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.     

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.     

Negative heterozygosity-fitness correlations observed with microsatellites located in functional areas of the genome

Genome-wide heterozygosity inferred from neutral markers such as microsatellites is often expected to (i) reflect individual inbreeding and (ii) covary positively with fitness, generating positive heterozygosity-fitness correlations (HFCs). The often forgotten other end of the inbreeding-outbreeding continuum is outbreeding depression: past a certain degree of heterozygosity, heterozygotes tend to have lower fitness than homozygotes. Outbreeding depression arises from the breakup of co-adapted gene complexes and/or the introgression of nonlocally adapted genes. Provided that a correlation in heterozygosity exists across loci, outbreeding depression will be reflected in negative HFCs. In this issue, Olano-Marin et al. (2011a) describe negative heterozygosity-fitness correlations (HFCs) in blue tits Cyanistes caeruleus (Fig. 1), whereby heterozygosity has a significant, negative effect on female hatching success and recruitment. This study, together with a similar study by the same authors published in Evolution (Olano-Marin et al. 2011b), forms an original contribution in two respects. First, in the same population, positive and negative HFCs were recorded, revealing both inbreeding and outbreeding depression depending on the trait studied (whereby both processes were reliant on unknown, and possibly different, sets of coding loci). Second, a large number of microsatellite markers were split into two functional groups: microsatellite markers were either designed using zebra finch expressed sequence tags (ESTs) or derived using traditional cloning methods and presumed to be neutral. Contrasting large classes of loci and their varying levels of polymorphism, rather than looking for one locus that would stand out among tens of randomly selected markers, pave the way for a more elegant and powerful approach to explore how HFCs vary across traits and among regions of the genome. [Figure: see text].     

Genetic diversity at neutral and adaptive loci determines individual fitness in a long-lived territorial bird

There is compelling evidence about the manifest effects of inbreeding depression on individual fitness and populations' risk of extinction. The majority of studies addressing inbreeding depression on wild populations are generally based on indirect measures of inbreeding using neutral markers. However, the study of functional loci, such as genes of the major histocompatibility complex (MHC), is highly recommended. MHC genes constitute an essential component of the immune system of individuals, which is directly related to individual fitness and survival. In this study, we analyse heterozygosity fitness correlations of neutral and adaptive genetic variation (22 microsatellite loci and two loci of the MHC class II, respectively) with the age of recruitment and breeding success of a decimated and geographically isolated population of a long-lived territorial vulture. Our results indicate a negative correlation between neutral genetic diversity and age of recruitment, suggesting that inbreeding may be delaying reproduction. We also found a positive correlation between functional (MHC) genetic diversity and breeding success, together with a specific positive effect of the most frequent pair of cosegregating MHC alleles in the population. Globally, our findings demonstrate that genetic depauperation in small populations has a negative impact on the individual fitness, thus increasing the populations' extinction risk.     

Seasonal variation in diet quality: antioxidants,invertebrates and blue tits Cyanistes caeruleus

Breeding success is often dictated by the degree to which parents can synchronize the maximum food requirements of offspring to the peak in abundance of invertebrate prey. Less studied is how the nutritional quality of individual diet items impacts on breeding. In the present study, we assessed the abundance and antioxidant concentrations of arboreal arthropods from oak woodland and provisioning behaviour of the blue tit Cyanistes caeruleus. Dietary antioxidants are important during development because they defend against oxidative stress. Operophtera caterpillars, Erannis caterpillars, and spiders contained significantly different levels of individual carotenoids and α‐tocopherol. Concentrations of lutein and β‐carotene in Operophtera caterpillars did not vary seasonally, although concentrations of zeaxanthin declined and α‐tocopherol increased with date. Blue tit broods hatched later in the season received significantly fewer caterpillars and more spiders per chick compared to earlier broods. Reflecting changes in prey composition, blue tit nestling plasma showed decreases in zeaxanthin and increases in α‐tocopherol with date. Thus, processes that shift the timing of breeding in birds and/or prey composition are likely to alter antioxidant intake and thus potentially influence the oxidative stress status of animals. The data obtained in the present study suggest a mechanism by which environmental change as a result of human activities could influence the health and fitness of individuals in natural populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 708–717.     

Genetic and environmental components of growth in nestling blue tits (Parus caeruleus)

We investigated the effect of brood‐size mediated food availability on the genetic and environmental components of nestling growth in the blue tit (Parus caeruleus), using a cross‐fostering technique. We found genetic variation for body size at most nestling ages, and for duration of mass increase, but not of tarsus growth. Hence, nestling growth in our study population seems to have the potential to evolve further. Furthermore, significant genotype–environment interactions indicated heritable variation in reaction norms of growth rates and growth periods, i.e. that our study population had a heritable plasticity in the growth response to environmental conditions. The decreasing phenotypic variance with nestling age indicated compensatory growth in all body traits. Furthermore, the period of weight increase was longer for nestlings growing up in enlarged broods, while there was no difference to reduced broods in the period of tarsus growth. At fledging, birds in enlarged broods had shorter tarsi and lower weights than birds in reduced broods, but there was no difference in wing length or body condition between the two experimental groups. The observed flexibility in nestling growth suggests that growing nestlings are able to respond adaptively to food constraint by protecting the growth of ecologically important traits.     

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.     

Heterozygosity is linked to the costs of immunity in nestling great tits (Parus major)

There is growing evidence that heterozygosity–fitness correlations (HFCs) are more pronounced under harsh conditions. Empirical evidence suggests a mediating effect of parasite infestation on the occurrence of HFCs. Parasites have the potential to mediate HFCs not only by generally causing high stress levels but also by inducing resource allocation tradeoffs between the necessary investments in immunity and other costly functions. To investigate the relative importance of these two mechanisms, we manipulated growth conditions of great tit nestlings by brood size manipulation, which modifies nestling competition, and simultaneously infested broods with ectoparasites. We investigated under which treatment conditions HFCs arise and, second, whether heterozygosity is linked to tradeoff decisions between immunity and growth. We classified microsatellites as neutral or presumed functional and analyzed these effects separately. Neutral heterozygosity was positively related to the immune response to a novel antigen in parasite‐free nests, but not in infested nests. For nestlings with lower heterozygosity levels, the investments in immunity under parasite pressure came at the expenses of reduced feather growth, survival, and female body condition. Functional heterozygosity was negatively related to nestling immune response regardless of the growth conditions. These contrasting effects of functional and neutral markers might indicate different underlying mechanisms causing the HFCs. Our results confirm the importance of considering marker functionality in HFC studies and indicate that parasites mediate HFCs by influencing the costs of immune defense rather than by a general increase in environmental harshness levels.     

Heterozygosity at neutral and immune loci is not associated with neonatal mortality due to microbial infection in Antarctic fur seals

Numerous studies have reported correlations between the heterozygosity of genetic markers and fitness. These heterozygosity–fitness correlations (HFCs) play a central role in evolutionary and conservation biology, yet their mechanistic basis remains open to debate. For example, fitness associations have been widely reported at both neutral and functional loci, yet few studies have directly compared the two, making it difficult to gauge the relative contributions of genome‐wide inbreeding and specific functional genes to fitness. Here, we compared the effects of neutral and immune gene heterozygosity on death from bacterial infection in Antarctic fur seal (Arctocephalus gazella) pups. We specifically developed a panel of 13 microsatellites from expressed immune genes and genotyped these together with 48 neutral loci in 234 individuals, comprising 39 pups that were classified at necropsy as having most likely died of bacterial infection together with a five times larger matched sample of healthy surviving pups. Identity disequilibrium quantified from the neutral markers was positive and significant, indicative of variance in inbreeding within the study population. However, multilocus heterozygosity did not differ significantly between healthy and infected pups at either class of marker, and little evidence was found for fitness associations at individual loci. These results support a previous study of Antarctic fur seals that found no effects of heterozygosity at nine neutral microsatellites on neonatal survival and thereby help to refine our understanding of how HFCs vary across the life cycle. Given that nonsignificant HFCs are underreported in the literature, we also hope that our study will contribute toward a more balanced understanding of the wider importance of this phenomenon.     

Heterozygosity is unrelated to adult fitness measures in a large, noninbred population of great tits (Parus major)

The extent to which heterozygosity-fitness correlations (HFCs) are expected in wild populations is an important and unresolved question in evolutionary biology, because it relates to our understanding of the genetic architecture of fitness. Here, we report a study of HFCs in a wild, noninbred population of great tits (Parus major), based on a sample comprising 281 individuals typed at 26 markers, resulting in a data set comprising over 5600 genotypes. We regressed pedigree-derived f-score and multilocus genetic diversity against eight life-history traits known to be associated with fitness in this population, including lifetime reproductive success (LRS), as well as several morphological traits under weak selection. We found no evidence for either multilocus or single-locus HFCs for any morphological or fitness trait, and further found no evidence that effect sizes were stronger for those life-history traits more closely associated with reproductive fitness. This result may, in part, be explained by the fact that we found no evidence that our set of 26 markers had any power to infer genome-wide heterozygosity in this population and that marker-derived heterozygosity was uncorrelated with pedigree-derived f-score. Overall, these results emphasize the fact that the often-reported strong HFCs detected in small, inbred populations do not reflect a general phenomenon of increasing individual reproductive fitness with increasing heterozygosity.     

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.     

Multiple responses to increasing spring temperatures in the breeding cycle of blue and great tits (Cyanistes caeruleus,Parus major)

Many bird species start laying their eggs earlier in response to increasing spring temperatures, but the causes of variation between and within species have not been fully explained. Moreover, synchronization of the nestling period with the food supply not only depends on first‐egg dates but also on additional reproductive parameters including laying interruptions, incubation time and nestling growth rate. We studied the breeding cycle of two sympatric and closely related species, the blue tit Cyanistes caeruleus and the great tit Parus major in a rich oak‐beech forest, and found that both advanced their mean first‐egg dates by 11–12 days over the last three decades. In addition, the time from first egg to fledging has shortened by 2–3 days, through a decrease in laying interruptions, incubation time (not statistically significant) and nestling development time. This decrease is correlated with a gradual increase of temperatures during laying, suggesting a major effect of the reduction in laying interruptions. In both species, the occurrence of second clutches has strongly decreased over time. As a consequence, the average time of fledging (all broods combined) has advanced by 15.4 and 18.6 days for blue and great tits, respectively, and variance in fledging dates has decreased by 70–75%. Indirect estimates of the food peak suggest that both species have maintained synchronization with the food supply. We found consistent selection for large clutch size, early laying and short nest time (laying to fledging), but no consistent changes in selection over time. Analyses of within‐individual variation show that most of the change can be explained by individual plasticity in laying date, fledging date and nest time. This study highlights the importance of studying all components of the reproductive cycle, including second clutches, in order to assess how natural populations respond to climate change.     

Heterozygosity–behaviour correlations in nine‐spined stickleback (Pungitius pungitius) populations: contrasting effects at random and functional loci

The study of heterozygosity‐fitness correlations (HFCs) has a long history in the fields of ecology and evolutionary biology but remains controversial. Recently, it has been shown that the genetic distance of markers from functional loci can be an important factor to be considered in addition to marker numbers and variability. In this study, we investigated the correlation between individual heterozygosity and behaviour (aggression, boldness and feeding activity) in nine‐spined stickleback (Pungitius pungitius) individuals originating from four populations in two contrasting environments. Offspring of full‐sib families raised in a common garden setting were assessed for behaviour and genotyped using 84 microsatellite markers that were either located within or near behaviourally or physiologically important genes (termed ‘functional’) or were randomly selected. No associations were detected with any behavioural trait in any population or over all populations when genetic variability was measured using all 84 markers combined. However, when the markers were separated into three functional categories (behavioural, physiological and random), several significant associations were observed both with functional markers and random markers in one of the four populations assessed. Interestingly, contrasting correlations with behaviour were observed when using physiological gene (negative) and random (positive) markers. Upon dividing the physiological gene markers into further subcategories based on their specific physiological functions, a strong relationship between the heterozygosity of markers linked to osmoregulation‐related genes, and behaviour was revealed in the brackish water population. Our results indicate that both local (physiological) and general (neutral) effects are important in shaping behaviour and that heterozygosity–behaviour correlations are population dependent.     

Causes and consequences of individual variation in the extent of post‐juvenile moult in the blue tit Cyanistes caeruleus (Passeriformes: Paridae)

Moult, comprising the growth or replacement of feathers in birds, is an energetically demanding process. As a result, in many species, the extent of the post‐juvenile moult can vary substantially. However, the reasons underlying this variation remain poorly understood, and the potential life‐history consequences of variation in moult extent are even less clear. In the present study, we aimed to use individual‐specific data to identify factors affecting the extent of the post‐juvenile moult in a population of over 2500 blue tits Cyanistes caeruleus Linnaeus 1758, and to assess the consequences of individual variation in moult extent on reproduction in the first year of life. There was a substantial sex difference in post‐juvenile moult extent, with males moulting more extensively than females. Putative immigrant birds had moulted on average less than those born locally. However, there was little evidence of carry‐over effects of the natal environment on moult extent because we found no relationship between moult extent and fledging date or nestling mass. Evidence that moult extent, and hence feather brightness, affected subsequent reproductive success was limited. Moult extent had no effect on recruitment in males, although female recruits had moulted significantly less than nonbreeders. Because it was not influenced by features of the natal environment, moult extent may not be an honest signal of individual quality in C. caeruleus. As a result, the potential consequences of variation in moult extent for fitness are likely to be small.     

Isolation and characterization of microsatellite loci from Chiasmia assimilis (Warren, 1899) (Lepidoptera: Geometridae)

Twelve microsatellite loci are presented for the biological control agent Chiasmia assimilis (Warren, 1899). These microsatellite loci were obtained through the construction of an enriched library, overcoming previous reported difficulties with obtaining microsatellites from other Lepidoptera due to the low frequency of microsatellites in their genomes.     

Colonisation and diversification of the blue tits (Parus caeruleus teneriffae-group) in the Canary Islands

The blue tit (Parus caeruleus teneriffae group) is proposed to have colonised the Canary Islands from North Africa according to an east-to-west stepping stone model, and today, the species group is divided into four subspecies, differing in morphological, acoustic, and ecological characters. This colonisation hypothesis was tested and the population structure between and within the islands studied using mitochondrial DNA sequences of the non-coding and relatively fast evolving control region. Our results suggest that one of the central islands, Tenerife, was colonised first and the other islands from there. Three of the presently recognised four subspecies are monophyletic, exception being the subspecies teneriffae, which consists of two monophyletic groups, the one including birds of Tenerife and La Gomera and the other birds of Gran Canaria. The Gran Canarian birds are well differentiated from birds of the other islands and should be given a subspecies status. In addition, the teneriffae subspecies group is clearly distinct from the European caeruleus group, and therefore the blue tit assemblage should be divided into two species.     

Ecological and morphological variation of Canary Island blue tits, Parus caeruleus (Aves: Paridae)

Canary Island blue tits have differentiated within the last million years. Compared with mainland populations they have short wings, large beaks and tarsi, and tend to be less variable in these parameters. Within the archipelago the eastern populations have the shortest wings and tarsi but the widest and deepest beaks. The eastern populations may have been derived from central island ones, following extinction of the original eastern populations during a warm period in the last Ice Age. New measurements have been used to test contrasting predictions of hypotheses that explain inter-island morphological variation in terms of either climatic or ecological influences. Neither hypothesis is sufficient to explain the variation. A model which combines mem is proposed as follows. Mild winter temperatures have selected for small body size and wing length, especially on the hot, dry, eastern islands. On the other pine-forested, islands, selection has favoured relatively long tarsi and long, slender, beaks for efficient foraging among pine foliage. The absence of die pine-dwelling coal tit from the islands has facilitated mis evolutionary shift. It is shown that blue tits in pines forage more in the needles and cones on the Canary Islands than they do on die North African mainland.     

Inbreeding depression in Lychnis flos-cuculi (Caryophyllaceae): effects of different levels of inbreeding

In a controlled crossing experiment on Lychnis flos-cuculi plants in the greenhouse, outbred and selfed maternal plants were each treated with pollen from unrelated plants, siblings and selves. The seeds thus obtained had expected inbreeding coefficients of 0, 0.25 and 0.5 for the outbred maternal plants, and 0, 0.5 and 0.75 for the selfed maternal plants. Seed abortion rate, seed weight and germination rate were estimated. Seedlings were transplanted to an outdoor garden, and monitored for survival, probability of flowering, number of capsules and area of capsules next spring. Inbred seeds germinated slower and in lower proportions than those less inbred, and seedlings had lower survival, flowering, fruit set and area of capsules if inbred. Combined fitness values were estimated from the survival and fecundity components, and severe inbreeding depression was detected for these estimates (0.51 and 0.56 for one generation of selfing). The fitness function decreased linearly with the increase in inbreeding coefficient, which is as expected if the inbreeding depression is additive among loci.     

Characterization of microsatellite loci in the subsocial spider Stegodyphus lineatus (Araneae: Eresidae)

Stegodyphus lineatus spiders live in groups consisting of closely related individuals. There appears to be no discrimination against related individuals as mates but females mate multiply, despite the fact that matings are shown to carry a cost. We have developed eight polymorphic dinucleotide microsatellite markers that allow us to assess levels of heterozygosity and relatedness among individuals of this species. These molecular markers are likely to prove highly effective tools for estimating levels of inbreeding and thus allow us to test hypotheses about the relationships between social structure, mating strategies and inbreeding avoidance.     

Development and characterization of EST‐SSR markers for the genus Rhododendron section Brachycalyx (Ericaceae)

Simple sequence repeat (SSR) markers were developed from expressed sequence tags (ESTs) for Rhododendron section Brachycalyx in order to elucidate its evolutionary processes and reproductive ecology. Nineteen polymorphic EST‐SSR markers were developed from EST libraries of R. amagianum and R. hyugaense. Polymorphisms for these markers were assessed using four species of section Brachycalyx. The number of alleles ranged from 1 to 14, and the observed and expected heterozygosity ranged from 0.000 to 0.931 and 0.000 to 0.904, respectively. The EST‐SSR markers developed in this study will be useful for elucidating population genetic structure and breeding systems in section Brachycalyx.