Fitness Effects of Ems-Induced Mutations on the X Chromosome of DROSOPHILA MELANOGASTER. I. Viability Effects and Heterozygous Fitness Effects

Drosophila melanogaster X chromosomes were mutagenized by feeding males sucrose solutions containing ethyl methanesulfonate (EMS); the concentrations of EMS in the food were 2.5 mM, 5.0 mM, and 10.0 mM. Chromosomes were exposed to the mutagen up to three times by treating males in succeeding generations. After treatment, the effective exposures were 2.5, 5.0, 7.5, 10.0, 15.0, and 30.0 mM EMS. X chromosomes treated in this manner were tested for effects on fitness in both hemizygous and heterozygous conditions, and for effects on viability in hemizygous and homozygous conditions. In addition, untreated X chromosomes were available for study. The viability and heterozygous fitness effects are presented in this paper, and the hemizygous fitness effects are discussed in the accompanying one (MITCHELL and SIMMONS 1977). Hemizygous and homozygous viability effects were measured by segregation tests in vial cultures. For hemizygous males, viability was reduced 0.5 percent per mM EMS treatment; for homozygous females, it was reduced 0.7% per mM treatment. The decline in viability appeared to be a linear function of EMS dose. The viabilities of males and females were strongly correlated. Heterozygous fitness effects were measured by monitoring changes in the frequencies of treated and untreated X chromosomes in discrete generation populations which, through the use of an X-Y translocation, maintained them only in heterozygous condition. Flies that were heterozygous for a treated chromosome were found to be 0.4% less fit per mM EMS than flies heterozygous for an untreated one.     

Heterozygous Effects on Fitness of Ems-Treated Chromosomes in DROSOPHILA MELANOGASTER

The heterozygous effects on fitness of second chromosomes carrying mutants induced with different doses of EMS were ascertained by monitoring changes in chromosome frequencies over time. These changes were observed in populations in which the treated chromosomes, as well as untreated competitors, remained heterozygous in males generation after generation. This situation was achieved by using a translocation which links the second chromosome to the X chromosome; however, only untranslocated second chromosomes were mutagenized. Chromosomes were classified according to their effects on viability in homozygous condition. A preliminary homozygosis identified completely lethal chromosomes; secondary tests distinguished between drastic (viability index < 0.1) and nondrastic chromosomes. Chromosomes that were nondrastic after treatment were found to reduce the fitness of their heterozygous carriers by 3-5%. The data show that flies homozygous for these chromosomes were about 2.7% less viable per treatment with 1 mm EMS than flies homozygous for untreated chromosomes. By comparing the fitness-depressing effects of nondrastic EMS-induced mutants in heterozygous condition with the corresponding viability-depressing effects measured by Temin, it is apparent that the total fitness effects are several times larger than the viability effects alone. Completely lethal chromosomes derived from the most heavily treated material reduced fitness by 11% in heterozygous condition; approximately half of this reduction was due to the lethal mutations themselves.     

Chromosome Interactions in DROSOPHILA MELANOGASTER. II. Total Fitness

In a large experiment, using nearly 200 population cages, we have measured the fitness of Drosophila melanogaster homozygous (1) for the second chromosome, (2) for the third chromosome, and (3) for both chromosomes. Twentyfour second chromosomes and 24 third chromosomes sampled from a natural population were tested. The mean fitness of the homozygous flies is 0.081 ± 0.014 for the second chromosome, 0.080 ± 0.017 for the third chromosome, and 0.079 ± 0.024 for both chromosomes simultaneously. Assuming that fitnesses are multiplicative (the additive fitness model makes no sense in the present case because of the large selection coefficients involved), the expected mean fitness of the homozygotes for both chromosomes is 0.0066; their observed fitness is more than ten times greater. Thus, it appears that synergistic interactions between loci are considerable; and that, consequently, the fitness function substantially departs from linearity. Two models are tentatively suggested for the fitness function: a "threshold" model and a "synergistic" model.—The experiments reported here confirm previous results showing that the concealed genetic load present in natural populations of Drosophila is sufficient to account for the selective maintenance of numerous polymorphisms (of the order of 1000).     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. II. Homozygous Effect of Polygenic Mutations

Polygenic mutations affecting viability were accumulated on the second chromosome of Drosophila melanogaster by treating flies with EMS in successive generations. The treated chromosomes were later made homozygous and tested for their effects on viability by comparison of the frequency of such homozygotes with that of other genotypes in the same culture. The treated wild-type chromosomes were kept heterozygous in Pm/+ males by mating individual males in successive generations to Cy/Pm females. The number of generations of accumulation was 1 to 30 generations, depending on the concentration of EMS. A similar experiment for spontaneous polygenic mutations was also conducted by accumulating mutations for 40 generations. The lower limit of the spontaneous mutation rate of viability polygenes is estimated to be 0.06 per second chromosome per generation, which is about 12 times as high as the spontaneous recessive lethal mutation rate, 0.005. EMS-induced polygenic mutations increase linearly with the number of treated generations and with the concentration of EMS. The minimum mutation rate of viability polygenes is about 0.017 per 10(-4)m, which is only slightly larger than the lethal rate of 0.013 per 10(-4) m. The maximum estimate of the viability reduction of a single mutant is about 6 to 10 percent of the normal viability. The data are consistent with a constant average effect per mutant at all concentrations, but this is about three times as high as that for spontaneous mutants. It is obvious that one can obtain only a lower limit for the mutation rate, since some mutants may have effects so near to zero that they cannot be detected. The possibility of measuring something other than the lower limit is discussed. The ratio of the load due to detrimental mutants to that caused by lethals, the D/L ratio, is about 0.2 to 0.3 for EMS-induced mutants, as compared to about 0.5 for spontaneous mutants. This is to be expected if EMS treatment produces a large fraction of small deletions and other chromosome rearrangements which are more likely to be lethal.     

Reduced X-linked rare polymorphism in males in comparison to females of Drosophila melanogaster

Natural selection is assumed to act more strongly on X-linked loci than on autosomal loci because the fitness effect of a recessive mutation on the X chromosome is fully expressed in hemizygous males. Therefore, selection is expected to fix or remove recessive mutations on the X chromosome more efficiently than those on autosomes. However, the assumption that hemizygosity of the X chromosome selectively accelerates changes in allele frequency has not been confirmed directly. To examine this assumption, we investigated current natural selection on X-linked chemoreceptor genes in a natural population of Drosophila melanogaster by comparing nucleotide diversity, linkage disequilibrium (LD), and departure from the neutrality in 4 chemoreceptor genes on 100 X chromosomes each from female and male flies. The general pattern of nucleotide diversity and LD for the genes investigated was similar in females and males. In contrast, males harbored significantly fewer rare polymorphisms defined as singletons and doubletons. When all the gene sequences were concatenated, Tajima's D showed a significant departure from the neutrality in both females and males, whereas Fu and Li's F* value revealed departure only in males. These results suggest that some rare polymorphisms on the X chromosome from females are recessively deleterious and are removed by stronger purifying selection when transferred to hemizygous males.     

Viabilities of Third Chromosomes of DROSOPHILA PSEUDOOBSCURA Differing in Relative Competitive Fitness

Arrowhead (AR) third chromosome arrangements of Drosophila pseudoobscura, whose competitive fitnesses had been determined in population cages, were tested for their genetic loads in homozygous, heterozygous (homokaryotypic), and heterokaryotypic (AR/CH) combinations. The results showed that their competitive population cage performances were correlated to their viabilities as homozygotes but were not correlated to their viabilities as heterozygotes or as heterokaryotypes. However, the results do not fit in too simply with the mutational model of population structure, since the improvement of homozygous viability with increased competitive fitness was not accompanied by a significant degree of dominance as measured by the regression of viabilities of heterozygotes on homozygotes. Only the AR chromosomes derived from the population with poorest competitive fitness showed marked partial dominance (h=.35). The viabilities of heterokaryotypes were markedly uniform for all chromosomes tested and produced significantly greater numbers of flies per culture than the homokaryotypes. In general, the results show that the ranking of relative competitive fitnesses of these chromosomes is not a simple extrapolation of their viabilities, although marked changes in the populations tested have occurred. It is proposed that the differences in competitive fitness, homozygous viability, and degree of dominance observed among these chromosomes, arise from differences in genetic variability which enable different linkage relationships to be established for genes affecting these attributes.     

Partial Dominance of Ems-Induced Mutations Affecting Viability in DROSOPHILA MELANOGASTER

More than 700 EMS-treated second chromosomes marked with either cn (cinnabar) or bw (brown), and derived from long-inbred stocks, were measured for their heterozygous effects on viability in both isogenic (homozygous) and nonisogenic (heterozygous) backgrounds. Each test was replicated five times. When the background was homozygous, flies heterozygous for a treated chromosome were an average of 2.1% less viable, per 0.005 m EMS, than flies heterozygous for an untreated chromosome. Classified according to their homogous effect in an accompanying series of crosses, the lethal-bearing chromosomes (L), which carry genes of less drastic effects as well, reduced the viability of their heterozygous carriers by 3.3%, severe detrimentals (D(s)) by 2.2%, and mild detrimentals (D(m)) by 1.2% at this dose. In the heterozygous background, the mean heterozygous disadvantage for the entire group was 1%, or about half as large.--When computed separately for each count from a single mating, the heterozygous disadvantage was consistently greatest for the earliest counts (4.8%), next highest for the middle count (0.8%), and lowest in the latest count (0.5%), in the homozygous background, indicating that mutant heterozygotes were delayed in time of emergence. The figures in the heterozygous background were, again, reduced, but in the same direction.-The relative viability disadvantage of the cn marker was about 2(1/2) times greater in the homozygous than in the heterozygous background, further supporting the conclusion that the homozygous background can accentuate differences. The enhancement of treatment and marker effects could be a direct result of the level of background heterozygosity per se or attributable to the reduced vigor of the inbred strain.-Dominance, a measure of the heterozygous effect of a mutant relative to its homozygous effect, is greater for genes with small homozygous disadvantage than for more drastic genes. In the homozygous background the average dominance for lethals was 0.019 in contrast to 0.183 for mild detrimentals, supporting other published results suggesting that genes with milder effect, because they occur more frequently, have a greater impact on a population.-The homozygous D:L ratio of EMS mutations was 0.266 and the D(m): L ratio, 0.092, which are lower than comparable load ratios for spontaneous mutations, but greater than for X-ray induced mutations.     

Estimation of Fitness Components in DROSOPHILA MELANOGASTER. II. Influence of Reduced Transmission Frequency

Results are presented of further analyses of the significant effects of reciprocal crosses reported by Katz and Cardellino (1978) in regard to viability indices of wild-type second chromosome heterozygotes. The observed differences between reciprocal crosses can be explained by the existence of reduced transmission frequencies of the wild-type homologue from Pm/+ and Cy/+ paternal parents. Mean estimates of transmission frequencies from Pm/+ and Cy/+ males in California and Japan populations are significantly less than the Mendelian expectation of 1/2. The transmission frequencies of +(i) chromosomes from Pm/+(i) and Cy/+(i) males are also found to be positively correlated in the California and pooled populations, suggesting that the degree of distortion is primarily due to the +(i) chromosome rather than to Cy or Pm. A sufficient estimator of relative viability that is independent of distorted transmission frequencies is derived for use in the Cy/Pm technique of viability estimation.     

Large Genetic Change at Small Fitness Cost in Large Populations of Drosophila Melanogaster Selected for Wind Tunnel Flight: Rethinking Fitness Surfaces

The fitness effects of extreme genetic change by selection were studied in large populations subjected to prolonged, intense selection. Two replicate populations of Drosophila melanogaster, with estimated effective sizes 500 <= N(e) <= 1000, were selected for increased performance in a wind tunnel, selecting on average the fastest 4.5% of flies. The mean apparent flying speed of both lines increased from ~2 to 170 cm/sec and continued to respond at diminishing rates, without reaching a plateau, for 100 generations. Competitive fitness tests in generations 50 and 85 showed minimal or no fitness loss in selected lines compared to controls. Sublines relaxed in generations 65 and 85 showed minimal or no regression in apparent flying speed. Hybrid lines, from a cross of selected X control lines in generation 75, responded to reselection saltationally, showing that the chromosomes of the selected lines had been assembled from alleles at many loci, from many different chromosomes in the base population. Thus, major genetic change was achieved, but without the costs usually associated with strong directional selection. Large population size has been interpreted, in opposing models, as either a brake or an accelerator in its effects on long-term change by selection. These results favor the second model, and challenge the concept of rugged fitness surfaces underlying the first model.     

Fertility Genes in Natural Populations of DROSOPHILA MELANOGASTER. I. Frequency, Allelism and Persistence of Sterility Genes

Three or four percent of the wild flies in natural populations of D. melanogaster have been found to be sterile. An analysis of sterility associated with the second chromosome revealed a much lower frequency of genetically sterile flies. The accumulation of sterility genes in a cage population was proportional to that of lethal genes, as were their equilibrium frequencies in several natural populations. Many sterile chromosomes were associated with low viability due to pleiotropic effects. The number of chromosomes leading to sterility in both sexes was larger than the expectation based on random combination of male and female sterility genes. This suggests that there is some linkage disequilibrium between male and female sterility genes, as well as a pleiotropic effect of single sterility genes. Some sterility genes were maintained in natural and cage populations, and the patterns of persistence of the sterility genes were very similar to those of lethal genes.     

Efficient Recovery of Enu-Induced Mutations from the Zebrafish Germline

We studied the efficiency with which two chemical mutagens, ethyl methanesulfonate (EMS) and N-ethyl-N-nitrosourea (ENU) can induce mutations at different stages of spermatogenesis in zebrafish (Brachydanio rerio). Both EMS and ENU induced mutations at high rates in post-meiotic germ cells, as indicated by the incidence of F(1) progeny mosaic for the albino mutation. For pre-meiotic germ cells, however, only ENU was found to be an effective mutagen, as indicated by the frequencies of non-mosaic mutant progeny at four different pigmentation loci. Several mutagenic regimens that varied in either the number of treatments or the concentration of ENU were studied to achieve an optimal ratio between the mutagenicity and toxicity. For the two most mutagenic regimens: 4 X 1 hr in 3 mM ENU and 6 X 1 hr in 3 mM ENU, the minimum estimate of frequencies of independent mutations per locus per gamete was 0.9-1.3 X 10(-3). We demonstrate that embryonic lethal mutations induced with ENU were transmitted to offspring and that they could be recovered in an F(2) screen. An average frequency of specific-locus mutations of 1.1 X 10(-3) corresponded to approximately 1.7 embryonic lethal mutations per single mutagenized genome. The high rates of mutations achievable with ENU allow for rapid identification of large numbers of genes involved in a variety of aspects of zebrafish development.     

The Zuker collection: a resource for the analysis of autosomal gene function in Drosophila melanogaster

The majority of genes of multicellular organisms encode proteins with functions that are not required for viability but contribute to important physiological functions such as behavior and reproduction. It is estimated that 75% of the genes of Drosophila melanogaster are nonessential. Here we report on a strategy used to establish a large collection of stocks that is suitable for the recovery of mutations in such genes. From approximately 72,000 F(3) cultures segregating for autosomes heavily treated with ethyl methanesulfonate (EMS), approximately 12,000 lines in which the treated second or third chromosome survived in homozygous condition were selected. The dose of EMS induced an estimated rate of 1.2-1.5 x 10(-3) mutations/gene and predicts five to six nonessential gene mutations per chromosome and seven to nine alleles per locus in the samples of 6000 second chromosomes and 6000 third chromosomes. Due to mosaic mutations induced in the initial exposure to the mutagen, many of the lines are segregating or are now fixed for lethal mutations on the mutagenized chromosome. The features of this collection, known as the Zuker collection, make it a valuable resource for forward and reverse genetic screens for mutations affecting a wide array of biological functions.     

Adding the heterochromatic YL arm to an X chromosome reduces reproductive fitnesses in Drosophila melanogaster: implications for the evolution of rDNA, heterochromatin, and reproductive isolation

For X-Y exchange to be of importance in the coevolution of X and Y rDNA, there must be a mechanism to maintain cytologically normal X chromosomes in the face of continual infusions of X.YL chromosomes produced by X-Y exchanges. Replicated populations were founded with different frequencies of isogenic X and X.YL chromosomes. The X.YL chromosome declined in frequency over time in all lines. Relative fitnesses, estimated from chromosome frequency trajectories, were 0.40, 1.01, and 1.0 for X.YL/X.YL, X.YL/X, and X/X females and 0.75 and 1.0 for X.YL/Y and X/Y males, respectively. The equilibrium frequency for the X.YL chromosome due to the balance between X-Y exchange and selection was predicted to be 4-16 x 10(-4). The results strengthen the evidence for the involvement of X-Y exchange in the coevolution of X and Y rDNA arrays. Conditions for the evolution of reproductive isolation by sex-chromosome translocation are much less probable than previously supposed since the X.YL translocation chromosome is at a selective disadvantage to cytologically normal X chromosomes. Additional heterochromatin was not neutral but was only deleterious beyond a threshold, as one dose of the heterochromatic XL arm did not reduce female reproductive fitness, but two doses did.     

Correlations between Chromosome Segments and Fitness in DROSOPHILA MELANOGASTER. I. the X Chromosome and Egg Production

Unmarked segments within the X chromosomes of four different Drosophila melanogaster isogenic lines were assessed with respect to egg production. By making a series of crosses among original and derived recombinant lines, it was possible to estimate parameters representing additive, dominance and interaction effects of the segments. It was shown that whereas most of the segments were additive for egg production when homozygous, they all displayed dominance in the heterozygous condition. Two of the strains were characterized by intersegmental interaction. A possible position effect was detected for these same two strains, with flies in the coupling phase laying more eggs than those in the repulsion configuration. There was no apparent relationship between the number of eggs laid and the amount of heterozygosity within the X chromosome.     

Darwinian fitness and adaptedness in experimental populations of Drosophila willistoni

Fifteen second chromosomes were extracted from Drosophila willistoni flies collected in four natural populations. The adaptedness of populations homozygous for each chromosome was measured by average population size and productivity. Six control populations were established with mixtures of the wild second chromosomes. The Darwinian fitness of flies homozygous for each wild second ehromosome, and of flies carrying random combinations of these chromosomes, was measured relative to the fitness of flies heterozygous for a wild and a marker chromosome. The Darwinian fitness of homozygotes for each second chromosome relative to the fitness of flies carrying random combinations of the natural chromosomes was then inferred. The estimated loss of fitness on making the natural second chromosomes homozygous was substantial, ranging from 39 to 83 pereent, with an average reduction in fitness of 66 percent. These results with D. willistoni are consistent with those from similar experiments with other drosophila species, and they are compatible with a significant role for heterosis in the maintenance of genetic variability.Populations homozygous for wild chromosomes differ in their adaptedness to the experimental environment. Population size and productivity are correlated, although the correlation is far from complete. Some populations have high productivity and low population size, or vice versa. The control populations, with greater genetic variability, were superior in adaptedness to the average of the single-chromosome populations. The Darwinian fitness and the adaptedness of the genotypes in this experiment were not significantly correlated. It follows that certain measures used by population geneticists, such as genetic load and average Darwinian fitness, cannot be taken as general indices of how well adapted a population is to its environment.This work was supported by U.S. Public Health Service Grant RO1-HDO5055, NSF grant GB-20694 (International Biological Program). AEC contract AT-(30-1) 3096, and PHS Career Development Award K3 GM 37265. The collection of the flies was supported by the Fundacão de Amparo a Pesquisa do Estado de São Paulo, Brazil. The senior author's stay in New York, where the experiments were conducted, was financed in part by Research Fellowship 2-12861 from the Panamerican Union.     

Genetic Load in Natural Populations: Is It Compatible with the Hypothesis That Many Polymorphisms Are Maintained by Natural Selection?

Recent studies of genetically controlled enzyme variation lead to an estimation that at least 30 to 60% of the structural genes are polymorphic in natural populations of many vertebrate and invertebrate species. Some authors have argued that a substantial proportion of these polymorphisms cannot be maintained by natural selection because this would result in an unbearable genetic load. If many polymorphisms are maintained by heterotic natural selection, individuals with much greater than average proportion of homozygous loci should have very low fitness. We have measured in Drosophila melanogaster the fitness of flies homozygous for a complete chromosome relative to normal wild flies. A total of 37 chromosomes from a natural population have been tested using 92 experimental populations. The mean fitness of homozygous flies is 0.12 for second chromosomes, and 0.13 for third chromosomes. These estimates are compatible with the hypothesis that many (more than one thousand) loci are maintained by heterotic selection in natural populations of D. melanogaster.     

Temperature-Sensitive Mutations in DROSOPHILA MELANOGASTER. IX. Dominant Cold-Sensitive Lethals on the Autosomes

Ethyl methanesulfonate-treated autosomes were screened for the presence of dominant cold-sensitive (DCS) lethal mutations in Drosophila melanogaster. None was found among 6,552 treated and 168 untreated third chromosomes. Twenty-three DCS-L chromosomes which caused death at 17 degrees C but survived at 22 degrees C and 29 degrees C were recovered from 5,046 mutagenized chromosome 2's.-The DCS-L mutations all mapped around dp and appeared to be functionally allelic. Lethality of heterozygotes for most of the DCS-L's occurred over a prolonged interval from the embryonic through the larval instars. Prolonged incubation at 17 degrees C did not demonstrate any maternal effect on zygotic survival.     

The genetics of Drosophila subobscura populations. 3. Inversion polymorphism and climatic factors

Summary The natural population of Drosophila subobscura of Mt. Parnes has been followed for two successive years and the frequency of the gene arrangements determined. Some changes occured in the first year but do not explain the existence of the geographical clines for these frequencies. Within the second year no changes were observed. For chromosomes J and U there is evidence for a greater fitness of the heterozygotes.Experiments on temperature effect on viability of adult male flies, on their temporal sterility, on the viability of flies during all their biological cycle and experiments on the effect of dryness on the viability of adult male flies did not show an important change of gene arrangement frequencies in the resistant individuals. Nor did diapausing females have very different gene arrangements, except for chromosome O. Cage experiments with temperature as a selective agent did not elucidate the action of this factor. It is concluded that temperature or dryness do not affect very much the polymorphic system, nor explain the existence of these clines.This polymorphic system is much more stable in D. subobscura than in D. pseudoobscura, its nearctic relative. It is also richer. It is concluded that it is historically older and helps the populations to cope with many environmental changes.     

Spontaneous and Ethyl Methanesulfonate-Induced Mutations Controlling Viability in DROSOPHILA MELANOGASTER. I. Recessive Lethal Mutations

The efficiency of the adult feeding method for EMS treatment in Drosophila melanogaster was studied by measuring the frequency of induced recessive lethals on the second chromosome. The treatment was most effective when mature spermatozoa or spermatids were treated and was much less effective on earlier stages. The number of mutations induced was proportional to the concentration except at the highest doses. The recessive lethal rate was estimated to be about 0.012 per second chromosome per 10(-4) M. In addition, about 0.004-0.005 recessive lethals per 10(-4) M were found in a later generation in chromosomes that had not shown the lethal effect in the previous generation. When the experiments are done in a consistent manner and gametes treated as mature sperm or spermatids are sampled, the results are highly reproducible. However, modifications of the procedure, such as starvation before EMS treatment, can considerably alter the effectiveness of the mutagen.     

X chromosome drive in a widespread Palearctic woodland fly,Drosophila testacea

Selfish genes that bias their own transmission during meiosis can spread rapidly in populations, even if they contribute negatively to the fitness of their host. Driving X chromosomes provide a clear example of this type of selfish propagation. These chromosomes have important evolutionary and ecological consequences, and can be found in a broad range of taxa including plants, mammals and insects. Here, we report a new case of X chromosome drive (X drive) in a widespread woodland fly, Drosophila testacea. We show that males carrying the driving X (SR males) sire 80–100% female offspring and possess a diagnostic X chromosome haplotype that is perfectly associated with the sex ratio distortion phenotype. We find that the majority of sons produced by SR males are sterile and appear to lack a Y chromosome, suggesting that meiotic defects involving the Y chromosome may underlie X drive in this species. Abnormalities in sperm cysts of SR males reflect that some spermatids are failing to develop properly, confirming that drive is acting during gametogenesis. By screening wild‐caught flies using progeny sex ratios and a diagnostic marker, we demonstrate that the driving X is present in wild populations at a frequency of ~ 10% and that suppressors of drive are segregating in the same population. The testacea species group appears to be a hot spot for X drive, and D. testacea is a promising model to compare driving X chromosomes in closely related species, some of which may even be younger than the chromosomes themselves.