Haploidy, diploidy and evolution of antifungal drug resistance in Saccharomyces cerevisiae

We tested the hypothesis that the time course of the evolution of antifungal drug resistance depends on the ploidy of the fungus. The experiments were designed to measure the initial response to the selection imposed by the antifungal drug fluconazole up to and including the fixation of the first resistance mutation in populations of Saccharomyces cerevisiae. Under conditions of low drug concentration, mutations in the genes PDR1 and PDR3, which regulate the ABC transporters implicated in resistance to fluconazole, are favored. In this environment, diploid populations of defined size consistently became fixed for a resistance mutation sooner than haploid populations. Experiments manipulating population sizes showed that this advantage of diploids was due to increased mutation availability relative to that of haploids; in effect, diploids have twice the number of mutational targets as haploids and hence have a reduced waiting time for mutations to occur. Under conditions of high drug concentration, recessive mutations in ERG3, which result in resistance through altered sterol synthesis, are favored. In this environment, haploids consistently achieved resistance much sooner than diploids. When 29 haploid and 29 diploid populations were evolved for 100 generations in low drug concentration, the mutations fixed in diploid populations were all dominant, while the mutations fixed in haploid populations were either recessive (16 populations) or dominant (13 populations). Further, the spectrum of the 53 nonsynonymous mutations identified at the sequence level was different between haploids and diploids. These results fit existing theory on the relative abilities of haploids and diploids to adapt and suggest that the ploidy of the fungal pathogen has a strong impact on the evolution of fluconazole resistance.     

Ploidy and recombination proficiency shape the evolutionary adaptation to constitutive DNA replication stress

In haploid budding yeast, evolutionary adaptation to constitutive DNA replication stress alters three genome maintenance modules: DNA replication, the DNA damage checkpoint, and sister chromatid cohesion. We asked how these trajectories depend on genomic features by comparing the adaptation in three strains: haploids, diploids, and recombination deficient haploids. In all three, adaptation happens within 1000 generations at rates that are correlated with the initial fitness defect of the ancestors. Mutations in individual genes are selected at different frequencies in populations with different genomic features, but the benefits these mutations confer are similar in the three strains, and combinations of these mutations reproduce the fitness gains of evolved populations. Despite the differences in the selected mutations, adaptation targets the same three functional modules in strains with different genomic features, revealing a common evolutionary response to constitutive DNA replication stress.     

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.     

Ploidy evolution in the yeast Saccharomyces cerevisiae: a test of the nutrient limitation hypothesis

The nutrient limitation hypothesis provides a nongenetic explanation for the evolution of life cycles that retain both haploid and diploid phases: differences in nutrient requirements and uptake allow haploids to override the potential genetic advantages provided by diploidy under certain nutrient limiting conditions. The relative fitness of an isogenic series of haploid, diploid and tetraploid yeast cells (Saccharomyces cerevisiae), which were also equivalent at the mating type locus, was measured. Fitness was measured both by growth rate against a common competitor and by intrinsic growth rate in isolated cultures, under four environmental conditions: (1) rich medium (YPD) at the preferred growth temperature (30 °C); (2) nutrient poor medium (MM) at 30 °C; (3) YPD at a nonpreferred temperature (37 °C); and (4) MM at 37 °C. In contrast to the predictions of the nutrient limitation hypothesis, haploids grew significantly faster than diploids under nutrient rich conditions, but there were no apparent differences between them when fitness was determined by relative competitive ability. In addition, temperature affected the relative growth of haploids and diploids, with haploids growing proportionately faster at higher temperatures. Tetraploids performed very poorly under all conditions compared. Cell geometric parameters were not consistent predictors of fitness under the conditions measured.     

The contribution of haploids, diploids and clones to fine-scale population structure in the seaweed Cladophoropsis membranacea (Chlorophyta)

Local populations of Cladophoropsis membranacea exist as mats of coalesced thalli composed of free-living haploid and diploid plants including clonally reproduced plants of either phase. None of the phases are morphologically distinguishable. We used eight microsatellite loci to explore clonality and fine-scale patch structure in C. membranacea at six sites on the Canary Islands. Mats were always composites of many individuals; not single, large clones. Haploids outnumbered diploids at all sites (from 2:1 to 10:1). In both haploid and diploid plants, genetic diversity was high and there was no significant difference in allele frequencies. Significant heterozygote deficiencies were found in the diploid plants at five out of six sites and linkage disequilibrium was associated with the haploid phase at all sites. Short dispersal distances of gametes/spores and small effective population sizes associated with clonality probably contribute to inbreeding. Spatial autocorrelation analysis revealed that most clones were found within a radius of approximately 60 cm and rarely further than 5 m. Dominance of the haploid phase may reflect seasonal shifts in the relative frequencies of haploids and diploids, but may alternatively reflect superiority of locally adapted and competitively dominant, haploid clones; a strategy that is theoretically favoured in disturbed environments. Although sexual reproduction may be infrequent in C. membranacea, it is sufficient to maintain both life history phases and supports theoretical modelling studies that show that haploid-diploid life histories are an evolutionarily stable strategy.     

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.     

Does Diploidy Increase the Rate of Adaptation?

Explanations of the evolution of diploidy have focused on the advantages gained from masking deleterious alleles. Recent theory has shown, however, that masking does not always provide an advantage to diploidy and would never favor diploidy in predominantly asexual organisms. We explore a neglected alternative theory which posits that, by doubling the genome size, diploids double the rate at which favorable mutations arise. Consequently, the rate of adaptation in diploids is presumed to be faster than in haploids. The rate of adaptation, however, depends not only on the rate of appearance of new favorable mutations but also on the rate at which these mutations are incorporated (which depends on the population size and on the dominance of favorable mutations). We show that, in both asexuals and sexuals, doubling the mutation rate via diploidy often does not accelerate the rate of adaptation. Indeed, under many conditions, diploidy slows adaptation.     

Differentiation of haploid and dihaploid rape plants at the cytological and morphological levels

Some cytological and morphological features of haploid and dihaploid winter rapó plants obtained via the anther cultivation approach have been studied. It was shown that in haploid plants the number of chloroplasts in stomatal guard cells, the size of the stomatal guard cells themselves were much smaller, and the number of stomata per square unit was greater than in doubled haploids and diploids. Haploids were also characterized by smaller sizes of petals and anthers and, in general, a smaller flower as compared to dihapliods and diploids.     

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.     

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.     

The interrelationship of cell growth and division in haploid and diploid cells of Saccharomyces cerevisiae

The coordination of cell growth and division has been examined in isogenic haploid and diploid strains of Saccharomyces cerevisiae. The average cell volume of the haploid and diploid cells was unaffected by a range of environmental conditions and generation times. For most environments and generation times the mean cell volume of diploid cells was between 1.52 and 1.83 of the haploid cell volume. Both haploid and diploid cell volumes were reduced drastically when the cells were grown in the chemostat with glucose as the limiting substrate. In this environment diploid cells have the same mean cell volume as haploid cells. Diploid cells are more elongated than haploid cells, and the characteristic shape (eccentricity) of the cells is unaffected by all environmental conditions and generation times tested. Mother cell volume increased during the cell cycle, although the pattern of this increase was affected by the environmental conditions. Under most growth conditions detectable mother cell volume increase occurred only during the budding phase, whereas under conditions of carbon limitation detectable increase only occurred during the unbudded phase. A consequence of this result is that the mean cell volume of haploids at bud initiation is relatively constant in all environments, including carbon limitation. This suggests that there is a critical size for bud initiation for haploids which is constant and independent of environmental conditions. The results for diploids are more complex. Coordination of growth and division in haploid cells can be explained by a simple model initially developed for prokaryotes by Donachie. A modification of this model is proposed to account for the results with diploids.     

Effects of the [PSI+] prion on rates of adaptation in yeast

The [PSI(+)] prion in yeast has been shown to improve short-term growth in some environments, but its effects on rates of adaptation have not been assessed before now. We adapted three yeast genotypes to three novel environments in the presence and the absence of the prion. There were significant differences in adaptation rates between lines with different combinations of genotype, environment, and prion status. We saw no consistent effect, however, of the prion on the rate of adaptation to new environments. A major factor affecting the rate of adaptation was initial fitness in the new environment: lines with low initial fitness evolved faster than lines with high initial fitness.     

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.     

Linking temperature dependence of fitness effects of mutations to thermal niche adaptation

Fitness effects of mutations may generally depend on temperature that influences all rate-limiting biophysical and biochemical processes. Earlier studies suggested that high temperatures may increase the availability of beneficial mutations (‘more beneficial mutations’), or allow beneficial mutations to show stronger fitness effects (‘stronger beneficial mutation effects’). The ‘more beneficial mutations’ scenario would inevitably be associated with increased proportion of conditionally beneficial mutations at higher temperatures. This in turn predicts that populations in warm environments show faster evolutionary adaptation but suffer fitness loss when faced with cold conditions, and those evolving in cold environments become thermal-niche generalists (‘hotter is narrower’). Under the ‘stronger beneficial mutation effects’ scenario, populations evolving in warm environments would show faster adaptation without fitness costs in cold environments, leading to a ‘hotter is (universally) better’ pattern in thermal niche adaptation. We tested predictions of the two competing hypotheses using an experimental evolution study in which populations of two model bacterial species, Escherichia coli and Pseudomonas fluorescens, evolved for 2400 generations at three experimental temperatures. Results of reciprocal transplant experiments with our P. fluorescens populations were largely consistent with the ‘hotter is narrower’ prediction. Results from the E. coli populations clearly suggested stronger beneficial mutation effects at higher assay temperatures, but failed to detect faster adaptation in populations evolving in warmer experimental environments (presumably because of limitation in the supply of genetic variation). Our results suggest that the influence of temperature on mutational effects may provide insight into the patterns of thermal niche adaptation and population diversification across thermal conditions.     

Translocations in DICTYOSTELIUM DISCOIDEUM

Fourteen translocations of independent origin were identified in Dictyostelium discoideum on the basis of segregation anomalies of diploids heterozygous for these chromosome rearrangements, all of which led to the cosegregation of unlinked markers. Many of these translocations were discovered in strains mutagenized with MNNG or in strains carrying mutations affecting DNA repair; however, spontaneous translocations were also obtained. Haploid mitotic recombinants of the rearranged linkage groups were produced from diploids heterozygous for the translocations at frequencies of up to 5% of viable haploid segregants; this is at least a ten-fold higher frequency than that seen with diploids not heterozygous for translocations (approximately 0.1%). These haploid recombinants included both translocated and nontranslocated strains. The T354(II, VII) translocation and possibly the T357(IV, VII) translocation reduce the chromosome number to n = 6; haploids carrying 11 other translocations all have karyotypes with n = 7. Genetic characterization of the T357(IV, VII) translocation showed that the bwnA and whiC loci normally found on linkage group IV were physically linked to the linkage group VII loci couA, phgA, bsgB and cobA.     

A comparison of sexual and asexual replication strategies in a simplified model based on the yeast life cycle

This paper develops simplified mathematical models describing the mutation-selection balance for the asexual and sexual replication pathways in Saccharomyces cerevisiae, or Baker’s yeast. The simplified models are based on the single-fitness-peak approximation in quasispecies theory. We assume diploid genomes consisting of two chromosomes, and we assume that each chromosome is functional if and only if its base sequence is identical to some master sequence. The growth and replication of the yeast cells is modeled as a first-order process, with first-order growth rate constants that are determined by whether a given genome consists of zero, one, or two functional chromosomes. In the asexual pathway, we assume that a given diploid cell divides into two diploids. For the sake of generality, our model allows for mitotic recombination and asymmetric chromosome segregation. In the sexual pathway, we assume that a given diploid cell divides into two diploids, each of which then divide into two haploids. The resulting four haploids enter a haploid pool, where they grow and replicate until they meet another haploid with which to fuse. In the sexual pathway, we consider two mating strategies: (1) a selective strategy, where only haploids with functional chromosomes can fuse with one another; (2) a random strategy, where haploids randomly fuse with one another. When the cost for sex is low, we find that the selective mating strategy leads to the highest mean fitness of the population, when compared to all of the other strategies. When the cost for sex is low, sexual replication with random mating also has a higher mean fitness than asexual replication without mitotic recombination or asymmetric chromosome segregation. We also show that, at low replication fidelities, sexual replication with random mating has a higher mean fitness than asexual replication, as long as the cost for sex is low. If the fitness penalty for having a defective chromosome is sufficiently high and the cost for sex sufficiently low, then at low replication fidelities the random mating strategy has a mean fitness that is a factor of larger than the asexual mean fitness. We argue that for yeast, the selective mating strategy is the one that is closer to reality, which if true suggests that sex may provide a selective advantage under considerably more relaxed conditions than previous research has indicated. The results of this paper also suggest that S. cerevisiae switches from asexual to sexual replication when stressed, because stressful growth conditions provide an opportunity for the yeast to clear out deleterious mutations from their genomes. That being said, our model does not contradict theories for the evolution of sex that argue that sex evolved because it allows a population to more easily adapt to changing conditions.     

Cyclical dynamics under constant selection against mutations in haploid and diploid populations with facultative selfing

We have found that constant selection against mutations can cause cyclical dynamics in a population with facultative selfing. When this happens, the distribution of the number of deleterious mutations per genotype fluctuates with the period approximately 1/sHe generations, where sHe is the coefficient of selection against a heterozygous mutation. The amplitude of oscillations of the mean population fitness often exceeds an order of magnitude. Cyclical dynamics can occur under intermediate selfing rates if selection against heterozygous mutations is weak and selection against homozygous mutations is much stronger. Cycling is possible without epistasis or with diminishing-returns epistasis, but not with synergistic epistasis. Under multiplicative selection, cycling might happen if the haploid mutation rate exceeds 1.9 in the case of selfing of haploids, and if this diploid mutation rate exceeds 4.5 in the case of selfing of diploids. We propose a heuristic explanation for cycling under facultative selfing and discuss its possible relevance.     

Parallel genetic changes and nonparallel gene-environment interactions characterize the evolution of drug resistance in yeast

Beneficial mutations are required for adaptation to novel environments, yet the range of mutational pathways that are available to a population has been poorly characterized, particularly in eukaryotes. We assessed the genetic changes of the first mutations acquired during adaptation to a novel environment (exposure to the fungicide, nystatin) in 35 haploid lines of Saccharomyces cerevisiae. Through whole-genome resequencing we found that the genomic scope for adaptation was narrow; all adapted lines acquired a mutation in one of four late-acting genes in the ergosterol biosynthesis pathway, with very few other mutations found. Lines that acquired different ergosterol mutations in the same gene exhibited very similar tolerance to nystatin. All lines were found to have a cost relative to wild type in an unstressful environment; the level of this cost was also strongly correlated with the ergosterol gene bearing the mutation. Interestingly, we uncovered both positive and negative effects on tolerance to other harsh environments for mutations in the different ergosterol genes, indicating that these beneficial mutations have effects that differ in sign among environmental challenges. These results demonstrate that although the genomic target was narrow, different adaptive mutations can lead populations down different evolutionary pathways, with respect to their ability to tolerate (or succumb to) other environmental challenges.     

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.