Statistical Methods for Analyzing Drosophila Germline Mutation Rates

Most studies of mutation rates implicitly assume that they remain constant throughout development of the germline. However, researchers recently used a novel statistical framework to reveal that mutation rates differ dramatically during sperm development in Drosophila melanogaster. Here a general framework is described for the inference of germline mutation patterns, generated from either mutation screening experiments or DNA sequence polymorphism data, that enables analysis of more than two mutations per family. The inference is made more rigorous and flexible by providing a better approximation of the probabilities of patterns of mutations and an improved coalescent algorithm within a single host with realistic assumptions. The properties of the inference framework, both the estimation and the hypothesis testing, were investigated by simulation. The refined inference framework is shown to provide (1) nearly unbiased maximum-likelihood estimates of mutation rates and (2) robust hypothesis testing using the standard asymptotic distribution of the likelihood-ratio tests. It is readily applicable to data sets in which multiple mutations in the same family are common.     

Increasing the data size to accurately reconstruct the phylogenetic relationships between nine subgroups of the Drosophila melanogaster species group (Drosophilidae, Diptera)

Previous phylogenetic analyses of the melanogaster species group have led to conflicting hypotheses concerning their relationship; therefore the addition of new sequence data is necessary to discover the phylogeny of this species group. Here we present new data derived from 17 genes and representing 48 species to reconstruct the phylogeny of the melanogaster group. A variety of statistical tests, as well as maximum likelihood mapping analysis, were performed to estimate data quality, suggesting that all genes had a high degree of contribution to resolve the phylogeny.Individual locus was analyzed using maximum likelihood (ML), and the concatenated dataset (12,988 bp) were analyzed using partitioned maximum likelihood (ML) and Bayesian analyses. Separated analysis produced various phylogenetic relationships, however, phylogenetic topologies from ML and Bayesian analysis based on concatenated dataset, at the subgroup level, were completely identical to each other with high levels of support. Our results recovered three major clades: the ananassae subgroup, followed by the montium subgroup, the melanogaster subgroup and the oriental subgroups form the third monophyletic clade, in which melanogaster (takahashii, suzukii) forms one subclade and ficusphila [eugracilis (elegans, rhopaloa)] forms another. However, more data are necessary to determine the phylogenetic position of Drosophila lucipennis which proved difficult to place.     

A screen for lethal mutations in the chromosomal region 59AB suggests that bellwether encodes the alpha subunit of the mitochondrial ATP synthase in Drosophila melanogaster

We report the isolation and complementation mapping of lethal mutations within the 59AB region on the second chromosome of Drosophila melanogaster. The newly induced lethal mutations in this region define four different complementation groups. Using existing and newly induced deficiencies, these loci can be assigned to three different chromosomal intervals. Moreover, complementation analysis with chromosomes carrying various P element insertions, in combination with a molecular characterization of the corresponding insertion sites, suggests that the previously described male sterile mutation bellwether is an allele of an essential gene that encodes the alpha subunit of the mitochondrial ATP synthase.     

The Distribution of Mutation Effects on Viability in Drosophila Melanogaster

Parameters of continuous distributions of effects and rates of spontaneous mutation for relative viability in Drosophila are estimated by maximum likelihood from data of two published experiments on accumulation of mutations on protected second chromosomes. A model of equal mutant effects gives a poor fit to the data of the two experiments; higher likelihoods are obtained with leptokurtic distributions or for models in which there is more than one class of mutation effect. Minimum estimates of mutation rates (events per generation) at polygenes affecting viability on chromosome 2 are 0.14 and 0.068, but estimates are strongly confounded with other parameters in the model. Separate information on rates of molecular divergence between Drosophila species and from rates of movement of transposable elements is used to infer the overall genomic mutation rate in Drosophila, and the viability data are analyzed with mutation rate as a known parameter. If, for example, a mutation rate for chromosome 2 of 0.4 is assumed, maximum likelihood estimates of mean mutant effect on relative viability are 0.4% and 1%, but the majority of mutations have very much smaller effects than these values as distributions are highly leptokurtic. The methodology is applied to estimate viability effects of single P element insertional mutations. The mean effect per insertion is found to be higher, and their distribution is found to be less leptokurtic than for spontaneous mutations. The equilibrium genetic variance of viability predicted by a mutation-selection balance model with parameters estimated from the mutation accumulation experiments is similar to laboratory estimates of genetic variance of viability from natural populations of Drosophila.     

Increased transmission of mutations by low-condition females: evidence for condition-dependent DNA repair

Evidence is mounting that mutation rates are sufficiently high for deleterious alleles to be a major evolutionary force affecting the evolution of sex, the maintenance of genetic variation, and many other evolutionary phenomena. Though point estimates of mutation rates are improving, we remain largely ignorant of the biological factors affecting these rates at the individual level. Of special importance is the possibility that mutation rates are condition-dependent with low-condition individuals experiencing more mutation. Theory predicts that such condition dependence would dramatically increase the rate at which populations adapt to new environments and the extent to which populations suffer from mutation load. Despite its importance, there has been little study of this phenomenon in multicellular organisms. Here, we examine whether DNA repair processes are condition-dependent in Drosophila melanogaster. In this species, damaged DNA in sperm can be repaired by maternal repair processes after fertilization. We exposed high- and low-condition females to sperm containing damaged DNA and then assessed the frequency of lethal mutations on paternally derived X chromosomes transmitted by these females. The rate of lethal mutations transmitted by low-condition females was 30% greater than that of high-condition females, indicating reduced repair capacity of low-condition females. A separate experiment provided no support for an alternative hypothesis based on sperm selection.     

Rates and Genomic Consequences of Spontaneous Mutational Events in Drosophila melanogaster

Because spontaneous mutation is the source of all genetic diversity, measuring mutation rates can reveal how natural selection drives patterns of variation within and between species. We sequenced eight genomes produced by a mutation-accumulation experiment in Drosophila melanogaster. Our analysis reveals that point mutation and small indel rates vary significantly between the two different genetic backgrounds examined. We also find evidence that ∼2% of mutational events affect multiple closely spaced nucleotides. Unlike previous similar experiments, we were able to estimate genome-wide rates of large deletions and tandem duplications. These results suggest that, at least in inbred lines like those examined here, mutational pressures may result in net growth rather than contraction of the Drosophila genome. By comparing our mutation rate estimates to polymorphism data, we are able to estimate the fraction of new mutations that are eliminated by purifying selection. These results suggest that ∼99% of duplications and deletions are deleterious—making them 10 times more likely to be removed by selection than nonsynonymous mutations. Our results illuminate not only the rates of new small- and large-scale mutations, but also the selective forces that they encounter once they arise.     

The effect of sexual harassment on lethal mutation rate in female Drosophila melanogaster

The rate by which new mutations are introduced into a population may have far-reaching implications for processes at the population level. Theory assumes that all individuals within a population have the same mutation rate, but this assumption may not be true. Compared with individuals in high condition, those in poor condition may have fewer resources available to invest in DNA repair, resulting in elevated mutation rates. Alternatively, environmentally induced stress can result in increased investment in DNA repair at the expense of reproduction. Here, we directly test whether sexual harassment by males, known to reduce female condition, affects female capacity to alleviate DNA damage in Drosophila melanogaster fruitflies. Female gametes can repair double-strand DNA breaks in sperm, which allows manipulating mutation rate independently from female condition. We show that male harassment strongly not only reduces female fecundity, but also reduces the yield of dominant lethal mutations, supporting the hypothesis that stressed organisms invest relatively more in repair mechanisms. We discuss our results in the light of previous research and suggest that social effects such as density and courtship can play an important and underappreciated role in mediating condition-dependent mutation rate.     

Mutagenicity of organophosphorus compounds in bacteria and drosophila

140 Organophosphorus compounds (OP's) have been tested for mutagenic activity in bacteria, principally by using two specially constructed sets of tester strains of the bacteria Salmonella typhimurium and Escherichia coli. It was found that 20% gave positive mutagenic responses and that this group of chemicals produce base substitutions rather than frame-shift mutations. In most cases the DNA repair genes exrA⁺ and recA⁺ were for mutagenic activity.Seven compounds were further tested in Drosophila melanogaster for the ability to induce recessive lethal mutations. In some of these cases the doses administered to the flies had to be very low due to the highly toxic nature of the compounds. To overcome this problem, the accumulation of recessive lethal mutations was measured in populations which were continually exposed to the compounds over a period of some 18 months. During this time the populations developed increased resistance to the compound and so the dose administered could gradually be increased. Six of the compounds were mutagenic.Of the compounds tested in both systems, those showing mutagenic activity in bacteria were also mutaganic in Drosophila, those mutagenic in bacteria were not mutagenic in Drosophila.     

Estimation of the neutral mutation rate in a finite population from DNA sequence data

The sampling theory for the infinite site model taking into account the phylogenetic relationship between the alleles is developed for those cases in which two or three alleles are observed in the sample. From this theory a maximum likelihood estimate of θ = 4Nμ can be obtained. Unlike the maximum likelihood estimate of θ based on the infinite allele model or the number of segregating sites, this estimate of θ is a function of the frequencies of the alleles. This method is used to estimate θ for mitochondrial DNA in Drosophila melanogaster and D. virilis from data obtained by Shah and Langley (1979. Nature (London)281, 696–699) using restriction endonucleases.     

Buthionine sulfoximine mediated enhancement of γ-radiation induced mutation frequency in Drosophila melanogaster

Experiments were carried out to investigate whether or not depletion of the glutathione (GSH) level in Drosophila melanogaster larvae with buthionine sulfoximine (BSO) treatment can result in the modulation of the frequency of sex-linked recessive lethal (SLRL) mutations induced by γ-radiation. Third instar larvae were fed on BSO for 24 h before exposure to 10 Gy γ-radiation. Immediately after this the larvae were divided into two batches, which were used to determining the GSH level and the induction of SLRL mutations respectively. The results obtained suggest that the depletion of the GSH level with BSO can lead to an enhancement in the frequency of SLRL mutations (significant at the 5% level). In a subsequent experiment in which adult Drosophila melanogaster male flies were fed on BSO for 72 h before irradiation, a significant increase was observed in the incidence of SLRL mutations.     

Anent the Genomics of Spermatogenesis in Drosophila melanogaster

An appreciable fraction of the Drosophila melanogaster genome is dedicated to male fertility. One approach to characterizing this subset of the genome is through the study of male-sterile mutations. We studied the relation between vital and male-fertility genes in three large autosomal regions that were saturated for lethal and male-sterile mutations. The majority of male-sterile mutations affect genes that are exclusively expressed in males. These genes are required only for male fertility, and several mutant alleles of each such gene were encountered. A few male-sterile mutations were alleles of vital genes that are expressed in both males and females. About one-fifth of the genes in Drosophila melanogaster show male-specific expression in adults. Although some earlier studies found a paucity of genes on the X chromosome showing male-biased expression, we did not find any significant differences between the X chromosome and the autosomes either in the relative frequencies of mutations to male sterility or in the frequencies of genes with male-specific expression in adults. Our results suggest that as much as 25% of the Drosophila genome may be dedicated to male fertility.     

Mutagenicity tests with metrifonate in Drosophila melanogaster

The organophosphorus insecticide metrifonate (O,O-dimethyl(1-hydroxy-2,2,2-trichloroethyl)phosphonate), also known as trichlorfon or Dipterex ®, was tested for its ability to induce sex-linked and autosomal recessive lethal mutations in Drosophila melanogaster. There was no evidence of an increase in the frequency of lethal mutations after feeding adult males with metrifonate, but the high toxicity of the compound meant that only low concentrations could be used in this test system.     

Screening of larval/pupal P-element induced lethals on the second chromosome in Drosophila melanogaster: clonal analysis and morphology of imaginal discs

We have carried out screens for lethal mutations on the second chromosome of Drosophila melanogaster that are associated with abnormal imaginal disc morphologies, particularly in the wing disc. From a collection of 164 P element-induced mutations with a late larva/pupa lethal phase we have identified 56 new loci whose gene products are required for normal wing disc development and for normal morphology of other larval organs. Genetic mosaics of these 56 mutant lines show clonal mutant phenotypes for 23 cell-viable mutations. These phenotypes result from altered cell parameters. Causal relationships between disc and clonal phenotypes are discussed.     

Inferring Genome-Wide Correlations of Mutation Fitness Effects between Populations

The effect of a mutation on fitness may differ between populations depending on environmental and genetic context, but little is known about the factors that underlie such differences. To quantify genome-wide correlations in mutation fitness effects, we developed a novel concept called a joint distribution of fitness effects (DFE) between populations. We then proposed a new statistic w to measure the DFE correlation between populations. Using simulation, we showed that inferring the DFE correlation from the joint allele frequency spectrum is statistically precise and robust. Using population genomic data, we inferred DFE correlations of populations in humans, Drosophila melanogaster, and wild tomatoes. In these species, we found that the overall correlation of the joint DFE was inversely related to genetic differentiation. In humans and D. melanogaster, deleterious mutations had a lower DFE correlation than tolerated mutations, indicating a complex joint DFE. Altogether, the DFE correlation can be reliably inferred, and it offers extensive insight into the genetics of population divergence.     

Hypomorphic lethal mutations and their implications for the interpretation of lethal complementation studies in Drosophila

In a small region of the X chromosome of Drosophila melanogaster, we have found that a third of the mutations that appear to act as lethals in segmental haploids are viable in homozygous mutant individuals. These viable mutations fall into four complementation groups. The most reasonable explanation of these mutations is that they are a subset of functionally hypomorphic alleles of essential genes: hypomorphic mutations with activity levels above a threshold required for survival, but below twice that level, should behave in this manner. We refer to these mutations as "haplo-specific lethal mutations." In studies of autosomal lethals, haplo-specific lethal mutations can be included in lethal complementation tests without being identified as such. Accidental inclusion of disguised haplo-specific lethals in autosomal complementation tests will generate spurious examples of interallelic complementation.     

A Comparison of Models to Infer the Distribution of Fitness Effects of New Mutations

Knowing the distribution of fitness effects (DFE) of new mutations is important for several topics in evolutionary genetics. Existing computational methods with which to infer the DFE based on DNA polymorphism data have frequently assumed that the DFE can be approximated by a unimodal distribution, such as a lognormal or a gamma distribution. However, if the true DFE departs substantially from the assumed distribution (e.g., if the DFE is multimodal), this could lead to misleading inferences about its properties. We conducted simulations to test the performance of parametric and nonparametric discretized distribution models to infer the properties of the DFE for cases in which the true DFE is unimodal, bimodal, or multimodal. We found that lognormal and gamma distribution models can perform poorly in recovering the properties of the distribution if the true DFE is bimodal or multimodal, whereas discretized distribution models perform better. If there is a sufficient amount of data, the discretized models can detect a multimodal DFE and can accurately infer the mean effect and the average fixation probability of a new deleterious mutation. We fitted several models for the DFE of amino acid-changing mutations using whole-genome polymorphism data from Drosophila melanogaster and the house mouse subspecies Mus musculus castaneus. A lognormal DFE best explains the data for D. melanogaster, whereas we find evidence for a bimodal DFE in M. m. castaneus.     

Comparison of lethal mutations of Drosophila melanogaster with D. simulans when detected by the attached-X method

The purpose of this study was to evaluate the attached-X method compared with the standard Basc method, and, using this method, to find out whether the observed differences in genetic polymorphisms are related to differences in lethal mutation rates in D. melanogaster and D. simulans. When EMS-treated Drosophila melanogaster males are mated to untreated attached-X females, a decrease in the progeny sex ratio (/+) is observed due to the induced lethal mutations on the X chromosome. The decrease in the frequency of male progeny were shown as the attached-X index. The expected male number is calculated from the control sex ratio. The difference between the expected and the observed male numbers, expressed as the ratio to the expected male number, defines the attached-X index. The index values for various EMS concentrations were compared to the lethal frequencies obtained by the standard Basc method for the same EMS treatments, and gave a highly positive correlation (=0.993, p<0.01, d.f.=2), thus providing an alternative method for evaluation of possible mutagens. The attached-X method was applied to D. simulans, of which natural populations are known to have relatively low genetic variation, and frequencies of the EMS-induced X chromosome lethal mutations were estimated and compared with those in D. melanogaster. The results indicate that D. melanogaster is slightly more sensitive in the sperm and spermatogonial stages, but less susceptible in the spermatid stage when compared with D. simulans. Since the spermatid stage occupies a relatively short period in spermatogenesis, a higher mutability of D. simulans during this stage probably does not make a significant contribution to the genetic variability of this species.     

The allele-frequency spectrum in a decoupled Moran model with mutation, drift, and directional selection, assuming small mutation rates

We analyze a decoupled Moran model with haploid population size N, a biallelic locus under mutation and drift with scaled forward and backward mutation rates θ₁=μ₁N and θ₀=μ₀N, and directional selection with scaled strength γ=sN. With small scaled mutation rates θ₀ and θ₁, which is appropriate for single nucleotide polymorphism data in highly recombining regions, we derive a simple approximate equilibrium distribution for polymorphic alleles with a constant of proportionality. We also put forth an even simpler model, where all mutations originate from monomorphic states. Using this model we derive the sojourn times, conditional on the ancestral and fixed allele, and under equilibrium the distributions of fixed and polymorphic alleles and fixation rates. Furthermore, we also derive the distribution of small samples in the diffusion limit and provide convenient recurrence relations for calculating this distribution. This enables us to give formulas analogous to the Ewens-Watterson estimator of θ for biased mutation rates and selection. We apply this theory to a polymorphism dataset of fourfold degenerate sites in Drosophila melanogaster.     

Gene Flow in Natural Populations of DROSOPHILA MELANOGASTER with Special Reference to Lethal Allelism Rates and Protein Variation

A simultaneous survey of 14 protein loci, together with frequencies and within- and between-population allelism rates of lethal chromosomes, was carried out in five (four Japanese and one Korean) natural populations and one cage population of Drosophila melanogaster. It was found that lethal allelism rates decrease rapidly as geographic distance between two populations increases, while variation at protein loci shows a remarkable similarity over all populations examined. These findings suggest that there are very high levels of gene flow in these natural populations and that selection at protein loci which can maintain substantial geographic variation, if present, is overshadowed by gene flow. There is no indication that invasion of D. melanogaster to the Far East occurred so recently that the frequencies of lethal chromosomes are still in nonequilibrium.