Interploidal hybridization and mating patterns in the Sphagnum subsecundum complex

Polyploidization is thought to result in instant sympatric speciation, but several cases of hybrid zones between one of the parental species and its polyploid derivative have been documented. Previous work showed that diploid Sphagnum lescurii is an allopolyploid derived from the haploids S. lescurii (maternal progenitor) and S. subsecundum (paternal progenitor). Here, we report the results from analyses of a population where allodiploid and haploid S. lescurii co-occur and produce sporophytes. We tested (i) whether haploids and diploids form hybrid triploid sporophytes; (ii) how hybrid and nonhybrid sporophytes compare in fitness; (iii) whether hybrid sporophytes form viable spores; (iv) the ploidy of any viable gametophyte offspring from hybrid sporophytes; (v) the relative viability of sporelings derived from hybrid and nonhybrid sporophytes; and (vi) if interploidal hybridization results in introgression between the allopolyploid and its haploid progenitor. We found that triploid hybrid sporophytes do occur and are larger than nonhybrid sporophytes, but exhibit very low germination percentages and produce sporelings that develop more slowly than those from nonhybrid sporophytes. All sporophytes attached to haploid gametophytes were triploid and were sired by diploid males, but all sporophytes attached to diploid gametophytes were tetraploid. This asymmetric pattern of interploidal hybridization is related to an absence of haploid male gametophytes in the population. Surprisingly, all sporelings from triploid sporophytes were triploid, yet were genetically variable, suggesting some form of aberrant meiosis that warrants further study. There was limited (but some) evidence of introgression between allodiploid and haploid S. lescurii.     

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.     

Genetic structure and genealogy in the Sphagnum subsecundum complex (Sphagnaceae: Bryophyta)

Allopolyploidy is probably the most extensively studied mode of plant speciation and allopolyploid species appear to be common in the mosses (Bryophyta). The Sphagnum subsecundum complex includes species known to be gametophytically haploid or diploid, and it has been proposed that the diploids (i.e., with tetraploid sporophytes) are allopolyploids. Nucleotide sequence and microsatellite variation among haploids and diploids from Newfoundland and Scandinavia indicate that (1) the diploids exhibit fixed or nearly fixed heterozygosity at the majority of loci sampled, and are clearly allopolyploids, (2) diploids originated independently in North America and Europe, (3) the European diploids appear to have the haploid species, S. subsecundum, as the maternal parent based on shared chloroplast DNA haplotypes, (4) the North American diploids do not have the chloroplast DNA of any sampled haploid, (5) both North American and European diploids share nucleotide and microsatellite similarities with S. subsecundum, (6) the diploids harbor more nucleotide and microsatellite diversity than the haploids, and (7) diploids exhibit higher levels of linkage disequilibrium among microsatellite loci. An experiment demonstrates significant artifactual recombination between interspecific DNAs coamplified by PCR, which may be a complicating factor in the interpretation of sequence-based analyses of allopolyploids.     

Differences in response between haploid and diploid isomorphic phases of Gracilaria verrucosa (Rhodophyta: Gigartinales) exposed to artificial environmental conditions

This study tests the responses of juvenile gametophytes and tetrasporophytes (holdfast stage) of the isomorphic alga Gracilaria verrucosa under different environmental conditions.Estimations of survival and growth of holdfasts of haploid and diploid juvenile individuals were performed in natural sea-water and artificial culture medium, and under stringent conditions using lead as a toxin and ultra violet radiation as a mutagen. Results indicate that (i) holdfasts of haploid juveniles grow better than diploids in non-optimal medium conditions; (ii) holdfasts of diploid juveniles have a better tolerance to lead than haploids; and (iii) slight advantage of holdfasts of diploid juveniles grow better than haploids under U.V. radiation.     

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.     

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.     

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.     

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.     

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.     

Meiotic hybridogenesis in triploid Misgurnus loach derived from a clonal lineage

Triploid loaches Misgurnus anguillicaudatus are derived from unreduced diploid gametes produced by an asexual clonal lineage that normally undergoes gynogenetic reproduction. Here, we have investigated the reproductive system of two types of triploids: the first type carried maternally inherited clonal diploid genomes and a paternally inherited haploid genome from the same population; the second type had the same clonal diploid genomes but a haploid genome from another, genetically divergent population. The germinal vesicles of oocytes from triploid females (3n=75) contained only 25 bivalents, that is, 50 chromosomes. Flow cytometry revealed that the majority of the progeny resulting from fertilization of eggs from triploid females with normal haploid sperm were diploid. This indicates that triploid females mainly produced haploid eggs. Microsatellite analyses of the diploid progeny of triploid females showed that one allele of the clonal genotype was not transmitted to haploid eggs. Moreover, the identity of the eliminated allele differed between the two types of triploids. Our results demonstrate that there is preferential pairing of homologous chromosomes as well as the elimination of unmatched chromosomes in the course of haploid egg formation, that is, meiotic hybridogenesis. Two distinct genomes in the clone suggest its hybrid origin.     

THE OCCURRENCE AND SIGNIFICANCE OF EPISTATIC VARIANCE FOR QUANTITATIVE CHARACTERS AND ITS MEASUREMENT IN HAPLOIDS

Epistatic genetic variance for quantitative traits may play an important role in evolution, but detecting epistasis in diploid organisms is difficult and requires complex breeding programs and very large sample sizes. We develop a model for detecting epistasis in organisms with a free-living haploid stage in their life cycles. We show that epistasis is indicated by greater variance among families of haploid progeny derived from individual diploids than among clonally replicated haploid sibs from the same sporophyte. Simulations show that the power to detect epistasis is linearly related to the number of sporophytes and the number of haploids per sporophyte in the dataset. We illustrate the model with data from growth variation among gametophytes of the moss, Ceratodon purpureus. The experiment failed to detect epistatic variance for biomass production, although there was evidence of additive variance.     

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

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

Mutational Load and the Transition between Diploidy and Haploidy in Experimental Populations of the Yeast Saccharomyces cerevisiae

Mutations were accumulated over hundreds of generations in a mutator strain of yeast in a constant laboratory environment. This ensured that mutations were frequent and that the quality of environment remained unchanged. Mutations were accumulated in asexual populations of diploids but their impact on fitness was tested both for the diploid clones and for haploid clones derived from them. Dozens of harmful and lethal mutations accumulated in diploids, but important phenotypic traits, such as maximum growth rate, did not deteriorate by more than 10%. There were no signs of decline in population size. In strong contrast, the populations of haploids derived from the diploids suffered from high mortality; their density was reduced by more than three orders of magnitude. These findings indicate how ineffective natural selection can be in removing deleterious mutations from populations of clonally reproducing diploids. They also suggest that phenotypic assays of heterozygous diploids may be of little value as indicators of increasing genetic degeneration.     

Sexual conflict and the alternation of haploid and diploid generations

Land plants possess a multicellular diploid stage (sporophyte) that begins development while attached to a multicellular haploid progenitor (gametophyte). Although the closest algal relatives of land plants lack a multicellular sporophyte, they do produce a zygote that grows while attached to the maternal gametophyte. The diploid offspring shares one haploid set of genes with the haploid mother that supplies it with resources and a paternal haploid complement that is not shared with the mother. Sexual conflict can arise within the diploid offspring because the offspring's maternal genome will be transmitted in its entirety to all other sexual and asexual offspring that the mother may produce, but the offspring's paternally derived genes may be absent from these other offspring. Thus, the selective forces favouring the evolution of genomic imprinting may have been present from the origin of modern land plants. In bryophytes, where gametophytes are long-lived and capable of multiple bouts of asexual and sexual reproduction, we predict strong sexual conflict over allocation to sporophytes. Female gametophytes of pteridophytes produce a single sporophyte and often lack means of asexual reproduction. Therefore, sexual conflict is predicted to be attenuated. Finally, we explore similarities among models of mate choice, offspring choice and segregation distortion.     

Mating system and gene flow in the red seaweed Gracilaria gracilis: effect of haploid-diploid life history and intertidal rocky shore landscape on fine-scale genetic structure

The impact of haploid-diploidy and the intertidal landscape on a fine-scale genetic structure was explored in a red seaweed Gracilaria gracilis. The pattern of genetic structure was compared in haploid and diploid stages at a microgeographic scale (< 5 km): a total of 280 haploid and 296 diploid individuals located in six discrete, scattered rock pools were genotyped using seven microsatellite loci. Contrary to the theoretical expectation of predominantly endogamous mating systems in haploid-diploid organisms, G. gracilis showed a clearly allogamous mating system. Although within-population allele frequencies were similar between haploids and diploids, genetic differentiation among haploids was more than twice that of diploids, suggesting that there may be a lag between migration and (local) breeding due to the long generation times in G. gracilis. Weak, but significant, population differentiation was detected in both haploids and diploids and varied with landscape features, and not with geographic distance. Using an assignment test, we establish that effective migration rates varied according to height on the shore. In this intertidal species, biased spore dispersal may occur during the transport of spores and gametes at low tide when small streams flow from high- to lower-shore pools. The longevity of both haploid and diploid free-living stages and the long generation times typical of G. gracilis populations may promote the observed pattern of high genetic diversity within populations relative to that among populations.     

Genetic analysis of polyauxotrophy and early mitotic progeny of Saccharomyces cerevisiae zygotes. II. Spore-forming segregants in the mitotic and meiotic progeny of polyauxotrophic clones

This article continues the investigation of polyauxotrophic (PA) clones formed in early mitotic progeny of zygotes. Cloning and segregation analysis of PA progeny suggest an unusual state of diploid genome in these strains, which is expressed as elimination of the dominance effect of the wild allele and as suppression or conversion of either of two loci of mating type. In PA progeny, except for recombinant haploids, sporulating diploids and unstable clones were detected. The tetrad analysis of the diploids points to homozygotization for individual markers. Over-replication of diploid set of chromosomes, prior to meiosis, and replacement of the haploid nucleus (the product of meiosis) for the diploid nucleus may explain the appearance of sporulating segregants in the diploid meiotic progeny. Unstable segregants may be considered as heterokaryons with complex interaction of nuclei.     

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, 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.     

Spontaneous deletions and reciprocal translocations in Saccharomyces cerevisiae: influence of ploidy

Studying spontaneous chromosomal rearrangements throws light on the rules underlying the genome reshaping events occurring in eukaryotic cells, which are part of the evolutionary process. In Saccharomyces cerevisiae, translocation and deletion processes have been frequently described in haploids, but little is known so far about these processes at the diploid level. Here we investigated the nature and the frequency of the chromosomal rearrangements occurring at this ploidy level. Using a positive selection screen based on a particular mutated allele of the URA2 gene, spontaneous diploid revertants were selected and analysed. Surprisingly, the diploid state was found to be correlated with a decrease in chromosome rearrangement frequency, along with an increase in the complexity of the rearrangements occurring in the target gene. The presence of short DNA tandem repeat sequences seems to be a key requirement for deletion and reciprocal translocation processes to occur in diploids. After discussing the differences between the haploid and diploid levels, some mechanisms possibly involved in chromosome shortening and arm exchange are suggested.     

Homologous recombination between direct repeats of chromosomal segments comprising heterozygotic duplications in Escherichia coli

In conjugational matings between double mutants for the deo operon of Escherichia coli, haploid recombinants and extended tandem duplications deoC deoD/deoA deoB::Tn5 with the DeoC+DeoA+DeoB+DeoD- phenotype are formed (the deoD+ allele is not expressed due to the polar effect of the Tn5 insertion). Selection for the expression of the recessive deoC deoD alleles (in the thyA genome) leads to the segregation of haploid clones by duplications and also of clones that retain the diploidy but that are homozygous for deoC deoD. In addition to haploids, diploid clones retaining the duplications have also been found among the DeoD+ segregants. The phenotype of segregants retaining the duplication shows that they were formed by an unequal exchange between sister chromosomes. A comparison of segregation frequency of haploid and diploid DeoD+ clones in rec+ and recBC sbcB sbcC strains shows that duplications in the rec+ genome are more stable. On this basis, it is assumed that the RecBCD pathway possibly makes a greater contribution than the RecF pathway to the preservation of heterozygous duplications playing an important role in the evolution of prokaryotes.