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.     

Inbreeding depression influences lifetime breeding success in a wild population of red deer (Cervus elaphus)

Evolutionary and conservation biologists have a long-standing interest in the consequences of inbreeding. It is generally recognized that inbred individuals may experience reduced fitness or inbreeding depression. By the same token, relatively outbred individuals can have greater than average fitness, i.e. heterosis. However, nearly all of the empirical evidence for inbreeding depression comes from laboratory or domestic species. Inbreeding depression and heterosis are difficult to detect in natural populations due to the difficulties in establishing pedigrees. An alternative method is to correlate heterozygosity, which is measured using genetic markers, with a trait related to fitness. The typically studied traits, such as juvenile survival and growth rates, either cover only early life or are weakly correlated with lifetime breeding success (LBS). In this paper we show that heterozygosity is positively associated with male and female adult LBS in a wild population of red deer (Cervus elaphus) on the Isle of Rum, Scotland. To the authors' knowledge, this is the first time that inbreeding depression and/or heterosis have been detected for a trait highly correlated with overall fitness in both sexes in a wild population.     

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.     

Inbreeding depression in a critically endangered carnivore

Harmful effects arising from matings between relatives (inbreeding) is a long‐standing observation that is well founded in theory. Empirical evidence for inbreeding depression in natural populations is however rare because of the challenges of assembling pedigrees supplemented with fitness traits. We examined the occurrence of inbreeding and subsequent inbreeding depression using a unique data set containing a genetically verified pedigree with individual fitness traits for a critically endangered arctic fox (Vulpes lagopus) population. The study covered nine years and was comprised of 33 litters with a total of 205 individuals. We recorded that the present population was founded by only five individuals. Over the study period, the population exhibited a tenfold increase in average inbreeding coefficient with a final level corresponding to half‐sib matings. Inbreeding mainly occurred between cousins, but we also observed two cases of full‐sib matings. The pedigree data demonstrated clear evidence of inbreeding depression on traditional fitness traits where inbred individuals displayed reduced survival and reproduction. Fitness traits were however differently affected by the fluctuating resource abundande. Inbred individuals born at low‐quality years displayed reduced first‐year survival, while inbred individuals born at high‐quality years were less likely to reproduce. The documentation of inbreeding depression in fundamental fitness traits suggests that inbreeding depression can limit population recovery. Introducing new genetic material to promote a genetic rescue effect may thus be necessary for population long‐term persistence.     

Inbreeding in Japanese quail estimated by pedigree and microsatellite analyses

Accurately estimating inbreeding is important because inbreeding reduces fitness and production traits in populations. We analyzed information from pedigrees and from microsatellite markers to estimate inbreeding in a line of Japanese quail derived from a randombred line (QO) and maintained for 17 generations by pedigreed matings of brothers to groups of sisters. Pedigree data were used to calculate the inbreeding coefficient (F(IT)), which is the level of inbreeding based on a reference ancestor. Data from analysis of 14 microsatellite markers in the inbred and QO lines were used to calculate the population differentiation (F(ST)) of the lines caused by inbreeding. The F(IT) was then calculated as F(IT) = F(IS) + (1 - F(IS)) x F(ST), where F(IS) is the level of inbreeding in the inbred line. Observed heterozygosity from analysis of the microsatellite markers of the QO and inbred lines was 0.43 and 0.21, respectively, and the number of alleles was 3.29 and 1.93, demonstrating a reduction of genetic diversity in the inbred line. The F(IT) of the inbred line calculated from the pedigree and microsatellite marker analyses was 0.69 +/- 0.07 and 0.57 +/- 0.33, respectively. These data suggest that pedigree analysis was more accurate than microsatellite marker analyses for estimating inbreeding in this line of Japanese quail.     

Comparing molecular measures for detecting inbreeding depression

Correlations between heterozygosity and components of fitness have been investigated in natural populations for over 20 years. Positive correlations between a trait of interest and heterozygosity (usually measured at allozyme loci) are generally recognized as evidence of inbreeding depression. More recently, molecular markers such as microsatellites have been employed for the same purpose. A typical study might use around five to ten markers. In this paper we use a panel of 71 microsatellite loci to: (1) Compare the efficacy of heterozygosity and a related microsatellite‐specific variable, mean d², in detecting inbreeding depression; (2) Examine the statistical power of heterozygosity to detect such associations. We performed our analyses in a wild population of red deer (Cervus elaphus) in which inbreeding depression in juvenile traits had previously been detected using a panel of nine markers. We conclude that heterozygosity‐based measures outperform mean d²‐based measures, but that power to detect heterozygosity‐fitness associations is nonetheless low when ten or fewer markers are typed.     

GENETIC VARIATION IN INBREEDING DEPRESSION IN THE RED FLOUR BEETLE TRIBOLIUM CASTANEUM

Inbreeding depression varies among species and among populations within a species. Few studies, however, have considered the extent to which inbreeding depression varies within a single population. We report on two experiments to provide evidence that inbreeding depression is genetically variable, such that within a single population some lineages suffer severe inbreeding depression, others suffer only mild inbreeding depression, and some lineages actually increase in phenotypic value at higher levels of inbreeding. We examine the effects of population structure on inbreeding depression for two traits in the first experiment (adult dry weight and female relative fitness), and for seven traits in the second experiment (female and male adult dry weight, female and male relative fitness, female and male developmental time, and egg-to-adult viability). In the first experiment, we collected data from 4 families within each of 38 lineages derived from a single ancestral stock population and maintained for four generations of full-sib mating. Both traits demonstrate significant inbreeding depression and provide evidence that even within a single lineage there is significant genetic variability in inbreeding depression. In the second experiment, we collected data from 5 replicates for each of 15 lineages derived from the same ancestral population used in the first experiment; these lineages were maintained for four generations of full-sib mating. We also collected data on outbred control beetles in each generation and incorporated these data into the analyses to account for environmental effects in an unbiased manner. All traits except female and male developmental time show significant inbreeding depression. All traits showing inbreeding depression are genetically variable in inbreeding depression, as is evident from a significant linear lineage-×-f component. For both experiments, the effect of population structure on inbreeding depression is further evident from the increasing amount of variation that can be explained by the models used to measure inbreeding depression when additional levels of population structure are included. Genetic variation in inbreeding depression has important implications for conservation biology and may be an important factor in mating-system evolution.     

Variability of individual genetic load: consequences for the detection of inbreeding depression

Inbreeding depression is a key factor affecting the persistence of natural populations, particularly when they are fragmented. In species with mixed mating systems, inbreeding depression can be estimated at the population level by regressing the average progeny fitness by the selfing rate of their mothers. We applied this method using simulated populations to investigate how population genetic parameters can affect the detection power of inbreeding depression. We simulated individual selfing rates and genetic loads from which we computed fitness values. The regression method yielded high statistical power, inbreeding depression being detected as significant (5?% level) in 92?% of the simulations. High individual variation in selfing rate and high mean genetic load led to better detection of inbreeding depression while high among-individual variation in genetic load made it more difficult to detect inbreeding depression. For a constant sampling effort, increasing the number of progenies while decreasing the number of individuals per progeny enhanced the detection power of inbreeding depression. We discuss the implication of among-mother variability of genetic load and selfing rate on inbreeding depression studies.     

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.     

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.     

Inbreeding,inbreeding depression and extinction

Inbreeding is unavoidable in small, isolated populations and can cause substantial fitness reductions compared to outbred populations. This loss of fitness has been predicted to elevate extinction risk giving it substantial conservation significance. Inbreeding may result in reduced fitness for two reasons: an increased expression of deleterious recessive alleles (partial dominance hypothesis) or the loss of favourable heterozygote combinations (overdominance hypothesis). Because both these sources of inbreeding depression are dependent upon dominance variance, inbreeding depression is predicted to be greater in life history traits than in morphological traits. In this study we used replicate inbred and control lines of Drosophila simulans to address three questions:1) is inbreeding depression greater in life history than morphological traits? 2) which of the two hypotheses is the major underlying cause of inbreeding depression? 3) does inbreeding elevate population extinction risk? We found that inbreeding depression was significantly greater in life history traits compared to morphological traits, but were unable to find unequivocal support for either the overdominance or partial dominance hypotheses as the genetic basis of inbreeding depression. As predicted, inbred lines had a significantly greater extinction risk.     

Genomic analysis reveals depression due to both individual and maternal inbreeding in a free‐living mammal population

There is ample evidence for inbreeding depression manifested as a reduction in fitness or fitness‐related traits in the focal individual. In many organisms, fitness is not only affected by genes carried by the individual, but also by genes carried by their parents, for example if receiving parental care. While maternal effects have been described in many systems, the extent to which inbreeding affects fitness directly through the focal individual, or indirectly through the inbreeding coefficients of its parents, has rarely been examined jointly. The Soay sheep study population is an excellent system in which to test for both effects, as lambs receive extended maternal care. Here, we tested for both maternal and individual inbreeding depression in three fitness‐related traits (birthweight and weight and hindleg length at 4 months of age) and three fitness components (first‐year survival, adult annual survival and annual breeding success), using either pedigree‐derived inbreeding or genomic estimators calculated using ~37 000 SNP markers. We found evidence for inbreeding depression in 4‐month hindleg and weight, first‐year survival in males, and annual survival and breeding success in adults. Maternal inbreeding was found to depress both birthweight and 4‐month weight. We detected more instances of significant inbreeding depression using genomic estimators than the pedigree, which is partly explained through the increased sample sizes available. In conclusion, our results highlight that cross‐generational inbreeding effects warrant further exploration in species with parental care and that modern genomic tools can be used successfully instead of, or alongside, pedigrees in natural populations.     

Inbreeding depression in development,survival, and reproduction in the adzuki bean beetle (<Emphasis Type="Italic">Callosobruchus chinensis</Emphasis>)

Inbreeding depression of an aspect of fitness is observed in many insects, but the traits that are of importance for inbreeding depression of fitness remain poorly understood. Here the magnitude of inbreeding depression of fitness-related traits in the development and adult stages was measured in a captive population of the adzuki bean beetle, Callosobruchus chinensis (Coleoptera: Bruchidae). Beetles produced by full-sib matings had 8% lower survival in the development stage than did beetles produced by unrelated matings. Although inbred and outbred offspring did not differ in body size after emergence, inbred offspring took 2–3% longer to develop to emergence. This indicates inbreeding depression of growth rate. At the adult stage, inbreeding had no significant effect on longevity, however lifetime offspring production was reduced by 11%. Thus, the magnitude of inbreeding depression was relatively large for offspring production. This suggests inbreeding depression of fitness manifests, to a particularly significant extent, in reduced productivity. This study shows the C. chinensis population, which has been in captivity for more than 100 generations, harbors genetic loads.     

Multigenerational effects of inbreeding in Cucurbita pepo ssp. texana (Cucurbitaceae)

The shape of the fitness function relating the decline in fitness with coefficient of inbreeding (f) can provide evidence concerning the genetic basis of inbreeding depression, but few studies have examined inbreeding depression across a range of f using noncultivated species. Futhermore, studies have rarely examined the effects of inbreeding depression in the maternal parent on offspring fitness. To estimate the shape of the fitness function, we examined the relationship between f and fitness across a range off from 0.000 to 0.875 for components of both male and female fitness in Cucurbita pepo ssp. texana. Each measure of female fitness declined with f, including pistillate flower number, fruit number, seed number per fruit, seed mass per fruit, and percentage seed germination. Several aspects of male fitness also declined with f, including staminate flower number, pollen number per flower, and the number of days of flowering, although cumulative inbreeding depression was less severe for male (0.34) than for female function (0.39). Fitness tended to decline linearly with f between f = 0.00 and f = 0.75 for most traits and across cumulative lifetime fitness (mean = 0.66), suggesting that individual genes causing inbreeding depression are additive and the result of many alleles of small effect. However, most traits also showed a small reduction in inbreeding depression between f = 0.75 and f = 0.875, and evidence of purging or diminishing epistasis was found for in vitro pollen-tube growth rate. To examine inbreeding depression as a maternal effect, we performed outcross pollinations on f = 0.0 and f = 0.5 mothers and found that depression due to maternal inbreeding was 0.07, compared to 0.10 for offspring produced through one generation of selfing. In at least some families, maternal inbreeding reduced fruit number, seed number and mass, staminate flower number, pollen diameter, and pollen-tube growth rate. Collectively these results suggest that, while the fitness function appears to be largely linear for most traits, maternal effects may compound the effects of inbreeding depression in multigenerational studies, though this may be partially offset by purging or diminishing epistasis.     

Effects of inbreeding on male function and self-fertility in the partially self-incompatible herb Campanula rapunculoides (Campanulaceae)

We examined the effect of inbreeding on fitness (through both male and female functions) and changes in self-fertility in the partially self-incompatible species Campanula rapunculoides. Individuals in natural populations of C. rapunculoides varied extensively in their strength of self-incompatibility (SI). We crossed 11 individuals that differed in their strength of SI to generate families with four levels of inbreeding (f = 0.0, 0.25, 0.5, and 0.75). Progeny were scored for three traits related to male fitness and for outcrossed and selfed seed production. Analyses of variance revealed significant inbreeding depression for the three male traits and seed set. Families with strong or weak SI differed in their response to inbreeding. Families with weak SI had lower levels of inbreeding depression for most traits than families with strong SI, but strong SI families had a greater increase in selfed seed set, but not self-fertility, with inbreeding. Finally, we found evidence of a significant linear response to inbreeding for all three male reproductive traits and outcrossed seed, indicating that inbreeding depression was primarily caused by partially or fully recessive deleterious alleles. Variation in genetic load was associated with variation in self-fertility, a finding that suggests an evolutionary role for partial self-fertility in natural populations of C. rapunculoides.     

Genetic rescue and inbreeding depression in Mexican wolves

Although inbreeding can reduce individual fitness and contribute to population extinction, gene flow between inbred but unrelated populations may overcome these effects. Among extant Mexican wolves (Canis lupus baileyi), inbreeding had reduced genetic diversity and potentially lowered fitness, and as a result, three unrelated captive wolf lineages were merged beginning in 1995. We examined the effect of inbreeding and the merging of the founding lineages on three fitness traits in the captive population and on litter size in the reintroduced population. We found little evidence of inbreeding depression among captive wolves of the founding lineages, but large fitness increases, genetic rescue, for all traits examined among F1 offspring of the founding lineages. In addition, we observed strong inbreeding depression among wolves descended from F1 wolves. These results suggest a high load of deleterious alleles in the McBride lineage, the largest of the founding lineages. In the wild, reintroduced population, there were large fitness differences between McBride wolves and wolves with ancestry from two or more lineages, again indicating a genetic rescue. The low litter and pack sizes observed in the wild population are consistent with this genetic load, but it appears that there is still potential to establish vigorous wild populations.     

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.     

Multigenerational inbreeding in <Emphasis Type="Italic">Succisa pratensis</Emphasis>: Effects on fitness components

We examined the effects of repeated inbreeding on fitness components of the long-lived perennial Succisa pratensis (Dipsacaceae). Plants from six populations differing in size were used to establish lines with expected inbreeding coefficients f of 0, 0.5 and 0.75. The effects of different inbreeding levels were measured for seed set, seed mass, percentage germination and seedling relative growth rate. Seed set decreased following one generation of inbreeding and seedling growth rate decreased after two generations of inbreeding. Our study indicated that the mutational load is difficult to purge and that continued inbreeding tends to affect important traits in S. pratensis. Although the partial dominance hypothesis for inbreeding depression seems to account for the results, the overdominance hypothesis cannot be ruled out completely. Overall, we conclude that the response of a long-lived plant, such as S. pratensis, to repeated inbreeding does not differ from that of other plant species with shorter life spans, surely because the mechanisms that account for inbreeding depression are universal for all plant species.     

Conservation genetic management of a critically endangered New Zealand endemic bird: minimizing inbreeding in the Black Stilt Himantopus novaezelandiae

For threatened species with small captive populations, it is advisable to incorporate conservation management strategies that minimize inbreeding in an effort to avoid inbreeding depression. Using multilocus microsatellite genotype data, we found a significant negative relationship between genetic relatedness (inbreeding) and reproductive success (fitness) in a captive population of the critically endangered Black Stilt or KakīHimantopus novaezelandiae. In an effort to avoid inbreeding depression in this iconic New Zealand endemic, we recommend re‐pairing closely related captive birds with less related individuals and pairing new captive birds with distantly related individuals.     

No relationship between microsatellite variation and neonatal fitness in Antarctic fur seals, Arctocephalus gazella

Published studies of wild vertebrate populations have almost universally reported positive associations between genetic variation measured at microsatellite loci and fitness, creating the impression of ubiquity both in terms of the species and the traits involved. However, there is concern that this picture may be misleading because negative results frequently go unpublished. Here, we analyse the relationship between genotypic variation at nine highly variable microsatellite loci and neonatal fitness in 1070 Antarctic fur seal pups born at Bird Island, South Georgia. Despite our relatively large sample size, we find no significant association between three different measures of heterozygosity and two fitness traits, birth weight and survival. Furthermore, increasing genetic resolution by calculating parental relatedness also yields no association between genetic variation and fitness. Our findings are consistent with necropsy data showing that most pups die from starvation or trauma, conditions that are unlikely to be influenced strongly by genetic factors, particularly if the benefits of high heterozygosity are linked to immune-related genes.