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.     

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.     

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.     

Evolution and maintenance of haploid–diploid life cycles in natural populations: The case of the marine brown alga Ectocarpus

The evolutionary stability of haploid–diploid life cycles is still controversial. Mathematical models indicate that niche differences between ploidy phases may be a necessary condition for the evolution and maintenance of these life cycles. Nevertheless, experimental support for this prediction remains elusive. In the present work, we explored this hypothesis in natural populations of the brown alga Ectocarpus. Consistent with the life cycle described in culture, Ectocarpus crouaniorum in NW France and E. siliculosus in SW Italy exhibited an alternation between haploid gametophytes and diploid sporophytes. Our field data invalidated, however, the long‐standing view of an isomorphic alternation of generations. Gametophytes and sporophytes displayed marked differences in size and, conforming to theoretical predictions, occupied different spatiotemporal niches. Gametophytes were found almost exclusively on the alga Scytosiphon lomentaria during spring whereas sporophytes were present year‐round on abiotic substrata. Paradoxically, E. siliculosus in NW France exhibited similar habitat usage despite the absence of alternation of ploidy phases. Diploid sporophytes grew both epilithically and epiphytically, and this mainly asexual population gained the same ecological advantage postulated for haploid–diploid populations. Consequently, an ecological interpretation of the niche differences between haploid and diploid individuals does not seem to satisfactorily explain the evolution of the Ectocarpus life cycle.     

DELETERIOUS MUTATION AND THE EVOLUTION OF GENETIC LIFE CYCLES

It is often proposed that the ability of diploids to mask deleterious mutations leads to an evolutionary advantage over haploidy. In this paper, we studied the evolution of the relative duration of haploid and diploid phases using a model of recurrent deleterious mutations across the entire genome. We found that a completely diploid life cycle is favored under biologically reasonable conditions, even when prolonging the diploid phase reduces a population's mean fitness. A haploid cycle is favored when there is complete linkage throughout the genome or when mutations are either highly deleterious or partially dominant. These results hold when loci interact multiplicatively and for synergistic epistasis. The strength of selection generated on the life cycle can be substantial because of the cumulative effect of selection against mutations across many loci. We did not find conditions that support cycles that retain both phases, such as those found in some plants and algae. Thus, selection against deleterious mutations may be an important force in the evolution of life cycles but may not be sufficient to explain all the patterns of life cycles seen in nature.     

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.     

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.     

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.     

CHPA, a cysteine- and histidine-rich-domain-containing protein, contributes to maintenance of the diploid state in Aspergillus nidulans

The alternation of eukaryotic life cycles between haploid and diploid phases is crucial for maintaining genetic diversity. In some organisms, the growth and development of haploid and diploid phases are nearly identical, and one might suppose that all genes required for one phase are likely to be critical for the other phase. Here, we show that targeted disruption of the chpA (cysteine- and histidine-rich-domain- [CHORD]-containing protein A) gene in haploid Aspergillus nidulans strains gives rise to chpA knockout haploids and heterozygous diploids but no chpA knockout diploids. A. nidulans chpA heterozygous diploids showed impaired conidiophore development and reduced conidiation. Deletion of chpA from diploid A. nidulans resulted in genome instability and reversion to a haploid state. Thus, our data suggest a vital role for chpA in maintenance of the diploid phase in A. nidulans. Furthermore, the human chpA homolog, Chp-1, was able to complement haploinsufficiency in A. nidulans chpA heterozygotes, suggesting that the function of CHORD-containing proteins is highly conserved in eukaryotes.     

Maintenance of Complex Life Cycles Via Cryptic Differences In The Ecophysiology Of Haploid And Diploid Spores Of An Isomorphic Red Alga1

Recognition of the wide diversity of organisms that maintain complex haploid–diploid life cycles has generated interest in understanding the evolution and persistence of such life cycles. We empirically tested the model where complex haploid–diploid life cycles may be maintained by subtle/cryptic differences in the vital rates of isomorphic haploid–diploids, by examining the ecophysiology of haploid tetraspores and diploid carpospores of the isomorphic red alga Chondrus verrucosus. While tetraspores and carpospores of this species did not differ in size or autofluorescence, concentrations of phycobiliproteins of carpospores were greater than that of tetraspores. However, tetraspores were more photosynthetically competent than carpospores over a broader range of photosynthetic photon flux densities (PPFDs) and at PPFDs found at both the depth that C. verrucosus is found at high tide and in surface waters in which planktonic propagules might disperse. These results suggest potential differences in dispersal potential and reproductive success of haploid and diploid spores. Moreover, these cryptic differences in ecological niche partitioning of haploid and diploid spores contribute to our understanding of some of the differences between these ploidy stages that may ultimately lead to the maintenance of the complex haploid–diploid life cycle in this isomorphic red alga.     

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.     

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.     

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.     

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.     

The evolution of life cycles with haploid and diploid phases

Sexual eukaryotic organisms are characterized by an alternation between haploid and diploid phases. In vascular plants and animals, somatic growth and development occur primarily in the diploid phase, with the haploid phase reduced to the gametic cells. In many other eukaryotes, however, growth and development occur in both phases, with substantial variability among organisms in the length of each phase of the life cycle. A number of theoretical models and experimental studies have shed light on factors that may influence life cycle evolution, yet we remain far from a complete understanding of the diversity of life cycles observed in nature. In this paper we review the current state of knowledge in this field, and touch upon the many questions that remain unanswered. BioEssays 20 :453–462, 1998. © 1998 John Wiley & Sons, Inc.     

Inheritance of extrachromosomal ribosomal DNA during the asexual life cycle of Dictyostelium discoideum: examination by use of DNA polymorphisms.

Wild-type isolates of Dictyostelium discoideum exhibited differences in the size of restriction fragments of the extrachromosomal 88-kilobase ribosomal DNA (rDNA) palindrome. Polymorphisms in rDNA also were found among strains derived solely from the NC4 wild-type isolate. These variations involved EcoRI fragments II, III, and V; they included loss of the EcoRI site separating fragments II and V and deletion and insertion of DNA. More than one rDNA form can coexist in the same diploid or haploid cell. However, one or another parental rDNA tended to predominate in diploids constructed, using the parasexual cycle, between haploid NC4-derived strains and haploid wild-type isolates. In some cases, most if not all of the rDNA of such diploids were of one form after ca. 50 generations of growth. Segregant haploids, derived from diploids that possessed predominantly a single rDNA allele, possessed the same allele as the diploid and did not recover the other form. This evidence implies that replication does not proceed from a single chromosomal or extrachromosomal copy of the rDNA during the asexual life cycle of D. discoideum.     

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.     

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.     

Ploidally antagonistic selection maintains stable genetic polymorphism

Understanding the maintenance of genetic variation in the face of selection remains a key issue in evolutionary biology. One potential mechanism for the maintenance of genetic variation is opposing selection during the diploid and haploid stages of biphasic life cycles universal among eukaryotic sexual organisms. If haploid and diploid gene expression both occur, selection can act in each phase, potentially in opposing directions. In addition, sex-specific selection during haploid phases is likely simply because male and female gametophytes/gametes tend to have contrasting life histories. We explored the potential for the maintenance of a stable polymorphism under ploidally antagonistic as well as sex-specific selection. Furthermore, we examined the role of the chromosomal location of alleles (autosomal or sex-linked). Our analyses show that the most permissible conditions for the maintenance of polymorphism occur under negative ploidy-by-sex interactions, where stronger selection for an allele in female than male diploids is coupled with weaker selection against the allele in female than male haploids. Such ploidy-by-sex interactions also promote allele frequency differences between the sexes. With constant fitness, ploidally antagonistic selection can maintain stable polymorphisms for autosomal and X-linked genes but not for Y-linked genes. We discuss the implications of our results and outline a number of biological settings where the scenarios modeled may apply.