Induction of Phase Variation Events in the Life Cycle of the Marine Coccolithophorid Emiliania huxleyi

Emiliania huxleyi is a unicellular marine alga that is considered to be the world's major producer of calcite. The life cycle of this alga is complex and is distinguished by its ability to synthesize exquisitely sculptured calcium carbonate cell coverings known as coccoliths. These structures have been targeted by materials scientists for applications relating to the chemistry of biomedical materials, robust membranes for high-temperature separation technology, lightweight ceramics, and semiconductor design. To date, however, the molecular and biochemical events controlling coccolith production have not been determined. In addition, little is known about the life cycle of E. huxleyi and the environmental and physiological signals triggering phase switching between the diploid and haploid life cycle stages. We have developed laboratory methods for inducing phase variation between the haploid (S-cell) and diploid (C-cell) life cycle stages of E. huxleyi. Plating E. huxleyi C cells on solid media was shown to induce phase switching from the C-cell to the S-cell life cycle stage, the latter of which has been maintained for over 2 years under these conditions. Pure cultures of S cells were obtained for the first time. Laboratory conditions for inducing phase switching from the haploid stage to the diploid stage were also established. Regeneration of the C-cell stage from pure cultures of S cells followed a predictable pattern involving formation of large aggregations of S cells and the subsequent production of cultures consisting predominantly of diploid C cells. These results demonstrate the ability to manipulate the life cycle of E. huxleyi under controlled laboratory conditions, providing us with powerful tools for the development of genetic techniques for analysis of coccolithogenesis and for investigating the complex life cycle of this important marine alga.     

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.     

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.     

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.     

PLOIDY ANALYSIS OF THE TWO MOTILE FORMS OF CHRYSOCHROMULINA POLYLEPIS (PRYMNESIOPHYCEAE)1

In some cultures of the flagellate Chrysochromulina polylepis Manton et Parke, established from cells isolated from the massive bloom in Skagerrak and Kattegat in 1988, we observed, two motile cell types. They were termed authentic and alternate cells and differed with respect to scale morphology. To investigate whether or not the two cell forms were joined in a sexual life cycle, the relative DNA content per cell and relative size of cells of several clonal cultures of C. polylepis were determined by flow cytometry. Percentages of authentic and alternate cells in the cultures were estimated by transmission electron microscopy. Pure authentic cultures (α) contained cells with the lowest level of DNA and were termed haploid. Two pure alternate cultures (β) contained cells with double the DNA content of authentic cells and were termed diploid. Other pure alternate cultures contained haploid cells only, or both haploid and diploid cells. Three cell types were observed, each capable of vegetative propagation: authentic haploid, alternate haploid, and alternate diploid cells. Both the haploid and diploid alternate cells were larger than the haploid authentic cells. Cultures containing diploid cells appeared unstable: cell type ratio and ploidy ratio changed during the experiment where this cell type was present, particularly when grown in continuous light. In contrast, cultures with only haploid cells remained unchanged at all growth conditions tested. Light condition may influence cell type ratio and ploidy ratio. Our attempt to induce syngamy by mixing different authentic haploid clones did not result in mating. Assuming that the authentic and alternate cell types are of the same species, the life cycle of C. polylepis includes three flagellated scale-covered cell forms. Two of the cell types are haploid and may function as gametes, and the third is diploid, possibly being the result of syngamy.     

Parasexually produced hybrids between female and male plants of Griffithsia tenuis C. Agardh,a red alga

Somatic cell fusion between vegetative cells of a male and a female isolate of Griffithsia tenuis, a marine red alga, has been obtained. Hybrid cells have been isolated and they have regenerated new plants. Almost all these hybrid plants made reproductive structures. In nearly half these cases the first 3–10 cells of the hybrid filament produced reproductive structures chracteristic of the tetrasporic (diploid) phase rather than the sexual (haploid) phase of the life cycle of this alga. However as these filaments continued to grow, cells further along the filament began to produce sexual, either female or male, reproductive structures. The observations suggest that the production of tetrasporangial branches does not require the fusion of male and female nucleic; rather, male and female nucleic remaining separate, act in concert to produce these structures, and in subsequent cell divisions the nuclei of one sex may be left behind allowing the nuclei of the remaining sex to direct the production of sexual branches.     

Selection and the Evolution of Genetic Life Cycles

The evolution of haploid and diploid phases of the life cycle is investigated theoretically, using a model where the relative length of haploid and diploid phases is under genetic control. The model assumes that selection occurs in both phases and that fitness in each phase is a function of the time spent in that phase. The equilibrium and stability conditions that allow for all-haploid, all-diploid, or polyphasic life cycles are considered for general survivorship functions. Types of stable life cycles possible depend on the form of the viability selection. If mortality rates are constant, either haploidy or diploidy is the only stable life cycle possible. Departures from constant mortality can give qualitatively different results. For example, when survivorship in each phase is a linear, decreasing function of the time spent in the phase, stable haploid, diploid or polyphasic life cycles are possible. The addition of genetic variation at a coevolving viability locus does not qualitatively affect the outcome with respect to the maintenance of polyphasic cycles but can lead to situations where more than one life cycle is concurrently stable. These results show that trade-offs between the advantages of being diploid and of being haploid may help explain the patterns of life cycles found in nature and that the type of selection may be critical to determining the results.     

The sensitivity of Emiliania huxleyi (Prymnesiophyceae) to ultraviolet-b radiation

Emiliania huxleyi (Lohm.) Hay et Miller is an important component of the phytoplankton in open ocean waters. The sensitivity of this cosmopolitan alga to natural levels of UVB radiation has never been tested. Since DNA is believed to be a major target of natural UVB radiation (UVBR: 280–315 nm) in living cells, experiments with E. huxleyi were performed using growth rate reduction and DNA damage as indicators of UVBR stress. Specific growth rate, cell volume, pigment content, and CPD (cyclobutane pyrimidine dimer) formation (a measure for DNA damage) were followed during and after prolonged exposure of a series of cultures to a range of UVBR levels. E. huxleyi was found to be very sensitive to UVBR: at a daily weighted UVBR dose of only 400 J·m⁻² ·d⁻¹ (BED_DNA300nm), growth was halted. At this UVBR level, both cell volume and contents of the major photosynthetic and photoprotective pigments had increased. The UVBR vulnerability of E. huxleyi cannot be explained by a high potential for cyclobutane thymine dimer formation (the most abundant CPD type) due to a high T content of nuclear DNA: the CG content of this E. huxleyi strain is high (68%) compared with other species. The high UVBR sensitivity may be related to the stage of the cell cycle during UVBR exposure, in combination with low repair capacity. It is concluded that E. huxleyi may experience UVBR stress through the formation of cyclobutane pyrimidine dimers, with subsequent low repair capacity and thereby arrest of the cell cycle.     

Heterozygote Advantage and the Evolution of a Dominant Diploid Phase

The life cycle of eukaryotic, sexual species is divided into haploid and diploid phases. In multicellular animals and seed plants, the diploid phase is dominant, and the haploid phase is reduced to one, or a very few cells, which are dependent on the diploid form. In other eukaryotic species, however, the haploid phase may dominate or the phases may be equally developed. Even though an alternation between haploid and diploid forms is fundamental to sexual reproduction in eukaryotes, relatively little is known about the evolutionary forces that influence the dominance of haploidy or diploidy. An obvious genetic factor that might result in selection for a dominant diploid phase is heterozygote advantage, since only the diploid phase can be heterozygous. In this paper, I analyze a model designed to determine whether heterozygote advantage could lead to the evolution of a dominant diploid phase. The main result is that heterozygote advantage can lead to an increase in the dominance of the diploid phase, but only if the diploid phase is already sufficiently dominant. Because the diploid phase is unlikely to be increased in organisms that are primarily haploid, I conclude that heterozygote advantage is not a sufficient explanation of the dominance of the diploid phase in higher plants and animals.     

Transcriptome analysis of functional differentiation between haploid and diploid cells of Emiliania huxleyi, a globally significant photosynthetic calcifying cell

Background  

Eukaryotes are classified as either haplontic, diplontic, or haplo-diplontic, depending on which ploidy levels undergo mitotic cell division in the life cycle. Emiliania huxleyi is one of the most abundant phytoplankton species in the ocean, playing an important role in global carbon fluxes, and represents haptophytes, an enigmatic group of unicellular organisms that diverged early in eukaryotic evolution. This species is haplo-diplontic. Little is known about the haploid cells, but they have been hypothesized to allow persistence of the species between the yearly blooms of diploid cells. We sequenced over 38,000 expressed sequence tags from haploid and diploid E. huxleyi normalized cDNA libraries to identify genes involved in important processes specific to each life phase (2N calcification or 1N motility), and to better understand the haploid phase of this prominent haplo-diplontic organism.     

The roles of the Chantransia phase of Lemanea (Lemaneaceae,Batrachospermales, Rhodophyta) and of the ‘Mushroom’ phase of Himanthalia (Himanthaliaceae,Fucales, Phaeophyta)

Summary

The life cycle of the Batrachospermales (freshwater florideophyte red such as Batrachospermum, Lemanea) is a shortened variant of the ‘normal’ marine florideophyte life cycle. The perennial Chantransia diploid phase is microscopic and encrusting. Each winter it produces one or more semi-erect haploid gametophytes by vegetative meiosis. Gamete production and fertilization is followed by production of diploid carposporophytes; these produce diploid carpospores which disperse, and regenerate the Chantransia phase. The question of the extent to which the Chantransia phase contributes resources to the gametophyte was approached by physiological-hydrodynamic modelling. These computations suggest that the photosynthetic rates in situ on an area basis are 20 times greater for the gametophyte than the Chantransia phase; this agrees with the observed ratios of peak biomass. The conclusion is that the Chantransia phase has a negligible role in provisioning the growing gametophyte, and that the role of the Chantransia phase is to occupy space with living biomass throughout the year, including exposure at summer drawdown, and (perhaps) by dispersal via production of monospores. A similar conclusion is arrived at on the basis of biomass data for the role of the perennial ‘mushroom’ phase of the semelparous marine Fucalean brown alga Himanthalia elongata in relation to the short-lived but much larger reproductive receptacles.     

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.     

TESTING THE EFFECTS OF ELEVATED PCO2 ON COCCOLITHOPHORES (PRYMNESIOPHYCEAE): COMPARISON BETWEEN HAPLOID AND DIPLOID LIFE STAGES1

The response of Emiliania huxleyi (Lohmann) W. W. Hay et H. Mohler, Calcidiscus leptoporus (G. Murray et V. H. Blackman) J. Schiller, and Syracosphaera pulchra Lohmann to elevated partial pressure of carbon dioxide (pCO₂) was investigated in batch cultures. We reported on the response of both haploid and diploid life stages of these three species. Growth rate, cell size, particulate inorganic carbon (PIC), and particulate organic carbon (POC) of both life stages were measured at two different pCO₂ (400 and 760 parts per million [ppm]), and their organic and inorganic carbon production were calculated. The two life stages within the same species generally exhibited a similar response to elevated pCO₂, the response of the haploid stage being often more pronounced than that of the diploid stage. The growth rate was consistently higher at elevated pCO₂, but the response of other processes varied among species. Calcification rate of C. leptoporus and of S. pulchra did not change at elevated pCO₂, whereas it increased in E. huxleyi. POC production and cell size of both life stages of S. pulchra and of the haploid stage of E. huxleyi markedly decreased at elevated pCO₂. It remained unaltered in the diploid stage of E. huxleyi and C. leptoporus and increased in the haploid stage of the latter. The PIC:POC ratio increased in E. huxleyi and was constant in C. leptoporus and S. pulchra. Elevated pCO₂ has a significant effect on these three coccolithophore species, the haploid stage being more sensitive. This effect must be taken into account when predicting the fate of coccolithophores in the future ocean.     

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.     

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.     

Corn Smut Dikaryon in Culture

A TYPICAL smut life cycle has three phases—diploid, haploid and dikaryon¹. Diploid spores (teliospores) formed in the host tissue are a resting phase. They undergo meiosis at germination to form haploid vegetative cells which are usually yeast-like. The dikaryon is the pathogenic phase and is made up of cells with two haploid nuclei. It is initiated by the fusion of two compatible non-pathogenic haploid cells and the formation of an infection hypha by the fusion product.     

The life cycle of the amoeboid alga Synchroma grande (Synchromophyceae, Heterokontophyta)--highly adapted yet equally equipped for rapid diversification in benthic habitats

Synchroma grande (Synchromophyceae, Heterokontophyta) is a marine amoeboid alga, which was isolated from a benthic habitat. This species has sessile cell stages (amoeboid cells with lorica and cysts) and non‐sessile cell stages (migrating and floating amoebae) during its life cycle. The different cell types and their transitions within the life cycle are described, as are their putative functions. Cell proliferation was observed only in cells attached to the substrate but not in free‐floating or migrating cells. We also characterised the phagotrophy of the meroplasmodium in comparison to other amoeboid algae and the formation of the lorica. The functional adaptations of S. grande during its life cycle were compared to the cell stages of other amoeboid algae of the red and green chloroplast lineages. S. grande was found to be highly adapted to the benthic habitat. One sexual and two asexual reproductive strategies (haplo‐diploid life cycle) support the ability of this species to achieve rapid diversification and high adaptivity in its natural habitat.     

Investigations on the cell cycle of haploid and diploid tissue cultures of Datura innoxia Mill. and its synchronization

Using a ¹⁴C/³H double-labelling technique, the influence of kinetic on the length of the cell cycle of meristematic cells in haploid and diploid callus cultures of Datura innoxia was determined. The total length of the cell cycle of haploid cells as compared to that of diploid cells was reduced by 2.3 h (-kinetin) or 1.4 h (+kinetin). Furthermore, the addition of kinetin to the nutrient solution also reduces cell cycle duration at both ploidy levels. For synchronization of the cell cycle, a fluorodesoxyuridine/thymidine system was successfully employed. Apparently, the reduction of total cell cycle duration of cycling cells due to treatment with kinetin occurred at the expense of the G₁phase. Nevertheless, kinetin seems to exert an influence on the transition of cells from the G₂ into the M phase as well.Abbreviations FUdR fluorodeoxyuridine - HU hydroxyurea - IAA nidole acetic acid