Made available in DSpace on 2017-07-10T18:13:20Z (GMT). No. of bitstreams: 1 Rafael Henrique da Rocha.pdf: 1457832 bytes, checksum: 601a9b8393782cd1f8fee92a45fbe86c (MD5) Previous issue date: 2016-04-06 / Fundação Araucária / Siluriforms is one of the most significant orders and greater diversity of species in the Neotropics, being Corydoras genus with the highest number of species. This group has color patterns and external morphology very similar among species. In this context, the present study used the cytogenetics and morphometry to characterize and differentiate Corydoras carlae and Corydoras sp. B of the Iguassu River basin. The diploid number found was 46 chromosomes, with karyotype formula of 22m + 22sm + 2st, and NF equal to 92. The impregnation with silver nitrate showed two bearing-NORs chromosomes and fluorescent hybridization with 18S ribosomal probe confirmed this result, charactering simple NORs system for species. Furthermore, the 5S rDNA was co-located with the 18S rDNA for Corydoras carlae and Corydoras sp. B. However, Corydoras sp. B have an extra marking 5S rDNA, located in interstitial position on the short arm of one submetacentric chromosome. The heterochromatin constitutive was found in centromeric and pericentomeric regions for both species, but with differences in the bearing chromosomes. The morphometric traits showed morphological differences between the two species, provided by indices: body height / standard length; interorbital distance / length of the head; horizontal diameter of orbit / length of the head. The results demonstrate that although both species have the same diploid number,e same karyotype formula and simple NORs system, C. carlae e Corydoras sp. B are different how much the location of the heterochromatin constitutive and in the 5S rDNA number bearing chromosomes, and present morphological differences which distinguish the two species. / Siluriformes é uma das ordens mais representativas e com maior diversidade de espécies da região Neotropical, sendo Corydoras o gênero com o maior número de espécies. Esse grupo apresenta padrões de coloração e morfologia externa muito semelhantes entre as espécies. Nesse contexto, o presente estudo utilizou a citogenética e a morfometria para caracterizar e diferenciar as espécies Corydoras carlae e Corydoras sp. B da bacia do rio Iguaçu. O número diplóide encontrado foi de 46 cromossomos, com fórmula cariotípica de 22m+22sm+2st e NF igual a 92 para ambas as espécies. A impregnação com o nitrato de prata revelou dois cromossomos portadores de RONs e a hibridização in situ fluorescente com sonda ribossomal 18S confirmou este resultado, caracterizando sistema de RONs simples para as espécies. Além disso, o DNAr 5S foi co-localizado com o DNAr 18S para Corydoras carlae e Corydoras sp. B. No entanto, Corydoras sp. B tem uma marcação extra de DNAr 5S, localizada em posição intersticial no braço curto de um cromossomo submetacêntrico. A heterocromatina constitutiva foi evidenciada em regiões centroméricas e pericentoméricas para ambas espécies, porém com diferenças nos cromossomos portadores. Os caracteres morfométricos avaliados demonstraram diferenças morfológicas entre as duas espécies, proporcionada pelos índices: altura do corpo/comprimento padrão; distância interorbital/comprimento da cabeça; diâmetro horizontal da orbita/comprimento da cabeça. Os resultados demonstram que, apesar das espécies apresentarem o mesmo número diplóide, mesma fórmula cariotípica e sistema de RONs simples, C. carlae e Corydoras sp. B são diferentes quanto a localização da heterocromatina constitutiva e o número de cromossomos portadores de DNAr 5S, além de apresentarem diferenças morfológicas que separam as duas espécies.

Des recherches en sciences judiciaires ont montré récemment une possible corrélation entre le temps d’entreposage d’échantillons de fluides corporels et la dégradation de l’ARN dans ceux-ci. Le moment où une tache a été déposée sur une scène de crime peut être important pour déterminer la pertinence d’un échantillon dans une enquête. Dans ce mémoire, nous rapportons les profils de dégradation de quatre ARN différents mesurés par RT-qPCR, soit l’ARN ribosomique 18S et les ARNm de la β-actine, de la glyceraldehyde-3-phosphate déhydrogénase et de la cyclophiline A, obtenus de taches de sang, de salive et de sperme, entreposés à la température de la pièce ou au congélateur à -80°C sur une période de 6 mois. Nos résultats montrent une faible variation interindividuelle pour le sang et le sperme, mais une différence importante entre les donneurs pour la salive. De plus, le profil de dégradation est semblable pour tous les transcrits, mais diffère entre les fluides. La congélation des échantillons stabilise les ARN avant leur analyse. Finalement, la quantité d’ARN détecté est en relation avec le temps d’entreposage et pourrait être utilisée afin d’estimer l’âge des échantillons lorsque l’impact des conditions d’entreposage sur la dégradation de l’ARN sera mieux connu. / Recent studies in forensic science have shown a possible correlation between the degradation rate of some RNA transcripts and the age of bloodstains. The time of deposition of a stain can be of major importance to determine the relevance of a sample in a forensic investigation. In this thesis, we describe the degradation profiles of the 18S ribosomal RNA and the β-actin, glyceraldehyde-3-phosphate dehydrogenase and cyclophilin A mRNAs, measured by RT-qPCR and obtained from dried blood, semen and saliva stains stored at room temperature or frozen at -80°C up to 6 months. Our results showed low inter-individual variation for blood and semen stains, but a high variation was observed between donors for saliva. Moreover, degradation profile of each transcripts was similar, but differed between fluids. Freezing samples prevented RNA degradation over time. Finally, RNA quantity was in relation with the time of storage and could be used to estimate the time since deposition of a stain when the effects of various storage conditions on RNA degradation profiles will be better documented.

Los Thraustochytriales o mejor conocidos como thraustochitridos son protistas pertenecientes al Grupo Chromista según los análisis del gen 18S rRNA. Considerados como una potencial fuente alternativa al aceite de pescado, estos microorganismos oleaginosos han sido aislados de diversos ambientes a nivel mundial encontrándose principalmente asociados a material vegetal en descomposición. Así, en este estudio se logró aislar cepas de thraustochitridos provenientes de todos los puntos muestreados en los manglares de Tumbes donde la caracterización bioquímica con acriflavina y rojo de nilo confirmaron su ocurrencia y los resultados de caracterización molecular permitieron determinar aislados pertenecientes a los géneros Aurantiochytrium (basónimo: Shizochytrium), Parietichytrium, Botryochytrium e Ulkenia sensu stricto. Asimismo se propone el posible descubrimiento de dos nuevas especies con potenciales biotecnológicos en base a información proporcionada de las características observadas en cultivo, de los análisis filogenéticos y de trabajos previos relacionadas a las cepas Aurantiochytrium sp. 15A-14a (Genbank Accesion N° AB811008) y Schizochytrium sp. S8 (Genbank Accesion N° DQ836630), los cuales dieron el mayor hit con los aislados 75, 507, 510, 512, 523, 526, 527 y C1, respectivamente. Finalmente, el presente estudio brindó los primeros reportes de la presencia de estos thraustochitridos en nuestro país.

Infertility is defined as not being able to conceive after 1 year of regular intercourse without use of contraception. Unexplained infertility is a diagnosis given to couples where the reason to infertility cannot be clarified even after the routine examination. Undefined infertility is a common and growing problem because most people are not aware of the fact that fertility decreases after the age of 35. Hyper- and hypothyroidism has been known to affect the menstrual cycle as well as increased risk of miscarriage. However, the specific effect of thyroid hormones on infertility has not yet been clarified. This study aims to compare the gene expression of two thyroid hormone receptors DIO2 and MOT8 in human endometrium and fallopian tube tissue from two phases of the menstruation cycle, follicular phase and lutheal phase. The methods used were RT-qPCR and immunohistochemistry, which showed a statistically significant difference in the expression of DIO2 and MOT8 between fallopian tube tissue and endometrium, but not between follicular and lutheal phase. However, MOT8 seemed to have a tendency to be down-regulated in the follicular phase but the results need to be validated with different endogenous controls and larger study groups.

Insect species complexes challenge entomologists in a variety of ways ranging from quarantine protection to pest management. Billbugs (Coleoptera: Curculionidae: <i>Sphenophorus</i> spp. Schönherr) represent one such species complex that has been problematic from a pest management perspective. These grass-feeding weevils reduce the aesthetic and functional qualities of turfgrass. Sixty-four species of billbugs are native to North America, and at least ten are associated with damage to turfgrass. Billbug species are sympatric in distribution and their species composition and seasonal biology varies regionally. Since their management relies heavily on proper choice of insecticide active ingredients and timing of insecticide applications that target specific life stages, understanding billbug seasonal biology underpins the development of efficient management programs. However, billbug seasonal biology investigations are currently hindered by our inability to identify the damaging larval stage to species level. DNA barcoding, which involves the use of short DNA sequences that are unique for each species, represents one potential tool that can aid these efforts. By combining DNA-based species identification with morphometric measures capable of serving as a proxy of larval development, it may be possible to gain a more holistic understanding of billbug seasonal biology. In this study, we developed a DNA barcoding reference library using cytochrome oxidase subunit 1 (COI) sequences from morphologically identified adult billbugs collected across Indiana, Missouri, Arizona, and Utah. Next, we applied our reference library for comparison and identification of unknown larval specimens collected across the growing season in Utah and Indiana. We then used a combination of DNA barcoding and larval head capsule diameters acquired from samples collected across a short span of the growing season to produce larval phenology maps. Adult billbug COI sequences varied within species, but the variation was not shaped by geography, indicating that this locus itself could resolve larval species identity. Overlaid with head capsule diameter data from specimens collected across the growing season, a better understanding of billbug species composition and seasonal biology emerged. This knowledge will provide researchers with the tools necessary to fill critical gaps in our understanding of billbug biology thereby improving turfgrass pest management. Using this approach researchers will be able to support efforts to provide growers with the information necessary to develop more prescriptive, location-based management programs and reduce the ecological footprint of turfgrass pest management.

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.

Le mycobiome digestif comprend l’ensemble des espèces de champignons contenu dans le tube digestif. Au cours de cette thèse, nous avons réalisé dans un premier temps une revue de la littérature sur le mycobiome digestif afin d’établir le répertoire des espèces de champignons décrites dans le tube digestif et leur implication dans les infections digestives et systémiques. Puis dans un second temps, notre travail expérimental a combiné l’approche culture et l’approche moléculaire basée sur le séquençage des clones pour explorer la diversité du mycobiome digestif. Nous avons répertorié une variété d’amorces PCR dans la littérature ciblant le gène 18S rRNA et ITS rRNA. En appliquant ces outils moléculaires et la culture à l’analyse d’une selle chez un sujet obèse nous avons détecté 16 espèces de champignons dont 11 espèces sont associées à l’alimentation avec 8 espèces de champignons observées pour la première fois dans les selles.Ensuite, la même approche appliquée à une selle collectée chez un sujet anorexique a permis de détecter 8 espèces de champignons dont 5 espèces associées au régime alimentaire du sujet et 3 espèces retrouvées pour la première fois dans les selles humaines.Dans un dernier temps en utilisant la même méthodologie, nous nous sommes intéressés à la diversité du mycobiome en fonction de l’origine géographique des sujets. Pour cela, nous avons exploré la communauté du mycobiome dans les selles provenant des quatre continents Afrique, Asie, Amérique et l’Europe. Dans ce travail, nous avons détecté 40 espèces de champignons provenant de l’environnement dont certains pathogènes opportunistes. / The human gut mycobiome, comprising of all fungal species detected in the gut. The Human Microbiome Project and the Metagenomics of the Human intestinal tract projects has led to new interest in the study of the human gut mycobiome. Recently, culture-independent approaches including PCR-based molecular clone libraries sequencing and high-throughput sequencing allowed to explore the diversity of gut mycobiota. In this thesis, firstly, we reviewed fungal species described in the human gut and their implication in systemic infections. We reported from literature fungal species described in healthy individuals compared to repertoire described in diseased individuals.Secondly, in our experimental work we used molecular and culture approaches to explore gut mycobiota diversity related to host physiology. We selected various set of PCR primers from literature targeting 18S rRNA gene and ITS rRNA gene. Combining molecular and culture tools in stool specimen from an obese individual we detected 16 fungal species, 11 were linked to food and 8 species were found for first in the human stools. Using the same approaches in an anorexic individual stool we identified 8 fungal species, five were associated to subjected diet collected and three fungal species were observed for the first time in the human stools. From these two studies, we observed that the gut mycobiome diversity is part associated to diet.Using the same methodology, we to explored gut mycobiota diversity according to different geographical locations. For this, fungal diversity was screened in stools samples from four continents Africa, America, Asia and Europe.

This thesis presents a comprehensive phylogenetic and taxonomic study of an acoel subgroup, the Childiidae. Members of this taxon are characterized by well-developed male copulatory organs with conical/cylindrical stylets. The phylogenetic analyses, by means of total evidence approach, based on three molecular markers (18S and 28S rRNA genes and Histone H3) and 50 morphological characters reaffirm the non-monophyly of the Childiidae sensu Dörjes, 1968 (Actinoposthiidae and Childia+Paraphanostoma). The total evidence phylogeny strongly support the Childia+Paraphanostoma clade separate from other former members of the Childiidae, which are now placed in Actinoposthiidae. The monophyly of Childia+Paraphanostoma is well corroborated by several morphological characters. A new taxon Childia is defined, in accordance with the PhyloCode, comprising all former Paraphanostoma species and a member of the monotypic genus C. groenlandica. A new diagnosis for the current members of Childia is provided. Several structures, shown to hold promising phylogenetic signals for unraveling acoel relationships, such as musculature pattern, sperm and male copulatory organs, are investigated, using a combination of traditional and modern techniques (ultrastructure, immunocytochemistry and confocal microscopy), with main focus on Childia and its closest relatives. New characters are described and their phylogenetic significance assessed. Morphological characters relating to body-wall musculature, statocyst muscles, male copulatory organ musculature and ultrastructure, and sperm cytoplasmic granules are shown to carry important phylogenetic signals at lower taxonomic levels, while most of the characters related to sperm ultrastructure are useful at higher taxonomic levels within the Acoela. The data obtained undermine the phylogenetic use of the seminal bursa in Childia. In addition to this, it is shown that most of the classical morphological characters used in acoel taxonomy, obtained using traditional histological methods, may be misleading in identifying monophyletic entities within the Acoela. The most corroborated synapomorphies, identified in this thesis, are used in determining the taxonomic placement of a new viviparous acoel, Childia vivipara, into the taxon Childia.

Les champignons sont des organismes hétérotrophes ubiquistes jouant des rôles pivots dans de nombreux écosystèmes (e.g. décomposeurs, symbiontes) et formant une lignée eucaryote majeure. Les Chytridiomycota constituent les branches basales du règne des Champignons, soit une position critique pour la compréhension de la radiation évolutive fongique. Or, ce groupe a été peu étudié, ce qui ne permet qu'une résolution partielle de ces relations évolutives. Ici, nous décrivons la diversité fongique, en ciblant principalement les Chytridiomycota via des analyses de génomique environnementale. Des échantillons de diverses natures ont été collectés au niveau de sources hydrothermales marines profondes qui sont connues pour être des hotspots de diversité, avec un fort taux d'endémisme. Pour l'analyse des séquences moléculaires obtenues, une base de données (PHYMYCO-DB) portant sur des marqueurs moléculaires fiables et dédiés à la phylogénie fongique, a été créée puis mise en ligne. Des outils moléculaires développés au cours de cette thèse ont permis de récupérer six phylotypes Chytridiomycota dont quatre produisent des branches non-décrites à ce jour. De plus, la diversité obtenue n'est pas limitée au clade des Opisthokonta (i.e. principalement les règnes des Animaux et des Champignons) puisqu'il a également été observé un phylotype Apusozoa et deux phylotypes ayant une position indéfinie à la base des Unikonta. Ces résultats offrent des perspectives pour la description de nouveaux organismes via le séquençage de génomes ou l'imagerie. Ces organismes sont également prometteurs pour la résolution des relations évolutives chez les Opisthokonta, les Unikonta, voire les Bikonta.

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.