Differences in patterns of species richness and taxonomic composition across continents are well documented. However, less is known about how these patterns originate from the fundamental processes that contribute to the assembly of continental biotas: speciation, extinction, immigration, and emigration. To truly decipher how these processes operate at a continental scale, it is crucial to understand how Earth-history events and environmental change shaped the biogeographical history of the taxa occurring in a region. The Pleistocene glacial cycles have been hypothesized to be a significant geological event which affected the Earth’s biota over the past three million years. During this period, multiple cycles, in which ice sheets covered a large portion of the Northern Hemisphere, have been hypothesized to facilitate allopatry and ecological differentiation. The central goal of this dissertation is to understand the assembly of continental biotas by integrating the role of Earth’s geological and environmental history and recent (late Quaternary) changes in distributions. Here, I studied the North American wood-warblers, which are passerine birds belonging to the family Parulidae. In the first chapter of this dissertation, I examined the extent to which recent diversification is underestimated by ignoring recently diverged taxa. To do so, I evaluated the effect of taxonomic delimitation on the inferred temporal patterns of diversification of wood-warblers in the genus Setophaga. My results show that species-level taxonomic delimitation in ecological and evolutionary studies is crucially important but is often ignored. Evaluating the effect of taxonomic delimitation in the genus Setophaga is particularly relevant because it has been widely cited as an ideal example of niche saturation, and previous studies suggested that lineages in this particular radiation exhibited an asymptotic accumulation of diversity through time. In this chapter, however, I demonstrate that this pattern was likely a consequence of the ways in which taxonomic diversity was sampled. In the second chapter, I examined how biotic and abiotic factors limit the distribution of species of the genus Oreothlypis at a continental scale. For most of the taxa examined, climatic- and biotic-based areas of suitability were equivalent, and therefore the relative importance of these factors could not be disentangled. However, in some cases, biotic variables limited the distribution in areas climatically suitable, and vice versa. The results of this chapter highlight the importance of considering potential effects of biotic interactions in the study of climate-driven range shifts. This paper is also an important methodological contribution to the general field of ecological niche modeling (ENM) by integrating climatic and palynological data to empirically estimate both abiotically and biotically suitable areas which that has only been done theoretically so far. In the third chapter, I integrated phylogenetic data, biotic and climatic ENMs, and reconstructions of environments during the LGM to test how cycles of fragmentation, differentiation, and expansion during the Pleistocene shaped the biogeographical patterns of the genus Oreothlypis in North America. Based on a time-calibrated phylogeny I identified two groups of taxa that diversified during the last million years and therefore were potentially affected by glacial cycles. My analyses suggest that there were rapid switches in the environmental conditions in which species of the genus occur and that glacial cycles resulted in dramatic range shifts from glacial maxima to interglacials. Distribution patterns during the LGM suggest that divergence in areas isolated by glaciers presumably contributed to the geographical structuring of Oreothlypis, as well as to their taxonomic and ecological diversity in the present. In summary, this work illustrates how wood-warblers are an ideal model system for examining the large-scale history of the North America biota and environment, particularly over the Pleistocene. They are highly diverse, have endemics in virtually all of North America's areas of endemism and ecosystems, and many of these patterns are repeated within and across clades. Furthermore, distributional patterns in warblers show high congruence with those of other organisms; thus inferences made about the history of this group likely have implications for understanding the biotic history of North America in general. In addition, many warblers have narrow ecological preferences in that they occupy forest environments with specific tree compositions. As a consequence, ecological models of their distributions, integrated with the North American pollen record of forest change, provide a new perspective on ecosystem change during glacial cycles, and its impact on the origin and maintenance of biodiversity in the continent.

Orientadores: João Semir, Lucia Garcez Lohmann / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T14:46:18Z (GMT). No. of bitstreams: 1 Firetti-Leggieri_Fabiana_D.pdf: 148843515 bytes, checksum: bfb6fa893bb9997ed92b1d321acb5e7a (MD5) Previous issue date: 2009 / Resumo: O complexo Anemopaegma arvense é constituído por espécies e variedades de difícil delimitação. As opiniões dos taxonomistas, baseadas em caracteres morfológicos, são controversas em considerálas uma única espécie altamente polimórfica ou separá-las. Com o intuito de auxiliar na circunscrição de tais táxons, realizou-se um estudo biossistemático que englobou os seguintes assuntos: morfologia e morfometria de caracteres vegetativos e reprodutivos, anatomia da lâmina foliolar, contagem cromossômica, aplicação de marcadores AFLP para a delimitação genética, fenologia, biologia floral e polinização, sistema reprodutivo e hibridação e, poliembrionia em espécies do gênero com distintos níveis de ploidia. As morfoespécies do complexo diferem principalmente na morfologia foliar, sendo os táxons de Anemopaegma acutifolium caracterizados por folíolos elípticos a estreitamente oblanceolados com razão comprimento/largura do folíolo 3,5 a 18,5, os de A. arvense por folíolos lineares a estreitamente oblanceolados (razão 22,2 a 45,5) e, A. glaucum por folíolos largamente oblanceolados, oblongos a obovados (razão entre 1,69 e 3,9). A partir de caracteres morfológicos, como crescimento indeterminado dos ramos, exclui-se A. scabriusculum do complexo. O estudo morfométrico de caracteres foliares se mostrou útil para a separação das espécies. Já a morfometria de caracteres reprodutivos não foi informativa para a delimitação de tais táxons. Anatomicamente, as espécies e morfoespécies do complexo diferem quanto à disposição dos estômatos, tipo de epiderme constituição do sistema vascular da nervura mediana e composição da bainha dos feixes vasculares das nervuras laterais. Dentre as morfoespécies de A. acutifolium, A. acutifolium "típica" difere das demais por possuir folíolos anfiestomáticos e A. acutifolium "sarmentosa" por apresentar epiderme da face adaxial com desdobramentos pontuais. Já A. arvense é caracterizada pela ausência de cordões floemáticos no sistema vascular da nervura mediana e por possuir a margem destituída de parênquima fundamental subepidérmico. As morfoespécies de A. glaucum, "típica" da Bahia e "não glauca", são diferenciadas das demais pela ausência de calotas de fibras sobre o xilema nas nervuras laterais de grande e médio calibre. A. scabriusculum difere das outras espécies por possuir extensão de bainha nos feixes vasculares de grande e médio calibre e estômatos agrupados nas regiões internervurais com câmaras subestomáticas unidas. A contagem cromossômica revelou a condição poliplóide das espécies e morfoespécies do complexo, tendo estas 2n = 80. Os marcadores AFLP, apesar de serem bastante utilizados para a separação de táxons em nível infra-específico, não se mostraram eficientes para a delimitação das espécies do complexo Anemopaegma arvense. Quanto ao comportamento fenológico, A. acutifolium, A. arvense e A. glaucum apresentaram eventos anuais de brotamento, floração e frutificação. Já os indivíduos de A. scabriusculum têm dois a três eventos de floração e frutificação por ano. As flores das espécies são bastante semelhantes quanto à morfologia e recursos produzidos e são polinizadas pelas mesmas espécies de abelhas. Através de polinizações controladas constatou-se que tais espécies são auto-compatíveis e interférteis, havendo, portanto, alta probabilidade de formação de híbridos em populações simpátricas destas espécies. Notou-se uma relação positiva entre poliploidia e poliembrionia nas espécies do gênero aqui abordadas, tendo as sementes das espécies poliplóides mais de um embrião e as da espécie diplóide, A. album, somente um embrião. / Abstract: Anemopaegma arvense complex is constituted by species and varieties of difficult delimiting. Taxonomists opinions based upon morphologic features are controversial as to considering them either an only highly polymorphic species or separating them into different taxa. In order to help with the circumscription of such taxa, a biosystematic study was conducted which included the following subjects: morphology and morphometry of vegetative and reproductive features, leaflet blade anatomy, chromosome counting, AFLP markers application for genetic delimitation, phenology , floral biology and pollination, reproductive system and hybridization and polyembryony of the genus species with different ploidy levels .The morphs of the complex differ mainly in leaf morphology where the Anemopaegma acutifolium taxa are characterized by elliptical leaflets with the lengh / width ratio of leaflet falling within the range 3,5 to 18,5; A. arvense characterized by linear leaflets to narrowly oblanceolate (ratio between 22,2 and 45,5) and, A. glaucum by leaflets broadly oblanceolate, from oblong to obovate (ratio between 1,69 and 3,9).Taking into account morphologic features such as undetermined growth of the branches, A. scabriusculum may be excluded off the complex. The morphometric study of the leaf features has been found useful for species separation. However, the morphometry of reproductive features were not informative enough for the delimitation of such taxa. Anatomically, the species and morphs of the complex differ from one another as to the stomata disposition, epidermis type, vascular system constitution of the midrib and, composition of the vascular bundle of the lateral veins. Among the A. acutifolium morphs, A. acutifolium "típica" differs from the others for possessing anphistomatic leaflets and, A. acutifolium "sarmentosa" for presenting the adaxial face epidermis with punctual unfoldings. As for A. arvense, it is characterized by the absence of phloematic strings in the midrib vascular system and parenchyma absent in the marginal region. As for the two morphs, A. glaucum "típica" of Bahia and "não glauca", they are differentiated from the others by the absence of fibers caps on the xilem on the lateral ribs of large and medium caliber. A. scabriusculum differs from the other species for possessing extension sheath in the vascular bundles of large and medium caliber and, stomata grouped between vascular bundles presenting substomatic cameras linked to one another. The chromosome counting revealed polyploidy condition of the species and the morphs of the complex, those presenting 2n = 80. The AFLP markers, in spite of being quite utilized for taxa separation in an infraspecific level, were not found efficient for the species delimitation of Anemopaegma arvense complex. With relation to the phenologic behavior, A. acutifolium, A. arvense and A. glaucum presented annual events of sprouting, blooming and fructification. However, A. scabriusculumindividuals presented two to three blooming and fructification events a year. The flowers of the species are very similar to one another as to their morphology and to the resources provided by them and are pollinated by the same species of bees. Through controlled pollinations, it could be verified, that such species are self- ompatible and inter fertile, bearing, therefore, high probability of hybrid formation in sympatric populations of those species. A positive relationship between polyploidy and polyembryony in the species of the genus studied here was observed, as well as the fact that more than one embryo were found for polyploidy species seeds whereas for diploid species, A. album, only one embryo has been registered. / Doutorado / Doutor em Biologia Vegetal

Orientador: Renato Goldenberg / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T02:40:56Z (GMT). No. of bitstreams: 1 Caddah_MayaraKrasinski_D.pdf: 44109301 bytes, checksum: 6f87fe47d630845de374495c24f66a41 (MD5) Previous issue date: 2013 / Resumo: Miconia é um é um dos maiores gêneros de angiospermas. Tradicionalmente, tem sido considerado complexo e mal circumscrito, assim como outros gêneros aparentados. Estudos filogenéticos recentes, baseados em marcadores moleculares, têm atestado a natureza polifilética de Miconia e suas onze seções. Neste trabalho, é apresentado um estudo filogenético-molecular aprofundado de um dos grupos naturais encontrados em estudos anteriores, o "subclado Miconia discolor", do "clado Miconia IV". Para tanto, foram utilizados sequências de quatro marcadores plastidias (psaI-accD; psnK-L; atpF-H; trnS-G) e dois nucleares (ITS e ETS). Os resultados obtidos permitiram esclarecer o relacionamento interno e externo do grupo. Por meio da otimização de caracteres morfológicos e biogeográficos, são sugeridas hipóteses evolutivas, como a evolução de inflorescências glomeruladas e pseudantos a partir de inflorescências com ramos escorpióides amplos, e a irradiação da diversidade na Floresta Atlântica a partir de poucas colonizações. Os resultados também permitiram a primeira proposição moderna de classificação ingra-genérica para Miconia, através da formalização e circunscrição de novos táxons: Miconia sect. Discolor, e subseções Albicans, Chrysophylla, Discolor e Multispicata, subordinadas à nova seção. Adicionalmente, uma revisão taxonômica é apresentada para a recém proposta subseção Discolor, incluindo descrições, ilustrações, imagens de Microscopia Eletrônica de Varredura, comentários, e uma chave dicotômica de identificação para as espécies. A subseção é composta 32 espécies, das quais três são novas. Trinta e dois nomes heterotípicos foram sinonimizados, e 39 lectotipificações foram propostas. A subseção pode ser reconhecida por apresentar as folhas densamente cobertas por tricomas ramificados na face abaxial (nunca formando um indumento aracnóide), inflorescências geralmente glomeruladas, raramente curtamente scorpióides, lobos do cálice caducos no fruto, e uma distribuição restrita ao sudeste da América do Sul, ocorrendo na Floresta Atlântica e no Cerrado / Abstract: Miconia is one of the biggest genera in angiosperms. It has been traditionally considered very complex and badly circumscribed, and other close genera as well. Phylogenetic studies based on molecular markers have been confirming the polyphily of Miconia and its eleven sections. Here I present a deeper molecular and phylogenetic study of a natural group found out in older studies, the "Miconia discolor subclade", from the "Miconia IV clade". I used sequences of four plastid (psaI-accD; psnK-L; atpF-H; trnS-G) and two nuclear markers (ITS and ETS). The results clarified the inner and outer relationship of that group. Evolutionary hypotheses are suggested by the optimization of morphological and biogeographical charcaters. For example, the evolution of glomerulate inflorescences and pseudanthia from inflorescences of wide, scorpioid branches, and the diversification of the genus in the Atlantic Forest from few colonizations. Additionally, the results allowed the first modern infra-generic rearrangement by the proposition and circumscription of new taxa: Miconia sect. Discolor, with the subsections Albicans, Chrysophylla, Discolor and Multispicata. A revision of the new Miconia subsect. Discolor is presented, with descriptions, illustrations, Scanning Electron Microscopy images, comments, and a taxonomic key for species. The subsection has 32 species and three of them are new. I synonymized 32 heterotypic names proposed 39 lectotipifications. The subsection can be recognized by the abaxial leaf surface usually densely covered by branched trichomes (never forming a cobweb indument), inflorescences usually glomerulate, rarely shortly scorpioid, calyx lobes caducous in fruit, and a distribution restricted to the southeast of South America, in the Atlantic Forest and Cerrado / Doutorado / Biologia Vegetal / Doutora em Biologia Vegetal

Baird's pocket gopher (Geomys breviceps) is found in eastern Texas, eastern Oklahoma, central and western Arkansas, and western Louisiana. The cytochrome-b gene was sequenced and analyzed for 16 pocket gophers from throughout the range of the species. Similar phylogenetic trees were obtained using maximum-parsimony, maximum-likelihood, neighbor-joining, and Bayesian analyses. Two major clades were formed with northern individuals belonging to clade I and southern individuals belonging to clade II. G. b. sagittalis was paraphyletic in relation to G. b. breviceps in all analyses. Based on inconsistencies between the taxonomic classification and systematic relationships within Baird's pocket gopher, a taxonomic restructuring appears warranted.

Bacteria regulate gene expression through coordinated interactions between cis-regulatory sequences and trans-regulatory factors. Understanding the molecular basis for the functions of these regulatory components is not only essential for deciphering complex biological processes in diverse bacteria but also critical for rational engineering of microbial phenotypes. However, systematically dissecting the sequence-function relationships of cis and trans regulatory components that underly gene expression is still a key challenge. Recent technological advances have provided novel tools and methods for mapping sequence-function relationships in high-throughput. This dissertation focuses on applying novel methods enabled through increased throughput and scalability of DNA synthesis and sequencing to elucidate the sequence-function relationships of cis and trans components that underlie bacterial gene regulation. In Chapter 2, evolutionary and functional diversity of primary σ70, a universally conserved global regulator in bacteria, is studied through comparative genomics, saturation mutagenesis, and transcriptomics. Through the combined efforts of these approaches, we demonstrate that sequence diversity of σ70 factors reflects functional differences which have been shaped by evolutionary constraints from co-evolving regulatory sequence targets during evolution. Chapter 3 discusses systematically mapping transcriptional activities of cis-regulatory sequences from Biosynthetic Gene Clusters (BGCs). Using a Streptomyces as a host, we found key regulatory features that affected gene expression, such as GC content, transcription start sites, and sequence motifs. We further explored regulation of BGC derived regulatory sequences by expressing global regulatory factors and screening for regulator sequences with altered expression levels. Finally, Chapter 4 highlights recent studies that made key contributions towards elucidating and modulating bacterial gene regulatory networks and reviews the current state of microbial systems biology and gene regulation. Together, the results and discussions presented in this dissertation seeks to further advance the current knowledge of sequence-function relationships of microbial regulatory components to enable better understanding, modeling, and rational engineering of bacterial gene regulation.

Over the past several years, the fields of synthetic biology and machine learning have demonstrated marked advances in the scale of their capabilities and the success of their applications. The work presented in this thesis focuses on the translation of recent advances in machine learning toward new applications in synthetic biology. In particular it is argued that the needs of synthetic biology researchers and practitioners are well met by a class of generative machine learning models, and that the scale of synthetic biology capabilities allows for their successful application across multiple domains of interest. In Chapter 1, a novel algorithm utilizing Markov Random Fields is used to, for the first time, design functional synthetic overlapping pairs of genes with potential applications for improved biological robustness and biosafety. In Chapter 2, motivated by a desire to extend the scope of protein sequence modeling to a greater range and diversity of protein sequences, a variant of a variational autoencoder model is used to project hundreds of millions of protein sequences into a continuous latent space with potentially useful representation features. Finally, in Chapter 3, we move beyond the realm of protein sequences to define a probabilistic species-specific model of regulatory sequences and explore this model’s utility for the challenging task of gene expression prediction for non-model bacterial organisms. Machine learning models presented in this thesis represent novel applications of models traditionally applied to data in the domains of images, text or sound toward addressing challenging problems in biology. Particular attention is devoted to the challenging task of utilizing large amounts of unlabeled data present in metagenomic sequences and the genomes of poorly characterized bacteria in the hope of improving researchers’ abilities to manipulate complex biological phenomena.

In eukaryotes, sequences that code for the amino acid structure of proteins represent a small fraction of the total sequence space in the genome. These are referred to as coding sequences, whereas the remaining majority of the genome is designated as noncoding. Studies of translation, the process in which a ribosome decodes a coding sequence to synthesize proteins, have primarily focused on coding sequences, mainly due to the belief that translation outside of canonical coding sequences occurs rarely and with little impact on a cell. However, recently developed techniques such as ribosome profiling have revealed pervasive translation in a diverse set of noncoding sequences, including long noncoding RNAs (lncRNAs), introns, and both the 5’ and 3’ UTRs of mRNAs. Although proteins with amino acid sequences derived partially or entirely from noncoding regions may be functional, they will often be nonfunctional or toxic to the cell and therefore need to be removed. Translation outside of canonical coding regions may further expose the noncoding genome to selective pressure at the protein level, leading to the generation of novel functional proteins over evolutionary timescales. Despite the potentially significant impact of these processes on the cell, the cellular mechanisms that function to detect and triage translation in diverse noncoding regions, as well as how peptides that escape triage may evolve into novel functional proteins, remain poorly understood.This thesis will describe novel findings that offer new insight into the process of noncoding translation mitigation revealed by a combination of high-throughput systems-based approaches and validated by biochemical and genetic approaches. Chapter 1 will discuss general concepts in the translation of noncoding sequences and the relevant cellular systems and impacts on human health. Chapter 2 will discuss the results of a high-throughput reporter assay investigating translation in thousands of noncoding sequences from diverse sources. The results discussed in this chapter revealed two factors involved in the mitigation of proteins derived from noncoding sequences: C-terminal hydrophobicity and proteasomal degradation. Chapter 3 will build on Chapter 2 and discuss the results of a genome-wide CRISPR/Cas9 knockout screen that identified the BAG6/TRC35/RNF126 membrane protein chaperone complex as a key cellular pathway in the detection and degradation of proteins with translated noncoding sequences. Having identified the BAG6 complex as targeting a specific reporter of translation of the 3’ UTR in the AMD1 gene, a series of knockout cell lines validated these results and demonstrated the participation of two additional genes, SGTA and UBL4A. Through coimmunoprecipitation western blots and rescue assays with flow cytometry as a readout, we confirmed physical interaction between BAG6 and the 3’ UTR of AMD1, and a similar experiment confirmed interaction between BAG6 and a readthrough mutant of the SMAD4 tumor suppressor gene. Finally, by combining our high-throughput reporter library with our BAG6 knockout cell line, we demonstrated that BAG6 targets hydrophobic C-terminal tails in many noncoding sequences of diverse origin. Finally, Chapter 4 will discuss the evolutionary perspective of noncoding translation through analyses of the sequence content of human and mouse genomes. The findings of this chapter demonstrate a significant trend for increased uracil content in noncoding regions of the genome, which frequently results in the translation of hydrophobic amino acids. We also find that many functional translated noncoding peptides localize to membranes, providing a theoretical link between the shuttling of translated noncoding sequences to a protein complex involved in membrane protein quality control and the emergence of newly evolving proteins from the noncoding genome.

This thesis studies the exhibition device, the structure that manages the appearance of what-is-shown. It focuses on the transition from taxonomic to immersive display, two modes with a particular visibility regime in each case. The first shows animals and plants according to a scientific classification, the second exhibits the specimens in a simulation of their original habitat through artificial environments built with a naturalistic appearance. I demonstrate that the transition between these two modes has transformed our visual culture in a similar way to cinema, photography or artistic collections and I propose an ontology of the exhibition device based on four of its main operations: the management of visibility, the regulation of the intensity of appearing, the camouflage of oneself and the generation of space. Patented by Carl Hagenbeck in 1896, the Panorama was the first immersive model and marks the entry of zoos into the entertainment industry. I explain how since then, living collections cannot show their specimens without previously building the illusion in which to insert them. With an emphasis on the material culture surrounding zoos and gardens and the architecture of their devices, I analyze three key materials: iron, artificial stone and glass, as well as their impact on various transatlantic living collections. Ultimately, the construction of the illusion connects living collections with the genealogy of the artificial and immersion in force since the 19th century, a crucial link of this research with fields of study such as environmental humanities and Anthropocene studies. Dispositivos de exhibición de colecciones vivas: La contribución de zoológicos y jardines botánicos a la cultura visual transatlántica, siglos XIX-XXI. Esta tesis estudia el dispositivo de exhibición, la estructura que gestiona el aparecer de lo-que-se-muestra. Se enfoca en la transición de la exhibición taxonómica a la inmersiva, dos modos con un régimen de visibilidad particular en cada caso. El primero muestra a los animales y las plantas de acuerdo a una clasificación científica, el segundo exhibe los ejemplares en una simulación de su hábitat original a través de entornos artificiales construidos con apariencia naturalística. Demuestro que la transición entre estos dos modos ha transformado nuestra cultura visual de forma similar al cine, la fotografía o las colecciones artísticas y propongo una ontología del dispositivo de exhibición basada en cuatro de sus operaciones principales: la gestión de la visibilidad, la regulación de la intensidad del aparecer, el camuflaje de sí mismo y la generación de espacio. Patentado por Carl Hagenbeck en 1896, el Panorama fue el primer modelo inmersivo y supone la entrada de los zoológicos en la industria del entretenimiento. Expongo cómo desde entonces, las colecciones vivas no pueden mostrar sus ejemplares sin construir previamente la ilusión en la cual insertarlos. Con énfasis en la cultura material que rodea a zoológicos y jardines y la arquitectura de sus dispositivos, analizo tres materiales claves: el hierro, la piedra artificial y el cristal, así como su impacto en diversas colecciones vivas transatlánticas. En definitiva, la construcción de la ilusión conecta a las colecciones vivas con la genealogía de lo artificial y la inmersión vigente desde el siglo XIX, un vínculo crucial de esta investigación con campos de estudio como las humanidades ambientales y los estudios del Antropoceno.

Os isolados de nematóides entomopatogênicos provenientes do solo de Monte Negro (RO) receberam o prefixo LPP seguido de um número sequencial. As linhagens de bactérias simbiontes receberam o prefixo MN como referência ao local de isolamento. As culturas dos isolados de nematóides entomopatogênicos LPP foram estabelecidas em nosso laboratório, sendo necessário cultivar também o lepidóptero Galleria mellonella para as infeções visando manter os nematóides em cultura. Os isolados, tanto de nematóides como das bactérias, foram identificados através do sequenciamento de genes ribossômicos (ITS 1 e 2 além do 5.8S para os nematóides e 16S para as bactérias). Análises integradas, abrangendo morfologia (em colaboração com a Dra. Claudia Dolinski e Inês Machado) e análises filogenéticas permitiram a identificação dos nematóides. Essas análises morfomoleculares mostram que as linhagens não podem ser descritas como novas espécies, baseadas apenas no local de isolamento, mas como pertencentes à espécies já descritas e bem caracterizadas. As linhagens de nematóides foram identificadas como pertencentes a duas espécies do gênero Heterorhabditis (Rhabditida): Heterorhabditis baujardi (LPP5, 7, 8, 10 e 11) Heterorhabditis indica (LPP1, 2, 3, 4, 9). A bactéria entomopatogênica simbionte isolada a partir do nematoide Heterorhabditis baujardi linhagem LPP7 foi identificada como pertencente à espécie Photorhabdus luminescens subsp. luminescens linhagem MN7. Análises filogenéticas mostram que há similaridade entre o isolado bacteriano e as outras linhagens já descritas de Photorhabdus luminescens subsp. luminescens. Ao mesmo tempo em que eram isoladas as bactérias simbiontes, ocorreu o isolamento de outra espécie de bactéria forética dos nematóides entomopatogênicos, identificada após sequenciamento parcial do 16S, como pertencentes ao gênero Ochrobactrum. As linhagens isoladas foram denominadas OMN2, OMN3, OMN4. / The entomopathogenic nematodes isolates from Monte Negro (RO) soils received the prefix LPP followed by a sequential number. The strains of bacterial symbionts received the prefix MN as a reference to its isolation site. Cultures of LPP entomopathogenic nematodes were established in our laboratory and the need for suitable hosts were supplied by cultivating the lepidopteran Galleria mellonella. Nematode and bacterial isolates were identified through ribosomal genes (ITS 1 and 2, 5.8S for nematodes and 16S for bacteria) sequencing. Integrated analysis, including morphology (in collaboration with Dr. Claudia Dolinski and Inês Machado) and a phylogenetic approach allowed the identification of nematodes. These morpho-molecular analysis clearly showed that our strains can not be described as new species, solely based on the isolation site critheria. It should be described as belonging to previously described and well characterized species. The nematode strains were identified as belonging to two species of the genus Heterorhabditis (Rhabditida): Heterorhabditis baujardi (LPP5, 7, 8, 10 and 11) Heterorhabditis indica (LPP1, 2, 3, 4, 9). The entomopathogenic bacterial symbiont isolated from the nematode Heterorhabditis baujardi strain LPP7 was identified as belonging to the species Photorhabdus luminescens subsp. luminescens strain MN7. Phylogenetic analysis showed similarities between isolated bacterial strains and other previously described Photorhabdus luminescens subsp. luminescens strains. In paralel work, along with bacterial symbionts isolation, other entomopathogenic nematodes foretic bacteria were isolated and identified after partial sequencing of 16S, as belonging to the genus Ochrobactrum. The isolated strains were named OMN2, OMN3, OMN4.

Classified in the Lissamphibia, modern amphibians are the only non-amniote tetrapods living today. They consist of three morphologically distinct groups: the tailless frogs and toads (Anura), the limbless caecilians (Gymnophiona), and the tailed salamanders and newts (Urodela). With 205 species, the caecilians are highly specialized worm-like forms that live a fossorial lifestyle, with a relatively narrow distribution in the tropic rainforests of South America, Africa and Asia (Duellman and Trueb, 1994; Amphibiaweb, 2015). Salamanders, with 683 species, are widely distributed in the North America, Asia and Europe, with a few plethodontids extending to Central and South America (Duellman and Trueb, 1994; Amphibiaweb, 2015). Frogs are the most diverse amphibian groups, with 6644 species distributed over all continents except Antarctica (Duellman and Trueb, 1994; Amphibiaweb, 2015). Both frogs and salamanders develop a wide array of lifestyles, ranging from terrestrial, aquatic, fossorial to aboreal lifestyles (Duellman and Trueb, 1994). During ontogeny, amphibian larvae usually undergo a drastic post-embryonic shift into an adult form, a term known as metamorphosis. In salamanders, another developmental pathway – neoteny – also occurs, in which the larval morphology is retained in sexually mature adults (Duellman and Trueb, 1994; Rose, 2003). Because of the diverse lifestyles and developmental pathways, frogs and salamanders are often used as model systems in many fields of biology (e.g., evo-devo). Over a century, but especially in the past two decades, a wealth of frog and salamander fossils has been discovered from the Mesozoic and Cenozoic of East Asia (e.g., Noble, 1924; Young, 1936; Borsuk-Bialynicka, 1978; Gao, 1986; Dong and Wang, 1998; Gao and Shubin, 2001, 2003, 2012; Gao and Wang, 2001; Gao and Chen, 2004; Wang and Rose, 2005; Wang and Evans, 2006b; Zhang et al., 2009; Chen et al., 2016; this study). Some of these fossils represent the earliest members of many crown clades, including the earliest crown salamanders from the Middle Jurassic (~165 Ma, Gao and Shubin, 2003), the earliest salamandroid from the Late Jurassic, the earliest sirenid from the Late Jurassic (this study), and the earliest spadefoot toads from the late Paleocence (Chen et al., 2016). Other fossils also bear important anatomical, temporal and geographical information in understanding their evolution. Unfortunately, the importance of many of these fossils remains obscure in a phylogenetic context. For example, an early-middle Oligocene Mongolian spadefoot toad Macropelobates osborni (Noble, 1924) was discovered outside the current distribution of spadefoot toads, yet its phylogenetic position and its implication on spadefoot toad biogeography remain not well understood. A major reason for the poor understanding of these fossils can be attributed to a trend of dichotomy between morphological and molecular phylogenies on amphibians. Whereas morphologists and paleontologists sometimes use a relatively small morphological dataset to reconstruct relationships (e.g., Gao and Shubin, 2012; Henrici, 2013), large-scale phylogenies are almost always conducted with molecular data with only living taxa (e.g., Roelants and Bossuyt, 2005; Pyron and Wiens, 2011). Very few studies on amphibian phylogeny have combined morphological and molecular data together, and even fewer also combined fossils. Because of this, the positions of many important fossils remains unclear, and the evolutionary scenarios inferred from only living species can sometimes be inconsistent with fossil evidence. In this thesis, I adopt a total-evidence approach to understand the evolution of amphibians, especially frogs and salamanders. I will incorporate information from fossils, morphology and molecules together to reconstruct the relationships. Compared with studies with each individual datasets, this approach incorporates all available data in a single analysis, with a goal to reach robust and congruent results that allow further discussions on character evolution and biogeographic reconstruction. The inclusion of fossils directly into the combined analysis provides the time dimension that is independent from molecular data (Norell, 1992). The anatomical combination of fossils can represent intermediate forms that help to solve the “long branch” problems caused by highly specialized modern taxa. The morphological dataset, despite its much smaller size with molecular data, is the only link between fossils and modern taxa. The inclusion of key morphological characters in both reconstructing phylogenetic hypotheses and examining character evolution provide consistent results that allow discussion on the homology/homoplasy of a certain character without ambiguity. The molecular sequence data provides overwhelmingly large data on modern taxa for phylogenetic reconstructions compared with morphological data, which helps to reach a robust hypothesis. Although fossils contain no molecular data, the inclusion of molecular sequence data into the combined analysis does have an effect on the positions of fossil taxa. By altering the relationship “framework” of modern taxa, the character optimization of fossils and other taxa of a combined analysis also varies compared with results of morphology-only analysis, thus changing the positions of fossils. In the following five chapters, I will describe a number of fossil amphibian species, reconstruct three combined phylogenies, and use the results for discussions on character evolution and biogeography. In Chapter 1 and Chapter 2, I focus on a frog clade called spadefoot toads (Anura: Pelobatoidea). In Chapter 1, I provide descriptions on three important fossil spadefoot toads from the Cenozoic of East Asia and North America: Macropelobates osborni from the early-middle Oligocene of Mongolia, Prospea holoserisca from the latest Paleocene of Mongolia, and Scaphiopus skinneri from the middle Oligocene of the United States. In Chapter 2, I conduct a combined phylogenetic analysis of archaeobatrachian frogs, and discuss the evolution of the bony spade and the historical biogeography of spadefoot toads based on the results of the phylogeny. In Chapter 3, I describe a new fossil frog from the Early Cretaceous of Inner Mongolia, China. The unique morphology of the new fossil is distinct from previous Early Cretaceous frogs from the Jehol Biota of China. Results of the combined analysis show that the new frog represents a basal member of the Pipanura. Comparisons between the Early Cretaceous frogs from China, Spain and Brazil show a high diversity of species coupled with a high degree of endemism during the Early Cretaceous. I discuss in the phylogenetic context how early frogs gradually reach their postcranial body plan with a shortened vertebral column, loss of ribs, and specialized pelvic regions. In Chapter 4, I provide a brief review of Mesozoic fossil salamanders from northern China, and describe a new fossil from the Late Jurassic of Liaoning Province, China. I conduct a combined phylogeny of higher-level relationships of salamanders. The new fossil, despite its general-looking appearance, represents a basal member of the highly specialized eel-like neotenic family Sirenidae on the cladogram. I discuss character evolutions in the Sirenidae, and how the neotenic developmental pathway evolved in early salamanders. In Chapter 5, I conduct a combined phylogenetic analysis of the salamander suborder Cryptobranchoidea, consisting of the neotenic giant salamanders (Cryptobranchidae) and the metamorphic Asiatic salamanders (Hynobiidae). The new morphological matrix includes new characters that were previously less sampled in the hynobranchial region. The monophyly of the Hynobiidae are confirmed by the new analysis, and four unequivocal synapomorphies are found for the clade. An S-DIVA biogeographic reconstruction is conducted to disscuss the distributional patterns of the Hynobiidae.