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.

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.

Temperature has a causal and plastic effect on timing of breeding in a small songbird.

Phenotypic plasticity is an important mechanism by which an individual can adapt its seasonal timing to predictable, short-term environmental changes by using predictive cues. Identification of these cues is crucial to forecast the response of species to long-term environmental change and to study their potential to adapt. Individual great tits (Parus major) start reproduction early under warmer conditions in the wild, but whether this effect is causal is not well known. We housed 36 pairs of great tits in climate-controlled aviaries and 40 pairs in outdoor aviaries, where they bred under artificial contrasting temperature treatments or in semi-natural conditions, respectively, for two consecutive years, using birds from lines selected for early and late egg laying. We thus obtained laying dates in two different thermal environments for each female. Females bred earlier under warmer conditions in climate-controlled aviaries, but not in outdoor aviaries. The latter was inconsistent with laying dates from our wild population. Further, early selection line females initiated egg laying consistently ∼9 days earlier than late selection line females in outdoor aviaries, but we found no difference in the degree of plasticity (i.e. the sensitivity to temperature) in laying date between selection lines. Because we found that temperature causally affects laying date, climate change will lead to earlier laying. This advancement is, however, unlikely to be sufficient, thereby leading to selection for earlier laying. Our results suggest that natural selection may lead to a change in mean phenotype, but not to a change in the sensitivity of laying dates to temperature.

Exploration of tissue-specific gene expression patterns underlying timing of breeding in contrasting temperature environments in a song bird.

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.

Between- and Within-Individual Variation of Maternal Thyroid Hormone Deposition in Wild Great Tits (Parus major).

Maternal hormones are often considered a mediator of anticipatory maternal effects; namely, mothers adjust maternal hormone transfer to prepare the offspring for the anticipated environment. The flexibility for mothers to adjust hormone transfer is therefore a prerequisite for such anticipatory maternal effects. Nevertheless, previous studies have focused only on the average differences of maternal hormone transfer between groups and neglected the substantial individual variation, despite the fact that individual plasticity in maternal hormone transfer is actually the central assumption. In this study, we studied the between- and within-individual variation of maternal thyroid hormones (THs) in egg yolk of wild great tits (Parus major) and estimated the individual plasticity of maternal yolk THs across environmental temperature, clutch initiation dates, and egg laying order using linear mixed effects models. Interestingly, our models provide statistical evidence that the two main THs-the main biologically active hormone T3 and T4, which is mostly considered a prohormone-exhibited different variation patterns. Yolk T3 showed significant between-individual variation on the average levels, in line with its previously reported moderate heritability. Yolk T4, however, showed significant between-clutch variation in the pattern over the laying sequence, suggesting a great within-individual plasticity. Our findings suggest that the role and function of the hormone within the endocrine axis likely influences its flexibility to respond to environmental change. Whether the flexibility of T4 deposition brings a fitness advantage should be examined along with its potential effects on offspring, which remain to be further investigated.

Fine-tuning of seasonal timing of breeding is regulated downstream in the underlying neuro-endocrine system in a small songbird.

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.

Temporally replicated DNA methylation patterns in great tit using reduced representation bisulfite sequencing.

Seasonal timing of reproduction is an important fitness trait in many plants and animals but the underlying molecular mechanism for this trait is poorly known. DNA methylation is known to affect timing of reproduction in various organisms and is therefore a potential mechanism also in birds. Here we describe genome wide data aiming to detect temporal changes in methylation in relation to timing of breeding using artificial selection lines of great tits (Parus major) exposed to contrasting temperature treatments. Methylation levels of DNA extracted from erythrocytes were examined using reduced representation bisulfite sequencing (RRBS). In total, we obtained sequencing data from 63 libraries over four different time points from 16 birds with on average 20 million quality filtered reads per library. These data describe individual level temporal variation in DNA methylation throughout the breeding season under experimental temperature regimes and provides a resource for future studies investigating the role of temporal changes in DNA methylation in timing of reproduction.

Seasonal Variation in Genome-Wide DNA Methylation Patterns and the Onset of Seasonal Timing of Reproduction in Great Tits.

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.

Artificial light at night shifts daily activity patterns but not the internal clock in the great tit (Parus major).

Artificial light at night has shown a dramatic increase over the last decades and continues to increase. Light at night can have strong effects on the behaviour and physiology of species, which includes changes in the daily timing of activity; a clear example is the advance in dawn song onset in songbirds by low levels of light at night. Although such effects are often referred to as changes in circadian timing, i.e. changes to the internal clock, two alternative mechanisms are possible. First, light at night can change the timing of clock controlled activity, without any change to the clock itself; e.g. by a change in the phase relation between the circadian clock and expression of activity. Second, changes in daily activity can be a direct response to light ('masking'), without any involvement of the circadian system. Here, we studied whether the advance in onset of activity by dim light at night in great tits (Parus major) is indeed attributable to a phase shift of the internal clock. We entrained birds to a normal light/dark (LD) cycle with bright light during daytime and darkness at night, and to a comparable (LDim) schedule with dim light at night. The dim light at night strongly advanced the onset of activity of the birds. After at least six days in LD or LDim, we kept birds in constant darkness (DD) by leaving off all lights so birds would revert to their endogenous, circadian system controlled timing of activity. We found that the timing of onset in DD was not dependent on whether the birds were kept at LD or LDim before the measurement. Thus, the advance of activity under light at night is caused by a direct effect of light rather than a phase shift of the internal clock. This demonstrates that birds are capable of changing their daily activity to low levels of light at night directly, without the need to alter their internal clock.

Recent natural selection causes adaptive evolution of an avian polygenic trait.

We used extensive data from a long-term study of great tits (Parus major) in the United Kingdom and Netherlands to better understand how genetic signatures of selection translate into variation in fitness and phenotypes. We found that genomic regions under differential selection contained candidate genes for bill morphology and used genetic architecture analyses to confirm that these genes, especially the collagen gene COL4A5, explained variation in bill length. COL4A5 variation was associated with reproductive success, which, combined with spatiotemporal patterns of bill length, suggested ongoing selection for longer bills in the United Kingdom. Last, bill length and COL4A5 variation were associated with usage of feeders, suggesting that longer bills may have evolved in the United Kingdom as a response to supplementary feeding.