Age-specific effect of heterozygosity on survival in alpine marmots, Marmota marmota

The fitness consequences of heterozygosity and the mechanisms underpinning them are still highly controversial. Using capture–mark–recapture models, we investigated the effects of individual heterozygosity, measured at 16 microsatellite markers, on age-dependent survival and access to dominance in a socially monogamous mammalian species, the alpine marmot. We found a positive correlation between standardized multilocus heterozygosity and juvenile survival. However, there was no correlation between standardized multilocus heterozygosity and either survival of older individuals or access to dominance. The disappearance of a significant heterozygosity fitness correlation when individuals older than juveniles are considered is consistent with the prediction that differences in survival among individuals are maximal early in life. The lack of a correlation between heterozygosity and access to dominance may be a consequence of few homozygous individuals attaining the age at which they might reach dominance. Two hypotheses have been proposed to explain heterozygosity-fitness correlations: genome-wide effects reflected by all markers or local effects of specific markers linked to genes that determine fitness. In accordance with genome-wide effects of heterozygosity, we found significant correlations between heterozygosities calculated across single locus or across two sets of eight loci. Thus, the genome-wide heterozygosity effect seems to explain the observed heterozygosity-fitness correlation in the alpine marmot.     

Adult survival and microsatellite diversity in possums: effects of major histocompatibility complex-linked microsatellite diversity but not multilocus inbreeding estimators

Adult survival is perhaps the fitness parameter most important to population growth in long-lived species. Intrinsic and extrinsic covariates of survival are therefore likely to be important drivers of population dynamics. We used long-term mark-recapture data to identify genetic, individual and environmental covariates of local survival in a natural population of mountain brushtail possums (Trichosurus cunninghami). Rainfall and intra-individual diversity at microsatellite DNA markers were associated with increased local survival of adults and juveniles. We contrasted the performance of several microsatellite heterozygosity measures, including internal relatedness (IR), homozygosity by loci (HL) and the mean multilocus estimate of the squared difference in microsatellite allele sizes within an individual (mean d ²). However, the strongest effect on survival was not associated with multilocus microsatellite diversity (which would indicate a genome-wide inbreeding effect), but a subset of two loci. This included a major histocompatibility complex (MHC)-linked marker and a putatively neutral microsatellite locus. For both loci, diversity measures incorporating allele size information had stronger associations with survival than measures based on heterozygosity, whether or not allele frequency information was included (such as IR). Increased survival was apparent among heterozygotes at the MHC-linked locus, but the benefits of heterozygosity to survival were reduced in heterozygotes with larger differences in allele size. The effect of heterozygosity on fitness-related traits was supported by data on endoparasites in a subset of the individuals studied in this population. There was no apparent density dependence in survival, nor an effect of sex, age or immigrant status. Our findings suggest that in the apparent absence of inbreeding, variation at specific loci can generate strong associations between fitness and diversity at linked markers.     

DOES LINKAGE DISEQUILIBRIUM GENERATE HETEROZYGOSITY‐FITNESS CORRELATIONS IN GREAT REED WARBLERS?

Heterozygosity-fitness correlations (HFCs) at noncoding genetic markers are commonly assumed to reflect fitness effects of heterozygosity at genomewide distributed genes in partially inbred populations. However, in populations with much linkage disequilibrium (LD), HFCs may arise also as a consequence of selection on fitness loci in the local chromosomal vicinity of the markers. Recent data suggest that relatively high levels of LD may prevail in many ecological situations. Consequently, LD may be an important factor, together with partial inbreeding, in causing HFCs in natural populations. In the present study, we evaluate whether LD can generate HFCs in a small and newly founded population of great reed warblers (Acrocephalus arundinaceus). For this purpose dyads of full siblings of which only one individual survived to adult age (i.e., returned to breed at the study area) were scored at 19 microsatellite loci, and at a gene region of hypothesized importance for survival, the major histocompatibility complex (MHC). By examining siblings, we controlled for variation in the inbreeding coefficient and thus excluded genome-wide fitness effects in our analyses. We found that recruited individuals had significantly higher multilocus heterozygosity (MLH), and mean d2 (a microsatellite-specific variable), than their nonrecruited siblings. There was a tendency for the survivors to have a more diverse MHC than the nonsurvivors. Single-locus analyses showed that the strength of the genotype-survival association was especially pronounced at four microsatellite loci. By using genotype data from the entire breeding population, we detected significant LD between five of 162 pairs of microsatellite loci after accounting for multiple tests. Our present finding of a significant within-family multilocus heterozygosity-survival association in a nonequilibrium population supports the view that LD generates HFCs in natural populations.     

On the correlation between heterozygosity and fitness in natural populations

Three primary hypotheses currently prevail for correlations between heterozygosity at a set of molecular markers and fitness in natural populations. First, multilocus heterozygosity-fitness correlations might result from selection acting directly on the scored loci, such as at particular allozyme loci. Second, significant levels of linkage disequilibrium, as in recently bottlenecked-and-expanded populations, might cause associations between the markers and fitness loci in the local chromosomal vicinity. Third, in partially inbred populations, heterozygosity at the markers might reflect variation in the inbreeding coefficient and might associate with fitness as a result of effects of homozygosity at genome-wide distributed loci. Despite years of research, the relative importance of these hypotheses remains unclear. The screening of heterozygosity at polymorphic DNA markers offers an opportunity to resolve this issue, and relevant empirical studies have now emerged. We provide an account of the recent progress on the subject, and give suggestions on how to distinguish between the three hypotheses in future studies.     

Heterozygosity-fitness correlations of conserved microsatellite markers in Kentish plovers Charadrius alexandrinus

Heterozygosity-fitness correlations (HFCs) are frequently used to examine the relationship between genetic diversity and fitness. Most studies have reported positive HFCs, although there is a strong bias towards investigating HFCs in genetically impoverished populations. We investigated HFCs in a large genetically diverse breeding population of Kentish plovers Charadrius alexandrinus in southern Turkey. This small shorebird exhibits highly variable mating and care systems, and it is becoming an ecological model species to understand breeding system evolution. Using 11 conserved and six anonymous microsatellite markers, we tested whether and how heterozygosity was associated with chick survival, tarsus and body mass growth controlling for nongenetic effects (chick sex, hatching date, length of biparental care and site quality) that influence survival and growth. There was no genome-wide effect of heterozygosity on fitness, and we did not find any significant effects of heterozygosity on growth rates. However, two of the 11 conserved markers displayed an association with offspring survival: one marker showed a positive HFC, whereas the other marker showed a negative HFC. Heterozygosity at three further conserved loci showed significant interaction with nongenetic variables. In contrast, heterozygosity based on anonymous microsatellite loci was not associated with fitness or growth. Markers that were correlated with chick survival were not more likely to be located in exons or introns than other markers that lacked this association.     

On the estimation of genome-wide heterozygosity using molecular markers

Coltman and Slate (2003) recently performed a meta-analysis on studies that investigated the association between genetic variation at microsatellite loci and phenotypic trait variation. One factor not explicitly addressed in their meta-analysis is the actual estimation of genome-wide heterozygosity via molecular markers. Many authors still associate marker-estimated heterozygosity with genome-wide heterozygosity, despite allozyme-based evidence that such correlations are usually very weak or nonexistent. Here, we show that genome-wide heterozygosity is poorly estimated not only by allozymes but also by microsatellite loci and by single-nucleotide polymorphisms (SNPs). Thus, associations between fitness (or other phenotypes) and heterozygosity should be established firmly on causative factors and not on simple correlations.     

Heterozygosity and survival in blue tits (Cyanistes caeruleus): contrasting effects of presumably functional and neutral loci

The relationship between genetic diversity and fitness has important implications in evolutionary and conservation biology. This relationship has been widely investigated at the individual level in studies of heterozygosity-fitness correlations (HFC). General effects caused by inbreeding and/or local effects at single loci have been used as explanations of HFC, but the debate about the causes of HFC in open, natural populations is still ongoing. Study designs that control for variation in the inbreeding level of the individuals, and knowledge on the function and location of the markers used to measure heterozygosity, are fundamental to understand the causes of HFC. Here we investigated correlations between individual heterozygosity and estimates of survival at different life-history stages in an open population of blue tits (Cyanistes caeruleus). For survival at the embryo, nestling and fledgling stage, we used a full-sibling approach, i.e. we controlled for the level of inbreeding. We genotyped 1496 individuals with 79 microsatellites mapped across 25 chromosomes in the zebra finch (Taeniopygia guttata) that were classified either as potentially functional (58 loci) or as neutral (21 loci). We found different effects of standardized multilocus heterozygosity (SH): SH(functional) had a negative effect on the probability of hatching and local recruitment of females, whereas SH(neutral) had a positive effect on adult survival. The negative effects of functional loci are better explained by local effects, whereas the positive effects of neutral markers could reflect inbreeding effects in the population. Our results highlight the importance of considering the characteristics of the markers used in HFC studies and confirm the mixed effects of heterozygosity in different contexts (e.g. sex and life-history stage).     

Heterozygosity and orange coloration are associated in the guppy (Poecilia reticulata)

The good‐genes‐as‐heterozygosity hypothesis predicts that more elaborate male sexual ornaments are associated with higher levels of heterozygosity. Recent theoretical work suggests that such associations are likely to arise in finite, structured populations. We investigated the correlation between multilocus heterozygosity (MLH), which was estimated using 13 microsatellite loci, and male coloration in a wild population of guppies (Poecilia reticulata), a model species in sexual selection research. We found that MLH was a significant predictor of the relative area of orange spots, a trait that is subject to strong female preference in this species. Neither the relative area of black spots nor the number of black or orange spots was significantly correlated with MLH. We found no statistical support for local effects (i.e. strong effects of heterozygosity at specific markers), which suggests that relative orange spots area reflects genome‐wide heterozygosity.     

Heterozygosity at a single locus explains a large proportion of variation in two fitness‐related traits in great tits: a general or a local effect?

In natural populations, mating between relatives can have important fitness consequences due to the negative effects of reduced heterozygosity. Parental level of inbreeding or heterozygosity has been also found to influence the performance of offspring, via direct and indirect parental effects that are independent of the progeny own level of genetic diversity. In this study, we first analysed the effects of parental heterozygosity and relatedness (i.e. an estimate of offspring genetic diversity) on four traits related to offspring viability in great tits (Parus major) using 15 microsatellite markers. Second, we tested whether significant heterozygosity–fitness correlations (HFCs) were due to ‘local’ (i.e. linkage to genes influencing fitness) and/or ‘general’ (genome‐wide heterozygosity) effects. We found a significant negative relationship between parental genetic relatedness and hatching success, and maternal heterozygosity was positively associated with offspring body size. The characteristics of the studied populations (recent admixture, polygynous matings) together with the fact that we found evidence for identity disequilibrium across our set of neutral markers suggest that HFCs may have resulted from genome‐wide inbreeding depression. However, one locus (Ase18) had disproportionately large effects on the observed HFCs: heterozygosity at this locus had significant positive effects on hatching success and offspring size. It suggests that this marker may lie near to a functional locus under selection (i.e. a local effect) or, alternatively, heterozygosity at this locus might be correlated to heterozygosity across the genome due to the extensive ID found in our populations (i.e. a general effect). Collectively, our results lend support to both the general and local effect hypotheses and reinforce the view that HFCs lie on a continuum from inbreeding depression to those strictly due to linkage between marker loci and genes under selection.     

The heterozygosity theory of extra-pair mate choice in birds: a test and a cautionary note

Although extra-pair fertilizations (EPF) are common in socially monogamous systems, the benefits to females remain elusive. One potential benefit that recently has begun to receive empirical attention is increased offspring heterozygosity. We tested the heterozygosity hypothesis in the black-throated blue warbler Dendroica caerulescens using a panel of five microsatellite loci. We did not find any evidence that male heterozygosity influenced female extra-pair mating patterns, that females choose genetically dissimilar extra-pair mates, nor that extra-pair offspring were more heterozygous than within-pair offspring. However, simple Monte Carlo simulations indicated that the number of microsatellite loci used in this study, and most other recent studies, would detect an effect of heterozygosity only if that effect is pronounced. Thus, we were unable to demonstrate any effect of offspring heterozygosity in our study species, but a more subtle effect remains possible. Researchers wishing to test this hypothesis should use a large number of genetic markers and interpret negative results cautiously.     

Understanding the relationship between the inbreeding coefficient and multilocus heterozygosity: theoretical expectations and empirical data

Geneticists have been interested in inbreeding and inbreeding depression since the time of Darwin. Two alternative approaches that can be used to measure how inbred an individual is involve the use of pedigree records to estimate inbreeding coefficients or molecular markers to measure multilocus heterozygosity. However, the relationship between inbreeding coefficient and heterozygosity has only rarely been investigated. In this paper, a framework to predict the relationship between the two variables is presented. In addition, microsatellite genotypes at 138 loci spanning all 26 autosomes of the sheep genome were used to investigate the relationship between inbreeding coefficient and multilocus heterozygosity. Multilocus heterozygosity was only weakly correlated with inbreeding coefficient, and heterozygosity was not positively correlated between markers more often than expected by chance. Inbreeding coefficient, but not multilocus heterozygosity, detected evidence of inbreeding depression for morphological traits. The relevance of these findings to the causes of heterozygosity--fitness correlations is discussed and predictions for other wild and captive populations are presented.     

Major histocompatibility complex heterozygosity enhances reproductive success

We investigated how heterozygosity at the major histocompatibility complex (MHC) affects fitness in wild-derived (F2) house mice (Mus musculus musculus). To compare and control for potential confounding effects from close inbreeding and genome-wide heterozygosity, we used mice that were systematically outbred. We assessed how heterozygosity at MHC and background loci (using 15 microsatellite markers on 11 different chromosomes) affects individual survival and reproductive success (RS) in large, semi-natural population enclosures. We found that overall heterozygosity significantly increased RS, and this correlation was entirely explained by heterozygosity at two MHC loci. Moreover, we found that the effects of MHC heterozygosity depend on the level of background heterozygosity, and the benefits of maximal MHC heterozygosity show a curvilinear effect with increasing background heterozygosity. The enhanced RS from MHC heterozygosity was not because of increased survival, and although MHC heterozygosity was correlated with body mass, body mass did not correlate with RS when heterozygosity is controlled. Breeders were more MHC heterozygous than nonbreeders for both sexes, indicating that MHC heterozygosity enhanced fecundity, mating success or both. Our results show that (i) MHC heterozygosity enhances fitness among wild, outbred as well as congenic laboratory mice; (ii) heterozygosity-fitness correlations can potentially be explained by a few loci, such as MHC; (iii) MHC heterozygosity can increase fitness, even without affecting survival, by increasing mating and RS; and (iv) MHC effects depend on background genes, and maximal MHC heterozygosity is most beneficial at intermediate or optimal levels of background heterozygosity.     

Heterozygosity-fitness correlations in a migratory bird: an analysis of inbreeding and single-locus effects

Studies in a multitude of taxa have described a correlation between heterozygosity and fitness and usually conclude that this is evidence for inbreeding depression. Here, we have used multilocus heterozygosity (MLH) estimates from 15 microsatellite markers to show evidence of heterozygosity-fitness correlations (HFCs) in a long-distance migratory bird, the light-bellied Brent goose. We found significant, positive heterozygosity-heterozygosity correlations between random subsets of the markers we employed, and no evidence that a model containing all loci as individual predictors in a multiple regression explained significantly more variation than a model with MLH as a single predictor. Collectively, these results lend support to the hypothesis that the HFCs we have observed are a function of inbreeding depression. However, we do find that fitness correlations are only detectable in years where population-level productivity is high enough for the reproductive asymmetry between high and low heterozygosity individuals to become apparent. We suggest that lack of evidence of heterozygosity-fitness correlations in animal systems may be because heterozygosity is a poor proxy measure of inbreeding, especially when employing low numbers of markers, but alternatively because the asymmetries between individuals of different heterozygosities may only be apparent when environmental effects on fitness are less pronounced.     

The imprecision of heterozygosity-fitness correlations hinders the detection of inbreeding and inbreeding depression in a threatened species

In nonpedigreed wild populations, inbreeding depression is often quantified through the use of heterozygosity-fitness correlations (HFCs), based on molecular estimates of relatedness. Although such correlations are typically interpreted as evidence of inbreeding depression, by assuming that the marker heterozygosity is a proxy for genome-wide heterozygosity, theory predicts that these relationships should be difficult to detect. Until now, the vast majority of empirical research in this area has been performed on generally outbred, nonbottlenecked populations, but differences in population genetic processes may limit extrapolation of results to threatened populations. Here, we present an analysis of HFCs, and their implications for the interpretation of inbreeding, in a free-ranging pedigreed population of a bottlenecked species: the endangered takahe (Porphyrio hochstetteri). Pedigree-based inbreeding depression has already been detected in this species. Using 23 microsatellite loci, we observed only weak evidence of the expected relationship between multilocus heterozygosity and fitness at individual life-history stages (such as survival to hatching and fledging), and parameter estimates were imprecise (had high error). Furthermore, our molecular data set could not accurately predict the inbreeding status of individuals (as 'inbred' or 'outbred', determined from pedigrees), nor could we show that the observed HFCs were the result of genome-wide identity disequilibrium. These results may be attributed to high variance in heterozygosity within inbreeding classes. This study is an empirical example from a free-ranging endangered species, suggesting that even relatively large numbers (>20) of microsatellites may give poor precision for estimating individual genome-wide heterozygosity. We argue that pedigree methods remain the most effective method of quantifying inbreeding in wild populations, particularly those that have gone through severe bottlenecks.     

Heterozygosity–fitness correlations and associative overdominance: new detection method and proof of principle in the Iberian wild boar

Heterozygosity-fitness correlations (HFC) may result from a genome-wide process — inbreeding — or local effects within the genome. The majority of empirical studies reporting HFCs have attributed correlations to inbreeding depression. However, HFCs are unlikely to be caused by inbreeding depression because heterozygosity measured at a small number of neutral markers is unlikely to accurately capture a genome-wide pattern. Testing the strengths of localized effects caused by associative overdominance has proven challenging. In their current paper, Amos and Acevedo-Whitehouse present a novel test for local HFCs. Using stochastic simulations, they determine the conditions under which single-locus HFCs arise, before testing the strength of the correlation between the neutral marker and a linked gene under selection in their simulations. They used insights gained from simulation to statistically investigate the likely cause of correlations between heterozygosity and disease status using data on bovine tuberculosis infections in a wild boar population. They discover that a single microsatellite marker is an excellent predictor of tuberculosis progression in infected individuals. The results are relevant for wild boar management but, more generally, they demonstrate how single-locus HFCs could be used to identify coding loci under selection in free-living populations.     

Genetic characteristics of 207 microsatellite markers in the Korean population and in other Asian populations

Microsatellites, short tandem repeats, are useful markers for genetic analysis because of their high frequency of occurrence over the genome, high information content due to variable repeat lengths, and ease of typing. To establish a panel of microsatellite markers useful for genetic studies of the Korean population, the allele frequencies and heterozygosities of 207 microsatellite markers in 119 unrelated Korean, Indian and Pakistani individuals were compared. The average heterozygosity of the Korean population was 0.71, similar to that of the Indian and Pakistani populations. More than 80% of the markers showed heterozygosity of over 0.6 and were valuable as genetic markers for genome-wide screening for disease susceptibility loci in these populations. To identify the allelic distributions of the multilocus genetic data from these microsatellite markers, the population structures were assessed by clustering. These markers supported, with the most probability, three clustering groups corresponding to the three geographical populations. When we assumed only two hypothetical clusters (K), the Korean population was separate from the others, suggesting a relatively deep divergence of the Korean population. The present 207 microsatellite markers appear to reflect the historical and geographical origins of the different populations as well as displaying a similar degree of variation to that seen in previously published genetic data. Thus, these markers will be useful as a reference for human genetic studies on Asians.     

Heterozygosity–fitness correlations in a wild mammal population: accounting for parental and environmental effects

HFCs (heterozygosity–fitness correlations) measure the direct relationship between an individual's genetic diversity and fitness. The effects of parental heterozygosity and the environment on HFCs are currently under‐researched. We investigated these in a high‐density U.K. population of European badgers (Meles meles), using a multimodel capture–mark–recapture framework and 35 microsatellite loci. We detected interannual variation in first‐year, but not adult, survival probability. Adult females had higher annual survival probabilities than adult males. Cubs with more heterozygous fathers had higher first‐year survival, but only in wetter summers; there was no relationship with individual or maternal heterozygosity. Moist soil conditions enhance badger food supply (earthworms), improving survival. In dryer years, higher indiscriminate mortality rates appear to mask differential heterozygosity‐related survival effects. This paternal interaction was significant in the most supported model; however, the model‐averaged estimate had a relative importance of 0.50 and overlapped zero slightly. First‐year survival probabilities were not correlated with the inbreeding coefficient (f); however, small sample sizes limited the power to detect inbreeding depression. Correlations between individual heterozygosity and inbreeding were weak, in line with published meta‐analyses showing that HFCs tend to be weak. We found support for general rather than local heterozygosity effects on first‐year survival probability, and g2 indicated that our markers had power to detect inbreeding. We emphasize the importance of assessing how environmental stressors can influence the magnitude and direction of HFCs and of considering how parental genetic diversity can affect fitness‐related traits, which could play an important role in the evolution of mate choice.     

Age-dependent genetic effects on a secondary sexual trait in male Alpine ibex, Capra ibex

Secondary sexual traits, such as horns in ungulates, may be good indicators of genetic quality because they are costly to develop. Genetic effects on such traits may be revealed by examining correlations between multilocus heterozygosity (MLH) and trait value. Correlations between MLH and fitness traits, termed heterozygosity-fitness correlations (HFC), may reflect inbreeding depression or associative overdominance of neutral microsatellite loci with loci directly affecting fitness traits. We investigated HFCs for horn growth, body mass and faecal counts of nematode eggs in wild Alpine ibex (Capra ibex). We also tested if individual inbreeding coefficients (f') estimated from microsatellite data were more strongly correlated with fitness traits than MLH. MLH was more strongly associated with trait variation than f'. We found HFC for horn growth but not for body mass or faecal counts of nematode eggs. The effect of MLH on horn growth was age-specific. The slope of the correlation between MLH and yearly horn growth changed from negative to positive as males aged, in accordance with the mutation accumulation theory of the evolution of senescence. Our results suggest that the horns of ibex males are an honest signal of genetic quality.     

Can a simple algebraic analysis predict markers-genome heterozygosity correlations?

A current algebraic analysis on genome-wide heterozygosity estimates suggests that correlations between molecular markers and genome-wide heterozygosity, rho, depend on the ratio between the number of markers used, r, and the number of genome loci, n; that is: rho approximately square root r/n. Hence, it is unfeasible to obtain reliable estimates of genome-wide heterozygosity in species of large genome using a few markers. We cast some doubts about this analysis as it assumed that the probability that an individual was heterozygous at a locus is equal to the average heterozygosity of this locus in the population. However, we believe that individual heterozygosity at a given locus depends on individual pedigree. Because the pedigree is common for all loci of an individual, their probabilities of heterozygosity are not independent within the genome. We first performed simulations generating random genomes for 100 individuals. Among these individuals, markers and genome-wide heterozygosities correlated as expected from the above equation. However, when we simulated random mating among these individuals and in successive generations including their descendents, as occur in real populations, the correlations between markers and genome-wide heterozygosity were much higher than those predicted from algebraic analyses, and estimates of genome-wide heterozygosity improved slightly with the increment of the number of loci in the genome.     

Negative effects of individual heterozygosity on reproductive success in a wild bird population

The evolutionary consequences of individual genetic diversity are frequently studied by assessing heterozygosity–fitness correlations (HFCs). The prevalence of positive and negative HFCs and the predominance of general versus local effects in wild populations are far from understood, partly because comprehensive studies testing for both inbreeding and outbreeding depression are lacking. We studied a genetically diverse population of blue tits in southern Germany using a genome‐wide set of 87 microsatellites to investigate the relationship between proxies of reproductive success and measures of multilocus and single‐locus individual heterozygosity (MLH and SLH). We used complimentary measures of MLH and partitioned markers into functional categories according to their position in the blue tit genome. HFCs based on MLH were consistently negative for functional loci, whereas correlations were rather inconsistent for loci found in nonfunctional areas of the genome. Clutch size was the only reproductive variable showing a general effect. We found evidence for local effects for three measures of reproductive success: arrival date at the breeding site, the probability of breeding at the study site and male reproductive success. For these, we observed consistent, and relatively strong, negative effects at one functional locus. Remarkably, this marker had a similar effect in another blue tit population from Austria (~400 km to the east). We suggest that a genetic local effect on timing of arrival might be responsible for most negative HFCs detected, with carry‐over effects on other reproductive traits. This effect could reflect individual differences in the distance between overwintering areas and breeding sites.