<p>Sexual reproduction in fungi is governed by a specialized genomic region called the mating-type locus (MAT). The ascomycetes, the largest phylum of fungi, primarily possess a bipolar mating system while the basidiomycetes, the second largest group, are mostly tetrapolar. The human fungal pathogen and basidiomycetous yeast Cryptococcus neoformans has evolved a bipolar mating system that encodes homeodomain (HD) and pheromone/receptor (P/R) genes. The MAT locus of C. neoformans is unusually large, spans greater than 100 kb, and encodes more than 20 genes. To understand how the pathogenic Cryptococcus species complex evolved this unique bipolar mating system, we investigated the evolution of MAT in closely and distantly related species and discovered an extant sexual cycle in Cryptococcus amylolentus. </p><p>Phylogenetic analysis using a six-gene multi-locus sequencing (MLS) approach identified the most closely related species to the pathogenic Cryptococcus species complex that are currently known. The two non-pathogenic sibling species, Tsuchiyaea wingfieldii and Cryptococcus amylolentus, and the more distantly related species Filobasidiella depauperata define the Filobasidiella clade. We also resolved the phylogeny of the species located in the sister clade, Kwoniella. A comprehensive tree dendrogram revealed that the 15 Tremellales species examined suggests a common saprobic ancestor. Moreover, the pathogenic Cryptococcus species have a saprobic origin but later emerged as pathogens. We further characterized the mating-type locus for T. wingfieldii and C. amylolentus by cloning and sequencing two unlinked genomic loci encoding the HD and P/R genes. Interestingly, linked and likely divergently transcribed homologs for SXI1 and SXI2 are present in T. wingfieldii and C. amylolentus, while the P/R alleles contain many genes also found in the MAT locus of the pathogenic Cryptococcus species. Also, hypothetical genes present in C. neoformans MAT are also MAT-linked in both species and indicate a possible translocation event between chromosomes 4 and 5 of C. neoformans. Our analysis of MAT in the sibling species indicates that T. wingfieldii is likely tetrapolar, and the C. amylolentus sequence comparison of the dimorphic SXI1 and SXI2 region and the pheromone receptor, STE3, suggests that C. amylolentus is also tetrapolar. The examination of MAT in these sibling species confirms the model for MAT evolution previously proposed in which this structure in C. neoformans and C. gattii evolved from an ancestral tetrapolar mating system. Moreover, the organization of MAT in these sibling species mirrors key aspects of the proposed intermediates in the evolution of MAT in the pathogenic Cryptococcus species, and for sex chromosomes in plants, animals, and alga in general. </p><p>We discovered an extant sexual cycle for C. amylolentus, a species previously thought to be asexual. Matings between two strains of opposite mating-types produce dikaryotic hyphae with fused clamp connections and uni- and bi-nucleate basidiospores. Genotyping of basidiospores using markers linked and unlinked to MAT revealed that genetic exchange (recombination) occurs during the sexual cycle of C. amylolentus, and it is likely that either aneuploids are generated during sex or more than one meiosis event occurs within each basidium. This is in contrast to C. neoformans, where only one meiotic event per basidium has been observed. Uniparental mitochondrial inheritance has also been observed in C. amylolentus progeny; similar to the pathogenic Cryptococcus species, mtDNA is inherited from the C. amylolentus MATa parent. Analysis of sex in C. amylolentus has provided insight into the mechanisms that phylogenetically related fungi employ in orchestrating sexual reproduction. </p><p>We also extended our analysis to include the distantly related tetrapolar basidiomycete Tremella mesenterica. We completed comparisons of MAT-specific genes between five strains of T. mesenterica and identified the regions that define its mating-type system. The HD locus is limited to the SXI1- and SXI2-like genes while the P/R locus is defined by STE3, STE12, STE20, and the pheromone gene, tremerogen a-13. Interestingly, many of the genes associated with the MAT locus of the pathogenic Cryptococcus species flank the HD and P/R locus and are not incorporated in MAT in T. mesenterica. The MAT region includes transposons and C. neoformans hypothetical genes also present in T. wingfieldii and C. amylolentus. The mating-type system in T. mesenterica reflects an ancestral intermediate in the evolution of the MAT locus in the pathogenic Cryptococcus species. In conclusion, this study provides an in-depth analysis on the structure, function, and evolution of an unusual mating-type locus with broader implications for the transitions in modes of sexual reproduction in fungi that impact gene flow in populations.</p> / Dissertation

Parasites are thought to have large effects on their host populations, driving genetic change, population density changes, speciation and be a major selective force maintaining sexual reproduction. Indirect signatures of parasite-mediated selection are common, but explicit examples of parasite-mediated selection in nature are lacking. In this thesis I examine parasite-mediated dynamics in a natural population of Daphnia magna that experiences an annual epidemic of the bacterial pathogen Pasteuria ramosa. I also test a novel hypothesis investigating the relationship between parasitism and the production of resting eggs. In chapter 2 a combined field study and laboratory infection experiment illustrates one of the best examples of parasite-mediated selection in a natural population, with Daphnia collected after a parasite epidemic having higher levels of parasite resistance than those collected before. This chapter also explored the relationship between parasitism and resting eggs, which are only produced during the sexual phase of reproduction. Daphnia that were reproducing sexually in the field prior to the parasite epidemic were more susceptible, supporting higher levels of parasite growth, than their asexual counterparts. This supports the idea that some genotypes invest in sex at the expense of parasite resistance. In chapter 3 I used molecular markers to investigate genotype frequency changes in the same population in relation to the parasite epidemic. The parasite epidemic was found to be associated with genetic change in the population, and a laboratory infection experiment revealed that the genotype most resistant to the parasite was also most common following the peak of the parasite epidemic. While chapter 2 explored a genetic relationship between susceptibility and resting eggs, chapter 4 explores whether crowding conditions, cues indicating parasite prevalence in the population, or direct exposure to parasite spores can induce resting egg production. I found that crowding conditions or parasite prevalence enhance levels of male and resting egg production, but patterns were entirely dependent on Daphnia genotypes. There was no indication that exposure to parasite spores affects levels of sexual reproduction.

Arbuscular mycorrhizal fungi (AMF) represent an ancient and critical symbiotic partner with the majority of land plants, understood to promote ecosystem productivity and biodiversity and are also important to ecologically sound land management practices. The fungus is thought to be over 400 million years old, and due to a lack of an observable sexual cycle, has been placed into a select group of eukaryotes called 'ancient asexuals', which seemingly defy evolutionary theory by persisting for an extended period of time in the absence of sexual reproduction. Recently however, molecular evidence has accumulated which may suggest AMF harbour a cryptic sexual cycle. In the first chapter of this thesis, entitled "Searching for clues of sexual reproduction in the genomes of arbuscular mycorrhizal fungi", I review evidence supporting this notion of a cryptic sexual cycle in AMF which includes: the presence of recombinational events, meiosis-specific genes as well as mating-specific transcription factors called SexM and SexP of the MATA_HMG protein family which are otherwise found only in the genomes of sexual fungi. In the second chapter, I present the main research of my MSc work where I used bioinformatic, population genetic, molecular and experimental approaches to build on this existing evidence of sexuality in AMF. These findings include the presence of a dramatically expanded family of MAT-HMG genes which are present in several isolates of the Rhizophagus irregularis and also harbour significant allele variation amongst these isolates, some of which resembles variation expected at MAT-genes in other fungi. Q-RT-PCR procedures revealed that at least some of these genes tend to increase in expression during crosses of R. irregularis isolates. We also uncovered the presence of a unique genomic region where at least three of these genes are located in tandem. Finally, several tests of recombination support the presence of intraisolate as well as interisolate recombination events occurring between these MAT-HMG genes.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv., 2007. / Härtill 5 uppsatser.

This thesis focused on the dynamics of the sexual reproduction of microcrustacean Daphnia (Cladocera, Anomopoda). Sex in Daphnia is induced environmentally after rounds of asexual parthenogenesis in favorable conditions. The onset of sexual reproduction and the occurrence of sexual individuals was investigated with respect to their distribution in the water column and clonal structure. The produced sexual eggs were genotyped and their density and distribution in sediment were analyzed. The impact of massive floods was investigated. The role of sexual reproduction in stable habitats and its relevance and importance for Daphnia communities is further discussed.

Como tentativa de elucidar os fatores ambientais que controlam a plasticidade fenotípica floral de Catasetum fimbriatum Lindley, iniciaram-se estudos fenológicos, vegetativos e anatômicos da espécie, através do acompanhamento de 45 indivíduos, sendo que trinta destes indivíduos foram coletados e posteriormente envasados, enquanto os demais foram mantidos e observados em seu ambiente natural, nas árvores do Campus ESALQ/USP, permitindo assim, traçar correlações ecológicas entre os três tratamentos instalados, com quinze indivíduos cada. O primeiro tratamento foi montado em casa de vegetação onde predominou intensa luminosidade, altas temperaturas e baixa umidade relativa do ar. O segundo tratamento foi submetido a baixa intensidade luminosa, menor temperatura e a uma umidade relativa do ar mais elevada em relação ao primeiro. O terceiro tratamento foi mantido nas condições naturais. Ao final do experimento, pôde-se constatar a grande adaptabilidade da espécie aos diferenciados ambientes, através da formação de ecótipos, os quais tornaram-se visíveis devido a diferenças apresentadas pelos individuos principalmente na quantidade de brotos. Quanto ao trimorfismo sexual, mesmo não tendo havido a ocorrência de flores diclinas femininas, o estudo anatômico constatou a presença de agregados celulares com características meristemáticas nos ovários e ginostêmios das flores, os quais, acredita-se, caso fossem estimulados pelo ambiente durante um período necessário para definirem a expressão sexual da espécie, possivelmente através de um aumento nos níveis endógenos de etileno, permitindo desta forma a formação de flores diclinas femininas e flores monoclinas. / As attempt of elucidating the environmental factors that they control the plasticity floral fenotipic of Catasetum fimbriatum Lindley, fenological, vegetative and anatomical studies of the species began, through of the accompaniment of 45 individuals. Thirty of these individuals were collected and put in the vases later. The other ones 15 already existed naturally in the trees of the Campus ESALQ/USP, allowing like this, to trace ecological correlations among the three installed treatments. The first fifteen individuals, they were conditioned at vegetation house where intense brightness prevailed, high temperatures and it lowers relative humidity of the air. Such treatment was entitled treatment 1. The other collected individuals that were put in the vase, and that they constituted the treatment 2, they were submitted the low luminous intensity, to a smaller temperature and a relative humidity of the highest air. The last fifteen individuals, presented the natural conditions of survival of the species, and they constituted in that way, the treatment 3. At the end of the experiment, the great adaptability could be verified of the species to the differentiated environments, through the ecotipes formation, which became visible due to differences presented mainly by the individuals in the amount of sprouts. With relationship to the sexual, same trimorfism not having had the occurrence of feminine diclinous flowers, the anatomical study verified the presence of cellular closters with meristematic characteristic in the ovaries and ginostemeus of the flowers, the ones which, If they were environmently stimulated by the period of necessary time, possibly through an increase in the levels etylene endogenous, they would interfere in the sexual expression of the species contributing to the appearance of feminine diclinous flowers and monoclinous flowers.

Arbuscular mycorrhizal fungi (AMF) represent an ancient and critical symbiotic partner with the majority of land plants, understood to promote ecosystem productivity and biodiversity and are also important to ecologically sound land management practices. The fungus is thought to be over 400 million years old, and due to a lack of an observable sexual cycle, has been placed into a select group of eukaryotes called 'ancient asexuals', which seemingly defy evolutionary theory by persisting for an extended period of time in the absence of sexual reproduction. Recently however, molecular evidence has accumulated which may suggest AMF harbour a cryptic sexual cycle. In the first chapter of this thesis, entitled "Searching for clues of sexual reproduction in the genomes of arbuscular mycorrhizal fungi", I review evidence supporting this notion of a cryptic sexual cycle in AMF which includes: the presence of recombinational events, meiosis-specific genes as well as mating-specific transcription factors called SexM and SexP of the MATA_HMG protein family which are otherwise found only in the genomes of sexual fungi. In the second chapter, I present the main research of my MSc work where I used bioinformatic, population genetic, molecular and experimental approaches to build on this existing evidence of sexuality in AMF. These findings include the presence of a dramatically expanded family of MAT-HMG genes which are present in several isolates of the Rhizophagus irregularis and also harbour significant allele variation amongst these isolates, some of which resembles variation expected at MAT-genes in other fungi. Q-RT-PCR procedures revealed that at least some of these genes tend to increase in expression during crosses of R. irregularis isolates. We also uncovered the presence of a unique genomic region where at least three of these genes are located in tandem. Finally, several tests of recombination support the presence of intraisolate as well as interisolate recombination events occurring between these MAT-HMG genes.

Current solutions of coral restoration rely mainly on fragmentation. Though a reliable technique, this asexual form of reproduction does not benefit the genetic diversity of the coral reef. With many global and local stressors threatening corals’ existence, the resiliency of corals to future ocean conditions depends highly on sexual reproduction to produce new genotypes. New technology allows coral spawning/larval release, larval settlement and rearing to be carried out in an aquarium system. Many of the techniques necessary to maintain coral recruits are well-established, however the effects of light intensity remain to be studied for these early life stages. Newly settled corals have been found on vertical surfaces and the undersides of ledges and crevices, suggesting full solar irradiance is detrimental to their health. Newly settled Porites astreoides and Agaricia agaricites recruits were placed under varying irradiance levels to test their survivorship, growth and pigmentation. In the first four weeks post-settlement, growth was significantly different between recruits under a PAR of 10 µmol quanta m-2 s-1 and 240 µmol quanta m-2 s-1. In a separate experiment, growth curves were significantly different between six different irradiance increase regimens in the first 14 weeks post-settlement. This study shows, for the first time, a definitive preference by newly settled coral recruits to lower intensity irradiance, devoid of ultraviolet radiation, in the first four weeks post-settlement, and that Porites astreoides recruits can acclimatize to higher intensities at a rate of ~ 11 µmol quanta m-2 s-1 per week for up to 15 weeks.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24455号 / 理博第4954号 / 新制||理||1707(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 教授 木庭 啓介, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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