoids are central to this idea. A major gap in our understanding is the neural basis for this link. Here we examine the stability and structure of behavioral, endocrine and neuroendocrine traits in a population of songbirds ( Parus major ). Upon identifying stable and covarying behavioral and endocrine traits, we test the hypothesis that risk-averse personalities exhibit a neuroendocrine stress axis that is systemically potentiated—characterized by stronger glucocorticoid reactivity and weaker negative feedback. We show high among-individual variation and covariation (i.e. personality) in risk-taking behaviors and demonstrate that four aspects of glucocorticoid physiology (baseline, stress response, negative feedback strength and adrenal sensitivity) are also repeatable and covary. Further, we establish that high expression of mineralocorticoid and low expression of glucocorticoid receptor in the brain are linked with systemically elevated plasma glucocorticoid levels and more risk-averse personalities. Our findings support the hypothesis that steroid hormones can exert pleiotropic effects that organize behavioral phenotypes and provide novel evidence that neuroendocrine factors robustly explain a large fraction of endocrine and personality variation. [ABSTRACT FROM AUTHOR]