INBREEDING INFLUENCES WITHIN‐BROOD HETEROZYGOSITY‐FITNESS CORRELATIONS (HFCS) IN AN ISOLATED PASSERINE POPULATION

Molecular estimates of inbreeding may be made using genetic markers such as microsatellites, however the interpretation of resulting heterozygosity‐fitness correlations (HFCs) with respect to inbreeding depression is not straightforward. We investigated the relationship between pedigree‐determined inbreeding coefficients (f) and HFCs in a closely monitored, reintroduced population of Stewart Island robins (Petroica australis rakiura) on Ulva Island, New Zealand. Using a full sibling design, we focused on differences in juvenile survival associated specifically with individual sibling variation in standardized multilocus heterozygosity (SH) when expected f was identical. We found that within broods, siblings with higher SH at microsatellite loci experienced a higher probability of juvenile survival. This effect, however, was detected primarily within broods that experienced inbreeding or when inbreeding had occurred in their pedigree histories (i.e., at the parents’ level). Thus we show, for the first time in a wild population, that the strength of an HFC is partially dependent on the presence of inbreeding events in the recent pedigree history. Our results illustrate the importance of realized effects of inbreeding on genetic variation and fitness and the value of full‐sibling designs for the study of HFCs in the context of small, inbred populations.     

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).     

Multilocus heterozygosity, parental relatedness and individual fitness components in a wild mountain goat, Oreamnos americanus population

Matings between relatives lead to a decrease in offspring genetic diversity which can reduce fitness, a phenomenon known as inbreeding depression. Because alpine ungulates generally live in small structured populations and often exhibit a polygynous mating system, they are susceptible to inbreeding. Here, we used marker-based measures of pairwise genetic relatedness and inbreeding to investigate the fitness consequences of matings between relatives in a long-term study population of mountain goats ( Oreamnos americanus ) at Caw Ridge, Alberta, Canada. We first assessed whether individuals avoided mating with kin by comparing actual and random mating pairs according to their estimated genetic relatedness, which was derived from 25 unlinked polymorphic microsatellite markers and reflected pedigree relatedness. We then examined whether individual multilocus heterozygosity H , used as a measure of inbreeding, was predicted by parental relatedness and associated with yearling survival and the annual probability of giving birth to a kid in adult females. Breeding pairs identified by genetic parentage analyses of offspring that survived to 1 year of age were less genetically related than expected under random matings. Parental relatedness was negatively correlated with offspring H , and more heterozygous yearlings had higher survival to 2 years of age. The probability of giving birth was not affected by H in adult females. Because kids that survived to yearling age were mainly produced by less genetically related parents, our results suggest that some individuals experienced inbreeding depression in early life. Future research will be required to quantify the levels of gene flow between different herds, and evaluate their effects on population genetic diversity and dynamics.     

Heterozygosity–fitness correlations and their relevance to studies on inbreeding depression in threatened species

The majority of reported multilocus heterozygosity–fitness correlations (HFCs) are from large, outbred populations, and their relevance to studies on inbreeding depression in threatened populations is often stressed. The results of such HFC studies conducted on outbred populations may be of limited application to threatened population management, however, as bottlenecked populations exhibit increased incidence of inbreeding, increased linkage disequilibrium, reduced genetic diversity and possible effects of historical inbreeding such as purging. These differences may affect both our ability to detect inbreeding depression in threatened species, and our interpretation of the underlying mechanisms for observed heterozygosity–fitness relationships. The study of HFCs in outbred populations is of interest in itself, but the results may not translate directly to threatened populations that have undergone severe bottlenecks.     

What should we weigh to estimate heterozygosity,alleles or loci?

The interest to study the effects of inbreeding in natural populations has increased in the last years. Several microsatellite-derived metrics have recently been developed to infer inbreeding from multilocus heterozygosity data without requiring detailed pedigrees that are difficult to obtain in open populations. Internal relatedness (IR) is currently the most widespread used index and its main attribute is that allele frequency is incorporated into the measure. However, IR underestimates heterozygosity of individuals carrying rare alleles. For example, descendants of immigrants paired with natives (normally more outbred) bearing novel or rare alleles would be considered more homozygous than descendants of native parents. Thus, the analogy between homozygosity and inbreeding that generally is carried out would have no logic in those cases. We propose an alternative index, homozygosity by loci (HL) that avoids such problems by weighing the contribution of each locus to the homozygosity index depending on their allelic variability. Under a wide range of simulated scenarios, we found that our index (HL) correlated better than both IR and uncorrected homozygosity (H(O)), measured as proportion of homozygous loci) with genome-wide homozygosity and inbreeding coefficients in open populations. In these populations, which are likely to prevail in nature, the use of HL instead of IR reduced considerably the sample sizes required to achieve a given statistical power. This is likely to have important consequences on the ability to detect heterozygosity fitness correlations assuming the relationship between genome-wide heterozygosity and fitness traits.     

Evidence of opposing fitness effects of parental heterozygosity and relatedness in a critically endangered marine turtle?

How individual genetic variability relates to fitness is important in understanding evolution and the processes affecting populations of conservation concern. Heterozygosity–fitness correlations (HFCs) have been widely used to study this link in wild populations, where key parameters that affect both variability and fitness, such as inbreeding, can be difficult to measure. We used estimates of parental heterozygosity and genetic similarity (‘relatedness’) derived from 32 microsatellite markers to explore the relationship between genetic variability and fitness in a population of the critically endangered hawksbill turtle, Eretmochelys imbricata. We found no effect of maternal MLH (multilocus heterozygosity) on clutch size or egg success rate, and no single‐locus effects. However, we found effects of paternal MLH and parental relatedness on egg success rate that interacted in a way that may result in both positive and negative effects of genetic variability. Multicollinearity in these tests was within safe limits, and null simulations suggested that the effect was not an artefact of using paternal genotypes reconstructed from large samples of offspring. Our results could imply a tension between inbreeding and outbreeding depression in this system, which is biologically feasible in turtles: female‐biased natal philopatry may elevate inbreeding risk and local adaptation, and both processes may be disrupted by male‐biased dispersal. Although this conclusion should be treated with caution due to a lack of significant identity disequilibrium, our study shows the importance of considering both positive and negative effects when assessing how variation in genetic variability affects fitness in wild systems.     

Population demography and heterozygosity–fitness correlations in natural guppy populations: An examination using sexually selected fitness traits

Heterozygosity–fitness correlations (HFCs) have been examined in a wide diversity of contexts, and the results are often used to infer the role of inbreeding in natural populations. Although population demography, reflected in population‐level genetic parameters such as allelic diversity or identity disequilibrium, is expected to play a role in the emergence and detectability of HFCs, direct comparisons of variation in HFCs across many populations of the same species, with different genetic histories, are rare. Here, we examined the relationship between individual microsatellite heterozygosity and a range of sexually selected traits in 660 male guppies from 22 natural populations in Trinidad. Similar to previous studies, observed HFCs were weak overall. However, variation in HFCs among populations was high for some traits (although these variances were not statistically different from zero). Population‐level genetic parameters, specifically genetic diversity levels (number of alleles, observed/expected heterozygosity) and measures of identity disequilibrium (g2 and heterozygosity–heterozygosity correlations), were not associated with variation in population‐level HFCs. This latter result indicates that these metrics do not necessarily provide a reliable predictor of HFC effect sizes across populations. Importantly, diversity and identity disequilibrium statistics were not correlated, providing empirical evidence that these metrics capture different essential characteristics of populations. A complex genetic architecture likely underpins multiple fitness traits, including those associated with male fitness, which may have reduced our ability to detect HFCs in guppy populations. Further advances in this field would benefit from additional research to determine the demographic contexts in which HFCs are most likely to occur.     

A high‐quality pedigree and genetic markers both reveal inbreeding depression for quality but not survival in a cooperative mammal

Inbreeding depression, the reduced fitness of offspring of closely related parents, is commonplace in both captive and wild populations and has important consequences for conservation and mating system evolution. However, because of the difficulty of collecting pedigree and life‐history data from wild populations, relatively few studies have been able to compare inbreeding depression for traits at different points in the life cycle. Moreover, pedigrees give the expected proportion of the genome that is identical by descent (IBD_g) whereas in theory with enough molecular markers realized IBD_g can be quantified directly. We therefore investigated inbreeding depression for multiple life‐history traits in a wild population of banded mongooses using pedigree‐based inbreeding coefficients (f_ped) and standardized multilocus heterozygosity (sMLH) measured at 35–43 microsatellites. Within an information theoretic framework, we evaluated support for either f_ped or sMLH as inbreeding terms and used sequential regression to determine whether the residuals of sMLH on f_ped explain fitness variation above and beyond f_ped. We found no evidence of inbreeding depression for survival, either before or after nutritional independence. By contrast, inbreeding was negatively associated with two quality‐related traits, yearling body mass and annual male reproductive success. Yearling body mass was associated with f_ped but not sMLH, while male annual reproductive success was best explained by both f_ped and residual sMLH. Thus, our study not only uncovers variation in the extent to which different traits show inbreeding depression, but also reveals trait‐specific differences in the ability of pedigrees and molecular markers to explain fitness variation and suggests that for certain traits, genetic markers may capture variation in realized IBD_g above and beyond the pedigree expectation.     

The strength of the association between heterozygosity and probability of interannual local recruitment increases with environmental harshness in blue tits

The extent of inbreeding depression and the magnitude of heterozygosity–fitness correlations (HFC) have been suggested to depend on the environmental context in which they are assayed, but little evidence is available for wild populations. We combine extensive molecular and capture–mark–recapture data from a blue tit (Cyanistes caeruleus) population to (1) analyze the relationship between heterozygosity and probability of interannual adult local recruitment and (2) test whether environmental stress imposed by physiologically suboptimal temperatures and rainfall influence the magnitude of HFC. To address these questions, we used two different arrays of microsatellite markers: 14 loci classified as neutral and 12 loci classified as putatively functional. We found significant relationships between heterozygosity and probability of interannual local recruitment that were most likely explained by variation in genomewide heterozygosity. The strength of the association between heterozygosity and probability of interannual local recruitment was positively associated with annual accumulated precipitation. Annual mean heterozygosity increased over time, which may have resulted from an overall positive selection on heterozygosity over the course of the study period. Finally, neutral and putatively functional loci showed similar trends, but the former had stronger effect sizes and seemed to better reflect genomewide heterozygosity. Overall, our results show that HFC can be context dependent, emphasizing the need to consider the role of environmental heterogeneity as a key factor when exploring the consequences of individual genetic diversity on fitness in natural populations.     

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.     

Heterozygosity-fitness correlations among wild populations of European tree frog (Hyla arborea) detect fixation load

Quantifying the impacts of inbreeding and genetic drift on fitness traits in fragmented populations is becoming a major goal in conservation biology. Such impacts occur at different levels and involve different sets of loci. Genetic drift randomly fixes slightly deleterious alleles leading to different fixation load among populations. By contrast, inbreeding depression arises from highly deleterious alleles in segregation within a population and creates variation among individuals. A popular approach is to measure correlations between molecular variation and phenotypic performances. This approach has been mainly used at the individual level to detect inbreeding depression within populations and sometimes at the population level but without consideration about the genetic processes measured. For the first time, we used in this study a molecular approach considering both the interpopulation and intrapopulation level to discriminate the relative importance of inbreeding depression vs. fixation load in isolated and non-fragmented populations of European tree frog (Hyla arborea), complemented with interpopulational crosses. We demonstrated that the positive correlations observed between genetic heterozygosity and larval performances on merged data were mainly caused by co-variations in genetic diversity and fixation load among populations rather than by inbreeding depression and segregating deleterious alleles within populations. Such a method is highly relevant in a conservation perspective because, depending on how populations lose fitness (inbreeding vs. fixation load), specific management actions may be designed to improve the persistence of populations.     

Inbreeding coefficient and heterozygosity–fitness correlations in unhatched and hatched song sparrow nestmates

Heterozygosity–fitness correlations use molecular measures of heterozygosity as proxy estimates of individual inbreeding coefficients (f) to examine relationships between inbreeding and fitness traits. Heterozygosity–fitness correlations partly depend on the assumption that individual heterozygosity and f are strongly and negatively correlated. Although theory predicts that this relationship will be strongest when mean f and variance in f are high, few studies of heterozygosity–fitness correlations include estimates of f based on pedigrees, which allow for more thorough examinations of the relationship between f, heterozygosity and fitness in nature. We examined relationships between pedigree‐based estimates of f, multilocus heterozygosity (MLH) and the probability of survival to hatch in song sparrow nestmates. f and MLH were weakly, but significantly negatively correlated. Inbreeding coefficient predicted the probability of survival to hatch. In contrast, MLH did not predict the probability of survival to hatch nor did it account for residual variation in survival to hatch after statistically controlling for the effects of f. These results are consistent with the expectation that heterozygosity–f correlations will be weak when mean and variance in f are low. Our results also provide empirical support for recent simulation studies, which show that variation in MLH among siblings with equal f can be large and may obscure MLH–fitness relationships.     

Age‐dependent,negative heterozygosity–fitness correlations and local effects in an endangered Caribbean reptile,Iguana delicatissima

Inbreeding depression can have alarming impacts on threatened species with small population sizes. Assessing inbreeding has therefore become an important focus of conservation research. In this study, heterozygosity–fitness correlations (HFCs) were measured by genotyping 7 loci in 83 adult and 184 hatchling Lesser Antillean Iguanas, Iguana delicatissima, at a communal nesting site in Dominica to assess the role of inbreeding depression on hatchling fitness and recruitment to the adult population in this endangered species. We found insignificant correlations between multilocus heterozygosity and multiple fitness proxies in hatchlings and adults. Further, multilocus heterozygosity did not differ significantly between hatchlings and adults, which suggests that the survivorship of homozygous hatchlings does not differ markedly from that of their heterozygous counterparts. However, genotypes at two individual loci were correlated with hatching date, a finding consistent with the linkage between specific marker loci and segregating deleterious recessive alleles. These results provide only modest evidence that inbreeding depression influences the population dynamics of I. delicatissima on Dominica.     

The different sources of variation in inbreeding depression, heterosis and outbreeding depression in a metapopulation of Physa acuta

Understanding how parental distance affects offspring fitness, i.e., the effects of inbreeding and outbreeding in natural populations, is a major goal in evolutionary biology. While inbreeding is often associated with fitness reduction (inbreeding depression), interpopulation outcrossing may have either positive (heterosis) or negative (outbreeding depression) effects. Within a metapopulation, all phenomena may occur with various intensities depending on the focal population (especially its effective size) and the trait studied. However, little is known about interpopulation variation at this scale. We here examine variation in inbreeding depression, heterosis, and outbreeding depression on life-history traits across a full-life cycle, within a metapopulation of the hermaphroditic snail Physa acuta. We show that all three phenomena can co-occur at this scale, although they are not always expressed on the same traits. A large variation in inbreeding depression, heterosis, and outbreeding depression is observed among local populations. We provide evidence that, as expected from theory, small and isolated populations enjoy higher heterosis upon outcrossing than do large, open populations. These results emphasize the need for an integrated theory accounting for the effects of both deleterious mutations and genetic incompatibilities within metapopulations and to take into account the variability of the focal population to understand the genetic consequences of inbreeding and outbreeding at this scale.     

Analysis of the relationship between allozyme heterozygosity and fitness in the rare Gentiana pneumonanthe L.

Especially for rare species occurring in small populations, which are prone to loss of genetic variation and inbreeding, detailed knowledge of the relationship between heterozygosity and fitness is generally lacking. After reporting on allozyme variation and fitness in relation to population size in the rare plant Gentiana pneumonanthe, we present a more detailed analysis of the association between heterozygosity and individual fitness. The aim of this study was to test whether increased fitness of more heterozygous individuals is explained best by the ‘inbreeding’ hypothesis or by the ‘overdominance’ hypothesis. Individual fitness was measured during 8 months of growth in the greenhouse as the performance for six life-history parameters. PCA reduced these parameters to four main Fitness Components. Individual heterozygosity was scored for seven polymorphic allozyme loci. For some of these loci (e.g. Aat3, Pgm1 and 6Pgdh2) heterozygotes showed a significantly higher relative fitness than homozygotes. To test the inbreeding model, regression analyses were performed between each Fitness Component and the number of heterozygous loci per individual. Multiple regressions with the adaptive distance of five loci as independent variables were used to test the overdominance model. Only the inbreeding model was a statistically significant explanation for the relationship between heterozygosity and fitness in G. pneumonanthe. The number of heterozygous loci was significantly negatively correlated with the coefficients of variation of three of the six initially measured fitness parameters. This suggests a lower developmental stability among more homozygous plants and may explain the higher phenotypic variation in small populations of the species observed earlier. The importance of the results for conservation biology is discussed.     

Rhh: an R extension for estimating multilocus heterozygosity and heterozygosity-heterozygosity correlation

Individual multilocus heterozygosity estimates based on a limited number of loci are expected to correlate only weakly with the inbreeding level of an individual. Before using multilocus heterozygosity estimates in studies of inbreeding, their ability to capture information on inbreeding in the given setting should be tested. A convenient method for this is to compute the heterozygosity-heterozygosity correlation, i.e. the mean correlation between multilocus heterozygosity estimates calculated from random samples of loci, which should be positive if multilocus heterozygosity carries a signature of inbreeding. Rhh is an extension package for the statistical software r that estimates this correlation and calculates three measures of individual multilocus heterozygosity: homozygosity by loci, internal relatedness and standardized heterozygosity. The extension package is available through the CRAN (http://cran.r-project.org) and has a homepage at http://www.helsinki.fi/biosci/egru/research/software.     

Do microsatellites reflect genome‐wide genetic diversity in natural populations? A comment on Väli et al. (2008)

A recent study by Väli et al. (2008) highlights that microsatellites will often provide a poor prediction of the genome‐wide nucleotide diversity of wild populations, but does not fully explain why. To clarify and stress the importance of identity disequilibrium and marker variability for correlations between multilocus heterozygosity and genome‐wide genetic variability, we performed a simple simulation with different types of markers, corresponding to microsatellites and SNPs, in populations with different inbreeding history. The importance of identity disequilibrium was apparent for both markers and there was a clear impact of marker variability.     

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.     

Problems in measuring among-family variation in inbreeding depression

Understanding the sources of variation in inbreeding depression within populations is important for understanding the evolution of selfing rates. At the population level, inbreeding depression is due to decreased heterozygosity caused by inbreeding, which decreases overdominance and increases the frequency of expression of recessive deleterious alleles. However, within individual families inbreeding has two distinct consequences: it reduces heterozygosity and it restricts the alleles present in offspring to those present in the parent. Outcrossing both increases heterozygosity and brings new alleles into a family (compared to the alleles present if the plant is self-pollinated). Both consequences of inbreeding affect offspring fitness, but the most common experimental design used to measure among-family variation in inbreeding depression cannot distinguish them. The result is that variance in inbreeding depression among families is confounded by genetic variation in the traits being measured. Also, correlations (among families) between measures of inbreeding depression or between inbreeding depression and mean trait values are confounded by genetic variation in the traits being measured. I conclude that more complex crossing designs that allow estimation of breeding values for individual families are required to accurately detect and measure among-family variation in inbreeding depression.     

Molecular and pedigree measures of relatedness provide similar estimates of inbreeding depression in a bottlenecked population

Individual‐based estimates of the degree of inbreeding or parental relatedness from pedigrees provide a critical starting point for studies of inbreeding depression, but in practice wild pedigrees are difficult to obtain. Because inbreeding increases the proportion of genomewide loci that are identical by descent, inbreeding variation within populations has the potential to generate observable correlations between heterozygosity measured using molecular markers and a variety of fitness related traits. Termed heterozygosity‐fitness correlations (HFCs), these correlations have been observed in a wide variety of taxa. The difficulty of obtaining wild pedigree data, however, means that empirical investigations of how pedigree inbreeding influences HFCs are rare. Here, we assess evidence for inbreeding depression in three life‐history traits (hatching and fledging success and juvenile survival) in an isolated population of Stewart Island robins using both pedigree‐ and molecular‐derived measures of relatedness. We found results from the two measures were highly correlated and supported evidence for significant but weak inbreeding depression. However, standardized effect sizes for inbreeding depression based on the pedigree‐based kin coefficients (k) were greater and had smaller standard errors than those based on molecular genetic measures of relatedness (RI), particularly for hatching and fledging success. Nevertheless, the results presented here support the use of molecular‐based measures of relatedness in bottlenecked populations when information regarding inbreeding depression is desired but pedigree data on relatedness are unavailable.