During the last decade high throughput DNA sequencing of single cells has evolved from an idea to one of the most high profile fields of research. Much of this development has been possible due to the dramatic reduction in costs for massively parallel sequencing. The four papers included in this thesis describe or evaluate technological advancements for high throughput DNA sequencing of single cells and single molecules. As the sequencing technologies improve, more samples are analyzed in parallel. In paper 1, an automated procedure for preparation of samples prior to massively parallel sequencing is presented. The method has been applied to several projects and further development by others has enabled even higher sample throughputs. Amplification of single cell genomes is a prerequisite for sequence analysis. Paper 2 evaluates four commercially available kits for whole genome amplification of single cells. The results show that coverage of the genome differs significantly among the protocols and as expected this has impact on the downstream analysis. In Paper 3, single cell genotyping by exome sequencing is used to confirm the presence of fat cells derived from donated bone marrow within the recipients’ fat tissue. Close to hundred single cells were exome sequenced and a subset was validated by whole genome sequencing. In the last paper, a new method for phasing (i.e. determining the physical connection of variant alleles) is presented. The method barcodes amplicons from single molecules in emulsion droplets. The barcodes can then be used to determine which variants were present on the same original DNA molecule. The method is applied to two variable regions in the bacterial 16S gene in a metagenomic sample. Thus, two of the papers (1 and 4) present development of new methods for increasing the throughput and information content of data from massively parallel sequencing. Paper 2 evaluates and compares currently available methods and in paper 3, a biological question is answered using some of these tools. / <p>QC 20160127</p>

La famille des Phytoseiidae comprend des espèces d'intérêt agronomique dont certaines sont utilisées pour lutter contre des acariens ravageurs appartenant principalement aux familles des Tetranychidae et Eriophyidae. L'identification et la description des espèces de Phytoseiidae sont essentiellement basées sur des caractères morphologiques. Cependant, plusieurs problèmes d'identification sont rencontrés du fait : (1) de leur petite taille (faible nombre de caractères observables), (2) du manque de recul sur la valeur diagnostique de certains caractères dits discriminants, (3) et enfin du diagnostic fiable uniquement au stade femelle. Dans ce contexte, ce travail de thèse a pour objectif de déterminer dans quelle mesure l'approche moléculaire peut assister le diagnostic traditionnel. Pour cela, nous avons étudié l'étendue des variations intra- et interspécifiques (moléculaires et morphologiques) de sept espèces de Phytoseiidae d'intérêt agronomique, ceci pour comparer la capacité discriminante de quatre marqueurs moléculaires (12S ARNr, Cytb, COI et ITS). Dans ce travail, nous mettons en évidence l'utilité de ces marqueurs pour le diagnostic de tous les stades de développement et montrons l'importance de la forme du calyx de la spermathèque pour différencier deux espèces du genre Euseius, ce qui nous a permis de décrire une nouvelle espèce. Nous montrons également que les marqueurs mitochondriaux et nucléaires peuvent différencier des espèces morphologiquement proches. Cependant, il faut rester prudent quand aux conclusions tirées (espèces différentes ou populations différentes au sein d'une même espèce) sur la base des résultats obtenus avec les marqueurs mitochondriaux. Enfin, nous montrons que les règles de décision (valeurs seuils) sont difficiles à (1) établir avec ces mêmes marqueurs et (2) surtout à généraliser pour la différenciation des espèces appartenant aux deux sous-familles étudiées. Pour conclure, ce travail met en évidence de façon claire la nécessité d'une approche taxonomique intégrative, incluant un va-et-vient entre données moléculaires, morphologiques et écologiques, pour la garantie d'un diagnostic fiable des espèces de Phytoseiidae. / The family Phytoseiidae includes numerous species of huge interest for biological control of mite pests, essentially belonging to the families Tetranychidae and Eriophyidae. Identification and description of Phytoseiidae species are mainly based on morphological characters. However, taxonomists have to face several problems for species diagnostic because of : (1) their small size (low number of visible characters), (2) little information on the diagnostic value of some characters considered as discriminant, and at last (3) diagnostic only reliable at female stage. In this context, the present works aims to determine how molecular approaches can assist traditionnal diagnostic. For this, we studied the intra- and interspecific variations (morphological and molecular) of seven agronomic important species, in order to compare the ability of four molecular markers (12S rRNA, Cytb mtDNA, COI mtDNA et ITS) to discriminate between species. We first show that molecular markers could be of great help for differentiating species at all life stages. Moreover, we show the importance of the spermatheca calyx shape for separating two species of the genus Euseius, and a new specie was then described. We also emphasize that both mitochondrial and nuclear markers differentiate morphological close species. However, some cautions have to be paid on the conclusions developped (different species or different populations within a same species) when only mitochondrial markers are used. At last, we underline that decision rules (treshold cutt-off values) are difficult (1) to establish with these latter markers and also (2) to generalise for the accurate differentiation of the Phytoseiidae species belonging to the two sub-families considered. To conclude, this work clearly emphasizes the importance of integrative taxonomy, including an « up-and-down » analysis of molecular, morphological and ecological data, to secure the specific diagnostic among the family Phytoseiidae

Correct species identification is fundamental to all areas of biology, but particularly the policy related areas of conservation and fisheries management. To enable guidelines to be developed for environmental management and conservation, such identifications need links to studies of the evolutionary history, biological factors and environmental influences driving species divergence and population dynamics for the target species. This study concerns two genera of gadiform fish, Muraenolepis and Macrourus, found in southern temperate and Antarctic waters, with a single species, Macrourus berglax, present in the North Atlantic. With similar distribution patterns to toothfish species, Dissostichus eleginoides and D. mawsoni, they are a major food source and by-catch of the toothfish fishery. Both are slow growing and long lived, with different evolutionary histories, life expectancies and strategies for reproduction. For both genera, the accuracy of morphological keys, number of described species and their distribution is under debate. This study has identified specimens to species level using both morphological and genetic techniques, redefining the range for morphological features and taxonomic keys. For Muraenolepis, this has clarified confusion over Mu. marmoratus and Mu. microps being a single species, confirmed some mis-identification from sexual dimorphism and provided genetic evidence for the recently described species Mu. evseenkoi. For Macrourus, this work has identified a new species, now named Ma. caml, and found that Ma. holotrachys and Ma. berglax are genetically identical, raising the question of bipolar distribution or recent divergence. The low level of genetic variation within both species suggests a recent evolution and expansion into Antarctic waters. Similar geographic species limits imply common processes influencing divergence, with the oceanographic fronts as potential barriers. Further investigation of niche overlap and fine scale population structure are required to fully understand the processes driving speciation and provide the underlying data required for fisheries management.

This diploma thesis is focused on species identification of sand flies belonging to two genera of the subfamily Phlebotominae, genus Phlebotomus and Sergentomyia. Genus Phlebotomus together with the genus Lutzomyia of New World include the only proven vectors of Leishmania parasites and they are also carriers of viral and bacterial infections. Species of the genus Sergentomyia are proven vectors of sister genus Sauroleishmania that infects reptiles, but for several decades there have been speculations about their possible involvement in the transmission of mammalian Leishmania species. These suspicions arise mainly from repeated findings of mammalian Leishmania parasites in their digestive system. Correct species determination of medically significant hematophagous arthropods is very important especially for purposes of epidemiological studies so that efficient vector control may be correctly set. Routine identification of sand flies is based on morphological characters located mainly on their heads and genitalia. However, these characters may be variable within a species, they require certain expertise and in the field samples they may be damaged, making proper species identification impossible. This thesis therefore presents two alternatives of sand fly identification based on molecular...

Barcode identification is a method that uses genetic information to differentiate species. Because of its general versatility it can be applied to contexts from archaeology to the food industry. Atlantic salmon (Salmo salar) is a fish species commonly hunted in modern times and has been found in archaeological settings. However, barcoding requires enough quality DNA for amplification and abiotic exposure tends to degrade it. High temperatures, such as when boiling, can diminish DNA quality. The extent of DNA degradation between fresh and boiled tissues and whether all tissues retain the same amount of DNA is unclear. In this study DNA was extracted from S. salar tissues fins, muscle, bones and scales without treatment and with boiling treatment. DNA concentrations between fresh and boiled bones were not significantly different, nor were comparisons between samples with the same treatments. Muscles had higher DNA concentrations when boiled and fins had higher when fresh. These findings show that regarding certain tissue types can be expected to better retain DNA concentrations after boiling.

A sistemática de Cnidaria Anthozoa foi revisada inúmeras vezes e por vários autores, sendo que os Ceriantharia sempre foram avaliados através de dados clássicos e muito pretéritos. O clado Ceriantharia, uma ordem dentre Hexacorallia, apresenta muitos caracteres dúbios e que poderiam colocar os animais em várias posições sistemáticas dentre Cnidaria. Ainda, as revisões de Ceriantharia são sempre derivadas de análises das mesmas espécies, principalmente do Mar Mediterrâneo. O presente trabalho tem como enfoque a discussão dos caracteres taxonômicos (morfológicos e moleculares) de Ceriantharia, sua posição sistemática, levantamento taxonômico de áreas no Oceano Atlântico praticamente inexploradas para o grupo e aspectos da biologia das espécies encontradas. Os resultados apontam para uma nova posição sistemática de Ceriantharia, levando o grupo à base de Anthozoa e como um ramo independente. A utilização de dados moleculares evidenciou o vigor do emprego de DNA barcoding na zoologia comparada de Ceriantharia. Aspectos de especiação e de revisão sistemática resultaram pelas análises moleculares. Ao mesmo tempo, os dados morfológicos são extremamente confusos e muito do que foi utilizado como caractere de delimitação de espécies pela tradição de se assumir homologias com Actiniaria é, aparentemente, inconsistente. O ciclo de vida de Isarachnanthus noturnus foi verificado, sendo que o seu estágio jovem, planctônico, e de dispersão é de longa duração. Dados sobre regeneração em pólipos também foi verificada e com a constatação de mecanismos divergentes entre as espécies. / The Cnidaria Anthozoa systematics was revised several times by a number of authors, and the Ceriantharia always evaluated using very classical and ancient data. The Ceriantharia clade, an Hexacorallia order, has many dubious characters that could place the animals in various systematic positions within Cnidaria. Also, the Ceriantharia reviews are always derived from analysis of the same species, mainly from the Mediterranean Sea. This work focuses on the discussion of taxonomic characters (morphological and molecular) of Ceriantharia, the systematic position, taxonomic survey of virtually unexplored Atlantic Ocean areas for the group and the biology of the observed species. The results suggest a new systematic position of Ceriantharia placing the group at the basis of Anthozoa and as an independent branch. The molecular data comparison showed the force of DNA barcoding use in Ceriantharia comparative zoology. The molecular data allowed the discussion of several aspects of speciation and systematics. At the same time, the morphological data are extremely confusing and much of it was used as delimiting species character by the tradition of assuming homologies with Actiniaria is, apparently, inconsistent. The life cycle of Isarachnanthus noturnus was observed, and the young, planktonic and dispersal stage is long-lived. Data on regeneration of polyps was also verified with the observation that mechanisms differ between species.

Background. Caryophyllaceae is a large and cosmopolitic flowering plant family, however the systematics of many of its basal groups has been unclear, due to a lack of unambiguous morphology. Some members of Caryophyllaceae are used medicinally, e.g. Corrigiola roots in Morocco. Monitoring the trade in medicinal plants is complicated due to the absence of useful identification characters in plant products such as roots, bark, and powders. This thesis aims at elucidating the systematics and the ethnobotany of some of the basal clades in Caryophyllaceae. Methods. A comprehensive sampling from herbarium as well as market and field collected material was used in systematic studies combining morphological investigation, molecular phylogenetic and molecular dating analyses. Results. The data show that Polycarpon is polyphyletic, that Sphaerocoma is sister to Pollichia and shows some intraspecific variation, that Sanctambrosia falls within the genus Spergularia, and that both Spergula and Spergularia are monophyletic. Preliminary data suggest that Polycarpaea is polyphyletic and should be split into three larger and several smaller genera, that the members of Paronychia subgen. Anaplonychia will need to be transferred to Herniaria to maintain monophyly, and that Caryophyllaceae emerged during the Paleocene. All the major extant lineages originated in the Oligocene and diversified later. Using molecular identification it was possible to identify around 50% of the Moroccan medicinal roots to species level and an additional 30% to genus level. Discussion and conclusions. The polyphyletic Polycarpon needs to be split into at least three separate genera, but no name changes were made pending further research. The two species of Sphaerocoma were merged into one species with two subspecies. The San Ambrosio Island endemic Sanctambrosia, the only tree-like plant in Caryophyllaceae, is probably the result of a long distance dispersal event and its woody habit and gynodioecy are probably caused by inbreeding depression. Sanctambrosia manicata is transferred to Spergularia. Molecular identification put into practice on traded medicinal roots has a somewhat lower success rate than most theoretical studies, indicating that a global barcoding database would need to include reference sequences from a broad range of populations for each species.

The oceans represent a wealth of biological diversity where many species remain to be discovered and described. Among seaweeds, a paucity of morphological features by which to differentiate species means that many genera harbour overlooked or cryptic species. Fucus, Porphyra and Ulva are three common genera of marine intertidal algae and all include species that are particularly difficult to distinguish morphologically. DNA barcoding has been championed as a revolutionary tool for species identification and discovery and applying this tool to algae was a logical step due to the difficulty of morphological identification of many algal species. This thesis is part of a significant initiative aimed at identification and discovery of all species of seaweeds in Canadian waters, using a DNA barcoding approach. The original concept of DNA barcoding relied on comparing the 5’ region of the mitochondrial cytochrome c oxidase 1 (COI-5P) gene among animal species. In this study, DNA barcoding with COI-5P was applied to the brown algal genus Fucus and worked as well as any other marker to assign morphologies to known species. The DNA barcoding results also uncovered substantial phenotypic diversity in Pacific F. distichus. Results were confirmed by comparison with sequences of the nuclear internal transcribed spacer region (ITS). For Porphyra, COI-5P DNA barcoding was compared with species identification using the chloroplast large rubisco subunit (rbcL) and the Universal Plastid Amplicon (UPA) in a floristic survey of Canadian Porphyra species. Two new species were discovered and described (Porphyra corallicola and Porphyra peggicovensis), and P. cuneiformis was synonymized with P. amplissima. The COI-5P emerged as the best marker for species discrimination despite difficulties with primer universality. To aid in choosing a marker for DNA barcoding for green algae, the universality and species discriminatory power of the rubisco large subunit (rbcL) (considering the 5’ and 3’ fragments independently), the UPA, the D2/D3 region of the nuclear large ribosomal subunit (LSU-D2/D3) and the ITS were evaluated. While the rbcL-3P highlighted several cryptic species, and worked well to distinguish Ulva species, more research is needed to recommend a marker for DNA barcoding generally in marine green macroalgae.

Orientador: Sérgio Nascimento Stampar / Resumo: Existe uma considerável abundância de espécies de cnidários no litoral do Brasil e uma das espécies mais intrigantes e pouco conhecidas é a Tamoya haplonema, a qual dentre suas características mais interessantes se destaca a presença de um padrão de bandas nos tentáculos, o que não é comum nos cubozoários, classe à qual pertence. Essa espécie é conhecida desde meados do século XIX, sendo uma das poucas espécies no Atlântico Sul capaz de causar acidentes graves em humanos. Em 2011 alguns registros de espécimes do mesmo gênero para o Mar do Caribe resultaram na descrição de uma nova espécie, Tamoya ohboya. Essa espécie foi delimitada, principalmente, por dados moleculares e devido à disponibilidade de poucos indivíduos a definição é dúbia em vários trechos. Já foi constatado que o cenário taxonômico do gênero Tamoya no Atlântico Ocidental não é consistente. Desta maneira, o principal objetivo deste estudo foi a comparação taxonômica do gênero Tamoya no Atlântico Sul e Mar do Caribe.Para tal, indivíduos do gênero Tamoya do Atlântico Sul foram analisados e comparados com T.ohboya, utilizando dados morfológicos/morfométricos e moleculares, incluindo o estudo da composição dos nematocistos através das medidas e análises comparativas. Os resultados morfológicos, bem como a análise do cnidoma e os resultados moleculares deixam claro que T.haplonema e T.ohboya são na realidade a mesma espécie. / Abstract: There is a considerable abundance of cnidarian species along the Brazilian coast and one of the most intriguing and little known species is the medusa Tamoya haplonema, which among its most interesting characteristics stands out the presence of a pattern of bands in the tentacles, which is not common in the Cubozoa, the class to which it belongs. This species has been known since the mid-nineteenth century, being one of the few species in the Southern Atlantic capable of causing serious stings to humans. In 2011 specimens of the same genus the Caribbean Sea resulting in the description of a new species, Tamoya ohboya. This species was delimited, mainly, by molecular data and due to the availability of few individuals the definition is dubious in several parts. It has already been verified that the taxonomic scenario of the genus Tamoya in the Western Atlantic is not consistent. Thus, the main objective of this study was the taxonomic comparison of the genus Tamoya in the South Atlantic and the Caribbean Sea. Thus individuals of the genus from the South Atlantic were analyzed and compared with T.ohboya, using morphological / morphometric and molecular data, including the study of the nematocysts composition through measurements and comparative analyzes. The morphological results as well as the analysis of the cnidome and molecular results make it clear that T.haplonema and T.ohboya are actually the same species. / Mestre

Black Cohosh (Actaea racemosa) is one of the highest selling medicinal plants, ranking as the sixth best seller in the US in 2015 (Smith et al., 2016). However, this popularity has been tarnished by claims of hepatotoxicity. The investigation of these reports has determined that implicated products did not contain Black Cohosh plant material. Other reports were shown to be incomplete or had other factors contributing. This has led to the suspicion that cases of adverse reactions may in fact be linked to cases of substitution or adulterations with Asian species of Actaea, rather than to A. racemosa. (Jordan et al., 2010). This shows the need for authentication of Black Cohosh products. In this study various DNA based authentication methods were developed. The first, PlantID is capable of discriminating between Actaea racemosa and four potential adulterant species; Actaea cimicifuga, Actaea cordifolia, Actaea podocarpa and Caulophyllum thalictroides, in a single PCR reaction. The resulting fragments are scrutinized using gel electrophoresis. Other platforms of analysis were trialled with little success. The second was a qPCR based method. These assays are competent in detecting A. racemosa, A. cimicifuga and A. dahurica species and are compared to a generic primer capable of amplification of ten Actaea species. This enables the user to detect specific species in comparison to how much Actaea species are present as a whole. This assay was extensively tested on many materials and products available in the UK and the USA. Out of 34 products assessed it was possible to extract DNA from 32. From the UK market it was found that five products contained undeclared species. From the US market it was found that six products contained undeclared species. All of the THR registered products were found to contain only the authentic species Actaea racemosa. This was a reassuring result from the analysis and adds further value to the scheme of THR. Sequence data from GenBank was used to assist in assigning species to sequenced DNA samples. The data contained on GenBank was scrutinised using various bioinformatics tools. Sequences were organised into molecular taxonomic units using tree diagram software. This showed efficiently and iii visually which sequence entries were reliable to use based upon grouping. This analysis showed that the nuclear internal transcribed spacer (nrITS) was an ideal barcoding region and that maturase K (MatK) was a poor choice for Actaea species. To address the issue of hepatotoxicity claims, cultured human hepatocyte derived cells were treated with 60% ethanol extracts of Actaea racemosa and Asian Actaea. A qPCR array was utilised to assess 84 genes associated with hepatotoxicity across various concentrations of extract. The collective array output gave a plethora of data which was analysed using bespoke online software from the manufacturer. Stringent quality controls were included on the arrays which gave confidence of results. There were small changes noted for Actaea racemosa and some activity for the Asian Actaea treated cells was also seen. An LDH and MTT assay were used to assess cell viability and toxicity in two human hepatocyte derived cell lines. Actaea racemosa showed no significant effects whereas the Asian Actaea extract showed a notable decrease in cell viability and significant release of LDH indicating toxicity. The Asian Actaea material used to manufacture extracts was of questionable species origin but determined to be either A. dahurica or A. cimicifuga. The results from these experiments were unfortunately not as conclusive as hoped, but did show some evidence of a more likely culprit of toxicity originating from Asian Actaea species.