Accurate identification of pathogens and vectors is essential in epidemiological studies of mosquito-borne pathogens. However, the members of the communis and punctor complexes are difficult to distinguish because they are highly cryptic species, with little to no species-specific morphological characters. The objective of this thesis is to develop molecular tools, including RFLP and DNA barcoding using cytochrome oxidase I (COI), internal transcribed spacer 2 (ITS2) and the intron of ribosomal protein S12 (RPS12) to facilitate identification of the members of these two complexes in Manitoba. A distinct interspecific distance for COI was found between the members of the communis complex included here, and diagnostic RFLP profiles were developed for Oc. communis and Oc. churchillensis. Relatively low average interspecific genetic distances using COI, ITS2 and RPS12 were observed between the members of the punctor complex, indicates no discernable boundaries between these species based on DNA barcoding.

The thesis investigates members of the crustose and largely calcified red algal family Peyssonneliaceae through molecular analyses and anatomical and ultrastructural observations. Mitochondrial CO1 DNA barcoding was implemented, in combination with fine-scale anatomy, to recognise species boundaries and identify complexes of cryptic species. Nuclear and organellar DNA markers were employed to construct a multigene phylogeny for Vanuatu and southern Australian members of the family facilitating the recognition of two undescribed genera Annea and Incendia.

Necrophagous insects plays an important role, especially in forensic practice, especially in determining the time of death. This work was aimed to monitor necrophagous species of the Diptera order in selected localities of South Moravian Region - Rakvice and Sokolnice. Furthermore, these necrophagous species were identified with anatomical-morphological and molecular genetic methods, which have been compared to each other. For this purpose it was necessary to isolate a DNA segment and amplify cytochrome oxidase I (COI) gene by the PCR method and with further processing to obtain the sequence of selected individuals from the mitochondrial genome. The results of the experiment more coincided with morphological identification database BOLD outputs than with outputs from the BLAST database. Anatomical and morphological identification cannot always accurately determine the genus and species necrophags, the assesment is not dependent not only on the development stage of the insect and its condition, but also on the expertise of the determinator. Most accurater results are achieved with use the combination of anatomical and morphological and molecular-genetic methods of determination, which is confirmed by the results of this thesis.

Biodiversity is an important element to conserve life on earth. We can divide it into three main categories: gene diversity, species diversity and the complex ecosystem diversity. In the world there are an estimated 10 million plant and animal species, but only about 1.5 million are described. DNA barcoding is a molecular method which helps to identify species by comparing the sequences of mitochondrial or nuclear genes. Within lime hawk-moth (Mimas tiliae) species could exist four different subspecies. The aim of this thesis is to verify usability of the nuclear genes CAD and EF-1a by the DNA barcoding method and to compare these results with the analysis of mitochondrial COI gene.

Orientador: Claudio de Oliveira / Abstract: The coastal drainages of Southern and Southeastern Brazil are part of the Eastern basin, currently composed by different drainages. The large distribution area and variety of habitats along these drainages have a direct influence on the species diversity and their high level of endemism. However, this region has been suffered intense exploration and loss of habitat due to anthropic actions. The present study aimed to identify Operational Taxonomic Units (OTUs) of the Loricariidae and to delimit ecoregions through their distribution patterns. A total of 499 partial sequences of the mitochondrial COI gene were analyzed, belonging to 47 Loricariidae species. 58 OTUs were delimited along 31 drainages of Southeastern and Southern coastal revealing a previously unrecognized genetic diversity for some groups. The 31 drainages based on Parsimony Analysis of Endemicity (PAE), were divided in five groups, characterizing areas that are favorable to the delimitation of ecoregions. The Jacuí, Ribeira de Iguape and Paraíba do Sul rivers presented fairly exclusive faunas in relation to morphological and genetic patterns, being essential for preservation. The stream capture and paleoclimatic events are largely responsible for the distribution of the Loricariidae along the drainages. / Resumo: Os rios costeiros da região Sul e Sudeste do Brasil fazem parte do complexo hidrográfico da bacia do Leste, que abriga diferentes drenagens isoladas atualmente, entre as quais destacamse, na região Sul e Sudeste as do Paraíba do Sul, Ribeira de Iguape, Itajaí e Jacuí. A grande área de distribuição e variedade de habitats ao longo dessas drenagens têm influência direta na diversidade de espécies e no seu alto nível de endemismo. No entanto, essa região vem sofrendo intensa exploração e perda de habitas por ações antrópicas. Trabalhos de zoneamento dessa região vêm sendo realizados com o intuito de fornecer melhores dados para a sua preservação. Nesse sentido, o presente trabalho teve como objetivo identificar unidades taxonômicas operacionais (OTUs) da família Loricariidae e delimitar ecorregiões através de seus padrões de distribuição. Foram analisadas 499 sequências parciais do gene mitocondrial Citocromo c Oxidase subunidade I (COI), representantes de 47 espécies de Loricariidae e encontradas 58 OTUs distribuídas ao longo de 31 drenagens da região costeira Sul e Sudeste do Brasil, revelando uma diversidade genética antes não reconhecida para alguns grupos. As 31 drenagens, com base na Análise de Parcimônia de Endemismo (PAE), foram divididas em cinco grupos, caracterizando áreas propícias à delimitação de ecorregiões. As drenagens Jacuí, Ribeira de Iguape e Paraíba do Sul, apresentaram faunas bastante exclusivas em relação a padrões morfológicos e genéticos, sendo imprescin... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Made available in DSpace on 2018-08-01T20:28:07Z (GMT). No. of bitstreams: 1 tese_11108_Dissertação_Verônica Luiza S. Fialho.pdf: 825449 bytes, checksum: 700df7d34ebe1579f26a45eb08933658 (MD5) Previous issue date: 2017-03-09 / Plantas das espécies Pfaffia spp. são amplamente utilizadas em território nacional e são popularmente conhecidas como Ginseng-brasileiro. As espécies mais conhecidas são a Pfaffia glomeratae a Pfaffia paniculata. Há indicações de uso como tônico e revigorante geral, no tratamento de fadiga física, esgotamento mental, falta de memória, como auxiliar no tratamento de distúrbios circulatórios, dentre outros. Embora as duas espécies sejam utilizadas com os mesmos propósitos, deve-se considerar que a diferença química entre elas pode interferir nos espectros de ações farmacológicas e toxicológicas. A identificação morfológica das raízes é tarefa difícil sobretudo devido a sua forma de comercialização. Por isso, faz-se necessário o desenvolvimento de novas metodologias de identificação das espécies comercializadas. O objetivo principal deste trabalho foi identificar, em nível de espécie, amostras de Ginseng-brasileiro vendidos no mercado brasileiro, utilizando, para isso, o código de barras de DNA (DNA barcode). O DNA de 60 amostras comerciais foi extraído e os genes matK e rbcL foram amplificados por PCR e sequenciados. Além disso, amostras referência, morfologicamente identificadas, das duas espécies foram utilizadas para comparação e submetidas ao mesmo protocolo. Posteriormente, as amostras foram confrontadas com amostras referências e com o banco de dados BOLD (Barcode of Life Data Systems). Dentre as amostras referências, 90% amplificaram, tanto para matK quanto para rbcL. As amostras comerciais obtiveram taxa de amplificação de 95% para ambos os genes. Todas amostras foram sequenciadas. Encontrou-se uma diferença interespecífica entre P. glomerata e P. paniculata de 1,92% para matK e 1,90% para rbcL. Não foi encontrada variação intraespecífica. Das 58 amostras avaliadas, 67,24% foram identificadas, sendo 61,54% P. glomerata e 38,46% P. paniculata. O restante, 32,76% não foram identificadas conforme o rótulo, caracterizando adulteração. Desses, 22,41% não foram identificadas e 10,34% apresentaram substituição de espécies. O método de identificação por DNA bacode possibilitou a identificação, em nível de espécie, das amostras comercializadas como Ginseng brasileiro obtidas do mercado brasileiro, como P. glomerata e P. paniculata.

Seagrass, mangrove, saltmarshes and macroalgae - the coastal vegetated habitats, offer a promising nature-based solution to climate change mitigation, as they sequester carbon in their living biomass and in marine sediments. Estimation of the macrophyte organic carbon contribution to coastal sediments is key for understanding the sources of blue carbon sequestration, and for establishing adequate conservation strategies. Nevertheless,identification of marine macrophytes has been challenging and current estimations are uncertain. In this dissertation, time- and cost-efficient DNA-based methods were used to fingerprint marine macrophytes and estimate their contribution to the organic pool accumulated in blue carbon habitats. First, a suitable short-length DNA barcode from the universal 18S gene was chosen among six barcoding regions tested, as it successfully recovered degraded DNA from sediment samples and fingerprinted marine macrophyte taxa. Second, an experiment was performed to test whether the abundance of eDNA represents the content of organic carbon within the macrophytes; results supported this notion, indicating a positive correlation (R2 = 0.85) between eDNA and organic carbon. Third, using the chosen barcode, eDNA of marine macrophyte was identified from sediments of seagrass meadows and mangrove forests in the Arabian Red Sea, to further estimate contributions to the organic carbon pools. Estimations based on eDNA were compared against estimations of organic carbon based on stable isotope analyses from the same sediments; results from both methods were similar. In addition, this research provided the first quantitative evidence of the contribution of macroalgae to coastal and oceanic carbon pools. Hitherto, macroalgae have been ignored in blue carbon assessments because their fingerprinting was challenging and there was no evidence of their carbon export. The results of this dissertation demonstrate that eDNA offers an unprecedent taxonomic discrimination, and resolve the contribution of marine macrophytes to the organic pools in blue carbon sediments.

This thesis deals with selection and verification of suitable mitochondrial markers for species identification of Cepaea molluscs. Optimisation of genomic DNA extraction method, PCR amplification using the 12S and 16S mitochondrial markers and automated sequencing of PCR products were performed. Data was evaluated using SeqScape Software v2.7, Sequence Scanner Software v1.0, MEGA7 programs and BLAST database. The aim of this thesis is to determinate whether the mitochondrial markers 12S and 16S are suitable for DNA barcoding of Cepaea.

Subterranean arthropod communities are important components of North American ecosystems, contributing numerous ecosystem services and essential food-web functions. Despite this, fundamental information about species diversity in these communities remains unknown, and their taxonomic composition and ecological diversity have scarcely been assessed. Subterranean pitfall traps are a commonly used method for sampling endogean and hypogean soil habitats in Europe but have never been widely implemented in North America. Here, I employed this method to sample subterranean arthropod communities in the Ridge and Valley and Blue Ridge ecoregions of Virginia, USA in the winter and spring. In total, 2,260 arthropod specimens were collected constituting 319 distinct species. I extracted and purified DNA and amplified the mitochondrial gene: cytochrome C oxidase subunit I (COI) from each recovered morphospecies and derived a unique COI barcode for each species sequenced. Objective sequence clustering was used to establish molecular operational taxonomic units (mOTUs) for downstream diversity analyses and establishment of dynamic identification resources. Total species richness and average species richness per site were assessed and compared for both regions and seasons. The Shannon-Wiener diversity index, Hutcheson's t-test, and effective numbers of species (ENS) were employed to compare regional subterranean arthropod diversity. The richness, Shannon-Wiener, and ENS comparisons indicated that both ecoregions encompass highly diverse subterranean arthropod communities with those of the Ridge and Valley being significantly more diverse than those of the Blue Ridge. / Master of Science in Life Sciences / Arthropods are invertebrate animals with hard exoskeletons, segmented bodies, and jointed paired appendages, and include insects, arachnids, crustaceans, and myriapods. Arthropods make up the majority of animal species on the planet and are important parts of ecosystems, making it important for researchers to study them. While we know a fair amount about North American arthropods that live above-ground, relatively little is known about those that live deep within the soil and underlying rock substrate. Subterranean pitfall traps are a common method used for collecting arthropods from subterranean habitats in various regions of the world but have rarely been used in the USA. As a result, much of the life beneath our feet may be unknown. I used these traps to collect subterranean arthropods in the Ridge and Valley and Blue Ridge regions of the Appalachian Mountains. A total of 2,260 individual arthropods were collected, belonging to 319 different species. I used DNA sequencing to establish a unique fingerprint-like "barcode" for each species. These barcodes serve as helpful identification resources, and will help name new species in the future. They also allowed me to measure the number of species (diversity) of subterranean arthropods collected from each region, and compare the two to determine which region is more diverse. I used common statistical metrics of diversity including species richness (number of species) and the Shannon-Wiener diversity index to compare regional subterranean arthropod diversity. My results show that both the Ridge and Valley and Blue Ridge regions of Virginia, USA are home to highly diverse subterranean arthropod communities and those of the Ridge and Valley are significantly more diverse than those of the Blue Ridge.

Mushrooms and insects are both integral components of ecosystems worldwide. Interactions between these two groups of organisms are particularly interesting to study due to the transient nature of mushroom fruiting bodies, which form for a short period of time when environmental conditions are optimal to allow the fungus to distribute its spores. Despite this unpredictability in where and when mushrooms will grow, a variety of insect species use mushrooms as a food source and a substrate on which to lay their eggs. Interactions between these two groups of organisms have been documented extensively in Europe. However, little is known about the diversity of insects that associate with mushrooms in Canada. In this study, 1,017 mushrooms were collected from forests in Hamilton and the Tillsonburg, Ontario regions between fall 2018 and fall 2019. Mushrooms and their associated insects were identified through DNA barcoding using sequences of the nuclear ITS region and the mitochondrial CO1 gene for the mushrooms and insects, respectively. In total, more than 100 insect species from at least 35 families and five orders were identified from the approximately 200 mushroom species collected. While some insect species displayed evidence of specificity in their choice of mushroom host, the larger network of associations was moderately generalized and many insect species inhabited mushrooms from multiple families and orders. This study highlights the incredible diversity of organisms that rely on mushrooms for survival and contributes to our overall understanding of mushroom-insect associations in this region of Southern Ontario. / Thesis / Master of Science (MSc) / Mushrooms and insects are both highly important organisms within ecosystems around the world. Mushrooms play key roles in breaking down organic matter within forests and contributing to plant health, while insects are important decomposers and pollinators. This project involved collecting over 1,000 mushrooms from two regions in Southern Ontario and identifying the insect species found living within these mushrooms in order to examine patterns in the interactions. To our knowledge, this is the first time a survey of mushroom-insect associations has been conducted in Canada. The collected mushrooms were inhabited by a diverse range of insect species, which included mostly flies and beetles, and each of the main sampling locations had their own unique mushroom and insect communities. Some insect species displayed preference in their choice of mushroom host. Overall, this study contributes greatly to our current understanding of mushroom and insect diversity in this part of Canada.