Asexual Life Cycles Research Articles

Impact of Phytopythium vexans on Plant Health: Hosts, Symptoms, Detection, and Management

Abstract Phytopythium vexans is an emerging phytopathogenic oomycete reported to be causing diseases in plants from multiple genera and families. It is a soil and water-inhabiting oomycete with sexual and asexual life cycles present in the environment. Sexual stages produce oospores whereas asexual stages produce zoospores. This pathogen has been reported to be infecting crops in Africa, Asia, Europe, North America, Oceania, and South America. Mainly root and crown regions of numerous perennial woody plants, herbaceous and weedy annual cereal crops, forest plants, ornamental plants, and fruit trees are reported to be infected. Certain names for the symptoms of pathogen infections associated with the host have been used such as replant disease, decline, and sadness syndrome. Young plants that are vegetatively propagated if infected die off in severe cases whereas in the case of older plants, the roots and crown regions are mainly affected by this pathogen. Infections typically display symptoms of brown necrosis in the roots and stem, with a soft, watery rotting mass inside in case of heavily infected samples. Upon gentle pulling, the outer bark sloughs off, revealing the extent of the damage. It also causes damping-off of seedlings. The pathogen moves within water and hence has the potential to induce chains of disease outbreaks in nurseries and greenhouses. A saturated soil for longer periods of time favors tissue colonization and disease progress since the water-loving infectious zoospores of this pathogen can swim to distances that directly favor the pathogen. Higher nitrogen content in the soil, reduced availability of oxygen in the soil, and decreased ability of the plant to grow at lower temperatures created by water stagnation are additional epidemiological factors that favor the disease development. An integrated approach, which includes early detection of the pathogen using culture-based assay/molecular approaches, use of preventive and curative fungicides/biofungicides, and cultural practices, is recommended for the management of Phytopythium . Use of cover crops, soil solarization, crop rotation with non-hosts, and biofumigation to suppress or kill the pathogen are recommended. Testing of irrigation water for the presence of pathogens and treatment using a combination of different measures such as filtration plus UV, ozone, and heat is also recommended. Information © The Authors 2023

Ergot Alkaloids Contribute to the Pathogenic Potential of the Fungus Aspergillus leporis.

Opportunistically pathogenic fungi have varying potential to cause disease in animals. Factors contributing to their virulence include specialized metabolites, which in some cases evolved in contexts unrelated to pathogenesis. Specialized metabolites that increase fungal virulence in the model insect Galleria mellonella include the ergot alkaloids fumigaclavine C in Aspergillus fumigatus (syn. Neosartorya fumigata) and lysergic acid α-hydroxyethylamide (LAH) in the entomopathogen Metarhizium brunneum. Three species of Aspergillus recently found to accumulate high concentrations of LAH were investigated for their pathogenic potential in G. mellonella. Aspergillus leporis was most virulent, A. hancockii was intermediate, and A. homomorphus had very little pathogenic potential. Aspergillus leporis and A. hancockii emerged from and sporulated on dead insects, thus completing their asexual life cycles. Inoculation by injection resulted in more lethal infections than did topical inoculation, indicating that A. leporis and A. hancockii were preadapted for insect pathogenesis but lacked an effective means to breach the insect's cuticle. All three species accumulated LAH in infected insects, with A. leporis accumulating the most. Concentrations of LAH in A. leporis were similar to those observed in the entomopathogen M. brunneum. LAH was eliminated from A. leporis through a CRISPR/Cas9-based gene knockout, and the resulting strain had reduced virulence to G. mellonella. The data indicate that A. leporis and A. hancockii have considerable pathogenic potential and that LAH increases the virulence of A. leporis. IMPORTANCE Certain environmental fungi infect animals occasionally or conditionally, whereas others do not. Factors that affect the virulence of these opportunistically pathogenic fungi may have originally evolved to fill some other role for the fungus in its primary environmental niche. Among the factors that may improve the virulence of opportunistic fungi are specialized metabolites--chemicals that are not essential for basic life functions but provide producers with an advantage in particular environments or under specific conditions. Ergot alkaloids are a large family of fungal specialized metabolites that contaminate crops in agriculture and serve as the foundations of numerous pharmaceuticals. Our results show that two ergot alkaloid-producing fungi that were not previously known to be opportunistic pathogens can infect a model insect and that, in at least one of the species, an ergot alkaloid increases the virulence of the fungus.

Toxoplasma invasion delayed by TgERK7 eradication.

Toxoplasma gondii causes serious clinical toxoplasmosis in humans mostly due to its asexual life cycles, which can be artificially divided into five tightly coterminous stages. Any radical or delay for the stage will result in tremendous changes immediately behind. We previously demonstrated that TgERK7 is associated with the intracellular proliferation of T. gondii, but during the process, other stages before were not meanwhile determined. To further clarify the function of ERK7 gene in T. gondii, the complemental strain of ΔTgERK7 tachyzoites created previously was engineered via electric transfection with the recombinant pUC/Tgerk7 plasmid, named pUC/TgERK7 strain in this study, and was used together with ΔTgERK7 and wild-type GT1 strains to retrospect the phenotypic changes including invasion and attachment. The results showed that TgERK7 protein can be re-expressed in the ΔTgERK7 tachyzoites and eradication of this protein leads to significantly lower invasion of T. gondii at 1 h and 2 h post-infection (P < 0.05), which is the key factor causing the following slow intracellular proliferation, in comparison with wild-type GT1 and pUC/TgERK7 parasites; noteworthily, at other early time points including 15 min for attachment assay was no statistical difference (P > 0.05). The data suggested that ERK7 protein in T. gondii is an important virulence factor that participates in the invasion of this parasite.

Pathogenesis of ocular toxoplasmosis.

Ocular toxoplasmosis is a retinitis -almost always accompanied by vitritis and choroiditis- caused by intraocular infection with Toxoplasma gondii. Depending on retinal location, this condition may cause substantial vision impairment. T. gondii is an obligate intracellular protozoan parasite, with both sexual and asexual life cycles, and infection is typically contracted orally by consuming encysted bradyzoites in undercooked meat, or oocysts on unwashed garden produce or in contaminated water. Presently available anti-parasitic drugs cannot eliminate T. gondii from the body. In vitro studies using T. gondii tachyzoites, and human retinal cells and tissue have provided important insights into the pathogenesis of ocular toxoplasmosis. T. gondii may cross the vascular endothelium to access human retina by at least three routes: in leukocyte taxis; as a transmigrating tachyzoite; and after infecting endothelial cells. The parasite is capable of navigating the human neuroretina, gaining access to a range of cell populations. Retinal Müller glial cells are preferred initial host cells. T. gondii infection of the retinal pigment epithelial cells alters the secretion of growth factors and induces proliferation of adjacent uninfected epithelial cells. This increases susceptibility of the cells to parasite infection, and may be the basis of the characteristic hyperpigmented toxoplasmic retinal lesion. Infected epithelial cells also generate a vigorous immunologic response, and influence the activity of leukocytes that infiltrate the retina. A range of T. gondii genotypes are associated with human ocular toxoplasmosis, and individual immunogenetics -including polymorphisms in genes encoding innate immune receptors, human leukocyte antigens and cytokines- impacts the clinical manifestations. Research into basic pathogenic mechanisms of ocular toxoplasmosis highlights the importance of prevention and suggests new biological drug targets for established disease.

The Cancer Aneuploidy Paradox: In the Light of Evolution.

Aneuploidy should compromise cellular proliferation but paradoxically favours tumour progression and poor prognosis. Here, we consider this paradox in terms of our most recent observations of chemo/radio-resistant cells undergoing reversible polyploidy. The latter perform the segregation of two parental groups of end-to-end linked dyads by pseudo-mitosis creating tetraploid cells through a dysfunctional spindle. This is followed by autokaryogamy and a homologous pairing preceding a bi-looped endo-prophase. The associated RAD51 and DMC1/γ-H2AX double-strand break repair foci are tandemly situated on the AURKB/REC8/kinetochore doublets along replicated chromosome loops, indicative of recombination events. MOS-associated REC8-positive peri-nucleolar centromere cluster organises a monopolar spindle. The process is completed by reduction divisions (bi-polar or by radial cytotomy including pedogamic exchanges) and by the release of secondary cells and/or the formation of an embryoid. Together this process preserves genomic integrity and chromosome pairing, while tolerating aneuploidy by by-passing the mitotic spindle checkpoint. Concurrently, it reduces the chromosome number and facilitates recombination that decreases the mutation load of aneuploidy and lethality in the chemo-resistant tumour cells. This cancer life-cycle has parallels both within the cycling polyploidy of the asexual life cycles of ancient unicellular protists and cleavage embryos of early multicellulars, supporting the atavistic theory of cancer.

UV-B response is modulated by cell-type specific signaling pathway in multicellular green algae Volvox carteri

A fundamental question in biology is how multicellular organisms regulate cellular and physiological processes in response to environmental signals in a tissue/cell type-specific manner. Light is one such cue but little is known about its effect on molecular mechanisms underlying cell-type specific signaling. The Volvox genus presents a Germ-Soma differentiation that can be used to understand the genetic mechanisms of evolutionary transition from single-cell to multicellular organisms. Here we report the transcriptional analysis throughout both asexual and sexual life cycles of Volvox carteri in two different cell types under UV-B light irradiation. Our data show that VcUVR8-V1, the main splice variant of the VcUVR8 transcript, accumulates during initiation of cleavage division. Moreover, the transcript level of VcUVR8-V1 increases in response to the sex inducer. VcUVR8 expression seems to remain the same in both cell types, while VcCOP1, the interacting partner of VcUVR8, is expressed in a cell-type specific manner. Interestingly, illumination with low doses of UV-B leads to an increase of VcCOP1 transcript levels only in the somatic cells. Our results indicate that UV-B signaling pathway is differentially regulated between two cell types and environmental UV-B could be involved in cell-type specific regulation of developmental and physiological processes.

An unusual sexual stage in the alkalophilic ascomycete Sodiomyces alkalinus

Exploring life cycles of fungi is insightful for understanding their basic biology and can highlight their ecology. Here, we dissected the sexual and asexual life cycles of the obligate alkalophilic ascomycete Sodiomyces alkalinus that thrives at extremely high pH of soda lakes. S.alkalinus develops acremonium-type asexual sporulation, commonly found in ascomycetous fungi. However, the sexual stage was unusual, featuring very early lysis of asci which release young ascospores inside a fruit body long before its maturation. In a young fruit body, a slimy matrix which originates from the combined epiplasm of asci and united cytoplasm of the pseudoparenchymal cells, surrounds pooled maturing ascospores. Upon maturity, the ascospores are forcibly released through a crack in the fruit body, presumably due to an increased turgor pressure. These features of the sexual stage development resemble the ones found in unrelated marine fungi, indicating convergent evolution of the trait. We hypothesise these developmental features of S. alkalinus to be adaptive in the conditions of periodically inundated rims of soda lakes where the fungus thrives.

Molecular characterization of Penicillium oxalicum 6R,8R-linoleate diol synthase with new regiospecificity

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn886, Arg707, and Arg934, are catalytic importance and belong to the catalytic groove. Phe794 and Gln889 were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.

Taxonomic revision of Chloromonas nivalis (Volvocales, Chlorophyceae) strains, with the new description of two snow-inhabiting Chloromonas species.

Chloromonas nivalis (Volvocales, Chlorophyceae) is considered a cosmopolitan species of a snow-inhabiting microalga because cysts morphologically identifiable as zygotes of the species are distributed worldwide. However, recent molecular data demonstrated that field-collected cysts identified as the zygotes consist of multiple species. Recently, we demonstrated that species identification of snow-inhabiting Chloromonas species is possible based on light and electron microscopy of asexual life cycles in strains and molecular phylogenetic analyses. Vegetative cells without eyespots and of inverted-teardrop shape have been reported once in North American material of C. nivalis; however, strains with such vegetative cells in snow-inhabiting species of Chloromonas have not been examined taxonomically in detail. Here, we used light and transmission electron microscopy together with molecular analyses of multiple DNA sequences to examine several C. nivalis strains. The morphological data demonstrated that one North American strain could be identified as C. nivalis, whereas three other strains should be re-classified as C. hoshawii sp. nov. and C. remiasii sp. nov. based on vegetative cell morphology, the number of zoospores within the parental cell wall during asexual reproduction, and whether cell aggregates (resulting from repeated divisions of daughter cells retained within a parental cell wall) were observed in the culture. This taxonomic treatment was supported by multigene phylogeny and comparative molecular analyses that included a rapidly evolving DNA region. Our molecular phylogenetic analyses also demonstrated that the North American strain of C. nivalis was phylogenetically separated from the Austrian and Japanese specimens previously identified as C. nivalis based on zygote morphology.

A fluorogenic C. neoformans reporter strain with a robust expression of m-cherry expressed from a safe haven site in the genome

C. neoformans is an encapsulated fungal pathogen with defined asexual and sexual life cycles. Due to the availability of genetic and molecular tools for its manipulation, it has become a model organism for studies of fungal pathogens, even though it lacks a reliable system for maintaining DNA fragments as extrachromosomal plasmids. To compensate for this deficiency, we identified a genomic gene-free intergenic region where heterologous DNA could be inserted by homologous recombination without adverse effects on the phenotype of the recipient strain. Since such a site in the C. neoformans genome at a different location has been named previously as “safe haven”, we named this locus second safe haven site (SH2). Insertion of DNA into this site in the genome of the KN99 congenic strain pair caused minimal change in the growth of the engineered strain under a variety of in vitro and in vivo conditions. We exploited this ‘safe’ locus to create a genetically stable highly fluorescent strain expressing mCherry protein (KN99mCH); this strain closely resembled its wild-type parent (KN99α) in growth under a variety of in vitro stress conditions and in the expression of virulence traits. The efficiency of phagocytosis and the proliferation of KN99mCH inside human monocyte-derived macrophages were comparable to those of KN99α, and the engineered strain showed the expected organ dissemination after inoculation, although there was a slight reduction in virulence. The mCherry fluorescence allowed us to measure specific association of cryptococci with leukocytes in the lungs and mediastinal lymph nodes of infected animals and, for the first-time, to assess their live/dead status in vivo. These results highlight the utility of KN99mCH for elucidation of host-pathogen interactions in vivo. Finally, we generated drug-resistant KN99 strains of both mating types that are marked at the SH2 locus with a specific drug resistant gene cassette; these strains will facilitate the generation of mutant strains by mating.

Sexual conflict and the evolution of asexuality at low population densities.

Theories for the evolution of sex rarely include facultatively sexual reproduction. Sexual harassment by males is an underappreciated factor: it should at first sight increase the relative advantage of asexual reproduction by increasing the cost of sex. However, if the same females can perform either sexual or asexual life cycles, then females trying to reproduce asexually may not escape harassment. If resisting male harassment is costly, it might be beneficial for a female to accept a mating and undertake a sexual life cycle rather than 'insist' on an asexual one. We investigate the effects of sexual harassment on the maintenance of sex under different population densities. Our model shows that resisting matings pays off at low population densities, which leads to the complete extinction of males, and thus to the evolution of completely asexual populations. Facultative sex persists in a narrow range of slightly higher densities. At high densities, selection favours giving up resisting male mating attempts and thus sexual reproduction takes over. These interactions between the outcomes of sexual conflict and population density suggest an explanation for the rarity of facultative sex and also patterns of geographical parthenogenesis, where marginal environments with potentially low densities are associated with asexuality.

TgERK7 is involved in the intracellular proliferation of Toxoplasma gondii.

Toxoplasma gondii uses a unique mechanism to fulfill its asexual life cycles by which the parasite can infect all the warm-blooded animals including humans. Mitogen-activated protein kinase (MAPK) or extracellular signal-regulated kinase (ERK) pathway widely existed in eukaryotic cells mediates the conversion of environmental stimuli to intracellular events such as proliferation and differentiation. Their counterparts have been identified in Apicomplexan parasites such as ERK7 in T. gondii. To confirm whether the unique mechanism of T. gondii is relevant to MAPK/ERK member, we created a mutant (ΔTgERK7) in GT1 tachyzoites using double homologous recombination method. Our results of virulence evaluation showed 100% survival of all the ΔTgERK7-infected mice until 35days post-challenge compared to no survival in wild-type GT1-infected group (10.6 ± 0.34days). Furthermore, lower parasite loads were detected in the peritoneal fluid of ΔTgERK7-infected mice (P < 0.05). To ensure whether or not ERK7 gene knockout leads to the growth deficiency of T. gondii, the intracellular proliferation of ΔTgERK7 was also examined in vitro. Our data indicated that the proliferation of ΔTgERK7 parasites was significantly prolonged in comparison with wild-type GT1 tachyzoites (P < 0.05). Therefore, we concluded that TgERK7 is important for the intracellular proliferation of T. gondii, which further emphasized that MAPK/ERK derived from T. gondii participates in the regulation of the asexual life cycles to ensure the survival and reinfections of this parasite.

Transcriptional analysis of Volvox photoreceptors suggests the existence of different cell-type specific light-signaling pathways.

Photosynthetic organisms, e.g., plants including green algae, use a sophisticated light-sensing system, composed of primary photoreceptors and additional downstream signaling components, to monitor changes in the ambient light environment towards adjust their growth and development. Although a variety of cellular processes, e.g., initiation of cleavage division and final cellular differentiation, have been shown to be light-regulated in the green alga Volvox carteri, little is known about the underlying light perception and signaling pathways. This multicellular alga possesses at least 12 photoreceptors, i.e., one phototropin (VcPhot), four cryptochromes (VcCRYa, VcCRYp, VcCRYd1, and VcCRYd2), and seven members of rhodopsin-like photoreceptors (VR1, VChR1, VChR2, VcHKR1, VcHKR2, VcHKR3, and VcHKR4), which display distinct light-dependent chemical processes based on their protein architectures and associated chromophores. Gene expression analyses could show that the transcript levels of some of the photoreceptor genes (e.g., VChR1 and VcHKR1) accumulate during division cleavages, while others (e.g., VcCRYa, VcCRYp, and VcPhot) accumulate during final cellular differentiation. However, the pattern of transcript accumulation changes when the alga switches to the sexual development. Eight photoreceptor genes, e.g., VcPhot, VcCRYp, and VcHKR1, are highly expressed in the somatic cells, while only the animal-type rhodopsin VR1 was found to be highly expressed in the reproductive cells/embryos during both asexual and sexual life cycles. Moreover, accumulation of VChR1 and VcCRYa transcripts is more sensitive to light and changes in response to more than one light quality. Obviously, different regulatory mechanisms underlying gene expression control transcript accumulation of photoreceptors not only during development, but also in a cell-type specific way and in response to various external signals such as light quality. The transcriptional patterns described in this study show that Volvox photoreceptors are mostly expressed in a cell-type specific manner. This gives reason to believe that cell-type specific light-signaling pathways allow differential regulation of cellular and developmental processes in response to the environmental light cues.

Investigating the life-cycle and growth rate of Pediastrum boryanum and the implications for wastewater treatment high rate algal ponds

The colonial alga Pediastrum boryanum has beneficial characteristics for wastewater treatment High Rate Algal Ponds (HRAP) including high biomass productivity and settleability. Our previous work has shown that these characteristics are enhanced when a portion of gravity harvested algae is recycled back to the pond. To help understand the mechanisms behind the improved performance of P. boryanum dominated HRAP with algal recycling, this study investigated the life-cycle of P. boryanum. Experiments determined the exact timing and growth rate of P. boryanum life-cycle stages (‘juvenile’, ‘growth’ and ‘reproductive’) under four combinations of light and temperature (250 or 120 μMol/m2/s; 20 or 10 °C). Single juvenile 16-celled colonies were grown in microcosms on an inverted microscope and photographed every 15 min until reproduction ceased. Two asexual life-cycles and a rarely occurring sexual life-cycle were observed. The time required to achieve asexual reproductive maturity increased from 52 h (high light and temperature) to 307 h (low light and temperature), indicating that the minimum hydraulic retention time or mean cell residence time (MCRT) must be higher than these values to sustain a P. boryanum HRAP culture under ambient conditions. The net growth rate of a P. boryanum colony varied between life-cycle stages (growth > juvenile > reproductive). This suggests that the higher biomass productivity measured in HRAP with algal recycling could be due to both the increased MCRT and an increase in the net growth rate of the HRAP culture by ‘seeding’ with faster growing colonies.

Possible algal origin and life cycle of Ediacaran Doushantuo microfossils with dextral spiral structure

In Ediacaran shallow-water dolomites of the Doushantuo Formation (ca. 570 Ma) of southern China, scarce phosphatized microfossils consisting of clusters of coil-like spheroids calledSpiralicellula bulbiferaand co-occurring spherical forms with helically arranged holes namedHelicoforamina wenganicaare interpreted to belong to the same taxon because both have a similar relative abundance and both, uniquely in the assemblage, exhibit a consistent dextral spiral feature—the oldest known fossil examples of fixed asymmetry. Thus, we interpret them as different stages of sexual and asexual life cycles in which the spiral structure was maintained throughout most of the developmental phases. While they can be placed with the acritarchs, we suggest they are a chlorophycean green alga, and like many Ediacaran macrofossils, may represent an extinct clade. This is compatible with the shoal-water marine depositional environment in which they lived, as it would have favored photosynthetic organisms over others kinds of encysting non-metazoan protists. This setting militates against their interpretation as putative embryos which has been put forward for a variety of forms co-occurring in the microfossil assemblage. The multiple affinities of the strikingly diverse biota remain far from resolved, but algal origins warrant further consideration.

Estimating inbreeding coefficients from NGS data: Impact on genotype calling and allele frequency estimation

Most methods for next-generation sequencing (NGS) data analyses incorporate information regarding allele frequencies using the assumption of Hardy–Weinberg equilibrium (HWE) as a prior. However, many organisms including those that are domesticated, partially selfing, or with asexual life cycles show strong deviations from HWE. For such species, and specially for low-coverage data, it is necessary to obtain estimates of inbreeding coefficients (F) for each individual before calling genotypes. Here, we present two methods for estimating inbreeding coefficients from NGS data based on an expectation-maximization (EM) algorithm. We assess the impact of taking inbreeding into account when calling genotypes or estimating the site frequency spectrum (SFS), and demonstrate a marked increase in accuracy on low-coverage highly inbred samples. We demonstrate the applicability and efficacy of these methods in both simulated and real data sets.

Reproductive effort of Mastocarpus papillatus (Rhodophyta) along the California coast.

Species with sexual and asexual life cycles may exhibit intraspecific differences in reproductive effort. The spatial separation of sexual and asexual lineages, called geographic parthenogenesis, is common in plants, animals and algae. Mastocarpus papillatus is a well-documented case of geographic parthenogenesis in which sexuals dominate southern populations, asexuals dominate northern populations, while mixed populations occur throughout central California. We quantified abundances and reproductive effort of sexual and asexual fronds and tetrasporophytes at eight sites in California to test the hypotheses that (1) reduced sexual reproduction at higher latitudes and tidal heights explains the observed geographic parthenogenesis and (2) reproductive effort (spore production per blade area) declines with increasing latitude. Abundances of all phases varied site-specifically. However, there was no geographic pattern of reproductive effort of fronds. Reproductive effort of fronds was greater in 2006 than in 2007 and correlated with sea surface temperatures. Sexual fronds exhibited greater reproductive effort than did asexual fronds and sexual reproductive effort was also inversely correlated with local upwelling index. Tetrasporophytes showed greater reproductive effort in northern sites, but total supply of tetraspores per m2 was greatest in the middle of the sampling range where crusts were more abundant. There was no decline of reproductive effort at higher latitudes. Geographic patterns of fecundity of life stages do not explain geographic parthenogenesis in M. papillatus. Site-specific differences in viability among spores or established thalli of different life cycles may explain their respective geographic distributions, as the sexual and asexual life cycles responded differently to environmental variations.

Heterothallism inSaccharomyces cerevisiaeisolates from nature: effect ofHOlocus on the mode of reproduction

Understanding the evolution of sex and recombination, key factors in the evolution of life, is a major challenge in biology. Studies of reproduction strategies of natural populations are important to complement the theoretical and experimental models. Fungi with both sexual and asexual life cycles are an interesting system for understanding the evolution of sex. In a study of natural populations of yeast Saccharomyces cerevisiae, we found that the isolates are heterothallic, meaning their mating type is stable, while the general belief is that natural S. cerevisiae strains are homothallic (can undergo mating-type switching). Mating-type switching is a gene-conversion process initiated by a site-specific endonuclease HO; this process can be followed by mother-daughter mating. Heterothallic yeast can mate with unrelated haploids (amphimixis), or undergo mating between spores from the same tetrad (intratetrad mating, or automixis), but cannot undergo mother-daughter mating as homothallic yeasts can. Sequence analysis of HO gene in a panel of natural S. cerevisiae isolates revealed multiple mutations. Good correspondence was found in the comparison of population structure characterized using 19 microsatellite markers spread over eight chromosomes and the HO sequence. Experiments that tested whether the mating-type switching pathway upstream and downstream of HO is functional, together with the detected HO mutations, strongly suggest that loss of function of HO is the cause of heterothallism. Furthermore, our results support the hypothesis that clonal reproduction and intratetrad mating may predominate in natural yeast populations, while mother-daughter mating might not be as significant as was considered.

Diploidy and the selective advantage for sexual reproduction in unicellular organisms

This article develops mathematical models describing the evolutionary dynamics of both asexually and sexually reproducing populations of diploid unicellular organisms. The asexual and sexual life cycles are based on the asexual and sexual life cycles in Saccharomyces cerevisiae, Baker's yeast, which normally reproduces by asexual budding, but switches to sexual reproduction when stressed. The mathematical models consider three reproduction pathways: (1) Asexual reproduction, (2) self-fertilization, and (3) sexual reproduction. We also consider two forms of genome organization. In the first case, we assume that the genome consists of two multi-gene chromosomes, whereas in the second case, we consider the opposite extreme and assume that each gene defines a separate chromosome, which we call the multi-chromosome genome. These two cases are considered to explore the role that recombination has on the mutation-selection balance and the selective advantage of the various reproduction strategies. We assume that the purpose of diploidy is to provide redundancy, so that damage to a gene may be repaired using the other, presumably undamaged copy (a process known as homologous recombination repair). As a result, we assume that the fitness of the organism only depends on the number of homologous gene pairs that contain at least one functional copy of a given gene. If the organism has at least one functional copy of every gene in the genome, we assume a fitness of 1. In general, if the organism has l homologous pairs that lack a functional copy of the given gene, then the fitness of the organism is kappa(l). The kappa(l) are assumed to be monotonically decreasing, so that kappa(0) = 1 > kappa(1) > kappa(2) > cdots, three dots, centered > kappa(infinity) = 0. For nearly all of the reproduction strategies we consider, we find, in the limit of large N, that the mean fitness at mutation-selection balance is max{2e(-mu) - 1,0} where N is the number of genes in the haploid set of the genome, epsilon is the probability that a given DNA template strand of a given gene produces a mutated daughter during replication, and mu = Nepsilon. The only exception is the sexual reproduction pathway for the multi-chromosomed genome. Assuming a multiplicative fitness landscape where kappa(l) = alpha(l) for alpha in (0, 1), this strategy is found to have a mean fitness that exceeds the mean fitness of all the other strategies. Furthermore, while other reproduction strategies experience a total loss of viability due to the steady accumulation of deleterious mutations once mu exceeds [Formula: see text] no such transition occurs in the sexual pathway. Indeed, in the limit as alpha --> 1 for the multiplicative landscape, we can show that the mean fitness for the sexual pathway with the multi-chromosomed genome converges to e(-2mu), which is always positive. We explicitly allow for mitotic recombination in this study, which, in contrast to previous studies using different models, does not have any advantage over other asexual reproduction strategies. The results of this article provide a basis for understanding the selective advantage of the specific meiotic pathway that is employed by sexually reproducing organisms. The results of this article also suggest an explanation for why unicellular organisms such as Saccharomyces cerevisiae (Baker's yeast) switch to a sexual mode of reproduction when stressed. While the results of this article are based on modeling mutation-propagation in unicellular organisms, they nevertheless suggest that, in more complex organisms with significantly larger genomes, sex is necessary to prevent the loss of viability of a population due to genetic drift. Finally, and perhaps most importantly, the results of this article demonstrate a selective advantage for sexual reproduction with fewer and much less restrictive assumptions than those of previous studies.

Identification of PpoA from Aspergillus nidulans as a Fusion Protein of a Fatty Acid Heme Dioxygenase/Peroxidase and a Cytochrome P450

The homothallic ascomycete Aspergillus nidulans serves as model organism for filamentous fungi because of its ability to propagate with both asexual and sexual life cycles, and fatty acid-derived substances regulate the balance between both cycles. These so-called psi (precocious sexual inducer) factors are produced by psi factor-producing oxygenases (Ppo enzymes). Bioinformatic analysis predicted the presence of two different heme domains in Ppo proteins: in the N-terminal region, a fatty acid heme dioxygenase/peroxidase domain is predicted, whereas in the C-terminal region, a P450 heme thiolate domain is predicted. To analyze the reaction catalyzed by Ppo enzymes, PpoA was expressed in Escherichia coli as an active enzyme. The protein was purified by 62-fold and identified as a homotetrameric ferric heme protein that metabolizes mono- as well as polyunsaturated C(16) and C(18) fatty acids at pH approximately 7.25. The presence of thiolate-ligated heme was confirmed on the basis of sequence alignments and the appearance of a characteristic 450 nm CO-binding spectrum. Studies on its reaction mechanism revealed that PpoA uses different heme domains to catalyze two separate reactions. Within the heme peroxidase domain, linoleic acid is oxidized to (8R)-hydroperoxyoctadecadienoic acid by abstracting a H-atom from C-8 of the fatty acid, yielding a carbon-centered radical that reacts with molecular dioxygen. In the second reaction step, 8-hydroperoxyoctadecadienoic acid is isomerized within the P450 heme thiolate domain to 5,8-dihydroxyoctadecadienoic acid. We identify PpoA as a bifunctional P450 fusion protein that uses a previously unknown reaction mechanism for forming psi factors.