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The epidemiology of blastomycosis remains poorly understood in part due to the lack of a robust and discriminatory strain typing method for Blastomyces dermatitidis. Here we describe the development of a multilocus sequence (MLST) method to study the genetic variation and population structure of B. dermatitidis. Eighty geographically diverse clinical and environmental isolates were examined. Thirty-six unique sequence types were identified. With a discriminatory index of 91.4%, MLST identifies significant genetic diversity for the characterization of local and global B. dermatitidis isolates. To test whether this fungus represented a single species throughout its geographic range we performed phylogenetic analyses, applying Genealogical Concordance Phylogenetic Species Recognition (GCPSR). Phylogenetic analysis revealed two distinct clades, with five of the eight gene phylogenies studied supporting the separation of these lineages, which were also geographically partitioned. Based on fulfillment of GCPSR, we propose the current species B. dermatitidis harbors two genetically distinct non-interbreeding phylogenetic species.

The epidemiology of blastomycosis remains poorly understood in part due to the lack of a robust and discriminatory strain typing method for Blastomyces dermatitidis. Here we describe the development of a multilocus sequence (MLST) method to study the genetic variation and population structure of B. dermatitidis. Eighty geographically diverse clinical and environmental isolates were examined. Thirty-six unique sequence types were identified. With a discriminatory index of 91.4%, MLST identifies significant genetic diversity for the characterization of local and global B. dermatitidis isolates. To test whether this fungus represented a single species throughout its geographic range we performed phylogenetic analyses, applying Genealogical Concordance Phylogenetic Species Recognition (GCPSR). Phylogenetic analysis revealed two distinct clades, with five of the eight gene phylogenies studied supporting the separation of these lineages, which were also geographically partitioned. Based on fulfillment of GCPSR, we propose the current species B. dermatitidis harbors two genetically distinct non-interbreeding phylogenetic species.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-22T12:28:36Z No. of bitstreams: 2 Dissertação - Laurine Lacerda Pigosso - 2012.pdf: 3120628 bytes, checksum: 05c24b721e04453fce0116ba2470c24a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-05-22T12:33:01Z (GMT) No. of bitstreams: 2 Dissertação - Laurine Lacerda Pigosso - 2012.pdf: 3120628 bytes, checksum: 05c24b721e04453fce0116ba2470c24a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-05-22T12:33:01Z (GMT). No. of bitstreams: 2 Dissertação - Laurine Lacerda Pigosso - 2012.pdf: 3120628 bytes, checksum: 05c24b721e04453fce0116ba2470c24a (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2012-07-31 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The genus Paracoccidioides comprises a complex of phylogenetic species of dimorphic pathogenic fungi, the etiologic agents of paracoccidioidomycosis (PCM), a disease confined to Latin America and of marked relevance in its endemic areas due to its high frequency and severity. The members of the Paracoccidioides genus are distributed in distinct phylogenetic species (S1, PS2, PS3 and 01-like) that potentially differ in their biochemical and molecular characteristics. In this work, we performed the proteomic characterization of different members of the genus Paracoccidioides. We compared the proteomic profiles of Pb01 (01-like), Pb2 (PS2), Pb339 (S1) and PbEPM83 (PS3) using 2D electrophoresis and mass spectrometry. The proteins/isoforms were selected based on the staining intensity of the spots as determined by image analysis. The proteins/isoforms were in-gel digested and identified by peptide mass fingerprinting and ion fragmentation. A total of 714 spots were detected, of which 343 were analyzed. From these spots, 301 represented differentially expressed proteins/isoforms among the four analyzed isolates, as determined by ANOVA. After applying the FDR correction, a total of 267 spots were determined to be differentially expressed. From the total, 193 proteins/isoforms were identified by PMF and confirmed by ion fragmentation. Comparing the expression profiles of the isolates, the proteins/isoforms that were related to glycolysis/gluconeogenesis and to alcohol fermentation were more abundant in Pb01 than in other representatives of the genus Paracoccidioides, indicating a higher use of anaerobic pathways for energy production. Those enzymes related to the oxidative stress response were more abundant in Pb01, Pb2 and Pb339, indicating a better response to ROS in these members of the Paracoccidioides complex. The enzymes of the pentose phosphate pathway were abundant in Pb2. Antigenic proteins, such as GP43 and a 27-kDa antigenic protein, were less abundant in Pb01 and Pb2. The proteomic profile indicates metabolic differences among the analyzed members of the Paracoccidioides genus. / O gênero Paracoccidioides compreende um complexo de espécies filogenéticas do fungo patogênico dimórfico, agente etiológico da paracoccidioidomicose (PCM), uma doença restrita à América Latina e de relevância acentuada em suas áreas endêmicas, devido à sua alta frequência e gravidade. Os membros do gênero Paracoccidioides são distribuídos em espécies filogenéticas distintas (S1, PS2, PS3 e 01-like) que diferem potencialmente nas suas características bioquímicas e moleculares. Neste trabalho, foi realizada a caracterização proteômica de diferentes membros do gênero Paracoccidioides. Foram comparados os perfis proteômicos de Pb01 (01-like), Pb2 (PS2), Pb339 (S1) e PbEPM83 (PS3) utilizando eletroforese 2D e espectrometria de massa. As proteínas / isoformas foram selecionados com base na intensidade de coloração dos spots conforme determinado por análise de imagem. As proteínas / isoformas foram excisadas do gel, digeridas e identificadas por PMF (Peptide mass fingerprinting) e fragmentação iônica. Um total de 714 spots foi detectado, 343 foram analisados. A partir destes spots, 301 apresentaram-se diferencialmente expressos entre os quatro isolados analisados, determinado por ANOVA. Depois de aplicar a correção FDR, um total de 267 spots foram diferencialmente expressos. Do total, 193 / isoformas proteínas foram identificadas por PMF e confirmadas por fragmentação iônica. Comparando-se os perfis de expressão dos isolados, as proteínas / isoformas que foram relacionados para a glicólise / gliconeogênese e fermentação alcoólica foram mais abundantes em Pb01 do que em outros representantes do gênero Paracoccidioides, indicando uma maior utilização das vias anaeróbias para a produção de energia. Enzimas relacionadas com a resposta ao estresse oxidativo foram mais abundantes em Pb01, Pb2 e Pb339, indicando uma melhor resposta às ROS nestes membros do complexo Paracoccidioides. As enzimas da via das pentoses foram abundantes em Pb2. Proteínas antigênicas, tal como GP43 e uma proteína antigênica de 27 kDa, foram menos abundantes em Pb01 e Pb2. O perfil proteômico indica diferenças metabólicas entre os membros analisados do gênero Paracoccidioides.

Species in the genus Calonectria (anamorph: Cylindrocladium) are euascomycetes in the order Hypocreales and are important pathogens of a wide range of plant hosts globally. At the outset, this thesis considers the literature pertaining to species of Calonectria and especially the importance of the biological, morphological and phylogenetic species concepts on the taxonomy of this group. It is clear that DNA sequence comparisons have revolutionised the taxonomy of Calonectria and literature also highlights the importance of a polyphasic approach to species identification. Studies in this thesis treat a number of forest nursery disease problems caused by Calonectria spp. and new species are consequently described based on DNA sequence comparisons, morphological characteristics and sexual compatibility tests. As a consequence several cryptic species were also identified in the genus. Therefore, a multigene genealogy was constructed for all Calonectriaspp. for which cultures were available and shown to group together in 13 subclades also supported by morphological similarities. As a consequence all Cylindrocladium spp. were circumscribed to the genus Calonectria, regardless whether the teleomorph state was present or not, based on new nomenclature regulations stated in Article 59. / Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / Unrestricted

Master of Science / Department of Plant Pathology / Robert L. Bowden / John F. Leslie / Fusarium graminearum Schwabe sensu lato (teleomorph: Gibberella zeae (Schwein.) Petch), a homothallic ascomycete fungus, is the causal agent of Fusarium head blight (FHB) of wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and other small grains. FHB occurs worldwide and serious outbreaks have been reported in North America, South America, Asia, and Europe. According to the phylogenetic species concept (PSC), F. graminearum is composed of at least 15 phylogenetic lineages known as the Fusarium graminearum species complex. Although F. graminearum is homothallic, some members of different phylogenetic lineages are known to intercross in the laboratory. It has been suggested that F. graminearum sensu lato fits the biological species concept (BSC). According to the BSC, “species are groups of actually or potentially interbreeding natural populations, which are reproductively isolated from other such groups”. Previous reports of intercrossing were qualitative, so the degree of reproductive isolation, if any, is not clear. Since intrinsic reproductive isolation is the key criterion to identify species by the BSC, more detailed quantitative information is needed. Chromosome rearrangements between fungal strains may reduce fertility in sexual crosses through the production of genetically inviable recombinant progeny. As such, rearrangements can be important postzygotic reproductive barriers between species. Following methods used in Neurospora crassa, ascospore tetrads were analyzed for patterns of ascospore viability. Crosses were made with three lineage 7 (F. graminearum sensu stricto according to PSC) strains as female. Each female was a MAT1-2 knockout mutant that rendered it obligately heterothallic. Males were several members of lineages 6 (F. asiaticum according to PSC) and lineage 7. Crosses with lineage 7 males formed complete asci with 8 ascospores indicating that their genomes are isosequential with the testers. Crosses with one strain from lineage 6 with two known inversions produced asci containing 8, 6, and 4 ascospores, consistent with it not being isosequential. However, three other strains of lineage 6 appeared to be isosequential with the testers. Therefore, chromosome rearrangements did not appear to be common to strains of lineage 6 and probably do not contribute significantly to reproductive isolation of lineage 6 and lineage 7. Interlineage fertility studies with the three lineage 7 tester strains were performed to quantify interlineage fertility parameters including the total number of ascospores produced, perithecial density, and perithecium internal development scores. All lineage 7 female testers successfully crossed to all 23 male strains from lineages 1 to 9. For total ascospore production, one female tester crossed equally well with all lineages and the other two testers showed statistically significant differences for a few lineages. For perithecial density, there was a significantly lower density with all three testers when crossed with lineage 6, but the other lineages were not statistically different from lineage 7. For perithecial development, there was large variation for every lineage. Therefore, in the crosses with reduced fertility, the reduction can be attributed to a postzygotic effect since mature perithecia and asci developed. All of the tested lineages of the Fusarium graminearum species complex can produce viable progeny with F. graminearum lineage 7, which was the taxonomic type of the original species before it was split into phylogenetic species. There are a few examples of reduced fertility with two lineage 7 testers, the remaining tester crossed equally well with all lineages. Therefore members of lineages 1-9 all should be considered members of Fusarium graminearum according to the BSC. The existing female testers could be used to identify members of the F. graminearum clade by performing test crosses in the laboratory. The PSC and BSC species concepts do not agree for this group of fungi. This disagreement indicates that the F. graminearum species complex is in the early stages of speciation. The lack of intrinsic reproductive barriers supports the hypothesis that these lineages have developed in geographic isolation. As the lineages have apparently been brought together through global trade, interlineage hybrids have been reported in the field. The discrepancy between PSC and BSC will eventually be resolved by whether the lineages fuse or remain separate in nature. Even if the lineages remain separate, this study demonstrates the potential for gene flow between lineage 7 and lineages 1 through 9.

Le genre Rumina Risso, 1926 (Subulinidae) est constitué de gastéropodes terrestres, hermaphrodites et capables de réaliser de l’autofécondation ainsi que de la fécondation croisée. Plusieurs espèces ont été décrites sur base de subtiles différences morphologiques telles que la forme et la taille de la coquille et la coloration du corps. Trois espèces sont actuellement toujours reconnue dans la littérature: Rumina decollata (Linnaeus, 1758), R. saharica Pallary, 1901 et R. paivae (Lowe 1861). Cependant, uniquement le statut spécifique de R. decollata et R. saharica a été confirmé par la morphologie de la coquille et l'anatomie génitale. L’objectif de la thèse est de clarifier la taxonomie du genre Rumina par une approche de taxonomie intégrative en associant des caractères moléculaires, morphologiques et anatomiques ainsi que les méthodes issues de la génétique des populations. Quatre gènes mitochondriaux et deux gènes nucléaires ont été séquencés pour reconstruire la phylogénie de Rumina. Les résultats confirment le statut d’espèce de R. saharica, excluent R. paivae en tant qu’espèce et proposent une nouvelle classification pour R. decollata en 6 espèces phylogénétiques. R. saharica est aussi confirmé en tant qu’espèce morphologique suite à une étude morphomètrique, cependant les nouvelles espèces de R. decollata n’ont pas pu être différenciées ni par les variables morphologiques testées, ni par des particularités dans l’anatomie génitale. Néanmoins, deux des espèces de R. decollata correspondent à deux morphotypes (clair et foncé) auparavant décrits et étudiés dans la région de Montpellier. L’étude de ces deux espèces phylogénétiques avec des microsatellites et allozymes permettent de confirmer le statut spécifique des deux morphotypes et révèlent que R. decollata pratique l’autofécondation croisée à un taux supérieur à celui rapporté dans la littérature, défiant ainsi la règle selon laquelle les hermaphrodites pratiqueraient exclusivement l’autofécondation ou exclusivement la fécondation croisée. L’espèce correspondante au morphotype foncé a été introduite en plusieurs endroits du monde et semble être l’espèce possédant la plus grande capacité invasive parmi les Rumina. Ainsi, nos résultats suggèrent que le genre Rumina, auparavant décrit comme étant composé de trois espèces, est en fait un complexe de sept espèces, qui doivent être davantage étudiées de façon à confirmer leur statut d’espèce par d’autres concepts d’espèce. / Rumina spp. Risso, 1826 (family Subulinidae) is a hermaphroditic terrestrial snail, performing both selfing and outcrossing. Several nominal species have been described based on subtle differences in the shape and size of the shell, and body coloration. Currently, three taxa are still recognized, viz. R. decollata (Linnaeus, 1758), R. saharica Pallary, 1901 and R. paivae (Lowe, 1860). Yet, species-specific differentiation has only been confirmed for R. decollata and R. saharica, based on shell and genital morphology. This work aims at resolving the taxonomy of the genus Rumina through an integrative taxonomic approach by combining molecular, morphological and anatomical characters, as well as population genetic methods. Four mitochondrial and two nuclear genes were used to infer Rumina’s phylogeny. Results suggest that R. saharica is a phylogentic species, R. paivae is not a phylogenetic species and R. decollata is composed by 6 phylogenetic species. The specific status of R. saharica was confirmed by a morphometric analysis, however the remaining phylogenetic species of R. decollata could not be differentiated neither by the shell characters analyzed nor by the genital anatomy. Nevertheless, two phylogenetic species of Rumina representing the dark and light colored strains previously described in the Montpellier region. The study of both these strains with microsatellites and allozymes confirmed their specific status and revealed that outcrossing might be more prevalent than was previously suggested in the literature, therefore defying the alleged rule that hermaphroditic species should be either strict self-fertilizers or strict outcrosser. The dark strain was introduced in several places through the world and seems to be the one with highest invasive character within the genus Rumina. Therefore, our results suggest that the genus Rumina, previously described as having three species, is in fact a complex of seven species that need to be further explored in order to confirm their species status under other species concepts. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished