Plumage yellowness predicts foraging ability in the blue tit Cyanistes caeruleus

Carotenoid‐based coloration in adult birds has been often regarded as an honest signal of individual quality. However, few studies have demonstrated a link between carotenoid display and the quantity or quality of resources provided to the offspring. The present study investigated the expression of a carotenoid‐based ornament, the breast plumage yellowness of the blue tit Cyanistes caeruleus, in relation to the level of parental provisioning effort and the amount of carotenoid‐rich prey provided to the young. The study was conducted in two forest types (evergreen and deciduous), which also allowed an exploration of the possible existence of habitat effects on the coloration of breeding birds. It was found that plumage colour intensity (carotenoid chroma) correlated positively with nestling provisioning rates of both males and females, supporting the good parent hypothesis. In addition, carotenoid chroma was positively related with the proportion of Lepidoptera larvae brought to the nest in both sexes. Female but not male coloration was positively linked to breeding success (proportion of fledged young). Nestling coloration did not correlate with that of their parents, nor the frequency with which they were fed. Hue and lightness of nestling's plumage correlated positively with body mass and tarsus length, respectively. The results obtained in the present study indicate that ventral plumage coloration in blue tits may advertise the ingested carotenoids (carotenoid foraging ability) and also their overall parental quality in terms of nestling provisioning rates. This suggests that plumage yellowness can be used as an indicator of foraging ability in this species. © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 106 , 418–429.     

Experimentally increased reproductive effort alters telomere length in the blue tit (Cyanistes caeruleus)

Telomeres have recently been suggested to play important role in ageing and are considered to be a reliable ageing biomarkers. The life history theory predicts that costs of reproduction should be expressed in terms of accelerated senescence, and some empirical studies do confirm such presumption. Thus, a link between reproductive effort and telomere dynamics should be anticipated. Recent studies have indeed demonstrated that reproduction may trigger telomere loss, but actual impact of reproductive effort has not received adequate attention in experimental studies. Here, we experimentally manipulated reproductive effort by increasing the brood size in the wild blue tit (Cyanistes caeruleus). We show that parents attending enlarged broods experienced larger yearly telomere decay in comparison to control birds attending unaltered broods. In addition, we demonstrate that the change in telomere length differs between sexes, but this effect was independent from our treatment. To our knowledge, this is the first experimental study in the wild revealing that telomere dynamics may be linked to reproductive effort. Thus, telomere shortening may constitute one of the potential proximate mechanisms mediating the costs of reproduction.     

A genome-wide set of 106 microsatellite markers for the blue tit (Cyanistes caeruleus)

We have characterized a set of 106 microsatellite markers in 26-127 individual blue tits (Cyanistes caeruleus), and assigned their location on the zebra finch (Taeniopygia guttata) and on the chicken (Gallus gallus) genome on the basis of sequence homology. Thirty-one markers are newly designed from zebra finch EST (expressed sequence tags) sequences, 22 markers were developed by others from EST sequences using different methods and the remaining 53 loci were previously designed or modified passerine markers. The 106 microsatellite markers are distributed over 26 and 24 chromosomes in the zebra finch and in the chicken genome respectively and the number of alleles varies between 2 and 49. Eight loci deviate significantly from Hardy-Weinberg equilibrium and show a high frequency of null alleles, and three pairs of markers located in the same chromosome appear to be in linkage disequilibrium. With the exception of these few loci, the polymorphic microsatellite markers presented here provide a useful genome-wide resource for population and evolutionary genetic studies of the blue tit, in addition to their potential utility in other passerine birds.     

MHC class I variation in a natural blue tit population (Cyanistes caeruleus)

The major histocompatibility complex (MHC) is central to the vertebrate immune system and its highly polymorphic genes are considered to influence several life-history traits of individuals. To characterize the MHC in a natural population of blue tits (Cyanistes caeruleus) we investigated the class I exon 3 diversity of more than 900 individuals. We designed two pairs of motif-specific primers that reliably amplify independent subsets of MHC alleles. Applying denaturing gradient gel electrophoresis (DGGE) we obtained 48 independently inherited units of unique band patterns (DGGE-haplogroups), which were validated in a segregation analysis within 105 families. In a second approach, we extensively sequenced 6 unrelated individuals to confirm that DGGE-haplogroup composition reflects individual allelic variation. The highest number of different DGGE-haplogroups in a single individual corresponded in 19 MHC exon 3 sequences, suggesting a minimum of 10 amplified MHC class I loci in the blue tit. In total, we identified 50 unique functional and 3 non-functional sequences. Functional sequences showed high levels of recombination and strong positive selection in the antigen binding region, whereas nucleotide diversity was comparatively low in the range of all passerine species. Finally, in a phylogenetic comparison of passerine MHC class I exon 3 sequences we discuss conflicting evolutionary signals possibly due to recent gene duplication, recombination events and concerted evolution. Our results indicate that the described method is suitable to effectively explore the MHC diversity and its ecological impacts in blue tits in future studies.     

Different population structures in northern and southern populations of the European blue tit (Parus caeruleus)

We sequenced the control region of the mitochondrial DNA from a sample of six European blue tit populations to investigate the phylogeography of Parus species. Along a transect from Barcelona, Spain to Oulu, Finland, the blue tit showed a different phylogeographic structure than the great tit and the willow tit. The southernmost sample from Barcelona consisted of two widely divergent maternal lineages (nucleotide divergence, π = 0.30%), a situation also found earlier in the French Alps. The more northern populations had a relatively uniform structure (π = 0.19%) with distinctive marks of a growing population, thus resembling the great tit populations (π = 0.19%). The amount of genetic variation among blue tits is lower than in the willow tit (π = 0.53%). This probably reflects a smaller long-term effective population size in the great tit and the blue tit than in the willow tit. The different genetic structure of the Barcelona population vs. the rest had an influence on the estimated population parameters, which are calculated based on the assumptions of genetic equilibrium of the populations.     

Bacterial species richness at three stages of the breeding season in Cyanistes caeruleus (blue tit)

Blue tits are exposed to a vast array of bacteria throughout their life cycle and are particularly exposed during a breeding attempt. Any pathogenic bacteria within their microbiome can have a detrimental effect on their fitness and that of the nestlings they are raising. This study aims to identify the bacterial species richness that birds of this species are exposed to during three key stages of the breeding cycle: nest build, clutch completion and immediately post fledging. Nests were swabbed at these time points across four deciduous woodland sites in the United Kingdom and genomic DNA extracted prior to T-RFLP analysis. This is the first known instance of this technique being used to assess the nest microbiome and the first culture independent assessment of nest microbiome within this species. This revealed 103 distinct OTUs (Operational Taxonomic Units) across all sites and stages with an increase in taxa richness at each stage. There were differences in the microbiomes of each nest across breeding stage and site with evidence suggesting the nest microbiome is largely determined by the local environment.     

Individual genetic diversity and probability of infection by avian malaria parasites in blue tits (Cyanistes caeruleus)

Understanding the importance of host genetic diversity for coping with parasites and infectious diseases is a long‐standing goal in evolutionary biology. Here, we study the association between probability of infection by avian malaria (Plasmodium relictum) and individual genetic diversity in three blue tit (Cyanistes caeruleus) populations that strongly differ in prevalence of this parasite. For this purpose, we screened avian malaria infections and genotyped 789 blue tits across 26 microsatellite markers. We used two different arrays of markers: 14 loci classified as neutral and 12 loci classified as putatively functional. We found a significant relationship between probability of infection and host genetic diversity estimated at the subset of neutral markers that was not explained by strong local effects and did not differ among the studied populations. This relationship was not linear, and probability of infection increased up to values of homozygosity by locus (HL) around 0.15, reached a plateau at values of HL from 0.15 to 0.40 and finally declined among a small proportion of highly homozygous individuals (HL > 0.4). We did not find evidence for significant identity disequilibrium, which may have resulted from a low variance of inbreeding in the study populations and/or the small power of our set of markers to detect it. A combination of subtle positive and negative local effects and/or a saturation threshold in the association between probability of infection and host genetic diversity in combination with increased resistance to parasites in highly homozygous individuals may explain the observed negative quadratic relationship. Overall, our study highlights that parasites play an important role in shaping host genetic variation and suggests that the use of large sets of neutral markers may be more appropriate for the study of heterozygosity–fitness correlations.     

Variation in growth in the blue tit (Parus caeruleus)

The pattern and amount of phenotypic and genetic variation in growth was analysed using the infinite-dimensional model in natural populations of the blue tit (Parus caeruleus). A size-index was used to analyse patterns of variation and covariation across ages (2, 5, 8, 11 and 14 days). Genetic variation was analysed using mother-, father-, and midparent-offspring regressions in the blue tit. Phenotypic variation in growth was limited to a few dimensions only, while heritabilities were close to zero at all ages except at 2 days when a significant mother-offspring relation was found. The phenotypic variance-covariance matrix over ages was found to be singular, which suggests that there are trajectories for which there is no phenotypic variation, and which therefore they cannot evolve. The phenotypic growth trajectory associated with the largest amount of variation basically showed a high to moderately high covariation across ages. These results have macroevolutionary implications in terms of the outcome of selection which generally will be in terms of overall size changes but with a slow rate of change due to low heritabilities; an outcome consistent with macro-evolutionary patterns of morphological variation in birds. A simulation study supported the conclusion that high covariances among traits and ages can impose considerable constraint on a microevolutionary scale.     

Intersexual niche differentiation in the blue tit (Parus caeruleus)

The intersexual niche differentiation hypothesis, according to which sexual size dimorphism (SSD) has evolved to facilitate niche separation between the sexes, has received equivocal support in studies of birds. One explanation for the inconsistency of the results is that other factors crucial for reproductive success, such as variation in food abundance among territories, may obscure food niche differentiation between parents. We studied the effects of SSD, food availability and food demands of broods on niche differentiation in the blue tit ( Parus caeruleus ). Parents attending experimentally enlarged broods delivered the same amount, but more diverse food items, to their offspring than parents attending reduced broods, although this effect was significant in only one of the two study years. The time-lag (deviation from the peak food abundance in habitat in days) and squared time-lag were the most important determinants of the diversity of food items delivered to young and their condition (fitness surrogate). SSD, as measured by principal component scores of morphological traits, did not explain any variation neither in the diversity or amount of food provided, nor in offspring condition. These results suggest that synchronization of breeding with peak food availability is the most important determinant of diversity of food delivered to young and, more importantly, Their condition. No evidence for intersexual food niche differentiation was found; at best, it is likely to constitute only a weak selection pressure on the maintenance or enhancement of SSD in this system.     

Sex‐specific heritability of cell‐mediated immune response in the blue tit nestlings (Cyanistes caeruleus)

Here, we aimed at estimating sex‐specific heritabilities of cell‐mediated immune response (CMI) in the blue tit nestlings (Cyanistes caeruleus). To separate genetic and environmental components of the phenotypic variance in CMI (measured using phytohaemagglutinin assay), we performed a cross‐fostering experiment. Additionally, controlled environmental variation was introduced by enlarging some broods. Our analyses revealed a significant genetic component (as approximated by the nest‐of‐origin term) of the phenotypic variance in immune response. More importantly, these genetic effects differed between sexes and experimentally manipulated brood sizes, as indicated by significant genotype‐by‐sex and genotype‐by‐environment interactions. We discuss possible causes of such sexual dimorphism in gene expression and suggest that sex‐ and environment‐specific genetic interactions may contribute to the maintenance of genetic variability in traits related to immune functions.     

Condition-dependent genetic benefits of extrapair fertilization in female blue tits Cyanistes caeruleus

In many socially monogamous birds, both partners perform extrapair copulations (EPC). As this behaviour potentially inflicts direct costs on females, they are currently hypothesized to search for genetic benefits for descendants, either as 'good' or 'complementary' genes. Although these hypotheses have found some support, several studies failed to find any beneficial consequence of EPC, and whether this behaviour is adaptive to females is subject to discussion. Here, we test these two hypotheses in a natural population of blue tits by accounting for the effect of most parameters known to potentially affect extrapair fertilization. Results suggest that female body mass affected the type of extrapair genetic benefits obtained. Heavy females obtained extrapair fertilizations when their social male was of low quality (as reflected by sexual display) and produced larger extrapair than within-pair chicks. Lean females obtained extrapair fertilizations when their social mate was genetically similar, thereby producing more heterozygous extrapair chicks. Our results suggest that mating patterns may be condition-dependent.     

Egg patterning is not a reliable indicator of intraspecific brood parasitism in the blue tit Cyanistes caeruleus

A recent study reported the incidence of intraspecific brood parasitism (IBP) in the blue tit, based on differences in pigmentation between eggs within clutches and the appearance of multiple eggs within a nest in a single day. Here we present data from another population of blue tits Cyanistes caeruleus in which we show that such evidence is not necessarily indicative of IBP. As in many other studies of birds, we found that the patterning and degree of pigmentation on eggs is individually highly characteristic of a female. As in the previous study of blue tits, a minority of clutches (about 3% of 904 clutches) contained one, two or three eggs that were distinctly different from the other eggs in the clutch, either in size or patterning, but not both. Molecular genetic analyses of parentage in a sample of these clutches with suspected IBP demonstrated unequivocally that all eggs within each clutch had in fact been laid by a single female, in all cases the social parent. We also found no evidence of IBP from paternity analyses of a sample of 3,529 offspring from 427 broods in this population, adding to previous evidence of the absence of IBP in this species. These findings cast doubt on the utility of purely observational approaches to provide evidence of intraspecific brood parasitism; our study suggests that egg morphology and records of laying patterns are inadequate to enable the identification of IBP.     

Correlations between heterozygosity and reproductive success in the blue tit (Cyanistes caeruleus): an analysis of inbreeding and single locus effects

To understand the mechanisms behind heterozygosity-fitness correlations (HFC), it is necessary to employ large numbers of markers with known function and independently estimate the variation in inbreeding in the population. Here we genotyped 794 blue tits with 79 microsatellites that were distributed across 25 chromosomes and that were classified either as "functional" (N= 58) or "neutral" (N= 21). We found a positive effect of individual heterozygosity at multiple loci on clutch size, on the number of eggs sired by males, and on the number of recruits produced by males and females. We documented the occurrence of some consanguineous matings and found evidence for a particular type of population structure that can contribute to the occurrence of inbreeding. As the set of "neutral" loci provided more power to detect HFC and identity disequilibrium, we argue that "neutral" markers are better predictors of the effects of inbreeding. The number of significant effects at single loci did not exceed the expected number of false positives and no strong effects were associated with heterozygosity at "functional" markers. Thus, the HFC found here cannot be attributed to strong effects of the loci under study.     

Paternity analysis reveals opposing selection pressures on crown coloration in the blue tit (Parus caeruleus)

In socially monogamous species, extra-pair paternity can increase the variance in reproductive success and thereby the potential for sexual selection on male ornaments. We studied whether male secondary sexual ornaments are selected through within- and/or extra-pair reproductive success in the blue tit (Parus caeruleus). Male blue tits display a bright blue crown plumage, which reflects substantially in the ultraviolet (UV) and previously has been indicated to be an important sexual signal. We show that males with a more UV-shifted crown hue were less cuckolded, which probably resulted from female preference for more ornamented mates. By contrast, however, older males and males with a less UV-shifted hue sired more extra-pair young. This probably did not reflect direct female preference, since cuckolders were not less UV-ornamented than the males they cuckolded. Alternatively, a trade-off between UV ornamentation and other traits that enhance extra-pair success could explain this pattern. Our results might reflect two alternative male mating tactics, where more UV-ornamented males maximize within-pair success and less UV-ornamented males maximize extra-pair success. Since crown colour was selected in opposite directions by within-pair and extra-pair paternity, directional selection through extra-pair matings seemed weak, at least in this population and breeding season. Reduced intensity of sexual selection due to alternative mating tactics constitutes a potential mechanism maintaining additive genetic variance of male ornaments.     

Heterozygosity-based assortative mating in blue tits (Cyanistes caeruleus): implications for the evolution of mate choice

The general hypothesis of mate choice based on non-additive genetic traits suggests that individuals would gain important benefits by choosing genetically dissimilar mates (compatible mate hypothesis) and/or more heterozygous mates (heterozygous mate hypothesis). In this study, we test these hypotheses in a socially monogamous bird, the blue tit (Cyanistes caeruleus). We found no evidence for a relatedness-based mating pattern, but heterozygosity was positively correlated between social mates, suggesting that blue tits may base their mating preferences on partner''s heterozygosity. We found evidence that the observed heterozygosity-based assortative mating could be maintained by both direct and indirect benefits. Heterozygosity reflected individual quality in both sexes: egg production and quality increased with female heterozygosity while more heterozygous males showed higher feeding rates during the brood-rearing period. Further, estimated offspring heterozygosity correlated with both paternal and maternal heterozygosity, suggesting that mating with heterozygous individuals can increase offspring genetic quality. Finally, plumage crown coloration was associated with male heterozygosity, and this could explain unanimous mate preferences for highly heterozygous and more ornamented individuals. Overall, this study suggests that non-additive genetic traits may play an important role in the evolution of mating preferences and offers empirical support to the resolution of the lek paradox from the perspective of the heterozygous mate hypothesis.     

Hormonal responses to male-male social challenge in the blue tit Cyanistes caeruleus: single-broodedness as an explanatory variable

The "challenge hypothesis" posits that when established social orders are challenged, plasma testosterone (T) in socially monogamous breeding male birds will temporarily increase to facilitate aggressive responses. However, not all birds conform to predictions. To expand upon past findings, we examined effects of direct territorial challenge on T levels in the blue tit (Cyanistes caeruleus). We found that simulated territorial intrusions caused a decline in plasma T during both territory establishment and laying/incubation. Conversely, corticosterone (CORT) levels dramatically increased. We also examined challenged blue tit males for levels of corticosterone-binding globulin (CBG), a carrier molecule that displays affinity for both CORT and T in birds. Although the CBG showed increased occupation by CORT during challenge, effects were not accompanied by a significant increase in the unbound T fraction. Thus, competitive hormone interactions on the CBG do not seem sufficient to explain changes in circulating T levels. To place our results within the context of past findings, we compared all socially monogamous birds tested to date for plasma levels of T during situational territorial intrusion experiments. We found that birds raising only one brood per season (e.g., the blue tit) consistently show no increase in plasma T but instead show elevations in circulating CORT. Thus, we suggest that single-broodedness plays an important role in determining patterns of hormone change and should be considered in future discussions of hormone-behavior interactions.     

Local specialization and maladaptation in the Mediterranean blue tit (Parus caeruleus)

Although the evolutionary interplay between gene flow and local adaptation of organisms in heterogeneous environments has been widely discussed from a theoretical point of view, few empirical studies have been designed to test predictions on the consequences of habitat patchiness on the evolution of life history traits. Using blue tits in Mediterranean habitat mosaics as a model, we defined two nested levels of habitat heterogeneity: an inter-regional level which compares two isolated landscapes (mainland, southern France vs the island of Corsica), and an intra-regional level which compares two habitat types within each landscape (deciduous vs evergreen trees). Deciduous habitats are more common than evergreen habitats on the mainland whereas the opposite is true on the island. Results suggest that: (1) on a regional scale, each population is specialized to the more common habitat, i.e. life history traits have evolved in such a way that breeding success is high; (2) in the less common habitats within each landscape, birds are clearly mistimed because they mismatch the best period of food availability, and hence their breeding success is lower; and (3) the density of the populations and the morphometry of the birds support the model of ideal despotic distribution. These results, which are supported by preliminary data on the genetic variation and gene flow of populations in the mainland landscape, are consistent with a source-sink model of population structure within each landscape. They are discussed in the framework of metapopulation theory and habitat selection models.     

Life history correlates of fecal bacterial species richness in a wild population of the blue tit Cyanistes caeruleus

Very little is known about the normal gastrointestinal flora of wild birds, or how it might affect or reflect the host's life‐history traits. The aim of this study was to survey the species richness of bacteria in the feces of a wild population of blue tits Cyanistes caeruleus and to explore the relationships between bacterial species richness and various life‐history traits, such as age, sex, and reproductive success. Using PCR‐TGGE, 55 operational taxonomic units (OTUs) were identified in blue tit feces. DNA sequencing revealed that the 16S rRNA gene was amplified from a diverse range of bacteria, including those that shared closest homology with Bacillus licheniformis, Campylobacter lari, Pseudomonas spp., and Salmonella spp. For adults, there was a significant negative relationship between bacterial species richness and the likelihood of being detected alive the following breeding season; bacterial richness was consistent across years but declined through the breeding season; and breeding pairs had significantly more similar bacterial richness than expected by chance alone. Reduced adult survival was correlated with the presence of an OTU most closely resembling C. lari; enhanced adult survival was associated with an OTU most similar to Arthrobacter spp. For nestlings, there was no significant change in bacterial species richness between the first and second week after hatching, and nestlings sharing the same nest had significantly more similar bacterial richness. Collectively, these results provide compelling evidence that bacterial species richness was associated with several aspects of the life history of their hosts.     

Unusual mitochondrial DNA polymorphism in two local populations of blue tit Parus caeruleus

Mitochondrial DNA (mtDNA) from 25 blue tits Parus caeruleus sampled from two populations of the Grenoble region (France) was assayed for polymorphism with 17 restriction endonucleases. Nine genotypes were found. Several mtDNA genotypes were also analysed by amplification via the polymerase chain reaction (PCR) and direct sequencing of 903 bp of the cytochrome b gene. The mtDNA polymorphism is greater in P. caeruleus than in other comparable bird species and results from the presence of two clearly differentiated mitochondrial lineages. Using the data of restriction polymorphism, the mean sequence divergence between individuals of the two lineages is 1.23%. Therefore, P. caeruleus should fall into the category II of phylogeographic pattern sensu Avise et al. (1987): discontinuous mtDNA genotypes which co-occur in the same region. P. caeruleus, like humans and other mobile species with high gene flow, seems to have lost its geographic structure in terms of mtDNA phylogeny. This unusual mitochondrial polymorphism can be explained by the recent admixture of two long-term isolated populations. This could be accounted for by two different scenarios. One assumes a simultaneous post-glacial colonization of the Grenoble region by two isolated European populations of P. caeruleus. Alternatively, hybridization between P. caeruleus and P. cyanus could have caused the observed pattern of mtDNA variation.