Despite decades of research on the properties of new mutations, we know relatively little about their effect on adult fitness. By quantifying the consequences of new X-chromosome mutations for the reproductive success of males and females, we have begun to address this gap. Our results indicate that selection against new mutations differs in magnitude between the sexes, though the direction of change appears broadly concordant. This may be a fundamental feature of many populations due to the ubiquity of sexual selection.
We estimated that the rate of decline in adult fitness due to MA was 0.53% per X chromosome per generation in homozygous females, 0.80% per X chromosome per generation in males, and 0.14% per X chromosome per generation in heterozygous females. Scaled up to the entire haploid genome, this would imply a 2.6% decline in fitness per generation in homozygous females, and a 3.9% decline per generation in homozygous males. The heterozygous female decline is predicted to be much lower, at 0.7% per haploid genome per generation. Thus, the MA treatment was associated with a rapid decline in fitness. Our estimates for the rates of mutational decline in adult fitness are greater than estimates from both classical  and recent MA studies using viability [21, 31, 32]. This is consistent with previous work performed with the IV population , which found that inbreeding depression for total fitness was mainly due to depression in adult fitness. If the IV population is at mutation-selection balance, it is possible that stronger inbreeding depression for adult fitness is reflective of increased mutational pressure. In any case, our results demonstrate that the total mutational load of populations could be much greater than measurements of viability alone would imply.
Potential sources of error in estimating the rate of mutational decline in fitness for a population include confounding factors that bias the rate of mutation-accumulation during MA and factors that bias the measured impact of mutation during fitness assays. We accumulated mutations on hemizygous X chromosomes in Drosophila males. In Drosophila, the rate of sequence change at neutral sites suggests that the mutation rate is not distinguishable from parity between the sexes , so we expect that the baseline rate of sequence change in our study was a fair representation of the normal mutation rate. Because we eliminated sexual selection by passing each MA line through single-X bottlenecks, the opportunity for selection within the MA lines was limited to differences in viability between siblings resulting from a single generation of MA. Under the low competition conditions employed, we expect little viability selection, and even less impact on the rate of mutation-accumulation for genes affecting adult fitness in the MA population.
The control lines were kept as small, effectively asexual populations (i.e.: no recombination between X chromosomes within a C-line) to minimize the possibility of adaptation. Adaptation in the control population would artificially inflate the measured decline in fitness due to MA and has been cited as a potentially major source of bias in estimating mutational parameters in other studies [9, 35]. Control-X chromosomes were expressed hemizygously and the opportunity for sexual selection existed in these populations; while this, along with a larger population size, will slow down the rate of MA, we cannot eliminate the possibility that some deleterious mutations have fixed in these lines. The presence of mutation-accumulation in the control lines will cause us to underestimate the rate of erosion in fitness due to MA. MA in the control population could also affect our estimates for the relative strength of selection in males and females, if these mutations have sex-specific effects on mean fitness. In particular, mutations in genes with female-limited expression could accumulate freely under our experimental design because C-line chromosomes are only exposed to selection in males. If mutations with larger effects on females had accumulated in the C-lines, this would diminish differences between control and MA females, making the male differential appear larger and inflating
. However, there is very little evidence for widespread female-limitation of gene-expression in the D. melanogaster genome , and the results presented here indicate that most mutations are selected against in both sexes. In addition, direct observations from experiments on the maintenance of control lines over several dozen generations show no evidence of female-specific mutational decline (additional file 1, Figure S1).
Another important consideration stems from the observation that the environmental conditions under which fitness assays are performed can profoundly affect the perceived decline in fitness due to MA. As an example, it is well known that highly competitive conditions exaggerate differences between the control and MA populations: the decline in viability with MA can be nearly 10-fold greater under harsh competitive conditions , and diluting the food has also been found to affect the relative performance of MA flies . Many MA studies have used wild-caught or recently domesticated populations; the fitness assays used are unlikely to encapsulate the relevant selective environment. The use of populations in novel environments may also increase the probability of adaptation in the control lines. When measuring the selective effects of mutations in a particular population, it therefore seems sensible to restrict our measures to the conditions that shaped its population-genetic structure. The IV population has been maintained under consistent culture conditions for over 700 generations, and we emulated the culture protocol in almost every detail for our fitness assays. We therefore do not anticipate substantial bias in our estimates of fitness decline resulting from the environmental conditions used.
Because we measured the performance of experimental flies as adults by counting their progeny, viability effects may have influenced our measurement of MA for adult fitness. In particular, the offspring of adults from the MA treatment may have suffered in terms of reduced viability, which would inflate our estimate of the effects of MA on adult fitness. This effect would be most pronounced for homozygous MA females, who pass on a full copy of their MA-X chromosomes to their sons, and who may also contribute adverse maternal effects to their offspring. A significant viability effect on the offspring of MA females would make our estimates of
conservative. Strong differences in viability due to MA-X chromosomes should manifest themselves in terms of skewed sex ratios, but we found no differential effect of MA on offspring sex ratio.
We estimated that the magnitude of mutational effects, X-chromosome wide, was approximately 1.4 times stronger in males than in females (1.06-2.14, a range that includes both experimental error and uncertainty around U). In addition, the intersexual genetic correlation for the MA lines was significant and positive. This suggests that, for the majority of new mutations on the X chromosome, selection operates in the same direction for both sexes. This is interesting because the X chromosome is the genomic location most likely to show mutations with sexually antagonistic or sex-independent effects [26, 27]. While we do not dispute this, our results nevertheless seem to indicate that most of the mutations we assayed had sexually concordant effects on adult fitness. However, even if all mutations had concordant effects on fitness, the range in
presented here (1.06-2.14) could be consistent with anything from modest benefits of sexual selection to females to a greater than two-fold fitness advantage compared to a hypothetical asexual competitor, depending on the mutation rate, so refining estimates for
through further study will be critical.
We found that the effects of new mutations were positively correlated between males and females when females expressed MA-X chromosomes homozygously, but new mutations will most often be expressed heterozygously in females. In the MA lines, we found a positive association between homozygous and heterozygous female fitness values suggesting that new mutations are partially additive. We estimate that the population-wide dominance coefficient for new mutations is about 0.3. As new mutations will be expressed hemizygously in males and heterozygously in females, the effectiveness of selection will be much greater for males. Based on the rate of heterozygous female decline in fitness, we estimate the 'effective'
to be greater than 5 for the X chromosome.
The hemizygosity of males should result in more efficient selection on recessive alleles on the X chromosome. When these alleles have concordant directional effects across the sexes, this will result in reduced genetic load, an expectation corroborated by the absence of detectable inbreeding depression for juvenile viability in several populations of Drosophila melanogaster [33, 37]. For adult fitness, however, our results indicate that there remains substantial standing deleterious genetic variation on the X chromosome, as evidenced by the presence of substantial inbreeding depression for female fitness in the control group.
There are several possible explanations for the high genetic load for adult fitness found on the X chromosomes of the IV population. First, adult fitness might represent a larger mutational target than juvenile viability. We believe this is likely because adult fitness will be influenced both by juvenile traits not captured by viability (for example larval condition upon pupation) and by mate competition. Second, sexually antagonistic alleles, though in the minority, may nonetheless exert considerable effects on net fitness because the genetic load associated with them tends to be greater than for concordantly selected alleles . In a separate population of Drosophila (LHm), the amount of sexually antagonistic variation on the X chromosome was sufficient to cause a negative intersexual genetic correlation for adult fitness . In that study, the X chromosome was estimated to account for about 45% of the total genetic variation in fitness, and nearly all of the sexually antagonistic variation. Even so, most new mutations in the LHm population are predicted to be under concordant selection , although the intersexual genetic correlation for fitness was not measured.
Similarly, the X chromosome appears to harbour a disproportionate amount of fitness variation in the IV population. Previous work with the IV population suggested that completely inbred IV-derived females were 36% as fit as their outbred counterparts . In this study, females inbred for the X chromosome were 57% as fit as outbred females. Assuming multiplicative fitness effects, we infer that females completely inbred for the autosomes would be 63% as fit as outbred females. The X chromosome therefore seems to contribute more than half of the total inbreeding depression for adult female fitness, despite accounting for only a fifth of the gene content. The presence of segregating sexually antagonistic alleles on the X chromosomes in the IV population would be consistent with the disproportionate amount of genetic load on this chromosome, and is supported by a lack of positive intersexual correlation in the control lines.
Moderate amounts of sexually antagonistic variation have the potential to reduce the benefits of sexual selection, but the extent to which it does so depends on the relative amount and intensity of sexually concordant and sex-specific selection on the genomic scale . Our results suggest that, at least for the X chromosome, the majority of mutations have sexually concordant effects, as our estimate of
is a global estimate. As long as the fraction of sexually antagonistic alleles generated by mutation is small, our results suggest that sexual selection could still yield net benefits to females, though estimating the precise fraction of mutations that are concordantly selected vs. sexually antagonistic should be a priority.
No study yet designed has been able to estimate all of the relevant properties of new mutations. Molecular methods are increasingly being used , but can only directly measure the total rate of sequence change; without contemporary fitness data these methods provide only indirect estimates of the deleterious mutation rate. Conversely, fitness estimates on their own, while providing important insight into the consequences and character of new mutations, do not produce reliable estimates of the mutation rate. Mutation-accumulation in well-defined and replicable experimental populations such as the IV population, combined with advances in sequencing, could provide a much clearer picture of the fate of new mutations in populations than either technique in isolation. For example, one approach could involve sequencing MA lines to obtain the actual changes having occurred during MA, and then allowing replicated populations to purge these mutations in the standard laboratory environment. The rate at which mutations are eliminated would permit estimation of the strength of selection against them.