The phylogenetic placement of the phylum Tardigrada among the Metazoa is somewhat uncertain. In analyses based on morphological characters, tardigrades are usually associated with arthropods but tardigrades have also been associated with a number of aschelminth phyla. We have sequenced the nearly complete 18S rRNA gene from a eutardigrade. Neighbor-Joining and Maximum-Parsimony analyses place tardigrades in a clade that includes arthropods and priapulids, but not other aschelminths.

A character data set suitable for cladistic analysis of tardigrades at the family level was developed. The data matrix consisted of 50 morphological characters from 15 families of tardigrades and was analyzed by maximum parsimony. Kinorhynchs, loriciferans and gastrotrichs were used as outgroups. The results agree with the currently accepted hypothesis that Eutardigrada and Heterotardigrada are distinct monophyletic groups. Among the eutardigrades, Eoyhypsibiidae was found to be a sister group to Macrobiotidae + Hypsibiidae, while Milnesiidae was the basal eutardigrade family. The basal heterotardigrade family was found to be Oreellidae. Echiniscoideans grouped with some traditional Arthrotardigrada (Renaudarctidae, Coronarctidae + Batillipedidae) suggesting that the arthrotardigrades are not monophyletic. An 18S rRNA phylogenetic hypothesis was developed and supports the monophyly of Heterotardigrada and of Parachela versus Apochela within the Eutardigrada. Mapping of habitat preference suggest that terrestrial tardigrades are the ancestral state. Molecular analysis of a sediment sample with an unusually large population of tardigrades had a higher diversity when compared to manual sorting and counting.

In the present study a character data set suitable for cladistic analysis at the family level was developed. A data matrix consisting of 50 morphological characters from 15 families of tardigrades was analyzed by maximum parsimony. Kinorhynchs, loriciferans, and gastrotrichs were used as outgroups. The results agree with the currently accepted hypothesis that Eutardigrada and Heterotardigrada are distinct monophyletic groups. Among the eutardigrades, Eohypsibiidae was found to be a sister group to Macrobiotidae + Hypsibiidae, while Milnesiidae was the basal eutardigrade family. The basal heterotardigrade family was found to be Oreellidae. Echiniscoideans grouped with some traditional Arthrotardigrada (Renaudarctidae, Coronarctidae + Batillipedidae) suggesting that the arthrotardigrades are not monophyletic. The 18S rRNA gene sequence of Batillipes mirus Richters, 1909 and Calohypsibius schusteri Nelson & McGlothlin, 1996 were obtained and their addition to a previously published dataset supports the monophyly of Heterotardigrada and of Parachela versus Apochela within the Eutardigrada.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第19960号 / 理博第4227号 / 新制||理||1607(附属図書館) / 33056 / 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 沼田 英治 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

No description available.

Apusozoa (Protozoa) is a phylum of heterotrophic gliding zooflagellates of unknown taxonomic affiliation, commonly observed in environmental samples. Almost nothing was previously known about the diversity and ecology of apusozoan species though, as bacterivores, they are probably important functional constituents within microbial assemblages. We explored apusozoan morphological and genetic diversity, ecology, and related methodological questions. By culturing environmental material from a range of habitats, we isolated and maintained monocultures of both previously described apusozoan orders, Apusomonadida (apusomonads) and Planomonadida (planomonads). For planomonads, we present a revised taxonomy based on morphology, ultrastructure, and 18S rDNA genetic differences. We describe nine new species and new genera Nutomonas and Fabomonas, and demonstrate ITS2 rDNA secondary structure analysis for species delineation. During our culturing effort, we also isolated two genotypes of a previously unknown flagellate group, shown here to belong to a novel third apusozoan order, Mantamonadida. We designed molecular probes specific to all three orders and applied them to environmental DNA, detecting novel 18S and ITS1 rDNA lineages in a range of habitats. We mined publically available metagenomic and metatranscriptomic sequence databases using 18S rDNA of described species as seeds, identifying hundreds of sequences with affinities to all three orders. Phylogenies featuring newly retrieved lineages with previously described species suggest that direct sequencing of transcriptomic material is more effective than amplification-dependent methods at detecting rare cells in mixed microbial assemblages. Finally, to test potential future applications of our newly isolated strains, we ran microcosm experiments examining the effect of protozoan (Cercozoa) grazing on the structure of bacterial assemblages, demonstrating that closely related and morphologically similar species can have different impacts on their prey base. Taken together, by combining traditional culturing and modern molecular methods, this thesis drastically improves our understanding of apusozoan diversity and sets the scene for future work using next-generation sequencing and ecologically driven functional experiments.

Os fungos micorrízicos arbusculares (FMAs, filo Glomeromycota) formam associações simbióticas com a maioria das plantas vasculares. Normalmente, as hifas dos FMAs crescem no solo e colonizam o interior das raízes. No entanto, não se sabe se as espécies mais abundantes detectadas no solo, por meio da identificação com base na morfologia dos esporos assexuais, são também as mais abundantes no interior das raízes, devido às dificuldades para a identificação dos FMAs com base nas estruturas intrarradiculares. Assim, o objetivo do presente trabalho foi avaliar a estrutura da comunidade de FMAs em cana-de-açúcar sob dois manejos de colheita por meio da identificação das espécies que estão no solo na forma de esporos assexuais e aquelas que estão nas raízes usando o sequenciamento de clones do gene rRNA 18S. Amostras de solo e raízes de cana-de-açúcar de três variedades e dois manejos de colheita: SEM QUEIMA prévia e COM QUEIMA prévia à colheita, foram coletadas em um experimento localizado no município de Novo Horizonte, SP. Foram utilizadas três abordagens para a identificação dos FMAs no interior das raízes: emprego de (1) iniciador específico para fungos em geral, (2) iniciador específico para FMAs e (3) iniciadores específicos para grupos de FMAs. O número de esporos por 50 g de solo, a riqueza de espécies observada e estimada e a diversidade de esporos não diferiram significativamente entre os manejos SEM QUEIMA e COM QUEIMA. Efeitos significativos de variedades de cana-de-açúcar ou na interação dos fatores manejo e variedade não foram observados. A análise de ordenação com base nos esporos identificados também não indicou separação das amostras em função dos tratamentos. Entretanto, plantas do tratamento sob manejo SEM QUEIMA apresentaram as maiores taxas de colonização micorrízica arbuscular, quando comparadas às plantas do tratamento sob manejo COM QUEIMA. Esses dados indicam que a taxa de colonização micorrízica arbuscular é um indicador mais sensível à mudança de manejo de colheita da cana-de-açúcar do que os outros indicadores avaliados. Após a extração de DNA das raízes, o uso dos iniciadores específicos para fungos em geral, para FMAs e iniciadores específicos para grupo de FMAs não resultou em sequências de Glomeromycota. Mesmo assim, a comunidade de fungos associados às raízes detectada por sequenciamento do gene rRNA 18S foi avaliada. Os resultados indicam que a estrutra da comunidade fúngica associada às raízes de cana-de-açúcar diferiu significativamente entre os manejos de colheita SEM QUEIMA e COM QUEIMA prévia, apesar de não haver diferenças na riqueza e índices de diversidade de unidades taxonômicas operacionais observadas. Em geral, estudos adicionais devem ser feitos para otimizar as condições para amplificação do gene rRNA 18S de FMAs para melhor entender a ecologia dos mesmos. / Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form mutualistic symbioses with most land plants. AMF hypha generally grow through the soil and colonize the cortical tissue of the plant roots. However, it is not known whether the most abundant species in the soil, determined based on the morphology of asexual spores are the most abundant inside the roots, due the difficulties in identifying AMF based on intraradical structures. Therefore, the aim of this study was to evaluate the AMF community structure in sugarcane rhizosphere and roots under two harvesting managements, based on spores in the soil and sequencing of 18S rRNA gene clones, respectively. Sugarcane rhizosphere soil and roots were sampled from three varieties, under two harvesting managements: without pre-harvesting burning and with pre-harvesting burning, at an experimental field located in Novo Horizonte (São Paulo, Brazil). Three approaches were used to identify AMF inside the roots: (1) using fungi-specific primers, (2) using AMF-specific primers and (3) using AMF group-specific primers. The number of spores in the soil, the observed and estimated species richness and the diversity of AMF spores in the treatments without and with pre-harvesting burning were not statistically different. Statistically significant effects of sugarcane varieties or the interaction of the factors Harvesting Management and Varieties were not observed. Ordination analysis based on the identified spores did not show clustering by treatments. However, intraradical root colonization rates were higher in the treatment without pre-harvesting burning, as compared to the treatment with pre-harvesting burning. These data indicate that intraradical colonization rate may be used as a more sensitive indicator of environmental changes due to harvesting management, as compared to the other indicators evaluated. The use of fungi-specific, AMF-specific and AMF group-specific primers did not allow the detection of Glomeromycota in the sugarcane roots sampled from the field experiment. Nonetheless, the fungal communities associated with sugarcane roots detected by 18S rRNA gene clone sequencing were evaluated. The results indicate that the fungal communities associated with sugarcane roots from the treatments without and with pre-harvesting burning were statistically different, even though no differences in operational taxonomic unit richness and diversity indices were observed. In general, additional studies are necessary to optimize AMF 18S rRNA gene amplification for a better understanding of their ecology.

Os fungos micorrízicos arbusculares (FMAs, filo Glomeromycota) formam associações simbióticas com a maioria das plantas vasculares. Normalmente, as hifas dos FMAs crescem no solo e colonizam o interior das raízes. No entanto, não se sabe se as espécies mais abundantes detectadas no solo, por meio da identificação com base na morfologia dos esporos assexuais, são também as mais abundantes no interior das raízes, devido às dificuldades para a identificação dos FMAs com base nas estruturas intrarradiculares. Assim, o objetivo do presente trabalho foi avaliar a estrutura da comunidade de FMAs em cana-de-açúcar sob dois manejos de colheita por meio da identificação das espécies que estão no solo na forma de esporos assexuais e aquelas que estão nas raízes usando o sequenciamento de clones do gene rRNA 18S. Amostras de solo e raízes de cana-de-açúcar de três variedades e dois manejos de colheita: SEM QUEIMA prévia e COM QUEIMA prévia à colheita, foram coletadas em um experimento localizado no município de Novo Horizonte, SP. Foram utilizadas três abordagens para a identificação dos FMAs no interior das raízes: emprego de (1) iniciador específico para fungos em geral, (2) iniciador específico para FMAs e (3) iniciadores específicos para grupos de FMAs. O número de esporos por 50 g de solo, a riqueza de espécies observada e estimada e a diversidade de esporos não diferiram significativamente entre os manejos SEM QUEIMA e COM QUEIMA. Efeitos significativos de variedades de cana-de-açúcar ou na interação dos fatores manejo e variedade não foram observados. A análise de ordenação com base nos esporos identificados também não indicou separação das amostras em função dos tratamentos. Entretanto, plantas do tratamento sob manejo SEM QUEIMA apresentaram as maiores taxas de colonização micorrízica arbuscular, quando comparadas às plantas do tratamento sob manejo COM QUEIMA. Esses dados indicam que a taxa de colonização micorrízica arbuscular é um indicador mais sensível à mudança de manejo de colheita da cana-de-açúcar do que os outros indicadores avaliados. Após a extração de DNA das raízes, o uso dos iniciadores específicos para fungos em geral, para FMAs e iniciadores específicos para grupo de FMAs não resultou em sequências de Glomeromycota. Mesmo assim, a comunidade de fungos associados às raízes detectada por sequenciamento do gene rRNA 18S foi avaliada. Os resultados indicam que a estrutra da comunidade fúngica associada às raízes de cana-de-açúcar diferiu significativamente entre os manejos de colheita SEM QUEIMA e COM QUEIMA prévia, apesar de não haver diferenças na riqueza e índices de diversidade de unidades taxonômicas operacionais observadas. Em geral, estudos adicionais devem ser feitos para otimizar as condições para amplificação do gene rRNA 18S de FMAs para melhor entender a ecologia dos mesmos. / Arbuscular mycorrhizal fungi (AMF, Glomeromycota) form mutualistic symbioses with most land plants. AMF hypha generally grow through the soil and colonize the cortical tissue of the plant roots. However, it is not known whether the most abundant species in the soil, determined based on the morphology of asexual spores are the most abundant inside the roots, due the difficulties in identifying AMF based on intraradical structures. Therefore, the aim of this study was to evaluate the AMF community structure in sugarcane rhizosphere and roots under two harvesting managements, based on spores in the soil and sequencing of 18S rRNA gene clones, respectively. Sugarcane rhizosphere soil and roots were sampled from three varieties, under two harvesting managements: without pre-harvesting burning and with pre-harvesting burning, at an experimental field located in Novo Horizonte (São Paulo, Brazil). Three approaches were used to identify AMF inside the roots: (1) using fungi-specific primers, (2) using AMF-specific primers and (3) using AMF group-specific primers. The number of spores in the soil, the observed and estimated species richness and the diversity of AMF spores in the treatments without and with pre-harvesting burning were not statistically different. Statistically significant effects of sugarcane varieties or the interaction of the factors Harvesting Management and Varieties were not observed. Ordination analysis based on the identified spores did not show clustering by treatments. However, intraradical root colonization rates were higher in the treatment without pre-harvesting burning, as compared to the treatment with pre-harvesting burning. These data indicate that intraradical colonization rate may be used as a more sensitive indicator of environmental changes due to harvesting management, as compared to the other indicators evaluated. The use of fungi-specific, AMF-specific and AMF group-specific primers did not allow the detection of Glomeromycota in the sugarcane roots sampled from the field experiment. Nonetheless, the fungal communities associated with sugarcane roots detected by 18S rRNA gene clone sequencing were evaluated. The results indicate that the fungal communities associated with sugarcane roots from the treatments without and with pre-harvesting burning were statistically different, even though no differences in operational taxonomic unit richness and diversity indices were observed. In general, additional studies are necessary to optimize AMF 18S rRNA gene amplification for a better understanding of their ecology.

A Molecular Approach to Assessing Meiofauna Diversity in Marine Sediments Heather C. Hamilton Abstract The purpose of this study was to determine if a molecular approach could be applied to calculating the diversity of meiofauna in marine sediments from two sites in Tampa Bay, FL, similar to the approach of McCaig et al, 1999 in calculating the diversity of microbes in pastureland soils. The approach includes extracting total DNA directly from the sediment and amplifying the 18S rRNA gene by PCR. Clone libraries from the 18S gene would be created for each site and 300 sequences from each clone library would be obtained. These sequences would then be phylogenetically analyzed and assigned to an OTU, from which diversity indices can be calculated. The phylogenetic analysis of the sequences from the two sites revealed that of the 102 OTUs assigned from the sequences, only 7 OTUs included sequences from both sites, while 93 OTUs contained sequences from one site or from the other. Thus the sites were phylogenetically different from each other. Shannon diversity indices calculated for each site showed a difference between the two sites and paralleled diversity indices for macrofauna data for each site collected by the Hillsborough County Environmental Protection Commission. Sequences from 30 OTUs were completely sequenced and identified by phylogenetic comparison with a metazoan reference alignment. A discrepancy between the sequence data and data collected from preserved samples taken at each site was evident upon analysis: roughly 60% of each preserved sample consisted of nematodes and 10% consisted of copepods, while roughly 30% of the identified OTUs consisted of copepods and 10% consisted of nematodes. This discrepancy could be explained if the OTUs that were not identified consisted of nematode sequences or if a primer bias were present in the PCR amplification such that the regions flanking the primer site in the nematode sequences inhibited primer annealing.

Botryococcus braunii (Chlorophyta, Botryococcaceae) is a colony-forming green microalga that produces large amounts of liquid hydrocarbons, which can be converted into transportation fuels. While B. braunii has been well studied for the chemistry of the hydrocarbon production, very little is known about the molecular biology of B. braunii. As such, this study developed both apparatus and techniques to culture B. braunii for use in the genetic and biochemical characterization. During genetic studies, the genome size was determined of a representative strain of each of the three races of B. braunii, A, B, and L, that are distinguished based on the type of hydrocarbon each produces. Flow cytometry analysis indicates that the A race, Yamanaka strain, of B. braunii has a genome size of 166.0 +/- 0.4 Mb, which is similar to the B race, Berkeley strain, with a genome size of 166 +/- 2.2 Mb, while the L race, Songkla Nakarin strain, has a substantially larger genome size at 211.3 +/- 1.7 Mb. Phylogenetic analysis with the nuclear small subunit (18S) rRNA and actin genes were used to classify multiple strains of A, B, and L races. These analyses suggest that the evolutionary relationship between B. braunii races is correlated with the type of liquid hydrocarbon they produce. Biochemical studies of B. braunii primarily focused on the B race, because it uniquely produces large amounts of botryococcenes that can be used as a fuel for internal combustion engines. C30 botryococcene is metabolized by methylation to generate intermediates of C31, C32, C33, and C34. Raman spectroscopy was used to characterize the structure of botryococcenes. The spectral region from 1600?1700 cm^-1 showed v(C=C) stretching bands specific for botryococcenes. Distinct botryococcene Raman bands at 1640 and 1647 cm^-1 were assigned to the stretching of the C=C bond in the botryococcene branch and the exomethylene C=C bonds produced by the methylations, respectively. A Raman band at 1670 cm^-1 was assigned to the backbone C=C bond stretching. Finally, confocal Raman microspectroscopy was used to map the presence and location of methylated botryococcenes within a living colony of B. braunii cells.