Extra-pair paternity in two passerine birds breeding in a gradient of urbanisation.

Urbanisation has been increasing worldwide in recent decades, driving environmental change and exerting novel selective pressures on wildlife. Phenotypic differences between urban and rural individuals have been widely documented in several taxa. However, the extent to which urbanisation impacts mating strategies is less known. Here, we investigated extra-pair paternity variation in great tits (Parus major) and blue tits (Cyanistes caeruleus) breeding in nestboxes set in a gradient of urbanisation in Warsaw, Poland, over three breeding seasons. Urbanisation was quantified as the amount of light pollution, noise pollution, impervious surface area (ISA) and tree cover within a 100-m radius around each nestbox. We obtained genotypes for 1213 great tits at 7344 SNP markers and for 1299 blue tits at 9366 SNP markers with a genotyping-by-sequencing method, and inferred extra-pair paternity by computing a genomewide relatedness matrix. We report higher extra-pair paternity in blue tits breeding in more urbanised areas, for example, with higher light pollution and ISA, and lower tree cover. However, no such trend was found in great tits. Late-stage survival of individual nestlings in both species was not associated with paternity or urbanisation proxies, thus we were not able to detect fitness benefits or drawbacks of being an extra-pair offspring in relation to urbanisation. Our results contribute to the growing body of knowledge reporting on the effects of urbanisation on avian ecology and behaviour, and confirm species-specific and population-specific patterns of extra-pair paternity variation.

Seasonal and environmental factors contribute to the variation in the gut microbiome: A large-scale study of a small bird.

Environmental variation can shape the gut microbiome, but broad/large-scale data on among and within-population heterogeneity in the gut microbiome and the associated environmental factors of wild populations is lacking. Furthermore, previous studies have limited taxonomical coverage, and knowledge about wild avian gut microbiomes is still scarce. We investigated large-scale environmental variation in the gut microbiome of wild adult great tits across the species' European distribution range. We collected fecal samples to represent the gut microbiome and used the 16S rRNA gene sequencing to characterize the bacterial gut microbiome. Our results show that gut microbiome diversity is higher during winter and that there are compositional differences between winter and summer gut microbiomes. During winter, individuals inhabiting mixed forest habitat show higher gut microbiome diversity, whereas there was no similar association during summer. Also, temperature was found to be a small contributor to compositional differences in the gut microbiome. We did not find significant differences in the gut microbiome among populations, nor any association between latitude, rainfall and the gut microbiome. The results suggest that there is a seasonal change in wild avian gut microbiomes, but that there are still many unknown factors that shape the gut microbiome of wild bird populations.

Measuring body temperature in birds - the effects of sensor type and placement on estimated temperature and metabolic rate.

Several methods are routinely used to measure avian body temperature, but different methods vary in invasiveness. This may cause stress-induced increases in temperature and/or metabolic rate and, hence, overestimation of both parameters. Choosing an adequate temperature measurement method is therefore key to accurately characterizing an animal's thermal and metabolic phenotype. Using great tits (Parus major) and four common methods with different levels of invasiveness (intraperitoneal, cloacal, subcutaneous, cutaneous), we evaluated the preciseness of body temperature measurements and effects on resting metabolic rate (RMR) over a 40°C range of ambient temperatures. None of the methods caused overestimation or underestimation of RMR compared with un-instrumented birds, and body or skin temperature estimates did not differ between methods in thermoneutrality. However, skin temperature was lower compared with all other methods below thermoneutrality. These results provide empirical guidance for future research that aims to measure body temperature and metabolic rate in small bird models.

Early-life environmental effects on mitochondrial aerobic metabolism: a brood size manipulation in wild great tits.

In avian species, the number of chicks in the nest and subsequent sibling competition for food are major components of the offspring's early-life environment. A large brood size is known to affect chick growth, leading in some cases to long-lasting effects for the offspring, such as a decrease in size at fledgling and in survival after fledging. An important pathway underlying different growth patterns could be the variation in offspring mitochondrial metabolism through its central role in converting energy. Here, we performed a brood size manipulation in great tits (Parus major) to unravel its impact on offspring mitochondrial metabolism and reactive oxygen species (ROS) production in red blood cells. We investigated the effects of brood size on chick growth and survival, and tested for long-lasting effects on juvenile mitochondrial metabolism and phenotype. As expected, chicks raised in reduced broods had a higher body mass compared with enlarged and control groups. However, mitochondrial metabolism and ROS production were not significantly affected by the treatment at either chick or juvenile stages. Interestingly, chicks raised in very small broods were smaller in size and had higher mitochondrial metabolic rates. The nest of rearing had a significant effect on nestling mitochondrial metabolism. The contribution of the rearing environment in determining offspring mitochondrial metabolism emphasizes the plasticity of mitochondrial metabolism in relation to the nest environment. This study opens new avenues regarding the effect of postnatal environmental conditions in shaping offspring early-life mitochondrial metabolism.

Individual response in body mass and basal metabolism to the risks of predation and starvation in passerines.

Wintering energy management in small passerines has focused on the adaptive regulation of the daily acquisition of energy reserves within a starvation-predation trade-off framework. However, the possibility that the energetic cost of living, i.e. basal metabolic rate (BMR), is being modulated as part of the management energy strategy has been largely neglected. Here, we addressed this possibility by experimentally exposing captive great tits (Parus major) during winter to two consecutive treatments of increased starvation and predation risk for each individual bird. Body mass and BMR were measured prior to and after each week-long treatment. We predicted that birds should be lighter but with a higher metabolic capacity (higher BMR) as a response to increased predation risk, and that birds should increase internal reserves while reducing their cost of living (lower BMR) when exposed to increased starvation risk. Wintering great tits kept a constant body mass independently of a week-long predation or starvation treatment. However, great tits reduced the cost of living (lower BMR) when exposed to the starvation treatment, while BMR remained unaffected by the predation treatment. Energy management in wintering small birds partly relies on BMR regulation, which challenges the current theoretical framework based on body mass regulation.

Disentangling the causes of age-assortative mating in bird populations with contrasting life-history strategies.

Age shapes fundamental processes related to behaviour, survival and reproduction, where age influences reproductive success, non-random mating with respect to age can magnify or mitigate such effects. Consequently, the correlation in partners' age across a population may influence its productivity. Despite widespread evidence for age-assortative mating, little is known about what drives this assortment and its variation. Specifically, the relative importance of active (same-age mate preference) and passive processes (assortment as a consequence of other spatial or temporal effects) in driving age assortment is not well understood. In this paper, we compare breeding data from a great tit and mute swan population (51- and 31-year datasets, respectively) to tease apart the contributions of pair retention, cohort age structure and active age-related mate selection to age assortment in species with contrasting life histories. Both species show age-assortative mating and variable assortment between years. However, we demonstrate that the drivers of age assortment differ between the species, as expected from their life histories and resultant demographic differences. In great tits, pair fidelity has a weak effect on age-assortative mating through pair retention; variation in age assortment is primarily driven by fluctuations in age structure from variable juvenile recruitment. Age-assortative mating is, therefore, largely passive, with no evidence consistent with active age-related mate selection. In mute swans, age assortment is partly explained by pair retention, but not population age structure, and evidence exists for active age-assortative pairing. This difference is likely to result from shorter life-spans in great tits compared with mute swans, leading to fundamental differences in their population age structure, whereby a larger proportion of great tit populations consist of a single age cohort. In mute swans, age-assortative pairing through mate selection may also be driven by greater age-dependent variation in fitness. The study highlights the importance of considering how different life histories and demographic differences arising from these affect population processes that appear congruent across species. We suggest that future research should focus on uncovering the proximate mechanisms that lead to variation in active age-assortative mate selection (as seen in mute swans); and the consequences of variation in age structure on the ecological and social functioning of wild populations.

Experimental light at night explains differences in activity onset between urban and forest great tits.

Artificial light at night (ALAN) is rapidly increasing and so is scientific interest in its ecological and evolutionary consequences. In wild species, ALAN can modify and disrupt biological rhythms. However, experimental proof of such effects of ALAN in the wild is still scarce. Here, we compared diel rhythms of incubation behaviour, inferred from temperature sensors, of female great tits (Parus major) breeding in urban and forest sites. In parallel, we simulated ALAN by mounting LED lights (1.8 lx) inside forest nest-boxes, to determine the potentially causal role of ALAN affecting diel patterns of incubation. Urban females had an earlier onset of activity compared to forest females. Experimentally ALAN-exposed forest females were similar to urban females in their advanced onset of activity, compared to unexposed forest birds. However, forest females exposed to experimental ALAN, but not urban females, were more restless at night than forest control females. Our findings demonstrate that ALAN can explain the early activity timing in incubating urban great tits, but its effects on sleep disturbance in the forest are not reflected in urban females. Consequently, future research needs to address potential effects of ALAN-induced timing on individual health, fitness and population dynamics, in particular in populations that were not previously affected by light pollution.

Urban tree composition is associated with breeding success of a passerine bird, but effects vary within and between years.

Birds breeding in urban environments have lower reproductive output compared to rural conspecifics, most likely because of food limitation. However, which characteristics of urban environments may cause this deficiency is not clear. Here, we investigated how tree composition within urban territories of passerine birds is associated with breeding probability and reproductive success. We used 7 years of data of breeding occupancy for blue and great tits (Cyanistes caeruleus; Parus major) and several reproductive traits for great tits, from 400 urban nest boxes located in 5 parks within the city of Malmö, Sweden. We found that tits, overall, were less likely to breed in territories dominated by either non-native trees or beech trees. Great tit chicks reared in territories dominated by non-native trees weighed significantly less, compared to territories with fewer non-native trees. An earlier onset of breeding correlated with increased chick weight in great tits. Increasing number of common oak trees (Quercus robur) was associated with delayed onset of breeding in great tits. Notably, as offspring survival probability generally increased by breeding earlier, in particular in oak-dominated territories, our results suggest that delayed onset of breeding induced by oak trees may be maladaptive and indicate a mismatch to this food source. Our results demonstrate that tree composition may have important consequences on breeding success of urban birds, but some of these effects are not consistent between years, highlighting the need to account for temporal effects to understand determinants of breeding success and inform optimal management in urban green spaces.

Effects of competition and predation operating at individual and population levels: an overview of results from a long-term field experiment.

After an overview of the discussion about the existence of intra- and interspecific competition that illustrates the contradictory opinions I conclude that long-term field experiments are needed for firm conclusions. I discuss in some detail the role of two factors that limit population size of secondary cavity nesting birds e.g. territorial behavior and adequate cavities. This is followed by an overview of experimental long-term field studies in Belgium showing that intra- and interspecific competition in a great tit-blue tit system exists. By using nestbox configurations with high densities of nestboxes that differ in the diameter of their entrance hole in replicate study plots it is possible to manipulate the breeding densities of great tit Parus major and blue tit Cyanistes caeruleus independently, thereby varying the intensity of intra- and interspecific competition between these two coexisting species. When blue tit densities are experimentally increased local recruitment of great tits increases, and adult great tit post-breeding dispersal to other study plots decreases, implying that great tits use blue tit density to evaluate habitat quality and that high blue tit density results in heterospecific attraction. The reverse is not true. An experimental increase in great tit density leading to an increase in interspecific competition in a plot where blue tit density was already high leads to a decrease in blue tit nestling mass (illustrating interspecific competition for food), but to a gradual increase in blue tit body size. Both are primarily caused by an increase in the body size of immigrants (caused by intraspecific competition for protected roosting holes) in contrast to the control plot, where neither is observed. I also summarize behavioral, ecological and possible evolutionary effects of sparrowhawks on blue tits after sparrowhawks settled in an isolated study plot halfway through the study: adult survival substantially decreased for both sexes, but more for females that laid large clutches, leading to selection for females that laid a smaller clutch. This led to a change in the reproduction/survival life-history trade-off. Adult winter weights and nestling weights decreased, and the heaviest fledglings were selected against. Furthermore the frequency of polygyny increased. The long-term experiments also document the role of the use of public information and that species that compete can be attracted to sites in which competitor density is high.

First evidence for development of Plasmodium relictum (Grassi and Feletti, 1891) sporozoites in the salivary glands of Culex modestus Ficalbi, 1889.

The competence of insect vectors to transmit diseases plays a key role in host-parasite interactions and in the dynamics of avian malaria and other haemosporidian infections (Apicomplexa, Haemosporida). However, the presence of parasite DNA in the body of blood-sucking insects does not always constitute evidence for their competence as vectors. In this study, we investigate the susceptibility of wild-caught mosquitoes (Culex spp.) to complete sporogony of Plasmodium relictum (cyt b lineage SGS1) isolated from great tits (Parus major L., 1758). Adult female mosquitoes were collected with a CO2 bait trap overnight. A set of 50 mosquitoes was allowed to feed for 3 h at night on a single great tit infected with P. relictum. This trial was repeated on 6 different birds. The bloodfed mosquitoes that survived (n = 68) were dissected within 1-2 days (for ookinetes, n = 10) and 10-33 days post infection (for oocysts and sporozoites, n = 58) in order to confirm the respective parasite stages in their organs. The experiment confirmed the successful development of P. relictum (cyt b lineage SGS1) to the stage of sporozoites in Culex pipiens L., 1758 (n = 27) and in Culex modestus (n = 2). Our study provides the first evidence that C. modestus is a competent vector of P. relictum isolated from great tits, suggesting that this mosquito species could also play a role in the natural transmission of avian malaria.

Global warming modifies the seasonal distribution of clutches on a Mediterranean great tit population.

Global warming has multiple effects on phenological events on a wide range of plants and animals. Specifically, many bird species have advanced the start of their breeding season, which could also imply an extension in its duration and also a change in the distribution of clutches throughout the breeding season. We have tested whether this occurred in a population of Great Tits (Parus major), in Sagunto (eastern Spain). The increase of March temperatures between 1986 and 2019 was related to an advancement of the breeding season. Although the breeding season was longer in years with higher June temperatures, the length did not show a temporal trend throughout the study period. The clutches were more concentrated at the beginning of the season (increase in the skewness), while the kurtosis ("tailedness" of the distribution) or the modality did not change significantly. Finally, the number of "equally good months" for breeding (a combined measure of length and distribution) has not changed throughout the years. Overall, all these phenological changes probably caused the observed increased proportion of pairs laying two clutches per year. It remains to be studied whether this increase in reproductive effort has positive or negative impact on fitness.

Risk-taking coping style correlates with SERT SNP290 polymorphisms in free-living great tits.

The coping style of an individual in relation to potentially dangerous situations has been suggested to be inherited in a polygenic fashion, SERT being one of the candidate genes. In this study, we assessed in free-living great tits (Parus major) the association between SNP290 in the SERT promoter and three standard fear-related behaviors: the response of the birds to a black-and-white flag fixed to the top of the nest box, distress calling rate of the birds in the hand once captured and the hissing call of incubating females when approached by a predator. We found a strong association between SNP290 polymorphism and the three risk-taking behaviors, with birds with genotype CT entering the nest box with the flag faster and displaying more distress calls and fewer hissing calls. CT birds could therefore be described as more proactive than CC individuals. These results also suggest that hissing behavior should be regarded as a fear-induced shy behavior, and confirm that SERT has an important function in relation to risk aversion behaviors and coping style.

Effect of prenatal glucocorticoids and thyroid hormones on developmental plasticity of mitochondrial aerobic metabolism, growth and survival: an experimental test in wild great tits.

Developmental plasticity is partly mediated by transgenerational effects, including those mediated by the maternal endocrine system. Glucocorticoid and thyroid hormones may play central roles in developmental programming through their action on metabolism and growth. However, the mechanisms by which they affect growth and development remain understudied. One hypothesis is that maternal hormones directly affect the production and availability of energy-carrying molecules (e.g. ATP) by their action on mitochondrial function. To test this hypothesis, we experimentally increased glucocorticoid and thyroid hormones in wild great tit eggs (Parus major) to investigate their impact on offspring mitochondrial aerobic metabolism (measured in blood cells), and subsequent growth and survival. We show that prenatal glucocorticoid supplementation affected offspring cellular aerobic metabolism by decreasing mitochondrial density, maximal mitochondrial respiration and oxidative phosphorylation, while increasing the proportion of the maximum capacity being used under endogenous conditions. Prenatal glucocorticoid supplementation only had mild effects on offspring body mass, size and condition during the rearing period, but led to a sex-specific (females only) decrease in body mass a few months after fledging. Contrary to our expectations, thyroid hormone supplementation did not affect offspring growth or mitochondrial metabolism. Recapture probability as juveniles or adults was not significantly affected by prenatal hormonal treatment. Our results demonstrate that prenatal glucocorticoids can affect post-natal mitochondrial density and aerobic metabolism. The weak effects on growth and apparent survival suggest that nestlings were mostly able to compensate for the transient decrease in mitochondrial aerobic metabolism induced by prenatal glucocorticoids.

Effects of nest-box environment on fledgling success rate and pathogen load.

Nest boxes have been used for many decades as tools for conservation and to study avian population dynamics. Plastic is increasingly used as a material for nest boxes, but no studies have investigated effects of this different material. Two consecutive studies were conducted to investigate effects of nest-box environment on nidicolous parasites, bacteria and fungi, as well as nest success, in blue tits Cyanistes caeruleus and great tits Parus major. The first compared microclimate and parasite and pathogen load in plastic and wooden nest boxes. The second tested the nest protection hypothesis ­ that birds naturally incorporate aromatic herbs into nests to decrease nest parasites and pathogens ­ by comparing parasite and pathogen load in plastic nest boxes to which aromatic or non-aromatic plant material was added. No significant difference in nest-box temperature or relative humidity was found between plastic and wooden boxes. Wooden boxes, however, contained 30-fold higher numbers of fleas and a higher total bacterial load on chicks. Fledging success for blue tit broods was significantly higher in wooden boxes. Parasites and bacteria did not decrease by the inclusion of aromatic herbs. The results increase the evidence base for nest-box design in support of plastic, which can provide an appropriate alternative nest-box material to wood, with apparently no difference in microclimate and no increase in the load of measured parasites and pathogens.

Seasonal changes in the hippocampal formation of hoarding and non-hoarding tits.

The hippocampal formation (HF) processes spatial memories for cache locations in food-hoarding birds. Hoarding is a seasonal behavior, and seasonal changes in the HF have been described in some studies, but not in others. One potential reason is that birds may have been sampled during the seasonal hoarding peak in some studies, but not in others. In this study, we investigate the seasonal changes in hoarding and HF in willow tits (Poecile montanus). We compare this to seasonal changes in HF in a closely related non-hoarding bird, the great tit (Parus major). Willow tits near Oulu, Finland, show a seasonal hoarding peak in September and both HF volume and neuron number show a similar peak. HF neuronal density also increases in September, but then remains the same throughout winter. Unexpectedly, the great tit HF also changes seasonally, although in a different pattern: the great tit telencephalon increases in volume from July to August and decreases again in November. Great tit HF volume follows suit, but with a delay. Great tit HF neuron number and density also increase from August to September and stay high throughout winter. We hypothesize that seasonal changes in hoarding birds' HF are driven by food-hoarding experience (e.g., the formation of thousands of memories). The seasonal changes in great tit brains may also be due to experience-dependent plasticity, responding to changes in the social and spatial environment. Large-scale experience-dependent neural plasticity is therefore probably not an adaptation of food-hoarding birds, but a general property of the avian HF and telencephalon.

Egg cooling associated with nest size in a passerine bird.

Breeders evolved adaptive responses to rapid changes in ambient temperature. In birds, nests are expected to reduce egg cooling when the incubator is temporarily off the eggs. Here we present the results of two complementary laboratory experiments aiming at testing the association between egg cooling and the thickness of the nest under and surrounding the eggs in a non-domesticated avian model species (great tit, Parus major). To simulate incubation behaviour, we exposed nests with 4-egg clutches to a heat source until the eggs reached a normal incubation temperature (ca. 39 °C) and then recorded egg cooling 8 min after removal of the heat source, which corresponds to the time females generally leave eggs unattended during the incubation period. Eggs cooled more quickly when the nest layer under the eggs was thinner and when ambient temperature was cooler. We also show that the wall around the nest cup is important to buffer egg cooling. It is hypothesised that in bird nests, both the thickness of the material under the eggs, and the wall surrounding the nest cup interact to maintain a heat envelope around the eggs for the time the incubating parent is foraging. This could explain why the thickness of the nest base and wall are adjusted to the ambient temperature the birds experience during the nest building phase, to anticipate the thermal conditions during incubation and preserve egg viability.

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.

Does Arsenic Contamination Affect DNA Methylation Patterns in a Wild Bird Population? An Experimental Approach.

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.

Human recreation decreases antibody titre in bird nestlings: an overlooked transgenerational effect of disturbance.

Outdoor recreational activities are booming and most animals perceive humans as predators, which triggers behavioural and/or physiological reactions [e.g. heart rate increase, activation of the hypothalamic-pituitary-adrenal (HPA) axis]. Physiological stress reactions have been shown to affect the immune system of an animal and therefore may also affect the amount of maternal antibodies a female transmits to her offspring. A few studies have revealed that the presence of predators affects the amount of maternal antibodies deposited into eggs of birds. In this study, using Eurasian blue and great tit offspring (Cyanistes caeruleus and Parus major) as model species, we experimentally tested whether human recreation induces changes in the amount of circulating antibodies in young nestlings and whether this effect is modulated by habitat and competition. Moreover, we investigated whether these variations in antibody titre in turn have an impact on hatching success and offspring growth. Nestlings of great tit females that had been disturbed by experimental human recreation during egg laying had lower antibody titres compared with control nestlings. Antibody titre of nestling blue tits showed a negative correlation with the presence of great tits, rather than with human disturbance. The hatching success was positively correlated with the average amount of antibodies in great tit nestlings, independent of the treatment. Antibody titre in the first days of life in both species was positively correlated with body mass, but this relationship disappeared at fledging and was independent of treatment. We suggest that human recreation may have caused a stress-driven activation of the HPA axis in breeding females, chronically increasing their circulating corticosterone, which is known to have an immunosuppressive function. Either, lower amounts of antibodies are transmitted to nestlings or impaired transfer mechanisms lead to lower amounts of immunoglobulins in the eggs. Human disturbance could, therefore, have negative effects on nestling survival at early life-stages, when nestlings are heavily reliant on maternal antibodies, and in turn lead to lower breeding success and parental fitness. This is a so far overlooked effect of disturbance on early life in birds.

Artificial light at night, in interaction with spring temperature, modulates timing of reproduction in a passerine bird.

The ecological impact of artificial light at night (ALAN) on phenological events such as reproductive timing is increasingly recognized. In birds, previous experiments under controlled conditions showed that ALAN strongly advances gonadal growth, but effects on egg-laying date are less clear. In particular, effects of ALAN on timing of egg laying are found to be year-dependent, suggesting an interaction with climatic conditions such as spring temperature, which is known have strong effects on the phenology of avian breeding. Thus, we hypothesized that ALAN and temperature interact to regulate timing of reproduction in wild birds. Field studies have suggested that sources of ALAN rich in short wavelengths can lead to stronger advances in egg-laying date. We therefore tested this hypothesis in the Great Tit (Parus major), using a replicated experimental set-up where eight previously unlit forest transects were illuminated with either white, green, or red LED light, or left dark as controls. We measured timing of egg laying for 619 breeding events spread over six consecutive years and obtained temperature data for all sites and years. We detected overall significantly earlier egg-laying dates in the white and green light vs. the dark treatment, and similar trends for red light. However, there was a strong interannual variability in mean egg-laying dates in all treatments, which was explained by spring temperature. We did not detect any fitness consequence of the changed timing of egg laying due to ALAN, which suggests that advancing reproduction in response to ALAN might be adaptive.