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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.

Climate change significantly impacts natural populations, particularly phenology traits, like the seasonal onset of reproduction in birds. This impact is mainly via plastic responses in phenology traits to changes in the environment, but the molecular mechanism mediating this plasticity remains elusive. Epigenetic modifications can mediate plasticity and consequently constitute promising candidates for mediating phenology traits. Here, we used genome-wide DNA methylation profiles of individual great tit (Parus major) females that we blood sampled repeatedly throughout the breeding season. We demonstrate rapid and directional variation in DNA methylation within the regulatory region of genes known to play key roles in avian reproduction that are in line with observed changes in gene expression in chickens. Our findings provide an important step towards unraveling the molecular mechanism mediating a key life history trait, an essential knowledge-gap for understanding how natural populations may cope with future climate change.IMPACT SUMMARYNatural populations are increasingly challenged by changing environmental conditions like global increases in temperature. A key way for species to adapt to global warming is via phenotypic plasticity, i.e. the ability to adjust the expression of traits to the environment. We, however, know little about how the environment can interact with an organism’s genetic make-up to shape its trait value. Epigenetic marks are known to vary with the environment and can modulate the expression of traits without any change in the genetic make-up and therefore have the potential to mediate phenotypic plasticity.To study the role of epigenetics for phenotypic plasticity, we here focus on the great tit (Parus major), a species that is strongly affected by global warming and plastic for temperature in an essential phenology trait, the seasonal onset of egg laying. As a first step, we investigated whether great tit females show within-individual and short-term variation in DNA methylation that corresponds to changes in the reproductive state of females. We therefore housed breeding pairs in climate-controlled aviaries to blood sample each female repeatedly throughout the breeding season and used these repeated samples for methylation profiling.We found rapid and directional variation in DNA methylation at the time females prepared to initiate egg laying that is located within the regulatory region of genes that have previously described functions for avian reproduction. Although future work is needed to establish a causal link between the observed temporal variation in DNA methylation and the onset of reproduction in female great tits, our work highlights the potential role for epigenetic modifications in mediating an essential phenology trait that is sensitive to temperatures.

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. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com.

International audience; 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.

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., Design Type(s)DNA methylation profiling by high throughput sequencing design • time series design • parallel group designMeasurement Type(s)DNA methylationTechnology Type(s)reduced representation bisulfite sequencing assayFactor Type(s)experimental conditionSample Characteristic(s)Parus major • blood • laboratory environment Machine-accessible metadata file describing the reported data (ISA-Tab format)

Epigenetic mechanisms can alter gene expression without a change in the nucleotide sequence and are increasingly recognized as important mechanisms that can generate phenotypic diversity. Most of our current knowledge regarding the origin and role of epigenetic variation comes from research on plants or mammals, often in controlled rearing conditions. Epigenetic research on birds in their natural habitats is still in its infancy, but is needed to answer questions regarding the origin of epigenetic marks and their role in phenotypic variation and evolution. Here we review the potential for studying epigenetic variation in natural bird systems. We aim to provide insights into (1) the origin of epigenetic variation, (2) the relationship between epigenetic variation and trait variation, and (3) the possible role of epigenetic variation in adaptation to changing environments. As there is currently little research on epigenetics in wild birds, we examine how findings on other taxa such as plants and mammals relate to birds. We also examine some of the pros and cons of the most commonly used methods to study patterns of DNA methylation in birds, and suggest some topics we believe need to be addressed to develop the field of wild avian epigenetics further.

The loss of Y-linked genes during sex chromosome evolution creates a potentially deleterious low gene dosage in males. Recent studies have reported different strategies of dosage compensation. Unfortunately, most of these studies investigated taxa with comparatively old sex chromosome systems, which may limit insights into the evolution of dosage compensation and thus into the causes of different compensation strategies. Using deep RNA sequencing, we investigate differential expression patterns along the young XY chromosomes of threespine sticklebacks. Our strata-specific analyses provide new insights into the spatial patterns during the early stages of the evolution of dosage compensation. In particular, our results indicate systematic upregulation of male gene expression in stratum II, which in turn causes female hypertranscription in the same stratum. These findings are consistent with theoretical predictions that selection during early stages of sex chromosome evolution is stronger for a compensating upregulation in males than for the countercompensation of female hyperexpression. In contrast, no elevated gene expression is detectable in stratum I. We argue that strata-specific differences in compensating male gene expression may evolve in response to differences in the prevailing mechanism of Y chromosome degeneration.

In the threespine stickleback, males and females are sexually dimorphic, having slight differences in body morphology but pronounced differences in reproductive behaviors and phenotype during the mating season. Recent interest in sex chromosome evolution and sex-specific gene expression has surged toward understanding the molecular basis of sexual dimorphism in this species. Sex-biased gene expression at the transcriptome level in liver tissue of nonreproductively active fish was reported recently, yet proteome level investigations in the species are scarce. Thus, proteome level investigations will supplement ongoing genomic studies in threespine stickleback. In this study, protein expression in liver was compared between the sexes using label-free quantitation. Over 300 proteins with a 95% confidence level were identified from fish that were not reproductively active. Common functions included metabolism related to amino acids and lipids and energy metabolism characteristic of normal liver function. Of the identified proteins, 5.8% were differentially expressed between the sexes. Male-biased genes had functions related to amino acid metabolism and signaling whereas female-biased genes were related to protein synthesis.

SUMMARYAlthough numerous studies on vertebrates suggest that inbreeding reduces their resistance against parasites and pathogens, studies in insects have found contradictory evidence. In this study we tested the effect of 1 generation of brother–sister mating (inbreeding) on potential and realized immune responses and other life-history traits inTenebrio molitor. We found that inbreeding reduced adult mass, pre-adult survival and increased development time, suggesting that inbreeding reduced the condition of the adults and thus potentially made them more susceptible to physiological stress. However, we found no significant effect of inbreeding on the potential immune response (encapsulation response), but inbreeding reduced the realized immune response (resistance against the entomopathogenic fungi,Beauveria bassiana). There was a significant family effect on encapsulation response, but no family effect on the resistance against the entomopathogenic fungi. Given that this latter trait showed significant inbreeding depression and that the sample size for the family-effect analysis was small it is likely that the lack of a significant family effect is due to reduced statistical power, rather than the lack of a heritable basis to the trait. Our study highlights the importance of using pathogens and parasites in immunoecological studies.

International audience; Evidence implicating differential gene expression as a significant driver of evolutionary novelty continues to accumulate, but our understanding of the underlying sources of variation in expression, both environmental and genetic, is wanting. Heritability in particular may be underestimated when inferred from genetic mapping studies, the predominant "genetical genomics" approach to the study of expression variation. Such uncertainty represents a fundamental limitation to testing for adaptive evolution at the transcriptomic level. By studying the inheritance of expression levels in 10,495 genes (10,527 splice variants) in a threespine stickleback pedigree consisting of 563 individuals, half of which were subjected to a thermal treatment, we show that 74-98% of transcripts exhibit significant additive genetic variance. Dominance variance is also prevalent (41-99% of transcripts), and genetic sources of variation seem to play a more significant role in expression variance in the liver than a key environmental variable, temperature. Among-population comparisons suggest that the majority of differential expression in the liver is likely due to neutral divergence; however, we also show that signatures of directional selection may be more prevalent than those of stabilizing selection. This predominantly aligns with the neutral model of evolution for gene expression but also suggests that natural selection may still act on transcriptional variation in the wild. As genetic variation both within- and among-populations ultimately defines adaptive potential, these results indicate that broad adaptive potential may be found within the transcriptome.

Recent research indicates that low genetic variation in individuals can increase susceptibility to parasite infection, yet evidence from natural invertebrate populations remains scarce. Here, we studied the relationship between genetic heterozygosity, measured as AFLP-based inbreeding coefficient fAFLP , and gregarine parasite burden from eleven damselfly, Calopteryx splendens, populations. We found that in the studied populations, 5-92% of males were parasitized by endoparasitic gregarines (Apicomplexa: Actinocephalidae). Number of parasites ranged from none to 47 parasites per male, and parasites were highly aggregated in a few hosts. Mean individual fAFLP did not differ between populations. Moreover, we found a positive association between individual's inbreeding coefficient and parasite burden. In other words, the more homozygous the individual, the more parasites it harbours. Thus, parasites are likely to pose strong selection pressure against inbreeding and homozygosity. Our results support the heterozygosity-fitness correlation hypothesis, which suggests the importance of heterozygosity for an individual's pathogen resistance.

The implications of transitioning to single nucleotide polymorphism (SNPs) from microsatellite markers (MSs) have been investigated in a number of population genetics studies, but the effect of genomic location on the amount of information each type of marker reveals has not been explored in detail. We developed novel SNP markers flanking 1 kb regions of 13 genic (within gene or1 kb away from gene) and 13 nongenic (10 kb from annotated gene) MSs in the threespine stickleback genome to obtain comparable data for both types of markers. We analysed patterns of genetic diversity and divergence on various geographic scales after converting the SNP loci within each genomic region into haplotypes. Marker type (SNP haplotype or MS) and location (genic or nongenic) significantly affected most estimates of population diversity and divergence. Between-lineage divergence was significantly higher in SNP haplotypes (genic and nongenic), however, within-lineage divergence was similar between marker types. Most divergence and diversity measures were uncorrelated between markers, except for population differentiation which was correlated between MSs and SNP haplotypes (both genic and nongenic). Broad-scale population structure and assignment were similarly resolved by both marker types, however, only the MSs were able to delimit fine-scale population structuring, particularly when genic and nongenic markers were combined. These results demonstrate that estimates of genetic variability and differentiation among populations can be strongly influenced by marker type, their genomic location in relation to genes and by the interaction of these two factors. This highlights the importance of having an awareness of the inherent strengths and limitations associated with different molecular tools to select the most appropriate methods for accurately addressing various ecological and evolutionary questions.

SUMMARYEcological immunology is based upon the notion that activation and use of the immune system is costly and should thus be traded off against other energy-demanding aspects of life history. Most of the studies on insects that have examined the possibility that mating results in trade-offs with immunity have shown that mating has immunosuppressive effects. The connection between mating and immunity has traditionally been investigated using indirect measures of immunity. However, studies that have assessed the effects of mating on the resistance against real pathogens have had conflicting results. A previous study on Tenebrio molitor showed that copulation decreases phenoloxidase activity in the haemolymph, and concluded that copulation corrupts immunity in this species. In the present study we tested whether mating also affects the ability of T. molitor to resist the entomopathogenic fungus, Beauveria bassiana. Interestingly, we found that mating enhanced resistance against the fungal infection and that the effect was stronger on males than females. Furthermore, we found that male beetles were overall more susceptible to the fungal infection than were females, indicating an immunological sex difference in immunity. Our study highlights the importance of the use of real pathogens and parasites in immuno-ecological studies.