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 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.     

What controls haploid—diploid ratio in the red alga,Gracilaria verrucosa?

The conditions for maintenance of a haploid—diploid life cycle in the species Gracilaria verrucosa were studied. This species is a red alga, where haploid plants have separate sexes. In the two natural populations studied, male and female haploid individuals were in equal proportions, and the frequency of diploid individuals reached 0.5. A two-fold advantage in viability for diploid relative to haploid juveniles was observed in the field. This advantage can account for a frequency of 0.5 of diploid individuals in natural populations. Different types of anomalies in the reproduction of diploid individuals were observed, all of which lead to a reduction of the haploid stage.     

Sexual biology of haploid and diploid males in the bumble beeBombus terrestris

Summary InB. terrestris diploid males develop normally into adults (Duchateau et al., 1994). The diploid males are similar in appearance to the haploid males, except that they are smaller. The size of the testis of diploid males, relative to the length of the radial cell, is smaller than that of haploid males. There is overlap in the frequency distribution with respect to body size and testis size. The spermatozoa of diploid males are larger than those of the haploids and the vasa deferentia contain fair less spermatozoa than those of haploid males of the same age. Countings and measurements of the spermatozoa, therefore, can give the best indication about the ploidy of the males. Diploid males are successful in mating. They mate at a younger age than haploid males and they die sooner. The number of vial offspring of diploid males, however, is very low. No queen that mated with a diploid male produced a colony, but a few queens did produce some progeny. These might have been triploid males and workers. InB. terrestris higher ploidy results in smaller individuals, whereas in several other species of the Hymenoptera it has been found to result in larger individuals.     

Growth and Survival of Early Juveniles of the Marine Sponge Hymeniacidon perlevis (Demospongiae) Under Controlled Conditions

To resolve “the supply problem” in sponge-derived drug development and other biotechnological applications, current research is exploring the possibility of obtaining an alternative sustainable supply of sponge biomass through intensive aquaculture of sponges utilizing artificial seed rearing. This study aimed to investigate the technology of early juvenile sponge cultivation under controlled conditions. The effects of food, temperature, water flow, and light on the growth and survival of early juveniles of the marine sponge Hymeniacidon perlevis were examined. The concentrations of four types of food elements [microalgae (Isochrysis galbana), photosynthetic bacteria (Rhodopseudomonas), Fe³⁺ (FeCl₃), and Si (Na₂SiO₃)] were investigated for early H. perlevis juvenile growth. Interestingly, temperature changes have striking effects on juvenile growth. Juvenile sponges grow faster when they are shifted to higher temperatures (18°C to 23°C) than when they are shifted to lower temperatures (18°C to 4°C to 23°C) or kept at a constant temperature (18°C). Periodic water flow and light cycles favor early juvenile sponge growth. Light was found to be a key factor in the color loss of early H. perlevis juveniles. Overall, size (area) increased as much as 29 times for H. perlevis juveniles under the tested controlled conditions.     

Relative competitiveness of haploid and diploid yeast cells growing in a mixed population

Saccharomyces cerevisiae was grown in a rich medium under the conditions of "quasi-continuous" cultivation and, after 200-300 generations, its diploid cells almost completely displaced haploid cells from the original mixed "haploid-diploid" population where the ratio between diploid and haploid strains was either 1:1 or 1:100. The cultivation at 40 degrees C did not change the relative competitive ability of haploids and diploids. When cells were cultivated in a rich medium at 6 degrees C or in a minimal medium at 30 degrees C, none of the strains showed an advantage over others for about 200 generations. Haploid cells had an advantage over diploid cells during "quasi-continuous" growth in the minimal medium at 30 degrees C. When the temperature was elevated to 40 degrees C, diploid cells displaced haploid cells from the mixed population. No advantage was found for diploid or haploid cells grown in a medium with an elevated KCl content (1.5 M). Haploid cells had an advantage over diploid cells when Pichia pinus was cultivated in a minimal medium. The results are discussed using the hypothesis about the diploid phase being fixed in the course of biological evolution.     

Ploidy chimeras induced in haploid sporophytes of <Emphasis Type="Italic">Osmunda claytoniana</Emphasis> and <Emphasis Type="Italic">Osmunda japonica</Emphasis>

Haploid sporophytes of Osmunda claytoniana (2n = x = 22) were apogamously produced from calli when cultivated on a hormone-free medium. Flow cytometric analysis showed that ploidy chimeras were spontaneously produced in a haploid sporophyte of O. claytoniana and those of O. japonica that were obtained in the previous study. In the haploid sporophyte of O. claytoniana, a diploid pinnule and a partially diploid terminal segment were produced in a haploid pinna. In O. japonica, a haploid sporophyte yielded a diploid pinna in a haploid frond, and another haploid sporophyte yielded a diploid pinnule in a haploid pinna. Diploid chimeras were large in size and could be readily distinguished from other haploid parts of the fronds. It is likely that the chimeras were produced clonally from a single diploid cell that established chromosome doubling.     

Gynogenesis in gentians (Gentiana triflora, G. scabra): production of haploids and doubled haploids

Gynogenesis was investigated on gentian (Gentiana triflora, G. scabra and their hybrids), which is an important ornamental flower. When unfertilized ovules were cultured in 1/2 NLN medium containing a high concentration of sucrose (100 g/l), embryo-like structures (ELS) were induced. Although genotypic variation was observed in ELS induction, all four genotypes produced ELSs ranging from 0.93 to 0.04 ELSs per flower bud. The ovules collected from flower buds of later stages (just before anthesis or flower anthesis) tended to exhibit higher response. The dark culture condition produced more than four times as many ELSs than in 16-h light condition. A significant number of plantlets were directly regenerated from ELSs on MS regeneration medium. The ploidy levels of 179 regenerated plants were determined by flow cytometry, revealing that the majority of them were diploid (55.9%) and haploid (31.3%). When a total of 54 diploid plants were examined by molecular genetic markers, 52 (96.3%) were considered as doubled haploids (DHs). This is the first report showing successful gynogenesis in gentian. The production of haploids and DHs by unfertilized ovule culture opens a novel prospect in gentian F1 hybrid breeding.     

Differential ecological responses to environmental stress in the life history phases of the isomorphic red alga Gracilaria chilensis (Rhodophyta)

In order to better understand the alternation of generations that characterizes haploid–diploid life cycles, we assessed the existence of ecological differences between the two phases (haploid gametophyte and diploid tetrasporophyte) in Gracilaria chilensis, a rhodophyte with a typical Polysiphonia-type life cycle. We investigated the effect of light intensity and salinity on viability and growth of both phases at different ontogenetic stages: juveniles and adults. In our study, the survival of juvenile gametophytes (n) was higher than the survival of juvenile tetrasporophytes (2n) despite culture conditions; however, low salinity had greater effect on carpospores (2n) than on tetraspores (n). On the other hand, a complex interaction between salinity and light intensity within each life history phase generated observed differences between juvenile growth rates. Low light was shown to trigger early onset of alteration of the holdfast growing pattern. In addition, adult tetrasporophytes showed, despite the conditions, a faster vegetative growth than female and male gametophytes. These differences between phases could have led to the complete dominance of tetrasporophyte fragments of fronds observed in G. chilensis farms. We hypothesize that Chilean fishers could have unknowingly selected for tetrasporophyte thalli during domestication of the species, thus enhancing the natural trend of tetrasporophytes dominance already present in estuarine natural populations of free-floating plants.     

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.     

Characterization of industrial strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> exhibiting filamentous growth induced by alcohols and nutrient deprivation

Summary The use of microorganisms in biotechnology is an important economic area of interest in Brazil, especially the use of Saccharomyces cerevisiae in the baking and alcohol fermentation industries. Dimorphism in S. cerevisiae (cell morphology alterations from budding cells to filamentous structures) has been observed in conditions of nitrogen and carbon deprivation and in the presence of fusel alcohols. This can be described as a defense mechanism that allows the yeast to forage for nutrients through cell elongation, hyphal formation and invasive growth. In this work fifteen industrial strains of S. cerevisiae (including haploid and diploid strains) isolated from the fermentative process for alcohol production were characterized for filamentation on solid culture media under growth conditions of carbon- and nitrogen-deprivation and in the presence of fusel alcohols. The majority of strains showed filamentation induced by isoamyl alcohol, butanol, isopropanol and isobutanol, but not by methanol. In rich medium (YEPD), both haploid and diploid strains showed invasive growth, although this kind of filamentous growth was more common in haploid strains. Similar results were observed when fructose or mannose was used as the sole carbon source. In nitrogen-deficient medium (SLAD) the strains did not filament. The results obtained indicate that the filamentation induced by higher alcohols and carbon deprivation (specially carbon) is a common process in industrial strains of S. cerevisiae contributing towards their maintenance/survival in adverse conditions.     

Higher flower bud formation in haploid tobacco is connected with higher peroxidase/IAA-oxidase activity,lower IAA content and ethylene production

Haploid tobacco plants (cv. Samsun) form inflorescences with a larger number of flowers than diploid plants. Leaves of haploid plants were shown to have lower free IAA level (by 40 %), higher peroxidase (by 160 %) and IAA-oxidase (by 70 %) activities and produce less ethylene (by 25 %) than leaves of corresponding diploid plants. The increase of peroxidase activity in haploids was due to the increase in the activity of the cathodic isozyme which is known to have high IAA-oxidase activity. It is proposed that higher peroxidase/IAA-oxidase activity in haploid plants may take part in IAA catabolism, at least duringin vitro culture of haploid explants. Lowered IAA level and ethylene production may then be directly correlated with a larger number of flower buds; as a higher IAA level is generally considered to act as a background inhibitor of flowering.     

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.     

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.     

Growth, peroxidase activity, and protein content in stem pith and callus tissues from haploid and diploid Nicotiana tabacum plants

Pith segments isolated from haploid and diploid Nicotiana tabacum cv. IAC-70 plants from five different heights in the stem, were cultured in vitro on MS-62 medium supplied with 0.5 mg/l IAA and 0.02 mg/l kinetin. Pith tissues of haploid plants showed greater growth potential than those of diploids; peroxidase activity was higher in the calluses of diploid tissues whereas protein content was higher in those of haploids. After three subcultures the growth pattern was inversed, as were the results for peroxidase activity and protein content. After the onset of culture, peroxidase activity in haploid explants dropped sharply, reaching values lower than in diploid tissues 40 d later, but after three subcultures these values were higher than those for diploid calluses. The results are discussed from the viewpoint of a possible relationship between greater polyploidization in haploid tissues on the one hand and growth decrease and peroxidase activity increase on the other.     

Haploidy from Hordeum interspecific crosses

Summary Hordeum arizonicum (2n=42) and H. lechleri (2n=42) were crossed with both H. bulbosum (2n=14 or 28) and H. vulgare (2n=14 or 28) and progeny plants were obtained through embryoculture. Crosses of arizonicum with diploid bulbosum invariably resulted in haploids (2n=21) of arizonicum, whereas arizonicum by tetraploid bulbosum or diploid vulgare crosses produced both hybrids and haploids of arizonicum. The lechleri by diploid bulbosum or diploid vulgare crosses resulted in haploids of lechleri, while lechleri by tetraploid bulbosum resulted in well differentiated embryos which failed to germinate.Hybrid embryos derived from the haploid producing crosses exhibit chromosome variability, suggesting that chromosome elimination leads to haploid formation.The results also indicate that the ratio of the parental genomes in the zygote is a critical factor which determines the chromosome elimination or stability in any cross combination. Furthermore, both arizonicum and lechleri appear to be of similar genetic strength in eliminating bulbosum and vulgare chromosomes. The possibility of stability factors in overcoming elimination and manipulation towards elimination are discussed.     

A review of the life history,reproduction and phenology of Gracilaria

The basic life history of the red alga Gracilaria is of the three-phase Polysiphonia type but a number of species show deviations. Plants can bear both gametangia and tetrasporangia, either on separate parts of the thallus or on the same. Explanations include the in situ germination of tetraspores (allowing gametophytic thalli to be epiphytic on tetrasporophytes), the coalescence of spores or developing discs (resulting in chimaeras), mitotic recombination during cell division in the mature diploid thallus (resulting in patches of diploid male and female cells on the tetrasporophyte), a mutation eliminating the repression of female expression allowing haploid male plants to be bisexual and initial failure of cell walls to form during the development of tetraspores. Polyploids can be produced from plants with diploid gametangia. The sexes and phases are usually morphologically identical but gametophytes or their parts may be smaller. The growth rates of the sexes may differ and diploid juveniles may survive better than haploid. Neither polyploidy nor hybridization results in superior growth. The sex ratio is probably 1:1 but females may appear to be more abundant. Diploid and haploid phases are usually either about equal or diploids predominate, often depending on the type of substratum. At high latitudes reproduction peaks in late summer whereas in the tropics it may be high all year. In temperate regions growth rate is fastest and biomass highest in late summer; in the tropics peak biomass is mainly in the winter. Spermatia are effective for only a few h. Spores vary in size around 25 \m, diploid ones usually being larger. Cystocarps or tetrasporangia in the field may not currently be releasing spores. In the laboratory spore release shows a diurnal rhythm, peaking during the night or day according to the species. All the above attributes are potentially important in planning and executing Gracilaria cultivation.     

Gametophyte-sporophyte coalescence in populations of the intertidal carrageenophyte <Emphasis Type="Italic">Mazzaella laminarioides</Emphasis> (Rhodophyta)

Mazzaella laminarioides has consistently been reported as a typical coalescent/clump species with a triphasic life history of the Polysiphonia-type in which the haploid gametophyte is the predominant phase with respect to the diploid sporophyte. Preliminary observations of intertidal populations revealed that, in some instances, cystocarpic and tetrasporic fronds emerged from the same clump (G-T clumps), implying a coalescent process of haploid and diploid thalli by fusion of their corresponding adjacent basal holdfasts. Population surveys at three sites in Coliumo Bay, central Chile, were carried out to characterize frond demography as well as to asses the frequency of gametophyte-tetrasporophyte (G-T) coalescence. Visual and resorcinol methods were employed to determine the phases of the fronds collected over central transects of 15 randomly sampled clumps. Coalescence of G-T clumps was infrequent, with gametophytes dominating over tetrasporophyte thalli.     

Isolated microspore culture of wheat (Triticum aestivum L.) in a defined media I. Effects of pretreatment,isolation methods,and hormones

Significant improvements were achieved in the production of haploid and doubled haploid plants from isolated microspore culture of wheat c.v. Chris on a defined media. Procedures found to be of benefit included: A 7-day pretreatment of anthers in 0.4M mannitol plus the macronutrients from FHG medium; the inclusion of 4.5 mg/liter abscisic acid in the pretreatment solution; the isolation of microspores from pretreated anthers by vortexing; and the use of phenylacetic acid (PAA) as the auxin source in MS medium. The best response was achieved with 4.0 mg/liter PAA in MS medium containing 90 g/liter maltose as the sugar source. Under these conditions, 68% of viable microspores underwent division, and an average of 93 embryos and 92 green plants were regenerated per 100 anthers used. The root-tip chromosome number and the fertility of 114 regenerating green plants revealed that 75% were completely fertile spontaneously doubled haploids.     

A simple wheat haploid and doubled haploid production system using anther culture

Summary Wheat (Triticum aestivum L.) haploids and doubled haploids have been used in breeding programs and genetic studies. Wheat haploids and doubled haploids via anther culture are usually produced by a multiple step culture procedure. We improved a wheat haploid and doubled haploid production system via anther culture in which plants are produced from microspore-derived embryos using one medium and one culture environment. In the improved protocol, tillers of donor plants were pretreated at 4°C for 1–2 wk before anthers were plated on a modified 85D12 basal medium with phenylacetic acid (PAA) and zeatin and cultured at 30°C with a 12-h daylength (43 μEs⁻¹m⁻²) in an incubator. Microspore-derived embryos developed in 2–3 wk and the plants were produced 3–4 wk after anther plating. In the improved system, as much as 53% of the anthers of Pavon 76 were responsive with multiple embryos. For plant regeneration, as many as 22 green and 25 albino plants were produced from 100 anthers. Sixty-five green plants were grown to maturity and 32 (49%) plants were fertile and produced seeds (indicating spontaneous chromosome doubling) while 33 plants did not produce seed. Of five Nebraska breeding lines tested using the protocol, NE96675 was very responsive and the other lines less so, indicating that the protocol is genotype-dependent.