The great tit HapMap project: A continental-scale analysis of genomic variation in a songbird.

A major aim of evolutionary biology is to understand why patterns of genomic diversity vary within taxa and space. Large-scale genomic studies of widespread species are useful for studying how environment and demography shape patterns of genomic divergence. Here, we describe one of the most geographically comprehensive surveys of genomic variation in a wild vertebrate to date; the great tit (Parus major) HapMap project. We screened ca 500,000 SNP markers across 647 individuals from 29 populations, spanning ~30 degrees of latitude and 40 degrees of longitude - almost the entire geographical range of the European subspecies. Genome-wide variation was consistent with a recent colonisation across Europe from a South-East European refugium, with bottlenecks and reduced genetic diversity in island populations. Differentiation across the genome was highly heterogeneous, with clear 'islands of differentiation', even among populations with very low levels of genome-wide differentiation. Low local recombination rates were a strong predictor of high local genomic differentiation (FST), especially in island and peripheral mainland populations, suggesting that the interplay between genetic drift and recombination causes highly heterogeneous differentiation landscapes. We also detected genomic outlier regions that were confined to one or more peripheral great tit populations, probably as a result of recent directional selection at the species' range edges. Haplotype-based measures of selection were related to recombination rate, albeit less strongly, and highlighted population-specific sweeps that likely resulted from positive selection. Our study highlights how comprehensive screens of genomic variation in wild organisms can provide unique insights into spatio-temporal evolutionary dynamics.

Sex-Specific Effects of Blood Serotonin on Reproductive Effort in a Small Passerine.

AbstractLaboratory animal models have shown that blood serotonin levels reflect consistent individual differences in behavioral decision-making and maternal behavior. Serotonin could also help to understand intraspecific variation in reproductive strategies, although the mechanisms are poorly understood. In this study, the relationships of plasma serotonin with breeding parameters and parental behavior were examined in wild great tits (Parus major). Females who laid eggs earlier had higher levels of serotonin in the second half of the nestling period, while no significant relationship of serotonin with clutch size, brood size, and body size was detected. In males, serotonin levels were negatively related to clutch size and brood size and positively related to body size. The association of serotonin with provisioning behavior was sex specific, and acute fear stress induced by a predator presentation did not change this relationship. Food provisioning was positively related to size-corrected serotonin levels in females and negatively related to size-corrected serotonin levels in males. These results suggest that peripheral serotonin is a sensitive marker of parental behavior and reproductive effort in wild birds, while the mechanisms linking this neurotransmitter to reproduction are probably mediated by interplay between the serotonergic system, sex hormones, and other neurotransmitters.

Population differences in the length and early-life dynamics of telomeres among European pied flycatchers.

Telomere length and shortening rate are increasingly being used as biomarkers for long-term costs in ecological and evolutionary studies because of their relationships with survival and fitness. Both early-life conditions and growth, and later-life stressors can create variation in telomere shortening rate. Studies on between-population telomere length and dynamics are scarce, despite the expectation that populations exposed to varying environmental constraints would present divergent telomere length patterns. The pied flycatcher (Ficedula hypoleuca) is a passerine bird breeding across Eurasia (from Spain to western Siberia) and migrating through the Iberian Peninsula to spend the nonbreeding period in sub-Saharan Africa. Thus, different populations show marked differences in migration distance. We studied the large-scale variation of telomere length and early-life dynamics in the pied flycatcher by comparing six European populations across a north-south gradient (Finland, Estonia, England and Spain) predicting a negative effect of migration distance on adult telomere length, and of nestling growth on nestling telomere dynamics. There were clear population differences in telomere length, with English birds from midlatitudes having the longest telomeres. Telomere length did not thus show consistent latitudinal variation and was not linearly linked to differences in migration distance. Early-life telomere shortening rate tended to vary between populations. Fast growth was associated with shorter telomeres in the early life, but faster nestling growth affected telomeres more negatively in northern than southern populations. While the sources of between-population differences in telomere-related biology remain to be more intensively studied, our study illustrates the need to expand telomere studies at the between-population level.

Repeated stimulation of the pituitary-adrenal axis alters offspring phenotype of a wild passerine.

Prolonged stress can have long-lasting effects on an individual's physiology and growth. However, the impact of chronically elevated glucocorticoids on the expression of early antipredator responses is still poorly documented. In this study, I simulated the effect of repeated acute stress on offspring phenotype in free-living pied flycatchers (Ficedula hypoleuca) by administering adrenocorticotropic hormone (ACTH) to nestlings for 6 days. The results showed that frequent induction of stress responses by ACTH injections, independent of parental care, adversely affected offspring final body size, wing length and baseline corticosterone levels. Nestling behavioural activity did not differ between ACTH- and saline-treated groups during exposure to control sounds, whereas behavioural activity during exposure to alarm calls was reduced in manipulated offspring only. I conclude that prolonged physiological stress may have short-term benefits to nest-bound offspring, such as more effective antipredator behaviour, but at the expense of negative effects on body size and developmental speed.

SERT gene polymorphisms are associated with risk-taking behaviour and breeding parameters in wild great tits.

Individual differences in coping with potentially dangerous situations are affected by a combination of genetic and environmental factors. How genetic polymorphisms and behavioural variations are related to fitness is unknown. One of the candidate genes affecting a variety of behavioural processes, including impulsivity, anxiety and mood fluctuations in both humans and other vertebrates, is the serotonin transporter gene (SERT/SLC6A). The aim of this study was to assess an association between SERT genotypes and novelty-seeking and risk-taking behaviours as well as breeding parameters of great tits (Parus major) in a natural environment. We associated polymorphisms in the promoter exonic regions of the SERT gene with parental risk-taking-related behaviour and fitness traits. Our results show that: (1) risk-taking behaviour in our great tit population is linked to single nucleotide polymorphisms in the SERT gene exon 3 and exon 8; (2) the genotype-behaviour associations are consistent with the presence of different stressors; and (3) polymorphisms in exon 8 could be associated with fitness-related traits, such as the start of egg-laying and hatching success. We showed for the first time that genetic variability of SERT plays an important role in shaping individual decision-making that affects fitness in a wild population. However, the results are based on one population and on the polymorphisms that are in a single gene. Therefore, replication studies are needed in order to confirm these preliminary results.

Corticosterone Response as an Age-Specific Mediator of Nestling Body Mass in a Wild Passerine.

The environment can affect individual performance directly via resource availability or indirectly through resource allocation among competing fitness components, such as body growth and maintenance activities related to short-term survival. Corticosterone (CORT), the main glucocorticoid in birds, may be an important mediator of energy allocation to different organismal functions, but its effect on the plasticity of fitness-related traits has rarely been investigated at different ontogenetic stages. Here, we evaluated the role of baseline and stress-induced CORT on nestling development of wild great tits (Parus major) under different growth conditions and at different developmental stages. We found that nestling mass and condition were positively related to stress-induced levels of total CORT and free CORT in the middle of nestling stage but negatively related at the prefledging stage. Our results also showed that stress-induced levels of total CORT can be used as a proxy for bioavailable free CORT. No significant relationship of body mass or condition in either stage was shown with levels of baseline CORT or stress-induced CORT-binding globulin capacity. None of these age-specific relationships depend on brood size manipulation. We conclude that the effects of glucocorticoids on nestling performance are stage specific. The ability to secrete CORT may have beneficial effects on nestling development in the middle of the nestling stage, while it can be adaptive to keep CORT levels at optimal ranges before fledging to give priority to growth-related processes that are important for long-term fitness.

Acute embryonic exposure to corticosterone alters physiology, behaviour and growth in nestlings of a wild passerine.

Maternally-derived glucocorticoids can modify the normal development of young animals. To date, little is known about maternal effects that are mediated by acute embryonic exposure to glucocorticoids. In birds, elevated maternal transmission of corticosterone (CORT) to egg albumen is mainly dependent on acute stress. In this study, we increased CORT levels in the egg albumen of a wild passerine, the great tit (Parus major), breeding in favourable deciduous and less suitable coniferous habitat. Subsequently we measured the somatic growth, baseline and acute glucocorticoid responses, immunity and behaviour of prenatally manipulated offspring with respect to control siblings. We found that prenatally CORT-exposed nestlings had lower baseline CORT levels, a more rapid decline in CORT during recovery from a standardized stressor, and a reduced heterophil/lymphocyte ratio compared with controls. Although stress-induced total CORT levels remained unchanged, free CORT levels were significantly lower and the levels of corticosteroid binding globulins (CBG) significantly higher in experimental offspring. Prenatally CORT-exposed offspring begged longer after hatching than controls. Stress-induced behavioural activity of fledglings did not differ between treatments, while its association with baseline CORT levels was significant in the control group only. The body mass and tarsus length of fledglings was positively affected by manipulation in unfavourable coniferous habitat only. We conclude that maternal effects related to elevated levels of albumen CORT modify diverse aspects of offspring phenotype and potentially increase offspring performance in resource poor environments. Moreover, our results indicate that maternal glucocorticoids may suppress the effect of hormones on behavioural responses.

Predator encounters have spatially extensive impacts on parental behaviour in a breeding bird community.

Predation risk has negative indirect effects on prey fitness, partly mediated through changes in behaviour. Evidence that individuals gather social information from other members of the population suggests that events in a community may impact the behaviour of distant individuals. However, spatially wide-ranging impacts on individual behaviour caused by a predator encounter elsewhere in a community have not been documented before. We investigated the effect of a predator encounter (hawk model presented at a focal nest) on the parental behaviour of pied flycatchers (Ficedula hypoleuca), both at the focal nest and at nearby nests different distances from the predator encounter. We show that nest visitation of both focal pairs and nearby pairs were affected, up to 3 h and 1 h, respectively. Parents also appeared to compensate initial disrupted feeding by later increasing nest visitation rates. This is the first evidence showing that the behaviour of nearby pairs was affected away from an immediate source of risk. Our results indicate that the impacts of short-term predator encounters may immediately extend spatially to the broader community, affecting the behaviour of distant individuals. Information about predators is probably quickly spread by cues such as intra- and heterospecific alarm calls, in communities of different taxa.

Does the interaction between glucocorticoids and insulin-like growth factor 1 predict nestling fitness in a wild passerine?

The crucial question in evolutionary ecology is to find out how physiological traits have coevolved so animals fit their stochastic environments. The plasticity of these different physiological mechanisms is largely mediated by hormones, like glucocorticoids and insulin-like growth factor 1 (IGF-1). Brood size manipulation with nestlings of free-living great tits (Parus major) was carried out to see the way in which plasma IGF-1 and feather corticosterone, a predictor of long-term sustained plasma corticosterone level, are associated across different nutritional conditions and how this association predicts survival during the nestling phase. We showed that the association between levels of IGF-1 and corticosterone depended on physiological condition of nestlings. Namely, there was a positive association between the hormones in nestlings from the decreased broods and a negative association in nestlings from the enlarged broods. Furthermore, we showed that the interaction between levels of IGF-1 and corticosterone was also related with the survival of the nestlings. Our results suggest that signalling pathways of IGF-1 and corticosterone most likely interact with each other in a nutrition-dependent way to maximize the rate of development and survival of nestlings in their stochastic environment.

Context-dependent effects of feather corticosterone on growth rate and fledging success of wild passerine nestlings in heterogeneous habitat.

Life history theory seeks answers to questions about how suites of traits, like growth rate, body mass and survival, have coevolved to maximize the fitness of individuals. In stochastic environments, individual fitness may be closely linked to environmental conditions experienced early in life. When conditions deteriorate, animals have to adapt physiologically to avoid detrimental effects to growth and survival. Hormones such as glucocorticoids are potentially important mediators of developmental plasticity, although their function is quite poorly understood in free-living animals to date. In this study, we used brood-size manipulation in wild great tits (Parus major) to see whether resource (e.g. food) availability can change feather corticosterone levels, somatic growth and fledging success in nestlings raised in habitats of different quality. Recent studies suggest that feather corticosterone offers a long-term hormonal measure for the main avian glucocorticoid by integrating the plasma levels of corticosterone over the whole nestling period. We showed that feather corticosterone, growth rate and fledging success were significantly affected by the treatment only in coniferous forests where growth conditions had a tendency to be poorer than in deciduous forests. We also found that feather corticosterone was negatively related to fledging success, and this effect was more pronounced in coniferous habitat. Our results suggest that feather corticosterone could offer an important physiological measure for nestling performance, mediated by a context-dependent developmental trade-off between immediate and future survival.

Large-scale geographical variation in eggshell metal and calcium content in a passerine bird (Ficedula hypoleuca).

Birds have been used as bioindicators of pollution, such as toxic metals. Levels of pollutants in eggs are especially interesting, as developing birds are more sensitive to detrimental effects of pollutants than adults. Only very few studies have monitored intraspecific, large-scale variation in metal pollution across a species' breeding range. We studied large-scale geographic variation in metal levels in the eggs of a small passerine, the pied flycatcher (Ficedula hypoleuca), sampled from 15 populations across Europe. We measured 10 eggshell elements (As, Cd, Cr, Cu, Ni, Pb, Zn, Se, Sr, and Ca) and several shell characteristics (mass, thickness, porosity, and color). We found significant variation among populations in eggshell metal levels for all metals except copper. Eggshell lead, zinc, and chromium levels decreased from central Europe to the north, in line with the gradient in pollution levels over Europe, thus suggesting that eggshell can be used as an indicator of pollution levels. Eggshell lead levels were also correlated with soil lead levels and pH. Most of the metals were not correlated with eggshell characteristics, with the exception of shell mass, or with breeding success, which may suggest that birds can cope well with the current background exposure levels across Europe.

Acute stress affects the corticosterone level in bird eggs: a case study with great tits (Parus major).

In vertebrates, stress experienced by mothers during the early stages of reproduction is an important source of epigenetic modifications in their offspring. Birds represent excellent models to test such effects as their maternal investment can be quantified in terms of egg quality. Recently, it has been demonstrated that corticosterone (CORT) can be transmitted from a female bird into its eggs. However, there is little published evidence about maternal effects that are mediated by acute stress. In this study, we demonstrated that female great tits Parus major facing an aerial predator during egg formation increased CORT concentration in eggs that were laid the morning after the treatment. By presenting a predator model to each experimental nest twice a day, we found that maternal stress influences corticosterone content in eggs during a time period from albumen production in the magnum until the initial phase of shell secretion, when additional water is added to the egg in the shell gland. We also found a positive correlation between the duration of parental alarm calls and CORT concentration in eggs. In conclusion, the response of female passerines to predatory cues brings about a maternal effect that may have negative consequences for offspring performance. We also suggest that the total duration of the behavioral response to a stressor is an important determinant of CORT levels in the albumen of bird eggs.

Geographical variation in egg mass and egg content in a passerine bird.

Reproductive, phenotypic and life-history traits in many animal and plant taxa show geographic variation, indicating spatial variation in selection regimes. Maternal deposition to avian eggs, such as hormones, antibodies and antioxidants, critically affect development of the offspring, with long-lasting effects on the phenotype and fitness. Little is however known about large-scale geographical patterns of variation in maternal deposition to eggs. We studied geographical variation in egg components of a passerine bird, the pied flycatcher (Ficedula hypoleuca), by collecting samples from 16 populations and measuring egg and yolk mass, albumen lysozyme activity, yolk immunoglobulins, yolk androgens and yolk total carotenoids. We found significant variation among populations in most egg components, but ca. 90% of the variation was among individuals within populations. Population however explained 40% of the variation in carotenoid levels. In contrast to our hypothesis, we found geographical trends only in carotenoids, but not in any of the other egg components. Our results thus suggest high within-population variation and leave little scope for local adaptation and genetic differentiation in deposition of different egg components. The role of these maternally-derived resources in evolutionary change should be further investigated.

Plumage bacterial assemblages in a breeding wild passerine: relationships with ecological factors and body condition.

Microorganisms have been shown to play an important role in shaping the life histories of animals, and it has recently been suggested that feather-degrading bacteria influence the trade-off between parental effort and self-preening behavior in birds. We studied a wild breeding population of great tits (Parus major) to explore habitat-, seasonal-, and sex-related variation in feather-degrading and free-living bacteria inhabiting the birds' yellow ventral feathers and to investigate associations with body condition. The density and species richness of bacterial assemblages was studied using flow cytometry and ribosomal intergenic spacer analysis. The density of studied bacteria declined between the nest-building period and the first brood. The number of bacterial phylotypes per bird was higher in coniferous habitat, while bacterial densities were higher in deciduous habitat. Free-living bacterial density was positively correlated with female mass; conversely, there was a negative correlation between attached bacterial density and female mass during the period of peak reproductive effort. Bacterial species richness was sex dependent, with more diverse bacterial assemblages present on males than females. Thus, this study revealed that bacterial assemblages on the feathers of breeding birds are affected both by life history and ecological factors and are related to body condition.

Geographical trends in the yolk carotenoid composition of the pied flycatcher (Ficedula hypoleuca).

Carotenoids in the egg yolks of birds are considered to be important antioxidants and immune stimulants during the rapid growth of embryos. Yolk carotenoid composition is strongly affected by the carotenoid composition of the female's diet at the time of egg formation. Spatial and temporal differences in carotenoid availability may thus be reflected in yolk concentrations. To assess whether yolk carotenoid concentrations or carotenoid profiles show any large-scale geographical trends or differences among habitats, we collected yolk samples from 16 European populations of the pied flycatcher, Ficedula hypoleuca. We found that the concentrations and proportions of lutein and some other xanthophylls in the egg yolks decreased from Central Europe northwards. The most southern population (which is also the one found at the highest altitude) also showed relatively low carotenoid levels. Concentrations of ß-carotene and zeaxanthin did not show any obvious geographical gradients. Egg yolks also contained proportionally more lutein and other xanthophylls in deciduous than in mixed or coniferous habitats. We suggest that latitudinal gradients in lutein and xanthophylls reflect the lower availability of lutein-rich food items in the northern F. hypoleuca populations and in montane southern populations, which start egg-laying earlier relative to tree phenology than the Central European populations. Similarly, among-habitat variation is likely to reflect the better availability of lutein-rich food in deciduous forests. Our study is the first to indicate that the concentration and profile of yolk carotenoids may show large-scale spatial variation among populations in different parts of the species' geographical range. Further studies are needed to test the fitness effects of this geographical variation.

Antioxidant protection, immune function and growth of nestling great tits Parus major in relation to within-brood hierarchy.

Differences in competitive abilities of siblings in birds can be caused by a combination of hatching asynchrony and intra-clutch variation in egg quality. However, very little is known how within-brood hierarchies affect the allocation of resources between different functions of the body. We examined the effects of within-brood hierarchy on growth of morphological parameters, blood plasma antioxidant protection and immune function of free-living great tit Parus major nestlings. To assure that competitive hierarchies occur, we experimentally delayed the start of incubation of the last two eggs in the clutch. At pre-fledging stage (day 13 post-hatch), late-hatched nestlings were smaller in body mass and wing length when compared to early-hatched nestlings, but no differences between siblings were found in tarsus length, plasma antioxidant potential, uric acid concentration, residual antioxidant potential (from regression with uric acid), hematocrit and response to phytohaemagglutinin injection. In early-hatched nestlings, the antioxidant potential and residual antioxidant potential measured in the middle of nestling period (day 6 post-hatch) were negatively related to body mass growth at early nestling stage, indicating that fast initial growth could reduce antioxidant properties of blood plasma.

Behavioral and physiological responses of nestling pied flycatchers to acoustic stress.

The extended secretion of stress hormones in fully developed animals is known to have profound consequences. However, little is known about the effects of stress on the behavior and physiology of free-living young animals, and how such responses relate to each other. We repeatedly (during 5 consecutive days, 1 h/day) exposed the nestlings of a passerine bird, the pied flycatcher (Ficedula hypoleuca), to recordings of nestling distress calls and examined their behavioral and physiological responses to the stressor on the first and the last day of the experiment (on days 9 and 13 post-hatch, respectively). In comparison with control siblings, stressed nestlings reduced the amount of time that they devoted to vocalization and locomotion and increased levels of circulating corticosterone. In 9-day-old nestlings, the level of stress-induced hormone was negatively related to locomotor activity, but not to the rate of vocalizations. The repeated presentation of the stressor increased the heterophile-to-lymphocyte ratio in nestlings but did not affect nestling growth rate. In 13-day-old nestlings, the level of stress-induced corticosterone was not related to behavioral activity. These results suggest that the high level of corticosterone released by immature nestlings in response to a stressor may promote anti-predator behavior (e.g., passive avoidance behavior). Moreover, repeatedly induced stress may have a cumulative and potentially negative effect on individual physiology.

Conception date affects litter type and foetal sex ratio in female moose in Estonia.

1. The Trivers-Willard model of optimal sex ratios predicts that in polygynous species mothers in better condition should produce more male than female offspring. However, empirical support for this hypothesis in mammals and especially ungulates has been equivocal. This may be because the fitness of mothers has been defined in different ways, reflecting morphological, physiological or behavioural measures of condition. In addition, factors other than maternal condition can influence a mother's fitness. Given that recent studies of wild ungulates have demonstrated the importance of the timing of conception and birth on offspring fitness, litters conceived at different stages of the rut might be expected to exhibit differences in types and embryonic sex ratio. 2. Based on a 6-year survey of the reproductive tracts of female moose harvested in Estonia, we investigated the effect of conception date on the types of litters produced and on the foetal sex ratio. 3. There was a clear relationship between conception date and litter characteristics. Overall, earlier conceived litters were more likely than those conceived late to contain multiple embryos and a high proportion of males. However, while foetal sex ratio varied nonlinearly with conception date in yearlings and subadults, no relationship was found in adults. 4. We conclude that female moose adjust foetal sex ratio and litter type/size depending on their age and the date of conception, and that these adjustments are in accordance with the Trivers-Willard hypothesis if females that conceive earlier are in better condition.

Development of stress response in nestling pied flycatchers.

Birds respond to unpredictable events by secreting corticosterone, which induces various responses to cope with stressful situations. However, the evidence is still elusive whether altricial nestlings perceive and respond to external stressors. We investigated the development of adrenocortical stress response to handling-related stressor in nestlings of a small passerine bird, the pied flycatcher (Ficedula hypoleuca). Nestlings were held in isolation from their parents during the experiment to ensure that they indeed respond to handling, not to parental alarm calls. We found that both 9- and 13-day-old nestlings were able to elicit hormonal stress response. Although baseline as well as stress-induced corticosterone levels rose slightly with age, the magnitude of difference between the control and stress-induced levels remained similar in both age groups. However, comparison with adults showed that the stress response of nestlings prior to fledging was still incomplete and significantly lower than in adults. Overall, our results indicate that altricial nestlings do respond to acute stressors, but on the contrary to previous predictions the development of corticosterone stress response during growth period is not gradual and varies remarkably between different passerine species.

Climatic effects on life-history traits of moose in Estonia.

Weather variables can influence life-history traits of ungulates. In this study, we assessed the suitability of regional climate indices including the NAO and two measures of local climate-the maximal extent of ice on the Baltic Sea (MIE) and absolute values of its annual deviations from the multi-year mean (VMIE)-to examine how density-independent processes influence moose body size and fecundity. We predicted that both winter severity (large values of MIE) and variability (large values of VMIE) depress moose traits (e.g., severe winters increase energy expenditure because of large snow depth or low temperatures, while the warmer than average winters may impose greater energetic demands on thermoregulation due to wet and windy weather, or may have indirect negative effects on summer foraging conditions). We estimated direct, delayed (lag) and cumulative effects of each climate measure. Both MIE and VMIE negatively affected jawbone length, with the effect size varying between the respective climatic indices and among age classes. In contrast to results obtained using local climatic variables, the NAO index had no significant effect on jawbone length. The probability of multiple ovulation was negatively influenced by direct effects of VMIE and delayed effects of MIE and NAO. We conclude that MIE and VMIE capture different aspects of the local climate and that these indices can be used in parallel as determinants of growth and fecundity of northern ungulates in coastal regions of the Baltic Sea.