Submitted by Renata Lopes (renatasil82@gmail.com) on 2018-06-12T11:58:26Z No. of bitstreams: 0 / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-06-14T12:44:23Z (GMT) No. of bitstreams: 0 / Made available in DSpace on 2018-06-14T12:44:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-02-26 / A família Cycloposthiidae (Ciliophora, Trichostomatia) inclui protozoários ciliados simbiontes intestinais de mamíferos herbívoros conhecidos por contribuir de forma significativa no metabolismo digestivo de seus hospedeiros. Atualmente, é representada por 69 espécies, divididas em 17 gêneros. Entretanto, devido à ausência de padrão metodológico para descrições de espécies, bem como diversas outras incongruências taxonômicas, a sistemática da família Cycloposthiidae necessita de ampla reformulação. Estudos recentes tem demonstrado que a família Cycloposthiidae não é monofilética, porém, há escassez de dados molecures para investigar tais relações. Esta dissertação encontra-se dividida em duas seções. A Seção 1 apresenta breve revisão taxonômica da família Cycloposthiidae, com dados sobre taxonomia, morfologia, checklist de espécies, hospedeiros e distribuição geográfica, e discute a validade dos táxons pertencentes à família, a fim de refletir sobre a sistemática do grupo. A Seção 2 apresenta dados sobre filogenia molecular de representantes da família Cycloposthiidae e fornece dez novas sequências, sendo nove de representantes do gênero Cycloposthium e uma do gênero Monoposthium, a fim de investigar a relação entre os membros da família Cycloposthiidae e demais ciliados tricostomatídeos, e investigar a evolução de alguns caracteres morfológicos dentro do gênero Cycloposthium. Deste modo, ressalta-se a importância de conhecer melhor os representantes da família Cycloposthiidae, a considerar sua diversidade, amplo espectro de hospedeiros nos quais estas espécies ocorrem e sua importância na digestão desses animais. / The family Cycloposthiidae (Ciliophora, Trichostomatia) includes symbiotic intestinal ciliates of herbivorous mammals known to contribute significantly to the digestive metabolism of their hosts. Currently, it is represented by 69 species, divided into 17 genera. However, because to the absence of a methodological pattern for species descriptions, as well as several other taxonomic incongruities, the systematics of the family Cycloposthiidae requires extensive reformulation. Recent studies have shown that the family Cycloposthiidae is not monophyletic, however, there is a paucity of molecular data to investigate such relationships. This dissertation is divided into two sections. Section 1 presents a brief taxonomic review of the family Cycloposthiidae, with data on taxonomy, morphology, species checklists, hosts and geographic distribution, and discusses the validity of taxa belonging to the family, with the objective to reflect on the group's systematics. Section 2 presents data on molecular phylogeny of representatives of the family Cycloposthiidae and provides ten new sequences, nine of which are representatives of the genus Cycloposthium and one of the genus Monoposthium, with the objective to investigate the relationships between the members of the family Cycloposthiidae and other tricostomatids ciliates, and to investigate the evolution of some characters within the genus Cycloposthium. In this way, it is emphasized the importance of knowing better the family Cycloposthiidae, to consider its diversity, wide spectrum of hosts where the family occurs and its importance in the digestion of these animals.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fungi represent an extremely important group of organisms in terrestrial ecosystems. Among their several important ecological roles, is the mutualistic association with plant roots, forming different types of mycorrhiza. Most studies carried out so far focused on epigeous ectomycorrhizal fungi, which occur above ground. On the other hand, below ground, hypogeous ectomycorrhizal fungi, are still poorly studied. This research aimed to study the diversity of hypogeous ectomycorrhizal fungi (Basidiomycetes) collected in Eucalyptus spp. plantations in the Central Region of Rio Grande do Sul, Brazil, based on morphological and molecular characters. Samples were taken between May 2009 and July 2010. A total of five species belonging to three families were identified. The material was analyzed to determine the morphological and molecular characters. Among the species identified are: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway and Setchelliogaster tenuipes (Setch.) Pouzar. Additionally a second species of Chondrogaster sp. was collected. However, no identity was determined for this species. Furthermore, it is believed it represents a new undescribed species to science. Among the species studied, Descomyces albus is reported for the first time in the State of Rio Grande do Sul. Hysterangium affine and H. inflatum are newly recorded species from Brazil while Chondrogaster pachysporus is recorded for the first in South America. / Os fungos representam um grupo de organismos extremamente importante nos ecossistemas terrestres. Entre as muitas funções por eles desempenhadas está a associação mutualística com as raízes dos vegetais, formando diferentes tipos de micorrizas. A maioria dos estudos realizados até o momento está principalmente focada nos fungos ectomicorrízicos epígeos, os quais ocorrem acima do solo. Por outro lado, os fungos ectomicorrízicos hipógeos, que vivem abaixo da superfície do solo, são ainda pouco estudados. A presente pesquisa tem como objetivo estudar a diversidade de fungos ectomicorrízicos hipógeos (Basidiomycetes) coletados em plantações de Eucalyptus spp. na Região Central do Rio Grande do Sul, Brasil, com base em características morfológicas e moleculares. O período de coleta dos fungos foi de maio de 2009 a julho de 2010. Um total de cinco espécies pertencentes a três famílias foram identificadas. Este material foi analisado para verificar suas características morfológicas e moleculares. Dentre as espécies identificadas estão: Chondrogaster pachysporus Maire, Descomyces albus (Berk.) Bougher & Castellano, Hysterangium affine Massee & Rodway in Massee, Hysterangium inflatum Rodway e Setchelliogaster tenuipes (Setch.) Pouzar. Adicionalmente identificou-se uma segunda espécie de Chondrogaster sp. porém não se chegou a uma espécie conhecida, acreditando tratar-se de uma espécie ainda desconhecida para a ciência. Dentre as espécies estudadas, destacam-se a Descomyces albus que é citada pela primeira vez para o Estado do Rio Grande do Sul, Hysterangium affine e H. inflatum que têm sua ocorrência registrada pela primeira vez no Brasil, além de Chondrogaster packysporus citado pela primeira vez para a América do Sul.

In dieser Arbeit wurde die progenetische Evolution der Dinophilidae innerhalb der Eunicida („Polychaeta“, Annelida) sowie der Ursprung weiterer vermutlich progenetischer Arten des Euniciden-Taxons „Dorvilleidae“ (Parapodrilus psammophilus und Microdorvillea sp. n.) mit Hilfe molekularer Daten untersucht. Ein etwa 1800 bp langer Abschnitt der 18S-rDNA wurde erfolgreich von den in Tabelle 1 aufgeführten Arten (s. S. 5-7), mit Ausnahme von Ophryotrocha puerilis und Pusillotrocha akessoni, sequenziert. Von der 28S-rDNA wurde ein etwa 2250 bp langer Abschnitt von Trilobodrilus heideri, Protodorvillea kefersteinii, Eunice sp. und Hyalinoecia tubicola sequenziert. Von den folgenden Arten wurden 488 bzw. 482 bp der CO I bestimmt: Rheomorpha neiswestonovae, Trilobodrilus axi, Trilobodrilus heideri, Ophryotrocha gracilis, Ophryotrocha puerilis, Protodorvillea kefersteinii, Schistomeringos rudolphi, Eunice sp., Marphysa sanguinea, Lumbrineris funchalensis, Hyalinoecia tubicola, Potamodrilus fluviatilis und Sabella crassicornis. Zusätzliche Sequenzen der 18S-rDNA, der 28S-rDNA und der CO I wurden der Datenbank GenBank entnommen. Weitere Sequenzen der 28S-rDNA und der CO I wurden freundlicherweise von Frau Jördens zur Verfügung gestellt. Vor den phylogenetischen Analysen wurden Bereiche unterschiedlicher Variabilität definiert. Unterschiede zwischen den Substitutions-, Transitions-, Transversions- und allgemeinen Mutationsraten sowohl untereinander als auch zwischen den Variabilitätsbereichen sowie Sättigungen wurden ermittelt. Das phylogenetische Signal wurde mittels Likelihood Mapping verdeutlicht. Phylogenetische Analysen der einzelnen Gene sowie in Kombination der beiden nukleären ribosomalen Gene und aller drei Gene wurden durchgeführt. Dabei wurden die Parsimonie-, die ML- und die Bayes´sche Analyse parallel angewendet. Soweit möglich wurden Signifikanztests durchgeführt. Die zum einen die beiden Hypothesen der Monophylie der Eunicida mit und ohne Dinophilidae gegeneinander und zum anderen die beste Lösung gegen diese beiden Hypothesen und die Hypothese einer Monophylie der Taxa der ehemaligen „Archiannelida“ verglichen. Die Voruntersuchungen an den Datensätzen der einzelnen Gene zeigen bei den drei Genen deutliche Unterschiede der Substitutionsraten sowohl zwischen den einzelnen Variabilitätsbereichen als auch untereinander. Die Muster in den beiden Genen der 28S‑rDNA und der 18S-rDNA sind sich relativ ähnlich, allerdings ist die 28S‑rDNA variabler. Die CO I unterscheidet sich deutlich von den beiden anderen Genen in ihrem Muster und in ihrer Variabilität. Es wurde in allen drei Analysen Substitutionsmodelle gewählt die diese Unterschiede adäquat berücksichtigten. In der ML- und der Bayes´schen Analyse wurden die Modelle mittels dem Programm Modeltest bzw. MrModeltest bestimmt. In den Analysen der einzelnen Gene zeichnet sich die 18S-rDNA für diese Fragestellungen durch die beste Auflösung aus. Dieses ist auf die niedrige Variabilität sowie die große Anzahl an OTUs zurückzuführen. Die 28S-rDNA ist in der Auflösung wesentlich besser als die CO I und etwa so gut wie die 18S-rDNA. Die CO I alleine ist für Fragestellungen, die die Phylogenie der höheren taxonomischen Einheiten der Polychaeten betreffen, nicht geeignet. Die Kombination meherer Gene führte ebenfalls zu einer Verbesserung der Auflösung. Dabei wird die Auflösung mit steigender Zahl der Gene besser. Auch in diesen Analysen unterstützen die „posterior probabilities“ mehr Gruppen mit signifikanten Werten und sind immer höher als die BS-Werte. Es konnte gezeigt werden, dass die Verwendung der besten Phylogenie der ML-Analyse als Startbaum in der Bayes´schen Analyse schneller und sicherer ins stabile optimale Gleichgewicht führt. Es wird daher empfohlen, diese Option wenigstens als Test für die Etablierung des stabilen optimalen Gleichgewichtes in zukünftigen Analysen zu verwenden. Die molekularen Daten lehnen eine nähere Verwandtschaft der Dinophilidae zu den Eunicida sowie zu den Taxa der ehemaligen „Archiannelida“ mit hoher Wahrscheinlichkeit, allerdings nicht signifikant, ab. Eine mögliche Verwandtschaft zu einem in die Analysen nicht eingegangenem Taxon der Eunicida kann nicht ausgeschlossen werden, da die Monophylie der Eunicida nur in den Analysen aller drei Gene bestätigt wird. Ebenfalls kann eine nähere Verwandtschaft der Dinophilidae zu einem anderen Taxon der „Polychaeta“ nicht mit signifikanter Unterstützung nachgewiesen werden. Da weder die molekularen noch die morphologischen Daten zurzeit eine eindeutige systematische Einordnung der Dinophilidae innerhalb der „Polychaeta“ erlauben, sollten die Dinophilidae wieder als eigenständiges Taxon innerhalb der „Polychaeta“ geführt und keinem anderen Taxon zugeordnet werden. Der progenetische Urspung der Dinophilidae ist aufgrund morphologischer Untersuchungen gut belegt. Die enge Verwandtschaft sowohl von Parapodrilus psammophilus als auch Microdorvillea sp. n. zu großen kiefertragenden Dorvilleiden mit polytrochen Larven wird mit signifikanten Werten in allen Analysen unterstützt. Die molekularen Daten unterstützen somit die vermutete progenetische Evolution von wenigstens Parapodrilus psammophilus. Dadurch dass die Dinophilidae mit hoher Wahrscheinlichkeit nicht in die „Dorvilleidae“ oder Eunicida eingeordnet werden können, ist auch die systematische Einordnung der Gattungen ohne muskulöses Pharynx-Organ (Apodotrocha und Apharyngtus) in die „Dorvilleidae“ nicht mehr mit eindeutiger Sicherheit gegeben. Sie wurden aufgrund der gleichen morphologischen Merkmale wie die Dinophilidae den „Dorvilleidae“ zu geordnet. Die molekular-phylogenetischen Analysen unterstützen nur in den kombinierten Analysen aller drei Gene die Monophylie der Eunicida. Dieser ist wahrscheinlich auf die „explosive Radiation“ dieses Taxons sowie der Taxa der Polychaeten im Allgemeinen zurückzuführen. Die nahe Verwandtschaft von Eunicidae und Onuphidae wird in allen Analysen, außer in denen mit der CO I, signifikant unterstützt. Die molekularen Daten unterstützen eine Monophylie der „Dorvilleidae“ nicht. Da der ctenognathe Kieferapparat der „Dorvilleidae“ sehr wahrscheinlich ein plesiomorphes Merkmal innerhalb der Eunicida ist, wird das Taxon auch morphologisch durch kein autapomorphes Merkmal charakterisiert. Die „Dorvilleidae“ sollten deshalb als parapyhletisch innerhalb der Eunicida betrachtet werden und mit Anführungsstrichen geführt werden. Die systematische Position der Histriobdellidae innerhalb der Eunicida kann basierend auf den Analysen der 18S-rDNA nicht eindeutig geklärt werden. Allerdings legt die Analyse, die eine Monophylie der Eunicida ohne Dinophilidae erzwingt, eine Verwandtschaft mit Ophryotrocha gracilis nahe. Zukünftige molekular-phylogenetische Analysen sowohl die Phylogenie der Annelida und im Besonderen der Eunicida als auch die systematische Einordnung der Dinophilidae betreffend sollten vor allem bei den Genen der 28S-rDNA und CO I noch mehr Taxa und Arten umfassen, um der geringen Auflösung der basalen Verzweigungen in allen Analysen und Problemen wie der „long branch attraction“ in zwei der Analysen mit der 28S-rDNA besser zu begegnen. Ferner sollte der Datensatz noch um andere Gene, wie zum Beispiel dem Elongationsfaktor 1a oder den Histonen, mit einer möglichst großen Zahl an Taxa erweitert werden.

The intestinal microbiota is composed of up to 100 trillion microorganisms of which bacteria are overwhelming majority. The microbiota affects the development of the immune system, defence against pathogens, host nutrition, vitamin synthesis or fat storage and its composition is changing throughout life. Some studies point to an association between microbiota composition and the development of inflammatory bowel disease. One of the treatment options is anti-TNFα antibodies therapy, which uptake or antagonize the TNFα cytokine that otherwise mediates inflammation in the intestinal mucosa. The aim of the thesis was to examine how this treatment affects the composition of the intestinal bacteriome in paediatric patients with Crohn's disease, and to find specific bacterial taxa, whose abundance changes during the treatment. By inclusion of patients with juvenile idiopathic arthritis, also treated with anti-TNFα, the study aims to discern specific effects of therapeutically induced intestinal restitution (observable in patients with Crohn's disease) from general effects of anti-TNFα therapy. Stool samples from healthy children were used to determine "healthy" bacteriome. The composition of the bacteriome was studied by profiling the variable region of the V4 gene of 16S rDNA from patients stool samples...

Mixosporídeos apresentam ampla distribuição geográfica e infectam principalmente peixes e invertebrados. Até o momento foram descritas aproximadamente 2.400 espécies. No entanto, para a América do Sul, onde se encontra a maior diversidade ictiofaunística, ainda existem poucos estudos. Esse estudo foi desenvolvido na Bacia do rio São Francisco a qual possui uma extraordinária ictiofauna com aproximadamente 158 espécies descritas. Entre estas, algumas espécies como aquelas da família Bryconidae merecem destaque pela importância econômica na pesca extrativista e potencial de cultivo. As analises morfológicas (microscopia de luz e análise ultraestrutural), biologia molecular (PCR e sequenciamento) foram utilizadas para descrever duas espécies novas de Myxobolus encontradas infectando um bryconídeo endêmicos do Rio São Francisco popularmente conhecido como matrinxã (Brycon orthotaenia). Myxobolus sp.1 foi encontrada no ovário e Myxobolus sp. 2 no fígado de B. orthotaenia. O estudo da interação parasito-hospedeiro permitiu a análise do processo de desenvolvimento do parasito e como interage com o hospedeiro. O estudo taxonômico foi realizado a partir da análise morfológica, utilizando microscopia de luz, microscopia eletrônica de transmissão e análise molecular através do sequenciamento do 18s rDNA. A análise filogenética também foi realizada. / Mixosporids have a wide geographical distribution and mainly infect fish and invertebrates. So far approximately 2,400 species have been described. However, for South America, where the greatest ichthyofaunistic diversity is found, there are still few studies. This study was developed in the São Francisco river basin where an extraordinary ichthyofauna has been reported with approximately 158 species described. Among these, some species such as the ones in the Bryconidae family deserve special attention because of their economic importance for extractive fisheries and potential for farming. Morphological analyses (light microscopy and ultrastructural analysis), molecular biology (PCR and sequencing) were used to describe two new species of Myxobolus found infecting an endemic bryconid, Brycon orthotaenia known as matrinxã, from São Francisco River. Myxobolus sp.1 was found in the ovary and Myxobolus sp. 2 in the liver of B. orthotaenia. The study of host-parasite interaction allowed an analysis of the developmental process of the parasite and how it interacts to the host. The taxonomic study was done by morphological analysis, using light microscopy, transmission electron microscopy and the molecular analysis was done through the sequencing of the 18S rDNA. The phylogenic tree was also performed.

Mixosporídeos apresentam ampla distribuição geográfica e infectam principalmente peixes e invertebrados. Até o momento foram descritas aproximadamente 2.400 espécies. No entanto, para a América do Sul, onde se encontra a maior diversidade ictiofaunística, ainda existem poucos estudos. Esse estudo foi desenvolvido na Bacia do rio São Francisco a qual possui uma extraordinária ictiofauna com aproximadamente 158 espécies descritas. Entre estas, algumas espécies como aquelas da família Bryconidae merecem destaque pela importância econômica na pesca extrativista e potencial de cultivo. As analises morfológicas (microscopia de luz e análise ultraestrutural), biologia molecular (PCR e sequenciamento) foram utilizadas para descrever duas espécies novas de Myxobolus encontradas infectando um bryconídeo endêmicos do Rio São Francisco popularmente conhecido como matrinxã (Brycon orthotaenia). Myxobolus sp.1 foi encontrada no ovário e Myxobolus sp. 2 no fígado de B. orthotaenia. O estudo da interação parasito-hospedeiro permitiu a análise do processo de desenvolvimento do parasito e como interage com o hospedeiro. O estudo taxonômico foi realizado a partir da análise morfológica, utilizando microscopia de luz, microscopia eletrônica de transmissão e análise molecular através do sequenciamento do 18s rDNA. A análise filogenética também foi realizada. / Mixosporids have a wide geographical distribution and mainly infect fish and invertebrates. So far approximately 2,400 species have been described. However, for South America, where the greatest ichthyofaunistic diversity is found, there are still few studies. This study was developed in the São Francisco river basin where an extraordinary ichthyofauna has been reported with approximately 158 species described. Among these, some species such as the ones in the Bryconidae family deserve special attention because of their economic importance for extractive fisheries and potential for farming. Morphological analyses (light microscopy and ultrastructural analysis), molecular biology (PCR and sequencing) were used to describe two new species of Myxobolus found infecting an endemic bryconid, Brycon orthotaenia known as matrinxã, from São Francisco River. Myxobolus sp.1 was found in the ovary and Myxobolus sp. 2 in the liver of B. orthotaenia. The study of host-parasite interaction allowed an analysis of the developmental process of the parasite and how it interacts to the host. The taxonomic study was done by morphological analysis, using light microscopy, transmission electron microscopy and the molecular analysis was done through the sequencing of the 18S rDNA. The phylogenic tree was also performed.

Made available in DSpace on 2017-07-10T14:17:13Z (GMT). No. of bitstreams: 1 Dissertacao PPRN Mariane_ Gavazzoni 2016.pdf: 1443094 bytes, checksum: 5fe99142fa5f6e62c58e029f07ceeebb (MD5) Previous issue date: 2016-02-22 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Astyanax is a polyphyletic taxon with richness of species and wide geographic distribution. Astyanax comprises species that are morphologically very similar with poorly detailed taxonomic delimitations, which makes it difficult to identify and to establish phylogenetic relationships. In order to provide data to contribute to cytogenetic, taxonomy and systematics of Astyanax, cytogenetics analyzes were carried out on ten Astyanax species from three river basins. Astyanax altiparanae and A. aff. fasciatus were collected on the Upper Paraná River basin; A. abramis and A. asuncionensis were collected on the Middle-low Paraná River basin; and A. cf. aramburui, A. eigenmanniorum, A. aff. fasciatus, A. jacuhiensis, A. aff. laticeps, A. cf. paris and Astyanax sp. were collected on the Upper-Middle Uruguai River basin. The results show interespecific variation in diploid number, 2n=46 chromosomes for A. cf. aramburui and A. aff. fasciatus, 2n=48 chromosomes for A. eigenmanniorum, and 2n=50 chromosomes for the remaining species. NORs (Ag-staining and 18S rDNA-FISH) showed species bearing single sites and species bearing multiple sites (up to 10 cistrons in Astyanax sp.), confirming the high variability reported for the genus. The species from A. bimaculatus complex (A. abramis, A. altiparanae, A. jacuhiensis e A. asuncionensis) showed single NORs, a plesiomorphic condition for the complex. FISH with 5S rDNA probes revealed a more conserved condition, with centromeric sites in at least one metacentric chromosome pair and one subtelocentric/acrocentric chromosome pair, however with interspecific variation, which proves it to be an important marker in the characterization and differentiation of these species. Heterochromatin distribution pattern was distinct for all species, except for A. cf. aramburui and A. aff. fasciatus (Ijuí River). This demonstrates that cytogenetic similarities may indicate closer relationship between each other than among to the other analyzed species; on the other hand, 5S rDNA genes showed to be important in differentiation of these cryptic species. The results reported the first cytogenetic data for A. cf. paris and reinforce their cytogenetic similarity with other congenus species, and we also report the occurrence of a Astyanax species that has not been taxonomically described yet. Thus, the results of this study provide data that assist taxonomy and systematic of Astyanax clade and Astyanax paris clade , reinforcing the need for extensive revisions, especially in A. bimaculatus and A. fasciatus complex, including markers such as 5S rRNA and heterochromatin distribution pattern, forthe better understanding of the phylogenetic relationships in Astyanax. / Astyanax é um taxon polifilético com grande riqueza de espécies e ampla distribuição geográfica. Compreende espécies com formas bastante semelhantes e delimitações taxonômicas pouco detalhadas que dificultam a identificação e o estabelecimento das relações filogenéticas. Com o objetivo de fornecer dados que contribuam com a citogenética, taxonomia e sistemática de Astyanax, foram realizadas análises citogenéticas em dez espécies de Astyanax de três bacias hidrográficas. Foram coletados exemplares de Astyanax altiparanae e A. aff. fasciatus na bacia do Alto rio Paraná; A. abramis e A. asuncionensis na bacia do Médio-Baixo rio Paraná; e A. cf. aramburui, A. eigenmanniorum, A. aff. fasciatus, A. jacuhiensis, A. aff. laticeps, A. cf. paris e Astyanax sp. na bacia do Alto-Médio rio Uruguai. Os resultados mostraram variação interespecífica no número diplóide, de 2n=46 cromossomos em A. cf. aramburui e A. aff. fasciatus, 2n=48 cromossomos em A. eigenmanniorum, e 2n=50 cromossomos nas demais espécies. As AgRONs, confirmadas pela 18S rDNA-FISH, evidenciaram espécies portando sítios simples e espécies portando sítios múltiplos (até 10 cístrons em Astyanax sp.), confirmando a alta variabilidade encontrada no gênero. Nas espécies do complexo A. bimaculatus (A. abramis, A. altiparanae, A. jacuhiensis e A. asuncionensis), foram evidenciadas RONs simples, característica plesiomórfica para o complexo. FISH com sonda de 5S rDNA evidenciou uma condição mais conservada, com cístrons centroméricos em pelo menos um par de cromossomos metacêntricos e em um par de cromossomos subtelocêntricos/acrocêntricos, porém com variação interespecífica, demostrando ser um importante marcador na caracterização e diferenciação destas espécies. O padrão de distribuição da heterocromatina mostrou-se distinto para as espécies, com exceção de A. cf. aramburui e A. aff. fasciatus (rio Ijuí), onde foi verificado semelhanças citogenéticas que podem indicar maior proximidade entre estas espécies quando comparadas com as demais analisadas, sendo que a distribuição dos genes 5S rDNA se mostrou importante na diferenciação destas espécies crípticas. Os resultados relatam os primeiros dados citogenéticos para A. cf. paris e reforçam sua semelhança citogenética com outras espécies congêneres, além de relatar a ocorrência de uma espécie de Astyanax ainda não descrita taxonomicamente. Em suma, os resultados do presente estudo fornecem dados que auxiliam na taxonomia e sistemática do clado Astyanax e clado Astyanax paris , reforçando a necessidade de revisões amplas, principalmente nos complexos A. bimaculatus e A. fasciatus que incluam marcadores como 5S rDNA e padrão de distribuição da heterocromatina, para melhor compreensão das relações filogenéticas em Astyanax.

A metagenômica trouxe novas perspectivas ao estudo de comunidades microbianas no ambiente, permitindo explorar tanto a diversidade taxonômica de microrganismos ainda não-cultivados, como o acesso direto a genes e vias metabólicas. Neste trabalho, foram construídas bibliotecas metagenômicas a partir de amostras de sedimentos de mangue da Baía de Guanabara (RJ), impactadas com hidrocarbonetos de petróleo e metais pesados. Proteobacteria (33,3%), bactérias afiliadas a redutoras-de-sulfato (29,7%) e Firmicutes (20%) representaram os grupos principais nas amostras ambientais, baseado em análises filogenéticas de rDNA 16S, ao passo que isolamentos seletivos utilizando diesel e naftaleno permitiram a recuperação preferencial de delta-Proteobacteria e actinomicetos. Bibliotecas metagenômicas dos sedimentos enriquecidos com óleo diesel, com insertos entre 25 e 35 Kb clonados em fosmídeos, foram triadas para detecção de genes catabólicos de monoxigenases (alkB1) e expressão de epóxido-hidrolases, esterases, lipases e monoxigenases em ensaios de alto desempenho (HTS, high throughput screening). Clones reativos a alkB1 foram detectados, porém não foram funcionais nas condições de HTS testadas. Nas bibliotecas de fosmídeos triadas, vários clones apresentaram atividade enzimática, sendo que dois apresentaram atividade de lipase-esterase com alta seletividade, elevada taxa de conversão de substratos e excesso enantiomérico (ee >99%). Os resultados de HTS comprovaram a eficiência do uso da clonagem direta de DNA ambiental na expressão de vias metabólicas de interesse com potencial de aplicação biotecnológica. / Metagenomics brought a new perspective to the study of microbial communities in the environment, enabling access to the taxonomic diversity of uncultured microorganisms, as well as direct access to genes and metabolic pathways. In the current study, metagenomic libraries were constructed from mangrove sediment samples of the Guanabara Bay (RJ, Brazil), impacted with oil hydrocarbons and heavy metals. Proteobacteria (33.3%), sulfate-reducing affiliated bacteria (29.7%) and Firmicutes (20%) represented the main groups in the environmental samples based upon 16S rDNA phylogenetic analysis, whereas selective isolation using diesel and naphtalene yielded delta-Proteobacteria and actinomycetes. Metagenomic libraries of diesel-enriched sediment samples, with 25 to 35 Kb fosmid inserts, were screened for detecting monooxigenase genes (alkB1) and expression of epoxide hydrolases, esterases, lipases and monooxigenases in high throughput screening (HTS) assays. Clones reactive to the alkB1 probe were detected, but were not functional under the HTS conditions used. Several functional clones were detected in the clone library, and two showed lipase-esterase activity with high rates of substrate conversion and enantiomeric ratio (ee >99%). The results obtained on HTS showed the efficiency of the direct cloning of environmental DNA for the expression of metabolic pathways with potential biotechnological application.

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 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.