Mode of selection and experimental evolution of antifungal drug resistance in Saccharomyces cerevisiae

We show that mode of selection, degree of dominance of mutations, and ploidy are determining factors in the evolution of resistance to the antifungal drug fluconazole in yeast. In experiment 1, yeast populations were subjected to a stepwise increase in fluconazole concentration over 400 generations. Under this regimen, two mutations in the same two chromosomal regions rose to high frequency in parallel in three replicate populations. These mutations were semidominant and additive in their effect on resistance. The first of these mutations mapped to PDR1 and resulted in the overexpression of the ABC transporter genes PDR5 and SNQ2. These mutations had an unexpected pleiotropic effect of reducing the residual ability of the wild type to reproduce at the highest concentrations of fluconazole. In experiment 2, yeast populations were subjected to a single high concentration of fluconazole. Under this regimen, a single recessive mutation appeared in each of three replicate populations. In a genome-wide screen of approximately 4700 viable deletion strains, 13 were classified as resistant to fluconazole (ERG3, ERG6, YMR102C, YMR099C, YPL056C, ERG28, OSH1, SCS2, CKA2, SML1, YBR147W, YGR283C, and YLR407W). The mutations in experiment 2 all mapped to ERG3 and resulted in the overexpression of the gene encoding the drug target ERG11, but not PDR5 and SNQ2. Diploid hybrids from experiments 1 and 2 were less fit than the parents in the presence of fluconazole. In a variation of experiment 2, haploids showed a higher frequency of resistance than diploids, suggesting that degree of dominance and ploidy are important factors in the evolution of antifungal drug resistance.     

Haploids adapt faster than diploids across a range of environments

Despite a great deal of theoretical attention, we have limited empirical data about how ploidy influences the rate of adaptation. We evolved isogenic haploid and diploid populations of Saccharomyces cerevisiae for 200 generations in seven different environments. We measured the competitive fitness of all ancestral and evolved lines against a common competitor and find that in all seven environments, haploid lines adapted faster than diploids, significantly so in three environments. We apply theory that relates the rates of adaptation and measured effective population sizes to the properties of beneficial mutations. We obtained rough estimates of the average selection coefficients in haploids between 2% and 10% for these first selected mutations. Results were consistent with semi-dominant to dominant mutations in four environments and recessive to additive mutations in two other environments. These results are consistent with theory that predicts haploids should evolve faster than diploids at large population sizes.     

Evolution of haploid–diploid life cycles when haploid and diploid fitnesses are not equal

Many organisms spend a significant portion of their life cycle as haploids and as diploids (a haploid–diploid life cycle). However, the evolutionary processes that could maintain this sort of life cycle are unclear. Most previous models of ploidy evolution have assumed that the fitness effects of new mutations are equal in haploids and homozygous diploids, however, this equivalency is not supported by empirical data. With different mutational effects, the overall (intrinsic) fitness of a haploid would not be equal to that of a diploid after a series of substitution events. Intrinsic fitness differences between haploids and diploids can also arise directly, for example because diploids tend to have larger cell sizes than haploids. Here, we incorporate intrinsic fitness differences into genetic models for the evolution of time spent in the haploid versus diploid phases, in which ploidy affects whether new mutations are masked. Life‐cycle evolution can be affected by intrinsic fitness differences between phases, the masking of mutations, or a combination of both. We find parameter ranges where these two selective forces act and show that the balance between them can favor convergence on a haploid–diploid life cycle, which is not observed in the absence of intrinsic fitness differences.     

Masking and purging mutations following EMS treatment in haploid, diploid and tetraploid yeast (Saccharomyces cerevisiae)

The yeast, Saccharomyces cerevisiae, was used as a model to investigate theories of ploidy evolution. Mutagenesis experiments using the alkylating agent EMS (ethane methyl sulphonate) were conducted to assess the relative importance that masking of deleterious mutations has on response to and recovery from DNA damage. In particular, we tested whether cells with higher ploidy levels have relatively higher fitnesses after mutagenesis, whether the advantages of masking are more pronounced in tetraploids than in diploids, and whether purging of mutations allows more rapid recovery of haploid cells than cells with higher ploidy levels. Separate experiments were performed on asexually propagating stationary phase cells using (1) prototrophic haploid (MAT alpha) and diploid (MATa/alpha) strains and (2) isogenic haploid, diploid and tetraploid strains lacking a functional mating type locus. In both sets of experiments, haploids showed a more pronounced decrease in apparent growth rate than diploids, but both haploids and diploids appeared to recover very rapidly. Tetraploids did not show increased benefits of masking compared with diploids but volume measurements and FACScan analyses on the auxotrophic strains indicated that all treated tetraploid strains decreased in ploidy level and that some of the treated haploid lines increased in ploidy level. Results from these experiments confirm that while masking deleterious mutations provides an immediate advantage to higher ploidy levels in the presence of mutagens, selection is extremely efficient at removing induced mutations, leading growth rates to increase rapidly over time at all ploidy levels. Furthermore, ploidy level is itself a mutable trait in the presence of EMS, with both haploids and tetraploids often evolving towards diploidy (the ancestral state of S. cerevisiae) during the course of the experiment.     

Mating systems and the evolutionary transition between haploidy and diploidy

According to the 'masking hypothesis', diploids gain an immediate fitness advantage over haploids because diploids, with two copies of every gene, are better able to survive the effects of deleterious recessive mutations. Masking in diploids is, however, a double-edged sword: it allows mutations to persist over tine. In contrast, deleterious mutations are revealed in haploid individuals and are more rapidly eliminated by selection, creating genetic associations that are favourable to haploidy. We model various mating schemes and show that assortative mating, selfing, and apomixis maintain the genetic associations that favour haploidy. These results suggest that a correlation should exist between mating system and ploidy level, with outcrossing favouring diploid life cycles and inbreeding or asexual reproduction favouring haploid life cycles. This prediction can be tested in groups, such as the Chlorophyta, with extensive variation both in life cycle and in reproductive system. Confirming or rejecting this prediction in natural populations would constitute the first empirical test of the masking hypothesis as a force shaping the evolution of life cycles.     

Cytotype variation and allopolyploidy in North American species of the Sphagnum subsecundum complex (Sphagnaceae)

Allopolyploid speciation is likely the predominant mode of sympatric speciation in plants. The Sphagnum subsecundum complex includes six species in North America. Three have haploid gametophytes, and three are thought to have diploid gametophytes. Microsatellite analyses indicated that some plants of S. inundatum and S. lescurii are heterozygous at most loci, but others have only one allele at each locus. Flow cytometry and Feulgen staining showed that heterozygous plants have twice the genome size as plants with one allele per locus; thus, microsatellite patterns can be used to survey the distribution and abundance of haploid and diploid gametophytes. Microsatellite analyses also revealed that S. carolinianum is consistently diploid, but S. lescurii and S. inundatum include both haploid and diploid populations. The frequency of diploid plants in S. lescurii increases with latitude. In an analysis of one population of S. lescurii, both cytotypes co-occurred but were genetically differentiated with no evidence of interbreeding. The degree of genetic differentiation showed that the diploids were not derived from simple genome duplication of the local haploids. Heterozygosity appears to be fixed or nearly so in diploids, strongly suggesting that although morphologically indistinguishable from the haploids, they are derived by allopolyploidy.     

The recognition and incidence of haploid and polyploid spermatozoa in man, rabbit and mouse.

The existence of polyploid mammalian spermatozoa has been inferred from studies of Feulgen-DNA absorption. Rabbit spermatozoa fell into two discrete groups with mean absorptions close to a 1:2 ratio (inferred to be haploids and diploids respectively); simple visual appraisal of the size of the head or nucleus gave an identical classification. The incidences of ploidy classes were 98-94% haploid, 1-06% diploid, 0-00% higher than diploid (N = 3010; from DNA measurements and visual appraisal of the size in a rabbit chosen to have a high incidence of diploids) and, correspondingly, 99-691%, 0-308%, 0-001% (N = 138001; from sixty-nine unselected rabbits, scored by visual appraisal of the size of the sperm head). In man also, virtually discrete groups with absorptions close to a 1:2 ratio existed and were inferred to be haploids and diploids respectively. A few human spermatozoa were found with absorptions corresponding to a ploidy of three and/or four. Visual appraisal of the size of the human sperm nucleus as Small, Medium or Large was only a partial guide to ploidy. All Small human spermatozoa measured for DNA absorption were found to be haploid. About two-thirds of Medium human spermatozoa were found, however, to be haploid, and some Large spermatozoa were haploid or diploid. The incidences of ploidy classes in the human were 99-37% haploid, 0-56% diploid, 0-07% higher than diploid (N = 5554; with consistency between duplicate slides and between two subjects; from DNA measurements and visual appraisal of nuclear size). The estimated incidence of diploid human spermatozoa is consistent with the known incidence oftriploid fetuses. In a mouse with a putatively high incidence of diploids, all 1000 DNA measurements were nevertheless within the haploid range, with one diploid encountered outside the main sampling.     

Cryptic fitness advantage: diploids invade haploid populations despite lacking any apparent advantage as measured by standard fitness assays

Ploidy varies tremendously within and between species, yet the factors that influence when or why ploidy variants are adaptive remains poorly understood. Our previous work found that diploid individuals repeatedly arose within ten replicate haploid populations of Saccharomyces cerevisiae, and in each case we witnessed diploid takeover within ~1800 asexual generations of batch culture evolution in the lab. The character that allowed diploids to rise in frequency within haploid populations remains unknown. Here we present a number of experiments conducted with the goal to determine what this trait (or traits) might have been. Experiments were conducted both by sampling a small number of colonies from the stocks frozen every two weeks (~ 93 generations) during the original experiment, as well through sampling a larger number of colonies at the two time points where polymorphism for ploidy was most prevalent. Surprisingly, none of our fitness component measures (lag phase, growth rate, biomass production) indicated an advantage to diploidy. Similarly, competition assays against a common competitor and direct competition between haploid and diploid colonies isolated from the same time point failed to indicate a diploid advantage. Furthermore, we uncovered a tremendous amount of trait variation among colonies of the same ploidy level. Only late-appearing diploids showed a competitive advantage over haploids, indicating that the fitness advantage that allowed eventual takeover was not diploidy per se but an attribute of a subset of diploid lineages. Nevertheless, the initial rise in diploids to intermediate frequency cannot be explained by any of the fitness measures used; we suggest that the resolution to this mystery is negative frequency-dependent selection, which is ignored in the standard fitness measures used.     

Ploidy controls the success of mutators and nature of mutations during budding yeast evolution

BACKGROUND: We used the budding yeast Saccharomyces cerevisiae to ask how elevated mutation rates affect the evolution of asexual eukaryotic populations. Mismatch repair defective and nonmutator strains were competed during adaptation to four laboratory environments (rich medium, low glucose, high salt, and a nonfermentable carbon source). RESULTS: In diploids, mutators have an advantage over nonmutators in all conditions, and mutators that win competitions are on average fitter than nonmutator winners. In contrast, haploid mutators have no advantage when competed against haploid nonmutators, and haploid mutator winners are less fit than nonmutator winners. The diploid mutator winners were all superior to their ancestors both in the condition they had adapted to, and in two of the other conditions. This phenotype was due to a mutation or class of mutations that confers a large growth advantage during the respiratory phase of yeast cultures that precedes stationary phase. This generalist mutation(s) was not selected in diploid nonmutator strains or in haploid strains, which adapt primarily by fixing specialist (condition-specific) mutations. In diploid mutators, such mutations also occur, and the majority accumulates after the fixation of the generalist mutation. CONCLUSIONS: We conclude that the advantage of mutators depends on ploidy and that diploid mutators can give rise to beneficial mutations that are inaccessible to nonmutators and haploid mutators.     

Parasexual Genetic Analysis of the Cellular Slime Mold DICTYOSTELIUM DISCOIDEUM A3

Haploid strain A3 of the cellular slime mold Dictyostelium discoideum is valuable for biochemical studies because it is capable of axenic growth. Mutants of A3 temperature-sensitive for growth and resistant to the drugs cycloheximide, acriflavin, or methanol were isolated.--Heterozygous diploid recombinants, formed at low frequency by cell and nuclear fusion, were isolated by selecting temperature-resistant progeny of mixed cultures of two nonallelic temperature-sensitive haploids (LOOMIS 1969). Each drug-resistant mutation was found to be recessive. Two independently isolated methanol-resistant mutants were in one complementation group.--Diploids of A3 heterozygous for drug resistance formed drug-resistant segregants with a frequency of approximately 10(-4). Segregants selected for resistance to a single drug were either haploid or diploid; the fraction which was haploid varied from 0.11 to 0.86, depending on the selected marker. Segregants selected for resistance to two or three drugs were almost all haploid.--Using this parasexual cycle of diploid formation and haploidization, linkage of these temperature-sensitive and drug-resistance mutations to each other and to mutations studied by KATZ and SUSSMAN (1972) and by WILLIAMS, KESSIN and Newell (1974b) was analyzed. The methanol-resistant mutants were found to be partially resistant to acriflavin, and unlinked to the mutant selected for acriflavin resistance, which was methanol-sensitive. Of the expected seven linkage groups in D. discoideum, five, and a possible sixth, have been marked.--Linkage analysis of a mutant abnormal in morphogenesis showed that its phenotype results from two unlinked chromosomal mutations.     

The effects of three rad genes on UV induced mutation rates in haploid and diploid Saccharomyces cells

Effects of the rad 2-20, rad 9-4, r1s, and the corresponding wild type RAD alleles in haploid and homozygous diploid Saccharomyces strains on UV induced mutation rates from adenine, lysine and histidine dependence to independence are reported. The UV induced mutation rates were similar for the RAD, r1s, and rad 9-4 haploids, whereas the rad 2-20 mutation causes a marked increase in the UV induced mutation rates. The diploid rad 2-20 strain also exhibits a marked increase in the UV induced mutation rates, whereas the rad 9-4 diploid has reduced mutation rates when compared to the wildtype. The UV induced mutation rates of haploid and diploid RAD strains are almost identical. For the rad 2-20 and rad 9-4 diploids, however, these rates are smaller than in the corresponding haploid strains. Differential effects of the rad genes on the ratio of locus to suppressor mutations were found. The implications of these findings on possible repair processes in yeasts are discussed.     

NUCLEAR GENE DOSAGE EFFECTS ON MITOCHONDRIAL MASS AND DNA

In order to assess the effect of nuclear gene dosage on the regulation of mitochondria we have studied serial sections of a set of isogenic haploid and diploid cells of Saccharomyces cerevisiae, growing exponentially in the absence of catabolite repression, and determined the amount of mitochondrial DNA per cell. Mitochondria accounted for 14% of the cytoplasmic and 12% of the total cellular volume in all cells examined regardless of their ploidy or their apparent stage in the cell cycle. The mean number of mitochondria per cell was 22 in the diploid and 10 in the haploids. The volume distribution appeared unimodal and identical in haploids and diploids. The mitochondrial DNA accounted for 12.6 ± 1.2% and 13.5 ± 1.3% of the total cellular DNA in the diploid and haploid populations, respectively. These values correspond to 3.6 x 10^-15 g, 2.2 x 10⁹ daltons, or 44 genomes (50 x 10⁶ daltons each) per haploid and twice that per diploid cell. On this basis, the average mitochondrion in these cells contains four mitochondrial genomes in both the haploid and the diploid.     

Selection on a modifier of recombination rate due to linked deleterious mutations

Several models have been suggested to explain the origin and maintenance of recombination. Here I present the results from computer simulations of multilocus haploid and diploid genotypes in small populations. Each chromosome consisted of 1001 loci where deleterious mutations occurred. At "equilibrium" for mutation-selection-genetic drift balance a single recombination variant was introduced to the population in the middle of a chromosome. On average 75,000 replicates for each combination of parameters were followed to fixation or loss of the modifier allele. The results show that, in a small population, increased recombination can be selected, even in the absence of epistasis or beneficial mutations. The effect of the mutation rate for deleterious mutations depends on the ploidy level and the recessiveness of deleterious mutations. A higher deleterious mutation rate is required for an increase in recombination rate to be favored in haploid populations. Increased recombination could not evolve in the case of strong associative overdominance.     

Mutational Load and the Transition between Diploidy and Haploidy in Experimental Populations of the Yeast Saccharomyces cerevisiae

Mutations were accumulated over hundreds of generations in a mutator strain of yeast in a constant laboratory environment. This ensured that mutations were frequent and that the quality of environment remained unchanged. Mutations were accumulated in asexual populations of diploids but their impact on fitness was tested both for the diploid clones and for haploid clones derived from them. Dozens of harmful and lethal mutations accumulated in diploids, but important phenotypic traits, such as maximum growth rate, did not deteriorate by more than 10%. There were no signs of decline in population size. In strong contrast, the populations of haploids derived from the diploids suffered from high mortality; their density was reduced by more than three orders of magnitude. These findings indicate how ineffective natural selection can be in removing deleterious mutations from populations of clonally reproducing diploids. They also suggest that phenotypic assays of heterozygous diploids may be of little value as indicators of increasing genetic degeneration.     

Mating system and gene flow in the red seaweed Gracilaria gracilis: effect of haploid-diploid life history and intertidal rocky shore landscape on fine-scale genetic structure

The impact of haploid-diploidy and the intertidal landscape on a fine-scale genetic structure was explored in a red seaweed Gracilaria gracilis. The pattern of genetic structure was compared in haploid and diploid stages at a microgeographic scale (< 5 km): a total of 280 haploid and 296 diploid individuals located in six discrete, scattered rock pools were genotyped using seven microsatellite loci. Contrary to the theoretical expectation of predominantly endogamous mating systems in haploid-diploid organisms, G. gracilis showed a clearly allogamous mating system. Although within-population allele frequencies were similar between haploids and diploids, genetic differentiation among haploids was more than twice that of diploids, suggesting that there may be a lag between migration and (local) breeding due to the long generation times in G. gracilis. Weak, but significant, population differentiation was detected in both haploids and diploids and varied with landscape features, and not with geographic distance. Using an assignment test, we establish that effective migration rates varied according to height on the shore. In this intertidal species, biased spore dispersal may occur during the transport of spores and gametes at low tide when small streams flow from high- to lower-shore pools. The longevity of both haploid and diploid free-living stages and the long generation times typical of G. gracilis populations may promote the observed pattern of high genetic diversity within populations relative to that among populations.     

Characterization of Dominant Resistance to Cobalt Chloride in DICTYOSTELIUM DISCOIDEUM and Its Use in Parasexual Genetic Analysis

Strains of Dictyostelium discoideum resistant to cobaltous chloride have been isolated at a frequency of approximately 10^-6. The resistant strains have one of three phenotypes, recessive to wild type, dominant to wild type and dominant to wild type but requiring the presence of cobaltous chloride to maintain resistance. Strains carrying a dominant cobaltous chloride resistance mutation and a recessive growth temperature-sensitive mutation can be mixed with wild-type haploid lines and then subjected to selection so that only diploid lines survive. Differential sensitivity to cycloheximide has also been observed. Hypersensitivity to cycloheximide in combination with dominant cobaltous chloride resistance provides a means of selecting diploids without the use of temperature-sensitive mutations.     

Two Multiallelic Mating Compatibility Loci Separately Regulate Zygote Formation and Zygote Differentiation in the Myxomycete PHYSARUM POLYCEPHALUM

The mating of Physarum polycephalum amoebae, the ultimate consequence of which is a "plasmodium," was recently shown to be governed by two compatibility loci, matA (or mt) and matB (Dee 1978; Youngmanet al. 1979). We present evidence that matA and matB separately regulate two discrete stages of mating: in the first stage, amoebae (which are normally haploid) fuse in pairs, with a specificity determined by matB genotype, to form diploid zygotes; subsequent differentiation of the zygotes into plasmodia is regulated by matA and is unaffected by matB. Mixtures of amoebae carrying unlike matA and matB alleles formed diploids to the extent of 10 to 15% of the cells present, and the diploids differentiated into plasmodia. When only the matB alleles differed, diploid cells still formed to a comparable (5 to 10%) extent, but rather than differentiating, these diploids remained amoebae. When strains carried the same alleles of matB, formation of diploid cells was greatly reduced: in like-matB, like-matA mixtures, none of 320 cells examined was diploid; in like-matB, unlike mat-A mixtures, differentiating diploids could be detected, but at only 10(-3) to 10(-2) the frequency of unlike-matB, unlike-matA mixtures. The nondifferentiating diploid amoebae recovered from unlike-matB, like-matA mixtures were genetically stable through extensive growth, even though they grew more slowly than haploids (10-hr vs. 8-hr doubling period), and could be crossed with both haploids and diploids. The results of such higher ploidy and mixed ploidy crosses indicate that karyogamy does not invariably accompany zygote formation and differentiation.     

Ribosomal RNA synthesis in haploid and diploid loach embryos]

rRNA synthesis was compared in the loach haploid (In) and diploid (2n) embryos. The relative intensity of synthesis was evaluated by 14C-uridine incorporation in 27S and 18S rRNA isolated from ribosomes taking into account label incorporation into total acid-soluble fraction and phosphrylated uridine derivatives. Label incorporation into rRNA, in reference with DNA content in 1n and 2n embryos, suggests that the level of rRNA synthesis per DNA unit in haploids is twice that in diploids whereas, in reference per cell, the same amount of ribosomes is synthesized both in haploids and diploids. The data obtained show that the amount of rRNAs synthesized in the loach embryogenesis does not depend on ploidy.     

Hybridization between two cryptic filamentous brown seaweeds along the shore: analysing pre‐ and postzygotic barriers in populations of individuals with varying ploidy levels

We aimed to study the importance of hybridization between two cryptic species of the genus Ectocarpus, a group of filamentous algae with haploid–diploid life cycles that include the principal genetic model organism for the brown algae. In haploid–diploid species, the genetic structure of the two phases of the life cycle can be analysed separately in natural populations. Such life cycles provide a unique opportunity to estimate the frequency of hybrid genotypes in diploid sporophytes and meiotic recombinant genotypes in haploid gametophytes allowing the effects of reproductive barriers preventing fertilization or preventing meiosis to be untangle. The level of hybridization between E. siliculosus and E. crouaniorum was quantified along the European coast. Clonal cultures (568 diploid, 336 haploid) isolated from field samples were genotyped using cytoplasmic and nuclear markers to estimate the frequency of hybrid genotypes in diploids and recombinant haploids. We identified admixed individuals using microsatellite loci, classical assignment methods and a newly developed Bayesian method (XPloidAssignment), which allows the analysis of populations that exhibit variations in ploidy level. Over all populations, the level of hybridization was estimated at 8.7%. Hybrids were exclusively observed in sympatric populations. More than 98% of hybrids were diploids (40% of which showed signs of aneuploidy) with a high frequency of rare alleles. The near absence of haploid recombinant hybrids demonstrates that the reproductive barriers are mostly postzygotic and suggests that abnormal chromosome segregation during meiosis following hybridization of species with different genome sizes could be a major cause of interspecific incompatibility in this system.