Genetic and epigenetic differentiation in response to genomic selection for avian lay date.

Anthropogenic climate change has led to globally increasing temperatures at an unprecedented pace and, to persist, wild species have to adapt to their changing world. We, however, often fail to derive reliable predictions of species' adaptive potential. Genomic selection represents a powerful tool to investigate the adaptive potential of a species, but constitutes a 'blind process' with regard to the underlying genomic architecture of the relevant phenotypes. Here, we used great tit (Parus major) females from a genomic selection experiment for avian lay date to zoom into this blind process. We aimed to identify the genetic variants that responded to genomic selection and epigenetic variants that accompanied this response and, this way, might reflect heritable genetic variation at the epigenetic level. We applied whole genome bisulfite sequencing to blood samples of individual great tit females from the third generation of bidirectional genomic selection lines for early and late lay date. Genomic selection resulted in differences at both the genetic and epigenetic level. Genetic variants that showed signatures of selection were located within genes mostly linked to brain development and functioning, including LOC107203824 (SOX3-like). SOX3 is a transcription factor that is required for normal hypothalamo-pituitary axis development and functioning, an essential part of the reproductive axis. As for epigenetic differentiation, the early selection line showed hypomethylation relative to the late selection line. Sites with differential DNA methylation were located in genes important for various biological processes, including gonadal functioning (e.g., MSTN and PIK3CB). Overall, genomic selection for avian lay date provided insights into where within the genome the heritable genetic variation for lay date, on which selection can operate, resides and indicates that some of this variation might be reflected by epigenetic variants.

Genotypes selected for early and late avian lay date differ in their phenotype, but not fitness, in the wild.

Global warming has shifted phenological traits in many species, but whether species are able to track further increasing temperatures depends on the fitness consequences of additional shifts in phenological traits. To test this, we measured phenology and fitness of great tits (Parus major) with genotypes for extremely early and late egg lay dates, obtained from a genomic selection experiment. Females with early genotypes advanced lay dates relative to females with late genotypes, but not relative to nonselected females. Females with early and late genotypes did not differ in the number of fledglings produced, in line with the weak effect of lay date on the number of fledglings produced by nonselected females in the years of the experiment. Our study is the first application of genomic selection in the wild and led to an asymmetric phenotypic response that indicates the presence of constraints toward early, but not late, lay dates.

An ecologist's guide for studying DNA methylation variation in wild vertebrates.

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.

Performance of methods to detect genetic variants from bisulphite sequencing data in a non-model species.

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.

Recent natural variability in global warming weakened phenological mismatch and selection on seasonal timing in great tits (Parus major).

Climate change has led to phenological shifts in many species, but with large variation in magnitude among species and trophic levels. The poster child example of the resulting phenological mismatches between the phenology of predators and their prey is the great tit (Parus major), where this mismatch led to directional selection for earlier seasonal breeding. Natural climate variability can obscure the impacts of climate change over certain periods, weakening phenological mismatching and selection. Here, we show that selection on seasonal timing indeed weakened significantly over the past two decades as increases in late spring temperatures have slowed down. Consequently, there has been no further advancement in the date of peak caterpillar food abundance, while great tit phenology has continued to advance, thereby weakening the phenological mismatch. We thus show that the relationships between temperature, phenologies of prey and predator, and selection on predator phenology are robust, also in times of a slowdown of warming. Using projected temperatures from a large ensemble of climate simulations that take natural climate variability into account, we show that prey phenology is again projected to advance faster than great tit phenology in the coming decades, and therefore that long-term global warming will intensify phenological mismatches.

Rapid changes in DNA methylation associated with the initiation of reproduction in a small songbird.

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.

Temporal changes in DNA methylation and RNA expression in a small song bird: within- and between-tissue comparisons.

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.