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.     

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.     

Mitotic recombination accelerates adaptation in the fungus Aspergillus nidulans

Understanding the prevalence of sexual reproduction in eukaryotes is a hard problem. At least two aspects still defy a fully satisfactory explanation, the functional significance of genetic recombination and the great variation among taxa in the relative lengths of the haploid and diploid phases in the sexual cycle. We have performed an experimental study to explore the specific advantages of haploidy or diploidy in the fungus Aspergillus nidulans. Comparing the rate of adaptation to a novel environment between haploid and isogenic diploid strains over 3,000 mitotic generations, we demonstrate that diploid strains, which during the experiment have reverted to haploidy following parasexual recombination, reach the highest fitness. This is due to the accumulation of recessive deleterious mutations in diploid nuclei, some of which show their combined beneficial effect in haploid recombinants. Our findings show the adaptive significance of mitotic recombination combined with flexibility in the timing of ploidy level transition if sign epistasis is an important determinant of fitness.     

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.     

Experimental studies on ploidy evolution in yeast

Variation in the prominence of haploidy and diploidy is a striking feature of eukaryote life cycles that has not been explained from an evolutionary point of view. the ease with which ploidy and other variables of population genetics may be manipulated in yeast make Saccharomyces cerevisiae an excellent subject for experiments on the fitness effects of ploidy. Several hypotheses have been advanced to explain the emphasis on diploidy in plants and animals, and yeast experiments have been particularly informative for a few. Evidence suggests that diploids may enjoy an immediate advantage over haploids in masking harmful mutations, avoiding the fitness cost such mutations impose on haploids. A convincing longer-term advantage for diploidy has proven elusive, and different evolutionary explanations for the origin and for the subsequent maintenance of diploidy may be required.     

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.     

Effect of triploid fitness on the coexistence of diploids and tetraploids

The conditions for the coexistence of diploids, triploids and tetraploids in a single population were investigated with a deterministic model under the assumptions that diploids might produce 2 n gametes, and that triploids had a lower fitness than other cytotypes and generated equal proportions of haploid and diploid gametes. When diploids produced only haploid gametes, the dynamics of the cytotypes were similar to that of heterozygote disadvantage with two alleles at a single locus, with triploids being equivalent to the heterozygotes. Production of 2 n gametes by diploids increased the pool of diploid gametes and created a stable equilibrium involving a majority of diploids and a minority of polyploids. When the fitness of tetraploids was equal to or higher than that of diploids, increased triploid fitness decreased the threshold of 2 n gametes necessary to deterministically fix tetraploids in the population. Conversely, when tetraploids were less fit than diploids, the rate of 2 n gamete production leading to the exclusion of diploids first decreases and then increased with increasing triploid fitness. Triploids are repeatedly found in diploid-tetraploid hybridizations and are rarely totally sterile. They might play a determinant role in the future of multiple cytotype populations. The effect of triploids depends on the relative fitness of diploids and tetraploids and is also a function of their fitness.     

Emergence and Maintenance of Sex among Diploid Organisms Aided by Assortative Mating

Using computer simulations I studied the simultaneous effect of variable environments, mutation rates, ploidy, number of loci subject to evolution and random and assortative mating on various reproductive systems. The simulations showed that mutants for sex and recombination are evolutionarily stable, displacing alleles for monosexuality in diploid populations mating assortatively under variable selection pressure. Assortative mating reduced excessive allelic variance induced by recombination and sex, especially among diploids. Results suggest a novel adaptive value for sex and recombination. They show that the adaptive value of diploidy and that of the segregation of sexes is different to that of sex and recombination. The results suggest that the emergence of sex had to be preceded by the emergence of diploid monosexual organisms and provide an explanation for the emergence and maintenance of sex among diploids and for the scarcity of sex among haploid organisms.     

Evolution of the alternation of haploid and diploid phases in life cycles. II. Maintenance of the haplo-diplontic cycle

The relative duration of the haploid and the diploid phases during the reproductive cycle varies greatly between organisms. This paper addresses the question of the evolution of haploid, diploid, and haplo-diplontic life cycles. When the life span of haploid and diploid individuals is constant whatever their cycle, we show that the haplo-diplontic cycle has an advantage, which depends on the sex-ratio in anisogamous species and on the probability of fertilization in isogamous species. This is because meiosis and fertilization occur half as often in the haplo-diplontic cycle as in haploid or diploid cycles, for the same number of generations of individuals. This argument is demonstrated using a model which considers a genetic determination of the cycle, and fixed haploid and diploid fitnesses. The relevance of measures of fitness of haploid and diploid individuals in predicting the evolution of life cycles is discussed. Measures obtained in algae are compared with theoretical predictions.     

Inter-embryo competition in the progeny of autotriploid Aloineae (Liliaceae)

In reciprocal crosses between diploid and triploid Aloineae the progeny are largely diploid or diploid plus one or two chromosomes, but in reciprocal crosses between triploids and tetraploids they are tetraploid or nearly so. Thus the triploids contribute circa haploid gametes to the progeny when crossed with diploids but circa diploid gametes when crossed with tetraploids. These results are compared with those of a number of earlier workers. It is concluded that the bias in the frequency of progeny types towards diploidy or tetraploidy, depending on the ploidy level of the plant which is crossed with the triploid, is caused by inter-embryo competition. Those embryos with an endosperm/embryo factor of 1.5, the value found in normal diploid/diploid crosses having triploid endosperms, are selected in preference to those with factors higher or lower than 1.5.Inter-gamete competition also occurs among the euploid and aneuploid gametes produced by the triploids. This is more pronounced on the male side, because the degree of survival of aneuploid pollen from the triploids into the next generation is much lower than that of aneuploid egg nuclei.Non-reduction in the triploids gives rise to occasional pentaploid progeny in crosses with tetraploids, but it is more probable that in diploid/triploid crosses tetraploid progeny are the products of non-reduction in the diploid.     

Population Studies in Microorganisms I. Evolution of Diploidy in SACCHAROMYCES CEREVISIAE

The relative adaptation of isogenic haploid and diploid strains of yeast was investigated in different sets of physiological conditions. When all nutrients were present in excess, no difference in the reproductive rates of isogenic haploid and diploid strains of yeast was detected in both optimal and non-optimal growth conditions. Competition between haploid and diploid strains of yeast was observed when growth was limited by the concentration of a single nutrilite. Under certain conditions when fitness (reproductive rate) is determined by transport of an essential nutrilite that exists in very low concentrations, diploid cells were selected against. These environmental conditions are similar to those found in offshore marine environments where nutrients are often present in extremely low concentrations. The fitness of diploid cells was estimated to be.93 +/-.02 (haploid fitness = 1). The reduced fitness of diploid cells in this environment can be explained by the reduced surface area/volume ratio possessed by diploid cells in comparison to haploid cells. The fitnesses of haploid and diploid cells in these environments are closely correlated with geometric variations in these strains. These results are consistent with the hypothesis that diploid cells are simply double haploids, and diploidy per se does not confer any direct adaptive advantage. The mechanism of the evolution of diploidy as a dominant phase in the life cycle of higher plants and animals remains obscure.     

Ploidy of Bacillus subtilis exfusants: the haploid nature of cells forming colonies with biparental or prototrophic phenotypes.

To investigate the relationship between DNA content and cell volume, we have attempted to repeat the construction of stable Bacillus subtilis diploid cells through protoplast fusion. Colonies with a biparental phenotype and those with a prototrophic phenotype were identified among exfusants of a cross between two polyauxotrophic strains. The ploidy of cells constituting such colonies was assessed by protoplast self-fusion, determination of the DNA to dry weight ratio of exponentially growing cells, and by quantitative DNA-DNA hybridization. Within the precision of these methods, all colonies were found to consist of haploid cells. A previously described non-complementing diploid was also found to be haploid. Therefore, the genetic evidence in favour of diploidy, based on continuing segregation of cells with a parental or recombinant phenotype, cannot be accounted for except by the maintenance of such cells as a minority population in mixed colonies through cross-feeding. Reconstruction experiments with mixtures of whole parental cells confirm that biparental colonies are indeed mixed colonies which arise either by sticking of parental cells or through coincidence, i.e. their plating within a distance of about 0.4 mm. The previously reported experimental results can be accounted for in the light of our results.     

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.     

The yield of experimental yeast populations declines during selection

The trade-off between growth rate and yield can limit population productivity. Here we tested for this life-history trade-off in replicate haploid and diploid populations of Saccharomyces cerevisiae propagated in glucose-limited medium in batch cultures for 5000 generations. The yield of single clones isolated from the haploid lineages, measured as both optical and population density at the end of a growth cycle, declined during selection and was negatively correlated with growth rate. Initially, diploid populations did not pay this cost of adaptation but haploidized after about 1000–3000 generations of selection, and this ploidy transition was associated with a decline in yield caused by reduced cell size. These results demonstrate the experimental evolution of a trade-off between growth rate and yield, caused by antagonistic pleiotropy, during adaptation in haploids and after an adaptive transition from diploidy to haploidy.     

Further Studies on the Role of Polyploidy in the Evolution of Meloidogyne

Two tetraploid isolates of Meloidogyne hapla, 86P and E289P, with haploid chromosome numbers of 34 and 28, respectively, were studied cytogenetically and biologically in relation to the diploid populations, 86-Va (n = 17) and E289-Taiwan (n = 14), from which they had been originally isolated. Both isolates were quite stable, converting to diploidy at the low rate of about 2.5%. The tetraploid isolate 86P maintained itself in competition with its diploid counterpart in mixed cultures, although an initial frequency of 50% polyploidy was reduced to about 9% at the end of the sixth generation. Both tetraploid isolates could maintain themselves in greenhouse cultures without artificial selection for at least 2 years. Crosses between diploid females and tetraploid males resulted in a few triploid females that produced mostly nonviable eggs, suggesting partial reproductive isolation between the two ploidy forms. Ten generations of propagation of only polyploid females of isolate 86P that were associated with males failed to yield an obligatorily amphimictic isolate that would not convert at all to diploidy. If one accepts a previous assumption that the present day amphimictic root-knot nematodes are tetraploids derived from diploid ancestors, results of the present study are not inconsistent with an evolutionary trend toward an even higher level of ploidy in Meloidogyne, presumably octaploidy.     

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.     

Ploidy reduction in Saccharomyces cerevisiae

Large-scale transitions in genome size from tetraploid to diploid were observed during a previous 1800-generation evolution experiment in Saccharomyces cerevisiae. Whether the transitions occurred via a one-step process (tetraploid to diploid) or through multiple steps (through ploidy intermediates) remained unclear. To provide insight into the mechanism involved, we investigated whether triploid-sized cells sampled from the previous experiment could also undergo ploidy loss. A batch culture experiment was conducted for approximately 200 generations, starting from four triploid-sized colonies and one contemporaneous tetraploid-sized colony. Ploidy reduction towards diploidy was observed in both triploid and tetraploid lines. Comparative genomic hybridization indicated the presence of aneuploidy in both the founder and the evolved colonies. The specific aneuploidies involved suggest that chromosome loss was not haphazard but that nearly full sets of chromosomes were lost at once, with some additional chromosome mis-segregation events. These results suggest the existence of a mitotic mechanism allowing the elimination of an entire set of chromosomes in S. cerevisiae, thereby reducing the ploidy level.     

Ploidy tug‐of‐war: Evolutionary and genetic environments influence the rate of ploidy drive in a human fungal pathogen

Variation in baseline ploidy is seen throughout the tree of life, yet the factors that determine why one ploidy level is maintained over another remain poorly understood. Experimental evolution studies using asexual fungal microbes with manipulated ploidy levels intriguingly reveals a propensity to return to the historical baseline ploidy, a phenomenon that we term “ploidy drive.” We evolved haploid, diploid, and polyploid strains of the human fungal pathogen Candida albicans under three different nutrient limitation environments to test whether these conditions, hypothesized to select for low ploidy levels, could counteract ploidy drive. Strains generally maintained or acquired smaller genome sizes (measured as total nuclear DNA through flow cytometry) in minimal medium and under phosphorus depletion compared to in a complete medium, while mostly maintained or acquired increased genome sizes under nitrogen depletion. Improvements in fitness often ran counter to changes in genome size; in a number of scenarios lines that maintained their original genome size often increased in fitness more than lines that converged toward diploidy (the baseline ploidy of C. albicans). Combined, this work demonstrates a role for both the environment and genotype in determination of the rate of ploidy drive, and highlights questions that remain about the force(s) that cause genome size variation.     

Evolutionary advantage of small populations on complex fitness landscapes

Recent experimental and theoretical studies have shown that small asexual populations evolving on complex fitness landscapes may achieve a higher fitness than large ones due to the increased heterogeneity of adaptive trajectories. Here, we introduce a class of haploid three-locus fitness landscapes that allow the investigation of this scenario in a precise and quantitative way. Our main result derived analytically shows how the probability of choosing the path of the largest initial fitness increase grows with the population size. This makes large populations more likely to get trapped at local fitness peaks and implies an advantage of small populations at intermediate time scales. The range of population sizes where this effect is operative coincides with the onset of clonal interference. Additional studies using ensembles of random fitness landscapes show that the results achieved for a particular choice of three-locus landscape parameters are robust and also persist as the number of loci increases. Our study indicates that an advantage for small populations is likely whenever the fitness landscape contains local maxima. The advantage appears at intermediate time scales, which are long enough for trapping at local fitness maxima to have occurred but too short for peak escape by the creation of multiple mutants.     

Unpredictable fitness transitions between haploid and diploid strains of the genetically loaded yeast Saccharomyces cerevisiae.

Mutator strains of yeast were used to accumulate random point mutations. Most of the observed changes in fitness were negative and relatively small, although major decreases and increases were also present. The average fitness of haploid strains was lowered by approximately 25% due to the accumulated genetic load. The impact of the load remained basically unchanged when a homozygous diploid was compared with the haploid from which it was derived. In other experiments a heterozygous diploid was compared with the two different loaded haploids from which it was obtained. The fitness of such a loaded diploid was much less reduced and did not correlate with the average fitness of the two haploids. There was a fitness correlation, however, when genetically related heterozygous diploids were compared, indicating that the fitness effects of the new alleles were not entirely lost in the heterozygotes. It is argued here that to explain the observed pattern of fitness transitions it is necessary to invoke nonadditive genetic interactions that go beyond the uniform masking effect of wild-type alleles. Thus, the results gathered with haploids and homozygotes should be extrapolated to heterozygotes with caution when multiple loci contribute to the genetic load.