O presente trabalho utiliza marcadores moleculares para auxiliar na caracterização e filogenia das espécies de macroalgas vermelhas do gênero Hypnea na costa do Brasil. O gênero Hypnea Lamouroux (1813) apresenta cerca de 67 espécies descritas e possui distribuição geográfica em águas quentes ao redor do mundo. Além disso, o gênero possui grande importância econômica e ecológica, como fonte de alimento e produção industrial de carragenana. Porém, a identificação das espécies de Hypnea com base exclusivamente em dados morfológicos é dificultada em virtude da plasticidade fenotípica presente no grupo, de sua morfologia relativamente simples e da ampla distribuição geográfica de suas espécies. Em vista disso, utilizamos a técnica de \"DNA barcoding\" que permite a análise de um grande número de amostras. Neste trabalho foram utilizados dois \"DNA barcodes\" (a região 5\' do gene mitocondrial cox1 e o \"universal plastid amplicon\" UPA), e para as análises filogenéticas foi utilizado o gene plastidial rbcL. Além disso, estudos morfológicos foram feitos a fim de delimitar o real valor dos caracteres morfo-anatômicos citados na literatura para a separação de espécies do gênero Hypnea. Ao todo, foram obtidas 230 amostras brasileiras de Hypnea, provenientes de 11 estados brasileiros, e 10 amostras de outros países. Um total de 367 sequências de marcadores moleculares foi obtido neste trabalho. Confirmamos a ocorrência de nove espécies para o gênero: H. cervicornis, \"H. flexicaulis\", \"H. musciformis\", H. spinella, \"H. stellulifera\", Hypnea sp. 1, Hypnea sp. 2, Hypnea sp. 3 e Hypnea sp. 4. As amostras coletadas e previamente identificadas em campo como H. cornuta revelaram-se, pelos estudos da biologia molecular, serem \"H. stellulifera\". As espécies H. musciformis, H. nigrescens, H. valentiae foram consideradas variações morfológicas de uma mesma espécie, denominada de \"H. musciformis\". A identificação das espécies com base apenas em características morfológicas mostrou-se insatisfatória, devido principalmente a plasticidade fenotípica presente no grupo, além da existência de espécies com morfologias convergentes. A técnica de \"DNA barcode\", principalmente com relação ao marcador cox1, mostrou-se essencial na identificação e delimitação das espécies, revelando cenários que passariam despercebidos com o uso apenas da morfologia / This study uses molecular markers to aid in the characterization and phylogeny of species of the genus Hypnea, a red macroalgae, on the coast of Brazil. The genus Hypnea Lamouroux (1813) presents about 67 described species and has geographical distribution in warm waters around the world. Furthermore, the genus has great economic and ecological importance as a source of food and industrial production of carrageenan. However, the identification of the species of Hypnea based solely on morphological data is difficult due to phenotypic plasticity present in this group, its relatively simple morphology and broad geographical distribution of its species. In view of this, we use the technique of \"DNA barcoding\" that allows the analysis of a large number of samples. In this work we used two \"DNA barcodes\" (the 5 \'region of the mitochondrial gene cox1 and universal plastid amplicon UPA), and for phylogenetic analysis the plastid gene rbcL was used. In addition, morphological studies were made in order to delimit the actual value of morpho-anatomical caracters cited in the literature for the separation of species of Hypnea. Altogether, 230 Hypnea samples were obtained from 11 Brazilian states, and 10 samples from other countries. A total of 367 sequences of molecular markers were obtained in this study. We confirm the occurrence of nine species of the genus: H. cervicornis, \"H. flexicaulis\", \"H. musciformis\", \"H. spinella, \"H. stellulifera\", Hypnea sp. 1, Hypnea sp. 2, Hypnea sp. 3 and Hypnea sp. 4. Samples collected in the field and previously identified as H. cornuta based on molecular data, proved to be \"H. stellulifera\". The species H. musciformis, H. nigrescens and H. valentiae were considered morphological variations of the same species, named \"H. musciformis\". The identification of species based on morphological characteristics proved unsatisfactory, mainly due to phenotypic plasticity in this group and the existence of species with convergent morphologies. The technique of \"DNA barcode\", especially with respect to cox1 marker, was essential for the identification and definition of species, revealing scenarios that would go unnoticed by using only morphology

Os Bombyliidae constituem uma das maiores famílias de Diptera, com mais de 5.000 espécies conhecidas em todo mundo, sendo o grupo de moscas com maior representatividade nas regiões desérticas da Terra, atualmente divididos em 17 subfamílias e 19 tribos. A subfamília Phthiriinae inclui duas tribos, 12 gêneros e 134 espécies válidas presentes em todas as regiões biogeográficas, são caracterizadas por serem moscas pequenas, variando de 3 mm a 12 mm de comprimento, com um acentuado dimorfismo sexual, pela presença de prolongamento dorsal no flagelo e presença de saco basal localizado na base do bulbo espermático na espermateca. Esta tese tem como objetivo central contribuir para a compreensão do padrão evolutivo em Bombyliidae por meio da realização de análise filogenética da subfamília Phthiriinae utilizando caracteres morfológicos. De modo específico, esta pesquisa realiza um estudo comparativo da morfologia externa e interna de Phthiriinae, testa a monofilia da subfamília, das tribos e dos gêneros incluídos, propõe uma nova classificação para a subfamília e delimita as espécies que apresentam dimorfismo sexual com base em ferramentas morfológicas e moleculares (DNA Barcoding). Foram estudados espécimes de todos os gêneros de Phthiriinae, totalizando 937 espécimes pertencentes a 74 espécies, das quais sete representam o grupo externo e 67 o grupo interno. A análise cladística baseada em 216 caracteres morfológicos, recupera a monofilia de Phthiriinae, com a inclusão do gênero Elektrophthiria + Nel, 2006 em uma nova subfamília aqui proposta, Elektrophthiriinae + subfam. nov., uma vez que este aparece fora do clado. Com essa alteração, a tribo Phthriini também passa a ser considerada monofilética. A maioria dos gêneros são recuperados como monofiléticos, no entanto Phthiria Meigen,1820, Poecilognathus Jaennicke, 1867, Relictiphthiria Evenhuis, 1986 e Tmemophlebia Evenhuis, 1986, com suas atuais composições, mostraram-se parafiléticos. Para recuperar a monofilia de Phthiria, um novo gênero é eregido para reunir as espécies Afrotropicais. Com relação ao gênero Poecilognathus, sua monofilia só pode ser recuperada após a remoção de Po. philippianus Rondani, 1863 e Po. xanthogaster Hall, 1976, que passam a compor dois novos gêneros monotípicos, pertencentes a Phthiriini e Poecilognathini, respectivamente, e, desse modo, a monofilia da tribo Poecilognathini também é recuperada. Além disso, Tmemophlebia é considerado sinônimo junior de Relictiphthiria. Esta tese propõe pela primeira vez a utilização do DNA Barcoding para a associação de machos e fêmeas de uma mesma espécie de Bombyliidae. Esse estudo possibilita a associação de machos e fêmeas pertencentes a 12 diferentes espécies sexualmente dimórficas, garantindo sua inclusão na análise cladística. / The Bombyliidae constitutes one of the largest family of Diptera, with more than 5.000 species known worldwide, being the group of flies with the largest representativeness in desertic regions of Earth, currently divided into 17 subfamilies and 19 tribes. The subfamily Phthiriinae comprises, two tribes, 12 genera and 134 valid species present in all biogeographic regions, are characterized by being small flies, varying from 3 mm to 12 mm, with accentuated sexual dimorphism, for the presence of dorsal prong on flagellum and presence of basal sac on spermathecae This thesis has the main goal to contribute for comprehension of the evolutionary pattern in Bombyliidae, performing a phylogenetic analysis of Phthiriinae based on morphological characters. Specifically, this research aims to realize a comparative study of external morphology of Phthiriinae, test the monophyly of the subfamily, tribes and included genera, propose a new classification for the subfamily and also delimit the species with sexual dimorphism based on molecular and morphological data (DNA Barcoding). Were studied specimens of all Phthriiinae genera, in a total of 937 specimens, belonging of 74 species, of which seven represents the outgroup and 67 the ingroup. The cladist analysis based in 216 morphological characters, recover the monophily of Phthirrinae, with the inclusion of the genus Elektrophthiria + Nel, 2006 in a new subfamily, Elektrophthiriinae + subfam. nov., once was positioned outside the group. With this change, Phthiriini tribe also becames monophyletic. Most genera are recovered as monophyletic, however, Phthiria Meigen,1820, Poecilognathus Jaennicke, 1867, Relictiphthiria Evenhuis, 1986 and Tmemophlebia Evenhuis, 1986, with the current composition, were shown to be paraphyletic. To recover the monophily of Phthiria, a new genus is erected to gather the Afrotropical species. Concerning the Poecilognathus genus, its monophyly can only be recovered with the withdrawal Po. philippianus Rondani, 1863 and Po. xanthogaster Hall, 1976, which begins to compose two new monotipic genera, belonging to Phthiriini and Poecilognathini, respectively, thus the monophily of Poecilognathini tribe is also recovered. Therefore, it is proposed Tmemophlebia as a junior synonym of Relictiphthiria. This thesis proposed for the first time the use of DNA Barcoding for the association of males and females of the same species of Bombyliidae. This study allows the association of males and females belonging to 12 different sexually dimorphic species, ensuring their inclusion in the cladistic analysis.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2017-10-27T12:45:54Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Geize.pdf: 2675219 bytes, checksum: 90ad2edda2ef72b5d73979929c6cca53 (MD5) / Made available in DSpace on 2017-10-27T12:45:54Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Dissertação Geize.pdf: 2675219 bytes, checksum: 90ad2edda2ef72b5d73979929c6cca53 (MD5) Previous issue date: 2017-02-24 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Loricariidae é uma das maiores famílias de peixes de água doce com 931 espécies válidas distribuídas em 100 gêneros. As espécies pertencentes a essa família são popularmente conhecidas como cascudos que possuem um corpo achatado, coberto por placas ósseas, e boca ventral em forma de ventosa. Pertencente à família Loricariidae e a subfamília Neoplecostominae, o gênero Neoplecostomus compreende espécies de tamanho pequeno que habitam riachos da região sul e sudeste do Brasil. O gênero apresenta interesse em estudos genéticos, visto que é composto por espécies vágeis e que, por raramente migrarem, podem acumular diferenças genéticas entre populações. Das dezesseis espécies válidas descritas até o momento para este gênero, oito foram descritas para a bacia do rio Paraná e há evidências da ocorrência de novas espécies para esta bacia. Sendo assim, este estudo teve como objetivo estudar a diferenciação cariotípica e a diversificação genética no gênero Neoplecostomus das bacias hidrográficas dos rios Iguaçu, Itararé, Cinzas e Tibagi com vistas ao entendimento da evolução cariotípica e descrição da biodiversidade. Para isso, foram realizas coletas em quatro localidades: rio Pinhão (Neoplecostomus sp. 1), rio Samambaias (Neoplecostomus cf. botucatu), rio das Pedras (Neoplecostomus sp. 2) e rio São João (Neoplecostomus yapo). Os dados citogenéticos revelaram um número diploide de 54 cromossomos para as quatro populações, poucas regiões heterocromáticas e sítios cromossomo independentes para os genes ribossomais 5 e 18S. Os resultados citogenéticos evidenciam uma alta estabilidade cariotípica, observado também na subfamília Neoplecostominae. Foram realizadas análises moleculares envolvendo o gene mitocondrial citocromo c oxidase subunidade 1 (COI) e todas as distâncias genéticas foram superiores a 4,7 %. Além disso, contrários à estabilidade cromossômica, os dados de distância genética evidenciaram ausência de fluxo gênico e estruturação populacional, além de sugerir uma provável espécie de Neoplecostomus não descrita para o rio Pinhão, bacia do rio Iguaçu. / The Loricariidae family is one of the largest families of freshwater fishes, with 931 species distributed in 100 genera. Loricariidae members are popularly known as armored catfishes and characterized by a depressed body, covered by bony plates and a ventral sucker mouth. Inside the subfamily Neoplecostominae, the genus Neoplecostomus comprises small species that inhabit streams of south and southeast regions of Brazil. This genus is interested in genetic studies because they rarely migrate and can accumulate genetic differences between populations. Of the sixteen valid species described so far for this genus, eight were described for the Paraná river basin and there is evidence of new species occurring in this basin. Therefore, the objective of this study was to analyze the karyotype differentiation and genetic diversification in the genus Neoplecostomus from Iguaçu, Itararé, Cinzas and Tibagi river basins, with the goal of understand the karyotype evolution and biodiversity. The specimens were captured at four localities: Pinhão river (Neoplecostomus sp. 1), Samambaias river (Neoplecostomus cf. botucatu), Pedras river (Neoplecostomus sp. 2) and São João river (Neoplecostomus yapo). Cytogenetic data revealed a diploid number of 54 chromosomes to the four populations, few heterochromatic regions and independent chromosome sites for 5S and 18S ribosomal genes. The cytogenetic results revealed a high karyotypic stability, which is also observed for the subfamily Neoplecostominae. Also, were performed molecular analyzes by mitochondrial cytochrome c oxidase subunit 1 gene (COI) amplification, where all genetic distances were higher than 4.7%. In addition, contrary to chromosome stability, genetic distance data also showed absence of gene flow, population structure and suggested a probable Neoplecostomus species not described yet for Pinhão river at Iguaçu basin.

Cette thèse s'inscrit dans le cadre du traitement des données issues de séquençage haut débit, et en particulier des données produites en metabarcoding ADN. Le metabarcoding ADN consiste à identifier des taxons ou des groupes taxinomiques à partir de l'ADN présent dans des échantillons environnementaux (eau, sol, fèces...). Après extraction de l'ADN, de courtes séquences utilisées comme marqueurs taxinomiques sont amplifiées par PCR puis séquencées en utilisant les nouvelles techniques de séquençage haut débit. De très importants volumes de données sont ainsi générés, le plus souvent, de plusieurs milliers à plusieurs centaines de milliers de séquences par échantillon. L'objectif principal de cette thèse était le développement de méthodes d'analyse de ces séquences. Les méthodes de classification permettent de traiter de nombreuses problématiques en metabarcoding ADN. La classification supervisée est utilisée pour assigner les séquences à des taxons en les comparant aux séquences de bases de données de référence. Les méthodes de classification non supervisée permettent de créer des groupes taxinomiques (MOTU) à partir des séquences, afin de faire des estimations de biodiversité. Ces méthodes sont aussi employées pour identifier les séquences erronées produites par la PCR et le séquençage notamment, où les séquences erronées dérivent souvent des vraies séquences et leur sont très similaires. Les méthodes de classification demandent une méthode de comparaison des séquences qui soit idéalement à la fois très rapide et exacte. Une telle méthode a été développée, en utilisant un algorithme d'alignement global de type Needleman-Wunsch calculant la longueur de la plus longue sous-séquence commune entre les séquences à aligner, associé à un filtre sans perte permettant d'éviter l'alignement de certaines paires de séquences n'ayant aucune chance de présenter une similarité supérieure à un seuil choisi. L'utilisation d'instructions Single Instruction, Multiple Data, de même que le multithreading optionnel des calculs, permettent d'associer rapidité et exactitude. Cette méthode de comparaison est implantée dans SUMATRA, un programme calculant toutes les similarités deux à deux d'un jeu de données ou entre deux jeux de données, avec possibilité de fixer un seuil de similarité en dessous duquel les similarités ne sont pas rapportées. Elle est aussi utilisée dans SUMACLUST. SUMACLUST est un programme regroupant les séquences en utilisant un algorithme de clustering en étoile, où chaque groupe possède une séquence représentative. Il peut être utilisé pour créer des MOTU, ou pour détecter les séquences erronées dérivant de vraies séquences. Plus spécialisé, le programme SUMACLEAN a été développé pour détecter les séquences contenant des erreurs ponctuelles de PCR. Pour cela, des graphes orientés acycliques sont générés, dont la topologie correspond parfaitement aux cascades d'erreurs générées par les erreurs ponctuelles de PCR. Par ailleurs, une réflexion a été menée pour le développement d'une nouvelle approche de classification supervisée pour l'assignation taxinomique des séquences. Aujourd'hui, la plupart des approches d'assignation utilisent des méthodes mal adaptées au polymorphisme important des marqueurs, et ne considèrent pas suffisamment l'incomplétude et les erreurs inhérentes aux bases de données de référence. Une nouvelle approche a été testée, basée sur l'idée d'un départ depuis la racine de l'arbre taxinomique, suivi d'une descente jusqu'à un arrêt possible lorsque descendre à un niveau taxinomique plus précis semble irraisonnable. Cela permettrait en théorie de mieux gérer les problèmes inhérents aux bases de données de référence, mais pose le problème de la représentation des séquences aux différents niveaux de l'arbre, et du modèle de choix du chemin à prendre, pour lesquels aucune solution complètement satisfaisante n'a été trouvée à ce jour. / This thesis positions itself in the context of the processing of high-throughput sequencing data, and specifically DNA metabarcoding data. DNA metabarcoding consists of the identification of taxa or taxonomic groups from DNA extracted from environmental samples (water, soil, animal feces). After extraction of the DNA, short sequences used as taxonomic markers are amplified by PCR, then sequenced using high-throughput sequencing technologies. Important volumes of data are produced that way, usually from several thousands to several hundreds of thousands sequences per sample. This thesis aimed for the development of methods for the analysis of these sequences. Classification methods allow the treatment of numerous problems in DNA metabarcoding. Supervised classification is used for the taxonomic assignment of sequences to taxa, by comparing them to the sequences of a reference database. Unsupervised classification methods are used to create taxonomic groups (MOTUs) from the sequences, in order to estimate biodiversity. They are also used to identify the erroneous sequences generated during the PCR and sequencing steps in particular, where erroneous sequences often derive from true sequences and remain very close to them. Classification approaches used in the context of DNA metabarcoding necessitate a sequence comparison method that should be both fast and exact. Such a method was developed, using a Needleman-Wunsch type global alignment algorithm computing the length of the longest common subsequence between the two sequences being aligned, associated with a lossless filter allowing to avoid the alignment of some pairs of sequences that have no chance to present a similarity superior to a chosen threshold. The use of Single Instruction, Multiple Data instructions, as well as the availability of multithreading speed up the calculations. This comparison method is implanted in SUMATRA, a program computing all the pairwise similarities of a dataset or between two datasets, with the possibility to set a threshold under which similarities are ignored. It is also used in SUMACLUST, a program grouping sequences using a star clustering algorithm, where each cluster possesses a representative sequence. This algorithm can be used to generate MOTUs, or to identify erroneous sequences deriving from true sequences, by using the fact that true sequences tend to end up as the representative sequences of their cluster. More specialized, the SUMACLEAN program was developed to identify sequences containing ponctual PCR errors. To that end, directed acyclic graphs are created, whose topology matches perfectly the successions of errors generated by ponctual errors during PCR. A new approach for the taxonomic assignment of sequences with a supervised classification method was also studied. Nowadays, most taxononomic assignment approaches use methods that are badly suited for the important polymorphism of markers, and don't take in account enough the incompleteness and errors inherent to reference databases. A new approach was tested, based on the idea of a start from the root of the taxonomic tree, and a descent in it with a possible stop before reaching a leaf, if descending to a more precise taxonomic level seems unreasonable. This approach would theoretically allow for a better handling of the problems inherent to reference databases, but poses a few issues, such as the representation of sequences at intermediate tree levels, and the model used to make choices regarding the path to take in the tree, for which no satisfying solutions have been found yet.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The mitochondrial gene COI is widely used as a molecular marker, and the region used for DNA barcoding is getting more and more popular to species delimitation and identification. However, the evolution patterns can vary along the genes depending on factors as the gene position in genome and taxonomic group, among others. Using one unique delimited region for all groups may not be suitable to a certain targeted taxon, or to answer its evolutionary questions. We examined the patterns of COI intra- and interspecific nucleotide divergence for aeglid crustaceans and we found highly variable divergence profiles along the COI gene; a possible evolutionary pattern for the Jerry-Pat region; lacking of evolutionary pressures by concentrated divergence; intra- and interspecific divergence levels overlap, but no overlapping in substitution profiles along the gene; a putative new COI region to access the total COI diversity in aeglids. We conclude that the barcoding region does not accurately reflect the evolution of the gene COI in aeglids. / O gene mitocondrial COI é amplamente utilizado como marcador molecular, sendo a região de DNA barcoding cada vez mais popular para delimitação e identificação de espécies. No entanto, os padrões de evolução variam ao longo dos genes dependendo de fatores como posição do gene no genoma, grupo taxonômico, entre outros. O uso de uma única região delimitada para qualquer grupo pode não ser adequado para o táxon de interesse ou para responder suas questões evolutivas. Nós examinamos os padrões de divergência nucleotídica intra e interespecíficas no gene COI de crustáceos eglídeos e obtivemos: perfis de divergência altamente variáveis ao longo de todo COI; possível padrão de evolução na região de Jerry-Pat; ausência de pressões evolutivas por divergência concentrada; sobreposição de níveis de divergência intraespecífica e com interespecífica, mas sem sobreposição dos perfis de substituição ao longo do gene; sugestão de nova região para acessar a diversidade total de COI em eglídeos. Concluímos que a região de barcoding não reflete acuradamente a evolução do gene mitocondrial COI em eglídeos.

O presente trabalho utiliza marcadores moleculares para auxiliar na caracterização e filogenia das espécies de macroalgas vermelhas do gênero Hypnea na costa do Brasil. O gênero Hypnea Lamouroux (1813) apresenta cerca de 67 espécies descritas e possui distribuição geográfica em águas quentes ao redor do mundo. Além disso, o gênero possui grande importância econômica e ecológica, como fonte de alimento e produção industrial de carragenana. Porém, a identificação das espécies de Hypnea com base exclusivamente em dados morfológicos é dificultada em virtude da plasticidade fenotípica presente no grupo, de sua morfologia relativamente simples e da ampla distribuição geográfica de suas espécies. Em vista disso, utilizamos a técnica de \"DNA barcoding\" que permite a análise de um grande número de amostras. Neste trabalho foram utilizados dois \"DNA barcodes\" (a região 5\' do gene mitocondrial cox1 e o \"universal plastid amplicon\" UPA), e para as análises filogenéticas foi utilizado o gene plastidial rbcL. Além disso, estudos morfológicos foram feitos a fim de delimitar o real valor dos caracteres morfo-anatômicos citados na literatura para a separação de espécies do gênero Hypnea. Ao todo, foram obtidas 230 amostras brasileiras de Hypnea, provenientes de 11 estados brasileiros, e 10 amostras de outros países. Um total de 367 sequências de marcadores moleculares foi obtido neste trabalho. Confirmamos a ocorrência de nove espécies para o gênero: H. cervicornis, \"H. flexicaulis\", \"H. musciformis\", H. spinella, \"H. stellulifera\", Hypnea sp. 1, Hypnea sp. 2, Hypnea sp. 3 e Hypnea sp. 4. As amostras coletadas e previamente identificadas em campo como H. cornuta revelaram-se, pelos estudos da biologia molecular, serem \"H. stellulifera\". As espécies H. musciformis, H. nigrescens, H. valentiae foram consideradas variações morfológicas de uma mesma espécie, denominada de \"H. musciformis\". A identificação das espécies com base apenas em características morfológicas mostrou-se insatisfatória, devido principalmente a plasticidade fenotípica presente no grupo, além da existência de espécies com morfologias convergentes. A técnica de \"DNA barcode\", principalmente com relação ao marcador cox1, mostrou-se essencial na identificação e delimitação das espécies, revelando cenários que passariam despercebidos com o uso apenas da morfologia / This study uses molecular markers to aid in the characterization and phylogeny of species of the genus Hypnea, a red macroalgae, on the coast of Brazil. The genus Hypnea Lamouroux (1813) presents about 67 described species and has geographical distribution in warm waters around the world. Furthermore, the genus has great economic and ecological importance as a source of food and industrial production of carrageenan. However, the identification of the species of Hypnea based solely on morphological data is difficult due to phenotypic plasticity present in this group, its relatively simple morphology and broad geographical distribution of its species. In view of this, we use the technique of \"DNA barcoding\" that allows the analysis of a large number of samples. In this work we used two \"DNA barcodes\" (the 5 \'region of the mitochondrial gene cox1 and universal plastid amplicon UPA), and for phylogenetic analysis the plastid gene rbcL was used. In addition, morphological studies were made in order to delimit the actual value of morpho-anatomical caracters cited in the literature for the separation of species of Hypnea. Altogether, 230 Hypnea samples were obtained from 11 Brazilian states, and 10 samples from other countries. A total of 367 sequences of molecular markers were obtained in this study. We confirm the occurrence of nine species of the genus: H. cervicornis, \"H. flexicaulis\", \"H. musciformis\", \"H. spinella, \"H. stellulifera\", Hypnea sp. 1, Hypnea sp. 2, Hypnea sp. 3 and Hypnea sp. 4. Samples collected in the field and previously identified as H. cornuta based on molecular data, proved to be \"H. stellulifera\". The species H. musciformis, H. nigrescens and H. valentiae were considered morphological variations of the same species, named \"H. musciformis\". The identification of species based on morphological characteristics proved unsatisfactory, mainly due to phenotypic plasticity in this group and the existence of species with convergent morphologies. The technique of \"DNA barcode\", especially with respect to cox1 marker, was essential for the identification and definition of species, revealing scenarios that would go unnoticed by using only morphology

The biting midges (family Ceratopogonidae) is one of the most species rich amongst the biting flies (Diptera) and has been recorded from most parts of the world. The species are mostly known for their capability to act as vectors for several important diseases, which have helped in shaping the focus to one of its genera, Culicoides Latreille, 1809.   This thesis gives an overview of the knowledge of the Swedish diversity, in the first paper (paper I) with a closer look at the species of Dasyhelea Kieffer, 1911 where all twenty species found in Sweden are presented with their associated localities, and two new species are described.  In the second paper (paper II) the biting midge diversity of Sweden is presented based on specimens collected from several localities. All these individuals were barcoded using the mitochondrial cytochrome oxidase I gene (COI). The analysis included 773 specimens that were assigned into 214 barcoding clusters (BINs) and sorted into 164 groups based on their morphology. The third paper (paper III) broadens the scale were the evolutionary relationships within the family are investigated by applying five protein coding genes (COI, CAD, TPI, AATS and PGD) and specimens from different parts of the World. The analysis recovers Ceratopogonini, Forcipomyia Meigen, 1818 and Bezzia Kieffer, 1899 as paraphyletic and Palpomyia Meigen, 1818 polyphyletic. In the last and fourth paper (paper IV) the family is used as a model organism together with Hymenoptera for an alternative analysis method for reducing the impact of saturation and long-branch attraction using non-synonymous coding (e.g. Degen1) on only parts of a dataset. The effectiveness of the method is compared to the removal of the faster evolving third codon position. The result yields a higher number of supported nodes as well as a higher median of support for the method as well as an ability to reduce long-branch attraction artifacts. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Manuscript.</p><p> </p>

Orientador: Claudio Oliveira / Resumo: A família Serrasalmidae é endêmica da região Neotropical, amplamente distribuída na América do Sul, e possui 97 espécies válidas distribuídas em 16 gêneros recentes e um fóssil. Embora a monofilia de Serrasalmidae seja bem estabelecida, assim como dos três clados que a compõe, várias relações entre os subgrupos são ainda conflitantes, há confusão sobre as posições dos gêneros dentro destes clados e restam dúvidas sobre a monofilia, composição e validade dos gêneros e espécies. Ainda, a imprecisão da descrição de muitas das espécies e seus atuais status taxonômicos dificultam o entendimento e o estudo para diversas áreas. Sendo assim, o presente trabalho visou elucidar as relações filogenéticas da família através de análises filogenômicas utilizando elementos ultraconservados (UCEs) e delimitar espécies de Catoprion e Pygocentrus através de análises integrativas, morfológicas e moleculares. Os resultados corroboram a monofilia da família, bem como dos três clados principais: (1) Colossoma, Mylossoma e Piaractus, (2) o “clado Myleus” formado por Acnodon, Myleus, Mylesinus, Myloplus, Ossubtus, Tometes e Utiaritichthys e (3) o “clado piranha”, composto por Catoprion, Metynnis, Pristobrycon, Pygocentrus, Pygopristis e Serrasalmus. As relações dentro do clado “Colossoma+Mylossoma+Piaractus” são bem suportadas, com Piaractus irmão do clado Colossoma e Mylossoma, sendo este clado grupo irmão de todos os outros serrasalmídeos. O “clado Myleus” apresenta gêneros não-monofiléticos, como... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Serrasalmidae is an endemic Neotropical fish family, broadly distributed in South America, with 97 valid species allocated in 16 extant genera and one fossil. Even though the monophyly of the family is well stablished, as the formation of three major clades composing it, several aspects of relationships between subgroups are discordant among authors and are observed conflicts in the interrelationships and monophyly of some genera, and in the validity of some species. Furthermore, the imprecision of the species description and its current taxonomic status make it difficult the understanding and study for several areas. Thus, the present study aimed to elucidate the phylogenetic relationships within the family through a phylogenomic analysis using ultraconserved elements (UCEs) and to delimit species of Catoprion and Pygocentrus through morphological and molecular integrative analysis. The results corroborate the monophyly of family as well as the three main clades: (1) Colossoma, Mylossoma and Piaractus, (2) the “Myleus clade” composed by Acnodon, Myleus, Mylesinus, Myloplus, Ossubtus, Tometes and Utiaritichthys, and (3) the “piranha clade", composed by Catoprion, Metynnis, Pristobrycon, Pygocentrus, Pygopristis and Serrasalmus. The relationships within the “Colossoma+Mylossoma+Piaractus” clade are well supported, with Colossoma and Mylossoma as sister group of Piaractus, and this clade sister to all other Serrasalmids. The “Myleus clade” contains several non-monophyletic gene... (Complete abstract click electronic access below) / Doutor

Blow flies (Diptera: Calliphoridae) are among the most dominant and conspicuous insects in the decomposition process. They are important in forensic entomology to determine time of death and, in certain situations, cause of death or relocation of a body. Insects are now included as standard operating procedures in crime scene investigations in many countries, however, this is not standard procedure in the Caribbean area due to lack of knowledge of insects involved in cadaveric decomposition. Successful application of forensic entomology depends on solid underlying data. Our main goal is to advance the knowledge of Calliphoridae in the Caribbean to enable forensic entomology studies. We performed a mega-transect across the Caribbean and extensively collected flies attracted to rotten meat baits during five years from 2011 to 2015. Overall we collected 61,332 flies of which 34,650 were Calliphoridae. We sampled 16 of the 18 species of forensically important Caribbean Calliphoridae and three continental species. We determine the diversity and distribution of Calliphoridae in the Caribbean. We also present a thorough DNA barcode dataset, covering the geographic range of most species in the region. Finally we established phylogenetic relationships among Calliphoridae species and test biogeographical hypotheses, and patterns of diversification and endemism in the Caribbean. In sum, this is the most comprehensive study of the family Calliphoridae from the Caribbean that will open the door for future research on forensic entomology in the region.

DNA barcoding is a valuable tool for fish species identification by food regulators, however, it does not perform well when multiple species are present within the same food product. PCR cloning has high potential to be used in combination with DNA barcoding to overcome this challenge. The objective of this study was to examine the use of PCR cloning combined with DNA barcoding to identify fish in a mixed-species product that cannot be identified with standard DNA barcoding. A total of 15 fish ball mixtures were prepared with known amounts of Nile tilapia (Oreochromis niloticus), Pacific cod (Gadus macrocephalus), and walleye pollock (Gadus chalcogrammus). The fish balls underwent DNA extraction in triplicate, followed by DNA barcoding across the full barcode (655 bp) and SH-E mini-barcode (226 bp) of the cytochrome c oxidase subunit 1 (CO1) region. Samples that did not pass sequencing according to regulatory standards were further analyzed with PCR cloning. Full barcoding enabled identification of at least one species in 80% of the fish ball mixtures compared to 51% for minibarcoding. The results of PCR cloning with samples that did not pass DNA barcoding showed identification success rates of 61% for clones (54 of 90) that underwent full barcoding and 51% for clones (111 of 220) that underwent mini-barcoding. All fish balls made of just one species tested positive for that species (i.e., tilapia, cod, or pollock).. The combination of standard full barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 6 of 12 (50%) of mixed-species fish balls. In comparison, the combination of standard mini-barcoding and PCR cloning enabled identification of Nile tilapia in all 12 mixed-species fish balls and Pacific cod in 9 of 12 (75%) of mixed-species fish balls. Overall, the results of this study show that PCR cloning may be an effective method to identify certain fish in mixed-species products when standard DNA barcoding fails. However, additional research is needed to understand the limitations associated with primer bias.