Orientador: Vadim R. Viviani / Banca: Marcia Regina Brochetto Braga / Banca: Mauricio Bacci Junior / Banca: Etelvino José Henriques Bechara / Banca: Cleide Costa / Resumo: A partir dos coleópteros luminescentes, mais de 20 luciferases já foram clonadas e seqüenciadas. Dessas, a maioria é de lampirídeos das regiões Neártica e Paleártica, quatro de elaterídeos jamaicanos e uma de um fengodídeo asiático. Apenas outras cinco são oriundas da América do Sul, o continente mais rico em espécies de coleópteros luminescentes e o provável berço evolutivo de Lampyridae, a família com maior número de coleópteros luminescentes. Espécies desta região apresentam a maior variedade de cores de bioluminescência. No presente trabalho clonamos e seqüenciamos luciferases dos gêneros Brasilocerus sp., Phrixothrix sp. e Taximastinocerus sp., da família Phengodidae. Com base na análise filogenética dessas luciferases e outras já publicadas, concluímos que as luciferases das lanternas laterais e cefálicas são codificadas por genes parálogos, e propusemos um modelo para a evolução das cores da bioluminescência nessa família. Também determinamos o genoma mitocondrial de Pyrophorus divergens, membro da família Elateridae. A partir desse genoma e outros já publicados, analisamos a evolução da bioluminescência na superfamília Elateroidea sensu Lawrence e Newton (1995) e concluímos que essa pode ter surgido três vezes independentemente nesse grupo. / Abstract: From luminescent Coleopteran's, more than 20 luciferases have already been cloned and sequenced. Among them, most part is from lampyrids of Neartic and Paleartic regions, four are from Jamaican elaterids and one is from Asiatic phengodids. Just five are from South American species, the richest continent in luminescent Coleopteran's and, probably, the evolutionary cradle of Lampyridae. Species from this region display the greatest range of bioluminescence colors. At the present work, we cloned and sequenced luciferases from the genera Brasilocerus sp., Phrixothrix sp. and Taximastinocerus sp., from Phengodidae family. Based on the phylogenetic analysis of these genes and other already published, we concluded that head and lateral lantern luciferases are coded by paralogous genes, and we also proposed a model for bioluminescence color evolution in this family. We also sequenced the mitochondrial genome of Pyrophorus divergens, an Elateridae member. Based on this genome and other already published, we analysed the evolutionary history of bioluminescence in Elateroidea superfamily sensu Lawrence e Newton (1995) and concluded that it could have appeared three times independently in this group. / Doutor

Abstract Mitochondria contain a maternally inherited 16,568bp genome (mtDNA) that encodes for 13 out of more than 70 subunits of complexes of the respiratory chain that produce ATP by oxidative phosphorylation (OXPHOS). As a byproduct of OXPHOS, reactive oxygen species (ROS) are formed, which may play a role in ageing. MtDNA has accumulated numerous polymorphisms during evolution, leading to haplogroups characterized by ancient polymorphisms and defined by letters. MtDNA polymorphisms are thought to be neutral, but some may be slightly deleterious or even advantageous and may influence phenotypes of complex traits. Interestingly, several complex traits such as longevity and maximal aerobic power show maternal inheritance. Associations between mtDNA polymorphisms and longevity have been reported, but no systematic study has been made of the role of mtDNA in longevity. In addition, there are no previous reports on mtDNA haplogroups in elite athletic performance. Associations are demonstrated here between mtDNA haplogroups J, K and U and longevity in Finns. Interestingly, subhaplogroup J2 and haplogroup K, which were found in increased frequency among the 225 very old subjects studied, were not found among the 52 endurance athletes but were present in 11% of the 89 sprint athletes Uncoupling of OXPHOS reduces ATP and ROS production. Thus, a mitochondrial genome with a higher level of uncoupling may promote longevity but may not be favourable in situations that require a high level of ATP production, such as elite endurance performance. A more detailed analysis also showed an association between a combination of three common mtDNA polymorphisms and longevity in both the Finns and the Japanese, providing the first epidemiological support for the assumption that the nature of a mutation is determined by interactions with other mutations in mtDNA. In addition, a systematic approach was applied to study the role of mtDNA in longevity. Association analyses of mtDNA allele combinations in longevity revealed that the mtDNA control region, the tRNA and rRNA genes and the nucleotide repeats in mtDNA may play a role in longevity, since the alleles and allele combinations that showed the strongest associations with longevity, either negative or positive, were among these genes. Differences in overall variation in mtDNA between the very old and their controls were also studied, revealing more differences at synonymous (silent) sites than at non-synonymous (amino acid altering) sites. The findings support previous data suggesting that certain mtDNA haplogroups are associated with longevity. In addition, those haplogroups that increased in frequency among the very old Finns were not found among Finnish endurance athletes. Also, a novel systematic approach was applied to study mtDNA alleles, allele combinations and overall sequence variation in longevity, suggesting that there are interactions between various mtDNA positions and that the tRNA and rRNA genes and short tandem repeats in mtDNA may play a role in longevity.

The Testis Specific Serine Kinases (Tssks) are a family of proteins that show testis and sperm-specific expression. Members of this family are most conserved among mammals, however there are homologs in vertebrates like birds and amphibians, chordates, and other invertebrates like insects and cnidarians. This specific expression suggests that these kinases are highly regulated. Analysis of murine and human Tssk1, Tssk2, and Tssk6 sequences show that these genes are comprised of one exon each, suggesting they are retrotransposons. The expression of these genes shows their importance, since many retrotransposons are silenced due to the foreign nature of the DNA, and knock-out mouse models have shown that these kinases are required for fertility. Understanding the properties of these kinases not only expands our scientific knowledge, but also lends itself to understanding fertility issues in men as well as being a contraceptive target. We looked at an epigenetic regulation factor, DNA methylation at CpG dinucleotides, to see if this caused the testis-specific gene expression we saw. Tssk2 and preliminary results from Tssk1 showed that there is no differential methylation at CpG dinucleotides or between tissues. Preliminary results for Tssk6 did show one site that may be differentially methylated, thus the tissue specific expression. We then started looking further into biochemically characterizing TSSK1 and TSSK2 to determine functionally relevant sites and new substrates. Understanding how these kinases function in sperm is relevant in our understanding in the fertility field and poses new targets for developing contraceptives.

Most phylogenetic trees are inferred using time-reversible evolutionary models that assume that the relative rates of substitution for any given pair of nucleotides are the same regardless of the direction of the substitutions. However, there is no reason to assume that the underlying biochemical mutational processes that cause substitutions are similarly symmetrical. Here, we evaluate the effect on phylogenetic inference in empirical viral and simulated data of incorporating non-reversibility into models of nucleotide substitution processes. I consider two non-reversible nucleotide substitution models: (1) a 6-rate nonreversible model (NREV6) that is applicable to analyzing mutational processes in double-stranded genomes in that complementary substitutions occur at identical rates; and (2) a 12-rate non-reversible model (NREV12) that is applicable to analyzing mutational processes in single-stranded (ss) genomes in that all substitution types are free to occur at different rates. Using likelihood ratio and Akaike Information Criterion-based model tests, we show that, surprisingly, NREV12 provided a significantly better fit than the General Time Reversible (GTR) and NREV6 models to 21/31 dsRNA and 20/30 dsDNA datasets. As expected, however, NREV12 provided a significantly better fit to 24/33 ssDNA and 40/47 ssRNA datasets. I tested how non-reversibility impacts the accuracy with which phylogenetic trees are inferred. As simulated degrees of non-reversibility (DNR) increased, the tree topology inferences using both NREV12 and GTR became more accurate, whereas inferred tree branch lengths became less accurate. I conclude that while non-reversible models should be helpful in the analysis of mutational processes in most virus species, there is no pressing need to use these models for routine phylogenetic inference. Finally, I introduce a web application, RpNRM, that roots phylogenetic trees using a non-reversible nucleotide substitution model. The phylogenetic tree is rooted on every branch and the likelihoods of each rooting are determined and compared with the highest likelihood tree being identified as that with the most plausible rooting. The rooting accuracy of RpNRM was compared to that of the outgroup rooting method, the midpoint rooting method and another non-reversible model-based rooting method implemented in the program IQTREE. I find that although the RpNRM and IQTREE reversible model-based methods are not as accurate on their own as outgroup or midpoint rooting methods, they nevertheless provide an independent means of verifying the root locations that are inferred by these other methods.

Ciliates provide a powerful system within microbial eukaryotes in which molecular genealogies can be compared to detailed morphological taxonomies. Two groups with such detailed taxonomies are the Colpodea and the Halteriidae. There are about 200 described Colpodea species that are found primarily in terrestrial habitats. In Chapters 1 and 2, taxon sampling is increased to include exemplars from all major subclades using nuclear small subunit rDNA (nSSU-rDNA) sequencing. Much of the morphological taxonomy is supported, but extensive non-monophyly is found throughout. The conflict between some nodes of the nSSU-rDNA genealogy and morphology-based taxonomy suggests the need for additional molecular marker. In Chapter 3, character sampling is increased using mitochondrial small subunit rDNA (mtSSU-rDNA) sequencing. The nSSU-rDNA and mtSSU-rDNA topologies for the Colpodea are largely congruent for well-supported nodes, suggesting that nSSU-rDNA work in other ciliate clades will be supported by mtSSU-rDNA as well. Chapter 4 compares the underlying genetic variation within two closely related species in the Halteriidae with increased taxon and molecular sampling using nSSU-rDNA and internally-transcribed spacer (ITS) region sequencing. The morphospecies Halteria grandinella shows extensive genetic variation that is consistent with either a large effective population size or the existence of multiple cryptic species. This pattern contrasts with the minimal of genetic variation in the morphospecies Meseres corlissi. Chapter 5 discusses the congruence and incongruence among morphological and molecular data in ciliates. Most of the incongruence occurs where there is little statistical support for the molecules, or where molecular data is consistent with alternative morphological hypotheses. Chapter 6 reviews the data for sex, or lack thereof, in the Colpodea, a potentially ancient asexual group where sex was regained in a derived species. In Chapter 7, four ciliate clades are redefined using the PhyloCode.

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Abordagens integrativas considerando diferentes dimensões da diversidade (p.ex., taxonômica, funcional, ou filogenética) cada vez mais estão sendo utilizadas para (1) avançar o nosso conhecimento sobre os mecanismos que criam e mantém a biodiversidade, e (2) elucidar a distribuição da biodiversidade tanto em áreas geográficas de interesse como dentro de áreas protegidas. De fato, entender como a biodiversidade se distribui no espaço e como ela é mantida ao longo do tempo é fundamental para embasar o planejamento de áreas protegidas e corredores ecológicos, assim como auxiliar no manejo de espécies invasoras, restauração de habitats degradados e manejo de ecossistemas. Nessa perspectiva, os objetivos centrais desta tese foram: (1) avaliar os mecanismos ecológicos e evolutivos, que potencialmente influenciam a diversidade beta taxonômica e filogenética de árvores nas florestas Atlânticas do sul do Brasil, e (2) avaliar como os componentes taxonômicos e filogenéticos se distribuem ao longo destas florestas, e como eles são representados dentro da rede regional de áreas protegidas. Para tal, utilizei modelagem de equações estruturais (capítulo 1) para testar a validade de uma rede de hipóteses ligando dados e teoria. No capítulo 1, avaliei a relação entre a diversidade beta taxonômica e filogenética, e como elas se relacionam com a riqueza de espécies, filtragem ambiental, espaço geográfico e estrutura filogenética (agrupamento filogenético). Nesse capítulo, concluí que a diversidade beta taxonômica é influenciada principalmente pelos gradientes altitudinais e climáticos, enquanto que a diversidade beta filogenética é determinada também pelo grau de agrupamento filogenético, em nível local, que provavelmente reflete o conservadorismo de nicho dentro das linhagens e distúrbio humano, que historicamente tem conduzido as florestas estudadas a um processo de homogeneização biótica. Em relação ao segundo objetivo, utilizei uma abordagem integrativa para predizer e mapear os componentes taxonômicos e filogenéticos da diversidade de árvores e, em seguida, avaliar a efetividade da rede de áreas protegidas em representar tais componentes nas florestas Atlânticas do sul Brasil. Nesse capítulo, concluí que as áreas protegidas são insuficientes para preservar adequadamente a biodiversidade de árvores nestas florestas. Sugeri que a expansão da rede em direção as áreas de alta singularidade taxonômica e filogenética, como definidas aqui, poderia aumentar, ao mesmo tempo, a representação da riqueza de espécies, da diversidade beta e da história evolutiva das espécies estudadas. Sugeri também que a inclusão de áreas de alta insubstituibilidade, em termos de história evolutiva, poderia ajudar a aumentar a proteção da diversidade de características e do potencial evolutivo das espécies. / Integrative approaches considering different dimensions of biodiversity are increasingly being used in ecology and conservation to (1) advance our knowledge about the mechanisms underlying current patterns of biological diversity, and (2) elucidate the distribution of biodiversity in geographical areas of interest, and within the protected areas. Indeed, understanding how biodiversity is distributed in space and how it is maintained over time is critical to support the planning of protected areas and ecological corridors as well as assist the management of invasive species, the restoration of degraded areas and ecosystem management. In this perspective, the central goals of this thesis were: (1) to evaluate the ecological and evolutionary mechanisms that potentially influence the tree taxonomic and phylogenetic beta diversity in Atlantic forests located in southern Brazil, and (2) to evaluate how the taxonomic and phylogenetic diversity components are distributed across these forests, and how they are represented within the regional network of protected areas. For this, I used structural equation modeling (chapter 1) to test the validity of a network of hypotheses linking data and theory. In the chapter 1, I evaluate the relationship between taxonomic and phylogenetic beta diversity, and how they are related to species richness, environmental filtering, geographical space and phylogenetic structure (phylogenetic clustering). In this chapter, I conclude that taxonomic beta diversity (at the study scale) is mainly driven by the altitudinal and climatic gradients, while phylogenetic beta diversity is also determined by the degree of phylogenetic clustering at local level, more likely reflecting niche conservatism within lineages and human disturbance that has historically conducted the studied forests to a process of biotic homogenization. In relation to the second goal, I used an integrative approach to predict and map the taxonomic and phylogenetic components of tree diversity, and to assess the effectiveness of the protected areas network in representing these components in the Atlantic forests. In this chapter, I conclude that protected areas are insufficient to adequately preserve the tree biodiversity in these forests. I suggest that expanding the network towards the areas of taxonomic and phylogenetic uniqueness, as defined here, could increase the representation of species richness, beta diversity and evolutionary history of angiosperm trees at the same time. Furthermore, the inclusion of areas of high irreplaceability in terms of evolutionary history could help to improve the protection of feature diversity and evolutionary potential of species.

The phylogenetic reconstruction has noted great development in recent decades. The development of computers and device for sequencing biopolymers have been an enormous amount od phylogenetic data from different sources and different types. The scientists are trying to reconstruct a comlet tree of life from these data. The phylogenetic supertree are theoretically this option because a supertree alow a combination of all information gathered so far – in contras to the phylogenetic trees. This thesis present the method of reconstruction supertrees using average konsensus method.