Testing the phenotype-linked fertility hypothesis in the presence and absence of inbreeding.

The phenotype-linked fertility hypothesis suggests that females can judge male fertility by inspecting male phenotypic traits. This is because male sexually selected traits might correlate with sperm quality if both are sensitive to factors that influence male condition. A recent meta-analysis found little support for this hypothesis, suggesting little or no shared condition dependence. However, we recently reported that in captive zebra finches (Taeniopygia guttata) inbreeding had detrimental effects both on phenotypic traits and on measures of sperm quality, implying that variation in inbreeding could induce positive covariance between indicator traits and sperm quality. Therefore, we here assess empirically the average strength of correlations between phenotypic traits (courtship rate, beak colour, tarsus length) and measures of sperm quality (proportion of functional sperm, sperm velocity, sperm length) in populations of only outbred individuals and in mixed populations consisting of inbreds (F = 0.25) and outbreds (F = 0). As expected, phenotype sperm-trait correlations were stronger when the population contained a mix of inbred and outbred individuals. We also found unexpected heterogeneity between our two study populations, with correlations being considerably stronger in a domesticated population than in a recently wild-derived population. Correlations ranged from essentially zero among outbred-only wild-derived birds (mean Fisher's Zr ± SE = 0.03 ± 0.10) to moderately strong among domesticated birds of mixed inbreeding status (Zr ± SE = 0.38 ± 0.08). Our results suggest that, under some conditions, the phenotype-linked fertility hypothesis might apply.

Meiotic recombination shapes precision of pedigree- and marker-based estimates of inbreeding.

The proportion of an individual's genome that is identical by descent (GWIBD) can be estimated from pedigrees (inbreeding coefficient 'Pedigree F') or molecular markers ('Marker F'), but both estimators come with error. Assuming unrelated pedigree founders, Pedigree F is the expected proportion of GWIBD given a specific inbreeding constellation. Meiotic recombination introduces variation around that expectation (Mendelian noise) and related pedigree founders systematically bias Pedigree F downward. Marker F is an estimate of the actual proportion of GWIBD but it suffers from the sampling error of markers plus the error that occurs when a marker is homozygous without reflecting common ancestry (identical by state). We here show via simulation of a zebra finch and a human linkage map that three aspects of meiotic recombination (independent assortment of chromosomes, number of crossovers and their distribution along chromosomes) contribute to variation in GWIBD and thus the precision of Pedigree and Marker F. In zebra finches, where the genome contains large blocks that are rarely broken up by recombination, the Mendelian noise was large (nearly twofold larger s.d. values compared with humans) and Pedigree F thus less precise than in humans, where crossovers are distributed more uniformly along chromosomes. Effects of meiotic recombination on Marker F were reversed, such that the same number of molecular markers yielded more precise estimates of GWIBD in zebra finches than in humans. As a consequence, in species inheriting large blocks that rarely recombine, even small numbers of microsatellite markers will often be more informative about inbreeding and fitness than large pedigrees.

Quantifying realized inbreeding in wild and captive animal populations.

Most molecular measures of inbreeding do not measure inbreeding at the scale that is most relevant for understanding inbreeding depression-namely the proportion of the genome that is identical-by-descent (IBD). The inbreeding coefficient FPed obtained from pedigrees is a valuable estimator of IBD, but pedigrees are not always available, and cannot capture inbreeding loops that reach back in time further than the pedigree. We here propose a molecular approach to quantify the realized proportion of the genome that is IBD (propIBD), and we apply this method to a wild and a captive population of zebra finches (Taeniopygia guttata). In each of 948 wild and 1057 captive individuals we analyzed available single-nucleotide polymorphism (SNP) data (260 SNPs) spread over four different genomic regions in each population. This allowed us to determine whether any of these four regions was completely homozygous within an individual, which indicates IBD with high confidence. In the highly nomadic wild population, we did not find a single case of IBD, implying that inbreeding must be extremely rare (propIBD=0-0.00094, 95% CI). In the captive population, a five-generation pedigree strongly underestimated the average amount of realized inbreeding (FPed=0.013

Carotenoid-based bill coloration functions as a social, not sexual, signal in songbirds (Aves: Passeriformes).

Many animals use coloration to communicate with other individuals. Although the signalling role of avian plumage colour is relatively well studied, there has been much less research on coloration in avian bare parts. However, bare parts could be highly informative signals as they can show rapid changes in coloration. We measured bill colour (a ubiquitous bare part) in over 1600 passerine species and tested whether interspecific variation in carotenoid-based coloration is consistent with signalling to potential mates or signalling to potential rivals in a competitive context. Our results suggest that carotenoid bill coloration primarily evolved as a signal of dominance, as this type of coloration is more common in species that live in social groups in the nonbreeding season, and species that nest in colonies; two socio-ecological conditions that promote frequent agonistic interactions with numerous and/or unfamiliar individuals. Additionally, our study suggests that carotenoid bill coloration is independent of the intensity of past sexual selection, as it is not related to either sexual dichromatism or sexual size dimorphism. These results pose a significant challenge to the conventional view that carotenoid-based avian coloration has evolved as a developmentally costly, condition-dependent sexual signal. We also suggest that bare part ornamentation may often signal different information than plumage ornaments.

Behaviour-related DRD4 polymorphisms in invasive bird populations.

It has been suggested that individual behavioural traits influence the potential to successfully colonize new areas. Identifying the genetic basis of behavioural variation in invasive species thus represents an important step towards understanding the evolutionary potential of the invader. Here, we sequenced a candidate region for neophilic/neophobic and activity behaviour - the complete exon 3 of the DRD4 gene - in 100 Yellow-crowned bishops (Euplectes afer) from two invasive populations in Spain and Portugal. The same birds were scored twice for activity behaviour while exposed to novel objects (battery or slice of apple) in captivity. Response to novel objects was repeatable (r = 0.41) within individuals. We identified two synonymous DRD4 SNPs that explained on average between 11% and 15% of the phenotypic variance in both populations, indicating a clear genetic component to the neophilic/neophobic/activity personality axis in this species. This consistently high estimated effect size was mainly due to the repeated measurement design, which excludes part of the within-individual nongenetic variance in the response to different novel objects. We suggest that the alternative alleles of these SNPs are likely introduced from the original population and maintained by weak or antagonistic selection during different stages of the invasion process. The identified genetic variants have not only the potential to serve as genetic markers of the neophobic/neophilic/activity personality axis, but may also help to understand the evolution of behaviour in these invasive bird populations.

Parental provisioning behaviour plays a key role in linking personality with reproductive success.

Repeatable behavioural traits ('personality') have been shown to covary with fitness, but it remains poorly understood how such behaviour-fitness relationships come about. We applied a multivariate approach to reveal the mechanistic pathways by which variation in exploratory and aggressive behaviour is translated into variation in reproductive success in a natural population of blue tits, Cyanistes caeruleus. Using path analysis, we demonstrate a key role for provisioning behaviour in mediating the link between personality and reproductive success (number of fledged offspring). Aggressive males fed their nestlings at lower rates than less aggressive individuals. At the same time, their low parental investment was associated with increased female effort, thereby positively affecting fledgling production. Whereas male exploratory behaviour was unrelated to provisioning behaviour and reproductive success, fast-exploring females fed their offspring at higher rates and initiated breeding earlier, thus increasing reproductive success. Our findings provide strong support for specific mechanistic pathways linking components of behavioural syndromes to reproductive success. Importantly, relationships between behavioural phenotypes and reproductive success were obscured when considering simple bivariate relationships, underlining the importance of adopting multivariate views and statistical tools as path analysis to the study of behavioural evolution.

Basal metabolic rate can evolve independently of morphological and behavioural traits.

Quantitative genetic analyses of basal metabolic rate (BMR) can inform us about the evolvability of the trait by providing estimates of heritability, and also of genetic correlations with other traits that may constrain the ability of BMR to respond to selection. Here, we studied a captive population of zebra finches (Taeniopygia guttata) in which selection lines for male courtship rate have been established. We measure BMR in these lines to see whether selection on male sexual activity would change BMR as a potentially correlated trait. We find that the genetic correlation between courtship rate and BMR is practically zero, indicating that the two traits can evolve independently of each other. Interestingly, we find that the heritability of BMR in our population (h(2)=0.45) is markedly higher than was previously reported for a captive zebra finch population from Norway. A comparison of the two studies shows that additive genetic variance in BMR has been largely depleted in the Norwegian population, especially the genetic variance in BMR that is independent of body mass. In our population, the slope of BMR increase with body mass differs not only between the sexes but also between the six selection lines, which we tentatively attribute to genetic drift and/or founder effects being strong in small populations. Our study therefore highlights two things. First, the evolvability of BMR may be less constrained by genetic correlations and lack of independent genetic variation than previously described. Second, genetic drift in small populations can rapidly lead to different evolvabilities across populations.

The genetic rescue of two bottlenecked South Island robin populations using translocations of inbred donors.

Populations forced through bottlenecks typically lose genetic variation and exhibit inbreeding depression. 'Genetic rescue' techniques that introduce individuals from outbred populations can be highly effective in reversing the deleterious effects of inbreeding, but have limited application for the majority of endangered species, which survive only in a few bottlenecked populations. We tested the effectiveness of using highly inbred populations as donors to rescue two isolated and bottlenecked populations of the South Island robin (Petroica australis). Reciprocal translocations significantly increased heterozygosity and allelic diversity. Increased genetic diversity was accompanied by increased juvenile survival and recruitment, sperm quality, and immunocompetence of hybrid individuals (crosses between the two populations) compared with inbred control individuals (crosses within each population). Our results confirm that the implementation of 'genetic rescue' using bottlenecked populations as donors provides a way of preserving endangered species and restoring their viability when outbred donor populations no longer exist.

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.

Testing for associations between candidate genes for circadian rhythms and individual variation in sleep behaviour in blue tits.

The regulation of sleep in animals is controlled by environmental factors, homeostatic mechanisms and endogenous circadian oscillators. The molecular mechanisms underlying such circadian oscillators have been described in detail and a variety of genes that are components of these molecular clocks have been reported. In addition to inter-specific variation in the temporal organization of sleep, there is significant intra-specific variation in different organisms. From numerous studies in humans it is known that polymorphisms in the regulatory clock genes are causing such variation but knowledge about associations between naturally occurring polymorphisms and sleep patterns in wild animals is scarce. In this study, we investigated the phenotypic sleep correlates of eleven previously described polymorphisms in seven candidate genes within a free-living blue tit Cyanistes caeruleus population. We detected associations between four single nucleotide polymorphisms and three of the nine tested sleep parameters representing temporal organization. Awakening time was associated with polymorphisms in AANAT and PERIOD2, morning latency with a polymorphism in CKIε and the duration of the longest sleep bout with a second polymorphism in AANAT. However, by a permutation procedure we showed that the number of significant results and the most significant association has a study-wide likelihood of 46.7 and 5.9 % respectively. Further replication studies are needed to evaluate the potential associations.

Determinants of distribution and prevalence of avian malaria in blue tit populations across Europe: separating host and parasite effects.

Although avian malarial parasites are globally distributed, the factors that affect the geographical distribution and local prevalence of different parasite lineages across host populations or species are still poorly understood. Based on the intense screening of avian malarial parasites in nine European blue tit populations, we studied whether distribution ranges as well as local adaptation, host specialization and phylogenetic relationships can determine the observed prevalences within populations. We found that prevalence differed consistently between parasite lineages and host populations, indicating that the transmission success of parasites is lineage specific but is partly shaped by locality-specific effects. We also found that the lineage-specific estimate of prevalence was related to the distribution range of parasites: lineages found in more host populations were generally more prevalent within these populations. Additionally, parasites with high prevalence that were also widely distributed among blue tit populations were also found to infect more host species. These findings suggest that parasites reaching high local prevalence can also realize wide distribution at a global scale that can have further consequences for host specialization. Although phylogenetic relationships among parasites did not predict prevalence, we detected a close match between a tree based on the geographic distance of the host populations and the parasite phylogenetic tree, implying that neighbouring host populations shared a related parasite fauna.

Search for informative polymorphisms in candidate genes: clock genes and circadian behaviour in blue tits.

The identification of functional polymorphisms in genes that underlie behavioural trait variation is a challenging but intriguing task in evolutionary biology. Given the wealth of genomic data and the increasing number of genotype-phenotype association studies in model organisms, one can ask whether and how this information can be used for non-model organisms. Here we describe two strategies to search for likely functional polymorphisms in candidate genes in a bird species that has been intensively studied by behavioural and population ecologists, the blue tit Cyanistes caeruleus. In the first approach we searched for repeating elements in coding regions of the genome using information about repeats in Gallus gallus genes. The rationale is that tandem-repeat elements have a high potential to be polymorphic and functional. The second strategy aimed to replicate reported genotype-phenotype association studies by extrapolating results from model organisms to our study species. Both strategies showed high success rates with respect to finding homologous gene regions and potentially informative genetic variants in the genes AANAT, ADCYAP1, CKIepsilon, CLOCK, CREB1, NPAS2 and PERIOD2.

Avian Clock gene polymorphism: evidence for a latitudinal cline in allele frequencies.

In comparison with most animal behaviours, circadian rhythms have a well-characterized molecular genetic basis. Detailed studies of circadian clock genes in 'model' organisms provide a foundation for interpreting the functional and evolutionary significance of polymorphic circadian clock genes found within free-living animal populations. Here, we describe allelic variation in a region of the avian Clock orthologue which encodes a functionally significant polyglutamine repeat (ClkpolyQcds), within free-living populations of two passerine birds, the migratory bluethroat (Luscinia svecica) and the predominantly nonmigratory blue tit (Cyanistes caeruleus). Multiple ClkpolyQcds alleles were found within populations of both species (bluethroat: 12 populations, 7 alleles; blue tit: 14 populations, 9 alleles). Some populations of both species were differentiated at the ClkpolyQcds locus as measured by F(ST) and R(ST) values. Among the blue tit, but not bluethroat populations, we found evidence of latitudinal clines in (i) mean ClkpolyQcds repeat length, and (ii) the proportions of three ClkpolyQcds genotype groupings. Parallel analyses of microsatellite allele frequencies, which are considered to reflect selectively neutral processes, indicate that interpopulation allele frequency variation at the ClkpolyQcds and microsatellite loci does not reflect the same underlying demographic processes. The possibility that the observed interpopulation ClkpolyQcds allele frequency variation is, at least in part, maintained by selection for microevolutionary adaptation to photoperiodic parameters correlated with latitude warrants further study.

A spatial genetic structure and effects of relatedness on mate choice in a wild bird population.

Inbreeding depression, as commonly found in natural populations, should favour the evolution of inbreeding avoidance mechanisms. If natal dispersal, the first and probably most effective mechanism, does not lead to a complete separation of males and females from a common origin, a small-scale genetic population structure may result and other mechanisms to avoid inbreeding may exist. We studied the genetic population structure and individual mating patterns in blue tits (Parus caeruleus). The population showed a local genetic structure in two out of four years: genetic relatedness between individuals (estimated from microsatellite markers) decreased with distance. This pattern was mainly caused by immigrants to the study area; these, if paired with fellow immigrants, were more related than expected by chance. Since blue tits did not avoid inbreeding with their social partner, we examined if individuals preferred less related partners at later stages of the mate choice process. We found no evidence that females or males avoided inbreeding through extra-pair copulations or through mate desertion and postbreeding dispersal. Although the small-scale genetic population structure suggests that blue tits could use a simple rule of thumb to select less related mates, females did not generally prefer more distantly breeding extra-pair partners. However, the proportion of young fathered by an extra-pair male in mixed paternity broods depended on the genetic relatedness with the female. This suggests that there is a fertilization bias towards less related copulation partners and that blue tits are able to reduce the costs of inbreeding through a postcopulatory process.

Carotenoid-based bill colour as an indicator of immunocompetence and sperm performance in male mallards.

Female mate choice is often based on exaggerated sexual traits, signals of male qualities that females cannot assess directly. Two such key qualities are male immune and/or sexual competence, whereby honesty in signalling could be maintained by physiological trade-offs. Carotenoid-based ornaments likely constitute such honest signals, as there is direct competition for (limited) carotenoids between ornament deposition and anti-oxidant support of immune or sperm functioning. Using spectrometry, we assessed the potential signalling function of the yellow, carotenoid-based colour of the bill of male mallards, a target of female mate choice. Here we demonstrate that bill reflectance varied with plasma carotenoid level, indicating antioxidant reserves. Moreover, lower relative UV reflectance during autumn pairing predicted immune responsiveness and correlated positively with sperm velocity during breeding, a trait that affects fertility. Our data provide support for current theories that females could use carotenoid-based sexual signals to detect immune vigour and fertilizing ability of prospective mates.

The effect of heavy metal exposure on egg size, eggshell thickness and the number of spermatozoa in blue tit Parus caeruleus eggs.

In this study we compared the egg size, the eggshell thickness and the number of spermatozoa trapped on the perivitelline layer of the eggs in three blue tit (Parus caeruleus) populations across a heavy metal pollution gradient. No significant differences were observed in egg characteristics among study sites. Eggs from the two most polluted sites however, had significantly less spermatozoa on the perivitelline layer than eggs from the least polluted site. Our results suggest a significant effect of pollution on blue tit sperm concentrations in the infundibulum. Determining the number of spermatozoa on the egg membranes is relatively cheap and straightforward, which renders this technique a new and useful tool in biomonitoring.

Bill morphology reflects female independence from male parental help.

The study of territorial polygyny in birds has been influential in the development of the theory of social mating systems. Alternative female mating options have been studied within the framework of the polygyny-threshold model and later as the outcome of conflicts of interest between individuals. However, little attention has been given to variations between individual females, and how this affects their mating behaviour. Here, we test the hypothesis that some females are better adapted to raise nestlings without male assistance, and thus to mate polygynously. Specifically, we investigate whether intraspecific variation in female bill morphology is related to mating behaviour. This hypothesis is derived from earlier studies showing that, in both intra- and interspecific comparisons, uniparental care by females is correlated with the catching of larger prey items than when both parents provision the young. Using the polygynous dusky warbler (Phylloscopus fuscatus) as a model species, we found that, in accordance with our prediction, females with deep bills were more likely to mate as a secondary female. Moreover, regardless of mating status, females with deep bills settled in territories with more food and they received less male assistance in feeding their offspring. We argue that females with stronger bills are better adapted to exploit the abundance of large food items in rich territories and thus to raise young on their own. Our results demonstrate the importance of studying variations between individual females, and provide evidence for an extended version of the 'constrained-female hypothesis'. As bill depth is a highly heritable trait, our study strongly suggests that variation in female mating behaviour is not only related to ecological factors and female condition (as shown elsewhere) but also to heritable morphological traits.

High levels of extra-pair paternity in an isolated, low-density, island population of tree swallows (Tachycineta bicolor).

Molecular genetic studies have suggested that apparently nonbreeding males ('floaters') may account for a significant proportion of extra-pair paternity (EPP) in avian populations. Attempts to determine the influence of breeding density on EPP are therefore confounded by the presence of a subpopulation of floaters whose numbers are difficult to estimate. To study EPP in a tree swallow (Tachycineta bicolor) population with few floaters, we chose a nestbox grid on an island with an excess of available breeding sites and very few floaters. We obtained DNA samples from 13 complete families and performed DNA profiling on them using four microsatellite loci. For comparison, we also obtained a sample of 58 extra-pair young (EPY) from a mainland population typed at five microsatellite loci. Paternity assignments among resident males in both populations were made using the microsatellite profiles and a likelihood-based statistical method. Of the 67 island nestlings typed, we found 37 (55%) nestlings from 11 (85%) different nests that were EPY. The proportion of nestlings that were EPY and the proportion of broods containing EPY did not differ significantly between island and mainland populations studied previously. There was no significant difference between island and mainland populations in the proportion of extra-pair paternities assigned among neighbouring resident males. Male breeding density does not appear to affect the ability of female tree swallows to obtain extra-pair fertilizations, at least over the range of densities studied so far. The rate of EPP has remained remarkably consistent over many years, studies and populations implying an important role of active female choice in determining EPP.