Individuals of the cnidarian genus Physalia are a common sight at New Zealand beaches and are the primary cause of jellyfish stings to beachgoers each year. The identity of the species and the environmental factors that determine its presence are unknown. Lack of knowledge of many marine species is not unusual, as pelagic invertebrates often lack detailed taxonomic descriptions as well as information about their dispersal mechanisms such that meaningful patterns of distribution and dispersal are almost impossible to determine. Molecular systematics has proven to be a powerful tool for species identification and for determining geographical distributions. However, other techniques are needed to indicate the causal mechanisms that may result in a particular species distribution. The aim of this study was to apply molecular techniques to the cnidarian genus Physalia to establish which species occur in coastal New Zealand, and to apply models to attempt to forecast its occurrence and infer some mechanisms of dispersal. Physalia specimens were collected from New Zealand, Australia and Hawaii and sequenced for Cytochrome c oxidase I (COI) and the Internal transcribed spacer 1 (ITS1). Three clans were found: a Pacific-wide clan, an Australasian clan and New Zealand endemic clan with a distribution confined to the Bay of Plenty and the East Coast of the North Island. Forecasting Physalia occurrence directly from presence data using artificial neural networks (ANN) proved unsuccessful and it was necessary to pre-process the presence data using a variable sliding window to reduce noise and improve accuracy. This modelling approach outperformed the time lagged based networks giving improved forecasts in both regions that were assessed. The ANN models were able to indicated significant trends in the data but would require more data at higher resolution to give more accurate forecasts of Physalia occurrence suitable for decision making on New Zealand beaches. To determine possible causal mechanisms of recorded occurrences and to identify possible origins of Physalia the presence and absence of Physalia on swimming beaches throughout the summer season was modelled using ANN and Naϊve Bayesian Classifier (NBC). Both models were trained on the same data consisting of oceanographic variables. The modelling carried out in this study detected two dynamic systems, which matched the distribution of the molecular clans. One system was centralised in the Bay of Plenty matching the New Zealand endemic clan. The other involved a dynamic system that encompassed four other regions on both coasts of the country that matched the distribution of the other clans. By combining the results it was possible to propose a framework for Physalia distribution including a mechanism that has driven clan divergence. Moreover, potential blooming areas that are notoriously hard to establish for jellyfish were hypothesised for further study and/or validation.

The Guiana Shield is a sub-region of Amazonia, one of the richest areas on earth in terms of species number. It is also one of the most pristine areas and is still largely unexplored. Species number, distribution, boundaries and their evolutionary histories remain at least unclear but most of the time largely unknown. This is the case for most Anurans, a group which is recognized as threatened globally and is disappearing even from pristine tropical forests. Given the pace of forest destruction and the growing concerns about climate change it is urgently necessary to obtain a better estimate of regional biodiversity in Amazonian frogs as well as a better understanding of the origin and distribution of Anuran diversity. Furthermore, given their sensitivity to climatic conditions, amphibians are a good model to investigate the influence of paleoclimatic events on Neotropical diversification which was supposedly the driving force on biotic evolution during Pleistocene in the Guiana Shield. I first test species boundaries in two species Scinax ruber and Rhinella margaritifera. These species are widely distributed, abundant and largely recognized as species complexes. I used an original species delineation method based on the combined use of mitochondrial and nuclear DNA in phylogenetic and phylogeographic analyses. Phylogenetic analyses demonstrated the polyphyly of Scinax ruber and Rhinella margaritifera. These species consist of multiple lineages that may all merit species status. Conflicting signals of mitochondrial and nuclear markers indicated the possibility of ongoing hybridization processes. Phylogeographic analyses added further information in support of the specific status of these lineages. Our results highlight the utility of combining phylogenetic and phylogeographic methods, as well as the use of both mitochondrial and nuclear markers within one study. This approach helped to better understand the evolutionary history of taxonomically complex groups of species. The assessment of the geographic distribution of genetic diversity in tropical amphibian communities can lead to conclusions that differ strongly from prior analyses based on the occurrence of currently recognized species alone. Such studies, therefore, hold the potential to contribute to a more objective assessment of amphibian conservation priorities in tropical areas. Subsequently, I tested if these first results on cryptic species are generalisable, questioning what would potentially be a minimum estimate of the number of cryptic frog species in Amazonia and the Guiana Shield, using mtDNA with multiple complementary approaches. I also combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs. In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contra to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher genetic distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species. Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas. As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised. The Rhinella margaritifera complex is characterisized by the presence of many cryptic species throughout its wide distribution, ranging from Panama to Bolivia and almost entire Amazonia. French Guiana has long been thought to harbor two species of this group, though molecular data analysed in previous chapters indicated as many as five lineages. I tested whether morphological measurements are correlated or not with genetic data using discriminant analysis and if diagnostic characteristics among the previously determined lineages can be used to describe these new species. This is a novel integrative method which can lead to a facilitation of the description of cryptic species that have been detected by phylogenetic and/or phylogeographic studies. These analyses, combined with published data of other Rhinella species, indicated that two of these lineages represent previously unnamed species. Two of the remaining are allocable to R. margaritifera while the status of the fifth is still unclear because so far it is morphologically indistinguishable from R. castaneotica. Determining if codistributed species responded to climate change in an independent or concerted manner is a basic objective of comparative phylogeography. Species boundaries, histories, ecologies and their geographical ranges are still to be explored in the Guiana Shield. According to the refugia hypothesis this region was supposed to host a forest refugium during climatic oscillations of the Pleistocene but the causes and timing for this have been criticized. We investigated patterns of genetic structure within 18 frog species in the eastern Guiana Shield to explore species boundaries and their evolutionary history. We used mtDNA and nuclear DNA and complementary methods to compare the genetic diversity spatially and temporally. With one exception all the species studied diversified repeatedly within the eastern Guiana Shield during the last 4 million years. Instead of one Pleistocene forest refugium the Guiana Shield has probably hosted multiple refugia during late Pliocene and Pleistocene. Most of these Pleistocene refugia were probably situated on the coast of French Guiana, Amapà, Suriname and Guyana. This diversification likely resulted from forest fragmentation. Many species deserve taxonomic revisions and their ranges to be reconsidered. The local endemism of the Anuran fauna of the Guiana Shield is likely to be much higher and some areas consequently deserve more conservation efforts. Specifically I questioned whether major intraspecific diversification started before the Pleistocene and occurred within the Guiana Shield or ex situ. According to ecological characteristics of the species involved I will test different diversification hypotheses. The consequences on the diversity and the endemism of the Guiana Shield will be explored. My results demonstrate that we have been grossly underestimating local biological diversity in the Guiana Shield but also in Amazonia in general. The order of magnitude for potential species richness means that the eastern Guiana Shield hosts one of the richest frog fauna on earth. In most of the species studied high levels of mtDNA differentiation between populations call for a reassessment of the taxonomic status of what is being recognised as single species. Most species display deep divergence between eastern Guiana Shield populations and Amazonian ones. This emphasizes that the local endemism in the Guiana Shield of these zones is higher than previously recognized and must be prioritised elements taken into account in conservation planning. Nevertheless, a few other species appear widely distributed showing that widespread species do exist. This underlines the fact that some species have efficient dispersal abilities and that the frog fauna of the eastern Guiana Shield is a mixture of old Guianan endemic lineages that diversified in situ mostly during late Pliocene and Pleistocene and more recently exchanged lineages with the rest of Amazonia. Recognizing this strong historical component is necessary and timely for local conservation as these zones are likely to be irremediably modified in the near future.

The work presented in this thesis describes methodologies developed for integration and accurate interpretation of barcoded DNA, to empower large-scale-omics analysis. The objectives mainly aim at enabling multiplexed proteomic measurements in high-throughput format through DNA barcoding and massive parallel sequencing. The thesis is based on four scientific papers that focus on three main criteria; (i) to prepare reagents for large-scale affinity-proteomics, (ii) to present technical advances in barcoding systems for parallel protein detection, and (iii) address challenges in complex sequencing data analysis. In the first part, bio-conjugation of antibodies is assessed at significantly downscaled reagent quantities. This allows for selection of affinity binders without restrictions to accessibility in large amounts and purity from amine-containing buffers or stabilizer materials (Paper I). This is followed by DNA barcoding of antibodies using minimal reagent quantities. The procedure additionally enables efficient purification of barcoded antibodies from free remaining DNA residues to improve sensitivity and accuracy of the subsequent measurements (Paper II). By utilizing a solid-phase approach on magnetic beads, a high-throughput set-up is ready to be facilitated by automation. Subsequently, the applicability of prepared bio-conjugates for parallel protein detection is demonstrated in different types of standard immunoassays (Papers I and II). As the second part, the method immuno-sequencing (I-Seq) is presented for DNAmediated protein detection using barcoded antibodies. I-Seq achieved the detection of clinically relevant proteins in human blood plasma by parallel DNA readout (Paper II). The methodology is further developed to track antibody-antigen interaction events on suspension bead arrays, while being encapsulated in barcoded emulsion droplets (Paper III). The method, denoted compartmentalized immuno-sequencing (cI-Seq), is potent to perform specific detections with paired antibodies and can provide information on details of joint recognition events. Recent progress in technical developments of DNA sequencing has increased the interest in large-scale studies to analyze higher number of samples in parallel. The third part of this thesis focuses on addressing challenges of large-scale sequencing analysis. Decoding of a huge DNA-barcoded data is presented, aiming at phase-defined sequence investigation of canine MHC loci in over 3000 samples (Paper IV). The analysis revealed new single nucleotide variations and a notable number of novel haplotypes for the 2nd exon of DLA DRB1. Taken together, this thesis demonstrates emerging applications of barcoded sequencing in protein and DNA detection. Improvements through the barcoding systems for assay parallelization, de-convolution of antigen-antibody interactions, sequence variant analysis, as well as large-scale data interpretation would aid biomedical studies to achieve a deeper understanding of biological processes. The future perspectives of the developed methodologies may therefore stem for advancing large-scale omics investigations, particularly in the promising field of DNA-mediated proteomics, for highly multiplex studies of numerous samples at a notably improved molecular resolution. / <p>QC 20150203</p>

The Guiana Shield is a sub-region of Amazonia, one of the richest areas on earth in terms of species number. It is also one of the most pristine areas and is still largely unexplored. Species number, distribution, boundaries and their evolutionary histories remain at least unclear but most of the time largely unknown. This is the case for most Anurans, a group which is recognized as threatened globally and is disappearing even from pristine tropical forests. Given the pace of forest destruction and the growing concerns about climate change it is urgently necessary to obtain a better estimate of regional biodiversity in Amazonian frogs as well as a better understanding of the origin and distribution of Anuran diversity. Furthermore, given their sensitivity to climatic conditions, amphibians are a good model to investigate the influence of paleoclimatic events on Neotropical diversification which was supposedly the driving force on biotic evolution during Pleistocene in the Guiana Shield. I first test species boundaries in two species Scinax ruber and Rhinella margaritifera. These species are widely distributed, abundant and largely recognized as species complexes. I used an original species delineation method based on the combined use of mitochondrial and nuclear DNA in phylogenetic and phylogeographic analyses. Phylogenetic analyses demonstrated the polyphyly of Scinax ruber and Rhinella margaritifera. These species consist of multiple lineages that may all merit species status. Conflicting signals of mitochondrial and nuclear markers indicated the possibility of ongoing hybridization processes. Phylogeographic analyses added further information in support of the specific status of these lineages. Our results highlight the utility of combining phylogenetic and phylogeographic methods, as well as the use of both mitochondrial and nuclear markers within one study. This approach helped to better understand the evolutionary history of taxonomically complex groups of species. The assessment of the geographic distribution of genetic diversity in tropical amphibian communities can lead to conclusions that differ strongly from prior analyses based on the occurrence of currently recognized species alone. Such studies, therefore, hold the potential to contribute to a more objective assessment of amphibian conservation priorities in tropical areas. Subsequently, I tested if these first results on cryptic species are generalisable, questioning what would potentially be a minimum estimate of the number of cryptic frog species in Amazonia and the Guiana Shield, using mtDNA with multiple complementary approaches. I also combined isolation by distance, phylogenetic analyses, and comparison of molecular distances to evaluate threshold values for the identification of candidate species among these frogs. In most cases, geographically distant populations belong to genetically highly distinct lineages that could be considered as candidate new species. This was not universal among the taxa studied and thus widespread species of Neotropical frogs really do exist, contra to previous assumptions. Moreover, the many instances of paraphyly and the wide overlap between distributions of inter- and intra-specific distances reinforce the hypothesis that many cryptic species remain to be described. In our data set, pairwise genetic distances below 0.02 are strongly correlated with geographical distances. This correlation remains statistically significant until genetic distance is 0.05, with no such relation thereafter. This suggests that for higher genetic distances allopatric and sympatric cryptic species prevail. Based on our analyses, we propose a more inclusive pairwise genetic distance of 0.03 between taxa to target lineages that could correspond to candidate species. Using this approach, we identify 129 candidate species, two-fold greater than the 60 species included in the current study. This leads to estimates of around 170 to 460 frog taxa unrecognized in Amazonia-Guianas. As a consequence the global amphibian decline detected especially in the Neotropics may be worse than realised. The Rhinella margaritifera complex is characterisized by the presence of many cryptic species throughout its wide distribution, ranging from Panama to Bolivia and almost entire Amazonia. French Guiana has long been thought to harbor two species of this group, though molecular data analysed in previous chapters indicated as many as five lineages. I tested whether morphological measurements are correlated or not with genetic data using discriminant analysis and if diagnostic characteristics among the previously determined lineages can be used to describe these new species. This is a novel integrative method which can lead to a facilitation of the description of cryptic species that have been detected by phylogenetic and/or phylogeographic studies. These analyses, combined with published data of other Rhinella species, indicated that two of these lineages represent previously unnamed species. Two of the remaining are allocable to R. margaritifera while the status of the fifth is still unclear because so far it is morphologically indistinguishable from R. castaneotica. Determining if codistributed species responded to climate change in an independent or concerted manner is a basic objective of comparative phylogeography. Species boundaries, histories, ecologies and their geographical ranges are still to be explored in the Guiana Shield. According to the refugia hypothesis this region was supposed to host a forest refugium during climatic oscillations of the Pleistocene but the causes and timing for this have been criticized. We investigated patterns of genetic structure within 18 frog species in the eastern Guiana Shield to explore species boundaries and their evolutionary history. We used mtDNA and nuclear DNA and complementary methods to compare the genetic diversity spatially and temporally. With one exception all the species studied diversified repeatedly within the eastern Guiana Shield during the last 4 million years. Instead of one Pleistocene forest refugium the Guiana Shield has probably hosted multiple refugia during late Pliocene and Pleistocene. Most of these Pleistocene refugia were probably situated on the coast of French Guiana, Amapà, Suriname and Guyana. This diversification likely resulted from forest fragmentation. Many species deserve taxonomic revisions and their ranges to be reconsidered. The local endemism of the Anuran fauna of the Guiana Shield is likely to be much higher and some areas consequently deserve more conservation efforts. Specifically I questioned whether major intraspecific diversification started before the Pleistocene and occurred within the Guiana Shield or ex situ. According to ecological characteristics of the species involved I will test different diversification hypotheses. The consequences on the diversity and the endemism of the Guiana Shield will be explored. My results demonstrate that we have been grossly underestimating local biological diversity in the Guiana Shield but also in Amazonia in general. The order of magnitude for potential species richness means that the eastern Guiana Shield hosts one of the richest frog fauna on earth. In most of the species studied high levels of mtDNA differentiation between populations call for a reassessment of the taxonomic status of what is being recognised as single species. Most species display deep divergence between eastern Guiana Shield populations and Amazonian ones. This emphasizes that the local endemism in the Guiana Shield of these zones is higher than previously recognized and must be prioritised elements taken into account in conservation planning. Nevertheless, a few other species appear widely distributed showing that widespread species do exist. This underlines the fact that some species have efficient dispersal abilities and that the frog fauna of the eastern Guiana Shield is a mixture of old Guianan endemic lineages that diversified in situ mostly during late Pliocene and Pleistocene and more recently exchanged lineages with the rest of Amazonia. Recognizing this strong historical component is necessary and timely for local conservation as these zones are likely to be irremediably modified in the near future.

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No. of bitstreams: 1 TeseCBMS.pdf: 4190969 bytes, checksum: 7f39aaee9acdf743fad7b9fcaec22326 (MD5) Previous issue date: 2016-09-30 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Salminus is a genus comprised of four Neotropical medium- and large-sized fishes species, top predators, with both recreational and commercial importance. The paucity of information on taxonomy, phylogeny and phylogeography make appropriate conservation policies difficult for the genus, which is in a significant population decline. For this reason, our goal was, by using mitochondrial (COI, Cytb and D-loop) and nuclear (RAG2 and S7 intron) molecular makers, to elucidate taxonomic uncertainties, identify cryptic diversity, investigate the phylogenetic relationship among the species and infer the historical processes that shaped the current Salminus distribution. To assist in taxonomic issues, we employed the traditional DNA barcoding and GMYC COI-based analyses in 110 specimens, representing the four valid species. In both methodologies, eight MOTUs (molecular operational taxonomic units) were identified. Only two species, Salminus affinis and Salminus franciscanus, represented a MOTU each. The species Salminus brasiliensis and Salminus hilarii were represented by two and four MOTU, respectively. These MOTUs are distributed in distinct hydrographic basins where morphological polymorphisms had already been described. The average intraspecific distances greater than the optimal threshold of 1.1% (S. brasiliensis – 3.6%, e S. hilarii – 5%), reinforce the idea of more taxonomic units in Salminus. The multiloci analysis recovered interesting information about the cryptic diversity: the paraphyletic mitochondrial lineages of S. brasiliensis, one from Upper Paraná river and another composed of specimens from the other regions of the Platina basin, formed a unique monophyletic group. For S. hilarii, despite the four MOTUs observed, only three of them were recovered. Therefore, based on multiloci analysis and phylogenetic species concept, we proposed a new taxonomic scenario for Salminus. The genus is now composed of six species: S. affinis, S. franciscanus, S. brasiliensis, S. hilarii, Salminus sp. Amazonas and Salminus sp. Araguaia. The phylogeny reconstruction refuted hypotheses previously proposed. S. affinis, the only trans-Andean species, was the sister species of the other Salminus, which formed two main groups: Northwest group, composed of S. sp. Amazonas and S. sp. Araguaia, and Southeast group, formed by S. brasiliensis, S. franciscanus and S. hilarii. The divergence processes among Salminus began in Later Miocene and it is associated with vicariance and geodispersion events that shaped the hydrological landscape in the past 12 million years. For the first time, it was used a model-based approach in order to test alternative biogeographic scenarios and to distinguish phylogeography signatures among the events responsible for Neotropical fishes’ diversification. We evidenced that the distinct vicariance and geodispersion signatures could be detected in our biological model. A clear description of these species brings in a valuable information for conservation, because, now, six biological units need to be protected. As these species are located in distinct hydrographic basins, each basin becomes one important biogeographic unit to maintain the evolutionary and ecological processes that sustain the species permanence and diversity. / Salminus é um gênero constituído por quatro espécies de peixes Neotropicais de médio e grande porte, predadores topo de cadeia, que possuem importância na pesca comercial e esportiva. Escassez de informações quanto a taxonomia, filogenia e filogeografia, dificultam medidas de conservações adequadas para o gênero, que se encontra em acentuado declínio populacional. Por essa razão, este estudo objetivou, através do emprego de marcadores moleculares mitocondriais (COI, Cytb e D-loop) e nucleares (RAG2 e íntron do S7), elucidar incertezas taxonômicas, identificar diversidade críptica, investigar as relações filogenéticas entre as espécies e inferir os processos históricos que modelaram a distribuição atual do gênero Salminus. Para auxiliar nas questões taxonômicas, nós empregamos análise de DNA barcoding tradicional e GMYC utilizando o marcador COI em 110 espécimes, representando as quatros espécies válidas. Em ambas metodologias, oito MOTUs (unidades taxonômicas operacionais moleculares) foram identificadas. Apenas duas espécies, Salminus affinis e Salminus franciscanus, apresentaram uma única MOTU cada. As espécies Salminus brasiliensis e Salminus hilarii foram representadas por duas e quatro MOTUs, respectivamente. Essas MOTUs estão distribuídas em distintas bacias hidrográficas onde polimorfismos morfológicos já haviam sido descritos. As médias das distâncias intraespecíficas superiores ao threshold ótimo de 1.1% (S. brasiliensis – 3.6%, e S. hilarii – 5%), reforçam a ideia de mais unidades taxonômicas em Salminus. A análise multiloci recuperou informações interessantes quanto à diversidade críptica: as linhagens mitocondriais parafiléticas de S. brasiliensis, uma proveniente do Alto rio Paraná e outra formada por espécimes das demais regiões da bacia Platina, formaram um único grupo monofilético. Para S. hilarii, apesar de quatros MOTUs observadas, apenas três foram recuperadas. Portanto, baseada na análise multiloci e no conceito filogenético de espécie, propomos um novo cenário taxonômico para Salminus. O gênero passa a ser constituído por seis espécies: S. affinis, S. franciscanus, S. brasiliensis, S. hilarii, Salminus sp. Amazonas e Salminus sp. Araguaia. A filogenia recriada refutou hipóteses previamente propostas. S. affinis, única espécie transandina, foi a espécie-irmã dos demais Salminus, que formam dois grandes grupos: grupo Noroeste, constituído por S. sp. Amazonas e S. sp. Araguaia, e grupo Sudeste, composto por S. brasiliensis, S. franciscanus e S. hilarii. Os processos de divergência entre as espécies do gênero se iniciou no Mioceno Superior e está associada a eventos de vicariância e geodispersão que modelaram a paisagem hidrogeológica nos últimos 12 milhões de anos. Pela primeira vez, foi utilizado uma abordagem baseada em modelos para testar cenários biogeográficos alternativos e distinguir se existem assinaturas filogeográficas entre os eventos responsáveis pelo processo de diversificação de peixes Neotropicais. Nós evidenciamos que distintas assinaturas filogeográficas entre vicariância e geodispersão puderam ser detectadas em nosso modelo biológico. A clara designação das espécies por si só já acarreta em uma informação valiosa para conservação, afinal seis unidades biológicas precisam ser protegidas. Como essas espécies estão localizadas em distintas bacias hidrográficas, cada bacia passa ser avaliada como uma unidade biogeográfica importante para a manutenção dos processos evolutivos e ecológicos que sustentam a diversidade e permanência das espécies.

Des prospections réalisées en Equateur (Amazonie et côte pacifique) ont permis la collecte d'un matériel entomologique abondant et diversifié, notamment chez les Psychodopygina. Nos travaux ont permis de réaliser plusieurs travaux de systématique, essentiellement moléculaire. Afin de tester les hypothèses phylogénétiques développées par Galati (2010), nous avons conduit une étude de phylogénie moléculaire chez les Psychodopygina. Basée sur les séquences des domaines D1, C2 et D2 de l'ADNr 28S et sur celles d'une partie du cytochrome b de l'ADNmt, elle inclut 49 espèces représentant les sept genres de la sous tribu et la majorité des sous-genres et séries. Les marqueurs ribosomiques sont mieux adaptés à la problématique que le marqueur mitochondrial. Le genre Psychodopygus est monophylétique. En raison du positionnement de Ny. richardwardi parmi les Trichophoromyia, nous concluons à la paraphylie des genres Nyssomyia et Trichophoromyia. Le genre Psathyromyia est également paraphylétique, tout comme le genre Martinsmyia. Le genre Bichromomyia serait le groupe frère du genre Psychodopygus et la validité du genre Vianniamyia, inclus dans le genre Psathyromyia doit être discutée. Des phylogénies moléculaires plus terminales ont été réalisées par comparaison de séquences de l'ITS2, de l'EF-1α et du cytochrome b.Chez les Psychodopygus de la série Guyanensis, une étude moléculaire couplée à une étude morphologique et morphométrique de morphotypes différents chez Ps. geniculatus, en sympatrie avec Ps. corossoniensis et Ps. luisleoni nous a conduit à décrire une espèce nouvelle pour la science : Ps. francoisleponti.Chez Pa. aragaoi, notre étude pilote basée sur l'analyse de morphotypes différents allopatriques et sympatriques renforce l'hypothèse de l'existence probable d'un complexe d'espèces chez ce taxon. Chez Ny. trapidoi, les analyses moléculaires et enzymatiques conduites sur des exemplaires clairs et foncés ne supportent pas la mise en évidence de deux populations comme cela avait été auparavant démontré. Nos approches épidémiologiques ont permis de mettre en évidence l'ADN d'Endotrypanum monterogeii chez plusieurs exemplaires de Ny. trapidoi. Si aucun phlebovirus n'a été détecté dans les échantillons étudiés, nous rapportons la présence d'un flavivirus chez Pa. abonnenci. Mots-clés: Psychodopygina, Equateur, ADN ribosomique, ADN mitochondrial, phylogénie, Endotrypanum. / Most Ecuadorian sand flies studied so far belong to Psychodopygina sub tribe and the present research uses morphometric and modern molecular techniques to answer many some questions regarding this taxon in Ecuador. We present phenetic and phylogenetic analyses based on the sequences of the domains D1, C2 and D2 of the 28S rDNA and cytochrome b mtDNA were used to test the classification of Psychodopygina sub tribe proposed by Galati (2010). Our study includes 49 species representing the seven genera included in the sub tribe and its main subgenera and series. The results support the monophyly of the genus Psychodopygus. The genera Psathyromyia, Nyssomyia and Trichophoromyia are paraphyletic. Bichromomyia is the sister group of Psychodopygus and the validity of the genus Viannamyia is doubtful because it is included inside the Psathyromyia genus. Our data strongly suggest the presence of two populations within Ps. geniculatus and the lack of intermediate forms between these two morphotypes incited us to describe a new sympatric species, Psychodopygus francoisleponti.We also carried out a pilot study based on the analysis of different allopatric and sympatric morphotypes of Pa. aragaoi which suggested the existence of a possible complex of species in this taxa.Finally, we analyzed of mitochondrial gene sequences and isoenzymes from Ny. trapidoi collecte from Ecuador and our result did not support the existence of two sibling species within as previously reported in the literature. From an epidemiological point of view, we emphasize the probable vectorial role of Nyssomyia trapidoi for Endotrypanum monterogeii. Moreover, no phlebovirus was detected in the processed sand flies whereas a flavivirus has been found in a pool of Psathyromyia. abonnenci females.Key words: Psychodopygina, Ecuador, ribosomal DNA, mitochondrial DNA, phylogeny, Endotrypanum.

Herbal medicines (HMs) have been used to supplement, maintain, and treat health conditions, and have inspired the development of many Western pharmaceuticals. Migrant South Asian (SA) communities in the UK have brought with them their own traditional forms of medicine, yet little is known about their current use of HMs in the UK. Consuming HMs alongside conventional Western medicines could affect pharmacological treatment and lead to herb-drug interactions; hence, healthcare professionals (HCPs) should be aware of their patients’ use of HMs. The import of HMs to the UK raises concerns over the quality, safety and regulation of HMs. Deoxyribonucleic acid (DNA) barcoding can be used to discriminate between different species, and identify contaminants and adulterants, thus can be used for the authentication of HMs. The South Asian Traditional Medicines (SATMED) questionnaire explored the knowledge and use of HMs by diasporic SA communities in the UK. It uncovered a vast range of HMs which were used by participants, where ingredients were sourced from, the concurrent use of herbal and Western medicines, and how minor ailments were treated. An online survey designed to investigate UK based practitioners’ views of HMs revealed that HCPs claimed to lack sufficient knowledge of HMs. HCPs said they needed more training on HMs to help them make better informed decisions. Tulsi (Ocimum tenuiflorum L.) was identified as a culturally and commercially valuable plant, which was used for molecular analysis. A variety of tulsi samples were collected for authentication: community samples from SA families in the UK, commercial samples, and referenced specimens. Both ITS and trnH-psbA regions were successfully used to distinguish between several Ocimum species, and identify a potential species substitution. This research represents the first time that DNA based methods have been used to authenticate medicinal plants species used by migrant SA communities living in the UK. The results of this multi-disciplinary study provide a unique contribution to the evolving discipline of ethnopharmacology.

The work deals with the fuzzy classification of DNA sequences. In the first part the theory summarized information about Fuzzy logic and methods of its use in the classification of biological sequence data. The second part is practically deal with the classification algorithm for assessing the similarity of sequences. Specifically, the dividing of coding and non-coding parts of the sequence and the use of fuzzy classification in DNA barcoding.

Most methods for analysis of genomic data work with symbolic sequences. Numerically represented genomic sequences can be analyzed by signal processing methods. A new method of numerical representation of DNA sequences, nucleotide density vectors, is proposed in this thesis. Usability of this method for purposes of molecular species identification is tested on DNA barcoding sequences. DNA barcoding is modern and popular methodology based on comparison of short mitochondrial DNA sequences. Beside species identification by proposed method based on nucleotide density vectors, higher taxa rank identification (e.g. families) was also tested. Furthermore, dendrograms were constructed from standardly used evolutionary distances and distances between nucleotide density vectors and the dendrograms were compared.

MSc (Zoology) / Department of Zoology / See the attached abstract below