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.
see also the Perspective by Jorg Tost
Phenology refers to the periodic appearance of life-cycle events and currently receives abundant attention as the effects of global change on phenology are so apparent. Phenology as a discipline ...observes these events and relates their annual variation to variation in climate. But phenology is also studied in other disciplines, each with their own perspective. Evolutionary ecologists study variation in seasonal timing and its fitness consequences, whereas chronobiologists emphasize the periodic nature of life-cycle stages and their underlying timing programmes (e.g. circannual rhythms). The (neuro-) endocrine processes underlying these life-cycle events are studied by physiologists and need to be linked to genes that are explored by molecular geneticists. In order to fully understand variation in phenology, we need to integrate these different perspectives, in particular by combining evolutionary and mechanistic approaches. We use avian research to characterize different perspectives and to highlight integration that has already been achieved. Building on this work, we outline a route towards uniting the different disciplines in a single framework, which may be used to better understand and, more importantly, to forecast climate change impacts on phenology.
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.
see also the Perspective by Melanie J. Heckwolf and Britta S. Meyer
1. Wind is an important abiotic factor that influences an array of biological processes, but it is rarely considered in studies on plant-herbivore interactions. 2. 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. 3. 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. 4. Wind exposure delayed flowering, decreased plant height and increased leaf concentrations of amino acids and glucosinolates. 5. 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. 6. 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. 7. 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.
Eavesdropping on interactions between conspecific animals provides a low‐cost method for assessing other individuals. Asymmetries in territorial counter‐singing interactions in songbirds provide a ...rich source of information for eavesdroppers about differences between the singers. Yet, little is known about the relationship between interactive singing in a natural, low‐arousal context among territorial neighbours and individual traits of males. We used a microphone array to monitor natural counter‐singing interactions in great tits (Parus major) during nest building, at the onset of the breeding season. We quantified song overlapping and song matching for 30 pairs (dyads) of interacting males, singing at their nest, respectively. We then compared these behaviours to five traits for 28 males: body condition, plumage ornamentation, offspring provisioning behaviour, offspring weight and breeding site quality. We found no relationship between a male song overlapping or matching behaviour and any of the measured traits. Therefore, our results do not support the idea that short‐term asymmetries in low‐arousal long‐range singing interactions among neighbours reflect differences in these fitness‐related traits. Instead, our findings suggest that such singing asymmetries have less signal value in the absence of an immediate conflict but instead reflect short‐term motivational differences, as shown in previous investigations.
Neighbouring great tits vocally interacting across territorial boundaries do not reveal fiteness relevant differences in their interactive singing. Eavesdroppers will need to integrate information from more contexts to obtain such information.
The environment experienced during early life is a crucial factor in the life of many organisms. This early life environment has been shown to have profound effects on morphology, physiology and ...fitness. However, the molecular mechanisms that mediate these effects are largely unknown, even though they are essential for our understanding of the processes that induce phenotypic variation in natural populations. DNA methylation is an epigenetic mechanism that has been suggested to explain such environmentally induced phenotypic changes early in life. To investigate whether DNA methylation changes are associated with experimentally induced early developmental effects, we cross‐fostered great tit (Parus major) nestlings and manipulated their brood sizes in a natural study population. We assessed experimental brood size effects on pre‐fledging biometry and behaviour. We linked this to genome‐wide DNA methylation levels of CpG sites in erythrocyte DNA, using 122 individuals and an improved epiGBS2 laboratory protocol. Brood enlargement caused developmental stress and negatively affected nestling condition, predominantly during the second half of the breeding season, when conditions are harsher. Brood enlargement, however, affected nestling DNA methylation in only one CpG site and only if the hatch date was taken into account. In conclusion, this study shows that nutritional stress in enlarged broods does not associate with direct effects on genome‐wide DNA methylation. Future studies should assess whether genome‐wide DNA methylation variation may arise later in life as a consequence of phenotypic changes during early development.
Social relationships can have important fitness consequences, and how well an individual is socially connected often correlates with other behavioral traits. Whether such correlations are caused by ...underlying individual differences in social attraction usually remains unclear, because to identify effects of individual traits on social attraction, it is essential to experimentally exclude the influence of the social partner. Using standardized high-definition video playback on captive great tits (Parus major), we effectively demonstrate the influence of individual traits on the motivation to be near a conspecific. We show that social attraction varied contrastingly with boldness and stimulus novelty. Shyer birds tended to show stronger social attraction when they were confronted with the stimulus bird for the first time. Lower ranked birds showed the overall strongest social attraction. This rank effect remained after experimentally changing dominance ranks by altering group compositions. Moreover, preference for social association tended to increase with a decrease in dominance rank, suggesting that birds plastically change their social preference in relation to their within-group dominance status. Our results provide insight into how social relations can form and change, processes that are key for understanding the long-term consequences of the social environment, and the role individuals might play in influencing this environment themselves.
Signals play a key role in the ecology and evolution of animal populations, influencing processes such as sexual selection and conflict resolution. In many species, sexually selected signals have a ...dual function: attracting mates and repelling rivals. Yet, to what extent males and females under natural conditions differentially respond to such signals remains poorly understood, due to a lack of field studies that simultaneously track both sexes. Using a novel spatial tracking system, we tested whether or not the spatial behavior of male and female great tits (Parus major) changes in relation to the vocal response of a territorial male neighbor to an intruder. We tracked the spatial behavior of male and female great tits (N = 44), 1 hr before and 1 hr after simulating territory intrusions, employing automatized Encounternet radio‐tracking technology. We recorded the spatial and vocal response of the challenged males and quantified attraction and repulsion of neighboring males and females to the intrusion site. We additionally quantified the direct proximity network of the challenged male. The strength of a male's vocal response to an intruder induced sex‐dependent movements in the neighborhood, via female attraction and male repulsion. Stronger vocal responders were older and in better body condition. The proximity networks of the male vocal responders, including the number of sex‐dependent connections and average time spent with connections, however, did not change directly following the intrusion. The effects on neighbor movements suggest that the strength of a male's vocal response can provide relevant social information to both the males and the females in the neighborhood, resulting in both sexes adjusting their spatial behavior in contrasting ways, while the social proximity network remained stable. This study underlines the importance of “silent” eavesdroppers within communication networks for studying the dual functioning and evolution of sexually selected signals.
Sexually selected signals are commonly assumed to have a dual function: attracting mates and repelling rivals; yet, these contrasting responses often remain untested in the field. Using a novel tracking system, we show that the spatial behavior of male and female great tits (Parus major) indeed changes in relation to long‐distance vocal signals of a male neighbor. Stronger signalers were also older and in better body condition, suggesting that the strength of a male's vocal response can simultaneously provide relevant public information to both the males and the females in the neighborhood.
Under sexual selection, mate preferences can evolve for traits advertising fitness benefits. Observed mating patterns (mate choice) are often assumed to represent preference, even though they result ...from the interaction between preference, sampling strategy and environmental factors. Correlating fitness with mate choice instead of preference will therefore lead to confounded conclusions about the role of preference in sexual selection. Here we show that direct fitness benefits underlie mate preferences for genetic characteristics in a unique experiment on wild great tits. In repeated mate preference tests, both sexes preferred mates that had similar heterozygosity levels to themselves, and not those with which they would optimise offspring heterozygosity. In a subsequent field experiment where we cross fostered offspring, foster parents with more similar heterozygosity levels had higher reproductive success, despite the absence of assortative mating patterns. These results support the idea that selection for preference persists despite constraints on mate choice.
Personality traits are heritable and respond to natural selection, but are at the same time influenced by the ontogenetic environment. Epigenetic effects, such as DNA methylation, have been proposed ...as a key mechanism to control personality variation. However, to date little is known about the contribution of epigenetic effects to natural variation in behaviour. Here, we show that great tit (Parus major) lines artificially selected for divergent exploratory behaviour for four generations differ in their DNA methylation levels at the dopamine receptor D4 (DRD4) gene. This D4 receptor is statistically associated with personality traits in both humans and nonhuman animals, including the great tit. Previous work in this songbird failed to detect functional genetic polymorphisms within DRD4 that could account for the gene–trait association. However, our observation supports the idea that DRD4 is functionally involved in exploratory behaviour but that its effects are mediated by DNA methylation. While the exact mechanism underlying the transgenerational consistency of DRD4 methylation remains to be elucidated, this study shows that epigenetic mechanisms are involved in shaping natural variation in personality traits. We outline how this first finding provides a basis for investigating the epigenetic contribution to personality traits in natural systems and its subsequent role for understanding the ecology and evolution of behavioural consistency.
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