Environmental stress increases selection against and dominance of deleterious mutations in inbred families of the Pacific oyster Crassostrea gigas

The deleterious effects of inbreeding are well documented and of major concern in conservation biology. Stressful environments have generally been shown to increase inbreeding depression; however, little is known about the underlying genetic mechanisms of the inbreeding-by-stress interaction and to what extent the fitness of individual deleterious mutations is altered under stress. Using microsatellite marker segregation data and quantitative trait locus (QTL) mapping methods, I performed a genome scan for deleterious mutations affecting viability (viability or vQTL) in two inbred families of the Pacific oyster Crassostrea gigas, reared in a stressful, nutrient-poor diet and a favourable, nutrient-rich diet, which had significant effects on growth and survival. Twice as many vQTL were detected in the stressful diet compared with the favourable diet, resulting primarily from substantially greater mortality of homozygous genotypes. At vQTL, estimates of selection (s) and dominance (h) were greater in the stressful environment (= 0.86 vs. 0.54 and = 0.35 vs. 0.18, in stressful and nonstressful diets, respectively). There was no evidence of interaction between vQTL. Individual vQTL differed across diets in selection only, or in both selection and dominance, and some vQTL were not affected by diet. These results suggest that stress-associated increases in selection against individual deleterious alleles underlie greater inbreeding depression with stress. Furthermore, the finding that inbreeding-by-environment interaction appears, to some extent, to be locus specific, helps to explain previous observations of lineage-specific expression of inbreeding depression and environment-specific purging, which have important implications for conservation and evolutionary biology.     

High genetic load in the Pacific oyster Crassostrea gigas

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2-3 months postfertilization in F(2) or F(3) hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8-14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena.     

QTL for resistance to summer mortality and OsHV‐1 load in the Pacific oyster (Crassostrea gigas)

Summer mortality is a phenomenon severely affecting the aquaculture production of the Pacific oyster (Crassostrea gigas). Although its causal factors are complex, resistance to mortality has been described as a highly heritable trait, and several pathogens including the virus Ostreid Herpes virus type 1 (OsHV‐1) have been associated with this phenomenon. A QTL analysis for survival of summer mortality and OsHV‐1 load, estimated using real‐time PCR, was performed using five F₂ full‐sib families resulting from a divergent selection experiment for resistance to summer mortality. A consensus linkage map was built using 29 SNPs and 51 microsatellite markers. Five significant QTL were identified and assigned to linkage groups V, VI, VII and IX. Analysis of single full‐sib families revealed differential QTL segregation between families. QTL for the two‐recorded traits presented very similar locations, highlighting the interest of further study of their respective genetic controls. These QTL show substantial genetic variation in resistance to summer mortality, and present new opportunities for selection for resistance to OsHV‐1.     

Recent approaches into the genetic basis of inbreeding depression in plants

Predictions for the evolution of mating systems and genetic load vary, depending on the genetic basis of inbreeding depression (dominance versus overdominance, epistasis and the relative frequencies of genes of large and small effect). A distinction between the dominance and overdominance hypotheses is that deleterious recessive mutations should be purged in inbreeding populations. Comparative studies of populations differing in their level of inbreeding and experimental approaches that allow selection among inbred lines support this prediction. More direct biometric approaches provide strong support for the importance of partly recessive deleterious alleles. Investigators using molecular markers to study quantitative trait loci (QTL) often find support for overdominance, though pseudo-overdominance (deleterious alleles linked in repulsion) may bias this perception. QTL and biometric studies of inbred lines often find evidence for epistasis, which may also contribute to the perception of overdominance, though this may be because of the divergent lines initially crossed in QTL studies. Studies of marker segregation distortion commonly uncover genes of major effect on viability, but these have only minor contributions to inbreeding depression. Although considerable progress has been made in understanding the genetic basis of inbreeding depression, we feel that all three aspects merit more study in natural plant populations.     

Genetic inviability is a major driver of type III survivorship in experimental families of a highly fecund marine bivalve

The offspring of most highly fecund marine fish and shellfish suffer substantial mortality early in the life cycle, complicating prediction of recruitment and fisheries management. Early mortality has long been attributed to environmental factors and almost never to genetic sources. Previous work on a variety of marine bivalve species uncovered substantial genetic inviability among the offspring of inbred crosses, suggesting a large load of early‐acting deleterious recessive mutations. However, genetic inviability of randomly bred offspring has not been addressed. Here, genome‐wide surveys reveal widespread, genotype‐dependent mortality in randomly bred, full‐sib progenies of wild‐caught Pacific oysters (Crassostrea gigas). Using gene‐mapping methods, we infer that 11–19 detrimental alleles per family render 97.9–99.8% of progeny inviable. The variable genomic positions of viability loci among families imply a surprisingly large load of partially dominant or additive detrimental mutations in wild adult oysters. Although caution is required in interpreting the relevance of experimental results for natural field environments, we argue that the observed genetic inviability corresponds with type III survivorship, which is characteristic of both hatchery and field environments and that our results, therefore, suggest the need for additional experiments under the near‐natural conditions of mesocosms. We explore the population genetic implications of our results, calculating a detrimental mutation rate that is comparable to that estimated for conifers and other highly fecund perennial plants. Genetic inviability ought to be considered as a potential major source of low and variable recruitment in highly fecund marine animals.     

Inbreeding reveals stronger net selection on Drosophila melanogaster males: implications for mutation load and the fitness of sexual females

Stronger selection on males has the potential to lower the deleterious mutation load of females, reducing the cost of sex. However, few studies have directly quantified the strength of selection for both sexes. As the magnitude of inbreeding depression (ID) is related to the strength of selection, we measured the cost of inbreeding for both males and females in a laboratory population of Drosophila melanogaster. Using a novel technique for inbreeding, we found significant ID for both juvenile viability and adult fitness in both sexes. The genetic variation responsible for this depression in fitness appeared to be recessive for adult fitness (h=0.11) and partially additive for juvenile viability (h=0.29). ID was identical across the sexes in terms of juvenile viability but was significantly more deleterious for males than females as adults, even though female X-chromosome homogamety should predispose them to a higher inbreeding load. We estimated the strength of selection on adult males to be 1.24 greater than on adult females, and this appears to be a consequence of selection arising from competition for mates. Combined with the generally positive intersexual genetic correlation for inbred lines, our results suggest that the mutation load of sexual females could be meaningfully reduced by stronger selection acting on males.     

Fixation, Segregation and Linkage of Allozyme Loci in Inbred Families of the Pacific Oyster Crassostrea Gigas (Thunberg): Implications for the Causes of Inbreeding Depression

The effect that inbreeding has on the fixation and segregation of genes has rarely been confirmed by direct observation. Here, fixation, segregation, and linkage of allozymes is investigated in the progeny of self-fertilized hermaphrodites of the normally outcrossing Pacific oyster Crassostrea gigas. The estimate of fixation pooled over loci, individuals, and families, F = 0.462, is significantly lower than the expected value of 0.5. Log-likelihood ratios reveal significant heterogeneity in fixation among individuals, among families, and among loci. In addition, the grand pooled segregation ratio, 127:243:54, deviates significantly from 1:2:1, with a bias against homozygotes for alleles of lesser frequency in the natural population. Segregation ratios for 11 of 14 loci are significantly heterogeneous among families, and exact tests for segregation within families reveal 16 significant results out of 51 tests. Thus, fixation and segregation of allozyme markers in inbred oyster families deviates from the expectations of neutral inbreeding theory. Di-genic disequilibria are significant for four of 74 di-locus pairs revealing two linkage groups. Strong viability selection is apparently conditional on the genotype of the hermaphrodite-founders and is largely focused on these two linkage groups. These genetic effects are explained by interaction between cis-linked factors and polymorphic regulatory backgrounds.     

The genetic covariance between life cycle stages separated by metamorphosis

Metamorphosis is common in animals, yet the genetic associations between life cycle stages are poorly understood. Given the radical changes that occur at metamorphosis, selection may differ before and after metamorphosis, and the extent that genetic associations between pre- and post-metamorphic traits constrain evolutionary change is a subject of considerable interest. In some instances, metamorphosis may allow the genetic decoupling of life cycle stages, whereas in others, metamorphosis could allow complementary responses to selection across the life cycle. Using a diallel breeding design, we measured viability at four ontogenetic stages (embryo, larval, juvenile and adult viability), in the ascidian Ciona intestinalis and examined the orientation of additive genetic variation with respect to the metamorphic boundary. We found support for one eigenvector of G (g_obsmax), which contrasted larval viability against embryo viability and juvenile viability. Target matrix rotation confirmed that while g_obsmax shows genetic associations can extend beyond metamorphosis, there is still considerable scope for decoupled phenotypic evolution. Therefore, although genetic associations across metamorphosis could limit that range of phenotypes that are attainable, traits on either side of the metamorphic boundary are capable of some independent evolutionary change in response to the divergent conditions encountered during each life cycle stage.     

GENETIC BASIS OF INBREEDING DEPRESSION IN ARABIS PETRAEA

Inbreeding depression may be caused by (partially) recessive or overdominant gene action. The relative evolutionary importance of these two modes has been debated; the former mode is emphasized in the “dominance hypothesis,” the latter in the “overdominance hypothesis.” We analyzed the genetic basis of inbreeding depression in the self-incompatible herb Arabis petraea (L.) Lam.: In the selfed progeny of twelve parental plants, we studied the proportion of chlorophyll-deficient seedlings, the genotypic distributions of marker genes, and associations of marker genotypes with viability and quantitative traits. Early components of fitness were examined by scoring seed size, germination time, and early growth rate and by observing the proportion of chlorophyll-deficient seedlings. Later components of fitness, flowering, and root and aboveground biomass were also measured. Marker genotypes of young seedlings were scored for 11 enzyme loci and three microsatellite markers. We found a high proportion (about 70%) of families with chlorophyll-deficient seedlings, indicating a high mutational load. We found six significant deviations from 1:2:1 ratio at marker loci of 60 tests in seedlings, with three of these significant at the experimentwide level. Deviations from the expected ratio were assumed to be due to linked viability loci. A graphical and a Bayesian method were used to distinguish between the overdominance and dominance hypotheses. Most of the deviant segregation ratios suggested overdominance instead of recessivity of the deleterious allele. Neither the early (seed size, germination time, or early growth trait) nor the late quantitative traits (flowering, and root and aboveground biomass) showed significant linkage to markers at the experimentwide level. Presence of significant associations between markers and early viability, but lack thereof for quantitative traits expressed late, suggests either that there may be relatively low inbreeding depression in later life stages or that individual quantitative trait loci may have smaller effects than loci contributing to early viability.     

Consequences of life history for inbreeding depression and mating system evolution in plants

Many plants are perennial, but most studies of inbreeding depression and mating system evolution focus on annuals. This paper extends a population genetic model of inbreeding depression due to recessive deleterious mutations to perennials. The model incorporates life history and mating system variation, and multiplicative selection across many genetic loci. In the absence of substantial mitotic mutation, perennials have higher mean fitness and lower, or even negative, inbreeding depression than annuals with the same mating system. As in annuals, self fertilization exposes deleterious recessive mutations to selection, increasing mean fitness and decreasing inbreeding depression. Including mitotic mutation decreases mean fitness while increasing inbreeding depression. Perenniality introduces a kind of selective sieve, such that strongly recessive mutations contribute disproportionately to mean fitness and inbreeding depression. In the presence of high mitotic mutation, this selective sieve may provide a mechanistic basis for high inbreeding depression observed in some long lived perennials. Without substantial mitotic mutation, it is difficult to reconcile genetically based models of inbreeding depression with the empirical generalization that perennials outcross while related annuals self fertilize.     

Segregation distortion for seed testa color in Mungbean (Vigna radiata L. Wilcek)

Genetic segregation experiments with plant species are commonly used for understanding the inheritance of traits. A basic assumption in these experiments is that each gamete developed from megasporogenesis has an equal chance of fusing with a gamete developed from microsporogenesis, and every zygote formed has an equal chance of survival. If gametic and/or zygotic selection occurs whereby certain gametes or zygotic combinations have a reduced chance of survival, progeny distributions are skewed and are said to exhibit segregation distortion. In this study, inheritance data are presented for the trait seed testa color segregating in large populations (more than 200 individuals) derived from closely related mungbean (Vigna radiata L. Wilcek) taxa. Segregation ratios suggested complex inheritance, including dominant and recessive epistasis. However, this genetic model was rejected in favor of a single-gene model based on evidence of segregation distortion provided by molecular marker data. The segregation distortion occurred after each generation of self-pollination from F1 thru F7 resulting in F7 phenotypic frequencies of 151:56 instead of the expected 103.5:103.5. This study highlights the value of molecular markers for understanding the inheritance of a simply inherited trait influenced by segregation distortion.     

Inbreeding uncovers fundamental differences in the genetic load affecting male and female fertility in a butterfly

Inbreeding depression is most pronounced for traits closely associated with fitness. The traditional explanation is that natural selection eliminates deleterious mutations with additive or dominant effects more effectively than recessive mutations, leading to directional dominance for traits subject to strong directional selection. Here we report the unexpected finding that, in the butterfly Bicyclus anynana, male sterility contributes disproportionately to inbreeding depression for fitness (complete sterility in about half the sons from brother-sister matings), while female fertility is insensitive to inbreeding. The contrast between the sexes for functionally equivalent traits is inconsistent with standard selection arguments, and suggests that trait-specific developmental properties and cryptic selection play crucial roles in shaping genetic architecture. There is evidence that spermatogenesis is less developmentally stable than oogenesis, though the unusually high male fertility load in B. anynana additionally suggests the operation of complex selection maintaining male sterility recessives. Analysis of the precise causes of inbreeding depression will be needed to generate a model that reliably explains variation in directional dominance and reconciles the gap between observed and expected genetic loads carried by populations. This challenging evolutionary puzzle should stimulate work on the occurrence and causes of sex differences in fertility load.     

Uneven segregation of sporophytic self-incompatibility alleles in Arabidopsis lyrata

Self-incompatibility in Arabidopsis lyrata is sporophytically controlled by the multi-allelic S-locus. Self-incompatibility alleles (S-alleles) are under strong negative frequency dependent selection because pollen carrying common S-alleles have fewer mating opportunities. Population genetics theory predicts that deleterious alleles can accumulate if linked to the S-locus. This was tested by studying segregation of S-alleles in 11 large full sib families in A. lyrata. Significant segregation distortion leading to an up to fourfold difference in transmission rates was found in six families. Differences in transmission rates were not significantly different in reciprocal crosses and the distortions observed were compatible with selection acting at the gametic stage alone. The S-allele with the largest segregation advantage is also the most recessive, and is very common in natural populations concordant with its apparent segregation advantage. These results imply that frequencies of S-alleles in populations of A. lyrata cannot be predicted based on simple models of frequency-dependent selection alone.     

GENETIC ARCHITECTURE FOR THE ADAPTIVE ORIGIN OF ANNUAL WILD RICE, ORYZA NIVARA

The wild progenitors of cultivated rice, Oryza nivara and Oryza rufipogon , provide an experimental system for characterizing the genetic basis of adaptation. The evolution of annual O. nivara from a perennial ancestor resembling its sister species, O. rufipogon , was associated with an ecological shift from persistently wet to seasonally dry habitats. Here we report a quantitative trait locus (QTL) analysis of phenotypic differentiation in life history, mating system, and flowering time between O. nivara and O. rufipogon . The exponential distribution of effect sizes of QTL fits the prediction of a recently proposed population genetic model of adaptation. More than 80% of QTL alleles of O. nivara acted in the same direction of phenotypic evolution, suggesting that they were fixed under directional selection. The loss of photoperiod sensitivity, which might be essential to the survival of the ancestral populations of O. nivara in the new environment, was controlled by QTL of relatively large effect. Mating system evolution from cross- to self-fertilization through the modification of panicle and floral morphology was controlled by QTL of small-to-moderate effect. The lack of segregation of the recessive annual habit in the F₂ mapping populations suggested that the evolution of annual from perennial life form had a complex genetic basis. The study captured the genetic architecture for the adaptive origin of O. nivara and provides a foundation for rigorous experimental tests of population genetic theories of adaptation.     

EFFECTIVENESS OF SELECTION IN REDUCING THE GENETIC LOAD IN POPULATIONS OF PEROMYSCUS POLIONOTUS DURING GENERATIONS OF INBREEDING

It has been hypothesized that natural selection reduces the “genetic load” of deleterious alleles from populations that inbreed during bottlenecks, thereby ameliorating impacts of future inbreeding. We tested the efficiency with which natural selection purges deleterious alleles from three subspecies of Peromyscus polionotus during 10 generations of laboratory inbreeding by monitoring pairing success, litter size, viability, and growth in 3604 litters produced from 3058 pairs. In P. p. subgriseus, there was no reduction across generations in inbreeding depression in any of the fitness components. Strongly deleterious recessive alleles may have been removed previously during episodes of local inbreeding in the wild, and the residual genetic load in this population was not further reduced by selection in the lab. In P. p. rhoadsi, four of seven fitness components did show a reduction of the genetic load with continued inbreeding. The average reduction in the genetic load was as expected if inbreeding depression in this population is caused by highly deleterious recessive alleles that are efficiently removed by selection. For P. p. leucocephalus a population that experiences periodic bottlenecks in the wild, the effect of further inbreeding in the laboratory was to exacerbate rather than reduce the genetic load. Recessive deleterious alleles may have been removed from this population during repeated bottlenecks in the wild; the population may be close to a threshold level of heterozygosity below which fitness declines rapidly. Thus, the effects of selection on inbreeding depression varied substantially among populations, perhaps due to different histories of inbreeding and selection.     

Genetic load in sexual and asexual diploids: segregation, dominance and genetic drift

In diploid organisms, sexual reproduction rearranges allelic combinations between loci (recombination) as well as within loci (segregation). Several studies have analyzed the effect of segregation on the genetic load due to recurrent deleterious mutations, but considered infinite populations, thus neglecting the effects of genetic drift. Here, we use single-locus models to explore the combined effects of segregation, selection, and drift. We find that, for partly recessive deleterious alleles, segregation affects both the deterministic component of the change in allele frequencies and the stochastic component due to drift. As a result, we find that the mutation load may be far greater in asexuals than in sexuals in finite and/or subdivided populations. In finite populations, this effect arises primarily because, in the absence of segregation, heterozygotes may reach high frequencies due to drift, while homozygotes are still efficiently selected against; this is not possible with segregation, as matings between heterozygotes constantly produce new homozygotes. If deleterious alleles are partly, but not fully recessive, this causes an excess load in asexuals at intermediate population sizes. In subdivided populations without extinction, drift mostly occurs locally, which reduces the efficiency of selection in both sexuals and asexuals, but does not lead to global fixation. Yet, local drift is stronger in asexuals than in sexuals, leading to a higher mutation load in asexuals. In metapopulations with turnover, global drift becomes again important, leading to similar results as in finite, unstructured populations. Overall, the mutation load that arises through the absence of segregation in asexuals may greatly exceed previous predictions that ignored genetic drift.     

Experimental studies on mating systems and genetic load in Onoclea sensibilis L. (Aspleniaceae: Athyrioideae)

Sporophytes from natural populations of Onoclea sensibilis L. from eastern North America and Japan were investigated to determine their mating system and the frequency of recessive deleterious genes (genetic load). Morphological and physiological criteria in the gametophyte generation indicate that this species has a high probability of intergametophytic mating in nature. All sporophytes exhibited some degree of heterozygosity for genetic load, expressed either as zygotic lethals or leaky lethals. Effects of antheridogen on gametophyte sex ratios and polyembryony are described.     

The genetic architecture of ecological speciation and the association with signatures of selection in natural lake whitefish (Coregonus sp. Salmonidae) species pairs

Adaptive evolutionary change is contingent on variation and selection; thus, understanding adaptive divergence and ultimately speciation requires information on both the genetic basis of adaptive traits as well as an understanding of the role of divergent natural selection on those traits. The lake whitefish (Coregonus clupeaformis) consists of several sympatric "dwarf" (limnetic) and normal (benthic) species pairs that co-inhabit northern postglacial lakes. These young species pairs have evolved independently and display parallelism in life history, behavioral, and morphological divergence associated with the use of distinct trophic resources. We identified phenotype-environment associations and determined the genetic architecture and the role of selection modulating population genetic divergence in sympatric dwarf and normal lake whitefish. The genetic architecture of 9 adaptive traits was analyzed in 2 hybrid backcrosses individually phenotyped throughout their life history. Significant quantitative trait loci (QTL) were associated with swimming behavior (habitat selection and predator avoidance), growth rate, morphology (condition factor and gill rakers), and life history (onset of maturity and fecundity). Genome scans among 4 natural sympatric pairs, using loci segregating in the map, revealed a signature of selection for 24 loci. Loci exhibiting a signature of selection were associated with QTL relative to other regions of the genome more often than expected by chance alone. Two parallel QTL outliers for growth and condition factor exhibited segregation distortion in both mapping families, supporting the hypothesis that adaptive divergence contributing to parallel reductions of gene flow among natural populations may cause genetic incompatibilities. Overall, these findings offer evidence that the genetic architecture of ecological speciation is associated with signatures of selection in nature, providing strong support for the hypothesis that divergent natural selection is currently maintaining adaptive differentiation and promoting ecological speciation in lake whitefish species pairs.     

Domestication reshaped the genetic basis of inbreeding depression in a maize landrace compared to its wild relative,teosinte

Inbreeding depression is the reduction in fitness and vigor resulting from mating of close relatives observed in many plant and animal species. The extent to which the genetic load of mutations contributing to inbreeding depression is due to large-effect mutations versus variants with very small individual effects is unknown and may be affected by population history. We compared the effects of outcrossing and self-fertilization on 18 traits in a landrace population of maize, which underwent a population bottleneck during domestication, and a neighboring population of its wild relative teosinte. Inbreeding depression was greater in maize than teosinte for 15 of 18 traits, congruent with the greater segregating genetic load in the maize population that we predicted from sequence data. Parental breeding values were highly consistent between outcross and selfed offspring, indicating that additive effects determine most of the genetic value even in the presence of strong inbreeding depression. We developed a novel linkage scan to identify quantitative trait loci (QTL) representing large-effect rare variants carried by only a single parent, which were more important in teosinte than maize. Teosinte also carried more putative juvenile-acting lethal variants identified by segregation distortion. These results suggest a mixture of mostly polygenic, small-effect partially recessive effects in linkage disequilibrium underlying inbreeding depression, with an additional contribution from rare larger-effect variants that was more important in teosinte but depleted in maize following the domestication bottleneck. Purging associated with the maize domestication bottleneck may have selected against some large effect variants, but polygenic load is harder to purge and overall segregating mutational burden increased in maize compared to teosinte.     

Theoretical aspects of extinction by inbreeding depression

Populational extinction due to inbreeding depression is analyzed with simple population genetic and population ecological models. Two alternative genetic mechanisms of inbreeding depression, i.e. recessive deleterious genes and overdominant genes, are assumed in separate analyses in order to examine their relative importance. With both mechanisms the population size and the coefficient of inbreeding are maintained at stable equilibria if there is no non-genetic demographic disturbance or stress. With a certain amount of demographic disturbance the population declines rapidly due to interaction between the decrease of population size and the increase of inbreeding coefficient. Such rapid extinction occurs with both genetic mechanisms. However, in the case of overdominant genes extinction happens only if the equilibrium population size is small and the selection coefficient is large such that segregation load is large. In nature, extinction due to overdominant genes is considered to be much less likely than extinction due to recessive deleterious genes.