Kees van Oers

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.

Several reduced-representation bisulfite sequencing methods have been developed in recent years to determine cytosine methylation de novo in nonmodel species. Here, we present epiGBS2, a laboratory protocol based on epiGBS with a revised and user-friendly bioinformatics pipeline for a wide range of species with or without a reference genome. epiGBS2 is cost- and time-efficient and the computational workflow is designed in a user-friendly and reproducible manner. The library protocol allows a flexible choice of restriction enzymes and a double digest. The bioinformatics pipeline was integrated in the Snakemake workflow management system, which makes the pipeline easy to execute and modular, and parameter settings for important computational steps flexible. We implemented bismark for alignment and methylation analysis and we preprocessed alignment files by double masking to enable single nucleotide polymorphism calling with Freebayes (epiFreebayes). The performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and great tit (Parus major), which confirmed its overall good performance. We provide a detailed description of the laboratory protocol and an extensive manual of the bioinformatics pipeline, which is publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.4764652).

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.

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls, and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations.

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.

Territorial animals often use signals to advertise territorial occupancy within their larger home ranges. Songbirds are among the best-studied territorial signaling taxa, and when competitors start singing during a territorial intrusion, residents usually show elevated spatial and vocal responses. These responses could be used by intruders and distant eavesdroppers to predict future responses or to compare responses across competitors. Yet, the extent to which responses of a resident to a territorial intrusion predict its future responses and its overall spatial behavior (home range) within a neighborhood is less well understood. We used wild great tits (Parus major) as a model species in repeated song playback trials, simulating territorial intrusions combined with radio-tracking before and during playback trials. The time spent close to the loudspeaker in response to an initial simulated intrusion predicted the same response variable during a second simulated intrusion on the next day, whereas singing activity during the first simulated intrusion did not predict singing during the second simulated intrusion. We also show that more explorative males (as determined by a novel environment test) and males with smaller home ranges sang more and spent more time near the loudspeaker in response to both simulated intrusions. Thus, by probing residents, intruders can obtain reliable information about subsequent response probabilities, while eavesdroppers from a distance, who can use auditory information only, would not receive sufficient predictive information. Our findings also suggest that males with larger home ranges are more tolerant toward intruders, which could reflect a trade-off between tendencies to respond strongly and to range widely. The lack of predictability of singing activity with regard to responses to future intrusions might explain why territorial animals continuously exchange vocal signals and regularly foray into neighboring territories, as a way to obtain regular information updates. Significance Statement: Animals use experience from interactions with conspecifics in their future decision making, such as mate choice and strategies for conflict resolution. The value of such information depends in part on the predictability of the future behavior of that conspecific. In songbirds, territorial individuals respond to intruders by approach and signaling. Here, we tested in radio-tagged great tits (Parus major) if territorial responses are predictable and are affected by individual and environmental factors. We show that the time spent near the simulated intruder was more predictable than singing activity and that birds with larger home ranges showed weaker responses. These findings suggest that information based on such spatial responses is more useful for future decision making, as compared to vocal information, and that distant eavesdroppers will thus receive less reliable information. Limited predictability may explain why territorial animals continuously exchange vocal signals and foray into neighboring territories, providing opportunities for regular information updates.

Globally increasing levels of artificial light at night (ALAN) are associated with shifting rhythms of behaviour in many wild species. However, it is unclear whether changes in behavioural timing are paralleled by consistent shifts in the molecular clock and its associated physiological pathways. Inconsistent shifts between behavioural and molecular rhythms, and between different tissues and physiological systems, disrupt the circadian system, which coordinates all major body functions. We therefore compared behavioural, transcriptional and metabolomic responses of captive great tits (Parus major) to three ALAN intensities or to dark nights, recording activity and sampling brain, liver, spleen and blood at mid-day and midnight. ALAN advanced wake-up time, and this shift was paralleled by advanced expression of the clock gene BMAL1 in all tissues, suggesting close links between behaviour and clock gene expression across tissues. However, further analysis of gene expression and metabolites revealed that clock shifts were inconsistent across physiological systems. Untargeted metabolomic profiling showed that only 9.7% of the 755 analysed metabolites followed the behavioural shift. This high level of desynchronization indicates that ALAN disrupted the circadian system on a deep, easily overlooked level. Thus, circadian disruption could be a key mediator of health impacts of ALAN on wild animals.

Territorial animals often respond less aggressively to neighbours than strangers. This ‘dear enemy’ effect is hypothesized to be adaptive by reducing unnecessary aggressive interactions with non-threatening individuals. A key prediction of this hypothesis, that individual fitness will be affected by variation in the speed and the extent to which individuals reduce their aggression towards neighbours relative to strangers, has never been tested. We used a series of song playbacks to measure the change in response of male great tits to a simulated establishment of a neighbour on an adjacent territory during early stages of breeding, as an assay of individuals’ tendencies to form dear enemy relationships. Males reduced their approach to the speaker and sang fewer songs on later playback repetitions. However, only some males exhibited dear enemy behaviour by responding more strongly to a subsequent stranger playback, and when the playback procedure was repeated on a subset of males, there was some indication for consistent differences among individuals in the expression of dear enemy behaviour. We monitored nests and analysed offspring paternity to determine male reproductive success. Individuals that exhibited dear enemy behaviour towards the simulated neighbour did not suffer any costs associated with loss of paternity, but there was also no evidence of reproductive benefits, and no net effect on reproductive fitness. The general ability to discriminate between neighbours and strangers is likely adaptive, but benefits are probably difficult to detect because of the indirect link between individual variation in dear enemy behaviour and reproductive fitness and because of the complex range of mechanisms affecting relations with territorial neighbours. Significance statement: The dear enemy effect, in which animals respond less aggressively to familiar neighbours compared to strangers, is probably beneficial because it reduces aggressive interactions with non-threatening individuals. However, no study has ever tested whether there actually are fitness benefits for individuals with a greater tendency to form dear enemy relationships. Our study used experimental playbacks to simulate neighbours and strangers, and we found no relationship between dear enemy behaviour and reproductive success in a songbird. However, our approach to test adaptive hypotheses of this widespread territorial behaviour and our longitudinal playback design to examine the development of familiarity towards a neighbour and discrimination of neighbours and strangers are likely to be important tools to advance our understanding of territorial behaviour and individual recognition.

Pollutants, such as toxic metals, negatively influence organismal health and performance, even leading to population collapses. Studies in model organisms have shown that epigenetic marks, such as DNA methylation, can be modulated by various environmental factors, including pollutants, influencing gene expression, and various organismal traits. Yet experimental data on the effects of pollution on DNA methylation from wild animal populations are largely lacking. We here experimentally investigated for the first time the effects of early-life exposure to environmentally relevant levels of a key pollutant, arsenic (As), on genome-wide DNA methylation in a wild bird population. We experimentally exposed nestlings of great tits (Parus major) to arsenic during their postnatal developmental period (3 to 14 days post-hatching) and compared their erythrocyte DNA methylation levels to those of respective controls. In contrast to predictions, we found no overall hypomethylation in the arsenic group. We found evidence for loci to be differentially methylated between the treatment groups, but for five CpG sites only. Three of the sites were located in gene bodies of zinc finger and BTB domain containing 47 (ZBTB47), HIVEP zinc finger 3 (HIVEP3), and insulin-like growth factor 2 mRNA binding protein 1 (IGF2BP1). Further studies are needed to evaluate whether epigenetic dysregulation is a commonly observed phenomenon in polluted populations and what are the consequences for organism functioning and for population dynamics.

Observing interactions between others can provide important information to individuals. Male songbirds often engage in singing contests where they vary the type and timing of signals and provide eavesdropping individuals with information about their competitiveness. How this information is used and its effect on subsequent spatial behaviour and reproductive decisions of eavesdroppers is not well understood. Here we tested whether great tits use information gathered by eavesdropping on male singing interactions to assess rivals and (potential) mates. We used interactive playback experiments to engage territorial males in song contests with either a more (song overlapping and more persistent singing) or less challenging (song alternating and less persistent singing) intruder. We followed male and female movements by automated radiotracking, determined paternity using microsatellite analysis and maternal investment by quantifying egg weights and provisioning behaviour. We expected that mates of males exposed to the challenging treatment would subsequently foray more often off territory to assess other males and potential extrapair mates and invest less in their broods. Moreover, we expected that neighbours would adjust their foraying behaviour according to information gained by eavesdropping. Females, however, did not alter their foraying behaviour or brood investment and neither female nor male neighbours changed their visiting behaviour to playback territories. Our results provide no evidence that females used information gathered by eavesdropping on asymmetric song interactions in reproductive decisions or that song interactions affected movements across territories in the neighbourhood. Overlapping or singing for a longer time on an intruded upon territory may not always be perceived as a higher level of threat, and reproductive decisions and assessment of familiar individuals are likely to be based on multiple sources of information rather than on a single interaction.

Anthropogenic pollution is known to negatively influence an organism’s physiology, behaviour, and fitness. Epigenetic regulation, such as DNA methylation, has been hypothesized as a potential mechanism to mediate such effects, yet studies in wild species are lacking. We first investigated the effects of early-life exposure to the heavy metal lead (Pb) on DNA methylation levels in a wild population of great tits (Parus major), by experimentally exposing nestlings to Pb at environmentally relevant levels. Secondly, we compared nestling DNA methylation from a population exposed to long-term heavy metal pollution (close to a copper smelter), where birds suffer from pollution-related decrease in food quality, and a control population. For both comparisons, the analysis of about one million CpGs covering most of the annotated genes revealed that pollution-related changes in DNA methylation were not genome wide, but enriched for genes underlying developmental processes. However, the results were not consistent when using binomial or beta binomial regression highlighting the difficulty of modelling variance in CpGs. Our study indicates that post-natal anthropogenic heavy metal exposure can affect methylation levels of development related genes in a wild bird population.

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.

Heterogeneous selection is often proposed as a key mechanism maintaining repeatable behavioral variation ("animal personality") in wild populations. Previous studies largely focused on temporal variation in selection within single populations. The relative importance of spatial versus temporal variation remains unexplored, despite these processes having distinct effects on local adaptation. Using data from >3,500 great tits (Parus major) and 35 nest box plots situated within five West-European populations monitored over 4 to 18 y, we show that selection on exploration behavior varies primarily spatially, across populations, and study plots within populations. Exploration was, simultaneously, selectively neutral in the average population and year. These findings imply that spatial variation in selectionmay represent a primarymechanism maintaining animal personalities, likely promoting the evolution of local adaptation, phenotype-dependent dispersal, and nonrandom settlement. Selection also varied within populations among years, which may counteract local adaptation. Our study underlines the importance of combining multiple spatiotemporal scales in the study of behavioral adaptation.

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.

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.